Retinoic acid promotes second heart field addition and regulates ventral aorta patterning in zebrafish

Retinoic acid (RA) signaling is used reiteratively during vertebrate heart development. Its earliest known role is to restrict formation of the earlier-differentiating first heart field (FHF) progenitors, while promoting the differentiation of second heart field (SHF) progenitors that give rise to the arterial pole of the ventricle and outflow tract (OFT). However, requirements for RA signaling at later stages of cardiogenesis remain poorly understood. Here, we investigated the role of RA signaling after the later differentiating SHF cells have begun to add to the OFT. We found that inhibiting RA production in zebrafish beginning at 26 hours post fertilization (hpf) produced embryos that have smaller ventricles with fewer ventricular cardiomyocytes, and reduced number of smooth muscle cells in the bulbus arteriosus (BA) of the OFT. Our results suggest that the deficiency of the ventricular cardiomyocytes is due to reduced SHF addition to the arterial pole. In contrast to smaller ventricles and BA, later RA deficiency also results in a dramatically elongated posterior branch of the adjacent ventral aorta, which is surrounded by an increased number of smooth muscle cells. Altogether, our results reveal that RA signaling is required during the period of SHF addition to promote addition of ventricular cardiomyocytes, partition smooth muscle cells onto the BA and posterior ventral aorta, and to establish proper ventral aorta anterior-posterior patterning.