Decreased contribution of Pitx2-positive cells to the Msx1-/- ; Msx2-/- mutant AV cushion mesenchyme. Panels C, D, G and H are enlarged views of the boxes in A, B (at E10.5) and E, F (at E11.5), respectively. Pitx2 immunostaining (green fluorescence) revealed a substantial reduction in the number of Pitx2-positive cells in the Msx1-/-; Msx2-/- mutant AV cushion mesenchyme compared with the control (compare the regions marked by yellow asterisks in C, D, G and H). White notched arrowheads in D and H indicate sparse Pitx2-positive mesenchymal cells in the Msx1/2 null mutant AV cushions. There was no detectable Pitx2 expression in the left atrial myocardium of the Msx1/2 double mutants shown in B and F (indicated by white arrows). I, Statistical analyses revealed significantly reduced percentages of Pitx2-positive cells in the AV cushion mesenchyme of the Msx1/2 double mutants at both E10.5 and E11.5 (n = 3 for each developmental stage, P < 0.01 at E10.5, P < 0.001 at E11.5, Student's t test). LA and LV, left atrium and ventricle; RA and RV, right atrium and ventricle. Scale bars: 0.1 mm in B (for A, B) and F (for E, F), 0.05 mm in D (for C, D) and H (for G, H).