小笠原 道生

Recent progress in multiple and automated-sequencing technology allows large-scale random cDNA sequencing, the so-called EST project, in various fields. In addition to the EST collection, the cDNA project requires analysis of spatiotemporal patterns of gene expression of a large number of clones by whole-mount in situ hybridization (WISH). To facilitate the multiple WISH procedures, we developed a protocol for rapid and uniform synthesis of multiple probes and multi-well based WISH processing. A DIG-labeled RNA probe for WISH was synthesized from a PCR-amplified template which contained an RNA promoter. All reactions of PCR and subsequent RNA synthesis were performed in a single tube by sequential addition of the reagents without phenol extraction or ethanol precipitation steps. An RNA probe was purified and condensed by a centrifugal ultrafilter to achieve high and stable purification efficiency. WISH of 96 samples were performed simultaneously in a 96-well plate attached to silent screen filters that were connected with a vacuum exhausting system. These processes eliminated the labor-intensive steps of WISH and provided opportunities to search for novel genes.

Among the transcription factor gene families, Pax genes play important and unique roles in morphological patterning of animal body plans. Of these, Group I Pax genes (Pax1 and Pax9) are expressed in the endodermal pharyngeal pouches in many groups of deuterostomes, and vertebrates seem to have acquired more extensive expression domains in embryos. To understand the evolution of Pax1/Pax9-related genes in basal groups of vertebrates, their cognates were isolated from the Japanese marine lamprey, Lampetra japonica. RT-PCR of larval lamprey cDNA yielded two different fragments containing vertebrate Pax1- and Pax9-like paired domains. The Pax9 orthologue was isolated and named LjPax9. Whole-mount in situ hybridization revealed that this gene was expressed in endodermal pharyngeal pouches, mesenchyme of the velum (the oral pumping apparatus) and the hyoid arch, and the nasohypophysial plate, but not in the somitic mesoderm of the lamprey embryo. These expression patterns could be regarded as a link between the basal chordates and the gnathostomes and are consistent with the phylogenetic position of the lamprey. Especially, the appearance of neural crest seemed to be the basis of velar expression. Homology of the velum and the jaw is also discussed based on the LjPax9 expression in the first pharyngeal pouch and in the velar mesenchyme. We conclude that Pax9 genes have sequentially expanded into new expression domains through evolution as more complicated body plans emerged.

The endostyle is a pharyngeal organ of urochordates, cephalochordates, and primitive vertebrates. This organ has iodine-concentrati ng and iodine-metabolism activities, and therefore the endostyle is considered to be homologous to the follicle of the thyroid gland. In higher vertebrates the genes for both thyroid transcription factor-1 (TTF-1) and thyroid peroxidase (TPO) are expressed in the thyroid gland follicle. TTF-1 regulates the expression of TPO, which encodes an iodinating enzyme associated with thyroid hormone synthesis. A recent study showed that the ascidian TTF-1 and TPO genes are specifically expressed in the endostyle, but that the expression domains of these genes are not overlapping, suggesting that ascidian TPO is not regulated by TTF-1. To investigate the molecular mechanisms involved in the formation and function of the endostyle, with special reference to the evolution of the follicle of the thyroid gland, I isolated and characterized cDNA clones for the amphioxus homologs of the TTF-1 gene (BbTTF-1) and TPO gene (BbTPO) from Branchiostoma belcheri. Reverse transcriptase-polymerase chain reaction/Southern blotting revealed that both amphioxus TTF-1 and TPO genes are expressed mainly in the adult endostyle. Spatial and temporal expression patterns assessed by in situ hybridization revealed that BbTTF-1 is expressed in the endodermal cells during early embryogenesis and is maintained in all zones of the adult endostyle. On the other hand, expression of BbTPO is chiefly in zones 5 and 6 of the adult endostyle where it overlaps with that of BbTTF-1, and to a lesser extent in zones and 3. This restriction of the expression of BbTTF-1 and BbTPO to the endostyle strongly suggests that the endostyle is homologous to the follicle of the thyroid gland. Moreover, the spatial and temporal expression patterns of these genes suggest that TTF-1 regulates TPO expression. The coexpression of these genes in amphioxus suggests that regulation of TPO by TTF-1 was present in the common ancestor of cephalochordates (acraniates) and craniates.

The endostyle is a pharyngeal organ of uro- chordates, cephalochordates, and primitive vertebrates. This organ has iodine-concentrating and iodine-metabolism activities, and therefore the endostyle is considered to be homologous to the follicle of the thyroid gland. In higher vertebrates the genes for both thyroid transcription factor-1 (TTF-1) and thyroid peroxidase (TPO) are expressed in the thyroid gland follicle. TTF-1 regulates the expression of TPO, which encodes an iodinating enzyme associated with thyroid hormone synthesis. A recent study showed that the ascidian TTF-1 and TPO genes are specifically expressed in the endostyle, but that the expression domains of these genes are not overlapping, suggesting that ascidian TPO is not regulated by TTF-1. To investigate the molecular mechanisms involved in the formation and function of the endostyle, with special reference to the evolution of the follicle of the thyroid gland, I isolated and characterized cDNA clones for the amphioxus homologs of the TTF-1 gene (BbTTF-1) and TPO gene (BbTPO) from Branchiostoma belcheri. Reverse transcriptase-polymerase chain reaction/Southern blotting revealed that both amphioxus TTF-1 and TPO genes are expressed mainly in the adult endostyle. Spatial and temporal expression patterns assessed by in situ hybridization revealed that BbTTF-1 is expressed in the endodermal cells during early embryogenesis and is maintained in all zones of the adult endostyle. On the other hand, expression of BbTPO is chiefly in zones 5 and 6 of the adult endostyle where it overlaps with that of BbTTF-1, and to a lesser extent in zones 1 and 3. This restriction of the expression of BbTTF-1 and BbTPO to the endostyle strongly suggests that the endostyle is homologous to the follicle of the thyroid gland. Moreover, the spatial and temporal expression patterns of these genes suggest that TTF-1 regulates TPO expression. The coexpression of these genes in amphioxus suggests that regulation of TPO by TTF-1 was present in the common ancestor of cephalochordates (acraniates) and craniates.

A cDNA library prepared from fertilized eggs of the ascidian Halocynthia roretzi was screened for prelocalized mRNAs in the early embryo by means of whole-mount in situ hybridization using a digoxigenin-labeled antisense RNA of each clone. Random mass screening of 150 cDNAs in a fertilized egg yielded six different clones which showed mRNA localization in the posterior-vegetal cytoplasm of the 8-cell embryo. An in situ hybridization study of the detailed spatial distribution of each mRNA in embryos of various stages revealed that there are, in contrast to the identical localization in embryos after the 16-cell stage, two distinct patterns of RNA distribution at earlier stages. One is colocalization with the myoplasm from the prefertilization stage to the 8-cell stage (type I postplasmic RNAs). The other is delayed accumulation of RNA at the posterior-vegetal cytoplasm after fertilization (type II postplasmic RNAs). We found that both types of RNAs associate with the cytoskeleton, but that they show different sensitivities to inhibitors of the cytoskeleton; translocation of the type I RNAs is dependent upon microfilaments during the first phase of ooplasmic segregation and dependent upon microtubules during the second phase of segregation, whereas translocation of the type II RNAs is dependent upon microfilaments throughout ooplasmic segregation. These results show that there are two pathways for the localization of the RNAs at the posterior-vegetal cytoplasm in the 8-cell embryo of the ascidian H. roretzi.

The gene family encoding RNA-binding proteins includes important regulators involved in the neurogenesis in both protostomes and deuterostomes. We isolated cDNAs of the ascidian homolog of one of the RNA-binding proteins, MUSASHI, from Halocynthia roretzi and Ciona intestinalis. The predicted amino acid sequences contained two RNA-recognition and RNA-binding motifs in the N-terminus and an ascidian-specific YG-rich domain in the C-terminus. Maternal transcripts of musashi were ubiquitous in early cleavage-stage embryos. Ascidian musashi had three domains of zygotic expression: the brain, nerve cord, and mesenchyma. The temporal order of the onset in these domains was highly divergent between the two species of ascidian examined.

Acorn worms are hemichordate deuterostomes that have remarkable gills thought to be homologous to pharyngeal ails in urochordates and cephalochordates, and pharyngeal pouches in vertebrates. In search of molecular keys to analyzing the origin and evolution of the anterior gut and neck region of the chordate body, the present study isolated cDNA clones for six gill-specific genes, designated PfG1 to PfG6, from Ptychodera flava using differential screening of a cDNA library of RNA from ails. Northern blotting confirmed that these genes were all expressed only in the gills. In situ hybridization showed that the expression of these genes is limited to the endodermally derived columnar epithelium of the pharynx. PfG1 encodes a 42-kDa polypeptide containing sequence similar to D-domains, protein domains characteristic of extracellular proteins. Expression of PfG1 is localized in a delimited pattern along the columnar epithelium of the inner all apparatus. Expression in the epibranchial ridge appears as two stripes running longitudinally in the epithelium just lateral of the midline, A stripe of expression also appears in a slightly posterior portion on the curve of each band of columnar epithelium on the pharyngeal surface of the secondary gill bars. The five other all-specific genes, PfG2 to PfG6, encode a family of C-type lectin polypeptides that appear to be secreted proteins. PfG2 to PfG6 are also expressed in the columnar epithelium of the epibranchial ridge as two parallel stripes, but at the lateral margin of the ridge. One of the genes, PfG6, is additionally expressed in the innermost curve of the epithelium on the pharyngeal surface of each secondary gill bar. The localization of expression of PfPax1/9, a all-specific transcription factor gene, was examined and shown to also be primarily in the endodermal columnar epithelium on the pharyngeal faces of the gill bars. On the secondary gill bars, where PfG1 and PfG6 are also expressed in the columnar epithelium, PfPax1/9 is expressed in the anterior and posterior portions but signal is not evident in the epithelium on the central, innermost curve of the gill bar. The anterior domain of PfPax1/9 expression is more extensive but overlaps the anterior domain of PfG1 expression, whereas its posterior domain of expression is more posterior and complementary to that of PfG6. Dev Dyn 2000;217;309-319. (C) 2000 Wiley-Liss, Inc.

The gene family encoding RNA-binding proteins includes important regulators involved in the neurogenesis in both protostomes and deuterostomes. We isolated cDNAs of the ascidian homolog of one of the RNA-binding proteins, MUSASHI, from Halocynthia roretzi and Ciona intestinalis. The predicted amino acid sequences contained two RNA-recognition and RNA-binding motifs in the N-terminus and an ascidian-specific YG-rich domain in the C-terminus. Maternal transcripts of musashi were ubiquitous in early cleavage-stage embryos. Ascidian musashi had three domains of zygotic expression: the brain, nerve cord, and mesenchyma. The temporal order of the onset in these domains was highly divergent between the two species of ascidian examined.

Acorn worms are hemichordate deuterostomes that have remarkable gills thought to be homologous to pharyngeal gills in urochordates and cephalochordates, and pharyngeal pouches in vertebrates. In search of molecular keys to analyzing the origin and evolution of the anterior gut and neck region of the chordate body, the present study isolated cDNA clones for six gill-specific genes, designated PfG1 to PfG6, from Ptychodera flava using differential screening of a cDNA library of RNA from gills. Northern blotting confirmed that these genes were all expressed only in the gills. In situ hybridization showed that the expression of these genes is limited to the endodermally derived columnar epithelium of the pharynx. PfG1 encodes a 42-kDa polypeptide containing sequence similar to D-domains, protein domains characteristic of extracellular proteins. Expression of PfG1 is localized in a delimited pattern along the columnar epithelium of the inner gill apparatus. Expression in the epibranchial ridge appears as two stripes running longitudinally in the epithelium just lateral of the midline. A stripe of expression also appears in a slightly posterior portion on the curve of each band of columnar epithelium on the pharyngeal surface of the secondary gill bars. The five other gill-specific genes, PfG2 to PfG6, encode a family of C-type lectin polypeptides that appear to be secreted proteins. PfG2 to PfG6 are also expressed in the columnar epithelium of the epibranchial ridge as two parallel stripes, but at the lateral margin of the ridge. One of the genes, PfG6, is additionally expressed in the innermost curve of the epithelium on the pharyngeal surface of each secondary gill bar. The localization of expression of PfPax1/9, a gill-specific transcription factor gene, was examined and shown to also be primarily in the endodermal columnar epithelium on the pharyngeal faces of the gill bars. On the secondary gill bars, where PfG1 and PfG6 are also expressed in the columnar epithelium, PfPax1/9 is expressed in the anterior and posterior portions but signal is not evident in the epithelium on the central, innermost curve of the gill bar. The anterior domain of PfPax1/9 expression is more extensive but overlaps the anterior domain of PfG1 expression, whereas its posterior domain of expression is more posterior and complementary to that of PfG6.

The endostyle is a pharyngeal organ for the internal filter feeding of urochordates, cephalochordates, and larval lamprey. This organ is also considered to be homologous to the follicular thyroid gland of higher vertebrates. Thyroglobulin (Tg) and thyroid peroxidase (TPO) are specifically expressed in the thyroid gland of higher vertebrates, and they play an important role in iodine metabolism for the synthesis of thyroid hormones. Previous histochemical observations showed that iodine-concentrating and peroxidase activities were detected in zones 7, 8, and 9 of the ascidian endostyle, suggesting that these zones contains cells that are equivalent to those in the vertebrate follicular thyroid. In order to investigate the molecular developmental mechanisms involved in the formation and function of the endostyle, with special reference to the evolution of the thyroid gland, in the present study, we isolated and characterized cDNA clones for TPO genes, CiTPO from Ciona intestinalis and HrTPO from Halocynthia roretzi. Northern blot and in situ hybridization analyses revealed that the expression of the ascidian TPO genes was restricted to zone 7, one of the elements equivalent to the thyroid. These results provide the first evidence at the gene expression level for shared function between a part of the ascidian endostyle and the vertebrate follicular thyroid gland. J. Exp. Zool. ( Mol. Dev. Evol. ) 285:158-169, 1999.

Expressed Sequence Tag (EST) research on the ascidian Halocynthia roretzi revealed that Hrsmad1/5, a homolog of smad genes, is expressed in H, roretzi eggs. A comparison of amino acid sequences of smad family members showed that the isolated clone was a homolog of smad1 and smad5 of vertebrates. A molecular phylogenetic analysis showed that Hrsmad1/5 was separated from the common ancestor with the group containing smad1 and smad5. A northern blot analysis showed that transcript of Hrsmad1/5 was abundant in the fertilized egg. The amount of the transcript remained constant until the gastrulae and then rapidly decreased at the neurulae. The spatial expression of Hrsmad1/5 was investigated by means of whole-mount in situ hybridization. Maternal transcripts of Hrsmad1/5 were detected in the entire fertilized egg. The signals were localized preferentially to the animal blastomeres of the 8-, 16-, 32- and 64-cell stages. The zygotic expression of Hrsmad1/5 started in prospective epidermal blastomeres in the animal hemisphere at the 64-cell stage but not in cells of the central nervous system, and it decreased rapidly around the neural-plate stage. At the tail-bud stage, signals were detected broadly all through the trunk and in a small part of the epidermis in the tail region. This is the first report of a maternal RNA that preferentially accumulates in the animal hemisphere in early ascidian embryos. Animal blastomeres of ascidian embryos differentiate mainly into epidermis in a cell-autonomous manner and partly differentiate into neural tissues by induction. The Hrsmad1/5 gene may play a role in the signal transduction process in epidermal precursor cells of ascidian embryos.

The endostyle is a pharyngeal organ for the internal filter feeding of urochordates, cephalochordates, and larval lamprey. This organ is also considered to be homologous to the follicular thyroid gland of higher vertebrates. Thyroglobulin (Tg) and thyroid peroxidase (TPO) are specifically expressed in the thyroid gland of higher vertebrates, and they play an important role in iodine metabolism for the synthesis of thyroid hormones. Previous histochemical observations showed that iodine-concentrating and peroxidase activities were detected in zones 7, 8, and 9 of the ascidian endostyle, suggesting that; these zones contains cells that are equivalent to those in the vertebrate follicular thyroid. In order to investigate the molecular developmental mechanisms involved in the formation and function of the endostyle, with special reference to the evolution of the thyroid gland, in the present study, we isolated and characterized cDNA clones for TPO genes, CiTPO from Ciona intestinalis and HrTPO from Halocynthia roretzi. Northern blot and in situ hybridization analyses revealed that; the expression of the ascidian TPO genes was restricted to zone 7, one of the elements equivalent to the thyroid. These results provide the first evidence at the gene expression level for shared function between a part of the ascidian endostyle and the vertebrate follicular thyroid gland. (C) 1999 Wiley-Liss, Inc.

Expressed Sequence Tag (EST) research on the ascidian Halocynthia roretzi revealed that Hrsmad1/5, a homolog of smad genes, is expressed in H. roretzi eggs. A comparison of amino acid sequences of smad family members showed that the isolated clone was a homolog of smad1 and smad5 of vertebrates. A molecular phylogenetic analysis showed that Hrsmad1/5 was separated from the common ancestor with the group containing smad1 and smad5. A northern blot analysis showed that transcript of Hrsmad1/5 was abundant in the fertilized egg. The amount of the transcript remained constant until the gastrulae and then rapidly decreased at the neurulae. The spatial expression of Hrsmad1/5 was investigated by means of whole-mount in situ hybridization. Maternal transcripts of Hrsmad1/5 were detected in the entire fertilized egg. The signals were localized preferentially to the animal blastomeres of the 8-, 16-, 32- and 64-cell stages. The zygotic expression of Hrsmad1/5 started in prospective epidermal blastomeres in the animal hemisphere at the 64-cell stage but not in cells of the central nervous system, and it decreased rapidly around the neural-plate stage. At the tail-bud stage, signals were detected broadly all through the trunk and in a small part of the epidermis in the tail region. This is the first report of a maternal RNA that preferentially accumulates in the animal hemisphere in early ascidian embryos. Animal blastomeres of ascidian embryos differentiate mainly into epidermis in a cell-autonomous manner and partly differentiate into neural tissues by induction. The Hrsmad1/5 gene may play a role in the signal transduction process in epidermal precursor cells of ascidian embryos.

The endostyle is a special organ in the pharynx of urochordates, cephalochordates and cyclostomes. During evolution of the primitive chordates, the endostyle was organized in their common ancestor(s) with a shift to internal feeding for extracting suspended food from the water. In addition, the endostyle has an iodine-concentrating activity and is therefore thought to be functionally homologous to the vertebrate thyroid gland. Human TITF1 and mouse titf1 are members of the Nkx-2.1/TTF-1 gene subfamily, which encode an NK-2 type homeodomain transcription factor. The genes are expressed in the thyroid gland and are essential for thyroid-specific structural gene expression. In the present study, we isolated cDNA clones for ascidian homologs of titf1 from Halocynthia roretzi and Ciona intestinalis, and examined whether the genes are expressed in the ascidian endostyle. Results clearly indicated that both the H. roretzi homolog Hrtitf1 and the C. intestinalis homolog Cititf1 are expressed specifically in the endostyle. The present finding therefore provide molecular evidence for the functional relationship between the ascidian endostyle and vertebrate thyroid gland. However, the genes are expressed in the supporting element regions but not in the putative iodine-concentrating regions of the endostyle.

The epithelium of the pharynx contributes to the formation of gills in hemichordates, urochordates, cephalochordates and primitive vertebrates, and is therefore a key structure for understanding developmental mechanisms underlying the establishment of chordate body plans. Pax1- and Pax9-related genes encode transcription factors which are expressed in the pharyngeal region of cephalochordates as well as in the vertebrate pharyngeal pouch epithelium that forms the thymus and parathyroid glands. To explore the molecular basis underlying the occurrence and modifications of the pharyngeal epithelium during evolution, we isolated cDNA clones for Pax1- and Pax9-related genes of urochordates (HrPax1/9 of Halocynthia roretzi and CiPax1/9 of Ciona intestinalis) and a hemichordate (PfPax1/9 of Ptychodera flava) from gill cDNA libraries. Each gene is present as a single copy per haploid genome. All of the cDNAs encode typical paired domains and octapeptides but not a homeodomain, as is also true of other Pax1- and Pax9-related genes. Molecular phylogenetic analysis based on comparison of the paired domain amino-acid sequences suggests that HrPax1/9, CiPax1/9 and PfPax1/9 belong to the Pax1/9 subfamily, and that they are descendants of a single precursor of Pax1/Pax9, Screening of HrPax1/9 cDNA clones yielded six different types of transcripts which were generated by alternative splicing. Northern blot, RT-PCR/Southern and in situ hybridization analyses revealed that HrPax1/9, CiPax1/9 and PfPax1/9 are not expressed during early embryogenesis but are expressed in the epithelia of differentiating gills, suggesting that these genes encode gill-specific transcription factors. The Pax1/9 genes therefore might provide the first developmental genetic corroboration of hypotheses of organ-level homology that unifies hemichordates, urochordates and cephalochordates.

The epithelium of the pharynx contributes to the formation of gills in hemichordates, urochordates, cephalochordates and primitive vertebrates, and is therefore a key structure for understanding developmental mechanisms underlying the establishment of chordate body plans. Pax1- and Pax9-related genes encode transcription factors which are expressed in the pharyngeal region of cephalochordates as well as in the vertebrate pharyngeal pouch epithelium that forms the thymus and parathyroid glands. To explore the molecular basis underlying the occurrence and modifications of the pharyngeal epithelium during evolution, we isolated cDNA clones for Pax1- and Pax9-related genes of urochordates (HrPax1/9 of Halocynthia roretzi and CiPax1/9 of Ciona intestinalis) and a hemichordate (PfPax1/9 of Ptychodera flava) from gill cDNA libraries. Each gene is present as a single copy per haploid genome. All of the cDNAs encode typical paired domains and octapeptides but not a homeodomain, as is also true of other Pax1- and Pax9-related genes. Molecular phylogenetic analysis based on comparison of the paired domain amino-acid sequences suggests that HrPax1/9, CiPax1/9 and PfPax1/9 belong to the Pax1/9 subfamily, and that they are descendants of a single precursor of Pax1/Pax9. Screening of HrPax1/9 cDNA clones yielded six different types of transcripts which were generated by alternative splicing. Northern blot, RT-PCR/Southern and in situ hybridization analyses revealed that HrPax1/9, CiPax1/9 and PfPax1/9 are not expressed during early embryogenesis but are expressed in the epithelia of differentiating gills, suggesting that these genes encode gill-specific transcription factors. The Pax1/9 genes therefore might provide the first developmental genetic corroboration of hypotheses of organ-level homology that unifies hemichordates, urochordates and cephalochordates.

The endostyle is a special organ in the pharynx of Urochordata, Cephalochordata, and Cyclostomata. It may have arisen in the common ancestor of these taxa, along with a shift to internal feeding for extracting suspended food from the water. In addition, the endostyle has a functional homology to the vertebrate thyroid gland. The endostyle is therefore one of the structures key to the understanding of the origin and evolution of chordates. In the present study, we isolated and characterized cDNA clones for four endo-style-specific genes, CiEnds1, CiEnds2, CiEnds3, and CiEnds4, of the ascidian Ciona intestinalis. Although the predicted amino acid sequences of the gene products CiENDS1, CiENDS2, and CiENDS3 showed no similarity to known proteins, their mean hydropathy profiles suggest that they are secretory proteins. In addition, CiENDS3 contained a unique repeat of 10 amino acids IR(QPCI)(RRPC)I]. CiEnds1 and CiEnds2 were expressed in zone 6, a protein-secreting glandular element of the endostyle, and CiEnds3 was expressed in zone 2, another secretory zone. CiEnds4, a cytoplasmic actin gene, was predominantly expressed in zones 3 and 5, which are supporting elements of the endostyle. The amino acid sequences of CiENDS1 and CiENDS2 resembled each other. In addition, they resembled a zone-6-specific gene product (HrENDS2) of another ascidian, Halocynthia roretzi. The results suggest that these genes are conserved among ascidian species, and therefore they las well as CiEnds3 for the protein with a unique motif) may be useful probes for further analyses of molecular mechanisms involved in endostyle development.

Maternally localized cytoplasmic determinants play important roles in the embryogenesis of many animals, including ascidians. Cytoplasmic determinants are particularly important in the determination of cell fates, and in the establishment of the embryonic axes. Ascidians, which show mosaic development, are good models for the study of maternal cytoplasmic determinants. Here we report the isolation and characterization of HrPOPK-1 (Halocynthia roretzi posterior protein kinase-1), a putative protein serine/threonine kinase. HrPOPK-1 cDNA was obtained from a Halocynthia roretzi fertilized egg cDNA library by screening for localized RNAs using whole-mount in situ hybridization. HrPOPK-1 mRNA is strongly localized at the posterior pole of embryos. The pattern of HrPOPK-1 mRNA localization during early embryogenesis is identical to that of HrWnt-5 in Halocynthia roretzi, and to those of the posterior end mark (pem) transcripts of Ciona savignyi. In addition, HrPOPK-1 shows zygotic expression in neural tissues at the tailbud stage. These results show that the temporal regulation of HrPOPK-1 transcription is complex.

The endostyle is a special organ in the pharynx of Urochordata, Cephalochordata, and Cyclostomata. It may have arisen in the common ancestor of these taxa, along with a shift to internal feeding for extracting suspended food from the water. In addition, the endostyle has a functional homology to the vertebrate thyroid gland. The endostyle is therefore one of the structures key to the understanding of the origin and evolution of chordates. In the present study, we isolated and characterized cDNA clones for four endostyle-specific genes, CiEnds1, CiEnds2, CiEnds3, and CiEnds4, of the ascidian Ciona intestinalis. Although the predicted amino acid sequences of the gene products CiENDS1, CiENDS2, and CiENDS3 showed no similarity to known proteins, their mean hydropathy profiles suggest that they are secretory proteins. In addition, CiENDS3 contained a unique repeat of 10 amino acids [R(QPCI)-(RRPC)I]. CiEnds1 and CiEnds2 were expressed in zone 6, a protein-secreting glandular element of the endostyle, and CiEnds3 was expressed in zone 2, another secretory zone. CiEnds4, a cytoplasmic actin gene, was predominantly expressed in zones 3 and 5, which are supporting elements of the endostyle. The amino acid sequences of CiENDS1 and CiENDS2 resembled each other. In addition, they resembled a zone-6-specific gene product (HrENDS2) of another ascidian, Halocynthia roretzi. The results suggest that these genes are conserved among ascidian species, and therefore they (as well as CiEnds3 for the protein with a unique motif) may be useful probes for further analyses of molecular mechanisms involved in endostyle development.

Ascidians show a highly determinate mode of development. In particular, components of the posterior-vegetal cytoplasm of fertilized eggs are responsible for the establishment of the embryonic axis. Recent studies have, however, also revealed significant roles of cell-cell interactions during embryogenesis. Proteins encoded by the Wntfamily of genes act as signals and have been shown to play important roles in a wide range of developmental processes. Here we have isolated and characterized an ascidian Wnt gene, HrWnt-5, from Halocynthia roretzi. HrWnt-5 mRNA is present in the vegetal cortex in unfertilized eggs. After fertilization, HrWnt-5mRNA moves to the equatorial region to form a crescent-like structure, after which the mRNA is concentrated in the posteriormost region of the embryo. This early pattern of HrWnt-5 mRNA localization coincides with another posterior-vegetally localized mRNA, pem, isolated from Ciona savignyi. In the gastrula, the zygotic HrWnt-5 mRNA is found in a variety of blastomeres, suggesting multiple roles of the gene.

Ascidians show a highly determinate mode of development. In particular, components of the posterior-vegetal cytoplasm of fertilized eggs are responsible for the establishment of the embryonic axis. Recent studies have, however, also revealed significant roles of cell-cell interactions during embryogenesis. Proteins encoded by the Wnt family of genes act as signals and have been shown to play important roles in a wide range of developmental processes. Here we have isolated and characterized an ascidian Wnt gene, HrWnt-5, from Halocynthia roretzi. HrWnt-5 mRNA is present in the vegetal cortex in unfertilized eggs. After fertilization, HrWnt-5 mRNA moves to the equatorial region to form a crescent-like structure, after which the mRNA is concentrated in the posteriormost region of the embryo. This early pattern of HrWnt-5 mRNA localization coincides with another posterior-vegetally localized mRNA, pem, isolated from Ciona savignyi. In the gastrula, the zygotic HrWnt-5 mRNA is found in a variety of blastomeres, suggesting multiple roles of the gene.

The endostyle is a special organ in the pharynx of Urochordata, Cephalochordata and Cyclostomata. This organ may have arisen in their common ancestor with a shift to internal feeding for extracting suspended food from the water. In addition, the endostyle has functional homology to the vertebrate thyroid gland. The endostyle is therefore another key structure in the understanding of the origin and evolution of chordates. Following a previous report of the pharyngeal gill-specific genes, we report here the isolation and characterization of cDNA clones for endostyle-specific genes HrEnds1 and HrEnds2 of the ascidian Halocynthia roretzi. These cDNA clones were obtained by differential screening of an endostyle cDNA library and a pharyngeal gill cDNA library with total endostyle cDNA probes. Both transcripts were abundant in the library; each represented about 10% of the cDNA clones of the library. The HrEnds1 transcript was small in size, about 600 bp in length. Although the predicted amino acid sequence of the gene product showed no similarity to known proteins, mean hydropathy profiles suggested that HrENDS1 is a type Ib protein or secreted protein. The HrEnds2 transcript was about 2.5 kb in length. Although the HrEnds2 gene product showed no sequence similarity to known proteins, mean hydropathy profiles suggested that HrENDS2 is a secreted protein. The transcripts of both genes were not detected in embryos, larvae and early juveniles but were evident in 1-month-old young adult after several compositional zones were organized in the endostyle. In situ hybridization revealed that distribution of transcripts of both genes was restricted to zone 6, the protein-secreting glandular element of the endostyle. These genes may be useful for further analysis of molecular mechanisms involved in endostyle development.

The most primitive chordates may have arisen with a shift to internal feeding through the use of the pharyngeal gill slits and endostyle for extracting suspended food from the water. Therefore, the pharyngeal gill and endostyle, in addition to notochord and nerve cord, are structures key to an understanding of the molecular developmental mechanisms underlying the origin and evolution of chordates. In this and a following study, isolation of cDNA clones for genes that are specifically expressed in the pharyngeal gill or endostyle in the ascidian Halocynthia roretzi was attempted. Differential screening of a pharyngeal gill cDNA library and an endostyle cDNA library with total pharyngeal-gill cDNA probes yielded cDNA clones for two pharyngeal gill-specific genes, HrPhG1 and HrPhG2. Northern blot analysis showed a 3.0-kb transcript of HrPhG1 and a 2.0-kb transcript of HrPhG2. Predicted amino acid sequences of the gene products suggested that both genes encode secretory proteins with no significant match to known proteins. In adults, both HrPhG1 and HrPhG2 genes were only expressed in the pharyngeal gill and not in other tissues including the endostyle, body-wall muscle, gonad, gut and digestive gland. HrPhG1 and HrPhG2 transcripts were undetectable in embryos and larvae, and were first detected in juveniles 3 days after initiation of metamorphosis. In situ hybridization revealed that the expression of HrPhG1 and HrPhG2 was restricted to differentiating pharyngeal-wall epithelium, with intense signals in the area surrounding the stigma or gill slit. These genes may serve as probes for further analyses of molecular mechanisms underlying the occurrence of pharyngeal gill and formation of gill slits during chordate evolution.