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Increased apoptosis of myoblasts in Drosophila model for the Walker-Warburg syndrome.

Ueyama M, Akimoto Y, Ichimiya T, Ueda R, Kawakami H, Aigaki T, Nishihara S - PLoS ONE (2010)

Bottom Line: We demonstrated that expression of RNA interference (RNAi) for the rt gene and the tw mutant was almost completely lethal and semi-lethal, respectively.Flies expressing RNAi had reduced lifespans.We then observed a high density of myoblasts with an enhanced degree of apoptosis in the tw mutant, which completely lost enzymatic activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioinformatics, Soka University, Hachioji, Tokyo, Japan.

ABSTRACT
Walker-Warburg syndrome, a progressive muscular dystrophy, is a severe disease with various kinds of symptoms such as muscle weakness and occasional seizures. The genes of protein O-mannosyltransferases 1 and 2 (POMT1 and POMT2), fukutin, and fukutin-related protein are responsible for this syndrome. In our previous study, we cloned Drosophila orthologs of human POMT1 and POMT2 and identified their activity. However, the mechanism of onset of this syndrome is not well understood. Furthermore, little is known about the behavioral properties of the Drosophila POMT1 and POMT2 mutants, which are called rotated abdomen (rt) and twisted (tw), respectively. First, we performed various kinds of behavioral tests and described in detail the muscle structures by using these mutants. The mutant flies exhibited abnormalities in heavy exercises such as climbing or flight but not in light movements such as locomotion. Defective motor function in mutants appeared immediately after eclosion and was exaggerated with aging. Along with motor function, muscle ultrastructure in the tw mutant was altered, as seen in human patients. We demonstrated that expression of RNA interference (RNAi) for the rt gene and the tw mutant was almost completely lethal and semi-lethal, respectively. Flies expressing RNAi had reduced lifespans. These findings clearly demonstrate that Drosophila POMT mutants are models for human muscular dystrophy. We then observed a high density of myoblasts with an enhanced degree of apoptosis in the tw mutant, which completely lost enzymatic activity. In this paper, we propose a novel mechanism for the development of muscular dystrophy: POMT mutation causes high myoblast density and position derangement, which result in apoptosis, muscle disorganization, and muscle cell defects.

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Larval body wall muscles in rt and tw mutant flies.Larval body wall muscles in abdominal segments 2–4 of wild-type flies (A), rtEP/rtKG flies (B), rt2/rt2 flies (C), and flies expressing RNAi for the tw gene (Act5C>tw-IR) (D). These muscles were stained by FITC-phalloidin. The arrowhead in (A) shows muscle 5. Deficient or thin muscle 5 were observed in rt mutants and in flies with ubiquitous expression of RNAi for the tw gene. We visualized live muscles by using a MHC-tauGFP marker in wild-type (MHC-tauGFP/Y) (E) and tw mutant (F–H) (tw, MHC-tauGFP/Y) larvae. The arrows indicate the absent muscles. A few muscles were absent in the mutant larvae (F and G), and many muscles were absent in rare cases of mutant larvae (H). The frequencies of abnormal patterning of larval body wall muscles in tw mutants (I). Error bars indicate standard error. The frequencies of abnormal patterning of the muscles were significantly higher in tw mutants. ***p<0.001 by Fisher's exact test.
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pone-0011557-g003: Larval body wall muscles in rt and tw mutant flies.Larval body wall muscles in abdominal segments 2–4 of wild-type flies (A), rtEP/rtKG flies (B), rt2/rt2 flies (C), and flies expressing RNAi for the tw gene (Act5C>tw-IR) (D). These muscles were stained by FITC-phalloidin. The arrowhead in (A) shows muscle 5. Deficient or thin muscle 5 were observed in rt mutants and in flies with ubiquitous expression of RNAi for the tw gene. We visualized live muscles by using a MHC-tauGFP marker in wild-type (MHC-tauGFP/Y) (E) and tw mutant (F–H) (tw, MHC-tauGFP/Y) larvae. The arrows indicate the absent muscles. A few muscles were absent in the mutant larvae (F and G), and many muscles were absent in rare cases of mutant larvae (H). The frequencies of abnormal patterning of larval body wall muscles in tw mutants (I). Error bars indicate standard error. The frequencies of abnormal patterning of the muscles were significantly higher in tw mutants. ***p<0.001 by Fisher's exact test.

Mentions: The behavioral data strongly suggest that Drosophila POMT mutants exhibit the muscle defect. Muscles of WWS patients also show structural abnormalities. Hence, we examined the effect of rt or tw on the patterning of muscles by checking the larval body wall muscles in mutants and in flies expressing RNAi for the rt gene. The normal structure of wild-type muscles is shown in Fig. 3A. Deficient or thin muscles were observed in the abdominal segment of rt mutants (Figs. 3B and C) and in flies expressing RNAi for the tw gene (Fig. 3D). The frequency of abnormal patterning of muscles detected by FITC-phalloidin staining in these mutants was approximately 10% (data not shown). In addition, we examined the frequency of abnormal patterning in larval muscles of tw mutants by using the MHC-tauGFP marker to observe live muscles. In MHC-tauGFP larvae, which did not show reduction of rt and tw transcripts (Fig. S3), the dorsal body wall muscles were structurally normal (Fig. 3E). A few muscles were absent in the mutant larvae (Figs. 3F and G), and many muscles were absent in rare cases of mutant larvae (Fig. 3H). Significantly higher frequencies of abnormal patterning were detected in the muscles of tw mutants (probabilities for all compared pairs were p<0.001, Fisher's exact test) (Fig. 3I).


Increased apoptosis of myoblasts in Drosophila model for the Walker-Warburg syndrome.

Ueyama M, Akimoto Y, Ichimiya T, Ueda R, Kawakami H, Aigaki T, Nishihara S - PLoS ONE (2010)

Larval body wall muscles in rt and tw mutant flies.Larval body wall muscles in abdominal segments 2–4 of wild-type flies (A), rtEP/rtKG flies (B), rt2/rt2 flies (C), and flies expressing RNAi for the tw gene (Act5C>tw-IR) (D). These muscles were stained by FITC-phalloidin. The arrowhead in (A) shows muscle 5. Deficient or thin muscle 5 were observed in rt mutants and in flies with ubiquitous expression of RNAi for the tw gene. We visualized live muscles by using a MHC-tauGFP marker in wild-type (MHC-tauGFP/Y) (E) and tw mutant (F–H) (tw, MHC-tauGFP/Y) larvae. The arrows indicate the absent muscles. A few muscles were absent in the mutant larvae (F and G), and many muscles were absent in rare cases of mutant larvae (H). The frequencies of abnormal patterning of larval body wall muscles in tw mutants (I). Error bars indicate standard error. The frequencies of abnormal patterning of the muscles were significantly higher in tw mutants. ***p<0.001 by Fisher's exact test.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2903483&req=5

pone-0011557-g003: Larval body wall muscles in rt and tw mutant flies.Larval body wall muscles in abdominal segments 2–4 of wild-type flies (A), rtEP/rtKG flies (B), rt2/rt2 flies (C), and flies expressing RNAi for the tw gene (Act5C>tw-IR) (D). These muscles were stained by FITC-phalloidin. The arrowhead in (A) shows muscle 5. Deficient or thin muscle 5 were observed in rt mutants and in flies with ubiquitous expression of RNAi for the tw gene. We visualized live muscles by using a MHC-tauGFP marker in wild-type (MHC-tauGFP/Y) (E) and tw mutant (F–H) (tw, MHC-tauGFP/Y) larvae. The arrows indicate the absent muscles. A few muscles were absent in the mutant larvae (F and G), and many muscles were absent in rare cases of mutant larvae (H). The frequencies of abnormal patterning of larval body wall muscles in tw mutants (I). Error bars indicate standard error. The frequencies of abnormal patterning of the muscles were significantly higher in tw mutants. ***p<0.001 by Fisher's exact test.
Mentions: The behavioral data strongly suggest that Drosophila POMT mutants exhibit the muscle defect. Muscles of WWS patients also show structural abnormalities. Hence, we examined the effect of rt or tw on the patterning of muscles by checking the larval body wall muscles in mutants and in flies expressing RNAi for the rt gene. The normal structure of wild-type muscles is shown in Fig. 3A. Deficient or thin muscles were observed in the abdominal segment of rt mutants (Figs. 3B and C) and in flies expressing RNAi for the tw gene (Fig. 3D). The frequency of abnormal patterning of muscles detected by FITC-phalloidin staining in these mutants was approximately 10% (data not shown). In addition, we examined the frequency of abnormal patterning in larval muscles of tw mutants by using the MHC-tauGFP marker to observe live muscles. In MHC-tauGFP larvae, which did not show reduction of rt and tw transcripts (Fig. S3), the dorsal body wall muscles were structurally normal (Fig. 3E). A few muscles were absent in the mutant larvae (Figs. 3F and G), and many muscles were absent in rare cases of mutant larvae (Fig. 3H). Significantly higher frequencies of abnormal patterning were detected in the muscles of tw mutants (probabilities for all compared pairs were p<0.001, Fisher's exact test) (Fig. 3I).

Bottom Line: We demonstrated that expression of RNA interference (RNAi) for the rt gene and the tw mutant was almost completely lethal and semi-lethal, respectively.Flies expressing RNAi had reduced lifespans.We then observed a high density of myoblasts with an enhanced degree of apoptosis in the tw mutant, which completely lost enzymatic activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioinformatics, Soka University, Hachioji, Tokyo, Japan.

ABSTRACT
Walker-Warburg syndrome, a progressive muscular dystrophy, is a severe disease with various kinds of symptoms such as muscle weakness and occasional seizures. The genes of protein O-mannosyltransferases 1 and 2 (POMT1 and POMT2), fukutin, and fukutin-related protein are responsible for this syndrome. In our previous study, we cloned Drosophila orthologs of human POMT1 and POMT2 and identified their activity. However, the mechanism of onset of this syndrome is not well understood. Furthermore, little is known about the behavioral properties of the Drosophila POMT1 and POMT2 mutants, which are called rotated abdomen (rt) and twisted (tw), respectively. First, we performed various kinds of behavioral tests and described in detail the muscle structures by using these mutants. The mutant flies exhibited abnormalities in heavy exercises such as climbing or flight but not in light movements such as locomotion. Defective motor function in mutants appeared immediately after eclosion and was exaggerated with aging. Along with motor function, muscle ultrastructure in the tw mutant was altered, as seen in human patients. We demonstrated that expression of RNA interference (RNAi) for the rt gene and the tw mutant was almost completely lethal and semi-lethal, respectively. Flies expressing RNAi had reduced lifespans. These findings clearly demonstrate that Drosophila POMT mutants are models for human muscular dystrophy. We then observed a high density of myoblasts with an enhanced degree of apoptosis in the tw mutant, which completely lost enzymatic activity. In this paper, we propose a novel mechanism for the development of muscular dystrophy: POMT mutation causes high myoblast density and position derangement, which result in apoptosis, muscle disorganization, and muscle cell defects.

Show MeSH
Related in: MedlinePlus