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Dpp signalling is a key effector of the wing-body wall subdivision of the Drosophila mesothorax

Florencia Cavodeassi^*, Isabel Rodríguez and Juan Modolell

Centro de Biología Molecular Severo Ochoa, CSIC and UAM, Cantoblanco, 28049 Madrid, Spain
^* Present address: Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, UK

View larger version (42K): [in a new window]   Fig. 1. Wg signalling does not delimit the notal Iro-C domain. (A-C) Domains of Iro-C expression in second, early third and late third instar wgCX3/wgCX4 wing discs, respectively. Arrows indicate ectopic nota; arrowhead, residual hinge territory. (D,E) Early third instar disc with clones of cells overexpressing UAS-Axin (red). The clones did not affect the distal border of Iro-C expression (arrowhead in green channel image, E). (F) Early third instar disc expressing UAS-dTCF driven by dppdisk-Gal4. The distal border of Iro-C expression (green) was not appreciably disturbed. Arrow points to region of maximal UAS-dTCF expression. Red channel, Wg counterstaining. (G) An older disc similarly expressing UAS-dTCF showed abnormal pattern of Wg (red, compare with Fig. 2G) in the wing pouch, indicating the activity of the construct. Control flies expressing this or the UAS-Axin construct showed typical wg insufficiency phenotypes. (H) Second instar wild-type disc stained for Tsh (red) and Iro-C (yellow or green; green channel shown at right). Note the absence of Tsh protein from the prospective wing pouch (arrowhead). (I) Older late second instar wgCX3/wgCX4 wing disc, stained as in H. Tsh is almost not removed from the distal part of the disc (asterisk), indicating the failure of the residual Wg to specify the wing territory (Wu and Cohen, 2002). Compare also with Fig. 4I.

View larger version (45K): [in a new window]   Fig. 2. Vn/EGFR signalling does not delimit the notal Iro-C domain. (A-C) Domains of vn expression in second, early third and mid third instar wgCX3/wgCX4 wing discs, respectively. Arrowhead in B indicates initial ectopic vn expression; arrow in C, vn expression in ectopic nota. Inset: late third instar wgCX3/wgCX4 wing disc showing symmetrical domains of vn expression (reduced magnification). (D,E) Extent of the Iro-C domain (green; arrowheads) is essentially not affected in UAS-vein; ap-Gal4 (misexpression in the D compartment) second and third instar discs, respectively (compare with Fig. 4A,E). Wg (wing pouch marker) is in red. In parallel experiments, no significant effects were observed using the C765, tsh-Gal4, omb-Gal4, dppdisk-Gal4 and MS1096 drivers. In E, the relatively large extent of the Wg domain in the anterior-posterior axis indicates that ap was already expressed in the dorsal compartment (Ng et al., 1996) and, therefore, that the ap-Gal4 driver was active in this disc. (F) Third instar disc overexpressing UAS-ras1V12 in the anterior compartment (ptc-Gal4 driver). Extent of notal Iro-C domain (green; arrowhead) is unaffected. Red: Ptc marker. (G) Notal Iro-C expression (green) is inhibited and notum territory is reduced (arrowhead, compare with E or F) in UAS-rafDN/MS-248Gal4 discs. Red: Wg marker. Inhibition of Iro-C was also observed using the driver dppdisk-Gal4. (H,I) Expression of the MS-248Gal4 driver, as revealed by UAS-lacZ (red; separate red channel is shown in H on the right), in second and late third instar wing discs, respectively. Counterstaining: Iro-C protein (green). Note strong expression of driver in the proximal region of the discs (arrowheads).

View larger version (60K): [in a new window]   Fig. 3. Complementary domains of Iro-C expression and Dpp activity in the second instar wild-type wing disc. Iro-C protein, green; Dpp pathway markers, red. (A,B) dpp expression (revealed by dpp-lacZ) preceeds that of Iro-C and it does not occur or it only occurs at very low levels within the Iro-C domain, the presumptive notum (arrowhead). (C,D) Dpp pathway activity (pMad protein) is reduced within the Iro-C domain (arrowhead). (E,F) Iro-C (yellow and green) is expressed within the domain of high accumulation of tkv-lacZ (red and yellow).

View larger version (116K): [in a new window]   Fig. 4. Insufficiency of dpp expands the Iro-C domain (green). (A) Second instar wild-type wing disc: notum (green, Iro-C), wing pouch (red, Nub protein) (Ng et al., 1995), hinge (unlabelled territory, arrowhead). (B-D) Second, early third and late third instar dppd12/dppd14 discs stained as in A. Iro-C ectopically accumulates in the hinge territory (arrowhead in B); later it is removed from part of it (arrowheads in C,D). (E,F) Tsh in wild-type and dppd12/dppd14 late third instar discs, respectively, accumulates most strongly in the hinge territory (arrowheads). (G,H) Sensory organ mother cells (anti-Senseless antibody, red) in wild-type and dppd12/dppd14 late third instar discs, respectively. Presumptive hinge groups (arrowheads) are shown at higher magnification in insets. (I,J) Tsh (red) and Iro-C in wild-type (I) and dppd12/dppd14 (J, merged and separate channels) second instar discs. To emphasize detail, discs are reproduced at different magnifications.

View larger version (101K): [in a new window]   Fig. 5. Dpp pathway inhibits Iro-C (green). (A,B) Early third instar wild-type and MS-248 Gal4; UAS-dpp discs, respectively. Iro-C is downregulated in the notum territory (arrowhead). Wing pouch marker: Wg (red). (C,D) UAS-tkvQD (red, lacZ marker) autonomously inhibits Iro-C in cell clones. Inhibition fails in a region of the lateral-posterior notum (arrowhead in C). Inhibition is also observed in late third instar discs (D, arrowhead). (E,F) Early and late third instar wild-type wing discs. dpp-lacZ expression (red) increases during the third larval instar in the most proximal region of the notum (arrows) and Iro-C is gradually inhibited (M, medial notum; L, lateral notum). (G) tkva12 cells (absence of red label) ectopically express Iro-C in the medial notum (asterisk, compare with F). In addition, Iro-C expression is increased within the clone, as compared with the contiguous wild-type territory (arrow). Arrowhead points to the anterior part of the border of the Iro-C domain, which is not modified by the presence of the clone. In contrast, expansion of the Iro-C domain has occurred in the region of the posterior border (red arrow; see also I). (H) Late induced UAS-tkvQD clones (lacZ marker, red) do not express Iro-C, except at the region within or close to a lateral Dpp source (circled, compare with F). (I) tkva12 clone (absence of blue marker) showing a distal expansion of Iro-C expression (arrow), as indicated by the overlap with the Tsh marker (red). Imaginary line between arrowheads would run along the normal border of the Iro-C domain.

View larger version (53K): [in a new window]   Fig. 6. Dpp signalling sequentially establishes the notum-hinge boundary in second instar wing disc (L2) and the medial-lateral subdivision of the notum in third instar wing disc (L3). A and P, anterior and posterior compartment. Dpp source is hatched in blue. During the second instar, Iro-C is activated by Vn/EFGR in the most proximal part of the wing disc (Wang et al., 2000; Zecca and Struhl, 2002a; Zecca and Struhl, 2002b). The distal border of the Iro-C domain, which defines the notum-hinge boundary (Diez del Corral et al., 1999), is established by repression by Dpp signalling, which at this stage only functions in the distal parts of the disc. In the third instar, dpp is expressed in the notum territory and again negatively regulates Iro-C in the medial notum. In the third instar disc, additional, uncharacterized factors (question marks) help maintain the notum-hinge border of Iro-C expression, since this border is generated, at least in part, under conditions of strong depletion of Dpp signalling (Fig. 4D, Fig. 5G).