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EGFR signalling inhibits Capicua-dependent repression during specification of Drosophila wing veins

Fernando Roch^1,*,, Gerardo Jiménez² and Jordi Casanova²

¹ University Museum of Zoology, Department of Zoology, University of Cambridge, Downing Street, Cambridge, UK, CB2 3EJ
² Institut de Biologia Molecular de Barcelona (CSIC). C/ Jordi Girona 18-26, 08034, Barcelona, Spain
Present address: Centre de Biologie du Développement, Université Paul Sabatier, Bât 4R3, 118 Route de Narbonne, 31062, Toulouse, France

View larger version (10K): [in a new window]   Fig. 1. Diagram of the cic locus and several mutations affecting the gene. The organisation of the major maternal transcript is shown; coding and untranslated regions are depicted by black and white boxes, respectively. The P(PZ)bwk8482 (P-element) and cic1 (hobo) transposon insertions are indicated by triangles. The cic2 allele maps 380 bp upstream of the HMG-box region (hatched) and consists of a deletion of 13 nucleotides, tctgattgtgtcc. Fragments deleted by three independent P(PZ)bwk8482-derived excisions are indicated by bars (open ends denote uncertainties in their limits). The genomic region included in the cic+ rescuing construct is also indicated.

View larger version (114K): [in a new window]   Fig. 2. Mutations in cic cause the development of ectopic veins. (A,B) Wild-type and cic2/bwk11 (C,D) adult wings showing the L2-L5 longitudinal veins. (B,D) Show a close view of the region corresponding to the L5 tip. Note the presence of cells showing different degrees of pigmentation, and bearing hairs of different length and thickness in D (small arrows). (E,F,H) Adult wing containing two cic2 M+ homozygous clones marked with the forked hair marker. (F) Close view of the anterior ventral clone shown in E, showing that cic mutant cells can differentiate either as ectopic vein tissue (white arrowhead) or as intervein tissue (black arrowhead). Note also a patch of cells outside the clone that differentiate as an ectopic vein (small arrow). (H) Close view of the wild-type dorsal surface opposite the ventral clone shown in F, displaying patches of ectopic veins (small arrows). (G) Adult wing containing a dorsal and ventral cic2 M+ clone occupying all the anterior compartment. In this mosaic wing, all the region between the margin and the L3 differentiates as vein tissue, whereas the region between L3 and the AP boundary differentiates as intervein, exactly as in the cic2/bwk11 mutants.

View larger version (143K): [in a new window]   Fig. 3. The levels of Cic protein are regulated post-transcriptionally in the wing imaginal disc. (A) Third larval instar imaginal wing disc stained with a cic antisense riboprobe. The cic transcript is uniformly distributed throughout the disc. (B-D) Third larval instar imaginal disc stained with an anti-Cic antibody; (B,C) a view of the main disc epithelium; (D) the peripodial membrane. (B) The Cic protein is expressed in the wing pouch but is absent in the notum region. Low Cic levels are observed in the prospective wing margin and along the L3 longitudinal vein. (C) A close view of the anterior D/V boundary region stained with anti-Cic (red) shows that Cic is clearly downregulated in the prospective wing margin. The cell nuclei are counterstained with nuclear GFP (green). The Cic protein is present in both the nucleus and the cytoplasm of most wing pouch cells, but is not present in the cell nucleolus that appears as a small black area. (D) Close view of the wing peripodial membrane stained as in C. In these large cells, Cic protein also accumulates in the nucleus (except the nucleolus, as in the wing epidermis, black arrowhead) and can be also observed in the perinuclear cytoplasm (white arrows). (E,F) Six hours APF wing discs stained as in B,C. At this stage Cic levels are decreasing in all the presumptive longitudinal veins. (F) Detail that includes the tip of the L5 vein. (G) Detail of the L5 vein tip in a 28-32 hours APF wing disc stained as in C. Cic is present at high levels in the intervein cells and expressed at much lower levels in the L5 and the posterior crossvein.

View larger version (122K): [in a new window]   Fig. 4. cic acts downstream of the Ras/Raf pathway. (A) The differentiation of all the wing veins is prevented in rhove vn1 homozygotes. (B) cic2/bwk11 flies differentiate ectopic veins throughout the wing. (C) In the triple mutant rhove vn1 cic2/ rhove vn1 bwk11, the loss of veins phenotype is completely suppressed and the extravein phenotype is completely epistatic. (D) Mutant clones Rasc40b M+ autonomously prevent vein differentiation. (E) Two anterior clones cic2 M+ cause ectopic veins (dorsal and ventral regions of the clone outlined in black and white, respectively). (F) Wing containing three M+ clones mutant for both cic2 and Rasc40b, differentiating extravein tissue. (G) Pupal wing staged 0-6 hours APF containing two large Rasc40b M+ clones and stained with anti-Cic (red). Mutant clones are marked as patches free of GFP staining (green). (H) Red channel in G. Cic is autonomously expressed at high levels in all the Rasc40b mutant cells. Cic is expressed at normal levels outside the mutant territories (white arrows) and is downregulated in the L3 vein (white line). (I) 0-6 hours APF pupal wing of a UASVn/Engrailed GAL4 fly stained with anti-Cic. Note the low levels of Cic present in the cells posterior to the L4 presumptive region (white line).

View larger version (158K): [in a new window]   Fig. 5. Aos is repressed in the intervein tissue by the activity of Cic. (A) Wild-type and (B) cic2/bwk11 third larval instar wing imaginal discs carrying the aos-lacZ reporter stained with anti ß-Gal. The aos reporter gene is expressed throughout the wing pouch in the mutant. (C,D) Wild-type and cic2/bwk11 wings (24-30 hours APF) carrying the aos-lacZ reporter stained as in A,B. Homogeneous levels of the aos reporter are found throughout the wing blade in the mutant. (E,F) Third larval instar imaginal wing disc expressing the aos-lacZ reporter and containing cic2 M+ mutant clones marked as cells lacking GFP (green in F). Staining with anti-ß-gal (red in F) shows that aos-lacZ is expressed autonomously in all the cic mutant cells.

View larger version (98K): [in a new window]   Fig. 6. (A,C,E,G,I) Wild-type wing and (B,D,F,H,J) cic2/bwk11 wings stained for various vein and intervein markers. (A,B) 6 hours APF wings stained with a Vvl antibody. At this stage, Vvl begins to fade away from the intervein tissue in the wild type. In the mutants, Vvl expression is maintained in the intervein tissue. (C-F) 24-30 hours APF, wings stained with antibodies against Vvl (green) and Bs (red). (E,F) A close view of the L4 vein. In the wild type, the patterns of Vvl and Bs are almost complementary, only a few nuclei abutting the vein tissue co-express both markers. In the mutant, most cells co-express variable levels of both proteins. (G,H) Pupal wings staged 24-30 hours APF stained with an antisense rho riboprobe. In the mutant, rho is expressed ectopically in the intervein tissue. (I,J) Pupal wings of the same age stained with a dpp antisense riboprobe. In the mutant, dpp is expressed at high levels in the future vein regions and at lower levels in the rest of the wing pouch.

View larger version (17K): [in a new window]   Fig. 7. The role of cic in the specification of Drosophila wing veins. Active genes and proteins are shown in black, whereas inactive genes and proteins are in red. EGFR and MAPK are active in vein cells and inactive in the intervein tissue from third larval instar onwards (see text). In vein cells, EGFR signalling downregulates Cic protein levels in the nucleus, thus relieving repression of vein-specific genes such as aos, vvl and dpp. In addition, EGFR signalling is required in the veins for the activation of rho and the repression of bs, in a cic-independent manner. rho expression in the veins further increases EGFR activation in a paracrine loop. Conversely, absence of EGFR signalling in the intervein territories allows these cells to maintain high levels of Cic throughout larval and pupal development, which represses the expression of vein-specific genes. In addition, these cells accumulate Bs protein that represses rho and promotes intervein differentiation.