Chen et al., 2017 - Imaging early embryonic calcium activity with GCaMP6s transgenic zebrafish. Developmental Biology   430(2):385-396 Full text @ Dev. Biol.

Fig. 1

GCaMP6s expression and fluorescencein 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. 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.

Fig. s3

Fig. S3. Comparison of calcium transient numbers in WT and mutants. (A) Quantification of total number of EVL calcium transients in WT, ventralized, and dorsalized embryos from 2.5 hpf to 6.5 hpf. N indicates the number of embryos in each group.(B) Quantification of EVL calcium transient numbers in each quadrant at 3.5–4 hpf from individual embryos. Arrows indicate the endogenousdorsal organizer.

Acknowledgments:
ZFIN wishes to thank the journal Developmental Biology for permission to reproduce figures from this article. Please note that this material may be protected by copyright.

Reprinted from Developmental Biology, 430(2), Chen, J., Xia, L., Bruchas, M.R., Solnica-Krezel, L., Imaging early embryonic calcium activity with GCaMP6s transgenic zebrafish, 385-396, Copyright (2017) with permission from Elsevier. Full text @ Dev. Biol.