Tunicate muscle actin genes. Structure and organization as a gene cluster.

We have isolated and determined the complete nucleotide sequences of two genes, HrMA4a and HrMA2, which encode the same muscle actin protein of the tunicate Halocynthia roretzi. HrMA4a and HrMA2 contain three exons, and the genes have intron-exon splice junctions at the same positions. The 5' flanking region of HrMA4a gene contains several potential regulatory elements. A TATA box is located at -30 and a CArG box found in regulatory region of vertebrate muscle-specific genes is located at -116. Seven E-box consensus sequences (CANNTG) known as binding sites for vertebrate myogenic determination factors are found within a 500 base-pair portion of the 5' flanking region of HrMA4a gene. HrMA4a and HrMA2 are separated by 1600 bases in genomic DNA and transcribed in the same direction. In addition to these genes, we have identified three other actin genes encoding muscle-type actins. All five actin genes are located in a 30 x 10(3) base-pair region of the genome and aligned in the same direction. This is the first report of a cluster of "vertebrate-type" muscle actin genes. The consensus sequences of 5' flanking region are conserved among these five genes, suggesting that the expression of the genes is controlled coordinately. This may be advantageous for the accumulation of considerable amounts of actin proteins in rapidly developing embryos of this animal.

A gene encoding a new ONECUT class homeodomain protein in the ascidian Halocynthia roretzi functions in the differentiation and specification of neural cells in ascidian embryogenesis.

Genes encoding a novel group of homeodomain transcription factors, ONECUT class homeodomain proteins, have previously been isolated from vertebrate and insect. Among them, vertebrate HNF-6 is expressed in hepatocytes and the central nervous system during embryogenesis. Although the functions of HNF-6 in hepatocytes have been well studied, the functions of HNF-6 in the central nervous system remain unknown. In this study, we isolated HrHNF-6, which encodes a new ONECUT class homeodomain protein, from an ascidian, Halocynthia roretzi. HrHNF-6 mRNA was expressed exclusively in neural cells, just posterior to the expression of Hroth during embryogenesis. One of the functions of HrHNF-6 in neural cells is the activation of the expression of HrTBB2, the ascidian beta-tubulin gene. Another is the restriction of the expression of HrPax-258 (which is expressed in the neural tube), suggesting that HrHNF-6 functions in the specification of the neural tube. These results indicate that HrHNF-6 functions in the differentiation and regional specification of neural cells during ascidian embryogenesis.

Maternally localized RNA encoding a serine/threonine protein kinase in the ascidian, Halocynthia roretzi.

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.

HrWnt-5: a maternally expressed ascidian Wnt gene with posterior localization in early embryos.

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.

Isolation of cDNA clones for genes that are expressed in the tail region of the ascidian tailbud embryo.

An ascidian tailbud embryo is comprised of the anterior trunk and posterior tail. We constructed cDNA libraries of the tail region and trunk region of the ascidian Halocynthia roretzi. The screening of the tail library by tail single-stranded cDNA minus the trunk library RNA as a probe, yielded cDNA clones for genes that are expressed in the tail epidermis, visceral ganglion, trunk lateral cells, muscle cells, and certain regions of the tail. Among them, a cDNA clone for a gene designated HrPost-1 is described in detail. HrPost-1 encodes a novel, possible secreted protein of 238 amino acids. The expression of the gene is zygotic. HrPost-1 transcript was first evident in the posterior B-line blastomeres including muscle cells and endodermal strand cells of the gastrula-stage embryo, and the expression in these regions disappeared by the early tailbud stage. Around neurulation, the HrPost-1 transcript appeared in epidermal cells of the posterior-most region of the embryo. As development proceeded, the gene expression spread anteriorly in the epidermal cells of the neurula and tailbud embryo, and thus at the early-to-mid tailbud stage, HrPost-1 expression appeared to define the boundary between the trunk and tail epidermis. These results suggest that, in addition to tissue-specific genes, the activities of a set of region-specific genes are associated with tail formation in the ascidian embryo.

Identification of a gene encoding a typical gamma-carboxyglutamic acid domain in the tunicate Halocynthia roretzi.

We report the identification of a gene capable of encoding a novel Gla (gamma-carboxyglutamic acid) protein from the tunicate Halocynthia roretzi, a primitive member of the phylum Chordata. We call this new hypothetical protein Gla-RTK; it has a Gla domain typical of human vitamin K-dependent coagulation factors, a transmembrane domain, and a receptor tyrosine kinase domain. The receptor tyrosine kinase domain is very similar to the ARK (adhesion-related kinase) family of receptor tyrosine kinases. The ARK family includes Axl, Tyro3, and c-Mer. This gene also encodes a propeptide that binds to the human gamma-glutamyl carboxylase within a range of affinities observed for mammalian propeptides. The cDNA for this putative protein is found distributed throughout the oocyte and embryo but the cDNA is apparently not transcribed except during oogenesis. One of the most interesting aspects of this hypothetical protein is that its Gla domain is highly homologous to the Gla domain of Gas6, a ligand for Axl, while its receptor tyrosine kinase domain is highly homologous to Axl.

A complementary DNA for an ascidian embryonic nuclear antigen Hgv2 encodes a protein closely related to the amphibian histone-binding protein N1.

We have isolated and sequenced a cDNA clone encoding an ascidian embryonic nuclear protein, Hgv2. An insert about 2 kb long covered almost the entire length of 2.3-kb Hgv2 mRNA. The amino acid sequence of Hgv2 deduced from the cDNA sequence showed that this protein is related to the amphibian karyophilic histone-binding protein N1, which is thought to be involved in nucleosome assembly. Homology between these two proteins is evident from their extremely similar amino acid compositions and hydropathy profiles. In addition, Hgv2 protein has sequences strikingly similar to the nuclear targeting signal of N1. This is therefore the first report of molecular cloning of a homologue of N1 in non-amphibian species. Putative histone-binding domains of N1 are composed of two acidic residue-rich clusters. Hgv2 polypeptide contains two highly acidic regions, but amino acid sequences of the regions are not conserved. Since Hgv2 protein exists in nuclei of every embryonic cell but disappears from nuclei of metamorphosed juvenile tissues, this protein may function as a nucleosome assembly factor during rapid embryonic cell divisions.

Expression of actin genes in the arrow worm Paraspadella gotoi (Chaetognatha).

Arrow worms (the phylum Chaetognatha), one of the major marine planktonic animals, exhibit features characteristic to both deuterostomes and protostomes, and their ancestry therefore remains unknown. As the first step to elucidate the molecular bases of arrow worm phylogeny, physiology and embryology, we isolated cDNA clones for three different actin genes (PgAct1, PgAct2 and PgAct3) from the benthic species Paraspadella gotoi, and examined their expression patterns in adults and juveniles. The amino acid sequences of the three actins resembled each other, with identities ranging from 86% to 92%. However, the patterns of the spatial expression of the genes were independent. The PgAct1 gene might encode a cytoplasmic actin and was expressed in oogenic cells, spermatogenic cells, and cells in the ventral ganglion. The PgAct2 and PgAct3 genes encoded actins of divergent types. The former was expressed in well-developed muscle of the head (gnathic) region and trunk muscle cells, whereas the latter was expressed in muscle of the trunk and tail regions and oogenic cells. These results suggest that, similarly to other metazoans, the chaetognath contains multiple forms of actins, which are expressed in various manners in the adult and juvenile arrow worm.

Isolation of an early neural maker gene abundantly expressed in the nervous system of the ascidian, Halocynthia roretzi.

Ascidian tadpole larvae possess a primitive nervous system, which is a prospective prototype of the chordate nervous system. It is composed of relatively few cells but sufficient for complex larval behavior. Here we report on HrETR-1, a gene zygotically expressed in a large proportion of the developing neural cells of the ascidian, Halocynthia roretzi. HrETR-1 is an early neural marker which can be used for analyzing neural differentiation. HrETR-1 expression intensified in most neural cells of genes isolated to date, in both central and peripheral nervous systems including palps as early as the 110-cell stage. Using this gene as a probe, we characterized neural cells in the nervous system as well as confirming their origins. Also, we recognized three types of peripheral epidermal neurons which presumably correlate to the larval neurons previously reported for another ascidian. Among these, five bilateral neurons located in the anterior region of the trunk appeared to be derived from a8.26 blastomeres.

Ascidian Wnt-5 gene is involved in the morphogenetic movement of notochord cells.

Wnt proteins play important roles in many developmental events. Wnts are divided into two groups according to biological function. The Wnt-5a class proteins function in morphogenetic movement during embryogenesis. Previously, a Wnt-5 homolog has been isolated from the ascidian, Halocynthia roretzi. HrWnt-5 is expressed in the notochord until the tail-bud stage, implying a role in the notochord. In this study, the function of HrWnt-5 was investigated. When HrWnt-5 mRNA was injected into fertilized eggs, the embryos showed morphologic defects at around the neurula stage. The anterior-posterior axis was shorter than in control embryos. These defects were caused by the abnormal movement of notochord cells. However, the overexpression of HrWnt-5 mRNA did not affect the differentiation of tissues, suggesting that HrWnt-5 solely regulates the morphogenetic movement. Although endogenous HrWnt-5 is expressed in the notochord, the overexpression of HrWnt-5 mRNA caused the defects, suggesting that the amount of HrWnt-5 mRNA in the notochord is strictly regulated. These results suggest that HrWnt-5 regulates the morphogenetic movement of notochord cells during ascidian embryogenesis.

Ascidian Wnt-7 gene is expressed exclusively in the tail neural tube of tailbud embryos.

In vertebrate embryogenesis, many Wnt genes are expressed in the neural tube and play important roles in regional specifications. There are many subfamilies of Wnt, and each subfamily shows distinct expression patterns in the neural tube. Ascidian larvae have a dorsal hollow neural tube similar to that of vertebrates. To date, the degree of correspondence between regionality of the neural tubes of ascidians and vertebrates remains unclear. To compare cellular differences in neural tubes, Wnt genes can be used as molecular probes. We report here that a new member of the ascidian Wnt gene family, HrWnt-7, was expressed in the tail neural tube at the early tailbud stage. Moreover, in cross-section, HrWnt-7 was expressed in the dorsal and ventral ependymal cells.

Phylogenetic Relationships between Solitary and Colonial Ascidians, as Inferred from the Sequence of the Central Region of their Respective 18S rDNAs.

Ascidians (tunicates) are primitive chordates. In spite of their elevated phylogenetic position in the animal kingdom, ascidians have evolved a varied reproductive repertoire; some of them live as individuals (solitary ascidians), while others form colonies (colonial ascidians). Colonial ascidians propagate asexually by budding and strobilation, and they have an extensive capacity for regeneration. However, the orthodox taxonomic classification of ascidians categorizes them into two major groups (the orders Enterogona and Pleurogona), irrespective of their solitary or colonial life style. To examine whether the orthodox classification of ascidians is substantiated by molecular phylogeny, the complete nucleotide sequence of a region of about 1000 base pairs in the central part of their respective 18S rDNAs was determined, and the sequences were compared among five solitary and three colonial ascidians. The phylogenetic tree deduced from these results suggests that the three species of Enterogona and the five species of Pleurogona examined form discrete and separate groups irrespective of their potential to form colonies. Therefore, a solitary or colonial life style is likely to have developed independently after the divergence of the two major groups of ascidians.

Binary specification of nerve cord and notochord cell fates in ascidian embryos.

In the ascidian embryo, the nerve cord and notochord of the tail of tadpole larvae originate from the precursor blastomeres for both tissues in the 32-cell-stage embryo. Each fate is separated into two daughter blastomeres at the next cleavage. We have examined mechanisms that are responsible for nerve cord and notochord specification through experiments involving blastomere isolation, cell dissociation, and treatment with basic fibroblast growth factor (bFGF) and inhibitors for the mitogen-activated protein kinase (MAPK) cascade. It has been shown that inductive cell interaction at the 32-cell stage is required for notochord formation. Our results show that the nerve cord fate is determined autonomously without any cell interaction. Presumptive notochord blastomeres also assume a nerve cord fate when they are isolated before induction is completed. By contrast, not only presumptive notochord blastomeres but also presumptive nerve cord blastomeres forsake their default nerve cord fate and choose the notochord fate when they are treated with bFGF. When the FGF-Ras-MAPK signaling cascade is inhibited, both blastomeres choose the default nerve cord pathway, supporting the results of blastomere isolation. Thus, binary choice of alternative fates and asymmetric division are involved in this nerve cord/notochord fate determination system, mediated by FGF signaling.

Two pathways of maternal RNA localization at the posterior-vegetal cytoplasm in early ascidian embryos.

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.

A maternal RNA encoding smad1/5 is segregated to animal blastomeres during ascidian development.

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.

Novel genes expressed differentially in ascidians with alternate modes of development.

We have used a subtractive procedure to isolate cDNA clones encoding genes expressed differentially in ascidian species with alternate modes of development. The ascidians used in this study were Molgula occulta, which develops a tailed (urodele) larva, and Molgula occulta, which develops a tailless (anural) larva. Two of the identified clones, Uro-2 and Uro-11, are described. Southern blots show that the Uro-2 and Uro-11 genes are present in both species, but the corresponding mRNAs are expressed preferentially in the urodele species. In situ hybridization showed that Uro-2 and Uro-11 transcripts accumulate in small oocytes during oogenesis. The maternal Uro-2 and Uro-11 transcripts were distributed throughout the oocyte cytoplasm. Transcript concentrations declined during vitellogenesis, but mature eggs still contain detectable levels of Uro-2 and Uro-11 mRNA. After fertilization, the maternal Uro-2 and Uro-11 transcripts were localized in the ectoplasm of uncleaved zygotes and mostly entered the ectoderm cells during cleavage. The Uro-2 gene appears to produce only maternal transcripts. In contrast, the Uro-11 gene may also produce zygotic transcripts, which accumulate between gastrulation and neurulation in posterior epidermis, neural and tail muscle cells. Zygotic expression of the Uro-11 gene was not detected in embryos of the anural species. The deduced amino acid sequences of the Uro-2 and Uro-11 cDNAs suggest that they encode novel basic proteins with distinctive structural features. The predicted Uro-2 protein contain, a leucine zipper motif, suggesting that it may dimerize with another protein. The predicted Uro-11 protein contains a nuclear localization signal, a region with similarity to part of the DNA-binding motif in the bacterial histone-like HU and IHF proteins, 12 repeats of the proposed DNA-binding motif S(T)PXX, and a potential zinc finger of the C6 or C6H2 class, suggesting that it may be a DNA-binding protein. The Uro-2 and Uro-11 proteins are candidates for regulatory factors involved in the evolutionary transition from urodele to anural development.

Molecular cloning and expression of a novel homeobox gene AHox1 of the ascidian, Halocynthia roretzi.

We have isolated a novel ascidian homeobox gene, designated AHox1, by screening the genomic DNA of Halocynthia roretzi with the Bombyx mori Antennapedia type homeobox as a probe. The AHox1 gene encodes a protein that consists of 741 amino acids. The homeobox of AHox1 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 AHox1 is one of the most diverged homeobox genes so far characterized. Northern blot hybridization with an AHox1 probe showed the presence of single transcripts approximately 2.8 kb in length in larvae, juveniles and some adult tissues. The expression of AHox1 is scarcely detected during the course of early development but it increases to a moderate level at the larval stage. After metamorphosis, the level of AHox1 expression increases as development proceeds. In situ hybridization to the juvenile 7 days after metamorphosis showed that the site of AHox1 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 AHox1. In gonad, body wall muscle and pharyngeal epithelium, the expression of AHox1 is relatively weak. These results suggest that AHox1 is primarily expressed in the tissues of endodermal origin and that the gene expression may be associated with differentiation of the endodermal tissues.

Expression of pharyngeal gill-specific genes in the ascidian Halocynthia roretzi.

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.

Expression of endostyle-specific genes in the ascidian Halocynthia roretzi.

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.