Fig. 3. Phenotypic analysis of compound mutants Tbx1^neo2/- and conditional rescue mutants Mesp1^Cre/+;Tbx1^E5/neo2. (A) Scheme of gene targeting strategy. H, HindIII. (B-M) Compound mutants Tbx1^neo2/-. (B'-M') Conditional rescue mutants Mesp1^Cre/+;Tbx1^E5/neo2. (B,B') Immunohistochemistry using an anti-Tbx1 antibody on sagittal sections of E9.5 embryos showed very low or undetectable signal in compound mutants (B) and reactivated expression in the mesoderm (arrows) of the rescued embryo (B'). (C,C') External ears (arrowhead) are hypoplastic in compound mutants but are of normal size in rescued mutants (arrow) at E18.5. (D,D') Absence of thymus indicated by asterisk in both compound and rescued mutants at E18.5. (E,E',F,F') The cardiac outflow tract phenotype was normal in rescued embryos at E18.5. (G,G') The hypoplasia of the 2nd pharyngeal arch of compound mutants (arrowhead) was corrected in rescued embryos. (H,H') Ink injection into the heart of E10.5 embryos revealed normalization of the 6th pharyngeal arch arteries but not of the 4th pharyngeal arch arteries in rescued mutants. This is also evident from histological sections (I,I'). (J,J') Whole-mount RNA in situ hybridization on E10 embryos with a PaxI probe revealed normalization of the 2nd pharyngeal pouch but only partial development of the 3rd pharyngeal pouch (arrowhead in J') in rescued embryos. (K,K') Whole-mount immunohistochemistry on E10 embryos with an anti-Hoxb1 antibody showed a more robust labeling of the 4th pouch (arrowhead) in rescued embryos. (L,L') Whole-mount RNA in situ hybridization on E10 embryos with a Crabp1 probe revealed only a marginal improvement of neural crest cell distribution in rescued embryos (compare with the normal pattern in Fig. 2H). Similarly, immunohistochemistry on E10 embryos using an anti neurofilament M antibody showed only modest improvement in rescued embryos, especially in the caudal region of the pharynx (M,M'), compare with normal pattern in Fig. 2I). Ph, pharynx; Ao, aorta; P, pulmonary trunk; Pa, pulmonary artery; VSD, ventricular septum defect; RV/LV, right/left ventricle; RSA/LSA, right/left subclavian artery; RCA/LCA, right/left carotid artery; I and II, 1st and 2nd pharyngeal arch; 3 and 6: 3rd and 6th pharyngeal arch artery; 1p, 2p, 3p and 4p, 1st, 2nd, 3rd and 4th pharyngeal pouches; V, trigeminal nerve; VII/VIII, facial/acoustic nerve; IX, glossopharyngeal nerve; X, vagus nerve; XI, accessory nerve. Scale bars: 2mm in C'; 1 mm in D',E',G',H',K',M'; 100 µm in B',F',I'.