PROJECT SUMMARY Organogenesis requires the execution of interwoven patterning processes that sculpt the distinct functional components of an organ with exquisite specificity. In the context of the embryonic heart, specific territories within each cardiac chamber take on unique attributes: for example, the pacemaker cells that reside within the atrial inflow tract (IFT) have particular conductive properties that are integral to their role in initiating the heartbeat. Cardiac pacemaking activity must be confined to a discrete region of the heart in order to avoid arrhythmia, but we do not yet fully understand the genetic pathways that define the dimensions of the IFT. How are an appropriate number of specialized cardiomyocytes established at the IFT? Prior studies have shown that IFT progenitor cells inhabit discrete outlying regions of the anterior lateral plate mesoderm (ALPM). Moreover, we have demonstrated that canonical Wnt signaling is active in these outlying regions and that the ligand Wnt5b acts to drive IFT differentiation. Thus, Wnt signaling plays a key role in promoting IFT development, but we do not yet understand how Wnt pathway activity is restricted to the edges of the ALPM. Here, we propose to utilize the suite of genetic and embryological approaches available in the zebrafish in order to identify essential patterning mechanisms that constrain IFT dimensions. Importantly, our preliminary studies suggest that the number of IFT cardiomyocytes is constrained through a two-phase process, with distinct signaling pathways operating at successive developmental stages. First, in the early embryo, we propose that Hedgehog (Hh) signaling restricts the allocation of progenitor cells into the IFT lineage. Later, in the ALPM, we propose that Fgf signaling reinforces constraints on the number of IFT cardiomyocytes by restricting the distribution of Wnt signaling. Together, our preliminary data highlight previously unappreciated roles for both Hh and Fgf signaling and suggest a novel model for the molecular mechanisms that restrict the size of the IFT. To test this model, we will employ loss- and gain-of-function analysis, fate mapping, and mosaic analysis in order to (1) determine whether Hedgehog signaling constrains specification of IFT progenitor cells and (2) ascertain whether Fgf signaling constrains differentiation of IFT cardiomyocytes. In addition, our model predicts that IFT progenitor cells possess distinct molecular characteristics prior to their overt differentiation into IFT cardiomyocytes. To test this, we will (3) define the developmental path of IFT progenitors by integrating spatial and transcriptomic data, thereby revealing how the signaling pathways that specify the IFT lineage set the stage for differentiation of the IFT myocardium. Taken together, our proposed studies will provide novel insight into the network of signaling pathways that control IFT dimensions, thereby illuminating new paradigms for the regulation of cardia...