Cardiac outflow tract formation requires the contribution of cell types from multiple embryological origins, and critically depends on the continued addition of cardiac progenitor cells from the anterior second heart field to ensure sufficient outflow tract growth. Neural crest cells are known to migrate into the outflow tract and later form structural components, such as the septum. Prior to entering the outflow tract, neural crest cells migrate in close apposition to the second heart field, and have historically been suggested to play important roles in regulating second heart field growth dynamics. However, the molecular mechanisms by which neural crest cells signal to the second heart field to control its growth have remained a critical unanswered question in the field.
We have discovered that removal of the ubiquitin ligase NEDD4 specifically in neural crest cells (Wnt1-Cre; Nedd4fl/fl) results in outflow tract defects reminiscent of those observed clinically (DORV, TGA). Lineage tracing reveals no deficiency of neural crest cells, however the second heart field exhibits premature cardiomyocyte differentiation, causing insufficient outflow tract lengthening. Laser capture microdissection and mRNAseq of the second heart field region revealed potential modulation of the Wnt signalling pathway, as well as an upregulation of the secreted Wnt signalling inhibitor DKK1. Spatial expression analysis revealed DKK1 is localised to the cardiac neural crest cells, and acts to inhibit canonical Wnt signalling in the adjacent second heart field. Modulation of Wnt signalling in vivo by injection of agonists/inhibitors into Wnt1-Cre; Nedd4fl/fl pregnant dams, or by genetic cross to a Dkk1+/- allele, revealed loss of NEDD4 in neural crest cells sensitises embryos to Wnt modulation. Furthermore, we discover DKK1 is a ubiquitinated target of NEDD4, and that a human congenital heart disease variant of NEDD4 (K1113R) has lost ability to ubiquitinate DKK1. We propose a new and unexpected role for neural crest cells as a rheostat of Wnt signalling in cardiac progenitors to influence the balance of progenitor maintence vs differentiation, identifying a new molecular pathway underpinning correct outflow tract morphogenesis, and new origins of congenital heart disease.