a, b, Organisation of distal vessels in the metaphysis of 4-week-old tibia. Endothelial cells were visualized by anti-Endomucin (Emcn, red) immunostaining (a) or GFP expression (green) in Cdh5(PAC)-CreERT2 Rosa26-mT/mG double transgenic mice (b) at 4 weeks of age. Note blunt appearance of most distal vessels (top) in proximity of growth plate chondrocytes. Nuclei, blue (DAPI). c, Maximum intensity projection showing the organization of distal, CD31-immunostained (type H) vessels in the tibial metaphysis at the indicated ages. Note emergence of blunt and blind-ended bulb-like protrusions (arrows) from arch vessels (arrowheads) at the distal end of endothelial columns. d, Confocal image showing expression of intercellular adhesion molecule 2 (ICAM2, red), a marker of lumenized vessels, in distal vessels of the tibial metaphysis. Nuclei, blue (DAPI). e, Podocalyxin (Podx, green), a sialoglycoprotein marking the apical surface of endothelial cells and thereby the vascular lumen (arrows), is present on the most distal, CD31-positive (red) vessel structures in the metaphysis. Nuclei, blue (DAPI). f, Transmission electron microscopy confirming the lumenized nature of distal vessels close to growth plate chondrocytes (ch). Yellow arrowheads indicate thin endothelial cells lining vessels. g, Quantitation of EdU labelled (proliferating) endothelial cells in the metaphysis of long bone. EdU+ endothelial cells were predominantly present in columnar vessels and were comparably rare (≤5%) in vascular arches (n = 5 mice from 3 independent experiments). Error bars, ± s.e.m. P values, two-tailed unpaired t-test.