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

描述（由申请人提供）：本提案旨在发展一种向前遗传方法，以确定在新型无脊椎刺胞动物模型系统Nematostella vectensis中调节上皮模式和形态发生的核心遗传成分。从神经管到内脏器官，上皮细胞的形态发生是结构形成和正常发育的关键组成部分。无脊椎动物模型系统是理想的工具，为经济有效的方法，以确定基因调节类似的生物过程，在脊椎动物的那些。Nematostella是一种独特的系统，具有简单的上皮体计划，基因组在基因内容和组织上比目前的无脊椎动物遗传系统更接近脊椎动物，并且具有高度再生能力。在线虫的发育过程中，触手的形成发生在动物的嘴周围，这与脊椎动物胚胎中上皮结构（如神经管、肢芽和器官）发育过程中观察到的模式和形态变化非常相似。虽然反向遗传方法现在已经很好地建立，允许在线虫发育过程中研究候选基因，但缺乏一种向前的无偏见的方法来识别普遍生物过程的新基因和途径。在这个新系统中研究触手形成的前沿方法的发展将改变和增强我们对上皮模式和形态发生的理解。我们已经制定了一项方案，通过使用常见诱变剂ENU诱导突变，并描述了一个标准的三代杂交方案，以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，以绘制与触手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汇集的突变动物感兴趣的基因组区域进行测序。序列数据将与亲本序列和线虫基因组测序进行比较，以确定导致所观察到的触须表型的突变。在这项研究中发现的突变将用于未来的表征和研究，重点是了解在动物发育和再生过程中控制上皮模式和形态发生的启动和执行的核心成分。在这项研究完成后，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和核心调节因子；(2)我们将在一种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首次基因筛选，从而为未来的研究人员利用这种新型模型系统提供蓝图，以提高我们对动物发育与人类健康和再生医学相关的理解。

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.