Anciently duplicated Broad Complex exons have distinct temporal functions during tissue morphogenesis

Broad Complex (BRC) is an essential ecdysone-pathway gene required for entry into and progression through metamorphosis in Drosophila melanogaster. Mutations of three BRC complementation groups cause numerous phenotypes, including a common suite of morphogenesis defects involving central nervous system (CNS), adult salivary glands (aSG), and male genitalia. These defects are phenocopied by the juvenile hormone mimic methoprene. Four BRC isoforms are produced by alternative splicing of a protein-binding BTB/POZ-encoding exon (BTB_BRC) to one of four tandemly duplicated, DNA-binding zinc-finger-encoding exons (Z1_BRC, Z2_BRC, Z3_BRC, Z4_BRC). Highly conserved orthologs of BTB_BRC and all four Z_BRC were found among published cDNA sequences or genome databases from Diptera, Lepidoptera, Hymenoptera, and Coleoptera, indicating that BRC arose and underwent internal exon duplication before the split of holometabolous orders. Tramtrack subfamily members, abrup, tramtrack, fruitless, longitudinals lacking (lola), and CG31666 were characterized throughout Holometabola and used to root phylogenetic analyses of Z_BRC exons, which revealed that the Z_BRC clade includes Z_abrupt. All four Z_BRC domains, including Z4_BRC, which has no known essential function, are evolving in a manner consistent with selective constraint. We used transgenic rescue to explore how different BRC isoforms contribute to shared tissue-morphogenesis functions. As predicted from earlier studies, the common CNS and aSG phenotypes were rescued by BRC-Z1 in rbp mutants, BRC-Z2 in b mutants, and BRC-Z3 in 2Bc mutants. However, the isoforms are required at two different developmental stages, with BRC-Z2 and -Z3 required earlier than BRC-Z1. The sequential action of BRC isoforms indicates subfunctionalization of duplicated Z_BRC exons even when they contribute to common developmental processes.