Name: induction and diagnosis of tumors in drosophila imaginal disc epithelia
Uploaded: 2017-07-25T14:00:00.000+00:00
Duration: 8 min 14 s
Description: Watch this Scientific Journal Video about Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia at JoVE.com

我们感谢J. Vaughen对手稿的批判性阅读。这项工作得到了JSPS KAKENHI Grant号26891025,15H01500和武田科学基金会YT研究奖学金的资助

飞越和克隆诱导<ol><li>在收集原始苍蝇之前12小时内取出小瓶中的所有苍蝇。 </li><li>通过注入CO 2气体和地方飞行到CO 2蝇垫麻醉在小瓶中的苍蝇。 </li><li>将10-20名女性和10名男性从CO 2飞虱转移到新鲜的小瓶中，并在25℃下孵育1天。 </li><li>将这些苍蝇转移到新鲜的小瓶中，并在25℃下孵育12小时。 注意:丢弃第一个小瓶，因为女性在第一天没有铺好足够的鸡蛋。 </li><li>对于增强子-GAL4系，除去成年苍蝇，并在25℃孵育小瓶，直到解剖。 </li><li>为了通过翻转-GAL4诱导马赛克克隆，取出成年苍蝇，并在25℃孵育小瓶2-4天。热休克前的孵化时间取决于实验目的。蛋沉积2天后的热休克（AED）会在未成熟的时间产生马赛克克隆时代上皮细胞。 3或4天后的热休克AED在分化的早期至中期三龄期的上皮样细胞中产生镶嵌克隆。 注意:重要的是检查改变的热休克时间对致瘤表型的影响。 </li><li>为了通过翻转GAL4诱导马赛克克隆，通过在37℃水浴中浸泡10-45分钟来热冲击小瓶，随后在25℃下孵育2-3天。 注意:每个实验中的热休克时间，时间和孵化时间的信息如图所示。 </li></ol> 2.解剖幼虫<ol><li>用木棍或钝镊子收集流浪的三龄幼虫，在荧光立体显微镜下通过适当的荧光标记（ 例如 EGFP）对它们进行基因分型，并将它们置于含有PBS（磷酸盐缓冲盐水）的解剖盘中。 </li><li>用2 mL塑料转移移液管移液冲洗PBS中的幼虫。 </li><li>用一个镊子夹住幼虫的中心，并用其他镊子将身体撕开一半。 </li><li>用一个镊子夹住前半部的嘴巴，用其他镊子将口朝向身体推动，将身体内部转出。 </li><li>用镊子去除不必要的物质，如唾液腺或脂肪体。 </li></ol>固定和抗体染色<ol><li>将解剖的幼体前体（包括图像翼盘）转移到1.5mL塑料管中，并在室温下，在暗中轻轻旋转，将其固定在1mL Fix溶液（PBS中4％甲醛）中10分钟。 小心:甲醛有毒，具有致癌潜力。戴防护手套和衣服以防止皮肤接触。 注意:在这部分中，除非另有说明，否则所有步骤均在室温下在暗室进行。 </li><li>删除Fix解决方案并丢弃。用1 mL PBT（0.3％Trit）洗涤组织在PBS中的X-100上）三次，每次15分钟。 </li><li>去除PBT，加入1 mL PBTG（0.2％牛血清白蛋白和5％正常山羊血清在PBT中），阻断并在室温下营养1小时或4℃过夜。 </li><li>取出PBTG，加入适量用PBTG（见材料表 ）稀释的一抗溶液，并在4℃下加入马蹄形苷过夜。 </li><li>取出一抗溶液，用1 mL PBT洗涤组织三次，每次15 min。 </li><li>取PBT并加入用PBTG（1:400）适当稀释的二抗溶液。在室温下营养2小时或在4℃过夜。 </li><li>去除二抗溶液，并用1 mL PBT洗涤组织两次，每次15 min。 </li><li>为了染色F-肌动蛋白，去除PBT，并加入适当稀释在PBS（1:40）中的鬼笔环肽溶液。然后营养20分钟。去除鲨鱼苷溶液，用1 mL PBT洗涤组织两次，每次15分钟。 </li><li>为了复染核DNA，除去PBT并加入DAPI（4&#39;，6-二脒基-2-苯基吲哚）溶液（0.5μg/ mL DAPI在PBS中）。然后营养10分钟 注意:DAPI具有致癌潜力。戴防护手套和衣服以防止皮肤接触。 </li><li>取出DAPI溶液，用1 mL PBT洗涤组织两次，每次15 min。 </li><li>在1 mL PBS中室温冲洗10分钟。 </li><li>移除PBS并加入500μL100％甘油作为预装介质。 </li></ol> 4.安装在显微镜幻灯片上<ol><li>使用2 mL塑料移液器将染色的组织放在显微镜载玻片上。 </li><li>使用镊子将组织转移到另一台显微镜载玻片上。 </li><li>用一个镊子压住解剖组织的末端，并用其他镊子拉开脑和眼睛的触角盘。 注意:将解剖后的大脑放置在机翼成像光盘附近。大脑作为防止盖玻片破碎机翼成像光盘的平台。 </li><li>为了隔离机翼成像盘，用一个镊子压住解剖组织的末端，轻轻地划伤体壁，并用其他镊子撕下椎间盘。 注意:如果难以找到翼形成像盘，请从后侧从前侧剥离气管。翼成像光盘粘在气管上。 </li><li>用盖玻片轻轻地覆盖图像盘，并用指甲油密封;储存于4°C。 </li></ol>共焦显微镜<ol><li>为了获得使用共焦显微镜设置的图像采集参数包括范围发光波长的，激光强度，增益，偏移，扫描速度和图像大小为24的共焦图像。 注意:有关图像采集设置的详细步骤，请参阅各显微镜制造商提供的使用说明书。 </li><li>捕捉单一公司整个机翼成像光盘的非焦点部分，使用20X物镜。 </li><li>使用40X或60X镜头通过z-stack扫描获得三维图像，步长为0.5 - 1.0μm。 注意:为了以高分辨率分析细胞表型，图像大小应大于512 x 512像素。 </li></ol> 6.使用ImageJ进行图像分析<ol><li>使用斐济，一个开源ImageJ软件专注于生物图像分析（https://fiji.sc/）25，获取和分析共聚焦Z-堆栈中的图像。 </li><li>要获取垂直部分，请打开ImageJ中的z-stack图像，然后选择菜单项"Image / Stacks / Reslice"。 注意:可以在Reslice菜单中选择XZ轴或YZ轴。也可以通过将直线或矩形绘制到z堆叠图像上，也可以在任意方向上获得垂直截面。 </li></ol> 7.肿瘤表型诊断<ol><li>打开垂直线（XZ或YZ轴），分析形态表型和抗体染色。 </li><li>对于肿瘤表型的诊断，主要集中在以下三点:（1）如果细胞质量包括敲除克隆偏离主要上皮层，（2）如果连接蛋白的亚细胞定位在该细胞质量中改变，和（3）如果该细胞团的直径大于4个细胞。 </li><li>根据图1所示的流程分类肿瘤表型。 </li></ol>

<ol>
	<li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hallmarks+of+cancer.">The hallmarks of cancer.</a> Cell. 100 (1), 57-70 (2000).</li><li>Xu, T., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+genetic+mosaics+in+developing+and+adult+Drosophila+tissues.">Analysis of genetic mosaics in developing and adult Drosophila tissues.</a> Development. 117 (4), 1223-1237 (1993).</li><li>Struhl, G., Basler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organizing+activity+of+wingless+protein+in+Drosophila.">Organizing activity of wingless protein in Drosophila.</a> Cell. 72 (4), 527-540 (1993).</li><li>Potter, C. J., Turenchalk, G. S., Xu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+in+cancer+research.+An+expanding+role.">Drosophila in cancer research. An expanding role.</a> Trends Genet. 16 (1), 33-39 (2000).</li><li>Miles, W. O., Dyson, N. J., Walker, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+tumor+invasion+and+metastasis+in+Drosophila.">Modeling tumor invasion and metastasis in Drosophila.</a> Dis Model Mech. 4 (6), 753-761 (2011).</li><li>Tipping, M., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+as+a+model+for+context-dependent+tumorigenesis.">Drosophila as a model for context-dependent tumorigenesis.</a> J Cell Physiol. 229 (1), 27-33 (2013).</li><li>Bilder, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+polarity+and+proliferation+control:+links+from+the+Drosophila+neoplastic+tumor+suppressors.">Epithelial polarity and proliferation control: links from the Drosophila neoplastic tumor suppressors.</a> Genes Dev. 18 (16), 1909-1925 (2004).</li><li>Humbert, P. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+tumourigenesis+by+the+Scribble/Dlg/Lgl+polarity+module.">Control of tumourigenesis by the Scribble/Dlg/Lgl polarity module.</a> Oncogene. 27 (55), 6888-6907 (2008).</li><li>Huang, L., Muthuswamy, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarity+protein+alterations+in+carcinoma:+a+focus+on+emerging+roles+for+polarity+regulators.">Polarity protein alterations in carcinoma: a focus on emerging roles for polarity regulators.</a> Curr Opin Genet Dev. 20 (1), 41-50 (2010).</li><li>Brumby, A. M., Richardson, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=scribble+mutants+cooperate+with+oncogenic+Ras+or+Notch+to+cause+neoplastic+overgrowth+in+Drosophila.">scribble mutants cooperate with oncogenic Ras or Notch to cause neoplastic overgrowth in Drosophila.</a> EMBO J. 22 (21), 5769-5779 (2003).</li><li>Igaki, T., Pastor-Pareja, J. C., Aonuma, H., Miura, M., Xu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+tumor+suppression+and+epithelial+maintenance+by+endocytic+activation+of+Eiger/TNF+signaling+in+Drosophila.">Intrinsic tumor suppression and epithelial maintenance by endocytic activation of Eiger/TNF signaling in Drosophila.</a> Dev Cell. 16 (3), 458-465 (2009).</li><li>Tamori, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Involvement+of+Lgl+and+Mahjong/VprBP+in+cell+competition.">Involvement of Lgl and Mahjong/VprBP in cell competition.</a> PLoS Biol. 8 (7), e1000422 (2010).</li><li>Cordero, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oncogenic+Ras+diverts+a+host+TNF+tumor+suppressor+activity+into+tumor+promoter.">Oncogenic Ras diverts a host TNF tumor suppressor activity into tumor promoter.</a> Dev Cell. 18 (6), 999-1011 (2010).</li><li>Yamamoto, M., Ohsawa, S., Kunimasa, K., Igaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ligand+Sas+and+its+receptor+PTP10D+drive+tumour-suppressive+cell+competition.">The ligand Sas and its receptor PTP10D drive tumour-suppressive cell competition.</a> Nature. 542 (7640), 246-250 (2017).</li><li>Vaughen, J., Igaki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slit-Robo+Repulsive+Signaling+Extrudes+Tumorigenic+Cells+from+Epithelia.">Slit-Robo Repulsive Signaling Extrudes Tumorigenic Cells from Epithelia.</a> Dev Cell. 39 (6), 683-695 (2016).</li><li>Tamori, Y., Suzuki, E., Deng, W. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+Tumors+Originate+in+Tumor+Hotspots,+a+Tissue-Intrinsic+Microenvironment.">Epithelial Tumors Originate in Tumor Hotspots, a Tissue-Intrinsic Microenvironment.</a> PLoS Biol. 14 (9), e1002537 (2016).</li><li>Ohsawa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elimination+of+oncogenic+neighbors+by+JNK-mediated+engulfment+in+Drosophila.">Elimination of oncogenic neighbors by JNK-mediated engulfment in Drosophila.</a> Dev Cell. 20 (3), 315-328 (2011).</li><li>Men&#233;ndez, J., P&#233;rez-Garijo, A., Calleja, M., Morata, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tumor-suppressing+mechanism+in+Drosophila+involving+cell+competition+and+the+Hippo+pathway.">A tumor-suppressing mechanism in Drosophila involving cell competition and the Hippo pathway.</a> Proc Natl Acad Sci U S A. 107 (33), 14651-14656 (2010).</li><li>Hogan, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+interface+between+normal+and+transformed+epithelial+cells.">Characterization of the interface between normal and transformed epithelial cells.</a> Nat Cell Biol. 11 (4), 460-467 (2009).</li><li>Kajita, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+with+surrounding+normal+epithelial+cells+influences+signalling+pathways+and+behaviour+of+Src-transformed+cells.">Interaction with surrounding normal epithelial cells influences signalling pathways and behaviour of Src-transformed cells.</a> J Cell Sci. 123 (Pt 2), 171-180 (2010).</li><li>Norman, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+Scribble+causes+cell+competition+in+mammalian+cells.">Loss of Scribble causes cell competition in mammalian cells.</a> J Cell Sci. 125 (1), 59-66 (2012).</li><li>Wagstaff, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+cell+competition+kills+cells+via+induction+of+lethal+p53+levels.">Mechanical cell competition kills cells via induction of lethal p53 levels.</a> Nat Commun. 7, 1-14 (2016).</li><li>Kajita, M., Fujita, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EDAC:+Epithelial+defence+against+cancer-cell+competition+between+normal+and+transformed+epithelial+cells+in+mammals.">EDAC: Epithelial defence against cancer-cell competition between normal and transformed epithelial cells in mammals.</a> J Biochem. 158 (1), 15-23 (2015).</li><li>North, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+is+believing?+A+beginners'+guide+to+practical+pitfalls+in+image+acquisition.">Seeing is believing? A beginners' guide to practical pitfalls in image acquisition.</a> J Cell Biol. 172 (1), 9-18 (2006).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nat Methods. 9 (7), 676-682 (2012).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118 (2), 401-415 (1993).</li><li>Jory, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+survey+of+6,300+genomic+fragments+for+cis-regulatory+activity+in+the+imaginal+discs+of+Drosophila+melanogaster.">A survey of 6,300 genomic fragments for cis-regulatory activity in the imaginal discs of Drosophila melanogaster.</a> Cell Rep. 2 (4), 1014-1024 (2012).</li><li>McGuire, S. E., Le, P. T., Osborn, A. J., Matsumoto, K., Davis, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatiotemporal+rescue+of+memory+dysfunction+in+Drosophila.">Spatiotemporal rescue of memory dysfunction in Drosophila.</a> Science. 302 (5651), 1765-1768 (2003).</li><li>Rodrigues, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+STAT+regulates+growth+and+induces+competitive+interactions+independently+of+Myc,+Yorkie,+Wingless+and+ribosome+biogenesis.">Activated STAT regulates growth and induces competitive interactions independently of Myc, Yorkie, Wingless and ribosome biogenesis.</a> Development. 139 (21), 4051-4061 (2012).</li><li>Khan, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+neoplasia+in+Drosophila+entails+switch+to+primitive+cell+states.">Epithelial neoplasia in Drosophila entails switch to primitive cell states.</a> Proc Natl Acad Sci U S A. 110 (24), E2163-E2172 (2013).</li><li>Pagliarini, R. A., Xu, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+screen+in+Drosophila+for+metastatic+behavior.">A genetic screen in Drosophila for metastatic behavior.</a> Science. 302 (5648), 1227-1231 (2003).</li><li>Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+melanogaster:+a+model+and+a+tool+to+investigate+malignancy+and+identify+new+therapeutics.">Drosophila melanogaster: a model and a tool to investigate malignancy and identify new therapeutics.</a> Nat Rev Cancer. 13 (3), 172-183 (2013).</li><li>Patel, P. H., Edgar, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+design:+how+Drosophila+tumors+remodel+their+neighborhood.">Tissue design: how Drosophila tumors remodel their neighborhood.</a> Semin Cell Dev Biol. 28, 86-95 (2014).</li><li>Nakajima, Y. -. I., Meyer, E. J., Kroesen, A., McKinney, S. A., Gibson, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+junctions+maintain+tissue+architecture+by+directing+planar+spindle+orientation.">Epithelial junctions maintain tissue architecture by directing planar spindle orientation.</a> Nature. 500 (7462), 359-362 (2013).</li><li>Colombani, J., Andersen, D. S., L&#233;opold, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secreted+peptide+Dilp8+coordinates+Drosophila+tissue+growth+with+developmental+timing.">Secreted peptide Dilp8 coordinates Drosophila tissue growth with developmental timing.</a> Science. 336 (6081), 582-585 (2012).</li><li>Garelli, A., Gontijo, A. M., Miguela, V., Caparros, E., Dominguez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaginal+discs+secrete+insulin-like+peptide+8+to+mediate+plasticity+of+growth+and+maturation.">Imaginal discs secrete insulin-like peptide 8 to mediate plasticity of growth and maturation.</a> Science. 336 (6081), 579-582 (2012).</li></ol>

作者没有什么可以披露的。

的GAL4-UAS系统是用于在果蝇 26靶基因表达的最有力的遗传工具之一，极大地方便了肿瘤细胞的诱导和分析在体内 4。该系统能够产生携带敲低肿瘤抑制基因的克隆或在野生型上皮组织内癌基因的过度表达，这种情况与人类癌症的初始阶段非常相似，其中转化的前肿瘤细胞被正常上皮细胞包围。由GAL4增强子捕获系或Janelia GAL4系27等增强子序列调节的GAL4驱动系具有独特的时空限制性表达模式。因此，这些增强子-GAL4系可用于在限制性的成像盘区域中一致地诱导肿瘤。除了本研究中显示的sd-GAL4和upd-GAL4之外 ，我们证实了lgl- Knockdown诱导b其他增强子陷阱GAL4系列包括切割的GAL4 （ en-GAL4 ）， hedgehog-GAL4 （ hh-GAL4 ）， optomotor-blind-GAL4 （ omb-GAL4 ）， 修补的GAL4 （ ptc-GAL4 ）和spalt- GAL4 （ salm-GAL4 ）可以在机翼成像盘的铰链区域产生肿瘤性肿瘤。如果增强子GAL4表达模式需要被暂时控制，GAL4的组合和GAL80的温度敏感的版本（GAL80 TS）是一种选择。 GAL80 TS在18℃而不是在29℃下，这允许UAS-转基因表达开启或关闭根据需要28被切换压制GAL4功能。 结合FLP-FRT系统和GAL4-UAS系统3的翻转式GAL4拼接系统在图像盘中生成随机克隆。通过结合热休克诱导型翻转酶（hsFLP）与翻转GAL4，克隆产生的时间离子是可控的。因为成虫盘细胞的致瘤潜力可以依赖于发育阶段，内源性信号事件，和/或细胞分化29，30时，检查改变的热休克定时对致瘤表型的影响是非常重要的。 先前已经表明，nTSG突变体克隆经历细胞依赖性竞争凋亡当由野生型细胞10，11，12，18所包围，这表明多个突变癌症发展过程中所需要的细胞转化。事实上，致癌Ras或YKI在LGL或Scrib的突变体克隆的过度表达将它们转换为侵袭性肿瘤10，18，31，而误Ras V12或Yki 3SA的表达在没有nTSG突变的情况下本身不诱导恶性肿瘤。如本文和以前16所示，在野生型翼片中产生的前肿瘤nTSG敲低克隆也经历凋亡，并且在翼囊16中不显示肿瘤发生。同时，位于铰链"热点"的nTSG敲低克隆发展成肿瘤性肿瘤，没有任何额外的致癌突变。在铰链区，nTSG缺陷型细胞劫持内源性的Janus激酶/信号转导和转录激活因子（JAK / STAT）信号传导致肿瘤发生16 。因此，铰链特异性GAL4如upd-GAL4是通过RNAi介导的肿瘤抑制基因敲低一贯诱导肿瘤肿瘤形成的有用工具。虽然翻转的GAL4诱导的nTSG敲除马赛克克隆也在铰链中诱导肿瘤性肿瘤胃癌，肿瘤发生发生依赖于初始克隆大小:通过20分钟的热休克产生的大克隆通常诱导肿瘤发生，而通过短于10分钟的热休克产生的大多数小克隆被完全消除（KM和YT，未公开的数据）。 最近的一些对肿瘤发展和癌症进展研究使用果蝇成虫盘作为模型系统32，33。这些研究的共同之处包括肿瘤肿瘤的几个标志物:畸形细胞块，变形的椎间盘形状，F-肌动蛋白的积累，增强的细胞周期（BrdU / EdU结合或PH3表达），JNK信号的激活及其下游靶标MMP1，基底膜破坏和/或上皮紊乱，包括参与顶端基底细胞极性的蛋白质的损失或亚细胞离域（Crumbs，Stardust，PatJ，aPKC，Bazooka / PAR-3或PAR-6），分隔结（Lgl，Scrib，Dlg或Coracle）或粘附连接（E-钙粘蛋白或犰狳）。这些表型通过常规抗体染色容易检测。虽然在肿瘤发生的早期阶段难以确定克隆表型是否是增生，发育不良，良性肿瘤或恶性肿瘤，但是在由单个上皮单层组成的果蝇成像盘上皮中应该更简单。在这项研究中，我们介绍了一种诊断方法来分类上皮单层诱导的肿瘤克隆表型（ 图1 ）。该方法需要仔细观察使用z-stack共聚焦图像的五个标准;可以通过常规抗体染色和共聚焦成像来检查每个诊断标准。我们在该方法中引入的新标准之一是"4细胞直径标准"，其区分成像光盘中的发育不良和肿瘤。肿瘤细胞如nTSG突变体克隆频繁从通过细胞竞争或主轴取向差15，16，34上皮层LY挤出。在大多数情况下，这些挤出的细胞凋亡，因此它们不能增殖。虽然前肿瘤细胞可能在挤出过程中进行一次细胞分裂，但是在挤出后可能会再次进行一次或两次细胞分裂，这使细胞质量达到2-3个细胞直径。因此，如果前肿瘤细胞偏离上皮层的细胞质量大于4细胞直径，我们可以确定它们作为上皮层外的肿瘤存活和增殖。 虽然这种诊断方法很简单，但随着肿瘤的发展和恶性肿瘤的发展，随着时间的流逝，临床表现是至关重要的。例如，发育不良是细胞学上异常的组织，是完全良性生长与前期生长之间的过渡状态恶性。因此，即使观察到的克隆被定义为根据流程图（ 图1 ）的发育异常，发育不良随后发展成肿瘤也是可能的。虽然幼虫阶段在时间上有限（25℃时为5-6天，在29℃AED为4-5天），但通过胰岛素样肽引起的瞳孔延迟，成像盘中的肿瘤生长延长了幼虫期35 ， 36 。因此，成像圆盘中的肿瘤肿瘤形成使得可以观察肿瘤克隆表型进展数天。我们简单的分类方法可能广泛适用于果蝇各种器官的肿瘤表型的克隆分析。

上皮组织是高度组织的系统，具有显着的稳态能力，通过发育和细胞周转维持其组织。然而，这种健壮的自组织系统在肿瘤发育过程中逐渐被破坏。在肿瘤发展开始时，由癌基因激活或肿瘤抑制基因失活引起的单个突变细胞出现在上皮层内。当这种转化的"前肿瘤细胞"逃避抑制环境时，会破坏上皮组织，并开始不受控制的增殖，发生肿瘤发生1 。在过去几十年中，遗传学和分子生物学方面的杰出技术进步在癌症研究方面取得了显着进展。特别是最近在黑腹果蝇中使用遗传马赛克分析工具的研究 ，如FLP-FRT（flippase重组酶/翻转酶重组酶靶）有丝分裂重组通货膨胀2和翻转出-GAL4-UAS（上游激活序列）系统3，已经极大地促进了更好地理解参与了肿瘤4，5，6的形成和转移的遗传机制。 一组保守的果蝇肿瘤抑制基因， 致死巨幼虫 （ lgl ）， 圆盘大 （ dlg ）和涂鸦 （ scrib ）的研究突出了上皮组织损失和肿瘤发展之间的关键关系，因为这些基因发挥关键作用在上皮组织中调节顶端细胞极性和细胞增殖7 。虽然果蝇成像圆盘通常是单层上皮，这三个基因中的任何一个的纯合突变导致细胞失去结构和极性，不能形成差异过度膨胀，最终形成与相邻组织融合的多层无定形块7 。同样，这些基因在哺乳动物的破坏是参与恶性肿瘤8,9的发展。由突变体组织表现出的表型的肿瘤已导致这三个基因的分类为保守的，肿瘤性的肿瘤抑制基因（nTSGs）7,8。然而，当纯合nTSG突变细胞开发使用FLP-FRT-介导的有丝分裂重组的野生型成虫盘被零散地生成，突变的细胞从组织至c-Jun N-末端激酶（JNK） -依赖性的细胞凋亡10,11消除，12，13，14，15挤压</suP&gt;， 如图16所示 ，或通过邻居17吞没和吞噬作用。在该镶嵌基因的上皮细胞凋亡主要是在位于克隆边界nTSG突变体细胞中检测到，这表明相邻正常细胞触发nTSG突变的细胞10，11，12，18的细胞凋亡。在哺乳动物细胞中最近的研究已经证实，有利于肿瘤细胞的这种细胞依赖性竞争淘汰是对抗癌症的19，20，21，22，23的进化上保守的上皮细胞的自我防御机制。 然而，最近对果蝇成像圆盘的研究表明，镶嵌nTSG敲低克隆诱导特异性的肿瘤性肿瘤机翼成像光盘的ic区域16 。初始肿瘤形成始终存在于外周"铰链"区域，并且从未在翼盘上皮的中央"囊"区域观察到，这表明nTSG敲低细胞的致瘤潜力取决于局部环境。中央袋区域作为"肿瘤冷点"，其中肿瘤细胞不显示发育不良的过度生长，而外周铰链区则表现为"肿瘤热点" 16 。在"冷点"小袋区域，nTSG敲低细胞从基底侧分层并进行细胞凋亡。相比之下，由于"热点"铰链细胞在其基底具有强大的细胞骨架结构网络，所以nTSG敲低细胞从上皮的顶端分层并引发致瘤性过度生长16 。因此，成像光盘中肿瘤表型的分析需要仔细考虑区域特异性致瘤性刺激易感性的降容。 在这里，我们描述了使用GAL4-UAS- RNAi系统在果蝇翼成像盘中诱导肿瘤肿瘤形成的方案，通过其在正常翼盘上皮细胞中产生nTSG敲低细胞。虽然这些实验系统对于研究癌症的早期阶段是有用的，但是尚未清楚地描述用于评估成像盘上皮细胞肿瘤进展阶段的明确分类方法。因此，我们还提出了一种将翼片上皮诱导的前肿瘤克隆表型分为三类:增生（增殖增多的正常出现细胞数量过多），异常增生（异常出现的恶化前组织细胞）和瘤形成（良性或恶性肿瘤由具有异常外观和异常增殖模式的细胞组成）。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Wako</td><td>162-19321</td><td></td></tr><tr><td>TritonX-100</td><td>Wako</td><td>168-11805</td><td></td></tr><tr><td>Formaldehyde</td><td>Wako</td><td>064-00406</td><td></td></tr><tr><td>bovine serum albumin</td><td>Sigma</td><td>A7906</td><td></td></tr><tr><td>normal goat serum</td><td>Sigma</td><td>G6767</td><td></td></tr><tr><td>mounting medium, Vectashield</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542</td><td></td></tr><tr><td>mouse-anti-Dlg 4F3</td><td>Developmental Studies Hybridoma Bank</td><td>4F3 anti-discs large, RRID:AB_528203</td><td>dilute in PBTG, 1:40</td></tr><tr><td>mouse-anti-MMP1</td><td>Developmental Studies Hybridoma Bank</td><td>3A6B4, RRID:AB_579780</td><td>3 mixed 1:1:1 and dilute in PBTG, 1:40</td></tr><tr><td>mouse-anti-MMP1</td><td>Developmental Studies Hybridoma Bank</td><td>3B8D12, RRID:AB_579781</td><td>3 mixed 1:1:1 and dilute in PBTG, 1:40</td></tr><tr><td>mouse-anti-MMP1</td><td>Developmental Studies Hybridoma Bank</td><td>5H7B11, RRID:AB_579779</td><td>3 mixed 1:1:1 and dilute in PBTG, 1:40</td></tr><tr><td>mouse-anti-&lt;span class=&quot;font13&quot;&gt;a&lt;/span&gt;&lt;span class=&quot;font8&quot;&gt;tubulin&lt;/span&gt;</td><td>Developmental Studies Hybridoma Bank</td><td>AA4.3, RRID:AB_579793</td><td>dilute in PBTG, 1:100</td></tr><tr><td>Alexa Fluor 546 Phalloidin</td><td>Molecular probes</td><td>A22283</td><td>dilute in PBS, 1:40</td></tr><tr><td>goat anti-mouse IgG antibody, Alexa Fluor 546</td><td>Molecular probes</td><td>A11030</td><td>dilute in PBTG, 1:400</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Fly strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;sd-Gal4&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#8609</td><td>recombined with &lt;em&gt;&lt;span class=&quot;font11&quot;&gt;UAS-EGFP&lt;/span&gt;&lt;/em&gt;</td></tr><tr><td>&lt;em&gt;upd-Gal4&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#26796</td><td>recombined with &lt;em&gt;&lt;span class=&quot;font11&quot;&gt;UAS-EGFP&lt;/span&gt;&lt;/em&gt;</td></tr><tr><td>&lt;em&gt;UAS-lgl-RNAi&lt;/em&gt;</td><td>Vienna Drosophila RNAi center</td><td>#51247</td><td></td></tr><tr><td>&lt;em&gt;UAS-scrib-RNAi&lt;/em&gt;</td><td>Vienna Drosophila RNAi center</td><td>#105412</td><td></td></tr><tr><td>&lt;em&gt;UAS-Ras&lt;span class=&quot;font12&quot;&gt;&lt;sup&gt;V12&lt;/sup&gt;&lt;/span&gt;&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#64196</td><td></td></tr><tr><td>&lt;em&gt;UAS-Yki&lt;span class=&quot;font12&quot;&gt;&lt;sup&gt;3SA&lt;/sup&gt;&lt;/span&gt;&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#28817</td><td></td></tr><tr><td>&lt;em&gt;hsFLP&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#6</td><td></td></tr><tr><td>&lt;em&gt;Act&gt;CD2&gt;GAL4 (flip-out GAL4)&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#4780</td><td>recombined with &lt;em&gt;&lt;span class=&quot;font11&quot;&gt;UAS-EGFP&lt;/span&gt;&lt;/em&gt;</td></tr><tr><td>&lt;em&gt;UAS-EGFP&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#5428</td><td>X chromosome</td></tr><tr><td>&lt;em&gt;UAS-EGFP&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#6658</td><td>third chromosome</td></tr><tr><td>&lt;em&gt;UAS-Dicer2&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#24650</td><td>second chromosome</td></tr><tr><td>&lt;em&gt;UAS-Dicer2&lt;/em&gt;</td><td>Bloomington Drosophila Stock Center</td><td>#24651</td><td>third chromosome</td></tr><tr><td>&lt;em&gt;vkg-GFP&lt;/em&gt;</td><td>Morin et al. 2001</td><td></td><td>GFP protein trap</td></tr></tbody></table>

induction and diagnosis of tumors in drosophila imaginal disc epithelia

在癌症的早期阶段，转化的突变细胞显示细胞学异常，开始不受控制的过度生长，并逐渐破坏组织组织。 黑腹果蝇已成为癌症生物学中流行的实验模型系统，用于研究肿瘤发生的遗传和细胞机制。特别地，用于果蝇成像盘（在幼虫中发育的上皮）的遗传工具使得能够在正常上皮组织内形成转化的前肿瘤细胞，这种情况类似于人类癌症的初始阶段。然而，最近关于果蝇翼成像圆盘中的肿瘤发生的研究表明，肿瘤起始取决于组织固有的细胞结构和局部微环境，这表明在评估肿瘤表型的成像中考虑到致瘤性刺激的区域特异性易感性是重要的光盘。促进肿瘤进展的表型分析在成像光盘中，我们描述了使用GAL4-UAS系统在机翼成像光盘中诱导肿瘤性肿瘤的遗传实验方案。我们进一步介绍一种诊断方法来分类在上皮细胞中诱导的克隆性病变的表型，因为以前没有描述过用于区分肿瘤进展的各个阶段（如增生，发育不良或瘤形成）的明确分类方法。这些方法可能广泛适用于果蝇各种器官的肿瘤表型的克隆分析。

为了证实通过RNAi介导的果蝇翼成像盘中的RNAi介导的nTSG敲低实验诱导的肿瘤肿瘤形成，使用三种不同的GAL4驱动子来表达用于lgl或scrib的 UAS-RNAi :（1） sd-GAL4，其驱动强的UAS表达翼袋和铰链区域的轻度表达（ 图2和图3A ）; （2）在铰链子域中驱动的upd-GAL4，包括在背内侧折叠的两个区域中的强表达，一个前外侧结构域中的轻度表达，以及在腹 - 后结构域中的弱表达（ 图2和图3C ）;和（3）热休克诱导的翻转式GAL4，其生成表示UAS-基因（ 图4和连接的随机镶嵌克隆5）。在所有实验中， UAS-EGFP与UAS-RNAi共表达以标记敲低克隆。 首先，使用sd-GAL4驱动的lgl-RNAi诱导翼囊和铰链区域中的lgl敲低。在没有UAS-lgl-RNAi的对照三龄期翼片中 ，没有发生形态学改变（ 图3A ）。相比之下，在铰链的某些部分（ 图3B ）中清楚地观察到表达lgl-RNAi的三龄期翼片，上皮变形和致瘤性过度生长。然而，在翼袋区域，没有观察到发育不良的过度生长。在肿瘤性铰链病变中观察到强的MMP1（基质金属蛋白酶-1）表达，但不在翼囊区域（ 图3B ）。由于MMP1表达涉及基底膜的降解es，这些铰链肿瘤可能具有转移能力（ 图3B ）。接下来，使用upd-GAL4在铰链区域诱导lgl敲低。在没有UAS-lgl-RNAi的对照三龄期翼片中 ，没有发生形态学改变（ 图3C ）。然而，在5天AED后，具有upd-GAL4和lgl-RNAi的第三龄期翼片显示突出的上皮组织，其中F-肌动蛋白积聚和从上皮层的克隆置换（ 图3D ）。在6天AED时，表达MMP1的肿瘤性肿瘤在铰链区域溢出（ 图3G ），尽管样品之间肿瘤大小的变化（ 图3E 和F ）。铰链区域的垂直部分确认肿瘤肿瘤在上皮层的顶端发生偏离的生长（ 为了监测由lgl敲低诱导的肿瘤生长的进展，使用热休克诱导的翻转GAL4镶嵌系统在翼片中产生表达lgl-RNAi的散发性克隆。热休克对第二龄幼虫（2天AED）后两天，位于铰链区域中的一些LGL -knockdown克隆显示偏离上皮层（ 图4A）的顶侧远离。热休克诱导后四天，发育异常的病变在铰链区变得清晰，这表明从顶端侧位移远LGL -knockdown克隆在管腔（ 图4B）增殖。热休克诱导后7天，致瘤性过度生长主导铰链区（ 图4C ）。 展示使用我们的诊断方法分类克隆表型，使用热休克诱发的翻转GAL4拼接系统在翼片中产生表达scrib-RNAi的散发性克隆。对二龄幼虫（2天AED）进行热休克后5天，某些GFP表达的划线克隆克隆显示出致瘤性表型（ 图5A ）。我们利用ImageJ分析了z-stack共焦图像的垂直截面中的四个克隆（ 图5A中的 B，C，D和E），因为难以获得2D共聚焦图像中详细的细胞学和结构表型（ 图4和图5A ）。克隆B和C被分类为发育异常，作为隔膜连接蛋白质盘大（Dlg）被错位置（ 图5B和C ）。在克隆B中，从上皮移位的细胞块的大小不大于直径的4个细胞r（ 图5B ），而克隆C没有偏离上皮（ 图5C ）;因此，克隆B和C不被认为是肿瘤。然而，还显示Dlg的错位的克隆D和E在上皮之外形成肿瘤细胞团（ 图5D 和5E ）。由于这些置换的肿瘤克隆的大小直径超过4个细胞，所以克隆被分类为肿瘤。 为了在机翼成像盘中诱导增生性肿瘤或囊肿形成，使用热休克诱导的产生了表达非特异性 Ras（ Ras V12 ）或组合型活性形式的约克的偶发性克隆（Yorkie），Hippo途径的转录共激活因子（ Yki 3SA ）翻转GAL4马赛克系统。热休克后4天，二龄幼虫（2天AED）， Ra由GFP标记的V12表达克隆在成像盘中变成圆形细胞块（ 图6A ）。表达Ras V12的克隆（ 图6A中的克隆B）的垂直部分显示从上皮层解离的细胞质量具有Dlg的适当定位，表明细胞质量在上皮层外部维持正常的上皮结构（ 图6B ） 。在这些Ras V12表达的克隆中没有观察到MMP1的表达（ 图6C ）。因此，将克隆B分类为囊肿形成。热休克后4天（2天AED），由GFP标记的Yki 3SA表达克隆在图像盘中形成大细胞块（ 图7A ）。我们集中在两个克隆（ 图7A中的 B和C）。克隆B显示整合在上皮层中，并将Dlg适当定位在上皮细胞的亚顶膜中，这表明Yki 3SA表达的克隆B维持上皮结构。因此，克隆B被分类为增生（ 图7B ）。位于铰链区的克隆C显示Dlg的错位，并在上皮层的基底侧形成多层结构。在Yki 3SA表达克隆中没有观察到MMP1的表达。总之，将克隆C分类为良性肿瘤（ 图7C和D ）。 <img alt="图1" class="xfigimg" src="/files/ftp_upload/55901/55901fig1.jpg" /> 图1:肿瘤表型分类流程图。该流程图表示在成像上皮细胞中分类克隆病变的诊断方法。分类（1）克隆与上皮层的偏差，（2）连接蛋白的亚细胞错位，（3）细胞增殖，（4）克隆大小和（5）MMP1表达。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55901/55901fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图2" class="xfigimg" src="/files/ftp_upload/55901/55901fig2.jpg" /> 图2 :( A ）对于Dlg（绿色）和核（DAPI，品红色）染色的野生翼型成像圆盘。 （ B ）显示翼袋（蓝色），铰链（橙色）和墨水（绿色）区域的果蝇翼成像盘的示意图。 （ C ）F-肌动蛋白（红色）和微管（绿色）染色的正常机翼成像圆盘。基底膜用胶原IV / Vkg-GFP（蓝色）标记。 （ D ）现场垂直剖面（ C ）中标有白色虚线。 （ E ）线图描绘（ D ）上皮层的顶点（红色）和基底（蓝色）侧。虚线追踪周边膜。指示背侧铰链的远端（Df），内侧（Mf）和近端（Pf）折叠。 详细基因型: A， w - CD， Vkg-GFP <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55901/55901fig2large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图3" class="xfigimg" src="/files/ftp_upload/55901/55901fig3.jpg" /> 图3:由加强剂-GAL4诱导的位点特异性肿瘤发生。 （ A ， B ）共聚焦图像显示从MMP1（红色）染色的指示基因型的第三龄期幼虫解剖的翼形成像圆盘。 sd-GAL4- expre通过GFP表达（绿色）标记ssing区域。 （ CG ）共焦图像显示在指定的时间点AED处从指定基因型的第三龄幼虫解剖的翼形图像圆盘。 F-肌动蛋白用（ CF ）中的鬼笔环肽（红色）染色。 （ G ）中的抗MMP1抗体（红色）对MMP1表达进行染色。通过GFP表达（绿色）标记upd-GAL4表达区域。 （ CF ）的底板显示了上图所示区域的放大视图。白色箭头表示由lgl- Knockdown克隆诱导的发育不良病变。 （ H ）（E）底面板上标有白色虚线的场地垂直剖面。白色虚线标记（A）和（B）中翅膀袋和铰链区之间的边界。核用DAPI（蓝色）标记。刻度棒=（AG）为100μm，（H）为50μm。详细基因型: A， w - ，sd-GAL4，UAS-EGFP; UAS-dicer2; B， w - ，sd-GAL4，UAS-EGFP; UAS-dicer2; UAS-LGL-RNA干扰; C， w - ，upd-GAL4，UAS-EGFP; UAS-dicer2; DH， w - ，upd-GAL4，UAS-EGFP; UAS-dicer2; UAS-lgl-RNAi <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55901/55901fig3large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图4" class="xfigimg" src="/files/ftp_upload/55901/55901fig4.jpg" /> 图4:随机马赛克克隆诱导的肿瘤表型。 （ AC ）共焦图像显示了在热休克诱导后的指定时间点，共同表达lgl-RNAi和GFP（绿色）的克隆的三龄幼虫的马赛克翼成像盘。核被DAPI（蓝色）标记。中间面板展示了左侧面板标记的白色正方形区域的放大视图。右侧面板<html>显示了上皮层（蓝色）顶端侧和GFP表达克隆（绿色）的示意图。位于上皮内的马赛克克隆以浅绿色描绘，而与上皮相反的肿瘤性肿瘤显示为品红色。左侧面板上的白色虚线表示机翼袋和铰链区域之间的边界。在中间的面板白色箭头指示不典型增生病变LGL -knockdown克隆诱导。刻度棒=100μm。详细基因型: AC， hsFLP; UAS-dicer2; GAL4，UAS-EGFP / UAS-lgl-RNAi （2天AED热休克30分钟，A组25℃孵育2天，B组培养4天，孵育7天，热休克后25℃）。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55901/55901fig4large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 5"class ="xfigimg"src ="/ files / ftp_upload / 55901 / 55901fig5.jpg"/&gt; 图5:发育不良的克隆表型的诊断检查。 （ A ）左图显示了热休克诱导后5天，共同表达scrib-RNAi和GFP（绿色）的克隆的三龄幼虫的马赛克翼成像盘，染色Dlg（红色）。右图示出上皮层（蓝色）顶端侧和GFP表达克隆（绿色）的示意图。上皮层中的马赛克克隆显示为浅绿色，而从上皮移位的肿瘤性肿瘤以品红色显示。 （ BE ）共焦图像显示（A）右图所示的划线 -克隆克隆的垂直截面。左边的第二个面板显示了Dlg定位模式（红色）。左侧第三个面板显示上皮层的顶端（红色）和基底（蓝色）侧的原始图线，GFP-expressing scrib- knockdown克隆（绿色）。上皮层中的发育异常克隆显示为浅绿色（B和C），而从上皮移位的肿瘤性克隆显示为品红色（D和E）。白色箭头表示显示致瘤性表型的划线 - 敲低克隆。核用DAPI（蓝色）标记。刻度棒=50μm。根据图1所示的流程图对每个克隆的肿瘤表型进行分类。详细基因型: AE， hsFLP; UAS-Scrib的，RNA干扰; act&gt; CD2&gt; GAL4，UAS-EGFP （在2天AED热休克30分钟，热休克后25℃孵育4天）。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55901/55901fig5large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图6" class="xfigimg" src="/files/ftp_upload/55901/55901fig6.jpg" /> 图6:诊断Ras V12表达克隆表型的tic检验。 （ A ）左侧面板显示了在热休克诱导后4天，共表达致癌性Ras V12和GFP（绿色）的克隆的三龄幼虫的马赛克翼成像盘，对Dlg（红色）染色。右图示出上皮层（蓝色）顶端侧和GFP表达克隆（绿色）的示意图。刻度棒表示50μm。 （ B ）（A）右图所示的Ras V12表达克隆的垂直切片。左边的第二个面板显示了Dlg定位模式（红色）。左侧第三组显示上皮层顶点（红色）和基底（蓝色）两侧以及Ras V12和GFP表达克隆（绿色）的示意图。比例尺= 25μm。 （ C ）三龄幼虫的马赛克翼成像盘与克隆共表达em&gt; Ras V12和GFP（绿色）热休克诱导后4天，染色MMP1（红色）。刻度棒=100μm。详细基因型: AC， hsFLP; UAS-Ras V12 ; GAL4，UAS-EGFP （2天AED热休克45分钟，热休克后25℃孵育4天）。 <a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55901/55901fig6large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> <img alt="图7" class="xfigimg" src="/files/ftp_upload/55901/55901fig7.jpg" /> 图7: Yki 3SA表达克隆表型的诊断检查。 （ A ）左图显示了在热休克诱导后4天共同表达组成型活性Yki 3SA和GFP（绿色）的克隆的三龄幼虫的马赛克翼成像盘，染色Dlg（红色）。右侧面板显示为<html>上皮层（蓝色）顶端侧的化学线条图和GFP表达克隆（绿色）。刻度棒=50μm。 （ BC ） Yki 3SA表达克隆的垂直切片在（A）的右图所示。左边的第二个面板显示了Dlg定位模式（红色）。左侧第三组显示上皮层顶点（红色）和基底（蓝色）两侧以及Yki和GFP表达克隆（绿色）的示意图 。比例尺= 25μm。 （ D ）在热休克诱导4天后，共同表达Yki 3SA和GFP（绿色）的克隆的三龄幼虫的马赛克翼成像盘被染色为MMP1（红色）。刻度棒=100μm。详细基因型: AD， hsFLP ;; UAS-YKI 3SA / act&gt; CD2&gt; GAL4，UAS-EGFP （2天AED热休克30分钟，25℃孵育4天）。5901fig7large.jpg"target ="_ blank"&gt;请点击此处查看此图的较大版本。

Watch this Scientific Journal Video about Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia at JoVE.com

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

果蝇成像盘上皮的马赛克克隆分析是研究肿瘤发生的遗传和细胞机制的有力模型系统。在这里，我们描述了使用GAL4-UAS系统在果蝇翼成像盘中诱导肿瘤的方案，并介绍了分类肿瘤表型的诊断方法。

诱导和诊断肿瘤<em&gt;果蝇</em&gt; Imaginal Disc Epithelia

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue ...

induction-diagnosis-tumors-drosophila-imaginal-disc

Nanyang Technological University

Research

JoVE Journal

Cancer Research

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

14.8K 次观看.  SOKENDAI.  果蝇成像盘上皮的马赛克克隆分析是研究肿瘤发生的遗传和细胞机制的有力模型系统。在这里，我们描述了使用GAL4-UAS系统在果蝇翼成像盘中诱导肿瘤的方案，并介绍了分类肿瘤表型的诊断方法。 

视频: Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

新一代测序为可操作的突变在固体和液体瘤检测

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

科研

癌症研究

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

该<em&gt;果蝇</em&gt;成虫盘瘤模型:基因的表达与肿瘤侵袭性的可视化和定量遗传马赛克

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

从3龄果蝇幼虫的成虫光盘夹层

生物学

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

乳腺癌细胞系外周血单核细胞破的人前临床模型的建立

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

病人衍生软组织肉瘤原代培养的建立

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Drosophila has illuminated our understanding of the genetic basis of normal development and disease for the past several decades and today it continues to contribute immensely to our understanding of complex diseases 1-7. Progression of tumors from a benign to a metastatic state is a complex event 8 and has been modeled in Drosophila to help us better understand the genetic basis of this disease 9. Here I present a simple protocol to genetically induce, observe and then analyze the progression of tumors in Drosophila larvae. The tumor induction technique is based on the MARCM system 10 and exploits the cooperation between an activated oncogene, RasV12 and loss of cell polarity genes (scribbled, discs large and lethal giant larvae) to generate invasive tumors 9. I demonstrate how these tumors can be visualized in the intact larvae and then how these can be dissected out for further analysis. The simplified protocol presented here should make it possible for this technique to be utilized by investigators interested in understanding the role of a gene in tumor invasion.

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Cooperation between an activated oncogene like RASV12 and mutations in cell polarity genes like scribbled, result in tumor growth in Drosophila where tumor cells also display invasive behaviors. Here a simple protocol for the induction and observation of the benign and invasive tumors is presented.

一个协议，用于基因诱导和良性和浸润性肿瘤可视化的头侧配合<em&gt;果蝇</em

Drosophila has illuminated our  understanding of the genetic basis of normal development and disease for the  past several decades and today it continues ...

医学

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

普帕尔阶段发育性皮瘤中组织定向和生长动力学的成像与分析

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

发育生物学

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

果蝇蛹腹部免疫

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

解剖和免疫染色成虫盘的<em&gt;果蝇</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

Drosophila Larva Imaginal Disc Dissection: A Method to Observe Developing Epithelia

Source: Morimoto, K. and Tamori, Y. <a href="https://www-jove-com.remotexs.ntu.edu.sg/video/55901/" target="_blank">Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia</a>. J. Vis. Exp. (2017).
When staining Drosophila imaginal discs, often a rough dissection is performed where the discs are exposed but remain attached to the body. Once stained, the discs of interest are isolated and mounted. The example protocol demonstrates a procedure for wing imaginal disc dissection and mounting.

果蝇幼虫假想盘清扫术:一种观察发育中的上皮细胞的方法

Source: Morimoto, K. and Tamori, Y. Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia. J. Vis. Exp. (2017).
When staining Drosophila ...

诱导和诊断肿瘤

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