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...[ Read more ]

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.