Fig. 2: Changes in vascularisation of the long bone in Clec14a^−/− mice.

A Representative flow-cytometry heatmap scatter plots showing Cd31^high Emcn^high EC (H-type) and Cd31^low Emcn^low (L-type) endothelial cells (EC) in Cd45⁻ Ter119^- cells isolated from the long bones of Clec14a^+/+ and Clec14a^−/− 2-week-old pups. B Relative abundance of H-type compared to L-type EC in 2-week-old mouse tibiae from Clec14a^−/− mice compared to Clec14a^+/+ (WT). Data was analysed with a Mann–Whitney test, results are presented as median ± IQR, *P < 0.05, ns = not significant. C High resolution 3D confocal microscopy of the juvenile (4 weeks old) Clec14a^+/+ (WT) and Clec14a^−/− mouse tibia capillary network. The panel shows representative maximum intensity projection images depicting organisation of H-type vessels in Clec14a^+/+ (WT) and Clec14a^−/− tibia. Vessels in the tibia section have been immunolabelled with Emcn (magenta). White line marks the limits of the type-H vessel front, note the shorter H capillary front in the Clec14a^−/− images. Scale bars are 200 µm. D Left graph: quantification of the length of H type columns in the metaphysis, data represents mean values for each biological replicate. Right graph: quantitative evaluation of the length of the type-H vessels front. In D and E, data is represented as mean ± SD, results were analysed by unpaired T test. **P < 0.01; *P < 0.05; ns = non-significant. E Representative enlarged images of the leading type-H vascular front (Emcn, magenta) in Clec14a^+/+ and Clec14a^−/− mice. Note dysregulation of the advancing H-type bulges protruding into the growth plate (highlighted by the white dashed ovals in the Clec14a^−/− image). Scale bars are 100 µm. F Representative immunofluorescent images of type-H vessels labelled with Emcn (blue) and counterstaining of cells and extracellular matrix in the metaphysis and growth plate with wheat germ agglutinin (WGA - orange). Note blunting of type-H vessel buds in Clec14a^−/−. Scale bars are 200 µm (main images) and 50 µm (high magnification insets). G Quantitative evaluation of the fraction of type-H vessels buds showing blunted phenotype in the bone metaphysis. H Maximum intensity projection images show immunostaining of Emcn (fire – colour gradient with high expression in orange and low expression in magenta) and nuclei (Hoechst, grey) in sections collected from 4-week-old Clec14a^+/+ and Clec14a^−/− male mice. Note absence of filopodia (white arrowhead) in Clec14a^−/− images. Scale bars are 20 µm. I, J Immunofluorescent images of the chondro-osseous interface (wheat germ agglutinin WGA - orange, Emcn - blue). Note the normal patterning of the growth plate in both Clec14a^+/+ and Clec14a^−/−. Scale bar = 200 µm (I) and 50 µm (J).