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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

Fusion of pax7a- and pax7b-reporter cells during wound repair. (A-C) Lateral confocal maximum intensity projection stacks of pre-wounded (A) and wounded (B,C) yolk extension somites of pax7a:GFP;pax7b:gal4;UAS:RFP (A,B) or single pax7a/b:GFP (C) larvae, anterior to left, dorsal to top. Scale bars: 50 µm. (A) At 3 dpf, pax7b:RFP fibres (white arrows) and presumptive mononucleate cells (cyan arrowheads) are present superficially (s/f) within the somite and differ from pax7a:GFP cells (blue arrowheads). Dual-labelled somite cells (magenta arrowheads) concentrate on VMZ. Note the lack of Pax7 cells in the deep myotome at this stage. The pax7b-reporter labelled cells strongly in somites, and also weakly in dorsal neural tube (NT). (B) Short stack of epaxial wounded region shown by white box in A with two small wounds (asterisks). At 1 dpw, pax7a:GFP;pax7b:RFP cells elongate in wound. By 2 dpw, time-lapse reveals several nascent fibres marked strongly by RFP and weakly by GFP. See Fig. S9 for separate monochrome images. (C) Time-lapse of pax7b:gal4;UAS:GFP reporter marks aligned cells (arrowheads) that form fibres (top) or disappear (centre). pax7a:GFP cells are frequently aligned with fibres, but more rarely assemble in rows. pax7a:GFP cells occasionally matured into nascent fibres (bottom). Asterisks mark the same cells at each time point. Note the stronger mononucleate cells and more abundant fibre labelling by the pax7b:GFP reporter, compared with the pax7b:RFP reporter in panel B. Arrow indicates a separate cell. (D) Counts of numbers (mean±s.e.m.) of red, green and dual-labelled cells in a single epaxial somite (or corresponding length of neural tube) by cell type in larvae transgenic (Tg) for pax7a:GFP (a, green), pax7b:GFP (b, green) or pax7b:RFP (b, red) as indicated by the Tg line letter code and colour. Larvae with (+) or without (−) a wound made at 3 dpf were analysed 1 dpw, at 4 dpf. Note the increase in labelled MPCs, decrease in fibres and constant number of xanthophores and neurons in wounded somites at 1 dpw. Letter groups (m,n,p,q) indicate difference at P<0.05 (t-test, n=3). (E) At 1 dpw, despite a similar fraction of total cells in myotome, there were more pax7b:GFP reporter cells in rows of two (≥2) or four (≥4) or more aligned cells, compared with pax7a:GFP cells. Mean±s.e.m., P-values show Mann–Whitney test of differences in proportions of total cells.
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DMM022251F5: Fusion of pax7a- and pax7b-reporter cells during wound repair. (A-C) Lateral confocal maximum intensity projection stacks of pre-wounded (A) and wounded (B,C) yolk extension somites of pax7a:GFP;pax7b:gal4;UAS:RFP (A,B) or single pax7a/b:GFP (C) larvae, anterior to left, dorsal to top. Scale bars: 50 µm. (A) At 3 dpf, pax7b:RFP fibres (white arrows) and presumptive mononucleate cells (cyan arrowheads) are present superficially (s/f) within the somite and differ from pax7a:GFP cells (blue arrowheads). Dual-labelled somite cells (magenta arrowheads) concentrate on VMZ. Note the lack of Pax7 cells in the deep myotome at this stage. The pax7b-reporter labelled cells strongly in somites, and also weakly in dorsal neural tube (NT). (B) Short stack of epaxial wounded region shown by white box in A with two small wounds (asterisks). At 1 dpw, pax7a:GFP;pax7b:RFP cells elongate in wound. By 2 dpw, time-lapse reveals several nascent fibres marked strongly by RFP and weakly by GFP. See Fig. S9 for separate monochrome images. (C) Time-lapse of pax7b:gal4;UAS:GFP reporter marks aligned cells (arrowheads) that form fibres (top) or disappear (centre). pax7a:GFP cells are frequently aligned with fibres, but more rarely assemble in rows. pax7a:GFP cells occasionally matured into nascent fibres (bottom). Asterisks mark the same cells at each time point. Note the stronger mononucleate cells and more abundant fibre labelling by the pax7b:GFP reporter, compared with the pax7b:RFP reporter in panel B. Arrow indicates a separate cell. (D) Counts of numbers (mean±s.e.m.) of red, green and dual-labelled cells in a single epaxial somite (or corresponding length of neural tube) by cell type in larvae transgenic (Tg) for pax7a:GFP (a, green), pax7b:GFP (b, green) or pax7b:RFP (b, red) as indicated by the Tg line letter code and colour. Larvae with (+) or without (−) a wound made at 3 dpf were analysed 1 dpw, at 4 dpf. Note the increase in labelled MPCs, decrease in fibres and constant number of xanthophores and neurons in wounded somites at 1 dpw. Letter groups (m,n,p,q) indicate difference at P<0.05 (t-test, n=3). (E) At 1 dpw, despite a similar fraction of total cells in myotome, there were more pax7b:GFP reporter cells in rows of two (≥2) or four (≥4) or more aligned cells, compared with pax7a:GFP cells. Mean±s.e.m., P-values show Mann–Whitney test of differences in proportions of total cells.

Mentions: To verify that Pax7+ cells contribute to muscle regeneration, we employed fish labelled with a pax7a:GFP BAC transgene (Mahalwar et al., 2014; S. Alsheimer, PhD thesis p. 249, Universität Tübingen, 2012). Prior to wounding, and in control and adjacent unwounded somites, the reporter labelled cells on the somite borders, as well as xanthophores and cells in the dorsal neural tube. To examine the response of MPCs specifically, pax7a:GFP was bred onto a pfeffer mutant background that substantially reduces xanthophore number (compare Fig. 4A with Fig. 5A) (Odenthal et al., 1996). In large wounds, most pax7a:GFP signal was lost at the wound site, consistent with ablation of many MPCs (Fig. S6A). pax7a:GFP cells re-accumulate at 1 dpw, divide and migrate, gradually invading the wound and contributing to fibres near the wound edge by 2 dpw (Fig. S6). Correlating with the extent of wound and time course of pax7a:GFP cell invasion, repair rate varied. However, by 6 dpw muscle seemed regenerated and some pax7:GFP cells remained undifferentiated after recovery (Figs S6, S7). Thus, pax7a-expressing MPCs participate in muscle wound repair.Fig. 4.


Cellular dynamics of regeneration reveals role of two distinct Pax7 stem cell populations in larval zebrafish muscle repair
Fusion of pax7a- and pax7b-reporter cells during wound repair. (A-C) Lateral confocal maximum intensity projection stacks of pre-wounded (A) and wounded (B,C) yolk extension somites of pax7a:GFP;pax7b:gal4;UAS:RFP (A,B) or single pax7a/b:GFP (C) larvae, anterior to left, dorsal to top. Scale bars: 50 µm. (A) At 3 dpf, pax7b:RFP fibres (white arrows) and presumptive mononucleate cells (cyan arrowheads) are present superficially (s/f) within the somite and differ from pax7a:GFP cells (blue arrowheads). Dual-labelled somite cells (magenta arrowheads) concentrate on VMZ. Note the lack of Pax7 cells in the deep myotome at this stage. The pax7b-reporter labelled cells strongly in somites, and also weakly in dorsal neural tube (NT). (B) Short stack of epaxial wounded region shown by white box in A with two small wounds (asterisks). At 1 dpw, pax7a:GFP;pax7b:RFP cells elongate in wound. By 2 dpw, time-lapse reveals several nascent fibres marked strongly by RFP and weakly by GFP. See Fig. S9 for separate monochrome images. (C) Time-lapse of pax7b:gal4;UAS:GFP reporter marks aligned cells (arrowheads) that form fibres (top) or disappear (centre). pax7a:GFP cells are frequently aligned with fibres, but more rarely assemble in rows. pax7a:GFP cells occasionally matured into nascent fibres (bottom). Asterisks mark the same cells at each time point. Note the stronger mononucleate cells and more abundant fibre labelling by the pax7b:GFP reporter, compared with the pax7b:RFP reporter in panel B. Arrow indicates a separate cell. (D) Counts of numbers (mean±s.e.m.) of red, green and dual-labelled cells in a single epaxial somite (or corresponding length of neural tube) by cell type in larvae transgenic (Tg) for pax7a:GFP (a, green), pax7b:GFP (b, green) or pax7b:RFP (b, red) as indicated by the Tg line letter code and colour. Larvae with (+) or without (−) a wound made at 3 dpf were analysed 1 dpw, at 4 dpf. Note the increase in labelled MPCs, decrease in fibres and constant number of xanthophores and neurons in wounded somites at 1 dpw. Letter groups (m,n,p,q) indicate difference at P<0.05 (t-test, n=3). (E) At 1 dpw, despite a similar fraction of total cells in myotome, there were more pax7b:GFP reporter cells in rows of two (≥2) or four (≥4) or more aligned cells, compared with pax7a:GFP cells. Mean±s.e.m., P-values show Mann–Whitney test of differences in proportions of total cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4920144&req=5

DMM022251F5: Fusion of pax7a- and pax7b-reporter cells during wound repair. (A-C) Lateral confocal maximum intensity projection stacks of pre-wounded (A) and wounded (B,C) yolk extension somites of pax7a:GFP;pax7b:gal4;UAS:RFP (A,B) or single pax7a/b:GFP (C) larvae, anterior to left, dorsal to top. Scale bars: 50 µm. (A) At 3 dpf, pax7b:RFP fibres (white arrows) and presumptive mononucleate cells (cyan arrowheads) are present superficially (s/f) within the somite and differ from pax7a:GFP cells (blue arrowheads). Dual-labelled somite cells (magenta arrowheads) concentrate on VMZ. Note the lack of Pax7 cells in the deep myotome at this stage. The pax7b-reporter labelled cells strongly in somites, and also weakly in dorsal neural tube (NT). (B) Short stack of epaxial wounded region shown by white box in A with two small wounds (asterisks). At 1 dpw, pax7a:GFP;pax7b:RFP cells elongate in wound. By 2 dpw, time-lapse reveals several nascent fibres marked strongly by RFP and weakly by GFP. See Fig. S9 for separate monochrome images. (C) Time-lapse of pax7b:gal4;UAS:GFP reporter marks aligned cells (arrowheads) that form fibres (top) or disappear (centre). pax7a:GFP cells are frequently aligned with fibres, but more rarely assemble in rows. pax7a:GFP cells occasionally matured into nascent fibres (bottom). Asterisks mark the same cells at each time point. Note the stronger mononucleate cells and more abundant fibre labelling by the pax7b:GFP reporter, compared with the pax7b:RFP reporter in panel B. Arrow indicates a separate cell. (D) Counts of numbers (mean±s.e.m.) of red, green and dual-labelled cells in a single epaxial somite (or corresponding length of neural tube) by cell type in larvae transgenic (Tg) for pax7a:GFP (a, green), pax7b:GFP (b, green) or pax7b:RFP (b, red) as indicated by the Tg line letter code and colour. Larvae with (+) or without (−) a wound made at 3 dpf were analysed 1 dpw, at 4 dpf. Note the increase in labelled MPCs, decrease in fibres and constant number of xanthophores and neurons in wounded somites at 1 dpw. Letter groups (m,n,p,q) indicate difference at P<0.05 (t-test, n=3). (E) At 1 dpw, despite a similar fraction of total cells in myotome, there were more pax7b:GFP reporter cells in rows of two (≥2) or four (≥4) or more aligned cells, compared with pax7a:GFP cells. Mean±s.e.m., P-values show Mann–Whitney test of differences in proportions of total cells.
Mentions: To verify that Pax7+ cells contribute to muscle regeneration, we employed fish labelled with a pax7a:GFP BAC transgene (Mahalwar et al., 2014; S. Alsheimer, PhD thesis p. 249, Universität Tübingen, 2012). Prior to wounding, and in control and adjacent unwounded somites, the reporter labelled cells on the somite borders, as well as xanthophores and cells in the dorsal neural tube. To examine the response of MPCs specifically, pax7a:GFP was bred onto a pfeffer mutant background that substantially reduces xanthophore number (compare Fig. 4A with Fig. 5A) (Odenthal et al., 1996). In large wounds, most pax7a:GFP signal was lost at the wound site, consistent with ablation of many MPCs (Fig. S6A). pax7a:GFP cells re-accumulate at 1 dpw, divide and migrate, gradually invading the wound and contributing to fibres near the wound edge by 2 dpw (Fig. S6). Correlating with the extent of wound and time course of pax7a:GFP cell invasion, repair rate varied. However, by 6 dpw muscle seemed regenerated and some pax7:GFP cells remained undifferentiated after recovery (Figs S6, S7). Thus, pax7a-expressing MPCs participate in muscle wound repair.Fig. 4.

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