(A-I) Metatarsal angiogenesis assays. RANKL (50 ng/ml) increased (B) and OPG (300 ng/ml) decreased (C) endothelial sprouting from WT metatarsals. PTHrP (100 nM) increased endothelial sprouting in WT metatarsals (D), which was inhibited by OPG (E). Neither RANKL nor PTHrP increased endothelial sprouting from Atf4−/− metatarsals (F-H). Quantitative data of each group (I). n = 5-6. Microscope: Olympus SZ61. Original magnification, x20. (J-R) TRAP staining. WT and Atf4−/− metatarsal sections treated as indicated were stained for TRAP activity. Quantitative analysis of TRAP-positive area, which was normalized to metatarsal section area (R). n = 5-6. Original magnification, x100. (S and T) ELISA. Metatarsals were dissected from WT and Atf4−/− E17.5 fetuses and cultured in α-MEM for 14d. Culture media were harvested for CTX and VEGF assays as described in Materials and Methods. n = 5-6, *P < 0.01, versus veh, #P < 0.01, versus WT, ^P < 0.01, versus PTHrP alone.