Kimio J. Tanaka

Dynamic redistribution of vasa homolog and exclusion of somatic cell determinants during germ cell specification in Ciona intestinalis.

Ascidian embryos sequester a specific cytoplasm, called the postplasm, at the posterior pole, where many maternal RNAs and proteins accumulate. Although the postplasm is thought to act as the germ plasm, it is also highly enriched in several factors essential for somatic cell development, and how the postplasm components regulate both germ and somatic cell differentiation remains elusive. Using a vasa homolog, CiVH, and other postplasmic components as markers, we found that the postplasm-containing blastomeres, the B7.6 cells, undergo an asymmetric cell division during gastrulation to produce two distinct daughter cells: B8.11 and B8.12. Most of the postplasmic components segregate only into the B8.11 cells, which never coalesce into the gonad. By contrast, the maternal CiVH RNA and protein are specifically distributed into the B8.12 cells, which divide further and are incorporated into the gonad in juveniles. In the B8.12 cells, CiVH production is upregulated from the maternal RNA source, resulting in the formation of perinuclear CiVH granules, which may be the nuage, a hallmark of germ cells in many animal species. We propose that the redistribution of specific maternal molecules into the B8.12 cells is essential for germ-cell specification in ascidians.

An Acidic Protein, YBAP1, Mediates the Release of YB-1 from mRNA and Relieves the Translational Repression Activity of YB-1

ABSTRACT Eukaryotic Y-box proteins are nucleic acid-binding proteins implicated in a wide range of gene regulatory mechanisms. They contain the cold shock domain, which is a nucleic acid-binding structure also found in bacterial cold shock proteins. The Y-box protein YB-1 is known to be a core component of messenger ribonucleoprotein particles (mRNPs) in the cytoplasm. Here we disrupted the YB-1 gene in chicken DT40 cells. Through the immunoprecipitation of an epitope-tagged YB-1 protein, which complemented the slow-growth phenotype of YB-1-depleted cells, we isolated YB-1-associated complexes that likely represented general mRNPs in somatic cells. RNase treatment prior to immunoprecipitation led to the identification of a Y-box protein-associated acidic protein (YBAP1). The specific association of YB-1 with YBAP1 resulted in the release of YB-1 from reconstituted YB-1-mRNA complexes, thereby reducing the translational repression caused by YB-1 in the in vitro system. Our data suggest that YBAP1 induces the remodeling of YB-1-mRNA complexes.

Expression patterns of musashi homologs of the ascidians, Halocynthia roretzi and Ciona intestinalis

Abstract The gene family encoding RNA-binding proteins includes important regulators involved in the neurogenesis in both protostomes and deuterostomes. We isolated cDNAs of the ascidian homolog of one of the RNA-binding proteins, MUSASHI, from Halocynthia roretzi and Ciona intestinalis. The predicted amino acid sequences contained two RNA-recognition and RNA-binding motifs in the N-terminus and an ascidian-specific YG-rich domain in the C-terminus. Maternal transcripts of musashi were ubiquitous in early cleavage-stage embryos. Ascidian musashi had three domains of zygotic expression: the brain, nerve cord, and mesenchyma. The temporal order of the onset in these domains was highly divergent between the two species of ascidian examined.

Visualization of the reconstituted FRGY2–mRNA complexes by electron microscopy

Xenopus oocytes store large quantities of translationally dormant mRNA in the cytoplasm as storage messenger ribonucleoprotein particles (mRNPs). The Y-box proteins, mRNP3 and FRGY2/mRNP4, are major RNA binding components of maternal storage mRNPs in oocytes. In this study, we show that the FRGY2 proteins form complexes with mRNA, which leads to mRNA stabilization and translational repression. Visualization of the FRGY2-mRNA complexes by electron microscopy reveals that FRGY2 packages mRNA into a compact RNP. Our results are consistent with a model that the Y-box proteins function in packaging of mRNAs to store them stably for a long time in the oocyte cytoplasm.

Interactions between cytoskeletal components during myoplasm rearrangement in ascidian eggs

Ooplasmic segregation in ascidian eggs consists of two phases of cytoplasmic movement, the first phase is mediated by the microfilament system and the second is mediated by the microtubule system. Recently, two novel proteins, p58 and myoplasmin‐C1, which are localized to the myoplasm, were suggested to have important roles in muscle differentiation. In order to analyze the molecular mechanisms underlying ooplasmic segregation, the interactions between actin, tubulin, p58 and myoplasmin‐C1 were examined. During the first segregation, microtubule meshwork in the unfertilized egg disappeared. At the second segregation, a novel structure of the microtubules that extended from the sperm aster and localized in the cortical region of the myoplasm was found. Moreover, uniform distribution of the cortical actin filament was observed at the second segregation. During the course of myoplasm rearrangement, p58 and myoplasmin‐C1 are colocalized and can form a molecular complex in vitro. This complex of p58 and myoplasmin‐C1 is a good candidate for a cytoskeletal component of the myoplasm, and is likely to be involved in the correct distribution of cytoplasmic determinants.

Expression of pharyngeal gill-specific genes in the ascidian Halocynthia roretzi.

Abstract The most primitive chordates may have arisen with a shift to internal feeding through the use of the pharyngeal gill slits and endostyle for extracting suspended food from the water. Therefore, the pharyngeal gill and endostyle, in addition to notochord and nerve cord, are structures key to an understanding of the molecular developmental mechanisms underlying the origin and evolution of chordates. In this and a following study, isolation of cDNA clones for genes that are specifically expressed in the pharyngeal gill or endostyle in the ascidian Halocynthia roretzi was attempted. Differential screening of a pharyngeal gill cDNA library and an endostyle cDNA library with total pharyngeal-gill cDNA probes yielded cDNA clones for two pharyngeal gill-specific genes, HrPhG1 and HrPhG2. Northern blot analysis showed a 3.0-kb transcript of HrPhG1 and a 2.0-kb transcript of HrPhG2. Predicted amino acid sequences of the gene products suggested that both genes encode secretory proteins with no significant match to known proteins. In adults, both HrPhG1 and HrPhG2 genes were only expressed in the pharyngeal gill and not in other tissues including the endostyle, body-wall muscle, gonad, gut and digestive gland. HrPhG1 and HrPhG2 transcripts were undetectable in embryos and larvae, and were first detected in juveniles 3 days after initiation of metamorphosis. In situ hybridization revealed that the expression of HrPhG1 and HrPhG2 was restricted to differentiating pharyngeal-wall epithelium, with intense signals in the area surrounding the stigma or gill slit. These genes may serve as probes for further analyses of molecular mechanisms underlying the occurrence of pharyngeal gill and formation of gill slits during chordate evolution.

Expression of endostyle-specific genes in the ascidian Halocynthia roretzi

Abstract The endostyle is a special organ in the pharynx of Urochordata, Cephalochordata and Cyclostomata. This organ may have arisen in their common ancestor with a shift to internal feeding for extracting suspended food from the water. In addition, the endostyle has functional homology to the vertebrate thyroid gland. The endostyle is therefore another key structure in the understanding of the origin and evolution of chordates. Following a previous report of the pharyngeal gill-specific genes, we report here the isolation and characterization of cDNA clones for endostyle-specific genes HrEnds1 and HrEnds2 of the ascidian Halocynthia roretzi. These cDNA clones were obtained by differential screening of an endostyle cDNA library and a pharyngeal gill cDNA library with total endostyle cDNA probes. Both transcripts were abundant in the library; each represented about 10% of the cDNA clones of the library. The HrEnds1 transcript was small in size, about 600 bp in length. Although the predicted amino acid sequence of the gene product showed no similarity to known proteins, mean hydropathy profiles suggested that HrENDS1 is a type Ib protein or secreted protein. The HrEnds2 transcript was about 2.5 kb in length. Although the HrEnds2 gene product showed no sequence similarity to known proteins, mean hydropathy profiles suggested that HrENDS2 is a secreted protein. The transcripts of both genes were not detected in embryos, larvae and early juveniles but were evident in 1-month-old young adult after several compositional zones were organized in the endostyle. In situ hybridization revealed that distribution of transcripts of both genes was restricted to zone 6, the protein-secreting glandular element of the endostyle. These genes may be useful for further analysis of molecular mechanisms involved in endostyle development.

An ascidian glycine-rich RNA binding protein is not induced by temperature stress but is expressed under a genetic program during embryogenesis.

We have cloned a putative ascidian glycine-rich RNA binding protein gene, CiGRP1. Its maternal transcript and protein are stored in the unfertilized egg. They are gradually decreased during the first few rounds of cleavage. The CiGRP1 zygotic transcript and protein start to accumulate at the gastrula stage. The CiGRP1 transcript is expressed in the brain precursor and mesenchyme precursor cells of the gastrula and the neurula stage, and the brain and mesenchyme cells of the tailbud stage embryo. The CiGRP1 protein is found in all nuclei and in the cytoplasm of brain and mesenchyme cells. Although many glycine-rich RNA binding protein homologs of plants and vertebrates are cold-inducible, CiGRP1 cannot be induced by cold shock or heat shock at the transcriptional and translational levels during embryogenesis. The temporal expression pattern and the tissue-restricted expression pattern of CiGRP1 suggest that it has important roles in the very early stage of development and in the brain and the mesenchyme tissue specification.

The transcript coding for an RNA-binding protein is localized in the anterior side of the ascidian 2-cell stage embryo

Abstract We have cloned a putative ascidian RNA-binding protein gene, CiRGG1, which has three RNA-recognition motifs and two arginine-glycine-glycine domains. Its deduced amino acid sequence has a weak similarity to nucleolin, which is one of the known components of messenger ribonucleoproteins. Its maternal transcript is transiently accumulated into the anterior side of 2-cell stage embryos. Zygotic expression of CiRGG1 is strongest in the brain, mesenchyme and endoderm of the trunk region of the tailbud stage embryo. This mRNA is the first example of an anterior-localized molecule in the ascidian 2-cell stage embryo.

Two Distinct Cell Types Identified in the Ascidian Notochord

Abstract We established a monoclonal antibody, designated CiNot-1, that recognizes ascidian Ciona notochordal cells in the early tailbud embryo. It distinguished two types of cells in the notochord, according to whether or not they expressed CiNot-1 antigen. Only A-line notochord cells expressed CiNot-1 antigen in cleavage-arrested embryos. Since A- and the B-line notochord precursor cells respond differently to inductive signals, CiNot-1 distinguished two specification mechanisms in the notochord. After the middle tailbud stage, CiNot-1 recognized the nerve cord and endodermal strand in addition to the notochord. In these tissues, several cells in the distal tip of the tail did not express CiNot-1 antigen. This revealed the distinct feature of unity in the distal tip of the larval tail and suggests the specification mechanism of this region.