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?...

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 。

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

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...

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

Universidad de Cartagena UDEC

Research

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

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 ...

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

的神经植体培养<em&gt;非洲爪蟾</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

神经科学

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

前子宫穿孔和整个半球的外植体早期皮质发育的研究：一个简单的实验方法

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

荧光钙成像及其后续原位杂交，用于异种性前体特征

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

生物学

Generation of Na&#239;ve Blastoderm Explants from Zebrafish Embryos

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. However, because of the complexity and redundancy of embryonic signals, it can be challenging to discern the complete role of any single signal during early embryogenesis. By explanting the animal region of the zebrafish blastoderm, relatively na&#239;ve clusters of embryonic cells are generated that can be easily cultured and manipulated ex vivo. By introducing a gene of interest by RNA injection before explantation, one can assess the effect of this molecule on gene expression, cell behaviors, and other developmental processes in relative isolation. Furthermore, cells from embryos of different genotypes or conditions can be combined in a single chimeric explant to examine cell/tissue interactions and tissue-specific gene functions. This article provides instructions for generating zebrafish blastoderm explants and demonstrates that a single signaling molecule - a Nodal ligand - is sufficient to induce germ layer formation and extension morphogenesis in otherwise na&#239;ve embryonic tissues. Due to their ability to recapitulate embryonic cell behaviors, morphogen gradients, and gene expression patterns in a simplified ex vivo system, these explants are anticipated to be of great utility to many zebrafish researchers.

Zebrafish blastoderm explants are generated by isolating embryonic cells from endogenous signaling centers within the early embryo, producing relatively na&#239;ve cell clusters easily manipulated and cultured ex vivo. This article provides instructions for making such explants and demonstrates their utility by interrogating roles for Nodal signaling during gastrulation.

从斑马鱼胚胎中产生幼稚的胚泡外植体

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. ...

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During this process, a portion of neural stem cells undergo differentiation and give rise to the first populations of differentiated primary neurons within the nascent central nervous system. Several vertebrate model organisms have been used to explore the mechanisms of neural cell fate specification, patterning, and differentiation. Among these is the African clawed frog, Xenopus, which provides a powerful system for investigating the molecular and cellular mechanisms responsible for primary neurogenesis due to its rapid and accessible development and ease of embryological and molecular manipulations. Here, we present a convenient and rapid method to observe the different populations of neuronal cells within Xenopus central nervous system. Using antibody staining and immunofluorescence on sections of Xenopus embryos, we are able to observe the locations of neural stem cells and differentiated primary neurons during primary neurogenesis.

Assessing Primary Neurogenesis in Xenopus Embryos Using Immunostaining

This article presents a convenient and rapid method for visualizing different neuronal cell populations in the central nervous system of Xenopus embryos using immunofluorescent staining on sections.

评估主要神经再生<em&gt;爪</em&gt;胚胎使用免疫染色

Primary neurogenesis is a dynamic and complex process during embryonic development that sets up the initial layout of the central nervous system. During ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

卵裂球外植体测试胚胎发育过程中细胞命运的承诺

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.

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的神经植体培养

前子宫穿孔和整个半球的外植体早期皮质发育的研究：一个简单的实验方法

荧光钙成像及其后续原位杂交，用于异种性前体特征

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾

从斑马鱼胚胎中产生幼稚的胚泡外植体

评估主要神经再生

卵裂球外植体测试胚胎发育过程中细胞命运的承诺

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