Interferon signaling promotes early neutrophil recruitment after zebrafish heart injury

Interferon signaling promotes early neutrophil recruitment after zebrafish heart injury | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Interferon signaling promotes early neutrophil recruitment after zebrafish heart injury View ORCID Profile Alexis V. Schmid , View ORCID Profile James A. Gagnon doi: https://doi.org/10.1101/2025.11.16.688717 Alexis V. Schmid 1 School of Biological Sciences, University of Utah , Salt Lake City, UT, 84112, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Alexis V. Schmid James A. Gagnon 1 School of Biological Sciences, University of Utah , Salt Lake City, UT, 84112, USA 2 Henry Eyring Center for Cell & Genome Science, University of Utah , Salt Lake City, 84112, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for James A. Gagnon For correspondence: james.gagnon{at}utah.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF ABSTRACT Heart injury triggers a robust cellular response in zebrafish, characterized by neovascularization, immune cell activation, and the infiltration of proliferative cardiomyocytes that collectively lead to scarless regeneration. Upon injury, damage-associated molecular patterns are released by dying cells and injured extracellular matrix. These molecules bind to pattern recognition receptors on various cell types, promoting inflammation and immune cell recruitment by upregulating chemokines and pro-inflammatory cytokines. We previously identified the activation of injury-induced interferon signaling as a distinguishing feature between the regenerative zebrafish and non-regenerative medaka. Here, we establish interferon-Φ1 (IFN□1) as the primary driver of interferon signaling after zebrafish heart injury. IFN□1 expression is induced hours after injury and directs interferon-stimulated gene expression, which peaks at 72 hours post-injury. This response is lost in ifnphi1 mutants, which disrupt IFN□1 expression. ifnphi1 mutants have reduced neutrophil recruitment to the injured myocardium, while macrophages and neovascularization are unaffected. By studying later stages of regeneration, we find that ifnphi1 mutant hearts have a modest defect in scar resolution. Collectively, these findings uncover a critical early signaling cascade during the inflammatory response to heart injury and provide new insights into the mechanisms that choreograph zebrafish heart regeneration. INTRODUCTION Myocardial infarction occurs when blood flow to a portion of the heart is blocked, leading to irreparable cell death and the formation of a large, inelastic scar that hinders heart function ( Salari et al., 2023 ). Nearly all mammals lose their regenerative capacity after the neonatal stage, whereas most fish and amphibians retain it throughout development and into adulthood. These regenerators replace collagenous scar tissue with fresh cardiomyocytes to restore heart function to the injured myocardium ( González-Rosa et al., 2011 ; González Rosa et al., 2017; Poss et al., 2002 ). Zebrafish have emerged as a powerful model to study cardiac regeneration ( Gemberling et al., 2013 ). Following cardiac injury, zebrafish employ a sequence of pro-inflammatory and anti-inflammatory immune signals to coordinate collagen deposition, revascularization, and eventual scar resolution. In contrast, the non-regenerating medaka fish exhibit a prolonged pro-inflammatory immune response after injury, leading to scar persistence ( Lai et al., 2017 ). Studies of regenerators may yield new therapeutic insights into the specific immune and cellular mechanisms required to resolve the scar and regenerate functional cardiac tissue. In zebrafish, heart regeneration is initiated by a choreographed immune response. The initial injury triggers the release of damage-associated molecular patterns (DAMPs) from necrotic tissue and injured extracellular matrix. These DAMPs induce inflammation by binding to pattern recognition receptors (PRRs) on multiple cell types to recruit waves of immune cells to the wound via the release of pro-inflammatory cytokines and chemoattractants ( Iribarne, 2021 ; Julier et al., 2017 ). Neutrophils are the first pro-inflammatory cells recruited to the injury zone, rapidly localizing to the wound as early as six hours post-injury (hpi) ( Julier et al., 2017 ). Neutrophils are vital for wound detection by helping to recruit monocytes, which differentiate into macrophages at the wound site ( Feng et al., 2025 ; Wilgus et al., 2013 ). In addition to promoting inflammation early after injury, neutrophils also secrete vascular endothelial growth factor (VEGF-A), which promotes early neovascularization of the injured myocardium ( El-Sammak et al., 2022 ; Gong & Koh, 2010 ; Lörchner et al., 2023 ). Neutrophils also activate epicardial cells to induce epithelial-to-mesenchymal transition, enabling epicardial infiltration and significant contributions to wound healing ( Peterson et al., 2024 ). Macrophages are the second immune cells recruited to the injury site, providing both a pro-inflammatory and an anti-inflammatory response ( Bevan et al., 2020 ; Sánchez-Iranzo et al., 2018 ). The numerous roles of macrophages are still being explored; however, we know they shape the inflammatory response by phagocytosing pro-inflammatory apoptotic neutrophils, clearing cellular debris, shaping new extracellular matrix and neovasculature, and depositing collagen alongside fibroblasts for scar formation ( Bevan et al., 2020 ; Constanty et al., 2025 ; Goumenaki et al., 2024 ; Julier et al., 2017 ). While immune cells are required for complete regeneration, the specific immune responses that distinguish regenerating and non-regenerating species are still being investigated ( Lai et al., 2017 ; Peterson et al., 2024 ). We previously compared the response to heart injury in two distantly related teleost species, medaka and zebrafish. Despite these fish having similar heart morphology and gene orthology, medaka cannot recover injured myocardium. The innate immune landscape in medaka is different from that of zebrafish, with higher neutrophil recruitment, less macrophage recruitment, and an overall more pro-inflammatory response ( Lai et al., 2017 ). Interestingly, stimulating macrophage recruitment with the TLR agonist, Poly I:C, stimulates heart regeneration in medaka, indicating a failure in the initial damage-sensing phase ( Lai et al., 2017 ). However, the specific signaling pathways underlying this process in zebrafish remain unclear. Using single-cell RNA sequencing, we identified a key difference in the inflammatory response after injury. We found that medaka lacked an injury-induced interferon signaling response to injury. Conversely, zebrafish display robust upregulation of interferon-stimulated genes (ISGs) across most cardiac cell types at three days post-injury (dpi) during the inflammatory stage ( Carey et al., 2024 ). Interferons are pro-inflammatory cytokines that are released by immune cells responding to infection or injury ( Cheng et al., 2008 ). There are three types of interferons in zebrafish: Type I (IFNΦ1-4), Type II (IFNγ1-2), and Type IV (IFNυ) ( Aggad et al., 2009 ; S. N. Chen et al., 2022 ; Cheng et al., 2008 ). IFNγ studies in mice and in cell culture have highlighted the importance of Type II interferon signaling in macrophage activation and successful skeletal muscle regeneration. However, prolonged IFNγ exposure in these models led to excessive fibrosis, reduced muscle contractility, and inhibited regeneration ( Adhikari et al., 2020 ; Z. Chen et al., 2021 ; Cheng et al., 2008 ). Our single-cell RNA sequencing analysis of injured zebrafish and medaka hearts revealed specific upregulation of ifnphi1 in injured zebrafish hearts ( Carey et al., 2024 ). Still, the role of Type I interferon signaling in heart regeneration remains unexamined. Interferons bind to various cell types in the heart, which leads to the expression of ISGs. ISGs have defined roles in pathogen clearance by recruiting innate immune cells to the infection ( Balla et al., 2020 ; Schneider et al., 2014 ); however, whether ISGs play a role in coordinating a pro-regenerative immune response during heart regeneration is unclear. Here, we establish IFN 1 as the principal driver of interferon signaling following heart injury in zebrafish. ifnphi1 gene expression is rapidly induced at the injury site, pointing to a role in early damage sensing and immune cell recruitment. We find that IFN 1 is required to recruit neutrophils to the injury site, an important early step for creating a pro-regenerative environment. Despite this key role in early inflammation, ifnphi1 -/- showed no defects in later regenerative processes, including macrophage recruitment and neovascularization. However, injured ifnphi1 -/- hearts still had a modest defect in scar resolution. Overall, ifnphi1 -driven interferon signaling orchestrates a discrete, essential role in the initiation of inflammation without being requisite for the subsequent complete regeneration of the zebrafish heart. RESULTS Interferon signaling is an immediate and injury-specific pathway activated during the inflammatory response We previously identified interferon-stimulated genes (ISGs) as significantly upregulated 3 dpi in zebrafish endocardial, epicardial, and leukocyte cell populations within the ventricle, and coincide with an increase in ifnphi1 positive cells ( Carey et al., 2024 ). In contrast, non-regenerative medaka lack an injury-induced interferon response, suggesting that interferon signaling may play an important role in creating a pro-regenerative environment in zebrafish early after heart injury. To better characterize this signaling pathway in zebrafish, we examined transcript levels of ifnphi1 mRNA in zebrafish heart ventricles in a time course following experimental cryoinjury ( Fig.1A ). Relative to the uninjured control hearts, ifnphi1 expression peaked 2 hours after cryoinjury and returned to baseline expression levels by 24 hpi ( Fig. 1A ). ifnphi 2-4 transcript levels were unchanged in injured hearts relative to uninjured hearts driving our focus on investigating ifnphi1 ( Fig. 1B ). Given the role of interferon signaling in pathogen clearance, and the common presence of infectious agents in fish facility water ( Balla et al., 2020 ), we tested for the upregulation of ifnphi1 after sham injury. This injury exposes the fish’s chest cavity to the fish facility water without damaging the ventricle. Here, no upregulation of ifnphi1 was observed, providing evidence that interferon signaling is specifically responding to heart cryoinjury and not chest cavity exposure alone ( Fig. 1C ). Download figure Open in new tab Figure 1: Interferon signaling is induced by zebrafish heart injury. A) A time course of ifnphi1 gene expression after cryoinjury (2 to 72 hpi) normalized to elf1a expression. Relative fold change calculated by normalizing to uninjured control ventricles. B) ifnphi1-4 gene expression in wildtype ventricles at 2 hpi relative to uninjured ventricles. C) A time course of ifnphi1 gene expression after sham injury (2 to 72 hpi) relative to uninjured ventricles. D) A time course of isg15 gene expression after cryoinjury (2 to 120 hpi) relative to uninjured ventricles. Each dot shown represents a single ventricle after dissection away from the atrium and bulbus arteriosus. Data are shown as the mean ±□s.d. To further characterize the production of ISGs downstream of interferon signaling, we used RT-qPCR to measure the relative fold change of interferon-stimulated gene-15 ( isg15 ) mRNA over an early injury time course in wild-type zebrafish. Relative to uninjured hearts, isg15 expression peaks at 72 hpi, well after the initial expression of interferon mRNA ( Fig. 1D ). Together, these data describe the tempo of interferon signaling after zebrafish heart injury: induced immediately after injury and received by endocardial, epicardial, and leukocyte cells to promote the expression of interferon-stimulated genes within the first three days after injury. Generating an ifnphi1 mutant zebrafish To study the role of interferon signaling in heart regeneration, we generated an ifnphi1 -/- zebrafish. Wildtype embryos were injected with four gRNAs targeting exon 1 and exon 3 of the ifnphi1 gene. Through crossing we established a stable line with a large deletion between these target sites which is predicted to disrupt protein function ( Fig. 2A and Supplementary Table). We assessed the functional disruption of interferon signaling in the mutant using RT-qPCR to evaluate the expression of isg15 in injured mutant and wild-type heart ventricles. ifnphi1 -/- fish had no induction of isg15 mRNA after injury, lacking the isg15 upregulation at 72 hpi observed in the wild-type zebrafish ( Fig. 2B ). As an orthogonal method to confirm that our mutant disrupted interferon signaling in the heart after injury, we visualized the production of isg15 transcripts using RNAscope at 3 dpi in ifnphi1 -/- and ifnphi1 +/+ hearts. This experiment revealed that ifnphi1 +/+ zebrafish had enrichment of isg15 mRNA in the injured zone of their ventricles, while ifnphi1 -/- fish lacked isg15 enrichment in the injured myocardium ( Fig. 2C,D and Supplementary Fig. 1). Download figure Open in new tab Figure 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 ifnphi zj7 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 (2 to 120 hpi) relative to uninjured 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). 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). The dots on the graph represent the uninjured or injured zones of individual ventricles. Data are shown as the mean ±□s.d.; n=4 wild-type and n=6 ifnphi1 -/- ventricles. Scale bar = 50 µm. E) ifnphi1-4 gene expression in ifnphi1 -/- ventricles at 2 hpi relative to uninjured ventricles. To ensure that the other Type I interferons were not compensating for the lack of ifnphi1 in mutants, RT-qPCR was used to test for the upregulation of ifnphi2-4 in both ifnphi1 +/+ and ifnphi1 -/- fish, relative to uninjured ifnphi1 +/+ fish ( Fig. 2E ). Here, ifnphi2-4 is not upregulated in ifnphi1 +/+ or ifnphi1 -/- conditions after injury. Collectively, these experiments provide strong evidence that ifnphi1 is the primary driver of interferon signaling after cardiac injury in zebrafish, and that ifnphi1 -/- fish have disrupted interferon signaling. ifnphi1 promotes early neutrophil recruitment to the injured myocardium Given the strong connections between interferon signaling and innate immune cell recruitment, we next examined the role of ifnphi1 in neutrophil and macrophage recruitment to the injury site. To study the role of interferon signaling in neutrophil recruitment, we labeled neutrophils with the mpx:GFP transgene in wildtype and ifnphi1 -/- hearts. We dissected and imaged injured hearts at 1 dpi, when neutrophil recruitment is peaking during heart regeneration ( Goumenaki et al., 2024 ; Julier et al., 2017 ). Because the impact of cryoinjury can vary between hearts, we calculated the total number of neutrophils and the percentage of neutrophils in the injured and uninjured zones by dividing the total number of neutrophils by the total number of nucleated cells in each injury zone ( Fig. 3A,B ). By both metrics, ifnphi1 -/- fish had significantly fewer neutrophils in the injury zone than ifnphi1 +/+ fish at one day post-injury. To observe if this defect persisted, we conducted the same comparison at 3 dpi. While three days after injury is after the peak neutrophil response in the injury zone, ifnphi1 +/+ fish still had a significantly higher number of neutrophils than ifnphi1 -/- fish ( Fig. 3C,D ). These data suggest that interferon signaling is important for the recruitment of neutrophils during the initial stage of zebrafish heart regeneration. Download figure Open in new tab Figure 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. 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) Representative images of wild-type Tg(mpx:GFP) and ifnphi1 -/- Tg(mpx:GFP) heart sections at 3 dpi. Sections were counterstained with DAPI (blue). D) Quantification of the percent of neutrophils (p=0.0590) and absolute number of neutrophils (p=0.0157) in the injured and uninjured zones of wild-type and ifnphi1 -/- hearts at 1 dpi was calculated as above. Each dot on the graphs represents the uninjured or injured zones calculated from individual sections of distinct heart ventricles. Data are shown as the mean ±□s.d.; n=6 wild-type and n=6 ifnphi1 -/- for one dpi, and n=6 wild-type and n=6 ifnphi1 -/- for 3 dpi. Statistical tests represent Welch’s t -test. Scale bar = 50 µm. Interferon signaling is dispensable for macrophage recruitment and neovascularization after heart injury Neutrophils directly interact with macrophages by secreting chemokines and cytokines, thereby influencing the pro-repair, anti-inflammatory identity of infiltrating macrophages ( Bouchery & Harris, 2019 ; Wilgus et al., 2013 ). In the wound, macrophages phagocytose cellular debris and dying neutrophils, contributing to the transition to an anti-inflammatory immune response ( Bouchery & Harris, 2019 ; Marwick et al., 2018 ). In addition to shaping the inflammatory response, macrophages closely associate with protruding cardiomyocytes at the border injury zone and help remodel the extracellular matrix to facilitate scar resolution ( Constanty et al., 2025 ). To estimate the number of macrophages that infiltrated the injury site in wildtype and ifnphi1 -/- mutant hearts, we measured mpeg expression, a classic marker of macrophages, using RNA-FISH. We found that both genotypes have similar amounts of mpeg expression ( Fig. 4A,B and Supplementary Fig. 2) suggesting that macrophage abundance was not impacted by the lack of interferon signaling in the mutants. Despite the implications of macrophage recruitment and TLR signaling demonstrated in a previous medaka study, our data suggest that ifnphi1 -driven interferon signaling does not play a role in macrophage recruitment in zebrafish ( Lai et al., 2017 ) ( Fig. 4A,B ). Download figure Open in new tab Figure 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). 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 bar = 50 µm. Neovascularization also occurs during the inflammatory response, beginning as early as 15 hpi ( Marín-Juez et al., 2016 ). Previous studies suggest that neutrophils play a role in initiating neovascularization in the wounded myocardium ( El-Sammak et al., 2022 ; Gong & Koh, 2010 ; Lörchner et al., 2023 ). We measured neovascularization at 3 dpi, 7 dpi, and in uninjured ifnphi1 +/+ and ifnphi1 -/- hearts using RNA-FISH for kdrl expression ( Fig. 5A-H ). The kdrl speckles were counted in both injured and uninjured zones of the myocardium and normalized to the number of nucleated cells in each zone ( Fig. 5 B,D,G). The enrichment of kdrl expression in the injured versus uninjured zones was calculated for each heart ( Fig. 5E,H ). Additionally, the number of kdrl speckles in the uninjured and injured zones for each heart was totaled (Supplementary Fig. 3). We found that uninjured ifnphi1 +/+ and ifnphi1 -/- hearts had very similar kdrl expression ( Fig. 5A,B ). Furthermore, at both 3 and 7 dpi, there were no significant differences in kdrl expression or enrichment between ifnphi1 +/+ and ifnphi1 -/- hearts ( Fig. 5C-H ). These data suggest that neovascularization in response to heart injury does not require interferon signaling. Download figure Open in new tab Figure 5: Interferon signaling is not required for neovascularization after heart injury. A, C, F) Representative images of RNA-FISH of kdrl (labels vasculature) and myl7 (labels cardiomyocytes) in uninjured, 3 dpi, and 7 dpi wild-type and ifnphi1 -/- ventricle cryosections. Sections were counterstained with DAPI (blue). B, D, G) 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, H) 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. 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=5 wild-type and n=5 ifnphi1 -/- for uninjured samples, n=4 wild-type and n=7 ifnphi1 -/- for 3 dpi samples, n=6 wild-type and n=4 ifnphi1 -/- for 7 dpi samples. Scale bar = 50 µm. Interferon signaling is not required for scarless regeneration Immune signaling is critical for scar resolution and scarless regeneration. When innate immune cells are depleted or removed from the inflammatory response to heart injury, collagen persists in the wounded area as a thick, irresolvable scar ( Lai et al., 2017 ; Peterson et al., 2024 ). We investigated whether interferon signaling is necessary for successful heart regeneration at later time points. After heart cryoinjury, adult zebrafish typically resolve their scar between 30 and 60 dpi. We used Acid Fuchsin Orange-G (AFOG) staining to visualize the extent of remaining scar tissue in ifnphi1 +/+ and ifnphi1 -/- hearts at 30 and 60 dpi ( Fig. 6A,B and Supplementary Fig. 4 and 5). We identified the most scarred heart section and quantified the size of the remaining scar by normalizing the area of the collagen staining (blue) to the area of the uninjured myocardium (yellow) in each injured heart ( Fig. 6C,D ). For the 30 dpi samples, the percent collagen in the ifnphi1 +/+ and ifnphi1 -/- injured hearts was similar ( Fig. 6C ), with somewhat more severe injuries in the wild-type hearts. Most hearts had healed from the injury, evidenced by a thicker cortical cardiomyocyte layer and minimal collagen near the injury site. At 60 dpi, the recovery continued in the population of ifnphi1 +/+ hearts, but stalled in the population of ifnphi1 -/- hearts. Download figure Open in new tab Figure 6: ifnphi1 mutant hearts are modestly impaired at resolving scars 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 scar resolution. C,D) Quantification of scar size (See Methods) in AFOG-stained hearts between wild-type and ifnphi1 -/- stained sections at each time point. (E,F) Scar severities for all 30 dpi and 60 dpi injured hearts were categorized into three groups: fully regenerated (0-2% collagen), mildly scarred (2-5% collagen), or severely scarred (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 wildtype and n=20 ifnphi1 -/- for 30 dpi samples, n=15 wildtype and n=20 ifnphi1 -/- for 60 dpi samples. Scale bars = 50 µm. The variation within and between groups at 60 dpi suggested a difference in healing trajectories between the ifnphi1 +/+ and ifnphi1 -/- injured hearts. To further investigate these apparent disparities, the injured hearts were categorized based on the percentage of collagen scar as calculated above, representing fully regenerated (0-2% collagen), mildly scarred (2-5% collagen), and severely scarred (>5% collagen) ( Fig. 6E,F ). This categorical analysis confirmed that at 30 dpi, more ifnphi1 +/+ hearts were severely scarred than ifnphi1 -/- hearts, although this difference was not statistically significant (p=0.1517). By 60 dpi, 95% of injured ifnphi1 +/+ hearts were either fully regenerated or mildly scarred, as expected ( Fig 6E,F ). However, regeneration in the ifnphi1 -/- hearts did not follow the same progression in recovery. Nearly half of the mutant hearts remained severely scarred at 60 dpi. The chi-squared statistical test revealed a modestly significant distinction between ifnphi1 +/+ and ifnphi1 -/- heart regeneration at this time point (p=0.0378). In summary, our data suggest that interferon signaling has a modest role in resolving the scar during zebrafish heart regeneration. DISCUSSION The inflammatory response to zebrafish heart injury clears cellular debris and promotes scar formation and resolution. Our study characterizes the role of Type I interferon signaling following cardiac injury in zebrafish, revealing its critical and specific contribution to early inflammation. We found that IFN 1, produced by the ifnphi1 gene, is the primary injury-responsive interferon in the heart, and directs upregulation of ISGs. This response is specific to heart injury, and not sham surgery alone, suggesting that ifnphi1 is activated by damage-associated molecular patterns (DAMPs) released from dying cells in the injured myocardium rather than pathogen-associated molecular patterns (PAMPs). This aligns with emerging evidence of non-canonical roles for interferon signaling in tissue repair ( Cheng et al., 2008 ; Lai et al., 2017 ; Leibowitz et al., 2021 ). The absence of an endogenous interferon response in injured medaka, which cannot regenerate after heart injury, further highlights its potential importance in creating a pro-regenerative environment ( Carey et al., 2024 ; Lai et al., 2017 ). We found that interferon signaling is required to attract neutrophils to the injury site early in zebrafish heart regeneration. Roles for neutrophils have been identified in several tissue regeneration contexts. In mouse skin and spinal cord injuries, when neutrophils were depleted, adult mice exhibited slower wound healing ( Nishio et al., 2008 ; Stirling et al., 2009 ). Zebrafish neutrophils control epicardial cell expansion just days after heart injury ( Peterson et al., 2024 ). When zebrafish neutrophils were depleted, there was significantly less epicardial cell expansion and less cardiomyocyte proliferation; however, the long-term effects of neutrophil depletion were not studied. Here, we found a correlation between interferon signaling, neutrophil recruitment, and timely scar resolution during zebrafish heart regeneration. We also found that disrupting interferon signaling did not impact macrophage recruitment to the site of injury or interfere with the re-growth of new blood vessels. We suspect that other pro-inflammatory cytokines, independent of the interferon signaling pathway, were likely sufficient to recruit macrophages and trigger vascular growth in the injured myocardium ( Luz et al., 2025 ; Nguyen-Chi et al., 2017 ). Interestingly, the artificial induction of interferon signaling in the medaka heart, via Toll-like receptor activation, enhanced macrophage recruitment, not neutrophil recruitment ( Lai et al., 2017 ). This highlights the differences in immune response to injury between species. We found a mild regeneration disparity between the ifnphi1 +/+ and ifnphi1 -/ - injured hearts, where regeneration stalls in the absence of interferon signaling. We hypothesize that the early impact on neutrophil recruitment in ifnphi1 -/- hearts may have resulted in slower wound healing. Elucidating the specific mechanisms that connect neutrophil activity with scar resolution remains an exciting avenue for study. Future work could interrogate these mechanisms using mutants or small-molecule inhibitors of neutrophil activity ( Isles et al., 2019 ; Kell et al., 2018 ; Peterson et al., 2024 ). Despite reduced neutrophil infiltration in all mutant hearts, approximately half of ifnphi1 -/- fish were still able to regenerate completely by 60 dpi. This suggests that the inflammatory process, including phagocytosis of cellular debris and remodeling of the extracellular matrix to facilitate regenerative scarring, was adequately executed in the absence of interferon signaling in these individuals. This variability between individuals may have many underlying reasons, including variation in the intensity of injury or more subtle differences in the tempo of damage sensing, cell recruitment, or tissue regrowth. Collectively, this study underscores the concept of regenerative robustness, where redundant mechanisms protect outcomes to the function of the organ and organism. Understanding how these mechanisms drive zebrafish heart regeneration may reveal novel therapeutic targets to promote repair in non-regenerative mammalian hearts, where key compensatory pathways may be lost or insufficient. MATERIAL AND METHODS Fish husbandry and zebrafish lines All zebrafish lines were maintained in accordance with standard husbandry practices at the CBRZ zebrafish facility at the University of Utah This study was conducted with approval of the Office of Institutional Animal Care and Use Committee (IACUC no. 20-07013) of the University of Utah’s animal care and use program. Generation of a zebrafish ifnphi1 mutant line To generate a stable ifnphi1 mutant line, ifnphi1 was targeted using CRISPR-Cas9 gene editing. 4 guide RNAs (gRNAs) targeting exons 1-3 of the ifnphi1 gene were designed and ordered from IDT. To generate the crRNA/gRNA duplex for each gRNA, a 100uM solution of crRNA and tracrRNA was made for each duplex in IDT gRNA buffer. Equal volume of tracrRNA (5 μl) and gRNA (5 μl) was combined annealed via PCR to generate the crRNA-gRNA duplex for each gRNA. The PCR program to anneal crRNA and gRNA is as follows: 95°C, 5 min; cool at 0.1°C/s to 25°C; 25°C, 5 min; cool to 4°C rapidly. Along with these gRNAs (800 ng/ µl), SpCas9 protein (from IDT), and phenol red were coinjected into WT embryos. Total volumes: crRNA/gRNA 1-4 (1.24 µl each), SpCas9 (0.5 µl at 10 µg/µl), and phenol red (1 µl). 1 nanoliter was injected into each embryo. Mosaic embryos were raised to adulthood and incrossed with each other before being outcrossed to wild-type fish. Primers amplifying the CRISPR-editing region were used to select outcrossed fish with large deletions. Specifically, embryos from this outcross carry large deletions, ∼800 bp. To establish a homozygous mutant line, these heterozygous mutant fish were incrossed. Three primers were used to distinguish between homozygous mutants, heterozygous fish, and wild-type fish in two separate PCR reactions (Supplementary Table 1). All experiments presented compare homozygous mutant zebrafish with WT siblings. This line was given the designation ifnphi zj7 . Mutant zebrafish were crossed into the Tg(mpx:GFP) line to label neutrophils ( Buchan et al., 2019 ). Cardiac cryoinjury Cryoinjuries were performed on the ventricular apex of anesthetized zebrafish as described previously ( González-Rosa et al., 2011 ). 0.02% Tricaine (MS-222) was used to anesthetize zebrafish. Zebrafish were positioned on a moist sponge. A small thoracic incision was made with forceps and dissecting scissors to expose the ventricular apex. Using a liquid nitrogen-chilled copper wire cryoprobe (0.5 mm in diameter, as previously described ( González-Rosa & Mercader, 2012 )), the ventricular apex was injured with the probe for 23 seconds. Following injury, the fish were revived in freshwater tanks and returned to the facility for monitoring. Histological methods Hearts were fixed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences) in 1X PBS for 24 hours, then sucrose-treated sequentially from 10% to 30% sucrose. Sucrose-treated hearts were then mounted in Optimal Cutting Temperature (O.C.T., Fisher HealthCare) embedding medium and frozen at -80 C. Hearts from Tg(mpx:GFP) animals were sectioned at 12 µm and mounted in Fluoroshield with DAPI (Sigma-Aldrich). For Acid Fuchsin Orange G (AFOG, Sigma) staining, O.C.T. mounted hearts were sectioned at 7 µm. Bouin’s solution (Sigma Aldrich) was used to fix heart tissue before staining with PT/PM (phosphotungstic acid/phosphomolybdic acid, Sigma-Aldrich) and AFOG stain. After staining, sections were washed twice with 0.5% acetic acid (30 seconds per wash), four times with 100% ethanol (1 minute per wash), and three times with xylenes (5 minutes per wash). For AFOG imaging, the ECHO Revolution 2.0 confocal microscope was used to capture brightfield images at 10X. RNAscope in situ hybridization Zebrafish hearts were fixed for 24 hours in 4% paraformaldehyde (PFA) in 1X PBS, sequentially sucrose-treated (10% to 30%), and O.C.T. embedded. Hearts were then cryosectioned at 12 µm. RNAscope (Advanced Cell Diagnostics, Hayward, CA, USA) was performed using the RNAscope® Multiplex Fluorescent Detection Kit v2 protocol, with previously published probes ( Carey et al., 2024 ). The Zeiss 880 confocal microscope was used to capture RNAscope images at 40X. Imaging quantification ImageJ was used to analyze images of heart sections probed for myl7 (cardiomyocytes), mpeg1.1 (macrophages), isg15 (interferon-stimulated gene-15), kdrl (vascular), and DAPI. To separate the wound from the uninjured ventricle, we set a threshold on the cardiomyocyte channel to create an uninjured ventricle mask. Using the DAPI channel and the difference between the uninjured ventricle mask and the DAPI mask, an injured area mask was created for every heart. Masked images of each probe in the injured and uninjured areas were uploaded to CellProfiler software (version 4.2.1) using a modified CellProfiler ‘Speckle Counting’ pipeline ( Stirling et al., 2021 ). Total number of nucleated cells in the DAPI channel and mpeg1.1, isg15 , and kdrl speckles were identified using the IdentifyPrimaryObjects module. Macrophages and nuclei were related using the RelateObjects CellProfiler module. The percent of macrophages relative to the total number of cells in injured and uninjured areas was calculated. Both isg15 and kdrl speckles were counted regardless of the nuclei relationship. Percent kdrl and isg15 speckles relative to the total number of cells in injured and uninjured areas was calculated. Furthermore, the enrichment of kdrl expression in the injured versus uninjured section was calculated for each heart. For Tg(mpx:GFP) neutrophil counts in injured wildtype and interferon 1Δ fish at 1- and 3-dpi, uninjured and injured masks were created using ImageJ as described above. A similar CellProfiler ‘Speckle Counting’ pipeline was used to count the number of neutrophils in the injured and uninjured areas. For scar quantification in AFOG images, ImageJ software was used. Here, the scar size was calculated by measuring the collagen (blue) stained area and divided by the total myocardium, and multiplied by 100 to generate a percentage. RT-qPCR RNA was purified from injured ventricles using PicoPure (Thermo Fisher) and converted to complementary DNA (cDNA) using QuantiTect Reverse Transcription Kit, using the included RT primer mix (Qiagen). cDNA was amplified using BrilliantII SYBR Green QPCR Master Mix (Agilent) with 10 pmol of each primer. Quantitative reverse-transcription PCR (qRT-PCR) was performed using the QuantStudio 5 (Thermo Fisher Scientific) protocol. Elongation factor 1-alpha ( ef1 α ) expression was measured for each biological triplicate in every experiment. The ΔCt method was used to analyze the data, with ef1 α as the normalization factor for each sample. The fold change was calculated using the ΔΔCt method, where 2-ΔΔCt of injured samples is relative to the average of 2-ΔΔCt of uninjured heart samples. Statistical analysis Statistical analyses for column and nested data were performed using GraphPad Prism (v.10.5.0). Data distribution was assessed using the Shapiro-Wilk normality test. The significance level was set to p=0.05, and the standard deviation from the mean is indicated on the figures. To determine the statistical significance of the normal data, Welch’s t-test was used. For the categorical scar analysis ( Figure 6E,F ), a chi-squared test statistic was calculated for both the 30 dpi and 60 dpi datasets. Then, using this chi-squared test statistic and degrees of freedom=2, a p-value was derived. Supplementary Figure 1: isg15 speckle count in ifnphi1 -/- versus wild-type injured ventricles at three dpi. The number of isg15 speckles counted using the CellProfiler pipeline is represented in the injured and uninjured zones of each fish at 3 dpi. Each dot represents the injured and uninjured zones of one ventricle. Differences in isg15 expression in the injured zones between ifnphi1 -/- and ifnphi1 +/+ were significant with p =0.0141 using Welch’s t-test. Supplementary Figure 2: mpeg speckle count in ifnphi1 -/- versus wild-type injured ventricles at three dpi. The number of mpeg speckles counted using the CellProfiler pipeline is represented in the injured and uninjured zones of each fish at 3 dpi. Each dot represents the injured and uninjured zones of one ventricle. Differences in mpeg expression in the injured zones between ifnphi1 -/- and ifnphi1 +/+ were not significant with p =0.2119 using Welch’s t-test. Supplementary Figure 3: kdrl speckle count in ifnphi1 -/- versus wild-type injured ventricles. A-C) The number of kdrl speckles counted using the CellProfiler pipeline is represented in the injured and uninjured zones of each fish at 3 and 7 dpi. Each dot represents the injured and uninjured zones of one ventricle. Differences in kdrl expression in the injured zones between ifnphi1 -/- and ifnphi1 +/+ were not significant at any post-injury time point. Supplementary Figure 4: All AFOG images at 30 dpi. A) All ifnphi1 +/+ hearts 30 dpi AFOG stained. B) All ifnphi1 -/- hearts 30 dpi AFOG stained. Scale bars = 500 µm. Supplementary Figure 5: All AFOG images at 60 dpi. A) All ifnphi1 +/+ hearts 60 dpi AFOG stained. B) All ifnphi1 -/- hearts 60 dpi AFOG stained. Scale bars = 500 µm. Supplementary Table 1: Sequence information for the ifnphi1 zj7 allele, ifnphi1 zj7 genotyping primers, RT-qPCR primer sequences, and gRNA sequences. FUNDING This project was supported by National Institutes of Health grants T32HD007491 (A.V.S.) and R35GM142950 (J.A.G.), and by startup funds from the University of Utah Henry Eyring Center for Cell and Genome Science (J.A.G.). CONTRIBUTIONS Conceptualization, methodology, software, formal analysis, and investigation: A.V.S.; Writing of the original draft: A.V.S.; Writing review and editing: A.V.S. and J.A.G.; Supervision: J.A.G.; Project administration: J.A.G.; Funding acquisition: J.A.G. ACKNOWLEDGMENTS We thank all members of the Gagnon laboratory for their assistance in completing this project, especially Drew Janik, Jenna Weber, and Kate Scuderi, for their contributions to surgical procedures, Connor Shrader for advice on statistics, and Hailey Hollins and Clay Carey for advice and technical support. We acknowledge the staff of the Centralized Zebrafish Animal Resource facility, ARUP Research Histology Core Laboratory for sectioning assistance, and the Cell Imaging Core at the University of Utah, specifically Xiang Wang. We thank Matthew Mulvey for the use of his microscope. These facilities did not provide financial support. Funder Information Declared NIH , T32HD007491 , R35GM142950 REFERENCES ↵ Adhikari , A. , Cobb , B. , Eddington , S. , Becerra , N. , Kohli , P. , Pond , A. , & Davie , J . ( 2020 ). IFN-γ and CIITA modulate IL-6 expression in skeletal muscle . Cytokine: X , 2 ( 2 ), 100023 . doi: 10.1016/j.cytox.2020.100023 OpenUrl CrossRef PubMed ↵ Aggad , D. , Mazel , M. , Boudinot , P. , Mogensen , K. E. , Hamming , O. J. , Hartmann , R. , Kotenko , S. , Herbomel , P. , Lutfalla , G. , & Levraud , J.-P . ( 2009 ). The Two Groups of Zebrafish Virus-Induced Interferons Signal via Distinct Receptors with Specific and Shared Chains . The Journal of Immunology , 183 ( 6 ), 3924 – 3931 . doi: 10.4049/jimmunol.0901495 OpenUrl Abstract / FREE Full Text ↵ Balla , K. M. , Rice , M. C. , Gagnon , J. A. , & Elde , N. C . ( 2020 ). Linking Virus Discovery to Immune Responses Visualized during Zebrafish Infections . Current Biology , 30 ( 11 ), 2092 – 2103 .e5. doi: 10.1016/j.cub.2020.04.031 OpenUrl CrossRef PubMed ↵ Bevan , L. , Lim , Z. W. , Venkatesh , B. , Riley , P. R. , Martin , P. , & Richardson , R. J . ( 2020 ). Specific macrophage populations promote both cardiac scar deposition and subsequent resolution in adult zebrafish . Cardiovascular Research , 116 ( 7 ), 1357 – 1371 . doi: 10.1093/cvr/cvz221 OpenUrl CrossRef PubMed ↵ Bouchery , T. , & Harris , N . ( 2019 ). Neutrophil–macrophage cooperation and its impact on tissue repair . Immunology & Cell Biology , 97 ( 3 ), 289 – 298 . doi: 10.1111/imcb.12241 OpenUrl CrossRef ↵ Buchan , K. D. , Prajsnar , T. K. , Ogryzko , N. V. , De Jong , N. W. M. , Van Gent , M. , Kolata , J. , Foster , S. J. , Van Strijp , J. A. G. , & Renshaw , S. A. ( 2019 ). A transgenic zebrafish line for in vivo visualisation of neutrophil myeloperoxidase . PLOS ONE , 14 ( 4 ), e0215592 . doi: 10.1371/journal.pone.0215592 OpenUrl CrossRef PubMed ↵ Carey , C. M. , Hollins , H. L. , Schmid , A. V. , & Gagnon , J. A . ( 2024 ). Distinct features of the regenerating heart uncovered through comparative single-cell profiling . Biology Open , 13 ( 4 ), bio060156. doi: 10.1242/bio.060156 OpenUrl CrossRef ↵ Chen , S. N. , Gan , Z. , Hou , J. , Yang , Y. C. , Huang , L. , Huang , B. , Wang , S. , & Nie , P . ( 2022 ). Identification and establishment of type IV interferon and the characterization of interferon-υ including its class II cytokine receptors IFN-υR1 and IL-10R2 . Nature Communications , 13 ( 1 ), 999 . doi: 10.1038/s41467-022-28645-6 OpenUrl CrossRef PubMed ↵ Chen , Z. , Li , B. , Zhan , R.-Z. , Rao , L. , & Bursac , N . ( 2021 ). Exercise mimetics and JAK inhibition attenuate IFN-γ–induced wasting in engineered human skeletal muscle . Science Advances , 7 ( 4 ), eabd9502. doi: 10.1126/sciadv.abd9502 OpenUrl CrossRef ↵ Cheng , M. , Nguyen , M.-H. , Fantuzzi , G. , & Koh , T. J . ( 2008 ). Endogenous interferon-γ is required for efficient skeletal muscle regeneration . American Journal of Physiology-Cell Physiology , 294 ( 5 ), C1183 – C1191 . doi: 10.1152/ajpcell.00568.2007 OpenUrl CrossRef PubMed Web of Science ↵ Constanty , F. , Wu , B. , Wei , K.-H. , Lin , I.-T. , Dallmann , J. , Guenther , S. , Lautenschlaeger , T. , Priya , R. , Lai , S.-L. , Stainier , D. Y. R. , & Beisaw , A . ( 2025 ). Border-zone cardiomyocytes and macrophages regulate extracellular matrix remodeling to promote cardiomyocyte protrusion during cardiac regeneration . Nature Communications , 16 ( 1 ), 3823 . doi: 10.1038/s41467-025-59169-4 OpenUrl CrossRef Dolejsi , T. , Delgobo , M. , Schuetz , T. , Tortola , L. , Heinze , K. G. , Hofmann , U. , Frantz , S. , Bauer , A. , Ruschitzka , F. , Penninger , J. M. , Campos Ramos , G. , & Haubner , B. J . ( 2022 ). Adult T-cells impair neonatal cardiac regeneration . European Heart Journal , 43 ( 28 ), 2698 – 2709 . doi: 10.1093/eurheartj/ehac153 OpenUrl CrossRef PubMed ↵ El-Sammak , H. , Yang , B. , Guenther , S. , Chen , W. , Marín-Juez , R. , & Stainier , D. Y. R . ( 2022 ). A Vegfc-Emilin2a-Cxcl8a Signaling Axis Required for Zebrafish Cardiac Regeneration . Circulation Research , 130 ( 7 ), 1014 – 1029 . doi: 10.1161/CIRCRESAHA.121.319929 OpenUrl CrossRef PubMed ↵ Feng , D. , Dong , Y. , Song , Y. , Yapundich , N. , Xie , Y. , Spurlock , B. , Lyu , T. , Kuehn , L. , Qian , L. , & Liu , J . ( 2025 ). Nr4a1 modulates inflammation and heart regeneration in zebrafish . Development , 152 ( 20 ), dev204395. doi: 10.1242/dev.204395 OpenUrl CrossRef ↵ Gemberling , M. , Bailey , T. J. , Hyde , D. R. , & Poss , K. D . ( 2013 ). The zebrafish as a model for complex tissue regeneration . Trends in Genetics , 29 ( 11 ), 611 – 620 . doi: 10.1016/j.tig.2013.07.003 OpenUrl CrossRef PubMed ↵ Gong , Y. , & Koh , D.-R . ( 2010 ). Neutrophils promote inflammatory angiogenesis via release of preformed VEGF in an in vivo corneal model . Cell and Tissue Research , 339 ( 2 ), 437 – 448 . doi: 10.1007/s00441-009-0908-5 OpenUrl CrossRef PubMed Web of Science González-Rosa , J. M. , Burns , C. E. , & Burns , C. G . ( 2017 ). Zebrafish heart regeneration: 15 years of discoveries . Regeneration , 4 ( 3 ), 105 – 123 . doi: 10.1002/reg2.83 OpenUrl CrossRef PubMed ↵ González-Rosa , J. M. , Martín , V. , Peralta , M. , Torres , M. , & Mercader , N . ( 2011 ). Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish . Development , 138 ( 9 ), 1663 – 1674 . doi: 10.1242/dev.060897 OpenUrl Abstract / FREE Full Text ↵ González-Rosa , J. M. , & Mercader , N . ( 2012 ). Cryoinjury as a myocardial infarction model for the study of cardiac regeneration in the zebrafish . Nature Protocols , 7 ( 4 ), 782 – 788 . doi: 10.1038/nprot.2012.025 OpenUrl CrossRef PubMed ↵ Goumenaki , P. , Günther , S. , Kikhi , K. , Looso , M. , Marín-Juez , R. , & Stainier , D. Y. R . ( 2024 ). The innate immune regulator MyD88 dampens fibrosis during zebrafish heart regeneration . Nature Cardiovascular Research , 3 ( 9 ), 1158 – 1176 . doi: 10.1038/s44161-024-00538-5 OpenUrl CrossRef PubMed ↵ Iribarne , M . ( 2021 ). Inflammation induces zebrafish regeneration . Neural Regeneration Research , 16 ( 9 ), 1693 – 1701 . doi: 10.4103/1673-5374.306059 OpenUrl CrossRef PubMed ↵ Isles , H. M. , Herman , K. D. , Robertson , A. L. , Loynes , C. A. , Prince , L. R. , Elks , P. M. , & Renshaw , S. A . ( 2019 ). The CXCL12/CXCR4 Signaling Axis Retains Neutrophils at Inflammatory Sites in Zebrafish . Frontiers in Immunology , 10 , 1784 . doi: 10.3389/fimmu.2019.01784 OpenUrl CrossRef PubMed ↵ Julier , Z. , Park , A. J. , Briquez , P. S. , & Martino , M. M . ( 2017 ). Promoting tissue regeneration by modulating the immune system . Acta Biomaterialia , 53 , 13 – 28 . doi: 10.1016/j.actbio.2017.01.056 OpenUrl CrossRef PubMed ↵ Kell , M. J. , Riccio , R. E. , Baumgartner , E. A. , Compton , Z. J. , Pecorin , P. J. , Mitchell , T. A. , Topczewski , J. , & LeClair , E. E . ( 2018 ). Targeted deletion of the zebrafish actin-bundling protein L-plastin (lcp1) . PLOS ONE , 13 ( 1 ), e0190353 . doi: 10.1371/journal.pone.0190353 OpenUrl CrossRef PubMed ↵ Lai , S.-L. , Marín-Juez , R. , Moura , P. L. , Kuenne , C. , Lai , J. K. H. , Tsedeke , A. T. , Guenther , S. , Looso , M. , & Stainier , D. Y . ( 2017 ). Reciprocal analyses in zebrafish and medaka reveal that harnessing the immune response promotes cardiac regeneration . eLife , 6 , e25605 . doi: 10.7554/eLife.25605 OpenUrl CrossRef PubMed ↵ Leibowitz , B. J. , Zhao , G. , Wei , L. , Ruan , H. , Epperly , M. , Chen , L. , Lu , X. , Greenberger , J. S. , Zhang , L. , & Yu , J . ( 2021 ). Interferon β drives intestinal regeneration after radiation . Science Advances , 7 ( 41 ), eabi5253. doi: 10.1126/sciadv.abi5253 OpenUrl CrossRef Li , L. , Yan , B. , Shi , Y.-Q. , Zhang , W.-Q. , & Wen , Z.-L . ( 2012 ). Live Imaging Reveals Differing Roles of Macrophages and Neutrophils during Zebrafish Tail Fin Regeneration * . Journal of Biological Chemistry , 287 ( 30 ), 25353 – 25360 . doi: 10.1074/jbc.M112.349126 OpenUrl Abstract / FREE Full Text ↵ Lörchner , H. , Cañes Esteve , L. , Góes , M. E. , Harzenetter , R. , Brachmann , N. , Gajawada , P. , Günther , S. , Doll , N. , Pöling , J. , & Braun , T . ( 2023 ). Neutrophils for Revascularization Require Activation of CCR6 and CCL20 by TNFα . Circulation Research , 133 ( 7 ), 592 – 610 . doi: 10.1161/CIRCRESAHA.123.323071 OpenUrl CrossRef PubMed ↵ Luz , R. B. D. S. , Paula , A. G. P. , Czaikovski , A. P. , Nunes , B. S. F. , De Lima , J. D. , Paredes , L. C. , Bastos , T. S. B. , Richardson , R. , & Braga , T. T. ( 2025 ). Macrophages and cardiac lesion in zebrafish: What can single-cell RNA sequencing reveal? Frontiers in Cardiovascular Medicine , 12 , 1570582 . doi: 10.3389/fcvm.2025.1570582 OpenUrl CrossRef PubMed ↵ Marín-Juez , R. , Marass , M. , Gauvrit , S. , Rossi , A. , Lai , S.-L. , Materna , S. C. , Black , B. L. , & Stainier , D. Y. R . ( 2016 ). Fast revascularization of the injured area is essential to support zebrafish heart regeneration . Proceedings of the National Academy of Sciences , 113 ( 40 ), 11237 – 11242 . doi: 10.1073/pnas.1605431113 OpenUrl Abstract / FREE Full Text ↵ Marwick , J. A. , Mills , R. , Kay , O. , Michail , K. , Stephen , J. , Rossi , A. G. , Dransfield , I. , & Hirani , N . ( 2018 ). Neutrophils induce macrophage anti-inflammatory reprogramming by suppressing NF-κB activation . Cell Death & Disease , 9 ( 6 ), 665 . doi: 10.1038/s41419-018-0710-y OpenUrl CrossRef PubMed Mitländer , H. , Yang , Z. , Krammer , S. , Grund , J. C. , Zirlik , S. , & Finotto , S . ( 2023 ). Poly I:C Pre-Treatment Induced the Anti-Viral Interferon Response in Airway Epithelial Cells . Viruses , 15 ( 12 ), 2328 . doi: 10.3390/v15122328 OpenUrl CrossRef PubMed ↵ Nguyen-Chi , M. , Laplace-Builhé , B. , Travnickova , J. , Luz-Crawford , P. , Tejedor , G. , Lutfalla , G. , Kissa , K. , Jorgensen , C. , & Djouad , F . ( 2017 ). TNF signaling and macrophages govern fin regeneration in zebrafish larvae . Cell Death & Disease , 8 ( 8 ), e2979 – e2979 . doi: 10.1038/cddis.2017.374 OpenUrl CrossRef PubMed ↵ Nishio , N. , Okawa , Y. , Sakurai , H. , & Isobe , K . ( 2008 ). Neutrophil depletion delays wound repair in aged mice . AGE , 30 ( 1 ), 11 – 19 . doi: 10.1007/s11357-007-9043-y OpenUrl CrossRef PubMed ↵ Peterson , E. A. , Sun , J. , Chen , X. , & Wang , J . ( 2024 ). Neutrophils facilitate the epicardial regenerative response after zebrafish heart injury . Developmental Biology , 508 , 93 – 106 . doi: 10.1016/j.ydbio.2024.01.011 OpenUrl CrossRef PubMed ↵ Poss , K. D. , Wilson , L. G. , & Keating , M. T . ( 2002 ). Heart Regeneration in Zebrafish . Science , 298 ( 5601 ), 2188 – 2190 . doi: 10.1126/science.1077857 OpenUrl Abstract / FREE Full Text ↵ Salari , N. , Morddarvanjoghi , F. , Abdolmaleki , A. , Rasoulpoor , S. , Khaleghi , A. A. , Hezarkhani , L. A. , Shohaimi , S. , & Mohammadi , M . ( 2023 ). The global prevalence of myocardial infarction: A systematic review and meta-analysis . BMC Cardiovascular Disorders , 23 ( 1 ), 206 . doi: 10.1186/s12872-023-03231-w OpenUrl CrossRef Sanceau , J. , Wijdenes , J. , Revel , M. , & Wietzerbin , J . ( 1991 ). IL-6 and IL-6 receptor modulation by IFN-gamma and tumor necrosis factor-alpha in human monocytic cell line (THP-1). Priming effect of IFN-gamma. Journal of Immunology (Baltimore , Md .: 1950 ) , 147 (8), 2630 – 2637 . OpenUrl ↵ Sánchez-Iranzo , H. , Galardi-Castilla , M. , Sanz-Morejón , A. , González-Rosa , J. M. , Costa , R. , Ernst , A. , Sainz De Aja , J. , Langa , X. , & Mercader , N. ( 2018 ). Transient fibrosis resolves via fibroblast inactivation in the regenerating zebrafish heart . Proceedings of the National Academy of Sciences , 115 ( 16 ), 4188 – 4193 . doi: 10.1073/pnas.1716713115 OpenUrl Abstract / FREE Full Text ↵ Schneider , W. M. , Chevillotte , M. D. , & Rice , C. M . ( 2014 ). Interferon-Stimulated Genes: A Complex Web of Host Defenses . Annual Review of Immunology , 32 ( 1 ), 513 – 545 . doi: 10.1146/annurev-immunol-032713-120231 OpenUrl CrossRef PubMed Web of Science ↵ Stirling , D. P. , Liu , S. , Kubes , P. , & Yong , V. W . ( 2009 ). Depletion of Ly6G/Gr-1 Leukocytes after Spinal Cord Injury in Mice Alters Wound Healing and Worsens Neurological Outcome . The Journal of Neuroscience , 29 ( 3 ), 753 – 764 . doi: 10.1523/JNEUROSCI.4918-08.2009 OpenUrl Abstract / FREE Full Text ↵ Stirling , D. R. , Swain-Bowden , M. J. , Lucas , A. M. , Carpenter , A. E. , Cimini , B. A. , & Goodman , A . ( 2021 ). CellProfiler 4: Improvements in speed, utility and usability . BMC Bioinformatics , 22 ( 1 ), 433 . doi: 10.1186/s12859-021-04344-9 OpenUrl CrossRef PubMed ↵ Wilgus , T. A. , Roy , S. , & McDaniel , J. C . ( 2013 ). Neutrophils and Wound Repair: Positive Actions and Negative Reactions . Advances in Wound Care , 2 ( 7 ), 379 – 388 . doi: 10.1089/wound.2012.0383 OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted November 16, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Interferon signaling promotes early neutrophil recruitment after zebrafish heart injury Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Interferon signaling promotes early neutrophil recruitment after zebrafish heart injury Alexis V. Schmid , James A. Gagnon bioRxiv 2025.11.16.688717; doi: https://doi.org/10.1101/2025.11.16.688717 Share This Article: Copy Citation Tools Interferon signaling promotes early neutrophil recruitment after zebrafish heart injury Alexis V. Schmid , James A. Gagnon bioRxiv 2025.11.16.688717; doi: https://doi.org/10.1101/2025.11.16.688717 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Developmental Biology Subject Areas All Articles Animal Behavior and Cognition (7629) Biochemistry (17660) Bioengineering (13881) Bioinformatics (41913) Biophysics (21436) Cancer Biology (18578) Cell Biology (25482) Clinical Trials (138) Developmental Biology (13372) Ecology (19889) Epidemiology (2067) Evolutionary Biology (24302) Genetics (15599) Genomics (22483) Immunology (17728) Microbiology (40365) Molecular Biology (17163) Neuroscience (88540) Paleontology (666) Pathology (2830) Pharmacology and Toxicology (4821) Physiology (7637) Plant Biology (15130) Scientific Communication and Education (2045) Synthetic Biology (4290) Systems Biology (9818) Zoology (2269)