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Hox cluster genes play crucial roles in development of the metazoan antero-posterior axis. Functions of Hox genes in patterning the central nervous system and limb buds are well known. They are also expressed in chordate endodermal tissues, where their roles in endodermal development are still poorly understood. In the invertebrate chordate, Ciona intestinalis, endodermal tissues are in a premature state during the larval stage, and they differentiate into the digestive tract during metamorphosis. In this study, we showed that disruption of a Hox gene, Ci-Hox10, prevented intestinal formation. Ci-Hox10-knock-down larvae displayed defective migration of endodermal strand cells. Formation of a protrusion, which is important for cell migration, was disrupted in these cells. The collagen type IX gene is a downstream target of Ci-Hox10, and is negatively regulated by Ci-Hox10 and a matrix metalloproteinase ortholog, prior to endodermal cell migration. Inhibition of this regulation prevented cellular migration. These results suggest that Ci-Hox10 regulates endodermal strand cell migration by forming a protrusion and by reconstructing the extracellular matrix.

Background Hox gene clusters with at least 13 paralog group (PG) members are common in vertebrate genomes and in that of amphioxus. Ascidians, which belong to the subphylum Tunicata (Urochordata), are phylogenetically positioned between vertebrates and amphioxus, and traditionally divided into two groups: the Pleurogona and the Enterogona. An enterogonan ascidian, Ciona intestinalis (Ci), possesses nine Hox genes localized on two chromosomes; thus, the Hox gene cluster is disintegrated. We investigated the Hox gene cluster of a pleurogonan ascidian, Halocynthia roretzi (Hr) to investigate whether Hox gene cluster disintegration is common among ascidians, and if so, how such disintegration occurred during ascidian or tunicate evolution. Results Our phylogenetic analysis reveals that the Hr Hox gene complement comprises nine members, including one with a relatively divergent Hox homeodomain sequence. Eight of nine Hr Hox genes were orthologous to Ci-Hox1, 2, 3, 4, 5, 10, 12 and 13. Following the phylogenetic classification into 13 PGs, we designated Hr Hox genes as Hox1, 2, 3, 4, 5, 10, 11/12/13.a, 11/12/13.b and HoxX. To address the chromosomal arrangement of the nine Hox genes, we performed two-color chromosomal fluorescent in situ hybridization, which revealed that the nine Hox genes are localized on a single chromosome in Hr, distinct from their arrangement in Ci. We further examined the order of the nine Hox genes on the chromosome by chromosome/scaffold walking. This analysis suggested a gene order of Hox1, 11/12/13.b, 11/12/13.a, 10, 5, X, followed by either Hox4, 3, 2 or Hox2, 3, 4 on the chromosome. Based on the present results and those previously reported in Ci, we discuss the establishment of the Hox gene complement and disintegration of Hox gene clusters during the course of ascidian or tunicate evolution. Conclusions The Hox gene cluster and the genome must have experienced extensive reorganization during the course of evolution from the ancestral tunicate to Hr and Ci. Nevertheless, some features are shared in Hox gene components and gene arrangement on the chromosomes, suggesting that Hox gene cluster disintegration in ascidians involved early events common to tunicates as well as later ascidian lineage-specific events.

Nodal signaling plays an essential role in the establishment of left–right asymmetry in various animals. However, it is largely unknown how Nodal signaling is involved in the establishment of the left–right asymmetric morphology. In this study, the role of Nodal signaling in the left–right asymmetric ocellus formation in the ascidian, Ciona intestinalis was dealt with. During the development of C. intestinalis , the ocellus pigment cell forms on the midline and moves to the right side of the midline. Then, the photoreceptor cells form on the right side of the sensory vesicle (SV). Ci-Nodal is expressed on the left side of the SV in the developing tail bud embryo. When Nodal signaling is inhibited, the ocellus pigment cell form but remain on the midline, and expression of marker genes of the ocellus photoreceptor cells is ectopically detected on the left side as well as on the right side of the SV in the larva. Furthermore, Ci-Rx , which is essential for the ocellus differentiation, turns out to be negatively regulated by the Nodal signaling on the left side of the SV, even though it is required for the right-sided photoreceptor formation. These results indicate that Nodal signaling controls the left–right asymmetric ocellus formation in the development of C. intestinalis .

Left-right asymmetric expression of Pitx as well as of Nodal has been observed in some ascidian species, but a mechanism that regulates the asymmetric expression of Pitx remains largely unclear. We addressed the transcription regulatory mechanism of the left-right asymmetric expression of Pitx gene in the ascidian, Ciona intestinalis. We first identified an intronic enhancer that drives Ci-Pitx left-sided expression in the epidermis and found that a single FoxH1 binding site present in this enhancer is essential for its activity. Also, we have shown that the enhancer requires Nodal signaling to drive the Ci-Pitx expression in the left epidermis. In C. intestinalis, left-sided expression of the Nodal gene has not been reported so far. We have confirmed that Ci-Nodal is expressed in the left epidermis of embryos at the tailbud stage only when they are allowed to develop within the chorion. Then, to test the importance of the FoxH1 binding site, we carried out knock down experiments using morpholino antisense oligonucleotides against Ci-FoxHa, the only FoxH gene that is expressed in the same stage as the left-sided expression of Ci-Pitx is observed. Knocking down of the function of Ci-FoxHa led to the down regulation of the expression of Ci-Pitx in the left epidermis. The present results suggest that the regulatory mechanism controlling the left-right asymmetric expression of the Pitx genes is well conserved between C. intestinalis and vertebrates.

Comparative studies on expression patterns of developmental genes along the anterior–posterior axis of the embryonic central nervous system (CNS) between vertebrates and ascidians led to the notion of “tripartite organization,” a common ground plan of the CNS, consisting of the anterior, central and posterior regions expressing Otx, Pax2/5/8 and Hox genes, respectively. In ascidians, however, descriptions and interpretations about expression of the developmental genes regarded as region specific have become not necessarily consistent. To address this issue, we examined detailed expression of key developmental genes for the ascidian CNS, including Otx, Pax2/5/8a, En, Fgf8/17/18, Dmbx, Lhx3 and Hox genes, in the CNS around the junction of the trunk and tail of three different tailbud-stage embryos of Ciona intestinalis, employing double-fluorescence in situ hybridization, followed by staining with DAPI to precisely locate expressing cells for each gene. Based on these observations, we have constructed detailed gene expression maps of the region at the tailbud stages. Our analysis shows that expression of several genes regarded as markers for specific domains in the ascidian CNS changes dynamically within a relatively short period. This motivates us to revisit to the tripartite ground plan and the origin of the midbrain–hindbrain boundary (MHB) region.

Ascidian embryos develop with a fixed cell lineage into simple tadpoles. Their lineage is almost perfectly conserved, even between the evolutionarily distant species Halocynthia roretzi and Ciona intestinalis,which show no detectable sequence conservation in the non-coding regions of studied orthologous genes. To address how a common developmental program can be maintained without detectable cis-regulatory sequence conservation, we compared in both species the regulation of Otx, a gene with a shared complex expression pattern. We found that in Halocynthia, the regulatory logic is based on the use of very simple cell line-specific regulatory modules, the activities of which are conserved,in most cases, in the Ciona embryo. The activity of each of these enhancer modules relies on the conservation of a few repeated crucial binding sites for transcriptional activators, without obvious constraints on their precise number, order or orientation, or on the surrounding sequences. We propose that a combination of simplicity and degeneracy allows the conservation of the regulatory logic, despite drastic sequence divergence. The regulation of Otx in the anterior endoderm by Lhx and Fox factors may even be conserved with vertebrates.

A screen for the systematic identification of cis-regulatory elements within large (>100 kb) genomic domains containing Hox genes was performed by using the basal chordate Ciona intestinalis . Randomly generated DNA fragments from bacterial artificial chromosomes containing two clusters of Hox genes were inserted into a vector upstream of a minimal promoter and lacZ reporter gene. A total of 222 resultant fusion genes were separately electroporated into fertilized eggs, and their regulatory activities were monitored in larvae. In sum, 21 separable cis-regulatory elements were found. These include eight Hox linked domains that drive expression in nested anterior–posterior domains of ectodermally derived tissues. In addition to vertebrate-like CNS regulation, the discovery of cis-regulatory domains that drive epidermal transcription suggests that C. intestinalis has arthropod-like Hox patterning in the epidermis.

Homeobox-containing genes play crucial roles in various developmental processes, including body-plan specification, pattern formation and cell-type specification. The present study searched the draft genome sequence and cDNA/EST database of the basal chordate Ciona intestinalis to identify 83 homeobox-containing genes in this animal. This number of homeobox genes in the Ciona genome is smaller than that in the Caenorhabditis elegans, Drosophila melanogaster, human and mouse genomes. Of the 83 genes, 76 have possible human orthologues and 7 may be unique to Ciona. The ascidian homeobox genes were classified into 11 classes, including Hox class, NK class, Paired class, POU class, LIM class, TALE class, SIX class, Prox class, Cut class, ZFH class and HNF1 class, according to the classification scheme devised for known homeobox genes. As to the Hox cluster, the Ciona genome contains single copies of each of the paralogous groups, suggesting that there is a single Hox cluster, if any, but genes orthologous to Hox7, 8, 9 and 11 were not found in the genome. In addition, loss of genes had occurred independently in the Ciona lineage and was noticed in Gbx of the EHGbox subclass, Sax, NK3, Vax and vent of the NK class, Cart, Og9, Anf and Mix of the Paired class, POU-I, III, V and VI of the POU class, Lhx6/7 of the LIM class, TGIF of the TALE class, Cux and SATB of the Cut class, and ZFH1 of the ZFH class, which might have reduced the number of Ciona homeobox genes. Interestingly, one of the newly identified Ciona intestinalis genes and its vertebrate counterparts constitute a novel subclass of HNF1 class homeobox genes. Furthermore, evidence for the gene structures and expression of 54 of the 83 homeobox genes was provided by analysis of ESTs, suggesting that cDNAs for these 54 genes are available. The present data thus reveal the repertoire of homeodomain-containing transcription factors in the Ciona genome, which will be useful for future research on the development and evolution of chordates.

Three groups of pepsinogens exist in vertebrates, namely, pepsinogen A, pepsinogen C, and prochymosin, which are produced at different developmental stages. In the chicken, prochymosin is expressed only in the embryonic stage, while pepsinogens A and C are secreted from adult chicken proventricular (glandular stomach) mucosa. In order to understand the mechanism of transcriptional regulation of these genes, we have cloned the genes encoding chicken pepsinogens A and C and analyzed the sequences possibly involved in their regulation. 5'-Upstream sequences of both genes contain putative binding motifs for transcription factors such as GATA, Sox, and HNF-3 beta, which are expressed in the chicken gut epithelium. Moreover, we found seven putative binding motifs for human MZF-1 in intron 8 of pepsinogen A gene. These transcription factors may act as regulators of expression of chicken pepsinogen genes.

Animals in each subgroup of the phylum Chordata exhibit a similar process by which they form a tubular central nervous system (CNS). However, little is known about spatial relationship among the CNSs of chordates; vertebrates, cephalochordates and urochordates (tunicates). Ascidians constitute a major animal group in the subphylum Urochordata. In the present study, we examined the expression patterns of labial and orthodenticle related genes of the ascidian, Halocynthia roretzi, in the developing larval CNS. These homeobox genes exhibited region-specific expression patterns that are strikingly similar to those of murine Hoxb-1 and Otx2. The regionalization as characterized by the expression of these genes supports the division of the ascidian larval CNS suggested by the previous morphological studies. Furthermore, conservation of the expression pattern of the homeobox genes suggests that such regionalization occurred in the CNS of a putative common ancestor of chordates.

The horizontal transfer of genes between distantly related organisms is undoubtedly a major factor in the evolution of novel traits. Because genes are functionless without expression, horizontally transferred genes must acquire appropriate transcriptional regulations in their recipient organisms, although the evolutionary mechanism is not known well. The defining characteristic of tunicates is the presence of a cellulose containing tunic covering the adult and larval body surface. Cellulose synthase was acquired by horizontal gene transfer from Actinobacteria. We found that acquisition of the binding site of AP-2 transcription factor was essential for tunicate cellulose synthase to gain epidermal-specific expression. Actinobacteria have very GC-rich genomes, regions of which are capable of inducing specific expression in the tunicate epidermis as the AP-2 binds to a GC-rich region. Therefore, the actinobacterial cellulose synthase could have been potentiated to evolve its new function in the ancestor of tunicates with a higher probability than the evolution depending solely on a spontaneous event.

Pitx genes play important roles in a variety of developmental processes in vertebrates. In an ascidian species, Halocynthia roretzi, Hr-Pitx, the only Pitx gene of this species, has been reported to be expressed in the left epidermis at the tailbud stage. In the present study, first, we have shown that Hr-Pitx is also expressed in the papilla-forming region at the neurula to tailbud stages, and then we addressed transcription regulatory mechanisms for the expression of Hr-Pitx in the papilla-forming region. We have identified the genomic region ranging from 850 to 1211 bp upstream from the translation start site of the Hr-Pitx gene as an enhancer region that drives the transcription of Hr-Pitx in the papilla-forming region. Within the enhancer region, putative transcriptional factor binding sites for Otx as well as Fox were shown to be required for its activity. Finally, we carried out knocking down experiments of Hr-Otx function using an antisense morpholino oligonucleotide, in which the knocking down of Hr-Otx function resulted in reduction of the enhancer activity and loss of the expression of Hr-Pitx in the papilla-forming region. In Xenopus laevis, it has been reported that Pitx genes are expressed downstream of Otx function during development of the cement gland, an adhesive organ of its larva. Taken together, it is suggested that the expression regulatory mechanism of Pitx, involving Otx as the upstream gene, in the developing adhesive organ is conserved between ascidians and vertebrates.

Embryonic chick pepsinogen (ECPg) is one of the pepsinogen isozymogens and its expression is restricted to epithelial cells of the embryonic chick proventriculus (glandular stomach). To examine whether DNA methylation is involved in the regulation of organ-specific and developmental stage-specific expression of ECPg gene, we analyzed the extent of methylation of ECPg gene in normal embryonic and hatched chick organs using methylation-sensitive restriction enzymes. In the proventriculus some CCGG sites underwent demethylation in the gene region after the onset of transcription of the ECPg gene. By contrast, these sites were kept methylated throughout the development in the other organs which do not express ECPg gene. GCGC sites in the gene region became methylated in organs which do not express the ECPg gene, after the initiation of transcription of the ECPg gene in the proventriculus. In the proventriculus, GCGC sites, which were methylated in other organs, were kept unmethylated throughout the development. The methylation state of CpG sites showed no change in the proventriculus of a chick 2 weeks after hatching when the expression of the ECPg gene had completely ceased. The data presented here demonstrate that the DNA methylation is involved in the regulation of organ-specific expression, but stage-specific expression might be brought about by some other mechanisms.

In animals, region specific morphological characters along the anteroposterior axis are controlled by a number of developmental genes, including Hox genes encoding homeodomain transcription factors. Although Hox genes have been regarded to play a key role in the evolution of morphological diversity, as well as in the establishment of the body plan, little is known about the function of Hox genes in invertebrates, except for in insects and nematodes. The present study addresses the role of Hox genes in body patterning during the larval development of the ascidian Ciona intestinalis conducting knockdown experiments of the seven Hox genes expressed during embryogenesis. Experimental results have demonstrated that Ci-Hox12 plays an important role in tail development through the maintenance of expression of Ci-Fgf8/17/18 and Ci-Wnt5 in the tail tip epidermis. Additionally, it has been shown that Ci-Hox10 is involved in the development of GABAergic neurons in the dorsal visceral ganglion. Surprisingly, knockdown of Ci-Hox1, Ci-Hox2, Ci-Hox3, Ci-Hox4 and Ci-Hox5 did not give rise to any consistent morphological defects in the larvae. Furthermore, expression of neuronal marker genes was not affected in larvae injected with MOs against Ci-Hox1, Ci-Hox3 or Ci-Hox5. In conclusion, we suggest that the contribution of Hox genes to the larval development of the ascidian C. intestinalis might be limited, despite the fact that Ci-Hox10 and Ci-Hox12 play important roles in neuronal and tail development.

The Hox genes and its evolutionary sister, the ParaHox genes, are widely distributed among animals. Although it has been expected that hemichordates and echinoderms have a single set of Hox genes and most likely a single set of ParaHox genes, it is not known whether the ortholog of Hox8 is absent in hemichordates, and in turn, consensus view about Hox/ParaHox gene complements in hemichordates has not been established. In this study, we isolated either complete or nearly complete coding sequences of 12 Hox genes, including the ortholog of the Hox8 that has not been reported in the previous studies, and three ParaHox genes from the recently discovered indirect-developing acorn worm, Balanoglossus simodensis. Our data suggest that the ancestral hemichordate had intact complements of ambulacrarian prototypical Hox and ParaHox genes, consisting of 12 and three members, respectively.

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 metamorphosis, 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.

CUT class homeobox genes, including CUX/CASP, ONECUT, SATB and COMPASS family genes, are known to exhibit diverse features in the homeodomain and the domain architecture. Furthermore, the intron/exon organization of CUX/CASP is different between vertebrates and protostomes, and SATB genes are only known for vertebrates, whereas COMPASS genes have only been found in protostomes. These observations suggest a complex evolutionary history for the CUT class homeobox genes, but the evolution of CUT class homeobox genes in the lineage to vertebrates remained largely unknown. To obtain clearer insights into this issue, we searched the genome of amphioxus, Branchiostoma floridae, a lower chordate, for CUT class homeobox genes by extensive BLAST survey and phylogenetic analyses. We found that the genome of Branchiostoma floridae encodes each single orthologue of CUX/CASP, ONECUT, and COMPASS, but not the SATB gene, and one atypical CUT gene likely specific to this species. In addition, the genomic structure of the amphioxus CUX/CASP gene turned out to be protostome-type, but not vertebrate-type. Based on these observations, we propose a model in which SATB is suggested to evolve at the expense of COMPASS and this change, together with the structural change in CUX/CASP, is supposed to take place in the lineage to vertebrates after divergence of the amphioxus and vertebrate ancestors. The present study provides an example of dramatic evolution among homeobox gene groups in the vertebrate lineage and highlights the ancient character of amphioxus, retaining genomic features shared by protostomes.

The epidermis of an ascidian larva derived from animal-hemisphere cells is regionalized along the anterior-posterior (AP) axis through inductive signals emanating from vegetal-hemisphere cells in early stages of the development. Previously, we showed by blastomere isolation and ablation experiments that the contact between the animal and vegetal hemispheres until the 32-cell stage is necessary for the proper AP patterning of the epidermis in the tailbud-stage embryo. We here addressed the patterning mechanism of the posteriormost epidermis using a tail-tip epidermis marker, HrTT-1. Employing blastomere isolation and ablation experiments along with knockdown of a master regulator gene for posterior mesoderm, we have demonstrated that presence of the posterior vegetal cells after the 32-cell stage is necessary for the expression of HrTT-1. To explore the timing and nature of the influence of the posterior vegetal cells, we treated the embryos with FGF signaling inhibitors at various developmental stages and found that HrTT-1 expression was lost from embryos treated with the inhibitors from stages earlier than the late neurula stage, just prior to the onset of HrTT-1 expression but not after the initial tailbud stage, at which the expression of HrTT-1 had started. In embryos lacking HrTT-1 expression, the expression domain of Hrcad, which would otherwise be localized anterior to that of HrTT-1, expanded to the tail-tip. These results suggest that FGF signaling from the neurula to initial tailbud stages is necessary for the initiation but not maintenance of HrTT-1 expression in the tail-tip epidermis. The contact with posterior vegetal cells until and after the 32-cell stage may be required for FGF signaling to occur in the posterior tail, which in turn regionalizes the tail-tip epidermal territory.

Ascidians, belonging to the subphylum Urochordata, the earliest branch from the lineage to the vertebrates, exhibit a prototypical morphogenesis of chordates in the larval development, although they subsequently metamorphose into adults with a unique body structure. Recent draft genome analysis of the ascidian Ciona intestinalis has identified 9 Hox genes, which, however, have been located on five scaffolds. Similarly, expression patterns of Ciona Hox genes are largely unknown, although some data have been available for a few Hox member genes. Thus, the cluster structure and colinearity of Hox genes are still an enigma in C. intestinalis . To address these issues, we used fluorescence in situ hybridization and whole-mount in situ hybridization techniques and examined the genomic organization and spatiotemporal expression of all Hox as well as extended Hox member genes (Evx and Mox) of C. intestinalis . We found that seven of nine Ciona Hox genes are located on a single chromosome with some ordering exceptions, and the other genes, including Evx and Mox, are located on three other chromosomes. Some Ciona Hox genes, if not all, exhibited spatially coordinated expression within the larval central nervous system and the gut of the juvenile. In light of these observations, we suggest that the cluster organization and colinearity of the Hox genes are under dispersing and disintegrating conditions in C. intestinalis .

The ascidian larva is often regarded as an organism close to the ancestral form of chordates, while it is generally accepted that the Spemann's organizer is absent from ascidian embryos. Not is one of the genes expressed in the organizer to execute functions in vertebrate embryos. To address the extent of conservation of Not gene expression among ascidians and vertebrates, we examined the structure and developmental expression of Not of the two distantly related ascidian species, Halocynthia and Ciona. Putative ascidian Not proteins were noted by the absence of one of the two motifs conserved among Not proteins of sea urchin and vertebrates. Analysis by in situ hybridization revealed that Not gene expression of ascidians could be categorized into three types: expression likely to be conserved between ascidians and vertebrates, that probably unique to ascidians, and that specific to ascidian species. Expression of ascidian Not in the posterior end of the tail as well as the notochord and a small part of the anterior neural tube at the tailbud stage is reminiscent of the expression of the vertebrate counterparts in the tailbud, which is regarded as a continuation of the organizer and the pineal gland, respectively. The expression of Not in the epidermis precursors during cleavage stage may be unique to ascidians. In the light of the present findings, evolutionary aspects of Not genes are discussed.

The draft genome of the ascidian Ciona intestinalis has been sequenced. Mapping of the genome sequence to the Ciona 14 haploid chromosomes is essential for future studies of the genome-wide control of gene expression in this basal chordate. Here we describe an efficient protocol for fluores-cent in situ hybridization for mapping genes to the Ciona chromosomes. We demonstrate how the locations of two BAC clones can be mapped relative to each other. We also show that this method is efficient for coupling two so-far independent scaffolds into one longer scaffold when two BAC clones represent sequences located at either end of the two scaffolds.

Solitary ascidian tadpole larvae develop two types of black pigment cells in the major sensory organs of the brain. Such pigment cells have been demonstrated to express the melanogenic genes, tyrosinase and Tyrp/TRP (tyrosinase-related protein). To understand the genetic and developmental mechanisms underlying the differentiation of chordate pigment cells, we examined the function of the promoter region of Tyrp/TRP gene, an ascidian (Halocynthia roretzi) tyrosinase family gene. The expression of the gene in pigment cell lineage starts at the early-mid gastrula stages. To identify the transcriptional regulatory region of the gene allowing cell-type-specific expression, a deletion series of the HrTyrp 5' flanking region fused to a lacZ reporter gene was constructed and microinjected into ascidian fertilized eggs. The region of 73 bp in HrTyrp was identified as sufficient for expression in pigment cell-precursors of tailbud stage embryos. It is noteworthy that there is no M-box element highly conserved in the promoters for vertebrate tyrosinase family genes such as tyrosinase, Tyrp1/TRP-1 and Tyrp2/TRP-2 (Dct). Although the regulatory system of ascidian pigment-cell development is likely to contain most factors critical to vertebrate pigment-cell development, there might be critical differences in the mode of regulation, such as the developmental timing of interactions of factors, proteins and genes, involved in pigment cell differentiation and pigmentation.

Mannose-binding lectin-associated serine proteases (MASPs) are involved in complement activation through the lectin pathway. To elucidate the phylogenetic origin of MASP and a primordial complement system, we cloned two MASP cDNAs from amphioxus (Branchiostoma belcheri) of the cephalochordates, considered to be the closest relative of vertebrates. The two sequences, orthologues of mammalian MASP-1 and MASP-3, were produced by alternative processing of RNA from a single gene consisting of a common H chain-encoding region and two L chain-encoding regions, a structure which is similar to that of the human MASP1/3 gene. We also isolated two MASP genes from the ascidian Halocynthia roretzi (urochordates) and found that each of them consists simply of an H chain-encoding region and a single L chain-encoding region. The difference in structure between the ascidian MASP genes and the amphioxus/mammalian MASP genes suggests that a prototype gene was converted to the MASP1/3-type gene possessing two L chain-encoding regions at an early stage of evolution before the divergence of amphioxus. This conclusion is supported by the presence of MASP-1 and MASP-3 homologues in almost all vertebrates, as demonstrated by the cloning of novel cDNA sequences representing lamprey (cyclostomes) MASP-1 and Xenopus MASP-3. The ancient origin of MASP-1 and MASP-3 suggests that they have crucial functions common to all species which emerged after cephalochordates.

Otx (otd in Drosophila) is a well-conserved homeobox gene throughout animal phylogeny and commonly expressed in the anterior part of the embryo. In embryos of the ascidian Halocynthia roretzi, Hroth, the otx homologue in this species, is expressed in the endoderm and the sensory vesicle, the anterior part of the larval ascidian central nervous system (CNS), which has been thought to be homologous to vertebrate forebrain and midbrain. The developmental expression pattern of Hroth is very similar to that of vertebrate counterparts, which leads to a possibility that a similar mechanism may exist in the patterning of the CNS between ascidians and vertebrates. To better understand the mechanism, we decided to undertake analysis of the transcriptional regulatory regions of Hroth. We isolated and determined the nucleotide sequence of the 11.4-kbp region upstream of the translation start site of Hroth. We found that Hroth transcripts are modified likely with spliced leader RNA; therefore, we could not determine the transcription start site. However, first, we identified three introns that are unknown with vertebrate otx genes. Second, we found two regions that are capable of functioning as a promoter through deletion analysis, one of which appeared to be an endogenous promoter of Hroth. We analyzed the 5' upstream region 5402-1473bp, the region between 1473 and 5402 base pairs upstream from the translation start site of Hroth, including the putative endogenous promoter. This region was capable of driving Hroth expression in the sensory vesicle lineage cells as well as some other lineages at the early tail bud stage. Deletion analysis of this region suggested that three regions, 1659-1650bp, 1628-1613bp, and 1542-1473bp are responsible for regulating Hroth expression in the sensory vesicle cells at the tail bud stage. Among these regions, no apparent sequence conservation was observed. The present study has revealed a complex organization of transcription regulatory regions for the ascidian otx.

Ascidians are primitive chordates. Their fertilized egg develops quickly into a tadpole-type larva, which consists of a small number but distinct types of cells, including those of epidermis, central nervous system with two sensory organs, endoderm and mesenchyme in the trunk, and notochord and muscle in the tail. This configuration of the ascidian tadpole is thought to represent the most simplified and primitive chordate body plan. In addition, the free-swimming and non-feeding larvae metamorphose into sessile and filter-feeding adults. The genome size of Ciona intestinalis is estimated to be about 160 Mb, and the number of genes approximately 15,500. The present Ciona cDNA projects focused on gene expression profiles of fertilized eggs, 32-110-cell stage embryos, tailbud embryos, larvae, and young adults. Expressed sequence tags (ESTs) of the 5'-most end and 3'-most end of more than 3000 clones were determined at each developmental stage, and the clones were categorized into independent clusters using the 3'-end sequences. Nearly 1000 clusters of them were then analyzed in detail of their sequences against a BLASTX search. This analysis demonstrates that, on average, half of the clusters showed proteins with sequence similarities to known proteins and the other half did not show sequence similarities to known proteins. Genes with sequence similarities were further categorized into three major subclasses, depending on their functions. Furthermore, the expression profiles of all of the clusters were analyzed by whole-mount in situ hybridization. This analysis highlights gene expression patterns characteristic to each developmental stage. As a result, the present study provides many new molecular markers for each of the tissues and/or organs that constitutes the Ciona tailbud embryo. This sequence information will be used for further comparative genome studies to explore molecular mechanisms involved in the formation of one of the most primitive chordate body plans. All of the data fully characterized may be viewed at the web site http://ghost.zool.kyoto-u.ac.jp.

Hroth, the ascidian homologue of vertebrate otx genes, is expressed in the anterior part of the larval ascidian central nervous System during its formation like vertebrate counterparts. The similarity of the expression pattern between Hroth and its vertebrate counterparts raise a possibility that a similar regulatory mechanism of otx transcription is present between aseidians and vertebrates. To understand the mechanism, we have analyzed transcription regulatory elements for the Hroth gene. We have demonstrated that at least three regulatory regions are present responsible for transcription of Hroth in the sensory vesicle, anterior trunk epidermis and a-line cells at the mid tailbud stage.

Ciotx, the otx homologue of the ascidian, Ciona intestinalis was isolated and examined for the structure and expression pattern in comparison with Hroth, the counterpart of Halocynthia roretzi. Ciotx shows significant similarity to Hroth throughout entire length. In endodermal and ectodermal lineages, the expression pattern of Ciotx was identical to that of Hroth. In other cell lineages, timing of the onset of Ciotx expression was the same as that of Hroth but Ciotx tended to be expressed longer than Hroth. Among these, expression in the b-line muscle lineage should be noted. The b-line cells of Ciona give rise to 2 muscle cells while those of Halocynthia give rise to 5 muscle cells. Ciotx is expressed in b-line cells at the 32-cell stage and the 92-cell stage, while Hroth is expressed only at the 32-cell stage. It has been demonstrated that Hroth represses muscle development through overexpression experiments. Assuming that the ascidian otx genes have a function of repressing muscle fate in common, difference in the number of b-line muscle cells may be attributed to the difference in the expression pattern of otx genes in the b-line lineage between Ciona and Halocynthia.

The origin of molecular mechanisms of cephalic development is an intriguing question in evolutionary and developmental biology. Ascidians, positioned near the origin of the phylum Chordata, share a conserved set of anteroposterior patterning genes with vertebrates. Here we report the cross-phylum regulatory potential of the ascidian Otx gene in the development of the Drosophila brain and the head vertex structures. The ascidian Otx gene rescued the embryonic brain defect caused by a null mutation of the Drosophila orthodenticle (otd) gene and enhanced rostral brain development while it suppressed trunk nerve cord formation. Furthermore, the ascidian Otx gene restored the head vertex defects caused by a viable otd mutation, ocelliless, via specific activation and repression of downstream regulatory genes. These cross-phylum regulatory potentials of the ascidian Otx gene are equivalent to the activities of the Drosophila and human otd/Otx genes in these developmental processes. These results support the notion that basal chordates such as ascidians have the same molecular patterning mechanism for the anterior structures found in higher chordates, and suggest a common genetic program of cephalic development in invertebrate, protochordate and vertebrate.

The long-standing question of how asymmetric development or asymmetric body structures in lancelets (amphioxus) are phylogenetically related to the body plan of other animals is still untouched. Three anterior structures, the preoral pit, club-shaped gland and mouth, are remarkable asymmetric features in developing lancelets that all open on the left side of the body. A Ptx-related gene, BbPtx is the first identified transcription factor gene with an asymmetrical expression pattern in lancelets similar to that in vertebrates, and thus it may provide a clue for the above question. Expression of the BbPtx gene is first detected at the dorsal margin of the blastopore in early mid-gastrulae and then becomes restricted to the left anterodorsal wall of the primitive gut and to the developing left somitocoelomic system. Expression continues on the left side in the developing preoral pit, club-shaped gland and mouth as well as in the mesoderm at the caudal end. Unlike D-Ptx1 in Drosophila, BbPtx is not coexpressed with a fork head gene in lancelets; instead the two genes are expressed in a complementary fashion on the left side of the embryo. The expression pattern of BbPtx is not compatible with the calcichordate hypothesis of Jefferies, in which the proposed ancestor of chordates rotated its tail 90° counterclockwise in relation to the head/trunk. The expression of both BbPtx and vertebrate Pitx2 in tissues derived from the coelom implies that the left-right asymmetric development has a common origin between cephalochordates and vertebrates. Considering the development of the coelom in deuterostomes, however, left-right asymmetric development involving Pitx2-related genes is rather likely to be a primitive character shared among deuterostomes.

The tyrosinase family in vertebrates consists of three related melanogenic enzymes: tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2. These proteins control melanin production in pigment cells and play a crucial role in determining vertebrate coloration. We have isolated a gene from the ascidian Halocynthia roretzi which encodes a tyrosinase-related protein (HrTRP) with 45–49% identity with vertebrate TRP-1 and TRP-2. The expression of the HrTRP gene in pigment lineage a8.25 cells starts at the early-mid gastrula stage, which coincides with the stage when these cells are determined as pigment precursor cells; therefore, it provides the earliest pigment lineage-specific marker, which enables us to trace the complete cell lineage leading to two pigment cells in the larval brain. In addition, the expression pattern of the HrTRP gene appears to share similar characteristics with the mouse TRP-2 gene although structurally the HrTRP gene is more closely related to mammalian TRP-1 genes. Based on these observations and on results from molecular phylogenetic and hybridization analyses, we suggest that triplication of the tyrosinase family occurred during the early radiation of chordates. Initially, duplication of an ancestral tyrosinase gene produced a single TRP gene before the urochordate and cephalochordate-vertebrate divergence, and a subsequent duplication of the ancestral TRP gene in the vertebrate lineage gave rise to two TRP genes before the emergence of teleost fishes. Evolution of the melanin synthetic pathway and possible phylogenetic relationships among chordate pigment cells that accommodate the metabolic process are discussed. Dev Dyn 1999;215:225–237. © 1999 Wiley-Liss, Inc.

It is now well established that epithelial-mesenchymal interactions are essential in the spatio-temporally regulated differentiation processes in organogenesis in higher vertebrates [1, 2]. The morphological and the functional differentiation of an organ necessarily involves the local expression or suppression of sets of genes in the cells constituting the organ.

We have isolated and cloned a gene, designated AMD1 (ascidian MyoD-related factor 1), and its cDNAs that encode a member of the family of myogenic basic helix-loop-helix (bHLH) factors from the ascidian Halocynthia roretzi. The AMD1 gene consists of four exons and is transcribed into at least two distinct mRNAs, which differ in their 3' untranslated region. The gene encodes a protein of 435 amino acids, which exhibits homology to the bHLH domain of other myogenic bHLH factors including vertebrate MyoDt. A reverse transcription-polymerase chain reaction (RT-PCR) assay revealed that transcripts of the gene were not detectable in fertilized eggs or in very early embryos. They were first detected at the 64-cell stage, a few hours prior to the accumulation of mRNAs for embryonic muscle-specific proteins. In addition, expression of the AMD1 gene was evident in adult body-wall muscle but not in heart and other nonmuscle tissues. These results suggest the possibility that, in ascidians as in vertebrates, the myogenic bHLH factor is involved in the specification of embryonic cells as myogenic cells.

Two histone H2B gene clones were isolated from macronuclei of Tetrahymena thermophila. Nucleotide sequences of the two clones were highly homologous within the coding region but not in the noncoding region. Comparison of the deduced amino acid sequences between the two clones showed three differences in a total of 121 amino acids. Each of the two clones contained a TAA triplet within the coding region, which appeared to code for a glutamine residue. To demonstrate the existence of histone mRNA containing UAA triplet, nuclease P1 protection mapping using total cellular RNA and nucleotide sequencing of primer extension products were carried out. The results clearly indicated that two cloned histone H2B genes were transcribed, giving rise to the major histone H2B mRNAs with a UAA triplet sequence in frame. The tentative 5'- and 3'-ends of histone H2B mRNAs were determined.

A set of 3423 expressed sequence tags derived from the Ciona intestinalis tailbud embryos was categorized into 1213 independent clusters. When compared with DNA Data Bank of Japan database, 502 clusters of them showed significant matches to reported proteins with distinct function, whereas 184 lacked sufficient information to be categorized (including reported proteins with undefined function) and 527 had no significant similarities to known proteins. Sequence similarity analyses of the 502 clusters in relation to the biosynthetic function, as well as the structure of the message population at this stage, demonstrated that 390 of them were associated with functions that many kinds of cells use, 85 with cell-cell communication and 27 with transcription factors and other gene regulatory proteins. All of the 1213 clusters were subjected to whole-mount in situ hybridization to analyze the gene expression profiles at this stage. A total of 387 clusters showed expression specific to a certain tissue or organ; 149 showed epidermis-specific expression; 34 were specific to the nervous system; 29 to endoderm; 112 to mesenchyme; 32 to notochord; and 31 to muscle. Many genes were also specifically expressed in multiple tissues. The study also highlighted characteristic gene expression profiles dependent on the tissues. In addition, several genes showed intriguing expression patterns that have not been reported previously; for example, four genes were expressed specifically in the nerve cord cells and one gene was expressed only in the posterior part of muscle cells.This study provides molecular markers for each of the tissues and/or organs that constitutes the Ciona tailbud embryo. The sequence information will also be used for further genome scientific approach to explore molecular mechanisms involved in the formation of one of the most primitive chordate body plans.