Project Summary/Abstract Congenital heart defects (CHDs) are the most common congenital malformations. However, the molecular etiology underlying most CHDs remain poorly understood. Furthermore, CHDs even following surgery can lead to complications later in life that result in arrhythmias, stroke, and premature death. In order to develop novel therapies able to prevent CHDs and target therapies to specific cardiovascular tissues, it is critical to garner understanding of fundamental mechanisms directing normal cardiac chamber development and regeneration. Therefore, long-term goals of our lab are to understand conserved mechanisms that direct the development of individual cardiac chambers and chamber-specific mechanisms utilized during regeneration in vertebrates. Few signals are known to be required that specifically direct atrial development, with specific regulators of atrial regeneration not being understood. The specific aims of this proposal are to elucidate the mechanisms by which a syntenic long non-coding RNA (lncRNA) family limits the expression of Nr2f transcription factors and decipher how Nr2f protein levels affect atrial heterogeneity during development and atrial regeneration in adult zebrafish. The studies in this proposal are relevant to human health as numerous genomic analyses now indicate that mutations in Nr2f2 are associated with CHDs, in particular ASDs in humans. While Nr2f2 knockout mice and in vitro studies with human stem cells have revealed requirements for both Nr2f1 and Nr2f2 in atrial development, the mechanisms by which Nr2f proteins direct proper atrial development are not completely understood. Importantly, there is currently no understanding of lncRNA-dependent mechanisms regulating Nr2f proteins. Our analysis of a lncRNA we call as-oca shows that in vivo it represses the translation of nr2f1a, the functional equivalent of mammalian Nr2f2. Moreover, we find that Nr2f1a levels regulate previously unrecognized heterogeneity of atrial cardiomyocytes in the embryonic atrium and atrial regeneration. In Aim 1, we will examine the specific mechanism that as-oca inhibits nr2f1a translation and the conservation of this mechanism among the NR2F-associated lncRNA family in human induced pluripotent stem cells. In Aim 2, we will examine the requirements of Nr2f1a and canonical Wnt signaling in generating atrial cardiomyocyte diversity and the transcriptional signature of a previously unrecognized atrial subpopulation. In Aim 3, we will examine the requirement of the epicardium in atrial regeneration and requirement of Nr2f1a within the atrial epicardium. Because Nr2f transcription factors play conserved roles in atrial development of all vertebrates, these studies will dramatically improve our understanding of post-transcriptional mechanisms regulating normal vertebrate atrial development and unique mechanisms employed during atrial regeneration. Ultimately, these studies will garner a foundation of knowledge that can be us...