Neural map of the larval central nervous system in the ascidian Ciona intestinalis.

We examined the distribution patterns and axonal pathways of cholinergic, GABAergic, and dopaminergic neurons in the central nervous system of Ciona intestinalis larvae, based on the expression patterns of two reporter genes (GFP and LacZ) driven by the promoters of several neuron-specific genes (vesicular acetylcholine transporter, glutamic acid decarboxylase, tyrosine 3-hydroxylase and dopa decarboxylase). Putative cholinergic and GABAergic cells were found in the sensory vesicle (SV) and visceral ganglion (VG), while putative dopaminergic cells were found only in the SV. The axons of almost all putative cholinergic and GABAergic cells in the SV extend posteriorly towards the VG and seem to connect with motor neurons. Some cells extend axons to the proximal region of the tail beyond the trunk-tail boundary. As this tail region contains several neurons, these cells may modulate larval behavior through the latter neurons. We also found that some putative cholinergic and GABAergic cells in the dorsal VG form a complex and extend axons anteriorly to the SV, posteriorly to the tail, and possibly ventrally to some motor neurons. Finally, we observed that one pair of the anterior most putative cholinergic cells in the ventral VG extends axons contralaterally to the right and left caudal axon tracts. We discuss the similarity of these cells to the Mauthner cells in vertebrates.

ERK- and JNK-signalling regulate gene networks that stimulate metamorphosis and apoptosis in tail tissues of ascidian tadpoles.

In ascidian tadpoles, metamorphosis is triggered by a polarized wave of apoptosis, via mechanisms that are largely unknown. We demonstrate that the MAP kinases ERK and JNK are both required for the wave of apoptosis and metamorphosis. By employing a gene-profiling-based approach, we identified the network of genes controlled by either ERK or JNK activity that stimulate the onset of apoptosis. This approach identified a gene network involved in hormonal signalling, in innate immunity, in cell-cell communication and in the extracellular matrix. Through gene silencing, we show that Ci-sushi, a cell-cell communication protein controlled by JNK activity, is required for the wave of apoptosis that precedes tail regression. These observations lead us to propose a model of metamorphosis whereby JNK activity in the CNS induces apoptosis in several adjacent tissues that compose the tail by inducing the expression of genes such as Ci-sushi.

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.

Development of Ciona intestinalis juveniles (through 2nd ascidian stage).

Following the reading of its draft genome sequence and the collection of a large quantity of cDNA information, Ciona intestinalis is now becoming a model organism for whole-genome analyses of the expression and function of developmentally relevant genes. Although most studies have focused on larval structures, the development of the adult form is also very interesting in relation to tissues and organs of vertebrate body. Here we conducted detailed observations of the development of tissues and organs in Ciona intestinalis larva and juveniles until so-called the 2nd ascidian stage. These observations included examination of the oral siphon, tentacle, oral pigments and atrial pigments, atrial siphon, ganglion and neural gland, longitudinal muscle, stigmata, transverse bar and languet, longitudinal bar and papilla, heart, digestive organ, gonad, endostyle, and stalk and villi. The findings from these observations make a new staging system for juvenile development possible. Based on the development of the internal organs, we propose here nine stages (stage 0-stage 8) starting with swimming larvae and proceeding through juveniles until the 2nd ascidian stage. These descriptions and staging system provide a basis for studying cellular and molecular mechanisms underlying the development of adult organs and tissues of this basal chordate.

Caenorhabditis elegans DAF-21 (HSP90) is characteristically and predominantly expressed in germline cells: spatial and temporal analysis.

Three monoclonal antibodies against antigens that exist in the Caenorhabditis elegans germ line have previously been described. In the present study, a full-length mRNA for one of these antigens was isolated, and by sequencing its corresponding cDNA, it was predicted that the protein would show a high homology with the 90 kDa heat shock protein (HSP90) in other species, and with the protein of daf-21, a previously identified hsp90 homologue. The spatial and temporal distribution of the antigen (DAF-21) was analyzed in C. elegans, and the localization of daf-21 mRNA, as detected by in situ hybridization, agreed with that detected by the monoclonal antibody. Under normal conditions, daf-21 mRNA is characteristically distributed in postembryonic germ cells derived from Z2 and Z3 cells in both hermaphrodites and males. Under heat stress conditions, however, daf-21 mRNA was not only detected in germ cells, but also apparently expressed all over the body. In addition, the DAF-21 protein seemed to be localized in the perinuclear region of somatic cells.

Primordial germ cells originate from the endodermal strand cells in the ascidian Ciona intestinalis.

The origin of germ cells in the ascidian is still unknown. Previously, we cloned a vasa homologue (CiVH) of Ciona intestinalis from the cDNA library of ovarian tissue by polymerase chain reaction and showed that its expression was specific to germ cells in adult and juvenile gonads. In the present study, we prepared a monoclonal antibody against CiVH protein and traced the staining for this antibody from the middle tailbud stage to young adulthood. Results showed that positive cells are present in the endodermal strand in middle tailbud embryos and larvae. When the larval tail was absorbed into the trunk during metamorphosis, the CiVH-positive cells migrated from the debris of the tail into the developing gonad rudiment, and appeared to give rise to a primordial germ cell (PGC) in the young juvenile. The testis rudiment separated from the gonad rudiment, the remainder of which differentiated into the ovary. PGCs of the testis rudiment and the ovary rudiment differentiated into spermatogenic and oogenic cells, respectively. When the larval tail containing the antibody-positive cells was removed, the juveniles did not contain any CiVH-positive cells after metamorphosis, indicating that the PGCs in the juvenile originated from part of the larval tail. However, even in such juveniles, positive cells newly appeared in the gonad rudiment at a later stage. This observation suggests that a compensatory mechanism regulates germline formation in C. intestinalis.

Developmental expression of ascidian neurotransmitter synthesis genes. I. Choline acetyltransferase and acetylcholine transporter genes.

To identify cholinergic neurons, we isolated a choline acetyltransferase (Ci-ChAT) gene from Ciona intestinalis by PCR methods. In the cloning process, we also obtained the gene encoding the vesicular acetylcholine transporter (Ci-vAChTP). These two genes shared the same 5'-UTR sequence as well as similar expression patterns. In both cases, the gene expression was first detected by whole-mount in situ hybridization in the anterior-dorsal region of the caudal nerve cord at the early tailbud stage. In the larva, the expression was seen in several cells of the visceral ganglion. These results suggest that ascidian larval motor neurons exist in the visceral ganglion.