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

项目总结： 这项建议旨在开发一种正向遗传方法来识别调节 新型无脊椎动物爬行动物模型系统Nematostella的上皮模式和形态发生 矢状病毒。上皮形态发生是形成和正常发展的重要组成部分。 从神经管到内脏的各种结构。无脊椎动物模型系统是计算成本的理想工具- 有效的正向方法来识别调控类似于人类基因组的基因 脊椎动物。线虫是一种独特的系统，具有简单的上皮体计划，其基因组更类似于 脊椎动物在基因含量和组织上比目前的无脊椎动物遗传系统要高 再生的。在线虫的发育过程中，动物的嘴周围会形成触角 与上皮细胞发育过程中观察到的花纹和形态变化非常相似 脊椎动物胚胎中的结构，如神经管、肢芽和器官。虽然是反向遗传 现在已经建立了很好的方法，可以研究线虫期间的候选基因 开发--一种为普遍生物学寻找新基因和途径的无偏见的前向方法 缺乏流程。在这一新系统中研究触角形成的前沿方法的发展将 改变和提高我们对上皮模式和形态发生的理解。我们已经开发出一种 通过用常见的诱变剂ENU处理来诱导突变的方案，并描述了标准的三种 世代杂交方案，筛选影响触角形成的隐性突变。我们有 生成了初步物理图，我们建议对其进行扩展以执行批量分离分析 绘制与触须发育表型连锁的突变基因组区域图。下一代测序 这项技术将被用于对池养突变动物的基因组感兴趣区域进行测序。该序列 数据将与亲本序列和线虫基因组序列进行比较，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 未来的表征和调查的重点是了解控制 在动物发育和发育过程中上皮图案化和形态发生的启动和执行 再生。完成这项研究后，我们将实现两个目标，这将促进我们的能力 在无脊椎动物模型系统中研究人类的健康和发育。(1)我们将确定一部小说 和上皮构型和形态发生的核心调节器；以及(2)我们将实施第一个 在一种与脊椎动物有惊人分子相似性的非双边海洋无脊椎动物中进行基因筛查， 从而为未来的研究人员开发这一新的模型系统提供了蓝图，以改进我们的 了解动物发育与人类健康和再生医学的关系。

Project summary: 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 similarities 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.