HORMONAL REQUIREMENTS FOR CNS REORGANIZATION--GENETIC AND IN VITRO APPROACHES

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Many cellular aspects of central nervous system (CNS) development in all complex animals, including birth, death, and remodeling, are regulated directly or indirectly by steroid hormones. This class of signaling molecules exerts many of its long-term effects by acting with DNA-binding nuclear receptors to alter transcriptional patterns of gene expression. As part of our efforts to dissect the molecular pathways underlying steroid hormone action on the developing CNS, we have embarked on a pair of complementary studies that combine classical and molecular genetic strategies with neural cell culture approaches. We are using insect metamorphosis as a model system because it provides the opportunity to investigate changes i neuronal architecture and postembryonic neurogenesis that are of critical importance to the developing adult, during a time interval with several predictable fluctuations in levels of a single predominant steroid hormone, 20-hydroxyecdysone (2)HE). Through both in vivo and in vitro experiments using Manduca, we have shown that 20HE regulates the size and shape of neuronal arbors in a stage- an cell type-specific manner. Our previous genetic experiments demonstrated that the hormone-activated regulatory gene, Broad Complex (BR-C), which encodes a family of transcription factors, is essential for CNS reorganization during Drosophila metamorphosis. We hypothesize that the defects in BR-C mutants arise because of misregulation of BRC target genes. Our preliminary experiments in cell culture document that Drosophila central neurons and neuroblasts respond to 20HE with enhanced growth and divisions, respectively. The goals of the proposed experiments are (i) to use in vitro cell and organ culture to study the effects of 20HE on neuronal growth and neurogenesis in the Drosophila CNS and (ii) to investigate the genetic basis of these steroid hormone effects. We will use a combination of regional dissection techniques, antibody markers, reporter-gene constructs, and mutants to identify the cell types that are sensitive to 20HE, and to determine which of the hormone effects are mediated by BRC transcription factors. Many of our experiments will focus on the mushroom bodies, an enigmatic region of the insect CNS implicated in learning and memory. We will use the enhancer-detector methodology in Drosophila as a means of identifying, cloning and characterizing BRC target genes in the CNS. The molecular mechanisms revealed by this project are likely to be relevant ot cellular events during normal and pathological hormone action on the vertebrate CNS.