作者要感谢GWU哈伦为支持蒙娜丽莎埃罗尔德的支持格兰特Paaqua和GWU·路德·赖斯奖学金的奖学金。这项工作是由美国国家科学基金会的资助MCB-1121711。

1。仪器仪表，文化传媒和菜肴的制备<ol><li>锐化钳使用氧化铝研磨胶片。在解剖显微镜下，监视针尖大小。作为一对钳子的背面的前端的情况下在操作过程中被损坏。高压灭菌所有四个钳子和存储在无菌容器中。 </li><li> 500毫升的0.1倍和1.0倍的培养基（马克的改良林格[MMR]或修改巴特的盐水[MBS];的食谱中15），和过滤消毒。我们经常使用MBS（1X = 88毫米氯化钠，氯化钾1毫米，0.7毫米氯化钙 ， 硫酸镁 1毫米，5毫米HEPES pH值7.8， 碳酸氢钠 2.5毫米）。在14-18°C的存储数月。 </li><li>在培养基中（2克电泳级琼脂糖1X在100毫升的培养基中的螺旋盖的玻璃瓶中）在2％琼脂糖溶液。高压釜中，溶解琼脂糖。这可以在4℃下储存几个月，和微波液化琼脂糖接下来需要时。 </li><li>虽然琼脂糖是液体，倒入约0.5毫升到无菌的24孔培养板的各孔中的底部上。这将防止外植体附着的塑料。 </li><li>虽然琼脂糖是液体，倒入约2-3毫升两至三个60毫米培养皿的底部上，并轻轻摇动以确保覆盖底部。这些将作为夹层菜肴和琼脂糖防止粘到塑料胚胎一旦它们的膜中除去。 </li><li>当琼脂糖已冷却并硬化，火焰6“巴斯德吸管的前端，直到它融化成球，并轻轻触摸它的表面上的培养板的各孔中的琼脂糖，这将创建一个浅凹陷处到其中外植体将被放置。 </li><li>填充各孔的培养板中的与1X无菌培养介质。 </li><li>当琼脂糖冷却和硬化，填补了解剖菜用稀释的培养液（0.1％MMR或0.1％MBS），以促进卵裂球分离。 </lI&gt; </ol> 2。胚胎选择<ol><li>获取受精卵和删除果冻外套根据标准协议15。将它们传输到0.5X 100毫米的陪替氏培养皿中的培养基中。 </li><li>当胚胎达到2 -细胞阶段，排序那些其中第一卵裂平分轻色素在动物半球的面积（ 图1）到一个单独的盘。这将是捐助者为外植体。保留剩余的2 - 细胞胚胎在一个单独的盘下整个过程，作为兄弟控制阶段外植体的囊胚。 </li></ol> 3。外植体的制备<ol><li>在一个夹层皿放置5-10胚胎，但在一个时间只能工作在一个胚胎。 </li><li>第一胚胎定位，以便在透明的卵黄膜中可以看出，它由明确的空间（围卵腔）的表面上方的动物极的胚胎分离。使用锋利的力量PS中的亚优势手（左手，如果其中一个是右撇子），掌握母细胞的卵黄膜上面。使用尖锐的镊子在另一方面，掌握接近的第1钳子小费膜，并轻轻地在相反的方向拉剥膜路程。使初始把握被解剖，使靶细胞除去膜期间不被损坏的电池，它是在距离。可以告诉大家，在膜已被删除，因为胚胎扁平化。 </li><li>带着一个相邻小区与下属音在一方面的钳子，并使用此单元格以被解剖的细胞作为一个“句柄”，所以还没有直接接触。在另一方面，随着钳子，轻轻将剩余的相邻小区距离所需的卵裂球。如果中线细胞，它们共享同样的命运，是有针对性的，他们可以将其作为对一起除去。最后，剖析了细胞的“手柄”。 </li><li>拿起卵裂球（或卵裂球的一对），用无菌巴斯德吸管，玻璃，避免气泡和过度抽吸。吸管可以火抛光，除去锐边，可以破坏细胞，但我们不经常这样做。放置的巴斯德吸移管的前端的表面下的培养介质中的孔中的外植体的培养皿中，并轻轻驱逐卵裂球。应该到浅的凹陷的重力滑动。相同卵裂球从一个以上的胚胎可以结合成一个单一的外植体。 </li><li>重复此过程，在解剖盘，一个接一个，其余的胚胎。经过约10个胚胎，解剖盘将充满细胞碎片。当这种情况发生时，改变到一个新的解剖菜。 </li><li>毕竟解剖完成后，检查是否将外植体在浅水洼地。如果没有问题，他们可以轻轻推入凹陷用吹风循环，已经在70％乙醇和空气干燥灭菌。 </li><li>大约一个小时后，最后清扫，清除杂物surrounding愈合的外植体，用无菌玻璃巴斯德吸管。 </li><li>培养板的外植体在14-20°C下的兄弟姐妹，控制胚胎的培养皿中。兄弟姐妹的胚胎将显示卵裂球外植体的发展阶段。 </li></ol> 4。收获外植体分析<ol><li>收获的兄弟姐妹时，外植体达到预期的发展阶段。这些外植体生存相当不错，即使一些细胞瓦解。因此，如果有大量的文化是个阴天，探索它用吹风循环或镊子，以确定是否是埋在一个健康的外植体。 </li><li>拿起外植体在小体积的培养液用玻璃巴斯德吸管，并轻轻驱逐他们到一种固定剂或适当的裂解缓冲液中进行测定。 </li></ol>

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没有利益冲突的声明。

成功培养的单个卵裂球的最关键的步骤是：1）正确识别的卵裂球的兴趣; 2）保持无菌，健康的文化; 3）解剖细胞在细胞周期中正确的部分; 4）制定必要的手册灵巧，以防止损坏在解剖和转让过程中的文化。 操作特定的卵裂球，关键的是要能够识别的大是大非轴。在爪蟾胚胎中，被识别的背侧可以通过三种方法：（1）标志着精子入口点，与一个重要的染料18，19，（...

非洲爪蟾胚胎已被广泛利用，以确定胚胎干细胞的机制，获得其特定的命运，因为他们的鸡蛋是大到足以允许显微外科方法。此外，它们的培养基中的营养补充品，而不需要为外部发展，因为每个小区包含一个提供了内在的能量存储的蛋黄血小板丰富的细胞内供给。研究细胞命运决定的机制，一个重要的资产，是一套全面的命运地图的卵裂阶段的卵裂球（2 -通过32细胞阶段）1，2，3，4，5，6。这些地图，构建了一个检测的分子显微注射到一个单一的，可识别的卵裂球，在发展监察组织填充标记的后代。一致的地图是可能的，因为命运的大是大非轴的胚胎可以可靠地识别在许多安莉芳操作系统。首先，在所有的野生型胚胎的动物半球色素，，而植物性半球是不。其次，该条目的精子受精导致朝向未来腹侧的动物半球色素沉着的收缩，在许多胚胎色素沉着的差异，因此，可以用来区分背侧和腹侧的两侧。第三，第一卵裂沟接近大多数胚胎中期矢状面，并因此可以被用来识别的左，右两侧的胚胎。的的命运地图也依赖于一个事实，即自然受精的卵子经常切割的规律，使每个识别在一个人口众多的胚胎的卵裂球。虽然有变异本文内和之间的描述有关的色素沉着和裂解模式，使用选择的程序的鸡蛋离合器允许细胞要确定与约90％的准确度与规定的命运。 可以阻止细胞的命运开采胚胎发育过程中，由几个机制。内在因素，如差异继承细胞质的mRNA或蛋白质，有助于早期图案的几个方面。例如，特定的母亲的基因，确定哪些单元格将有助于生殖系，成为内胚层，或有助于背的身体轴（7检讨）。相邻小区或更远的胚胎信号中心提供本地的外在因素，是诱导特定的组织类型和的图案几乎每一个器官系统。信号中心的实例包括组织者/节点的原肠胚诱导的神经外胚层和图案中胚层，和偏振活性区域的模式的前后轴的肢芽。虽然命运地图的前体的不同器官和他们的后裔在正常的胚胎，揭示了发展的道路，他们无法区分内在和外在影响这些细胞的。他们也无法揭示的充分发展潜力的一个细胞，其后代分化的胚胎在复杂的信号环境。两个实验的方法可以测试是否一个细胞的命运是确定的内在因素或者是后来影响的外部因素：1）移植的细胞在胚胎中一个新的位置，或2）去除细胞从胚胎随后由文化在外源信号的情况下。 这两个实验的方法是可行的，因为细胞是在非洲爪蟾大到足以被人工分离。例如，许多研究已删除单到新的地点在宿主胚胎细胞的胚胎（改变细胞间的相互作用）或移植的细胞命运的变化（审查，7，8，9）测试。另外，第二种方法的小数量的细胞的胚胎来自不同区域的explanting我置身于文化，以澄清感性的组织在没有外源性因素的相互作用可能是因为非洲爪蟾胚胎的细胞都充满了细胞内的营养物质店，蛋黄血小板。因此，它们可以被培养在一个定义的盐介质中无营养或生长因子的培养基中补充了几天。我们用这种方法来背动物卵裂球的的自主能力产生神经和背中胚层组织，以母系遗传的基因10，11，和其他人表明，32 -细胞卵裂球中胚层规范依赖于内在和外在的信息12 。作为外植体的培养细胞的一个优点是，该介质还可以补充有定义信令因素，以便确定哪个可能影响细胞-细胞间的通信路径的取出的单元12，13，14的命运。此外，可以注入卵裂球公关的ior文化与mRNA过度表达的基因，或与反义寡核苷酸，以防止内源性的mRNA的翻译。这些收益和损失的功能分析，可以识别的分子所需要的自主表达的命运。要分析外植体的命运，识别特定的细胞类型可以通过标准的基因表达（ 例如，原位杂交，RT-PCR）和免疫组化检测。该协议提供了一个简单，但功能强大的方法来区分之间内在的和外在的机制，调节胚胎细胞发育成特定的组织。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论（可选） </td></tr><tr><td>氧化铝研磨胶片</td><td>托马斯科学</td><td> ＃6775E-38 </td><td>场（12微米），主要维修镊子提示</td></tr><tr><td>氧化铝研磨胶片</td><td>托马斯科学</td><td> ＃6775E-46 </td><td>中等（3微米），，精细锐化的镊子提示</td></tr><tr><td>氧化铝研磨胶片</td><td>托马斯科学</td><td> ＃6775E-54 </td><td>精细（0.3微米），抛光镊子提示</td></tr><tr><td>镊子：杜蒙，Dumoxel Biologie＃5 </td><td>精细科学工具</td><td> ＃11252-30 </td><td>这些具有优良的技巧，不需要锐化在第一次购买时。耐腐蚀，使他们能够可高压灭菌。 </td></tr><tr><td>庆大霉素解决方案</td><td>西格玛</td><td> G1397 </td><td>添加到介质上使用同一天， </td></tr></tbody></table>

blastomere explants to test for cell fate commitment during embryonic development

命运的地图，所有的胚胎细胞谱系追踪构造，揭示下降，从每一个细胞的胚胎的组织。虽然命运地图是非常有用的，确定的前体器官移植和澄清的后裔细胞的发展路径填充该机构在正常的胚胎，他们没有说明发展潜力的前体细胞，或识别机制它的命运是确定的。为了测试细胞命运的承诺，一个细胞的正常剧目的后裔比较完整的胚胎的命运（图）实验操作后，所表达的。固定的细胞的命运（提交），无论周围的细胞环境，或者是其邻国所提供的外部因素的影响？非洲爪蟾胚胎使用全面命运的地图，我们将介绍如何识别，分离和培养单个卵裂阶段的前体，称为囊胚meres。这种方法允许1评估这些早期的细胞是否致力于获得的命运在完整的胚胎在其正常的环境中，需要与它们相邻的细胞的相互作用，或者可以影响表达如果暴露于其它类型的信号的备用命运。

在此测定法来准确地评估细胞的发育潜能的能力依赖于解剖出正确的卵裂球的基础上的命运地图1，2，3，4，5，6。因此，关键是选择正确的色素沉着在2 -细胞阶段模式中，作为示出在图1中 ，随后与符合正常卵裂模式，如在图2中示出的胚胎。如果一个人观察的排序的胚胎，因为他们达到所需的卵裂阶段和分离出来呈现出不规则的分裂的胚胎，精度大大提高。正如?...

Watch this Scientific Journal Video about Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development at JoVE.com

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

单个胚胎细胞的命运可以继承的分子和/或通过来自相邻小区的信号的影响。利用爪蟾胚胎的卵裂阶段的命运地图，单个卵裂球可确定为文化隔离的贡献进行评估的遗传分子与细胞间的相互作用。

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 ...

blastomere-explants-to-test-for-cell-fate-commitment-during-embryonic

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Cell Biology

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Unit 1

A Hierarchical Stem-Cell System: Blood Cell Formation

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15.2K 次观看.  The George Washington University. 单个胚胎细胞的命运可以继承的分子和/或通过来自相邻小区的信号的影响。利用爪蟾胚胎的卵裂阶段的命运地图，单个卵裂球可确定为文化隔离的贡献进行评估的遗传分子与细胞间的相互作用。

视频: Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

文化和维护人类胚胎干细胞

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

科研

生物学

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

人类胚胎干细胞冷冻和解冻

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

一个实用的方法遗传诱导的命运映射：一个视觉引导标记和跟踪细胞在体内</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

一个反向遗传学方法测试胚胎发育过程中的功能冗余

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

人类胚胎干细胞畸胎瘤形成命运的映射

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

单细胞在斑马鱼的命运映射

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

使用LysoTracker检测及鉴别胚胎干细胞在胚胎细胞程序性死亡

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

在斑马鱼发育的细胞追踪使用Photoconvertible蛋白

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...