The Role of PINCH-1 in Regulating Cardiac Neural Crest Behavior

PINCH-1 在调节心脏神经嵴行为中的作用

• 批准号: 7074094
• 负责人: Jamie L. Lohr
• 金额: $ 7.48万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-12 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7074094
• 关键词: behavior; neural crest; role

DESCRIPTION (provided by applicant): Congenital heart defects are responsible for significant morbidity and mortality in infancy, and long-term survivors of surgical repair often have chronic medical and developmental disabilities. Approximately 1/3rd of congenital heart defects are related to abnormal formation of the outflow tract of the heart, a region that includes a portion of the ventricular septum and the great vessels. The normal formation of these structures is dependent on the presence and migration of cardiac neural crest (CMC) cells during development. We have isolated genes enriched in cardiac neural crest by comparing cardiac neural crest gene expression patterns to those of other axial levels of neural crest. A gene highly expressed in the CNC was identified as the LIM family member PINCH-1. We have determined that chick PINCH-1 is expressed in the forming neural folds and is downregulated at the time of neural crest cell migration. It is reexpressed in the branchial arches, cardiac outflow tract and myocardium later in development at a time when neural crest cells are populating these structures. Lastly, overexpression of PINCH-1 causes failure of cardiac neural crest cell migration in an in vitro assay system. Based on this data, we hypothesize that PINCH-1 is an important regulator of cardiac neural crest cell behavior, and is required for normal cardiac outflow tract development. To test this hypothesis, we propose the following Specific Aims: Aim 1: To determine if PINCH-1 regulates chick neural crest cell migration, proliferation or survival in an in vitro neural crest explant assay system; and Aim 2: To determine if PINCH-1 regulates the cardiac neural crest contribution to normal heart development in whole chick embryos. The role of PINCH-1 in neural crest cell behavior (Aim 1) will be investigated by altering PINCH-1 expression in cardiac neural crest explants using electroporation of an siRNA vector, an overexpression vector containing wild-type PINCH-1 DNA or an overexpression vector containing targeted mutations in PINCH-1 ILK and Nck-2 binding sites. The effect of alterations of PINCH-1 on cardiac development (Aim 2) will be performed by electroporating the same vectors as in Aim 1 into the cardiac neural crest of whole chick embryos and evaluating neural crest cell behavior and cardiac development. The work done in this application will be used as the basis for an R01 application to further study the biology of PINCH-1 and its binding partners in cardiac disease. This research is relevant to public health because congenital heart disease (CHD) effects 1% of live-born infants. CHD causes significant death and disability, and treatment incurs high costs, both financial and emotional. The goal of this project is to learn more about PINCH-1 and other genes involved in normal heart formation in order to develop methods of prevention or better treatment in the future.

描述（由申请人提供）：先天性心脏缺陷导致婴儿期的显着发病率和死亡率，而手术修复的长期幸存者通常患有慢性医学和发育障碍。大约1/3的先天性心脏缺陷与心脏流出道的异常形成有关，该区域包括部分室间隔和大血管。这些结构的正常形成取决于发育过程中心脏神经嵴 (CMC) 细胞的存在和迁移。通过将心脏神经嵴基因表达模式与神经嵴其他轴向水平的基因表达模式进行比较，我们分离出了心脏神经嵴中富集的基因。 CNC中高表达的基因被鉴定为LIM家族成员PINCH-1。我们已经确定小鸡 PINCH-1 在形成的神经褶皱中表达，并在神经嵴细胞迁移时下调。当神经嵴细胞填充这些结构时，它在发育后期的鳃弓、心脏流出道和心肌中重新表达。最后，PINCH-1 的过度表达导致体外测定系统中心脏神经嵴细胞迁移失败。基于这些数据，我们假设 PINCH-1 是心脏神经嵴细胞行为的重要调节因子，并且是正常心脏流出道发育所必需的。为了检验这一假设，我们提出以下具体目标： 目标 1：在体外神经嵴外植体测定系统中确定 PINCH-1 是否调节鸡神经嵴细胞迁移、增殖或存活；目标 2：确定 PINCH-1 是否调节心脏神经嵴对整个鸡胚正常心脏发育的贡献。将通过使用 siRNA 载体、包含野生型 PINCH-1 DNA 的过表达载体或包含 PINCH-1 ILK 和 Nck-2 结合位点的靶向突变的过表达载体的电穿孔改变心脏神经嵴外植体中的 PINCH-1 表达来研究 PINCH-1 在神经嵴细胞行为中的作用（目标 1）。 PINCH-1 的改变对心脏发育（目标 2）的影响将通过将与目标 1 中相同的载体电穿孔到整个鸡胚胎的心脏神经嵴中并评估神经嵴细胞行为和心脏发育来进行。该应用中完成的工作将用作 R01 应用的基础，以进一步研究 PINCH-1 及其结合伴侣在心脏病中的生物学。这项研究与公共卫生相关，因为 1% 的活产婴儿患有先天性心脏病 (CHD)。先心病会导致严重的死亡和残疾，治疗会带来高昂的经济和情感成本。该项目的目标是更多地了解 PINCH-1 和其他参与正常心脏形成的基因，以便在未来开发预防或更好的治疗方法。