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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

Rapid recovery of Pax7-expressing cells in wounded somites through proliferation and relocation enhances differentiation in central myotome. Wild-type zebrafish larvae wounded at 3 dpf in epaxial somites 16-18 (yellow brackets) were analysed at the indicated times post-wounding by confocal immunodetection of Pax7 with EdU (A-F) or Myogenin (G,I) or in situ mRNA hybridisation for myf5 (H), shown in lateral view, anterior to left, dorsal to top. Blue boxes are magnified. (A-C) Diminished numbers of Pax7+ cells after wounding (A) are rapidly replaced (B) and show increased proliferation (B,C). (D-G). Pax7+ cells were counted in 2-4 wounded and 2-4 adjacent unwounded somite regions per larva and averaged to yield a value for each animal. VMZ, vertical myoseptum. Mean±s.e.m values from four larvae (D,E,G) or the number indicated (F). Statistical analysis is shown in Fig. S3A. (H) myf5 mRNA adjacent to a hypaxial wound (outlined by dots). Note the lack of myf5 mRNA in unwounded somites at this stage. (I) Pax7+Myog+ nuclei (white arrowheads) generally have lower Myog signal than Pax7−Myog+ cells (yellow arrowheads). Scale bars: 50 µm.
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DMM022251F3: Rapid recovery of Pax7-expressing cells in wounded somites through proliferation and relocation enhances differentiation in central myotome. Wild-type zebrafish larvae wounded at 3 dpf in epaxial somites 16-18 (yellow brackets) were analysed at the indicated times post-wounding by confocal immunodetection of Pax7 with EdU (A-F) or Myogenin (G,I) or in situ mRNA hybridisation for myf5 (H), shown in lateral view, anterior to left, dorsal to top. Blue boxes are magnified. (A-C) Diminished numbers of Pax7+ cells after wounding (A) are rapidly replaced (B) and show increased proliferation (B,C). (D-G). Pax7+ cells were counted in 2-4 wounded and 2-4 adjacent unwounded somite regions per larva and averaged to yield a value for each animal. VMZ, vertical myoseptum. Mean±s.e.m values from four larvae (D,E,G) or the number indicated (F). Statistical analysis is shown in Fig. S3A. (H) myf5 mRNA adjacent to a hypaxial wound (outlined by dots). Note the lack of myf5 mRNA in unwounded somites at this stage. (I) Pax7+Myog+ nuclei (white arrowheads) generally have lower Myog signal than Pax7−Myog+ cells (yellow arrowheads). Scale bars: 50 µm.

Mentions: In addition to muscle fibres, at the time of wounding, somites contained mononucleate cells, many of which are marked by the muscle stem/precursor cell marker Pax7 (Hammond et al., 2007; Hollway et al., 2007; Minchin et al., 2013; Stellabotte et al., 2007; Windner et al., 2012). These Pax7+ cells are originally distributed on the lateral myotome surface and concentrate at the dorsal and ventral edges of the somite and the horizontal and vertical myosepta (HZM and VMZ; Windner et al., 2012). Subsequently, small numbers of Pax7+ cells arise in the deep central myotome (Minchin et al., 2013) (Fig. 3A). Upon making a large wound, the number of Pax7+ cells was rapidly reduced and then recovered by 1 dpw (Fig. 3A,B,D; Fig. S3A). At 1-3 dpw, an increased proportion of Pax7+ cells were in S-phase, as assayed by EdU pulse labelling (Fig. 3B,C,F). To demonstrate that proliferative cells contribute to new muscle fibres in wounds, larvae marked with membrane-targeted GFP were continuously exposed to EdU from 3 hpw to 3 dpw. At regenerating wounds, large numbers of nuclei were observed, most of which were EdU+ and new fibres at the wound contained multiple EdU+ nuclei (Fig. S4). Fibres in adjacent unwounded somites contained few EdU-labelled and many unlabelled nuclei (Fig. S4), indicative of a low rate of MPC fusion to muscle fibres during growth. Most nuclei in regenerating muscle wounds had undergone S-phase after wounding. Thus, proliferation of Pax7+ cells contributes to recovery in somite cell numbers.Fig. 3.


Cellular dynamics of regeneration reveals role of two distinct Pax7 stem cell populations in larval zebrafish muscle repair
Rapid recovery of Pax7-expressing cells in wounded somites through proliferation and relocation enhances differentiation in central myotome. Wild-type zebrafish larvae wounded at 3 dpf in epaxial somites 16-18 (yellow brackets) were analysed at the indicated times post-wounding by confocal immunodetection of Pax7 with EdU (A-F) or Myogenin (G,I) or in situ mRNA hybridisation for myf5 (H), shown in lateral view, anterior to left, dorsal to top. Blue boxes are magnified. (A-C) Diminished numbers of Pax7+ cells after wounding (A) are rapidly replaced (B) and show increased proliferation (B,C). (D-G). Pax7+ cells were counted in 2-4 wounded and 2-4 adjacent unwounded somite regions per larva and averaged to yield a value for each animal. VMZ, vertical myoseptum. Mean±s.e.m values from four larvae (D,E,G) or the number indicated (F). Statistical analysis is shown in Fig. S3A. (H) myf5 mRNA adjacent to a hypaxial wound (outlined by dots). Note the lack of myf5 mRNA in unwounded somites at this stage. (I) Pax7+Myog+ nuclei (white arrowheads) generally have lower Myog signal than Pax7−Myog+ cells (yellow arrowheads). Scale bars: 50 µm.
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Related In: Results  -  Collection

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DMM022251F3: Rapid recovery of Pax7-expressing cells in wounded somites through proliferation and relocation enhances differentiation in central myotome. Wild-type zebrafish larvae wounded at 3 dpf in epaxial somites 16-18 (yellow brackets) were analysed at the indicated times post-wounding by confocal immunodetection of Pax7 with EdU (A-F) or Myogenin (G,I) or in situ mRNA hybridisation for myf5 (H), shown in lateral view, anterior to left, dorsal to top. Blue boxes are magnified. (A-C) Diminished numbers of Pax7+ cells after wounding (A) are rapidly replaced (B) and show increased proliferation (B,C). (D-G). Pax7+ cells were counted in 2-4 wounded and 2-4 adjacent unwounded somite regions per larva and averaged to yield a value for each animal. VMZ, vertical myoseptum. Mean±s.e.m values from four larvae (D,E,G) or the number indicated (F). Statistical analysis is shown in Fig. S3A. (H) myf5 mRNA adjacent to a hypaxial wound (outlined by dots). Note the lack of myf5 mRNA in unwounded somites at this stage. (I) Pax7+Myog+ nuclei (white arrowheads) generally have lower Myog signal than Pax7−Myog+ cells (yellow arrowheads). Scale bars: 50 µm.
Mentions: In addition to muscle fibres, at the time of wounding, somites contained mononucleate cells, many of which are marked by the muscle stem/precursor cell marker Pax7 (Hammond et al., 2007; Hollway et al., 2007; Minchin et al., 2013; Stellabotte et al., 2007; Windner et al., 2012). These Pax7+ cells are originally distributed on the lateral myotome surface and concentrate at the dorsal and ventral edges of the somite and the horizontal and vertical myosepta (HZM and VMZ; Windner et al., 2012). Subsequently, small numbers of Pax7+ cells arise in the deep central myotome (Minchin et al., 2013) (Fig. 3A). Upon making a large wound, the number of Pax7+ cells was rapidly reduced and then recovered by 1 dpw (Fig. 3A,B,D; Fig. S3A). At 1-3 dpw, an increased proportion of Pax7+ cells were in S-phase, as assayed by EdU pulse labelling (Fig. 3B,C,F). To demonstrate that proliferative cells contribute to new muscle fibres in wounds, larvae marked with membrane-targeted GFP were continuously exposed to EdU from 3 hpw to 3 dpw. At regenerating wounds, large numbers of nuclei were observed, most of which were EdU+ and new fibres at the wound contained multiple EdU+ nuclei (Fig. S4). Fibres in adjacent unwounded somites contained few EdU-labelled and many unlabelled nuclei (Fig. S4), indicative of a low rate of MPC fusion to muscle fibres during growth. Most nuclei in regenerating muscle wounds had undergone S-phase after wounding. Thus, proliferation of Pax7+ cells contributes to recovery in somite cell numbers.Fig. 3.

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