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

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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.