Imaging early embryonic calcium activity with GCaMP6s transgenic zebrafish.

Intracellular Ca signaling regulates cellular activities during embryogenesis and in adult organisms. We generated stable Tg[βactin2:GCaMP6s] and Tg[ubi:GCaMP6s] transgenic lines that combine the ubiquitously-expressed Ca indicator GCaMP6s with the transparent characteristics of zebrafish embryos to achieve superior in vivo Ca imaging. Using the Tg[βactin2:GCaMP6s] line featuring strong GCaMP6s expression from cleavage through gastrula stages, we detected higher frequency of Ca transients in the superficial blastomeres during the blastula stages preceding the midblastula transition. Additionally, GCaMP6s also revealed that dorsal-biased Ca signaling that follows the midblastula transition persisted longer during gastrulation, compared with earlier studies. We observed that dorsal-biased Ca 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 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 transients specifically in the dorsal forerunner cells. The GCaMP6s transgenic lines described here enable unprecedented visualization of dynamic Ca events from embryogenesis through adulthood, augmenting the zebrafish toolbox.