We thank Kristen Kwan for the pCS2-H2A.F/Z-EGFP and pCS2-mCherry-CAAX vectors. We thank Chris Rodesch for tutoring us in live imaging in zebrafish. We thank Shawn Williams, Erik Malarkey and Brad Yoder for assistance in confocal imaging at UAB and the High Resolution Imaging Facility at UAB. The High Resolution Imaging Facility is supported by the UAB Comprehensive Cancer Center Support Grant (P30CA013148) and the Rheumatic Disease Core Center (P30 AR048311). J.M.P. is supported by the National Institute of Neurological Disease and Stroke (NIH R21 NS092105), and pilot grants from American Cancer Society (ACS IRG-60-001-53-IRG) and the UAB Comprehensive Cancer Center (P30CA013148). S.M.P. is supported by the Cell and Molecular Biology T32 Training Grant (5T32GM008111-28).

1. 体外转录<ol><li>线性化PCS2-H2A.F / Z-EGFP和/或PCS2-mCherry-CAAX载体通过的NotI限制性内切酶消化31。 体外转录试剂盒使用RNA，产生从每个模板5&#39;加帽mRNA产物，根据制造商的协议。 </li><li>净化用纯化试剂盒的皑皑的mRNA。按照制造商的说明。用无RNase H 2 O洗脱</li><li>确定在使用分光光度计260nm处吸光度的RNA的浓度。 （OD 260×稀释×40微克/毫升）。 </li><li>稀释的RNA至100毫微克/微升的每个H2A.F / Z-EGFP和mCherry-CAAX无RNA酶的H 2 O如果RNA浓度过低，荧光会被减少或不存在。光明的样本将逐渐减少光毒性和光漂白的关注。另一方面，过多的RNA可能是有毒的和/或引起脱靶效应。 注:存储剩余纯化的mRNA在-80℃的冰箱中。 </li></ol> 2.斑马鱼繁育，胚胎收集，和mRNA注射36-38 <ol><li>组装养殖坦克的障碍，坦克分成两个区域，并填写在斑马鱼设施所用养殖系统中的水每一个养殖池。 </li><li>为了防止过早配种，将两个雄鱼的屏障的一侧，两名女鱼放在另一边的夜滋生了。 </li><li>第二天，在冰上解冻预先制备的mRNA混合物中。用新鲜的水产养殖​​系统中的水替换养殖箱水和消除障碍。的障碍被拉到后，立即温热注射模具至28.5℃，并设置在设备的显微注射。 注:有关注塑模具的信息，请参考格拉赫36。 </li><li>收集鸡蛋每10 - 用滤茶15分钟，冲洗鸡蛋放到一个干净的100×15毫米的培养皿有E3蓝（5毫米氯化钠，0.17毫米氯化钾，0.33毫米氯化钙2，0.33mm的硫酸镁 ，10 -5％亚甲蓝）。 E3蓝用于防止真菌生长和确保仔鱼的适当发展。有关配套和胚胎收集更多信息，请参阅格拉赫36和37 Porazinkski。 </li><li>嵌入单细胞期胚在温热注塑模具和注入RNA进入蛋黄中的胚胎（ 图1A）的所需量。账户自然胚胎死亡和未受精的胚胎通过在15％以上的胚胎比实验所需进行胚胎注射。有关斑马鱼的胚胎显微注射的更多详细信息，请参考格拉赫36和37 Porazinkski。 注:对于第一次使用的mRNA，进行剂量曲线分析，以确定荧光和活力（定义为无毛发育缺陷最多5 DPF）执行5D成像之前的最佳剂量。 150 - 200纳克/微升注入胚胎往往是最佳浓度，因此它是为最终浓度的良好的起点。 </li><li>仔细提取采用改良9"玻璃巴斯德吸管模具注入胚胎。要修改吸管，熔体使用本生灯，直到它形成了一个球结束。 </li><li>将注射胚胎在E3蓝100×15毫米的培养皿和房屋在28.5℃培养箱。 </li><li>六小时后注射，从平板除去任何死亡或未受精的胚胎，并添加干净E3蓝。房子胚胎在28.5℃。 </li></ol> 3.制备及成像活斑马鱼胚胎嵌入（图1B） <ol><li>成像前两小时，屏幕采用了荧光解剖显微镜对GFP注入胚胎。将鲜绿色的绿色荧光蛋白表达的胚胎在一个新的100×15毫米培养皿E3蓝。 </li><li>通过加入1g低熔点的琼脂糖至100毫升的E3蓝煮沸1％低熔点琼脂糖的原液。使用琼脂糖后，用铝箔覆盖的烧瓶中。原液保持为长达一个月有用。 </li><li>等分试样将3ml熔化的琼脂成17×100毫米的培养管中。通过将培养管在42℃水浴，直到准备使用保持琼脂糖温暖。制备在去离子水中的15mM的三卡因溶液来麻醉斑马鱼胚胎36。 注意:如果在较早的时间点成像是期望的，琼脂的浓度可以降低低到0.3％39。 </li><li>把15毫三卡因，筛选胚胎，低熔点琼脂糖，E3蓝，和35毫米的玻璃盖玻片底培养皿到解剖光学显微镜。仔细＃5镊子取出胚胎的绒毛。做到这一点的三个胚胎。 </li><li>放置在一个单独的容器中的dechorionated胚胎被麻醉。盖玻片底培养皿的盖通常用于此目的。用移液管，加三滴（约150微升）15毫米三卡因到5ml E3蓝盘（如果使用盖玻片底部盘的盖子），或直到胚​​胎已被充分麻醉。此外，加3-4滴 - 15毫米三卡因溶液（约150 200微升）到1％琼脂糖融化管。 </li><li>使用1厘米切断了枪头的P200移液器;在麻醉的胚胎转移到盖玻片底培养皿。删除多余的E3蓝:三卡因溶液。 </li><li>慢慢加入5 - 10微升低熔点琼脂糖的:三卡因溶液在胚胎中，保持每个液滴分开，保证了胚胎不小心漂移彼此接近。 警告:如果琼脂糖太热，会损坏胚胎。一个良好的温度维持在琼脂为42℃。 </li><li>使用21g的1个半针，轻轻定向在琼脂糖到所需位置的胚胎。当使用时间推移成像倒置显微镜，东方感兴趣区域（ROI）尽量靠近盖玻片作为possib地区勒。 注意:对于一般用途，尾部区域提供易于取向和清晰度由于相对薄组织（ 图1A）。其他组织，如围绕蛋黄和鳍褶皱上皮细胞层，可以使用28,29。这些组织提供了极大的清晰度，但是这些地区只有少数细胞层厚。对于这个协议的目的，是形象有利于获得尽可能多的细胞分裂尽可能尾部区域。 </li><li>允许琼脂局部凝固几分钟。使用针掰开一小块琼脂，以测试其固化。当一块琼脂可以从下拉被拉远，进行到下一个步骤。 </li><li>覆盖低熔琼脂形成了嵌入式胚胎圆顶整个盖玻片。允许琼脂到移动的菜对于共焦成像（ 图1A）之前固化。 </li><li>在琼脂凝固过程（大约需要10分钟），制备3-毫升邻同（约250微升）的15mM三卡因五滴˚FE3蓝溶液到在成像期间被放置在嵌入胚胎。 </li></ol>活斑马鱼胚胎40,41 4. 5D聚焦成像注:请参阅有贺40和41奥布莱恩关于如何使用其他共聚焦系统来执行5D影像细节。对于Z-间隔，Z堆叠，Z深度，时间间隔，和5D定义，请参见图1C。 <ol><li>打开成像软件并设置显微镜60X 1.4 NA物镜。适用浸油物镜，并放置在显微镜舞台上的滑动架的培养皿。使用轴控制器，居中的物镜上述感兴趣的胚胎，并把物镜向上满足培养皿。 </li><li>点击目镜图标并切换到显微镜上的GFP过滤器。专注于投资回报率。着眼于组织最接近盖玻片便邻FFER最佳的成像效果。 </li><li>拆下互锁。选择GFP和mCherry通道（软件预先设定的波长）并设置行均选择正常。 </li><li>使用"查看/采集控制/ A1扫描区域"命令打开A1扫描区域的工具。 </li><li>开始扫描。使用轴控制器，定位胚胎使得扫描区域填充有尽可能多的斑马鱼尽可能的。激光功率并不需要在这一点上最佳的。降低激光功率，以避免不必要的光漂白。 </li><li>使用"查看/采集控制/ ND收购"命令打开ND采集控制面板。 </li><li>开始扫描设置Z-栈参数。设置Z-堆栈上限至细胞，其中未聚焦和下限，其中细胞不再可见。允许一个3微米空间中的采样生长和细胞运动之上，其可以扩展到成像领域。的Z堆叠对该亲所示的附图母育酚在胚胎尾覆盖40μm的Z深度。 </li><li>的Z区间的步长设置为2微米。平均来说，一个细胞的直径为10微米;因此2微米会产生对每个小区进行分析成像数据五个间隔。 注:可获取的图像的深度取决于在Z间隔。 Z轴分辨率牺牲来获得在Z尺度整体深度以图像为许多细胞有丝分裂经历尽可能（大Z深度）。逆是真实的，在通过减小步长，Z轴分辨率的不断积累，而Z向深度处死（小Z深度）。 </li><li>调整图像的激光功率，HV，和偏移水平。对于这个协议证明实验中，使用下面的激光功率，高压和偏移，分别设置在相应的水平，为GFP通道电平; 2 - 5 120 - 140，和-9至-11。对于mCherry通道，使用下面的激光功率，高压和失调的水平，分别为; 3 - 6，120 - 140，和-3至-8。一旦参数被设定，关闭扫描，以防止不必要的激光曝光可能导致样品的光毒性和漂白。 </li><li>选择2倍线平均图标。 "不平均"产生颗粒感的图像，而"4倍平均线"极大地提高了扫描时间。 2X线平均的使用提供最佳的图像质量和最快的扫描时间。 </li><li>选择所需的实验适当的时间间隔和持续时间。对于野生型师，2小时两分钟的时间间隔是最佳的，用于确定有丝分裂的持续时间（在图1，图2，图3A和3B中）。该激活纺锤体装配检验点的时间超过30分钟，司更适合于四小时间隔五分钟，以便如在图3C中展示了以保持荧光。 </li><li>勾选"保存到文件"框并命名文件自动保存文件作为被收购它。仔细检查所有的PArameters设置正确，然后点击"开始→运行"。 </li><li>收购完成后，要查看三维格式的文件，点击量门槛图标。 </li></ol>

The authors have nothing to disclose.

该方法的使用允许人们推断核膜破裂，通过微管的着丝点的附件形成中期板的，和姐妹染色单体的分离，以形成在体内和一个时间依赖性两个新的细胞。在斑马鱼中观察到的有丝分裂的能力是在固定的样品和细胞培养系统有利的，因为细胞在自然生理被成像，组织是透明其允许要使用的荧光蛋白质，他们开发比较快，和延时成像可以获取。成年斑马鱼的这个协议的使用是由于可能因被收购?...

有丝分裂为生长，分化和再生在活生物体中的关键的细胞过程是必不可少的。在准确的准备和在间DNA的复制，使细胞被起动来划分。有丝分裂，前期，第一阶段是由细胞周期蛋白B / CDK1的活化启动。前期的特征在于染色材料的缩合到染色体。核膜破裂发生在前期和前中期之间的过渡。在早中期，中心体，主轴形成成核中心，开始迁移到对立的两极，而在寻找着丝点附件延伸的微管。一旦附件转换到最终的微管附着和张力定位形成中期板1染色体。如果所有染色体正确连接时，纺锤体装配检验点满足，黏着​​环抱着姐妹染色单体一起被切开，微管缩短拉妹妹染色单体到对立的两极后期2,3时。最后阶段，末期，涉及围绕两个新的核核膜的细胞和改革的延伸。胞质分裂完成通过分离两个新的子细胞4-6的细胞质分裂过程。关键有丝分裂途径（ 即纺锤体装配检验点，中心体复制，姐妹染色单体凝聚力等 ）的改变可能会导致中期逮捕，染色体missegregation和基因组不稳定性7-10。最终，在通路控制有丝分裂的缺陷会导致发育障碍和癌症，有丝分裂的迫使可视化和其在现场，脊椎动物，多细胞生物体10-16缺陷。 斑马鱼的胚胎作为实时成像一个伟大的模式生物，由于透明组织，易于注射的，快速发展。使用斑马鱼，这份手稿的总体目标是描述的活5D有丝分裂17（ 图1C）的（尺寸的X，Y，Z，时间和波长）成像的方法。利用斑马鱼突变体在不同有丝分裂的途径有缺陷的论证这种缺陷的结果。对于这个协议，极光B和Esco2突变体被选择来说明这些缺陷。极光B是一种激酶是参与纺锤体形成和微管附着的染色体乘客复合物（CPC）的一部分。它也需要在胞质18,19分裂沟的形成。在斑马鱼中，极光B缺乏导致皱纹的诱导，细胞分裂和染色体分离20的缺陷。 Esco2，另一方面，是一种乙酰这对姐妹染色单体凝聚力21,22是必不可少的。它乙酰化上从而稳定黏结，以确保正确的染色体分离的中期-后期过渡23环的SMC3部黏着。在斑马鱼Esco2的丧失导致为chromosome missegregation，过早姐妹染色单体分离，基因组不稳定和p53依赖性和独立的凋亡24,25。由于可用性，auroraB hi1045 和 esco2 hi2865突变斑马鱼（以下简称为aurB M / M和esco2 米/分 ，分别地）将被用来说明此技术25-27。 用荧光标记的细胞的机械耦合共焦显微镜使研究人员有丝分裂25,28,29中可视化染色和细胞膜动力学。荧光标记的组蛋白在历史上被用于可视化的染色质。组蛋白是四种不同的对（H2A，H2B，H3和H4）是负责该构成染色体30中的核小体结构组成的核蛋白质。尽管H2B可以说是在荧光蛋白中最常用的组蛋白小鼠和细胞培养，用组蛋白2A的，家庭Z（H2A.F / Z）已在斑马鱼31,32使用证明很好。伴刀豆球蛋白A和酪蛋白激酶1-γ，例如，定位于细胞膜和先前已显示有效的可视化细胞膜在海胆和果蝇33,34。其它研究已经表明，CAAX荧光标记蛋白标记的细胞膜并成功地斑马鱼31。 CAAX是受翻译后修饰的酶如farnesyltransferases和geranylgeranyltransferases识别的基序。由这些酶修饰导致蛋白质成为膜相关，从而标记的细胞膜35。 由于在斑马鱼的先前使用，该协议选择使用H2A.F / Z和CAAX标记染色质和细胞膜。这种方法的应用将允许研究者在单个细胞水平监测的有丝分裂，观察个人染色体动力学，以及同时监测可能影响组织的分化和发育的多个细胞分裂。本文将着眼于在单个单元格级别的有丝分裂过程中染色体成像分离的动态。在这个手稿，观察几个有丝分裂，分裂计算时间和破译的有丝分裂表型的能力进行说明和讨论。通过使用这些参数，生理学相关的数据可以被收集并施加到受有丝分裂的缺陷数的疾病状态。

<table border="0" cellpadding="0" cellspacing="0" width="1094">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">pCS2 vectors</td>
			<td style="width:175px;">Gift from K. Kwan</td>
			<td style="width:129px;"></td>
			<td style="width:460px;">For plasmid of interest</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">NotI-HF restriction enzyme</td>
			<td style="width:175px;">New England BioLabs</td>
			<td>R3189S</td>
			<td>For restriction digest of plasmid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">mMessage SP6 kit</td>
			<td style="width:175px;">Life Technologies</td>
			<td>AM1340</td>
			<td>For in vitro transcription</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">RNeasy Mini kit</td>
			<td style="width:175px;">Qiagen</td>
			<td style="width:129px;">74104</td>
			<td style="width:460px;">For purifying mRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">100 x 15 mm petri dishes</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:129px;">FB0875712</td>
			<td>For housing embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">microinjection mold</td>
			<td style="width:175px;">homemade</td>
			<td style="width:129px;"></td>
			<td>For holding embryos during microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Agarose II</td>
			<td style="width:175px;">Amresco</td>
			<td style="width:129px;">0815-25G</td>
			<td>For embedding embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Tricaine</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td>E10521-10G</td>
			<td>For anesthetizing embryos</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Sodium Chloride</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:129px;">S9888</td>
			<td>For embryo water (E3 Blue), dissolved in UltraPure H2O</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Potassium Chloride</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:129px;">P3911</td>
			<td>For embryo water (E3 Blue), dissolved in UltraPure H2O</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Calcium Chloride Dihydrate</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:129px;">C8106</td>
			<td>For embryo water (E3 Blue), dissolved in UltraPure H2O</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Magnesium Sulfate</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:129px;">M7506</td>
			<td>For embryo water (E3 Blue), dissolved in UltraPure H2O</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:331px;">Methylene Blue Hydrate</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:129px;">MB1</td>
			<td>For embryo water (E3 Blue), dissolved in UltraPure H2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">100 mm culture tube</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td>50-819-812</td>
			<td>For melted agar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">35 mm glass coverslip bottom culture dish&#160;</td>
			<td style="width:175px;">MatTek Corp</td>
			<td>P35G-0-20-C&#160;</td>
			<td>No. 0, 20 mm glass, For embedding embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">#5 tweezers</td>
			<td style="width:175px;">Dumont</td>
			<td style="width:129px;">72701-D</td>
			<td>For dechorionating embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">21G 1 1/2 gauge needle&#160;</td>
			<td style="width:175px;">Becton Dickinson</td>
			<td style="width:129px;">305167</td>
			<td>For positioning embryos in agar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Dissecting microscope</td>
			<td style="width:175px;">Nikon AZ100</td>
			<td style="width:129px;"></td>
			<td>For screening and embedding embryos, any dissecting scope will do</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Confocal microscope</td>
			<td style="width:175px;">Nikon A1+</td>
			<td style="width:129px;"></td>
			<td>For time-lapse imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:331px;">Confocal software</td>
			<td style="width:175px;">NIS Elements AR 4.13.00</td>
			<td style="width:129px;"></td>
			<td>For image acquisition and processing</td>
		</tr>
	</tbody>
</table>

observing mitotic division and dynamics in a live zebrafish embryo

有丝分裂为有机体的生长和分化至关重要。这个过程是高度动态的，需要有序的事件来完成适当的染色质凝聚，微管连接着丝粒，染色体分离和胞质分裂在小时间框架。在微妙的过程中的错误可能导致人类疾病，包括出生缺陷和癌症。传统的方法研究人类疾病的有丝分裂状态往往依赖于细胞培养系统，研究人类疾病时，缺乏自然的生理和发育/组织特异性环境有利。该协议提供了一种可视化，高分辨率，在脊椎动物系统染色体动态，斑马鱼克服许多障碍。该协议将详细可用于获得分裂的细胞，其包括的动态图像的方法: 在体外转录，斑马鱼养殖/收集，胚胎嵌入，和延时成像。优化和改性的该协议的ications进行了探讨。使用H2A.F / Z-EGFP（标签染色质）和mCherry-CAAX（标签细胞膜）的mRNA注射的胚胎，有丝分裂在AB野生型，auroraB hi1045 和 esco2 hi2865突变斑马鱼被可视化。在斑马鱼高分辨率实时成像允许一个观察多个核分裂进行统计量化有丝分裂的缺陷和有丝分裂进程的时机。此外，定义不当的有丝分裂过程（ 即，板集合缺陷，染色体missegregation 等 ）和不适当的染色体的结果（ 即，非整倍体，多倍体，微核等 ）质量方面观察被观察。该测定可用于组织分化/发展的观察，是适合于使用突变斑马鱼和药理学试剂的。在有丝分裂的缺陷如何导致癌症和发育障碍将极大地可视化提高疾病的发病机理的理解。

图2显示，观察使用AB野生型斑马鱼尾巴的宽视场多细胞分裂的能力。超过七有丝分裂细胞成像在14分钟的时间内（ 电影1）。在两个小时的时间，当然，超过40的有丝分裂事件被抓获。平均来说，在AB观察50分裂的细胞，并在AUR B M /米胚（ 图2B）30分裂的细胞。考虑到成像单元的数量，有丝分裂的细胞，以成像的计?...

Watch this Scientific Journal Video about Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo at JoVE.com

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

观察有丝分裂和动力学在现场斑马鱼胚胎

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

observing-mitotic-division-and-dynamics-in-a-live-zebrafish-embryo

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11.9K 次观看.  University of Alabama at Birmingham. Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

视频: Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

肾再生的成年斑马鱼庆大霉素性损伤

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

科研

发育生物学

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

微珠植入斑马鱼胚胎

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

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 ...

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 ...

Use of Immunolabeling to Analyze Stable, Dynamic, and Nascent Microtubules in the Zebrafish Embryo

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods are described here to analyze distinct populations of MTs in the developing neural tube of the zebrafish embryo. While the focus is on neural tissue, this methodology is broadly applicable to other tissues. The procedures are optimized for early to mid-somitogenesis-stage embryos (1 somite to 12 somites), however they can be adapted to a range of other stages with relatively minor adjustments. The first protocol provides a method to assess the spatial distribution of stable and dynamic MTs and perform a quantitative analysis of these populations with image-processing software. This approach complements existing tools to image microtubule dynamics and distribution in real-time, using transgenic lines or transient expression of tagged constructs. Indeed, such tools are very useful, however they do not readily distinguish between dynamic and stable MTs. The ability to image and analyze these distinct microtubule populations has important implications for understanding mechanisms underlying cell polarization and morphogenesis. The second protocol outlines a technique to analyze nascent MTs specifically. This is accomplished by capturing the de novo growth properties of MTs over time, following microtubule depolymerization with the drug nocodazole and a recovery period after drug washout. This technique has not yet been applied to the study of MTs in zebrafish embryos, but is a valuable assay for investigating the in vivo function of proteins implicated in microtubule assembly.

Immunolabeling methods to analyze distinct populations of microtubules in the developing zebrafish brain are described here, which are broadly applicable to other tissues. The first protocol outlines an optimized method for immunolabeling stable and dynamic microtubules. The second protocol provides a method to image and quantify nascent microtubules specifically.

利用 Immunolabeling 分析斑马鱼胚胎中的稳定、动态和初生的微管

Microtubules (MTs) are dynamic and fragile structures that are challenging to image in vivo, particularly in vertebrate embryos. Immunolabeling methods ...

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; ...

AFM and Microrheology in the Zebrafish Embryo Yolk Cell

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. Various propositions claim to have been important contributions to the understanding of the mechanical properties of cells and tissues in their spatiotemporal organization in different developmental and morphogenetic processes. However, due to the lack of reliable and accessible tools to measure material properties and tensional parameters in vivo, validating these hypotheses has been difficult. Here we present methods employing atomic force microscopy (AFM) and particle tracking with the aim of quantifying the mechanical properties of the intact zebrafish embryo yolk cell during epiboly. Epiboly is an early conserved developmental process whose study is facilitated by the transparency of the embryo. These methods are simple to implement, reliable, and widely applicable since they overcome intrusive interventions that could affect tissue mechanics. A simple strategy was applied for the mounting of specimens, AFM recording, and nanoparticle injections and tracking. This approach makes these methods easily adaptable to other developmental times or organisms.

The lack of tools to measure material properties and tensional parameters in vivo prevents validating their roles during development. We employed atomic force microscopy (AFM) and nanoparticle-tracking to quantify mechanical features on the intact zebrafish embryo yolk cell during epiboly. These methods are reliable and widely applicable avoiding intrusive interventions.

斑马鱼胚胎卵黄细胞中的 AFM 和 Microrheology

Elucidating the factors that direct the spatio-temporal organization of evolving tissues is one of the primary purposes in the study of development. ...

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

斑马鱼原代细胞的培养与转染

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

果蝇胚胎肌肉收缩的实时成像和分析

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with development, tissue homeostasis, and disease processes. Drosophila spermatocytes undergoing meiosis are ideal for this analysis because (1) whole Drosophila testes containing spermatocytes are relatively easy to prepare for microscopy, (2) the spermatocytes&#8217; large size makes them well suited for high resolution imaging, and (3) powerful Drosophila genetic tools can be integrated with in vivo analysis. Here, we present a readily accessible protocol for the preparation of whole testes from Drosophila third instar larvae and early pupae. We describe how to identify meiotic spermatocytes in prepared whole testes and how to image them live by time-lapse microscopy. Protocols for fixation and immunostaining whole testes are also provided. The use of larval testes has several advantages over available protocols that use adult testes for spermatocyte analysis. Most importantly, larval testes are smaller and less crowded with cells than adult testes, and this greatly facilitates high resolution imaging of spermatocytes. To demonstrate these advantages and the applications of the protocols, we present results showing the redistribution of the endoplasmic reticulum with respect to spindle microtubules during cell division in a single spermatocyte imaged by time-lapse confocal microscopy. The protocols can be combined with expression of any number of fluorescently tagged proteins or organelle markers, as well as gene mutations and other genetic tools, making this approach especially powerful for analysis of cell division mechanisms in the physiological context of whole tissues and organs.

Preparation of Drosophila Larval and Pupal Testes for Analysis of Cell Division in Live, Intact Tissue

The goal of this protocol is to analyze cell division in intact tissue by live and fixed cell microscopy using Drosophila meiotic spermatocytes. The protocol demonstrates how to isolate whole, intact testes from Drosophila larvae and early pupae, and how to process and mount them for microscopy.

用于活、完整组织中细胞分部分析的果蝇拉瓦尔和普帕尔睾试验的制备

Experimental analysis of cells dividing in living, intact tissues and organs is essential to our understanding of how cell division integrates with ...