Cardiac progenitors instruct second heart field fate through Wnts

The Wnt signalling pathway has multiple, well-characterised roles in mammalian heart development (1). One of these roles is in the second heart field (SHF), where Wnt signalling is required to maintain proliferation (ref). However, since there are 19 Wnt ligands (2), and these secreted molecules are capable of diffusing some distance from their source, the identity and origin of the Wnt ligands required for SHF development are not well established. In this paper, Miyamoto and colleagues use an elegant approach to address this problem. Secretion of Wnt ligands requires the Wntless (Wls) protein (2), thus conditional deletion of the Wls gene in different regions of the developing embryos can be used to address the origin of Wnt ligands. Broad deletion of Wls in precardiac mesoderm using Mesp1-Cre resulted in a lack of right ventricle and outflow tract structures at embryonic day (E) 9.5, conforming an essential role for Wnt signalling in SHF development. Likewise, deletion using Isl1-Cre (expressed in both FHF and SHF) gave the same phenotype, confirming that Wnt ligands expressed in the cardiac progenitors are required for SHF development. However, deletion in just the anterior SHF (which contributes cells to the RV and OFT), using Tbx1-Cre, did not lead to cardiac defects. This suggests that the source of the Wnt must be outside the anterior SHF.

To investigate further, the authors performed scRNASeq of Mesp1+ cells to compare the effects of Wls deletion at W8.0. E8.5 and E9.5. After unsupervised clustering, they examined Wnt ligand expression levels. In normal cells, only the FHF cells expressed appreciable levels of Wnt ligands, in particular Wnt2. They then analysed the effects of Wls deletion on all clusters, using trajectory analysis comparing the progression from E8.0 to E9.5. This pointed to decreased cell proliferation in the aSHF as a key mechanism for the knockout heart phenotype, which they then verified in vivo. This supports previous work using ß-catenin knockout in the anterior SHF (3). Next, they used basic ligand-receptor analysis of the scRNASeq data to identify Fzd4 as the most likely receptor in the anterior SHF. Finally, they validated that Wnt2-Fzd signalling was able to stimulate SHF proliferation in an in vitro precardiac organoid system.

This paper provides further evidence for a key role of canonical Wnt signalling in maintining the anterior SHF cells in a proliferative state. Surprisingly, the source of the Wnt signal is likely to be the adjacent FHF cells, which have already begun the differentiation process into cardiomyocytes.