a, Quantitative real time PCR analysis of transcripts encoding vessel guidance molecules in type H and type L endothelial cells isolated by FACS from 4-week-old femur. Note high levels of transcripts for Netrin-1 (Ntn1), Netrin-3 (Ntn3), Neuropilin 1 (Nrp1), Plexin D1 (Plxnd1), Unc5b and Robo4 in type H relative to type L endothelium (n = 5 mice from 4 independent experiments). Error bars, ± s.e.m. P values, two-tailed unpaired t-test. b–f, Confocal images showing immunostaining of vessel guidance molecules such as Neuropilin 1 (b, Nrp1), Neuropilin 2 (c, Nrp2), Plexin D1 (d), Unc5b (e), Robo4 (f) in metaphyseal (type H) vessels and surrounding mesenchymal cells. High levels of Nrp1 were detected in type H endothelial cells (arrows), but comparably lower expression was also observed in vessel-associated osteoprogenitors and growth plate chondrocytes (ch). Nrp2 is highly expressed in perivascular osteoprogenitors (arrowheads). High expression of PlexinD1 and Unc5b, and lower levels of Robo4 were detected in type H vessel bulbs and columns (arrows). g, Maximum intensity projection showing high expression of VEGFR3 in endothelial bulbs and arches (white arrows) compared to columns. VEGFR3 was absent in the arteries. h, Image showing Dll4 expression in type H endothelial cells forming bulbs and arches in 4-week-old tibial metaphysis. With the exception of arteries (arrows), Dll4 expression was low in vessels of the diaphysis. i, Jag1 expression was detected in endothelial cells (white arrows) and surrounding mesenchymal cells.