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Fig. 3. Tgfbr2 is not required for AV cushion mesenchyme formation. (A) The conditional knockout embryo (right) isolated at E10.5 was grossly normal compared with the control littermate (left). Embryos were stained with X-gal. The Tie2cre-mediated recombination in the mutant embryo occurred at the same level and with the same pattern as that in the control embryo (as judged from the R26R locus). (B) Mutant embryo isolated at E9.0, immediately before the onset of EMT in the AVC region, stained with X-gal and sectioned. (C-F) Control (C,E) and mutant (D,F) embryos isolated at E9.5 (C,D) and E10.5 (E,F), stained with X-gal and sagittally sectioned. The AV cushion mesenchymal cells formed normally in mutant embryonic hearts. (G) Count of the number of AV cushion mesenchymal cells in AVC regions of mutant and control embryonic hearts (E9.5). Data were averaged from four embryos of each genotype. Error bars indicate the s.d. (H) Sagitally sectioned mutant embryo isolated at E10.5, in which growth was arrested at around the 25 somite stage. Mesenchymal cells (arrowheads) can be observed in the AV cushions. (I) Quantitative analysis of the recombined Tgfbr2^loxp allele amplified by quantitative real-time PCR analysis from genomic DNA isolated from AV endocardial/mesenchymal cells of control or mutant embryonic hearts (Fig. 1C). Lanes 1-3 represent a positive control in which a plasmid containing the recombined Tgfbr2^loxp allele was used as a template, the copy number of which ranged from 10⁴ to 10². Lane 4 is a no template control. Lanes 5-7 and lanes 8-10 represent the mutant and control samples, respectively, from three independent experiments. (J) The PCR product of the recombined Tgfbr2^loxp allele could only be detected in the mutant samples. (K) Semi-quantitative RT-PCR analysis was performed with RNA isolated from the AV endocardial/mesenchymal cells. Lanes 1-3 are control samples with 100, 5 and 10% of input RNA. Lane 4 is the mutant sample with 100% of input RNA. GAPDH was used as a loading control. A, atrium; cko, Tie2cre;Tgfbr2^loxp/loxp;R26R; ctrl, Tie2cre;Tgfbr2^loxp/+;R26R; IC, inferior cushion; SC, superior cushion; V, ventricle.

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