Pitx2: Molecular mechanisms in eye development & disease

Pitx2：眼睛发育的分子机制

• 批准号: 6652638
• 负责人: PHILIP J GAGE
• 金额: $ 31.89万
• 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 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6652638
• 关键词: developmental disease /disorder; developmental genetics; epidermal growth factor; eye; eye disorder; fluorescence; functional /structural genomics; gene dosage; gene expression; gene mutation; gene targeting; genetic mapping; genetically modified animals; growth factor receptors; homeobox genes; in situ hybridization; laboratory mouse; mesoderm; molecular cloning; neural crest; regulatory gene; syndrome; terminal nick end labeling; transcription factor

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.

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。眼前段虹膜和房水引流系统缺陷引起的眼压升高常常伴随青光眼。导致青光眼的视网膜神经节细胞死亡显然是慢性高眼压的结果，但其潜在的分子机制尚不清楚。控制眼前段结构正常发育的遗传调控网络也鲜为人知。小鼠为确定促进眼睛正常发育的基本机制和对遗传性眼病进行分子分析提供了理想的模型。双基因相关同源框基因PITX2的突变导致了阿森菲尔德-里格综合征(ARS)，这是一种常染色体显性疾病，导致先天性眼前节缺陷和青光眼。我已经从小鼠身上克隆了Pitx2，并使用基因打靶技术在小鼠身上生成了Pitx2的等位基因序列(零、亚型和条件)。对空等位基因的初步分析确定了在眼睛发育中对Pitx2的需求比从人类表型预测的更广泛。纯合子突变的人在视神经和眼睛后部有缺陷。这项建议的目的是利用这些小鼠来分析在正常眼睛发育中Pitx2功能的基本机制，并确定Pitx2活性部分或完全丧失的分子后果。我们将评估Pitx2在眼神经脊和中胚层谱系中具有不同功能的假设。在使用二元转基因系统标记每个细胞谱系后，将确定每个谱系中Pitx2的表达。这些标记系统也将被用来比较野生型和Pitx2-/-眼的每个血统的命运，并确定需要Pitx2的遗传机制。ARS的结果是改变了PITX2的剂量，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亚型等位基因来改变PITX2基因的剂量，以确定对PITX2水平变化最敏感的特定发育功能，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-/-小鼠的致死性，以便稍后分析Pitx2在眼睛发育中的功能。这种多方面的方法应该提供关于Pitx2在眼睛发育中的多种功能的具体机制细节，也有望为眼周间充质在发育中的更一般的基本机制提供洞察力。这些基本信息对于了解眼病是必不可少的。