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Fig. 6. Analysis of reporter expression in transgenic E9.5 embryos. (A) Chimeric embryos were generated with the BRE-lac1 ES cell line by injection into blastocysts of ß-actin-GFP mice and analyzed at E9.5. ß-galactosidase staining was present in the heart, the optic vesicle (op), the ventral forelimb (l), neural crest (nc, blue arrowhead) and in the branchial arches (ba). Adult male chimeras generated with BRE-lac1 and BRE-lac2 ES lines were crossed with wild-type females and the progeny analyzed at E9.5. BRE-lac1 (B) and BRE-lac2 (C) transgenic embryos showed reporter expression pattern similar to that in transient chimeras. ß-Galactosidase staining was also detectable in the roof of the midbrain neuroepithelium (green arrowheads), in the hindbrain, where the region around rhombomere 5 showed very low ß-Galactosidase staining, in the posterior mesenchyme and in the aorta (white arrows). (D-E) Transversal sections through the heart of the embryo shown in A. ß-Galactosidase staining is found in the outflow tract (ot), the bulbus cordis (bc) and the ventricle (v), as well as in the atrioventricular canal (avc, red arrowhead). (F) In the optic vesicle, reporter expression is restricted to the dorsal side, while low expression was observed in the overlying ectoderm. (G,H) Transverse sections through the heart of BRE-lac2 transgenic embryos showed reporter expression in endocardium, pericardium and myocardium, with stronger expression in the outflow tract and the atrioventricular canal. Frontal (I) and lateral (J) views of a transgenic embryo showing ß-galactosidase expression in the cardiac crescent and in the amnion at E7.5. (K-N) Co-staining for phosphorylated Smad1/5/8 (PS1 antibody) and ß-galactosidase in E9.5 transgenic embryos. ß-Galactosidase expression coincides with PS1 staining in the heart and in the foregut (K,L), while in the neural tube and neural crest some cells are stained for PS1 and not ß-galactosidase (M, black arrowheads). High correlation between phosphorylated Smad1/5/8 and ß-galactosidase was observed in the branchyal arches (N). (O) Quantification of the incidence of ß-galactosidase and PS1 staining. Sections containing posterior body wall (pbw), heart and foregut (h+f), neurectoderm (nt) and branchyal arches (ba) were scored for double or single ß-galactosidase and PS1 staining. Results are presented as the mean of the percentage of single or double stained versus total of stained cells within three sections per count. Bars, 500 µm (A-C,I-J) and 100 µm (D-H,K-N). Abbreviations: a, atrium; am, amnion; as, aortic sac; cc, cardiac crescent; f, foregut; l, forelimb bud; p, pericardium; r5, rhombomere 5.
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