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

Abstract

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.

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

“…The spatiotemporal change in the expression pattern of Epith-1 and -2 from the very early period of cleavage to the total loss from the cell surface during migration in the blastocoel 4,8,61 strongly suggests the importance of the involvement of substances on the cell surface in the EMT signaling mechanism. Because the gene structures of Epith-1 and -2 have not been elucidated, further genomic analysis will enable the evaluation of the molecular regulatory mechanism of the morphogenetic role of these cell surface proteins.…”

“…They are the neuronal mesenchyme cells (NMC) that were initially introduced as the tertiary mesenchyme cells, 4 which were discovered to be new group of mesenchymal cells in the last couple of years. However, because the cells are characterized by the expression of neuronal genes or proteins, such as glutamate decarboxylase [GAD; g-aminobutyric acid (GABA) synthetase], serotonin receptors 4 and encephalopsin, 5 they are called NMCs in this Review. The morphogenetic analysis of PMC formation has been incorporated in sea urchin developmental biology as a classical model of the EMT (Fig.…”

“…144 The EMT of NMCs Recent neurogenetic studies of H. pulcherrimus have detected mesenchymal cells that express genes and proteins including encephalopsin, 5 5HThpr, 4 GAD, 4,128 and GABA (a neurotransmitter) 128 during the swimming blastula stage, in which PMC ingression is not yet occurred. Among these cell types, 5HThpr cells cannot be eliminated completely by removing the entire archenteron by microsurgery at the early gastrula stage, 13 indicating that NSMCs alone are not the ancestors of these cells.…”

“…The presence of another source of 5HThpr cells has been suggested. The succeeded study of the following GADC formation, 5HThpr is expressed in a 94 , and gene transcription and protein expression patterns in NMC of H. pulcherrimus (Hp-ecpn, 5 Hp-5HThpr, 12 Hp-gad 4 ). EMT, EMT in S. purpuratus; Blue letter EMT, EMT in H. pulcherrimus; Light gray screen, before ingression; Dark gray screen, after ingression; Red screen, EMT-related genes; Green screen, NMC-related genes in H. pulcherrimus.…”

“…According to whole-mount in situ hybridization analysis, a majority of GADC precursors are located in the animal ectoderm region that is a descendant of the mesomeres in 16-cell embryos. 4 They are delaminated from the animal ectoderm along with other ectodermal regions during the swimming blastula stage until the gastrula stage by losing Epith-2 protein from their cell surface (Fig. 11).…”