We have cloned and studied Hp-ECPN, an encephalopsin orthologue of the sea urchin Hemicentrotus pulcherrimus. Hp-ecpn cDNA was produced and found to contain a 1461-bp open reading frame that encodes 486 amino acids. Accumulation of Hp-ecpn mRNA and protein expression occurred at the 14 h postfertilization (hpf) swimming blastula stage and thereafter. The Hp-ECPN protein was N-glycosylated, and the amino acid sequence was similar to that of vertebrate encephalopsins. Whole-mount immunohistochemistry revealed the presence of Hp-ECPN in cells (ECPN cells) that appeared initially around the tip of the archenteron in 20 hpf early gastrulae. By the 54 hpf pluteus stage, ECPN cells had spread through the aboral ectoderm, and, by the eight-arm pluteus stage, were restricted to the tips of the larval arms and the posterior end of the body. The number of ECPN cells increased under conditions of continuous light, but decreased under continuous dark. Knockdown of Hp-ecpn mRNA using morpholino antisense oligonucleotides decreased the number of ECPN cells considerably, and inhibited the vertical swimming of the larvae. This suggested that Hp-ECPN plays a role in photosensitive larval swimming vertical migration. In adult tissues, the ECPN cells were detected exclusively in tube feet.

SummaryThe ontogenetic origin of blastocoelar glutamate decarboxylase (GAD)-expressing cells (GADCs) in larvae of the sea urchin Hemicentrotus pulcherrimus was elucidated. Whole-mount in situ hybridisation (WISH) detected transcription of the gene that encodes GAD in H. pulcherrimus (Hp-gad) in unfertilised eggs and all blastomeres in morulae. However, at and after the swimming blastula stage, the transcript accumulation was particularly prominent in clumps of ectodermal cells throughout the embryonic surface. During the gastrula stage, the transcripts also accumulated in the endomesoderm and certain blastocoelar cells. Consistent with the increasing number of Hp-gad transcribing cells, immunoblot analysis indicated that the relative abundance of Hp-Gad increased considerably from the early gastrula stage until the prism stage. The expression pattern of GADCs determined by immunohistochemistry was identical to the pattern of Hp-gad transcript accumulation determined using WISH. In early gastrulae, GADCs formed blastocoelar cell aggregates around the blastopore with primary mesenchyme cells. The increase in the number of blastocoelar GADCs was inversely proportional to the number of ectodermal GADCs ranging from a few percent of total GADCs in early gastrulae to 80% in late prism larvae; this depended on ingression of ectodermal GADCs into the blastocoel. Some of the blastocoelar GADCs were fluorescein-positive in the larvae that developed from the 16-cell stage chimeric embryos; these comprised fluorescein-labeled mesomeres and unlabelled macromeres and micromeres. Our finding indicates that some of the blastocoelar GADCs are derived from the mesomeres and thus they are the new group of mesenchyme cells, the tertiary mesenchyme cells.

The molecular structure and role of two splice-isoforms of Unc-5 (Hp-Unc-5v1 and v2) in Unc-5/netrin interaction during serotonergic axonal projection were elucidated in this study. HpUnc-5v1 was found to be comprised of two immunoglobulin-like domains, two thrombospondin domains in the extracellular region, and ZU-5, DB, and Death domains in the cytoplasmic region, whereas Hp-Unc-5v2 lacked one thrombospondin domain, the transmembrane domain, and all cytoplasmic domains. Hp-Unc-5v1 was transcribed in unfertilized eggs, which continued until the 3-day post-fertilization (-dpf) 2-arm pluteus stage, but was suspended at the mesenchyme blastula stage (mBl), whereas Hp-Unc-5v2 was not transcribed in unfertilized eggs, but was from after fertilization to the same developmental stage of mBl as Hp-Unc-5v1. Relative accumulation of transcripts of both splice-isoforms peaked at the prism stage and declined thereafter, and they were localized at the vegetal pole region of early gastrulae, around the blastopore in mid-to late gastrulae, at fore-and mid-gut regions and on the basal side of dorsal ectoderm in 28-hour postfertilization prism larvae, and within axons at and after the 2-dpf pluteus stage. Hp-Unc-5v2:GFP was detected in the entire serotonergic cell body and extracellularly on the basal surface of oral ectoderm in 2-dpf plutei and exclusively within axons in 4-dpf plutei. Overexpression of Hp-Unc-5v2 resulted in decreased axonal projection in plutei. Knockdown of Hp-unc-5v1 and v2 by morpholino antisense oligonucleotide resulted in severe deficiency of axonal projection. Interference of Unc-5/ netrin interaction using an exogenous synthetic IFKSQDFGKWQPY peptide from the VI domain in Hp-netrin, inhibited axonal projection and larval swimming.

Little is known about the molecular mechanisms governing gonadal developmental processes in abalones. Here, we conducted transcriptome analysis of Pacific abalone Haliotis discus discus for gene discovery in the brain, ovary, testis, and unfertilized eggs. Among the annotated unigenes, 48.6% of unigenes were identified by Venn diagram analysis as having universal or tissue-specific expression. Twenty-three genes with gonad-biased gene ontology (GO) terms were first obtained. Secondly, 36 genes were found by screening known gene names related to germ cell development. Finally, 17 genes were obtained by querying the annotated unigene database for zygotically expressed gonadal genes (ovary and testis) and maternally expressed gonadal genes (ovary, testis, and unfertilized eggs) using keywords related to reproduction. To further verify tissue distribution pattern and subcellular localization of these genes, RT-PCR and in situ hybridization were performed using a unigene encoding a germ cell marker, vasa, as control. The results showed that vasa was expressed mainly in the early developmental stages of germ cells in both sexes. One of the candidate genes, vitelline envelope zona pellucida domain protein 12 (ZP12), was expressed in the primordial germ cells of immature gonad and early developmental stages of germ cells of the adult female. The results obtained from the present study suggest that vasa and ZP12 are involved in germ cell development of Pacific abalone and that ZP12 is an especially useful germ cell-specific marker in immature adults. The current gonadal transcriptome profile is an extensive resource for future reproductive molecular biology studies of this species.