Fig. 3: Defining an osteocyte transcriptome signature.
a Experimental design used to identify genes with enriched expression in osteocytes. Diagram illustrating the strategy used to identify genes enriched for expression in osteocytes in bone samples enriched with osteocytes relative to intact bone samples in which bone marrow and cells lining bone were retained (i). Experimental workflow used to identify genes enriched for expression in osteocytes (ii). b Gene-enrichment in osteocyte-enriched bone samples relative to whole-bone samples distinguished genes known to be expressed in osteocytes (blue) from housekeeping genes (green) and genes expressed in marrow cell populations (yellow) (i). Enrichment for each individual gene is shown as a dot and plotted as a function of normalised gene expression (log2-FPKM). The four component gaussian mixture model fitted to the density distribution of gene-enrichment in (i) used to define the osteocyte enrichment threshold (ii). Each component is denoted by a separate colour. The top two Gene Ontology biological processes associated with genes in each component are illustrated in coloured boxes with p-values (Bonferroni adjusted) calculated by hypergeometric test. (ii). 1777 genes above the osteocyte-enrichment threshold (indicated by the red dashed line) were significantly enriched for expression in osteocytes (iii). LFC = log2 fold-change. c Volcano plots comparing the expression of osteocyte-enriched genes (from b) with blood (i), bone marrow (ii) and muscle (iii)13. Dashed lines represent p < 0.05 cutoff. Genes significantly enriched in these tissues relative to osteocytes are identified as red dots. P-values were calculated in the original publishing article13 by F-test with Benjamini–Hochberg adjustment. d Filtering pipeline used to define the 1239-gene osteocyte transcriptome signature (OTS). The number of genes remaining at each filtering stage is indicated. e Expression specificity29 (Tau) of OTS genes relative to other organs and tissues28. Genes with Tau <0.15 = low expression specificity in osteocytes (green), 0.15 ≤ Tau ≤ 0.85 = moderate expression specificity (orange), while Tau > 0.85 = high expression specificity (red). f OTS genes were enriched for expression in osteocytes relative to osteoblasts and bone-lining cells isolated by laser-capture microdissection. Tukey boxplots show a summary of median OTS gene expression values in each cell type calculated across n = 3 biological replicates from a single experiment. Boxes indicate median and interquartile range (IQR) of scaled, normalised gene expression values, whiskers denote values ±1.5 × IQR and outlier values beyond this range are shown as individual points. P-values (two-tailed, Benjamini and Hochberg adjusted) were calculated by competitive gene set test accounting for inter-gene correlation (CAMERA)105.
