Fig. 6: Single-cell transcriptomic profiling of mature osteoblasts and differentiating osteocytes highlights a key role for Sp7.

a Long bones were subjected to serial collagenase/EDTA digestions (see methods), and cells from fractions 5, 7, and 8 were collected for flow cytometry. Viable (DAPI-negative) tdTomato-positive cells were sorted followed by single-cell RNA-seq library construction. See also Supplementary Figs. 5 and 6. Cell-clustering from WT mice was performed. b–e Feature plots showing the expression of cluster-specific markers: Tnc (c3); Kcnk2 (c4); Dpysl3 (c5); Fbln7 (c6). f–j RNA in situ hybridization of cluster-specific markers: Tnc, Kcnk2, Dpysl3, and Fbln7. Kcnk2 expression (red triangle) is primarily noted in endosteal cells that form a canopy around the osteoblast. Dpysl3 expression is noted in osteocytes close to bone surfaces (green) and a subpopulation of endosteal cells (blue). Fbln7 expression is noted only in embedded osteocytes (brown). Negative control: Dabp. k Integrated analysis of cells from WT (blue) and Sp7^OcyKO (red) mice after down-sampling of WT library to match cellular representation seen in Sp7 mutant mice. l Feature plot showing Sp7 expression across 8 Ob/Ocy clusters. As expected, Sp7 expression is reduced in mutant mice. m Relative proportions (out of 1.0) in WT vs Sp7^OcyKO mice. Sp7 mutants show reduced canonical osteoblasts (cluster 1), increased cells in clusters 3–5, and reduced cells in cluster 6 (mature osteocytes). Barnard’s exact test was used to determine the significance between genotypes. n Monocle3 analysis showing pseudotime trajectory mapping across tdTomato-positive cells in WT (left) and Sp7^OcyKO (right) mice. Red arrows indicate Sp7-specific subpopulations in clusters 3 and 5, demonstrating apparently arrested differentiation. o Left: the expression of top 150 mature osteocyte markers (c6) was analyzed in a mouse brain single-cell RNA-seq atlas, where significant enrichment is found in aggregated expression values for neurons (p = 0.022) as compared against all other major cell type aggregated expression values. Right: the expression of the top 150 osteoblast marker (c1 + 2) was analyzed in a mouse brain single-cell RNA-seq atlas. OPC oligodendrocyte progenitor cell, Mitotic mitotic cells, Neurogenesis neurogenesis-associated cells. p Left: the expression of 77 osteocyte-specific Sp7 targets was analyzed in a mouse brain scRNA-seq atlas, where significant enrichment is found in aggregated expression values for neurons (p = 0.047) as compared against all other major cell type aggregated expression values. Right: the expression of 134 POB-specific Sp7 targets was analyzed in a mouse brain scRNA-seq atlas, where significant enrichment is found in aggregated expression values for neurons (p = 0.045) as compared against all other major cell type aggregated expression values. Wilcoxon rank-sum test was used in o and p, exact p values are listed above.