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

Ethics statement: All animal work, including husbandry, breeding and sacrifice was approved by Memorial Sloan Kettering Cancer Center&#39;s Institutional Animal Care and Use Committee (IACUC), protocol #03-12-017.
1. Embryo Collection
Note: All animal work must have been approved by institutional and local authorities and conform to local and institutional rules.
<ol>
	<li>Mate a virgin female mouse with a fertile stud male of the desired genotype...

<ol>
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The present protocol describes a method to perform a quantitative analysis of whole-mount immunofluorescence on preimplantation stage mouse embryos. A robust immunofluorescence protocol 22 is followed by high-resolution, whole-mount confocal imaging and by image segmentation using a tailored piece of software 4. While the choice of immunofluorescence protocol is not critical, we find the one presented here 22 to be fast, reliable and to provide robust signal for many of the antibodies we ...

The mouse preimplantation embryo is a paradigm to study the emergence and maintenance of pluripotency in vivo, as well as a model for the study of cell fate specification and de novo epithelialization in mammals. The preimplantation stages of mammalian development are dedicated to the establishment of the three cell lineages that make up the blastocyst, namely the pluripotent epiblast - which gives rise to most somatic tissues and germ cells - and two extraembryonic lineages, the trophectoderm (TE) and the primitive endoderm (PrE) (Figure 1A) 1,2. This protocol describes the procedures to (1) harvest and fix preimplantation stage mouse embryos, (2) perform immunofluorescence to label proteins of interest, (3) carry out whole-mount imaging using a confocal microscope with z-sectioning capabilities and (4) perform nuclear segmentation of confocal images and subsequent quantitative image analyses. This pipeline allows the unbiased measurement of protein levels for the assignment of cell identities to characterize subpopulations of cells in situ. This protocol can be carried out in as little as 3 - 4 days for a single litter (generally up to 10 mouse embryos), from embryo collection to the data analysis (Figure 1B). The simultaneous analysis of several litters would increase the imaging and data analysis time burden, thus extending the overall length of the protocol.
The preimplantation stage mouse embryo is an experimentally tractable system which, given its small size and stereotypical morphology 3, is well suited for in toto imaging of cellular processes with single-cell resolution. To carry out an unbiased, systems-level analysis of a statistically relevant number of embryos, an automated, quantitative analysis pipeline is desirable. However, due to the high nuclear density of the inner cell mass (ICM) of the blastocyst (Figure 1A, 2D), conventional image segmentation platforms fail to provide sufficient accuracy to establish an automated or semi-automated workflow. On the other hand, manual segmentation, while accurate, does not allow the processing of large cohorts of cells and embryos, nor is it suitable for a reproducible, unbiased determination of cell identities - which is especially critical when studying developmental stages where patterns of marker expression have not fully resolved (e.g., do not exhibit a binary distribution across a population). We have recently developed and validated an image segmentation method tailored for mouse preimplantation stage embryos and for mouse embryonic stem cells (ESCs) that achieves high accuracy, while requiring minimal user input 4-8.
The analysis pipeline presented here revolves around the MATLAB-based image segmentation tool Modular Interactive Nuclear Segmentation (MINS) 4. MINS performs unsupervised nuclear segmentation on large batches of confocal Z-stacks after the user has established a minimal number of image properties, using a graphical user interface (GUI) (Table 1) 4. This pipeline has proven efficient for the generation of high throughput data on protein expression and cell localization in both wild type, experimentally treated and genetically modified embryos and ESCs 5-7. In the present protocol, we describe the application of MINS to the segmentation of preimplantation-stage embryo images. For examples of MINS performance on ESCs please refer to 4,7. The automated nuclear segmentation step significantly reduces the time burden of the cell identification process, whereas the spatial and fluorescence intensity measurements allow an unbiased determination of cell identities and the generation of three-dimensional maps of gene expression domains and cell position in the embryo (Figure 1C). Moreover, the scalability of this workflow makes it applicable to the analysis of individual litters through large cohorts of experimentally treated embryos, or embryos of different genetic backgrounds 5,6. MINS is freely available at&#160; http://katlab-tools.org (the software requires a MATLAB license).
No approach developed to date allows the generation of such in-depth data on protein expression and cell localization in mouse preimplantation embryos. All attempts thus far at quantifying these types of data have been restricted to the manual determination and quantitation of cell numbers for different populations in the embryo (either entirely manually, or software-assisted) 9-19. This approach (incorporating MINS software) has been tailored for and tested on mouse preimplantation embryos and ESCs; nevertheless its performance on other systems with high nuclear density, although yet untested, is expected to be equivalent.

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

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

To facilitate data interpretation and presentation, care should be taken not to damage the embryos during collection and manipulation, so that all cells and their relative position can be analyzed. Figure 2A - D shows examples of intact blastocysts at different stages with an expanded cavity. Should damage occur, extra care should be taken when analyzing and interpreting results.
The quality and reliability of t...

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

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

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

The overall goal of this protocol is to quantify protein levels in situ in mouse preimplantation embryos. So this method allows us to perform high-throughput analysis of confocal microscopy images with single-cell resolution in a semi-automated way. This method can be used to quantify protein concentrations on confocal images where there is high nuclear density and when one wants to study heterogeneities within cell populations.To begin, use an open flame on a glass pasteur pipette to draw a capillary end at the tip. Gently rub the far end of the pipette against the capillary, to break it while creating a blunt end. After sacrificing a pregnant female mouse on the desired embryonic day of development, expose the abdominal viscera according to the text protocol and locate the uterus.Hold the cervical end, cut through the cervix, and gently pull up to stretch both uterine horns. Trim the fat from it, taking care not to pierce the uterine wall. Cut above the oviducts, below the ovaries, to release the entire uterus, and place on a petri dish.Next, cover the uterus with PBS, and separate both uterine horns by cutting through the proximal end on both sides of the cervix. Then separate each oviduct from the uterus by cutting below the isthmus that connects them. Under a dissection microscope, use a one mL syringe with a hypodermic needle to flush the blastocysts out of the uterus and into manipulation medium, by forcing 0.5-1 mL of medium through each horn from the cervical opening.Allow up to one to two minutes for blastocysts to sink to the bottom of the dish after flushing. Then locate the blastocysts, and using the mouth-controlled glass pasteur pipette with a capillary end, collect the blastocysts and transfer to a fresh drop of medium. After rinsing the blastocysts, remove the zona pellucida by using Acidic Tyrode's Solution to briefly wash the embryos.As soon as the zona is no longer visible, transfer the blastocysts back to manipulation medium. Wash the blastocysts in room temperature PBS, and fix them by transferring to 4%PFA in PBS for 10 minutes at room temperature. After fixation, transfer the blastocysts back to PBS for storage at four degrees Celsius.After carrying out imunoflourescence according to the text protocol, use a fine mouth pipette to make microdrops of PBS or nuclear stain solution on the glass surface of a 35mm glass-bottom dish, and cover them with mineral oil. Place the embryos in the microdrops and arrange in a consistent manner, preferably with the ICM cavity access parallel to the glass surface, then set up the dish on the microscope holder. To carry out confocal imagining, use the lowest laser power that provides a strong signal-to-noise ratio without bleaching the fluorophores.Adjust the gain and off set to obtain the widest dynamic range without overexposing the sample. Capturing most of or the entire grayscale range will facilitate the detection of small differences in intensity between images. Follow the additional details outlined in the text protocol to image embryos throughout the entire Z axis.Follow the graphic user interface of the MATLAB-based segmentation tool Modular Interactive Nuclear Segmentation, or MINS, to load a confocal image or an entire Z stack of raw data generated by the microscope. To view the outcome of each step, click the corresponding View"button. A yellow tag above the button indicates operation in process.A green tag indicates operation completed. Assess the outcome of the detection step before proceeding to segmentation. If it is not satisfactory, modify the detection parameters and rerun it.From the Batch Mode"run menu, click Add Files"and load all files to be processed at once. Click Start Batch Mode Run"and allow time for the software to process the files. Segmentation output will be saved in the same directory as the original files.Identify false positives such as apoptotic vesicles or other elements that are not intact nuclei but which may have been identified as such by MINS. Delete the corresponding records for false positives from the starstatistics. csv file.Preserve the original starstatistics. csv file for future reference and edit only a copy of it. If a nucleus has been over segmented and presented as two or more nuclei, either merge the records by averaging their intensity level, or keep one of the records for that cell and discard the rest.To resolve under segmentation, identify events where MINS has failed to detect the border between two or more nuclei and segmented them as a single one, or where it has failed to detect a nucleus altogether. Use ImageJ to measure the average gray level for each channel in the under or unsegmented cells. Next, find a medial section of the under or unsegmented nucleus on the DNA channel, using the freehand selection tool, outline the perimeter of the under or unsegmented nucleus.Then, press Control M"or go to the Analyze"menu and select Measure"This will record the mean gray value for the area outlined and will display it on a new window. Using the same outlined area just selected in the DNA channel, repeat the measurement of each of the fluorescence channels of interest. The results will be upended to the previous one on the measurements results window.Then use the measurements obtained to replace the erroneous records in the starstatistics. csv file. If the nucleus has been undersegmented duplicate its row, assign different cell IDs to each new cell, and introduce the values obtained in ImageJ under the corresponding column.In this case, both cells will share spacial coordinates. When mitotic nuclei are to be considered separately in the analysis, manually score them and add the information to the data file. Refer to the text protocol for additional image processing instructions.This figure shows examples of intact blastocysts at different stages with an expanded cavity. Shown here are examples of good antibodies for a number of nuclear proteins, including CDX2, GATA4, GATA6, NANOG, and OCT4. This sample was labeled with the cytoplasmic protein DAB2 that gives a high signal-to-noise ratio.In this panel, examples of a bad staining for GATA4 with a low signal-to-noise ratio are shown, where the samples were only fixed for 10 minutes. This particular anti-GATA4 antibody requires overnight fixation to provide a strong signal. These embryos were imaged with a 40x oil immersion objective with an NA of 1.30 and a 0.21 mm working distance The middle panels show magnifications of the ICMs or individual nuclei and the border between them can be distinguished.The bottom panels illustrate how the more advanced the embryo, the higher the nuclear density, and thus the greater the chance of segmentation errors. These images show errors that MINS can commit, such as detecting apoptotic nuclei as live cells, over segmentation, or under segmentation. This sequence of Z slices reveals an under segmentation event, where two cells have been identified as one.Once mastered, this protocol can be carried out in three to four days from beginning to end, with a time burden of two to four hours per day. While performing this procedure, it is important to remember to be very consistent with experiment conditions, that is immunofluorescent protocols and imaging parameters. After its development, this technique paved the way for researchers in the field of mouse developmental biology to perform in situ quantitative analysis of gene and protein expression in single cells.After watching this video, you shall have a good understanding of how to acquire image data that is appropriate for quantitative in situ expression analysis.

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Research

JoVE Journal

Developmental Biology

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.

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

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.

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.

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.

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.

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.

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

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.

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).

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).

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.

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.

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