Dual-receptor switch bifurcates Kitlga for Nile tilapia pigmentation

The Kit signaling pathway governs vertebrate pigmentation. However, the mechanisms governing cell fate coordination by duplicated ligand-receptor pairs in teleosts remain elusive. In the present study, we elucidated this network in Nile tilapia ( Oreochromis niloticus ) by generating single and double mutants of kitlga , kitlgb , kita , and kitb via CRISPR/Cas9-mediated gene editing. Kitlga was identified as the dominant ligand. While kitlgb −/− and kitb −/− single mutants showed no detectable pigment defects, and kita −/− mutants exhibited only partial melanophore reduction, kitlga deficiency caused a severe loss of melanophores. kita −/− ; kitb −/− double mutants recapitulated this severe kitlga phenotype, confirming that Kitlga signals through both receptors. Beyond melanophore loss, kitlga −/− mutants developed a “red tilapia”-like appearance with metallic luster. This phenotype arose from chromatophore population remodeling; the suppression of melanophores triggered the compensatory proliferation of erythrophores (quantified by elevated erythrophore numbers and carotenoid levels) and structural reorganization of iridophores. Transmission electron microscopy demonstrated that these mutants developed three layers of guanine crystal platelets, in contrast to the single layer found in wild-type fish. Mechanistically, double-color fluorescence in situ hybridization analysis revealed that, while both receptor genes were co-expressed in melanophores, transcripts in kita were significantly more abundant than those in kitb . Consistent with this, our data suggest that Kitlga acts as a molecular dual switch; it preferentially engages abundant Kita to drive differentiation via mitogen-activated protein kinase (MAPK) while recruiting Kitb to support survival via phosphatidylinositol 3-kinase-protein kinase B (PI3K-Akt). Both pathways converged on mitfa , which was validated by the rescue of defects via mitfa mRNA injection. Collectively, these findings identify Kitlga as an important regulator that orchestrates pigment cell plasticity and structural coloration, thus providing a genetic basis for cichlid color diversity. Graphical Abstract: A Dual-Receptor Mechanism for Pigmentation Diversity . • Deciphered a dual-receptor switch decoupling melanophore differentiation (via Kita-MAPK) from survival (via Kitb-PI3K-Akt). • Established the Kitlga-Kita/b axis as a key regulator of Nile tilapia pigmentation via systematic paralog knockouts. • Revealed kitlga functions beyond melanophores: erythrophore expansion and iridophore remodeling for metallic luster. • Produced two ornamental tilapia strains by mutation of kitlga and kita.