Heart fields auto-regulate second heart field cell fate via Wnt secretion

The purpose of this study was to identify the source of Wnt signals that lead to development/proliferation of the heart fields, particularly the second heart field. To this end, we knocked out Wntless (Wls), which is necessary for Wnt secretion, in Mesp1+ lineage cells. We performed single cell RNA-seq to better identify the source of Wnt signals and the effects of the loss of Wnt secretion in mesoderm. We isolated Mesp1+ cells from Mesp1-Cre; Ai9; Wlsfl/+ and Wlsfl/fl mice at three developmental timepoints (E8.0, E8.5, and E9.5) and performed scRNA-seq. As separating control and mutant cells into separate capture runs can lead to potential downstream batch effects, we multiplexed and pooled samples using lipid-tagged indices through MULTI-seq. We subsequently performed fluorescence-activated cell sorting to isolate RFP+ cells, and generated sequencing libraries through the 10x Chromium platform.