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.

器官形成的一个基本问题是构成组织如何达到其正确的大小以及细胞结构。单个组织中的缺陷会影响整个器官的形成和经典这就是眼睛的例子，视网膜组织发生的破坏会导致微观恐惧症，这是严重的眼异常，其特征是小眼睛和先天性失明。突变视觉系统同源蛋白盒2（VSX2）基因引起微心心脏。视网膜的明确标记规范，VSX2在残留的祖细胞中起作用以定义组织身份。同时，VSX2是祖细胞增殖和神经源程序的几个方面所必需的神经发生（神经源性时机）和双极细胞的命运规范。我们的两个差距理解是由VSX2调节的祖细胞特性的机械互连性以及祖细胞在组织发生过程中使用VSX2的方式是否改变。解决这些差距，我们在小鼠中产生了两个新的VSX2等位基因，一个带有敲门记者/敲除配置，另一种用于条件基因失活。在前两个目标中，我们将表征这些突变等位基因的视网膜表型，并确定它们与A的表型相比天然无效等位基因和两个与人类引起疾病突变相对应的错义等位基因。有条件的基因失活将使用他莫昔芬诱导的Cre/Lox重组来确定祖细胞中VSX2利用的临时窗口，并检验了VSX2控制的假设视网膜身份，神经源性时序和增殖是分开的。我们还预测每个常规细胞类别（神经源输出）中细胞的平衡产生将被揭露神经发生后的暂时失活。在第三个目标中，我们检验了仍然存在的假设身份控制从依赖VSX2的独立状态转移，该状态在某些方面是表观遗传定义的早期目标。在第四个目标中，我们将纳入离体培养范式来测试候选人在先前的目标中确定的基因在促进或干扰方面的功能意义视网膜发展。这些研究的完成将为VSX2如何编排提供新的见解视网膜祖细胞特性以及视网膜祖细胞如何驱动视网膜组织发生，这是必不可少的眼器官发生的成分。