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Imaging Cell Competition in Drosophila Imaginal Discs
C H A P T E R
T W E N T Y
Imaging Cell Competition in Drosophila Imaginal Discs Shizue Ohsawa,* Kaoru Sugimura,† Kyoko Takino,* and Tatsushi Igaki*,‡ Contents 407 409 409 410 411 412
1. Introduction 2. Live Imaging of Cell Competition 2.1. Preparation of chambers 2.2. Imaginal disc culture 2.3. Live imaging of imaginal discs References
Abstract Cell competition is a process in which cells with higher fitness (“winners”) survive and proliferate at the expense of less fit neighbors (“losers”). It has been suggested that cell competition is involved in a variety of biological processes such as organ size control, tissue homeostasis, cancer progression, and the maintenance of stem cell population. By advent of a genetic mosaic technique, which enables to generate fluorescently marked somatic clones in Drosophila imaginal discs, recent studies have presented some aspects of molecular mechanisms underlying cell competition. Now, with a live-imaging technique using ex vivo-cultured imaginal discs, we can dissect the spatiotemporal nature of competitive cell behaviors within multicellular communities. Here, we describe procedures and tips for live imaging of cell competition in Drosophila imaginal discs.
1. Introduction Cell–cell interactions in multicellular organisms play crucial roles in coordination of cell proliferation, differentiation, and cell death during normal development and homeostasis. “Cell competition” is a form of cell–cell * Department of Cell Biology, G-COE, Kobe University Graduate School of Medicine, Kobe, Japan Institute for Integrated Cell-Material Sicences (iCeMS), Kyoto University iCeMS Complex 2, Sakyo-ku, Kyoto, Japan { PRESTO, Japan Science and Technology Agency (JST), Saitama, Japan {
Methods in Enzymology, Volume 506 ISSN 0076-6879, DOI: 10.1016/B978-0-12-391856-7.00044-5
#
2012 Elsevier Inc. All rights reserved.
407
408
Shizue Ohsawa et al.
interaction in which cells with higher fitness (“winners”) survive and proliferate at the expense of neighboring cells with lower fitness (“losers”) (Morata and Ripoll, 1975). Loser cells, but otherwise viable cells, undergo apoptosis when confronted with winner cells. Winner cells then proliferate to occupy the space, resulting in the size of the population unchanged. Thus, cell competition is a process in which fitter cells are selected among otherwise homogeneous population. It has been suggested that cell competition is involved in a variety of biological processes such as organ size control, tissue homeostasis, cancer progression, and the maintenance of stem cell population (Baker, 2011; Johnston, 2009; Morata and Martin, 2007; Moreno, 2008). Studies in Drosophila imaginal discs have indicated that the “cellular fitness” can be determined by several factors: (i) the amount of ribosomal proteins, (ii) the expression level of a proto-oncogene myc, (iii) the activity of the Hippo pathway, and (iv) the integrity of apico-basal polarity. Cells with heterozygous mutation in any one of the ribosomal protein genes (called Minute mutants) are eliminated from the tissue as losers when confronted with wild-type cells (Morata and Ripoll, 1975; Moreno et al., 2002; Simpson, 1979). Cells expressing higher level of Drosophila myc (dmyc) become winners when confronted with cells with relatively lower dmyc expression (de la Cova et al., 2004; Moreno and Basler, 2004). Similarly, cells with inactivated Hippo tumor-suppressor pathway components behave as “super-competitors” that eliminate neighboring wild-type cells (Tyler et al., 2007). In addition, epithelial cells seem to compete the integrity of the polarized structure with one another; cells with disrupted apico-basal polarity are eliminated as losers when confronted with normally polarized wild-type cells (Bilder, 2004; Hariharan and Bilder, 2006). This elimination of polarity-deficient cells by cell competition seems to work as an “intrinsic tumor suppression” (Igaki, 2009) (see below). Most cancers originate from epithelium. Loss of apico-basal polarity in epithelial cells is frequently associated with cancer progression (Bissell and Radisky, 2001; Fish and Molitoris, 1994). Similarly, mutant flies deficient for evolutionarily conserved apico-basal polarity genes such as scribble (scrib) or discs large (dlg) develop overgrown tumors in their imaginal epithelium (Bilder, 2004; Hariharan and Bilder, 2006). Intriguingly, when surrounded by wild-type cells, these polarity-deficient mutant cells do not overgrow but are eliminated from the tissue (Brumby and Richardson, 2003; Igaki et al., 2006, 2009; Pagliarini and Xu, 2003; Woods and Bryant, 1991). This suggests that normal epithelium possesses an intrinsic tumor-suppression mechanism that eliminates oncogenic polarity-deficient cells by cell competition. The polarity-deficient cells that are confronted with wild-type cells result in c-Jun N-terminal kinase (JNK)-dependent cell death (Brumby and Richardson, 2003), which is triggered by endocytic activation of Eiger (Drosophila tumor necrosis factor, TNF) (Igaki et al., 2009). Surrounding normal cells also activate Eiger–JNK signaling, which does not cause cell death but enhances the elimination of neighboring polarity-deficient cells
Imaging Cell Competition
409
by activating the PVR-ELMO–Mbc-mediated engulfment pathway (Ohsawa et al., 2011). The JNK activation in polarity-deficient cells has also been shown to be triggered by Eiger expressed in hemocytes (Cordero et al., 2010). Thus, Eiger–JNK signaling plays a central role in competition between polarized and nonpolarized epithelial cells. Interestingly, Minute clones are still eliminated by cell competition in the absence of eiger (Ohsawa et al., 2011), suggesting that competitive interactions in different cellular contexts are regulated by different mechanisms. About 30 years after the first discovery (Morata and Ripoll, 1975), progress has been made in understanding the molecular mechanism of cell competition with some technical advances especially for producing fluorescently labeled somatic clones in imaginal discs (Lee and Luo, 1999; Xu and Rubin, 1993). Recent studies have identified both positive (JNK, hid, Flower, Eiger, and the engulfment genes) and negative (Dpp, the Hippo pathway components, and Sparc) regulators of cell competition (Brumby and Richardson, 2003; de la Cova et al., 2004; Igaki et al., 2009; Li and Baker, 2007; Moreno et al., 2002; Neto-Silva et al., 2010; Ohsawa et al., 2011; Rhiner et al., 2010; Tyler et al., 2007; Ziosi et al., 2010). Yet, the underlying mechanism by which these regulators act, if exists, in a common cell competition pathway and the upstream mechanisms that regulate these factors still remain to be elucidated. Spatiotemporal analysis in live tissues could provide new insights into understanding cell competition. Here, we describe procedures and tips for live imaging of cell competition using ex vivo-cultured Drosophila imaginal discs.
2. Live Imaging of Cell Competition Live imaging, in conjunction with genetic manipulations, is a powerful tool for dissecting dynamic cellular processes within multicellular communities. Here, we overview a recently established live-imaging system for visualizing cell competition in ex vivo-cultured Drosophila imaginal discs.
2.1. Preparation of chambers The chambers for culturing imaginal discs are prepared by assembling following materials (Fig. 20.1). Glass bottom dishes for inverted microscopes are also commercially available. For upright microscopes (chamber A)
60-mm petri dish 35-mm dish bottom Glass ring as a spacer (e.g., height: 4 mm: outside diameter: 20 mm) Nontoxic glue paste SILPOT 184 W/C (DOW CORNING TORAY)
410
Shizue Ohsawa et al.
A
Objective lens Imaginal disc
B Cover glass Schneider’s medium Glass ring
Observation hole 35-mm dish bottom
60-mm dish
Imaginal disc
35-mm dish bottom
Schneider’s medium Objective lens
Figure 20.1 Chamber preparation for live imaging of imaginal discs using an upright (A) or an inverted (B) confocal laser scanning microscope.
For inverted microscopes (chamber B) 35-mm glass bottom dish (MATSUNAMI Glass No. 1S #D111300) The commercially available glass bottom dishes should be rinsed with 2 ml of 100% ethanol for 2–3 times to remove toxicity before use.
2.2. Imaginal disc culture As cell competition is a phenomenon observed in proliferating imaginal epithelium, the cultured discs must be kept with constant cell proliferation rate during the imaging. In addition, the imaginal discs must be tightly attached to the dish/glass to prevent out-of-focus imaging. The following procedure allows to perform live imaging of cell competition for at least 3 h in proliferating imaginal epithelium. Imaginal discs (eye-antennal discs or wing discs) with genetically manipulated somatic clones (labeled with fluorescent proteins) are dissected out from third-instar larvae in phosphate-buffered saline (PBS) and are set on the prepared chambers as follows: 1. Dissect larvae in PBS and take imaginal discs out from the larvae using forceps. 2. Place the dissected imaginal disc, with peripodial-side up, on a chamber with a drop of PBS. PBS facilitates nonspecific adhesion of imaginal disc to dish/glass in the chamber. 3. Pour Schneider’s Drosophila medium (GIBCO) containing heat-inactivated 10% FBS into the chamber and culture the disc at room temperature.
Imaging Cell Competition
411
2.3. Live imaging of imaginal discs The imaginal disc culture is subjected to the time-lapse imaging using an upright or inverted confocal laser scanning microscope. Here, we describe an example of live imaging of cell competition between polarized and nonpolarized cells in eye-antennal discs using both upright and inverted microscopes. Clones of cells mutant for scrib were induced in eye-antennal discs using the genetic mosaic technique (Lee and Luo, 1999; Xu and Rubin, 1993). Homozygous (scrib/scrib; polarity deficient) and heterozygous (scrib/þ; polarized) mutant clones were visualized by 2Ubi-GFP and 1UbiGFP (green), respectively, and Eiger-overexpressing polarized cells were visualized by actin-Gal4/UAS-myrRFP (magenta). The eye-antennal disc was cultured in chamber A, and images were acquired at 5-min interval for 3 h using an upright confocal microscope (FV1000, Olympus) equipped with an Olympus 60/NA1.1 LUMFL water-immersion objective lens. A polarity-deficient scrib/ cell (arrowhead) is eliminated by cell competition when confronted with polarized cells (Fig. 20.2 and Movie 20.1) (http://www.elsevierdirect.com/companions/9780123918567) (Ohsawa et al., 2011). Similar images were also obtained using an inverted confocal microscope (LSM700, Zeiss) equipped with an Zeiss Plan-Apochromat 40/NA1.3 oil-immersion objective lens (data not shown).
Figure 20.2 Live imaging of cell competition between polarized and nonpolarized cells in imaginal epithelium. Homozygous scrib mutant cells (labeled with 2Ubi-GFP; “losers”) are eliminated when confronted with normally polarized Eiger-expressing cells (labeled with actin-Gal4/UAS-myrRFP; “winners”) in an ex vivo-cultured eye-antennal disc. A polarity-deficient scrib/ cell is eliminated from the tissue after it is incorporated into “polarized” cell population (arrowheads). Eiger was overexpressed in normal cells to enhance cell competition. Four frames of Movie 20.1 are shown. Image processing was done with ImageJ. Scale bar, 10 mm. Genotypes is: y,w, eyFLP1; G454, Act > yþ > Gal4, UAS-myrRFP/UAS-Eigerþ W; FRT82B/FRT82B, ubi-GFP, Tub-Gal80, scrib1. This figure was modified from the data previously published in Ohsawa et al. (2011).
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Shizue Ohsawa et al.
REFERENCES Baker, N. E. (2011). Cell competition. Curr. Biol. 21, R11–R15. Bilder, D. (2004). Epithelial polarity and proliferation control: Links from the Drosophila neoplastic tumor suppressors. Genes Dev. 18, 1909–1925. Bissell, M. J., and Radisky, D. (2001). Putting tumours in context. Nat. Rev. Cancer 1, 46–54. Brumby, A. M., and Richardson, H. E. (2003). Scribble mutants cooperate with oncogenic Ras or Notch to cause neoplastic overgrowth in Drosophila. EMBO J. 22, 5769–5779. Cordero, J. B., Macagno, J. P., Stefanatos, R. K., Strathdee, K. E., Cagan, R. L., and Vidal, M. (2010). Oncogenic Ras diverts a host TNF tumor suppressor activity into tumor promoter. Dev. Cell 18, 999–1011. de la Cova, C., Abril, M., Bellosta, P., Gallant, P., and Johnston, L. A. (2004). Drosophila myc regulates organ size by inducing cell competition. Cell 117, 107–116. Fish, E. M., and Molitoris, B. A. (1994). Alterations in epithelial polarity and the pathogenesis of disease states. N. Engl. J. Med. 330, 1580–1588. Hariharan, I. K., and Bilder, D. (2006). Regulation of imaginal disc growth by tumorsuppressor genes in Drosophila. Annu. Rev. Genet. 40, 335–361. Igaki, T. (2009). Correcting developmental errors by apoptosis: Lessons from Drosophila JNK signaling. Apoptosis 14, 1021–1028. Igaki, T., Pagliarini, R. A., and Xu, T. (2006). Loss of cell polarity drives tumor growth and invasion through JNK activation in Drosophila. Curr. Biol. 16, 1139–1146. Igaki, T., Pastor-Pareja, J. C., Aonuma, H., Miura, M., and Xu, T. (2009). Intrinsic tumor suppression and epithelial maintenance by endocytic activation of Eiger/TNF signaling in Drosophila. Dev. Cell 16, 458–465. Johnston, L. A. (2009). Competitive interactions between cells: Death, growth, and geography. Science 324, 1679–1682. Lee, T., and Luo, L. (1999). Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22, 451–461. Li, W., and Baker, N. E. (2007). Engulfment is required for cell competition. Cell 129, 1215–1225. Morata, G., and Martin, F. A. (2007). Cell competition: The embrace of death. Dev. Cell 13, 1–2. Morata, G., and Ripoll, P. (1975). Minutes: Mutants of Drosophila autonomously affecting cell division rate. Dev. Biol. 42, 211–221. Moreno, E. (2008). Is cell competition relevant to cancer? Nat. Rev. Cancer 8, 141–147. Moreno, E., and Basler, K. (2004). dMyc transforms cells into super-competitors. Cell 117, 117–129. Moreno, E., Basler, K., and Morata, G. (2002). Cells compete for decapentaplegic survival factor to prevent apoptosis in Drosophila wing development. Nature 416, 755–759. Neto-Silva, R. M., de Beco, S., and Johnston, L. A. (2010). Evidence for a growthstabilizing regulatory feedback mechanism between Myc and Yorkie, the Drosophila homolog of Yap. Dev. Cell 19, 507–520. Ohsawa, S., Sugimura, K., Takino, K., Xu, T., Miyawaki, A., and Igaki, T. (2011). Elimination of oncogenic neighbors by JNK-mediated engulfment in Drosophila. Dev. Cell 20, 315–328. Pagliarini, R. A., and Xu, T. (2003). A genetic screen in Drosophila for metastatic behavior. Science 302, 1227–1231. Rhiner, C., Lopez-Gay, J. M., Soldini, D., Casas-Tinto, S., Martin, F. A., Lombardia, L., and Moreno, E. (2010). Flower forms an extracellular code that reveals the fitness of a cell to its neighbors in Drosophila. Dev. Cell 18, 985–998.
Imaging Cell Competition
413
Simpson, P. (1979). Parameters of cell competition in the compartments of the wing disc of Drosophila. Dev. Biol. 69, 182–193. Tyler, D. M., Li, W., Zhuo, N., Pellock, B., and Baker, N. E. (2007). Genes affecting cell competition in Drosophila. Genetics 175, 643–657. Woods, D. F., and Bryant, P. J. (1991). The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions. Cell 66, 451–464. Xu, T., and Rubin, G. M. (1993). Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117, 1223–1237. Ziosi, M., Baena-Lopez, L. A., Grifoni, D., Froldi, F., Pession, A., Garoia, F., Trotta, V., Bellosta, P., and Cavicchi, S. (2010). dMyc functions downstream of Yorkie to promote the supercompetitive behavior of hippo pathway mutant cells. PLoS Genet. 6, e1001140.
T W E N T Y
Imaging Cell Competition in Drosophila Imaginal Discs Shizue Ohsawa,* Kaoru Sugimura,† Kyoko Takino,* and Tatsushi Igaki*,‡ Contents 407 409 409 410 411 412
1. Introduction 2. Live Imaging of Cell Competition 2.1. Preparation of chambers 2.2. Imaginal disc culture 2.3. Live imaging of imaginal discs References
Abstract Cell competition is a process in which cells with higher fitness (“winners”) survive and proliferate at the expense of less fit neighbors (“losers”). It has been suggested that cell competition is involved in a variety of biological processes such as organ size control, tissue homeostasis, cancer progression, and the maintenance of stem cell population. By advent of a genetic mosaic technique, which enables to generate fluorescently marked somatic clones in Drosophila imaginal discs, recent studies have presented some aspects of molecular mechanisms underlying cell competition. Now, with a live-imaging technique using ex vivo-cultured imaginal discs, we can dissect the spatiotemporal nature of competitive cell behaviors within multicellular communities. Here, we describe procedures and tips for live imaging of cell competition in Drosophila imaginal discs.
1. Introduction Cell–cell interactions in multicellular organisms play crucial roles in coordination of cell proliferation, differentiation, and cell death during normal development and homeostasis. “Cell competition” is a form of cell–cell * Department of Cell Biology, G-COE, Kobe University Graduate School of Medicine, Kobe, Japan Institute for Integrated Cell-Material Sicences (iCeMS), Kyoto University iCeMS Complex 2, Sakyo-ku, Kyoto, Japan { PRESTO, Japan Science and Technology Agency (JST), Saitama, Japan {
Methods in Enzymology, Volume 506 ISSN 0076-6879, DOI: 10.1016/B978-0-12-391856-7.00044-5
#
2012 Elsevier Inc. All rights reserved.
407
408
Shizue Ohsawa et al.
interaction in which cells with higher fitness (“winners”) survive and proliferate at the expense of neighboring cells with lower fitness (“losers”) (Morata and Ripoll, 1975). Loser cells, but otherwise viable cells, undergo apoptosis when confronted with winner cells. Winner cells then proliferate to occupy the space, resulting in the size of the population unchanged. Thus, cell competition is a process in which fitter cells are selected among otherwise homogeneous population. It has been suggested that cell competition is involved in a variety of biological processes such as organ size control, tissue homeostasis, cancer progression, and the maintenance of stem cell population (Baker, 2011; Johnston, 2009; Morata and Martin, 2007; Moreno, 2008). Studies in Drosophila imaginal discs have indicated that the “cellular fitness” can be determined by several factors: (i) the amount of ribosomal proteins, (ii) the expression level of a proto-oncogene myc, (iii) the activity of the Hippo pathway, and (iv) the integrity of apico-basal polarity. Cells with heterozygous mutation in any one of the ribosomal protein genes (called Minute mutants) are eliminated from the tissue as losers when confronted with wild-type cells (Morata and Ripoll, 1975; Moreno et al., 2002; Simpson, 1979). Cells expressing higher level of Drosophila myc (dmyc) become winners when confronted with cells with relatively lower dmyc expression (de la Cova et al., 2004; Moreno and Basler, 2004). Similarly, cells with inactivated Hippo tumor-suppressor pathway components behave as “super-competitors” that eliminate neighboring wild-type cells (Tyler et al., 2007). In addition, epithelial cells seem to compete the integrity of the polarized structure with one another; cells with disrupted apico-basal polarity are eliminated as losers when confronted with normally polarized wild-type cells (Bilder, 2004; Hariharan and Bilder, 2006). This elimination of polarity-deficient cells by cell competition seems to work as an “intrinsic tumor suppression” (Igaki, 2009) (see below). Most cancers originate from epithelium. Loss of apico-basal polarity in epithelial cells is frequently associated with cancer progression (Bissell and Radisky, 2001; Fish and Molitoris, 1994). Similarly, mutant flies deficient for evolutionarily conserved apico-basal polarity genes such as scribble (scrib) or discs large (dlg) develop overgrown tumors in their imaginal epithelium (Bilder, 2004; Hariharan and Bilder, 2006). Intriguingly, when surrounded by wild-type cells, these polarity-deficient mutant cells do not overgrow but are eliminated from the tissue (Brumby and Richardson, 2003; Igaki et al., 2006, 2009; Pagliarini and Xu, 2003; Woods and Bryant, 1991). This suggests that normal epithelium possesses an intrinsic tumor-suppression mechanism that eliminates oncogenic polarity-deficient cells by cell competition. The polarity-deficient cells that are confronted with wild-type cells result in c-Jun N-terminal kinase (JNK)-dependent cell death (Brumby and Richardson, 2003), which is triggered by endocytic activation of Eiger (Drosophila tumor necrosis factor, TNF) (Igaki et al., 2009). Surrounding normal cells also activate Eiger–JNK signaling, which does not cause cell death but enhances the elimination of neighboring polarity-deficient cells
Imaging Cell Competition
409
by activating the PVR-ELMO–Mbc-mediated engulfment pathway (Ohsawa et al., 2011). The JNK activation in polarity-deficient cells has also been shown to be triggered by Eiger expressed in hemocytes (Cordero et al., 2010). Thus, Eiger–JNK signaling plays a central role in competition between polarized and nonpolarized epithelial cells. Interestingly, Minute clones are still eliminated by cell competition in the absence of eiger (Ohsawa et al., 2011), suggesting that competitive interactions in different cellular contexts are regulated by different mechanisms. About 30 years after the first discovery (Morata and Ripoll, 1975), progress has been made in understanding the molecular mechanism of cell competition with some technical advances especially for producing fluorescently labeled somatic clones in imaginal discs (Lee and Luo, 1999; Xu and Rubin, 1993). Recent studies have identified both positive (JNK, hid, Flower, Eiger, and the engulfment genes) and negative (Dpp, the Hippo pathway components, and Sparc) regulators of cell competition (Brumby and Richardson, 2003; de la Cova et al., 2004; Igaki et al., 2009; Li and Baker, 2007; Moreno et al., 2002; Neto-Silva et al., 2010; Ohsawa et al., 2011; Rhiner et al., 2010; Tyler et al., 2007; Ziosi et al., 2010). Yet, the underlying mechanism by which these regulators act, if exists, in a common cell competition pathway and the upstream mechanisms that regulate these factors still remain to be elucidated. Spatiotemporal analysis in live tissues could provide new insights into understanding cell competition. Here, we describe procedures and tips for live imaging of cell competition using ex vivo-cultured Drosophila imaginal discs.
2. Live Imaging of Cell Competition Live imaging, in conjunction with genetic manipulations, is a powerful tool for dissecting dynamic cellular processes within multicellular communities. Here, we overview a recently established live-imaging system for visualizing cell competition in ex vivo-cultured Drosophila imaginal discs.
2.1. Preparation of chambers The chambers for culturing imaginal discs are prepared by assembling following materials (Fig. 20.1). Glass bottom dishes for inverted microscopes are also commercially available. For upright microscopes (chamber A)
60-mm petri dish 35-mm dish bottom Glass ring as a spacer (e.g., height: 4 mm: outside diameter: 20 mm) Nontoxic glue paste SILPOT 184 W/C (DOW CORNING TORAY)
410
Shizue Ohsawa et al.
A
Objective lens Imaginal disc
B Cover glass Schneider’s medium Glass ring
Observation hole 35-mm dish bottom
60-mm dish
Imaginal disc
35-mm dish bottom
Schneider’s medium Objective lens
Figure 20.1 Chamber preparation for live imaging of imaginal discs using an upright (A) or an inverted (B) confocal laser scanning microscope.
For inverted microscopes (chamber B) 35-mm glass bottom dish (MATSUNAMI Glass No. 1S #D111300) The commercially available glass bottom dishes should be rinsed with 2 ml of 100% ethanol for 2–3 times to remove toxicity before use.
2.2. Imaginal disc culture As cell competition is a phenomenon observed in proliferating imaginal epithelium, the cultured discs must be kept with constant cell proliferation rate during the imaging. In addition, the imaginal discs must be tightly attached to the dish/glass to prevent out-of-focus imaging. The following procedure allows to perform live imaging of cell competition for at least 3 h in proliferating imaginal epithelium. Imaginal discs (eye-antennal discs or wing discs) with genetically manipulated somatic clones (labeled with fluorescent proteins) are dissected out from third-instar larvae in phosphate-buffered saline (PBS) and are set on the prepared chambers as follows: 1. Dissect larvae in PBS and take imaginal discs out from the larvae using forceps. 2. Place the dissected imaginal disc, with peripodial-side up, on a chamber with a drop of PBS. PBS facilitates nonspecific adhesion of imaginal disc to dish/glass in the chamber. 3. Pour Schneider’s Drosophila medium (GIBCO) containing heat-inactivated 10% FBS into the chamber and culture the disc at room temperature.
Imaging Cell Competition
411
2.3. Live imaging of imaginal discs The imaginal disc culture is subjected to the time-lapse imaging using an upright or inverted confocal laser scanning microscope. Here, we describe an example of live imaging of cell competition between polarized and nonpolarized cells in eye-antennal discs using both upright and inverted microscopes. Clones of cells mutant for scrib were induced in eye-antennal discs using the genetic mosaic technique (Lee and Luo, 1999; Xu and Rubin, 1993). Homozygous (scrib/scrib; polarity deficient) and heterozygous (scrib/þ; polarized) mutant clones were visualized by 2Ubi-GFP and 1UbiGFP (green), respectively, and Eiger-overexpressing polarized cells were visualized by actin-Gal4/UAS-myrRFP (magenta). The eye-antennal disc was cultured in chamber A, and images were acquired at 5-min interval for 3 h using an upright confocal microscope (FV1000, Olympus) equipped with an Olympus 60/NA1.1 LUMFL water-immersion objective lens. A polarity-deficient scrib/ cell (arrowhead) is eliminated by cell competition when confronted with polarized cells (Fig. 20.2 and Movie 20.1) (http://www.elsevierdirect.com/companions/9780123918567) (Ohsawa et al., 2011). Similar images were also obtained using an inverted confocal microscope (LSM700, Zeiss) equipped with an Zeiss Plan-Apochromat 40/NA1.3 oil-immersion objective lens (data not shown).
Figure 20.2 Live imaging of cell competition between polarized and nonpolarized cells in imaginal epithelium. Homozygous scrib mutant cells (labeled with 2Ubi-GFP; “losers”) are eliminated when confronted with normally polarized Eiger-expressing cells (labeled with actin-Gal4/UAS-myrRFP; “winners”) in an ex vivo-cultured eye-antennal disc. A polarity-deficient scrib/ cell is eliminated from the tissue after it is incorporated into “polarized” cell population (arrowheads). Eiger was overexpressed in normal cells to enhance cell competition. Four frames of Movie 20.1 are shown. Image processing was done with ImageJ. Scale bar, 10 mm. Genotypes is: y,w, eyFLP1; G454, Act > yþ > Gal4, UAS-myrRFP/UAS-Eigerþ W; FRT82B/FRT82B, ubi-GFP, Tub-Gal80, scrib1. This figure was modified from the data previously published in Ohsawa et al. (2011).
412
Shizue Ohsawa et al.
REFERENCES Baker, N. E. (2011). Cell competition. Curr. Biol. 21, R11–R15. Bilder, D. (2004). Epithelial polarity and proliferation control: Links from the Drosophila neoplastic tumor suppressors. Genes Dev. 18, 1909–1925. Bissell, M. J., and Radisky, D. (2001). Putting tumours in context. Nat. Rev. Cancer 1, 46–54. Brumby, A. M., and Richardson, H. E. (2003). Scribble mutants cooperate with oncogenic Ras or Notch to cause neoplastic overgrowth in Drosophila. EMBO J. 22, 5769–5779. Cordero, J. B., Macagno, J. P., Stefanatos, R. K., Strathdee, K. E., Cagan, R. L., and Vidal, M. (2010). Oncogenic Ras diverts a host TNF tumor suppressor activity into tumor promoter. Dev. Cell 18, 999–1011. de la Cova, C., Abril, M., Bellosta, P., Gallant, P., and Johnston, L. A. (2004). Drosophila myc regulates organ size by inducing cell competition. Cell 117, 107–116. Fish, E. M., and Molitoris, B. A. (1994). Alterations in epithelial polarity and the pathogenesis of disease states. N. Engl. J. Med. 330, 1580–1588. Hariharan, I. K., and Bilder, D. (2006). Regulation of imaginal disc growth by tumorsuppressor genes in Drosophila. Annu. Rev. Genet. 40, 335–361. Igaki, T. (2009). Correcting developmental errors by apoptosis: Lessons from Drosophila JNK signaling. Apoptosis 14, 1021–1028. Igaki, T., Pagliarini, R. A., and Xu, T. (2006). Loss of cell polarity drives tumor growth and invasion through JNK activation in Drosophila. Curr. Biol. 16, 1139–1146. Igaki, T., Pastor-Pareja, J. C., Aonuma, H., Miura, M., and Xu, T. (2009). Intrinsic tumor suppression and epithelial maintenance by endocytic activation of Eiger/TNF signaling in Drosophila. Dev. Cell 16, 458–465. Johnston, L. A. (2009). Competitive interactions between cells: Death, growth, and geography. Science 324, 1679–1682. Lee, T., and Luo, L. (1999). Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22, 451–461. Li, W., and Baker, N. E. (2007). Engulfment is required for cell competition. Cell 129, 1215–1225. Morata, G., and Martin, F. A. (2007). Cell competition: The embrace of death. Dev. Cell 13, 1–2. Morata, G., and Ripoll, P. (1975). Minutes: Mutants of Drosophila autonomously affecting cell division rate. Dev. Biol. 42, 211–221. Moreno, E. (2008). Is cell competition relevant to cancer? Nat. Rev. Cancer 8, 141–147. Moreno, E., and Basler, K. (2004). dMyc transforms cells into super-competitors. Cell 117, 117–129. Moreno, E., Basler, K., and Morata, G. (2002). Cells compete for decapentaplegic survival factor to prevent apoptosis in Drosophila wing development. Nature 416, 755–759. Neto-Silva, R. M., de Beco, S., and Johnston, L. A. (2010). Evidence for a growthstabilizing regulatory feedback mechanism between Myc and Yorkie, the Drosophila homolog of Yap. Dev. Cell 19, 507–520. Ohsawa, S., Sugimura, K., Takino, K., Xu, T., Miyawaki, A., and Igaki, T. (2011). Elimination of oncogenic neighbors by JNK-mediated engulfment in Drosophila. Dev. Cell 20, 315–328. Pagliarini, R. A., and Xu, T. (2003). A genetic screen in Drosophila for metastatic behavior. Science 302, 1227–1231. Rhiner, C., Lopez-Gay, J. M., Soldini, D., Casas-Tinto, S., Martin, F. A., Lombardia, L., and Moreno, E. (2010). Flower forms an extracellular code that reveals the fitness of a cell to its neighbors in Drosophila. Dev. Cell 18, 985–998.
Imaging Cell Competition
413
Simpson, P. (1979). Parameters of cell competition in the compartments of the wing disc of Drosophila. Dev. Biol. 69, 182–193. Tyler, D. M., Li, W., Zhuo, N., Pellock, B., and Baker, N. E. (2007). Genes affecting cell competition in Drosophila. Genetics 175, 643–657. Woods, D. F., and Bryant, P. J. (1991). The discs-large tumor suppressor gene of Drosophila encodes a guanylate kinase homolog localized at septate junctions. Cell 66, 451–464. Xu, T., and Rubin, G. M. (1993). Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117, 1223–1237. Ziosi, M., Baena-Lopez, L. A., Grifoni, D., Froldi, F., Pession, A., Garoia, F., Trotta, V., Bellosta, P., and Cavicchi, S. (2010). dMyc functions downstream of Yorkie to promote the supercompetitive behavior of hippo pathway mutant cells. PLoS Genet. 6, e1001140.