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.

現在のプロトコルは、着床前段階のマウス胚の全マウント免疫蛍光の定量的な分析を実行する方法について説明します。堅牢な免疫蛍光プロトコル22は、高解像度、ホールマウント共焦点イメージングによっておよびソフトウェア4の仕立て片を用いて画像分割が続いています。免疫蛍光プロトコルの選択は重要ではないが、我々は、高速で信頼性が高いことが、我々がテスト...

マウスの着床前胚は、細胞の運命の仕様および哺乳動物におけるデノボ上皮の研究のための出現とin vivoでの多能性の維持だけでなく、モデルを研究するためのパラダイムです。そして2胚体外系統、栄養外胚葉（TE）と - ほとんどの体細胞組織と生殖細胞を生じさせる - 哺乳類の発生の着床前段階は胚盤胞、すなわち多能性胚盤葉上層を構成する3つの細胞系統の確立に専念しています原始内胚葉（PRE）（ 図1A）1,2。このプロトコルは、（1）収穫に手順を説明し、着床前段階のマウス胚を固定し、（2）目的のタンパク質を標識するために免疫蛍光を行う、（3）のzセクショニング機能を備えた共焦点顕微鏡を用いて全マウントイメージングを行い、（4）共焦点画像とその後の定量的画像分析の核セグメンテーションを行います。このパイプラインは、トンを可能にします彼は、 その場での細胞の亜集団を特徴づけるために、セルIDの割り当てのためのタンパク質レベルの測定に公正。データ解析（ 図1B）に胚コレクションから、（一般的に10マウス胚まで）単一のごみのために4日-このプロトコルはわずか3で行うことができます。いくつかの同腹仔の同時分析は、このように、プロトコルの全体の長さを延長する、画像化およびデータ分析の時間負担を増加させます。 着床前段階のマウス胚は、その小さなサイズとステレオタイプの形態3与えられた、実験的に扱いやすいシステムであり、単一セルの解像度を持つ細胞プロセスのトト撮影に適しています。胚の統計学的に関連する多数の公平な、システムレベルの分析を行うために、自動化され、定量分析パイプラインが望ましいです。しかし、胚盤胞の内部細胞塊（ICM）（高核密度に起因<st栄&gt;図1A、図2D）、従来の画像分割プラットフォームは、自動化または半自動化されたワークフローを確立するために十分な精度が得られません。一方、マニュアルセグメンテーションは、正確な一方で、細胞や胚の大コホートの処理を許可していません。また、セルアイデンティティの再現性、公平な決意するのに適している - 発達段階を研究する際に特に重要であるマーカーのパターン式は完全に（ 例えば 、人口全体のバイナリディストリビューションを示さない）解決しません。最近開発され、最小限のユーザ入力4-8を必要としつつ、高精度を実現マウス着床前段階の胚およびマウス胚性幹細胞（ESC）に合わせた画像分割方法を検証しています。 ここで紹介する解析パイプラインは、MATLABベースの画像分割ツールモジュラー対話型原子力セグメンテーション（MINS）4を中心に展開。 MINS Pユーザは、グラフィカル・ユーザー・インターフェース（GUI）（ 表1）4 を使用して 、画像特性の最小数を確立した後、共焦点のZスタックの大きなバッチの教師なし核セグメンテーションをerforms。このパイプラインは、野生型、実験的に処理し、遺伝的に改変された胚とのESC 5-7の両方でタンパク質の発現および細胞局在化の高スループットデータの生成のために効率的であることが証明されました。現在のプロトコルでは、我々は、着床前段階の胚の画像のセグメンテーションにMINSのアプリケーションを記述します。 ESCの上MINS性能の例については4.7を参照してください。空間的な蛍光強度の測定は、セル識別情報の公正な決意胚（ 図中の遺伝子発現ドメイン及び細胞位置の三次元マップの生成を可能にする一方、自動化された核のセグメント化ステップが大きく、セル識別プロセスの時間の負担を軽減1C </strong&gt;）。また、このワークフローの拡張性は、実験的に処理された胚の大コホートを通じて個々リットルの分析、または異なる遺伝的背景5,6の胚にそれが適用になります。 MINSは（ソフトウェアは、MATLABのライセンスが必要）http://katlab-tools.orgで自由に利用可能です。 今日までに開発なしのアプローチは、マウスの着床前胚におけるタンパク質発現および細胞局在化に対するこのような綿密なデータの生成を可能にしません。すべての試みはこれまでに、これらのタイプのデータを定量化で9月19日 （いずれか完全に手動で、またはソフトウェアアシスト）胚における異なる集団のために細胞数のマニュアル決意および定量に制限されています。 （MINSソフトウェアを組み込んだ）このアプローチは、に合わせ、マウスの着床前胚およびESCの上でテストされています。それにもかかわらず、高核密度を有する他のシステムでは、その性能は、まだテストされていないが、同等であると予想されます。

<table><tbody><tr><td>&lt;strong&gt;Embryo collection&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Blunt probe for plug checking</td><td>Roboz</td><td>RS-9580</td><td></td></tr><tr><td>Forceps</td><td>Roboz</td><td>RS-4978</td><td></td></tr><tr><td>Surgery scissors</td><td>Roboz</td><td>RS-5910</td><td></td></tr><tr><td>Glass Pasteur pipettes</td><td>Fisher</td><td>13-678-20C</td><td></td></tr><tr><td>Pre-assembled aspirator tube &amp;amp; mouthpiece</td><td>Sigma</td><td>A5177</td><td></td></tr><tr><td>Longer rubber tubing</td><td>Fisher</td><td>14-178-2AA&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>M2</td><td>Millipore</td><td>MR-015-D</td><td></td></tr><tr><td>FHM</td><td>Millipore</td><td>MR-024-D</td><td></td></tr><tr><td>Acid Tyrode&#039;s solution</td><td>Millipore</td><td>MR-004-D</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140</td><td></td></tr><tr><td>Bovine Serum Albumin&amp;nbsp;</td><td>Sigma</td><td>A9647</td><td></td></tr><tr><td>4-well plates</td><td>Nunc/Thermo-Fisher</td><td>12-566-300&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;Immunofluorescence&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>96-well U-bottom plates</td><td>Fisher</td><td>14-245-73</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>G7403</td><td></td></tr><tr><td>Horse serum</td><td>Sigma</td><td>H0146</td><td></td></tr><tr><td>&lt;strong&gt;Primary antibodies&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Concentration&lt;/strong&gt;</td></tr><tr><td>CDX2</td><td>Biogenex</td><td>AM392-5M</td><td>1:200</td></tr><tr><td>GATA6</td><td>R&amp;amp;D</td><td>AF1700</td><td>1:100</td></tr><tr><td>GATA4</td><td>Santa Cruz</td><td>sc-9053</td><td>1:100, 10 min fixation</td></tr><tr><td>GATA4</td><td>Santa Cruz</td><td>sc-1237</td><td>1:100, O/N fixation</td></tr><tr><td>SOX17</td><td>R&amp;amp;D</td><td>AF1924</td><td>1:100</td></tr><tr><td>Nanog</td><td>ReproCELL</td><td>RCAB0002P-F</td><td>1:500</td></tr><tr><td>OCT4</td><td>Santa Cruz</td><td>sc-5279</td><td>1:100, 10 min to O/N fixation</td></tr><tr><td>DAB2</td><td>BD</td><td>BD-610464</td><td>1:200</td></tr><tr><td>Secondary antibodies</td><td>Life Technologies</td><td>Various</td><td>1:500</td></tr><tr><td>Hoechst 33342</td><td>Life Technologies</td><td>H3570</td><td></td></tr><tr><td>&lt;strong&gt;Imaging&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>35 mm glass-bottom dishes</td><td>MatTek</td><td>P35G-1.5-14-C</td><td></td></tr><tr><td>&lt;strong&gt;Segmentation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>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><td>MATLAB (software)</td><td>Mathworks</td><td>n/a</td><td></td></tr><tr><td>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

このプロトコルは、系統の仕様を研究するために、タンパク質発現のインサイチュ分析で定量、単セルを実行するための方法を提供 マウスの着床前胚インチ胚コレクション、免疫蛍光、共焦点顕微鏡で画像化し、画像分割および分析のために必要な手順が記載されています。この方法は、複数の核マーカーの発現と空間胚内のすべてのセルの（XYZ）座標の定量を可能にします。それはMINS、特に着床前胚および胚性幹細胞（ESC）のコロニーの共焦点画像の解析のために開発された画像セグメンテーションのソフトウェアツールを利用しています。 MINSはX、YおよびZ次元を横切る教師なし核セグメンテーションを行い、最小限のユーザ入力で、すべてのチャンネルについて、三次元空間内のセルの位置情報、ならびに核蛍光レベルを生成します。このプロトコルは、画像の解析のために最適化されているが着床前段階のマウス胚の、容易に良好な信号対雑音比を示す任意の他のサンプルの分析に適合し、ここで、高核密度は、画像分割（ 例えば 、胚性幹細胞（ESCの発現解析に障害をもたらすことができます）コロニーを、培養物中の細胞を分化、他の種または段階の胚、 等 ）。

すべての細胞およびそれらの相対的な位置を分析することができるように、データの解釈とプレゼンテーションを容易にするために、ケアは、収集および操作中の胚を傷つけないように注意すべきである。図2A - 。Dは、拡張キャビティと異なる段階で無傷の胚盤胞の例を示しています。発生する損傷べき結果を分析し、解釈する際に、余分な注意が必要?...

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

マウス着床前胚でリネージュの仕様を研究するためのタンパク質発現の定量分析

このプロトコルは、マウス着床前胚において系統の仕様を研究するために、タンパク質発現のin situ分析 、定量的、単一のセルを実行するための方法を提示します。胚盤胞のコレクションのために必要な手順、全マウント免疫蛍光タンパク質の検出、共焦点顕微鏡での試料の画像化、および核分割及び画像解析について説明します。

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

10.8K ビュー.  Sloan Kettering Institute. このプロトコルは、マウス着床前胚において系統の仕様を研究するために、タンパク質発現のin situ分析 、定量的、単一のセルを実行するための方法を提示します。胚盤胞のコレクションのために必要な手順、全マウント免疫蛍光タンパク質の検出、共焦点顕微鏡での試料の画像化、および核分割及び画像解析について説明します。 

ビデオ: 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.

loss-が及び着床前マウス胚の初期の細胞運命決定要因を調査するための機能獲得型のアプローチ

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

発生生物学

Temporal Ordering of Dynamic Expression Data from Detailed Spatial Expression Maps

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a strict species-specific periodicity. The periodicity of the process is regulated by a molecular oscillator, known as the "segmentation clock," acting in the PSM cells. This clock drives the oscillatory patterns of gene expression across the PSM in a posterior-anterior direction. These so-called clock genes are key components of three signaling pathways: Wnt, Notch, and fibroblast growth factor (FGF). In addition, Notch signaling is essential for synchronizing intracellular oscillations in neighboring cells. We recently gained insight into how this may be mechanistically regulated. Upon ligand activation, the Notch receptor is cleaved, releasing the intracellular domain (NICD), which moves to the nucleus and regulates gene expression. NICD is highly labile, and its phosphorylation-dependent turnover acts to restrict Notch signaling. The profile of NICD production (and degradation) in the PSM is known to be oscillatory and to resemble that of a clock gene. We recently reported that both the Notch receptor and the Delta ligand, which mediate intercellular coupling, themselves exhibit dynamic expression at both the mRNA and protein levels. In this article, we describe the sensitive detection methods and detailed image analysis tools that we used, in combination with the computational modeling that we designed, to extract and overlay expression data from distinct points in the expression cycle. This allowed us to construct a spatio-temporal picture of the dynamic expression profile for the receptor, the ligand, and the Notch target clock genes throughout an oscillation cycle. Here, we describe the protocols used to generate and culture the PSM explants, as well as the procedure to stain for the mRNA or protein. We also explain how the confocal images were subsequently analyzed and temporally ordered computationally to generate ordered sequences of clock expression snapshots, hereafter defined as "kymographs," for the visualization of the spatiotemporal expression of Delta-like1 (Dll1) and Notch1 throughout the PSM.

The segmentation clock drives oscillatory gene expression across the pre-somitic mesoderm (PSM). Dynamic Notch activity is key to this process. We use imaging and computational analyses to extract temporal dynamics from spatial expression data to demonstrate that Delta ligand and Notch receptor expression oscillate in the vertebrate PSM.

詳細な空間式マップからダイナミック発現データの時間的順序

During somitogenesis, pairs of epithelial somites form in a progressive manner, budding off from the anterior end of the pre-somitic mesoderm (PSM) with a ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

タンパク質レベルの組織や標準化された定量ウェスタンブロットによる開発全体の堅牢な比較

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

生化学

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.

マウス胚における心臓前駆細胞集団の定量的ホール マウント免疫染色解析

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.

全体のマウス胚における β-ガラクトシダーゼ活性の検出によって遺伝子発現をトレース

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. ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

C.エレガンス胚発生を解析するセミハイスループットイメージング法とデータ可視化ツールキット

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

マウス胚細胞の多重化単細胞mRNAシーケンシング解析

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

初期マウス胚からのシーケンシングのための小型RNAライブラリーの調製

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

発生中の(カエル)胚における細胞系譜ガイド質量分析プロテオミクス

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design ...

化学

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

初期段階のマウス胚を用いて組織特異的遺伝子のためのクロマチン免疫沈降アッセイ

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

マウス着床前胚でリネージュの仕様を研究するためのタンパク質発現の定量分析

要約

さらに動画を探す

この動画の章

loss-が及び着床前マウス胚の初期の細胞運命決定要因を調査するための機能獲得型のアプローチ

詳細な空間式マップからダイナミック発現データの時間的順序

タンパク質レベルの組織や標準化された定量ウェスタンブロットによる開発全体の堅牢な比較

マウス胚における心臓前駆細胞集団の定量的ホールマウント免疫染色解析

全体のマウス胚における β-ガラクトシダーゼ活性の検出によって遺伝子発現をトレース

C.エレガンス胚発生を解析するセミハイスループットイメージング法とデータ可視化ツールキット

マウス胚細胞の多重化単細胞mRNAシーケンシング解析

初期マウス胚からのシーケンシングのための小型RNAライブラリーの調製

発生中の(カエル)胚における細胞系譜ガイド質量分析プロテオミクス

初期段階のマウス胚を用いて組織特異的遺伝子のためのクロマチン免疫沈降アッセイ

マウス着床前胚でリネージュの仕様を研究するためのタンパク質発現の定量分析

要約

さらに動画を探す

この動画の章

loss-が及び着床前マウス胚の初期の細胞運命決定要因を調査するための機能獲得型のアプローチ

詳細な空間式マップからダイナミック発現データの時間的順序

タンパク質レベルの組織や標準化された定量ウェスタンブロットによる開発全体の堅牢な比較

マウス胚における心臓前駆細胞集団の定量的ホール マウント免疫染色解析

全体のマウス胚における β-ガラクトシダーゼ活性の検出によって遺伝子発現をトレース

C.エレガンス胚発生を解析するセミハイスループットイメージング法とデータ可視化ツールキット

マウス胚細胞の多重化単細胞mRNAシーケンシング解析

初期マウス胚からのシーケンシングのための小型RNAライブラリーの調製

発生中の(カエル)胚における細胞系譜ガイド質量分析プロテオミクス

初期段階のマウス胚を用いて組織特異的遺伝子のためのクロマチン免疫沈降アッセイ

マウス胚における心臓前駆細胞集団の定量的ホールマウント免疫染色解析