PROJECT SUMMARY The sinoatrial node (SAN) is a tiny structure consisting of a precise arrangement of specialized pacemaker cardiomyocytes (PCs) that trigger each heartbeat. Sinus node dysfunction (SND), resulting from loss or malfunction of PCs, is a common and morbid disease that is not well understood. There is currently no treatment that can delay or prevent SND, so sufferers must undergo permanent pacemaker implantation. This proposal will leverage novel tools and experimental approaches to determine how a multipotent population of SAN progenitor cells are allocated to different fates within the SAN, and to define how transcriptional hierarchies govern gene expression programs and spatial organization in the SAN. Ultimately, we hope that our findings will lead to new approaches to prevent or reverse SND by targeting the biological pathways that control SAN formation and maintenance. Previous efforts in this area have been hampered by the lack of specific genetic tools to mark the SAN progenitor population and the lack of an in vitro system that can model SAN development. In the past, our group has co- discovered a key activating role for the transcription factor Isl1 in SAN development and we have identified an Isl1 SAN Enhancer (ISE) that is specifically active in the SAN and its progenitor population. We have used this enhancer to generate a new mouse line, ISE-CreERT2, that allows conditional genetic modification of the SAN progenitor population. Clonal fate mapping with this mouse line and single cell sequencing analysis have defined the dynamics of the SAN progenitor population and allowed us to develop a system to explore how SAN progenitors are allocated among several possible fates. To gain further insight into how this fate allocation occurs, we developed a novel in vitro protocol using hiPSCs that recapitulates key aspects of SAN differentiation, including diversification of SAN progenitors into anatomically and functionally distinct pacemaker cardiomy