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Groucho mediates a Ci-independent mechanism of hedgehog repression in the anterior wing pouch

¹ Institute of Molecular Biology and Biotechnology, Fo.R.T.H., Heraklion, Greece
² Department of Biology, University of Crete, Heraklion, Greece
³ Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada

View larger version (100K): [in a new window]   Fig. 1. Response of hh-lacZ (A-C) and en-lacZ (D-E) to gro loss of function in the wing. Df(3R)Espl22 clones are shown, marked by increased GFP expression (bright green) in A-C and by absence of GFP expression (absence of green) in D-E. The gro+/+ twin spots (clones arising from the sister cell bearing the reciprocal recombination product) lack GFP and hh-lacZ (A-C, see Materials and Methods) or have twice the level of GFP (D-E). Anti-ß-galactosidase staining is shown in red. (A) A large anterior clone abutting the boundary (blue line) expresses hh-lacZ strongly (type I) – note comparable expression levels in a posterior clone, where hh-lacZ copy number is also 2 (arrow). (B) Large anterior clones further away from the AP boundary also express hh-lacZ strongly, but the intensity drops away from the DV boundary (type II/III). (C) Examples of small type II (short arrows) and III clones (long arrows) that express hh-lacZ only when located within an anterior domain close to the AP boundary. Several non-expressing clones (type IV-arrowheads) are seen in C, away from the AP boundary. (D,E) These large type I/II clones shown should express hh strongly throughout the clone (compare with A,B); however, en-lacZ derepression is more restricted. (A'-E') Red channel only. (A''-E'') Green channel only. The blue line in A,D E marks the AP boundary. Anterior is towards the left and dorsal is upwards.

View larger version (114K): [in a new window]   Fig. 2. Response of dpp-lacZ to gro loss of function in the wing. groE48 clones are marked by loss of GFP (green A,C) or Myc epitope (green, B) and ß-galactosidase is shown in red (red channel shown separately in A'-C'). A subset of gro- clones in A (some clones are outlined in blue) express dpp ectopically. This effect is non-autonomous, as neighbouring wild-type cells are also induced to express dpp (arrows). Ectopic dpp-lacZ is never seen anterior to its normal stripe in hhE23 groE48 clones (C), suggesting that its ectopic expression is mediated by hh activity. The few ß-galactosidase-positive cells in the anterior clones in C are within the normal dpp expression stripe. dpp-lacZ is autonomously derepressed in anterior clones posterior to the normal expression stripe (B), where it would be normally downregulated by En. This can happen in gro- (A,B) as well as in hh- gro- (C) clones – marked by arrowheads. All disks are oriented with anterior towards the left and dorsal upwards. The straight blue line indicates the position of the AP boundary.

View larger version (113K): [in a new window]   Fig. 3. Response of hh-lacZ to gro loss of function in a ciCe2/+ background. Df(3R)Espl22 clones are marked by increased GFP, as in Fig. 1A-C. (A',B') Red channel only (hh-lacZ). (A'',B'') Green channel only (GFP). Only clones near the DV boundary show good hh-lacZ derepression (white arrows). Type I clones (blue arrows), which would normally express high levels of hh-lacZ, show weak or no expression, whereas type III clones (arrowheads) are fully repressed.

View larger version (28K): [in a new window]   Fig. 4. (A) Schematic of the different mutant versions of gro transgenes. The hatched box in GroWD40 corresponds to 21 foreign amino acids added as a consequence of the construction strategy. (B-G) S2 cells transfected with different gro mutant expression vectors and stained with the anti-Gro (red – all panels except D) or pan-TLE monoclonal antibody (red – D) and TOPROIII (blue – overlap with Gro immunostaining seen as magenta) as a DNA counterstain – co-transfection with GFP was used to identify the transfected cells (not shown). (B) Grocdc2-, (C) GroQ, (D) GroGCS, (E) GroNLS- and (F,G) GroWD40. Note that transfected Gro is overexpressed with respect to endogenous; G is the same panel as F, except that the detection sensitivity is increased for red and endogenous Gro immunoreactivity is detectable in the three untransfected cells (arrows). As exogenous Gro is in excess, the localization observed is not influenced by interaction with endogenous Gro. Wild-type Gro also accumulates in the nucleus: in H, UAS-gro+ (red) was co-transfected with HA-tagged full length ci and counterstained with GFP (green), which localizes predominantly in the nucleus, but also in the cytoplasm. I shows another cell from the same transfection stained with an anti-HA antibody to detect Ci (red), which is exclusively cytoplasmic, thus providing no evidence for Ci-Gro interaction.

View larger version (65K): [in a new window]   Fig. 5. (A-D) Df(3R)Espl22 clones marked with increased GFP (bright green) and stained for hh-lacZ (red) in the background of UAS-gro(mutant) transgene expression using omb-Gal4; overexpressed Gro is visualized in the blue channel. (A'-D') green (GFP) and red (hh-lacZ) channels only. (A''-D'') red channel only (hh-lacZ). (A) UAS-grocdc2- rescues the gro- defect, as it does not allow hh-lacZ derepression in anterior clones (arrow). (B) UAS-groWD40 and C, D: UAS-groQ do not restore hh repression. The position of the AP boundary is indicated by a blue line. Anterior is towards the left and dorsal upwards.

View larger version (19K): [in a new window]   Fig. 6. Transcriptional inputs proposed to regulate hh in the A compartment. (A) The wild-type situation. The levels of Ci[rep] (blue) and Ci[act] (brown) are complementary, as these forms are reciprocally controlled by Hh signalling at the AP boundary (black vertical line). Ci[act] induces the expression or the activity of repressor X, which together with Gro forms a repressive complex (green) – thus, the green line follows the brown, except in cases of gro loss of function (C). As there is some repressor complex (blue or green) present at any given position in the A compartment, hh expression (red) is nil. B shows two ci- clones (grey bars on the AP axis), where both forms of Ci (blue and brown) are eliminated. They both contain low basal levels of hh transcription. This is because of the absence of both repressor and activator complexes within the clones. Note that the brown and blue curves have been shifted anteriorly (to the left) – this is because the loss of ci near the boundary disables the induction of ptc and therefore posterior Hh can diffuse further (Chen and Struhl, 1996) anteriorly to generate Ci[act] and suppress Ci[rep]. C shows two gro- clones (cross-hatched bars on the AP axis). Those affect only the X-gro repressor activity (drop in green line). As a result of high levels of Ci[act] activator (brown) and lack of repressor (green or blue) within the more posterior (rightmost) clone, hh is expressed at high levels. No effect is expected in the more anterior clone. Again, note that the brown and blue curves have been shifted anteriorwards as a result of high ectopic Hh activity that influences the relative amounts of Ci[act] and Ci[rep]. In B, we assume that the low basal hh levels within the clones are not sufficient to produce a similar effect.