Name: quantitative analysis of protein expression to study lineage specification in mouse preimplantation embryos
Uploaded: 2016-02-22T13:00:00.000+00:00
Duration: 11 min 25 s
Description: Watch this Scientific Journal Video about Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos at JoVE.com

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.

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The authors declare no conflicts of interest.

본 프로토콜은 착상 전 단계 마우스 배아에서 전체 마운트 면역의 정량 분석​​을 수행하는 방법을 설명합니다. 강력한 면역 프로토콜 (22)는 고해상도의 뒤에, (4)의 소프트웨어 맞춤 부재를 사용하는 공 초점 이미지 및 이미지 분할을하여 전체를 실장. 면역 프로토콜의 선택은 중요하지 않지만, 우리는 빠르고 안정적으로 우리는 시험 한 항체의 많은 강력한 신호를 제공하기 위?...

마우스 착상 전 배아 세포 운명 명세서 및 포유 동물에서 상피 새로이 연구 출현 생체 능성의 유지뿐만 아니라, 모델을 학습하는 패러다임이다. 두 배외 (extraembryonic) 계통, trophectoderm (TE)와 - 대부분의 체세포 조직 및 생식 세포에 상승을 제공 - 포유류 개발의 착상 전 단계는 배반포, 즉 만능 epiblast을 구성하는 세 가지 세포 계통의 확립에 최선을 다하고 있습니다 원시 내배엽 (예정) (그림 1A) 1,2. 이 프로토콜은, (2), 관심의 단백질을 라벨 면역 형광을 수행 (3) Z-절편 기능과 함께 공 초점 현미경을 사용하여 이미지를 마운트 전체 수행 (1) 수확 및 수정 착상 전 단계 마우스 배아에 대한 절차를 설명 (4) 공 초점 이미지와 이후의 양적 이미지 분석의 핵 분할을 수행합니다. 이 파이프 라인은 t 수 있습니다그 자리에서 세포의 개체군을 특성화 셀 아이덴티티 할당을 위해 단백질 수준을 측정 바이어스. 데이터 분석 (도 1b)에 배아 컬렉션 (일반적으로 10 마우스 배아까지) 한 쓰레기 4 일 -이 프로토콜은 적은 3로 수행 될 수있다. 여러 리터의​​ 동시 분석에 따라서 프로토콜의 전체 길이를 연장하는, 촬상 데이터 분석 시간 부담을 증가시킨다. 착상 전 단계 마우스 배아는 작은 크기와 진부한 형태 3 제공, 단일 셀 해상도 세포 과정의 토토 영상에 적합하다, 실험적으로 다루기 쉬운 시스템입니다. 배아의 통계적으로 중요한 수의 편향, 시스템 레벨 분석을 수행하는, 자동화 된 정량 분석​​ 파이프 바람직하다. 그러나, 내부 세포괴 높은 핵 밀도의 배반포 (ICM)에 의한 ( <st룽&gt;도 1a,도 2D), 종래의 이미지 분할 플랫폼은 자동화 또는 반 자동화 된 작업 흐름을 확립하기에 충분한 정확도를 제공하지 못한다. 한편, 수동 세분화 정확하면서 세포 및 배아 큰 집단의 처리를 허용하지 않으며, 셀 아이덴티티 재현성 편향 판에 적합 - 발달 단계를 연구 할 때 특히 중요 여기서 마커 패턴 표현은 완전히 (예를 들면, 인구에 걸쳐 진 분포를 나타내지 않는다)가 해결되지. 최소한의 사용자 입력 4-8을 필요로하는 동안 우리는 최근에, 마우스 착상 전 단계의 배아과 높은 정확도를 달성 마우스 배아 줄기 세포 (는 ESC)에 맞춰 이미지 분할 방법을 개발하고 검증했다. 여기에 제시된 분석 파이프 라인은 MATLAB 기반의 영상 분할 도구 모듈 형 인터랙티브 핵 분할 (분) (4)를 중심으로 돌아 가지. MINS 피사용자는 그래픽 사용자 인터페이스 (GUI) (표 1) (4)를 이용하여, 이미지 속성의 최소 수를 설정 한 후 촛점 Z-스택 대규모 배치에서 자율 핵 분할 erforms. 이 파이프 라인은 모두 야생형 단백질의 발현 및 세포 국산화 높은 처리량 데이터의 생성 효율이 입증 된 실험적 치료 및 유전자 배아는 ESC 5-7 변성. 본 프로토콜에서, 우리는 착상 전 단계의 배아 이미지의 분할에 MINS의 응용 프로그램을 설명합니다. 는 ESC에 MINS 성능의 예를 들어 4,7 참조하십시오. 자동화 핵 분할 단계는 크게 공간 형광 세기 측정은 배아 세포 아이덴티티 바이어스 결정 및 유전자 발현 도메인 및 셀 위치의 3 차원지도를 생성 할 수있는 반면 (도를 셀 구분 처리시의 부담을 줄일 1C </strong&gt;). 또한,이 워크 플로의 확장 성을 실험적으로 처리 된 배아, 또는 다른 유전 적 배경 5,6의 배아의 큰 집단을 통해 개별 새끼의 분석은 적용한다. 분 (소프트웨어 MATLAB 라이센스가 필요합니다) http://katlab-tools.org에서 자유롭게 사용할 수 있습니다. 현재까지 개발 된 어떤 접근 방식은 마우스 착상 전 배아의 단백질 발현 및 세포 현지화에 같은 깊이있는 데이터의 생성을 허용하지 않습니다. 모든 시도는 지금까지 이러한 유형의 데이터를 정량화 수동 판정 다른 집단에 대한 세포 수를 정량 배아 (전체가 수동으로, 또는 소프트웨어 보조 식) 9-19로 제한되어왔다. (MINS 소프트웨어를 포함)이 접근 방식에 맞춰 마우스 착상 전 배아와는 ESC에서 테스트되었습니다; 그럼에도 불구하고 높은 핵 밀도가 다른 시스템의 성능은 아직 검증되지 않은 있지만, 상당 할 것으로 예상된다.

<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

이 프로토콜은 방법은 정량적 수행하기 위해 제공, 현장에서 단일 셀 혈통 사양을 연구하는 단백질 발현 분석 마우스 착상 전 배아에서. 배아 수집, 면역, 공 초점 현미경 영상 및 이미지 세분화 및 분석에 필요한 절차를 설명한다. 이 방법은 여러 핵 마커의 발현 및 공간 (XYZ)의 정량은 태아의 모든 셀의 좌표를 허용한다. 그것은 MINS, 특히 착상 전 배아 및 배아 줄기 세포 (ESC) 식민지의 공 초점 이미지의 분석을 위해 개발 한 이미지 분할 소프트웨어 툴을 활용합니다. 분 X, Y 및 Z 차원 걸쳐 자율 핵 세그멘테이션을 수행하고, 최소한의 사용자 입력을 모든 채널에 대해 3 차원 공간에서 셀의 위치에 대한 정보뿐만 아니라, 핵 형광 레벨을 생성한다. 이 프로토콜은 화상의 분석을 위해 최적화되어 있지만착상 전 단계의 마우스 배아, 그것은 쉽게 양호한 신호 대 잡음비 및 여기서 높은 핵 밀도 (이미지 분할에 장애물 포즈 등., 배아 줄기 세포의 발현 분석을 나타내는 임의의 다른 시료의 분석 (ESC에 적용 할 수있다 ) 식민지 문화의 셀, 다른 종 또는 단계 등)의 배아를 차별화.

데이터 해석 및 프리젠 테이션을 용이하게하기 위해주의가 모든 세포의 상대적인 위치를 분석 할 수 있도록, 수집 및 조작시 배아를 손상시키지 않도록주의해야한다도 2a를 -. D는 팽창 캐비티와 다른 단계에서 그대로 포배의 예를 나타낸다. 발생하는 손상 경우, 별도의주의를 분석하고 결과를 해석 할 때주의해야한다. <p class="jove_content" fo:keep-together.within-p...

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

단백질 발현의 정량 분석​​은 마우스 착상 전의 배아에서 리니지 사양을 공부하기

이 프로토콜은 마우스 착상 전 배아 계통 명세서를 연구하는 단백질 발현의 정량적 분석 시츄, 단일 셀을 수행하는 방법을 제시한다. 포배의 수집에 필요한 절차, 단백질의 면역 검출을 전체 마운트, 공 초점 현미경, 핵 세분화 및 이미지 분석에 샘플 영상은 설명한다.

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 San Sebastian

Research

JoVE Journal

Developmental Biology

10.8K 조회수.  Sloan Kettering Institute.  이 프로토콜은 마우스 착상 전 배아 계통 명세서를 연구하는 단백질 발현의 정량적 분석 시츄, 단일 셀을 수행하는 방법을 제시한다. 포배의 수집에 필요한 절차, 단백질의 면역 검출을 전체 마운트, 공 초점 현미경, 핵 세분화 및 이미지 분석에 샘플 영상은 설명한다. 

비디오: 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.

착상 전의 마우스 배아의 초기 세포 운명 결정 요인을 조사 할 Loss- 및 이득 -의 - 기능 접근

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

연구

발생학

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.

자세한 공간 식지도에서 동적 식 데이터의 시간적 순서

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.

단백질 수준 및 개발 표준화 양적 부 럽을 사용 하 여 전체 조직에 걸쳐 강력한 비교

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 ...

생화학

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.

마우스 배아에서 심장 조상 인구의 양적 전체 마운트 면역 형광 분석

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.

전체 마우스 배아에서 β-galactosidase 활동의 감지를 통해 유전자 발현 추적

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. elegans 배아 개발을 분석하는 세미 고처리량 이미징 방법 및 데이터 시각화 툴킷

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.

마우스 배아 세포의 다중화 단세포 mRNA 염기서열 분석

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.

초기 마우스 배아에서 시퀀싱을 위한 작은 RNA 라이브러리 의 준비

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.

발달 중인 (개구리) 배아의 세포 계통 유도 질량 분석법 단백질체학

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

화학

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.

초기 마우스 배아를 사용하는 조직 특정 유전자에 대한 염색질의 Immunoprecipitation 분석

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

단백질 발현의 정량 분석은 마우스 착상 전의 배아에서 리니지 사양을 공부하기

요약

더 많은 비디오 탐색

이 비디오의 챕터

착상 전의 마우스 배아의 초기 세포 운명 결정 요인을 조사 할 Loss- 및 이득 -의 - 기능 접근

자세한 공간 식지도에서 동적 식 데이터의 시간적 순서

단백질 수준 및 개발 표준화 양적 부 럽을 사용 하 여 전체 조직에 걸쳐 강력한 비교

마우스 배아에서 심장 조상 인구의 양적 전체 마운트 면역 형광 분석

전체 마우스 배아에서 β-galactosidase 활동의 감지를 통해 유전자 발현 추적

C. elegans 배아 개발을 분석하는 세미 고처리량 이미징 방법 및 데이터 시각화 툴킷

마우스 배아 세포의 다중화 단세포 mRNA 염기서열 분석

초기 마우스 배아에서 시퀀싱을 위한 작은 RNA 라이브러리 의 준비

발달 중인 (개구리) 배아의 세포 계통 유도 질량 분석법 단백질체학

초기 마우스 배아를 사용하는 조직 특정 유전자에 대한 염색질의 Immunoprecipitation 분석

단백질 발현의 정량 분석​​은 마우스 착상 전의 배아에서 리니지 사양을 공부하기

요약

더 많은 비디오 탐색

이 비디오의 챕터

착상 전의 마우스 배아의 초기 세포 운명 결정 요인을 조사 할 Loss- 및 이득 -의 - 기능 접근

자세한 공간 식지도에서 동적 식 데이터의 시간적 순서

단백질 수준 및 개발 표준화 양적 부 럽을 사용 하 여 전체 조직에 걸쳐 강력한 비교

마우스 배아에서 심장 조상 인구의 양적 전체 마운트 면역 형광 분석

전체 마우스 배아에서 β-galactosidase 활동의 감지를 통해 유전자 발현 추적

C. elegans 배아 개발을 분석하는 세미 고처리량 이미징 방법 및 데이터 시각화 툴킷

마우스 배아 세포의 다중화 단세포 mRNA 염기서열 분석

초기 마우스 배아에서 시퀀싱을 위한 작은 RNA 라이브러리 의 준비

발달 중인 (개구리) 배아의 세포 계통 유도 질량 분석법 단백질체학

초기 마우스 배아를 사용하는 조직 특정 유전자에 대한 염색질의 Immunoprecipitation 분석

단백질 발현의 정량 분석은 마우스 착상 전의 배아에서 리니지 사양을 공부하기