A forward genetics approach to identify novel core regulators of epithelial patte

描述(申请人提供)：这项建议旨在开发一种正向遗传学方法，以确定在新的无脊椎动物线虫模式系统中调节上皮模式和形态发生的核心遗传成分。上皮形态发生是从神经管到内脏的各种结构形成和正常发展的关键组成部分。无脊椎动物模型系统是具有成本效益的前沿方法的理想工具，用于识别调控与脊椎动物相似的生物过程的基因。线虫是一个独特的系统，具有简单的上皮体计划，与现有的无脊椎动物遗传系统相比，其基因组在基因内容和组织结构上更类似于脊椎动物，并且具有高度的再生性。在线虫的发育过程中，触角的形成发生在动物的嘴周围，与脊椎动物胚胎中观察到的上皮结构(如神经管、肢芽和器官)的发育过程中观察到的图案和形态变化非常相似。尽管现在已经建立了反向遗传方法来研究线虫发育过程中的候选基因，但缺乏一种正向的、无偏见的方法来识别新的基因和普遍生物学过程的途径。在这一新系统中研究触角形成的前沿方法的发展将改变和加强我们对上皮模式和形态发生的理解。我们已经开发了一种通过用常见的诱变剂ENU处理来诱导突变的方案，并描述了一种标准的三代交叉方案来筛选影响触须形成的隐性突变。我们已经生成了一个初步的物理图谱，我们建议对其进行扩展，以便进行批量分离分析，以定位与触须发育表型相关的突变基因组区域。下一代测序技术将用于对池养突变动物的基因组感兴趣区域进行测序。序列数据将与亲本序列和已测序的线虫基因组进行比较，以确定导致观察到的触须表型的突变(S)。这项研究中发现的突变将用于未来的表征和调查，重点是了解在动物发育和再生过程中控制上皮图案化和形态发生的启动和执行的核心成分。这项研究完成后，我们将实现两个目标，这将促进我们在无脊椎动物模型系统中研究人类健康和发展的能力。(1)我们将确定上皮模式和形态发生的新的核心调控因子；以及(2)我们将在与脊椎动物具有惊人分子相似性的非双边海洋无脊椎动物中进行第一次基因筛查，从而为未来的研究人员提供一个蓝图，利用这一新的模型系统来提高我们对动物发育的理解，因为它与人类健康和再生医学有关。 公共卫生相关性：这项工作与人类健康相关，因为它确定了未来研究的核心基因，这些基因调控被称为上皮组织的细胞片的图案、生长和形态(形状)变化。在几乎所有的身体结构(神经管、四肢和器官等)的发育过程中，都会出现上皮组织的形态变化和受控生长。而这一过程中的缺陷往往会导致出生缺陷，如脊柱裂或腭裂。识别和研究控制上皮组织模式、生长和形态的基因将增强我们开发对广泛出生缺陷的检测和治疗的能力。

DESCRIPTION (provided by applicant): This proposal aims to develop a forward genetic approach to identify core genetic components that regulate epithelial patterning and morphogenesis in the novel invertebrate cnidarian model system Nematostella vectensis. Epithelial morphogenesis is a critical component that underlies formation and proper development of structures ranging from the neural tube to internal organs. Invertebrate model systems are ideal tools for cost- effective forward approaches to identify genes that regulate similar biological processes to those in vertebrates. Nematostella is a unique system that has simple epithelial body plan, a genome more similar to vertebrates in gene content and organization than current invertebrate genetic systems, and is highly regenerative. During development of Nematostella, tentacle formation occurs around the mouth of the animal and is remarkably similar to the patterning and morphological changes observed in development of epithelial structures, such as the neural tube, limb buds, and organs, in vertebrate embryos. Though reverse genetic approaches are now well established allowing for investigations of candidate genes during Nematostella development, a forward non-biased approach to identify novel genes and pathways for universal biological processes is lacking. Development of forward approaches to study tentacle formation in this novel system will transform and enhance our understanding of epithelial patterning and morphogenesis. We have developed a protocol to induce mutations via treatment with the common mutagen ENU and describe a standard three generation crossing scheme to screen for recessive mutations that affect tentacle formation. We have generated a preliminary physical map, which we propose to expand in order to perform bulk segregant analysis to map mutant genomic regions linked to tentacle development phenotypes. Next-generation sequencing technology will be used to sequence the genomic region of interest in pooled mutant animals. The sequence data will be compared to the parental sequences and the sequenced Nematostella genome to identify the mutation(s) that lead to the observed tentacle phenotype. Mutations identified in this study will be used for future characterization and investigations focused on understanding the core components that control the initiation and execution of epithelial patterning and morphogenesis during animal development and regeneration. Upon completion of this study, we will have accomplished two goals that will promote our ability to investigate human health and development in invertebrate model systems. (1) We will have identified novel and core regulators of epithelial patterning and morphogenesis; and (2) We will have carried out the first genetic screen in a non-bilaterian marine invertebrate that has striking molecular similarity to vertebrates, thus providing a blueprint for future researchers to exploit this novel model system to improve our understanding of animal development as it relates to human health and regenerative medicine. PUBLIC HEALTH RELEVANCE: This work is relevant to human health because it identifies core genes for future study that regulate patterning, growth, and morphological (shape) changes in sheets of cells called epithelial tissue. Morphological changes and controlled growth of epithelial tissue occurs in the development of nearly all body structures (neural tube, limbs and organs etc.) and defects in this process often lead to birth defects such as spina bifida or cleft palate. Identifying and studying genes that control epithelial tissue patterning, growth, and morphology will enhance our ability to develop detection and treatments for a wide range of birth defects.