POU FACTORS AND NEURONAL DEVELOPMENT

拟议项目的主要目标是（i）确定 果蝇POU因子Cf 1a在CNS中的体内功能 （ii）下游靶基因和上游靶基因的鉴定 Cf 1a基因的调节子和（iii）鉴定和表征 编码蛋白质的基因可能与Cf 1a直接相互作用 蛋白质作为异源二聚体或共激活剂。拟议的具体目标是 通过EMS诱变产生Cf 1a突变等位基因的集合。 收集的亚型、温度敏感性或组织特异性突变 在这样的筛选将有助于表征Cf 1a突变表型， 详细说明并推导出Cf 1a蛋白的特定细胞需求， 胚胎发育将通过免疫学方法分析突变菌株 标记各种细胞标记物。半变形或温度- 诱变筛选产生的敏感Cf 1a等位基因也将被 诱变以在编码以下的基因中产生增强子或抑制子突变 这些蛋白质可能与Cf 1a蛋白直接相互作用。蛋白质- 还将研究涉及Cf 1a蛋白的蛋白质相互作用 使用32 P-标记的Gt 11文库的直接结合筛选， Cf 1a蛋白片段作为探针分离潜在的共激活因子， 检测异二聚体形成。与其他三种果蝇合作， 在爱荷华州的实验室，将产生增强子陷阱插入文库 并筛选潜在的Cf 1a靶基因。预计候选人将 使用Cf 1a突变体等位基因检测Cf 1a依赖性表达， 来自Hsp 70-Cf 1a插入的Cf 1a蛋白的遍在表达 株Cf 1a基因的上游调控因子将通过一个 Cf 1a调控序列的详细表征， 克隆的基因组序列。胚胎细胞核DNA酶足迹法 提取物，修饰的Cf 1a-lacZ融合基因的转基因表达和 将结合进化上保守的序列元件的比较 以鉴定序列元件和相应的DNA结合因子， 是正确的时间和细胞特异性表达所必需的， Cf 1a基因。 将检测可能直接调节Cf 1a表达的已知基因 通过分析不同突变背景下Cf 1a蛋白的表达， 影响中线和气管发育， 特征性Cf 1a抗血清。POU结构域转录因子已经被 在其他物种中显示在细胞谱系的指定中是重要的 身份通过在一个高度适应的系统中检验POU因子函数， 到基因和分子操纵，如果蝇，信息 收集有关转录因子的相互作用， 发展可以外推到更高阶的系统， 容易检查。Cf 1a基因与肿瘤发生之间的潜在关系 发育中的CNS中神经胶质细胞和神经元细胞的分化应 研究各种人类神经系统的临床医生感兴趣 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.