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A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish.

Radev Z, Hermel JM, Elipot Y, Bretaud S, Arnould S, Duchateau P, Ruggiero F, Joly JS, Sohm F - PLoS ONE (2015)

Bottom Line: We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA.These symptoms worsened with ageing as described in patients with collagen VI deficiency.Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders.

View Article: PubMed Central - PubMed

Affiliation: UMS 1374, AMAGEN, INRA, Jouy en Josas, Domaine de Vilvert, France; UMS 3504, AMAGEN, CNRS, Gif-sur-Yvette, France.

ABSTRACT
Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders.

No MeSH data available.


Related in: MedlinePlus

Age-dependent progressive disorganization of muscle fibers of col6a1ama605003 mutants.Light photomicrographs of Richardson’s stained semi-thin 1-μm sections from wild type (WT, A1-4), heterozygous (HT, B1-4) and homozygous (HM, C1-4) col6a1ama605003 mutants at 2 days, 3 weeks and 4 months post-fertilization (2 dpf, 3wpf, 4 mpf, respectively). Right from 2 dpf in HT (B1) as in HM (C1) mutants, we observed abnormal vacuoles (red arrowheads) in the centre of some of the muscle fibers. Muscle fibers adjacent to abnormal ones remained similar to the ones WT (A1). At 3 wpf and at 4 mpf, in HT (B2-4) as in HM (C2-4), the number and the size of abnormal vacuoles in myofibers varied from one area to another. Abnormal myofibers with vacuoles were scattered among unaffected myofibers identical to those observed in WT (A2-4). In sagittal sections of muscle from 3 wpf HT we observed breaks in fiber tethering (B2, red arrowhead) or cell in advanced degradation (C2, red arrowheads). Sections were cut according to transversal plane, except A2, B2, and C2 which were sagittal sections. Scale bars, 10 μm.
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pone.0133986.g005: Age-dependent progressive disorganization of muscle fibers of col6a1ama605003 mutants.Light photomicrographs of Richardson’s stained semi-thin 1-μm sections from wild type (WT, A1-4), heterozygous (HT, B1-4) and homozygous (HM, C1-4) col6a1ama605003 mutants at 2 days, 3 weeks and 4 months post-fertilization (2 dpf, 3wpf, 4 mpf, respectively). Right from 2 dpf in HT (B1) as in HM (C1) mutants, we observed abnormal vacuoles (red arrowheads) in the centre of some of the muscle fibers. Muscle fibers adjacent to abnormal ones remained similar to the ones WT (A1). At 3 wpf and at 4 mpf, in HT (B2-4) as in HM (C2-4), the number and the size of abnormal vacuoles in myofibers varied from one area to another. Abnormal myofibers with vacuoles were scattered among unaffected myofibers identical to those observed in WT (A2-4). In sagittal sections of muscle from 3 wpf HT we observed breaks in fiber tethering (B2, red arrowhead) or cell in advanced degradation (C2, red arrowheads). Sections were cut according to transversal plane, except A2, B2, and C2 which were sagittal sections. Scale bars, 10 μm.

Mentions: Histology of the trunk skeletal white muscle was evaluated by Richardson’s staining from transversal and sagittal semi-thin sections. We studied HT and HM col6a1ama605003 mutants and WT siblings at 2 days, 3 weeks and 4 months post-fertilization (2 dpf, 3 wpf, 4 mpf). At 2 dpf, we observed a mild disorganization of the skeletal muscle tissue in transverse sections of HT (Fig 5B1) and HM (Fig 5C1) col6a1ama605003 fry as compared to WT (Fig 5A1): the vast majority of the myofibers were normal but a few of them already presented abnormal intracellular vacuoles. At 3 wpf, the phenotype worsened with noticeable abnormal intracellular vacuoles in the myofibers of mutants (red arrowheads, Fig 5B2-3 and 5C2-3). While the vacuoles were indeed present in myofibers of HT fish (Fig 5B2-3), they were clearly more numerous in the myofibers of HM fish (red arrowheads, Fig 5C2–5C3). At 4 mpf, myofibers with abnormal vacuoles persisted (Fig 5B4 and 5C4). We observed a relatively low number of altered myofibers in the representative sections we analyzed, which reflects the patchy distribution of abnormal myofibers amongst normal ones in mutant fish. In contrast, we were unable to find any abnormal vacuoles in myofibers in any muscle section from the WT fish we analyzed (Fig 5A1-4). Additionally, in col6a1ama605003 mutants, cell-to-cell contact appeared to be weaker: we observed large gaps between pathological fibers and their neighbours in sagittal section (red arrowheads; Fig 5B2–5C2), although healthy myofibers were in close contact. This was probably due to a greater fragility of mutant myofibers towards the contraction-induced fixation artifact [48]. Generally, the muscular tissue of col6a1ama605003 mutants appeared more fragile and the myofibers were not tethered to each other as well as in WT. The Richardson’s staining appeared also weaker in the altered cells than in the healthy ones (Fig 5B and 5C). The presence of apoptosis was assessed by TUNEL on cryostat sections of trunk skeletal muscles at 3 wpf and 4 mpf on WT as well as on HT and HM col6a1ama605003. No obvious difference in TUNEL was found between WT and either col6a1ama605003 mutant (data not shown). Finally, at 5 mpf, hematoxylin-eosin-safran or Masson’s trichrome stainings of transversal paraffin sections of white muscles were performed to visualize cell nuclei and collagen content respectively. Indeed, col6a1ama605003 mutant muscles showed a increase in the number of nuclei, most probably due to the presence of numerous fibroblasts, and associated abundant extracellular matrix indicating the development of fibrosis in damaged muscles (violet; Fig 6B, 6C, 6E and 6F). In HM, the increase in number of nuclei was amplified (arrows, Fig 6C) and was associated with the development of areas with accumulation of extracellular material (arrowheads, Fig 6C). Finally, we also observed in HM collagen-rich areas with numerous nuclei (star, Fig 6F) most probably due to fibrosis that was unseen in WT or in HT (Fig 6).


A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish.

Radev Z, Hermel JM, Elipot Y, Bretaud S, Arnould S, Duchateau P, Ruggiero F, Joly JS, Sohm F - PLoS ONE (2015)

Age-dependent progressive disorganization of muscle fibers of col6a1ama605003 mutants.Light photomicrographs of Richardson’s stained semi-thin 1-μm sections from wild type (WT, A1-4), heterozygous (HT, B1-4) and homozygous (HM, C1-4) col6a1ama605003 mutants at 2 days, 3 weeks and 4 months post-fertilization (2 dpf, 3wpf, 4 mpf, respectively). Right from 2 dpf in HT (B1) as in HM (C1) mutants, we observed abnormal vacuoles (red arrowheads) in the centre of some of the muscle fibers. Muscle fibers adjacent to abnormal ones remained similar to the ones WT (A1). At 3 wpf and at 4 mpf, in HT (B2-4) as in HM (C2-4), the number and the size of abnormal vacuoles in myofibers varied from one area to another. Abnormal myofibers with vacuoles were scattered among unaffected myofibers identical to those observed in WT (A2-4). In sagittal sections of muscle from 3 wpf HT we observed breaks in fiber tethering (B2, red arrowhead) or cell in advanced degradation (C2, red arrowheads). Sections were cut according to transversal plane, except A2, B2, and C2 which were sagittal sections. Scale bars, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4519248&req=5

pone.0133986.g005: Age-dependent progressive disorganization of muscle fibers of col6a1ama605003 mutants.Light photomicrographs of Richardson’s stained semi-thin 1-μm sections from wild type (WT, A1-4), heterozygous (HT, B1-4) and homozygous (HM, C1-4) col6a1ama605003 mutants at 2 days, 3 weeks and 4 months post-fertilization (2 dpf, 3wpf, 4 mpf, respectively). Right from 2 dpf in HT (B1) as in HM (C1) mutants, we observed abnormal vacuoles (red arrowheads) in the centre of some of the muscle fibers. Muscle fibers adjacent to abnormal ones remained similar to the ones WT (A1). At 3 wpf and at 4 mpf, in HT (B2-4) as in HM (C2-4), the number and the size of abnormal vacuoles in myofibers varied from one area to another. Abnormal myofibers with vacuoles were scattered among unaffected myofibers identical to those observed in WT (A2-4). In sagittal sections of muscle from 3 wpf HT we observed breaks in fiber tethering (B2, red arrowhead) or cell in advanced degradation (C2, red arrowheads). Sections were cut according to transversal plane, except A2, B2, and C2 which were sagittal sections. Scale bars, 10 μm.
Mentions: Histology of the trunk skeletal white muscle was evaluated by Richardson’s staining from transversal and sagittal semi-thin sections. We studied HT and HM col6a1ama605003 mutants and WT siblings at 2 days, 3 weeks and 4 months post-fertilization (2 dpf, 3 wpf, 4 mpf). At 2 dpf, we observed a mild disorganization of the skeletal muscle tissue in transverse sections of HT (Fig 5B1) and HM (Fig 5C1) col6a1ama605003 fry as compared to WT (Fig 5A1): the vast majority of the myofibers were normal but a few of them already presented abnormal intracellular vacuoles. At 3 wpf, the phenotype worsened with noticeable abnormal intracellular vacuoles in the myofibers of mutants (red arrowheads, Fig 5B2-3 and 5C2-3). While the vacuoles were indeed present in myofibers of HT fish (Fig 5B2-3), they were clearly more numerous in the myofibers of HM fish (red arrowheads, Fig 5C2–5C3). At 4 mpf, myofibers with abnormal vacuoles persisted (Fig 5B4 and 5C4). We observed a relatively low number of altered myofibers in the representative sections we analyzed, which reflects the patchy distribution of abnormal myofibers amongst normal ones in mutant fish. In contrast, we were unable to find any abnormal vacuoles in myofibers in any muscle section from the WT fish we analyzed (Fig 5A1-4). Additionally, in col6a1ama605003 mutants, cell-to-cell contact appeared to be weaker: we observed large gaps between pathological fibers and their neighbours in sagittal section (red arrowheads; Fig 5B2–5C2), although healthy myofibers were in close contact. This was probably due to a greater fragility of mutant myofibers towards the contraction-induced fixation artifact [48]. Generally, the muscular tissue of col6a1ama605003 mutants appeared more fragile and the myofibers were not tethered to each other as well as in WT. The Richardson’s staining appeared also weaker in the altered cells than in the healthy ones (Fig 5B and 5C). The presence of apoptosis was assessed by TUNEL on cryostat sections of trunk skeletal muscles at 3 wpf and 4 mpf on WT as well as on HT and HM col6a1ama605003. No obvious difference in TUNEL was found between WT and either col6a1ama605003 mutant (data not shown). Finally, at 5 mpf, hematoxylin-eosin-safran or Masson’s trichrome stainings of transversal paraffin sections of white muscles were performed to visualize cell nuclei and collagen content respectively. Indeed, col6a1ama605003 mutant muscles showed a increase in the number of nuclei, most probably due to the presence of numerous fibroblasts, and associated abundant extracellular matrix indicating the development of fibrosis in damaged muscles (violet; Fig 6B, 6C, 6E and 6F). In HM, the increase in number of nuclei was amplified (arrows, Fig 6C) and was associated with the development of areas with accumulation of extracellular material (arrowheads, Fig 6C). Finally, we also observed in HM collagen-rich areas with numerous nuclei (star, Fig 6F) most probably due to fibrosis that was unseen in WT or in HT (Fig 6).

Bottom Line: We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA.These symptoms worsened with ageing as described in patients with collagen VI deficiency.Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders.

View Article: PubMed Central - PubMed

Affiliation: UMS 1374, AMAGEN, INRA, Jouy en Josas, Domaine de Vilvert, France; UMS 3504, AMAGEN, CNRS, Gif-sur-Yvette, France.

ABSTRACT
Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders.

No MeSH data available.


Related in: MedlinePlus