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Drosophila myb exerts opposing effects on S phase, promoting proliferation and suppressing endoreduplication

Department of Molecular Genetics, University of Illinois at Chicago, College of Medicine, Chicago, IL 60607-7170, USA

View larger version (18K): [in a new window]   Fig. 1. Topographies of vertebrate and Drosophila Myb proteins. Schematic representations of the mouse and chicken c-Myb proteins and v-Myb protein from avian myeloblastosis virus (AMV; derived from the chicken gene) (Oh and Reddy, 1999). Abbreviations: R1, R2 and R3, three imperfect tandem repeats that comprise the DNA binding domain (region I); TA, transcriptional activator domain; LZ, leucine zipper; NR, negative regulatory domain. Also depicted is an additional region encoded by an alternatively spliced exon that contains the majority of conserved region II. The DNA-binding domain is also indicated. Regions of the c-Myb protein that are highly conserved between mouse and chicken are shown in the chicken protein in black. The four regions of conservation shared between vertebrate and Drosophila Myb proteins are indicated by Roman numerals (hatched in the DMyb proteins). The v-Myb protein has suffered both N- and C-terminal truncations, but the latter has been shown to be sufficient to activate transcriptional activation and transformation potentials of the Myb protein. Below are schematic representations of the two DMyb proteins produced by the fragments cloned into pUAST: DMyb, the full-length protein; and DMyb, the C-terminally truncated protein which is expected to be hyperactive by analogy to the v-Myb protein.

View larger version (112K): [in a new window]   Fig. 2. Ectopic expression of DMyb causes malformation in the larval wing disc. Imaginal wing discs dissected from wandering third instar larvae, posterior to the right in all figures. (A-C) Fluorescent micrographs showing the expression patterns of the en-Gal4 and sd-Gal4 drivers: (A) en-Gal4/UAS-GFP, (B) en-Gal4/UAS-DMyb and (C) sd-Gal4/UAS-GFP. Red, DAPI-staining to visualize nuclei; green, either (A,C) GFP fluorescence or (B) staining with an antibody raised against the DNA-binding domain of DMyb (Jackson et al., 2001). Note the difference in the shape of the disc when DMyb is expressed in the posterior compartment. (D-F) Micrographs using differential interference contrast (DIC) optics show a comparison between the appearance of (D) a control en-Gal4/+ wing disc and discs in which DMyb has been ectopically expressed; (E) en-Gal4/UAS-DMyb; and (F) sd-Gal4/UAS-DMyb. Scale bars: in A, 0.05 mm for A-C; in D, 0.05 mm in D-F.

View larger version (77K): [in a new window]   Fig. 3. Ectopic expression of DMyb promotes increased S phase in imaginal discs. Dissected wing (A,B), haltere (C,D) and leg (E,F) imaginal discs from wandering third instar larvae were labeled for DNA synthesis by BrdU incorporation (white dots). S-phase cells were similarly distributed in the anterior and posterior compartments of a control en-Gal4/+ disc (A,C,E). By contrast, higher levels of BrdU incorporation were observed in the posterior compartment of each disc when en-Gal4 was used to drive expression of either UAS-DMyb (B,F) or UAS-DMyb (D) in posterior compartments. White lines indicate compartment boundaries, posterior towards the right. Scale bars: in A 0.05 mm for A,B; in C, 0.05 mm for C-F shown at same magnifications.

View larger version (172K): [in a new window]   Fig. 4. Ectopic expression of DMyb promotes S phase in the ZNC of larval wing discs. Views of the dorsoventral boundaries of wing discs that were double-stained with DAPI to visualize nuclei (left panels) and for DNA synthesis by BrdU incorporation (right panels). In wild-type control discs, en-Gal4/+, BrdU incorporation was not detected in the zone of non-proliferating cells (ZNC), which is composed of cells at the dorsoventral boundary (A). However, when en-Gal4 was used to drive expression of either UAS-DMyb (B) or UAS-DMyb (C) in the posterior compartment, BrdU incorporation could be detected in the posterior ZNC (indicated by arrows); and when sd-Gal4 was used to drive expression of UAS-DMyb throughout the wing pouch, no ZNC could be detected (D). Scale bar: 0.05 mm.

View larger version (65K): [in a new window]   Fig. 5. Ectopic expression of DMyb promotes mitosis in imaginal discs. Mitotic cells identified with the PH3 antibody were similarly distributed in the anterior and posterior compartments of a control sd-Gal4/+ disc (A), but were more frequent in the posterior compartment when en-Gal4 was used to drive expression of UAS-DMyb (B). White line indicates the compartment boundary, posterior towards the right. (C,D) Higher magnification views of the anterior ZNC in wing discs triply stained with DAPI to visualize nuclei (blue), PH3 antibody to identify mitotic cells (green) and Cyclin B antibody (red). In the control sd-Gal4/+ disc, no mitotic cells were detected in the ventral and dorsal domains of the anterior ZNC, where Cyc B accumulates to high levels (C). By contrast, mitotic cells could be detected in these domains when sd-Gal4 was used to drive expression of UAS-DMyb in the wing pouch (D). Scale bars: in A, 0.05 mm for A,B; in D, 0.05 mm in C,D.

View larger version (133K): [in a new window]   Fig. 6. Ectopic expression of DMyb induces small increases in apoptosis in wing discs. A low level of apoptosis was detected with in a control wing disc (A). Small increases were observed when en-Gal4 was used to drive ectopic expression of either DMyb (not shown) or DMyb (B) in the posterior compartment, and when sd-Gal4 was used to drive ectopic expression of DMyb (C). Higher levels of apoptosis were observed when sd-Gal4 was used to drive DMyb expression (D). Results were similar with Acridine Orange and TUNEL staining. The former is shown in A-C and the latter in D. Scale bar: 0.05 mm.

View larger version (110K): [in a new window]   Fig. 7. Ectopic DMyb activity inhibits endoreduplication and growth in salivary glands. (A,B) Fluorescent micrographs of salivary glands dissected from (A) fkh-Gal4/UAS-GFP and (B) sd-Gal4/UAS-GFP third instar larvae show that both Gal4 lines drive expression in endocycling salivary gland cells (sg), but not in fat body (fb) or imaginal ring cells (ir), which are shown in an inset at higher magnification. (C) Graphical representation for each indicated genotype of the average ratio of the DNA signal from ‘expressing’ salivary gland nuclei to non-expressing fat body nuclei [using the method of Weiss et al. (Weiss et al., 1998)]. Standard deviations are shown. (D-H) DNA staining of salivary glands and representative nuclei from larvae that were approximately 120 hours AED. Genotypes were: (D) sd-Gal4/+ control; (E) sd-Gal4/UAS-DMyb; (F) sd-Gal4/UAS-DMyb; (G) sd-Gal4/UAS-DMyb; HS-dE2F, HS-dDP/+, which had been subjected to a 30 minute heat-shock treatment every 24 hours after collection; and (H) sd-Gal4/UAS-RBF. Panels from left to right show the relative sizes of complete glands and (at a higher magnification), relative sizes of imaginal ring nuclei (ir), fat body nuclei (fb) and salivary gland nuclei (sg) that represent the mean for each genotype. The DNA signal ratio of that nucleus to the average fat body nucleus indicated. (I,J) DNA staining of salivary glands from (I) sd-Gal4/UAS-DMyb and (J) sd-Gal4/UAS-DMyb; HS-dE2F, HS-dDP/+ larvae, which had been subjected to 30 minute heat-shock treatments every 12 hours after collection, and which were 144 hours AED. Arrowhead in J indicates enlarged fat body nuclei resulting from excess endoreduplication in these cells driven by HS-dE2F/DP. Scale bars: in A, 0.1 mm for A,B; in A (inset), 0.025 mm for A,B; in D, 0.1 mm (low magnification) for D-J; in D, 0.025 mm (high magnification) for D-H.

View larger version (59K): [in a new window]   Fig. 8. BrdU incorporation is strongly inhibited in salivary glands when DMyb, but not DMyb, is ectopically expressed. Salivary glands dissected from larvae between 72 and 96 hours AED were double-stained with DAPI to visualize nuclei (left panels) and for DNA synthesis by BrdU incorporation (right panels). Imaginal ring cells (ir) are shown at higher magnification in inset panels. BrdU incorporation was detected in both imaginal ring and salivary gland (sg) nuclei in (A) control sd-Gal4/+ glands and in (B) sd-Gal4/UAS-DMyb glands. However, BrdU incorporation was only detected in imaginal ring nuclei and not in salivary gland nuclei in (C) sd-Gal4/UAS-DMyb and (D) fkh-Gal4/UAS-DMyb glands. (E) Heat-shock induction of E2F/DP expression in sd-Gal4/UAS-DMyb; HS-E2F, HS-DP/+ glands was able to override the inhibition of DNA synthesis in salivary gland nuclei, but also induced increased levels of BrdU incorporation in fat body (fb) nuclei, leading to excess endoreduplication in these cells. Examples of these fat body nuclei are indicated with arrowheads. Scale bars: in A (left), 0.05 mm for A-E; in A (inset), 0.025 mm for A-E.

View larger version (13K): [in a new window]   Fig. 9. The roles played by three transcription factors involved in cell cycle regulation in Drosophila melanogaster. The E2F/DP and DREF transcription factors have been shown to promote DNA replication (progression from G1 into S) in mitotic and endocycling cells. Data from previous (Fung et al., 2002; Katzen et al., 1998) and current investigations of the function of Dm myb have demonstrated that although in mitotic cells, DMyb shares the function of being a positive regulator of progression from G1 into S with E2F/DP and DREF, DMyb also acts as a negative regulator of endoreduplication and is able to promote progression from G2 into M. We propose that the ability of DMyb to inhibit endoreduplication is an important aspect of maintaining genomic integrity in proliferating cells. We have also shown that while the abilities of full-length and truncated (activated) DMyb proteins to promote cell cycle progression in proliferating cells were similar, the truncated protein was considerably more potent at inhibiting S phase in endoreduplicating cells. As indicated in the figure, we hypothesize that one or more factors, to which we refer as DMyb activating factors (DMyb AF), must interact with full-length DMyb to activate its potential as a transcriptional regulator, and that these factors are present in proliferating cells, but absent in endocycling cells.