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Wingless blocks bristle formation and morphogenetic furrow progression in the eye through repression of Daughterless

¹ Howard Hughes Medical Institute and the Department of Developmental Biology, Stanford University School of Medicine, Beckman Center,Stanford, California 94305, USA
² Department of Molecular, Cellular and Developmental Biology, University of Michigan, Natural Sciences Building, Ann Arbor, Michigan 48109, USA

View larger version (186K): [in a new window]   Fig. 1. da dominantly enhances the P[sev-wgts] bristle phenotype. Scanning electron micrographs of adult fly heads. P[sev-wgts]/+ flies reared at 25°C (A) or 17.6°C (B). P[sev-wgts]/da11B6 (C) and P[sev-wgts]/da3 (D) flies reared at 17.6°C. P[sev-wgts]/+ flies reared at the lower temperature had about 150 bristles/eye (compared to approximately 600 observed at 25°C) grouped in the dorsal third of the eye (large arrows) with a few bristles in the ventral half (small arrows in B). When heterozygous for strong da alleles, bristle number was reduced to 10-25 bristles/eye, and no bristles were seen in the ventral half of the eye.

View larger version (112K): [in a new window]   Fig. 2. Wg represses Da expression, which is required for proneural gene expression and bristle formation. (A) Micrograph of an adult fly head containing a small clone of da5B5 cells (marked by absence of pigment). Bristles fail to form inside the clone. (B,C) Confocal images of pupal eye (B) and larval wing imaginal disc (C) containing da5B5 clones and immunostained for Ac. Clonal boundaries were marked by the absence of ß-gal staining (not shown; indicated by white lines). Ac expression is markedly reduced inside the da clones in both tissues. (D) Wing imaginal disc containing a da5B5 clone stained for Ac protein (orange) and sc mRNA (blue). The approximate location of the da clone is indicated by the reduction of Ac expression (white arrows). This area also contains reduced sc transcript. (E-H) Pupal eyes stained for Da protein. (E) Wild type; (F) P[sev-wg]; (G) P[GMR-Gal4], P[UAS-wg]; (H) sc10-1 (deficiency removing the ac and sc loci). While Da expression is significantly repressed by ectopic Wg (F,G), it is only mildly affected by removal of ac and sc (H). All pupal eyes were at 3h APF.

View larger version (110K): [in a new window]   Fig. 3. Endogenous Wnt signaling represses Da and Ac expression and bristle formation at the periphery of the eye. (A) Wild-type pupal eye (3h APF) stained for Wg (red) and Ac (green). (B) P[wg-lacZ] pupal eye stained for ß-gal (red) and Da (green). In both cases, Ac and Da expression is absent from the edge of eye, where Wg is expressed. The Da signal observed at the edge of the eye in B is from peripodal membrane staining. (C-E) Micrographs of adult fly heads containing clones of wgCX4 (C), Df(2L)RF (D) or arm25B (E). Inside the wg clone, bristles are still inhibited at the edge of the eye. In the Df(2L)RF or arm clones shown, bristles are found up to the edge of the eye. At the top of the arm clone, ectopic bristles are present but are out of focus. See Results for further description of the clonal analysis.

View larger version (100K): [in a new window]   Fig. 4. Coexpression of a dominant negative version of DTcf (DTcfDN), da or sc suppresses Wg-induced bristle inhibition. Scanning electron micrographs of adult fly heads (A,D,G,J,M) and pupal eyes (6 h APF) immunostained for Ac (B,E,H,K,N) or Da (C,F,I,L,O). (A-C) P[GMR-Gal4], P[UAS-lacZ]; (D-F) P[GMR-Gal4], P[UAS-wg], P[UAS-lacZ]; (G-I) P[GMR-Gal4], P[UAS-wg], P[UAS-TCFDN]; (J-L) P[GMR-Gal4], P[UAS-wg], P[UAS-da]; (M-O) P[GMR-Gal4], P[UAS-wg], P[UAS-sc]. Ectopic expression of wg via the GMR promoter causes a severe reduction in eye size and completely blocks bristle formation (D). GMR/wg causes a huge decrease in Ac (E) and Da (F) expression. Co-expression of wg with TCFDN partially suppresses the small eye and loss of bristles (G), as well as the Ac (H) and Da (I) inhibition. Co-expression of wg with da or sc does not significantly alter the small eye phenotype, but fully suppresses the bristle loss (J,M). GMR/wg/da causes a modest increase in Ac levels (K; compared with E). GMR/wg/sc also causes a modest increase in Ac (N) and Da (O) levels. All cultures were maintained at 25°C.

View larger version (99K): [in a new window]   Fig. 5. Expression of da and ato, but not hh and dpp, can rescue the block in MF progression caused by ectopic activation of Wg signaling. Wandering third instar larval eye discs stained for ß-gal (all samples have a P[dpp-lacZ] transgene) and Elav. (A) WT; (B) P[dpp-Gal4], P[UAS-wg]; (C) P[dpp-Gal4], P[UAS-wg], P[UAS-TCFDN]; (D-F) P[dpp-Gal4], P[UAS-wg], P[UAS-da]; (G-I) P[dpp-Gal4], P[UAS-wg], P[UAS-hh]; (J) P[dpp-Gal4], P[UAS-wg], P[UAS-ato]; (K) P[dpp-Gal4], P[UAS-armact]; (L) P[dpp-Gal4], P[UAS-armact], P[UAS-da]. (A) At this stage, the MF is normally more than halfway across the eye primordia. (B) Ectopic expression of wg at the posterior edge of the eye completely blocks MF progression and differentiation of Elav-positive cells. (C) Coexpression of TCFDN with wg significantly rescues MF progression. (D-F) Coexpression of da with wg always resulted in some MF progression, with approximately 40% of the eyes similar to D, 40% similar to E and the remainder rescued to the extent shown in F. Coexpression of dpp with wg resulted in no rescue (data not shown). (G-I) Coexpression of hh with wg did not rescue MF progression from the posterior edge, but did result in usually one (occasionally more) MF being initiated in the interior of the eye primordia and progressing concentrically. These ectopic MFs had progressed to different extents at the time of fixation, with some recent (G), some several rows of photoreceptors (H) and some progressed to the periphery of the eye (I; note that dpp-lacZ is at the periphery, indicating the leading edge of the MF). Coexpression of ato with wg resulted in considerable rescue of MF progression, though less than seen with da coexpression (the eye in J is one of the best rescues). Expression of an activated form of arm (armact) blocked MF progression, though not as well as wg (note some Elav-positive cells in K). Coexpression of da with armact resulted in significant rescue of MF progression (L). All crosses were maintained at 29°C before fixation.

View larger version (75K): [in a new window]   Fig. 6. Wg does not appear to induce Emc or H expression in the eye, but emc and/or h is required for Wg-dependent repression of Da expression and bristle formation. (A-D) Wandering third instar eyes; (E-G) pupal eyes (6 h APF) and adult P[sev-wg] eye with emc1, hIL clones (H). (A,C) P[dpp-Gal4], P[UAS-lacZ]; (B,D) P[dpp-Gal4], P[UAS-wg]; (E) P[GMR-Gal4], P[UAS-lacZ]; (F) P[GMR-Gal4], P[UAS-wg]; (G,H) clones of emc1, hIL in a P[sev-wg] (G) or P[GMR-Gal4], P[UAS-wg] (H) background. (A,B,E,F) H expression; (C,D) Emc expression; (G,H) Da expression. Clonal boundaries were marked with ß-gal (not shown). H is normally expressed in a stripe several cell diameters ahead of the MF (marked with arrows). Emc is expressed in a stripe between the H stripe and MF. In Dpp/wg eyes, Emc is at the posterior edge of the eye (D) and H just anterior. This is consistent with the lack of MF progression in this background. Behind the MF, ectopic expression of wg (via the GMR promoter) does not induce H expression (F) or Emc (data not shown). Removal of emc and h blocks P[sev-wg] from inhibiting Da expression (G) but not P[GMR-Gal4], P[UAS-wg]-dependent Da repression (H). Clonal boundaries are indicated by white lines.