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First published online 11 January 2006
doi: 10.1242/dev.02249

Development 133, 601-610 (2006)
Published by The Company of Biologists 2006
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Loss of myogenin in postnatal life leads to normal skeletal muscle but reduced body size

Jennifer R. Knapp1,2, Judith K. Davie1, Anita Myer1, Eric Meadows1,2, Eric N. Olson3 and William H. Klein1,2,*

1 Department of Biochemistry and Molecular Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.
2 Graduate Training Program in Genes and Development, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030, USA.
3 Department of Molecular Biology, The University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.


Figure 1
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  		Fig. 1. Generation of the Myogflox allele, targeted ES cells and heterozygous mice. (A) The top line shows the myogenin locus with its three exons (black rectangles). The second line shows the targeting construct. The region of homologous DNA (thicker line) spans 3.8 kb and is flanked by EcoRI sites. Also shown are loxP sites (red triangles) and a neomycin cassette placed into the BamHI site in the first intron (gray rectangle). A thymidine kinase (TK) cassette (white rectangle) lies upstream of the homology region. The next two lines depict the targeted Myog locus before and after Cre recombinase-mediated deletion of the first exon and neomycin cassette. (B) Southern genome hybridization of ES cell DNA digested with BamHI and HindIII, confirming proper targeting. A 3' probe derived from the third exon immediately 3' to the EcoRI site outside of the homology region was used in the hybridization analysis. The 2 kb band shown on the figure represents the wild-type BamHI fragment bounding the second and third exons (Myog+). The 6 kb band represents the targeted BamHI-HindIII fragment (Myogflox) containing the neomycin cassette. The left and right lanes show DNA from targeted and wild-type ES cell lines, respectively. (C) Southern genome hybridization of tail DNA digested with EcoRI and hybridized with a Myog cDNA probe. Tail DNA was obtained from P10 pups resulting from mating Myog+/- and Myog+/flox mice. The left, idle and right lanes show DNA from Myog+/flox, Myog+/- and Myogflox/- mice, respectively. The 2 kb band representing the Myog- allele is the result of a EcoRI site in the neomycin cassette that is not present in the neomycin cassette of the Myogflox allele. The origins of the 4 kb and 6 kb bands associated with the wild-type and Myogflox alleles, respectively, are shown in A.

 

Figure 2
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  		Fig. 2. Deletion of floxed Myog sequences using CMV-cre and analysis of embryonic skeletal muscle at E18.5. (A) Southern genome hybridization of yolk-sac DNA digested with EcoRI from embryos resulting from a Myogflox/+;CMV-cre/+ intercross and probed with Myog cDNA. Deletion of the floxed myogenin sequences produced a 3 kb EcoRI fragment representing the Myogflox{Delta} allele. The 4 kb EcoRI fragment represents the wild-type allele (Myog+). Lanes 1 and 2 show DNA from Myogflox{Delta}/flox{Delta} embryos; lanes 3 and 4 show Myogflox{Delta}/+ embryos; and lanes 5 and 6 show wild-type embryos. (B) Diaphragms of E18.5 Myog+/-, Myog-/- and Myogflox{Delta}/flox{Delta} embryos. The genotype is shown above each image. Scale bar: 100 µm. (C) Hindlimb sections from E18.5 Myogflox{Delta}/+ and Myogflox{Delta}/flox{Delta} embryos immunostained with polyclonal M225 anti-myogenin antibody. Arrows indicate positively stained cells. Scale bar: 100 µm.

 

Figure 3
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  		Fig. 3. Perinatal lethality associated with Myogflox{Delta}/flox{Delta} mice. Mice with the genotype Myogflox/flox;+/+ were mated to Myogflox/flox;CAGGCre-ERTM/+ mice and pregnant females resulting from the cross were injected with tamoxifen at E15.5 or E17.5 days of pregnancy. (A,B) Histograms show the number of mice surviving to P10 after tamoxifen injection of pregnant females at E17.5 (A) or E15.5 (B). Genotypes are shown below each figure.

 

Figure 4
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  		Fig. 4. Deletion of Myog and attenuation of myogenin transcripts and protein in Mygflox{Delta}/flox{Delta} mice. (A) Quantitative PCR of tail DNA to determine the extent of deletion of floxed Myog sequences in Myogflox/flox and Myogflox{Delta}/flox{Delta} mice at P10. The ratio of Myog to Myf5 genomic DNA from one Myogflox/flox mouse was arbitrarily set to 1 and all other ratios from both Myogflox/flox and Myogflox{Delta}/flox{Delta} genotypes were normalized to that value. (B) Southern genome hybridization with EcoRI-digested DNA extracted from the hindlimb skeletal muscle of 10-week-old mice from a Myogflox/flox;+/+ x Myogflox/flox;CAGGCre-ERTM/+ cross and probed with Myog cDNA. The 6 kb and 3 kb bands represent the Myogflox and Myogflox{Delta} alleles, respectively, as depicted in Fig. 1. The skeletal DNA in lanes 1 and 3 contained very little of the Myogflox allele and therefore represents a Myogflox{Delta}/flox{Delta} genotype, while the skeletal DNA in lanes 2, 4, 5 and 6 contained no Myogflox{Delta} allele and therefore represents a Myogflox/flox genotype. (C) Quantitative RT-PCR to determine the levels of Myog transcript expression in hindlimb skeletal muscle from mice at 3 days (right panel) and 2 weeks (left panel) of age. The genotypes are shown below the histograms. The ratio of Myog to ribosomal protein L7 RNA for one RNA sample with a Myogflox/flox genotype was arbitrarily set to 1, and all other ratios from both Myogflox/flox and Myogflox{Delta}/flox{Delta} genotypes were normalized to that value. The median value for each genotype is shown as a black dot. Asterisks indicate significant differences between the two genotypes. The arrow bars indicate one s.d. from the median. (D) Hindlimb sections from Myogflox/flox (right panel) and Myogflox{Delta}/flox{Delta} (left panel) mice at 3 days of age immunostained with the monoclonal F5D anti-myogenin antibody. Arrows indicate positively stained cells. Scale bar: 100 µm.

 

Figure 5
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  		Fig. 5. Skeletal muscle from Myogflox/flox and Myogflox{Delta}/flox{Delta} mice at 10 weeks of age. (A-D) Cross-sections (A,B) and longitudinal sections (C,D) of hindlimbs. (E,F) Sections through tongues. (G-J) Sections through diaphragms. One Myogflox{Delta}/flox{Delta} mouse has a thin diaphragm (H). Thin diaphragms were found in only a single pair of mice from a single litter. All other Myogflox{Delta}/flox{Delta} mice had normal diaphragms (J). Scale bar: 100 µm. (K) Identical myofiber diameters were observed in Myogflox/flox and Myogflox{Delta}/flox{Delta} mice. Myofiber diameters were determined from cross-sections of hindlimbs of mice at 10 weeks of age. Each column represents the diameters determined for individual myofibers from a single hindlimb section (squares). The genotypes are shown below the graph.

 

Figure 6
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  		Fig. 6. Skeletal muscle gene expression of Mck, Mrf4, Myod1 and Myf5 in Myogflox/flox and Myogflox{Delta}/flox{Delta} mice. Quantitative RT-PCR was performed on RNA prepared from hindlimb muscle at 3 days (A) and 2 weeks (B) of age. In each case, the ratio of myogenin to ribosomal protein L7 RNA for one RNA sample with a Myogflox/flox genotype was arbitrarily set to 1, and all other ratios from both Myogflox/flox and Myogflox{Delta}/flox{Delta} genotypes were normalized to that value. The median value for each genotype is shown as a black dot. Asterisks indicate significant differences between the two genotypes. Error bars indicate one s.d. from the median.

 

Figure 7
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  		Fig. 7. Body size and weight differences between Myogflox/flox and Myogflox{Delta}/flox{Delta} mice during postnatal growth. (A) Image of Myogflox/flox and Myogflox{Delta}/flox{Delta} littermates at 12 weeks of age. (B) Growth curves for two litters of Myogflox/flox and Myogflox{Delta}/flox{Delta} mice from 1.5 to 9 weeks of age. Each data point represents one individual mouse. (C) Range in body weights for the two genotypes at 12 weeks of age. Asterisk indicates significant mean weight difference between the two genotypes. Error bars indicate one s.d. from the mean.

 

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© The Company of Biologists Ltd 2006