The formation of Ca 2+ gradients at the cleavage furrows during cytokinesis of Zebrafish embryos

In dividing embryos, a localized elevation in intracellular Ca 2+ ([Ca 2+ ] i ) at the cleavage furrow has been shown to be essential for cytokinesis. However, the underlying mechanisms for generating and maintaining these [Ca 2+ ] i gradients throughout cytokinesis are not fully understood. In the present study, we analyzed the role of inositol 1,4,5-trisphosphate receptors (IP 3 Rs) and endoplasmic reticulum (ER) distribution in determining the intracellular Ca 2+ gradients in early zebrafish blastomeres. Application of the injected Ca 2+ indicator, Indo-1, showed that during the first cell division a standing Ca 2+ gradient was formed ∼35 min after fertilization, with the [Ca 2+ ] i spatially decaying from 500–600 nmol/L at the cleavage furrow to 100–200 nmol/L around the nucleus. While the IP3R immunohistochemical fluorescence was relatively concentrated in the peri-furrow region, ER labeling was relatively enriched in both peri-furrow and peri-nuclear regions. Numeric simulation suggested that a divergence in the spatial distribution of IP3R and the locations of Ca 2+ uptake within the ER was essential for the formation of a standing Ca 2+ gradient, and the Ca 2+ gradient could only be well-established under an optimal stoichiometry of Ca 2+ uptake and release. Indeed, while inhibition of IP3R Ca 2+ release blocked the generation of the Ca 2+ gradient at a lower [Ca 2+ ] i level, both Ca 2+ release stimulation by inositol 1,4,5-trisphosphate (IP3) injection and ER Ca 2+ pump inhibition by cyclopiazonic acid also eliminated the Ca 2+ gradients at higher [Ca 2+ ] i levels. Our results suggest a dynamic relationship between ER-mediated Ca 2+ release and uptake that underlies the maintenance of the perifurrow Ca 2+ gradient and is essential for cytokinesis of zebrafish embryos.