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Fig. 3 Quantification analysis reveals differences between sox11a/b knock-out and control embryos. All staining images were after “Max Intensity” z-stack projection. (A) Antibody and HCR staining comparing the expression levels of Phox2b, HuC/D, and adcyap1b in sox11a/b knock-out embryos (Top) with the control embryos (Bottom) at 4.5 dpf. On the right, hindgut regions that showed a significant difference in cells expressing Phox2b and HuC/D were zoomed in. A significant difference in adcyap1b expression was observed throughout the whole gut. (Scale bar, 100 μm, and 50 μm for zoomed region.) (B) Antibody and HCR staining comparing the expression levels of adcyap1b, nos1, and vip in sox11a/b knock-out embryos with the control embryos at 6.5 dpf. On the right, corresponding regions that showed a significant difference in cells expressing nos1 and vip were zoomed in. A significant difference in adcyap1b expression was observed throughout the whole gut. (Scale bar, 100 μm, and 50 μm for zoomed region.) (C) At 4.5 dpf, an overall developmental delay was observed in sox11a/b knock-out embryos (Nsox11a/b = 11, Ntyr = 14, replicates = 3). (D) At 6.5 dpf, a specific reduction of the inhibitory motor neuron cluster was observed in sox11a/b knock-out embryos, particularly in adcyap1b expression (for Phox2b and HuC/D, Nsox11a/b = 7, Ntyr = 7, replicates = 3; For other neuronal markers, Nsox11a/b = 8, Ntyr = 12, replicates = 3). (E) A significant increase of incoherent gut movement was found in sox11a/b knock-out embryos. (Nsox11a/b = 7, Ntyr = 8). (F) Kymographs showing the passage of coherent contraction from proximal to distal in tyr knock-out embryos, and little movement at the length of the gut in sox11a/b knock-out embryos. Arrows indicate gut contraction.