Canonical BMP signaling is required for endothelial MSX1 induction upon limb ischemia in vivo. (A) IF staining for pSMAD1/5/8 (green), isolectin GS-IB4 (red), αSMA (white), and DAPI (blue) in unligated (left) and 7-d ligated (right) adductors. (B) Staining for pSMAD1/5/8 (red), ENG (green), and DAPI (blue) in adductor muscle 7 d after ligation. (C) IF staining for MSX1 (green), pSMAD1/5/8 (red), isolectin GS-IB4 (white), and DAPI (blue) in adductor muscles 7 d after ligation. The asterisks indicate double-positive arterial ECs, and arrowheads point at venous ECs only positive for pSMAD1/5/8. A, artery; V, vein. (D) Staining for MSX1 (green), αSMA (red), and DAPI (blue) in the adductor muscle of tamoxifen-treated Cdh5-CreERT2;Smad1^fl/fl:Smad5^fl/fl mice 7 d after ligation. The arrowheads and asterisks indicate ECs positive and negative for MSX1, respectively. Quantification is represented as percent MSX1-positive collateral ECs. Error bars represent the SEM. n = 7 Cdh5-Cre−;Smad1^fl/fl:Smad5^fl/fl mice and n = 9 Cdh5-Cre+;Smad1^fl/fl:Smad5^fl/fl mice. *, P < 0.05 versus Cdh5-Cre−;Smad1^fl/fl:Smad5^fl/fl mice. Bars: (A–C) 10 µm; (D) 25 µm; (magnifications in D) 12 µm.