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Fig. 4. prrx1b−/− hearts contain excessive amounts of pro-fibrotic fibroblasts. (A) tSNE map of the single-cell sequencing data as shown in Fig. 3C, indicating ten transcriptionally distinct cell populations. (B) tSNE map showing the contribution of wild-type cells (cyan) and prrx1b−/− cells (red). (C) Stacked bar graph showing the relative cell contribution to major clusters in wild-type and prrx1b−/− hearts. (D) Pie charts showing the contribution of wild-type and prrx1b−/− cells per cluster. (E) Differential gene expression analysis using the DESeq algorithm between fibroblast clusters 2 and 3. Enriched genes were selected for either cluster 2 or 3 with a P-value cut-off of <0.05 (red). Gene Ontology analysis was performed using the online tool DAVID. Gene and full Gene Ontology lists are provided in Tables S2 and S3. (F,G) Characterization of cluster 3. Left: tSNE maps visualizing log2-transformed read-counts for genes with high expression in the indicated cluster (circled). Middle: In situ hybridization for the cluster 3-enriched genes in wild-type and prrx1b−/− hearts at 7 dpi. Dashed line indicates injury border. Scale bars: 100 μm. Right: Magnifications of the boxed regions in the injury area with arrowheads pointing to cells with high expression. Scale bars: 25 μm. Three hearts analysed per condition. (H) Sirius Red staining showing collagen in red on sections of wild-type and prrx1b−/− hearts at 7 dpi. Right-hand panels show magnifications of the boxed regions in the sub-epicardial layer and further inside the injury area. Scale bars:100 μm (left); 50 μm (right). (I) Quantification of Sirius Red (collagen) staining in wild-type (n=6) and prrx1b−/− (n=7) hearts showing significantly more fibrosis in prrx1b−/− hearts inside and around the injury area (mean±s.d., P=0.012, unpaired t-test).