Evolution of MAF and OTX families during the emergence and stabilization of rod photoreceptors across Metazoans

A central question in evolutionary developmental biology is how gene regulatory networks are rewired to generate new cell types. In mammals, rod specification and maintenance depend on the MAF-family transcription factor NRL; yet, non-mammalian vertebrates also possess NRL-independent rods, suggesting that alternative regulatory mechanisms may underlie rod evolution across lineages. Here, we investigate the evolutionary history and functional diversification of the MAF transcription factor family across metazoans. Phylogenetic and comparative genomic analyses reveal that large and small MAFs expanded through ancient duplication events, whereas the ancestral large and small MAF genes predate metazoan diversification. We uncover signatures of episodic positive selection and markedly elevated expression of NRL in mammals compared to non-mammalian orthologs. This upregulation is associated with cis-regulatory elements that include binding sites for the OTX-family homeodomain proteins, OTX2 and CRX. Furthermore, we identify evidence of co-evolution between the DNA-binding domain of NRL and its interacting partner CRX, suggesting coordinated evolution of transcription factor complexes together with their target cis -regulatory sequences. Our findings support a model in which gene duplication, regulatory innovation, and protein co-evolution collectively drive the emergence and stabilization of mammalian rod photoreceptor identity, with NRL playing a pivotal role in enabling mammalian survival through the Mesozoic era. These studies highlight context-specific deployment of conserved transcription factor networks to generate new features and functions during evolution of sensory systems in vertebrates.