Nr2f-dependent allocation of ventricular cardiomyocyte and pharyngeal muscle progenitors

Multiple syndromes share congenital heart and craniofacial muscle defects, indicating there is an intimate relationship between the adjacent cardiac and pharyngeal muscle (PM) progenitor fields. However, mechanisms that direct antagonistic lineage decisions of the cardiac and PM progenitors within the anterior mesoderm of vertebrates are not understood. Here, we identify that retinoic acid (RA) signaling directly promotes the expression of the transcription factor Nr2f1a within the anterior lateral plate mesoderm. Using zebrafish nr2f1a and nr2f2 mutants, we find that Nr2f1a and Nr2f2 have redundant requirements restricting ventricular cardiomyocyte (CM) number and promoting development of the posterior PMs. Cre-mediated genetic lineage tracing in nr2f1a; nr2f2 double mutants reveals that tcf21(+) progenitor cells, which can give rise to ventricular CMs and PM, more frequently become ventricular CMs potentially at the expense of posterior PMs in nr2f1a; nr2f2 mutants. Our studies reveal insights into the molecular etiology that may underlie developmental syndromes that share heart, neck and facial defects as well as the phenotypic variability of congenital heart defects associated with NR2F mutations in humans. Author summary Many developmental syndromes include both congenital heart and craniofacial defects, necessitating a better understanding of the mechanisms underlying the correlation of these defects. During early vertebrate development, cardiac and pharyngeal muscle cells originate from adjacent, partially overlapping progenitor fields within the anterior mesoderm. However, signals that allocate the cells from the adjacent cardiac and pharyngeal muscle progenitor fields are not understood. Mutations in the gene NR2F2 are associated with variable types of congenital heart defects in humans. Our recent work demonstrates that zebrafish Nr2f1a is the functional equivalent to Nr2f2 in mammals and promotes atrial development. Here, we identify that zebrafish nr2f1a and nr2f2 have redundant requirements at earlier stages of development than nr2f1a alone to restrict the number of ventricular CMs in the heart and promote posterior pharyngeal muscle development. Therefore, we have identified an antagonistic mechanism that is necessary to generate the proper number of cardiac and pharyngeal muscle progenitors in vertebrates. These studies provide evidence to help explain the variability of congenital heart defects from NR2F2 mutations in humans and a novel molecular framework for understanding developmental syndromes with heart and craniofacial defects.