Essential functions of PITX2 in cornea, iris, and iridocorneal angle development

描述（由申请人提供）：我们的目标是确定控制眼前段发育的遗传网络，并了解这些网络的破坏如何导致眼前段结构和功能的缺陷。我们的目的是测试同位结构域转录因子PITX2在人类PITX2突变患者中最常受影响的前段组织中的功能。我们的中心假设是，PITX2调节指定正常角膜细胞命运所需的遗传网络，从发育和成熟的角膜中排除血管，调节虹膜和虹膜-角膜角结构内的细胞增殖和谱系规范。我们的假设是根据对颞叶基因敲除小鼠的分析产生的初步数据制定的。本研究的基本原理是了解PITX2在角膜、虹膜和虹膜-角膜角的正常发育中调控的遗传网络，将促进我们对前段发育不良和相关病理（包括眼压升高）的理解。我们将通过三个具体目标来验证我们的假设：1)验证Pitx2是规范和维持角膜细胞命运所必需的假设；2)验证Pitx2是阻止血管生长进入发育和成熟角膜所必需的假设；3)验证Pitx2是虹膜和虹膜-角膜角结构正常发育所必需的假设。在前两个目标下，申请人手中已经可行的颞叶敲除策略将用于在角膜发育初期切除Pitx2，之后将使用成熟的方法评估角膜谱系和血管生长。在第三个目标下，一种类似的颞部敲除方法，也在申请人的手中建立了可行性，将用于在虹膜和虹膜角膜角发育开始时切除Pitx2，并确定对结构发育的影响。预期的结果是确定PITX2在角膜、虹膜和虹膜-角膜角发育中的基本功能。该方法具有创新性，因为它利用时间基因敲除策略克服了全局和组织特异性Pitx2敲除动物的局限性，从而使我们能够研究前段重要的后期形成结构。提出的研究意义重大，因为它将垂直推进和扩大对发育过程中前段结构如何形成的理解。最终，这些知识将为前段发育不良如何导致视力丧失提供见解。

DESCRIPTION (provided by applicant): Our goal is to identify the genetic networks governing ocular anterior segment development and to understand how disruption of these networks leads to defects in anterior segment structure and function. Our objective is to test the functions of the homeodomain transcription factor PITX2 in anterior segment tissues that are most commonly affected in human patients with PITX2 mutations. Our central hypothesis is that PITX2 regulates genetic networks that are required to specify normal corneal cell fates, exclude blood vessels from the developing and mature cornea, and regulate cell proliferation and lineage specification within the iris and structures of the iridocorneal angle. Our hypothesis was formulated on the basis of preliminary data generated by analysis of temporal knockout mice. The rationale for the proposed research is that knowledge of the genetic networks regulated by PITX2 in normal development of the cornea, iris, and iridocorneal angle will advance our understanding of anterior segment dysgenesis and associated pathologies, including elevated intraocular pressure. We will test our hypothesis by pursuing three specific aims: 1) Test the hypothesis that Pitx2 is required for specification and maintenance of corneal cell fates, 2) Test the hypothesis that Pitx2 is required to prevent vascular growth into the developing and mature cornea, and 3) Test the hypothesis that Pitx2 is required for normal development of the iris and structures of the iridocorneal angle. Under the first two aims, a temporal knockout strategy, which is already feasible in the applicant's hands, will be used to ablate Pitx2 at the beginning of corneal development, after which corneal lineages and vascular growth will be assessed using well-established approaches. Under the third aim, an analogous temporal knockout approach, also established as feasible in the applicant's hands, will be used to ablate Pitx2 at the beginning of iris and iridocorneal angle development and the consequences on development of the structures will be determined. The expected outcome is that essential functions of PITX2 in the development of the cornea, iris, and iridocorneal angle will be identified. The approach is innovative because it utilizes a temporal gene knockout strategy to overcome the limitations of global and tissue-specific Pitx2 knockout animals, thereby permitting us to study important later-forming structures in the anterior segment. The proposed research is significant because it will vertically advance and expand understanding of how anterior segment structures are formed during development. Ultimately, such knowledge will provide insights into how anterior segment dysgenesis contributes to vision loss.