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. ? ? ?
