Shh signaling synchronizes tissue formation and mitochondria network maturation. (A) Pharmacological experiments design: agents were administered at 10 hpf, images captured at 24, 28, and 48 hpf. (B–D) Confocal imaging of mitochondrial network (Tomm20, T20; green) counterstained with Phalloidin (P; red) and Hoescht (H; blue) after administration of pharmacological agents to activate Shh signaling (SAG), inhibit Shh signaling (Cyclopamine, Cyc) or antagonize BMP signaling (DMH1) at 24 hpf (B), 28 hpf (C), and 48 hpf (D) in embryos submitted to the different drugs and in controls (Ctrl). (E) Electron micrographs of longitudinal sections at 48 hpf in Ctrl and Cyc treated embryo. Mitochondria are overlaid in green. (F) Quantification of mitochondria number, mean area and circularity at 48 hpf in Cyc treated embryos and Ctrl from longitudinal electron micrographs (n = 5 micrographs per group). (G) Confocal imaging of embryonic cells cultured from 10 hpf bud stage embryos either transfected with empty vector (Mock) or expressing Cherry-Shh. Mitochondria network is labeled with the component of complex IV MTCO1 (green), counterstained by Cherry (red) and Hoechst (H; blue). Bars are mean ± SEM. ∗P < 0.05, ∗∗P < 0.01, unpaired T-test. See Supplementary Figure S7.
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