Tissue-specific developmental regulation and isoform usage underlie the role of doublesex in sex differentiation and mimicry in Papilio swallowtails

Adaptive phenotypes often arise by rewiring existing developmental networks. Co-option of transcription factors in novel contexts has facilitated the evolution of ecologically important adaptations such as eyespots and thoracic horns. doublesex (dsx), a transcription factor that has diverse pleiotropic effects, governs sexual differentiation during embryonic and pupal development in holometabolous insects. In Papilio polytes, dsx regulates female-limited mimetic polymorphism, resulting in mimetic and non-mimetic forms. To understand how a critical gene such as dsx regulates novel wing patterns while maintaining its basic function in sexual differentiation, we traced its expression through metamorphosis in P. polytes using developmental transcriptome data. We found three key dsx expression peaks: (i) eggs, pre- and post-ovisposition; (ii) developing wing discs and body in final larval instar, and (iii) 3-day pupae. We identified potential dsx targets using co-expression and differential expression analysis, and found distinct, non-overlapping sets of genes, containing putative dsx binding sites, in developing wings versus abdominal tissue, and in mimetic versus non-mimetic individuals. This suggests that dsx regulates distinct downstream targets in different tissues and female morphs, and has perhaps acquired new, previously unknown targets, for regulating mimetic polymorphism. Additionally, we observed that the three female isoforms of dsx were differentially expressed across stages and tissues, and differed in their protein structure. This may promote differential protein-protein interactions for each isoform and facilitate subfunctionalization of dsx activity across its isoforms. Our findings suggest that dsx employs tissue-specific downstream effectors and partitions its functions across multiple isoforms to regulate sexually dimorphic traits through insect development.