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Vsx2 Dependent Regulation of Retinal Progenitor Cell Properties

视网膜祖细胞特性的 Vsx2 依赖性调节

基本信息

批准号：
10667540
负责人：
EDWARD M LEVINE
金额：
$ 54.31万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-08-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10667540
关键词：
ATAC-seq Address Affect Alleles Blindness CRISPR/Cas technology Candidate Disease Gene Cells Complete Blindness Congenital Abnormality Data Defect Development Disease Electroporation Embryo Epigenetic Process Eye Foundations Gene Expression Gene Expression Regulation Gene Silencing Generations Genes Genetic Models Genetic Recombination Harvest Homeobox Human Hyperpigmentation Inherited Injury Knock-in Knock-out Knowledge LacZ Genes LoxP-flanked allele Microphthalmos Mus Mutation Natural regeneration Organ Organogenesis Output Phenotype Process Production Proliferating Property Regulation Reporter Retina Role Specific qualifier value Structural defect Tamoxifen Testing Time Tissues Visual System Visual impairment bisulfite sequencing disease-causing mutation early onset eye formation histogenesis in vivo inducible Cre insight mutant neurogenesis postmitotic prevent progenitor programs retinal progenitor cell single-cell RNA sequencing stem cells transcription factor transcriptome sequencing whole genome

项目摘要

A fundamental question in organ formation is how the constituent tissues achieve their correct sizes and cytoarchitectures. Defects in a single tissue can affect the formation of an entire organ and a classic example of this is the eye, where disruptions in retinal histogenesis can cause microphthalmia, a severe ocular anomaly characterized by small, poorly formed eyes and congenital blindness. Mutations in the Visual System Homeobox 2 (Vsx2) gene cause microphthalmia. A definitive marker of retinal specification, Vsx2 functions in retinal progenitor cells to define tissue identity. Concurrently, Vsx2 is required for progenitor proliferation and several aspects of the neurogenic program including the timing of neurogenesis onset (neurogenic timing), and the fate specification of bipolar cells. Two gaps in our understanding are the mechanistic interconnectedness of the progenitor properties regulated by Vsx2 and whether progenitors change in how they utilize Vsx2 over the course of histogenesis. To address these gaps, we generated two new Vsx2 alleles in mice, one with a knock-in reporter/knock-out configuration and the other for conditional gene inactivation. In the first two aims, we will characterize the retinal phenotypes of these mutant alleles and determine how they compare to phenotypes caused by a natural null allele and two missense alleles that correspond to disease-causing mutations in humans. Conditional gene inactivation will be done with tamoxifen-inducible Cre/lox recombination to determine the temporal windows of Vsx2 utilization in progenitors and test the hypothesis that Vsx2’s control of retinal identity, neurogenic timing, and proliferation are separable. We also predict that additional roles in the balanced production of cells in each retinal cell class (neurogenic output) will be unmasked by temporal inactivation after the start of neurogenesis. In the third aim, we test the hypothesis that retinal identity control shifts from a Vsx2-dependent to independent state that is epigenetically defined for some of the earlier targets. In the fourth aim, we will incorporate an ex vivo culture paradigm to test candidate genes identified in the previous aims for their functional significance in promoting or interfering with retinal development. Completion of these studies will provide new insights into how Vsx2 orchestrates retinal progenitor properties and how retinal progenitors drive retinal histogenesis, an essential component of eye organogenesis.

器官形成中的一个基本问题是组成组织如何达到其正确的大小和细胞结构。单个组织中的缺陷会影响整个器官和经典组织的形成这方面的例子是眼睛，视网膜组织发生的中断会导致小眼炎，一种严重的眼畸形以小而畸形的眼睛和先天失明为特征的眼畸形基因突变视觉系统同源盒2(Vsx2)基因可引起小眼球。视网膜的明确标志规范中，Vsx2在视网膜祖细胞中发挥功能，以定义组织特性。同时，Vsx2是祖细胞增殖所需的时间和神经发生计划的几个方面，包括时间神经发生的开始(神经发生的时间)，以及双极细胞的命运指定。我们的两个差距理解是受Vsx2调控的前体属性的机械性互连以及祖细胞在组织发生过程中是否改变了他们利用Vsx2的方式。致信地址在这些缺口中，我们在小鼠身上产生了两个新的Vsx2等位基因，其中一个带有敲入报告/敲除配置和另一个用于条件基因失活。在前两个目标中，我们将描述这些突变等位基因的视网膜表型，并确定它们与由与人类致病突变相对应的自然零等位基因和两个错义等位基因。条件基因失活将通过三苯氧胺诱导的Cre/lox重组来确定 Vsx2在祖细胞中利用的时间窗，并检验Vsx2的S控制的假说视网膜的同一性、神经源性的时间和增殖是可分离的。我们还预测，在每一类视网膜细胞的平衡生产(神经源性输出)将被神经发生开始后的暂时性失活。在第三个目标中，我们测试了视网膜身份控制从Vsx2依赖状态转变为独立状态，这是从表观上为某些人定义的早些时候的目标。在第四个目标中，我们将结合体外培养范式来测试候选人在先前的目的中确定的基因在促进或干扰视网膜发育。这些研究的完成将为Vsx2如何协调提供新的见解视网膜前体细胞的特性以及视网膜前体细胞如何驱动视网膜组织发生眼睛器官发生的组成部分。