Fibroblast growth factor 6 regulates sizing of the muscle stem cell pool

Abstract

Skeletal muscle stem cells, i.e., satellite cells (SCs), are the essential source of new myonuclei for skeletal muscle regeneration following injury or chronic degenerative myopathies. Both SC number and regenerative capacity diminish during aging. However, molecular regulators that govern sizing of the initial SC pool are unknown. We demonstrate that fibroblast growth factor 6 (FGF6) is critical for SC pool scaling. Mice lacking FGF6 have reduced SCs of early postnatal origin and impaired regeneration. By contrast, increasing FGF6 during the early postnatal period is sufficient for SC expansion. Together, these data support that FGF6 is necessary and sufficient to modulate SC numbers during a critical postnatal period to establish the quiescent adult muscle stem cell pool. Our work highlights postnatal development as a time window receptive for scaling a somatic stem cell population via growth factor signaling, which might be relevant for designing new biomedical strategies to enhance tissue regeneration.

Keywords: FGF6; development; fibroblast growth factor; regeneration; satellite cells; skeletal muscle stem cells.

Figure 1

SC hypoplasia in Fgf6 mutant mice (A–H) Representative dual anti-PAX7/anti-Laminin IF stainings on 10-μm cryosections from diaphragm (A and B) and hindlimb muscle groups: soleus (C and D), TA (E and F), and EDL (G and H) from 3-month-old male Wt (A, C, E, and G) and Fgf6^−/− mice (B, D, F, and H) at 40× magnification; arrows indicate PAX7 (red)-positive nuclei (blue) within the basal lamina (green); scale bar in (H), 50 μm. (I and J) Quantification of average SC numbers per 0.155-mm² area of 10-μm cryosections (I) and normalized as fold difference (J) from male Wt and Fgf6^−/− diaphragm, soleus, TA, and EDL muscles, and female Wt and Fgf6^−/− TA muscles (n = 4 adult mice for each genotype); paired two-tailed Student’s t test: ^∗p < 0.05; ^∗∗p < 0.01.

Figure 2

No histological anomalies or myofiber size differences in Fgf6 mutant mice under baseline conditions (A–H′) Representative images of H&E-stained 10-μm cryosections from diaphragm (A and B′), TA (C and D′), soleus (E and F′), and EDL (G and H′) from adult (3-month-old) male Wt (A, C, E, and G) and Fgf6^−/− mice (B, D, F, and H) at 10× magnification (A–H) and 40× magnification (A′–H′); scale bar for 10× images in (H), 100 μm, and for 40× images in (H′), 10 μm. (I) Average muscle fiber sizes in μm² for diaphragm (Dia), soleus, TA, and EDL muscles; n = 6 adult mice for each genotype. (J) Average minimum Feret diameter in μm for diaphragm (Dia), soleus, TA, and EDL muscles; n = 6 adult mice for each genotype. (K) Total EDL muscle fiber number; n = 4 3-month-old mice for each genotype; paired two-tailed Student’s t test: not significant (ns), p > 0.05. (L) Myonuclei count normalized to muscle fibers for diaphragm (Dia), soleus, TA, and EDL muscles; n = 4 3-month-old mice for each genotype; paired two-tailed Student’s t test: not significant (ns), p > 0.05.

Figure 3

SC hypoplasia emerges during perinatal stages in Fgf6 mutant mice (A–D‴) Representative dual anti-PAX7/anti-Laminin IF stainings on 10-μm cryosections from Wt (A–A‴, C–C‴) and Fgf6^−/− (B–B‴, D–D‴) lower hindlimb muscles at postnatal day 7 (P7, A and B‴) and day 14 (P14, C and D‴). Individual images for DAPI (blue, (A–D), Laminin (green, A′–D′), Pax7 (A″–D″), and the merged overlay (A‴–D‴) are shown at 20× magnification; scale bar in (D‴), 50 μm. (E–H) Average SC numbers per 0.155-mm² area of 10-μm hindlimb muscle cryosections (E and G) and normalized as fold difference (F and H) from P7 (E and F) and P14 (G and H); n = 4 adult male mice for each genotype; paired two-tailed Student’s t test: not significant (ns), p > 0.05; ^∗∗p < 0.01.

Figure 4

Reduced SC proliferation in Fgf6 mutant mice at P14 (A–D″) Representative images of anti-PAX7 IF and EdU stainings on 10-μm cryosections from Wt (A–A″, C–C″) and Fgf6^−/− (B–B″, D–D″) lower hindlimb muscles at postnatal day 7 (P7, A–B″) and day 14 (P14, C–D″). Individual images for EdU (blue, (A–D), PAX7 (red, A′–D′), and the merged overlay (A″, B″, C″, D″) are shown at 20× magnification; arrows indicate nuclei labeled with both EdU and PAX7; scale bar in (D″), 50 μm. (E and F) Quantification of PAX7⁺ SCs positive for EdU per 0.155-mm² area of 10-μm cryosections at P7 (E) and P14 (F) from Wt and Fgf6^−/− hindlimb muscles; n = 4 early postnatal mice for each genotype; paired two-tailed Student’s t test: ^∗p < 0.05.

Figure 5

Injury-induced muscle regeneration is compromised in Fgf6 mutant muscles (A–F) Representative images of H&E-stained 10-μm cryosections from Wt (A–C) and Fgf6^−/− (D–F) TA muscles at 3.5 DPI (A, D), 5 DPI (B, E), and 21 DPI (C, F) post injury via intra-muscular cardiotoxin delivery; scale bar for (A, D) in (D), 20 μm; scale bar for (B, C, E, and F) in (F), 50 μm. (G–L) Quantification of regenerative TA myofiber sizes in μm² (G–I) and minimum Feret in μm (J–L) of Wt and Fgf6^−/− mice at 3.5 DPI (G and J), 5 DPI (H and K), and 21 DPI (I–L); n = 3–8 adult male mice per genotype and regeneration time point; paired two-tailed Student’s t test: not significant (ns), p > 0.05; ^∗p < 0.05; ^∗∗p < 0.01.

Figure 6

SC hypoplasia persists after injury-induced muscle regeneration (A and B) Representative dual anti-PAX7/anti-Laminin IF stainings on 10-μm cryosections from 21 DPI TA muscles from Wt (A) and Fgf6^−/− (B) mice at 20× magnification; arrows indicate PAX7 (red)-positive nuclei (blue) within the basal lamina (green). (C and D) Quantification of SCs in regenerated tissue (identified via centrally located myonuclei); average SC numbers per 0.155-mm² area of 10 μm regenerated TA cryosections (C) and normalized as fold difference (D) are displayed; n = 4 adult male mice per genotype; scale bar in (B), 50 μm; paired two-tailed Student’s t test: ^∗∗∗p < 0.001. (E and F) Representative images of H&E-stained 10-μm cryosections of TA muscles from Wt (E) and Fgf6^−/− (F) mice at 20× magnification following three consecutive cardiotoxin-induced injury/regeneration cycles, each 21 days apart. (G and H) Quantification of average triple-regenerated TA muscle fiber size in μm² (G) and minimum Feret in μm (H) from Wt and Fgf6^−/− mice; n = 4 four adult male mice for both genotypes tested; scale bar in (F), 50 μm.

Figure 7

SC increase after perinatal intra-muscular FGF6 protein delivery (A) Schematic for experimental design: 2 μg of recombinant FGF6 was delivered via injection into the right TA, while the left TA received normal saline as control at P14; TA muscles were harvested at 2 months. (B and C) Representative dual anti-PAX7/anti-Laminin IF stainings on 10-μm cryosections from FGF6- (B) and control saline-injected (C) TA muscles at 20× magnification; arrows indicate PAX7 (red)-positive nuclei (blue) within the basal lamina (green); scale bar in (C), 50 μm. (D and E) Quantification of average SC numbers per 0.155-mm² area of 10-μm cryosections (D) and normalized as fold change (E) from saline and FGF6 perinatally injected TA muscles; n = 6 2-month-old mice for each group tested; paired two-tailed Student’s t test: ^∗∗p < 0.01. (F) Quantification of average muscle fiber size in μm² from saline and FGF6 perinatally injected TA muscles; n = 6 2-month-old mice for each group tested; paired two-tailed Student’s t test: not significant (ns), p > 0.05. (G) Myonuclei count normalized to muscle fibers for saline and FGF6 perinatally injected TA muscles; n = 6 2-month-old mice for each genotype; paired two-tailed Student’s t test: not significant (ns), p > 0.05. (H) Schematic for experimental design: 5 μg of recombinant FGF6 was delivered via injection into the right TA, while the left TA received normal saline as control at P90; TA muscles were harvested 2 weeks later (P104). (I and J) Representative dual anti-PAX7/anti-Laminin IF stainings on 10-μm cryosections from FGF6- (I) and control saline-injected (J) TA muscles at 20× magnification; arrows indicate PAX7 (red)-positive nuclei (blue) within the basal lamina (green); scale bar in (J), 50 μm. (K) Quantification of average SC numbers per 0.155-mm² area of 10-μm cryosections from saline and FGF6-injected TA muscles; n = 5 adult male mice for each group tested; paired two-tailed Student’s t test: not significant (ns), p > 0.05. See also Figure S1.