Establishment of a transitory dorsal-biased window of localized Ca2+ signaling in the superficial epithelium following the mid-blastula transition in zebrafish embryos

Abstract

Using complementary luminescent- and fluorescent-based Ca(2+) imaging techniques, we have re-examined the Ca(2+) dynamics that occur during the Blastula Period (BP) of zebrafish development. We confirm that aperiodic, localized Ca(2+) transients are generated predominately in the superficial epithelial cells (SECs). At the start of the BP, these Ca(2+) transients are distributed homogeneously throughout the entire superficial epithelium. Following the mid-blastula transition (MBT), however, their distribution becomes asymmetrical, where a significantly greater number are generated in the presumptive dorsal quadrant of the superficial epithelium. This asymmetry in Ca(2+) signaling lasts for around 60 min, after which the total number of transients generated from the entire superficial epithelium falls to less than one per minute until the end of the BP. We have thus called this asymmetry the "dorsal-biased Ca(2+) signaling window". The application of pharmacological agents indicates that the post-MBT SEC Ca(2+) transients are generated via the phosphatidylinositol (PI) signaling pathway. This suggests that the previously reported ventralizing function attributed to the homogeneously distributed PI pathway-generated SEC Ca(2+) transients is most likely to occur earlier in development, prior to the MBT.

Chemokine GPCR Signaling Inhibits β-Catenin during Zebrafish Axis Formation

“…Second, we tested whether Ccr7, as expected from a chemokine GPCR, signals through G-protein coupling in the process of axis formation. Consistent with this notion, treating Ccr7 overexpressing embryos with GDP-β-S, a GPCR/heterotrimeric G protein activation inhibitor [56], suppressed the ventralizing effect of Ccr7, as revealed by szl expression (Figure 4B). …”

“…Also consistent with the antagonism between Ca 2+ and the Wnt/β-catenin pathway during axis formation is the report of increased intracellular Ca 2+ in the ventralized maternal-effect zebrafish mutant, hecate [70]. A number of studies using pharmacological inhibitors have implicated a signal transduction pathway dependent on the phosphatidylinositol (PI) cycle upstream of Ca 2+ release from intracellular organelles in limiting dorsal axis formation [56],[58]. Moreover, three genes encoding IP 3 receptors that mobilize intracellular Ca 2+ stores upon activation by IP 3 are expressed in zebrafish blastula [19], and IP 3 concentration increases after 32-cell stage during axis specification [20].…”

“…Likewise, GDP-β-S suppressed the ventralizing effect of Ccr7 overexpression (Figure 4B). Further, we showed that during early cleavage stages, Ccr7 and/or Ccl19.1 overexpression increased frequencies of Ca 2+ transients that occur homogenously in the superficial cells of early zebrafish blastula [20],[56], while depleting Ccr7 and/or Ccl19.1 had the opposite effect (Figures 4F and S4A,B). Our observations that treatments with thapsigargin and 2-APB, drugs that inhibit Ca 2+ transients (Figures 4F and S4A,B), elevate β-catenin levels (Figure 4E) and promote expression of β-catenin-dependent organizer genes (Figure S4C) suggest that Ccr7 inhibits β-catenin and axis formation, via its GPCR activity, to promote intracellular Ca 2+ transients.…”

“…Aperiodic, localized Ca 2+ transients occur starting at 32 to 1K cell stage (1.75–3 hpf) and are uniformly distributed in the superficial blastomeres of zebrafish blastulae [20],[56], concurrent with the nuclear localization of β-catenin on the prospective dorsal side [48]. Compared to the WT control, the frequency of Ca 2+ transients was significantly higher in embryos overexpressing Ccr7 as well as in embryos co-expressing Ccr7 and its Ccl19.1 chemokine ligand (see below), correlating with a reduction of β-catenin in embryos overexpressing Ccr7 (Figures 2G, 2E, and 3D,E).…”

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).…”