Fig 1.
Identification of Wnt3a regulated transcriptome in osteoblasts.
A) Experimental setup. Osteoblasts were isolated from calvaria of 5–6 days old mice and treated with Wnt3a recombinant protein. RNA was isolated from Wnt3a treated and sham treated cultures 24h post treatment, and sequenced using Illumina high-throughput sequencing technology. B) Genes up- and down-regulated by Wnt3a in osteoblasts. C) Heatmap shows the expression of differentially regulated Wnt pathway members in sham treated WT OBs (WT) and Wnt3a treated WT OBs (WT+Wnt3a). D) Enriched biological processes associated with up- and down-regulated genes. Figure shows the number of up- and down-regulated genes associated with relevant biological processes.
Table 1.
Top 25 up- and down-regulated genes with highest log2 fold changes.
Table 2.
Key signaling pathways regulated by Wnt3a.
Table 3.
Wnt3a targets associated with GO term ‘ossification’.
Table 4.
Wnt3a target genes associated with bone phenotypes in mice.
Fig 2.
Expression profiles of Wnt3a targets during the differentiation of pre-osteoblast to mature osteoblasts.
A) Expression profile of osteocalcin, a mature osteoblast marker. Osteocalcin expression starts around day 10. B) Expression profiles of genes up-regulated by Wnt3a. A large number of genes up-regulated by Wnt3a were highly expressed in early stage (D2-D8) osteoblasts (highlighted with *). C) Expression profiles of genes down-regulated by Wnt3a. Most of the genes down-regulated by Wnt3a were highly expressed in mature (D10-D18) osteoblasts (highlighted with #).
Fig 3.
Effect of Lrp5 and Lrp6 ablation in osteoblasts.
A) Expression of Lrp5 in WT and Lrp5KO OBs. Expression values are given in counts per million (CPM) mapped reads. B) Expression of Lrp6 in controls (Lrp6fl/fl+TMX) and Lrp6KO OBs (Lrp6fl/fl;UBC-Cre-ERT2+TMX). Expression values indicate the number of RNA-seq reads mapped to exon2 (deleted exon) in Lrp6 (in CPM). C) Expression of Lrp5 and Lrp6 in controls (Lrp5fl/fl;Lrp6fl/fl+TMX) and Lrp5/6KO OBs (Lrp5fl/fl;Lrp6fl/fl; UBC-Cre-ERT2+TMX). Expression values indicate the number of RNA-seq reads mapped to exon2 in Lrp5 and Lrp6 (in CPM). D) Venn diagram showing overlap between genes up-regulated in Lrp5KO OBs, Lrp6KO OBs and Lrp5/6KO OBs compared to respective controls. E) Venn diagram showing overlap between genes down-regulated in Lrp5KO OBs, Lrp6KO OBs and Lrp5/6KO OBs compared to respective controls.
Fig 4.
Expression profiles of Lgals3bp and Irgm2 during osteoblast differentiation.
Expression profiles of Lgals3bp and Irgm2 compared to pre-osteoblast marker Sp7 and mature osteoblast marker Bglap (Osteocalcin). Expression values were obtained from the RNA-seq dataset GSE54461. Both Lgals3bp and Irgm2 were robustly expressed on osteoblasts and showed highest expression at day 8 (D8).
Fig 5.
Wnt3a regulated genes in Lrp5KO osteoblasts.
A) Venn diagram showing overlap between genes up-regulated in Wnt3a treated Lrp5KO OBs (Lrp5KO+Wnt3a) compared to sham treated WT OBs (WT) and sham treated Lrp5KO OBs (Lrp5KO). B) Venn diagram showing overlap between genes down-regulated in Wnt3a treated Lrp5KO OBs (Lrp5KO+Wnt3a) compared to sham treated WT OBs (WT) and sham treated Lrp5KO OBs (Lrp5KO). C) Heatmap showing genes up- or down-regulated by Wnt3a in both WT OBs (WT +Wnt3a) and Lrp5KO OBs (Lrp5KO+Wnt3a) compared to WT (WT) controls. D) Heatmap showing genes up- or down-regulated by Wnt3a in WT OBs (WT +Wnt3a) but, not in Lrp5KO OBs (Lrp5KO+Wnt3a) compared to WT (WT) controls. These genes either had a fold change < 1.5 or FDR corrected p-value > 0.05.
Fig 6.
Overlap between Wnt3a targets identified in WT, Lrp5KO, Lrp6KO and Lrp5/6KO osteoblasts.
A) Overlap between genes up-regulated by Wnt3a. B) Overlap between genes down-regulated by Wnt3a.