QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 12 August 2008
doi: 10.1242/jcs.026534

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Otto, A. Articles by Patel, K. Search for Related Content PubMed PubMed Citation Articles by Otto, A. Articles by Patel, K. Social Bookmarking                         What's this?

Canonical Wnt signalling induces satellite-cell proliferation during adult skeletal muscle regeneration

¹ School of Biological Sciences, AMS Building, University of Reading, Whiteknights, PO Box 228, Reading, Berkshire RG6 6AJ, UK
² Institute of Anatomy, Ludwigs-Maximilians-University of Munich, Pettenkoferstr. 11, 80336 Munich, Germany
³ Department of Veterinary Basic Sciences, Royal Veterinary College, London NW1 0TU, UK
⁴ Dubowitz Neuromuscular Unit, Imperial College, South Kensington Campus, London SW7 2AZ, UK
⁵ Brighton and Sussex Medical School, Falmer Campus, Brighton BN1 9PX, UK

View larger version (61K): [in this window] [in a new window]   Fig. 1. Isolated adult muscle fibres and the Wnt-signalling pathway. (A) RT-PCR of Wnt genes from single fibres cultured for 48 hours. Lane 1, Wnt11; Lane 2, Wnt5a; Lane 3, Wnt3a; Lane 4, Wnt1. The position of the 500 bp size marker is indicated. (B) TOPFlash Wnt reporter activity of isolated single muscle fibres. Control activity was normalised to 1. Isolated fibres gave 2.15 times the control stimulation (*P<0.01). LiCl (positive control) gave stimulation of 2.5 times control (**P<0.001). (C) EGCG, a compound that promotes β-catenin degradation inhibits satellite-cell proliferation. Control fibres cultured for 72 hours resulted in 75.3±7.8 (s.e.m.) satellite cells per myofibre, compared with 31.9±3.2 on myofibres treated with EGCG (*P<0.0001, n=25 control and n=35 EGCG-treated myofibres). (D) Pan β-catenin expression on freshly isolated myofibres (t=0 hours). Pan β-catenin was localised at the periphery of the satellite cell (green arrowhead). (E) Double staining for pan β-catenin and the nucleus. Pan β-catenin localised around the edge of the nucleus at t=0 (green arrowhead). (F) Double staining for Act-β-Cat and the nuclei at t=0 hours. Act-β-Cat was not detected in freshly isolated fibres. (G) Double staining for Act-β-Cat and MyoD after 6 hours in culture. MyoD-positive satellite cells do not show the presence of Act-β-Cat (red arrowhead). Adjacent myonucleus (MyoD-negative) shows faint Act-β-Cat expression (green arrowhead). (H) Double staining for Act-β-Cat and MyoD after 24 hours in culture. MyoD-positive satellite cells coexpress low levels of Act-β-Cat (white arrowhead). (I) Satellite cells prior to division, double stained for Act-β-Cat and MyoD after 48 hours in culture. MyoD-positive satellite cells robustly coexpress Act-β-Cat (white arrowheads). (J) Double staining for Act-β-Cat and MyoD after 48 hours in culture in satellite cells after division. MyoD-positive nuclei show weak coexpression of Act-β-Cat (red arrowhead). However, strong Act-β-Cat expression localised between two divided cells (green arrowhead). (K) Double staining for Act-β-Cat and myogenin after 72 hours in culture. Rare large cluster of satellite cells expressing Act-β-Cat but not myogenin is shown. (L) Image shown in K overlaid with DAPI. Large cluster of satellite cells express Act-β-Cat localised to the nuclei. (M) Double staining for Act-β-Cat and myogenin after 72 hours in culture. A small cluster showing mutually exclusive expression of Act-β-Cat (green arrowhead) and myogenin (red arrowhead). (N) Same image shown in M overlaid with DAPI, confirming the nuclear localisation. Myogenin was expressed in cells that failed to express Act-β-Cat (red arrowhead). Adjacent cells show two profiles of Act-β-Cat expression, either at the membrane (green arrowhead), reminiscent of J, or predominantly in the nucleus (white arrowhead) similar to I. (O) Double staining for Act-β-Cat and BrdU after 36 hours in culture. Single activated satellite cell shows coexpression of both markers (yellow arrowhead). (P) Same image shown in O overlaid with DAPI, confirming the nuclear localisation of Act-β-Cat and BrdU (white arrowhead). (Q) Satellite-cell progeny at 72 hours stained for BrdU and DAPI. Culture was pulsed with BrdU at 48 hours. Two BrdU-positive nuclei are indicated (red arrowheads). (R) Same image shown in Q overlaid with Act-β-Cat expression, which localises predominantly to the BrdU-positive nuclei (white arrowheads). (S) Percentage of satellite-cell nuclei positive for BrdU alone, BrdU and Act-β-Cat and neither marker was quantified at 48 and 72 hours of culture. At 48 hours, 91.8% of satellite-cell nuclei were Act-β-Cat+ BrdU+, 6.3% were Act-β-Cat– BrdU+, and 1.5% were Act-β-Cat– BrdU–. At 72 hours, 74.5% of satellite-cell nuclei were Act-β-Cat+ BrdU+, 24.8% were Act-β-Cat– BrdU+, and 0.7% were Act-β-Cat– BrdU–. n=24 myofibres for each time point.

View larger version (29K): [in this window] [in a new window]   Fig. 2. Effect of ectopic Wnt proteins on satellite-cell proliferation. (A,B) Quantification of satellite-cell proliferation after a 36 hour culture period in the presence of Wnt1, Wnt3a, Wnt4, Wnt5a and Wnt6. All data are given as averages ± s.e.m. (A) Average cluster size following culture period: Wnt1 (2.6±0.09), Wnt3a (2.3±0.21) and Wnt5a (2.4±0.24) treated cultures all gave a greater number of satellite-cell progeny per cluster than LacZ controls (1.35±0.03), #P<0.0001, ¶P=0.0001 and P=0.0002, n=834, n=463, n=66 and n=51 clusters for LacZ, Wnt1, Wnt3a and Wnt5a, respectively. Wnt4 (1.03±0.01) and Wnt6 (1.13±0.02) showed significantly smaller cluster sizes than LacZ control cultures, *P<0.0001 and **P<0.0001, n=231 and n=548 clusters for Wnt4 and Wnt6, respectively. (B) Average total number of satellite cells per myofibre following culture period: Wnt1 (12.83±0.82) and Wnt3a (12.25±0.75) treated cultures both gave a greater number of satellite-cell progeny per myofibre than LacZ controls (10.45±0.57), #P=0.028 and ¶P<0.05, n=124 and n=78 myofibres for Wnt1 and Wnt3a, respectively). Wnt5a gave a higher number of progeny cells per myofibre than control (11.09±1.1); however, this was not statistically significant. Wnt4 (7.18±0.47) and Wnt6 (7.83±0.53) both showed a significantly lower number of satellite cells per myofibre than LacZ control cultures, *P<0.001 and **P=0.0015, n=32 and n=92 myofibres for Wnt4 and Wnt6, respectively. (C-H) Single fibres cultured for 36 hours in Wnt1 (C,F), Wnt6 (D,G) and control cells (E,H). Wnt1 resulted in Pax7-expressing clusters of multiple cells (green arrows). By contrast, Wnt6 rarely showed Pax7-positive clusters and typically gave only single cells (green arrow). Control cells gave rise to small clusters (green arrow). (I) Quantification of satellite-cell activation by Wnt proteins. No statistical difference was detected in the number of MyoD+ cells per myofibre following coculture in Wnt1, Wnt6 or LacZ control conditions for 2 hours. n=21, n=25 and n=50 myofibres for Wnt1, Wnt6 and LacZ cultures, respectively. P=0.44 for Wnt1 and P=0.2 for Wnt6 when compared with the control. All results are mean ± s.e.m.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Reversible action of inhibitory Wnt proteins on satellite-cell proliferation. Quantification of satellite-cell progeny on fibres cultured in two consecutive 36 hour stages. Fibres cultured for 36 hours in Wnt6 and then transferred to another dish of Wnt6-expressing cells for an additional 36 hours gave significantly lower number of satellite-cell progeny compared with control (*P<0.02). Fibres cultured initially with control cells and then transferred to Wnt6-expressing cells gave fewer progeny than fibres cultured in control cells alone (although this was not statistically significant). Fibres cultured in Wnt6 and then transferred to Wnt1-expressing cells gave significantly more progeny compared with fibres cultured in Wnt6 alone (P<0.005) or those in control or Wnt6-expressing cell conditions (P<0.05). Fibres cultured in control cells and then transferred to Wnt1 (control-Wnt1) gave significantly more satellite-cell progeny compared with Wnt6-Wnt1 (*P<0.02), control-Wnt6 (P<0.00001), Wnt6-Wnt6 (P<0.00001) and control-control (¶P<0.001). (B) Myogenic profiling after two consecutive 36 hour periods with control cells gave medium-sized clusters that either expressed myogenin (red arrowhead) or nuclear Pax7 (green arrowhead). (C) Profiling after incubation with control cells followed by Wnt1 cells gave rise to large clusters that predominantly expressed Pax7 (green arrowhead) with a minority of myogenin-positive cells (red arrowhead). (D) Incubation of fibres in two consecutive periods with Wnt6 gave small clusters of myogenic progeny expressing either myogenin (red arrowhead) or Pax7 (green arrowhead).

View larger version (55K): [in this window] [in a new window]   Fig. 4. Activated satellite cells show nuclear localisation of Act-β-Cat with Wnt1 but not Wnt6. Isolated fibres were cultured in the presence of Wnt1, Wnt6 or control cells for 2 hours. (A,D,G,J) Satellite cells following culture with Wnt1. (A) Robust expression of Act-β-Cat (green arrowhead). (D) Robust expression of MyoD (red arrowhead). (G) Colocalisation of Act-β-Cat and MyoD (white arrowhead). (J) Confocal slice-scan between points indicated by asterisks in G showing nuclear colocalisation of MyoD and Act-β-Cat. (B,E,H,K) Satellite cells following culture with Wnt6. (B) Weak expression of Act-β-Cat (green arrowhead). (E) Robust expression of MyoD (red arrowhead). (H) Mutually exclusive expression of Act-β-Cat (green arrowhead) and MyoD (red arrowhead). (K) Confocal slice-scan between points indicated by asterisks in H showing Act-β-Cat localisation flanking nuclear MyoD. (C,F,I,L) Satellite cells following culture with control cells. (C) No expression of Act-β-Cat in satellite cells (green arrowhead) but weak expression in adjacent myonuclei (blue arrowhead). (F) Robust expression of MyoD in satellite cell (red arrowhead). (I) Myonuclei express Act-β-Cat (green arrowhead) whereas satellite cells express MyoD (red arrowhead). (L) Confocal slice-scan between points indicated by asterisks in I showing no Act-β-Cat expression in the MyoD-positive satellite cell. (M) Quantification of BrdU+ Act-β-Cat+ satellite cell nuclei following 2 hours of culture in Wnt1, Wnt6 and LacZ control conditions. Myofibres exposed to Wnt1 conditions showed a significantly higher number of satellite cells expressing BrdU and nuclear Act-β-Cat (1.52±0.31) compared with LacZ controls (0.04±0.03), *P<0.0001, n=23 and n=27 myofibres for Wnt1 and LacZ, respectively. BrdU+ Act-β-Cat+ satellite-cell nuclei were never seen on Wnt6-treated myofibres (n=15 myofibres).

View larger version (102K): [in this window] [in a new window]   Fig. 5. Activated β-catenin expression during in vivo skeletal muscle regeneration. (A-P) Acute muscle regeneration following cardiotoxin injection of the tibialis anterior muscle in mouse. (A-H) 6 days after injection. (I-P) 15 days after injection. (A) Haematoxylin and eosin staining showing heterogeneously sized muscle fibres containing centrally located nuclei. (B) Enlarged region of adjacent section to that in A showing Act-β-Cat localisation to centrally (red arrowhead) or peripherally (yellow arrowheads) located nuclei. (C) Act-β-Cat localisation to centrally (red arrowhead) or peripherally (yellow arrowheads) located nuclei within laminin-expressing myofibre. (D) Clusters of robustly expressing Pax7-positive cells (green arrowheads). (E) Weak expression of Caveolin 3 to mark the regenerating sarcolemma. Note variable fibre diameter and shapes. (F) High-power confocal image showing expression of Act-β-Cat (yellow arrowheads), (G) the nucleus (red arrowheads) and (H) an overlay showing predominantly nuclear localisation of Act-β-Cat (white arrowheads). (I) 15 days after injury, haematoxylin and eosin staining showed muscle with mostly uniform sized fibres with very few displaying centrally located nuclei. (J) Weak expression of Act-β-Cat located outside of the nuclei (yellow arrowhead). (K) Localisation of Act-β-Cat adjacent to laminin expression. (L) Pax7 expression becomes infrequent. (M) Robust expression of Caveolin 3. On rare occasions, Act-β-Cat was found adjacent to a nuclei but high-power confocal imaging showed its expression (N) in relation to the nucleus (O) was complimentary (P). (Q-F1) Human muscle regeneration. (Q-T) Chronic muscle regeneration. (Q) Variably sized fibres with centrally located nuclei. (R) Nuclear localisation of Act-β-Cat (white arrowheads). (S) Centrally (red arrowheads) and peripherally (yellow arrowhead) located nuclei showed expression of Act-β-Cat. (T) Robust expression of Pax7 (green arrowheads). (U-X) Control human muscle showing no signs of regeneration. (U) Haematoxylin and eosin staining showing peripheral nuclei and a lack of tissue infiltrate. (V) Sparse non-nuclear Act-β-Cat expression (green arrowheads). (W) Rare Act-β-Cat within laminin-positive muscle fibres (green arrowhead). (X) Rare Pax7 expression beneath muscle fibre lamina (green arrowhead). (Y-B1) Chronic human muscle regeneration. (Y) Robust Pax7 and Ki67 double-positive nuclei showing dividing satellite cells (yellow arrowheads). (Z) Overlay of image shown in Y with DAPI confirming nuclear localisation (white arrowheads). (A1) Strong expression of Act-β-Cat in Ki67-positive cells (yellow arrowheads). (B1) Overlay of the image shown in A1 with DAPI confirming nuclear localisation of the Act-β-Cat and Ki67 signal (white arrowheads). (C1-F1) Control human muscle. (C1) Rare expression of Pax7 in a quiescent satellite cell negative for Ki67 (green arrowhead). (D1) overlay of the image shown in C1 with DAPI confirming the nuclear localisation of Pax7 (white arrowhead). (E1) Rare pockets/membranous regions of Act-β-Cat expression are noted along with Ki67-negative muscle tissue (green arrowhead). (F1) Overlay of the image shown in E1 with DAPI, confirming the non-nuclear localisation of Act-β-Cat (green arrowhead).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter