Fig. 5: RNA sequencing of molecular changes in Clec14a−/− lysates.
A–E Clustering and gene ontology analysis of Clec14a+/+ (WT) and Clec14a−/− pup calvaria lysates. A, B Unsupervised hierarchical clustering, (dendrogram – A; principal component analysis – B) of log2 normalised counting reads over exons (RPM-reads per million) for all detected genes, N = 4 for both groups. Clec14a−/− samples (blue), Clec14a+/+ samples (WT - orange). Each sample represents a different individual animal. C) Differentially expressed gene counts. Raw counts were analysed with DESeq2, significantly expressed genes were cut off at log2FC values > 0.5, adjusted p (padj) < 0.05, p value was corrected for multiple comparisons (Benjamini–Hochberg); Orange = genes upregulated in Clec14a−/− samples; Blue = genes downregulated in Clec14a−/− samples. D GSEA analysis enrichment score for pathways related to endochondral ossification. ES-enrichment score, FDR -false discovery rate. Gene expression is presented as a Z score (scale). E GSEA heatmap showing the list of leading-edge genes enriched at the top of the Wikipathways ‘Endochondral Ossification’ gene set. Heatmap presents upregulation of transcripts relevant to endochondral ossification in Clec14a−/− (blue) samples compared to WT control (orange). Gene expression is presented as a Z score. Transcripts presented were identified as statistically significant by GSEA analysis, FDR (q-value) < 0.05, nominal p < 0.01. F Analysis of expression of genes linked to osteoblast maturation and osteogenic activity. P4 pup calvarial lysates were subjected to RT qPCR analysis to dissect expression patterns of genes known to regulate commitment to osteoblast lineage and osteoblast maturation, gene expression was normalised to Gapdh, shown as arbitrary units (2^-ΔCT). Data is presented as median ± IQR, a Mann–Whitney test was used to analyse the results. Plots represent results from two independent experiments (n = 7 Clec14a+/+, n = 4–5 Clec14a−/−), data points in each graph are biological replicates.
