Heme oxygenase 1 activity mediates hemoglobin clearance and tail fin regeneration in zebrafish larvae

Heme oxygenase 1 activity mediates hemoglobin clearance and tail fin regeneration in zebrafish larvae | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Heme oxygenase 1 activity mediates hemoglobin clearance and tail fin regeneration in zebrafish larvae Ashmair Mirza, Carlos Muñoz-Montecinos, Graham J. Lieschke, Miguel L. Allende, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7799531/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Tissue injury triggers a tightly regulated cascade of events that transitions from inflammation to resolution and ultimately tissue remodeling. Although the cellular dynamics of immune cells during these phases are increasingly well-characterized, the molecular mediators orchestrating the response to injury are yet to be fully elucidated. Based on a zebrafish model of tissue injury and using proteomic, in situ RNA expression analyses, and novel transgenic fluorescent reporters, we aimed to uncover relevant molecular mediators of tissue inflammation, resolution, and regeneration. We found that hemoglobin accumulated in the injury site after tail fin amputation in zebrafish larvae, reaching its peak during the inflammatory phase and decreasing together with the resolution of inflammation. Furthermore, we observed that the heme scavenger and cytoprotective enzyme heme oxygenase 1 (Hmox1) is expressed in the injury site of amputated tail fins, and that macrophages were the main source of the functional hmox1a paralog. Pharmacological inhibition of Hmox1 activity impaired hemoglobin clearance and tail fin regeneration. In addition, depletion of macrophages led to impaired hemoglobin clearance, phenocopying Hmox1 inhibition. Altogether, our findings reveal a novel role for Hmox1 in shaping the regenerative microenvironment and identify hemoglobin and hmox1a -expressing macrophages as previously overlooked players in the zebrafish injury response. This work underscores a new link between heme metabolism, immune regulation, and tissue regeneration in vivo . Biological sciences/Biochemistry Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Cell biology Biological sciences/Molecular biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The tissue response to injury is composed of an orchestrated cascade of events, including wound closure, inflammation, proliferation, and tissue remodeling. In non-regenerative systems, these events normally culminate in scar formation, whereas in fully regenerative systems, the outcome is the formation of a tissue that is architecturally and functionally identical to the tissue before injury. Immune cells are key players in the cascade of events following tissue damage. Neutrophils and macrophages are rapidly recruited to the injured tissue and promote local pro-inflammatory responses primarily to protect the tissue from potential pathogenic infection [ 1 , 2 ]. Macrophages, on the other hand, are responsible for clearing apoptotic and damaged cells in the injury site, counteract pro-inflammatory signals, and produce growth factors that initiate wound healing and tissue regeneration [ 1 ]. To perform these tasks, macrophages undergo phenotypic and functional changes that endow them with anti-inflammatory and pro-regenerative functions [ 3 ]. However, the temporal dynamics of these anti-inflammatory programs in macrophages and their precise contribution to the resolution of inflammation and tissue regeneration remain poorly understood. Zebrafish ( Danio rerio ) has become a well-established animal model for studying inflammatory responses thanks to its optical transparency during larval stages, which allows intravital and real-time visualization of immune responses following injury. Additionally, its high regenerative capacity has facilitated studying the impact of immune cells during tissue regeneration. The zebrafish larva possesses functional hematopoietic and innate immune cells [ 4 – 6 ], with the latter being critical orchestrators of tissue inflammation and regeneration. Using the tail fin amputation model in zebrafish larvae, we and others have previously shown that macrophages are the predominant immune cell type contributing to tissue regeneration [ 7 – 10 ]. Mechanistically, recruited macrophages promote the resolution of inflammation by attenuating local production of pro-apoptotic Il1b [ 11 ] and suppressing ROS production [ 10 ], while they activate regenerative programs on stromal cells through Tnfa signaling [ 9 ]. However, which anti-inflammatory proteins are expressed by zebrafish macrophages and play a role in the resolution of inflammation and tissue regeneration remains under investigation. Studies from mammals show that expression of the heme scavenger and cytoprotective enzyme heme oxygenase 1 (HO-1, encoded by the HMOX1 gene) confers macrophages with anti-inflammatory [ 12 ] and pro-regenerative functions [ 13 ]. Zebrafish possess two orthologs of the mammalian HMOX1 gene: hmox1a and hmox1b . Although hmox1a has been associated with macrophage function [ 14 ], it remains unclear whether hmox1a is expressed by zebrafish macrophages during injury and plays a role in the resolution of inflammation and tissue regeneration. In this work, using an unbiased proteomic analysis, we found that hemoglobin (Hb) accumulated in the injury site after tail fin amputation in zebrafish larvae. Hb levels in the injury site peaked during the inflammatory phase and decreased before regeneration took place. By performing RNA expression analysis and using a novel fluorescent transgenic line, we observed the expression of the functional hmox1a paralog in macrophages at the injury site of amputated tail fins. Depletion of macrophages led to impaired Hb clearance from the injury site, whereas pharmacological inhibition of Hmox1 activity following tail fin amputation impaired Hb clearance and tail fin regeneration. Altogether, our findings reveal a novel role for Hmox1 in shaping the regenerative microenvironment and identify Hb and hmox1a -expressing macrophages as relevant players in the zebrafish injury response. Results Accumulation of hemoglobin in the injury site after tail fin amputation Using our previously described model of tail fin amputation [ 10 ], we analyzed the recruitment of neutrophils and macrophages to the injury site in 72 hours post fertilization (hpf) double transgenic reporter Tg(mpeg1:GFP; mpx:mCherry) larvae. In line with previous reports [ 7 , 15 ], we observed an “inflammation” phase at 6 hours post amputation (hpa), characterized by a peak of neutrophils recruited to the injury site (Fig. 1 A-B) and a modest increase in the recruitment of macrophages. After 6hpa, the number of neutrophils in the injury site decreased, while the number of recruited macrophages continued increasing. By 24hpa, we observed a “resolution” phase where macrophages were predominant over neutrophils (Fig. 1 A-B). We selected 6hpa (inflammation) and 24hpa (resolution) to perform proteomic analyses of injured tissues and uninjured time-matched controls (Fig. 1 C, Table S1 ). After filtering and removal of duplicated proteins (see methods), we sought to identify the proteins that were exclusively expressed during inflammation and resolution. By comparing with their respective time-matched controls, we identified 256 proteins that were exclusively present in injured tissues during inflammation, while 296 proteins were detected exclusively in the injured tissue at the resolution phase (Fig. 1 D, Table S2 ). Next, we compared the proteins exclusive to injured tissues in inflammation versus resolution, and we found 219 inflammation-specific and 259 resolution-specific proteins (Fig. 1 E, Table S2 ). Functional classification analysis of identified phase-specific proteins using DAVID bioinformatic resources [ 16 ] gave us insights into proteins and biological processes that were active during inflammation and resolution (Fig. 1 F). Among them, we found targets previously described during resolution and important for tissue regeneration, such as hsp60/GroEL activity [ 17 ] and Coronin, the latter expressed by myeloid cells during tissue damage [ 7 ]. During inflammation, we found increased expression of mitochondrial cytochrome c oxidase proteins (Cox5aa and Cox5ab), supporting increased inflammation and suggesting higher mitochondrial activity. Interestingly, we found increased levels of the hemoglobin (Hb) proteins Hbbe2, Ba1 (Hbba1), and Hbbe3 during inflammation (Fig. 1 F, Table S3 ). To validate these findings, we performed Hb stainings with o-dianisidine in tail fin amputated larvae during inflammation and resolution. We found accumulation of o-dianisidine + dots at the injury site during inflammation (6hpa), which decreased sharply at resolution (24hpa, Fig. 1 G-H). Since these proteins are common markers for erythroid cells, we analyzed the expression of the coding genes for these proteins ( hbbe2 , hbba1 and hbbe3 ) in a previously published single-cell dataset of the developing zebrafish embryo [ 18 ] ( Fig S1 A-B ). We found that expression of these genes is restricted to red blood cells (RBCs) and are not expressed by immune cell types in homeostasis ( Fig. S1 C ). Altogether, these results indicate that Hb, likely contained in RBCs, accumulate in the injury site during the inflammatory phase after tail fin amputation in zebrafish larvae. Expression of heme oxygenase 1 is detected in the injury site of amputated larvae The decrease of Hb in the injury site during resolution led us to hypothesize that an active mechanism oversees the removal of Hb from the injury site after tissue damage. The degradation of Hb involves the breakdown of globin proteins into amino acids, and the catalysis of the heme ring by the Heme oxygenase 1 (Hmox1) enzyme into carbon monoxide (CO), iron (Fe), and biliverdin [ 19 ]. We therefore analyzed the expression of Hmox1 in the injury site after damage. Through whole-mount in situ hybridization (WISH) we found enriched expression of the zebrafish Hmox1-coding paralogs hmox1a and hmox1b in amputated tail fins, in a time frame that coincided with the reduction of Hb from the injury site from 6hpa to 24hpa (Fig. 2 A). Interestingly, we found different expression patterns for the Hmox1 paralogs within the injury site: while the hmox1a signal was discrete and seemed restricted to individual cells, hmox1b expression was broadly expressed in the edge of the injured tissue (Fig. 2 A). These results suggest that hmox1b expression is locally induced in the injured tissue, whereas hmox1a expression takes place in specific cells supporting the response to injury. As it has been previously described that hmox1a is the functional ortholog of the human HMOX1 gene and that hmox1b is likely a pseudogene [ 20 ], we decided to focus on the characterization of hmox1a in the injury site. Macrophages are the source of hmox1a in the injury site of amputated larvae It has been previously reported that hmox1a is expressed in hematopoietic tissues of larvae [ 21 , 22 ], and that is required for the proper function of macrophages [ 14 ], which led us to hypothesize that macrophages are the source of hmox1a in the injury site of amputated larvae. By performing RT-PCR from FACS-sorted neutrophils and macrophages from the triple transgenic Tg(mpx:GFP; mpeg1:Gal4FF; UAS:nfsB-mCherry) at 72hpf, we observed that macrophages, but not neutrophils, expressed hmox1a in homeostatic conditions (Fig. 2 B, Fig. S2 ). Single-cell analysis from the developing zebrafish atlas confirmed macrophages as the main source of hmox1a among immune and hematopoietic cells ( Fig. S3 A ). To further explore the kinetics of hmox1a + cells in vivo , we generated two transgenic fluorescent reporters for hmox1a expression. For this, we cloned the 2.1kb genomic region immediately upstream of the initial ATG from the hmox1a in a Tol2-flanking plasmid containing the fluorescent proteins Dendra2 or membrane mCherry (mCherryCAAX) ( Fig. S3 B , see methods). We injected these constructs into WT embryos, and after 2 generations we obtained the stable transgenic lines Tg(-2.1hmox1a:Dendra2) and Tg(-2.1hmox1a:mCherryCAAX) . We observed fluorescence expression of hmox1a in the posterior blood island (PBI) at 30hpf and caudal hematopoietic tissue (CHT) at 72hpf and 120hpf, in addition to expression in the lens and liver ( Fig. S3 C ), in line with observations made in a previously generated reporter for hmox1a [ 22 ]. Next, we crossed the generated hmox1a reporters with Tg(lyz:DsRed ) and Tg(mpeg1:Dendra2) transgenic fish to analyze hmox1a expression in neutrophils and macrophages, respectively. We observed that macrophages were positive for hmox1a at 72hpf, while neutrophils were not ( Fig. 2Ci , Fig. S3 D-F ). Interestingly, we found variable expression levels of hmox1a in macrophages, as well as a group of macrophages that were negative for hmox1a ( Fig. 2Cii ). A more detailed analysis of the tail of 72hpf Tg(mpeg1:Dendra2; -2.1hmox1a:mCherryCAAX) larvae showed that 81.22% of tail macrophages expressed hmox1a , while the remaining 18.78% of tail macrophages were negative ( Fig. S3 E ). Next, we performed tail fin amputations in the double transgenic Tg(mpeg1:Dendra2; -2.1hmox1a:mCherryCAAX) at 72hpf to analyze hmox1a expression by macrophages in the injury site. We found that 90.28% of macrophages recruited to the injury site at 6hpa expressed hmox1a , but this proportion was reduced to 80.79% at 24hpa (Fig. 2 D-E). Of note, the expression of hmox1a in the injury site was restricted to mpeg1 + macrophages in both timepoints, indicating that macrophages were the source of hmox1a expression in the injury site. Inhibition of Hmox1 activity impairs Hb clearance and tail fin regeneration To determine the functional consequences of Hmox1 activity after tail fin amputation, we took advantage of the Hmox1 inhibitor Tin protoporphyrin XI (SnPP), previously tested in zebrafish [ 14 , 23 ], and we treated larvae for 24 hours immediately after amputation (Fig. 3 A). Since a previous report suggested that macrophage recruitment to injury was impaired after Hmox1 activity inhibition [ 14 ], we analyzed macrophage recruitment in our experimental settings. Although we did not observe differences in the number (Fig. 3 B-C) nor their sphericity (Fig. 3 D, left ), we observed that recruited macrophages in SnPP-treated larvae had a smaller average volume compared to control larvae (Fig. 3 D, right ), suggesting that SnPP may affect macrophage physiology. Next, we analyzed the clearance of Hb from the injury site after tail fin after amputation in SnPP-treated larvae. Inhibition of Hmox1 activity by SnPP led to an increased number of o-dianisidine + dots at 24hpa (Fig. 3 E-F), indicating impaired Hb clearance from the injury site. We finally assessed whether inhibiting Hmox1 activity during the first 24h after amputation has long-term consequences and affects tail fin regeneration (Fig. 3 G). We found that the regenerating tail fins of SnPP-treated larvae at 3 days post amputation (3dpa) were smaller in area when compared to their respective controls (Fig. 3 H-I). These findings indicate that Hmox1 activity in the injury site promotes the clearance of hemoglobin from the injury site and the regeneration of the tail fin in larvae. Macrophage depletion increases Hb in the injury site during the resolution of inflammation The importance of macrophages in the clearance of damage and pro-inflammatory cues, and our observations indicating that macrophages were the source of hmox1a in the injury site, led us to hypothesize that depleting macrophages may impair Hb clearance from the injury site. To test this, we depleted macrophages by injecting 2nL of 1:10 (0.5mg/mL) Lipo-clodronate and Lipo-PBS in the bloodstream of 54hpf zebrafish embryos (Fig. 4 A), which we previously showed to deplete ~ 60% of macrophages in the tail and impair tail fin regeneration after amputation at 72hpf [ 10 ]. As expected, the recruitment of macrophages in Lipo-clodronate-injected larvae was markedly reduced at 6hpa and 24hpa compared to Lipo-PBS controls (Fig. 4 B-C). Additionally, the number of o-dianisidine + dots at the injury site was higher in the Lipo-clodronate group, compared to Lipo-PBS controls, at 24hpa (Fig. 4 D - E ), phenocopying the pharmacological inhibition of Hmox1 activity. In conclusion, our findings support macrophages as regulators of Hb levels in the injury site after tail fin amputation in zebrafish larvae. Discussion The availability of transgenic reporter lines and their optical transparency has made zebrafish larvae an ideal model to explore innate immune responses in vivo . While the focus has been directed toward neutrophils and macrophages, the potential involvement of additional hematopoietic-derived mediators during tissue injury has remained unexplored. Using an unbiased proteomic approach, we identified the accumulation of hemoglobin (Hb) in the injury site during inflammation, which subsequently cleared during resolution. In parallel, we observed the expression of the Hmox1 coding genes hmox1a and hmox1b in the injury site, and we identified macrophages as the source of hmox1a in the injury site after tail fin amputation. Functionally, both pharmacological inhibition of Hmox1 activity with SnPP and the depletion of macrophages led to increased Hb levels at the injury site, supporting the role of macrophages in Hb clearance. Additionally, we found that the inhibition of Hmox1 activity led to impaired tail fin regeneration, similar to what has been previously observed upon macrophage depletion [8–10]. Altogether, our findings show that Hmox1 is important for the macrophage-dependent clearance of Hb from the injury site and promotes tissue regeneration. Our results show that zebrafish Hb accumulates in the injury site after tail fin amputation. Given that Hb is a protein whose expression is restricted to red blood cells (RBCs), we propose that RBCs accumulate in the injury site after tail fin amputation. However, non-erythroid cells can also express hemoglobin under specific conditions (reviewed by [24, 25]), as reported previously for immune cells such as macrophages or peripheral blood mononuclear cells in response to inflammatory stimuli [26, 27]. Therefore, further analyses are required, e.g., in vivo tracking of fluorescently labelled RBCs using fluorescence reporters for the erythroid lineage marker gata1a [28, 29] or the LCR locus controlling globin expression [30], to confirm that RBCs are the source of the Hb found in the injury site of zebrafish larvae. While the primary function of Hb from RBCs is to provide oxygen to the injured tissue, oxygen exchange in zebrafish larvae occurs independently of RBCs [31, 32], which raises the question of what the role of Hb, and potentially RBCs, is in this context. It is known that RBCs are highly susceptible to hemolysis in extravascular spaces [33], and the heme released after hemolysis to the extracellular space may act as a DAMP capable of inducing oxidative stress, cytotoxicity, and amplifying inflammation through activation of toll-like receptor 4 (TLR4) signaling pathways [34]. Thus, while Hb accumulation could be relevant to the initial steps of inflammation and likely protect the injured tissue from external pathogens [35], the release of heme in extravascular tissues poses a significant risk of cytotoxicity and exacerbation of inflammation, and therefore must be cleared to avoid chronic inflammation. The temporal dynamics of Hb at the injury site tissue suggests that active Hb clearance is part of the resolution of inflammation, a required step for tissue regeneration. We observed the expression of the Hmox1-coding genes hmox1a and hmox1b in the injury site, which correlated with the peak and reduction of Hb in the injury site, and led us to propose that the function of Hmox1 in the injured tissue is the degradation of heme from the Hb accumulated following injury. However, it is also possible that the anti-oxidant function of Hmox1 activity directly regulates the inflammatory responses in the injury site, as previous studies have suggested [36, 37], and further analysis of pro-inflammatory cytokine expression and reactive oxygen species (ROS) production are required to better understand the relationship between Hmox1 activity and inflammatory responses. Since a previous study showed that hmox1a is the functional ortholog of the human HMOX1 gene [20], we focused our attention on characterizing the source of hmox1a expression following injury. However, we cannot rule out that hmox1b expression in the injury site can play regulatory functions such as small interfering RNA, competitive endogenous RNA, or antisense transcripts [38], which could ultimately modulate the tissue response to injury at a transcriptional level. Our results showed that macrophages are an important, but not exclusive, source of hmox1a in zebrafish during steady state conditions. However, macrophages were the sole source of hmox1a in the injury site after tail fin amputation, with most of the macrophages being hmox1a + . The use of a novel hmox1a fluorescent reporter line in our study provided an unprecedented view of hmox1a expression dynamics in vivo . A previous study described a reporter for hmox1a [22], and the expression pattern described in the rostral blood island, liver, and retina matched our observations. However, they attributed hmox1a expression to erythroid lineages and not to macrophages, as we showed in this study. Our observations are supported by previous evidence showing functional defects of macrophages in hmox1a mutants [14] and single-cell transcriptomic analysis showing expression of hmox1a in macrophages at the CHT [39]. Thus, our reporter enabled real-time visualization and spatial resolution of hmox1a -expressing macrophages in the context of tissue injury, and offers a valuable advantage for future studies aiming to dissect the precise temporal and cellular dynamics of heme metabolism and immune regulation during homeostasis, tissue injury, and repair. Our findings showed that 1) macrophages were identified as the sole source of hmox1a expression at the injury site, 2) pharmacological inhibition of Hmox1 activity using SnPP led to an impaired clearance of Hb from the injury site, and 3) depletion of macrophages also led to impaired Hb clearance. Therefore, we propose that hmox1a -expressing macrophages are critical mediators of Hb clearance and resolution of inflammatory responses after tissue injury. Crucially, SnPP-treated larvae exhibited impaired tail fin regeneration following amputation, supporting an effector role of Hmox1a in promoting tissue regeneration after injury. This interpretation is further supported by cumulative evidence from murine models of injury showing that HMOX1 expression drives macrophage polarization towards anti-inflammatory profiles [12] and endows them with pro-regenerative functions [13]. However, it remains unclear whether the phenotype of hmox1a -expressing macrophages is predetermined before their recruitment to the injury site, or is modulated in response to local cues — such as the presence of Hb. Furthermore, whether the clearance of Hb relies on phagocytosis by macrophages must be confirmed by employing RBC and macrophage fluorescent reporters, which will allow us to clarify the cellular choreography underpinning resolution of inflammation and tissue regeneration. Despite the insights acquired from our study, several limitations should be considered. First, our reliance on o-dianisidine staining to detect Hb in zebrafish tissues presents inherent limitations. Since o-dianisidine detects Hb based on peroxidase activity [40], it is unclear whether certain subpopulations of erythroid cells or Hb degradation products escape detection, potentially leading to underestimation or spatial misrepresentation of Hb accumulation. A more quantitative or marker-specific approach, such as RBC-specific fluorescent transgenic reporters, may improve future resolution. Second, although we inferred the inflammatory and resolution phases following injury based on immune cell dynamics — specifically neutrophil presence — we relied on using specific timepoints associated with inflammation and resolution (6hpa and 24hpa), omitting a comprehensive kinetic evaluation of Hb at multiple timepoints. Additionally, assessing the expression of common pro- and anti-inflammatory cytokines (such as tnfa , il1b , and il10 ) would further aid in delineating the inflammatory and resolution phases following injury. A better-defined characterization of the inflammatory milieu would help to further clarify timing and polarization of inflammatory responses, and validate the proposed role of Hmox1a in inflammation resolution. Furthermore, interpretative caveats must be considered with the expression of the hmox1a fluorescent reporters to track hmox1a -expressing cells, as fluorescence proteins could persist in a cell even after hmox1a transcription has ceased. As such, the reporter may reflect prior, but not necessarily current, transcriptional activity. Conversely, during early stages of hmox1a induction, the reporter may not yet have accumulated to a detectable threshold—leading to potential underrepresentation of actively transcribing cells. This temporal disconnect between transcriptional dynamics and reporter signal could obscure transient or low-level expression events and must be considered in the interpretation of the data. Collectively, our findings emphasize hmox1a as a macrophage-associated effector essential for the resolution of inflammation after injury and the promotion of tissue regeneration in zebrafish. Additionally, it uncovers hemoglobin as a likely mediator of inflammatory responses, which may have implications in the study of immune responses of patients suffering from hemoglobinopathies. Methods Fish husbandry and lines Zebrafish ( Danio rerio , strains TAB5, AB, and Tübingen) were reared and kept, according to standard procedures, in the zebrafish facilities at the Facultad de Ciencias of Unversidad de Chile, the FishCore at Monash University, and the Karolinska Institutet Zebrafish Core Facility. Adult zebrafish, used to obtain embyros for experiments, were maintained at 28 °C in recirculating, filtered water systems (Techniplast, Italy; Aquaneering, USA), on a regimen of 14 hours of light and 10 hours of darkness. The fish lines used in this work were: Tg(mpeg1:GFP) and Tg(mpeg1:Gal4FF; UAS:nfsB-mCherry) [42]; Tg(mpeg1:Dendra2) [43]; TgBAC(mpx:GFP) i114 , referred to as Tg(mpx:GFP) [44]; Tg(mpx:mCherry) [45]; Tg(lyz:DsRed2) [46]. Zebrafish embryos were collected by natural spawning of adults and were kept at 28°C in E3 water (NaCl 5mM, KCl 0.17mM, CaCl 2 0.33mM, MgSO 4 0.33mM). Embryos were checked daily, and specimenes showing any sings of malformations or developmental delays were discarded and not used for experiments. All procedures were performed in accordance with the “Guidelines for the Use of Fishes in Research Use” of the American Fisheries Society (www.fisheries.org) and complied with local regulations. Husbandry, breeding of zebrafish stocks for the collection of embryos, and procedures performed in zebrafish older than 5 days post fertilization were approved by the Animal Ethics Committee of the Universidad de Chile (approval: 2015-04-20). Husbandry and breeding of zebrafish stocks for the collection of embryos were approved by the Monash University Animal Ethics Committee (protocol MAS-2010-18) and the Stockholm Ethical Committee from the Swedish Board of Agriculture (diary number 15591-2023). This study is reported in accordance with the ARRIVE guidelines. Tail fin amputation and in vivo imaging At 72 hours post fertilization (hpf), larvae were anesthetized with 0.016% MS-222, and the tail fin was amputated with a scalpel, using the caudal vein loop and the posterior section of the ventral pigmentation gap in the tail as references [10]. The amputation performed did not cause damage to the caudal vein loop. Immediately after amputation, larvae were rinsed and incubated in E3 medium at 28°C. Quantifications, imaging of recruited immune cells to the injury site (up to ∼200μm from the amputation site), and imaging of the regenerating tail fin were performed using an Olympus MVX10 stereomicroscope, Olympus IX81 epifluorescence microscope, or a Zeiss Axiovert 200 widefield microscope. Quantifications from images were performed using the “Cell Counter” plugin in Fiji/ImageJ software (NIH, USA). After imaging, larvae were euthanized by incubation with an overdose of MS-222 (0.1%) for 30 minutes, followed by freezing of individuals. Tissue collection and proteomic analyses At 6 and 24 hours post amputation (hpa), larvae were euthanized with an overdose of MS-222, and tail tissue pieces were collected by performing a second transection at approximately 200-300µm from the injury site, using the narrowest region of the larval fin fold as a reference. Single replicates containing tail tissue pieces from 200 larvae/condition/timepoint were collected in 1.5mL Eppendorf tubes, which were spun before removing all the media, snap-frozen in dry ice-cooled methanol, and stored at -80°C. At each time point, tissues from non-amputated controls were collected for comparisons. Samples were subsequently submitted to a global proteomic profiling of 10.000 sequencing events, followed by sequence library searching (service provided by Bioproximity LLC, USA). Briefly, proteins from samples were extracted and sequenced by Shotgun proteomics followed by Tandem Mass Spectrometry (MS/MS) in a Q-Exactive HF-X Orbitrap mass spectrometer. Identified peptides (using OMSSA and X!Tandem algorithms) were aligned to the zebrafish proteome, and UniProt (Universal Protein Resource) identification codes and gene names were obtained. The identification of proteins exclusively present in the injury site at the studied timepoints was performed in 3 steps: 1) First, identified UniProt IDs were converted to Entrez ID using the DAVID Bioinformatic resource v6.8 [16, 47], and redundant Entrez IDs were excluded; 2) common and sample-specific Entrez IDs were identified in samples from injured tissues (TFA) were compared to their respective time-matched non-injured controls using an online Venn diagram generator (https://bioinformatics.psb.ugent.be/webtools/Venn/), and injury-specific Entrez-ID were selected; and 3) common and timepoint-specific Entrez IDs (inflammation or resolution) were identified. Finally, functional annotation clustering of the proteins exclusively present in the injury site at the specific time point was performed using the DAVID Bioinformatic resource v6.8, in which functional annotations, gene ontology (GO), pathways (KEGG [48, 49], permission granted by Kanehisa Laboratories), and protein domains were considered. The classification was performed using the highest stringency settings. Hemoglobin staining in zebrafish larvae Zebrafish larvae were stained with o-dianisidine (Sigma) before or after fixation with PFA 4%, based on previously described protocols [50, 51]. Before imaging, larvae were depigmented with a solution of H 2 O 2 3% v/v and KOH 1% w/v in deionized water. Images were acquired in an Olympus MVX10 stereoscope (Olympus, Japan), and the number of o-dianisidine + dots in the injury site was quantified using the “Cell Counter” plugin in Fiji/ImageJ. Whole-mount in situ hybridization For in situ hybridization experiments, larvae were incubated from 24hpf in E3 supplemented with phenyl thiourea (PTU), and amputations were performed using larvae collected before amputation (72hpf) and after 6h, 10h, and 24h post-amputation. Larvae were fixed in PFA 4%, dehydrated in methanol, and incubated for at least 1 night at -20°C. Whole-mount in situ hybridizations were performed as previously described [52]. For imaging, stained larvae were transferred to glycerol and imaged with an MVX10 stereoscope (Olympus). The pBSKII SK plasmid containing the probe against hmox1a was kindly provided by Dr. Makoto Kobayashi [53], whereas the probe for hmox1b was generated by cloning a portion of the hmox1b gene into a pBSK vector, as previously described [54]. Primers used for the cloning of the hmox1b probe are found in Table S4. Analysis of single-cell RNAseq databases For expression analysis, we took advantage of the single-cell atlas of the developing zebrafish embryo (Farnsworth et al., 2020). The Seurat file containing the clustered single cells was downloaded from https://www.adammillerlab.com/resources. Hematopoietic cells, including neutrophils (cluster 150), macrophages (clusters 71, 184, and 212), thymocytes (cluster 59), RBCs (clusters 6, 38, 48, 63, 85, and 157), and RBC progenitors (cluster 153) were selected for further analysis. Gene expression analyses were performed using Seurat v4.0 in RStudio. Chemical treatments The Hmox1 inhibitor Tin protoporphyrin XI (SnPP, Cat. #0747, Tocris) was resuspended in DMSO to a working stock of 5mM. Immediately after tail fin amputations, larvae were randomly separated in two groups. The first group (treatment) was treated with SnPP at a concentration of 5µM (1:1000 from working stock). The second group of larvae (control) was treated with E3 supplemented with DMSO 0.1%. Treatments were performed for 24 hours, after which larvae were rinsed three times with E3. Sorting of immune cells from larvae and RT-PCR At 72hpf, transgenic reporter larvae Tg(mpx:GFP; mpeg1:Gal4FF; UAS:nfsB-mCherry) were disaggregated to single-cell suspensions as previously reported (Paredes-Zuñiga et al., 2017). Sorting of neutrophils (GFP + ) and macrophages (mCherry + ) was performed using an Influx2 Cell Sorter (BD Biosciences) at the Monash University FlowCore unit. Total RNA was isolated from sorted cells using the RNeasy mini kit (Qiagen), and RT-PCR analyses were performed using the SuperScript III one-step RT-PCR system (Invitrogen). Primers used for RT-PCR are shown in Table S4. Generation of the hmox1a transgenic reporter line The 2,297bp genomic sequence containing the upstream and first 45 nucleotides of the hmox1a coding sequence (Gene ID: 791518) was amplified and cloned into a pCRII vector. A second PCR was performed on the previously cloned sequence to amplify a 2,151bp sequence containing the upstream and first nine nucleotides of the hmox1a coding sequence, flanked by restriction enzyme sites for XhoI and Cfr9I. Primers used for PCR amplifications are shown in Table S4. All the PCRs were performed using the Platinum Taq DNA polymerase high fidelity (Invitrogen). The plasmid Tol2_ mpeg1:Dendra2 (Addgene, plasmid #51462)[43], containing the Dendra2 fluorescent protein flanked by Tol2 elements, was co-digested by XhoI/Cfr9I (Thermo Fisher) to remove the original mpeg1 promoter and ligate the cloned 2.1kb hmox1a promoter, thus generating the Tol2-based -2.1hmox1a:Dendra2 construct. The cloning strategy used left the first 3 amino acids of the hmox1a coding sequence in-frame with the Dendra2 coding sequence. Cloning was verified by restriction enzyme analysis and Sanger sequencing (Macrogen, South Korea). For the generation of the Tol2-based -2.1hmox1a:mCherryCAAX construct, the Dendra2 coding sequence of the generated vector was excised using Cfr9I and MunI restriction enzymes, and replaced by a PCR product containing the mCherryCAAX coding sequence flanked by Cfr9I and MunI. For the generation of the hmox1a transgenic lines, 1-2nL of injection solution containing the generated plasmids (25ng/µL) and the Tol2 transposase mRNA (35ng/µL) was injected into 1-cell stage wild-type zebrafish zygotes. Positive larvae for Dendra2 or mCherryCAAX (F0 generation) were raised to adults, and founders were identified after outcrossing F0 adults with non-fluorescent wild-type fish. Larvae used for experiments were from the F2 generation onwards. Depletion of macrophages with clodronate liposomes Clodronate-loaded liposomes (www.clodronateliposomes.com) were used to reduce the macrophage pool of zebrafish larvae. At 54hpf, zebrafish larvae were randomly separated in two groups. The first group of larvae was injected with 2nL of 0.5mg/mL clodronate-loaded liposomes (Lipo-clodronate, 1:10 from stock) into the bloodstream through the circulation valley. The second group of larvae was injected with an equivalent dilution of PBS-loaded liposomes (Lipo-PBS). Amputations were conducted 18 hours after Lipo-clodronate or Lipo-PBS injections. Statistical analysis Statistical tests were performed using Prism 8.0 software (GraphPad, USA). Differences between samples were considered significant when the obtained p-value was lower than 0.05. Declarations Acknowledgements We thank Dr. Oscar A. Peña for early contributions to the project; staff from the zebrafish facilities at the Facultad de Ciencias of Universidad de Chile, the FishCore at Monash University, and the Karolinska Institutet Zebrafish Core Facility for expert fish care; staff from the FlowCore at Monash University for services and technical assistance; Florencio Espinoza and Dr. Vahid Pazhakh for expert technical help and support; and Dr. Myra N. Chávez for expert revision of the manuscript. Authors’ contributions G.J.L., M.L.A., and R.A.M.C. conceived the idea and designed experiments. A.M., C.M-M., and R.A.M.C. performed experiments. A.M. and R.A.M.C. wrote the manuscript. All authors revised and approved the final version of the manuscript. Funding This work was supported by grants conferred to M.L.A. (FONDAP 15090007, ICN 2021_044, FONDECYT 1140702 and 1221360, REDES 150094) and R.A.M.C. (CONICYT/ANID scholarship 21130458, Åke Wiberg Foundation M24-0035). Availability of data and materials Shotgun proteomic raw files are publicly available in the MassIVE proteomic repository (accession number: MSV000092719). Additional data will be made available on reasonable request. Competing interests The authors declare no competing interests. References Koh TJ, DiPietro LA. Inflammation and wound healing: the role of the macrophage. Expert Rev Mol Med. 2011;13:e23. https://doi.org/10.1017/S1462399411001943. Wilgus TA, Roy S, McDaniel JC. Neutrophils and Wound Repair: Positive Actions and Negative Reactions. Adv Wound Care (New Rochelle). 2013;2:379–88. https://doi.org/10.1089/wound.2012.0383. Wynn TA, Vannella KM. Macrophages in Tissue Repair, Regeneration, and Fibrosis. Immunity. 2016;44:450–62. https://doi.org/10.1016/j.immuni.2016.02.015. Ellett F, Lieschke GJ. Zebrafish as a model for vertebrate hematopoiesis. Curr Opin Pharmacol. 2010;10:563–70. https://doi.org/10.1016/j.coph.2010.05.004. van der Vaart M, Spaink HP, Meijer AH. Pathogen recognition and activation of the innate immune response in zebrafish. Adv Hematol. 2012;2012:159807. https://doi.org/10.1155/2012/159807. Franza M, Varricchio R, Alloisio G, De Simone G, Di Bella S, Ascenzi P, et al. Zebrafish (Danio rerio) as a Model System to Investigate the Role of the Innate Immune Response in Human Infectious Diseases. Int J Mol Sci. 2024;25:12008. https://doi.org/10.3390/ijms252212008. Li L, Yan B, Shi Y-Q, Zhang W-Q, Wen Z-L. Live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration. J Biol Chem. 2012;287:25353–60. https://doi.org/10.1074/jbc.M112.349126. Petrie TA, Strand NS, Yang C-T, Rabinowitz JS, Moon RT. Macrophages modulate adult zebrafish tail fin regeneration. Development. 2014;141:2581–91. https://doi.org/10.1242/dev.098459. Nguyen-Chi M, Laplace-Builhé B, Travnickova J, Luz-Crawford P, Tejedor G, Lutfalla G, et al. TNF signaling and macrophages govern fin regeneration in zebrafish larvae. Cell Death Dis. 2017;8:e2979. https://doi.org/10.1038/cddis.2017.374. Morales RA, Allende ML. Peripheral Macrophages Promote Tissue Regeneration in Zebrafish by Fine-Tuning the Inflammatory Response. Front Immunol. 2019;10:253. https://doi.org/10.3389/fimmu.2019.00253. Hasegawa T, Hall CJ, Crosier PS, Abe G, Kawakami K, Kudo A, et al. Transient inflammatory response mediated by interleukin-1β is required for proper regeneration in zebrafish fin fold. Elife. 2017;6:e22716. https://doi.org/10.7554/eLife.22716. Zhang M, Nakamura K, Kageyama S, Lawal AO, Gong KW, Bhetraratana M, et al. Myeloid HO-1 modulates macrophage polarization and protects against ischemia-reperfusion injury. JCI Insight. 2018;3:e120596, 120596. https://doi.org/10.1172/jci.insight.120596. Patsalos A, Tzerpos P, Halasz L, Nagy G, Pap A, Giannakis N, et al. The BACH1-HMOX1 Regulatory Axis Is Indispensable for Proper Macrophage Subtype Specification and Skeletal Muscle Regeneration. J Immunol. 2019;203:1532–47. https://doi.org/10.4049/jimmunol.1900553. Luo K, Ogawa M, Ayer A, Britton WJ, Stocker R, Kikuchi K, et al. Zebrafish Heme Oxygenase 1a Is Necessary for Normal Development and Macrophage Migration. Zebrafish. 2022;19:7–17. https://doi.org/10.1089/zeb.2021.0058. Gray C, Loynes CA, Whyte MKB, Crossman DC, Renshaw SA, Chico TJA. Simultaneous intravital imaging of macrophage and neutrophil behaviour during inflammation using a novel transgenic zebrafish. Thromb Haemost. 2011;105:811–9. https://doi.org/10.1160/TH10-08-0525. Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4:44–57. https://doi.org/10.1038/nprot.2008.211. Pei W, Tanaka K, Huang SC, Xu L, Liu B, Sinclair J, et al. Extracellular HSP60 triggers tissue regeneration and wound healing by regulating inflammation and cell proliferation. NPJ Regen Med. 2016;1:16013-. https://doi.org/10.1038/npjregenmed.2016.13. Farnsworth DR, Saunders LM, Miller AC. A single-cell transcriptome atlas for zebrafish development. Dev Biol. 2020;459:100–8. https://doi.org/10.1016/j.ydbio.2019.11.008. Ryter SW, Alam J, Choi AMK. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. Physiol Rev. 2006;86:583–650. https://doi.org/10.1152/physrev.00011.2005. Luo K, Stocker R, Britton WJ, Kikuchi K, Oehlers SH. Haem oxygenase limits Mycobacterium marinum infection-induced detrimental ferrostatin-sensitive cell death in zebrafish. FEBS J. 2022;289:671–81. https://doi.org/10.1111/febs.16209. Craven SE, French D, Ye W, de Sauvage F, Rosenthal A. Loss of Hspa9b in zebrafish recapitulates the ineffective hematopoiesis of the myelodysplastic syndrome. Blood. 2005;105:3528–34. https://doi.org/10.1182/blood-2004-03-1089. Holowiecki A, O’Shields B, Jenny MJ. Spatiotemporal expression and transcriptional regulation of heme oxygenase and biliverdin reductase genes in zebrafish (Danio rerio) suggest novel roles during early developmental periods of heightened oxidative stress. Comp Biochem Physiol C Toxicol Pharmacol. 2017;191:138–51. https://doi.org/10.1016/j.cbpc.2016.10.006. Li Z-X, Chen J-W, Yuan F, Huang Y-Y, Zhao L-Y, Li J, et al. Xyloketal B exhibits its antioxidant activity through induction of HO-1 in vascular endothelial cells and zebrafish. Mar Drugs. 2013;11:504–22. https://doi.org/10.3390/md11020504. Saha D, Patgaonkar M, Shroff A, Ayyar K, Bashir T, Reddy KVR. Hemoglobin expression in nonerythroid cells: novel or ubiquitous? Int J Inflam. 2014;2014:803237. https://doi.org/10.1155/2014/803237. Reed EC, Kim JD, Case AJ. Non-canonical hemoglobin: An updated review on its ubiquitous expression. Redox Biol. 2025;82:103602. https://doi.org/10.1016/j.redox.2025.103602. Liu L, Zeng M, Stamler JS. Hemoglobin induction in mouse macrophages. Proc Natl Acad Sci U S A. 1999;96:6643–7. https://doi.org/10.1073/pnas.96.12.6643. Brunyanszki A, Erdelyi K, Szczesny B, Olah G, Salomao R, Herndon DN, et al. Upregulation and Mitochondrial Sequestration of Hemoglobin Occur in Circulating Leukocytes during Critical Illness, Conferring a Cytoprotective Phenotype. Mol Med. 2015;21:666–75. https://doi.org/10.2119/molmed.2015.00187. Long Q, Meng A, Wang H, Jessen JR, Farrell MJ, Lin S. GATA-1 expression pattern can be recapitulated in living transgenic zebrafish using GFP reporter gene. Development. 1997;124:4105–11. https://doi.org/10.1242/dev.124.20.4105. Traver D, Paw BH, Poss KD, Penberthy WT, Lin S, Zon LI. Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants. Nat Immunol. 2003;4:1238–46. https://doi.org/10.1038/ni1007. Ganis JJ, Hsia N, Trompouki E, de Jong JLO, DiBiase A, Lambert JS, et al. Zebrafish globin switching occurs in two developmental stages and is controlled by the LCR. Dev Biol. 2012;366:185–94. https://doi.org/10.1016/j.ydbio.2012.03.021. Pelster B, Burggren WW. Disruption of hemoglobin oxygen transport does not impact oxygen-dependent physiological processes in developing embryos of zebra fish (Danio rerio). Circ Res. 1996;79:358–62. https://doi.org/10.1161/01.res.79.2.358. Rombough P, Drader H. Hemoglobin enhances oxygen uptake in larval zebrafish (Danio rerio) but only under conditions of extreme hypoxia. J Exp Biol. 2009;212 Pt 6:778–84. https://doi.org/10.1242/jeb.026575. Jeney V, Eaton JW, Balla G, Balla J. Natural history of the bruise: formation, elimination, and biological effects of oxidized hemoglobin. Oxid Med Cell Longev. 2013;2013:703571. https://doi.org/10.1155/2013/703571. Dutra FF, Bozza MT. Heme on innate immunity and inflammation. Front Pharmacol. 2014;5:115. https://doi.org/10.3389/fphar.2014.00115. Anderson HL, Brodsky IE, Mangalmurti NS. The evolving erythrocyte: RBCs as modulators of innate immunity. J Immunol. 2018;201:1343–51. https://doi.org/10.4049/jimmunol.1800565. Wagener FADTG, Volk H-D, Willis D, Abraham NG, Soares MP, Adema GJ, et al. Different faces of the heme-heme oxygenase system in inflammation. Pharmacol Rev. 2003;55:551–71. https://doi.org/10.1124/pr.55.3.5. Willis D, Moore AR, Frederick R, Willoughby DA. Heme oxygenase: a novel target for the modulation of the inflammatory response. Nat Med. 1996;2:87–90. https://doi.org/10.1038/nm0196-87. Tasnim M, Wahlquist P, Hill JT. Zebrafish: unraveling genetic complexity through duplicated genes. Dev Genes Evol. 2024;234:99–116. https://doi.org/10.1007/s00427-024-00720-6. Wattrus SJ, Smith ML, Rodrigues CP, Hagedorn EJ, Kim JW, Budnik B, et al. Quality assurance of hematopoietic stem cells by macrophages determines stem cell clonality. Science. 2022;377:1413–9. https://doi.org/10.1126/science.abo4837. O’brien BRA. Identification of Haemoglobin by its Catalase Reaction with Peroxide and o-Dianisidine. Stain Technology. 1961;36:57–61. https://doi.org/10.3109/10520296109113244. Westerfield M. The zebrafish book: a guide for the laboratory use of zebrafish Danio (Brachydanio) rerio. 4th ed. Eugene, Or: ZFIN; 2000. Ellett F, Pase L, Hayman JW, Andrianopoulos A, Lieschke GJ. mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish. Blood. 2011;117:e49-56. https://doi.org/10.1182/blood-2010-10-314120. Harvie EA, Green JM, Neely MN, Huttenlocher A. Innate immune response to Streptococcus iniae infection in zebrafish larvae. Infect Immun. 2013;81:110–21. https://doi.org/10.1128/IAI.00642-12. Renshaw SA, Loynes CA, Trushell DMI, Elworthy S, Ingham PW, Whyte MKB. A transgenic zebrafish model of neutrophilic inflammation. Blood. 2006;108:3976–8. https://doi.org/10.1182/blood-2006-05-024075. Yoo SK, Deng Q, Cavnar PJ, Wu YI, Hahn KM, Huttenlocher A. Differential regulation of protrusion and polarity by PI3K during neutrophil motility in live zebrafish. Dev Cell. 2010;18:226–36. https://doi.org/10.1016/j.devcel.2009.11.015. Hall C, Flores MV, Storm T, Crosier K, Crosier P. The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish. BMC Dev Biol. 2007;7:42. https://doi.org/10.1186/1471-213X-7-42. Huang DW, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009;37:1–13. https://doi.org/10.1093/nar/gkn923. Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28:27–30. https://doi.org/10.1093/nar/28.1.27. Kanehisa M, Furumichi M, Sato Y, Matsuura Y, Ishiguro-Watanabe M. KEGG: biological systems database as a model of the real world. Nucleic Acids Res. 2025;53:D672–7. https://doi.org/10.1093/nar/gkae909. Paffett-Lugassy NN, Zon LI. Analysis of hematopoietic development in the zebrafish. Methods Mol Med. 2005;105:171–98. https://doi.org/10.1385/1-59259-826-9:171. Chu C-Y, Cheng C-H, Chen G-D, Chen Y-C, Hung C-C, Huang K-Y, et al. The zebrafish erythropoietin: functional identification and biochemical characterization. FEBS Lett. 2007;581:4265–71. https://doi.org/10.1016/j.febslet.2007.07.073. Thisse C, Thisse B. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat Protoc. 2008;3:59–69. https://doi.org/10.1038/nprot.2007.514. Nakajima H, Nakajima-Takagi Y, Tsujita T, Akiyama S-I, Wakasa T, Mukaigasa K, et al. Tissue-restricted expression of Nrf2 and its target genes in zebrafish with gene-specific variations in the induction profiles. PLoS One. 2011;6:e26884. https://doi.org/10.1371/journal.pone.0026884. Fuse Y, Nakajima H, Nakajima-Takagi Y, Nakajima O, Kobayashi M. Heme-mediated inhibition of Bach1 regulates the liver specificity and transience of the Nrf2-dependent induction of zebrafish heme oxygenase 1. Genes Cells. 2015;20:590–600. https://doi.org/10.1111/gtc.12249. Additional Declarations No competing interests reported. Supplementary Files 20250901hmox1apaperSupplementaryFigures.pdf Supplementary figure legends Figure S1. Single-cell expression analysis of coding genes for globin proteins identified by proteomics. (A) UMAP from the zebrafish embryo single-cell atlas (Farnsworth et al., 2020) showing the distribution of hematopoietic and immune cell clusters. (B) Markers per studied cluster. (C) Volcano plot showing expression of the hemoglobin genes hbba1 , hbbe2 , and hbbe3 by hematopoietic and immune clusters. Figure S2. RT-PCR analysis of sorted zebrafish neutrophils and macrophages at 72 hours post fertilization. (A) Raw gel image corresponding to Fig. 2B. Specific DNA ladder bands between 100-1000bp are indicated (ladder used was 1kb plus DNA ladder from Invitrogen, Cat. #10787-018). Μϕ = RNA from sorted macrophages, Νϕ = RNA from sorted neutrophils, (-) = no-template PCR control. (B) Expected PCR product sizes for each gene tested, based on primers sequences listed in Table S4. Figure S3. The hmox1a gene is expressed by zebrafish macrophages. (A) Expression of hmox1a by hematopoietic and immune clusters from the zebrafish embryo single-cell atlas. (B) Maps of the Tol2-based -2.1hmox1a:Dendra2 and -2.1hmox1a:mCherryCAAX plasmids used for transgenesis in zebrafish. (C) Fluorescence expression pattern of the generated hmox1a transgenic reporters at the indicated timepoints. White arrowheads indicate expression in the rostral blood island/caudal hematopoietic tissue, yellow arrowheads indicate fluorescence in the retina, and sky blue arrowheads show expression in the liver. Scale bar = 500μm (D) Representative picture of the tail of 72hpf Tg(mpeg1:Dendra2; -2.1hmxo1a:mCherryCAAX) double transgenic zebrafish larvae. (E) Quantification of tail macrophages positive and negative for hmox1a . (F) Representative image from a tail section of Tg(-2.1hmox1a:Dendra2; lyz:DsRed) double transgenic zebrafish larva at 72hpf. Scale bar = 200μm. TableS1proteomicanalysisdata.xlsx Table S1. Proteomic profile of tail portions from control and tail fin amputated zebrafish embryos. TableS2VenndiagramsEntrezdata.xlsx Table S2. Venn diagram analyses of identified protein-coding genes at inflammatory and resolution phases following tail fin amputation. TableS3DAVIDFunctionalclusteringEntrezresults.xlsx Table S3. DAVID functional classification of protein-coding genes exclusively expressed in amputated larvae during inflammation and resolution phases. TableS4Listofprimersv2.pdf Table S4. List of primers used in this study. 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1","display":"","copyAsset":false,"role":"figure","size":641999,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHemoglobin accumulates in the injury site following tail fin amputation in zebrafish larvae\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Representative images of double transgenic reporter \u003cem\u003eTg(mpeg1:GFP; mpx:mCherry)\u003c/em\u003e after tail fin amputation. Scale bar = 100μm. (\u003cstrong\u003eB\u003c/strong\u003e) Quantification of neutrophils and macrophages after tal fin amputation (mean ± SD; n = 40 larvae per condition/timepoint). (\u003cstrong\u003eC\u003c/strong\u003e) Schematic for proteomic analysis after tail fin amputation. (\u003cstrong\u003eD\u003c/strong\u003e) Venn diagram showing proteins exclusively identified in amputated larvae versus control during inflammation (6hpa) and resolution (24hpa). The number of identified proteins is indicated in the figure. (\u003cstrong\u003eE\u003c/strong\u003e) Identification of proteins exclusively expressed during inflammation (6hpa) and resolution (24hpa). Numbers of proteins per group are indicated in the Venn diagram. (\u003cstrong\u003eF\u003c/strong\u003e) DAVID functional classification of proteins exclusively identified in amputated tail fins during inflammation (6hpa) and resolution (24hpa). The top 5 classifications per studied time point are plotted. (\u003cstrong\u003eG\u003c/strong\u003e) Representative pictures of control and amputated tail fins of zebrafish larvae stained with o-dianisidine. Scale bar = 200μm. (\u003cstrong\u003eH\u003c/strong\u003e) Quantification of o-dianisidine\u003csup\u003e+\u003c/sup\u003e dots in the injury site of tail fin amputated larvae versus time-matched non-amputated controls (mean ± SD; n = 23-24 larvae per condition/timepoint).\u003c/p\u003e\n\u003cp\u003eUnpaired t-tests were performed in H\u003cstrong\u003e \u003c/strong\u003eat the indicated timepoints. ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"20250901hmox1apaperMainFigures1.png","url":"https://assets-eu.researchsquare.com/files/rs-7799531/v1/a5a436f1a7bc10c2c2c989c8.png"},{"id":95226121,"identity":"2ece2ff0-7ae0-41a9-ab7d-647bb2498a1c","added_by":"auto","created_at":"2025-11-05 16:26:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":998019,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of Hmox1 paralogs at the injury site of tail fin amputated zebrafish larvae. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) \u003cem\u003eIn situ \u003c/em\u003ehybridization showing mRNA expression of the zebrafish paralogs \u003cem\u003ehmxo1a\u003c/em\u003e and \u003cem\u003ehmox1b\u003c/em\u003e at different timepoints following tail fin amputation. Representative images from 2 independent experiments are shown. Scale bar = 200μm. (\u003cstrong\u003eB\u003c/strong\u003e) Expression of \u003cem\u003ehmox1a\u003c/em\u003e by RT-PCR from neutrophils (ΝΦ) and macrophages (ΜΦ) sorted from \u003cem\u003eTg(mpx:GFP; mpeg1:Gal4FF; UAS:nfsB-mCherry)\u003c/em\u003e larvae at 72hpf. Specific markers for neutrophils (\u003cem\u003empx\u003c/em\u003e), macrophages (\u003cem\u003empeg1.1\u003c/em\u003e), and a housekeeping control (\u003cem\u003eef1a\u003c/em\u003e) are also shown. A representative result from 3 independent experiments is shown. (\u003cstrong\u003eC\u003c/strong\u003e) Confocal images from the tail of the double transgenic reporter \u003cem\u003eTg(mpeg1:Dendra2; -2.1hmox1a:mCherryCAAX)\u003c/em\u003e. A zoom of the CHT is shown in \u003cstrong\u003ei\u003c/strong\u003e. Blue arrowheads indicate macrophages positive for \u003cem\u003ehmox1a\u003c/em\u003e (ΜΦ \u003cem\u003ehmox1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e), whereas white arrowheads indicate macrophages negative for \u003cem\u003ehmox1a\u003c/em\u003e (ΜΦ \u003cem\u003ehmox1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e-\u003c/em\u003e\u003c/sup\u003e). Scale bar = 200μm for full picture (left) and 50μm for zoomed region (right). (\u003cstrong\u003eD\u003c/strong\u003e) Representative images showing recruitment of \u003cem\u003ehmox1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e macrophages to the injury site following tail fin amputation. Scale bar = 200μm. (\u003cstrong\u003eE\u003c/strong\u003e) Quantification of \u003cem\u003ehmox1a\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e macrophages recruited to the injury site following amputation (mean ± SD; n = 10 larvae per timepoint).\u003c/p\u003e","description":"","filename":"20250901hmox1apaperMainFigures2.png","url":"https://assets-eu.researchsquare.com/files/rs-7799531/v1/35efa3ba780a10ed03ba728d.png"},{"id":95115012,"identity":"211e896b-33c1-45b0-94a2-e19e1aceadd2","added_by":"auto","created_at":"2025-11-04 12:40:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":710804,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of Hmox1 activity impairs hemoglobin clearance from the injury site and affects tail fin regeneration. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Experimental design for quantification of amputated larvae treated with the Hmox1 inhibitor SnPP. (\u003cstrong\u003eB\u003c/strong\u003e) Quantification of macrophages recruited to the injury site in SnPP-treated amputated larvae (1 dot = 1 larva, 20 larvae per condition/timepoint). (\u003cstrong\u003eC\u003c/strong\u003e) Representative images of SnPP-treated \u003cem\u003eTg(mpeg1:Dendra2)\u003c/em\u003e amputated larvae at 24 hours post-amputation (hpa). Scale bar = 200μm. (\u003cstrong\u003eD\u003c/strong\u003e) Shape descriptors (sphericity and volume) of \u003cem\u003empeg1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e macrophages recruited to the injury site in SnPP-treated larvae at 24hpa. Average values of recruited macrophages per larva are plotted (1 dot = 1 larva, 6-8 larvae per condition). (\u003cstrong\u003eE\u003c/strong\u003e) Representative o-dianisidine staining images of SnPP-treated amputated larvae at 24hpa. Scale bar = 200μm. (\u003cstrong\u003eF\u003c/strong\u003e) Quantification of o-dianisidine\u003csup\u003e+\u003c/sup\u003e dots in the injury site of SnPP-treated larvae at 24hpa (1 dot = 1 larva, 27-28 larvae per condition). (\u003cstrong\u003eG\u003c/strong\u003e) Experimental design for tail fin regeneration analyses performed in SnPP-treated larvae. (\u003cstrong\u003eH\u003c/strong\u003e) Representative tail fins of amputated control and SnPP-treated larvae at 3 days post-amputation (3dpa). Scale bar = 200μm. (\u003cstrong\u003eI\u003c/strong\u003e) Quantification of the tail fin area of control and SnPP-treated larvae at 3dpa (1 dot = 1 larva, 30 larvae per condition).\u003c/p\u003e\n\u003cp\u003eUnpaired t-tests were performed in B, D, F, and I at specified timepoints. *p\u0026lt;0.05; **p\u0026lt;0.01; ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"20250901hmox1apaperMainFigures3.png","url":"https://assets-eu.researchsquare.com/files/rs-7799531/v1/296cb89a71b2ab957c3fa619.png"},{"id":95224564,"identity":"017640e6-3489-4717-a0ad-3075c11e823f","added_by":"auto","created_at":"2025-11-05 16:23:54","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":503549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMacrophage depletion impairs hemoglobin clearance from the injury site after tail fin amputation in larval zebrafish\u003c/strong\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Experimental design for the depletion of macrophages using clodronate-loaded liposomes (Lipo-clodro). (\u003cstrong\u003eB\u003c/strong\u003e) Recruitment of macrophages in control (Lipo-PBS) and macrophage-depleted (Lipo-clodro) zebrafish at 24hpa. Representative images of the studied conditions are shown. Scale bar = 100μm. (\u003cstrong\u003eC\u003c/strong\u003e) Quantification of macrophages recruited to the injury site (mean ± SD; n = 25-26 larvae per condition/timepoint). (\u003cstrong\u003eD\u003c/strong\u003e) Representative o-dianisidine stainings of amputated Lipo-PBS and Lipo-clodro larvae at 24hpa. Scale bar = 200μm. (\u003cstrong\u003eE\u003c/strong\u003e) Quantification of o-dianisidine\u003csup\u003e+\u003c/sup\u003e dots in the injury site at 24hpa (1 dot = 1 larva, 32-34 larvae per condition).\u003c/p\u003e\n\u003cp\u003eUnpaired t-tests were performed in C and E at specified timepoints. *p\u0026lt;0.05; ***p\u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"20250901hmox1apaperMainFigures4.png","url":"https://assets-eu.researchsquare.com/files/rs-7799531/v1/b9450b4ce788651cdb0f3dac.png"},{"id":95230499,"identity":"051bf043-4872-4a10-b797-e3c4d202118a","added_by":"auto","created_at":"2025-11-05 16:37:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3773523,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7799531/v1/28d6e1a4-8fb7-4825-a78d-fa04963bf9f1.pdf"},{"id":95115014,"identity":"1019e162-8d98-46cc-8f0a-34869ce2926c","added_by":"auto","created_at":"2025-11-04 12:40:51","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":448843,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary figure legends\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S1. Single-cell expression analysis of coding genes for globin proteins identified by proteomics.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) UMAP from the zebrafish embryo single-cell atlas (Farnsworth et al., 2020) showing the distribution of hematopoietic and immune cell clusters. (\u003cstrong\u003eB\u003c/strong\u003e) Markers per studied cluster. (\u003cstrong\u003eC\u003c/strong\u003e) Volcano plot showing expression of the hemoglobin genes\u003cem\u003e hbba1\u003c/em\u003e, \u003cem\u003ehbbe2\u003c/em\u003e, and \u003cem\u003ehbbe3\u003c/em\u003e by hematopoietic and immune clusters.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S2. RT-PCR analysis of sorted zebrafish neutrophils and macrophages at 72 hours post fertilization.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Raw gel image corresponding to Fig. 2B. Specific DNA ladder bands between 100-1000bp are indicated (ladder used was 1kb plus DNA ladder from Invitrogen, Cat. #10787-018). Μϕ = RNA from sorted macrophages, Νϕ = RNA from sorted neutrophils, (-) = no-template PCR control. (\u003cstrong\u003eB\u003c/strong\u003e) Expected PCR product sizes for each gene tested, based on primers sequences listed in Table S4.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S3. The \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ehmox1a\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene is expressed by zebrafish macrophages.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) Expression of \u003cem\u003ehmox1a\u003c/em\u003e by hematopoietic and immune clusters from the zebrafish embryo single-cell atlas. (\u003cstrong\u003eB\u003c/strong\u003e) Maps of the Tol2-based \u003cem\u003e-2.1hmox1a:Dendra2\u003c/em\u003eand \u003cem\u003e-2.1hmox1a:mCherryCAAX\u003c/em\u003e plasmids used for transgenesis in zebrafish. (\u003cstrong\u003eC\u003c/strong\u003e) Fluorescence expression pattern of the generated \u003cem\u003ehmox1a\u003c/em\u003e transgenic reporters at the indicated timepoints. White arrowheads indicate expression in the rostral blood island/caudal hematopoietic tissue, yellow arrowheads indicate fluorescence in the retina, and sky blue arrowheads show expression in the liver. Scale bar = 500μm (\u003cstrong\u003eD\u003c/strong\u003e) Representative picture of the tail of 72hpf \u003cem\u003eTg(mpeg1:Dendra2; -2.1hmxo1a:mCherryCAAX) \u003c/em\u003edouble transgenic zebrafish larvae. (\u003cstrong\u003eE\u003c/strong\u003e) Quantification of tail macrophages positive and negative for \u003cem\u003ehmox1a\u003c/em\u003e. (\u003cstrong\u003eF\u003c/strong\u003e) Representative image from a tail section of\u003cem\u003e Tg(-2.1hmox1a:Dendra2; lyz:DsRed)\u003c/em\u003e double transgenic zebrafish larva at 72hpf. Scale bar = 200μm.\u003c/p\u003e","description":"","filename":"20250901hmox1apaperSupplementaryFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7799531/v1/6d729fe38cf34e8eb095deb2.pdf"},{"id":95224490,"identity":"606aad38-a815-4b26-bd4b-bcd3484c707d","added_by":"auto","created_at":"2025-11-05 16:23:49","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":513012,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S1.\u003c/strong\u003e Proteomic profile of tail portions from control and tail fin amputated zebrafish embryos.\u003c/p\u003e","description":"","filename":"TableS1proteomicanalysisdata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7799531/v1/44e64bc4584018ec701f7254.xlsx"},{"id":95115019,"identity":"6c575442-398b-41ba-850c-1e942d3c069e","added_by":"auto","created_at":"2025-11-04 12:40:51","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":36243,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S2.\u003c/strong\u003e Venn diagram analyses of identified protein-coding genes at inflammatory and resolution phases following tail fin amputation.\u003c/p\u003e","description":"","filename":"TableS2VenndiagramsEntrezdata.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7799531/v1/b81051e9316d7d611f836ff8.xlsx"},{"id":95115018,"identity":"03ecea08-990b-4654-a508-6e95c2d27f32","added_by":"auto","created_at":"2025-11-04 12:40:51","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":30504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S3.\u003c/strong\u003e DAVID functional classification of protein-coding genes exclusively expressed in amputated larvae during inflammation and resolution phases.\u003c/p\u003e","description":"","filename":"TableS3DAVIDFunctionalclusteringEntrezresults.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7799531/v1/b8c74f66e5d9ef5eb6320c38.xlsx"},{"id":95226092,"identity":"5e17ee34-406e-42ac-af67-6f0defc279f3","added_by":"auto","created_at":"2025-11-05 16:26:13","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":184814,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable S4.\u003c/strong\u003e List of primers used in this study.\u003c/p\u003e","description":"","filename":"TableS4Listofprimersv2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7799531/v1/07917e63c1068e4a04a71ba6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eHeme oxygenase 1 activity mediates hemoglobin clearance and tail fin regeneration in zebrafish larvae\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe tissue response to injury is composed of an orchestrated cascade of events, including wound closure, inflammation, proliferation, and tissue remodeling. In non-regenerative systems, these events normally culminate in scar formation, whereas in fully regenerative systems, the outcome is the formation of a tissue that is architecturally and functionally identical to the tissue before injury. Immune cells are key players in the cascade of events following tissue damage. Neutrophils and macrophages are rapidly recruited to the injured tissue and promote local pro-inflammatory responses primarily to protect the tissue from potential pathogenic infection [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Macrophages, on the other hand, are responsible for clearing apoptotic and damaged cells in the injury site, counteract pro-inflammatory signals, and produce growth factors that initiate wound healing and tissue regeneration [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. To perform these tasks, macrophages undergo phenotypic and functional changes that endow them with anti-inflammatory and pro-regenerative functions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, the temporal dynamics of these anti-inflammatory programs in macrophages and their precise contribution to the resolution of inflammation and tissue regeneration remain poorly understood.\u003c/p\u003e\u003cp\u003eZebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) has become a well-established animal model for studying inflammatory responses thanks to its optical transparency during larval stages, which allows intravital and real-time visualization of immune responses following injury. Additionally, its high regenerative capacity has facilitated studying the impact of immune cells during tissue regeneration. The zebrafish larva possesses functional hematopoietic and innate immune cells [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], with the latter being critical orchestrators of tissue inflammation and regeneration. Using the tail fin amputation model in zebrafish larvae, we and others have previously shown that macrophages are the predominant immune cell type contributing to tissue regeneration [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Mechanistically, recruited macrophages promote the resolution of inflammation by attenuating local production of pro-apoptotic Il1b [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and suppressing ROS production [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], while they activate regenerative programs on stromal cells through Tnfa signaling [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, which anti-inflammatory proteins are expressed by zebrafish macrophages and play a role in the resolution of inflammation and tissue regeneration remains under investigation. Studies from mammals show that expression of the heme scavenger and cytoprotective enzyme heme oxygenase 1 (HO-1, encoded by the \u003cem\u003eHMOX1\u003c/em\u003e gene) confers macrophages with anti-inflammatory [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and pro-regenerative functions [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Zebrafish possess two orthologs of the mammalian \u003cem\u003eHMOX1\u003c/em\u003e gene: \u003cem\u003ehmox1a\u003c/em\u003e and \u003cem\u003ehmox1b\u003c/em\u003e. Although \u003cem\u003ehmox1a\u003c/em\u003e has been associated with macrophage function [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], it remains unclear whether \u003cem\u003ehmox1a\u003c/em\u003e is expressed by zebrafish macrophages during injury and plays a role in the resolution of inflammation and tissue regeneration.\u003c/p\u003e\u003cp\u003eIn this work, using an unbiased proteomic analysis, we found that hemoglobin (Hb) accumulated in the injury site after tail fin amputation in zebrafish larvae. Hb levels in the injury site peaked during the inflammatory phase and decreased before regeneration took place. By performing RNA expression analysis and using a novel fluorescent transgenic line, we observed the expression of the functional \u003cem\u003ehmox1a\u003c/em\u003e paralog in macrophages at the injury site of amputated tail fins. Depletion of macrophages led to impaired Hb clearance from the injury site, whereas pharmacological inhibition of Hmox1 activity following tail fin amputation impaired Hb clearance and tail fin regeneration. Altogether, our findings reveal a novel role for Hmox1 in shaping the regenerative microenvironment and identify Hb and \u003cem\u003ehmox1a\u003c/em\u003e-expressing macrophages as relevant players in the zebrafish injury response.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAccumulation of hemoglobin in the injury site after tail fin amputation\u003c/h2\u003e\u003cp\u003eUsing our previously described model of tail fin amputation [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], we analyzed the recruitment of neutrophils and macrophages to the injury site in 72 hours post fertilization (hpf) double transgenic reporter \u003cem\u003eTg(mpeg1:GFP; mpx:mCherry)\u003c/em\u003e larvae. In line with previous reports [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], we observed an \u0026ldquo;inflammation\u0026rdquo; phase at 6 hours post amputation (hpa), characterized by a peak of neutrophils recruited to the injury site (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B) and a modest increase in the recruitment of macrophages. After 6hpa, the number of neutrophils in the injury site decreased, while the number of recruited macrophages continued increasing. By 24hpa, we observed a \u0026ldquo;resolution\u0026rdquo; phase where macrophages were predominant over neutrophils (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). We selected 6hpa (inflammation) and 24hpa (resolution) to perform proteomic analyses of injured tissues and uninjured time-matched controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). After filtering and removal of duplicated proteins (see methods), we sought to identify the proteins that were exclusively expressed during inflammation and resolution. By comparing with their respective time-matched controls, we identified 256 proteins that were exclusively present in injured tissues during inflammation, while 296 proteins were detected exclusively in the injured tissue at the resolution phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, \u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). Next, we compared the proteins exclusive to injured tissues in inflammation versus resolution, and we found 219 inflammation-specific and 259 resolution-specific proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, \u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). Functional classification analysis of identified phase-specific proteins using DAVID bioinformatic resources [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] gave us insights into proteins and biological processes that were active during inflammation and resolution (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). Among them, we found targets previously described during resolution and important for tissue regeneration, such as hsp60/GroEL activity [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] and Coronin, the latter expressed by myeloid cells during tissue damage [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. During inflammation, we found increased expression of mitochondrial cytochrome c oxidase proteins (Cox5aa and Cox5ab), supporting increased inflammation and suggesting higher mitochondrial activity. Interestingly, we found increased levels of the hemoglobin (Hb) proteins Hbbe2, Ba1 (Hbba1), and Hbbe3 during inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, \u003cb\u003eTable \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e\u003c/b\u003e). To validate these findings, we performed Hb stainings with o-dianisidine in tail fin amputated larvae during inflammation and resolution. We found accumulation of o-dianisidine\u003csup\u003e+\u003c/sup\u003e dots at the injury site during inflammation (6hpa), which decreased sharply at resolution (24hpa, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-H). Since these proteins are common markers for erythroid cells, we analyzed the expression of the coding genes for these proteins (\u003cem\u003ehbbe2\u003c/em\u003e, \u003cem\u003ehbba1\u003c/em\u003e and \u003cem\u003ehbbe3\u003c/em\u003e) in a previously published single-cell dataset of the developing zebrafish embryo [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] (\u003cb\u003eFig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA-B\u003c/b\u003e). We found that expression of these genes is restricted to red blood cells (RBCs) and are not expressed by immune cell types in homeostasis (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC\u003c/b\u003e). Altogether, these results indicate that Hb, likely contained in RBCs, accumulate in the injury site during the inflammatory phase after tail fin amputation in zebrafish larvae.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eExpression of heme oxygenase 1 is detected in the injury site of amputated larvae\u003c/h3\u003e\n\u003cp\u003eThe decrease of Hb in the injury site during resolution led us to hypothesize that an active mechanism oversees the removal of Hb from the injury site after tissue damage. The degradation of Hb involves the breakdown of globin proteins into amino acids, and the catalysis of the heme ring by the Heme oxygenase 1 (Hmox1) enzyme into carbon monoxide (CO), iron (Fe), and biliverdin [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. We therefore analyzed the expression of Hmox1 in the injury site after damage. Through whole-mount \u003cem\u003ein situ\u003c/em\u003e hybridization (WISH) we found enriched expression of the zebrafish Hmox1-coding paralogs \u003cem\u003ehmox1a\u003c/em\u003e and \u003cem\u003ehmox1b\u003c/em\u003e in amputated tail fins, in a time frame that coincided with the reduction of Hb from the injury site from 6hpa to 24hpa (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Interestingly, we found different expression patterns for the Hmox1 paralogs within the injury site: while the \u003cem\u003ehmox1a\u003c/em\u003e signal was discrete and seemed restricted to individual cells, \u003cem\u003ehmox1b\u003c/em\u003e expression was broadly expressed in the edge of the injured tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). These results suggest that \u003cem\u003ehmox1b\u003c/em\u003e expression is locally induced in the injured tissue, whereas \u003cem\u003ehmox1a\u003c/em\u003e expression takes place in specific cells supporting the response to injury. As it has been previously described that \u003cem\u003ehmox1a\u003c/em\u003e is the functional ortholog of the human \u003cem\u003eHMOX1\u003c/em\u003e gene and that \u003cem\u003ehmox1b\u003c/em\u003e is likely a pseudogene [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], we decided to focus on the characterization of \u003cem\u003ehmox1a\u003c/em\u003e in the injury site.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eMacrophages are the source of\u003c/em\u003e hmox1a \u003cem\u003ein the injury site of amputated larvae\u003c/em\u003e\u003c/p\u003e\u003cp\u003eIt has been previously reported that \u003cem\u003ehmox1a\u003c/em\u003e is expressed in hematopoietic tissues of larvae [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and that is required for the proper function of macrophages [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], which led us to hypothesize that macrophages are the source of \u003cem\u003ehmox1a\u003c/em\u003e in the injury site of amputated larvae. By performing RT-PCR from FACS-sorted neutrophils and macrophages from the triple transgenic \u003cem\u003eTg(mpx:GFP; mpeg1:Gal4FF; UAS:nfsB-mCherry)\u003c/em\u003e at 72hpf, we observed that macrophages, but not neutrophils, expressed \u003cem\u003ehmox1a\u003c/em\u003e in homeostatic conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cb\u003eFig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e\u003c/b\u003e). Single-cell analysis from the developing zebrafish atlas confirmed macrophages as the main source of \u003cem\u003ehmox1a\u003c/em\u003e among immune and hematopoietic cells (\u003cb\u003eFig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eA\u003c/b\u003e). To further explore the kinetics of \u003cem\u003ehmox1a\u003c/em\u003e\u003csup\u003e+\u003c/sup\u003e cells \u003cem\u003ein vivo\u003c/em\u003e, we generated two transgenic fluorescent reporters for \u003cem\u003ehmox1a\u003c/em\u003e expression. For this, we cloned the 2.1kb genomic region immediately upstream of the initial ATG from the \u003cem\u003ehmox1a\u003c/em\u003e in a Tol2-flanking plasmid containing the fluorescent proteins Dendra2 or membrane mCherry (mCherryCAAX) (\u003cb\u003eFig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eB\u003c/b\u003e, see methods). We injected these constructs into WT embryos, and after 2 generations we obtained the stable transgenic lines \u003cem\u003eTg(-2.1hmox1a:Dendra2)\u003c/em\u003e and \u003cem\u003eTg(-2.1hmox1a:mCherryCAAX)\u003c/em\u003e. We observed fluorescence expression of \u003cem\u003ehmox1a\u003c/em\u003e in the posterior blood island (PBI) at 30hpf and caudal hematopoietic tissue (CHT) at 72hpf and 120hpf, in addition to expression in the lens and liver (\u003cb\u003eFig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eC\u003c/b\u003e), in line with observations made in a previously generated reporter for \u003cem\u003ehmox1a\u003c/em\u003e [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Next, we crossed the generated \u003cem\u003ehmox1a\u003c/em\u003e reporters with \u003cem\u003eTg(lyz:DsRed\u003c/em\u003e) and \u003cem\u003eTg(mpeg1:Dendra2)\u003c/em\u003e transgenic fish to analyze \u003cem\u003ehmox1a\u003c/em\u003e expression in neutrophils and macrophages, respectively. We observed that macrophages were positive for \u003cem\u003ehmox1a\u003c/em\u003e at 72hpf, while neutrophils were not (\u003cb\u003eFig.\u0026nbsp;2Ci\u003c/b\u003e, \u003cb\u003eFig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eD-F\u003c/b\u003e). Interestingly, we found variable expression levels of \u003cem\u003ehmox1a\u003c/em\u003e in macrophages, as well as a group of macrophages that were negative for \u003cem\u003ehmox1a\u003c/em\u003e (\u003cb\u003eFig.\u0026nbsp;2Cii\u003c/b\u003e). A more detailed analysis of the tail of 72hpf \u003cem\u003eTg(mpeg1:Dendra2; -2.1hmox1a:mCherryCAAX)\u003c/em\u003e larvae showed that 81.22% of tail macrophages expressed \u003cem\u003ehmox1a\u003c/em\u003e, while the remaining 18.78% of tail macrophages were negative (\u003cb\u003eFig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003eE\u003c/b\u003e). Next, we performed tail fin amputations in the double transgenic \u003cem\u003eTg(mpeg1:Dendra2; -2.1hmox1a:mCherryCAAX)\u003c/em\u003e at 72hpf to analyze \u003cem\u003ehmox1a\u003c/em\u003e expression by macrophages in the injury site. We found that 90.28% of macrophages recruited to the injury site at 6hpa expressed \u003cem\u003ehmox1a\u003c/em\u003e, but this proportion was reduced to 80.79% at 24hpa (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-E). Of note, the expression of \u003cem\u003ehmox1a\u003c/em\u003e in the injury site was restricted to \u003cem\u003empeg1\u003c/em\u003e\u003csup\u003e\u003cem\u003e+\u003c/em\u003e\u003c/sup\u003e macrophages in both timepoints, indicating that macrophages were the source of \u003cem\u003ehmox1a\u003c/em\u003e expression in the injury site.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eInhibition of Hmox1 activity impairs Hb clearance and tail fin regeneration\u003c/h3\u003e\n\u003cp\u003eTo determine the functional consequences of Hmox1 activity after tail fin amputation, we took advantage of the Hmox1 inhibitor Tin protoporphyrin XI (SnPP), previously tested in zebrafish [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and we treated larvae for 24 hours immediately after amputation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Since a previous report suggested that macrophage recruitment to injury was impaired after Hmox1 activity inhibition [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], we analyzed macrophage recruitment in our experimental settings. Although we did not observe differences in the number (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C) nor their sphericity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, \u003cb\u003eleft\u003c/b\u003e), we observed that recruited macrophages in SnPP-treated larvae had a smaller average volume compared to control larvae (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, \u003cb\u003eright\u003c/b\u003e), suggesting that SnPP may affect macrophage physiology. Next, we analyzed the clearance of Hb from the injury site after tail fin after amputation in SnPP-treated larvae. Inhibition of Hmox1 activity by SnPP led to an increased number of o-dianisidine\u003csup\u003e+\u003c/sup\u003e dots at 24hpa (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-F), indicating impaired Hb clearance from the injury site. We finally assessed whether inhibiting Hmox1 activity during the first 24h after amputation has long-term consequences and affects tail fin regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). We found that the regenerating tail fins of SnPP-treated larvae at 3 days post amputation (3dpa) were smaller in area when compared to their respective controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003eH-I). These findings indicate that Hmox1 activity in the injury site promotes the clearance of hemoglobin from the injury site and the regeneration of the tail fin in larvae.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eMacrophage depletion increases Hb in the injury site during the resolution of inflammation\u003c/h3\u003e\n\u003cp\u003eThe importance of macrophages in the clearance of damage and pro-inflammatory cues, and our observations indicating that macrophages were the source of \u003cem\u003ehmox1a\u003c/em\u003e in the injury site, led us to hypothesize that depleting macrophages may impair Hb clearance from the injury site. To test this, we depleted macrophages by injecting 2nL of 1:10 (0.5mg/mL) Lipo-clodronate and Lipo-PBS in the bloodstream of 54hpf zebrafish embryos (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eA), which we previously showed to deplete\u0026thinsp;~\u0026thinsp;60% of macrophages in the tail and impair tail fin regeneration after amputation at 72hpf [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. As expected, the recruitment of macrophages in Lipo-clodronate-injected larvae was markedly reduced at 6hpa and 24hpa compared to Lipo-PBS controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C). Additionally, the number of o-dianisidine\u003csup\u003e+\u003c/sup\u003e dots at the injury site was higher in the Lipo-clodronate group, compared to Lipo-PBS controls, at 24hpa (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003eD\u003cb\u003e- E\u003c/b\u003e), phenocopying the pharmacological inhibition of Hmox1 activity. In conclusion, our findings support macrophages as regulators of Hb levels in the injury site after tail fin amputation in zebrafish larvae.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe availability of transgenic reporter lines and their optical transparency has made zebrafish larvae an ideal model to explore innate immune responses \u003cem\u003ein vivo\u003c/em\u003e. While the focus has been directed toward neutrophils and macrophages, the potential involvement of additional hematopoietic-derived mediators during tissue injury has remained unexplored. Using an unbiased proteomic approach, we identified the accumulation of hemoglobin (Hb) in the injury site during inflammation, which subsequently cleared during resolution. In parallel, we observed the expression of the Hmox1 coding genes \u003cem\u003ehmox1a\u003c/em\u003e and \u003cem\u003ehmox1b\u003c/em\u003e in the injury site, and we identified macrophages as the source of \u003cem\u003ehmox1a\u003c/em\u003e in the injury site after tail fin amputation. Functionally, both pharmacological inhibition of Hmox1 activity with SnPP and the depletion of macrophages led to increased Hb levels at the injury site, supporting the role of macrophages in Hb clearance. Additionally, we found that the inhibition of Hmox1 activity led to impaired tail fin regeneration, similar to what has been previously observed upon macrophage depletion [8\u0026ndash;10]. Altogether, our findings show that Hmox1 is important for the macrophage-dependent clearance of Hb from the injury site and promotes tissue regeneration.\u003c/p\u003e\n\u003cp\u003eOur results show that zebrafish Hb accumulates in the injury site after tail fin amputation. Given that Hb is a protein whose expression is restricted to red blood cells (RBCs), we propose that RBCs accumulate in the injury site after tail fin amputation. However, non-erythroid cells can also express hemoglobin under specific conditions (reviewed by [24, 25]), as reported previously for immune cells such as macrophages or peripheral blood mononuclear cells in response to inflammatory stimuli [26, 27]. Therefore, further analyses are required, e.g., \u003cem\u003ein vivo\u003c/em\u003e tracking of fluorescently labelled RBCs using fluorescence reporters for the erythroid lineage marker \u003cem\u003egata1a\u003c/em\u003e [28, 29] or the LCR locus controlling globin expression [30], to confirm that RBCs are the source of the Hb found in the injury site of zebrafish larvae. While the primary function of Hb from RBCs is to provide oxygen to the injured tissue, oxygen exchange in zebrafish larvae occurs independently of RBCs [31, 32], which raises the question of what the role of Hb, and potentially RBCs, is in this context. It is known that RBCs are highly susceptible to hemolysis in extravascular spaces [33], and the heme released after hemolysis to the extracellular space may act as a DAMP capable of inducing oxidative stress, cytotoxicity, and amplifying inflammation through activation of toll-like receptor 4 (TLR4) signaling pathways [34]. Thus, while Hb accumulation could be relevant to the initial steps of inflammation and likely protect the injured tissue from external pathogens [35], the release of heme in extravascular tissues poses a significant risk of cytotoxicity and exacerbation of inflammation, and therefore must be cleared to avoid chronic inflammation. The temporal dynamics of Hb at the injury site tissue suggests that active Hb clearance is part of the resolution of inflammation, a required step for tissue regeneration.\u003c/p\u003e\n\u003cp\u003eWe observed the expression of the Hmox1-coding genes \u003cem\u003ehmox1a\u003c/em\u003e and \u003cem\u003ehmox1b\u003c/em\u003e in the injury site, which correlated with the peak and reduction of Hb in the injury site, and led us to propose that the function of Hmox1 in the injured tissue is the degradation of heme from the Hb accumulated following injury. However, it is also possible that the anti-oxidant function of Hmox1 activity directly regulates the inflammatory responses in the injury site, as previous studies have suggested [36, 37], and further analysis of pro-inflammatory cytokine expression and reactive oxygen species (ROS) production are required to better understand the relationship between Hmox1 activity and inflammatory responses. Since a previous study showed that \u003cem\u003ehmox1a\u003c/em\u003e is the functional ortholog of the human \u003cem\u003eHMOX1\u003c/em\u003e gene [20], we focused our attention on characterizing the source of \u003cem\u003ehmox1a\u003c/em\u003e expression following injury. However, we cannot rule out that \u003cem\u003ehmox1b\u003c/em\u003e expression in the injury site can play regulatory functions such as small interfering RNA, competitive endogenous RNA, or antisense transcripts [38], which could ultimately modulate the tissue response to injury at a transcriptional level. Our results showed that macrophages are an important, but not exclusive, source of \u003cem\u003ehmox1a\u003c/em\u003e in zebrafish during steady state conditions. However, macrophages were the sole source of \u003cem\u003ehmox1a\u003c/em\u003e in the injury site after tail fin amputation, with most of the macrophages being\u003cem\u003e\u0026nbsp;hmox1a\u003csup\u003e+\u003c/sup\u003e\u003c/em\u003e. The use of a novel \u003cem\u003ehmox1a\u003c/em\u003e fluorescent reporter line in our study provided an unprecedented view of \u003cem\u003ehmox1a\u003c/em\u003e expression dynamics \u003cem\u003ein vivo\u003c/em\u003e. A previous study described a reporter for \u003cem\u003ehmox1a\u003c/em\u003e [22], and the expression pattern described in the rostral blood island, liver, and retina matched our observations. However, they attributed \u003cem\u003ehmox1a\u003c/em\u003e expression to erythroid lineages and not to macrophages, as we showed in this study. Our observations are supported by previous evidence showing functional defects of macrophages in \u003cem\u003ehmox1a\u003c/em\u003e mutants [14] and single-cell transcriptomic analysis showing expression of \u003cem\u003ehmox1a\u003c/em\u003e in macrophages at the CHT [39]. Thus, our reporter enabled real-time visualization and spatial resolution of \u003cem\u003ehmox1a\u003c/em\u003e-expressing macrophages in the context of tissue injury, and offers a valuable advantage for future studies aiming to dissect the precise temporal and cellular dynamics of heme metabolism and immune regulation during homeostasis, tissue injury, and repair.\u003c/p\u003e\n\u003cp\u003eOur findings showed that 1) macrophages were identified as the sole source of \u003cem\u003ehmox1a\u003c/em\u003e expression at the injury site, 2) pharmacological inhibition of Hmox1 activity using SnPP led to an impaired clearance of Hb from the injury site, and 3) depletion of macrophages also led to impaired Hb clearance. Therefore, we propose that \u003cem\u003ehmox1a\u003c/em\u003e-expressing macrophages are critical mediators of Hb clearance and resolution of inflammatory responses after tissue injury. Crucially, SnPP-treated larvae exhibited impaired tail fin regeneration following amputation, supporting an effector role of Hmox1a in promoting tissue regeneration after injury. This interpretation is further supported by cumulative evidence from murine models of injury showing that \u003cem\u003eHMOX1\u003c/em\u003e expression drives macrophage polarization towards anti-inflammatory profiles [12] and endows them with pro-regenerative functions\u003cem\u003e\u0026nbsp;\u003c/em\u003e[13]. However, it remains unclear whether the phenotype of \u003cem\u003ehmox1a\u003c/em\u003e-expressing macrophages is predetermined before their recruitment to the injury site, or is modulated in response to local cues \u0026mdash; such as the presence of Hb. Furthermore, whether the clearance of Hb relies on phagocytosis by macrophages must be confirmed by employing RBC and macrophage fluorescent reporters, which will allow us to clarify the cellular choreography underpinning resolution of inflammation and tissue regeneration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDespite the insights acquired from our study, several limitations should be considered. First, our reliance on o-dianisidine staining to detect Hb in zebrafish tissues presents inherent limitations. Since o-dianisidine detects Hb based on peroxidase activity [40], it is unclear whether certain subpopulations of erythroid cells or Hb degradation products escape detection, potentially leading to underestimation or spatial misrepresentation of Hb accumulation. A more quantitative or marker-specific approach, such as RBC-specific fluorescent transgenic reporters, may improve future resolution. Second, although we inferred the inflammatory and resolution phases following injury based on immune cell dynamics \u0026mdash; specifically neutrophil presence \u0026mdash; we relied on using specific timepoints associated with inflammation and resolution (6hpa and 24hpa), omitting a comprehensive kinetic evaluation of Hb at multiple timepoints.\u003cem\u003e\u0026nbsp;\u003c/em\u003eAdditionally, assessing the expression of common pro- and anti-inflammatory cytokines (such as \u003cem\u003etnfa\u003c/em\u003e, \u003cem\u003eil1b\u003c/em\u003e, and \u003cem\u003eil10\u003c/em\u003e) would further aid in delineating the inflammatory and resolution phases following injury. \u0026nbsp;A better-defined characterization of the inflammatory milieu would help to further clarify timing and polarization of inflammatory responses, and validate the proposed role of Hmox1a in inflammation resolution. Furthermore, interpretative caveats must be considered with the expression of the \u003cem\u003ehmox1a\u003c/em\u003e fluorescent reporters to track \u003cem\u003ehmox1a\u003c/em\u003e-expressing cells, as fluorescence proteins could persist in a cell even after \u003cem\u003ehmox1a\u003c/em\u003e transcription has ceased. As such, the reporter may reflect prior, but not necessarily current, transcriptional activity. Conversely, during early stages of \u003cem\u003ehmox1a\u003c/em\u003e induction, the reporter may not yet have accumulated to a detectable threshold\u0026mdash;leading to potential underrepresentation of actively transcribing cells. This temporal disconnect between transcriptional dynamics and reporter signal could obscure transient or low-level expression events and must be considered in the interpretation of the data.\u003c/p\u003e\n\u003cp\u003eCollectively, our findings emphasize \u003cem\u003ehmox1a\u003c/em\u003e as a macrophage-associated effector essential for the resolution of inflammation after injury and the promotion of tissue regeneration in zebrafish. Additionally, it uncovers hemoglobin as a likely mediator of inflammatory responses, which may have implications in the study of immune responses of patients suffering from hemoglobinopathies.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003eFish husbandry and lines\u003c/em\u003e\u003c/p\u003e\n\n\u003cp\u003eZebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e, strains TAB5, AB, and T\u0026uuml;bingen) were reared and kept, according to standard procedures, in the zebrafish facilities at the Facultad de Ciencias of Unversidad de Chile, the FishCore at Monash University, and the Karolinska Institutet Zebrafish Core Facility. Adult zebrafish, used to obtain embyros for experiments, were maintained at 28 \u0026deg;C in recirculating, filtered water systems (Techniplast, Italy; Aquaneering, USA), on a regimen of 14 hours of light and 10 hours of darkness. The fish lines used in this work were: \u003cem\u003eTg(mpeg1:GFP)\u003c/em\u003e and \u003cem\u003eTg(mpeg1:Gal4FF; UAS:nfsB-mCherry)\u003c/em\u003e [42]; \u003cem\u003eTg(mpeg1:Dendra2) \u003c/em\u003e[43]; \u003cem\u003eTgBAC(mpx:GFP)\u003csup\u003ei114\u003c/sup\u003e\u003c/em\u003e, referred to as \u003cem\u003eTg(mpx:GFP)\u003c/em\u003e [44]; \u003cem\u003eTg(mpx:mCherry) \u003c/em\u003e[45]; \u003cem\u003eTg(lyz:DsRed2)\u003c/em\u003e [46]. Zebrafish embryos were collected by natural spawning of adults and were kept at 28\u0026deg;C in E3 water (NaCl 5mM, KCl 0.17mM, CaCl\u003csub\u003e2\u003c/sub\u003e 0.33mM, MgSO\u003csub\u003e4\u003c/sub\u003e 0.33mM). Embryos were checked daily, and specimenes showing any sings of malformations or developmental delays were discarded and not used for experiments. All procedures were performed in accordance with the \u0026ldquo;Guidelines for the Use of Fishes in Research Use\u0026rdquo; of the American Fisheries Society (www.fisheries.org) and complied with local regulations. Husbandry, breeding of zebrafish stocks for the collection of embryos, and procedures performed in zebrafish older than 5 days post fertilization were approved by the Animal Ethics Committee of the Universidad de Chile (approval: 2015-04-20). Husbandry and breeding of zebrafish stocks for the collection of embryos were approved by the Monash University Animal Ethics Committee (protocol MAS-2010-18) and the Stockholm Ethical Committee from the Swedish Board of Agriculture (diary number 15591-2023). This study is reported in accordance with the ARRIVE guidelines.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eTail fin amputation and in vivo imaging\u003c/em\u003e\u003c/p\u003e\n\n\u003cp\u003eAt 72 hours post fertilization (hpf), larvae were anesthetized with 0.016% MS-222, and the tail fin was amputated with a scalpel, using the caudal vein loop and the posterior section of the ventral pigmentation gap in the tail as references [10]. The amputation performed did not cause damage to the caudal vein loop. Immediately after amputation, larvae were rinsed and incubated in E3 medium at 28\u0026deg;C. Quantifications, imaging of recruited immune cells to the injury site (up to \u0026sim;200\u0026mu;m from the amputation site), and imaging of the regenerating tail fin were performed using an Olympus MVX10 stereomicroscope, Olympus IX81 epifluorescence microscope, or a Zeiss Axiovert 200 widefield microscope. Quantifications from images were performed using the \u0026ldquo;Cell Counter\u0026rdquo; plugin in Fiji/ImageJ software (NIH, USA). After imaging, larvae were euthanized by incubation with an overdose of MS-222 (0.1%) for 30 minutes, followed by freezing of individuals.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eTissue collection and proteomic analyses\u003c/em\u003e\u003c/p\u003e\n\n\u003cp\u003eAt 6 and 24 hours post amputation (hpa), larvae were euthanized with an overdose of MS-222, and tail tissue pieces were collected by performing a second transection at approximately 200-300\u0026micro;m from the injury site, using the narrowest region of the larval fin fold as a reference. Single replicates containing tail tissue pieces from 200 larvae/condition/timepoint were collected in 1.5mL Eppendorf tubes, which were spun before removing all the media, snap-frozen in dry ice-cooled methanol, and stored at -80\u0026deg;C. At each time point, tissues from non-amputated controls were collected for comparisons. Samples were subsequently submitted to a global proteomic profiling of 10.000 sequencing events, followed by sequence library searching (service provided by Bioproximity LLC, USA). Briefly, proteins from samples were extracted and sequenced by Shotgun proteomics followed by Tandem Mass Spectrometry (MS/MS) in a Q-Exactive HF-X Orbitrap mass spectrometer. Identified peptides (using OMSSA and X!Tandem algorithms) were aligned to the zebrafish proteome, and UniProt (Universal Protein Resource) identification codes and gene names were obtained. The identification of proteins exclusively present in the injury site at the studied timepoints was performed in 3 steps: 1) First, identified UniProt IDs were converted to Entrez ID using the DAVID Bioinformatic resource v6.8 [16, 47], and redundant Entrez IDs were excluded; 2) common and sample-specific Entrez IDs were identified in samples from injured tissues (TFA) were compared to their respective time-matched non-injured controls using an online Venn diagram generator (https://bioinformatics.psb.ugent.be/webtools/Venn/), and injury-specific Entrez-ID were selected; and 3) common and timepoint-specific Entrez IDs (inflammation or resolution) were identified. Finally, functional annotation clustering of the proteins exclusively present in the injury site at the specific time point was performed using the DAVID Bioinformatic resource v6.8, in which functional annotations, gene ontology (GO), pathways (KEGG [48, 49], permission granted by Kanehisa Laboratories), and protein domains were considered. The classification was performed using the highest stringency settings.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eHemoglobin staining in zebrafish larvae\u003c/em\u003e\u003c/p\u003e\n\n\u003cp\u003eZebrafish larvae were stained with o-dianisidine (Sigma) before or after fixation with PFA 4%, based on previously described protocols [50, 51]. Before imaging, larvae were depigmented with a solution of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 3% v/v and KOH 1% w/v in deionized water. Images were acquired in an Olympus MVX10 stereoscope (Olympus, Japan), and the number of o-dianisidine\u003csup\u003e+\u003c/sup\u003e dots in the injury site was quantified using the \u0026ldquo;Cell Counter\u0026rdquo; plugin in Fiji/ImageJ.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eWhole-mount in situ hybridization\u003c/em\u003e\u003c/p\u003e\n\n\u003cp\u003eFor \u003cem\u003ein situ\u003c/em\u003e hybridization experiments, larvae were incubated from 24hpf in E3 supplemented with phenyl thiourea (PTU), and amputations were performed using larvae collected before amputation (72hpf) and after 6h, 10h, and 24h post-amputation. Larvae were fixed in PFA 4%, dehydrated in methanol, and incubated for at least 1 night at -20\u0026deg;C. Whole-mount \u003cem\u003ein situ\u003c/em\u003e hybridizations were performed as previously described [52]. For imaging, stained larvae were transferred to glycerol and imaged with an MVX10 stereoscope (Olympus). The pBSKII SK plasmid containing the probe against \u003cem\u003ehmox1a\u003c/em\u003e was kindly provided by Dr. Makoto Kobayashi [53], whereas the probe for \u003cem\u003ehmox1b\u003c/em\u003e was generated by cloning a portion of the \u003cem\u003ehmox1b\u003c/em\u003e gene into a pBSK vector, as previously described [54]. Primers used for the cloning of the \u003cem\u003ehmox1b\u003c/em\u003e probe are found in Table S4. \u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eAnalysis of single-cell RNAseq databases\u003c/em\u003e\u003c/p\u003e\n\n\u003cp\u003eFor expression analysis, we took advantage of the single-cell atlas of the developing zebrafish embryo (Farnsworth et al., 2020). The Seurat file containing the clustered single cells was downloaded from https://www.adammillerlab.com/resources. Hematopoietic cells, including neutrophils (cluster 150), macrophages (clusters 71, 184, and 212), thymocytes (cluster 59), RBCs (clusters 6, 38, 48, 63, 85, and 157), and RBC progenitors (cluster 153) were selected for further analysis. Gene expression analyses were performed using Seurat v4.0 in RStudio.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eChemical treatments\u003c/em\u003e\u003c/p\u003e\n\n\u003cp\u003eThe Hmox1 inhibitor Tin protoporphyrin XI (SnPP, Cat. #0747, Tocris) was resuspended in DMSO to a working stock of 5mM. Immediately after tail fin amputations, larvae were randomly separated in two groups. The first group (treatment) was treated with SnPP at a concentration of 5\u0026micro;M (1:1000 from working stock). The second group of larvae (control) was treated with E3 supplemented with DMSO 0.1%. Treatments were performed for 24 hours, after which larvae were rinsed three times with E3.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eSorting of immune cells from larvae and RT-PCR\u003c/em\u003e\u003c/p\u003e\n\n\u003cp\u003eAt 72hpf, transgenic reporter larvae \u003cem\u003eTg(mpx:GFP; mpeg1:Gal4FF; UAS:nfsB-mCherry)\u003c/em\u003e were disaggregated to single-cell suspensions as previously reported (Paredes-Zu\u0026ntilde;iga et al., 2017). Sorting of neutrophils (GFP\u003csup\u003e+\u003c/sup\u003e) and macrophages (mCherry\u003csup\u003e+\u003c/sup\u003e) was performed using an Influx2 Cell Sorter (BD Biosciences) at the Monash University FlowCore unit. Total RNA was isolated from sorted cells using the RNeasy mini kit (Qiagen), and RT-PCR analyses were performed using the SuperScript III one-step RT-PCR system (Invitrogen). Primers used for RT-PCR are shown in Table S4. \u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eGeneration of the hmox1a transgenic reporter line\u003c/em\u003e\u003c/p\u003e\n\n\u003cp\u003eThe 2,297bp genomic sequence containing the upstream and first 45 nucleotides of the \u003cem\u003ehmox1a\u003c/em\u003e coding sequence (Gene ID: 791518) was amplified and cloned into a pCRII vector. A second PCR was performed on the previously cloned sequence to amplify a 2,151bp sequence containing the upstream and first nine nucleotides of the \u003cem\u003ehmox1a\u003c/em\u003e coding sequence, flanked by restriction enzyme sites for XhoI and Cfr9I. Primers used for PCR amplifications are shown in Table S4. All the PCRs were performed using the Platinum \u003cem\u003eTaq\u003c/em\u003e DNA polymerase high fidelity (Invitrogen). The plasmid Tol2_\u003cem\u003empeg1:Dendra2\u003c/em\u003e (Addgene, plasmid #51462)[43], containing the Dendra2 fluorescent protein flanked by Tol2 elements, was co-digested by XhoI/Cfr9I (Thermo Fisher) to remove the original \u003cem\u003empeg1\u003c/em\u003e promoter and ligate the cloned 2.1kb \u003cem\u003ehmox1a\u003c/em\u003e promoter, thus generating the Tol2-based \u003cem\u003e-2.1hmox1a:Dendra2\u003c/em\u003e construct. The cloning strategy used left the first 3 amino acids of the \u003cem\u003ehmox1a\u003c/em\u003e coding sequence in-frame with the Dendra2 coding sequence. Cloning was verified by restriction enzyme analysis and Sanger sequencing (Macrogen, South Korea). For the generation of the Tol2-based \u003cem\u003e-2.1hmox1a:mCherryCAAX\u003c/em\u003e construct, the Dendra2 coding sequence of the generated vector was excised using Cfr9I and MunI restriction enzymes, and replaced by a PCR product containing the mCherryCAAX coding sequence flanked by Cfr9I and MunI. For the generation of the \u003cem\u003ehmox1a\u003c/em\u003e transgenic lines, 1-2nL of injection solution containing the generated plasmids (25ng/\u0026micro;L) and the Tol2 transposase mRNA (35ng/\u0026micro;L) was injected into 1-cell stage wild-type zebrafish zygotes. Positive larvae for Dendra2 or mCherryCAAX (F0 generation) were raised to adults, and founders were identified after outcrossing F0 adults with non-fluorescent wild-type fish. Larvae used for experiments were from the F2 generation onwards.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eDepletion of macrophages with clodronate liposomes\u003c/em\u003e\u003c/p\u003e\n\n\u003cp\u003eClodronate-loaded liposomes (www.clodronateliposomes.com) were used to reduce the macrophage pool of zebrafish larvae. At 54hpf, zebrafish larvae were randomly separated in two groups. The first group of larvae was injected with 2nL of 0.5mg/mL clodronate-loaded liposomes (Lipo-clodronate, 1:10 from stock) into the bloodstream through the circulation valley. The second group of larvae was injected with an equivalent dilution of PBS-loaded liposomes (Lipo-PBS). Amputations were conducted 18 hours after Lipo-clodronate or Lipo-PBS injections.\u003c/p\u003e\n\n\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\n\n\u003cp\u003eStatistical tests were performed using Prism 8.0 software (GraphPad, USA). Differences between samples were considered significant when the obtained p-value was lower than 0.05.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. Oscar A. Pe\u0026ntilde;a for early contributions to the project; staff from the zebrafish facilities at the Facultad de Ciencias of Universidad de Chile, the FishCore at Monash University, and the Karolinska Institutet Zebrafish Core Facility for expert fish care; staff from the FlowCore at Monash University for services and technical assistance; Florencio Espinoza and Dr. Vahid Pazhakh for expert technical help and support; and Dr. Myra N. Ch\u0026aacute;vez for expert revision of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.J.L., M.L.A., and R.A.M.C. conceived the idea and designed experiments. A.M., C.M-M., and R.A.M.C. performed experiments. A.M. and R.A.M.C. wrote the manuscript. All authors revised and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants conferred to M.L.A. (FONDAP 15090007, ICN 2021_044, FONDECYT 1140702 and 1221360, REDES 150094) and R.A.M.C. (CONICYT/ANID scholarship 21130458, \u0026Aring;ke Wiberg Foundation M24-0035).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShotgun proteomic raw files are publicly available in the MassIVE proteomic repository (accession number: MSV000092719). Additional data will be made available on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKoh TJ, DiPietro LA. Inflammation and wound healing: the role of the macrophage. Expert Rev Mol Med. 2011;13:e23. https://doi.org/10.1017/S1462399411001943.\u003c/li\u003e\n\u003cli\u003eWilgus TA, Roy S, McDaniel JC. Neutrophils and Wound Repair: Positive Actions and Negative Reactions. Adv Wound Care (New Rochelle). 2013;2:379\u0026ndash;88. https://doi.org/10.1089/wound.2012.0383.\u003c/li\u003e\n\u003cli\u003eWynn TA, Vannella KM. Macrophages in Tissue Repair, Regeneration, and Fibrosis. Immunity. 2016;44:450\u0026ndash;62. https://doi.org/10.1016/j.immuni.2016.02.015.\u003c/li\u003e\n\u003cli\u003eEllett F, Lieschke GJ. Zebrafish as a model for vertebrate hematopoiesis. Curr Opin Pharmacol. 2010;10:563\u0026ndash;70. https://doi.org/10.1016/j.coph.2010.05.004.\u003c/li\u003e\n\u003cli\u003evan der Vaart M, Spaink HP, Meijer AH. Pathogen recognition and activation of the innate immune response in zebrafish. Adv Hematol. 2012;2012:159807. https://doi.org/10.1155/2012/159807.\u003c/li\u003e\n\u003cli\u003eFranza M, Varricchio R, Alloisio G, De Simone G, Di Bella S, Ascenzi P, et al. Zebrafish (Danio rerio) as a Model System to Investigate the Role of the Innate Immune Response in Human Infectious Diseases. Int J Mol Sci. 2024;25:12008. https://doi.org/10.3390/ijms252212008.\u003c/li\u003e\n\u003cli\u003eLi L, Yan B, Shi Y-Q, Zhang W-Q, Wen Z-L. Live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration. J Biol Chem. 2012;287:25353\u0026ndash;60. https://doi.org/10.1074/jbc.M112.349126.\u003c/li\u003e\n\u003cli\u003ePetrie TA, Strand NS, Yang C-T, Rabinowitz JS, Moon RT. Macrophages modulate adult zebrafish tail fin regeneration. Development. 2014;141:2581\u0026ndash;91. https://doi.org/10.1242/dev.098459.\u003c/li\u003e\n\u003cli\u003eNguyen-Chi M, Laplace-Builh\u0026eacute; B, Travnickova J, Luz-Crawford P, Tejedor G, Lutfalla G, et al. TNF signaling and macrophages govern fin regeneration in zebrafish larvae. Cell Death Dis. 2017;8:e2979. https://doi.org/10.1038/cddis.2017.374.\u003c/li\u003e\n\u003cli\u003eMorales RA, Allende ML. Peripheral Macrophages Promote Tissue Regeneration in Zebrafish by Fine-Tuning the Inflammatory Response. Front Immunol. 2019;10:253. https://doi.org/10.3389/fimmu.2019.00253.\u003c/li\u003e\n\u003cli\u003eHasegawa T, Hall CJ, Crosier PS, Abe G, Kawakami K, Kudo A, et al. Transient inflammatory response mediated by interleukin-1\u0026beta; is required for proper regeneration in zebrafish fin fold. Elife. 2017;6:e22716. https://doi.org/10.7554/eLife.22716.\u003c/li\u003e\n\u003cli\u003eZhang M, Nakamura K, Kageyama S, Lawal AO, Gong KW, Bhetraratana M, et al. Myeloid HO-1 modulates macrophage polarization and protects against ischemia-reperfusion injury. JCI Insight. 2018;3:e120596, 120596. https://doi.org/10.1172/jci.insight.120596.\u003c/li\u003e\n\u003cli\u003ePatsalos A, Tzerpos P, Halasz L, Nagy G, Pap A, Giannakis N, et al. The BACH1-HMOX1 Regulatory Axis Is Indispensable for Proper Macrophage Subtype Specification and Skeletal Muscle Regeneration. J Immunol. 2019;203:1532\u0026ndash;47. https://doi.org/10.4049/jimmunol.1900553.\u003c/li\u003e\n\u003cli\u003eLuo K, Ogawa M, Ayer A, Britton WJ, Stocker R, Kikuchi K, et al. Zebrafish Heme Oxygenase 1a Is Necessary for Normal Development and Macrophage Migration. Zebrafish. 2022;19:7\u0026ndash;17. https://doi.org/10.1089/zeb.2021.0058.\u003c/li\u003e\n\u003cli\u003eGray C, Loynes CA, Whyte MKB, Crossman DC, Renshaw SA, Chico TJA. Simultaneous intravital imaging of macrophage and neutrophil behaviour during inflammation using a novel transgenic zebrafish. Thromb Haemost. 2011;105:811\u0026ndash;9. https://doi.org/10.1160/TH10-08-0525.\u003c/li\u003e\n\u003cli\u003eHuang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 2009;4:44\u0026ndash;57. https://doi.org/10.1038/nprot.2008.211.\u003c/li\u003e\n\u003cli\u003ePei W, Tanaka K, Huang SC, Xu L, Liu B, Sinclair J, et al. Extracellular HSP60 triggers tissue regeneration and wound healing by regulating inflammation and cell proliferation. NPJ Regen Med. 2016;1:16013-. https://doi.org/10.1038/npjregenmed.2016.13.\u003c/li\u003e\n\u003cli\u003eFarnsworth DR, Saunders LM, Miller AC. A single-cell transcriptome atlas for zebrafish development. Dev Biol. 2020;459:100\u0026ndash;8. https://doi.org/10.1016/j.ydbio.2019.11.008.\u003c/li\u003e\n\u003cli\u003eRyter SW, Alam J, Choi AMK. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. Physiol Rev. 2006;86:583\u0026ndash;650. https://doi.org/10.1152/physrev.00011.2005.\u003c/li\u003e\n\u003cli\u003eLuo K, Stocker R, Britton WJ, Kikuchi K, Oehlers SH. Haem oxygenase limits Mycobacterium marinum infection-induced detrimental ferrostatin-sensitive cell death in zebrafish. FEBS J. 2022;289:671\u0026ndash;81. https://doi.org/10.1111/febs.16209.\u003c/li\u003e\n\u003cli\u003eCraven SE, French D, Ye W, de Sauvage F, Rosenthal A. Loss of Hspa9b in zebrafish recapitulates the ineffective hematopoiesis of the myelodysplastic syndrome. Blood. 2005;105:3528\u0026ndash;34. https://doi.org/10.1182/blood-2004-03-1089.\u003c/li\u003e\n\u003cli\u003eHolowiecki A, O\u0026rsquo;Shields B, Jenny MJ. Spatiotemporal expression and transcriptional regulation of heme oxygenase and biliverdin reductase genes in zebrafish (Danio rerio) suggest novel roles during early developmental periods of heightened oxidative stress. Comp Biochem Physiol C Toxicol Pharmacol. 2017;191:138\u0026ndash;51. https://doi.org/10.1016/j.cbpc.2016.10.006.\u003c/li\u003e\n\u003cli\u003eLi Z-X, Chen J-W, Yuan F, Huang Y-Y, Zhao L-Y, Li J, et al. Xyloketal B exhibits its antioxidant activity through induction of HO-1 in vascular endothelial cells and zebrafish. Mar Drugs. 2013;11:504\u0026ndash;22. https://doi.org/10.3390/md11020504.\u003c/li\u003e\n\u003cli\u003eSaha D, Patgaonkar M, Shroff A, Ayyar K, Bashir T, Reddy KVR. Hemoglobin expression in nonerythroid cells: novel or ubiquitous? Int J Inflam. 2014;2014:803237. https://doi.org/10.1155/2014/803237.\u003c/li\u003e\n\u003cli\u003eReed EC, Kim JD, Case AJ. Non-canonical hemoglobin: An updated review on its ubiquitous expression. Redox Biol. 2025;82:103602. https://doi.org/10.1016/j.redox.2025.103602.\u003c/li\u003e\n\u003cli\u003eLiu L, Zeng M, Stamler JS. Hemoglobin induction in mouse macrophages. Proc Natl Acad Sci U S A. 1999;96:6643\u0026ndash;7. https://doi.org/10.1073/pnas.96.12.6643.\u003c/li\u003e\n\u003cli\u003eBrunyanszki A, Erdelyi K, Szczesny B, Olah G, Salomao R, Herndon DN, et al. Upregulation and Mitochondrial Sequestration of Hemoglobin Occur in Circulating Leukocytes during Critical Illness, Conferring a Cytoprotective Phenotype. Mol Med. 2015;21:666\u0026ndash;75. https://doi.org/10.2119/molmed.2015.00187.\u003c/li\u003e\n\u003cli\u003eLong Q, Meng A, Wang H, Jessen JR, Farrell MJ, Lin S. GATA-1 expression pattern can be recapitulated in living transgenic zebrafish using GFP reporter gene. Development. 1997;124:4105\u0026ndash;11. https://doi.org/10.1242/dev.124.20.4105.\u003c/li\u003e\n\u003cli\u003eTraver D, Paw BH, Poss KD, Penberthy WT, Lin S, Zon LI. Transplantation and in vivo imaging of multilineage engraftment in zebrafish bloodless mutants. Nat Immunol. 2003;4:1238\u0026ndash;46. https://doi.org/10.1038/ni1007.\u003c/li\u003e\n\u003cli\u003eGanis JJ, Hsia N, Trompouki E, de Jong JLO, DiBiase A, Lambert JS, et al. Zebrafish globin switching occurs in two developmental stages and is controlled by the LCR. Dev Biol. 2012;366:185\u0026ndash;94. https://doi.org/10.1016/j.ydbio.2012.03.021.\u003c/li\u003e\n\u003cli\u003ePelster B, Burggren WW. Disruption of hemoglobin oxygen transport does not impact oxygen-dependent physiological processes in developing embryos of zebra fish (Danio rerio). Circ Res. 1996;79:358\u0026ndash;62. https://doi.org/10.1161/01.res.79.2.358.\u003c/li\u003e\n\u003cli\u003eRombough P, Drader H. Hemoglobin enhances oxygen uptake in larval zebrafish (Danio rerio) but only under conditions of extreme hypoxia. J Exp Biol. 2009;212 Pt 6:778\u0026ndash;84. https://doi.org/10.1242/jeb.026575.\u003c/li\u003e\n\u003cli\u003eJeney V, Eaton JW, Balla G, Balla J. Natural history of the bruise: formation, elimination, and biological effects of oxidized hemoglobin. Oxid Med Cell Longev. 2013;2013:703571. https://doi.org/10.1155/2013/703571.\u003c/li\u003e\n\u003cli\u003eDutra FF, Bozza MT. Heme on innate immunity and inflammation. Front Pharmacol. 2014;5:115. https://doi.org/10.3389/fphar.2014.00115.\u003c/li\u003e\n\u003cli\u003eAnderson HL, Brodsky IE, Mangalmurti NS. The evolving erythrocyte: RBCs as modulators of innate immunity. J Immunol. 2018;201:1343\u0026ndash;51. https://doi.org/10.4049/jimmunol.1800565.\u003c/li\u003e\n\u003cli\u003eWagener FADTG, Volk H-D, Willis D, Abraham NG, Soares MP, Adema GJ, et al. Different faces of the heme-heme oxygenase system in inflammation. Pharmacol Rev. 2003;55:551\u0026ndash;71. https://doi.org/10.1124/pr.55.3.5.\u003c/li\u003e\n\u003cli\u003eWillis D, Moore AR, Frederick R, Willoughby DA. Heme oxygenase: a novel target for the modulation of the inflammatory response. Nat Med. 1996;2:87\u0026ndash;90. https://doi.org/10.1038/nm0196-87.\u003c/li\u003e\n\u003cli\u003eTasnim M, Wahlquist P, Hill JT. Zebrafish: unraveling genetic complexity through duplicated genes. Dev Genes Evol. 2024;234:99\u0026ndash;116. https://doi.org/10.1007/s00427-024-00720-6.\u003c/li\u003e\n\u003cli\u003eWattrus SJ, Smith ML, Rodrigues CP, Hagedorn EJ, Kim JW, Budnik B, et al. Quality assurance of hematopoietic stem cells by macrophages determines stem cell clonality. Science. 2022;377:1413\u0026ndash;9. https://doi.org/10.1126/science.abo4837.\u003c/li\u003e\n\u003cli\u003eO\u0026rsquo;brien BRA. Identification of Haemoglobin by its Catalase Reaction with Peroxide and o-Dianisidine. Stain Technology. 1961;36:57\u0026ndash;61. https://doi.org/10.3109/10520296109113244.\u003c/li\u003e\n\u003cli\u003eWesterfield M. The zebrafish book: a guide for the laboratory use of zebrafish Danio (Brachydanio) rerio. 4th ed. Eugene, Or: ZFIN; 2000.\u003c/li\u003e\n\u003cli\u003eEllett F, Pase L, Hayman JW, Andrianopoulos A, Lieschke GJ. mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish. Blood. 2011;117:e49-56. https://doi.org/10.1182/blood-2010-10-314120.\u003c/li\u003e\n\u003cli\u003eHarvie EA, Green JM, Neely MN, Huttenlocher A. Innate immune response to Streptococcus iniae infection in zebrafish larvae. Infect Immun. 2013;81:110\u0026ndash;21. https://doi.org/10.1128/IAI.00642-12.\u003c/li\u003e\n\u003cli\u003eRenshaw SA, Loynes CA, Trushell DMI, Elworthy S, Ingham PW, Whyte MKB. A transgenic zebrafish model of neutrophilic inflammation. Blood. 2006;108:3976\u0026ndash;8. https://doi.org/10.1182/blood-2006-05-024075.\u003c/li\u003e\n\u003cli\u003eYoo SK, Deng Q, Cavnar PJ, Wu YI, Hahn KM, Huttenlocher A. Differential regulation of protrusion and polarity by PI3K during neutrophil motility in live zebrafish. Dev Cell. 2010;18:226\u0026ndash;36. https://doi.org/10.1016/j.devcel.2009.11.015.\u003c/li\u003e\n\u003cli\u003eHall C, Flores MV, Storm T, Crosier K, Crosier P. The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish. BMC Dev Biol. 2007;7:42. https://doi.org/10.1186/1471-213X-7-42.\u003c/li\u003e\n\u003cli\u003eHuang DW, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 2009;37:1\u0026ndash;13. https://doi.org/10.1093/nar/gkn923.\u003c/li\u003e\n\u003cli\u003eKanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28:27\u0026ndash;30. https://doi.org/10.1093/nar/28.1.27.\u003c/li\u003e\n\u003cli\u003eKanehisa M, Furumichi M, Sato Y, Matsuura Y, Ishiguro-Watanabe M. KEGG: biological systems database as a model of the real world. Nucleic Acids Res. 2025;53:D672\u0026ndash;7. https://doi.org/10.1093/nar/gkae909.\u003c/li\u003e\n\u003cli\u003ePaffett-Lugassy NN, Zon LI. Analysis of hematopoietic development in the zebrafish. Methods Mol Med. 2005;105:171\u0026ndash;98. https://doi.org/10.1385/1-59259-826-9:171.\u003c/li\u003e\n\u003cli\u003eChu C-Y, Cheng C-H, Chen G-D, Chen Y-C, Hung C-C, Huang K-Y, et al. The zebrafish erythropoietin: functional identification and biochemical characterization. FEBS Lett. 2007;581:4265\u0026ndash;71. https://doi.org/10.1016/j.febslet.2007.07.073.\u003c/li\u003e\n\u003cli\u003eThisse C, Thisse B. High-resolution in situ hybridization to whole-mount zebrafish embryos. Nat Protoc. 2008;3:59\u0026ndash;69. https://doi.org/10.1038/nprot.2007.514.\u003c/li\u003e\n\u003cli\u003eNakajima H, Nakajima-Takagi Y, Tsujita T, Akiyama S-I, Wakasa T, Mukaigasa K, et al. Tissue-restricted expression of Nrf2 and its target genes in zebrafish with gene-specific variations in the induction profiles. PLoS One. 2011;6:e26884. https://doi.org/10.1371/journal.pone.0026884.\u003c/li\u003e\n\u003cli\u003eFuse Y, Nakajima H, Nakajima-Takagi Y, Nakajima O, Kobayashi M. Heme-mediated inhibition of Bach1 regulates the liver specificity and transience of the Nrf2-dependent induction of zebrafish heme oxygenase 1. Genes Cells. 2015;20:590\u0026ndash;600. https://doi.org/10.1111/gtc.12249.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7799531/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7799531/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTissue injury triggers a tightly regulated cascade of events that transitions from inflammation to resolution and ultimately tissue remodeling. Although the cellular dynamics of immune cells during these phases are increasingly well-characterized, the molecular mediators orchestrating the response to injury are yet to be fully elucidated. Based on a zebrafish model of tissue injury and using proteomic, \u003cem\u003ein situ\u003c/em\u003e RNA expression analyses, and novel transgenic fluorescent reporters, we aimed to uncover relevant molecular mediators of tissue inflammation, resolution, and regeneration. We found that hemoglobin accumulated in the injury site after tail fin amputation in zebrafish larvae, reaching its peak during the inflammatory phase and decreasing together with the resolution of inflammation. Furthermore, we observed that the heme scavenger and cytoprotective enzyme heme oxygenase 1 (Hmox1) is expressed in the injury site of amputated tail fins, and that macrophages were the main source of the functional \u003cem\u003ehmox1a\u003c/em\u003e paralog. Pharmacological inhibition of Hmox1 activity impaired hemoglobin clearance and tail fin regeneration. In addition, depletion of macrophages led to impaired hemoglobin clearance, phenocopying Hmox1 inhibition. Altogether, our findings reveal a novel role for Hmox1 in shaping the regenerative microenvironment and identify hemoglobin and \u003cem\u003ehmox1a\u003c/em\u003e-expressing macrophages as previously overlooked players in the zebrafish injury response. This work underscores a new link between heme metabolism, immune regulation, and tissue regeneration \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Heme oxygenase 1 activity mediates hemoglobin clearance and tail fin regeneration in zebrafish larvae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-04 12:40:46","doi":"10.21203/rs.3.rs-7799531/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-19T10:31:32+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-11T16:55:37+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-27T11:39:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"146552555276977436447102676364259307039","date":"2025-10-27T11:13:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"38909048306218868258456306201554546099","date":"2025-10-23T11:24:21+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-23T09:18:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-22T17:23:04+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-22T17:00:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-20T15:24:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-10-20T15:20:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2969a453-8725-472e-9840-bfeab221029c","owner":[],"postedDate":"November 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":57414681,"name":"Biological sciences/Biochemistry"},{"id":57414682,"name":"Biological sciences/Biological techniques"},{"id":57414683,"name":"Biological sciences/Biotechnology"},{"id":57414684,"name":"Biological sciences/Cell biology"},{"id":57414685,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-05-21T08:55:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-04 12:40:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7799531","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7799531","identity":"rs-7799531","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}