Design of the F₂ mosaic screen to recover suppressors of the de2f1 mutant phenotype.

Mitotic marker phospho-H3 in clones of de2f1 Su(E1) double mutant cells. Eye imaginal discs were dissected from third-instar larvae. Clones of wild-type cells, identified by the presence of GFP (green), and homozygous mutant cells, identified by the absence of GFP (green), were induced by ey-FLP. Genotypes are shown, and posterior is to the right. (A–I) Eye imaginal discs were labeled with anti-phospho-H3 (magenta) to mark cells in mitosis. (A) Wild-type disc showing only phospho-H3. (B–I) de2f1⁷²⁹ l(3)mbt^3d33 (B), de2f1⁷²⁹ Su(E1)-A^7d13 (C), de2f1⁷²⁹ CG31133^7d21 (D), de2f1⁷²⁹ gfzf^6a27 (E), Su(E1)-A^7d13 (F), de2f1⁷²⁹ CG31133^7d21 (G), de2f1⁷²⁹ gfzf^6a27 (H), and Doa^KG09056 (I) cells progress through mitosis as evidenced by phospho-H3 (magenta)-positive cells in clones of mutant tissue.

Effect of Su(E1)'s on E2F-dependent transcription in tissue culture cells. (A) Depletion of dE2F1 represses expression of a PCNA-luc reporter. Codepletion of L(3)MBT, or dE2F2, or RBF1 (R1) and RBF2 (R2), together with dE2F1, derepresses expression of the E2F reporter. S2R+ cells were incubated with either control or experimental double-stranded RNA (dsRNA) as indicated. Cells were then incubated for an additional day with either control (NS) or dE2F1 dsRNA before being cotransfected with a PCNA-luc reporter and a β-Gal expression plasmid in duplicates. (B) Codepletion of L(3)MBT or dE2F2 (E2) with dE2F1 (E1) derepresses expression of the endogenous PCNA gene. S2 cells were incubated with either control (NS) or corresponding dsRNAs as indicated. After 4 days, the steady-state level of the PCNA mRNA was determined by real-time reverse transcription–PCR in triplicates. (C) The basal level of expression of a PCNA-luc reporter is elevated in cells depleted of either GFZF or CG31133 proteins. S2R+ cells were incubated with control (NS) or corresponding dsRNAs as indicated. After 4 days, cells were transfected with a PCNA-luc reporter in duplicates. (D) S2R+ cells were incubated with control (NS) or corresponding dsRNAs as indicated. After 4 days cells were lysed and the level of dE2F1 was determined by Western blot analysis. Tubulin was used as a loading control. (E) Codepletion of GFZF or CG31133 with RBF1 (R1) and RBF2 (R2) synergistically activates a PCNA-luc reporter. After 4 days, cells were cotransfected with a PCNA-luc reporter and a β-Gal expression plasmid in duplicates.

Suppression of the de2f1 mutant phenotype in adult eyes and wing imaginal discs. (A–J) Mitotic recombination was induced in the eyes of heterozygous animals by ey-FLP. Wild-type chromosomes carry the mini-white gene and therefore wild-type clones (red) can be distinguished from homozygous mutant tissue (white). (A and B) Adult eyes of ey-FLP; FRT42D/FRT42D P[mini-white] (wild type) and ey-FLP; FRT42D dDP^a3/FRT42D P[mini-white] (DP). Arrow indicates the location of the dDP clone in B. (C and D) dDP mutant tissue in the cell lethal background. Adult eyes of ey-FLP; FRT42D /FRT42D l(2)cl-R11¹ P[mini-white] (wild-type CL) and ey-FLP; FRT42D dDP^a3/FRT42D l(2)cl-R11¹ P[mini-white] (DP CL) are shown. (E) No de2f1 mutant tissue can be seen on the cell lethal background in ey-FLP; FRT42D de2f1⁷²⁹ /FRT42D l(3)cl-R3¹ P[mini-white]. Thus, de2f1 mutant tissue is not able to contribute to the size of the fly eye and the wild-type tissue is not able to compensate for this lack of tissue, due to the cell lethal background, which is why this eye appears smaller than the eyes in other panels. (F–J) Mutation of suppressors l(3)mbt^3d33 (F), Su(E1)-A^7d13 (G), CG31133^7d21 (H), gfzf^6a27 (I), and Doa^6d2 (J) is able to rescue proliferation of de2f1 mutant cells as evidenced by the presence of double mutant tissue (white). (K–Q) Wing imaginal discs were dissected from wandering third-instar larvae. Clones of wild-type cells, identified by the presence of GFP (green), and homozygous mutant cells, identified by the absence of GFP (green), were induced by Ubx-FLP. DAPI is shown in blue. (K) Control experiment in the wing disc showing clones of cells homozygous for wild-type chromosomes. Without any mutations there is approximately an equal distribution of GFP (green)-positive tissue and tissue lacking GFP (green). (L) Almost no de2f1⁷²⁹ mutant tissue, identified by the lack of GFP (green), can be seen. Arrows point to representative clones of de2f1 mutant cells. Note the small size of the clones. Folds in the tissue do not have a GFP signal and are identified by asterisks (*). (M–Q) Mutation of suppressors l(3)mbt^3d33 (F), Su(E1)-A^7d13 (G), CG31133^7d21 (H), gfzf^6a27 (I), and Doa^6d2 (J) is able to rescue proliferation of de2f1 mutant cells as evidenced by the presence of more double mutant tissue than tissue generated by de2f1 single mutant cells (L). Double mutant tissue is identified by the lack of GFP (green). (K′–Q′) The same corresponding images as K–Q showing only GFP (green).

The SMW in clones of de2f1 Su(E1) double mutant cells. Eye imaginal discs were dissected from third-instar larvae. Clones of wild-type cells, identified by the presence of GFP (green), and homozygous mutant cells, identified by the absence of GFP (green) were induced by ey-FLP. Genotypes are shown, the morphogenetic furrow (MF) is marked by an arrowhead, and posterior is to the right. (A–J) Eye imaginal discs were labeled with BrdU (red) to visualize cells in S phase. (A) Wild-type disc showing only BrdU incorporation. (B and C) Suppressors l(3)mbt^3d33 (B) and Su(E1)-A^7d13 (C) strongly rescue the SMW in de2f1 mutant cells as evidenced by the entry into and exit from the SMW in double mutant cells at approximately the same time as adjacent wild-type cells. (D–F) In contrast, suppressors CG31133^7d21 (D), gfzf^6a27 (E), and Doa^6d2 (F) only weakly rescue the SMW in de2f1 mutant cells as evidenced by BrdU incorporation beginning and ending several columns more posterior in double mutant cells than in adjacent wild-type cells. (G–J) BrdU incorporation in clones of single mutant suppressors Su(E1)-A^7d13 (G), CG31133^7d21 (H), and gfzf^6a27 (I) shows no SMW defects as evidenced by the entry into and exit from the SMW in single mutant cells at approximately the same time as adjacent wild-type cells. Doa^KG09056 single mutant clones show a slightly delayed SMW entry (J), but the delay is less extreme than the delay in de2f1⁷²⁹ Doa^6d2 double mutant cells (F). (B′–J′) The same corresponding images as B–J showing only BrdU (red). Clones of mutant cells are outlined.

Neuronal marker ELAV in clones of de2f1 Su(E1) double mutant cells. Eye imaginal discs were dissected from third-instar larvae. Clones of wild-type cells, identified by the presence of GFP (green), and homozygous mutant cells, identified by the absence of GFP (green), were induced by ey-FLP. Genotypes are shown, and posterior is to the right. Cells were stained with the neuronal marker ELAV (red). (A–D) The onset of neuro-nal differentiation in de2f1⁷²⁹ l(3)mbt^3d33 (A), de2f1⁷²⁹ Su(E1)-A^7d13 (B), de2f1⁷²⁹ CG31133^7d21 (C), and de2f1⁷²⁹ gfzf^6a27 (D) double mutant cells and Su(E1)-A^7d13 (F), CG31133^7d21 (G), and gfzf^6a27 (H) single mutant cells initiates at the same time as in the adjacent wild-type cells. (E and I) There is a slight delay in the onset of neuronal differentiation in de2f1⁷²⁹ Doa^6d2 (E) double mutant cells and in Doa^KG09056 (I) single mutant cells. (A′–I′) The same corresponding images as A–I showing only ELAV (red). Clones of mutant cells are outlined.