Imaging early embryonic calcium activity with GCaMP6s transgenic zebrafish

Abstract

Intracellular Ca²⁺ 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 Ca²⁺ indicator GCaMP6s with the transparent characteristics of zebrafish embryos to achieve superior in vivo Ca²⁺ imaging. Using the Tg[βactin2:GCaMP6s]^stl351 line featuring strong GCaMP6s expression from cleavage through gastrula stages, we detected higher frequency of Ca²⁺ transients in the superficial blastomeres during the blastula stages preceding the midblastula transition. Additionally, GCaMP6s also revealed that dorsal-biased Ca²⁺ signaling that follows the midblastula transition persisted longer during gastrulation, compared with earlier studies. We observed that dorsal-biased Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ transients specifically in the dorsal forerunner cells. The GCaMP6s transgenic lines described here enable unprecedented visualization of dynamic Ca²⁺ events from embryogenesis through adulthood, augmenting the zebrafish toolbox.

Keywords: Calcium transients; Dorsal forerunner cells; Embryonic cleavages; Gastrulation; Nodal; β-catenin.