The Ciona intestinalis genome: when the constraints are off

The recent genome sequencing of a non-vertebrate deuterostome, the ascidian tunicate Ciona intestinalis, makes a substantial contribution to the fields of evolutionary and developmental biology.1 Tunicates have some of the smallest bilaterian genomes, embryos with relatively few cells, fixed lineages and early determination of cell fates. Initial analyses of the C. intestinalis genome indicate that it has been evolving rapidly. Comparisons with other bilaterians show that C. intestinalis has lost a number of genes, and that many genes linked together in most other bilaterians have become uncoupled. In addition, a number of independent, lineage-specific gene duplications have been detected. These new results, although interesting in themselves, will take on a deeper significance once the genomes of additional invertebrate deuterostomes (e.g. echinoderms, hemichordates and amphioxus) have been sequenced. With such a broadened database, comparative genomics can begin to ask pointed questions about the relationship between the evolution of genomes and the evolution of body plans.     

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.     

Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc-finger nucleases

Zinc‐finger nucleases (ZFNs) are engineered nucleases that induce DNA double‐strand breaks (DSBs) at target sequences. They have been used as tools for generating targeted mutations in the genomes of multiple organisms in both animals and plants. The DSB induced by ZFNs is repaired by non‐homologous end joining (NHEJ) or by homologous recombination (HR) mechanisms. Non‐homologous end joining induces some errors because it is independent of a reference DNA sequence. Through the NHEJ mechanism, ZFNs generate insertional or deletional mutations at the target sequence. We examined the usability, specificity and toxicity of ZFNs in the basal chordate Ciona intestinalis. As the target of ZFNs, we chose an enhanced green fluorescent protein (EGFP) gene artificially inserted in the C. intestinalis genome because this locus is neutral for the development and growth of C. intestinalis, and the efficiency of mutagenesis with ZFNs can thus be determined without any bias. We introduced EGFP ‐ZFN mRNAs into the embryos of an EGFP ‐transgenic line and observed the mutation frequency in the target site of EGFP . We also examined the effects of the EGFP ‐ZFNs at off‐target sites resembling the EGFP target sequence in the C. intestinalis genome in order to examine the specificity of ZFNs. We further investigated the influence of ZFNs on embryogenesis, and showed that adequate amounts of ZFNs, which do not disrupt embryogenesis, can efficiently induce mutations on the on‐target site with less effect on the off‐target sites. This suggests that target mutagenesis with ZFNs will be a powerful technique in C. intestinalis.     

Spermatozoa of Ciona intestinalis and analysis of ascidian fertilization

The complex array of vestments which surrounds the ascidian oocyte indicates that spermatozoa of these urochordates may be highly modified to effect penetration through the egg-envelopes and to achieve successful fusion with the female gamete. Examination of the spermatozoa of Ciona intestinalis reveals, however, that they lack an acrosomal vesicle and do not possess detectable amounts of actin. Furthermore, the spermatozoa do not undergo observable morphological alterations when exposed to ammoniated seawater, a treatment known to produce aggregation and an acrosome reaction in other organisms. Ciona spermatozoa do contain significant quantities of proteases. It is hypothesized that the localization of these enzymes may be related to the ridge-like surface ornamentation which is observed as a component of the head region of Ciona spermatozoa.     

Studies on the receptors in Ciona intestinalis

Summary A photoreceptor type structure not previously described has been found in the dorsal wall of the cerebral vesicle of the tadpole larva of Ciona intestinalis. The membranes of this receptor are organised as tubules some 60–100 nm in diameter and up to 1.5 m long. The tubules are confined in bundles about 1.5 m in diameter, which extend from the cell surface into the cavity of the cerebral vesicle. These tubules are similar to those in the rhabdomeric type of photoreceptor. However, in the cells from which the tubule processes arise are structures typical of the bases of cilia, and found in ciliary type photoreceptors.I should like to thank Professor J. Z. Young, F. R. S. for his continuing encouragement and help, and Dr. R. Bellairs for the use of electron microscope facilities. Mr. R. Moss and Mrs. J. Hamilton gave excellent technical assistance.     

EST analysis of gene expression in testis of the ascidian Ciona intestinalis

To explore the gene expression underlying spermatogenesis, a large-scale analysis has been done on the cDNAs from testis of the ascidian, Ciona intestinalis. A set of 5,461 expressed sequence tags was analyzed and grouped into 2,806 independent clusters. Approximately 30% of the clusters showed significant sequence matches to the proteins reported in DDBJ/GenBank/EMBL database including a set of proteins closely related to the gene regulation during spermatogenesis, functional and morphological changes of spermatogenic cells during spermiogenesis, and physiological functions of sperm, as well as those with housekeeping functions commonly expressed in other cells. Some clones show similarities to the proteins present in vertebrate lymphocytes, suggesting a primitive immune system in ascidians. We have also found some genes that are known to participate in hormonal regulation of spermatogenesis in vertebrates. The large majority of the genes expressed in Ciona testis show no significant matches to known proteins and the further analysis of these genes may shed new light on the molecular mechanism of spermatogenesis and sperm functions.     

Immunological Maturation in the Tunicate Ciona intestinalis

The least explored areas in the phylogenetic development ofimmunity are the primitive chordate subphyla. The limited studieson tunicate internal defense mechanisms are presented and discussed.Primary and secondary immune responses and immunological maturationto vertebrate erythrocytes in Ciona intestinalis suggest thatits internal defense mechanisms may be closely allied to boththe invertebrates and vertebrates.     

Morphological evidence that the molecularly determined Ciona intestinalis type A and type B are different species: Ciona robusta and Ciona intestinalis

Ciona intestinalis is considered a widespread and easily recognizable tunicate, the sister group of vertebrates. In recent years, molecular studies suggested that C. intestinalis includes at least two cryptic species, named ‘type A’ and ‘type B’, morphologically indistinguishable. It is dramatic to certify that two different species may be hidden under the name of a species widely used as a model species in biological researches. This raised the problem of identifying diagnostic morphological characters capable of distinguishing these types. We compared the morphology of specimens belonging to the two types and found that only type A specimens possess tunic tubercular prominences, allowing unambiguous discrimination. Remarkably, these structures were already described as distinctive of the Japanese species Ciona robusta, Hoshino and Tokioka, 1967; later synonymized under C. intestinalis (sensu Millar, 1953). In this study, we have confirmed that C. intestinalis type A corresponds to C. robusta. Based on the geographic distribution of C. intestinalis type B, and considering that the original C. intestinalis species was described from North European waters, we determined that C. intestinalis type B corresponds to C. intestinalis as described by Millar in 1953 and possibly to Linnaeus' Ascidia intestinalis L., 1767 for which we have deposited a neotype (from Roscoff, France) and for which we retain the name Ciona intestinalis (Linnaeus, 1767).     

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.     

Sensing charges of the Ciona intestinalis voltage-sensing phosphatase

Voltage control over enzymatic activity in voltage-sensitive phosphatases (VSPs) is conferred by a voltage-sensing domain (VSD) located in the N terminus. These VSDs are constituted by four putative transmembrane segments (S1 to S4) resembling those found in voltage-gated ion channels. The putative fourth segment (S4) of the VSD contains positive residues that likely function as voltage-sensing elements. To study in detail how these residues sense the plasma membrane potential, we have focused on five arginines in the S4 segment of the Ciona intestinalis VSP (Ci-VSP). After implementing a histidine scan, here we show that four arginine-to-histidine mutants, namely R223H to R232H, mediate voltage-dependent proton translocation across the membrane, indicating that these residues transit through the hydrophobic core of Ci-VSP as a function of the membrane potential. These observations indicate that the charges carried by these residues are sensing charges. Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing.     

An integrated database of the ascidian, Ciona intestinalis: towards functional genomics

An integrated genome database is essential for future studies of functional genomics. In this study, we update cDNA and genomic resources of the ascidian, Ciona intestinalis, and provide an integrated database of the genomic and cDNA data by extending a database published previously. The updated resources include over 190,000 ESTs (672,396 in total together with the previous ESTs) and over 1,000 full-insert sequences (6,773 in total). In addition, results of mapping information of the determined scaffolds onto chromosomes, ESTs from a full-length enriched cDNA library for indication of precise 5'-ends of genes, and comparisons of SNPs and indels among different individuals are integrated into this database, all of these results being reported recently. These advances continue to increase the utility of Ciona intestinalis as a model organism whilst the integrated database will be useful for researchers in comparative and evolutionary genomics.     

Identification of neuron-specific promoters in Ciona intestinalis

We isolated 5' flanking regions of four genes, Ci-Galphai1, Ci-arr, Ci-vAChTP, and Ci-vGAT, each of which is expressed in distinct sets of neurons in the central nervous system of the ascidian Ciona intestinalis, and we examined their function by introducing green fluorescent protein (GFP)-fusion constructs into Ciona embryos. The reporter gene driven by the 5' flanking region of Ci-Galphai1, Ci-arr, and Ci-vAChTP recapitulated the endogenous gene expression patterns, while that of Ci-vGAT can drive GFP expression in particular subsets of neurons expressing the endogenous gene. Deletion analysis revealed that the Ci-Galphai1 promoter consists of multiple regulatory modules controlling the expression in different types of cells. The GFP fluorescence enabled visualization of cell bodies and axons of different sets of neurons in ascidian larvae. These promoters can be a powerful tool for studying molecular mechanisms of neuronal development as well as neuron networks and functions in ascidians.