Figure 1.
Frequency and duration of mitosis in cultivated wing imaginal discs.
(A) Numbers of mitotic cells were determined in wing imaginal discs expressing Lac-YFP. The central pouch region delimited by the nearest circumferential fold (dashed line in left panel) was analyzed. Mitotic cells (asterisks) were identified based on their characteristic rounded shape as illustrated by the region (white square in left panel) shown at higher magnification (right panel). Scale bars correspond to 50 µm (left) and 5 µm (right), respectively. (B) The average numbers of mitotic cells in discs fixed immediately after dissection at either 100, 104, or 118 hours AED (in vivo) were compared to the average numbers observed in discs cultured after dissection at 100 hours AED for the indicated time (in vitro). Numbers of mitotic cells were normalized to the reference region (average size of the central pouch at 100 hours AED). n≥8 discs per time point; s.d. indicated. t = 0 in the graph corresponds to 100 hours AED. (C, D) Time lapse imaging as illustrated (C; scale bar = 5 µm) was used to determine the duration of mitosis in discs during the first two hours in culture (0–2 hours) as well as during later times. Bars indicate average duration with s.d., *** p<0.001 (t test).
Figure 2.
Progression through S phase in cultured wing imaginal discs.
(A) Wing imaginal discs dissected 100 hours AED were pulse labeled with EdU (30 minutes) either immediately (0 h) or after 7 hours of cultivation (7 h). After fixation and EdU visualization, discs were double labeled with Hoechst (DNA). EdU signals in wing pouch regions after identical acquisition and processing are shown in the second row. Central regions (white frames in the second row) are shown at higher magnification after contrast maximization in the third row. Scale bar = 40 µm. (B) EdU signal intensities integrated over the pouch region were quantified. Bars indicate average integrated intensity and whiskers s.d.; n≥14 discs; *** p<0.001 (t test).
Figure 3.
A novel RGB cell cycle tracker reveals inhibition of cell cycle progression in cultured discs.
(A) The transcript expressed from the RGB cell cycle tracker transgene generates three separate proteins because of the intervening T2A cis-acting hydrolase elements. The three proteins are fused to different fluorescent proteins, EBFP2 (blue), tdTomato (red) and EGFP (green). Their intracellular localization is dictated by nuclear localization signals (nls). Moreover, the PCNA part enforces localization in a subnuclear pattern during S phase. Degradation signals result in proteolysis during S phase (Cdt11–101) or late M and G2 (CycB1–96 and CycB1–285), respectively. (B) Time lapse in vivo imaging of peripodial cells in cultured en-GAL4 UAS-RGB wing imaginal discs. In the left series of still frames, a cell progressing from G1 through S into G2 is displayed. In the right series of still frames, a cell progressing from G2 through M into G1 is shown. Time is given in hours∶minutes. Scale bars = 5 µm. (C) The effect of culture on cell cycle progression was analyzed with nub-GAL4 UAS-RGB wing imaginal discs. Discs fixed immediately after dissection at 100, 107 or 118 hours AED are labeled "in vivo", while those dissected at 100 hours AED followed by cultivation for 7 or 18 hours before fixation are labeled "in vitro". Representative images of the central pouch region at either an apical focal plane containing mitotic cells (left side) or a basal focal plane with interphase cells in the disc proper (right side) are shown. The dashed line indicates the future wing margin. Scale bar = 10 µm.
Figure 4.
Effects of E2f1/Dp overexpression on cell cycle progression during wing imaginal disc culture.
Imaginal discs were dissected from larvae with single copies of the transgenes en-GAL4, UAS-E2f1, UAS-Dp, and tub-GAL80ts. These larvae were either grown constantly at 23°C (A, B; - E2f1/Dp overexpression) or at 29°C for the final 18 hours before disc dissection (C, D; + E2f1/Dp overexpression). Dissected wing imaginal discs were labeled with EdU (EdU), anti-PH3 (PH3) and a DNA stain (DNA) either immediately (0 h) or after 7 hours of cultivation at 25°C (7 h). Complete imaginal discs (A, C; scale bar = 50 µm) and high magnification views of the central pouch region (B, D; scale bar = 20 µm) are shown with dashed lines indicating the boundary between anterior and posterior compartment, in which en-GAL4 driven UAS transgene expression occurs at 29°C.
Figure 5.
Effects of culture condition on induction of UAS-EGFP expression in wing imaginal discs.
(A) After development at 23°C, an aliquot of en-GAL4 UAS-EGFP tub-GAL80ts larvae were shifted to 29°C for the indicated times before dissection and microscopic analysis of wing imaginal discs (in vivo). Another aliquot of these larvae was used for dissection of wing imaginal discs that were either immediately shifted to 29°C (in vitro early) or after 12 hours of cultivation at 23°C (in vitro late) before fixation at the indicated times. (B) Wing pouch region of imaginal discs from en-GAL4 UAS-EGFP tub-GAL80ts larvae after induction of UAS-EGFP expression by shifts from 23°C to 29°C as described in (A). Insets display higher contrast to reveal weak signals. Scale bar = 50 µm. (C) Quantification of EGFP signals in the posterior compartment of the pouch region of imaginal discs from en-GAL4 UAS-EGFP tub-GAL80ts larvae after induction of UAS-EGFP expression by shifts from 23°C to 29°C as described in (A) and illustrated in (B).
Figure 6.
Cell proliferation in wing imaginal discs after transplantation in female abdomina.
Wing imaginal discs were isolated from early L3 larvae, but instead of in vitro culture they were immediately transplanted into the abdomen of adult female host flies. Transplanted discs were re-isolated at the indicated times of cultivation in the host and cell counts were determined; cell counts were also determined just before transplantation. Black lines and diamonds (average cell number +/− s.d.; n≥11 per time point) illustrate cell proliferation after transplantation. For comparison cell counts were also determined immediately after isolation of discs from larvae aged for different times (73, 86.5, 98, 110, 120, 125 hours AED). Grey lines and squares (average cell number, n≥8 per time point, s.d. completely covered by the squares) illustrate cell proliferation in situ. The difference in cell numbers at the start of the curves presumably reflects limitations in staging precision.
Figure 7.
Effects of Ras85DV12 expression on cell cycle progression during wing imaginal disc cultivation.
(A) en-GAL4 tub-GAL80ts UAS-Ras85DV12 larvae were used for spatially and temporally controlled hyperactivation of the Ras signaling pathway as illustrated schematically. (B, C) Wing imaginal discs from larvae without (B; -Ras85DV12) or with (C; +Ras85DV12) preceding UAS-Ras85DV12 expression in the posterior compartment were isolated and fixed either immediately (0 h) or after seven hours of cultivation in vitro (7 h) before EdU labelling and staining with anti-PH3 and a DNA dye. Scale bar = 50 µm.
Figure 8.
Comparative analysis of different wing imaginal disc culture conditions.
(A, B) Effects of medium supplementation with candidate growth promoting factors. Wing imaginal discs were isolated (100 hours AED) and cultured in Mcl8 (A) or WM1 (B) culture media with the indicated supplements: none (Mcl8 only), 25% larval hemolymph (+ hemolymph; n = 12), 4% BME (+ BME), 35 µM ( = 0.2 mg/ml) bovine insulin (+ Insulin), none (WM1 only), 35 µM bovine insulin (+ Insulin), 0.5 µM Dilp2 (+ Dilp2). After cultivation for 7 hours or in some cases for 14 hours as indicated (14 h), discs were fixed and stained with anti-PH3 for determination of the number of mitotic cells. Bars indicate average number of mitotic cells +/− s.d. (C) To confirm the activity of chemically synthesized Dilp2, Kc cells were treated for 30 minutes as indicated before analysis of total cell extracts by immunoblotting with an antibody against active Akt1 (phospho-Akt1) and with anti-Tubulin (α-Tub) to control loading.(D) Effect of co-cultivation of larval tissues. Wing imaginal discs isolated from larvae 100 hours AED were cultured for 7 hours in Mcl8 without (control) or with additional larval tissues from 10 larvae (fat body, brain, as indicated). The number of mitotic cells was determined after anti-PH3 staining. Bars indicate average +/− s.d.; the number of analyzed discs (n) is indicated. (E, F) Effect of partial dissection of wing imaginal discs. (E) To reduce mechanical stress during isolation of wing imaginal discs and retain larval tissues that might produce endocrine signals, partial disc dissection was performed as schematically illustrated. With a first cut (1., solid line) the very anterior most region of larvae was removed resulting in extrusion of wing imaginal discs, while the larvae was fixed with a forceps (dashed line). With a second cut (2., solid line) the posterior two thirds of larvae were removed. Bright field image of a pair of partially dissected disc is displayed on the right. Scale bar = 100 µm. (F) Cultures with wing imaginal discs from larvae 100 hours AED were set up in different ways (culture type I-V). In type I, completely dissected discs were cultured for control. In type II, partially dissected discs were analyzed. In type III, completely dissected discs were analyzed after co-culture with partially dissected discs. In type IV, partially dissected discs were separated from the other larval parts before culture. In type V, discs with intact stalks and connections to the tracheal trunk were cultured without any additional larval tissues. The number of mitotic figures present after 7 hours of cultivation in Mcl8 was determined after fixation and anti-PH3 staining. Bars indicate average number of mitotic cells +/− s.d.; the number of discs (n) is indicated. (G) Effect of partial dissection in combination with high insulin. Wing imaginal discs isolated from larvae 100 hours AED either by complete or partial dissection were cultured for 7 or 14 hours in WM1 with or without 0.2 mg/ml bovine insulin as indicated. The number of mitotic cells was determined after anti-PH3 staining. Bars indicate average +/− s.d.; the number of analyzed discs (n) is indicated.