Abstract
The microphthalmia-associated transcription factor (Mitf) family is central to melanocyte biology. Teleosts possess two mitf paralogs, mitfa and mitfb, yet their functional divergence remains incompletely understood. Here, we characterized the coding sequences, phylogeny, tissue distribution, developmental expression, cellular localization, and stress responsiveness of mitfa and mitfb in Congjiang natural mutant crucian carp (NMCC). Both genes encoded proteins containing the characteristic bHLH-ZIP domain. Phylogenetic analysis revealed that teleost mitfb clustered with the tetrapod mitf clade, indicating closer evolutionary affinity than mitfa. mitfa was predominantly expressed in skin and eyes, whereas mitfb showed broad tissue distribution. Both paralogs were constitutively expressed in skin across developmental stages; mitfa levels remained consistently lower than mitfb. Both transcripts were localized to melanocyte-containing regions of the skin. Transcriptomic analysis following high temperature (34 °C), UV radiation, and combined stress (48 h) revealed that mitfa was significantly upregulated by thermal stress, while mitfb showed no significant changes. Profiling of DNA damage response pathways uncovered extensive, stress-specific transcriptional remodeling. High temperature broadly suppressed apoptotic genes while upregulating tsc2, gadd45b, and cdip1; UV triggered widespread apoptotic gene activation. Combined stress elicited a hybrid signature. Notably, robust DNA Damage Response pathway remodeling contrasted with muted mitf responses, suggesting that DNA Damage Response-to-pigmentation signaling may operate predominantly at post-transcriptional levels. These findings reveal a substantial regulatory divergence between mitfa and mitfb, with mitfa maintaining a conserved melanogenic role while mitfb has been co-opted for broader functions in non-pigmentary tissues. The differential stress responses of the two paralogs further provide new insights into tissue-specific genotoxic stress signaling and melanogenic regulation in teleosts.