Fig. 4. The expression of crucial genes associated with melanophores, xanthophores, and granular glands was reduced or lost in mitf^−/−Xenopus tropicalis.

a Whole-mount in situ hybridization analysis of stage 33/34 Xenopus tropicalis embryos demonstrated a reduction or absence of mRNA hybridization signals in mitf^−/− embryos compared to the WT. The black arrows indicated some weaker mRNA hybridization signals. At least five embryos were used for in situ hybridization of each gene in every genotype embryo. b–e Representative results of RT-PCR analysis for several melanophores-related genes, including mitf, tyr, pmel, mlana, ednrb1, slc45a2, and dct, as well as xanthophores-related genes, trpm1, pax3, and gid2, were presented in b. The expression of aquaporin-related genes, such as aqp5 and aqp8, as well as Ca²⁺-activated chloride channel anoctamin 1 (ano1), and antimicrobial peptides-related genes, including nmb and pgq, in the granular glands were displayed in c and d, respectively. Several hormone-related genes of granular glands, including trh, xt6l, and tph1, were also evaluated (e). The representative results of this analysis were presented in panels c–e. odc was used as the RNA loading control and c–e shared the RNA loading control in c. For RT-PCR analysis, each gene was subjected to three repetitions using three replicates of total RNA samples, while each total RNA sample was extracted from the skin of three individual frogs. The original data for the mentioned RT-PCR can be found in Supplementary Fig. 32 and Supplementary Fig. 33. Additionally, the original data for the RT-PCR in Supplementary Fig. 25 can be found in Supplementary Fig. 34. DS, dorsal skin; VS, ventral skin. The black scale bar in a is 0.5 mm.