Molecular Mechanism of C. elegans gonadogenesis

描述（由申请人提供）：一个复杂器官的形成需要多个发育过程，每个过程必须正确执行，并且彼此协调。研究这3个过程的细胞和分子基础为了解正常发育机制提供了重要的见解，这将扩大我们对发育缺陷的理解，并有助于在培养用于医疗用途的组织和器官方面取得进展。此外，由于许多疾病是由正常的细胞过程出错引起的，基础发育生物学研究为疾病的发生、发展和潜在的治疗提供了关键的见解。这一建议的重点是阐明秀丽隐杆线虫NUD-1蛋白和功能相关蛋白在特定器官性腺发育中的作用。性腺是研究器官发生的良好模型系统，然而，性腺形成的许多特征尚不清楚。NUD-1是一种保守的新蛋白，它对秀丽隐杆线虫的促性腺作用的了解较少。首先，将使用两种不同的方法确定与nud1缺失相关的性腺表型。NUD-1敲低突变体将使用rna介导干扰（RNAi）的反向遗传方法产生，并使用细胞类型特异性标记进行表征。为了研究与nud1缺失相关的原发性性腺缺陷，将进行基因筛选，试图获得可遗传的nud1突变。第二个目的是表征nudi的性腺表达模式。同样，我们将采用双管齐下的方法，生成并分析NUD- 1::GFP表达构建体和NUD- 1特异性抗体。最后，其他具有Nud表型的突变将在遗传筛选中确定并表征。将进行实验以确定新发现的基因座在性腺发育中是否与nud1或相互作用。总的来说，拟议的工作旨在增加我们对器官形成机制的了解，并阐明nud1发挥其作用的手段。

DESCRIPTION (provided by applicant): The formation of a complex organ requires multiple developmental processes that must each be properly executed and also coordinated with one another. Investigating the cellular and molecular basis of these 3rocesses provides important insight into normal developmental mechanisms that will expand our understanding of developmental defects and contribute to progress in culturing tissues and organs for medical use. Furthermore, as many diseases result from a normal cellular process gone awry, basic developmental biology research provides key insights into disease initiation, progression, and potential treatments. This proposal is focused on elucidating the role of the Caenorhabditis elegans NUD-1 protein and functionally-related proteins in the development of a specific organ, the gonad. The gonad is a well established model system for studying organogenesis, however, many features of gonad formation are not understood. NUD-1 is a well conserved, but novel protein that provides an inroad into less understood aspects of C. elegans gonadogenesis. First, the gonadal phenotype associated with depletion of NUD-1 will be determined using two distinct approaches. NUD-1 knock-down mutants will be generated using the reverse genetic approach of RNA-mediated interference (RNAi) and characterized using cell-type specific markers. To allow study of the primary gonadal defect associated with loss of NUD-1, a genetic screen will be performed in an attempt to obtain a heritable nud-1 mutation. The second aim is to characterize the gonadal expression pattern of NUD-I. Again, a two-pronged approach will be employed and both NUD- 1::GFP expression constructs and a NUD-l-specific antibody will be generated and analyzed. Finally, other mutations with a Nud phenotype will be identified in the genetic screen and characterized. Experiments will be performed to determine if the newly identified loci function with nud-1, or with each other, in gonad development. Overall, the proposed work is designed to increase our knowledge of mechanisms used in organ formation and to shed light on the means by which NUD-1 exerts its effects.