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A characterization of the effects of Dpp signaling on cell growth and proliferation in the Drosophila wing

Fred Hutchinson Cancer Research Center, Division of Basic Sciences, 1100 Fairview Avenue North, Seattle, WA 98109, USA

View larger version (40K): [in a new window]   Fig. 1. (A) Activation of Dpp signaling using the activated receptor TkvQ253D produces large clones with smooth borders that minimize contact with neighboring wild-type cells. Left panels show digital camera images of the disc morphology by Hoechst 33258 DNA stain. Right panels show Flip-out/Gal4 clones in the same tissue marked with GFP. TkvQD clones are round (GFP), compare TkvQD clones (bottom right) with wild-type clones (upper right). TkvQD clones seem also larger than control clones. This is most obvious in areas of the tissue far from the AP boundary. For all the imaginal disc images, dorsal is upwards and anterior is leftwards. Scale bar: 100 µm. (B) TkvQ253D overexpression alters cell cycle phasing but not cell size. A parallel experiment, in which only GFP was expressed, was used as an external control (wt, top panels). Cells in the same tissue not expressing GFP (and therefore no transgene) were used as an internal control. Black traces correspond to the internal controls and gray filled traces correspond to the GFP-expressing experimental populations. Numbers in the top right corner of the size histograms show the ratio of the mean size of GFP+ cells (experimental population) versus the mean size of GFP– cells (control population) from the forward scatter data (FSC). Expression of the activated receptor TkvQD causes a decrease in the G1 population and an increase in G2 population. TkvQD does not dramatically affect cell size. TkvQD has the same effects in notum and wing regions (data not shown).

View larger version (49K): [in a new window]   Fig. 2. TkvQ253D-expressing cells proliferate and grow faster than do wild-type cells. (A) Area definition for doubling time and clone area measurement experiments. dpp expression is indicated by the black line down the center of the disc. For wing definition, the longest fold along the AP axis was used as a border. For mediolateral definition, the most lateral folds were used. Clones exterior to them were considered lateral clones, and clones interior to them were considered medial clones. (B) Cell doubling times of TkvQD-expressing cells and control cells expressing GFP alone. The caspase inhibitor p35 was co-expressed in both experiments. Clones were scored in the entire wing (total), and in medial and lateral areas. Numbers of clones counted in each area are noted inside the bars. Black bars represent the control experiment and gray bars the TkvQD samples. Numbers on top of each bar correspond to the cell doubling time. P values are noted below each category. TkvQD cell doubling time is significantly reduced and lateral cells are dividing with a 20% shorter cell cycle (2.7 hours shorter, from 13.5 to 10.8 hours doubling time). (C) Clone size of TkvQD and wild-type Flip-out/Gal4 clones shown as pixels per clone. Nomenclature and color code as in B. Numbers on top of the gray bars correspond to the increase in TkvQD clone size compared with control clones. TkvQD clones are significantly larger than control clones and lateral clones are more than 3.5 times larger. (D) S-phase cells visualized by BrdU incorporation in TkvQD Flip-out/Gal4 clones. Left panel shows clone position by GFP signal and right panel shows BrdU incorporation. Lateral clones show a strong BrdU incorporation.

View larger version (52K): [in a new window]   Fig. 3. Inhibition of Dpp signaling pathway by overexpression of Dad impairs cell proliferation and tissue growth. (A) Cell doubling times of Dad-expressing cells and control cells. Left panels correspond to confocal images showing GFP marked cells. Clones in the medial area of the tissue were recovered only when the apoptotic inhibitor p35 was co-expressed. The big cell clone in the -p35 experiment corresponds to a clone in the peripodial membrane and not to a clone in the columnar epithelium. Histogram nomenclature and color code as in Fig. 2B. Dad-expressing cells (gray bars) divide more slowly than do wild-type cells (black bars). Medial cells divided with a 22% longer cell cycle (3.4 hours longer, from 15.3 to 18.7 hours doubling time). (B) A9 Gal4 driver was used to express Dad or TkvQD throughout most of the wing disc. Cells were visualized by Hoechst 33258 DNA stain. Dad expression causes a reduction of the disc size (58% of wild-type size, n=7; Dad n=10, P<0.001), mainly along the AP axis. Conversely, TkvQD expression promotes enlargement of the disc (154% compared with wild-type size, n=7; TkvQD n=5, P<0.001). Deltavision images were acquired at the same magnification. Scale bars: 100 µm.

View larger version (59K): [in a new window]   Fig. 4. tkv mutant cells are proliferation and growth impaired. (Left panels) Confocal images of mitotic recombination clones of the indicated genotype visualized by GFP. A Minute background was used. GFP– cells correspond to the experimental populations, wild-type or tkv7 cells. Cells in the same disc expressing GFP and therefore M–/+ (wild-type experiment) or M–/+ tkv7/+ (tkv7 experiment) were used as an internal controls. tkv7 cells colonize much smaller an area than do wild-type cells and they are found mainly in the lateral areas (arrows, compare GFP– population in wt and tkv7 experiments). Some tkv7 discs show an abnormal morphology (lower panel in tkv7 experiment). Scale bar: 100µm. (Right panels) FACS analysis of the same experiment as in left panels. Gray filled traces correspond to the internal GFP+ control cells, and black traces correspond to the GFP– experimental populations. Numbers in the top right-hand corner in the size histograms show the ratio of the mean size of GFP– cells (experimental population) versus the mean size of GFP+ cells (control population) from FSC data. tkv7 cells show a severe reduction in the S phase and G2 populations, and an increase in G1 population (DNA content histogram). Cell size is not affected. tkv7 has the same effects in notum and wing regions (data not shown).

View larger version (61K): [in a new window]   Fig. 5. RBF expression blocks TkvQ253D induced proliferation. (Left panels) Confocal images of Flip-out/Gal4 lateral clones of the indicated genotype visualized by GFP signal. (Right panel) FACS analysis of a similar timing experiment. Black traces show the GFP– internal control population, and gray filled traces show the GFP+ experimental population. Numbers in the top right-hand corner in the size histograms show the ratio of the mean FSC of GFP+ cells (experimental population) versus the mean FSC of GFP– cells (control population). Clones expressing RBF and TkvQD contain as few cells as RBF-expressing clones (arrows). These cells do not show the characteristic cell cycle profile of TkvQD-expressing cells (DNA content histogram) and are bigger than cells expressing RBF alone (size histogram). Scale bar: 50 µm.

View larger version (53K): [in a new window]   Fig. 6. PI3K/Dp110-dependent and -independent effects of TkvQ253D overexpression. (Left panels) Confocal images of Flip-out/Gal4 lateral clones of the indicated genotype visualized by GFP signal. (Right panel) FACS analysis of the same experiment. Nomenclature and color code as in Fig. 5. Clones co-expressing p60 and TkvQD contain as few cells as p60-expressing clones (arrows). The same phenotype was observed when cell death was blocked by co-expressing p35 (data not shown). These cells are slightly larger than p60 cells (size histogram) but show a wild-type cell cycle profile (DNA content histogram). Scale bar: 50 µm.

View larger version (50K): [in a new window]   Fig. 7. TkvQ253D requires Vg to promote tissue growth but does not induce Vg expression. (A) Digital camera images of imaginal wing disc overexpressing GFP alone or with TkvQD in a vg mutant background. vg83b27R heterozygous animals develop normally and wing discs reach normal size (upper left panel). TkvQD induces wing disc over-growth in a vg83b27R heterozygous background, just as in a wild-type background (top right panel). The growth effect of TkvQD is blocked in a vg83b27R homozygous animal (lower right panel); the size of the wing discs is similar to the size of vg83b27R homozygous discs (lower left panel). (B) Digital camera images of Flip-out/Gal4 clones expressing GFP alone (lower left) or with TkvQD (lower right). Normal Vg expression pattern is shown in the upper left panel. TkvQD expression does not induce Vg protein upregulation, regardless of clone position (top right panel). Scale bars: 100 µm.