Morphological and functional effects of insulin signaling and the bHLH transcription factor Dimmed on different neuron types in Drosophila

Yiting Liu

Morphological and functional effects of insulin signaling and the bHLH transcription factor Dimmed on different neuron types in Drosophila

Yiting Liu

©Yiting Liu, Stockholm University 2016 Cover picture: ‘‘a fly in neon lights’’ ISBN 978-91-7649-283-3 Printed in Sweden by Holmbergs, Malmö, Stockholm 2016 Distributor: Department of Zoology, Stockholm University

List of papers This thesis is based on the following papers and manuscripts and will be refered by their Roman numerals.

I. Luo J, Liu Y, Nässel DR: Insulin/IGF-regulated size scaling of neuroendocrine cells expressing the bHLH transcription factor Dimmed in Drosophila. PLoS Genetics 9: e1004052 (2013).

II. Liu Y, Luo J, Nässel DR: Multiple effects of the transcription

factor Dimmed and the insulin receptor on growth and differentiation vary among neuron types and developmental stages in Drosophila. Manuscript

III. Luo J*, Liu Y*, Nässel DR: Transcriptional reorganization

of Drosophila motor neurons and their muscular junctions towards a neuroendocrine phenotype by the bHLH protein DIMM. Manuscript (* equal contribution)

IV. Liu Y, Liao S, Veenstra JA, Nässel, DR: Drosophila insulin-like

peptide 1 (DILP1) is transiently expressed and plays roles during non-feeding stages and reproductive dormancy. Sci. Rep. (under revision)

Publications not included in the thesis

1. Liu Y*, Luo J*, Carlsson MA, Nässel DR. Serotonin and insulin-like peptides modulate leucokinin-producing neurons that affect feeding and water homeostasis in Drosophila. J. Comp. Neurol. (2015). (* equal contribution)

2. Nässel, DR, Liu, Y., and Luo, J. (2015). Insulin/IGF signaling and its

regulation in Drosophila. Gen Comp Endocrinol 221, 255-266.

3. Nässel DR, Kubrak OI, Liu Y, Luo J, Lushchak OV (2013) Factors that regulate insulin producing cells and their output in Drosophila. Frontiers in physiology 4: 252.

Contents

Introduction .................................................................................................... 9Insulin-like peptides and growth ................................................................................... 10Insulin signaling pathway and growth ........................................................................... 14Fat body and growth ..................................................................................................... 17Glial cells and growth ................................................................................................... 17Dimmed and its functions ............................................................................................. 18

Aim of the thesis .......................................................................................... 21Material and Methods .................................................................................. 22

Drosophila husbandry ................................................................................................... 22Immunocytochemistry ................................................................................................... 22Image analysis .............................................................................................................. 23Quantitative real-time PCR (qPCR) .............................................................................. 24Stress assays ............................................................................................................... 24Capillary feeding (CAFE) assay ................................................................................... 25Locomotor activity recording ......................................................................................... 26Statistical Analysis ........................................................................................................ 26

Discussion .................................................................................................... 27Conclusions ................................................................................................. 32References ................................................................................................... 34Acknowledgement ........................................................................................ 40

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Introduction The Insulin/insulin-like growth factor signaling (IIS) pathway is evolutionarily

conserved in animals, and functions as a nutrient-sensing pathway,

mediating dietary conditions to control growth and development, metabolism,

reproduction, stress responses, and life span (Baker and Thummel, 2007;

Broughton et al., 2005; Geminard et al., 2009; Giannakou and Partridge,

2007; Gronke et al., 2010; Tatar et al., 2001; Teleman, 2010; Wu and Brown,

2006). Multiple roles of insulin have been unraveled, since the first discovery

and pioneering work on this peptide hormone. Frederick Banting and

Charles Best injected a ground-up extract of pancreas into a dog, from which

pancreas had been removed. The dog regained health. Later, this extract

named “isletin” was extracted and purified from cows, and is known as

insulin today, and was used for treating children with hypoglycemia (Banting

et al., 1922). The effect of insulin was very successful. For this great

contribution, Banting and his mentor John Macleod were awarded the 1923

Nobel Prize in Medicine and Physiology. A lot of investigations followed and

illuminated a variety of roles of insulin. Mammalian insulin released from

pancreatic beta cells regulates the metabolism of carbohydrates and lipids

by reducing the glucose level in the blood, by promoting the glucose uptake

from the blood to skeletal muscles and adipose tissue. In the brain,

mammalian insulin can modulate neuronal function in the hypothalamic

arcuate nucleus and influence food intake (Fernandez and Torres-Aleman,

2012). Memory in recognition tasks and odor discrimination can be improved

with the mediation of insulin signaling in the olfactory bulb (Marks et al.,

2009). In addition to the classical roles of insulin, it is also involved in

development and growth functions. Peptides of the insulin family have

various functions in cell growth, neuronal proliferation and neurite outgrowth.

Cell cycle and cell cycle reentry in the embryonic cerebral cortex can be

promoted by insulin-like growth factor-I (IGF-I) (Hodge et al., 2004), as well

as neuronal survival and differentiation in the developmental rat cerebellum

(Torres-Aleman et al., 1994). IGF-I is also involved in the axon outgrowth of

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corticospinal motor neurons (Ozdinler and Macklis, 2006), and acts

chemoattractively for the axon guidance of olfactory neurons in the olfactory

bulb during rat development (Scolnick et al., 2008).

The insulin-signaling pathway has been studied in a number of model

organisms, both in invertebrates and vertebrates. The three most well

studied organism groups are the nematode Caenorhabditis elegans, the fruit

fly Drosophila melanogaster and various mammals. Studies found homologs

of insulin signaling components across those organisms, and showed the

evolutionally conservation of this important pathway (Garofalo, 2002; Gronke

et al., 2010). Drosophila melanogaster, because of its easy culturing in the

laboratory, short generation time, genetic tractability and huge molecular

toolbox (Mohr et al., 2014), has been used and improved as an invertebrate

model organism since its first use in the hands of Thomas Hunt Morgan in

1906 (Wangler et al., 2015). Drosophila has been well studied in various

biological roles, and for human disease, including the unraveling of the entire

insulin-signaling pathway (Altintas et al., 2015; Brookheart and Duncan,

2015; Donelson and Sanyal, 2015; Jimenez-Del-Rio and Velez-Pardo, 2015;

Lepesant, 2015; Panagi et al., 2015).

Insulin-like peptides and growth Eight insulin-like peptides, DILP1–8, have been identified in Drosophila

melanogaster, mostly based on their sequence homology to the mammalian

insulin and the typical B-C-A domain structure and positions of cysteins

necessary for forming disulfide bridges, as observed in mammalian insulin

(Brogiolo et al., 2001; Colombani et al., 2012; Garelli et al., 2012; Gronke et

al., 2010). Dilp1-8 encoding precursor proteins have structures similar to

preproinsulin containing a signal peptide, a B chain, a C peptide, and an A

chain. The active peptides consist of two polypeptide chains (B and A) joined

by disulfide bridges, which is comparable with vertebrate insulins (Brogiolo et

al., 2001; Colombani et al., 2012; Garelli et al., 2012).

The eight insulin-like peptides (DILP1-8) are differentially expressed

during the developmental stages and in tissues: in larvae DILP2, DILP4, and

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DILP7 are expressed in the mesoderm and midgut; DILP2, DILP3 and DILP5

are expressed in a set of 14 median neurosecretory cells (insulin producing

cells, IPCs) of the brain that send axons to the ring gland, heart and foregut.

DILP2 is expressed in imaginal discs and salivary glands, and may be also

in glial cells of the CNS at the early larval stages. DILP3 is expressed in

adult midgut muscle. DILP5 is expressed in ovarian follicle cells and

Malpighian tubules. DILP6 is expressed in the fat body, larval salivary

glands, and heart and in surface glial cells of the CNS. DILP7 is expressed

in neurons of the abdominal neuromeres of the ventral ganglion, a subset of

which innervates the adult hindgut and another that makes possible contact

with IPCs. DILP8 is expressed in the imaginal discs in the larvae and ovaries

in the adult flies (Brogiolo et al., 2001; Broughton et al., 2005; Chell and

Brand, 2010; Cognigni et al., 2011; Colombani et al., 2012; Garelli et al.,

2012; Gorczyca et al., 1993; Ikeya et al., 2002; Miguel-Aliaga et al., 2008;

Okamoto et al., 2009; Rulifson et al., 2002; Slaidina et al., 2009; Veenstra et

al., 2008). A summary is shown for the main distributions and axon

terminations of DILPs in Drosophila (Fig. 1).

DILP1 and DILP4 have been very little studied, both in terms of temporal

and spatial expression, and with respect to functional roles. Dilp1 was

detected by in situ hybridization in larval IPCs (Lee et al., 2008) and dilp4 in

the anterior larval midgut (Brogiolo et al., 2001). Thus, Paper IV in this thesis

presents a first comprehensive study of dilp1/DILP1 expression, how

expression is regulated, and some putative functions, especially in relation to

adult reproductive dormancy (diapause).

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Fig. 1. Distribution of DILP1-7 in the central nervous system and periphery.

A set of 14 IPCs (blue, producing DILP2, 3, 5) localized in pars intercerebralis of the

brain send axons to the tritocerebrum close to the subesophageal ganglion (SEG), to

the corpora cardiaca (CC), anterior aorta, proventriculus (PV) and crop. Another set

of 20 neurons in the ventral nerve cord (VNC) produces DILP7 (red). Their axons

extend to the hindgut including the rectal papillae (RP), and branches from two of the

neuron set project to the SEG. DILP5 is produced in the principal cells (green) of the

renal tubules (RT). DILP6 is produced in the fat body cells in both head and abdomen

(Nässel et al., 2013).

Even though dilps show highly differentiated temporal and spatial

expression patterns, ubiquitous overexpression of dilp1-7 is sufficient to

promote organismal growth (Brogiolo et al., 2001; Ikeya et al., 2002).

Ubiquitous expression of each of the dilp genes results in body size

increase. Overexpression of dilp2 leads to a 51% weight gain in male flies.

Both the number of ommatidia in eyes and cell size and number in the wing

blade show an increase in the bigger flies (Brogiolo et al., 2001; Ikeya et al.,

2002). Single null mutants of some dilps also lead to loss of body weight in

both male and female adult flies. Triple mutation of dilp2-3,5 or dilp1-4,5

displays even smaller flies with 47% and 53% reduction of body weight.

Wing size and cell number, and fat body cell size are reduced in dilp1-5

mutant flies, which gives rise to smaller larvae, pupae and adult flies (Zhang

et al., 2009).

Targeted ablation of IPCs leads to severely smaller larvae with 58% of

normal length, seven days developmental delay to reach wandering third

instar and puparium formation. The adult wings show reductions in both cell

size and number (Rulifson et al., 2002). The ovary size strongly decreases

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after ablation of IPCs. Compared to wild type with multiple vitellogenic

oocytes in each ovariole, IPC-ablated female possess at most a single

vitellogenic oocyte, with which a reduced fecundity can be suspected (Ikeya

et al., 2002).

Dilp6 was found in a temporally regulated expression pattern. It reaches

remarkably high levels during the late third instar and pupa-adult non-feeding

stages, whereas other dilps were at relatively very low levels at the same

time (Okamoto et al., 2009; Slaidina et al., 2009). Dilp6 mutants have a

~10% reduced adult body size and weight (Okamoto et al., 2009; Slaidina et

al., 2009; Zhang et al., 2009). There is a reduction in wing area, a decrease

in cell number, but no reduction in the cell size (Okamoto et al., 2009).

DILP6 acts as an IGF-like peptide that regulates the utilization efficiency of

nutrient stores and growth during the post-feeding stage. Fat-body specific

expression of dilp6 could rescue the dilp6 mutant phenotype (Okamoto et al.,

2009; Slaidina et al., 2009). Furthermore, DILP6 acts locally in the glial cells

within the central nervous system (Avet-Rochex et al., 2012; Chell and

Brand, 2010; Sousa-Nunes et al., 2011). This aspect will be introduced in the

later section on the function of glial cells in growth.

DILP7 is expressed in several sets of neurons (dMP2) in abdominal

neuromeres A6-A9 and one pair of DP interneurons in A1 neuromeres

(Miguel-Aliaga et al., 2008; Yang et al., 2008). Some of the abdominal DILP7

expressing neurons are postmitotically differentiated from the surviving

dMP2 neuroblasts. Specific combinatorial codes and the retrograde bone

morphogenetic protein (BMP) signaling are involved in this postmitotic

transition from dMP2 pioneer neurons to DILP7 expressing neurons (Miguel-

Aliaga et al., 2008). The recently discovered new member of the DILP family, DILP8, is a

divergent insulin/relaxin-like peptide (Colombani et al., 2012; Garelli et al.,

2012). DILP8 is secreted by damaged or tumorous imaginal discs and leads

to the delay of pupariation through c-Jun N-terminal kinase (JNK) signaling,

a stress-activated signaling pathway and ecdysone signaling (Colombani et

al., 2012; Garelli et al., 2012; Hackney and Cherbas, 2014; Katsuyama et al.,

2015). The relaxin family receptor Lgr3 has been shown as DILP8 receptor

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in very recent studies (Garelli et al., 2015; Katsuyama et al., 2015; Vallejo et

al., 2015). A pair of bilateral Lgr3 positive neurons in larval CNS connects

with IPCs and prothoracicotropic hormone (PTTH) producing neurons to

attenuate growth and maturation. The DILP8 signal coordinates via this

circuitry to maintain organismal size (Colombani et al., 2015; Vallejo et al.,

2015).

Insulin signaling pathway and growth The Drosophila insulin receptor (dInR) and its downstream effectors are

expressed ubiquitously, whereas the eight insulin-like peptides are

expressed spatio-temporarily in different cells and tissues, presumably in

response to different inputs (Brogiolo et al., 2001; Ikeya et al., 2002).

Activated insulin receptors (receptor tyrosine kinase) trigger a conserved

phosphorylation cascade involving the pathway components (see Fig 2): the

insulin receptor substrate Chico (Bohni et al., 1999), phosphoinositide-3

kinase (PI3K), and Akt/protein kinase B (PKB) (Oldham and Hafen, 2003).

An active PI3K complex consists of a catalytic subunit (p110) and an adaptor

subunit (p60) (Leevers et al., 1996; Weinkove et al., 1999). The p110

catalytic subunit catalyzes the conversion of phosphatidylinositol 4,5-

bisphosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3) in the

cell membrane, which can be catalytically reversed by the lipid phosphatase

(PTEN) by removing the D3 phosphate from PIP3 (Gao et al., 2000;

Goberdhan et al., 1999; Oldham et al., 2002). Accumulation of PIP3 in cell

membrane recruits phosphoinositide-dependent protein kinase 1 (dPDK1)

and Akt1/PKB (Rintelen et al., 2001; Verdu et al., 1999). Activation of Akt by

phosphorylation (p-Akt) subsequently leads to phosphorylation of various

downstream proteins (Montagne et al., 1999; Verdu et al., 1999). One of the

downstream targets of Akt is a transcription factor FOXO. Phosphorylation of

FOXO inhibits its nuclear localization, thus preventing its transcriptional

activity (Junger et al., 2003).

Target of rapamycin (TOR) protein kinase is another branch of signaling

cascade mediating nutrient sensing and cell growth, which is closely

associated with insulin signaling. Activation of TOR promotes cell growth by

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enhancing global translation and ribosome biogenesis through

phosphorylation of the translation initiation factor 4E-binding protein (4EBP)

and ribosomal protein S6 kinase (S6K), respectively (Hay et al., 2004;

Montagne et al., 1999). Each branch of IIS and TOR can function either

independently (Radimerski et al., 2002) or together as linear insulin/Akt/TOR

signaling network (Banerjee et al., 2012; Dann and Thomas, 2006; Oldham

et al., 2000). TOR can be activated cell-autonomously in response to nutrient

availability, especially at the level of cellular amino acids (Oldham et al.,

2000). Moreover, insulin signaling can indirectly activate the TOR pathway.

Activation of Akt by insulin signaling promotes the TOR pathway by

suppressing the complex formed by tuberous sclerosis complex 2 (TSC2),

an inhibitor of TOR activity (Gao and Pan, 2001; Potter et al., 2001).

Complete loss-of-function mutations in the dInR lead to embryonic

lethality, whereas some partial loss-of-function mutations (heteroallelic

combinations) yield viable animals that are sterile, and with abnormal head

structures and cuticle (Chen et al., 1996; Fernandez et al., 1995; Tatar et al.,

2001). Loss-of-function mutant embryos lack neuroblasts and fail to

complete germ band retraction and dorsal closure, and are defective in

ventral nerve cord condensation and commissure formation. One mutation in

the dInR (InRE19) (Brogiolo et al., 2001; Chen et al., 1996) has a 10 to 20

day prolongation of the developmental time and a severely but proportionally

reduced body size. Mutants display reduced cell density in the wing, less

average number of ommatidia (photoreceptive units) in the compound eye

and smaller eye tissue in the head capsule of mutant male flies. The head

size is dependent on the allele (the strongest reduction was in mutants

InR339, a putative null allele), these results revealed that the small body size

results from a reduction in cell size and cell number and from dInR regulation

autonomously in a cell- and tissue-specific manner. Fat body-specific

activation of dInR promotes triglyceride storage, by increasing fat cell

number and lipid content (DiAngelo and Birnbaum, 2009). Mutations in

downstream components of the IIS pathway, Chico, PTEN and catalytic

subunit Dp110 of PI3K affect both cell number and cell size (Bohni et al.,

1999; Goberdhan et al., 1999; Leevers et al., 1996), whereas downstream

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factor S6K mutant flies display only reduced cell and body size without

changes in cell number (Montagne et al., 1999). dPDK1 controls cellular and

organism growth by activating Akt1 and S6 kinase, dS6k (Rintelen et al.,

2001). Loss of dPDK1 results in reduced head size. TSC 1/2 are negative

regulators of insulin signaling in cell growth. Heterozygosity of TSC1 or

TSC2 is sufficient to rescue the lethality of loss-of-function dInR mutants

(Gao and Pan, 2001). Overexpression of dFOXO, another target of Akt

signaling, inhibits cell proliferation and causes smaller organ size and

reduction of cell number (Puig et al., 2003; Wang et al., 2005). FOXO limits

growth and extends life span in conditions of poor nutrition or oxidative

stress (Wang et al., 2005).

Fig. 2. The IIS and TOR-signaling pathways in Drosophila melanogaster. The insulin/PI3K/Akt branch exerts its downstream effects through various ways. Akt phosphorylates FOXO transcription factor, prevents its nuclear localization, and thus inhibits the expression of a variety of FOXO target genes. Akt can also activate TOR by inhibition of TSC1/2, the negative regulators of TOR, thereby allowing the connection between IIS and TOR branch. The Alk signaling pathway is activated only at nutritional restriction (NR) and inhibits the two other pathways to ensure CNS growth as a super sparing of this tissue at the cost of other tissues. However at low nutrients conditions the Alk signaling activates the pathway downstream the dInR (PI3K). Arrows depict activating signals and the T-shaped connectors inhibitory ones. The two pathways intersect at the level of Akt and TSC1/2 and converge on regulation of protein synthesis and growth (via ribosome biogenesis and translation apparatus) (Luo et al., 2013).

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Fat body and growth The fat body plays a central role in systemic amino acid sensing and

regulation of growth (Colombani et al., 2003; Geminard et al., 2009;

Hietakangas and Cohen, 2009; Rajan and Perrimon, 2012). Fat body

secretes several factors into the hemolymph and circulation, which affects

insulin signaling and modulates systemic growth, such as Unpaired 2 (Upd2)

(Colombani et al., 2003; Geminard et al., 2009; Rajan and Perrimon, 2012),

the acid labile subunit (ALS) protein (Arquier et al., 2008; Boisclair et al.,

2001; Honegger et al., 2008), Neural Lazarillo (NLaz) (Hull-Thompson et al.,

2009; Pasco and Leopold, 2012) and Adiponectin (Kadowaki et al., 2006;

Kwak et al., 2013; Matsuzawa et al., 2004). Upd2 act on GABAergic neurons

in the vicinity of the IPCs, and eventually inhibits the tonic inhibition of IPCs,

and facilitates the release of DILPs into the hemolymph (Geminard et al.,

2009). dALS forms a ternary complex with DILP2 and Imaginal

morphogenesis protein - Late 2 (Imp-L2) (Honegger et al., 2008), a homolog

to human IGF binding protein 7 (IGFBP-7) (Alic et al., 2011). This complex

appears to antagonize the activity of DILPs. NLaz suppresses insulin

signaling (Hull-Thompson et al., 2009; Pasco and Leopold, 2012).

Drosophila adiponectin receptor (dAdipoR) is expressed in the IPCs in both

larval and adult brains and plays a role in the regulation of DILP2 secretion

(Kwak et al., 2013).

Glial cells and growth

Glial cells in the central nervous system are also involved in modulation of

organismal growth in Drosophila melanogaster. Recently a new CNS glia-

derived protein in the hemolymph, secreted decoy of InR (SDR), has been

identified as a suppressor of systemic insulin signaling and body growth

(Okamoto et al., 2013). Due to high similarity to a sequence of the

extracelluar domain of dInR, SDR competes with dInR to bind DILPs and

thus suppresses the activity of DILPs (Okamoto et al., 2013). DILP2 and

DILP6 expressed in blood brain barrier (BBB) glia or DILP6 from underlying

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cortex glia are required for inducing growth and thus reactivation of

proliferation in quiescent neuroblasts (Chell and Brand, 2010; Hindle and

Bainton, 2014; Sousa-Nunes et al., 2011; Speder and Brand, 2014).

Neuroblast reactivation happens during the first and second larval instars.

DILPs from glial cells, rather than from IPCs regulate the exit of neuroblasts

from quiescence (Sousa-Nunes et al., 2011). This nutritional checking point

requires dietary amino acids. However, larval CNS supplied with amino acids

cannot induce neuroblast reactivation, whereas adding the fat body together

reactivates neuroblasts (Sousa-Nunes et al., 2011). Upd 2 from fat body

seems to activate the surface glia in the CNS to trigger production and

secretion of DILPs to reactivate neuroblasts (Chell and Brand, 2010; Rajan

and Perrimon, 2012).

In summary, growth of cells including cell number and cell size, tissues

and whole organisms can be regulated via insulin signaling pathway and

TOR pathway, combined with cell autonomous nutrient sensing. Fat body

and glial cells are involved in this mechanism and secrete different IIS

interacting factors for systemic growth or neuroblast reactivation. But insulin

signaling and its possible sources and nutritional conditions for regulation of

post-mitotic cell growth and differentiation have been less investigated. This

aspect was studied in Paper I-III in this thesis, where also the requirement of

Dimmed is established (see next section).

Dimmed and its functions Dimmed (Dimm) is a basic helix-loop-helix (bHLH) transcription factor in

Drosophila, which is the ortholog of mammalian Mist1 (Lemercier et al.,

1998). Dimm is expressed in a set of 300 neuroendocrine cells in the larval

central nervous system, and coexpressed with at least 24 amidated

neuropeptides from early post-mitotic stage through adulthood (Hewes and

Taghert, 2001; Nassel and Winther, 2010; Park et al., 2008b). DIMM directly

activates transcription of the amidating enzyme peptidylglycine alpha-

hydroxylating monooxygenase (PHM) (Park et al., 2008a). To maintain the

secretory properties, Dimm controls the level of regulated secretory activity

in neuroendocrine cells, regulating production, packaging and releasing

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large amounts of neuropeptide/peptide hormone (Hamanaka et al., 2010;

Hewes et al., 2003; Park et al., 2008b). Thus, Dimm is a master regulator of

peptidergic cell fate. A previous report demonstrated that Dimm-

misexpression induces an altered phenotype in histaminergic photoreceptors

of the Drosophila compound eye (Hamanaka et al., 2010). Photoreceptor

cells after Dimm-expression produce no histamine, and display highly

accumulated large dense core vesicles (LDCVs), loss of presynaptic active

zone, and capitate projections (Hamanaka et al., 2010; Kittel et al., 2006).

Moreover, ectopic Dimm expression induces aggregation of peptidergic

vesicles in axonal boutons. Dimm seems to transform boutons at type Ib

terminals towards a morphology more similar to type III boutons and in turn

enhance presynaptic dense core vesicle (DCV) capture (Bulgari et al., 2014).

Furthermore, Dimm was also found recently to maintain levels of two

synaptotagmin proteins in Dimm positive cells. These two vesicle-associated

synaptotagmin proteins are involved in controlling release of neuropeptide

(Park et al., 2014). Besides, Dimm can be induced transiently and sensitively

in the enteroendocrine cells of the adult midgut to protect against infection

by Gram-negative pathogen Pseudomonas entomophila (Pe) (Beebe et al.,

2015). Genome wide analysis via in vivo chromatin immunoprecipitation

(ChiP) and deep sequencing of purified Dimm neurons identifies a number of

direct Dimm binding targets that are associated with key roles in

determination of important properties of neuroendocrine cells. Most of these

target genes are linked to features of LDCVs and the secretory pathway

(Hadzic et al., 2015).

In addition to the function as master regulator of secretory capacity, it also

acts as a part of a combinatorial code for terminal differentiation of neurons

(Allan et al., 2005; Hewes et al., 2006; Hewes et al., 2003; Miguel-Aliaga et

al., 2008). As a bHLH transcription factor, Dimm can cooperate with other

transcription factors in a cascade to determine specification of cells. Dimm

together with the homeodomain transcription factor Apterous and the zinc-

finger transcription factor Squeeze forms a combinatorial code to control the

expression of the neuropeptide FMRFamide (Allan et al., 2005; Allan et al.,

2003). Ectopic expression of FMRFamide results from misexpression of

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these three factors. The dMP2 neurons require the Hox gene abdominal A

(adb-A), Hb9, Fork Head and dimm to go through the postembryonic

apoptosis, survival and insulinergic differentiation (Miguel-Aliaga et al.,

2008). Different combinatorial codes contribute to specification of different

neuropeptidergic identities in neurons, and different expression levels of

each combinatorial code component is required in a cell type specific

manner (Gauthier and Hewes, 2006; Herrero et al., 2003). The different

types of leucokinin (LK) producing neurons are differentially regulated by

Dimm during development. Dimm mutants display an increase of transcript

level of Lk in LHLK and SELK neurons, but a reduced level of LK peptide

(Gauthier and Hewes, 2006). On the contrary, Lk transcript level is

downregulated in ABLKs. Combining with the temporal gene grainy head,

Dimm can trigger ectopic expression of FMRFamide peptide in ABLK

neurons (Baumgardt et al., 2009; Benito-Sipos et al., 2010).

In this thesis the effect of ectopic expression of Dimm and dInR in various

sets of neurons was studied in order to understand their actions and

interactions. Neurons of the main types were selected for analysis in this

thesis. Thus we investigated peptidergic and non-peptidergic neurons,

including interneurons, neurosecretory cells, sensory cells, motor neurons

and gut endocrine cells (enteroendocrine cells). Many of these neurons/cells

are Dimm negative. Therefore some of the Dimm effects listed above, such

as the master regulation of neuropeptidergic properties and the possible cell

fate determination, could be results of ectopic expression of Dimm in Dimm

negative neurons.

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Aim of the thesis How the size is regulated in organisms and their substructures such as

organs, tissues and the smallest units – the cells, still remains unresolved to

a large extent. Among neurons and other cells, there is a large variation in

size in an organism. The regulation of cell growth is a combination of genetic

programming and extrinsic factors, including systemic signaling. To what

extent growth of individual post-mitotic cells can be regulated has not been

clearly investigated. One primary aim of this thesis is to unravel mechanisms

behind size scaling of individual neurons, and the role of insulin/IGF-

signaling (IIS) and the transcription factor Dimm in the regulation of the size

of individual neurons in the Drosophila central nervous system. We also

asked what might be the origin of DILP(s) acting on the dInR in this growth

regulation. Furthermore, we try to understand how dInR and Dimm

cooperate with each other in size regulation, as well as in neuron

differentiation and possible regulation of cell determination. In order to find

further components of insulin signaling involved in cell growth, we also

investigated one of the little known insulin like peptides, DILP1. Its regulation

of expression and possible functions was studied.

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Material and Methods

Drosophila husbandry Flies were reared at 12h:12h Light:Dark conditions at 25 °C, on a yeast, corn

meal, agar medium (according to recipe from Bloomington Drosophila Stock

Center (BDSC)). For the conditional gene interference experiments, flies

were raised at 20 °C and then transferred to 29 °C. Flies were backcrossed

into w1118 background for four generations before experiments and w1118 flies

were used as controls in crosses in all experiments.

Adult diapause was induced with 10:14 Light:Dark cycle at 11°C. In the

starvation experiment on dilp1 mutant / w1118, both 2S1Y (sugar:yeast 2:1)

food medium and a modified diet were used for raising flies. The modified

diet contained 50g/L sucrose, 100g/L yeast, 12g/L agar, 3mL/L propionic

acid and 3g/L nipagin (1S2Y food medium).

Immunocytochemistry The central nervous system of first, second and third instar larvae, as well as

adult CNS was fixed with ice-cold 4% paraformaldehyde (4% PFA) in 0.1M

sodium phosphate buffer (PB; pH 7.4) for 2-4 h. After washing up with PB 3

x 15 min, CNSs were dissected in PB. Third instar larval body wall muscles

were firstly dissected out in and fixed with 4%PFA in PB for 10 minutes.

Muscles were rinsed with PB 3 x15 min. All tissues were washed finally in

0.01M PBS with 0.25% Triton-X (PBS-Tx) for 15 min. Tissues were then

incubated in primary antibodies for 24-48h at 4 °C with gentle agitation.

Tissues were taken out and kept at room temperature (RT) for 30 min, and

then washed 4 x 15 min in PBS-Tx. Secondary antibodies were applied for

overnight or 48h at 4 °C. Tissues were then washed in PBS-Tx 7 x 10 min,

rinsed in 0.01M PBS and mounted with 80% glycerol in 0.01M PBS.

For embryo analysis eggs were collected on food plates with apple juice

at 25 °C. After washing, embryos were dechorionated with 3% sodium

hypochlorite for 2-3 min and blotted on paper to remove excess liquid.

23

Embryos were fixed for 20 min with a 1:1 mixture of heptane and fresh 4%

formaldehyde and devitellinized with ice-cold methanol for 30 sec to 1 min.

For immunostaining, embryos were rehydrated in 50% methanol for 5 min

and washed 3 x 5 min in PBST and then 4 x 20 min in PBST with 0.5% NGS

(normal goat serum). Incubation of primary antiserum was performed over

night at 4 °C with gentle agitation. Embryos were washed 3 x 5 min and then

4 x 20 min in PBST, and finally 30 min in PBS followed by application of

secondary antiserum overnight at 4 °C. For mounting, embryos were

dehydrated 10 min each in 50%, 70%, 80% and 2 x 99% ethanol. Then

embryos were clarified by replacing ethanol with methyl salicylate and

settled for 30 min. Finally embryos were mounted in newly exchanged

methyl salicylate.

Image analysis Specimens were scanned with Zeiss LSM 510 META and Zeiss LSM 780

confocal microscopes (Jena, Germany) using 20x, 40x oil, and 63x oil

immersion objectives. Images were processed with Zeiss LSM software for

either projection of z-stacks or single optical sections. Images were edited

for contrast and brightness in Adobe Photoshop CS3 Extended version 10.0.

For determination of CNS and cell body size, as well as axon and

arborization size in third instar larval body wall muscle, staining of a region of

interest were outlined manually and measured by using Image J 1.40 from

NIH, Bethesda, Maryland, USA (http://rsb.info.nih.gov/ij/).

For quantification of immunofluorescence, confocal images of neurons

from different genotypes were carried out with identical settings of laser

intensity and other scan parameters. Immunofluorescence intensity of cell

bodies was carried out using Image J. Mean gray value and area sizes of

cell bodies and corresponding background from three different regions were

measured. The final immunofluorescence intensity in cell bodies was

conducted by subtracting the average intensity of the background from mean

gray value of cell bodies. Total fluorescence of cell bodies or nuclei was

calculated by multiplying mean gray value by cell body size or nuclei size.

24

For measurement of whole body, wing and abdomen of 3-4 day old adult

flies, images of flies were taken with a Leica EZ4HD light microscope

(Wetzlar, Germany). The outline of different parts of body were extracted

and measured using ImageJ.

Quantitative real-time PCR (qPCR) Total RNA was extracted from whole flies or separated heads and bodies of

experimental virgin female flies using Trizol-chloroform (Sigma-Aldrich) from

three independent biological replicates with 15-25 flies in each replicate.

Quality and concentration of the RNA were determined with a NanoDrop

2000 spectrophotometer (Thermo Scientific). For cDNA synthesis reactions

2 µg of total RNA, 0.4 uL random hexamer primer (Thermo Scientific) and 2

µl of M-MuLV reversible transcriptase (Thermo Scientific) were used. The

cDNA was then applied for quantitative real-time PCR (qPCR) using a

StepOnePlus™ System (Applied Biosystem, USA) instrument and

SensiFAST SYBR Hi-ROX Kit (Bioline) according to the manufacturer. For

each sample triplicate reactions of the total volume of 20 µl were conducted

with a primer concentration of 400 nM and 4 ul of diluted 1:10 cDNA

template. The mRNA levels were normalized to rp49 levels in the same

samples. Relative expression values were determined by the 2-ΔΔCt method

(Livak and Schmittgen, 2001).

Stress assays For non-conditional experiments, 4-6 day old male flies were placed in the

empty vials for desiccation (without water and food), or in the vials containing

0.5% aqueous agarose for starvation (no access to food) in an incubator at

25°C with 12:12 h light:dark (LD) conditions and controlled humidity. For

conditional experiments, flies aged 4-6 days were raised at 20°C, and then

transferred to 29°C for assays. Each vial contained 15 flies to avoid a

crowded environment. Dead flies were counted in every 12 hours.

For starvation resistance experiments, flies were placed in vials

containing 0.5% aqueous agarose for starvation (no access to food) in an

25

incubator at 25°C with 12:12 h light:dark (LD) conditions and controlled

humidity. Each vial contained 15 flies to avoid a crowded environment. Dead

flies were counted in every 12 hours. For desiccation resistance the

experiment was the same, except that flies obtained no food and no water.

The above experiments were run on flies raised on two different diets, and

with three different ages of flies tested (3, 4-5, and 8 d old at onset of stress).

For heat knockdown experiment, 4-5 day old flies were placed into empty

vials. Each vial contained 15 flies. A transparent incubator (Heidolph

incubator 1000, Unimax 1010, Schwabach, Germany) was adjusted to 30°C,

with three thermometers inside to check the fluctuation of real time

temperature. Vials with flies were put into the air incubator and number of

flies knocked down was measured at 5-min intervals.

For chill coma recovery experiment, 4-5 day old flies were placed in

empty vials, which were put into an icebox inside a refrigerator (2°C) to

induce chill coma for 4 hours. The recovery at room temperature was

recorded at 5-min intervals. Each vial contained 15 flies.

All these experiments were performed in three replicates with at least 30

flies of each genotype per replicate. Survival curves were produced in Prism

GraphPad 6.0.

Capillary feeding (CAFE) assay The capillary feeding (CAFE) assay was performed according to Ja and

others (Ja et al., 2007) with moderate modifications. 4-6 d old male flies

were placed into 1.5 ml Eppendorf microcentrifuge tubes with an inserted 5

µl capillary (Sigma, VWR) containing 5% sucrose, 2% yeast extract and

0.1% propionic acid. Three to five tubes with food-filled capillaries but

without flies were used as controls for measuring the possible evaporation of

the food or other errors during the experiments. All tubes were placed into a

test tube rack, and the rack was kept into a transparent plastic box with

water embedded tissues at the bottom for humidity. The box was covered

with plastic wrap and placed in an incubator at 25°C with 12:12 h light:dark

(LD) conditions. The food consumption was recorded and the capillaries

were refulfilled for new recording every 24h. The final consumption of each

26

tubes was determined as subtracting the average decrease in control

capillaries. Cumulated food consumption was calculated through four days

and for statistical analysis. These experiments were applied in three

replicates with 5-10 flies of each genotype per replicate.

Locomotor activity recording The Drosophila Activity Monitor System manufactured by TriKinetics Inc.

USA was used to record locomotor activity and sleep duration. Flies were

placed individually in glass tubes plugged with agar/sucrose medium (5%

sucrose, 2% agar) as food at one end and sealed with paraffin wax, and at

the other end tubes were filled with small pieces of sponge. For each set of

experiments, 32 glass tubes were inserted into a holder with infrared

detectors. The software DAMsystem308 from TriKinetics Inc. was used for

recording the locomotor activity, which is the number of infrared beam cross

per minute. The sleep-like resting state is defined as inactivity for 10 min.

The interval time for collecting the sleep amount was 60 minutes. Average

locomotor activity, total sleep amount and average sleep bout duration were

calculated by using an Excel macros file produced by Dr.Taishi Yoshii

(Würzburg, Germany).

Statistical Analysis All statistical analyses were performed using Prism GraphPad 5.0. Survival

data were analyzed by Log rank test with Mantel-Cox posttest. For the

remaining experiments, data was first checked with Shapiro-Wilk normality

test and then analyzed with unpaired Students’ t test or ANOVA with

Dunnett’s or Bonferroni’s multiple comparisons tests. If data was not

normally distributed, non-parametric tests, Mann Whitney or Kruskal-Wallis

test was performed.

27

Discussion In Paper I, we genetically manipulated dInR expression in a variety of

neuron types and found that cell size, axon terminations, as well as Golgi

apparatus, are affected only in neuroendocrine cells specified by Dimmed.

After overexpression of dInR and downstream components of IIS like PI3

Kinase, Akt1, as well as the components from nutrient-dependent TOR

pathway, Rheb, TOR and S6K, Dimm-positive neurons (LK-, PDF-, DILP2-,

DILP7- and FMRFamide positive neurons) display enlarged cell bodies,

whereas DIMM-negative neurons (OK6-Gal4 expressing motor neurons,

PTTH- and 5-HT expressing neurons) do not respond by growth to dInR

manipulation, but respond to manipulation of PI3K and Rheb. Knockdown of

dInR and these components leads to diminished cell bodies. The size

change takes place already at the end of the first instar larval stage and

continues along the adult stage (up to 35d old flies). This developmental

effect may be the factor that leads to physiological function changes of LK

neurons after non-conditional dInR manipulation. Both knockdown and

overexpression of dInR in the LK neurons result in extended life span at

desiccation and decreased feeding.

Since only Dimm positive neurons displayed size changes after dInR

manipulation, we asked whether there is a functional correlation between

Dimm and dInR-regulated neuronal development and growth, and if the

growth of Dimm neurons is nutrient dependent. Cell body size is reduced

after knockdown of Dimm in ABLKs, whereas no change of size occurs after

overexpression of Dimm. Dimm knockdown also leads to diminishing dInR

labeling in ABLKS and overexpression gives rise to increased dInR

immunofluroscence intensity. We also did the reverse manipulation: dInR

knockdown decreases total DIMM immunolevel and the size of Dimm

labeled nuclei, whereas dInR overexpression increases the size of DIMM

labeled nuclei but not total DIMM immunolevel. Flies with two copies of UAS-

Dimm-RNAi display extremely small ABLKs. But the double transgene

together with knockdown of dInR does not result in a smaller cell body, and

28

overexpression of dInR in Dimm knockdown flies cannot bring back the cell

size to normal level. These data indicate that there is a strong effect of DIMM

on dInR expression, and without Dimm, dInR has little effect on cell size.

Larvae reared under nutrient restriction display significantly smaller cell body

size after overexpression of dInR compared to those reared in the normal

food. This indicates that dInR mediated cell size regulation is protein

dependent.

In Paper II, we extended our previous analysis in Paper I by asking

whether growth of Dimm negative neurons can be triggered by IIS if Dimm is

ectopically expressed. Our study shows that Dimm overexpression or

ectopic Dimm expression together with dInR can regulate cell body size of

several groups of neurons including both Dimm positive and negative

neurons in the larval stage. However, cell body size of more than half of the

tested neuron types is not affected by expressing Dimm alone. When

coexpressing Dimm and dInR in Dimm positive and negative neurons, 83%

of the tested neuron types display larger cell body size. Furthermore, in all

the Dimm negative neurons, coexpression of Dimm and dInR results in

increase of cell body size in larvae. In adult flies, combined Dimm/dInR

promotes cell body growth of both Dimm positive and Dimm negative

neurons. Dimm alone gives rise to larger cell bodies in eight out of 10 types

of neurons. In five neuron types expression of Dimm blocks apoptosis, and

coexpression of Dimm and dInR results in enlarged cell body size of the

surviving neurons. These neuron types are PTTH, SELK, abdominal CRZ,

EH, and subesophageal CCAP neurons that normally undergo programmed

cell death during metamorphosis, or several hours after adult eclosion.

Conditional targeting of Dimm and Dimm/dInR to LK expressing neurons

results in different phenotypes in different subsets of these neurons. This

reveals a complexity in responses to Dimm and dInR in regulation of cell

body size. The ABLK cell bodies grow after either dInR expression or Dimm

expression, and the effect is amplified after coexpressing both factors. In

contrast, none of the conditional manipulations affect cell body size of

29

LHLKs. The SELKs, which are Dimm negative, display larger cell body size

only after coexpression of Dimm and dInR.

We found that in adult flies DILP6 secreted from glial cells can regulate

the cell body size in Dimm positive neurons, but not in DIMM negative ones.

This result provides novel evidence of a niche regulating neuron growth in

adult flies mediated by DILP6 and the dInR. Moreover, we also show by

expressing DILP2-FLAG in fat body or glial cells that DILP2 can diffuse

within the CNS and be sequestered by certain neurons. This indicates that

both circulating and glial-derived DILPs can act within the CNS to mediate

dInR mediated cell growth.

Since we found earlier (Paper II) that ectopic Dimm and Dimm/dInR

expression in motor neurons resulted in increased size of cell bodies and

strongly reduced dendrites, we further investigated the properties of NMJ

site of the manipulated motor neurons in Paper III. Increased branching of

axon terminations with enlarged synaptic boutons was observed at the NMJ.

These transformed features taken together result in morphology similar to

that of efferent neuroendocrine cells, rather than typical motor neurons.

Several glutamatergic markers such as vGluT, GluRIIA and GluRIIB and as

well as pre- and postsynaptic proteins (Brp, synapsin, synaptotagmin and

Dlg) were absent or strongly reduced after ectopic Dimm expression. These

markers are commonly absent in efferent neuroendocrine cells. Interestingly,

filopodia-like extensions associated with the enlarged axon terminations

were observed frequently with Dimm and Dimm/dInR expression, suggesting

a change in interactions between motor neuron and target muscle. Such a

phenotype can be related to wingless signaling (Packard et al., 2002).

Indeed, we found that wingless/frizzled immunolabeling was strongly

reduced in the Dimm mis-expressing axon terminations and at postsynaptic

sites. Furthermore, expression of an exosome-associated protein, Evi, which

is important for wingless exocytosis (Koles et al., 2012; Korkut et al., 2013),

was diminished. It has been shown that the proteins Rab11 and Ykt-6,

involved in Evi-exosome release at the NMJs (Gross et al., 2012; Koles et

al., 2012) are direct Dimm targets.

30

Both Paper II and Paper III showed dInR mediated cell growth in the

presence of Dimm, and DILP6 as one candidate ligand for this mechanism.

Since DILP1 displays some similarities to DILP6 in temporal expression

profile we investigated the expression and function of DILP1 in Paper IV. We

demonstrate that dilp1/DILP1 is transiently expressed in the brain insulin

producing cells from early pupa until a few days of adult life. However, in

adult flies kept under diapause conditions the dilp1/DILP1 expression

remains high for at least 9 weeks, and incidence of diapause is increased in

dilp1 mutant flies. The dilp1 mRNA level is upregulated in dilp2, 3, 5 and

dilp6 mutant flies. Also, the DILP1 expression is under stimulatory regulation

of short neuropeptide F and presence of larval adipocytes. Furthermore,

blocking juvenile hormone production increases DILP1 levels. Finally, dilp1

mutants display increased food intake, whereas median lifespan, as well as

starvation and desiccation resistance are reduced. However, no effect of

dilp1 knockdown or dilp1 null mutation on growth of neurons in the brain was

detected (Fig. 3).

31

Fig.3. Cell size and intensity in dilp1 null mutants and dilp1 knockdown flies. A-C. Cell body size of adult IPCs was not affected in dilp1 mutants. IPCs were labeled with

anti DILP2 antibody. Immunofluorescence intensity increased significantly in dilp1

mutants. D-F. Leucokinin producing cells in adult abdominal segment (ABLKs)

showed no significant difference in cell body size and immunofluorescence intensity

between dilp1 mutants and control flies. Anti Leucokinin (LK) antibody was applied

for labeling ABLKs. G-I. Knockdown of dilp1 in IPCs (dilp1-Gal4) did not alter the cell

body size or LK intensity of ABLKs. Data are presented as means ± S.E.M, n = 6-10

flies for each genotype from three independent crosses (*p<0.05, ns - not significant

as assessed by unpaired Students’ t-test). Scale bar = 20 µm.

32

Conclusions Insulin receptor-mediated signaling may contribute to the plasticity in size of

secretory neurons independent on growth of other neurons. This regulation

requires the presence of DIMM. In order to be flexible for demands of

peptide signaling and responding to changes in environment and organismal

homeostasis, secretory cells are scaled up during differentiation. Dimm

functions in the up-scaling of the secretory pathway and the size of the cell

bodies. The activation of the dInR may trigger signaling that converges on

scaling events in the Dimm positive neurons. We performed a

comprehensive screening of the effects of Dimm and dInR on growth and

differentiation of a broad set of Dimm positive and negative neuron types,

including sensory cells, interneurons, motor neurons, and enteroendocrine

cells in midgut. This analysis confirmed that dInR mediated cell body growth

occurs in a Dimm dependent manner in the CNS. Expressing both Dimm

and dInR in Dimm negative neurons induced cell body growth, whereas dInR

alone did not. On the other hand we found that Dimm alone can regulate cell

growth in a differential way depending on specific cell types. This effect is

likely to be associated with the function of Dimm as part of a combinatorial

code for terminal cell differentiation. In certain neurons, Dimm alone could

contribute to cell growth during metamorphosis and/or in adult stage. In other

cases Dimm may act in maintenance of neuron identity in the adult CNS and

inhibit apoptosis in the neurons destined for programmed cell death. Taken

together our results suggest that Dimm plays different roles during different

developmental stages and in different neuron types.

Among different neuron types, we find that dimm misexpression leads to

a loss of glutamatergic phenotype in motor neurons and changes of pre- and

postsynaptic structures at the NMJ. Thus, the neurons lose presynaptic

vesicular glutamate transporter (vGluT), synapse proteins such as

bruchpilot, synapsin, synaptotagmin, and postsynaptic discs large and

glutamate receptors GluRIIA and B, all characteristic of Drosophila motor

neurons and NMJ. Interactions between the axon termination and muscle

cell appears disrupted resulting in loss of wingless signaling components

33

and formation of presynaptic filopodia-like structures. Furthermore, the

Dimm-misexpressing motor neurons display morphologies similar to

peptidergic efferent neuroendocrine cells and start to express several

proteins known to be transcriptional targets of Dimm. In summary, we

demonstrate in detail how Dimm triggers a transformation of the NMJ

towards a neuroendocrine phenotype both at the molecular and the

morphological levels through transcriptional orchestration.

We also show that one source of DILP(s) for dInR mediated cell growth in

the CNS is DILP6 from glia cells. Another candidates of DILPs, DILP1 is

shown not to be involved in this regulation on cell growth. We demonstrate

that dilp1/DILP1 expression is seen in the brain IPCs in the pupa and newly

hatched adult fly. However, in virgin female flies that had been subjected to

low temperature and short photoperiod (inducing reproductive diapause), we

found that the dilp1/DILP1 expression remained high for at least 9 weeks of

adult life. This expression declined after one week of recovery from low

temperature and short photoperiod conditions. The DILP1 expression

correlated to some extent with the persistence of larval/pupal fat body and is

dependent on juvenile hormone, other DILPs and the neuropeptide short

neuropeptide F (sNPF). Furthermore, dilp1 mutant flies displayed increased

food intake, diminished lifespan and stress resistance. This is opposite to the

effects of ablation of IPCs or knockdown of the other DILPs of these cells

(see (Broughton et al., 2005)). Thus, the hitherto enigmatic DILP1 seems to

play a role distinct from other DILPs. In summary, DILP1 is expressed in

non-feeding stages and in the adult diapausing fly, known to feed at a

strongly reduced level, and dilp1/DILP1 expression is dependent on

feedback from other DILPs.

34

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Acknowledgement

I would like to express my deepest gratitude and appreciation to my

supervisor Dr. Dick R. Nässel for your guidance, patience, encouragement

and support throughout my PhD study. Thank you for your constant and

careful supervision for my project. I am also very grateful for your patience

and great help in finalizing the thesis during these months. I enjoyed a lot

with you during academic discussion and life conversation, and I always felt

comforted and encouraged by your enthusiasm and preciseness for science,

as well as your modesty and amiableness.

Thanks my co-supervisor Dr. Mikael Carlsson for introducing me the

Calcium imaging and other practical lab work as well as the cooperation. The

wine you recommended was really good! Dr. Heinrich Dircksen, thank you

for your generous help with chemicals, devices and advice for my

experiment and PhD progress. I still remember the day when we

accomplished to change the broken fan of your IBM laptop. It was really fun!

Sincerely thanks Dr. Rafael Cantera for providing the great ideas for Paper

III, and patiently teaching me data mining and analysis. Thanks Bertil Borg

for carefully reading my thesis and giving many useful comments. Your

glögg is the best! Siw Gustafsson, Anette Lorents, Minna Miettinen and Berit

Strand, thank you very much for your patience helping me deal with many

trivial office stuffs.

Thanks my previous colleagues Dr. Neval Kapan, Dr. Oleh V. Lushchak,

Dr. Yi Ta Shao for the kind help and advice with formalities and university

facilities when I came to Sweden as a newcomer. Dr. Jiangnan Luo, great

thanks for your tremendous support and cooperation for the project. Thanks

Timm Kress for always perfectly organizing the barbecue and surströmming

party in GYLLENE SPIGGEN. Lovely and nice guys in our division: Dr. Olga

Kubrak, sincere appreciation for kindly guiding me into qPCR and

biochemical analysis techniques. I was always inspirited by your persistence,

confidence, energy, passion and optimism. There are so many valuable and

interesting moments with you. Thanks for your support and accompanying.

Dr. Jonas Bengtsson, thanks for your useful suggestion on the thesis cover

41

and casual talk about life and films. Great appreciation to your effort and

time for fixing my old laptop! Dr. Meet R Zandawala, thanks for the decisive

remarks for the thesis cover. Your rigorousness always gives us a new

direction of thinking, which is really helpful. Chrysoula Roufidou, thanks for

all the help in and outside the lab. The Greek barbecue and dessert you

made were so delicious! Good luck you're your PhD. Sifang Liao, many

thanks for the cooperation in Paper IV. Good luck with your PhD and best

wish to your family, brand new father!

Thanks Diana, Helene, Severine, Alex, Sandra, Märta, Naomi, Peter,

Shreyas and everyone at Zootis, I enjoyed a lot with you in different activities

and many thanks for the warm atmosphere and your support!

To all my thesis committee members, thanks for your valuable time and

comments.

Thanks all my friends in Stockholm and in Germany for the precious and

happy time we spent together.

Many thanks to my family for always being there supporting me and for

your endless love.