Blocking Rab11 in the adult muscle precursors results in lethality at different stages of development.

<p>The vertical bars represent mean (<u>+</u> S.E) of mean % survivors in three replicates. Percentage of pupae (red bars) and pharate pupae (green bars) were significantly low in <i>Rab11</i>^<i>mo</i>/<i>Rab11</i>^<i>mo</i>, <i>1151</i>/<i>+; UAS-Rab11</i>^<i>N124I</i>/<i>+</i> and <i>1151</i>/<i>+; Rab11</i>^<i>RNAi</i>/<i>+</i> as compared to the control. The percentage survival of flies after one day of eclosion (blue bars) showed significant decrease in <i>1151</i>/<i>+; UAS-Rab11</i>^<i>N124I</i>/<i>+</i> and <i>1151</i>/<i>+; Rab11</i>^<i>RNAi</i>/<i>+</i>. Asterisk (*) represents significance at p<0.05.</p

Loss of Rab11 reduces the myoblast expanse and notum size of the wing imaginal disc.

<p>(A) Wild type wing imaginal disc notum showing adult muscle precursors; (B) <i>1151</i>/<i>+; UAS-Rab11</i>^<i>N124I</i>/<i>+</i> and (C) <i>1151</i>/<i>+</i>; UAS<i>-Rab11</i>^<i>RNAi</i>/<i>+</i>; (F) The area occupied by the myoblast in the imaginal disc of both <i>1151</i>/<i>+; UAS-Rab11</i>^<i>N124I</i>/<i>+</i> and <i>1151</i>/<i>+</i>; UAS<i>-Rab11</i>^<i>RNAi</i>/<i>+</i> is significantly reduced. (E) In addition the density occupied by the myoblst is also reduced. ***p< 0.001 in both cases. Scale bar represent 20 µm.</p

Flight assay and Wing phenotype.

<p>Flight assay and Wing phenotype.</p

Wing position phenotype in adult flies on expressing dominant negative Rab11 and <i>Rab11</i>^<i>RNAi</i>

<p>(A) Normal wing position observed in a Wild type fly (B) Abnormal wing position phenotype (wing held out at different angles to the axis) on overexpression of dominant negative Rab11 using the <i>1151GAL4</i> driver (C) <i>UAS-Rab11</i>^<i>N124I</i>/<i>+; Actin88F</i>-GAL4/+ flies showing held out wings (D) <i>mhc-GAL4> UAS-Rab11</i>^<i>N124I</i> flies with wings held out at almost 45^° angle to the axis.</p

Myoblast acquire migratory characteristics on altering Rab11.

<p>Optical line sections of everting wing disc at 2.5h APF, at an interval of 0.75µm. The sections are displayed in the order of basal to apical polarity of the disc. Rab11 in white and DAPI in red. Pseudo colours have been used in both channels for better contrast; (A–C) Myoblast in the everting wing disc of controls (1151-GAL4/+) showing rounded morphology; (D–F) <i>1151</i>/<i>+; UAS-Rab11</i>^<i>DN</i>/<i>+</i>AMPs showing filopodial extensions at their polar ends along with increased Rab11 localization along the length of the filopodial extensions.</p

<i>de novo</i> formation of individual DVM fiber fails to occur in the absence of Rab11

<p>(A) Schematic showing hemithorax of an adult <i>Drosophila</i> containing dorsal ventral muscles that are present as three individual bundles consisting of three, two and two muscles each indicated by red asterisks. The third muscle of the DVM I is usually not visible as it lies in a different plane. Anterior is to the left and dorsal is to the top in this and all subsequent images. (B) Wild type hemithorax showing DVM I, II and III marked with asterisk in yellow (C) <i>Rab11</i>^<i>mo</i><i>/TM6B</i> homozygotes showing presence of a single DVM I and DVM II muscle fibre instead of two muscles observed in the control thoraces. The DVMs also appear to be noticeably thin. (D) <i>1151</i>/<i>+; UAS-Rab11</i>^<i>N124I</i>/+ hemithorax showing complete absence of the second muscle of DVM III. Single muscle in DVM III bundle has been marked with red asterisk (E) <i>1151</i>/<i>+</i>; UAS<i>-Rab11</i>^<i>RNAi</i>/<i>+</i> hemithoraces showing single muscle fibre in DVM I and DVM III bundle. Inter-filament spaces present between fibres are marked with yellow arrows. Scale bar represent 100 µm (F) Summary showing the effect on fibre number by comparing the number of fibres present in each hemithorax of an adult fly. Wildtype in blue bars, Rab11^mo/Rab11^mo shown in pink bars, <i>1151</i>/<i>+; UAS-Rab11</i>^<i>N124I</i>/<i>+</i> in yellow and <i>1151</i>/<i>+</i>; UAS<i>-Rab11</i>^<i>RNAi</i>/<i>+</i>in red. Standard error bars are shown for each of genotypes.</p

Rab11 is mislocalized in the AMPs resulting in altered morphology and loss of cell-cell contacts.

<p>Optical line sections of wing imaginal disc notum at an interval of 1µm, harbouring the AMPs. The sections are displayed in the order of basal to apical polarity of the disc; (A-A’’’) <i>1151-GAL4/+</i> notum showing punctuate localization of Rab11 in green; (B-B’’’) merged image showing DAPI in red and Rab11 in green; (C–C’) Ectopic expression of <i>UAS-Rab11</i>^<i>N124I</i> shows complete loss of punctuate localization of Rab11 in <i>1151</i>/<i>+; UAS-Rab11</i>^<i>DN</i>/<i>+</i> AMPs. Instead the protein is localized at the apical ends of the AMPs that show ‘tear drop’ morphology; (C’’’) Myoblast in the basal layers are transformed to spindle like cells and show complete absence of cell-cell contacts; (D-D’’’) merged image; (E-E’’’) <i>1151</i>/<i>+; UAS-Rab11</i>^<i>RNAi</i>/<i>+</i> wing disc notum showing completely disorganized AMPs population and loss of Rab11 punctuate localization. Scale bar represents 10µm.</p

Inhibiting Rab11 function in AMPs prevents differentiation of the dorsal longitudinal muscles.

<p>(A) Schematic showing hemithorax of an adult <i>Drosophila</i> with six dorsal longitudinal muscles (DLMs in green) indicated by red asterisks. Anterior is to the left and dorsal is to the top in this and all subsequent images. (B) Thoracic preparation of a hemithorax from a wild type fly showing six, well differentiated DLMs marked with asterisks in red; (C) <i>Rab11</i>^<i>mo</i><i>/TM6B</i> homozygotes showing undifferentiated and reduced DLMs. The DLMs in <i>Rab11</i>^<i>mo</i> homozygotes can be observed as compact structures occupying only half of the thorax as compared to their wild type control; (D) In <i>1151</i>/<i>+; UAS-Rab11</i>^<i>N124I</i>/<i>+</i> thoraces, three DLM fibres can be observed, showing complete failure of differentiation as the larval templates fail to split into adult fibres (E) <i>1151</i>/<i>+</i>; UAS<i>-Rab11</i>^<i>RNAi</i>/<i>+</i>fly thoraces, occasionally show four or five DLM fibres that are thinner as compared to the controls and are often present as ‘wavy’ structures, (F) Graph shows the number of DLM fibres per hemithorax in different genotypes. Presence of all six DLMs are observed in very few hemithoraces of Rab11 mutant, <i>1151</i>/<i>+; UAS-Rab11</i>^<i>N124I</i>/<i>+</i> and <i>1151</i>/<i>+</i>; UAS<i>-Rab11</i>^<i>RNAi</i>/<i>+</i>, compared to 96% control hemithoraces with intact six DLMs. Wildtype in blue bars, <i>Rab11</i>^<i>mo</i>/<i>Rab11</i>^mo in red bars, <i>1151</i>/<i>+; UAS-Rab11</i>^<i>N124I</i>/<i>+</i> in navy blue and <i>1151</i>/<i>+</i>; UAS<i>-Rab11</i>^<i>RNAi</i>/<i>+</i> in green bars. Scale bar represent 20 µm.</p

Impaired proliferation of the adult muscle progenitors in Rab11 altered condition

<p>Mitotic AMP population in the wing disc notum were immunostained with anti-phospho-histone H3 antibody. The twist positive myoblast (white) cross labelled with anti-phospho-histone (red) and DAPI in blue (A) <i>1151 GAL4</i> control (B) <i>1151</i>/<i>+; UAS-Rab11</i>^<i>DN</i>/<i>+</i> and (C) <i>1151</i>/<i>+</i>; UAS<i>-Rab11</i>^<i>RNAi</i>/<i>+</i> wing disc. The total number of Mitotic phase cells in a representative area of 4500 µm² were counted during the period of active proliferation of myoblast. (D) The number of myoblast positive for phospho-histoneH3 are significantly lower in both <i>1151</i>/<i>+; UAS-Rab11</i>^<i>N124I</i>/<i>+</i>, <i>1151</i>/<i>+</i>; UAS<i>-Rab11</i>^<i>RNAi</i>/<i>+</i> than control. ***p< 0.001 by t-test. Error bars indicate standard error.</p

Rab11 function in the adult muscle precursors during early adult myogenesis is crucial for IFM development.

<p>(A) When progenies of the genotype <i>1151-GAL4; UAS-Rab11</i>^<i>N124I</i><i>; tubGAL80</i>^<i>ts</i> were raised at 29.5^°C during the embryonic and larval stages and then moved to 18^°C for the pupal phase, the DLMs were reduced in number and showed absence of splitting; (B) individual DVMs fibres were reduced in number in <i>1151-GAL4; UAS-Rab11</i>^<i>N124I</i><i>; tubGAL80</i>^<i>ts</i> progenies.</p