Cardiac progenitors auto-regulate second heart field cell fate via Wnt secretion

Proper heart formation requires coordinated development of two anatomically distinct groups of cells - the first and second heart fields (FHF and SHF). Given that congenital heart defects are often restricted to derivatives of the FHF or SHF, it is crucial to understand the mechanisms controlling their development. Wnt signaling has previously been implicated in SHF proliferation; however, the source of Wnts remains unknown. Through comparative gene analysis, we found upregulation of Wnts and Wnt receptor/target genes in the FHF and SHF, respectively, raising the possibility that early cardiac progenitors may secrete Wnts to influence SHF cell fate. To probe this further, we deletedWntless (Wls), a gene required for Wnt ligand secretion, in various populations of precardiac cells. Deletion of Wls in Mesp1+cells resulted in formation of a single chamber heart with left ventricle identity, implying compromised SHF development. This phenotype was recapitulated by deleting Wls in cells expressing Islet1, a pan-cardiac marker. Similarly, Wls deletion in cells expressing Nkx2.5, a later-expressed pan-cardiac marker, resulted in hypoplastic right ventricle, a structure derived from the SHF. However, no developmental defects were observed when deleting Wls in SHF progenitors. To gain mechanistic insights, we isolated Mesp1-lineage cells from developing embryos and performed single-cell RNA-sequencing. Our comprehensive single cell transcriptome analysis revealed that Wls deletion dysregulates developmental trajectories of both anterior and posterior SHF cells, marked by impaired proliferation and premature differentiation. Together, these results demonstrate a critical role of local precardiac mesodermal Wnts in SHF fate decision, providing fundamental insights into understanding heart field development and chamber formation.Significance StatementThere is significant interest in understanding the mechanisms underlying heart formation to develop treatments and cures for patients suffering from congenital heart disease. In particular, we were interested in the intricacies of first (FHF) and second heart field (SHF) development, as many congenital heart defects present with heart field-specific etiologies. Here, we uncovered a novel relationship between specified cardiac progenitor cells and second heart field progenitors. Through genetic manipulation of Wnt secretion in developing mouse embryos, we identified a population of cardiac progenitor cells that acts as a local source of Wnts which are necessary for proper SHF development. Our single cell transcriptomic analysis of developing anterior mesoderm showed cardiac progenitor-secreted Wnts function through regulation of differentiation and proliferation among SHF progenitors. Thus, this study provides insight into the source and timing of Wnts required for SHF development, and points to the crucial role of co-developing cell populations in heart development.