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

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、死亡和重塑，都受到调控 直接或间接地由类固醇激素引起。这类信号 分子通过与DNA结合而发挥其许多长期作用 核受体改变基因表达的转录模式。AS 我们努力剖析类固醇分子途径的一部分 荷尔蒙对发育中的中枢神经系统的作用，我们已经着手对 结合经典遗传学和分子遗传学的互补性研究 采用神经细胞培养方法的策略。我们用的是昆虫 变形作为一个模型系统，因为它提供了机会 研究神经元结构和胚胎后神经发生的变化 在一段时间内，对发育中的成年人至关重要的 具有几个可预测的波动的时间间隔 主要类固醇激素，20-羟基蜕皮酮(2)。 通过使用Manduca的体内和体外实验，我们已经证明 20HE在一个阶段调节神经元乔木的大小和形状-一个 特定于单元类型的方式。我们之前的基因实验证明 激素激活的调节基因广泛复合体(BR-C)，它 编码一系列转录因子，对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突变体中的缺陷是由于BRC靶基因的错误调控而产生的。 我们在细胞培养中的初步实验证明，果蝇 中枢神经元和神经母细胞对20HE有促进生长和 分别为分部。拟议实验的目标是：(I) 用体外细胞和器官培养方法研究20He对小鼠肾小管上皮细胞生长的影响 果蝇中枢神经系统内神经元的生长和神经发生 研究这些类固醇激素效应的遗传基础。 我们将结合使用局部解剖技术，抗体 用于识别细胞类型的标记、报告基因结构和突变体 对20HE敏感的人，并确定哪种激素影响 是由BRC转录因子介导的。我们的许多实验将 关注蘑菇体，这是昆虫中枢神经系统的神秘区域 与学习和记忆有关。我们将使用增强剂探测器 果蝇的方法学作为一种识别、克隆和 中枢神经系统中BRC靶基因的特征。分子机制 这个项目揭示的可能与细胞事件相关 在脊椎动物中枢神经系统的正常和病理激素作用过程中。

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.