A regulatory protein thought to be crucial for maintaining the muscle stem-cell pool throughout life is shown to be dispensable in the adult. Muscle biologists are left wondering what fundamental things apply as time goes by.
When, as we grow up, we abandon our beloved teddy bear, we don't throw it away — we merely ignore it, a desertion that has parallels in skeletal-muscle development. Lepper et al.1 (page 627 of this issue) show that the Pax7 transcription factor, which is required for early juvenile muscle growth in mice, becomes inessential in the adult, although it is still expressed.
The sequence of cellular events that gives rise to skeletal muscle in vertebrates is similar throughout prenatal development and postnatal growth, and during muscle regeneration in the adult. The proliferation of precursor cells is followed by the differentiation and fusion of most cells into muscle fibres containing multiple nuclei. In all instances, some undifferentiated cells remain as reserves that can give rise to muscle tissue. In mice, just before birth, most of these undifferentiated cells become enclosed in the basement membrane that develops around each muscle fibre, forming a reservoir of muscle stem cells known as satellite cells. This reservoir is the main source of cells for postnatal muscle growth and repair.
The similarities in cellular events in the different phases of muscle development are paralleled by strong similarities in molecular mechanisms. The transcription factors Pax3 and Pax7, which activate genes that are essential for muscle development, perform an intriguing pas de deux throughout embryonic and fetal muscle development2. Their functions seem largely to overlap, although Pax3 seems to be essential for some processes, such as migration of precursor muscle cells from their site of origin in the somites (the tissue that develops on either side of the embryonic central axis) to sites of muscle formation in the limbs and body wall2(Fig. 1a). More recently, Pax7 was shown to be required for the maintenance of the satellite-cell population after birth. In mice that lack the Pax7 gene (Pax7 germline-null mice), muscle develops normally up to birth, but fails to grow or regenerate after this3. It was initially proposed3 that Pax7 is necessary to specify satellite cells, but it is now believed that it is required for satellite-cell survival4,5. How Pax7 performs this function is unknown, but it is generally thought that, in limb skeletal muscle, Pax7 takes over from Pax3 to support satellite-cell survival in postnatal life (Fig. 1b). Indeed, Pax7 expression is used as a definitive marker of satellite cells.
The work by Lepper et al.1 profoundly upsets this status quo. The authors use an elegant approach to knock out Pax7 after birth in mice. In these animals, only one of the two Pax7 genes is functional, and the functional Pax7 gene is engineered to be ablated, solely in cells that express it (satellite cells), on giving the drug tamoxifen. The same mechanism that ablates Pax7 simultaneously induces expression of a reporter molecule, allowing this specific satellite-cell population to be tracked.
When Pax7 was ablated at birth, limb skeletal muscle failed to grow or regenerate after injury, similar to mice that lack Pax7 from conception (the germline-null mutants). However, surprisingly, when Pax7 was ablated in mice at 2 to 3 weeks of age and beyond, limb skeletal-muscle growth and regeneration was normal. And by detecting the reporter molecule, the authors showed1 that the cells undertaking muscle regeneration were satellite cells that had expressed Pax7 at the time of the tamoxifen-induced knockout. This last point is significant, because several studies have shown that muscle cells can arise from non-satellite cells associated with blood vessels or interstitial tissue, prompting suggestions6 that such cells are important for regeneration and are a precursor population of the satellite cell. Both notions are challenged, at least in the short term, by the finding that virtually all satellite cells in regenerated muscle were marked as being derived from Pax7-ablated progenitors.
An alternative explanation for the retention of the regenerative ability of Pax7-deficient satellite cells is that these cells might re-express Pax3, which may compensate for the absence of Pax7. But Lepper and colleagues1 disproved this by showing that ablating both Pax3 and Pax7 in adult mice did not impair limb skeletal-muscle regeneration.
Thus, it seems that the cells that give rise to limb skeletal muscle, having been dependent on either Pax3 or Pax7 throughout most of prenatal life, abruptly cast off these developmental shackles towards the end of the postnatal growth period. Moreover, this liberation from Pax dependence seems to be intrinsic to the satellite cells, as ablation of Pax7 in tissue culture leads to impaired muscle formation in precursor muscle cells isolated from young but not older muscle.
Intriguingly, the transition of satellite cells to Pax7 independence occurs during the period when there was shown to be a sharp decrease in fusion of Pax7-expressing cells into the growing muscle fibres1. It perhaps marks the time when muscle fibres begin to respond predominantly to environmental signals, and to undertake regeneration in response to injury, or enlargement in response to the demands of physiological work, as opposed to processes driven predominantly by developmental mechanisms.
The development of adult muscle cells in the absence of Pax7 and Pax3 has left researchers with a lot of explaining to do. Given the increasing appreciation of the diversity of different types of skeletal muscle, it would be worthwhile investigating the effects of Pax3 and Pax7 ablation in some muscles of the head, which are not Pax3 dependent7, and in muscles of the diaphragm and body wall, where satellite cells continue to express Pax3 in the adult5 (Fig. 1b). It is also important to examine the longer-term effects of ablation of the Pax genes and to try to relate the phases of muscle development in the mouse to the quite different time frame of muscle development and use in humans.
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Journal of Developmental Origins of Health and Disease (2014)
Gene Expression Patterns (2013)