Figure 1.
The absence of emc delays cell cycle progression in the G2 phase.
(A, B) Third instar wing imaginal discs containing GFP-labelled (green) control (A) and emc1 (B) mitotic recombinant clones. Phalloidin (Phal) staining is shown in red. The emc1 mutant clones were always smaller than control clones (compare B to A). (C, D) Phenotype of ap-Gal4 UAS-GFP/+ (C) and ap-Gal4 UAS-GFP/+; UAS-emcRNAi/+ (D) adult wings. ap-Gal4 UAS-GFP/+; UAS-emcRNAi/+ wings were smaller than the control wings, with vein fusions and extra bristles in the dorsal compartment (compare D to C). (E, F) FACS analysis of third instar wing imaginal discs from ap-Gal4 UAS-GFP/+ (E) and ap-Gal4 UAS-GFP/+; UAS-emcRNAi/+ (F) genotypes. Note the accumulation of cells in the G2 phase among the GFP + cells in the emcRNAi expressing discs (F, n = 4 independent experiments, p-value<0.05).
Figure 2.
Overexpression of da mimics the phenotype induced by the absence of emc.
(A, B) Third instar wing imaginal discs with Flip-out control (A) and UAS-da (B) clones labelled with GFP (green) and stained for Wingless (Wg, in red). Clones of da-expressing cells were smaller than the control clones (compare B to A). (C, D) Third instar salEPv-Gal4 UAS-GFP/+ (C) and salEPv-Gal4 UAS-GFP/UAS-da (D) wing imaginal discs stained for the mitotic marker phospho-histone-3 (PH3). This marker was strongly diminished in the salEPv expression domain (in green) of da-expressing discs. Accordingly, this region was smaller in mutant versus control discs (compare D with C). (E, F) FACS analysis of third instar wing imaginal discs of the salEPv-Gal4 UAS-GFP/+ (E) and salEPv-Gal4 UAS-GFP/UAS-da (F) genotypes. Note the accumulation of GFP + cells from the UAS-da expressing discs in the G2 phase (F, n = 3 independent experiments, p-value<0.05).
Figure 3.
Da regulates the expression of string.
(A, B) Expression of Cyclin B (CycB) in salEPv-Gal4 UAS-GFP/+ (A, A′) and salEPv-Gal4 UAS-GFP/UAS-da (B, B′) third instar imaginal wing discs revealed with anti-CycB (red in A and B and grey in A′–B′). The expression of CycB in salEPv-Gal4 UAS-GFP/UAS-da discs was comparable to that of the control discs (compare B′ to A′). (C, D) In situ hybridization to string mRNA in salEPv-Gal4 UAS-GFP/+ control (C) and salEPv-Gal4 UAS-GFP/UAS-da (D) third instar wing imaginal discs. The salEPv-Gal4 presumptive area is marked with a white dotted line. Note that the stg expression was strongly reduced when da was overexpressed (D). (E) Quantitative Real-Time PCR of cDNA from imaginal wing discs of genotypes WT, ap-Gal4/+; UAS-emcRNAi and en-Gal4/UAS-da; tubG80ts/+. emc reduction and da overexpression caused a significant reduction in stg mRNA when da was overexpressed (n = 3 independent experiments, **p<0.01 vs WT).
Figure 4.
The ectopic expression of string is sufficient to restore the mitotic defects induced by the elimination of emc or the ectopic expression of da.
(A–D) Adult wings of salEPv-Gal4 UAS-GFP/+ (A), salEPv-Gal4 UAS-GFP/UAS-stg (B), salEPv-Gal4 UAS-GFP UAS-da/UAS-GFP (C), and salEPv-Gal4 UAS-GFP UAS-da/UAS-stg (D). Note that the defects in wing size observed following da overexpression were reverted by stg overexpression (compare C to D). (E, F) Phospho-histone-3 (PH3) staining (in red) in salEPv-Gal4 UAS-GFP UAS-da/UAS-GFP (E) and salEPv-Gal4 UAS-GFP UAS-da/UAS-stg (F) third instar wing imaginal discs. (E′, F′) CycB expression was reduced when stg was overexpressed (alone or in combination with UAS-da), due to the rapid G2/M transition of these cells. (G, H, I) Third instar wing imaginal discs containing control (G), emc1 (H) and UAS-stg; emc1 (I) clones were, positively labelled by GFP (in green) and stained with Phalloidin (Phal, red). The smaller size of the emc1 mutant clones was reverted by overexpressing stg (compare H to I). (J) Quantitative analysis of the size of control, emc1 and UAS-stg; emc1 clones. Note that the emc1 mutant clones were always smaller than 20 cells, whereas UAS-stg; emc1 clones were of a similar size to the control clones (n = 40 clones per group).
Figure 5.
Da can bind string regulatory region.
(A) Scheme representing string regulatory region. The LacZ lines generated by B. Edgar lab stg-ß, stg_ ß-E3.2 and stg_ ß-E2.2 are indicated in grey. The results obtained by the modENCODE ChIP project for Daughterless transcription factor are indicated in blue. Every blue line indicates a fragment of DNA that was immunoprecipitated with an anti-Da antibody. The orange lines marked as “ChIP-qPCR” represent the fragments studied by us in our ChIP experiment. The Gal4 lines generated by the Janelia Farm covering the stg regulatory region are shown in black. We highlighted the lines with expression in the imaginal wing disc (marked by GFP expression, in green in the discs), which are the GMR_32B06, GMR_32C11 line and the GMR_32F08 line. The stg-107–112-LacZ line, generated in F. Casares lab was also included, and a disc stained with anti- ß-Gal (in red) was shown. Note the similarity in the expression of this line with the GMR_32C11 line. (B) Detail of a fragment of the stg regulatory region contained by the Janelia GMR_32C11 and GMR_32F08 lines and the stg-107–112-LacZ line. All Da putative binding sites present in this region are indicated with a red arrowhead. We also show the fragment found in the modENCODE project as a target for Da binding (blue line) and the fragment found by us (orange line). (C) Graphical representation of our ChIP-qPCR experiment. Each bar represents the relative DNA quantity immunoprecipitated with the anti-Da antibody. The gene CG12255 was studied as negative control (see Matherials & Methods). achaete promoter was used as an internal positive control for the experiment. The string fragments indicated in (A) and (B) with an orange line were also represented. Note that Da binding to the stg promoter was very similar to that which was observed for the achaete promoter.
Figure 6.
The over-expression of da down-regulates the expression of different stg-reporters.
(A, B, D, E, G and H) Third instar larval wing discs showing the expression of UAS-mCD8-GFP (in grey) driven by the Janelia Gal4 lines GMR_32C11 (A), GMR_32F08 (D) and GMR_32B06 (G) in control discs or in discs that over-express UAS-da, GMR_32C11-Gal4 UAS-mCD8-GFP/UAS-da (B), GMR_32F06-Gal4 UAS-mCD8-GFP/UAS-da (E) and GMR_32B06-Gal4 UAS-mCD8-GFP/UAS-da (H). (C, F, and I) Bar charts show the average levels of mCD8-GFP expression in control (UAS-GFP) and discs over-expresing UAS-da (UAS-GFP UAS-da) driven by GMR_32C11, GMR_32F08 and GMR_32B06 lines. For each experiments at least 9 wing discs were quantified. (B) The over-expression of UAS-da under the control of GMR_32C11 Gal4 caused the down-regulation of the levels of UAS-GFP expressed in the wing pouch. The band of cells expressing this reporter along the A/P boundary was also reduced (compared B to A, *** p-value = 0.001). (E) The over-expression of UAS-da under the regulation of GMR_32F08 Gal4 line reduced the levels of expression of GFP throughout the wing disc, compared with the control in D (*** p-value<0.001). (H) The expression of UAS-GFP reporter was also reduced in the proximal region of the wings discs when UAS-da was over-expressed with the GMR_32B06 line (compared H to control G, * p-value<0.05). (J-K) Third instar larval wing discs stained with anti-ß-Gal to reveal the pattern of expression of the stg-107–112-LacZ reporter (in red in J, K and in grey in J′ K′). In stg-107–112-LacZ/UAS-da; ap-Gal4 wing discs (K–K′) the expression of this reporter was strongly reduced throughout the dorsal compartment (marked by the expression of UAS-GFP in green), compared to control discs J–J′. Note the strong reduction of the expression of this reporter in the dorsal band of cells along the A/P boundary.
Figure 7.
The ectopic expression of da alters the parameters of cell proliferation in the absence of the E(spl)C genes.
(A) Expression of Da (red in A and grey in A′) in en-Gal4 UAS-GFP/UAS-sc third instar imaginal wing discs. The expression of Da was upregulated by the overexpression of sc (compare posterior GFP+ cells to anterior cells). (B–D) Third instar wing imaginal discs containing GFP+ (green) control (B), UAS-da (C) and UAS-da; E(spl)b32.2 (D) mitotic recombinant clones. The discs were also stained with anti-Da (red) (B) and Phalloidin (Phal, red: C, D). UAS da and UAS da; E(spl)C clones were always smaller than control clones (compare C and D to B). (E) Quantification of the size of control, E(spl)b32.2, UAS-da and UAS- da; E(spl)b32.2 clones (n = 37, 29, 91 and 63 analysed clones, respectively). Note that most control and E(spl)C clones contained more than 100 cells (40%–50% of clones), whereas only around 6% of the UAS-da and UAS-da; E(spl)b32.2 reached this size.
Figure 8.
Da directly binds to the string promoter region and acts as a transcriptional repressor.
(A) Scheme of the 0.7 Kb region of the stg promoter. The two E-boxes with the canonical Da binding sites CAG/CCTG contained in this region are written in red. The highlighted red region of 21 nucleotides corresponds to the probe used to perform the band shift experiments. (B) Band shift assay showing the physical binding of a Da-GST fusion protein to the stg promoter region. The DNA binding properties of Da were tested using cyc5 stg-prom (red), cyc3 stg-prom (green) and cyc5 stg-prom* (red) probes. The putative Da binding sites were mutated in the latter probe. Da bound to the probe containing the E-box CAGCTG but not to the mutated probe. This binding was specific, as oligonucleotides with a mutated binding site did not compete for binding. The gel was cut to show only the specific band formed by DNA-protein complexes. (C) Luciferase reporter assay of S2 cells. The 0.7 Kb region of the stg promoter shown in A was cloned into a pGL2 vector containing a minimal promoter (HS43), a construct that induced the activation of luciferase. The presence of the stg regulatory region (pGL2 stg) increased the basal activity of the pGL2 vector, which was strongly repressed by overexpression of Da (***p<0.001 vs pGL2 stg). When Da binding sites were ablated from the promoter (pGL2 stg*), no activation of the luciferase activity was observed.