Drosophila POGLUT1 Mutations and Muscle Development

The effect of mutations on POGLUT1 activity was assessed in terms of the development of indirect flight muscles in Drosophila [16 (link)]. Rescue experiments were performed with Mef2-GAL4 driven overexpression of POGLUT1^WT and POGLUT1^mutant in rumi^79/79 animals, as reported previously [35 (link)]. Animals were raised at 25°C until puparium formation. After 25% pupal development, pupal indirect flight muscles were dissected and stained using standard methods. Antibodies used were mouse 22C10 1:50 (DSHB), mouse α‐FLAG M2 1:100 (Sigma), donkey α-rabbit-Cy3 1:500, donkeyα-mouse-Cy3 1:500 (Jackson Immuno Research Laboratories). Confocal imaging was performed on a Leica TCS-SP8 microscope. Myotube length measurements were performed with Amira5.2.2. Mean myotube lengths and standard deviations (SD) were calculated for pupae with the following genotypes: WT control (y w; n=3), rumi^79/79 (n=3), and rumi^79/79 overexpressing POGLUT1^WT (n=4), POGLUT1^D233E (n=3), POGLUT1^R183W (n=3), POGLUT1^Y57C (n=3) and POGLUT1^C102F (n=3). One-way ANOVA with Tukey’s multiple comparisons test was used to determine the P-values. Images were processed with Adobe Photoshop CS5; figures were assembled in Adobe Illustrator CS5.

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Servián-Morilla E., Cabrera-Serrano M., Johnson K., Pandey A., Ito A., Rivas E., Chamova T., Muelas N., Mongini T., Nafissi S., Claeys K.G., Grewal R.P., Takeuchi M., Hao H., Bönnemann C., Neto O.A., Medne L., Brandsema J., Topf A., Taneva A., Vilchez J.J., Tournev I., Haltiwanger R.S., Takeuchi H., Jafar-Nejad H., Straub V, & Paradas C. (2020). POGLUT1 biallelic mutations cause a myopathy with reduced satellite cells, α-dystroglycan hypoglycosylation and a distinctive radiological pattern. Acta neuropathologica, 139(3), 565-582.

Publication 2020

mouse α‐FLAG M2 donkey α-rabbit-Cy3 donkeyα-mouse-Cy3 TCS-SP8 microscope Illustrator CS5

Animals Anova Antibodies Development muscles Donkey Drosophila Genotypes Microscope Mouse Muscles Mutations Myotube Pupae Rabbit

Corresponding Organization :

Other organizations : Instituto de Biomedicina de Sevilla, Universidad de Sevilla, Newcastle University, Baylor College of Medicine, University of Georgia, Biomedical Research Networking Center on Neurodegenerative Diseases, Medical University of Sofia, Centro de Investigación Biomédica en Red, Centre for Biomedical Network Research on Rare Diseases, Instituto de Investigación Sanitaria La Fe, University of Turin, Tehran University of Medical Sciences, Shariati Hospital, KU Leuven, Saint Francis Medical Center, Seton Hall University, National Institute of Neurological Disorders and Stroke, Children's Hospital of Philadelphia

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Variable analysis

independent variables

POGLUT1WT overexpression
POGLUT1D233E overexpression
POGLUT1R183W overexpression
POGLUT1Y57C overexpression
POGLUT1C102F overexpression

dependent variables

Indirect flight muscle development in Drosophila

control variables

Temperature (25°C)
Developmental stage (25% pupal development)

positive control

WT control (y w)

negative control

rumi79/79

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