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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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Excessive apoptosis of myoblasts in the wing imaginal disc of tw mutant larva.(A, C, and E) The wild-type fly (1151>GFPnls). (B, D, and F) The tw mutant fly (tw, 1151>GFPnls). (A) and (B) Myoblasts in the wing imaginal disc of larvae. The GFP localize in myoblast nuclei. (C) and (D) Myoblasts stained by caspase-3 antibody, a marker of apoptotic cells. (E) and (F) Merged images of (A) and (C) and of (B) and (D), respectively. The arrowheads and arrows show the co-localization of GFP and caspase-3. The arrowheads show the nuclei just before breakdown. The arrows show degraded nuclei in more of a progressive apoptotic stage than the nuclei shown by arrowheads. (G) The number of myoblasts positive for caspase-3 in the wing imaginal disc of wild-type and tw mutant larvae. Error bars indicate standard error. The number of apoptotic myoblasts in tw mutant larvae was significantly lower than that in wild-type larvae. ***p<0.001 by t test.
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pone-0011557-g008: Excessive apoptosis of myoblasts in the wing imaginal disc of tw mutant larva.(A, C, and E) The wild-type fly (1151>GFPnls). (B, D, and F) The tw mutant fly (tw, 1151>GFPnls). (A) and (B) Myoblasts in the wing imaginal disc of larvae. The GFP localize in myoblast nuclei. (C) and (D) Myoblasts stained by caspase-3 antibody, a marker of apoptotic cells. (E) and (F) Merged images of (A) and (C) and of (B) and (D), respectively. The arrowheads and arrows show the co-localization of GFP and caspase-3. The arrowheads show the nuclei just before breakdown. The arrows show degraded nuclei in more of a progressive apoptotic stage than the nuclei shown by arrowheads. (G) The number of myoblasts positive for caspase-3 in the wing imaginal disc of wild-type and tw mutant larvae. Error bars indicate standard error. The number of apoptotic myoblasts in tw mutant larvae was significantly lower than that in wild-type larvae. ***p<0.001 by t test.

Mentions: Changing of the density of myoblasts is a result of an alteration in cell death or cell division. Therefore, we checked the number of apoptotic and dividing myoblasts. The nuclei of myoblasts were visualized by GFP (Figs. 8A and B), and we observed the apoptotic myoblasts by using the cleaved caspase-3 antibody (Figs. 8C and D). Both wild-type flies and tw mutants had apoptotic myoblasts (Figs. 8E and F); however, the number of apoptotic myoblasts in the wing discs of tw mutants was 2.4-fold higher than that in the wing discs of wild-type flies (Fig. 8G). We also observed dividing myoblasts by using phospho-histone H3 antibody. The number of dividing myoblasts in the wing disc did not differ between wild-type flies and tw mutants (Fig. S4). These results showed that apoptosis was enhanced in myoblasts of the tw mutant while the number of dividing cells was not altered. Excessive apoptosis of myoblasts during muscle differentiation should lead to muscle disorganization, including muscle cell defects.


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)

Excessive apoptosis of myoblasts in the wing imaginal disc of tw mutant larva.(A, C, and E) The wild-type fly (1151>GFPnls). (B, D, and F) The tw mutant fly (tw, 1151>GFPnls). (A) and (B) Myoblasts in the wing imaginal disc of larvae. The GFP localize in myoblast nuclei. (C) and (D) Myoblasts stained by caspase-3 antibody, a marker of apoptotic cells. (E) and (F) Merged images of (A) and (C) and of (B) and (D), respectively. The arrowheads and arrows show the co-localization of GFP and caspase-3. The arrowheads show the nuclei just before breakdown. The arrows show degraded nuclei in more of a progressive apoptotic stage than the nuclei shown by arrowheads. (G) The number of myoblasts positive for caspase-3 in the wing imaginal disc of wild-type and tw mutant larvae. Error bars indicate standard error. The number of apoptotic myoblasts in tw mutant larvae was significantly lower than that in wild-type larvae. ***p<0.001 by t test.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0011557-g008: Excessive apoptosis of myoblasts in the wing imaginal disc of tw mutant larva.(A, C, and E) The wild-type fly (1151>GFPnls). (B, D, and F) The tw mutant fly (tw, 1151>GFPnls). (A) and (B) Myoblasts in the wing imaginal disc of larvae. The GFP localize in myoblast nuclei. (C) and (D) Myoblasts stained by caspase-3 antibody, a marker of apoptotic cells. (E) and (F) Merged images of (A) and (C) and of (B) and (D), respectively. The arrowheads and arrows show the co-localization of GFP and caspase-3. The arrowheads show the nuclei just before breakdown. The arrows show degraded nuclei in more of a progressive apoptotic stage than the nuclei shown by arrowheads. (G) The number of myoblasts positive for caspase-3 in the wing imaginal disc of wild-type and tw mutant larvae. Error bars indicate standard error. The number of apoptotic myoblasts in tw mutant larvae was significantly lower than that in wild-type larvae. ***p<0.001 by t test.
Mentions: Changing of the density of myoblasts is a result of an alteration in cell death or cell division. Therefore, we checked the number of apoptotic and dividing myoblasts. The nuclei of myoblasts were visualized by GFP (Figs. 8A and B), and we observed the apoptotic myoblasts by using the cleaved caspase-3 antibody (Figs. 8C and D). Both wild-type flies and tw mutants had apoptotic myoblasts (Figs. 8E and F); however, the number of apoptotic myoblasts in the wing discs of tw mutants was 2.4-fold higher than that in the wing discs of wild-type flies (Fig. 8G). We also observed dividing myoblasts by using phospho-histone H3 antibody. The number of dividing myoblasts in the wing disc did not differ between wild-type flies and tw mutants (Fig. S4). These results showed that apoptosis was enhanced in myoblasts of the tw mutant while the number of dividing cells was not altered. Excessive apoptosis of myoblasts during muscle differentiation should lead to muscle disorganization, including muscle cell defects.

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