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First published online June 28, 2004
doi: 10.1242/10.1242/dev.01217

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Armadillo/ß-catenin-dependent Wnt signalling is required for the polarisation of epidermal cells during dorsal closure in Drosophila

Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK

View larger version (111K): [in a new window]   Fig. 1. Ubiquitous overexpression of Wg or activated Arm rescues the polarity of DME cells in wingless mutant embryos. Confocal images of stage 13 embryos showing the subcellular localisation of Fmi and Dlg (A-D, green and red, respectively), ßTubulin and Fas3 (A'-D', green and red, respectively), and Myosin (A''-D''; green, C'',D'', red is Fas3). (A-A'') In wild-type embryos, the DME cells (outlined by Fas3 in A') are elongated in the DV direction. Fmi (A) localises at the membrane, but is excluded from the LE (arrow), and accumulates at the level of the ANCs (arrowhead). The microtubules bundle and orient in the DV direction (green in A') and Myosin shows a `beads on a string' pattern at the LE (A''). (B-B'') In wg– embryos, the cells are stretched in the AP direction and Fmi, which weakly labels the membrane, fails to accumulate at the ANCs (B, green). The microtubule bundles are stretched in the main direction of the cells (B') and the weak Myosin staining is characterised by the lack of accumulation at the LE (B''). In embryos ubiquitously overexpressing Wg (C-C'') or Armact (D-D''), although few cells show a triangular shape, the elongation of the DME and the more ventrally located epidermal cells (C,D) is rescued. The localisation of Fmi at the membrane and its concentration in ANCs (green C,D), the orientation of the microtubule bundles (green C',D') and the `beads on a string' pattern of Myosin (green, C'',D'', red is Fas3) are comparable with wild type. In all the figures, anterior is leftwards and dorsal upwards. as, amnioserosa, ep, epidermis. (A-A'',B',B'') Modified, with permission, from Kaltschmidt et al. (Kaltschmidt et al., 2002). Scale bar: 20 µm.

View larger version (38K): [in a new window]   Fig. 2. Rescue of `canonical' Wg pathway features following overexpression of Wg, Armact or Dsh in wg– embryos. Analysis of the different cuticle phenotypes from the following crosses: females wgCX4/CyoftzZ;daGal4 x males wgCX4,UASWg/CyoftzZ (wg>da>Wg), wgCX4,UASArmact/CyoftzZ (wg>da>Armact) and wgCX4/CyoftzZ;;UASDsh (wg>da>Dsh). The embryos shown above are stained for Engrailed. wg>da>Wg: 44.5% of the embryos overexpress Wg and present a fully naked cuticle, and are organised in two categories of different strength (30%, 14.5%). 21% of the embryos overexpress Wg and have a wild-type phenotype (out of the 54% showing a wild-type phenotype, 33% do not overexpress Wg). Wg overexpression in wg– embryos is thus able to rescue the naked cuticle identity and to some extent suppress the denticules specification. En expression is rescued to normal. wg>da> Armact: 39.4% of the embryos overexpress Armact and show a fully naked cuticle, and 30.6% of the embryos overexpress Armact and show a wild-type phenotype. Armact overexpression is thus able to rescue the naked cuticle identity. En expression, as shown by the above embryo, is normal. wg>da>Dsh: all the embryos from this cross overexpress Dsh. A mild rescue of the naked cuticle identity (see Fig. 5C') is observed in 21% of the embryos (33% are wg–). A partial rescue of En expression is observed.

View larger version (101K): [in a new window]   Fig. 3. Activation of the `canonical' Wg pathway is required in the epidermal cells but not in the amnioserosa. Subcellular localisation of Fmi, Dlg, Myosin, Fas3 and Wg in wg– embryos overexpressing either Wg or Armact in the amnioserosa. (A,E) Stage 13 embryos stained for Fmi (green) and Dlg (red). Although the DME cells are oriented in the DV direction in the wg>AS>Wg embryos (A), they do not elongate and even sometimes show a triangular shape (arrowhead). The ventrally located epithelial cells present an isotropic shape (asterisk). In these embryos, however, Fmi localises at the membrane and concentrates at the level of the ANCs as in wild type (compare with Fig. 1A). By contrast, in wg>AS>Armact embryos (E), DME cells are not oriented in the DV direction but rather stretched in the AP direction as in wg– embryos. In these embryos, Fmi shows a `dotty' staining similar to that observed in wg– embryos (compare with Fig. 1B). (B,C,F,G) Subcellular localisation of Myosin in stage 13 (B,F) and stage 15 (C,G) embryos. At a stage when Myosin already shows accumulation at the LE in wild type (compare with Fig. 1C), no accumulation is observed in wg>AS>Wg embryos (B). However, when the zippering process has been initiated at both ends, accumulation is observed at the LE (C). By contrast, neither at stage 13 (F) nor at stage 15 (G) does Myosin accumulate at the LE of wg>AS>Armact embryos. (D) Subcellular localisation of Wg (green) in a stage 13 wg>AS>Wg embryo. After overexpression, a strong Wg expression is observed in the amnioserosa and Wg-containing dots are seen on the apical side of the epithelium up to six cells away from the LE (the cells are outline in red by Dlg). All panels are single confocal sections, dorsal is upwards and anterior leftwards. Scale bar: 20 µm.

View larger version (94K): [in a new window]   Fig. 4. Rescue of dpp expression levels following overexpression of Wg or Armact. Lateral view of stage 13 embryos stained by in situ hybridisation against decapentaplegic. dpp is expressed in the DME cells of wild-type embryos (A), and in one or two additional cells per segment ventrally localised, compare with the LE. In wg– (B), expression levels are lower and dpp expression is often lost in the posterior DME cells. After ubiquitous overexpression of Wg (C) or Armact (D), dpp expression levels are restored to normal. Interestingly, although the overexpression is ubiquitous, the expression pattern in stage 13 embryos is not altered. (E) In wg>AS>Wg embryo, dpp expression levels are rescued, in contrast to wg>AS> Armact embryos (F), which show a weak expression of dpp similar to the levels observed in wg– embryos.

View larger version (139K): [in a new window]   Fig. 5. Contribution of the Dsh domains to the rescue of `canonical' features in wg– embryos. (A'-E') Magnification of the ventral side of the cuticle shown in A-E. (A'',B'',C'',E'') Lateral views; (D'') dorsal view. Cuticle (A-A') and En expression pattern (A'') of wild-type embryos. (B-B'') wg– embryos show a short cuticle (B) and a lawn of denticules on the ventral side (B'). The expression in stripe of En in the lateral and dorsal ectoderm is lost, except for very few cells (B''). Upon ubiquitous overexpression of Dsh (C,C') or DshDEP (D,D'), the length of the cuticle and the presence of naked cuticle on the ventral side are rescued. A mild rescue of En expression is observed in wg>da>Dsh embryos (C'') and to a lesser extent in wg>da>DshDEP (D''). No rescue of either the length of the cuticle (E), the presence of naked cuticle (E') or the expression of En (E'') is observed after ubiquitous overexpression of DshDIX. Moreover, the cuticle of wg>da>DshDIX embryos is shorter than the cuticle of wg– embryos (compare E with B).

View larger version (171K): [in a new window]   Fig. 6. Contribution of the Dsh domains to the rescue of dorsal closure features in wg– embryos. (A-D') Dorsal view of the cuticles shown in Fig. 5. Confocal high magnification pictures of late stage 13 embryos (A'',B'',D'') and early stage 14 (C''), oriented dorsal upwards and anterior leftwards. (A-A') Cuticle of a wg– embryo showing an anterior and a anterodorsal hole (arrowheads), as well as a short and folded cuticle on the dorsal side. After ubiquitous overexpression of Dsh (B) or DshDEP (C) in wg– embryos, the length of the dorsal side of the cuticle is rescued and the anterodorsal hole suppressed. The dorsal cuticle of wg>da>Dsh embryos (B') shows some warts in addition to a puckering on the midline, while the dorsal cuticle of wg>da>DshDEP embryos (C') is disorganised with occasional warts. By contrast, the dorsal cuticle of wg>da>DshDIX embryos is very short and a strong dorsal hole is observed (D-D', arrowheads). The cell organisation, as revealed by Fmi (green) and Dlg (red) staining (A''-D''), is partially rescued after overexpression of Dsh (B'') or DshDEP (C''), with DME cells showing a mild elongation in the DV direction and accumulation of Fmi at the membrane and weakly at ANCS. No rescue of cell shape or Fmi localisation is observed following overexpression of DshDIX (compare D'',A'').

View larger version (129K): [in a new window]   Fig. 7. Dsh is not required for dorsal closure to proceed when the `canonical' Wg pathway is activated. Cuticles (A-C'), Fmi (green) and Dlg (red) antibody staining (A''-C'') of dshGLC (A-A''), dshGLC>da>Dsh (B-B'') and sgg,dshGLC (C-C'') embryos. The cuticle of dshGLC embryos presents an anterodorsal hole (A', arrowheads) and is very short with most of the dorsal epidermis missing (A). In dshGLC>da>Dsh embryos, the length and the hole of the cuticle are rescued (B) and a strong puckering is now observed dorsally (B'). The cell morphology and polarity in dshGLC embryos is very similar to that observed in wg– embryos (compare A'' with Fig. 1B): the DME cells are stretched in the AP direction while the other dorsal epidermal cells present a rather isotropic shape. Fmi does not localise at the membrane but shows a `dotty' localisation in the epidermis. Upon ubiquitous overexpression of Dsh in dshGLC, the DME cells elongation in the DV direction is rescued (B''). Fmi does not localise at the membrane in stage 13 embryos (not shown), but shows membrane localisation and ANCs accumulation in late stage 13-early stage 14 embryos (B''). The cuticle of sgg,dshGLC embryos (C), which lacks both dsh and sgg function, presents a strong retraction defect and a ventral naked cuticle characteristic of the constitutive activation of the `canonical' Wg pathway associated with the loss of sgg function. They present an anterior hole (arrowhead) and a puckering of the dorsal midline (C'). Although the retraction defect makes the observations difficult, the cuticle is longer than in dshGLC embryos (compare with A). Fmi localises at the membrane and concentrates at ANCs in sgg,dshGLC embryos (C'') and the main direction of DME cells is restored to the DV axis.

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