The evolutionary loss of ednrb2 in specific vertebrate lineages, such as mammals and cypriniform fish, raises fundamental questions about its functional necessity and potential redundancy or synergy with paralogous endothelin receptors in pigment cell development. In teleosts possessing both ednrb1a and ednrb2 (e.g., Nile tilapia), their respective and combined roles in regulating neural crest-derived pigment cell precursors remains unresolved. Using CRISPR/Cas9, we generated single and double ednrb mutants to dissect their functions. We demonstrated that ednrb1a and ednrb2 synergistically govern the differentiation and migration of iridophore precursors. While ednrb1a is broadly essential for iridophore development, ednrb2 plays a unique and indispensable role in the colonization of iridophores in the dorsal iris. Double mutants exhibit near-complete iridophore loss; severe depletion of melanophores, xanthophores, and erythrophores; and a striking, fertile, transparent phenotype. Crucially, this iridophore deficiency does not impair systemic guanine synthesis pathways. mRNA rescue experiments confirmed mitfa as a key downstream effector within the Ednrb signaling cascade. This work resolves the synergistic regulation of pigment cell fates by Ednrb receptors and establishes a mechanism for generating transparent ermplasm.

Adenylate cyclase 5 (Adcy5) generates cyclic adenosine monophosphate (cAMP) downstream of G protein-coupled receptor signaling, yet its role in vertebrate pigmentation remains incompletely understood. Here, we generated a CRISPR/Cas9-mediated adcy5 knockout line in Nile tilapia (Oreochromis niloticus) to investigate its function in chromatophore biology. Loss of adcy5 resulted in a pronounced disruption of body coloration, characterized by the absence of vertical black bars and a global reduction in pigmentation. Despite this, chromatophore number was largely unaffected, indicating that Adcy5 is not required for pigment cell specification but is essential for functional pigmentation. At the cellular level, pigment granules in melanophores and erythrophores failed to undergo dispersion and instead remained constitutively aggregated, revealing a primary defect in intracellular pigment granule transport. Consistently, adcy5 mutants exhibited reduced expression of key melanogenesis-associated genes, including mitfa, tyrb, tyrp1a, and tyrp1b, accompanied by decreased melanin content across multiple tissues. Pharmacological activation of cAMP signaling partially rescued the pigment dispersion defect, whereas stimulation of upstream α-MSH signaling produced only limited effects, placing Adcy5 upstream of intracellular cAMP production within the pigment regulatory cascade. Importantly, we further demonstrate that Adcy5 is required for erythrophore pigment granule dispersion, extending its functional role beyond melanophore biology. Together, these findings identify Adcy5 as a conserved regulator integrating cAMP-dependent pigment synthesis and granule transport across multiple chromatophore types in teleost fish.

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.