Vsx2 Dependent Regulation of Retinal Progenitor Cell Properties

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， 细胞结构单个组织的缺陷会影响整个器官的形成， 这方面的例子是眼睛，其中视网膜组织发生的破坏可引起小眼症，一种严重的 以小而不成形的眼睛和先天性失明为特征的眼睛异常。突变 视觉系统同源框2（Vsx 2）基因导致小眼球。视网膜病变的决定性标志物 在本说明书中，Vsx 2在视网膜祖细胞中起作用以定义组织身份。同时，VSX 2是 所需的祖细胞增殖和几个方面的神经原性程序，包括时间 神经发生的开始（神经发生的时间），和双极细胞的命运规范。我们的两个差距 理解是由Vsx 2调节的祖细胞特性的机械相互联系 以及祖细胞在组织发生过程中如何利用Vsx 2是否发生变化。解决 这些缺口，我们在小鼠中产生了两个新的Vsx 2等位基因，一个具有敲入报告基因/敲除 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，并确定它们如何比较表型所造成的 天然无效等位基因和两个错义等位基因，其对应于人类中的致病突变。 将用他莫昔芬诱导的Cre/lox重组进行条件性基因失活，以确定 Vsx 2在祖细胞中利用的时间窗口，并检验Vsx 2控制细胞增殖的假设。 视网膜的特性、神经发生的时间和增殖是可分离的。我们还预测， 每种视网膜细胞类别中细胞的平衡产生（神经源性输出）将通过 神经发生开始后的暂时失活。在第三个目标中，我们测试了视网膜的假设， 身份控制从Vsx 2依赖性转变为独立状态，这是表观遗传学定义的， 早期的目标。在第四个目标中，我们将结合离体培养范例来测试候选物。 在先前的目标中鉴定的基因，其在促进或干扰 视网膜发育这些研究的完成将为VSX 2如何编排提供新的见解 视网膜祖细胞的特性以及视网膜祖细胞如何驱动视网膜组织发生， 眼器官发生的组成部分。

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.