A genomewide survey of developmentally relevant genes in Ciona intestinalis. VI. Genes for Wnt,TGFbeta, Hedgehog and JAK/STAT signaling pathways

Cell-cell interactions play important roles in a variety of developmental processes, and therefore molecules involved in the signaling pathways have been studied extensively. Recently, the draft genome sequence of the basal chordate, Ciona intestinalis, was determined. Here we annotated genes for the signaling pathways of Wnt, transforming growth factor beta (TGFbeta), Hedgehog, and JAK/STAT in the genome of Ciona intestinalis. The Ciona genome contains ten wnt genes, six frizzled genes, four sFRP genes, ten TGFbeta family member genes, five TGFbeta-receptor genes, and five Smad genes; most of the genes were found with less redundancy than in vertebrate genomes. The other genes in the signaling pathways are present as a single copy in the Ciona genome. In addition, all of the identified genes for the signaling pathway, except for a few genes, have EST evidence, and their cDNAs are available from the Ciona intestinalis gene collection. Therefore, Ciona intestinalis may provide an experimental system for exploring the basic genetic cascade associated with the signaling pathways in chordates.     

A genomewide survey of developmentally relevant genes in Ciona intestinalis. V. Genes for receptor tyrosine kinase pathway and Notch signaling pathway

In the present survey, we identified most of the genes involved in the receptor tyrosine kinase (RTK), mitogen activated protein kinase (MAPK) and Notch signaling pathways in the draft genome sequence of Ciona intestinalis, a basal chordate. Compared to vertebrates, most of the genes found in the Ciona genome had fewer paralogues, although several genes including ephrin, Eph and fringe appeared to have multiplied or duplicated independently in the ascidian genome. In contrast, some genes including kit/flt, PDGF and Trk receptor tyrosine kinases were not found in the present survey, suggesting that these genes are innovations in the vertebrate lineage or lost in the ascidian lineage. The gene set identified in the present analysis provides an insight into genes for the RTK, MAPK and Notch signaling pathways in the ancient chordate genome and thereby how chordates evolved these signaling pathway.     

A genome-wide survey of genes for enzymes involved in pigment synthesis in an ascidian, Ciona intestinalis

The draft genome sequence and a large quantity of EST and cDNA information are now available for the ascidian Ciona intestinalis. In the present study, genes involved in pigment synthesis pathways were identified in the decoded genome of Ciona, and information about these genes was obtained from available EST and cDNA sequences. It was found that the Ciona genome contains orthologous genes for each enzyme of the melanin, pteridine, ommochrome, papiliochrome, and heme synthesis pathways. Several appear as independent duplications in the Ciona genome. Because cDNA clones for all but two of these genes have already been isolated by the cDNA project, C. intestinalis will provide an experimental system to explore molecular mechanisms underlying color patterns, through future genome-wide studies.     

Three insulin-relaxin-like genes in Ciona intestinalis

The Ciona intestinalis genome harbors three insulin-like genes: INS-L1, -L2 and -L3. Conserved synteny between the Ciona-human genomes predicts that Ciona INS-Ls are orthologous to the vertebrate insulin-relaxin family, but this relation cannot be inferred from molecular phylogeny. A conserved protein core with six cysteines; typical arrangement of B-, C- and A-protein domains; pro-protein maturation mode; and putative insulin receptor-binding sites were identified in Ciona INS-L proteins. ESTs used to assemble exonic sequences of INS-Ls combined with qRT-PCR analysis provided evidence that the predicted genes are expressed in the developing and adult Ciona. Our results support that Ciona INS-L1 is orthologous to the vertebrate insulin-like/relaxin genes, INS-L2 to insulin genes and INS-L3 to IGF genes. Our analysis also implies that the insulin-like/relaxin ancestor switched receptor type from tyrosine kinase- to GPCR-type, whereas insulin-IGF subfamily retained the tyrosine kinase-type of receptor. We propose that this receptor-switch occurred after the time when urochordates branched from the common chordate lineage, but before the two genome-duplications at the root of the vertebrates.     

Fgf genes in the basal chordate <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

In vertebrates, a number of fibroblast growth factors (FGFs) have been shown to play important roles in developing embryos and adult organisms. However, the molecular relationships of the vertebrate FGFs are not yet completely understood, partly due to the divergence of their amino acid sequences. To solve this problem, we have identified six FGF genes in a basal chordate, the ascidian Ciona intestinalis. A phylogenetic analysis confidently assigned two of them to vertebrate FGF8/17/18 and FGF11/12/13/14, respectively. Based on the presence of the conserved domains within or outside of the FGF domains, we speculate that three of the other genes are orthologous to vertebrate FGF3/7/10/22, FGF4/5/6 and FGF9/16/20, respectively, although we cannot assign the sixth member to any of the vertebrate FGFs. A survey of the raw whole genome shotgun sequences of C. intestinalis demonstrated the presence of no FGF genes other than the six genes in the genome. The identification of these six FGF genes in the basal chordate gave us an insight into the diversification of specific subfamilies of vertebrate FGFs.     

On the origins of the extracellular matrix in vertebrates.

Extracellular matrix (ECM) is a key metazoan characteristic. In addition to providing structure and orientation to tissues, it is involved in many cellular processes such as adhesion, migration, proliferation and differentiation. Here we provide a comprehensive analysis of ECM molecules focussing on when vertebrate specific matrices evolved. We identify 60 ECM genes and 20 associated processing enzymes in the genome of the urochordate Ciona intestinalis. A comparison with vertebrate and protostome genomes has permitted the identification of both a core set of metazoan matrix genes and vertebrate-specific innovations in the ECM. We have identified a few potential cases of de novo vertebrate ECM gene innovation, but the majority of ECM genes have resulted from duplication of pre-existing genes present in the ancestral vertebrate. In conclusion, the modern complexity we see in vertebrate ECM has come about largely by duplication and modification of pre-existing matrix molecules. Extracellular matrix genes and their processing enzymes appear to be over-represented in the vertebrate genome suggesting that these genes played an active role enabling and underpinning the evolution of vertebrates.     

A genomewide survey of developmentally relevant genes in Ciona intestinalis. IX. Genes for muscle structural proteins

Ascidians are simple chordates that are related to, and may resemble, vertebrate ancestors. Comparison of ascidian and vertebrate genomes is expected to provide insight into the molecular genetic basis of chordate/vertebrate evolution. We annotated muscle structural (contractile protein) genes in the completely determined genome sequence of the ascidian Ciona intestinalis, and examined gene expression patterns through extensive EST analysis. Ascidian muscle protein isoform families are generally of similar, or lesser, complexity in comparison with the corresponding vertebrate isoform families, and are based on gene duplication histories and alternative splicing mechanisms that are largely or entirely distinct from those responsible for generating the vertebrate isoforms. Although each of the three ascidian muscle types - larval tail muscle, adult body-wall muscle and heart - expresses a distinct profile of contractile protein isoforms, none of these isoforms are strictly orthologous to the smooth-muscle-specific, fast or slow skeletal muscle-specific, or heart-specific isoforms of vertebrates. Many isoform families showed larval-versus-adult differential expression and in several cases numerous very similar genes were expressed specifically in larval muscle. This may reflect different functional requirements of the locomotor larval muscle as opposed to the non-locomotor muscles of the sessile adult, and/or the biosynthetic demands of extremely rapid larval development.     

A genome-wide survey of the genes for planar polarity signaling or convergent extension-related genes in Ciona intestinalis and phylogenetic comparisons of evolutionary conserved signaling components

Non-canonical Wnt signals similar to planar cell polarity (PCP) signaling in the fly control convergent extension (CE) of the dorsal mesoderm during gastrulation in vertebrates. Using the Ciona complete genome sequence and EST sequence data, we present here an initial and exhaustive search in non-vertebrate chordates, Ciona intestinalis for the family members as well as homologs or orthologs that are involved in PCP/CE signaling cascades. We clarified 7 cardinal gene families, including the MAPK, STE20 group kinase, Rho small GTPase, STAT, Glypican, Fz and Wnt gene families, as well as gene homologs or orthologs for known PCP/CE signaling components with their phylogenetic nature. As a result, we characterized 62 Ciona component genes. Among them, 59 genes were novel and functional genes which were supported by EST expressions and 15 genes belonged to PCP/CE component orthologs of other organisms or common ancestor genes. Moreover, from the phylogenetic point of view, we compared these components genome-widely with the PCP signaling components of fly and the CE signaling components of vertebrates. We then discovered not only that ascidians contain the basic ancestral signaling pathway components in chordates but also that several signaling components have not found in ascidian, indicating that ascidian CE pathway might have several gaps from vertebrate CE pathway. The present study provides an initial step for the subsequent analysis of CE in the non-vertebrate chordates, ascidians. In addition, this phylogenetic approach will help to facilitate understanding of the relationship between fly PCP signaling and the vertebrate CE pathway.     

A genomewide survey of developmentally relevant genes in Ciona intestinalis. VIII. Genes for PI3K signaling and cell cycle

Cell growth and cell divisions are two fundamental biological processes for cells in multi-cellular organisms. The molecules involved in these biological processes are highly conserved within eukaryotes, including plants and unicellular organisms such as yeast. However, some regulatory molecules seem to be innovated during animal evolution. Therefore, to understand how the ubiquitous systems have evolved or have been conserved, we examined genes for the phosphoinositide 3-kinase (PI3K) pathway that is important for cell growth, and genes for cell cycle regulation in the genome of Ciona intestinalis. It was found that the Ciona intestinalis genome contains all the essential constituents of the PI3K pathway. In addition, the class IB PI3K catalytic and regulatory subunits, which had not previously been known in animals other than mammals, were found in the Ciona genome. Similarly, all essential cyclins and CDKs were found in the Ciona genome, while cyclin G and cyclin L were likely to be independently lost in the ascidian lineage, which may be dispensable for the cell cycle. Cyclin F, which was previously known only in vertebrates, was not found in the Ciona genome. Therefore, this gene was probably innovated during the evolution of vertebrates to be involved in vertebrate-specific cell cycle regulation. Since Ciona is regarded as one of the most primitive extant chordates, the present analysis gives us an insight into how these fundamental biological genes are evolved or are conserved during chordate evolution.     

Computational analysis of Ciona intestinalis operons

Operons are clusters of genes that are co-regulated from a common promoter. Operons are typically associated with prokaryotes, although a small number of eukaryotes have been shown to possess them. Among metazoans, operons have been extensively characterized in the nematode Caenorhabditis elegans in which ～15% of the total genes are organized into operons. The most recent genome assembly for the ascidian Ciona intestinalis placed ～20% of the genes (2909 total) into 1310 operons. The majority of these operons are composed of two genes, while the largest are composed of six. Here is reported a computational analysis of the genes that comprise the Ciona operons. Gene ontology (GO) terms were identified for about two-thirds of the operon-encoded genes. Using the extensive collection of public EST libraries, estimates of temporal patterns of gene expression were generated for the operon-encoded genes. Lastly, conservation of operons was analyzed by determining how many operon-encoded genes were present in the ascidian Ciona savignyi and whether these genes were organized in orthologous operons. Over 68% of the operon-encoded genes could be assigned one or more GO terms and 697 of the 1310 operons contained genes in which all genes had at least one GO term. Of these 697 operons, GO terms were shared by all of the genes within 146 individual operons, suggesting that most operons encode genes with unrelated functions. An analysis of operon gene expression from nine different EST libraries indicated that for 587 operons, all of the genes that comprise an individual operon were expressed together in at least one EST library, suggesting that these genes may be co-regulated. About 50% (74/146) of the operons with shared GO terms also showed evidence of gene co-regulation. Comparisons with the C. savignyi genome identified orthologs for 1907 of 2909 operon genes. About 38% (504/1310) of the operons are conserved between the two Ciona species. These results suggest that like C. elegans, operons in Ciona are comprised of a variety of genes that are not necessarily related in function. The genes in only 50% of the operons appear to be co-regulated, suggesting that more complex gene regulatory mechanisms are likely operating.     

Loss of ancestral genes in the genomic evolution of Ciona intestinalis

Comparison of the predicted protein sets encoded by the complete genomes of two vertebrates (human and pufferfish), the urochordate Ciona intestinalis, three nonchordate animals, and two fungi were used to reconstruct a set of gene families present in the common ancestor of chordates. These ancestral families were much more likely to be lost in Ciona than in either vertebrate. In addition, of 256 duplicate gene pairs that arose by duplication prior to the most recent common ancestor of vertebrates and insects, one of the duplicate genes was four times as likely to be lost in Ciona as in the vertebrates. These results show that the genome of Ciona is not representative of the ancestral chordate genome with respect to gene content but rather shows derived features that may reflect adaptation of the specific ecological niche of urochordates.     

A cDNA resource from the basal chordate Ciona intestinalis

The genome of the basal choradate Ciona intestinalis contains a basic set of genes with less redundancy compared to the vertebrate genome. Extensive EST analyses, cDNA sequencing, and clustering yielded "Ciona intestinalis Gene Collection Release 1," which contains cDNA clones for 13,464 genes, covering nearly 85% of the Ciona mRNA species. This release is ready for use in cDNA cloning, micro/macroarray analysis, and other comprehensive genome-wide analyses for further molecular studies of basal chordates.     

A genomewide survey of developmentally relevant genes in Ciona intestinalis. X. Genes for cell junctions and extracellular matrix

Cell junctions and the extracellular matrix (ECM) are crucial components in intercellular communication. These systems are thought to have become highly diversified during the course of vertebrate evolution. In the present study, we have examined whether the ancestral chordate already had such vertebrate systems for intercellular communication, for which we have searched the genome of the ascidian Ciona intestinalis. From this molecular perspective, the Ciona genome contains genes that encode protein components of tight junctions, hemidesmosomes and connexin-based gap junctions, as well as of adherens junctions and focal adhesions, but it does not have those for desmosomes. The latter omission is curious, and the ascidian type-I cadherins may represent an ancestral form of the vertebrate type-I cadherins and desmosomal cadherins, while Ci-Plakin may represent an ancestral protein of the vertebrate desmoplakins and plectins. If this is the case, then ascidians may have retained ancestral desmosome-like structures, as suggested by previous electron-microscopic observations. In addition, ECM genes that have been regarded as vertebrate-specific were also found in the Ciona genome. These results suggest that the last common ancestor shared by ascidians and vertebrates, the ancestor of the entire chordate clade, had essentially the same systems of cell junctions as those in extant vertebrates. However, the number of such genes for each family in the Ciona genome is far smaller than that in vertebrate genomes. In vertebrates these ancestral cell junctions appear to have evolved into more diverse, and possibly more complex, forms, compared with those in their urochordate siblings.     

Globin genes are present in Ciona intestinalis

The key position of the Ciona intestinalis basal to the vertebrate phylogenetic tree brings up the question of which respiratory proteins are used by the tunicate to facilitate oxygen transport and storage. The publication of the Ciona draft genome sequence suggests that globin genes are completely missing and that-like some molluscs and arthropods-the sea squirt uses hemocyanin instead of hemoglobin for respiration. However, we report here the presence and expression of at least four distinct globin gene/protein sequences in Ciona. This finding is in agreement with the ancestral phylogeny of the vertebrate globins. Moreover, it seems likely that the Ciona hemocyanin-like sequences have enzymatic instead of respiratory functions.