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

中枢神经系统重组的激素需求——遗传和体外方法

• 批准号: 6243683
• 负责人: LINDA L RESTIFO
• 金额: $ 10.66万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6243683
• 关键词: Drosophilidae; central nervous system; developmental genetics; developmental neurobiology; genetic enhancer element; genetic regulation; hormone regulation /control mechanism; intercellular connection; invertebrate hormone; molecular cloning; neuroanatomy; neurogenetics; tissue /cell culture; transcription factor

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.

中枢神经系统（CNS）发育的许多细胞方面 复杂的动物，包括出生，死亡和改造，受到调节 直接或间接通过类固醇激素。 这类信号传导 分子通过与DNA结合作用发挥其许多长期作用 核受体改变基因表达的转录模式。 作为 我们剖析类固醇基础途径的一部分努力 激素对发育中心的CNS作用，我们踏上了一对 结合经典和分子遗传的互补研究 具有神经细胞培养方法的策略。 我们正在使用昆虫 变形为模型系统，因为它提供了机会 研究变化I神经元结构和胚胎后神经发生 在一段时间内，这对于发展中的成年人至关重要 间隔单个水平的几个可预测的波动 主要的类固醇激素，20-羟基甲虫（2）He）。 通过使用Manduca的体内和体外实验，我们已经显示了 20人调节一个阶段中神经元乔木的大小和形状 - 细胞类型特异性的方式。 我们以前的基因实验证明了 激素激活的调节基因，广泛的复合物（BR-C）， 编码转录因子家族，对于中枢神经系统至关重要 果蝇变态过程中的重组。 我们假设 BR-C突变体的缺陷由于BRC靶基因的不正体而出现。 我们在果蝇细胞培养文献中的初步实验 中央神经元和神经细胞对20HE的反应，增长增长和 分别分别。 拟议实验的目标是（i） 使用体外细胞和器官培养研究20HE的影响 果蝇中枢神经系统的神经元生长和神经发生和（ii） 研究这些类固醇激素作用的遗传基础。 我们将结合区域解剖技术，抗体 标记，报告基因构建体和突变体以识别细胞类型 对20HE敏感，并确定哪些激素效应 由BRC转录因子介导。 我们的许多实验将 专注于蘑菇体，这是昆虫中枢神经系统的神秘区域 与学习和记忆有关。 我们将使用增强器检测器 果蝇中的方法论是识别，克隆和 表征中枢神经系统中的BRC靶基因。 分子机制 该项目揭示的可能是相关的细胞事件 在正常和病理激素对脊椎动物中枢神经系统的作用。