Extended Data Fig. 6: Alx3^Pos cells are a distinct subset of the AHF population.

(A) Representative images from RNA in situ hybridization for Armh4 (green) and Alx3 (red) in an E10.5 embryo from VEH treated female. The scale bar represents 500 µm. (B) Representative images from whole mount RNA in situ hybridization of E10.5 embryos using light sheet microscopy. Armh4 (green) and Alx3 (red) expression is shown from the dorsal view (D – left) and the right oblique view (O – right). A white bracket (left) highlights the anterior part of Alx3^Pos cells. A white dotted oval (right) highlights the Alx3^Pos cell streak on left side of the embryo from outflow tract (OFT) towards the posterolateral region. Still images were extracted from Supplementary video 2. Scale bar represents 100 µm. PA2, pharyngeal arch 2; BW, body wall. (C) The distribution of Alx3 positive cells by scRNA-seq between E7.75 and E9.25. (D) scRNA-seq UMAP of cardiac progenitor cells at E9.25 from the same data as (C) color coded by cell type annotation. AHF, anterior heart field; BM progenitors, branchiomeric muscle progenitors; pSHF, posterior second heart field. (E) Expression of Alx3 on the same UMAP as (D). (F) Heatmap of differentially expressed genes (DEGs) between Alx3^Neg AHF and Alx3^Pos AHF at E9.25. All detected DEGs that attained adjusted p-val < 0.05 and Log₂FC > 0.25 are shown. Top GO terms enriched in upregulated or downregulated DEGs are shown with representative genes composing each GO (Fisher’s exact test, corrected for multiple testing using the Benjamini-Hochberg method). Scale bar indicates z-scored expression values. (G) Heatmap presentation of DEGs between AHF1 and AHF2 at E10.5 using only VEH cells in the scRNA-seq data. All detected DEGs that attained adjusted p-val < 0.05 and Log₂FC > 0.25 are shown. Top GO terms enriched in upregulated or downregulated DEGs are shown with representative genes composing each GO (Fisher’s exact test, corrected for multiple testing using the Benjamini-Hochberg method). Scale bar indicates z-scored expression values. (H) Venn diagram representing the intersect between DEGs shown in (F-G).