Towards long term cultivation of Drosophila wing imaginal discs in vitro

The wing imaginal disc of Drosophila melanogaster is a prominent experimental system for research on control of cell growth, proliferation and death, as well as on pattern formation and morphogenesis during organogenesis. The precise genetic methodology applicable in this system has facilitated conceptual advances of fundamental importance for developmental biology. Experimental accessibility and versatility would gain further if long term development of wing imaginal discs could be studied also in vitro. For example, culture systems would allow live imaging with maximal temporal and spatial resolution. However, as clearly demonstrated here, standard culture methods result in a rapid cell proliferation arrest within hours of cultivation of dissected wing imaginal discs. Analysis with established markers for cells in S- and M phase, as well as with RGB cell cycle tracker, a novel reporter transgene, revealed that in vitro cultivation interferes with cell cycle progression throughout interphase and not just exclusively during G1. Moreover, quantification of EGFP expression from an inducible transgene revealed rapid adverse effects of disc culture on basic cellular functions beyond cell cycle progression. Disc transplantation experiments confirmed that these detrimental consequences do not reflect fatal damage of imaginal discs during isolation, arguing clearly for a medium insufficiency. Alternative culture media were evaluated, including hemolymph, which surrounds imaginal discs during growth in situ. But isolated larval hemolymph was found to be even less adequate than current culture media, presumably as a result of conversion processes during hemolymph isolation or disc culture. The significance of prominent growth-regulating pathways during disc culture was analyzed, as well as effects of insulin and disc co-culture with larval tissues as potential sources of endocrine factors. Based on our analyses, we developed a culture protocol that prolongs cell proliferation in cultured discs

A genetically encoded fluorescent probe for imaging of oxygenation gradients in living

Oxygen concentrations vary between tissues of multicellular organisms and change under certain physiological or pathological conditions. Multiple methods have been developed for measuring oxygenation of biological samples and However, most require complex equipment, are laborious and have significant limitations. Here we report that oxygen concentration determines the choice between two maturation pathways of DsRed FT (Timer). At high oxygen levels, this DsRed derivate matures predominantly into a red fluorescent isoform. By contrast, a green fluorescent isoform is favored by low oxygen levels. Ratiometric analysis of green and red fluorescence after a pulse of Timer expression in larvae provides a record of the history of tissue oxygenation during a subsequent chase period, for the whole animal with single-cell precision. Tissue spreads revealed fine differences in oxygen exposure among different cells of the same organ. We expect that the simplicity and robustness of our approach will greatly impact hypoxia research, especially in small animal models

Towards Long Term Cultivation of <i>Drosophila</i> Wing Imaginal Discs <i>In Vitro</i>

<div><p>The wing imaginal disc of <i>Drosophila melanogaster</i> is a prominent experimental system for research on control of cell growth, proliferation and death, as well as on pattern formation and morphogenesis during organogenesis. The precise genetic methodology applicable in this system has facilitated conceptual advances of fundamental importance for developmental biology. Experimental accessibility and versatility would gain further if long term development of wing imaginal discs could be studied also in vitro. For example, culture systems would allow live imaging with maximal temporal and spatial resolution. However, as clearly demonstrated here, standard culture methods result in a rapid cell proliferation arrest within hours of cultivation of dissected wing imaginal discs. Analysis with established markers for cells in S- and M phase, as well as with <i>RGB</i> cell cycle tracker, a novel reporter transgene, revealed that in vitro cultivation interferes with cell cycle progression throughout interphase and not just exclusively during G1. Moreover, quantification of EGFP expression from an inducible transgene revealed rapid adverse effects of disc culture on basic cellular functions beyond cell cycle progression. Disc transplantation experiments confirmed that these detrimental consequences do not reflect fatal damage of imaginal discs during isolation, arguing clearly for a medium insufficiency. Alternative culture media were evaluated, including hemolymph, which surrounds imaginal discs during growth in situ. But isolated larval hemolymph was found to be even less adequate than current culture media, presumably as a result of conversion processes during hemolymph isolation or disc culture. The significance of prominent growth-regulating pathways during disc culture was analyzed, as well as effects of insulin and disc co-culture with larval tissues as potential sources of endocrine factors. Based on our analyses, we developed a culture protocol that prolongs cell proliferation in cultured discs.</p></div

Effects of culture condition on induction of <i>UAS-EGFP</i> expression in wing imaginal discs.

<p>(A) After development at 23°C, an aliquot of <i>en-GAL4 UAS-EGFP tub-GAL80^ts</i> 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 <i>en-GAL4 UAS-EGFP tub-GAL80^ts</i> larvae after induction of <i>UAS-EGFP</i> 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 <i>en-GAL4 UAS-EGFP tub-GAL80^ts</i> larvae after induction of <i>UAS-EGFP</i> expression by shifts from 23°C to 29°C as described in (A) and illustrated in (B).</p

Comparative analysis of different wing imaginal disc culture conditions.

<p>(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.</p

Effects of E2f1/Dp overexpression on cell cycle progression during wing imaginal disc culture.

<p>Imaginal discs were dissected from larvae with single copies of the transgenes <i>en-GAL4</i>, <i>UAS-E2f1</i>, <i>UAS-Dp</i>, and <i>tub-GAL80^ts</i>. 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 <i>en-GAL4</i> driven <i>UAS</i> transgene expression occurs at 29°C.</p

Progression through S phase in cultured wing imaginal discs.

<p>(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).</p

Cell proliferation in wing imaginal discs after transplantation in female abdomina.

<p>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.</p

Effects of Ras85D^V12 expression on cell cycle progression during wing imaginal disc cultivation.

<p>(A) <i>en-GAL4 tub-GAL80^ts UAS-Ras85D^V12</i> 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; -Ras85D^V12) or with (C; +Ras85D^V12) preceding <i>UAS-Ras85D^V12</i> 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.</p