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

Podsumowanie

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.

Streszczenie

The Escherichia coli LacZ gene, encoding β-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, β-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 β-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 β-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.

Wprowadzenie

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 β-galactosidase gene (LacZ) of Escherichia coli (E. coli) is one of the most widely used^1,2,3,4. β-galactosidase (β-gal) catalyzes the hydrolysis of β-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-β-D-galactopyranoside), a glycoside that is hydrolyzed by β-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 ago^7,8. 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 β-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 β-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 expression^8,12.

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.

Protokół

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 Autónoma de Madrid to ensure minimal animal suffering.

1. Collection of Embryos from Pregnant Mice (from E8.5 to E12.5)

1. 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).
2. 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.
3. Pinch and lift the abdominal skin using mouse-tooth forceps.
4. Make an incision through the skin and the abdominal wall, 2 to 4 cm vertically across the midline of the abdomen using surgical scissors.
5. 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.
6. 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).
7. 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' 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.
8. Collect the littermate embryos from pregnant mice at early stages (from E8.5 to E12.5) (Figure 2).
9. 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.
10. 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' forceps.
    NOTE: LacZ genotyping is determined by polymerase chain reaction (PCR) primers: forward, 5´-ATCGTGCGGTGGTTGAACTG-3´; reverse, 5´-CGAGGCGTTAACCGTCAGCA -3´.

2. Fixation of Mouse Embryos

1. 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.
2. 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.
3. 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.
4. 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.

3. Whole Mount β-galactosidase Histochemistry of Mouse Embryos

1. 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.
2. Incubate the embryos in X-Gal Rinse buffer for 10 min at room temperature (Figure 2).
3. Incubate the embryos in X-Gal Staining Solution from 2 h to overnight at 37 °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.
4. 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.
5. Transfer stained embryos to 1 mL 4% paraformaldehyde (PFA) to re-fix them, for 1 h to overnight at 4 °C.
   NOTE: Embryos can be stored in 4% PFA/PBS for longer times at 4 °C or can be immediately processed for sectioning. This final fixation step is crucial for the long-term conservation of the staining pattern.
6. Wash the embryos in 1 mL 1x PBS twice for 10 min at room temperature.
7. 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 °C at this step (Figure 2). Adding a very small amount of sodium azide ora crystal of thymol to the embryos stored at 4 °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).
8. 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).

4. Photography of Whole Mount Embryos

1. 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.
2. Cover the embryo completely with 10 mL 1x PBS in the agarose-coated dishes to prevent dehydration and reflection while photographing through the liquid.
3. 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.
4. Place the dish on the stereomicroscope stage, using the transmitted (under-stage) light. Change between the 'dark-field' and 'bright-field' 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.

5. Paraffin Embedding and Sectioning of X-gal Stained Embryos

1. Paraffin wax embedding
   1. Remove the X-gal stained embryos from 4 °C (step 3.5) and wash them with 1 mL 1x PBS three times to eliminate the excess of fixative.
   2. Transfer each embryo to a histology cassette using forceps.
   3. 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 °C until starting the dehydration process.
   4. Incubate the whole cassette in isopropanol for 30 min at room temperature.
   5. By using forceps, transfer the cassettes to a glass staining trough containing pre-warmed isopropanol and leave in an oven at 60 °C for 30 min.
   6. 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 °C oven.
   7. Incubate the cassettes with the embryos with two changes of molten paraffin wax, 30 min at 60 °C each.
   8. 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.
   9. Fill the well with molten paraffin wax at 60 °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.
   10. 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.
   11. 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 °C until sectioning¹⁴.
2. Paraffin Sectioning
   1. Orient the blade on the microtome and set up 5-7 µm of thickness. 5.2.2. Cool the paraffin block on the ice and trim it to produce a cube or a pyramid.
   2. Attach the block to the microtome holder by using a small amount of molten wax.
   3. Orient the block for optimal cutting. Section the paraffin blocks into 5-7 µm thick slices at room temperature using standard microtome.
   4. Using a fine paintbrush, place the sections in a water bath at room temperature for manipulating and selecting slices.
   5. Pick up sections from the water bath with a microscope slide and transfer them into a water bath at 42 °C for stretching.
   6. Remove sections from the water bath and gently place them onto adhesion microscope slides.
   7. Dry the slides well in an oven at 37 °C overnight to allow the sections to completely adhere, otherwise, they may get lost during the staining.
      NOTE: Store the slides at 4 °C until starting staining procedures.
3. Deparaffinization and Counterstaining of X-Gal-stained Paraffin Sections
   1. Put the slides on a microscope-slide tray in an oven at 60 °C for at least 45 min to make the paraffin wax more fluid.
   2. 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 °C; isopropanol for 3 min; 100% ethanol for 2 min; 96% ethanol for 1 min; 70% ethanol for 1 min at room temperature.
   3. Rinse sections in distilled water twice to make sure that there are no alcohol residues.
   4. 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.
   5. 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.
   6. 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.
   7. 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.

Wyniki

Here we show the results from applying the standard protocol for the β-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...

Dyskusje

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 β-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...

Materiały

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