POU-FACTORS AND NEURONAL DEVELOPMENT

拟议项目的主要目标是：(1)确定 果蝇Pou因子Cf1a在中枢神经系统中的体内作用 开发，(Ii)确定下游靶基因和上游 Cf1a基因的调控及(III)鉴定与鉴定 编码可能与Cf1a直接相互作用的蛋白质的基因 作为异二聚体或共激活剂的蛋白质。拟议的具体目标是 通过EMS诱变产生一组Cf1a突变等位基因。 收集的亚型、温度敏感型或组织特异性突变 这将有助于确定Cf1a突变的表型。 详细说明并推断细胞对Cf1a蛋白的特定需求 胚胎发育。突变菌株将通过免疫学分析 标记各种细胞标志物。亚象或温度- 由诱变筛选产生的敏感Cf1a等位基因也将 突变以产生编码基因的增强子或抑制子突变 可能与Cf1a蛋白直接相互作用的蛋白质。蛋白质- 还将研究涉及Cf1a蛋白的蛋白质相互作用 使用使用32P标记的lambda-gt11文库的直接结合屏幕 以CF1a蛋白片段为探针分离潜在的辅活化子或 检测杂二聚体的形成。与其他三只果蝇合作 在爱荷华州的实验室，将产生一个增强子-陷阱插入文库 并筛选潜在的Cf1a靶基因。可能的候选人将是 使用Cf1a突变等位基因和 Hsp70-Cf1a插入的Cf1a蛋白的表达 紧张。Cf1a基因的上游调控因子将通过一种 使用先前的方法对Cf1a调节序列进行详细表征 克隆的基因组序列。利用胚胎细胞核进行DNA酶足迹研究 修饰的Cf1a-lacZ融合基因的提取、转基因表达和 将结合进化保守的序列元件的比较 鉴定序列元件和相应的DNA结合因子 对于正确的时间和细胞特定的表达是必不可少的 CF1a基因。 可能直接调节Cf1a表达的已知基因将被检测 通过分析Cf1a蛋白在不同突变背景下的表达 影响中线和气管发育 鉴定Cf1a抗血清。POU结构域转录因子已经被 在其他物种中显示出在细胞谱系的指定中很重要 身份。通过考察高度服从的系统中的Pou因子函数 对遗传和分子的操纵，如果蝇，信息 收集有关转录因子相互作用的信息 发展可以外推到更高阶的系统，而不能像 很容易检查。Cf1a基因与骨肉瘤的潜在关系 中枢神经系统发育过程中神经胶质细胞和神经细胞的分化 对研究各种人类神经学的临床医生很有兴趣 涉及神经元变性或髓鞘形成的综合征 少突胶质细胞。

The primary objectives of the proposed project are (i) determination of the in vivo function of the Drosophila POU factor, Cf1a, during CNS development, (ii) identification of downstream target genes and upstream regulators of the Cf1a gene and (iii) identification and characterization of genes encoding proteins which may interact directly with the Cf1a protein as heterodimers or coactivators. The proposed specific aims are to generate a collection of Cf1a mutant alleles by ems mutagenesis. Hypomorphic, temperature-sensitive or tissue-specific mutations collected in such screens will help to characterize the Cf1a mutant phenotype in detail and deduce specific cellular requirements for Cf1a protein during embryonic development. Mutant strains will be analyzed by immunological labeling for various cellular markers. Hypomorphic or temperature- sensitive Cf1a alleles resulting from mutagenic screens will also be mutagenized to generate enhancer or suppressor mutations in genes encoding proteins which may interact directly with the Cf1a protein. Protein- protein interactions involving the Cf1a protein will also be investigated using a direct binding screen of a lambda-gt11 library using 32P-labeled Cf1a protein fragments as probe to isolate potential coactivators or detect heterodimer formation. In collaboration with three other Drosophila laboratories at Iowa, an enhancer-trap insertion library will be generated and screened for potential Cf1a target genes. Putative candidates will be tested for Cf1a-dependent expression using Cf1a mutant alleles and the ubiquitous expression of the Cf1a protein from an Hsp70-Cf1a insertion strain. Upstream regulators of the Cf1a gene will be identified by a detailed characterization of Cf1a regulatory sequences using previously cloned genomic sequences. DNase footprinting using embryonic nuclear extracts, transgenic expression of modified Cf1a-lacZ fusion genes and a comparison of evolutionarily conserved sequence elements will be combined to identify sequence elements and corresponding DNA-binding factors which are essential for the correct temporal and cell-specific expression of the Cf1a gene. Known genes which may directly regulate Cf1a expression will be examined by analyzing Cf1a protein expression in various mutant backgrounds affecting midline and tracheal development using a previously characterized Cf1a antiserum. POU-domain transcription factors have been shown in other species to be important in the designation of cell lineage identities. By examining POU-factor function in a system highly amenable to genetic and molecular manipulation, such as Drosophila, information gathered concerning the interactions of transcription factors during development can be extrapolated to higher order systems which cannot be as easily examined. A potential relationship between the Cf1a gene and the differentiation of glial and neuronal cells in the developing CNS should be of interest for clinicians investigating various human neurological syndromes which involve degeneration of neurons or myelinating oligodendrocytes.