spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 9 May 2006
doi: 10.1242/jcs.02952

Journal of Cell Science 119, 2185-2195 (2006)
Published by The Company of Biologists 2006
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rooney, J. E.
Right arrow Articles by Burkin, D. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rooney, J. E.
Right arrow Articles by Burkin, D. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Severe muscular dystrophy in mice that lack dystrophin and {alpha}7 integrin

Jachinta E. Rooney1,*, Jennifer V. Welser1,*, Melissa A. Dechert1, Nichole L. Flintoff-Dye1, Stephen J. Kaufman2 and Dean J. Burkin1,3,{ddagger}

1 Department of Pharmacology, University of Nevada, Reno, NV 89557, USA
2 Department of Cell and Developmental Biology, University of Illinois, Urbana, IL 61801, USA
3 Nevada Transgenic Center, University of Nevada, Reno, NV 89557, USA


Figure 1
View larger version (54K):

[in a new window]
  		Fig. 1. mdx/{alpha}7-/- mice lack dystrophin and {alpha}7 integrin. (A) Multiplex PCR and ARMS assays were performed on genomic tail DNA to detect wild-type versus {alpha}7-integrin-targeted allele and the wild-type versus mdx mutation, respectively. The wild-type {alpha}7-integrin allele (727 bp) was detected in wild-type and mdx mice, whereas the targeted {alpha}7-integrin allele (482 bp) was detected in the {alpha}7-/- and double-knockout mice. Wild-type dystrophin (275 bp) was detected in wild-type and {alpha}7-/- mice, whereas the mdx mutation (275 bp) was present in the mdx and double-knockout mice. Neg, negative control lacking DNA. (B) Western blotting with anti-{alpha}7A-integrin and {alpha}7B-integrin antibodies in gastrocnemius muscle of 3-week-old mice confirmed the absence of {alpha}7-integrin protein in {alpha}7-/- and mdx/{alpha}7-/- mice. (C) Immunofluorescence with anti-CA5.5 antibody, which recognizes {alpha}7 integrin, demonstrated loss of the {alpha}7 integrin in triceps muscle from 3-week-old {alpha}7-/- and mdx/{alpha}7-/- mice. The absence of dystrophin in mdx and mdx/{alpha}7-/- mice was verified by immunofluorescence with an anti-dystrophin antibody. Bar, 10 µm.

 

Figure 2
View larger version (40K):

[in a new window]
  		Fig. 2. Muscle-wasting and reduced viability in mdx/{alpha}7-/- mice. (A) Photograph of mdx and mdx/{alpha}7-/- littermates aged 18 days. Double-knockout mice appear smaller than mdx littermates and show outward signs of kyphosis. These mice also exhibit joint contractures, reduced mobility and tremors (see supplementary material, Movie 1). (B) The average weight of 10-day-old male mdx/{alpha}7-/- mice was indistinguishable from that of mdx littermates. The weight of both mdx and mdx/{alpha}7-/- mice was significantly different from wild-type and {alpha}7-/- animals (*P<0.05). (C) The average weight of 21-day-old male mdx/{alpha}7-/- mice was significantly different from wild-type, mdx, and {alpha}7-/- animals (*P<0.05), indicating that double-knockout mice failed to gain weight. (D) The viability of mdx/{alpha}7-/- mice (n=21) is severely reduced compared with wild-type (n=12), {alpha}7-/- (n=148) and mdx (n=19) mice. All mdx/{alpha}7-/- mice died at 16-26 days.

 

Figure 3
View larger version (32K):

[in a new window]
  		Fig. 3. Reduced sarcolemmal integrity in muscle from mdx/{alpha}7-/- mice. (A) Evan's Blue dye (EBD) uptake was used to assess membrane integrity of the tibialis anterior skeletal muscle. Myofibers were outlined with Oregon Green-488-conjugated WGA (green). EBD uptake (red) was not detected in wild-type and {alpha}7-/- myofibers. By contrast, significant EBD uptake was observed in muscle from mdx and mdx/{alpha}7-/- animals. Bar, 10 µm. (B) The number of EBD-positive muscle fibers was assayed. Wild-type and {alpha}7-/- muscle had less than 1% EBD-positive fibers. By contrast, 18.9% of fibers were positive for EBD in mdx/{alpha}7-/- mice, but this was not significantly different from the number of EBD-positive fibers seen in mdx animals.

 

Figure 4
View larger version (69K):

[in a new window]
  		Fig. 4. Severe muscle pathology in mdx/{alpha}7-/- mice. (A) Hematoxylin and eosin staining of tibialis anterior muscle from 3-week-old mice shows the characteristically uniform fiber diameters in wild-type mice. Small necrotic regions and variations in myofiber size were observed in muscle of 3-week-old mdx mice. At this age, muscle from {alpha}7-/- mice appeared to be similar to wild-type. By contrast, muscle from mdx/{alpha}7-/- mice exhibited extreme variations myofiber size and large areas of mononuclear cell infiltration (arrow). Bar, 10 µm. (B) Inflammation in the muscle of mdx/{alpha}7-/- mice was quantified by counting the number of CD4-positive immune cells per field of view in triceps muscle of 3-week-old mice. Muscle of wild-type, mdx and {alpha}7-/- animals showed less than 1% CD4-positive cells. By contrast, an average of 43.88 CD4-positive immune cells per field was observed in the triceps muscle of mdx/{alpha}7-/- mice, which was significantly higher when compared with control genotypes (*P<0.05).

 

Figure 5
View larger version (45K):

[in a new window]
  		Fig. 5. Enhanced myofiber regeneration in mdx/{alpha}7-/- mice. (A) Hematoxylin and eosin staining of tibialis anterior muscle of 3-week-old mice to analyze the localization of nuclei within myofibers. Centrally localized nuclei were observed in the muscle of mdx/{alpha}7-/- mice. Compared with control genotypes they indicate extensive muscle regeneration, which follows degeneration of diseased myofibers. Central nuclei are indicated by arrows. Bar, 10 µm. (B) Quantitation of centrally-located nuclei in triceps and tibialis anterior muscles. As expected, wild-type mice exhibited peripherally located nuclei in both muscle groups. Centrally located nuclei were observed in muscle fibers from mdx and {alpha}7-/- mice compared with wild-type (2.8% and 0.32%, respectively, for triceps muscle; 4.1% and 3.2%, respectively, for tibialis anterior muscle). By contrast, mdx/{alpha}7-/- mice showed 40.1% and 17.5% centrally located nuclei in triceps and tibialis anterior muscle, respectively, which are significantly higher than controls (*P<0.05).

 

Figure 6
View larger version (33K):

[in a new window]
  		Fig. 6. Increased expression of eMyHC in mdx/{alpha}7-/- mice. (A) Immunofluorescence was used to detect eMyHC expression in tibialis anterior muscle fibers from 3-week-old mice. Muscle from wild-type, mdx and {alpha}7-/- mice showed few eMyHC-positive fibers (green). Many muscle fibers from mdx/{alpha}7-/- mice were positive for eMyHC. Rhodamine-labeled WGA (red) was used to define muscle fibers. Bar, 10 µm. (B) Quantitation of eMyHC-positive fibers. Few eMyHC-positive muscle fibers were counted in wild-type, mdx or {alpha}7-/- mice. By contrast, an average of 7.46 fibers per field were positive for eMyHC in mdx/{alpha}7-/- mice, which is significantly different from the controls (*P<0.05).

 

Figure 7
View larger version (65K):

[in a new window]
  		Fig. 7. Altered expression of laminin-{alpha}2 chain in the skeletal muscle of mdx/{alpha}7-/- mice. (A) Immunofluorescence was used to detect the localization of ß1D and laminin-{alpha}2 chain in triceps muscle of 3-week-old mice. A similar pattern of ß1D localization at the myofiber sarcolemma was observed in wild-type, {alpha}7-/-, mdx and mdx/{alpha}7-/- mice. Immunofluorescence revealed a decrease in laminin-{alpha}2 chain in the muscle of {alpha}7-/- and mdx/{alpha}7-/- mice compared with both wild-type and mdx mice. Bar, 10 µm. (B) Laminin-{alpha}2 expression in the gastrocnemius muscle was quantitated by western analysis. mdx mice showed a significant increase in laminin-{alpha}2 expression compared with wild-type. {alpha}7-/- and mdx/{alpha}7-/- mice showed a decrease in laminin-{alpha}2-chain expression compared with wild-type and mdx animals (*P<0.05).

 

Figure 8
View larger version (48K):

[in a new window]
  		Fig. 8. Increased utrophin expression in mdx but not mdx/{alpha}7-/- mice. (A) Utrophin localization was examined by immunofluorescence in the gastrocnemius muscle of 3-week-old wild-type, mdx, {alpha}7-/- and mdx/{alpha}7-/- mice. As expected, utrophin was localized at the NMJs of muscle from wild-type mice and at neuromuscular and extrajunctional sites of muscle from mdx animals, confirming previous studies. Utrophin localization appeared at both junctional and extrajunctional sites in {alpha}7-/- or mdx/{alpha}7-/- mice. Bar, 10 µm. (B) Utrophin expression in the gastrocnemius muscle was quantitated by western analysis. mdx mice showed a statistically significant increase in utrophin expression compared with wild-type (*P<0.05). {alpha}7-/- and mdx/{alpha}7-/- mice showed no increase in utrophin compared with wild-type animals.

 

Figure 9
View larger version (210K):

[in a new window]
  		Fig. 9. Ultrastructure of MTJs in muscle from 3-week-old wild-type, mdx, {alpha}7-/- and mdx/{alpha}7-/- mice. The sarcolemma of the MTJs of wild-type and mdx mice is highly folded, increasing the surface area between muscle and tendon (arrows); the extracellular matrix at this site is highly organized and electron-dense. By contrast, MTJs of {alpha}7-/- and mdx/{alpha}7-/- mice show little or no sarcolemmal folding (arrows) and the extracellular matrix appeared less dense and organized compared with those of wild-type and mdx mice (arrowheads). Bar, 1 µm.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2006