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Fig. 2. OFT lengthening and conotruncal cushion development is abnormal in Nkx2.5^Cre/+;Shh^flox/- embryos due to cell death. (A-D) Comparing E10.5 OFT and right ventricle length between wild-type (A,C) and Nkx2.5^Cre/+; Shh^flox/- (B,D) embryos reveals a reduction in length of the mutant OFT and right ventricle (all bars are identical in adjacent images and quantified in G), whereas the left ventricle is unchanged. Paired t-test P=0.002 for OFT and P=0.025 for the right ventricle. (E-F') OFT conotruncal cushions (F,F', arrows) are reduced at E10.5 compared with Nkx2.5^Cre/+; Shh^flox/+ (E,E'). Immunohistochemistry markers in E and F are PECAM (red) and AP2 (green). (H-K'') Levels of cell death, as revealed by Lysotracker Red analysis, at E10.5 (H-I', sagittal views; J-K'', frontal views) demonstrate increased cell death in the pharyngeal endoderm (K,K'), in the splanchnic mesoderm (I,K') and within the core-arch mesoderm (I') of mutants. In all panels, arrows and arrowheads mark abnormal and normal findings, respectively, in mutants as compared with control embryos. A, atrium; OFT, outflow tract; PE, pharyngeal endoderm; SM/NCC, splanchnic mesoderm/neural crest cells; V, ventricle; PS, pharyngeal space; 1-4, pharyngeal arch 1-4.

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