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>

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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 border="0" cellpadding="0" cellspacing="0" width="1104">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:316px;">Name of Material/Equipment</td>
			<td style="width:197px;">Company</td>
			<td style="width:124px;">Catalog Number</td>
			<td style="width:467px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">(Z)-4-Hydroxytamoxifen&#160;</td>
			<td>Sigma Aldrich</td>
			<td>H7904</td>
			<td>dilute in ethanol and store in the dark at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20x Objective</td>
			<td>Zeiss</td>
			<td></td>
			<td>20x/0,5 Ph2 &#8734;/0,17</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40x Objective</td>
			<td>Zeiss</td>
			<td></td>
			<td>40x/1,2W Korr &#8734;/0,14-0,18</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">63x Objective</td>
			<td>Zeiss</td>
			<td></td>
			<td>63x/1.4 Oil Ph3 &#8734;/0,17</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BamHI</td>
			<td>New England Biolabs</td>
			<td>R0136S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>LSM 510 META</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover glass</td>
			<td>VWR</td>
			<td>631-0171</td>
			<td>Diameter 25mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethylpolysiloxane</td>
			<td>Sigma Aldrich</td>
			<td>DMPSV</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dow Corning high-vacuum silicone grease&#160;</td>
			<td>Sigma Aldrich</td>
			<td>MKBL4135V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">Fluorescent stereoscope&#160;</td>
			<td>Leica</td>
			<td></td>
			<td>M205 FA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kwik-Fill Brosilicate Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100F-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microinjection mold TU-1</td>
			<td>Adaptive Science Tools</td>
			<td>TU-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microloader tip</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td>Axiovert 200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mMESSAGE mMACHINE Kit</td>
			<td>Ambion</td>
			<td>AM1348</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease-free water</td>
			<td>Invitrogen</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pneumatic pico pump</td>
			<td>World Precision Instruments&#160;</td>
			<td>PV820&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pneumatic pico pump</td>
			<td>Warner Instruments</td>
			<td>PLI-90A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">QIAprep Spin Miniprep Kit</td>
			<td>Quiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rhodamine B isothiocyanate-Dextran</td>
			<td>Sigma Aldrich</td>
			<td>R8881</td>
			<td>10mg/&#956;l in water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereoscope</td>
			<td>Leica</td>
			<td></td>
			<td>M80</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tg(lysC:dsRed2)nz50Tg&#160;</td>
			<td></td>
			<td></td>
			<td>BMC developmental biology 7, 42, doi:10.1186/1471-213X-7-42 (2007)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tg(UAS:eGFP-HRASG12V)io006</td>
			<td></td>
			<td></td>
			<td>PloS one 5 (12), e15170, doi:10.1371/journal.pone.0015170 (2010)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tricaine/MS-222</td>
			<td>Sigma Aldrich</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UltraPure Agarose</td>
			<td>Invitrogen</td>
			<td>16500-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UltraPure Low Melting Point Agarose&#160;</td>
			<td>Invitrogen</td>
			<td>16520-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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://www.jove.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

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

3D器官联合培养模型支持胸腺髓质上皮细胞增殖，分化和混杂基因表达

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

科研

发育生物学

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

在GFP-转基因斑马鱼使用Photoconvertible PSmOrange系统追踪单元

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

生物传感运动神经元膜电位在活斑马鱼胚胎

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

成年转基因斑马鱼视网膜外植体培养由多光子显微镜活细胞成像

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

体外在拟南芥中受精卵分化和胚模式的活体细胞成像的胚珠培养

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

小鼠第二腭融合的实时成像

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

斑马鱼幼虫动物模型的建立研究锥虫运动性体内

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Electroretinogram Recording in Larval Zebrafish using A Novel Cone-Shaped Sponge-tip Electrode

The zebrafish (Danio rerio) is commonly used as a vertebrate model in developmental studies and is particularly suitable for visual neuroscience. For functional measurements of visual performance, electroretinography (ERG) is an ideal non-invasive method, which has been well established in higher vertebrate species. This approach is increasingly being used for examining the visual function in zebrafish, including during the early developmental larval stages. However, the most commonly used recording electrode for larval zebrafish ERG to date is the glass micropipette electrode, which requires specialized equipment for its manufacture, presenting a challenge for laboratories with limited resources. Here, we present a larval zebrafish ERG protocol using a cone-shaped sponge-tip electrode. The novel electrode is easier to manufacture and handle, more economical, and less likely to damage the larval eye than the glass micropipette. Like previously published ERG methods, the current protocol can assess outer retinal function through photoreceptor and bipolar cell responses, the a- and b-wave, respectively. The protocol can clearly illustrate the refinement of visual function throughout the early development of zebrafish larvae, supporting the utility, sensitivity, and reliability of the novel electrode. The simplified electrode is particularly useful when establishing a new ERG system or modifying existing small-animal ERG apparatus for zebrafish measurement, aiding researchers in the visual neurosciences to use the zebrafish model organism.

Here, we present a protocol that simplifies the measurement of light evoked electroretinogram responses from larval zebrafish. A novel cone-shaped sponge-tip electrode can help to make the study of visual development in larval zebrafish using the electroretinogram ERG easier to achieve with reliable outcomes and lower cost.

一种新型锥形海绵尖电极在幼虫斑马鱼中的电录记录

The zebrafish (Danio rerio) is commonly used as a vertebrate model in developmental studies and is particularly suitable for visual neuroscience. For ...