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.     

Studies on fertilization in the Ascidans. I. Self-sterility and specific recognition between gametes of Ciona intestinalis

Density dependent cell death is shown to be an important process during adjustment of stationary cell densities in 3T3 cell populations under certain conditions. The present data suggest a common mechanistic basis for density dependent cell death and density dependent inhibition of cell division. In SV40-3T3 cell populations density dependent growth-inhibiting processes may occur transiently.     

Protochordate immunity. I. Primary immune response of the tunicate Ciona intestinalis to vertebrate erythrocytes

Serum from the tunicate Ciona intestinalis contains a low titer, natural agglutinin for a variety of vertebrate erythrocytes. Primary injections of 8.5 × 10³ human erythrocytes or 7.0 × 10² duck erythrocytes into the tunic tissues or perivisceral cavity were agglutinated and then cleared by phagocytosis within 24 hr. Higher concentrations of human or duck erythrocytes injected into the tunic tissues were encapsulated. Concomitant with clearance or encapsulation, serum agglutinin levels decreased and then returned to normal within 24 hr. Tunicates are classified taxonomically with the vertebrates in the phylum Chordata. Since phagocytosis and encapsulation reactions are typical of most invertebrates, the results of this study suggest that tunicate internal defense mechanisms are more closely allied to invertebrates than to 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.     

Studies on fertilization in the ascidians : Fucosyl sites on vitelline coat of Ciona intestinalis

The vitelline coat (originally called chorion) of the ascidian egg is the site where species-specific recognition and binding of spermatozoa occurs. Recent findings from this laboratory have suggested that fucosyl residues are present on the vitelline coat of Ciona intestinalis eggs and play an important role in the process of fertilization. The results reported in this paper confirm and extend those findings. With Fucose Binding Protein (FBP) and Fucosyl-Ferritin as markers, fucosyl sites have been localized on the fibrillar tufts emerging from the outer surface of the vitelline coat, both on glycerol-treated eggs and on living eggs deprived of follicle cells at pH 5. Observations on isolated vitelline coats have shown that the inner surface does not contain fucosyl sites. Studies performed with ¹²⁵I-FBP on glycerol-treated eggs have indicated that two distinct classes of fucosyl sites are present on the vitelline coat. The association constants for FBP of the high affinity and of the low affinity class are 2.3×10⁶ and 4.1×10⁵ respectively. The gel electrophoresis of the proteins extracted in sodium dodecyl sulfate (SDS) from sonicated vitelline coats has shown three fucosyl-containing polypeptide bands.     

Germline transgenesis of the ascidian Ciona intestinalis by electroporation

Microinjection of the Minos transposon is the only reported technique for generating stable transgenic lines in the cosmopolitan ascidian, Ciona intestinalis. To establish a more amenable method for generating stable transgenic Ciona, we examined the possibility of using electroporation of DNA into eggs. From 0-44.4% of electroporated individuals transmitted transgenes to the next generation. The transgene was integrated into one chromosome and multiple copies of the transgene were inserted into one site of the chromosome, indicating that electroporation is an easy and powerful technique for achieving stable transgenesis in C. intestinalis. Together with possible inland culture of this ascidian, this technique will be useful for generating stable lines which have reporter gene expression in a specific tissue or organ and the generation of transposase-expressing stable transgenic (jump-starter) lines and mutator lines which contain a lot of Minos transposons in an insertion position.     

Genetic and Genomic Toolbox of the Chordate Ciona intestinalis

The experimental malleability and unique phylogenetic position of the sea squirt Ciona intestinalis as part of the sister group to the vertebrates have helped establish these marine chordates as model organisms for the study of developmental genetics and evolution. Here we summarize the tools, techniques, and resources available to the Ciona geneticist, citing examples of studies that employed such strategies in the elucidation of gene function in Ciona. Genetic screens, germline transgenesis, electroporation of plasmid DNA, and microinjection of morpholinos are all routinely employed, and in the near future we expect these to be complemented by targeted mutagenesis, homologous recombination, and RNAi. The genomic resources available will continue to support the design and interpretation of genetic experiments and allow for increasingly sophisticated approaches on a high-throughput, whole-genome scale.     

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.     

Blue-green algalike cells associated with the tunic of Ciona intestinalis L.

Summary Certain organisms resembling blue-green algae embedded in the tunic of the solitary ascidian Ciona intestinalis L. are described. Their probable symbiotic role as related to the peculiar habitat of this ascidian is suggested.     

Construction of BAC libraries derived from the ascidian Ciona intestinalis

Large insert genomic bacterial artificial chromosome (BAC) libraries were constructed from a basal chordate, the ascidian Ciona intestinalis. Insert analyses of randomly selected clones indicated that in the first library the mean insert size was 135 kb and predicted a 15-fold coverage of the Ciona genome, and in the second library the mean insert size was 165 kb and predicted a 5-fold coverage of the genome. These first large insert genomic libraries of the ascidian should increase the speed of genomic analyses of basal chordates.     

The evolution of the vertebrate metzincins; insights from Ciona intestinalis and Danio rerio

Background

The metzincins are a large gene superfamily of proteases characterized by the presence of a zinc protease domain, and include the ADAM, ADAMTS, BMP1/TLL, meprin and MMP genes. Metzincins are involved in the proteolysis of a wide variety of proteins, including those of the extracellular matrix. The metzincin gene superfamily comprises eighty proteins in the human genome and ninety-three in the mouse. When and how the level of complexity apparent in the vertebrate metzincin gene superfamily arose has not been determined in detail. Here we present a comprehensive analysis of vertebrate metzincins using genes from both Ciona intestinalis and Danio rerio to provide new insights into the complex evolution of this gene superfamily.

Results

We have identified 19 metzincin genes in the ciona genome and 83 in the zebrafish genome. Phylogenetic analyses reveal that the expansion of the metzincin gene superfamily in vertebrates has occurred predominantly by the simple duplication of pre-existing genes rather than by the appearance and subsequent expansion of new metzincin subtypes (the only example of which is the meprin gene family). Despite the number of zebrafish metzincin genes being relatively similar to that of tetrapods (e.g. man and mouse), the pattern of gene retention and loss within these lineages is markedly different. In addition, we have studied the evolution of the related TIMP gene family and identify a single ciona and four zebrafish TIMP genes.

Conclusion

The complexity seen in the vertebrate metzincin gene families was mainly acquired during vertebrate evolution. The metzincin gene repertoire in protostomes and invertebrate deuterostomes has remained relatively stable. The expanded metzincin gene repertoire of extant tetrapods, such as man, has resulted largely from duplication events associated with early vertebrate evolution, prior to the sarcopterygian-actinopterygian split. The teleost repertoire of metzincin genes in part parallels that of tetrapods but has been significantly modified, perhaps as a consequence of a teleost-specific duplication event.     

Test cell migration and tunic formation during posthatching development of the larva of the ascidian, Ciona intestinalis

Morphological changes in the tunic layers and migration of the test cells during swimming period in the larva of the ascidian, Ciona intestinalis , were observed by light and electron microscopy. The swimming period was divided into three stages. In stage 1, further formation of juvenile tunic layer started only in the larval trunk and neck region. In stage 2, the layer became swollen in the ventral and dorsal sides of the neck region and in stage 3, the swelling expanded backward. Concomitantly with these changes, the outermost larval tunic layer (outer cuticular layer), which had been formed before hatching, also swelled in the neck region in stage 2 and formed two humps in stage 3, although the layer did not change in the tail region during the swimming period. Test cells that were present over the entire larval tunic layer in stage 1 began to move from the surface of the fin toward that of the side of the body in stage 2, and finally gathered to form six bands running radially from the anterior end to the posterior end of the trunk region and aligned along the lateral sides of body in the tail region in stage 3. In electron microscopic observations, pseudopodia protruding from the test cells invaded the larval tunic, following which they extended proximate to the juvenile tunic in the trunk region. In the tail region, which had no juvenile tunic layer as that described, the pseudopodia invaded and remained adjacent to the surface of the epidermis or the sensory cilia protruded from the epidermis. Metamorphosis of the larvae, further tunic formation, degradation of adhesive papilla, attachment of larva to the substratum and tail resorption commenced after these morphological changes occurred. The possible role of the test cells in metamorphosis is discussed.     

Primary genetic linkage maps of the ascidian, Ciona intestinalis

For whole-genome analysis in a basal chordate (protochordate), we used F1 pseudo-testcross mapping strategy and amplified fragment length polymorphism (AFLP) markers to construct primary linkage maps of the ascidian tunicate Ciona intestinalis. Two genetic maps consisted of 14 linkage groups, in agreement with the haploid chromosome number, and contained 276 and 125 AFLP loci derived from crosses between British and Neapolitan individuals. The two maps covered 4218.9 and 2086.9 cM, respectively, with an average marker interval of 16.1 and 18.9 cM. We observed a high recombinant ratio, ranging from 25 to 49 kb/cM, which can explain the high degree of polymorphism in this species. Some AFLP markers were converted to sequence tagged sites (STSs) by sequence determination, in order to create anchor markers for the fragmental physical map. Our recombination tools provide basic knowledge of genetic status and whole genome organization, and genetic markers to assist positional cloning in C. intestinalis.     

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.     

Identification of testis‐specific ubiquitin‐conjugating enzyme in the ascidian Ciona intestinalis

The ubiquitin–proteasome system is known to play a key role in fertilization in ascidians, sea urchins, and mammals. To obtain insights into the ubiquitin‐conjugating enzymes (Ube2) involved in reproductive systems, we systematically explored Ube2 enzymes expressed in the testis of the ascidian Ciona intestinalis. Here, we report cDNA cloning and characterization of a novel type of Ube2r (Ci0100152677) that is capable of making a thiolester bond with ubiquitin. Northern analysis, whole‐mount in situ hybridization and immunocytochemistry indicate that this enzyme is exclusively expressed in the testis, mainly in the germ cells during the late stage of spermatogenesis, and is localized in the sperm head and tail, suggesting possible participation in fertilization or spermatogenesis/spermiogenesis. Mol. Reprod. Dev. 77: 640–647, 2010. © 2010 Wiley‐Liss, Inc.