Calcium signaling during the early meroblastic cleavages of zebrafish and medaka embryos

Abstract

The regulation of cytokinesis in “giant” embryonic cells (i.e.,> 500 μm in diameter) presents exacting challenges that include long-range signaling with respect to time and space; the transport and assembly, followed by disassembly, of an extensive contractile apparatus; and the remodeling and addition of new surface membrane to the resulting daughter cells. As methods have been developed to visualize intracellular Ca signaling in a nondestructive and reproducible manner, it is becoming clear that this versatile and ubiquitous signaling system (Berridge et al., 2003) plays a necessary and dynamic role in regulating early embryonic cytokinesis (Webb and Miller, 2008). The large embryos of fish of the Cyprinidae family have proved to be excellent systems in which to explore the role of Ca during cytokinesis. This is due to a number of complementary factors such as their ex utero development, large cell size, ease of manipulation, and optical clarity (Gamo and Terajima, 1963; Kimmel et al., 1995). In these large embryos there are a number of distinct and sequential steps that contribute to successful cytokinesis. These include the initial positioning of the cleavage furrow within the embryonic cell cortex, followed by the propagation (without significant deepening) of the cleavage furrow across the cell surface; the furrow then begins to deepen (i.e., furrow ingression) toward the underlying yolk cell, which results in two daughter cells (and their nuclei) being separated by a distinct extracellular groove; and finally furrow apposition occurs, where the daughter cells “zip together” following the disassembly of the contractile apparatus (Webb et al., 1997; Chang and Lu, 2000). Furrow apposition is a feature of cytokinesis in the early blastoderm of cyprinids, where the daughter cells do not completely separate from each other as in other dividing systems such as bacteria (Uehara et al., 2010) and yeast cells (Roncero and Sanchez, 2010); however, they remain apposed thus forming a classical hemispherical blastoderm. Cleavage furrow apposition thus represents the final step in this distinct type of embryonic cytokinesis (Fluck et al., 1991; Webb et al., 1997; Lee et al., 2003). Owing to the large size of their embryos and the fact that different sequential cytokinetic elements (positioning, propagation, deepening, and apposition) are separated both temporally and spatially, the first a few meroblastic cleavages of zebrafish embryos have provided an excellent opportunity to explore the mechanisms responsible for generating cytoplasmic Ca signals (Lee et al., 2003), as well as their relationship to the rearrangements of cytoskeletal elements and membrane components specific to each stage in the embryonic cytokinetic process (Li et al., 2006; Li et al., 2008). The earliest reports via direct measurement that [Ca]i might increase during embryonic cell division were reported from medaka (Oryzias latipes) using both the Ca-sensitive bioluminescent reporter aequorin (Ridgway et al., 1977) and Ca sensitive microelectrodes (Schantz et al., 1985), respectively. The first direct visualization of Ca transients during embryonic cytokinesis was once again obtained from medaka embryos using aequorin (Fluck et al., 1991). This group reported that the cytokinetic Ca transients took the form of two sequential propagating Ca waves that accompanied, firstly the progression of the cleavage furrow across the surface of the blastodisc, and secondly its subsequent ingression. This groundbreaking report by Fluck et al. was followed by a number of subsequent reports from zebrafish (Danio rerio), using either fluorescence-based Ca reporters (Chang and Meng, 1995; Chang and Lu, 2000) or a variety of aequorins (Webb et al., 1997; Creton et al., 1998; Lee et al., 2003; Lee et al., 2006). These subsequent reports confirmed that the Ca transients associated with embryonic cytokinesis consisted of a number of sequential Received July 21, 2010; accepted July 23, 2010