Supporting Information
Figure S1  Chondrogenic Differentiation in Bmp2C/C; Bmp4C/C; Prx1::cre Animals
(A–C) In BMP2, BMP4–deficient limbs, there is only one zeugopod present, and it is often fused with the stylopod. All panels are from E17.5 Bmp2C/C; Bmp4C/C; Prx1::cre embryos. (A) Hematoxylin and eosin–stained section of the forelimb. Alcian blue– and Alizarin red–stained skeletons shown in (B) (forelimb) and (C) (hindlimb). Black arrow shows the fusion of the zeugopod and stylopod in BMP2, BMP4–deficient limbs.
(D and E) Acridine orange–stained limbs from E11.5 wild-type (D) and Bmp2C/C; Bmp4C/C; Prx1::cre embryos (E).
(F and G) Acridine orange–stained hindlimbs from E13.5 wild-type (F) and Bmp2C/C; Bmp4C/C; Prx1::cre (G) embryos.
(H–M) In the absence of BMP2 and BMP4, condensation begins in the limb normally. Whole mount in situ staining of Sox9 mRNA in embryonic limb buds from E10.5 (H and I) and E11.5 (J–M) embryos. (H, J, and L) From wild-type embryos, (I, K, and M) from Bmp2C/C; Bmp4C/C; Prx1::cre embryos. (H, I, J, and K) Forelimbs, (L and M) hindlimbs.
(N and O) In situ hybridizations with Col II mRNA probes of sections derived from E12.5 wild-type (N) and Bmp2C/C; Bmp4C/C; Prx1::cre (O) embryos.
(P–S) Bmp6 mRNA expression is not increased in E13.5 Bmp2C/C; Bmp4C/C; Prx1::cre cartilage. Bmp6 in situ on sagittal sections from wild-type (Q) and Bmp2C/C; Bmp4C/C; Prx1::cre (S) embryonic forelimbs. (P and R) Bright field images of (Q) and (S), respectively.
(23 MB TIF)
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Figure S2  Skeletal Differentiation Occurs Normally in Absence of BMP2
(A) Toluidine blue–stained section of newborn femurs from Bmp2C/C animals.
(B–D) Marker analyses. Sections of femurs from Bmp2C/C animals hybridized with Col II (B), Col X (C), and Col I (D) mRNA probes.
(E) Toluidine blue–stained section of newborn femurs from Bmp2C/C; Prx1::cre animals.
(F–H) Marker analyses. Sections of femurs from Bmp2C/C; Prx1::cre animals hybridized with Col II (F), Col X (G), and Col I (H) mRNA probes.
(15 MB TIF)
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Figure S3  BMP2, BMP4–Deficient Limb Skeleton Is Resorbed
Alcian blue– and Alizarin red–stained skeletons of forelimbs (A–D) and hindlimbs (E–F) from 1-wk-old (A, B, E, and F) and 3-wk-old (C and D) animals. (A, C, and E) Wild-type animals, (B, D, and F) Bmp2C/C; Bmp4C/C; Prx1::cre animals. The limbs of Bmp2C/C; Bmp4C/C; Prx1::cre (H) animals are severely defective compared to a wild-type (G) animal at 1 wk of age.
Please note that the proximal part of the femur is present at 1 wk of age but missing at 3 wks (refer to Figure 6L).
(12 MB TIF)
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Figure S4  Osteogenesis Occurs in Mice Lacking Three of Four Alleles of Bmp2 and Bmp4 Combined
Toluidine blue–stained sections of femurs from adult control, Bmp2C/+; Bmp4C/C; Prx1::cre, and Bmp2C/C; Bmp4C/+; Prx1::cre animals.
(11 MB TIF)
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