Asymmetric Subfunctionalization of alx4a and alx4b Regulates Iridophore Differentiation and Cranial Development in Nile Tilapia

Skin transcriptomic and quantitative PCR analyses revealed marked downregulation of pnp4a and tfec, which are associated with iridophore differentiation and coloration, whereas the iridophore survival-related genes ltk and mpv17 remained largely unchanged. AlphaFold-based protein–DNA modeling further predicted a stronger potential interaction between ALX4A and the pnp4a promoter with a binding energy of −6.6 kcal mol⁻¹, consistent with a potential direct regulatory relationship.

In contrast, the development and coloration of melanophores, xanthophores, and erythrophores, as well as dorsal-fin spine formation, were not detectably affected. Together, these findings support asymmetric subfunctionalization of alx4a and alx4b, with alx4a acting as the dominant paralog in iridophore differentiation and both paralogs contributing cooperatively to cranial ossification, and identify pnp4a as a candidate downstream target of alx4a.