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 is aimed at understanding the role of connexins in the modulation of mouse cardiovascular development. An integration of cell biological and molecular approaches are used for these studies. We recently found a surprising role for connexin43 gap junctions in modulating stem cell survival, including survival of primordial germ cell and hematopoietic stem cells. These studies have led to the hypothesis of a role for hematopoeitic cells in supporting embryonic myocardial development. A related second area of research interest is in elucidating the cell signaling function of connexin43 in modulating cardiac neural crest and epicardial cell motile behavior. These studies have revealed an unexpected role for connexin43 in modulating the actin and tubulin cytoskeleton. These findings suggest a possible mechanism through which connexins may affect cell signaling function independent of the channel. Our findings suggest such cell signaling function could have an important role in the regulation of cell proliferation and cell survival. A third project involves the use of a discovery approach to identify novel genes essential for mammalian cardiovascular development. Noninvasive prenatal ultrasound imaging has been used to screen ENU mutagenized mice for congenital cardiovascular defects. Nearly 13,000 mouse fetuses have been ultrasound scanned from over 450 ENU mutagenized families. Mutants have been recovered exhibiting phenotypes that include all of the major congenital heart defects seen clinically. ENU induced mutation in 15 families have been mapped, three of which have been identified - one being semaphorin 3C, another in connexin43, and a third in p53 binding protein, Trp53bp1. Surprisingly, 7 of the 15 mutant families show heart and limb anomalies. These findings suggest conservation of shared cell signaling pathways that modulate both cardiovascular and limb morphogenesis. We are now embarking on the use of microarray analysis to elucidate the gene regulatory networks that modulate heart/limb development. Together these studies will help to elucidate the genetic pathways that play a critical role in congenital heart disease. A fourth project entails the development of ultrasound phenotyping approaches for the analysis of cardiovascular structure and function in mouse models. These studies have entailed the use of ultra-high frequency ultrasound instrumentation for imaging both fetal and adult mice. Through these studies we have developed a large database of cardiac and hemodynamic measurements for tracking mouse cardiovascular function. Through collaborative studies of the KLF2 KO mouse model, we recently obtained surprising evidence that vascular tone in the embryo is modulated from embryonic day 11.5, which is prior to septation of the outflow tract and ventricles. This modulation of vascular tone requires KLF2 function and is essential for survival of the embryo. 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. 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. In addition, a cryo EFIC imaging system is being developed, that would allow the integration of EFIC imaging with the recovery of tissue samples for RNA expressioni profiling. This would provide the means to integrate gene expression patterns from microarray analysis with 2D and 3D reconstructed images of the embryo. A new effort is also underway using MRI and EFIC imaging to develop an Altas of the Human Embryo from Carnegie Stage 13 to stage 23. This Atlas will provide details of development of all of the major organ systems. The analysis of the normal human embryos will be followed by analysis of abnormal human embryos with a variety of congenital defects. The latter studies will complement data obtained from the mouse ENU mutagenesis screen. Such mouse/human comparisons will provide novel insights into the underlying etiology of human congenital malformations.
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