Pax3/Pax7 mark a novel population of primitive myogenic cells during development

Abstract

Skeletal muscle serves as a paradigm for the acquisition of cell fate, yet the relationship between primitive cell populations and emerging myoblasts has remained elusive. We identify a novel population of resident Pax3+/Pax7+, muscle marker-negative cells throughout development. Using mouse mutants that uncouple myogenic progression, we show that these Pax+ cells give rise to muscle progenitors. In the absence of skeletal muscle, they apoptose after down-regulation of Pax7. Furthermore, they mark the emergence of satellite cells during fetal development, and do not require Pax3 function. These findings identify critical cell populations during lineage restriction, and provide a framework for defining myogenic cell states for therapeutic studies.

Figure 1.

Pax3/Pax7 and MRF expression identify distinct cell populations in the somite. (A) Pax3 and Pax7 are coexpressed in almost all dermomyotomal (DM), but not myotomal (My) cells at E9.75; transverse section of interlimb somites. Pax3 also marks dorsal root ganglion (DRG) cells. (B) Scheme representing proposed cell order in skeletal muscle. MRFs (Myf5, Mrf4, Myod) are expressed in embryonic progenitors as well as myoblasts (precursors), but not in the presumed stem cell. (C) X-gal staining of E11.5 Myf5^nlacZ/+ and Myf5^nlacZ/nlacZ embryos showing absence of a myotome (My) in the latter. (D-F) Immunohistochemistry of cryostat sections of E11.5 embryos showing merged images of antibody stainings in low magnifications (high magnifications are shown in boxed areas). Pax⁺ cells enter the myotome dorsally (“parachute,” arrows). (D) anti-Pax7, anti-βgal stainings show Pax7⁺/β-gal^- cells in the myotome in Myf5^nlacZ/+ embryo as well as coexpression. (E) anti-Pax3, anti-βgal stainings show release of the Pax⁺ population into presumed myotome space in the absence of a myotome in Myf5^nlacZ/nlacZ. Boxed area shows coexpression of Pax3 and β-gal in developmentally arrested MPCs in ventral dermomyotome lip in Myf5^nlacZ/nlacZ. (F) anti-Pax7, anti-MyHC stainings show exclusion in the myotome of Pax3^Sp/Sp at E11.5. (G) The majority of Pax7⁺ cells are excluded from Desmin⁺ myoblasts and Myogenin⁺ (Myog) differentiating cells at E11.5. (H) Identification of distinct cell states in Myf5^nlacZ/nlacZ embryos. In the absence of Desmin⁺ precursors, Pax⁺/MRF^- (stem) and Pax⁺/MRF⁺ (progenitors) are identified in E. Images on sections are confocal optical sections. Arrowheads (C; low magnifications, D-F) indicate dorsal (red) and ventral (white) dermomyotome. (NT) Neural tube; (Sc) sclerotome; (DIC) differential interference contrast microscopy. Bars: A, 20 μM; D-F, low magnifcations, 50 μM; D-G, high magnifications, 10 μM.

Figure 2.

Embryonic and fetal muscle progenitors are genetically distinct. (A) Immunohistochemistry with anti-MyHC antibody shows that fetal skeletal muscles are lacking at E18.5 in Myf5^GFP-P/GFP-P:Myod^-/- mutants (arrowhead, residual embryonic muscle) compared with the Myf5^nlacZ/+ control (arrowhead, diaphragm). (B) Western blot; anti-Mrf4 antibody does not cross-react with the other MRFs when overexpressed in HEK293T cells. (C) Pax3 and Mrf4 proteins are coexpressed in a nucleus of an embryonic progenitor in the dermomyotome (DM) at E10.5 (arrowhead). Mrf4⁺ cells are also located in the myotome (My). GFP autofluorescence from Myf5^GFP-P is not detected under these fixation conditions. (D) X-gal staining (dark nuclei) of E15.5 Mrf4^nlacZ/+:Myf5^loxP/+ muscles shows that Mrf4 is not coexpressed with Pax7 (arrowheads) in stem/progenitors in the fetus (see E). Myogenin (arrowheads) is observed in X-gal-negative cells; therefore, Mrf4 is expressed later in differentiation. (E) Proposed model for generation of progenitors from stem cells. (h) Heart; (li) liver. Bars: A, 1 mm; B,C, 10 μM.

Figure 3.

Uncoupling fetal from embryonic myogenesis points to a hierarchical cell order in the muscle lineage. (A) Immunohistochemistry of cryostat sections showing Pax7⁺/β-gal^- cells in trunk muscles of Myf5^nlacZ/+ embryo at E14 (arrowheads). Asterisks indicate coexpressing cells. (B) Individual Pax7⁺/Desmin^- cells (red) are associated with residual intercostal Desmin⁺ embryonic fibers in the absence of fetal myogenesis at E16.5 in Myf5^GFP-P/GFP-P:Myod^-/- fetus. (C) Some of these cells are negative with an anti-GFP antibody (arrowheads). Anti-Pax7, anti-GFP stainings reveal coexpression in some MPCs (asterisks). (D) Anti-activated Caspase3 antibody in E16.5 control Myf5^GFP-P/+ (left, intercostal; inset, mesenchyme with apoptotic cells) and E14.5 Myf5^GFP-P/GFP-P:Myod^-/- fetuses. No apoptotic cells in control muscle, and abundant apoptotic cells in the mutant; the latter shows no colocalization with Pax7⁺ cells (see inset, right). (E) Antibodies against activated Caspase3 and anti-GFP reveal apoptosis in myogenic cells in E14.5 Myf5^GFP-P/GFP-P:Myod^-/- fetuses (arrowhead). GFP autofluorescence from Myf5^GFP-P is not detected under these fixation conditions. (F) Scheme indicating that differentiating skeletal muscles are required for maintenance of the primitive myogenic cells. Bars, 10 μM.

Figure 4.

Resident Pax7⁺ cells mark the emergence of satellite cells and do not require Pax3. (A) Immunohistochemistry of cryostat sections shows Pax7⁺ (anti-Pax7; arrowhead) cells associated with the muscle fiber by anti-laminin (green) staining in trunk muscles of Myf5^GFP-P/+ fetus at E16.5. (B) E18.5 Pax7⁺ satellite cell in trunk muscle coexpressing the cell proliferation marker PM-Histone3 (green). (C) Freshly isolated forelimb fiber from a Myf5^nlacZ/+ new-born shows a Pax7⁺/β-gal^- satellite cell. On section and single fibers at birth, this fraction comprises <1% of mononucleated β-gal^- cells. (D) Pax7⁺ satellite cells at E17 in Pax3^Sp/Sp:Myf5^nlacZ/+ mutant. Some remain X-gal (dark nuclei) negative (arrowhead). Bars, 10 μM.