The role of intra- and intercellular Ca[superscript]2+ transients in the differentiation of enveloping layer cells during the blastula period of zebrafish (danio rerio) development

Abstract

It has been reported that zebrafish (Danio rerio) enveloping layer (EVL) cells, but not the underlying deep cells (DCs), undergo an apical to basolateral thinning during the Blastula Period, and that this morphological transformation is accompanied by the generation of both intra- and intercellular Ca2+ transients restricted to this specific cellular domain. No data, however, have been presented to clearly link these Ca2+ signals with EVL cell thinning. Using fluorescent-based confocal Ca2+ imaging, I confirmed that these Ca2+ signals are generated primarily by EVL cells. Analysis of luminescent-based Ca2+ imaging data reveals that between ~2.5 and 4.0 hpf, almost every EVL cell (i.e., 92% ± 1%) generated at least one Ca2+ transient. To investigate the possible role of the Ca2+ signals in the EVL cell thinning process, embryos were treated with a Ca2+ chelator (5, 5’-difluoro BAPTA AM; DFB), or a Ca2+ ionophore (A23187), to down- and up-regulate the signals, respectively, then imaged via confocal microscopy. The shape of both EVL cells and DCs was measured using an ‘Ellipse Shape Factor’ (ESF) function. To investigate the source of these Ca2+ signals and the possible involvement of the phosphoinositide (PI) and the Wnt/Ca2+ pathway in the cell thinning process, embryos were treated with antagonists (i.e., thapsigargin, 2-APB and U73122) or with an agonist (i.e., Wnt-5A) of these Ca2+ signaling pathways. I report that A23187 and Wnt-5A facilitated the EVL cell thinning process, while DFB, thapsigargin, 2-APB and U73122 all significantly slowed EVL cell thinning. Thus, the EVL Ca2+ signals generated by Ca2+ release via IP3Rs appear to play a necessary role in the EVL cell thinning process. In addition, cytochalasin B treatment retarded EVL cell thinning in a dose-dependent manner, suggesting the involvement of Ca2+ -sensitive components of the actin-based cytoskeleton as possible downstream targets of the Ca2+ transients.

Date of Award	2009
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology