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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

Altered ultrastructure of mitochondria in col6a1ama605003 mutant fish myofibers.TEM pictures of sagittal sections of muscles from wild type (WT, A1-4), heterozygous (HT, B1-4) and homozygous (HM, C1-4) col6a1ama605003 mutants, at 2 dpf (A1, B1, C1), 3 wpf (A2, B2, C2), and 4 mpf (A3-4, B3-4, C3-4). In WT, the mitochondria constantly exhibited a dense and well-delineated lamination of the inner membrane and a well-defined outer membrane (A), attesting of the good quality of the fixation-inclusion procedures. In HT and HM muscles, the morphology of mitochondria was diversiform. Similarly to observations in Figs 7 and 8, some mitochondria presented a matrix partly cleared, or even devoid, of electron dense material (B, C arrow) and with severely dilated external membrane detached from the inner condensed membrane (B2-3, C3-4, arrows). These abnormalities were often associated with figures of vacuole/autophagic vesicles (C2, C4, asterisk). In the same area of HT or HM sections, we often observed a normal mitochondrion close to another one exhibiting either swollen cristae (B4, C4, arrows), or even cristae reduced to a few vesicles (B1-2, C2). In a few mitochondria the cristae were even ultra-condensed to a dense core separated from the outer membrane by a vesicle (arrows, B3.1–2, C1). The abnormal mitochondria most often bounded the limits between the crystal-like organized (o) and disorganized (d) myofibrils.
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pone.0133986.g009: Altered ultrastructure of mitochondria in col6a1ama605003 mutant fish myofibers.TEM pictures of sagittal sections of muscles from wild type (WT, A1-4), heterozygous (HT, B1-4) and homozygous (HM, C1-4) col6a1ama605003 mutants, at 2 dpf (A1, B1, C1), 3 wpf (A2, B2, C2), and 4 mpf (A3-4, B3-4, C3-4). In WT, the mitochondria constantly exhibited a dense and well-delineated lamination of the inner membrane and a well-defined outer membrane (A), attesting of the good quality of the fixation-inclusion procedures. In HT and HM muscles, the morphology of mitochondria was diversiform. Similarly to observations in Figs 7 and 8, some mitochondria presented a matrix partly cleared, or even devoid, of electron dense material (B, C arrow) and with severely dilated external membrane detached from the inner condensed membrane (B2-3, C3-4, arrows). These abnormalities were often associated with figures of vacuole/autophagic vesicles (C2, C4, asterisk). In the same area of HT or HM sections, we often observed a normal mitochondrion close to another one exhibiting either swollen cristae (B4, C4, arrows), or even cristae reduced to a few vesicles (B1-2, C2). In a few mitochondria the cristae were even ultra-condensed to a dense core separated from the outer membrane by a vesicle (arrows, B3.1–2, C1). The abnormal mitochondria most often bounded the limits between the crystal-like organized (o) and disorganized (d) myofibrils.

Mentions: For the behavioral studies on adult fish, data distributions were not tested to determine normal distribution, therefore they were analyzed through nonparametric tests. Chi-square tests were used to compare speed distributions and were performed with R. Mann-Whitney tests were used for all other pairwise comparisons and were performed with Statview. In the behavior analysis Figs (9 and 10), symbols *, ** and **** correspond to p < 0.05, p < 0.01 and p < 0.0001 respectively.


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)

Altered ultrastructure of mitochondria in col6a1ama605003 mutant fish myofibers.TEM pictures of sagittal sections of muscles from wild type (WT, A1-4), heterozygous (HT, B1-4) and homozygous (HM, C1-4) col6a1ama605003 mutants, at 2 dpf (A1, B1, C1), 3 wpf (A2, B2, C2), and 4 mpf (A3-4, B3-4, C3-4). In WT, the mitochondria constantly exhibited a dense and well-delineated lamination of the inner membrane and a well-defined outer membrane (A), attesting of the good quality of the fixation-inclusion procedures. In HT and HM muscles, the morphology of mitochondria was diversiform. Similarly to observations in Figs 7 and 8, some mitochondria presented a matrix partly cleared, or even devoid, of electron dense material (B, C arrow) and with severely dilated external membrane detached from the inner condensed membrane (B2-3, C3-4, arrows). These abnormalities were often associated with figures of vacuole/autophagic vesicles (C2, C4, asterisk). In the same area of HT or HM sections, we often observed a normal mitochondrion close to another one exhibiting either swollen cristae (B4, C4, arrows), or even cristae reduced to a few vesicles (B1-2, C2). In a few mitochondria the cristae were even ultra-condensed to a dense core separated from the outer membrane by a vesicle (arrows, B3.1–2, C1). The abnormal mitochondria most often bounded the limits between the crystal-like organized (o) and disorganized (d) myofibrils.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133986.g009: Altered ultrastructure of mitochondria in col6a1ama605003 mutant fish myofibers.TEM pictures of sagittal sections of muscles from wild type (WT, A1-4), heterozygous (HT, B1-4) and homozygous (HM, C1-4) col6a1ama605003 mutants, at 2 dpf (A1, B1, C1), 3 wpf (A2, B2, C2), and 4 mpf (A3-4, B3-4, C3-4). In WT, the mitochondria constantly exhibited a dense and well-delineated lamination of the inner membrane and a well-defined outer membrane (A), attesting of the good quality of the fixation-inclusion procedures. In HT and HM muscles, the morphology of mitochondria was diversiform. Similarly to observations in Figs 7 and 8, some mitochondria presented a matrix partly cleared, or even devoid, of electron dense material (B, C arrow) and with severely dilated external membrane detached from the inner condensed membrane (B2-3, C3-4, arrows). These abnormalities were often associated with figures of vacuole/autophagic vesicles (C2, C4, asterisk). In the same area of HT or HM sections, we often observed a normal mitochondrion close to another one exhibiting either swollen cristae (B4, C4, arrows), or even cristae reduced to a few vesicles (B1-2, C2). In a few mitochondria the cristae were even ultra-condensed to a dense core separated from the outer membrane by a vesicle (arrows, B3.1–2, C1). The abnormal mitochondria most often bounded the limits between the crystal-like organized (o) and disorganized (d) myofibrils.
Mentions: For the behavioral studies on adult fish, data distributions were not tested to determine normal distribution, therefore they were analyzed through nonparametric tests. Chi-square tests were used to compare speed distributions and were performed with R. Mann-Whitney tests were used for all other pairwise comparisons and were performed with Statview. In the behavior analysis Figs (9 and 10), symbols *, ** and **** correspond to p < 0.05, p < 0.01 and p < 0.0001 respectively.

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