ROLE OF POU-FACTORS IN NEURONAL DEVELOPMENT

拟议项目的主要目标是：(1)确定 果蝇因子Cf1a等POU因子在发育中的作用(II) Cf1a基因是如何被调控的及其与 其他模式形成基因和(Iii)推定基因的确认 Cf1a与多巴胺神经元特异性表达的关系 脱羧酶(DDC)基因。提议的具体目标是将 通过对CF1a基因组进行定位和测序获得Cf1a转录单位 P-元件转化株的克隆与构建 Cf1a/β-半乳糖苷酶融合基因。Cf1a/β-半乳糖苷酶的分析 Cf1a调控序列内有突变的融合基因应该 鉴定野生型表达所需的序列。野性的人 Cf1a基因在发育过程中的表达模式将是 其特点是研制了一种Cf1a-抗血清 免疫组织化学标记Cf1a/-半乳糖苷酶融合蛋白的表达 P-元件转化株中的基因和In的继续应用 使用地高辛标记的Cf1a探针进行原位杂交(见初步 结果)。我们将分析Cf1a基因突变的表型。 利用杂交种产生Cf1a P元件插入突变体 发育不全以及重叠的基因特征 移动Cf1a基因的缺陷。改变的表型效应 通过普遍表达Cf1a来限制Cf1a的表达 热休克P元件中HSP70启动子调控的蛋白质 转化子菌株将决定限制表达是否既 在空间和时间上是Cf1a正确功能所必需的。一个 确凿证据表明Cf1a直接调节DDC的表达 特定的神经元将特别重要，因为它希望 与调控下游基因有关的谱系决定因素 一种神经元细胞类型的表型表达。无处不在的表达 Cf1a蛋白的表达也可能对DDC产生直接影响 基因表达异常的CF1a导致DDC的表达 不适当的神经元。如果这种效果依赖于交互作用 有了CF1结合位点，转化子中就看不到它了 只包含一个DDC基因的菌株，在 CF1结合部位，使其不能结合Cf1a蛋白。一个 关于Cf1a如何被调控的研究结果与 突变体中Cf1a基因高表达和低表达的表型效应 菌株将暗示POU因子的可能发育功能，如 作为Cf1a。人类与人类之间显著的结构守恒 果蝇Pou-Domes表明，来自于 果蝇可以很容易地外推到更具临床意义的 申请。Cf1a基因在骨肉瘤发生发展中的可能作用 临床医生应该对多巴胺能神经元的研究感兴趣 各种人类病理，如帕金森氏症，涉及憎恶 特定神经元的过早退化的发展。

The primary objectives of the proposed project are (i) determination of the role of POU factors such as the Drosophila factor Cf1a in development, (ii) characterization of how the Cf1a gene is regulated and its relationship to other pattern formation genes and (iii) confirmation of the putative relationship between Cf1a and the neuron-specific expression of the dopa decarboxylase (Ddc)gene. The proposed specific aims are to characterize the Cf1a transcription unit by mapping and sequencing of CF1a genomic clones and construction of P-element transformant strains expressing Cf1a/beta-galactosidase fusion genes. Analysis of Cf1a/beta-galactosidase fusion genes with mutations within Cf1a regulatory sequences should identify sequences necessary for wild-type expression. The wild-type expression pattern of the Cf1a gene during development will be characterized both by the development of a Cf1a-antiserum for immunohistological labeling, the expression of Cf1a/-galactosidase fusion genes in P-element transformant strains and by continued application of in situ hybridization using digoxigenin-labeled Cf1a probes (see Preliminary Results). The phenotype of mutations within the Cf1a gene will be analyzed by the generation of Cf1a P-element insertion mutants using hybrid dysgenesis as well as a thorough genetic characterization of overlapping deficiencies which move the Cf1a gene. The phenotypic effects of altering the restricted expression of Cf1a by ubiquitously expressing the Cf1a protein under the control of the hsp70 promoter in heat-shocked P-element transformant strains will determine whether restricted expression both spatially and temporally is required for the correct function of Cf1a. A conclusive demonstration that Cf1a directly regulates Ddc expression in specific neurons would be especially significant since it would like a lineage determinant to regulation of a downstream gene involved in the phenotypic expression of a neuronal cell-type. The ubiquitous expression of Cf1a protein could also potentially demonstrate a direct effect upon Ddc gene expression of abherrant CF1a expression causes Ddc expression in inappropriate neurons. If this effect is dependent upon an interaction with the Cf1 binding site, then it would not be seen in transformant strains containing only a Ddc gene with a clustered point mutation in the Cf1 binding site, rendering it incapable of binding the Cf1a protein. A correlation of results from studies on how Cf1a is regulated with the phenotypic effects of over- and under-expression of the Cf1a gene in mutant strains will suggest possible developmental functions for POU factors such as Cf1a. The remarkable conservation of structure between human and Drosophila POU-domains suggests that results from molecular studies in Drosophila can be readily extrapolated to more clinically relevant applications. The suspected role of the Cf1a gene in the development of dopaminergic neurons should be of interest for clinicians investigating various human pathologies such as Parkinsonism which involve the abherrant development of premature degeneration of specific neurons.