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， 直接或间接通过类固醇激素。 这类信号 分子通过与DNA结合而发挥其许多长期作用。 核受体来改变基因表达的转录模式。 作为 我们研究类固醇的分子途径 激素对发育中的中枢神经系统的作用，我们已经开始对 联合收割机结合经典和分子遗传学的互补研究 与神经细胞培养方法的策略。 我们用昆虫 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后神经发生的变化 对发育中的成年人来说至关重要， 在一个单一的水平上有几个可预测的波动的间隔 优势甾体激素20-羟基蜕皮激素（2）HE）。 通过使用Manduca的体内和体外实验，我们已经表明 20 HE在一个阶段调节神经元乔木的大小和形状， 细胞类型的特定方式。 我们之前的基因实验证明 这种激活的调节基因，广泛复合物（BR-C）， 编码一个转录因子家族，是中枢神经系统必需的， 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靶基因的错误调节而产生的。 我们在细胞培养中的初步实验证明， 中枢神经元和神经母细胞对20 HE的反应是生长增强， 分，分别。 拟议实验的目标是（i） 采用体外细胞和器官培养方法，研究20 HE对 果蝇CNS中的神经元生长和神经发生，以及（ii） 研究这些类固醇激素效应的遗传基础。 我们将结合局部解剖技术，抗体 标记物、突变基因构建体和突变体，以鉴定细胞类型 对20 HE敏感的细胞，并确定哪些激素影响 由BRC转录因子介导。 我们的许多实验 重点放在蘑菇体上，这是昆虫中枢神经系统的一个谜 与学习和记忆有关 我们将使用增强探测器 方法学在果蝇中作为鉴定、克隆和 表征CNS中的BRC靶基因。 的分子机制 可能与细胞事件有关 在正常和病理性激素作用于脊椎动物CNS期间。

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.