Fig. 4

Cardiomyocyte clones carrying biallelic mutations in tnnt2a, cmlc2, or amhc survive to adulthood intermingled with wild-type cells (A) Schematic representation of the experimental approach to test whether labeled myocardial clones carrying biallelic mutations can be recovered in adult hearts. (B–D) Representative images of dissected hearts from cardiodeleter+ animals injected with the indicated guide shuttles. (B′–D′) mKate signal from hearts shown in (B–D), indicating cardiomyocyte clones that have incorporated the corresponding guide shuttle. The GFP fluorescence from the cardiodeleter transgene is shown in the images located in the bottom left corner. (E–G) Histological sections from hearts shown in (B–D), immunostained with antibodies to identify guide shuttle+ cardiomyocytes (mKate+, magenta) and the corresponding protein encoded by the targeted gene (cyan). (E′–G′) Consecutive sections from hearts shown in (E–G) stained using the AFOG technique to identify collagen (blue) and myocardium (orange/brown). Yellow asterisks indicate large mKate+ clones that show complete absence of the corresponding protein. (H–J) Combined immunofluorescence-RNAScope in consecutive sections from hearts shown in (E–G). Signal from mRNA ISH appears in yellow. White arrows indicate guide shuttle (mKate)+ cardiomyocytes with biallelic mutations, leading to the reduction of the corresponding mRNA and the complete loss of the corresponding protein. Orange arrowheads point to guide shuttle– cardiomyocytes that maintain high levels of mRNA and protein expression. at: atrium; v, ventricle. Scale bars, 200 μm (B–D), 100 μm (E–I and E′–G′), and 20 μm (H′–J′).