Figure 3.

Fractional ablation of embryonic CMs by chimeric complementation. (A) Schematic diagram of the eGFP/DTA construct before and after αMHC-Cre-mediated recombination (left panel). Brightfield and GFP fluorescence images of ROSA26^eGFP-DTA control and αMHC^Cre/+;ROSA26^eGFP-DTA transgenic embryos (right panel) at E8.5, E9.5, and E10.5 of development. Note the compound transgenic embryo shows a progressive defect in cardiac morphogenesis by E9.5 and is completely missing a heart structure at E10.5. (B) Generation of αMHC^Cre/+;ROSA26^eGFP-DTA ESC lines. Brightfield and GFP fluorescence microscopy images are shown for the three ESC lines used. (C) Fractional CM ablation by injection of αMHC^Cre/+;ROSA26^eGFP-DTA ESCs into wild-type CD-1 blastocysts (upper panel). The summary table demonstrates the complementation efficiency for each ESC line tested based upon the total number of animals collected (lower panel). (D) Brightfield and GFP fluorescence images of E10.5 chimeric αMHC^Cre/+;ROSA26^eGFP-DTA embryos. Note that embryos with >50% ablation are developmentally arrested and exhibit impaired cardiac morphogenesis. A pericardial effusion is occasionally observed. (E) eGFP, troponin T (cTnT), and DAPI staining of E9.5 chimeric embryos with progressively increasing amounts of cardiomyocyte ablation. Some residual unablated eGFP⁺ cardiomyocytes remain in the hearts at this stage. Scale bars: 500 μm for (A); 200 μm for (B and E); 1 mm for (D).