The author thanks Hugo Juraver-Geslin, Marion Wassef and Anne H&#233;l&#232;ne Monsoro-Burq for their help and advice, and the Animal Facility of the Institut Curie. The author thanks Paul Johnson for his editing work on the manuscript. This work was supported by the Centre National de la Recherche Scientifique (CNRS UMR8197, INSERM U1024) and by grants from the "Association pour la Recherche sur le Cancer" (ARC 4972 and ARC 5115; FRC DOC20120605233 and LABEX Memolife) and the Fondation Pierre Gilles de Gennes (FPGG0039).

实验遵守有关用于科学目的，并与更换，减少和完善国际公认的原则动物保护国家和欧洲法规。 1.平安装非洲爪蛙蝌蚪前神经管后，整个安装原位杂交 <ol><li>获得十根据标准程序4和老化，直到它们到达神经胚级26及以上（根据新科普和法伯发育表17 蟾胚胎。 </li><li>修复X.通过将它们在4％低聚甲醛（PFA）的溶液蟾蝌蚪。岩石并旋转的胚胎在2 ml玻璃瓶中，1至1.5小时，在室温级26到级38的胚胎，2小时，在室温下的胚胎在稍后阶段。 注意:PFA是通过接触和可疑致癌物质的毒性。它应的罩下被操纵。 备注:一个X.蟾育雏是通常是固定在一个2毫升的玻璃小瓶，但较大的玻璃小瓶都可以使用。 </li><li>洗用1×PBS中的0.1％吐温（PBT）在同一小瓶中的胚，3分钟在室温下，两次。 注意:除非另有指定使用的PBS是有或没有钙和镁。 </li><li>脱水在100％的甲醇（MeOH）的胚胎在至少12小时，在RT在同一小瓶中。使用微量的塑料罩下至少两次更改甲醇100％。 注:甲醇变成微黄色的脂质溶解在其中。小心的甲醇是通过吸入有毒和一个引擎盖下应被操纵。 </li><li>的MeOH 75％在PBS中，甲醇在PBS 50％，在PBS，PBS中的MeOH 25％两次，在室温下每浴5-10分钟:通过分级系列的MeOH浴再水合使用相同的管形瓶中的胚胎。该甲醇溶液使用微量的塑料罩下改变。 </li><li>使用塑料移液管，转移到胚胎在60毫米的培养皿填充到与PBT顶端被解剖。使用两个细镊子小心通过插入眼睛和神经管之间的一个细镊子，在视柄的水平完全卸下的眼睛。从神经管分离的眼睛。仔细介绍外胚层下方覆盖的神经管钳。小心，剥离从头部后方开始外胚层并丢弃。 </li><li>执行使用双或单ISH地高辛标记或荧光标记探针如前所述18,19。 </li><li>后ISH转移在一个新的60毫米培养皿填充到使用PBT塑料吸管顶端的胚胎。 </li><li>用细镊子仔细分离从胚胎的其余部分的神经管。在这个阶段中，前神经管从后神经管分离。仔细分离外胚层其余部分覆盖在神经管，其松散地附着在神经管，耳囊泡和中胚层的其余部分之下脊索。确保神经管是没有任何附录在这个阶段。 注:为避免损坏神经管在必要时执行所有神经管/胚胎移植用塑料移液管或微量尖形切割在其结束。前端的直径调节成神经管的大小。 </li><li>传送解剖神经管（见注步骤1.9）在1.5ml管中填充有50％的甘油在PBS中稀释。等到神经管已经下降到了甘油溶液的底部，通常是O / N在4℃。 </li><li>除去50％甘油的溶液/用PBS将微量P1000和相同的1.5毫升管中添加90％甘油/ PBS中使用微量P1000的溶液。等到神经管下降到90％甘油/ PBS溶液的底部，通常是O / N在4℃。 注意:长期储存，使用90％甘油/ PBS加抗生素预防细菌生长。 </li><li>转移在玻璃板上，或者用塑料移液管载玻片神经管。 </li><li>派息教派沿背鳍和使用钨针中线腹侧神经管。在该步骤中，神经管被保持在90％甘油/ PBS中。 </li><li>使用覆盖有玻璃盖玻片加强环安装神经管的两侧在90％甘油/ PBS中。 </li><li>修正了清漆的盖玻片，并保持在4℃。 </li></ol> 2.动物帽外植体诱导采用阴离子交换树脂颗粒<ol><li>转移100微升阴离子交换树脂珠到2ml管使用微量P1000。至少连续五次用无菌蒸馏水洗珠。允许珠沉降在管的底部，并使用微量P1000取代无菌蒸馏水。请勿触摸珠。 </li><li>让珠浸泡O / N在2 ml管中，在4℃下填充用无菌蒸馏水与牛血清白蛋白（BSA）（10毫克/毫升）。 </li><li>收集的AC，地方一半的珠到一个新的1.5毫升前两小时管使用微量P1000。取代无菌蒸馏水，用500μL含有分子介质的要为它的感应电势进行测试，或已知诱导特定的命运。保持珠的另一半，因为它们将被用作阴性对照。 </li><li>孵育小珠在4℃下至少2小时。 </li><li>深入开展体视显微镜下的所有后续步骤，并执行所有交流传输用微量。 </li><li>囊胚或非常早期原肠胚转移到PBS中钙和镁补充用塑料移液管的0.2％BSA中。 注:胚胎发展在12℃范围囊胚和原肠胚阶段，20至24小时。 </li><li>使用镊子如前所述20取出胚胎的卵黄膜。修复的胚胎有一个镊子。捏卵黄膜与其他钳的一侧。握住膜，慢慢剥开胚胎。在VEN执行此过程胚胎的二尖瓣（植物）的一面。不要损坏胚胎的兽性的一面。 </li><li>隔离小的AC如前所述21。动物极是胚胎的色素一部分。用细镊子切出组织的小广场出来的动物极了。只有交流组织含有外胚层细胞。确保该组织是均匀的厚度。如果不是，从分析中删除的交流。 </li><li>将每个交流的寺崎多孔板的0.5倍修改巴特的生理盐水（MBS）4。将交流入井与其着色动物的一面朝下，与井的圆底接触。 注:涂布移液器用0.1％BSA溶液防止小片粘合剂的组织到塑料的粘合性。 </li><li>放置一个珠子上使用微量P20每个帽。如果需要的话，可以使用镊子小心地将珠在盖的中心。 注:在条件培养基浸泡，珠子的颜色为粉红色。选择最合lored珠。 </li><li>在室温下孵育（18至22℃）6小时，而无需移动寺崎多孔板。珠粘在​​AC在不到2小时。 </li><li>放置寺崎多孔板对浸泡在水中的文件上面。覆盖板的塑料容器。 注:此"恒温恒湿试验箱"防止井过早蒸发。 </li><li>文化的AC的0.5倍MBS或3/4 NAM（见步骤4.1）中的湿度试验箱在15-20℃，直到兄弟的胚胎已经达到正确的阶段，以测试你的因素的影响。 </li><li>修复AC的1小时在新鲜配制的4％的煤灰。脱水的AC在100％MeOH中如前所述（步骤1.4）。 </li></ol> 3.动物帽细胞分离和再聚集在嫁接在非洲爪蟾神经胚<ol><li>涂层培养皿（60毫米）或12孔平板用3％的琼脂糖在无菌水或在PBS无钙和镁。加入足够的琼脂糖覆盖将bottom培养皿或井。预温所述板在室温（18-22℃）。任选地，存储所述板为两天，在4℃，以防止脱水。 </li><li>制备无钙Holtfreter的盐水（60毫摩尔NaCl，0.7毫米氯化钾，4.6毫米的HEPES，0.1％BSA（A-7888西格玛pH值7.6））并Holtfreter的盐水（60毫摩尔NaCl，0.7毫米氯化钾，0.9毫氯化钙 ，4.6毫HEPES，0.1％BSA的pH7.6）中5。注: 氯化钙的溶液不能高压灭菌。 </li><li>填充琼脂糖包被的孔与无钙Holtfreter的盐水的顶部。 </li><li>制备囊胚或非常早期原肠胚以分离其的AC如前所述（步骤2.5至2.7）。 </li><li>隔离至少15或最多30个，小的AC 21。用细镊子切出组织的小广场出来的动物极了。交流的组织只包含外胚层细胞，因此是均匀的厚度。如果不是，从分析中删除的交流。 注:动物极是安莉芳Ø的色素一部分。 </li><li>传输的AC成琼脂糖覆盖的孔填充用无钙Holtfreter的盐水的顶部。放置无线控制器与色素一面朝上。 注:在分解过程中无钙Holtfreter的盐水迅速启动。 </li><li>等待几分钟的细胞开始游离。通过组织分解注意这一点。用细镊子，从交流的其余部分分隔开着色层和用微量P20丢弃它们。完整细胞解离过程指示细胞彼此分离。 注:一个或两个着色层可以重新聚集植内留下来帮助移植过程中的可视化。 </li><li> Center中使用的板材圆运动细胞。用微量P1000小心地取出尽可能多介质成为可能。要小心，不要碰到细胞。 </li><li>加入1毫升Holtfreter的盐水中钙到孔中。转移分离的AC入1.5ml试管。 注:2ml管，不能因为使用他们的圆底。 </li><li>沉淀细胞通过离心，5分钟的2,000rpm下一个工作台离心机（500×g离心）的最大速度。用微量P1000小心地取出上清。 </li><li>添加到解离细胞20微升Holtfreter的盐水与钙（60毫摩尔NaCl，0.7毫米氯化钾，0.9毫米氯化钙2，4.6毫米的HEPES，0.1％BSA（A-7888西格玛pH值7.6）5。 </li><li>保持分离的AC，3至6个小时，在室温（18-22℃）。 注意:这是必要的时间为细胞重新聚集。的再聚集是可见的细胞形成一个小球在管的底部。 </li><li>通过加入1毫升的1毫升3/4 NAM 0.5X MBS或从管底部仔细分离的外植体（参见步骤4.1）。传送中使用塑料移液管的未涂覆的板的外植体。 </li><li>舞台利用其控制的兄弟姐妹外植体。对于长期培养使用的抗生素:卡那霉素霉素（50微克/毫升），氨苄青霉素（50微克/ ml）和庆大霉素（50微克/毫升）。 </li></ol> 4.嫁接动物帽外植体X的神经板蟾胚胎<ol><li>制备3/4 NAM（110毫摩尔NaCl，2mM的氯化钾，1毫米的Ca（NO 3）2，1mM的MgSO 4干燥，0.1毫摩尔EDTA，1毫碳酸氢钠 ，0.2×PBS，50μg/ ml的庆大霉素）。不要将超过1周解剖和储存在4℃。旧版NAM可用于制备琼脂糖包被清扫菜（下同）。 </li><li>大衣皮氏培养皿（60毫米），3％琼脂糖在3/4 NAM成小孔已使用一个硅胶模具，或乒乓球拍的封面。 注:琼脂糖覆盖培养皿的底部。留有一定的空间，这样的培养皿中，以填补3/4 NAM。或者使清扫菜不干橡皮泥。在粘土，解剖过程中轻轻挤压胚胎。挖小孔琼脂糖采用了圆润的镊子这种"夹层菜&#39;可以帮助清扫过程中保持胚胎。 </li><li>收集清扫工具:塑料移液管，一个"眉刀"22，两罚解剖钳，P1000微量和技巧，用放大倍数8-40X一个stereosmicroscope（与眉毛石蜡包埋在巴斯德玻璃吸管的尖端制造），明亮的光源与光纤导向。保留所有解剖工具清洁;每次实验后，冲洗他们两次用蒸馏水，一旦用100％乙醇和晾干。灰尘，如有必要，反应釜中的金属解剖工具保存好。 </li><li>让离解和重新聚集的AC（协议3），和他们的兄弟姐妹X.蟾胚胎发育，直至达到13级（神经胚）根据新科普和麦嘉华发展表17。用于移植的神经板台13内到阶段15胚胎被使用。 </li><li>进行所有体视显微镜下的后续步骤。 </li><li>使用塑料移液管，转移胚胎到PBS中钙和镁补充有0.2％BSA的。除去胚胎的卵黄膜如前所述20。修复的胚胎有一个镊子。捏卵黄膜与其他钳的一侧。紧紧握住膜，慢慢剥开胚胎。 注:请在胚胎的腹侧（植物）侧此过程。不要损坏胚胎神经板。如果胚胎已期间卵黄膜去除步骤被损坏，转移的胚胎成含有3/4 NAM使用塑料移液管60毫米培养皿，并等待15分钟，一个足够的时间以发生愈合过程。 </li><li>在解剖盘与新鲜的3/4 NAM上填写。 </li><li>用塑料移液管，转移胚胎没有他们的卵黄膜进入解剖盘。将胚胎背侧成的孔中。在传输过程中不允许胚胎与自X的空气-液体界面接触输入蟾由表面张力裂解。 </li><li>转移外植体的AC被嫁接到用塑料移液管或微量吸管P1000的夹层板。一旦移液管内的液体，使植慢慢下沉通过重力，或推非常轻柔。 </li><li>保持与圆形镊子胚胎。随着眉刀做一个切口进入神经板，你打算移植的植的作品。 注:在阶 ​​段14神经板的前弯曲可以用作一个里程碑，它标志着间脑领土（ 图4）。 </li><li>切出一小块神经上皮，采用了圆润的镊子和眉毛knive。剪一小块用眉刀外植体。 注:片外植体应当是大约相同的尺寸和形状与神经上皮烧蚀区域。 </LI&gt; <li>放置一块外植体到使用眉刀和细镊子神经上皮切口。确保植迅速附着于胚胎。 </li><li>或者放置一块玻璃盖玻片上接枝胚胎保持地方移植。要做到这一点，切细的玻璃盖玻片到使用粗钳非常小的碎片。确保该尺寸是大约1.5mm 2。沉浸件到含有3/4 NAM或PBS使用镊子培养皿。选择一块玻璃大于胚胎，以避免损坏它。胚胎将是一点点夷为平地。 </li><li>等待至少30分钟不动，或仅将解剖板平缓。让胚胎复苏30分钟至2小时，并在必要时轻轻取出盖玻片。认真吸取的胚胎移植到一个干净的菜充满了3/4的不结盟运动，用塑料移液管。 注:嫁接胚胎倾向于开发细菌或真菌污染。对于长期Çulture使用抗生素:卡那霉素（50微克/毫升），氨苄青霉素（50微克/ ml）和庆大霉素（50微克/毫升）。 </li></ol>

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The authors have nothing to disclose.

神经发育是通过从周围组织（来自在3,31,32）蜂窝发展方案和信号之间的复杂的相互作用编排。在这里，我们描述了一组协议，可以在十中使用蟾胚胎探索外在和参与神经命运决定和在体外和体内神经形态发生内在因素。这些协议可以用作例如在 X上热带胚胎，但是X.热带胚胎小四倍然后X.蟾胚胎。这两种需要使用的镊子和钨针更细。如果可能的话，使用X.蟾 。 十，精确的可视化蟾 和 X. 热带神经histogenic字段可以使用来自WM-ISH解剖神经管的组织学平面安装来实现。这项技术是在 X上成功地进行蟾胚胎阶段26级45（ 图1）和 在 X上从热带阶段30胚胎阶段45 8,33。旧的胚胎更容易剖析那么年轻的胚胎。这种技术是容易执行。它允许的基因表达模式，单独或组合，在不同发育阶段的神经管分析。与此协议很容易神经管的一侧与另一比较。这是在非洲爪蟾 GOF和LOF实验非常有用的。在神经管的注射侧观察到的缺陷可以用在控制侧观察到的正常发育事件直接比较。这种策略已经被用于分析GOF和LOF表型X.蟾胚胎8,33。在这个协议中一个重要的步骤是胚胎组织的正确固定。一个不完整的固定防止神经管的外胚层的高效分离。预解剖神经管的有助于在仰卧的渗透ü探头。然而，一旦分离，就容易失去神经管。是可取的ISH过程期间保持胚胎的一部分，以防止任何损失。 用于所有这些实验胚胎需要是坚固的需要愈合良好。丢弃任何批次不健康的胚胎。为了解剖AC和接枝植台13和台15之间有可能生长X.蟾植和胚胎在各种温度下（12-18℃，18-20℃）。值得注意的X.蟾胚胎细胞含有足够的蛋黄，让他们的生存数日不添加任何外部的营养。为了不损害胚胎组织，胚胎，交流，或植体的转让过程有件事要认真落实。最好是使用一个塑料移液管或微量如有必要，用尖切口在其端部。前端的直径调节至胚胎的大小，或植体的尺寸，要传送。它是值得注意的，涂层移液器用0.1％BSA溶液防止小块组织的在塑料的粘合性。 交流植可以编程通过强制的基因表达，或通过暴露于外在因素以时间和空间控制的方式。我们描述了一种技术，允许通过外植体和珠之间的直接接触当地的感应。如有必要，该珠子可以进一步分片使用镊子。相对于其他先前描述的策略，该技术允许本地诱导用纯因素，单独或组合，在精确的时间窗口中的检测。 使用细胞解离和重新关联34的先前描述的方法，有可能以调查夹持并混合交流植8细胞运动性的能力，细胞分离行为，并形成隔室边界。在细胞分离过程开始于小于15分钟。避免沙金克中的解离步骤中的板，以防止细胞的损失。交流着色层不分离良好。建议在解离步骤将其删除。一对夫妇的交流色素层留在重新聚集植。它有助于可视化的移植。它不会干扰分析，在以后的发展阶段。 在这份手稿的协议移植混植成神经板的详细介绍。在果蝇胚胎 ，神经板是13级和15级这些阶段，因此适合于神经上皮内移植期间开放。X.蟾组织粘合性能随时 ​​间而发生变化。它接枝交流植入宿主以相似发育阶段是重要的。在早期发育阶段，X.蟾在3/4 NAM维修成长速度极快的胚胎。如果一些胚胎已除去卵黄膜的过程中被破坏，可能是有用的等待15分钟，这是必要的时间发生愈合过程。插入可以通过将一块玻璃盖玻片上接枝宿主组织受到青睐，但它不是必需的。通过在愈合过程中产生的力可挤出移植细胞。宿主胚的形态保存较好时接枝被强制进入它与外部的压力，例如用盖玻片。为了评估移植物的成功，荧光示踪剂可注入的外植体。荧光细胞的接枝胚胎内的存在可被可视化，直到神经管闭合。在交流培养和移植实验的主要缺点是细菌或真菌污染的频率。使用干净或无菌材料和无菌溶液尽可能多地避免污染的菜在协议的每一步。对于接枝的胚胎的长期培养总是使用抗生素和15℃的温度下进行。避免交流外植体或胚胎琼脂糖长期的联系。我们observ编，它可以减少外植体的预期寿命。嫁接技术是强大的，但耗费时间。这是不可取的筛选与它命运的决心线索，但要获得你所选择的组织上有足够的事前的信息。 除了 ​​它的传统优势， 在非洲爪蟾的最近的遗传进展为此开辟了道路 模型系统研究是否可使用大规模基因组分析深RNA测序和Chromatide免疫沉淀测序（芯片起）5,35基因调控网络。有优秀的RNAseq和芯片起涉及胚胎发育早期的参考数据集。一个芯片起分析缺点是需要收集大量的材料。交流植编程允许生产大量的特定神经组织，并且至少部分地，有助于克服这种技术限制。 该发展计划基础神经管机构ogenesis很大程度上保守的，特别是在脊椎动物。使用两栖动物获取的信息有助于潜在的脊椎动物发育25,32,36-38细胞和分子过程的理解。在诱导多能干细胞（iPS细胞）的领域中的最新进展打开了网关的研究在再生医学和药物开发。 iPSCs的是从使用的转录因子的结合体细胞进行编程。编程线索干细胞的搜索正在进行中。该试验描述在这里提供的平均，以产生在体外神经衍生的细胞类型，可以在药物筛选中使用。它们还提供了一种廉价和快速的方式来测试可用于iPSCs的39,40的进一步重新编程神经命运的决定因素。

脊椎动物神经系统出现从神经板作为神经上皮细胞的均质层。了解如何发展方案被诱导，编码，并建立了神经板的区域化过程中，目前，在发育生物学的一个主要目标。与其他系统相比，实验上适合爪蟾胚胎是选择用于分析早期步骤的神经发育1,2-一个模型。这是很容易获得的胚胎的大量涌现，以及外部发展允许访问神经胚3的第一个步骤。许多工具都可以通过实验操作非洲爪蟾 （ 非洲爪蟾）胚胎发育。微注射的mRNA或吗啉（MO），包括诱导型MOS管，以及生化和药理学工具，允许控制增益功能（GOF）和损失函数（LOF）和信号通路4,5的具体变化。该BLAstocoel屋顶外胚层，周围囊胚，或非常早期原肠胚的动物极，和被称为"动物帽&#39;（AC）是多能干细胞的来源，可以由操纵基因在表达前的被编程外植体的准备。在这份手稿是用X的详细协议蟾交流植测试在体外和体内的分子机制和细胞过程底层神经发育。 一种技术被提出，允许精细观察的基因表达模式的非洲爪蟾蝌蚪的神经管，在命运决定的线索识别的预备步骤。而观察扁平式组织通常在鸡胚6的研究中使用的，它并没有被正确地在爪蟾说明。基因表达由注射合成mRNA或MO成的2个或4细胞期胚胎的卵裂球操纵允许交流的编程外植体4。为骨形态发生蛋白（BMP）通路被抗BMP因子头蛋白的表达的例子抑制，给出了一个神经同一性交流细胞3。该协议是详细说明用于通过用阴离子交换树脂珠粒直接接触进行交流外植体和本地时间控制暴露于外在线索。最后一个技术描述了由不同的编程细胞的分离和重新编写相关的混植体移植试验在体内的神经祖细胞发育功能。 青蛙胚胎是一个功能强大的模型来研究脊椎动物的早期神经发育。结合操纵基因表达的外植在体外培养物提供了在神经上皮区域化，增殖和形态7-12的研究的重要信息。交流外植体的编程允许的官能心脏体外 13,14发展。使用外植体移植的15导致的最小转录开关诱导神经嵴分化方案 16的识别。 透明带limitans intrathalamica（ZLI）是信令中心分泌音猬（SHH），以控制尾侧前脑的成长和区域化。当持续暴露于嘘，神经上皮细胞共表达三个转录因子基因- BARH样同源盒-2（barhl2），orthodenticle-2（OTX2）及易洛魁-3（irx3） -获取ZLI车厢的两个特点:有权表达SHH，和由前神经板细胞分离的能力。作为一个模型系统，感应一个ZLI命运的成神经上皮细胞将提交8。 这些协议旨在提供简单，廉价，​​有效的发育生物学家和其他研究人员的工具来探索的基本MEC关键的神经细胞行为的hanisms。这些协议是非常通用的，并允许大范围的外在和内在的神经判定线索调查。它允许长期在神经系承诺，诱导的相互作用和细胞行为的体内分析 。

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			<td>Paraformaldehyde</td>
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			<td>&#160;20909.290</td>
			<td>Toxic</td>
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			<td>anion exchange resin beads</td>
			<td>Biorad</td>
			<td>140- 1231</td>
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			<td>Bovine Serum Albumin&#160;</td>
			<td>SIGMA</td>
			<td>A-7888</td>
			<td>For culture of animal cappH 7.6</td>
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			<td>Gentamycine&#160;</td>
			<td>GIBCO</td>
			<td>15751-045&#160;</td>
			<td>antibiotic</td>
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			<td>Bovine Serum Albumin</td>
			<td>SIGMA</td>
			<td>A7906</td>
			<td>&#160;for bead preparation</td>
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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.

基于形态的考虑在不同的物种，胚胎操作和调控基因的表达模式，一个概念模型认为神经板被分成横向和纵向分段限定发育网格生成不同histogenic字段。在神经板，前脑，中脑，后脑和脊髓的原基都已经沿前-后（AP）轴原肠胚形成（在23-25 ​​综述）期间建立。期间神经胚，所述histogenic字段可以被检测为基因表达的空间受限制的领域。基于调节基因的表达模式，前脑原基分为六个横向分段部分产生明显的称为prosomeres（p）的histogenic字段。每个prosomere被分成腹侧（基础）和一个背部（鼻翼）部分（来自于26,27）。在两栖动物中，prosomere 2（P2）产生该上丘脑和丘脑和其组织者在 Z 奥纳 大号 imitans 我 ntrathalamica（ZLI）8（在28,29中综述）。 在图1上示出的平板式安装X的蟾 和 X. 热带前神经管在阶段30，32，和35的WM-DISH之后。 WM-ISH是根据协议1使用探头的转录因子fezf2标志着端脑和P3，barhl2标志着P2，皮质折边和中脑，易洛魁-1（IRX1）及易洛魁-3（irx3）进行分别标记尾p2和鼻翼p2 ​​和用于形态发生SHH用于标记ZLI，基底前脑和脑（图1E）。不同的基因表达模式的比较分析表明，前限制 barhl的2 p2的表达邻接fezf2表达的p3的尾部极限（ 图1A）。irx3共表达用 Shh在未来ZLI（图1B）。 P2内IRX1表达域是互补的SHH（ 图1C）8。 barhl2的X中的表达模式热带在阶段35被示于图1D。的这些基因在显影水陆两用车辆前脑的表达领土的示意图在图1E提供。 采用方案2的浸泡在嘘阴离子交换树脂珠诱导SHH表达AC的外植体进行了研究的能力。X.蟾胚胎注入4动物卵裂球在4和8细胞阶段用的mRNA的前神经上皮导流头蛋白与barhl2，同时编码öTX2，irx3 和 GFP作为注射示踪。抗BMP因子头蛋白的表达抑制BMP通路并给出了一个神经同一性交流细胞3。我们指的是AC表达头蛋白作为Anteriorised动物帽（AAC）。囊胚屋顶外植体如在与阴离子交换树脂珠粒浸泡在条件培养基（CM）中接触协议描述2.气冷中培养48小时，在18℃下制备含，或否，分泌的N-末端部分从大鼠的形态发生音速刺猬（N-嘘）。内源Shh的表达通过ISH（图2）进行分析。的爪蟾SHH（Xshh）表达被检测的细胞中与浸泡在CM中与N- Shh的但不是在细胞与不具有N- Shh的（图2B）浸过的CM珠接触的珠接触。 使用协议3种不同的细胞类型的能力，从一个anoth偏析呃了调查。X.蟾胚胎注入4动物卵裂球在4和8细胞的mRNA为头蛋白编码具有或不OTX2，barhl2和irx3阶段，所指示的，使用 ßGal或GFP作为示踪剂（ 图3A）。舞台8/9 ACS患者分离和重新聚集产生的外植体含有ßGal-和表达GFP的细胞混合神经上皮细胞组成。所述reaggregated外植体培养48小时，在18℃。根据30（图3A）ßGal活性显露为红色。观察由以下的再聚集的外植体在其定位细胞的行为。在植其中气冷细胞用气冷细胞表达OTX2（红色）混合时，该ßGal表达细胞不从表达GFP的细胞分离这两种类型的细胞可自由混合的（图3B，3C）。与此相反，在外植体，其中OTX2表达细胞（GFP）与Barhl2，OTX2，Irx3（BOI3）表达细胞（ßGal）混合，将ßGal表达细胞（红色），形成凝聚力的补丁，不统一的重新聚集植内扩散（ 图3D，3E）。 使用协议4，编程交流细胞的行为进行了研究在体内 。X.蟾胚胎注入4动物卵裂球在4和8细胞阶段用的mRNA为头蛋白与OTX2 单独或连同 barhl2和irx3编码。ßGal用作示踪剂。平行地，胚胎类似注射的mRNA编码为头蛋白和形态发生Shh的来自大鼠的分泌N-末端部分（N- SHH）。在阶段8/9，交外植体分离，并立即重新聚集以生成neuroepit组成混合植helial细胞无论是表达的N- Shh和OTX2，或表达的N- Shh和OTX2，Barhl2 和Irx3（BOI3）小件混合植体移植到X的神经板蟾胚胎阶段14.移植细胞对标记ßGal染色（红色）。在阶段35内源性Shh的表达通过ISH分析。当移植到X的前神经板蟾胚胎，BOI3和N- Shh的表达细胞组成的混合植制定了具体的ZLI细胞两个特征:接枝细胞表达Xshh和由前神经上皮细胞 （图3C）隔离。相反，当混合植OTX2组成-和N- Shh-表达细胞被接枝在十蟾神经板，接枝细胞不表达Shh和没有从他们的邻居（图3B）8分离。 图1.扁平式的 X. 神经管 蟾 和 X. 热带 胚胎整个安装双ISH（A - D）。WM-ISH使用进行 fezf2，barhl2，IRX1，irx3， 和 Shh作为（A - C）探针X.蟾胚胎阶段30，32，和35 和 （D）X。热带胚胎在阶段35所指示的。代表性的胚胎的神经管解剖并如在方案1中描述的解剖神经管由一个侧视图，背起来，前左所示平面安装。标记和阶段表示。 p2的延髓和尾部边界用箭头表示。 （AC）比例尺代表0.5毫米。 （四）在S凯奥条代表0.1毫米。 。前脑标记在st.30（E）示意图barhl2表达域显示在阴影线;表达的区域显示为fezf2（粉红色），SHH（红色），irx3（黄色）和IRX1（绿色）。 p表示prosomere。位于P2顶部的松果体显示。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53474/53474fig1large.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/53474/53474fig2.jpg" /> 图 2. 通过与阴离子交换树脂珠接触编程，外植体 诱导 SHH 表达 的 。小的AC从胚胎注射了头蛋白 ，barhl2，OTX2和irx3的mRNA编码，以绿色荧光蛋白作为示踪准备。AAC代表˚F或Anteriorised动物帽。表达Barhl2，OTX2和Irx3（AAC BOI3）的AAC外植体48小时与浸泡在条件培养基的珠（CM）或者含有N-Shh的，或不是，所指示的接触培养。将外植体通过ISH为非洲爪蟾 （X）的Shh的表达进行分析。比例尺代表0.5毫米。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53474/53474fig2large.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/53474/53474fig3.jpg" /> 在重新聚集外植体细胞分离行为 图3. 分析（一 ） &#160;非洲爪蟾胚胎注射了头蛋白 ，OTX2，barhl2和irx3的mRNA编码表示。 ßGal和GFP用作示踪剂。在阶段8/9小的AC准备<html> ，解离和重新聚集以生成制成GFP /ßGal混合细胞类型的外植体。所述外植体培养48小时，在18℃和ßGal活性显露（红色）。 （B - E）混合细胞中表达的蛋白质所示显示组成的代表外植体。 AAC代表Anteriorised动物帽。 （B，C）的AC表达头蛋白和OTX2（ßGal）随机地分布在与AC的表达头蛋白（GFP）混合。 （C）（B）的放大图。 （ 四）气冷表达Barhl2，OTX2和Irx3（BOI3）（ßGal）重新集结，从气冷表达OTX2（GFP）隔离。 （E）（D）的放大图。 （B，D）比例尺代表0.5毫米。 （C，E），比例尺表示0.2毫米。G"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图4" src="/files/ftp_upload/53474/53474fig4.jpg" /> 图4. 编程植表现出其发展的特点时，在一个阶段14胚胎的神经板移植 （A）的实验计划:气冷从胚胎注射的mRNA准备所示。在阶段8/9细胞从囊胚的屋顶被离解和重新聚集以生成发表达ßGal（红色）混合细胞类型的外植体。将外植体培养，直到他们的兄弟姐妹到达阶段14.小块混合植（绿色矩形）被放入切口前神经板内。操作胚生长，直到级35运行的胚胎是由WM-ISH使用X.分析蟾 SHH（Xshh）为探针。 ßGal活动揭示了红色的ACC奥尔丁30。 （B，C）阶段的嫁接侧面35代表神经管显示，侧视图，背了起来，前左。 （B）接枝混合气冷表达OTX2和N-嘘。 （C）的接枝混合气冷表达Barhl2，OTX2，Irx3（BOI3）和N- Shh的。移植细胞都标有一个红色的明星。粉红色的星号表示未来的间脑。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53474/53474fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo at JoVE.com

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, ...

stem-cell-like-xenopus-embryonic-explants-to-study-early-neural

Nanyang Technological University

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JoVE Journal

Developmental Biology

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

7.9K 次观看.  Institut Curie. 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. 

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

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.

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 ...

科研

发育生物学

Zebrafish Keratocyte Explants to Study Collective Cell Migration and Reepithelialization in Cutaneous Wound Healing

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell migration. However, when explants are established, these cells also move collectively, maintaining many of the features which make individual keratocytes an attractive model to study migration: rapid rates of motility, extensive actin-rich lamellae with a perpendicular actin cable, and relatively constant speed and direction of migration. In early explants, the rapid interconversion of cells migrating individually with those migrating collectively allows the study of the role of cell-cell adhesions in determining the mode of migration, and emphasizes the molecular links between the two modes of migration. Cells in later explants lose their ability to migrate rapidly and collectively as an epithelial to mesenchymal transition occurs and genes associated with wound healing and inflammation are differentially expressed. Thus, keratocyte explants can serve as an in vitro model for the reepithelialization that occurs during cutaneous wound healing and can represent a unique system to study mechanisms of collective cell migration in the context of a defined program of gene expression changes. A variety of mutant and transgenic zebrafish lines are available, which allows explants to be established from fish with different genetic backgrounds. This allows the role of different proteins within these processes to be uniquely addressed. The protocols outlined here describe an easy and effective method for establishing these explant cultures for use in a variety of assays related to collective cell migration.

Zebrafish keratocytes migrate in cell sheets from explants and provide an in vitro model for the study of the mechanisms of collective cell migration in the context of epithelial wound healing. These protocols detail an effective way to establish primary explant cultures for use in collective cell migration assays.

斑马鱼角膜植研究集体细胞迁移和再上皮化在皮肤伤口愈合

Due to their unique motile properties, fish keratocytes dissociated from explant cultures have long been used to study the mechanisms of single cell ...

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

操作和<em&gt;在体外</em中&gt;成熟<em&gt;非洲爪蟾</em&gt;卵母细胞，其次是胞浆内单精子注射，研究胚胎发育

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

小鼠胚胎干细胞的无血清单层培养神经细胞分化

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

生成CRISPR / Cas9介导的单等位基因缺失来研究小鼠胚胎干细胞功能增强

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

转录组分析]<em&gt;活体</em&gt;生产牛胚胎植入前胚胎采用双色微阵列平台

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

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 ...

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

在体外和体内模型来研究角膜内皮-间充质转换

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

胚胎血管生成的可视化和定量分析<em&gt;热带非洲爪蟾</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

使用体内和组织和细胞外植体方法研究胚胎和围产期主动脉的形态和发病机制

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...