Fig. 2.

SHF development is dysregulated through decreased proliferation without Mesp1⁺ Wnts. (A) UMAP clustering based on cell type. (B) Expression of Wnts across cell populations. (C ) Quantification of differentially expressed genes (DEGs) across time points and cell populations. (D) aSHF developmental trajectories from control and knockout embryos and heatmaps identifying differential gene trends with expression dynamics of cell cycle and cardiomyogenesis genes. (E ) Quantification of percentage of Mesp1⁺/Tbx5⁻ cells undergoing proliferation. White boxes highlight the Tbx5-positive regions which were excluded from quantification. Control average = 2.77%. Knockout average = 1.71%. *P = 0.039. (F ) Differentially expressed ligand–receptor pairs in knockout embryos in the aSHF. (G) Frontal (Top) and lateral (Bottom) views of whole-mount in situ hybridization of candidate Wnts with Tbx5 cohybridization in 4ss embryos (HF, head fold; CC, cardiac crescent; A, anterior; P, posterior). The white scale bar indicates 500 μM. Auto fluorescent debris is noted in Wnt5a/5b/11 embryos. (H ) Flow cytometry quantifications of Hcn4-GFP; Tbx1-RFP-positive cells at day 7 of differentiation after IWP-2 and IWP-2+Wnt2 treatment. IWP-2 %GFP⁺ average = 9%; %RFP⁺ average = 20.44%. IWP-2+Wnt2 %GFP⁺ average = 8.61%; %RFP⁺ average = 27.43%. GFP⁺ cells P = 0.86 (NS = not significant); RFP⁺ cells *P = 0.016.