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UNIVERSIDADE DE LISBOA
Faculdade de Ciências
Departamento de Biologia Vegetal
Analysis of teashirt mutants affecting cell
proliferation in
Drosophila melanogaster
Silvia Alexandra Barbosa Jacome Pimentel
DOUTORAMENTO EM BIOLOGIA (Genética)
Lisboa 2010
UNIVERSIDADE DE LISBOA
Faculdade de Ciências
Departamento de Biologia Vegetal
Analysis of teashirt mutants affecting cell
proliferation in
Drosophila melanogaster
Silvia Alexandra Barbosa Jacome Pimentel
DOUTORAMENTO EM BIOLOGIA (Genética)
Supervisors: Rui Gomes
Laurent Fasano
Lisboa 2010
II
Por ti, Alexandre.
The most important thing in life is not the triumph but the struggle.
IV
This PhD work was support by my PhD grant (SFRH/BD4861/2001 and
SFRH/BD/13233/2003) from the POCI 2010 (Fundo Social Europeu_ FSE) approved by
Fundação para a Ciência e a Tecnologia (Portugal), and also by the collaboration between the
Rui Gomes lab and Laurent Fasano labs (CNRS and the Association pour la Recherche Contre le
Cancer and La Ligue, France).
V
VI
Acknowledgements
I would like to thank to all the people that directly or indirectly contributed for the
concretisation of this work:
Ao meu Orientador, Professor Rui Gomes, por me ter acolhido no seu grupo, pela
oportunidade que me deu em realizar este trabalho no seu laboratório, pelo seu esforço em
arranjar alternativas que permitissem ultrapassar as nosssas limitações financeiras e técnicas,
por todas as sugestões e discussões. E pela sua boa disposição e humor.
Au Laurent Fasano, je lui remercie pour m’avoir accueilli dans son labo, pour toute sa
disponibilité et patience avec moi, pour me faire confiance et me donner confiance dans mes
résultats, pour toutes les suggestions et discussions et pour me montrer une autre forme de
faire sience.
À Fundação para a Ciência e Tecnologia (FCT), pelo apoio financeiro, sem o qual teria
sido impossível a realização deste trabalho.
To the Centro de Genética e Biologia Molecular (CGBM) and to the Institut de Biologie
du Développement du Luminy (IBDML) for giving me conditions for the development of this
work.
To all the people that contributed with the necessary reagents needed for the
elaboration of this work, mainly to Dr. Alvaro Tavares, Dr. Claudio Sunkel, Dr.Roger Karess,
Dr. Steve Kerridge, Dr. Stephen Cohen, Dr. Armel Gallet, Dra. Ruth Lehmann, Dr. E. McCarthy
and to Bloomington Drosophila stock Centres.
Aos meus colegas do CGBM, pela entre-ajuda, pela amizade, pelo convívio e pelas
“brincadeiras”, que tornaram o nosso dia-a-dia mais agradável e produtivo. Um OBRIGADO
especial para os colegas de grupo, Cristina e Paulo, e para a minha “mamã” científica, Isabel,
que me revelou o maravilhoso mundo das moscas e me fez dar os primeiros passos no ciclo
celular, com muita paciência e enorme boa disposição.
À toutes les personnes qui m’ont aidé dans l’IBDML, sans oublier les personnes des
plate-formes techniques qui ont facilité énormément mon séjour à Marseille. Un gros MERCI à
tous les membres de l’équipe KF que j’ai connus (Laurent, Natalie, Xavier, Christine, Pierre,
Virginia, Hervé, Elise, Steve, Delphine, Mylene, Bernard, Sébastien, Lucile et Fred), pour votre
Acknowledgements
accueil et patience d’essayer entendre mon luso-français, pour tous les conseils et discussions
et pour votre amitié. Je ne peux pas oublier de vous remercier pour tous les bons moments,
les blagues, les rigolades et les petites fêtes qui m’ont fait rester presque un année de plus
que le prévu! Je vous remercie la magnifique soirée que vous avez organisée avant mon
départ et que je ne l’oublierai jamais. Je veux exprimer ma gratification à Laurent et à
Christine pour tout le temps dépensé à regarder et à discuter mes résultats et surtout me
papier, à Delphine pour tous les protocoles, les anticorps, les solutions et à Steve pour tous les
stocks de moches. Un Gros Merci à vous tous !
Ao Dr. Álvaro Tavares e a todos os membros da sua Equipe que tive o prazer de
conhecer (à Susana, à Célia, à Mariana, à Cláudia e ao Paulo), gostaria de agradecer o facto de
nos terem aberto a porta sempre que precisámos, inclusive para a realização das experiências
de RNAi.
A toda a minha família e amigos, inclusive aos que infelizmente já partiram, um
obrigado pelos telefonemas e mails que encurtaram a distância e pelo apoio que prestaram.
Um agradecimento aos meus pais e irmão por gostarem de mim e pelo carinho que sempre me
deram, cada um à sua maneira. Um agradecimento aos quatro avos do Alex por todas as
vezes que ficaram com ele para que eu terminasse a tese. E à minha prima Sara pelas
enumeras vezes que fiquei em sua casa, nesta minha vida de viagens.
Finalmente, porque os últimos são os primeiros, obrigada César, por me aturares, pelo
teu apoio, por estares sempre a meu lado, pelo teu amor e ao Alex, apesar de todo o trabalho
e desgaste associado, obrigado por teres nascido e seres meu filho. Os teus sorrisos e
beijinhos são a motivação para não desistir e continuar lutando para ultrapassar a pouco e
pouco os obstàculos que vão surgindo.
VIII
Objectives, Methodological Approach and Structure of the Thesis
The main goal of this thesis was to clarify the role of the transcription factor Teashirt
(Tsh) during cell cycle progression and proliferation. Previous work in our lab showed that the
late larval lethal mutants tshA3-2-66 and tshNG1 present atrophied brains and strong reduced
imaginal discs with a strong mitotic arrest in a metaphase-like stage (Salazar, 2000). We
performed a better characterisation of the mitotic phenotype by immunostaining and by the
analysis of the phenotype of tsh in double mutant constructs. The depletion of tsh in S2 cells
by the interference RNA technique (RNAi) was performed in order to compare with the
phenotype observed in tsh mutant neuroblasts. The analysis of the expression pattern in
mutant brains by microarrays was also performed in order to identify an obvious Tsh target
among cell cycle genes. Unfortunately, no canonical cell cycle target gene was found to be
particularly upregulated or downregulated. Furthermore, our detailed characterisation of the
mitotic phenotype revealed only a few structural abnormalities or defects, not enough to cause
such a strong mitotic arrest. All these results are presented in chapter II (part IV) of this
manuscript.
In order to prove that tsh is the gene responsible for the mitotic arrest observed in
tshA3-2-66 neuroblasts we performed P-element excision mutagenesis to get a revertant. During
this mutagenesis scheme, we obtained news alleles and, among themselves, we identified a
potential tsh female sterile mutations causing atrophied ovaries and which do not lay eggs.
This interesting phenotype led us to study the tsh role during Drosophila oogenesis. The 5’
region of tsh gene was sequenced in three of the six female sterile lines obtained. It was
characterised the ovary phenotype and functional studies have been performed in vivo using
the UAS/Gal4 system. The mutagenesis was made in Rui Gomes’ lab and the characterisation
of the sterile female mutants was mostly performed in Laurent Fasano lab. All the results are
presented in chapter II (part I, II and III) of this manuscript.
Curiously, these independent studies of tsh function had given convergent results. We
concluded that tsh is required for the normal progression of the cell cycle in L3 neuroblasts and
depleted S2 cells, as in the proliferation of the follicle stem cells during oogenesis (this is
detailed discussed in chapter III).
During all this PhD work, the main concern was to prove that the tsh gene was required
for cell proliferation since the progression of the cell cycle was affected in the three Drosophila
systems (mutant neuroblasts, S2 cells and ovaries). Thus, the introduction of this manuscript
will start with a description of the tsh gene and their reported functions during Drosophila
development (Chapter I part I). The introduction will be followed by a overview of oogenesis,
with special incidence in the principal pathway required for the proliferation and maintenance
of the three type of stem cells found in the Drosophila germarium (germline stem cells, follicle
Objectives, Methodological Approach and Structure of the Thesis
stem cells and escort stem cells; chapter I part II). Finally, a brief review about the cell cycle
focalised in the metaphase/ anaphase transition will end the introduction (chapter I, part III).
The results will not be presented by chronological order. The major results were
obtained during the study of tsh in the follicle stem cells proliferation. Thus, chapter II
(results) will start with P-element excision mutagenesis performed to obtain the new tsh alleles
(part I). Next, it will be characterised and described the tsh function in follicle stem cells (part
II). A reference about the mutual repression between tsh and their paralog tiptop during
oogenesis will be presented afterwards (part III). The results will end with the presentation of
all the evidence implicating tsh in mitosis progression (part IV).
A final discussion will be presented in the chapter III. Here, the major conclusion will be
discussed, as well as the new questions raised with the results presented in this PhD work.
In this manuscript, I did not dedicated one chapter exclusively to the material and
methods used during my PhD work, since they are presented in each part of the results
(chapter II).
All the references mentioned in chapters I, II and III are presented in the end of this
manuscript.
Objective
The main goal of this thesis was to clarify the role of Tsh in cell cycle progression and to
identify putative cell cycle target. For that, it was studied the requirement of Tsh to the
progression of cell cycle using three Drosophila model systems: (1) ovaries to study follicle
stem cells proliferation during oogenesis and, (2) neuroblasts and (3) S2 cells to analyse the
progression of mitosis onset. In addition, during the development of this work, I proposed to
characterise, for the first time, the Tsh function in Drosophila oogenesis.
The results presented in this manuscript give evidence that Tsh is required for cell
proliferation in Drosophila neuroblasts, S2 cells and ovaries. Furthermore, it was showed that
Tsh acts through the Hedgehog pathway to mediate follicle stem cells proliferation in ovaries.
Finally, we demonstrated that both in neuroblasts or S2 cells the absence of Tsh arrest cells in
metaphase/ anaphase transition.
X
Table of Contents
Acknowledgements ....................................................................................................................................... VII
Objectives, Methodological Approach and Structure of the Thesis ........................................................................ IX
Table of Contents .......................................................................................................................................... XI
Liste of Figures............................................................................................................................................XIII
Liste of Tables.............................................................................................................................................. XV
Liste of Abbreviatures .................................................................................................................................. XVI
Resumo..................................................................................................................................................... XVII
Palavras Chave: ........................................................................................................................................... XX
Abstract .................................................................................................................................................... XVII
Key Word: .................................................................................................................................................. XXI
CHAPTER I_ INTRODUCTION
I- The teashirt (tsh) gene............................................................................................................................ 3
I_1- Characterisation of the tsh gene .................................................................................................................3
I_2- Expression patterning of the tsh mRNA and protein in Drosophila ...................................................................4
I_3- Modulators of the tsh expression during embryogenesis ................................................................................5
I_4- Tsh functions during Drosophila development...............................................................................................5
I_4.1- Homeotic function of Tsh: the identity of the trunk and the specification of the prothoracic identity..................6
I_4.2- Tsh in the segmental polarity: modulation of the Wingless pathway and regulation of the hedgehog pathway
target genes ...................................................................................................................................................8
I_4.3- Tsh and the midgut morphogenesis........................................................................................................11
I_4.4- Tsh in the Drosophila Pax6/ So/ Eya network during eye development .......................................................13
I_4.5.2- tsh promotes the identity of the proximal structures of wing and halteres discs ........................................16
I_5- tiptop as a tsh paralog ............................................................................................................................18
I_6- The vertebrate tsh family.........................................................................................................................20
II- Overview of Drosophila Oogenesis ....................................................................................................... 23
II_1- The Drosophila reproductive system ........................................................................................................23
II_2- Drosophila germarium offers the unique opportunity of to study three types of stem cells and their niches .......25
II_3- Origin of GSC, TF and CC........................................................................................................................27
II_4- Ovary stem cells and their molecular mechanisms of regulation ..................................................................28
II_4.1- Genes and Pathways that control GSC self-renewal, proliferation and differentiation ...................................28
II_4.1.1- BMP, Piwi and Yb from the niche to regulate the maintenance and division of the GSC..............................29
II_4.1.2- External factors: Nutrition.................................................................................................................32
II_4.1.3- Intrinsic factors in GSC maintenance and differentiation .......................................................................33
II_4.1.3.1- Translational factor: Pumilio (Pum) , Nanos (Nos), Vasa (Vas) and Pelota (Pelo) ...................................33
II_4.1.3.2- MicroRNA (miRNA) and repeat-associated small interfering RNA (rasiRNA) is important for GSC proliferation
and maintenance...........................................................................................................................................33
II_4.1.3.3- Junctional proteins: DE-cadherin, Armadillo (Arm) and Zero population growth (Zpg)............................34
II_4.1.3.4- Cell cycle regulators: Cyclin B (CycB) and Imitation switch (ISWI) ......................................................35
II_4.1.3.5- Differentiation promoting factors: Bag of marbles (Bam), Benign germ cell neoplasm (Bgcn), Orb, Sex
lethal (Sxl) and ovarian tumor (Otu) ................................................................................................................36
II_4.2- Genes and Pathways that control FSC self-renewal, proliferation and differentiation....................................38
II_4.2.1- Molecular mechanisms shared by GSC and FSC for self-renewal and proliferation ....................................38
II_4.2.2- Hedgehog (Hh) and Wingless (Wg), two critical signals for FSC maintenance and proliferation...................38
Table of Contents
II_4.2.2.1- Wingless (Wg) signalling is essential for FSC maintenance and proliferation .........................................40
II_4.2.2.2- Hedgehog (Hh) signalling is essential for FSC maintenance and proliferation ........................................40
II_4.3- Genes and Pathways controlling the maintenance of ESC and EC..............................................................43
II_5- Drosophila ovary as a model system in stem cells studies. .........................................................................43
III- Overview of Drosophila Cell Cycle....................................................................................................... 44
III_1- The Cell Cycle......................................................................................................................................44
III_2- M-phase..............................................................................................................................................45
III_2.1- Mitosis Promoting Factor (MPF) and Mitosis entry ..................................................................................48
III_2.1.1- CdK1 family ...................................................................................................................................49
III_2.1.2- Cyclin A.........................................................................................................................................50
III_2.1.3- Cyclin B.........................................................................................................................................51
III_3- The bipolar spindle and the kinetocore-microtubule attachment until the formation of the metaphase plate.....53
III_3.1- The Kinetochore ................................................................................................................................55
III_4- The anaphase promoting complex/ Cyclosome (APC/C).............................................................................56
III_4.1- The APC/C activators and substrate recognition.....................................................................................57
III_4.2- APC/C regulation ...............................................................................................................................57
III_5- The regulation of the cell cycle: the checkpoints ......................................................................................58
III_5.1- DNA damage checkpoint ....................................................................................................................59
III_5.2- Metaphase or Spindle assembly checkpoint (SAC) .................................................................................61
III_5.2.1- Models for spindle assembly checkpoint sense the defects ...................................................................63
III_5.2.1.1- Microtubule attachment model.......................................................................................................64
III_5.2.1.2- Tension model .............................................................................................................................65
III_5.2.2- Inhibition of APC/C by the spindle assembly checkpoint.......................................................................66
III_5.2.2.1- Recruitment of spindle checkpoint proteins to unattached/ untensed kinetochores ...............................68
CHAPTER II_ RESULTS
Part I: ........................................................................................................................................................73
New teashirt alleles obtained by P-element excision mutagenesis ........................................................................75
Part II: .......................................................................................................................................................91
Regulation of cell proliferation and patterning in Drosophila oogenesis by Hedgehog signalling depends on teashirt ...93
Part III:.................................................................................................................................................... 117
Teashirt and Tiptop repress each other in Terminal Filament Cells during Drosophila oogenesis............................. 119
Part IV: .................................................................................................................................................... 137
The knock-down of the teashirt gene causes a metaphase-like arrest in Drosophila larval neuroblasts and S2 cells.. 139
CHAPTER III_ DISCUSSION AND PERSPECTIVES
1- Tsh and its paralog Tio repress each other in the tip of the Drosophila ovary, and both are able to regulate En
expression.................................................................................................................................................. 173
2-Tsh regulates the expression of Hh and En at the Tip of the Drosophila germarium........................................... 174
3-Tsh mediating Hh pathway is required for FSC proliferation ........................................................................... 175
4-The metaphase/anaphase transition is compromised in the absence of Tsh...................................................... 177
5- Tsh/Hh signalling from flies to vertebrates.................................................................................................. 179
Concluding Remarks: ................................................................................................................................... 180
REFERENCE.............................................................................................................................................. 183
APPENDIX ............................................................................................................................................... 211
XII
List of Figures
CHAPTER I_ INTRODUCTION
Figure I_1- Tsh expression patterning during Drosophila embryogenesis ..............................................................4
Figure I_2- Tsh as a Hox co-factor in the segment identity of the trunk................................................................7
Figure I_3- Summary of gene interactions in the cells surrounding the middle constriction ...................................12
Figure I_4- Schematic representation of Drosophila leg imaginal discs and the adult leg.......................................15
Figure I_5- Schematic representation of Drosophila wing imaginal discs and the adult wing ..................................17
Figure I_6- Embryonic expression of Tio and Tsh.............................................................................................19
Figure I_7- The Teashirt family in Drosophila and vertebrates ...........................................................................21
Figure II_1- Diagram and a Drosophila ovary and ovarioles ..............................................................................24
Figure II_2- Drosophila germarium cross section showing the locations of germ line stem cells (GSC), follicle stem
cells (FSC), and their niches............................................................................................................................26
Figure II_3- Diagrams of the identified signalling pathways and intrinsic factors that regulate GSC maintenance and
differentiation in the Drosophila ovary..............................................................................................................31
Figure II_4- Diagrams of the identified signalling pathways and intrinsic factors that regulate follicle stem cells (FSC)
and escort stem cells (ESC) maintenance and differentiation in the Drosophila ovary .............................................39
Figure II_5- The Hedgehog pathway based on what is known in Drosophila ........................................................41
Figure III_1- Cell cycle phases. ....................................................................................................................45
Figure III_2- Diagram of mitosis in animal cells ..............................................................................................47
Figure III_3-The regulation of the MPF activity by phosporilation ......................................................................48
Figure III_4- Schematic representation of the G2 checkpoint............................................................................60
Figure III_5- Relationship of the SAC with the cell-cycle machinery...................................................................62
Figure III_6- The attachment process............................................................................................................63
Figure III_7- A Model for the Regulation of APC/CCdc20 by the Spindle Checkpoint.............................................69
CHAPTER II_ RESULTS
Part I
Figure 1- Diagram of the flies matting during P-element excision mutagenesis....................................................79
Figure 2- No mitotic anomalies were detected in neuroblasts from 8 P-element excision lines L3 lethal. ..................82
Figure 3- No mitotic anomalies were detected in neuroblasts from P-element excision lines lethal during pupae stage.
...................................................................................................................................................................82
Figure 4- Adult ∆P48/tshA3-2-66 and ∆P127/tshA3-2-66 ovaries showed a very strong and early arrest in oogenesis........83
Figure 5- Amplified and sequenced regions of the PlacW in tshA3-2-66 and ∆P126. ..................................................86
Figure 6- Morphologic aspects of ∆P126/ tshA3-2-66 flies. .....................................................................................88
Part II
Figure 1- en co-localises with a tsh-subset domain in Drosophila ovaries........................................................... 102
Figure 2- Expression level variation of tsh in ∆126/tshA3-2-66 ovarioles. .............................................................. 103
Figure 3- ∆126/ tshA3-2-66 ovaries display abnormal germline cyst encapsulation. ............................................... 105
Figure 4- Cell cycle progression is affected in ∆126/tshA3-2-66 ovarioles. ............................................................. 107
Figure 5- Tsh is required for the proper expression of En and Hh in the germarium. ........................................... 109
Figure 6- Ectopic expression of hh and tsh induces similar effects. ................................................................... 110
Figure 7- Tsh rescues the imperfect proliferation phenotype of ∆126/tshA3-2-66 mutants and allows the progression of
oogenesis until stage S5/S6.......................................................................................................................... 111
Supplementary Figure 1- Rescue of the ∆126/tshA3-2-66 proliferation phenotype by ectopic Hh............................ 116
List of Figures
Part III
Figure 1- Tsh and Tio co-repress each other. ................................................................................................. 124
Figure 2- Tsh and Tio rescue the imperfect proliferation phenotype of the ∆126/tshA3-2-66 mutant and allow the
progression of oogenesis until stage S5/S6..................................................................................................... 126
Figure 3- Localisation of En in wild-type, ∆126/tshA3-2-66 and tio473 germaria....................................................... 127
Figure 4- Ectopic expression of hh, tsh and tio induces a similar phenotype....................................................... 129
Supplementary Figure 1- Tsh and En in wild-type, ∆126/tshA3-2-66, tio473 and ∆126/tshA3-2-66;UAS-tio/hsGAL4
germaria. ................................................................................................................................................... 135
Part IV
Figure 1- Molecular organisation in tshNG1 and tshA3-2-66 mutants. ...................................................................... 150
Figure 2- Expression analysis of Tsh. ............................................................................................................ 151
Figure 3- Mitotic phenotypes in tsh mutant larval brains. ................................................................................ 152
Figure 4- Mitotic figures in preparations of larval CNS cells from wild-type and tsh homozygotes. ........................ 153
Figure 5- Cyclin A and B levels in wild-type and tsh mutant larval CNS cells. ..................................................... 154
Figure 6- Distribution of centromere CID and kinetochore components Bub1 and Rod in wild-type and tshNG1
neuroblast cells. .......................................................................................................................................... 156
Figure 7- Time course analysis of Tsh RNAi in Drosophila S2 Cells. ................................................................... 158
Figure 8- Abnormal mitotic figures observed in S2 TSH-RNAi cells.................................................................... 159
Supplementary Figure 1- Immunolocalisation of the Tsh protein in larval CNS cells.......................................... 166
Supplementary Figure 2- In situ cell death detection.................................................................................... 167
Supplementary Figure 3- Cuticules phenotype............................................................................................. 168
Supplementary Figure 4- Immunolocalisation of Polo in larval CNS cells ......................................................... 169
Supplementary Figure 5- Analisys of Polo expression by western blot............................................................. 169
Supplementary Figure 6- A CNS from L1, L2 and L3 larvae instars expressing tshlacZ stained with X-Gal. .......... 170
CHAPTER III_ DISCUSSION AND PERSPECTIVES
Figure 1- Tsh signalling to Follicle Stem cells (FSC) proliferation mediating Hh expression................................... 175
Figure 2- Proposed model to explain Tsh role in cell proliferation, mediating Hh expression signal in FSC and
neuroblasts................................................................................................................................................. 183
XIV
List of Tables
CHAPTER II_ RESULTS
Part I
Table1- Counting of F2 male that lost P-element based in the colour of the eyes. .................................................81
Table 2- Classification of the P-element excision line based in their lethality in trans-heterozygous with tshA3-2-66. ....81
Table 3- Fertility of the seven P-element excision lines with viable escapers, in Trans with the tshA3-2-66. .................84
Table 4- Fertility of the combination of the seven P-element excision lines with viable escapers in Trans between
them............................................................................................................................................................85
Table 5- List of the tsh alleles crossed with ∆P126 and tshA3-2-66 to test for the viability and fertility of the adult female.
...................................................................................................................................................................87
Supplementary table 1- PlacW excision results obtained in two independents mutagenesis from the tshA-3-2-66 allele
by Salazar (2000, refered in chapter IV of the results of this manuscript) and Pimentel (presented in this chapter). ..90
Part II
Table 1- List of the tsh alleles crossed with P{lacW}A3-3-66∆126 and tshA3-2-66 to test for the viability and fertility of the
adult female. .............................................................................................................................................. 104
Table 2- Quantification of the ∆126/ tshA3-2-66and the ∆126/ tshA3-2-66;UAS-tsh13/hs-Gal4 ovaries phenotype. ........ 112
Supplementary Table 1- Quantification of the positive P-Histone H3 FC in the first 5 egg chambers of wild-type (n=69)
and ∆126/ tshA-3-2-66(n=98) ovaries................................................................................................................ 116
Part IV
Table 1- Mitotic index (number of mitotic cells per optic field) and percentage of anaphase figures in the larval CNS of
wild-type, tsh, rod x5, bub1, tsh rodx5 and tsh bub1 mutants........................................................................... 157
Table 2- List of the most significant gene culters altered in tshNG1 homozygous neuroblats microarrays with the
respectived P value and total number of genes include. .................................................................................... 160
Table 3- The most up- and down-regulated genes in tshNG1 homozygous neuroblats microarrays, the putative Tsh
target genes are also listed with the respective lower bound of fold change (LBFC), The positive LBFC represent the up
regulated genes while the negative LBFC represent the downregulated genes. .................................................... 161
APPENDIX
Table I- Results of the up- or down-regulation genes in tshNG1 homozygous neuroblats which have been ordered by
lower bound of fold change (LBFC), after using a cut-off of 1.3. The positive LBFC represent the up regulated genes
while the negative LBFC represent the downregulated genes. Only the genes with the absolute value of LBFC upper
than 1.3 are represented. ............................................................................................................................. 213
Table II- Results of the gene ontology clusters with a P-value <0.001 in tshNG1 homozygous neuroblats microarrays
after using a cut-off of 1.3. The positive lower bound of fold change (LBFC) represents the up regulated genes while
the negative LBFC represent the downregulated genes. Only the genes with the absolute value of LBFC upper than 1.3
are represented........................................................................................................................................... 227
Liste of Abbreviatures
µg microgram µl microliter
µm micrometer APC/C Anaphase promoting complex/ cyclosome
bp base pair CC Cap cells
Cdc genes Cell division cycle genes Cdk Cyclin-dependent kinase CNS Central nervous system
D. melanogaster Drosophila melanogaster DAPI 4’,6’-Diamidino-2-phenilindole DNA Desoxiribonucleic acid
dsRNA double-stranded RNA en engrailed
ESC Escort stem cells EC Escort cells
FSC Follicle stem cells FC Follicle cells
FITC Flurescein isothiocyanate GSC Germline stem cells
hh hedeghog hs heat-shock promoter
IGS Inner germarial sheath cells ihog interference hedgehog KDa Kilo-Dalton (1KDa=1000 Da) MPF Mitosis promoter factor
mRNA messenger Ribonucleic acid MT Microtubule
NEBD Nuclear envelope breakdown PBS Phosphate buffer saline pH=7.4
PBST PBS with 0,1% Triton X-100 PCR Polymerase chain reaction
PPH3 Phospho-histone H3 RNA Ribonucleic acid RNAi RNA interference SAC Spindle assemby checkpoint
SC Stem cells SDS Sodium dodecyl sulphate Ser Serine TF Terminal Filament cells
Thr Threonine tio tiptop tsh teashirt Tyr Tyrosine
UAS Upstream activator sequence X-Gal X-gal-5-bromo-4-chloro-3-indolyl-beta-D-galactopiranosideo
WT Wild-type
Resumo
O gene teashirt (tsh) de Drosophila codifica para uma proteína de 116 KD com três
motivos atípicos do tipo Zinc finger (Cx2Cx12HMx4H) espaçados. Este gene foi, inicialmente,
referido como sendo necessário para a correcta especificação dos segmentos do tronco,
durante a embriogénese de Drosphila, em colaboração com os genes Hox. Adicionalmente, em
Drosophila, o gene tsh foi implicado na morfogénese do intestino médio, durante o
desenvolvimento da parte proximal dos apêndices e durante a especificação dos olhos no
adulto.
Este trabalho teve como principal objectivo clarificar a função do gene tsh na
proliferação celular em Drosophila. Para tal, estudou-se o efeito da perda de função tsh em
três contextos celulares de Drosophila: (1) nos ovários, com o intuito de estudar a proliferação
das células estaminais foliculares (FSC) do germarium; (2) nos neuroblastos e (3) nas células
S2, com o fim de analisar a progressão da mitose propriamente dita. Caracterizou-se as
anomalias apresentadas em neuroblastos e ovários de mutantes hipomorficos tsh e em células
S2 defectivas para o mesmo gene através da técnica de interferência de RNA (RNAi). A análise
do padrão de expressão de cérebros mutantes para tsh foi obtida, utilizando a técnica de
microarrays.
Durante este trabalho, observou-se que o gene tsh é co-expresso com o gene
hedgehog (hh) na extremidade anterior do germário de Drosophila: nas células do filamento
terminal (TF) e nas células cap (CC), ambas são localizadas adjacentemente às células
estaminais germinais (GSC) e são descritas como parte integrante do nicho das GSC e FSC.
Nos ovários de Drosophila, a via de sinalização Hh é essencial para a auto-renovação e
proliferação das FSC. Uma nova mutação tsh, causadora da paragem precoce em oogénese
(no estado S2/S3), permitiu mostrar que a proteína Tsh regula a expressão dos genes
engrailed (en) e hh na extremidade do germário, tornando-se, desta forma, crucial para a
normal proliferação das FSC. De acordo com esta observação, as experiências de sobre
expressão do gene tsh nos ovários, originaram um fenótipo que se asssemelha ao obtido
aquando da sobre expressão do gene hh. As FSC proliferam de forma excessiva, originando
um excesso de descendência que se acumula entre as câmaras do ovo, formando as estruturas
semelhantes a stalks gigantes. Em adição, nos mutantes defectivos para o tsh, a reposição
deste gene, usando o sistema UAS/ hsGal4, permitiu recuperar parcialmente o fenótipo
associado à proliferação anormal das FSC, possibilitando a progressão da oogénese até ao
estado S5/S6. Consistentemente, a expressão do gene hh através do mesmo sistema UAS/
Gal4, também promoveu uma melhoria do fenótipo nos mutantes tsh, mas de forma menos
eficiente.
Resumo
Assim, conclui-se que o Tsh desempenha um papel crítico na regulação da expressão do
hh, contribuindo, desta forma, para a proliferação e especificação da identidade das FSC nos
ovários de Drosophila. Estes resultados estão de acordo com as interacções entre o gene tsh e
a via de sinalização Hh, previamente descritas em embriões de Drosophila. Infelizmente, os
resultados obtidos não permitiram concluir se o Tsh actua directamente na regulação da
expressão dos genes hh e en, ou se por outro lado, se trata de uma regulação indirecta. Mais,
continua por se clarificar, neste caso, se o gene tsh actua como um repressor ou um activador.
Recentemente, o gene tiptop (tio) foi descrito como sendo um novo membro da família
tsh em Drosophila. Tio codifica para uma proteína de 1024 amino ácidos, a qual apresenta um
quarto motivo, tipo zinc finger na região C-terminal. Durante a embriogénese, foi demonstrado
que os dois membros da família tsh em Drosophila se reprimem mutuamente. Durante a
oogénese, observou-se uma complementaridade na expressão de tsh e tio nas TF: Tio localiza-
se nas TF apicais, enquanto o Tsh é preferencialmente detectado nas TF proximais. No
entanto, a expressão de tio é ectópica em todas as celulas do TF em ovários ∆126/tshA3-2-66,
assim como a expressão de tsh é generalizasa a todas as TF células em ovários tio473
homozigotas. A expressão de hsGal4>UAS-tioFL permitiu a recuperação do fenótipo dos
ovários ∆126/tshA3-2-66, tal como foi acima mencionado para o hsGal4>UAS-tsh13. De forma
idêntica, a expressão ectópica de tio induz efeitos similares aos acima descritos para a sobre
expressão de tsh, especialmente num contexto mutante para tsh. Desta forma, tal como foi
descrito durante a embriogénese, tsh e tio reprimem-se mutuamente na extremidade apical do
germário. Sugere-se ainda que Tio desempenha uma função redundante, completamente
restituída pela presença de Tsh nos ovários tio473.
Durante este trabalho, reuniu-se várias evidências que indicam que o gene Tsh é
indispensável para a progressão em mitose. Duas mutações hipomórficas no gene tsh (tshNG1
and tshA3-2-66) causam, em neuroblastos, uma paragem em metaphase, com a maioria das
figuras mitóticas exibindo cromossomas altamente condensados e associados a fusos mitóticos
aparentemente normais e a centrossomas regulares. A análise dos esfregaços destes
neuroblastos, com proteínas específicas do ponto de controlo do fuso (como Bub1 e Rod),
revelou a normal localização destas proteínas. A imunomarcação com anticorpos contra a
proteína CID (homologa da CENP-A) excluiu a ocorrência da separação prematura de
cromatídeos irmãos. Mais ainda, nos neuroblastos parados em mitose, a ciclina A aparece
normalmente degradada, enquanto que a a ciclina B continua a ser detectada. Duplos
mutantes para tsh rod e tsh bub1 apresentam um fenótipo semelhante ao observado nos
mutantes tsh simples. Assim, as mutações rod e bub1 não conseguem ultrapassar a forte
paragem imposta pela perda de função tsh, continuando inibida a progressão para anáfase.
Um papel geral para o Tsh é desconhecido, considerando o seu padrão de expressão localizado
no sistema nervoso central (CNS) e as suas bem documentadas funções durante a
especificação de estruturas particulares. No entanto, não foi identificado nenhum tipo de
XVIII
Resumo
regionalização específica dos defeitos mitóticos no CNS. Além disso, o silenciamento do gene
tsh em células S2 pela técnica de RNAi provoca uma paragem em mitose, imitando o fenótipo
descrito em neuroblastos mutantes para o tsh. Durante as experiências de RNAi, a maioria das
células pararam numa configuração semelhante à metafase, o que originou um incrível
aumento no índice mitótico. Assim, estes resultados levam a propor que o gene tsh
desempenha um papel indispensável na transição metafase/anafase, uma vez que na ausência
deste gene, o ponto de controlo do fuso e/ou a actividade do complexo promotor da anafase/
ciclossoma (APC/C) são pertubados. A análise do padrão de expressão de cérebros mutantes
para tsh, comparativamente ao selvagem, não permitiu identificar um óbvio gene mitótico
alvo. Curiosamente, foi possível estabelecer uma ligação putativa com a via de sinalização Hh
através do interference–hedgehog gene (ihog), o terceiro gene com níveis de downregulation
mais significativos. IHog é uma proteína transmembranar intermediária na resposta ao sinal
activo de Hh, actuando como amplificador deste sinal upstream do receptor negativo desta via
de sinalização: Patched (Ptc). O mecanismo, pelo qual o Hh induz a proliferação, depende do
tecido em causa, mas inclui a indução e a regulação de componentes do ciclo celular como o
das ciclinas: D1, D2, B1 e E, a Cdc25 e o N-Myc. Teoricamente, a Cdc25 (homóloga do String)
pode também ser um alvo do Hh nos neuroblastos, uma vez que, de todos os alvos mitóticos
conhecidos para a via Hh, a Cdc25 é o unico que se encontra ligeiramente downregulated nos
mutantes tsh. No entanto, o sincronismo de paragem em metafase, observado nos
neuroblastos tsh, está mais de acordo com alterações nos níveis de expressão do gene polo
(um alvo mitótico com níveis de downregulation idênticos aos registados para a Cdc25). De
facto, a observação dos neuroblastos tsh faz lembrar o fenótipo mitótico apresentado por
mutantes polo (por exemplo homozigotas para o alelo polo9). No entanto, a redução dos níveis
de expressão de polo não é por si só suficiente, para explicar a forte paragem em metafase
apresentada pelos mutantes tsh e, por outro lado, o polo nunca foi implicado como sendo um
alvo da via Hh. Neste ponto, os resultados aqui expostos falham para identificar um alvo
mitótico, no entanto, os significantes níveis de downregulation registados para o iHog sugerem
uma interligação entre a forte paragem em mitose e a via de sinalização Hh, fazendo lembrar
os resultados observados durante a oogénese. Assim, propõe-se que o gene tsh é necessário
para a transcrição do iHog e que este ligando está implicado na modulação da sinalização Hh
durante a mitose.
Com o trabalho aqui apresentado, pretende-se demonstrar que o gene tsh afecta a
proliferação das FSC através da regulação da expressão do en e hh. Em adição, poder-se-á
especular que em neuroblastos e células S2, o Tsh poderá também afectar a progressão da
mitose através da sinalização Hh, uma vez que, pelo menos em neuroblastos, pela técnica dos
microarrys, verificou-se que o gene ihog é o terceiro mais donwregulated. Assim, propõe-se
uma nova função para o gene tsh na proliferação celular, através da via de sinalização Hh.
XIX
Resumo
Palavras Chave: teashirt; tiptop; via de signalização do hedgehog; células
estaminais somáticas; neuroblastos; mitose, ponto de controlo do fuso; transição metafase/
anafase, complexo promotor da anafase/ ciclossoma (APC/C).
XX
Abstract
Drosophila teashirt (tsh) encodes a zinc finger protein essential during Drosophila
development.
My PhD aimed to clarify the tsh role in cell proliferation studying tsh function in three
Drosophila systems: follicle stem cells (FSC), neuroblasts and S2 cells.
I found that Tsh and Hh were co-expressed in the Drosophila terminal filament and cap
cells. A new tsh female sterile mutation exhibiting early oogenesis arrest allowed me to show
that Tsh acts via Hh-mediated signalling pathway to control FSC proliferation; tsh
overexpression mimics the hh overexpression associated phenotype. Additionally, in tsh
background, the hsGal4 driven expression of tsh or hh partially rescues the abnormal
proliferation phenotype allowing oogenesis progression. These results suggest that Tsh is
critical to control hh expression contributing for the regulation of FSC proliferation and
specification of somatic cell identity.
In neuroblasts, hypomorphic tsh mutation leads to a metaphase-like arrest with highly
condensed chromosomes associated with apparently normal mitotic spindles and centrosomes.
Additionally, checkpoint proteins Bub1 and ROD were localised normally and CID
imunodetection excluded an eventual premature sister chromatid separation. Furthermore,
cyclin-A appears normally degraded, whereas cyclin-B remains detectable. Double tsh rod and
tsh bub1 mutants phenotypes resemble the tsh single mutant. Finally, tsh depletion in S2 cells
mimics the tsh mutant phenotype in neuroblasts. These results suggest that Tsh is critical
during mitosis progression and I propose that Tsh loss maintains active the spindle checkpoint
and/or unable the APC/C activity.
We proposed a tsh acts through Hh signalling in cell proliferation. During oogenesis, tsh
affects FSC proliferation mediating Hh expression. In neuroblasts, tsh could acts through Hh
signalling, since expression of interference hedgehog, a positive mediator of Hh signal, was
severely downregulated in microarrays-based analysis.
Key Word: teashirt; tiptop; hedgehog signalling; follicle stem cells; neuroblasts;
mitosis, spindle assembly checkpoint; metaphase-anaphase transition, anaphase-promoting
complex/ cyclosome (APC/C).
XXII
CHAPTER I
INTRODUCTION
I-The teashirt (tsh) gene
2
I-The teashirt (tsh) gene
I- The teashirt (tsh) gene
I_1- Characterisation of the tsh gene
The tsh gene was firstly identified by the enhancer trap technique, in a screen for β-
galactosidase activity during embryogenesis, (Fasano et al., 1991). The tsh gene is located at
the left arm of chromosome 2 in the cytogenetic region 40A5.
Two size classes of tsh transcripts were detected in Drosophila: a predominant class
consisted of transcripts of 5.4 kb, which are expressed throughout development, and a large
size class of 8.5 kb transcripts that are detected predominantly, though not exclusively, during
embryogenesis (Fasano et al., 1991). The differential splicing is indicated as the responsible,
at least in part, for these two different classes of transcript.
Tsh codes for a protein of 993 amino acids, with an estimated molecular mass of 106
KDa (Fasano et al., 1991). The tsh protein (Tsh) is characterised by the presence of three
atypical widely-spaced C2-H2 zinc finger motifs, corresponding to the consensus sequence
CX2CX3FX5(L,M)X2HMX4H, in which the two histidines are separated by five amino acids and
the size of the loop between the second C and the first H is of twelve residues in all Tsh zinc
fingers (Fasano et al., 1991). In addition, the protein contains a consensus motif (PLDLS) for
the interaction with the C terminal Binding Protein (CtBP) in a complex with Brinker (Brk) and
three domains rich in alanine, in the N-terminal (Fasano et al., 1991; Manfroid et al., 2004;
Saller et al., 2002). Finally, the acidic rich domain, identified in Tsh, is required for their
interaction with Scr (Sex combs reduced) for the identity of the prothorax (Taghli-Lamallem et
al., 2007).
These structural properties are compatible with a role on the modulation of the
chromatin. In fact, the gene Su(var)3-7, which encodes a protein with seven zinc of the Tsh
zinc finger type, play a role in the modification of the chromatin structure (Cleard and Spierer,
2001). It was shown that Tsh is able to bind to DNA behaving like a transcription factor. In
vitro, Tsh binds specifically to a fragment on the promoter of the gene modulo (mod) and
recognises two specific binding sites in this mod fragment (Alexandre et al., 1996). The mod
expression is repressed by Tsh in the trunk (Alexandre et al., 1996). Moreover, cell culture
analyses showed that tsh acts as a transcriptional repressor of mod (Waltzer et al., 2001).
Recently, Tsh was identified as a protein that interact with FE65 (Kajiwara et al., 2009). FE65
is an adapter protein that binds to the amyloid protein precursor and this complex can
modulated gene expression (Duilio et al., 1991). FE65 could simultaneously recruit SET (a
component of the inhibitor of acetyl transferase) and Tsh (that in turn recruit histone
deacetylase to produced a powerful gene silencing complex; Kajiwara et al., 2009). The
primate specific Caspase 4 (CASP 4) was identified as a target of this FE65/ SET or FE65/ Tsh
complex. In addition, chromatin immunoprecepitation showed a direct interaction between
FE65 and Teashirt3 with the promoter region of the CASP 4 (Kajiwara et al., 2009).
I-The teashirt (tsh) gene
I_2- Expression patterning of the tsh mRNA and protein in Drosophila
The tsh transcripts are first detected at the beginning of cellularisation (stage 4-5), in a
central ring of cells corresponding to the primordium of the thorax (Fig. I_1; Fasano et al.,
1991). At the end of cellularisation/ beginning of gastrulation, tsh expression is observed in
five bands transient in the central region of the embryo (the three posterior bands are weaker
then the two anterior ones). During gastrulation (stage 6), the striped pattern gives a more
uniform labelling in a domain of cells that correspond to the presumptive trunk region of the
embryo (Fasano et al., 1991). At the beginning of the gastrulation, tsh present a transient
expression in 5 bands of the embryo trunk. Than, at stage 9, tsh transcripts present a
homogeny distribution in all the presumptive domain of the trunk (to parasegments 3 to 13).
This staining is maintained until the end of the embryogenesis, with the tsh transcripts
presenting a particular segmental pattern: they are stronger expressed in the posterior region
of each trunk segments (Fasano et al., 1991).
Figure I_1- Tsh expression patterning during Drosophila embryogenesis (in <flybase.bio.indiana.edu
/reports/FBgn0003866.html>).
At stage 4-5 tsh transcripts are first dectect in the primordium of the thorax.
At stage 6 (beginning of gastrulation) tsh expression is observed transiently in five band in the central region of the
embryo; that correspond to the presumptive trunk region of the embryo.
At stage 9 (the extended germband stage), cells specific to parasegments 3 to 13 are labelled, that correspond to the
presumptive domain of the trunk. The trunk staining is maintained until the end of the embryogenesis with the tsh
transcripts stronger expressed in the posterior region of each trunk segments.
After stage 15 of embryogenesis, tsh expression is also observed in the ventral nervous cord, in the anterior part of
the pharynx, in the visceral mesoderm, in the anal opening and in stellate cells of the Mapighi tubes.
Tsh transcripts are also detected in ventral nervous cord, with a strong intensity in the
three thoracic segments, in the anterior part of the pharynx, in the visceral mesoderm and in
the anal opening, after the 15 stage of embryogenesis (Fasano et al., 1991).
More recently, other domains of tsh expressions have been reported. The tsh transcript
is also detected in stellate cells, a group of specific cells in the Mapighian tubes (Denholm et
4
I-The teashirt (tsh) gene
5
al., 2003). Tsh is also present in the proximal region of the imaginal discs of the halteres,
wings and legs (Bhojwani et al., 1997; Erkner et al., 1999).
During embryogenesis, the tsh transcript and protein present sensibly the same
distribution. The protein is detected within the cytoplasm or the nucleus during the first stages
of development. After stage 7 of embryogenesis, the localisation is predominantly in the
nucleus (Alexandre et al., 1996).
I_3- Modulators of the tsh expression during embryogenesis
tsh expression is regulated during embryogenesis by Hox genes in ectoderm and
mesoderm derivatives (Roder et al., 1992; Mathies et al., 1994). Moreover, some of these
homeodomain transcription factors, including Antennapedia (Antp), Ultrabithorax (Ubx) and
abdominal-A (abd A), bind directly to specific enhancers in the tsh regulatory region
(McCormik et al., 1995). However, Hox proteins are not required for initiation of the tsh
expression, but rather modulate and maintain the expression pattern in a segment-specific
manner. Tsh in turn regulates the expression of Hox genes active in the gnathal segment and
head: Sex combs reduced (Scr), Deformed (Dfd) and labial (lab; Roder et al., 1992). Thus, tsh
works at the same level as Hox.
spalt (sal) and grunge (gug, homologue of vertebrates atrophine genes), are known to
regulate tsh expression (Erkner et al., 2002; Röder et al., 1992). Sal protein acts like a
transcriptional repressor of tsh: in the absence of sal, tsh is expressed ectopically in
parasegment 2, in the labial and in the tail region (parasegments 14-15, stage 10-11; Röder et
al., 1992). Grunge acts positively to regulate teashirt expression in proximoventral parts of the
leg. Grunge has other regulatory functions in the leg, including the patterning of ventral parts
along the entire proximodistal axis and the proper spacing of bristles in all regions (Erkner et
al., 2002). In Gug loss-mutants, the embryo segmentation is affected via a failure in the
repression of at least four segmentation genes known to regulate tsh [hunchback (hb), Krüppel
(Kr), knirps (kni) and fushi tarazu (ftz)]; in these embryos, tsh appears in bands pattern in the
dorsal region, and the ventral tsh expression is lost (Coré et al., 1997; Erkner et al., 2002;
Röder et al., 1992).
I_4- Tsh functions during Drosophila development
Tsh is crucial for the patterning of the trunk identity in collaboration with the Hox genes
(Fasano et al., 1991; Roder et al., 1992). Tsh also acts on the Wingless (Wg) and Hedgehog
(Hh) pathways (Angelats et al., 2002; Gallet et al., 1998, 1999) for the specification of the
naked cuticule during embryogenesis. In addition, tsh function is required for the midgut
morphogenesis (Mathies et al., 1994) and for the development of adult appendages (Bessa et
al., 2002; Erkner et al., 1999; Pan and Rubin, 1998; Soanes et al., 2001; Wu and Cohen,
I-The teashirt (tsh) gene
6
2000). The molecular mechanisms underlying tsh function are poorly understood. However, in
vitro experiments have shown that, when bound to DNA, Tsh could repress transcription
(Alexandre et al., 1996; Waltzer et al., 2001; Saller et al., 2002). Tsh binds to a specific
enhancer of the modulo (mod) gene, a known target of Scr and Ubx (Graba et al., 1994). Tsh
inhibits mod expression in the epidermis of the prothorax (or T1, the first thoracic segment)
(Alexandre et al., 1996). In the midgut mesoderm, tsh is required for the transcriptional
repression of Ubx that is mediated by high levels of Wg in collaboration with the co-repressors
Brk and CtBP (Waltzer et al., 2001; Saller et al., 2002). In this case, no sequence similar to
the mod enhancer bound by Tsh has been identified. Instead, Brk binds the Ubx enhancer and
then recruits Tsh and CtBP into a ternary repressor complex (section I_4.3 of this chapter).
I_4.1- Homeotic function of Tsh: the identity of the trunk and the specification
of the prothoracic identity
The absence of any single homeotic gene results in a change in the identity of a specific
segment or segments into another type, generating a typical homeotic transformation. Trunk
morphology in the Drosophila embryo also depends on the normal function of the tsh gene.
However, tsh is different from the classical homeotic genes in at least two aspects: (1) it codes
for a zinc finger protein and (2) the analysis of the phenotype of mutations at this locus affect
the entire trunk (Roder et al., 1992).
tsh is critically required for the identity of the T1 segment and globally for segmental
identity throughout the entire trunk, whereas the “classical” homeotic genes have more
specific roles (Fig. I_2; de Zulueta et al., 1994; Fasano, 1991; Roder et al., 1992).
The specific identity of the anterior prothorax is determined by the simultaneous
activities of the tsh and Scr gene products. First, in the absence of the Hox genes from the
trunk [Scr, Antp, Ubx, abdA and abdominal B (AbdB)], each thoracic and abdominal segment
adopts a T1 identity conferred by the presence of tsh, which is active in all this region (Struhl,
1983). Moreover, in tsh mutants, the T1 segment is transformed in labial, since Scr is unable
to promote the T1 identity in the absence of tsh (de Zulueta et al., 1994; Fasano et al., 1991).
In the absence of tsh, the Scr expression is affected: Scr is still present in the labial and the
dorsal part of T1 segment, but it is ectopic in the ventral region of this T1 segment, showing
that tsh controls the posterior limit of the Scr expression in the ventral ectoderm (Fasano et
al., 1991).
I-The teashirt (tsh) gene
Figure I_2- Tsh as a Hox co-factor in the segment identity of the trunk (in Laugier, 2005 modified
from Robertson et al., 2004).
In the trunk segments, the Tsh protein repress the expression of disco and target the segments for a trunk identity.
Sal (Spalt protein) defines the boundary between head and trunk (broken line) repressing tsh expression in the
gnathal segments. In the head segments, disco is expressed and activates the acquisition of gnathal fate. Thus, the
distribution of Tsh and Disco regionalize the embryo. The combination of these proteins with Hox genes determines the
segment identity. Scr (Sex combs reduced) is expressed in the gnathal domain as well as in the trunk, and the
acquisition of different segment identities depend on the co-factor present. Dfd (Deformed) can establish the maxilar
ou mandibular identity in function of the presence of the protein cap-n-collar (Cnc; Mohler et al., 1995). Ma:
mandibular segment; Mx: maxilar segment; Lb: labial segment; T1: first thoracic segment (or prothorax) and T2:
second thoracic segment.
In contrast, the ectopic expression of tsh induces the inverse: the head-to-trunk
transformation. The labial segment is transformed in T1 and the maxilar and antennal
segments acquire a trunk identity, illustrated by the presence of naked cuticule, without clear
segmental identity (de Zulueta et al., 1994). In this genetic context, Scr expression remains
unchanged. When tsh and Scr are simultaneously ectopically expressed, the labial, maxilar and
antennal segments acquire a T1 phenotype.
Recently, there has been a new insight into the mechanism by which Tsh, in concert
with Scr, specifies the prothoracic identity (Taghli-Lamallem et al., 2007). It was shown that
Tsh interacts directly with Scr, and this interaction depends, at least in part, on the presence
of a short “acidic domain” located on the N-terminal half of Tsh. In vivo, the expression of full
length Tsh can rescue the tsh null phenotype throughout the trunk, whereas Tsh lacking the
Scr interacting domain rescues all the trunk defects except in the prothorax (Taghli-Lamallem
et al., 2007). Thus, direct interaction between tsh and Scr is indispensable to define the T1
identity (de Zulueta et al., 1994; Taghli-Lamallem et al., 2007).
7
I-The teashirt (tsh) gene
8
Since tsh activity is required for segment identity of the entire trunk region, it has
characteristics in common with the “region specific” homeotic genes spalt and fork head
(Jürgens, 1988; Jürgens and Weigel, 1988).
It has been shown that Split ends (Spen), tsh and Antp function in a combinatorial
manner to repress the development of head-like sclerites and to promote the development of
thoracic identity (Wiellette et al., 1999).
In tsh Antp double mutant embryos, the first two thoracic denticle belts are completely
absent and replaced by cuticle typical of the head skeleton (Roder et al., 1992). Conversely,
ectopic expression of Antp induces a strong transformation of T1 to T2 (Gibson and Gehring,
1988; Gibson et al., 1990). In vitro, it was recently shown that Tsh interacts directly with the
Hox protein Antp (Taghli-Lamallem et al., 2007).
If Tsh is a co-factor for Hox proteins in the trunk, how can different Hox proteins
establish different segment identities with the same co-factor (for example, Scr and Antp with
Tsh)?
One role for the Tsh–Scr and Tsh–Antp complexes could be to control specific subset of
target genes, in order to specify the identity of the T1 and T2 segments. Moreover, the
capacity of Tsh∆acid (defective for the acidic domain) to rescue the T2 defects of a tsh8 null
mutant suggests that the collaborative activity of Tsh/Scr and Tsh/Antp does not involve the
same domain of Tsh (Taghli-Lamallem et al., 2007).
I_4.2- Tsh in the segmental polarity: modulation of the Wingless pathway and
regulation of the hedgehog pathway target genes
The segment-polarity genes, which encode a diversified group of proteins including
transcription factors and components of the Hedgehog (Hh; Ingham, 1998) and Wingless (Wg)
signal transduction pathways (Klingensmith and Nusse, 1994; Van den Heuvel et al., 1989;
Willert and Nusse, 1998) are required for intrasegmental patterning (Baker, 1988; Bejsovec
and Martinez-Arias, 1991; DiNardo et al., 1988; Dougan and DiNardo, 1992; Heemskerk et
al., 1991; Martinez-Arias et al., 1988; Noordermeer et al., 1992; Perrimon, 1994).
The wg gene (Wnt in vertebrates), encodes secreted glycoproteins (Klingensmith and
Nusse, 1994; Kühl and Wedlich, 1997; Miller and Moon, 1996; Van den Heuvel et al., 1989;
Willert and Nusse, 1998). A conserved signal transduction pathway transmits the signal
promoted by Wg following its secretion (Klingensmith and Nusse, 1994; Kühl and Wedlich,
1997; Miller and Moon, 1996; Willert and Nusse, 1998). At the end of this pathway, two
proteins are responsible for Wg output: Armadillo (Arm; homologue to vertebrate β-catenin;
Kühl and Wedlich, 1997; Miller and Moon, 1996; Peifer and Wieschaus , 1990; Peifer et al.,
1991; Riggleman et al., 1990; Willert and Nusse, 1998), and the Drosophila T-cell factor
(dTcf), which is also known as Pangolin and is analogous to the vertebrate T-cell factor (Tcf) or
lymphocyte enhancer factor-1 (Lef-1; Behrens et al., 1996; Brunner et al., 1997; Huber et al.,
I-The teashirt (tsh) gene
9
1996; Riese et al., 1997; van de Wetering et al., 1997). Tcf is a member of the family of high
mobility group (HMG) DNA-binding proteins required for DNA architecture and gene regulation.
Both, Arm and dTcf, seem to be crucial for the transmission of all known Wg signalling events.
Two pools of Arm exist within cells: one at the cell membrane that is required for cell adhesion
(Orsulic and Peifer, 1996; see also section II_4.1.3.3 of this chapter), and which is
independent of the Wg signal, and the other, a cytoplasmic pool for transmission of the Wg
signal (Pai et al., 1997; Peifer et al., 1991; Riggleman et al., 1990). In the plasmic membrane,
Arm binds to E-cadherin and α-catenin in order to maintain the cellular adhesion (Willert and
Nusse, 1998). In the cytoplasm, upon reception of Wg, Arm is stabilized and recruited for the
wg transduction signalling (Peifer, 1995).
In the absence of Wg, cytoplasmic Arm is ubiquitinated and degraded by the
proteossome, thereby blocking the transmission of the Wg signal (Aberle et al., 1997). In Wg-
receiving cells, cytoplasmatic Arm is stabilised and associated with dTcf. The Arm–dTcf
complex is transported to the nucleus, where it is thought to regulate the transcription of
genes required for cellular patterning (Brunner et al., 1997; Riese et al., 1997; van de
Wetering et al., 1997).
Tsh acts like a modulator of the Wg signalling pathway, being required to maintain the
expression of the late target (like wg itself), but not the early target (like engrailed, in stages
7–10 of embryogenesis (the cell-stabilisation phase; Gallet et al., 1998). Moreover, ectopic Tsh
expression maintains the Wg expression in the gnathal labial and maxillary segments at the
12-13 stage, whereas at this stage, in a wild embryo, wg is no longer detected in these two
segments (Gallet et al., 1998; Manfroid et al., 2004). In addition, tsh mutants present a
phenotype similar to the one observed in absence of the late components of the Wg pathway
and, in fact, wg is not maintained ventrally in the trunk segments of the embryo (Gallet et al.,
1998).
Epistasis tests and in vitro interactions showed that Tsh modulates Wg signalling by a
direct interaction with the C-terminal domain of Arm (Gallet et al., 1998; 1999). The
expression pattern of both proteins is modulated in a similar way depending on Wg. In fact,
around stage 10/11, both (Tsh and Arm proteins) accumulate to a high level in the nuclei of
posterior cells, receiving Wg signal that will form the naked cuticle. By contrast, in the anterior
part of the segment, where Wg does not signal, lower levels of nuclear Tsh are detected, which
in part contribute to the patterning of denticles (Gallet et al., 1998). Very high-level of Tsh
replaces denticles with naked cuticle. Moreover, the maintenance of wg expression is
controlled by tsh in the ventral part of the trunk (Gallet et al., 1998). The intracellular
localisation of Tsh depends on phosphorylation: Tsh is hyperphosphorylated in the nucleus and
hypophosporylated in the cytoplasm. This phosphorylation and consequent nuclear
accumulation seems to depend partially on Wg signalling. Probably, the stabilization of Arm by
Wg occurs first and favours interaction between Arm and Tsh in the cytoplasm prior to the
I-The teashirt (tsh) gene
10
translocation of Tsh to the nucleus. In fact, the accumulation of Arm precedes Tsh
accumulation in nuclei (Gallet et al., 1999).
Recently the interaction between Tshz and Wnt signalling has been demonstrated in
Xenopus (Koebernick, et al., 2006). XTsh1 is not expressed in the hindbrain, but was found to
influence spatially restricted hindbrain genes. Embryos injected with XTsh1 antisense
morpholino oligonucleotides (MO) showed reduced Wnt-4 expression in brain and spinal cord.
However, ectopic expression of XTsh1 in Xenopus had no effect on Wnt-4 expression
(Koebernick, et al., 2006).
The alternate expression of wg, hh, patched (ptc) and rhomboid (rho) is responsible for
cellular identity and polarity of the embryonic segments (Alexandre et al., 1999; Gritzan et al.,
1999). Hh is required to maintain the expression of wg in a single row of cells (Ingham and
Hidalgo, 1993; Hidalgo and Ingham, 1990). The stabilisation of wg expression allows the
maintenance of hh transcription in the adjacent cell row and, thus, Hh and Wg reinforce each
others expression. In a later step, these secreted molecules will specify the cell fate choices.
Wg is directly responsible for the naked cell fate with the requirement of Tsh (as mentioned
above), whereas Hh will indirectly govern some of the denticle identities by activating rho
transcription in rows of cells posterior to the Hh-secreting cells (Sanson et al., 1999; Gritzan et
al., 1999). rho encodes a transmembrane protein implicated in EGF signal activation and it is
required for denticle diversity during embryogenesis (Szuts et al., 1997; O’Keefe et al., 1997).
In addition, to transduce the Hh signal, a third target gene expression, the Hh receptor Ptc,
must be maintained by Hh on both sides of its expression domain, thus limiting the range of
Hh action by restricting its diffusion (Chen and Struhl, 1996; Hidalgo and Ingham, 1990;
Hooper and Scott, 1989; Nakano et al.,1989).
Loss of hh function abolishes wg, rho and ptc expression and gives rise to larvae
without any naked cuticle and none denticle diversity. By contrast, hh overexpression
promotes the expansion of wg, rho and ptc expression and the larvae displays mirror image
duplication of their denticle belts (Alexandre et al., 1999, Gallet et al., 2000; Ingham, 1993;
Tabata and Kornberg, 1994).
Cubittus interruptus (Ci; belonging to the vertebrate Gli family) is the most downstream
component so far identified in the Hh pathway (reviewed by Aza-Blanc and Kornberg, 1999). It
encodes a zinc-finger transcription factor that mediates the majority of the Hh cellular
responses in the imaginal discs and embryo. In the presence of Hh, Ci is converted into an
active form (155-kDa form, full-length), while the absence of Hh promotes the cleavage of Ci
into a repressive form (75-kDa form; Alexandre et al., 1996a; Aza-Blanc et al., 1997;
Dominguez et al., 1996; Hepker et al., 1997; Méthot and Basler, 1999; Ohlmeyer and
Kalderon, 1998; Von Ohlen et al., 1997; Wang and Holmgren, 1999; see also section II_4.2.2
of this chapter). Thus, Ci is required for both activation and repression of Hh target genes.
However, Ci does not mediate all Hh functions during embryogenesis: it is only required for ptc
I-The teashirt (tsh) gene
11
regulation and for late wg maintenance, but not for early neither for rho expression (Gallet et
al., 2000).
It appears that Tsh is also an actor in Hh signalling, controlling the expression of certain
target genes in the embryonic epidermis (Gallet et al., 2000). Moreover, before stage 11, Tsh
and Ci are necessary in a redundant way, for the regulation of Wg dependent on Hh (Gallet et
al. 2000). In addition, the expression of the gene rho requires the presence of Tsh.
Different epistasis tests showed that different combinations of the three proteins (Tsh,
Ci, and Arm) are required for the specification of the naked cuticle at different positions along
the anterior–posterior (A/P) axis (Angelats et al., 2002). Biochemical analyses revealed the
presence of complexes including the proteins Tsh, Ci and Arm (Angelats et al., 2002). In vitro,
it was previously shown that Ci is able to directly interact with the hyperphosphorylated form
of Tsh, while hypophosphorylated Tsh specifically immunoprecipitates with Arm (Gallet et al.,
1999). In addition, Arm does not bind directly to Ci, at least in vitro. Therefore, it was
suggested that Arm, Tsh, and Ci form protein complexes, with Tsh interacting directly with
both Ci and Arm (Angelats et al., 2002).
Tsh takes part in the regulation of target genes of two essential pathways, responsible
for the correct polarisation patterning of the ventral part of the segments in the embryo trunk,
appearing as an essential regulator for the development of the ventral epidermis. Tsh is a good
candidate to link segmental polarity and homeotic segmental identity in the trunk, since Tsh
participates in these both aspects of embryogenesis.
I_4.3- Tsh and the midgut morphogenesis
The Drosophila midgut is formed late in embryogenesis and is a tube consisting of two
cell layers: an inner endodermal layer and an outer mesodermal layer, encasing the yolk
(Skaer, 1993). The gut becomes divided into several compartments through the constriction of
the midgut in three places and four tubes.
Drosophila midgut development is regulated by four homeotic genes expressed in the
visceral mesoderm, where two of their identified target genes encode secreted proteins (Bienz
and Tremml, 1988; Tremml and Bienz, 1989; Reuter and Scott, 1990). The Ubx gene activates
transcription of the decapentaplegic (dpp) gene, while in adjacent mesoderm cells the abd-A
gene activates transcription of the wg gene. Dpp is a secreted molecule, homologue to TGFβ
(Transforming growth factor β) familly and it is implicated in some processes of proliferation
and differentiation (Hoodless et Wrana, 1998). The homeotic genes Antp and Scr act in more
anterior midgut regions. Both target genes, dpp and wg, are transcribed in the mesoderm, but
their secreted signalling molecules move into the endoderm (van den Heuvel et al., 1989;
Panganiban et al., 1990b; Reuter et al., 1990). In the endoderm, both are required for normal
expression of the homeotic gene labial (lab; Immerglück et al., 1990; Reuter et al., 1990;
Tremml and Bienz, 1992).
I-The teashirt (tsh) gene
Figure I_3- Summary of gene interactions in the cells surrounding the middle constriction (adapted
from Mathies et al., 1994).
Ubx and abd-A are expressed in adjacent cells in parasegments parasegment 7 and parasegment 8 respectively. abd-A
represses the transcription of Ubx in the cells posterior to parasegment 7, and so no cells express both Ubx and abd-A
(Tremml and Bienz, 1989). Ubx activates expression of dpp in parasegment 7, while abd-A activates expression of wg
in parasegment 8. abd-A prevents Ubx from activating dpp in the cells posterior to parasegment 7 (Reuter et al.,
1990). wg and dpp are both required for activation of tsh in the cells overlying and posterior to the central
constriction. dpp influences tsh expression through the activation of wg.
During embryogenesis, perturbations in the tsh expression affect the formation of the
midgut. Tsh is required for the formation of the anterior and central structures of the midgut
(the 1st and 2sd midgut constrictions) in the visceral mesoderm (Fig. I_3; Mathies et al., 1994).
In this domain, tsh transcription is controled by the Hox genes Antp, Ubx and abdA, and by the
Wg and Dpp signalling pathways (Mathies et al., 1994; McCormick et al., 1995). Antp activates
tsh in anterior midgut mesoderm, while in the central midgut mesoderm Ubx, abd-A, dpp, and
wg are required for proper tsh expression (Mathies et al., 1994). During embryogenesis,
perturbations in the tsh expression affect the formation of the midgut. Tsh is required for the
formation of the anterior and central structures of the midgut (the 1st and 2sd midgut
constrictions) in the visceral mesoderm (Fig. I_3; Mathies et al., 1994). In this domain, tsh
transcription is controled by the Hox genes Antp, Ubx and abdA, and by the Wg and Dpp
signalling pathways (Mathies et al., 1994; McCormick et al., 1995). Antp activates tsh in
anterior midgut mesoderm, while in the central midgut mesoderm Ubx, abd-A, dpp, and wg
are required for proper tsh expression (Mathies et al., 1994).
In the embryonic midgut, Ubx and labial are stimulated by low levels of Wg and
repressed by high levels of Wg (Bienz 1997). It was shown, that Tsh is also required for this
transcriptional repression of Ubx. In response to high levels of Wg, Brinker (Brk) binds to the
regulatory sequences of Ubx and then recruits Tsh to form a repressor complex; after that, the
12
I-The teashirt (tsh) gene
13
two proteins can recruit the corepressor C-terminal Binding Protein (CtBP) and repress the Ubx
expression (Waltzer et al., 2001; Saller et al., 2002).
I_4.4- Tsh in the Drosophila Pax6/ So/ Eya network during eye development
The eyes of the fly arise from a larval structure called the eye-antennal imaginal disc.
tsh is also crucial for eye development, which is controlled by the ‘eye selector’ genes: the
Pax6 paralogs eyeless (ey) and twin of eyeless (toy) (Czerny et al., 1999; Gehring, 2002).
Both ey and toy have the ability to induce ectopic eye formation when ectopically expressed
during larval development (Halder et al., 1995; Czerny et al., 1999).
The eye primordium specification starts with the expression of the early retinal genes:
eyes absent (eya), sine ocullis (so) and Dachshund (Dac). Their coexpression is necessary to
lock-in the eye fate within the eye field, possibly by acting together as a transcriptional
complex (Desplan, 1997; Kenyon et al., 2003; Kumar and Moses, 2001; Pichaud et al., 2001).
Then, retinogenesis is triggered by hh and the Hh target (Dpp/Bmp4) signals that are
produced by the surrounding posterior margin cells, at the so-called ‘firing point’ (Treisman
and Heberlein, 1998). The expression of hh and hh-dependent dpp transcription, at the
posterior margin of the disc, is a key for the definition of the eye primordium, since they
activate the expression of eya and so. The eye-inducing functions of dpp, also include the
posterior repression of wg, that by they turn, could repress eye development by promoting the
alternative head-capsule fate (Dominguez and Casares, 2005).
Retinal differentiation begins in the posterior region of the eye primordium and
proceeds as a wave in a posterior-to-anterior direction (Curtiss and Mlodzik, 2000; Dominguez
and Hafen, 1997; Heberlein et al., 1993). During wave progression, undifferentiated cells are
anterior to the morphogenetic furrow (MF; an indentation of the main epithelium that mark the
border of differentiated/ undifferentiated cells), while differentiating cells are posterior to it
(Treisman and Heberlein, 1998). The progression of the MF is driven by the joint action of dpp,
expressed within the furrow, and by hh, expressed in cells posterior to the furrow. The
induction of the proneural gene atonal (ato) by hh, is the first step towards the definition of
the R8 photoreceptor, the founder neuron of the mature eye units or ommatidia (Dominguez,
1999; Dominguez and Hafen, 1997).
tsh expression starts during L2 and is restricted to one of the two epithelial layers that
compose the disc (the ME). tsh expression in L3 eye discs is very similar to that of ey/toy,
whose expression is activated anterior to the MF and repressed posterior to it (Bessa et al.,
2002; Fasano et al., 1991). The tsh territory can be further subdivided into two domains: a
domain far from the MF, in which Homotorax (hth, a transcription factor member of Meis
family homeobox gene) expression maintains cells in an undifferentiated state and represses
retinal selector gene expression (such as eya); and a domain more close to the MF, in which
hth is repressed leading to eya upregulation (Bessa et al., 2002). The latter is also known as
I-The teashirt (tsh) gene
14
the pre-proneural domain, since it precedes the onset of retinal differentiation (Greenwood and
Struhl, 1999). Note that Tsh is not expressed in the two rows of cells immediatly anterior to
the MF, which stop to divide and are synchronized to start differentiation once the MF has
moved on (Bessa et al., 2002).
tsh overexpression in the eye disc can induce ectopic eye development or block its
normal formation, depending on the Gal4-promoter used (Manfroid et al., 2004; Pan and
Rubin, 1998; Singh et al., 2004; Singh et al., 2002).
It has been postulated that Tsh is required to prevent the premature expression of
downstream transcription factors: So, Eya and Dac (Bessa et al., 2002). In addition, tsh and
ey, mutually induce each others expression in the ventral region in the antenna disc (Pan and
Rubin, 1998; Bessa et al., 2002; Singh et al., 2002). This genetic pathway seems conserved
during the evolution: the vertebrate Pax6 genes (homologue to toy and ey) can mimic the
overexpression of toy and ey promoting the formation of ectopic eyes in the Drosophila
(Quiring et al., 1994).
These asymmetric Tsh functions depend on the presence of other factors implicated
early in the dorso-ventral regionalisation of the eye. Thus, the expression of Iroquois complex
(Iro-C) and Delta (Dl) genes in the dorsal region of the discs are necessary and sufficient for
dorsal function of tsh, while ventral tsh role in the eye suppression requires Wg and Serrete
(Ser) (Singh et al., 2004).
More recently it was shown that tsh induces eye specification, at least in part, by
allowing the activation of eye specification genes by the Wg and Dpp signalling pathways
(Bessa and Casares, 2005).
I_4.5- Tsh during the development of proximal region of adult’s appendixes
The appendages of Drosophila developed from invaginations of the larval ectoderm to
form imaginal discs. In leg and wing imaginal discs the distal-most tip of the appendage is
formed by cells in the center of the disc, with peripheral tissue contributing to more proximal
structures. Proximal-distal (PD) axis must be created de novo for each appendage during
embryogenesis.
I_4.5.1- tsh promotes the identity of the proximal structures of legs
The Drosophila leg is divided in: the proximal leg segments (coxa and trochanter) and
the distal region, which forms the remaining leg segments (femur, tibia, basitarsus and tarsus;
Fig. I_4).
I-The teashirt (tsh) gene
Figure I_4- Schematic representation of Drosophila leg imaginal discs and the adult leg (Schubiger,
1968; modified from Fristrom and Fristrom, 1993).
(A) A dorsal–ventral (d-v) cross section through a third-instar leg imaginal disc, which is made up of a folded
epithelium inside the larva, attached to larval structures (black shading). The peripodial membrane (pm) overlays the
distal epithelium (arrowhead) and corresponds to the most proximal structure of the disc, which will form parts of the
future body wall. The green-shaded regions of the epithelium will give rise to the body wall, coxa, and trochanter. The
dotted line represents the focus on the P-D boundary (between trochanter and femur).
(B) Third-instar imaginal disc with an apical view (under the peripodial membrane). The arrowhead indicates the distal
tip. The proximal regions (green) derive from the ring on the periphery at the disc. The dotted line corresponds to the
anterior(a)–posterior(p) compartment boundary. The blue region represents the expression domain of wg, in the
ventral anterior sector. Wg is secreted on either side of its expression domain to organize ventral and proximal distal
(P-D) patterning.
(C) A Drosophila adult leg representing the different leg segments along the P-D axis: the proximal segments (green)
are the coxa (co) and the trochanter (tr). The distal segments are called the femur (fe), tibia (ti), and tarsus (ts). The
ventral region is shown by the blue shading. The leg is attached by the coxa to the body wall.
The leg PD axis is elaborated by Wg and Dpp, which induce the expression of
downstream transcription factors at different positions along the PD axis in the leg imaginal
discs (Lecuit and Cohen 1997; Abu-Shaar andMann 1998). The secreted signaling proteins Wg
and Dpp instruct cells to adopt distal identity. Distal leg segments are lost in wg or dpp
mutants (Diaz-Benjumea et al., 1994; Held et al., 1994). By mechanisms that are still not well
understood, high levels of both Dpp and Wg are required to activate Distal-less (Dll)
expression, while lower levels of the same two signals activate another transcription factor:
dachshund (dac) (Lecuit and Cohen 1997). Dll protein is crucial for the formation of specific
distal parts of the legs (Cohen, 1990), as loss of function gives rise to an excess of proximal
leg tissue at the expense of distal patterns. The result of the Wg/ Dpp signalling is the
formation of three domains of gene expression along the PD axis: Dll in distal cells, dac in
medial cells, and hth/ tsh in proximal cells (Lecuit and Cohen 1997; Abu-Shaar and Mann
1998).
15
I-The teashirt (tsh) gene
16
Tsh expression is restricted to a proximal ring and the peripodial membrane (Gonzalez-
Crespo and Morata, 1996) destined to make the body wall, coxa, and trochanter. Studies
making use of hypomorphic mutations (Erkner et al., 1999; Wu and Cohen, 2000) confirmed
that tsh function is required for the normal development of proximal leg segments. The
hypomorphic mutants show a reduction in the size of the proximal structures of the leg (coxa,
where lacks most of the bristles and sense organs, trochanter and femur), while in stronger
mutant combination the trochanter is no longer detectable as a discrete segment and the coxa
appears to articulate directly with the femur, with both (coxa and femur) reduced in size and
no sense organs recognised on the coxa and trochanter rudiment (Erkner et al., 1999; Wu and
Cohen, 2000).
It was shown a complex regulatory relationship between Hth and Tsh, in which Hth acts
both directly and indirectly to modulate Tsh levels and to define the distal limit of tsh
expression (Wu and Cohen, 2000). Although, hth and tsh influence each others expression, tsh
mutants produce quite different phenotypes from hth mutants in the leg. Hth limits the
proximal extent of dac expression and is required for the affinity boundary between trochanter
and femur. Tsh limits the proximal extent of Dll expression and is required for proper growth
and differentiation of proximal segments, but does not appear to have a role in PD boundary
formation (Wu and Cohen, 2000).
More recently, it was shown that Tsh plays a role, not only in proximal leg
morphogenesis, but also in the boundary formation between Tsh-expressing and -
nonexpressing cells; as hypomorphic mutants affect, not only the development of the proximal
leg parts, but also the morphology of the region just adjacent to Tsh-expressing cells (Erkner
et al., 1999;2002).
In the leg, Dll acts as a repressor of tsh in Wg active cells, which keeps the boundary
intact in ventral positions (Erkner et al., 1999). In contrast, it seems that Hth acts as tsh
activator in a cell autonomic manner (Azpiazu et Morata, 2002).
I_4.5.2- tsh promotes the identity of the proximal structures of wing and
halteres discs
In the Drosophila wing imaginal disc, different domains along the PD axis are present as
concentric regions, each with a unique genetic address. The centre cells of the disc originate
the distal region of the mature wing, while the peripheral tissues contribute to the formation of
the more proximal structures (Fig. I_5).
The tsh function is required for the proper development of the proximal structures of
the wing and halteres. In fact, the tsh loss of function mutations cause an abnormal wing
posture phenotype called aeroplane (ae): the wings present a paralysis and the halteres are
falling. This is the consequence of the the proximal ventral radius fusion of the wing hinge to
I-The teashirt (tsh) gene
the pleural wing process of the thorax (Soanes and Bell, 1999 ; Soanes et al., 2001; Soanes et
Bell, 2001).
tsh and hth are also co-expressed in all wing disc. However, tsh and hth expression, in
the early of the second and third instars, are restricted to the proximal cells in order to allow
the correct development of the distal region (Azpiazu et Morata, 2000; Wu et Cohen, 2002).
Although tsh and hth showed the same expression pattern in the wing disc, they don’t share
the same mechanisms of regulation. In the wing disc, like in the leg disc, Wg and Dpp signal
transduction also appear to be necessary to complete hth repression in the pouch.
Nevertheless, wg activity is only required for hth repression in a subset of the wing pouch
(Azpiazu and Morata, 2000). The relationship between Dpp signaling and tsh regulation
remains unclear. Dpp pathway in early-induced clones only represses tsh in the most lateral
regions of the disc (Wu and Cohen, 2002) and hypomorphic dpp mutant larvae still show some
tsh repression in the distal wing disc (Cavodeassi et al., 2002).
Figure I_5- Schematic representation of Drosophila wing imaginal discs and the adult wing (in
Laugier, 2005).
(A) Schematic representation of a wing disc. The dorsal region and the ventral wing pouch are represented by (d) and
(v), respectively, from each size of the dorsoventral boundary. (n) Represent the notum and (h) the hinge. The dotted
line indicate the limit of the (A) anterior and (P) posterior compartments.
(B) The adult wing with the indication of the region correspondent to the (n) notum and the (h) hinge.
Wg is required for the early establishment of tsh repression in distal wing disc (Ng et
al., 1996). In contrast, later, Notch-dependent expression of wg at the Dorsoventral (D/V)
boundary is dispensable for tsh repression (Wu and Cohen, 2002). Instead, the maintenance of
tsh repression requires Polycomb Group (PcG) mediated gene silencing (Zirin and Mann,
2004).
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I-The teashirt (tsh) gene
18
Soon after tsh is repressed, distal wing disc cells activate nubbin (nub), which encodes
a POU domain transcription factor required for wing patterning and growth (Cifuentes and
Garcia-Bellido, 1997; Ng et al., 1995). nub expression is indirectly induced by vestigial (vg) in
the wing pouch, but extends more proximally into the wing hinge (Liu et al., 2000;Rodriguez
et al., 2002). tsh expression is incompatible with distal appendage development. Ectopic Tsh
expression in distal cells blocks nub activation and proximalises the adult wing (Azpiazu and
Morata, 2002; Casares and Mann, 2000;Wu and Cohen, 2002; Zirin and Mann, 2007). Thus,
Tsh acts as a repressor of wg expression in the hinge region of the wing disc (Zirin and Mann,
2004).
Recently, it was proposed the division of the wing disc in three domains, whose
boundaries restrict the pattern of cell divisions along the PD axis. These three regions are
comprised of cells that are: Nub+ and Tsh− (the Nub domain), Nub− and Tsh− (the gap
domain), and Nub− and Tsh+ (the Tsh domain; Zirin and Mann, 2007). In this model, tsh
repressed state does not define a compartment using the strict definition of this term (lack
their stringent and immobile boundaries), but the authors defend an analogy with
vein/intervein divisions in the wing disc (de Celis et al., 1995; Gonzalez- Gaitan et al., 1994),
and the rhombomere divisions in the development of the vertebrate brain (Fraser et al., 1990;
Kiecker and Lumsden, 2005).
I_5- tiptop as a tsh paralog
The tiptop (tio), a paralogue of tsh, was identified by a bioinformatics approach. In
addition to the three zinc finger domain common to tsh, tio has a supplementary zinc finger
type Cx2Cx12Hx4H domain in the C-terminal (Laugier et al., 2005). Curiously, the zinc finger
domains organisation present in tio is more closely related to the other tsh member family
than to tsh itself. The mosquito (Anopheles gambiae) and the bee (Apis mellifera), only
present a unique tsh member that codes for a protein containing four zinc fingers domains,
closer to Tio than to Tsh. These various results suggest that in the arthropods, the duplication
event responsible for the generation of the genes tsh and tio occurred within the drosophilae
and that the tsh prototypical gene was closely related to tio (Laugier et al., 2005). The best tsh
and tio sequences homology (51%) is located into the zing finger domain (Laugier et al.,
2005).
Tio is first detected at stage 10 (Campos-Ortega and Hartenstein, 1985) in two
posterior regions of the embryo: Malpighian tubule primordia and a sub-region of the hindgut
primordia (Fig. I_6A). At stage 11, Tio is found in the Malpighian tubule buds and part of the
hindgut (Fig. I_6B). In the head domain, the expression is initiated in the clypeolabrum, in two
bilaterally symmetric clusters of cells (Fig. I_6B). At stage 12, Tio expression appears in the
central nervous system (CNS) of the trunk and the epidermis (Fig. I_6C). The staining in the
hindgut is maintained throughout embryogenesis (black arrowhead).
I-The teashirt (tsh) gene
Figure I_6- Embryonic expression of Tio and Tsh (in Laugier et al., 2005).
Lateral (A, C– F), dorsal (B, H) and ventral (G) views. (A–F) Tio staining. Arrows: Malpighian tubules (MTs);
arrowhead: part of the hindgut; black asterisk: part of the clypeolabrum corresponding to future pharyngeal cells;
gray asterisk: neurons in the brain; white arrowheads: second constriction of the gut. (G, H) Tio (red) and Tsh (green)
immunostainings. Arrowhead: second constriction of the gut; arrow: central nervous system (G) and stellate cells of
the MTs (H). In all panels, the anterior is towards the left and the dorsal towards the top.
At stage 13, Tio staining is present in elongating Malpighian tubules (Fig. I_6D). The
anterior staining is detected in cells that invaginate into the stomodeum (Fig. I_6D) and by
stage 15 onwards, in cells close to the pharynx (Figs. I_6E, F). Tio is also detected in cells of
the brain, the second constriction of the gut, the trunk epidermis, the anterior segments of the
CNS (the three thoracic and the first two abdominal segments) and in the Malpighian tubules
(Figs. I_6E, F; Laugier et al., 2005). Thus, tsh and tio initiate and maintain exclusive
expression domain, however later they present common expression patterns in certain tissues.
At the functional level, the ectopic expression of tsh or tio induce similar effects: both promote
a homeotic transformation of the head into trunk, but tio is less effective than tsh to induce
19
I-The teashirt (tsh) gene
20
the naked cuticle identity. Moreover, the ectopic expression of tsh is more qualified than the
one of tio for the modelling of the trunk, because it restores the diversity of the denticules
more effectively than tio, in a tsh mutant (Laugier et al., 2005).
The simultaneous loss of tsh and tio has a stronger effect than the tsh loss alone,
showing that tio is active in absence of tsh and can partially replace the activity of tsh in the
trunk. In fact, tio and tsh repress each other: in the clypeolabrum, tio represses tsh
expression, while in the trunk, tsh functions as a negative regulator of tio (Laugier et al.,
2005). Consequently, the crucial role of tsh in the patterning of the ventral skin of the trunk is
partly masked by the ectopic expression of tio in absence of tsh (Laugier et al., 2005).
Recently, the interaction between the two paralogs was described during imaginal
development (Bessa et al., 2009; Datta et al., 2009). In the imaginal discs, tsh and tio have
coincident expression domain and in addition both genes show similar functional properties,
when expressed ectopically. Furthermore, tio is able to rescue the development of tsh
mutants, indicating that both genes are functionally equivalent during imaginal development
(Bessa et al., 2009). In addition, these genes share the ability of being able to induce ectopic
eye formation when forcibly expressed in the antennae tissue (Datta et al., 2009). Curiously,
tio appears to be a more effective inducer of retinal formation than tsh (Datta et al., 2009).
Interestingly, the transcriptional regulation of tio and tsh is linked by a negative feedback loop,
which is required to maintain a tight control on the total levels of Tio/Tsh and could explain the
apparently maintenance of a dispensable gene in Drosophila (Bessa et al.,2009).
I_6- The vertebrate tsh family
Tsh plays an essential role during the Drosophila development. As it was explained
before (sections I_4.1 to I_4.5.2 of this chapter), Tsh acts as a co-factor of Hox proteins, that
have been highly conserved during evolution, but Tsh also functions as a modulator of Wg and
Hh signallings, which are homologue to the vertebrates Wnt and Hh proteins, respectivelly.
Three Tshz-related genes, Tshz1, Tshz2 and Tshz3, have been isolated in vertebrates
(chick, mouse and human) based on sequence homology. The conservation, between
Drosophila and murine Tsh amino acid sequences is very low (35%) and essentially restricted
to the region of the three atypical zinc-finger motifs (Cx2Cx12HMx4H) and the acidic domain
towards the N terminus. The murine Tsh proteins possess in addition, two more classical
(Cx2Cx12Hx3-4H) zinc-finger motifs at their C-terminal end, which are not found in Tsh (Fig.
I_7; Manfroid et al., 2004). Thus, murine Tsh proteins are structurally more close to Tio than
to Tsh protein.
I-The teashirt (tsh) gene
Figure I_7- The Teashirt family in Drosophila and vertebrates
The two representants of teashirt family in Drosophila: Teashirt (Tsh) and Tiptop (Tio) are characterized by the
presence of 3 zinc finger motifs (blue box ZnF) in the N-terminus region and an additional Zinc finger motifs (blue box
ZnF) in the C terminus of Tio. The 3 members of Tsh family in vertebrates presents a structure more close to Tio, with
an extra Zinc fingers (3 Zinc fingers in the N-terminus and 2 Zinc fingers in the C terminus represented by the blue
box ZnF) and a acid domain (pink box HD) in the C terminus. The N-terminus is to the left and the C-terminus is to
the right. The numbers in the right correspond to the predict total amino acids number.
The expression patterns of Tshz1 and Tshz2, at different stages of mouse
embryogenesis, are reminiscent of tsh as they appear in restricted regions of the trunk, the
gut and the limbs (Caubit et al., 2000; Coré et al., 2007). Tshz3 is detected in a temporal and
spatial pattern that is similar, though not identical, to Tshz1 (Caubit et al., 2005; Caubit not
published data). These preliminary observations suggest a conserved function for tsh genes, in
the patterning of the anterior/posterior axis.
Murine tsh-related genes are in many aspects functionally equivalent to Drosophila tsh.
The ectopic expression of the three mouse Tsh proteins in Drosophila, like tsh, results in
homeotic transformation of the labial head segment into the T1 trunk segment (Manfroid et
al., 2004). Following ectopic Tshz (or tsh) expression, one or two other head segments take a
trunk identity, as they differentiate denticles and naked cuticle in place of certain head
structures. More importantly, Tshz genes efficiently (although, not completely in the T1
segment) rescue the phenotype of tsh8 null mutants in a very similar way to that described for
tsh: the segment polarity and the T1 to labial homeotic phenotypes are rescued (de Zulueta et
al., 1994; Manfroid et al., 2004). Both murine and Drosophila tsh, also affect the expression of
wg in the ventral labium at stage 11 and repress the expression of the direct tsh target gene
mod (Manfroid et al., 2004). Moreover, Tshz proteins behave as transcriptional repressor: they
21
I-The teashirt (tsh) gene
22
can reduce mod expression and they inhibit transcription in a heterologous test in mammalian
cells (Manfroid et al., 2004; Waltzer et al., 2001).
The ability of tsh and Tshz genes to induce T1 identity indicates that both collaborate
with the same genes and/or proteins that determine segment identity. The Drosophila tsh
directly interacts with the Hox gene Scr to promote the T1 identity (see section I_4.1 of this
chapter; de Zulueta et al., 1994; Röder et al., 1992; de Zulueta et al., 1994; Taghli-Lamallem
et al., 2007). The fact that, ectopic expression of Tshz1, Tshz2 or Tshz3 can transform the
labial segment into T1, provides strong evidence that murine Tshz can specify T1 identity in
cooperation with Scr. Interestingly, the analysis of the skeleton of HoxA5 (an orthologue of
Scr) mutant mice reveals that the most frequent morphological abnormality is the posterior
transformation of the last cervical vertebra (C7) into the likeness of a thoracic vertebra
(Jeannotte et al., 1993). Curiously, this position corresponds to the anterior limit of Tshz2
expression, suggesting its requirement to restrict the domain of activity of HoxA5 in the
somites and to set a morphological border. Even more, ectopic expression of HoxA5 in
Drosophila embryos activates ectopic expression of salivary gland target genes, just like its
functional homologue Scr (Zhao et al., 1993a). This ectopic expression leads also to the
transformation of the larval thoracic segments T2 and T3 toward T1 (Zhao et al., 1993a),
suggesting that, the mammalian protein not only recognizes the correct Scr target sites on
DNA, but also interacts with the proper Scr cofactors. Given that, the interaction between Tsh
and the Hox protein Scr in Drosophila seems to be conserved in vertebrates, at least in the
trunk patterning.
All together these data support that, in flies, the three putative mouse Tshz genes
behave in a similar way as the fly tsh gene (Manfroid et al., 2004) suggesting that, the results
obtained during Drosophila embryogenesis could be applicable to vertebrates embryogenesis.
In addition, the two murine genes Tshz1 and Tshz2 show regionalised patterns of expression,
in the trunk, in the developing limbs and the gut of mice, reforcing the hypothesis that
Teashirt function may have been conserved for patterning the primary axis and specification of
limb structures (Caubit et al., 2000). Thus, it seems that, at least some of the functions
promoted by tsh in Drosophila are conserved in vertebrates
The human tsh genes are localised at the 18q22.3-23 (TSHZ1), 20q13.2 (TSHZ2) and
19q13-11 (TSHZ3). In a screen to identified potential genes implicated in the colon cancer, a
partial cDNA, correspondent to TSHZ1, was isolated (Scanlan et al., 1998). Interestingly, the
human TSHZ1 (SDCCAG33) gene maps in the critical interval (18q22.3–18q23) frequently
reported with chromosomal anomalies in patients with congenital aural atresia (CAA), a rare
developmental anomaly of the ear, which is characterised by a conductive hearing loss with
variable degree (Cody et al., 1999; Core et al., 2007; Cremers et al., 1988; Keppler-Noreuil et
al., 1998; Kline et al., 1993; Strathdee et al., 1995). In addition, the inactivation of Tshz1 in
mice leads to a severe middle ear phenotype, mimicking defects observed in the CAA
syndrome in humans, suggesting that TSHZ1 is a good candidate gene for CAA syndrome
I-The teashirt (tsh) gene
(Core et al., 2007). In the region of TSHZ2, many genes coding for proteins with zinc finger
motifs (for example, ZNF217) are overexpressed in the breast carcinoma (Nonet et al., 2001).
Rencently, it was demonstrated that Tshz3 plays a critical role in mammalian ureteric
development, by controlling smooth muscle (SM) differentiation (Caubit et al., 2008). Tshz3 is
expressed in mesenchymal cells during ureter development, within the SM cells and stromal
cell layers. Furthermore, homozygous Tshz3 null mutant mice display dilatation of the fetal
renal pelvis and proximal ureter and this phenotype correlates with the early, aberrant down-
regulation of genes encoding contractile SM cells markers. In fact, myocardin (Myocd)
expressed in developing wild-type ureteric SMCs, it is downregulated in Tshz3lacZ/lacZ ureters,
suggesting that Tshz3 may control SM differentiation by regulating Myocd. Thus, Tshz3 null
mutant mice have congenital hydronephrosis and hydroureter affecting the proximal section of
the ureter only. This model of ‘functional’ urinary obstruction resembles human congenital
pelvi-ureteric junction obstruction, which occurs in 0.5% gestations, suggesting that TSHZ, or
related, gene variants may contribute to this disorder (Caubit et al., 2008). The implication of
Tshz3 in urinary tract in mice could reflect also a conserved tsh function. In Drosophila it was
previously reported the presence of tsh and tio in the stellate cells, sub-population of the renal
(or Malpighian) tubules, the major excretory and osmoregulatory organs in Drosophila (Laugier
et al., 2005).
Thus, Tsh and Tsh-related are zinc finger protein implicated in the same several
processes both in Drosophila and in vertebrates.
II- Overview of Drosophila Oogenesis
During my PhD work, we identified and analysed some tsh mutants that revealed
problems during oogenesis progression, specifically in follicle cell stem cells (FSC) proliferation.
Thus, we take advantage of the Drosophila ovary to study FSC proliferation.
Drosophila oogenesis represents an excellent system to analyse regulatory intercellular
signalling, it produces ovarian follicle continually throughout adult life and it involves a high
degree of coordination between the proliferation and differentiation programs of two lineages
cells: soma and germ line (Narbonne-reveau et al., 2006).
II_1- The Drosophila reproductive system
The internal reproductive system of the adult female of Drosophila melanogaster is
comprised of the paired ovaries, the efferent genital duct system and its diverticula (King,
1970). Each ovary consists of a cluster of parallel ovarioles (Fig.II_1A). The average number of
23
II-Overview of Drosophila Oogenesis
ovarioles is relatively constant (15-20). Each ovarioles is differentiated into an anterior,
sausage-shape germarium and a posterior vetellarium.
In the germarium take a place the consecutive mitosis to produce a cluster of 16-cell
cyst which one is enveloped by follicle cells. However, the major growth of the ovary and its
accompanying cells occurs within the vitelarium (King, 1970).
Figure II_1- Diagram and a Drosophila ovary and ovarioles (in King, 1970).
(A) A dorsal view of the internal reproductive system of a Drosophila adult female. Two ovarioles have been pulled
loose from the left ovary. Sperm are stored in the ventral seminal receptacle (which is drawn and uncoiled) and in
paired spermathecae. The uterus is drawn expanded as it would be when contains a mature egg.
(B) A Diagram of a single ovariole and its investing membranes. The nurse cells-oocyte complex is representative of
the first 6 stages of oogenesis. Within the vitellarium, sectioned egg chambers are drawn so as to show the
morphology of the nuclei of the oocyte and six of the fifteen nurse cells.
Attached to the surface of the germarium and to all developing egg chamber, there is
an acellular membrane, with 50 µm thick, called the tunica propria (Fig.II_1B). The germarium
and vitellarium reside within a tubular multinucleated epithelial sheath: the peritoneal sheath.
This sheath is a contractile structure containing numerous smooth muscle fibrils and abundant
trachaeoles. Apically, the tubular epithelial sheath tapers to a point where its lumen disappears
and, its inner surface becomes confluent with the tunica propria, surrounding the anterior tip
of the germarium. Anterior to this region, the epithelial sheath transforms into a solid
cytoplasmic cylinder which attaches the ovarioles to the inner apex of the peritoneal sheath.
Each ovarioles is a linear string of gradual maturing follicle. In the germarium, the eggs
chambers are made up in the anterior tip of each ovarioles, maturing progressively through
successive stages, as they move towards posterior part of this autonomous unit of the ovary,
24
II-Overview of Drosophila Oogenesis
to form a polarized oocyte surrounded by a patterned epithelium of somatic follicle cells
(Zhang and Kalderon, 2000; Lin and Spradling, 1993).
II_2- Drosophila germarium offers the unique opportunity of to study three
types of stem cells and their niches
In the germarium, three types of stem cells was identified.
The apical portion of the germarium (region 1) contains 2-3 germline stem cells (GSC)
identified by clonal analysis (Wieschaus and Szabad, 1979) and laser ablation studies (Lin and
Spradling, 1993). They are marked by the presence of the spectrosome. The terminal filament
cells (TF) and the cap cells (CC), which are both none dividing somatic cells, support the GSC
niche (Fig.II_2). The CC directly contacts with GSC by adherent junctions enriched in DE-
cadherin and Armadillo (β-catenin). CC signal for stem cells (SC) maintenance, regulation of
proliferation and differentiation of the SC progeny. GSC divides asymmetrically every 20 hours
to produce a SC and a daughter cystoblast. The differentiating cystoblast undergoes four
rounds of mitosis, with incomplete cytokinesis, to generate a 16-cell cyst (Spradling, 2005).
One of the 16 cyst cells becomes the oocyte while the other 15 cells differentiate as nurse cells
(reviewed in King, 1970).
A recent study, has implicated a newly identified escort stem cells (ESC) as a part of
GSC niche (Decotto and Spradling, 2005). ESC was identified as the most anterior inner
germarial sheath (IGS) cells that directly contacts CC and surround GSC with their cellular
processes (Fig.II_2). These groups of four to six IGS cells are also capable of dividing and
generating differentiated IGS cells. Their progeny are renamed as escort cells (EC). It has
been proposed that these ESCs divide synchronously with GSC to generate differentiated EC,
which tightly wrap early germline cells and move posteriorly with the germline cells. Once
germline cysts pass through region 2a of the germarium, they lose their connections to EC and
become surrounded by the follicle cells (Decotto and Spradling, 2005; Kirilly and Xie, 2007).
In the region 2a/2b, two follicle stem cells (FSC) divides, in average every 10 hours, to
generate 16 cells that initially cover each cyst by an epithelial cell-like follicle cells and then
bud off him from the germarium to form individual egg chambers separated by 5-7stalk cells
(Spradling, 2005). The follicle cells go through approximately five rounds of mitotic division
before leaving the germarium and than more three to four rounds of mitosis (until stage 6),
before switching to the endocycle since stage S7 until stage S9, in which they become
polyploid by cycling through S phase without the intervention of cell division (Calvi et al.,
1998, Nystul and Spradling, 2010).
Previously, Clonal analysis has revealed that, the follicle cells are already divided into two
populations in the germarium. One lineage gives rise to the stalk cells and the polar cells,
which are a pair of follicle cells at each end off the egg chamber that connects to the stalk.
25
II-Overview of Drosophila Oogenesis
Once the egg chamber is formed, both cells types are never divided (Tworoger et al., 1999;
Lopez-Schier and Johnston, 2001). The rest of the follicle cells derive from a separate lineage
and form the epithelium around the cyst (epithelial follicle cells or main-body).
Figure II_2- Drosophila germarium cross section showing the locations of germ line stem cells
(GSC), follicle stem cells (FSC), and their niches (adapted from Lie and Xie, 2005).
Two or three GSC (red cells, left) are situated in their niche, composed of cap cells (CC, green cells, left) and terminal
filament cells (TF, light blue cells, left tip), whereas their differentiated progeny, including cystoblasts (CB) and
differentiated cysts (yellow cells, middle), are surrounded by inner germarial sheath (IGS) cells recently renamed by
escort cells EC, purple cells and green cells, bottom and top). As the CB divides it becomes a 16-cell developing cyst
(DC) and it passes through region 2a, after which it will be surrounded by follicle cells (FC, orange cells on right),
generated by the follicle stem cells (FSC, red cells, bottom and top). Two or three FSC directly contact the posterior
group of inner sheath cells (green cells, bottom and top) forming their niche. The three regions of the germarium are
indicated below the diagram.
Nystul and Spradling, (2010) by examining clones induced at low frequency, shown
recently that FC type specification is independent of cell lineage. Instead, follicle cells are
multipotent prior to polar or stalk specification. This fits well with recent studies showing that
many additional cells in the germarium can be induced to take on a polar cell fate by strong
Notch signalling, while high levels of JAK-STAT signalling can induce more stalk cells (Assa-
Kunik et al. 2007). In addition, they map the specification of the first anterior and posterior
polar cells, when cysts are associated with 8-16 FC (in mid to late region 2b), what is in
agreement with previous studies (Margolis and Spradling, 1995; Besse and Pret, 2003) which
found that polar cells were first specified at the 14-cell stage.
26
II-Overview of Drosophila Oogenesis
The two distinct populations of stem cells, FSC and GSC, work in a coordinated fashion
to efficiently produce egg chambers and share some molecular mechanisms to control self-
renewal and proliferation. First, like the GSC, the FSC divides to generate a self-renewing FSC
(which remains in the niche) and a differentiating daughter cell (which one moves away from
the niche for further proliferation and differentiation). Second, like the GSC, the FSC is also
anchored to niche cells (the posterior EC) through DE-cadherin-mediated cell adhesion (Song
and Xie, 2002).
II_3- Origin of GSC, TF and CC
The pole cells, which are equivalent to primordial germ cells of vertebrate embryos,
constitute the first distinct cell lineage established in the Drosophila embryo. Pole cells (a
group of cells set aside at the start of embryogenesis) are the precursor of the GSC and
initially are located at the posterior pole of the blastoderm embryo. At the beginning of
gastrulation, they move dorsally and are carried inside the embryo by posterior midgut
invagination. Next, pole cells migrate across the midgut to contact the embryonic mesoderm
and subsequently divide into two groups, with interact with the gonadal mesoderm to form the
primitive gonads (Starz-Gaiano and Lehmann, 2000). Each female embryonic gonad hosts 12
pole cells on average. During embryogenesis and larval stage, these cells proliferate to make
80-110 pole cells per larval ovary. At the end of the larval development, pole cells stop their
proliferation and the first sign of oogenic differentiation is observed (King, 1970; Lin and
Spradling, 1997). At the larval-pupal transition, pole cells become refractory to hybrid
dysgenesis, which suggests that larval and pupal germline cells possess different properties
(Bhat and Schedl, 1997).
The somatic cells of the gonad also proliferate during the larval stages, remaining
relatively undifferentiated until the end of the larval phase of the development, when they
ultimately subdivide the gonad into 15-20 ovarioles. At this stage, TF and a group of cells, that
at least on morphological grounds, look like CC, form the anterior structure of the germarium
(Chen et al., 2001; Godt and Laski, 1995). In close contact with these presumptive CC are two
or three germline-derived cells behaving like stem cells in mature ovaries: they possess a
spherical fusome located apically, near the boundary with the CC and they divide, so that one
of the daughter cells always remains in contact with the CC (Gonzales-Reyes, 2003).
Considering that germ cells become refractory to hybrid dysgenesis at the larval-pupal
transition, and that this coincides with the differentiation of the TF and presumably CC, it is
tempting to speculate that it is the apposition of CC and pole cells that push these last ones
into acquiring a stem cell fate. In this model, pole cells proliferate throughout larval stages to
make the pool of germline cells present in third instar larvae. Once the TF and CC of the gonad
are differentiated and organised into a niche, they induce the germline cells in contact with the
27
II-Overview of Drosophila Oogenesis
CC to adopt a stem cell fate. The newly formed GSC will then initiate asymmetric divisions to
sustain oogenesis (Gonzales-Reyes, 2003).
II_4- Ovary stem cells and their molecular mechanisms of regulation
II_4.1- Genes and Pathways that control GSC self-renewal, proliferation and
differentiation
In the last years, it was identified a number of genes with critical functions in GSC self-
renewal, proliferation and differentiation. Some of these genes encode intrinsic factors,
including nuclear factors and translational regulators that function inside the GSC, while the
others produce proteins that function to generate signals and regulators of signal production
within the niche.
In the apical tip of the germarium, two or three GSC can be easily identified by their
localisation (directed anchored to posterior side of the CC), size (the biggest cell at the tip of
the germarium) and anterior localisation of a round spectrossome. The spectrossome and its
counterpart in the mitotic cyst (the branched fusome) are germ cell-specific organelles that are
rich in membrane skeletal protein such as α-Spectrin and Hu li tai shao (Hts). Germ cells,
including GSC, also express the Vasa protein (Lin et al., 1994; Cuevas et al., 1996).
Instead of asymmetrically deposited of intracellular factor, observed for neuronal stem
cell-like neuroblasts in embryo and larval brain, the asymmetric division of GSC is
accomplished by differential signalling resulting from an asymmetric organisation of the GSC
niche (Song et al., 2002; Jan and Jan, 2001). Normally, the GSC is anchored to CC by
adherent junctions formed between the two cell types, which are enriched in DE-cadherin and
Armadillo (more details in II_4.1.3.3). As the GSC divides to generate two daughters cells, one
remains in contact with the CC and the other one is placed one cell away from the CC. The
anterior daughter continues to receive the signals from GSC niche and self-renew as a stem
cell, while the posterior daughter differentiates into a cystoblast due to differential signalling
from their microenvironment. Following loss of one neighbouring GSC, the two stem cells
daughter’s stay in the niche, due to available niche space, and becomes GSC (symmetric
division in this particular case; Xie and Spradling, 2000). If they move away from their niche
due to the loss of adherens junctions, both daughters can differentiated into cystoblasts
resulting in the loss of GSC (Song et al., 2002).
Significant progress has been made in the past few years in gaining a better
understanding of how the niche controls GSC function by identifying signalling pathways and
genes that regulate these pathways (Fig.II_3).
28
II-Overview of Drosophila Oogenesis
II_4.1.1- BMP, Piwi and Yb from the niche to regulate the maintenance and
division of the GSC
The GSC asymmetric cell division can be partially attributed to the extrinsics factors
emanating from the niche. Regulatory molecules: bone morphogenetic protein (BMPs) and Piwi
are produced from CC to modulate ovarian GSC maintenance and division via intracellular
signal pathway (Fig. II_3).
The pathway best studied for control GSC function is the BMP pathway, which is both
necessary and sufficient for GSC self-renewal and proliferation (Xie and Spradling, 1998; Song
et al., 2004). Two BMP ligands, Decapentaplegic (Dpp) and Glassbottomed boat (Gbb) are
expressed in CC and TF (Xie and Spradling, 1998; Song et al., 2004). They serve as a short
range signal to activate BMP signalling in GSC through type I [Thickveins (Tkv) and Sax] and
TypeII (Punt) receptors. When Dpp reaches a GSC, it interacts with its receptors. Within this
ligand-receptor complex, the constitutively actived receptor Punt phosphorylates Tkv, which in
turn phosphorylates the receptor SMAD (R-SMAD): mother against dpp (MAD). Phosphorylated
MAD (p-MAD) subsequently interacts with the common-mediator-SMAD (co-SMAD, also know
as SMAD4, with only a single member in D. melanogaster encode by the Medea gene). This
heteromeric complex (p-MAD and Medea) is translocated into the nucleus for: (I) to participate
in the activation of a BMP response gene, Daughters against dpp (Dad), primarily in the GSC
(Chen and McKearin, 2003; Kai and Spradling, 2003; Casanneuva and Ferguson, 2004; Song
et al., 2004; Affolter and Basler, 2007), and (II) to controls GSC self-renewal by directly
repressing the expression of differentiation-promoting genes such as bag of marbles (bam)
(Chen and McKearin, 2003; Song et al., 2004). Bam acts with its partner, benign gonial cell
neoplasm (BGCN; which is a large DExH box protein), to initiate differentiation of the GSC
daughters that leave the niche by an unknown mechanism (Fuller and Spradling 2007).
One of the two proteins domains of the SMAD proteins, the so called SMAD-homology
domain I, behaves as a DNA-binding domain that recognises a GC-rich motif, such sequence
elements were identified in Dpp-responsive genes (Affolter and Basler, 2007).
Mutations in dpp, gbb or downstream components lead to the GSC loss by premature
differentiation and slower division rates (Xie and Spradling, 1998; Xie and Spradling, 2000;
Song et al., 2004). Overexpression of dpp, but not gbb, completely blocks cystoblast
differentiation, resulting in proliferation of GSC-like tumors throughout the germarium (Xie and
Spradling, 1998; Song et al., 2004). These studies indicated that BMP signalling is necessary
and sufficient for GSC self-renewal. Since Gbb and Dpp use common signal transduction, it is
still to be determinate why overexpression of dpp, but not gbb, is sufficient to block germline
cell differentiation (Haerry et al., 1998; Khalsa et al, 1998). BMP signalling also have an
important role in controlling the division of primordial germ cells (PGSC) in the developing
female gonad and the recruitment of GSC to their niche (Zhu and Xie, 2003). Surprisingly,
partially differentiated 2-, 4- and 8-cells cysts are capable to revert back to GSC, when the
29
II-Overview of Drosophila Oogenesis
niche signal dpp is provided (Kai and Spradling, 2004). How Dpp signalling can completely turn
off the active differentiation program in cystoblasts is not clear. Understanding this
phenomenon would help us to know how GSC self-renewal and differentiation is regulated, and
it may allow the regeneration of stem cells from differentiated cells, in future regenerative
medicine, if such cell fate reversal also exists in mammalian stem cells. How the short range
effects of BMP signal are controlled and whether or not there is other repressed target gene for
beyond bam; they are some major question about BMP pathway in GSC without answer for
now.
BMP and Janus kinase–signal transducers and activators of transcription (JAK-STAT)
signalling are indispensable for GSC self-renewal in Drosophila testis (Kigger et al., 2001;
Tulina and Matunis, 2001; Shivdasani and Ingham, 2002; Kawase et al., 2004). Germ cell
regulation by somatic BMP signals is probably an ancient mechanism (Gilboa and Lehmann,
2005; Li and Xie, 2005); however, bam and bgcn orthologs are unknown outside Drosophila.
The roles of Bam, Bgcn, and BMP signalling are all substantially different in males, where Bam
and Bgcn function to end rather than initiate mitotic cyst divisions. BMP signal reception is still
required to maintain GSC and repress bam transcription, but gbb appears to have a stronger
effect on GSC maintenance than dpp in testis, which is consistent with their relative expression
levels (Shivdasani and Ingham, 2002; Kawase et al., 2004; Fuller and Spradling, 2007). gbb is
expressed in cyst cells and the hub, and bam is repressed in gonialblasts as well as in GSC.
BAM misexpression can be toxic to male GSC, rather than just promoting differentiation, as it
does in females (Schulz et al., 2004). The primary task of controlling GSC self-renewal in
males is performed by a different signal, the cytokine-like ligand Unpaired (UPD) (Kigger et al.,
2001; Tulina and Matunis, 2001). UPD, expressed by hub cells, activates the JAK-STAT
pathway in GSC to specify stem cell self-renewal. When a male GSC divides, one of the
daughter retains contact with the hub maintaining the stem cell identity, while the other
daughter displaced away experiences a weaker signal and initiates differentiation (Decotto and
Spradling, 2005; Li and Xie, 2005). Therefore, JAK-STAT signalling plays an instructive role in
GSC self-renewal in the testis, similar to the Dpp/BMP signalling pathway in the ovary. JAK-
STAT pathway is also able to revert back differentiated germ cells into GSC (Brawley and
Matunis, 2004). In contrast, female GSC have no autonomous requirement for JAK-STAT
signalling (Decotto and Spradling, 2005; Li and Xie, 2005).
Like BMP, piwi and fs(I)Yb (Yb) are involved in GSC maintenance. Piwi and Yb are required in
somatic cells, such as TF and CC. Loss-of-function mutations in piwi or Yb cause rapid loss of
GSC, while Yb and piwi overexpression increases GSC-like or cystoblast-like germ cells (Lin
and Spradling, 1997; Cox et al., 1998; King and Lin, 1999; Cox et al., 2000; King et al.,
2001). Yb encodes a novel intracellular protein that regulates piwi and hedgehog (hh)
expression (King and Lin, 1999; King et al., 2001). piwi, is the founding member of the piwi
family genes coding for proteins containing conserved PAZ and Piwi domains (which bind to
30
II-Overview of Drosophila Oogenesis
RNA), and it is involved in the regulation of microRNA (miRNA) production (Cox et al., 1998;
Cox Figure II_3- Diagrams of the identified signalling pathways and intrinsic factors that regulate GSC
maintenance and differentiation in the Drosophila ovary (in Kirilly and Xie, 2007).
Terminal Filament cells (TF)/ cap cells (CC) express Yb, Piwi and BMPs (Dpp and Gbb), which control germline stem
cell (GSC) self-renewal and/ or division in a non-cell-autonomous fashion, while the insulin pathway controls GSC
division. Also, gap junctions formed by Zpg and adherens junctions formed by E-cadherin encoded by shg controls
GSC maintenance and anchorage, respectively. The translational regulators (Pum, Nos and Pelo), the cell cycle
regulator CycB, the chromatin remodeling factor Iswi are required intrinsically in the GSC to control its self-renewal,
while the GSC-expressed Piwi and miRNA pathway components (Dcr-1 and Loqs) are required intrinsically for
controlling GSC division. In the cystoblast (CB), Bgcn, Bam, Sxl and Otu control CB differentiation.
31
II-Overview of Drosophila Oogenesis
RNA), and it is involved in the regulation of microRNA (miRNA) production (Cox et al., 1998;
Cox et al., 2000; Grivna et al., 2006a; Grivna et al., 2006b; Kotaja et al., 2006; Megosh et al.,
2006).
Piwi-mediated pathway is absolutely essential for GSC self-renewal, while Hh signalling
play a non essential role in controlling GSC self-renewal by having redundant function with the
piwi-mediated pathway (King et al., 2001).
Recently, two studies indicated that Piwi also controls bam repression in GSC. Like it
was reported for dpp and gbb mutants, the specific removal of piwi function from GSC niche,
results in the derepression of bam in GSC. Remains unclear if piwi pathway could regulate BMP
production, secretion or activation or, for other hand, it could control the production of a new
signal that is also essential for controlling GSC self-renewal and bam repression independently
from BMP signalling (Kirilly and Xie, 2007).
II_4.1.2- External factors: Nutrition
Several external factors influence the rate of egg production such as nutrition,
humidity, availability of males and of egg laying sites (Ashburner, 1989; Bownes and Blair,
1986; King, 1970). In particular, the rate of proliferation of germline and follicle stem cells
(SC), as well as of their mitotically dividing progeny, responds to nutritional input (Drummond-
Barbosa and Spradling, 2001). In females kept on a protein-rich diet, the follicle cell
proliferation rate is 3 to 4-fold higher compared to the one of females feet with a protein-poor
diet. Follow switching from a rich to poor food source, or vice versa, the adjustment of
proliferation rates occurs within 18–24 h. This rapid and reversible change of proliferation
rates in response to nutritional input requires the insulin pathway (Fig.II_3), based on the
analysis of a female sterile mutation of the insulin receptor substrate (IRS)-like gene chico,
which is a major insulin pathway mediator (Drummond-Barbosa and Spradling, 2001). The
physiology of insulin action during the proliferative response to nutrition is poorly understood,
although female sterility results from mutations of several components of the insulin pathway
(Bohni et al., 1999; Chen et al., 1996; Montagne et al., 1999).
Insulin is not produced by cells in the germarium, instead by fat cells in the brain.
Insulin is a well-know signal for regulating sugar metabolism and is modulated by nutritional
conditions. Two recent studies demonstrated that, Drosophila α-endosulfine (dendos) gene and
the neuronal-derived Drosophila insulin-like peptides (DILPs) directly regulate GSC divisions’
rates, demonstrating that signals mediator of the ovarian response to nutritional input can
modify stem cells activity in a niche-independent manner (Drummond-Barbosa and Spradling,
2004; LaFever and Drummond-Barbosa, 2005). The GSC defective in insulin divides much
slower than wild-type ones, but insulin signals pathway only affects stem cells division and not
self-renewal in Drosophila ovary.
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II-Overview of Drosophila Oogenesis
II_4.1.3- Intrinsic factors in GSC maintenance and differentiation
II_4.1.3.1- Translational factor: Pumilio (Pum) , Nanos (Nos), Vasa (Vas) and
Pelota (Pelo)
Two classes of intrinsic factors control GSC: self-renewing factors or differentiation
factors (Fig.II_3).
Translational regulation by the intrinsic factors plays a critical role in GSC self-renewal.
The translational repressors Pumilio (Pum) and Nanos (Nos), are required to maintain GSC in
adult ovary as well as to prevent precocious differentiation of PGC in the developing female
gonad, by repressing the translation of mRNA that encode for differentiation factors (Lin and
Spradling, 1997; Forbes and Lehmann, 1998; Gilboa and Lehmann, 2004; Wang and Lin,
2004). They are RNA biding proteins, which form proteins complexes that repress the
translation of mRNA in Drosophila embryos (Baker et al., 1992). Mutations in both (pum and
nos), cause precocious differentiation of PGC as well as premature GSC loss (Gilboa and
Lehmann, 2004; Wang and Lin, 2004). In addition, the germ cell-specific factor, vasa (vas),
encoding a Drosophila homologue of eukaryotic initiation factor 4A, is also required for ovarian
GSC self-renewal. vas mutants germaria contain few degenerate or growth-arrested germ cells
(Styhler et al., 1998). Recently, another gene was identified for its essential role in controlling
GSC self-renewal and proliferation: pelota (pelo). pelo encodes a translation release factor-like
protein and, pelo mutants completely lose GSC in the first week after eclosion and also slow
down GSC division (Xi et al., 2005).
The identification of the mRNA target for Pum, Nos, Vas and Pelo will help to
understand how GSC self-renewal is controlled.
II_4.1.3.2- MicroRNA (miRNA) and repeat-associated small interfering RNA
(rasiRNA) is important for GSC proliferation and maintenance
One of the key characteristics of stem cells (SC) is their capacity to divide for long
periods of time in an environment where most of the cells are quiescent. Therefore, a critical
question in stem cell biology is how stem cells escape from cell division stop signals.
Recently, it was reported the necessity of the microRNA (miRNA) pathway for proper
control of GSC division in Drosophila melanogaster (Fig.II_3; Rochat et al., 1994; Zhang et al.,
2003; Hatfield et al., 2005; Clarke and Fuller, 2006). miRNAs and short interfering RNAs
(siRNAs), processed by the type III double-stranded RNase Dicer, function in an RNA-based
mechanism of gene silencing (Rochat et al., 1994; Zhang et al., 2003; Hatfield et al., 2005;
Clarke and Fuller, 2006). Most characterised miRNAs repress gene expression by blocking the
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II-Overview of Drosophila Oogenesis
translation of complementary messenger RNAs into protein. Experimental evidence has
suggested that small RNAs regulate stem cell character in plants and animals (Bjerknes and
Cheng, 1999; Hatfield et al., 2005; Xie et al., 2005). Moreover, some miRNAs are differentially
expressed in stem cells, suggesting a specialised role in stem cell regulation (Lin, 2002;
Hatfield et al., 2005).
MicroRNAs (miRNAs) are 21 to 23 nucleotide single stranded RNAs that are encoded in
the chromosomal DNA and repress cognate mRNA targets (Forstemann et al., 2005; Clarke
and Fuller, 2006). They are transcribed as long hairpin-containing precursors by RNA
polymerase II and processed in the nucleus by the multidomain RNase III endonuclease
Drosha (Rochat et al., 1994; Bjerknes and Cheng, 1999; Lin, 2002; Zhang et al., 2003;
Forstemann et al., 2005; Xie et al., 2005). Drosha converts pri-miRNA transcripts to precursor
miRNA (pre-miRNA); in the cytoplasm, a second RNase III endonuclease, Dicer, converts pre-
miRNA into mature miRNA (Xie and Spradling, 1998; King and Lin, 1999; King et al., 2001;
Song et al., 2004; Forstemann et al., 2005). In Drosophila, two Dicer paralogs define parallel
pathways for small RNA biogenesis. Dicer-1 (Dcr-1) liberates miRNA from pre-miRNA, whereas
Dicer-2 (Dcr-2) excises small interfering RNA (siRNA) from long double-stranded RNA (dsRNA)
(Cox et al., 1998; Cox et al., 2000; Forstemann et al., 2005).
Dcr-1 mutant GSC exhibit normal identity but are defective in cell cycle control, due to
delay in the G1 to S transition, which is dependent on the cyclin dependent kinase inhibitor
Decapo, suggesting that miRNAs are required for SC to bypass the normal G1/S checkpoint
(Hatfield et al., 2005).
The dsRBD protein Loquacious (Loqs, also known as R3D1), a paralog of R2D2, it was
identified as the partner of Dcr-1. Mutation of loqs disrupt normal pre-miRNA processing, in
adittion, loss of Loqs function in the ovary disrupts GSC maintenance, rendering loqs mutant
females sterile (Jiang et al., 2005; Forstemann et al., 2005).
It is intriguing, that two interacting protein partners (Dcr-1 and Logs), that are
essential for miRNA production, have distinct role in the control of the GSC function. One
explanation is that the dcr-1 mutations, used in the study, are not null alleles and retain
enough function to control GSC self-renewal but not enough to GSC division. Alternatively,
Loqs could signal for controlling self-renewal in the niche and for GSC division in SC, similar to
piwi (Kirilly and Xie 2007). In the future, it will be important to distinguish between these two
hypotheses and to clarify miRNAs functions produced by niche and GSC.
Piwi is known to be involved in generating 29-31 nucleotides repeat-associated small
interfering RNA (rasiRNA) for silencing retrotransposons and repeat sequences (66). In piwi
mutant GSC divides much slower than wild-type but have normal self-renewal capacity,
indicating that rasiRNA pathway is also required to control GSC division (Cox et al., 2000).
II_4.1.3.3- Junctional proteins: DE-cadherin, Armadillo (Arm) and Zero
population growth (Zpg)
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II-Overview of Drosophila Oogenesis
Junctional proteins are also important for GSC anchorage in the niche and for the
communication between the niche and GSC (Fig. II_3). Germline cells come into contact with
the specialised somatic cells that form the tip of the germarium during late larval development.
The juxtaposition of germline cells with future CC seems to initiate the establishment of
adherent junctions that are maintained throughout pupal development and adult life.
Cadherin-mediated adhesion, probably in concert with other factors, is necessary to recruit SC
to their developmental microenvironment. In addition, DE-cadherin accumulates at the
anterior side of GSC forming adherens junction with CC in the adult ovary, which supports a
role for this adhesion system in anchoring GSC to their niche. Mutations in Armadillo (Arm,
β−catenin) and shotgun (E-cadherin), the two key components of adherens junctions, cause
rapid GSC loss (Song et al., 2002). However, a lack of DE-cadherin or Armadillo does not seem
to compromise the identity or behaviour of SC, since the mutant SC persist for at least three
weeks after removal of DE-cadherin or Armadillo. As long as these cells remain in the
appropriate environment, even in the absence of DE-cadherin or Armadillo proteins, they
retain the self-renewal capacity and asymmetric division typical of SC. (Song et al., 2002). The
main function of adherens junctions is to keep GSC in close proximity to niche cells to receive
maximal BMP signalling for self-renewal. In Drosophila testis, adherent junctions also function
as a focal point for regulating spindle orientation of SC division, assuring the asymmetric cell
division (Yamashita et al., 2003). Cadherin also accumulates between blood SC and their
niche, suggesting that cadherin-mediated cell adhesion may be a general mechanism for
anchoring SC in their niche (Zhang et al., 2003).
In addition, gap junctions are also formed between GSC and CC. Zero population
growth (Zpg), a component of gap junctions (inexin-4) in the ovary and testis, is required to
maintain GSC and to promote germ cell differentiation (Tazuke et al., 2002; Gilboa et al.,
2003). Loss of zpg function results in partial GSC loss due to cell death and accumulation of a
few single cells. Because zpg is a gap junction components, it helps in the transport of small
molecules from supporting somatic cells to control the survival of GSC and the differentiation
of their progeny. zpg may function in parallel with bam to regulate germ cell differentiation.
II_4.1.3.4- Cell cycle regulators: Cyclin B (CycB) and Imitation switch (ISWI)
Cyclin B (Cyc B) is a G2 cyclin that acts in complexes with cyclin dependent kinase 1
(Cdk1), and it is specifically required in germ cells for promoting division of PGC and GSC (Fig.
II_3). Cyc B mutation doesn’t have obvious effect on somatic development but, severely
delays PGC proliferation and affects GSC maintenance. Curiously, the overexpression of Cyclin
A (Cyc A) and Cyclin B3 (Cyc B3), which are others G2 cyclins partners of Cdk1 (see sections
III_2.1.2 and III_2.1.3 of this chapter), can not rescue the cyc B mutant defects. However,
35
II-Overview of Drosophila Oogenesis
remain unclear how this general cell cycle regulator specifically affect PGC and GSC self-
renewal and proliferation (Wang and Lin, 2005).
ATP-dependent chromatin remodelling factors are required to control stem cell self-
renewal. Imitation switch (ISWI) changes the conformation of the chromatin, making the DNA
accessible to transcription factors for gene activation or repression (Tsukiyama et al., 1995;
Corona et al., 1999). In Drosophila ovary, at least in part, ISWI controls GSC self-renewal
through the regulation of the responses of SC to BMP niche signals. Mutants GSC for iswi are
defective to repress bam expression and are lost rapidly (Xi and Xie, 2005).
II_4.1.3.5- Differentiation promoting factors: Bag of marbles (Bam), Benign
germ cell neoplasm (Bgcn), Orb, Sex lethal (Sxl) and ovarian tumor (Otu)
Several differentiation-promoting factors are involved in cystoblast differentiation in the
ovary; these include Bag of marbles (Bam), Benign germ cell neoplasm (Bgcn), ovarian tumor
(Otu), Orb and Sex lethal (Sxl) (Fig. II_3; Boop et al., 1993; Christerson and McKearin, 1994;
Lantz et al., 1994; McKearin and Ohlstein 1995; Lavoie et al., 1999; Ohlstein et al., 2000).
Bam and Bgcn are the best studied (McKearin and and Spradling, 1990; Gateff et al., 1996;
Lavoie et al., 1999; Ohlstein et al., 2000). Mutations in both, bam and bgcn germline cells,
abolish cystoblast differentiation leading to a cystoblast like germ cell tumor phenotype (Gateff
et al., 1996; McKearin and and Spradling, 1990; Ohlstein et al., 2000). However, bam
overexpression (but not bgcn in GSC) causes their premature differentiation and consequently
germ cell depletion (Ohlstein and McKearin, 1997). Bam acts as a differentiation-promoting
factor in cystoblasts. One of the bam function is to downregulate Dpp signaling downstream of
Dpp receptor activation. This Bam role is functionally redundant with the degradation of the
target phosphorilated Mad (a BMP specific SMAD) by action of the ubiquitin protein ligase
Smurf (Casaneuva and Ferguson, 2004).
Mutations in Orb, Sxl and Out, cause an accumulation of GSC-like or cystoblast-like
cells mixed with early differentiated cysts, suggesting that they regulate, but are not
absolutely required for cystoblast differentiation and early cyst division (Boop et al., 1993;
Christerson and McKearin, 1994; Lantz et al., 1994). Orb encodes a protein involved in the
regulation of mRNA polyadenylation (Christerson and McKearin, 1994). Sxl is a RNA biding
protein, which is known to be involved in the regulation of the mRNA splicing and translational
regulation (Christerson and McKearin, 1994; Lantz et al., 1994). Otu is also a putative mRNA
binding protein but, its function at the level of the mRNA stability, splicing or translation, is not
clear. It remains unclear whether overexpression of otu would be sufficient to cause GSC
differentiation, like bam (Kirilly and Xie, 2007).
Self-renewal and differentiation are two antagonists process for stem cell and must be
correctly balanced in a normal situation. We expected that the two mechanisms neutralise
36
II-Overview of Drosophila Oogenesis
each other, in order to assure the normal switch between SC maintenance and differentiation
of their descendent. In fact, BMP and Piwi signal from the niche are required to repress bam
expression in GSC since, defects in BMP or Piwi signalling result in the upregulation of bam in
GSC (Szakmary et al., 2005). In other hand, Bam seems to repress BMP signal in GSC.
Removal of bam function, completely suppress the loss of GSC cause by defective BMP and
Piwi pathway. In addition, the loss of GSC phenotype, caused by bam overexpression, could be
partially suppressed by elevated BMP expression (Casaneuva and Fergunson, 2004; Kai and
Spradling, 2004). As BMP and Piwi are redundant to repress bam, this last one, also has a
redundant function with Smurf in the downregulation of the Dpp signalling (Casaneuva and
Fergunson, 2004).
However bam, doesn’t seems to be the only differentiating factor to induced cystoblast
differentiation. In pum and pelo GSC mutants, Bam is not upregulated indicating that, Pum
and Pelo normally repress the (same or different) Bam-independent pathway (Szakmary et al.,
2005; Xi et al., 2005; Chen and McKearin, 2005). Consistently, bam pum and bam pelo double
mutants are able to differentiate in germ cell cyst, while bam mutations alone arrested cells as
pre-cystoblasts (Chen and McKearin, 2005; Szakmary et al., 2005; Xi et al., 2005).
In resume, these signals could be integrated and balanced by the following model. GSC
are retained as SC because Pum:Nos complexes repress translation of a pool of mRNAs that
induce cystoblasts differentiation.
Dpp/BMP signal from the niche is received by GSC through its receptors Punt and TKV,
and it is transduced by pMad to silence bam transcription in these cells. The silencing of bam
transcription blocks the formation of Bam:Bgcn complexes and allows maintaining active the
Pum:Nos translational repression, thus maintaining the SC fate. Piwi in niche cells has an
essential and cooperative function to repress bam expression. Piwi and Dpp signalling
pathways converge at some unknown point upstream of bam, in either niche cells or GSC.
After a GSC division, the strength of Dpp signalling falls to levels that cannot longer efficiently
silence bam transcription in the cell displaced to the posterior and away from CC. This could be
due to declining Dpp levels or diminished piwi-dependent signals, which leads to the reduction
of p-Mad levels. As bam transcription increases, Bam:Bgcn complexes antagonise Pum:Nos
action and cause expression of cystoblasts-promoting mRNAs, initiating the events of
cystoblasts differentiation. Therefore, Pum/Nos can be considered as a switch between self-
renewal and differentiation, whereas niche signalling through Bam/Bgcn regulates this switch
at a single cell level (Chen and McKearin, 2005; Szakmary et al., 2005).
However, it is important continuing the search of molecular links between self-renewal
and differentiation pathway and learn more about biochemical functions of these factors, to
clarify how self-renewing factors and differentiation-promoting factors interact with each other
to control the balance between the antagonistic self-renewal and differentiation processes in
GSC.
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II-Overview of Drosophila Oogenesis
II_4.2- Genes and Pathways that control FSC self-renewal, proliferation and
differentiation
II_4.2.1- Molecular mechanisms shared by GSC and FSC for self-renewal and
proliferation
The continuous production of egg chambers in the Drosophila female depends on the
coordinated work of two types of stem cells: GSC and FSC, which are responsible for producing
differentiated germ cells and somatic follicle cells, respectively. Thus, these two types of SC
share some molecular mechanisms controlling self-renewal and proliferation.
Just like GSC, FSC are also anchored to their neighbouring supporting cells (posterior
escort cells, EC), through DE-cadherin-mediated cell adhesion (Fig.II_4; Song and Xie, 2002).
Such anchorage is important for maintaining FSC identity. DE-cadherin and Armadillo (Arm,
Drosophila β-catenin) accumulate in the junctions between FSC and posterior EC. Removal of
DE-cadherin from FSC results in SC loss in the adult ovary. Furthermore, the cadherin-
mediated adhesion is also important for maintaining FSC in their niches before adulthood
(Song and Xie, 2002).
As observed for GSC, when FSC divides one daughter (that retains stem cell identity)
remains in its position and the other daughter moves posteriorly to proliferate and then
generates differentiated follicle cells and stalk cells.
BMP signalling is also necessary and sufficient for promoting self-renewal and
proliferation of FSC in the Drosophila ovary (Fig.II_4). BMP signalling (by Gbb) is required in
FSC to directly control their maintenance and division, but it is dispensable for proliferation of
their differentiated progeny (Kirilly et al., 2005).
Finally, like the GSC that requires ISWI (see section II_4.1.3.4 of this chapter) for its
self-renewal, the FSC requires Domino (Dom; Xi and Xie, 2005). Dom is a member of
SWI2/SNF2 family of DNA-dependent ATPases, which function as a transcriptional repressor by
interfering with the chromatin structure (Ruhf et l., 2001). FSC mutants for dom, are lost
rapidly due to precocious differentiation, but not apoptosis (Fig.II_4; Xi and Xie, 2005).
II_4.2.2- Hedgehog (Hh) and Wingless (Wg), two critical signals for FSC
maintenance and proliferation
Despite the similarities in mechanisms shared by GSC and FSC, there are significant
differences between these two SC type. The organization of the FSC niche is quite different
from that of the GSC niche. The GSC niche is composed by the neighbouring somatic cells (TF,
CC and ESC).
38
II-Overview of Drosophila Oogenesis
Figure II_4- Diagrams of the identified signalling pathways and intrinsic factors that regulate
follicle stem cells (FSC) and escort stem cells (ESC) maintenance and differentiation in the Drosophila
ovary (adapted from Kirilly and Xie, 2007).
Wg and Hh produced by terminal filament cells (TF)/ cap cells (CC) and Gbb produced by posterior escort cells (EC)
control FSC maintenance, while the Jak-Stat pathway controls ESC maintenance. E-cadherin-mediated cell adhesion is
required for anchoring FSC, while the chromatin remodeling factor, Dom, is required for controlling FSC maintenance.
39
II-Overview of Drosophila Oogenesis
from that of the GSC niche. The GSC niche is composed by the neighbouring somatic cells (TF,
CC and ESC). In contrast, beyond the posterior EC, located adjacently to the FSC, the TF and
CC, located a few cells away, are also integrant part of the FSC niche.
II_4.2.2.1- Wingless (Wg) signalling is essential for FSC maintenance and
proliferation
Wingless (Wg) is essential for FSC, but is dispensable for GSC (Fig.II_4). Wg is involved
in regulation of many different cellular processes during Drosophila development. Wg signal is
transduced by Frizzled receptors, Frizzled (Fz) and Frizzled 2 (Fz2), resulting in the
phosphorilation of the cytoplasmic protein Dishevelled (Dsh), inhibition of Shaggy (Sgg) and
Axin (Axn), stabilization of Arm and activation of target genes expression (reviewed by Peifer
and Polakis, 2000).
In Drosophila ovary, Wg protein is expressed in TF and CC and functions as a long-
range signal to directly act on the FSC controlling its self-renewal. Disruption of Wg signalling
in FSC abolishes SC self-renewal (Song and Xie, 2003). Hyperactive Wg signalling, resulting
from removal of negative regulators, such as Axin and sgg, causes over-proliferation and
improper differentiation of follicle cells, and intriguingly also destabilizes FSC (Song and Xie,
2003; Kirilly et al., 2005).
II_4.2.2.2- Hedgehog (Hh) signalling is essential for FSC maintenance and
proliferation
Hh signalling is essential for self-renewal and proliferation of the FSC, while it plays a
non-essential role in maintaining the GSC by having redundant function with the piwi–
mediated pathway (Fig.II_4; King et al., 2001). Hh, is produced by TF and CC, and also
functions as a long-range signal to control FSC self-renewal and proliferation and for budding
of egg chambers (Forbes et al., 1996; King et al., 2001; Zhang and Kalderon, 2001).
Hh signalling contributes to pattern formation and regulates proliferation in a number of
settings in Drosophila and other animals (Ingham, 1998). The Hh signalling pathway has been
well characterised, and the same pathway is employed in many different tissues (Murone et
al., 1999). Upon binding of Hh to its receptor, Patched (Ptc), this last one derepresses a
second 7-transmembrane domain protein, Smoothened (Smo) and ultimately leading to the
activation of Cubitus interruptus (Ci) (Fig.II_5). Ci is a transcription factor that transmits the
Hh signal from the cytoplasm to the nucleus, where it is primarily responsible for the activation
and repression of Hh target genes (Aza- Blanc and Kornberg, 1999; Ingham, 1998). Ci exists
in at least two forms. In the absence of the Hh signal, Ci is proteolytically cleaved to a 75 kDa
repressor form. In the presence of Hh, this cleavage is prevented, and Ci functions instead as
40
II-Overview of Drosophila Oogenesis
a transcriptional activator. Consistent with this model, effects of Hh signalling are observed
only in the presence of Ci (Methot and Basler, 2001). The regulation of Ci is complex and
involves the function of positive, as well as negative regulators. The negative regulators
required for repression of this pathway include the transmembrane protein Ptc (Hooper and
Scott, 1989; Nakano et al., 1989), protein kinase A (PKA) (Lepage et al., 1995; Li et al., 1995;
Pan and Rubin, 1995), and a kinesin-related protein, Costal2 (COS2; COS – FlyBase; Robbins
et al., 1997; Sisson et al., 1997). Loss of either ptc, cos2 or Pka stimulates intracellular Hh
signalling, even in the absence of Hh.
Figure II_5- The Hedgehog pathway based on what is known in Drosophila (in Wicking et al., 1999).
In the absence of hedgehog (hh), the patched protein (ptc) inhibits signalling by smoothened (smo). Full-length
Cubitus interruptus (Ci) forms a complex with Fused (Fu), Costal 2 (Cos-2) and Supressor of fused [Su(fu)], via which
it associates with microtubules. This association leads to targeting of Ci to the proteasome where it is cleaved to
release the transcriptional repressing form Ci75. Upon binding of hedgehog to ptc, the inhibitory effect on Smo is
suppressed leading to the dissociation of the Fu–Cos-2–Ci complex from microtubules and blocking the cleavage of
Ci155. This results in the release and stabilization of Ci, which translocate to the nucleus, initiates Hedgehog target
gene expression. In vertebrates the role of ci is achieved by complex interactions involving three Gli genes (Gli1, Gli2
and Gli3).
(Hh) hedgehog, (Ptc) patched, (Smo) smoothened, (Fu) fused, [Su(Fu)] suppressor of fused, (cos-2) costal-2, (Ci)
Cubitus interruptus.
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II-Overview of Drosophila Oogenesis
In Drosophila ovary, hyperactivation of hh signalling causes the overproduction of
somatic follicle cells, increases the number of FSC (Forbes et al., 1996a; Forbes et al., 1996b),
mis-position the oocyte within egg chambers and generates ectopic polar cells (Forbes et al.,
1996a; Forbes et al., 1996b Liu and Montell, 1999; zhang & kalderon). The mechanism by
which overexpression of Hh signalling promotes ectopic polar cell fate is not known. Ectopic Hh
signalling is able to transform other follicle cells into ectopic polar cells through the
suppression of eye absent (eya) expression (Bai and Montell, 2002). Disruption of the Hh
signalling cascade results in rapid FSC loss (King et al., 2001; Zhang and Kalderon, 2001).
This gives rise to compound egg chambers that contain multiple germline cysts in a single
follicle epithelium. It as therefore been postulated, that Hh signalling regulates follicle cells
proliferation at an early stage in the germarium and may also regulated oocyte positioning,
perhaps via effects on follicle cell-type determination.
The differentiated somatic follicle cell types have similar characteristics to epithelial
cells in mammals, such as basal-lateral and basal-apical polarities. Interestingly, in mammals,
Wnt and Shh has been implicated in the regulation of epithelial stem cell/precursor, cell
maintenance and proliferation in the intestine and airway (He et al., 2004; Korinek et al.,
1998; Watkins et al., 2003).In addition, in mice and humans, hyperactivation of the shh
pathway, either by mutations or overexpression, causes over-proliferation of skin cells,
resulting in the formation of skin tumors (Johnson et al., 1996; Fan et al., 1997; Oro et al.,
1997). These studies, along with those from Drosophila, suggest that the molecular
mechanisms regulating epithelial stem cells are likely conserved from Drosophila to humans.
Thus, FSC in the Drosophila ovary could represent an effective system to study epithelial stem
cell self-renewal, proliferation, and differentiation (Song and Xie, 2002).
Interestingly, in germarium, where all germline cells were removed, EC enter in
apoptosis and are lost. In this situation, FSC moves to anterior and occup the empty niche
continuing their SC prolifereation due to the presence of Hh, Wg and BMP signal in adjacent TF
and CC (Margolis and Spradling, 1995; Xie and Spradling, 2000; Kai and Spradling, 2003).
Some studies in the last few years have been improving our current understanding and
knowledge about regulation of FSC self-renewal, maintenance and proliferation, although this
is only the beginning and many questions persist. How BMP, Hh and Wg are integrating in FSC
to regulate expression of target genes that are important for FSC self-renewal and
proliferation? Although dom is ubiquitous present in the germarium, it is only required in FSC
for their self-renewal. Can dom helps to regulate the expression of target genes of the BMP,
Hh and Wg signalling pathways (Xi and Xie, 2005)?
The answers to these and other questions will provide novel insight into how SC self-
renewal and differentiation are controlled in general.
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II-Overview of Drosophila Oogenesis
II_4.3- Genes and Pathways controlling the maintenance of ESC and EC
Recently, a new type of somatic stem cell was identified in the Drosophila germarium. A
the escort cells are maintained by 4-6 anteriorly located escort stem cells (ESC) that
extensively contact GSC within the niche. ESC strongly resembles the cyst progenitor cells of
the testis in morphology, location and behaviour (Decotto and Spradling, 2005).
It has been proposed, that these ESC divides synchronously with GSC to generate
differentiated escort cells, which tightly wrap early germline cells and move posteriorly with
the germ cell, until they are lost by apoptosis and replaced by follicle cells in the 2a/2b
boundary(Decotto and Spradling, 2005).
In Drosophila, JAK/STAT signalling initiates when the ligand unpaired (upd; or its close
relatives) binds to the domeless receptor and activates hopscotch (the Janus kinase; JAK). In
turn, the activated JAK kinase phosphorylates the Stat92E transcriptional activator factor that
translocates into the nucleus (reviewed in Rawlings et al., 2004). Mutations in stat cause the
degeneration of the germarium probably due to loss of escort cells, while overexpression of
upd dramatically expands the escort cells population. Therefore, JAK/STAT signalling controls
the maintenance and the proliferation of ESC (Fig.II_4; Decotto and Spradling, 2005). In
germarium, where escort cells are mutants for stat, GSC are also lost indicating that ESC
participates of the GSC niche (Decotto and Spradling, 2005).
There are 40-50 EC in the germarium (Xie and Spradling, 2000; Decotto and Spradling,
2005). Like escort cells, also IGS cells have protuberances that contact germline cysts.
Germline cyst fail to differentiated in mutants where the production of these protuberances are
affected, like in mutation in stem cell tumor (stet), which is a rhomboid-like membrane
protease that activates Egfr ligands in the signalling cell. In stet mutant ovaries, the cellular
processes of IGS cells are severely reduced, and spectrosome-containing single germ cells
accumulate, suggesting that stet is required for cystoblast differentiation. Supporting a role of
EGF signalling in EC, the activated MAP kinase accumulates in wild-type EC, but is reduced in
stet mutant EC (Schultz et al., 2002).
In the future, it will be important to demonstrate whether EGFR signalling is required
for the production or maintenance of the cellular processes of the escort cells, and also clarifies
how escort control germ cell differentiation.
II_5- Drosophila ovary as a model system in stem cells studies.
The extreme conservation of the relation between SC and their niche led the
investigators to confidently use Drosophila as a model system to study SC regulation. In
addition, the molecular mechanisms regulators of the epithelial SC are likely conserved from
Drosophila to humans (Song and Xie, 2002). Thus, Drosophila ovary offers several unique
advantages to study molecular and genetic networks that control SC regulation.
43
II-Overview of Drosophila Oogenesis
First, the Drosophila ovary offers the unique opportunities for simultaneously study
three types of SC and respective niches: GSC, FSC and ESC (Decotto and Spradling, 2005;
Kirilly and Xie, 2007).
Second, Drosophila ovary is a relatively simple and well-characterized structure; in
addition, the linear arrangement between SC and their progeny simplifies qualitative and
quantitative analyses (Lin, 2002; Gilboa and Lehmann, 2004; Xie et al., 2005). All the three
SC types are present in small number (2-3 GSC, 2-3 FSC and 4-6 ESC) and they are located in
a simple tubular structure, also known as the germarium, in which SC and their surrounding
cells are well defined and easily distinguishable from one to another, specially GSC based on
their morphology and size (Lin, 2002; Xie et al., 2005).
Third, powerful genetic and molecular tools are available to manipulate easily and
precise gene function in SC and their niche (Xie et al., 2005).
Fourth, more than 50% of the Drosophila genes mutations are available. In addition,
microarray and proteomic reagents are available due to the finished Drosophila genome, which
dramatically enhance our ability to discover genes that encode intrinsic factors and niche
signals, important for controlling self-renewal, proliferation and differentiation of SC.
Finally, comparative studies on the SC in the Drosophila ovary and in the testis, help to
find general SC regulatory mechanisms, because the Drosophila testis also contains easily
identified SC and niche cells (Wong et al., 2005). Therefore, the Drosophila ovary represents
one of the best systems for studying basic SC biology.
III- Overview of Drosophila Cell Cycle
My PhD work started with the challenge of clarifying why some tsh mutants cause late
larval lethality with neuroblasts arrested in a metaphase-like state. To understand better the
results presented in chapter IV, we will make a brief overview about the phases of the cell
cycle, specially the progression during mitosis and the regulation of their critical steps.
III_1- The Cell Cycle
The cell cycle is an ordered and sequential process by which cells duplicate, grow, and
prepare themselves to divide again (Fig. III_1).
The cell cycle was divided in two steps, based in microscopes studies: the phase M,
and the interphase. Many morphologic changes are observed in the cells and culminate with
the cell division during M phase. In somatic cells the period of division is called mitosis or M-
phase, while in germline cells the cell cycle ends in meiosis (the process that produces
44
II-Overview of Drosophila Cell Cycle
gametes with haploid nuclei). The interphase is the period between two M phase, in which the
cell improves their size and mass. Studies in cellular biology and biochemistry led to the
classification of the interphase in three distinct steps: G1, S phase and G2. The DNA
replication, important to assure the distribution of equal genetic material (DNA) to the two
daughter cells, occurs during S (synthesis) phase. G1 and G2 are gaps where cells prepare to
S phase and M phase, respectively (Fig. III_1).
Figure III_1- Cell cycle phases.
After a cell division (M-phase) a new cell cycle starts at G1 phase. During G1 cell gets prepared to duplicate its DNA
contents in S phase (synthesis). In G2, cells prepare the machinery needed to divide in the next M-phase. Some cells
(like fibroblasts) leave the cell cycle after mitosis and just before S-phase, being able to remain in this G0 stage for
long periods. Key control points (checkpoints signalised by the red star), ensure the correct order of these events.
The cell cycle has been the subject of intense research. Rudolf Virchow set forth the
first cell theory, in which he proposed that the cell was the basic unit of all living organisms.
We now know that this theory is true; cells make up all living things, whether they are plants,
animals, or microorganisms. In the late 1970s and 1980s, the molecular biology together with
the already achieved development in biochemistry and genetics allowed the cell biologists to
better understand the working of a cell at the molecular level.
III_2- M-phase
M-phase can be divided in Mitosis and Cytokinesis (Nigg, 2001). The word mitosis came
from the Greek mitos that means thread. It was originally used by Walter Flemming in the
early 1880s and it was based on the shape of the chromosomes during cell division (Flemming,
1965).
45
III-Overview of Drosophila Cell Cycle
The main purpose of mitosis is to segregate sister chromatids into two daughters cells,
ensuring that each daughter cell will receive a complete set of chromosomes. Other organelles,
such as the centrosomes are also segregate at this time. Cytokinesis is the process that
promotes the physical separation of the two daughter cells and occurs just after last mitotic
phase, the telophase.
Mitosis has been divided in five distinct phases, in accord with the changes observed in
the structure and behaviour of the spindle and the chromosomes (Fig. III_2):
Prophase: at the beginning of mitosis, cells start to condense interphase chromatin
into well defined mitotic chromosomes. At the same time, the already duplicated centrosomes
move apart, defining the poles of the future mitotic spindle and the nuclear envelope breaks
down (NEBD). Centrosomes nucleate highly dynamic microtubules (MT)
Prometaphase: the highly dynamics microtubules are captured by kinetochores
(proteinaceous structures that are associated with the centromere DNA of the mitotic
chromosomes). The attachment of microtubules with kinetochores will become stable if the
chromosomes are bipolar attached (attached to both poles) otherwise, the monopolar
attachment (the attachment to only one pole) is unstable.
Metaphase: Bipolar attached chromosomes will congress to the equatorial region of
the cell to form the metaphase plate. Chromosomes will oscilate in the metaphase plate, a
dance that finishes when all are under tension.
Anaphase: Loss of sister chromatids cohesion triggers the anaphase onset and the
sister chromatids are pulled apart to opposite poles (anaphase A). The poles also move apart
leading to the elongation of the spindle and cell (anaphase B).
Telophase: After reaching the poles, chromatin decondenses and nuclear envelop
reforms. The formation of an actomyosin contractile ring in the equatorial region of the cells
leads to the invagination of the membrane, originating the cleavage furrow that separates the
two daughters cells.
Cytokinesis begins with the formation of a MT-based midbody near the original spindle
equator.
M-phase has to be tightly regulate to ensure that one event just take place after the
end of the previous one in a concerned fashion. Like other cellular process, M-phase is mainly
regulated by two distinct post-translational mechanisms: protein phosphorylation/
dephosphorylation and proteolysis. In fact, mitotic kinases are the major regulators of cell
division, where cyclin dependent Kinase 1 (Cdk1), a member of cyclin dependent kinase
family, is the most prominent.
46
III-Overview of Drosophila Cell Cycle
Figure III_2- Diagram of mitosis in animal cells (in <eapbiofield.wikispaces.com>).
During interphase the DNA is doubled in preparation for cell division. According with structure and behaviour of the
chromosomes and spindle, Mitosis has been divided in five consecutive phases: Profase, Promethaphase, Methapase,
Anaphase (with two sub-phase Aand B) and Telophase. In Prophase, the nuclear envelope breaks down and a spindle
forms between two centrioles. In prometaphase the chromosomes attach to the spindle fibers and then will undergo
movements until all chromosomes to be aligned in the equator of the cell (the metaphase plate). In anaphase A the
sister chromatids start to be separated and then, the poles also move apart leading to the elongation of the cell
(anaphase B). In telophase, the chromosomes reached the poles and the chromatin decondense and nuclear envelop
reforms. After the end of the mitosis, the cytoplasmic content of the mother cells is separated by cytokinesis and the
two daughters cells are formed.
47
III-Overview of Drosophila Cell Cycle
III_2.1- Mitosis Promoting Factor (MPF) and Mitosis entry
Progression through mitosis is governed by activation and inactivation of M-phase
promoting factor (MPF), a complex comprising the kinase CdK1 and a cyclin B regulatory
subunit (Fig. III_3).
Figure III_3-The regulation of the MPF activity by phosporilation (adaptated from Ohi and Gould,
1999).
Activatction of Cdc2-Cyclin B by Cdc25-mediated Thr14/ Tyr15 desphosphorylation of Cdc2 is promoted by activation
of Cdc25 by Polo. The Plkk is involved in the activation of Polo. The cdc2-Cyclin B complex is inactivated by the
phosphorylation of Thr14 and Tyr15 by the kinases Myt1 and Wee1, respectively.
The first evidence for the existence of a factor capable of inducing mitosis was
described in the 1970s. Some embryologists observed that the transfer of the cytoplasm from
a mature oocyte to a recipient immature oocyte could induce oocyte maturation even without
hormonal stimulation or protein synthesis (Mausiand Market, 1971; Smith and Ecker, 1971;
Kishimoto and Kanatani, 1976). This unidentified cytoplasmic component, responsible for
meiotic maturation, was called Maturation Promoting Factor (MPF). Around 1980, MPF was
renamed M-Promoting Factor, after their activity being also identified in mitotic cells (see
Khishimoto et al., 1988 for review).
MPF purifications revealed that it consisted of two subunits, one was identified as a
protein kinase with homology to Cdc2 (Labbe et al., 1988). The other subunit was found to be
a mitotic cyclin, the cyclin B protein (Lohka et al., 1988). This protein is called cyclin because
48
III-Overview of Drosophila Cell Cycle
embryos synthesized and destroyed it in a cyclic and synchronized way with cell cycle division
(Evans et al., 1983). At the ends of 1980s it was showed that the MPF is a universal regulator
of the M-phase onset (Nurse, 1990).
III_2.1.1- CdK1 family
The Cdc2/Cdc28 proteins are members of the conserved family of ser/thr protein
kinases; know as Cyclin-dependent kinases (Cdk). These kinases are inactive as monomers
and need to bind to a Cyclin partner in order to become active.
Cdk1 is involved in many stages of the cell cycle, including mitosis (for review see
Porter and Donoghue, 2003). Cdk1 can bind cyclin A or cyclin B, depending on the stage of the
cell cycle. Cdk1 binding to cyclin A controls the entry and progression through the G2 phase,
whereas Cdk1-cyclin B1 complex regulates the G2-M transition. Once bounded and activated in
the nucleus, Cdk1/ cyclin B1 phosphorylates multiple targets that initiate mitotic entrance,
regulates its progression, and controls mitotic exit, at which point cyclin B1 degradation is
necessary (Schmit and Ahmad, 2007).
Cdk1 (Cdc2 in Drosophila) protein level is stable throughout the cell cycle; therefore,
Cdk1 activity is controlled directly by phosphorylation and indirectly through regulation of its
cyclin binding partners. Inactivation of Cdk1 is maintained by phosphorylation of Thr14 and
Tyr15 by the kinases Myt1 and Wee1, respectively (Porter and Donoghue, 2003). These
residues reside in the ATP-binding site of Cdk1; thus, phosphorylation inhibits ATP binding
(Porter and Donoghue, 2003). Myt1 and Wee1 are active during the S and G2 phases of the
cell cycle (Porter and Donoghue, 2003). At the mitotic transition, Myt1 is phosphorylated by
Plk1 (only one member in Drosophila: Polo) and Cdk1 in vitro, resulting in lowered kinase
activity, indicating a possible feedback loop (Porter and Donoghue, 2003). Wee1 is
phosphorylated by the kinases Chk1 and Cds1, resulting in kinase inactivation, the 14-3-3
binding decreased and protein stability (Porter and Donoghue, 2003).
Activation of Cdk1 occurs through the combination of three required steps. First,
phosphorylation of Thr161 opens up the catalytic region of Cdk1. Phosphorylation of Thr161 is
done by Cdk-activating kinase (CAK), which occurs late in G2 and is not removed until the
cyclin B1 degradation in late mitosis (Fesquet et al., 1993). Second, nuclear localization of
Cdk1/cyclin B1 is promoted by Plk1 phosphorylation of Ser147 on Cdk1 (Porter and Donoghue,
2003). The final activation step of Cdk1 involves dephosphorylation of Thr14 and Tyr15 by
members of the Cdc25 phosphatase family, Cdc25A, Cdc25B, and Cdc25C (only one member
in Drosophila: String; Porter and Donoghue, 2003).
Cdc25A seems to be involved in the G1-S phase transition, whereas Cdc25B and
Cdc25C are involved in dephosphorylation and activation of Cdk1 in G2, with Cdc25C being the
primary phosphatase at the G2-M transition. Activation of Cdc25C is achieved by
49
III-Overview of Drosophila Cell Cycle
phosphorylation of Ser198 by Plk1 (Roshak et al., 2000). Ser198 lies within the nuclear export
signal of Cdc25C, and phosphorylation of this residue promotes localization of Cdc25C to the
nucleus. Once Cdc25C is activated and localised to the nucleus, it becomes
hyperphosphorylated by Cdk1/cyclin B1, creating a positive feedback loop, increasing Cdc25C
and Cdk1/ cyclin B1 activities. Inactivation of Cdc25C is achieved through phosphorylation of
Ser216, creating a binding site for 14-3-3 protein, sequestering Cdc25C in the cytoplasm, and
preventing Cdc25C and Cdk1 interaction. Phosphorylation of Ser216 has been attributed to
Chk1, Chk2, C-TAK1, and Plk3 (Porter and Donoghue, 2003; Ouyang et al., 1999).
Deregulation or mutations of Cdk1 itself have not been reported in any cancers.
However, inhibition of Cdk1 has been showed to induce cell cycle arrest and apoptosis. Cdk
inhibitors may impart their effects through either inhibiting the catalytic subunit, preventing
downstream phosphorylation, or through inhibiting the ability of Cdk1 to control transcription
through phosphorylation of RNA polymerase II. Many of the Cdks, including Cdk1,
phosphorylate the COOH-terminal domain of RNA polymerase II, inhibiting mRNA production
during mitosis (Oelgeschlager, 2002). Tumor cells seem to be particularly sensitive to RNA
polymerase II inhibition, indicating that the proapoptotic response to Cdk inhibition may be
due to both blocking its kinase activity on traditional targets and altering its affects on
transcription (Koumenis and Giaccia, 1997).
The basic biochemistry of the regulation of the Cdk-Cyclin B activation is conserved
from yeasts to humans.
In all known eukaryotes, the successive activation and inactivation of different Cdk-
Cyclin complex control the correct cell cycle progression (Pines, 1999). In lower eukaryotes,
only one or two cdc2/ cdc28-like genes products are found, while in mammals nine Cdks
(cdk1-9) have been already identified (Johnson and Walker, 1999). In budding yeast, the main
Cdk involved in cell cycle control is Cdc28, which is activated by nine different cell cycle
specific Cyclins (Levine et al.,1999). Three CLN or G1 Cyclins are involved in cell cycle events
that occur during G1 or at the start point (the point where a cell is committed to enter S-
phase). Six other cyclins know as Clb are required for initiation of DNA replication during S-
phase and subsequent mitosis (Nasmyth, 1996). In mammals, seventeen cyclins have been
identified: A, B1, B2, B3, C, D1, D2, D3, E, F, G1, G2, H, I, K, T1 and T2. All cyclin contain a
common region of homology, known as cyclin box, which is the domain used to bind Cdks
(Hunt, 1991).
III_2.1.2- Cyclin A
Cdk-Cyclin B is the major activator/regulator of mitosis, although another Cdk complex
is also present, at least during the firs stage of the mitosis, the Cdk-Cyclin A complex. Cdk1-
Cyclin A is formed in late G1 and is required for S-phase and G2/M transition. Little information
50
III-Overview of Drosophila Cell Cycle
is knowed about the importance of CdK1-Cyclin A complex in initiating S-phase. It has been
proposed that this complex is required for maintaining DNA synthesis once S-phase has been
initiated by another complex, the Cdk1-Cyclin E (Resnitzky et al., 1995). If the role of Cdk1-
Cyclin A is not well understood in S-phase, the activity of this complex during G2 and M-phase
is worse known. Drosophila cyclin A mutants can develop until cycle 14 of embryogenesis due
to the presence of maternal mRNA, however, after the exhaustation of maternal contribution,
cells no longer enter in mitosis, which suggests that Cyclin A is required for the initiation of the
M-phase (Lehner and O’Farrel, 1989). During early embryonic Drosophila cycles, Cyclin A is
mainly nuclear and the reduction of Cyclin A amount results in a increase in the normal
duration of the nuclear cycles (Stiffler et al., 1999). Further, the injection of anti-Cyclin A
antibodies in G2 tissues cultured cells, stops the cell cycle progression and cells are arrested
prior to mitosis onset, indicating that Cyclin A is required to initiate the mitosis (Pagano et al.,
1992). In addition, microinjections of exogenous Cdk1-Cyclin A in G2 vertebrate’s cells induce
a premature entry in mitosis and eliminate G2 phase. However, if the injections are made
during S-phase no effect on the progression to the next mitosis is observed (Furuno et al.,
1999). These authors suggested that Cdk1-Cyclin A complexes are required for cells to
complete prophase, since inhibition of this complex in mid prophase causes cell return to G2
phase. Cyclin A is degraded ahead of Cyclin B and it is degraded by proteolysis at the end of
pro-metaphase suggesting, that Cyclin A destruction is required for the subsequent alignment
of the chromosomes at the metaphase plate and for anaphase (Elzen and Pines, 2001; Geley
et al., 2001).
III_2.1.3- Cyclin B
Cyclin B is the mitotic cyclin with the clearest cell cycle role. Together with Cdk1, form a
primary mitotic Cdk which phosphorylates a large number of mitotic substrates (Nigg, 1993).
Among these substrates were identified nuclear lamins, cytoskeleton proteins, such as actin
biding proteins, and microtubules-associated proteins (MAPs). Therefore, it is easy to
understand that this complex has to be correctly activated in order to prevent drastic
consequences for cell.
In vertebrates cells, the cellular localisation of the Cdc2-Cyclin B complexes is
determined by the Cyclin B variant in which Cdc2 is bound. Cdc2-Cyclin B1 complexes co-
localise with cytoplasmic microtubules during interface and abruptly translocate to the nucleus
upon entry into mitosis (Heald et al., 1993; Jackman et al., 1995; Ookata et al., 1992; Pines
and Hunter, 1991). Cdc2-Cyclin B2 complexes co-localise with Golgi apparatus (Jackman et al.,
1995), and Cyclin B3, which share properties with both A and B-types cyclins, is constitutively
nuclear during cell cycle. In Drosophila, the localisation patterns of Cyclin B and B3 are similar
51
III-Overview of Drosophila Cell Cycle
to those of vertebrates Cyclin B1 and B3, respectively (Jacobs et al., 1998; Lehner and
O’Farrell, 1990).
The contribution of each class of MPF to the mitotic progression is not understood at
this point. Analyses of mices that are nullizygous for either Cyclin B1 or B2 have showed that
Cyclin B1, but not Cyclin B2, is essential for viability and fertility (Brandeis et al., 1998). This
indicates that Cdc2-Cyclin B2 is dispensable for mitotic progression, or alternatively, Cdc2-
Cyclin B1 is capable of compensating the Cdc2-Cyclin B2 loss. Cdc2-Cyclin B2 probably is
required for mitotic fragmentation of the Golgi apparatus (Lowe et al., 1998). It will be
interesting to determine the consequence of deleting Cyclin B3 in mice and investigate the
potential functional redundancies with Cyclin B1 and B2. In Drosophila, genetic studies have
demonstrated that, surprisingly, neither Cyclin B nor B3 are essential for cell division, however
removal of both Cyclins results in delayed entry into mitosis and formation of aberrant and
functionally compromised spindle (Jacobs et al., 1998). The Drosophila cyclin B3 is required for
female fertility, but not for male fertility (Jacobs et al., 1998).
Cdc2-Cyclin B1 activity is regulated by Wee1 and Cdc25C: Wee1 is nuclear until the
onset of mitosis while, Cdc25C is imported into the nucleus at G2/M transition (Heald et al.,
1993). Perhaps, Myt1 and Cdc25B function to coordinate cytoplasmic mitotic events, which are
mediated through Cdc2-Cyclin B1 (and possibly Cdc2-Cyclin B3). The division of labour
between the various MPF complexes may not be this straightforward; however, a cytoplasmic
accumulation of Cdc25B has been implicated in triggering centrosomal microtubule nucleation
in HeLa cells (Gabrielli et al., 1996).
Investigations about Cyclin B1 in Xenopus and HeLa cells have demonstrated a more
dynamic localisation; Cyclin B1 shuttles continuously between the cytoplasm and nucleus
during interphase (Hagting et al., 1998; Toyoshima et al., 1998; Yang et al., 1998). Import of
Cdc2-cyclin B1 into the nucleus occurs through binding of cyclin B1 to importin-β; Cdc2 is
dispensable for cyclin B1 nuclear import (Moore et al., 1999). Phosphorylation of four serine
residues located within the cyclin B, has been implicated in the nuclear retention of Cdc2-cyclin
B1 during the onset of mitosis (Li et al., 1997). Remains to be identified the kinase which
phosphorylated cyclin B1 and regulates its nuclear retention. Prior to the G2/M transition,
cdc2-cyclin B1 is maintained in the cytoplasm by the activity of the nuclear export factor CRMI
(also know as a exportin 1; Pines and Hunter, 1994).
In S. cerevisae, six B-type cyclins were identified (clb1-6; for review see Nasmyth,
1993). Clb1 and Clb2 are a closely related pair of B-type cyclins and are expressed in late S-
phase (Surana et al., 1991). Clb3 and Clb4 are expressed in early S-phase, and as Clb1 and
Clb2, are also expressed until late mitosis (Dahmann and Futcher, 1995). Clb5 and Clb6 are
the less similar to metazoan B-type cyclins (Mendenhall and Hodge, 1998).
The activation of the protein kinase by cyclin partner normally induces the entry into a
new cell cycle stage. Very often, the exit of a particular stage also requires the inactivation of
52
III-Overview of Drosophila Cell Cycle
the Cdk-Cyclin complexes. In mitosis, the Cdk-cyclin B complexes are normally inactivated by
proteolysis of the cyclin B. This destruction is thought to control progression during mitosis.
Immunofluorescence studies on tissue culture cells have showed that cyclin B
degradation starts during anaphase (Girard et al., 1995). Live analysis of cell proteins (Clute
and Pines, 1999) showed that in HeLa cells, cycB 1-GFP fusion proteins start to be degraded
30min before the metaphase/ anaphase transition, as soon as the last chromosomes is
attached to the spindle MTs. Cyclin B localisation was detected at the spindle poles during
prophase and prometaphase, but not in metaphase. Cyclin B was also seen associated with
chromosomes during prometaphase but again not in metaphase. These authors proposed a
pattern for Cyclin B degradation: Cyclin B starts to be degraded first from the spindle poles
and then from the chromosomes, just after the attachment of the last chromosome on the
metaphase plate.
A Cyclin B-GFP fusion protein was also studied in living Drosophila embryos (Huang and
Raf 1999). These authors showed that Cyclin B in interphase is placed at centrosomes (the
organelles that organise microtubules) and during mitosis, it is located at the mitotic spindle in
prometaphase and at the microtubules that overlap in the spindle mid-zone during metaphase.
A spatial regulation of Cyclin B destruction was also reported. Cyclin B disappears at the end of
metaphase, in a wave that started at the spindle poles and spreads to the spindle equator.
There is almost no Cyclin B-GFP at the moment that chromosomes enter in anaphase.
These two independent studies suggest that a substantial fraction of Cyclin B is being
degraded while the cell is still in metaphase, which is in contradiction with previous
immunostained studies that, reported an abrupt degradation of the protein at the
metaphase/anaphase transition. Moreover, the disappearance of Cyclin B seems to be
regulated spatially in both Drosophila and vertebrates cells.
In Addition, in Drosophila embryos it was showed that centrosomes (the organelles that
organise microtubules) have a role in the destruction of cyclin B (Wakefield et al., 2000). Using
a mutation called cfo (centrosome fall off), which produces embryos where the centrosomes
detach from formed spindle (centrosomeless spindles); Cyc B degradation was only detected at
the centrosome level and not on the spindle. These observations lead the authors to suggest
that cyclin B degradation starts at the poles and a connection between centrosomes and
spindle is required for the normal destruction of the cyclin B associated with the spindle.
III_3- The bipolar spindle and the kinetocore-microtubule attachment until
the formation of the metaphase plate
After nuclear envelope breakdown (NEBD), at prometaphase, each sister chromatid is
exposed and thus capable of capturing the spindle microtubules.
53
III-Overview of Drosophila Cell Cycle
Microtubules are metastable polymers of α- and β-tubulin that switch between phases
of growth and shrinkage, a phenomenon known as ‘dynamic instability’ (Mitchison and
Kirschner, 1984). Microtubule plus ends attach to the chromosome through a macromolecular
protein complex known as the kinetochore (see section III_3.1 of this chapter; reviewed in
Maiato et al., 2004). The minus ends of spindle microtubules are crosslinked to each other and
to interdigitated microtubules which is stretched from pole to pole, creating the spindle poles
(reviewed in Zimmerman and Doxsey, 2000). Anchorage of kinetochore-bound microtubules at
spindle poles, allows the force applied to a chromosome to be translated into directional
movement (Gordon et al., 2001).
Spindle poles and centrosomes anchor microtubules in distinct manners. Spindle poles
provide anchorage through crosslinking of microtubules by proteins such as NuMA, LGN, and
Dynein (Gaglio et al., 1996; Merdes et al., 1996; Heald et al., 1997; Du et al., 2001). In the
absence of centrosomes, spindle poles are sufficient to anchor microtubules allowing for high-
fidelity chromosome segregation.
Centrosomes nucleate and anchor microtubules.The γ-tubulin ring complex is directly
responsible for the centrosomes nucleating activity (Zheng et al., 1995) and also caps the
minus ends of microtubules (Wiese and Zheng, 2000; Moritz et al., 2000). Appendages on the
mother centriole within the centrosome, and a growing list of proteins, have been implicated in
the anchorage of microtubule minus ends at the centrosome, although the mechanism of this
activity is still unclear (reviewed in Bornens, 2002).
The kinetochore that has successfully captured a MT laterally is usually subjected to
rapid poleward motion. It is believed that, the poleward movement enables the kinetochore to
run into more MTs emanated from the approaching pole, to facilitate the stable, or “end-on”,
MT attachment (Rieder et al., 1998; Cleveland et al., 2003). Chromosomes with such
monooriented kinetochores, then experience oscillations toward poleward and away from their
attaching pole until their unattached kinetochores capture microtubules from the opposite pole.
For monooriented kinetochores, their poleward movement is attributed to the poleward
pulling force at the attached kinetochore, whereas their away pole movement is likely a
composite effect of both the pushing force at the kinetochore and the polar ejection force
acting on chromosome arms through non attached kinetochore- microtubules (Cleveland et al.,
2003; Kapoor and Compton, 2002). These antagonizing forces appear to act alternatively to
enable the oscillation, during which kinetochore- microtubules (k-fibers) remain attached but,
assemble and disassemble accordingly (Cleveland et al., 2003; Kapoor and Compton, 2002).
Full chromosome alignment is a prerequisite for equal segregation of sister chromatids into
daughter cells. The spatial location of each chromosome must differ from cell to cell after
nuclear envelope breakdown. Therefore, it is amazing that usually metaphase is accomplished
within 20 min (Alberts et al., 1994). How cells achieve chromosome alignment so efficiently is
still not fully understood.
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III-Overview of Drosophila Cell Cycle
The bi-oriented chromosomes move towards the metaphase plate and finally align
there, waiting for inactivation of the spindle checkpoint (for more detail see section III_5.2 of
this chapter) to allow anaphase onset mediated by the Anaphase promoter complex or
clyclosome (APC/C for more detail see section III_4 of this chapter; Cleveland et al., 2003;
Kapoor and Compton, 2002; Rieder et al., 1998).
III_3.1- The Kinetochore
Kinetochores of animal cells can be subdivided into two regions. The inner kinetochore
normally forms on highly repetitive DNA sequences and, assembles into a specialised form of
chromatin that persists throughout the cell cycle. The outer kinetochore is a proteinaceous
structure with many dynamic components, that assembles and functions only during mitosis.
Kinetochore functions include: (1) attachment of the chromosomes to the dynamic plus-ends
of spindle microtubules, (2) mediation of mitotic chromosomes movements, and (3) the
control of the metaphase/anaphase transition by inhibiting sisters chromatids separation until
completion of a proper bipolar attachment.
The kinetochore is the physical location where the checkpoint signal is produced (detail
in section III_5.2 of this chapter). They also contain motor proteins that are involved in their
attachment and mobility.
The histone variant CENP-A and additional constitutive proteins mark the inner plate of
mammalian kinetochores, which is located at the periphery of centromeric chromatin
(Cleveland et al., 2003; DeLuca and Salmon, 2004; Meraldi et al., 2006). Before mitosis, other
evolutionarily conserved proteins, including Spc105 (KNL-1 in Caenorhabditis elegans), the
Mtw1 (MIS12 in humans), Ndc80 (HEC1 in humans) and minichromosome maintenance
protein-21 (MCM21) complexes, are recruited to the kinetochore outer plate to form
attachment sites for bundles of spindle microtubules, which are known as the kinetochore
microtubules (Cheeseman et al., 2006; Cleveland et al., 2003; DeLuca and Salmon, 2004;
DeLuca et al., 2006; Meraldi et al., 2006). Several other proteins, including the SAC and motor
proteins, bind within a region of the kinetochore that is known as the fibrous corona, and is
found at the periphery of the outer plate. The temporal order and specific requirements for the
recruitment of kinetochore and SAC proteins are complex (for review see Chan et al., 2005;
Liu et al., 2006; Johnson et al., 2004; Maiato et al., 2004; Taylor et al., 2004; Vigneron et al
2004; Wojcik et al., 2001).
The attachment of microtubules to multiple points within the spindle is a critical, yet
poorly understood, process. Two types of kinetochore-microtubule attachments have been
described: one dependent of Cenp-E and Dynein, and the other involving the Ndc80 complex.
The microtubule motors Cenp-E and Dynein initially form lateral attachments with
microtubule polymers and use their motor activities to move chromosomes (Wood et al., 1997;
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III-Overview of Drosophila Cell Cycle
Hyman and Mitchison, 1991). These lateral or ‘‘side-on’’ attachments mature into ‘‘end-on’’
attachments consisting of bundles of spindle microtubules, termed kinetochore fibers (K-
fibers), embedded in the kinetochore’s outer plate. Mature K-fibers remain dynamic,
continuously adding tubulin subunits at their plus ends, but are resistant to depolymerization
treatments such as low doses of nocodazole and cold temperatures (reviewed in Maiato et al.,
2004).
Dynein is widely involved in cellular functions requiring MT-based motility, for instances,
mitosis, cell migration, and cargo transport (Alberts et al., 1994; Gupta et al., 2002; Karki and
Holzbaur, 1999). A pool of dynein is located to the kinetochore in M phase through dynactin
(Echeverri et al., 1996; King et al., 2000). The multiprotein complex, dynactin, increases the
processivity of dynein and anchors dynein to certain cargos or target sites. Dynein is recruited
to kinetochores by dynactin that binds the ZW10/Rod complex (Karess, 2005; Starr et
al.,1998). ZW10 may thus serve as an ideal target for regional elimination of dynein/dynactin.
Kinetochore dynein is generally believed to play a role in congression by driving the poleward
kinetochore movement (Cleveland et al., 2003; Maiato et al., 2004; Rieder et al., 1998).
The hetero-tetrameric Ndc80 complex residing in the kinetochore outer plate is required
for mature microtubule attachment and is a second type of kinetochore-microtubules
attachment (He et al., 2001; Wigge and Kilmartin, 2001; Deluca et al., 2002; McCleland et al.,
2003, 2004). In higher eukaryotes, the Mis12 and Zwint complexes also localise to the outer
plate and are also involved in kinetochore-microtubule attachment (Cheeseman et al., 2004;
Emanuele et al., 2005; Kline et al., 2006). Outer kinetochore proteins are thought to interact
with microtubule-associated proteins (MAPs) to facilitate mature microtubule binding.
III_4- The anaphase promoting complex/ Cyclosome (APC/C)
The anaphase promoting complex (APC/C) was first described as an ubiquitin ligase,
essential to the proteolytic degradation of the mitotic cyclins (King et al., 1995). Ubiquitin-
mediated proteolysis ensures the specific and rapid protein degradation in such a way that cell
cycle events become irreversible.
Ubiquitination of proteins is mediated by three types of enzymatic activity: an ubiquitin-
activating enzyme (E1), an ubiquitin-conjugated enzyme (E2) and an ubiquitin-ligase enzyme
(E3). The attachment of ubiquitin molecules to a target proteins are catalysed by the Ubiquitn
ligase. This will signal the target proteins for degradation by the 26S proteosome (Hershko,
1999). The 26S proteosome has a highly complex structure, it is composed by a 20S catalytic
core and a 19S regulatory cap and, it is present in the cytoplasm and nuclei of higher
eukaryotes (Wilkinson, 1999). The APC/C is an E3 ligase composed for eight to thirteen sub-
unities highly conserved (Peter, 1999). The APC/C is required for the degradation of mitotic
proteins such as mitotic cyclins A and B and securins (King et al., 1996; Morgan, 1999).
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III-Overview of Drosophila Cell Cycle
III_4.1- The APC/C activators and substrate recognition
The specificity of the APC/C substrate is in part regulated by the activator associated to
the APC in a cell cycle dependent manner. APC/C bind to one of the two highly conserved WD
40 repeat proteins: Cdc20 (Fizzy/Slp1/p55Cdc) and cdh1 (hct1/Fizzy-related/Srw1/ste9). Cdc20
activates APC/C during early-mid mitosis (including mitotic cyclins and securins degradation)
and Cdh1 directs APC/C activity at final steps of mitosis and early G1 (Fang. et al., 1999).
In Cyclin B and securins, it was identified a sequence of nine amino acids (RXXLXXXXN)
called destruction box (D-box) which are essential for APC/C-mediated degradation (Glotzer et
al., 1991; King et al., 1996). Some models have been proposed to explain the contribution of
the Cdc20 to the D-box dependent recognition by the APC/C. A second motif, known as KEN
box, was identified in Cdc20, which is a target of APC/Ccdh1 that does not possess a D-box
motif (Pfleger and Kirschner, 2000). In Xenopus extracts, it was shown that APC/CCdc20 has a
strong affinity to D-boxes substrate, while APC/CCdh1 binds preferentially to a sequence of three
residues of amino acids known as KEN-boxes substrates (Pfleger et al., 2001a). PlK1 and ARK-
2 (kinase member of Aurora kinase family) are also APC/CCdh1 substrates during anaphase and
G1. Nevertheless, the efficient degradation of some proteins requires both motifs (Burton and
Solomon 2001).
III_4.2- APC/C regulation
The anaphase-promoting complex/ cyclosome (APC/C) is under complex control via
phosphorylation (Rudner and Murray, 2000) and by binding one of two WD 40 repeat co-
factors: Cdc20 (Fizzy/Slp1/p55Cdc) and Cdh1 (hct1/Fizzy-related/Srw1/ste9), which functions
as rate-limiting and cell cycle stage-specific activators of the APC/C (Morgan, 1999).
During the entry into mitosis, APC/C components are phosphorylated by Plk1 (Plk in
Drosophila). This phosphorylation acivates the APC/C complex and improves their affinity to
the Cdc20. At the same time, the phosphorylation of the Cdh1 components reduces its affinity
to the APC/C. At this moment, the APC/CCdc20 is able to signal to destruction proteins with D-
box motif, like Cyclin B and Securins (Pds1 in S. cerevisae and Cut2p in S. pombe). Cdc20 is
negatively regulated by the binding to the checkpoint proteins Mad2 and BubR1 (for details see
section III_5.2 of this chapter).
During late anaphase, the Cdc14 phosphatase removes the inhibitory phosphate from
the co-factor Cdh1. The desphosphorylation of Cdh1 promotes the replacement of Cdc20 by
Cdh1, and now the APC/CCdh1 targets to destruction Ken-box motif substrates, like Cdc20 (not
binding to the APC/C), PlK1 and AIRK-2 (kinase member of Aurora kinase family). The
APC/CCdh1 complex is maintained until G1.
In Drosophila, both the Cdc20 and the Cdh1 homolog (Fizzy and Fizzy related,
respectively), promote the degradation of cyclin B in a temporal and spatial way during
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III-Overview of Drosophila Cell Cycle
mitosis. Early in mitosis, Fzy/Cdc20 (concentrated at kinetochores and centrosomes) initiates
the degradation of the spindle-associated cyclin B by the centrosomes and spreads to the
spindle equator. Slightly later in mitosis, Fzr/Cdh1 (concentrated at centrosomes throughout
the cell cycle) degrades the remaining cytoplasmic cyclin B (Huang and Raff, 1999, 2002; Raff
et al., 2002). In syncytial embryos, only Fzy/Cdc20 is present, and only the spindle-associated
cyclin B is degraded at the end of mitosis.
Cyclin A is also targeted for destruction by Fzy (Cdc20)–APC/C complexes, but it is not
concentrated on spindles and it seems unlike that Fzy/Cdc20 also catalyses the destruction of
cyclin A only on the spindle (Pines and Hunter, 1991; Dawson et al., 1995). Therefore, it was
proposed that, there must exist separate pools of Fzy/ Cdc20 responsible for the degradation
of cyclins A (in cytoplasm) and B (on the spindle). This hypothesis has important implications
in the target control by the spindle checkpoint. Only the pool of Fzy/Cdc20 that passes through
the kinetochore is inhibited from activating the APC/C by the spindle checkpoint system, and
only this pool of Fzy/Cdc20 is competent to catalyse the destruction of cyclin B. Thus, the
spindle checkpoint in Drosophila inhibits only the cyclin B degradation, whereas the destruction
of cyclin A is not (Whitfield et al., 1990; denElzen and Pines, 2001; Geley et al., 2001).
III_5- The regulation of the cell cycle: the checkpoints
Two major events, S-phase and mitosis are required for all cell cycles (except
endoreplication, where cells duplicate their DNA without mitosis); they ensure that both
daughter cells receive a full complement of chromosomes. For example, cells cannot divide
their DNA content in mitosis before proper duplication in S-phase. If these events are not
properly completed in the correct sequence, then the newly divided cells will not receive the
full set of genes required for all cellular activities; daughter cells then either die or sustain
genetic damage. During the cell cycle, control step are necessary to bring an orderly
progression through S-phase and mitosis and to couple these two processes with cell growth.
The concept of checkpoint arose from the observation that different mutants for cdc (cell
division cycle) arrest in different phases of the cell cycle, preventing the subsequential events
to occur (Weinert and Hartwell, 1988).
Nowadays, checkpoints are regulatory pathways at critical transition points, where
progression to the next cell-cycle step can be delayed or arrested by negative signals until the
correct conditions arise.
Two independent well-established checkpoints ensure the maintenance of the genetic
stability during the cell cycle: the “DNA damage checkpoint” that arrest cell in G2/M transition
in response to DNA damage or DNA replication errors, and “Spindle assembly checkpoint” that
prevents anaphase onset until all chromosomes are bipolarly attached. The components of
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III-Overview of Drosophila Cell Cycle
these checkpoint pathways are conserved in all eukaryotes, enhancing the regulatory
machinery of the cell cycle.
III_5.1- DNA damage checkpoint
In response to DNA damage, such as that caused by ionizing radiation, the most
prominent arrest is in the G2 phase (Fig. III_4; Iliakis et al., 2003).However, in cells that have
a functional p53 pathway, both a G1 and a G2 arrest can take place in response to DNA
damage (Crosby e al., 2007; Wahl and Carr, 2001). It has been established that the G2
checkpoint initiation is primarily regulated through the control of Cdk1 (Cdc2) activity, which is
regulated at multiple levels (Fig. III_4), including periodic association with the B-type Cyclins,
reversible phosphorylation, and intracellular compartmentalisation (Atherton et al., 1993).
Cyclin B1, the prototypical cyclin B (B2 and B3 are less abundant), reaches maximum levels in
G2, when it enters into the nucleus to form a complex with Cdk1 in a phosphorylation-
dependent manner (Hagting et al., 1998). The complex is activated by phosphorylation of
Cdk1 on threonine 161 (T161) by Cdk-activating kinase (CAK), but is kept inactive by its
phosphorylation on threonine 14 (T14) and tyrosine 15 (Y15) by the Myt1 and Wee1 kinases,
respectively (explained in section III_2.1.1 of this chapter; Parker and Piwnica-Worms, 1992).
Following DNA damage, Chk1 and Chk2 kinases are phosphorylated and thus activated
by ATM (ataxia telangiectasia-mutated) and ATR (A-T- and rad3-related) kinases, related
members of the phosphatidylinositol 3-kinase (PI3K) family of proteins (Kastan and Bartek,
2004; Ray et al., 2007). The activacted Chk1 and Chk2 kinases phosphorylate the Cdc25C
protein phosphatase at serine 216 (Blasina et al., 1999; Furnari et al., 1999). Once
phosphorylated, Cdc25C can then to be bound by 14-3-3s and to be sequestered in the
cytoplasm (Blasina et al., 1999). Cdc25C, only when it is in the nucleus, dephosphorylates the
Thr14 and Tyr15 residues and thus activates the Cdk1/cyclin B1 complex (Peng et al., 1997;
Sanchez et al., 1997; Solomon et al., 1990). Additional phosphatases, Wip1 (PPM1D)
(Shreeram et al., 2006) and PP2A, (Margolis et al., 2006) may also regulate the checkpoint by
dephosphorylating other factors that contribute to the activation of Cdk1. Cdk1/cyclin B can
phosphorylate Cdc25C, further activating it and initiating a positive feedback loop (Izumi et
al.,1992). A nuclear export signal (NES) in Cyclin B1 facilitates rapid export of the Cdk1/cyclin
B1 complex from the nucleus (Pines and Hunter, 1994). Phosphorylation of the NES blocks the
nuclear export by interfering with the binding of the nuclear export receptor.
In response to incomplete DNA replication, another kinase is activated instead (Elledge,
1996). Chk1 and Cds1 kinases modulate the activity of Cdk1 by activating Wee1/Myt1 kinases
or inhibiting Cdc25 phosphatase. Both kinases can phosphorylate Cdc25 in vitro, thereby
inhibiting their activity (Furnari et al., 1997).
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III-Overview of Drosophila Cell Cycle
Figure III_4- Schematic representation of the G2 checkpoint (in Plesca et al., 2007).
Cdk1 is the key control point for G2 checkpoint initiation, which is dependent on both positive and negative regulators.
To be active, Cdk1 requires binding of its catalytic partner, cyclin B1 (also B2 and B3 in some circumstances), which is
regulated transcriptionally and by its nuclear localization. Cdk1 is regulated by its phosphorylation, as determined by a
balance of kinase and phosphatase activities, as well as at the level of transcription. G2 progression to mitosis is
triggered by the Cdc25 mediated dephosphorylation of the Cyclin B/Cdk1 complex. Cyclin B/Cdk1 is activated by
phosphorylation of Thr161 by CAK (Cyc H/Cdk7 complex) and the dephosphorylation of Thr14 and Tyr15 by Cdc25C.
The complex is kept in an inactive state due to the phosphorylation of Thr14 and Tyr15 by the Myt1 and Wee1 kinases
that can in turn be regulated by Plk1. The stable maintenance of the G2 arrest is determined by the activity of E2F,
Rb, or p53 family of transcription factors through their transcriptional targets, which include many of the components
described here. p53 regulates the stability of the checkpoint through levels of its mitosis target genes cdk1, cyclin B1,
but also p21 (CDKN1A) and 14-3-3s. The activity of Cdc25 is also regulated by Chk1 or Chk2-mediated
phosphorylation, leading to its inactivation through binding to and sequestration by 14-3-3s. ATM/ATR kinases
transduce the DNA damage signal to the effector kinases Chk1 and Chk2. Chk1 and Plk1, by regulating Wee1, also
play a role in the recovery from the arrest.
More recent studies suggest that Plk is also an important target of DNA damage
checkpoint. In human cells, DNA damage inhibits the activation of Plk and CdK1-Cyclin B in an
ATM-dependent manner. Moreover, G2/M arrest induced by DNA damage could be overcome
by constitutive expression of a Plk1 active form (Smits et al., 2000). In addition, a decrease in
Plk1 was observed in an ATM-dependent response to radiomimetic drugs and in an ATR-
dependent response to ultraviolet treatment and to topoisomerase-II inhibitors (Van Vugt et
al., 2001; Deming et al., 2002). Furthermore, Plk1 was identified as a putative substrate of the
Chk2 kinase (Seo et al., 2003). All theses studies indicated Plk1 is also a target of the DNA
damage checkpoint cascade.
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III-Overview of Drosophila Cell Cycle
III_5.2- Metaphase or Spindle assembly checkpoint (SAC)
We will make a more detailed description of this control step, once identified mitotic
mutants (see chapter II part IV of this manuscript) revealed problems at the metaphase-
anaphase transition/regulation.
The spindle assembly (or metaphase) checkpoint is a quality control mechanism that
prevents chromosome segregation errors. The spindle assembly checkpoint detects spindle
assembly defects and, loss or impairment of functional connections between kinetochores and
spindle microtubules during mitosis, disseminating inhibitory signals that delays anaphase
onset (Fig. III_5). This signal is presumably amplified and ultimately acts to arrest the APC/C
(reviewed by Hoyt, 2001; Shah and Cleveland, 2000; Amon, 1999).
In 1991, two independent screens identified various genes mutation, of which bypassed
the ability of wild-type S. cerevisiae cells to arrest in mitosis in the presence of spindle poisons
(Hoyt et al., 1991; Li and Murray, 1991). The genes identified in these screens included the
Mad (mitotic-arrest deficient) genes mad1, mad2 and mad3 (bubR1 in humans), and the Bub
(budding uninhibited by benzimidazole) gene bub1 (Hoyt et al., 1991; Li and Murray, 1991).
Another bub gene, bubB3, was identified as an extra-copy suppressor of the bub1-1 mutant1.
These genes are conserved in all eukaryotes, and they are collectively involved in a spindle-
assembly checkpoint (SAC) pathway that is actived in prometaphase and which prevents the
precocious separation of sister chromatids (Musacchio and Hardwick, 2002; Taylor et al.,
2004).
The target of the complex intracellular mechanisms of the spindle assembly checkpoint
is the protein Cdc20, a co-factor of the ubiquitin ligase APC/C (Hwang et al.1998; Kim et al.,
1998). Specifically, the SAC negatively regulates the ability of Cdc20 to activate the APC/C-
mediated polyubiquitynation of two key substrates, cyclin B and securin, thereby preventing
their destruction by the 26S proteasome. Securin is a stoichiometric inhibitor of a protease
known as separase. Separase is required to cleave the cohesin complex that holds sister
chromatids together, and cohesin cleavage is required to execute anaphase (Peters, 2007).
The inhibition of the APC/C causes high cyclin levels, sustained cyclin-dependent kinase 1
(cdk1) activity, and prolonged mitotic arrest. The mitotic arrest allows time for the correction
of the chromosome connections to the spindle. Only when all chromosomes have become bi-
orientated between separated spindle poles on the metaphase plate, the checkpoint is silenced
relieving the mitotic arrest and allowing anaphase to proceed (Bhalla, 2003; Jordan and
Wilson, 2004; Kops et al., 2005).
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III-Overview of Drosophila Cell Cycle
Figure III_5- Relationship of the SAC with the cell-cycle machinery (in Musacchio and Salmon, 2007).
Mitosis is traditionally subdivided into five consecutive and morphologically distinct phases: prophase, prometaphase,
metaphase, anaphase and telophase. To enter mitosis, the cell requires the activity of the master mitotic kinase,
cyclin-dependent kinase-1 (CDK1), which depends strictly on the binding of cyclin B to CDK1 (Peters, 2006). Separase
is a protease, the activity of which is required to remove sister-chromatid cohesion at the metaphase-to anaphase
transition (cohesin is indicated in yellow on the expanded view of the chromosome). Prior to anaphase, separase is
kept inactive by the binding of a protein known as securin (SEC). Unattached kinetochores (red hemi-circles)
contribute to the creation of the mitotic checkpoint complex (MCC), which inhibits the ability of CDC20 to activate the
anaphase-promoting complex/cyclosome (APC/C). The attachment of all sister-kinetochore pairs to kinetochore
microtubules, and their bi-orientation — which produces congression to the spindle equator — negatively regulates the
spindle-assembly checkpoint (SAC) signal. This releases CDC20, which can now activate the APC/C. This results in the
polyubiquitylation of anaphase substrates such as cyclin B and securin, and their subsequent proteolytic destruction by
the proteasome. The degradation of SEC results in the activation of separase, which targets the cohesin ring that is
holding the sister chromatids together, thus causing the loss of sister-chromatid cohesion and the separation of sister
chromatids. The degradation of cyclin B at this stage also inactivates the master mitotic kinase CDK1–cyclin B,
initiating cytokinesis and the mitotic-exit programme.
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III-Overview of Drosophila Cell Cycle
III_5.2.1- Models for spindle assembly checkpoint sense the defects
Two models are proposed to explain how spindle assembly checkpoint detects a single
spindle defects. One model proposes that checkpoint machinery is sensitive to the tension
exerted in kinetochore by the opposite spindle microtubules forces. The other explains that
metaphase checkpoint monitors the availability of kinetochores unattached.
Figure III_6- The attachment process (in Musacchio and Salmon, 2007).
(a) Unattached kinetochores generate a ‘wait’ signal and recruit the spindle assembly checkpoint (SAC) proteins. The
levels of mitotic-arrest deficient homologue-2 (MAD2) are high at unattached kinetochores (left) and moderately high
at attached kinetochores in a monotelic pair (right). Under these conditions, the Aurora-B kinase concentrates at
centromeres and is believed to be activated by the lack of tension between the sister chromatids. Bi-orientation
depletes MAD2 (and budding uninhibited by benzimidazole (BUBR1) from kinetochores and promotes the acquisition of
tension in the centromere area, which is visualized as an increase in the inter-kinetochore distance between sister
chromatids. When all chromosomes have achieved this situation, the SAC signal is extinguished and anaphase ensues
thanks to the activation of separase, which removes sister-chromatid cohesion by proteolysing cohesin. At entry into
anaphase, Aurora-B is also depleted from the centromere region.
(b) Correct and incorrect attachments can occur during mitosis, and correction mechanisms exist to prevent incorrect
chromosome inheritance, which would occur if improper attachment persisted until anaphase. Monotelic attachment is
a normal condition during prometaphase before bi-orientation. In syntelic attachment, the both sisters in a pair
connect to the same pole. A mechanism that corrects this improper attachment depends on the Aurora-B/Ipl1 kinase.
Merotelic attachment occurs quite frequently and is also corrected by the Aurora-B kinase. The SAC might be able to
sense syntelic attachment, but it is unable to detect merotelic attachment.
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III-Overview of Drosophila Cell Cycle
The first arguments in favour of tension model come from experiments of
micromanipulation performed in the spermatocytes of praying mantids, which have three
sexual chromosomes (two X chromosomes and one Y chromosome; Gorbsky and Ricketts,
1993; Li and Nicklas, 1995; Li and Nicklas, 1997; Nicklas et al., 1995). In this system, only
one chromosome attached to a single spindle pole is sufficient to inhibit the progression to
anaphase. When mono-attach X-chromosome is pulled with a microneedle, the tension is re-
established. In this case, the inhibition of the progression of mitosis is cancelled and the cells
progress normally to anaphase.
However, tension don’t explain everything, the laser ablation of the last unattached
kinetochore silence the spindle checkpoint and allows the progression to anaphase, despite the
lack of tension in the monooriented sister chromatid (Rieder et al., 1995).
III_5.2.1.1- Microtubule attachment model
The SAC monitors the attachment of kinetochores to microtubule plus-ends during an
unperturbed mitosis. Ideally, SAC activity at sister kinetochores should remain active until bi-
orientation, the only condition that ensures accurate segregation at anaphase (Fig. III_6a).
Cyclin B and securin start to be degraded after the last chromosome has been aligned, and
they become largely depleted prior to anaphase (Clute and Pines, 1999; Hagting, A. et al.,
2002). Early during this process, the addition of spindle poisons stops proteolysis immediately
and blocks anaphase onset in a SAC-dependent manner (Clute and Pines, 1999). Interference
with kinetochore assembly impairment of microtubule motors (for example, dynein or CENP-E)
and interference with microtubule dynamics also activate the SAC (Rieder and Maiato, 2004).
Certain SAC proteins are immediately removed after the attachment of microtubule plus-ends
and formation of kinetochore microtubules. For example, Mad2 localises to unattached
kinetochores during prometaphase or in cells treated with nocodazole, which induce MT
depolymerization and prevent formation of MT–kinetochore end-on attachments (Chen et al.,
1996; Waters et al., 1998). Conversely, the amount of Mad2 becomes highly reduced at
metaphase kinetochores (50–100-fold comparatively with unattached prometaphase
kinetochores). Therefore, kinetochore localization of Mad2 decreases substantially as
kinetochores fill more of their attachment sites with kinetochore microtubules (occupancy).
Because Mad1 and Mad2 accumulate at unattached kinetochores and are removed on
attachment, their kinetochore localisation is interpreted to signify lack of attachment (Howell
et al., 2001; Skoufias et al., 2001; Waters et al., 1998). In metazoans, Mad2 depletion from
kinetochores depends not only on MT attachment, but also on cytoplasmic dynein motility
along MT. The inhibition of the dynein at metaphase kinetochores results in the return to
~25% of the level of Mad2 at unattached kinetochores, without a loss in kinetochore-
microtubule number (Howell et al., 2001; Wojcik et al., 2001). This indicates that,
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III-Overview of Drosophila Cell Cycle
kinetochore-MT formation promotes Mad1 and Mad2 dissociation by providing high local
concentrations of MT, in order to promote the ATP-dependent motility of dynein along
microtubules (Howell et al., 2001; Wojcik et al., 2001).
III_5.2.1.2- Tension model
In addition to microtubule–kinetochore attachment, tension is important for SAC
inactivation (Nicklas et al., 1995; Nicklas, 1997). Stretching of centromeric chromatin on bi-
orientation, increases kinetochore-to-kinetochore distance and, consequently, kinetochore
tension. MT–kinetochore attachment is normally destabilised at low kinetochore tension and
stabilised by high tension between bi-orientated sisters kinetochores (Nicklas, 1997; Nicklas et
al., 2001). Therefore, tension might provide a fundamental criterion to discriminate against
incorrect attachments. For example, if both sister kinetochores attach to microtubules from the
same pole (syntelic attachment; Fig. III_6b), not enough tension is generated and MT–
kinetochore attachment is destabilised to correct the problem. This destabilisation depends on
Aurora-B/Ipl1 kinase (Hauf et al., 2003; Lampson et al., 2004; Pinsky and Biggins, 2005;
Tanaka et al., 2002). Aurora-B/Ipl1 is also critical for correcting merotelic attachments, which
are not sensed by the SAC. Merotelic attachment occurs when one sister kinetochore becomes
attached to MT from opposite poles. Bi-orientation of chromosomes with merotelic
kinetochores produces sufficient occupancy and tension to turn off SAC activity (Fig. III_6b).
As a result, merotelic kinetochores, if left uncorrected, can produce lagging chromatids and
potential chromosome mis-segregation in anaphase (Cimini and Degrassi, 2005). Tension and
centromere stretch also seem to have a significant role in turning off SAC activity by inhibiting
the association rate of SAC proteins at kinetochores. For example, SAC activity is turned on by
adding taxol or low doses of vinblastine to metaphase HeLa cells to stop kinetochore-MT
dynamics and reduce tension. (Taxol decreases inter-kinetochore tension in bi-orientated
sisters, while stabilising the number of metaphasic kinetochore-MT.) In cells undergoing these
treatments, SAC activation correlates with a significant increase in the concentration of BubR1
and Bub1 at kinetochores, as well with the phosphorylation of an unknown phosphoepitope by
Plk1 kinase that is detected by the antibody 3F3/2 (Gorbsky and Ricketts, 1993; Nicklas,
1997; Waters et al., 1998). This type of experiment does not prove that lack of tension alone
is sufficient to activate the SAC, once that, metaphase cells treated with taxol were shown to
contain one or a few Mad2-positive kinetochores (possibly owing to low occupancy), which
could be sufficient to trigger a SAC arrest (Waters et al., 1998). However, 3F3/2
phosphorylation and re-recruitment of BubR1 and Bub1, but not Mad2 to all kinetochores,
imply that the reduction in tension changes the kinetochore in a significant way, independently
of any change in occupancy. Therefore, irrespective of whether the checkpoint is indeed
regulated by tension, independently of occupancy, kinetochore localisation of checkpoint
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III-Overview of Drosophila Cell Cycle
components is differentially regulated by occupancy and tension. Even so, it must be
remembered that, distinguishing the relative contributions of tension and attachment, when
manipulating spindles is difficult, since interfering with the creation of tension probably affects
attachment (Pinsky and Biggins, 2005; Zhou et al., 2002).
We remain unaware of any SAC or SAC-related protein for which activity is directly
regulated by tension in an adequately reconstituted system. SAC activity during the successive
phases of bi-orientation, which macroscopically define the attainment of attachment and
tension, seems to result in the generation of the same cell-cycle inhibitor, the MCC18.
Consistently, Mad2 is required to sustain the SAC, even after its substantial depletion from
kinetochores (Howell et al. 2001; Shannon et al., 2002). Similarly, although kinetochore Mad2
remains low in taxol-treated cells, Mad2 is critically required to maintain the taxol-dependent
arrest (Howell et al. 2001; Shannon et al., 2002; Skoufias et al., 2001; Waters et al., 1998).
The separation at the molecular level between attachment and tension will be clarified through
a better understanding of the physical connections at the kinetochore between the MT-binding
machinery and the SAC.
III_5.2.2- Inhibition of APC/C by the spindle assembly checkpoint
Although the nature of the direct molecular interactions between SAC proteins and
kinetochores and, the interdependencies of SAC proteins have not been determined
(Musacchio and Hardwick, 2002), the basic plan of the signalling cascade is established (Fig.
III_7).
APC/CCdc20 is a key molecular target of the spindle checkpoint (Bharadwaj and Yu,
2004). Biochemical analysis revealed that (Mad)1-3 and (Bub)1-3, the components of the
spindle checkpoint (Bharadwaj and Yu, 2004) use multiple strategies to inhibit APC/CCdc20 upon
checkpoint activation. Stoichiometric binding of either Mad2 or BubR1 (the vertebrate ortholog
of yeast Mad3) to Cdc20, inhibits APC/CCdc20 in vitro (Fang et al., 1998a; Tang et al., 2001a).
Mad2, Mad3/BubR1, Bub3, and Cdc20 can form a single mitotic checkpoint complex in vivo
(Sudakin et al., 2001). In addition, Bub1 phosphorylates Cdc20 inhibiting catalytically
APC/CCdc20 (Tang et al., 2004). Finally in yeast, Mad2 and Mad3 binding to Cdc20, triggers the
auto-ubiquitination of Cdc20 and reduces the protein level of Cdc20 (Pan and Chen, 2004),
although it remains to be determined whether this mechanism is conserved in other
organisms.
Fluorescence recovery after photobleaching (FRAP) experiments revealed that, the
kinetochore-bound pools of BubR1, Mad2, and Cdc20 exchange rapidly with their cytosolic
pools (Howell et al., 2004; Shah et al., 2004). Furthermore, structural and biochemical
analysis of Mad1 and Mad2 suggests that the kinetochore-bound Mad1 promotes a
conformational change of Mad2, which is required for its efficient binding to Cdc20 (reviewed in
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III-Overview of Drosophila Cell Cycle
Yu, 2006). Finally, in Xenopus egg extracts, phosphorylation of Cdc20 by MAPK and Cdk1 is
required for efficient Mad2 binding and, for the spindle checkpoint (Chung and Chen, 2003;
D’Angiolella et al., 2003). These findings support an attractive model, in which the unattached
kinetochore facilitates the formation of APC/C-inhibitory checkpoint complexes containing
BubR1, Bub3, Mad2, and Cdc20: by increasing the concentrations of these proteins locally, by
triggering a conformational change of Mad2 and, by posttranslational modifications on Cdc20.
In addition, Bub1 bound to chromosomes exhibiting higher autophosphorylation activity as
compared to cytosolic Bub1 (Chen, 2004). This suggests that unattached kinetochores might
also stimulate the kinase activity of Bub1 toward Cdc20 facilitating inhibition of APC/CCdc20. It
remains to be determined, whether phosphorylation of Cdc20 by Bub1 also enhances its
binding to Mad2 or BubR1, similar to phosphorylation of Cdc20 by MAPK and Cdk1 (Chung and
Chen, 2003; D’Angiolella et al., 2003).
In an alternative model, it was proposed that the mitotic checkpoint complex forms
constitutively during the cell cycle, but it is only capable of associating with the APC/C core
modified during mitosis by unattached kinetochores and spindle checkpoint signalling (Sudakin
et al., 2001). This model is inconsistent with the fact that, the concentrations of mitotic
checkpoint complex are much higher in mitosis than during interphase in several organisms
(Chen, 2002; Millband and Hardwick, 2002). Furthermore, no such spindle checkpoint-
dependent modifications of APC/C, which allow mitotic checkpoint complex binding in mitosis,
have been discovered so far.
It has been suggested that Mad2 binding to Cdc20 prevents the release of substrates
(presumably ubiquitinated substrates, i.e., products of the ubiquitination reaction), thereby
inhibiting APC/CCdc20 (Pfleger et al., 2001b). It is unclear, how binding of BubR1 to Cdc20 or,
Bub1-mediated phosphorylation of Cdc20, inhibits APC/CCdc20. Both Mad2 and BubR1 can be
coimmunoprecipitated with core subunits of APC/C, such as Cdc27 in nocodazole-arrested
mitotic human cells (Fang et al., 1998a;Sudakin et al., 2001), suggesting that binding of Mad2
or BubR1 to APC/C does not dissociate Cdc20 from APC/C. Likewise, phosphorylated Cdc20
remains bound to APC/C in mitosis. Thus, Mad2, BubR1, and Bub1 inhibit APC/CCdc20 through
the interference with the function of Cdc20 at a step that is subsequent to Cdc20 binding to
APC/C, possibly by substrate recruitment and/or product release.
Though counterintuitive, this mode of APC/CCdc20 inhibition by the spindle checkpoint is
in fact advantageous for two reasons. (1) While the spindle checkpoint inhibits the ubiquitin
ligase activity of APC/C toward securin and cyclin B, it does not block degradation of certain
APC/CCdc20 substrates, such as cyclin A and Nek2A (Geley et al., 2001; Hayes et al., 2006). (2)
The association of Mad2- and BubR1-bound Cdc20 with APC/C provides an opportunity for
Cdc20 autoubiquitination by APC/C. In one hand, Cdc20 autoubiquitination, at least in yeast,
reduce the protein levels of Cdc20, facilitating inhibition of APC/CCdc20 by the spindle checkpoint
67
III-Overview of Drosophila Cell Cycle
(Pan and Chen, 2004). In other hand, Cdc20 autoubiquitination dissociates Mad2 from Cdc20
to inactivate the spindle checkpoint (Reddy et al., 2007; Stegmeier et al., 2007).
III_5.2.2.1- Recruitment of spindle checkpoint proteins to unattached/
untensed kinetochores
Regardless of the competing models, it is generally agreed upon that ultimately,
checkpoint signal has to originate from the kinetochores of misaligned chromatids and is then
distributed and amplified throughout the cell to exert inhibition of APC/C (Fig. III_7).
A signalling network that recruits spindle checkpoint proteins to unattached
kinetochores has been established, by examining their interdependency at kinetochores in
mammalian cells and in Xenopus egg extracts.
The chromosome passenger complex (CPC), containing Aurora B, INCENP, survivin and
Dasra/Borealin, recruits the Bub1-Bub3 complex and Mps1 to the kinetochores (Johnson et al.,
2004; Vigneron et al., 2004). The CPC and Bub1 collaborate to recruit Plk1 to kinetochores
through Bub1-Plk1 and INCENP-Plk1 interactions (Goto et al., 2006; Qi et al., 2006). Plk1 then
facilitates the kinetochore recruitment of Cdc20 and the BubR1-Bub3 and Mad1-Mad2
complexes (Ahonen et al., 2005; Wong and Fang, 2005). Plk1 phosphorylates and targets
Plk1-interacting checkpoint helicase (PICH, which is a member of the SNF2 family of DNA-
binding ATPases) to kinetochores during prometaphase and in anaphase to stretched
centromeric chromatin that contains concatenated DNA (Baumann et al., 2007). PICH is
specifically required for the kinetochore localisation of Mad2, but not of Mad1, suggesting that
PICH maintains the Mad1-Mad2 interaction at the kinetochores in response to the lack of
tension across the kinetochores (Baumann et al., 2007). Interestingly, depletion of Nek2A also
causes delocalisation of Mad2 from kinetochores without affecting the kinetochore localisation
of Mad1 (Lou et al., 2004). Recently, Draviam and collegues identified the protein kinase TAO1
as a novel spindle checkpoint protein, that was required for the kinetochore localisation of
Mad2, but not other spindle checkpoint proteins examined, including Mad1 (Draviam et al.,
2007).
Therefore, upon checkpoint activation, an elaborate signaling network targets the Mad
and Bub proteins to unattached/untensed kinetochores, which then promotes the formation of
APC/C-inhibitory complexes.
Recently, Cdc20 autoubiquitination was suggested as an important mechanism for
regulating the Mad2-Cdc20 interaction and for checkpoint inactivation. New antagonistic
proteins UbcH10 and USP44 was recently identified and implicated in Cdc20 autoubiquitination.
The UbcH10 (UBC) was implicated in the checkpoint inactivation (Reddy et al., 2007). It
was showed that UBC promotes Ccd20 autoubiquitination, disrupting Mad2-Cdc20 interaction
and activating APC/C (Reddy et al., 2007). Consistent with earlier studies that established a
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III-Overview of Drosophila Cell Cycle
role of p31 comet (a conformation-specific Mad2-binding protein) in checkpoint inactivation
(Xia et al., 2004), p31 comet and UBC showed a synergistic effect on checkpoint inactivation
through Cdc20 autoubiquitination (Reddy et al., 2007).
Figure III_7- A Model for the Regulation of APC/CCdc20 by the Spindle Checkpoint (in Yu, 2007).
Upon checkpoint activation, an elaborate signaling network targets the spindle checkpoint proteins to unattached
kinetochores. The chromosome passenger complex (CPC) containing Aurora B and INCENP recruits Bub1-Bub3.
Phosphorylation of INCENP and Bub1 by Cdk1 creates docking sites for the polo-box domain of Plk1, targeting Plk1 to
kinetochores. Plk1 then facilitates the kinetochore localization of PICH, Mad1-Mad2, BubR1-Bub3, and Cdc20. The
close proximity of Cdc20 and the checkpoint proteins at kinetochores and a Mad1-triggered conformational change of
Mad2 facilitate the formation of the mitotic checkpoint complex (MCC) containing BubR1, Bub3, Mad2, and Cdc20,
which associates and inhibits APC/C. Phosphorylation of Cdc20 by Bub1 also inhibits APC/CCdc20. The concentration of
Mad2-Cdc20-containing checkpoint complexes is controlled by two opposing dynamic processes: kinetochore-
facilitated formation and p31comet- and UbcH10-dependent disassembly through Cdc20 autoubiquitination. Protectin
deubiquitinates Cdc20 and maintains the Mad2-Cdc20 interaction, contributing to checkpoint activation. When the
kinetochores are captured by spindle microtubules and are under tension, the concentrations of checkpoint proteins at
kinetochores and possibly their activities in certain cases decrease. p31comet binds to Mad1-bound Mad2, preventing
the generation of the active conformer of Mad2. These mechanisms reduce the rate of formation of Mad2-Cdc20.
Meanwhile, p31comet binds to Cdc20-bound Mad2 and, along with UbcH10, stimulates the autoubiquitination of Cdc20
and the disassembly of Mad2-Cdc20. This leads to checkpoint inactivation and activation of APC/CCdc20, which
indirectly activates APC/CCdh1. APC/CCdh1 then targets several spindle checkpoint proteins for degradation, including
Plk1,Aurora B, Mps1, and Bub1, and prevents the reestablishment of the spindle checkpoint following mitotic exit.
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III-Overview of Drosophila Cell Cycle
The USP44 protein, an ubiquitin isopeptidase, was identified as a novel spindle
checkpoint protein (Stegmeier et al., 2007). It was shown that USP44 has a role in overlap the
mitotic arrest induced by taxol treatment, and is also required for the proper timing of mitotic
events during the normal cell cycle, a function shared with Mad2 and BubR1 (Meraldi et al.,
2004). In addition, USP44 antagonised the ability of UbcH10 and p31 comet to induce the
disruption of the Mad2-Cdc20 interaction and APC/C activation in vitro. Importantly, the
isopeptidase activity of USP44 is required for spindle checkpoint signaling in cells and appears
to be activated by phosphorylation during mitosis. Activated recombinant USP44 directly
removed ubiquitin from ubiquitinated Cdc20. The USP44 protein was renamed protectin to
reflect its role in reversing Cdc20 autoubiquitination and consequently protecting the Mad2-
Cdc20-containing checkpoint complexes from disassembly (Stegmeier et al., 2007).
Collectively, these two studies demonstrated that cellular concentrations of Mad2-Cdc20
and the activation status of the spindle checkpoint depend on the delicate balance between
two opposing dynamic processes, providing a fast on-or-off switch for the spindle checkpoint
(Reddy et al., 2007; Stegmeier et al., 2007). It is unclear whether UbcH10- and p31comet-
dependent Cdc20 autoubiquitination and the activity of protectin are directly regulated by the
spindle checkpoint.
70
CHAPTER II
RESULTS
72
RESULTS_Part I:
RESULTS_PART I
74
New teashirt alleles obtained by P-element excision mutagenesis
Silvia Pimentel1,2, Isabel Salazar2, Christine Vola2, Rui Gomes 1 and Laurent Fasano2
1 Departamento de Biologia Vegetal, Faculdade de Ciências da Universidade de Lisboa,
Edifício C2-4º, Campo Grande, 1749-016 Lisboa, Portugal
2 Institut de Biologie du Développement de Marseille Luminy (IBDML), UMR 6216,
CNRS-Université de la Méditerranée, Campus de Luminy, Case 907, 13288 Marseille Cedex 09,
France.
RESULTS_PART I
76
RESULTS_PART I
Abstract
The hypomorphic allele tshA3-2-66 corresponds to a P{lacW} insertion 1652 base pairs
(bp) upstream the translation start of the teashirt (tsh) gene. New mutations had been
generated by mobilization of the P-lacW transposon, which is marked with a mini-white+ gene.
The endogenous white locus was the w1118 allele. Precise and imprecise excisions (E{P}) of
the tshA3-2-66 transposon were induced in the presence of the Sb P[ry+ 2-3] source of
transposase and male selected in F2 on the basis of the loss of the eye color marker.
Out of 760 F2 males screened, we obtained 145 E{P}/CyO P-element-excised
revertants lines. These revertant were tested with the original P{lacW}A3-3-66 mutant, 20 were
embryonic lethal, 101 died during larval stages and 2 didn’t survive the pupal stage. One line
that gave fertile adults might correspond to a precise P-element excision event. We observed
female escaper sterile in 6 of the F2 lines, probably resulting from imprecise P-element
excision events. This female failed to lay eggs and presented atrophied ovaries with a strong
arrest in early oogenesis. The P-element excision from tsh locus were confirmed. We identified
6 lines that was homozygous lethal (∆P30, ∆P99, ∆P104, ∆P123, ∆P126 and ∆P128) and gave
a severe female sterile phenotype when in Trans with tshA3-2-66, probably resulting from
imprecise P-element excision events. This female show a modifications in the eye patterned
white expression observed in the tshA3-2-66 adult eyes. Absence of re-integration of the P-
element was tested by PCR.
Key words: Drosophila, P-element excision, P-lacW transposon, teashirt
77
RESULTS_PART I
Materials and methods
Drosophila strains
All genetic markers and balancers are described by Lindsley and Zimm (1992) and
FlyBase (http://flybase.bio.indiana.edu) except as noted.
The hypomorphic allele tshA3-2-66 (P{lacW}A3-3-66; Bloomington stock 10842) have a
P{lacW} insertion at 1652 base pairs (bp) upstream the translation start of the teashirt (tsh)
gene and was identified after the screening of the Bloomington P-element insertions at the
38A6-40B1 cytogenetic region (Salazar and Gomes, 1999). The lethal P{lacW} insertion allele
tshA3-2-66 fail to complement the deficiencies Df(2L)TW161, Df(2L)305 and tsh8 allele .
P-element excision
For mobilisation of the P{lacW}A3-3-66 insert, w/w;T(2;3)CyOTM6,Tb/ P{lacW}A3-3-66;+
females were crossed to males carrying P{ry+, ∆2-3} on a Stubble (Sb) marked third
chromosome (Robertson et al., 1988) of genotype w/Y; T(2;3)CyOTM6,Tb/S Sp Bl bwD; ∆2-
3,Sb (Fig.1). All the strains used in the reversion scheme, carried X-chromosomes with the
eye-marker white1118 (w1118), and thus individuals carrying the P{lacW} chromosome (F2 w/Y;
T(2;3)CyOTM6Tb/P{lacW}A3-3-6) had coloured eyes due to the presence of the extra wild-type
copy w+ on the second chromosome, while lines having lost the P{lacW} (F2 w/Y;
T(2;3)CyOTM6Tb/∆P{lacW}A3-3-6) showed white eyes due to the white background on the X-
chromosome. The F1 males of genotype w/Y;P{lacW}A3-3-66/S Sp Bl bwD;+/∆2-3Sb were
crossed to females of genotype w/w;T(2;3)CyOTM6Tb/P{lacW}A3-3-66;+. 145 white-eyed males
(w/Y; T(2;3)CyOTM6Tb/∆P{lacW}A3-3-6) were found amongst 760 F2 males examined (Fig.1;
Table1). The 145 putative P-element excision chromosomes were then independently re-
balanced over T(2;3)CyOTM6Tb to establish P-element excision lines and test for viability and
fertility. For simplify, the F2 males were numbered in ascending order according to the order
that they have been gotten. The progeny of each F2 male correspond to a reversion line. Thus,
we analysed 760 lines of which 145 correspond to excision lines (∆P).
Staining of neuroblast with acetic orcein
For orcein acetic staining, the third instar larval brain were dissected in 0.7% NaCl and
fixed for 30 seconds in a 45% acetic acid and then transferred individually to be labelled in a
4% orcein in 45% acetic acid, for more 30 seconds. The brains were then washed in 45%
acetic acid and mounted in a drop of 4% orcein between slide and coverslip. The preparation
was squashed using filter paper and observed in the Zeiss Axioplan II microscope. Images
were acquired with a SPOT-2 camera (Diagnostics Instruments, Inc.) and processed with
Adobe Photoshop 6.0 (Adobe System).
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RESULTS_PART I
Figure 1- Diagram of the flies matting
during P-element excision mutagenesis.
tshA3-2-66 flies carry the P{lacW} chromosome, while
∆PtshA3-2-66 represents the loss of the P{lacW}
chromosome. ∆2-3 was used as the source of the
transposase. In F2, the male were individually
matted (each male was matted with two virgin
female).
Staining of ovaries
The ovaries were dissected in PBS and then fixed in 3,7% formaldehyde in PBS during
10 minutes (adapted from Ashburner, 1989). The ovaries were washed with PBST (PBS with
0,1% Triton X-100) during 1 hour and then the DNA was stained with 1µg/ml of Hoechst
33258 during 20 minutes. After 1 hour of washes with PBST, the ovaries were mounted in
Vectashield medium (Vectors). The preparations were observed in fluorescence using a Zeiss
Axioplan II microscope. Images were acquired with a SPOT-2 camera (Diagnostics
Instruments, Inc.) and processed with Adobe Photoshop 6.0 (Adobe System).
Wing Flies preparation
Wings from adult flies were dissected in 100% etanol and then transferred to xylene. In
a slide, the wings were placed in the top of a drop of Eukitt mounting medium (Electron
Microscopy Sciences). Finally, in the top of this was placed a coverslip and some weight during
the drying of EuKitt. The preparations were observed in the LEICA MZ FL III.
5’P and 3’P PCR amplification and contiguous regions of the PlacWA3-2-66
For tshA3-2-66 and in ∆P126, 5' and 3' P-element ends (5'P and 3'P) and the contiguous
regions of the P{lacW}A3-3-66 were PCR amplified. The 5’P was amplified with the primers:
ATTCAGTGCACGTTTGCTTG (5P_Fwd); TTTGGGAGTTTTCACCAAGG (5P_Rev);
AACGACGCATTTCGTACTCC (5P_Rev1); GCCTCTTCGCTATTACGCC (5P_Rev2);
TGGGCGCATCGTAACCGT (5P_Rev3) and GCAGGCTTCTGCTTCAATCAG (PlacW_1). The primers
5P_Rev2 and 5P_Rev3 are localise downstream the 5’P end of the P{lacW}A3-3-66 and in pair
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RESULTS_PART I
with the 5P_Fwd amplified part of the 5’P end region and the contiguous downstream region.
The 3’P end was amplified with the primers: AAGGATTTCCTTTGCCCAGT (3P_Fwd) and
TCCGTGGGGTTTGAATTAAC (3P_Rev).
Absence of re-integration of the P-element was confirmed by PCR.
Genomic DNA was isolated from L3 larvae homozygous for tsh126 allele after treatment
with a phenol/chloroform/isoamyl alcohol mixture to remove protein contaminants and
precipitation with 100% ethanol. The DNA was re-suspended in water for further
experimentation.
DNA sequencing of the ∆P99, ∆P104, ∆P123 and ∆P126 lines
Genomic DNA was isolated from L3 larvae homozygous for the ∆P99, ∆P104, ∆P123 and
∆P126 revertant lines after treatment with a phenol/chloroform/isoamyl alcohol mixture to
remove proteins contaminants and precipitation with 100% ethanol. The DNA was re-
suspended in water for further experimentation.
We sequenced and analysed 3913bp upstream of the translation start of tsh in the
∆P99, ∆P104, ∆P123 and ∆P126 lines for comparison. This region was amplified by PCR and
fragments were purified and sent for sequencing (GenomExpress). The following PCR primers
were used: 5’CACAAACATTGACACCTTGCC3’; 5’GACTACGCCGAACCGCCAA3’;
5’CCTTCTTTTGTCCTATTCCG3’;5’GCCAAATCTGAGCCTTAGAC3’;5’CCCAGCACCTCTTCTTTAGG3’;
5’CGTCACCCTTCTTTTGTCC3’;5’GCAGCCAAAAAGAAAAGTGG3’;5’TCGTCGTCACTACTCGAACG3’;5
’GTATGGTTAAATATTGTATATGCAA3’;5’CAAAATTTAGTTGCGAGGAGT3’;5’CGTTGTTGTTGTTTGTC
AGC3’ and 5’TTAGTTGGATTTTGGAACTTAAC. Sequences were compared and BLAST with the
tsh sequence reported in flybase (http://flybase.bio.indiana.edu) and the genomic sequence
that surround the insertion site of other transposable elements inserted in the same region
(P{RS3}CB-5359-3; P{XP}cl00773; P{lacW}l(2)k16406; P{XP}cl05049).
Results and Discussion
1-Excision Rate of the PlacW excision mutagenesis
The hypomorphic allele tshA3-2-66 (Bloomington stock 10842) was identified as a tsh
allele (tshA3-2-66) while we screened for P-element insertions at the 38A6-40B1 interval that fail
to complement the deficiencies Df(2L)TW161 and Df(2L)305, the tshNG1 and the tsh8 alleles
(Salazar and Gomes, 1999). We found that tshA3-2-66/tsh8 heterozygous were lethal, the tshA3-2-
66 homozygous dye at pupal stage and tshA3-2-66/Df(2L)TW161 also dye at pupal stage with very
few escapers.
After mobilizing the P element in tshA3-2-66, we isolated 145 F2 males lines with white
eyes. The screening for excision or modification of the transposon was based on the loss of the
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RESULTS_PART I
white+ eye colour marker. F2 males that had lost the P{lacW} and concomitantly had lost the
extra wild-type copy w+ on the second chromosome, shown in consequence white eyes due to
the white background on the X-chromosome. The 145 F2 males were obtained out of 760 F2
screened males (Table 1). This results points out the excision rate around 19% for that
mutagenesis.
Table1- Counting of F2 male that lost P-element based in the colour of the eyes.
Eyes colour F2 males coloured eyes 615 white eyes 145 Total 760
2-Classificatation based in the lethality of the 145 F2 P-excision lines in Trans
with the tshA3-2-66 allele
For better classified the F2 white eyes males, we grouped in class according with their
lethality in Trans with the tshA3-2-66 allele. Several broad categories could be defined on the
basis of their lethality. We obtained lines lethal at different developmental stages and lines
that give viable adults escapers in Trans with the tshA3-2-66 allele (Table 2). The majority of P-
jump out lines (69,7%) were lethal in larvae stage and should corresponds to internal
deletions within the P-element (P*). Probably, these P internal deletions include, at least, the
partial elimination of the white+ eye colour marker.
Table 2- Classification of the P-element excision line based in their lethality in Trans-heterozygous with tshA3-
2-66.
Viability F2 males Dead 11
Sterile 4
Embryo stage 20
Larvae stage 101
Leth
al
Pupae stage 2
Escapers 7
Total 145
In order to obtain alleles with abnormal mitotic phenotype, we randomly picked eight
L3 lethal lines and we stained the DNA of their homozygous neuroblasts with acetic orcein (Fig.
2). No abnormal mitotic figures were observed indicating that apparently none of these
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RESULTS_PART I
analysed lines represent a new mitotic tsh allele. In addition, the no detection of new mitotic
tsh allele suggest that the mitotic defects observed in tshA3-2-66 are due to the presence of the
P{lacW A3-2-66} in the predicted region of the tsh promoter.
Figure 2- No mitotic anomalies were detected in neuroblasts from
8 P-element excision lines L3 lethal.
L3 neuroblasts from (A, A’) Wild-type; (B, B’) homozygous ∆P6; (C, C’)
homozygous ∆P97; (D, D’) homozygous ∆P118; (E, E’) homozygous ∆P124; (F, F’)
homozygous ∆P131; (G, G’) homozygous ∆P132; (H, H’) homozygous ∆P137 and
(I, I’) homozygous ∆P139 stained with acetic orcein. All these ∆P lines are L3
lethal in Trans with tshA3-2-66 but, no mitotic anomalies were detected. The scale
bar represents 10µm.
Figure 3- No mitotic anomalies were detected in neuroblasts from
P-element excision lines lethal during pupae stage.
L3 neuroblasts from (A, A’) Wild-type; (B, B’) homozygous ∆P48 and (C, C’)
homozygous ∆P127 stained with acetic orcein. Both ∆P lines are pupae lethal,
when in Trans with tshA3-2-66. No mitotic anomalies were detected. The scale bar
represents 10µm.
The L3 neuroblasts of the two P-element excisions lines lethal at the pupal stage (∆P48
and ∆P127 lines) were also stained with acetic orcein (Fig. 3). No abnormal mitotic figures
were observed for this class of lines, similar to the results obtained for the randomly studies L3
lethal lines.
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RESULTS_PART I
3- Preliminary oogenesis characterization of the ∆P48/tshA3-2-66 and the
∆P127/tshA3-2-66 ovaries
To eliminate the possible contribution of the genetic background of the chromosome 3
to the lethality at the pupal stage, the ∆P48 and the ∆P127 lines (balanced over T(2:3) CyO
TM6 Tb e) were re-balanced over CyL. From re-balanced lines, we obtained a few ∆P48/tshA3-2-
66 and ∆P127/tshA3-2-66 escapers, showing that the change in the genetic background allowed
the development of some trans-heterozygous flies until adult stage. The preliminary analyses
of the ∆P48 and the ∆P127 ovaries showed strong oogenesis defects (Fig.4). The ∆P48/tshA3-2-
66 and the ∆P127/tshA3-2-66 ovaries resemble a big ovarian tumor. The ovaries seem an amount
of cells, where it was difficult to delimitate the germarium, none cellular types (germline and
somatic cells) were distinguished and none individualised egg chamber was identified, not even
S1 stage in the germarium. This suggests that oogenesis is severely affected in these lines. In
wild-type, pupae ovaries already presented a functional germarium and 2 or 3 egg chambers
perfectly formed in the vitelarium. The phenotype displayed by the ovaries from ∆P48 and
∆P127 revertant lines, indicates a premature oogenesis arrest, perhaps during ovaries
formation in pupa stage.
Figure 4- Adult ∆P48/tshA3-2-66 and ∆P127/tshA3-2-66 ovaries showed a very strong and early arrest
in oogenesis.
Ovaries from (A) wild-type, (B) ∆P48/tshA3-2-66 and (C) ∆127P/tshA3-2-66 in which, the DNA was stained with
Hoechst 33258. No individualised egg chambers were observed in lines ∆P48 and ∆P127 indicating that oogenesis
arrests before S1 stage in theses germarium. The scale bar represents 50µm and anterior is to the right and posterior
is to the left.
Mutations in fused, female sterile and narrow genes are known to produce ovarian
tumours but not as generalized as in ∆48/tshA3-2-66 and ∆127/tshA3-2-66 females, where all the
ovaries display a tumor-like phenotype.
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RESULTS_PART I
4- Preliminary characterization of the ∆P28, ∆P30, ∆P99, ∆P104, ∆P123,
∆P126 and ∆P128 lines
4.1- Based in viability and male/ female fertility
The seven F2 male showing viable escapers (in Trans with the tshA3-2-66) were classified
according to their fertility (Table 3). The ∆P28/tshA3-2-66 male and female were fertile, and
probably corresponds to a precise excision of P{lacW}. The other six lines showed female
sterile, apparently due to imprecise excisions of P{lacW}. In addition the ∆P30/tshA3-2-66 and
∆P99/tshA3-2-66 males were also sterile. In all the seven lines homozygous are lethal.
Table 3- Fertility of the seven P-element excision lines with viable escapers, in Trans with the tshA3-2-66.
Lines Female Male ∆P28 Fertile Fertile
∆P30 Sterile Sterile
∆P99 Sterile Sterile
∆P104 Sterile Fertile
∆P123 Sterile Fertile
∆P126 Sterile Fertile
∆P128 Sterile Fertile
We tested the viability and fertility of the trans-heterozygous of the ∆P30, ∆P99,
∆P104, ∆P123, ∆P126 and ∆P128 lines combined between them (table4). Curiously, trans-
heterozygous females resultant from the combination of the ∆P99 with the other ∆P104,
∆P123, ∆P126 and ∆P128 lines were viable and sterile, while combinations between the last
other four lines, produce trans-heterozygous lethal before achieved the adult stage. (1) This
behaviour of the trans-heterozygous ∆P/∆P (table 4) and (2) the proximity of the timing when
the F2 male were obtained (illustrated by the proximity between the number of these lines)
make us suspect that ∆P30, ∆P99 revertant lines correspond to an event while, ∆P104, ∆P123,
∆P126 and ∆P128 revertants lines correspond to other independent event. In addition, the
preliminary ovaries phenotype characterization, no revealed significant differences between
ovaries from the 6 lines (30, 99, 104, 123, 126 and 128; data not show): all these female are
sterile: fail to lagg eggs and present atrophied ovaries (Fig.6E), where oogenesis is arrested
before S3 stage (for details see chapter II part II of this manuscript).
All the seven lines produce homozygous lethal. The existence of ∆P28/tshA3-2-66 viable
and fertile, suggested that the lethality observed in the homozygous of the revertant lines is
due to other mutation in chromosome 2, which occurred during P excision mutagenesis and
was not present in the initial chromosome 2 of the tshA3-2-66 allele.
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RESULTS_PART I
Table 4- Fertility of the progeny (female) resultant from the combination between the seven P-element
excision lines with viable escapers.
∆P30 ∆P99 ∆P104 ∆P123 ∆P126 ∆P128
∆P28 nd nd nd nd nd nd
∆P30 L nd nd nd nd nd
∆P99 nd L L L L nd
∆P104 nd VS L L L nd
∆P123 nd VS L L L L
∆P126 nd VS L L L L
∆P128 nd nd nd L L L
F= fertile; L= lethal; nd= not determined; S= sterile and V= viable
4.2- Confirmation of the P{lacW} excision by PCR (tested for ∆P126)
In order to confirm the P{lacW} excision from the ∆P126, we designed primers insight
the P{lacW}: the forward prime was designed in 5’P region and the three reversed primers
(5P-Rev2, 5P-Rev3 and PlacW_1) were designed downstream at 639bp (119bp downstream
5’P),at 811bp (290bp downstream 5’P) and at 1498bp (978bp downstream 5’P), respectively.
These regions were amplified in tshA3-2-66 but not in ∆P126, as expected, apparently indicating
the absent of the PlacW from the ∆P126 genome (Fig.5B).
Since, sometimes during the excision of a P element, this one could undergo some
rearrangements and then re-integrates the genome, we tested the presence of 5P and 3P
regions in ∆P126 genome, for note, these regions have to be intact to allow the P-element re-
integration.
A fragments of 217 bp within the 5’P and another of 211 bp within 3’P regions of the
P{lacW} was amplified by PCR. Unexpectedly, we obtained DNA amplification from the tshA3-2-
66 and the ∆P126 DNA with the pair primers designed into the 5’P and 3’P region of the
P{lacW} (Fig.5A, C and D, respectively). These fragments was then sequence and no
modification was detected comparatively to the reported in flybase database
(http://flybase.bio.indiana.edu; data not show). This suggests that, the ∆P126 allele conserved
in their genome intacts 5’P and 3’P sequences. This could be a result (1) of the duplication of
P-element extremities during an ancient P-element excision. (2) In other hand, ∆P126 could
have a residual P-element. This residual P-element could be the PlacW which, after excision
from tsh locus went through a rearrangement and, it was re-integrated in another region of
the ∆P126 genome. (3) Other hypothesis can be an insertion of other P-element (beyond the
PlacW) already present in tshA3-2-66 genome when the P-element excision mutagenesis was
performed. (4) Finally, since we only amplified 217 bp of the 5P regin and 211 bp from de 3P
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RESULTS_PART I
region, the 5P and 3P regions in ∆P126 could have a mutation in the non sequenced region,
indicating that this is a defective P-element. Only in this last case, we could exclude completely
the possibility of a PlacW re-insertion during P-excision mutagenesis.
Some of these assays were performed with DNA from other ∆P lines with the same
results. However, we choose to present here only the results obtained with ∆P126 DNA (the
justification for this choice is explained below in the item 5.2 of this part).
Figure 5- Amplified and sequenced regions of the PlacW in tshA3-2-66 and ∆P126.
(A and B) PlacW fragments amplified and migrated in 0,7% agarose gel at 100 volts. (M) 1Kb Ladder DNA marker;
water was used as a control.
(A) ∆P126 and tshA3-2-66 DNA amplification of 512 bp fragment of the 5 Prime region using the 5P-Fwd and the 5P-Rev1
primers pair.
(B) In the left of the marker (M), ∆P126 and tshA3-2-66 DNA amplification of the 639bp fragment (521 bp from 5P region
and 118bp of the contiguous downstream region) using the 5P-Fwd and the 5P-Rev2 primers pair. In the right of the
marker (M), ∆P126 and tshA3-2-66 DNA amplification of the 811bp fragment (521 bp from 5P region and 290 bp of the
contiguous downstream region) using the 5P-Fwd and the 5P-Rev3 primers pair. No amplification was obtained for
∆P126 DNA using the combination of the primer that amplified 5P region and the contiguous downstream PlacWA3-2-66
DNA.
(C and D) 5’P ends and 3’P ends fragments amplified by PCR and migrated in 1,5% agarose gel at 100 volts. (M) 100
bp Ladder DNA marker; for control was used water.
(C) ∆P126 and tshA3-2-66 DNA amplification of 217 bp of the 5 Prime region using the 5P-Fwd and the 5P-Rev primers
pair;
(D) ∆P126 and tshA3-2-66 DNA amplification of 211 bp of the 3’P ends region using the 3P-Fwd and the 3P-Rev primers
pair. Both regions were amplified with ∆P126 and tshA3-2-66 DNA.
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RESULTS_PART I
4.3-Sequence of the upstream region contiguous to the tsh mRNA and allelism
tests (tested in ∆P99, ∆P104, ∆P123 and ∆P126)
We sequenced and analysed 3913bp upstream of the translation start of tsh in ∆P99,
∆P104, ∆P123 and ∆P126 lines for comparison with the tsh sequence published in FlyBase
(http://flybase.bio.indiana.edu) and the genomic sequence that surrounds the insertion site of
other transposable elements inserted in the same region (P{RS3}CB-5359-3; P{XP}cl00773;
P{lacW}l(2)k16406; P{XP}cl05049). We were unable to identify any mutation. Instead, we
identified a 6 nucleotide sequence (AACTGC) located 1297bp upstream of the translation start
site, which is absent from the current release of the Drosophila melanogaster genome but
present in all sequences that we analyzed (Supplementary Fig. 1).
In the absence of a mutation identified, we cross the ∆P126 with some reported tsh
alleles in order to found a combination that give a sterile female phenotype, resemble the
trans-heterozygous ∆P126/tshA3-2-66 (table 5). To simplify, we only presented the allelism test
performed with the ∆P126 line, but the results was the same for the other ∆P lines.
Unfortunately, behind the tshA3-2-66, all other combinations with ∆P126 give viable and fertile
adult, disabling us to demonstrate that ∆P126 is a new tsh allele.
Table 5- List of the tsh alleles crossed with ∆P126 and tshA3-2-66 to test for the viability and fertility of the
adult female.
Alelle Characterization Viability/Fertility in Trans with:
References
Bloomington stock
symbol P-element insertion ∆P126 tshA3-2-66
167 Df(2L)TW161 nd L
Df(2L)305 VF L induced by B. Wakimoto
tsh 8 --- VF L Fasano et al., 1991
tsh 2757 R7 --- VF L Andrew et al., 1994
tsh NG1 --- VF L Salazar and Gomes, 1999
10842 tshA3-2-66 1652 bp upstream ATG VS L
1018 P{A92}tsh[i71] VF VF
1296 P{WA[R]}tsh[4-3] VF VF
3040 P{GAWb}tsh[md621] VF VF
11178 P{lacw} l(2)K16406[K16406] 1317 bp upstream ATG VF nd
11370 P{PZ}tsh[04319] 720 bp upstream ATG VF nd
F= fertile; L= lethal; nd= not determined; S= sterile and V= viable
Our sequence data and allelism results, doesn’t allow us to confirm that ∆P99, ∆P104,
∆P123 or ∆P126 are a new tsh allele, however, in the Trans with tshA3-2-66, we observed other
morphologic defects that could be co-related with a diminution of Tsh.
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RESULTS_PART I
5- Other morphologic defects observed in ∆P30/tshA3-2-66, ∆P99/tshA3-2-66,
∆P104/tshA3-2-66, ∆P123/tshA3-2-66, ∆P126/tshA3-2-66 and ∆P128/tshA3-2-66 flies co-
related with a diminution of Tsh expression
5.1-Wings defects
All Trans-heterozygous viable female for ∆PlacW/tshA3-2-66 behind the oogenesis defects
show difficulties in to move their wings. In addition, their wings were smaller than wild-type,
and sometimes it was possible to observe ectopic veins (Fig.6). This wing phenotype could be
related with some tsh deregulation. For note, one of the reported tsh loss of function
mutations cause an abnormal wing posture phenotype called aeroplane characterized by a
wings paralysis among other defects (Soanes and Bell, 1999 ; Soanes et al., 2001; Soanes et
Bell, 2001).
Figure 6- Morphologic aspects of
DP126/ tshA3-2-66 flies.
(A) Wild-type female Oregon-R with red eyes
(arrowhead) and ∆P/ tshA3-2-66 female with orange
eyes (arrow) due to the presence of the white
background on the X-chromosome and a extra wild-
type copy w+ on the second chromosome (tshA3-2-66
allele). (B) Drosophila eyes from W;tshA3-2-66/CyO
(left side) and W;∆P126/tshA3-2-66∆126 (right side)
adults. ∆P126/tshA3-2-66 have white eyes, despite the
presence of one copy of the P{lacW}A3-3-66
responsible for the orange eyes phenotype of the
W;tshA3-2-66/CyO female.
(C) Wing from wild-type female and (D) wing from
∆P126/tshA3-2-66 female. The dotted in (C) represents
the contours of the wild-type wing that doesn’t
coincide with the contours of the ∆P126/ tshA3-2-66
wing. ∆P126/ tshA3-2-66 wings are a little smaller and
showed sometimes ectopic veins (arrow in panel D).
(E) wild-type ovaries and (F) ∆P126/ tshA3-2-66
ovaries from female with the same age and grow at
the same temperature. The ∆P126/tshA3-2-66 ovaries
are atrophied and smaller than wild-type.
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RESULTS_PART I
5.2-Reduction in eyes colour patterning
In the sterile female line, white was expressed as previously reported for other P{lacW}
inserted in the tsh locus (Fig. 6B; Sun et al., 1995; Bhojwani et al., 1995; FL unpublished
data). The tshA3-2-66 adults show the anterior half of the eye coloured, reporting the tsh
expression eye domain, in virtue of the presence of the mini-white in the promoter of the tsh
gene. The carefully observation of the ∆P/tshA3-2-66 adults eyes revealed a slight reduction in
the coloured eye domain, suggesting that tsh expression is reduced in the eyes of these
mutants (Fig. 6A).
During the analyses of the tsh alellism, and in order to eliminated the possible genetic
interaction between tsh gene and the CyO (Curly of oyster) balancer, the tshA3-2-66 and the
∆P126 chromosomes were balanced with the Cy L (curly lobe) balancer. From the cross
between w;tshA3-2-66/CyL female and the w; ∆P126 /CyL male we obtained 2/3 of the w; ∆P126/
tshA3-2-66 adults with white eyes, indicating that ∆P126 modifies the patterned white expression
observed in the tshA3-2-66 adult eyes (Fig. 6B). As the expression patterns of white may reflect
the activity of regulatory elements belonging to the tsh gene, we hypothesised that the ∆P126
chromosome could bare a mutation in the tsh locus even if we were incapable of identify it
(Sun et al., 1995; Bhojwani et al., 1995; FL unpublished data).
In conclusion, we fail to clearly prove that we isolated a new tsh allele (∆P126), but we
can argue that ∆P126/ tshA3-2-66 display a phenotype co-related with a diminution of Tsh
suggesting that Tsh expression is affected in this revertent line. These observations, prompted
us to investigate whether the w; ∆P126/ tshA3-2-66 female sterile phenotype could be associated
to an altered expression of tsh in the ovaries (for details see chapter II part II of this
manuscript). Subsequently, the ∆P126 line will be referred as ∆126.
For note, the mutagenesis data display in part II of this chapter included the results
described above in this mutagenesis chapter and the results obtained during a independent
mutagenesis performed by Salazar (Salazar, 2000) in order to ensure that the lethality of the
tshA3-2-66 allele was directly attributable to the P{lacW}A3-3-66 insertion (for resume see
supplementary table 1).
89
Supplementary Data
Supplementary table 1- PlacW excision results obtained in two independents P-excision mutagenesis from
the tshA-3-2-66 allele by Salazar (2000, refered in chapter IV of the results of this manuscript) and by Pimentel
(presented in this chapter).
Salazar data Pimentel data Total
Males F2 analysed 1622 760 2382
Revertant line with escapers 10 7 17
Escapers viable 6 1 7
Escapers with sterile female 4 6 10
RESULTS_Part II:
RESULTS_PART II
92
Regulation of cell proliferation and patterning in Drosophila
oogenesis by Hedgehog signalling depends on teashirt
Silvia Pimentel1,2, Isabel Salazar1, Christine Vola2, Rui Gomes 1 and Laurent Fasano2†
1 Universidade de Lisboa, Faculdade de Ciências, Departamento de Biologia Vegetal,
Campo Grande, 1749-016 Lisboa, Portugal 2 Institut de Biologie du Développement de Marseille Luminy (IBDML), UMR 6216, CNRS-
Université de la Méditerranée, Campus de Luminy, Case 907, 13288 Marseille Cedex 09,
France.
†Authors for correspondence: (emails: <[email protected]>)
RESULTS_PART II
Abstract
Interaction between intrinsic factors with extrinsic signals plays a central role to control
stem cell self-renewal, proliferation and differentiation. In Drosophila ovaries, Hedgehog (Hh)
signalling is essential for self-renewal and proliferation of follicle stem cells (FSC). Here, we
show that Teashirt (Tsh) is co-expressed with Hh at the tip of Drosophila germarium, in
terminal filament cells (TFs) and in cap cells (CC). The latter are a subset of hh-expressing
cells located adjacently to germline stem cells (GSC) and are described as a niche for GSC and
FSC. We demonstrated that overexpression of tsh mimics the phenotype associated with hh
overexpression. Phenotypic characterisation of ovaries into which tsh expression was perturbed
revealed an early oogenesis arrest (stage S2/S3 of oogenesis) and a perturbed expression of
hh and engrailed (en). In these mutant ovaries, ectopically provided Tsh partially rescued the
defects and restored hh and en expression. Here we explore for the first time the expression
and the function of tsh during oogenesis. Our data suggest that in adult ovaries tsh expression
might be required to regulate hh expression, contributing for the regulation of FSC proliferation
and the specification of somatic cell identity.
Key words: Drosophila, oogenesis, follicle stem cells, follicle cells, teashirt, hedgehog
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RESULTS_PART II
Introduction
Drosophila oogenesis represents an excellent model system for studying how
proliferation and patterning of both germline and somatic cell lineages are controlled by
signalling pathways. It produces ovarian follicle continually throughout adult life and it involves
a high degree of coordination between the proliferation and differentiation programs of two
lineages cells, soma and germ line (Narbonne-Reveau et al., 2006).
Each of the two ovaries in adult fly typically contains 16 ovarioles. Eggs chambers are
made up within the germarium, a distinct anterior region of every ovariole. Egg chambers
leave the germarium and mature as they move posteriorward within the ovariole. They
develop through successive stages to form a polarized oocyte, surrounded by a patterned
epithelium of somatic follicle cells (Zhang and Kalderon, 2000; Lin and Spradling, 1993).
In the germarium, three types of stem cells have been identified: the germline stem
cells (GSC), the follicle stem cells (FSC) and the escort stem cells (ESCs) (Fig. 1A) (Decotto
and Spradling, 2005; Lin and Spradling, 1993; Margolis and Spradling 1995; Wieschaus and
Szabad, 1979). The apical portion of the germarium (region 1) contains 2-3 GSC identified by
clonal analysis (Wieschaus and Szabad, 1979) and laser ablation studies (Lin and Spradling,
1993). At the anterior end of the germarium, the terminal filament cells (TFs) and the cap cells
(CC), which are both none dividing somatic cells, support the GSC niche. The CC directly
contact with GSC by adherent junctions enriched in DE-cadherin and Armadillo (β-catenin)
(Song et al., 2002). CC signals for stem cell (SC) maintenance, regulation of proliferation and
differentiation of the SC progeny (Xie and Spradling, 1998; Song et al. 2004; Cox et al.,
2000). GSC divide asymmetrically every 20 hours to produce a SC and a daughter cystoblast.
The differentiating cystoblast undergoes four rounds of mitosis with incomplete cytokinesis, to
generate a 16-cell cyst (kai et al., 2005). One of the 16 cyst cells becomes the oocyte while
the other 15 cells differentiate as nurse cells (reviewed in King, 1970). The escort stem cells
(ESCs) were identified as the most anterior cells until that time known by inner germarial
sheath (IGS) cells that directly contacts CC and surrounds GSC with their cellular processes
(Decotto and Spradling, 2005). Their progeny, the squamous escort cells (ES) are responsible
to enclose the cystoblasts in a thin covering and help him to move posterior (Fig. 1A). At the
junction between region 2a and region 2b, cysts are forced into single file, lose their escort cell
covering, which by turn enter in apoptosis, and begin to acquire a follicular layer.
In the region 2a/2b, two somatic follicle stem cells (FSC) divide, in average every 10
hours, to give rise to all polar cells, stalk cells and main body cells needed to form the
epithelial cell-like follicle cells (FC) that cover up each cyst and mold them into a “lens shape”
characteristic of region 2b. Under the influence of continued somatic cell growth, cysts and
their surrounding cells round up, enter in region 3 (also known as stage 1), and bud off from
the germarium to form individual egg chambers connected to their neighbours by 5-7 stalk
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RESULTS_PART II
cells (Kai et al., 2005; Nystul and Spradling, 2010). Before leaving the germarium, FC undergo
approximately five rounds of mitotic division. Three to four mitotic cycles later, in stage 6,
division cease following three endocycles (from stage 7-10) to generate a follicle’s monolayer
of around 650 polyploid FC comprising multiple subtypes (Nystul and Spradling, 2010)
Previously, clonal analysis has revealed that the FC are already divided into two
populations in the germarium (Besse and Pret, 2003). One lineage gives rise to the stalk cells
and the polar cells, which are a pair of follicle cells at each end of the egg chamber that
connects to the stalk (Tworoger et al., 1999; Lopez-Schier and Johnston, 2001). The rest of
the follicle cells derive from a separate lineage and form the epithelium around the cyst; they
are called epithelial follicle cells or main-body. Nystul and Spradling (2010) by examining
clones induced at low frequency, shown recently that FC types specification is independent of
cell lineage. Instead, FC are multipotent prior to polar or stalk specification. This fits well with
recent studies showing that many additional cells in the germarium can be induced to take on
a polar cell fate by strong Notch signalling, while high levels of JAK-STAT signalling can induce
more stalk cells (Assa-Kunik et al. 2007). In addition, they map the specification of the first
anterior and posterior polar cells, when cysts are associated with 8-16 FC (in mid to late region
2b), what is in agreement with previous studies (Margolis and Spradling, 1995; Besse and
Pret, 2003) which found that polar cells were first specified at the 14-cell stage.
The two distinct populations of stem cells, FSC and GSC, work in a coordinated fashion
to efficiently produce egg chambers and share some molecular mechanisms to control self-
renewal and proliferation (Song and Xie, 2002). Despite these similarities, it is worth noting
that the organization of the FSC niche is different from that of the GSC niche. The FSC niche
includes not only neighbouring posterior EC but also TF cells and CC located over a few cells
away. As a consequence, proliferation and self-renewal of GSC and FSC depend on different
combination of niche signals, but both depend on Hedgehog (Hh) signalling (for a review see
Kirilly and Xie, 2007).
In adult Drosophila ovaries, Hh is detected in TF cells and CC (Fig. 1C). Hh functions as
a long-range signal to control FSC self-renewal, proliferation and budding of egg chambers
(Forbes et al., 1996a; Forbes et al., 1996b; King et al., 2001; Zhang and Kalderon, 2001). Hh
signalling is essential for self-renewal and proliferation of the FSC, while it plays a non-
essential role in maintaining the GSC, by having redundant function with the piwi–mediated
pathway (King et al., 2001). Hh acts by binding to its receptor, Patched (Ptc), a
transmembrane protein that normally acts to restrict the activity of Smoothened (Smo), a
seven-transmenbrane domain protein (Hooper and Scott, 2005). Upon binding of Hh to Ptc,
Smo becomes active, ultimately leading to the activation of the transcription factor, Cubitus
interruptus (Ci), which in turn regulates the expression of Hh target genes (Ingham, 1998;
Aza-Blanc and Kornberg, 1999). Disruption of the Hh signalling results in rapid FSC loss and
gives rise to compound egg chambers that contain multiple germline cysts in a single follicle
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RESULTS_PART II
epithelium (King et al., 2001; Zhang and Kalderon, 2001). Hyperactivation of Hh signalling
increases the number of FSC, causes the overproduction of somatic FC that accumulates
between egg chambers, induces mis-positioning of oocyte within egg chambers and generates
ectopic polar cells (Forbes et al., 1996a; Forbes et al., 1996b; Liu and Montell, 1999; Zhang
and kalderon, 2000). It is therefore postulated that Hh signalling in the germarium regulates
somatic FC proliferation and contributes to oocyte positioning.
The Teashirt (tsh) gene was first identified in Drosophila as a region-specific homeotic
gene specifying embryonic trunk identity (Fasano et al 1991; Röder et al 1992; Andrew et al
1994; Taghli-Lamallem et al 2007). The zinc-finger protein Teashirt (Tsh) was also
characterized as one of the factors necessary for Hh target gene regulation and biochemical
approaches suggested that Tsh and Ci interact directly in protein complexes (Gallet et al.,
2000; Angelats et al., 2002). Based on sequence homology, mouse tsh-like genes (Tshz1-3)
were identified and, their specific expression patterns raised the possibility that they may play
developmental roles in mammals (Caubit et al. 2000 and 2005). Indeed, each substitutes for
fly tsh in genetic rescue experiments (Manfroid et al 2004). Recently, we reported that Tshz3
plays a key role for ureteral smooth muscle differentiation downstream of Sonic Hedgehog
(Caubit et al., 2008).
In this study, we characterize for the first time the expression of tsh during oogenesis
and our data suggest that the expression of tsh might be necessary for controlling hh
expression in ovary and, as a consequence, for controlling the follicle stem cells proliferation.
Materials and Methods
Genetics
The enhancer trap tsh1 (Fasano et al., 1991) was used to perform X-Gal staining.
The lethal P{lacW} insertion allele tshA3-2-66 (P{lacW}A3-3-66) was identified after the
screening of the Bloomington P-element insertions at the 38A6-40B1 cytogenetic region. The
P{lacW}A3-3-66 insertion was mapped 1652 base pairs (bp) upstream the translation start of the
teashirt (tsh) gene. Northern blot analysis revealed that tshA2-3-66 causes a significant reduction
on the tsh transcript levels (data not shown). We found that tshA3-2-66/tsh8 heterozygotes were
lethal, tshA3-2-66/tshA3-2-66 all dye at pupal stage and tshA3-2-66/Df(2L)TW161 also dye at pupal
stage with very few escapers.
We performed a P-element excision mutagenesis from the tshA3-2-66 allele. The
endogenous white locus was the w1118 allele. Precise and imprecise excisions (E{P}) of the
transposon were induced in the presence of the Sb P[ry+ 2-3] source of transposase
(Robertson, et al., 1988). E{P}/CyO P-element-excised revertants can be distinguished by
their white eye color and were tested with the original P{lacW}A3-3-66mutant lines. Analysis of
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RESULTS_PART II
2382 males allowed us to identify 17 lines that gave escapers when in Trans with tshA3-2-66: out
of 17 revertants, seven were fully viable and fertile and the other ten were also viable but
female sterile (two were male sterile).
Absence of re-integration of the P-element was confirmed by PCR with specific primers
designed for the 5’P of the P{lacW}; tshA3-2-66 was used as a positive control for the PCR
conditions.
The revertant line ∆126 and the tshA3-2-66 allele were balanced over T(2;3)CyOTM6B,Tb.
Females (or males) tshA3-2-66/T(2;3)CyOTM6B,Tb were crossed with male (or females)
∆126/T(2;3)CyOTM6B,Tb to get ∆126/tshA3-2-66 females used in this study.
All genetic markers and balancers are described by Lindsley and Zimm (1992) and
FlyBase (http://flybase.bio.indiana.edu) except as noted.
Rescue of ∆126 phenotype
For rescue and overexpression experiments the constructs UAS-tsh13 (Gallet et al.,
1998) and UAS-hh-M4 (Ingham and Fietz, 1995) were crossed to the driver P{w+mC=GAL4-
Hsp70.PB}TR2 (Bloomington).
DNA sequencing of the ∆126 revertant
Genomic DNA was isolated from L3 larvae homozygous for ∆126 allele after treatment
with a phenol/chloroform/isoamyl alcohol mixture to remove protein contaminants and
precipitation with 100% ethanol. The DNA was re-suspended in water for further
experimentation.
We sequenced and analysed 3913bp upstream of the translation start of tsh in ∆126
revertant. This region was amplified by PCR and fragments were purified and sent for
sequencing (GenomExpress). The following PCR primers were used:
5’CACAAACATTGACACCTTGCC3’;5’GACTACGCCGAACCGCCAA3’;5’CCTTCTTTTGTCCTATTCCG3’;
5’GCCAAATCTGAGCCTTAGAC3’;5’CCCAGCACCTCTTCTTTAGG3’;5’CGTCACCCTTCTTTTGTCC3’;5’
GCAGCCAAAAAGAAAAGTGG3’;5’TCGTCGTCACTACTCGAACG3’;5’GTATGGTTAAATATTGTATATGC
AA3’;5’CAAAATTTAGTTGCGAGGAGT3’;5’CGTTGTTGTTGTTTGTCAGC3’ and
5’TTAGTTGGATTTTGGAACTTAAC. Comparison of the ∆126 sequence with tsh sequence
published in FlyBase (http://flybase.bio.indiana.edu) and the genomic sequence that surrounds
the insertion site of other transposable elements inserted in the same region (P{RS3}CB-
5359-3; P{XP}cl00773; P{lacW}l(2)k16406; P{XP}cl05049) did not allowed us to identify any
mutation. Instead, we identified a 6 nucleotide sequence (AACTGC) located 1297bp upstream
of the translation start site, which is absent from the current release of the Drosophila
melanogaster genome but present in all sequences we analyzed.
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RESULTS_PART II
Staining ovaries
β-galactosidase activity staining was performed as described previously (Fasano and
Kerridge, 1988), but ovaries were fixed with 2.5% Formaldehyde in PBS for 10 minutes. X-Gal
stained ovaries were analysed under an Axiophot Zeiss microscope.
Antibody staining was performed based on the protocol described by Zhang and
Kalderon (2000), with the following modifications: ovaries were fixed in 1:3 heptane: 10%
DMSO and 4% paraformaldehyde in PBS for 20 minutes. The ovaries were blocked in TNB
(blocking reagent from the Tyramide signal amplification kit (PerkinElmer) for 1hour then
incubated with the primary antibody at 4°C, overnight.
Primary and secondary antibodies and their dilutions were as follows: anti-Tsh (rabbit
Tsh, a gift from S Cohen, EMBL Heidelberg, 1:1000 dilution); mouse 4-D9 anti-Engrailed
invected (Developmental Studies Hybridome bank, 1:10); anti-Hh (rabbit anti-serum against
Hh, a gift from A. Gallet, Nice University, France, 1:200); mouse anti-Arm (Developmental
Studies Hybridome bank, 1:5);mouse 7G10 anti-Fasciclin III (Developmental Studies
Hybridome bank, 1:200); rabbit anti-Vasa (a gift from R. Lehmann 1:1000); rabbit anti-
phospho-Histone H3 (Upstate Biotech, 1:500); rat anti-BrdU (abcam, 1:100); and rabbit anti-
Caspase 3 (Cell Signalling Tecnology; 1:200) Secondary are conjugated with Texas red and
FITC (Molecular Probes, 1:500).
For En and Hh staining, the signal was amplified with the Tyramide Signal Amplification
Kit (PerkinElmer) using biotinylated secondary antibodies (Jackson Laboratories, 1:500). In
these cases, the ovaries were submitted to a peroxidase treatment (3% H2O2 for 10 minutes)
to reduce the background before their blocking.
Stained ovaries were mounted in Vectashield medium with DAPI (Vectors) and analyzed
under a LSM 510 Zeiss confocal microscope.
BrdU incorporation
Follicle cells were labelled with BrdU, as described previously (Lilly and Spradling, 1996;
Shcherbata et al., 2004) with slight modifications. Ovaries from 7 days old female were
dissected in Grace’s insect medium and then incubated with 10µm BrdU (Upstate Biotech) in
the same medium for 15 minutes. To stop BrdU incorporation, ovaries were washed twice with
fresh Grace’s insect medium and then maintained in these medium for 90 minutes or 270
minutes at room temperature. The ovaries were then fixed in 3.7% Formaldehyde during 20
minutes, washed with PBST (PBS with 0.1% Triton X-100), then treated with 2N HCl for 30
minutes. Neutralization was performed in 100mM Borax for 2 minutes. Tissues were rinsed
with PBST three times (10 minutes), blocked and stained as described above.
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RESULTS_PART II
Rescue of ∆126/ tshA-3-2-66 mutant phenotype
∆126; UAS-tsh13/T(2;3)CyOTM6B,Tb or ∆126; UAS-hh-M4/T(2;3)CyOTM6B,Tb males
were crossed to tshA-3-2-66; hsGal4/T(2;3)CyOTM6B,Tb females. In the day of the eclosion,
hsGal4>UAS-tsh13; ∆126/ tshA-3-2-66 or hsGal4>UAS-hh-M4; ∆126/ tshA-3-2-66 females were
submitted to a heat-shock: tubes were immersed in a water bath at 37°C, for 1hour, twice a
day, in 3 consecutive days. The 4th day, ovaries were dissected and stained with FasIII and
Vasa antibodies. Females were grown at 18°C or at 25°C, until the adult stage and during the
period between heat-shocks.
Tsh and Hh overexpression
UAS-tsh13/T(2;3)CyOTM6B,Tb or UAS-hh-M4/T(2;3)CyOTM6B,Tb males were crossed
to hsGal4/T(2;3)CyOTM6B,Tb females. In the day of the eclosion, hsGal4>UAS-tsh13 or
hsGal4>UAS-hh-M4 females were heat-shocked at 37°C, for 1hour, twice a day, in 3
consecutive days. In the 4th day, ovaries were dissected and stained with FasIII and Vasa or,
Tsh and En antibodies. Females were grown at 25°C, until adult stage and between heat-
shocks.
Results
1-Tsh is expressed in Escort cells and co-expressed with En in Terminal
Filament cells and Cap cells
To analyse expression of the tsh gene in ovaries, we first took advantage of the
enhancer trap line tsh-LacZ, which was shown to faithfully reproduce the pattern of the
endogenous tsh gene in embryos and imaginal discs (Fasano et al., 1991). ß-galactosidase (ß-
gal) activity was detected in terminal filament (TF) and cap cells (CC) at the tip of the adult
germarium (Fig. 1B). Thus, in adult ovary, Tsh expression pattern resembles expression
patterns previously reported for Hh (Fig. 1D; Forbes et al., 1996a). To better characterise the
Tshz-positive cell population, we next performed double immunostaining for Tsh and Engrailed
(En). Expression of the Tsh protein was confirmed in the nuclei of TF and CC where it
colocalizes with En (Fig. 1D-D’). Unlike En, Tsh was also strongly detected in a group of
somatic cells, in region 1, 2b, and in the boundary of the 2a/2b region of the adult germarium
(Fig. 1D-D’), The location and the morphology of theses cells let us think that they are
probably the escort cells (EC) and the posterior EC (Fig. 1D arrow), respectively, which
participate in FSC niche (Kirilly and Xie, 2007). In addition, low level of Tsh was observed in
recently produced FC that surround germline in region 3 of the germarium (stage S1) and,
sometimes, this weak staining was still detectable in FC belonging to S2 egg chambers. Tsh
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RESULTS_PART II
was never observed in germline cells. As flies age, Tsh staining becomes weaker in the apical
half of the TF, but remains high in more proximal TF cells and CC, especially in flies older than
9 days. This fading coincides with the condensation of the CC and some proximal TF cells into
a cluster within the germarium tip (data not shown).
Figure 1- en co-localises with a tsh-
subset domain in Drosophila ovaries.
(A) Diagram of the Drosophila germarium and the
location of stem cells (in Kirilly and Xie, 2007). The
four sub-regions of the germarium are indicated
below the diagram. (B) X-Gal staining (blue) of tsh1
adult ovariole. (B’) Close up of the anterior end of
the germarium shown in B.
Germarium of late pupae (C and C’), and 7-day old
adult (D and D’) stained for Tsh (green), En (red)
and nuclei (DAPI, blue). (C and C’) Tsh positive and
En negative CC are observed in germaria from late
pupae (arrow). (D) In adult, Tsh is strongly detected
in the terminal filament cells (TF), the cap cells (CC;
arrowhead), the escort cells (EC) including escort
stem cells (ESC) and the posterior EC (arrow). (C’
and D’) Staining for Tsh, in the same confocal plane
as in C and D, respectively. In all panels, anterior is
to the left. In B and C, the scale bars represent
20µm and is the same in panels C-D’. CB, cystoblast;
CC, cap cell; DC, 16-cell developing cyst; ESC, escort
stem cell; FC, follicle cell; GSC, germline stem cell;
IGS (EC), inner germarial sheath cell, or escort cell;
SS, spectrosome; SSCs, somatic follicle stem cell,
(also know by FSC).
To investigate the onset of tsh expression in the germarium, late pupal gonads were
double stained with anti-En and anti-Tsh. As in the adult ovaries, Tsh and En were co-
expressed in TF and in CC precursors (Fig. 1C-C’) but Tsh was absent from the EC and newly
born FC. The presence of Tsh positive and En negative CC precursors suggests that Tsh
expression precedes En expression (Fig. 1C-C’).
2- teashirt expression is perturbed in P{lacW}A3-3-66∆126/tshA-3-2-66 ovaries
The cytogenetic location of tsh (40A) has precluded the analysis of tsh function during
oogenesis by germline clone analysis. To analyze the function of tsh in the adult ovary we
sought after female sterile alleles of tsh. To this aim, we first screened for P-element insertions
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RESULTS_PART II
at the 38A6-40B1 interval that fail to complement the deficiencies Df(2L)TW161, Df(2L)305
and the tshNG1 and tsh8 alleles (Salazar and Gomes, 1999; GR unpublished data). Based on this
criteria, we selected the lethal P{lacW}A3-3-66 (tshA3-2-66). Moreover, in this line white was
expressed as previously reported for other P{lacW} inserted in the tsh locus (Fig. 2A) (Sun et
al., 1995; Bhojwani et al., 1995; FL unpublished data).
Figure 2- Expression level variation
of tsh in ∆126/tshA3-2-66 ovarioles.
(A) Drosophila eyes from W;tshA3-2-66/CyO (left
side) and W;∆126/tshA3-2-66∆126 (right side)
adults. ∆126/tshA3-2-66 have white eyes, despite
the presence of one copy of the P{lacW}A3-3-66
responsible for the orange eyes phenotype of
the W;tshA3-2-66/CyO female.
(B-D') ovaries were stained for Tsh (green) and
nuclei (DAPI, blue). (B, C and D) confocal plane
and (B’, C’ and D’) projection of a confocal
image stack. In all panels, anterior is to the left.
The scale bar (in panels B) represents 20µm
and is the same for all panels.
(E) Relative abundance of the three classes of
Tsh staining patterns in trans-heterozygous
∆126/tshA3-2-66 (n=260 ovarioles) and wild-type
ovarioles (n=315 ovarioles) from three to seven
days old females grown at 25°C.
To ensure that the lethality was directly attributable to the P{lacW}A3-3-66 insertion, P-
element excisions (E{P}) of the transposon were induced by the ∆2-3 transposase. Among
2382 white-eye E{P}-jump-out lines (P{lacW}A3-3-66∆), 17 were viable in Trans with tshA3-2-66,
indicating that no second lethality exists in the tshA3-2-66 line. Out of 17 revertants, seven were
fully viable and fertile and the other ten were also viable but female sterile (two were male
sterile). Among these ten lines, we decided to further characterize the P{lacW}A3-3-66∆ (∆126)
line because 2/3 of the ∆126/tshA3-2-66 adults have white eyes, indicating that ∆126 modifies
the eye patterned white expression observed in the tshA3-2-66 adult eyes (Fig. 2A). As the
expression patterns of white may reflect the activity of regulatory elements belonging to the
tsh gene, we hypothesised that the ∆126 chromosome could bare a mutation in the tsh locus
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RESULTS_PART II
(Sun et al., 1995; Bhojwani et al., 1995; FL unpublished data). This observation, prompted us
to investigate whether the ∆126/tshA3-2-66 female sterile phenotype could be associated to an
altered expression of tsh in the ovaries.
To this aim, we analyzed the expression of Tsh in ∆126/tshA-3-2-66 ovaries (Fig. 2B-E).
We observed that Tsh was abnormal in 75% of the ovaries (Fig. 2E). Analysis of the variability
in Tsh staining allowed us to distinguish three types of patterns (type I, II and III) (Fig. 2E).
25,4% of ∆126/tshA-3-2-66 ovarioles presented an apparent wild-type distribution of Tsh in TF,
CC, EC and in newly born FC (type I, Fig. 2B-B’). In 50,0% of ∆126/tshA-3-2-66 ovarioles Tsh
was mostly detected in the TF and CC (type II; Fig. 2C-C’). Type III corresponds to 24,6% of
the ovarioles into which Tsh was absent from the germarium (Fig. 2D-D’).
Table 1- List of the tsh alleles crossed with P{lacW}A3-3-66∆126 and tshA3-2-66 to test for the viability and fertility
of the adult female.
Alelle Characterization Viability/Fertility in Trans with:
References
Bloomington stock
symbol P-element insertion
∆126 tshA3-2-66
Df(2L)tsh 8 VF L Fasano et al., 1991
tshNG1 VF L Salazar & Gomes, 1999
167 Df(2L)TW161 nd L
Df(2L)305 nd L induced by B. Wakimoto
10842 tshA3-2-66 1652 bp upstream ATG VS L
F= fertile; L= lethal; nd= not determined; S= sterile and V= viable
∆126 is a homozygous lethal mutation and a PCR-based analysis failed to reveal that
the P{lacW}A3-3-66 element reinserted. The allelism to tsh was tested by crossing ∆126 with tsh
alleles (Table 1) and we performed a sequence analysis of about 4 kbp upstream of the
translation start of tsh from the ∆126 chromosome; both analysis failed to validate that ∆126
is a new tsh allele and future experiments may help to answer this question. Nevertheless, the
∆126/tshA-3-2-66 females gave us the opportunity to investigate for the first time the
consequence of the perturbed expression of tsh on ovaries development.
3- Wrapping of the germ line cysts by the somatic cells is defective in
P{lacW}A3-3-66∆126/tshA-3-2-66 ovaries
To analyze the function of tsh in adult ovary, we used ∆126/tshA-3-2-66 trans-
heterozygous sterile females. ∆126/tshA-3-2-66 trans-heterozygous females didn’t lag eggs and
show atrophied ovaries.
Ovaries were stained for Vasa and Fasciclin III (FasIII), which respectively mark the
germline and follicular cells (Fig. 3; Ruohola et al., 1991). In wild-type ovaries, FasIII stained
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RESULTS_PART II
all the FC between the 16 cell cysts that leave the germarium and becomes restricted to the
polar cells around stage 3 (Fig. 3A).
Figure 3- ∆126/ tshA3-2-66 ovaries
display abnormal germline cyst
encapsulation.
Ovarioles from wild-type (A, B) and ∆126/tshA3-
2-66 flies (C-F) stained for FasIII (membrane of
the follicle cells; green), Vasa (germline; red)
and nuclei (DAPI, blue). (A) In wild-type
ovaries, FasIII becomes restricted to the polar
cells around stage 3 (arrows). (B) germarium
shown in panel A. (C) in ∆126/tshA3-2-66ovaries
oogenesis is arrested in S2/S3 stage. (D)
∆126/tshA3-2-66 swollen germaria showing
aberrant germline accumulation in region 1-2b.
Arrows indicate polyploid nurse cells typical of a
later stage. (E) Compound egg chambers with
30 nurse cells and two oocytes suggesting that
the follicle cells surrounded two independent 16
cell-cysts. In this confocal plane, the possible
position of each oocyte is indicated (O1 and O2).
(E’) 30 nurse cells (numbered from1-30) were
counted in a projection of a confocal image
stack of the same egg chamber shown in E. (F)
Example of a fused egg chamber. An egg
chamber has failed to bud off the germarium
and remained connected to the next one. Notice
the absence of stalk cells.
The three regions of the germarium are
indicated in panels B and D. In all panels,
anterior is to the left. The bar (in panels A, B, E,
E’ and F) represents 20µm. The panels A and C
and, B and D are at the same scale. GSC,
germline stem cell; FC, follicle cell.
Analysis of ∆126/tshA-3-2-66 ovaries revealed that the level of FasIII remains abnormally
high in mutant follicle cells (Fig. 3C). In the vitelarium, the follicle cells that packing the egg
chambers resemble the wild-type precursor for this population in the S1 stage, based in the
FasIII contents. Moreover, in ∆126/tshA-3-2-66 ovaries no polar cells were identified, the apical
localisation of the main body nucleus was perturbed and the stalk cells fail to maintain the
alignment in an organized monolayer of 5-7 cells.
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RESULTS_PART II
The germline stem cells (GSC) were identified as the 2-3 biggest Vasa-expressing cells
at the tip of the germarium (Fig. 3B). In tsh mutants, the number and morphology of GSC was
apparently normal (Fig. 3D), but ovaries shown swollen germaria with an increase in the size
of both (anterior/posterior and specially the dorsa/ventral) axis. Germline cysts abnormally
accumulate inside the germarium, especially prior to the 2a/2b border. In addition, within
region 3, cysts maintained the “lens shape” characteristic of region 2b and some cystoblasts
display a size comparable to the GSC, suggesting polyploidy levels characteristic of later stages
(Fig. 3D_arrows). Finally, in S2/S3 egg chambers, nurse cells display low levels of polyploidy.
All these observations suggested an oogenesis arrested before S3 stage.
Beside the arrested before S3 stage, the ∆126/tshA-3-2-66 ovaries displayed more than 5
consecutive egg chambers per ovarioles. In 24,4% of the ∆126/tshA-3-2-66 ovarioles, we
observed abnormal egg chambers (Table 2), 50% of which contain more than 16 germline cells
that could result from the abnormal wrapping of more than one 16-cell cyst (compound egg
chambers, Fig. 3E and E’). These compound egg chambers contain two or more groups of 16
germ line cells. In a compound egg chamber corresponding to a 32-cell cyst (Fig. 3E’), analysis
of confocal stack images and DAPI staining suggested the presence of two oocyte nuclei (Fig.
3E). This result supports that the nurse cell to oocyte ratio (15:1) is normal and that these egg
chambers result from the packaging of two consecutive cysts by the same epithelium, rather
than from an extra round of germline nuclear division.
The other 50% defective egg chambers correspond to fused egg chambers resulting
from normal encapsulation of 16-cell cyst, which failed to bud off the germarium (Fig. 3F). As
a consequence, they remain connected to each other and are not separated by stalk cells.
We noticed that encapsulation defects were more severe when trans-heterozygous flies
were grown for three to six days at 29°C, whereas under the same conditions wild-type flies
were fertile and have normal ovaries (data not shown).
Our data suggest that in ∆126/tshA-3-2-66 ovaries the wrapping of the cysts by the follicle
cells is perturbed and/or the budding-off from the germarium is delayed.
4- P{lacW}A3-3-66∆126/tshA-3-2-66 follicle cells are delayed in cell cycle
progression
To evaluate whether a diminution of Tsh activity has an effect on the proliferative
response of FSC and progeny, ∆126/tshA-3-2-66 ovaries were stained for phospho-histone H3
(PPH3), a mitotic cell marker. In the ∆126/tshA-3-2-66 ovaries, we observed ovarioles containing
more than 5 egg chambers but we never observed egg chambers beyond the S3 stage of
maturation. Thus, we performed the quantification and comparison of PPH3 positive FC in the
first three consecutive egg chambers (S1 to S3), since only these egg chambers display
comparable maturation stage in wild type and ∆126/tshA-3-2-66 ovaries. This analysis revealed
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RESULTS_PART II
that the number of the PPH3 positive cells was higher in ∆126/tshA-3-2-66 ovaries compared to
control ovaries (Fig. 4A-B and E).
Figure 4- Cell cycle progression is
affected in ∆126/tshA3-2-66 ovarioles.
(A, B) Mitotic follicle cells stained for PPH3 (green)
and nuclei (DAPI, blue); in wild-type (A) and in
∆126/tshA3-2-66 (B) ovarioles (projection of confocal
image stack). (C, D) Ovaries from wild-type (C) and
∆126/tshA3-2-66 (D) stained for activated-Caspase-3
(green) and nuclei (DAPI, blue). (E) Distribution of
the mitotic follicle cells labelled by Phospho-histone
H3 (PPH3) in the first three consecutive egg
chambers starting from stage S1 in the germarium.
PPH3 positive follicle cells were counted in wild-type
(n=69) and ∆126/tshA3-2-66 (n=98) ovarioles. (F, G,
H, I) Wild-type (F, G) and ∆126/tshA3-2-66 (H, I)
ovarioles stained for BrdU (green) and nuclei (DAPI,
blue) (projection of confocal image stack). Ovarioles
cultured for 90 minutes (F, H) or 270 minutes (G, I);
BrdU (10µM) was added for 15 minutes for pulse
labelling. In all panels, anterior is to the left. The bar
in panels A, C and F represents 50µm; the scale is
the same for all panels.
In the ∆126/tshA-3-2-66 ovaries, the number of PPH3 positive FC was always higher in the
egg chambers analysed (S1-S3) and this difference was maximize in the S1 stage, where we
observed three more mitotic FC compare to control (3,2 versus 0,8 FC/egg chamber ;
Supplementary Table 1). This result suggested that the perturbed expression of tsh in the
∆126/tshA-3-2-66 ovaries afected FC proliferation. The increase in mitotic FC number could result
(1) from a FC overproliferation or (2) from a mitotic delay of the FC. In the ∆126/tshA-3-2-66 egg
chambers, the FC nucleus seems significantly less compact and the S2/S3 egg chambers size
seems comparable to wild-type. Thus, no evidences of an increase of total FC cells, was
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RESULTS_PART II
observed in ∆126/tshA-3-2-66 ovaries to justify the FC overproliferation, suggesting that an
increase in mitotic FC result from a mitotic delay.
The global analysis of the distribution of the mitotic FC suggested that, in ∆126/tshA-3-2-
66 ovaries, the number of mitotic FC increase until the 4rd consecutive egg chambers showing
an abrupt reduction in the 5th egg chamber (Supplementary Table 1). This reduction could be
related with an excess of apoptosis observed in ∆126/tshA-3-2-66 ovaries beyond the 4th egg
chamber, as revealed by analysis of activated Caspase-3 activity (Fig. 4C, D). In addition, high
level of activated caspase-3 was also visible in germline.
To test for a delay in cell cycle progression in ∆126/tshA-3-2-66 ovaries, we performed 5-
bromo-2-deoxyuridine (Brdu) incorporation experiments (Fig. 4F-I). Dissected wild-type
(control) and mutant ovarioles were pulse labelled with BrdU for 15 minutes and then cultured
for 90 minutes or 270 minutes. In ∆126/tshA-3-2-66 ovaries cultured for 90 minutes, the BrdU
staining appears brighter and there was more BrdU positive FC compared to the control (Fig. 4
F, H). This result supports an extension in the length of the cell cycle in the ∆126/tshA-3-2-66
ovaries.
When wild-type ovarioles were cultured for 270 minutes, the majority of FC that have
incorporated the BrdU undergoes further division, which results in the dilution of the BrdU. In
the ∆126/tshA-3-2-66 ovaries, we observed more BrdU positive FC, especially in the first egg
chambers, showing that some FC in ∆126/tshA-3-2-66 are cycling very slowly or are cell cycle
arrested (Fig 4G, I).
All together, these results suggest that in follicular cells, tsh is required for the cell
cycle progression.
5- Down-regulation of teashirt during Drosophila oogenesis leads to the loss of
engrailed and hedgehog expression
In ∆126/tshA-3-2-66 ovarioles, proliferation of the FC was reduced and, as a consequence,
the wrapping of the cysts by the FC was perturbed and/or the budding-off from the germarium
was delayed. In the germarium, Tsh was detected with Hh and En in the TF cells and CC and,
Hh signalling has been shown to be critical to modulate proliferation of FC (Forbes et al.,
1996a; Zhang and Kalderon, 2000).
To investigate a possible regulatory relationship between tsh, hh and en in the adult
ovary, we examined the distribution of En and Hh in tsh mutants. In all ∆126/tshA-3-2-66
ovarioles, En was strongly reduced or totally absent in CC, nevertheless CC were correctly
enriched in Arm; En was normally detected in TF cells (Fig. 5B). In all ∆126/tshA-3-2-66 ovaries,
Hh staining was lost from the tip of the germarium (Fig. 5D).
These data suggest that expression of Tsh has to be tightly controlled to guarantee the
correct expression of en and hh respectively in CC and TF cells during Drosophila oogenesis.
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RESULTS_PART II
Figure 5- Tsh is required for the proper
expression of En and Hh in the germarium.
(A, B) Wild-type (A) and ∆126/tshA3-2-66 ovaries (B),
stained for Arm (green), En (red) and nuclei (DAPI, blue).
(C, D) Wild-type (C) and ∆126/tshA3-2-66 ovaries (D),
stained for Hh (green) and nuclei (DAPI, blue). Only the
TF, region 1 and region 2a of a single germarium are
shown. In all panels, anterior is to the left. The bar in
panel A represents 20µm; all panels are at the same scale.
- Ectopic tsh expression in the ovary induces ectopic En and phenocopies
ectopi
the role played by tsh in egg chamber formation, we ectopically
expres
excess of FSC derivatives
betwee
vitellog
and Hh lead to similar phenotypes, we tested whether ectopic
expres
6
c hh expression
To further analyze
sed tsh in wild-type adult ovaries. To this aim, hs-Gal4>UAS-tsh13 flies that carry one
copy of the UAS-tsh13 and hs-Gal4 constructs were heat-shocked.
Ectopic expression of tsh induces the accumulation of an
n egg chambers forming giant “stalk” like structures (Fig. 6A-A’). A similar phenotype
was observed when hh was ectopically expressed under the same conditions (Fig. 6B-B’;
Forbes et al., 1996a; Forbes et al., 1996b; Zhang & Kalderon 2000; Zhang & Kalderon, 2001).
In control egg chamber, FasIII is expressed in all FC during the germarial and early
enic stages but becomes restricted to the two polar follicles cells at each end of the egg
chamber by the stage 4 (Fig. 6C-C’). In contrast, in hs-Gal4>UAS-tsh13 and hs-Gal4>UAS-hh-
M4, FasIII was detected in FC at later stages and in all the excessive FSC progeny
accumulated between egg chambers (Fig. 6A-B’). These results suggest that ectopic tsh, like
ectopic hh, promotes cell proliferation and affects FasIII expression in FC, delaying follicle cells
fate acquisition. Of note, tsh was less efficient than hh in inducing giant “stalk” like structures
(Fig. 6A-B’); the giant “stalks” were smaller and less frequent in UAS-tsh13 (37%) than in
UAS-hh-M4 (62%) ovaries.
Because ectopic Tsh
sion of tsh may impact on the Hh signalling pathway. We analyzed the expression of En
in hs-Gal4>UAS-tsh13 heat-shocked ovaries and found that ectopic expression of tsh induces
ectopic expression of En (Fig. 6E-F’; compare to Fig. 6D-D’).
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RESULTS_PART II
Figure 6- Ectopic expression of hh and tsh
induces similar effects.
Ovarioles
hs-Gal4/+ (C, C’), were heat-
-Rescue of the P{lacW}A3-3-66∆126/tshA-3-2-66 phenotype
order to demonstrate that the perturbed expression of tsh contribute to the defects
observ ectopically provided Tsh
could rA-3-2-
from hs-Gal4>UAS-tsh13 (A, A’), hs-Gal4>UAS-
hh-M4 (B, B’) and control
shocked (kept at 25°C between heat-shocks) and stained
for FasIII (green), Vasa (red) and nuclei (DAPI, blue). (A’,
B’, C’) Enlargements of the stalk between the 1st and 2nd
egg chambers of the vitellarium signal with a arrow in the
respective left panels (B, C, D). Ectopic expression of tsh
(A, A’) and hh (B, B’) result in hyperproliferation of somatic
cells and formation of giant interfollicular stalk-like
structures (arrows). (C, C’) In control, consecutive egg
chambers are separated by 5-7 stalk cells (arrows). (D, D’)
heat-shocked hs-Gal4/+ and (E-F’) heat-shocked hs-
Gal4>UAS-tsh13 ovarioles were kept at 18°C between heat
shock and stained for Tsh (green), En (red) and nuclei
(DAPI, blue); (D’, E’ and F’) only show En (red) and nuclei
(DAPI, blue) staining. (F and F’) enlargements of the S3
egg chambers framed in E and E’, respectively. The
majority of the follicle cells into which Tsh was ectopically
produced were En positive (arrows). Anterior is to the right
and posterior is to the left. The bar (in panels A, A’, D, F)
represents 20µm. The panels (A, B, C), (A’, B’, C’), (D-E’)
and (F, F’) are at the same scale.
7
In
ed in the ∆126/tshA-3-2-66 ovaries, we decided to test whether
escue the ∆126/tshA-3-2-66 phenotype. To this aim, we ectopically expressed tsh in hs-
Gal4>UAS-tsh13; ∆126/tsh 66 mutant ovaries.
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RESULTS_PART II
Figure 7- Tsh rescues the imperfect proliferation phenotype of ∆126/tshA3-2-66 mutants and allows
the progression of oogenesis until stage S5/S6.
hs-Gal4/+ heat-shocked ovaries used as a control (A, D, G), ∆126/tshA3-2-66 (B, E, H) and ∆126/tshA3-2-66;UAS-
tsh13/hs-Gal4 (C,F, I). (A, B, C) ovaries (from flies maintained at 25°C) stained for FasIII (green) and Vasa (red).
Insets on bottom left show S5/S6 (A’ and C’) or S2/S3 (B’) egg chambers. Insets on bottom right show the stalk
between consecutive egg chambers (A’’, B’’ and C’’). (D-F) ovaries (from flies maintained at 18°C) stained for En
(red). (G-I) ovaries stained for Hh (green). Arrow in panel F and I indicate the restoration of En in CC and Hh
expression in TF cells, respectively.
In all panels, nuclei are stained with DAPI (blue), anterior is to the right and posterior is to the left, the scale bars in
(A-A’’) represent 50 µm and is the same for the panels (A-C), (A’-C’) and (A’’-C’’) respectively. The scale bar in (D and
G) represent 20µm and is the same for panels (D-F) and (G-I), respectively. CC, cap cells; TF, terminal filament.
This rescue assay is not ideal because (1) hsGAL4 produce a generalised tsh ectopic
expression (even out of the normal wild-type tsh context, what could have a toxic effect) and
(2) not guarantee the simultaneous and continue ectopic tsh expression in the TF and CC in all
ovarioles of the same ovary, (bab-GAL4, for example restrict expression to TF and CC).
Independently of the Gal4 driver used, the Tiptop (Tio) and the Tsh endogenous could
contribute, at least partially, to repress the ectopic tsh expression.
Previously, it was reported that tio and tsh repress each other during embryogenesis
(Laugier et al., 2005). In ∆126/tshA-3-2-66 ovaries Tio expression is increase to all TF cells, while
in wild-type it’ is restrict to the apical TF cells only (data not show). Thus it is predicable that
tsh and tio maintain the mutual repression in the tip of the germarium. It’s tempting to
suspect that ectopic tsh expression in TF cells will be affected by the presence of Tio in all
∆126/tshA-3-2-66 TF cells.
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RESULTS_PART II
Table 2- Quantification of the ∆126/ tshA3-2-66 and the ∆126/ tshA3-2-66;UAS-tsh13/hs-Gal4 ovaries phenotype.
∆126/tsh A3-2-66 ∆126/tsh A3-2-66; UAS-tsh13/hsGal4
Total ovarioles (n=) 275 188 Ovarioles with swollen germarium (%) 100 75,0 Ovarioles with compound and fused egg chambers (%) 24,4 4,2 Ovarioles with S3/S4 egg chambers (%) 0 32,4 Ovarioles with S4/S5 egg chambers (%) 0 9,6 Ovarioles with S5/S6 egg chambers (%) 0 4,8 Ovarioles with En in CC in the tip of the germarium (%) 0 22,2 (1)
(1) n=18 ovarioles
Beside the limitations of our rescue assay, the ectopic expression of tsh in ∆126/tshA-3-2-
66 ovaries partially rescued the tsh proliferation phenotype (Fig. 7C; compare with Fig. 7B). In
∆126/tshA-3-2-66 ovaries oogenesis was arrested before S3 stage (in 100% of the ovarioles) with
poorly polyploid nurse cells and a disorganised FC epithelium. In contrast, in the rescued
ovaries, oogenesis progressed beyond the S2/S3 stage in 46,8% of the ovarioles, the
polyploidy of the nurse cells was improved and the oocyte was located close to the posterior
polar cell (Table 2 and Fig. 7C; compare with Fig.7B and 3C,D). In addition, follicle cell
epithelium nuclei localisation (apical) and density were similar to the wild-type. Stalk cells
were correctly intercalated and formed normal stalks with 5 to 7 cells. Furthermore, in 4,8% of
the ovarioles, the maturation of the egg chambers reached the S5/S6 stage and, the frequency
of ovarioles with compound or fused egg chambers was reduce to only 4,2% (Table 2 and Fig.
7C’-C’’). In 24,5% of the hs-Gal4>UAS-tsh13; ∆126/tshA-3-2-66 ovarioles, the size of the
germaria was improved, but remains slightly bigger than in wild-type. These results show that
expression of tsh can rescue the ∆126/tshA-3-2-66 mutant phenotype.
We then asked whether the rescued ∆126/tshA-3-2-66 mutant phenotype could depend on
the capacity of the ubiquitously provided tsh to restore hh and/or en expression. In heat-
shocked hs-Gal4>UAS-tsh13; ∆126/tshA-3-2-66 ovaries, we found that tsh restored en and hh
expression (Fig. 7 F and I; Table2). This result is consistent with the previous finding that tsh
is required for the expression of hh and en respectively in the TF cells and CC. These data
suggest that the absence of hh expression in ∆126/tshA-3-2-66 ovaries could be due to the down
regulation of tsh. However Hh that was loss in both, TF and CC, in ∆126/tshA-3-2-66 ovaries, it
was restored only in CC of the hs-Gal4>UAS-tsh13; ∆126/tshA-3-2-66 ovaries. In ∆126/tshA-3-2-66
ovaries Tio expression is extended to all TF cells, while in wild-type it’ is restrict to the apical
TF cells only, indicated a mutual repression between the two paralogue, as explained above
(data not show, for details see chapter II part II of this manuscript). Thus, it is predicable that
in hs-Gal4>UAS-tsh13; ∆126/tshA-3-2-66 ovaries, the presence of Tio in all TF cells could
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RESULTS_PART II
diminish the tsh ectopic expression in all TF, justifying the presence of ectopic hh expression
only in CC, where no Tio was observed.
Finally, to test whether ectopic Hh could rescue the tsh mutant phenotype, ∆126/tshA-3-
2-66;hs-Gal4/UAS-hh-M4 flies were heat-shocked (Ingham and Fietz, 1995). Under these
conditions, the germline/somatic cells distribution resembles the wild-type and the germarium
size was comparable to hs-Gal4>UAS-tsh13; ∆126/tshA-3-2-66 (Supplementary Fig. 1).
Furthermore, the distribution of the follicle epithelium nuclei and stalk cells intercalation were
also improved. Nevertheless, oogenesis was unable to proceed beyond the S3/S4 stage and
egg chambers contained nurse cells with low levels of polyploidy and absence of differentiated
polar cells. Thus, the ∆126/tshA-3-2-66 mutant phenotype can also be rescued by over
expressing hh, suggesting that Tsh is acting upstream of hh.
Discussion
Whereas the function of tsh has been extensively studied during embryogenesis and
imaginal discs development, the function of tsh in the ovaries was never investigated. Here we
showed that Tsh is co-expressed with Hh at the tip of Drosophila germarium, in terminal
filament (TF) cells and in cap cells (CC). We demonstrated that overexpression of tsh mimics
the phenotype associated with hh overexpression. Phenotypic characterisation of ovaries into
which tsh expression is perturbed allowed us to suggest that hh and engrailed (en) expression
might depend on tsh. In these mutant ovaries, ectopically provided Tsh partially rescues the
defects and restores hh and en expression in CC. In addition, ectopic expression of Tsh induce
ectopic En expression. Our results suggest that in adult ovaries expression of Tsh might play a
critical role in regulating hh expression contributing for the regulation of FSC proliferation and
the specification of somatic cell identity.
Control of FSC proliferation upstream of the Hh pathway
At the tip of the Drosophila adult ovary, the germarium contains three identified stem
cell populations [germline stem cells (GSC), follicle stem cells (FSC) and escort stem cells
(ESCs)], making it an excellent model system for studying stem cell biology. Our data suggest
that Tsh might play a role in FSC proliferation. We showed that Tsh, like Hh, is probably
required for the proper packing of 16-cell germ-line cyst; the absence of Tsh correlates with de
disruption of the coordination between the 16-cell germ-line cyst formation and FSC
proliferation. Our data support that the frequency of the encapsulation defects increases with
the lowering of the Tsh level in the germarium.
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RESULTS_PART II
The Hh pathway has been implicated in the FSC proliferation (Forbes et al., 1996a and
1996b; Zhang and Kalderon, 2000 and 2001). We show that in wild-type ovaries the ectopic
expression of tsh mimics the constitutive Hh phenotype, that is the accumulation of an excess
of FSC derivates between egg chambers as a result of FSC over-proliferation. This result
supports that Tsh positively affects FSC proliferation and, either by a direct or indirect manner,
negatively affects FC differentiation, as previously reported for Hh. In our experimental
conditions, we noticed that the hs-Gal4>UAS-hh-M4 ovaries showed a more penetrating
phenotype, when compared to hs-Gal4>UAS-tsh13 ovaries; the giant “stalks” structures are
bigger and more frequent.
Ubiquitous expression of tsh in ∆126/tshA-3-2-66 ovaries partially rescues the proliferation
defect, significantly improved the encapsulation process and the progression of oogenesis.
More important, in the rescued ovarioles, hh and en expression was restored in the CC,
suggesting that the low levels of Tsh detected in ∆126/tshA-3-2-66 ovaries could account for the
proliferation defect.
The presence of polyploid nurse cells characteristic of a later stage in S1 egg chambers
indicates a delayed budding off from the germarium. Accordingly, PPH3 staining and BrdU
incorporation revealed that ∆126/tshA-3-2-66 ovaries exhibit a delay in cell cycle progression.
The quantification of the PPH3 positive FC per egg chamber in wild-type and ∆126/tshA-3-2-66
ovaries supports the idea of a delay to complete the sequence of approximately five mitotic
cycles downstream from the stem cell until leave the germarium and probably an incapacity
for the FC to undergo the next three to four mitotic cycles from 2-6 stage. In the Drosophila
eye, a recent study indicated that blocking the Hh pathway reduces the number of retinal
progenitors that take up BrdU as a consequence of a slower cell cycle kinetics (Locker et al.,
2006). Thus it is attempting to propose that the delay in cell cycle progression observed in
∆126/tshA-3-2-66 ovaries could be a consequence of the down-regulation of hh, which is known
to promote follicle-cell proliferation (Forbes et al., 1996a; Zhang and Kalderon, 2000). In
vertebrates, mutations in the SHH pathway have been shown to affect cell cycle progression
(Adolphe et al., 2006; Barnes et al., 2001). In the future, it would be interesting to analyze
whether vertebrate orthologues of tsh affect cell cycle progression.
Regulation of En and Hh expression in the Tip of the germarium
In adult Drosophila ovary, Tsh was detected in proximal TF, CC, ESs (including ESC)
and FC in region 3 of the germarium and the expression levels of Tsh in TF cells and CC vary
with aging. In the TF cells and CC, Tsh was co-detected with Hh, and Tsh and En proteins were
detected in the same group of somatic cells at the tip of the germarium. In the absence of Tsh,
hh expression was drastically reduced in TF and CC whereas En was absent from CC but was
still detectable in TF cells. In the ovaries, the regulatory relationships between hh and en
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RESULTS_PART II
remain poorly understood. Our analysis propose that en expression is maintained in the TF
cells in absence of hh, suggesting that in the TF cells en expression might not depend on hh.
It has been shown that the ectopic expression of hh and en in the adult ovary cannot
induce the expression of the other outside its normal domain of expression (Forbes et al.,
1996). Here we show that ectopic expression of tsh induces expression of en in FC. These
results suggest that tsh can act upstream of en and hh, and that tsh could participate to the
regulation of en and hh expression in Drosophila ovaries. Interestingly, we noticed that, as
flies age, while En continues to be strongly expressed in all non-somatic cells in the tip of the
germarium, Tsh was down regulated in the most distal TF cells. This observation suggests that
Tsh not only co-localises with, but also displays the same dynamic expression in TF and CC
reported for Hh (Forbes et al., 1996a; Forbes et al., 1996b). Our experiments suggest that the
down regulation of hh may be tsh-dependent and, because of the absence of a suitable tsh
temperature-sensitive allele and the impossibility to make tsh clones, we were unable to
further analyze these regulatory mechanisms.
The mechanisms by which Tsh may regulate hh and/or en expression are still poorly
understood. In vertebrate and Drosophila, Tsh factors can recrute the corepressor CtBP and in
vertebrate TSHZ3 has been shown to recrute HDAC1/2 activities (Manfroid et al., 2004;
Kajiwara et al., 2009). Moreover, in Drosophila, Tsh has been shown to behave as a repressor
(Taghli-Lamallem et al., 2007). Thus, if in the TF cells and CC tsh regulates the expression of
hh and/or en, it is likely that this regulation might not be direct but rather indirect through the
control of the activity of a repressor.
In the future, our findings may help to further identify and characterize factors that
participate to the exquisite control of Hh expression during embryonic development and adult
tissue homeostasis.
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RESULTS_PART II
Supplementary Data
Supplementary Figure 1- Rescue of the ∆126/tshA3-2-66 proliferation phenotype by ectopic Hh
(A) hs-Gal4/+ (B) ∆126/ tshA3-2-66; UAS-hh-M4/ hs-Gal4 ovarioles stained for FasIII (green), Vasa (red) and
nuclei (DAPI; blue).
In (B) ∆126/ tshA3-2-66; UAS-hh-M4/ hs-Gal4 ovarioles the germline/somatic cells distribution and the germarium size
are approximated to the observed in wild-type. Furthermore, some improvements are also observed in the distribution
of the follicle epithelium nuclei and stalk cells intercalation. Nevertheless, oogenesis was unable to proceed beyond the
S3/S4 stage and egg chambers contained nurse cells with low levels of polyploidy and absence of differentiated polar
cells.
In all panels, anterior is to the right and posterior is to the left. The scale bar (in panel A) represents 50 µm and is the
same for both panels.
Supplementary Table 1- Quantification of the positive P-Histone H3 FC in the first 5 egg chambers of wild-
type (n=69) and ∆126/ tshA-3-2-66(n=98) ovaries.
Egg Chamber position
FC/ovariole in(1):
WT ∆126/tshA-3-
2-66
1 0,8 3,2 2 1 2,2 3 2,6 3,2 4 5,5 2,6 5 3,7 0,8
(1)FC/ovarioles= Total FC counted in the 1st (2nd, 3rd, 4th or 5th) egg chamber/ total number of ovarioles
screened
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RESULTS_Part III:
118
Teashirt and Tiptop repress each other in Terminal Filament Cells
during Drosophila oogenesis
Silvia Pimentel1,2, Isabel Salazar1, Christine Vola2, Rui Gomes 1 and Laurent Fasano2†
1 Universidade de Lisboa, Faculdade de Ciências, Departamento de Biologia Vegetal, Campo
Grande, 1749-016 Lisboa, Portugal 2 Institut de Biologie du Développement de Marseille Luminy (IBDML), UMR 6216, CNRS-
Université de la Méditerranée, Campus de Luminy, Case 907, 13288 Marseille Cedex 09,
France.
†Authors for correspondence: (emails: <[email protected]>)
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RESULTS_PART III
Abstract
In Drosophila, the teashirt (tsh) gene encodes a zinc finger protein known to be
required for proper specification of the trunk segments in the developing embryo. The Tsh
protein is expressed throughout development and within a number of different tissues.
Recently, it was described a new member of the tsh family in Drosophila, call Tiptop (Tio),
which encodes a protein of 1024 amino acids with a fourth Zinc finger motif in the C-terminal
part. Priviously was described the mutual repression between Tsh and Tio during
embryogenesis, as the ectopic expression of Tio masked a crucial role for Tsh in trunk
patterning. Here we report that, during oogenesis, Tsh and Tio present a complementary
expression pattern in terminal filament cells (TF). In ∆126/tshA3-2-66 ovaries, which do not
express Tsh properly, Tio is expressed ectopically in all TF, and the reverse is observed for Tsh
in tio473 homozygous ovaries. The construct hsGal4>UAS-tioFL rescues the ∆126/tshA3-2-66
phenotype as efficiently as the hsGal4>UAS-tsh13. The ectopic expression of tio induces
similar effects to those described after overexpression of tsh, but preferentially in background
defective for tsh. We found that, like it was reported during Drosophila embryogenesis, Tsh
and Tio repress each other in Drosophila germarium. During oogenesis, Tio has, at least, a
partial redundant function, since the lack of Tio may be rescued by the tsh expression in
tio473mutants.
Key words: Drosophila; oogenesis; terminal filament cells; teashirt; tiptop.
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RESULTS_PART III
Materials and Methods
Drosophila strains
All genetic markers and balancers are described by Lindsley and Zimm (1992) and
FlyBase (http://flybase.bio.indiana.edu) except as noted.
The hypomorphic allele tshA3-2-66 (Bloomington) has a P{lacW} insertion at 1652bp
upstream of the tsh translation start (see chapter II part I of this manuscript).
The ∆126 line results from a tshA3-2-66 P-element excision mutagenesis (for details see
chapter II part I and part II of this manuscript). Among 2382 revertants, 17 were viable in
Trans with tshA3-2-66. From this 17 viable revertants, seven were fully viable and fertile and the
other ten were also viable but female sterile. The P{lacW}A3-3-66∆ (∆126) was one of the ten
lines that presented females viables but sterile. In addition, 2/3 of the ∆126/tshA3-2-66 adults
have white eyes, indicating that ∆126 modifies the eye patterned white expression observed in
the tshA3-2-66 adult eyes (for more details see chapter II part I and part II of this manuscript).
Our analysis failed to validate that ∆126 is a new tsh allele, nevertheless, in ∆126/tshA-3-2-66
ovaries tsh expression is perturbed, which constitute a opportunity to investigate the effect of
tsh in the expression of his paralogue tio during oogenesis.
Females (or males) tshA3-2-66/T(2;3)CyOTM6B,Tb were crossed with male (or females)
∆126/T(2;3)CyOTM6B,Tb to get ∆126/tshA3-2-66 females used in this study.
The allele tio473 was identified as a tio deletion that removes tio and surrounding DNA,
3,6Kb upstream of the start codon and 0,9Kb downstream of the stop codon. Neither tio mRNA
nor protein are detected (Laugier et al., 2005).
For rescue and overexpression experiments the UAS-tsh13 construct (Gallet et al.,
1998), the UAS-tio construct (Laugier et al., 2005) or the UAS-hh-M4 construct (Ingham and
Fietz, 1995) were crossed to the driver P{w+mC=GAL4-Hsp70.PB}TR2 (Bloomington).
Staining ovaries
Antibody staining, was performed as described by (Zhang and Kalderon, 2000), with
the modifications previously reported in chapter II partII of this manuscript.
Primary and secondary antibodies and their dilutions were the following: anti-Tsh
(rabbit Tsh, a gift from S Cohen, EMBL Heidelberg, 1:1000 dilution); anti-Tio 1:500 (rat Tio,
Laugier et al., 2005, 1:500 dilution) mouse 4-D9 anti-Engrailed invected (Developmental
Studies Hybridome bank, 1:10 dilution); mouse 7G10 anti-Fasciclin III (Developmental Studies
Hybridome bank, 1:200 dilution) and rabbit anti-Vasa (gift from Lehaman, 1:1000 dilution).
Secondary were conjugated with Texas red or FITC (Molecular Probes, 1:500 dilution).
For En and Tio staining, the signal was amplified with the Tyramide Signal Amplification
Kit (PerkinElmer) using biotinylated secondary antibodies (Jackson Laboratories, 1:500). In
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RESULTS_PART III
these cases, the ovaries were submitted to a peroxidase treatment (3% H2O2 for 10 minutes)
to reduce the background, before their blocking.
Stained ovaries were mounted in Vectashield medium with DAPI (Vectors) and analyzed
under a LSM 510 Zeiss confocal microscope.
Rescue of ∆126/ tshA-3-2-664 mutant phenotype
∆126;UAS-tsh13/T(2;3)CyOTM6B,Tb or ∆126;UAS-tioFL/T(2;3)CyOTM6B,Tb males
were crossed with tshA-3-2-66;hsGal4/T(2;3)CyOTM6B,Tb females. In the day of the enclosion,
∆126/tshA-3-2-66;UAS-tsh13/hsGal4 or ∆126/tshA-3-2-66;UAS-tioFL/hsGal4 females were
submitted to heat-shock; tubes were immersed in a water bath at 37°C, for 1hour, twice a
day, during 3 consecutive days. In the 4th day, ovaries were dissected and stained with FasIII
and Vasa antibodies. Females were grown at 25°C until the adult stage and between the heat-
shocks.
Tsh, Tio and Hh overexpression
UAS-tsh13/T(2;3)CyOTM6B,Tb, UAS-tioFL/T(2;3)CyOTM6B,Tb or UAS-hh-
M4/T(2;3)CyOTM6B,Tb males were crossed with hsGal4/T(2;3)CyOTM6B,Tb females. In the
day of the enclosion, UAS-tsh13/hsGal4, UAS-tioFL/hsGal4 or UAS-hh-M4/hsGal4 females were
submitted to a heat-shock at 37°C, for 1hour, which was repeated twice a day, during 3
consecutive days. In the 4th day, ovaries were dissected and stained with FasIII and Vasa.
Females were grown at 25°C, until adult stage and between heat-shock.
Results
1-Tsh and Tio show complementary localisation in Terminal Filament cells
Previous results have shown that, during early embryogenesis, tsh and tio expression is
detected in distinct domains (Laugier et al., 2005). Tsh is located in the trunk and Tio in parts
of the head and tail. In later embryogenesis, they present both common and tissue-specific
expression patterns. The loss of tio caused ectopic expression of tsh in the clypeolabrum,
where tio is normally expressed (Laugier et al., 2005). Similarly, loss of tsh caused ectopic
expression of tio in the trunk. In different mutant situation it was showed that in the trunk and
head, they repress each other’s expression (Laugier et al., 2005). In contrast, during adult
development, it was recently shown that tsh and tio were co-expressed in imaginal discs
(Besssa et al., 2009).
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RESULTS_PART III
In order to find out whether Tsh and Tio behave during oogenesis, in a similar way to
embryos or to imaginal discs development, we looked for Tsh and Tio localisation in wild-type
and mutant Drosophila ovaries.
In wild-type, Tsh is present in terminal filaments (TF) cells, cap cells (CC) and in escort
cells (EC) including escort stem cells (ESC) and recently formed follicle cells (FC; see chapter
II part II of this manuscript and Fig. 1A). Tio is only observed in TF (Fig. 1A). Double staining
in wild-type adult ovaries, with anti-Tsh and anti-Tio, show a complementary localisation of
both proteins in the nuclei of TF. Tio is localised in more apical TF, where Tsh staining is weak,
while Tsh is stronger in more proximal TF cells, where Tio is not detected (Fig. 1A). Although
the complementar expression in TF, tsh has a more extense domain of expression suggesting a
more important role in oogenesis. Interestingly, a precise deletion of tio is homozygous viable
and fertile (Laugier et al., 2005). tio473 homozygotes show perfectly normal ovaries, without
detectable anomalies (data not show). While ∆126/tshA3-2-66 female are sterile, with atrophied
ovaries, in wich oogenesis is arrested before S3 stage (Fig. 2B, for more details see also
chapter II part II of this manuscript).
Figure 1- Tsh and Tio co-repress each other.
(A) wild-type; (B) ∆126/tshA3-2-66 and (C) tio473 homozygous germarium stained with Tsh (green) and Tio (red)
antibodies. tsh is expressed in cap cells (CC), escort cells (EC) and also, in new follicle cells in S1 stage. (A) In
terminal filament, Tsh is observed in more proximal cells (TFp), while tio is expressed only in more apical cells (TFa).
(B) Tsh is lost from all germarium ∆126/tshA3-2-66 (phenotype III) while, Tio is detected in all terminal filament cells
(TF). (C) In tio473 homozygous germarium, Tsh is observed in all terminal filament cells (TF) and in cap cells (CC),
escort cells (EC) and also, in new follicle cells in S1 stage.
Anterior is to the right and posterior is to the left. The scale bar (in panel A) represents 20µm and is the same for all
panels.
In order to avalaible the behavoir of Tsh in the absence of Tio and vice-versa, we
analysed the Tsh expression in the germarium of tio473 homozygotes, and the Tio expression in
the germarium of ∆126/tshA3-2-66. We observed ectopic Tsh staining in the apical TF cells of the
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RESULTS_PART III
tio473 germaria; Tsh was detected in all TF, CC and EC, and a weak Tsh staining continues to be
observed in recently produced follicles cells (Fig.1C). In ∆126/tshA3-2-66 ovarioles, Tio was
detected in all TF but never in CC or EC, nor even in ovarioles where Tsh is missing in all the
germarium (phenotype type III; Fig.1B; for more detail see chapter II part II of this
manuscript). For note, we suspected that ∆126/tshA3-2-66 ovaries are an allele of regulation, and
a residual quantity of Tsh (not detectable by immunostainig) could be enough to repress tio in
CC of ∆126/tshA3-2-66ovaries.
2- The gene tio show similar functional properties to tsh in the tip of the
ovaries and can partially substitute it during oogenesis
As tio and tsh have been shown to be functionally similar during embryogenesis and
imaginal development (Laugier et al., 2005; Bessa et al, 2009), we dicide to investigated if
this was also the case during oogenesis. We preformed three experiments to test this point.
2.1-UAS-tioFL expression can partially rescue ∆126/tshA3-2-66phenotype
We tested the ability of tio to rescue the ∆126/tshA3-2-66 phenotype using the UAS/Gal4
system (Brand and Perrimon, 1993). We used the hsGal4 driver, where Gal4 is under the
control of the heat-shock promoter hsp70, because no other Gal4 driver mimicking tsh
expression in ovaries was available. Flies carrying a single copy of UAS-tsh13 or UAS-tioFL and
hsGal4 will be referred subsequently as ∆126/tshA3-2-66;UAS-tsh13/hsGal4 or ∆126/tshA3-2-
66;UAS-tioFL/hsGal4, respectively.
In ∆126/tshA3-2-66 ovaries, oogenesis was arrested before S3 stage (in 100% of the
ovarioles) with poorly polyploid nurse cells and a disorganised FC epithelium. These ovaries
present enlarged germaria, with frequent abnormal egg chamber (24,4% of the ovaioles
presented at least one compound or fused egg chambers; Fig.2B, for more detail see chapter
II part II of this manuscript ). If Tio/Tio were functionally equivalent, we expect that Tio can
rescue the tsh loss of function, when expressed in the tsh domain.
The ectopic expression of tio in ∆126/tshA3-2-66 ovaries partially rescues the proliferation
phenotype (Fig.2C-D). In these flies, oogenesis progress until S5/S6 stage, nurse cells improve
ther ploidy levels and it is possible to distinguish the oocyte close to the posterior polar cell
(Fig.2 D arrowhead and arrow, respectivelly). Follicle epithelium nuclei are perfectly placed in
the apical part of the epithelium, and their density resembles the wild-type. Stalk cells are
correctly intercalated forming normal stalks. The size of the germaria is smaller than in
∆126/tshA3-2-66.
Unexpectedly, in some aspects Tio seems more efficient than Tsh in the rescue of the
∆126/tshA3-2-66 phenotype. The S5/S6 egg chambers were more frequent whenever we
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RESULTS_PART III
ectopically expressed tio. In addition, and unlike tsh ectopic expression, in ∆126/tshA3-2-
66;UAS-tioFL/hsGal4 ovaries, we also observed an improvement in the germ-line/ somatic cells
organization in the germarium.
Together, these results suggest that, ectopic tio is able to restore the absence of tsh in
∆126/tshA3-2-66ovaries, and surprisingly, in some aspects, tio was even more efficient than tsh
to rescue the proliferation phenotype observed in ∆126/tshA3-2-66ovaries.
Figure 2- Tsh and
Tio rescue the imperfect
proliferation phenotype
of the ∆126/tshA3-2-66
mutant and allow the
progression of oogenesis
until stage S5/S6.
(A) hsGal4/+; (B)
∆126/tshA3-2-66; (C)
∆126/tshA3-2-66; UAS-
tsh13/hsGal4 and (D)
∆126/tshA3-2-66;UAS-
tioFL/hsGal4 ovarioles were
stained with antibodies to
FasIII (green) in the
membrane of the follicle
cells and Vasa (red) in
germ-line. DNA (blue) was
labelled with DAPI.
(C, D) Tsh and Tio ectopic
expression rescues the
imperfect proliferation
phenotype displayed by
∆126/tshA3-2-66 ovaries; it is
possible to observe S5/S6
egg chambers (insert in
bottom right in C and
biggest egg chamber in D)
with the oocyte (arrowhead)
located close to the
posterior polar cell (arrow), whereas nurse cells improved their polyploidy. The density and the arrangement of the
nuclei of the follicle epithelium, in both cases (C, D) are comparable to the wild-type (A). Stalk cells are correctly
intercalated. The size of the ∆126/tshA3-2-66;UAS-tsh13/hsGal4 (C) and ∆126/tshA3-2-66;UAS-tioFL/hsGal4 (D) germaria
are smaller than in ∆126/tshA3-2-66(B).
Anterior is to the right and posterior is to the left. The scale bar (in panel A) represents 50µm and is the same for all
panels and insert.
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RESULTS_PART III
2.2- Tio maintained engrailed (en) expression in all TF cells of ∆126/tshA3-2-66
germarium, while in tio473 homozygous ovaries en expression is maintened by Tsh.
We found that Tsh is required for proper engrailed (en) and hedgehog (hh) expression
in the tip of the germarium (see chapter II part II of this manuscript and Fig.3). In the wild-
type, En is co-localised with Hh in the nuclei of the same group of somatic cells. Hh is
expressed selectively in the membrane of specialized non–proliferating somatic TF and CC, at
the anterior tips of the germarium (Forbes et al., 1996a; Forbes et al., 1996b Fig.3A). To
evaluate the possibility of tio to be able to replace tsh in the regulation of En expression in TF,
and vice versa, we stained ovaries from ∆126/tshA3-2-66 and tio473 with antibodies against En
and Armadillo (Arm; Fig. 3).
En is detected in all TF and CC in homozygous tio473 germaria (Fig.3C). In this
background, Tsh replaces Tio in apical TF and, in consequence, En is detected like it was
observed in wild-type ovaries. Since Tsh can completely replace the absence of tio, at least in
the maintenance of En expression, homozygous tio473 ovaries are perfectly normal and female
fertile.
Figure 3- Localisation of En in wild-type, ∆126/tshA3-2-66 and tio473 germaria.
(A) wild-type; (B) ∆126/tshA3-2-66and (C) tio473 homozygous germaria stained with Arm (green) and En (red)
antibodies. DNA (blue) was labelled with DAPI. (A, C) In wild-type and tio473 germaria, En is detected in terminal
filament (TF) cells and cap cells (CC). (B) In ∆126/tshA3-2-66 germarium En is lost from cap cells (CC) that continue to
display normal levels of Arm.
Anterior is to the right and posterior is to the left. The scale bar (in panel A) represents 20µm and is the same for all
panels.
Thus, ectopic tsh expression perfectly mimics tio expression in TF in tio473 ovaries and
apparently could replace the absence of Tio, since tio473 homozygous female are perfectely
fertile.
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RESULTS_PART III
In ∆126/tshA3-2-66 ovaries, Hh is completely absent from the tip of the germarium. En is
strongly reduced or totally absent in CC (although these last cells are correctly enriched in
Arm). Curiously all TF cells still expressed en (Fig.3B and for more detail see chapter II part II
of this manuscript). The absence of Hh from the TF and CC at the tip of ∆126/tshA3-2-66 ovaries
suggests that Tio alone is insufficient to substitute Tsh in the maintenance of Hh expression
(data show, see chapter II part II of this manuscript).
The maintenaice of En in TF but not in CC suggest that Tio can replace tsh in all TF, and
maintain En expression in this domain of the ∆126/tshA3-2-66 germarium. However, since tio is
not ectopically expressed outside TF, En expression is not maintained in CC. Thus, Tio is
expressed in all TF cells (being ectopic only in proximal TF cells) of ∆126/tshA3-2-66ovaries and it
is insufficient to matained En in this domain in the absence of tsh.
These show that Tsh can replace Tio but not the inverse and suggests that Tio may
have a redundant function with Tsh during oogenesis, resembling their relation during
embryogenesis. However, at least in ∆126/tshA3-2-66;UAS-tioFL/hsGal4 ovaries the ectopic
expression of tio induce ectopic en expression out of their wild-type domain.
2.3- Ectopic expression of tio mimic ectopic expression of tsh specially in a
mutant background context
In embryos the ectopic expression of Tsh and Tio in embryo and in imaginal discs
induced similar effects (Laugier et al., 2005; Bessa et al., 2009). It was showed that Tio is less
efficient than Tsh to induce naked cuticle identity but, from stage 13 onwards, ectopic tsh and
tio activated and maintained wg transcription in a similar way (Laugier et al., 2005).
The ectopic expression of tsh generated giants stalk-like structures, (see chaper II part
II of this manuscript and Fig.4C) and mimic the ectopically expression of hh (Forbes et al.,
1996a; Forbes et al., 1996b; Zhang & Kalderon 2000; Zhang & Kalderon, 2001).
In wild-type, two consecutive egg chambers are separated by 5-7 stalk cells (Decotto
and Spradling, 2005; Fig.4A). In hsGal4>UAS-tsh13 ovaries, we observed the overproliferation
of the follicle stem cells (FSC) and/ or progeny, which accumulated between egg chambers to
give giants stak-like structures (Fig.4D). In hsGal4>UAS-tioFL ovaries the giants stak-like
structures was rarely observed (Fig.4E). However, in ∆126/tshA3-2-66;UAS-tioFL/hsGal4 these
abnormal stalks were frequent and particularly enriched in FasIII, resemble the observed for
hsGal4>UAS-tsh13 and for hsGal4>UAS-hh-M4 giants stak-like structures(Fig.4F).
These data suggest that ectopic tio can induce the same Hh overactivation phenotype,
preferentially, in the absence of tsh.
Unlike to hsGal4>UAS-tsh13 and hsGAL4>UAS-hh-M4, the ectopic expression of tio
produced bigger germaria specially increased in region 1-2b. These bigger germaria seem to
have extra germ-line cysts, at least two rows of disc shaped cysts surrounded by follicle cells
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RESULTS_PART III
in region 2b. However, these germaria conserved the germline organization observed in wild-
type. Unlike to ∆126/tshA3-2-66 germaria, the hsGal4>UAS-tioFL germaria presented typical disc
shaped cysts in region 2b and were correctly intercalated by the somatic follicle cells.
Moreover, in region 3, the germ-line cysts were perfectly circular and well surrounded by the
follicle cells (Fig.4E-inset). In these ovaries (hsGal4>UAS-tioFL), we failed to detect
encapsulation problems, compound or fused egg chambers. In a wild-type background, the
ectopic expression presented a more evident effect in GSC rather than in FSC proliferation.
More work is need to confirme this possible implication of Tio in GSC proliferation, for exemple,
specific GSC staining and posterior counting, should be performed.
Figure 4- Ectopic expression of
hh, tsh and tio induces a similar
phenotype.
(A) hsGal4/+; (B) ∆126/tshA3-2-66; (C)
hsGal4>UAS-tsh13; (D) hsGal4>UAS-hh-
M4; (E) hsGal4>UAS-tioFL and (F)
∆126/tshA3-2-66;UAS-tioFL/hsGal4 stained
with antibodies to FasIII (green) and Vasa
(red) and with DAPI for DNA (blue).
In wild-type (A), consecutive egg
chambers are separated by 5-7 stalk cells.
Hyperactivation of tsh (C) or hh (D) results
in hyperproliferation of somatic cells and
the formation of giant interfollicular stalk-
like structures (arrowheads in panels C, D
and F)). The same phenotype, caused by
tio overexpression, is more frequent in a
tsh mutant background (F); giants stak-
like structures are rare in hsGal4>UA-tioFL
(E).
An enlargement of each germarium is
shown in the bottom right inset of each
panel. In hsGal4>UAS-tioFL ovarioles (F)
the bigger germaria seem to result from an
increase in germline-cyst production. It is
possible to observe at least two rows of
disc shaped cysts (arrows in the bottom
right inset in panel E). In contrast to
∆126/tshA3-2-66germaria (B), the
hsGal4>UAS-tioFL germaria (F) maintain
the germline/ soma organization
characteristic of wild-type (A).
Anterior is to the right and posterior is to
the left. The bar (in panels A) represents
50µm. All the panels are at the same scale.
129
RESULTS_PART III
Discussion
Teashirt and Tiptop are localised in the left arm of chromosome 2 and are transcribed in
the sam
Tio and Tsh proteins is about 28%. The best
percen
other duplicated genes in the Drosophila genome are important during its
develo
uplication event
characteristic of
utual repression between teashirt and tiptop
tio473 ovaries shows that Tsh
and Tio TF) cel
s
orm
e orientation 5’-3’. These genes are spaced 183 Kb from each other and code for a
protein with zinc finger (Znf) C2H2 type motifs.
The global percentage of identity between
tage of identity coincides with the three Znf motifs and is around 51%. Tio has a fourth
Znf motif conserved in the C-terminal part, which is also present in all other Tsh family
members, except in Tsh (Caubit et al., 2000; Laugier et al., 2005; Herke et al., 2005; Onai et
al., 2007).
Many
pment. For example, engrailed (en) and invected (inv; Poole et al., 1985), knirps (kni)
and knirps-related (knrl; Rothe et al., 1989), sloppy paired 1 (slp1) and sloppy paired 2 (slp2;
Grossniklaus et al., 1992), zerknüllt 1 (zen1) and zerknüllt 2 (zen2; Rushlow et al., 1987),
disconnected (disco) and disco-related (disco-r; Mahaffey et al., 2001) or distal antenna (dan)
and distal antenna related (danr; Emerald et al., 2003). These genes are characterised by
close genetic linkage, by extensive sequence homology and, similarly to the duplication of tsh
tio, they are also duplicated in tandem (Laugier et al., 2005; Laugier, 2005).
The tsh tio gene pair seems to have arisen from a recent d
Drosophilidae. Phylogenetic analysis of the sequence of Tsh/Tio family protein
in the arthropods showed that Drosophila Tio protein is closer to the other arthropod proteins
than to Drosophila Tsh (Laugier et al., 2005; Laugier, 2005). It seems that tio is closer to the
ancestral gene than to tsh, and as is often the case for duplicated genes in Drosophila, one
plays a dominant role as compared to the other (Gonzalez-Gaitan et al., 1994; Gustavson et
al., 1996); in this case tsh played the dominant role (Laugier et al., 2005; Laugier, 2005). The
duplication arose only after the separation of the Drosophila and Glossina lineages, between 60
and 140 million years ago. The fact that the embryonic pattern of expression of the single Tc
tiotsh gene in the flour beetle roughly resembles the summation of the patterns of tsh and tio
in Drosophila embryo points to a possible subfunctionalization of tsh and tio after the
duplication (Lynch and Force, 2000; Shippy et al., 2008; Bessa et al., 2009).
M
The expression pattern analysis in ∆126/tsh A3-2-664 and
repress each other in the terminal filament ( ls. In tio473 ovaries, Tsh is detected
in all TF cells. When tsh activity is removed (∆126/tshA3-2-66), Tio i detected in all TF cells,
including the more proximal TF cells, where Tsh is n ally detected, showing that Tsh
represses the expression of tio and the inverse. This is in accordance with the reports during
130
RESULTS_PART III
embryogenesis and eye development, where the loss of one gene leads to the transcriptional
de-repression of the paralog resulting in the compensation by one paralog for the loss of the
other (Laugier et al., 2005; Bessa et al., 2009; Datta et al., 2009).
Curiously, Tio was never detected in cap cells (CC) of ∆126/tshA3-2-66 ovaries, even in
the cas
fertile, ∆126/tshA3-2-66 flies are sterile
and ar
at, as fly age, Tsh was down regulated in the most distal TF cells. Could
be inte
ess each other but also act synergistically
in som
to what have been reported for embryos (Laugier et al., 2005; Laugier, 2005),
our ob
e of ovarioles, where no Tsh is detectable by imunostainig in the germarium (phenotype
III, see chaper II part II of this manuscript). For note, in contrast to the homozygous tio473,
the trans-heterozygote ∆126/tshA3-2-66 is not a null mutant. Thus, this may reflect that even a
tiny amount of tsh expression is enough to repress tio.
Although homozygous tio473 flies are viable and
rest oogenesis before S3 stage. This reinforces Tsh as having the major role of the two
duplicated genes during oogenesis. And suggests that, tio plays at least in part, a commum
function with tsh during Drosophila oogenesis, similar to what it happens during embrogenesis
and eye formation.
We noticed th
resting available if this downregulation is related with Tio, since this is the late Tio
domain. Our Tio/Tsh immunostainig was performed in flies with around 8 days old. In future
would be interesting performed these analyses in younger flies. In addition, the Tio/ Tsh
behaviour during late pupal stage should also be interesting to study, since Tsh is expressed in
all TF at this stage. Tio start to be expressed after Tsh start to be donwregulated, or initial the
two paralogue share the same expression domain?
In embryos, we may say that tsh and tio repr
e way, and that this may fine tune the development (Laugier et al., 2005; Laugier,
2005). It was showed that Tio can partially replace the loss of tsh function, while homozygous
tio473 embryos are viable, the tsh8 embryos (double mutants for tio and tsh) present a more
severe phenotype than tsh mutants alone (for example tsh2757R8; Laugier et al., 2005; Laugier,
2005). In order to find out if the absence of both proteins aggravates the ∆126/tshA3-2-664
ovary phenotype, we tried to deplete simultaneously tsh and tio during oogenesis using UAS-
RNAi-tsh in tio473 ovaries (a gift from Casares). However, the absence of a specific Gal4 driver,
that mimic tsh expression in their wild-type domain during all oogenesis unable us to efficienly
depleted Tsh.
Similarly
servations did not allowed us to distinguish if the regulatory mechanism between Tsh
and Tio repression is direct or indirect. In a simple model, Tsh may repress tio and vice versa.
The molecular similarities between the two paralogs support a possibility of a negative auto
and cross-regulatory loop (Bessa et al., 2009).
131
RESULTS_PART III
Tio show similar functional properties to tsh in the tip of the ovaries and can
partially substitute it during oogenesis.
We showed that the ectopic expression of tio is able to rescue the ∆126/tshA3-2-66
phenotype as efficiently, or even better, than the tsh ectopic expression. This suggests that, in
the ectopic situation, when tio is expressed out of its normal expression domain, he is able to
compensate the loss of tsh and restore the normal morphology of the ∆126/tshA3-2-66 ovarioles
allowing the progression of the oogenesis until S5/S6 stage.
The lower toxicity of the ubiquitus expression of the UAS-tio construct, comparatively to
the ubiquitus expression of the UAS-tsh construct could justify the differences in the efficacy of
the 2 constructs. Accordingly to what has been described for embryogenesis, Tsh may have
more number of target genes than Tio during oogenesis, and in consequence of that, the
ectopic expression of tio affect the expression of a more restrict number of target gene and
therefore is less toxic in ovaries than the ectopic expression of tsh. Other simple, explanation
could be just a mater of efficiency of the construct, independently of the gene associated.
The overexpression of tio is able to mimic the overactivity of Tsh mediating Hh in FSC
proliferation during oogenesis, preferencially in a mutant context for tsh. The production of
giants stalk-like structures, delayed in their fate acquisition, was more frequently observed in
∆126/tshA3-2-66;UAS-tioFL/hsGAL4 than in hsGAL4>UAS-tioFL.
Interestingly, the presence of Tsh seems to prevent ectopic tio to induce giants stalks-
like structures. In a mutant context for tsh, the ectopic tio was able to mimic overproliferation
of FSC and delaying FC fate acquisition, as described for overexpression of Hh signalling. This
suggests that, in hsGAL4>UAS-tioFL ovaries, the endogenous Tsh is able to repress the ectopic
expression of Tio or it is capable to inhibit the activation of Hh by ectopic Tio. Tsh and/or Tio
levels are critical for normal development. Therefore, it would be predicted the tightly
regulation of the total concentration of Tio and Tsh presented in a cell. Transcriptional cross
regulation between tio and tsh ensure that their protein levels are kept constant as has been
show in the embryo (Laugier et al., 2005) and imaginal discs (Bessa et al., 2009). Thus in
hsGal4>UAS-tioFL ovaries is probable that the endogenous Tsh prevent a generalised ectopic
expression of Tio, and in consequence a few giants stalk-like structures are observed. In
contrast, in a mutant context background, the ubiquitous expression of Tio will be effective,
and more giants stalk-like structures enriched in FasIII will be produced.
On the other hand, these results indicated that ectopic Tio is able to positively affect
the expression of Hh, at least in the absence of tsh, suggesting that Tio can induce the same
target known for tsh in oogenesis, which is Hh and En. In addition, it was observed ectopic en
(out of their normal domain: TF and CC) in ∆126/tshA3-2-66;UAS-tioFL/hsGal4 (supplementary
Fig.1J).
In the TF cells of the ∆126/tshA3-2-66 ovaries, Tio is ectopic in the Tsh domain, localizing
in all TF cells but never in CC, curiously En display the same domain of expression in this
132
RESULTS_PART III
context of Tsh absence (∆126/tshA3-2-66;Fig 3 and Supplementary Fig. 2D-F). Thus, it is
tempting propose that Tio can contributed to the maintenance of en expression in the
∆126/tshA3-2-66 ovaries. However the absence of Hh from the all TF cells and CC in this ovaries
(see chapter II part II of this manuscript) indicating clearly that in a tsh reduction context, Tio
was unable to maintain hh in TF. It is not clear whether Tio contribute to the maintenance of
en expression in ∆126/tshA-3-2-66 mutant but this result suggest that Tsh might regulate hh
expression in different ways or in a regulatory mutant context (like we suppose that is the case
of the ∆126/tshA3-2-66 ovaries) the reminiscent Tsh prevent the Hh maintenance by Tio, for
example, preventing tio expression in CC. We question, if in a tsh null mutant context, Tio
could not keep En and Hh in all TF and CC. Further experimental work must be performed to
clarify how Tsh and Tio regulates en and hh expression during oogenesis.
In addition, we observed that, unlike the overexpression of tsh or hh, the ectopic
expression of tio produced bigger germarium suggesting extra GSC and germ-line cysts, that
conserve the germline organization observed in wild-type. Unlike to the ∆126/tshA3-2-66
germaria, the hsGal4>UAS-tioFL germaria presented a normal germline/ somatic cell
organization. It is know, that beside the importance of the Hh function for self-renewal and
proliferation of the FSC, this signalling plays a non-essential role in maintaining the GSC, by
having redundant function with the piwi–mediated pathway (King et al., 2001). However the
overexpression of hh stimulates a slight increase in the number of GSC (King et al., 2001).
Thus, it is tempting to suppose that the apparent increase of the germline cyst, in the ovaries
where tio was ectopically expressed, could be the result of the effect of Tio in Hh signalling
favoring GSC instead FSC proliferation. For note, tio is normally expressed only in TF, also
component of the GSC niche. The ectopic tio expression could have affected also the number
of GSC or the rate of germ-line cyst production, originating the accumulation of the cyst in
region 1-2b of the germarium. We speculated that Tsh could mediates the FSC proliferation
while Tio could have a more preponderant role in mediates the GSC proliferation, in both
cases, through Hh signalling.
In a tsh mutant background, may be the overexpression of tio produces germaria with
normal size because the increase in GSC number and or proliferation rate was accomplished by
an increase in follicle production, documented by the more frequent presence of giants stalk-
like structures in hsGal4>UAS-tioFL /tshA3-2-66;UAS-tioFL/hsGal4 ovaries, comparatively to
hsGal4>UAS-tioFL ovaries. This suggests that, in some level, endogenous Tsh represses the
ectopic expression of tio or the ectopic effect of Tio in tsh targets during oogenesis.
In conclusion, we confirmed that tsh and tio repressed each other during oogenesis,
and we verified that tio is able to perform a redundant function with tsh in ovaries; Tio and Tsh
share some targets (perhaps indirect targets) like en and hh. We speculate that the ability to
regulate these targets may be assigned to a few sequence similarities between Tio and Tsh,
particularly to the three zinc finger region. The maintenance of En by tsh and tio has been
133
RESULTS_PART III
already described in Drosophila embryos and imaginal development, as well as a commum
function of the two paralogs (Laugier et al., 2005; Laugier, 2005; Bessa et al., 2009). Here we
show that these functions are maintained during Drosophila oogenesis, opening the way to
clarify the relationships of this signalling pathway and its regulation within the GSC and FSC.
134
RESULTS_PART III
Supplementary figures
Supplementary Figure 1- Tsh and En in wild-type, ∆126/tshA3-2-66, tio473 and ∆126/tshA3-2-66;UAS-
tio/hsGAL4 germaria.
(A, B, C) wild-type; (D, E, F) ∆126/tshA3-2-66, (G, H, I) tio473 homozygous germaria and (J) ∆126/tshA3-2-66;UAS-
tio/hsGAL4 stained with Tsh (green) and En (red) antibodies. DNA (blue) was labelled with DAPI. (A, B, C, G, H, I). In
wild-type and tio473 germaria, En is detected in all terminal filament and cap cells. (D, E, F) In ∆126/tshA3-2-
66germarium En is lost in cap cells but maintained in all terminal filament cells. (J) In ∆12 6/tshA3-2-66;UAS-tio/hsGAL4
germarium, en is ectopic out of their wild-type pattern of expression.
Anterior is to the right and posterior is to the left. The scale bar (in panel A) represents 20µm and is the same for all
panels.
135
RESULTS_Part IV:
138
The knock-down of the teashirt gene causes a metaphase-like
arrest in Drosophila larval neuroblasts and S2 cells
Isabel Salazar1, Sílvia Pimentel1,2, Jorg D. Becker3, Laurent Fasano2, Stephen Kerridge2
and Rui Gomes1 *
1 Universidade de Lisboa, Faculdade de Ciências, Departamento de Biologia Vegetal,
Campo Grande, 1749-016 Lisboa, Portugal 2 Institut de Biologie du Développement de Marseille Luminy (IBDML), UMR 6216,
CNRS-Université de la Méditerranée, Campus de Luminy, Case 907, 13288 Marseille Cedex 09,
France. 3 Affymetrix Core Facility, Instituto Gulbenkian de Ciência, Rua Quinta Grande Nº6,
2780-156-Oeiras, Portugal.
Authors for correspondence: (emails: <[email protected]>)
139
RESULTS_PART IV
Abstract
In Drosophila, the teashirt (tsh) gene encodes a zinc finger protein known to be
required for proper specification of the trunk segments of the developing embryo. Tsh is
expressed throughout development and within a number of different tissues. Here we report
the characterisation of two new hypomorphic mutations of teashirt that cause a metaphase-
like arrest in larval neuroblasts, with the majority of mitotic figures exhibiting highly
condensed chromosomes. Metaphase-arrested figures are associated with apparently normal
mitotic spindles and regular centrosomes. Analysis of squashed preparations of tsh mutant
larval neuroblasts with specific antibodies to the checkpoint proteins (Bub1 and Rod) and to
the CENP-A homologue CID revealed normal localisation of these proteins and no premature
sister chromatid separation. Furthermore, in metaphase-arrested neuroblasts cyclin-A appears
to be degraded whereas cyclin-B remains detectable. Double mutants tsh rod and tsh bub1
display phenotypes that resemble the phenotype of the tsh single mutant, as their neuroblasts
present an elevated mitotic index and a strong metaphase-like arrest, with highly condensed
chromosomes associated with detectable levels of cyclin-B. Therefore, rod and bub mutations
cannot override the strong mitotic arrest produced by the mutant tsh alleles and progression
to anaphase is inhibited. Similarly, the depletion of tsh in Drosophila S2 cells by double-
stranded RNA interference (RNAi) disrupts mitotic progression and mimics the tsh mutant
phenotype in neuroblasts. Throughout the course of RNAi experiments, most cells arrest in a
metaphase-like configuration leading to a remarkable increase in the metaphase and mitotic
indices. Microarray data identified ihog as a significantly downregulated gene in tsh mutants.
Our results strongly suggest that Tsh is required for mitosis progression possibly acting trough
the Hedgehog-pathway.
Key words: Drosophila, teashirt, mitosis, metaphase-anaphase transition, anaphase-
promoting complex/cyclosome (APC/C), neuroblasts, mitotic mutant, hedgehog, iHog.
Introduction
The teashirt (tsh) gene encodes a protein with three atypical widely-spaced C2-H2 zinc
finger motifs and it is required for the patterning of the trunk segments in Drosophila
embryogenesis in collaboration with the Hox genes (Fasano et al., 1991; Roder et al., 1992).
Loss-of-function mutations in tsh lead to trunk-to-head transformation, whereas ectopic
expression of tsh results in head-to-trunk transformation (Fasano et al., 1991; Roder et al.,
1992; de Zulueta et al., 1994). More recently, it was shown that Tsh directly interact with the
Hox gene Sex combs reduced (Scr; Taghli-Lamallem et al., 2007).
Mutants in tsh also resemble segment-polarity genes, revealing that tsh acts as a
temporal and spatial modulator of the Wingless (Wg) pathway in the ventral ectoderm, and
that it is required for development of the proximal parts of the adult appendages (Erkner et
al., 1999; Wu and Cohen, 2000) and for specification of hinge structures of the wing (Azpiazu
and Morata, 2000; Bessa et al., 2002; Casares and Mann, 2000; Pan and Rubin, 1998; Soanes
et al., 2001; Pan and Rubin, 1998).
Interestingly, vertebrate Tsh proteins were isolated and their expression patterns
suggest that function has been conserved from Drosophila to the vertebrates (Caubit et al.,
2000; Manfroid et al., 2004).
Progression into and through mitosis is controlled by activation and inactivation of M-
phase promoting factor (MPF), a complex comprising the Cyclin-dependent kinase 1 (Cdk1,
Cdc2 in Drosophila) and a Cyclin regulatory subunit. In animal cells, the mitotic Cyclins A (Cyc
A) and B (Cyc B), which bind as regulatory subunits to Cdk1, are required for entry into
mitosis.
MPF activity is also modulated by post-translational mechanisms, notably
phosphorylation on inhibitory sites of CdK1, (Thr14 and Tyr15) by the kinases Myt1 and Wee1,
respectively (Porter and Donoghue, 2003). Activation of Cdk1 occurs through the combination
of three required steps: phosphorylation of Thr161, in late G2 by the Cdk-activating kinase
(CAK); nuclear localization of Cdk1/cyclin B1 is promoted by Plk1 phosphorylation of Ser147
and dephosphorylation of the Cdk1 in the Thr14 and Tyr15 by members of the Cdc25
phosphatase family (only one member in Drosophila: String; Porter and Donoghue, 2003;
Fesquet et al., 1993). Activation of Cdc25C is achieved by phosphorylation of Ser198 by Plk1
(Roshak et al., 2000).
Polo-like kinases (PlK) are well established modulators of cell cycle progressionin a wide
range of organisms participating in many aspects of cell division such as mitotic entry, bipolar
spindle formation, chromosome segregation, mitotic exit and cytokinesis (Glover et al., 1998;
Nigg, 1998).
142
A series of phosphorylations catalysed directly or indirectly by MPF provokes profound
structural reorganisation of the cell and drives the formation of the metaphase spindle, while
inactivation of MPF triggers the completion of mitosis and the return to the interphase state.
During mitosis is essential to ensure the equal distribution of a complete set of
chromosomes by each daughter cell. The spindle assembly checkpoint (SAC) or metaphase
checkpoint is a quality control mechanism that prevents chromosome segregation errors. It
acts to restrain cells from entering anaphase, until all replicated chromatids have formed
proper attachments to a functional bipolar spindle. The checkpoint pathway transduces an
inhibitory signal generated by improperly attached kinetochores that delays anaphase onset.
This signal is presumably amplified and ultimately acts to halt the action of the APC (reviewed
by Hoyt, 2001; Shah and Cleveland, 2000; Amon, 1999).
The SAC acts via a signal transduction pathway composed of the Mad2 and Bub
proteins to inactivate Cdc20 (Alexandru et al., 1999; Fang et al., 1998; Hwang et al., 1998;
Kim et al., 1998). This prevents the degradation of Securin and consequent chromosome
segregation until all the chromosomes be attached to both spindle poles.
The Securins must be degraded to initiate sister chromatid separation and to allow the
onset of anaphase. All these proteins are degraded by ubiquitin-mediated delivery to the
proteosome 26S, which requires an ubiquitin ligase called the anaphase-promoting
complex/cyclosome (APC/C) (for review see Peters, 1999; Peters, 2006; Zachariae and
Nasmyth, 1999).
The APC/C is under complex control via phosphorylation Rudner and Murray, 2000) and
by binding one of two WD 40 repeat proteins: Cdc20 (Fizzy/Slp1/p55Cdc) and Cdh1 (hct1/Fizzy-
related/Srw1/ste9), which function as rate-limiting and cell cycle stage-specific activators of
the APC/C (for review see Morgan, 1999). In Drosophila, as in animal cells, Cdc20Fizzy initiates
APC/C-dependent degradation at metaphase-to-anaphase transition (Dawson et al., 1995;
Leismann et al., 2000; Sigrist et al., 1995).
The proteolytic degradation of Cyc A and B is essential for the exit from mitosis (Jacobs
et al., 2001; Jacobs et al., 1998; Lehner and O'Farrell, 1989; Parry and O'Farrell, 2001; Sigrist
et al., 1995). Once Cyclins are destroyed, activation of the APC/C by Cdc20 declines and is
replaced by Cdh1Fizzy-related until cells enter S phase (Sigrist and Lehner, 1997).
Here we describe two new tsh alleles (tshNG1and tshA3-2-66) and their molecular and
phenotypic characterization. Our analysis revealed that the Tsh protein is essential for mitotic
progression in larval neuroblasts and S2 cells. In tshNG1 and tshA3-2-66 mutants, the absence of
tsh function causes a metaphase-like arrest in neuroblasts, and the depletion of tsh in S2 cells
by RNAi mimics this mutant phenotype. Here, we discuss potential roles of this transcription
factor in mitosis regulation.
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Materials and Methods
Drosophila strains and identification of tshNG1 and tshA3-2-66 mutants
All genetic markers and balancers are described by Lindsley and Zimm (1992) and
FlyBase (http://flybase.bio.indiana.edu) except as noted. The tshNG1 mutant allele was isolated
after MNNG (N-methyl-N’-nitro-N-nitrosoguanidine) mutagenesis using standard conditions
(Roberts, 1998), and lines carrying an induced lethal on the second chromosome were then re-
balanced over T(2;3)CyO-TM6B,Tb. Since TM6B carries the dominant larval/pupal marker
Tubby (Tb), the use of this translocation allows larvae homozygous for any gene of interest on
the balanced second or third chromosomes to be recognised by virtue of being Tb+. Available
deficiencies for chromosome 2 in Bloomington and Umea Drosophila Stock Centres delimited
the tshNG1 mutation to the cytogenetic interval 39D3-E1; 40A1-F on the basis of lack of
complementation with deficiencies Df(2L)305 (breakpoints 39A-E7; 40A1-F) and Df(2L)TW161
(breakpoints 38A6-B1; 40A4-B1) and full complementation with Df(2L)TW84 (37F5-38A1;
39D3-E1). tshNG was subsequently identified as a tsh mutant allele that fails to complement
tsh8 (a deficiency that removes tsh and the distal breakpoint was localised at 2Kb upstream of
the start codon of tiptop; Fasano et al., 1991; Laugier et al., 2005). The lethal P{lacW}
insertion allele tshA3-2-66 (P{lacW}A3-3-66) was identified after the screening of the Bloomington
P-element insertions at the 38A6-40B1 interval that fail to complement the tshNG1 allele, and
subsequently shown to be unable to complement deficiencies Df(2L)TW161, Df(2L)305 and
tsh8. For rescue experiments, the UAS-tsh13 construct (Gallet et al., 1998) was crossed to the
driver P{w+mC=GAL4-Hsp70.PB}TR2.
To test for embryonic lethality, tshNG1/In(2LR)CyL and tshA3-2-66/In(2LR)CyL stocks were
outcrossed to wild type and Cy+L+ male progeny were backcrossed to the balanced stocks.
Eggs collected over a 24-hour period were aged for 48 hours and examined. No embryonic
lethality of tshNG1 or tshA3-2-66 was observed. On the other hand, embryonic lethality was
detected when these alleles were combined with Df(2L)305 or tsh8. Crosses between tshNG1/+
males or tshA3-2-66/+ males and Df(2L)305/+ females or tsh8/+ females produced 24%
(tshNG1/Df(2L)305), 23% (tshNG1/tsh8), 23% (tshA3-2-66/Df(2L)305) or 22% (tshA3-2-66/tsh8) of
dead eggs (n=200 in each case).
tsh rod and tsh bub1 double mutants were constructed by standards means, using the
genetically null allele rodX5 (Karess et al., 1989, Scaerou et al., 1999, Basto et al., 2000) and
bub1 allele (l(2)k02113 (Basu et al., 1999). Both alleles were a kind gift from R. Karess
(CNRS, Gif-sur-Yvette) and the double mutant constructs were confirmed by segregation and
complementation tests.
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Nucleic acid procedures and analysis
Standard protocols were used for nucleic acid preparation and in situ hybridisation
(Gonzalez and Glover, 1993; Sambrook et al., 1989). The pπ 12.20 plasmid (O'Hare and
Rubin, 1983) was used as a probe to confirm the cytological location of the P{lacW} tshA3-2-66
insertion to the interval 39E-40A. A genomic DNA fragment next to the P{lacW} insertion was
isolated by plasmid rescue: DNA from heterozygous tshA3-2-66 flies was digested with EcoRI,
self-ligated, and transformed into TOP10 E. coli competent cells (Invitrogen). The fly genomic
DNA from the chimeric plasmid was completely sequenced starting from primer P1 (5’-GAC
CAC CTT ATG TTA TTT CA-3’) and primer lacW8465 (5’-AAT AGG CGT ATC ACG AGG C-3’),
both specific for the P{lacW} transposon. Manual DNA sequencing was performed with the
Sequenase 2.0 Kit (Amersham Pharmacia) and sequences were independently confirmed by an
automated ABI377 v3.0 DNA Sequencer (Toplab, Germany). Homology searches using the
BLASTN 2.0 programs (Warren Gish, unpublished; Altschul et al., 1990) identified a fragment
within the sequence AE003781 (GenBank) which includes the teashirt gene. The structure of
the mutant allele tshNG1 was determined by primer walking and sequencing of PCR-generated
fragments starting from the primers: (5’-AGC AGG ACG GCG CAT GCG A-3’) and (5’-GGC GGT
CTT CTC CTT CTT CA-3’). These fragments were subcloned into pCR2.1-TOPO (Invitrogen) and
double–stranded sequenced by an automated ABI377 v3.0 DNA Sequencer (Toplab, Germany).
For Northern analysis, poly(A)+ RNA was extracted from wild type, revertant,
transformant and homozygous tsh third instar larvae using the QuickPrep Micro mRNA Kit
(Amersham Pharmacia). Twenty microgram samples of poly(A)+ RNA were subjected to
electrophoresis in 1.2% agarose gels containing formaldehyde. The tsh probe used for
Northern was a mixture in equal parts of the following cDNA clones (ESTs) from the Berkeley
Drosophila Genome Project (BDGP) and Research Genetics: CK00422, CK01473, LP11282,
LP07848 and LP44646. As a control for loading, the Northern was re-probed with specific ESTs
for the NUCB1 gene (Otte et al., 1999) made from an equal mixture of the following cDNA
clones from BDGP and Research Genetics: LD40692, LP03575, LP08122, GH05163 and
CK01389.
Rescue of tsh mutants
Males from transformed lines carrying the transgene on the third chromosome (w;
CyO/Sp; UAS-Tsh13/ UAS-Tsh13) (Gallet et al., 1998) were crossed to T(2;3)CyO
TM6,Tb/tshNG1. In parallel, females In(2L)Cy In(2R)Cy, al2 Cy lt3 cn2 L4 sp/S Sp Bl bwD were
crossed to w;TM3,P{w+mC=GAL4-Hsp70.PB}TR2,Ser/Sb males. Among the F1 progeny,
females CyO/tshNG1; UAS-Tsh13/+ were selected and mated to bwD/+; TM3, P{w+mC=GAL4-
Hsp70.PB}TR2, Ser /+. F2 males bwD/tshNG1; TM3, P{w+mC=GAL4-Hsp70.PB}TR2, Ser / + or
UAS-Tsh13 were crossed to CyO/tshNG1; UAS-Tsh13/+ females. Offspring were heat shocked in
145
a water bath at 37ºC for 30 minutes twice every day and allowed to develop at 25ºC. Animals
bearing the same construct without heat shock treatment were prepared in parallel. An
analogous procedure was followed to examine the ability of the transgene to rescue the mitotic
defects and viability of tshA3-2-66 flies.
The production of ectopic Tsh protein rescued the mitotic defects in the central nervous
system (CNS) in third instar larvae of tshNG1/ tshNG1; TM3, P{w+mC=GAL4-Hsp70.PB}TR2, Ser /
UAS-Tsh13 and tshA3-2-66/ tshA3-2-66 ; TM3, P{w+mC=GAL4-Hsp70.PB}TR2, Ser / UAS-Tsh13, and
the development proceeded shortly after the eclosion from the pupal case. Animals bearing the
same constructs without heat shock treatment failed to rescue the abnormal phenotypes, as
well as control tshA3-2-66 and tshNG1 mutants carrying either UAS-tsh13 or the hs-GAL4 driver
when subjected to the same heat shock treatment.
Cytology and Immunofluorescence Observations
Larval brain squashes and immunostaining of whole-mount third instar larval CNS and
imaginal discs were carried out as described (Gonzalez and Glover, 1993). Microtubules were
detected with rat anti-α−tubulin antibody, YL1/2 (1:100, Sera-Lab) and Alexa 488-conjugated
anti-rat (1:200, Molecular Probes). Centrosomes were revealed with mouse anti-γ-tubulin
GTU88 (1:100, Sigma) and an FITC-conjugated horse anti-mouse secondary antibody (1:50;
FI-2001, Vector Laboratories). Cyclin A and B (Whitfield et al., 1990; a kind gift from A.
Tavares, IGC, Lisboa), Polo Ma294 (Llamazares et al., 1991; a kind gift from A. Tavares, IGC,
Lisboa), Rod (Scaerou et al., 1999, Scaerou et al., 2001), Bub1 (Basu et al., 1999), CID
(Blower & Karpen; a kind gift from R. Karess, CNRS, Gif-sur-Yvette) were detected with
specific rabbit antibodies and used at 1:100, 1:200, 1:100, 1:500, 1:1000 and 1:1000
respectively. Secondary antibodies were made in goat and conjugated to FITC or rhodamine
(Jackson ImmunoResearch Laboratories). DNA was stained with 1µg/ml of propidium iodide or
Hoechst 33258 and the brains were mounted in Vectashield (Vector Laboratories).
Immunoblotting
Standard protein and immunological techniques were followed (Harlow and Lane, 1988;
Sambrook et al., 1989). For immunoblotting, secondary antibodies were all peroxidase-coupled
and detection was performed using the Lumi-Light Plus Western Blotting Kit (Roche). Anti-rat-
POD was bought from Sigma. Total protein samples from Drosophila third instar larvae and
Schneider S2 cells were prepared by homogenisation in SDS sample buffer. We used the
follow primary antibodies: rat anti-Tsh (1:500); mouse anti-Fizzy (1:20; a kind gift from C.
Lehner) and rabbit anti-Polo MA294 (1:500, a kind gift from A. Tavares, IGC, Lisboa).
146
RNAi treatment and analysis of S2 Tissue culture cells
RNAi was performed according to the methods of Clemens et al. (2000) using target
sequences that exhibited minimal homology with others genes as determined by BLAST
comparison. The template for in vitro transcription was generated using the primer pairs: 5´-
TAA TAC GAC TCA CTA TAG GGA GAC CAC AGC GGG TCA ATT CCC TGA CT-3´ and 5´-TAA TAC
GAC TCA CTA TAG GGA GAC CAC GAT TTG CAT GGC CTG TTC CA-3´. The Purified PCR product
(~650 bp in length) was used to produce double-strand RNA (dsRNA) using Megascript T7
transcription kit (Ambion) according to the manufacturer’s instructions. Schneider S2 cells
were grown in Schneider’s Drosophila medium (Gibco BRL) supplemented with 10% fetal calf
serum (FC, Gibco) and 50 µg/ ml streptomycin and penicillin at 25 ºC. To perform RNAi cells
were treated with 15 µg dsRNA as described by Somma et al., 2002. Control cultures were
prepared in the same way with GFP-dsRNA. Both cultures were grown for 144 h at 25ºC and
then processed for biochemical and cytological analysis. For immunofluorescence, cells were
processed as previously described (Adams et al., 2001). Immunostaining of microtubules and
centrosomes was performed with antibodies against α−tubulin and γ-tubulin (Sigma-Aldrich).
Secondary antibodies were FITC-α-rat and Texas red-α-mouse (Jackson ImmunoResearch
Laboratories).
Microscopy images
Larval CNS and S2 cells were examined under phase contrast or fluorescence using a
Zeiss Axioplan II microscope with a 100x/1.30 oil immersion Plan-Neofluar objective. Images
were acquired with a SPOT-2 camera (Diagnostics Instruments, Inc.) and processed with
Adobe Photoshop 6.0 (Adobe System).
RNA Isolation, Target Synthesis and Hybridization to Affymetrix GeneChips
Total RNA was extracted from an appropriate number of brains (2x150 wild type brains
and 2x300 mutant brains) with 71,85 ng/µl (70,2 ng/µl-73 ng/µl) of RNA in average per
sample using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Concentration and purity was
determined by spectrophotometry and integrity was confirmed using an Agilent 2100
Bioanalyzer (Agilent Technologies, Palo Alto, CA).
RNA was processed for use on Affymetrix (Santa Clara, CA, USA) Drosophila Genome
Arrays, according to the manufacturer’s Small Sample Labeling Protocol VII described in the
technical note “GeneChip Eukaryotic Small Sample Target Labeling Assay Version II”
(http://www.affymetrix.com/support/technical/technotes/smallv2_technote.pdf). Briefly, 100
ng of total RNA was used in a reverse transcription reaction (SuperScript II; Invitrogen,
Paisley, UK) to generate first-strand cDNA. After second-strand synthesis, double-strand cDNA
147
was used in an in vitro transcription (IVT) reaction to generate cRNA (MEGAscript T7 kit;
Ambion, Austin, TX). 400 ng of this cRNA was used for a second cDNA synthesis, followed by a
second IVT reaction to generate biotinylated cRNA (ENZO BioArray HighYield RNA Transcript
Labeling Kit; ENZO Diagnostics, Farmingdale, NY). Size distribution of the cRNA and
fragmented cRNA, respectively, was assessed using an Agilent 2100 Bioanalyzer.
15 µg of cRNA was used in a 300-µl hybridization containing added hybridization
controls. 200 µl of mixture was hybridized on arrays for 16 h at 45°C. Standard post
hybridization wash and double-stain protocols were used on an Affymetrix GeneChip Fluidics
Station 400. Arrays were scanned on an Affymetrix GeneChip scanner 2500.
Data Analysis of the Affymetrix GeneChips
Scanned arrays were analyzed first with Affymetrix MAS 5.0 software to obtain
Absent/Present calls and for subsequent analysis with dChip 1.3 (http://www.dchip.org, Wong
Lab, Harvard). The four arrays used in this study (biological replicates) were normalized
applying an Invariant Set Normalization Method (Li and Wong, 2001b). Then the normalized
probe cell intensities of the 4 arrays were used to obtain model-based gene expression indices
based on a PM-only model (Li and Hung Wong, 2001a). Replicate data for the same sample
were weighted gene-wise by using inverse squared standard error as weights. Only genes
called Present in at least 1 of the 4 arrays and within replicate arrays called Present within a
variation of 0 < Median (Standard Deviation/Mean) < 0.5 were kept for downstream analysis
(7099 genes). Thus, genes called absent in all arrays and genes with inconsistent expression
levels within replicate arrays were excluded. Finally, all genes compared were considered to be
differentially expressed if they were called Present in at least one of the arrays and if the 90%
lower confidence bound of the fold change between experiment and baseline was above 1.2.
The lower confidence bound criterion means that we can be 90% confident that the fold
change is a value between the lower confidence bound and a variable upper confidence bound.
Li and Wong (2001b) have shown that the lower confidence bound is a conservative estimate
of the fold change and therefore more reliable as a ranking statistic for changes in gene
expression. This criterion has been used in other gene expression studies (Becker et al., 2003;
Ramalho-Santos et al., 2002). A LCB threshold of 1.2 seems to be justified, because
comparisons with real-time PCR data showed that Affymetrix Genechips analyses are reliable
and underestimate differences in gene expression (Yuen et al., 2002).
Annotations for the ~13.500 genes represented on the Drosophila Genome Array were
obtained from the NetAffx database (www.affymetrix.com) as of June 2003.
148
Results
1-Molecular analysis of the tsh mutant alleles
The tshNG1 allele was selected amongst a collection of second chromosome late larval
lethals after nitrosoguanidine mutagenesis (Materials and Methods) and a non-complementing
allele (tshA3-2-66) was further identified as a P{lacW} insertion within the promoter of the tsh
gene. A 2 kb EcoRI genomic fragment adjacent to the P{lacW}A3-2-66 insertion was cloned by
plasmid rescue and used as a probe to confirm by in situ hybridisation its localisation to 40A on
wild-type polytene chromosomes (data not shown). DNA sequencing revealed that the
P{lacW}A3-2-66 element is inserted 439 bp upstream of the putative transcription start site
(GTCAGTCG), i.e., 1652bp upstream the translation start (Fig. 1). DNA sequencing of PCR-
generated fragments of tshNG1 allele revealed a deletion of 7 bp at {-18, -26bp} from the
larger exon, which deletes the putative branch site consensus and affects the splicing (Fig. 1).
Northern blot analysis in wild-type third instar larvae confirmed the expression of tsh
transcripts of two size classes: a predominant class of transcripts of 5.4 kb and a minor class
of 8.5 kb transcripts corresponding to the previously reported 5-5.4 kb and 8.1-8.5 kb tsh
transcripts expressed throughout development (Fig. 2A, lane 5;Fasano et al., 1991). In tshA3-2-
66 homozygotes the 8.5 kb transcripts are missing and the 5.4 kb are scarcely expressed,
suggesting that the reduction in the tsh transcripts is associated with the mutant phenotype
(Fig. 2A, lane 2). Transcript expression was restored in those lines showing full reversion to
the wild-type, and in which remobilization of P{lacW}A3-3-66 with the ∆2-3 transposase source
(supplementary material) resulted in its precise excision (Fig. 2A, lane 3). In larvae
homozygous for tshNG1 only an abnormal 7Kb transcript is observed (Fig. 2A, lane 1).
Presumably the absence of the normal transcripts of 5,4 Kb and 8,5 Kb and, the presence of
these abnormal tsh mRNA of 7kb, are responsible for the defects observed in tshNG1 mutants.
To confirm that the tsh transcription unit was responsible for the tsh phenotype, we undertook
to rescue the mutant phenotype by expressing the tsh cDNA using the hs-Gal4/UAS system
(Brand and Perrimon, 1993) in the mutant backgrounds of tshNG1 and tshA3-2-66 (see materials
and methods and Fig. 2A, lane 4). We found that a 30-min heat-shock at 37ºC twice a day
gave the rescue of all mitotic defects observed in both tsh mutant homozygotes and they
reached the adult stage. The lethality was shifted from larval to adult stage.
Expression of Tsh in mutant larval central nervous system (CNS) was also assessed by
Western blotting (Fig. 2B) using a previously described anti-Tsh antibody (Gallet et al., 1998).
On Western-blots this antibody detected a 116 kDa isoform of Tsh in wild-type third instar
larval CNS (Fig. 2B, lane 1). In contrast, the amount of Tsh protein is undetectable in the
tshNG1 and tshA3-2-66 mutants (Fig. 2B, lanes 2 and 3 and Supplementary fig.1). Thus, tshNG1
and tshA3-2-66 mutants appear to be protein nulls or severe hypomorphic alleles. The same blot
was re-probed with anti-Fizzy as a loading control (Fig. 2B, bottom line).
149
Figure 1- Molecular organisation in tshNG1 and tshA3-2-66 mutants.
(A) Schematic overview of the tsh region. Sites of P-element insertion causing tshA3-2-66 mutation and genomic deletion
associated with the lethal tshNG1 allele are explicitly indicated. The tsh transcription unit (open and black boxes) is
interrupted by one intron (kinked lines). Non-coding 5’ and 3’ regions of the transcript are shown in open boxes. The
tshA3-2-66 insertion site maps at –439 nucleotides upstream the transcription start site i.e., 1652bp upstream the
translation start, possibly within the promoter region. The tshNG1 allele deletes 7 bp at {–18, -26 bp} upstream the
second exon. (B) Excerpts from the DNA sequence AE003781 (GenBank) showing relevant segments of the tsh gene,
the tshA3-2-66 insertion site (indicated by a triangle) and the deleted nucleotides in tshNG1 (in bold). The start site of tsh
transcription is in bold and indicated by an arrow.
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Figure 2- Expression analysis of Tsh.
(A) Northern analysis of tsh expression in tshNG1 and tshA3-2-66 mutants. Lanes 1-5 were loaded with 20 µg of poly(A)+
RNA extracted from homozygous third instar larvae of tshNG1, tshA3-2-66, tshRev1 , tshhs-GAL4:UAS-Tsh and wild-type,
respectively. At the bottom is shown a loading control of the same filter with the NUCB1 probe. (B) Western analysis
of Tsh and Fizzy in CNS cells from tshNG1 and tshA3-2-66 homozygotes. Lanes 1-3 were loaded with proteins extracted
from 12 wild-type third instar larvae, 24 tshNG1 third instar larvae and 18 tshA3-2-66 third instar larvae respectively. The
116 kDa Tsh protein is not detectable in tsh alleles. The same blot probed with anti-Cdc20 (Fizzy) shows equal
amounts of this protein in tsh mutants by comparison with the wild-type.
2- tshNG1 and tshA3-2-66 mutations cause a metaphase-like arrest
tshNG1 homozygotes die as third instar larvae with abnormal trunk. Dissection of these
larvae revealed that imaginal discs are absent or very small and the size of the CNS is also
reduced. This phenotype suggests that tsh gene activity is essential for cell proliferation.
Observations of squashed preparations of the larval CNS from tshNG1 homozygotes revealed
several mitotic defects (Fig.3C, D, G and H). In contrast to wild-type cells (Fig. 3A, B and G),
where hyperploids are never seen, 85% of total mitotic tshNG1 cells (n=1000) contained more
than a diploid complement of chromosomes (Fig. 3C and D). The overall mitotic index in tshNG1
larval CNS was almost three times higher than wild-type (Fig. 3G) and the frequency of
anaphases is very low (0.2%, Fig. 3H), indicative of a metaphase-like arrest. Moreover, in
most mitotic cells, chromosomes are much more highly condensed than that seen in wild-type,
indicating that cells have been delayed in mitosis for some time. A proportion of cells show a
high ploidy number (15-20%, Fig. 3D), suggesting that some metaphase-arrested cells are
capable of reverting to interphase and undergo DNA replication in the absence of chromosome
segregation or cytokinesis.
151
Figure 3- Mitotic phenotypes in tsh
mutant larval brains.
Wild-type control (Oregon-R) metaphase (A)
and anaphase (B) figures. (C) Aneuploid
metaphase figure with hypercondensed
chromosomes from a tshNG1 homozygote. (D)
Aneuploid overcondensed mitotic figure from a
tshNG1 homozygote. (E) Hypercondensed
metaphase figure from a tshA3-2-66 larval
neuroblast. (F) Anaphase figure with lagging
chromatids (arrows) from a tshNG1/tshA3-2-66
trans-heterozygote. (G and H) Mitotic
parameters in larval brains. Error bars
represent standard error of the means. At least
500 fields were counted. (G) Mitotic index is
calculated as the number of mitotic cells per
microscope field using a 100 x objective and 10x/25 eyepieces. (H) Percentage of anaphases is calculated as 100x
(number of anaphases)/ (number of mitotic cells). Bar in A represent 10 µm and is the same for all panels from A-F.
tshA3-2-66 homozygotes also die in third instar larvae with very reduced imaginal discs
and a smaller CNS. tshA3-2-66 squashed CNS display mitotic abnormalities such as aneuploidy
and/or hypercondensed chromosomes (Fig. 3E). The proportion of anaphase figures in tshA3-2-
66 is higher (10%) than in tshNG1 (0.2%), but 3.5 times lower than in wild-type (Fig. 3H),
indicative of a significant delay in the metaphase-anaphase transition. Moreover, 49% of tshA3-
2-66 homozygotes anaphases are abnormal: 24% are disorganised, 16% show lagging
chromatids and 9% are overcondensed (n=1000). Paradoxically, the mitotic index of tshA3-2-66
brains is 2.4 times lowers than wild-type, while the tshNG1 allele causes a significant increase
(Fig. 3G). For note, tshA3-2-66 neuroblasts display higher levels of apoptosis, compared to the
wild-type (Supplementary fig.2).Trans-heterozygotes tshNG1/ tshA3-2-66 die as late larvae and
CNS cells show an abnormal phenotype comparable to tshA3-2-66 homozygous (Fig. 3F, G and
H).
To elucidate if the homozygous tshNG1 or tshA3-2-66 larvae were dying earlier, a simple
test for embryonic lethality was carried out (see Materials and Methods). Trans-heterozygotes
tshNG1/tsh8 or tshA3-2-66/tsh8 die at embryogenesis, before hatching, as does the tsh8 null mutant
(Supplementary fig. 3; Fasano et al., 1991; Gallet et al., 1998; Roder et al., 1992). We
examined such eggs but were unable to detect obvious mitotic defects either in syncytial or
cellularized embryos. This may result from the fact that tsh transcripts are initially detected at
the end of 13th nuclear division of the syncytial embryo (Fasano et al., 1991). We conclude
that the mitotic role of tsh occurs later in the development as compared with the homeotic
role, and that tshNG1 and tshA3-2-66 are hypomorphic alleles.
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Figure 4- Mitotic figures in preparations of larval CNS cells from wild-type and tsh homozygotes.
Microtubules revealed with anti-α-tubulin are shown in green (A-F), centrosomes revealed with anti-γ-tubulin are
shown in green (G-I) and DNA stained with propidium iodide is shown in red. (A and G) Wild-type metaphase. (D)
Wild-type anaphase. Normal bipolar spindles in a tshNG1 polyploid hypercondensed metaphase (B) and in a tshA3-2-66
overcondensed metaphase (C). (E) tshNG1 polyploid cell containing a normal bipolar spindle in an anaphase-like
configuration. (F) Bipolar spindle in an asymmetric anaphase from tshA-3-2-66. Wild-type (G), tshNG1 (H) and tshA-3-2-66 (I)
metaphase cells containing two normal centrosomes. Bar represent 5 µm and is the same for all panels.
3- tsh mutants organise apparently normal spindles
We analysed mitotic spindle structure in tsh mutants by immunostaining whole-mount
preparations of the larval CNS to visualise microtubules and centrosomes. We found that the
majority (80%, n=1000) of mitotic cells observed in tsh mutants develop an apparently
normal bipolar spindle; the aneuploid and polyploid overcondensed metaphases and anaphases
were in association with bipolar focused spindles (Fig. 4B, C, E and F). Moreover, the majority
of the abnormal mitotic figures have a normal number of centrosomes at the two poles (one at
each pole; Fig. 4H and I). Rarely, only 3-5% (n=300) of metaphase-arrested cells of tshNG1
mutant have a monopolar spindle, where the condensed chromosomes are arranged around a
single centrosome (data not shown).
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Figure 5- Cyclin A and B levels in wild-type and tsh mutant larval CNS cells.
In Cyclin A panel (leftt panel), microtubules are in green, cyclin A is in red and DNA (blue) is stained with Hoechst
33258 in merged (left) images. The monochromatic image on the right is of cyclin A alone, as revealed with the
antibody Rb270. DNA staining alone can be seen in the inserts on bottom right of the merged figures. In cyclin B panel
(revealed with the antibody Rb271; right panel), cyclin B is in green and DNA in red was stained with propidium
iodide, (A and B) Cyclin A in a wild-type (WT) prophase and metaphase cell, respectivaly. (C and D) Absence of cyclin
A staining in tshA3-2-66 and tshNG1 mutant neuroblasts, respectively. (E) Cyclin B in a wild-type (WT) metaphase cell. (F)
Cyclin B in a wild-type (WT) anaphase cell. (G and H) High levels of cyclin B in tshA3-2-66 and tshNG1 mutant condensed
metaphases, respectively. (I and J) Elevated levels of cyclin B in tsh rod and tsh bub1 polyploid metaphases,
respectively.
In Drosophila, degradation of mitotic Cyclins A, B and B3 are required to allow
inactivation of the associated Cdk1, exit from mitosis and restoration of an interphase stage
(Sigrist et al., 1995). Cyclin A and Cyclin B proteins are targeted for destruction during mitosis
by the anaphase-promoting complex (APC/C)-fzy (Charles et al., 1998; Kotani et al., 1998).
We therefore examined whether Cyclin A and B degradation could take place in tshNG1 and
tshA3-2-66 neuroblasts. In wild-type, Cyclin A is abundant in prophase (Fig. 5A) but is degraded
during the prometaphase/ metaphase transition, ahead of Cyclin B, which one is still abundant
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at metaphase (Fig. 5D; Parry and O'Farrell, 2001; Sigrist et al., 1995). After the progressive
Cyclin B degradation at various stages of the metaphase-anaphase transition, wild-type
anaphases have low levels of this mitotic Cyclin (Fig. 5E). In metaphase-arrested tshNG1 (Fig.
5C and G) and tshA3-2-66 neuroblasts (Fig. 5B and F) Cyclin A has been normally degraded but
Cyclin B is still detected.
4- Strong tsh mitotic mutations do not disrupt the association of centromere
and kinetochore proteins
Rough Deal (Rod) is a checkpoint protein that is required for the metaphase arrest in
response to spindle damage, and is also required during normal mitosis to delay anaphase
onset until all chromosomes are properly attached to the spindle (Scaerou et al., 2001; Basto
et al., 2000; Scaerou et al., 1999). During mitosis, the Rod protein accumulates on
kinetochores until cells reach a pro-metaphase state. In metaphase, when chromosomes are
aligned at the spindle equator, Rod decorates kinetochore spindle fibers in an irregular pattern
(Fig. 6A) and remains associated with separating kinetochores throughout anaphase (Scaerou
et al., 2001; Basto et al., 2000). We examined the localisation of this checkpoint protein on
tshNG1 neuroblast cells. On all the bipolar spindles, Rod protein was localised as in wild-type,
with prominent kinetochore signal in prometaphases, and distributed along spindle
microtubules in metaphase plates (Fig. 6B and C).
Furthermore, we examined the localisation of the kinetochore-associated kinase Bub1
on tshNG1 neuroblast cells. Bub 1 is a classical checkpoint protein that is required for the
metaphase arrest in response to spindle damage (Taylor & McKeon, 1997, Basu et al., 1999).
In wild-type, the Bub1 protein is associated with kinetochores in metaphase cells from the
larval CNS (Fig. 6D) and is barely detected after anaphase onset. We found that in metaphase-
like arrested tshNG1 cells, Bub1 was present on all chromosomes and stained kinetochores pairs
as intensively as in control metaphase cells (Fig. 6E). Moreover, Bub 1 was also localised on
kinetochores of tshNG1 anaphase-like cells (Fig. 6F) clearly indicating that sister chromatids did
not disjoin. To more accurately determine the state of chromosomes in these cells, we looked
at the distribution of Cid protein that binds to the centromeric regions of chromosomes (Blower
& Karpen, 2001). In control cells, the centromeres were closely paired in metaphase (Fig. 6G)
but well separated in anaphase. In the majority of tshNG1 neuroblasts, all the centromeres are
tightly clustered in a metaphase-like alignment (Fig. 6H). Furthermore, in anaphase-like cells
Cid staining revealed chromosomes aligned as pairs, rather than chromatids, that are being
pulled apart by tension on the spindle (Fig. 6I). Together this results indicate that mutant
tshNG1 cells fail to undergo sister chromatid separation even in cells that displayed an
anaphase-like configuration.chromome
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Figure 6- Distribution of centromere CID and kinetochore components Bub1 and Rod in wild-type
and tshNG1 neuroblast cells.
In Rod panel, DNA is in blue, Rod isin red and microtubules are in green. In Bub and CID panels, DNA is in red and
Bub1 or Cid are in green. (A) Rod in a wild-type metaphase cell. (B and C) tshNG1 metaphase-like cells where Rod
localises both at kinetochores and is associated with KMTs. (D) Bub1 in a wild-type metaphase cell. (E) tshNG1
metaphase-like cell showing normal Bub1 staining at kinetochores. (F) tshNG1 anaphase-like cell showing strong
abnormal staining at both kinetochores revealing that sister chromatid separation did not occur. (G) Cid in a wild-type
metaphase. (H) tshNG1metaphase-like cell showing normal Cid staining at centromeres. (I) tshNG1 anaphase-like cell
stained to reveal Cid, confirming that sister chromatid did not disjoin.pair in metaphase (Fig. 6G) but w
5- tshNG1 mutant cells are in a state of metaphase arrest even in the absence of
the checkpoint proteins Bub1 and Rod
To analyse whether tshNG1 cells were in a state of checkpoint arrest, we examined the
mitotic progression in the larval CNS of tsh cells, which were also homozygous-mutant for the
rod or bub1 genes.
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Table 1- Mitotic index (number of mitotic cells per optic field) and percentage of anaphase figures in the
larval CNS of wild-type, tsh, rod x5, bub1, tsh rodx5 and tsh bub1 mutants.
Mitotic Index % Anaphase
Wild-type 1.8 17
teashirt (tshNG1) 5.1 0.2
rodx5 0.9 24
teashirt rod x5 5.5 0.6
bub1 0.7 38
teashirt bub1 3.9 0.4
Double mutants tsh rodx5 display a high mitotic index and a phenotype similar to the
tsh mutation alone (Table 1). Neuroblasts show a metaphase-like arrest with very condensed
chromosomes and detectable levels of Cyclin-B (Fig. 5H). Therefore, even a checkpoint
mutation as rod cannot override the arrest imposed by tsh. Like tsh rodx5, tsh bub1 mutants
show a high mitotic index, detectable Cyclin-B levels and a metaphase-like arrest similar to tsh
mutation alone (Table 1 and Fig. 5I). These results indicate that neither Rod or Bub1 are able
to override the metaphase arrest imposed by tsh. Even after relieving the metaphase
checkpoint in tshNG1 mutants, the anaphase-promoting complex (APC) is unable to promote
anaphase onset in tsh mutants. Together, these data strongly suggest that, in the absence of
tsh activity, cells arrest in metaphase even in the presence of an active checkpoint, essentially
because progression is inhibited.
6- Depletion of Tsh function in S2 cells also affects mitotic exit as seen in
mutants
To determine whether there were similar effects on mitotic progression, we performed
Tsh-dsRNAi in Drosophila S2 cells. The S2 tsh-dsRNA-treated cells will be referred
subsequently as Tsh-depleted cells and the control GFP-dsRNA-treated cells will be referred
subsequently as control cells. To analyse the Tsh depleting effects on mitotic S2 cells, we
stained every 24h Tsh-depleted cells and control cells with antibodies against α−tubulin and
γ−tubulin and the DNA.
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Figure 7- Time course analysis of Tsh RNAi in Drosophila S2 Cells.
(A) Western Blot showing the levels of Tsh in S2 dsRNAi-GFP(+) or dsRNAi-Tsh treated (-) cells over time. C
represented the untreated S2 cells. These blots were also probed with anti γ-tubulin antibody as loading control. (B)
Mitotic index after Tsh RNAi is represented as the average percentage of mitotic cells (n > 1000) in the total
population. (C and C´) Mitotic parameters quantification during the experiment, in control and in cells after Tsh RNAi
(n>1000). (D and D´) Quantification of the observed mitotic abnomalities in control and RNAi depleted cells (n>1000).
As judged by Western Blotting the Tsh levels decreased significantly (90 %) within 72h
after the addition of dsRNA (Fig. 7A). Compared with control cells, Tsh-depleted cells show an
increased of three-four folds, in the mitotic index by 96 h (the maximum of RNAi mitotic
effects; Fig. 7B). Later the mitotic index, decreased, coincident with a reappearance of Tsh
protein (Fig.7A lane 9 and Fig.7B). Examination of Tsh–depleted mitotic cells revealed a high
frequency of metaphases from 72 to 96 h (66%-76% compared with 44%-58% in control
cells; Fig. 7C' and C) and a concomitant decrease in anaphases and telophases, indicating a
prolonged metaphase arrest as in tsh mutant neuroblasts. Moreover, throughout the course of
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the experiment, the mitotic apparatus in most control cells was well organized with a bipolar
spindle with a single centrosome at each pole (Fig. 8A). However, in cells exposed to tsh
dsRNA from 72 to 120 h, there was a significant increase in mitotic abnormalities. The most
significant aberrant phenotype was the observation of mitotic cells aligned at the equator of a
typical metaphase-like spindle with condensed chromosomes (~25% of mitotic cells; Fig. 7D'
and 8C). This phenotype was maximal at 96 h (Fig. 7C'). A second class of defects, increased
during RNAi experiment, was the assembling of monopolar spindles containing a single or
clustered centrosome at only one pole (Fig. 7D' and 8B; ~10-17% compared with 1-3 % in
control mitotic cells). The third category of defects, which increased after 72 h of Tsh-RNAi,
were the anaphase-like configurations exhibiting lagging chromosomes (Fig. 7D-7D’ and 8F ;
~7 % compared with ~1.5 % of control mitotic cells). Thus, the ablation of Tsh protein
produced a similar metaphase arrest phenotype and revealed striking similar defects in mitotic
cells by comparison with those seen in mutants. These results are consistent and further
support the observation that tsh plays a crucial role in mitotic exit.
Figure 8- Abnormal mitotic figures observed in
S2 TSH-RNAi cells.
Cells were stained for α−tubulin (green), DNA (blue) and γ-
tubulin (red). DNA staining alone can be seen in the inserts on
bottom right of the merged figures. (A) Normal metaphase
displaying a bipolar spindle in a control cell. (B) Polyploid
metaphase displaying a monopolar spindle with a large
centrosome, observed 120 h after tshRNAi. (C) Condensed
metaphase displaying a bipolar spindle observed 96 h after
tshRNAi. (D) Monopolar spindle associated with a metaphase-like
plate exhibiting condensed chromosomes observed 96 h after
tsh
RNAi. (E) Normal anaphase in a control cell. (F) Anaphase-like
cell showing abnormal lagging chromosomes (arrow). Bar, 5
µm.
7- Microarrays data points to some ontology clusters and putative targets of
the tsh gene
In order to get a rough estimate of gene expression differences in tsh mutant brains as
compared to wild-type brains, we screened the Drosophila set of Affimetrix chips. We detected
up- or down-regulation in several genes which have been ordered by lower bound of fold
change (LBFC), after using a cut-off of 1.3 (appendix I).
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Table 2- List of the most significant gene culters altered in tshNG1 homozygous neuroblats microarrays with
the respectived P value and total number of genes include.
Gene cluster P= N°
genes
include
extracellular region" genes 0.000000 34
imaginal disc pattern formation" genes 0.000007 5
"cell fate specification" genes 0.000019 6
"lysozyme activity" genes 0.000021 5
"defense response" genes 0.000087 30
"Notch signaling pathway" genes 0.000165 7
"antimicrobial humoral response (sensu Protostomia)" genes 0.000329 5
"chymotrypsin activity" genes 0.000674 15
"wing disc anterior/posterior pattern formation" genes 0.000780 4
The obtained results are in agreement with previously published results regarding Tsh
function, but suggest some interesting new connections.
In terms of function we found 9 gene ontology clusters with a P-value <0.001
(appendix II and Table 2), namely "extracellular region" genes (P=0.000000; 1); "imaginal
disc pattern formation" genes (P=0.000007; 2); "cell fate specification" genes (P= 0.000019;
3); "lysozyme activity" genes (P=0.000021; 4); "defense response" genes (P=0.000087; 5);
"Notch signaling pathway" genes (P=0.000165; 6); "antimicrobial humoral response (sensu
Protostomia)" genes (P=0.000329; 7); "chymotrypsin activity" genes (P= 0.000674; 8) and
"wing disc anterior/posterior pattern formation" genes (P=0.000780; 9). In brief, ontology
data significantly indicates a role in both developmental signal transduction and immune
response.
In terms of individual genes (Table 3 and Appendix I), we found Immune induced
molecule 1 as the most up-regulated gene (7.0 of LBFC). We were unable to identify any
known mitotic or cell cycle gene amongst the most up-regulated genes.
The most down-regulated gene is Ecdysone-inducible gene E2 (-11.7 of LBFC) whose
function is unknown. The fourth most down-regulated target is the recently characterised
interference–Hedgehog gene (iHog, -3.0 of LBFC).
Regarding cell-cycle genes we were unable to identify anyone with an outstanding
down-regulated value: candidates with stronger values of LBFC are Cbl (-1.66), Cdc25-string
(-1.63) and Polo (-1.63).
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Table 3- The most up- and down-regulated genes in tshNG1 homozygous neuroblats microarrays, the putative
Tsh target genes are also listed with the respective lower bound of fold change (LBFC), The positive LBFC represent
the up regulated genes while the negative LBFC represent the downregulated genes.
Gene LBFC
Immune induced molecule 1 7,0
Hedgehog -1,3
Cdc25-String -1,6
Polo -1,6
Cbl -1,7
Engrailed -1,7
interference Hedgehog -3,0
Ecdysone-inducible gene E2 -11,7
Having in mind that the depletion of tsh seems to cause problems during mitosis, we
looked at Polo levels by immunofluorescence in tsh mutant brains and in westerns blot of S2
cells after the Tsh-depletion (Supplementary fig. 4 and 5). We found that Polo localisation in
mitotic cells occurs in the right places in the right time; however, several mitotic figures do not
show staining in all the expected places, as if Polo levels were not enough to occupy all the
sub-cellular locations and to the whole tissue. Accordingly, we noticed that Polo levels are
significantly down-regulated in S2 cells exposed to the tsh dsRNA from 72 to 120 h. Curiously
this observation is coincident with the maximum of RNAi mitotic effects observed.
Discussion
In this study we report that tsh is required for cell proliferation. Loss of tsh function in
tshNG1 and tshA3-2-66 alleles leads to lethality at third instar larvae. Examination of neuroblasts
dissected from dying third instar tshNG1 and tshA3-2-66 homozygous larvae revealed that cells
became arrested in a metaphase-like state in mitosis. Such a general role to Tsh is unusual,
considering its localised expression pattern and well-documented functions on the specification
of particular structures. Regarding the localisation of the mitotic defects on the larval CNS, we
were unable to detect any regional specificity. Although tsh is not ubiquitous express in the
CNS (Supplementary fig. 6), the same range of mitotic abnormalities was observed throughout
the brain hemispheres and the trunk CNS. Moreover, the same range of mitotic defects occurs
in the imaginal discs of the mutants and was also detected in S2 Drosophila culture cells after
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tsh-RNAi, re-enforcing the idea of a universal mitotic role for Tsh. Such a role could be
mediated by a long-range diffusible factor responding to the localised action of the tsh
transcription factor.
Our detailed characterisation of the mitotic phenotype revealed only a few structural
abnormalities or defects, not enough to cause such a strong mitotic arrest. Probably, these are
secondary effects of cells escaping from a strong restriction to proliferation after a long delay.
The metaphase-like arrest phenotype observed in mutants, or after the knock-down of tsh,
does not seem to result from defects in the integrity of the mitotic organising centres. Both γ-
tubulin and Centrosomin (data not shown) show a normal localisation to the centrosomes in
mitosis. Accordingly, centrosomes seem to nucleate normal microtubules and are capable of
assembling focused bipolar spindles and organize a metaphase-like array of chromosomes.
Checkpoint proteins localise properly in mutants as if cells were time-frozen in metaphase. The
spindle checkpoint seems to be active and, weirdly, bub neither rod checkpoint mutants are
unable to override the block imposed by the tsh mutation in double mutant constructs. We
conclude that tsh-deficient cells are restricted to bypass metaphase either because they lack or
over-express a potent regulator of cell proliferation. This may act independently of the
checkpoint machinery or known effectors required for the metaphase-anaphase transition. In
fact, it should be stressed that APC/C mutants, thus lacking fundamental effectors proteins
required for this transition, are able to override the checkpoint in double mutant constructs
with bub (Donaldson et al., 2001a). Therefore, even though small changes in mitotic
parameters may activate the metaphase checkpoint they do not explain the strong restriction
to enter in anaphase. Quite probably the mitotic phenotype reflects an indirect role of Tsh in
cell proliferation acting through a potent regulator.
An overview of the expression pattern by microarray analysis of mutant brains failed to
detect obvious alterations in cell cycle candidate genes. However, a putative link to cell
proliferation control mediated by Hedgehog signalling was found, as we identified the recently
characterised interference–Hedgehog gene (iHog) to be significantly down-regulated.
Interestingly, we also got evidence that Hedgehog signalling is a tsh target in ovaries while
carrying out the characterisation of female-sterile tsh mutants in a independent work (see
chapter II part II of this manuscript).
The Hedgehog (Hh) pathway regulates proliferation in a variety of tissues, but its
specific effects on the cell cycle are unclear. Its role has been controversial, with studies
variably suggesting a stimulatory or an inhibitory effect on proliferation. Recently, a general
role of Hh in the nervous system and other tissues has been proposed (Agathocleous et al.,
2007): hh may convert quiescent stem cells into fast cycling transient amplifying progenitors
that are closer to cell cycle exit and differentiation. The fast cycling cells in Hh gain of function
experiments are pushed out of the cell cycle prematurely, while slow dividing progenitors, with
blocked Hh signalling, exhibit delayed cell cycle exit.
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The mechanism of Hh induced proliferation varies depending on the tissues, but
includes the induction and regulation of cell cycle components, including Cyclins D1, D2, B1
and E, CDC25 and N-Myc (Barnes et al., 2001; Benazeraf et al., 2006; Kenney and Rowitch,
2000; Kenney et al., 2004). In the case of CyclinD1 and N-Myc, this effect may be direct as
the Gli transcription factors have been shown to bind to sequences in the promoters of these
genes (Hu et al., 2006).
Work in the Drosophila eye has implicated Hh in the activation of cyclin-D and cyclin-E
(Heberlein et al. 1995; Duman-Scheel et al. 2002; Vrailas and Moses 2006). Moreover, it was
demonstrated that Ci (the most downstream element of the Hh pathway) specifically binds to
the cyclin-E promoter, directly inducing Cyc-E expression (Duman-Scheel et al. 2002).
A recent study in retinal precursor cells points to a conserved function of Hh in the
control of cell cycle kinetics, through the regulation of G1 and G2 length (Locker et al., 2006).
It was reported that Hh is both necessary and sufficient to induce G1 and G2 acceleration
explained by a decrease in both G1 and G2 duration (Locker et al. 2006). These cell cycle
accelerating effects of Hh may be brought about because the expression of key cell cycle
stimulators is influenced by Hh signaling. Cyclin-D1 regulates G1/S transition, and, consistent
with a slower G1 transition, its expression is decreased upon Hh inhibition, as already reported
in the mouse retina (Wang et al. 2005) and other areas of the vertebrate central nervous
system (Kenney and Rowitch 2000). In the Locker and colleagues study (2006), cyclin-B1 and
cdc25C, crucial regulators of G2/M transition and cyclin A, involved in both S- and G2-phase
progression (Collins and Garrett 2005), were proposed as key candidates for Hh effects on G2
length duration. The related cdc25B phosphatase was recently found to be a target of Hh
signalling in the neural tube (Benazeraf et al. 2006).
Theoretically, the Drosophila CDC25 homologue String could also be an hh target in
Drosophila neuroblasts, but our microarray data indicates just a slight reduction on the mitotic
target String. Furthermore, this would provoke problems at the G2/M transition, which we
were unable to find as we only detected problems at exiting mitosis. Therefore other targets
must be involved. The slight reduction on the mitotic target Polo (microarray and RNAi data) is
more consistent with the timing of the observed metaphase arrest (Donaldson et al., 2001b).
However, reduced levels of Polo are not enough to justify such a strong metaphase arrest, and
Polo has never been implicated in the Hedgehog pathway.
Our results failed to identify an obvious cell-cycle target. However, the significant
down-regulation of iHog strongly suggests a link between the observed mitotic arrest and Hh
signalling. A number of receptors facilitate Hh binding to its main receptor Patched (Ptc),
including members of the cell adhesion molecule–related/down-regulated by oncogenes (Cdo)
family (reviewed in Jacob and Lum, 2007). A targeted mouse mutation in Cdo, a gene
encoding an Ig-family transmembrane protein, causes microform holoprosencephaly, a
condition associated with loss of Hh signalling (Cole and Krauss, 2003). The Drosophila
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homolog of this gene, iHog, was identified in an RNAi screen to be a positive regulator of Hh
signalling (Lum et al., 2003a), which is consistent with the mouse mutant phenotype. The
extracellular protein CDO, IHOG and its relate BOC have been found to positively regulate
hedgehog signalling through the binding and sequestration of the Hh ligand Ptc, resulting in
locally enhanced signalling (Zhang et al., 2006; Yao et al, 2006). Cdo and Ihog associate with
Hh through a fibronectin type III (FnIII) repeat, a motif with potential for binding sulfate ions
(reviewed in Jacob and Lum, 2007). Indeed, dimerization of Ihog and its conversion from a
weak to a high-affinity Hh-binding molecule can be induced by heparin, a protein with
sulphated polysaccharide modifications. How Ihog and Cdo proteins promote Hh-mediated
responses in coordination with Ptc and other Hh receptors, particularly the heparan sulfate–
modified Dally-like protein (Dlp), which also contributes to the Hh response, remains to be
addressed.
A plausible explanation for our results is that the Hh pathway controls a strong
regulator of the cell cycle progression acting at the metaphase-anaphase transition. Even
though Polo levels are affected in our mutants, the phenotype is not ameliorated by
metaphase checkpoint mutations, and do not fully explain the strong block to progression. This
potent regulator could confer a cytoplasmic state similar to the CSF-cytostatic activity blocking
mitotic progression. We speculate that tsh is required for the transcription of iHog and that,
this ligand is required to modulate Hh signalling in mitosis progression. In the absence of iHog
cells are unable to receive the Hh signal to enter in anaphase and exit mitosis. Further work
will be needed to support our speculative model of Hh signalling in mitosis progression.
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Supplementary Material
Reversion analysis and P{lacW} remobilization to generate new tsh alleles
For mobilisation of the P{lacW}A3-3-66 insert, w / w; T(2;3)CyO TM6, Tb / P{lacW}A3-3-
66;+ females were crossed to males carrying P{ry+, ∆2-3} on a Stubble marked third
chromosome (Robertson et al., 1988) of genotype w/ Y; T(2;3)CyO TM6, Tb/ S Sp Bl bwD; ∆2-
3, Sb. All the strains used in the reversion scheme carried X-chromosomes with the eye-
marker white (w), and thus individuals carrying the P{lacW} chromosome had coloured eyes
due to the presence of the extra wild-type copy w+ on the second chromosome, while lines
having lost the P{lacW} showed white eyes due to the white background on the X-
chromosome. The F1 males of genotype w/ Y ; P{lacW}A3-3-66/ S Sp Bl bwD ; +/ ∆2-3, Sb were
crossed to w/ w ; T(2;3)CyO TM6, Tb/ P{lacW}A3-3-66; + females. Ten white-eyed males were
found amongst 1622 F2 males examined. The ten putative revertant chromosomes were then
independently re-balanced over T(2;3)CyO TM6, Tb to establish revertant lines and test for
viability and fertility. Molecular analysis of six fertile excising lines demonstrated precise
excision of P{lacW} (data not shown) indicating that the transposon insertion is responsible for
the teashirt phenotype. The other four excision lines were infertile apparently due to imprecise
excisions of P{lacW} (data not shown).
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Tsh protein is absent in tsh mutant larval CNS
We also performed immunostaining of third instar larval CNS from tshNG1 and tshA3-2-66
mutants using the anti-Tsh antibody and compared with the staining pattern observed in wild-
type larval CNS. Tsh was detected using rat anti-Tsh (1:50, (Gallet et al., 1998) and Alexa
488-conjugated anti-rat (1:200, Molecular Probes). Although both methods yielded similar
results, immunostaining of squashed CNS (Supplementary Fig. 1) gave clearer expression
profiles as compared to whole-mount staining. In cells of tsh mutant homozygotes, no specific
Tsh staining was observed at any stage of the cell cycle consistent with the fact that these
alleles behave as genetic nulls (Supplementary Fig. 1D-I). Otherwise, in wild-type CNS, Tsh
protein specifically localises in both the nuclear and cytoplasmic compartments throughout
interphase to prophase (Supplementary Fig. 1A-C). With nuclear envelope breakdown at
prometaphase, Tsh appears to spread over the cytoplasm until the end of mitosis
(Supplementary Fig. 1A-C, cell in the upper right corner). Thus, the localisation of Tsh in CNS
is basically the same as that observed in wild-type embryos at stage 8 of development, where
Tsh is detected as a diffuse labelling within the nuclei and cytoplasm of the cells in the region
of the future thoracic and abdominal segments (Alexandre et al., 1996; Gallet et al., 1999).
Supplementary Figure 1.-
Immunolocalisation of the Tsh protein in
larval CNS cells.
Tsh protein revealed with anti-Tsh (Gallet et al., 1998) is
shown in green and DNA stained with propidium iodide is
shown in red in the merged images. In the wild-type, Tsh
localises both in nuclear and cytoplasmic compartments
during interphase (A-C). In tshNG1 (D-F) and tshA3-2-66 (G-I)
mutant cells Tsh staining is below the level of detection.
166
Apoptose levels in tsh mutant larval CNS
Supplementary Figure 2- In situ cell death detection.
Labelling of apoptotic nuclei in third instar larval CNS by TdT-mediated dUTP nick end labelling (TUNEL) with FICT anti-
dUTP conjugates was performed using the In Situ Cell Death Detection Kit (Roche) according to the manufacturer’s
instructions. Brains were mounted in Vectashield (Vector Laboratories).
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Cuticle phenotypes
For cuticle preparations, eggs were allowed to develop at 25ºC for 48 hours and
mounted as described (Andrew et al., 1994). Cuticle preparations from Trans combinations of
the mitotic alleles showed characteristic phenotype defects of tsh mutants (Supplementary Fig.
3). The alleles tshNG1 and tshA3-2-66 in Trans with tsh8 revealed similar abnormalities to tsh8
homozygotes, such as defects in labial mouth derivatives and disruption and sclerotization of
the anal opening (Supplementary Fig. 3C-H). Ventral trunk segments are disrupted in
tshNG1/tsh8 embryos, which show segment fusion within restricted parts of the abdomen and
disruption of the denticle belt (Supplementary Fig. 3C and D); tshA3-2-66/tsh8 display smaller
trunk segments and fewer denticles in the abdominal segments (Supplementary Fig. 3E and
F).
Supplementary Figure 3- Cuticules phenotype.
A comparison of the embryo cuticle abnormalities of wild-type (A and B), tshNG1/tsh8 trans-heterozygotes (C and D),
tshA3-2-66/tsh8 trans-heterozygotes (E and F) and tsh8 homozygotes (G and H). Panels B, D, F and H are enlarged views
of A, C, E and G, respectively. Bar represents 50 µm.
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Polo expression in tshNG1 larval CNS and in dsRNAi- Tsh treated cells
Supplementary Figure 4-
Immunolocalisation of Polo in larval CNS cells.
Polo protein revealed with anti-Polo (a gift from A.
Tavares) is shown in green and DNA is shown in red.
(A, C and E) In the wild-type metaphase (A), Polo
localises in the kinetochore, in late metaphase (C)
and anaphase (E) the polo protein is also detected in
the poles of the spindle. (B, D and F) In neuroblasts
from tshNG1 third instar larvae, Polo is localised in the
right places and in the right time; however, more
than 50% of the mitotic figures do not show staining
in all the expected places. In some mitotic figures
Polo is not detectable (B), and in other mitotic
figures that resemble an anaphase-like, Polo is only
detectable in the poles of the spindle (D) or only in
the kinetochores (F).
Supplementary Figure 5- Analisys of Polo expression by western blot.
(A) Levels of Polo in S2 dsRNAi-GFP(+) or dsRNAi- TSH treated (-) cells 96h after transfection. These blots were also
probed with anti γ-tubulin antibody as loading control. (B) Western analysis of Polo and α-tubulin in CNS cells from
wild-type and tshNG1 homozygotes. Lanes 1-2 were loaded with proteins extracted from 9 wild-type third instar larvae
and 24 tshNG1 third instar larvae, respectively. The polo protein display significantly lower levels in dsRNAi-Tsh treated
cells and in tshNG1 CNS compared to the control. Mouse Polo antibody (a gift from A. Tavares) was used at 1:1000.
169
Expressition patterning of the tshlacZ in CNS during larvae development
Supplementary Figure 6- A CNS from L1, L2 and L3 larvae instars expressing tshlacZ stained with
X-Gal.
The central chord is strongly labelled as well some particular cells in each lobe. The L1, L2 and L3 represent the first,
the second and the third larvae instar. All painels are at the same amplification.
170
CHAPTER III
DISCUSSION AND PERSPECTIVES
DISCUSSION AND PERSPECTIVES
The objective of this PhD was to explain the teashirt (tsh) function during cell cycle
progression. For that, it was studied the requirement for tsh in two independent processes in
Drosophila melanogaster: mitosis of neuroblasts and S2 cells, and Follicle stem cells (FSC)
proliferation during oogenesis. The results obtained in Drosphila oogenesis, larval neuroblasts
and S2 cells are in accordance and suggest a conserved tsh function through Hh signalling
during the progression of the cell cycle.
1-Tsh and its paralog Tio repress each other in the tip of the Drosophila ovary,
and both are able to regulate En expression.
Tsh and Tiptop (tio) are localised in the left arm of chromosome 2 and are transcribed
in the same orientation (5’-3’). These genes are spaced 183 Kb apart and encode a protein
with zinc finger (Znf) motifs of the C2H2 type. The tsh tio gene pair seems to have arisen from
a recent duplication event characteristic of Drosophilidae. It seems that tio is more close to the
ancestral gene (Laugier et al., 2005; Laugier, 2005) and also to the murine tsh genes (Caubit
et al., 2000) than tsh.
We found that tsh and tio are co-repressed in the tip of the wild-type ovaries. In tio473
ovaries, Tsh is detected in all TF, independently of flies age. When tsh activity is specifically
removed, Tio is ectopically detected in the more proximal TF (Tsh normal expression domain),
showing that, in wild-type, Tsh represses the expression of tio in these particular cells.
Curiously, Tio was never detected in CC of ∆126/tshA3-2-66 ovaries, even in the case of
ovarioles, where Tsh is totally missing from the germarium (phenotype III). However, this is
not the ideal condition to verify the repression of Tio by Tsh, since ∆126/tshA3-2-66 is not a null
mutant. Although no Tsh protein has been detected by immunostaining, this does not mean
that no Tsh at all has been produced. We speculate that a small amount of Tsh protein may
repress tio expression. Another possibility is the presence of another unknown protein that
represses tio, but not tsh in CC.
According to our data, besides the mutual repression between the two members of tsh
family in Drosophila, Tio can also partially replace the loss of tsh during oogenesis, although
less efficiently.
Tsh and Tio show similar functional properties, when expressed ectopically.
Furthermore, tio is able to rescue the oogenetic defects of ∆126/tshA3-2-66 mutants, indicating
that both genes are functionally equivalent during oogenesis, similar to the reported during
imaginal development (Bessa et al., 2009). In addition, these genes share the ability of being
able to induce giant stalk-like structures when ectopic express.Thus, we conclude that Tsh and
Tio have a redundant function during oogenesis, as described during embryogenesis and
imaginal development (Laugier et al., 2005; Laugier, 2005; Bessa et al., 2009; Datta et al.,
173
DISCUSSION AND PERSPECTIVES
2009). In accordance with the reported phenotype during embryogenesis (Laugier et al., 2005;
Laugier, 2005), the loss of tsh seems more dramatic for oogenesis than the lost of tio, thus tsh
is maintaining as the more effective of the two duplicated genes also during oogenesis. In the
future will be interesting investigate the behaviour of Tio in late pupa stage, during ovary
formation. In this stage, Tsh is already present in all TF and in the precursors of CC, preceding
En expression. And Tio? tio is already express in TF at this stage; tio sharing the same
expression domain with Tsh? Or, tio expression only started after the downregulation of tsh in
the anterior TF cells? These are some of the interesting aspects to better understand the
transcriptional regulation of tio/ tsh and explain the maintenance of a apparently dispensable
gene in Drosophila.
2-Tsh regulates the expression of Hh and En at the Tip of the Drosophila
germarium
The results obtained during this PhD work showed that, in ovaries, tsh affects FSC
proliferation by mediating Hh expression in TF cells and CC. A reduction in Tsh activity resulted
in the loss of Hh in TF and CC. We also showed that, in tsh mutant ovary, En was absent from
CC, but maintained in TF cells. In addition, the observed phenotype in tsh ovaries resembled
the phenotype of ovaries in which hh was absent (Forbes et al, 1996a; 1996b). Inversely, the
ectopic expression of tsh induced the ectopic expression of en and mimicked the phenotype
resultant from hh-ectopic expression (Forbes et al, 1996a; 1996b). These results support the
proposition that tsh regulates both en and hh expression, and that the transcriptional
regulatory mechanisms in TF and CC are different. In addition, they are in agreement with the
interaction between Tsh and Hh pathways previously described during the cellular identity and
polarity of the embryonic segments (Angelats et al., 2002; Gallet et al., 2000). These studies
showed the requeriment of Tsh and Ci in a redundant manner for the Hh dependent regulation
of wg before stage 10 of embryogenesis. Additionally, it was shown that Tsh does not regulate
the transcription of ptc (the Hh receptor), but is required for the expression of rho, a Hh target
gene. Moreover, it was identified a complex containing both proteins: Tsh and Ci (Angelats et
al., 2002). It is important to notice that tio473 tshRNAi embryos lost En expression in the
ventral epidermis, from embryonic stage 11 onwards (Laugier et al., 2005). The parallel
between our observation during oogenesis and those reported during embryogenesis,
suggested a conserved function of the tsh genes family in the regulation of hh expression, at
least in some Drosophila tissue. However our results don’t allow us to conclude about the
nature of the regulation.
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DISCUSSION AND PERSPECTIVES
3-Tsh mediating Hh pathway is required for FSC proliferation
The results obtained during the characterisation of the ovary phenotype of tsh female
sterile mutants, allow us to conclude that tsh (through Hh) is required for the proper FSC
and/or early FC proliferation. In the absence of tsh, FSC fail to proliferate correctly, giving rise
to the accumulation of germline cells and swollen germarium. In contrast, in a wild-type
background, tsh overexpression stimulated FSC proliferation, resulting in the excess of stalk
cells and the developing of giants stak-like structures.
Figure 1- Tsh signalling to Follicle Stem cells (FSC) proliferation mediating Hh expression (adapted
from Kirilly and Xie, 2007).
This proposed model shows that Teashirt (Tsh) is required upstream of hedgehog (Hh) expression for follicle stem cells
(FSC; violet cells) proliferation. In terminal filament (TF brown cells) and cap cells (CC, green cells), Tsh acts upstream
of Hh pathway and positively regulates both en and hh expression. In TF, Tiptop (Tio) can also positively affect
engrailed (en) expression. Hh together with Wingless (Wg), (both from CC), and Glassbottomed boat (Gbb) produced
by posterior escort cells (EC; dark blue cells), will signal for FSC proliferation and maintenance.
We speculate that Tsh in EC cells could also signal for FSC proliferation and/or maintenance. In escort stem cells (ESC;
light blue cells), the presence of Tsh suggests a possible role during germline cyst migration.
(CC) cap cells, (ESC) escort stem cells, (GSC) germline stem cell, [EC (IGS)] inner germarial sheath renamed by
escort cells), (FSC) Follicle stem cell, (TF) terminal filament cells. Teashirt (Tsh), Tiptop (Tio), Hedgehog (Hh),
Engrailed (En), Wingless (Wg), Glassbottomed boat (Gbb), Anterior is to the left and posterior is to the right.
175
DISCUSSION AND PERSPECTIVES
We propose a model to explain the effect of Tsh in FSC proliferation (Fig.1). Hh and Wg
proteins are both expressed in the tip of the ovary and then diffuse to signal for the proper
proliferation and maintenance of FSC, together with Gbb (which one is produced by posterior
EC). In our model, Tsh regulates En and Hh expression at the tip of the ovary. In TF, Tio is
able to regulate the expression of en, but apparently not hh. The role of Hh signalling in FSC
proliferation is well documented (Forbes et al., 1996a; 1996b; King et al., 2001; Liu and
Montell, 1999; Zhang and kalderon, 2000; 2001). We speculate that Tsh, in the posterior EC,
could be upstream of a signal emanating from these cells for FSC proliferation and/or
maintenance. Also, a speculative role for Tsh in the migration of the cyst may be suggested by
the presence of Tsh in the ESC and progeny.The clarification of the Tsh role in posterior EC and
ESC needs more specific work.
Our finding opens new insights about Tsh function. In the ectopic eye formation, Tsh
promote cell proliferation (Bessa et al. 2002), but our results show by first time the implication
of Tsh in stem cell proliferation (probably by an indirect way through Hh). In combination with
the interaction of the Tshz3 with β-Catenin, (a gene mutated in the majority of the colon
cancer cases), this new tsh function could help us to understand why Tshz1 have been
identified in a screen for potential genes implicated in colon cancer (Scanlan et al., 1998).
In our model, Tsh is presented as a positive regulator. However, we do not know the
mechanisms by which Tsh positively regulates the expression of En and Hh: is it directly or
indirectly? The mechanisms by which Tsh controls the expression of targets genes (Alexandre
et al., 1996a; Graba et al., 1994; Waltzer et al., 2001; Saller et al., 2002) are still poorly
understood. So far, Tsh has been shown to directly repress the expression of modulo
(Alexandre et al., 1996a) which suggested that Tsh may be a repressor. Recently, Tsh was
implicated in a repressor complex with FE65/SET and HDAC that directly silence the expression
of the primate specific caspase-4 gene, which is associated with the progression of the
Alzheimer disease (Kajiwara et al., 2009). Thus, it is tempting to propose that, in TF cells and
CC, Tsh may repress a repressor of hh and/or en. Thus, the positive regulation of hh by Tsh
could be indirect. However, an activater role for Tsh is not completely exclude.
The implication of Tsh in the proliferation of epithelial cells could be broadened to other
epithelium type cells and not be restrict to only FSC and progeny.
One characteristic effect of tsh overexpression during embryogenesis is the homeotic
transformation of the labial segment (the more posterior segment of the embryo head) in T1
segment, easily identified by the presence of the beard (additional group of denticles, specific
of the T1 segment; de Zulueta et al., 1994). Curiously, this duplicated T1 segment is bigger
when compared to a wild-type labial segment, suggesting that the enlargement results from
an increase in the number, or in the size, of the cells. In contrast, among with other
developmental specific defects, the trunk segments of tsh larvae are shorter than the normal
(Fasano et al 1991). Based on our finding during oogenesis, we question, whether tsh could
176
DISCUSSION AND PERSPECTIVES
also have a role in the proliferation of epidermal cells during embryogenesis. To answer this
question we need to be able of compare the total number of cells, in the transformed T1
homeotic segment when tsh is ectopically expressed, with the total number of cells in the wild-
type labial segment and in the equivalent segment in tsh embryo. Unfortunately, we do not
have the appropriate tools to perform an accurate counting of the total cell number. However,
the size of the epidermal cells seems to be the same in the wild-type T1 and in the labial
transformed T1 segment (data not shown). This observation make us suspected, that the
homeotic transformation of the labial segment in T1 segment is accomplished by an increse in
the total number of epidermal cells. If we could prove this increase in the labial homeotic
transformed T1 segment and, on the other hand, a decrease in the total number of epidermal
cells in the T1 tsh- segment, we will be able to propose a conserved tsh function in the
proliferation of epithelial cells, (since embryonic epidermis and follicle cells are both of the
epithelium type cells).
4-The metaphase/anaphase transition is compromised in the absence of Tsh
This PhD started with the observation that tsh was required for mitosis progression. The
loss of tsh function in tshNG1 and tshA3-2-66 alleles, leads to lethality at third instar larvae. The L3
neuroblasts from tshNG1 and tshA3-2-66 homozygous larvae revealed that cells became arrested
in a metaphase-like state in mitosis. Such a general role for Tsh is unusual, considering its
localised expression pattern and well-documented functions on the specification of particular
structures. Despite the preferential Tsh expression in the ventral chord, we were unable to
detect any regional specificity of the mitotic defects in the CNS. Moreover, the same range of
mitotic defects occurred in S2 Drosophila culture cells after tsh-RNAi, re-enforcing the idea of a
universal mitotic role for Tsh. Such a role could be mediated by a long-range diffusible factor
responding to the localised action of the Tsh transcription factor. Showing some similarities
with the role of Tsh (expressed in TF cell and CC) through Hh, in the proliferation of the FSC
and progeny in Drosophila ovaries (described above).
We question if Tsh metaphase arrest could be a secondary effect of cells escaping from
a strong restriction for proliferation after a long delay. The few mitotic structural defects
observed in tsh mutants are not sufficient to explain the strong metaphase arrest imposed by
the loss of tsh. We speculate that, tsh-deficient cells are restricted to bypass metaphase,
because they lack or over-express a potent regulator of cell proliferation. This may act
dependently of the checkpoint machinery and/or known effectors, required for the metaphase-
anaphase transition. However, metaphase checkpoint can be activated by small changes in
mitotic parameters, but this does not explain the strong restriction to enter anaphase. In other
hand, in contrast to what we observed in tsh mutants, APC/C mutants that lack fundamental
effectors proteins required for metaphase/anaphase transition are able to override the
177
DISCUSSION AND PERSPECTIVES
checkpoint in double mutant constructions with bub (Donaldson et al., 2001a; 2001b). Thus,
we speculate that the tsh mitotic phenotype reflects an indirect role of Tsh in cell proliferation
acting through a potent regulator.
The analysis of the expression pattern of the mutant’s brains did not show an obvious
cell cycle target gene. Interestingly, the third most significantly down-regulated gene in our
microarray analysis was interference–Hedgehog (iHog), providing a link to cell proliferation
control mediated by Hh signalling.
The Cyclins D1, D2, B1 and E, Cdc25 and N-Myc are some of the cell cycle components
regulated by Hh (Barnes et al., 2001; Benazeraf et al., 2006; Kenney and Rowitch, 2000;
Kenney et al., 2004). In addition, there is evidence of a direct binding between Hh effectors
and the promoter of these genes (cyclin D1, N-Myc, cyclin-D and cyclin-E; Duman-Scheel et al.
2002; Heberlein et al. 1995; Hu et al., 2006; Vrailas and Moses 2006). Another interesting
work reported the importance of Hh in the cell cycle kinetics in mouse retina, relating that to
the expression of key cell stimulators (cyclin-D1, Cyclin-B1 and Cdc25C; Locker et al., 2006).
We failed to identify an obvious cell-cycle target. The significant down-regulation of
ihog, strongly suggests a link between the Tsh-dependent mitotic arrest and the Hh signalling.
iHog, positively regulates hedgehog signalling, resulting in a locally enhanced signalling (Zhang
et al., 2006; Yao et al, 2006). We speculated that Hedgehog pathway controls a strong
regulator of cell cycle progression acting at the metaphase-anaphase transition or provoking
arrest at that stage. One potential candidate could be Polo but methaphase ckeckpoint
mutations no override the strong metaphase arrest displayed by tsh neuroblasts. In addition,
Polo was never implicated in the Hh signalling. Other possibility is that, Hh regulates another
potent regulator that could confer a cytoplasmic state, similar to the CSF-cytostatic activity,
blocking mitotic progression. The abnormalities observed in tsh mutations are similar to the
morula phenotypes (Reed and Orr-Weaver, 1997), in which the metaphase arrest associated
with the stabilisation of a set of mitotic proteins, including Cyclin B, was explained as a failure
to maintain active APC/C. In addition, Cyclin B doesn’t seem to be normal degraded in tsh
mutant’s neuroblasts. Thus, we are tempting to hypothesie that Tsh protein is may required to
promote same aspects of APC/C activity that mediated cyclin B degradation. Again the protein
kinases related to the Drosophila gene polo have been implicated as positive regulators of the
APC/C in budding yeast (Shirayama et al., 1998) Drosophila (Donaldson et al., 2001a) and
mammals (Kotani et al., 1998), but, double mutants tsh bub1 doesn’t override the metaphase
arrest in contrast to the observed in polo bub1 mutants, as explain above.
In our microaarys, the Cbl gene, which is donwregulated with a LBFC of -1.7, could be
a candidate to interfere with the APC/C activity. In fact the Cbl proteins are a family of proteins
conserved throughout the animal kingdom (Thien and Langdon, 2001; Nau and Lipkowitz,
2003). Cbl proteins function as ubiquitin protein ligases (E3s), which mediate the
ubiquitination of activated tyrosine kinases, such as the EGFR, and target them for degradation
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DISCUSSION AND PERSPECTIVES
(Thien and Langdon, 2001). We question, in what whether, the reduction in the Cbl activity
could not avoid the degradation of some unknow (until now) factor that confer a cytoplasmic
state of the tsh neuroblasts? And in that case, cbl is a new target of Hh signalling? Or Cbl is a
Tsh target independently of the effect of Tsh in Hh pathway?
We propose that tsh is required for the transcription of ihog and that, this ligand is
required to modulate Hh signalling during mitosis progression. In the absence of iHog, cells are
less sensible to receive the Hh signal, which by an unknown target, is reflected in the
incapacity in to enter in anaphase and exit mitosis. Further work will be needed to investigate
the Tsh and/or Hh signalling target (or targets) that confere to the mitotic tsh neuroblasts this
metaphase arrest described in this manuscript and similar to the CSF-cytostatic activity.
5- Tsh/Hh signalling from flies to vertebrates.
Several characteristics of normal and deregulated development of vertebrate epidermis
and hair follicles, suggested that Sonic hedgehog (Shh) might also act on stem cells in those
tissues. Shh is required for hair follicle development in mice, and Shh mutant phenotypes
include reduced proliferation of ectodermal stem cell derivatives (Chiang et al, 1999; St
Jacques et al., 1998). Conversely, inappropriate mutational activation of the Shh signal
transduction pathway is universally observed in human familial and sporadic basal-cell
carcinomas (BCC; Dahmane et al., 1997; Gailani et al., 1996; Johnson et al., 1996; Xie et al.,
1998). Several observations proposed that activation of this pathway might be obligatory in
the aetiology of BCC and can suffice to initiate this cancer (Fan et al., 1997; Grachtchouk et
al., 2000; Nilsson et al., 2000; Oro et al., 1997; Xie et al., 1998). Moreover, it was suggested
that proliferation of follicular stem cells might normally require Shh, among other factors, and
that mutational activation of the Shh signalling pathway in BCC acts specifically on these stem
cells to increase their cycling frequency or their cell number, or perhaps even to expand their
niche into or towards the surrounding epithelium (Zhang and Kalderon, 2001). Any of these
changes could produce a sustainable large increase in a population of transiently proliferating
cells, which would constitute the bulk of the carcinoma.
Interestingly, preliminary studies in mouse revealed that Tshz3 is expressed in
mesenchyme cells that contribute to form the hairs, closely interacting with epithelia cells that
express Shh (Caubit, X. and Fasano, L., not published). Thus, Tshz could conserve a function
upstream of Hh signalling related to the one that plays in TF and CC during Drosophila
oogenesis.
Unlike to that observed in flies, in vertebrate epidermis and hair Follicles, tsh does not
co-localise with Hh, but we speculate that Tshz3 in mesenchyme could signal neighbour
epithelial cells to express Hh. A relation between Tshz3 and Shh was also demonstrated in the
urinary tract in mice, however, in this case, Tshz3 acts downstream of Shh and BMP4 (Caubit
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DISCUSSION AND PERSPECTIVES
et al., 2008). During the embryonic development of the mouse urinary tract, Tshz3,
(expressed in mesenchymal populations), is required for proximal ureteric smooth muscle cells
(SMC) differentiation. Inactivation of Tshz3 causes hydroureter and SMC malformation (Caubit
et al., 2008). Shh in the urinary tract, has a dual role: a proliferative effect on ureteric
mesenchyme and a BMP4 inducer in peri-urothelial mesenchymal cells for differentiation of
ureteric mesenchyme into SMC (Yu et al., 2002). This exclusive, but close localisation of Tshz
and Shh is also observed in other mouse tissue. In that cases, Tshz1 and Tshz3 are expressed
in the same cells that express Ptc, which contacts Shh cells (Caubit, X., Core, N. and Fasano,
L., not published). According to our findings of iHog expression in neuroblasts, TshZ and Ptc
co-localisation could indicate the needs of Tsh to one accurate Shh response. We question if
Tshz could not contribute to make these cells capable of answering to Shh signal.
According to what we found in Drosophila Tshz upregulation in mouse precedes the
satellite cells proliferation in response to a muscle injury (Faralli H. and Fasano L., not
publish). Shh has never been implicated in the proliferation of satellite cells. In opposite, Tshz
probably interacts with the Pax genes, similarly to what has been described during Drosophila
eye development (Bessa et al., 2002).
Despite the differences in some aspects, the examples describe above reveal a positive
TshZ interaction with Shh pathways and/or stem cell proliferation, suggesting some conserved
function, at this level, from flies to vertebrates.
Concluding Remarks:
The analysis of tsh function during Drosophila oogenesis, as well as in neuroblasts and
S2 cells mitosis, suggests that Tsh is required for proper cell proliferation, potentially through
Hh signalling. tsh loss of function induces a strong delay or arrest in cell proliferation.
Moreover, in neuroblasts and S2 cells, the absence of Tsh prevented the exit of mitosis and
induced a strong metaphase arrest. Additionally, hh (in TF and CC) is downregulated in tsh
ovaries, while ihog is significantly downregulated in tsh neuroblasts. Thus, in the two systems,
the loss of tsh is followed by a decrease in the activity of the Hh signalling pathway, affecting
the expression of the hh itself or of the Hh receptor-like ihog. Based in all these similarities
found in both systems, we propose a speculative model where Tsh, acting upstream of Hh
signalling, could affect the progression of cell proliferation (Fig.2).
We propose a general role for Tsh acting upstream Hh signalling for proper progression
of cell proliferation in Drosophila. We proved that during oogenesis, Tsh controls Hh expression
in the tip of the ovary. Unfortunately, our results do not allow us to conclude much about the
nature of this regulation. Further work is needed to explain whether Tsh affects directly or
indirectly hh expression, and the nature of the regulation (repression or activation).
180
DISCUSSION AND PERSPECTIVES
Figure 2- Proposed model to explain
Tsh role in cell proliferation, mediating Hh
expression signal in FSC and neuroblasts.
This speculative model proposes a role for Tsh in
cell proliferation both in follicle stem cells (FSC, in
ovaries; left circle gray) and in neuroblasts (in CNS;
right circle gray). In FSC, Tsh at the tip of the
germarium, regulates positively Hh expression,
which in turn will positively affect FSC proliferation
through unknown target(s) gene(s). In neuroblast
cells, Tsh affects cell cycle progression probably
through a diffusible factor. Hh is a potential
candidate, because it was shown that Tsh positively
affects iHog expression. We speculate that Polo
could be a new Hh target.
Although the positive regulation of Hh pathway in FSC proliferation is well documented,
some questions remain: How does Hh signals for FSC proliferation? What are the Hh targets in
FSC?
In neuroblasts, we speculate that Tsh is required for the transcription of iHog that by its
turn, is required to enhance Hh signalling and promote mitosis progression through the
regulation of one or more potent cell cycle regulators. Additionally, we question whether Polo
could be a new, but not the only, mitotic target controlled by Hh signalling, at least in
Drosophila neuroblasts.
The present work has shed some light in the biological Tsh function on cell proliferation,
but opens up several questions: How does Tsh regulates hh (in ovaries) and ihog (in
neuroblasts)? What is the nature of this regulation (direct or indirect)? Is Tsh an activator or a
repressor, or both? Is it polo a new mitotic target gene of Hh? Why do egg chambers stop
exactly at stage S2/S3 in absence of tsh and at stage S5/S6 when rescued by ubiquitously
provided tsh? Do tsh mutants allow to identify two checkpoints in egg chamber development?
In the follicular cells, the endocycles begin at stage S6/S7, when N pathway mediates the
downregulation of cut, string and hh, promoting the M/E switch. Could tsh have a later role in
the regulation of this process? Future works are required to try answer all these questions.
181
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210
APPENDIX
Appendix I- Microarays Results for expression patterning of tshNG1 neuroblasts expression comparing to wild-type (ordered
by lower bound of fold change (LBFC), after using a cut-off of 1.3)
Table I- Results of the up- or down-regulation genes in tshNG1 homozygous neuroblats which have been ordered by lower bound of
fold change (LBFC), after using a cut-off of 1.3. The positive LBFC represent the up regulated genes while the negative LBFC represent
the downregulated genes. Only the genes with the absolute value of LBFC upper than 1.3 are represented.
Gene LBFC
Immune induced molecule 1 7,0
FB:FBgn0034330 /sym=CG18107 /name= /prod= /func= /map=55C9-55C9 /transc=CT40695 /len=248 /GB:AE003799 5,5
FB:FBgn0036833 /sym=CG3819 /name= /prod= /func= /map=75E4-75E4 /transc=CT12789 /len=1272 /GB:AE003518 4,4
FB:FBgn0034294 /sym=CG5765 /name= /prod= /func= /map=55B1-55B1 /transc=CT18116 /len=1528 /GB:AE003801 4,0
FB:FBgn0040735 /sym=CG16836 /name= /prod= /func= /map=55C9-55C9 /transc=CT37415 /len=298 /GB:AE003799 3,3
Drosomycin 3,3
FB:FBgn0031246 /sym=CG11592 /name= /prod= /func=structural protein /map=21B6-21B6 /transc=CT33137 /len=1668 /GB:AE003590 3,3
FB:FBgn0030072 /sym=CG7090 /name= /prod= /func=enzyme /map=8C3-8C3 /transc=CT21911 /len=763 /GB:AE003445 3,2
FB:FBgn0040734 /sym=CG15065 /name= /prod= /func= /map=55C9-55C9 /transc=CT34936 /len=123 /GB:AE003799 3,2
FB:FBgn0036393 /sym=CG17362 /name= /prod= /func= /map=70C9-70C9 /transc=CT33207 /len=552 /GB:AE003536 3,2
Lysozyme A /// Lysozyme C 3,1
FB:FBgn0034900 /sym=CG12491 /name= /prod= /func= /map=59F7-59F7 /transc=CT32938 /len=474 /GB:AE003461 3,1
Attacin-A 3,0
Odorant-binding protein 56a 3,0
FB:FBgn0034296 /sym=CG10912 /name= /prod= /func= /map=55B2-55B2 /transc=CT30553 /len=875 /GB:AE003801 /note=3prime sequence from clone BDGP:GH07575.3prime-hit
3,0
FB:FBgn0038074 /sym=CG6188 /name= /prod= /func= /map=87C7-87C7 /transc=CT19392 /len=941 /GB:AE003696 3,0
Esterase 6 2,9
FB:FBgn0028920 /sym=BG:DS00941.12 /name= /prod= /func= /map=34D4-34D4 /transc=CT25774 /len=826 /GB:AE003641 2,8
FB:FBgn0034512 /sym=CG18067 /name= /prod= /func= /map=57A7-57A7 /transc=CT40493 /len=773 /GB:AE003791 2,8
FB:FBgn0028534 /sym=BG:DS00941.13 /name= /prod= /func= /map=34D4-34D4 /transc=CT23894 /len=864 /GB:AE003641 2,7
FB:FBgn0039419 /sym=CG12290 /name= /prod=G-protein coupled receptor-like /func=G protein linked receptor /map=97A6-97A6 /transc=CT19320 /len=3056 /GB:AE003755 /note=3prime sequence from clone BDGP:GH12381.3prime-hit
2,7
Drosocin 2,7
APPENDIX I
FB:FBgn0038241 /sym=CG8087 /name= /prod= /func= /map=88C7-88C7 /transc=CT24214 /len=461 /GB:AE003705 2,6
FB:FBgn0039316 /sym=CG11893 /name= /prod= /func= /map=96C7-96C7 /transc=CT33087 /len=1301 /GB:AE003751 2,6
FB:FBgn0031560 /sym=CG16713 /name= /prod= /func= /map=24A4-24A4 /transc=CT37195 /len=298 /GB:AE003579 2,5
Immune induced molecule 23 2,5
FB:FBgn0030262 /sym=CG2081 /name= /prod= /func= /map=10A3-10A3 /transc=CT6756 /len=712 /GB:AE003485 2,5
FB:FBgn0028533 /sym=BG:DS00941.14 /name= /prod= /func= /map=34D4-34D4 /transc=CT23898 /len=913 /GB:AE003641 2,5
FB:FBgn0031561 /sym=CG16712 /name= /prod= /func= /map=24A4-24A4 /transc=CT37183 /len=336 /GB:AE003579 2,5
FB:FBgn0032209 /sym=CG18144 /name= /prod= /func= /map=31D4-31D4 /transc=CT40888 /len=365 /GB:AE003628 2,5
FB:FBgn0035611 /sym=CG13285 /name= /prod= /func= /map=64E1-64E2 /transc=CT32571 /len=597 /GB:AE003565 2,5
FB:FBgn0037181 /sym=CG11370 /name= /prod= /func= /map=79F2-79F2 /transc=CT21093 /len=1173 /GB:AE003598 2,4
Trypsin 2,4
Henna 2,4
Glutathione S transferase E6 2,3
Ecdysone-dependent gene 91 2,2
FB:FBgn0032282 /sym=CG7299 /name= /prod= /func= /map=32A1-32A1 /transc=CT22515 /len=534 /GB:AE003629 2,2
FB:FBgn0036419 /sym=CG13482 /name= /prod= /func= /map=70D5-70D5 /transc=CT32848 /len=309 /GB:AE003535 2,2
FB:FBgn0028921 /sym=BG:DS00941.11 /name= /prod= /func= /map=34D4-34D4 /transc=CT37476 /len=1217 /GB:AE003641 /note=3prime sequence from clone BDGP:GH07914.3prime-hit
2,2
FB:FBgn0031908 /sym=CG5177 /name= /prod= /func=enzyme /map=27F6-27F6 /transc=CT16575 /len=980 /GB:AE003617 /note=3prime sequence from clone BDGP:LD18740.3prime-hit
2,2
Juvenile hormone-inducible protein 26 2,2
FB:FBgn0033820 /sym=CG4716 /name= /prod= /func= /map=50A1-50A1 /transc=CT15231 /len=783 /GB:AE003819 /note=3prime sequence from clone BDGP:GH06388.3prime-hit
2,2
Alcohol dehydrogenase 2,2
FB:FBgn0038783 /sym=CG4367 /name= /prod= /func= /map=92D9-92D9 /transc=CT14252 /len=642 /GB:AE003730 2,1
upheld 2,1
Cytochrome b5-related 2,1
FB:FBgn0028542 /sym=BG:DS00180.8 /name= /prod= /func=cell adhesion /map=34E1-34E1 /transc=CT37470 /len=3394 /GB:AE003642 2,1
Troponin C at 73F 2,1
FB:FBgn0031562 /sym=CG3604 /name= /prod=trypsin inhibitor-like /func=enzyme inhibitor /map=24A4-24A4 /transc=CT11958 /len=536 /GB:AE003579 2,1
FB:FBgn0034203 /sym=CG17288 /name= /prod= /func= /map=54A3-54A3 /transc=CT35735 /len=996 /GB:AE003804 2,1
FB:FBgn0028510 /sym=BG:DS07851.3 /name= /prod= /func= /map=35C4-35C4 /transc=CT35205 /len=502 /GB:AE003647 2,1
FB:FBgn0034899 /sym=CG13560 /name= /prod= /func= /map=59F7-59F7 /transc=CT32937 /len=546 /GB:AE003461 2,1
FB:FBgn0033589 /sym=CG13227 /name= /prod= /func= /map=47D8-47D8 /transc=CT32471 /len=363 /GB:AE003826 2,0
FB:FBgn0033593 /sym=CG9080 /name= /prod= /func= /map=47E1-47E1 /transc=CT26066 /len=435 /GB:AE003826 2,0
FB:FBgn0036181 /sym=CG18331 /name= /prod= /func= /map=68C9-68C9 /transc=CT41625 /len=360 /GB:AE003544 2,0
Jonah 74E 2,0
214
APPENDIX I
Rhythmically expressed gene 3 2,0
FB:FBgn0034364 /sym=CG5493 /name= /prod= /func= /map=55E4-55E4 /transc=CT17422 /len=967 /GB:AE003799 2,0
FB:FBgn0039896 /sym=CG1629 /name= /prod= /func= /map=102A3-102A3 /transc=CT4279 /len=1158 /GB:AE003844 2,0
FB:FBgn0038420 /sym=CG10311 /name= /prod= /func= /map=89B22-89B22 /transc=CT28967 /len=719 /GB:AE003713 2,0
Misexpression suppressor of ras 4 1,9
FB:FBgn0040733 /sym=CG15068 /name= /prod= /func= /map=55C9-55C9 /transc=CT34939 /len=189 /GB:AE003799 1,9
Lysozyme A /// Lysozyme D 1,9
/// 1,9
Lysozyme E 1,9
FB:FBgn0033928 /sym=CG13941 /name= /prod= /func=signal transduction /map=50F6-50F6 /transc=CT33487 /len=582 /GB:AE003815 1,9
Tissue inhibitor of metalloproteases 1,9
FB:FBgn0032726 /sym=CG10621 /name= /prod= /func= /map=37B8-37B8 /transc=CT29750 /len=1470 /GB:AE003661 1,9
Pherokine 3 1,9
FB:FBgn0030138 /sym=CG2996 /name= /prod= /func=enzyme /map=8E9-8E10 /transc=CT7882 /len=12908 /GB:AE003447 /note=3prime sequence from clone BDGP:SD01877.3prime-hit
1,9
Odorant-binding protein 18a 1,9
Tetraspanin 42El 1,9
FB:FBgn0031558 /sym=CG16704 /name= /prod= /func= /map=24A4-24A4 /transc=CT37177 /len=318 /GB:AE003579 1,9
FB:FBgn0035743 /sym=CG15829 /name= /prod=diazepam-binding inhibitor-like protein /func=enzyme inhibitor /map=65E5-65E5 /transc=CT39291 /len=249 /GB:AE003560
1,9
BDGP:GH07188.complete-hit /ESTpos=maps 3prime of FB:FBgn0039613 1,8
Punch 1,8
FB:FBgn0032785 /sym=CG10026 /name= /prod=alpha-tocopherol transfer relative protein /func=transcription factor /map=37E3-37E3 /transc=CT28189 /len=1143 /GB:AE003663 /note=3prime sequence from clone BDGP:SD01558.3prime-hit
1,8
FB:FBgn0038243 /sym=CG8066 /name= /prod=cystatin-like /func=cysteine protease inhibitor /map=88C7-88C7 /transc=CT24182 /len=418 /GB:AE003705 1,8
Glutathione S transferase E7 1,8
Troponin C at 47D 1,8
Esterase-7 1,8
FB:FBgn0031220 /sym=CG4822 /name= /prod=ATP-binding cassette transporter-like /func=enzyme /map=21B1-21B1 /transc=CT41972 /len=2894 /GB:AE003590
1,8
FB:FBgn0035742 /sym=CG8629 /name= /prod=diazepam-binding inhibitor-like protein /func=enzyme inhibitor /map=65E5-65E5 /transc=CT14596 /len=255 /GB:AE003560
1,8
Larval cuticle protein 3 1,8
FB:FBgn0036616 /sym=CG4893 /name= /prod= /func= /map=72F1-72F1 /transc=CT15728 /len=716 /GB:AE003527 1,8
Elastin-like 1,8
FB:FBgn0036996 /sym=CG5932 /name= /prod=triacylglycerol lipase-like /func=enzyme /map=77C1-77C1 /transc=CT18587 /len=1461 /GB:AE003591 /note=3prime sequence from clone BDGP:LP10120.3prime-hit
1,8
FB:FBgn0034289 /sym=CG10910 /name= /prod= /func=structural protein /map=55B1-55B1 /transc=CT30545 /len=1407 /GB:AE003801 1,8
215
APPENDIX I
FB:FBgn0032427 /sym=CG5453 /name= /prod= /func= /map=33D4-33D4 /transc=CT17294 /len=819 /GB:AE003636 /note=3prime sequence from clone BDGP:GH02216.3prime-hit
1,8
FB:FBgn0034583 /sym=CG10527 /name= /prod= /func= /map=57B20-57B20 /transc=CT29543 /len=1133 /GB:AE003452 /note=3prime sequence from clone BDGP:LD46156.3prime-hit
1,8
FB:FBgn0038242 /sym=CG14852 /name= /prod= /func= /map=88C7-88C7 /transc=CT34668 /len=525 /GB:AE003705 1,8
Myosin light chain 2 1,8
FB:FBgn0031692 /sym=CG6514 /name= /prod= /func=ligand binding or carrier /map=25C10-25C10 /transc=CT20253 /len=536 /GB:AE003609 1,8
FB:FBgn0038113 /sym=CG11668 /name= /prod=serine protease /func=endopeptidase /map=87D11-87D11 /transc=CT36689 /len=1197 /GB:AE003698 1,8
FB:FBgn0030968 /sym=CG7322 /name= /prod= /func=enzyme /map=17E4-17E4 /transc=CT22587 /len=729 /GB:AE003510 1,7
Cyclic-nucleotide-gated ion channel protein 1,7
Lysozyme B 1,7
FB:FBgn0036787 /sym=CG4306 /name= /prod= /func= /map=75C6-75C6 /transc=CT14001 /len=989 /GB:AE003520 1,7
FB:FBgn0034709 /sym=CG3074 /name= /prod= /func=endopeptidase /map=58C5-58C7 /transc=CT10308 /len=1982 /GB:AE003456 /note=3prime sequence from clone BDGP:GM06507.3prime-hit
1,7
FB:FBgn0022341 /sym=CG17467 /name= /prod= /func= /map=102E3-102E3 /transc=CT38621 /len=1002 /GB:AE003846 1,7
FB:FBgn0030237 /sym=CG15209 /name= /prod= /func= /map=9F4-9F4 /transc=CT35141 /len=444 /GB:AE003484 1,7
globin 1 1,7
brother of tout-velu 1,7
Myosin alkali light chain 1 1,7
FB:FBgn0032773 /sym=CG15825 /name= /prod= /func= /map=37D7-37E1 /transc=CT38769 /len=1906 /GB:AE003662 /note=3prime sequence from clone BDGP:HL01053.3prime-hit
1,7
Jonah 65Aiii 1,7
FB:FBgn0010070 /sym=Msp-300 /name=Muscle-specific protein 3 /prod= /func=actin binding /map=25C8-25C8 /transc=CT41354 /len=2016 /GB:AE003608 /note=3prime sequence from clone BDGP:SD10272.3prime-hit
1,7
Galactose-specific C-type lectin 1,7
BDGP:LD47230.3prime-hit /ESTpos=maps in FB:FBgn0034661 /sym=CG4386 /name= /prod=serine protease /func=endopeptidase /map=58A2-58A2 /transc=CT14320 /len=559
1,7
FB:FBgn0038243 /sym=CG8066 /name= /prod=cystatin-like /func=cysteine protease inhibitor /map=88C7-88C7 /transc=CT24182 /len=418 /GB:AE003705 1,7
FB:FBgn0038647 /sym=CG14302 /name= /prod= /func= /map=91C1-91C2 /transc=CT33932 /len=268 /GB:AE003723 1,7
Transferrin 1 1,7
FB:FBgn0031565 /sym=CG10030 /name= /prod= /func=DNA replication factor /map=24A5-24A5 /transc=CT28231 /len=4588 /GB:AE003579 /note=3prime sequence from clone BDGP:HL03084.3prime-hit
1,7
Myosin heavy chain 1,6
FB:FBgn0030341 /sym=CG1967 /name= /prod= /func= /map=10F1-10F2 /transc=CT6115 /len=953 /GB:AE003487 1,6
FB:FBgn0015714 /sym=Cyp6a17 /name= /prod=cytochrome P450, CYP6A17 /func=cytochrome P45 ; EC:1.14.14.1 | inferred from sequence similarity /map=51D2-51D2 /transc=CT28783 /len=1519 /GB:AE003813 /note=3prime sequence from clone BDGP:GH10635.3prime-hit
1,6
FB:FBgn0033565 /sym=CG18003 /name= /prod= /func= /map=47C6-47C6 /transc=CT40270 /len=1255 /GB:AE003828 /note=3prime sequence from clone BDGP:GH14288.3prime-hit
1,6
FB:FBgn0036234 /sym=CG17824 /name= /prod=peritrophin-like /func=cell adhesion /map=68E3-68E3 /transc=CT39558 /len=2415 /GB:AE003543 1,6
FB:FBgn0038009 /sym=CG17738 /name= /prod= /func= /map=87B8-87B8 /transc=CT34537 /len=333 /GB:AE003695 1,6
216
APPENDIX I
FB:FBgn0038115 /sym=CG7966 /name= /prod=selenium-binding protein-like /func=ligand binding or carrier /map=87D11-87D11 /transc=CT23978 /len=1498 /GB:AE003698 /note=3prime sequence from clone BDGP:GH14316.3prime-hit
1,6
Tropomyosin 2 1,6
FB:FBgn0032283 /sym=CG7296 /name= /prod= /func= /map=32A1-32A1 /transc=CT22519 /len=575 /GB:AE003629 1,6
Pre-intermoult gene 1 1,6
Jonah 25Bii 1,6
FB:FBgn0031937 /sym=CG13795 /name= /prod= /func=neurotransmitter transporter /map=28C2-28C2 /transc=CT33284 /len=927 /GB:AE003618 1,6
Dromyosuppressin 1,6
pugilist 1,6
FB:FBgn0030484 /sym=CG1681 /name= /prod=glutathione transferase-like /func=enzyme /map=11F1-11F1 /transc=CT4698 /len=1098 /GB:AE003492 /note=3prime sequence from clone BDGP:GH16740.3prime-hit
1,6
Muscle protein 20 1,6
Jonah 25Bi 1,6
FB:FBgn0032897 /sym=CG9336 /name= /prod= /func= /map=38F1-38F1 /transc=CT5238 /len=660 /GB:AE003668 1,6
FB:FBgn0036525 /sym=CG13451 /name= /prod= /func= /map=71E3-71E3 /transc=CT32813 /len=195 /GB:AE003530 1,6
FB:FBgn0035917 /sym=CG6416 /name= /prod= /func=enzyme /map=66D9-66D10 /transc=CT20013 /len=1175 /GB:AE003554 /note=3prime sequence from clone BDGP:GH19182.3prime-hit
1,6
FB:FBgn0032426 /sym=CG15493 /name= /prod= /func= /map=33D4-33D4 /transc=CT35594 /len=468 /GB:AE003636 1,6
FB:FBgn0032638 /sym=CG6639 /name= /prod= /func=endopeptidase /map=36C2-36C2 /transc=CT20606 /len=1493 /GB:AE003655 1,6
FB:FBgn0036422 /sym=CG3868 /name= /prod= /func=signal transduction /map=70D7-70D7 /transc=CT12881 /len=1161 /GB:AE003534 /note=3prime sequence from clone BDGP:GH23259.3prime-hit
1,6
FB:FBgn0039644 /sym=CG11897 /name= /prod=ATP-binding cassette transporter /func=transporter /map=99A2-99A3 /transc=CT37062 /len=4458 /GB:AE003768 /note=3prime sequence from clone BDGP:LD17001.3prime-hit
1,6
Cystatin-like 1,6
Calpain-A 1,6
FB:FBgn0035112 /sym=CG13877 /name= /prod= /func= /map=61A6-61A6 /transc=CT33404 /len=324 /GB:AE003467 1,6
FB:FBgn0030679 /sym=CG8206 /name= /prod= /func= /map=13E16-13E16 /transc=CT24425 /len=997 /GB:AE003500 /note=3prime sequence from clone BDGP:GH04557.3prime-hit
1,6
FB:FBgn0036146 /sym=CG14141 /name= /prod= /func=protein kinase /map=68A8-68A8 /transc=CT33742 /len=444 /GB:AE003545 1,6
FB:FBgn0038644 /sym=CG7710 /name= /prod= /func= /map=91B8-91B8 /transc=CT23373 /len=460 /GB:AE003723 1,6
FB:FBgn0039208 /sym=CG6643 /name= /prod= /func=enzyme /map=96A7-96A7 /transc=CT20638 /len=2769 /GB:AE003748 /note=3prime sequence from clone BDGP:GH21511.3prime-hit
1,6
Sorbitol dehydrogenase 1 1,6
FB:FBgn0040741 /sym=CG14486 /name= /prod= /func= /map=54E3-54E3 /transc=CT34197 /len=171 /GB:AE003802 1,6
FB:FBgn0035742 /sym=CG8629 /name= /prod=diazepam-binding inhibitor-like protein /func=enzyme inhibitor /map=65E5-65E5 /transc=CT14596 /len=255 /GB:AE003560
1,5
FB:FBgn0039529 /sym=CG5612 /name= /prod= /func= /map=98A4-98A4 /transc=CT17752 /len=1352 /GB:AE003761 1,5
FB:FBgn0036116 /sym=CG7888 /name= /prod=amino-acid permease-like /func=transporter /map=68A2-68A2 /transc=CT36915 /len=1827 /GB:AE003546 /note=3prime sequence from clone BDGP:GH09436.3prime-hit
1,5
UDP-glycosyltransferase 35a 1,5
217
APPENDIX I
Lysozyme S 1,5
FB:FBgn0034638 /sym=CG10433 /name= /prod= /func= /map=57F3-57F3 /transc=CT29298 /len=1071 /GB:AE003454 /note=3prime sequence from clone BDGP:GH10517.3prime-hit
1,5
FB:FBgn0032955 /sym=CG2201 /name= /prod=choline kinase-like /func=enzyme /map=39E3-39E3 /transc=CT7236 /len=1914 /GB:AE003781 /note=3prime sequence from clone BDGP:LD18613.3prime-hit
1,5
FB:FBgn0033205 /sym=CG2064 /name= /prod= /func=enzyme /map=43E7-43E7 /transc=CT6708 /len=996 /GB:AE003840 /note=3prime sequence from clone BDGP:SD07613.3prime-hit
1,5
FB:FBgn0033683 /sym=CG18343 /name= /prod= /func= /map=48E10-48E10 /transc=CT41671 /len=463 /GB:AE003823 1,5
FB:FBgn0036262 /sym=CG6910 /name= /prod= /func= /map=68F7-68F7 /transc=CT21406 /len=1459 /GB:AE003542 1,5
hugin 1,5
FB:FBgn0034501 /sym=CG13868 /name= /prod= /func= /map=56F17-57A1 /transc=CT33390 /len=3604 /GB:AE003792 /note=3prime sequence from clone BDGP:SD03066.3prime-hit
1,5
Trypsin 1,5
/// /// Trypsin /// Trypsin 1,5
Lobe 1,5
FB:FBgn0027544 /sym=BcDNA:GH11688 /name= /prod= /func= /map=99F6-99F6 /transc=CT6804 /len=4019 /GB:AE003773 /note=3prime sequence from clone BDGP:GH11688.3prime-hit
1,5
spatzle 1,5
Ejaculatory bulb protein III /// 1,5
ABC transporter expressed in trachea 1,5
FB:FBgn0037817 /sym=Cyp12e1 /name= /prod=cytochrome P450, CYP12E1 /func=cytochrome P45 /map=86A7-86A7 /transc=CT34465 /len=1518 /GB:AE003687
1,5
FB:FBgn0032774 /sym=CG17549 /name= /prod= /func= /map=37E1-37E1 /transc=CT38763 /len=1051 /GB:AE003662 /note=3prime sequence from clone BDGP:GH19142.3prime-hit
1,5
FB:FBgn0038774 /sym=CG5023 /name= /prod=calponin-like /func=ligand binding or carrier /map=92D2-92D2 /transc=CT16114 /len=697 /GB:AE003729 /note=3prime sequence from clone BDGP:GH21596.3prime-hit
1,5
FB:FBgn0036145 /sym=CG7607 /name= /prod= /func=cell adhesion /map=68A8-68A8 /transc=CT23219 /len=660 /GB:AE003545 1,5
FB:FBgn0038160 /sym=CG9759 /name= /prod= /func= /map=87F5-87F5 /transc=CT27579 /len=685 /GB:AE003701 1,5
lethal (2) essential for life 1,5
FB:FBgn0039754 /sym=CG9747 /name= /prod= /func=enzyme /map=99E3-99E3 /transc=CT27555 /len=2124 /GB:AE003772 /note=3prime sequence from clone BDGP:GH07782.3prime-hit
1,5
FB:FBgn0038664 /sym=CG5449 /name= /prod= /func=endopeptidase /map=91E1-91E1 /transc=CT17110 /len=2563 /GB:AE003724 /note=3prime sequence from clone BDGP:GH21941.3prime-hit
1,5
FB:FBgn0037267 /sym=CG1054 /name= /prod= /func= /map=82D5-82D5 /transc=CT1108 /len=4410 /GB:AE003605 /note=3prime sequence from clone BDGP:GH25780.3prime-hit
1,5
Annexin IX 1,5
FB:FBgn0033817 /sym=CG4688 /name= /prod= /func=enzyme /map=49F15-49F15 /transc=CT15137 /len=699 /GB:AE003819 1,5
/// Fat body protein 1 1,5
cAMP-dependent protein kinase 1,5
FB:FBgn0028526 /sym=BG:DS01759.2 /name= /prod= /func= /map=34E5-34E5 /transc=CT35241 /len=1144 /GB:AE003642 1,5
Jonah 65Aiv 1,5
218
APPENDIX I
FB:FBgn0039031 /sym=CG17244 /name= /prod= /func= /map=94D8-94D9 /transc=CT33347 /len=480 /GB:AE003741 1,5
FB:FBgn0030309 /sym=CG1572 /name= /prod= /func= /map=10C4-10C4 /transc=CT4080 /len=955 /GB:AE003486 /note=3prime sequence from clone BDGP:LD04844.3prime-hit
1,5
Esterase-1 1,5
FB:FBgn0035321 /sym=CG1275 /name= /prod=cytochrome b561-like /func=electron transfer /map=62D4-62D5 /transc=CT2537 /len=1681 /GB:AE003474 /note=3prime sequence from clone BDGP:LD36721.3prime-hit
1,5
FB:FBgn0032422 /sym=CG6579 /name= /prod= /func= /map=33C3-33C4 /transc=CT20347 /len=834 /GB:AE003635 1,5
FB:FBgn0033775 /sym=Cyp9h1 /name= /prod=cytochrome P450, CYP9H1 /func=cytochrome P45 /map=49D1-49D1 /transc=CT33386 /len=1557 /GB:AE003820 1,5
FB:FBgn0034098 /sym=CG15707 /name= /prod= /func=RNA binding /map=52F11-52F11 /transc=CT35928 /len=2230 /GB:AE003807 /note=3prime sequence from clone BDGP:LD30829.3prime-hit
1,5
FB:FBgn0038353 /sym=CG5399 /name= /prod= /func= /map=89A3-89A3 /transc=CT17118 /len=762 /GB:AE003710 1,5
FB:FBgn0032413 /sym=CG16997 /name= /prod= /func=endopeptidase /map=33C1-33C1 /transc=CT34780 /len=822 /GB:AE003635 1,4
FB:FBgn0035552 /sym=CG11350 /name= /prod= /func= /map=64B12-64B12 /transc=CT31662 /len=1101 /GB:AE003481 1,4
Trehalose-6-phosphate synthase 1 1,4
Drosulfakinin 1,4
longitudinals lacking 1,4
FB:FBgn0026414 /sym=Kaz1 /name= /prod= /func=serine protease inhibitor /map=61A6-61A6 /transc=CT42525 /len=626 /GB:AE003467 1,4
FB:FBgn0033600 /sym=CG9077 /name= /prod=cuticle protein /func=structural protein /map=47E1-47E1 /transc=CT26058 /len=396 /GB:AE003826 1,4
FB:FBgn0033789 /sym=CG13324 /name= /prod= /func= /map=49F1-49F1 /transc=CT32640 /len=339 /GB:AE003820 1,4
Insulin-related peptide 1,4
FB:FBgn0035187 /sym=CG9122 /name= /prod=tryptophan 5-monooxygenase /func=enzyme /map=61F3-61F3 /transc=CT9937 /len=2160 /GB:AE003470 /note=3prime sequence from clone BDGP:GH12537.3prime-hit
1,4
FB:FBgn0038721 /sym=CG16718 /name= /prod= /func= /map=92A13-92A13 /transc=CT37197 /len=4061 /GB:AE003727 /note=3prime sequence from clone BDGP:LD10322.3prime-hit
1,4
Saccharomyces cerevisiae UAS construct a of Raisin 1,4
Imaginal disc growth factor 4 1,4
FB:FBgn0031405 /sym=CG4267 /name= /prod=lipase-like /func=enzyme /map=22D3-22D3 /transc=CT11345 /len=1301 /GB:AE003583 1,4
FB:FBgn0031936 /sym=CG13794 /name= /prod= /func= /map=28C2-28C2 /transc=CT33283 /len=213 /GB:AE003618 1,4
FB:FBgn0032284 /sym=CG7294 /name= /prod= /func= /map=32A1-32A1 /transc=CT22525 /len=527 /GB:AE003629 1,4
FB:FBgn0034885 /sym=CG4019 /name= /prod=water transporter-like /func=transporter /map=59F1-59F2 /transc=CT38997 /len=1089 /GB:AE003461 1,4
FB:FBgn0038198 /sym=CG3153 /name= /prod= /func= /map=88A10-88A10 /transc=CT10556 /len=814 /GB:AE003703 1,4
FB:FBgn0033875 /sym=CG6357 /name= /prod= /func=endopeptidase /map=50C13-50C13 /transc=CT19866 /len=1418 /GB:AE003817 /note=3prime sequence from clone BDGP:GM07827.3prime-hit
1,4
/// Trypsin /// Trypsin 1,4
phantom 1,4
FB:FBgn0033384 /sym=CG8788 /name= /prod= /func= /map=45B1-45B1 /transc=CT25336 /len=1291 /GB:AE003834 1,4
antisense 1,4
Saccharomyces cerevisiae UAS construct a of Daborn 1,4
219
APPENDIX I
FB:FBgn0036710 /sym=CG6479 /name= /prod= /func= /map=74B1-74B1 /transc=CT20177 /len=1858 /GB:AE003524 /note=3prime sequence from clone BDGP:GM01209.3prime-hit
1,4
HMG Coenzyme A synthase 1,4
FB:FBgn0029766 /sym=CG15784 /name= /prod= /func= /map=4F10-4F10 /transc=CT36052 /len=1665 /GB:AE003434 1,4
FB:FBgn0037128 /sym=CG14572 /name= /prod= /func= /map=79A1-79A1 /transc=CT34303 /len=774 /GB:AE003595 1,4
FB:FBgn0040718 /sym=CG15353 /name= /prod= /func= /map=22B8-22B8 /transc=CT35362 /len=162 /GB:AE003584 1,4
FB:FBgn0040718 /sym=CG15353 /name= /prod= /func= /map=22B8-22B8 /transc=CT35362 /len=162 /GB:AE003584 1,4
FB:FBgn0038878 /sym=CG3301 /name= /prod= /func=enzyme /map=93D4-93D4 /transc=CT11093 /len=913 /GB:AE003734 /note=3prime sequence from clone BDGP:GH01837.3prime-hit
1,4
FB:FBgn0037083 /sym=CG5656 /name= /prod=alkaline phosphatase-like /func=enzyme /map=78D5-78D5 /transc=CT17844 /len=1770 /GB:AE003594 /note=3prime sequence from clone BDGP:LP05865.3prime-hit
1,4
FB:FBgn0032218 /sym=CG5381 /name= /prod= /func=transcription factor /map=31D8-31D8 /transc=CT17078 /len=1884 /GB:AE003628 1,4
FB:FBgn0036394 /sym=CG9040 /name= /prod= /func= /map=70C9-70C9 /transc=CT25958 /len=677 /GB:AE003536 1,4
FB:FBgn0037807 /sym=CG6293 /name= /prod=sodium-dependent L-ascorbic acid transporter /func=transporter /map=86A2-86A2 /transc=CT19686 /len=2088 /GB:AE003686 /note=3prime sequence from clone BDGP:LD30822.3prime-hit
1,4
FB:FBgn0039073 /sym=CG4408 /name= /prod=zinc carboxypeptidase A /func=peptidase /map=94F1-94F1 /transc=CT14368 /len=1651 /GB:AE003743 /note=3prime sequence from clone BDGP:LD41739.3prime-hit
1,4
Gelsolin 1,4
FB:FBgn0039800 /sym=CG11314 /name= /prod= /func= /map=100A5-100A5 /transc=CT31575 /len=605 /GB:AE003775 1,4
FB:FBgn0034755 /sym=CG3746 /name= /prod= /func= /map=58F2-58F2 /transc=CT10065 /len=557 /GB:AE003458 1,4
insulin-like peptide 3 1,4
FB:FBgn0037239 /sym=CG11739 /name= /prod= /func= /map=82B2-82B2 /transc=CT36785 /len=1274 /GB:AE003606 1,4
FB:FBgn0038343 /sym=CG14871 /name= /prod= /func= /map=88F6-88F6 /transc=CT34690 /len=327 /GB:AE003709 1,4
BDGP:LP01187.3prime-hit /ESTpos=maps in FB:FBgn0034226 /sym=CG4837 /name= /prod=5'-nucleotidase /func=enzyme /map=54C7-54C7 /transc=CT15539 /len=414
1,4
FB:FBgn0032899 /sym=CG9338 /name= /prod= /func= /map=38F1-38F1 /transc=CT5240 /len=660 /GB:AE003668 /note=3prime sequence from clone BDGP:GH07967.3prime-hit
1,4
FB:FBgn0039102 /sym=CG16705 /name= /prod=serine protease-like /func=endopeptidase /map=95B1-95B1 /transc=CT37173 /len=1288 /GB:AE003744 /note=3prime sequence from clone BDGP:GH28857.3prime-hit
1,4
Thor 1,4
Lamin C 1,4
Cytochrome c proximal 1,4
Glutamine synthetase 2 1,4
FB:FBgn0031563 /sym=CG10031 /name= /prod=trypsin inhibitor-like /func=enzyme inhibitor /map=24A4-24A4 /transc=CT28233 /len=333 /GB:AE003579 1,4
Imaginal disc growth factor 2 1,4
Rhythmically expressed gene 5 1,4
Paxillin 1,4
Multiple drug resistance 65 1,4
FB:FBgn0029639 /sym=CG14419 /name= /prod= /func= /map=3C3-3C3 /transc=CT34076 /len=588 /GB:AE003425 1,4
220
APPENDIX I
methuselah-like 4 1,4
/// Glutathione S transferase E3 1,4
FB:FBgn0032287 /sym=CG6415 /name= /prod=aminomethyltransferase /func=enzyme /map=32A2-32A2 /transc=CT20030 /len=1241 /GB:AE003629 /note=3prime sequence from clone BDGP:GH04419.3prime-hit
1,4
FB:FBgn0039464 /sym=CG6330 /name= /prod=uridine phosphorylase /func=enzyme /map=97D10-97D11 /transc=CT19812 /len=1469 /GB:AE003758 /note=3prime sequence from clone BDGP:GH09623.3prime-hit
1,4
FB:FBgn0034648 /sym=CG15675 /name= /prod= /func= /map=57F6-57F7 /transc=CT35860 /len=1440 /GB:AE003454 /note=3prime sequence from clone BDGP:GM01014.3prime-hit
1,4
Muscle LIM protein at 84B 1,4
/// 1,4
FB:FBgn0030929 /sym=CG15043 /name= /prod= /func= /map=17B4-17B4 /transc=CT34909 /len=492 /GB:AE003509 1,4
FB:FBgn0032237 /sym=CG5362 /name= /prod= /func=enzyme /map=31E1-31E1 /transc=CT17038 /len=1183 /GB:AE003628 1,4
yellow-d 1,4
FB:FBgn0036600 /sym=CG13043 /name= /prod= /func= /map=72E3-72E3 /transc=CT32262 /len=447 /GB:AE003527 1,4
Glutathione S transferase D10 1,4
Phosphoglyceromutase 1,4
subunit of type II geranylgeranyl transferase 1,4
FB:FBgn0036298 /sym=CG10627 /name= /prod= /func=phosphoacetylglucosamine mutase /map=69C4-69C4 /transc=CT13770 /len=1866 /GB:AE003541 1,3
FB:FBgn0010578 /sym=Eb1 /name= /prod= /func= /map=42C3-42C3 /transc=CT10957 /len=2073 /GB:AE003789 1,3
FB:FBgn0030040 /sym=CG15347 /name= /prod= /func= /map=7E6-7E7 /transc=CT35350 /len=824 /GB:AE003444 1,3
FB:FBgn0032318 /sym=CG14072 /name= /prod= /func= /map=32C3-32C4 /transc=CT33642 /len=366 /GB:AE003630 1,3
FB:FBgn0034757 /sym=CG13514 /name= /prod= /func= /map=58F3-58F3 /transc=CT32885 /len=1437 /GB:AE003458 1,3
FB:FBgn0036471 /sym=CG13460 /name= /prod= /func= /map=71B1-71B1 /transc=CT32823 /len=654 /GB:AE003532 1,3
FB:FBgn0038973 /sym=CG18594 /name= /prod= /func= /map=94B4-94B4 /transc=CT42527 /len=598 /GB:AE003739 1,3
FB:FBgn0039149 /sym=CG18428 /name= /prod= /func= /map=95D10-95D10 /transc=CT29930 /len=568 /GB:AE003746 1,3
FB:FBgn0040968 /sym=CG14933 /name= /prod= /func= /map=33B2-33B2 /transc=CT34761 /len=618 /GB:AE003634 1,3
FB:FBgn0035452 /sym=CG10359 /name= /prod=fibrinogen-like /func=structural protein /map=63E3-63E4 /transc=CT29060 /len=1675 /GB:AE003478 /note=3prime sequence from clone BDGP:GM10015.3prime-hit
1,3
FB:FBgn0032603 /sym=CG17928 /name= /prod=cytochrome b5-like /func=electron transfer /map=36A9-36A9 /transc=CT39948 /len=1326 /GB:AE003652 1,3
FB:FBgn0034140 /sym=CG8317 /name= /prod= /func= /map=53C7-53C7 /transc=CT24573 /len=693 /GB:AE003806 1,3
methuselah-like 3 1,3
/// 1,3
FB:FBgn0020642 /sym=Lcp65Ac /name= /prod=larval cuticle protein 65Ac /func=structural protein of larval cuticle (Drosophila) /map=65A5-65A5 /transc=CT21559 /len=330 /GB:AE003563
1,3
FB:FBgn0037288 /sym=CG14661 /name= /prod= /func= /map=82E7-82E7 /transc=CT34439 /len=618 /GB:AE003604 1,3
FB:FBgn0037552 /sym=CG7800 /name= /prod= /func=cell adhesion /map=84F5-84F5 /transc=CT23295 /len=1745 /GB:AE003678 /note=3prime sequence from clone BDGP:GH11496.3prime-hit
1,3
Larval serum protein 1 1,3
221
APPENDIX I
hikaru genki 1,3
Acetyl Coenzyme A synthase 1,3
FB:FBgn0034117 /sym=CG7997 /name= /prod= /func=enzyme /map=53B1-53B1 /transc=CT24026 /len=1725 /GB:AE003807 /note=3prime sequence from clone BDGP:LD13649.3prime-hit
1,3
spire 1,3
Cytochrome P450-18a1 1,3
Actin 57B 1,3
FB:FBgn0036449 /sym=CG5295 /name= /prod= /func= /map=70F5-70F5 /transc=CT16881 /len=2429 /GB:AE003533 /note=3prime sequence from clone BDGP:LD21785.3prime-hit
1,3
FB:FBgn0037515 /sym=CG3066 /name= /prod=monophenol monooxygenase activator /func=serine carboxypeptidase /map=84D11-84D11 /transc=CT10270 /len=1830 /GB:AE003676 /note=3prime sequence from clone BDGP:SD07170.3prime-hit
1,3
Adenylate kinase-1 1,3
FB:FBgn0030251 /sym=CG2145 /name= /prod=serine proteinase /func=endopeptidase /map=10A1-10A1 /transc=CT7008 /len=2045 /GB:AE003484 /note=3prime sequence from clone BDGP:GH10845.3prime-hit
1,3
FB:FBgn0032512 /sym=CG9305 /name= /prod= /func=transcription factor /map=34B6-34B6 /transc=CT26505 /len=2150 /GB:AE003639 -1,3
GTP-binding-protein -1,3
FB:FBgn0036848 /sym=CG10424 /name= /prod= /func= /map=75F6-75F6 /transc=CT29264 /len=1194 /GB:AE003518 -1,3
optic ganglion reduced -1,3
Chip -1,3
FB:FBgn0035872 /sym=CG7185 /name= /prod=RNA binding protein-like /func=nucleic acid binding /map=66C6-66C7 /transc=CT22187 /len=2779 /GB:AE003556 /note=3prime sequence from clone BDGP:LD25239.3prime-hit
-1,3
Kruppel homolog 1 -1,3
anterior open -1,3
hedgehog -1,3
FB:FBgn0030093 /sym=CG7055 /name= /prod= /func=DNA binding /map=8C17-8D1 /transc=CT21811 /len=2652 /GB:AE003446 /note=3prime sequence from clone BDGP:LD22190.3prime-hit
-1,3
dacapo -1,3
pannier -1,3
E(spl) region transcript m4 /// /// /// -1,3
FB:FBgn0031453 /sym=CG9894 /name= /prod= /func= /map=23B1-23B1 /transc=CT9918 /len=725 /GB:AE003582 -1,3
FB:FBgn0033089 /sym=CG17266 /name= /prod=peptidylprolyl isomerase /func=chaperone /map=42B4-42B4 /transc=CT38243 /len=552 /GB:AE003789 -1,3
FB:FBgn0038583 /sym=CG7183 /name= /prod= /func= /map=90F1-90F1 /transc=CT22177 /len=2552 /GB:AE003721 /note=3prime sequence from clone BDGP:GM01008.3prime-hit
-1,3
FB:FBgn0015805 /sym=Rpd3 /name= /prod=histone deacetylase /func=histone deacetylase /map=64B17-64B17 /transc=CT1177 /len=2141 /GB:AE003482 /note=3prime sequence from clone BDGP:GM14158.3prime-hit
-1,3
FB:FBgn0034631 /sym=CG10496 /name= /prod= /func= /map=57E6-57E8 /transc=CT29466 /len=2755 /GB:AE003454 /note=3prime sequence from clone BDGP:LD41005.3prime-hit
-1,3
mitochondrial ribosomal protein L36 -1,3
methuselah-like 8 -1,3
gliolectin -1,3
222
APPENDIX I
ballchen -1,3
FB:FBgn0034801 /sym=CG11716 /name= /prod= /func= /map=59B6-59B6 /transc=CT36757 /len=614 /GB:AE003459 -1,4
Saccharomyces cerevisiae UAS construct a of O'Keefe -1,4
BDGP:LD27862.3prime-hit /ESTpos=maps in FB:FBgn0037027 /sym=CG3680 /name= /prod= /func= /map=77E8-77E8 /transc=CT12245 /len=575 -1,4
brain tumor -1,4
FB:FBgn0034354 /sym=CG5224 /name= /prod= /func=enzyme /map=55D3-55D3 /transc=CT16701 /len=734 /GB:AE003799 /note=3prime sequence from clone BDGP:LD18692.3prime-hit
-1,4
FB:FBgn0034063 /sym=CG8389 /name= /prod=monocarboxylate transporter-like /func=transporter /map=52E1-52E1 /transc=CT18579 /len=1865 /GB:AE003808 /note=3prime sequence from clone BDGP:SD04201.3prime-hit
-1,4
tailless -1,4
FB:FBgn0032705 /sym=CG10346 /name= /prod= /func=chaperone /map=37A4-37A4 /transc=CT29062 /len=1986 /GB:AE003660 -1,4
FB:FBgn0038610 /sym=CG7675 /name= /prod= /func=enzyme /map=91A2-91A2 /transc=CT30419 /len=1100 /GB:AE003721 /note=3prime sequence from clone BDGP:GH26851.3prime-hit
-1,4
Kinesin-like protein at 61F -1,4
cdc2c -1,4
FB:FBgn0035235 /sym=CG7879 /name= /prod= /func=RNA binding /map=62A7-62A7 /transc=CT23834 /len=3692 /GB:AE003472 /note=3prime sequence from clone BDGP:SD09402.3prime-hit
-1,4
twins -1,4
klumpfuss -1,4
similar -1,4
FB:FBgn0029755 /sym=CG4202 /name= /prod= /func= /map=4F2-4F2 /transc=CT13852 /len=1568 /GB:AE003433 /note=3prime sequence from clone BDGP:GH08670.3prime-hit
-1,4
FB:FBgn0027949 /sym=msb1l /name= /prod= /func= /map=37F2-37F2 /transc=CT29112 /len=1340 /GB:AE003663 /note=3prime sequence from clone BDGP:LD32040.3prime-hit
-1,4
FB:FBgn0037382 /sym=CG2031 /name= /prod= /func= /map=83C3-83C3 /transc=CT6554 /len=2288 /GB:AE003601 /note=3prime sequence from clone BDGP:LD43883.3prime-hit
-1,4
muscleblind -1,4
Microtubule-associated protein 60 -1,4
DNA replication-related element factor -1,4
FB:FBgn0038666 /sym=CG5451 /name= /prod= /func=enzyme /map=91E1-91E1 /transc=CT16994 /len=1627 /GB:AE003724 -1,4
FB:FBgn0035076 /sym=CG10142 /name= /prod=peptidyl-dipeptidase (inactive) /func=peptidase /map=60E3-60E4 /transc=CT9828 /len=2078 /GB:AE003465 /note=3prime sequence from clone BDGP:GH05754.3prime-hit
-1,4
FB:FBgn0034438 /sym=CG9416 /name= /prod= /func= /map=56D3-56D3 /transc=CT26702 /len=2758 /GB:AE003796 -1,4
FB:FBgn0034939 /sym=CG2980 /name= /prod= /func=motor /map=60A13-60A13 /transc=CT40308 /len=555 /GB:AE003462 /note=3prime sequence from clone BDGP:LD21694.3prime-hit
-1,4
FB:FBgn0039130 /sym=CG5854 /name= /prod= /func= /map=95C12-95C12 /transc=CT18371 /len=1549 /GB:AE003745 /note=3prime sequence from clone BDGP:LD23561.3prime-hit
-1,4
Epidermal growth factor receptor -1,4
FB:FBgn0033367 /sym=CG8193 /name= /prod=monophenol oxidase /func=enzyme /map=45A6-45A7 /transc=CT2817 /len=2272 /GB:AE003835 -1,4
E(spl) region transcript m7 /// /// -1,4
223
APPENDIX I
FB:FBgn0038321 /sym=CG6218 /name= /prod= /func= /map=88F1-88F1 /transc=CT19478 /len=1523 /GB:AE003708 /note=3prime sequence from clone BDGP:GH07590.3prime-hit
-1,4
FB:FBgn0037670 /sym=CG8436 /name= /prod= /func= /map=85D4-85D4 /transc=CT24729 /len=940 /GB:AE003682 /note=3prime sequence from clone BDGP:LD32555.3prime-hit
-1,4
broad -1,4
deltex -1,4
yemanuclein -1,4
FB:FBgn0035030 /sym=CG3541 /name= /prod= /func= /map=60D7-60D8 /transc=CT11882 /len=2417 /GB:AE003464 /note=3prime sequence from clone BDGP:GH12163.3prime-hit
-1,4
Bearded -1,5
heat shock construct of Sigrist -1,5
FB:FBgn0033182 /sym=CG1621 /name= /prod= /func=transcription factor /map=43C7-43C7 /transc=CT4338 /len=1240 /GB:AE003841 /note=3prime sequence from clone BDGP:LD05592.3prime-hit
-1,5
FB:FBgn0038476 /sym=CG5175 /name= /prod= /func=transcription factor /map=89E13-89E13 /transc=CT16563 /len=2309 /GB:AE003716 /note=3prime sequence from clone BDGP:LD09231.3prime-hit
-1,5
FB:FBgn0029515 /sym=CG3038 /name= /prod= /func= /map=1A5-1A5 /transc=CT10170 /len=1680 /GB:AE003417 /note=3prime sequence from clone BDGP:LD16783.3prime-hit
-1,5
FB:FBgn0030710 /sym=CG8924 /name= /prod= /func=transcription factor /map=13F14-13F14 /transc=CT40370 /len=2155 /GB:AE003500 /note=3prime sequence from clone BDGP:LD19131.3prime-hit
-1,5
gluon -1,5
FB:FBgn0033998 /sym=CG8092 /name= /prod= /func=nucleic acid binding /map=51E7-51E8 /transc=CT22077 /len=4271 /GB:AE003811 /note=3prime sequence from clone BDGP:LD41072.3prime-hit
-1,5
abnormal spindle -1,5
FB:FBgn0034564 /sym=CG9344 /name= /prod=LSM6; U6 snRNP core protein /func=nucleic acid binding /map=57B14-57B14 /transc=CT26549 /len=240 /GB:AE003452
-1,5
cleavage and polyadenylation specificity factor -1,5
FB:FBgn0030762 /sym=CG4453 /name= /prod=nucleoporin /func=endopeptidase /map=14F2-14F2 /transc=CT14464 /len=7261 /GB:AE003502 /note=3prime sequence from clone BDGP:LD46585.3prime-hit
-1,5
E(spl) region transcript m /// /// -1,5
glial cells missing -1,5
FB:FBgn0027528 /sym=BcDNA:LD21405 /name= /prod= /func=endopeptidase /map=15A3-15A3 /transc=CT27236 /len=3597 /GB:AE003503 /note=3prime sequence from clone BDGP:LD21405.3prime-hit
-1,5
FB:FBgn0037377 /sym=CG1218 /name= /prod= /func= /map=83C1-83C1 /transc=CT2292 /len=1479 /GB:AE003601 /note=3prime sequence from clone BDGP:LD28626.3prime-hit
-1,5
dachs -1,5
FB:FBgn0031097 /sym=CG17052 /name= /prod=peritrophin-like /func=structural protein /map=19C1-19C1 /transc=CT37860 /len=1383 /GB:AE003572 -1,5
Serine protease inhibitor 43Aa -1,5
doublesex -1,6
FB:FBgn0032693 /sym=Cyp310a1 /name= /prod=cytochrome P450, CYP310A1 /func=cytochrome P45 /map=37A3-37A3 /transc=CT29164 /len=1628 /GB:AE003659 /note=3prime sequence from clone BDGP:LD44491.3prime-hit
-1,6
BDGP:SD10469.3prime-hit /ESTpos=maps 3prime of FB:FBgn0033913 -1,6
FB:FBgn0039717 /sym=CG7854 /name= /prod= /func= /map=99C7-99C8 /transc=CT23808 /len=1888 /GB:AE003771 /note=3prime sequence from clone -1,6
224
APPENDIX I
BDGP:GH10942.3prime-hit
18 wheeler -1,6
FB:FBgn0037228 /sym=CG1092 /name= /prod= /func= /map=82A4-82A4 /transc=CT1569 /len=1388 /GB:AE003607 /note=3prime sequence from clone BDGP:HL02811.3prime-hit
-1,6
grainy head -1,6
Saccharomyces cerevisiae UAS construct a of Struhl -1,6
polo -1,6
FB:FBgn0027521 /sym=BcDNA:LD21675 /name= /prod= /func= /map=14B13-14B14 /transc=CT12345 /len=2719 /GB:AE003501 /note=3prime sequence from clone BDGP:LD21675.3prime-hit
-1,6
string -1,6
wingless -1,6
Trithorax-like -1,6
FB:FBgn0029584 /sym=CG11509 /name= /prod= /func= /map=2B6-2B6 /transc=CT36379 /len=742 /GB:AE003421 /note=3prime sequence from clone BDGP:GH13132.3prime-hit
-1,6
odd paired -1,6
FB:FBgn0039229 /sym=CG6995 /name= /prod= /func=RNA binding /map=96B1-96B2 /transc=CT21632 /len=3029 /GB:AE003749 /note=3prime sequence from clone BDGP:LP03039.3prime-hit
-1,6
engrailed -1,7
FB:FBgn0039709 /sym=CG7805 /name= /prod=cadherin /func=cell adhesion /map=99C6-99C6 /transc=CT4930 /len=5083 /GB:AE003771 /note=3prime sequence from clone BDGP:LD23052.3prime-hit
-1,7
FB:FBgn0020224 /sym=Cbl /name= /prod= /func=cell cycle regulator /map=66C12-66C13 /transc=CT21779 /len=2675 /GB:AE003555 -1,7
echinoid -1,7
Ras-related protein -1,7
E(spl) region transcript m /// /// -1,7
Hemolectin -1,7
fringe -1,8
forkhead domain 96Cb -1,8
Wrinkled -2,0
BDGP:LD29477.3prime-hit /ESTpos=maps in FB:FBgn0039021 /sym=CG17138 /name= /prod= /func= /map=94C6-94C6 /transc=CT38064 /len=443 -2,0
FB:FBgn0037305 /sym=CG12173 /name= /prod= /func=protein phosphatase /map=83A1-83A1 /transc=CT8961 /len=763 /GB:AE003603 -2,0
Enhancer of split /// -2,1
FB:FBgn0031961 /sym=CG7102 /name= /prod=Sm-E snRNP core protein /func=RNA binding /map=28D5-28D5 /transc=CT21947 /len=1180 /GB:AE003619 /note=3prime sequence from clone BDGP:LD40565.3prime-hit
-2,1
FB:FBgn0038826 /sym=CG17838 /name= /prod=RNA binding protein /func=RNA binding /map=92F10-92F10 /transc=CT39628 /len=1802 /GB:AE003732 /note=3prime sequence from clone BDGP:GH28335.3prime-hit
-2,1
FB:FBgn0039704 /sym=CG7802 /name= /prod= /func= /map=99C5-99C5 /transc=CT4915 /len=2950 /GB:AE003771 /note=3prime sequence from clone BDGP:GH22837.3prime-hit
-2,1
BDGP:LD32760.3prime-hit /ESTpos=maps 3prime of FB:FBgn0039851 -2,5
FB:FBgn0039539 /sym=CG12880 /name= /prod= /func= /map=98A8-98A8 /transc=CT32024 /len=1649 /GB:AE003762 /note=3prime sequence from clone BDGP:LD29228.3prime-hit
-2,5
225
APPENDIX I
FB:FBgn0022342 /sym=CG4844 /name= /prod= /func=ligand binding or carrier /map=54C7-54C8 /transc=CT15561 /len=1202 /GB:AE003803 /note=3prime sequence from clone BDGP:LD10058.3prime-hit
-2,5
FB:FBgn0039484 /sym=CG6124 /name= /prod= /func=cell adhesion /map=97E6-97E6 /transc=CT19227 /len=2673 /GB:AE003759 -2,9
interference Hedgehog -3,0
FB:FBgn0031956 /sym=CG7106 /name= /prod=C-type lectin-like /func=ligand binding or carrier /map=28D3-28D3 /transc=CT21973 /len=789 /GB:AE003619 -5,4
FB:FBgn0039760 /sym=CG9682 /name= /prod= /func= /map=99E5-99E5 /transc=CT27378 /len=1449 /GB:AE003773 /note=3prime sequence from clone BDGP:LP01629.3prime-hit
-6,0
Ecdysone-inducible gene E2 -11,7
226
227
Appendix II- Microarays Results for expression patterning of tshNG1 neuroblasts expression comparing to wild-type
(ordered by ontology cluster)
Table II- Results of the gene ontology clusters with a P-value <0.001 in tshNG1 homozygous neuroblats microarrays after using a
cut-off of 1.3. The positive lower bound of fold change (LBFC) represents the up regulated genes while the negative LBFC represent the
downregulated genes. Only the genes with the absolute value of LBFC upper than 1.3 are represented.
Gene
LBFC
Gene Ontology "extracellular region" genes in a list with 267 annotated genes (all: 326/8391, PValue: 0.000000) ********* FB:FBgn0028542 /sym=BG:DS00180.8 /name= /prod= /func=cell adhesion /map=34E1-34E1 /transc=CT37470 /len=3394 /GB:AE003642 2,1 FB:FBgn0031097 /sym=CG17052 /name= /prod=peritrophin-like /func=structural protein /map=19C1-19C1 /transc=CT37860 /len=1383 /GB:AE003572 -1,5 Drosulfakinin 1,4 Trypsin 1,5 wingless -1,6 Lysozyme B 1,7 Lysozyme E 1,9 Lysozyme S 1,5 hedgehog -1,3 Trypsin 2,4 /// Trypsin /// Trypsin 1,4 /// /// Trypsin /// Trypsin 1,5 Drosomycin 3,3 Drosocin 2,7 Ejaculatory bulb protein III /// 1,5 Transferrin 1 1,7 Imaginal disc growth factor 4 1,4 FB:FBgn0031558 /sym=CG16704 /name= /prod= /func= /map=24A4-24A4 /transc=CT37177 /len=318 /GB:AE003579 1,9 Saccharomyces cerevisiae UAS construct a of Struhl -1,6 Immune induced molecule 23 2,5 Immune induced molecule 1 7 Odorant-binding protein 56a 3 insulin-like peptide 3 1,4
APPENDIX II
FB:FBgn0036234 /sym=CG17824 /name= /prod=peritrophin-like /func=cell adhesion /map=68E3-68E3 /transc=CT39558 /len=2415 /GB:AE003543 1,6 Insulin-related peptide 1,4 Imaginal disc growth factor 2 1,4 Hemolectin -1,7 Larval serum protein 1 1,3 Hikaru genki 1,3 Tissue inhibitor of metalloproteases 1,9 Ecdysone-inducible gene E2 -11,7 Attacin-A 3 spatzle 1,5 Gelsolin 1,4 Gene Ontology "imaginal disc pattern formation" genes in a list with 267 annotated genes (all: 10/8391, PValue: 0.000007) ****** twins -1,4 wingless -1,6 hedgehog -1,3 Epidermal growth factor receptor -1,4 Engrailed -1,7 Gene Ontology "cell fate specification" genes in a list with 267 annotated genes (all: 19/8391, PValue: 0.000019) ***** Bearded -1,5 E(spl) region transcript m4 /// /// /// -1,3 E(spl) region transcript m /// /// -1,7 tailless -1,4 wingless -1,6 fringe -1,8 Gene Ontology "lysozyme activity" genes in a list with 267 annotated genes (all: 12/8391, PValue: 0.000021) ***** Lysozyme B 1,7 Lysozyme A /// Lysozyme C 3,1 Lysozyme A /// Lysozyme D 1,9 Lysozyme E 1,9 Lysozyme S 1,5 Gene Ontology "defense response" genes in a list with 267 annotated genes (all: 449/8391, PValue: 0.000087) ***** FB:FBgn0033367 /sym=CG8193 /name= /prod=monophenol oxidase /func=enzyme /map=45A6-45A7 /transc=CT2817 /len=2272 /GB:AE003835 -1,4 Glutathione S transferase E6 2,3 Glutathione S transferase E7 1,8 Twins -1,4 Multiple drug resistance 65 1,4 Drosomycin 3,3
228
APPENDIX II
Drosocin 2,7 glial cells missing -1,5 Galactose-specific C-type lectin 1,7 Transferrin 1 1,7 FB:FBgn0033089 /sym=CG17266 /name= /prod=peptidylprolyl isomerase /func=chaperone /map=42B4-42B4 /transc=CT38243 /len=552 /GB:AE003789 -1,3 FB:FBgn0033817 /sym=CG4688 /name= /prod= /func=enzyme /map=49F15-49F15 /transc=CT15137 /len=699 /GB:AE003819 1,5 Immune induced molecule 23 2,5 Immune induced molecule 1 7 Glutathione S transferase D10 1,4 lethal (2) essential for life 1,5 FB:FBgn0034638 /sym=CG10433 /name= /prod= /func= /map=57F3-57F3 /transc=CT29298 /len=1071 /GB:AE003454 /note=3prime sequence from clone BDGP:GH10517.3prime-hit
1,5
FB:FBgn0037552 /sym=CG7800 /name= /prod= /func=cell adhesion /map=84F5-84F5 /transc=CT23295 /len=1745 /GB:AE003678 /note=3prime sequence from clone BDGP:GH11496.3prime-hit
1,3
FB:FBgn0038115 /sym=CG7966 /name= /prod=selenium-binding protein-like /func=ligand binding or carrier /map=87D11-87D11 /transc=CT23978 /len=1498 /GB:AE003698 /note=3prime sequence from clone BDGP:GH14316.3prime-hit
1,6
FB:FBgn0030484 /sym=CG1681 /name= /prod=glutathione transferase-like /func=enzyme /map=11F1-11F1 /transc=CT4698 /len=1098 /GB:AE003492 /note=3prime sequence from clone BDGP:GH16740.3prime-hit
1,6
Hemolectin -1,7 18 wheeler -1,6 hikaru genki 1,3 FB:FBgn0035452 /sym=CG10359 /name= /prod=fibrinogen-like /func=structural protein /map=63E3-63E4 /transc=CT29060 /len=1675 /GB:AE003478 /note=3prime sequence from clone BDGP:GM10015.3prime-hit
1,3
Thor 1,4 FB:FBgn0039644 /sym=CG11897 /name= /prod=ATP-binding cassette transporter /func=transporter /map=99A2-99A3 /transc=CT37062 /len=4458 /GB:AE003768 /note=3prime sequence from clone BDGP:LD17001.3prime-hit
1,6
FB:FBgn0034354 /sym=CG5224 /name= /prod= /func=enzyme /map=55D3-55D3 /transc=CT16701 /len=734 /GB:AE003799 /note=3prime sequence from clone BDGP:LD18692.3prime-hit
-1,4
UDP-glycosyltransferase 35a 1,5 FB:FBgn0037515 /sym=CG3066 /name= /prod=monophenol monooxygenase activator /func=serine carboxypeptidase /map=84D11-84D11 /transc=CT10270 /len=1830 /GB:AE003676 /note=3prime sequence from clone BDGP:SD07170.3prime-hit
1,3
spatzle 1,5 Gene Ontology "Notch signaling pathway" genes in a list with 267 annotated genes (all: 38/8391, PValue: 0.000165) **** Enhancer of split /// -2,1 Bearded -1,5 deltex -1,4 E(spl) region transcript m4 /// /// /// -1,3 E(spl) region transcript m /// /// -1,7 E(spl) region transcript m /// /// -1,5 Fringe -1,8 Gene Ontology "antimicrobial humoral response (sensu Protostomia)" genes in a list with 267 annotated genes (all: 20/8391, PValue:
229
APPENDIX II
0.000329) **** Lysozyme B 1,7 Lysozyme A /// Lysozyme C 3,1 Lysozyme A /// Lysozyme D 1,9 Lysozyme E 1,9 Lysozyme S 1,5 Gene Ontology "chymotrypsin activity" genes in a list with 267 annotated genes (all: 182/8391, PValue: 0.000674) **** Trypsin 1,5 Trypsin 2,4 /// Trypsin /// Trypsin 1,4 /// /// Trypsin /// Trypsin 1,5 Jonah 25Bi 1,6 Jonah 25Bii 1,6 FB:FBgn0032413 /sym=CG16997 /name= /prod= /func=endopeptidase /map=33C1-33C1 /transc=CT34780 /len=822 /GB:AE003635 1,4 FB:FBgn0032638 /sym=CG6639 /name= /prod= /func=endopeptidase /map=36C2-36C2 /transc=CT20606 /len=1493 /GB:AE003655 1,6 Jonah 65Aiv 1,5 Jonah 74E 2 FB:FBgn0038113 /sym=CG11668 /name= /prod=serine protease /func=endopeptidase /map=87D11-87D11 /transc=CT36689 /len=1197 /GB:AE003698 1,8 BDGP:LD47230.3prime-hit /ESTpos=maps in FB:FBgn0034661 /sym=CG4386 /name= /prod=serine protease /func=endopeptidase /map=58A2-58A2 /transc=CT14320 /len=559 1,7 Jonah 65Aiii 1,7 FB:FBgn0039102 /sym=CG16705 /name= /prod=serine protease-like /func=endopeptidase /map=95B1-95B1 /transc=CT37173 /len=1288 /GB:AE003744 /note=3prime sequence from clone BDGP:GH28857.3prime-hit 1,4 FB:FBgn0037515 /sym=CG3066 /name= /prod=monophenol monooxygenase activator /func=serine carboxypeptidase /map=84D11-84D11 /transc=CT10270 /len=1830 /GB:AE003676 /note=3prime sequence from clone BDGP:SD07170.3prime-hit 1,3 Gene Ontology "wing disc anterior/posterior pattern formation" genes in a list with 267 annotated genes (all: 14/8391, PValue: 0.000780) **** wingless -1,6 hedgehog -1,3 engrailed -1,7 FB:FBgn0039709 /sym=CG7805 /name= /prod=cadherin /func=cell adhesion /map=99C6-99C6 /transc=CT4930 /len=5083 /GB:AE003771 /note=3prime sequence from clone BDGP:LD23052.3prime-hit -1,7
230
231