Ascidians as a vertebrate-like model organism for physiological studies of Rho GTPase signaling

GTPases of the Rho family are evolutionarily conserved proteins that control cell shape dynamics during physiological processes as diverse as cell migration and polarity, axon outgrowth and guidance, apoptosis and phagocytosis. In mammals, 18 Rho proteins are distributed in 7 subfamilies. Rho, Rac and Cdc42 are the best-characterized ones, benefiting from the use of worm and drosophila, which only express these 3 subfamilies. An additional model would therefore help understand the physiological role of other mammalian subfamilies. We identified in genome databases the complete Rho family of two ascidians, Ciona intestinalis and Ciona savignyi, and showed that these families contain single ancestors of most mammalian Rho subfamilies. In Ciona intestinalis, all Rho genes are expressed and display specific developmental variations of mRNA expression during tadpole formation. Although C. intestinalis expresses five additional Rac compared to the closely related Ciona savignyi, only two appeared fully active in functional assays. Last, we identified in Ciona intestinalis database more than 50 Rho regulators (RhoGEFs and RhoGAPs) and 20 effector targets, whose analysis further supports the notion that Rho signaling components are of comparable complexity in mammals and ascidians. Since the tadpole of ascidians combines vertebrate-like developmental features with reduced cell number, particularly adapted to evolutionary and developmental biology studies, our data advocate this model for physiological studies of Rho signaling pathways.     

Novel genes involved in canonical Wnt/beta-catenin signaling pathway in early Ciona intestinalis embryos

We report here characterization of five genes for novel components of the canonical Wnt/ β -catenin signaling pathway. These genes were identified in the ascidian Ciona intestinalis through a loss-of-function screening for genes required for embryogenesis with morpholinos, and four of them have counterparts in vertebrates. The five genes we studied are as follows: Ci-PGAP1 , a Ciona orthologue of human PGAP1 , which encodes GPI (glycosylphosphatidylinositol) inositol-deacylase, Ci-ZF278 , a gene encoding a C2H2 zinc-finger protein, Ci-C10orf11 , a Ciona orthologue of human C10orf11 that encodes a protein with leucine-rich repeats, Ci-Spatial/C4orf17 , a single counterpart for two human genes Spatial and C4orf17 , and Ci-FLJ10634 , a Ciona orthologue of human FLJ10634 that encodes a member of the J-protein family. Knockdown of each of the genes mimicked β -catenin knockdown and resulted in suppression of the expression of β -catenin downstream genes ( Ci-FoxD , Ci-Lhx3 , Ci-Otx and Ci-Fgf9/16/20 ) and subsequent endoderm formation. For every gene, defects in knockdown embryos were rescued by overexpression of a constitutively active form, but not wild-type, of Ci- β -catenin. Dosage-sensitive interactions were found between Ci-β-catenin and each of the genes. These results suggest that these five genes act upstream of or parallel to Ci- β -catenin in the Wnt/ β -catenin signaling pathway in early Ciona embryos.     

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.     

Cytoplasmic intermediate filament protein expression in tunicate development: a specific marker for the test cells

The urochordate Ciona intestinalis is a well established system for embryological studies, and large scale EST sequences begin to emerge. We cloned five cytoplasmic intennediate filament (IF) cDNAs and made specific antibodies to the recombinant proteins. Self-assembly studies and immunofluorescence microscopy were used to study these proteins and their distribution. Confirming and extending previous studies in Styela, we found that Ciona protein IF-A is expressed in muscle and forms homopolymeric filaments while proteins IF-C and IF-D, which form only obligatory heteropolymeric filaments, resemble a keratin pair exclusively found in the entire epidermis. Protein IF-B and the new protein IF-F potentially reflect tunicate-specific IF proteins. They are found in the entire internal epithelia including the neural gland. We also extended the analysis to earlier developmental stages of Ciona. Protein IF-A is expressed in muscle from larval stages, whereas proteins IF-C and IF-D are found only in the tail epidermis. Protein IF-F is detected abundantly in the test cells of eggs, embryos and premetamorphic larvae. Our studies show that IF proteins could prove very useful markers in the study of cell fate determination in Ciona. They also support previous findings on the evolutionary relationships of different IF proteins. Non-vertebrate chordates have IF proteins which represent orthologs of vertebrate type I to III proteins, but also IF proteins that do not seem to fit into these classes. However, the intron positions of all tunicate IF genes are conserved with vertebrate type I to III genes, pointing to a common evolutionary origin.     

Characterization of novel GPCR gene coding locus in amphioxus genome: gene structure, expression, and phylogenetic analysis with implications for its involvement in chemoreception

Chemosensation is the primary sensory modality in almost all metazoans. The vertebrate olfactory receptor genes exist as tandem clusters in the genome, so that identifying their evolutionary origin would be useful for understanding the expansion of the sensory world in relation to a large-scale genomic duplication event in a lineage leading to the vertebrates. In this study, I characterized a novel GPCR (G-protein-coupled receptor) gene-coding locus from the amphioxus genome. The genomic DNA contains an intronless ORF whose deduced amino acid sequence encodes a seven-transmembrane protein with some amino acid residues characteristic of vertebrate olfactory receptors (ORs). Surveying counterparts in the Ciona intestinalis (Asidiacea, Urochordata) genome by querying BLAST programs against the Ciona genomic DNA sequence database resulted in the identification of a remotely related gene. In situ hybridization analysis labeled primary sensory neurons in the rostral epithelium of amphioxus adults. Based on these findings, together with comparison of the developmental gene expression between amphioxus and vertebrates, I postulate that chemoreceptive primary sensory neurons in the rostrum are an ancient cell population traceable at least as far back in phylogeny as the common ancestor of amphioxus and vertebrates.     

Expression cloning in ascidians: isolation of a novel member of the asctacin protease family

The small genome size and gene number of ascidians makes them an ideal model system in which to screen for conserved genes that regulate the development of chordates. Expression cloning has proven to be an effective strategy for isolating genes that play a role in embryogenesis. We have taken advantage of the large size and ease of manipulation of Xenopus embryos for use as an assay system to screen for developmental regulatory genes from the ascidian Ciona intestinalis. Many invertebrate genes have been shown to function in vertebrates, providing us with precedent for our cross-species analysis. The first clone isolated from this screen is an astacin class metalloprotease. This ascidian astacin, named no va, causes a gastrulation defect in Xenopus. In C. intestinalis, no va is expressed both maternally and zygotically. The zygotic expression is seen in the mesenchyme of gastrula and neurula staged embryos.     

Further characterization of genes expressed during Ciona intestinalis metamorphosis

Metamorphosis of ascidians is a dynamic event by which a nonfeeding, mobile tadpole larva is transformed into a filter-feeding, fixed juvenile. This process usually begins with the settlement of the larva and is followed by a series of coordinated morphogenetic movements that rearrange organs, tissues, and cells. To identify genes that are involved in the initiation of metamorphosis, we conducted differential screening between mRNAs of swimming larvae and those of juveniles in Ciona intestinalis. This screening permitted the isolation of cDNA clones for genes whose expression is upregulated during metamorphosis, and the characterization of four such genes (Ci-meta3, Ci-meta4, Ci-meta5 and Ci-meta6) is reported here. Ci-meta3 encodes a protein with a domain found in Sp1a and the RYanodine receptor. This gene is not expressed in early swimming larvae but is expressed in the endoderm region and part of the retractile tail region in metamorphosing juveniles. The predicted proteins encoded by Ci-meta4, Ci-meta5 and Ci-meta6 do not contain any known consensus motifs, nor do they show any similarity to known proteins. Ci-meta4 and Ci-meta5 are expressed weakly in mesenchyme cells of the early larva and strongly in the metamorphosing juvenile, while Ci-meta6 is expressed in the mesenchyme in the late larva. In addition, we characterized 53 independent cDNA clones whose expression was downregulated during the period from early swimming larvae to metamorphosing juveniles by taking advantage of the Ciona intestinalis cDNA project database and BLAST searches. The expression patterns of some of these clones were changed during the larval period.     

Characterization of Brachyury-downstream notochord genes in the Ciona intestinalis embryo

The notochord has two major roles during chordate embryogenesis, as a source of inductive signals for the patterning of neural tube and paraxial mesoderm and as a supportive organ of the larval tail. Despite the recent identification of mutations that affect the notochord development in vertebrate embryos, little is known about genes that are expressed in the differentiating notochord itself. In the urochordate ascidian Ciona intestinalis, Brachyury (Ci-Bra) plays a key role in notochord differentiation. In a previous study, we isolated cDNA clones for nearly 40 potential Ci-Bra target genes that are expressed in notochord cells (H. Takahashi et al., 1999, Genes Dev. 13, 1519-1523). Here we characterized 20 of them by determining the complete nucleotide sequences of the cDNAs. These genes encode a broad spectrum of divergent proteins associated with notochord formation and function. Two genes encode ascidian homologs of the Drosophila Prickle LIM domain proteins and another encodes the ERM protein, all 3 of which appear to be involved in the control of cytoskeletal architecture. In addition, genes for netrin, leprecan, cdc45, ATP:citrate lyase, ATP sulfurylase/APS kinase, protein tyrosine phosphatase, beta4-galactosyltransferase, fibrinogen-like protein, divergent tropomyosin-like proteins, and Drosophila Pellino-like protein were identified. The observation of the netrin gene expression in the notochord may provide the first molecular evidence that the ascidian notochord is a source of signals as in vertebrates. In addition, the present information should be used to identify nonchordate deuterostome tissues homologous to the notochord as well as genes which are expressed in the notochord cells of vertebrate embryos.     

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.