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First published online 1 June 2005
doi: 10.1242/dev.01889

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Hox-controlled reorganisation of intrasegmental patterning cues underlies Drosophila posterior spiracle organogenesis

¹ Laboratoire de Génétique et Physiologie du Développement, IBDM, CNRS, Université de la méditerranée, Parc Scientifique de Luminy, Case 907, 13288, Marseille Cedex 09, France
² Biozentrum der Universität Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
³ University of Cambridge, Department of Zoology, Downing Street, Cambridge CB2 3EJ, UK
⁴ Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide, Carretera de Utrera, Km 1, Seville, 41013, Spain

View larger version (120K): [in a new window]   Fig. 1. Localisation of Wg, Hh and Egf signalling sources in the dorsal A8 ectoderm. (A) Electron micrograph of a stage 11 embryo [lateral view, from FlyBase (FlyBase Consortium, 2003)]. (A') Magnified view of posterior spiracle primordia, showing prospective stigmatophore (stained in green by anti-Sal antibody) and spiracular chamber (stained in red by anti-Cut antibody) territories. (B) Electron micrograph of a stage 13 embryo (dorsal view, from Flybase). (B') Magnified view of the posterior spiracle, stained in green for stigmatophore and in red for spiracular chamber cells. (C-H) In situ hybridisation to hh transcripts (green) in wild-type embryos at stage 10 (C), mid-stage 11 (D-F) and stage 13 (G,H). A7 and A8 mark the position of the seventh and eighth abdominal segments. (I-N) In situ hybridisation to wg transcripts (green) in wild-type embryos at stage 10 (I), mid-stage 11 (J-L) and stage 13 (M,N). At stage 13, three cells express wg, compared with two at stage 11. Circles in I and J indicate the expression of wg in the dorsal ectoderm of A8. (O-T) In situ hybridisation to rho transcripts (green) in wild-type embryos at stage 10 (O), mid-stage 11 (P-R) and stage 13 (S,T). Arrows in O and P indicate rho expression in the tenth tracheal placode. The circle indicates the appearance at stage 11 of a more dorsal and posterior cluster of rho-expressing cells in A8. Spiracular chamber cells are identified by Cut immunolabelling, in red in E,G,K,M,Q,S. Stigmatophore cells are identified by Sal immunolabelling, in red in F,H,L,N,R,T. In all magnified views, anterior is towards the right and posterior towards the left at stages 10 and 11; this orientation is reversed at stage 13 following germ-band retraction.

View larger version (140K): [in a new window]   Fig. 2. Requirement of Wg, Hh and Egfr signalling for posterior spiracle formation. (A) Cuticle of a wild-type larva. (B) Magnified view of the posterior spiracle. The spiracular chamber cells are differentiated into filzkörpers (white arrow), which are located within a protruding dome-shaped structure, the stigmatophore. (C,E,G) No posterior spiracles are present in wgCX4 (C), hhIJ35 (E) and Egfrf2 (G) mutants. (D,F,H) Cuticles of thermosensitive alleles of wg, hh, Egfr shifted to restrictive temperature from 5-8 hours of development. Differentiated filzkörpers are present in wgIL114 embryos (D), stigmatophores are present in hhts2 embryos (F) and complete posterior spiracles are present in Egfrtsla embryos (H), despite severe segment polarity defects. In wgIL114 embryos, filzkörpers are often at abnormal positions, which reflects the absence of attachment to the disorganised mutant tracheal network.

View larger version (99K): [in a new window]   Fig. 3. Wg, Hh and Egfr signalling are dispensable for posterior spiracle primordia specification. Expression at stage 11 of Cut (A,D,G,J), ems (B,E,H,K) and Sal (C,F,I,L) in wild-type (A-C) or wg (D-F), Egfr (G-I) and hh (J-L) mutant embryos. Arrows and circles indicate the sites of posterior spiracle formation. Cut and Sal expression is maintained in mutant contexts, while ems transcription is absent in wg and Egfr mutant embryos.

View larger version (118K): [in a new window]   Fig. 4. Wg, Hh and Egfr signalling are required in spiracular chamber and stigmatophore cells for posterior spiracle organogenesis. (A-D) ems-Gal4 or sal-Gal4 promote expression at late stage 11. ems-Gal4/UAS-GFP (A,B) and sal-Gal4/UAS-GFP embryos (C,D), immunostained in red for GFP, and in green for Cut (A,B) and Sal (C,D) at early (A,C) or late (B,D) stage 11. GFP is detected in late stage 11 embryos, roughly 1 hour after the onset of Sal and Cut protein accumulation in the dorsal A8 ectoderm.(E-J) Effects of DN-TCF (E,H), DN-Egfr (F,I) and DN-Ci (G,J) expression driven by ems-Gal4 or sal-Gal4. Blocking either signalling by ems-Gal4 impairs filzkörper formation, and not that of stigmatophore (E-G). Reciprocally, blocking either signalling by sal-Gal4 impairs stigmatophore development, leaving filzkörper differentiation unaffected (H-J). The stigmatophore phenotype resulting from the loss of Hh signalling (J) is weaker than those resulting from the loss of Wg and Egfr signalling (H,I).

View larger version (98K): [in a new window]   Fig. 5. Wg, Hh and Egfr signalling are required for maintenance of Cut and Sal expression in the posterior spiracle. (A) Schematic representation of the delayed effects of DN molecules expression on Sal and Cut expression, and correspondence between embryonic stages and times of development at 25°C. (B-E) Sal expression in sal-Gal4/UAS-DN-TCF (C) and Cut expression in ems-Gal4/UAS-DN-TCF (E) in late stage 12 embryos are not affected compared with expression in wild-type embryos (B,D). (F-I) Anti-Sal staining at stage 13. Wild-type embryos (F), and embryos carrying sal-Gal4 and UAS-DN-TCF (G), UAS-DN-Egfr (H) or UAS-DN-Ci (I). Black brackets indicate cells close to the spiracular opening and white brackets indicate more distant cells, in which Sal is no longer detected upon signalling inhibition. (J-M) Anti-Cut staining at stage 13. Wild-type embryos (J), or embryos carrying ems-Gal4 and UAS-DN-TCF (K), UAS-DN-Egfr (L) or UAS-DN-Ci (M). The discontinuous lines encircle spiracular chamber cells, where Cut expression is significantly reduced upon signalling inhibition.

View larger version (139K): [in a new window]   Fig. 6. AbdB and Hh signalling control A8-specific patterns of wg and rho. (A-C) In situ hybridisation (green) to rho (A), wg (B) and hh (C) transcripts in mid-stage 11 AbdB mutant embryos. rho and wg fail to adopt A8-specific patterns (circles; compare A with Fig. 1P and B with Fig. 1J). (D,E) 69B-Gal4/UAS-AbdB embryos stained in red for Cut protein and in green for rho (D) or wg (E) RNA. The expression of wg and rho in all trunk segments mimics expression found in wild-type A8. However, wg is expressed in more than three cells (compare with Fig. 1M). (F,G). In situ hybridisation (green) to rho (F) or wg (G) transcripts in mid-stage 11 hhIJ35 mutant embryos. As in AbdB mutant embryos, rho and wg patterns do not acquire A8-specific properties (circles). (H-K) Magnified views of ems-lacZ embryos stained for ß-Gal (red) and wg (H,I) or rho (J,K) transcripts (green). ß-Gal is detected before (H) and after (I) wg has adopted an A8-specific pattern. The transition of the rho expression pattern occurs slightly later than that of wg, as ß-gal is not detected prior to rho expression (J), but concomitantly with its expression in the posterior dorsal cell cluster (K).

View larger version (91K): [in a new window]   Fig. 7. Local requirements of Wg and Egfr signalling for posterior spiracle organogenesis. Effects of Wg and SpiS expression in spiracular chamber or stigmatophore cells on the morphology of the posterior spiracle. ems-Gal4/UAS-wg (A,A') or ems-Gal4/UAS-spiS (B,B') and sal-Gal4/UAS-wg (C,C') or sal-Gal4/UAS-spiS (D,D'). In all genotypes, embryos harbour filzkörpers elongation defects and reduced sized stigmatophores. Some variability in filzkörper defects is seen and illustrated in A',B',C',D', which show extreme phenotypes for each genotypes. Filzkörpers and stigmatophore defects are more pronounced when Wg and SpiS expression is driven by sal-Gal4.

View larger version (114K): [in a new window]   Fig. 8. A8-specific regulation of hh and en at stages 12 and 13. (A) Schematic representation of the `delay experiment' used to detect de novo En expression in the anterior compartment of A8. En production requires a single step (1) but that of ß-Gal requires three steps (1-3). The en introns, the small size of which does not induce a significant delay in En production, have not been represented. (B) Wild-type expression of En in abdominal segments at stage 11, shown here for A6-A8. In order to align the segments with those of a stage 13 embryo that has completed germ-band retraction, the magnified view shown in B comes from a stage 11 embryo oriented with anterior towards the left. (C) Stage 13 en-Gal4/UAS-lacZ embryo stained in blue for En and brown for ß-gal. En and ß-gal are co-expressed in A6 and A7. In A8, co-expression only occurs in cells lying in the posterior part of the stigmatophore (white arrow), while anterior stigmatophore cells express En but not yet ß-gal (black arrowhead). Cells located ventrally in A8 do not express En, but ß-gal is still detected because of the transcriptional delay and ß-gal stability (white arrowhead, out of focus). (D-F) En is required for stigmatophore development. Cuticle of an enE embryo (D) shows loss of stigmatophores, while filzkörper-like structures are still present (black arrows). enE embryos bearing the sal-Gal4 and UAS-en transgenes display developed stigmatophores (E, white bracket) comparable in size with those observed in embryos containing the two transgenes but in an otherwise wild-type context (F, white bracket). (G-I) hh is expressed in an A8-specific pattern from stage 12 onwards (G). In A8, but not in more anterior segments (arrow in A7), hh (blue) is expressed in cells (arrow in A8) that border the En segmental stripe (brown). This expression is lost (arrow) in AbdB mutants (H) and still occurs in en mutants (I).

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