(A) A 158kb human transgene spanning the Sost and Meox1 transcript was used to generate the Sost^tg gain-of-function allele (Loots et al., 2005). (B) Skeletal preparations of E17.5 wild type and Sost^tg embryos where Sost gain-of-function results in progressive loss of skeletal elements from posterior to anterior. A limb was scored as abnormal if it fell outside of the range observed in wild type samples; 117 total animals were scored consisting of 77 Sost^tg and 40 wild type. (d digit, ra radius, ul ulna, tg transgenic, wt wild type). (C-F) Human Sost expression was visualized by in situ hybridization using a human specific Sost probe, in a time-course panel of E9.5 to E12.5 Sost^tg embryos. At each time point whole embryo (C-F), and dorsal limb and AER views are provided (c-f). Dorsal limb bud views are provided for two different Sost^tg littermate embryos at each time point, where the top limb corresponds to a severely affected embryos and the bottom limb is from a Sost^tg embryo that is morphologically indistinguishable from a wild type limb bud). Asterisk highlights developmentally delayed mutant limb bud; arrowhead points to AER. fl forelimb, hl hindlimb, nf neural folds, sc spinal cord, mb midbrain, fb forebrain, def defective, tg transgenic

(A-H) Skeletal preparations of wild type or Lrp6^+/- (A), Sost^tg (B), Grem1^-/- (C), Sost^tg;Grem1^-/- (D), Lrp6^-/- (E), Sost^tg;Lrp6^-/- (F), Sost^-/- (G) and Sost^-/-;Lrp6^-/- (H) forelimbs. 40% of Sost^tg;Grem1^-/- (D) forelimbs lacked an elbow, where the humerus, radius and ulna have formed one skeletal element (arrow) and the wrist and autopod were highly reduced and lack most digits (bracket). While Lrp6 heterozygous forelimbs were indistinguishable from wild type, Sost^tg modified the Lrp6 phenotype (E) such that Sost^tg;Lrp6^+/- forelimbs were indistinguishable from Lrp6^-/- (F). Removal of Sost in the absence of Lrp6 receptor resulted in dramatic rescue of Lrp6^-/- limb phenotype (H).

(A-F) WNT activity in the limb was examined in BatGal WNT reporter mice. LacZ expression from the BatGal WNT reporter transgene was examined in E10.5 wild type (A-F) and Sost^tg (A′-F′) embryos in whole mount (A, A′), AER limb views (B, B′), transverse limb sections (C, C′, F, F′), dorsal (D, D′) and ventral (E, E′) limb bud views. Ectodermal derived SOST (G) repressed BatGal LacZ reporter expression in the dorsal (D, D′) and ventral ectoderm (E, E′) in a pattern consistent with human SOST transgene expression (yellow dashed region). On the ventral side all reporter activity was lost (E′, F′; red dashed region). AER LacZ expression was unaffected or enhanced in the presence of SOST (B′, D′, F′). Black arrows indicate the AER, the region where SOST is absent (G), orange arrows indicate morphogenic activity of ectodermal derived SOST to suppress WNT signaling in the underlining mesenchyme.

(A-D) Shh, (E-H) Fgf4 and (I-L) Fgf8 were significantly down-regulated in Sost^tg E10.5 limb buds. Shh was down to 20% its normal expression level in Sost^tg, but unaffected in Sost^-/- embryos (D). Fgf4 and Fgf8 were down-regulated in both Sost^tg and Sost^-/- embryos (H, L); the AER width was dependent on Sost levels, such that high levels of Sost limited its domain (J′, red arrow), while the absence of Sost disorganized and expanded it dorso-ventrally (K′, green arrow). (M-P) Grem1, a mesenchymal marker, while quantitatively appeared unaffected (P), its expression domain (N, O) was altered in both Gain- and Loss-of-Function embryos. In Sost^tg embryos Grem1 domain was expanded posteriorly (N′, red arrow), along with an anterior reduction on the ventral side (N′, green arrow). In Sost^-/- embryos similar asymmetric, but more subtle changes were observed (O′). (Q) Sost expression is restricted to the limb bud ectoderm. (R) Limb bud sections were carried out as illustrated.

(A-C) Limb bud RNA was isolated from wild type, Sost^tg and Lrp6^-/- E10.5 limb buds. Normal expression of Shh (A) and its downstream transcriptional target dHand (B) was dramatically reduced in both Sost^tg and Lrp6^-/- embryos. Similarly, Axin2 expression, a downstream target of canonical WNT signaling, was less than 50% of normal levels in both Sost^tg and Lrp6^-/- embryos (C).