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.

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.

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.

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. 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.