Limits...
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 to existing myotubes during wound repair. Extended orthogonal projection views of an epaxial somite wound in a pax7a:GFP;pax7b:gal4;UAS:RFP 4 dpf larva showing individual pax7a:GFP;pax7b:RFP dual-labelled (yellow) MPCs fusing to existing unlabelled (A) or RFP+ (B) fibres from Movies 2 and 3. The whole image was non-linearly enhanced and brightness corrected to compensate for bleaching and facilitate tracking of individual cells, as described in Materials and Methods. (A) At 25.5 hpw, prior to fusion, an MPC had uniform cytoplasmic GFP and diffuse cytoplasmic and vesicular RFP (arrow). 10 min later, cytoplasmic GFP and RFP have now filled the whole cytoplasm of a large adjacent previously unlabelled fibre (brackets), whereas the vesicular RFP remains localised (arrowhead) and integrates into the fibre in the succeeding 50 min (see Movie 2, blue MPC). Transverse II shows the same view as Transvere I, but with the fusing fibre marked (dots). (B) At 39.5 hpw, two MPCs (magenta and yellow arrows), fuse simultaneously to the same large adjacent myotube (arrowheads; Movie 2, magenta and yellow MPCs). (C) At 32.3 hpw, a dual-labelled MPC (arrowhead; Movie 3, white MPC) that originated from the anterior somite border, divided and then migrated along a recently formed GFP+ nascent myofibre (arrow). 10 min later the MPC has fused into the nascent fibre, as shown by RFP loss from the MPC and increase in the fibre. The fused cell remains distinct at 10 min, but merges into the nascent fibre by 20 min. Process shown in merge and single colour lateral (dorsal up, anterior left) and transverse (dorsal up, medial left) views. Blue lines indicate range of extended orthogonal projection views.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4920144&req=5

DMM022251F6: Fusion of pax7a- and pax7b-reporter cells to existing myotubes during wound repair. Extended orthogonal projection views of an epaxial somite wound in a pax7a:GFP;pax7b:gal4;UAS:RFP 4 dpf larva showing individual pax7a:GFP;pax7b:RFP dual-labelled (yellow) MPCs fusing to existing unlabelled (A) or RFP+ (B) fibres from Movies 2 and 3. The whole image was non-linearly enhanced and brightness corrected to compensate for bleaching and facilitate tracking of individual cells, as described in Materials and Methods. (A) At 25.5 hpw, prior to fusion, an MPC had uniform cytoplasmic GFP and diffuse cytoplasmic and vesicular RFP (arrow). 10 min later, cytoplasmic GFP and RFP have now filled the whole cytoplasm of a large adjacent previously unlabelled fibre (brackets), whereas the vesicular RFP remains localised (arrowhead) and integrates into the fibre in the succeeding 50 min (see Movie 2, blue MPC). Transverse II shows the same view as Transvere I, but with the fusing fibre marked (dots). (B) At 39.5 hpw, two MPCs (magenta and yellow arrows), fuse simultaneously to the same large adjacent myotube (arrowheads; Movie 2, magenta and yellow MPCs). (C) At 32.3 hpw, a dual-labelled MPC (arrowhead; Movie 3, white MPC) that originated from the anterior somite border, divided and then migrated along a recently formed GFP+ nascent myofibre (arrow). 10 min later the MPC has fused into the nascent fibre, as shown by RFP loss from the MPC and increase in the fibre. The fused cell remains distinct at 10 min, but merges into the nascent fibre by 20 min. Process shown in merge and single colour lateral (dorsal up, anterior left) and transverse (dorsal up, medial left) views. Blue lines indicate range of extended orthogonal projection views.

Mentions: To understand whether disappearance of dual-labelled cells reflected loss of markers, cell death, migration or fusion, we performed continuous time-lapse analysis between 1 and 2 dpw. Dual-labelled MPCs were highly dynamic and frequently fused to pre-existing fibres (Fig. 6; Movie 2). Upon fusion, GFP and some RFP immediately filled the host fibre, as predicted from the rapid cytoplasmic GFP diffusion in fibres (Bajanca et al., 2015). However, the fortuitous localisation of a portion of the RFP in punctate structures within some MPCs allowed us to track the location of the fusing MPC and its integration over an hour into the fibre outline (Fig. 6A,B). pax7a:GFP-only MPCs also moved over and between pre-existing fibres, frequently extending and retracting processes and dividing. In continuous 3D time-lapse, two pax7a:GFP-only MPCs can be seen to extend processes to each somite border and form a nascent fibre (Movie 3). Simultaneously, a pax7a:GFP;pax7b:RFP dual-labelled cell leaves the VMZ, divides and one daughter then fuses to the nascent green fibre, which gradually accumulates RFP. Thus, migratory dual-labelled MPCs fuse into nascent fibres (Fig. 6C; Movie 3). Fibre initiation by dual-labelled MPCs was not observed. We conclude that, within wounds, dual-labelled MPCs are rapidly dividing, differentiating and fusing to fibres, consistent with the abundant labelling of fibres by pax7b:RFP (and weak pax7a:GFP), whereas pax7a-only cells behave differently, aligning less in wounds and initiating fibre formation.Fig. 6.


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 to existing myotubes during wound repair. Extended orthogonal projection views of an epaxial somite wound in a pax7a:GFP;pax7b:gal4;UAS:RFP 4 dpf larva showing individual pax7a:GFP;pax7b:RFP dual-labelled (yellow) MPCs fusing to existing unlabelled (A) or RFP+ (B) fibres from Movies 2 and 3. The whole image was non-linearly enhanced and brightness corrected to compensate for bleaching and facilitate tracking of individual cells, as described in Materials and Methods. (A) At 25.5 hpw, prior to fusion, an MPC had uniform cytoplasmic GFP and diffuse cytoplasmic and vesicular RFP (arrow). 10 min later, cytoplasmic GFP and RFP have now filled the whole cytoplasm of a large adjacent previously unlabelled fibre (brackets), whereas the vesicular RFP remains localised (arrowhead) and integrates into the fibre in the succeeding 50 min (see Movie 2, blue MPC). Transverse II shows the same view as Transvere I, but with the fusing fibre marked (dots). (B) At 39.5 hpw, two MPCs (magenta and yellow arrows), fuse simultaneously to the same large adjacent myotube (arrowheads; Movie 2, magenta and yellow MPCs). (C) At 32.3 hpw, a dual-labelled MPC (arrowhead; Movie 3, white MPC) that originated from the anterior somite border, divided and then migrated along a recently formed GFP+ nascent myofibre (arrow). 10 min later the MPC has fused into the nascent fibre, as shown by RFP loss from the MPC and increase in the fibre. The fused cell remains distinct at 10 min, but merges into the nascent fibre by 20 min. Process shown in merge and single colour lateral (dorsal up, anterior left) and transverse (dorsal up, medial left) views. Blue lines indicate range of extended orthogonal projection views.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

DMM022251F6: Fusion of pax7a- and pax7b-reporter cells to existing myotubes during wound repair. Extended orthogonal projection views of an epaxial somite wound in a pax7a:GFP;pax7b:gal4;UAS:RFP 4 dpf larva showing individual pax7a:GFP;pax7b:RFP dual-labelled (yellow) MPCs fusing to existing unlabelled (A) or RFP+ (B) fibres from Movies 2 and 3. The whole image was non-linearly enhanced and brightness corrected to compensate for bleaching and facilitate tracking of individual cells, as described in Materials and Methods. (A) At 25.5 hpw, prior to fusion, an MPC had uniform cytoplasmic GFP and diffuse cytoplasmic and vesicular RFP (arrow). 10 min later, cytoplasmic GFP and RFP have now filled the whole cytoplasm of a large adjacent previously unlabelled fibre (brackets), whereas the vesicular RFP remains localised (arrowhead) and integrates into the fibre in the succeeding 50 min (see Movie 2, blue MPC). Transverse II shows the same view as Transvere I, but with the fusing fibre marked (dots). (B) At 39.5 hpw, two MPCs (magenta and yellow arrows), fuse simultaneously to the same large adjacent myotube (arrowheads; Movie 2, magenta and yellow MPCs). (C) At 32.3 hpw, a dual-labelled MPC (arrowhead; Movie 3, white MPC) that originated from the anterior somite border, divided and then migrated along a recently formed GFP+ nascent myofibre (arrow). 10 min later the MPC has fused into the nascent fibre, as shown by RFP loss from the MPC and increase in the fibre. The fused cell remains distinct at 10 min, but merges into the nascent fibre by 20 min. Process shown in merge and single colour lateral (dorsal up, anterior left) and transverse (dorsal up, medial left) views. Blue lines indicate range of extended orthogonal projection views.
Mentions: To understand whether disappearance of dual-labelled cells reflected loss of markers, cell death, migration or fusion, we performed continuous time-lapse analysis between 1 and 2 dpw. Dual-labelled MPCs were highly dynamic and frequently fused to pre-existing fibres (Fig. 6; Movie 2). Upon fusion, GFP and some RFP immediately filled the host fibre, as predicted from the rapid cytoplasmic GFP diffusion in fibres (Bajanca et al., 2015). However, the fortuitous localisation of a portion of the RFP in punctate structures within some MPCs allowed us to track the location of the fusing MPC and its integration over an hour into the fibre outline (Fig. 6A,B). pax7a:GFP-only MPCs also moved over and between pre-existing fibres, frequently extending and retracting processes and dividing. In continuous 3D time-lapse, two pax7a:GFP-only MPCs can be seen to extend processes to each somite border and form a nascent fibre (Movie 3). Simultaneously, a pax7a:GFP;pax7b:RFP dual-labelled cell leaves the VMZ, divides and one daughter then fuses to the nascent green fibre, which gradually accumulates RFP. Thus, migratory dual-labelled MPCs fuse into nascent fibres (Fig. 6C; Movie 3). Fibre initiation by dual-labelled MPCs was not observed. We conclude that, within wounds, dual-labelled MPCs are rapidly dividing, differentiating and fusing to fibres, consistent with the abundant labelling of fibres by pax7b:RFP (and weak pax7a:GFP), whereas pax7a-only cells behave differently, aligning less in wounds and initiating fibre formation.Fig. 6.

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