Functional analysis of the ecdysone receptor (ecr) gene in drosophila melanogaster
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In Drosophila, the steroid hormone ecdysone coordinates molting, metamorphosis, and aspects of embryonic development and adult reproduction. The ecdysone receptor is a heterodimer of two nuclear receptors, USP and EcR. The EcR gene produces 3 protein isoforms (EcR-A, EcR-B1, and EcR-B2) through alternative splicing and the alternative usage of promoters EcR-A and EcR-B. Mutations that inactivate all EcR isoforms are embryonic lethal, hindering analysis of EcR function during later development. Using transgenes in which a heatshock promoter drives expression of an EcR cDNA, EcR null mutants were rescued to later stages to analyze EcR function. The results show that EcR is required for hatching, larval molting, and the initiation of metamorphosis. In EcR mutants arrested prior to metamorphosis, expression of ecdysone responsive genes is blocked, imaginal tissues fail to develop, and larval tissues fail to degenerate. These results implicate EcR in the reorganization of imaginal and larval tissues at the onset of metamorphosis. To analyze the genetic regulatory components downstream of EcR, transcriptional profiling of more than 7,500 genes was undertaken using gene microarrays. New genes were identified whose expression was altered in EcR null mutants arrested prior to metamorphosis. 82 genes were identified that were up-regulated by more than 3-fold in the wild-type but clearly failed to be up-regulated in the EcR mutants, while 109 genes were identified that were down-regulated by more than 3-fold in the wild-type but clearly failed to be down-regulated in the EcR mutants. To analyze the regulation of the EcR gene, a transgene was constructed in which the EcR-B1 cDNA is driven by an EcR-B upstream DNA fragment of 7,896 bp. This transgene was found to be sufficient to rescue EcR-B1 mutants and a lacZ reporter gene linked to the same upstream fragment was found to be sufficient to recapitulate the wild-type expression pattern of the EcR-B1 gene at the onset of metamorphosis. In EcR null mutants, expression of the reporter gene was absent from gastric caeca and the posterior portion of the midgut, suggesting EcR-B auto-induction in those tissues. Sequencing and computer analysis identified 3 potential ecdysone receptor binding sites within the fragment.