Transient inflammatory response mediated by interleukin-1β is required for proper regeneration in zebrafish fin fold
Progress in regenerative medicine depends on unraveling the mechanisms underlying tissue and organ regeneration . The zebrafish is a powerful model species for investigation of regeneration mechanisms because it exhibits high regenerative capacity . Zebrafish can regenerate complex structures such as fins , heart , brain , retina , and other tissues ( Gemberling et al . , 2013 ) . The caudal fin of zebrafish , in particular , has been used as a model for analyzing epimorphic regeneration , a type of regeneration in the urodele limb and fish fin ( Poss et al . , 2003 ) . During epimorphic regeneration , two characteristic tissues , the wound epidermis and the blastema , are induced in response to tissue amputation , and their coordinated actions regulate cell proliferation and morphogenesis and thus lead to tissue regeneration . Studies conducted using the caudal fin regeneration model have identified numerous genes and molecular signaling pathways that are critical for fin regeneration ( Yoshinari and Kawakami , 2011; Wehner and Weidinger , 2015 ) . In addition to the classical regeneration model developed using the adult caudal fin , a zebrafish larval fin fold model has been developed ( Kawakami et al . , 2004; Mateus et al . , 2012 ) , allowing identification of additional genes and signaling pathways required for regeneration ( Mathew et al . , 2009; Ishida et al . , 2010; Yoo et al . , 2012 ) . Specifically , the fin fold regeneration model has facilitated genetic analysis of the regeneration mechanism by exploiting a number of mutant zebrafish resources ( Rojas-Muñoz et al . , 2009; Yoshinari et al . , 2009 ) , because several zebrafish lethal mutants survive beyond 7 days post fertilization ( dpf ) —an adequate period for assaying tissue regeneration—because of nutrient supply from the yolk . Previously , we reported that zebrafish mutants , cloche ( clo ) ( Stainier et al . , 1995; Reischauer et al . , 2016 ) and tal1/scl ( Bussmann et al . , 2007 ) , showed a unique regenerative defect: these mutants could not regenerate their fin fold because of apoptosis of the regenerative cells ( Hasegawa et al . , 2015 ) . Our analyses of the clo mutant revealed that a diffusible survival factor from myeloid-lineage cells is required to prevent apoptosis of the primed regenerative cells . That study suggested that the regenerative cells are sensitive to apoptosis during tissue regeneration , but the mechanism of this sensitization and the identity of the survival factor remain unknown . Myeloid cells are widely recognized to play a role in protection against pathogens and microorganisms after tissue injury . Neutrophils are first recruited to sites of inflammation , and these cells encounter pathogens and phagocytose and kill microorganisms by producing reactive oxygen species and/or antibacterial proteins ( Kolaczkowska and Kubes , 2013 ) . Subsequently , macrophages infiltrate the inflammation site , produce pro- or anti-inflammatory cytokines , remove tissue debris , secrete growth factors , and support tissue restoration ( Koh and DiPietro , 2011 ) . Several recent studies have used the zebrafish model and addressed the roles of myeloid cells in response to tissue injury: Li and coworkers ( Li et al . , 2012 ) showed that knockdown of macrophage differentiation delayed fin fold regeneration and resulted in formation of an abnormal fin fold featuring large vacuoles . Furthermore , Petrie and coworkers ( Petrie et al . , 2014 ) showed that genetic ablation of macrophages in adult zebrafish impaired fin outgrowth and often induced an abnormal fin . These studies suggest a crucial role for macrophages in tissue regeneration; however , the mechanism by which myeloid cells affect regeneration remains to be elucidated . Here , we show that a pro-inflammatory cytokine gene , interleukin 1 beta ( il1b ) , is aberrantly upregulated in the clo mutant , and that the resulting excessive and prolonged inflammation causes apoptosis of the regenerative cells . Moreover , although myeloid cells are widely regarded as the main cells that secrete pro-inflammatory cytokines such as Il1b and thereby promote inflammation , we demonstrate that epidermal cells surrounding the amputated tissue are the source of Il1b in both wild-type ( WT ) and clo larvae . Notably , il1b expression in the regenerating epidermis is usually quenched by the action of macrophages , and a lack of this anti-inflammatory effect of the macrophages results in apoptosis . Lastly , we show that Il1b signaling plays an indispensable role in normal tissue regeneration by activating expression of regeneration-induced genes . Thus , our study highlights the function of Il1b and the inflammatory response in tissue regeneration . A proper level of il1b expression and the inflammatory response , which are tuned by macrophages , are necessary for progression of tissue regeneration .
To understand the molecular pathway leading to apoptosis of regenerative cells in the clo mutant , we performed a transcriptome analysis of amputated larval tail tissues of WT and clo mutant zebrafish at approximately 6 hr post amputation ( hpa ) , just before the stage at which apoptosis is first detectable in the clo mutant ( Hasegawa et al . , 2015 ) . Compared with WT larvae , the clo larvae showed marked upregulation of regeneration-induced genes such as junba , junbb , matrix metallopeptidase 9 ( mmp9 ) , fibronectin 1b ( fn1b ) , and fgf20a ( Yoshinari et al . , 2009; Shibata et al . , 2016 ) ( Figure 1A and Supplementary file 1 ) . In addition to these genes , expression of a group of genes including il1b and tumor necrosis factor-b ( tnfb ) , which are involved in the inflammatory response , was also upregulated in the clo mutant during fin fold regeneration . Excluding enzymes and uncharacterized proteins , il1b was the most upregulated gene . il1b expression in the clo mutant was also confirmed using reverse transcription polymerase chain reaction ( RT-PCR ) analysis ( Figure 1B ) . Notably , prostaglandin2a ( ptgs2a ) , a downstream gene in Il1b signaling ( Dinarello , 2009 ) , was also upregulated in the clo mutant ( Figure 1A ) , which clearly indicated that Il1b signaling was activated in the clo mutant . 10 . 7554/eLife . 22716 . 003Figure 1 . Prolonged activation of il1b expression in the clo mutant after fin fold amputation . ( A ) Transcriptome analysis of posterior fin fold tissues from uncut WT , amputated WT , and amputated clo mutant at 6 hpa . The ordinate , final RPKM , indicates the reads per kilobase of exon model per million mapped reads . Expression of regeneration-induced genes such as fn1b , junba , junbb , mmp9 , and fgf20a was upregulated in the clo mutant . Notably , expression of inflammation-related genes such as il1b , tnf1b , and ptgs2a was highly upregulated in the amputated clo mutant . ( B ) RT-PCR analysis of il1b expression in WT and clo larvae at 3 , 6 , and 12 hpa . actb1: actinb1 . ( C ) ISH analysis of il1b expression at corresponding stages of regeneration in WT and clo . Arrowheads indicate myeloid-like cells expressing il1b . Scale bars , 25 μm . ( D ) Quantification of il1b expression . The level of il1b expression was divided into four groups according to the ISH signal; the numbers in the bars indicate the ratios of the respective groups . Data are the sum of three experiments ( total n > 13 for each sample ) . The data suggest that il1b expression extended into later stages in clo than in WT . ( E ) ISH analysis of il1b expression after LPS injection . Phenol red: negative control containing only phenol red; hpi: hours post injection . Scale bars , 200 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 22716 . 00310 . 7554/eLife . 22716 . 004Figure 1—source data 1 . il1b expression at respective time points after fin fold amputation . The table shows the numbers of larvae used for evaluating the il1b expression levels in WT or clo mutant at respective time points after fin fold amputation . DOI: http://dx . doi . org/10 . 7554/eLife . 22716 . 004 Next , we performed in situ hybridization ( ISH ) analysis of il1b to reveal its spatiotemporal expression during regeneration . At 3 hpa , strong il1b expression was observed in both WT and clo mutant larvae ( Figure 1C and D ) . In WT larvae , il1b was expressed in a group of scattered cells that appeared to be myeloid cells ( Figure 1C; arrowheads ) and also expressed at the distal edge of the fin fold . After 3 hpa , il1b expression was rapidly diminished in WT larvae , and most larvae displayed very weak or no il1b expression at 6 hpa . In contrast to the expression in WT , il1b expression in the clo mutant was maintained at 6 hpa and was still detectable at 12 hpa , indicating a prolonged inflammatory reaction in the clo mutant . Bacterial infection is recognized to trigger an inflammatory response that accompanies the induction of il1b expression ( Dinarello , 2009 ) . To investigate whether the same mechanism underlies the induction of il1b expression by fin fold amputation and bacterial infection , we injected zebrafish with Salmonella typhosa lipopolysaccharide ( LPS ) , a reagent whose injection mimics bacterial infection ( Lu et al . , 2008 ) . WT larvae injected with LPS displayed systemic induction of il1b in cells that appeared to be myeloid cells ( Figure 1E ) . By contrast , the clo mutant larvae injected with LPS showed few il1b-positive cells , which indicated that tissue injury and bacterial infection induce il1b expression through distinct mechanisms . The absence of myeloid cells in the clo mutant suggests that the il1b is expressed in non-myeloid cells . Accordingly , examination of tissue cryosections of the clo fin fold after il1b ISH analysis suggested that il1b was expressed in epidermal cells ( Figure 2A ) . 10 . 7554/eLife . 22716 . 005Figure 2 . Spatiotemporal expression of il1b during fin fold regeneration . ( A ) Tissue cryosection of an amputated clo fin fold in which il1b expression was detected using ISH analysis . Arrowheads indicate the epidermal cells expressing il1b . Scale bar , 10 μm . ( B ) Live imaging of il1b expression in the Tg ( il1b:egfp ) line in WT and clo mutant at corresponding stages of regeneration . As in the case of il1b expression observed in ISH analysis ( Figure 1C ) , EGFP fluorescence of the Tg ( il1b:EGFP ) was strongly upregulated in the clo mutant . The EGFP fluorescence in WT decreased more slowly than did the il1b mRNA signal , because of the comparatively longer half-life of the protein EGFP . Scale bars , 100 μm . ( C ) Immunofluorescence detection of EGFP and E-cadherin in Tg ( il1b:EGFP ) at 3 and 12 hpa . Confocal longitudinal and transverse optical sections are shown at the top and right side , respectively . Vertical and horizontal red and green lines indicate the approximate sites of the optical sections . Scale bars , 50 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 22716 . 00510 . 7554/eLife . 22716 . 006Figure 2—figure supplement 1 . Reproduction of il1b expression in the Tg ( il1b:egfp ) line . ( A ) EGFP expression in Tg ( il1b:egfp ) at 2 dpa . Arrowheads , EGFP expression in the pectoral fins and the posterior tip of the notochord . Arrow , EGFP expression in the lens by crystalline alpha A:egfp inserted in the Tol2 vector . Scale bar , 300 μm . ( B ) ISH analysis of egfp and endogenous il1b expression in Tg ( il1b:egfp ) . The two genes were expressed in the same pattern . Scale bar , 50 μm . ( C ) EGFP fluorescence in Tg ( il1b:egfp ) after injection of LPS or control solution containing phenol red . Arrowheads indicate some of the EGFP-positive myeloid cells in LPS-injected larvae at 2 dpf; hpi: hours post injection . Scale bar , 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 22716 . 006 To further investigate the identity of the il1b-expressing cells , we generated a transgenic ( Tg ) line , Tg ( il1b:egfp ) , by introducing the egfp-nitroreductase fusion gene into the il1b locus of a bacterial artificial chromosome ( BAC ) clone , CH211-147H23 . The larvae of Tg ( il1b:egfp ) showed faint EGFP expression in the entire epidermis and weak EGFP expression in the pectoral fins and at the end of the notochord ( Figure 2—figure supplement 1A; arrowheads ) . After fin fold amputation , egfp expression was induced in a similar pattern to endogenous il1b expression ( Figure 2B; Figure 2—figure supplement 1B ) , although EGFP fluorescence was detected at later stages of regeneration than il1b mRNA , probably as a result of the relatively longer half-life of the protein EGFP . LPS injection also induced EGFP fluorescence in myeloid cells ( Figure 2—figure supplement 1C ) , which indicated that the il1b:egfp transgene recapitulated endogenous il1b expression in myeloid cells and also in the injured fin fold . Next , using the Tg ( il1b:egfp ) reporter line , we confirmed that the EGFP-positive cells in the transgenic line mostly colocalized with the epithelial marker E-cadherin ( Figure 2C ) , in both WT and the clo mutant; this finding indicated that il1b expression was principally induced in epithelial cells in response to tissue amputation . In addition to detecting il1b expression in the injured tissue , we detected il1b expression in WT larvae in migrating cells that are likely to be the myeloid cells ( Video 1 ) . 10 . 7554/eLife . 22716 . 007Video 1 . Time-lapse imaging of EGFP fluorescence in Tg ( il1b:egfp ) from 1 to 11 hpa . Images were acquired once every 15 min . The epidermal cells at the injury site and the migrating cells , which are considered to be myeloid cells , expressed EGFP . DOI: http://dx . doi . org/10 . 7554/eLife . 22716 . 007 Excessive inflammation has been suggested to directly or indirectly induce apoptosis ( Wallach et al . , 2014 ) . Therefore , because elevated expression of the pro-inflammatory cytokine Il1b leads to chronic inflammation ( Dinarello , 2011 ) , we suspected that excessive il1b expression in the clo mutant could be a cause of the apoptosis of regenerative cells . First , we used the Tg ( il1b:egfp ) reporter line to examine the relationship between il1b-expressing cells and apoptotic cells . Although many of the il1b-expressing cells did not overlap with the apoptotic cells that were mainly distributed in the mesenchyme ( Hasegawa et al . , 2015 ) , the cells lay in close apposition ( Figure 3A ) . 10 . 7554/eLife . 22716 . 008Figure 3 . il1b knockdown or Dex treatment rescued the clo from apoptosis . ( A ) Simultaneous detection of apoptosis by TdT-mediated dUTP nick end labeling ( TUNEL ) and the il1b-expressing cell at 12 hpa in the clo mutant carrying the transgene il1b:egfp . Most of the TUNEL-positive cells were detected in close association with the il1b-expressing epithelial cells . Vertical and horizontal lines indicate the approximate sites of the optical sections . Scale bar , 100 μm . ( B ) RT-PCR analysis of il1b and actinb1 expression in zebrafish larvae injected with il1b MO . The il1b MO was targeted to the splice donor site . Arrowhead indicates the aberrant transcript . ( C ) TUNEL analysis in amputated WT or clo larvae ( 12 hpa ) after il1b knockdown . Injection of il1b MO substantially reduced the apoptosis of regenerative cells in the clo mutant , whereas the apoptosis was unaffected by std MO . Scale bars , 50 μm . ( D ) Quantification of TUNEL staining in the areas bracketed in ( C ) . ( E ) ISH analysis of il1b expression at 3 hpa in larvae treated with Dex . DMSO: dimethyl sulfoxide , used as the vehicle . Dex treatment abolished il1b expression . Scale bar , 50 μm . ( F ) TUNEL staining in the amputated fin fold at 12 hpa in Dex-treated WT and clo larvae . Scale bars , 50 μm . ( G ) Quantification of TUNEL staining in the areas bracketed in ( F ) . In ( D ) and ( G ) , data are presented as means ± SEM . Student’s t test , **p<0 . 01; ***p<0 . 001; N . S . , not significant ( p=0 . 172 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 22716 . 00810 . 7554/eLife . 22716 . 009Figure 3—figure supplement 1 . Rescue from apoptosis by il1b MO2 . ( A ) TUNEL analysis of amputated clo larvae that were injected with il1b MO2 . The il1b MO2 rescued the regenerative cells from apoptosis in the clo mutant , whereas the apoptosis was unaffected by std MO . Scale bars , 50 μm . ( B ) Quantification of TUNEL-positive cells in the areas bracketed in ( A ) . Data are presented as means ± SEM . Student’s t test , ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 22716 . 009 Next , to demonstrate the role of Il1b in the apoptosis occurring in the clo mutant , we knocked down il1b expression using an antisense morpholino oligonucleotide ( MO ) . The il1b MO1 targeted to the splice sites ( Nguyen-Chi et al . , 2014 ) effectively inhibited il1b mRNA splicing ( Figure 3B ) . Notably , il1b knockdown in the clo mutant resulted in a reduction in apoptosis of regenerative cells , whereas in the clo mutant injected with the standard control MO ( std MO ) , no reduction in apoptosis was detected ( Figure 3C and D ) . A similar anti-apoptosis effect of il1b knockdown was also obtained using another MO against il1b ( Yan et al . , 2014 ) ( Figure 3—figure supplement 1 ) . To examine whether excessive inflammation is a cause of the apoptosis in the clo mutant , we used dexamethasone ( Dex ) , a synthetic glucocorticoid that has been reported to suppress inflammation and il1b expression ( Kern et al . , 1988 ) . Our results showed that Dex treatment abolished il1b expression in the clo mutant starting from an early stage of regeneration ( Figure 3E ) , which indicated that the treatment effectively suppressed inflammation and il1b expression . In the Dex-treated clo larvae , the regenerative cells were rescued from apoptosis , much as in the il1b-knockdown larvae ( Figure 3F and G ) . Collectively , these data support the notion that excessive Il1b signaling and inflammation in response to tissue injury represent a cause of apoptosis in the clo mutant . We have previously shown that the presence of myeloid cells is essential for survival of regenerative cells ( Hasegawa et al . , 2015 ) . Myeloid cells such as neutrophils and macrophages have been shown to transiently migrate to the injured fin fold ( Li et al . , 2012 ) . Actually , we observed that the myeloid cell accumulation in the injured fin fold reached a maximum between 3 and 6 hpa ( Figure 4A and B ) . The timing coincides with the stage at which the initial il1b expression immediately after wounding was downregulated ( Figure 1C and D ) . This suggests that myeloid cell migration could play a role in attenuating il1b expression , although we cannot conclude that myeloid cell migration is necessary for il1b downregulation . 10 . 7554/eLife . 22716 . 010Figure 4 . Macrophages are responsible for survival of regenerative cells . ( A ) ISH analysis of lcp1 expression , a macrophage marker , during fin fold regeneration in WT . Scale bar , 50 μm . ( B ) ISH analysis of mpx expression , a neutrophil marker , during fin fold regeneration in WT . Scale bar , 50 μm . ( C ) TUNEL analysis in the amputated fin fold at 12 hpa after injection of spi1b , csf3r , and irf8 MOs . Injection of spi1b MO and irf8 MO markedly increased apoptosis of regenerative cells . Scale bar , 100 μm . ( D ) Quantification of TUNEL-positive cells in the areas bracketed in ( A ) . In ( B ) , data are presented as means ± SEM . Student’s t test , ***p<0 . 001; N . S . , not significant ( p=0 . 981 ) . DOI: http://dx . doi . org/10 . 7554/eLife . 22716 . 01010 . 7554/eLife . 22716 . 011Figure 4—figure supplement 1 . Efficacy of MO-mediated macrophage and/or neutrophil depletion . ( A ) Sudan Black staining in std , spi1b , and csf3r morphants . Scale bar , 300 μm . Knockdowns by the spi1b and csf3r MOs decreased the number of neutrophils . ( B ) Quantification of Sudan Black staining ( A; posterior to the yolk extension ) . Data are presented as means ± SEM . Student’s t test , ***p<0 . 001 . ( C ) Analysis of apoeb expression in std and irf8 morphants . The irf8 MO inhibited differentiation of glial cells marked by apoeb . Scale bar , 100 μm . ( D ) ISH analysis of mpx expression , a neutrophil marker , at 2 hpa in amputated fin fold of larvae injected with std , spi1b , or csf3r MOs . mpx-positive cells were not detected in the spi1b or csf3r morphants . Arrowheads indicate representative neutrophils . Scale bar , 25 μm . ( E ) ISH analysis of lcp1 , a macrophage marker , at 6 hpa in amputated fin fold of larvae injected with std , spi1b , or irf8 MOs . Arrowheads indicate representative macrophages which have migrated to the injured site . lcp1-positive macrophages are rarely observed in amputated fin fold of the spi1b or irf8 morphants . Scale bar , 25 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 22716 . 011 Myeloid cells comprise several different cell types , including neutrophils and macrophages , but the cell type required for the survival of regenerative cells remains to be determined . Here , we used csf3r , irf8 , and spi1b ( pu . 1 ) MOs , which inhibited the differentiation of neutrophils , macrophages , and both types of cells , respectively , and examined whether the depletion of neutrophils or macrophages induces apoptosis of regenerative cells as in the clo mutant . The efficacy of csf3r knockdown was confirmed by Sudan Black staining of neutrophils and ISH analysis using the mpx probe . In both the csf3r morphants and the spi1b morphants , fewer neutrophils were detected than in the control morphants ( Figure 4—figure supplement 1A , B , D ) . In the irf8 morphants , the expression of apoeb , a marker for microglia—the resident macrophages in the central nervous system—was drastically decreased ( Figure 4—figure supplement 1C ) . The decrease of macrophages was also evident in the wounded fin fold as revealed by lcp1 ISH analysis ( Figure 4—figure supplement 1E ) . None of these morphants showed any morphological abnormalities . These data indicated that the irf8 MO effectively inhibited macrophage differentiation . To examine apoptosis , we performed TUNEL staining in the aforementioned morphants after fin fold amputation , and the results showed that irf8 morphants exhibited aberrant apoptosis of the regenerative cells , as did the spi1b morphants ( Hasegawa et al . , 2015 ) ( Figure 4C ) . Importantly , similar numbers of TUNEL-positive cells were observed in the irf8 and the spi1b morphants ( Figure 4D ) . In sharp contrast to the irf8 morphants , the csf3r morphants showed no increase in the apoptosis of regenerative cells . These results suggest that macrophages are responsible for supporting the survival of regenerative cells . We next investigated the mechanism by which macrophages support the survival of regenerative cells . First , we examined il1b expression at 6 hpa in the spi1b , csf3r , and irf8 morphants . Whereas normal il1b expression was detected in the csf3r morphants , as in WT larvae , il1b expression remained elevated in the spi1b and irf8 morphants , as in the clo mutant ( Figure 5A and B ) . This result suggests that macrophages attenuate il1b expression and thereby prevent apoptosis of regenerative cells in WT larvae . Furthermore , the apoptosis induced in the spi1b or irf8 morphants was rescued following il1b knockdown or Dex treatment ( Figure 5C–E ) , which suggests that elevated il1b expression in the spi1b and irf8 morphants is responsible for the observed apoptosis . 10 . 7554/eLife . 22716 . 012Figure 5 . Prolonged il1b expression and apoptosis are induced by macrophage loss during fin fold regeneration . ( A ) ISH analysis of il1b expression in spi1b , csf3r , and irf8 morphants . The spi1b and irf8 morphants displayed prolonged il1b expression at 6 hpa . Scale bar , 50 μm . ( B ) Quantification of il1b expression detected using ISH analysis in ( A ) . The level of il1b expression was evaluated as in Figure 1D . Data are the sum of two experiments ( total n > 16 for each MO ) . ( C ) TUNEL analysis of the amputated fin fold at 12 hpa after il1b knockdown or Dex treatment in larvae in which macrophages were depleted using the spi1b or irf8 MO . Scale bar , 100 μm . Apoptosis caused by spi1b and irf8 MOs was rescued by il1b MO administration or Dex treatment . ( D ) Quantification of TUNEL staining in the spi1b morphants ( C; bracketed areas ) . ( E ) Quantification of TUNEL staining in the irf8 morphants in ( C ) . In ( D ) and ( E ) , data are presented as means ± SEM . Student’s t test , ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 22716 . 01210 . 7554/eLife . 22716 . 013Figure 5—source data 1 . il1b expression in larvae injected with std , spi1b , csf3r , or irf8 MOs . The table shows the numbers of larvae used for evaluating the il1b expression levels in larvae that were injected with respective MOs . DOI: http://dx . doi . org/10 . 7554/eLife . 22716 . 013 To further demonstrate that excessive il1b expression induces apoptosis , we generated and used the construct pTol2 ( hsp70l:mCherry-T2a-il1b ) , which we hereafter refer to as pTol2 ( hsp70l:il1b ) . The precise cleavage site of the zebrafish Il1b pro-peptide has not yet been identified ( Vojtech et al . , 2012 ) , but based on comparing the zebrafish and human sequences , we estimated that the human IL-1β cleavage site ( D116 ) corresponds to the threonine residue at the 124th amino acid ( a . a . ) position in zebrafish Il1b ( Figure 6—figure supplement 1A ) . Furthermore , to obtain constitutive secretion of this Il1b protein lacking a canonical signal-peptide sequence , the Il1b sequence after a . a . 125 was fused with the human IL-1β receptor antagonist signal sequence ( Wingren et al . , 1996; Tu et al . , 2008 ) and placed under the control of the promoter of heat shock protein 70l ( Figure 6A ) . In the obtained Tg ( hsp70l:il1b ) line , mCherry fluorescence and il1b expression were observed after heat shock treatment ( Figure 6B; Figure 6—figure supplement 1B ) , but not after stress caused by fin fold amputation ( Figure 6—figure supplement 1C ) . 10 . 7554/eLife . 22716 . 014Figure 6 . il1b overexpression induces aberrant apoptosis . ( A ) A schematic diagram of the construct used; in this construct , the zebrafish heat shock protein 70l promoter drives the expression of mCherry and il1b harboring a signal sequence . ( B ) mCherry fluorescence in Tg ( hsp70l:il1b ) at 4 hr after heat shock . WT , sibling WT . Scale bar , 250 μm . ( C ) TUNEL analysis in the amputated fin fold of Tg ( hsp70l:il1b ) at 2 dpa . The diagram depicts the experimental procedure . HS , heat shock . Scale bar , 100 μm . ( D ) Quantification of TUNEL staining in ( C ) . ( E ) Fin fold regeneration in Tg ( hsp70l:il1b ) and sibling WT larvae . Dotted lines indicate amputation planes and fin fold outlines . Scale bar , 100 μm . ( F ) Quantification of the length of the regenerate ( length posterior to the amputation plane; shown in ( E ) ) . In ( D ) and ( F ) , data are presented as means ± SEM . Student’s t test , ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 22716 . 01410 . 7554/eLife . 22716 . 015Figure 6—figure supplement 1 . Generation of Tg ( hsp:il1b ) . ( A ) Comparison of zebrafish Il1b and human IL-1β amino acid sequences . Arrows indicate the human IL-1β cleavage site ( D116 ) and the estimated zebrafish Il1b cleavage site ( T124 ) . ( B ) ISH analysis of il1b expression in Tg ( hsp70l:il1b ) at 6 hr after heat shock ( HS ) . HS induced systemic il1b expression . Scale bar , 250 μm . ( C ) Absence of mCherry induction by fin fold amputation in the Tg ( hsp70l:il1b ) . A stress caused by fin fold amputation did not induce mCherry expression . Scale bar , 100 μm . ( D ) TUNEL analysis of Tg ( hsp70l:il1b ) at 24 hpf after induction of il1b overexpression . The diagram depicts the experimental procedure . Scale bar , 500 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 22716 . 015 When TUNEL staining was performed after heat shock induction at 2 days post amputation ( dpa ) , several TUNEL-positive cells were detected in the injured fin fold ( Figure 6C and D ) , which indicated that the primed regenerative cells were susceptible to the excess Il1b signal . Furthermore , systemic and aberrant apoptosis was also observed when il1b was overexpressed in WT embryo from 12 to 24 hr post fertilization ( hpf ) , at which stage myeloid cells have not fully differentiated ( Figure 6—figure supplement 1D ) . This indicated that the excessive Il1b signaling potentially induces apoptosis not only in fin fold but also in other parts of the body . In addition to inducing the aberrant apoptosis , il1b overexpression suppressed the extension of the fin fold ( Figure 6E and F ) . Our data thus far suggest that excessive Il1b-mediated inflammation negatively affects the survival of regenerative cells and tissue regeneration in the clo mutant . However , inflammation has also been suggested to be essential for complete tissue regeneration ( Mathew et al . , 2007; King et al . , 2012; Kyritsis et al . , 2012 ) . To examine the role of tissue inflammation and Il1b during normal fin fold regeneration , we used Dex and il1b MO1 and tested whether the inflammation mediated by Il1b affects normal fin fold regeneration . To assess the role of inflammation in tissue regeneration , we administered Dex to WT larvae and assessed fin fold regeneration . The Dex-treated larvae displayed apparent retardation of regeneration at 3 dpa ( Figure 7A and B ) and a statistically significant reduction in cell proliferation in the distal fin fold region ( Figure 7C and D ) . Notably , the expression of regeneration-induced genes such as junba ( Yoshinari et al . , 2009 ) and fgf20a ( Whitehead et al . , 2005; Shibata et al . , 2016 ) was downregulated in the Dex-treated larvae ( Figure 7E ) . These data suggest that inflammation is required for activation of regeneration-induced gene expression which is a prerequisite for normal regeneration . 10 . 7554/eLife . 22716 . 016Figure 7 . Il1b-mediated inflammation is required for normal regeneration . ( A ) Fin fold regeneration in WT larvae treated with Dex . Regeneration was retarded following treatment with Dex but not vehicle DMSO ( arrowheads ) . Scale bar , 100 μm . ( B ) Quantification for ( A ) ( posterior to the notochord ) . ( C ) BrdU incorporation in larvae treated with DMSO or Dex . BrdU labeling , 0–24 hpa . Scale bar , 50 μm . ( D ) Quantification for ( C ) ( bracketed areas ) . BrdU-positive cells were significantly reduced following Dex treatment . ( E ) Detection of junba expression using ISH and fgf20a expression using the HGn21A enhancer-trap line ( Shibata et al . , 2016 ) in DMSO- or Dex-treated larvae . The expression of junba and fgf20a was also significantly downregulated following Dex treatment . Arrowheads indicate EGFP expression at amputation sites . Scale bars , 50 μm ( F ) Fin fold regeneration in larvae injected with std or il1b MO . The knockdown of il1b reduced fin fold regeneration . Scale bar , 100 μm . ( G ) Quantification for ( F ) ( posterior to the notochord ) . ( H ) BrdU incorporation in larvae injected with std or il1b MO . BrdU labeling , 0–24 hpa . Scale bar , 50 μm . ( I ) Quantification for ( H ) ( bracketed areas ) . ( J ) Detection of junba expression using ISH and fgf20a expression using the HGn21A line in larvae injected with std or il1b MO . The expression of junba and fgf20a was downregulated following il1b knockdown as in the Dex-treated larvae . Arrowheads indicate EGFP expression atamputation sites . Scale bars , 50 μm . ( K ) Schematic of the procedure of il1b overexpression in uninjured WT larvae . Heat shock was applied twice before fixation . ( L ) ISH analysis of fn1b , junba , and junbb in WT and Tg ( hsp70l:il1b ) . il1b overexpression stimulated ectopic expression of regeneration-induced genes in uninjured larvae . Scale bar , 50 μm . In ( A ) and ( F ) , dotted lines indicate amputation planes and fin fold outlines . In ( B ) , ( D ) , ( G ) , and ( I ) , data are presented as means ± SEM . Student’s t test , **p<0 . 01; ***p<0 . 001 . DOI: http://dx . doi . org/10 . 7554/eLife . 22716 . 01610 . 7554/eLife . 22716 . 017Figure 7—figure supplement 1 . Absence of apoptosis after two HS inductions in the Tg ( hsp70l:il1b ) . TUNEL analysis after two HS inductions of il1b overexpression ( Figure 6K ) . At this stage , apoptosis has not been induced . Scale bar , 100 μm . DOI: http://dx . doi . org/10 . 7554/eLife . 22716 . 017 Next , we used il1b knockdown in WT zebrafish to demonstrate the role of Il1b signaling in normal regeneration: il1b knockdown induced a similar phenotype to Dex treatment ( Figure 7F–I ) , although the phenotype was slightly milder than that of the Dex-treated larvae , probably because of lower penetrance of the MO knockdown . Importantly , similar to the Dex-treated larvae , the il1b morphants showed attenuated expression of junba and fgf20a ( Figure 7J ) . Collectively , these results suggest that the inflammatory reaction mediated by Il1b plays a necessary role in normal fin fold regeneration . Lastly , we tested whether il1b overexpression induces a marked increase in expression of the regeneration-induced genes junba , junbb , and fn1b in uninjured tissue ( Figure 7K ) . As expected , il1b overexpression induced the ectopic expression of fn1b , junba , and junbb , both in the uncut fin fold ( Figure 7L ) and in several other tissues such as the pectoral fins . Importantly , the apoptosis induced by il1b overexpression was only detectable at 2 days after multiple heat shocks ( Figure 6C and D ) , possibly because of the anti-inflammatory function of macrophages in WT . But , induction of junba , junbb , and fn1b was observed at 12 hr after two heat shocks , a stage at which the apoptosis induced by il1b overexpression has not taken place ( Figure 7—figure supplement 1 ) , indicating that expression of the regeneration-induced genes was not caused by an indirect regenerative response to cell death . These data indicate that Il1b signaling is required tostimulate expression of regeneration-induced genes and regulate the initiation of fin fold regeneration .
It has been suggested that myeloid cells play crucial roles in the inflammatory responses of injured tissues . The pro-inflammatory cytokines ( such as Il1b ) produced by myeloid cells , including macrophages , induce early responses against infection or injury ( Dinarello , 2009 ) . Thus , myeloid cells are considered to trigger inflammation by providing pro-inflammatory cytokines . Here , we demonstrated that the pro-inflammatory cytokine Il1b is provided by epidermal cells in response to tissue injury . We showed that apoptosis occurred if the Il1b action at the injury site was prolonged , and that macrophages were responsible for attenuation of il1b expression and resolution of inflammation . Furthermore , our data suggest that normal il1b expression and inflammatory response are necessary in tissue regeneration to activate expression of regeneration-induced genes . Thus , our study has revealed that Il1b signaling and tissue inflammation act as a double-edged sword: they are required for regeneration , but in excess , they impair tissue regeneration ( Figure 8 ) . 10 . 7554/eLife . 22716 . 018Figure 8 . Schematic of the role of Il1b and macrophages during fin fold regeneration . ( A and B ) Tissue injury induces il1b expression in epithelial cells . The Il1b acts on the surrounding cells to prime them for regeneration by triggering expression of regeneration-induced genes such as fn1b , junba , junbb , and fgf20a . ( C ) The macrophage-derived factor subsequently attenuates il1b expression during normal regeneration . ( D ) In the absence of macrophages , apoptosis is induced in the primed regenerative cells . Thus , the Il1b function is a double-edged sword in tissue regeneration . DOI: http://dx . doi . org/10 . 7554/eLife . 22716 . 018 Myeloid cells are considered to be the principal producers of Il1b , although certain studies have reported il1b expression in melanoma cells ( Okamoto et al . , 2010 ) and human keratinocytes ( Kupper et al . , 1986 ) . In this study , we showed that il1b is expressed by epidermal cells around the injury site . Notably , LPS injection induced il1b expression in myeloid cells , but not in the injured epidermal cells , suggesting that il1b induction by tissue injury occurs through a mechanism distinct from the mechanism underlying induction in myeloid cells following bacterial infection . Although it remains unknown how tissue injury stimulates il1b expression , a subject for future study , a substance released from disrupted cells ( Rock and Kono , 2008 ) or a mechanical signal could be responsible for inducing il1b expression ( Kanjanamekanant et al . , 2013 ) . We also showed that in the clo mutant apoptosis is caused by prolonged il1b expression and inflammatory response . The precise mechanism by which Il1b induces apoptosis is unclear , but Tnf signaling could be a downstream mediator for triggering apoptosis . Tnfα signaling is known to stimulate apoptosis through activation of Caspase 8 ( Sedger and McDermott , 2014 ) . Tissue inflammation has been suggested to induce tnfα expression , and zebrafish tnfb , one of the zebrafish homologs of tnfα , was upregulated in the clo mutant ( Figure 1A ) ; therefore , Tnfα signaling potentially serves as a direct mediator for inducing apoptosis in the clo mutant . Alternatively , the clo apoptosis might occur because of ER stress signaling: Il1b administration into primary rat β-cells and MIN6 cells was reported to increase ER stress through intracellular release of Ca2+ and to induce apoptosis in a JNK-dependent manner ( Eizirik and Mandrup-Poulsen , 2001; Wang et al . , 2009 ) . Thus , excessive ER stress evoked by Il1b around the injury site could induce apoptosis in the clo mutant . In this study , we further demonstrated that macrophages play a crucial role in fin fold regeneration by attenuating il1b expression and supporting survival of regenerative cells . Recent studies have shown that knockdown of macrophage differentiation delayed fin fold regeneration and resulted in an abnormal fin fold featuring large vacuoles ( Li et al . , 2012 ) , and genetic ablation of macrophages in adult zebrafish was demonstrated to affect fin ray patterning and fin growth ( Petrie et al . , 2014 ) . These studies suggest that macrophages play a critical role in tissue regeneration , but the precise function of the cells remains unknown . Our study has , for the first time , elucidated a role of macrophages in attenuating il1b expression and inflammation during tissue regeneration . In accordance with our observation , macrophage-depleted mice and salamanders were found to display elevated il1b expression ( Goren et al . , 2009; Godwin et al . , 2013 ) , which suggests that the macrophage function of attenuating il1b transcription is conserved in other vertebrate species . Our observation of macrophage accumulation in the injured fin fold suggests that the macrophage migration could play a role in attenuating il1b expression and quenching inflammation . However , we cannot conclude that the macrophage migration is necessary for attenuating the il1b expression , because our preceding study showed that an activity that rescues the clo apoptosis exists in the WT body extract prior to tissue amputation . We previously suggested that the effect of myeloid cells is mediated by a factor that is diffusible and heat-stable ( Hasegawa et al . , 2015 ) , but its identity remains unknown . The factor could be an anti-inflammatory protein/molecule such as IL-10 , Tgf-β , or a lipid mediator ( Serhan et al . , 2008; Ortega-Gómez et al . , 2013 ) . It is speculated that signals such as nFkB and/or STAT3 , which are activated by tissue injury , are involved in inducing il1b expression ( Fang et al . , 2013; Ogryzko et al . , 2014; Karra et al . , 2015 ) . The anti-inflammatory molecules bind to cell surface receptors to activate intracellular pathways and antagonize the nFkB and/or STAT3 signals to attenuate il1b transcription ( Yu et al . , 2009; Saraiva and O'Garra , 2010; Liao et al . , 2012 ) . Most importantly , our study suggests that inflammation mediated by Il1b is also necessary for normal fin regeneration . Previously , we showed that genes such as fn1b , junba , and junbb were induced in response to tissue injury ( Yoshinari et al . , 2009 ) , but the upstream mechanism that regulates the expression of these genes remains unknown . Here , we have demonstrated that Il1b signaling is necessary and sufficient for triggering expression of regeneration-induced genes . Although the primary factors involved in tissue regeneration , tentatively named alarmins ( Bianchi , 2007 ) , remain to be identified , Il1b could represent one of the key upstream mediators for initiating expression of regeneration-induced genes and advancing tissue regeneration . Given that macrophages are required for resolving inflammation , an impairment or reduction of this function will lead to the diseases that accompany chronic inflammation , such as rheumatoid arthritis , inflammatory bowel disease , and auto-inflammatory diseases . Therefore , elucidation of the regulatory mechanism of the Il1b signal and the action of anti-inflammatory substances released from macrophages will enhance our understanding of the pathogenic processes of chronic-inflammation diseases and lead to development of suitable therapeutics .
Zebrafish of a WT line , which has been maintained in our facility for >10 years through inbreeding , were housed in a recirculating system in a 14 hr day/10 hr night cycle at 28 . 5°C . When necessary , larvae were incubated in egg water ( 0 . 06% artificial marine salt , 0 . 0002% methylene blue ) containing 0 . 003% phenylthiourea to prevent pigment formation . The zebrafish mutant strain clom39 ( RRID:ZFIN_ZDB-ALT-980203-381 ) , the fgf20a enhancer-trap line HGn21A ( Shibata et al . , 2016 ) , the BAC Tg ( il1b:egfp ) line , and Tg ( hsp70l:il1b ) were used . The clom39 mutant was genotyped as previously described ( Hasegawa et al . , 2015 ) . Zebrafish were subject to heat-shock induction at 38°C for 1 hr in a small water bath and then cooled to 28 . 5°C over time . Fin fold fin amputation was performed as previously described ( Kawakami et al . , 2004 ) . Briefly , zebrafish larvae at 2 dpf were anesthetized with 0 . 04% 3-amino benzoic acid ethylester ( tricaine ) in egg water and their fin fold was amputated using a surgical razor blade . For reproducible quantification , the fin fold was carefully amputated at sites just posterior to the notochords . For quantification of fin fold regeneration in Figure 6F , Figure 7B and G , the length from the notochord end to the posterior tip of fin fold was measured . Larval posterior tissues ( ~1 mm from the distal end , approximately the tissue posterior to the yolk extension ) from uncut WT , amputated WT , and amputated clo mutant , which were obtained from more than five independent batches of incrosses of clo heterozygotes , were collected on dry ice and stored at −80°C . Total RNAs from approximately 500 posterior tissue samples were extracted using TRIzol ( Thermo Fisher Scientific , Waltham , MA ) according to the manufacturer’s instructions . RNA sequencing analysis was performed by Dragon Genomics ( Kyoto , Japan ) . Briefly , purified and fragmented PolyA+ RNAs were prepared from 3 μg each of the total RNAs using the TruSeq RNA sample preparation kit ( Illumina , San Diego , CA ) , and the respective cDNAs were synthesized according to the manufacturer’s instructions . The cDNAs fused with an adaptor on both ends were PCR-amplified ( 15 cycles ) and used for sequencing analysis ( Illumina HiSeq2000 ) . The obtained sequence reads ( 60927373 reads for WT uncut , 59284653 reads for amputated WT at 6 hpa , 58507894 reads for amputated clo mutant at 6 hpa ) were aligned to the zebrafish reference sequence using Bowtie software ( version 0 . 12 . 7; RRID:SCR_005476 ) ( Langmead et al . , 2009 ) . The normalized expression level of the respective genes , RPKM ( reads per kilobase of exon model per million mapped reads ) , was calculated using ERANGE software ( version 3 . 2; RRID:SCR_005240 ) ( Mortazavi et al . , 2008 ) . MOs ( Gene Tools , Philomath , OR ) were dissolved in Danieau solution . Fertilized zebrafish eggs were dechorionated by incubating them with 2% pronase ( Roche ) and then microinjected at the 1–2 cell stages . The following MOs were used in this study: irf8 MO: 5ʹ-AATGTTTCGCTTACTTTGAAAATGG-3ʹ ( Hall et al . , 2014 ) csf3r MO1: 5ʹ-ATTCAAGCACATACTCAC-TTCCATT-3ʹ ( Hall et al . , 2014 ) csf3r MO2: 5ʹ- GAACTGGCGGATCTGTAAAGACAAA −3ʹ ( Halloum et al . , 2016 ) spi1b MO: 5ʹ-GATATACTGATACTCCATTGGTGGT-3ʹ ( Rhodes et al . , 2005 ) il1b MO1: 5ʹ-CCCACAAACTGCA-AAATATCAGCTT-3ʹ ( Nguyen-Chi et al . , 2014 ) il1b MO2: 5ʹ- AAACGTAAAATAACTCACCATTGCA −3ʹ ( Yan et al . , 2014 ) std MO: 5ʹ-CCTCTTACCTCAGTTACAATTTATA-3ʹ The csf3r MO2 ( 0 . 25 mM ) and the rest of MOs ( 1 mM ) were injected at approximately 0 . 5 nl per egg . Under these conditions , no apparent side effect was observed . Injected embryos were incubated in 0 . 3× Niu-Twitty solution . Total RNAs were extracted from larval tail tissues ( 200 for each ) posterior to the yolk extension using TRIzol and further purified using the RNeasy kit ( Qiagen , Venlo , Netherlands ) . Among the purified total RNAs , aliquots that corresponded to 90 larval tails were used for cDNA synthesis . The cDNAs were synthesized using the Thermoscript RT-PCR kit ( Thermo Fisher Scientific ) with random hexamers as the primer . The synthesized cDNAs were diluted to 0 . 1 μg/μl and stored at −30°C . PCR was performed according to a standard procedure using Paq5000 DNA polymerase ( Agilent Technologies , Santa Clara , CA ) , and the products were analyzed using 2% agarose gels . The following primers were used for PCR: il1b Fw: 5ʹ- GCAGAGGAACTTAACCAGCT −3ʹ il1b Rv: 5ʹ- TGCCGGTCTCCTTCCTGA −3ʹ actb1 Fw: 5ʹ- ATGGATGAGGAAATCGCTGCCCTGGTCGTTGACAA −3ʹ actb1 Rv: 5ʹ- AGAGAGAGCACAGCCTGGATGGCCACATACATGGC −3ʹ Whole-mount ISH analysis was performed as described previously ( Hasegawa et al . , 2015 ) , except that 5% polyvinyl alcohol was included in the buffer during the color reaction . After detection of the ISH signal , the samples were fixed with 4% paraformaldehyde ( PFA ) in phosphate-buffered saline ( PBS ) for color preservation , equilibrated with 80% glycerol , and then mounted on glass slides and photographed . The antisense junba , junbb , fn1b , apoeb , mpx , and lcp1 ( l-plastin ) probes used here were described previously ( Yoshinari et al . , 2009; Hall et al . , 2012 ) . The egfp probe was synthesized from the construct pCS2-egfp , which contained the full-length egfp coding sequence . The il1b probe was directly synthesized from the il1b PCR product using the T7 promoter ( underlined in the primer sequence ) ( Thisse and Thisse , 2008 ) . il1b probe Fw: 5ʹ-ATGGCATGCGGGCAATATGA-3ʹ il1b probe Rv: 5ʹ-TAATACGACTCACTATAGGGCTAGATGCGCACTTTATCCT-3ʹ For preparing tissue sections , samples were incubated in 20% sucrose in PBS for overnight at 4°C , embedded in Tissue-Tek compound ( Miles ) and 16 μm sections were obtained using a cryostat . S . typhosa LPS ( Sigma–Aldrich , St . Louis , MO ) was dissolved in water at 10 mg/ml and stored in aliquots at −30°C . A solution containing LPS ( 5 mg/ml ) and phenol red ( 0 . 5% ) was injected at 0 . 5 nl per larva into the pericardial cavity of anesthetized larvae at 2 dpf . The construct for generating Tg ( hsp70l:mCherry-2a-il1b ) was prepared by replacing the creERt2 sequence of pT2 ( hsp70l:mCherry-2a-creERt2 ) ( Yoshinari et al . , 2012 ) with the il1b cassette . The il1b cassette was prepared by PCR-amplifying the il1b sequence a . a . 125–273 . The signal sequence of the human IL-1β receptor antagonist ( Wingren et al . , 1996 ) and the HA tag were fused at the N-terminus and C-terminus , respectively ( underlined in the primer sequences ) . Fw il1b primer: 5ʹ-GCTAGCATGGAAATCTGCAGAGGCCTCCGCAGTCACCTAATCACTCTCCTCCTCTTCCTGTTCCATTCAGAGACGATCTGCAAAAACGTCTTGCAATGCACGATTTGCG-3ʹ Rv il1b primer: 5ʹ-AGATCTCTAAGCGTAGTCTGGGACGTCGTATGGGTAGATGCGCACTTTATCCTGCAGCTCGAAG-3ʹ The construct was injected into fertilized eggs . To identify germ-line transmission , the F0 fish were crossed with each other or with WT , and the embryos produced were screened for mCherry fluorescence after heat shock . To generate Tg ( il1b:egfp ) , the iTol2 cassette was first introduced into the BAC clone CH211-147H23 using BAC recombineering ( Suster et al . , 2011 ) . Next , the egfp-nitroreductase cassette ( Grohmann et al . , 2009 ) was introduced into the site of the initiation codon of il1b . Lastly , a mixture of purified BAC DNA ( 125 ng/μl ) and the transposase mRNA ( 25 ng/μl ) was injected into 1-cell-stage zebrafish embryos ( 1 nl/embryo ) . The Tg was screened for EGFP expression in the lens , which is driven by the crystalline alpha promoter , and further tested for EGFP induction in response to fin fold amputation . Zebrafish larvae were fixed with 4% PFA in PBS for 2 hr at room temperature ( RT ) or overnight at 4°C , washed thrice with PBS containing 0 . 1% Triton X-100 ( PBTx ) , dehydrated with methanol , and stored at −30°C . Samples were rehydrated with PBTx and then incubated ( overnight , 4°C ) with anti-E-cadherin ( 1:1000; BD Biosciences , Franklin Lakes , NJ; RRID:AB_397580 ) and anti-EGFP ( 1:1000; Nacalai Tesque , Kyoto , Japan; RRID:AB_10013361 ) antibodies in blocking buffer ( 5% serum and 0 . 2% bovine serum albumin in PBTx ) . After extensive washing with PBTx at RT , the samples were incubated ( overnight , 4°C ) with anti-mouse Alexa Fluor 568 and anti-rat Alexa Fluor 488 antibodies ( both 1:1000; Invitrogen ) . After washing with PBTx , the tail regions were isolated and mounted in 80% glycerol containing 2 . 5% 1 , 4-diazabicyclo[2 . 2 . 2]octane ( Nacalai Tesque ) as an anti-fading reagent . Fluorescence images were acquired using a confocal microscope ( FV-1000 , Olympus , Tokyo , Japan ) . Zebrafish larvae of the BAC Tg ( il1b:egfp ) line at 1 hpa were placed on a 2% agarose-gel stage in egg water and embedded in 0 . 7% low-melting agarose . Time-lapse images were acquired once every 15 min using the confocal microscope equipped with a 20× water-immersion objective lens; the acquired images were z-stacks containing 20 optical slices . Larvae were fixed with 4% PFA for 2 hr at RT or overnight at 4°C , dehydrated with methanol , and stored at −30°C . Apoptosis was examined using an in situ apoptosis detection kit ( Roche , Basel , Switzerland ) . Briefly , samples were rehydrated with PBTx , treated with 10 μg/ml Proteinase K in PBTx ( 5 min , RT ) , washed with PBTx , and refixed with 4% PFA in PBS ( 20 min ) . The samples were further incubated ( 15 min , on ice ) in a freshly prepared 0 . 1% sodium citrate buffer containing 0 . 1% Triton X-100 , washed with PBTx , and reacted with the TUNEL reaction mixture at 37°C for 1 . 5 hr . The reaction was terminated by washing with PBTx . The samples were mounted in 80% glycerol and fluorescence images were acquired using confocal microscopy . The TUNEL-positive cells were quantified by counting the number of cells in the area posterior to the notochords . Dex ( D-2915; Sigma ) was dissolved in dimethyl sulfoxide ( DMSO ) at 100 mM and stored at −30°C . The Dex solution was diluted to 100 μM with egg water and administered to zebrafish larvae at least 1 hr before fin fold amputation . Larvae were fixed with 4% PFA for 1 hr at RT , rinsed with PBS , and incubated in 0 . 03% Sudan black B ( Sigma ) . After extensive washing with 70% ethanol and rehydration with PBS containing 0 . 1% Tween 20 ( PBT ) , samples were mounted in 80% glycerol . Proliferating cells were labeled with 5-bromo-2-deoxyuridine ( BrdU , 5 mM ) during 0–12 hpa . The labeled larvae were fixed with 4% PFA for 2 hr at RT or overnight at 4°C , dehydrated with methanol , and stored at −30°C . Immunochemical detection was performed as described ( Yoshinari et al . , 2009 ) and BrdU-positive cells were quantified from the acquired confocal images . Similar to the quantification of TUNEL-positive cells , the BrdU-positive cells were quantified by counting the number of cells in the area posterior to the notochords . Most assessments of phenotypes and expression patterns were replicated in at least two independent experiments with comparable results . Larvae were collected from independent crosses , and experimental processing ( injection , heat shock , and/or staining ) was carried out on independent occasions . Exceptions to this include data presented in Figure 1E , Figure 7L , Figure 2—figure supplement 1 , and Figure 6—figure supplement 1B–D . In each of these cases , multiple larvae were processed , and the obtained phenotypes were the same in all or most cases . The n is reported within the respective figures . Data are presented as means ± SEM . Statistical analyses were performed using Microsoft Excel 2013 . For normally distributed data , differences were analyzed using Student’s t tests; p<0 . 05 was considered to be statistically significant .
Cellular responses to injury are crucial for complete tissue regeneration , but their underlying processes remain incompletely elucidated . We have previously reported that myeloid-defective zebrafish mutants display apoptosis of regenerative cells during fin fold regeneration . Here , we found that the apoptosis phenotype is induced by prolonged expression of interleukin 1 beta ( il1b ) . Myeloid cells are considered to be the principal source of Il1b , but we show that epithelial cells express il1b in response to tissue injury and initiate the inflammatory response , and that its resolution by macrophages is necessary for survival of regenerative cells . We further show that Il1b plays an essential role in normal fin fold regeneration by regulating expression of regeneration-induced genes . Our study reveals that proper levels of Il1b signaling and tissue inflammation , which are tuned by macrophages , play a crucial role in tissue regeneration .
Animals and other multicellular organisms all have at least some ability to regenerate lost or wounded tissues . Zebrafish are particularly good at this to the extent that they can replace damaged or lost body parts with exact replicas of the originals . In 2015 , a team of researchers found that some mutant zebrafish that lack blood cells including immune cells are unable to regenerate lost tissues . This is because the cells that are primed to regenerate die instead , but it was not clear why this happens . Many immune cells have roles in fighting infection and in responding to tissue damage . When a tissue is damaged , the area often becomes inflamed as white blood cells called macrophages flock to the damaged area to protect it from infection and remove damaged cells . Hasegawa et al . – who include several researchers involved in the 2015 study – used genetic approaches to investigate the role of inflammation in tissue regeneration in zebrafish . The experiments show that several genes involved in inflammation – including one called interleukin 1b – were active over longer periods of time in the mutant fish compared with normal zebrafish . The gene produces a signal protein and this prolonged activity causes the primed regenerative cells to die . However , the cells can survive if interleukin 1b activity is quickly suppressed by macrophages . The experiments also show that , in order for tissues to regenerate properly , interleukin 1b needs to be active for only a short period of time . The findings reveal that some inflammation is needed for tissues to regenerate , but that a more severe inflammatory response can block the process . A future challenge will be to identify the signals that macrophages produce to suppress inflammation to allow tissues to regenerate . These anti-inflammatory signals may have the potential to be used as drugs to cure chronic inflammatory diseases and boost tissue regeneration potential in humans .