This laboratory's research program is focused on the genetics and cell and molecular biology of cardiovascular development. An integration of whole animal imaging, microscopy, and genomic and proteomic approaches are being utilized to elucidate the cell signaling pathways that regulate cardiovascular development. One project entails studies aimed at understanding the role of two extracardiac cell populations, the cardiac neural crest and the proepicardially derived cells, in the modulation of cardiovascular development and function. These studies will help elucidate cellular and molecular mechanisms that regulate outflow tract morphogenesis and coronary artery development. An integration of cell biological and molecular approaches together with animal imaging studies with ultrasound and episcopic fluorescence image capture are used for these studies. In addition, we have recently incorporated the use of microarray analysis for gene expression profiling. Such studies have revealed an essential role for connexin43 gap junctions in coronary vascular patterning and remodeling. A related second area of research interest is elucidating the cell signaling function of connexin43 gap junction protein, and gain insight into its role in modulating proepicardial and neural crest cell migration. Present studies are focused on identifying protein-protein interactions essential in this novel connexin mediated cell signaling function. These studies entail the use of transgenic animal models and cultured cells together with proteomic methods, including mass spectrometry, to identify the interacting proteins. A third project involves the use of a discovery approach to identify novel genes essential for mammalian cardiovascular development. Noninvasive prenatal ultrasound imaging is being used to screen ENU mutagenized mice for congenital cardiovascular defects. Nearly 9,000 mouse fetuses have been ultrasound scanned thus far from over 300 ENU mutagenized families. More then 150 of these families were identified with congenital heart defects and together they show phenotypes that include all of the major congenital heart defects seen clinically. Heritability testing and genome scans with microsatellite DNA markers are under way to map the mutations and identify candidate genes. The ENU induced mutation in 8 families have been mapped, three of which have been identified - one is in semaphorin 3C, another in connexin43, and a third in p53 binding protein, Trp53bp1. Studies are underway to identify the mutation in the other 5 families, even as more mutations are being recovered through the screen. These studies will help to identify novel genes and reveal novel gene functions essential for cardiovascular development and function. A fourth project entails using a genotype based screen of EMS mutagenized embryonic stem cells to generate new mouse models harboring connexin gene mutations have been completed. Over a dozen connexin mutations have been identified, and ES cell injections show 2 of 4 mutant clones provided germ line transmission. A fifth project entails the development of episcopic fluorescence image capture (EFIC) for phenotyping mouse cardiovascular development using 3 dimensional (3D) histological reconstructions. Using EFIC, we have created an interactive web based mouse cardiovascular development atlas that details mouse cardiovascular morphogenesis from E9.5 to birth using 2D image stacks and 3D reconstructions. In addition, in this same atlas, we have detailed the cardiovascular defects in a number of the new mutant mouse models recovered in our ENU mutagenesis screen. This Atlas is soon to be released in published form on line and in print. Through this high resolution and high throughput 3D imaging tool, we hope to gain further insights into the developmental defects underlying the congenital heart malformations exhibited in our novel mutant mouse models.
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