Human Molecular Genetics

This Article Abstract FREE 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 ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (30) Request Permissions Google Scholar Articles by Takeda, S. Articles by Toda, T. Search for Related Content PubMed PubMed Citation Articles by Takeda, S. Articles by Toda, T. Social Bookmarking          What's this?

Human Molecular Genetics, 2003, Vol. 12, No. 12 1449-1459
DOI: 10.1093/hmg/ddg153
© 2003 Oxford University Press

Fukutin is required for maintenance of muscle integrity, cortical histiogenesis and normal eye development

Satoshi Takeda¹, Mari Kondo¹, Junko Sasaki², Hiroki Kurahashi², Hiroki Kano², Ken Arai³, Kazuyo Misaki⁴, Takehiko Fukui⁵, Kazuhiro Kobayashi², Masaji Tachikawa², Michihiro Imamura⁶, Yusuke Nakamura⁷, Teruo Shimizu³, Tatsufumi Murakami⁸, Yoshihide Sunada⁸, Takashi Fujikado⁵, Kiichiro Matsumura³, Toshio Terashima⁴ and Tatsushi Toda^2,*

¹Otsuka GEN Research Institute, Otsuka Pharmaceutical Co. Ltd, Tokushima, Japan, ²Division of Functional Genomics, Department of Post-Genomics and Diseases, Osaka University Graduate School of Medicine, 2-2-B9 Yamadaoka, Suita, Osaka 565-0871, Japan, ³Department of Neurology, Teikyo University School of Medicine, Tokyo, Japan, ⁴Department of Anatomy and Neurobiology, Kobe University Graduate School of Medicine, Kobe, Japan, ⁵Department of Ophthalmology and Visual Science, Osaka University Graduate School of Medicine, Osaka, Japan, ⁶Institute of Neuroscience, National Center for Neurology and Psychiatry, Tokyo, Japan, ⁷Human Genome Center, Institute of Medical Science, University of Tokyo, Tokyo, Japan and ⁸Department of Neurology, Kawasaki Medical School, Kurashiki, Japan

Received December 5, 2002; Revised March 21, 2003; Accepted April 10, 2003

DDBJ/EMBL/GenBank accession no. AB077383

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Fukuyama-type congenital muscular dystrophy (FCMD), one of the most common autosomal-recessive disorders in Japan, is characterized by congenital muscular dystrophy associated with brain malformation due to a defect during neuronal migration. Through positional cloning, we previously identified the gene for FCMD, which encodes the fukutin protein. Here we report that chimeric mice generated using embryonic stem cells targeted for both fukutin alleles develop severe muscular dystrophy, with the selective deficiency of -dystroglycan and its laminin-binding activity. In addition, these mice showed laminar disorganization of the cortical structures in the brain with impaired laminin assembly, focal interhemispheric fusion, and hippocampal and cerebellar dysgenesis. Further, chimeric mice showed anomaly of the lens, loss of laminar structure in the retina, and retinal detachment. These results indicate that fukutin is necessary for the maintenance of muscle integrity, cortical histiogenesis, and normal ocular development and suggest the functional linkage between fukutin and -dystroglycan.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Since the discovery of the Duchenne muscular dystrophy (DMD) gene product dystrophin (1), many studies have focused on understanding the pathophysiology of muscular dystrophies and on developing therapeutic approaches. Structural defects in the dystrophin–glycoprotein complex (DGC) can result in a loss of linkage between laminin-2 (merosin) in the extracellular matrix and actin in the subsarcolemmal cytoskeleton, and this can lead to various muscular dystrophies (2). Of these, -dystroglycan is a heavily glycosylated mucin-type glycoprotein on the surface of muscle cells (3–5). It is the key component of the DGC, providing a tight linkage between the cell and basement membranes by binding laminin via its carbohydrate residues (3–5). -Dystroglycan plays an active role in the basement membrane assembly itself (6).

Fukuyama-type congenital muscular dystrophy (FCMD), one of the most common autosomal-recessive disorders in Japan, is characterized by congenital muscular dystrophy associated with brain malformation (polymicrogyria) due to a defect during neuronal migration (7). Patients with FCMD manifest weakness of facial and limb muscles, and general hypotonia which usually appears before 9 months of age. Functional disability is more severe in FCMD patients than in DMD patients; usually the maximum level of motor function achieved is sliding while sitting on the buttocks, and most FCMD patients are never able to walk. Patients usually become bedridden before 10 years of age due to generalized muscle atrophy and joint contracture, and most of them die by 20 years of age. Another manifestation observed in all cases is severe mental retardation; IQ scores in most FCMD patients lie between 30 and 50. Seizures occur in nearly half of the cases, in association with abnormal electroencephalograms (7).

Through positional cloning, we previously identified the gene responsible for FCMD, which encodes the fukutin protein (8–10). Most FCMD patients carry an ancestral mutation (11), which arose as a consequence of the integration of a 3 kb retrotransposon element into the 3' untranslated region of the fukutin gene (10). FCMD is the first known human disease to be caused by an ancient retrotransposal integration. No FCMD patients have been identified with non-founder (point) mutations on both alleles, suggesting that such patients are embryonic-lethal and that fukutin is essential for normal development (12). There are no reported naturally occurring mice carrying mutations in the fukutin gene. Targeted homozygous mutation of this gene in mice leads to lethality at embryonic day 6.5–7.5, prior to development of skeletal muscle, cardiac muscle or mature neurons, suggesting that fukutin is essential for early embryonic development (Kurahashi et al., unpublished data). To test the hypothesis that fukutin is necessary for maintenance of muscle integrity or histiogenesis of cerebral and cerebellar cortices, we here generated fukutin-deficient chimeric mice using embryonic stem (ES) cells targeted for both fukutin alleles. Interestingly, they also showed anomaly of the lens, loss of laminar structure in the retina, and retinal detachment. Our results indicate that fukutin is necessary for the maintenance of muscle integrity, cortical histiogenesis and normal ocular development, and suggest functional linkage between fukutin and -dystroglycan.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Generation of fukutin-deficient chimeric mice
We used targeting vectors to generate ES cells in which both fukutin alleles have been disrupted by homologous recombination. To design a targeting vector for the generation of fukutin-deficient chimeric mice, we characterized a cosmid clone spanning exons 1–5 of the mouse fukutin gene (13) isolated from a 129/SvEv mouse library. The 2.3 kb KpnI–BamHI fragment containing the first coding exon (exon 2) was replaced with either a neomycin or a puromycin resistance gene (Fig. 1A), thereby removing the coding sequence corresponding to amino acids 1–35, as well as the splicing donor and acceptor sites. Targeted fukutin gene disruption was confirmed by Southern blot analysis of genomic DNA from ES cells (Fig. 1B).

View larger version (22K): [in this window] [in a new window]   Figure 1. Targeted disruption of both copies of fukutin in ES cells. (A) Physical map of the fukutin wild-type locus containing exon 2, targeting vector harboring the neomycin (neor) or puromycin (puror) resistant gene, and the disrupted fukutin locus. The arrowhead indicates the orientation of the drug-resistance genes. (B) Southern blot of BamHI-digested ES cell genomic DNA with the 3' probe depicted in (A). A 5.0 kb wild-type fragment is detected in the single copy neor targeted clone (lane 2) as in parental ES cells (lane 1). In the double-copy targeted clone (lane 3), only a 6.4 kb band corresponding to the mutant allele was detected. (C) Contribution of fukutin-disrupted ES cells to brain and skeletal muscle. 129/SvEv and C57BL/6J mouse strains from which the fukutin-disrupted and wild-type tissues were derived have distinct electrophoretic variants of the two subunits of the glucose phosphate isomerase (129/SvEv, Gpi-1a; C57BL/6J, Gpi-1b). In correspondence with the coat color of the chimeric mice, the contribution of fukutin-deficient cells was high (lanes 1–3), medium (lanes 4 and 5), and low (lanes 6 and 7). (D) Survival rate of chimeric mice. The number of mice examined is shown in parentheses.

 
After seven rounds of blastocyst injections, a total of 62 mice were born that were determined to be highly chimeric because of their coat color (agouti). Glucose phosphate isomerase isozyme (Gpi) assay revealed that the extent of chimerism in various tissues corresponded well with that determined by coat color (Fig. 1C). Therefore, we classified chimeric mice into three groups depending on chimerism of coat color (high, over 70% contribution of fukutin^-/- cells; medium, 70–20%; and low, under 20%). Body weight was lower in the agouti mice (highly chimeric), and some died within one month. From 12 months of age, survival rates of high and medium chimeras gradually decreased. At 21 months survival rates of high, medium and low chimeras were 48, 67 and 94%, respectively (Fig. 1D).

Behavioral abnormalities in fukutin-deficient chimeric mice
Chimeric mice were dystrophic, although those with 50% or greater contribution from heterozygous ES cells showed no obvious phenotype, consistent with the lack of phenotype in fukutin^+/- mice (Kurahashi et al., unpublished data). Agouti mice (high) developed clasping when suspended by the tail (Fig. 2A and B). Analysis of hind footprints showed that they were unable to walk in a straight line and dragged their feet (Fig. 2C and D). Chimeras also showed muscle weakness in the hanging wire grip test (Fig. 2E) and positional instability in the rotor-rod test (Fig. 2F). These features first appeared at about 1 month but were not progressive.

View larger version (33K): [in this window] [in a new window]   Figure 2. Behavioral abnormalities of fukutin-deficient chimeric mice. (A and B) When lifted by the tail, wild-type (C57BL/6J) mice extended their limbs (A), whereas chimeric mice folded their limbs towards the trunk (B). (C and D) Walking footprints. In contrast with wild-type mice (C), chimeric mice could not walk along a straight line and dragged their feet (D). (E and F) Hanging wire grip test (E) and Rotor-rod test (F). In both tests, the chimeric mice with coat colors indicating a high contribution of fukutin-deficient cells (high) fell with a shorter latency than either wild-type mice or chimeric mice with a lower contribution of fukutin-deficient cells (medium and low). The number of mice examined is shown in parentheses. *P<0.05.

 
Muscular dystrophic changes in fukutin-deficient chimeric mice
Figure 3A shows the histology of skeletal muscle in the hindlimbs of fukutin-deficient chimeric mice (high, lanes 1–3 in Fig. 1C). Massive necrosis with phagocytosis, mononuclear cell infiltration, basophilic regenerating muscle fibers, and increase of interstitial connective tissue were present at 1 month after birth. At later stages (7–9 months), a large number of small-sized muscle fibers contained central nuclei, indicating active regeneration. A small number of muscle fibers were undergoing degeneration and phagocytosis by macrophages. Connective tissue hyperplasia and fat cell deposition were also present.

View larger version (131K): [in this window] [in a new window]   Figure 3. Muscular dystrophic changes in fukutin-deficient chimeric mice. (A) HE stained quadriceps muscle of chimeric mice. Massive necrosis with phagocytosis (asterisk), mononuclear cell infiltration (arrowhead), basophilic regenerating fibers (arrow), and an increase in connective tissue mass was present at 1 month of age. At the late stages of 7–9 months, a large number of small-sized fibers were found to have central nuclei (arrowhead), while a small number of fibers were undergoing active degeneration (arrow). Scale bar, 100 µm. (B) Immunohistochemical analysis of sarcolemmal proteins in quadriceps muscle from normal (wt) and chimeric mice. Lam-2, laminin 2 chain; DG, dystroglycan; Dys, dystrophin; SG, sarcoglycan; Utr, utrophin. Selective and scattered deficiency of -dystroglycan was observed in chimeric mice, while the other proteins were preserved. Scale bar, 100 µm. (C) Immunoblot and laminin blot overlay analyses of quadriceps muscle from the two normal (wt) and three chimeric mice. -Dystroglycan immunoreactivity, as well as its laminin-binding activity, was greatly reduced in chimeric mice, while ß-dystroglycan and laminin 2 chain were preserved. The deficiency of -dystroglycan revealed by laminin blot overlay paralleled that revealed by monoclonal antibody IIH6C4 and was more prominent than that revealed by monoclonal antibody VIA4-1

 
-Dystroglycan, a component of the DGC, is a heavily glycosylated mucin-type glycoprotein on the surface of muscle cells (3–5). It is the key component of the DGC, providing a tight linkage between the cell and basement membranes by binding laminin via its carbohydrate residues (3–5). -Dystroglycan plays an active role in the basement membrane assembly itself (6). It was demonstrated recently that -dystroglycan was selectively deficient in skeletal muscle from FCMD patients (14). We thus investigated the expression of -dystroglycan, as well as the other components of the DGC, in skeletal muscle from fukutin-deficient chimeric mice by immunohistochemistry. -Dystroglycan was greatly reduced in the sarcolemma of most muscle fibers in chimeric mice, while the other proteins, including ß-dystroglycan and laminin 2 chain, were preserved (Fig. 3B). Interestingly, all regenerating muscle fibers were deficient in -dystroglycan, suggesting that the fukutin deficiency did not significantly interfere with muscle fiber regeneration. Anti-fukutin antibody reacts with Golgi only in normal cell lines transfected with fukutin, not in non-transfected normal cells or normal human muscle, suggesting that the expression level of endogenous fukutin may be low (Kobayashi et al., unpublished data). As muscle fibers are multi-nucleated, in situ hybridization for the targeting vector neo^r did not show complete correspondence between neo^r-positive and -dystroglycan-negative fibers, although most neo^r-positive fibers were -dystroglycan-negative (data not shown).

Immunoblot analysis confirmed the reduction of -dystroglycan, especially in the antibody against the laminin-binding sugar chain of -dystroglycan (IIH6; Fig. 3C). In addition, laminin blot overlay analysis revealed a deficiency in the laminin-binding activity of -dystroglycan in chimeric mice (Fig. 3C). These results indicate that the linkage between laminin-2 and -dystroglycan on the sarcolemma is disrupted in chimeric mice.

Brain anomalies in fukutin-deficient chimeric mice
We found markedly disorganized laminar structures in the cerebral and cerebellar cortices and hippocampus of fukutin-deficient chimeric mice. In the cerebral cortex, the normal six-layered structure was not clearly discernible (Fig. 4A and B). In some areas, cortical neurons had overmigrated, and the molecular layer (layer I) of the cerebral cortex had disappeared. The midline interhemispheric fissure was partially deficient, with fusion of medial surfaces of the cerebral cortex (Fig. 4C and D). A small number of pyramidal cells in the CA3 sector of the hippocampus were not laminated, although the majority of pyramidal cells were aggregated in a compact lamina. Granule cells in the dentate gyrus were aggregated in a distorted, wavy distribution (Fig. 4E and F). In the cerebellum, the development of folia was defective, and the cerebellar fissure between the adjoining folia were partially fused. The granular layer was disorganized along the fusion lines of adjacent folia or at the pia matter, and Purkinje cells were sporadically malpositioned. Fusions between the caudal surface of the inferior colliculus and the rostral surface of the cerebellum were also observed (Fig. 4G and H).

View larger version (306K): [in this window] [in a new window]   Figure 4. Brain anomalies in fukutin-deficient chimeric mice. (A–H) HE-stained sections of brain from normal (A, C, E and G) and chimeric (B, D, F and H) mice. (A and B) cerebral cortex; (C and D) interhemispheric fissure; (E and F) hippocampus and dentate gyms; (G and H) cerebellum. In chimeric mice, cerebral cortical neurons overmigrated, and the molecular layer was absent (arrowhead) (B). The midline interhemispheric fissure was absent due to the fusion of the adjoining medial surfaces of the cerebral cortex (arrowhead) (D). A single laminar distribution of pyramidal neurons was disorganized in the CA3 sector of the hippocampus (asterisk), and dentate granular cells were aggregated in a distorted wavy distribution (arrowhead) (F). The granular cell layer in the cerebellar cortex was disorganized along the fused cerebellar folia (asterisk) (H). (I–L) HRP retrograde labeling of cerebral cortex from normal (I and K) and chimeric (J and L) mice. In chimeric mice, HRP-labeled corticospinal neurons were not localized in layer V but scattered throughout all the depths of the motor cortex. (M and N) Laminin immunostaining of cerebral surface from normal (M) and chimeric (N) mice. In chimeric mice, the basement membrane was interrupted (arrowhead), and laminin was deposited in sporadic granules (asterisk) in the cerebral cortex. Scale bar, 100 µm. (O) Immunoblot and laminin blot overlay analyses of cerebral surface from the three normal (wt) and four chimeric mice. -dystroglycan immunoreactivity, as well as its laminin-binding activity, was greatly reduced in chimeric mice. The results were similar to those in skeletal muscle (Fig. 3C).

 
After the injection of horseradish peroxidase (HRP) into the lumbar cord, corticospinal neurons with pyramidal somata were retrogradely labeled. While the somata of HRP-labeled corticospinal neurons were situated exclusively in layer V in the motor-sensory cortex of control mice (Fig. 4I and K), they were not distributed in any specific zone but were scattered diffusely throughout all depths of the cortex in chimeric mice (Fig. 4J and L).

Immunostaining with anti-laminin antibody revealed irregular interruptions of the meningeal basement membrane and granular deposits of laminin in the disorganized cerebral cortex (Fig. 4M and N). The meningeal basement membrane was also deficient over the fused medial cortex along the midline interhemispheric fissure or the fused cerebellar folia along the cerebellar sulci (data not shown). In contrast, the basement membrane surrounding blood vessels in the brain parenchyma was preserved. These findings indicate that fukutin is required for the assembly and/or remodeling of the meningeal basement membrane during the developmental period of brain cortical structures.

Immunoblot analysis revealed that -dystroglycan was greatly reduced in the cerebral surface in chimeric mice and laminin blot overlay analysis revealed a deficiency in the laminin-binding activity of -dystroglycan in chimeric mice (Fig. 4O). These results were similar to those in skeletal muscle and suggest that abnormal laminin–dystroglycan complex possibly cause brain anomalies in chimeric mice.

Eye anomalies in fukutin-deficient chimeric mice
Eye findings were quite remarkable. Fukutin-deficient chimeric mice showed corneal opacification with vascular infiltration (Fig. 5A and B). Microscopic analysis revealed a number of anomalies, including anomalous formation of the eyeball and lens (Fig. 5C and D); loss of laminar structure of the retina and retinal detachment (Fig. 5E and F); extensive folding of the retina (Fig. 5G); and thickened cornea with granular tissue formation, adhesion of the lens and cornea, and corneal inflammation and degeneration (Fig. 5H). Electroretinography (ERG) revealed extinction of the b-wave (Fig. 5I).

View larger version (105K): [in this window] [in a new window]   Figure 5. Eye anomalies in fukutin-deficient chimeric mice. (A and B) Gross appearance of eyes from normal (A) and chimeric (B) mice. In chimeric mice, corneal and/or lens opacification was present (B). (C–H) HE-stained sections of eyeballs from normal (C and E) and chimeric (D, F–H) mice. In chimeric mice, the lens had a peculiar shape, with the sclera bent and intercalated between the lens and retina (D). The laminar structure of the retina was completely disorganized and retinal detachment was present (F). The retina was misfolded extensively in a wavy pattern (arrowhead) (G). The cornea was thickened by granular tissue formation (asterisk) and adhered to the lens with inflammation (arrowhead) (H). Scale bar, C and D, 1.3 mm; E–H, 100 µm. (I) ERG revealed extinction of the b-wave.

 

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Here we have generated chimeric mice deficient in fukutin and shown that, similar to FCMD patients, the mutant mice develop neuronal migration disorder and ocular abnormality in addition to severe muscular dystrophy. At present, the function of fukutin remains unknown, and the mechanism by which its deficiency causes defects in multiple organs has not been clarified. In this respect, it should be noted that sequence analysis predicts fukutin to be an enzyme that modifies cell-surface glycoproteins or glycolipids (15). There are several lines of indirect but significant evidence to support this. First, it was reported that highly glycosylated -dystroglycan was selectively deficient in the skeletal muscle of FCMD patients (14). Second, we have reported that muscle–eye–brain disease (MEB), an autosomal-recessive disorder having skeletal muscle, eye and brain defects similar to FCMD (16), is caused by mutations in the gene encoding the protein O-linked mannose ß1, 2-N-acetylglucosaminyltransferase (POMGnT1), which cause the loss of the enzyme activity (17). Moreover, we have found the selective deficiency of -dystroglycan in the skeletal muscle of MEB patients (18). Finally, defective glycosylation of -dystroglycan has also been reported in another form of congenital muscular dystrophy, MDC1C, caused by mutations in the gene encoding the putative glycosyltransferase named FKRP (fukutin-related protein) (19) and in myd mice, an animal model of congenital muscular dystrophy, caused by the mutation in the gene encoding a putative glycosyltransferase named Large (20), although brain and eye anomalies are not the hallmarks of MDC1C. Quite recently 20% of Walker–Warburg syndrome patients have been found to have mutations in POMT1, a putative human counterpart of yeast O-mannosyltransferase (21). Moreover, Michele et al. (22) showed, in MEB, FCMD and myodystrophy mouse, that -dystroglycan is expressed at the muscle membrane, but similar hypoglycosylation in the diseases directly abolishes binding activity of dystroglycan for the ligands laminin, neurexin and agrin. Together with the present results, these findings suggest that defective glycosylation of -dystroglycan due to the primary genetic defects of glycosyltransferases may be the common denominator causing muscle cell degeneration in these diseases.

Strikingly, the deficiency of -dystroglycan revealed by laminin blot overlay paralleled that revealed by the monoclonal antibody IIH6C4, which recognizes the laminin-binding carbohydrate residues of -dystroglycan, and was more prominent than that revealed by the monoclonal antibody VIA4-1, which recognizes carbohydrate residues unrelated to the binding of laminin (4,23) (Figs 3C and 4O). It is of course possible that -dystroglycan is still present but in a hypoglycosylated form, as was suggested to occur in FCMD by Michele et al. (22). This raises the possibility that fukutin may be involved in the modification of laminin-binding carbohydrate residues in -dystroglycan and, in the absence of fukutin, the linkage between laminin and -dystroglycan may never be established on the muscle cell surface. This scenario is also consistent with the report that chimeric mice lacking skeletal muscle dystroglycan developed muscular dystrophy similar to the mice described here (24).

Finally, the role of dystroglycan in the pathogenesis of brain and eye defects in FCMD remains unclear. Brain and/or eye defects similar to those reported here have recently been observed in mice lacking dystroglycan in the brain via Cre/loxP-mediated gene inactivation (25) and in myd mice, although the retina was apparently morphologically normal (26). Interestingly, brain and/or eye defects similar to those reported here have also been observed in mice lacking integrin ß1 subunit in brain via Cre/loxP-mediated gene inactivation (27) and in mice carrying a targeted mutation in the integrin 6 subunit gene (28), respectively. We can envisage that the defects in dystroglycan have similar consequences as those caused by integrin deficiency, because these two receptors are thought to work in concert. Alternatively, fukutin might be involved in the modification of the carbohydrate residues of integrin subunits in brain and eye, and integrin might not function normally in the absence of fukutin, although immunostaining for integrin 7 on skeletal muscle cryosections showed no difference (data not shown).

Our data indicates that fukutin is essential for maintenance of muscle integrity, cortical histiogenesis, and normal eye development and suggest the functional linkage between fukutin and -dystroglycan. Fukutin-deficient chimeric mice are suitable models for studying not only the biological function of fukutin but also the molecular pathogenesis of and therapeutic approaches to complex disorders exhibiting the simultaneous occurrence of central nervous, ocular and muscular abnormalities seen in FCMD and its related diseases.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Targeted disruption of fukutin
A cosmid clone spanning exons 1–5 of the mouse fukutin gene was isolated from a 129/SvEv mouse library. Two targeting vectors were constructed with a 12 kb KpnI–BlpI fragment containing the first coding exon (exon 2) and flanking introns. The 2.3 kb KpnI–BamHI fragment containing exon 2 was replaced with either a neomycin or a puromycin resistance gene (pKO-Select Neo or pKO-Select Puro, Lexicon), thereby removing the coding sequence corresponding to amino acids 1–35, as well as the splicing donor and acceptor sites. Targeting vectors were linearized using NotI, and AB2.2 prime ES cells (Lexicon) were first electroporated with the neo^r vector. We picked, expanded and screened Geneticin-resistant (Sigma) clones for homologous recombination. One clone (no. 8) was chosen and electroporated with the puro^r vector. Puromycin-resistant clones were screened to identify homologous recombinant clones. The double copy-targeted ES cell clone P182 was injected into E3.5 blastocysts of the C57BL/6 mouse strain using standard methods. Mice were maintained in accordance with the Animal Care guidelines of Otsuka Pharmaceutical Co. Ltd.

Evaluation of fukutin-null ES cell contribution to chimeric mice
Mice were sacrificed between 1 and 9 months of age. Protein extracts from tissue samples were subjected to Gpi electrophoresis, and the Gpi variants were stained on the cellulose acetate gels (Helena).

Behavioral phenotyping
Mice were tested during the light phase of their light-dark cycle between 13:00 and 17:00. Mice 7–9 months old were placed on the rotor-rod treadmill apparatus (rod diameter, 3 cm, MK-600, Muromachi Kikai) for a maximum of 60 s, and the latency to fall off the rod within the time period was recorded. Five consecutive trials were performed at 12 rpm. The hanging wire test was carried out by placing 5- to 7-month-old mice on top of the wire cage lid and turning the lid upside down. The latency to fall off the wire was measured up to 60 s. To record the footprint pattern, hindpaws were dipped in India ink, and mice were allowed to walk along a 35 cm long, 10 cm wide runway with 6 cm high walls. The footprints were recorded on a clean sheet of white paper placed on the floor of the dark tunnel. All mice were given one training run per week for 3 weeks before being subjected to a test run.

Histology and immunohistochemistry
Animals were anesthetized with ether and exsanguinated. Brains and eyes were removed and fixed with 10% formalin in 10 mM phosphate buffer (pH 7.2) for 4 days. After paraffin embedding, cerebrums were sectioned serially to 8 µm thickness at coronale. Cerebellums and eyeballs were sectioned to 8 µm thickness at sagittal. Every tenth section was counterstained with hematoxylin and eosin (HE) or by Klüver–Barrera's (KB) methods. Several adjoining sections were immunostained with a polyclonal anti-laminin antibody (Harber Bio-products). For skeletal muscle, serial frozen cryosections (8 µm) were stained with HE or immunostained with the monoclonal -dystroglycan antibody VIA4-1 (Upstate) or antisera against laminin 2 chain (29), ß-dystroglycan (30), dystrophin (31), -sarcoglycan (29), ß-sarcoglycan (32), and utrophin (30). After incubation with Alexa Fluor 488-labeled secondary antibodies (Molecular Probes), sections were examined under a fluorescent microscope.

Immunoblot and laminin blot overlay
SDS–PAGE, 3–12%, and immunoblotting of skeletal muscle and cerebral surface cryosections were performed as described previously (33), using the monoclonal antibodies IIH6C4 and VIA4-1 against -dystroglycan (Upstate), the antiserum against ß-dystroglycan, and the monoclonal antibody 2D9 against the laminin 2 chain (34). The laminin blot overlay was performed using 3 nM laminin-1 (Koken), and the laminin bound to -dystroglycan on the polyvinylidene difluoride membrane was detected using a polyclonal anti-laminin antibody (Sigma).

HRP labeling
One microliter of 10% aqueous solution of HRP (type VI, Sigma) was injected into both sides of the upper lumbar cord. After 48 h, the animals were transcardially perfused with 1.25% glutaraldehyde and 1% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). The brain and spinal cord were immersed in 30% phosphate-buffered sucrose overnight at 4°C, sectioned coronally at 40 µm thickness on a freezing microtome, and reacted for the presence of HRP using the chromagen tetramethylbenzidine (TMB) (35).

Electroretinography
Following 15 min of dark adaptation, ERGs were recorded from the corneal surface electrode after mydriasis. Stimulation with white flash light (10 J) was used (Photostimulator 3G21, NEC-Sanei).

	ACKNOWLEDGEMENTS

 
We thank Drs Masato Horie, Ei-ichi Takahashi, Kenji Araishi, Yukiko K. Hayashi and Eva Engvall for discussion and antibodies; Sawako Muroi, Takashi Wadatsu, Noritaka Koseki, Norihiro Miyazawa, Yuji Fujimori, Tomoyuki Iwanaga, Mai Okano and Kuniko Ohmori for assistance; and Dr Jennifer Logan for editing the manuscript. This study was supported by a Health Science Research Grant, ‘Research on Brain Science’ (H12-Brain-017) and by a Research Grants for Nervous and Mental Disorders (14B-4), both from the Ministry of Health, Labor and Welfare of Japan.

	FOOTNOTES

 
^* To whom correspondence should be addressed. Tel: +81 668793380; Fax: +81 668793389; Email: toda{at}clgene.med.osaka-u.ac.jp

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

1. Hoffman, E.P., Brown, R.H. Jr. and Kunkel, L.M. (1987) Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell, 51, 919–928.[CrossRef][Web of Science][Medline]

2. Campbell, K.P. (1995) Three muscular dystrophies: loss of cytoskeleton–extracellular matrix linkage. Cell, 80, 675–679.[CrossRef][Web of Science][Medline]

3. Ibraghimov-Beskrovnaya, O., Ervasti, J.M., Leveille, C.J., Slaughter, C.A., Sernett, S.W. and Campbell, K.P. (1992) Primary structure of dystrophin-associated glycoproteins linking dystrophin to the extracellular matrix. Nature, 355, 696–702.[CrossRef][Medline]

4. Ervasti, J.M. and Campbell, K.P. (1993) A role for the dystrophin-glycoprotein complex as a transmembrane linker between laminin and actin. J. Cell. Biol., 122, 809–823.[Abstract/Free Full Text]

5. Chiba, A., Matsumura, K., Yamada, H., Inazu, T., Shimizu, T., Kusunoki, S., Kanazawa, I., Kobata, A. and Endo, T. (1997) Structures of sialylated O-linked oligosaccharides of bovine peripheral nerve -dystroglycan. The role of a novel O-mannosyl-type oligosaccharide in the binding of -dystroglycan with laminin. J. Biol. Chem., 272, 2156–2162.[Abstract/Free Full Text]

6. Henry, M.D. and Campbell, K.P. (1998) A role for dystroglycan in basement membrane assembly. Cell, 95, 859–870.[CrossRef][Web of Science][Medline]

7. Fukuyama, Y., Osawa, M. and Suzuki, H. (1981) Congenital progressive muscular dystrophy of the Fukuyama types—clinical, genetic and pathological considerations. Brain Dev., 3, 1–29.[Web of Science][Medline]

8. Toda, T., Segawa, M., Nomura, Y., Nonaka, I., Masuda, K., Ishihara, T., Sakai, M., Tomita, I., Origuchi, Y., Suzuki, M. et al. (1993) Localization of a gene for Fukuyama type congenital muscular dystrophy to chromosome 9q31-33. Nat. Genet., 5, 283–286.[CrossRef][Web of Science][Medline]

9. Toda, T., Miyake, M., Kobayashi, K., Mizuno, K., Saito, K., Osawa, M., Nakamura, Y., Kanazawa, I., Nakagome, Y., Tokunaga, K. and Nakahori, Y. (1996) Linkage-disequilibrium mapping narrows the Fukuyama-type congenital muscular dystrophy (FCMD) candidate region to <100 kb. Am. J. Hum. Genet., 59, 1313–1320.[Web of Science][Medline]

10. Kobayashi, K., Nakahori, Y., Miyake, M., Matsumura, K., Kondo-lida, E., Nomura, Y., Segawa, M., Yoshioka, M., Saito, K., Osawa, M. et al. (1998) An ancient retrotransposal insertion causes Fukuyama-type congenital muscular dystrophy. Nature, 394, 388–392.[CrossRef][Medline]

11. Colombo, R., Bignamini, A.A., Carobene, A., Sasaki, J., Tachikawa, M., Kobayashi, K. and Toda, T. (2000) Age and origin of the FCMD 3'-untranslated-region retrotransposal insertion mutation causing Fukuyama-type congenital muscular dystrophy in the Japanese population. Hum. Genet., 107, 559–567.[CrossRef][Web of Science][Medline]

12. Kondo-lida, E., Kobayashi, K., Watanabe, M., Sasaki, J., Kumagai, T., Koide, H., Saito, K., Osawa, M., Nakamura, Y. and Toda, T. (1999) Novel mutations and genotype-phenotype relationships in 107 families with Fukuyama-type congenital muscular dystrophy (FCMD). Hum. Mol. Genet., 8, 2303–2309.[Abstract/Free Full Text]

13. Horie, M., Kobayashi, K., Takeda, S., Nakamura, Y., Lyons, G.E. and Toda, T. (2002) Isolation and characterization of the mouse ortholog of the Fukuyama-type congenital muscular dystrophy gene. Genomics, 80, 482–486.[CrossRef][Web of Science][Medline]

14. Hayashi, Y.K., Ogawa, M., Tagawa, K., Noguchi, S., Ishihara, T., Nonaka, I. and Arahata, K. (2001) Selective deficiency of -dystroglycan in Fukuyama-type congenital muscular dystrophy. Neurology, 57, 115–121.[Abstract/Free Full Text]

15. Aravind, L. and Koonin, E.V. (1999) The fukutin protein family—predicted enzymes modifying cell-surface molecules. Curr. Biol., 9, R836–837.[CrossRef][Web of Science][Medline]

16. Santavuori, P., Somer, H., Sainio, K., Rapola, J., Kruus, S., Nikitin, T., Ketonen, L. and Leisti, J. (1989) Muscle–eye–brain disease (MEB). Brain Dev., 11, 147–153.[Web of Science][Medline]

17. Yoshida, A., Kobayashi, K., Manya, H., Taniguchi, K., Kano, H., Mizuno, M., Inazu, T., Mitsuhashi, H., Takahashi, S., Takeuchi, M. et al. (2001) Muscular dystrophy and neuronal migration disorder caused by mutations in a glycosyltransferase, POMGnT1. Dev. Cell, 1, 717–724.[CrossRef][Web of Science][Medline]

18. Kano, H., Kobayashi, K., Herrmann, R., Tachikawa, M., Manya, H., Nishino, I., Nonaka, I., Straub, V., Talim, B., Voit, T. et al. (2002) Deficiency of -dystroglycan in muscle-eye-brain disease. Biochem. Biophys. Res. Commun., 291, 1283–1286.[CrossRef][Web of Science][Medline]

19. Brockington, M., Blake, D.J., Prandini, P., Brown, S.C., Torelli, S., Benson, M.A., Ponting, C.P., Estournet, B., Romero, N.B., Mercuri, E. et al. (2001) Mutations in the fukutin-related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin 2 deficiency and abnormal glycosylation of -dystroglycan. Am. J. Hum. Genet., 69, 1198–1209.[CrossRef][Web of Science][Medline]

20. Grewal, P.K., Holzfeind, P.J., Bittner, R.E. and Hewitt, J.E. (2001) Mutant glycosyltransferase and altered glycosylation of -dystroglycan in the myodystrophy mouse. Nat. Genet., 28, 151–154.[CrossRef][Web of Science][Medline]

21. Beltrán-Valero de Bernabé, D., Currier, S., Steinbrecher, A., Celli, J., van Beusekom, E., van der Zwaag, B., Kayserili, H., Merlini, L., Chitayat, D., Dobyns, W.B. et al. (2002) Mutations in the O-mannosyltransferase gene POMT1 give rise to the severe neuronal migration disorder Walker–Warburg syndrome. Am. J. Hum. Genet., 71, 1033–1043.[CrossRef][Web of Science][Medline]

22. Michele, D., Barresi, R., Kanagawa, M., Saito, F., Cohn, R.D., Satz, J.S., Dollar, J., Nishino, I., Kelley, R.I., Somer, H. et al. (2002) Post-translational disruption of dytroglycan–ligand interactions in congenital muscular dystrophies. Nature, 418, 417–422.[CrossRef][Medline]

23. Matsumura, K., Chiba, A., Yamada, H., Fukuta-Ohi, H., Fujita, S., Endo, T., Kobata, A., Anderson, L.V., Kanazawa, I., Campbell, K.P. and Shimizu, T. (1997) A role of dystroglycan in schwannoma cell adhesion to laminin. J. Biol. Chem., 272, 13904–13910.[Abstract/Free Full Text]

24. Cote, P.D., Moukhles, H., Lindenbaum, M. and Carbonetto, S. (1999) Chimaeric mice deficient in dystroglycans develop muscular dystrophy and have disrupted myoneural synapses. Nat. Genet., 23, 338–342.[CrossRef][Web of Science][Medline]

25. Moore, S.A., Saito, F., Chen, J., Michele, D.E., Henry, M.D., Messing, A., Cohn, R.D., Ross-Barta, S.E., Westra, S., Williamson, R.A. et al. (2002) Deletion of brain dystroglycan recapitulates aspects of congenital muscular dystrophy. Nature, 418, 422–425.[CrossRef][Medline]

26. Holzfeind, P.J., Grewal, P.K., Reitsamer, H.A., Kechvar, J., Lassmann, H., Hoeger, H., Hewitt, J.E. and Bittner, R.E. (2002) Skeletal, cardiac and tongue muscle pathology, defective retinal transmission, and neuronal migration defects in the Large^myd mouse defines a natural model for glycosylation-deficient muscle–eye–brain disorders. Hum. Mol. Genet., 11, 2673–2687.[Abstract/Free Full Text]

27. Graus-Porta, D., Blaess, S., Senften, M., Littlewood-Evans, A., Damsky, C., Huang, Z., Orban, P., Klein, R., Schittny, J.C. and Muller, U. (2001) ß1-class integrins regulate the development of laminae and folia in the cerebral and cerebellar cortex. Neuron, 31, 367–379.[CrossRef][Web of Science][Medline]

28. Georges-Labouesse, E., Mark, M., Messaddeq, N. and Gansmuller, A. (1998) Essential role of 6 integrins in cortical and retinal lamination. Curr. Biol., 8, 983–986.[CrossRef][Web of Science][Medline]

29. Araishi, K., Sasaoka, T., Imamura, M., Noguchi, S., Hama, H., Wakabayashi, E., Yoshida, M., Hori, T. and Ozawa, E. (1999) Loss of the sarcoglycan complex and sarcospan leads to muscular dystrophy in ß-sarcoglycan-deficient mice. Hum. Mol. Genet., 8, 1589–1598.[Abstract/Free Full Text]

30. Imamura, M., Araishi, K., Noguchi, S. and Ozawa, E. (2000) A sarcoglycan–dystroglycan complex anchors Dp116 and utrophin in the peripheral nervous system. Hum. Mol. Genet., 9, 3091–3100.[Abstract/Free Full Text]

31. Hagiwara, Y., Sasaoka, T., Araishi, K., Imamura, M., Yorifuji, H., Nonaka, I., Ozawa, E. and Kikuchi, T. (2000) Caveolin-3 deficiency causes muscle degeneration in mice. Hum. Mol. Genet., 9, 3047–3054.[Abstract/Free Full Text]

32. Noguchi, S., Wakabayashi, E., Imamura, M., Yoshida, M. and Ozawa, E. (2000) Formation of sarcoglycan complex with differentiation in cultured myocytes. Eur. J. Biochem., 267, 640–648.[Web of Science][Medline]

33. Matsumura, K., Tome, F.M., Collin, H., Azibi, K., Chaouch, M., Kaplan, J.C., Fardeau, M. and Campbell, K.P. (1992) Deficiency of the 50K dystrophin-associated glycoprotein in severe childhood autosomal recessive muscular dystrophy. Nature, 359, 320–322.[CrossRef][Medline]

34. Hori, H., Kanamori, T., Mizuta, T., Yamaguchi, N., Liu, Y. and Nagai, Y. (1994) Human laminin M chain: epitope analysis of its monoclonal antibodies by immunoscreening of cDNA clones and tissue expression. J. Biochem., 116, 1212–1219.[Abstract/Free Full Text]

35. Mesulam, M.M. (1978) Tetramethyl benzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction product with superior sensitivity for visualizing neural afferents and efferents. J. Histochem. Cytochem., 26, 106–117.[Abstract]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				J. S. Satz, A. R. Philp, H. Nguyen, H. Kusano, J. Lee, R. Turk, M. J. Riker, J. Hernandez, R. M. Weiss, M. G. Anderson, et al. Visual Impairment in the Absence of Dystroglycan J. Neurosci., October 21, 2009; 29(42): 13136 - 13146. [Abstract] [Full Text] [PDF]

				M. Kanagawa, A. Nishimoto, T. Chiyonobu, S. Takeda, Y. Miyagoe-Suzuki, F. Wang, N. Fujikake, M. Taniguchi, Z. Lu, M. Tachikawa, et al. Residual laminin-binding activity and enhanced dystroglycan glycosylation by LARGE in novel model mice to dystroglycanopathy Hum. Mol. Genet., February 15, 2009; 18(4): 621 - 631. [Abstract] [Full Text] [PDF]

				M. R. Ackroyd, L. Skordis, M. Kaluarachchi, J. Godwin, S. Prior, M. Fidanboylu, R. J. Piercy, F. Muntoni, and S. C. Brown Reduced expression of fukutin related protein in mice results in a model for fukutin related protein associated muscular dystrophies Brain, February 1, 2009; 132(2): 439 - 451. [Abstract] [Full Text] [PDF]

				J. S. Satz, R. Barresi, M. Durbeej, T. Willer, A. Turner, S. A. Moore, and K. P. Campbell Brain and Eye Malformations Resembling Walker-Warburg Syndrome Are Recapitulated in Mice by Dystroglycan Deletion in the Epiblast J. Neurosci., October 15, 2008; 28(42): 10567 - 10575. [Abstract] [Full Text] [PDF]

				P. Thornhill, D. Bassett, H. Lochmuller, K. Bushby, and V. Straub Developmental defects in a zebrafish model for muscular dystrophies associated with the loss of fukutin-related protein (FKRP) Brain, June 1, 2008; 131(6): 1551 - 1561. [Abstract] [Full Text] [PDF]

				R. Barresi and K. P. Campbell Dystroglycan: from biosynthesis to pathogenesis of human disease J. Cell Sci., January 15, 2006; 119(2): 199 - 207. [Abstract] [Full Text] [PDF]

				P. K. Grewal, J. M. McLaughlan, C. J. Moore, C. A. Browning, and J. E. Hewitt Characterization of the LARGE family of putative glycosyltransferases associated with dystroglycanopathies Glycobiology, October 1, 2005; 15(10): 912 - 923. [Abstract] [Full Text] [PDF]

				T. Willer, B. Prados, J. M. Falcon-Perez, I. Renner-Muller, G. K. H. Przemeck, M. Lommel, A. Coloma, M. C. Valero, M. H. de Angelis, W. Tanner, et al. Targeted disruption of the Walker-Warburg syndrome gene Pomt1 in mouse results in embryonic lethality PNAS, September 28, 2004; 101(39): 14126 - 14131. [Abstract] [Full Text] [PDF]

				C. Longman, M. Brockington, S. Torelli, C. Jimenez-Mallebrera, C. Kennedy, N. Khalil, L. Feng, R. K. Saran, T. Voit, L. Merlini, et al. Mutations in the human LARGE gene cause MDC1D, a novel form of congenital muscular dystrophy with severe mental retardation and abnormal glycosylation of {alpha}-dystroglycan Hum. Mol. Genet., November 1, 2003; 12(21): 2853 - 2861. [Abstract] [Full Text] [PDF]

				P. K. Grewal and J. E. Hewitt Glycosylation defects: a new mechanism for muscular dystrophy? Hum. Mol. Genet., October 15, 2003; 12(90002): R259 - 264. [Abstract] [Full Text] [PDF]

This Article Abstract FREE 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 ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (30) Request Permissions Google Scholar Articles by Takeda, S. Articles by Toda, T. Search for Related Content PubMed PubMed Citation Articles by Takeda, S. Articles by Toda, T. Social Bookmarking          What's this?

Online ISSN 1460-2083 - Print ISSN 0964-6906

Copyright © 2009 Oxford University Press

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics