Effectors of Hedgehog Signaling in the Etiology of Coloboma

Hedgehog 信号传导在缺损病因学中的效应器

• 批准号: 10020181
• 负责人: Sarah Lusk
• 金额: $ 3.2万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10020181
• 关键词: Axon; Biological Assay; Blindness; Cells; Cellular Morphology; Childhood; Coloboma; Data; Defect; Development; Embryo; Erinaceidae; Etiology; Eye; Eye Development; Family; Fissural; Four-dimensional; Fresh Water; Gene Expression; Genes; Genetic; Genetic Epistasis; Genetic Transcription; Goals; Human; Hyperactive behavior; Image; Individual; Intrinsic factor; Label; Lead; Ligands; Link; Modeling; Molecular; Morphogenesis; Mosaicism; Movement; Mutation; Newborn Infant; Optic vesicle; Optics; Pathway interactions; Pattern; Phenotype; Process; Proteins; Publications; Reporter; Retinal Ganglion Cells; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Structural defect; Structure; Testing; Time; Transcript; Transgenes; Transgenic Organisms; Transplantation; Up-Regulation; Visual impairment; Work; Zebrafish; base; blastocyst; cell behavior; cell dimension; cell motility; differential expression; experimental study; gain of function; loss of function; loss of function mutation; mutant; new therapeutic target; optic stalk; overexpression; receptor; single cell sequencing; single-cell RNA sequencing; smoothened signaling pathway; teleost fish; transcriptome

Project Summary Developmental defects in eye structure commonly account for visual impairment in newborns. One such defect, uveal coloboma, is caused by failed development of the optic fissure and is a significant cause of pediatric blindness worldwide. One pathway central to this process is the Hedgehog (Hh) signaling pathway: loss-of-function mutations in the Hh receptor ptch2, which produce overactive Hh signaling, can result in coloboma. In our recent publication (Gordon and Lusk et al., 2018), using zebrafish, we determined the cellular basis of normal optic fissure formation, and identified specific defects in optic fissure and stalk formation in ptch2 mutants, by utilizing timelapse imaging, 4-dimensional cell tracking, and quantitative analysis of individual cell behaviors. We determined through transplantation experiments that both cell-intrinsic and intercellular signaling molecules are involved in the ptch2 mutant phenotype and that this occurs through a Gli-dependent mechanism. I hypothesize that overactivation of Hh signaling results in the upregulation of transcriptional targets that directly disrupt cell movements and morphogenesis, leading to coloboma. The goal of the work proposed here is to identify these targets and dissect their role in the etiology of coloboma. I propose to use a targeted candidate approach in parallel with an unbiased sequencing approach in an effort to identify factors responsible for the ptch2 mutant coloboma phenotype. Based on my preliminary data, I am investigating Netrin as a cell-extrinsic downstream effector of Hh signaling. In Aim 1, I will dissect the genetic interaction between Netrin and Hedgehog signaling during optic fissure and stalk formation, using gain- and loss-of-function approaches. Because I hypothesize at least one intrinsically-acting molecule downstream of Hh signaling is involved in causing coloboma, in Aim 2, I will use unbiased single-cell RNA-sequencing and functional analysis to identify Hh downstream genes that are responsible for the ptch2 mutant phenotype. I will identify specific transcriptomes from cells responding to overactive Hh signaling and directly test the role of upregulated genes in optic fissure and stalk formation. The work proposed here will allow me to uncover molecular pathways and potentially novel therapeutic targets regulating formation of the optic fissure and stalk, with the ultimate goal being a mechanistic understanding of coloboma.

项目摘要 眼睛结构的发育缺陷通常是新生儿视力障碍的原因。 其中一种缺陷，葡萄膜缺损，是由视神经裂发育失败引起的， 是全球儿童失明的重要原因。这一过程的一个核心途径是 Hedgehog（Hh）信号通路：Hh受体ptch 2的功能缺失突变， 产生过度活跃的Hh信号，可导致缺损。在我们最近的出版物（戈登和 Lusk等人，2018），使用斑马鱼，我们确定了正常视裂的细胞基础 形成，并确定了ptch 2突变体中视裂和柄形成的特定缺陷， 利用时间推移成像、4维细胞跟踪和个体的定量分析， 细胞行为我们通过移植实验确定， 细胞间信号分子参与ptch 2突变表型， 通过依赖于神经胶质细胞的机制。我假设Hh信号的过度激活导致 直接破坏细胞运动的转录靶点的上调， 形态发生导致缺损这里提出的工作目标是确定这些 目标和剖析他们的作用，病因的缺损。我建议用一个目标候选人 方法与无偏排序方法并行，以确定因素 负责ptch 2突变体缺损表型。根据我的初步数据，我 研究Netrin作为Hh信号传导的细胞外源性下游效应物。在目标1中，我将 剖析视裂期间Netrin和Hedgehog信号传导之间的遗传相互作用， 茎形成，使用功能获得和功能丧失方法。因为我假设至少有一个 Hh信号传导下游的一种内在作用分子参与引起缺损， 2、我将使用无偏的单细胞RNA测序和功能分析来鉴定Hh 下游基因负责ptch 2突变表型。我会确定具体的 转录组从细胞响应过度活跃的Hh信号，并直接测试的作用， 上调视裂和柄形成的基因。这里提出的工作将使我能够 揭示调节肿瘤形成的分子途径和潜在的新型治疗靶点， 视裂和柄，最终目标是对缺损的机械理解。