Gene expression profiles in Ciona intestinalis tailbud embryos.

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.

Cross-phylum regulatory potential of the ascidian Otx gene in brain development in Drosophila melanogaster.

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.

Hremx, the ascidian homologue of ems/emx, is expressed in the anterior and posterior-lateral epidermis but not in the central nervous system during embryogenesis.

Recent comparative studies on expression patterns of homeobox genes in the development between ascidians and vertebrates have come to suggest a possibility that a common basic mechanism may exist in the patterning of the central nervous system (CNS). The ems/emx genes have been demonstrated to be involved in the formation and patterning of the anterior CNS in Drosophila and vertebrate embryos. In the present study, we have isolated and analyzed expression of Hremx, the ascidian homologue of ems/emx with particular attention to whether it is expressed in the larval ascidian CNS. Expression of Hremx was detected in the anterior trunk and lateral tail epidermis but not in the anterior CNS. The two expression domains of the epidermis responded in different ways upon treatment with retinoic acid: the anterior expression domain was unaltered, while the posterior expression domain extended to the anterior. The present result suggests that Hremx may have a function in anterior patterning but not in the patterning of the CNS in the ascidian embryo. We suggest the possibility that the function of ems/emx genes in the patterning of the anterior CNS in Drosophila and vertebrate embryos might have been acquired independently in the lineages to Drosophila and vertebrates.

Early expressed genes showing a dichotomous developing pattern in the lancelet embryo.

Lancelets (amphioxus), although showing the most similar anatomical features to vertebrates, never develop a vertebrate-like head but rather several structures specific to this animal. The lancelet anatomical specificity seems to be traceable to early developmental stages, such as the vertebrate dorsal and anterior-posterior determinations. The BMP and Wnt proteins play important roles in establishing the early basis of the dorsal structures and the head in vertebrates. The early behavior of BMP and Wnt may be also related to the specific body structures of lancelets. The expression patterns of a dpp-related gene, Bbbmp2/4, and two wnt-related genes, Bbwnt7 and Bbwnt8, have been studied in comparison with those of brachyury and Hnf-3beta class genes. The temporal expression patterns of these genes are similar to those of vertebrates; Bbbmp2/4 and Bbwnt8 are first expressed in the invaginating primitive gut and the equatorial region, respectively, at the initial gastrula stage. However, spatial expression pattern of Bbbmp2/4 differs significantly from the vertebrate cognates. It is expressed in the mid-dorsal inner layer of gastrulae and widely in the anterior region, in which vertebrates block BMP signaling. The present study suggests that the lancelet embryo may have two distinct developmental domains from the gastrula stage, the domains of which coincide later with the lateral diverticular and the somitocoelomic regions. The embryonic origin of the anterior-specific structures in lancelets corresponds to the anterior domain where Bbbmp2/4 is continuously expressed.

Left-right asymmetric expression of BbPtx, a Ptx-related gene, in a lancelet species and the developmental left-sidedness in deuterostomes.

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 degrees 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.

Anteroposterior patterning of the epidermis by inductive influences from the vegetal hemisphere cells in the ascidian embryo.

Patterning along the anteroposterior axis is a critical step during animal embryogenesis. Although mechanisms of anteroposterior patterning in the neural tube have been studied in various chordates, little is known about those of the epidermis. To approach this issue, we investigated patterning mechanisms of the epidermis in the ascidian embryo. First we examined expression of homeobox genes (Hrdll-1, Hroth, HrHox-1 and Hrcad) in the epidermis. Hrdll-1 is expressed in the anterior tip of the epidermis that later forms the adhesive papillae, while Hroth is expressed in the anterior part of the trunk epidermis. HrHox-1 and Hrcad are expressed in middle and posterior parts of the epidermis, respectively. These data suggested that the epidermis of the ascidian embryo is patterned anteroposteriorly. In ascidian embryogenesis, the epidermis is exclusively derived from animal hemisphere cells. To investigate regulation of expression of the four homeobox genes in the epidermis by vegetal hemisphere cells, we next performed hemisphere isolation and cell ablation experiments. We showed that removal of the vegetal cells before the late 16-cell stage results in loss of expression of these homeobox genes in the animal hemisphere cells. Expression of Hrdll-1 and Hroth depends on contact with the anterior-vegetal (the A-line) cells, while expression of HrHox-1 and Hrcad requires contact with the posterior-vegetal (the B-line) cells. We also demonstrated that contact with the vegetal cells until the late 32-cell stage is sufficient for animal cells to express Hrdll-1, Hroth and Hrcad, while longer contact is necessary for HrHox-1 expression. Contact with the A-line cells until the late 32-cell stage is also sufficient for formation of the adhesive papillae. Our data indicate that the epidermis of the ascidian embryo is patterned along the anteroposterior axis by multiple inductive influences from the vegetal hemisphere cells and provide the first insight into mechanisms of epidermis patterning in the chordate embryos.

Ascidian tail formation requires caudal function.

Although the tail is one of the major characteristics of animals of the phylum Chordata, evolutionary aspects of the molecular mechanisms involved in its formation are not clear. To obtain insights into these issues, we have isolated and investigated the caudal gene of an ascidian, one of the lower animal groups among chordates. Ascidian caudal is expressed from the midgastrula stage onward in the lateral walls of the posterior neural tube cell lineage and also in the posterior epidermal cells from the neurula stage. Thus, ascidian caudal expression is restricted to the ectoderm of a tail-forming region throughout embryogenesis. Suppression of caudal function by an antisense oligonucleotide or a dominant negative construct caused inhibition of the cell movement required for tail formation. Overexpression of wild-type caudal mRNA in an ascidian animal cap, an animal half explant prepared at the eight-cell stage, caused elongation of the cap. Furthermore, Xenopus embryos injected with dominant negative ascidian caudal exhibited defects in elongation, suggesting a conserved caudal function among chordates. These results indicate that caudal function is required for chordate tail formation and may play a key role in its evolution.

Cloning and embryonic expression of Hrsna, a snail family gene of the ascidian Halocynthia roretzi: implication in the origins of mechanisms for mesoderm specification and body axis formation in chordates.

snail family genes encode a transcription factor with specific zinc finger motifs. In vertebrates, they are expressed in the entire mesoderm in early embryogenesis and later in the paraxial mesoderm and the tail-bud, suggesting roles in specification and morphogenesis of the paraxial mesoderm. In the present study, a snail family gene Hrsna from a member of the chordates, an ascidian (Halocynthia roretzi), was cloned to obtain an insight into the origin of the mechanisms of mesoderm specification and body axis formation as observed in vertebrates. Expression of Hrsna during ascidian embryogenesis was found to be quite similar to that of vertebrate snail genes. First, before gastrulation, Hrsna was initially expressed in most precursors of mesodermal tissues including the notochord where As-T, the ascidian homolog of brachyury, was expressed. Hrsna expression persisted in the paraxial mesoderm, the mesenchyme and muscle, but not in the notochord precursors. Also, just as vertebrate snail family genes are expressed in the border of the neural plate that develops into dorsal neural tube and neural crest cells, so Hrsna expression was detected in the precursors of lateral and dorsal regions of the neural tube. However, Hrsna expression was not detected in the tip of the tail, unlike in vertebrate counterparts. In the light of the present findings, similarity and dissimilarity of mechanisms governing mesoderm specification and body axis formation between ascidians and vertebrates are discussed.

Structure and developmental expression of the ascidian TRP gene: insights into the evolution of pigment cell-specific gene expression.

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.

Vegetal cell fate specification and anterior neuroectoderm formation by Hroth, the ascidian homologue of orthodenticle/otx.

To obtain insights into the mechanisms of gastrulation and neural tube formation, we studied the function and regulation of expression of Hroth, the ascidian homologue of orthodenticle/otx, during embryogenesis. Microinjection of synthetic Hroth mRNA into fertilized eggs led to embryos with an expanded trunk and a reduced tail. In these embryos, development of notochord and muscle was effected. Also, Hroth overexpression caused ectopic formation of anterior neuroectoderm, along with suppression of epidermis development, even in the absence of cell-cell interaction. Furthermore, we demonstrated that ectodermal expression of Hroth requires an inductive influence from the vegetal hemisphere cells. These data suggest roles of Hroth in both specification of mesoendodermal cells and anterior neuroectoderm formation.

Opsonic complement component C3 in the solitary ascidian, Halocynthia roretzi.

The recent identification of two mannose-binding lectin-associated serine protease clones from Halocynthia roretzi, an ascidian, suggested the presence of a complement system in urochordates. To elucidate the structure and function of this possibly primitive complement system, we have isolated cDNA clones for ascidian C3 (AsC3) and purified AsC3 protein from body fluid. The deduced primary structure of AsC3 shows overall similarity to mammalian C3, including a typical thioester site with the His residue required for nucleophilic activation of the thioester. AsC3 has a two-subunit chain structure, and the alpha-chain is cleaved at a specific site near to the N terminus upon activation. Ascidian body fluid contains an opsonic activity which enhances phagocytosis of yeast by ascidian blood cells, and Ab against AsC3 inhibits this opsonic activity. These results indicate that the complement system played a pivotal role in innate immunity by enhancing phagocytosis before the emergence of the vertebrates and well ahead of the establishment of adaptive immunity, which is believed to have occurred at about the time of the appearance of cartilaginous fish.

Two lineages of mannose-binding lectin-associated serine protease (MASP) in vertebrates.

Mannose-binding lectin-associated serine protease (MASP) is a newly identified member of the serine protease superfamily. MASP is involved in host defense against pathogens through a novel system of complement activation, designated the lectin pathway. To elucidate the origin of the lectin pathway and the molecular evolution of MASP, we cloned six MASP cDNAs from five vertebrate species going from mammal to cyclostome. An alignment of the amino acid sequences deduced from the cDNAs revealed the presence of two different lineages of the MASP gene. This classification was supported by the precise correlation with two types of exon organization for the protease domain. One of the two lineages is unique in that a single exon encodes the protease domain, unlike most other serine proteases. All members of this group, termed the AGY type, have an AGY codon at the active site serine. A phylogenetic tree suggests that the AGY type diverged from another lineage, termed the TCN type, before the emergence of primitive vertebrates. Furthermore, the presence of MASP or MASP-like sequences in most vertebrate species suggests that the lectin pathway functions extensively in vertebrates and that its origin is traced back to the invertebrate stage.

Analysis of temporal expression pattern and cis-regulatory sequences of chicken pepsinogen A and C.

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.

Retinoic acid affects patterning along the anterior-posterior axis of the ascidian embryo.

Because retinoic acid (RA) is known to affect anterior posterior patterning in vertebrate embryos, it was questioned whether it shows similar effects in a more primitive chordate, the ascidian Halocynthia roretzi. Ascidian embryos treated with RA exhibited truncated phenotypes in a dose-dependent manner similar to the anterior truncations seen in vertebrate embryos. The most severely affected larvae possessed a round trunk without the papillae characteristic of the anterior terminal epidermis. Retinoic acid also altered the expression of HrHox-1 and Hroth in a dose-dependent manner. Expression of HrHox-1 increased, whereas expression of Hroth decreased with increasing levels of RA. In treated embryos, HrHox-1 was first expressed pan-ectodermally, then degraded in all but specific regions of the embryo. By contrast, initiation of Hroth expression was not affected, but epidermal expression was lost while expression in the neural tube narrowed toward the anterior in tail-bud embryos. These alterations in the expression of homeobox genes appear to correlate closely to the morphological defects elicited by RA treatment, suggesting broad conservation of developmental patterning mechanisms within the Phylum Chordata.

Tripartite organization of the ancestral chordate brain and the antiquity of placodes: insights from ascidian Pax-2/5/8, Hox and Otx genes.

Ascidians and vertebrates belong to the Phylum Chordata and both have dorsal tubular central nervous systems. The structure of the ascidian neural tube is extremely simple, containing less than 400 cells, among which less than 100 cells are neurons. Recent studies suggest that, despite its simple organization, the mechanisms patterning the ascidian neural tube are similar to those of the more complex vertebrate brain. Identification of homologous regions between vertebrate and ascidian nervous systems, however, remains to be resolved. Here we report the expression of HrPax-258 gene: an ascidian homologue of vertebrate Pax-2, Pax-5 and Pax-8 genes. Molecular phylogenetic analyses indicate that HrPax-258 is descendant from a single precursor gene that gave rise to the three vertebrate genes. The expression pattern of HrPax-258 suggests that this subfamily of Pax genes has conserved roles in regional specification of the brain. Comparison with expression of ascidian Otx (Hroth) and a Hox gene (HrHox1) by double-staining in situ hybridizations indicate that the ascidian brain region can be subdivided into three regions; the anterior region marked by Hroth probably homologous to the vertebrate forebrain and midbrain, the middle region marked by HrPax-258 probably homologous to the vertebrate anterior hindbrain (and maybe also midbrain) and the posterior region marked by Hox genes which is homologous to the vertebrate hindbrain and spinal cord. Later expression of HrPax-258 in atrial primordia implies that basal chordates such as ascidians have already acquired a sensory organ that develops from epidermal thickenings (placodes) and expresses HrPax-258; we suggest it is homologous to the vertebrate ear. Therefore, placodes are not likely to be a newly acquired feature in vertebrates, but may have already been possessed by the earliest chordates.

Early specification of intestinal epithelium in the chicken embryo: a study on the localization and regulation of CdxA expression.

CdxA, a chicken homeobox-containing gene related to caudal in Drosophila, has been implicated in the regionalization of endoderm. It is reported here that, in the development of the chicken embryo, CdxA expression appears in the endoderm at day 1.5 of development as bilateral bands on either side of the splanchnopleure which later contribute to intestinal epithelium. The CdxA-expressing area extends medially and caudally as formation of the gut tube progresses. It is also shown that the rostral limit of CdxA expression demarcates the boundary between stomach and duodenum after day 3 of development. CdxA is not expressed in digestive tract appendages which open into the intestine, such as pancreas, liver and allantois. Early restriction of CdxA expression in intestinal lineage suggests that the intestinal specification involving CdxA expression commences before the gut tube is formed. The expression of CdxA in epithelial-mesenchymal tissue recombinants suggests that mesenchymal influence regulating CdxA expression plays an important role in confirming the boundary between the stomach and intestine. Chronological change in the spatial distribution of CdxA transcripts and the results of tissue recombination experiments, together with precise fate maps of early endoderm and splanchnic mesoderm, lead to a model of mechanisms by which intestinal specification is brought about.

Hroth an orthodenticle-related homeobox gene of the ascidian, Halocynthia roretzi: its expression and putative roles in the axis formation during embryogenesis.

To obtain insight into the axis-forming mechanism in ascidian embryogenesis, Hroth, an ascidian counterpart of orthodenticle/otx, was isolated from Halocynthia roretzi and its expression in embryogenesis was examined by whole mount in situ hybridization. It was revealed that Hroth is expressed in both involuting mesoendoderm and anterior ectoderm during gastrulation while later expression is restricted to the sensory vesicle and anterior epidermis. Expression pattern of Hroth around gastrulation was compared with that of Hrlim, the ascidian LIM class homeobox gene that is known to be expressed during gastrulation. In the light of the present findings on the expression of Hroth, properties of the axis-forming mechanism in ascidian embryogenesis are discussed.

Homeobox genes exhibit evolutionary conserved regionalization in the central nervous system of an ascidian larva.

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.

Expression of the labial group Hox gene HrHox-1 and its alteration induced by retinoic acid in development of the ascidian Halocynthia roretzi.

Ascidian embryogenesis shares several developmental features with vertebrates. Thus, it is presumed that some molecular mechanisms that are critical for vertebrate development may also act in the early development of ascidians. Here, we investigated expression of the ascidian labial group Hox gene HrHox-1 in the development of Halocynthia roretzi. HrHox-1 showed a spatially restricted expression pattern along the anterior-posterior axis, which is remarkably similar to that of the vertebrate gene, Hoxb-1. The expression of HrHox-1, however, was exclusively in tissues of ectoderm origin unlike its vertebrate counterpart. Exposure of the embryos to 10(-6) M all-trans retinoic acid induced a larval phenotype with elimination of the anteriormost structures, the papillae. In this phenotype, the level of HrHox-1 expression was enhanced and ectopic expression was observed at the anterior terminal epidermis where the papillae are otherwise formed. These observations suggest that there are some conserved mechanisms in the spatial regulation of expression of labial group genes in embryogenesis of ascidians and vertebrates.

Spatially and temporally regulated expression of the LIM class homeobox gene Hrlim suggests multiple distinct functions in development of the ascidian, Halocynthia roretzi.

Hrlim is a LIM class homeobox gene that was first isolated from the ascidian Halocynthia roretzi. To assess its roles in early development of the ascidian, spatial and temporal expression of Hrlim was examined by whole mount in situ hybridization. This revealed that transcription of Hrlim is activated at the 32-cell stage specifically in the endoderm lineage. Hrlim is also transiently expressed in all notochord precursor cells. Expression in the endoderm lineage continues through to the middle of gastrulation. After gastrulation, Hrlim is expressed in certain lineages that give rise to subsets of cells in the brain and spinal cord. Based on these observations, it is suggested that Hrlim plays multiple distinct roles in ascidian embryogenesis.