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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+/
(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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-actin in transverse sections of E10.0 embryos of wild-type and mutant
embryos. (K-P) Electron microscopic analysis of the perivascular walls.
(M,N) Higher magnification images of the insets in K and L, respectively.
White arrowhead indicates a mural cell; black arrowheads indicate endothelial
cells; arrows indicate tight junctions. (O,P) Higher-magnification views of
the tight junction between endothelial cells in M and N. da, dorsal aorta.
Scale bars: 200 µm in A-H; 20 µm in I,J; 10 µm in K-N; 1.0 µm in
O,P.
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