We would like to thank the Histopathological Service for their technical assistance at the Centro Nacional de Investigaciones Cardiovasculares (CNIC). We also thank Dr. Motoharu Seiki for kindly providing Mt4-mmpLacZ mice, and Dr. Alicia G. Arroyo for supporting our project and for her critical reading of the manuscript. We wish to thank Peter Bonney for proofreading this article. This work was supported by Universidad Europea de Madrid by means of a grant (# 2017UEM01) awarded to C.S.C.

All experimental procedures were approved by the Committee on the Ethics of Animal Experiments of the CNIC (Centro Nacional de Investigaciones Cardiovasculares) and the Comunidad Aut&#243;noma de Madrid to ensure minimal animal suffering.
1. Collection of Embryos from Pregnant Mice (from E8.5 to E12.5)
<ol>
	<li>Sacrifice pregnant mice by either cervical dislocation or CO2 inhalation. The day of the first observed vaginal plug was considered embryonic day 0.5 (E0.5).</li>
	<li>Lay the animal in the supine position on the absorbent pad and clean the abdominal skin of the mouse with 70% ethanol. 
	NOTE: Sterility is not required in the following steps.</li>
	<li>Pinch and lift the abdominal skin using mouse-tooth forceps.</li>
	<li>Make an incision through the skin and the abdominal wall, 2 to 4 cm vertically across the midline of the abdomen using surgical scissors.</li>
	<li>Expose the abdomen and remove the uterine horns from the mother with forceps cutting blood vessels along the inner curvature of the uterus with surgical scissors.</li>
	<li>Remove the string of embryos and transfer it to a Petri dish on ice containing 10 mL ice-cold 0.01 M phosphate buffer saline pH 7.4 (PBS).</li>
	<li>Carefully dissect the embryos out from the uterus under a stereomicroscope in a Petri dish with 10 mL fresh ice-cold PBS. Grasp the muscle wall with watchmakers&#39; forceps to expose the amniotic sac, and then tear the amniotic membrane to remove the enclosed embryo and the yolk sac using the tips of the forceps.</li>
	<li>Collect the littermate embryos from pregnant mice at early stages (from E8.5 to E12.5) (Figure 2).</li>
	<li>Transfer the embryos to a fresh dish with 10 mL cold 1x PBS using curved forceps or, for very small embryos (E8.5-E9.5), use plastic transfer pipettes to collect the samples without embryo damage.</li>
	<li>Before starting the fixation, take an embryonic sample in a microcentrifuge tube for genotyping by cutting a small piece of the embryo or taking the amniotic membrane in the smallest embryos (from E8.5 to E9.5) with watchmakers&#39; forceps. 
	NOTE: LacZ genotyping is determined by polymerase chain reaction (PCR) primers: forward, 5&#180;-ATCGTGCGGTGGTTGAACTG-3&#180;; reverse, 5&#180;-CGAGGCGTTAACCGTCAGCA -3&#180;.</li>
</ol>
2. Fixation of Mouse Embryos
<ol>
	<li>Transfer each embryo into a 2 mL microcentrifuge tube with a rounded base containing 1 mL 1x PBS. 
	NOTE: Perform all steps in the protocol in these tubes with agitation using a rolling shaker.</li>
	<li>Wash the embryos in 1x PBS for 1 min at room temperature to eliminate the remaining blood. Use plastic transfer pipettes to change liquids in the tubes.</li>
	<li>Fix the embryo in 1 mL of 0.125% glutaraldehyde on ice. 
	NOTE: The time of fixation depends on the size of the embryo: 20 min for E8.5-E9.5 embryos, 30 min for E10.5 embryos and 1 h for E12.5 embryos.</li>
	<li>Remove the fixative and wash the embryos in 1x PBS twice for 10 min at room temperature before processing the sample for X-gal staining.</li>
</ol>
3. Whole Mount &#946;-galactosidase Histochemistry of Mouse Embryos
<ol>
	<li>Prepare X-Gal Rinse buffer and X-Gal Staining Solution before starting the procedure (see Table 1 and Table of Materials). Use a wild-type (WT) littermate embryos as negative controls for the enzymatic reaction.</li>
	<li>Incubate the embryos in X-Gal Rinse buffer for 10 min at room temperature (Figure 2).</li>
	<li>Incubate the embryos in X-Gal Staining Solution from 2 h to overnight at 37 &#176;C protected from light. Monitor the color reaction periodically via a dissecting microscope until sufficient intensity of blue staining is observed without the background in the embryo. Keep the pH of the solution between 8 and 9 to reduce background during the whole staining. If the staining solution begins to turn light, replace it with fresh substrate solution to extend the enzymatic reaction.</li>
	<li>Stop the reaction when the desired signal is obtained by washing embryos in a microcentrifuge tube with 1 mL 1x PBS twice for 10 min each at room temperature.</li>
	<li>Transfer stained embryos to 1 mL 4% paraformaldehyde (PFA) to re-fix them, for 1 h to overnight at 4 &#176;C. 
	NOTE: Embryos can be stored in 4% PFA/PBS for longer times at 4 &#176;C or can be immediately processed for sectioning. This final fixation step is crucial for the long-term conservation of the staining pattern.</li>
	<li>Wash the embryos in 1 mL 1x PBS twice for 10 min at room temperature.</li>
	<li>Option 1: Clear embryos by immersion in a series of solutions with increasing concentrations of glycerol (20%, 40%, 60% and 80% glycerol in 1x PBS) for 1 h at room temperature in each solution. 
	NOTE: Wash embryos in 80% glycerol for longer times, even days, until they are cleared, and then store them in 80% glycerol at 4 &#176;C at this step (Figure 2). Adding a very small amount of sodium azide ora crystal of thymol to the embryos stored at 4 &#176;C either in glycerol or 1x PBS is recommended to prevent mold growth. After clearing, whole mount embryos can be used for photographing (step 4).</li>
	<li>Option 2: Process embryos for paraffin embedding and sectioning using a microtome (Figure 2). Document the expression pattern in whole stained embryos before sectioning (steps 4 and 5).</li>
</ol>
4. Photography of Whole Mount Embryos
<ol>
	<li>To photograph whole mount stained embryos prior to sectioning, transfer them to a Petri dish prepared with a base of solidified 1-2% agarose in 1x PBS.</li>
	<li>Cover the embryo completely with 10 mL 1x PBS in the agarose-coated dishes to prevent dehydration and reflection while photographing through the liquid.</li>
	<li>Orientate the embryo immersed in 1x PBS using forceps. For older embryos (E10.5-E12.5), make a small hole in the agarose with forceps to place and position the specimen within it at different angles and to avoid the free-floating during photography.</li>
	<li>Place the dish on the stereomicroscope stage, using the transmitted (under-stage) light. Change between the &#39;dark-field&#39; and &#39;bright-field&#39; adjustments to set the desired illumination during photography and to optimize the translucency of the sample. 
	NOTE: Use fiber optic illumination for later-stage embryos to avoid reflection at the surface of the embryo. When photographing cleared embryos, follow the same procedure but transfer them to a Petri dish containing 100% glycerol and fully immerse them.</li>
</ol>
5. Paraffin Embedding and Sectioning of X-gal Stained Embryos
<ol>
	<li>Paraffin wax embedding
	<ol>
		<li>Remove the X-gal stained embryos from 4 &#176;C (step 3.5) and wash them with 1 mL 1x PBS three times to eliminate the excess of fixative.</li>
		<li>Transfer each embryo to a histology cassette using forceps.</li>
		<li>Place the cassettes containing the embryos in a beaker and then dehydrate by passing them through a graded ethanol series at room temperature: 70% ethanol, once for 30 min; 96% ethanol, twice for 30 min each; 100% ethanol, twice for 30 min each. 
		NOTE: Embryos can be stored in 70% ethanol for an undetermined time at 4 &#176;C until starting the dehydration process.</li>
		<li>Incubate the whole cassette in isopropanol for 30 min at room temperature.</li>
		<li>By using forceps, transfer the cassettes to a glass staining trough containing pre-warmed isopropanol and leave in an oven at 60 &#176;C for 30 min.</li>
		<li>Place the cassettes in a new glass staining trough containing a mixture of 50% isopropanol/ 50% melted paraffin wax and leave for 4 h in a 60 &#176;C oven.</li>
		<li>Incubate the cassettes with the embryos with two changes of molten paraffin wax, 30 min at 60 &#176;C each.</li>
		<li>At the end of the final paraffin wash, open the cassette and transfer each embryo to a separate tissue embedding mold to view the sample under a stereomicroscope.</li>
		<li>Fill the well with molten paraffin wax at 60 &#176;C. Use heated forceps to keep the wax molten and carefully orientate the embryo in the mold. 
		NOTE: The proper orientation of the sample is a critical step for sectioning the embryo in the desired plane.</li>
		<li>Before paraffin solidifies in the mold, place a cassette without the lid on the top of the block and fill it with molten paraffin wax. Let the remaining wax cool until solidified overnight at room temperature.</li>
		<li>Once the paraffin is completely solidified, remove the solid block from the mold and store the paraffin blocks either at room temperature or at 4 &#176;C until sectioning14.</li>
	</ol>
	</li>
	<li>Paraffin Sectioning
	<ol>
		<li>Orient the blade on the microtome and set up 5-7 &#181;m of thickness. 5.2.2. Cool the paraffin block on the ice and trim it to produce a cube or a pyramid.</li>
		<li>Attach the block to the microtome holder by using a small amount of molten wax.</li>
		<li>Orient the block for optimal cutting. Section the paraffin blocks into 5-7 &#181;m thick slices at room temperature using standard microtome.</li>
		<li>Using a fine paintbrush, place the sections in a water bath at room temperature for manipulating and selecting slices.</li>
		<li>Pick up sections from the water bath with a microscope slide and transfer them into a water bath at 42 &#176;C for stretching.</li>
		<li>Remove sections from the water bath and gently place them onto adhesion microscope slides.</li>
		<li>Dry the slides well in an oven at 37 &#176;C overnight to allow the sections to completely adhere, otherwise, they may get lost during the staining. 
		NOTE: Store the slides at 4 &#176;C until starting staining procedures.</li>
	</ol>
	</li>
	<li>Deparaffinization and Counterstaining of X-Gal-stained Paraffin Sections
	<ol>
		<li>Put the slides on a microscope-slide tray in an oven at 60 &#176;C for at least 45 min to make the paraffin wax more fluid.</li>
		<li>Transfer the slides onto a rack and use glass staining dishes to perform the following steps: submerge the rack in the staining jars containing alcohols of decreasing concentrations for complete paraffin removal and hydration of the tissues: isopropanol for 5 min at 60 &#176;C; isopropanol for 3 min; 100% ethanol for 2 min; 96% ethanol for 1 min; 70% ethanol for 1 min at room temperature.</li>
		<li>Rinse sections in distilled water twice to make sure that there are no alcohol residues.</li>
		<li>Counterstain sections with a stain (e.g. Nuclear-Fast Red solution) for 5 min (usually between 3-8 min) at room temperature (Figure 2). 
		NOTE: This staining helps to reveal overall tissue structure in the sections.</li>
		<li>After staining, wash the slides in distilled water for 1 min and check section staining in the microscope. 
		NOTE: If the desired staining is too weak, counterstain sections again for a longer time.</li>
		<li>Dehydrate sections in the following series with increasing concentrations of ethanol: two washes in 95% ethanol for 2 min each, two washes in 100% ethanol for 2 min each, and, to avoid dissolving the blue color precipitates, just one quick wash in xylene.</li>
		<li>Remove the slides from the rack one by one and place them on a piece of paper. Then mount and coverslip each slide using a synthetic, permanent mounting medium. 
		NOTE: Xylene is a flammable product whose vapors are irritating and harmful to human health and to aquatic organisms. It needs to be manipulated within a hood and requires the use of proper protective equipment.</li>
	</ol>
	</li>
</ol>

The authors declare that they have no competing financial interest.

The E. coli LacZ gene has been widely used as a reporter in studies of gene expression patterns because of its high sensitivity and ease of detection. The present protocol describes a classic method for detecting &#946;-gal expression based on an enzymatic reaction that is easy and quick to perform as well as inexpensive. This method can be also applied without major modifications in whole mount embryos, intact organs, cryostat tissue sections or cultured cells.
Accurate application o...

In order to describe specific gene expression patterns, the use of reporter genes as markers has been paramount from Drosophila to mammals. In experiments involving transgenic and knockout animals, the bacterial &#946;-galactosidase gene (LacZ) of Escherichia coli (E. coli) is one of the most widely used1,2,3,4. &#946;-galactosidase (&#946;-gal) catalyzes the hydrolysis of &#946;-galactosides (such as lactose) into its monosaccharides (glucose and galactose)5. Its most commonly used substrate is X-gal (5-bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside), a glycoside that is hydrolyzed by &#946;-galactosidase giving rise to 5-bromo-4-chloro-3-hydroxyindole and galactose. The first is oxidized into a dimer that, when used combined with potassium ferri-and ferro-cyanide, produces a characteristic insoluble, blue color precipitate (Figure 1)6.
The LacZ gene started to be used as a reporter gene over thirty years ago7,8. Usually, LacZ is inserted downstream of an endogenous promoter in the place of the open reading frame, so it can be used in bacterial and cell culture to visualize cells containing a particular insert, as well as in transgenic animals as a tracer of endogenous gene expression patterns during development9. In this regard, the visualization of &#946;-galactosidase activity has been extensively used in Drosophila to understand the developmental and cellular processes from single cells to whole tissues. Drosophila genetics favor the generation of stable lines in which a modified P-element construct containing the reporter gene LacZ is inserted at random locations in the genome. Thus, when placed under the influence of enhancer elements it may drive its expression in a tissue specific manner, which has allowed the systematic analysis of the expression patterns of many genes during the past two decades10. In addition, the use of transgenic mice to monitor LacZ gene expression also allows detection of gene recombination events by Cre-loxP mediated recombination, and localization of the mutant embryonic stem cell derivatives in chimeric analyses11, which facilitates the control of LacZ expression in specific tissues as well as temporally. Also, in whole embryos, detection of the &#946;-galactosidase activity may produce differential staining patterns at different intensities that can be conveniently observed across different developmental stages to analyze temporal changes in gene expression8,12.
In this article, we present a protocol to visualize gene expression through X-gal staining in the whole mount tissue at early developmental stages of mouse embryos. We present this histochemical method as a highly sensitive and inexpensive technique that favors accurate detection of the labeled cells either in whole mount specimens or at the cellular level after paraffin embedded tissues or embryos. The method allows for the direct visualization of staining in the mouse tissue with the minimum background when compared with other methods13.

<table><tbody><tr><td>&lt;strong&gt;REAGENTS&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2-Propanol</td><td>SIGMA-ALDRICH</td><td>24137-1L-R</td><td></td></tr><tr><td>Agarose</td><td>SCHARLAU</td><td>50004/ LE3Q2014</td><td></td></tr><tr><td>Aqueous mounting medium</td><td>VECTOR LABS</td><td>H-5501</td><td></td></tr><tr><td>Synthetic mounting media</td><td>MERCK</td><td>100579</td><td></td></tr><tr><td>96% Ethanol</td><td>PROLABO</td><td>20824365</td><td></td></tr><tr><td>99.9% Ethanol absolute</td><td>SCHARLAU</td><td>ET00021000</td><td></td></tr><tr><td>50% Glutaraldehyde solution</td><td>SIGMA-ALDRICH</td><td>G6403-100ml</td><td></td></tr><tr><td>85% Glycerol</td><td>MERCK</td><td>104094</td><td></td></tr><tr><td>99.9% Glycerol</td><td>SIGMA-ALDRICH</td><td>G5516</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>SIGMA-ALDRICH</td><td>63064</td><td></td></tr><tr><td>Nonionic surfactant (Nonidet P-40)</td><td>SIGMA-ALDRICH</td><td>542334</td><td></td></tr><tr><td>Nuclear Fast Red counterstain</td><td>SIGMA-ALDRICH</td><td>N3020</td><td></td></tr><tr><td>Paraffin pastilles</td><td>MERCK</td><td>111609</td><td></td></tr><tr><td>Paraformaldehyde</td><td>SIGMA-ALDRICH</td><td>158127-500g</td><td></td></tr><tr><td>Phosphate buffered saline (tablets)</td><td>SIGMA-ALDRICH</td><td>P4417-50TAB</td><td></td></tr><tr><td>Potassium ferrocyanate</td><td>MERCK</td><td>1049840500</td><td></td></tr><tr><td>Potassium ferrocyanide</td><td>MERCK</td><td>1049731000</td><td></td></tr><tr><td>Sodium azide</td><td>SIGMA-ALDRICH</td><td>S8032</td><td></td></tr><tr><td>Sodium deoxycholate</td><td>SIGMA-ALDRICH</td><td>30970</td><td></td></tr><tr><td>Sodium dihydrogen phosphate monohydrate</td><td>SIGMA-ALDRICH</td><td>106346</td><td></td></tr><tr><td>Sodium phosphate dibasic dihydrate</td><td>SIGMA-ALDRICH</td><td>71638</td><td></td></tr><tr><td>Thymol</td><td>SIGMA-ALDRICH</td><td>T0501</td><td></td></tr><tr><td>Tris hydrochloride (Tris HCl)</td><td>SIGMA-ALDRICH</td><td>10812846001 (Roche)</td><td></td></tr><tr><td>X-GAL</td><td>VENN NOVA</td><td>R-0004-1000</td><td></td></tr><tr><td>Xylene</td><td>VWR CHEMICALS</td><td>VWRC28973.363</td><td></td></tr><tr><td>&lt;strong&gt;EQUIPMENT&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Disposable plastic cryomolds 15x15x5 mm</td><td>SAKURA</td><td>4566</td><td></td></tr><tr><td>Rotatory Microtome</td><td>Leica</td><td>RM2235</td><td></td></tr><tr><td>Cassettes</td><td>Oxford Trade</td><td>OT-10-9046</td><td></td></tr><tr><td>Microscope Cover Glasses 24x60 mm</td><td>VWR</td><td>ECN631-1575</td><td></td></tr><tr><td>Microscope slides</td><td>Thermo Scientific, MENZEL-GL&amp;Auml;SER</td><td>AGAA000001#12E</td><td></td></tr><tr><td>Adhesion microscope slides</td><td>Thermo Scientific, MENZEL-GL&amp;Auml;SER</td><td>J1820AMNZ</td><td></td></tr><tr><td>Flotation Water bath</td><td>Leica</td><td>HI1210</td><td></td></tr><tr><td>Disposable Low Profile Microtome Blades</td><td>Feather</td><td>UDM-R35</td><td></td></tr><tr><td>Paraffin oven</td><td>J.R. SELECTA</td><td>2000205</td><td></td></tr><tr><td>Wax Paraffin dispenser</td><td>J.R. SELECTA</td><td>4000490</td><td></td></tr><tr><td>Stereomicroscope</td><td>Leica</td><td>DM500</td><td></td></tr><tr><td>Polypropylene microcentrifuge tubes 2.0 mL</td><td>SIGMA-ALDRICH</td><td>T2795</td><td></td></tr><tr><td>Polypropylene microcentrifuge tubes 1.5 mL</td><td>SIGMA-ALDRICH</td><td>T9661</td><td></td></tr><tr><td>Orbital shaker</td><td>IKA Labortechnik</td><td>HS250 BASIC</td><td></td></tr><tr><td>Stirring Hot Plate</td><td>Bibby</td><td>HB502</td><td></td></tr><tr><td>Vortex Shaker</td><td>IKA Labortechnik</td><td>MS1</td><td></td></tr><tr><td>Laboratory scale</td><td>GRAM</td><td>FH-2000</td><td></td></tr><tr><td>Precision scale</td><td>Sartorius</td><td>ISO9001</td><td></td></tr><tr><td>pHmeter</td><td>Crison</td><td>Basic 20</td><td></td></tr><tr><td>Optic fiber</td><td>Optech</td><td>PL2000</td><td></td></tr></tbody></table>

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 show the results from applying the standard protocol for the &#946;-galactosidase histochemical reaction using X-gal as the substrate in whole mouse embryos (Figure 1 and Figure 2). By using this protocol, we examine Membrane type 4-matrix metalloproteinase (Mt4-mmp) expression at different embryonic developmental stages (E9.5, E11.5, and E12.5) using Mt4-mmp mutant mice that express the LacZ reporter under the control of...

Watch this Scientific Journal Video about Tracing Gene Expression Through Detection of Î²-galactosidase Activity in Whole Mouse Embryos at JoVE.com

Tracing Gene Expression Through Detection of &#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. ...

tracing-gene-expression-through-detection-galactosidase-activity

Research

JoVE Journal

Developmental Biology

26.1K Views.  Universidad Europea de Madrid. Here we describe the standard protocol for the detection of β-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.

Video: Tracing Gene Expression Through Detection of β-galactosidase Activity in Whole Mouse Embryos

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.).

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 ...

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 ...

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

Genetics

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we describe a method to label in situ a subset of dorsal root ganglion (DRG) axon projections to assess their phenotypic characteristics using several genetically manipulated mouse lines. The TrkA-positive neurons are nociceptor neurons, dedicated to the transmission of pain signals. We utilize a TrkAtaulacZ mouse line to label the trajectories of all TrkA-positive peripheral axons in the intact mouse embryo. We further breed the TrkAtaulacZ line onto a Bax null background, which essentially abolishes neuronal apoptosis, in order to assess growth-related questions independently of possible effects of genetic manipulations on neuronal survival. Subsequently, genetically modified mice of interest are bred with the TrkAtaulacZ/Bax null line and are then ready for study using the techniques described herein. This presentation includes detailed information on mouse breeding plans, genotyping at the time of dissection, tissue preparation, staining and clearing to allow for visualization of full-length axonal trajectories in whole-mount preparation.

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

Neuroscience

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.

Capitalizing on a binary genetic strategy we provide a detailed protocol for neural circuit tracing in mice that express complementary transsynaptic tracers after Cre-mediated recombination. Because cell-specific tracer production is genetically encoded, our experimental approach is suitable to study the formation and maturation of neural circuitry during murine embryonic brain development at a single cell resolution.

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits ...

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.

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

Biology

Isolation and Genotyping of Gastrulating Mouse Embryo for Single-Cell Sequencing

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.

Author Spotlight: A Pipeline to Analyze Lineage-Specific Mutant Embryos at Single-Cell Resolution

This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states ...

Laser-assisted Lentiviral Gene Delivery to Mouse Fertilized Eggs

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the host chromosome and is passed on to progeny. Thus, they are ideal vectors for creation of stable cell lines, in vivo delivery of indicators, and transduction of single cell fertilized eggs to create transgenic animals. However, mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against lentiviral gene delivery. Lentiviruses are too large to penetrate the zona and are typically delivered by microinjection of viral particles into the perivitelline cavity, the space between the zona and the embryonic cells. The requirement for highly skilled technologists and specialized equipment has minimized the use of lentiviruses for gene delivery to mouse embryos. This article describes a protocol for permeabilizing the mouse fertilized eggs by perforating the zona with a laser. Laser-perforation does not result in any damage to embryos and allows lentiviruses to gain access to embryonic cells for gene delivery. Transduced embryos can develop into blastocyst in vitro, and if implanted in pseudopregnant mice, develop into transgenic pups. The laser used in this protocol is effective and easy to use. Genes delivered by lentiviruses stably incorporate into mouse embryonic cells and are germline transmittable. This is an alternative method for creation of transgenic mice that requires no micromanipulation and microinjection of fertilized eggs.

Mouse fertilized eggs and early stage embryos are protected by the zona pellucida, a glycoprotein matrix that forms a barrier against gene delivery. This article describes a protocol for perforating the zona with a laser to transduce embryonic cells with lentiviral vectors and to create transgenic mice.

Lentiviruses are efficient vectors for gene delivery to mammalian cells. Following transduction, the lentiviral genome is stably incorporated into the ...

Tracing Gene Expression Through Detection of β-galactosidase Activity in Whole Mouse Embryos

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