Pax7a expressing muscle precursor cells preferentially generate new fibres during larval muscle growth

Technical Summary: Pax7a-expressing muscle precursor cells preferentially generate new fibres during larval muscle growth

Problem Statement
The mechanisms controlling skeletal muscle size in vertebrates remain poorly understood, specifically regarding how muscle precursor cells (mpcs) decide between two distinct growth modes: hyperplasia (forming new muscle fibres) and hypernucleation (fusing with pre-existing fibres to increase nuclear content). While distinct mpc subpopulations with founder cell (FC) and fusion-competent cell (FCM) roles are well-characterized in Drosophila, similar molecularly distinct subpopulations in vertebrates have not been clearly defined or linked to specific growth outcomes. In zebrafish, although it is known that mpcs contribute to both new fibre formation and the addition of nuclei to existing fibres, the specific identities and fates of these subpopulations during early larval development remain unclear.

Methodology
The study utilized zebrafish (Danio rerio) larvae to perform high-resolution, time-lapse imaging of muscle growth. The authors employed a combination of genetic tools and molecular techniques:

• Transgenic Reporters: The study used existing transgenic lines (pax7a:GFP and pax7b:RFP) and generated a new knock-in line (pax7a:kalTA4) to track the expression of the two zebrafish pax7 paralogs.
• Lineage Tracing: A Kaede photoconversion strategy (using Tg(acta:Gal4);Tg(UAS:Kaede)) was used to distinguish pre-existing muscle fibres from newly formed ones by converting green fluorescent Kaede to red, allowing the visualization of new green-only fibres.
• Genetic Manipulation: The study utilized myogenin (myog) mutants to block myocyte fusion, tbx6 mutants, and mesp-b antisense morpholinos to disrupt early somite patterning and assess the dependence of specific mpc populations on these pathways.
• Imaging and Quantification: Confocal microscopy was used to track cell numbers, locations, and division rates (via time-lapse) in specific somites (15–18) from 1 to 5 days post-fertilization (dpf). Immunodetection of Pax7, Pax3, and sarcomeric MyHC was used to validate reporter expression.

Key Results
The research identified three distinct populations of muscle precursor cells based on their expression of pax7a and/or pax7b, which exhibit differential spatial distributions, proliferation rates, and fates:

1. Pax7b-only mpcs:

   • Expression & Location: These cells appear early (before pax7a) and are concentrated in the central dermomyotome near the horizontal myoseptum.
   • Dynamics: They contribute to the initial wave of fast muscle fibre formation but subsequently show little proliferation or differentiation. Their numbers do not increase significantly during larval growth.
   • Dependency: The formation and maintenance of this population depend on the Tbx6-Mesp-b signaling axis; disruption of this pathway leads to a specific loss of Pax7b-only cells and fibres.
   • Fate: They contribute minimally to larval myotome growth after the embryonic stage.

2. Pax7a-only mpcs:

   • Expression & Location: These cells are concentrated at the epaxial (dorsal) and hypaxial (ventral) extremes of the myotome.
   • Dynamics: They undergo rapid proliferation, increasing approximately sevenfold between 1 and 5 dpf.
   • Fate: This population is the primary driver of hyperplasia (new fibre formation). They preferentially form new, thin, oligonucleate fibres at the dorsoventral edges of the myotome.
   • Mechanism: Using myogenin mutants, the authors demonstrated that the formation of these new fibres requires the fusion of Pax7a-only mpcs with one another. In the absence of Myogenin, the number of nascent fibres increases (due to a failure to fuse into multinucleate fibres), confirming that these cells normally fuse to form oligonucleate nascent fibres.

3. Dual-marked (Pax7a+ and Pax7b+) mpcs:

   • Expression & Location: Found in intermediate regions, including vertical myosepta and central areas.
   • Dynamics: They proliferate rapidly (approx. fivefold increase) and are highly motile, entering the deep myotome.
   • Fate: Their primary contribution to larval growth is hypernucleation. They frequently fuse with pre-existing muscle fibres rather than initiating new ones.

Significance and Claims
The paper claims to demonstrate that muscle stem cell diversity arises early in primary myogenesis and underpins the regulation of muscle growth. The authors conclude that:

• Molecularly distinct mpc subtypes, defined by pax7a and pax7b expression, have non-interconvertible fates.
• Pax7a-only mpcs act as the primary founders of new muscle fibres (hyperplasia) at the myotome edges, a process dependent on Myogenin-mediated fusion among themselves.
• Pax7b-only mpcs represent a quiescent-like population located at the horizontal myoseptum that contributes to early fibre formation but largely ceases activity during larval growth.
• Dual-marked mpcs primarily contribute to muscle growth by adding nuclei to existing fibres (hypernucleation).
• The data suggest that this diversity of mpc populations may be an evolutionarily ancient vertebrate synapomorphy, potentially homologous to distinct subpopulations in amniotes, and that the sub-functionalization of the duplicated pax7 genes in teleosts allows for the distinct regulation of these growth modes.

The authors explicitly state that they do not claim to have identified the mechanism for the decision between hyperplasia and hypernucleation, nor do they claim that Pax7b-only cells are definitively quiescent satellite cells, though they share characteristics with such populations. The study focuses on characterizing the spatial and behavioral heterogeneity of these cells during normal larval development.