ROLE OF POU-FACTORS IN NEURONAL DEVELOPMENT

拟议项目的主要目标是（i）确定 POU因子如果蝇因子Cf 1a在发育中的作用，（ii） Cf 1a基因是如何调控的及其与 其他模式形成基因和（iii）确认推定的 Cf 1a与多巴胺神经元特异性表达的关系 脱羧酶（Ddc）基因。 拟议的具体目标是， 通过对CF 1a基因组定位和测序确定CF 1a转录单位 克隆和构建表达P-元件的菌株 Cf 1a/β-半乳糖苷酶融合基因。 Cf 1a/β-半乳糖苷酶的分析 在Cf 1a调控序列内具有突变的融合基因应该 鉴定野生型表达所必需序列。 野生型 Cf 1a基因在发育过程中的表达模式将是 其特征在于开发了Cf 1a抗血清， 免疫组织化学标记，Cf 1a/-半乳糖苷酶融合蛋白的表达 基因在P-元件突变株和继续应用， 使用地高辛标记的Cf 1a探针进行原位杂交（见初步 结果）。 将分析Cf 1a基因内突变的表型 通过使用杂交产生Cf 1a P元件插入突变体， 发育不全以及重叠的彻底遗传特征 Cf 1a基因的缺陷。 改变基因的表型效应 通过广泛表达Cf 1a，限制Cf 1a的表达， 热激P元件中hsp 70启动子控制下的蛋白质 限制性表达菌株将决定是否限制性表达 Cf 1a的正确功能需要在空间和时间上的变化。 一 Cf 1a直接调节Ddc表达的结论性证明， 特定的神经元将是特别重要的，因为它会喜欢一个 谱系决定子调控下游基因参与的 神经元细胞类型的表型表达。 无处不在的表达 Cf 1a蛋白的表达也可能对Ddc产生直接影响。 异常CF 1a表达的基因表达导致Ddc表达， 不合适的神经元 如果这种效应依赖于 与Cf 1结合位点，那么它将不会被视为在 仅含有Ddc基因的菌株，其基因组中具有成簇的点突变， Cf 1结合位点，使其不能结合Cf 1a蛋白。 一 Cf 1a如何调节的研究结果与 突变体中Cf 1a基因过表达和低表达的表型效应 菌株将提示POU因子可能的发育功能， Cf1a。 人类和动物之间结构的显著保守性 果蝇POU结构域表明， 果蝇可以很容易地外推到更临床相关的 应用. Cf 1a基因在肿瘤发生中的作用 多巴胺能神经元应该引起临床医生的兴趣， 各种人类疾病，如帕金森氏症， 特定神经元的过早变性的发展。

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.