Extended Data Figure 3. Analysis of c-kit lineage labeling in the heart at P0 (birth).

a, Diagram of the timing whereby newborn Kit^+/Cre × R-GFP mice were analyzed for all subsequent experiments in this figure. b, Histological sections for eGFP fluorescence (green) from the ileum and lung at P0 showing the characteristic c-kit labeling pattern as observed at other time points or in other studies when antibodies were employed. Blue shows nuclei c, Histological section for eGFP fluorescence (green) from the heart at P0. Blue shows nuclei and magnification was 40X. d, Immunohistochemical tissue section from the P0 heart of Kit^+/Cre × R-GFP mice stained with sarcomeric α-actin (red) to show all underlying cardiomyocytes (right panel) or with eGFP expression in green (left panel) as being c-kit derived. The green cells noted by the arrows are non-myocytes that do not express sarcomeric α-actin. e, eGFP expression alone (left) or eGFP with co-staining for cardiomyocytes in red (sarcomeric α-actin) from heart sections at P0 of Kit^+/Cre × R-GFP mice. Blue staining depicts nuclei. The cardiomyocyte that is shown has clear striations in the eGFP staining pattern, while the 2 non-myocytes do not show striated eGFP and also lack sarcomeric α-actin staining. f, eGFP expression alone in green (left) with nuclei in blue or eGFP with sarcomeric α-actin co-staining (red) from heart sections at P0 of Kit^+/Cre × R-GFP mice. All eGFP⁺ cells shown lack striations and are non-myocytes although the 2 cells in the center sit directly on top of cardiomyocytes and could be easily mis-interpreted. Great care is needed in scoring myocytes in the P0 heart because they are small and often the same size as eGFP⁺ non-myocytes. g, eGFP expression (green) with nuclei in blue and cardiomyocytes identified in red with sarcomeric α-actin antibody from heart histological sections at P0 of Kit^+/Cre × R-GFP mice. Here the data show c-kit lineage derived cardiomyocytes that appear in a loose cluster (arrows), presumably from a clonal expansion event earlier in development.