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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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Genetic interaction between rt or tw and Dg in the wing.(A) Normal wing shape in wild-type flies. (B) Blistered phenotype in the wings of knockdown flies. (C) Penetrances of the blistered phenotype in knockdown flies. At least 30 individuals were observed in each knockdown group. At 28°C, the penetrances of the blistered phenotype with the double knockdowns rt-Dg and tw-Dg were significantly higher than those with single knockdown. *p<0.05; ***p<0.001 by Fisher's exact test. n.s., not significant.
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pone-0011557-g013: Genetic interaction between rt or tw and Dg in the wing.(A) Normal wing shape in wild-type flies. (B) Blistered phenotype in the wings of knockdown flies. (C) Penetrances of the blistered phenotype in knockdown flies. At least 30 individuals were observed in each knockdown group. At 28°C, the penetrances of the blistered phenotype with the double knockdowns rt-Dg and tw-Dg were significantly higher than those with single knockdown. *p<0.05; ***p<0.001 by Fisher's exact test. n.s., not significant.

Mentions: Dg is one of the putative core proteins that are O-mannosylated by RT and TW. Wild-type flies had normal-shaped wings (Fig. 13A); however, knockdown of Dg in the posterior region of the wing disc resulted in a blistered phenotype (Fig. 13B). The blistered phenotype results from cell adhesion failure in the 2 cell layers during wing development. Actually, Dg expression was dramatically decreased in the posterior region (Fig. S5), indicating that Dg contributed the attachment of the 2 cell layers during wing development. We examined the genetic interaction between rt or tw and Dg by using this phenotype. At 25°C, the penetrances of the blistered phenotype in wings of single knockdown flies of rt, tw, and Dg were 0, 0, and 0.06, respectively. The penetrances of the phenotype in double knockdown flies of rt-Dg and tw-Dg were 0.14 and 0.08, respectively, but they were not significantly higher than those of the single knockdown flies (Fisher's exact test). As mentioned in the above section, “Lethality in tw mutants and flies expressing RNAi for the rt gene,” knockdown at 28°C is more efficient than that at 25°C. Thus, we performed the knockdown at 28°C. At 28°C, the penetrances of the blistered phenotype in wings of single knockdown flies of rt, tw, and Dg were 0, 0, and 0.34, respectively. The penetrances of double knockdown flies of rt-Dg and tw-Dg were 0.86 and 0.48, and they were significantly higher than those of single knockdown flies (p<0.001 and p<0.05, Fisher's exact test) (Fig. 13C). These data showed that rt or tw genetically interact with Dg to contribute to cell adhesion in the wings. Together with the high density of myoblasts observed in the tw mutant (Fig. 7F), these results suggest that both epidermal cells and muscle progenitor cells of Drosophila POMT mutants give rise to cell adhesion derangement.


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)

Genetic interaction between rt or tw and Dg in the wing.(A) Normal wing shape in wild-type flies. (B) Blistered phenotype in the wings of knockdown flies. (C) Penetrances of the blistered phenotype in knockdown flies. At least 30 individuals were observed in each knockdown group. At 28°C, the penetrances of the blistered phenotype with the double knockdowns rt-Dg and tw-Dg were significantly higher than those with single knockdown. *p<0.05; ***p<0.001 by Fisher's exact test. n.s., not significant.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2903483&req=5

pone-0011557-g013: Genetic interaction between rt or tw and Dg in the wing.(A) Normal wing shape in wild-type flies. (B) Blistered phenotype in the wings of knockdown flies. (C) Penetrances of the blistered phenotype in knockdown flies. At least 30 individuals were observed in each knockdown group. At 28°C, the penetrances of the blistered phenotype with the double knockdowns rt-Dg and tw-Dg were significantly higher than those with single knockdown. *p<0.05; ***p<0.001 by Fisher's exact test. n.s., not significant.
Mentions: Dg is one of the putative core proteins that are O-mannosylated by RT and TW. Wild-type flies had normal-shaped wings (Fig. 13A); however, knockdown of Dg in the posterior region of the wing disc resulted in a blistered phenotype (Fig. 13B). The blistered phenotype results from cell adhesion failure in the 2 cell layers during wing development. Actually, Dg expression was dramatically decreased in the posterior region (Fig. S5), indicating that Dg contributed the attachment of the 2 cell layers during wing development. We examined the genetic interaction between rt or tw and Dg by using this phenotype. At 25°C, the penetrances of the blistered phenotype in wings of single knockdown flies of rt, tw, and Dg were 0, 0, and 0.06, respectively. The penetrances of the phenotype in double knockdown flies of rt-Dg and tw-Dg were 0.14 and 0.08, respectively, but they were not significantly higher than those of the single knockdown flies (Fisher's exact test). As mentioned in the above section, “Lethality in tw mutants and flies expressing RNAi for the rt gene,” knockdown at 28°C is more efficient than that at 25°C. Thus, we performed the knockdown at 28°C. At 28°C, the penetrances of the blistered phenotype in wings of single knockdown flies of rt, tw, and Dg were 0, 0, and 0.34, respectively. The penetrances of double knockdown flies of rt-Dg and tw-Dg were 0.86 and 0.48, and they were significantly higher than those of single knockdown flies (p<0.001 and p<0.05, Fisher's exact test) (Fig. 13C). These data showed that rt or tw genetically interact with Dg to contribute to cell adhesion in the wings. Together with the high density of myoblasts observed in the tw mutant (Fig. 7F), these results suggest that both epidermal cells and muscle progenitor cells of Drosophila POMT mutants give rise to cell adhesion derangement.

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