The authors would like to acknowledge the CIHR for fellowship support of Steve Deimling and the Department of Paediatrics, University of Western Ontario for support of Steve Deimling, Rami Halabi and Stephanie Grover. This work was supported by the NSERC grant R2654A11 and an NSERC Discovery Accelerator Supplement

1.胚胎准备<ol><li>如果不是作为胚胎培养，脱果冻用2.5％半胱氨酸，pH 8.0中之前定影8胚胎的一部分经常做。虽然这不是绝对必要的，它然后手动使用细镊子固定之前​​除去受精信封是有用的。 <ol><li>使用玻璃巴斯德吸管转移的胚胎。移液器是不够宽传送胚胎所以使用金刚石笔来切割玻璃吸管在一个点宽到足以拿起一个胚胎。通过快速传递切断端通过本生灯的火焰来熔化尖锐边缘切割后消除移液器的锐边。 注：护理需要采取，因为即使它似乎已经冷却目测玻璃仍然很热，会造成烫伤。 </li></ol></li><li>在执行阶段，胚胎固定。首先，准备玻璃小瓶中的胚胎固定使用。使用这些小瓶的所有步骤，包括最后的存储。用小瓶清楚，在盖子的好聚四氟乙烯密封，允许在所有加工步骤的监测胚胎。标签使用永久性记号笔适当的实验信息的小瓶，然后盖上透明胶带标签，因为即使记号笔将失去在诉讼过程中由于使用酒精等溶剂。 <ol><li>使用Mempfa修复的胚胎。使在50-100毫升批次8％多聚甲醛的库存的时间。使用所需的H 2 O和热的约75％至50〜60℃，这是必要的，以获得多聚甲醛溶液中。 注意：此解决方案比使用旧的发布的协议版本Memfa，它采用多聚甲醛，而不是甲醛略有不同。 </li><li>加入2-3滴10N的NaOH或直到pH值大约为7.5（用pH试纸来检查pH值）。多聚甲醛的毒性很大，因此产生的原液在通风橱。一旦第甲醛是在溶液中，过滤通过Whatman纸上的溶液到一个新的容器中，添加的H 2 O来最终体积。如果需要的话，储存在4℃下的多聚甲醛溶液为1-2周。 </li><li>组装Mempfa定影溶液（ 表1）的剩余部分。确保多聚甲醛的最终工作浓度是4％。存储在固定解决方案的所有部件，在4℃为股票。 </li><li>使用切玻璃吸管中，加入胚胎与标记的玻璃小瓶已填充有大约3-4毫升Mempfa溶液（ 表1）定出的胚胎。避免定影每瓶超过20-30胚胎。添加的胚胎以最小从胚胎介质液体传输。固定在Mempfa溶液胚胎2小时，在室温下过夜或在4℃下。 </li><li>对于后期的内胚层结构上手动隔离肠道和内胚层衍生工具9,10进行的拓扑，其允许探针的良好渗透和还避免空腔染色。 </li><li>存储100％的甲醇在-20℃的溶液中。的多聚甲醛固定后，替换为约4毫升的-20℃，100％的甲醇用于存储的胚胎的Mempfa溶液。 </li><li>加入甲醇后摇动小瓶，以防止胚粘到玻璃或其它胚胎。另外，确保小瓶密封，因为甲醇可如果松动，在冷冻蒸发随着时间的推移。 注：胚胎可以存储在至少一年的时间，并有可能更长，在以染色与原位杂交质量没有损失的现有的甲醇。 </li></ol></li></ol> 2.探针制备<ol><li>用1-2微克模板DNA，使高辛标记的探针。含有合适的DNA序列在用限制性感兴趣的基因的5&#39;端切割酶的质粒适于塔T载体，产生反义RNA探针。 注：质粒应当具有适当的RNA聚合酶结合位点（ 例如 T7，T3或SP6）。 </li><li>允许该DNA，水，NTP混合和聚合酶缓冲温热至室温，组装探针合成反应之前。添加的顺序的RNA合成反应到1.5ml微量离心管中的成分如表2中列出。调整水的量加入，使所述探针合成反应的总体积为20微升。装配在室温下将反应因聚合酶缓冲的部件可以沉淀在高浓度和冷的模板DNA时。 </li><li>孵育2小时，在37℃的转录反应。 注：稍长于两个小时的孵育导致无不良影响，而且还显示很小增加的产量。如果温育1小时，产率会降低，但仍足以使一个好的探针。 <ol><li>在等待转录反应到结束，使将要用于测试探针的质量的琼脂糖凝胶。熔体1微克琼脂糖在100毫升1×TAE缓冲液的（ 见表1）通过加热该溶液到沸点。起锅的解决方案时，琼脂糖粉完全溶解。 </li><li>添加2微升溴化乙锭原液（10毫克/毫升），以约100毫升琼脂糖凝胶时琼脂糖已冷却至约60℃，以允许的RNA在紫外线（UV）光可视化。 注意：此琼脂糖凝胶溶液，可以保持在60℃的培养箱中，使得将来的凝胶可以在不重复的熔化浇。小心处理溴化乙锭由于潜在的毒性。 </li></ol></li><li>添加1μl的DNA酶I（无RNA酶的级）的转录反应的2小时温育后，孵育另外10分钟，在37℃下，以消除模板DNA。 </li><li>除去1微升反应混合物中，以检查在1％琼脂糖TAE凝胶和其余（20微升）中的TE缓冲液（10mM的Tris pH值8.0，1mM EDTA）中，加入10μl5M NH 4醋酸纤维，和220微升添加80μl的1％SDS的冷乙醇。涡大力混合，并预留冰上直到RNA质量检查的结果是已知的。 <ol><li>运行1微升RNA沉淀在1％琼脂糖凝胶TAE前去除。为了缓解加载在凝胶上，加4-5微升水和1μl的标准样染料与RNA。查看使用UV光透射​​凝胶中的RNA，以检查探针的质量（见讨论）。 </li></ol></li><li>沉淀被搁置在冰上，在步骤2.5通过纺丝在微量全速进行10至15分钟，剩余的RNA。抽出上清液用抽出玻璃吸管，并允许简单干燥。 <ol><li>重悬探针用1ml的RNA杂交缓冲液（ 见表1），在微量离心管中。涡和BRiefly再次加热该管至37℃并涡旋。探头溶液转移到一个15毫升的螺丝帽聚苯乙烯管，并填写7-10毫升RNA杂交缓冲液。注意：该探针可以进一步稀释（10倍进一步稀释仍然可以工作）常常导致低的背景，但在染色反应也将花费更长的时间。 </li></ol></li></ol> 3. 在原位杂交<ol><li>取被储存在-20℃的甲醇，该胚胎，并允许温热至室温。在玻璃小瓶中执行整个过程。如果从一组不同的胚胎要由不同的探针来看待，使他们在一个单一的小瓶中加入之前的探针，以减少在第一天的变异和劳动罚金。 <ol><li>再水化通过甲醇系列胚胎根据在制备例表3（见表1对洗涤配方）概述的加成探针。轻轻摇动安莉芳每次更改后的OS，以确保它们不会附着在侧面或彼此，并且通过安装在小瓶一个nutator摇动胚胎。当传送的液体，检查盖的内侧，以确保胚胎尚未被困在那里。 </li><li>一旦在探针溶液，如表3中所述杂交胚胎过夜。 </li></ol></li><li>拆除探头的解决方案。减所使用的探针通过存储在15ml螺丝帽的聚苯乙烯管中，标有日期和时间的探针已被用于数，在-20℃下。 注意：相同的探针可以重复使用多次为原位杂交随后直至比色反应开始采取异常长的时间以达到期望的强度。 <ol><li>一旦探针被去除，制备了胚胎对通过串联在表4中概述洗涤的探针抗体染色。改变温度的要求（ 表4）通过移动吨他章动器，附加的胚胎的小瓶，直接进入被设置为适当的温度的杂交炉。 </li><li>弥补了MAB适当体积+ HTSS + BR +抗DIG抗体（ 表4），在该胚胎被培养在MAB + HTSS + BR封闭液，使抗体之前加入到阻止时间胚胎。使阻断溶液新鲜上使用的当天。 </li></ol></li><li>除去抗体溶液，并开始如以下过夜孵育抗体在表5中概述的洗涤。为了染色准备与碱性磷酸酶底物，并减少背景尽可能执行至少12 30分钟洗涤。使用任一MAB缓冲或TBT溶液（ 表1），用于洗涤步骤。 注：TBT溶液是TTW具有2mg / ml的牛血清白蛋白的补充。 <ol><li>替换为BM紫碱性磷酸酶底的最后洗涤液。进行染色REAC灰在室温或37℃。 注：染色是在37℃，更迅速，但如果放置过夜，不可接受的背景染色往往是一个结果。染色反应往往需要通宵染色和室温下是最安全的了。 </li><li>如果进一步染色是必需的，对BM紫溶液将具有一种蓝色，替换以新鲜的BM紫染色溶液，并把管放入37℃。如果靶mRNA是非常丰富，放于染色溶液中的胚胎在4℃下过夜，然后将胚胎至室温或37℃，以允许更好地监测该染色反应。 </li><li>仔细监测新的反应，随着时间的最终结果，不同的目标的RNA之间有很大的差别。为了保持一致性，停止染色反应（见3.4）时，有较长的潜伏期而产生的表达没有新的站点。重要的是，如果在原位杂交被用作测定对于实验看不同的治疗组，使用相同的时间进行治疗和对照胚胎之间的颜色反应。 </li></ol></li><li>停止染色反应和制备的胚胎用于存储和图像记录通过如在表6中概述改变的液体。在此阶段，不要摇动胚胎因为去除污渍可以被可视化为围绕胚胎的甲醇紫色着色。 注意：通常胚胎有从染色溶液和冷的甲醇可以至少部分地移除光蓝染色，虽然这是不足够的，以除去重背景或空腔染色。冷甲醇是指保持在-20℃的冷冻的甲醇。 <ol><li>再水合胚胎和修复Mempfa（ 表6）的污点。一旦固定，取下Mempfa和洗涤，用25％甲醇的胚胎。 </li><li>除去25％的甲醇和如果添加漂白溶液除去内源性颜料是必须的。小心处理漂白解决方案，它可引起灼伤。密切观察漂白，因为它发生相对迅速和漂白的程度可以变化为不同的效果（ 图2）。 </li><li>通过甲醇系列至100％甲醇于长期贮存以下漂白脱水胚胎，或转移到PBS中于短期存储和随后的成像（ 见表6）。 </li></ol></li></ol> 4.成像胚胎<ol><li>一旦胚胎已完成染色的过程中，图像中的胚胎，使上，其中感兴趣的基因表达的信息被捕获为更广泛的观众。琼脂糖使用1％作为背景，查看未清除的胚胎。 注：琼脂糖给出一个蓝色/灰色的背景，与胚胎和染色反应的蓝色对比井。它还有助于弥漫出现阴影和转移注意力从胚胎反射。 <ol><li>琼脂糖加入水，然后煮沸，直到琼脂糖是在溶液中，然后放凉到50倒入培养皿之前。由于与TAE凝胶液，保存在55℃培养箱用于多种用途的琼脂糖溶液。倒入琼脂糖至约2mm的深度到陪替氏培养皿中。如果需要的话，调整的琼脂糖的深度以产生稍微不同的色光的背景。 </li><li>以下从存储在甲醇补液的水溶液（PBS或TTW），使用甲醇系列，放置在培养皿中的琼脂糖基的胚胎（ 见表6）。保持清洁的解决方案，如果需要，可以使用简单的过滤，消除小颗粒物，能破坏的良好形象干净的背景。 </li><li>图像从备用视图，如从腹侧胚胎，切细的通道中使用的琼脂糖细镊子以适合胚胎并放置在胚胎中的那些信道对的方向（ 图3中 </strong&gt;的）。小心在操纵胚胎，因为它们很容易损坏。 注意：大多数阶段都有自己承担时，放入溶液中的特征位置。例如，囊胚期胚胎倾向于坐动物朝上。当胚胎开始拉长，他们躺在自己的身边。 </li></ol></li><li>使用光纤光源从一个浅角度照亮胚胎，创建提供深度图像上的阴影胚胎，并有助于辨别表面结构。由于漂白可以消除色素，提供有用的标志性建筑，用阴影的强烈漂白的胚胎。 </li><li>清除胚胎染色图像是深的胚胎内，如在脊索，肺，或脑部，（ 图4）的区域。为了实现这一点，把通过甲醇系列的胚胎直到在100％甲醇。 <ol><li>在甲醇中完成浸泡后，将胚转移到溶液1份苄醇和2份苄基是nzoate（BABB）。胚胎最初将浮在表面上，但由于与BABB甲醇混合，它们将沉入BABB。每次执行与BABB处理玻璃小瓶或菜肴一步; BABB会融化任何塑料或油漆。 </li><li>一旦清除，查看与透射光从胚胎下方传来的胚胎。调节光的强度，以及所述角度从下方，以提高对比度，并提供最好的颜色。 </li><li>当观看清除胚胎提高玻璃培养皿关闭基地。只需使用另外两个培养皿的盖子，以提高它，以便与胚胎菜的区域升高做到这一点。 注意：这具有服用碱失焦和消除从在显微镜基，可以与图像干扰的缺陷或污迹分散效果的优点。 </li></ol></li></ol> 5.双击原位杂交<ol><li>为了同时查看总重量的表达模式o在单个胚胎不同的基因，合成两个探头，一个用于每个不同的基因。合成使用DIG-11-UTP作为标签如上所述一个探针。稀释在核糖核酸杂交缓冲液的转录反应的产物，以产生3倍更浓缩的探针比用于单原位杂交。 <ol><li>合成其它探针的利益使用相同的协议，作为DIG标记探测不同的是荧光素-12-UTP，必须取代DIG-11-UTP。稀释在核糖核酸杂交缓冲液的转录反应的产物，以产生3倍更浓缩的探针比用于单原位杂交。 </li><li>混合两种浓缩探针以1：1的比例。为了达到最佳效果，使用荧光素标记探针，显示了强大的表达在单原位杂交协议的基因。 </li></ol></li><li> 原位杂交协议使用相同的描述单原位&lt;/ em&gt;的杂交，除了使用双探针（探针含地高辛标记和荧光标记的探针的1.5倍浓缩混合物）在第一天的代替单个原位杂交探针的末端。 <ol><li>按照对双原位杂交协议的第二天单杂交协议，除了在一个1使用抗荧光素-AP Fab片段：4000稀释代替抗DIG-AP Fab片段。从胚胎中洗出过量的抗体作为单原位协议，并进行第一色反应使用BM-紫色的AP底物。 </li></ol></li><li>继第一显色反应，灭活荧光素抗体的0.1M甘氨酸pH值2.0 40分钟后万分之五分钟洗的人与生物圈计划。阻止MAB + HTSS + BR胚胎90分钟。添加抗DIG抗体以1：2000稀释MAB + HTSS + BR和孵化在4℃过夜。 <ol><li>次日，洗涤胚胎第oroughly在生物圈（12次洗涤30分钟），以除去过量的抗体。 </li><li>洗胚胎中的AP缓冲液10分钟（ 表1），然后染色用BCIP（为0.5mg / ml，在AP缓冲液）。 注： 在原位组合应该得到暗蓝紫色染色剂用于第一色反应和用于第二（ 图5）为淡蓝色的显色反应。 </li><li>停止最终色反应通过除去AP缓冲液和冲洗三次生物圈。固定用Mempfa胚胎10分钟。与5速洗的MAB或TBT洗胚胎。 </li><li>与此颜色组合，在后染色处理使用甲醇不再可能，因为这将消除BCIP单独颜色。染色和固定在PBS中与0.02％叠氮化钠后存储所述胚胎。染色强度可以与疲软的双重所在地 。如果这是一个问题，减少洗涤至4，每2个小时的持续时间。 </li></ol></li></ol>

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf+is+required+to+regulate+anterior-posterior+patterning+in+the+Xenopus+lateral+plate+mesoderm.">Fgf is required to regulate anterior-posterior patterning in the Xenopus lateral plate mesoderm.</a> Mech Dev. , (2011).</li><li>Fletcher, R. B., Harland, R. 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+role+of+FGF+signaling+in+the+establishment+and+maintenance+of+mesodermal+gene+expression+in+Xenopus.">The role of FGF signaling in the establishment and maintenance of mesodermal gene expression in Xenopus.</a> Dev Dyn. 237, 1243-1254 (2008).</li><li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Early development of Xenopus laevis : a laboratory manual. , (2000).</li><li>Park, E. C., Hayata, T., Cho, K. W., Han, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+cDNA+microarray+identification+of+genes+with+endodermal+organ+expression.">Xenopus cDNA microarray identification of genes with endodermal organ expression.</a> Dev Dyn. 236, 1633-1649 (2007).</li><li>Horb, M. E., Slack, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoderm+specification+and+differentiation+in+Xenopus+embryos.">Endoderm specification and differentiation in Xenopus embryos.</a> Dev Biol. 236, 330-343 (2001).</li><li>Nieuwkoop, P. D., Faber, 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> Normal table of Xenopus laevis (Daudin) : a systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis. , (1994).</li><li>Gebhardt, D. O., Nieuwkoop, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Influence+of+Lithium+on+the+Competence+of+the+Ectoderm+in+Ambystoma+Mexicanum.">The Influence of Lithium on the Competence of the Ectoderm in Ambystoma Mexicanum.</a> J Embryol Exp Morphol. 12, 317-331 (1964).</li><li>Nieuwkoop, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+formation+in+artificially+activated+ectoderm+(Rana+pipiens+and+Ambystoma+punctatum).">Pattern formation in artificially activated ectoderm (Rana pipiens and Ambystoma punctatum).</a> Dev Biol. 6, 255-279 (1963).</li><li>Sato, S. M., Sargent, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+approach+to+dorsoanterior+development+in+Xenopus+laevis.">Molecular approach to dorsoanterior development in Xenopus laevis.</a> Dev Biol. 137, 135-141 (1990).</li><li>Smith, S. J., Kotecha, S., Towers, N., Latinkic, B. V., Mohun, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XPOX2-peroxidase+expression+and+the+XLURP-1+promoter+reveal+the+site+of+embryonic+myeloid+cell+development+in+Xenopus.">XPOX2-peroxidase expression and the XLURP-1 promoter reveal the site of embryonic myeloid cell development in Xenopus.</a> Mech Dev. 117, 173-186 (2002).</li><li>Drysdale, T. A., Tonissen, K. F., Patterson, K. D., Crawford, M. J., Krieg, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+troponin+I+is+a+heart-specific+marker+in+the+Xenopus+embryo:+expression+during+abnormal+heart+morphogenesis.">Cardiac troponin I is a heart-specific marker in the Xenopus embryo: expression during abnormal heart morphogenesis.</a> Dev Biol. 165, 432-441 (1994).</li><li>Carroll, T., Wallingford, J., Seufert, D., Vize, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+regulation+of+pronephric+development.">Molecular regulation of pronephric development.</a> Curr Top Dev Biol. 44, 67-100 (1999).</li><li>Tonissen, K. F., Drysdale, T. A., Lints, T. J., Harvey, R. P., Krieg, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XNkx-2.5,+a+Xenopus+gene+related+to+Nkx-2.5+and+tinman:+evidence+for+a+conserved+role+in+cardiac+development.">XNkx-2.5, a Xenopus gene related to Nkx-2.5 and tinman: evidence for a conserved role in cardiac development.</a> Dev Biol. 162, 325-328 (1994).</li><li>Davidson, L. A., Ezin, A. M., Keller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+wound+healing+by+apical+contraction+and+ingression+in+Xenopus+laevis.">Embryonic wound healing by apical contraction and ingression in Xenopus laevis.</a> Cell Motil Cytoskeleton. 53, 163-176 (2002).</li><li>Hurtado, R., Mikawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+sensitivity+and+stability+in+two-color+in+situ+hybridization+by+means+of+a+novel+chromagenic+substrate+combination.">Enhanced sensitivity and stability in two-color in situ hybridization by means of a novel chromagenic substrate combination.</a> Dev Dyn. 235, 2811-2816 (2006).</li></ol>

Authors have no competing financial interests to disclose.

使用原位杂交技术来可视化特定基因的表达模式的能力仍然是最常用的方法，以确定在非洲爪蟾胚胎的特定器官或细胞类型。这是因为通过该技术提供若干优点。的基因的表达可以前充分分化的任何组织学征兆确定具体的结构，例如在之前的那些细胞18的任何明确划界的心脏祖细胞NKX2.5表达的情况。一旦所有的试剂是在手，它也是非常符合成本效益。许多个别的探针，...

The expression pattern of a specific gene is an important piece of information in determining the potential role for that gene in the development of a specific organ or cell type. Simply put, if it is not expressed at the right time and place it is unlikely to play a key role. In Xenopus, as in most early embryos, the most commonly used assay for detecting the expression of a gene is whole mount in situ hybridization using labeled antisense RNA probes. The use of antibody staining to assess expression of a gene in Xenopus is becoming more common as researchers discover antibodies, usually raised against mammalian proteins, that cross react to the Xenopus homologue or generate their own 1-3. However, the vast majority of studies on Xenopus organogenesis still utilize antisense RNA probes. When antibodies are used, each individual antibody often requires optimization for the primary antibody concentration or fixation protocols. In contrast, the protocol for in situ hybridizations is essentially invariant for different probes. The basic concept is relatively simple and an excellent standard protocol has been well established 4. Our protocol is a streamlined version of the original protocol 4 that still provides excellent detection of gene expression patterns in the early embryo. The embryos are fixed and then prepared for hybridization by changing solutions and temperatures such that it allows for high stringency binding of the labeled antisense RNA probe to its target mRNA. The unbound probe is washed away and the embryos are then prepared for binding of an antibody against the label on the RNA probes. Excess antibody is then washed away and an enzymatic color reaction is used to localize where the RNA probe is bound in the embryo. There are now a number of Xenopus transgenic lines that drive expression of fluorescent proteins in specific tissues and these are available at the Xenopus stock centers such as the National Xenopus Resource in Woods Hole. While very useful for many experiments that require examining organogenesis in living embryos, this option requires separate housing for the transgenic lines. 
In situ hybridization can clearly delineate where specific organs or cell types will form in the early embryo (Figure 1). The technique is remarkably sensitive given that one can detect gene expression in a small number of cells in a single embryo 5. However, in situ hybridization using the intensity of colorimetric staining is not considered quantifiable because the color reaction is not a linear one. Despite difficulty in quantifying staining intensity, changes in expression are often quite noticeable; particularly when the in situ hybridization shows quantifiable increases or decreases in the size of expression domains 6,7. 
The clear advantages of whole mount in situ hybridization make it a critical assay in the study of early development. However, it is a time consuming one that requires many steps over several days. This protocol is a simplified version of the standard protocol that eliminates several steps without reducing the quality of the in situ result. The simplification also eliminates sources of variability, making trouble shooting easier if an in situ hybridization is not optimal. Specifically, we have eliminated the use of proteinase K and RNAse treatments of the embryo, two steps that can depend on reagent quality and can also reduce signal intensity if overdone. The protocol also provides some degree of cost saving due to eliminating the use of several reagents. Finally, this protocol also provides some simple guidelines for improved capturing of images of in situ hybridization results. Although this protocol is optimized for work in Xenopus embryos, it is likely that at least some of the simplifications will be applicable to in situ hybridization work in other embryo systems.

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understanding early organogenesis using a simplified in situ hybridization protocol in xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

利用组织特异性探针可以在关于开发特定器官的状态提供了优秀的信息。在下列实施例中，胚胎的阶段是基于新科普和费伯临时表11。若用分化后表达的探针形式的基因， 心肌肌钙蛋白I在阶段28-30，例如（ 图1C），分化的器官的存在或大小可以在任何阶段交分化进行评估。几年前，胚胎学家们能够基于早期胚胎12,13的组织学显着的知识，做这样的分析，但这些?...

Watch this Scientific Journal Video about Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus at JoVE.com

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

了解早期器官使用简体<em&gt;原位</em&gt;杂交协议<em&gt;爪</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

understanding-early-organogenesis-using-simplified-situ-hybridization

Research

JoVE Journal

Developmental Biology

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

38.9K 次观看.  Hospital for Sick Children. The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

视频: Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

干细胞样<em&gt;爪</em&gt;胚胎外植体，研究早期神经发育特点<em&gt;体外</em&gt;和<em&gt;在体内</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

科研

发育生物学

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

功能性克隆使用<em&gt;爪</em&gt;卵母细胞表达系统

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

技术目标显微注射到显影<em&gt;爪</em&gt;肾

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are expressed in spatial relation to each other in situ. Multi-target chromogenic whole-mount in situ hybridization (MC-WISH) greatly facilitates the instant comparison of gene expression patterns, as it allows distinctive visualization of different mRNA species in contrasting colors in the same sample specimen. This provides the possibility to relate gene expression domains topographically to each other with high accuracy and to define unique and overlapping expression sites. In the presented protocol, we describe a MC-WISH procedure for comparing mRNA expression patterns of different genes in Drosophila embryos. Up to three RNA probes, each specific for another gene and labeled by a different hapten, are simultaneously hybridized to the embryo samples and subsequently detected by alkaline phosphatase-based colorimetric immunohistochemistry. The described procedure is detailed here for Drosophila, but works equally well with zebrafish embryos.

Multi-target Chromogenic Whole-mount In Situ Hybridization for Comparing Gene Expression Domains in Drosophila Embryos

We describe a multi-target chromogenic whole-mount in situ hybridization (MC-WISH) procedure in intact Drosophila embryos allowing simultaneous and specific detection of three different mRNA distribution patterns by contrasting color precipitates.

多目标显色全安装<em&gt;原位</em&gt;在比较基因表达域杂交<em&gt;果蝇</em&gt;胚胎

To analyze gene regulatory networks active during embryonic development and organogenesis it is essential to precisely define how the different genes are ...

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

全贴装荧光原位杂交和免疫荧光法在斑马鱼中 Multiciliated 细胞的可视化

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

利用荧光原位杂交 (fish) 监测中期和中期 I. 型果蝇卵母细胞的臂部凝聚状态

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

Two-step Approach to Explore Early- and Late-stages of Organ Formation in the Avian Model: The Thymus and Parathyroid Glands Organogenesis Paradigm

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the formation of quail-chicken chimeras and the use of the chorioallantoic membrane (CAM) to sustain the development of ectopic tissues date back to the last century. Nowadays, the combination of these classical techniques with recent in vitro methodologies offers novel prospects to further explore organ formation.
Here we describe a two-step approach to study early- and late-stages of organogenesis. Briefly, the embryonic region containing the presumptive territory of the organ is isolated from quail embryos and grown in vitro in an organotypic system (up to 48 h). Cultured tissues are subsequently grafted onto the CAM of a chicken embryo. After 10 days of in ovo development, fully formed organs are obtained from grafted tissues. This method also allows the modulation of signaling pathways by the regular administration of pharmacological agents and tissue genetic manipulation throughout in vitro and in ovo developmental steps. Additionally, developing tissues can be collected at any time-window to analyze their gene-expression profile (using quantitative PCR (qPCR), microarrays, etc.) and morphology (assessed with conventional histology and immunochemistry).
The described experimental procedure can be used as a tool to follow organ formation outside the avian embryo, from the early stages of organogenesis to fully formed and functional organs.

This article provides an updated approach to the classical quail-chicken chimera system to study organ formation, by combining novel in vitro and in ovo experimental procedures.

鸟类模型中器官形成早期和晚期的两步探讨: 胸腺和甲状旁腺器官发生范式

The avian embryo, as an experimental model, has been of utmost importance for seminal discoveries in developmental biology. Among several approaches, the ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Wholemount In Situ Hybridization for Astyanax Embryos

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of genes. Prior research into this emerging model system capitalized on comprehensive genome-wide investigations that have identified numerous quantitative trait loci (QTL) associated with various cave-associated phenotypes. However, the ability to connect genes of interest to the heritable basis for phenotypic change remains a significant challenge. One technique that can facilitate deeper understanding of the role of development in troglomorphic evolution is whole-mount in situ hybridization. This technique can be implemented to directly compare gene expression between cave- and surface-dwelling forms, nominate candidate genes underlying established QTL, identify genes of interest from next-generation sequencing studies, or develop other discovery-based approaches. In this report, we present a simple protocol, supported by a flexible checklist, that can be widely adapted for use well beyond the presented study system. It is hoped that this protocol can serve as a broad resource for the Astyanax community and beyond.

Wholemount In Situ Hybridization for Astyanax Embryos

This protocol enables visualization of gene expression in embryonic Astyanax cavefish. This approach has been developed with the goal of maximizing gene expression signal, while minimizing non-specific background staining.

阿斯蒂亚纳克斯胚胎的全原位杂交

In recent years, a draft genome for the blind Mexican cavefish (Astyanax mexicanus) has been released, revealing the sequence identities for thousands of ...

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.

Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

斑马鱼原位杂交染色的基因分型与定量

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...