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First published online 15 September 2004
doi: 10.1242/dev.01380

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Activation and repression activities of ash2 in Drosophila wing imaginal discs

Departament de Genètica, Universitat de Barcelona, Diagonal 645, Barcelona 08028, Spain

View larger version (101K): [in a new window]   Fig. 1. Wing phenotypes of ash2 alleles. (A) Wild-type (wt) adult wing. Intervein regions (A-E, in blue), longitudinal veins (L1-L6) and anterior (a-cv) and posterior (p-cv) crossveins are marked. (B) Adult wing of ash2112411 homozygous mutant at the same magnification as A, showing anomalous L2 in which the distal half is crooked, ectopic crossvein tissue between L2-L3 and L4-L5, and a notch in the posterior margin. (C) Wing disc of wild-type third instar. (D) Wing disc of homozygous ash2112411 third instar. (E) Wing disc of homozygous ash2I1 third instar. (F,G) Mitotic clones homozygous for ash2I1 in Minute background. (F) Ventral clone where L2 appears thickened and intervein region A is reduced. (G) Dorsal clone showing ectopic vein tissue between the proximal region of L4-L5.

View larger version (79K): [in a new window]   Fig. 2. ash2 downregulates the Egfr pathway during vein development. (A) Lack of wing veins in rhove vn1 homozygous flies. (B) In the triple mutant rhove vn1 ash2112411, the loss of veins is rescued. (C) top1/top3C81 adult wing that lacks the a-cv and the middle part of L4. (D) The combination top1/top3C81 with one copy of the ash2I1 allele restores the wild-type vein pattern. (E) The gain-of-function ElpB1 results in ectopic vein tissue in distal L2. (F) ElpB1; ash2112411 individuals show a reduction in wing size, with notches appearing in the posterior margin and extra vein tissue (extra crossveins and a proximal L2-L3 fusion). (G) rlSem/+ flies develop wings with ectopic branches of veins. (H) Ectopic vein tissue is enhanced in rlSem; ash2I1 transheterozygous flies. All wings are shown at the same magnification.

View larger version (83K): [in a new window]   Fig. 3. In situ hybridisation for rho in discs and pupal wings. (A) Wild-type disc. (B) rho organisation is lost in ash2I1 homozygous discs. (C) Wild-type pupal wing. (D) High magnification of a wild-type vein. (E) ash2112411 homozygous pupal wing. Veins L2 and L3 are wider. (F) High magnification of a severe ash2112411 wing showing vein thickening.

View larger version (126K): [in a new window]   Fig. 4. ash2 is required for net activation. (A-C) Expression of net mRNA in third instar wing discs. (A) Wild-type discs showing net transcripts in intervein regions. The position of veins (lacking net mRNA) is indicated. (B) net expression is reduced in discs ash2112411/ash2112411. However, the central domain of the wing pouch shows expression of net. (C) Absence of net mRNA in ash2I1 wing discs. (D) Wing of net1 homozygous mutant with ectopic vein tissue. (E) net1/net1; ash2I1 heterozygous flies show posterior wing blistering and enhanced ectopic vein connections, especially between L2-L3. (F) Wings of net1; ash2112411 homozygous flies exhibit a total fusion of L2-L3 and L4-L5 with the corresponding loss of intervein regions B and D. The size of the wing is also reduced and the posterior margin is partially lost. (G) High magnification of L2 and L3 fusion, which is mostly associated with L2 thickening, as L5 is for L4-L5 fusion. Note the ventral pattern of corrugation in L2 and the sensillia campaniforme, and dorsal corrugation in L3.

View larger version (99K): [in a new window]   Fig. 5. ash2 is required for activation of bs. (A) Wings of heterozygous bs03267 flies display a small ectopic vein in intervein region D. (B,C) bs03267/+; ash2112411/ash2112411wings show blisters with extra vein tissue (B) and reduction of interveins B and D alongside partial fusion of L2-L3 and L4-L5 (C). (D) Detail of extra crossveins connecting L2 and L3. (E) rho expression in a bs03267/+; ash2112411/ash2112411 pupal wing. (F) High magnification of the same image. (G) Bs staining in a wild-type third instar wing disc. The vein territories and the DV boundary are devoid of antibody staining, which is only associated with intervein territories. (H) ash2I1 clones in a wing imaginal disc (visualised as black spots by the absence of GFP). (I) Bs staining of the same disc. The reduction of Bs staining is more pronounced in the clone located between L2 and L3 (arrow) than in the one between L3 and L4 (arrowhead). (J) Merged green and red channels. (K-M) 24-30 hours APF wings stained with anti-Bs. (K) Wild type. (L) ash2112411/ash2112411 where extra vein tissue is restricted to wider p-cv and an extra proximal crossvein (arrow). (M) ash2112411/ash2112411 pupal wing of an individual with thicker vein tissues. In both L and M, some interveins are reduced and show some negatively stained cells within the interveins. (N) ash2I1 mitotic clones in pupal wings. (O) Bs staining of the same wing. (P) Merged green and red channels. (Q-S) High magnification of an ash2I1 clone.

View larger version (83K): [in a new window]   Fig. 6. Patterns of sal (A-H) and brk (I-L) expression in wing discs. (A) Wild type. (B) ash2I1/ash2I1 showing reduced sal expression. (C) ash2112411/ash2112411. (D) net1/net1; ash2112411/ash2112411. (E) bs03267/+; ash2112411/ash2112411. (F-H) ash2I1 mutant clones, marked by the absence of GFP. (G) salm expression is reduced in ash2I1 clones. (H) Merged green and red channels. (I) brk expression is visualised by means of the brk-lacZ reporter X47 in a wild-type wing disc. (J) High magnification of the boxed area indicated in I, ash2I1 clones lack GFP. (K) There is no change in ß-galactosidase expression in ash2I1 mutant cells. (L) Merged images of J and K. Scale bars: 20 µm.

View larger version (86K): [in a new window]   Fig. 7. ash2 regulates kni expression. kni in wild-type (A), ash2I1/ash2I1 (B) and ash2112411/ash2112411 (C) wing discs. (D) Minute+ clones homozygous for ash2I1 induced at 110±4 hours AEL are marked by the absence of ß-galactosidase. (E) Expression of kni is cell-autonomously upregulated by the loss of ash2 function (arrow). There is residual kni expression in heterozygous tissue. (F) Merged images of C and D. (G) Clones homozygous for ash2I1 induced at 52±4 hours AEL are marked as black spots by the absence of GFP and twin clones of wild-type cells appear as intense green spots. (H) kni expression is upregulated in homozygous ash2I1 mutant cells and residual kni disappears in cells homozygous for GFP. (I) Merged green and red channels of F and G. (J) Detail of a clone. Homozygous GFP cells are outlined in white (HMZ) and homozygous ash2I1 cells are outlined in yellow. HTZ. heterozygous tissue. (K) Anti-Kni staining of the same disc. One mutant copy of ash2 is enough to de-repress kni expression. (L) Merged images of I and J, and an additional channel with nuclear labelling (propidium iodide, shown in blue). (M) Detail of endogenous Kni (red) showing nuclear localisation (blue) in the L2 domain (pink in lower panel corresponds to nuclear labelling). (N) Detail of an ash2I1 clone; Kni is mainly cytoplasmic, although nuclei also contain some protein. (O) Adult kniri–1/kniri–1 wing. (P) kniri–1/kniri–1 ash2112411/ash2112411 wing showing rescue of L2 vein. (Q) rho expression in pupal kniri–1/kniri–1 wing. (R) rho expression in pupal kniri–1/kniri–1 ash2112411/ash2112411 wing. There is distal expression in L2. (S) RT-PCR showing increased kni mRNA in ash2I1 homozygous larvae compared with wild type. Rp49 is shown as a control. Scale bars: 20 µm.

View larger version (37K): [in a new window]   Fig. 8. kni upregulation by ash2I1 is independent of sal-C downregulation. (A) Overexpression of sal-C from UAS-sal64d using a nubbin-Gal4 driver results in loss of L2 and L5 and severe reduction of wing size. (B) Wing imaginal disc showing ectopic expression of salm throughout the wing pouch. (C) ash2I1 homozygous clones are devoid of GFP. (D) kni expression is also upregulated in ash2I1 mutant clones even, when salm is overexpressed. (E) Merged images. Scale bars: 20 µm.