Broad Complex Evolution, Function and Expression: Insights From Tissue Reorganization During Metamorphosis

Abstract

Broad Complex (BRC) is an ecdysone-pathway gene essential for entry into and progression through metamorphosis in D. 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. Alternative splicing, of a protein-binding BTB-encoding exon (BTBBRC) to one of four tandemly duplicated, DNA-binding zinc-finger-encoding exons (Z1BRC, Z2BRC, Z3BRC, Z4BRC), produces four BRC isoforms. Highly conserved orthologs of BTBBRC and all four ZBRC were found in silico from Diptera, Lepidoptera, Hymenoptera and Coleoptera, indicating that BRC arose and underwent internal exon duplication before the split of holometalolous orders. Five Tramtrack subfamily members were characterized throughout Holometabola and used to root phylogenetic analyses of ZBRC exons, revealing that Z3BRC is the basal member. All four ZBRC domains, including Z4BRC which has no known essential function, are evolving in a manner consistent with selective constraint. Transgenic rescue and immunohistochemistry were used to explore how different BRC isoforms contribute to their shared tissue-morphogenesis functions at the onset of metamorphosis, when BRC is required for CNS reorganization. As predicted, the common CNS and aSG phenotypes were rescued by BRC-Z1 in rbp mutants, BRC-Z2 in br mutants, and BRC-Z3 in 2Bc mutants. However, the isoforms are required at two developmental stages, with BRC-Z2 and -Z3 required earlier than BRC-Z1. Each isoform had a unique expression pattern in the CNS, with no substantial three-way overlap among them. Z4 is strongly expressed in a novel subset of CNS neurons. The most prominent localizations of BRC-Z1, -Z2, -Z3 corresponded with glia, neuroblasts and neurons, respectively. There appears to be a switch from BRC-Z2 in proliferating cells to BRC-Z1 and BRC-Z3 in differentiating cells. The temporal-requirement and spatial-distribution data suggest that BRC-dependent CNS morphogenesis is the result of multicellular interactions among different cell types at different times. BRC-Z1-expressing glia in prepupae may mediate the final steps of CNS morphogenesis. Lastly, BRC is required for migration and programmed cell death of the ring gland, the site of ecdysone and juvenile hormone production. Therefore, BRC may function in ecdysone auto-regulation.