Pitx2: Molecular mechanisms in eye development & disease

描述（由申请人提供）：青光眼是美国第二大致盲原因，在非裔美国人中排名第一。青光眼常伴随虹膜缺损及前段房水引流系统的眼压升高。视网膜神经节细胞死亡导致青光眼显然是由于长期高IOP的损伤，但其潜在的分子机制尚不清楚。控制前节结构正常发育的遗传调控网络也知之甚少。小鼠为确定正常眼睛发育的基本机制和遗传眼病的分子分析提供了理想的模型。双头体相关的同型框基因PITX2突变可导致Axenfeld-Rieger综合征（ARS），这是一种常染色体显性遗传病，可导致先天性前段缺损和青光眼。我从小鼠中克隆了Pitx2，并使用基因靶向为小鼠中的Pitx2生成等位基因系列（null， hypoomorphic和conditional）。对空等位基因的初步分析表明，Pitx2在眼睛发育中的需求比人类表型预测的更为广泛。纯合子突变体在视神经和眼睛后部有缺陷。本研究的目的是利用这些小鼠分析Pitx2功能在正常眼睛发育中的基本机制，并确定Pitx2活性部分或完全丧失的分子后果。我们将评估Pitx2在眼神经嵴和中胚层谱系中具有不同功能的假设。使用二元转基因系统标记每个细胞系后，将确定每个细胞系中Pitx2的表达。这些标记系统还将用于比较每个谱系在野生型和Pitx2-/-眼中的命运，并确定需要Pitx2的遗传机制。ARS是由Pitx2剂量改变引起的，这表明眼睛发育的某些步骤对不同的Pitx2蛋白水平高度敏感。我们将使用空等位基因和次胚等位基因来改变Pitx2基因剂量，以确定与Pitx2水平变化最相关的特定发育功能，并确定潜在的分子机制。最后，我们将使用嵌合小鼠来挽救Pitx2-/-小鼠的致病性，以便分析Pitx2在后期眼睛发育中的功能。这种多方面的研究方法将提供Pitx2在眼睛发育中多种功能的具体机制细节，并有望为眼周间充质在发育中的更普遍的基本机制提供见解。这些基本信息对于了解眼病是必不可少的。

DESCRIPTION (provided by applicant): Glaucoma is the second leading cause of blindness overall in the United States and first among African Americans. Elevated intraocular pressure from defects in the iris and aqueous humor drainage system in the anterior segment frequently accompanies glaucoma. Retinal ganglion cell death leading to glaucoma apparently results from the insult of chronically high IOP but the underlying molecular mechanisms are not understood. The genetic regulatory networks governing normal development of anterior segment structures are also poorly understood. Mice provide ideal models for determining the basic mechanisms contributing to normal eye development and for molecularly analyzing genetic eye disease. Mutations in the bicoid-related homeobox gene PITX2 result in Axenfeld-Rieger Syndrome (ARS), an autosomal dominant disease resulting in congenital anterior segment defects and glaucoma. I have cloned Pitx2 from mice and used gene targeting to generate an allelic series (null, hypomorphic, and conditional) for Pitx2 in mice. Initial analysis of the null allele established a more widespread requirement for Pitx2 in eye development than predicted from the human phenotype. Homzygotes mutants have defects in the optic nerve and posterior parts of the eye. The goal of this proposal is to use these mice to analyze the basic mechanisms of Pitx2 function in normal eye development and to identify the molecular consequences of partial or complete loss of Pitx2 activity. We will assess the hypothesis that Pitx2 has distinct functions in the ocular neural crest and mesoderm lineages. Pitx2 expression in each lineage will be determined after using binary transgenic systems to mark each cell lineage. These marking systems will also be used to compare the fates of each lineage in wild type and Pitx2-/- eyes, and determine the genetic mechanisms that require Pitx2. ARS results from altered Pitx2 dosage, indicating certain steps in eye development are highly sensitive to varied PITX2 protein levels. We will vary Pitx2 gene dosage using the null and hypomorphic alleles to identify the specific developmental functions that are most to variations in PITX2 levels and determine the underlying molecular mechanisms. Finally, we will use chimeric mice to rescue the lethality of Pitx2-/- mice in order to analyze Pitx2 functions later in eye development. This multifaceted approach should provide specific mechanistic details about the multiple functions of Pitx2 in eye development and also promises to provide insight into more general fundamental mechanisms of periocular mesenchyme in development. This basic information is essential for understanding eye disease.