Fig. 3: Changes in vessel phenotype and skeletal progenitor distribution following postnatal development.
From: Quantitative 3D imaging of the cranial microvascular environment at single-cell resolution
A, B MIP of vessels and Osterix+ progenitors from calvaria of 4-weeks-old (A, A’) and 12-weeks-old (B, B’) mice. C 40-μm-thick sagittal section showing vessels (C) and Osterix+ progenitors (C, C’) along the thickness of the calvarium displayed in B. 12-weeks-old calvaria have more bone marrow vessels (CD31hiEmcnhi and CD31loEmcnhi/lo) and fewer periosteal and meningeal vessels compared to 4-weeks-old calvaria. Osterix+ progenitors in 12-weeks-old calvaria are spatially restricted to transcortical canals (white arrowheads), bone marrow cavities, and osteogenic fronts adjacent to the sutures (yellow arrowheads), and are absent from the periosteum and dura mater (yellow arrows). D, E MIP of vessels and Gli1+ progenitors of calvaria from 4-weeks-old (D) and 12-weeks-old (E) mice. F Zoomed-in region from E. Gli1+ cells in 12-weeks-old calvaria (E, E’, F, F’) are concentrated at and nearby the transcortical canals (white arrowheads) and the sutures (yellow arrowheads). Results were replicated in 3 calvaria for each experimental group and staining combination (12 total calvaria). G–I Comparison of total skeletal progenitor number (G), total vessel volume (H), and fractional vessel phenotype volumes (I) between 4- and 12-weeks-old calvaria (n = 3 for G; n = 6 for H, I). J Plots representing the spatial correlation of Osterix+ and Gli1+ progenitors to vessel phenotypes (n = 3). Both cell types maintain a preferential relationship with CD31hiEmcnhi vessels in 12-weeks-old mice. Osterix+ cells demonstrate a higher spatial affinity to CD31hiEmcnhi vessels in 12-weeks-old versus 4-weeks-old calvaria. K, L MicroCT 3D volume projections of calvaria at 4 weeks (K) and 12 weeks (L) of age. M MicroCT quantification of bone volume to total volume (BV/TV) percentage and bone volume (BV) in the parietal and posterior frontal bones of 4- and 12-weeks-old mice (n = 4). Data are mean ± SD. Statistics were performed using a two-way ANOVA with Bonferroni post-hoc test (G–J) or two-tailed t-test (H, M). ***p < 0.001, **p < 0.01, *p < 0.05 where designated or between CD31hiEmcnhi and CD31hiEmcn− vessels in J; +++p < 0.001 between CD31loEmcnhi and CD31hiEmcn− vessels (J). Exact p-values for two-tailed t-tests: H p = 0.3321, M p = 0.0213 (top left), 0.0005 (top right), 0.0001 (bottom left), 0.0005 (bottom right). Scale bars: 1000 μm (A, B, D, E, K, L); 700 μm (C); 300 µm (C, inset); 200 μm (F). Colors: Red: Endomucin (Emcn), Gray: Osterix (A–C) or Gli1 (D–F), Green: CD31.
