Loss of Ptpn11 (Shp2) drives satellite cells into quiescence
- Max Delbrück Center for Molecular Medicine (MDC) in the Helmholtz Society, Germany
- Experimental and Clinical Research Center, Charité Medical Faculty and Max Delbrück Center for Molecular Medicine Berlin, Germany
- Children's Cancer Therapy Development Institute, United States
- Leibniz Institute for Molecular Pharmacology, Germany
Figures
Figure 1 with 1 supplement
Conditional Ptpn11 mutation leads to a deficit in postnatal muscle growth.
(a) Western blot analysis of Ptpn11 in YFP-positive cells isolated by FACS from limbs of control and coPtpn11 mutant mice that carry the RosaeYFP allele; YFP-positive cells from E15.5 and P0 animals …
Figure 1—figure supplement 1
Genetic strategy used to mutate Ptpn11 and isolation of myogenic stem cells.
(a) Genetic strategy used to mutate Ptpn11 using Pax7Cre and Ptpn11flox alleles. (b) Gating strategy to isolate YFP+ and YFP- cells from E14.5 and P0 Pax7Cre;RosaeYFP animals by FACS. YFP …
Figure 2 with 2 supplements
Ptpn11 is essential for proliferation of muscle stem cells in neonatal mice.
(a) Immunohistological analysis of Pax7 (red) and laminin (green) in muscle of control and coPtpn11 mutant mice; DAPI (blue) was used as a counterstain. Arrowheads point to Pax7+ cells. (b) …
Figure 2—figure supplement 1
Ptpn11 mutation leads to decreased numbers of myogenic progenitor cells but does not affect apoptosis.
(a) Gating strategy used to isolate VCAM1+ CD31- CD45- Sca1- myogenic cells from P0 animals. VCAM1 and the sum of CD31, CD45, and Sca1 fluorescence intensities are displayed on the x- and y-axis, …
Figure 2—figure supplement 2
Ptpn11 mutation impairs proliferation of satellite cells.
(a) Quantification of mRNA transcripts related to cell cycle, myogenesis, autophagy, transdifferentiation in YFP+ cells isolated by FACS from control and coPtpn11 mutant limb muscle at P0. (b) …
Figure 3
Loss of Ptpn11 does not affect myogenic differentiation.
(a) Cytospins of freshly isolated muscle stem cells from control and coPtpn11 mice (P0); cells were immunostained for Pax7 (red) and MyoG (green), and nuclei were counterstained with DAPI (blue). (b)…
Figure 4 with 1 supplement
Ptpn11 controls Mapk/Erk1/2 activation in myogenic cells.
(a) Western blot analysis of phosphorylation of Mapk/Erk1/2, PI3K/Akt and Mapk/p38 in C2C12 cells cultured in the presence of either vehicle or GS493 (5, 10, 20 and 30 µM). (b) Western blot analysis …
Figure 4—figure supplement 1
Efficient recombination of the Ptpn11flox allele in adult satellite cells and floating fiber cultures.
(a) Genetic strategy used to induce tamoxifen-dependent mutation of Ptpn11 and to activate Map2k1DD expression in adult satellite cells. (b) Gating strategy to isolate VCAM1+ CD31- CD45- Sca1- …
Figure 5 with 1 supplement
Activation of Mapk/Erk1/2 signaling rescues proliferation of Ptpn11-mutant satellite cells.
Fibers from adult Txcontrol and TxcoPtpn11 mice were cultured for the indicated times, and analyzed for VCAM1 (red) and Ki67 (green) protein; the inset depicts Ki67 staining (green). Nuclei were …
Figure 5—figure supplement 1
Ptpn11/Mapk/Erk signaling affects satellite cell differentiation in floating single myofiber cultures.
(a) Immunostaining of VCAM1 (red) and MyoG (green) on fibers from Txcontrol and TxcoPtpn11 mice cultured for 72 hrs. Nuclei were counterstained with DAPI (blue). (b) Quantification of VCAM1+ cells …
Figure 6 with 1 supplement
Ptpn11 is essential for long-term maintenance of satellite cells in sedentary adult muscle.
(a) Outline of the experiment. (b) Immunostaining for VCAM1 (red), Pax7 (green), and laminin (blue) of tibialis anterior muscle from Txcontrol and TxcoPtpn11 mice 10 days after tamoxifen; arrowheads …
Figure 6—figure supplement 1
Ptpn11 is essential for long-term maintenance of satellite cells and muscle regeneration.
(a) Outline of the experiment. (b) Quantification of Pax7+VCAM1+ satellite cells/100 fibers in tibialis anterior muscle before (t0), 10 (t1), and 90 (t2) days after tamoxifen administration. (c) …
Figure 7 with 1 supplement
Ptpn11 is essential for skeletal muscle repair.
(a) Outline of the regeneration experiment. (b) Immunostaining for laminin (blue) of injured muscle from Txcontrol and TxcoPtpn11 animals seven days after injury (7dpi). Nuclei were counterstained …
Figure 7—figure supplement 1
Mutation of Ptpn11 in satellite cells leads to a severe muscle regeneration deficit and accumulation of fibrotic and adipose tissue.
(a) Haematoxylin/eosin staining of adult tibialis anterior muscle seven days after injury. (b) Staining for C/EBPα (green), collagen IV (ColIV, red) and DAPI (blue) of muscle from Txcontrol and …
Figure 8 with 1 supplement
Pharmacological inhibition of Ptpn11 leads to a premature cell cycle withdrawal of satellite cells at late stages of muscle regeneration.
(a) Outline of the experiment. (b) Immunostaining for Pax7 (red), Ki67 (green) and collagen IV (CollV; blue) in regenerating tibialis anterior muscle seven days after injury (7dpi). Mice were …
Figure 8—figure supplement 1
Pharmacological inhibition of Ptpn11 at late stages of muscle regeneration does not induce apoptosis or adipogenesis.
(a) Outline of the experiment. (b) Immunostaining for Pax7 (red), TUNEL (green) and laminin (blue) on regenerating muscle after treatment with vehicle or GS493. (c) Quantification of …
Tables
Table 1
Gene expression changes in Ptpn11 mutant satellite cells.
| Cell cycle associated genes | FC | FDR |
|---|---|---|
| Ccnd1 Cyclin D1 | -2.58 | 0.00363 |
| Ccnb1 Cyclin B1 | -2.33 | 0.00144 |
| Prc1 Protein regulator of cytokinesis 1 | -2.32 | 0.01522 |
| Ranbp1 Ran-binding protein 1 | -2.18 | 0.00081 |
| Cdc2a Cyclin dependent kinase 1a | -2.11 | 0.00402 |
| Immediate early genes | ||
| Egr4 Early growth response 4 | -3.00 | 0.00300 |
| Tnfrsf12a FGF-inducible 14/ Tweak-receptor | -2.65 | 0.00144 |
| Egr2 Early growth response 2 | -2.58 | 0.00318 |
| Plk2 Polo-like-kinase 2 | -2.57 | 0.00004 |
| Extracellular matrix/ secreted proteins | ||
| Adamts2 ADAM Metallopeptidase 2 | 3.15 | 0.00144 |
| Thbs2 Thrombospondin 2 | 2.89 | 0.00519 |
| Mfap5 Microfibrillar-associated protein 4 | 2.84 | 0.01179 |
| Col1a1 Collagen 1a1 | 2.75 | 0.00374 |
| Mfap2 Microfibrillar-associated protein 2 | 2.62 | 0.00179 |
| Col1a2 Collagen 1a2 | 2.29 | 0.00082 |
| Col16a1 Collagen 16a1 | 2.17 | 0.00681 |
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FC: fold-change; FDR: false discovery rate; corrected p-value (q-value) determined as described (Hochberg and Benjamini, 1990).
Additional files
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Supplementary file 1
List of all deregulated genes in neonatal Ptpn11 mutant muscle stem cells.
Transcripts labeled in colors are also shown in Table 1 and are associated with particular GO terms. Green: cell cycle-associated; yellow: immediate early gene; purple: extracellular matrix and matrix remodeling. FDR: false discovery rate. Corrected p-values (q-value) was determined as described (Hochberg and Benjamini, 1990).
- https://doi.org/10.7554/eLife.21552.019
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Supplementary file 2
List of enriched GO terms.
Enriched GO terms in deregulated genes in Ptpn11 mutant muscle stem cells. FDR: false discovery rate. Corrected p-values (q-value) was determined as described (Hochberg and Benjamini, 1990).
- https://doi.org/10.7554/eLife.21552.020
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Supplementary file 3
Statistical data for each experiment shown in Figures 1–8 and Figure 1—figure supplement 1; Figure 2—figure supplement 1; Figure 2—figure supplement 2; Figure 4—figure supplement 1; Figure 5—figure supplement 1; Figure 6—figure supplement 1; Figure 7—figure supplement 1; Figure 8—figure supplement 1.
Shown are the definition of the center, the error bars, the statistical test used, n-numbers for each genotype or treatment for a specific developmental stage and the corresponding p-value for each comparison. S.E.M.: standard error of the mean.
- https://doi.org/10.7554/eLife.21552.021
