Direct examination of chromosomal clustering of organ-specific genes in the chordate Ciona intestinalis

One of challenges in the field of developmental biology is to understand how spatially and/or temporally coordinated expression of genes is controlled at the chromosomal level. It remains controversial whether genes expressed in a given tissue are randomly distributed throughout a given animal genome, or instead resolve into clusters. Here we used microarray analysis to identify more than 1,700 genes that are expressed preferentially in each of 11 organs of the chordate Ciona intestinalis adult, and determined the location of these genes on the 14 pairs of Ciona chromosomes. In spite of extensive mapped gene analysis, we only confirmed small clusters containing two or three genes. Our result indicates that organ-specific genes are distributed rather evenly all over chromosomes, suggesting that the notion of clustering of organ-specific genes in animal genomes is not generally applicable to this chordate.     

Large-scale characterization of genes specific to the larval nervous system in the ascidian Ciona intestinalis

The central and peripheral nervous systems (CNS and PNS) of the ascidian tadpole larva are comparatively simple, consisting of only about 350 cells. However, studies of the expression of neural patterning genes have demonstrated overall similarity between the ascidian CNS and the vertebrate CNS, suggesting that the ascidian CNS is sufficiently complex to be relevant to those of vertebrates. Recent progress in the Ciona intestinalis genome project and cDNA project together with considerable EST information has made Ciona an ideal model for investigating molecular mechanisms underlying the formation and function of the chordate nervous system. Here, we characterized 56 genes specific to the nervous system by determining their full-length cDNA sequences and confirming their spatial expression patterns. These genes included those that function in the nervous systems of other animals, especially those involved in photoreceptor-mediated signaling and neurotransmitter release. Thus, the nervous system-specific genes in Ciona larvae will provide not only probes for determining their function but also clues for exploring the complex network of nervous system-specific genes.     

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.     

Calcitonin-like immunochemical staining in the alimentary tract of Ciona intestinalis L.

Summary Calcitonin-like immunoreactivity has been found with the peroxidase-anti-peroxidase (PAP) method in cells of the epithelium of the alimentary tract as well as in nerve cells and nerve fibers in the connective tissue underlying the epithelium of the alimentary tract of Ciona intestinalis L. The nature of these cells is discussed with reference to endocrine-like cells found in the alimentary tract of other protochordates and to the possible dual role of calcitonin occurring in the gastroenteropancreatic system, on the one hand, and in the nervous system, on the other.     

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.     

脑红蛋白和细胞红蛋白：携氧蛋白质家族2个新成员

脑红蛋白(neuroglobin, Ngb)和细胞红蛋白(cytoglobin, Cygb)是新发现的2个携氧蛋白家族的成员.脑红蛋白主要存在于脑中,而细胞红蛋白在全身各个组织都含有,它们和另外2个携氧蛋白——血红蛋白和肌红蛋白的同源性<25%,但它们在种属之间的同源性很高(>95%).脊椎动物脑红蛋白基因定位于14q24,细胞红蛋白基因定位于17q25,都含有4个外显子和3个内含子.2种蛋白在生理条件下含有6个配位键,不同于血红蛋白和肌红蛋白的5个配位键结构.这2种新蛋白和氧都具有很高的亲和力,在缺氧条件下其基因及蛋白表达都有明显的提升,对细胞的存活有一定保护作用.对于脑红蛋白和细胞红蛋白的功能研究,有助于更好地了解机体氧代谢和氧利用过程,并为临床在缺氧损伤时的治疗提供新的观点和途径.     

Characterization of highly polymorphic nuclear microsatellite loci from the ascidian Ciona intestinalis

Eight nuclear polymorphic microsatellite markers were characterized from the ascidian Ciona intestinalis whole genome sequence. The behaviour of these loci was investigated against two geographically distinct populations: one from Plymouth, UK the other from the Fusaro Lagoon, Italy, both belonging to the type A Ciona cryptic species. The markers exhibited six to 29 alleles and average observed heterozygosity ranging from 0.06 to 0.73. These new microsatellite loci demonstrated to be valuable tools for both population genetic analysis at different scales and genetic identification of mutant phenotypes frequently encountered in Mediterranean populations of C. intestinalis.     

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).     

The acrosomal region of the spermatozoon of Ciona intestinalis: Its relationship with the binding to the vitelline coat of the egg

This paper describes a study of the apical region of the spermatozoon of Ciona intestinalis before and during its binding to the vitelline coat of the egg. A combination of the techniques of thin sectioning, negative staining, and freeze fracture has revealed that in the apical-most region, where a small acrosomal vesicle lies on the flat tip of the nucleus, there is a cap-like region almost completely free of particles on the P face of the plasma membrane. The particle-free area is surrounded by two circlets of orderly arranged particles. Upon binding to the vitelline coat the particles of the distal circlet show a partial displacement, while the particles of the apical circlet remain unaltered. The relationship between the apical circlet and the binding process is discussed. The final step of the acrosome reaction, which occurs in only a few of the bound spermatozoa, consists in the fusion of the plasma membrane with the acrosomal membrane, in the dehiscence of the acrosomal contents and finally in the formation of membrane tubules.     

Induction of anterior neural fates in the ascidian Ciona intestinalis

The sensory vesicle of ascidians is thought to be homologous to the vertebrate forebrain and midbrain (Development 125 (1998) 1113). Here we report the isolation of two sensory vesicle markers in the ascidian Ciona intestinalis, which are homologs of vertebrate otx and gsx homeobox genes. By using these markers to analyze the induction of anterior neural tissue in Ciona, we find that the restriction of anterior neural fate to the progeny of the anterior animal blastomeres is due to a combination of two factors. The vegetal blastomeres show a differential inducing activity along the anterior-posterior axis, while the competence to respond to this inducing signal is markedly higher in the anterior animal blastomeres than in the posterior animal blastomeres. This differential competence to respond is also observed in response to bFGF, a candidate neural inducer in ascidians (J. Physiol. 511.2 (1998) 347) and can be detected by the gastrula stage. Our results, however, indicate that bFGF can only induce a subset of the responses of the endogenous inducer, suggesting that additional signals in the embryo are necessary to induce a fully patterned nervous system.     

ciCD94-1, an ascidian multipurpose C-type lectin-like receptor expressed in Ciona intestinalis hemocytes and larval neural structures

Abstract C-type lectins play an important role in the immune system and are part of a large superfamily that includes C-type lectin-like domain (CTLD)-containing proteins. Divergent evolution, acting on the CTLD fold, has generated the Ca²⁺-dependent carbohydrate-binding lectins and molecules, as the lectin-like natural killer (NK) receptors that bind proteins, rather than sugars, in a Ca²⁺-independent manner. We have studied ciCD94-1, a CTLD-containing protein from the tunicate Ciona intestinalis , which is a homolog of the CD94 vertebrate receptor that is expressed on NK cells and modulates their cytotoxic activity by interacting with MHC class I molecules. ciCD94-1 shares structural features with the CTLD-containing molecules that recognize proteins, suggesting that it could be located along the evolutionary pathway leading to the NK receptors.
ciCD94-1 was up-regulated in response to inflammation induced by lipopolysaccharide (LPS) acting on a blood cell type present in both the tunic and circulating blood. Furthermore, an anti-ciCD94-1 antibody specifically inhibited the phagocytic activity of these cells. ciCD94-1 was also expressed during development in the larva and in the early stages of metamorphosis in structures related to the nervous system, and loss of its function affected the correct differentiation of these territories. These findings suggest that ciCD94-1 has different roles in immunity and in development, thus strengthening the concept of gene co-option during evolution and of an evolutionary relationship between the nervous and the immune systems.     

No chromosomal clustering of housekeeping genes in the marine chordate Ciona intestinalis

Housekeeping genes, widely expressed genes that are required for the basal function of most cell types, are clustered in the human and worm genomes. This arrangement suggests coordinate control of housekeeping gene expression at the chromosomal level. Here we examined whether this notion is applicable to a marine chordate, Ciona intestinalis. Using microarrays, we analyzed genes that were expressed in 11 organs of the adult, including the neural complex, branchial sac, esophagus, stomach, endostyle, intestine, body-wall muscle, heart, blood cells, ovary and testis. This analysis identified 158 genes that are expressed ubiquitously in these organs. These housekeeping genes could be classified into a range of Gene Ontology categories, in particular, ribosomal protein components. Of these 158 genes, we were able to map 141 genes onto the 14 pairs of the C. intestinalis chromosomes. They were distributed rather evenly over all the chromosomes, except for small clusters containing two or three genes. Therefore, the notion of chromosomal clustering of housekeeping genes is not applicable in this chordate.     

Inducible galectins are expressed in the inflamed pharynx of the ascidian Ciona intestinalis

Although ascidians belong to a key group in chordate phylogenesis, amino acid sequences of Ciona intestinalis galectin-CRDs (CiLgals-a and -b) have been retained too divergent from vertebrate galectins. In the present paper, to contribute in disclosing Bi-CRD galectin evolution a novel attempt was carried out on CiLgals-a and -b CRDs phylogenetic analysis, and their involvement in ascidian inflammatory responses was shown. CiLgals resulted aligned with Bi-CRD galectins from vertebrates (Xenopus tropicalis, Gallus gallus, Mus musculus, Homo sapiens), cephalochordates (Branchiostoma floridae), echinoderms (Strongylocentrotus purpuratus) and a mono-CRD galectin from the ascidian Clavelina picta. The CiLgals-a N-terminal and C-terminal CRDs contain the signature sequence involved in carbohydrate binding, whereas the CiLgals-b C-CRD presents only three out of seven key aminoacids and it could not be suitable as sugar binding motif. Sequence similarity between clusters suggests an evolutionary model based on CRD domain gene duplication and sequence diversification. In particular CiLgals-b N-CRD and C-CRD were similar to each other and both grouped with the ascidian C. picta mono-CRD. Homology modeling process shows a CiLgals molecular structure superimposed to chicken and mouse galectins. The CiLgals-a and CiLgals-b genes were upregulated by LPS inoculation suggesting that they are inducible and expressed in the inflamed pharynx as revealed by real-time PCR analysis. Finally, in situ hybridization and immunohistochemical assays showed their localization in the inflamed tissues, while immunoblotting analysis indicated that CiLgals can form oligomers.