to a lesser extent in endostyle. All of these organs are of endodermal origin. In addition, a considerable level of. AHoxl transcription was detected in coelomic ...
Development 111, 821-828 (1991) Printed in Great Britain © The Company of Biologists Limited 1991
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Molecular cloning and expression of a novel homeobox gene AHoxi of the ascidian, Halocynthia roretzi HIDETOSHI SAIGA 1 *, 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 of Halocynthia roretzi with the Bombyx mori Antennapedia type homeobox as a probe. The AHoxl gene encodes a protein that consists of 741 amino acids. The homeobox of AHoxl is interrupted by 2 introns each of which is about 300 bp in length and it shows about 70 % similarity at a deduced amino acid level to that of Drosophila H2.0. This suggests that AHoxl is one of the most diverged homeobox genes so far characterized. Northern blot hybridization with an AHoxl probe showed the presence of single transcripts approximately 2.8 kb in length in larvae, juveniles and some adult tissues. The expression of AHoxl is scarcely detected during the course of early development but it increases to a moderate level at the larval stage. After metamor-
phosis, the level of AHoxl expression increases as development proceeds. In situ hybridization to the juvenile 7 days after metamorphosis showed that the site of AHoxl expression is the epithelium of digestive tract. Among the adult tissues examined, digestive tract, digestive gland and coelomic cells were the major sites of the expression of AHoxl. In gonad, body wall muscle and pharyngeal epithelium, the expression of AHoxl is relatively weak. These results suggest that AHoxl is primarily expressed in the tissues of endodermal origin and that the gene expression may be associated with differentiation of the endodermal tissues.
Introduction
Protochordata, is exceptionally autonomous. This feature enabled Conklin, Ortolani and Nishida and Satoh to trace the fate of each blastomere (Conklin, 1905; Ortolani, 1955; Nishida and Satoh, 1983, 1985). The cell lineage in early development has been established completely up to the 110-cell stage in Halocynthia roretzi (Nishida, 1987). It is thought that the autonomous development of the ascidians is due to factors, named determinants, which appear to be involved in the determination of the fate of each blastomere and are localized heterogeneously in the fertilized egg cytoplasm (for recent reviews see Satoh, 1987 and Satoh et al. 19906; Uzman and Jeffery, 1986; Whittaker, 1987; Venuti and Jeffery, 1989). The molecular identity of a determinant, however, is still an enigma (see reviews by Jeffery, 1985; Satoh et al. 1990a). We are interested in studying the homeobox genes of ascidians for two reasons. (1) Since ascidians belong to the class Protochordata located phylogenetically between invertebrates and vertebrates, the structure of
The homeobox was originally discovered as a small segment of conserved nucleotide sequence among the Drosophila genes that are involved in the formation of axes and body segments, and in the specification of the body segments (for review see Gehring, 1987; Akam, 1987). The homeodomain, the translated form of the homeobox, has a helix-turn-helix motif, which confers a DNA binding capacity to the homeobox gene product and therefore it has been suggested that homeobox genes encode transcription factors. There are several lines of evidence supporting this hypothesis (Quian et al. 1989; Ingham, 1988; Scott et al. 1989, references therein). Homeobox genes have been found to be widely distributed among various animal species, from nematodes to insects and vertebrates on the phylogenetic 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 early development of ascidians, which belong to the class
Key words: ascidian, homeobox, molecular cloning, gene expression, developmental expression, in situ hybridization.
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H. Saiga and others
the homeobox genes of ascidians should shed some light on the evolutionary aspect of homeobox genes. (2) Since it is highly Likely that expression of homeobox genes is involved in determining cell differentiation, the expression patterns of homeobox genes in ascidians or nematodes, which exhibit typical mosaic development, are of particular interest. This might lead to better understanding of the molecular identity of the so-called determinants. Therefore, we have started isolating homeobox genes from the ascidian, Halocynthia roretzi- In this report, we describe the isolation and molecular characterization of a homeobox gene from the Halocynthia genome. Although we used the Antennapedia (Antp) type homeobox as a probe, the isolated homeobox gene, designated AHoxl, was not of this type but, unexpectedly, it has a similar homeobox to that of H2.0. This is a Drosophila homeobox gene showing tissue-specific expression rather than regionspecific expression (Barad et al. 1988). Likewise the expression of the isolated ascidian homeobox gene, AHoxl, is primarily detected in the endoderm during the course of development.
Materials and methods Ascidian Adult ascidians, Halocynthia roretzi, were purchased from fishermen near Asamushi Marine Biological Station, Tohoku University, Mutsu Bay, Aomori, Japan. Adult specimens were dissected and tissues were frozen quickly with liquid nitrogen. The frozen samples were kept at —80°C until use. Naturally spawned eggs were fertilized with a suspension of non-self sperm, and fertilized eggs were raised in filtered sea water at 13—15 °C. Embryogenesis proceeded synchronously amongst batches of eggs. Tadpole larvae hatched about 33 h after fertilization. They were allowed to accomplish metamorphosis naturally. Juveniles that adhered to plastic dishes were cultured for 13 days in aquaria with circulating natural seawater. Samples at appropriate stages were collected by low speed centrifugation and were frozen with liquid nitrogen. Construction of genomic and subgenomic libraries Halocynthia roretzi genomic DNA was prepared from the gonad of one adult individual according to Blin and Stafford (1976). Genomic DNA was digested partially with Sau3A and ligated to the BamFfl-digested AEMBL3 vector. To construct a subgenomic library, genomic DNA was digested completely with EcoRI and separated on a 1 % preparative agarose gel. The DNA fragments of about 2.7 kb in length were recovered by electroelution and ligated to the £coRI-digested Agtll vector. Ligated materials were packaged and propagated in NM539 and Y1088 for genomic and subgenomic libraries, respectively. The probes used for screening of the libraries were as follows; a Hindi-Avail fragment of pBm3.6 about 250 bp in length, which is the genomic clone of a Bombyx mori Antptype homeobox gene (Hara and Suzuki, unpublished), and a Psd-TaqI fragment of pHBl about 200 bp in length, which contains a sea urchin homeobox as described by Dolecki et al. (1986). For screening of the iibrary, hybridization was carried out under reduced stringency conditions essentially as described
by McGinnis et al. (1984). In brief, hybridization was carried out 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 phosphate pH7.0, 20^gml" 1 E. coli DNA and 1-5xK^ctsminimi" 1 probe labelled by random priming using [a--32?] dCTP as the labelled nucleotide. After hybridization, the filters were washed in 2xSSC, 0.2% SDS at 37°C for 30min twice. Preparation of poly (A) RNA Total RNA from embryos or adult tissues was prepared essentially according to Ullrich et al. (1977). H. roretzi embryos were developed at 15 °C, collected by low-speed centrifugation and kept at -80°C. Frozen packed embryos or adult tissues were suspended in 4 M guanidine thiocyanate, 0.1M sodium acetate, 5mM EDTA, pH5.0 and were homogenized using a Polytron homogenizer (Kinematics). After low-speed centrifugation to remove cell debris, the homogenate was layered into a tube for a Beckman SW4OTi rotor one-third filled with 5 M CsCl, 0.1M sodium acetate, 5mM EDTA, pH5.0 and subjected to centrifugation at 35000revsmin~^ for 15 h at 20 °C. The pellet was dissolved in a small volume of 10 mM Tris-HCl pH7.6, 10 mM EDTA, 0.5% SDS, extracted with phenol and ether, and the RNA was recovered by ethanol precipitation. Poly(A) RNA was selected using Oligotex-dT30 Latex beads (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 that prepared using the ordinary oligo(dT)-cellulose column. cDNA library construction cDNA was synthesized from gonad poly(A) RNA using a cDNA synthesis kit (Pharmacia). Synthesized cDNA was ligated to the Agtll vector. The ligated materials were packaged in vitro as described in 'Construction of genomic and subgenomic libraries'. Southern and northern blotting 2 ng of genomic DNA digested with Eco RI, Hindlll or BgUl was separated on a 1 % agarose gel. After electrophoresis, the gel was soaked in 0.5 M NaOH, 1.5 M NaCl for 15min and subjected 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 gel containing 6 % formaldehyde and were transferred to a nylon membrane filter using a Vac Gene apparatus and IOXSSC as transferring buffer. Southern or northern blot hybridization was carried out according to the standard procedure (Maniatis etal. 1982). In situ hybridization In situ hybridization using RNA probes and autoradiography was carried out according to the method described by Tomlinson et al. (1987). [35S]ATP-labelled probes were prepared 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 sense and antisense RNAs were transcribed with T7 and SP6 RNA polymerases, after linearization of the plasmid DNA with Hindlll and EcoRI, respectively. Juveniles 7 days after metamorphosis were fixed in ice-cold 95 % ethanol:acetic acid (3:1, v/v), and embedded in Tissue-Prep (Fisher Scientific Co.). The specimens were sectioned at 8/an and attached to subbed glass slides. After treatment with 1 /igmF 1 proteinase K, the specimens were postfixed in 4% paraformaldehyde,
A homeobox gene of an ascidian treated with freshly prepared 0.25% acetic anhydride, 0.1M triethanolamine, and air dried. Approximately lxH^ctsmin" 1 of 35S-labelled RNA probe was applied to each slide in 50^/1 hybridization buffer (50% formamide, lxDenhardt's solution, 10% dextran sulfate, 2xSET, 0.01M DTT, 500/igmT 1 TRNA, 500/igmT1 poly(A)). Hybridization reaction was carried out at 45 °C approximately for 12 h, and the slides were washed at high stringency (in 0.2xSSC at 45 °C for 45min). The washed slides were dehydrated with 95% ethanol, air dried and subjected to autoradiography. The autoradiographs were stained through the emulsion with haematoxylin-eosin. Results
Isolation of a homeobox gene Prior to library screening, we carried out Southern blotting analysis of H. roretzi genomic DNA to determine the conditions for hybridization so as to give signals with reasonable strength. This was done using a genomic DNA probe of the Antp-type homeobox of silk worm or sea urchin. As shown in Fig. 1, under reduced stringency hybridization conditions, both probes resulted in several bands on a membrane filter but most coincided with those detected by ethidium bromide staining of the gel. A 2.7 kb band detected upon EcoKl digestion, however, was distinct from those detected by ethidium bromide staining. To facilitate efficient cloning, the ZscoRI-digested genomic DNA fragments
823
of about 2.7 kb in length were recovered, and from these a subgenomic library was constructed. We screened the subgenomic library under the reduced stringency hybridization conditions using the Bombyx homeobox probe, since it gave lower background than the sea urchin probe. We were able to isolate a positive clone, designated 62, which had a 2.7 kb insert. By determination of the nucleotide sequence, it was identified as a clone containing a part of the homeobox. Using DNA fragments from the clone 62, we have further isolated three cDNA clones corresponding to the genomic DNA fragment included in the clone 62 from the adult gonad cDNA library as well as genomic clones covering the cDNAs from the H. roretzi genomic library.
Structure of the ascidian homeobox gene, AHoxl Fig. 2 shows the nucleotide sequence of cDNA of the ascidian homeobox gene designated AHoxl. It has a single long open reading frame consisting of 741 amino acid residues and including a highly diverged homeodomain. One structural feature of the AHoxl homeobox is the presence of two introns of 319 and 314 bp in length (data not shown) located at position 9 and after position 44, respectively. This is in contrast to the engrailed (en), invested (inv) and labial (lab) which have one intron in the homeobox locating at position 17 (en and inv) or after position 44 (lab). Comparison of the amino acid sequence of the AHoxl homeodomain with those of several representative Drosophila homeobox genes is shown in Fig. 3. The amino acid sequence of the AHoxl homeodomain is highly diverged from all of E H Bg E H Bg the known homeodomains with less than 50 % identity over 60 residues. The only one exception is the homeodomain of H2.0 of Drosophila to which the kb AHoxl homeodomain shows 70% identity, though 21.2 " outside the homeodomain, there is no significant similarity. AHoxl also lacks the sequences corresponding to the M repeat and the YPWM peptide, which are conserved upstream of the homeobox in many of the 5.02 homeobox genes of Drosophila and vertebrates. 3.53 To see whether the Halocynthia genome contains i other genes possessing a similar homeobox to that of AHoxl, Southern genomic hybridization under 2.03 _ reduced stringency conditions was carried out using the DNA fragment from the clone 62 as a probe, which contained the last one-third of the AHoxl homeobox. 1.38 — As shown in Fig. 4A, a 2.7 kb band was detected in the 0.85 — ZscoRI-digested genomic DNA as expected. This and intense bands seen in the Hindlll- and BglH-digested DNA correspond to the AHoxl gene. Other than these, no significant band was detected. Upon Hindlll Fig. 1. Detection of homeobox homology in the genomic digestion, two bands were detected as shown more DNA of the ascidian Halocynthia roretzi. 10 /ig of genomic clearly in Fig. 4B. These results suggest that two copies DNA digested with EcoKl (E), Hindlll (H), or Bglil (Bg) of AHoxl are present in the genome, possibly was separated on a 1% agarose gel. Genomic Southern representing two allelic forms, and it is unlikely that a blots were hybridized under reduced stringency conditions gene containing a similar homeobox to that of AHoxl is with the Bombyx homeobox probe (left) and the sea urchin present in the Halocynthia genome. homeobox probe (right). An arrowhead indicates the 2.7kb band, which was distinct from the ethidium bromideExpression of AHoxl stained bands. The size marker used was end-labelled ADNAfragmentsdigested with EcoRI and HindlU. We have examined expression of the AHoxl gene in
B
1
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H. Saiga and others 1
TTT TGA AAT CAT TTA TTG ACT TCT ATT ATA TCT AAT TAT CTA TTC ATA CAT ACA GCT AGT ACA TTT TTT ACT TTT
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T S L G G L k M S 1 P R p S N S T C S V E L C A E ACA TCC TTA GGA CCA CTC CCA AAA TCA TCC ACT ATT CCC ACC TTC GCA GTA CCA GAA TGC GAA TCA ATG CGT AAC
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k I E E E S R H T N N M P I R L D A I T T R L M Q AAA CAT ATC GAA GAA CAA AGA ACC AAT AAC ATC ACA ACA ATG CCA ATG AAC AGA CGC TTC TTA GAT CCC CAA AAT
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k k N k R R E Y F H S S S E N S E Q A E F S Q Q AAA AAC AAG CAA AAC AGA TTT GAA ACG TAT TCT AGT TCA AAC CAT CCC CAA GAA CAA TCT TCA CAC CAA AAT TTT
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V k k C D k I R T L S I k I S D F G k N A E S N L TGC AGA ACC AAG AAA GAT AGC ACC GTT TTG AAG TTT GGA ATT GAC AGC ATA CTT AAA AAT AAA AAC CCA CAA AAA
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V P A R S G R G 1 I I T N F N D E T S A T E S C Q GTA CCA AAA GCA ATA TCT AAC CCC GGA AGG ATT CAG GAT GAA AGA TTC ACA CAG GCA TCT ACT AAC ACA ACC TCC
144 525
14 5 526 170 601
Q A T T A R C R s AAT GAC CAG TTA GGT CCA CCA AGA ACT GCT ACA s TCT ACT F S S S S E I N k K A S D S S C T Y B E N C E Y A TTT TCA TCC AGC TCA GAA GCA AGT GAC AGT AAA TCA TGC TGT ACA AAT AAC AAT GAA GAA GCC CCT TAC AAC TAT
194 675
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I R I R k V D V N D C D T D S A D D S E S A A P E ACA GTT ATT GAC AAG CGG AAG AGC GCG GAT TCA GAC TGC AGT GAA GAC GCA ACT GGA AAC CAA CCA CAC GAT CCT
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I R D D N H N T L V D I E L A E S L C D A S I Q Q GAC GAT CAT ATT AAT CAA GAT AAC TCT GAT TTA GCC AGC ACA CTT GAG CAA ACT CGT ATT GTT GCT TTG GAA ATA
244 825
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k k P R R L N L L S T L L D N A Y D R E S L D S Q TTG AAA AAC AAA AGA CTT CGA CTT GAC ACC AGT GAA GCT CTT AAC GAC CTT ACA CCA TAT GAT CAA TTG TCA CCT
269 900
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I N T D I N N E V E F U A R M H E A M S E R Q Q Q ACC GAA GAT CAG CAG ATA AGT ACC AGA GTG GAA ATC ATC AAT CAC CAA GCC TTC GCC AGA CAA AAC AAC CAA TCG
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R P F Y P I N L P P S F S S F N L A F A L C D H Q CCA AGA ACT TTT TCT TCT GGA TTG CAA GAT CCA TTT GCA AAA AAT CTA CCT AAC GCA TTC CTG CCC TTT TAC ATG
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k N I R N H I P Y I L R D L Y Y Y Y A L H R Q I Q CAA CCC TAT TTG AGG GCT TAT TAC AAT ATT CAA AAA TAT ATT TAC CAC AAA AAC TTG TTA AAT CGA AAT GAC AGG
344 1125
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R P Y R E S T k I V N N E A F N S E E L Y E D T S TTC TAC AGA GAA GCA AAC GTT GAA AAT GAT AAT TAC AAG ACT GAG GAA TCA TTG AGG TCT CCG AGC GAA ACA AAA
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V R I I D L V k D G N E E G D D V L F E S S A L C GAT GTA GAC GAG GGC GAC AAG GAA GCA AAC AAA TTC TTC AAA GAT CTT TCT GTC AGC GTT GGC GAC AGA TTA TCA
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R I T K L S Y N A G F L L T S R D N G Y S G S N Q AAT GCC TTC TCT TAT GGT CGA AAG GAC TAC AAC GGT CTG TCA ACC TCT CAA ACC TCA GGG AAT CGT TTT CTA AAT
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I P N N D Y N N D E T S L Q G I T I L S I E M Q Q GAG GAC AAT AAT CCT CAA ACG ATT GGG AGC GAA TAC CAA ATT AAT ACA CAA CGA AGC ATG AAA GAT AAT TTA CTC
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P V N H G A D H Y L F G A A P A P T G S N N N Q CAT CCT CCA CAC TAT GGA GCG GCG CAA GTT AAT CCG ACA GCA GGC AAC AAT TTC AAG TCT ATG TTA CCG GGT AAT
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669 2100
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694 2175
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719 2250
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742 2325
2326
CGA TTC CTC ATT CCA AAA TCA TGC CGC CAC CGA ACC CAG ACA GTA CCA ATA AAG TTC ACT AAT TTC ACG GTT TCC
2400
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Fig. 2. The structure of the homeobox gene AHoxl of H. roretzi- The nucleotide sequence and deduced amino acid sequence of AHoxl are shown, numbered left to right. The amino acids are numbered starting with the putative initiating methionine residue. The homeobox region indicated by underlining is interrupted by two introns (arrowheads).
A homeobox gene of an ascidian AHoxi
RKWNRAVFSLMQRRGLEKSFQSQKYVAKPERRKLADALSLTDAQVKIWFQNRRMKWRQEI
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Fig. 3. Comparison of the homeodomain of AHoxl with those of the Drosophila homeobox genes. The identical residues are shown by a dash (—). Similarity between the homeodomains of AHoxl and other homeobox genes is indicated by the percentage on the right margin.
M
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Fig. 4. Southern blot hybridization of Halocynthia genomic DNA with the AHoxl probe. (A) 2^g of DNA isolated from one adult individual was digested with EcoBI (E), Hindlll (H), and BgUl (Bg), and transferred to a nylon membrane and hybridized with a 250 bp genomic DNA fragment encompassing the second intron-exon boundary and the end of the homeobox. The size marker (M) was end-labelled ADNA fragments digested with EcoBI and Hindlll. B is an autoradiogram from the same filter as in A but with a shorter exposure to show more clearly the two bands in lane H in A.
early embryos, juveniles and adult tissues. Poly(A) RNAs were prepared from embryos at various stages and subjected to northern blot hybridization. As shown in Fig. 5, a single band of 2.8 kb in length was detected. During early development, the 2.8 kb transcript was scarcely detectable (Fig. 5A). The multiple bands seen in the higher molecular weight region in Fig. 5A may be non-specific hybridization to abundant classes of mRNAs which became apparent due to a long exposure time. When embryos reached the larval stage, the level of expression of AHoxl obviously increased. In Fig. 5B, AHoxl expression was followed in juveniles up to approximately 2 weeks after metamorphosis. The level of AHoxl expression increased as development progressed up to day 7 and remained high thereafter.
The expression of AHoxl in various adult tissues was also examined. As shown in Fig. 5C, intense expression was detected in digestive tract and digestive gland, and to a lesser extent in endostyle. All of these organs are of endodermal origin. In addition, a considerable level of AHoxl transcription was detected in coelomic cells, which are thought to be derived from embryonic mesenchyme cells. Very weak expression was also detected in pharyngeal epithelium (ectodermal tissue) and in gonad and body wall muscle (mesodermal tissues). Following this, we carried out in situ hybridization to localize the site of expression on sections from juveniles 7 days after metamorphosis. As shown in Fig. 6A,B, the AHoxl expression was detected in endodermal cells that were differentiating to form the digestive system. Hybridization signals were observed only on the epithelium of the digestive system, but on both the outside surface, which faces to the future pharyngeal epithelium, and the inside surface, which faces the coelom. No accumulated grains above background were observed in epidermal cells, the nervous system or pharyngeal epithelium (Fig. 6C,D). At this stage, hybridization signals did not accumulate on coelomic cells (Fig. 6C,D). In situ hybridization with the sense RNA probe showed no significant accumulation of grains in those particular regions (Fig. 6E,F).
Discussion Homology researches employing zoo-blot hybridization have demonstrated that the genomes of a variety of animals including ascidians contain regions crossreacting with the Drosophila Antp-type homeobox (McGinnis et al. 1984; Holland and Hogan, 1986). However, little was known about the homeobox genes of ascidians. One exception is an Antp-typ& homeobox gene isolated from the genomic DNA of Phallusia mammilata (Gehring, personal communication), though it is uncertain whether this Phallusia homeobox gene is expressed in embryos, larvae, juveniles and adult organisms. In the present study, we have isolated an ascidian homeobox gene AHoxl from H. roretziThe nucleotide sequence of AHoxl shows only a limited degree of similarity to the Antp-type homeobox sequence, though we used the Antp-type homeobox for
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H, Saiga and others
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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 includes oocytes, sperm and somatic cells (Go), fertilized eggs (F), 8- and 16-cell embryos (16), 64-cell embryos (64), gastrulae (Ga), neurulae (N), earlytailbud embryos (eTb), middle-tailbud embryos (mTb) and larvae (La). The blot was hybridized with a AHoxl probe (nucleotides 1504-1760 in Fig. 2) and washed in O.lxSSPE at 60°C. (B) 10//g of poly(A) RNA isolated 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) after metamorphosis, was loaded on the lanes indicated. The blot was hybridized 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. Washing was carried out as in A. The size marker used was end-labelled ADNA fragments digested with EcoRl and HindUl. Note that exposure was carried out for 10 days with A and for 20 h with B and C. Therefore the intensities of the bands for larvae or gonad poly(A) RNA are different in A, B and C, so can be used as controls for comparison of the relative intensity 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 region enabled us to isolate AHoxl. On the contrary, the isolated homeobox gene has an unexpectedly similar homeodomain to that of H2.0 of Drosophila. Since it has recently been pointed out that the AntR-type homeobox genes clustering on the chromosome(s) of Drosophila and vertebrates have a common ancestor (Graham et al. 1989), and both Antp- and en-type homeobox genes have been isolated in the sea urchin genome (Dolecki et al. 1986; Dolecki and Humphreys, 1988), one would expect an ascidian to also possess genes containing the Antp-type homeobox. However, we have not been successful in isolating an Antp-type homeobox gene from the H. roretzi genome using the Antp homeobox of a silkworm, a sea urchin or Drosophila and the Scr homeobox of Drosophila as probes. At the present stage, a possible explanation for this may be the difference in codon usage between the probes and the H. roretzi genome so that isolation by virtue of cross hybridization was not possible. Expression of AHoxl was very restricted in early embryos, but became obvious during the later stages of development. Transcripts were evident in larvae and juveniles. The results of in situ hybridization with the
juveniles suggest that the primary site of expression is in endoderm. There are considerable variations among ascidian species in the patterns of temporal differentiation in juvenile tissues. H. roretzi is one of the species that exhibit retarded differentiation. Although the endoderm is evident in embryos at later stages, it still appears to contain undifferentiated cells. Differentiation of the endoderm into the digestive tract begins during metamorphosis (e.g. Mita-Miyazawa et al. 1987). Such temporal development of the endoderm seems to be compatible with the developmental expression pattern of AHoxl. Furthermore, relatively large amounts of the AHoxl mRNA were found in adult endodermal tissues such as the digestive tract and digestive gland. These results suggest that the AHoxl expression is associated with differentiation of the endodermal tissues. Together with the similarity of the AHoxl homeobox to that of Drosophila H2.0, these observations are reminiscent of those concerning the expression 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 been identified either, it is possible that the function of AHoxl in endoderm of a deuterostome is similar to that of H2.0 in visceral mesoderm of a protostome. On the other hand, intense expression of AHoxl was
A homeobox gene of an ascidian observed in coelomic or blood cells. In ascidians, six to nine different types of blood cells are found within the open circular system (reviewed by Goodbody, 1974; Wright, 1981). Each type of blood cell has a characteristic morphology. Various functions such as coagulation, nutrition, immune responses, heavy metal accumulation, gonad and germ cell formation, and
827
tunic formation have been attributed to either one or several types of blood cells. In spite of extensive studies on the structure and function of ascidian blood cells, little is known about their embryological origin, though it is suggested that coelomic cells originate from embryonic mesenchyme cells (Cowden, 1968). Recent studies have shown that the A7.6 cells of the 64-cell
as
ep
bs ns
ds
ph
^.
/
CO
en
tu
r
^co
Ph
ds
ep
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 the midline of the juvenile and (B) dark-field image of A. Hybridization signals above background are evident only on the epithelium of the digestive system (ds). (C) Light photomicrograph of paracross section of the juvenile and (D) dark-field image of C, showing the grains are restricted to the epithelium of the digestive system (ds). (E) Light photomicrograph of parasagittal section of the juvenile and (F) dark-field image of E. By contrast to the antisense probe, hybridization with the sense 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 specific to TLCs stains coelomic cells of juvenile and adult basophilic blood cells, suggesting the developmental relationship between TLCs and a type of blood cell (Mishide et al. 1989). At present, however, we are not certain which type(s) of blood cells expresses AHoxl or of the embryonic origin of the blood cells with AHoxl expression. In mammalian hematopoietic cell lineages, it has been reported that a variety of homeobox genes are expressed and their expression patterns are different depending on the cell lineage (Kongsuwan et al. 1988; Shen etal. 1989). The cell lineage in which AHoxl is expressed is also a subject for further research. The authors thank Drs W. Hara and Y. Suzuki, G. Dolecki and T. Humphreys and A. Kuroiwa for kindly providing us with pBM3.6, the genomic clone of Bombyx mori Antp-Xyyz homeobox, pHBl, the sea urchin homeobox clone and Drosophila homeobox probes, respectively. Thanks are also due to Drs W. R. Jeffery and B. Swalla for technical advice with in situ hybridization, and we are grateful to Drs T. Matsui, M. Nomoto and A. Ison for comments and critical reading of the manuscript. This work was supported by Grants-in-Aid for Priority Area No. 62124047 and No. 02221101 from Ministry of Education, Science and Culture of Japan to H.S.
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