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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 HM fish swam around twice the distance of WT and HT fish.We video-recorded free-swimming WT, HT and HM col6a1ama605003 fish at 3 mpf and 9 mpf for one hour in the horizontal plane, then we calculated by triangulation the total distance swum (A, C) and the maximum instantaneous speed (B, D). (A) At 3 mpf, there was no significant difference in the total distance swam between WT and HT, between WT and HM or between HT and HM. (C) At 9 mpf, there was a significant difference in the total swum distance between WT and HM (C, p-value 0.0015) and between HT and HM (C, p-value 0.0077), but not between WT and HT, indicating the late development of impairment in HM. There was no difference in the maximum speed between genotypes at the two ages studied (B, D). (E-J) We represented in pie-charts the speed activity profile (SAP) distributions in percentage of 3 classes and according to the fish bl. i.e. rest SAP (horizontal lines, speed slower than 1 cm/s for 3 mpf, E, and 2 cm/s for 9 mpf, H), middle range SAP (squares, speed comprise between 1 to 6 cm/s for 3 mpf, F and 2 to 12 cm/s for 9 mpf, I) and fast SAP (open dots, speed superior to 6 cm/s for 3 mpf, G and to 12 cm/s for 9 mpf, J). This representation allowed us to show that at 9 mpf, there is a highly significant difference between WT versus HM (H vs. J, p-value 9.421x10-11) and between HT and HM (I vs. J, 9.83x10-14). We further analyzed in a pairwise manner the histograms of the time (s) fish swum in the 3 SAP classes (speed distribution) described above for 3 mpf (K, L and M for rest, middle and fast SAP respectively) and 9 mpf (N, O and P for rest, middle and fast SAP respectively). This analysis showed that there was no difference in SAP within any of the 3 mpf groups. But the analysis showed that 9 mpf HM swam significantly more time in middle range SAP at the expense of their resting time i.e. in the rest SAP the difference is significant between WT and HM (N, p-value 0.0035); between HT and HM (N, p-value 0.0182). In the middle range SAP, the difference was significant between WT and HM (O, p-value 0.0035) and between HT and HM (O, p-value 0.0082). For 3 mpf fish, n = 12, 16 and 11 for WT, HT and HM respectively; for 9 mpf fish, n = 15, 19 and 7 for WT, HT and HM respectively. For each histogram and pie-chart, we performed either a Mann-Whitney (MW) or Chi-square test respectively, with *, ** and **** indicating p-values of <0.05, 0.01 and 0.0001 respectively.
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pone.0133986.g010: The 9 mpf col6a1ama605003 HM fish swam around twice the distance of WT and HT fish.We video-recorded free-swimming WT, HT and HM col6a1ama605003 fish at 3 mpf and 9 mpf for one hour in the horizontal plane, then we calculated by triangulation the total distance swum (A, C) and the maximum instantaneous speed (B, D). (A) At 3 mpf, there was no significant difference in the total distance swam between WT and HT, between WT and HM or between HT and HM. (C) At 9 mpf, there was a significant difference in the total swum distance between WT and HM (C, p-value 0.0015) and between HT and HM (C, p-value 0.0077), but not between WT and HT, indicating the late development of impairment in HM. There was no difference in the maximum speed between genotypes at the two ages studied (B, D). (E-J) We represented in pie-charts the speed activity profile (SAP) distributions in percentage of 3 classes and according to the fish bl. i.e. rest SAP (horizontal lines, speed slower than 1 cm/s for 3 mpf, E, and 2 cm/s for 9 mpf, H), middle range SAP (squares, speed comprise between 1 to 6 cm/s for 3 mpf, F and 2 to 12 cm/s for 9 mpf, I) and fast SAP (open dots, speed superior to 6 cm/s for 3 mpf, G and to 12 cm/s for 9 mpf, J). This representation allowed us to show that at 9 mpf, there is a highly significant difference between WT versus HM (H vs. J, p-value 9.421x10-11) and between HT and HM (I vs. J, 9.83x10-14). We further analyzed in a pairwise manner the histograms of the time (s) fish swum in the 3 SAP classes (speed distribution) described above for 3 mpf (K, L and M for rest, middle and fast SAP respectively) and 9 mpf (N, O and P for rest, middle and fast SAP respectively). This analysis showed that there was no difference in SAP within any of the 3 mpf groups. But the analysis showed that 9 mpf HM swam significantly more time in middle range SAP at the expense of their resting time i.e. in the rest SAP the difference is significant between WT and HM (N, p-value 0.0035); between HT and HM (N, p-value 0.0182). In the middle range SAP, the difference was significant between WT and HM (O, p-value 0.0035) and between HT and HM (O, p-value 0.0082). For 3 mpf fish, n = 12, 16 and 11 for WT, HT and HM respectively; for 9 mpf fish, n = 15, 19 and 7 for WT, HT and HM respectively. For each histogram and pie-chart, we performed either a Mann-Whitney (MW) or Chi-square test respectively, with *, ** and **** indicating p-values of <0.05, 0.01 and 0.0001 respectively.

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)

The 9 mpf col6a1ama605003 HM fish swam around twice the distance of WT and HT fish.We video-recorded free-swimming WT, HT and HM col6a1ama605003 fish at 3 mpf and 9 mpf for one hour in the horizontal plane, then we calculated by triangulation the total distance swum (A, C) and the maximum instantaneous speed (B, D). (A) At 3 mpf, there was no significant difference in the total distance swam between WT and HT, between WT and HM or between HT and HM. (C) At 9 mpf, there was a significant difference in the total swum distance between WT and HM (C, p-value 0.0015) and between HT and HM (C, p-value 0.0077), but not between WT and HT, indicating the late development of impairment in HM. There was no difference in the maximum speed between genotypes at the two ages studied (B, D). (E-J) We represented in pie-charts the speed activity profile (SAP) distributions in percentage of 3 classes and according to the fish bl. i.e. rest SAP (horizontal lines, speed slower than 1 cm/s for 3 mpf, E, and 2 cm/s for 9 mpf, H), middle range SAP (squares, speed comprise between 1 to 6 cm/s for 3 mpf, F and 2 to 12 cm/s for 9 mpf, I) and fast SAP (open dots, speed superior to 6 cm/s for 3 mpf, G and to 12 cm/s for 9 mpf, J). This representation allowed us to show that at 9 mpf, there is a highly significant difference between WT versus HM (H vs. J, p-value 9.421x10-11) and between HT and HM (I vs. J, 9.83x10-14). We further analyzed in a pairwise manner the histograms of the time (s) fish swum in the 3 SAP classes (speed distribution) described above for 3 mpf (K, L and M for rest, middle and fast SAP respectively) and 9 mpf (N, O and P for rest, middle and fast SAP respectively). This analysis showed that there was no difference in SAP within any of the 3 mpf groups. But the analysis showed that 9 mpf HM swam significantly more time in middle range SAP at the expense of their resting time i.e. in the rest SAP the difference is significant between WT and HM (N, p-value 0.0035); between HT and HM (N, p-value 0.0182). In the middle range SAP, the difference was significant between WT and HM (O, p-value 0.0035) and between HT and HM (O, p-value 0.0082). For 3 mpf fish, n = 12, 16 and 11 for WT, HT and HM respectively; for 9 mpf fish, n = 15, 19 and 7 for WT, HT and HM respectively. For each histogram and pie-chart, we performed either a Mann-Whitney (MW) or Chi-square test respectively, with *, ** and **** indicating p-values of <0.05, 0.01 and 0.0001 respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0133986.g010: The 9 mpf col6a1ama605003 HM fish swam around twice the distance of WT and HT fish.We video-recorded free-swimming WT, HT and HM col6a1ama605003 fish at 3 mpf and 9 mpf for one hour in the horizontal plane, then we calculated by triangulation the total distance swum (A, C) and the maximum instantaneous speed (B, D). (A) At 3 mpf, there was no significant difference in the total distance swam between WT and HT, between WT and HM or between HT and HM. (C) At 9 mpf, there was a significant difference in the total swum distance between WT and HM (C, p-value 0.0015) and between HT and HM (C, p-value 0.0077), but not between WT and HT, indicating the late development of impairment in HM. There was no difference in the maximum speed between genotypes at the two ages studied (B, D). (E-J) We represented in pie-charts the speed activity profile (SAP) distributions in percentage of 3 classes and according to the fish bl. i.e. rest SAP (horizontal lines, speed slower than 1 cm/s for 3 mpf, E, and 2 cm/s for 9 mpf, H), middle range SAP (squares, speed comprise between 1 to 6 cm/s for 3 mpf, F and 2 to 12 cm/s for 9 mpf, I) and fast SAP (open dots, speed superior to 6 cm/s for 3 mpf, G and to 12 cm/s for 9 mpf, J). This representation allowed us to show that at 9 mpf, there is a highly significant difference between WT versus HM (H vs. J, p-value 9.421x10-11) and between HT and HM (I vs. J, 9.83x10-14). We further analyzed in a pairwise manner the histograms of the time (s) fish swum in the 3 SAP classes (speed distribution) described above for 3 mpf (K, L and M for rest, middle and fast SAP respectively) and 9 mpf (N, O and P for rest, middle and fast SAP respectively). This analysis showed that there was no difference in SAP within any of the 3 mpf groups. But the analysis showed that 9 mpf HM swam significantly more time in middle range SAP at the expense of their resting time i.e. in the rest SAP the difference is significant between WT and HM (N, p-value 0.0035); between HT and HM (N, p-value 0.0182). In the middle range SAP, the difference was significant between WT and HM (O, p-value 0.0035) and between HT and HM (O, p-value 0.0082). For 3 mpf fish, n = 12, 16 and 11 for WT, HT and HM respectively; for 9 mpf fish, n = 15, 19 and 7 for WT, HT and HM respectively. For each histogram and pie-chart, we performed either a Mann-Whitney (MW) or Chi-square test respectively, with *, ** and **** indicating p-values of <0.05, 0.01 and 0.0001 respectively.
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