Specific induction of self-discrimination by follicle cells in Ciona intestinalis oocytes

Self-incompatibility, a mechanism that prevents self-fertilization in ascidians, is based on the ability of the oocyte vitelline coat to distinguish and accept only heterologous spermatozoa. In Ciona intestinalis self-discrimination is established during late oogenesis and is contributed or controlled by products of the overlying follicle cells. In this study we have further investigated the role of the follicle cells in the onset of self-discrimination by using in vitro maturation of ovarian oocytes deprived of the follicle cells and incubated with either autologous or heterologous follicle cells. Fertilization assays demonstrate that the action of the follicle cells is exerted even when they are detached from the vitelline coat and that only autologous follicle cells can promote the induction of self-sterility on the egg coat. Electron microscopy of the oocytes during maturation reveals that the switch from self-fertility to self-sterility is accompanied by the appearance of a thin electron-dense layer on the outer surface of the vitelline coat. We suggest that the formation of this layer is the result of the interaction between products of the follicle cells and the autologous vitelline coat.     

Blood System Formation in the Urochordate Ciona intestinalis Requires the Variable Receptor vCRL1

Adaptive immune systems are present only in vertebrates. How do all the remaining animals withstand continuous attacks of permanently evolving pathogens? Even in the absence of adaptive immunity, every organism must be able to unambiguously distinguish "self" cells from any imaginable "nonself." Here, we analyzed the function of highly polymorphic gene vCRL1, which is expressed in follicle and blood cells of Ciona intestinalis, pointing to possible recognition roles either during fertilization or in immune reactions. By using segregation analysis, we demonstrate that vCRL1 locus is not involved in the control of self-sterility. Interestingly, genetic knockdown of vCRL1 in all tissues or specifically in hemocytes results in a drastic developmental arrest during metamorphosis exactly when blood system formation in Ciona normally occurs. Our data demonstrate that vCRL1 gene might be essential for the establishment of a functional blood system in Ciona. Presumably, presence of the vCRL1 receptor on the surface of blood cells renders them as self, whereas any cell lacking it is referred to as nonself and will be consequently destroyed. We propose that individual-specific receptor vCRL1 might be utilized to facilitate somatic self/nonself discrimination.     

Re-examination of sibling cross-sterility in the ascidian,Ciona intestinalis: genetic background of the self-sterility

Self-sterility of solitary ascidians is a typical example of the allogeneic recognition, though its molecular mechanism remains an open question. In this paper we analyze the fertility between siblings from selfed and crossed eggs to understand the genetic basis of self-sterility in the ascidian, Ciona intestinalis. First, we show that the self-sterility is strict and stable, and the individuality expressed in gametes is highly diversified in the wild population that we used. Secondly, we show one-way cross-sterility and reciprocal cross-sterility within the siblings that are self-sterile but fertile with non-siblings. Thirdly, we show self-sterility and cross-sterility share some natures and both are closely related to the sperm capacity not to bind to the vitelline coat of the autologous eggs or the eggs sterile to the sperm concerned. In all, this paper shows that the self-sterility is genetically governed by a multiple-locus system, and that most probably individual-specific determinants are haploid expression in sperm and diploid expression in eggs, given they recognize self but not non-self.     

Chymotrypsin-like enzymes are involved in sperm penetration through the vitelline coat of Ciona intestinalis egg

In Ciona intestinalis, sperm penetration through the egg vitelline coat is an essential event of fertilization. We investigated whether trypsin- and chymotrypsin-like enzymes are involved in this event. Inhibitors and peptide substrates for chymotrypsin-like enzymes blocked the overall process of fertilization in a concentration-dependent manner. The inhibitory activity was specifically exerted on the step of sperm penetration. Chymotrypsin-like protease activity was identified in spermatozoa with the fluorogenic synthetic substrate Suc-Ala-Ala-Phe-AMC, which was the most effective substrate in blocking sperm penetration. These data indicate that a chymotrypsin-like protease activity is a sperm lysin of Ciona intestinalis.     

Allorecognition mechanisms during ascidian fertilization

Ascidians (primitive chordates) are hermaphroditic animals, releasing sperm and eggs nearly simultaneously. But, many ascidians, including Ciona intestinalis and Halocynthia roretzi, show self-sterility or preference for cross-fertilization rather than self-fertilization. The molecular mechanisms underlying this allorecognition process are only poorly understood. We recently identified the genes responsible for self-incompatibility in C. intestinalis by a positional cloning: sperm-borne polycystin 1-like receptor, referred to as s-Themis, and its fibrinogen-like ligand called v-Themis on the vitelline coat (VC) are highly polymorphic and appear to be responsible for allorecognition in the fertilization of C. intestinalis. In H. roretzi, on the other hand, we revealed that HrVC70, a 70-kDa main component of the VC consisting of 12 epidermal-growth-factor (EGF)-like repeats, is a candidate allorecognition protein, since the attachment of this protein to the VC during oocyte maturation and its detachment by weak acid are closely linked to the gain and the loss of self-sterility, respectively, and also since nonself-sperm rather than self-sperm efficiently bound to HrVC70-agarose. As a binding partner of HrVC70, a 35-kDa GPI-anchored glycoprotein in sperm lipid rafts, referred to as HrUrabin, was identified: HrUrabin appears to play a key role in allorecognizable sperm binding to HrVC70 during fertilization. In the present review, we describe the current progress on the molecular bases of allorecognition, or self-incompatibility, during ascidian fertilization, by considering the SI systems in another organisms including fungies and flowering plants.     

Helper Function of Follicle Cells in Sperm-Egg Interactions of the Ascidian, Ciona intestinalis

Studies were made on the involvement in sperm-egg interactions of follicle cells of Ciona intestinalis , which are tall, vacuolated cells attached to the outer surface of the egg vitelline coat. The basal surface of the follicle cells is polygonal. The borders between cells could easily be observed by the binding of fluorescent SBA (soy bean agglutinin), a lectin recognizing N-acetylgalactosamine (GaINAc) residues. At fertilization many spermatozoa aggregate along these polygonal borders of cells on the vitelline coat, through which they entered the perivitelline space. The removal of follicle cells was sometimes associated with loss of SBA-binding sites, and in such cases the sperm did not show a hexagonal pattern of aggregation, but became dispersed all over the vitelline coat. Removal of the follicle sometimes delayed fertilization. Examination of sections of gametes stained with DAPI, a fluorescent dye staining DNA, showed that removal of the follicle reduced the number of spermatozoa bound to the vitelline coat and, more especially, the number of spermatozoa penetrating through the vitelline coat. The blockage of GalNAc residues on the vitelline coat with SBA did not appreciably affect the time course of fertilization or the number of sperm associated with eggs. These findings are discussed in relation to the role of follicle cells in facilitating sperm aggregation on the vitelline coat and their penetration through it.     

Membrane hyperpolarization by sperm-activating and -attracting factor increases cAMP level and activates sperm motility in the ascidian Ciona intestinalis.

In the ascidian Ciona intestinalis (and C. savignyi), sperm-activating and -attracting factor (SAAF) is released from the egg at fertilization and stimulates both Ca(2+) influx and a transient increase in cAMP level of the sperm, leading to the activation of sperm motility (M. Yoshida et al., 1994, Dev. Growth Differ. 36, 589-595). In this paper we show in C. intestinalis that valinomycin, a potassium-selective ionophore, as well as SAAF, activated sperm motility, and this activation was suppressed by extracellular high K(+). Membrane potential measurements showed that both SAAF and valinomycin increase K(+) permeability of sperm and induce membrane hyperpolarization, the amplitude of which depends on the external K(+) concentration. The membrane potential and intracellular K(+) concentration of Ciona sperm without SAAF were estimated to be about -50 mV and 560 +/- 40 mM, respectively. After treatment with SAAF or valinomycin the membrane potential became almost equal to the equilibrium potential of K(+) (-100 mV), and the cAMP level increased in artificial seawater. A potent voltage-dependent K(+) channel blocker, MCD peptide, at the concentration of 10 microM blocked SAAF-induced hyperpolarization of the cells, increase in cAMP, and sperm motility. These results suggest that membrane hyperpolarization produced by the opening of K(+) channels elevates cAMP synthesis and leads to the activation of sperm motility in Ciona.     

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.     

Minos transposon causes germline transgenesis of the ascidian Ciona savignyi

An ascidian, Ciona savignyi, is regarded as a good experimental animal for genetics because of its small and compact genome for which a draft sequence is available, its short generation time and its interesting phylogenic position. ENU-based mutagenesis has been carried out using this animal. However, insertional mutagenesis using transposable elements (transposons) has not yet been introduced. Recently, one of the Tc1/mariner superfamily transposons, Minos, was demonstrated to cause germline transgenesis in the related species Ciona intestinalis. In this report, we show that Minos has the ability to transpose from DNA to DNA in Ciona savignyi in transposition assays. Although the activity was slightly weaker than in Ciona intestinalis, Minos still caused germline transgenesis in Ciona savignyi. In addition, one insertion seemed to have caused an enhancer trapping. These results indicate that Minos provides a potential tool for transgenic techniques such as insertional mutagenesis in Ciona savignyi.     

Segregation during cleavage of a factor determining endodermal alkaline phosphatase development in ascidian embryos.

Localized alkaline phosphatase activity (EC 3.1.3.1) develops progressively in endodermal tissues of the presumptive digestive system in Ciona intestinalis embryos. It was first detected histochemically at late gastrulation, and a puromycin sensitivity period coincident with this time suggests that new alkaline phosphatase is synthesized. Embryos in which cell division was blocked with cytochalasin B at early cleavage stages up to the 64-cell stage, eventually differentiated strong alkaline phosphatase activity in certain cells at each cleavage-arrested stage. The maximum cell numbers and their positions were identical to those of the previously known endodermal cell lineage. Actinomycin D did not prevent development of endodermal alkaline phosphatase when administered from fertilization onwards, nor did other inhibitors of RNA synthesis (chromomycin A3, cordycepin, and daunomycin). There is probably a preformed maternal mRNA for endodermal alkaline phosphatase present in the unfertilizec Ciona egg. Either this RNA itself, or some related translation factor, is localized in the egg cytoplasm and segregated during early cleavages into the endodermal cell lineage of the embryo.     

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.     

Effects of egg size on the development time of non-feeding larvae

The evolution of egg size in marine invertebrates remains a topic of central importance for life-history biologists, and the pioneering work of Vance has strongly influenced our current views. Vance's model and most models developed since have assumed that increases in egg size result in an increase in the prefeeding period of marine invertebrate larvae. For lecithotrophic species, this means that the entire development period should be correlated with egg size. Despite the importance of this assumption, it has not been tested at the appropriate scale-within species. We investigated the effects of egg size on development time for three lecithotrophic species from two phyla: the ascidians Phallusia obesa and Ciona intestinalis, and the echinoid Heliocidaris erythrogramma. We found that within individual broods of eggs, larger eggs took longer than smaller eggs to develop or become metamorphically competent larvae. It has long been recognized that producing larger eggs decreases fecundity, but our results show that increasing egg size also carries the extra cost of an extended planktonic period during which mortality can occur. The substantial variation in egg sizes observed within broods may represent a bet-hedging strategy by which offspring with variable dispersal potentials are produced.     

Purification and characterization of a vitelline coat lysin from Ciona intestinalis spermatozoa.

In Ciona intestinalis a chymotrypsin-like activity is involved in sperm penetration of the egg vitelline coat. A chymotrypsin-like enzyme has been purified from spermatozoa by a protocol including ion exchange chromatography, gel filtration, and native polyacrylamide gel electrophoresis. The purified enzyme resulted homogeneous when analyzed by SDS-PAGE. The molecular weight of the chymotrypsin-like enzyme was estimated to be 35 kDa by gel filtration and 24 KDa by SDS-PAGE in nonreducing conditions. The pH optimum of the enzyme is 8.4 and its activity is enhanced by Ca2+. It shows the highest activity towards the synthetic substrate Suc-Ala-Ala-Pro-Phe-AMC. Furthermore, by electron microscopy, the purified enzyme affects the structure of egg vitelline coat, and thus it fulfills one of the criteria of a lysin.     

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.     

Mechanism of the block to hybridization and selfing between the sympatric ascidians Ciona intestinalis and Ciona savignyi

The solitary ascidians Ciona intestinalis and Ciona savignyi co-occur in southern California harbors, but no hybrids have been recognized in nature. Numerous differences in their egg morphology were detected. Homologous (normal outcross) fertilization yielded 96-99% cleavage, where autologous (self) fertilization showed 3% and heterologous (hybrid) fertilization showed 0-1%. Acid treatment (pH 3.2) removed the block to selfing (P < 0.0001) but not hybridization for both species. Heterologous sperm bind to the vitelline coat (VC), but fail to penetrate. Enzymatic removal of the VC resulted in 91-97% cleavage with autologous and heterologous sperm (P < 0.0001). The vitelline coats of the two species differ in lectin binding to surface glycosides. Fertilization in both species is significantly inhibited by the lectins, fucose binding protein (P < 0.0001) and concanavalin A (P < 0.0001), and wheat germ agglutinin inhibits fertilization in C. intestinalis (P < 0.0001) but is without effect on C. savignyi fertilization. Self and hybrid blocks employ different mechanisms including glycoside composition and acid sensitivity.     

A genomewide survey of developmentally relevant genes in Ciona intestinalis. VII. Molecules involved in the regulation of cell polarity and actin dynamics

In the present study, genes involved in the pathways that establish cell polarity and cascades regulating actin dynamics were identified in the completely sequenced genome of Ciona intestinalis, a basal chordate. It was revealed that the Ciona genome contains orthologous genes of each component of aPKC-Par and PCP pathways and WASP/WAVE/SCAR and ADF/cofilin cascades, with less redundancy than the vertebrate genomes, suggesting that the conserved pathways/cascades function in Ciona development. In addition, the present study found that the orthologous proteins of five gene groups (Tc10, WRCH, RhoD, PLC-L, and PSKH) are conserved in humans and Ciona but not in Drosophila melanogaster, suggesting a similarity in the gene composition of Ciona to that of vertebrates. Ciona intestinalis, therefore, may provide refined clues for the study of vertebrate development and evolution.     

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.     

A thermodynamic study of sperm-egg interaction.

We have studied the binding of spermatozoa to the receptor sites on the vitelline coat (VC) of glycerol-treated eggs (ghost eggs) of the Ascidian, Ciona intestinalis (Protochordate). Glycerol treatment cytolyses the egg without affecting the ability of the VC to bind spermatozoa in a species-specific manner; however, in this system binding is not followed by the acrosome reaction. The ghost eggs are metabolically inert. As a base line for our analysis, we have studied the concentration-dependent heat evolved and oxygen consumption of spermatozoa when diluted in sea water. The process has been analyzed on the basis of equations derived by Liquori and Tripiciano to describe cell growth. Upon binding to the ghost eggs, the spermatozoa produce an explosive heat evolution (excess heat) which is not accompanied by oxygen consumption. The excess heat produced plotted against sperm concentration (at constant egg concentrations) gives an asymmetric bell-shaped curve. This is interpreted as being due to the competitive effect of sperm agglutination at a high sperm concentration. It is concluded that only spermatozoa that attach singly (monomeric spermatozoa) to the egg undergo metabolic activation.