The Role of Pax6 in Mammalian Lens Development

描述：发育中的脊椎动物眼睛提供了一个强大的系统，用于研究器官发生的诱导组织相互作用。在之前的研究期间，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成分。我们发现Pax6调控一个保守的从属基因网络，但在脊椎动物的透镜中，它并不完美。然而，值得注意的是，我们还发现，这种遗传学上古老的调控网络在进化中被重新部署，在整个脊椎动物器官发生中发挥作用。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。我们鉴定了三种不同的Pax6眼部增强子，它们在不同的视网膜细胞类型中直接表达。然后，我们将重点放在Pax6外胚层增强剂或EE上，其与第二个SIMO增强剂一起指导发育中的透镜外胚层中的Pax6表达。我们将EE缩小到一个107 bp的最小增强子，该增强子仍然足以驱动发育中的透镜中的稳健表达，并且我们已经鉴定了Meis同源异型蛋白家族的成员作为关键EE调节剂。我们现在还定义了EE活性所需的其他顺式调节元件，并确定了结合它们的因子，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信号在早期透镜形成中的明确作用。然而，很少有人知道BMP信号通路如何调节EE，或者如果它调节的转录因子，调节EE。因此，这个更新的应用程序试图纠正这些差距，我们的知识，进一步阐明如何Meis，Sox和POU转录因子调节Pax6在发展中的透镜的表达，并建立BMP信号通路如何与它们相互作用。通知这个补助金的基本假设是，通过鉴定这些遗传和分子的相互关系，我们可以为透镜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节Pax6 EE的Meis辅因子从发育的角度来看表现适当。第二，我们将使用小鼠遗传学来确定如何Sox因子，通过与辅因子Oct1的协同相互作用，调节Pax6 EE.Third，我们将确定EE和SIMO增强子是否由Meis，Sox和POU因子协调调节，以及它们的表达特性如何相互关联。最后，我们将采用小鼠突变体，实验胚胎学和透镜上皮细胞系，以测试是否BMP信号通路收敛于转录因子，如Sox 2调节Pax6 EE。总的来说，这些实验有可能显着扩大我们的上游调控和调控网络，控制Pax6表达在早期透镜发展的知识。

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.