Unc-5/netrin-mediated axonal projection during larval serotonergic nervous system formation in the sea urchin, Hemicentrotus pulcherrimus

Abstract

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.

Characterization and Endocytic Internalization of Epith-2 Cell Surface Glycoprotein during the Epithelial-to-Mesenchymal Transition in Sea Urchin Embryos

“…Instead, a smaller but distinctive new band appeared at the 126 kDa region (Figure 4A, lane 12), which is similar to our previous observations in T. hardwicki in which the major 160 kDa band shift to a smaller 142 kDa at this stage (10). This finding also suggests that the post-maternal message of the Epith-2 protein was switched on during the period between the 29- and 40-hpf larva stages as was previously reported regarding the Hp-Unc-5 expression pattern in H. pulcherrimus (39). However, further elucidative studies ought to be done.…”

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

“…Whole-mount in situ hybridisation (WISH) and whole-mount fluorescent in situ hybridisation (WFISH) were performed as described (Abe et al, 2013). Unfertilised eggs (Ufe), eggs 30 minutes post-fertilisation (Fe), 6-hour post-fertilisation (hpf) morulae (Morula), 15-hpf swimming blastulae (sBl), 17-hpf mesenchyme blastulae (mBl), 18-hpf early gastrulae (eG), 22-hpf late gastrulae (lG) and 24-hpf prism larvae (Prism) were fixed with 4% paraformaldehyde diluted in mixture of FSW (4% PFA), 10 mmol l −1 3-morpholinopropanesulphonic acid (MOPS) buffer and 0.1% Tween 20 at ambient temperature (AT) for one hour.…”

Mechanisms of the epithelial-to-mesenchymal transition in sea urchin embryos

“…The chemotrophic guidance factor is also involved in serotonergic axon guidance in sea urchin larvae. 146 Although the GADC streak remains structurally stable until the onset of the metamorphosis that deforms the entire larval body, the GADCs in the GADC streak do not express Epith protein. This morphogenetic process could be a variant of the MET.…”