High-Resolution Studies Link Vascular Endothelial Growth Factor Signaling with Endocardial-Myocardial Dynamics Controlling Heart Ventricle Development
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Determining how coordinated gene expression changes direct embryonic heart development is paramount to understanding the genetic causes and developmental origins of congenital cardiomyopathies. Towards this goal, I present an optimized protocol for mouse thiouracil tagging (TU-tagging), a novel transcriptomics methodology for defining dynamic and cell specific gene expression programs, and validate TU-tagging for cardiovascular research. I then apply related and additional high-resolution approaches to characterize how vascular endothelial growth factor (VEGF) signaling coordinates cell and gene expression dynamics underlying heart ventricle development. TU-tagging is a powerful approach to study dynamic gene expression programs of defined cell types while they are natively embedded within a complex organ. TU-tagging integrates genetic and chemical approaches to provide temporally controlled in vivo covalent labeling of cell type–specific RNA. Here, I describe two significant optimizations of the TU-tagging molecular biology and bioinformatics workflows that improve the method’s ability to identify differentially expressed genes and expand the technology’s utility. Next, using chemical inhibition of VEGF signaling in combination with high-resolution imaging and transcriptomic methods, I show that VEGF signaling directly promotes formation of the trabeculae that uniquely develop in the heart ventricle. By RNA-Seq, I identify VEGF-dependent target genes, including Gpr126 and Bmp10, which encode additional signaling proteins. I further show that myocardial Bmp10 expression and resulting endocardial Bmp10-driven pSMAD1/5/8 signaling is under sustained control by endocardial VEGF signaling. This continuous VEGF-BMP signaling interplay between endocardial and myocardial cells led me to examine the dynamic tissue arrangements between the two cell types during early stages of trabeculation. By extensive staining and high resolution imaging, I show that endocardial cells can be subdivided into three classes; 1) quiescent cavity cells that are well-separated from the myocardium, 2) proliferative and signal responsive transition zone/stalk cells that directly interact with myocardium to coordinate both cell types’ activities, and 3) CD34-expressing migratory tip-like cells uniquely found at the base of forming trabeculae. VEGF promotes trabeculation by 1) driving proliferation of endocardial transition zone/stalk cells and, secondarily, neighboring myocardium, and 2) directing the outward migration of endocardial tip cells that causes myocardial tissue to accumulate within individual and extensive ridge-like trabeculae. Defining these multiple roles of VEGF signaling during ventricle development reveals a novel conceptual framework for understanding trabeculation mechanisms and therefore processes likely to be disrupted in common congenital cardiomyopathies. This dissertation includes previously published and unpublished co-authored material.