This work was funded by a Wellcome Trust Sir Henry Dale Fellowship to Y. F. 100104/Z/12/Z.
We would like to thank Dr. Carl Tucker and the team of the fish facility at the Queen&#8217;s Medical Research Institute, University of Edinburgh. We like to thank Michael Millar of the immunohistology and imaging facility of the MRC Centre for Reproductive Medicine, University of Edinburgh for support.
We thank Prof. Dr. Matthias Hammerschmidt and Dr. Heiko Loehr of the Institute of Developmental Biology, University of Cologne for providing the p5E-krt4 entry vector. We also would like to thank Dr. Dirk Sieger of the Centre of Neuroregeneration, Univerity of Edinburgh for providing the pDestTol2CG destination vector.

下面的实验过程是在严格按照动物（科学程序）1986年法令进行。 1.制备质粒DNA的显微注射<ol><li>根据制造商的说明eGFP的用市售的质粒DNA纯化试剂盒和稀质粒DNA至最终原液100毫微克/微升浓度:;:纯化质粒pTol2-krt4 cmlc2 KalTA4-ER T2。 </li><li>线性化含有质粒pT3TS / TOL2 14由BamHI限制性消化的转座，按照制造商的协议。这一步可以在一夜之间被延长。通过苯酚/氯仿萃取沉淀出的DNA线性化，按照标准协议。 </li><li> 在体外 ，根据制造商的说明从线性化质粒用市售转录试剂盒用于大量加帽的RNA的转录转座的mRNA，稀释并等分至100毫微克/微升的无核酸酶水的最终原液浓度。确保纯化的RNA的质量与天然1-2％TAE（40毫摩尔Tris，20mM的醋酸，1mM EDTA）中的琼脂糖凝胶电泳（80-120Ⅴ）。存储RNA在-80℃。 </li></ol> 2.显微注射质粒DNA的瞬态基因表达<ol><li>准备琼脂糖注入板块注入期间举行的受精卵。倒液3％琼脂糖成90毫米的培养皿。让琼脂糖冷却至40-50℃并添加楔形注射模具的TU-1上的液体琼脂糖的顶部。硬化后在室温下，让其余琼脂糖，在4℃下30分钟去除模具之前。商店显微注射模具，在4℃和重用。 </li><li>制备的注射剂溶液的等分试样。执行在冰上以下步骤。吸管1微升质粒DNA（10毫微克最终浓度/微升），2微升转座的mRNA（终浓度20纳克/微升），2的微升10毫克/微升罗丹明B异硫氰酸酯-葡聚糖，2微升5×Danieau溶液（290 mM氯化钠，3.5 mM的氯化钾，2mM的硫酸镁 ，3毫米的Ca（NO 3）2，25毫米的HEPES，pH值7.6）至一个的10微升在无核酸酶水最终体积。 </li><li> 1毫米直径的拉毛细管相反的方向用微量普勒含有一个内灯丝硼硅玻璃毛细管准备针。使用下面的程序:530热，拉200，速度80和时间150。 </li><li>填充一个针与2微升的注射溶液（保持在冰上）。通过仔细取出微加载尖端同时释放溶液进入毛细管避免气泡。小心打破用钳子将针的尖端。 </li><li>调节用气动微微泵的液滴尺寸。测量水滴直径/体积（V = 4/3πR3）中，用下在二甲基聚硅氧烷一个立体镜的目镜测微调节液滴尺寸。我们推荐100微米，相当于0.5 NL。这保证了每个每个注射胚胎统一和可重复的浓度。滴定注入的浓度，并调整每个结构和质粒的准备。 </li><li>放置从Tg的交获得的受精卵（ 图1A）（UAS:EGFP-HRAS G12V）io006 15 与TG（型lysC:的DsRed2）nz50Tg 16进入注入板的模具通过使用3毫升pastette的。同时，保持在预制备的注射针的尖端在二甲基聚硅氧烷，以避免从干涸和凝血的溶液。 </li><li>注入下一个立体镜的预定量下降（100微米相当于0.5 NL）通过绒毛膜进入胚盘17之前，所述第一切割（ 图1A）。注射入单细胞阶段增加了质粒DNA和RNA中的显影单元的质量和的可能性种系传播的均匀分布的可能性。这是值得注意的是结构的表达往往趋于马赛克。 </li></ol>由4-羟基3.有条件的皮肤细胞转化（4-OHT） <ol><li>转移注射的胚胎成0.3X Danieau溶液并保持在28.5℃的培养箱中培养。从盘，在球体阶段17取出未受精的卵子。在共注射罗丹明B异硫氰酸酯 - 葡聚糖可以用作下一个荧光立体镜的注塑控制。 </li><li>屏幕胚胎在24 HPF（小时受精后）的绿色荧光蛋白的表达心脏。继续提高CMLC:绿色荧光蛋白阳性的斑马鱼胚胎，直到72高倍视野。仔细监测水质（0.3X Danieau）。 </li><li>除去那些没有在72 HPF斜线与钳胚胎绒毛膜。小心捅绒毛膜1镊子和利用其他把它分开。利用荧光体视镜的DsRed2表达:为lysC的选择胚胎。 </li><li>加入10微升10毫米库存（Z）-4-羟基他至20毫升0.3×Danieau溶液（5μM终浓度）。使用一个标准的培养皿（90 mm）输入4-OHT感应解决方案上一批40-60胚胎。 </li><li>胚胎转移到含有20毫升新鲜的感应解决方案，新的培养皿中。的4-OHT必须存储并在黑暗中处理。表达式的第一个迹象可能会检测到2个小时后的诱导开始。归纳步可以在一夜之间被扩展，牢记最初的宿主细胞:癌前细胞间的相互作用可以治疗后已经发生了几个小时。 </li></ol> 4.安装和斑马鱼幼虫的实时成像<ol><li>通过引入孔的直径为18-20毫米制备60毫米培养皿。使用高真空硅脂以覆盖和密封的培养皿25毫米玻璃盖（ 图1B）的外底部的孔中。 </li><li>制备1％的低熔点（LMP）琼脂糖。保留一个1.5毫升管的LMP琼脂糖，在37℃的加热块中。 </li><li>预选的Tg（krt4:KalTA4-ER T2; UAS:EGFP-HRAS G12V; lysC的:的DsRed2）幼虫的绿色荧光皮肤细胞和积极的DsRed2先天免疫细胞表达，在荧光体视镜。通过加入1毫升的0.4％三卡因18向20毫升感应溶液麻醉幼虫。 </li><li>传输与pastette进液LMP琼脂糖预选的幼虫。让幼虫沉入LMP琼脂糖，并将它们转移到玻璃盖（ 图1B）。定向下一个立体镜的胚胎。幼虫必须直接放置在玻璃，以减少工作距离（最多6个幼虫）。 </li><li>覆盖LMP琼脂糖含感应解决方案后，已硬化（ 图1B箭头）三卡因。 </li><li>图像使用倒置荧光，激光扫描或旋转盘共聚焦显微镜的嵌入式幼虫。使用40X或63X目标与长工作距离目标启用集中在整个幼虫。 </li></ol>

<ol>
	<li>Kajita, M., Sugimura, K., 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=Filamin+acts+as+a+key+regulator+in+epithelial+defence+against+transformed+cells.">Filamin acts as a key regulator in epithelial defence against transformed cells.</a> Nature communications. 5, 4428 (2014).</li><li>Feng, Y., Santoriello, C., Mione, M., Hurlstone, A., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+innate+immune+cell+sensing+of+transformed+cells+in+zebrafish+larvae:+parallels+between+tumor+initiation+and+wound+inflammation.">Live imaging of innate immune cell sensing of transformed cells in zebrafish larvae: parallels between tumor initiation and wound inflammation.</a> PLoS Biology. 8 (12), e1000562 (2010).</li><li>Feng, Y., Renshaw, S., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+Tumor+Initiation+in+Zebrafish+Larvae+Reveals+a+Trophic+Role+for+Leukocyte-Derived+PGE+2.">Live Imaging of Tumor Initiation in Zebrafish Larvae Reveals a Trophic Role for Leukocyte-Derived PGE 2.</a> Current Biology. 22 (13), 1253-1259 (2012).</li><li>Halpern, M. E., Rhee, J., Goll, M. G., Akitake, C. M., Parsons, M., Leach, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gal4/UAS+transgenic+tools+and+their+application+to+zebrafish.">Gal4/UAS transgenic tools and their application to zebrafish.</a> Zebrafish. 5 (2), 97-110 (2008).</li><li>Gong, Z., Ju, 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=fluorescent+protein+expression+in+germ-line+transmitted+transgenic+zebrafish+under+a+stratified+epithelial+promoter+from+keratin8.">fluorescent protein expression in germ-line transmitted transgenic zebrafish under a stratified epithelial promoter from keratin8.</a> Developmental Dynamics an Official Publication of the American Association of Anatomists. 223 (2), 204-215 (2002).</li><li>Distel, M., Wullimann, M. F., K&#246;ster, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+Gal4+genetics+for+permanent+gene+expression+mapping+in+zebrafish.">Optimized Gal4 genetics for permanent gene expression mapping in zebrafish.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (32), 13365-13370 (2009).</li><li>Feil, R., Wagner, J., Metzger, D., Chambon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Cre+recombinase+activity+by+mutated+estrogen+receptor+ligand-binding+domains.">Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains.</a> Biochemical and Biophysical Research Communications. 237 (3), 752-757 (1997).</li><li>Gerety, S. S., Breau, M. a., Sasai, N., Xu, Q., Briscoe, J., Wilkinson, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inducible+transgene+expression+system+for+zebrafish+and+chick.">An inducible transgene expression system for zebrafish and chick.</a> Development. 140 (10), 2235-2243 (2013).</li><li>Yuan, S., Sun, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+mRNA+and+Morpholino+Antisense+Oligonucleotides+in+Zebrafish+Embryos.">Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.</a> J. Vis. Exp. (27), e1113 (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+Zebrafish+Embryos+to+Analyze+Gene+Function.">Microinjection of Zebrafish Embryos to Analyze Gene Function.</a> J. Vis. Exp. (25), e1115 (2009).</li><li>Eisenhoffer, G. T., Rosenblatt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+Imaging+of+Cell+Extrusion+from+the+Epidermis+of+Developing+Zebrafish.">Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish.</a> J. Vis. Exp. (52), e2689 (2011).</li><li>Kague, E., Weber, C., Fisher, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mosaic+Zebrafish+Transgenesis+for+Evaluating+Enhancer+Sequences.">Mosaic Zebrafish Transgenesis for Evaluating Enhancer Sequences.</a> J. Vis. Exp. (41), e1722 (2010).</li><li>Balciuniene, J., Gene Balciunas, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trapping+Using+Gal4+in+Zebrafish.">Trapping Using Gal4 in Zebrafish.</a> J. Vis. Exp. (79), e50113 (2013).</li><li>Balciunas, D., Wangensteen, K. 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=Harnessing+a+high+cargo-capacity+transposon+for+genetic+applications+in+vertebrates.">Harnessing a high cargo-capacity transposon for genetic applications in vertebrates.</a> PLoS Genetics. 2 (11), e169 (2006).</li><li>Santoriello, C., Gennaro, E., 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=Kita+driven+expression+of+oncogenic+HRAS+leads+to+early+onset+and+highly+penetrant+melanoma+in+zebrafish.">Kita driven expression of oncogenic HRAS leads to early onset and highly penetrant melanoma in zebrafish.</a> PloS One. 5 (12), e15170 (2010).</li><li>Hall, C., Flores, M. V., Storm, T., Crosier, K., Crosier, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+lysozyme+C+promoter+drives+myeloid-specific+expression+in+transgenic+fish.">The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish.</a> BMC Developmental Biology. 7, 42 (2007).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Developmental Dynamics : an Official Publication of the American Association of Anatomists. 203 (3), 253-310 (1995).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> THE ZEBRAFISH BOOK: A guide for the laboratory use of zebrafish (Danio rerio). , (2007).</li><li>Le Guellec, D., Morvan-Dubois, G., Sire, J. -. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+development+in+bony+fish+with+particular+emphasis+on+collagen+deposition+in+the+dermis+of+the+zebrafish+(Danio+rerio).">Skin development in bony fish with particular emphasis on collagen deposition in the dermis of the zebrafish (Danio rerio).</a> The International Journal of Developmental Biology. 48 (2-3), 217-231 (2004).</li><li>Kwan, K. M., Fujimoto, E., 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=The+Tol2kit:+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs.">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Developmental Dynamics : an Official Publication of the American Association of Anatomists. 236 (11), 3088-3099 (2007).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MultiSite+Gateway+Three-+Fragment+Vector+Construction+Kit.">MultiSite Gateway Three- Fragment Vector Construction Kit.</a> Protocols and Product Manuals. 12537-023, (2010).</li><li>Petersen, L. K., Stowers, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Gateway+MultiSite+Recombination+Cloning+Toolkit).">A Gateway MultiSite Recombination Cloning Toolkit).</a> PLoS ONE. 6 (9), e10 (2011).</li><li>Magnani, E., Bartling, L., Hake, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Gateway+to+MultiSite+Gateway+in+one+recombination+event.">From Gateway to MultiSite Gateway in one recombination event.</a> BMC Molecular Biology. 7, 46 (2006).</li><li>Robertson, D. W., Katzenellenbogen, 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=Synthesis+of+the+(E)+and+(Z)+isomers+of+the+antiestrogen+tamoxifen+and+its+metabolite,+hydroxytamoxifen,+in+tritium-labeled+form.">Synthesis of the (E) and (Z) isomers of the antiestrogen tamoxifen and its metabolite, hydroxytamoxifen, in tritium-labeled form.</a> The Journal of Organic Chemistry. 47 (12), 2387-2393 (1982).</li><li>Murphy, C., Langan-Fahey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-function+relationships+of+hydroxylated+metabolites+of+tamoxifen+that+control+the+proliferation+of+estrogen-responsive+T47D+breast+cancer+cells+in+vitro.">Structure-function relationships of hydroxylated metabolites of tamoxifen that control the proliferation of estrogen-responsive T47D breast cancer cells in vitro.</a> Molecular. 38 (5), 737-743 (1990).</li><li>Furr, B. J. A., Jordan, V. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pharmacology+and+clinical+uses+of+tamoxifen.">The pharmacology and clinical uses of tamoxifen.</a> Pharmacology &amp; Therapeutics. 25 (2), 127-205 (1984).</li><li>Akerberg, A. a., Stewart, S., Stankunas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+Temporal+Control+of+Transgene+Expression+in+Zebrafish.">Spatial and Temporal Control of Transgene Expression in Zebrafish.</a> PloS One. 9 (3), e92217 (2014).</li><li>Hans, S., Kaslin, J., Freudenreich, D., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporally-controlled+site-specific+recombination+in+zebrafish.">Temporally-controlled site-specific recombination in zebrafish.</a> PloS One. 4 (2), e4640 (2009).</li></ol>

The authors declare that they have no competing financial interests.

在胚胎和幼虫发育，胚胎斑马鱼的皮肤是由两个上皮的层，所述表面层被称为周皮和一层基底角质形成细胞的附着到底层基底膜19（ 图1C，一 ）。这里介绍的协议描述了一个简单的方法来有条件地诱导在斑马鱼幼体的浅表皮肤层癌前细胞转化，并允许后续的交互分析先天免疫细胞的实时成像。在我们的协议的基本方法，遵循公认的斑马鱼技巧9 - 13，和我们的协议可以很容易地调整和修改。 所述krt4启动子5此处使用驱动KalTA4-ER T2表达特别是在最外层皮肤层（ 图1C中的B）。然而，模块化多点会议网关克隆策略，用于生成krt4:KalTA4-ER T2构建体（ 图1D，a）中 ，提供了克隆中KalTA4-ER T2的前面任何感兴趣的启动子在单克隆步骤的可能性。本文中所呈现的方法的一个适应因此，有可能对胚胎和幼虫阶段期间大多数组织中。启动子序列的可用性现的限制因素。此外，克隆的后面一个的UAS兴趣几乎任何基因可以有条件通过使用在空间和时间上受控KalTA4-ER T2表达的过表达在不同的发育阶段。因此，我们的协议可以适于在斑马鱼幼体感兴趣的任何组织执行条件的基因表达，在镶嵌的方式。你也可以优化显微镜为了生活图像深层组织如肝胰脏或设置。 我们已经描述了使用协议，他一个应用程序再，同样地，人们可以过表达其他炎症调节剂使用该协议来研究的炎症反应的调节，因为可以很容易地实时图像的中性粒细胞和下列中一个候选炎症调节剂的表达的诱导和巨噬细胞阻滞行为变化。此外，适于协议也将是有益的，谁想要跟踪期间组织形态发生和器官的形成和不同阶段的细胞运动和行为改变，最后实时图像与其他特定细胞谱系的先天免疫细胞相互作用的相互作用，可以有针对性由可诱导的KalTA4-ER T2 / UAS表达系统。 我们使用pDestTol2CG向量从斑马鱼Tol2kit 20 - 23，其中包含一个cmlc2:EGFP-pA的录像带（ 图1D，一个 ），它允许随后选择胚胎的绿色荧光积极心中的本质经文标记20，简化了F0的筛选过程。感兴趣进基因组的转座子侧翼基因的稳定插入由共注射所述质粒DNA一起转座基因促进。质粒DNA的制备和转座基因显微注射遵循标准协议。然而，该构建入基因组的成功整合取决于TOL2基于换位14。这凸显支付的特别注意无菌条件下冰的工作，以避免污染和退化由RNA酶和DNA酶。显微注射到单细胞阶段的胚胎是一个强大的和成熟的技术用于增益和功能研究的损失在斑马鱼9,10。虽然一个训练有素的人可以很容易地注入到超过1000的胚胎在1小时的蛋黄中，注入胚盘（ 图1A，星号）需要更多的经验和训练。在此过程中关键的IS中的针的处理。针必须非常薄，以穿透无损伤的细胞，因此具有仅在它的顶端被打破。它定期控制和喷射期间测量液滴尺寸，以保证均匀的注入到每个胚胎是重要的。小心处理，针可被用来旋转胚胎。这通常需要找到胚盘和最优穿透角。 DNA和转座的mRNA用于注射几乎一定的浓度影响表达效率。更高的剂量可导致细胞表达该转基因的增加的比例，但是具有更高的浓度的DNA（&gt; 50纳克/微升）和转座的mRNA（&gt; 100毫微克/微升）也之间注射的胚胎毒性效应的潜力真的出现。我们倾向于使用10纳克/微升DNA和20纳克/微升转座的mRNA注射，其对应于50皮克质粒DNA和每注入EMB​​ 100pg的核糖核酸的坂。胚胎注射这些浓度为100％的人正常发育，和40-70％之间显示EGFP-HRAS G12V表达后的4-OHT诱导，这取决于注入效率的单电池。之中阳性胚胎，我们通常发现，具有1-10个克隆约30％，40％具有10-50个克隆和30％的具有多于50个克隆。由于我们的研究目的，我们赞成使用胚胎表达EGFP-HRAS G12V较少的克隆。我们选择用胚胎克隆10-50为我们短暂的实验。对于转基因鱼创始人的产生，我们建议，只选择胚胎两者的eGFP阳性心脏标志物，并在其表皮的大量的eGFP-HRAS G12V阳性克隆。 （Z）-4-羟基他经历顺-反式（EZ）互当暴露于光24。结果发现，顺式异构体（E）的100倍以下的抗雌激素比其反式对口25,26。 Exposur因此E要轻，必须避免。溶解在乙醇中的4-OHT原液可以储存在-20℃下在黑暗中在很长一段时间。我们建议使用一箱保护培养皿从光孵化器和传输中的靶基因诱导过程。虽然成像的面积是非常小的和4-OHT于UV光曝光被降低到最低限度，感应溶液不断地交流，每2小时，同时成像在更长的时间周期，建议以保持最大表达水平。的4-OHT有效permeabilizes通过绒毛膜和期间斑马鱼胚胎发生的最外皮肤层，因此它可诱导基因表达的非常迅速。我们可以在2小时的诱导后容易观察的eGFP发射并且已经报道了靶基因表达的最初迹象可以在1小时高原后3小时治疗8后进行观察。该差异可能是由于在我们的研究中所使用的不同的启动子。由于一直公关eviously报道，4-OHT处理是剂量依赖性的8,27，我们选择使用5μM的最高报道的浓度，以实现我们的瞬时方法鲁棒和可再现的表达水平。这应该保证携带外源基因的所有单元格将诱导靶基因的表达。 它必须被考虑到，这里给出的瞬态方法可能会导致不同的表达水平由于多次和随机基因组插入事件的可能性。此外，在的Tg（UAS:EGFP-HRAS G12V）的TOL2转基因背景中使用的转座的mRNA io006可导致所述转基因的UAS可能导致基因沉默或破坏的潜在换位。然而，如这里介绍的方法是一种瞬时的方式，预先选择后续胚胎注射是强制性的。许多实验室已发现的UAS序列往往被沉默在转基因后代，因此在jecting的pTol2-krt4:KalTA4-ER T2; cmlc2:绿色荧光蛋白构建成的Tg（UAS:EGFP-HRAS G12V）io006胚胎可能不一样有效，注射UAS构建成的Tg（krt4:KalTA4-ER T2; cmlc2:GFP ）胚胎。使用稳定的转基因系的Tg:与TG（UAS:EGFP-HRAS G12V）（krt4 KalTA4-ER T2）交叉io006将允许一个精确的监测时间剂量依赖性的4-OHT基因激活的通/断的动力学。 幼虫的安装过程中的一个关键步骤是转移到LMP琼脂糖并到达盖玻璃（ 图1B）。只存在于液体LMP琼脂糖温度短时间帧，其允许安全预选幼虫转移和取向，而不由任何热冲击或损害鱼硬化的凝胶。幼虫应直接定向到盖玻璃，以减少用于随后微观ANALY工作距离SIS。因为这可以显微镜中是一个限制因素的适当目标的选择是必需的。一旦安装，幼虫可维持从数小时到数天。 本文介绍的模型系统的条件允许通过4-OHT激活转基因的重复改造。表达系统依赖于4-OHT的存在，因此，所关注的基因的KalTA4控制的表达是可逆的（ 图1D，b）中 。与此相反的酶Cre-ER T2 / lox系统，这有利于一个不可逆转的基因组重组事件28，KalTA4-ER T2 / UAS可以被激活并在加入或除去的4-OHT停用并且此潜在的重复感应是的一个优点该技术在酶Cre-ER T2 /液氧系统。然而，对于4-OHT恒定水平的要求是一个限制，如果在实验，必须从几天延长到几个星期。

一个癌前细胞后代的其他正常上皮片内的过度生长在很大程度上取决于其微环境的相互作用。其宿主组织的相互作用是可能的癌前细胞是否能够建立一个克隆利基进一步发展成完全成熟的癌症的主要决定因素。尽管在发展中的重要性，癌症进展的初始阶段是不可访问中最常用的模型系统， 体内 。 斑马鱼， 斑马鱼 ，是一种行之有效的模式生物，因为它的整个发展的透明度，有可能为遗传操作和可访问性的水溶性药物实时成像研究。利用幼虫斑马鱼的模型，过表达人癌基因HRAS G12V转化上皮细胞最近的研究，结果表明宿主细胞衍生的信号，尤其是H 2 O 2引发招聘先天免疫细胞。有趣的是，招募免疫细胞，中性粒细胞和巨噬细胞，显示出具有在支持癌前细胞增殖2,3-一个营养作用。 为了了解肿瘤起始和进展的早期事件，以及炎症应答的贡献，需要能够从图像中最早的时间点起，这些事件。因此，有必要控制细胞转化中的时间和组织特异性方式。我们利用Gal4的/ UAS系统已成功地适应从果蝇 4有条件地表达在皮肤特异性启动子的角蛋白4（krt4）的控制下的人类癌基因HRAS G12V 5。转录激活Gal4的，即KalTA4 6的修改后的版本，使得上游活化序列的控制（UAS下HRAS G12V的表达）。有条件地诱导的转录活化因子的表达，KalTA4已融合的人雌激素受体​​α的突变体的配体结合结构域（ER T2）7，其结合特异性4-羟基（4-OHT）1。在不存在的4-OHT对ER T2由热休克蛋白结合的细胞质中。当4-OHT结合热休克蛋白解离，允许KalTA4-ER T2的核易位和随后的激活控制的兴趣8基因的UAS的（ 图1C，b）中 。作为这种表达系统依赖于4-OHT的存在下，所关注的基因的KalTA4控制的表达是可逆的。与此相反的酶Cre-ER T2 / lox系统，这导致一个不可逆的重组事件，转录激活KalTA4-ER T2的感应是可逆通过加入或除去的4-OHT。 以下协议异体<html>皮肤细胞在斑马鱼幼体WS条件改造和后续监测与先天免疫细胞荧光蛋白表达的相互作用。在这个协议中采用的基本方法是相似的一些斑马鱼社区内9常用技术- 13。 的产生和组合的使用的转基因系与转基因构建体的显微注射一起使瞬变的方法来在镶嵌方式仅转化的单个细胞。胚胎和幼虫斑马鱼的皮肤的氧气渗透率提高的可能性，通过从小时高分辨率显微镜来天的时间推移实时成像研究。此外，其可以水溶性药物将允许未来的机理研究和体内细胞生物学事件，它可能导致一个更好的了解癌症发展的早期阶段的监测。 _content"&gt;此协议可以适于在斑马鱼幼体感兴趣的任何组织执行条件的基因表达，在镶嵌的方式，其中一个还可以优化显微镜为了生活图象深层组织比皮肤的其它设置。

<table><tbody><tr><td>(Z)-4-Hydroxytamoxifen</td><td>Sigma Aldrich</td><td>H7904</td><td>dilute in ethanol and store in the dark at -20&amp;nbsp;&amp;deg;C</td></tr><tr><td>20X Objective</td><td>Zeiss</td><td></td><td>20X/0.5 Ph2 &amp;infin;/0.17</td></tr><tr><td>40X Objective</td><td>Zeiss</td><td></td><td>40X/1.2W Korr &amp;infin;/0.14-0.18</td></tr><tr><td>63X Objective</td><td>Zeiss</td><td></td><td>63X/1.4 Oil Ph3 &amp;infin;/0.17</td></tr><tr><td>BamHI</td><td>New England Biolabs</td><td>R0136S</td><td></td></tr><tr><td>Confocal Microscope</td><td>Zeiss</td><td></td><td>LSM 510 META</td></tr><tr><td>Cover glass</td><td>VWR</td><td>631-0171</td><td>Diameter 25 mm</td></tr><tr><td>Dimethylpolysiloxane</td><td>Sigma Aldrich</td><td>DMPSV</td><td></td></tr><tr><td>Dow Corning high-vacuum silicone grease&amp;nbsp;</td><td>Sigma Aldrich</td><td>MKBL4135V</td><td></td></tr><tr><td>Flaming/Brown P-97 Micropipette Puller</td><td>Sutter Instruments Co.</td><td>P-97</td><td>Program: heat 530, pull 200, velocity 80, and time 150</td></tr><tr><td>Fluorescent stereoscope&amp;nbsp;</td><td>Leica</td><td></td><td>M205 FA</td></tr><tr><td>Kwik-Fill Brosilicate Glass Capillaries</td><td>World Precision Instruments</td><td>1B100F-4</td><td></td></tr><tr><td>Microinjection mold TU-1</td><td>Adaptive Science Tools</td><td>TU-1</td><td></td></tr><tr><td>Microloader tip</td><td>Eppendorf</td><td>930001007</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td></td><td>Axiovert 200</td></tr><tr><td>mMESSAGE mMACHINE Kit</td><td>Ambion</td><td>AM1348</td><td></td></tr><tr><td>Nuclease-free water</td><td>Invitrogen</td><td>AM9937</td><td></td></tr><tr><td>Pneumatic pico pump</td><td>World Precision Instruments&amp;nbsp;</td><td>PV820&amp;nbsp;</td><td></td></tr><tr><td>Pneumatic pico pump</td><td>Warner Instruments</td><td>PLI-90A</td><td></td></tr><tr><td>pTol2-krt4:KalTA4-ERT2;cmlc2:eGFP</td><td></td><td></td><td>Dr. Thomas Ramezani/Yi Feng Lab/The University of Edinburgh</td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Quiagen</td><td>27106</td><td></td></tr><tr><td>Rhodamine B isothiocyanate-Dextran</td><td>Sigma Aldrich</td><td>R8881</td><td>10 mg/&amp;mu;l in water</td></tr><tr><td>Stereoscope</td><td>Leica</td><td></td><td>M80</td></tr><tr><td>&lt;em&gt;Tg(lysC:dsRed2)nz50Tg&lt;/em&gt;</td><td></td><td></td><td>&lt;em&gt;BMC Developmental Biology&lt;/em&gt; &lt;strong&gt;7&lt;/strong&gt;, 42, doi:10.1186/1471-213X-7-42 (2007)</td></tr><tr><td>&lt;em&gt;Tg(UAS:eGFP-HRASG12V)io006&lt;/em&gt;</td><td></td><td></td><td>&lt;em&gt;PloS One&lt;/em&gt; &lt;strong&gt;5&lt;/strong&gt; (12), e15170, doi:10.1371/journal.pone.0015170 (2010)</td></tr><tr><td>Tricaine/MS-222</td><td>Sigma Aldrich</td><td>A5040</td><td></td></tr><tr><td>UltraPure Agarose</td><td>Invitrogen</td><td>16500-100</td><td></td></tr><tr><td>UltraPure Low Melting Point Agarose&amp;nbsp;</td><td>Invitrogen</td><td>16520-050</td><td></td></tr><tr><td>UltraPure Phenol : Chloroform : Isoamyl Alcohol</td><td>Invitrogen</td><td>15593-031</td><td>25:24:1, v/v</td></tr></tbody></table>

live imaging of innate immune and preneoplastic cell interactions using an inducible gal4/uas expression system in larval zebrafish skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

io006和TG（型lysC:的DsRed2）:Tg的（EGFP-HRAS G12V UAS）之间的交叉的受精卵nz50Tg收集10-15分钟受精后（ 图1A），以得到1-细胞期的胚胎。注射后的胚胎被升高直到3旦，诱导4-OHT和安装用于实时成像（ 图1B）之前。 2-6小时后的感应所安装的胚胎置于任一倒置荧光显微镜下（ 图2A）或倒共聚焦显微镜（ 图2B-C）和成像2-3小时用1.5分钟的间隔。癌前浅表皮肤细胞可以通过它们更圆的形态相比正常角质形成细胞，以及局部的eGFP-HRAS G12V膜的绿色荧光发射来识别。被选定为共定位癌前皮肤细胞和先天免疫细胞（ 图2A箭头）的成像领域。由于快速MIG时延间隔被选择的嗜中性粒细胞的定量方法是1-1.5分钟以Z堆叠的步长大小的0.7-1.7微米之间，以获得最大强度投影（ 图2中，视频信号1）。有一个广泛的行为，可以同时实时成像观察:明年中性粒细胞迁移到下癌前细胞（ 图2C，视频1），形成直接接触到细胞（ 图2B和C），去除癌前细胞进行残存凋亡（视频1），或积极吞噬癌前细胞份（未示出）。捕捉相互作用的全谱，最好是由延时实时成像遵循他们的行为。 <img alt="图1" src="/files/ftp_upload/52107/52107fig1highres.jpg" width="700px" /> 图1:原理图中的斑马鱼幼体有条件的皮肤细胞转化。 （A）的Tg的（UAS合子的图像:EGFP-HRAS G12V; lysC的:的DsRed2）在斑马鱼10分钟后受精（MPF）。受精卵被绒毛膜（箭头）所包围。站点注射是胚盘（星号），通过从蛋黄（箭头）细胞质流形成，它定义了胚胎的动物极。斑马鱼幼体（B）的安装。 60毫米培养皿具有18-20毫米的孔（箭头），其由25毫米玻璃盖密封。斑马鱼幼体被固定并安装到盖板玻璃1％低熔点琼脂糖。含有5μM的4-OHT 0.3X Danieau溶液（箭头）被用于覆盖LMP琼脂糖（℃）4-OHT诱导浅表皮肤细胞的幼虫皮肤的（一）示意图的横截面轮廓的变换。胚胎和幼虫皮肤是由两个上皮细胞层，从表面细胞层和被连接到下面的基底膜（BM）的角质形成细胞的基底细胞层。（二）的瞬时表达系统吨Ø诱发癌前细胞克隆之间浅表的皮肤细胞。 KalTA4-ER T2被专门表达于皮肤最外层由krt4子（黄色）驱动。 KalTA4-ER T2的马赛克表达激活UAS控制（蓝色）EGFP-HRAS G12V表达的表层细胞，导致癌前细胞（绿色）的克隆。瞬时表达系统（D）示意图。（一）TOL2的盒式pTol2-krt4:KalTA4-ER T2; cmlc2:瞬时使用绿色荧光蛋白表达载体（二）条件诱导转录激活因子的表达。 KalTA4融合至人类雌激素受体​​α（ER T2），其结合特异性4-羟基（4-OHT）的突变体的配体结合结构域。在不存在的4-OHT对ER T2由热休克蛋白（热休克蛋白90）结合的细胞质中。当4-OHT结合的Hsp90解离，允许KalTA4-ER的核转T2和控制的EGFP-HRAS G12V表达UAS的后续激活。比例尺A = 500微米。 <a href="/files/ftp_upload/52107/52107fig1highres.jpg" target="_blank">请点击此处查看图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/52107/52107fig2highres.jpg" width="700px" /> 图2:癌前和先天免疫细胞相互作用的现场影像。交流（AC）4单丝旦的Tg图像（UAS:EGFP-HRAS G12V;型lysC:的DsRed2）胚胎，这已被注射pTol2-krt4:KalTA4-ER T2; cmlc2:EGFP和转座的mRNA在一个小区的阶段。 6小时后的4-OHT感应拍摄图像（A）的横向在6小时时尾翼区域的视图后处理，显示了EGFP-HRAS G12V补丁表达的皮肤细胞（箭头），和lysC:的DsRed2 +嗜中性粒细胞（箭头）。 （BC）代表图像TA肯从共焦时间推移电影，呈现中性粒细胞（红色）与EGFP-HRAS G12V表达皮肤细胞（绿色）的相互作用。（B）一种癌前细胞形成短丝状伪足的扩展与接触中性粒细胞（箭头）。（C）中性粒细胞（红色）是紧密联系，癌前皮肤细胞（绿色）的克隆。在比例尺= 100微米; B / C = 50微米。 <a href="/files/ftp_upload/52107/52107fig2highres.jpg" target="_blank">请点击此处查看图的放大版本。</a> <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/52107/Video_1.mov" target="_blank">视频1</a> : 癌前和先天免疫细胞相互作用的定时实时成像 。延时4 DPF 的Tg（UAS:EGFP-HRAS G12V; lysC的:的DsRed2）的胚胎，注射pTol2-krt4:KalTA4-ER T2; cmlc2:绿色荧光蛋白转座及基因在一个细胞阶段。从6-9豪焦时间推移电影URS后的4-OHT感应出的lysC:+的DsRed2中性粒细胞（红色）与EGFP-HRAS G12V表达皮肤细胞（绿色）的相互作用。旁边的中性粒细胞迁移到下癌前细胞和删除发生凋亡细胞癌前残存。 1.5分钟超过3小时的延时间隔以Z堆栈步长1.3微米。

Watch this Scientific Journal Video about Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin at JoVE.com

使用诱导型 Gal4/UAS 表达系统对斑马鱼幼虫皮肤中先天免疫和肿瘤前细胞相互作用进行实时成像 |发育生物学|视频

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

研究癌前细胞发展和先天免疫细胞相互作用的最早事件是举足轻重的理解和治疗癌症。在这里，我们描述了一个方法，有条件地诱导上皮细胞转化和先天免疫细胞相互作用与HRAS G12V表达的皮肤细胞在斑马鱼幼虫随后的实时成像。

先天免疫和癌前细胞间的相互作用使用可诱导的Gal4 / UAS表达系统在斑马鱼幼体皮肤实时成像

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

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11.9K 次观看.  The University of Edinburgh. 研究癌前细胞发展和先天免疫细胞相互作用的最早事件是举足轻重的理解和治疗癌症。在这里，我们描述了一个方法，有条件地诱导上皮细胞转化和先天免疫细胞相互作用与HRAS G12V表达的皮肤细胞在斑马鱼幼虫随后的实时成像。 

视频: Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

播散性念珠菌在斑马鱼幼虫的非侵入性成像

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

科研

免疫与感染

Facilitating Drug Discovery: An Automated High-content Inflammation Assay in Zebrafish

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a &lt;1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed3-6, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos7 or tailfin amputation3,5,6. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay8 eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED26,9 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes.
In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area occupied by red fluorescent leukocytes within an empirically defined area surrounding injured green fluorescent neuromasts. Furthermore, we automated data processing, handling, visualization, and storage all based on custom developed MATLAB and Python scripts.
In brief, we introduce an automated HC/HT screen that allows testing of chemical compounds for their effect on initiation, progression or resolution of a granulocytic inflammatory response. This protocol serves a good starting point for more in-depth analyses of drug mechanisms and pathways involved in the orchestration of an innate immune response. In the future, it may help identifying intolerable toxic or off-target effects at earlier phases of drug discovery and thereby reduce procedural risks and costs for drug development.

Here we describe a novel high-content chemically induced inflammation assay aiming at the identification of immune-modulatory bioactives. We have successfully combined automated microscopy with custom developed software scripts enabling automated quantification of the inflammatory response as well as further data processing, analysis, mining, and storage.

促进药物发现:斑马鱼在一个自动化的高含量检测炎症

Zebrafish larvae are particularly amenable to whole animal small molecule screens1,2 due to their small size and relative ease of manipulation and ...

Visualization of Cellular Electrical Activity in Zebrafish Early Embryos and Tumors

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling processes of excitable neuronal and muscular cells and many other biological processes, such as embryonic developmental patterning. However, there is a need for in vivo electrical activity monitoring in vertebrate embryogenesis. The advances of genetically encoded fluorescent voltage indicators (GEVIs) have made it possible to provide a solution for this challenge. Here, we describe how to create a transgenic voltage indicator zebrafish using the established voltage indicator, ASAP1 (Accelerated Sensor of Action Potentials 1), as an example. The Tol2 kit and a ubiquitous zebrafish promoter, ubi, were chosen in this study. We also explain&#160;the processes of Gateway site-specific cloning, Tol2 transposon-based zebrafish transgenesis, and the imaging process for early-stage fish embryos and fish tumors using regular epifluorescent microscopes. Using this fish line, we found that there are cellular electric voltage changes during zebrafish embryogenesis, and fish larval movement. Furthermore, it was observed that in a few zebrafish malignant peripheral nerve sheath tumors, the tumor cells were generally polarized compared to the surrounding normal tissues.

Here, we show the process of creating a cellular electric voltage reporter zebrafish line to visualize embryonic development, movement, and fish tumor cells in vivo.

斑马鱼早期胚胎和肿瘤细胞电活动的可视化研究

Bioelectricity, endogenous electrical signaling mediated by ion channels and pumps located on the cell membrane, plays important roles in signaling ...

发育生物学

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

先天免疫反应中的斑马鱼幼体型号无创成像<em&gt;链球菌iniae</em&gt;感染

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

在斑马鱼胚胎的单个神经元和神经嵴细胞的细胞运动和肌动蛋白细胞骨架的实时成像

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

生物学

Live Imaging of Cell Extrusion from the Epidermis of Developing Zebrafish

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function. Our studies have found that dying cells send signals to their live neighbors to form and contract a ring of actin and myosin that ejects it out from the epithelial sheet while closing any gaps that might have resulted from its exit, a process termed cell extrusion1. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe a method to induce and image extrusion in the larval zebrafish epidermis. To visualize extrusion, we inject a red fluorescent protein labeled probe for F-actin into one-cell stage transgenic zebrafish embryos expressing green fluorescent protein in the epidermis and induce apoptosis by addition of G418 to larvae. We then use time-lapse imaging on a spinning disc confocal microscope to observe actin dynamics and epithelial cell behaviors during the process of apoptotic cell extrusion. This approach allows us to investigate the extrusion process in live epithelia and will provide an avenue to study disease states caused by the failure to eliminate apoptotic cells.

Dying cells are extruded from epithelial tissues by concerted contraction of neighboring cells without disrupting barrier function. The optical clarity of developing zebrafish provides an excellent system to visualize extrusion in living epithelia. Here we describe methods to induce and image extrusion in the larval zebrafish epidermis at cellular resolution.

发展中国家斑马鱼的表皮细胞挤压实时成像

Homeostatic maintenance of epithelial tissues requires the continual removal of damaged cells without disrupting barrier function.  Our studies have found ...

Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage where they execute crucial antimicrobial functions. Their trafficking to these loci is orchestrated by several inflammatory chemoattractants, including chemokines. At the molecular level, chemoattractant signaling is regulated by the intracellular trafficking of the corresponding receptors. However, it remains unclear how subcellular changes in chemokine receptors affect leukocyte migration dynamics at the cell and tissue level. Here we describe a methodology for live imaging and quantitative analysis of chemokine receptor dynamics in neutrophils during inflammatory responses to tissue damage. These tools have revealed that differential chemokine receptor trafficking in zebrafish neutrophils coordinates neutrophil clustering and dispersal at sites of tissue damage. This has implications for our understanding of how inflammatory responses are self-resolved. The described tools could be used to understand neutrophil migration patterns in a variety of physiological and pathological settings and the methodology could be expanded to other signaling receptors.

Here we describe protocols to perform live imaging and quantitative analysis of chemoattractant receptor dynamics in zebrafish neutrophils

在伤口反应期间斑马鱼中性粒细胞中趋化因子受体的实时成像

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage ...

In vivo Imaging of Fully Active Brain Tissue in Awake Zebrafish Larvae and Juveniles by Skull and Skin Removal

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at cellular and subcellular resolution. Advances in molecular and imaging technologies have allowed us to gain numerous detailed insights into cellular and molecular mechanisms of brain development in the transparent zebrafish embryo. Recently, processes of refinement of neuronal connectivity that occur at later larval stages several weeks after fertilization, which are for example control of social behavior, decision making or motivation-driven behavior, have moved into focus of research. At these stages, pigmentation of the zebrafish skin interferes with light penetration into brain tissue, and solutions for embryonic stages, e.g., pharmacological inhibition of pigmentation, are not feasible anymore.
Therefore, a minimally invasive surgical solution for microscopy access to the brain of awake zebrafish is provided that is derived from electrophysiological&#160;approaches. In teleosts, skin and soft skull cartilage can be carefully removed by micro-peeling these layers, exposing underlying neurons and axonal tracts without damage. This allows for recording neuronal morphology, including synaptic structures and their molecular contents, and the observation of physiological changes such as Ca2+ transients or intracellular transport events. In addition, interrogation of these processes by means of pharmacological inhibition or optogenetic manipulation is feasible. This brain exposure approach provides information about structural and physiological changes in neurons as well as the correlation and interdependence of these events in live brain tissue in the range of minutes or hours. The technique is suitable for in vivo brain imaging of zebrafish larvae up to 30 days post fertilization, the latest developmental stage tested so far. It, thus, provides access to such important questions as synaptic refinement and scaling, axonal and dendritic transport, synaptic targeting of cytoskeletal cargo or local activity-dependent expression. Therefore, a broad use for this mounting and imaging approach can be anticipated.

Here we present a method to image the zebrafish embryonic brain in vivo upto larval and juvenile stages. This microinvasive procedure, adapted from electrophysiological approaches, provides access to cellular and subcellular details of mature neuron and can be combined with optogenetics and neuropharmacological studies for characterizing brain function and drug intervention.

在体内成像完全活跃的脑组织在清醒斑马鱼幼虫和青少年由骷髅和皮肤去除

Understanding the ephemeral changes that occur during brain development and maturation requires detailed high-resolution imaging in space and time at ...

神经科学

<meta charset="utf8"></head><body>阐明斑马鱼视盖发育中的小胶质细胞行为

Live Imaging and Characterization of Microglia Dynamics in the Zebrafish Embryo

Microglia are highly dynamic cells and their migration and colonization of the brain parenchyma is a crucial step for proper brain development and function. Externally developing zebrafish embryos possess optical transparency, which along with well-characterized transgenic reporter lines that fluorescently label microglia, make zebrafish an ideal vertebrate model for such studies. In this paper, we take advantage of the unique features of the zebrafish model to visualize the dynamics of microglia cells in vivo and under physiological conditions. We use confocal microscopy to record a timelapse of microglia cells in the optic tectum of the zebrafish embryo and then, extract tracking data using the IMARIS 10.0 software to obtain the cells' migration path, mean speed, and distribution in the optic tectum at different developmental stages. This protocol can be a useful tool to elucidate the physiological significance of microglia behavior in various contexts, contributing to a deeper characterization of these highly motile cells.

<meta charset="utf8"></head><body>作者聚焦：使用 Zebrafish 探索大脑发育过程中的小胶质细胞迁移

We demonstrate a method that takes advantage of fast scanning confocal microscopy to perform live imaging of microglia cells in the developing zebrafish optic tectum, allowing for the analysis of the dynamics of these cells in vivo.

先天免疫和癌前细胞间的相互作用使用可诱导的Gal4 / UAS表达系统在斑马鱼幼体皮肤实时成像

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