Disappearance of an epithelial cell surface‐specific glycoprotein (Epith‐1) associated with epithelial–mesenchymal conversion in sea urchin embryogenesis

Abe

et al. 2013

Biology Open

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.

Section: Methodsmentioning

confidence: 99%

Mesomere-derived glutamate decarboxylase-expressing blastocoelar mesenchyme cells of sea urchin larvae

Abe

et al. 2013

Biology Open

“…Epithelial cell-specific Epith-2 monoclonal antibody was produced in our laboratory (Kanoh et al, 2001). Anti-DRD1 antiserum was kindly provided by Dr Suyemitu, Saitama University, Japan.…”

Section: Primary Antibodiesmentioning

confidence: 99%

Development of the γ-amino butyric acid (GABA)-ergic signaling system and its role in larval swimming in sea urchin

Abe

Journal of Experimental Biology

et al. 2013

SUMMARYThe present study aimed to elucidate the development and γ-amino butyric acid (GABA)-ergic regulation of larval swimming in the sea urchin Hemicentrotus pulcherrimus by cloning glutamate decarboxylase (Hp-gad), GABA A receptor (Hp-gabrA) and GABA A receptor-associated protein (Hp-gabarap), and by performing immunohistochemistry. The regulation of larval swimming was increasingly dependent on the GABAergic system, which was active from the 2days post-fertilization (d.p.f.) pluteus stage onwards. GABA-immunoreactive cells were detected as a subpopulation of secondary mesenchyme cells during gastrulation and eventually constituted the ciliary band and a subpopulation of blastocoelar cells during the pluteus stage. Hp-gad transcription was detected by RT-PCR during the period when Hp-Gad-positive cells were seen as a subpopulation of blastocoelar cells and on the apical side of the ciliary band from the 2d.p.f. pluteus stage. Consistent with these observations, inhibition of GAD with 3-mercaptopropioninc acid inhibited GABA immunoreactivity and larval swimming dose dependently. Hp-gabrA amplimers were detected weakly in unfertilized eggs and 4d.p.f. plutei but strongly from fertilized eggs to 2d.p.f. plutei, and Hp-GabrA, together with GABA, was localized at the ciliary band in association with dopamine receptor D1 from the two-arm pluteus stage. Hpgabarap transcription and protein expression were detected from the swimming blastula stage. Inhibition of the GABA A receptor by bicuculline inhibited larval swimming dose dependently. Inhibition of larval swimming by either 3-mercaptopropionic acid or bicuculline was more severe in older larvae (17 and 34d.p.f. plutei) than in younger ones (1d.p.f. prism larvae).

“…Prior to antibody treatment, embryos were hydrated in phosphatebuffered saline with 0.1% Tween-20 (PBST), and then incubated for 24-48h at 4°C with rabbit antibodies against DA (catalog number DA-1140, BIOMOL International LP, Plymouth Meeting, PA, USA or catalog number DOP11-S, Alpha Diagnostic International, San Antonio, TX, USA; diluted 1:150, the latter antibody was used only as an alternative to the DA-1140 antibody as stated in the text), rabbit antibodies against serotonin (Sigma-Aldrich Japan; 1:500), monoclonal antibody against -tubulin (EXBIO Praha, a.s. Vestec, Czech Republic; 1:1000), monoclonal antibody against acetylated -tubulin (Sigma-Aldrich Japan; 1:500), rabbit antibodies against 5Ј-bromo-2Ј-deoxyuridine (BrdU; Sigma-Aldrich Japan; 1:250), monoclonal antibody Epith-2 against sea urchin epithelium plasma membrane (Kanoh et al, 2001), monoclonal antibody 1E11 against synaptotagmin, rabbit anti-Hp-DRD1 antiserum (1:500), or mouse antibodies against serotonin receptor (5HThpr) (Katow et al, 2004) (1:500), either diluted in PBST or undiluted in the case of the monoclonal antibodies. Anti-BrdU antibodies were applied after 1h of treatment with 2moll -1 HCl to reveal incorporated BrdU.…”

Section: Whole-mount Immunohistochemistrymentioning

confidence: 99%

“…To examine how closely the pattern of distribution of the DAG stripes resembled that of the cilia, the cytological localization of the DAGs was examined by immunostaining with anti-DA antibody and an Epith-2 monoclonal antibody, which is specific to the lateral plasma membrane of epithelial cells (Kanoh et al, 2001). This staining revealed that each DAG was positioned apically (Fig.2A, inset) in the middle of a single ectodermal cell (Fig.2A).…”

Section: Formation Of Dagsmentioning

confidence: 99%

Development of a dopaminergic system in sea urchin embryos and larvae

Journal of Experimental Biology

Suyemitsu

Ooka

et al. 2010

SUMMARYThe mechanisms that regulate the organized swimming movements of sea urchin blastulae are largely unknown. Using immunohistochemistry, we found that dopamine (DA) and the Hemicentrotus pulcherrimus homolog of the dopamine receptor D1 (Hp-DRD1) were strongly co-localized in 1-2m diameter granules (DA/DRD1 granules). Furthermore, these granules were arranged across the entire surface of blastulae as they developed locomotory cilia before hatching, and remained evident until metamorphosis. DA/DRD1 granules were associated with the basal bodies of cilia, and were densely packed in the ciliary band by the eight-arm pluteus stage. The transcription of Hp-DRD1 was detected from the unfertilized egg stage throughout the period of larval development. Treatment with S-(-)-carbidopa, an inhibitor of aromatic-L-amino acid decarboxylase, for 20-24h (i) from soon after insemination until the 20h post-fertilization (20hpf) early gastrula stage and (ii) from the 24hpf prism larva stage until the 48hpf pluteus stage, inhibited the formation of DA granules and decreased the swimming activity of blastulae and larvae in a dose-dependent manner. Exogenous DA rescued these deprivations. The formation of DRD1 granules was not affected. However, in 48hpf plutei, the serotonergic nervous system (5HT-NS) developed normally. Morpholino antisense oligonucleotides directed against Hp-DRD1 inhibited the formation of DRD1 granules and the swimming of larvae, but did not disturb the formation of DA granules. Thus, the formation of DRD1 granules and DA granules occurs chronologically closely but mechanically independently and the swimming of blastulae is regulated by the dopaminergic system. In plutei, the 5HT-NS closely surrounded the ciliary bands, suggesting the functional collaboration with the dopaminergic system in larvae. Supplementary material available online at