Ectopic expression of hh in the absence of mtv activity. (A) Wing disc labelled to visualize expression of the mtv-lacZ reporter gene (antibody to β-Gal, green) in an hh-GAL4;UAS-GFP background (GFP, blue). (F) Wing disc labelled to visualize expression of a hh-lacZ reporter gene (antibody to β-gal, blue) and Mtv protein expression (green or white). (B–E,G,H) Clones of cells mutant for mtv (mtv⁶; B–E) or for both engrailed/invected and mtv (Df(2R)en[E] mtv⁶; G,H) and labelled by the absence of the GFP marker (green). The contour of the clone is marked by a red or white line. hh-lacZ (blue or white; B,C,G), Engrailed (En, red; C), Hedgehog (Hh, purple; D) or Patched (Ptc, purple or white; E,H) is ectopically expressed in a clone abutting the anterior–posterior (ap) and dorsal–ventral (dv; dashed lines) boundaries. Note in (D) that the marked clone with ectopic Hh protein expression was born in the anterior (a) compartment (twin clone is marked by an arrowhead) but crosses into the posterior (p) compartment. A neighbouring p clone (white asterisk) and its twin (white arrow) are shown. Violation of the ap compartment was very frequently observed in these clones (data not shown). (I,J) Two different mechanisms to repress hh expression in a cells. (I) En expression in p cells induces hh expression and represses cubitus interruptus (ci) expression. Hh activity in a cells stabilizes the transcriptional activator form of Ci (Ci^act). In the absence of Hh signal, Ci is transformed to a transcriptional repressor (Ci^rep). Ci^rep represses Hh expression. (J) Proposed model illustrating the role of Mtv and Gro in the repression of hh in a cells. GFP, green fluorescent protein; Gro, Groucho; Mtv, master of thickveins.

Gro and Mtv interact in vivo and in vitro. (A,D) Cuticle preparations of gro¹/gro^E48 (A) and mtv⁶/+;gro¹/gro^E48 (D) adult wings. Note in (D) ectopic veins L2 and L3 (L2^*, L3^*), and the enlargement of the anterior compartment; compartments a and p are indicated. (B,C,E,F) gro¹/gro^E48 (B,C) and mtv⁶/+;gro¹/gro^E48 (E,F) wing discs labelled to visualize expression of hh messenger RNA (purple; B,E), Araucan (Ara, blue; C,F) and Engrailed (red; C,F). (G) Co-immunoprecipitation (IP) of Myc-tagged Mtv (Mtv–Myc) by binding to Gro. S2 cells were co-transfected to express Gro and Mtv–Myc or transfected to express Gro or Mtv–Myc alone; empty vector was co-transfected as control in the latter cases. Right panels show 1/30 of Gro and Mtv–Myc proteins in cell lysates; cell lysates were immunoprecipitated with Gro antibody (left). Mtv–Myc was co-immunopreciptitated in significant amounts in cells co-transfected with Gro and Mtv–Myc (lane 1). Mtv–Myc was also co-immunopreciptitated in lesser amount in cells transfected with Mtv–Myc alone (lane 3) as a result of the endogenous Gro present in S2 cells (lanes 11, 12, 15 and 16). In the absence of Gro antibody, Mtv–Myc was not co-immunopreciptitated (data not shown). a, anterior; Gro, Groucho; Hh, hedgehog; Mtv, master of thickveins; p, posterior.

Genetic interactions between mtv and the hh gain-of-function allele Moonrat. (A–C) Cuticle preparations of wild type (A), hh^Mrt/+ (B) and mtv⁶/+; hh^Mrt/+ (C) adult wings. The adult wing is decorated by four longitudinal veins (L2–L5) and sensory organs along the margin. Note in (B) and (C) the ectopic vein L3 (L3^*, marked by an arrow). In (C) the anterior compartment is enlarged. As shown in the magnification of the anterior wing margin (red box) at the bottom of the panel, the sensory organs located in the most anterior wing margin (Triple Row Bristles, TR) are transformed into more posteriorly located bristles (Double Row Bristles, DR). Anterior (a) and posterior (p) compartments are indicated. (D–K) Wild-type (D,G,J), hh^Mrt/+ (E,H) and mtv⁶/+; hh^Mrt/+ (F,I,K) wing discs labelled to visualize expression of hh messenger RNA (purple; D–F), Araucan (Ara, blue; G–I), Engrailed (red in the top panels, white in the bottom panels; G–I), Delta (Dl, blue; J,K) and Ci (red; K). Note ectopic expression of Dl in the anterior compartment, presumably marking the presumptive ectopic vein L3 (L3^*, marked by an arrow) visualized in adult flies (C). Hh, Hedgehog; Mtv, master of thickveins.

Anterior hh expression depends on the endogenous activity of the Hh and Notch signalling pathways. Clones of cells mutant for mtv (mtv⁶; A,B), ptc and mtv (ptc^S2mtv⁶; C–E), gro (Df(3R)Espl22; F,J), ci (ci⁹⁴; G) or gro and ci (Df(3R)Espl22, ci⁹⁴; H,I) labelled by the absence (A,C,G), expression (B,D,E) or presence of two copies of the GFP marker (green; F,H–J). Expression of hh-lacZ (light blue, top; white, bottom) was monitored. The endogenous dorsal–ventral boundary is labelled by a dashed line or by Wingless (Wg, red; D) expression. The Hh signalling pathway was blocked by overexpression of a dominant-negative version of Smoothened in all wing cells (Smo-5A) in (A). Using the MARCM technique, the activity of the Hh or Notch signalling pathways was blocked or activated by means of full-length Ptc (B), a dominant-negative form of Mastermind (mam-dn; D) or by an activated form of the Notch receptor (Nintra; E). In (J), Ci^Cell was expressed under the control of MS1096^Gal4 control. The MS1096^Gal4 domain of expression is depicted by a white line. Gal4 expression levels are higher in the dorsal (d) wing pouch. Ci, Cubitus interruptus; GFP, green fluorescent protein; Gro, Groucho; Hh, Hedgehog; MARCM, mosaic analysis with a repressible cell marker; Mtv, Master of thickveins.