Attenuation of epicardial activation and myofibroblast abundance via the Fbln2–Nupr1b axis stimulates cardiac regeneration in zebrafish

Figures

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  Fig. 1. fbln2 is upregulated in EPDCs and endothelial cells after cardiac cryoinjury in zebrafish. a,b, In situ HCR for fbln2 (green) and immunostaining for mCherry (magenta), and DAPI (DNA, blue), on sections of uninjured (a) and 4 dpci (b) TgBAC(tcf21:mCherry) ventricles and DAPI (DNA, blue). c, In situ HCR for fbln2 (green) and cdh5 (magenta) on sections of wild-type ventricles at 4 dpci. High-magnification images show border-zone EPDCs. d, In situ HCR for fbln2 (green) and immunostaining for mCherry (magenta) and EGFP (white), and DAPI (DNA, blue), on sections of 4 dpci TgBAC(tcf21:mCherry); Et(krt4:EGFP) ventricles. White arrowheads point to fbln2+ EPDCs and yellow arrowheads point to fbln2+ endocardial cells. e, In situ HCR for fbln2 (green) and immunostaining for RFP (magenta) on sections of 7 dpci Tg(-0.8flt1:RFP) ventricles. High-magnification images show border-zone coronary endothelial cells expressing fbln2. f, In situ HCR for fbln2 (green) and immunostaining for mCherry (magenta), and DAPI (DNA, blue), on 7 dpci TgBAC(tcf21:mCherry) ventricles. g, In situ HCR for fbln2 (green) and DAPI (DNA, blue) on wild-type ventricles at 14 dpci. High-magnification images of border-zone…

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  Fig. 2. fbln2PL−/− shows reduced TGFβ signaling and decreased numbers of epicardial myofibroblasts. a, Schematic representation of the fbln2 locus showing gRNAs (pink lines) targeting 5′ UTR and exon 1 to generate a promoter-less (fbln2PL) allele. Protein-coding exons are shown as gray boxes and UTRs as white boxes. The arrow indicates the transcription start site. The gray dashed line represents the deleted sequence. b, RT-qPCR analysis of fbln2, fbln1 and efemp2a mRNA levels of fbln2+/+ and fbln2PL−/− siblings at 5 dpf; n = 5 (pools of 5). Data are shown as means ± s.d. and bars represent medians. Statistical test: two-tailed Student’s t-test. c, Immunostaining of sections of cryoinjured ventricles of fbln2+/+ and fbln2PL−/− siblings at 7 dpci stained for pSmad3 (green), Cav1 (magenta) and DAPI (DNA, blue). Arrowheads point to epicardial pSmad3+ cells. d, Number of total, intraventricular (intrav.) and epicardial pSmad3+ cells per mm2 injured area in fbln2+/+ and fbln2PL−/− ventricles at 7 dpci; n = 7. Box plots show the median and interquartile range (IQR; 25th–75th percentiles), with whiskers spanning minimum (min) to maximum (max) values. Statistical test: two-tailed Student’s…

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  Fig. 3. fbln2PL−/− ventricles present reduced cardiomyocyte and endothelial proliferation and retain smaller collagen deposits. a, Immunostaining of sections of cryoinjured ventricles of fbln2+/+ and fbln2PL−/− siblings at 7 dpci stained for Mef2 (myocyte enhancer factor 2; CMs, magenta), PCNA (proliferating cell nuclear antigen; proliferation marker, green) and DAPI (DNA, blue). Arrowheads point to PCNA+ CMs. b, Percentage of PCNA+ CMs in the injury border zone of fbln2+/+ (n = 6) and fbln2PL−/− (n = 8) siblings at 7 dpci. Statistical test: two-tailed Student’s t-test. c, Immunostaining of sections of cryoinjured ventricles of fbln2+/+ and fbln2PL−/− siblings at 7 dpci stained for Mef2 (CMs, magenta), N2.261 (embryonic MyHC, green) and DAPI (DNA, blue). Arrowheads point to N2.261+ CMs. d, Percentage of N2.261+ CMs in the injury border zone of fbln2+/+ (n = 5) and fbln2PL−/− siblings (n = 5) at 7 dpci. Statistical test: two-tailed Student’s t-test. e, Immunostaining of sections of cryoinjured ventricles of fbln2+/+ and fbln2PL−/− siblings at 4 dpci stained for Fli1 (endothelial cells, magenta), PCNA (proliferation marker, green) and DAPI (DNA, blue). Arrowheads point to PCNA+ endothelial…

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  Fig. 4. TGFβ signaling is further decreased and fibrosis resolution altered in cryoinjured ventricles of fbln2 full-locus fbln2 deletion mutants. a, Schematic representation of the fbln2 locus showing gRNAs (pink lines) targeting the 5′ UTR and 3′ UTR to create a full-locus deletion (fbln2FLD) allele. Protein-coding exons are shown as gray boxes and UTRs as white boxes. The arrow indicates the transcription start site. The gray dashed line represents the deleted sequence. b, RT-qPCR analysis of fbln2 mRNA levels of fbln2+/+ (n = 4) and fbln2FLD−/− (n = 5) siblings at 5 dpf. Data are shown as means ± s.d. and bars represent medians. Statistical test: two-tailed Student’s t-test c, Immunostaining of sections of cryoinjured ventricles of fbln2+/+ and fbln2FLD−/− siblings at 7 dpci stained for Mef2 (CMs, magenta), PCNA (proliferation marker, green) and DAPI (DNA, blue). Arrowheads point to PCNA+ CMs. d, Percentage of PCNA+ CMs in the injury border zone of fbln2+/+ (n = 6) and fbln2FLD−/− (n = 7) siblings at 7 dpci. Box plots show the median and IQR (25th–75th percentiles), with whiskers spanning min–max values. Statistical test: two-tailed Student’s t-test. e, Immunostaining of sections of…

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  Fig. 5. Reduced activation of EPDCs in cryoinjured fbln2PL−/− ventricles. a, UMAP representation of all samples combined and individual groups with inferred cellular identity after scRNA-seq analysis. Lympho, lymphocytes; neutro, neutrophils; mes, mesenchymal cells; macro, macrophages. b, Dot plot depicting average expression (avg. exp) and abundance (Pct. exp) of marker genes across all cell types. c, Proportion of cells per cluster within each sample. d, UMAP representation of EPDC subclusters. Purple dashed lines indicate epithelial EPDC clusters (podxl+), blue dashed lines indicate mesenchymal EPDC clusters (hapln1a+) and orange dashed lines indicate activated EPDC clusters. e, Dot plot depicting average expression and abundance (Pct. exp) of marker genes across all EPDC subclusters. f, Normalized expression of selected marker genes of Ep0 (ptx3a and stra6), Ep4 (manf and hyou1) and Ep10 (top2a) clusters in UMAPs (all samples combined). g, UMAP representation of pseudotime along the inferred trajectory of EPDCs. White circles represent leaf nodes, and black circles represent branch points. h, Comparison of pseudotime distribution of EPDCs per group at the single-cell level (each…

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  Fig. 6. Epicardial overexpression of nupr1b rescues the number of epicardial myofibroblasts in fbln2PL−/− ventricles after cryoinjury. a, RT-qPCR analysis of nupr1b mRNA levels in uninjured ventricles at 2, 4 and 21 dpci (n = 4 or 5, pools of 2 ventricles). Data are shown as means ± s.d. and bars represent medians. Statistical test: one-way ANOVA followed by Tukey’s post hoc test. b, In situ HCR for nupr1b (green) and immunostaining for mCherry (magenta) on sections of uninjured TgBAC(tcf21:mCherry) ventricles and at 4 dpci. Arrowheads point to nupr1b+ EPDCs. c, Immunostaining of sections of cryoinjured ventricles of nupr1b+/+ and nupr1bFLD−/− siblings at 7 dpci stained for Mef2 (CMs, magenta), PCNA (proliferation marker, green) and DAPI (DNA, blue). Arrowheads point to PCNA+ CMs. d, Percentage of PCNA+ CMs in the injury border zone of nupr1b+/+ and nupr1bFLD−/− siblings at 7 dpci (n = 8). Box plots show the median and IQR (25th–75th percentiles), with whiskers spanning min–max values. Statistical test: two-tailed Student’s t-test. e, Immunostaining of sections of cryoinjured ventricles of nupr1b+/+ and nupr1bFLD−/− siblings at 7 dpci stained for αSMA (green), MyHC (magenta) and DAPI…

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  Extended Data Fig. 1. Generation of the fbln2 loss-of-function alleles. a. Schematic representation of the fbln2 locus showing the location of gRNAs targeting the 5’UTR and exon 1 (pink lines) to create the promoter-less allele. Primers used for genotyping are shown in green. DNA gel electrophoresis analysis of PCR products amplified from DNA extracted from fin-clips. Inner primers are located within the deleted region and amplify the WT allele, outer primers span the genomic deletion and amplify the mutant allele. DNA ladder: 100 bp. b. Schematic representation of the fbln2 locus showing the location of gRNAs targeting the 5’UTR and 3’UTR (pink lines) to create a full-locus deletion allele. Primers used for genotyping are shown in green. DNA gel electrophoresis analysis of PCR products amplified from DNA extracted from fin-clips. Inner primers are located within the deleted region and amplify the WT allele, outer primers span the genomic deletion and amplify the mutant allele. DNA ladder: 100 bp. c. RT-qPCR analysis of fbln2 mRNA levels in uninjured fbln2+/+ (n = 3) and fbln2FLD+/- (n = 4) ventricles (pools of 2 per sample). Data shown as mean ± SD and bars representing median. Statistical test:…

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  Extended Data Fig. 2. Generation of the epicardial-specific nupr1b overexpression line. a. Schematic representation of the 4-OHT and heat-shock treatments applied to fbln2PL-/-, TgBAC(tcf21:CreERT2, cryaa:EGFP); Tg(hsp70l:LBL-nupr1b-t2A-mCherry) larvae. Wholemount images of 6 dpf larvae showing lens-specific EGFP expression (cryaa:EGFP, green), global BFP expression (hsp70:BFP, blue) and mCherry expression (floxed in tcf21+ cells, red). b. RT-qPCR analysis of nupr1b mRNA levels in 5 dpf larvae treated with EtOH (n = 4) or 4-OHT (n = 4) and subjected to heat-shocks (HS). Data shown as mean ± SD and bars representing median. Statistical test: Mann-Whitney test. Scale bar: 200 μm. c. In situ HCR for nupr1b (magenta) and ptx3a (green) on 7 dpci ventricular sections of fbln2PL-/-, TgBAC(tcf21:CreERT2, cryaa:EGFP); Tg(hsp70l:LBL-nupr1b-t2A-mCherry) treated with EtOH or 4-OHT (DAPI: DNA, blue). Arrowheads point to nupr1b+ ptx3a+ nuclei. Scale bar: 50 μm. Source data

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  Extended Data Fig. 3. Expression of EPDCs markers. a. UMAPs showing normalized expression levels of selected marker genes of mesenchymal EPDCs (hapln1a), epithelial EPDCs (podxl), Ep6 (cdh5 and spock3), Ep9 (mpeg1.1), Ep11 (actc1a) and Ep12 clusters (hbegfb and admb) in all samples combined. b. In situ HCR for ptx3a (magenta) and DAPI (DNA, blue) on sections of cryoinjured ventricles of fbln2+/+, fbln2PL-/- and fbln2FLD-/- at 7 dpci. Arrowheads point to ptx3a+ cells. Scale bar: 20 μm.

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  Extended Data Fig. 4. Rescue of TGF-β targets in fbln2FLD mutants. RT-qPCR analysis of twist2, scxa and gsc mRNA levels in 3 dpf larvae of fbln2+/+, fbln2FLD-/- and fbln2 mRNA-injected fbln2FLD-/- (pools of two larvae per sample). Statistical test: one-way ANOVA followed by Tukey’s post-hoc test. Error bars represent mean ± SD and bars represent median values. Source data

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  Extended Data Fig. 5. Expression of nupr1b in fbln2 mutants. a. Heatmap showing aggregated expression levels across samples and cell types of top downregulated genes in fbln2PL-/- at 4 dpci compared with wild types. b. RT-qPCR analysis of nupr1b mRNA levels in fbln2+/+ (n = 4), fbln2PL-/-(n = 5) and fbln2FLD-/- (n = 5) cryoinjured ventricles at 4 dpci (pools of 2 ventricles per sample). Error bars represent mean ± SD and bars represent median values. Statistical test: one-way ANOVA followed by Tukey’s post-hoc test. c. In situ HCR of nupr1b (green) and DAPI (DNA, blue) on sections of cryoinjured fbln2+/+, fbln2PL-/- and fbln2FLD-/- ventricles at 7 dpci. Arrowheads point to nupr1b+ cells. Scale bar: 20 μm. Source data

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  Extended Data Fig. 6. nupr1b full-locus deletion mutant generation and analyses. a. In situ HCR of nupr1b (green) and immunostaining for mCherry (magenta) and EGFP (white), and DAPI (DNA, blue) on sections of cryoinjured TgBAC(tcf21:mCherry); Et(krt4:EGFP) ventricles at 4 dpci. Arrowheads point to nupr1b+ cells. White dotted lines delineate the border of the injured tissue. Scale bar: 20 μm. b. Schematic representation of the nupr1b locus showing the location of gRNAs (pink lines) targeting the 5’UTR and 3’UTR (white boxes) to create a full-locus deletion allele. Primers used for genotyping are shown in green. DNA gel electrophoresis analysis of PCR products amplified from DNA extracted from fin-clips. Primers span the genomic deletion, and the mutant allele appears as a shorter product. DNA ladder: 100 bp. c. RT-qPCR analysis of nupr1b and nupr1a mRNA levels in uninjured nupr1b+/+ (n = 4) and nupr1bFLD-/- (n = 5) ventricles (pools of 2 ventricles per sample). Error bars represent mean ± SD and bars represent median values. Statistical test: Mann-Whitney test. Source data