Imaging early embryonic calcium activity with GCaMP6s transgenic zebrafish

Intracellular Ca(2+) signaling regulates cellular activities during embryogenesis and in adult organisms. We generated stable Tg[βactin2:GCaMP6s](stl351) and Tg[ubi:GCaMP6s](stl352) transgenic lines that combine the ubiquitously-expressed Ca(2+) indicator GCaMP6s with the transparent characteristics of zebrafish embryos to achieve superior in vivo Ca(2+) imaging. Using the Tg[βactin2:GCaMP6s](stl351) line featuring strong GCaMP6s expression from cleavage through gastrula stages, we detected higher frequency of Ca(2+) transients in the superficial blastomeres during the blastula stages preceding the midblastula transition. Additionally, GCaMP6s also revealed that dorsal-biased Ca(2+) signaling that follows the midblastula transition persisted longer during gastrulation, compared with earlier studies. We observed that dorsal-biased Ca(2+) signaling is diminished in ventralized ichabod/β-catenin2 mutant embryos and ectopically induced in embryos dorsalized by excess β-catenin. During gastrulation, we directly visualized Ca(2+) signaling in the dorsal forerunner cells, which form in a Nodal signaling dependent manner and later give rise to the laterality organ. We found that excess Nodal increases the number and the duration of Ca(2+) transients specifically in the dorsal forerunner cells. The GCaMP6s transgenic lines described here enable unprecedented visualization of dynamic Ca(2+) events from embryogenesis through adulthood, augmenting the zebrafish toolbox.