The authors would like to thank Stefano Di Talia, Alberto Puliafito, Venkatraman Seshan and Panagiotis Xenopoulos, for input on data handling, analysis and representation, Berenika Plusa for assistance in the design of the immunofluorescence protocol and antibody testing and members of the Hadjantonakis lab for comments on the manuscript and on the development of this protocol. Work in our lab is supported by the National Institutes of Health R01-HD052115 and R01-DK084391, and by NYSTEM N13G-236.

<ol>
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The authors declare no conflicts of interest.

本协议描述在植入前阶段的小鼠胚胎进行全挂载免疫荧光定量分析的方法。一个强大的免疫协议22后跟高分辨率，整个安装使用定制的软件4聚焦成像和图像分割。而免疫协议的选择不是关键，我们发现这里呈现22是快速，可靠，并为许多我们已经测试了抗体的提供可靠的信号之一。其它协议可遵循，条件是它们的应用是在整个等效实验一致。虽然许多因素会影响每个单独?...

鼠标植入前胚胎是一个范例来研究细胞命运说明书和哺乳动物从头上皮的研究出现和体内的多能性的维持，以及一个模型。哺乳动物发育的胚胎植入前阶段致力于建立三种细胞谱系构成囊胚，即多能性外胚层 ​​- 这引起了大多数体细胞组织和生殖细胞 - 和两个胚谱系，滋养外胚层（TE）和原始内胚层（PRE）（图1A）1,2。这个协议描述的步骤（1）收获并修复植入前阶段的小鼠胚胎，（2）执行免疫荧光标记的感兴趣的蛋白质，（3）进行整个安装使用共聚焦显微镜用z-切片功能和成像（4）执行共聚焦图像和随后的定量图像分析的核分割。这条管道可以牛逼他无偏蛋白水平的测量小区标识的分配来表征原位细胞亚群。这种协议可以在少至3来进行-第4天为一个单一垫料（通常最多10个小鼠胚胎），从胚胎收集到数据分析（图1B）。几个窝的同时分析会增加成像和数据分析时间负担，从而延长了协议的总长度。 植入前阶段的小鼠胚胎是一个听话的实验系统，其中，由于其体积小，定型形态3， 在单细胞分辨率的细胞过程TOTO成像非常适合。开展胚胎的统计相关数字的公正，制度层面的分析，自动的，定量分析的管道是可取的。然而，由于内细胞团的囊胚（ICM）的高核密度（ <st荣&gt;图1A，2D），传统的图像分割平台不能提供足够的精度，以建立一个自动化或半自动化的工作流程。在另一方面，手动分割准确，还不允许细胞和胚胎的大型队列的处理，也不是适合重复性的，公正的决心小区身份的 - 学习发育阶段时，这是特别重要的地方标记模式表达还没有完全解决的（例如，不表现出跨越人口二进制分布）。我们最近开发并验证了小鼠植入前阶段的胚胎，并为实现高精度的小鼠胚胎干细胞（ES细胞）的量身定制的图像分割方法，同时需要很少的用户输入4-8。 这里提出的分析管道围绕基于MATLAB的图像分割工具，模块化交互式核分割（分）4。敏思performs无监督核分割上共焦的Z堆叠的大批量的用户已经建立图片属性的最少数量后，利用图形用户界面（GUI）（表1）4。这条管道已被证明有效的高通量数据对野生型蛋白表达和细胞定位的产生，处理实验和转基因胚胎和胚胎干细胞5-7。在本协议中，我们描述的MIN到植入前阶段的胚胎图像分割中的应用。有关胚胎干细胞的敏思性能的例子请参考4,7。自动核分割步骤显著降低了小区识别处理时的负担，而空间和荧光强度测量允许小区身份的无偏测定和基因表达结构域和细胞位置的三维地图的产生在胚胎（图1C </strong&gt;）。此外，该工作流程的可扩展性使其适用于个别窝的分析，通过实验处理的胚胎，或不同的遗传背景5,6胚胎的大同伙。敏思是免费提供的http://katlab-tools.org（软件需要MATLAB许可证）。 没有开发迄今方法允许对小鼠植入前胚胎蛋白质表达和细胞定位这样深入的数据的生成。所有的尝试到目前为止在量化这些类型的数据已被限制为手动判定和对于不同人群的细胞数的定量在胚胎（或者完全手动，或软件辅助）9-19。 （结合敏思软件）这种方法已经被量身定做和鼠标胚胎植入前胚胎干细胞和测试;不过其上具有高的核密度的其它系统的性能，虽然尚未检验，预计是等价的。

<table border="0" cellpadding="0" cellspacing="0" width="1365">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Embryo collection</td>
			<td style="width:157px;"></td>
			<td style="width:143px;"></td>
			<td style="width:743px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blunt probe for plug checking</td>
			<td>Roboz</td>
			<td>RS-9580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps</td>
			<td>Roboz</td>
			<td>RS-4978</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgery scissors</td>
			<td>Roboz</td>
			<td>RS-5910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Pasteur pipettes</td>
			<td>Fisher</td>
			<td>13-678-20C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pre-assembled aspirator tube &#38; mouthpiece</td>
			<td>Sigma</td>
			<td>A5177</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Longer rubber tubing</td>
			<td>Fisher</td>
			<td>14-178-2AA&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">M2</td>
			<td>Millipore</td>
			<td>MR-015-D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FHM</td>
			<td>Millipore</td>
			<td>MR-024-D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acid Tyrode&#39;s solution</td>
			<td>Millipore</td>
			<td>MR-004-D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin&#160;</td>
			<td>Sigma</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-well plates</td>
			<td>Nunc/Thermo-Fisher</td>
			<td>12-566-300&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Immunofluorescence</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96-well U-bottom plates</td>
			<td>Fisher</td>
			<td>14-245-73</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycine</td>
			<td>Sigma</td>
			<td>G7403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Horse serum</td>
			<td>Sigma</td>
			<td>H0146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primary antibodies</td>
			<td></td>
			<td></td>
			<td>Concentration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CDX2</td>
			<td>Biogenex</td>
			<td>AM392-5M</td>
			<td>1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GATA6</td>
			<td>R&#38;D</td>
			<td>AF1700</td>
			<td>1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GATA4</td>
			<td>Santa Cruz</td>
			<td>sc-9053</td>
			<td>1:100, 10 min fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GATA4</td>
			<td>Santa Cruz</td>
			<td>sc-1237</td>
			<td>1:100, overnight fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SOX17</td>
			<td>R&#38;D</td>
			<td>AF1924</td>
			<td>1:100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nanog</td>
			<td>ReproCELL</td>
			<td>RCAB0002P-F</td>
			<td>1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OCT4</td>
			<td>Santa Cruz</td>
			<td>sc-5279</td>
			<td>1:100, 10 min to overnight fixation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DAB2</td>
			<td>BD</td>
			<td>BD-610464</td>
			<td>1:200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Secondary antibodies</td>
			<td>Life Technologies</td>
			<td>Various</td>
			<td>1:500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33342</td>
			<td>Life Technologies</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm glass-bottom dishes</td>
			<td>MatTek</td>
			<td>P35G-1.5-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Segmentation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Computer running 64-bit Windows OS</td>
			<td></td>
			<td>n/a</td>
			<td>Verify minimal system requirements at http://katlab-tools.org and in Lou et al., (2014) Stem Cell Reports</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MATLAB (software)</td>
			<td>Mathworks</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MINS (software)</td>
			<td>Free</td>
			<td>n/a</td>
			<td>http://katlab-tools.org</td>
		</tr>
	</tbody>
</table>

quantitative analysis of protein expression to study lineage specification in mouse preimplantation embryos

这个协议提供了一个方法来进行定量，原位单细胞蛋白表达的分析，以研究谱系规范 在小鼠植入前胚胎。描述所需胚胎收集，免疫荧光，在共聚焦显微镜成像，和图像分割和分析的过程。此方法允许在胚胎的所有单元中的多个核标志物的表达和空间（XYZ）的定量的坐标。这需要MINS，专门为着床前胚胎和胚胎干细胞（ESC）的菌落的共焦图像的分析开发出一种图像分割的软件工具的优点。 MINS执行通过X，Y和Z维度的无监督核分割，并以最小的用户输入的所有信道产生在三维空间上单元位置信息，以及核荧光水平。虽然该协议已经为图像的分析而优化植入前阶段的小鼠胚胎的，它可以容易地适应于呈现良好信噪比并且其中高核密度带来了障碍的图像分割（例如，胚胎干细胞中的表达分析的任何其他样品的分析（ESC ）菌落，分化培养中的细胞，其它物种或阶段等 ）的胚胎。

为了便于数据解释和介绍，应注意不要收集和操作过程中损坏的胚胎，使所有的细胞和它们的相对位置可分析图2A - D示出了在具有扩展腔的不同阶段的完整胚泡的例子。如果出现损坏，格外小心，应注意分析和解释结果时。 使用该协议生成的数据的质量和可靠性依赖于固定的质量和用于检测所关注的蛋白质的抗体...

Watch this Scientific Journal Video about Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos at JoVE.com

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

蛋白表达的定量分析来研究宗族规范小鼠植入前胚胎

该协议提出对蛋白质表达的原位分析进行定量，单细胞研究在小鼠植入前胚胎沿袭规范的方法。必要的囊胚的汇编程序，整个安装蛋白的免疫检测，在一个共焦显微镜，和核分割和图像分析的样品的成像进行描述。

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

quantitative-analysis-protein-expression-to-study-lineage

Research

JoVE Journal

Developmental Biology

10.8K 次观看.  Sloan Kettering Institute. 该协议提出对蛋白质表达的原位分析进行定量，单细胞研究在小鼠植入前胚胎沿袭规范的方法。必要的囊胚的汇编程序，整个安装蛋白的免疫检测，在一个共焦显微镜，和核分割和图像分析的样品的成像进行描述。 

视频: Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

损失 - 和增益的功能角度进行调查研究早期细胞命运决定在植入前小鼠胚胎

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

科研

发育生物学

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

在嵌合斑马鱼胚胎的个别视网膜祖细胞转基因表达的体内成像来研究细胞非自治的影响

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

斑马鱼视网膜蛋白定位的相关超分辨和电镜观察

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines retain the ability to either self-renew or differentiate under specific conditions. Due to these properties, mESC are a useful tool in regenerative medicine, disease modeling, and tissue engineering studies. This article describes a simple protocol to obtain mESC lines with high derivation efficiencies (60-80%) by culturing blastocysts from permissive mouse strains on feeder cells in defined medium supplemented with leukemia inhibitory factor. The protocol can also be applied to efficiently derive mESC lines from non-permissive mouse strains, by the simple addition of a cocktail of two small-molecule inhibitors to the derivation medium (2i medium). Detailed procedures on the preparation and culture of feeder cells, collection and culture of mouse embryos, and derivation and culture of mESC lines are provided. This protocol does not require specialized equipment and can be carried out in any laboratory with basic mammalian cell culture expertise.

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

从小鼠胚胎植入前胚胎干细胞系的推导

Mouse embryonic stem cell (mESC) derivation is the process by which pluripotent cell lines are established from preimplantation embryos. These lines ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Tracing Gene Expression Through Detection of &#946;-galactosidase Activity in Whole Mouse Embryos

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. The classical histochemical reaction is based on the hydrolysis of the substrate X-gal in combination with ferric and ferrous ions, which produces an insoluble blue precipitate that is easy to visualize. Therefore, &#946;-galactosidase activity serves as a marker for the expression pattern of the gene of interest as the development proceeds. Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the subsequent method for paraffin sectioning and counterstaining. Additionally, a procedure for clarifying whole embryos is provided to better visualize X-gal staining in deeper regions of the embryo. Consistent results are obtained by performing this procedure, although optimization of reaction conditions is needed to minimize background activity. Limitations in the assay should be also considered, particularly regarding the size of the embryo in whole mount staining. Our protocol provides a sensitive and a reliable method for &#946;-galactosidase detection during the mouse development that can be further applied to the cryostat sections as well as whole organs. Thus, the dynamic gene expression patterns throughout development can be easily analyzed by using this protocol in whole embryos, but also detailed expression at the cellular level can be assessed after paraffin sectioning.

Here we describe the standard protocol for the detection of &#946;-galactosidase activity in early whole mouse embryos and the method for paraffin sectioning and counterstaining. This is an easy and quick procedure to monitor gene expression during development that can also be applied to tissue sections, organs or cultured cells.

通过检测全小鼠胚胎β乳糖酶活性追踪基因表达

The Escherichia coli LacZ gene, encoding &#946;-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

成人小鼠成纤维细胞对红细胞生成子细胞的直接线重编程

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Isolation and Staining of Mouse Skin Keratinocytes for Cell Cycle Specific Analysis of Cellular Protein Expression by Mass Cytometry

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the epidermis of mouse skin. There are seven important steps: separation of the epidermis from the dermis, digestion of the epidermis, staining of the epidermal cell populations with cisplatin, sample barcoding, staining with metal tagged antibodies for cell cycle markers and proteins of interest, detection of metal-tagged antibodies by mass cytometry, and the analysis of expression in the various cell cycle phases. The advantage of this approach over histological methods is the potential to assay the expression pattern of &#62;40 different markers in a single cell at different phases of the cell cycle. This approach also allows for the multivariate correlation analysis of protein expression that is more quantifiable than histological/imaging methods. The disadvantage of this protocol is that a suspension of single cells is needed, which results in the loss of location information provided by the staining of tissue sections. This approach may also require the inclusion of additional markers to identify different cell types in crude cell suspensions. The application of this protocol is evident in the analysis of hyperplastic skin disease models. Moreover, this protocol can be adapted for the analysis of specific sub-type of cells (e.g., stem cells) by the addition of lineage-specific antibodies. This protocol can also be adapted for the analysis of skin cells in other experimental species.

This protocol describes how to isolate skin keratinocytes from mouse models, to stain with metal-tagged antibodies, and to analyze stained cells by mass cytometry in order to profile the expression pattern of proteins of interest in the different cell cycle phases.

小鼠皮肤角质细胞的分离和染色,用于细胞周期特定分析细胞蛋白表达的量度细胞测定

The goal of this protocol is to detect and quantify protein expression changes in a cell cycle-dependent manner using single cells isolated from the ...

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. The high transfection efficiency permits at least 4 distinct mRNAs to be co-transfected with ~87% efficiency. Most of the electroporated mRNAs are degraded during the first 2 h post-electroporation, permitting time-sensitive experiments to be carried out in the developing embryo. Finally, we describe how to dynamically image live embryos electroporated with mRNAs that encode various subcellular targeted fluorescent proteins.

We report messenger RNA (mRNA) electroporation as a method that permits fast and efficient expression of multiple proteins in the quail embryo model system. This method can be used to fluorescently label cells and record their in vivo movements by time-lapse microscopy shortly after electroporation.

使用mRNA电穿孔快速有效地表达Avian胚胎中的多种蛋白质

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...