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Cellular dynamics of regeneration reveals role of two distinct Pax7 stem cell populations in larval zebrafish muscle repair

View Article: PubMed Central - PubMed

ABSTRACT

Heterogeneity of stem cells or their niches is likely to influence tissue regeneration. Here we reveal stem/precursor cell diversity during wound repair in larval zebrafish somitic body muscle using time-lapse 3D confocal microscopy on reporter lines. Skeletal muscle with incision wounds rapidly regenerates both slow and fast muscle fibre types. A swift immune response is followed by an increase in cells at the wound site, many of which express the muscle stem cell marker Pax7. Pax7+ cells proliferate and then undergo terminal differentiation involving Myogenin accumulation and subsequent loss of Pax7 followed by elongation and fusion to repair fast muscle fibres. Analysis of pax7a and pax7b transgenic reporter fish reveals that cells expressing each of the duplicated pax7 genes are distinctly localised in uninjured larvae. Cells marked by pax7a only or by both pax7a and pax7b enter the wound rapidly and contribute to muscle wound repair, but each behaves differently. Low numbers of pax7a-only cells form nascent fibres. Time-lapse microscopy revealed that the more numerous pax7b-marked cells frequently fuse to pre-existing fibres, contributing more strongly than pax7a-only cells to repair of damaged fibres. pax7b-marked cells are more often present in rows of aligned cells that are observed to fuse into a single fibre, but more rarely contribute to nascent regenerated fibres. Ablation of a substantial portion of nitroreductase-expressing pax7b cells with metronidazole prior to wounding triggered rapid pax7a-only cell accumulation, but this neither inhibited nor augmented pax7a-only cell-derived myogenesis and thus altered the cellular repair dynamics during wound healing. Moreover, pax7a-only cells did not regenerate pax7b cells, suggesting a lineage distinction. We propose a modified founder cell and fusion-competent cell model in which pax7a-only cells initiate fibre formation and pax7b cells contribute to fibre growth. This newly discovered cellular complexity in muscle wound repair raises the possibility that distinct populations of myogenic cells contribute differentially to repair in other vertebrates.

No MeSH data available.


Related in: MedlinePlus

Time-course of muscle wound repair. (A,B) Large needle incision wounds (boxed regions) in the indicated somites of zebrafish 3.5 dpf larvae carrying transgenes expressed in slow (A; smyhc1:gfp) or (B; mylz2:gfp) fast fibres were repeatedly imaged in the same live fish by confocal fluorescence microscopy over 7 dpw. Larvae are shown anterior to left, dorsal up. Note the brighter fluorescence of newly synthesised unbleached GFP in regenerated region. s15-s19, somite 15 to somite 19. (C) Rate of recovery (mean±s.e.m.) of GFP fluorescence in epaxial somite of slow smyhc1:gfp and fast mylz2:gfp muscle of n larvae. (D-D″) smyhc1:gfp larvae showing slow fibres (white arrows) in deep somite, viewed from dorsal (D; 3 dpw) and lateral (D′) and corresponding transversal (D″) views at 4 dpw. The red and green crosshairs indicate planes, red arrows indicate elongated fibre-associated nuclei. (E) To investigate the source of new fibres, two adjacent somites in embryos injected with Kaede RNA were photoconverted from green to red at 2.5 dpf, then wounded in the epaxial domain and followed for 6 dpw. Representative confocal slices in lateral view show loss of KaedeRed without replacement by KaedeGreen. (F) Loss and gain of nuclei (mean±s.e.m.) in epaxial somites of Tg(h2afva:H2AFVA-GFP)kca66 larvae wounded at 3.5 dpf and imaged until 12 dpf (ANOVA, n=4). Scale bars: 50 µm.
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DMM022251F1: Time-course of muscle wound repair. (A,B) Large needle incision wounds (boxed regions) in the indicated somites of zebrafish 3.5 dpf larvae carrying transgenes expressed in slow (A; smyhc1:gfp) or (B; mylz2:gfp) fast fibres were repeatedly imaged in the same live fish by confocal fluorescence microscopy over 7 dpw. Larvae are shown anterior to left, dorsal up. Note the brighter fluorescence of newly synthesised unbleached GFP in regenerated region. s15-s19, somite 15 to somite 19. (C) Rate of recovery (mean±s.e.m.) of GFP fluorescence in epaxial somite of slow smyhc1:gfp and fast mylz2:gfp muscle of n larvae. (D-D″) smyhc1:gfp larvae showing slow fibres (white arrows) in deep somite, viewed from dorsal (D; 3 dpw) and lateral (D′) and corresponding transversal (D″) views at 4 dpw. The red and green crosshairs indicate planes, red arrows indicate elongated fibre-associated nuclei. (E) To investigate the source of new fibres, two adjacent somites in embryos injected with Kaede RNA were photoconverted from green to red at 2.5 dpf, then wounded in the epaxial domain and followed for 6 dpw. Representative confocal slices in lateral view show loss of KaedeRed without replacement by KaedeGreen. (F) Loss and gain of nuclei (mean±s.e.m.) in epaxial somites of Tg(h2afva:H2AFVA-GFP)kca66 larvae wounded at 3.5 dpf and imaged until 12 dpf (ANOVA, n=4). Scale bars: 50 µm.

Mentions: Zebrafish larvae expressing GFP in specific muscle fibre types were wounded by unilateral needle insertion into the epaxial somite. Tg(9.7kb smyhc1:gfp)i104, in which the slow myosin heavy chain 1 enhancer drives GFP labelling of ∼20 mononucleate superficial slow muscle fibres in each somite (Elworthy et al., 2008), and Tg(-2.2mylz2:gfp)i135, which labels underlying multinucleate fast fibres (von Hofsten et al., 2008), were used to analyse fibre loss and repair in individual fish over time (Fig. 1A-C). Upon lesion, GFP fluorescence rapidly diminished in the disrupted fibres. At 1 day post-wounding (1 dpw), significant loss of labelled fibres was observed in one to three somites in each transgenic line. Contralateral and adjacent somites seemed unaffected (Fig. 1A,B and data not shown). By 2 dpw, small smyhc1:GFP and mylz2:GFP fibres were observed spanning the wound region. Reappearance of GFP in both slow fibre monolayer and underlying fast muscle was significant by 3 dpw (Fig. 1A-C). Although fibres generally re-integrated correctly into the original somite structure, some misplaced slow fibres were observed deep in the wound site (Fig. 1D-D″). Kaede photoconversion-based cell tracking revealed that the vast majority of labelled cells at the injury site were lost and replaced by weakly fluorescent cells between 2-4 dpw (Fig. 1E), thereby showing that Kaede tracing was not suitable to determine the source of regenerated muscle fibres (Fig. 1E). Analysis of wounded larvae stained with phalloidin and Hoechst 33342 confirmed the loss of structural components of muscle (Fig. S1A). Nuclei within the lesion were rapidly lost and then re-accumulated at the wound site from 2 dpw onwards (Fig. S1B; Fig. 1F). Thus, damage to somitic muscle fibres is rapidly repaired, consistent with previous reports (Rodrigues et al., 2012; Seger et al., 2011). These findings show that the cell biology of muscle wound repair is open to time-lapse analysis in zebrafish embryos.Fig. 1.


Cellular dynamics of regeneration reveals role of two distinct Pax7 stem cell populations in larval zebrafish muscle repair
Time-course of muscle wound repair. (A,B) Large needle incision wounds (boxed regions) in the indicated somites of zebrafish 3.5 dpf larvae carrying transgenes expressed in slow (A; smyhc1:gfp) or (B; mylz2:gfp) fast fibres were repeatedly imaged in the same live fish by confocal fluorescence microscopy over 7 dpw. Larvae are shown anterior to left, dorsal up. Note the brighter fluorescence of newly synthesised unbleached GFP in regenerated region. s15-s19, somite 15 to somite 19. (C) Rate of recovery (mean±s.e.m.) of GFP fluorescence in epaxial somite of slow smyhc1:gfp and fast mylz2:gfp muscle of n larvae. (D-D″) smyhc1:gfp larvae showing slow fibres (white arrows) in deep somite, viewed from dorsal (D; 3 dpw) and lateral (D′) and corresponding transversal (D″) views at 4 dpw. The red and green crosshairs indicate planes, red arrows indicate elongated fibre-associated nuclei. (E) To investigate the source of new fibres, two adjacent somites in embryos injected with Kaede RNA were photoconverted from green to red at 2.5 dpf, then wounded in the epaxial domain and followed for 6 dpw. Representative confocal slices in lateral view show loss of KaedeRed without replacement by KaedeGreen. (F) Loss and gain of nuclei (mean±s.e.m.) in epaxial somites of Tg(h2afva:H2AFVA-GFP)kca66 larvae wounded at 3.5 dpf and imaged until 12 dpf (ANOVA, n=4). Scale bars: 50 µm.
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Related In: Results  -  Collection

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DMM022251F1: Time-course of muscle wound repair. (A,B) Large needle incision wounds (boxed regions) in the indicated somites of zebrafish 3.5 dpf larvae carrying transgenes expressed in slow (A; smyhc1:gfp) or (B; mylz2:gfp) fast fibres were repeatedly imaged in the same live fish by confocal fluorescence microscopy over 7 dpw. Larvae are shown anterior to left, dorsal up. Note the brighter fluorescence of newly synthesised unbleached GFP in regenerated region. s15-s19, somite 15 to somite 19. (C) Rate of recovery (mean±s.e.m.) of GFP fluorescence in epaxial somite of slow smyhc1:gfp and fast mylz2:gfp muscle of n larvae. (D-D″) smyhc1:gfp larvae showing slow fibres (white arrows) in deep somite, viewed from dorsal (D; 3 dpw) and lateral (D′) and corresponding transversal (D″) views at 4 dpw. The red and green crosshairs indicate planes, red arrows indicate elongated fibre-associated nuclei. (E) To investigate the source of new fibres, two adjacent somites in embryos injected with Kaede RNA were photoconverted from green to red at 2.5 dpf, then wounded in the epaxial domain and followed for 6 dpw. Representative confocal slices in lateral view show loss of KaedeRed without replacement by KaedeGreen. (F) Loss and gain of nuclei (mean±s.e.m.) in epaxial somites of Tg(h2afva:H2AFVA-GFP)kca66 larvae wounded at 3.5 dpf and imaged until 12 dpf (ANOVA, n=4). Scale bars: 50 µm.
Mentions: Zebrafish larvae expressing GFP in specific muscle fibre types were wounded by unilateral needle insertion into the epaxial somite. Tg(9.7kb smyhc1:gfp)i104, in which the slow myosin heavy chain 1 enhancer drives GFP labelling of ∼20 mononucleate superficial slow muscle fibres in each somite (Elworthy et al., 2008), and Tg(-2.2mylz2:gfp)i135, which labels underlying multinucleate fast fibres (von Hofsten et al., 2008), were used to analyse fibre loss and repair in individual fish over time (Fig. 1A-C). Upon lesion, GFP fluorescence rapidly diminished in the disrupted fibres. At 1 day post-wounding (1 dpw), significant loss of labelled fibres was observed in one to three somites in each transgenic line. Contralateral and adjacent somites seemed unaffected (Fig. 1A,B and data not shown). By 2 dpw, small smyhc1:GFP and mylz2:GFP fibres were observed spanning the wound region. Reappearance of GFP in both slow fibre monolayer and underlying fast muscle was significant by 3 dpw (Fig. 1A-C). Although fibres generally re-integrated correctly into the original somite structure, some misplaced slow fibres were observed deep in the wound site (Fig. 1D-D″). Kaede photoconversion-based cell tracking revealed that the vast majority of labelled cells at the injury site were lost and replaced by weakly fluorescent cells between 2-4 dpw (Fig. 1E), thereby showing that Kaede tracing was not suitable to determine the source of regenerated muscle fibres (Fig. 1E). Analysis of wounded larvae stained with phalloidin and Hoechst 33342 confirmed the loss of structural components of muscle (Fig. S1A). Nuclei within the lesion were rapidly lost and then re-accumulated at the wound site from 2 dpw onwards (Fig. S1B; Fig. 1F). Thus, damage to somitic muscle fibres is rapidly repaired, consistent with previous reports (Rodrigues et al., 2012; Seger et al., 2011). These findings show that the cell biology of muscle wound repair is open to time-lapse analysis in zebrafish embryos.Fig. 1.

View Article: PubMed Central - PubMed

ABSTRACT

Heterogeneity of stem cells or their niches is likely to influence tissue regeneration. Here we reveal stem/precursor cell diversity during wound repair in larval zebrafish somitic body muscle using time-lapse 3D confocal microscopy on reporter lines. Skeletal muscle with incision wounds rapidly regenerates both slow and fast muscle fibre types. A swift immune response is followed by an increase in cells at the wound site, many of which express the muscle stem cell marker Pax7. Pax7+ cells proliferate and then undergo terminal differentiation involving Myogenin accumulation and subsequent loss of Pax7 followed by elongation and fusion to repair fast muscle fibres. Analysis of pax7a and pax7b transgenic reporter fish reveals that cells expressing each of the duplicated pax7 genes are distinctly localised in uninjured larvae. Cells marked by pax7a only or by both pax7a and pax7b enter the wound rapidly and contribute to muscle wound repair, but each behaves differently. Low numbers of pax7a-only cells form nascent fibres. Time-lapse microscopy revealed that the more numerous pax7b-marked cells frequently fuse to pre-existing fibres, contributing more strongly than pax7a-only cells to repair of damaged fibres. pax7b-marked cells are more often present in rows of aligned cells that are observed to fuse into a single fibre, but more rarely contribute to nascent regenerated fibres. Ablation of a substantial portion of nitroreductase-expressing pax7b cells with metronidazole prior to wounding triggered rapid pax7a-only cell accumulation, but this neither inhibited nor augmented pax7a-only cell-derived myogenesis and thus altered the cellular repair dynamics during wound healing. Moreover, pax7a-only cells did not regenerate pax7b cells, suggesting a lineage distinction. We propose a modified founder cell and fusion-competent cell model in which pax7a-only cells initiate fibre formation and pax7b cells contribute to fibre growth. This newly discovered cellular complexity in muscle wound repair raises the possibility that distinct populations of myogenic cells contribute differentially to repair in other vertebrates.

No MeSH data available.


Related in: MedlinePlus