这项工作的支持，R01-HD058629 IEZ

1。制备ECM包被的培养皿或盖玻片<ol><li>准备ECM原液中溶解:ECM，衬底在PBS（Dulbecco氏磷酸盐缓冲盐水，＃14040-133，Invitrogen公司）。使用为0.2mm的注射器过滤器（VWR，＃28145-495）和冻结以等分试样在-80℃下进行筛选</li><li> ECM原液稀释在PBS中，以所需的浓度。对于MaxGel，DMEM（Dulbecco改良的Eagle培养基，Invitrogen公司，＃11995-065）中稀释。 </li><li>如果将进行免疫荧光染色，灭菌通过浸渍在乙醇中的12毫米的圆形玻璃盖玻片（VWR，＃89015-724）和在空气中晾干。这项工作应在无菌层流罩。如果免疫荧光法，将不会执行跳到步骤1.5。 </li><li>地点灭菌盖玻片在24孔板的各孔中（Corning公司，＃3526）。 </li><li> 0.5毫升稀释的底物溶液加入到24孔板，在室温下孵育3小时温度的各孔中。 </li><li>吸出的ECM解决方案，并用THRE在PBS的e时代。 </li><li>吸干PBS及立即使用或包裹在封口膜，并储存于4℃下进行长达2个星期。 </li></ol> 2。准备解剖工具 Sylgard的，添加木炭准备每板制造商的协议。这些板的耐药黑色背景提供支持牵制胚胎和颜色提供良好的对比度，便于可视化的透光性的胚胎。 <ol><li>为了准备板，权衡直接进入三倒入烧杯中的平衡150 G部分的液体15克B部分木炭粉和1 g（世界精密仪器，＃SYLG184）。混合在一起，用一个木制的冰棒棍或压舌板。倒入玻璃或塑料100 cm培养皿。用一个小丁烷火炬，举行了约10至12英寸以上的菜烧的气泡。将平板上的盖子，让坐在至少24小时来设置。 </li><li>准备清扫工具插入一个Minutien针（0.1毫米diameteR）进入的销保持器（精细科学工具，＃26002-10和＃26018-17），分别交替26表针（BD，＃309597），与借助钳子弯曲成直角，可以用作解剖仪器。锋利的学生杜蒙＃5镊子（精细的科学工具，＃91150-20）用磨刀石（精细的科学工具，＃29008-22）。 </li><li>所有清扫工具，在使用前用70％乙醇消毒。 </li></ol> 3。收集胚胎<ol><li>根据使用动物委员会条例安乐死的时间为8.5天coitum孕鼠和取出子宫。 </li><li>将子宫在塑料培养皿中，用无菌PBS。 </li><li>用无菌PBS洗净子宫一次。 </li><li>在解剖显微镜下小心地取出胚胎蜕膜组织，用一个钳子。 </li><li>删除胚外膜和基因分型，如果需要保存。 </li><li>计数体节的胚胎阶段。理想的阶段，使外植体，分析细胞BEHavior在神经褶海拔之间的6和10体节阶段时，神经褶才刚刚开始提升的颅间充质细胞的主要形态发生之前。 </li><li>胚胎在PBS中洗涤。在一个黑色的SYLGARD板用DMEM包含没有酚红（Invitrogen公司，＃21063-029）的地点。删除的胚外膜和基因分型，如果需要保存。 </li></ol> 4。外植体的制备<ol><li>再聚焦在头部区域的胚胎在最高可能的放大倍率的显微镜。 </li><li>用解剖针在每手削减。使用一针保持在地方和其他组织作出削减。 </li><li>剪去头的菱前的胚胎。 </li><li>切沿中线平分头，分成两等分。 </li><li>切的头部和中线周围的外边缘。这些削减将删除的premigratory的的波峰和外边框和表面外胚层的在中线prechordal板。其余的生活将是外植体，将包含迁移颅神经嵴旁轴中胚层和表面外胚层。 </li><li>当组织被切断，除去解剖的碎片从培养皿用无菌巴斯德吸管。这可以防止混淆了外植体解剖的碎片。 </li><li>轻轻冲洗外植体，由pipeting向上和向下20微升移液器吸头和PIPETMAN。这将删除松散贴壁细胞和防止外植体边缘呈锯齿状。 </li><li>小心地拿起每个外植体，在约10微升的液体，使用移液管尖20微升和PIPETMAN和地点的中心处的包被的培养以及。 </li><li>中加入20μl含15％FBS补充的培养基中，以覆盖外植体。 </li><li>地点板在湿润的组织培养箱（5％CO 2，37℃）1-2小时，以允许附加到外植体的培养皿的底部。定期查看，以确保外植体不干燥了。 </li><li>外植体已经附着时，小心地加至每个孔中加入250μl含10％FBS DMEM培养液已被温热至37℃的水浴中。一旦外植体连接时不会移动板在显微镜下轻轻摇动。 </li><li>返回菜组织培养的孵化器。 </li><li> 24小时后，将外植体牢固地附着和细胞就会开始移民。此时药理剂可添加到介质中。 </li><li> 48小时后，电镀，已经从外植体细胞。可以可视化和成像的距离迁移。我们通常会停止在这一点上，因为文化外植体开始出现较长的孵育的生存能力下降。如吖啶橙或台盼蓝甲活力染色也可用于判断是否将外植体的可行性。 </li><li>在这一点上，可以获取图像文件从外植体的细胞迁移的距离和数量。另外，如果外植体涂布于盖玻片上，它们可以是固定的，并执行的可视化感兴趣的蛋白质的免疫组织化学分析。 </li></ol>在协议中的关键步骤<ol><li>把外植体的大致相同的大小。 </li><li>清除所有清扫污物解剖板解剖，以避免混淆所需的移植组织碎片。 </li><li>当电镀外植体井或盖玻片上是一定要放在液滴的媒体中心以及与外植体。 </li><li>允许植足够的时间来连接洪水以及与媒体之前，。 </li><li>每一个测试基板和药理活性剂处理条件应该是标准化的。 </li><li>两个每个胚胎外植体可以由（一个从左边和从右侧的神经管中的一个）。 </li><li>当测试迁移的细胞突变小鼠品系不同实验条件下，把一个突变的外植体在对照组和治疗组。 </li></ol>故障排除<ol><li>如果外植体无法连接，增加附件规定的时间。根据我们的经验，约一半的外植体不附加和较大的外植体更有效地附着。 </li><li>如果外植体附加到井的周围，它们将难以图像。请务必将外植体中心的良好。 </li><li>理想的情况下，外植体是大致相同的尺寸。然而，外植体大小的差异可以被校正为在quanitating从外植体的细胞的迁移时，使用的外植体的直径作为校正因子。 </li><li>如果发生污染确保消毒工作条件正在使用。所有的工具和工作表面应用70％乙醇消毒。移液管和培养皿应该是新的和不育。 </li><li>如果外植体迷路的解剖过程中，要定期清除杂物，以防止丢失的外植体。 </li><li>掌握解剖非常耗时，而且需要练习。优化正常的放大倍率，锐性剥离的工具和针帮助。 </li></ol>

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没有利益冲突的声明。

采用的方法提供了一个功能强大的检测，检查颅骨间充质细胞的行为。在除了这里介绍的静态分析，在明亮的字段或组合住成像实验与GFP-标记的蛋白质的表达，可以采用实时检查细胞的行为，因为它们从外植体中迁移。对于实时成像实验中，外植体可以用DiI标记或分化的神经嵴细胞的迁移，从近轴中胚层，ROSA-YFP Mesp1创建或ROSA-YFP; WNT1中铁快运或其他转基因株系可以使用。颅间充质细胞-ECM相互作用也可以利用此外植体的分析研究。在这里，我们，本的颅间充质中表明，细胞对这些迁移到不同的程度，ECM的影响的细胞的形态的不同的ECM基板的板外植体对。此外，外植体可以被嵌入在一个三维矩阵存取的行为大公LS在这种情况下。此外植体检测是可以改变的遗传学和药理学的操作的颅间充质行为来分析内在的和外在的线索，可以调节和修改迁移的影响分析。例如，我们在我们的研究中利用该测定辨别Hectd1突变颅间充质15中的异常细胞的行为的过量的热休克蛋白90（Hsp90）分泌的作用。在这些实验中，我们处理的外植体与抗-Hsp90的抗体，格尔德霉素和DMA（二甲基amelioride），热休克蛋白90（Hsp90）蛋白阻止分泌的热休克蛋白90（Hsp90），表明异常行为的Hectd1突变的细胞是由于过量的细胞外热休克蛋白90（Hsp90）15。类似的方法可用于药理学解剖颅间充质细胞的正常或异常的形态发生的基本的附加通路。测定完成后，外植体和迁移的细胞可以分析由一个主机的方法。例如，免疫组化分析C可检查本地化的关键细胞运动的蛋白质。转录组分析也可以治疗，以确定如何影响基因的表达。 这种技术的一个显着的限制是，它不会在三维模型的行为，因为它们将在体内发生。的协议，其中外植体被嵌入在一个三维的矩阵（ 例如，基质胶）以及表达荧光蛋白标记的细胞区室的变形例，可以采用必要的，以解决这个问题。即使进行这些修改，这与在体内的体外测定中看到的行为相关联的进一步的实验来将是必要的。其他限制包括大量的胚胎需要产生足够数量的来生成静态显著数据。最重要的是，夹层是相对简单的，它需要练习才能熟练掌握。

在胚胎8.5天（E8.5）和9.5在小鼠胚胎神经管闭合发生在颅地区。未能正确关闭的神经管头无脑儿，一个共同的结构在人类出生缺陷和不符合生活。驱动头的神经管闭合的力量，会产生神经组织本身和周围的表皮细胞和间充质3。特别是扩大的颅间充质被认为是必不可少的海拔的颅神经褶1,2。的颅间充质是含有丰富的细胞外基质蛋白，特别是糖基化蛋白，硫酸肝素糖蛋白，硫酸软骨素，透明质酸4-8。 不像在鸡胚胎神经嵴细胞的移民从背神经管，神经管闭合后，在小鼠胚胎神经嵴迁移的神经褶的同时，开始为rISE（后5体节期）。因此，在小鼠胚胎神经胚形成过程中，脑神经细胞来源于神经嵴和近轴中胚层间充质细胞组成的。诱导神经嵴旁轴中胚层的人口在不同时期的发展定位在不同的位置在胚胎发育成不同的结构9,10。近轴中胚层起源于原始的条纹，并迁移到前牙区的胚胎的的推定神经板的基础。交界处的神经板和表皮外胚层的神经嵴细胞是诱导，经历了一个上皮细胞向间质细胞转变，脱层只是之前的啮齿类动物胚胎的神经褶海拔。神经嵴细胞迁移以及刻板的路径中的subectodermal的旁轴中胚层的鳃弓，额鼻和眼周的间充质。近轴中胚层，将有助于一些的头骨穹窿的骨骼和面部肌肉;而日E神经嵴将有助于其他的头骨和面部骨骼，除了颅神经9-11。差异的近轴中胚层和神经嵴细胞谱系标记的Mesp1创建和WNT1的表达Cre重组酶转基因小鼠的线，分别为9。 治疗的ECM干扰剂，如透明质酸，硫酸软骨素酶ABC，肝素酶或重氮-氧代-亮氨酸（DON）在神经胚形成减值的神经管闭合7啮齿动物胚胎神经管闭合的颅间充质细胞中的重要作用已推断实验， 12-14。在这些实验中，后的静态部分神经胚形成的组织学分析显示相关颅间充质7,12-14 dysmorphogeneis。然而，由于致畸剂获得多个组织，但它仍然被确定，如果颅间充质是真正的靶组织。在支持的结论，该组织是必不可少的神经胚形成，的颅间充质出现异常后，一些的小鼠突变与exencephaly 15日至17日组织学分析。尽管如此，在大多数情况下，突变的颅间充质细胞的细胞行为的效果没有得到解决。 我们已经制定了一个体外植试验，直接研究基因突变的结果或药物处理的行为的的颅间充质细胞15。此法的基础，是类似访问的颅间充质细胞的分化潜能公布的2003年Tzahor 等的菱18，除非我们修改了外植体解剖学习的迁移性能更前的人群的颅骨间充质前神经网络的基础板。我们的方法是在鸡胚进行修改外植体实验分析的神经嵴细胞的迁移行为与科Ÿ差异。前的准备工作外植神经嵴或更后的近轴中胚层19,20。此外，在海拔在鸡胚神经褶，神经嵴尚未移居海外，从背神经管和，从而植前的近轴中胚层将不包含神经嵴细胞。在我们的试验中，颅间质组成的近轴的中胚层，神经嵴和表面外胚层的外植体被制备和镀在基板上。可以进行实验操作，包括隔离的外植体，基因突变，电镀外植体，在不同的ECM或药物治疗。与细胞迁移的外植体和的距离，数量和行为可以分析和治疗组之间的比较。此外，该制剂是可以改变的，以通过实时成像技术中的细胞迁移分析。迁移实验后，外植体可以被固定，并进行免疫组化分析，裘THER阐明的治疗效果。从总体上看，这里介绍的协议是一个简单的体外实验调查的颅间充质细胞的行为。作为一个代表性的实验中，我们利用这个试验，以检查不同的细胞外基质的的颅间充质迁移。

an explant assay for assessing cellular behavior of the cranial mesenchyme

是来自中枢神经系统的神经板，经过一系列复杂的形态发生变动导致的神经管形成一个被称为神经胚形成过程中。在神经胚形成过程中，被认为是推动神经褶海拔形态的间充质细胞，神经板的基础。颅间充质是由近轴中胚层和神经嵴细胞。细胞的颅间充质形式组成星状状细胞和细胞外基质（ECM）股神经褶支持混杂的一个pourous小梁。在神经胚形成过程中，的颅间充质进行定型重排，从而导致其扩张和神经褶海拔，这些动作被认为是提供动力。然而，途径和细胞的行为，带动颅间充质形态仍然很差研究。 ECM的的颅间充质underly THES的细胞之间的相互作用的E细胞的行为。在这里，我们描述了一个简单的体外植试验设计的特点，这些细胞的行为。此法是可以改变的药理操作解剖的信号通路参与和实时成像分析，以进一步鉴定这些细胞的行为。我们提出了一个代表性的实验表明本测定中的实用程序的特征的颅间充质细胞的迁移性能上的各种ECM组分。

从颅间充质细胞的外植体的细胞迁移的外观如图2所示。我们测试了在不同的的ECM基板，是在颅间充质细胞在神经胚形成包括纤维连接蛋白，层粘连蛋白（100毫克/毫升;西格玛L2020），（100毫克/毫升;西格玛F1141）MaxGel ECM（1:500稀释的DMEM迁移;西格玛＃E0282）和透明质酸（1毫克/毫升，Sigma公司，＃53747）。细胞不能迁移从外植体时，没有ECM存在（数据未示出）和测试的所有基板支持的迁移的细胞。的形状，以及从外植体的细胞迁移的大小取决于基板。这是预期的，因为以前的研究表明，细胞与不同的ECM的相互作用所产生的机械力的影响的形状和迁徙性的细胞21。 <img alt="图1" src="/files/ftp_upload/4245/4245fig1.jpg" fo:contenT-宽度="5.5英寸"的FO:src ="/ files/ftp_upload/4245/4245fig1highres.jpg"/&gt; 图1。外植体的制备。（A）编制外植体，E8.5胚胎蜕膜组织和胚外组织的解剖。 （B）胚胎头从身体左前菱解剖。外植体的制备是通过从中线和头部的外边缘的移除prechordal的板分别在中线和premigratory的神经嵴和在边界表面外胚层，除去组织。 （C）的外植体的制备是通过切去组织从中线边缘和外边缘的头部移除prechordal板中线和premigratory的神经嵴和在边界表面外胚层。 （D）的外植体放置在一个小体积培养基在ECM涂覆的菜/盖玻片和允许连接之前，除了更多的介质。 <img alt="图2" src="/files/ftp_upload/4245/4245fig2.jpg" /> 图2。从不同衬底上镀的外植体的颅间充质细胞的迁移。外植体接种于纤维连接蛋白，层粘连蛋白，MaxGel ECM或透明质酸和文化拍下后48小时。大小酒吧= 200毫米。

Watch this Scientific Journal Video about An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme at JoVE.com

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

颅间充质经历了戏剧性的形态发生变动，可能提供的驱动力升高的神经褶<sup1,2</sup&gt;。在这里，我们描述一个简单的<em&gt;体外</em&gt;外植体试验，在神经胚形成的颅间充质细胞的行为特征。此法有许多应用，包括适合药物处理和实时成像分析。

为外植体含量的颅间充质细胞行为评定

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

an-explant-assay-for-assessing-cellular-behavior-cranial

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.7K 次观看.  Children's National Medical Center. 颅间充质经历了戏剧性的形态发生变动，可能提供的驱动力升高的神经褶

视频: An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

一种酶和无血清神经干细胞培养模型EMT研究适合药物发现

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

科研

发育生物学

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

躯干神经嵴细胞迁移使用一个修改过的Zigmond室法分析

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

神经科学

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

小鼠原发性颅神经冠细胞的解剖、培养与分析，用于神经冠细胞脱角和迁移研究

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals. Here, we describe an in vitro assay, "the reorientation assay", that has been used extensively to study the effect of extrinsic and intrinsic signals on the orientation of commissural axons2. This assay was developed by numerous people in the laboratories of Jane Dodd, Thomas Jessell and Andrew Lumsden (see acknowledgements for more details) and versions of this assay were used to demonstrate the reorientation activities of key axon guidance molecules, including the BMP chemorepellent in the roof plate3,4 and the chemoattractive activities of Netrin15 and Sonic Hedgehog (Shh)6 in the floor plate in the spinal cord.
Explants comprising 2-3 segments of the dorsal two-thirds of spinal cord are dissected from embryonic day (E) 11 rats and cultured in three dimensional collagen gels7. E11 dorsal spinal explants contain newly born commissural neurons, which can be identified by their axonal expression of the glycoprotein, Tag18. Over the course of 30-40 hours in culture, the commissural axon trajectory is recapitulated in these dorsal explants with a time course similar to that seen in vivo. This axonal trajectory can be challenged by placing either test tissues or a COS cell aggregate expressing a candidate signaling molecule in contact with one of the lateral edges of the dorsal explant. Commissural axons extending in the vicinity of the appended tissue will grow under the influence of both the endogenous roof plate and signals from the ectopic lateral tissue. The degree to which commissural axons are reoriented under these circumstances can be quantified. Using this assay, it is possible both to examine the sufficiency of a particular signal to reorient commissural axons3,4 as well the necessity for this signal to direct the commissural trajectory9.

This assay assesses the ability of a signaling molecule, here Bone Morphogenetic Protein 7 (BMP7), to reorient commissural axons. An explant of embryonic dorsal spinal cord is cultured adjacent to an aggregate of COS cells secreting the candidate growth factors. Reoriented commissural axons growing within the explant are visualized by immunohistochemistry.

含量测定扩散的信号分子能够重新调整胚胎脊髓连合轴突

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals.  Here, we ...

生物学

Isolation and Time-Lapse Imaging of Primary Mouse Embryonic Palatal Mesenchyme Cells to Analyze Collective Movement Attributes

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and fusion above the tongue. Defects in this process lead to cleft palate, a common birth defect. Recent studies have shown that palatal shelf elevation involves a remodeling process that transforms the orientation of the shelf from a vertical to a horizontal one. The role of the palatal shelf mesenchymal cells in this dynamic remodeling has been difficult to study. Time-lapse-imaging-based quantitative analysis has been recently used to show that primary mouse embryonic palatal mesenchymal (MEPM) cells can self-organize into a collective movement. Quantitative analyses could identify differences in mutant MEPM cells from a mouse model with palate elevation defects. This paper describes methods to isolate and culture MEPM cells from E13.5 embryos-specifically for time-lapse imaging-and to determine various cellular attributes of collective movement, including measures for stream formation, shape alignment, and persistence of direction. It posits that MEPM cells can serve as a proxy model for studying the role of palatal shelf mesenchyme during the dynamic process of elevation. These quantitative methods will allow investigators in the craniofacial field to assess and compare collective movement attributes in control and mutant cells, which will augment the understanding of mesenchymal remodeling during palatal shelf elevation. Furthermore, MEPM cells provide a rare mesenchymal cell model for investigation of collective cell movement in general.

We present a protocol for isolation and culture of primary mouse embryonic palatal mesenchymal cells for time-lapse imaging of two-dimensional (2D) growth and wound-repair assays. We also provide the methodology for analysis of the time-lapse imaging data to determine cell-stream formation and directional motility.

原代小鼠胚胎腭间充质细胞的分离和延时成像，以分析集体运动属性

Development of the palate is a dynamic process, which involves vertical growth of bilateral palatal shelves next to the tongue followed by elevation and ...

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment.

鸡颅神经嵴细胞培养物的制备及形态学分析

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

Neural crest cells (NCCs) are highly migratory cells that originate from the dorsal region of the neural tube. The emigration of NCCs from the neural tube is an essential process for NCC production and their subsequent migration toward target sites. The migratory route of NCCs, including the surrounding neural tube tissues, involves hyaluronan (HA)-rich extracellular matrix. To model NCC migration into these HA-rich surrounding tissues from the neural tube, a mixed substrate migration assay consisting of HA (average molecular weight: 1,200-1,400 kDa) and collagen type I (Col1) was established in this study. This migration assay demonstrates that NCC cell line, O9-1, cells are highly migratory on the mixed substrate and that the HA coating is degraded at the site of focal adhesions in the course of migration. This in vitro model can be useful for further exploration of the mechanistic basis involved in NCC migration. This protocol is also applicable for evaluating different substrates as scaffolds to study NCC migration.

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

This protocol outlines an in vitro migration experiment suitable for the functional analysis of the molecules involved in the in vivo migration of neural crest cells into the hyaluronan-rich extracellular matrix.

体外 富含透明质酸的细胞外基质对神经嵴细胞迁移的影响研究

Neural crest cells (NCCs) are highly migratory cells that originate from the dorsal region of the neural tube. The emigration of NCCs from the neural tube ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

评估外胚层上皮细胞在颅面发育的信号属性

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

颅面间质细胞的电

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Cranial Neural Crest Cells Three-Dimensional In Vitro Differentiation Protocol for Multiplexed Assay

With their remarkable capacity to generate both ectodermal and mesenchymal derivatives, cranial neural crest cells (CNCC) have attracted a lot of interest in studying the mechanisms regulating cell fate decisions and plasticity. Originating in the dorsal neuroepithelium, this cell population is transient and relatively rare in the developing embryo - making functional tests, genomic screens, and biochemistry assays challenging to perform in vivo. To overcome these limitations, several methods have been developed to model CNCC development in vitro. Neurosphere (NS) based culturing methods provide a complex microenvironment that recapitulates the developing anterior neuroepithelium in 3D. These systems allow the growth of many NS in the same plate to generate a large amount of CNCC, but the produced NS present a high variability in shape, size, and number of CNCC formed - making quantitative assays difficult to perform. This protocol outlines a reproducible method for generating NS from mouse embryonic stem cells (mESC) in a 96-well format. NS generated in 96-well plates produce cranial neural crest cells (CNCC), which can be further cultured. By controlling the number of starting cells, this approach reduces variability in the size and shape between NS and increases reproducibility across experiments. Finally, this culture system is adaptable to several applications and offers a higher degree of flexibility, making it highly customizable and suitable for multiplexing experimental conditions.

We present a three-dimensional (3D) in vitro differentiation protocol generating neurospheres of reproducible size to produce cranial neural crest cells from mouse embryonic stem cells. We show that this methodology reduces variability compared to previous protocols and how it can be used for multiplexed assay to study cranial neural crest cell development.

用于多重测定的颅神经嵴细胞三维 体外 分化方案

With their remarkable capacity to generate both ectodermal and mesenchymal derivatives, cranial neural crest cells (CNCC) have attracted a lot of interest ...

为外植体含量的颅间充质细胞行为评定

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一种酶和无血清神经干细胞培养模型EMT研究适合药物发现

躯干神经嵴细胞迁移使用一个修改过的Zigmond室法分析

小鼠原发性颅神经冠细胞的解剖、培养与分析，用于神经冠细胞脱角和迁移研究

含量测定扩散的信号分子能够重新调整胚胎脊髓连合轴突

原代小鼠胚胎腭间充质细胞的分离和延时成像，以分析集体运动属性

鸡颅神经嵴细胞培养物的制备及形态学分析

体外富含透明质酸的细胞外基质对神经嵴细胞迁移的影响研究

评估外胚层上皮细胞在颅面发育的信号属性

颅面间质细胞的电

用于多重测定的颅神经嵴细胞三维体外分化方案

为外植体含量的颅间充质细胞行为评定

摘要

探索更多视频

此视频中的章节

一种酶和无血清神经干细胞培养模型EMT研究适合药物发现

躯干神经嵴细胞迁移使用一个修改过的Zigmond室法分析

小鼠原发性颅神经冠细胞的解剖、培养与分析，用于神经冠细胞脱角和迁移研究

含量测定扩散的信号分子能够重新调整胚胎脊髓连合轴突

原代小鼠胚胎腭间充质细胞的分离和延时成像，以分析集体运动属性

鸡颅神经嵴细胞培养物的制备及形态学分析

体外 富含透明质酸的细胞外基质对神经嵴细胞迁移的影响研究

评估外胚层上皮细胞在颅面发育的信号属性

颅面间质细胞的电

用于多重测定的颅神经嵴细胞三维 体外 分化方案

体外富含透明质酸的细胞外基质对神经嵴细胞迁移的影响研究

用于多重测定的颅神经嵴细胞三维体外分化方案