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

一种识别上皮细胞新型核心调节因子的正向遗传学方法

• 批准号: 8669580
• 负责人: MARK q MARTINDALE
• 金额: $ 15.21万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8669580
• 关键词: forward; genetics; approach; identify; novel

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.

项目概要： 该提案旨在开发一种正向遗传方法，以确定调节 无脊椎动物刺胞动物模式系统Nematostella的上皮结构和形态发生 vectensis。上皮形态发生是一个重要的组成部分，是形成和适当发展的基础。 从神经管到内部器官的结构。无脊椎动物模型系统是降低成本的理想工具- 有效的前向方法来识别调节与那些在基因组中类似的生物过程的基因。 脊椎动物Nematostella是一个独特的系统，具有简单的上皮体计划，基因组更类似于 脊椎动物的基因内容和组织比目前的无脊椎动物遗传系统， 再生的在Nematostella的发育过程中，在动物的口部周围形成触手 并且与在上皮细胞发育中观察到的模式和形态学变化非常相似， 脊椎动物胚胎中的神经管、肢芽和器官等结构。虽然反向遗传 现在已经建立了一些方法，可以研究线虫病期间的候选基因， 开发，一个向前的无偏见的方法，以确定新的基因和途径，为普遍的生物 过程缺乏。在这个新的系统中，研究张力形成的前向方法的发展将 改变并增强我们对上皮细胞模式和形态发生的理解。我们已经开发出一种 通过用常见诱变剂ENU处理来诱导突变的方案，并描述了标准的三个 世代杂交方案，以筛选影响触角形成的隐性突变。我们有 产生了一个初步的物理图，我们建议扩大，以执行批量分离子分析 绘制与触角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中的感兴趣的基因组区域进行测序。序列 数据将与亲本序列和测序的Nematostella基因组进行比较，以确定 突变导致观察到的tenglomerase表型。本研究中鉴定的突变将用于 未来的表征和研究集中在了解控制 在动物发育过程中启动和执行上皮图案化和形态发生， 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健康和发育。(1)我们将发现新的 和上皮图案和形态发生的核心调节器;（2）我们将进行第一次 在一种与脊椎动物有着惊人分子相似性的非两侧对称海洋无脊椎动物中进行遗传筛选， 从而为未来的研究人员提供了一个蓝图，利用这种新的模型系统来改善我们的 了解动物的发展，因为它涉及到人类健康和再生医学。