E93 controls adult differentiation by repressing in .

In , successful development relies on the precise coordination of both spatial and temporal regulatory axes. The temporal axis governs stage-specific identity and developmental transitions through a number of genes, collectively forming the . Among these, Ecdysone inducible protein 93F (E93) serves as the critical determinant for adult specification, but its mechanism of action remains unclear. Here, we found that, rather than acting mainly as an instructive signal, E93 promotes adult differentiation through the repression of the pupal specifier (). In the absence of E93, sustained high levels of Br during the pupal stage strongly represses pupal-specific enhancers that are essential for the terminal differentiation of the wing. Notably, RNA-seq analysis confirmed that the majority of E93-dependent transcriptomic changes in pupal wings are primarily driven by repression. In addition, we also show that Br represses the pupal-enhancers during the larval and prepupal stages preventing the premature implementation of the adult genetic program, and that it also dampens the activity of larval enhancers during the latter stages of larval development. This mechanism of action seems to be a derived feature acquired in Diptera, as in the coleopteran , repression of by E93 is not sufficient to allow adult differentiation. In summary, our study elucidates the crucial role of the intricate interplay between E93 and Br as the governing mechanism in the process of terminal differentiation in . This finding holds significant implications for advancing our understanding of the evolution of insect metamorphosis.