Fig. 5. Mitochondria hyper-fusion causes a transcriptional shift to cross-striated muscle type.

a–k Adult wild-type (a, e, i) as well as Mef2::Marf-1 flight muscles (b, f, j) and wild-type leg muscles (c, g, k) expressing GFP-tagged muscle-type specific proteins Actin 88F-GFP (a–c), Flightin-GFP (e–g) and Kettin-GFP (i–k); samples were fixed and actin was visualised with phalloidin. Relative GFP fluorescence levels are represented via a pixel intensity scale (white represents higher intensity). d, h, l GFP fluorescence was quantified with quantitative confocal microscopy (see Methods section) and plotted relative to control flight muscle levels (in d n = 7, 8 and 5 animals, respectively; in h n = 9, 6 and 7 animals, respectively; in l n = 5, 12 and 5 animals, respectively). Note that Marf over-expression in flight muscle converts the expression levels towards wild-type leg muscle levels. m–o Spalt protein levels in developing flight muscle myotubes at 24 h APF were quantified using immunostaining and quantitative confocal microscopy comparing wild type (m, o; n = 12 animals)) to Mef2::Marf-1 (n, o; n = 14 animals). Actin was visualised with phalloidin, nuclei with DAPI. Note the comparable expression levels. p–r Bruno protein levels in developing flight muscle myotubes at 24 h APF were quantified using immunostaining and quantitative confocal microscopy comparing wild type (p, r; n = 6 animals) to Mef2::Marf-1 (q, r; n = 7 animals). Actin was visualised with phalloidin, nuclei with DAPI. In all plots the mean ± standard-deviation (SD) is indicated, each dot the value from single animals, and significance from two-tailed unpaired t-tests is denoted as p-values = 0.0287 (*), ***p ≤ 0.001. n.s. non-significant. Scale bars are 5 µm.