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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

Disorganized Cohnheim’s fields, swollen reticulum and altered mitochondria in col6a1ama605003 mutant fish muscle.TEM pictures of transversal sections of muscle from wild type (WT, A1-3) and col6a1ama605003 heterozygous (HT, B1-3) and homozygous (HM, C1-3) mutants at 2 dpf, 3 wpf and 4 mpf. At 2dpf (A1, B1, C1), 3 wpf (A2, B2, C2) and 4 mpf (A3, B3, C3) in HT and HM mutants, we observed in a few muscle fibers, abnormal mitochondria with areas of the matrix devoid of electron dense material (B1-3, C1-3, arrows). In these myofibers, the abnormal mitochondria were often located in close contact with enlarged sarcoplasmic reticulum (B1-2, C1-3, arrowheads). The altered mitochondria often marked the limit between crystal-like organized (o) Cohnheim’s field and pathologic disorganized (d) myofibrils (B1-3, C1-2). In HM (C1), some fibers appeared more affected than in HT, with drastically disorganized (d) areas separated from organized myofibrils (o) with mitochondria having swollen cristae (C1-3, arrows). The sarcoplasm area of the more affected fibers appeared devoid of material (B3, C1-2, asterisks). (B3) In a few muscle fibers of HM, the reticulum appeared drastically dilated (asterisk) with numerous blebs or autophagic vacuoles localized nearby or within myofibrillar disarray foci (C2) that were also in close contact with mitochondria with enlarged cristae (C2-3, arrows). None abnormal mitochondrion, myofibril disorganization or enlarged reticulum was seen in WT (A1-3).
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pone.0133986.g007: Disorganized Cohnheim’s fields, swollen reticulum and altered mitochondria in col6a1ama605003 mutant fish muscle.TEM pictures of transversal sections of muscle from wild type (WT, A1-3) and col6a1ama605003 heterozygous (HT, B1-3) and homozygous (HM, C1-3) mutants at 2 dpf, 3 wpf and 4 mpf. At 2dpf (A1, B1, C1), 3 wpf (A2, B2, C2) and 4 mpf (A3, B3, C3) in HT and HM mutants, we observed in a few muscle fibers, abnormal mitochondria with areas of the matrix devoid of electron dense material (B1-3, C1-3, arrows). In these myofibers, the abnormal mitochondria were often located in close contact with enlarged sarcoplasmic reticulum (B1-2, C1-3, arrowheads). The altered mitochondria often marked the limit between crystal-like organized (o) Cohnheim’s field and pathologic disorganized (d) myofibrils (B1-3, C1-2). In HM (C1), some fibers appeared more affected than in HT, with drastically disorganized (d) areas separated from organized myofibrils (o) with mitochondria having swollen cristae (C1-3, arrows). The sarcoplasm area of the more affected fibers appeared devoid of material (B3, C1-2, asterisks). (B3) In a few muscle fibers of HM, the reticulum appeared drastically dilated (asterisk) with numerous blebs or autophagic vacuoles localized nearby or within myofibrillar disarray foci (C2) that were also in close contact with mitochondria with enlarged cristae (C2-3, arrows). None abnormal mitochondrion, myofibril disorganization or enlarged reticulum was seen in WT (A1-3).

Mentions: In order to better characterize myofibre defects, we studied transverse and sagittal, ultra-thin sections of muscles from col6a1ama605003 mutant and WT zebrafish, at 2 dpf, 3 wpf and 4 mpf (Figs 7–9) by transmission electron microscopy (TEM). These results confirm and complete those obtained by light microscopy (Figs 5 and 6) such as the patchy distribution of abnormal myofibers and the presence of abnormal intracellular vacuoles, and finally emphasize structural defects in mitochondria, myofibrils and the sarcoplasmic reticulum (SR).


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)

Disorganized Cohnheim’s fields, swollen reticulum and altered mitochondria in col6a1ama605003 mutant fish muscle.TEM pictures of transversal sections of muscle from wild type (WT, A1-3) and col6a1ama605003 heterozygous (HT, B1-3) and homozygous (HM, C1-3) mutants at 2 dpf, 3 wpf and 4 mpf. At 2dpf (A1, B1, C1), 3 wpf (A2, B2, C2) and 4 mpf (A3, B3, C3) in HT and HM mutants, we observed in a few muscle fibers, abnormal mitochondria with areas of the matrix devoid of electron dense material (B1-3, C1-3, arrows). In these myofibers, the abnormal mitochondria were often located in close contact with enlarged sarcoplasmic reticulum (B1-2, C1-3, arrowheads). The altered mitochondria often marked the limit between crystal-like organized (o) Cohnheim’s field and pathologic disorganized (d) myofibrils (B1-3, C1-2). In HM (C1), some fibers appeared more affected than in HT, with drastically disorganized (d) areas separated from organized myofibrils (o) with mitochondria having swollen cristae (C1-3, arrows). The sarcoplasm area of the more affected fibers appeared devoid of material (B3, C1-2, asterisks). (B3) In a few muscle fibers of HM, the reticulum appeared drastically dilated (asterisk) with numerous blebs or autophagic vacuoles localized nearby or within myofibrillar disarray foci (C2) that were also in close contact with mitochondria with enlarged cristae (C2-3, arrows). None abnormal mitochondrion, myofibril disorganization or enlarged reticulum was seen in WT (A1-3).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4519248&req=5

pone.0133986.g007: Disorganized Cohnheim’s fields, swollen reticulum and altered mitochondria in col6a1ama605003 mutant fish muscle.TEM pictures of transversal sections of muscle from wild type (WT, A1-3) and col6a1ama605003 heterozygous (HT, B1-3) and homozygous (HM, C1-3) mutants at 2 dpf, 3 wpf and 4 mpf. At 2dpf (A1, B1, C1), 3 wpf (A2, B2, C2) and 4 mpf (A3, B3, C3) in HT and HM mutants, we observed in a few muscle fibers, abnormal mitochondria with areas of the matrix devoid of electron dense material (B1-3, C1-3, arrows). In these myofibers, the abnormal mitochondria were often located in close contact with enlarged sarcoplasmic reticulum (B1-2, C1-3, arrowheads). The altered mitochondria often marked the limit between crystal-like organized (o) Cohnheim’s field and pathologic disorganized (d) myofibrils (B1-3, C1-2). In HM (C1), some fibers appeared more affected than in HT, with drastically disorganized (d) areas separated from organized myofibrils (o) with mitochondria having swollen cristae (C1-3, arrows). The sarcoplasm area of the more affected fibers appeared devoid of material (B3, C1-2, asterisks). (B3) In a few muscle fibers of HM, the reticulum appeared drastically dilated (asterisk) with numerous blebs or autophagic vacuoles localized nearby or within myofibrillar disarray foci (C2) that were also in close contact with mitochondria with enlarged cristae (C2-3, arrows). None abnormal mitochondrion, myofibril disorganization or enlarged reticulum was seen in WT (A1-3).
Mentions: In order to better characterize myofibre defects, we studied transverse and sagittal, ultra-thin sections of muscles from col6a1ama605003 mutant and WT zebrafish, at 2 dpf, 3 wpf and 4 mpf (Figs 7–9) by transmission electron microscopy (TEM). These results confirm and complete those obtained by light microscopy (Figs 5 and 6) such as the patchy distribution of abnormal myofibers and the presence of abnormal intracellular vacuoles, and finally emphasize structural defects in mitochondria, myofibrils and the sarcoplasmic reticulum (SR).

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