Abstract: The goal of this research project is to identify the signature genes that define the ontogenic niche for cardiac endocardial cells to generate the critical heart structures, valves and coronary arteries, using mouse genetics and single cell transcriptomics. Our previous studies showed that cardiac endocardial cells are the progenitor cells for heart valves and coronary arteries, arising from different cardiac regions and at different stages through distinct mechanisms. Endocardial cells at the cardiac outflow tract and atrioventricular canal give rise to heart valves, whereas those at the ventricle generate coronary arteries. These observations document that the endocardial cells are a heterogeneous population with distinct developmental fates and functions. However, the intrinsic factors temporally regulating the endocardial ontogenic niche to distinguish some cells from the others and to specify cell fate and function are currently unknown, due at least to the lack of experiment models and methods to isolate and interrogate individual endocardial cells. This research program will fill this critically missing knowledge gap using a synergistic approach of mouse genetic models, developmental heart anatomy, advanced single cell transcriptome technology, and systems biology to study the expression profiles of individual cells in order to understand cell heterogeneity and uncover subpopulations. The study will lead to the identification of potential regulators for cell fate determination and for initiating coronary angiogenesis, which can be further validated and functionally confirmed. We have generated genetic mouse models that allow us to isolate ventricular versus valve endocardial cells at different ontogenic stages. In this research program we will use single cell sorting and RNA-seq on these models to identify the genetic factors differentially expressed by subpopulations of endocardial cells at valve- or coronary-forming regions at different stages (Aim 1). We will then use integrated morphologic, cellular, and molecular analysis to reveal their potential functions in heart valve formation and coronary artery development (Aim 2). The new information will shed light on endocardial biology in heart development and disease.