Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Macrophages modulate adult zebrafish tail fin regeneration

Macrophages modulate adult zebrafish tail fin regeneration © 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 406 doi:10.1242/dev.120642 CORRECTION Timothy A. Petrie, Nicholas S. Strand, Chao-Tsung Yang, Jeremy S. Rabinowitz and Randall T. Moon There was an error published in Development 141, 2581-2591. Author Chao-Tsung Yang was incorrectly listed as Chao Tsung-Yang and, as such, appeared in the Table of Contents for the issue as Tsung-Yang, C. instead of Yang, C.-T. The corrected author list appears above. The authors apologise to readers for this mistake. DEVELOPMENT © 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 RESEARCH ARTICLE STEM CELLS AND REGENERATION 1,2, 1,2 3 1,2 1,2 Timothy A. Petrie *, Nicholas S. Strand , Chao Tsung-Yang , Jeremy S. Rabinowitz and Randall T. Moon ABSTRACT Mammals have a limited capacity for regeneration (Porrello et al., 2011; Seifert et al., 2012). In light of evidence that tissue regeneration Neutrophils and macrophages, as key mediators of inflammation, is an evolutionarily conserved response to injury (Morrison et al., have defined functionally important roles in mammalian tissue 2006), this has provided an incentive to identify useful models repair. Although recent evidence suggests that similar cells exist in relevant to mammalian inflammation for the study of regeneration. zebrafish and also migrate to sites of injury in larvae, whether Zebrafish have become a powerful vertebrate model for understanding these cells are functionally important for wound healing or the cellular and molecular mechanisms of regeneration (Goldsmith regeneration in adult zebrafish is unknown. To begin to address + + and Jobin, 2012) based on their regenerative ability, their simple but these questions, we first tracked neutrophils (lyzC , mpo )and relevant anatomy, in vivo imaging capability and genetic advantages. macrophages (mpeg1 ) in adult zebrafish following amputation of The adult zebrafish tail (caudal) fin has become a model of choice the tail fin, and detailed a migratory timecourse that revealed for studying analogous appendage regeneration in mammals. The conserved elements of the inflammatory cell response with caudal fin is composed of bony rays, mesenchymal tissue, blood mammals. Next, we used transgenic zebrafish in which we could vessels and nerves, enclosed by epidermis and can fully regenerate all selectively ablate macrophages, which allowed us to investigate tissues after resection. Regeneration of the caudal fin after amputation whether macrophages were required for tail fin regeneration. We (resection) entails three regenerative stages: (1) wound healing identified stage-dependent functional roles of macrophages in [0-1 days post amputation (dpa)]; (2) formation of the regeneration mediating fin tissue outgrowth and bony ray patterning, in part blastema (1-3 dpa), a mass of highly proliferative lineage-restricted through modulating levels of blastema proliferation. Moreover, we mesenchymal progenitor cells; and (3) regenerative outgrowth and also sought to detail molecular regulators of inflammation in adult patterning of new tissue (>3 dpa) (Echeverri et al., 2001; Han et al., zebrafish and identified Wnt/β-catenin as a signaling pathway that 2005; Kintner and Brockes, 1984; Stoick-Cooper et al., 2007a,b). regulates the injury microenvironment, inflammatory cell migration Several signaling pathways are known to control different aspects and macrophage phenotype. These results provide a cellular and of the regenerative process. Of particular note is Wnt/β-catenin molecular link between components of the inflammation response signaling, which is necessaryand sufficient forcaudal fin regeneration and regeneration in adult zebrafish. (Kawakami et al., 2006; Stoick-Cooper et al., 2007a,b). Given the KEY WORDS: Regeneration, Inflammation, Zebrafish, Fin, crucial role of Wnt/β-catenin signaling in zebrafish fin regeneration, Macrophages, Neutrophils, Wnt as well as evidence that this pathway regulates macrophage chemotaxis, recruitment and inflammatory diseases in several INTRODUCTION mammalian models (Newman and Hughes, 2012; Matzelle et al., In mammals, distinct cells of the inflammatory response play crucial 2012; Baker-LePain et al., 2011; Whyte et al., 2012), Wnt/β-catenin roles in determining the level of repair of injured organs. signaling is a candidate for linking inflammation and regeneration in Neutrophils contribute to the initial defense against foreign zebrafish. However, it is still relatively unclear how this key pathway microbes and their ultimate removal (resolution) is essential for is activated and how Wnt/β-catenin signaling affects specific cells and optimal tissue repair (Martin and Feng, 2009; Novoa and Figueras, stages of the regenerative process. 2012). Macrophages, comprising distinct subpopulations of M1 or Importantly, zebrafish share many features with the mammalian M2 subtypes, secrete growth factors and cytokines that may attract immune system, including the existence of cells analogous to keratinocytes and fibroblasts to trigger either tissue repair or scar neutrophils, macrophages, dendritic cells and B and T cells formation (Leibovich and Ross, 1975; Serhan and Savill, 2005; Sica (Renshaw and Trede, 2012). Zebrafish neutrophils rapidly and Mantovani, 2012; Murray and Wynn, 2011). Neutrophils and accumulate at wounds in larvae through various injury cues and macrophages can have pro- or anti-repair effects after injury, engulf small dead cell debris, much like their mammalian counterparts depending on the tissue and injury context (Dovi et al., 2003; (Renshaw et al., 2006; Loynes et al., 2010; Mathias et al., 2007; Brancato and Albina, 2011; Marrazzo et al., 2011; Walters et al., Li et al., 2012; Yoo et al., 2011; Colucci-Guyon et al., 2011). Larval 2009). Therefore, it is evident that modulating inflammation could zebrafish macrophages appear at wound sites later than neutrophils, be a useful therapeutic approach to augment tissue healing. exhibit phagocytic behavior in response to bacterial infiltration and, as in mammals, may exist as different subsets of differing function (Herbomel et al., 1999; Lieschke et al., 2011; Redd et al., 2006; 1 2 HHMI, Chevy Chase, MD 20815, USA. Department of Pharmacology, University 3 Mathias et al., 2009; Volkman et al., 2010). These larval studies of Washington, Seattle, WA 98109, USA. Department of Microbiology, University of indicate that these inflammatory cells may behave similarly after Washington, Seattle, WA 98105, USA. injury to their mammalian counterparts. A number of transgenic lines *Author for correspondence ([email protected]) have been developed that express fluorescent reporters under the This is an Open Access article distributed under the terms of the Creative Commons Attribution control of neutrophil [myeloperoxidase (mpo; mpx – ZFIN); lysozyme License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, C (lyzC)] and macrophage-driven [macrophage expressed 1 (mpeg1)] distribution and reproduction in any medium provided that the original work is properly attributed. promoters in order to better characterize the injury response of these cells (Mathias et al., 2006, 2009; Ellett et al., 2011). Received 30 April 2013; Accepted 23 April 2014 DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 Nonetheless, the functional role of these cells in adult zebrafish appeared to be driven by departure from the vasculature near the tissue regeneration is still unclear. Intriguingly, inflammation may amputation plane, followed by migration to the injured area be a positive regulator of zebrafish neuronal regeneration in (supplementary material Movie 1). A similar accumulation pattern traumatic brain injury (Kyritsis et al., 2012), which is contrary to was seen in experiments with the alternative neutrophil tracking fish, findings in mammals. Dissecting out the effect(s) of individual Tg(lyzC:dsRed) (supplementary material Fig. S2). inflammatory components on regeneration is a more useful Using the same strategy as above, we amputated caudal fins of the approach to understanding how inflammation may be involved Tg(mpeg1:mCherry) fish to track macrophage behavior during in the regenerative process. Moreover, detailing the cellular regeneration. In contrast to neutrophils, macrophages were resident inflammatory response after injury, its effect on zebrafish in greater density than neutrophils in uninjured fin tissue and showed regeneration, and the molecular mechanisms involved is crucial little localized accumulation through 3 dpa (Fig. 1D,E). Macrophages in driving forward the study of vertebrate immunity in general. began accumulating near the injured edge at 3-4 dpa, reached their The present study uses transgenic cell tracking and genetic peak numbers at ∼6-8 dpa and gradually decreased through 14 dpa ablation technology to identify the in vivo post-injury response of (Fig. 1D-F). Again contrasting with neutrophils, macrophages neutrophils and macrophages, as well as delineating functional roles appeared to accumulate primarily in newly regenerated tissue of macrophages in zebrafish caudal fin regeneration. Our findings (Fig. 1D,E, 4-7 dpa, green arrows mark the proximal boundary of provide evidence for stage-dependent functional roles of new fin tissue) and maintained elevated levels even at 14 dpa (Fig. 1F). macrophages in the regenerative process, shed light on possible Both neutrophils and macrophages accumulated more quickly and at signaling cues that modulate this response, and provide a context- greater densities in the more proximal (faster regenerating) resection specific functional link between inflammation and regeneration in compared with distally amputated tissue (Fig. 1C,F). adult zebrafish. Although no published means exists to inhibit macrophage recruitment, we did investigate how reducing neutrophil RESULTS recruitment after injury might affect fin regeneration. Incubation Neutrophils and macrophages are differentially recruited in diphenyliodonium chloride (DPI), a hydrogen peroxide inhibitor during fin regeneration previously shown to inhibit neutrophil recruitment to injury (Deng In order to characterize the cellular inflammatory response that occurs et al., 2012; Yoo et al., 2011), reduced neutrophil accumulation to during adult caudal fin regeneration in zebrafish, we used transgenic the injury site through 3 dpa, yet yielded no difference in the rate of fish to track the two most prominent types of inflammatory cells, fin regeneration compared with untreated fish (supplementary namely neutrophils and macrophages. Neutrophils were visualized material Fig. S4). with Tg(mpo:GFP) and Tg(lyzC:dsRed) fish, in which cellular In summary, both neutrophils and macrophages are present at the fluorescence is driven by the mpo and lyzC promoters, respectively right time and location to be functionally involved fin regeneration, (Mathias et al., 2006; Renshawet al., 2006; Hall et al., 2007), and these as we examine below. largely label the same cells (supplementary material Fig. S1). Macrophages were visualized using Tg(mpeg1:mCherry) fish, with Genetic ablation of macrophages reveals a functional role mCherry expression driven by the mpeg1 promoter (Ellett et al., during regeneration 2011). Recent studies have extensively characterized the specificity of To investigate the functional role of macrophages in fin regeneration these neutrophil and macrophage promoters (Ellett et al., 2011; we developed a transgenic fish Tg(mpeg1:NTR-eYFP) that Mathias et al., 2006, 2009). utilizes an eYFP-tagged, human codon-optimized version of the To visualize these inflammatory cells throughout regeneration, Escherichia coli enzyme nitroreductase (NTR) downstream of the caudal fins were amputated and live images were taken at various time mpeg1 promoter. NTR converts an exogenously delivered pro-drug points starting from 6 h post amputation (hpa) and continuing through metronidazole (MTZ) into a cytotoxic agent capable of killing the 14 dpa. In addition to characterizing general inflammation throughout cell. NTR-MTZ ablation technology has been used in zebrafish to adult fin regeneration, we compared inflammatory responses in tissue successfully ablate a variety of specific cells and tissues in both undergoing differing rates of regeneration within the same fin in order larval and adult zebrafish with negligible neighboring effects (Chen to better understand how inflammation correlates with regeneration. et al., 2011; Curado et al., 2007; Singh et al., 2012) (supplementary To accomplish this, we used the inherent positional memory of material Fig. S5A). amputated fins (Lee et al., 2005; Nachtrab et al., 2013) and performed After 36 h of MTZ treatment, the numbers of cells showing mpeg1- both proximal (rapid growth) and distal (slow growth) resections driven fluorescence in Tg(mpeg1:NTR-eYFP) fish were, upon visual within individual fish fins. During regeneration, undamaged cells inspection, dramatically reduced throughout most discernible tissues retain or actively use information that may dictate morphological including the eye, pectoral fin and caudal fin. We quantified the pattern, a phenomenon termed positional memory. Quantification of reduction of macrophages in the caudal fin by flow cytometry, and inflammatory cells was by total fluorescence intensity normalized to consistently obtained ∼80-90% reduction of eYFP cells in MTZ- the injured area (see Materials and Methods). treated Tg(mpeg1:NTR-eYFP) fish compared with untreated fish Consistent with an early role in response after injury, neutrophil (supplementary material Fig. S5B,C and Fig. S6). eYFP cells were accumulation began at 6 hpa in adult Tg(mpo:GFP) fish (Fig. 1A-C). morphologically identical to mCherry cells in Tg(mpeg1:mCherry) Peak accumulation was achieved by 3 dpa, with the number of fish, and the migrational timeline of eYFP cells during fin regeneration localized neutrophils rapidly declining by 5 dpa. Pre-amputation was also identical to that of mCherry cells, indicating that the Tg levels of neutrophils were reached by 7 dpa and maintained through (mpeg1:NTR-eYFP) line is macrophage specific (supplementary 14 dpa (Fig. 1A-C). Proximal amputations recruited over twice the material Fig. S5A,D). We did not observe any unusual behavior, number of neutrophils as distal amputations, but both injuries including aberrant swimming or eating behavior, in these animals. followed the same pattern of accumulation throughout regeneration. Macrophage recovery was initiated by washing out the MTZ with Similar to larval fins and most mammalian tissues, few neutrophils regular fish water. Washout resulted in a return to normal macrophage were resident in uninjured adult fin tissue. Neutrophil recruitment levels, which is indicative of a constant renewal model of macrophage DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 Fig. 1. Leukocyte recruitment in regenerating caudal fins follows distinct timelines and aligns with positional memory. (A,B) Representative images detailing a regenerative timecourse of neutrophil accumulation in Tg(mpo:GFP) amputated fish, from uncut through 14 dpa. Fish received a dorsal proximal cut (indicated by ‘P’) and a ventral distal cut (‘D’). Fluorescent images were acquired and converted to grayscale for visualization. (C) Neutrophil density was quantified separately for the resected edge of both the proximal and distal cuts (n=9). Total fluorescence intensity of GFP-positive cells was normalized to the injured fin area and used as a correlation for cell number (see Materials and Methods). TFI, total fluorescence intensity. (D,E) Using the same strategy as above, macrophages were tracked in Tg(mpeg1:mCherry) fish during 14 days of regeneration. Boxes indicate regions magnified. (F) Quantification of macrophages near the amputation planes for proximal and distal cuts (n=10). Both neutrophils and macrophages accumulate in greater numbers in more proximal (faster regenerating) compared with distally cut tissue. Error bars indicate s.e.m. averages of each experiment. Scale bars: 200 µm. replacement (supplementary material Fig. S6B and Fig. S9). normally, at a rate significantly higher (56%) than in WT+MTZ Continuous drug treatment daily for up to 14 dpa resulted in >80% (13.4%)orNTR−MTZ (7.8%) (Fig. 2D). We conducted a similar ablation during and at the end of the timecourse (supplementary experiment using a larval fold fin amputation model and observed material Fig. S9). We tested for deleterious unintended effects of a slight decrease in new tissue at 5 dpa (supplementary material MTZ drug treatment by first quantifying the number of caspase 3 Fig. S10), which is suggestive of at least a partially conserved role (apoptotic) cells in the caudal fin in wild-type adult fish before and in regeneration from larvae to adults. after continuous MTZ treatment and no difference was found Since each bony ray can regenerate independently of others, we (supplementary material Fig. S7A). Moreover, no morphological also examined how macrophage depletion alters individual bone ray differences in new fin tissue after caudal fin amputation were length segment morphology and ray branching. Quantitative image observed after treatment with MTZ in wild-type fish (data not analysis at 10 dpa revealed that NTR+MTZ fish exhibited a significant shown). Finally, inflammation was not affected by MTZ treatment in reduction in the average number of segments in the regenerated wild-type fish that had undergone fin amputation (supplementary ray (P<0.04, Fig. 3A,C), although bone segment width was not material Fig. S7B,C and Fig. S8). Thus, this macrophage ablation significantly altered (Fig. 3D). Bone ray branching (as measured by model exhibits minimal off-target effects. the number of bifurcations) was also altered in NTR+MTZ fish To examine the regenerative capacity of the tail fin after (P<0.03, Fig. 3B), and joint specification (bifurcation position) was substantial macrophage loss, we amputated caudal fins from wild- unchanged. These latter data specify direct measures of bone type and Tg(mpeg1:NTR-eYFP) fish and continuously treated patterning, since osteoblast activity can only partially affect these both with MTZ for 14 dpa. In transgenic fish in which measures (Knopf et al., 2011). We further investigated bone quality, macrophages were ablated (NTR+MTZ), the extent of new fin via mineralization formation, using in vivo calcein labeling to tissue growth was decreased compared with wild-type fish given examine actively mineralizing surfaces in newly regenerated bone drug daily (WT+MTZ) (Fig. 2A,B). Tg(mpeg1:NTR-eYFP) fish segments. Qualitatively, NTR+MTZ fish exhibited greater inter-ray that were fin amputated but did not receive MTZ treatment had heterogeneity and weaker calcein labeling than WT+MTZ fish in the regeneration rates similar to those of wild type (Fig. 2B). regenerated tissue (Fig. 3E). We quantified calcein intensities in Moreover, new fin tissue growth was often non-homogeneous in individual bone segments. Quantification of the coefficient of NTR+MTZ fish. These fish often displayed scattered, distinct variation of intensity (Fig. 3G), which is a measure of dispersion, areas of aberrant tissue growth along the fin (Fig. 2A, green arrows supported the qualitative assessment that NTR+MTZ induced a mark areas of comparatively reduced growth), which can occur greater heterogeneity and reduced intensity of labeling (Fig. 3F). DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 Fig. 2. Macrophages modulate caudal fin regeneration rate and phenotype. (A) Macrophages were continuously ablated after fin resection (up to 14 dpa) using the macrophage ablation fish line Tg(mpeg1:NTR-eYFP). Fin images are representative of macrophage-ablated (NTR+MTZ) and control (WT+MTZ) fish in at least three independent experiments. Green arrows point to areas of unusually reduced tissue growth and formation; red arrowheads indicate the original fin cut line. (B) Quantification of regenerated tissue as a percentage of original fin area for NTR+MTZ (n=11), WT+MTZ (n=18) and control fish (NTR−MTZ, n=14). Full regeneration to the original fin area is considered 100% regeneration. Data are compiled and averaged over three separate experiments using identical conditions. 10 dpa, *P=0.0124; 14 dpa, *P=0.0262; two-tailed t-test. Error bars indicate s.e.m. averages of each experiment. (C) Representative images at 4 dpa and 10 dpa of MTZ-treated Tg(mpeg1:NTR-eYFP) caudal fins displaying aberrant tissue phenotypes. (D) Summary of percentage of fish qualitatively assessed for aberrant phenotypes at 14 dpa. Scale bars: 300 µm. Taken together, these data indicate that macrophage depletion impairs that a loss of macrophages did not significantly affect gross bone ray patterning and the quality of bone formation. blastema morphology and size (Fig. 4A,C), but did result in a We next investigated how macrophages might affect key significant decrease in actively proliferating cells, particularly in regenerative processes. We concentrated on possible effects of the mesenchymal region (Fig. 4B,D). We also assayed gene macrophages on blastema phenotype and function, particularly expression levels from blastema regions of macrophage-depleted proliferative capacity. We amputated caudal fins from wild-type fins and detected reduced levels of regeneration-associated genes, and Tg(mpeg1:NTR-eYFP) fish and continuously treated both along with various injury-response genes, particularly at 4 dpa with MTZ for 3 dpa throughout blastema formation. We observed (supplementary material Fig. S11). To investigate whether Fig. 3. Macrophages modulate bony ray patterning and formation during tissue outgrowth. Macrophages were continuously ablated up to 10 dpa. (A) Representative fin images of NTR+MTZ (ii) versus control (i) for at least two independent experiments. Red bars indicate bifurcation points on each ray. Black arrowheads indicate the original fin cut line. (B) Total bifurcations in regenerated tissue are decreased in NTR+MTZ fish compared with wild-type fish. *P=0.030 (two-tailed t-test, error bars indicate s.e. m.). (C) The average number of total segments in each regenerated bony ray is decreased in NTR+MTZ fish compared with WT+MTZ fish. *P=0.040 (two-tailed t-test, error bars indicate s.e.m.). (D) Average segment width for NTR+MTZ and control fins. No significant differences were observed. (E) Fluorescent images of calcein staining in (ii) WT+MTZ and (i) NTR+MTZ fish. Note the less intense and more scattered staining in NTR+MTZ fins compared with WT+MTZ fins. (F) Mean calcein intensity is decreased in NTR +MTZ fish compared with WT+MTZ fish. *P=0.044 (two-tailed t-test, error bars indicate s.e.m.). (G) Coefficient of variation (C.O.V.; a measure of dispersion) for calcein intensity is significantly increased in NTR+MTZ fish compared with wild-type fish. *P=0.047 (two-tailed t-test, three separate experiments, error bars indicate s.e.m.). DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 regeneration. To test their requirement during blastema formation and wound healing, we ablated macrophages beginning 2 days before amputation through 3 dpa, followed by washout until 14 dpa (Fig. 5A), during which new macrophages were produced and migrated to the fin (supplementary material Fig. S6B, Fig. S9). When macrophages were ablated through blastema formation (−2 to 3 dpa), regeneration was inhibited to a similar extent as ablating macrophages for the entire 14-day post-resection period (Fig. 5A-C). Moreover, aberrant fin phenotypes persisted in macrophage-depleted fish (Fig. 5D). To test macrophage requirement during tissue outgrowth, we ablated from 3 dpa through 14 dpa (Fig. 5E); the regeneration rate was not significantly affected (Fig. 5F,G). The occurrence of the aberrant phenotype was still elevated in macrophage-depleted fish (33%, NTR+MTZ) over controls (16%, WT+MTZ; 9%, NTR −MTZ). Thus, there is a functional requirement for macrophages during the wound healing and blastema formation stage that directly affects subsequent tissue growth, whereas during the tissue outgrowth stage macrophages mainly modulate only tissue patterning. Wnt/β-catenin signaling modulates the recruitment and resolution of inflammatory cells Since Wnt/β-catenin signaling is required for blastema formation and regenerative outgrowth in zebrafish caudal fins (Ito et al., 2007; Kawakami et al., 2006; Poss et al., 2000; Stoick-Cooper et al., 2007a,b), but also modulates inflammatory processes including scar formation, fibrosis, wound healing and tissue remodeling in mammals (French et al., 2004; Ren et al., 2013; Koch et al., 2011), we investigated whether there might be a role for Wnt/β-catenin signaling in regulating inflammation during fin regeneration. Using a transcriptional reporter line of Wnt/β-catenin signaling, ia5 Tg(7xTCF-Xla.Siam:nlsmCherry) [designated Tg(TCFsiam: mCherry); Moro et al., 2012], which expresses nuclear-localized mCherry driven by a multimerized TCF response element and Fig. 4. Macrophages modulate the proliferative capacity of the minimal siamois promoter, we tracked cells undergoing active regeneration blastema. (A) Hematoxylin-stained sections of tail fin Wnt/β-catenin signaling. We discovered that a greater density of regenerates (blastemal region) at 3 dpa. Macrophage-depleted fins (right) these cells resides in proximal (faster regenerating) than distal display slightly reduced numbers of deep mesenchymal cells of the blastema. (slower regenerating) resections, similar to the trend of neutrophil Arrowheads indicate the plane of amputation. (B) Blastemal and macrophage and macrophage densities (Fig. 1 and Fig. 6A). In order to directly proliferation assessed by staining 2 (iii,iv) or 3 (i,ii) dpa regenerates for PCNA assess the effect of Wnt/β-catenin signaling on the injury response, (i-iv) or L-plastin (i,ii), a marker for leukocytes (mostly macrophages), and with we assessed gene expression levels in blastema fin tissue in DAPI. Scale bars: 20 µm. (C) Quantification of the length of the blastema in macrophage-depleted (NTR+MTZ; n=7) and wild-type (n=6) fins at 3 dpa. a transgenic line expressing heat shock-inducible Dickkopf Macrophage-depleted fins displayed slightly decreased blastemal size (hsDKK1:GFP), a secreted inhibitor of Wnt/β-catenin signaling, compared with wild-type fins. (D) Cell proliferation (PCNA cells) quantified in and Wnt8a (hsWnt8a:GFP). Genes characteristic of the early injury the blastema is reduced in NTR+MTZ compared with wild-type controls. response (tnfa, il1b, mmp13) were upregulated in DKK1- PCNA cell number was averaged among all sections spanning the entire overexpressing fish over wild-type controls, either during fin width, and normalized to DAPI counts in the image. WT+MTZ, n=10; continuous Wnt inhibition or after a 12 h pulse (Fig. 6B). Levels NTR−MTZ, n=8; NTR+MTZ, n=9. *P=0.0425 (two-tailed t-test, error bars indicate s.e.m.). were unchanged when Wnt8a was overexpressed for 12 h (Fig. 6B), implying that a Wnt/β-catenin signaling threshold macrophages affect other components of inflammation, we might modulate the injury microenvironment. continuously depleted macrophages before and after injury in To determine if Wnt/β-catenin signaling acts directly on Tg(lyzC:dsRed) and Tg(mpeg1:NTR-eYFP; lyzC:dsRed) fish and inflammatory cells in this context, we crossed the Tg(TCFsiam: did not observe significantly altered neutrophil accumulation or mCherry) Wnt reporter fish line with the neutrophil-tracking resolution (supplementary material Fig. S7B and Fig. S8). Taken Tg(mpo:GFP) fish line and separately with the Tg(mpeg1:\TR- together, these data indicate that macrophages affect the rate of eYFP) macrophage ablation line. Inflammatory cells accumulated caudal fin regeneration possibly through impacting the near siam cells distally, but did not appear to express mCherry proliferative capacity of the blastema. (Fig. 6C). Using flow cytometry on pooled, dissociated fins, we found that fewer than 1% of neutrophils and 3% of macrophages Macrophages exhibit stage-dependent effects on fin exhibited activated Wnt reporter fluorescence at 3, 7 or 10 dpa, regeneration indicating that the substantial majority of inflammatory cells do not We took advantage of the cell recovery utility of this model to explore display elevated Wnt/β-catenin signaling (Fig. 6D,E). Hence, the when macrophages are required for complete fin regeneration. We effects of Wnt signaling on cytokine expression are mediated ablated macrophages at two distinct time frames during fin through a non-leukocyte, as yet unidentified, cell population. DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 Fig. 5. Macrophages exhibit stage- dependent effects on fin regeneration. (A) Experimental scheme. Macrophages were ablated after fin resection through 3 dpa, then allowed to repopulate normally via MTZ washout. (B) Representative fin images at 7 and 14 dpa, which is 4 and 11 days after macrophage repopulation initiation, respectively. Green arrow indicates irregular fin phenotype, as dictated by non-homogenous growth areas; red arrows indicate original resection plane. (C) Macrophage reduction through 3 dpa largely recapitulated the reduction in regenerative outgrowth seen with 14 days ablation. Rate of tissue regeneration was reduced in NTR+MTZ (n=11) fish compared with WT+MTZ (n=7) and NTR-MTZ (n=10) fish. Data are averaged over two separate experiments using identical conditions. 7 dpa, **P=0.0455; 10 dpa, **P=0.0278; 14 dpa, **P=0.0220; two-tailed t-test. (D) Quantification of percentage of fish displaying any aberrant phenotype at 14 dpa. Total quantification is cumulative from two separate experiments. (E) Experimental scheme. Macrophages were ablated beginning at 3 dpa through 14 dpa. (F) Representative images at 7 and 14 dpa, which is 4 and 11 days after the ablation of macrophages had begun, respectively. (G) Delayed macrophage reduction did not significantly reduce the rate of regeneration. Data are averaged over two separate experiments using the same conditions. (H) Quantification of the percentage of fish displaying any aberrant phenotype at 14 dpa. Data are cumulative from two separate experiments. Error bars indicate s.e.m. Scale bars: 300 µm. In order to assess the effect of Wnt/β-catenin signaling on subjected to a 12 h pulse of DKK1 resulted in gene expression inflammatory events, we crossed a transgenic line for heat shock- profiles of known inflammation-associated cytokines [il8 (cxcl8), inducible Dickkopf (hsDKK1:GFP) with the Tg(lyzC:dsRed) il10, il12] that differed from wild-type control profiles (supplementary neutrophil-tracking or Tg(mpeg1:mCherry) macrophage-tracking material Fig. S13). lines. Macrophage accumulation within the injured area was almost Taken together, these data suggest that Wnt/β-catenin signaling completely inhibited in Tg(hsDKK1:GFP) fish compared with wild- might be necessary for normal progression of the injury response type fish (Fig. 7A,B). Moreover, unlike wild-type fish, in hsDKK1: during regeneration. Moreover, this pathway may exert its effects GFP fish there was no significant statistical difference between mechanistically through modulating macrophage activity and proximal and distal resections in macrophage accumulation at any phenotype at various time points. time period. The heat shock protocol by itself did not perturb inflammatory cell migration (Fig. 7B,D). Inhibition of Wnt/β- DISCUSSION catenin signaling delayed neutrophil resolution and prolonged Although wound healing has been extensively studied in mammals, neutrophil number in the injury area compared with wild-type fish, we have a limited understanding of the injury-induced cellular taking twice as long (12 dpa) in DKK1-overexpressing fins to reach response in a regenerative context. In this study, we utilized a the level of neutrophils observed at 6 dpa for wild-type fins in adults combination of cell tracking and genetic cell ablation approaches to (Fig. 7C,D). No cell accumulation differences were observed in detail the course and role of cellular components of inflammation in gain-of-function Wnt8a fish compared with wild-type controls. To zebrafish fin regeneration. Our data suggest that the relative time disassociate initial regenerative events from leukocyte migration frame of inflammatory cell movement to and from sites of injury is later in the process, Wnt inhibition was delayed, beginning after similar for adult zebrafish and mammals, where neutrophils are tissue outgrowth initiation (at 3 and 5 dpa). Delayed Wnt inhibition attracted to the wound first through ‘homing’ from the circulation, again decreased macrophage accumulation near the site of injury followed by circulation-based or resident macrophages (Sadik et al., (supplementary material Fig. S14). Furthermore, Wnt inhibition 2011; Yoo and Huttenlocher, 2011; Li et al., 2012). Cell tracking data decreased the density of proliferating macrophages (5 dpa) in indicate that activated neutrophils are circulation derived, whereas the regenerating area (Fig. 7E,F; supplementary material Fig. S12). most macrophages are resident in the fin, in contrast to both larval Subsequent gene profiling of macrophages sorted from tissue zebrafish and mammalian appendages. Macrophage accumulation DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 Fig. 6. Wnt/β-catenin signaling by non-leukocytes affects the injury environment in regenerating fins. (A) Representative images detailing cells undergoing Wnt/β-catenin signaling (siam , red) for proximal and distal fin resections in Tg(TCFsiam:mCherry) fish. Siam cell number is increased in proximal cuts. 4 dpa, *P=0.0329; 7 dpa, *P=0.0296 (two-tailed t-test, error bars indicate s.e.m.). (B) Gene expression levels (4 dpa) of pooled blastemal fin tissue (n>5) as assessed by qRT- PCR for wild-type and for the Tg(hsDKK1:GFP) loss-of- function and Wnt8a (hsWnt8a:GFP) gain-of-function Wnt/β-catenin signaling fish lines. Levels were normalized to fold over non-heat shock control. Data were averaged over two separate experiments. One group included daily heat shock following amputation; the other group included a single heat shock pulse at 84 hpa with tissue extraction 12 h later at 4 dpa. mpx is mpo. (C) Representative images of distal resections from Tg(mpo:GFP; TCFsiam:mCherry) fish and Tg(mpeg1:NTR-YFP; TCFsiam:mCherry) fish at 6 dpa. Little colocalization is evident between neutrophils + + (mpo ) and siam cells. Scale bar: 40 µm; 100 µm in bottom panel. (D) Quantification of flow cytometry sorted cells from pooled resected fins (n=8) from Tg(mpo:GFP; TCFsiam:mCherry) fish indicating the presence of few + + mpo siam cells. (E) Quantification of flow cytometry sorted cells from pooled resected fins (n=7) from Tg (mpeg1:NTR-eYFP; TCFsiam:mCherry) fish indicating + + the presence of few mpeg1 siam cells. (D,E) Error bars indicate s.e.m. of the average of three experiments. mainly occurred after the blastema formation stage, suggesting that occurred after the tissue outgrowth phase (>3 dpa). These data zebrafish macrophages respond to events well after the wound healing advocate a model whereby spatially close resident macrophages phase of fin regeneration. Therefore, we describe a fast-moving and modulate events initially, but during later regenerative stages either fast-responding neutrophil population and a correspondingly slow- newly proliferated macrophages or slowly migrating macrophages moving resident macrophage population in adult zebrafish. affect the regenerative response in a different manner than the early We present evidence that macrophages may have differential macrophage population. Cataloguing the composition of this stage-dependent effects on the extent of tail fin regeneration. population over the injury timecourse using single-cell lineage Although mammalian macrophages serve unique, specific functions tracing or Brainbow technology would be useful to delineate the at distinct phases during tissue repair (Liu et al., 1999; Lucas et al., level of macrophage heterogeneity. 2010), zebrafish macrophages seem to function differently at In contrast to recent evidence that neutrophil deficiency analogous stages after wounding. Whereas in mice macrophage (neutropenia) increases the regeneration rate in larval fins (Li et al., depletion during tissue outgrowth can result in severe hemorrhage in 2012), ourcreation of a neutropenic environment in adult zebrafish did the wound (Mirza et al., 2009), ablation during tissue outgrowth in not affect the fin regeneration rate. Moreover, it is unlikely that zebrafish only affects fin patterning, not growth. Moreover, neutrophils have an inhibitory effect on regeneration because although macrophage depletion has not been found to negatively neutrophils accumulated in markedly greater numbers in faster affect wound closure rates and endothelial repair in mammals (Dovi regenerating tissue throughout the regenerative process. Since et al., 2003; Martin and Feng, 2009; Evans et al., 2013), neutrophils may either promote or inhibit wound healing and tissue macrophage depletion reduced tissue growth in adult zebrafish. repair in mice depending on the tissue and injury context (Dovi et al., We also found no evidence that zebrafish macrophages modulate 2003; Harty et al., 2010; Marrazzo et al., 2011; Rieger et al., 2012), neutrophil recruitment or resolution, whereas macrophages have neutrophil function in zebrafish might be highly injury- and time- been found to modulate these cellular responses in mouse limb dependent. Given the proven utility of the genetic macrophage wounds (Cailhier et al., 2006). These data provide further ablation model in this study, the creation of a similar mpo- and/or lyzC- justification for the view that macrophages have different roles driven ablation fish would more conclusively clarify the supportive or after appendage injury in mammals versus adult zebrafish. reductive role of neutrophils in various regenerative contexts. This study supports the existence of either (1) a single We further establish that Wnt/β-catenin signaling partially macrophage population that has different roles in the regenerative modulates the time frame and degree of leukocyte response in tail course over time, or (2) multiple, functionally distinct macrophage fin regeneration. Wnt/β-catenin signaling inhibition ‘arrested’ the cell populations, similar to in mammals. It is also possible that other and cytokine environment at a stage similar to the early injury myeloid-like cells might migrate from non-fin sites over the course environment. Importantly, this effect was still observed when Wnt/β- of injury, although rapid macrophage movement was not observed catenin signaling was impaired after the initial regenerative events had either in vasculature or interstitial tissue. Macrophages mainly begun, supporting a more direct role of Wnt signaling in determining exerted effects on tissue growth during the initial regenerative macrophage movement. Active Wnt signaling might mitigate early stages, but aberrant phenotypes, including impaired bony ray stage inflammation and act as a molecular switch to proceed to later patterning and bone formation, were still observed when depletion stages of the immune response (neutrophil resolution/macrophage DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 Fig. 7. Wnt/β-catenin signaling regulates leukocyte response to injury. (A) The loss-of-function Wnt/β-catenin signaling line Tg(hsDKK1:GFP) crossed to the Tg(mpeg1:mCherry) line was used to track macrophages after Wnt modulation. Resected wild-type or loss-of-function Wnt/β-catenin signaling (hsDKK) fins received a proximal cut and a distal cut. Representative images are shown of macrophage accumulation through 12 dpa. Fluorescent images were acquired and converted to grayscale for ease of visualization. (B) Macrophage accumulation was reduced in DKK1-overexpressing fins at every time point from 3 dpa until 14 dpa and no significant difference in macrophage number was observed between proximal and distal resections. Data are representative of at least three independent experiments with at least six to eight fish per time point. HsDKK-PROX versus hsWT-PROX, WT-PROX: 6 dpa, *P=0.0083; 8 dpa, *P=0.0072; 12 dpa, P=0.0175. HsDKK-DIST versus WT-DIST, WT-DIST; 6 dpa, **P=0.0140; 8 dpa, **P=0.0195; 12 dpa, **P=0.0361; two-tailed t-test. (C) Tg(hsDKK1:GFP) was crossed to a neutrophil promoter-driven Tg(lyzC:dsRed) line in order to visualize neutrophil accumulation following Wnt inhibition. Representative images indicate that neutrophil accumulation remains elevated longer in DKK1-overexpressing fins compared with wild-type controls. (D) Neutrophil accumulation was higher in DKK1-overexpressing fins compared with wild-type controls after 5 dpa. Data are representative of three independent experiments with at least six to eight fish per time point/condition. hsDKK1 versus hsWT, WT: 6 dpa, *P=0.0075; 8 dpa, *P=0.0112; 10 dpa, *P=0.0105; two-tailed t-test. (E) Proliferation of wild- type and DKK1-overexpressing regenerates at 5 dpa as assessed by anti-PCNA (red), anti-L-plastin (green) and DAPI (blue) staining. Red arrowheads indicate + + original cut site; white arrowheads indicate double-stained (PCNA LP ) cells. The boxed regions are magnified beneath. (F) Proliferating macrophages as a percentage of total cells and total macrophages (LP cells). Numbers were averaged over at least seven sections of each sample. Data are representative of three independent experiments (n>5). hsDKK1 versus hsWT: *P=0.0475; **P=0.0349 (two-tailed t-test, error bars indicate s.e.m.). Scale bars: 200 µm in A; 300 µm in C; 20 µm in E. DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 by bacterial nitroreductase (NTR) was described previously (Curado et al., enrichment). This idea shares similarities with the situation in 2007). A DNA fragment containing EYFP-NTR was subcloned into a Tol2 mammals, in which timely neutrophil removal (resolution) after injury vector that contained the zebrafish mepg1 promoter (Ellett et al., 2011). The is essential to the termination of inflammation – delayed apoptosis or Tol2 construct and transposase RNA were microinjected into 1- to 4-cell impaired clearance of neutrophils can aggravate and prolong tissue stage embryos and the transgenic line was isolated by the specific injury (Sadik et al., 2011). The idea that Wnt/β-catenin signaling may expression of YFP in macrophages in the next generation. Tg(hsDKK1: restrict several aspects of inflammation is supported in several GFP;mpeg1:mCherry), Tg(hsWnt8a:GFP;mpeg1:mCherry), Tg(7xTCF- ia5 mammalian models of disease and injury. For example, high Dkk1 Xla.Siam:nlsmCherry;mpo:GFP) (Moro et al., 2012), Tg(lyzC:dsRed; activity is associated with pro-inflammatory bone loss in mouse mpo:GFP) and Tg(mpeg1:NTR-EYFP;7xTCF-Xla.Siam:nlsmCherry) fish myelomas (Tian et al., 2003), and inhibition of Dkk1 activity in a were made by crossing individual transgenic homozygotes with the mouse model of rheumatoid arthritis results in greater bone formation corresponding transgenic complement. (Diarra et al., 2007). The role of Wnt/β-catenin signaling in modulating the injury response might indeed be similarly context- Adult zebrafish fin amputation surgeries Zebrafish of ∼6-12 months of age were used for all studies. Fin amputation specific in zebrafish; further study in other anatomical injury models surgeries were performed as previously described (Stoick-Cooper et al., would be beneficial in this context. 2007a,b). Two amputation cut schemes were employed: (1) a single cut was The cellular basis of the effects of Wnt signaling on inflammation made traversing the entire dorsoventral length of the caudal fin in each fish; is unclear, in part because cells responding to Wnt ligands had or (2) two separate cuts were made on each fish, one closer to the body of the remained unidentified until very recently (Wehner et al., 2014); it fish (‘proximal’, ventral) and one further away from the body (‘distal’, was determined that a population of actinotrichia-forming cells and dorsal) (Lee et al., 2005). osteoblast progenitors undergo Wnt signaling during blastemal specification, regulating epidermal patterning and osteoblast Live image analysis differentiation indirectly through secretion of factors. Given that The injured adult zebrafish were anesthetized as previously described with Wnt/β-catenin signaling inhibition eliminated the differential Tricaine (Stoick-Cooper et al., 2007a,b), placed on their side and imaged positional memory aspect of macrophage recruitment, and that under a Nikon TiE inverted widefield fluorescence high-resolution microscope. Full fin images were assembled from 30-50 stitched images delayed inhibition reduced longer term migration, it is likely that (20×) encompassing the entire fin, with the fish under constant anesthetization. Wnt/β-catenin signaling also indirectly affects macrophage Live fin images were taken for each fish periodically post amputation. phenotype and activity through a similar regulation of secretion factors. Additionally, the similar accumulation patterns of Wnt- + Analysis of cell density in the injured area of amputated fins responsive cells (siam ) and neutrophils/macrophages suggests that To ascertain the timecourse of cell recruitment to the fin injury area, a measure both inflammatory and Wnt signaling cells might respond to the of cell density near the resected fin edge was utilized. An ‘injured area’ was same injury signals. This idea is further supported by the fact that defined as the area spanning two set dimensions: one dimension being the amputating more proximally also involves the damage of a greater distal-ventral boundary of the fin; the other dimension being defined as from volume of tissue and, therefore, may result in more robust levels of perpendicular to the distal-ventral axis, one-quarter of the fin length proximal paracrine ‘injury signals’, including H O , redox and the Src family 2 2 to the original amputation plane. Using Image-Pro software (Media kinase Lyn, all previously identified in zebrafish (Pase et al., 2012; Cybernetics), the total fluorescence intensity (TFI) from promoter-driven Yoo et al., 2012; Niethammer et al., 2009). Wnt-responding cells in fluorescent cells in the injury area from fin images at each time point was quantified. The TFI was normalized to the pixel area of the injured area for that mammals have recently been linked to modulating angiogenic fin to obtain a measure of cell density in the injured area. This analysis was factors, which can in turn affect the injury response (Kitajewski, used based on the assumption that the fluorescence intensity of each labeled 2003); examining whether Wnt inhibition and macrophage depletion cell was similar on average in each fish as verified by flow cytometry. regulate angiogenesis might shed more light on their mechanistic effects on inflammation and regeneration. Identifying which Wnt- Fin regeneration measurements modulated signals directly affect macrophage proliferation, cytokine Total regeneration was gauged by a percent regeneration metric. Briefly, this release and migration would assist in further developing this measurement required phase-contrast full-fin images be taken before mechanistic insight into how Wnt/β-catenin signaling modulates amputation and at each time point after amputation. The full area (in inflammation and regeneration. pixels) of the caudal fin, from the proximal end of the fin rays to the distal fin Our findings detail the cellular events in the normal injury edge/cut, was quantified from the pre-amputation images for each fish using response during zebrafish epimorphic regeneration. We reveal that ImageJ (NIH). The new tissue area, from the new distal fin edge to the macrophages regulate aspects of appendage regeneration in adult amputation plane, was also quantified. Percent regeneration for each fin at each time point was defined as: % regeneration=100×(new tissue area/ zebrafish. We also provide evidence that Wnt/β-catenin signaling original fin area amputated). may in turn modulate cellular and biochemical inflammatory events during the regenerative process. Our findings, coupled with Macrophage ablation recent research detailing pro-repair roles of inflammatory cells in For all macrophage ablation experiments, Tg(mpeg1:NTR-eYFP) fish were zebrafish brain regeneration, advocate some degree of anatomical housed in static tanks of fish water (five fish/liter) supplemented with or conservation of the role of injury components in regenerative without 2.5 mM metronidazole (MTZ) for the duration of the experiment. process in zebrafish. Finally, macrophages may indeed form part of During ablation experiments, fish were kept on a 12 h light/12 h dark cycle, a cellular bridge between robustly regenerative organisms such as since MTZ is sensitive to long exposure to light. Water was changed daily zebrafish and the less regenerative mammals that could potentially and fresh MTZ was added daily. Two control groups were used: NTR be manipulated for mammalian regenerative therapies. transgenic fish housed in fish water without MTZ, and wild-type fish housed in fish water with MTZ (2.5 mM) under the same daily light/dark cycle. MATERIALS AND METHODS Transgenic lines Flow cytometry and sorting w202 + + + The Tg(mpeg1:NTR-EYFP) line was created using the Tol2 Flow cytometry and partial FACS analysis to isolate siam , mpo , mpeg1 , + + lyzC and YFP (NTR+) cells from various transgenic fish was performed transposon system (Urasaki et al., 2006). Targeted cell ablation mediated DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 Author contributions beginning with isolation of the injured area fin tissue. Once isolated, this T.A.P. and N.S.S. conducted the experiments, T.A.P. analyzed the data, T.A.P., tissue was immediately placed in a tissue disassociation solution of 2 mg/ml J.S.R. and R.T.M. designed the experiments and wrote the paper; and C.T.-Y. collagenase (Sigma-Aldrich) and 0.3 mg/ml protease (type XIV, Sigma- assisted in the generation of the mpeg1:NTR-YFP line. Aldrich) in Hanks solution. The solution was moderately shaken at 30°C for 1 h with gentle trituration performed every 10 min with an 18 gauge needle. Funding After 1 h, the solution was incubated for 5 min in 0.05% trypsin in PBS. T.A.P. and J.S.R. were supported by postdoctoral fellowships from the Howard Before flow cytometry, disassociated cells were washed in 2% (fetal bovine Hughes Medical Institute. C.T.-Y. was supported by a postdoctoral fellowship from serum) FBS in cell disassociation solution. Disassociated cells from wild- the Taiwan National Science Council [NSC97-2917-I-564-109] and his contribution type fish at an identical time point were used to set up the lower limit to this work is also supported by National Institutes of Health (NIH) RO1 grants (background) of fluorescence in each experiment. For cleaved caspase 3 [AI54503 and AI036396] to Lalita Ramakrishnan at the University of Washington. analysis, caspase 3 antibody (Sigma-Aldrich, AV00021; 1:200 in 2% FBS) N.S.S. was supported by a T32 grant [GM00727] and a P01 grant [GM081619] from the National Institutes of Health (NIH). R.T.M. is an investigator of the Howard was added to the suspension for 30 min on ice. After three successive Hughes Medical Institute, which supported this research. Deposited in PMC for washes with 2% FBS, fluorescently labeled secondary antibody was added immediate release. (Alexa Fluor 647, Gt anti-mouse IgG; Life Technologies, A21236; 1:1000) for 20 min on ice. After three further washes (the last including Supplementary material 1:600 DAPI), the suspension was strained and read. Supplementary material available online at http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.098459/-/DC1 Immunohistochemistry Whole adult fin stumps (encompassing the entire fin plus 1-2 mm of the References body girdle) were harvested and fixed in 4% formaldehyde in PBS overnight Baker-LePain, J. C., Nakamura, M. C. and Lane, N. E. (2011). Effects of inflammation on bone: an update. Curr. Opin. Rheumatol. 23, 389-395. at 4°C. Tissue was then washed for 30 min at room temperature with 5% Brancato, S. K. and Albina, J. E. (2011). Wound macrophages as key regulators of sucrose in PBS, followed by two washes for 1 h each in 5% sucrose in PBS, repair: origin, phenotype, and function. Am. J. Pathol. 178, 19-25. and an overnight wash in 30% sucrose in PBS at 4°C. After another Cailhier, J. F., Sawatzky, D. A., Kipari, T., Houlberg, K., Walbaum, D., Watson, S., overnight wash in a 1:1 ratio of 30% sucrose:100% O.C.T. compound Lang, R. A., Clay, S., Kluth, D., Savill, J. et al. (2006). Resident pleural (Tissue-Tek, VWR #25608-930) at 4°C, the tissue was embedded directly in macrophages are key orchestrators of neutrophil recruitment in pleural inflammation. 100% OCT in embedding wells and stored at −80°C before sectioning. Am. J. Respir. Crit. Care Med. 173, 540-547. Embedded tissue was sectioned in a cryostat and the entire dorsoventral span Chen, C.-F., Chu, C.-Y., Chen, T.-H., Lee, S.-J., Shen, C.-N. and Hsiao, C.-D. of the fin cut into 14 µm transverse sections and adhered to Superfrost Plus (2011). Establishment of a transgenic zebrafish line for superficial skin ablation slides (VWR) overnight at 40°C. and functional validation of apoptosis modulators in vivo. PLoS ONE 6, e20654. Colucci-Guyon, E., Tinevez, J.-Y., Renshaw, S. A. and Herbomel, P. (2011). Rabbit L-plastin antibody (a gift of Anna Huttenlocher; 1:300) or PCNA Strategies of professional phagocytes in vivo: unlike macrophages, neutrophils antibody (Sigma-Aldrich, P8825; 1:250) were added in antibody solution engulf only surface-associated microbes. J. Cell Sci. 124, 3053-3059. (0.5% Triton X-100, 5% goat serum, 0.2% BSA in PBS) for 2 h at room Curado, S., Anderson, R. M., Jungblut, B., Mumm, J., Schroeter, E. and temperature in the dark. Slides were washed six times for 15 min each in Stainier, D. Y. R. (2007). Conditional targeted cell ablation in zebrafish: a new tool antibody solution with gentle shaking, and goat anti-rabbit Alexa Fluor 647 for regeneration studies. Dev. Dyn. 236, 1025-1035. secondary antibody (Life Technologies, A21244; 1:1000) added for 2 h at Deng, Q., Harvie, E. A. and Huttenlocher, A. (2012). Distinct signalling room temperature in the dark. After six more washes in antibody solution the mechanisms mediate neutrophil attraction to bacterial infection and tissue slides were sealed with a coverslip with Prolong Gold Antifade Reagent injury. Cell Microbiol. 14, 517-528. Diarra, D., Stolina, M., Polzer, K., Zwerina, J., Ominsky, M. S., Dwyer, D., Korb, (Life Technologies). EdU staining was performed according to Click-iT A., Smolen, J., Hoffmann, M., Scheinecker, C. et al. (2007). Dickkopf-1 is a assays (Life Technologies, C10428). EdU was added 6 h prior to tissue master regulator of joint remodeling. Nat. Med. 13, 156-163. extraction at 14 dpa. Dovi, J. V., He, L.-K. and DiPietro, L. A. (2003). Accelerated wound closure in For calcein-AM fluorochrome labeling, fish were immersed in 0.05% neutrophil-depleted mice. J. Leukoc. Biol. 73, 448-455. calcein-AM (Life Technologies, C3099) and rinsed for 10 min in fresh Echeverri, K., Clarke, J. D. W. and Tanaka, E. M. (2001). In vivo imaging indicates water. For analysis, the midpoint coordinates for all regenerated bone muscle fiber dedifferentiation is a major contributor to the regenerating tail segments were manually identified and the corresponding calcein intensities blastema. Dev. Biol. 236, 151-164. were used to compute mean intensity (μ), standard deviation of intensity (σ), Ellett, F., Pase, L., Hayman, J. W., Andrianopoulos, A. and Lieschke, G. J. (2011). mpeg1 promoter transgenes direct macrophage-lineage expression in and coefficient of variation (σ/μ) for each fish. zebrafish. Blood 117, e49-e56. Evans, M. A., Smart, N., Dubé, K. N., Bollini, S., Clark, J. E., Evans, H. G., Taams, Quantitative RT-PCR L. S., Richardson, R., Lévesque, M., Martin, et al. (2013). Thymosin Total RNA was extracted from zebrafish fin regenerates using TRIZOL β4-sulfoxide attenuates inflammatory cell infiltration and promotes cardiac according to the manufacturer’s protocol (Invitrogen). Tissue incorporating wound healing. Nat. Commun. 4, 2081. all new tissue as well as one or two bone rays proximal to the original cut site French, D. M., Kaul, R. J., D’Souza, A. L., Crowley, C. W., Bao, M., Frantz, G. D., Filvaroff, E. H. and Desnoyers, L. (2004). WISP-1 is an osteoblastic regulator was extracted. Equal amounts of total RNA from each sample were reverse expressed during skeletal development and fracture repair. Am. J. Pathol. 165, transcribed with Thermoscript reverse transcriptase (Invitrogen) using oligo 855-867. (dT) and random hexamer primers. All levels were normalized to β-actin Goldsmith, J. R. and Jobin, C. (2012). Think small: zebrafish as a model system of (18S levels were similar) and fold induction was calculated by setting human pathology. J. Biomed. Biotech. 2012, 817341. control conditions to 1. Primers are listed in supplementary material Hall, C., Flores, M. V., Storm, T., Crosier, K. and Crosier, P. (2007). The zebrafish Table S1. lysozyme C promoter drives myeloid-specific expression in transgenic fish. BMC Dev. Biol. 7, 42. Acknowledgements Han, M., Yang, X., Taylor, G., Burdsal, C. A., Anderson, R. A. and Muneoka, K. We thank Jerry Ament, Stanley Kim and Jeanot Muster for fish care and technical (2005). Limb regeneration in higher vertebrates: developing a roadmap. Anat. assistance; Jeff Mumm from Georgia Health Science University (Augusta, USA) for Rec. B New. Anat. 287B, 14-24 providing a construct containing the eYFP-NTR fragment; Graham Lieschke at the Harty, M. W., Muratore, C. S., Papa, E. F., Gart, M. S., Ramm, G. A., Gregory, S. H. Australian Regenerative Medicine Institute (Melbourne, Australia) for providing DNA and Tracy, T. F., Jr (2010). Neutrophil depletion blocks early collagen degradation containing mpeg1; Francesco Argenton at the University of Padova (Padova, Italy) in repairing cholestatic rat livers. Am. J. Pathol. 176, 1271-1281. for the Tg(TCFsiam:mCherry) line; and Anna Huttenlocher at the University of Herbomel, P., Thisse, B. and Thisse, C. (1999). Ontogeny and behavior of early Wisconsin-Madison for providing L-plastin antibody. macrophages in the zebrafish embryo. Development 126, 3735-3745. Ito, M., Yang, Z., Andl, T., Cui, C., Kim, N., Millar, S. E. and Cotsarelis, G. (2007). Competing interests Wnt-dependent de novo hair follicle regeneration in adult mouse skin after The authors declare no competing financial interests. wounding. Nature 447, 316-320. DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 Kawakami, Y., Rodriguez Esteban, C., Raya, M., Kawakami, H., Marti, M., Niethammer, P., Grabher, C., Look, A. T. and Mitchison, T. J. (2009). A tissue- scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish. Dubova, I. and Izpisua Belmonte, J. C. (2006). Wnt/beta-catenin signaling regulates vertebrate limb regeneration. Genes Dev. 20, 3232-3237. Nature 459, 996-999. Novoa, B. and Figueras, A. (2012). Zebrafish: model for the study of inflammation Kintner, C. R. and Brockes, J. P. (1984). Monoclonal antibodies identify blastemal cells derived from dedifferentiating muscle in newt limb regeneration. Nature 308, and the innate immune response to infectious diseases. Adv. Exp. Med. Biol. 946, 253-275. 67-69. Pase, L., Layton, J. E., Wittmann, C., Ellett, F., Nowell, C. J., Reyes-Aldasoro, C. C., Kitajewski, J. (2013). Wnts heal by restraining angiogenesis. Blood 121, Varma, S., Rogers, K. L., Hall, C. J., Keightley, M. C. et al. (2012). Neutrophil- 2381-2382. delivered myeloperoxidase dampens the hydrogen peroxide burst after tissue Knopf, F., Hammond, C., Chekuru, A., Kurth, T., Hans, S., Weber, C. W., wounding in zebrafish. Curr. Biol. 22, 1818-1824. Mahatma, G., Fisher, S., Brand, M., Schulte-Merker, S. et al. (2011). Bone Porrello, E. R., Mahmoud, A. I., Simpson, E., Hill, J. A., Richardson, J. A., regenerates via dedifferentiation of osteoblasts in the zebrafish fin. Dev. Cell 20, Olson, E. N. and Sadek, H. A. (2011). Transient regenerative potential of the 713-724. neonatal mouse heart. Science 331, 1078-1080. Koch, S., Nava, P., Addis, C., Kim, W., Denning, T. L., Li, L., Parkos, C. A. and Poss, K. D., Shen, J. and Keating, M. T. (2000). Induction of lef1 during zebrafish Nusrat, A. (2011). The Wnt antagonist Dkk1 regulates intestinal epithelial fin regeneration. Dev. Dyn. 219, 282-286. homeostasis and wound repair. Gastroenterology 141, 259-268. e8. Redd, M. J., Kelly, G., Dunn, G., Way, M. and Martin, P. (2006). Imaging Kyritsis, N., Kizil, C., Zocher, S., Kroehne, V., Kaslin, J., Freudenreich, D., macrophage chemotaxis in vivo: studies of microtubule function in zebrafish Iltzsche, A. and Brand, M. (2012). Acute inflammation initiates the regenerative wound inflammation. Cell Motil. Cytoskeleton 63, 415-422. response in the adult zebrafish brain. Science 338, 1353-1356. Ren, S., Johnson, B. G., Kida, Y., Ip, C., Davidson, K. C., Lin, S.-L., Kobayashi, A., Lee, Y., Grill, S., Sanchez, A., Murphy-Ryan, M. and Poss, K. D. (2005). Fgf Lang, R. A., Hadjantonakis, A.-K., Moon, R. T. et al. (2013). LRP06 is a signaling instructs position-dependent growth rate during zebrafish fin coreceptor for multiple fibrogenic signaling pathways in pericytes and myofibroblasts regeneration. Development 132, 5173-5183. that are inhibited by DKK-1. Proc. Natl. Acad. Sci. U.S.A. 110, 1440-1445. Leibovich, S. J. and Ross, R. (1975). The role of macrophage in wound repair: a Renshaw, S. A. and Trede, N. S. (2012). A Model 450 million years in the making: study with hydrocortisone and antimacrophage serum. Am. J. Pathol. 78, 71-100. zebrafish and vertebrate immunity. Dis. Mod. Mech. 5, 38-47. Li, L., Yan, B., Shi, Y.-Q., Zhang, W.-Q. and Wen, Z.-L. (2012). Live imaging Renshaw, S. A., Loynes, C. A., Trushell, D. M. I., Elworthy, S., Ingham, P. W. and reveals differing roles of macrophages and neutrophils during zebrafish tail fin Whyte, M. K. B. (2006). A transgenic zebrafish model of neutrophilic regeneration. J. Biol. Chem. 287, 25353-25360. inflammation. Blood 108, 3976-3978. Lieschke, G. J., Oates, A. C., Crowhurst, M. O., Ward, A. C. and Layton, J. E. Rieger, A. M., Konowalchuk, J. D., Grayfer, L., Katzenback, B. A., Havixbeck, J. J., (2011). Morphologic and functional characterization of granulocytes and Kiemele, M. D., Belosevic, M. and Barreda, D. R. (2012). Fish and mammalian macrophages in embryionic and adult zebrafish. Blood 98, 3087-3096. phagocytes differentially regulate pro-inflammatory and homeostatic responses in Liu, Y., Cousin, J. M., Hughes, J., Van Damme, J., Seckl, J. R., Haslett, C., vivo. PLoS ONE 7, e47070. Dransfield, I., Savill, J. and Rossi, A. G. (1999). Glucocorticoids promote Sadik, C. D., Kim, N. D. and Luster, A. D. (2011). Neutrophils cascading their way nonphlogistic phagocytosis of apoptotic leukocytes. J. Immunol. 162, 3639-3646. to inflammation. Trends Immunol. 32, 452-460. Loynes, C. A., Martin, J. S., Robertson, A., Trushell, D. M. I., Ingham, P. W., Seifert, A. W., Kiama, S. G., Seifert, M. G., Goheen, J. R., Palmer, T. M. and Whyte, M. K. B. and Renshaw, S. A. (2010). Pivotal advance: pharmacological Maden, M. (2012). Skin shedding and tissue regeneration in African spiny mice manipulaton of inflammation resolution during spontaneously resolving tissue (Acomys). Nature 489, 561-565. neutrophilia in the zebrafish. J. Leukoc. Biol. 87, 203-212. Serhan, C. N. and Savill, J. (2005). Resolution of inflammation: the beginning Lucas, T., Waisman, A., Ranjan, R., Roes, J., Krieg, T., Mü ller, W., Roers, A. and programs the end. Nat. Immunol. 6, 1191-1197. Eming, S. A. (2010). Differential roles of macrophages in diverse phases of skin Sica, A. and Mantovani, A. (2012). Macrophage plasticity and polarization: in vivo repair. J. Immunol. 184, 3964-3977. veritas. J. Clin. Invest. 122, 787-795. Marrazzo, G., Bellner, L., Halilovic, A., Li Volti, G., Drago, F., Dunn, M. W. and Singh, S. P., Holdway, J. E. and Poss, K. D. (2012). Regeneration of amputated Schwartzman, M. L. (2011). The role of neutrophils in corneal wound healing in zebrafish fin rays from de novo osteoblasts. Dev. Cell 22, 879-886. HO-2 null mice. PLoS ONE 6, e21180. Stoick-Cooper, C. L., Moon, R. T. and Weidinger, G. (2007a). Advances in Martin, P. and Feng, Y. (2009). Inflammation: wound healing in zebrafish. Nature signaling in vertebrate regeneration as a prelude to regenerative medicine. Genes 459, 921-923. Dev. 21, 1292-1315. Mathias, J. R., Perrin, B. J., Liu, T.-X., Kanki, J., Look, A. T. and Huttenlocher, A. Stoick-Cooper, C. L., Weidinger, G., Riehle, K. J., Hubbert, C., Major, M. B., (2006). Resolution of inflammation by retrograde chemotaxis of neutrophils in Fausto, N. and Moon, R. T. (2007b). Distinct Wnt signaling pathways have transgenic zebrafish. J. Leukoc. Biol. 80, 1281-1288. opposing roles in appendage regeneration. Development 134, 479-489. Mathias, J. R., Dodd, M. E., Walters, K. B., Rhodes, J., Kanki, J. P., Look, A. T. Tian, E., Zhan, F., Walker, R., Rasmussen, E., Ma, Y., Barlogie, B. and and Huttenlocher, A. (2007). Live imaging of chronic inflammation casued by Shaughnessy, J. D., Jr (2003). The role of the Wnt-signaling antagonist DKK1 in mutation of zebrafish Hai1. J. Cell Sci. 120, 3372-3383. the development of osteolytic lesions in multiple myeloma. N. Engl. J. Med. 349, Mathias, J. R., Dodd, M. E., Walters, K. B., Yoo, S. K., Ranheim, E. A. and 2483-2494. Huttenlocher, A. (2009). Characterization of zebrafish larval inflammatory Urasaki, A., Morvan, G. and Kawakami, K. (2006). Functional dissection of the macrophages. Dev. Comp. Immunol. 33, 1212-1217. Tol2 transposable element identified the minimal cis-sequence and a highly Matzelle, M. M., Gallant, M. A., Condon, K. W., Walsh, N. C., Manning, C. A., repetitive sequence in the subterminal region essential for transposition. Genetics Stein, G. S., Lian, J. B., Burr, D. B. and Gravallese, E. M. (2012). Resolution of 174, 639-649. inflammation induces osteoblast function and regulates the Wnt signaling Volkman, H. E., Pozos, T. C., Zheng, J., Davis, J. M., Rawls, J. F. and pathway. Arthritis. Reum. 64, 1540-1550. Ramakrishnan, L. (2010). Tuberculous granuloma induction via interaction of a Mirza, R., DiPietro, L. A. and Koh, T. J. (2009). Selective and specific macrophage bacterial secreted protein with host epithelium. Science 327, 466-469. ablation is detrimental to wound healing in mice. Am. J. Pathol. 175, 2454-2462. Walters, K. B., Dodd, M. E., Mathias, J. R., Gallagher, A. J., Bennin, D. A., Moro, E., Ozhan-Kizil, G., Mongera, A., Beis, D., Wierzbicki, C., Young, R. M., Rhodes, J., Kanki, J. P., Look, A. T., Grinblat, Y. and Huttenlocher, A. (2009). Bournele, D., Domenichini, A., Valdivia, L. E., Lum, L. et al. (2012). In vivo Wnt Muscle degeneration and leukocyte infiltration caused by mutation of zebrafish signaling tracing through a transgenic biosensor fish reveals novel activity Fad24. Dev. Dyn. 238, 86-99. domains. Dev. Biol. 366, 327-340. Wehner, D., Cizelsky, W., Vasudevaro, M. D., Ozhan, G., Haase, C., Kagermeier- Morrison, J. I., Lö ö f, S., He, P. and Simon, A. (2006). Salamander limb Schenk, B., Rö der, A., Dorsky, R. I., Moro, E., Argenton, F. et al. (2014). Wnt/ regeneration involves the activation of a multipotent skeletal muscle satellite cell β-catenin signaling defines organizing centers that orchestrate growth and population. J. Cell Biol. 172, 433-440. differentiation of the regenerating zebrafish caudal fin. Cell Rep. 6, 467-481. Murray, P. J. and Wynn, T. A. (2011). Obstacles and opportunities for understanding Whyte, J. L., Smith, A. A. and Helms, J. A. (2012). Wnt signaling and injury repair. macrophage polarization. J. Leukoc. Biol. 89, 557-563. Cold Spring Harb. Perspect. Biol. 4, a008078. Nachtrab, G., Kikuchi, K., Tornini, V. A. and Poss, K. D. (2013). Transcriptional Yoo, S. K. and Huttenlocher, A. (2011). Spatiotemporal photolabeling of neutrophil components of anteroposterior positional information during zebrafish fin trafficking during inflammation in live zebrafish. J. Leukoc. Biol. 89, 661-667. regeneration. Development 140, 3754-3764. Yoo, S. K., Freisinger, C. M., LeBert, D. C. and Huttenlocher, A. (2012). Early Newman, A. C. and Hughes, C. C. W. (2012). Macrophages and angiogenesis: a redox, Src family kinase, and calcium signaling integrate wound responses and role for Wnt signaling. Vasc. Cell 4, 13. tissue regeneration in zebrafish. J. Cell Biol. 199, 225-234. DEVELOPMENT http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Development The Company of Biologists

Macrophages modulate adult zebrafish tail fin regeneration

Download PDF
12 pages

Loading next page...
 
/lp/the-company-of-biologists/macrophages-modulate-adult-zebrafish-tail-fin-regeneration-ml0INbBKzQ

References (142)

Publisher
The Company of Biologists
Copyright
© 2021 The Company of Biologists. All rights reserved.
ISSN
0950-1991
eISSN
0950-1991
DOI
10.1242/dev.098459
Publisher site
See Article on Publisher Site

Abstract

© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 406 doi:10.1242/dev.120642 CORRECTION Timothy A. Petrie, Nicholas S. Strand, Chao-Tsung Yang, Jeremy S. Rabinowitz and Randall T. Moon There was an error published in Development 141, 2581-2591. Author Chao-Tsung Yang was incorrectly listed as Chao Tsung-Yang and, as such, appeared in the Table of Contents for the issue as Tsung-Yang, C. instead of Yang, C.-T. The corrected author list appears above. The authors apologise to readers for this mistake. DEVELOPMENT © 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 RESEARCH ARTICLE STEM CELLS AND REGENERATION 1,2, 1,2 3 1,2 1,2 Timothy A. Petrie *, Nicholas S. Strand , Chao Tsung-Yang , Jeremy S. Rabinowitz and Randall T. Moon ABSTRACT Mammals have a limited capacity for regeneration (Porrello et al., 2011; Seifert et al., 2012). In light of evidence that tissue regeneration Neutrophils and macrophages, as key mediators of inflammation, is an evolutionarily conserved response to injury (Morrison et al., have defined functionally important roles in mammalian tissue 2006), this has provided an incentive to identify useful models repair. Although recent evidence suggests that similar cells exist in relevant to mammalian inflammation for the study of regeneration. zebrafish and also migrate to sites of injury in larvae, whether Zebrafish have become a powerful vertebrate model for understanding these cells are functionally important for wound healing or the cellular and molecular mechanisms of regeneration (Goldsmith regeneration in adult zebrafish is unknown. To begin to address + + and Jobin, 2012) based on their regenerative ability, their simple but these questions, we first tracked neutrophils (lyzC , mpo )and relevant anatomy, in vivo imaging capability and genetic advantages. macrophages (mpeg1 ) in adult zebrafish following amputation of The adult zebrafish tail (caudal) fin has become a model of choice the tail fin, and detailed a migratory timecourse that revealed for studying analogous appendage regeneration in mammals. The conserved elements of the inflammatory cell response with caudal fin is composed of bony rays, mesenchymal tissue, blood mammals. Next, we used transgenic zebrafish in which we could vessels and nerves, enclosed by epidermis and can fully regenerate all selectively ablate macrophages, which allowed us to investigate tissues after resection. Regeneration of the caudal fin after amputation whether macrophages were required for tail fin regeneration. We (resection) entails three regenerative stages: (1) wound healing identified stage-dependent functional roles of macrophages in [0-1 days post amputation (dpa)]; (2) formation of the regeneration mediating fin tissue outgrowth and bony ray patterning, in part blastema (1-3 dpa), a mass of highly proliferative lineage-restricted through modulating levels of blastema proliferation. Moreover, we mesenchymal progenitor cells; and (3) regenerative outgrowth and also sought to detail molecular regulators of inflammation in adult patterning of new tissue (>3 dpa) (Echeverri et al., 2001; Han et al., zebrafish and identified Wnt/β-catenin as a signaling pathway that 2005; Kintner and Brockes, 1984; Stoick-Cooper et al., 2007a,b). regulates the injury microenvironment, inflammatory cell migration Several signaling pathways are known to control different aspects and macrophage phenotype. These results provide a cellular and of the regenerative process. Of particular note is Wnt/β-catenin molecular link between components of the inflammation response signaling, which is necessaryand sufficient forcaudal fin regeneration and regeneration in adult zebrafish. (Kawakami et al., 2006; Stoick-Cooper et al., 2007a,b). Given the KEY WORDS: Regeneration, Inflammation, Zebrafish, Fin, crucial role of Wnt/β-catenin signaling in zebrafish fin regeneration, Macrophages, Neutrophils, Wnt as well as evidence that this pathway regulates macrophage chemotaxis, recruitment and inflammatory diseases in several INTRODUCTION mammalian models (Newman and Hughes, 2012; Matzelle et al., In mammals, distinct cells of the inflammatory response play crucial 2012; Baker-LePain et al., 2011; Whyte et al., 2012), Wnt/β-catenin roles in determining the level of repair of injured organs. signaling is a candidate for linking inflammation and regeneration in Neutrophils contribute to the initial defense against foreign zebrafish. However, it is still relatively unclear how this key pathway microbes and their ultimate removal (resolution) is essential for is activated and how Wnt/β-catenin signaling affects specific cells and optimal tissue repair (Martin and Feng, 2009; Novoa and Figueras, stages of the regenerative process. 2012). Macrophages, comprising distinct subpopulations of M1 or Importantly, zebrafish share many features with the mammalian M2 subtypes, secrete growth factors and cytokines that may attract immune system, including the existence of cells analogous to keratinocytes and fibroblasts to trigger either tissue repair or scar neutrophils, macrophages, dendritic cells and B and T cells formation (Leibovich and Ross, 1975; Serhan and Savill, 2005; Sica (Renshaw and Trede, 2012). Zebrafish neutrophils rapidly and Mantovani, 2012; Murray and Wynn, 2011). Neutrophils and accumulate at wounds in larvae through various injury cues and macrophages can have pro- or anti-repair effects after injury, engulf small dead cell debris, much like their mammalian counterparts depending on the tissue and injury context (Dovi et al., 2003; (Renshaw et al., 2006; Loynes et al., 2010; Mathias et al., 2007; Brancato and Albina, 2011; Marrazzo et al., 2011; Walters et al., Li et al., 2012; Yoo et al., 2011; Colucci-Guyon et al., 2011). Larval 2009). Therefore, it is evident that modulating inflammation could zebrafish macrophages appear at wound sites later than neutrophils, be a useful therapeutic approach to augment tissue healing. exhibit phagocytic behavior in response to bacterial infiltration and, as in mammals, may exist as different subsets of differing function (Herbomel et al., 1999; Lieschke et al., 2011; Redd et al., 2006; 1 2 HHMI, Chevy Chase, MD 20815, USA. Department of Pharmacology, University 3 Mathias et al., 2009; Volkman et al., 2010). These larval studies of Washington, Seattle, WA 98109, USA. Department of Microbiology, University of indicate that these inflammatory cells may behave similarly after Washington, Seattle, WA 98105, USA. injury to their mammalian counterparts. A number of transgenic lines *Author for correspondence ([email protected]) have been developed that express fluorescent reporters under the This is an Open Access article distributed under the terms of the Creative Commons Attribution control of neutrophil [myeloperoxidase (mpo; mpx – ZFIN); lysozyme License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, C (lyzC)] and macrophage-driven [macrophage expressed 1 (mpeg1)] distribution and reproduction in any medium provided that the original work is properly attributed. promoters in order to better characterize the injury response of these cells (Mathias et al., 2006, 2009; Ellett et al., 2011). Received 30 April 2013; Accepted 23 April 2014 DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 Nonetheless, the functional role of these cells in adult zebrafish appeared to be driven by departure from the vasculature near the tissue regeneration is still unclear. Intriguingly, inflammation may amputation plane, followed by migration to the injured area be a positive regulator of zebrafish neuronal regeneration in (supplementary material Movie 1). A similar accumulation pattern traumatic brain injury (Kyritsis et al., 2012), which is contrary to was seen in experiments with the alternative neutrophil tracking fish, findings in mammals. Dissecting out the effect(s) of individual Tg(lyzC:dsRed) (supplementary material Fig. S2). inflammatory components on regeneration is a more useful Using the same strategy as above, we amputated caudal fins of the approach to understanding how inflammation may be involved Tg(mpeg1:mCherry) fish to track macrophage behavior during in the regenerative process. Moreover, detailing the cellular regeneration. In contrast to neutrophils, macrophages were resident inflammatory response after injury, its effect on zebrafish in greater density than neutrophils in uninjured fin tissue and showed regeneration, and the molecular mechanisms involved is crucial little localized accumulation through 3 dpa (Fig. 1D,E). Macrophages in driving forward the study of vertebrate immunity in general. began accumulating near the injured edge at 3-4 dpa, reached their The present study uses transgenic cell tracking and genetic peak numbers at ∼6-8 dpa and gradually decreased through 14 dpa ablation technology to identify the in vivo post-injury response of (Fig. 1D-F). Again contrasting with neutrophils, macrophages neutrophils and macrophages, as well as delineating functional roles appeared to accumulate primarily in newly regenerated tissue of macrophages in zebrafish caudal fin regeneration. Our findings (Fig. 1D,E, 4-7 dpa, green arrows mark the proximal boundary of provide evidence for stage-dependent functional roles of new fin tissue) and maintained elevated levels even at 14 dpa (Fig. 1F). macrophages in the regenerative process, shed light on possible Both neutrophils and macrophages accumulated more quickly and at signaling cues that modulate this response, and provide a context- greater densities in the more proximal (faster regenerating) resection specific functional link between inflammation and regeneration in compared with distally amputated tissue (Fig. 1C,F). adult zebrafish. Although no published means exists to inhibit macrophage recruitment, we did investigate how reducing neutrophil RESULTS recruitment after injury might affect fin regeneration. Incubation Neutrophils and macrophages are differentially recruited in diphenyliodonium chloride (DPI), a hydrogen peroxide inhibitor during fin regeneration previously shown to inhibit neutrophil recruitment to injury (Deng In order to characterize the cellular inflammatory response that occurs et al., 2012; Yoo et al., 2011), reduced neutrophil accumulation to during adult caudal fin regeneration in zebrafish, we used transgenic the injury site through 3 dpa, yet yielded no difference in the rate of fish to track the two most prominent types of inflammatory cells, fin regeneration compared with untreated fish (supplementary namely neutrophils and macrophages. Neutrophils were visualized material Fig. S4). with Tg(mpo:GFP) and Tg(lyzC:dsRed) fish, in which cellular In summary, both neutrophils and macrophages are present at the fluorescence is driven by the mpo and lyzC promoters, respectively right time and location to be functionally involved fin regeneration, (Mathias et al., 2006; Renshawet al., 2006; Hall et al., 2007), and these as we examine below. largely label the same cells (supplementary material Fig. S1). Macrophages were visualized using Tg(mpeg1:mCherry) fish, with Genetic ablation of macrophages reveals a functional role mCherry expression driven by the mpeg1 promoter (Ellett et al., during regeneration 2011). Recent studies have extensively characterized the specificity of To investigate the functional role of macrophages in fin regeneration these neutrophil and macrophage promoters (Ellett et al., 2011; we developed a transgenic fish Tg(mpeg1:NTR-eYFP) that Mathias et al., 2006, 2009). utilizes an eYFP-tagged, human codon-optimized version of the To visualize these inflammatory cells throughout regeneration, Escherichia coli enzyme nitroreductase (NTR) downstream of the caudal fins were amputated and live images were taken at various time mpeg1 promoter. NTR converts an exogenously delivered pro-drug points starting from 6 h post amputation (hpa) and continuing through metronidazole (MTZ) into a cytotoxic agent capable of killing the 14 dpa. In addition to characterizing general inflammation throughout cell. NTR-MTZ ablation technology has been used in zebrafish to adult fin regeneration, we compared inflammatory responses in tissue successfully ablate a variety of specific cells and tissues in both undergoing differing rates of regeneration within the same fin in order larval and adult zebrafish with negligible neighboring effects (Chen to better understand how inflammation correlates with regeneration. et al., 2011; Curado et al., 2007; Singh et al., 2012) (supplementary To accomplish this, we used the inherent positional memory of material Fig. S5A). amputated fins (Lee et al., 2005; Nachtrab et al., 2013) and performed After 36 h of MTZ treatment, the numbers of cells showing mpeg1- both proximal (rapid growth) and distal (slow growth) resections driven fluorescence in Tg(mpeg1:NTR-eYFP) fish were, upon visual within individual fish fins. During regeneration, undamaged cells inspection, dramatically reduced throughout most discernible tissues retain or actively use information that may dictate morphological including the eye, pectoral fin and caudal fin. We quantified the pattern, a phenomenon termed positional memory. Quantification of reduction of macrophages in the caudal fin by flow cytometry, and inflammatory cells was by total fluorescence intensity normalized to consistently obtained ∼80-90% reduction of eYFP cells in MTZ- the injured area (see Materials and Methods). treated Tg(mpeg1:NTR-eYFP) fish compared with untreated fish Consistent with an early role in response after injury, neutrophil (supplementary material Fig. S5B,C and Fig. S6). eYFP cells were accumulation began at 6 hpa in adult Tg(mpo:GFP) fish (Fig. 1A-C). morphologically identical to mCherry cells in Tg(mpeg1:mCherry) Peak accumulation was achieved by 3 dpa, with the number of fish, and the migrational timeline of eYFP cells during fin regeneration localized neutrophils rapidly declining by 5 dpa. Pre-amputation was also identical to that of mCherry cells, indicating that the Tg levels of neutrophils were reached by 7 dpa and maintained through (mpeg1:NTR-eYFP) line is macrophage specific (supplementary 14 dpa (Fig. 1A-C). Proximal amputations recruited over twice the material Fig. S5A,D). We did not observe any unusual behavior, number of neutrophils as distal amputations, but both injuries including aberrant swimming or eating behavior, in these animals. followed the same pattern of accumulation throughout regeneration. Macrophage recovery was initiated by washing out the MTZ with Similar to larval fins and most mammalian tissues, few neutrophils regular fish water. Washout resulted in a return to normal macrophage were resident in uninjured adult fin tissue. Neutrophil recruitment levels, which is indicative of a constant renewal model of macrophage DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 Fig. 1. Leukocyte recruitment in regenerating caudal fins follows distinct timelines and aligns with positional memory. (A,B) Representative images detailing a regenerative timecourse of neutrophil accumulation in Tg(mpo:GFP) amputated fish, from uncut through 14 dpa. Fish received a dorsal proximal cut (indicated by ‘P’) and a ventral distal cut (‘D’). Fluorescent images were acquired and converted to grayscale for visualization. (C) Neutrophil density was quantified separately for the resected edge of both the proximal and distal cuts (n=9). Total fluorescence intensity of GFP-positive cells was normalized to the injured fin area and used as a correlation for cell number (see Materials and Methods). TFI, total fluorescence intensity. (D,E) Using the same strategy as above, macrophages were tracked in Tg(mpeg1:mCherry) fish during 14 days of regeneration. Boxes indicate regions magnified. (F) Quantification of macrophages near the amputation planes for proximal and distal cuts (n=10). Both neutrophils and macrophages accumulate in greater numbers in more proximal (faster regenerating) compared with distally cut tissue. Error bars indicate s.e.m. averages of each experiment. Scale bars: 200 µm. replacement (supplementary material Fig. S6B and Fig. S9). normally, at a rate significantly higher (56%) than in WT+MTZ Continuous drug treatment daily for up to 14 dpa resulted in >80% (13.4%)orNTR−MTZ (7.8%) (Fig. 2D). We conducted a similar ablation during and at the end of the timecourse (supplementary experiment using a larval fold fin amputation model and observed material Fig. S9). We tested for deleterious unintended effects of a slight decrease in new tissue at 5 dpa (supplementary material MTZ drug treatment by first quantifying the number of caspase 3 Fig. S10), which is suggestive of at least a partially conserved role (apoptotic) cells in the caudal fin in wild-type adult fish before and in regeneration from larvae to adults. after continuous MTZ treatment and no difference was found Since each bony ray can regenerate independently of others, we (supplementary material Fig. S7A). Moreover, no morphological also examined how macrophage depletion alters individual bone ray differences in new fin tissue after caudal fin amputation were length segment morphology and ray branching. Quantitative image observed after treatment with MTZ in wild-type fish (data not analysis at 10 dpa revealed that NTR+MTZ fish exhibited a significant shown). Finally, inflammation was not affected by MTZ treatment in reduction in the average number of segments in the regenerated wild-type fish that had undergone fin amputation (supplementary ray (P<0.04, Fig. 3A,C), although bone segment width was not material Fig. S7B,C and Fig. S8). Thus, this macrophage ablation significantly altered (Fig. 3D). Bone ray branching (as measured by model exhibits minimal off-target effects. the number of bifurcations) was also altered in NTR+MTZ fish To examine the regenerative capacity of the tail fin after (P<0.03, Fig. 3B), and joint specification (bifurcation position) was substantial macrophage loss, we amputated caudal fins from wild- unchanged. These latter data specify direct measures of bone type and Tg(mpeg1:NTR-eYFP) fish and continuously treated patterning, since osteoblast activity can only partially affect these both with MTZ for 14 dpa. In transgenic fish in which measures (Knopf et al., 2011). We further investigated bone quality, macrophages were ablated (NTR+MTZ), the extent of new fin via mineralization formation, using in vivo calcein labeling to tissue growth was decreased compared with wild-type fish given examine actively mineralizing surfaces in newly regenerated bone drug daily (WT+MTZ) (Fig. 2A,B). Tg(mpeg1:NTR-eYFP) fish segments. Qualitatively, NTR+MTZ fish exhibited greater inter-ray that were fin amputated but did not receive MTZ treatment had heterogeneity and weaker calcein labeling than WT+MTZ fish in the regeneration rates similar to those of wild type (Fig. 2B). regenerated tissue (Fig. 3E). We quantified calcein intensities in Moreover, new fin tissue growth was often non-homogeneous in individual bone segments. Quantification of the coefficient of NTR+MTZ fish. These fish often displayed scattered, distinct variation of intensity (Fig. 3G), which is a measure of dispersion, areas of aberrant tissue growth along the fin (Fig. 2A, green arrows supported the qualitative assessment that NTR+MTZ induced a mark areas of comparatively reduced growth), which can occur greater heterogeneity and reduced intensity of labeling (Fig. 3F). DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 Fig. 2. Macrophages modulate caudal fin regeneration rate and phenotype. (A) Macrophages were continuously ablated after fin resection (up to 14 dpa) using the macrophage ablation fish line Tg(mpeg1:NTR-eYFP). Fin images are representative of macrophage-ablated (NTR+MTZ) and control (WT+MTZ) fish in at least three independent experiments. Green arrows point to areas of unusually reduced tissue growth and formation; red arrowheads indicate the original fin cut line. (B) Quantification of regenerated tissue as a percentage of original fin area for NTR+MTZ (n=11), WT+MTZ (n=18) and control fish (NTR−MTZ, n=14). Full regeneration to the original fin area is considered 100% regeneration. Data are compiled and averaged over three separate experiments using identical conditions. 10 dpa, *P=0.0124; 14 dpa, *P=0.0262; two-tailed t-test. Error bars indicate s.e.m. averages of each experiment. (C) Representative images at 4 dpa and 10 dpa of MTZ-treated Tg(mpeg1:NTR-eYFP) caudal fins displaying aberrant tissue phenotypes. (D) Summary of percentage of fish qualitatively assessed for aberrant phenotypes at 14 dpa. Scale bars: 300 µm. Taken together, these data indicate that macrophage depletion impairs that a loss of macrophages did not significantly affect gross bone ray patterning and the quality of bone formation. blastema morphology and size (Fig. 4A,C), but did result in a We next investigated how macrophages might affect key significant decrease in actively proliferating cells, particularly in regenerative processes. We concentrated on possible effects of the mesenchymal region (Fig. 4B,D). We also assayed gene macrophages on blastema phenotype and function, particularly expression levels from blastema regions of macrophage-depleted proliferative capacity. We amputated caudal fins from wild-type fins and detected reduced levels of regeneration-associated genes, and Tg(mpeg1:NTR-eYFP) fish and continuously treated both along with various injury-response genes, particularly at 4 dpa with MTZ for 3 dpa throughout blastema formation. We observed (supplementary material Fig. S11). To investigate whether Fig. 3. Macrophages modulate bony ray patterning and formation during tissue outgrowth. Macrophages were continuously ablated up to 10 dpa. (A) Representative fin images of NTR+MTZ (ii) versus control (i) for at least two independent experiments. Red bars indicate bifurcation points on each ray. Black arrowheads indicate the original fin cut line. (B) Total bifurcations in regenerated tissue are decreased in NTR+MTZ fish compared with wild-type fish. *P=0.030 (two-tailed t-test, error bars indicate s.e. m.). (C) The average number of total segments in each regenerated bony ray is decreased in NTR+MTZ fish compared with WT+MTZ fish. *P=0.040 (two-tailed t-test, error bars indicate s.e.m.). (D) Average segment width for NTR+MTZ and control fins. No significant differences were observed. (E) Fluorescent images of calcein staining in (ii) WT+MTZ and (i) NTR+MTZ fish. Note the less intense and more scattered staining in NTR+MTZ fins compared with WT+MTZ fins. (F) Mean calcein intensity is decreased in NTR +MTZ fish compared with WT+MTZ fish. *P=0.044 (two-tailed t-test, error bars indicate s.e.m.). (G) Coefficient of variation (C.O.V.; a measure of dispersion) for calcein intensity is significantly increased in NTR+MTZ fish compared with wild-type fish. *P=0.047 (two-tailed t-test, three separate experiments, error bars indicate s.e.m.). DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 regeneration. To test their requirement during blastema formation and wound healing, we ablated macrophages beginning 2 days before amputation through 3 dpa, followed by washout until 14 dpa (Fig. 5A), during which new macrophages were produced and migrated to the fin (supplementary material Fig. S6B, Fig. S9). When macrophages were ablated through blastema formation (−2 to 3 dpa), regeneration was inhibited to a similar extent as ablating macrophages for the entire 14-day post-resection period (Fig. 5A-C). Moreover, aberrant fin phenotypes persisted in macrophage-depleted fish (Fig. 5D). To test macrophage requirement during tissue outgrowth, we ablated from 3 dpa through 14 dpa (Fig. 5E); the regeneration rate was not significantly affected (Fig. 5F,G). The occurrence of the aberrant phenotype was still elevated in macrophage-depleted fish (33%, NTR+MTZ) over controls (16%, WT+MTZ; 9%, NTR −MTZ). Thus, there is a functional requirement for macrophages during the wound healing and blastema formation stage that directly affects subsequent tissue growth, whereas during the tissue outgrowth stage macrophages mainly modulate only tissue patterning. Wnt/β-catenin signaling modulates the recruitment and resolution of inflammatory cells Since Wnt/β-catenin signaling is required for blastema formation and regenerative outgrowth in zebrafish caudal fins (Ito et al., 2007; Kawakami et al., 2006; Poss et al., 2000; Stoick-Cooper et al., 2007a,b), but also modulates inflammatory processes including scar formation, fibrosis, wound healing and tissue remodeling in mammals (French et al., 2004; Ren et al., 2013; Koch et al., 2011), we investigated whether there might be a role for Wnt/β-catenin signaling in regulating inflammation during fin regeneration. Using a transcriptional reporter line of Wnt/β-catenin signaling, ia5 Tg(7xTCF-Xla.Siam:nlsmCherry) [designated Tg(TCFsiam: mCherry); Moro et al., 2012], which expresses nuclear-localized mCherry driven by a multimerized TCF response element and Fig. 4. Macrophages modulate the proliferative capacity of the minimal siamois promoter, we tracked cells undergoing active regeneration blastema. (A) Hematoxylin-stained sections of tail fin Wnt/β-catenin signaling. We discovered that a greater density of regenerates (blastemal region) at 3 dpa. Macrophage-depleted fins (right) these cells resides in proximal (faster regenerating) than distal display slightly reduced numbers of deep mesenchymal cells of the blastema. (slower regenerating) resections, similar to the trend of neutrophil Arrowheads indicate the plane of amputation. (B) Blastemal and macrophage and macrophage densities (Fig. 1 and Fig. 6A). In order to directly proliferation assessed by staining 2 (iii,iv) or 3 (i,ii) dpa regenerates for PCNA assess the effect of Wnt/β-catenin signaling on the injury response, (i-iv) or L-plastin (i,ii), a marker for leukocytes (mostly macrophages), and with we assessed gene expression levels in blastema fin tissue in DAPI. Scale bars: 20 µm. (C) Quantification of the length of the blastema in macrophage-depleted (NTR+MTZ; n=7) and wild-type (n=6) fins at 3 dpa. a transgenic line expressing heat shock-inducible Dickkopf Macrophage-depleted fins displayed slightly decreased blastemal size (hsDKK1:GFP), a secreted inhibitor of Wnt/β-catenin signaling, compared with wild-type fins. (D) Cell proliferation (PCNA cells) quantified in and Wnt8a (hsWnt8a:GFP). Genes characteristic of the early injury the blastema is reduced in NTR+MTZ compared with wild-type controls. response (tnfa, il1b, mmp13) were upregulated in DKK1- PCNA cell number was averaged among all sections spanning the entire overexpressing fish over wild-type controls, either during fin width, and normalized to DAPI counts in the image. WT+MTZ, n=10; continuous Wnt inhibition or after a 12 h pulse (Fig. 6B). Levels NTR−MTZ, n=8; NTR+MTZ, n=9. *P=0.0425 (two-tailed t-test, error bars indicate s.e.m.). were unchanged when Wnt8a was overexpressed for 12 h (Fig. 6B), implying that a Wnt/β-catenin signaling threshold macrophages affect other components of inflammation, we might modulate the injury microenvironment. continuously depleted macrophages before and after injury in To determine if Wnt/β-catenin signaling acts directly on Tg(lyzC:dsRed) and Tg(mpeg1:NTR-eYFP; lyzC:dsRed) fish and inflammatory cells in this context, we crossed the Tg(TCFsiam: did not observe significantly altered neutrophil accumulation or mCherry) Wnt reporter fish line with the neutrophil-tracking resolution (supplementary material Fig. S7B and Fig. S8). Taken Tg(mpo:GFP) fish line and separately with the Tg(mpeg1:\TR- together, these data indicate that macrophages affect the rate of eYFP) macrophage ablation line. Inflammatory cells accumulated caudal fin regeneration possibly through impacting the near siam cells distally, but did not appear to express mCherry proliferative capacity of the blastema. (Fig. 6C). Using flow cytometry on pooled, dissociated fins, we found that fewer than 1% of neutrophils and 3% of macrophages Macrophages exhibit stage-dependent effects on fin exhibited activated Wnt reporter fluorescence at 3, 7 or 10 dpa, regeneration indicating that the substantial majority of inflammatory cells do not We took advantage of the cell recovery utility of this model to explore display elevated Wnt/β-catenin signaling (Fig. 6D,E). Hence, the when macrophages are required for complete fin regeneration. We effects of Wnt signaling on cytokine expression are mediated ablated macrophages at two distinct time frames during fin through a non-leukocyte, as yet unidentified, cell population. DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 Fig. 5. Macrophages exhibit stage- dependent effects on fin regeneration. (A) Experimental scheme. Macrophages were ablated after fin resection through 3 dpa, then allowed to repopulate normally via MTZ washout. (B) Representative fin images at 7 and 14 dpa, which is 4 and 11 days after macrophage repopulation initiation, respectively. Green arrow indicates irregular fin phenotype, as dictated by non-homogenous growth areas; red arrows indicate original resection plane. (C) Macrophage reduction through 3 dpa largely recapitulated the reduction in regenerative outgrowth seen with 14 days ablation. Rate of tissue regeneration was reduced in NTR+MTZ (n=11) fish compared with WT+MTZ (n=7) and NTR-MTZ (n=10) fish. Data are averaged over two separate experiments using identical conditions. 7 dpa, **P=0.0455; 10 dpa, **P=0.0278; 14 dpa, **P=0.0220; two-tailed t-test. (D) Quantification of percentage of fish displaying any aberrant phenotype at 14 dpa. Total quantification is cumulative from two separate experiments. (E) Experimental scheme. Macrophages were ablated beginning at 3 dpa through 14 dpa. (F) Representative images at 7 and 14 dpa, which is 4 and 11 days after the ablation of macrophages had begun, respectively. (G) Delayed macrophage reduction did not significantly reduce the rate of regeneration. Data are averaged over two separate experiments using the same conditions. (H) Quantification of the percentage of fish displaying any aberrant phenotype at 14 dpa. Data are cumulative from two separate experiments. Error bars indicate s.e.m. Scale bars: 300 µm. In order to assess the effect of Wnt/β-catenin signaling on subjected to a 12 h pulse of DKK1 resulted in gene expression inflammatory events, we crossed a transgenic line for heat shock- profiles of known inflammation-associated cytokines [il8 (cxcl8), inducible Dickkopf (hsDKK1:GFP) with the Tg(lyzC:dsRed) il10, il12] that differed from wild-type control profiles (supplementary neutrophil-tracking or Tg(mpeg1:mCherry) macrophage-tracking material Fig. S13). lines. Macrophage accumulation within the injured area was almost Taken together, these data suggest that Wnt/β-catenin signaling completely inhibited in Tg(hsDKK1:GFP) fish compared with wild- might be necessary for normal progression of the injury response type fish (Fig. 7A,B). Moreover, unlike wild-type fish, in hsDKK1: during regeneration. Moreover, this pathway may exert its effects GFP fish there was no significant statistical difference between mechanistically through modulating macrophage activity and proximal and distal resections in macrophage accumulation at any phenotype at various time points. time period. The heat shock protocol by itself did not perturb inflammatory cell migration (Fig. 7B,D). Inhibition of Wnt/β- DISCUSSION catenin signaling delayed neutrophil resolution and prolonged Although wound healing has been extensively studied in mammals, neutrophil number in the injury area compared with wild-type fish, we have a limited understanding of the injury-induced cellular taking twice as long (12 dpa) in DKK1-overexpressing fins to reach response in a regenerative context. In this study, we utilized a the level of neutrophils observed at 6 dpa for wild-type fins in adults combination of cell tracking and genetic cell ablation approaches to (Fig. 7C,D). No cell accumulation differences were observed in detail the course and role of cellular components of inflammation in gain-of-function Wnt8a fish compared with wild-type controls. To zebrafish fin regeneration. Our data suggest that the relative time disassociate initial regenerative events from leukocyte migration frame of inflammatory cell movement to and from sites of injury is later in the process, Wnt inhibition was delayed, beginning after similar for adult zebrafish and mammals, where neutrophils are tissue outgrowth initiation (at 3 and 5 dpa). Delayed Wnt inhibition attracted to the wound first through ‘homing’ from the circulation, again decreased macrophage accumulation near the site of injury followed by circulation-based or resident macrophages (Sadik et al., (supplementary material Fig. S14). Furthermore, Wnt inhibition 2011; Yoo and Huttenlocher, 2011; Li et al., 2012). Cell tracking data decreased the density of proliferating macrophages (5 dpa) in indicate that activated neutrophils are circulation derived, whereas the regenerating area (Fig. 7E,F; supplementary material Fig. S12). most macrophages are resident in the fin, in contrast to both larval Subsequent gene profiling of macrophages sorted from tissue zebrafish and mammalian appendages. Macrophage accumulation DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 Fig. 6. Wnt/β-catenin signaling by non-leukocytes affects the injury environment in regenerating fins. (A) Representative images detailing cells undergoing Wnt/β-catenin signaling (siam , red) for proximal and distal fin resections in Tg(TCFsiam:mCherry) fish. Siam cell number is increased in proximal cuts. 4 dpa, *P=0.0329; 7 dpa, *P=0.0296 (two-tailed t-test, error bars indicate s.e.m.). (B) Gene expression levels (4 dpa) of pooled blastemal fin tissue (n>5) as assessed by qRT- PCR for wild-type and for the Tg(hsDKK1:GFP) loss-of- function and Wnt8a (hsWnt8a:GFP) gain-of-function Wnt/β-catenin signaling fish lines. Levels were normalized to fold over non-heat shock control. Data were averaged over two separate experiments. One group included daily heat shock following amputation; the other group included a single heat shock pulse at 84 hpa with tissue extraction 12 h later at 4 dpa. mpx is mpo. (C) Representative images of distal resections from Tg(mpo:GFP; TCFsiam:mCherry) fish and Tg(mpeg1:NTR-YFP; TCFsiam:mCherry) fish at 6 dpa. Little colocalization is evident between neutrophils + + (mpo ) and siam cells. Scale bar: 40 µm; 100 µm in bottom panel. (D) Quantification of flow cytometry sorted cells from pooled resected fins (n=8) from Tg(mpo:GFP; TCFsiam:mCherry) fish indicating the presence of few + + mpo siam cells. (E) Quantification of flow cytometry sorted cells from pooled resected fins (n=7) from Tg (mpeg1:NTR-eYFP; TCFsiam:mCherry) fish indicating + + the presence of few mpeg1 siam cells. (D,E) Error bars indicate s.e.m. of the average of three experiments. mainly occurred after the blastema formation stage, suggesting that occurred after the tissue outgrowth phase (>3 dpa). These data zebrafish macrophages respond to events well after the wound healing advocate a model whereby spatially close resident macrophages phase of fin regeneration. Therefore, we describe a fast-moving and modulate events initially, but during later regenerative stages either fast-responding neutrophil population and a correspondingly slow- newly proliferated macrophages or slowly migrating macrophages moving resident macrophage population in adult zebrafish. affect the regenerative response in a different manner than the early We present evidence that macrophages may have differential macrophage population. Cataloguing the composition of this stage-dependent effects on the extent of tail fin regeneration. population over the injury timecourse using single-cell lineage Although mammalian macrophages serve unique, specific functions tracing or Brainbow technology would be useful to delineate the at distinct phases during tissue repair (Liu et al., 1999; Lucas et al., level of macrophage heterogeneity. 2010), zebrafish macrophages seem to function differently at In contrast to recent evidence that neutrophil deficiency analogous stages after wounding. Whereas in mice macrophage (neutropenia) increases the regeneration rate in larval fins (Li et al., depletion during tissue outgrowth can result in severe hemorrhage in 2012), ourcreation of a neutropenic environment in adult zebrafish did the wound (Mirza et al., 2009), ablation during tissue outgrowth in not affect the fin regeneration rate. Moreover, it is unlikely that zebrafish only affects fin patterning, not growth. Moreover, neutrophils have an inhibitory effect on regeneration because although macrophage depletion has not been found to negatively neutrophils accumulated in markedly greater numbers in faster affect wound closure rates and endothelial repair in mammals (Dovi regenerating tissue throughout the regenerative process. Since et al., 2003; Martin and Feng, 2009; Evans et al., 2013), neutrophils may either promote or inhibit wound healing and tissue macrophage depletion reduced tissue growth in adult zebrafish. repair in mice depending on the tissue and injury context (Dovi et al., We also found no evidence that zebrafish macrophages modulate 2003; Harty et al., 2010; Marrazzo et al., 2011; Rieger et al., 2012), neutrophil recruitment or resolution, whereas macrophages have neutrophil function in zebrafish might be highly injury- and time- been found to modulate these cellular responses in mouse limb dependent. Given the proven utility of the genetic macrophage wounds (Cailhier et al., 2006). These data provide further ablation model in this study, the creation of a similar mpo- and/or lyzC- justification for the view that macrophages have different roles driven ablation fish would more conclusively clarify the supportive or after appendage injury in mammals versus adult zebrafish. reductive role of neutrophils in various regenerative contexts. This study supports the existence of either (1) a single We further establish that Wnt/β-catenin signaling partially macrophage population that has different roles in the regenerative modulates the time frame and degree of leukocyte response in tail course over time, or (2) multiple, functionally distinct macrophage fin regeneration. Wnt/β-catenin signaling inhibition ‘arrested’ the cell populations, similar to in mammals. It is also possible that other and cytokine environment at a stage similar to the early injury myeloid-like cells might migrate from non-fin sites over the course environment. Importantly, this effect was still observed when Wnt/β- of injury, although rapid macrophage movement was not observed catenin signaling was impaired after the initial regenerative events had either in vasculature or interstitial tissue. Macrophages mainly begun, supporting a more direct role of Wnt signaling in determining exerted effects on tissue growth during the initial regenerative macrophage movement. Active Wnt signaling might mitigate early stages, but aberrant phenotypes, including impaired bony ray stage inflammation and act as a molecular switch to proceed to later patterning and bone formation, were still observed when depletion stages of the immune response (neutrophil resolution/macrophage DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 Fig. 7. Wnt/β-catenin signaling regulates leukocyte response to injury. (A) The loss-of-function Wnt/β-catenin signaling line Tg(hsDKK1:GFP) crossed to the Tg(mpeg1:mCherry) line was used to track macrophages after Wnt modulation. Resected wild-type or loss-of-function Wnt/β-catenin signaling (hsDKK) fins received a proximal cut and a distal cut. Representative images are shown of macrophage accumulation through 12 dpa. Fluorescent images were acquired and converted to grayscale for ease of visualization. (B) Macrophage accumulation was reduced in DKK1-overexpressing fins at every time point from 3 dpa until 14 dpa and no significant difference in macrophage number was observed between proximal and distal resections. Data are representative of at least three independent experiments with at least six to eight fish per time point. HsDKK-PROX versus hsWT-PROX, WT-PROX: 6 dpa, *P=0.0083; 8 dpa, *P=0.0072; 12 dpa, P=0.0175. HsDKK-DIST versus WT-DIST, WT-DIST; 6 dpa, **P=0.0140; 8 dpa, **P=0.0195; 12 dpa, **P=0.0361; two-tailed t-test. (C) Tg(hsDKK1:GFP) was crossed to a neutrophil promoter-driven Tg(lyzC:dsRed) line in order to visualize neutrophil accumulation following Wnt inhibition. Representative images indicate that neutrophil accumulation remains elevated longer in DKK1-overexpressing fins compared with wild-type controls. (D) Neutrophil accumulation was higher in DKK1-overexpressing fins compared with wild-type controls after 5 dpa. Data are representative of three independent experiments with at least six to eight fish per time point/condition. hsDKK1 versus hsWT, WT: 6 dpa, *P=0.0075; 8 dpa, *P=0.0112; 10 dpa, *P=0.0105; two-tailed t-test. (E) Proliferation of wild- type and DKK1-overexpressing regenerates at 5 dpa as assessed by anti-PCNA (red), anti-L-plastin (green) and DAPI (blue) staining. Red arrowheads indicate + + original cut site; white arrowheads indicate double-stained (PCNA LP ) cells. The boxed regions are magnified beneath. (F) Proliferating macrophages as a percentage of total cells and total macrophages (LP cells). Numbers were averaged over at least seven sections of each sample. Data are representative of three independent experiments (n>5). hsDKK1 versus hsWT: *P=0.0475; **P=0.0349 (two-tailed t-test, error bars indicate s.e.m.). Scale bars: 200 µm in A; 300 µm in C; 20 µm in E. DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 by bacterial nitroreductase (NTR) was described previously (Curado et al., enrichment). This idea shares similarities with the situation in 2007). A DNA fragment containing EYFP-NTR was subcloned into a Tol2 mammals, in which timely neutrophil removal (resolution) after injury vector that contained the zebrafish mepg1 promoter (Ellett et al., 2011). The is essential to the termination of inflammation – delayed apoptosis or Tol2 construct and transposase RNA were microinjected into 1- to 4-cell impaired clearance of neutrophils can aggravate and prolong tissue stage embryos and the transgenic line was isolated by the specific injury (Sadik et al., 2011). The idea that Wnt/β-catenin signaling may expression of YFP in macrophages in the next generation. Tg(hsDKK1: restrict several aspects of inflammation is supported in several GFP;mpeg1:mCherry), Tg(hsWnt8a:GFP;mpeg1:mCherry), Tg(7xTCF- ia5 mammalian models of disease and injury. For example, high Dkk1 Xla.Siam:nlsmCherry;mpo:GFP) (Moro et al., 2012), Tg(lyzC:dsRed; activity is associated with pro-inflammatory bone loss in mouse mpo:GFP) and Tg(mpeg1:NTR-EYFP;7xTCF-Xla.Siam:nlsmCherry) fish myelomas (Tian et al., 2003), and inhibition of Dkk1 activity in a were made by crossing individual transgenic homozygotes with the mouse model of rheumatoid arthritis results in greater bone formation corresponding transgenic complement. (Diarra et al., 2007). The role of Wnt/β-catenin signaling in modulating the injury response might indeed be similarly context- Adult zebrafish fin amputation surgeries Zebrafish of ∼6-12 months of age were used for all studies. Fin amputation specific in zebrafish; further study in other anatomical injury models surgeries were performed as previously described (Stoick-Cooper et al., would be beneficial in this context. 2007a,b). Two amputation cut schemes were employed: (1) a single cut was The cellular basis of the effects of Wnt signaling on inflammation made traversing the entire dorsoventral length of the caudal fin in each fish; is unclear, in part because cells responding to Wnt ligands had or (2) two separate cuts were made on each fish, one closer to the body of the remained unidentified until very recently (Wehner et al., 2014); it fish (‘proximal’, ventral) and one further away from the body (‘distal’, was determined that a population of actinotrichia-forming cells and dorsal) (Lee et al., 2005). osteoblast progenitors undergo Wnt signaling during blastemal specification, regulating epidermal patterning and osteoblast Live image analysis differentiation indirectly through secretion of factors. Given that The injured adult zebrafish were anesthetized as previously described with Wnt/β-catenin signaling inhibition eliminated the differential Tricaine (Stoick-Cooper et al., 2007a,b), placed on their side and imaged positional memory aspect of macrophage recruitment, and that under a Nikon TiE inverted widefield fluorescence high-resolution microscope. Full fin images were assembled from 30-50 stitched images delayed inhibition reduced longer term migration, it is likely that (20×) encompassing the entire fin, with the fish under constant anesthetization. Wnt/β-catenin signaling also indirectly affects macrophage Live fin images were taken for each fish periodically post amputation. phenotype and activity through a similar regulation of secretion factors. Additionally, the similar accumulation patterns of Wnt- + Analysis of cell density in the injured area of amputated fins responsive cells (siam ) and neutrophils/macrophages suggests that To ascertain the timecourse of cell recruitment to the fin injury area, a measure both inflammatory and Wnt signaling cells might respond to the of cell density near the resected fin edge was utilized. An ‘injured area’ was same injury signals. This idea is further supported by the fact that defined as the area spanning two set dimensions: one dimension being the amputating more proximally also involves the damage of a greater distal-ventral boundary of the fin; the other dimension being defined as from volume of tissue and, therefore, may result in more robust levels of perpendicular to the distal-ventral axis, one-quarter of the fin length proximal paracrine ‘injury signals’, including H O , redox and the Src family 2 2 to the original amputation plane. Using Image-Pro software (Media kinase Lyn, all previously identified in zebrafish (Pase et al., 2012; Cybernetics), the total fluorescence intensity (TFI) from promoter-driven Yoo et al., 2012; Niethammer et al., 2009). Wnt-responding cells in fluorescent cells in the injury area from fin images at each time point was quantified. The TFI was normalized to the pixel area of the injured area for that mammals have recently been linked to modulating angiogenic fin to obtain a measure of cell density in the injured area. This analysis was factors, which can in turn affect the injury response (Kitajewski, used based on the assumption that the fluorescence intensity of each labeled 2003); examining whether Wnt inhibition and macrophage depletion cell was similar on average in each fish as verified by flow cytometry. regulate angiogenesis might shed more light on their mechanistic effects on inflammation and regeneration. Identifying which Wnt- Fin regeneration measurements modulated signals directly affect macrophage proliferation, cytokine Total regeneration was gauged by a percent regeneration metric. Briefly, this release and migration would assist in further developing this measurement required phase-contrast full-fin images be taken before mechanistic insight into how Wnt/β-catenin signaling modulates amputation and at each time point after amputation. The full area (in inflammation and regeneration. pixels) of the caudal fin, from the proximal end of the fin rays to the distal fin Our findings detail the cellular events in the normal injury edge/cut, was quantified from the pre-amputation images for each fish using response during zebrafish epimorphic regeneration. We reveal that ImageJ (NIH). The new tissue area, from the new distal fin edge to the macrophages regulate aspects of appendage regeneration in adult amputation plane, was also quantified. Percent regeneration for each fin at each time point was defined as: % regeneration=100×(new tissue area/ zebrafish. We also provide evidence that Wnt/β-catenin signaling original fin area amputated). may in turn modulate cellular and biochemical inflammatory events during the regenerative process. Our findings, coupled with Macrophage ablation recent research detailing pro-repair roles of inflammatory cells in For all macrophage ablation experiments, Tg(mpeg1:NTR-eYFP) fish were zebrafish brain regeneration, advocate some degree of anatomical housed in static tanks of fish water (five fish/liter) supplemented with or conservation of the role of injury components in regenerative without 2.5 mM metronidazole (MTZ) for the duration of the experiment. process in zebrafish. Finally, macrophages may indeed form part of During ablation experiments, fish were kept on a 12 h light/12 h dark cycle, a cellular bridge between robustly regenerative organisms such as since MTZ is sensitive to long exposure to light. Water was changed daily zebrafish and the less regenerative mammals that could potentially and fresh MTZ was added daily. Two control groups were used: NTR be manipulated for mammalian regenerative therapies. transgenic fish housed in fish water without MTZ, and wild-type fish housed in fish water with MTZ (2.5 mM) under the same daily light/dark cycle. MATERIALS AND METHODS Transgenic lines Flow cytometry and sorting w202 + + + The Tg(mpeg1:NTR-EYFP) line was created using the Tol2 Flow cytometry and partial FACS analysis to isolate siam , mpo , mpeg1 , + + lyzC and YFP (NTR+) cells from various transgenic fish was performed transposon system (Urasaki et al., 2006). Targeted cell ablation mediated DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 Author contributions beginning with isolation of the injured area fin tissue. Once isolated, this T.A.P. and N.S.S. conducted the experiments, T.A.P. analyzed the data, T.A.P., tissue was immediately placed in a tissue disassociation solution of 2 mg/ml J.S.R. and R.T.M. designed the experiments and wrote the paper; and C.T.-Y. collagenase (Sigma-Aldrich) and 0.3 mg/ml protease (type XIV, Sigma- assisted in the generation of the mpeg1:NTR-YFP line. Aldrich) in Hanks solution. The solution was moderately shaken at 30°C for 1 h with gentle trituration performed every 10 min with an 18 gauge needle. Funding After 1 h, the solution was incubated for 5 min in 0.05% trypsin in PBS. T.A.P. and J.S.R. were supported by postdoctoral fellowships from the Howard Before flow cytometry, disassociated cells were washed in 2% (fetal bovine Hughes Medical Institute. C.T.-Y. was supported by a postdoctoral fellowship from serum) FBS in cell disassociation solution. Disassociated cells from wild- the Taiwan National Science Council [NSC97-2917-I-564-109] and his contribution type fish at an identical time point were used to set up the lower limit to this work is also supported by National Institutes of Health (NIH) RO1 grants (background) of fluorescence in each experiment. For cleaved caspase 3 [AI54503 and AI036396] to Lalita Ramakrishnan at the University of Washington. analysis, caspase 3 antibody (Sigma-Aldrich, AV00021; 1:200 in 2% FBS) N.S.S. was supported by a T32 grant [GM00727] and a P01 grant [GM081619] from the National Institutes of Health (NIH). R.T.M. is an investigator of the Howard was added to the suspension for 30 min on ice. After three successive Hughes Medical Institute, which supported this research. Deposited in PMC for washes with 2% FBS, fluorescently labeled secondary antibody was added immediate release. (Alexa Fluor 647, Gt anti-mouse IgG; Life Technologies, A21236; 1:1000) for 20 min on ice. After three further washes (the last including Supplementary material 1:600 DAPI), the suspension was strained and read. Supplementary material available online at http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.098459/-/DC1 Immunohistochemistry Whole adult fin stumps (encompassing the entire fin plus 1-2 mm of the References body girdle) were harvested and fixed in 4% formaldehyde in PBS overnight Baker-LePain, J. C., Nakamura, M. C. and Lane, N. E. (2011). Effects of inflammation on bone: an update. Curr. Opin. Rheumatol. 23, 389-395. at 4°C. Tissue was then washed for 30 min at room temperature with 5% Brancato, S. K. and Albina, J. E. (2011). Wound macrophages as key regulators of sucrose in PBS, followed by two washes for 1 h each in 5% sucrose in PBS, repair: origin, phenotype, and function. Am. J. Pathol. 178, 19-25. and an overnight wash in 30% sucrose in PBS at 4°C. After another Cailhier, J. F., Sawatzky, D. A., Kipari, T., Houlberg, K., Walbaum, D., Watson, S., overnight wash in a 1:1 ratio of 30% sucrose:100% O.C.T. compound Lang, R. A., Clay, S., Kluth, D., Savill, J. et al. (2006). Resident pleural (Tissue-Tek, VWR #25608-930) at 4°C, the tissue was embedded directly in macrophages are key orchestrators of neutrophil recruitment in pleural inflammation. 100% OCT in embedding wells and stored at −80°C before sectioning. Am. J. Respir. Crit. Care Med. 173, 540-547. Embedded tissue was sectioned in a cryostat and the entire dorsoventral span Chen, C.-F., Chu, C.-Y., Chen, T.-H., Lee, S.-J., Shen, C.-N. and Hsiao, C.-D. of the fin cut into 14 µm transverse sections and adhered to Superfrost Plus (2011). Establishment of a transgenic zebrafish line for superficial skin ablation slides (VWR) overnight at 40°C. and functional validation of apoptosis modulators in vivo. PLoS ONE 6, e20654. Colucci-Guyon, E., Tinevez, J.-Y., Renshaw, S. A. and Herbomel, P. (2011). Rabbit L-plastin antibody (a gift of Anna Huttenlocher; 1:300) or PCNA Strategies of professional phagocytes in vivo: unlike macrophages, neutrophils antibody (Sigma-Aldrich, P8825; 1:250) were added in antibody solution engulf only surface-associated microbes. J. Cell Sci. 124, 3053-3059. (0.5% Triton X-100, 5% goat serum, 0.2% BSA in PBS) for 2 h at room Curado, S., Anderson, R. M., Jungblut, B., Mumm, J., Schroeter, E. and temperature in the dark. Slides were washed six times for 15 min each in Stainier, D. Y. R. (2007). Conditional targeted cell ablation in zebrafish: a new tool antibody solution with gentle shaking, and goat anti-rabbit Alexa Fluor 647 for regeneration studies. Dev. Dyn. 236, 1025-1035. secondary antibody (Life Technologies, A21244; 1:1000) added for 2 h at Deng, Q., Harvie, E. A. and Huttenlocher, A. (2012). Distinct signalling room temperature in the dark. After six more washes in antibody solution the mechanisms mediate neutrophil attraction to bacterial infection and tissue slides were sealed with a coverslip with Prolong Gold Antifade Reagent injury. Cell Microbiol. 14, 517-528. Diarra, D., Stolina, M., Polzer, K., Zwerina, J., Ominsky, M. S., Dwyer, D., Korb, (Life Technologies). EdU staining was performed according to Click-iT A., Smolen, J., Hoffmann, M., Scheinecker, C. et al. (2007). Dickkopf-1 is a assays (Life Technologies, C10428). EdU was added 6 h prior to tissue master regulator of joint remodeling. Nat. Med. 13, 156-163. extraction at 14 dpa. Dovi, J. V., He, L.-K. and DiPietro, L. A. (2003). Accelerated wound closure in For calcein-AM fluorochrome labeling, fish were immersed in 0.05% neutrophil-depleted mice. J. Leukoc. Biol. 73, 448-455. calcein-AM (Life Technologies, C3099) and rinsed for 10 min in fresh Echeverri, K., Clarke, J. D. W. and Tanaka, E. M. (2001). In vivo imaging indicates water. For analysis, the midpoint coordinates for all regenerated bone muscle fiber dedifferentiation is a major contributor to the regenerating tail segments were manually identified and the corresponding calcein intensities blastema. Dev. Biol. 236, 151-164. were used to compute mean intensity (μ), standard deviation of intensity (σ), Ellett, F., Pase, L., Hayman, J. W., Andrianopoulos, A. and Lieschke, G. J. (2011). mpeg1 promoter transgenes direct macrophage-lineage expression in and coefficient of variation (σ/μ) for each fish. zebrafish. Blood 117, e49-e56. Evans, M. A., Smart, N., Dubé, K. N., Bollini, S., Clark, J. E., Evans, H. G., Taams, Quantitative RT-PCR L. S., Richardson, R., Lévesque, M., Martin, et al. (2013). Thymosin Total RNA was extracted from zebrafish fin regenerates using TRIZOL β4-sulfoxide attenuates inflammatory cell infiltration and promotes cardiac according to the manufacturer’s protocol (Invitrogen). Tissue incorporating wound healing. Nat. Commun. 4, 2081. all new tissue as well as one or two bone rays proximal to the original cut site French, D. M., Kaul, R. J., D’Souza, A. L., Crowley, C. W., Bao, M., Frantz, G. D., Filvaroff, E. H. and Desnoyers, L. (2004). WISP-1 is an osteoblastic regulator was extracted. Equal amounts of total RNA from each sample were reverse expressed during skeletal development and fracture repair. Am. J. Pathol. 165, transcribed with Thermoscript reverse transcriptase (Invitrogen) using oligo 855-867. (dT) and random hexamer primers. All levels were normalized to β-actin Goldsmith, J. R. and Jobin, C. (2012). Think small: zebrafish as a model system of (18S levels were similar) and fold induction was calculated by setting human pathology. J. Biomed. Biotech. 2012, 817341. control conditions to 1. Primers are listed in supplementary material Hall, C., Flores, M. V., Storm, T., Crosier, K. and Crosier, P. (2007). The zebrafish Table S1. lysozyme C promoter drives myeloid-specific expression in transgenic fish. BMC Dev. Biol. 7, 42. Acknowledgements Han, M., Yang, X., Taylor, G., Burdsal, C. A., Anderson, R. A. and Muneoka, K. We thank Jerry Ament, Stanley Kim and Jeanot Muster for fish care and technical (2005). Limb regeneration in higher vertebrates: developing a roadmap. Anat. assistance; Jeff Mumm from Georgia Health Science University (Augusta, USA) for Rec. B New. Anat. 287B, 14-24 providing a construct containing the eYFP-NTR fragment; Graham Lieschke at the Harty, M. W., Muratore, C. S., Papa, E. F., Gart, M. S., Ramm, G. A., Gregory, S. H. Australian Regenerative Medicine Institute (Melbourne, Australia) for providing DNA and Tracy, T. F., Jr (2010). Neutrophil depletion blocks early collagen degradation containing mpeg1; Francesco Argenton at the University of Padova (Padova, Italy) in repairing cholestatic rat livers. Am. J. Pathol. 176, 1271-1281. for the Tg(TCFsiam:mCherry) line; and Anna Huttenlocher at the University of Herbomel, P., Thisse, B. and Thisse, C. (1999). Ontogeny and behavior of early Wisconsin-Madison for providing L-plastin antibody. macrophages in the zebrafish embryo. Development 126, 3735-3745. Ito, M., Yang, Z., Andl, T., Cui, C., Kim, N., Millar, S. E. and Cotsarelis, G. (2007). Competing interests Wnt-dependent de novo hair follicle regeneration in adult mouse skin after The authors declare no competing financial interests. wounding. Nature 447, 316-320. DEVELOPMENT RESEARCH ARTICLE Development (2014) 141, 2581-2591 doi:10.1242/dev.098459 Kawakami, Y., Rodriguez Esteban, C., Raya, M., Kawakami, H., Marti, M., Niethammer, P., Grabher, C., Look, A. T. and Mitchison, T. J. (2009). A tissue- scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish. Dubova, I. and Izpisua Belmonte, J. C. (2006). Wnt/beta-catenin signaling regulates vertebrate limb regeneration. Genes Dev. 20, 3232-3237. Nature 459, 996-999. Novoa, B. and Figueras, A. (2012). Zebrafish: model for the study of inflammation Kintner, C. R. and Brockes, J. P. (1984). Monoclonal antibodies identify blastemal cells derived from dedifferentiating muscle in newt limb regeneration. Nature 308, and the innate immune response to infectious diseases. Adv. Exp. Med. Biol. 946, 253-275. 67-69. Pase, L., Layton, J. E., Wittmann, C., Ellett, F., Nowell, C. J., Reyes-Aldasoro, C. C., Kitajewski, J. (2013). Wnts heal by restraining angiogenesis. Blood 121, Varma, S., Rogers, K. L., Hall, C. J., Keightley, M. C. et al. (2012). Neutrophil- 2381-2382. delivered myeloperoxidase dampens the hydrogen peroxide burst after tissue Knopf, F., Hammond, C., Chekuru, A., Kurth, T., Hans, S., Weber, C. W., wounding in zebrafish. Curr. Biol. 22, 1818-1824. Mahatma, G., Fisher, S., Brand, M., Schulte-Merker, S. et al. (2011). Bone Porrello, E. R., Mahmoud, A. I., Simpson, E., Hill, J. A., Richardson, J. A., regenerates via dedifferentiation of osteoblasts in the zebrafish fin. Dev. Cell 20, Olson, E. N. and Sadek, H. A. (2011). Transient regenerative potential of the 713-724. neonatal mouse heart. Science 331, 1078-1080. Koch, S., Nava, P., Addis, C., Kim, W., Denning, T. L., Li, L., Parkos, C. A. and Poss, K. D., Shen, J. and Keating, M. T. (2000). Induction of lef1 during zebrafish Nusrat, A. (2011). The Wnt antagonist Dkk1 regulates intestinal epithelial fin regeneration. Dev. Dyn. 219, 282-286. homeostasis and wound repair. Gastroenterology 141, 259-268. e8. Redd, M. J., Kelly, G., Dunn, G., Way, M. and Martin, P. (2006). Imaging Kyritsis, N., Kizil, C., Zocher, S., Kroehne, V., Kaslin, J., Freudenreich, D., macrophage chemotaxis in vivo: studies of microtubule function in zebrafish Iltzsche, A. and Brand, M. (2012). Acute inflammation initiates the regenerative wound inflammation. Cell Motil. Cytoskeleton 63, 415-422. response in the adult zebrafish brain. Science 338, 1353-1356. Ren, S., Johnson, B. G., Kida, Y., Ip, C., Davidson, K. C., Lin, S.-L., Kobayashi, A., Lee, Y., Grill, S., Sanchez, A., Murphy-Ryan, M. and Poss, K. D. (2005). Fgf Lang, R. A., Hadjantonakis, A.-K., Moon, R. T. et al. (2013). LRP06 is a signaling instructs position-dependent growth rate during zebrafish fin coreceptor for multiple fibrogenic signaling pathways in pericytes and myofibroblasts regeneration. Development 132, 5173-5183. that are inhibited by DKK-1. Proc. Natl. Acad. Sci. U.S.A. 110, 1440-1445. Leibovich, S. J. and Ross, R. (1975). The role of macrophage in wound repair: a Renshaw, S. A. and Trede, N. S. (2012). A Model 450 million years in the making: study with hydrocortisone and antimacrophage serum. Am. J. Pathol. 78, 71-100. zebrafish and vertebrate immunity. Dis. Mod. Mech. 5, 38-47. Li, L., Yan, B., Shi, Y.-Q., Zhang, W.-Q. and Wen, Z.-L. (2012). Live imaging Renshaw, S. A., Loynes, C. A., Trushell, D. M. I., Elworthy, S., Ingham, P. W. and reveals differing roles of macrophages and neutrophils during zebrafish tail fin Whyte, M. K. B. (2006). A transgenic zebrafish model of neutrophilic regeneration. J. Biol. Chem. 287, 25353-25360. inflammation. Blood 108, 3976-3978. Lieschke, G. J., Oates, A. C., Crowhurst, M. O., Ward, A. C. and Layton, J. E. Rieger, A. M., Konowalchuk, J. D., Grayfer, L., Katzenback, B. A., Havixbeck, J. J., (2011). Morphologic and functional characterization of granulocytes and Kiemele, M. D., Belosevic, M. and Barreda, D. R. (2012). Fish and mammalian macrophages in embryionic and adult zebrafish. Blood 98, 3087-3096. phagocytes differentially regulate pro-inflammatory and homeostatic responses in Liu, Y., Cousin, J. M., Hughes, J., Van Damme, J., Seckl, J. R., Haslett, C., vivo. PLoS ONE 7, e47070. Dransfield, I., Savill, J. and Rossi, A. G. (1999). Glucocorticoids promote Sadik, C. D., Kim, N. D. and Luster, A. D. (2011). Neutrophils cascading their way nonphlogistic phagocytosis of apoptotic leukocytes. J. Immunol. 162, 3639-3646. to inflammation. Trends Immunol. 32, 452-460. Loynes, C. A., Martin, J. S., Robertson, A., Trushell, D. M. I., Ingham, P. W., Seifert, A. W., Kiama, S. G., Seifert, M. G., Goheen, J. R., Palmer, T. M. and Whyte, M. K. B. and Renshaw, S. A. (2010). Pivotal advance: pharmacological Maden, M. (2012). Skin shedding and tissue regeneration in African spiny mice manipulaton of inflammation resolution during spontaneously resolving tissue (Acomys). Nature 489, 561-565. neutrophilia in the zebrafish. J. Leukoc. Biol. 87, 203-212. Serhan, C. N. and Savill, J. (2005). Resolution of inflammation: the beginning Lucas, T., Waisman, A., Ranjan, R., Roes, J., Krieg, T., Mü ller, W., Roers, A. and programs the end. Nat. Immunol. 6, 1191-1197. Eming, S. A. (2010). Differential roles of macrophages in diverse phases of skin Sica, A. and Mantovani, A. (2012). Macrophage plasticity and polarization: in vivo repair. J. Immunol. 184, 3964-3977. veritas. J. Clin. Invest. 122, 787-795. Marrazzo, G., Bellner, L., Halilovic, A., Li Volti, G., Drago, F., Dunn, M. W. and Singh, S. P., Holdway, J. E. and Poss, K. D. (2012). Regeneration of amputated Schwartzman, M. L. (2011). The role of neutrophils in corneal wound healing in zebrafish fin rays from de novo osteoblasts. Dev. Cell 22, 879-886. HO-2 null mice. PLoS ONE 6, e21180. Stoick-Cooper, C. L., Moon, R. T. and Weidinger, G. (2007a). Advances in Martin, P. and Feng, Y. (2009). Inflammation: wound healing in zebrafish. Nature signaling in vertebrate regeneration as a prelude to regenerative medicine. Genes 459, 921-923. Dev. 21, 1292-1315. Mathias, J. R., Perrin, B. J., Liu, T.-X., Kanki, J., Look, A. T. and Huttenlocher, A. Stoick-Cooper, C. L., Weidinger, G., Riehle, K. J., Hubbert, C., Major, M. B., (2006). Resolution of inflammation by retrograde chemotaxis of neutrophils in Fausto, N. and Moon, R. T. (2007b). Distinct Wnt signaling pathways have transgenic zebrafish. J. Leukoc. Biol. 80, 1281-1288. opposing roles in appendage regeneration. Development 134, 479-489. Mathias, J. R., Dodd, M. E., Walters, K. B., Rhodes, J., Kanki, J. P., Look, A. T. Tian, E., Zhan, F., Walker, R., Rasmussen, E., Ma, Y., Barlogie, B. and and Huttenlocher, A. (2007). Live imaging of chronic inflammation casued by Shaughnessy, J. D., Jr (2003). The role of the Wnt-signaling antagonist DKK1 in mutation of zebrafish Hai1. J. Cell Sci. 120, 3372-3383. the development of osteolytic lesions in multiple myeloma. N. Engl. J. Med. 349, Mathias, J. R., Dodd, M. E., Walters, K. B., Yoo, S. K., Ranheim, E. A. and 2483-2494. Huttenlocher, A. (2009). Characterization of zebrafish larval inflammatory Urasaki, A., Morvan, G. and Kawakami, K. (2006). Functional dissection of the macrophages. Dev. Comp. Immunol. 33, 1212-1217. Tol2 transposable element identified the minimal cis-sequence and a highly Matzelle, M. M., Gallant, M. A., Condon, K. W., Walsh, N. C., Manning, C. A., repetitive sequence in the subterminal region essential for transposition. Genetics Stein, G. S., Lian, J. B., Burr, D. B. and Gravallese, E. M. (2012). Resolution of 174, 639-649. inflammation induces osteoblast function and regulates the Wnt signaling Volkman, H. E., Pozos, T. C., Zheng, J., Davis, J. M., Rawls, J. F. and pathway. Arthritis. Reum. 64, 1540-1550. Ramakrishnan, L. (2010). Tuberculous granuloma induction via interaction of a Mirza, R., DiPietro, L. A. and Koh, T. J. (2009). Selective and specific macrophage bacterial secreted protein with host epithelium. Science 327, 466-469. ablation is detrimental to wound healing in mice. Am. J. Pathol. 175, 2454-2462. Walters, K. B., Dodd, M. E., Mathias, J. R., Gallagher, A. J., Bennin, D. A., Moro, E., Ozhan-Kizil, G., Mongera, A., Beis, D., Wierzbicki, C., Young, R. M., Rhodes, J., Kanki, J. P., Look, A. T., Grinblat, Y. and Huttenlocher, A. (2009). Bournele, D., Domenichini, A., Valdivia, L. E., Lum, L. et al. (2012). In vivo Wnt Muscle degeneration and leukocyte infiltration caused by mutation of zebrafish signaling tracing through a transgenic biosensor fish reveals novel activity Fad24. Dev. Dyn. 238, 86-99. domains. Dev. Biol. 366, 327-340. Wehner, D., Cizelsky, W., Vasudevaro, M. D., Ozhan, G., Haase, C., Kagermeier- Morrison, J. I., Lö ö f, S., He, P. and Simon, A. (2006). Salamander limb Schenk, B., Rö der, A., Dorsky, R. I., Moro, E., Argenton, F. et al. (2014). Wnt/ regeneration involves the activation of a multipotent skeletal muscle satellite cell β-catenin signaling defines organizing centers that orchestrate growth and population. J. Cell Biol. 172, 433-440. differentiation of the regenerating zebrafish caudal fin. Cell Rep. 6, 467-481. Murray, P. J. and Wynn, T. A. (2011). Obstacles and opportunities for understanding Whyte, J. L., Smith, A. A. and Helms, J. A. (2012). Wnt signaling and injury repair. macrophage polarization. J. Leukoc. Biol. 89, 557-563. Cold Spring Harb. Perspect. Biol. 4, a008078. Nachtrab, G., Kikuchi, K., Tornini, V. A. and Poss, K. D. (2013). Transcriptional Yoo, S. K. and Huttenlocher, A. (2011). Spatiotemporal photolabeling of neutrophil components of anteroposterior positional information during zebrafish fin trafficking during inflammation in live zebrafish. J. Leukoc. Biol. 89, 661-667. regeneration. Development 140, 3754-3764. Yoo, S. K., Freisinger, C. M., LeBert, D. C. and Huttenlocher, A. (2012). Early Newman, A. C. and Hughes, C. C. W. (2012). Macrophages and angiogenesis: a redox, Src family kinase, and calcium signaling integrate wound responses and role for Wnt signaling. Vasc. Cell 4, 13. tissue regeneration in zebrafish. J. Cell Biol. 199, 225-234. DEVELOPMENT

Journal

Development – The Company of Biologists

Published: Jul 1, 2014

There are no references for this article.