Identification of PITX2-dependent mechanisms in the developing and mature cornea

我们的目标是确定控制胚胎发育和角膜发育的调控网络 出生后角膜愈合，并确定这些网络的破坏如何导致视力限制缺陷 在角膜功能方面。作为实现这一目标的关键一步，我们的目标是确定PITX2在 角膜发育和伤口愈合。我们的中心假设是Cited2和 抑制BMP2和BMP3的表达是PITX2调节角膜的重要机制 此外，研究还发现，Pitx2基因剂量的减少会对成熟角膜的伤口愈合产生不利影响。 我们的假设是基于数据显示Cited2表达丢失，而BMP2和 在没有PITX2的情况下，BMP3在发育中的角膜中表达增加，而成人角膜中BMP3的表达增加 Pitx2+/-小鼠浑浊，在微妙的侮辱后出现新生血管。这一事件的意义 建议的研究是关于PITX2在正常发育和发育中调控的遗传网络的知识 角膜的愈合将促进我们对这些过程的总体理解，这一结果可能会在 未来有助于设计改进的治疗方法来预防和治疗角膜造成的视力损失 疾病或伤害。我们将通过三个具体目标来测试我们的假设：1)测试Cited2是一个 PITX2在角膜发育过程中的重要下游效应，2)检验PITX2- 介导抑制BMP2和BMP3的表达是角膜所需的额外基本机制 3)检验成熟角膜伤口愈合对Pitx2基因敏感的预测 剂量。根据目标1，申请人的实验室已经提供了有条件的淘汰赛策略，该策略将 用于在角膜发育过程中特异性地消融眼神经脊或表面外胚层中的Cited2， 并使用成熟的方法评估角膜血统和血管生长。在目标2下，a 条件策略将被用来在基因上激活BMP2或BMP3在眼神经脊的表达 利用我们培育的小鼠进行角膜发育，由此产生的突变将由 与目标1中的标准相同。在目标3中，申请人开发的有缺陷的Pitx2等位基因将用于 确定Pitx2基因剂量对成熟角膜伤口愈合和新生血管的影响(S)。这个 预期的结果是，PITX2在发育和成熟的角膜中的基本功能将被确定。 由于PITX2的基本功能在不断发展和成熟，因此本文的研究具有创新性 将确定角膜，包括首次证明Cited2的表达和BMP信号的活性 抑制是角膜发育和伤口愈合的基本要求。最终，这样的 知识可能为预防和治疗视力丧失提供富有成效的新疗法的见解 由于角膜感染和受伤。更广泛地说，这些结果也可能适用于理解 某些癌症中新生血管的机制。

Our goal is to identify the regulatory networks that govern corneal development during embryogenesis and corneal healing post-natally, and to determine how disruption of these networks leads to sight-limiting defects in cornea function. As a key step towards this goal, our objective is to identify essential functions of PITX2 in corneal development and wound healing. Our central hypotheses are that activation of Cited2 and repression of Bmp2 and Bmp3 expression are essential mechanisms by which PITX2 regulates corneal development, and that reductions in Pitx2 gene dose adversely affect wound healing in the mature cornea. Our hypothesis was formulated on the basis of data showing that Cited2 expression is lost, and Bmp2 and Bmp3 expression is elevated in the developing cornea in the absence of PITX2, and that corneas of adult Pitx2+/- mice are cloudy and neovascularization is present following subtle insults. The significance of the proposed research is that knowledge of the genetic networks regulated by PITX2 in normal development and healing of the cornea will advance our understanding of these processes in general, an outcome that may in the future contribute to rationale design of improved therapies to prevent and treat vision loss due to corneal disease or injury. We will test our hypotheses via three specific aims: 1) Test the hypothesis that Cited2 is an essential downstream effector of Pitx2 during cornea development, 2) Test the hypothesis that PITX2- mediated suppression of Bmp2 and Bmp3 expression are additional essential mechanisms required for corneal development, and 3) Test the prediction that wound healing in the mature cornea is sensitive to Pitx2 gene dose. Under aim 1, a conditional knockout strategy, which is already available in the applicant’s laboratory, will be used to specifically ablate Cited2 in ocular neural crest or surface ectoderm during corneal development, and assess corneal lineages and vascular growth using well-established approaches. Under aim 2, a conditional strategy will be used to genetically activate Bmp2 or Bmp3 expression in ocular neural crest during corneal development using mice that we have generated, and the resulting mutants will be analyzed by the same criteria as in Aim 1. Under aim 3, the defective Pitx2- allele that the applicant developed will be used to determine the effect(s) of Pitx2 gene dose on wound healing and neovascularization in the mature cornea. The expected outcome is that essential functions of PITX2 in the developing and mature cornea will be identified. The proposed research is innovative because essential functions of PITX2 in the developing and mature cornea will be determined, including the first demonstration that Cited2 expression and BMP signaling activity suppression are essential requirements for corneal development and wound healing. Ultimately, such knowledge may provide insights into productive new therapies for the prevention and treatment of vision loss due to corneal infection and injury. More broadly, the results may also have applicability to understanding mechanisms of neovascularization in certain cancers.