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Fig. 3. Ubx expression and function during cardiac tube remodelling. (A-H) Ubx expression pattern during cardiac tube remodelling. A magnified view of the cardiac tube at the indicated stages is provided. (A,C,E,G) Anti-Ubx staining, revealed with Alkaline Phosphatase secondary antibody. (B,D,F,G; left) Fluorescent immunostaining against Ubx (green) and either ß-Gal (red, D,F,G) in the svp-lacZ reporter strain or Tin (red, B). Cardiac tube limits are highlighted. Arrowheads indicate svp-expressing cells; arrows indicate Tin-expressing cells. (Right) Schematic drawing of the Ubx, Tin or Svp expression pattern at indicated stages. (A,B) Ubx expression pattern in the adult cardiac tube. Adult Ubx expression is restricted to the A1-A5 svp-expressing myocytes (arrowheads) that correspond to the ostium-forming cells. Arrows indicate unstained Tin-expressing myocytes, recognised by the size of the nucleus in A. (C,D) At 24 hours APF, Ubx is expressed in all Tin- (arrows) and svp-expressing (arrowheads) myocytes of segments A1-A4. (E,F) At 30 hours APF, Ubx expression becomes progressively repressed in some Tin-expressing myocytes (black arrows, E) but remains expressed in others (red arrows in E). pc, pericardiac cell unspecifically stained. (G,H) At 48 hours APF, Ubx expression is maintained in svp-expressing myocytes (arrowheads) but is not detected in Tin-expressing myocytes. pc, pericardiac cell; im, Ubx-expressing imaginal muscle cell nuclei. (I-M) Effects of Ubx overexpression during cardiac tube remodelling on myocyte differentiation (I-L) and Ih expression (M). In all cases, individuals were shifted to 29°C at the onset of metamorphosis. (I-L) Cardiac tubes from Gal80^ts, 24B>Gal4; UAS>mcd8-GFP alone (I,K) or Gal80^ts, 24B>Gal4; UAS>mcd8-GFP, UAS>Ubx (J,L) pharate adults. (I,J) Detection of membrane-bound GFP demonstrates that Ubx overexpression profoundly perturbs cardiac tube remodelling. (K,L) Phalloidin staining. Ubx overexpression has no visible effect on ostium differentiation (arrowhead in L), but perturbs the Tin-expressing myocyte remodelling that differentiates longitudinal myofibrils (arrow). (M) Cardiac tube from a Gal80^ts, 24B>Gal4; UAS>Ubx pharate adult stained for Ih transcripts (green) and dMef2 protein (red). Ubx overexpression prevents Ih expression in adult heart Tin-expressing myocytes (arrows). Asterisk indicates non-specific staining of the fat body; arrowhead indicates svp-expressing myocytes. (N) Ubx is required for wg expression in the svp-expressing myoctes at metamorphosis. Df(3R)Ubx-109; 24B>Gal4; UAS> dsRNA>Ubx cardiac tube at 30 hours APF, stained for Wg and detected with anti-mouse alkaline phosphatase-conjugated secondary antibody. Individuals were shifted to 29°C at the onset of metamorphosis. dsRNA>UAS-Ubx prevents Wg activation in svp-expressing cells (arrowheads). (B,D,F,H,M) Confocal images. Scale bars: 50 µm.

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