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

描述（由申请人提供）：本提案旨在开发一种正向遗传方法，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组分。上皮形态发生是从神经管到内脏器官的结构形成和正常发育的基础。无脊椎动物模型系统是成本有效的正向方法的理想工具，以确定基因调控类似的生物过程中的脊椎动物。Nematostella是一个独特的系统，具有简单的上皮体计划，基因组在基因内容和组织上比目前的无脊椎动物遗传系统更类似于脊椎动物，并且具有高度的再生性。在Nematostella的发育过程中，触手的形成发生在动物的嘴周围，并且与脊椎动物胚胎中上皮结构（如神经管、肢芽和器官）发育中观察到的模式和形态变化非常相似。虽然反向遗传学的方法，现在已经很好地建立了允许的候选基因的调查Nematostella的发展，一个正向的无偏见的方法来确定新的基因和途径的普遍的生物过程是缺乏的。在这个新的系统中，研究tendonucleus形成的前向方法的发展将改变和提高我们对上皮图案化和形态发生的理解。我们已经开发了一个协议，通过与常见的诱变剂ENU处理诱导突变，并描述了一个标准的三代杂交方案，以筛选影响tenetransferase形成的隐性突变。我们已经产生了一个初步的物理图谱，我们建议扩大，以进行批量分离子分析，以映射与tenglomerate发展表型的突变基因组区域。下一代测序技术将用于对合并突变动物中的目标基因组区域进行测序。将序列数据与亲本序列和测序的Nematostella基因组进行比较，以鉴定导致观察到的线虫表型的突变。本研究中鉴定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图案形成和形态发生的启动和执行的核心成分。完成这项研究后，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育的能力。(1)我们将确定新的和核心的调节上皮图案和形态发生;和（2）我们将进行第一次遗传筛选在一个非双侧的海洋无脊椎动物，具有惊人的分子相似性脊椎动物，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，以提高我们的理解动物的发展，因为它涉及到人类健康和再生医学。 公共卫生相关性：这项工作与人类健康有关，因为它确定了未来研究的核心基因，这些基因调节称为上皮组织的细胞片的模式，生长和形态（形状）变化。上皮组织的形态变化和受控生长发生在几乎所有身体结构（神经管、四肢和器官等）的发育中。而这一过程中的缺陷往往导致出生缺陷，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提高我们开发检测和治疗各种出生缺陷的能力。

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.