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

鉴定发育和成熟角膜中 PITX2 依赖性机制

• 批准号: 9381229
• 负责人: PHILIP J GAGE
• 金额: $ 44.31万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9381229
• 关键词: Ablation; Adult; Affect; Aggressive behavior; Alleles; BMP2 gene; BMP3 gene; Blindness; Blood Vessels; Cell Lineage; Cells; Cicatrix; Cornea; Corneal Diseases; Corneal Injury; Corneal Neovascularization; Data; Defect; Development; Dose; Ectoderm; Embryonic Development; Essential Genes; Eye; Future; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Models; Genetic Transcription; Goals; Grant; Growth; Human; Infection; Injury; Knock-out; Knockout Mice; Knowledge; Laboratories; Lead; Life; Ligands; Lymphatic vessel; Maintenance; Malignant Neoplasms; Mediating; Modeling; Mus; Mutation; Neural Crest; Outcome; Pathway interactions; Prevention therapy; Process; Repression; Research; Risk Factors; Role; Signal Transduction; Surface Ectoderm; TFAP2A gene; Testing; Thick; Tissues; Vision; World Health Organization; Wound Healing; cell type; corneal scar; design; healing; homeodomain; improved; induced pluripotent stem cell; innovation; insight; mutant; neovascularization; novel therapeutics; prevent; repaired; response; transcription factor; translational impact

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.

我们的目标是确定胚胎发育过程中控制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 在角膜功能上。作为实现这一目标的关键一步，我们的目标是确定PITX 2的基本功能， 角膜发育和伤口愈合。我们的中心假设是，激活Cited 2和 抑制Bmp 2和Bmp 3的表达是PITX 2调节角膜上皮细胞增殖的重要机制。 Pitx 2基因剂量的减少不利地影响成熟角膜中的伤口愈合。 我们的假设是基于数据表明Cited 2表达丢失，Bmp 2和 在缺乏PITX 2的情况下，发育中的角膜中Bmp 3表达升高，而成人角膜中Bmp 3表达升高 Pitx 2 +/-小鼠是浑浊的，并且在轻微损伤后存在新血管形成。的意义 拟议的研究是在正常发育中由PITX 2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理解，这一结果可能会影响我们对这些过程的理解。 未来有助于合理设计改进的治疗方法，以预防和治疗由于角膜炎引起的视力丧失， 疾病或损伤。我们将通过三个具体目标来检验我们的假设：1）检验Cited 2是一个 角膜发育过程中Pitx 2的重要下游效应物，2）检验PITX 2- 介导的Bmp 2和Bmp 3表达的抑制是角膜炎所需的另外的基本机制。 3）测试成熟角膜中伤口愈合对Pitx 2基因敏感的预测 次给药结束根据aim 1，申请人的实验室中已经可用的条件敲除策略将 用于在角膜发育过程中特异性消融眼神经嵴或表面外胚层中的Cited 2， 并使用成熟的方法评估角膜谱系和血管生长。在目标2下，a 条件策略将用于在眼神经嵴中遗传激活Bmp 2或Bmp 3表达， 角膜发育使用我们已经产生的小鼠，并产生的突变体将由 与目标1相同的标准。根据目标3，申请人开发的缺陷Pitx 2等位基因将用于 确定Pitx 2基因剂量对成熟角膜中伤口愈合和新血管形成的影响。的 预期的结果是将鉴定PITX 2在发育和成熟角膜中的基本功能。 PITX 2在发展和成熟过程中的基本功能， 角膜将被确定，包括第一次证明，Cited 2表达和BMP信号活性， 抑制是角膜发育和伤口愈合的基本要求。最终，这样的 知识可以为预防和治疗视力丧失的有效新疗法提供见解 因为角膜感染和受伤更广泛地说，这些结果也可能适用于理解 某些癌症中的新血管形成机制。