PROJECT SUMMARY Illuminating how the multi-layered ventricular wall is created entails investigating the earliest cellular events that control the allocation of cardiomyocytes between these wall layers. Because its complex vertebrate embryonic heart is comprised of 200-300 cardiomyocytes during cardiac chamber formation and furthermore is amenable to high resolution in vivo live-imaging, the zebrafish embryo offers a unique opportunity to interrogate in detail how individual cardiomyocytes dynamically organize to create the nascent structures of the embryonic ventricular wall. As a result, recent zebrafish studies have shown that distinct cardiomyocytes extend from the embryonic ventricular wall into the lumen to form cardiac trabeculae, whereas others remain within this outer wall. Yet, how these cardiomyocytes are selected to form these distinct ventricular myocardial wall layers still remains to be fully elucidated. Although previous studies have shown that endocardial Notch signaling non-cell autonomously promotes myocardial trabeculation through Erbb2 and BMP signaling, we have discovered a new and exciting role for myocardial Notch signaling within the ventricular myocardium to coordinate the sorting of cardiomyocytes between the ventricular wall layers. Because Notch signaling has been shown to control receptor tyrosine kinase (RTK) signaling through lateral inhibition pathways in order to regulate the social interactions between cells that assign cell fate position during epithelial branching, we hypothesize that a similar lateral inhibition mechanism between myocardial Notch and Erbb2 signaling may guide the selection, allocation and assembly of cardiomyocytes to morphologically create the distinct wall layers of the ventricular chamber. To further explore this hypothesis, we propose to: 1) investigate the underlying mechanisms regulating myocardial Notch signaling activity; 2) examine how myocardial Notch signaling regulates ventricular cardiomyocyte allocation; and 3) explore how the myocardial Notch-Erbb2 signaling axis regulates the dynamic cellular events guiding the morphogenesis of the ventricular wall layers.!