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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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Related in: MedlinePlus

Crosses for examining tw and rt mutant fly viability.(A) The cross scheme for examining tw mutant fly viability. Female tw/+ flies were crossed with male tw mutants (tw/Y). Genotypes of F1 progeny are tw/tw, tw/Y, tw/+, and +/Y. Individuals with genotype tw/tw or tw/Y have the twisted abdominal phenotype, which is represented as [tw−]. On the other hand, individuals with genotype tw/+ or +/Y have the normal abdominal phenotype, which is represented as [tw+]. The number of F1 progeny with phenotype [tw−] or [tw+] is shown in Table 6. (B) The cross scheme for the rescue experiment of the tw mutant. Female Act5C-Gal4 driver flies with tw mutation were crossed with male UAS-tw flies with tw mutation. In the F1 progeny, rescued and non-rescued individuals are born and are described as [Cy−, GFP−] and [Cy+, GFP+], respectively. The number of F1 progeny with phenotypes [Cy−, GFP−] or [Cy+, GFP+] is shown in Table 7. (C) The cross scheme for examining the viability of flies expressing RNAi for the rt gene. Female Act5C-Gal4 driver flies were crossed with male UAS-rt-IR flies. In the F1 progeny, flies expressing RNAi for the rt gene, Act5C-Gal4 driver, and UAS-rt-IR individuals were born. Flies expressing RNAi for the rt gene do not have CyO GFP balancer; thus, the phenotype is described as [Cy−, GFP−]. Act5C-Gal4 driver and UAS-rt-IR flies have CyO GFP balancer; thus, the phenotype is described as [Cy+, GFP+]. The number of F1 progeny with ([Cy−, GFP−]) or without ([Cy+, GFP+]) ubiquitous expression of RNAi for the rt gene is shown in Table 8.
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pone-0011557-g010: Crosses for examining tw and rt mutant fly viability.(A) The cross scheme for examining tw mutant fly viability. Female tw/+ flies were crossed with male tw mutants (tw/Y). Genotypes of F1 progeny are tw/tw, tw/Y, tw/+, and +/Y. Individuals with genotype tw/tw or tw/Y have the twisted abdominal phenotype, which is represented as [tw−]. On the other hand, individuals with genotype tw/+ or +/Y have the normal abdominal phenotype, which is represented as [tw+]. The number of F1 progeny with phenotype [tw−] or [tw+] is shown in Table 6. (B) The cross scheme for the rescue experiment of the tw mutant. Female Act5C-Gal4 driver flies with tw mutation were crossed with male UAS-tw flies with tw mutation. In the F1 progeny, rescued and non-rescued individuals are born and are described as [Cy−, GFP−] and [Cy+, GFP+], respectively. The number of F1 progeny with phenotypes [Cy−, GFP−] or [Cy+, GFP+] is shown in Table 7. (C) The cross scheme for examining the viability of flies expressing RNAi for the rt gene. Female Act5C-Gal4 driver flies were crossed with male UAS-rt-IR flies. In the F1 progeny, flies expressing RNAi for the rt gene, Act5C-Gal4 driver, and UAS-rt-IR individuals were born. Flies expressing RNAi for the rt gene do not have CyO GFP balancer; thus, the phenotype is described as [Cy−, GFP−]. Act5C-Gal4 driver and UAS-rt-IR flies have CyO GFP balancer; thus, the phenotype is described as [Cy+, GFP+]. The number of F1 progeny with ([Cy−, GFP−]) or without ([Cy+, GFP+]) ubiquitous expression of RNAi for the rt gene is shown in Table 8.

Mentions: It is known that WWS has high lethality rates during early development. Therefore, we determined whether Drosophila POMT genes play an important role in viability. We crossed females heterozygous for tw with male hemizygous for tw and checked the number of F1 progenies (Fig. 10A). If all the eggs of F1 progenies hatched and developed normally, the ratio of the number of individuals having a twisted abdomen, the tw phenotype, to those having a normal abdomen is expected to be 1. The ratio was found to be 0.2, significantly lower than the expected ratio (p<0.001, chi-square test) (Table 6). In addition, in order to determine the necessity of the tw gene for viability, we performed a rescue experiment by expressing the tw gene in tw mutants (Fig. 10B). The ratio was found to be 1.25 and was significantly increased (p<0.001, chi-square test) (Table 7). These data indicated that the tw gene plays an important role in viability and normal development. We also examined fly viability after knockdown of the rt gene under 3 different temperature conditions. Knockdown at high temperature is more efficient than that at low temperature because yeast transcriptional factor GAL4 binds strongly to UAS sequences. Ratios of the number of knockdown flies to the number of non-knockdown flies were 0.26, 0.12, and 0.00 at 18, 25, and 28°C, respectively (Fig. 10C, Table 8). Growing flies at a higher temperature resulted in higher lethality, indicating that the rt gene also contributed to normal development. The abovementioned results showed that the tw and rt genes were essential for the viability of the embryo, larva, and/or pupa. Elucidation of the reason for the lethal phenotype in these mutants could clarify the mechanism of low viability in human patients.


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)

Crosses for examining tw and rt mutant fly viability.(A) The cross scheme for examining tw mutant fly viability. Female tw/+ flies were crossed with male tw mutants (tw/Y). Genotypes of F1 progeny are tw/tw, tw/Y, tw/+, and +/Y. Individuals with genotype tw/tw or tw/Y have the twisted abdominal phenotype, which is represented as [tw−]. On the other hand, individuals with genotype tw/+ or +/Y have the normal abdominal phenotype, which is represented as [tw+]. The number of F1 progeny with phenotype [tw−] or [tw+] is shown in Table 6. (B) The cross scheme for the rescue experiment of the tw mutant. Female Act5C-Gal4 driver flies with tw mutation were crossed with male UAS-tw flies with tw mutation. In the F1 progeny, rescued and non-rescued individuals are born and are described as [Cy−, GFP−] and [Cy+, GFP+], respectively. The number of F1 progeny with phenotypes [Cy−, GFP−] or [Cy+, GFP+] is shown in Table 7. (C) The cross scheme for examining the viability of flies expressing RNAi for the rt gene. Female Act5C-Gal4 driver flies were crossed with male UAS-rt-IR flies. In the F1 progeny, flies expressing RNAi for the rt gene, Act5C-Gal4 driver, and UAS-rt-IR individuals were born. Flies expressing RNAi for the rt gene do not have CyO GFP balancer; thus, the phenotype is described as [Cy−, GFP−]. Act5C-Gal4 driver and UAS-rt-IR flies have CyO GFP balancer; thus, the phenotype is described as [Cy+, GFP+]. The number of F1 progeny with ([Cy−, GFP−]) or without ([Cy+, GFP+]) ubiquitous expression of RNAi for the rt gene is shown in Table 8.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2903483&req=5

pone-0011557-g010: Crosses for examining tw and rt mutant fly viability.(A) The cross scheme for examining tw mutant fly viability. Female tw/+ flies were crossed with male tw mutants (tw/Y). Genotypes of F1 progeny are tw/tw, tw/Y, tw/+, and +/Y. Individuals with genotype tw/tw or tw/Y have the twisted abdominal phenotype, which is represented as [tw−]. On the other hand, individuals with genotype tw/+ or +/Y have the normal abdominal phenotype, which is represented as [tw+]. The number of F1 progeny with phenotype [tw−] or [tw+] is shown in Table 6. (B) The cross scheme for the rescue experiment of the tw mutant. Female Act5C-Gal4 driver flies with tw mutation were crossed with male UAS-tw flies with tw mutation. In the F1 progeny, rescued and non-rescued individuals are born and are described as [Cy−, GFP−] and [Cy+, GFP+], respectively. The number of F1 progeny with phenotypes [Cy−, GFP−] or [Cy+, GFP+] is shown in Table 7. (C) The cross scheme for examining the viability of flies expressing RNAi for the rt gene. Female Act5C-Gal4 driver flies were crossed with male UAS-rt-IR flies. In the F1 progeny, flies expressing RNAi for the rt gene, Act5C-Gal4 driver, and UAS-rt-IR individuals were born. Flies expressing RNAi for the rt gene do not have CyO GFP balancer; thus, the phenotype is described as [Cy−, GFP−]. Act5C-Gal4 driver and UAS-rt-IR flies have CyO GFP balancer; thus, the phenotype is described as [Cy+, GFP+]. The number of F1 progeny with ([Cy−, GFP−]) or without ([Cy+, GFP+]) ubiquitous expression of RNAi for the rt gene is shown in Table 8.
Mentions: It is known that WWS has high lethality rates during early development. Therefore, we determined whether Drosophila POMT genes play an important role in viability. We crossed females heterozygous for tw with male hemizygous for tw and checked the number of F1 progenies (Fig. 10A). If all the eggs of F1 progenies hatched and developed normally, the ratio of the number of individuals having a twisted abdomen, the tw phenotype, to those having a normal abdomen is expected to be 1. The ratio was found to be 0.2, significantly lower than the expected ratio (p<0.001, chi-square test) (Table 6). In addition, in order to determine the necessity of the tw gene for viability, we performed a rescue experiment by expressing the tw gene in tw mutants (Fig. 10B). The ratio was found to be 1.25 and was significantly increased (p<0.001, chi-square test) (Table 7). These data indicated that the tw gene plays an important role in viability and normal development. We also examined fly viability after knockdown of the rt gene under 3 different temperature conditions. Knockdown at high temperature is more efficient than that at low temperature because yeast transcriptional factor GAL4 binds strongly to UAS sequences. Ratios of the number of knockdown flies to the number of non-knockdown flies were 0.26, 0.12, and 0.00 at 18, 25, and 28°C, respectively (Fig. 10C, Table 8). Growing flies at a higher temperature resulted in higher lethality, indicating that the rt gene also contributed to normal development. The abovementioned results showed that the tw and rt genes were essential for the viability of the embryo, larva, and/or pupa. Elucidation of the reason for the lethal phenotype in these mutants could clarify the mechanism of low viability in human patients.

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