Deletions recovered in gro. (A) Diagram of the domain structure of Drosophila Gro. The two predicted amphipathic α-helices in the Q domain (AH1 and AH2) are indicated. The coding regions of MB36, MB5 and MB12 transcripts are also shown. (B) Alignment of the amino-acid sequences of the mutated region in MB5 to the corresponding wild-type region (+). AH1 is underlined; amino-acid residues deleted in MB5 that lie outside AH1 are boxed with the corresponding sequence from hTLE1 shown above. (C) Western blots of extracts from gro mutant embryos (from gro mothers) run on SDS–polyacrylamide gel electrophoresis gels. MB36 is not detected and levels of MB12 are reduced. β-Tubulin is shown as a loading control. Gro, Groucho.

Expression of vgQ-lacZ in gro mutant clones. Third instar imaginal wing disks (anterior to the left and dorsal to the top) stained for GFP and β-galactosidase. Loss of GFP (green) marks clones of homozygous gro mutant cells. Ectopic vgQ-lacZ expression is detected in clones of (A) E48, (B) MB36 and (C) MB12 at the anterior and posterior edges of the wing pouch but not in clones of (D) MB5. Arrows indicate positions of gro clones at the edge of the wing pouch; GFP, green fluorescent protein; Gro, Groucho.

Genetic screen for modifiers of ectopic hairy expression in the eye of Drosophila. (A–D) Light photomicrographs showing dominant suppression of the eye phenotype by gro alleles; weak for MB5 and strong for the null allele MB36. Genotypes are as indicated below. (E) Schematic representation of the screening strategy. F₁ progeny homozygous for GMR-Gal4,UAS-hairy and heterozygous for a mutagenized FRT 80B FRT 82B chromosome were scored for the suppression of the eye phenotype. e, ebony; EMS, ethyl methane sulphonate; Gro, Groucho; h, hairy; w, white.

Phenotypic analysis of gro alleles. (A–D) Cuticles of wild-type (+) and gro mutant embryos. MB5 embryos show a weaker phenotype (loss of two or three even-numbered denticle belts) than MB12 or MB36. MB12 embryos are neurogenic but retain more body mass than MB36. Expression of (E–H) tll, (I–L) kni and (M–P) ftz transcripts in blastoderm embryos, showing that MB5 and MB12 retain activity. tll expression is wild type in MB5, slightly expanded in MB12 and throughout the embryo in MB36, indicating residual activity in MB12. Expression of the central stripe of kni is reduced in most MB12 embryos, but not completely abolished as in MB36. Stripes of ftz expression are irregular in MB5, broadened in MB12 and completely fused in MB36. (Q–T) Expression of sna transcripts is expanded towards the posterior pole in MB12 or MB36 embryos (indicated by arrows) in a manner resembling the sna expression pattern in hkb mutants (Goldstein et al, 1999), indicating that repression mediated by Hkb is lost in MB12 embryos. (U–X) Staining of stage 13 wild type and gro for the neural marker Hb. MB12 and MB36 mutants show ectopic expression of Hb protein that is characteristic of excessive neural development. ftz, fushi tarazu; Gro, Groucho; Hb, Hunchback; Hkb, Huckebein; kni, knirps; sna, snail; tll, tailless.

Q-domain-mediated oligomerization of Gro variants. (A) Autoradiograph of ³⁵S-Met-labelled Gro mutant proteins run on SDS–polyacrylamide gel electrophoresis. MB12 migrates slightly faster than wild type (+), which is consistent with an amino-terminal truncation. MB5 and L36P,L87P variants run similar to wild type. (B) Autoradiograph of the translation reactions shown in (A) run on BN-PAGE. MB12 and L36P,L87P do not form a tetrameric complex. (C) Western blot of wild type and MB5 extracts run on BN-PAGE. MB5 forms a high-molecular-weight, tetrameric band. (D,E) Pull-down analysis of ³⁵S-Met-labelled full-length, translated Gro⁺ and (D) MB5 or (E) L36P,L87P or MB12 protein on GST–wild-type and GST–MB5 Q domains. Neither MB5 nor MB12 binds to an isolated Q domain in this assay. (F) Pull-down analysis of translated Gro⁺, MB5 and MB12 proteins on GST fusions of full-length Gro⁺, MB5 and MB12 proteins, showing that immobilized, full-length MB5 retains the ability to bind to translated Gro⁺. (G) Disruption of oligomerization in two different Q domain mutants, MB12 and L38P,L87P, reduces binding to the WRPW motif. MB41 is a missense mutation in the WD domain that abolishes binding to WRPW (Jennings et al, 2006). Control input tracks represent 10% of labelled Gro protein. BN-PAGE, blue native-polyacrylamide gel electrophoresis; Gro, Groucho; GST, glutathione-S-transferase.