JCS015354 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Genotyping genomic DNA using a primer mismatch assay (see Materials and Methods) gave reliable results. WT and M indicate wild-type and mutant primer combinations, and +/+, +/− and −/− indicate wild-type, heterozygous and homozygous nmf417 DNA samples. respectively.

• Supplemental Figure S2 -

  Fig. S2. A growth curve of body weights from five nmf417 homozygote mice, five unaffected littermates and a strain-matched control are shown. Weights do not diverge significantly until 5 weeks of age (P<0.01), markedly later than severe alleles such as dy^W or dy^3K (null) mice, suggesting that the nmf417 mutation is a hypomorph.

• Supplemental Figure S3 -

  Fig. S3. The anticipated enrichment of extracellular matrix molecules at the NMJ is not altered in nmf417 homozygotes. NMJs were identified by counterstaining with α-bungarotoxin (BTX, middle column). Merged images are shown at the right. Laminin α2, α4, α5 and β2 were examined as well as agrin (agr). Scale bar: 15 µm.

• Supplemental Figure S4 -

  Fig. S4. The muscle pathology observed in nmf417 was consistent in both the plantaris (Fig. 3) and the soleus, a muscle composed of <50% slow, type I myosin fibers. Low- and high-magnification images indicate that nmf417 homozygotes again show a severity similar to dy^2J/dy^2J, which is milder than dy/dy. Scale bar: 300 µm for low-magnification images; 75 µm for the higher magnification series.

• Supplemental Figure S5 -

  Fig. S5. The regenerative capacity of nmf417/nmf417 plantaris muscle was assessed by counting total muscle-fiber number and the percentage of fibers with central nuclei. Regenerative capacity depends on BL integrity. Comparisons were therefore made to dy/dy and dy^2J/dy^2J muscles. The plantaris in dy/dy mice had a reduction in total fiber number compared with controls at P59 (P=0.059 with only three dy/dy animals analyzed), whereas the reduction in fiber number for dy^2J/dy^2J and nmf417/nmf417 was not significant. The percentage of fibers with central nuclei was significantly higher than control in all Lama2 alleles; the dy/dy allele had a lower percentage of fibers with central nuclei, although the difference from dy^2J/dy^2J and nmf417/nmf417 was not significant. These results are consistent with dy^2J/dy^2J and nmf417/nmf417 retaining more regenerative capacity than dy/dy. All mice were P59 when examined; n=7 control, 4 dy^2J/dy^2J, 5 nmf417/nmf417 and 3 dy/dy samples.

• Supplemental Figure S6 -

  Fig. S6. The dy^2J phenotype is much more variable than that of nmf417. Ventral roots of the L5 spinal nerves from three additional animals are shown. The root from animal dy^2J/dy^2J #2 is comparable to that shown in Fig. 6 and to nmf417/nmf417 samples. However, #1 has a much higher percentage of myelinated fibers and #4 has many thinly myelinated fibers within the axon bundles. This thin myelination was confirmed by electron microscopy (bottom panels). The roots of nmf417 homozygotes were all comparable in their amyelination phenotype.