Necessary role for intracellular Ca2+ transients in initiating the apical-basolateral thinning of enveloping layer cells during the early blastula period of zebrafish development

Abstract

During the early blastula period of zebrafish embryos, the outermost blastomeres begin to undergo a significant thinning in the apical/basolateral dimension to form the first distinct cellular domain of the embryo, the enveloping layer (EVL). During this shape transformation, only the EVL-precursor cells generate a coincidental series of highly restricted Ca(2+) transients. To investigate the role of these localized Ca(2+) transients in this shape-change process, embryos were treated with a Ca(2+) chelator (5,5'-difluoro BAPTA AM; DFB), or the Ca(2+) ionophore (A23187), to downregulate and upregulate the transients, respectively, while the shape-change of the forming EVL cells was measured. DFB was shown to significantly slow, and A23187 to significantly facilitate the shape change of the forming EVL cells. In addition, to investigate the possible involvement of the phosphoinositide and Wnt/Ca(2+) signaling pathways in the Ca(2+) transient generation and/or shape-change processes, embryos were treated with antagonists (thapsigargin, 2-APB and U73122) or an agonist (Wnt-5A) of these pathways. Wnt-5A upregulated the EVL-restricted Ca(2+) transients and facilitated the change in shape of the EVL cells, while 2-APB downregulated the Ca(2+) transients and significantly slowed the cell shape-change process. Furthermore, thapsigargin and U73122 also both inhibited the EVL cell shape-change. We hypothesize, therefore, that the highly localized and coincidental Ca(2+) transients play a necessary role in initiating the shape-change of the EVL cells.