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Development 111, 821-828 (1991)Printed in Great Britain © The Company of Biologists Limited 1991
821
Molecular cloning and expression of a novel homeobox gene AHoxi of the
ascidian, Halocynthia roretzi
HIDETOSHI SAIGA1*, ATSUSHI MIZOKAMI1, KAZUHIRO W. MAKABE2, NORIYUKI SATOH2 and
TAKASHI MITA1
^Department of Molecular Biology, University of Occupational and Environmental Health, Yahatanishi-ku, 1-1 Iseigaoka, Kitakyushu 807,Japan^Department of Zoology, Kyoto University, Kyoto 606, Japan
* Author for correspondence
Summary
We have isolated a novel ascidian homeobox gene,designated AHoxl, by screening the genomic DNA ofHalocynthia roretzi with the Bombyx mori Antennapediatype homeobox as a probe. The AHoxl gene encodes aprotein that consists of 741 amino acids. The homeoboxof AHoxl is interrupted by 2 introns each of which isabout 300 bp in length and it shows about 70 %similarity at a deduced amino acid level to that ofDrosophila H2.0. This suggests that AHoxl is one of themost diverged homeobox genes so far characterized.Northern blot hybridization with an AHoxl probeshowed the presence of single transcripts approximately2.8 kb in length in larvae, juveniles and some adulttissues. The expression of AHoxl is scarcely detectedduring the course of early development but it increasesto a moderate level at the larval stage. After metamor-
phosis, the level of AHoxl expression increases asdevelopment proceeds. In situ hybridization to thejuvenile 7 days after metamorphosis showed that the siteof AHoxl expression is the epithelium of digestive tract.Among the adult tissues examined, digestive tract,digestive gland and coelomic cells were the major sites ofthe expression of AHoxl. In gonad, body wall muscleand pharyngeal epithelium, the expression of AHoxl isrelatively weak. These results suggest that AHoxl isprimarily expressed in the tissues of endodermal originand that the gene expression may be associated withdifferentiation of the endodermal tissues.
Key words: ascidian, homeobox, molecular cloning, geneexpression, developmental expression, in situ hybridization.
Introduction
The homeobox was originally discovered as a smallsegment of conserved nucleotide sequence among theDrosophila genes that are involved in the formation ofaxes and body segments, and in the specification of thebody segments (for review see Gehring, 1987; Akam,1987). The homeodomain, the translated form of thehomeobox, has a helix-turn-helix motif, which confers aDNA binding capacity to the homeobox gene productand therefore it has been suggested that homeoboxgenes encode transcription factors. There are severallines of evidence supporting this hypothesis (Quian etal. 1989; Ingham, 1988; Scott et al. 1989, referencestherein). Homeobox genes have been found to bewidely distributed among various animal species, fromnematodes to insects and vertebrates on the phylogen-etic tree (McGinnis et al. 1984; Holland and Hogan,1986) where they play key roles in development.
Since the last century, it has been noted that the earlydevelopment of ascidians, which belong to the class
Protochordata, is exceptionally autonomous. Thisfeature enabled Conklin, Ortolani and Nishida andSatoh to trace the fate of each blastomere (Conklin,1905; Ortolani, 1955; Nishida and Satoh, 1983, 1985).The cell lineage in early development has beenestablished completely up to the 110-cell stage inHalocynthia roretzi (Nishida, 1987). It is thought thatthe autonomous development of the ascidians is due tofactors, named determinants, which appear to beinvolved in the determination of the fate of eachblastomere and are localized heterogeneously in thefertilized egg cytoplasm (for recent reviews see Satoh,1987 and Satoh et al. 19906; Uzman and Jeffery, 1986;Whittaker, 1987; Venuti and Jeffery, 1989). Themolecular identity of a determinant, however, is still anenigma (see reviews by Jeffery, 1985; Satoh et al.1990a).
We are interested in studying the homeobox genes ofascidians for two reasons. (1) Since ascidians belong tothe class Protochordata located phylogenetically be-tween invertebrates and vertebrates, the structure of
822 H. Saiga and others
the homeobox genes of ascidians should shed some lighton the evolutionary aspect of homeobox genes. (2)Since it is highly Likely that expression of homeoboxgenes is involved in determining cell differentiation, theexpression patterns of homeobox genes in ascidians ornematodes, which exhibit typical mosaic development,are of particular interest. This might lead to betterunderstanding of the molecular identity of the so-calleddeterminants. Therefore, we have started isolatinghomeobox genes from the ascidian, Halocynthia ror-etzi- In this report, we describe the isolation andmolecular characterization of a homeobox gene fromthe Halocynthia genome. Although we used theAntennapedia (Antp) type homeobox as a probe, theisolated homeobox gene, designated AHoxl, was not ofthis type but, unexpectedly, it has a similar homeoboxto that of H2.0. This is a Drosophila homeobox geneshowing tissue-specific expression rather than region-specific expression (Barad et al. 1988). Likewise theexpression of the isolated ascidian homeobox gene,AHoxl, is primarily detected in the endoderm duringthe course of development.
Materials and methods
AscidianAdult ascidians, Halocynthia roretzi, were purchased fromfishermen near Asamushi Marine Biological Station, TohokuUniversity, Mutsu Bay, Aomori, Japan. Adult specimenswere dissected and tissues were frozen quickly with liquidnitrogen. The frozen samples were kept at —80°C until use.
Naturally spawned eggs were fertilized with a suspension ofnon-self sperm, and fertilized eggs were raised in filtered seawater at 13—15 °C. Embryogenesis proceeded synchronouslyamongst batches of eggs. Tadpole larvae hatched about 33 hafter fertilization. They were allowed to accomplish metamor-phosis naturally. Juveniles that adhered to plastic dishes werecultured for 13 days in aquaria with circulating naturalseawater. Samples at appropriate stages were collected by lowspeed centrifugation and were frozen with liquid nitrogen.
Construction of genomic and subgenomic librariesHalocynthia roretzi genomic DNA was prepared from thegonad of one adult individual according to Blin and Stafford(1976).
Genomic DNA was digested partially with Sau3A andligated to the BamFfl-digested AEMBL3 vector. To constructa subgenomic library, genomic DNA was digested completelywith EcoRI and separated on a 1 % preparative agarose gel.The DNA fragments of about 2.7 kb in length were recoveredby electroelution and ligated to the £coRI-digested Agtllvector. Ligated materials were packaged and propagated inNM539 and Y1088 for genomic and subgenomic libraries,respectively.
The probes used for screening of the libraries were asfollows; a Hindi-Avail fragment of pBm3.6 about 250 bp inlength, which is the genomic clone of a Bombyx mori Antp-type homeobox gene (Hara and Suzuki, unpublished), and aPsd-TaqI fragment of pHBl about 200 bp in length, whichcontains a sea urchin homeobox as described by Dolecki et al.(1986).
For screening of the iibrary, hybridization was carried outunder reduced stringency conditions essentially as described
by McGinnis et al. (1984). In brief, hybridization was carriedout at 37 °C for 30-40 h in the mixture consisting of 38%formamide, 6xSSC, 0.2% polyvinylpyrrolidone, 0.2%Ficoll, 0.1% BSA, 0.5% SDS, 50mM sodium phosphatepH7.0, 20^gml"1 E. coli DNA and 1-5xK^ctsminimi"1
probe labelled by random priming using [a--32?] dCTP as thelabelled nucleotide. After hybridization, the filters werewashed in 2xSSC, 0.2% SDS at 37°C for 30min twice.
Preparation of poly (A) RNATotal RNA from embryos or adult tissues was preparedessentially according to Ullrich et al. (1977). H. roretziembryos were developed at 15 °C, collected by low-speedcentrifugation and kept at -80°C. Frozen packed embryos oradult tissues were suspended in 4 M guanidine thiocyanate,0.1M sodium acetate, 5mM EDTA, pH5.0 and werehomogenized using a Polytron homogenizer (Kinematics).After low-speed centrifugation to remove cell debris, thehomogenate was layered into a tube for a Beckman SW4OTirotor one-third filled with 5 M CsCl, 0.1M sodium acetate,5mM EDTA, pH5.0 and subjected to centrifugation at35000revsmin~^ for 15 h at 20 °C. The pellet was dissolved ina small volume of 10 mM Tris-HCl pH7.6, 10 mM EDTA,0.5% SDS, extracted with phenol and ether, and the RNAwas recovered by ethanol precipitation.
Poly(A) RNA was selected using Oligotex-dT30 Latexbeads (Roche Japan) according to manufacturer's procedure.Normally 1-3 % of the total RNA was recovered as poly(A)RNA. Quality of the poly (A) RNA was comparable to thatprepared using the ordinary oligo(dT)-cellulose column.
cDNA library constructioncDNA was synthesized from gonad poly(A) RNA using acDNA synthesis kit (Pharmacia). Synthesized cDNA wasligated to the Agtll vector. The ligated materials werepackaged in vitro as described in 'Construction of genomicand subgenomic libraries'.
Southern and northern blotting2 ng of genomic DNA digested with Eco RI, Hindlll or BgUlwas separated on a 1 % agarose gel. After electrophoresis, thegel was soaked in 0.5 M NaOH, 1.5 M NaCl for 15min andsubjected to blotting onto a nylon membrane (Hy-bond N,Amersham) using a low-pressure vacuum blotting apparatus(Vac Gene, LKB-Pharmacia). For northern blotting, poly(A)RNAs (10 ^g per lane) were separated on a 1 % agarose gelcontaining 6 % formaldehyde and were transferred to a nylonmembrane filter using a Vac Gene apparatus and IOXSSC astransferring buffer. Southern or northern blot hybridizationwas carried out according to the standard procedure (Maniatisetal. 1982).
In situ hybridizationIn situ hybridization using RNA probes and autoradiographywas carried out according to the method described byTomlinson et al. (1987). [35S]ATP-labelled probes wereprepared from a 1108 bp fragment of AHoxl cDNA(corresponding to nucleotides 209-1317 in Fig. 2 in Results)subcloned in the pGEM-Blue vector (Promega). The senseand antisense RNAs were transcribed with T7 and SP6 RNApolymerases, after linearization of the plasmid DNA withHindlll and EcoRI, respectively. Juveniles 7 days aftermetamorphosis were fixed in ice-cold 95 % ethanol:acetic acid(3:1, v/v), and embedded in Tissue-Prep (Fisher ScientificCo.). The specimens were sectioned at 8/an and attached tosubbed glass slides. After treatment with 1 /igmF1 proteinaseK, the specimens were postfixed in 4% paraformaldehyde,
A homeobox gene of an ascidian 823
treated with freshly prepared 0.25% acetic anhydride, 0.1Mtriethanolamine, and air dried. ApproximatelylxH^ctsmin"1 of 35S-labelled RNA probe was applied toeach slide in 50 /1 hybridization buffer (50% formamide,lxDenhardt's solution, 10% dextran sulfate, 2xSET, 0.01MDTT, 500/igmT1 TRNA, 500/igmT1 poly(A)). Hybridiz-ation reaction was carried out at 45 °C approximately for 12 h,and the slides were washed at high stringency (in 0.2xSSC at45 °C for 45min). The washed slides were dehydrated with95% ethanol, air dried and subjected to autoradiography.The autoradiographs were stained through the emulsion withhaematoxylin-eosin.
Results
Isolation of a homeobox genePrior to library screening, we carried out Southernblotting analysis of H. roretzi genomic DNA todetermine the conditions for hybridization so as to givesignals with reasonable strength. This was done using agenomic DNA probe of the Antp-type homeobox of silkworm or sea urchin. As shown in Fig. 1, under reducedstringency hybridization conditions, both probesresulted in several bands on a membrane filter but mostcoincided with those detected by ethidium bromidestaining of the gel. A 2.7 kb band detected upon EcoKldigestion, however, was distinct from those detected byethidium bromide staining. To facilitate efficientcloning, the ZscoRI-digested genomic DNA fragments
BE H Bg E H Bg
1kb
21.2 "
5.02 -
3.53 -i
2.03 _
1.38 —
0.85 —
Fig. 1. Detection of homeobox homology in the genomicDNA of the ascidian Halocynthia roretzi. 10 /ig of genomicDNA digested with EcoKl (E), Hindlll (H), or Bglil (Bg)was separated on a 1% agarose gel. Genomic Southernblots were hybridized under reduced stringency conditionswith the Bombyx homeobox probe (left) and the sea urchinhomeobox probe (right). An arrowhead indicates the2.7kb band, which was distinct from the ethidium bromide-stained bands. The size marker used was end-labelledADNA fragments digested with EcoRI and HindlU.
of about 2.7 kb in length were recovered, and fromthese a subgenomic library was constructed. Wescreened the subgenomic library under the reducedstringency hybridization conditions using the Bombyxhomeobox probe, since it gave lower background thanthe sea urchin probe. We were able to isolate a positiveclone, designated 62, which had a 2.7 kb insert. Bydetermination of the nucleotide sequence, it wasidentified as a clone containing a part of the homeobox.Using DNA fragments from the clone 62, we havefurther isolated three cDNA clones corresponding tothe genomic DNA fragment included in the clone 62from the adult gonad cDNA library as well as genomicclones covering the cDNAs from the H. roretzi genomiclibrary.
Structure of the ascidian homeobox gene, AHoxlFig. 2 shows the nucleotide sequence of cDNA of theascidian homeobox gene designated AHoxl. It has asingle long open reading frame consisting of 741 aminoacid residues and including a highly diverged homeo-domain. One structural feature of the AHoxl homeo-box is the presence of two introns of 319 and 314 bp inlength (data not shown) located at position 9 and afterposition 44, respectively. This is in contrast to theengrailed (en), invested (inv) and labial (lab) which haveone intron in the homeobox locating at position 17 (enand inv) or after position 44 (lab). Comparison of theamino acid sequence of the AHoxl homeodomain withthose of several representative Drosophila homeoboxgenes is shown in Fig. 3. The amino acid sequence ofthe AHoxl homeodomain is highly diverged from all ofthe known homeodomains with less than 50 % identityover 60 residues. The only one exception is thehomeodomain of H2.0 of Drosophila to which theAHoxl homeodomain shows 70% identity, thoughoutside the homeodomain, there is no significantsimilarity. AHoxl also lacks the sequences correspond-ing to the M repeat and the YPWM peptide, which areconserved upstream of the homeobox in many of thehomeobox genes of Drosophila and vertebrates.
To see whether the Halocynthia genome containsother genes possessing a similar homeobox to that ofAHoxl, Southern genomic hybridization underreduced stringency conditions was carried out using theDNA fragment from the clone 62 as a probe, whichcontained the last one-third of the AHoxl homeobox.As shown in Fig. 4A, a 2.7 kb band was detected in theZscoRI-digested genomic DNA as expected. This andintense bands seen in the Hindlll- and BglH-digestedDNA correspond to the AHoxl gene. Other thanthese, no significant band was detected. Upon Hindllldigestion, two bands were detected as shown moreclearly in Fig. 4B. These results suggest that two copiesof AHoxl are present in the genome, possiblyrepresenting two allelic forms, and it is unlikely that agene containing a similar homeobox to that of AHoxl ispresent in the Halocynthia genome.
Expression of AHoxlWe have examined expression of the AHoxl gene in
824 H. Saiga and others
1
176
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5701801
5951876
6201951
6452026
6702101
6952176
7202251
2326
2401
2476
TTT
ATA
TACA
kAAA
kAAA
CTGC
VGTA
AAT
FTTT
RACA
DGAC
LTTG
TACC
PCCA
QCAA
FTTC
QCAA
DGAT
NAAT
FTTT
kAAG
EGAG
ACA
WTGG
DCAT
FTTT
ATG
RCGA
VGTC
NAAT
DGAT
CGA
ATA
GTA
TGA
CTC
STCC
HCAT
kAAC
RAGA
PCCA
GTT
STCA
VGTT
DGAT
kAAA
EGAA
RAGA
PCCC
YTAC
YTAC
VGTA
AGCC
STCC
NAAT
DGAC
GCG
PCCA
ACCT
EGAG
SAGC
kAAG
AGCA
RAGA
HCAC
TTC
GCA
TTA
AAT
GCA
LTTA
IATC
kAAG
SACC
IAAA
AAC
STCC
IATT
HCAT
NAAC
DGAT
SACT
YTAT
RAGA
STCT
EGAC
LTTC
DGAC
STCA
NAAT
AAA
FTTT
PCCA
NAAC
SAGT
WTGG
IAAA
RCGG
EGAA
CTC
ATC
AAT
CAT
TTA
GGGA
EGAA
QCAA
kAAG
GGCA
CCA
SAGC
DGAC
IATT
kAAA
QCAG
FTTT
LTTG
EGAA
PCCA
EGAG
STCT
IAAA
GGGA
NAAT
TTA
AGCA
HCAC
PCCA
STCC
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PCCG
HATC
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ATT
AAC
TAC
TTA
AAA
GCCA
EGAA
NAAC
kAAA
IATA
TTG
STCA
IAAG
NAAT
RAGA
QCAG
STCT
RAGG
AGCA
DGAT
GGGC
YTAT
GGGG
MATG
PCCT
TTA
AGCA
YTAT
YTAT
QCAA
RCGA
EGAA
kAAG
EGAA
CCA
AAC
GTA
TTG
AGC
LCTC
ECAA
RAGA
DGAT
STCT
C
AGT
EGAA
RCGG
QCAA
LCTT
IATA
STCT
AGCT
NAAC
AGCA
DGAC
GGGT
IATA
SAGC
QCAA
GAA
STCA
GGGA
FTTT
NAAC
AGCA
RAGA
WTGG
IAAA
AAA
AAT
TTT
ACT
HATG
RCCA
RAGA
FTTT
SAGC
NAAC
AAA
AGCA
kAAG
DGAT
RCGA
SAGT
CGGA
YTAT
VGTT
SAGT
kAAG
RCGA
QCAG
FTTT
TACG
NAAC
VGTT
AGCG
FTTC
PCCA
VCTT
RAGG
RAGG
EGAA
TCA
AAA
GTA
TCT
EGAA
kAAA
TACC
EGAA
TACC
ACCC
TAT
SAGT
SAGC
NAAC
LCTT
IACC
LTTG
YTAC
EGAA
TACT
EGAA
IAAG
AGCT
DGAT
IATT
EGAA
RAGA
AGCG
NAAT
HCAT
FTTC
IAAC
QCAA
IAAA
TGC
CTG
CAC
ATT
iAAA
STCA
SAAT
RACG
VGTT
GGGA
TTC
DGAC
AGCG
CTCT
DGAC
RAGA
QCAA
NAAT
NAAT
FTTC
AGCA
DGAC
C,GGT
FTTC
GGGG
GCT
IAAA
QCAA
EGAG
STCT
SAGC
CTC
EGAG
TACG
CGC
CAA
C
ATA
,1ATG
STCC
NAAC
YTAT
LTTG
RAGG
AAG
SAGT
DGAT
DGAT
SACC
VGTG
DGAT
IATT
DGAT
YTAC
NAAC
YTAC
SAGT
PCCC
SAGC
AAA
SAGT
VGTT
LCTT
ECAA
LCTT
AGCG
IATA
QCAA
CAC
TCC
TCT
HCAC
SACT
IATC
STCT
kAAG
1ATT
CCA
IAAA
STCA
LTTA
SAGT
EGAA
PCCA
QCAA
NAAT
PCCT
IAAA
KAAC
YTAT
PCCT
EGAA
TTG
YTAC
NAAT
NAAC
TACA
MATG
DGAT
kAAA
TACC
CGA
ATC
AAT
STCA
1ATT
TACA
SAGT
FTTT
QCAG
TCG
STCA
DGAC
AGCC
EGAA
MATC
FTTT
kAAA
YTAC
IATA
LTTC
GGGT
YTAC
IAAA
YTAC
CCT
DCAT
PCCG
TACA
QCAA
QCAA
ACCC
HATG
PCCT
ACC
AAC
TAT
kAAA
PCCC
TACA
STCA
GGGA
DGAT
sTCTC
TGC
VTGC
SAGC
AGCT
MATC
AGCA
YTAT
IAAG
RAGA
FTTC
LCTG
QCAA
FTTC
QCAA
TAT
PCCA
TACA
LTTG
NAAC
RCGT
LCTA
kAAA
STCT
CAG
AAG
CTA
STCT
TACC
MATG
AAC
IATT
EGAA
AAT
CTGT
SAGT
TACA
LCTT
NAAT
IAAA
IATT
TACT
TACT
IAAA
STCA
TACT
GGGA
IATT
GGGG
AGCA
AGCA
DGAC
RAGA
RCGT
SAGT
NAAT
DGAT
ACA
AGA
TTC
VCTT
LTTC
ACCA
HCAT
DGAC
RAGA
GAC
TACA
EGAA
LCTT
NAAC
HCAC
NAAT
YTAC
EGAG
EGAG
DGAT
TACC
GGGA
SAGT
NAAT
NAAC
LTTG
GGGC
TACT
SAGT
GGGA
LTTG
RCGC
ECAA
GTA
TTA
ATA
STCA
AGCA
MATG
ACCC
SAGC
FTTC
QCAG
KAAT
DGAC
EGAG
DGAC
QCAA
LCTA
HCAC
EGAA
DGAT
LCTT
STCT
EGAA
NAAC
TACA
ATC
RAGA
NAAC
TACC
EGAA
LCTT
TACC
CGGT
GGGG
CCA
TTA
CAT
PCCT
VGTA
IAAC
QCAA
IATA
TACA
TTA
NAAC
AGCA
QCAA
LCTT
AGCC
PCCT
IAAA
STCA
SAGT
CTCT
QCAA
RAGA
NAAC
QCAA
GTA
sTCC
NAAT
GGGT
EGAA
EGAA
DGAT
LCTG
EGAA
ATA
TAT
ACA
VGTC
pCCA
RAGA
EGAA
LCTT
ECAG
CGGT
NAAT
TACT
SACT
TACA
FTTC
NAAC
IAAC
LTTG
KAAT
VGTC
TACC
NAAT
STCA
ICGA
TACA
YTAT
FTTC
FTTC
VGTA
IAAA
AGCA
VGTG
VGTG
AAG
TTT
GCT
PCCA
EGAA
RCGC
QCAA
kAAA
AGCA
ACCA
EGAA
CGGA
RCGT
PCCA
AGCC
AGCA
LTTG
RAGG
GGGG
SAGC
STCA
DGAT
STCA
SAGC
QCAA
FTTC
AAG
LCTT
RCGC
SAGT
QCAG
CCA
IATC
TTC
TAT
AGT
FTTC
CTGC
LTTC
STCT
NAAT
CTCT
RCCA
EGAA
NAAC
IATT
YTAT
RAGA
FTTC
LTTA
STCT
SAGC
VGTT
GGGG
STCT
TACC
MATG
TAT
SAGT
STCT
SAGT
GCGT
FTTT
VCTT
ICTA
NAAC
ACT
ATC
ACA
NAAC
EGAA
LTTA
STCA
IAAA
TACT
RAGA
AGCC
ECAA
VGTT
DGAT
ECAA
LCTG
HAAT
PCCG
RAGA
GGGC
NAAT
LCTT
DGAC
IAAA
CCA
RCGA
HATG
RCGT
TACT
QCAA
IAAA
HCAT
VGTC
AAT
ATA
TTT
sAAC
STCA
DGAT
ECAC
NAAC
NAAC
TACT
BCCT
ACCA
AGCT
QCAA
NAAC
PCCC
RCGA
SAGC
NAAT
DGAC
RCGT
AGCG
kAAG
DGAT
RAGA
FTTC
LTTA
QCAA
VGTC
SAGT
1ATC
IATT
DGAT
TTC
CAT
TTT
SACC
MATG
PCCC
ECAA
ACCA
TACA
AGCT
YTAC
DCAC
LTTG
LTTG
NAAC
FTTT
NAAT
EGAA
LTTG
RAGA
FTTT
GGGA
PCCA
NAAT
CCT
NAAT
PCCG
YTAC
kAAA
QCAA
WTCG
LCTT
TAA
ACG
ATT
ACT
NAAC
CCGT
QCAA
NAAT
ECAA
SACC
TACA
kAAC
DGAT
EGAA
STCA
ECAA
YTAC
DGAC
TACA
kAAA
LTTA
LCTA
PCCA
EGAG
LTTA
TTT
NAAT
GGGT
CCGC
kAAA
kAAA
FTTC
GCGT
TGA
GTT
ATT
TTT
NAAC
NAAC
NAAT
FTTT
kAAA
STCC
sACTY
TAT
PCCT
IATA
RCCT
UTCG
HATG
RAGG
kAAA
VGTC
STCA
NAAT
LCTC
QCAA
LCTC
TCA
SAGT
NAAT
HCAT
RACA
YTAC
QCAG
QCAA
AAA
TCC
CTA
75
19150
44225
69300
94375
1 19450
144525
1 69600
194675
219750
244825
269900
294975
3191050
3441125
3691200
3941275
4191350
4441425
4691500
4941575
5191650
1725
5691800
5941875
6191950
6442025
6692100
6942175
7192250
7422325
2400
2475
2500
Fig. 2. The structure of the homeobox gene AHoxl of H. roretzi- The nucleotide sequence and deduced amino acidsequence of AHoxl are shown, numbered left to right. The amino acids are numbered starting with the putative initiatingmethionine residue. The homeobox region indicated by underlining is interrupted by two introns (arrowheads).
A homeobox gene of an ascidian 825
AHoxi RKWNRAVFSLMQRRGLEKSFQSQKYVAKPERRKLADALSLTDAQVKIWFQNRRMKWRQEI 100 %
H2.0 -S-S NL—K IQ—Q IT—D AR-N V HTR 7 0Antp —RG-QTYTRY-TLE E-HFNR-LTRRR-IEI-H--C — ER-I KK-N 46 .6Scr T-RQ-TSYTRY-TLE E-HFNR-LTRRR-IEI-H—C--ER-I LKK-H 43.3Abd-B VRKK-KPY-KF-TLE E-L—A—S-QK-WE — RN-Q--ER NKKNS 50Dfd P-RQ-TAYTRH-ILE E-HYNR-LTRRR-IEI-HT-V-SER-I KKDN 38.3Lab NNSG-TN-TNK-LTE E-HFNR-LTRAR-IEI-NT-Q-NFN QKKRV 4 0Eve VRRY-TA-TRE-LGR E-YKEN — SR-R-CE—AQ-N-PESTI-V DKRQR 4 0En E-RP-TA—SE-LAR-KRE-NENR-LTERR-QQ-SSE-G-NE--I K-A-IKKST 36.7Prd QRRC-TT—AS-LDE—RA-ERTQ-PDIYT-EE--QRTN—E-RIQV--S ARL-KQH 33.3
Fig. 3. Comparison of the homeodomain of AHoxl with those of the Drosophila homeobox genes. The identical residuesare shown by a dash (—). Similarity between the homeodomains of AHoxl and other homeobox genes is indicated by thepercentage on the right margin.
M E H Bg
kb
21.2 -
6 . 0 2 ^
3.53 -
2 . 0 3 "
1.38 —
Fig. 4. Southern blot hybridization of Halocynthia genomicDNA with the AHoxl probe. (A) 2^g of DNA isolatedfrom one adult individual was digested with EcoBI (E),Hindlll (H), and BgUl (Bg), and transferred to a nylonmembrane and hybridized with a 250 bp genomic DNAfragment encompassing the second intron-exon boundaryand the end of the homeobox. The size marker (M) wasend-labelled ADNA fragments digested with EcoBI andHindlll. B is an autoradiogram from the same filter as inA but with a shorter exposure to show more clearly thetwo bands in lane H in A.
early embryos, juveniles and adult tissues. Poly(A)RNAs were prepared from embryos at various stagesand subjected to northern blot hybridization. As shownin Fig. 5, a single band of 2.8 kb in length was detected.During early development, the 2.8 kb transcript wasscarcely detectable (Fig. 5A). The multiple bands seenin the higher molecular weight region in Fig. 5A may benon-specific hybridization to abundant classes ofmRNAs which became apparent due to a long exposuretime. When embryos reached the larval stage, the levelof expression of AHoxl obviously increased.
In Fig. 5B, AHoxl expression was followed injuveniles up to approximately 2 weeks after metamor-phosis. The level of AHoxl expression increased asdevelopment progressed up to day 7 and remained highthereafter.
The expression of AHoxl in various adult tissues wasalso examined. As shown in Fig. 5C, intense expressionwas detected in digestive tract and digestive gland, andto a lesser extent in endostyle. All of these organs are ofendodermal origin. In addition, a considerable level ofAHoxl transcription was detected in coelomic cells,which are thought to be derived from embryonicmesenchyme cells. Very weak expression was alsodetected in pharyngeal epithelium (ectodermal tissue)and in gonad and body wall muscle (mesodermaltissues).
Following this, we carried out in situ hybridization tolocalize the site of expression on sections from juveniles7 days after metamorphosis. As shown in Fig. 6A,B,the AHoxl expression was detected in endodermal cellsthat were differentiating to form the digestive system.Hybridization signals were observed only on theepithelium of the digestive system, but on both theoutside surface, which faces to the future pharyngealepithelium, and the inside surface, which faces thecoelom. No accumulated grains above backgroundwere observed in epidermal cells, the nervous system orpharyngeal epithelium (Fig. 6C,D). At this stage,hybridization signals did not accumulate on coelomiccells (Fig. 6C,D). In situ hybridization with the senseRNA probe showed no significant accumulation ofgrains in those particular regions (Fig. 6E,F).
Discussion
Homology researches employing zoo-blot hybridizationhave demonstrated that the genomes of a variety ofanimals including ascidians contain regions cross-reacting with the Drosophila Antp-type homeobox(McGinnis et al. 1984; Holland and Hogan, 1986).However, little was known about the homeobox genesof ascidians. One exception is an Antp-typ& homeoboxgene isolated from the genomic DNA of Phallusiamammilata (Gehring, personal communication),though it is uncertain whether this Phallusia homeoboxgene is expressed in embryos, larvae, juveniles andadult organisms. In the present study, we have isolatedan ascidian homeobox gene AHoxl from H. roretzi-
The nucleotide sequence of AHoxl shows only alimited degree of similarity to the Antp-type homeoboxsequence, though we used the Antp-type homeobox for
826 H, Saiga and others
A BGo F 16 64 Ga N eTB mTb La La 1 3 5 7 9 11 13
V
- 3.53 -* * '• * flu
-2 .03 _
_0.95
cGo Ph Dt Dg En Mu Be
kb
- 3.53
_ 2.03
_ 0.95
Fig. 5. Expression of AHoxl examined by northern blot hybridization.(A) Northern blot of 10 fig of poly(A) RNA from gonad which includesoocytes, sperm and somatic cells (Go), fertilized eggs (F), 8- and 16-cellembryos (16), 64-cell embryos (64), gastrulae (Ga), neurulae (N), early-tailbud embryos (eTb), middle-tailbud embryos (mTb) and larvae (La).The blot was hybridized with a AHoxl probe (nucleotides 1504-1760 inFig. 2) and washed in O.lxSSPE at 60°C. (B) 10//g of poly(A) RNAisolated from larvae (La) and juveniles at 1 day (1), 3 days (3), 5 days(5), 7 days (7), 9 days (9), 11 days (11) and 13 days (13) aftermetamorphosis, was loaded on the lanes indicated. The blot washybridized and washed as in A. (C) 10 fig of poly(A) RNA from gonad(Go), pharyngeal epithlium (Ph), digestive tract (Dt), digestive gland(Dg), endostyle (En), body wall muscle (Mu) and coelomic (blood) cells(Be) of adult animals was hybridized with the AHoxl probe. Washingwas carried out as in A. The size marker used was end-labelled ADNAfragments digested with EcoRl and HindUl. Note that exposure wascarried out for 10 days with A and for 20 h with B and C. Therefore theintensities of the bands for larvae or gonad poly(A) RNA are different inA, B and C, so can be used as controls for comparison of the relativeintensity of AHoxl expression between the blots.
the screening. Two uninterrupted matches to the probe,one of 14 base pairs and the other of 10 base pairs,present in the last one-third of the homeobox regionenabled us to isolate AHoxl. On the contrary, theisolated homeobox gene has an unexpectedly similarhomeodomain to that of H2.0 of Drosophila. Since ithas recently been pointed out that the AntR-typehomeobox genes clustering on the chromosome(s) ofDrosophila and vertebrates have a common ancestor(Graham et al. 1989), and both Antp- and en-typehomeobox genes have been isolated in the sea urchingenome (Dolecki et al. 1986; Dolecki and Humphreys,1988), one would expect an ascidian to also possessgenes containing the Antp-type homeobox. However,we have not been successful in isolating an Antp-typehomeobox gene from the H. roretzi genome using theAntp homeobox of a silkworm, a sea urchin orDrosophila and the Scr homeobox of Drosophila asprobes. At the present stage, a possible explanation forthis may be the difference in codon usage between theprobes and the H. roretzi genome so that isolation byvirtue of cross hybridization was not possible.
Expression of AHoxl was very restricted in earlyembryos, but became obvious during the later stages ofdevelopment. Transcripts were evident in larvae andjuveniles. The results of in situ hybridization with the
juveniles suggest that the primary site of expression is inendoderm. There are considerable variations amongascidian species in the patterns of temporal differen-tiation in juvenile tissues. H. roretzi is one of the speciesthat exhibit retarded differentiation. Although theendoderm is evident in embryos at later stages, it stillappears to contain undifferentiated cells. Differen-tiation of the endoderm into the digestive tract beginsduring metamorphosis (e.g. Mita-Miyazawa et al.1987). Such temporal development of the endodermseems to be compatible with the developmentalexpression pattern of AHoxl. Furthermore, relativelylarge amounts of the AHoxl mRNA were found inadult endodermal tissues such as the digestive tract anddigestive gland. These results suggest that the AHoxlexpression is associated with differentiation of theendodermal tissues. Together with the similarity of theAHoxl homeobox to that of Drosophila H2.0, theseobservations are reminiscent of those concerning theexpression of the Drosophila H2.0 homeobox gene,which is restricted to visceral mesoderm (Barad et al.1988). Although the function of H2.0 has not beenidentified either, it is possible that the function ofAHoxl in endoderm of a deuterostome is similar to thatof H2.0 in visceral mesoderm of a protostome.
On the other hand, intense expression of AHoxl was
A homeobox gene of an ascidian 827
observed in coelomic or blood cells. In ascidians, six tonine different types of blood cells are found within theopen circular system (reviewed by Goodbody, 1974;Wright, 1981). Each type of blood cell has a character-istic morphology. Various functions such as coagu-lation, nutrition, immune responses, heavy metalaccumulation, gonad and germ cell formation, and
tunic formation have been attributed to either one orseveral types of blood cells. In spite of extensive studieson the structure and function of ascidian blood cells,little is known about their embryological origin, thoughit is suggested that coelomic cells originate fromembryonic mesenchyme cells (Cowden, 1968). Recentstudies have shown that the A7.6 cells of the 64-cell
bs ns
as
ph
en
ep
ds. /
CO
tu
r Ph
ep
cods
tu
CO
ep
cite"
Fig. 6. In situ hybridization to sectioned specimens of 7-day-old juveniles with antisense (A-D) and sense RNA probes (E,F). as, atrial siphon; bs, buccal siphon; co, coelom; ds, digestive system; en, endostyle; ep, epidermis; ns, nervous system;oc, ocellus; ot, otolith; ph, pharynx; tu, tunic. Scale bars, 100 pm. (A) Light photomicrograph of a section through themidline of the juvenile and (B) dark-field image of A. Hybridization signals above background are evident only on theepithelium of the digestive system (ds). (C) Light photomicrograph of paracross section of the juvenile and (D) dark-fieldimage of C, showing the grains are restricted to the epithelium of the digestive system (ds). (E) Light photomicrograph ofparasagittal section of the juvenile and (F) dark-field image of E. By contrast to the antisense probe, hybridization with thesense probe did not show any significant accumulation of grains. Bright spots down left in the photograph are nonspecific,irrespective of grains.
828 H. Saiga and others
embryo of H. roretzi give rise to the trunk-lateral cells(TLCS) of a tadpole larva (Nishida and Satoh, 1985;Nishida, 1987) and that a monoclonal antibody specificto TLCs stains coelomic cells of juvenile and adultbasophilic blood cells, suggesting the developmentalrelationship between TLCs and a type of blood cell(Mishide et al. 1989). At present, however, we are notcertain which type(s) of blood cells expresses AHoxl orof the embryonic origin of the blood cells with AHoxlexpression. In mammalian hematopoietic cell lineages,it has been reported that a variety of homeobox genesare expressed and their expression patterns are differ-ent depending on the cell lineage (Kongsuwan et al.1988; Shen etal. 1989). The cell lineage in which AHoxlis expressed is also a subject for further research.
The authors thank Drs W. Hara and Y. Suzuki, G. Doleckiand T. Humphreys and A. Kuroiwa for kindly providing uswith pBM3.6, the genomic clone of Bombyx mori Antp-Xyyzhomeobox, pHBl, the sea urchin homeobox clone andDrosophila homeobox probes, respectively. Thanks are alsodue to Drs W. R. Jeffery and B. Swalla for technical advicewith in situ hybridization, and we are grateful to Drs T.Matsui, M. Nomoto and A. Ison for comments and criticalreading of the manuscript.
This work was supported by Grants-in-Aid for PriorityArea No. 62124047 and No. 02221101 from Ministry ofEducation, Science and Culture of Japan to H.S.
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(Accepted 12 December 1990)