Name: blastomere explants to test for cell fate commitment during embryonic development
Uploaded: 2013-01-26T13:00:00.000+00:00
Duration: 14 min 8 s
Description: Watch this Scientific Journal Video about Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development at JoVE.com

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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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

Biology

Unit 1

A Hierarchical Stem-Cell System: Blood Cell Formation

SCIENTISTS IN ACTION

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

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

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

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

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

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

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

科研

发育生物学

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has quickly become a commonplace amongst researchers pursuing a wide variety of biological questions. Users most often employ the Cas9 protein derived from Streptococcus pyogenes in a complex with an easily reprogrammed guide RNA (gRNA). These components are introduced into cells, and through a base pairing with a complementary region of the double-stranded DNA (dsDNA) genome, the enzyme cleaves both strands to generate a double-strand break (DSB). Subsequent repair leads to either random insertion or deletion events (indels) or the incorporation of experimenter-provided DNA at the site of the break.
The use of a purified single-guide RNA and Cas9 protein, preassembled to form an RNP and delivered directly to cells, is a potent approach for achieving highly efficient gene editing. RNP editing particularly enhances the rate of gene insertion, an outcome that is often challenging to achieve. Compared to the delivery via a plasmid, the shorter persistence of the Cas9 RNP within the cell leads to fewer off-target events. 
Despite its advantages, many casual users of CRISPR gene editing are less familiar with this technique. To lower the barrier to entry, we outline detailed protocols for implementing the RNP strategy in a range of contexts, highlighting its distinct benefits and diverse applications. We cover editing in two types of primary human cells, T cells and hematopoietic stem/progenitor cells (HSPCs). We also show how Cas9 RNP editing enables the facile genetic manipulation of entire organisms, including the classic model roundworm Caenorhabditis elegans and the more recently introduced model crustacean, Parhyale hawaiensis.

Utilizing a preassembled Cas9 ribonucleoprotein complex (RNP) is a powerful method for precise, efficient genome editing. Here, we highlight its utility across a broad range of cells and organisms, including primary human cells and both classic and emerging model organisms.

增强基因组编辑与 Cas9 Ribonucleoprotein 在不同的细胞和有机体

Site-specific eukaryotic genome editing with CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems has ...

遗传学

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

协议小鼠胚胎成纤维细胞直接转化为滋养层干细胞

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

在小鼠胚胎干细胞结构功能研究使用重组酶介导的盒式交换

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. This tissue complexity confounds the identification of causal link between extrinsic cues and cellular dynamics. A synthetically reconstituted multicellular system overcomes this problem by enabling researchers to test for a specific cue while eliminating others. Here, we present a method to reconstitute cell contact patterns with isolated Caenorhabditis elegans blastomere and adhesive polystyrene beads. The procedures involve eggshell removal, blastomere isolation by disrupting cell-cell adhesion, preparation of adhesive polystyrene beads, and reconstitution of cell-cell or cell-bead contact. Finally, we present the application of this method to investigate the orientation of cellular division axes that contributes to the regulation of spatial cellular patterning and cell fate specification in developing embryos. This robust, reproducible, and versatile in vitro method enables the study of direct relationships between spatial cell contact patterns and cellular responses.

In Vitro Reconstitution of Spatial Cell Contact Patterns with Isolated Caenorhabditis elegans Embryo Blastomeres and Adhesive Polystyrene Beads

Tissue complexities of multicellular systems confound the identification of causal relationship between extracellular cues and individual cellular behaviors. Here, we present a method to study the direct link between contact-dependent cues and division axes using C. elegans embryo blastomeres and adhesive polystyrene beads.

在Vitro重建空间细胞接触模式与孤立的Caenorhabditis elegans胚胎冲击器和粘性聚苯乙烯珠

In multicellular systems, individual cells are surrounded by the various physical and chemical cues coming from neighboring cells and the environment. ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

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. ...

Protocol for Human Blastoids Modeling Blastocyst Development and Implantation

A model of the human blastocyst formed from stem cells (blastoid) would support scientific and medical advances. However, its predictive power will depend on its ability to efficiently, timely, and faithfully recapitulate the sequences of blastocyst development (morphogenesis, specification, patterning), and to form cells reflecting the blastocyst stage. Here we show that na&#239;ve human pluripotent stem cells cultured in PXGL conditions and then triply inhibited for the Hippo, transforming growth factor- &#946;, and extracellular signal-regulated kinase pathways efficiently undergo morphogenesis to form blastoids (&gt;70%). Matching with developmental timing (~4 days), blastoids unroll the blastocyst sequence of specification by producing analogs of the trophoblast and epiblast, followed by the formation of analogs of the primitive endoderm and the polar trophoblasts. This results in the formation of cells transcriptionally similar to the blastocyst (&gt;96%) and a minority of post-implantation analogs. Blastoids efficiently pattern by forming the embryonic-abembryonic axis marked by the maturation of the polar region (NR2F2+), which acquires the specific potential to directionally attach to hormonally stimulated endometrial cells, as in utero. Such a human blastoid is a scalable, versatile, and ethical model to study human development and implantation in vitro.

A protocol outlining the formation of human blastoids that efficiently, timely, and sequentially generate blastocyst-like cells.

人爆破模拟胚发育和植入的协议

A model of the human blastocyst formed from stem cells (blastoid) would support scientific and medical advances. However, its predictive power will depend ...

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a promising model organism for laboratory studies of development, providing a useful outgroup comparison to the well studied arthropod model organism Drosophila melanogaster. In contrast to the syncytial cleavages of Drosophila, the early cleavages of Parhyale are holoblastic. Fate mapping using tracer dyes injected into early blastomeres have shown that all three germ layers and the germ line are established by the eight-cell stage. At this stage, three blastomeres are fated to give rise to the ectoderm, three are fated to give rise to the mesoderm, and the remaining two blastomeres are the precursors of the endoderm and germ line respectively. However, blastomere ablation experiments have shown that Parhyale embryos also possess significant regulatory capabilities, such that the fates of blastomeres ablated at the eight-cell stage can be taken over by the descendants of some of the remaining blastomeres. Blastomere ablation has previously been described by one of two methods: injection and subsequent activation of phototoxic dyes or manual ablation. However, photoablation kills blastomeres but does not remove the dead cell body from the embryo. Complete physical removal of specific blastomeres may therefore be a preferred method of ablation for some applications. Here we present a protocol for manual removal of single blastomeres from the eight-cell stage of Parhyale embryos, illustrating the instruments and manual procedures necessary for complete removal of the cell body while keeping the remaining blastomeres alive and intact. This protocol can be applied to any Parhyale cell at the eight-cell stage, or to blastomeres of other early cleavage stages. In addition, in principle this protocol could be applicable to early cleavage stage embryos of other holoblastically cleaving marine invertebrates.

Ablation of a Single Cell From Eight-cell Embryos of the Amphipod Crustacean Parhyale hawaiensis

The amphipod Parhyale hawaiensis is a promising model organism for studies of crustacean embryology and comparative arthropod development and evolution. This protocol describes a method for manual removal of single blastomeres from early cleavage stage embryos of Parhyale.

一个单细胞消融从甲壳动物端足八细胞胚胎<em&gt; Parhyale hawaiensis</em

The amphipod Parhyale hawaiensis is a small crustacean found in intertidal marine habitats worldwide. Over the past decade, Parhyale has emerged as a ...

Fate Mapping

Fate mapping is a technique used to understand how embryonic cells divide, differentiate, and migrate during development. In classic fate mapping experiments, cells in different areas of an embryo are labeled with a chemical dye and then tracked to determine which tissues or structures they form. Technological improvements now allow for individual cells to be marked and traced throughout embryonic development and adulthood.
This video reviews the concepts behind fate mapping, and then details a fate mapping protocol in zebrafish using photoactivatable fluorescent proteins. Finally, specific applications and modifications of this unique technique are discussed.

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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