Molecular Mechanisms and Environmental Influences Underlying Heart Development

Heart development is an intricate process that involves complex morphological events controlled by transcriptional/signaling networks. Any alterations in these processes can result in congenital heart defects (CHDs), the most common structural birth defect in the world. While some genetic risk factors for CHDs have been identified, there is evidence that various environmental factors influence heart development, including maternal diet during pregnancy. The relationship between the genetic and environmental factors on heart development remains poorly understood.;CHDs can be caused by improper establishment of the left-right (L-R) body axis. Leftward fluid flow in the mouse node generated by cilia is critical for L-R axis asymmetry and alteration in this process leads to situs inversus. The mouse model for Coiled-coil domain containing-40 ( Ccdc40) deficiency display altered motile cilia and abnormal establishment of the L-R axis, yet the underlying molecular mechanism is unknown. Ccdc40lnks mutants show delayed induction of markers of the left-lateral plate mesoderm (L-LPM) including Lefty1, Lefty2 and Nodal. Upstream of this altered of L-LPM expression, perinodal expression of Cerberus like-2 (Cerl2) and Nodal is delayed than randomized. Thus, I propose the model that the defective motile cilia of Ccdc40lnks mutants results in symmetric Cerl2 expression and therefore Nodal is antagonized equally on both sides of the node. This effectively reduces Nodal activation bilaterally, leading to reduced and delayed activation of Nodal and its antagonists in the LPM. Further reduction in Nodal gene dosage in these Ccdc40lnks mutants results in failure to establish Nodal expression in the L-LPM, causing a predominance of right not left isomerism. Together these results suggest a model in which cilia generated fluid flow in the node functions to ensure robust Nodal activation and a timely left sided developmental program in the LPM.;Another vital signaling pathway in heart development is controlled by retinoic acid (RA) signaling. RA is required throughout heart development; specifically, it is vital for proper secondary heart field (SHF), artery and ventricle formation. Hectd1opm/opm mutants display embryonic lethal phenotypes, including peripheral edema, indicating cardiac failure. Further analysis of Hectd1opm/opm mutants show alignment defects of the arterial pole of the heart, including double outlet right ventricle (DORV) and ventricular septal defects (VSD), along with malformations of the great artery network. These defects are preceded by alterations in SHF patterning and pharyngeal arch artery (PAA) formation, respectively. These types of CHDs are consistent with decreased RA signaling, which is demonstrated in vivo in Hectd1opm/opm mutants through decreased activation of a retinoic acid response element (RARE) reporter. This supports previous unpublished work from the Zohn laboratory that Hectd1 binds to and ubiquitinates the retinoic acid receptor alpha (RARalpha), therefore providing evidence that Hectd1 positively regulates RA signaling. Interestingly, Hectd1opm heterozygous mutants (Hectd1opm/+) do not display any changes in SHF patterning or PAA formation, but reductions in RA exposure either genetically or through alteration of maternal diet cause CHD phenotypes.;The mutation of Hectd1 also affects the developing ventricles, as demonstrated by reductions in the compact myocardium, ventricle size and proliferation. The epicardium plays a major role in ventricle growth, acting as a signaling center to induce proliferation of the adjacent myocardium through pathways like RA signaling. The epicardium forms normally in Hectd1 opm/opm mutants and Hectd1 is highly expressed in the epicardium, indicating that it may be a player in epicardial signal transduction. Importantly, decreased RARE activation is found in the epicardium of Hectd1 opm/opm mutants along with decreased expression of downstream targets of RA signaling that promote proliferation of the adjacent myocardium. As in artery development, reduction of RA exposure in Hectd1opm/+ mutants through feeding dams a vitamin A deficient (VAD) diet causes a noncompact myocardium phenotype and further exacerbates the phenotype in Hectd1opm/opm mutants. Finally, conditional loss and reactivation of Hectd1 expression in various heart-related lineages indicates that Hectd1 is required in the heart and placenta during ventricle development, supporting a newly proposed model for the role of extracardiac organs in promoting epicardial signaling required for ventricle development. Overall, these results identify Hectd1 is a novel CHD gene and a novel regulator of RA signaling during heart development. I have also established a model of genetic and environmental influences on heart development by looking at Hectd1 and Vitamin A deficiency (VAD), providing further understanding of this complex relationship.