The early gene Broad is involved in the ecdysteroid hierarchy governing vitellogenesis of the mosquito Aedes aegypti

Abstract

The broad (br ) gene, encoding a family of C2H2 type zinc-finger DNA-binding proteins, has been shown to act as a crucial member of the 20-hydroxyecdysone (20E) regulatory hierarchy in the fruitfly, Drosophila melanogaster and the moth, Manduca sexta. In this study, we have shown that the br gene is involved in the 20E-regulatory hierarchy controlling vitellogenesis in the mosquito, Aedes aegypti. Unlike E74 and E75 early genes, expression of br was activated in previtellogenic females, during a juvenile hormone (JH)-dependent period. The levels of Z1, Z2 and Z4 isoform mRNA were elevated in the fat body of 2-day-old females after in vitro exposure to JH III. However, JH III repressed 20E activation of br in 3-to 5-day-old females, indicating a switch in hormonal commitment. Expression of Z1, Z2 and Z4 was stimulated after blood feeding in both vitellogenic tissues, the fat body and the ovary, corresponding to peaks of ecdysteroid titers. In the fat body, the mRNA profiles of these three isoforms correlated well with those of yolk protein precursor (YPP) genes. These BR isoforms were activated by 20E in fat bodies cultured in vitro and behaved as early genes, with a self-repressive autoregulatory loop that can be blocked by the protein inhibitor, cyclohexamide. Multiple binding sites for all four BR isoforms were present in the 5′-regulatory region of the major YPP gene, vitellogenin (Vg). Effects of BR isoforms on the expression of Vg have been demonstrated by cell transfection analysis. In particular, BR isoforms by themselves had no effects on the Vg promoter. However, Z1 and Z4 each repressed Aedes aegypti ecdysone receptor (EcR)/Ultraspiracle (USP)-mediated 20E activation of the Vg promoter, while Z2 enhanced activation of the Vg promoter by AaEcR/AaUSP in the presence of 20E. Z3 had no obvious effect in the same experiment. These results suggested that BR isoforms are essential for proper activation and termination of the Vg gene in response to 20E. Overall, our study implicated br in the regulation of mosquito vitellogenesis.