We thank O. Subach for providing the original H2B-PSmOrange plasmid and our fish facility team for fish care. We are grateful to the Nikon Imaging Center at the University of Heidelberg for access to microscopy equipment and analysis software. We acknowledge the support of the Core Facility Live Cell Imaging Mannheim at the CBTM (DFG INST 91027/10-1 FUGG). This work was supported by the Excellenzcluster CellNetworks, EcTop Spatio-temporal coordination of signaling processes (EcTop 2), University of Heidelberg to C.A.B. and the Medical Faculty Mannheim of the University Heidelberg and the DFG (FOR 1036/2, 298/3-1 and 298/6-1) to M.C.

1. H2B-PSmOrange基因体外转录 mRNA纯化<ol><li>线性化使用根据制造商的说明的NotI限制性内切酶含有PCS2 +质粒H2B-PSmOrange。 注意:请使用适当的保护措施，如手套和白大褂，以防止污染的mRNA降解，并在接下来的步骤。 </li><li>纯化使用根据制造商的协议的PCR纯化试剂盒或苯酚 - 氯仿基础的方法的线性化的DNA。 </li><li>根据制造商的说明使用线性模板DNA 1微克了SP6-mRNA的转录。 </li><li>通过在37℃下加入1.0单位RNAse游离DNA酶I 10-20分钟除去的DNA。 </li><li>使用的RNA，根据制造商的协议清理试剂盒清理的mRNA。 </li><li>以增加mRNA的纯度和浓度，通过加入6微升乙酸钠（3.0M，pH5.2）和150微升96％乙醇沉淀出的mRNA。孵育并购在-20℃ixed溶液至少30分钟，长达24小时。 </li><li>通过在4℃下纺丝在18.407 XG的溶液45分钟收集的mRNA。弃去液体，并在150微升70％乙醇重悬的mRNA。混合和离心的溶液在4℃额外的15分钟。弃去液体，干燥在冰上5分钟，将沉淀，并在20微升超纯无RNase水重悬的mRNA。 </li><li> 100的mRNA稀释液（在99微升超纯无RNase水1微升mRNA）的:由1的光度测量评估mRNA的浓度和纯度。 </li><li>对于短期存储，保持在-20℃的mRNA的溶液。对于长期存储，保持的mRNA在-80℃。 </li></ol> 2. H2B-PSmOrange基因微量注射的斑马鱼胚胎<ol><li>设置使用TG注射前对交配夜（FOXD3:GFP）; TG（FLH:GFP）的双转基因鱼（或任何其他的GFP转基因鱼）。离开鱼放置一个隔板隔开在它们之间，以控制交配的时间。 </li><li>除去在注射的早晨隔板和让鱼交配20分钟。 </li><li>在交配时，淡化H2B-PSmOrange的mRNA 130皮克的最终浓度/ NL（在无RNA酶的水），并转让6微升注射溶液成使用微加载尖端的显微注射毛细管。检查注射量与校准的载玻片。调节注射压力注射2 NL的mRNA溶液（260皮克）。 </li><li>收集鸡蛋放入含有1x胚胎（E3）中无菌塑料培养皿。 </li><li>转移20到30的胚胎，使用一个塑料巴斯德吸管的喷射盘。 </li><li>注入含有溶液到细胞或正下方的细胞进入蛋黄在单细胞阶段11 2 NL的mRNA。 </li><li>转印注射胚胎成含有1x E3介质的培养皿，除去未受精或不能正常发育中的胚胎，并在28℃下生长它们。 注:轻保障oF中的胚胎，不需要在整个实验。 </li><li>提高在1×E3介质斑马鱼的胚胎和原肠胚形成后添加0.2mM的PTU（1-苯基-2-硫脲），以抑制色素沉着。改变介质每天两次，以减少细菌污染的危险。 </li></ol> 3.胚胎嵌入<ol><li>选择的GFP（绿色）和PSmOrange使用装有荧光灯和适当的发射滤光片双目显微镜（橙色/红色）共表达的胚胎。转移的积极胚胎成含有1x E3介质的无菌培养皿。 </li><li> Dechorionate胚胎使用镊子12在立体显微镜下。 </li><li>在含有1.0毫升1.5毫升管传送一个胚胎预热用的切口尖端吸管（P200）在超纯水中制备1.0％低熔点琼脂糖（LMA）。注:在预热至LMA 80℃和嵌入胚胎前冷却在室温下3-5分钟。 </li><li>转移胚胎在150微升LMA成腔玻璃罩和调节胚胎的取向，例如背侧中为倒共聚焦激光扫描显微镜A1R +（或任何其它合适的显微镜）中描述的实验下来，用细塑料小费。当向LMA是聚合的，用含有0.2mM的PTU和0.02％3-氨基苯甲酸乙酯甲磺酸盐（三卡因）麻醉鱼水填充腔室。 </li><li>使样品成像之前，等待15分钟，以确定三卡因的效果。麻醉防止胚胎运动，可以使用装备有明照明立体显微镜进行监测。 注意:室可两次重复使用。 </li></ol> 4. PSmOrange光转化<ol><li>放置共焦显微镜下的样品和扫描试样，以确定该地区使用488纳米和561纳米激光器分别成像GFP和PSmOrange到photoconvert。 注意:使用倒置激光共聚焦显微镜A1R +上的倒站GE TiEcontrolled用显微镜成像软件，并配备了488纳米，561纳米和640纳米激光器光转化和成像。然而，应用程序当然没有限制，只要转换和成像激光线路可用限于此显微镜。 <ol><li>把20X空中目标到位（NA:0.75; WD 1.0毫米; FOV:0.64点¯x0.64毫米）。 </li><li>使用下面的显微镜设置光转化之前检测PSmOrange蛋白质:561纳米的激光，在聚焦平面上的目标上述测定0.74毫瓦。 注意:这对应于40％的在该系统上（在聚焦平面的测量是确定有效功率的唯一方式）。根据实验的需要为H2B-PSmOrange注射的效率可以变化调整激光功率。 </li><li>添加缩放因子，突出感兴趣的区域。放大倍率越高，降低图像采集时间，因此光毒性。 </li></ol></li><li>收购Z堆栈覆盖结构的S使用488纳米和在顺序模式（线模式1-&gt; 4）561纳米激光器的兴趣。修正1.0和2.0微米之间的z步骤。线平均和扫描频率可用于优化图像质量。 </li><li>扫描样本并选择感兴趣（ROI）工具，该地区要突出以photoconvert的区域。修复投资回报率的刺激区域。选择照片激活/漂白模块，通过检查各个框激活488纳米激光器和488nm的激光设定为80％（在目标测量1毫瓦的激光功率）。设置扫描速度0.5秒/帧。 </li><li>打开光转化工具（ND刺激），并指定光转换设置。 <ol><li>点击"添加"命令指定的光转化协议。 </li><li>在"采集/刺激"菜单"获取"设置"相位"1和表示在"循环"菜单光转换之前获得的图像的数量。 </li><li>设置"相位"2"圣要执行imulation"和输入的刺激的事件的数量。 </li><li>调整"相位"3作为"相位"1可选描述，插入一个等待"相位"通过输入时间上轮图像采集前要等待"相位"后2。 </li><li>一旦所有的参数都设置，应用刺激设置和运行该光转化。 注意:激光功率，扫描和迭代次数为每一轮光转化可以变化的频率和从胚胎不同胚胎。的设置来启动与表1中示出。 </li></ol></li><li>收购使用488纳米，561纳米和640纳米的激光应用设置如上一个最终的Z堆栈。设置640纳米的激光来可视化使用高的激光功率的转换PSmOrange（高达4.5毫瓦）。 </li></ol> 5. LMA胚胎下马<ol><li>除去从显微镜容纳室胚胎。小心地从琼脂糖ü取出胚胎唱镊子。 </li><li>转移胚胎成新灭菌塑料培养皿或进入无菌含有1x E3和0.2mM的PTU 6-孔板。孵育在28℃的胚胎直至发展所需的阶段。 </li></ol> 6.分析光转换PSmOrange蛋白表达细胞的命运<ol><li>下点3.3和3.4中所述重新嵌入胚胎。 </li><li>放置共聚焦显微镜下的样品和扫描样本，以确定的利息（488纳米）的GFP转基因结构光转换细胞（640纳米）。 </li><li>收购Z堆栈覆盖感兴趣的结构采用488纳米，561纳米和连续模式（行模式1-&gt; 4）640 nm激光。修正1.0和2.0微米之间的z步骤。线平均和扫描频率可用于优化图像质量。 </li><li>使用下列方法之一来识别细胞，其共表达GFP和所述光转换蛋白。 <ol><li>定制自动斐济图片Ĵ宏3 <ol><li>卷积使用"高斯模糊"插件原来栈（→处理→过滤器→高斯模糊），并减去从原来的使用"图像计算器"插件（→处理→图像计算器）顺利堆栈。使用此步骤可视化感兴趣的结构，并减少计算时间用于分析。 </li><li>适用于绿色和远红外通道特定的阈值，以突出的光转换单元（→图像→调整→阈值）。使用"分析粒子"工具，使用合适的设置来检测阈值的区域（→分析→分析颗粒）。 </li><li>打开"图像计算器"插件和黄（→处理→图像计算器）显示在绿色和远红外通道重叠的投资回报。 </li></ol></li><li>显微镜图像处理软件的3D数据评估<ol><li>显示在堆栈3D使用show量视图选项（→三维可视化菜单→显示卷视图）。使用图形界面来选择绿色，红色和远红色频道，调节亮度，对比度和作物的三维堆叠以突出显示光转换区域（ 图1）。 注意:类似的方法来分析在大多数用于图像分析的通用软件的数据是可用的。 </li></ol></li></ol></li></ol>

<ol>
	<li>Lombardo, V. A., Sporbert, A., Abdelilah-Seyfried, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+Tracking+Using+Photoconvertible+Proteins+During+Zebrafish+Development.">Cell Tracking Using Photoconvertible Proteins During Zebrafish Development.</a> J. Vis. Exp. (4350), (2012).</li><li>Subach, O. 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=A+photoswitchable+orange-to-far-red+fluorescent+protein,+PSmOrange.">A photoswitchable orange-to-far-red fluorescent protein, PSmOrange.</a> Nature Methods. 8, 771-777 (2011).</li><li>Beretta, C. A., Dross, N., Bankhead, P., Carl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ventral+habenulae+of+zebrafish+develop+in+prosomere+2+dependent+on+Tcf7l2+function.">The ventral habenulae of zebrafish develop in prosomere 2 dependent on Tcf7l2 function.</a> Neural Development. 8, 19 (2013).</li><li>Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 12651-12656 (2002).</li><li>Habuchi, S., Tsutsui, H., Kochaniak, A. B., Miyawaki, A., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mKikGR,+a+monomeric+photoswitchable+fluorescent+protein.">mKikGR, a monomeric photoswitchable fluorescent protein.</a> PLoS One. 3, e3944 (2008).</li><li>McKinney, S. A., Murphy, C. S., Hazelwood, K. L., Davidson, M. W., Looger, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bright+and+photostable+photoconvertible+fluorescent+protein.">A bright and photostable photoconvertible fluorescent protein.</a> Nature Methods. 6, 131-133 (2009).</li><li>Chudakov, D. M., Lukyanov, S., Lukyanov, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+intracellular+protein+movements+using+photoswitchable+fluorescent+proteins+PS-CFP2+and+Dendra2.">Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2.</a> Nature Protocols. 2, 2024-2032 (2007).</li><li>Subach, F. V., 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=Photoactivatable+mCherry+for+high-resolution+two-color+fluorescence+microscopy.">Photoactivatable mCherry for high-resolution two-color fluorescence microscopy.</a> Nature Methods. 6, 153-159 (2009).</li><li>Patterson, G. H., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photoactivatable+GFP+for+selective+photolabeling+of+proteins+and+cells.">A photoactivatable GFP for selective photolabeling of proteins and cells.</a> Science. 297, 1873-1877 (2002).</li><li>Subach, F. V., Patterson, G. H., Renz, M., Lippincott-Schwartz, J., Verkhusha, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright+monomeric+photoactivatable+red+fluorescent+protein+for+two-color+super-resolution+sptPALM+of+live+cells.">Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.</a> J Am Chem Soc. 132, 6481-6491 (2010).</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>Concha, M. 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=Local+tissue+interactions+across+the+dorsal+midline+of+the+forebrain+establish+CNS+laterality.">Local tissue interactions across the dorsal midline of the forebrain establish CNS laterality.</a> Neuron. 39, 423-438 (2003).</li><li>Auer, T. O., Duroure, K., Concordet, J. P., Del Bene, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-mediated+conversion+of+eGFP-+into+Gal4-transgenic+lines+in+zebrafish.">CRISPR/Cas9-mediated conversion of eGFP- into Gal4-transgenic lines in zebrafish.</a> Nature Protocols. 9, 2823-2840 (2014).</li><li>Auer, T. O., Duroure, K., De Cian, A., Concordet, J. P., Del Bene, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+CRISPR/Cas9-mediated+knock-in+in+zebrafish+by+homology-independent+DNA+repair.">Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair.</a> Genome Research. 24, 142-153 (2014).</li><li>Keller, P. J., Schmidt, A. D., Wittbrodt, J., Stelzer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+zebrafish+early+embryonic+development+by+scanned+light+sheet+microscopy.">Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy.</a> Science. 322, 1065-1069 (2008).</li></ol>

The authors have nothing to disclose.

携带荧光记者转基因胚胎有助于从根本上了解胚胎发育。然而，仍然有启动子的基本需要，以促进特定结构的特定的可视化。在他们的缺席，研究人员依靠技术，如荧光蛋白的光转化，了解他们的利益结构的起源和发展。这又是鉴定参与在其发展的分子机制的关键先决条件。在斑马鱼领域的技术进步，现在允许更换FP在方法14,15使用CRISPR-Cas9介导的敲除转基因系与，例如，photoconvertible蛋白?...

Exponentially improving imaging techniques allow following developmental processes over time periods of up to about four consecutive days3. In zebrafish and many other animal model systems, specific cells, tissues, axonal or vascular structures are marked by transgenic green or sometimes red or yellow fluorescent proteins to facilitate visualization. However, in most transgenic lines the transgene is not specifically expressed in the cells of interest but also additional structures, which hinders the precise tracking of for instance single cells or groups of cells.
Fluorescent photoconvertible proteins are well suited for cell tracking during embryonic development. The prerequisites for the application of such proteins are a long-lived nature, a well-separated emission range upon conversion and bright fluorescence. Available photoconvertible proteins comprise those that change their emission range upon conversion such as Kaede4, KiGR5, mEos26, PS-CFP2 or Dendra27 and others which are only fluorescent when photoactivated (PAmCherry8, PAGFP9 or PATagRFP10). Their applications to track cells in existing FP-transgenic animals are however limited as they often involve a green fluorescent state or do not fulfill all of the above criteria. Only recently, Subach and colleagues reported the PSmOrange protein, which changes its emission from orange/red to far red upon photoconversion and was successfully applied in cells in culture and cultured cells injected into mice2.
To investigate the protein's suitability for cell tracking in a living embryo, we generated an expression construct for the microinjection of nuclear-tagged H2B-PSmOrange into zebrafish embryos. We find that the protein fulfills all prerequisites for successful cell tracking in GFP transgenic backgrounds during the first 4 (and possibly more) days of zebrafish embryonic development. During this time, most of the cell migratory events are completed in fish making the PSmOrange system an excellent addition to the zebrafish toolkit.

<table border="0" cellpadding="0" cellspacing="0" width="821">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">PCR Purification Kit</td>
			<td style="width:233px;">Qiagen</td>
			<td align="right" style="width:119px;">28104</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mMESSAGE mMACHINE SP6 Transcription kit</td>
			<td>Ambion</td>
			<td>AM1340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy MiniElute Cleanup kit</td>
			<td>Qiagen</td>
			<td align="right">74204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic Pasteur</td>
			<td>alpha laboratories</td>
			<td>LW4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Original H2B-PSmOrange Plasmid</td>
			<td>Addgene</td>
			<td>31920</td>
			<td>The plasmid described in the paper is available in the Carl lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FemtoJet Microinjector</td>
			<td>Eppendorf</td>
			<td>5247 000.013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps (5 Inox)</td>
			<td>NeoLab</td>
			<td>2-1633</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lab-Tek II Chambered #1.5 German Coverglass System&#160;</td>
			<td>Nunc</td>
			<td align="right">155382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon A1R+</td>
			<td>Nikon GmbH Germany</td>
			<td>No Number</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon PLAN Apo &#955; 20x air objective&#160;</td>
			<td>Nikon GmbH Germany</td>
			<td>No Number</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NIS Elements AR Software (v. 4.30.02)</td>
			<td>Nikon GmbH Germany/Laboratory Imaging</td>
			<td>No Number</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

图1示出了PSmOrange光转换系统的一个例子。松果体复杂的是在脊椎动物背间脑的保守结构。像在许多其他脊椎动物中，此复合体由在间脑和左双面parapineal细胞的中心松果体器官。优雅但费时解笼锁的实验表明，parapineal细胞松果体器官13的前部产生。在TG（FOXD3:GFP）; TG（FLH:GFP）转基因胚胎，无论松果体和parapineal细胞发育过程中的标签。为了评估PSmOrange?...

Watch this Scientific Journal Video about Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System at JoVE.com

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

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 ...

tracking-cells-gfp-transgenic-zebrafish-using-photoconvertible

Research

JoVE Journal

Developmental Biology

8.5K 次观看.  Heidelberg University. 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.

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

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.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

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 ...

科研

发育生物学

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

使用提顿体系的研究斑马鱼再生的分子机制

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

多能干细胞的海藻酸钠封装使用同轴喷嘴

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

从小鼠羊水细胞多能干细胞使用转座子系统的生产

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

使用由内皮结肠形成细胞和间质干细胞形成的共培养球体的三维血管生成测定系统

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Visualizing Ocular Morphogenesis by Lightsheet Microscopy Using rx3:GFP Transgenic Zebrafish

Vertebrate eye development is a complex process that begins near the end of embryo gastrulation and requires the precise coordination of cell migration, proliferation, and differentiation. Time-lapse imagining offers unique insight to the behavior of cells during eye development because it allows us to visualize oculogenesis in vivo. Zebrafish are an excellent model to visualize this process due to their highly conserved vertebrate eye and their ability to develop rapidly and externally while remaining optically transparent. Time-lapse imaging studies of zebrafish eye development are greatly facilitated by use of the transgenic zebrafish line Tg(rx3:GFP). In the developing forebrain, rx3:GFP expression marks the cells of the single eye field, and GFP continues to be expressed as the eye field evaginates to form an optic vesicle, which then invaginates to form an optic cup. High resolution time lapse imaging of rx3:GFP expression, therefore, allows us to track the eye primordium through time as it develops into the retina. Lightsheet microscopy is an ideal method to image ocular morphogenesis over time due to its ability to penetrate thicker samples for fluorescent imaging, minimize photobleaching and phototoxicity, and image at a high speed. Here, a&#160;protocol is&#160;provided&#160;for time-lapse imaging of ocular morphogenesis using a commercially available lightsheet microscope and an image processing workstation to analyze the resulting data. This protocol details the procedures for embryo anesthesia, embedding in low melting temperature agarose, suspension in the imaging chamber, setting up the imaging parameters, and finally analyzing the imaging data using image analysis software. The resulting dataset can provide valuable insights into the process of ocular morphogenesis, as well as perturbations to this process as a result of genetic mutation, exposure to pharmacological agents, or other experimental manipulations.

Here, a&#160;protocol is&#160;provided&#160;for time-lapse imaging of ocular morphogenesis using a commercially available lightsheet microscope and an image processing workstation to analyze the resulting data. This protocol details the procedures for embryo anesthesia, embedding in low melting temperature agarose, suspension in the imaging chamber, setting up the imaging parameters, and finally analyzing the imaging data using image analysis software.&#160;

使用rx3:GFP转基因斑马鱼通过光片显微镜可视化眼部形态发生

Vertebrate eye development is a complex process that begins near the end of embryo gastrulation and requires the precise coordination of cell migration, ...