我们感谢托马斯·贝茨的批判性阅读和改善手稿。我们也感谢克里斯蒂娜·艾伯特和SabrinaStötzer鱼类维护。

所有动物实验均根据"实验动物护理的原则"以及关于动物保护的当前版本的德国法进行。 1.反义吗啉的制备（MO） <ol><li>要准备3毫米MO原液，加100微升灭菌，超纯水的冻干MO（在玻璃小瓶300纳摩尔）。完全溶解，加热储备溶液至65℃5分钟。密封用封口膜的小瓶中并在室温下储存（RT）下将MO原液。不冷却在MO原液，因为这可能会导致在MO的关联与药瓶墙壁。 </li><li>通过稀释的MO原液用无菌，超纯水制备1mM的MO工作溶液，并添加0.5％酚红溶液，以0.05％的终浓度。 <ol><li>要获得10微升MO工作液中，加入3.3微升MO原液和1微升0.5％酚红溶液5.7微升水。是前准备一批新的工作溶液加热MO原液至65℃5分钟。 </li></ol></li><li>在使用之前，离心机将MO工作溶液，以防止针堵塞。例如，旋转10微升的MO原液以15,000 xg离心5分钟（RT）并取下8微升上清液用于注射。 注:随着时间的推移11吗啉的解决方案，可能会出现活性损失。为了排除此，可以按照制造商提供的协议测试通过UV光谱法的解决方案，在吗啉降低的有效性尚未观察到在存储的长时间的使用wt1a的情况下，（3毫工作液储存超过一个长年）。 </li></ol> 2.设置接合对受精卵的收集<ol><li>之前显微注射下午，建立5对的Tg（wt1b:GFP）线路，每在装有可拆卸的筛子以分离繁殖容器夜间男性和女性。 注:要确保有足够的鸡蛋，可为成功注入实验中，作为接合对鱼应该是预先筛选和评价为良好"生产者"。 </li><li>第二天早上，开灯后，将男性和女性在一起，防止鱼类吃了他们的鸡蛋筛以上。 </li><li>观看产卵。一旦被放置的蛋可以在4细胞期之前被注入的量再次达到从女性（约50），单独的男性。用巴氏吸管转移将鸡蛋打入培养皿装满胚胎水用于第一轮注射。为了让鸡蛋的额外回合，把一对交配在一起，分开几次。 </li></ol> 3.显微注射准备工作注:提前执行步骤3.1和3.2。 <ol><li>要准备，注射（注射菜）期间保持在原位胚胎菜插入GL在40-45°角的屁股滑入94毫米的培养皿充满液体，透明，纯净，食品级硅。取出幻灯片当硅硬化。产生在硅层的槽。 注:另外，用1.5-3％琼脂糖代替硅和菜储存在4℃。 </li><li>产生从用针拔出器的玻璃毛细管注射针。 <ol><li>使用含有内部细丝毛细血管。 注:灯丝将有助于回填（见3.3）后向针尖的MO解决运输。 </li><li>在针拉马建立适当的设置。良好的针注射到斑马鱼应该有长而薄的技巧12。产生具有大约10毫米的长度，并在其最末端的直径为1.5-2微米的提示。例如，使用一个水平针车夫，设置如下:热= 330，拉= 0，速度= 40，时间= 100。 </li><li>检查针的下一个质量解剖显微镜和梳理那些短或过长提示。 注:针短提示趋向而这种非常长的和薄的提示弯曲触摸绒毛膜时损坏胚胎和不穿透它。 </li></ol></li><li>通过使用微加载尖端回填2微升的MO工作溶液的针，并将其插入到注射装置的针保持器。 </li><li>作为针在前端封闭，产生由捏背面用解剖显微镜下急剧镊子针的最末端的开口。另外也可以使用显微注射设备尖端轻轻推到坚硬的表面（ 如小金属块或镊子的背面）。 </li><li>校准通过注入在MO工作液入放置在刻度矿物油的液滴的注射体积。调节喷射的持续时间用于产生75微米的水滴直径以获得约200复注射体积。 </li></ol><p类="jove_title"&gt; 4。显微注射过程<ol><li>安排沿着具有朝向从在针来（朝向槽的45°角）的方向上的植物极显微注射培养皿的槽1-细胞期的胚胎。 </li><li>穿孔绒毛膜和与针蛋黄和注入1mM的MO工作溶液（0.2皮摩尔）200复成1-蛋黄至2-细胞胚胎。 </li><li>使用巴斯德吸管具有宽开口，收集所有的注射的胚胎在培养皿中填充有胚胎水中，并在28.5℃培养它们。 </li><li>当天下午，通过使用巴斯德吸管用广口取出死胚和替换胎水。 </li></ol> 5.准备胚胎的体内成像<ol><li>第二天早晨，用含有0.003％N-苯基硫脲（PTU）抑制黑化胚胎水取代胚胎水。原肠胚形成，因为它INTERF结束前不要使用PTUERES与早期胚胎发育。 注意:PTU是毒性和致畸。在任何时候都戴上手套，避免吸入以及直接接触皮肤。 </li><li> Dechorionate在所需发育阶段解剖显微镜下用尖锐的镊子的胚胎。抓住绒毛膜一个镊子，并试图抓住绒毛膜的另一侧与第二镊子。小心拉在相反方向上镊子而不破坏胚胎。 </li><li>通过用含有PTU（0.003％）和0.02％3-氨基苯甲酸乙酯甲磺酸盐（三卡因）胚胎水代替包含PTU胚胎水（0.003％）麻醉的胚胎。 </li><li>琼脂糖嵌入在一个玻璃罩底μ-菜与一个50微米搬迁电网低熔点胚胎。适当的嵌入需要一些练习。 <ol><li>制备0.7％琼​​脂糖的1ml等份在1.5ml反应管，并把它们设定为36℃的加热块上。使用巴斯德吸管具有广泛的开放TRAnsfer胚胎μ-菜。删除使用巴斯德吸管采用超薄尖多余胚胎的水和覆盖与回火琼脂糖胚胎。 </li><li>而琼脂糖仍然液体，定向在所希望的位二凝胶加载提示胚胎，关于感兴趣的结构以及对重定位网格的距离。放置的兴趣尽可能接近玻璃底部（这里:背侧向下）的结构（此处为前肾），并在紧挨着的网格。 </li><li>为获得最佳的图像质量通过放置的胚胎尽可能接近底部的感兴趣结构最小化的胚胎和玻璃底之间的琼脂糖层。除此之外，定位在靠近附近向电网胚胎但照顾该网格将不与感兴趣的结构叠加。嵌入3-4胚胎在不同的μ-菜每使用定位最好的之一。 </li><li>当琼脂糖很难足以容纳胚胎等待2-3分钟，并覆盖嵌入胚胎用含有0.003％PTU和0.02％三卡因胚胎水。倒出多余的胚胎的水或通过使用巴氏吸管超薄尖端移除。在锁定位置，以防止蒸发关闭μ-培养皿的盖子。 注:避免气泡的发生在μ-菜的，因为它们会损害图像记录。 </li></ol></li></ol> 6.显微镜注:使用配备通过结构照明的光学切片显微镜。图像记录包含以下模块软件:多通道，Z-Stack的，时间序列，软件自动对焦和扩展对焦。 <ol><li> Z-栈收购 <ol><li>反转μ-菜。玻璃底现在朝向物镜和μ-培养皿用作成像室。 </li><li>切换滑块到网格位置和使能在软件控制结构照明模式。 </li><li>校准通过下面的焦点校准向导的指示进行检查电网重点选择的通道（S）与试样（嵌入式胚胎）。 </li><li>激活的Z-Stack采集模式，并将其设置为中心的模式。聚焦到试样的中间，并设置为通过点击中心 Z-堆栈的中心平面。通过定义围绕中心位置的范围内的z堆栈的尺寸。 注:对象结构不应被第一和Z堆叠的最后面以外急剧看到。 </li><li>调整光部分之间的间距。为了达到最佳的z分辨率点击最佳按钮。基于该目标的景深的软件将设置以下奈奎斯特绕圈（50％重叠）建议的距离。 注意:如果瞄准较小的文件大小和结构许可，手动设置间距较大尺寸。设置非推荐较小的步骤只会导致更大的文件大小不增加以后人的信息。 </li></ol></li><li> 时间推移成像 <ol><li>打开时间序列工具，并设置时间序列实验的时间间隔。例如记录Z堆栈图像在一段5小时每隔30分钟。 </li><li>随着时间的推移修正焦点漂移，建立一个自动聚焦战略。检查聚焦战略对话中，从下拉列表中选择软件自动对焦并选择TL-明场通道作为参考通道。 </li><li>先从自动对焦每个时间点。为了保证优化的时间框架内一个可靠的搜索范围，设定一个固定的200微米的相对的搜索范围。这个范围是完全在扫描最大步长（采样参​​数）（覆盖范围参数）基本品质（Quality参数）。 </li><li>以提高自动聚焦精度，激活的兴趣（聚焦ROI），并位置的对焦区域内的计量所述周围的感兴趣结构的实时取景窗口焦点投资回报率。 注:如果系统缺乏软件自动对焦模块延时录制过程中自动更新中心z位置，暂停试验在给定的时间和手动调焦​​，并继续实验。 </li><li>以时间序列记录期间补偿潜在的xy漂移，定位印迹格（的成像室）的一个特定点到软件的十字线并且通过使屏幕截图保存的定位。 </li><li>开始的时间系列实验。的时间序列区间结束之前，暂停实验和，如果需要的话，定位根据截图重新调整。与实验继续。 </li></ol></li></ol> 7.图像处理<ol><li>切换到处理选项卡，选择对焦（EDF）在方法对话框中的扩展深度。由于光学切片被收购，应用最大投影算法对图像系列。 注:这里的算法将投影BRightest或从堆的复合2D图像中的第一步骤中的最暗的像素，并最终使用具有最高方差为每个时间点的结果的图像。 </li><li>使用影片导出方法了EDF图像系列创建一个时间的推移电影（ 例如 AVI或移动文件）。 </li></ol>

<ol>
	<li>Lieschke, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+human+disease:+zebrafish+swim+into+view.">Animal models of human disease: zebrafish swim into view.</a> Nat Rev Genet. 8 (5), 353-367 (2007).</li><li>Drummond, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney+development+and+disease+in+the+zebrafish.">Kidney development and disease in the zebrafish.</a> J Am Soc Nephrol. 16 (2), 299-304 (2005).</li><li>Drummond, I. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+development+of+the+zebrafish+pronephros+and+analysis+of+mutations+affecting+pronephric+function.">Early development of the zebrafish pronephros and analysis of mutations affecting pronephric function.</a> Development. 125 (23), 4655-4667 (1998).</li><li>Wingert, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cdx+genes+and+retinoic+acid+control+the+positioning+and+segmentation+of+the+zebrafish+pronephros.">The cdx genes and retinoic acid control the positioning and segmentation of the zebrafish pronephros.</a> PLoS Genet. 3 (10), 1922-1938 (2007).</li><li>Kramer-Zucker, A. G., Wiessner, S., Jensen, A. M., Drummond, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+the+pronephric+filtration+apparatus+in+zebrafish+requires+Nephrin,+Podocin+and+the+FERM+domain+protein+Mosaic+eyes.">Organization of the pronephric filtration apparatus in zebrafish requires Nephrin, Podocin and the FERM domain protein Mosaic eyes.</a> Dev Biol. 285 (2), 316-329 (2005).</li><li>Wingert, R. A., Davidson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+pronephros:+a+model+to+study+nephron+segmentation.">The zebrafish pronephros: a model to study nephron segmentation.</a> Kidney Int. 73 (10), 1120-1127 (2008).</li><li>Kreidberg, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wt-1+Is+Required+for+Early+Kidney+Development.">Wt-1 Is Required for Early Kidney Development.</a> Cell. 74 (4), 679-691 (1993).</li><li>Bollig, F., 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=Identification+and+comparative+expression+analysis+of+a+second+wt1+gene+in+zebrafish.">Identification and comparative expression analysis of a second wt1 gene in zebrafish.</a> Dev Dyn. 235 (2), 554-561 (2006).</li><li>Perner, B., Englert, C., Bollig, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Wilms+tumor+genes+wt1a+and+wt1b+control+different+steps+during+formation+of+the+zebrafish+pronephros.">The Wilms tumor genes wt1a and wt1b control different steps during formation of the zebrafish pronephros.</a> Dev Biol. 309 (1), 87-96 (2007).</li><li>Schnerwitzki, D., 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=Alternative+splicing+of+Wilms+tumor+suppressor+1+(Wt1)+exon+4+results+in+protein+isoforms+with+different+functions.">Alternative splicing of Wilms tumor suppressor 1 (Wt1) exon 4 results in protein isoforms with different functions.</a> Dev Biol. 393 (1), 24-32 (2014).</li><li>Bedell, V. M., Westcot, S. E., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+from+morpholino-based+screening+in+zebrafish.">Lessons from morpholino-based screening in zebrafish.</a> Brief Funct Genomics. 10 (4), 181-188 (2011).</li><li>Rembold, M., Lahiri, K., Foulkes, N. S., Wittbrodt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenesis+in+fish:+efficient+selection+of+transgenic+fish+by+co-injection+with+a+fluorescent+reporter+construct.">Transgenesis in fish: efficient selection of transgenic fish by co-injection with a fluorescent reporter construct.</a> Nat Protoc. 1 (3), 1133-1139 (2006).</li><li>Conchello, J. A., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+sectioning+microscopy.">Optical sectioning microscopy.</a> Nat Methods. 2 (12), 920-931 (2005).</li><li>Jensen, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Types+of+imaging,+Part+2:+an+overview+of+fluorescence+microscopy.">Types of imaging, Part 2: an overview of fluorescence microscopy.</a> Anat Rec (Hoboken). 295 (10), 1621-1627 (2012).</li><li>Chasles, F., Dubertret, B., Boccara, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+and+characterization+of+a+structured+illumination+microscope.">Optimization and characterization of a structured illumination microscope.</a> Opt Express. 15 (24), 16130-16140 (2007).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203 (3), 253-310 (1995).</li><li>Hsu, H. J., Lin, G., Chung, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+early+development+of+zebrafish+interrenal+glands+and+pronephros:+differential+control+by+wt1+and+ff1b.">Parallel early development of zebrafish interrenal glands and pronephros: differential control by wt1 and ff1b.</a> Development. 130 (10), 2107-2116 (2003).</li><li>O'Brien, L. 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=Wt1a,+Foxc1a,+and+the+Notch+mediator+Rbpj+physically+interact+and+regulate+the+formation+of+podocytes+in+zebrafish.">Wt1a, Foxc1a, and the Notch mediator Rbpj physically interact and regulate the formation of podocytes in zebrafish.</a> Dev Biol. 358 (2), 318-330 (2011).</li></ol>

作者迈克尔·格拉夫是卡尔·蔡司显微镜有限公司产生本条法器的员工。

斑马鱼已成为脊椎动物发育和建模人类疾病的研究流行的模式生物。因为斑马鱼胚胎保持透明，如大脑和心脏显影器官可以使用标准制备显微镜观察到。以转基因品系的优势与器官特异性荧光使荧光显微镜下活胎内的整个开发过程中的不同阶段器官的评估。与标准荧光显微镜成像整个器官的结构细节一个限制是信号从上方和下方的聚焦平面的物体的影响。外的焦点该光不仅导致降低图像的对比度和分辨率，它也可能会掩盖感兴趣13,14重要结构。几种技术如激光扫描confocal-，旋转盘confocal-或多光子显微镜已开发，以减少外的焦点的信息，并由此人口增加Ë图像对比度以及轴向分辨率。的替代方法，以获得光学切片是被激光和扫描自由结构照明显微镜，宽基于场的照明技术，该技术是简单的，定期显微镜15来实现。此处呈现的结果表明，光学切片，由结构照明来实现，在解剖显微镜来实现，并与扩展聚焦图像重建相结合，使得能够正常结构细节的可视化，并在斑马鱼干扰肾发展。特别是，在wt1a morphants GFP阳性细胞被分散在不同的聚焦深度，并与失焦模糊仅在一个平面的图像会低估表型的三维的复杂性。 跟随器官组织，重排或中断的动态，时间推移的荧光显微镜是一种强大尽管复杂的技术。在延时成像轻微的振动或轻微的温度波动引起的x，y或z方向上漂移。这是为了获得稳定的结果需要额外的设备（环境室，抗震动表）。该方法采用解剖显微镜的能力与录制时间推移图像，而无需额外的设备电动对焦驱动。保持稳定的聚焦，自动聚焦策略建立和用于x的校正，y轴不稳定印迹到成像室的底部的重定位网格。 胚胎的适当嵌入是在协议中的一个关键步骤。需要一些实践以放置感兴趣尽可能接近到玻璃底和中紧挨给电网的结构，而没有与它重叠。 该方法的主要优点是，它是对发育过程如在活生长和迁移的直接观察实惠和简单的工具胚胎在几个小时。此外，解剖显​​微镜特定收益如观察和扩展工作距离大视场更大的方便样本，包括整个器官的检查。然而，一些限制必须牢记。实验的暂停来检查和重新调整的定位使得费时和缺乏可靠的温度控制的改变"标准"发育时间，这是在28.5℃的斑马鱼16定义为小时受精后的过程。另一个潜在的问题是成象的受限制的深度到动物，尤其是当感兴趣结构位于胚胎或幼虫的内部。这样的结构是斑马鱼pronephric肾小球，这是位于该体节和卵黄囊之间。成像肾小球限制深度被发现是大约200微米，以后发展5天达到该距离。相反，荧光结构，其为1-ocated接近动物的表面能够被成像为一个较长的时间。例如肝细胞成像访问至少要等到9 DPF。进一步的限制是，在琼脂糖和三卡因治疗胚胎或幼虫的长期固定诱导发育迟缓和心脏水肿，分别。因为这可能与正常发展干扰，建议时间推移记录的持续时间限制到感兴趣的一定期间。以确保在感兴趣的成像结构正常发展是有帮助的比较（在末端）与该动物的整体外观保持相同的实验条件下，但没有嵌入和麻醉。 记录光学部分的Z堆叠在一段5小时和聚焦图像的扩展深度，随后计算每30分钟提供有关正常的早期事件的空间和时间信息和干扰肾发展其诱导的BŸ吗啉介导wt1a的击倒。先前执行啉拦截实验已经表明，Wt1a满足在前肾形成的早期和必不可少的作用。在肾标记17,18和转基因wt1b就业原位杂交:在固定的时间用于成像前肾发展的GFP线指向9,10-揭示这wt1a缺乏导致肾小球破坏形态和失败的足规范。相对于这些静态的方法，定时录音，直接可视化控制胚胎早期nephrogenesis和GFP阳性细胞从pronephric区域客场wt1a morphants迁移的动态。随着时间的推移跟踪错误路由的细胞的可能性允许的更详细的表型分析。 在一般情况下，在这里提出的方法提供了一种廉价和易于使用的替代复杂的，并且较少可访问本身tups通常用于延时成像如激光扫描显微镜（装备有环境舱和抗振动台）或光片的显微镜。所描述的程序来解决漂移问题，也可以应用到没有光学切片的选项，如荧光立体显微镜上执行设置时间推移的图像。 该技术不仅适用于研究肾发展，但也可以适用于监控其他器官，如心脏或肝脏的正常和有缺陷的形态。此外，该方法可以用来观察在胚胎和成年模式生物的各种更多的动态过程，如伤口​​愈合或再生。

以下原肠胚形成，器官是个体的生命周期的下一阶段。它涉及到重排，互动和经常细胞的迁移，以产生组织和器官。误差在器官的发育所依据的过程往往导致可立即或也表现出来以后的生活中的疾病。因此，了解器官已经发育生物学和生物医学研究的重大努力。为了能够调查特定器官的发育，它具有即使是通过胚胎的体壁可见。这使得能够在开发过程中发生的空间和时间变化的记录。也为了分析参与器官发生因素的重要性，它需要容易操纵。 一个生物体是非常适合于体内器官的调查是斑马鱼。其transpar与使用的荧光转基因系在组合胚胎发育过程中ency允许蛋白定位和表达动力学的观察，以及内部器官1的可视化。这提供了有关相比，哺乳动物模型，其器官都无法进入微观分析器官发育的实时调查的独特优势。此外，不同的工具可用来操纵斑马鱼的胚胎发育。旁的突变系的产生，反义吗啉代寡核苷酸（MO）可以用来敲除所涉及的某些器官的发育特定基因的活性。无论是材料订单目标剪接位点或翻译起始密码子（AUG），从而与mRNA前体或翻译的拼接干涉。 斑马鱼的胚胎肾中，前肾，是一种解剖学上简单而有价值的模型来研究肾脏发育和根的功能与肾脏疾病2上课。它仅由两个被称为肾功能单元。每个肾单位，其中包括血液过滤发生3肾小球的。进一步组件是短颈部区域以及分割小管分泌和溶质的重吸收和管道，在泄殖腔4中结束。不管其简单组合物，组织和不同细胞类型的斑马鱼前肾的非常相似的哺乳动物肾5,6。 一个因素，这是极其参与肾发展，由肾母细胞肿瘤抑制基因WT1 7编码。斑马鱼具有两个旁系称为wt1a和wt1b以重叠但不相同的模式的前肾8的发育过程中表达。通过使用GFP标记前肾结构转基因斑马鱼，它已被证明的wt1a &lt;即完整击倒/ em&gt;的或特定剪接形式导致9,10分别胚胎肾严重或轻微畸形。 这里所描述的方法允许在斑马鱼胚胎正常和受损nephrogenesis的时间推移分析通过使用配备用于经由结构照明光学切片荧光解剖显微镜。一般光学切片允许收购其中只包含焦点对准信息图像。外的焦点可通过各种方法，如数学算法（ 例如去卷积），光学设计（ 例如共聚焦激光扫描显微镜）或两者的组合（ 例如 ，结构照明）来避免信息。 诱导肾发育缺陷，我们使用的反义MO针对注入的转基因斑马鱼线wt1a（TG（wt1b:GFP））。这条线表示在肾小球，颈部和前的GFP荧光细管9,10的一部分。相比于更复杂的设置时间推移的录音与光学切片，如激光扫描或光片的显微镜，变焦显微镜是易于处理和更便宜。此外，结构照明设备可以很容易地与传统的荧光设备（ 如荧光灯，过滤器），并提供特定的解剖显微镜等优势，如延长工作距离和大视场相结合。带漂移问题解决了没有使用通常需要稳定业绩的补充设备（环境试验箱，抗震动表）。为了校正成立自动聚焦策略和与压印搬迁网格成像室中利用重新调整之后在x或y方向上的漂移的定位焦点的漂移。 所提出的方法也可以应用到显微镜没有光学切片的选项，如荧光立体声米icroscopes并提供更复杂的设备通常用于发育组织和器官的动态时移记录的替代品。

<table border="0" cellpadding="0" cellspacing="0" width="762">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Material</td>
			<td style="width:147px;"></td>
			<td style="width:148px;"></td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Control Morpholino</td>
			<td style="width:147px;">Gene tools, LLC</td>
			<td style="width:148px;">Standard control oligo</td>
			<td style="width:252px;">Prepared&#160;control&#160;oligo</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">wt1a Morpholinos&#160;</td>
			<td style="width:147px;">Gene tools, LLC</td>
			<td style="width:148px;">customized</td>
			<td style="width:252px;">Designted to target the first splice donor site, sequence as published in Ref. Nr. 9&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0,5% Phenol red solution</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:148px;">P0290</td>
			<td style="width:252px;">Injection tracer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">peqGOLD universal agarose</td>
			<td style="width:147px;">peqlab</td>
			<td style="width:148px;">35-1020</td>
			<td style="width:252px;">To prepare microinjection dish</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Glass capillaries&#160; (GC100F-10)</td>
			<td style="width:147px;">Hugo Sachs Elektronik</td>
			<td style="width:148px;">30-0019</td>
			<td style="width:252px;">To generate injection needles&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microloader tips</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:148px;">5242956003</td>
			<td style="width:252px;">To load the microinjection needle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dumont forceps&#160;</td>
			<td style="width:147px;">Fine Sience Tools</td>
			<td style="width:148px;">91150-20</td>
			<td style="width:252px;">To generate an opening at the needle tip&#160;</td>
		</tr>
		<tr height="88">
			<td height="88" style="height:88px;width:216px;">Embryo water (0.3x Danieaus&#180; solution)</td>
			<td style="width:147px;"></td>
			<td style="width:148px;"></td>
			<td style="width:252px;">1x: 58 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO4,&#160; 0.6 Ca(NO3)2, 5 mM HEPES, pH: 7,5; Do not add methylene blue!</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Graticule 5mm / 0.05mm</td>
			<td style="width:147px;">Science Services</td>
			<td style="width:148px;">PY68039-02</td>
			<td style="width:252px;">For calculation of injection amount</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pasteur pipette 137 mm, 4.8 ml</td>
			<td style="width:147px;">Roth</td>
			<td style="width:148px;">E305.1</td>
			<td style="width:252px;">To collect eggs, to transfer embryos and to remove dead embryos</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pasteur pipette with ultra-thin tip 157 mm, 3.5 ml</td>
			<td style="width:147px;">Roth</td>
			<td style="width:148px;">E304.1</td>
			<td style="width:252px;">To remove excess of water&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N-Phenylthiourea (PTU)</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:148px;">P7629</td>
			<td style="width:252px;">For suppression of melanization</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td>
			<td style="width:147px;">Sigma</td>
			<td style="width:148px;">E10521</td>
			<td style="width:252px;">To anethesize zebrafish</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Low melting agarose</td>
			<td style="width:147px;">Biozym</td>
			<td style="width:148px;">850111</td>
			<td style="width:252px;">For embedding&#160;</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:216px;">&#181;-dish 35 mm, high Glass Bottom Grid-50</td>
			<td style="width:147px;">ibidi</td>
			<td style="width:148px;">81148</td>
			<td style="width:252px;">Imaging chamber</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gel loader tips</td>
			<td style="width:147px;">Hartenstein</td>
			<td style="width:148px;">GS05</td>
			<td style="width:252px;">To orient the embryo for proper embedding</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Equipment</td>
			<td style="width:147px;"></td>
			<td style="width:148px;"></td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micropipette puller (model P-97)</td>
			<td style="width:147px;">Sutter Instrument</td>
			<td style="width:148px;"></td>
			<td style="width:252px;">To generate injection needles&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micromanipulator</td>
			<td style="width:147px;">Saur Laborbedarf</td>
			<td style="width:148px;"></td>
			<td style="width:252px;">For microinjection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thermomixer copact</td>
			<td style="width:147px;">Eppendorf</td>
			<td style="width:148px;"></td>
			<td style="width:252px;">Thermoblock for tempering the low melting agarose</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SZ61 (Stereomicroscope)</td>
			<td style="width:147px;">Olympus</td>
			<td style="width:148px;"></td>
			<td style="width:252px;">For injection control</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Axio Zoom. V16</td>
			<td style="width:147px;">Zeiss</td>
			<td style="width:148px;"></td>
			<td style="width:252px;">For embedding and imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ApoTome.2 slider</td>
			<td style="width:147px;">Zeiss</td>
			<td style="width:148px;"></td>
			<td style="width:252px;">For optical sectioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">AxioCam MRm</td>
			<td style="width:147px;">Zeiss</td>
			<td style="width:148px;"></td>
			<td style="width:252px;">For image acquisition&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ZEN 2012</td>
			<td style="width:147px;">Zeiss</td>
			<td style="width:148px;"></td>
			<td style="width:252px;">Imaging software</td>
		</tr>
	</tbody>
</table>

analysis of zebrafish kidney development with time-lapse imaging using a dissecting microscope equipped for optical sectioning

为了了解器官，需要记录组织发育过程中出现的空间和时间的改变。这里所描述的方法允许在斑马鱼胚胎正常和受损肾发展的时间推移分析使用装备结构照明和z堆栈采集荧光解剖显微镜。为了显现nephrogenesis，转基因斑马鱼（TG（wt1b:GFP））用于与荧光标记的肾脏结构。肾缺陷是由对Wilms肿瘤基因wt1a，已知可用于肾发展的关键因素的反义吗啉代寡核苷酸的注射触发。 实验装置的优点是用在X，Y或Z方向上没有附加设备重新调整的动作简单的策略的变焦显微镜的组合。规避是受温度变化和机械振动引起的焦距漂移，自动聚焦策略应用，而不是利用通常需要环境室。为了重新调整由于XY-漂移位置的变化，采用了与印迹搬迁改造成像室。 相比于更复杂的设置为与光学切片的时间推移记录如共焦激光扫描或光片的显微镜，变焦显微镜是容易处理。此外，它提供了具体的解剖显微镜效益，如高景深和扩展工作距离。 研究这里提出器官的方法，也可以用荧光立体显微镜不能光学切片的使用。虽然不限于高通量的，这种技术提供了更为复杂的设备通常用于发育组织和器官的动态时移记录的替代品。

时间推移显微镜是一种强大的技术在一段较长的时间来观看动态生物过程。时间推移成像过程中经常发生的问题是，在X，Y或Z方向的移动，被称为漂移，这是由温度变化和机械振动引起的。这里介绍的方法实现定时录制在解剖显微镜的斑马鱼胚胎或幼虫的荧光标记的结构不受环境室和抗振动台。 为XY-漂移的补偿，将试样包埋在成像室带有印迹重定位网格（ 图1）。相关的软件工具的十字线网格的一个特定点的位置被用于检体返回到预调整的位置（ 图2A）。所呈现的结果是通过使用放大显微镜获得为经由结构照明（ 图2B）的光学切片的集成滑块。对于连续焦点校正，自动对焦的战略正式成立。在这种策略中，软件自动对焦使用基于各时间点之前最大对比度找到的焦点。这使定义焦平面随后被设置为Z堆栈购书中心的计划。为了最大限度地减少在样品中光毒性，透射光明信道被用作参考信道，因为它在自动对焦步骤（ 图2C）可以足够短的曝光时间。 已申请在转基因斑马鱼线的控制和wt1a morphant胚胎（TG（wt1b:GFP））肾脏发育的比较分析的方法。在早期nephrogenesis此行显示在中间的中胚层绿色荧光，被肾脏祖细胞从9,10和后来出现在成形管和肾原基（ 图3）。 隔时图像记录表明nephrogenesis在控制啉注射的胚胎相比是未注射的那些未改变的（结果未显示），并如下早期斑马鱼肾发展3所描述的步骤。在20 HPF显影pronephric管是可见的，在其前部的提示，细胞球形聚集，代表成形肾原基可以被检测出来。在接下来的时间，小管和肾原基生长和稍后的原基开始在中线（ 图4，视频2）融合。相比之下，nephrogenesis严重的wt1a morphant胚胎破坏。尽管GFP阳性的管状结构是在20高倍视野可见的，它们显得更弥漫和欠发达国家。此外，已形成​​没有合适的肾原基。最显着的区别为t他在这个时间点控制胚胎，但是，是在显影前肾（ 图4，视频2）以外的大量的荧光细胞的外观。随后，这些细胞离开pronephric字段和腹侧迁移（ 视频2）。 在控制胚胎和转基因wt1b线wt1a morphants肾脏发育都通过不同的固定时间点9,10拍摄图像以前相比。与此相反的静态方法，定时录音允许按照正常nephrogenesis而造成wt1a枯竭肾脏祖细胞的错打的动态。 <img alt="图1" src="/files/ftp_upload/53921/53921fig1.jpg" /> 图1:市售商会与搬迁网格嵌入式胚胎的示意图 。这道菜有四宫格，每个细分为50μm的重复距离，印迹到玻璃盖玻片底部。要成像的胚胎被嵌入倒挂，在靠近观察肾方结构，但没有与电网覆盖。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53921/53921fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53921/53921fig2.jpg" /> 图2:调整对漂移的补偿时间推移成像和结构照明的网格投影搬迁电网的不同位置被带进图像中心（黄色十字标记），并作为一个参考点以后的XY对准在小变化的情况下，。红色矩形表示感兴趣区域（ROI）的自动对焦策略（A）使用的区域。光学切片WA■通过结构照明获得。该图像示出了在正确的校准（B）的后焦平面伸出的格子结构。对于自动对焦策略（红色矩形）的投资回报率被设定为涵盖独特的胚胎结构的高对比度（在这里个体节），允许可靠的自动对焦到感兴趣（C）的结构。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53921/53921fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53921/53921fig3.jpg" /> 图3:转基因 wt1b:GFP胚胎绿色荧光在发展中肾背（A）或横向（B）传输和荧光图像叠加显示有前左侧。 NP，肾原基; PT，pronephric肾小管;一般般螨。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53921/53921fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53921/53921fig4.jpg" /> 图4:wt1a敲除 打乱胚胎肾发展代表，从时间推移录音对焦的图像扩展焦深。在控制啉注射胚胎，肾发展呈现出不断增长的肾小管和肾原基开始其在中线融合的正常进展。与此相反，wt1a morphants不能形成适当的肾原基和GFP阳性细胞的巨量是pronephric字段之外。 （NP，肾原基; PT，pronephric肾小管）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53921/53921fig4large.jpg" target="_blank">请点击此处查看该网络的放大版本</a>古尔。 <img alt="电影1" src="/files/ftp_upload/53921/53921movie1.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53921/co-MO.mov" target="_blank">补充视频1.在控制啉注射胚胎正常肾发展延时录制。 （点击下载）。</a>视频显示管如何生长和肾原基开始融合。在20 HPF开始，图像30分钟间隔取一段5小时。 <img alt="电影2" src="/files/ftp_upload/53921/53921movie2.jpg" /> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53921/wt1a-MO.mov" target="_blank">在wt1a morphant胚胎感到不安nephrogenesis的补充视频2.定时录制。 （点击下载）。</a>视频显示GFP阳性细胞的迁移出pronephric地区。在20 HPF开始，图像拍摄于30英里N个间隔了一段5小时。

Watch this Scientific Journal Video about Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning at JoVE.com

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

这里介绍的方法允许在斑马鱼胚胎器官发育的时间推移分析，通过使用荧光解剖能够执行光学切片和调整的简单的策略来纠正焦点和平面漂移的显微镜。

配备了光学切片与成像定时使用解剖显微镜斑马鱼肾脏发展分析

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

analysis-zebrafish-kidney-development-with-time-lapse-imaging-using

Research

JoVE Journal

Developmental Biology

10.7K 次观看.  Leibniz Institute on Aging - Fritz Lipmann Institute (FLI). 这里介绍的方法允许在斑马鱼胚胎器官发育的时间推移分析，通过使用荧光解剖能够执行光学切片和调整的简单的策略来纠正焦点和平面漂移的显微镜。 

视频: Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

捕获组织修复的斑马鱼幼虫与时间推移明视立体显微镜

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

科研

发育生物学

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 ...

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 ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

水下滤纸三明治长期<em&gt;防爆大毛</em&gt;早期鸡胚时间推移成像

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune cells, and the coordination with lymphatic vessels and nerves. The multi-cell, multi-system interactions necessitate the investigation of angiogenesis in a physiologically relevant environment. Thus, while the use of in vitro cell-culture models have provided mechanistic insights, a common critique is that they do not recapitulate the complexity associated with a microvascular network. The objective of this protocol is to demonstrate the ability to make time-lapse comparisons of intact microvascular networks before and after angiogenesis stimulation in cultured rat mesentery tissues. Cultured tissues contain microvascular networks that maintain their hierarchy. Immunohistochemical labeling confirms the presence of endothelial cells, smooth muscle cells, pericytes, blood vessels and lymphatic vessels. In addition, labeling tissues with BSI-lectin enables time-lapse comparison of local network regions before and after serum or growth factor stimulation characterized by increased capillary sprouting and vessel density. In comparison to common cell culture models, this method provides a tool for endothelial cell lineage studies and tissue specific angiogenic drug evaluation in physiologically relevant microvascular networks.

An Ex Vivo Method for Time-Lapse Imaging of Cultured Rat Mesenteric Microvascular Networks

Angiogenesis involves multi-cell, multi-system interactions that need to be investigated in a physiologically relevant environment. The objective of this study is to demonstrate the ability of the rat mesentery culture model to make time-lapse comparisons of intact microvascular networks during angiogenesis.

一个<em&gt;离体</em&gt;方法培养大鼠肠系膜微血管网络的时间推移成像

Angiogenesis, defined as the growth of new blood vessels from pre-existing vessels, involves endothelial cells, pericytes, smooth muscle cells, immune ...

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

Here, we present a versatile mounting method that allows for the long-term time-lapse imaging of the posterior body development of live zebrafish embryos without perturbing normal development.

斑马鱼发展长期成像的多功能安装方法

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

精确的细胞消融方法建模发展中的斑马鱼急性肾损伤

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

多光子时间推移成像实时可视化发展: 迁移神经嵴细胞在斑马鱼胚胎中的可视化

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ellipsoid shape during development. This developmental process is called oogenesis and is crucial to generating functional mature eggs to secure the next fly generation. For these reasons, egg chambers have served as an ideal and relevant model to understand animal organ development.
Several in vitro culturing protocols have been developed, but there are several disadvantages to these protocols. One involves the application of various covers that exert an artificial pressure on the imaged egg chambers in order to immobilize them and to increase the imaged acquisition plane of the circumferential surface of the analyzed egg chambers. Such an approach may negatively influence the behavior of the thin actomyosin machinery that generates the power to rotate egg chambers around their longer axis.
Thus, to overcome this limitation, we culture Drosophila egg chambers freely in the media in order to reliably analyze actomyosin machinery along the circumference of egg chambers. In the first part of the protocol, we provide a manual detailing how to analyze the actomyosin machinery in a limited acquisition plane at the local cellular scale (up to 15 cells). In the second part of the protocol, we provide users with a new Fiji-based plugin that allows the simple extraction of a defined thin layer of the egg chambers&#8217; circumferential surface. The following protocol then describes how to analyze actomyosin signals at the tissue scale (&#62;50 cells). Finally, we pinpoint the limitations of these approaches at both the local cellular and tissue scales and discuss its potential future development and possible applications.

Analysis of Actomyosin Dynamics at Local Cellular and Tissue Scales Using Time-lapse Movies of Cultured Drosophila Egg Chambers

This protocol provides a Fiji-based, user-friendly methodology along with straightforward instructions explaining how to reliably analyze actomyosin behavior in individual cells and curved epithelial tissues. No programming skills are required to follow the tutorial; all steps are performed in a semi-interactive manner using the graphical user interface of Fiji and associated plugins.

使用培养的果蝇卵室延时电影对局部细胞和组织尺度的actomyosin动力学的分析

Drosophila immature eggs are called egg chambers, and their structure resembles primitive organs that undergo morphological changes from a round to an ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

亚细胞分辨率的各向同性光片显微镜及细胞自动动力学分析

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...