The role of the Fox gene fd64a in Drosophila embryonic development

描述(申请人提供)：细胞迁移发生在所有多细胞生物体中。由于细胞迁移可能对有机体有益，如在发育过程中，也可能有害于癌症，因此在细胞和分子水平上了解这一过程的细微差别是很重要的。这项提议的目的是确定转录因子Fd64a在果蝇胚胎发育中的作用。Fd64a是Fox转录因子家族的成员，具有多种不同的作用，包括调节参与迁移的基因的表达。Fd64a在直接接触迁移唾液腺的果蝇肌肉的子集上表达，这是在器官背景下研究细胞迁移的理想和独特的模型系统。在缺乏fd64a的果蝇中，唾液腺表现出一系列的迁移缺陷。这些初步发现导致假设Fd64a调节引导唾液腺迁移的分子的表达。为了进一步研究fd64a在细胞迁移中的作用，具体目标1的A部分将通过以下方式创建fd64a的敲除 同源重组，然后分析唾液腺迁移和肌肉缺陷的这个零等位基因。具体目标1的B部分将测试fd64a的过度表达是否会影响唾液腺的迁移。此外，这一目标将决定是否可以通过以组织特异性的方式提供野生型fd64a功能来挽救敲除缺陷。因为fd64a在果蝇胚胎的多个不同组织中都有表达，所以特定目标2的目标是首先确定表达fd64a的组织，然后检查这些组织在fd64a缺失等位基因中的发育和形态。具体目标3将调查Fd64a的下游靶点。A部分将使用候选基因方法。Semaphorin 2A是一种已知影响多种其他细胞类型迁移的分子，它的表达与fd64a的表达重叠，表明可能存在调节相互作用。我将使用原位杂交、蛋白质表达和表型分析来确定信号素2A是否是Fd64a的一个有效靶点。B部分将通过比较野生型和fd64a缺失型胚胎中基因表达的变化，通过微阵列分析来确定Fd64a的其他靶点。潜在的靶标将通过原位分析、染色质IP和突变-等位基因表型分析来验证。对果蝇胚胎中fd64a的分子和遗传分析将有助于深入了解周围组织提供的线索如何影响特定器官的迁移。这是对拓宽细胞迁移领域的一个重要贡献。 与公共卫生相关：细胞迁移是一个普遍而必要的过程，可用于癌症等疾病，肿瘤利用细胞迁移来转移和侵袭其他组织。Fd64a是一种在果蝇肌肉中表达的转录因子，它会影响附近组织的迁移，并改变其最终位置。了解改变组织迁移的因素具有重要的医学意义，例如了解肿瘤如何以及为什么能够在全身移动。

DESCRIPTION (provided by applicant): Cell migration occurs in all multicellular organisms. Because cell migration can be beneficial to an organism, as in development, or detrimental, as in cancer, it is important to understand the nuances of this process both at the cellular and molecular level. The goal of this proposal is to determine the role of the transcription factor, Fd64a, in Drosophila embryonic development. Fd64a is a member of the Fox family of transcription factors, which have multiple different roles, including modulating the expression of genes involved in migration. fd64a is expressed in a subset of Drosophila muscles that directly contact the migrating salivary gland, an ideal and unique model system for studying cell migration in the context of an organ. In flies deficient for fd64a, the salivary gland exhibits a range of migration defects. These preliminary findings led to the hypothesis that Fd64a regulates expression of molecules that direct salivary gland migration. To further investigate the role of fd64a in cell migration, Part A of Specific Aim 1 will be to create a knock-out of fd64a by homologous recombination, and then analyzing this null allele for salivary gland migration and musculature defects. Part B of Specific Aim 1 will test if over-expression of fd64a can affect salivary gland migration. Additionally, this aim will determine if the knock-out defects can be rescued by providing wild-type fd64a function in a tissue-specific manner. Because fd64a is expressed in multiple different tissues of the Drosophila embryo, the goals of Specific Aim 2 will be to first positively identify the tissues that express fd64a and then to examine the development and morphology of these tissues in the fd64a null allele. Specific Aim 3 will investigate the downstream targets of Fd64a. Part A will use a candidate gene approach. Expression of Semaphorin 2A, a molecule known to affect migration of multiple other cell types, overlaps that of fd64a, suggesting a potential regulatory interaction. I will use in situ hybridization, protein expression, and phenotypic analysis to determine if semaphorin 2A is a viable target of Fd64a. Part B will identify other targets of Fd64a via microarray analysis comparing changes of gene expression in wild-type and fd64a-null embryos. Potential targets will be verified by in situ analysis, chromatin IP, and mutant-allele phenotype analysis. The molecular and genetic analysis of fd64a in the Drosophila embryo will provide insight into how cues provided by surrounding tissues influence migration of specific organs. This represents an important contribution to the broadening field of cell migration. PUBLIC HEALTH RELEVANCE: Cell migration is a ubiquitous and necessary process that can be exploited in diseases such as cancer, where tumors use cell migration to metastasize and invade other tissues. Fd64a, a transcription factor expressed in a subset of Drosophila muscles, affects the migration of a nearby tissue and changes its final placement. Understanding the factors that alter tissue migration has important medical implications, such as learning how and why tumors are able to move throughout the body.