Asymmetric Functional Divergence of alx4a and alx4b in Iridophore Differentiation and Cranial Development in Nile Tilapia.
Neural crest cells give rise to the craniofacial skeleton and multiple pigment cell lineages, yet how duplicated developmental regulators partition their ancestral functions after teleost-specific whole-genome duplication remains unclear. Here, we employed CRISPR/Cas9 to generate alx4a and alx4b single and double mutants in Nile tilapia (Oreochromis niloticus). By integrating phenotype, skeleton, transcriptome, quantitative PCR, and AlphaFold-based structural modeling analyses, we revealed their functional divergence. Loss of alx4a caused a regionally restricted reduction in iridophore-derived reflectance and abnormal cranial morphology, whereas alx4b single mutants showed no obvious phenotype under the conditions examined. By contrast, double mutants exhibited an almost complete loss of iridophore-derived structural coloration and substantially more severe cranial defects, accompanied by reduced calcein labeling in the opercular region, consistent with altered cranial mineralization. Skin transcriptomic and quantitative PCR analyses revealed marked downregulation of pnp4a and tfec, which are associated with iridophore differentiation and coloration, whereas no significant expression differences were detected for the iridophore survival-related genes ltk and mpv17. AlphaFold2-assisted HDOCK protein-DNA modeling yielded more favorable docking metrics for Alx4a than for Alx4b with the pnp4a promoter, supporting a potential Alx4a-pnp4a promoter interaction that requires experimental validation. In contrast, no significant genotype-dependent differences were detected in the measured abundance of melanophores, xanthophores, or erythrophores, and no obvious difference in gross dorsal-fin spine formation was observed under the conditions examined. Together, these findings reveal unequal functional contributions of alx4a and alx4b, with alx4a acting as the dominant paralog in iridophore-associated structural coloration and both paralogs contributing unequally to cranial development, and support pnp4a as a candidate downstream gene associated with Alx4a activity.