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First published online 10 November 2004
doi: 10.1242/dev.01538

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Repression of Dpp targets in the Drosophila wing by Brinker

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA

View larger version (41K): [in a new window]   Fig. 2. Mutated/modified Brk protein and activity. (A, part i) Domains/motifs in the Brk protein. DBD, DNA binding domain; Q, poly-glutamine; H, histidine rich; A, poly-alanine; 3R, independent repression domain. CiM and GiM, interaction motifs for the co-repressors CtBP and Gro, respectively. (A, parts ii-v) Properties and effects of modified/mutated Brk proteins shown in B-D. (A, part ii) Domains present: present (+), deleted (–) or mutated (M). (A, part iii) Nuclear (N) or cytoplasmic (C) localization (blank spaces, here and in other columns, indicates that they were not tested). (A, part iv) Activity level assessed by effect on the phenotype of adult wings (`–', no repressor activity; `++++', maximal activity; see Materials and methods for details on assigning activity level). (A, part v) The ability of each protein to repress the endogenous sal gene, an omb-lacZ line, and two reporters, vg-QE and UbxB. Y, repressed; N, not repressed; U, we were unable to detect reliable differences in UbxB expression between wild-type and mutant embryos of any genotype. (B) EMS point mutants in the endogenous brk gene. A protein truncated within the DBD, brkM68, or immediately after it, brkE427, has no activity, whereas a truncation producing a longer protein, but which still does not include the CiM or GiM, brkF138, has significant activity and can repress omb, but not the other targets. An amino acid substitution within the DBD, brkF124, also abolishes activity. (C) Point mutants in a UAS-brk transgene, A438. Four result in amino acid substitutions in the DBD and reduce activity. The fifth mutation, A438-53, results in a truncation immediately after the DBD and has no activity. (D) In vitro mutated/modified UAS-brk transgenes. There are two basic requirements for these transgenes to repress gene expression: the DBD and a repression domain/motif. In addition to the CiM and GiM, there is another independent repressor domain, 3R, located between the DBD and CiM. The DBD plus any one of the three repressor domains/motifs is sufficient to repress some Brk targets, although there is some variability in the ability of individual repressor domains to repress different targets.

View larger version (45K): [in a new window]   Fig. 3. Mutations in UAS-brkA438, which originally contained a wild-type brk transgene. (A) Wild-type adult wing. (B) Misexpression of wild-type A438 results in a dramatically reduced wing. (C-G) Misexpression of mutated forms of A438, however, results in larger wings: from completely wild-type in the case of A438-53, indicating a complete loss of activity, to small in the case of A438-S4 and A438-C, indicating significant, but less than wild-type, activity. Wings produced with A438-D44 and A438-F2 are almost wild type in size, but have venation defects indicating that some activity still remains.

View larger version (52K): [in a new window]   Fig. 1. Sequence comparison of Brk from Drosophila melanogaster and from three other insects, D. pseudoobscura, Anopheles gambiae and Bombyx mori. The DNA-binding domain (boxed in red) is strongly conserved in all four species. Outside of the DNA-binding domain there is only one other region of significant similarity, centered on the Groucho interaction motif (GiM, blue), although the short CtBP interaction motif (CiM, blue) is also conserved. The third repression domain, 3R, of D. melanogaster, identified by functional studies described here, is outlined in pink. This domain shares some limited sequence identity with D. pseudoobscura, but this does not extend to the other two species, although it is rich in Ala and His residues in Anopheles, as well as in the two Drosophila species.

View larger version (51K): [in a new window]   Fig. 5. Repression of sal, omb and vg-QE following misexpression of modified/mutated forms of Brk in the posterior of wing discs using en-Gal4. The interface between posterior (p) and anterior (a) is marked by a dashed line (i.e. transgene expression was driven in cells to the right). (A) Wild-type Brk3PF3 at 20°C completely represses sal (green) and omb (red). Brk3PF3 is tagged with the HA epitope, shown in blue in part iii. (B) At lower temperatures (resulting in lower levels of transgene expression), wild-type Brk3PF3 still completely represses sal (green) but some omb expression remains (arrow). (C,D) BrkNLS (at 30°C) has no effect on the expression of sal or omb. (E) Higher magnification image focused on the large peripodial membrane cells, stained for HA, showing that expression of BrkNLS is exclusively nuclear. (F) By contrast, BrkNLSW (at 30°C) can repress sal completely and omb (arrow) almost completely. (G) BrkStop1NAC (at 25°C), which possesses only a CiM, completely represses sal and almost completely represses omb (arrow). (H) At 25°C, BrkStop1, which lacks both the GiM and CiM, represses sal and omb completely. (I) However, at 20°C, although the lower levels of BrkStop1 are sufficient to almost completely repress omb (there is some residual expression, arrow in ii), Sal is still expressed at high levels in the posterior (arrow in i). (J-L) Repression of vg-QE expression. (J) Brk3PF3 drastically reduces the level and width of the vg-QE expression domain. (K) BrkNLSW has no effect on vg-QE expression. (L) BrkStop1 almost completely represses vg-QE.

View larger version (19K): [in a new window]   Fig. 6. Repression of the UbxB reporter following the misexpression of modified/mutated forms of Brk in the visceral mesoderm of embryos (X-gal stained), using the 24B Gal4 driver. (A) No brk transgene. At this stage, UbxB drives lacZ expression in parasegments 6-9 of the midgut mesoderm. (B) Wild-type Brk3PF3 completely represses UbxB. (C) BrkNLS has no effect on UbxB expression. (D,E) However, BrkNLSW dramatically reduces, and BrkStop1 completely represses, UbxB expression.

View larger version (25K): [in a new window]   Fig. 7. Phenotype of adult wings following the misexpression of modified/mutated forms of brk. UAS-brk transgenes (see Fig. 2D) were driven by en-Gal4 in the posterior compartment and C765 ubiquitously (C765 is weaker than en-Gal4). (A) Wild-type wings shaded to indicate the expression domains of the Gal4 drivers. (B-F) The activity level of different Brk proteins was classified into five categories from `++++' (full, wild-type level) to `–' (no repressor activity; see Materials and methods); examples of each category are shown, for comparison, the temperature must be taken into account (Gal4 is cold sensitive). (B) Full activity of 3PF3 (wild type). With en-Gal4, no animals survived to adult; with C765 the wing size is drastically reduced at 20°C. (C-F) Mutated/modified Brk proteins have weaker activity than wild type. (C) Stop1, 20°C (top); CM, 20°C (bottom). (D) 3M, 25°C (top); 3M, 30°C (bottom). (E) A2, 30°C. (F) NLS, 30°C (top); F124, 30°C (bottom). At high temperatures, ectopic veins are produced with F124, which we attribute to gain of function, as it has no effect on Brk target gene expression.

View larger version (55K): [in a new window]   Fig. 4. Phenotype of brk point mutants and CtBP and gro mutants. Third instar wing discs containing mutant clones, marked by the loss of a ubiquitous GFP transgene, and stained for omb (lacZ, anti-ßgal), Sal (antibody) and/or vg-QE (lacZ, anti-ßgal) expression. (A,B) brkF124 clones show ectopic expression of Sal and omb within the wing pouch (arrow), and expansion of the vg-QE domain. (C) brkF138 clones. (D) Detail from boxed area in C, showing a large clone in the posterior compartment. The edge of the normal Sal and omb domains are shown in red and blue, respectively, in part i, and in white in parts ii and iii (these are approximations). If this were a null mutant clone, Sal and omb would be expressed in all of the mutant cells in the wing pouch, and omb would extend outside of the pouch (see A). However, there is no ectopic omb expression in brkF138 mutant cells, apart from possibly an expansion to one or two cells wider than normal (note, omb is on the same chromosome as brk, so that omb expression is upregulated in brk mutant cells within its endogenous domain because these cells are now homozygous for the omb enhancer trap). There is some ectopic Sal expression, but only in mutant cells within the endogenous omb domain (arrow in part ii) and not more laterally. (E) vg-QE expression is expanded laterally in some brkF138 clones (arrow). (F-H) CtBP gro double mutant clones are similar to those of brkF138, only showing ectopic Sal expression (F, arrow) immediately adjacent to the endogenous domain (when located in the omb domain, not shown). By contrast, Sal is not ectopically expressed in any CtBP single mutant clones (G), whereas there is an occasional, minor deregulation of Sal in gro clones (H, arrow).