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First published online 2 June 2004
doi: 10.1242/dev.01195

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The dachsous gene, a member of the cadherin family, is required for Wg-dependent pattern formation in the Drosophila wing disc

Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Cantoblanco, 28049 Madrid, Spain

View larger version (45K): [in a new window]   Fig. 1. DNW phenotype is caused by abnormal subdivision along the PD and AP axis. Adult structures (A-F) and wing imaginal discs (G-K) of wild-type (C,D,G) and ds38k mutant (A,B,E,F,H,I,J,K) specimens are shown. (A) Reduced wing (black arrowhead) associated with an ectopic notum (n'). (B) Magnification of an ectopic notum shows the scutum (sc) and scutelum (sct) structures next to a minimal winglet that is exclusively formed by a duplicated tegula (black arrowhead). (C) Wild-type hinge (square) and the wing blade regions divided by the AP boundary (black line). The asterisk marks the position of the anterior wing margin. (D) High magnification view of the hinge in C, showing anterior proximal structures such as the tegula (tg), humeral sclerite (hsc) and costa (co). (E) Detail of a winglet composed of anterior proximal structures arranged in a mirror-image duplication. (F) Detail of a rudimentary wing blade in which only anterior wing margin bristles (normally formed at the position marked by the asterisk in C) are present. (G) Third instar wild-type discs stained for (a) Nub (green) and Iro (red) to delimit the territories along the PD axis. wp, wing pouch; n, notum. The AP and DV compartments are marked by (b) Ci (red) and Wg (green); (c) En (green) and Vg (red); and (d) En (green) and Ap (red). The white line highlights the border between the A and P compartments. (H-K) Late DNW wing discs show (H) a reduced wing pouch (Nub; green) and an expansion of the notum territory (n and n') (Iro; red). (I) The notal duplication, visualised by a double notal band of Wg (green), is expanded into the hinge territory and is formed by A (Ci; red) and P cells. (J) P cells (En; green) are only present in the notum territory. The wing pouch (Vg; red) cells are confined in the A compartment. The inset in J represent early discs of wild-type (left) and ds38k (right) larvae. The comparison shows that the DNW phenotype occurs very early. The dorsal (red) and ventral compartments are apparently normal (K).

View larger version (117K): [in a new window]   Fig. 2. ds is expressed in the wing disc from early stages. (A-E) Wild-type imaginal wing discs. (A,B) In early- (A) and mid-second (B) instar discs, ds-lacZ expression (red) is confined to the distal part of the wing disc, except in those anterior cells that express Wg (arrowhead). (C) At late second instar, ds-lacZ expression fades away from the P cells adjacent to the AP border (En; green). (D) At early third instar, the ds-lacZ domain forms a ring around the wing pouch that spans the whole hinge territory delimited by Nub (green) and Iro (blue) domains. (E) Spatial distribution of Ds protein is similar to ds-lacZ expression. A cross-section shows that Ds protein is accumulated apically at the plasma membrane. (F) At late third instar, ds-lacZ is expanded into the lateral regions of the notum territory.

View larger version (81K): [in a new window]   Fig. 3. The hinge territory is not specified in DNW discs. Wild-type (A-G) and DNW mutant (H-M) wing discs are shown. (A) In early wild-type discs, the spatial pattern of expression of ds-lacZ (red) and Wg (green) is complementary. At the periphery of the Wg domain, cells express ds-lacZ (red) at low levels, and Tsh expression (green; B) is excluded from those cells. At early third instar, ds-lacZ (red; C,D,E), Hth (green; C) and Zfh2 (green; E) are highly expressed in a ring of cells around the wing pouch where Tsh expression (green; D) is excluded. (F) In late third instar, a cross-section along the AP border (dotted line) shows a ring of cells abutting the Nub domain (green) that express ds-lacZ (red) at high levels (yellow arrow); (G) Tsh expression (red) is absent (yellow arrow). In DNW discs, early Wg expression is apparently normal but restricted to the A cells (H), and Ds expression is almost undetectable (I, compare with the control in 2E). (J,K) Only a narrow ring of cells that surround the wing pouch (Nub, red) express specific hinge markers, such as Hth (green; J), and ds-lacZ (green; K). By contrast, Tsh (L) is excluded from the two-cell wide ring (as in G), indicating that Wg activity is decreased. (M) Zfh2 is expressed in a broader ring around the wing pouch cells. (N) Schematic representation summarising the changes observed in DNW (ds–/–) with respect to wild-type (wt) discs. Different stripes represent the spatial distribution of genes involved in the specification of territories along the PD axis. Low (light) and high (dark) levels of expression are indicated for each gene in different colours. A/P, anterior/posterior; P, proximal wing. Numbers 1 to 5 indicate the folds of a mature wing imaginal disc. RI, RII and RIII correspond to the three concentric rings around Nub domain.

View larger version (107K): [in a new window]   Fig. 4. An increase of Wg activity rescues the DNW phenotype. (A-D) Third instar ds38k mutant wing discs. (A,B) Expression of UAS-wg (red) under the control of dpp-Gal4 driver rescues the DNW morphology to wild type. (B) The hinge territory is specified and the expression pattern of Hth (green) is restored. The hinge is enlarged in respect to wild type due to an increase in cell proliferation caused by ectopic Wg. (C,D) Ectopic expression of Dpp driven by omb-Gal4 increases the proliferation in the wing pouch but fails to rescue the notal duplication. (C) The size of wing pouch is increased. In some cases wing duplications can be recovered (wp and wp'); however, the double notal Wg (red) stripe indicates that the notum duplication is still retained. Dpp activity was monitored by Sal (green), a target gene of the Dpp pathway. (D) The symmetrical location of the AP border into the wing pouch territory is restored in ds38k wing discs, as visualised by Ci (green) expression in A cells. Red channels (insets) show the notal Wg stripes.

View larger version (102K): [in a new window]   Fig. 5. Ds function is required for the early activation of zfh2, a target gene of Wg pathway. (A-D) Third instar wing discs containing dsD36 clones marked by absence of lacZ expression (red). (A,B) Early-induced dsD36 clones (36±12 hours AEL) eliminate zfh2 expression in the hinge region. Observe that the ds mutant territory overproliferates with respect to the wild type. Inset shows Zfh2 expression in wild-type wing disc. (C,D) Late-induced dsD36 clones (60±12 hours AEL) only downregulate zfh2 expression at the proximal hinge region (asterisk in D), whereas in the pleura region its expression looks unaffected (arrowheads in D). (B,D) Green channels show the expression of Zfh2.

View larger version (72K): [in a new window]   Fig. 6. The sub-cellular distribution of Wg protein in the Wg-producing cells is altered in ds mutant cells. (A,B) Third instar discs containing ds38k clones generated by the Minute technique. Clones were induced during second instar and are marked by the absence of armZ (red). (A) In ds38k cells, the gradient of extracellular Wg (green) is expanded with respect to the adjacent wild-type cells (arrowheads). (B) A cross-section from the region marked by the white line in A, showing a higher accumulation of Wg protein at the apical surface of ds mutant cells (compare with wild-type cells, red marker). Wg is uniformly distributed along the apical-basal axis. Note that the mislocalization of Wg also affects the wild-type cells adjacent to ds38k mutant cells (bracket). Wg expression is shown in the green channel.

View larger version (75K): [in a new window]   Fig. 7. Ds regulates Wg signaling during leg development. Removal of one dose of several genes that participate in the Wg pathway modifies the ds phenotype in the leg. Proximal leg of a wild-type (A) and ds mutant (B,C,D,E) specimens. Right panels show a higher magnification of the third tarsal joint for each genotype. In all panels, proximal is to the right and distal to the left. (A) The wild-type tarsus is divided into five segments connected by four tarsal joints (asterisks). (B) In dsZ/ds1 mutants, tarsal segments are shortened and some tarsal joints are incomplete. (C) Insufficiency of the Wg pathway, caused by removal of one dose of dsh, increases the severity of the dsZ/ds1 phenotype. The length of the segments is reduced and most of the tarsal joints are almost eliminated (arrowhead, right). In several cases the tarsal joints are completely absent (not shown). Reduction of nkd, by one dose, produces an increase in Wg pathway signalling. (D) The leg phenotype of ds38k/ds1 is similar that shown in B. (E) Elimination of one dose of nkd in ds38k/ds1 background completely rescues the tarsal joints and the size of the tarsal segments is recovered almost to that of wild type. dsZ/ds1 and ds38k/ds1 are considered mild allelic ds combinations with respect to the leg phenotype. A representative phenotype for each genotype was illustrated. dshV6 or nkd1E89 are null alleles.

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