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

描述(由申请人提供)：先天性心脏缺陷是婴儿期发病率和死亡率的主要原因，手术修复的长期幸存者通常患有慢性医学和发育障碍。约1/3的先天性心脏病与心脏流出道的异常形成有关，流出道包括部分室间隔和大血管。这些结构的正常形成依赖于发育过程中心脏神经脊(CMC)细胞的存在和迁移。我们通过比较心脏神经脊基因的表达模式与其他轴向水平的神经脊基因表达模式，分离出了心脏神经脊丰富的基因。其中一个在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，Chick Pinch-1在形成的神经褶中表达，并在神经脊细胞迁移时下调。在发育后期的颧弓、心流出道和心肌中，当神经脊细胞充填这些结构时，它重新表达。最后，在体外检测系统中，过表达PINCH-1会导致心脏神经脊细胞迁移失败。基于这一数据，我们假设PINCH-1是心脏神经脊细胞行为的重要调节因子，是正常心脏流出道发育所必需的。为了验证这一假说，我们提出了以下具体目标：目的1：确定在体外神经脊移植实验系统中，Pinch-1是否调控鸡神经脊细胞的迁移、增殖或存活；以及目标2：确定Pinch-1是否调控整个鸡胚胎心脏神经脊对正常心脏发育的贡献。通过电穿孔siRNA载体、含有野生型Pinch-1 DNA的过表达载体或含有Pinch-1 ILK和Nck-2结合位点靶向突变的过表达载体，研究Pinch-1在神经脊细胞行为中的作用(Aim 1)。PINCH-1基因的改变对心脏发育的影响(目标2)将通过将与目标1相同的载体电穿孔到整个鸡胚的心脏神经脊并评估神经脊细胞的行为和心脏发育来实现。在本申请中所做的工作将被用作R01应用的基础，以进一步研究PINCH-1及其结合伙伴在心脏病中的生物学。这项研究与公共卫生有关，因为先天性心脏病(CHD)影响了1%的活产婴儿。CHD会导致严重的死亡和残疾，而且治疗会产生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，以便开发未来的预防方法或更好的治疗方法。

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.