Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on June 22, 2004

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Human Molecular Genetics, 2004, Vol. 13, No. 16 1775-1784
DOI: 10.1093/hmg/ddh190
Human Molecular Genetics, Vol. 13, No. 16 © Oxford University Press 2004; all rights reserved

Laminin 1 chain reduces muscular dystrophy in laminin 2 chain deficient mice

Kinga Gawlik¹, Yuko Miyagoe-Suzuki², Peter Ekblom¹, Shin'ichi Takeda² and Madeleine Durbeej^1,*

¹Department of Cell and Molecular Biology, Section for Cell and Developmental Biology, University of Lund, Lund, Sweden and ²Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Kodaira, Tokyo, Japan

Received April 14, 2004; Accepted June 11, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Laminin (LN) 2 chain deficiency in humans and mice leads to severe forms of congenital muscular dystrophy (CMD). Here, we investigated whether LN1 chain in mice can compensate for the absence of LN2 chain and prevent the development of muscular dystrophy. We generated mice expressing a LN1 chain transgene in skeletal muscle of LN2 chain deficient mice. LN1 is not normally expressed in muscle, but the transgenically produced LN1 chain was incorporated into muscle basement membranes, and normalized the compensatory changes of expression of certain other laminin chains (4, ß2). In 4-month-old mice, LN1 chain could fully prevent the development of muscular dystrophy in several muscles, and partially in others. The LN1 chain transgene not only reversed the appearance of histopathological features of the disease to a remarkable degree, but also greatly improved health and longevity of the mice. Correction of LN2 chain deficiency by LN1 chain may serve as a paradigm for gene therapy of CMD in patients.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Laminins (LN), major components of basement membranes, are heterotrimers of -, ß- and -chains. The five -, three ß- and three -chains give rise to at least 15 different protein isoforms that differ in their tissue distribution (1,2). Mutations in the LAMA2 gene encoding the LN2 chain—the main chain in skeletal muscle—cause congenital muscular dystrophy (CMD) with LN2 chain deficiency. In European populations this accounts for about 50% of the classical CMDs (3). This disorder shows autosomal recessive inheritance and is characterized by neonatal onset of muscle weakness, hypotonia, early muscle fiber degeneration and white matter abnormalities (4–6).

Two knock-out mouse models (dy^w/dy^w, dy^3K/dy^3K) and three spontaneous mutant mouse strains (dy/dy, dy^2J/dy^2J, dy^Pas/dy^Pas) representing animal models for CMD with LN2 chain deficiency have been reported (7–12). The dy^w/dy^w mice still express small amounts of a truncated LN2 chain, whereas the dy^3K/dy^3K mice are completely deficient in LN2 chain. Both strains develop early and severe clinical signs of muscular dystrophy (7–9). In addition, LN2 chain deficiency in mice results in defects in multiple tissues including peripheral and central nervous systems (7,8,13–15).

The development of therapies for muscular dystrophy involves in vivo strategies aiming to introduce a normal copy of the defective gene (16). Indeed, it has been demonstrated that a human LN2 chain transgene can rescue the dystrophic symptoms in the dy^w/dy^w mouse (8). However, one major obstacle of gene transfer is the tendency of the immune system to reject novel antigens (16). Instead, delivery of homologous genes already expressed at other sites in the body could eradicate these concerns. Utrophin can compensate for dystrophin deficiency and prevent the development of muscular dystrophy in a mouse model for Duchenne muscular dystrophy (17). Yet, there is no evidence that homologous gene therapy would work in CMD.

In several mouse models for LN deficiency other LN chains are upregulated. The LN4 chain is upregulated in the LN2 chain deficient muscle, but this upregulation is inadequate to prevent muscular dystrophy (18). Similarly, the upregulation of LNß1 chain in the glomerular basement membrane of LNß2 chain deficient kidneys does not prevent nephrosis (19). In addition, some basement membranes in LN5 chain deficient mice are ultrastructurally defective despite ectopic deposition of other -chains (20). Thus, whether LN chains functionally can compensate for each other in vivo remains to be determined.

Here, we analyzed whether LN1 chain, which is mainly expressed in epithelial cells (21,22), could compensate for LN2 chain deficiency and rescue the dystrophic symptoms in LN2 chain deficient dy^3K/dy^3K mice. LN1 chain was chosen as a therapeutic protein, because this -chain is structurally most similar to LN2 chain (1,23). Furthermore, LN-1, which contains the 1-chain, can significantly promote myogenesis in vitro (24), perhaps by binding to integrins (25) or dystroglycan (26). Yet, there are also notable differences between the LN1 and LN2 chains. The 2-chain binds much more efficiently to dystroglycan than the 1-chain (26). Myoblast spreading is significantly faster on 2LNs than on 1LNs (27), and 2LNs have been reported to be specifically required for myotube stability and survival in vitro (28). Therefore, it was by no means clear from previous studies that LN1 chain would compensate for lack of 2-chain in muscles in vivo.

We demonstrate that expression of LN1 chain transgene in skeletal muscles of dy^3K/dy^3K mice reduces the dystrophy symptoms in these animals as evaluated by histology of muscle, weight gain and longevity of the animals. Our data also illustrate for the first time that LN chains can functionally compensate for each other in vivo.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Generation and characterization of LN1 chain transgenic mice
LN1 chain is mainly limited to some epithelial basement membranes in adult mice (21). To achieve broad expression of LN1 chain as a transgene, the cDNA for mouse LN1 chain was inserted into a vector driven by the cytomegalovirus (CMV) enhancer and the chicken ß-actin promoter, followed by the rabbit ß-globin polyadenylation signal (29) (Fig. 1A). Fifty-one mice were born from microinjected fertilized eggs. Thirteen of the mice carried the transgene as detected by Southern blot analyses (data not shown). Our primary goal was to study the effects of the LN1 transgene in LN2 chain deficient muscle. Thus, we selected mice expressing LN1 chain in skeletal muscle. Five of the 13 mice showed immunofluorescence staining of LN1 chain to a varying degree in skeletal muscles. Skeletal muscles from line No. 12 contained high expression of LN1 chain, and were selected for further analysis and for production of dy^3K mice lacking LN2 chain but expressing LN1 chain. The data presented were obtained with mice derived from line No. 12. Reverse transcription–polymerase chain reaction (RT–PCR) reactions yielded a 532 bp amplicon corresponding to a LN1 chain product in transgenic mice (Fig. 1B). No LN1 chain was detected in skeletal muscle of wild-type mice (Fig. 1C–E) (30). In contrast, immunofluorescence staining demonstrated the presence of LN1 chain in basement membranes of skeletal and cardiac muscle in line No. 12 (Fig. 1C). Also, blood vessels within muscle, which normally do not express LN1 chain (21), were positively stained for LN1 chain (Fig. 1C). In skeletal muscle, LN1 chain was also detected in the neuromuscular and myotendinous junctions (Fig. 1D and E). LN1 chain expression was noted in other organs (e.g. salivary gland, pancreas and thymus) where it is normally not expressed (data not shown). However, LN1 chain was not present in the sciatic nerve of line No. 12 (Fig. 1C). Importantly, overexpression of LN1 chain in mice revealed no discernible pathological phenotypes.

View larger version (45K): [in this window] [in a new window]   Figure 1. Overexpression of LN1 chain in transgenic mice. (A) cDNA encoding LN1 chain was subcloned into the pCAGGS expression vector. Restriction sites used to engineer the construct are shown. (B) PCR amplification of reverse transcribed mRNA from transgenic (LN1TG) and wild-type (WT) skeletal muscle. (C) Expression of LN1 chain in transgenic mice from the No. 12 line. LN1 chain was expressed in the skeletal muscle (SM) (tibialis anterior) and the heart, but not in the peripheral nerve (PN) of transgenic mice or in the corresponding wild-type tissues. The arrow denotes a LN1 chain positively stained blood vessel. (D) Localization of LN1 chain in neuromuscular junction. Sections from skeletal muscle containing neuromuscular junctions of LN1 chain transgenic and wild-type mice were doubly stained with antibodies against LN1 chain (red) and fluorescein -bungarotoxin (-BTX, green). (E) Localization of LN1 chain at the myotendinous junction (arrows). Bar, 50 µm.

 
We next produced mice heterozygous for the transgene and homozygous for the dy^3K mutation, hereafter called dy^3KLN1TG. The LN1 transgene was expressed in these mice in the same manner as the transgenic line No. 12 (Fig. 3).

View larger version (84K): [in this window] [in a new window]   Figure 3. Immunostaining of LNs, perlecan, collagen IV and dystroglycan. Cross-sections of skeletal muscles (tibialis anterior) from 2-week-old wild-type, dy3K/dy3K and dy3KLN1TG mice were stained with antibodies against LN1, LN2, LN4, LN5 and LNß2 chains; perlecan; collagen IV; -dystroglycan and ß-dystroglycan. Bar, 50 µm.

 
Dy^3K/dy^3K mice with LN1 transgene are healthy and long-lived
Dy^3K/dy^3K mice are characterized by growth retardation and severe muscular dystrophy symptoms (7). As shown in Figure 2A and B, the overall health of dy^3KLN1TG mice was significantly improved compared with dy^3K/dy^3K mice. First, dy^3KLN1TG mice are bigger than dy^3K/dy^3K mice. At 2 weeks of age, dy^3K/dy^3K mice can be identified owing to their growth retardation, whereas dy^3KLN1TG mice appeared outwardly normal (data not shown). Weight gain for dy^3K/dy^3K mice was greatly delayed in 5-week-old mice, whereas the weight gain for dy^3KLN1TG mice was significantly increased compared with dy^3K/dy^3K mice (Fig. 2C). In addition, the average body weight of 10-week-old dy^3KLN1TG mice was close to that of wild-type mice (Fig. 2D). Second, dy^3KLN1TG mice live longer. On an average, dy^3K/dy^3K mice died at the age of 4–5 weeks (Fig. 2E). Besides the death of a single dy^3KLN1TG mouse, dy^3KLN1TG mice survived beyond 10 weeks (Fig. 2E). Currently, our oldest mouse is 11 months old.

View larger version (47K): [in this window] [in a new window]   Figure 2. Overall phenotype of LN2 chain deficient animals expressing the LN1 chain transgene. (A) The 5-week-old dy3KLN1TG mice (arrow) are larger than the emaciated dy3K/dy3K mice. (B) The 5-month-old dy3KLN1TG mice (arrow) remain alert and lively with good muscle tone. A dy3K/+LN1TG littermate is shown for comparison. (C) Whole body weights of 5-week-old female wild-type, dy3K/dy3K and dy3KLN1TG mice. Each bar represents the mean±SEM of six (WT), four (dy3K/dy3K) and five (dy3KLN1TG) mice. Note that dy3KLN1TG mice weigh significantly more than dy3K/dy3K mice (P<0.001). (D) Whole body weights of 10-week-old female wild-type and dy3KLN1TG mice. Each bar represents the mean±SEM of six (WT) and five (dy3KLN1TG) mice (P>0.05). (E) Survival curves of dy3K/dy3K (n=10) and dy3KLN1TG mice (n=10). One death occurred in the group of dy3KLN1TG mice (at the age of 10 weeks), whereas most of the dy3K/dy3K mice died between 4 and 5 weeks. (F) Exploratory locomotion of 10–17-week-old female mice in an open field test. Each value represents the mean±SEM of 4 mice.

 
Previous studies have shown that 4-week-old LN2 chain deficient mice display a significantly reduced locomotory activity (31). Here, we analyzed the activity of older dy^3KLN1TG mice (10–17-week-old). Exploratory locomotion studies revealed that dy^3KLN1TG mice appeared as active as wild-type mice (Fig. 2F). An additional indication for the improved health is that both male and female dy^3KLN1TG mice are able to produce offspring (data not shown). Dy^3K/dy^3K mice die before reaching reproductive age, however, dy/dy mice survive longer but do not reproduce (32) (www.jax.org).

Localization of basement membrane components in muscles of dy^3KLN1TG mice
As expected, LN2 chain was completely absent in dy^3KLN1TG mice (Fig. 3). In wild-type mice, LN4 and LN5 chains were mainly expressed in blood vessels. In agreement with previous studies, the expression of the LN4 chain was strongly increased at the muscle basement membrane area in dy^3K/dy^3K mice, whereas the LN5 chain was weakly upregulated (18,31) (Fig. 3). In dy^3KLN1TG mice, the muscle basement membrane expression of LN4 chain was down-regulated to some extent, whereas the expression of LN5 chain remained unchanged. Cohn et al. (33) have previously reported a reduction of LNß2 staining in skeletal muscle membranes of CMD patients with LN2 deficiency. Similarly, we noted a moderate reduction of LNß2 chain in dy^3K/dy^3K mice. Interestingly, the expression of LNß2 chain in the skeletal muscle membrane of dy^3KLN1TG mice was also normalized to expression levels seen in wild-type mice (Fig. 3). Other basement membrane components including type IV collagen and perlecan were similarly expressed in wild-type, dy^3K/dy^3K and in dy^3KLN1TG mice (Fig. 3). Dystroglycan (composed of - and ß-subunits) is a major receptor for several LNs (26,34). It was recently suggested on the basis of immunofluorescence that the amount of -dystroglycan is decreased, whereas the expression of ß-dystroglycan is increased in dy^w/dy^w mice compared with controls (31). Yet, using similar assays but different antibodies, we found no differences in the expression patterns of - or ß-dystroglycan between normal mice and dy^3K/dy^3K or dy^3KLN1TG mice (Fig. 3).

LN1 transgene reduces dystrophic pathology of skeletal muscles
We next examined the morphology of dy^3K/dy^3K and dy^3KLN1TG skeletal muscle. Histological features of dystrophic muscle in 2-week-old dy^3K/dy^3K mice included large groups of centrally nucleated small-caliber muscle fibers revealing the process of active regeneration (7). In contrast, muscles from 2-week-old dy^3KLN1TG mice had a near normal morphology with significantly fewer central nuclei (Fig. 4A and B). Hence, LN1 chain expression protected myofibers from degeneration. In 2-week-old dy^3K/dy^3K mice there was a nearly complete absence of basement membrane around muscle fibers as revealed by electron microscopy studies (Fig. 4C) (7). The LN1 chain transgene restored the basement membrane in dy^3KLN1TG mice (Fig. 4C).

View larger version (100K): [in this window] [in a new window]   Figure 4. Analyses of skeletal muscles from different mice. (A) H&E staining of tibialis anterior muscles of 2-week-old wild-type, dy3K/dy3K and dy3KLN1TG mice. Dystrophic changes with large groups of centrally nucleated small-caliber muscle fibers were detected in dy3K/dy3K mice. Muscle degeneration was prevented in dy3KLN1TG mice. Bar, 50 µm. (B) Quantification of central nucleation in tibialis anterior (TA) and quadriceps femoris (QUAD) muscles from 2-week-old mice. The number of fibers examined for each sample is given in parenthesis. (C) Transmission electron microscopy of tibialis anterior muscle. The basement membrane, clearly present along the sarcolemma in wild-type mouse, was almost absent in muscle fibers of 2-week-old dy3K/dy3K mice (indicated by asterisks). The basement membrane (arrows) was restored in dy3KLN1TG mice. Bar, 300 nm.

 
To investigate whether the LN1 chain also prevented pathological changes in older mice we analyzed skeletal muscles (quadriceps femoris, gluteus maximus, tibialis anterior, triceps brachii and diaphragm) from 4-month-old mice. No dy^3K/dy^3K mice survive till that age. Skeletal muscles of 3–4-week-old dy^3K/dy^3K mice display signs of a severe dystrophy with pronounced fibrosis characteristic of LN2 chain deficient CMD (Fig. 5) (7). In addition, dy/dy mice show extensive fibrosis in various muscles (35). Fibrous tissue is believed to replace muscle when the myogenic satellite cell pool becomes exhausted and consequently fail to maintain muscle regeneration (36). Noticeably, no pathological fibrous tissue was detected in skeletal muscles of 4-month-old dy^3KLN1TG mice (Fig. 5), whereas pronounced fibrosis was detected in all muscles of 3.5-week-old dy^3K/dy^3K mice (Fig. 5). In quadriceps femoris of dy^3KLN1TG mice most fibers were of polygonal shape and normal size, and very few fibers had internally placed nuclei (Fig. 5). A very mild myopathy was detected in gluteus maximus of dy^3KLN1TG mice, with occasional areas of fibers with centrally located nuclei (Fig. 5). In tibialis anterior and triceps brachii of dy^3KLN1TG mice we noted larger areas with centrally located nuclei but no fibrosis (Fig. 5). In contrast, diaphragm of dy^3KLN1TG mice had a near normal morphology with no fibrosis, regular myofibre size and virtually no centralized nuclei, whereas severe fibrosis was detected in diaphragm of dy^3K/dy^3K mice (Fig. 5).

View larger version (123K): [in this window] [in a new window]   Figure 5. H&E staining of various muscles from 4-month-old wild-type mice, 3.5-week-old dy3K/dy3K mice and 4-month-old dy3KLN1TG mice. Cryosections of quadriceps femoris (Quad), gluteus maximus (Glut), tibialis anterior (TA), triceps brachii (Tri) and diaphragm (Dia) were stained with H&E and with antibodies against LN1 chain (staining in red in the last column). Dia magn. represents higher magnification of diaphragm. Bar, 50 µm.

 
In mature dy/dy muscle, the expression of tenascin-C is upregulated and extended to the interstitium between muscle fibers, especially within focal lesions, whereas in control muscle, tenascin-C expression is restricted to the myotendinous junction (37). In sharp contrast to this, very little tenascin-C expression was noted in skeletal muscles of dy^3KLN1TG mice (Fig. 6 and data not shown).

View larger version (59K): [in this window] [in a new window]   Figure 6. Immunostaining of tenascin-C. Cross-sections of triceps brachii (Tri) and diaphragm (Dia) from 4-month-old dy3KLN1TG mice and 3.5-week-old dy3K/dy3K mice were stained with antibodies against tenascin-C. Bar, 50 µm.

 
LN2 chain deficiency also results in dysmyelination of peripheral nerve (38–40), a phenotype that was not corrected in dy^3KLN1TG mice, as the LN1 chain was not expressed in peripheral nerve (Fig. 1C). Injury to peripheral nerve causes neurogenic atrophy of muscle fibers. Accordingly, we noted occasional shrunken angular muscle fibers indicating neurogenic lesions in several muscles of 4-month-old dy^3KLN1TG mice (Fig. 7).

View larger version (64K): [in this window] [in a new window]   Figure 7. H&E staining of muscles from 4-month-old dy3KLN1TG mice reveals occasional neurogenic lesions. Cryosections of quadriceps femoris (Quad), gluteus maximus (Glut), tibialis anterior (TA) and triceps brachii (Tri) were stained with H&E. Arrows denote angular atrophic muscle fibers. Bar, 50 µm.

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
We report that LN2 chain deficient mice expressing a LN1 chain transgene in skeletal muscle display a prolonged life, better health and improved muscle morphology. The greatly improved health of the LN2 chain deficient mice induced by the transgene was remarkable for several reasons. First, although some classical studies showed that the LN1 chain can promote short-term myogenesis in vitro (24,41), 2 chain LNs are much better than 1 chain LNs as in vitro stimulators of myoblast spreading (27) and myotube stability and survival (28). Second, detailed studies of LN receptors have revealed that LN1 chain binds to dystroglycan with about 10-fold lower affinity than LN2 chain or agrin (26), and dystroglycan is an essential link between the extracellular matrix and the cytoskeleton in muscle (42). Third, compensatory upregulation of other LN chains in mouse knock-out models of other LN chains appears to be of no apparent help (1).

Recently, it was reported that an agrin minigene similarly can rescue dystrophic symptoms in the dy^W/dy^W mouse model of CMD. In these experiments, LN5 chain expression was significantly enhanced, suggesting an indirect mechanism of rescue (31). No enhanced expression of LN5 chain was seen in the present study. Hence, we consider it likely that the neo-expression of LN1 in muscles is the major cause of the rescue, although we cannot exclude that expression in other tissues also was beneficial.

Our results indicate that early gene transfer of LN1 chain into skeletal muscles constitute promising therapeutic strategies for LN2 chain deficient CMD. This could be achieved by introducing LN1 chain into myofibers by the usage of viral vectors. Because the LN1 chain cDNA is fairly large (9 kb), gutted adenoviral vectors, which have a cloning capacity>30 kb, would be the viral vector of choice (43). Investigations aiming at up-regulating endogenous LN1 chain in skeletal muscle are also merited. For example, constitutively active Akt-1 induces transcription and synthesis of LN1 chain in embryonic stem cells (44). However, Akt controls numerous transcription factors and is considered to be a hot drug target for the treatment of cancer, diabetes and stroke (45). Thus, it is unlikely to be specific for the treatment of CMD. Interestingly, the identification of an upstream enhancer in the mouse LN1 chain was recently reported (46). This enhancer activates LN1 chain expression in parietal endoderm cells, and activating this enhancer in muscle cells could be a possible strategy in the treatment of LN2 chain deficient CMD.

Peripheral nerve is also involved in LN2 chain deficient CMD (6,38–40). In addition, dy^3KLN1TG mice flexed their hind legs to the trunk when lifted by the tail, an indication of a neurological problem. They also displayed hind leg paralysis, but to a varying degree (data not shown). In addition, we detected angular atrophic muscle fibers (indicating neurogenic lesions) in rescued muscles although they appeared rarely. As the LN1 chain was not expressed in peripheral nerves of LN1 chain transgenic mice, the nerve defect was not expected to be rescued. In this context, it is noteworthy that several attempts to express a LN2 transgene in peripheral nerve have failed (9). Interestingly, loss of LN2 chain in sciatic nerve of dy^2J/dy^2J mice was recently found to be accompanied by variable expression of LN1 chain (47). Thus, it remains possible that overexpression of LN1 chain can compensate for LN2 chain deficiency also in peripheral nerves.

Apart for muscular dystrophy and peripheral neuropathy, LN2 chain deficient mice also display central nervous system myelination defects, hearing loss and abnormal thymocyte and odontoblast development (13–15,48). It will now be interesting to investigate whether LN1 chain can compensate for the absence of LN2 chain in central nervous system, inner ear, thymus and tooth.

In conclusion, we have established that LN1 chain significantly reduces muscular dystrophy in LN2 chain deficient mice. Hence, our data suggest that LN1 chain should be considered in the design of therapies to treat LN2 chain deficient CMD. We also provide the first evidence that LN chains functionally can compensate for each other in vivo.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Transgenic construct
Full-length mouse LN1 cDNA was generously provided by Dr P. Yurchenco (Robert Wood Johnson Medical School, Piscataway, NJ, USA) (49). EcoRI adaptors (Amersham-Pharmacia) were ligated to the ends of LN1 chain cDNA, which was subsequently inserted to the EcoRI site of the expression vector pCAGGS containing a CMV enhancer and chicken ß-actin promoter (generously provided by Dr J. Miyazaki, Osaka University Medical School, Osaka, Japan) (29). A 12 kb transgenic vector was released with SalI and HindIII and used for one-cell embryo microinjection.

Production of LN1 chain transgenic mice on wild-type background
Transgenic mice were generated by microinjections of transgene DNA into the pronucleus of fertilized single-cell CBAxC57BL/6 embryos (Karolinska Center of Transgene Technologies, Stockholm, Sweden). LN1 transgenic mice were identified by Southern blot analysis using genomic DNA prepared from mouse tails and the probe indicated in Figure 1A. LN1 positive founder transgenic mice were maintained in animal facilities according to animal care guidelines. The use of animals complied with national guidelines, and permission was given by the regional ethical board.

Production of transgenic mice on dy^3K background
Heterozygous LN1 transgenic mice were bred to heterozygous dy^3K/+ mice (7), followed by sib breeding to generate mice heterozygous for the transgene and homozygous for dy^3K.

Genotyping
PCR on tail DNA was performed with LN1 cDNA primer 5'-GGCATTGGGCGTGTCGAACAG-3' and chicken ß-actin primer 5'-GGTTCGGCTTCTGGCGTGTGA-3', amplifying a 400 bp product for LN1 chain positive transgenic mice. Dy^3K PCR was performed with primers 5'-CTTTCAGATTGCATTGCAAGC-3' and 5'-CAATGCAGCTTTTTGATCTTAC-3', which anneal to Lama2 intron sequences flanking an exon (encoding a part of domain VI of LN2 chain) that is disrupted by the insertion of a neo cassette in dy^3K/dy^3K mice (7). The PCR product is 1 kb for wild-type mice; 1 and 2.5 kb for heterozygous dy^3K/+ mice and 2.5 kb for homozygous dy^3K/dy^3K mice.

RT–PCR
Total RNA from skeletal muscle was isolated using TRIzol reagent (Invitrogen) according to manufacturer specifications. First strand cDNA synthesis from total RNA was performed using Superscript^TM II RT (Invitrogen). PCR was perfomed using primers directed against LN1 chain: 5'-ATATCACACGCAATCGATGG-3' and 5'-AGTAATAACGTCTTGTG-3'.

Exploratory locomotion
Exploratory locomotion was examined in an open field test. In each experiment a mouse was placed into a new cage and allowed to explore the cage for 5 min. The time that the mouse spent moving around was measured manually. For all experiments, each dy^3KLN1TG animal (n=4) was compared with a wild-type (n=4) sibling of the same sex from the same litter.

Immunofluorescence
Tissues were immersed in Tissue Tek and frozen in liquid nitrogen. Cryosections (7 µm) were either stained with hemotoxylin and eosin (H&E) or analyzed by immunofluorescence. Primary antibodies were: anti-LN1 chain mAb200 (50), anti tenascin-C MTn15 (50), anti-LN2 chain 4H8-2 (Alexis Biochemicals), anti-LN4 chain (generously provided by Dr R. Timpl), anti-LN5 chain (generously provided by Dr R. Timpl), anti-LNß2 chain (generously provided by Dr T. Sasaki) (51), anti-collagen type IV (Chemicon), anti-perlecan (generously provided by Dr R. Timpl), anti--dystroglycan IIH6C4 (Upstate Biotechnology). A rabbit polyclonal antibody was generated against the C-terminal 15 amino acids (KNMTPYRSPPPYVPPC) of ß-dystroglycan and affinity purified. For staining of NMJs, samples were simultaneously incubated with FITC-conjugated -bungarotoxin (Molecular Probes). Images of sections analyzed by fluorescence microscopy (Zeiss Axioplan) were captured using an ORCA 1394 ER digital camera with Openlab 3 software. Images were prepared for publication using Adobe Photoshop software.

Transmission electron microscopy
Tibialis anterior muscles were fixed for 2 h with 2.5% glutaraldehyde, rinsed in Sörensen's phosphate buffer, post-fixed in 1% OsO₄ and then embedded in Epon. Ultra-thin sections were stained with uranyl acetate and lead citrate. Specimens were examined by transmission electron microscopy (Philips CM 10).

	ACKNOWLEDGEMENTS

 
We thank P. Yurchenco, J. Miyazaki, T. Sasaki and the late R. Timpl for gifts of reagents and T. Hjalt and V. Allamand for critical reading of the manuscript. VR, Kungliga Fysiografiska Sällskapet, and Crafoord, Lars Hiertas Minne, Magnus Bergwall, and Åke Wibergs foundations funded this work.

	FOOTNOTES

 
^* To whom correspondence should be addressed at: Department of Cell and Molecular Biology, Section for Cell and Developmental Biology, University of Lund, BMC B12, 221 84 Lund, Sweden. Tel: +46 462220812; Fax: +46 462220855; Email: madeleine.durbeej_hjalt{at}medkem.lu.se

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TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

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