STERIOD HORMONE-REGULATED GENES IN THE CNS

拟议的实验是一项长期计划的一部分， 在发育过程中神经系统重塑的遗传控制。 因为 分子和经典遗传学方法的结合力量， 发育问题，果蝇中枢神经系统变态已被选为 实验模型系统 类固醇激素影响中枢神经系统的许多方面 在脊椎动物和无脊椎动物的发展。 确定机制 介导激素对CNS形态发生以及对神经元 出生、分化和死亡对于理解 人类神经系统发育异常。 与类固醇激素对基因表达的已知作用一致， 由类固醇蜕皮激素20- 羟蜕皮激素（20 HE）在中枢神经系统局部表现出特异性缺陷 形态发生和神经组织。 这个基因，宽复合体（BR- C），在许多组织中表达，包括CNS，并调节 许多其他场所的活动。 建议的实验是基于 假设突变体中CNS缺陷是由于 BR-C在CNS中的靶基因。 因此，该项目的主要目标是 分离和鉴定在小鼠中表达的20个HE诱导基因， CNS，特别强调那些由BR-C监管的。 最初，BR-C在CNS中的转录将通过原位杂交来研究。 杂交和共聚焦显微镜。 结构和激素- 通过标准RNA印迹分析这些转录物的诱导 和映射方法。 从大肠杆菌中分离大肠杆菌诱导基因的方案 CNS RNA结合了历史悠久的差异杂交方法 使用允许体外合成cRNA的更新的cDNA克隆载体。 这种扩增的cRNA为文库提供了高灵敏度的试剂 构建和分析。 一个消减cDNA文库，富集 优先在暴露于病毒的CNS组织中表达的序列， 构建和筛选。 各种标准，包括监管不当 在BR-C突变体中，将用于确定哪些20 HE诱导型克隆 代表在CNS变态过程中可能具有重要作用的基因。 这些基因的表达模式和DNA序列将被确定 并通过突变体的产生和分析评估其功能。

The proposed experiments are part of a long-term plan to investigate the genetic control of nervous system remodeling during development. Because of the combined power of molecular and classical genetic approaches to developmental problems, Drosophila CNS metamorphosis has been chosen as an experimental model system. Steroid hormones influence many aspects of CNS development in vertebrates and invertebrates. Determining the mechanisms mediating hormonal effects on CNS morphogenesis, as well as on neuronal birth, differentiation, and death, are critical to understanding abnormalities of human nervous system development. In keeping with the known action of steroid hormones on gene expression, mutants of a gene regulated by the steroid molting hormone 20- hydroxyecdysone (20HE) exhibit specific defects in regional CNS morphogenesis and neural organization. this gene, the Broad Complex (BR- C), is expressed in many tissues, including the CNS, and regulates the activities of many other loci. The proposed experiments are based on the hypothesis that CNS defects in the mutants result from the misregulation of BR-C target genes in the CNS. Thus, the major goal of this project is the isolation and characterization of 20HE-inducible genes expressed in the CNS, with particular emphasis on those regulated by the BR-C. Initially, BR-C transcription in the CNS will be studied by in situ hybridization and confocal microscopy. The structure and hormone- inducibility of these transcripts will be analyzed by standard RNA blotting and mapping methods. The scheme for isolating hormone-inducible genes from CNS RNA combines the time-honored approach of differential hybridization with newer cDNA cloning vectors which allow in vitro synthesis of cRNA. This amplified cRNA provides high-sensitivity reagents for library construction and analysis. A subtracted cDNA library, enriched for sequences preferentially expressed in hormone-exposed CNS tissue, will be constructed and screened. A variety of criteria, including misregulation in BR-C mutants, will be used to determine which 20HE-inducible clones represent genes with probable important roles during CNS metamorphosis. The expression patterns and DNA sequence of these genes will be determined and their function assessed by the generation and analysis of mutants.