Supplementary Figure 3: FISH validation for predicted FHP and SHP-specific markers.

(a) Violin plots and FISH validation of the expression for indicated predicted FHP and SHP-specific genes. Violin plots represent the distribution of the expression of indicated genes in defined cell clusters. Sample size of the violin plots (number of cells in each cell identity) is provided in Supplementary Table 6 (Data sheet: cell identity and number). mRNAs are visualized by whole mount fluorescent in situ hybridization (green). Nuclei of TVCs are marked by Mesp>nls::LacZ and revealed by anti-beta-galactosidase antibody (red). Mesp-driven hCD4::mCherry accumulates in the cell membrane and was revealed by anti-mCherry antibody (blue). Anterior to the left. Scale bar, 10 μm. Solid arrowheads, ASM; open arrowheads, SHPs; arrows, FHPs; M, midline (dotted line). The numbers of embryos showing the demonstrated gene expression pattern and numbers of the observed embryos are indicated at the right bottom corner of each image. (b) Dach and Tbx1/10 regulates the SHP fate specification in the juvenile heart (Unprocessed raw images of Fig. 7c). Dach and Tbx1/10 is required to limit the proportion of SHP-derived cells forming Mhc2+ cardiomyocytes in juveniles. Representative image of analysed embryos. Control^CRISPR, 4 embryos; Dach^CRISPR, 8 embryos; Tbx1/10^CRISPR, 6 embryos. Grey: Mhc2 mRNA visualized by in situ hybridization. SHP-derived cells are labelled with 3XT12>H2B::mCherry (green), B7.5 lineage cells are labelled with Mesp>nls::LacZ (red). Scale bar, 10µm.