Conditional Ablation of Ezh2 in Murine Hearts Reveals Its Essential Roles in Endocardial Cushion Formation, Cardiomyocyte Proliferation and Survival
Figure 1
Conditional knockout of Ezh2 gene in murine hearts caused perinatal lethality.
(A) Schematic illustration showing the annealing sites of primer pairs of mouse Ezh2 and Ezh1 (a homolog of Ezh2). (B) Ezh2 expression in the murine embryonic hearts. Semi-quantitative RT-PCR with two different numbers of amplification cycles (30 and 35) was performed on RNA samples purified from WT hearts of E9.5, E10.5, and E11.5. 1 µl or 0.5 µl cDNA from reverse transcription reaction was used for detection of Ezh2 or GAPDH (as a control), respectively. (C) qRT-PCR analysis revealed ∼70% decrease in Ezh2 mRNA of Ezh2-cKO heart (n = 3, p<0.001), but Ezh1 mRNA was not affected, compared with that of the control littermate hearts. Data are presented as mean ± SD of 3 replicate samples for each strain/gene analyzed. (D) The level of H3K27Me3 was reduced in the E18.5 Ezh2-cKO hearts, compared to that of control. The total Histone H3 and GAPDH both serve as controls. The asterisks indicate H3K27Me3. NS, non-specific. (E) The neonatal mortality rate (%) and (F) the P1 frequency of four different genotypes resulting from the crossbreeding between Ezh2fl/+:Nkx2.5-cre+ and Ezh2fl/fl mice. Data in panel E & F represent the average of 12 litters comprising a total of 99 animals. (G) The number and recovery rate (in parenthesis) of Ezh2fl/+, Ezh2fl/+:Nkx2.5-cre+, Ezh2fl/fl, and Ezh2fl/fl:Nkx2.5-cre+ embryos collected at various embryonic stages are shown. Offspring from intercrossing between genotypes of Ezh2fl/fl and Ezh2fl/+:Nkx2.5-cre+ mice. Note that the expected Mendelian rate of embryo recovery for each of these four genotypes is 25%. (H) Embryonic demise of Ezh2 mutant mice at a later gestational stage. The representative images of E16.5 WT and dead Ezh2 mutant embryos are shown.