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Connecting Hh, Dpp and EGF signalling in patterning of the Drosophila wing; the pivotal role of collier/knot in the AP organiser

Centre de Biologie du Développement, UMR 5547 CNRS/UPS, 118 route de Narbonne, 31062 Toulouse Cedex, France

View larger version (72K): [in a new window]   Fig. 1. col/kn mutant wings: abnormal vein pattern and reduced size. (A) Wild-type wing with the longitudinal L2 to L5 veins indicated. The small arrowhead and arrow point to veins L3 and L4, respectively. (B) col1 /kn1 mutant wing; veins L3 and L4 are closer to each other than in wild-type wing and partly apposed proximally. (C) col1/col1 wing (abbreviated col1 in subsequent figures). L2 and L5 veins are normal, but a central larger L3-type vein is present (L3m vein, large arrowhead), whereas only a small, proximal region of L4 vein forms (vertical arrow). col wings are smaller than wild type, owing to (partial) loss of the region between L3 and L4 veins and a reduced size of the posterior compartment. In addition, col1 wings display a wider L2-L3 intervein than do wild type or col1 /kn1. (D) Schematic superimposition of wild type (grey) and col1 (black) wings adjusted so as to align the proximal L4 vein. All wings are shown at the same magnification.

View larger version (69K): [in a new window]   Fig. 2. Loss of L4 vein and posteriorwards shift of L3 vein in col1 mutant wings. (A,B) BS (D-SRF in figure) expression in 24 hours APF pupae. The central BS labelling which prefigures the L3-L4 intervein in wild type wings (A) is missing in col1 pupae (B), confirming the presence of a single central provein in col1 mutants. (C,D) Ci expression in pupal wings 28 hours APF. The posterior limit of Ci expression bisects the L3-L4 intervein in wild-type wings (C) but coincides with the posterior limit of L3m vein in col1 wings, indicating a posterior shift in its position (D). Positions of the L3, (residual) L4 and L3m veins are indicated as in Fig. 1.

View larger version (88K): [in a new window]   Fig. 3. col requirement for emc upregulation and cell proliferation in the L3-L4 intervein primordium. Cell proliferation in wild-type (A) and col1 (B) pupal wings, 18-20 hours APF, as visualised by the phosphorylation pattern of Histone H3 (H3P). (A) In wild type, a wave of cell proliferation occurs along veins in each intervein region at this stage, including the L3-L4 presumptive intervein (black frame) No such wave occurs in the medial region of a col1 wing (B). (C,D) emc expression in third instar wing discs. Upregulation in the AP organiser of wild-type discs (C) is specifically lost in col1 mutant discs (D). The small arrowhead and arrow indicate proveins L3 and L4, respectively.

View larger version (76K): [in a new window]   Fig. 4. Changes in rho and iro expression in larval discs prefigure the abnormal vein pattern of col1 wings. (A,B) rho expression in wild-type and col1 third instar wing discs. In col1 discs (B), rho expression in the presumptive L3 vein is wider than in wild type (A), while it is strongly downregulated dorsally or absent ventrally (white arrow) in the L4 vein primordium. The L3 and residual L4 primordia are closer to each other than in wild type. (C,D) Double in situ hybridisation for rho (blue) and col (pink, red bar) transcripts. In wild-type discs, there is no overlap between rho and col expression (C). The overlap observed in col1 discs (D) indicates a posterior shift of L3 vein. (E,F) Double in situ hybridisation for ara (blue, grey bar) and col (pink, red bar) transcripts. In col1 discs (F), the anterior border of ara expression is shifted closer to the AP boundary. (G,H) Dpp signalling in wing discs, revealed by the distribution of phosphorylated P-Mad. In wild-type discs (G), Dpp signalling is strongly downregulated in the AP organiser (white bar) and peaks on either side (black bars). In col1 discs (H), a uniform labelling is observed in the centre of the disc (black bar). (I) Schematic diagram of the expression domains of En (orange box), Col (red box), iro (grey box), rho (heavy grey hatching) and high Dpp signalling (Mad*, blue) in wild-type (top) and col1 (bottom) wing discs, based on data presented in A-H (see Blair, 1992; Gomez-Skarmeta et al., 1996; de Celis and Barrio, 2000). The L3 provein is indicated in light grey hatching and the AP border as a vertical black bar. This scheme postulates that the anterior shift of iro expression in col1 mutants is due to changes in sal/salr activity in response to changes in Dpp signalling, while increased rho expression reflects the loss of repression by Col in posterior-most iro-expressing cells.

View larger version (73K): [in a new window]   Fig. 5. Col-mediated upregulation of vn transcription in the AP organiser is required but not sufficient for L4 vein formation. (A) kn1/kn1 and (B) kn1/kn1; vnM2/+ adult wings. In kn1/kn1; vnM2/+ wings, a large region of L4 vein is missing (white arrow). (C,D) vn transcription is strongly downregulated in the L3-L4 intervein primordium in col1 (D) relative to wild type (C) third instar wing discs, except in cells close to the presumptive hinge; (E-H) col1 and UAS-Vn/dpp-Gal4; col1 adult wings (E,F) and corresponding larval discs (G,H). In the absence of col, vn expression in the AP organiser does not rescue formation of L4 vein, while vein L3m is widened (F). Double in situ hybridisation for rho (blue) and col (pink) transcripts in wing discs shows that rho expression is not activated in posterior L4 vein cells but is upregulated in cells corresponding to L3m vein or the posterior margin (H). The broken black line indicates the position of the AP boundary. (I) col1 mutant clones in the wing, marked by shavenoid (sha). L4 vein forms posterior to mutant clone spanning three to four rows of cells along the AP border (blue line); L4 vein is missing and L3 is wider and shifted posteriorly when the col1 clone fills the entire L3-L4 region (red line). (J) UAS-TkvDN/ptc-Gal4 adult wings showing that sequestering Dpp by overexpression of TkvDN in the AP organiser results in wings smaller than wild type and specific loss of the L4 vein.

View larger version (50K): [in a new window]   Fig. 6. Proposed model for the interplay between the Hh, Dpp and EGF signalling pathways in the AP organiser and patterning of the medial region of the wing. Anterior cells that receive high doses of Hh (the AP wing organiser) activate Col, which, in turn, upregulates expression of BS, vn, emc and mtv, and represses expression of EGFR (aMohler et al., 2000). BS (D-SRF) is required for commitment of Col-expressing cells to an intervein fate (L3-L4 intervein) and Emc for their proliferation. Repression of tkv by Mtv leads to low levels of Dpp signalling in the AP organiser cells and high levels in both A and P flanking cells (bFunakoshi et al., 2001). Vn diffuses and activates EGF signalling in posterior cells (visualised by rho expression; heavy grey hatching). Col-mediated upregulation of Vn expression and modulation of Dpp signalling are both required for activation of EGF signalling in posterior cells and formation of L4 vein. rho repression in posterior-most iro-expressing cells positions the L3 vein competent domain anterior to the AP organiser (see Fig. 4I for the role of sal/salr in positioning iro expression). The signals triggering EGF activation in L3 vein and positioning rho expression at the centre of L3 and L4 provein domains (Biehs et al., 1998) remain unknown.