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

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.

Regulation and phylogeny of skeletal muscle regeneration

“…In zebrafish larvae, muscle injury by puncture wounds to the ventral myotome induces proliferation of SLCs, differentiation and fusion to repair damaged myofibres (Knappe et al, 2015). Of note, the Pax7 gene is duplicated in zebrafish (Pax7a and Pax7b), and they differ in expression pattern and function: Pax7a-cells participate in repair of the first wave of nascent fibres whereas Pax7b-cells generate larger fibres (Pipalia et al, 2016). The ablation of one population or the other results in deficits in repair suggesting lack of compensation (Pipalia et al, 2016).…”

“…Of note, the Pax7 gene is duplicated in zebrafish (Pax7a and Pax7b), and they differ in expression pattern and function: Pax7a-cells participate in repair of the first wave of nascent fibres whereas Pax7b-cells generate larger fibres (Pipalia et al, 2016). The ablation of one population or the other results in deficits in repair suggesting lack of compensation (Pipalia et al, 2016). Similarly, it has been shown in the adult electric fish (S. macrurus) that muscle repair following tail amputation also involves Pax7-positive SLCs, but not myofibre dedifferentiation (Weber et al, 2012).…”

Transposons As Tools for Functional Genomics in Vertebrate Models

“…7). Since the body of a zebrafish larva is transparent through all developmental stages, such transgenic fish have been used to visualize the vascular system [108], the central and peripheral nervous systems [109, 110], the regenerating processes of the sensory system and fin [111, 112], the cellular process of muscle wound repair [113], and proteolytic cleavage of the extracellular domain of a membrane protein (Neuregulin) in the motor neurons [114], among others. Also, the transgenic fish were applied to image the activity of specific neuronal populations or endothelial cells by targeted expression of a calcium indicator GCaMP [115, 116].…”

“…( E ) Caudal trunk (and the wound epidermis) in HGn21A embryo at 1 dpf [112]. ( F ) Fgf7b-positive muscle cells in gSAIzGFFD164A at 1 dpf [113]. ( G ) Central nervous system and motor neurons in the spinal cord in SAIGFF213A at 1 dpf [114].…”

Myotome adaptability confers developmental robustness to somitic myogenesis in response to fibre number alteration

“…In Drosophila, distinct molecular pathways create founder myoblasts, which initiate fibres, and fusion competent myoblasts, which augment fibre growth (Abmayr and Pavlath, 2012). Our recent analyses of zebrafish muscle repair (Knappe et al, 2015;Pipalia et al, 2016) revealed two Pax7-expressing myoblast subpopulations with similarities to founder and fusion-competent cells (Pipalia et al, 2016). Whether such myoblast diversity underlies fibre formation during development and thereby determines muscle size in vertebrates is unknown.…”