Interferon signaling is required for early neutrophil recruitment after zebrafish heart injury

Figures

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  Fig. 1.. Interferon signaling is induced by zebrafish heart injury. (A) A time course of ifnphi1 gene expression after cryoinjury 2, 6, and 24 hpi normalized to elf1a expression. Relative fold change calculated by normalizing to uninjured control ventricles. Each dot on the plot represents a pool of three uninjured or three injured zones of injured ventricles. Welch's t-test was used to determine statistical significance between the relative fold change of ifnphi1 in the injured zone relative to the uninjured zone, with P=0.010 at 2 hpi. (B) ifnphi1-4 gene expression in wild-type ventricles at 2 hpi relative to uninjured ventricles. (C) A time course of ifnphi1 gene expression after sham injury 2, 18, and 24 h post sham (hps) relative to uninjured ventricles. (D) A time course of isg15 gene expression after cryoinjury 1, 3, and 7 dpi relative to uninjured ventricles. Welch's t-test was used to determine statistical significance between the relative fold change of isg15 in the injured zone relative to the uninjured zone, with *P=0.019 at 3 dpi. For A and D, each dot on the plot represents a pool of three uninjured or three injured zones of injured ventricles. For B and C, each dot shown…

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  Fig. 2.. A zebrafish mutant in ifnphi1 disrupts interferon signaling after heart injury. (A) A design for mutating the ifnphi1 gene, with flanking guide RNAs shown (above) and the ifnphizj7 allele with a large deletion that removes part of exon 1, all of exon 2, and part of exon 3 (see Table S1 for sequences). (B) A time course of isg15 gene expression after cryoinjury in ifnphi1−/− ventricles 1, 3, and 7 dpi relative to uninjured ventricles. Each dot on the plot represents a pool of three uninjured or three injured zones of injured ventricles. (C) Representative images of RNA fluorescence in situ hybridization (RNA-FISH) of isg15 (interferon stimulated gene-15) and myl7 (labels cardiomyocytes) in wild-type and ifnphi1−/− ventricle cryosections at 3 dpi. Sections were counterstained with DAPI (blue). BA, bulbus arteriosus; V, uninjured ventricle; W, wound. Injury border is shown as a dashed line. (D) Percent isg15 calculated via the number of isg15 speckles quantified in CellProfiler divided by the number of nucleated cells in the injured and uninjured zones. Welch's t-test was used to determine statistical significance between the isg15 speckle percent in the injured zones (P=0.0023).…

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  Fig. 3.. Interferon signaling promotes neutrophil recruitment to injured myocardium. (A) Representative images of wild-type Tg(mpx:GFP) and ifnphi1−/− Tg(mpx:GFP) heart sections at 1 dpi. Sections were counterstained with DAPI (blue). Neutrophils are shown in white. Injury border is shown as a dashed line. (B) Quantification of the percent of neutrophils and absolute number of neutrophils in the injured and uninjured zones of wild-type and ifnphi1−/−hearts at 1 dpi. The total number of neutrophils was counted in the uninjured and injured zones in wild-type and ifnphi1−/− hearts (P=0.0137). The percent of neutrophils was calculated by dividing the number of neutrophils by the number of nucleated cells in the uninjured and injured zones in wild-type and ifnphi1−/− hearts (P=0.0012). (C) The sum of neutrophils in uninjured and injured zones in wild-type and ifnphi1−/− hearts at 1 DPI (P=0.0035). (D) Representative images of wild-type Tg(mpx:GFP) and ifnphi1−/− Tg(mpx:GFP) heart sections at 3 dpi. Sections were counterstained with DAPI (blue). Injury border is shown as a dashed line. (E) Quantification of the percent of neutrophils (P=0.0590) and absolute number of neutrophils (P=0.0157) in…

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  Fig. 4.. Interferon signaling is not required for macrophage recruitment to the heart injury zone. (A) Representative images of RNA-FISH of mpeg (labels macrophages) and myl7 (labels cardiomyocytes), in wild-type and ifnphi1−/− ventricle cryosections at 3 dpi. Sections were counterstained with DAPI (blue). BA, bulbus arteriosus; A, atrium; V, uninjured ventricle; W, wound. Injury border is shown as a dashed line. (B) Percent macrophages were calculated by dividing the number of mpeg speckles by the number of nucleated cells in the injured and uninjured zones. The dots on the graph represent the uninjured or injured zones calculated from individual sections of distinct heart ventricles. Data are shown as the mean±s.d.; n=7 wild type and n=9 ifnphi1−/−. Scale bars: 50 μm.

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  Fig. 5.. Interferon signaling is not required for endothelial regeneration after heart injury. (A,C) Representative images of RNA-FISH of kdrl (labels vasculature) and myl7 (labels cardiomyocytes) in uninjured and 7 dpi wild-type and ifnphi1−/− ventricle cryosections. Sections were counterstained with DAPI (blue). Injury border is shown as a dashed line. (B,D) Percent kdrl was calculated by dividing the number of kdrl speckles by the number of nucleated cells in the injured and uninjured zones at each time point. (E) kdrl enrichment was calculated by dividing the normalized kdrl speckle count in the injured zone by the normalized kdrl speckle count in the uninjured zone of the same heart section, for each heart. (F) Representative images of wild-type Tg(kdrl:GFP) and ifnphi1−/− Tg(kdrl:GFP) heart sections at 7 dpi. Sections were counterstained with DAPI (blue). Cardiac endothelial cells are in white. Injury border is shown as a dashed line. Middle and right panels are kdrl:GFP channel only. Arrowheads indicate modest endothelial regrowth in the injury zone. (G) Percent kdrl pixels in injured or uninjured area. The dots on the graph represent the uninjured or injured zones calculated from…

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  Fig. 6.. ifnphi1 mutant hearts are modestly impaired at resolving fibrotic tissue after injury. (A) Experimental design for heart injury and collection at 30 and 60 dpi. (B) Images of AFOG-stained sections of wild-type and ifnphi1−/−hearts at 30 and 60 dpi, showing representatives of mild and severe categories of fibrotic tissue resolution. (C,D) Quantification of fibrotic tissue (see Materials and Methods) in AFOG-stained hearts between wild-type and ifnphi1−/− stained sections at each time point. (E,F) Fibrosis severities for all 30 dpi and 60 dpi injured hearts were categorized into three groups: fully regenerated (0-2% collagen), mildly fibrotic (2-5% collagen), or severely fibrotic (greater than 5% collagen). Chi-squared test for categorical data showed that 30 dpi categories between wild type and ifnphi1−/− are not significantly different (P=0.1517), while 60 dpi categories between wild type and ifnphi1−/− are modestly statistically significant (P=0.0378). The dots on the graph represent the uninjured or injured zones calculated from individual sections of distinct heart ventricles. Data are shown as the mean±s.d.; n=19 wild type and n=20 ifnphi1−/− for 30 dpi samples, n=15 wild…