Quantitative 3D light-sheet imaging of murine calvaria
To visualize vessel phenotypes and skeletal progenitors in the murine calvarium, we developed and optimized an imaging pipeline comprised of whole-mount immunostaining, optical tissue clearing, and light-sheet imaging (Supplementary Fig. 1a). We adapted a staining regimen used in long-bone sections14 to achieve adequate antibody penetration and labeling of up to three molecular markers: CD31 and Emcn for vessels and Osterix or Gli1 for skeletal progenitor subpopulations. Osterix is a marker for skeletal progenitors that are restricted to the osteoblast lineage15, while Gli1 marks less-differentiated skeletal stem cells16,17. To achieve high-quality visualization of each marker, we used fluorophores spanning the red to the infrared spectrum, as ultraviolet and green dyes resulted in high levels of background and light scattering during imaging. We cleared the calvaria by removing the blood prior to staining and incubating in a graded series of 2,2-thiodiethanol (TDE) following staining. This method did not require us to decalcify bone to achieve adequate bone tissue clearing and maintained the geometry of the calvarium (Supplementary Fig. 1B, C). Following clearing, we imaged calvaria using light-sheet microscopy, which allowed us to rapidly acquire high-resolution data and minimize photo-bleaching through the duration of the scan (~5 h per sample). Our resulting images captured the 3D distribution of different vessel phenotypes and skeletal progenitors in the calvarium with high axial and lateral resolution (Fig. 1A–D and Supplementary Video 1).
A Maximum intensity projection (MIP) of vessels and Osterix+ skeletal progenitors in the parietal and posterior frontal bones of the calvarium. B Images from the boxed region in A demonstrating the high resolution and signal quality obtained in each channel (i–iv). The dyes used for each channel are designated above (A) and (B): AF555 for Endomucin (i), AF647 plus for Osterix (ii), and AF800 plus for CD31 (iii). C, D Sagittal (C) and frontal (D) 40-μm-thick optical sections demonstrating the location of vessels and Osterix+ cells along the thickness of the calvarium. Boxed regions from (Ci) and (Di) are shown in Cii and Dii. Similar results were achieved for all the calvaria imaged in this study (n = 45 calvaria). Scale bars: 1000 μm (A, Di), 800 μm (Ci), 300 μm (Cii), 100 μm (Bi–iv, Dii). Colors: Red: Endomucin (Emcn), Gray: Osterix, Green: CD31.
To analyze vessel phenotypes and skeletal progenitors, we developed a quantitative pipeline to enable 3D spatial characterization of high-resolution datasets (~500 GB raw, ~200 GB compressed, Supplementary Fig. 2A). We applied the spots and surfaces modules in Imaris software to segment blood vessels and skeletal progenitors, and then performed a series of masking and filtering algorithms to denote three-vessel phenotypes: CD31hiEmcn− arteries and arterioles, CD31hiEmcnhi capillaries, and CD31loEmcnhi capillaries and sinusoids18,19 (Supplementary Fig. 2B–D). CD31loEmcnlo sinusoids were not segmented due to their low signal-to-noise ratio. We exported the spots and surfaces statistics into GraphPad Prism and XiT software14 to analyze vessel volume, skeletal progenitor number, and vessel-skeletal progenitor spatial distances (Supplementary Fig. 2E). The resulting analysis provides a comprehensive characterization of the spatial coupling between vessels and skeletal progenitors by reflecting the native 3D environment across large tissue volumes (cm3).
3D map of calvarial vessels and skeletal progenitors
Using our imaging pipeline, we generated high-resolution 3D maps of vessel phenotypes, Osterix+ skeletal progenitors, and Gli1+ skeletal progenitors in the parietal and posterior frontal bones of juvenile 4-weeks-old mice. Vessels were located in the periosteum, dura mater, transcortical canals, marrow cavities, and osteogenic fronts adjacent to the sutures (Fig. 2A–D and Supplementary Fig. 3A, B). Marrow vessels in the parietal and frontal bones were observed near the sutures, while only periosteal and meningeal vessels were observed at the center of each bone. Vessel phenotypes were also differentially distributed in the calvarium: Most CD31hiEmcn− arteries and arterioles were present in the periosteum and dura mater, while CD31loEmcnhi/lo sinusoids were restricted to the marrow cavities. CD31hiEmcnhi capillaries were present in the periosteum, dura mater, marrow cavities, and osteogenic fronts, connecting CD31hiEmcn− periosteal arterioles to CD31loEmcnhi sinusoids. Expression of CD31 and Emcn in CD31hiEmcnhi capillaries was most intense at the transcortical canals—the regions that enable arterioles in the periosteum and dura mater to connect to venous sinusoids.
A, B MIP displaying calvarial vessels (Ai, Bi) and Osterix+ (Ai, Ai’) or Gli1+ skeletal progenitors (Bi, Bi’) in the parietal and posterior frontal bones of the calvarium. Zoomed-in images demonstrate the distribution of vessel phenotypes (Aii–vi, Bii–vi) and skeletal progenitors (Aii–vi, Aii’–vi’, Bii–vi, Bii’–vi’) in different regions. C, D 50-μm-thick sagittal sections showing the locations of vessels Ci–ii, Di–ii) and skeletal progenitors (Ci–ii, Ci’–ii’, Di–ii, Di’–ii’) along the thickness of the calvarium. CD31hiEmcn− and CD31hiEmcnhi vessels are prevalent in the periosteum and dura mater (yellow arrows), while CD31hiEmcnhi and CD31loEmcnhi vessels make up the majority of vessels in the bone marrow cavities (white arrows). Osterix+ progenitors (A, C) are distributed throughout the periosteum and dura mater (yellow arrows), transcortical canals (white arrowheads), and osteogenic fronts nearby the sutures (yellow arrowheads), while Gli1+ progenitors (B, D) are mainly restricted to the transcortical canals and sutures. Results were replicated in 3 calvaria for each staining combination (CD31/Emcn/Osterix, CD31/Emcn/Gli1). E Plots showing the nearest distance of Osterix+ (top) and Gli1+ (bottom) progenitors to each vessel phenotype (n = 3). Both cell types exhibit a preferential spatial relationship to CD31hiEmcnhi vessels compared to the other phenotypes. CD31loEmcnlo vessels were not quantified due to their low fluorescence signal-to-noise ratio. Data are mean ± SD. Statistics were performed using a two-way ANOVA and Bonferroni post-hoc test. ***p < 0.001, **p < 0.01, *p < 0.05 between CD31hiEmcnhi and CD31hiEmcn− vessels; +++p < 0.001 between CD31loEmcnhi and CD31hiEmcn− vessels; Scale bars: 1000 μm (Ai, Bi), 100 μm (Aii–vi, Bii–vi), 700 μm (Ci), 300 μm (Cii), 500 μm (Di), 200 μm (Dii). Colors: Red: Endomucin (Emcn), Gray: Osterix (A, C) or Gli1 (B, D), Green: CD31.
Similar to vessel phenotypes, skeletal progenitors varied in their spatial distribution. Osterix+ osteoprogenitors were prevalent in the periosteum and dura mater, osteogenic fronts nearby sutures, transcortical canals, and marrow cavities of the parietal and frontal bones (Fig. 2A, C). Gli1+ progenitors—a marker for less-differentiated skeletal stem cells16,17—were concentrated at the sutures, transcortical canals, and marrow cavities adjacent to transcortical canals, but they were mostly absent from the periosteum and dura mater (Fig. 2B, D). Interestingly, expression of Gli1 was visibly more intense at the transcortical canals compared to the sutures.
To determine whether skeletal progenitors exhibited a preferential spatial relationship to specific vessel phenotypes, we quantified the distribution of Osterix+ and Gli1+ progenitors relative to each vessel type. We found that both progenitor populations were preferentially associated with CD31hiEmcnhi vessels compared to other vessel phenotypes (Fig. 2E). This relationship was most apparent at the transcortical canals, where we observed the highest protein expression of CD31 and Emcn in vessels and Osterix or Gli1 in skeletal progenitors.
Postnatal growth shifts the distribution of vessel phenotypes and Osterix+ progenitors
Next, we compared the calvaria of juvenile (4-weeks-old) and adult (12-weeks-old) mice to determine how vessel phenotype and skeletal progenitor distribution change following postnatal growth. In adult mice, there were fewer CD31hiEmcn− periosteal and meningeal vessels and visible increases in CD31loEmcnlo sinusoids (not quantified due to low fluorescence intensity, Fig. 3A–F, I). These changes were corroborated by microCT data, which showed greater development of bone marrow cavities in adult calvaria (Fig. 3K–M). There were no significant changes in the volume of CD31hiEmcnhi or CD31loEmcnhi sinusoids, although fewer CD31hiEmcnhi vessels were observed in the periosteum and dura mater (Fig. 3A–F, I). Additionally, the total vessel volume remained the same (Fig. 3H).
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.
Along with changes in vessel phenotypes, the numbers of Osterix+ and Gli1+ skeletal progenitors decreased in adult calvaria, and their distribution was mainly restricted to the sutures, transcortical canals, and bone marrow cavities (Fig. 3A–F, G). While a decrease in progenitors was observed in different regions of the calvarium, the most significant decline occurred in the parietal bones (Supplementary Fig. 4A). Moreover, Osterix+ cells were mostly absent in the periosteum and dura mater of adult calvaria (Fig. 3A–C). These results correlated with differences in vessel-progenitor relationships: The fraction of Osterix+ cells within 5 μm of the nearest vessel in adult versus juvenile calvaria was significantly higher for CD31hiEmcnhi and CD31loEmcnhi vessels and lower for CD31hiEmcn− vessels (Fig. 3J). These trends held across different regions of the calvarium (Supplementary Fig. 4B). There were no significant changes in the relationship of Gli1+ cells to vessel phenotypes between juvenile and adult calvaria (Fig. 3J). Nonetheless, both progenitor cell types maintained a preferential spatial association with CD31hiEmcnhi vessels at 4 and 12 weeks of age.
PTH stimulates Osterix+ progenitor proliferation, but does not alter vessel phenotype distribution
To provide insight on how vessel phenotypes and skeletal progenitors interact during calvarial bone remodeling, we administered a parathyroid hormone analog (PTH 1–34) daily for 1 month, a regimen previously shown to increase osteoblast number and bone mineral deposition in murine long bone20. We found that PTH administration did not significantly change the fractional volume for each vessel phenotype or total vessel volume; although, there were areas of increased Emcn signal intensity in sinusoids near the transcortical canals (Fig. 4A–D, G–J, K, L). Furthermore, we observed increased marrow cavities and CD31loEmcnlo sinusoids in the parietal bone with PTH administration—a finding complemented by decreased bone volume to total volume percentage (BV/TV) and increased bone surface area (SA) (Fig. 4O–Q).
A–D MIP of calvarial vessels (A–D) and Osterix+ progenitors (A–D, A’–D’) in 12-weeks-old mice with (B, B’, D, D’) or without (A, A’, C, C’) 4 weeks of PTH administration. Increased Osterix+ cells are present within the periosteum and dura mater of PTH-treated calvaria (yellow arrows). E–J 40-μm-thick sagittal sections demonstrating the expansion of Osterix+ progenitors in the periosteum and dura mater (yellow arrows) in PTH-treated calvaria (E) compared to the control (F). G–J MIP of calvarial vessels (G–J) and Gli1+ skeletal progenitors (G–J, G’–J’) in control (G, G’, I, I’) and PTH-treated (H, H’, J, J’) groups. More Gli1+ cells are observable in the marrow cavities nearby the transcortical canals (white arrows), as well as in the transcortical canals (white arrowheads) and periosteum (yellow arrows) nearby the sutures in PTH-treated calvaria. Results were replicated in 3 calvaria for each experimental group and staining combination (12 total calvaria). K–M Total vessel volume (K), fractional vessel phenotype volume (L), and skeletal progenitor number (M) in control and PTH-treated calvaria (n = 3 for M; n = 6 for K, L). N 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 following PTH treatment, but fewer Osterix+ cells are associated with CD31hiEmcnhi vessels compared to control calvaria. O MicroCT quantification of bone volume (BV), bone volume to tissue volume (BV/TV) percentage, and bone surface area (SA) in the parietal and posterior frontal bones of control and PTH-treated calvaria (n = 4). P, Q MicroCT 3D volume projections of control (P) and PTH-treated calvaria (Q). Data are mean ± SD. Statistics were performed using a two-way ANOVA with Bonferroni post-hoc test (L–N) or two-tailed t-test (K, O). ***p < 0.001, **p < 0.01, *p < 0.05 where designated or between CD31hiEmcnhi and CD31hiEmcn− vessels in N; +++p < 0.001 between CD31loEmcnhi and CD31hiEmcn− vessels (N). Exact p-values for two-tailed t-tests: K p = 0.7493, O p = 0.7771 (top left), 0.2299 (top right), 0.0021 (middle left), 0.7862 (middle right), 0.0021 (bottom left), 0.2111 (bottom right). Scale bars: 1000 μm (A, B, G, H, P, Q); 700 μm (E, F); 300 μm (I, J); 200 μm (C, D). Colors: Red: Endomucin (Emcn), Gray: Osterix (A–F) or Gli1 (G–J), Green: CD31.
Despite a lack of significant change in vessel phenotypes, we found differences in the skeletal progenitor populations with PTH administration. PTH significantly increased the total number of Osterix+ progenitors, especially in the periosteum and dura mater (Fig. 4A–F, M). By contrast, PTH did not increase the number of Gli1+ progenitors (Fig. 4G–J, M). However, there were some changes in Gli1+ progenitor distribution. Cells moderately expressing Gli1 expanded in the marrow cavities adjacent to transcortical canals—particularly near vessels with high Emcn expression—and in periosteal and transcortical canals nearby the coronal suture (Fig. 4J). Both Osterix+ and Gli1+ progenitors remained preferentially associated with CD31hiEmcnhi vessels following PTH administration, but the fraction of Osterix+ cells within 5 μm of a CD31hiEmcnhi vessel was significantly reduced compared to the control (Fig. 4N).
Loss of preosteoclast PDGF-BB secretion decreases the spatial affinity of skeletal progenitors to CD31hiEmcnhi vessels
Preosteoclast-derived PDGF-BB is required for angiogenesis with coupled osteogenesis during normal bone homeostasis and in disease conditions21,22. To determine the phenotypic changes in calvarial blood vessels and skeletal progenitors in response to decreased bone remodeling activity, we used Trap+ osteoclast lineage-specific conditional Pdgfb deletion mice (PdgfbcKO) by crossing Trap-Cre mice with Pdgfb-floxed mice. In the calvaria of 4-weeks-old mice, we found that CD31hiEmcnhi fractional volume and total vessel volume decreased, while fractional CD31hiEmcn− vessel volume increased in PdgfbcKO mice relative to Pdgfb-floxed (WT) mice (Fig. 5A–H, K–L). While we did not find any statistical differences in the number of Osterix+ or Gli1+ cells, we found that the preferential association of these cells to CD31hiEmcnhi vessels was significantly reduced in PdgfbcKO calvaria (Fig. 5I–J, M). This effect was most apparent at the transcortical canals and osteogenic fronts, where the concentration of Osterix+ and Gli1+ cells was visibly lower in PdgfbcKO versus WT calvaria (Fig. 5C, D, G–H). We also observed alterations in bone microarchitecture: There was less bone marrow cavity development in the frontal bones of PdgfbcKO calvaria, as demonstrated by a higher BV/TV percentage and lower bone SA (Fig. 5N–P). To determine whether osteoclasts resided in close proximity to these regions, we stained for Vpp3—a marker known to exclusively stain osteoclasts in bone23. Most osteoclasts were found adjacent to CD31hiEmcnhi and CD31loEmcnhi vessels in the marrow cavities and within proximity to the transcortical canals (Supplementary Fig. 5A–C).
A–H MIP of vessels (A–H), Osterix+ progenitors (A–D, A’–D’), and Gli1+ progenitors (E–H, E’–H’) from the calvaria of 4-weeks-old WT and PdgfbcKO mice. Protein expression of CD31 and Emcn in vessels and Osterix or Gli1 in skeletal progenitors is markedly reduced in PdgfbcKO calvaria at the transcortical canals (arrowheads) and osteogenic fronts nearby the sutures. Results were replicated in 4 WT calvaria and 3 PdgfbcKO calvaria for each staining combination (14 total calvaria). I Spatial distribution of Osterix+ and Gli1+ cells relative to each vessel phenotype in WT and PdgfbcKO calvaria (WT: n = 4 Osterix, n = 4 Gli1; PdgfbcKO: n = 3 Osterix, n = 3 Gli1). J Fraction of Osterix+ and Gli1+ progenitors within 5 μm of their nearest vessel in WT and PdgfbcKO calvaria (WT: n = 4 Osterix, n = 4 Gli1; PdgfbcKO: n = 3 Osterix, n = 3 Gli1). Significantly fewer Osterix+ cells are associated with CD31hiEmcnhi vessels in PdgfbcKO calvaria compared to the WT control. K–M Total vessel volume (K), fractional vessel phenotype volume (L), and skeletal progenitor number (M) in the calvaria of WT versus PdgfbcKO mice (n = 8 for WT and n = 6 for PdgfbcKO in K–L; M: n is the same as in I and J). N MicroCT quantification of bone volume (BV), bone volume to tissue volume (BV/TV) percentage, and bone surface area (SA) in the parietal and posterior frontal bones of WT and PdgfbcKO calvaria (WT: n = 6; PdgfbcKO: n = 5). O, P MicroCT 3D volume projections of WT (O) and PdgfbcKO calvaria (P). Data are mean ± SD. Statistics were performed using a two-way ANOVA with Bonferroni post-hoc test (I, J, L, M) or two-tailed t-test (K, N). ***p < 0.001, **p < 0.01, *p < 0.05 where designated or between CD31hiEmcnhi and CD31hiEmcn− vessels in l; +++p < 0.001 between CD31loEmcnhi and CD31hiEmcn− vessels (I). Exact p-values for two-tailed t-tests: K p = 0.0112, N p = 0.2565 (top left), 0.0736 (bottom left), 0.2183 (top middle), 0.0152 (bottom middle), 0.6635 (top right), 0.0151 (bottom right). Scale bars: 1000 μm (A, B, E, F, O, P); 300 μm (C, D, G, H). Colors: Red: Endomucin (Emcn), Gray: Osterix (A–D) or Gli1 (E–H), Green: CD31.
CD31hiEmcnhi vessels and Gli1+ progenitors infiltrate into calvarial defect following injury
In addition to remodeling, we investigated the contribution of vessel phenotypes and skeletal progenitors to calvarial bone healing. We created 1-mm subcritical-sized defects in the parietal bone of adult mice and evaluated healing at 21- and 56-days following fracture (PFD21, PFD56; PFD: post-fracture day). At PFD21, defects were highly vascularized, and the majority of vessels were CD31hiEmcnhi (Fig. 6A, B, E, F, L, N). Gli1+ cells were highly concentrated across the entire defect region, while Osterix+ cells resided in regions of active bone formation (Fig. 6A, B, E, F and Supplementary Fig. 6A). In addition, there was a substantial expansion of Osterix+ and Gli1+ cells in the periosteum extending from the defect to nearby sutures (Fig. 6A, B, E, F, I, J). This effect was unique to the periosteum, as there were few Osterix+ and Gli1+ cells detected in the dura mater—the only layer that remained uninjured following the creation of the defect (Fig. 6I, J). By PFD56, total vessel volume, fractional CD31hiEmcnhi volume, Osterix+ cell number, and Gli1+ cell number decreased in the defect relative to PFD21, but the vessel and Gli1+ cell density remained higher relative to the surrounding uninjured bone (Fig. 6C, D, G, H, L–N). Furthermore, there was no significant change in defect bone volume, suggesting that most healing happened within the first 3 weeks of injury (Fig. 6O and Supplementary Fig. 6A, B). Nevertheless, both Osterix+ and Gli1+ progenitors remained preferentially associated with CD31hiEmcnhi vessels at PFD21 and PFD56 (Fig. 6K).
A–H Blood vessels (A–H), Osterix+ progenitors (A–D, A’–D’), and Gli1+ progenitors (E–H, E’–H’) inside and around 1-mm parietal bone defects at PFD21 (A, B, E, F) and PFD56 (C, D, G, H). The dotted circle marks the defect region. I, J Expansion of Osterix+ and Gli1+ progenitors in the periosteum (yellow arrows) nearby the defect region (dotted lines) and sutures (yellow arrowheads) at PFD21. Progenitors were sparsely populated in the dura mater—the only portion of the calvarium that remained intact following defect injury. Results were replicated in 3 calvaria for each timepoint and staining combination (12 total calvaria). K Spatial relationship of skeletal progenitors to different vessel phenotypes at PFD21 and PFD56 (n = 3). L–N Fractional vessel phenotype volume (L), skeletal progenitor number (M), and total vessel volume (N) in the defect at PFD21 and PFD56 (n = 6 for L, N; n = 3 for M). O MicroCT quantification of defect to contralateral bone volume ratio at PFD21 and PFD56 (n = 4). Data are mean ± SD. Statistics were performed using a two-way ANOVA with Bonferroni post-hoc test (K–M) or two-tailed t-test (N–O). ***p < 0.001, **p < 0.01, *p < 0.05 where designated or between CD31hiEmcnhi and CD31hiEmcn− vessels in K; +++p < 0.001 between CD31loEmcnhi and CD31hiEmcn− vessels. Exact p-values for two-tailed t-tests: N p = 0.0094, O p = 0.5660. Scale bars: 500 μm (I, J); 300 μm (A, C, E, G); 200 μm (B, D, F, H). Colors: Red: Endomucin (Emcn), Gray: Osterix (A–D, I, J) or Gli1 (E–H), Green: CD31.
Since there was a significant expansion of skeletal progenitors around the defect region, we evaluated whether there was a systemic response to injury. We quantified skeletal progenitors in the ipsilateral and contralateral sides of the parietal bone and compared them to the number of progenitors in uninjured adult mice. Surprisingly, there were elevated levels of Gli1+ cells in both the ipsilateral and contralateral sides of the injured calvaria at PFD21 compared to the uninjured calvaria (Supplementary Fig. 6D–S). Most of this expansion occurred in the periosteum, especially in the regions near the sutures (Supplementary Fig. 5D, G, J, M). By PFD56, Gli1+ and Osterix+ cell number significantly decreased to levels comparable to the uninjured calvaria (Supplementary Fig. 6P–S). However, alterations in bone surface topography and regions of excess mineral formation remained at PFD56 (Supplementary Fig. 6A–C).
