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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

The 9 mpf col6a1ama605003 mutant fish presented a hypoxia behavior.We video-recorded for 30 min (25 frames/sec) and tracked the swimming trajectories in the vertical plane of WT, HT and HM col6a1ama605003 fish at 9 mpf in a tank. The position of the fish every 6 sec (1 frame every 150 frames) was plotted. A representative trajectory of each genotype is presented, WT (A), HT (B) and HM (C). From these videos (n = 5 of each genotype) the cumulated time fish swam in the upper quarter of the tank (D) was calculated and was significantly different between WT and HT (p-value 0.009), WT and HM (p-value 0.0283) and HT and HM (p-value 0.0472). HT and HM mutants spent visibly more time in the oxygen-richer part of the tank. The respiratory rate (oral/opercular movements, min-1) was determined for mutants and controls, n = 5 (E) and this rate was significantly different between WT and HM (p-value 0.009) and HT and HM (p-value 0.009). As a test for centrophobia, we measured the time fish spent in the centre of the tank in the horizontal plane. The centre corresponds to the half centre area of the tank. There was no difference of occupancy between the three genotypes. n = 15, 19 and 7 for WT, HT and HM respectively. For each histogram, a Mann-Whitney test was performed; *, ** indicate p-values of < 0.05 and < 0.01 respectively.
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pone.0133986.g011: The 9 mpf col6a1ama605003 mutant fish presented a hypoxia behavior.We video-recorded for 30 min (25 frames/sec) and tracked the swimming trajectories in the vertical plane of WT, HT and HM col6a1ama605003 fish at 9 mpf in a tank. The position of the fish every 6 sec (1 frame every 150 frames) was plotted. A representative trajectory of each genotype is presented, WT (A), HT (B) and HM (C). From these videos (n = 5 of each genotype) the cumulated time fish swam in the upper quarter of the tank (D) was calculated and was significantly different between WT and HT (p-value 0.009), WT and HM (p-value 0.0283) and HT and HM (p-value 0.0472). HT and HM mutants spent visibly more time in the oxygen-richer part of the tank. The respiratory rate (oral/opercular movements, min-1) was determined for mutants and controls, n = 5 (E) and this rate was significantly different between WT and HM (p-value 0.009) and HT and HM (p-value 0.009). As a test for centrophobia, we measured the time fish spent in the centre of the tank in the horizontal plane. The centre corresponds to the half centre area of the tank. There was no difference of occupancy between the three genotypes. n = 15, 19 and 7 for WT, HT and HM respectively. For each histogram, a Mann-Whitney test was performed; *, ** indicate p-values of < 0.05 and < 0.01 respectively.

Mentions: At 3 mpf, no significant changes were observed between WT and col6a1ama605003 mutant fish when the total distance swum or the maximum swimming speed (Fig 10A and 10B) was measured. All statistical analyses are indicated in the corresponding tables in Figs 10 and 11. At 9 mpf, maximal instantaneous speed remained similar between genotypes (Fig 10D). Strikingly, however, HM fish swam significantly longer distances, roughly double those swam by WT and HT (Fig 10C; p-value WT vs. HM, 0.0015; HT vs. HM, 0.0077, Mann-Whitney, MW). This result was rather unexpected since HM fish had worse myofibre defects than HT. Thus, we further analyzed the distribution of instantaneous speed from 1 to 21 cm/s of 3 and 9 mpf mutants and WT sibling fish (see S7 Fig). Since the average fish body length (bl.) of 3 mpf (about 2 cm) is the half of the bl. of the fish at 9 mpf (about 4 cm), we defined three classes of speed activity profile (SAP) according to the fish bl.: rest, middle range and fast SAP. Rest SAP was defined as speed below 0.5 bl./s, middle range SAP as speed ranging from 0.5 to 3 bl./s and fast SAP as speed greater than 3 bl./s (see Materials and methods and Fig 10E–10J).


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)

The 9 mpf col6a1ama605003 mutant fish presented a hypoxia behavior.We video-recorded for 30 min (25 frames/sec) and tracked the swimming trajectories in the vertical plane of WT, HT and HM col6a1ama605003 fish at 9 mpf in a tank. The position of the fish every 6 sec (1 frame every 150 frames) was plotted. A representative trajectory of each genotype is presented, WT (A), HT (B) and HM (C). From these videos (n = 5 of each genotype) the cumulated time fish swam in the upper quarter of the tank (D) was calculated and was significantly different between WT and HT (p-value 0.009), WT and HM (p-value 0.0283) and HT and HM (p-value 0.0472). HT and HM mutants spent visibly more time in the oxygen-richer part of the tank. The respiratory rate (oral/opercular movements, min-1) was determined for mutants and controls, n = 5 (E) and this rate was significantly different between WT and HM (p-value 0.009) and HT and HM (p-value 0.009). As a test for centrophobia, we measured the time fish spent in the centre of the tank in the horizontal plane. The centre corresponds to the half centre area of the tank. There was no difference of occupancy between the three genotypes. n = 15, 19 and 7 for WT, HT and HM respectively. For each histogram, a Mann-Whitney test was performed; *, ** indicate p-values of < 0.05 and < 0.01 respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133986.g011: The 9 mpf col6a1ama605003 mutant fish presented a hypoxia behavior.We video-recorded for 30 min (25 frames/sec) and tracked the swimming trajectories in the vertical plane of WT, HT and HM col6a1ama605003 fish at 9 mpf in a tank. The position of the fish every 6 sec (1 frame every 150 frames) was plotted. A representative trajectory of each genotype is presented, WT (A), HT (B) and HM (C). From these videos (n = 5 of each genotype) the cumulated time fish swam in the upper quarter of the tank (D) was calculated and was significantly different between WT and HT (p-value 0.009), WT and HM (p-value 0.0283) and HT and HM (p-value 0.0472). HT and HM mutants spent visibly more time in the oxygen-richer part of the tank. The respiratory rate (oral/opercular movements, min-1) was determined for mutants and controls, n = 5 (E) and this rate was significantly different between WT and HM (p-value 0.009) and HT and HM (p-value 0.009). As a test for centrophobia, we measured the time fish spent in the centre of the tank in the horizontal plane. The centre corresponds to the half centre area of the tank. There was no difference of occupancy between the three genotypes. n = 15, 19 and 7 for WT, HT and HM respectively. For each histogram, a Mann-Whitney test was performed; *, ** indicate p-values of < 0.05 and < 0.01 respectively.
Mentions: At 3 mpf, no significant changes were observed between WT and col6a1ama605003 mutant fish when the total distance swum or the maximum swimming speed (Fig 10A and 10B) was measured. All statistical analyses are indicated in the corresponding tables in Figs 10 and 11. At 9 mpf, maximal instantaneous speed remained similar between genotypes (Fig 10D). Strikingly, however, HM fish swam significantly longer distances, roughly double those swam by WT and HT (Fig 10C; p-value WT vs. HM, 0.0015; HT vs. HM, 0.0077, Mann-Whitney, MW). This result was rather unexpected since HM fish had worse myofibre defects than HT. Thus, we further analyzed the distribution of instantaneous speed from 1 to 21 cm/s of 3 and 9 mpf mutants and WT sibling fish (see S7 Fig). Since the average fish body length (bl.) of 3 mpf (about 2 cm) is the half of the bl. of the fish at 9 mpf (about 4 cm), we defined three classes of speed activity profile (SAP) according to the fish bl.: rest, middle range and fast SAP. Rest SAP was defined as speed below 0.5 bl./s, middle range SAP as speed ranging from 0.5 to 3 bl./s and fast SAP as speed greater than 3 bl./s (see Materials and methods and Fig 10E–10J).

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