Heart regeneration and border zone cardiomyocyte proliferation is impaired in prrx1b^−/− zebrafish. (A) AFOG staining on 30 dpi wild-type and prrx1b^−/− heart sections showing fibrin in red, collagen in blue and remaining muscle tissue in orange. Scale bars: 100 μm. (B) Quantification of the remaining scar size at 30 dpi in prrx1b^−/− hearts (n=6) and wild-type sibling hearts (n=9) (mean±s.d., P=0.0082, unpaired t-test). (C) AFOG staining on sections of 90 dpi hearts. Scars were completely resolved in wild-type hearts (n=12), whereas incomplete scar resolution was observed in prrx1b^−/− four out of seven hearts. Scale bars: 100 μm. (D) Immunofluorescence staining on 7 dpi wild-type and prrx1b^−/− heart sections using an anti-Mef2 antibody as a marker for cardiomyocyte nuclei, and an anti-PCNA antibody as a nuclear proliferation marker. Insets show higher magnifications of the boxed areas. Arrowheads indicate proliferating cardiomyocytes. Scale bars: 100 μm (main panels); 10 μm (insets). (E) Quantification of the percentage of proliferating (PCNA⁺) border zone cardiomyocytes in prrx1b^−/− hearts (n=8) and wild-type sibling hearts (n=7) (mean±s.d., P<0.0001, unpaired t-test).

Prrx1 is expressed in the epicardium/EPDCs and follows epicardial dynamics post-injury. (A) Schematic illustrating the experimental procedures. (B-F) Immunofluorescence staining on 1, 3, 7, 14 and 30 dpi tcf21:mCherry⁺ wild-type heart sections staining Prrx1 (green) and mCherry (magenta). Areas in the coloured boxes are shown at higher magnification below. Arrowheads indicate tcf21:mCherry⁺/Prrx1⁺ cells. Scale bars: 100 μm (low-magnification images); 10 μm (high-magnification images). BZ epicardium, border zone epicardium; Rm epicardium, remote epicardium. Six hearts analysed per condition. Dashed line indicates the border between myocardium and injury area. (G) Schematic of Prrx1 dynamics upon injury. Prrx1⁺ cells are in green. Dark colour at the apex represents the injury area. (H) Quantification of the distribution of tcf21:mCherry⁺/Prrx1⁺ cells per time point. Size of the dots represents the percentage of tcf21:mCherry⁺/Prrx1⁺ cells and absolute count number is visualized by a colour gradient.

Single-cell sequencing identifies epicardial-derived cell populations in the injured zebrafish heart. (A,B) Workflow of the isolation (A) and sorting (B) of tcf21:mCherry⁺ cells in wild-type and prrx1b^−/− hearts at 7 dpi. (C,D) tSNE plotting of the data results in ten transcriptionally distinct clusters (C), as also indicated by the heatmap (D). (E) tSNE maps visualizing log2-transformed read-counts for tcf21, tbx18, aldh1a2 and wt1b. (F-I) Characterization of the different cell clusters. Left: Panels show tSNE maps visualizing log2-transformed read-counts for genes with high expression in the indicated cluster (circled). Middle: In situ hybridization for the cluster-enriched genes in wild-type hearts at 7 dpi. Dashed line indicates injury border. Scale bars: 100 μm. Right: Magnifications of the boxed regions in remote (RE) and injury epicardium (IE) with arrowheads pointing to cells with high expression. Scale bars:10 μm. Three hearts analysed per condition. Gene lists are provided in Table S1.

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).

Prrx1b stimulates Nrg1 expression. (A) RNAscope in situ hybridization for nrg1 co-detected with Prrx1 antibody on 7 dpi wild-type hearts. Arrowheads indicate colocalization of nrg1 and Prrx1. Dashed line marks edge of the border zone. Insets show higher magnifications of the boxed areas. Scale bars: 100 μm (main panels); 10 μm (insets). Four hearts analysed. (B) RNAscope in situ hybridization for nrg1 on 7 dpi wild-type and prrx1b^−/− hearts. Dashed line marks edge of the border zone. Insets show higher magnifications of the boxed areas. Scale bars: 100 μm (main panels); 10 μm (insets). (C) Quantification of nrg1 RNAscope dots in the BZ epicardium in 7 dpi wild-type (n=6) and prrx1b^−/− (n=5) hearts. BZ epicardium is defined as a 100-μm-wide strip, 100 μm up and 100 μm down from where the edge of intact myocardium meets the epicardium (mean±s.d., P=0.0051, unpaired t-test). (D) Schematic of the workflow used for NRG1 injection experiments shown in E. (E) Quantification of the percentage of proliferating (PCNA⁺) BZ cardiomyocytes (mean±s.d., wild-type −NRG1 n=7; wild-type +NRG1 n=8; prrx1b^−/− −NRG1 n=4; prrx1b^−/− +NRG1 n=7; wild-type −NRG1 versus prrx1b^−/−−NRG1 P=0.0118; prrx1b^−/−−NRG1 versus prrx1b^−/−+NRG1 P=0.0013; ns, not significant; one-way ANOVA followed by multiple comparisons analysis using Tukey's test).

PRRX1 promotes NRG1 expression in human EPDCs. (A) Schematic of the workflow for the experiments shown in B and C. After isolation, human fetal epicardial cells are cultured in the presence of the ALK4/5/7 kinase inhibitor SB-431542. Cells transform from cobble- to spindle-shape upon removal of SB-431542. (B) Representative brightfield pictures of cobble- and spindle-shaped human fetal epicardial cells. Scale bars: 100 µm. (C) qPCR results for POSTN, FN1, PRRX1 and NRG1 in human fetal cobble (n=3) and spindle (n=3) epicardial cells (mean±s.d.; POSTN P<0.001, FN1 P=0.001, PRRX1 P=0.01, NRG1 P<0.001, unpaired t-tests). (D) Schematic of the workflow for the experiments shown in E and F. (E) Western blot for PRRX1 in U87 cells. Vinculin was used as a loading control. (F) qPCR results for PRRX1 and NRG1 in human fetal spindle epicardial cells after PRRX1 siRNA treatment (non-transfected cells n=4, CTRL siRNA n=4, PRRX1 siRNA n=4) (mean±s.d., PRRX1 CTRL siRNA versus PRRX1 siRNA P=0.003, NRG1 CTRL siRNA versus PRRX1 siRNA P=0.04, unpaired t-tests) (G) ELISA results for secreted NRG1-β1 in the conditioned cell culture medium of human fetal spindle epicardial cells between 24 and 48 h after PRRX1 siRNA treatment (non-transfected cells n=3, CTRL siRNA n=3, PRRX1 siRNA n=3) (mean±s.d., CTRL siRNA versus PRRX1 siRNA P=0.0061, unpaired t-tests).

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.