Mutual regulation underlies paralogue functional diversification

Gene duplication followed by functional divergence eliminates potential redundancy, but to what extent does either paralogue retain the ancestral function? Insect Hox3/zen genes represent an evolutionary hotspot, with orthologues required either for early specification or late morphogenesis of the protective extraembryonic tissues. The zen paralogues of the beetle Tribolium castaneum present a unique opportunity to investigate both functions in a single species. We show that despite high sequence similarity the paralogues have diverged substantially in function. High-resolution analyses of expression dynamics (transcript and protein) and transcriptional targets (RNA-seq after RNAi) demonstrate that the paralogues act non-redundantly, specifically in the serosal tissue. Together, they comprise an evolutionarily novel regulatory unit, with an unexpected early role whereby Tc-Zen2 inhibits its own activator, Tc-Zen1. We further link persistent Tc-Zen2 protein with ongoing roles in the serosa that culminate in late morphogenesis. While complementary roles and mutual regulation underpin paralogue retention, this very functional divergence also resulted in both beetle paralogues now differing from single orthologues in other species.