Pitx2: Molecular mechanisms in eye development & disease

Pitx2：眼睛发育的分子机制

• 批准号: 6786578
• 负责人: PHILIP J GAGE
• 金额: $ 31.87万
• 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 2006-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6786578
• 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，在非洲裔美国人中居首位。由于眼前节虹膜和房水引流系统的缺陷而导致的眼内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由慢性高IOP的损伤引起的，但其潜在的分子机制尚不清楚。对于控制眼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分子分析遗传性眼病提供了理想的模型。在bicoid相关的同源框基因PITX 2中的突变导致Axenomy-Rieger综合征（ARS），一种常染色体显性疾病，导致先天性眼前节缺陷和青光眼。我已经从小鼠中克隆了Pitx 2，并使用基因靶向技术在小鼠中产生Pitx 2的等位基因系列（无效，亚型和条件）。无效等位基因的初步分析建立了比从人类表型预测的更广泛的Pitx 2在眼睛发育中的需求。纯合子突变体在视神经和眼睛后部有缺陷。该提案的目标是使用这些小鼠来分析Pitx 2在正常眼睛发育中的基本功能机制，并确定Pitx 2活性部分或完全丧失的分子后果。我们将评估的假设，Pitx 2在眼神经嵴和中胚层谱系有不同的功能。在使用二元转基因系统标记每个细胞谱系后，将确定每个谱系中的Pitx 2表达。这些标记系统也将用于比较野生型和Pitx 2-/-眼睛中每个谱系的命运，并确定需要Pitx 2的遗传机制。ARS由改变的PITX 2剂量引起，表明眼睛发育中的某些步骤对不同的PITX 2蛋白水平高度敏感。我们将使用无效等位基因和亚纯型等位基因改变Pitx 2基因剂量，以确定PITX 2水平变化最多的特定发育功能，并确定潜在的分子机制。最后，我们将使用嵌合小鼠来挽救Pitx 2-/-小鼠的致死性，以分析Pitx 2在眼睛发育后期的功能。这种多方面的方法应该提供具体的机制细节Pitx 2在眼睛发育中的多种功能，也有望提供更一般的眼周间充质发育的基本机制的见解。这些基本信息对于了解眼疾至关重要。