Fig. 6. Augmentation of the Smad2/3 pathway in Man1-deficient embryos. (A-P) Confocal microscopic analysis of the branchial regions of wild-type and mutant embryos at E9.5 for phospho-Smad1 (PS1) and phospho-Smad2 (PS2). Nuclei were visualized with DAPI. All pictures were recorded under the same conditions and quantified without any modifications. (B,F,J,N) Higher magnification views of the mesenchymal cells in A,E,I,M. (Q,R) For phospho-Smads quantitation, mesenchymal cells (MC, ^+/+, n=50; ^/, n=100), endothelial cells (EC, ^+/+, n=20; ^/, n=20) and neural cells (NC, ^+/+, n=50; ^/, n=50) were randomly picked up, and the intensities of PS1, PS2 and DAPI-positive areas were quantified using the NIH Image-J software. Quantitative results are expressed as integrated density (IntDen)/pixel² at the mean±standard deviations. Statistical differences were determined using the Student's t-test and the p values are indicated. The difference in the PS1 calculation (P>0.05) between the wild-type and mutant embryos was not significant.