Lixy Yamada

Mechanism of Self-Sterility in a Hermaphroditic Chordate

Hermaphroditic organisms avoid inbreeding by a system of self-incompatibility (SI). A primitive chordate (ascidian) Ciona intestinalis is an example of such an organism, but the molecular mechanism underlying its SI system is not known. Here, we show that the SI system is governed by two gene loci that act cooperatively. Each locus contains a tightly linked pair of polycystin 1–related receptor (s-Themis) and fibrinogen-like ligand (v-Themis) genes, the latter of which is located in the first intron of s-Themis but transcribed in the opposite direction. These genes may encode male- and female-side self-recognition molecules. The SI system of C. intestinalis has a similar framework to that of flowering plants but utilizing different molecules.

A genomewide survey of developmentally relevant genes in Ciona intestinalis. V. Genes for receptor tyrosine kinase pathway and Notch signaling pathway.

In the present survey, we identified most of the genes involved in the receptor tyrosine kinase (RTK), mitogen activated protein kinase (MAPK) and Notch signaling pathways in the draft genome sequence of Ciona intestinalis, a basal chordate. Compared to vertebrates, most of the genes found in the Ciona genome had fewer paralogues, although several genes including ephrin, Eph and fringe appeared to have multiplied or duplicated independently in the ascidian genome. In contrast, some genes including kit/flt, PDGF and Trk receptor tyrosine kinases were not found in the present survey, suggesting that these genes are innovations in the vertebrate lineage or lost in the ascidian lineage. The gene set identified in the present analysis provides an insight into genes for the RTK, MAPK and Notch signaling pathways in the ancient chordate genome and thereby how chordates evolved these signaling pathway.

macho-1-related genes in Ciona embryos

Abstract. In ascidians, maternal factor(s) localized in the myoplasm of the egg are essential for specification and subsequent differentiation of larval muscle cells. The macho-1 gene of Halocynthia roretzi encodes a zinc-finger protein: the gene is only expressed maternally, the resultant maternal mRNA is localized to the myoplasm, and the gene function is essential for muscle cell differentiation. Here we have characterized macho-1 homologues, Ci-macho1 of Ciona intestinalis and Cs-macho1 of Ciona savignyi. Interestingly, we found that the Cionamacho-1 genes are expressed both maternally and zygotically: their maternal transcript is localized to the myoplasm while their zygotic expression is seen after neurulation in cells of the central nervous system. Functional suppression of Cs-macho1 with morpholino antisense oligonucleotide resulted in inhibition of the initiation of zygotic expression of a muscle-specific actin gene. We propose a possible evolutionary scenario in which an ancestral Zic-related gene gave rise to both the macho-1-like muscle determinant gene as well as neuronal Zic genes.

Postplasmic/PEM RNAs: A class of localized maternal mRNAs with multiple roles in cell polarity and development in ascidian embryos

Ascidian is a good model to understand the cellular and molecular mechanisms responsible for mRNA localization with the discovery of a large family of localized maternal mRNAs, called postplasmic/PEM RNAs, which includes more than 40 members in three different ascidian species (Halocynthia roretzi, Ciona intestinalis, and C. savignyi). Among these mRNAs, two types (Type I and Type II) have been identified and show two different localization patterns from fertilization to the eight‐cell stage. At the eight‐cell stage, both types concentrate to a macromolecular cortical structure called CAB (for Centrosome Attracting Body) in the posterior‐vegetal B4.1 blastomeres. The CAB is responsible for unequal cleavages and the partitioning of postplasmic/PEM RNAs at the posterior pole of embryos during cleavage stages. It has also been suggested that the CAB region could contain putative germ granules. In this review, we discuss recent data obtained on the distribution of Type I postplasmic/PEM RNAs from oogenesis to late development, in relation to their localization and translational control. We have first regrouped localization patterns for Type I and Type II into a comparative diagram and included all important definitions in the field. We also have made an exhaustive classification of their embryonic expression profiles (Type I or Type II), and analyzed their functions after knockdown and/or overexpression experiments and the role of the 3′‐untranslated region (3′UTR) controlling both their localization and translation. Finally, we propose a speculative model integrating recent data, and we also discuss the relationship between postplasmic/PEM RNAs, posterior specification, and germ cell formation in ascidians. Developmental Dynamics 236:1698–1715, 2007.

The Lingula genome provides insights into brachiopod evolution and the origin of phosphate biomineralization.

The evolutionary origins of lingulid brachiopods and their calcium phosphate shells have been obscure. Here we decode the 425-Mb genome of Lingula anatina to gain insights into brachiopod evolution. Comprehensive phylogenomic analyses place Lingula close to molluscs, but distant from annelids. The Lingula gene number has increased to ∼34,000 by extensive expansion of gene families. Although Lingula and vertebrates have superficially similar hard tissue components, our genomic, transcriptomic and proteomic analyses show that Lingula lacks genes involved in bone formation, indicating an independent origin of their phosphate biominerals. Several genes involved in Lingula shell formation are shared by molluscs. However, Lingula has independently undergone domain combinations to produce shell matrix collagens with EGF domains and carries lineage-specific shell matrix proteins. Gene family expansion, domain shuffling and co-option of genes appear to be the genomic background of Lingulas unique biomineralization. This Lingula genome provides resources for further studies of lophotrochozoan evolution.

Comprehensive Egg Coat Proteome of the Ascidian Ciona intestinalis Reveals Gamete Recognition Molecules Involved in Self-sterility

Despite central roles of egg coat proteins in gamete recognition, their functions and composition are poorly understood. Here, we report that the proteome of the egg coat in the solitary ascidian Ciona intestinalis, called vitelline coat (VC) fraction, contains more than 800 proteins identified by mass spectrometry-based analyses. Over 100 proteins were enriched in the VC fraction compared with the VC-free egg proteome. The most abundant component in the VC was an apolipoprotein-like protein. The VC contained multiple homologs of mammalian zona pellucida (ZP) proteins, the number of which was unexpectedly large and most of which possessed epidermal growth factor-like repeats. Furthermore, the present study revealed that two fibrinogen-like proteins, v-Themis-A and -B, both of which are expressed in the VC, are the molecules responsible for the two self-sterility loci that were identified by our previous genetic study in this species.

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.

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.

CIPRO 2.5: Ciona intestinalis protein database, a unique integrated repository of large-scale omics data, bioinformatic analyses and curated annotation, with user rating and reviewing functionality

The Ciona intestinalis protein database (CIPRO) is an integrated protein database for the tunicate species C. intestinalis. The database is unique in two respects: first, because of its phylogenetic position, Ciona is suitable model for understanding vertebrate evolution; and second, the database includes original large-scale transcriptomic and proteomic data. Ciona intestinalis has also been a favorite of developmental biologists. Therefore, large amounts of data exist on its development and morphology, along with a recent genome sequence and gene expression data. The CIPRO database is aimed at collecting those published data as well as providing unique information from unpublished experimental data, such as 3D expression profiling, 2D-PAGE and mass spectrometry-based large-scale analyses at various developmental stages, curated annotation data and various bioinformatic data, to facilitate research in diverse areas, including developmental, comparative and evolutionary biology. For medical and evolutionary research, homologs in humans and major model organisms are intentionally included. The current database is based on a recently developed KH model containing 36 034 unique sequences, but for higher usability it covers 89 683 all known and predicted proteins from all gene models for this species. Of these sequences, more than 10 000 proteins have been manually annotated. Furthermore, to establish a community-supported protein database, these annotations are open to evaluation by users through the CIPRO website. CIPRO 2.5 is freely accessible at http://cipro.ibio.jp/2.5.

Self-incompatibility response induced by calcium increase in sperm of the ascidian Ciona intestinalis

Many hermaphroditic organisms possess a self-incompatibility system to avoid self-fertilization. Recently, we identified the genes responsible for self-sterility in a hermaphroditic primitive chordate (ascidian), Ciona intestinalis: sperm-side polycystin 1-like receptors s-Themis-A/B and egg-side fibrinogen-like ligands on the vitelline coat (VC) v-Themis-A/B. Here, we investigated the sperm behavior and intracellular Ca2+ concentration ([Ca2+]i) in response to self/nonself-recognition. We found that sperm motility markedly decreased within 5 min after attachment to the VC of self-eggs but not after attachment to the VC of nonself-eggs and that the apparent decrease in sperm motility was suppressed in low Ca2+ seawater. High-speed video analysis revealed that sperm detached from the self-VC or stopped motility within 5 min after binding to the self-VC. Because s-Themis-B contains a cation channel domain in its C terminus, we monitored sperm [Ca2+]i by real-time [Ca2+]i imaging using Fluo-8H-AM (AAT Bioquest, Inc.). Interestingly, we found that sperm [Ca2+]i rapidly and dramatically increased and was maintained at a high level in the head and flagellar regions when sperm interacted with the self-VC but not when the sperm interacted with the nonself-VC. The increase in [Ca2+]i was also suppressed by low-Ca2+ seawater. These results indicate that the sperm self-recognition signal triggers [Ca2+]i increase and/or Ca2+ influx, which elicits a self-incompatibility response to reject self-fertilization in C. intestinalis.