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Prenatal muscle development in a mouse model for the secondary dystroglycanopathies.

Kim J, Hopkinson M, Kavishwar M, Fernandez-Fuente M, Brown SC - Skelet Muscle (2016)

Bottom Line: In addition, the total number of Pax7(+) progenitor cells in the FKRP(KD) tibialis anterior at E15.5 was significantly reduced, and myotube cluster/myofibre size showed a significant reduction in size.These data identify an early reduction of laminin α2, reduction of myogenicity and depletion of Pax7(+) progenitor cells which would be expected to compromise subsequent postnatal muscle growth and its ability to regenerate postnatally.These findings are of significance to the development of future therapies in this group of devastating conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Biomedical Sciences, Royal Veterinary College, University of London, London, UK.

ABSTRACT

Background: The defective glycosylation of α-dystroglycan is associated with a group of muscular dystrophies that are collectively referred to as the secondary dystroglycanopathies. Mutations in the gene encoding fukutin-related protein (FKRP) are one of the most common causes of secondary dystroglycanopathy in the UK and are associated with a wide spectrum of disease. Whilst central nervous system involvement has a prenatal onset, no studies have addressed prenatal muscle development in any of the mouse models for this group of diseases. In view of the pivotal role of α-dystroglycan in early basement membrane formation, we sought to determine if the muscle formation was altered in a mouse model of FKRP-related dystrophy.

Results: Mice with a knock-down in FKRP (FKRP(KD)) showed a marked reduction in α-dystroglycan glycosylation and reduction in laminin binding by embryonic day 15.5 (E15.5), relative to wild type controls. In addition, the total number of Pax7(+) progenitor cells in the FKRP(KD) tibialis anterior at E15.5 was significantly reduced, and myotube cluster/myofibre size showed a significant reduction in size. Moreover, myoblasts isolated from the limb muscle of these mice at E15.5 showed a marked reduction in their ability to form myotubes in vitro.

Conclusions: These data identify an early reduction of laminin α2, reduction of myogenicity and depletion of Pax7(+) progenitor cells which would be expected to compromise subsequent postnatal muscle growth and its ability to regenerate postnatally. These findings are of significance to the development of future therapies in this group of devastating conditions.

No MeSH data available.


Related in: MedlinePlus

Immunolabelling of α- and β-dystroglycan, dystrophin and perlecan. a Transverse sections through the TA and EDL at E15.5 and P0 immunolabelled for α- (A-D) and β-dystroglycan (E-H), dystrophin (Mandys1, I-L) and perlecan (M-P). α-dystroglycan (IIH6) showed a clear delineation of the basement membrane around clusters of primary and secondary myotubes at E15.5 (A) and individual muscle fibres at P0 (C), although some clusters are still evident. There was an almost total absence of IIH6 in the FKRPKD at both time points (B,D). Residual staining of the neuromuscular junction in the FKRPKD at P0 is shown in D as an inserted image. β-dystroglycan, dystrophin and perlecan immunolabelling were not markedly different between FKRPKD and wild type at either time point (E-P)
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Fig2: Immunolabelling of α- and β-dystroglycan, dystrophin and perlecan. a Transverse sections through the TA and EDL at E15.5 and P0 immunolabelled for α- (A-D) and β-dystroglycan (E-H), dystrophin (Mandys1, I-L) and perlecan (M-P). α-dystroglycan (IIH6) showed a clear delineation of the basement membrane around clusters of primary and secondary myotubes at E15.5 (A) and individual muscle fibres at P0 (C), although some clusters are still evident. There was an almost total absence of IIH6 in the FKRPKD at both time points (B,D). Residual staining of the neuromuscular junction in the FKRPKD at P0 is shown in D as an inserted image. β-dystroglycan, dystrophin and perlecan immunolabelling were not markedly different between FKRPKD and wild type at either time point (E-P)

Mentions: Transverse sections through the entire lower hindlimb at E15.5 and P0 of both FKRPKD and wild type immunolabelled for slow myosin heavy chain showed no alteration in muscle patterning (Fig. 1d). In the tibialis anterior (TA) and extensor digitorum longus (EDL), a population of the larger primary myotubes could be clearly identified. The majority of these primaries at E15.5 were surrounded by smaller secondaries forming myotube clusters which were enclosed within a single basement membrane delineated by immunolabelling for laminin, perlecan or collagen type IV. In wild type mice immunolabelling for IIH6 (identifies the laminin binding epitope of α-dystroglycan) followed that of other basement membrane markers (Fig. 2a). However, in the FKRPKD at E15.5 and P0, although there was some variation between pups most likely due to the model being a hypomorph, overall, there was a marked reduction in IIH6 immunolabelling. These observations therefore indicate that glycosylated α-dystroglycan is present from the earliest stages of myotube formation in wild type mice and is markedly reduced in the FKRPKD. However, β-dystroglycan was unchanged between wild type and FKRPKD as was dystrophin which marks the sarcolemma of both primary and the early secondaries (Fig. 2a). Perlecan, an additional ligand of α-dystroglycan also dependent on its proper glycosylation showed no difference between mutant and wild type (Fig. 2a).Fig. 2


Prenatal muscle development in a mouse model for the secondary dystroglycanopathies.

Kim J, Hopkinson M, Kavishwar M, Fernandez-Fuente M, Brown SC - Skelet Muscle (2016)

Immunolabelling of α- and β-dystroglycan, dystrophin and perlecan. a Transverse sections through the TA and EDL at E15.5 and P0 immunolabelled for α- (A-D) and β-dystroglycan (E-H), dystrophin (Mandys1, I-L) and perlecan (M-P). α-dystroglycan (IIH6) showed a clear delineation of the basement membrane around clusters of primary and secondary myotubes at E15.5 (A) and individual muscle fibres at P0 (C), although some clusters are still evident. There was an almost total absence of IIH6 in the FKRPKD at both time points (B,D). Residual staining of the neuromuscular junction in the FKRPKD at P0 is shown in D as an inserted image. β-dystroglycan, dystrophin and perlecan immunolabelling were not markedly different between FKRPKD and wild type at either time point (E-P)
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Related In: Results  -  Collection

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Fig2: Immunolabelling of α- and β-dystroglycan, dystrophin and perlecan. a Transverse sections through the TA and EDL at E15.5 and P0 immunolabelled for α- (A-D) and β-dystroglycan (E-H), dystrophin (Mandys1, I-L) and perlecan (M-P). α-dystroglycan (IIH6) showed a clear delineation of the basement membrane around clusters of primary and secondary myotubes at E15.5 (A) and individual muscle fibres at P0 (C), although some clusters are still evident. There was an almost total absence of IIH6 in the FKRPKD at both time points (B,D). Residual staining of the neuromuscular junction in the FKRPKD at P0 is shown in D as an inserted image. β-dystroglycan, dystrophin and perlecan immunolabelling were not markedly different between FKRPKD and wild type at either time point (E-P)
Mentions: Transverse sections through the entire lower hindlimb at E15.5 and P0 of both FKRPKD and wild type immunolabelled for slow myosin heavy chain showed no alteration in muscle patterning (Fig. 1d). In the tibialis anterior (TA) and extensor digitorum longus (EDL), a population of the larger primary myotubes could be clearly identified. The majority of these primaries at E15.5 were surrounded by smaller secondaries forming myotube clusters which were enclosed within a single basement membrane delineated by immunolabelling for laminin, perlecan or collagen type IV. In wild type mice immunolabelling for IIH6 (identifies the laminin binding epitope of α-dystroglycan) followed that of other basement membrane markers (Fig. 2a). However, in the FKRPKD at E15.5 and P0, although there was some variation between pups most likely due to the model being a hypomorph, overall, there was a marked reduction in IIH6 immunolabelling. These observations therefore indicate that glycosylated α-dystroglycan is present from the earliest stages of myotube formation in wild type mice and is markedly reduced in the FKRPKD. However, β-dystroglycan was unchanged between wild type and FKRPKD as was dystrophin which marks the sarcolemma of both primary and the early secondaries (Fig. 2a). Perlecan, an additional ligand of α-dystroglycan also dependent on its proper glycosylation showed no difference between mutant and wild type (Fig. 2a).Fig. 2

Bottom Line: In addition, the total number of Pax7(+) progenitor cells in the FKRP(KD) tibialis anterior at E15.5 was significantly reduced, and myotube cluster/myofibre size showed a significant reduction in size.These data identify an early reduction of laminin α2, reduction of myogenicity and depletion of Pax7(+) progenitor cells which would be expected to compromise subsequent postnatal muscle growth and its ability to regenerate postnatally.These findings are of significance to the development of future therapies in this group of devastating conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Biomedical Sciences, Royal Veterinary College, University of London, London, UK.

ABSTRACT

Background: The defective glycosylation of α-dystroglycan is associated with a group of muscular dystrophies that are collectively referred to as the secondary dystroglycanopathies. Mutations in the gene encoding fukutin-related protein (FKRP) are one of the most common causes of secondary dystroglycanopathy in the UK and are associated with a wide spectrum of disease. Whilst central nervous system involvement has a prenatal onset, no studies have addressed prenatal muscle development in any of the mouse models for this group of diseases. In view of the pivotal role of α-dystroglycan in early basement membrane formation, we sought to determine if the muscle formation was altered in a mouse model of FKRP-related dystrophy.

Results: Mice with a knock-down in FKRP (FKRP(KD)) showed a marked reduction in α-dystroglycan glycosylation and reduction in laminin binding by embryonic day 15.5 (E15.5), relative to wild type controls. In addition, the total number of Pax7(+) progenitor cells in the FKRP(KD) tibialis anterior at E15.5 was significantly reduced, and myotube cluster/myofibre size showed a significant reduction in size. Moreover, myoblasts isolated from the limb muscle of these mice at E15.5 showed a marked reduction in their ability to form myotubes in vitro.

Conclusions: These data identify an early reduction of laminin α2, reduction of myogenicity and depletion of Pax7(+) progenitor cells which would be expected to compromise subsequent postnatal muscle growth and its ability to regenerate postnatally. These findings are of significance to the development of future therapies in this group of devastating conditions.

No MeSH data available.


Related in: MedlinePlus