The Role of Pax6 in Mammalian Lens Development

描述：发育中的脊椎动物眼睛为研究器官发生基础上的诱导组织相互作用提供了一个强大的系统。在之前的资助期内，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分。我们发现Pax6调节了一个保守的从属基因网络，但在脊椎动物晶状体中，它的调节并不完美。然而，值得注意的是，我们还发现这种系统发育上古老的调节网络已经在进化中重新部署，在整个脊椎动物器官发生中发挥作用。此外，我们在理解Pax6上游调控的本质方面也取得了重大进展。我们确定了三种不同的Pax6眼增强剂，直接表达在不同的视网膜细胞类型。然后我们将重点放在Pax6外胚层增强子或EE上，它与第二个SIMO增强子一起指导Pax6在发育中的晶状体外胚层中的表达。我们将EE缩小到107 bp的最小增强子，这仍然足以在发育晶体中驱动稳健的表达，并且我们已经确定了Meis同源蛋白家族的成员作为关键的EE调节因子。我们现在还定义了EE活动所需的其他顺式调控元件，并确定了约束它们的因素，包括Sox和POU家族的成员。包括我们自己在内的几个小组的工作也揭示了bmp信号在早期晶状体形成中的明确作用。然而，关于bmp信号通路如何调节EE，或者它是否调节调节EE的转录因子，我们知之甚少。因此，本更新申请旨在通过进一步阐明Meis， Sox和POU转录因子如何调节发育透镜中的Pax6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。这项资助的基本假设是，通过识别这些遗传和分子的相互关系，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，这一相互竞争的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，从发育的角度确定Meis转录因子和一个尚未确定的调节Pax6 EE的Meis辅助因子的行为是适当的。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的合作相互作用来调节Pax6 EE。第三，我们将确定EE和SIMO增强子是否受到Meis、Sox和POU因子的协调调节，以及它们的表达特性如何相互关联。最后，我们将利用小鼠突变体、实验胚胎学和晶状体上皮细胞系来测试bmp信号通路是否会聚于调节Pax6 EE的Sox2等转录因子上。总的来说，这些实验有可能显著扩展我们对早期晶状体发育中控制Pax6表达的上游调控和调控网络的认识。

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.