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.

Fig. 1

GCaMP6s expression and fluorescence in Tg[βactin2:GCaMP6s]^{stl351/stl351} and Tg[ubi:GCaMP6s]^{stl352/stl352} transgenic zebrafish during early embryogenesis. (A) Schematics of the Tol2[βactin2:GCaMP6s] or Tol2[ubi:GCaMP6s] constructs. (B) GCaMP6s fluorescent confocal microscope images in Tg[βactin2:GCaMP6s]^{stl351/stl351} and Tg[ubi:GCaMP6s]^{stl352/stl352} embryos at several developmental stages. a, a′, d, d′, lateral view; b, b′, c, c′, animal pole view. Asterisks indicate the yolk, and arrowheads point to the blastodisc in a, a′. Arrows point to the heart in d, d′. (C-D) RT-PCR and qRT-PCR analyses of GCaMP6s RNA expression levels in Tg[βactin2:GCaMP6s]^{stl351/stl351} and Tg[ubi:GCaMP6s]^{stl352/stl352} embryos in the course of embryogenesis. The qRT-PCR results were normalized to β-actin. Error bars represent standard deviation; N=3.

Fig. 2

Dynamics of Ca²⁺ signaling in Tg[βactin2:GCaMP6s]^{stl351/stl351} embryos at cleavage and blastula stages. (A–L) Still images of GCaMP6s signals during cleavage furrow progression from 2-cell stage to 16-cell stage. (M) Quantification of Ca²⁺ transient duration before and after MBT. Error bars represent standard deviation; N=4 embryos. ns, not significant. (N) Comparison of Ca²⁺ transient numbers before and after MBT; N=6 embryos. (O–P) Still images and traces of Ca²⁺ transients in a time-lapse series at blastula stages. (Q) Time-lapse overlay of GCaMP6s signal from 3.7 hpf to 4 hpf from a single z-section in lateral view.

Fig. 3

Dorsal bias of the EVL Ca²⁺ transients from midblastula to late blastula stage. (A) Quantification of total Ca²⁺ transient numbers at 30-min intervals from 2.5 hpf to 8 hpf. Error bars represent standard deviation; ns, not significant. ****; P≤0.0001; N=8–10 embryos. (B) Representative Ca²⁺ transient traces from a single spot in each quadrant between 3.5 hpf and 4 hpf. (C) Manual quantification of Ca²⁺ transient frequency in four blastoderm quadrants from 2.5 hpf to 8 hpf; N=8–10 embryos. (D). Comparisons of Ca²⁺ transient numbers in individual quadrant between manual and automated quantifications at 2.5–3 hpf and 3.5–4 hpf. (E) Automated quantification of Ca²⁺ transient frequency from 2.5 hpf to 7 hpf. Error bars represent S.E.M. *, P≤0.05. ***, P≤0.001. ****, P≤0.0001; N=4–6 embryos.

Fig. 4

EVL Ca²⁺ signaling pattern in ventralized and dorsalized embryos. (A-B) Automated quantification of EVL Ca²⁺ transient frequency in ichabod/β-catenin2 ventralized embryos between 2.5 hpf and 6.5 hpf. Error bars represent S.E.M.; N=6 embryos. (C) Automated quantification of EVL Ca²⁺ transient frequency in β-catenin1-injected dorsalized embryos. Induced dorsal organizer is oriented to the right. Error bars represent S.E.M. *, P≤0.05. **, P≤0.01***, P≤0.001. ****, P≤0.0001; N=6 embryos. (D) Comparison of Ca²⁺ transient number in individual quadrants among WT, ventralized, and dorsalized embryos at 3.5–4 hpf. Error bars represent standard deviation. ns, not significant. *, P≤0.05. ****, P≤0.0001; (E) Representative 30-min time-lapse overlay of GCaMP6s signals in WT, ventralized, and dorsalized embryos from 2.5 hpf to 6.5 hpf.

Fig. 5

Excess Nodal signaling prolongs Ca²⁺ transient duration specifically in the DFCs. (A) Representative images of a single Ca²⁺ transient in a DFC at 6.5 hpf. (B) Quantification of Ca²⁺ transient duration in WT and Cyc/Ndr2-misexpressing embryos. Error bars represent standard deviation. ****, P≤0.0001. N=3 embryos in control and Cyc/Ndr2-misexpressing embryos, respectively. (C) Still images of Ca²⁺ transients in the DFCs of uninjected control and Cyc/Ndr2-misexpressing embryo in a time-lapse series. Arrowhead points to the DFC region that was imaged. (D) Quantification of DFC Ca²⁺ transient duration between DFCs and EVL cells in cyc/ndr2 RNA-injected embryos after CBX treatment. ****, P≤0.0001. N=2 embryos. (E) Quantification of DFC Ca²⁺ transient duration in Cyc/Ndr2-misexpressing embryos before and after CBX treatment. ns, not significant; N=4 embryos.