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Figure 2


Fig. 2. Generation of Man1-deficient mice and developmental expression of Man1. (A) The exon-intron organization of Man1 with the genetrap vector (upper) and a schematic structure of Man1 (middle) are depicted. The LEM domain (red), transmembrane domains (black) and the Smad-interacting domain (green) are indicated. The genetrap vector, which contains an engrailed 2 splice acceptor (SA, white) and ßgeo (blue), was inserted in intron 4 to produce a Man1-ßgeo fusion protein (lower) lacking the C-terminal region containing the Smad-interacting domain. Numbers indicate exons. Genomic DNA was digested with PstI (Ps) for Southern blotting using a probe (yellow) outside the insertion site. P1 and P2 are probes for whole-mount in situ hybridization analysis. Double-headed arrows indicate the positions of the cDNA fragments amplified by RT-PCR in B. (B) Genotyping by RT-PCR using yolk sacs from embryos at E8.5. (C) Genotyping by Southern blotting of tail DNA from embryos at E9.5. The wild-type allele yielded a 7.9 kb band and the mutant allele yielded a 3.6 kb band. (D) A sequencing result of the Man1-ßgeo transcript shown in B. Three other independent clones showed the same results. The deduced amino acid sequence is shown. (E) Western blot of lysates from E9.5 embryos and MEFs with antibodies to ß-galactosidase and Man1. ß-Tubulin was used as a loading control. (F-J,M,O,P) Whole-mount in situ hybridization with the sense (F,H) or antisense (G,I,J,M,O,P) P1 and P2 probes. No signals were observed in the mutant embryo probed with the antisense P2 probe (J). (P) A cryosection of the E9.0 embryo shown in O. Anterior to the left in F-O. Whole-mount X-gal staining in wild-type (K) and Man1+/{Delta} (L,N,Q,R) embryos and mice. (Q) A section through the yolk sac at E9.5. (R) The dorsal aorta of an 11-week-old mouse. In the inset, black arrowheads indicate endothelial cells; white arrowheads indicate VSMCs. Genotypes of the embryos and stages are indicated. al, allantois; ba, branchial arch; ec, ectoplacental cone; fg, foregut; h, heart; hm, head mesenchyme; pm, paraxial mesoderm; sv, sinus venosus; tm, tail mesenchyme. Scale bars: 200 µm in F-P; 50 µm in Q,R.





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