Localized Calcium Transients Accompany Furrow Positioning, Propagation, and Deepening during the Early Cleavage Period of Zebrafish Embryos

Abstract

Through the injection of f-aequorin (a calcium-specific luminescent reporter) and the use of an imaging photon detector, we see a distinct localized elevation of intracellular calcium that accompanies the appearance of the first furrow arc at the blastodisc surface: the furrow positioning signal. As the leading edges of the arc progress outward toward the margins of the blastodisc, they are accompanied by two subsurface slow calcium waves moving at about 0.2 micron/s: the furrow propagation signal. As these wave fronts approach the edge of the blastodisc, another calcium signal arises in the central region where the positioning signal originally appeared. Like the propagation signal, it extends outward to the margins of the blastodisc, but in this case it also moves downward, accompanying the deepening process that separates the daughter cells: the furrow deepening signal. Both of these furrow deepening progressions move at around 0.1 to 0.2 micron/s. The deepening signal begins to diminish from the center outward, returning to precleavage resting levels on completion of cytokinesis. The signaling sequence is repeated during the second cell division cycle. These localized transients do not require external calcium and they can be dissipated after they have begun by introducing calcium shuttle buffers, resulting in furrow delocalization and regression. They also occur in parthenogenetically activated eggs in which, in an attenuated form, they accompany abortive cleavages.

A Global, Myosin Light Chain Kinase-dependent Increase in Myosin II Contractility Accompanies the Metaphase–Anaphase Transition in Sea Urchin Eggs

“…Calcium has been shown to be required for cytokinesis in several embryonic cell types (Fluck et al, 1991;Miller et al, 1993;Chang and Meng, 1995;Muto et al, 1996;Webb et al, 1997;Saul et al, 2004;Wong et al, 2005), and myosin activation (Shuster and Burgess, 2002a;Stack et al, 2006) as well as membrane addition (Shuster and Burgess, 2002a) are candidate downstream effectors. Imaging of calcium dynamics in dividing L. pictus eggs revealed that there is a mobilization of calcium at the metaphase-anaphase transition that is necessary for cytokinesis (Groigno and Whitaker, 1998), raising the possibility that this calcium transient may partially serve as the "timer" for the activation of myosin II.…”

Advances in Cytokinesis Research. Contractile Ring Formation in Xenopus Egg and Fission Yeast.

“…However, data regarding the relevance of Ca waves in cleavage furrow formation is contradictory or incomplete. In fish eggs, it has been suggested that the cleavage furrow formation is accompanied by a localized Ca wave which propagates in the subcortical layer along the growing end of the cleavage furrow (Fluck et al, 1993;Chang and Meng, 1995;Webb et al, 1998). However, in these studies, the actual positions of the furrow ends have not been identified when the Ca waves have been observed.…”

Expression and reconstitution of the bioluminescent Ca2+ reporter aequorin in human embryonic stem cells, and exploration of the presence of functional IP3 and ryanodine receptors during the early stages of their differentiation into cardiomyocytes

“…Indeed, well-defined Ca 2+ signals have been demonstrated to correlate with, and sometimes to directly initiate and/or regulate, distinct morphological events (Homa et al, 1993;Swann et al, 1994;Chang and Meng, 1995;Webb et al, 1997;Leung et al, 1998;Créton et al, 2000;Porter et al, 2003;Ma et al, 2009;Lohmann, 2009;Cheung et al, 2011). In addition, when errors occur in the normal pattern of Ca 2+ signaling, these might result in the onset of a particular disorder or disease (Somlo and Erlich, 2001;Splawski et al, 2004;Allen et al, 2010;Woods and Padmanabhan, 2012).…”

“…Together, these various characteristics of bioluminescent Ca 2+ reporters such as aequorin allow the ready detection and/or visualization of even the smallest changes in intracellular [Ca 2+ ] continually over the relatively long time periods that are required to monitor ESC differentiation. Aequorin has long been the method of choice for recording Ca 2+ signals in large cells such as muscle fibers and axons (Ridgway and Ashley, 1967;Baker et al, 1971), and in eggs and early embryos of fish, frogs and sea urchins (Ridgway et al, 1977;Eisen and Reynolds, 1985;Webb et al, 1997;Leclerc et al, 2000), which can be loaded with the reporter via microinjection. However, the protein component of aequorin (i.e., apo-aequorin) can also be genetically introduced into cells, after which active holo-aequorin is reconstituted in vivo by simple incubation with its prosthetic co-factor, coelenterazine (Rizzuto et al, 1992).…”