Western blot analysis of the temporal expression pattern of the short Gα_s isoform (Gα_sS) in whole wild-type zebrafish from 24 to 96 h post-fertilization (hpf). (a) Representative blot of the Gα_sS expression level using GAPDH as a loading control. Whole zebrafish tissue lysates were used to extract total protein (~40 µg loaded per well). (b) The band intensities were quantified using ImageJ software, followed by normalization of the Gα_sS band intensity to that of GAPDH, yielding the relative Gα_sS expression level in arbitrary units. One-way ANOVA followed by Tukey’s multiple comparisons test were performed (* p < 0.05). Each bar represents the mean ± SEM (n = 3).

Western blot analysis of the knockdown efficiency of the three gnas Morpholino (MO) doses at 48 hpf. (a) Representative blot of the Gα_sS expression level in non-injected control (UI Ctrl) and standard control (Std Ctrl) embryos compared to those injected with 1, 3, and 5 ng of gnas MOs. The Std Ctrl embryos were injected with 5 ng of Std Ctrl MO. Whole zebrafish tissue lysates were used to extract total protein (~40 µg loaded per well) and GAPDH served as a loading control. (b) The band intensities were quantified using ImageJ software, followed by normalization of the Gα_sS band intensity to that of GAPDH. The Gα_sS expression level was expressed as a percentage of that in the UI Ctrl embryos. One-way ANOVA followed by Tukey’s multiple comparisons test were performed (**** p < 0.0001). Each bar represents the mean ± SEM (n = 4).

The survival rates at 24 and 48 hpf of the zebrafish embryos (n ≈ 20 per group) injected with 5 ng of Std Ctrl MO or different doses (1, 3, or 5 ng) of gnas MOs. The survival rates were normalized to those of the non-injected controls (UI Ctrl). Two-way ANOVA and Tukey’s post hoc test were performed, indicating no significant differences between the survival rates at either timepoints. Each bar represents the mean ± SEM (n = 4).

The rate of tail flicks at 24 hpf of the zebrafish embryos (n ≈ 10 per group) injected with 5 ng of Std Ctrl MO or different doses (1, 3, or 5 ng) of gnas MOs, compared to non-injected controls (UI Ctrl). The frequency of this spontaneous movement was measured by the DanioScope software as the mean burst count per minute. One-way ANOVA and Tukey’s multiple comparisons test were performed, indicating no significant differences between the mean tail-flicking rates. Each bar represents the mean ± SEM (n = 4).

The hatching rates at 48 hpf of the zebrafish embryos (n ≈ 20 per group) injected with 5 ng of Std Ctrl MO or different doses (1, 3, or 5 ng) of gnas MOs, compared to non-injected controls (UI Ctrl). One-way ANOVA and Tukey’s multiple comparisons test were performed, indicating no significant differences between the mean hatching rates. Each bar represents the mean ± SEM (n = 4).

Quantification of the Oil Red O staining at 120 hpf of whole zebrafish larvae (n ≈ 20 per group) injected with 5 ng of Std Ctrl MO or two doses (3 or 5 ng) of gnas MOs. Staining was quantified by the absorbance of the extracted stain at 495 nm and normalized to that of non-injected controls (UI Ctrl). One-way ANOVA and Tukey’s multiple comparisons test were performed (* p < 0.05 and ** p < 0.01). Each bar represents the mean ± SEM (n = 5).

Yolk sac areas of zebrafish larvae (n = 10 per group), at 72 and 120 hpf, injected with 5 ng of Std Ctrl MO or two doses (3 or 5 ng) of gnas MOs, compared to those of non-injected controls (UI Ctrl). Two-way ANOVA and Tukey’s multiple comparisons test were performed, indicating significant increases in yolk size of gnas morphants at 120 hpf compared to non-injected controls (* p < 0.05). Each bar represents the mean ± SEM (n = 3).

Relative change in fluorescence after incubation in alamarBlue^TM assay buffer of zebrafish larvae injected with 5 ng of Std Ctrl MO or two doses (3 or 5 ng) of gnas MOs, normalized to that of non-injected controls (UI Ctrl). The larvae (n ≈ 12 per group) were incubated from 72 to 96 hpf. One-way ANOVA and Tukey’s multiple comparisons test were performed (** p < 0.01). Each bar represents the mean ± SEM (n = 4).

The mean body lengths of zebrafish larvae (n = 10 per group), at 72 and 120 hpf, injected with 5 ng of Std Ctrl MO or two doses (3 or 5 ng) of gnas MOs, compared to those of non-injected controls (UI Ctrl). Two-way ANOVA and Tukey’s multiple comparisons test were performed, indicating no significant differences. Each bar represents the mean ± SEM (n = 3).

Representative images of the lateral views of standard control and gnas (3 and 5 ng) morphants at 120 hpf. Spine curvature and yolk sac enlargement of the gnas morphants are indicated by arrows and arrowheads, respectively. The images were captured at 20× magnification following straightening of the larvae in methylcellulose as much as possible.

The mean larval masses of zebrafish larvae (n ≈ 20 per group), at 120 hpf, injected with 5 ng of Std Ctrl MO or two doses (3 or 5 ng) of gnas MOs, compared to those of non-injected controls (UI Ctrl). One-way ANOVA and Tukey’s multiple comparisons test were performed, indicating no significant differences. Each bar represents the mean ± SEM (n = 3).

Hatching rates at 72 hpf of zebrafish larvae (n ≈ 25 per group) injected with 5 ng of Std Ctrl MO or two doses (3 or 5 ng) of gnas MOs, compared to those of non-injected controls (UI Ctrl). One-way ANOVA and Tukey’s multiple comparisons test were performed, indicating no significant differences. Each bar represents the mean ± SEM (n = 3).

Concentrations of different molecules in whole zebrafish larvae at 72 hpf, determined by ELISA. (a) Triglyceride, (b) leptin, and (c) cAMP levels in larvae (n ≈ 35 per group) injected with 5 ng of Std Ctrl MO or two doses (3 or 5 ng) of gnas MOs, relative to those of non-injected controls (UI Ctrl). One-way ANOVA and Tukey’s multiple comparisons test were performed, indicating no significant differences. Each bar represents the mean ± SEM (n = 3).