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First published online November 10, 2005
doi: 10.1242/10.1242/dev.02091

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Steroid-dependent modification of Hox function drives myocyte reprogramming in the Drosophila heart

Laboratoire de Génétique et Physiologie du Développement, UMR 6545 CNRS-Université, IBDM-CNRS-Université de la Méditerranée, Campus de Luminy, Case 907, 13288 Marseille Cedex 09, France

View larger version (45K): [in a new window]   Fig. 1. Drosophila melanogaster cardiac tube remodelling. (A,B) Larval (A) and adult (B) cardiac tubes, stained with phalloidin for polymerised actin (F-actin), are shown at the same magnification. Scale bars: 50 µm. (C) Scheme of larval and adult cardiac tube. (Top) Larval cardiac tube. The svp, tin, Ubx and abdA expression patterns are indicated. (Bottom) Adult cardiac tube. svp and tin expression are indicated according to Molina and Cripps (Molina and Cripps, 2001) and to our own observations. The adult organ is formed by larval myocytes from T1 to A5 segments. Cardiac myocyte morphological modifications (including size, myofibril content and orientation, and innervations) are schematized (see Fig. S2 in the supplementary material). a.m., aorta myocytes; h.m., heart myocytes; t-c.m., terminal chamber myocyte.

View larger version (41K): [in a new window]   Fig. 2. Cardiomyocyte transcriptional reprogramming. (A,D) Embryonic expression of wg (A) and Ih (D) in stage 16 embryos. In situ hybridization (green), and anti-dMef2 staining (A,D, red) or anti-Tin staining (D, inset, red). (A) At stage 16, wg is expressed in svp-expressing cells in segments A5 to A7 (arrowheads) (Lo et al., 2002). (D) In the embryonic cardiac tube, Ih is expressed in the two posterior-most pairs of Tin-myocytes in segment A4, and in the four pairs of Tin-myocytes in segments A5 and A6 (arrows). Ih is not expressed in svp-expressing cells (which also do not express Tin, arrowheads), or in segment A7, or in the anterior and posterior aorta (segments T1 to A3 and anterior part of segment A4). (B,C) Anti-Wg staining of a 30-hour-APF pupal cardiac tube. Five pairs of cells express Wg. (C) At higher magnification and based on their morphology, Wg-expressing cells are identified as the svp-expressing myocytes of segments A1 to A5 (arrowhead). (E,F) Ih expression in the adult cardiac tube. Ih is expressed in all Tin-expressing myocytes of segments A1 to A4 (white arrows), but not in svp-expressing myocytes (arrowhead). Ih is no longer detected in segment A5 Tin-expressing myocytes (red arrow). (F) Detail of the posterior part of an adult cardiac tube. Ih is not expressed in svp-expressing myocytes (arrowhead), nor in segment A5 Tin-expressing myocytes (red arrow). (H) Ndae1 expression in the adult heart. Detail of the posterior part of an adult cardiac tube. Ndae1 is not expressed in svp-expressing myocytes (arrowhead), nor in segment A5 Tin-myocytes (red arrow), but is expressed in Tin-expressing myocytes of segments A1 to A4 (only A4 is shown, white arrows). Asterisk in E and H indicates fat body non-specific staining. (G) Schematic representation of svp, Ih, Ndae1, Tin and Wg expression in the embryonic and adult cardiac tubes. (A,D,E,F,H) Confocal images. Scale bars: 50 µm.

View larger version (97K): [in a new window]   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 Gal80ts, 24B>Gal4; UAS>mcd8-GFP alone (I,K) or Gal80ts, 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 Gal80ts, 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.

View larger version (106K): [in a new window]   Fig. 4. AbdA expression and function during cardiac tube remodelling. (A,B) Adult heart stained for AbdA (green), polymerised actin (phalloidin, white) and dMef2 (B, red). AbdA is expressed in segment A5 Tin-expressing myocytes (arrow) and in the posterior-most pair of myocytes that correspond to the anterior-most segment A6 svp-expressing myocytes (arrowhead). (C) Schematic drawing of AbdA expression in the adult cardiac tube. (D-L) In all cases, individuals were shifted to 29°C at the onset of metamorphosis. (D) Posterior tip of a Gal80ts, 24B>Gal4; dsRNA>abdA; UAS>mcd8-GFP adult cardiac tube, stained for Synaptotagmin (green), GFP (mb GFP, white) and 22C10 (red). 22C10 staining shows that neurones accurately extend to the posterior cardiac tube (red arrows), but abdA loss of function during metamorphosis prevents nerve termination to form synaptic contacts on A5 myocytes, as judged by the absence of sinaptotagmin staining on A5 myocytes (compare with Fig. S2M in the supplementary material). (E,F) Phalloidin staining of the posterior tip of wild-type (E) and Gal80ts, 24B>Gal4; dsRNA>abdA (F) adult cardiac tubes. Arrows point to Tin-expressing myocytes; arrowheads indicate svp-expressing cells. Loss of abdA function during metamorphosis prevents segment A5 remodelling. Myofibrils are oriented transversally instead of longitudinally and the A5 segment is thicker than in wild type. In F, note that loss of abdA function does not affect histolysis in segments A6-A7. (G,H) Phalloidin staining of a wild-type (G) or Gal80ts, 24B>Gal4; UAS>abdA (H) adult cardiac tube. In G, magnification of A3-A4 segments illustrates the transversal orientation of myofibrils. (H) The cardiac tube is thinner following AbdA overexpression during metamorphosis and myofibrils are longitudinal, instead of transversal as in the wild type. (I-L) Effects of abdA dosage on Ih expression in the cardiac tube during embryogenesis (I,J) and metamorphosis (K,L). In all cases, anterior is left, Ih transcripts are in green and dMef2 protein in red. Arrows indicate Tin-expressing myocytes, arrowheads svp-expressing myocytes. (I,J) In the embryo, loss of abdA function (I, abd-AM1) prevents Ih expression in the cardiac tube, whereas abdA misexpression (J, 24B>Gal4; UAS>abdA) induces ectopic expression in Tin-expressing myocytes (red arrows, compare with Fig. 2D). (K) Gal80ts, 24B>Gal4; dsRNA>abdA adult cardiac tube. Loss of abdA function during metamorphosis induces Ih expression in segment A5 Tin-expressing myocytes (red arrows). (L) Gal80ts, 24B>Gal4; UAS>abdA adult. abdA overexpression represses Ih transcription in A1-A4 Tin-expressing myocytes (open arrow). Asterisk indicates non-specific fat body staining. Scale bars: 50 µm.

View larger version (77K): [in a new window]   Fig. 5. Ecdysone signalling cell autonomously controls all aspects of cardiac tube remodelling. In all cases, individuals were shifted to 29°C at the onset of metamorphosis. (A) Phalloidin staining of a Gal80ts, 24B>Gal4; UAS>EcRDN pharate adult cardiac tube. Inhibition of EcR function prevents heart remodelling, including A6/A7 histolysis (red asterisk), A5 transformation (arrowhead) and posterior aorta remodelling, which remains thin anterior to A5 (arrow). (B) Gal80ts, 24B>Gal4; UAS>dsRNA-EcR cardiac tube at 30 hours APF, stained for Wg, detected with anti-mouse alkaline phosphatase-conjugated secondary antibody. wg is not expressed in the svp-expressing myocytes (arrowheads). (C) Phalloidin (white) and anti-Ubx (red) staining of NP5169-Gal4; UAS>dsRNA-EcR pharate adult cardiac tubes. EcR inactivation in Tin-expressing myocytes prevents Ubx repression in segment A1-A4 Tin-expressing myocytes (arrows). pc, pericardial cell. (D) Anti-synaptotagmin (green), 22C10 (red) and phaloidin staining of Gal80ts, 24B>Gal4; UAS>EcrDN pharate adult cardiac tubes. (E) Anti-AbdA (green) and phalloidin staining on Gal80ts, 24B>Gal4; UAS>EcRDN pharate adult cardiac tubes. In both cases, myofibrils are transversal (open arrow), indicating that EcR loss of function prevents terminal chamber differentiation. (D) Nerve terminations do not make synaptic contact with the myocytes, as judged by the absence of synaptotagmin staining. (E) EcR inactivation does not affect AbdA expression. (F,G) Phaloidin staining of Gal80ts, 24B>Gal4; UAS>AbdA, UAS>EcRDN pharate adult cardiac tubes. Inhibition of ecdysone signalling prevents AbdA from inducing terminal chamber structure both in A1-A4 segments (F) and in A5 (G). (H) Schematic representation of a cardiac tube in which EcR function has been inhibited during metamorphosis. Ubx and abdA expression are represented (see C and E). Scale bars: 50 µm.

View larger version (19K): [in a new window]   Fig. 6. Model of the ecdysone-dependent control of cardiac tube remodelling during metamorphosis. Ecdysone signalling triggers adult heart formation throughout the transcriptional regulation of Ubx. By contrast, ecdysone modifies AbdA function, whose activity is switched during remodelling towards a new genetic program that leads to differentiated cells with function, cell behaviour and transcriptional activity characteristic of the adult. Segment A6-A7 programmed cell death does not depends on abdA function. a.m., aorta myocytes; h.m., heart myocytes; t-c.m., terminal chamber myocyte.