The Role of Pax6 in Mammalian Lens Development

描述：发育中的脊椎动物的眼睛为研究作为器官发生基础的诱导组织相互作用提供了一个强大的系统。在之前的资助期间，我们研究了Pax6基因在小鼠眼睛发育中的功能，并确定了Pax6遗传途径的上游和下游组成部分。我们发现，Pax6调控着一个保守的从属基因网络，但在脊椎动物的晶状体中，它的作用并不完美。然而，值得注意的是，我们也发现，这个在系统发育上古老的调控网络在进化中被重新部署，在脊椎动物器官发生过程中发挥作用。此外，我们还在理解Pax6上游调控的性质方面取得了重大进展。我们确定了三种不同的Pax6眼睛增强剂，它们直接在不同类型的视网膜细胞中表达。然后我们重点研究了Pax6外胚层增强子或EE，它与第二个SIMO增强子一起在发育中的晶状体外胚层中指导Pax6的表达。我们将EE缩小到107个碱基的最小增强子，该增强子仍然足以在发育中的晶状体中驱动强劲的表达，我们已经确定Meis同源蛋白家族的成员是关键的EE调节因子。我们现在还定义了EE活动所需的其他顺式调节元件，并确定了结合它们的因素，包括SOX和POU家族的成员。包括我们自己在内的几个研究小组的工作也揭示了BMP信号在晶状体形成早期的明确作用。然而，关于BMP信号通路如何调控EE，或者它是否调控调控EE的转录因子，人们知之甚少。因此，这个更新应用程序试图通过进一步阐明MEIS、SOX和POU转录因子如何调控发育中的晶状体中Pax6的表达，并通过建立BMP信号通路如何与它们相互作用来纠正我们知识中的这些空白。提供这项资助的基本假设是，通过识别这些基因和分子之间的相互关系，我们可以为晶状体发育的不同阶段建立不同的调控网络。为此，这一竞争性更新提出了四个具体目标。首先，我们将利用小鼠遗传学的力量来确定MEIS转录因子和一个尚未确定的调控Pax6EE的MEIS辅助因子从发育的角度来看是适当的。其次，我们将使用小鼠遗传学来确定SOX因子如何通过与辅助因子Oct1的协同作用来调节Pax6EE。第三，我们将确定EE和SIMO增强子是否受MEIS、SOX和POU因子的协调调控，以及它们的表达特性如何相互关联。最后，我们将使用小鼠突变体、实验胚胎学和晶状体上皮细胞系来测试BMP信号通路是否会聚到调节Pax6EE的转录因子如Sox2上。总而言之，这些实验有可能显著扩展我们对上游调控因子和调控网络的知识，这些调控因子和调控网络在晶状体发育的早期控制Pax6的表达。

DESCRIPTION: The developing vertebrate eye provides a powerful system for studying the inductive tissue interactions that underlie organogenesis. In the prior grant period, we investigated the function of the Pax6 gene in murine eye development, and identified both upstream and downstream components of the Pax6 genetic pathway. We found that Pax6 regulates a conserved network of subordinate genes, but that in the vertebrate lens it does so imperfectly. Remarkably, however, we also found that this phylogenetically ancient regulatory network has been re-deployed in evolution to function throughout vertebrate organogenesis. In addition, we have also made major inroads into understanding the nature of Pax6 upstream regulation. We identified three distinct Pax6 ocular enhancers that direct expression in different retinal cell types. We then focused on the Pax6 Ectodermal Enhancer or EE, that together with a second SIMO enhancer directs Pax6 expression in developing lens ectoderm. We narrowed the EE to a 107 bp minimal enhancer that remains sufficient to drive robust expression in the developing lens, and we have identified members of the Meis homeoprotein family as key EE regulators. We have now also defined additional cis-regulatory elements that are required for EE activity, and have identified factors that bind them, including members of the Sox and POU families. Work from several groups, including our own, has also revealed a clear role for BMP-signaling in early lens formation. However, little is known about how the BMP-signaling pathway regulates the EE, or if it regulates the transcription factors that regulate the EE. Thus, this renewal application seeks to rectify these gaps in our knowledge by further elucidating how Meis, Sox and POU transcription factors regulate Pax6 expression in the developing lens, and by establishing how the BMP-signaling pathway interacts with them. The underlying hypothesis that informs this grant is that by identifying these genetic and molecular inter- relationships, we can establish distinct regulatory networks for different phases of lens development. To this end, this competing renewal proposes four Specific Aims. First, we will use the power of mouse genetics to establish that the Meis transcription factors and a yet to be identified Meis co-factor that regulate the Pax6 EE behave appropriately from a developmental standpoint. Second, we will use mouse genetics to determine how Sox factors, through cooperative interaction with the co-factor Oct1, regulate the Pax6 EE. Third, we will determine whether the EE and SIMO enhancers are coordinately regulated by Meis, Sox and POU factors, and how their expression properties inter-relate. Lastly, we will employ mouse mutants, experimental embryology and lens epithelial cell lines to test whether the BMP-signaling pathway converges upon transcription factors such as Sox2 that regulate the Pax6 EE. Collectively, these experiments have the potential to significantly expand our knowledge of the upstream regulators and regulatory networks that control Pax6 expression in early lens development.