DEV029926 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Impaired vascular invasion and mineralization in Smad1/5^CKO mice. (A,B) Von Kossa staining of WT (A) and mutant (B) E16.5 proximal tibiae, showing disorganized mineralization in the thickened perichondrium in mutants. (C,D) Von Kossa staining of WT (C) and mutant (D) marrow space. The blood cells are non-specifically stained, and can be seen in discrete blood vessels within the marrow cavity (arrows). In the mutant, the junction between the cartilage and marrow space is evident (arrowhead), but terminal hypertrophic chondrocytes are not present, nor is there any evidence of vascular invasion or mineralization.

• Supplemental Figure S2 -

  Fig. S2. Ihh and Pthrp expression in midgestation Smad1/5^CKO mice. (A-D) Ihh expression. Bright-field (A,C) and corresponding dark-field (B,D) images of E14.5 tibiae and fibulae hybridized to an antisense Ihh probe. (E-H) Pthrp expression. Bright-field (E,G) and corresponding dark-field (F,H) images of E14.5 tibiae and fibulae hybridized to an antisense Pthrp probe. (I-L) Bright-field (I,K) and corresponding dark-field (J,L) images of E14.5 limbs hybridized to antisense Ptch1 probe. All sections are from a single WT and mutant limb.

• Supplemental Figure S3 -

  Fig. S3. Ihh expression in phalanges of E14.5 Smad1/5^CKO mice. (A,B) Bright- and dark-field images, respectively, of an E14.5 WT autopod hybridized to an antisense Ihh probe. (C,D) Bright- and dark-field images, respectively, of an E14.5 Smad1/5^CKO autopod hybridized to an antisense Ihh probe.

• Supplemental Figure S4 -

  Fig. S4. Smad1 linker phosphorylation is not altered by FGF in growth plates. Limbs cultured in the presence or absence of FGF18 were analyzed by immunofluorescence (red) for pSmad1L expression. Sections are counterstained with DAPI (blue). (A) Control and (B) FGF18-treated limbs exhibit indistinguishable patterns of primarily nuclear pSmad1L localization.