The role of the Fox gene fd64a in Drosophila embryonic development

描述（由申请人提供）：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生物体有益，如在发育中，或有害，如在癌症中，因此重要的是要在细胞和分子水平上了解这一过程的细微差别。本研究的目的是确定转录因子Fd64a在果蝇胚胎发育中的作用。Fd64 a是Fox家族转录因子的成员，其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亚群中表达，这是研究器官背景下细胞迁移的理想且独特的模型系统。在缺乏fd64a的果蝇中，唾液腺表现出一系列迁移缺陷。这些初步发现导致了Fd64a调节指导唾液腺迁移的分子表达的假设。为了进一步研究fd64a在细胞迁移中的作用，具体目标1的A部分将通过以下方法产生fd64a的敲除： 同源重组，然后分析该无效等位基因的唾液腺迁移和肌肉组织缺陷。特定目标1的B部分将检测fd64 a的过表达是否会影响唾液腺迁移。此外，该目标将确定是否可以通过以组织特异性方式提供野生型fd64a功能来挽救敲除缺陷。由于fd64a在果蝇胚胎的多种不同组织中表达，因此特定目标2的目标将是首先明确鉴定表达fd64a的组织，然后检查fd64a无效等位基因中这些组织的发育和形态。具体目标3将研究Fd64a的下游靶标。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，其表达与fd64a重叠，表明存在潜在的调控相互作用。我将使用原位杂交，蛋白表达和表型分析，以确定是否semaphorin 2A是一个可行的目标Fd64a。部分B将通过微阵列分析比较野生型和fd64 a缺失胚胎中基因表达的变化来鉴定Fd64 a的其他靶标。将通过原位分析、染色质IP和多等位基因表型分析验证潜在靶点。果蝇胚胎中fd64a的分子和遗传分析将提供对周围组织提供的线索如何影响特定器官迁移的深入了解。这是对拓宽细胞迁移领域的重要贡献。 公共卫生相关性：细胞迁移是一个普遍存在的必要过程，可以在癌症等疾病中利用，其中肿瘤利用细胞迁移转移和侵入其他组织。Fd64a是一种在果蝇肌肉中表达的转录因子，它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.