We thank Sophie Gournet for help with illustrations, Julie Chaumeil for reading the manuscript, the animal housing facility and the imaging platform of the Unit. This work has received support under the program &#171;Investissements d&#39;Avenir&#187; launched by the French Government and implemented by ANR with the references ANR-10-LABX-0044 and ANR-10-IDEX-0001-02 PSL, the EpiGeneSys FP7 no. 257082 Network of Excellence to E.H., an ERC Advanced Investigator award no. 250367 and EU FP7 SYBOSS grant no. 242129 to E.H. The authors would like to acknowledge the Cell and Tissue Imaging Platform of the Genetics and Developmental Biology Department&#160;(UMR3215/U934)&#160;of Institut Curie,&#160;member of France-Bioimaging (ANR-10-INSB-04),&#160;for help with light microscopy.

Animal procedures were performed in accordance with the approved institutional animal care and use committee of the Institut Curie (CEEA-IC) protocols (C 75-05-18). The work has also been conducted under the approval from the French Ministry of Higher Education and Research for the use of Genetically Modified Organisms (agreement number 5549CA-I).
1. Preparation of Cryostat Sections
<ol>
	<li>Collect embryos from naturally ovulating F1 C57BL/6 x DBA/2J mice as described in Shea et al.,12. On day 7 of gestation (E7), sacrifice 8-12 week old mouse by cervical dislocation. Collect the entire conceptus i.e decidua12.</li>
	<li>Isolate E7 conceptuses, as described in Shea et al.,12. Place them in a 60 mm Petri dish containing PBS.</li>
	<li>Freeze the E7 mouse conceptus for cryostat sections.
	<ol>
		<li>Prepare adapted aluminum foil wells 1cm in height (&#39;home-made&#39; using a glass Pasteur pipet). Deposit a drop of tissue freezing medium on the bottom of this small container. Deposit the conceptus in the correct orientation (i.e.,&#160;for longitudinal sections the conceptus should maintained in its horizontal orientation).</li>
		<li>Fill the well containing the conceptus with tissue freezing medium. Suspend it with forceps in vapor above liquid N2 in order to allow it to freeze slowly&#160;&#8211; then immerse the block in the liquid N2 for a few sec until it turns white. Subsequently, transfer the block into a freezing vial and store at -80 &#176;C (can store for several months).</li>
	</ol>
	</li>
	<li>Before cryo-sectioning, place the frozen block containing the conceptus at -20 &#176;C in the cryostat for 30 min. Perform cryosections of 8 &#181;m thickness. Deposit 4 sections on a slide to enable efficient attachment. Place sections close enough to fit under an 18 x 18 mm2 coverslip.</li>
	<li>Check the quality of the sections (intact and without scratches) and the orientation of the embryo (longitudinal sections) with a stereomicroscope and choose the sections suitable for further analysis. As quickly as possible deposit them in a coplin jar and perform RNA FISH (See 3.3.2).</li>
</ol>
2. Preparation of Secondary TGCs
<ol>
	<li>Isolate the Conceptus (see 1.1 and 1.2).</li>
	<li>Dissect the E7 Ectoplacental Cone (EPC)
	<ol>
		<li>Use a stereomicroscope and Petri dishes containing sterile PBS. Dissect the decidua with forceps. Pierce the sample and open forceps to tear the two sides of the decidua apart.</li>
		<li>Shell out the embryo. Use tips of closed fine forceps (Dumont No. 5) in a scissor-like action to separate the EPC from the embryo proper. Use special care to obtain a perfectly clean sample, separate from maternal tissue as well as from chorion and yolk sac. Wash out EPC explants in PBS.</li>
		<li>With a little spoon, transfer individual dissected EPC to a 4-well plate containing a sterile coverslip in medium.</li>
	</ol>
	</li>
	<li>Derive TGCs from EPCs Short-term Cultures.
	<ol>
		<li>Prepare 4-well plates containing coverslips. Sterilize 12 mm diameter round glass coverslips by immersing in ethanol, followed by flame-drying.</li>
		<li>Add 0.5 ml EPC medium to each well (EPC medium: RPMI 1640 supplemented with 15% FCS, 0.1mM 2-mercaptoethanol and antibiotics).</li>
		<li>Deposit single, dissected-EPCs at the center of the coverslip in the well along with culture medium. Use fine forceps to apply the EPC on the glass coverslip. Culturefor 3-5 days at 37 &#176;C, 5% CO2. Individual explant forms an outgrowth that spreads as a monolayer of flattened TGCs (Figure 2A).</li>
	</ol>
	</li>
</ol>
3. RNA FISH
NOTE: The protocols are based on those described for embryonic stem cells (ESCs) in Chaumeil et al.,8 and Pollex and Heard13.
<ol>
	<li>Advanced Preparation of Stock Solutions
	<ol>
		<li>Prepare a 3M sodium acetate buffer pH 5.2.</li>
		<li>Prepare 2x hybridization buffer containing 40 % (w/v) sodium dextran sulfate, 20x BSA, 400 mM Vanadyl Ribonucleoside Complex (VRC) in 4x Saline Sodium Citrate (SSC).</li>
		<li>Prepare mounting medium containing 90% (v/v) glycerol, 0.1% (w/v) p-phenylenediamine, pH 9 in PBS.</li>
	</ol>
	</li>
	<li>Freshly Prepared Solutions
	<ol>
		<li>Prepare fixative solution consisting of 3% freshly prepared paraformaldehyde (PFA) in PBS. Prepare permeabilization solution containing 0.5 % Triton-X-100 in PBS supplemented with 2 mM VRC. Prepare wash buffer by mixing 50% formamide (FA) and 2x SSC and adjust the pH to 7.2-7.4.</li>
		<li>Prepare DNA counter-staining solution consisting of 1 &#181;g/ml DAPI (4&#8242;,6-diamidino-2-phenylindole dihydrochloride) in 2x SSC.</li>
	</ol>
	</li>
	<li>Fixation and Permeabilization in Preparation for FISH
	<ol>
		<li>For cells on coverslips, wash cells in 1x PBS for 5 min. Fix cells on coverslips, or embryonic sections on slides, for 10 min in fixative (3% PFA) solution at RT.</li>
		<li>Rinse cells three times with 1x PBS. Permeabilize cells for 5 min in ice-cold permeabilization solution on ice. Wash cells three times with 70% ethanol. Store coverslips in 4-well plates and slides in 4-slide transport box in 70% ethanol at -20 &#176;C. 
		NOTE: Coverslips and slides can be stored for several months at -20 &#176;C. Plates and boxes containing them have to be sealed with parafilm to avoid ethanol evaporation.</li>
	</ol>
	</li>
	<li>DNA Probe Labeling
	<ol>
		<li>Label DNA probes by nick translation using fluorescent nucleotides. Follow the manufacturer&#39;s instructions (see Table 1)8.</li>
		<li>For a 50 &#181;l reaction mix, add 1-2 &#181;g of plasmid, bacterial artificial chromosomes (BAC) or multiple displacement amplification (MDA) (for DNA amplification; see 3.4.4) DNA to 17.5 &#181;l water, 2.5 &#181;l 0.2 mM SR-, SG-, Cy5-dUTP, 10 &#181;l 10 mM each dNTP mix (dGTP, dATP, dCTP), 5 &#181;l 10 mM dTTP, 5 &#181;l 10x nick translation buffer and 8 &#181;l nick translation enzyme.</li>
		<li>Incubate for 16 hr at 15 &#176;C in the dark. Inactivate the reaction by freezing at -20 &#176;C. Store probes for months at -20 &#176;C.</li>
		<li>MDA 
		NOTE: For BAC DNA, quantity of DNA obtained after classical preparation is low, therefore one can use a step of DNA amplification before labeling by using MDA. Use a commercial kit and follow the manufacturer&#39;s instructions (see Table 1).</li>
		<li>Mix 0.5 &#181;l BAC DNA with 9.5 &#181;l sample buffer. Heat 3 min at 95 &#176;C. Immediately quench on ice; leave on ice 10 min. Prepare reaction buffer/enzyme mix: (9.5 &#181;l + 0.5 &#181;l enzyme mix) and keep on ice.</li>
		<li>Mix 10 &#181;l DNA mix + 10 &#181;l reaction buffer/enzyme mix. Incubate at 30 &#176;C at least 20 hr. Inactivate enzyme by heating sample 10 min at 65 &#176;C. Cool sample to 4 &#176;C before storing at -20 &#176;C.</li>
		<li>Check MDA by digestion. Add 1 &#181;l MDA, 2 &#181;l 10x buffer, 2 &#181;l Hind III and 15 &#181;l H2O. Incubate at 37 &#176;C overnight. Run slowly on a 0.8-1% agarose gel to confirm accurate DNA amplification. Clear DNA fragments of high molecular weight should be observed on the gel.</li>
	</ol>
	</li>
	<li>Probe Preparation
	<ol>
		<li>Use 0.1 or 1 &#181;g probe per coverslip or slide. 
		NOTE: Add 2-5 &#181;g of Cot-1 DNA if competition is required e.g., most probes as they can contain repeat sequences which otherwise cross hybridize and increase background.
		<ol>
			<li>For precipitation, add 5 &#181;g salmon sperm DNA, 1/10 volume 3 M sodium acetate pH 5.2 and 3 volumes ethanol. Spin at 16,000 x g and 4 &#176;C for 25 min. Wash pellets with 70% ethanol and spin down again for 5 min.</li>
		</ol>
		</li>
		<li>Dry pellet for 2 min in a concentrator/speed vac. Resuspend in 100% formamide in half the volume required for hybridization (e.g., 2.5 &#181;l for coverslip or 7 &#181;l for embryonic sections on slide). Place 30 min at 37 &#176;C with shaking in a thermomixer. Denature for 7 min at 75 &#176;C.</li>
		<li>Quench on ice, or if competition is required put directly at 37 &#176;C for 30-60 min. Mix the probe solution with an equal volume of 2x hybridization solution.</li>
	</ol>
	</li>
	<li>Hybridization and Washes
	<ol>
		<li>Dehydrate; coverslips in wells, and slides in a Coplin jar, by sequential washing in 1x 80%, 1x 95% and 2x 100% ethanol for 5 min each. Dry coverslips and slides completely.</li>
		<li>For hybridization, apply the 5 &#181;l probe hybridization mix (see 3.5) onto a slide and lower the coverslip, with cells facing into the hybridization mix. For sections on slide, apply 14 &#181;l probe hybridization mix (see 3.5) and cover with an 18 x 18 mm2 coverslip.</li>
		<li>Place slides in a humid chamber (tissue paper soaked in 50% FA/2x SSC) and incubate at 37 &#176;C overnight, in the dark.</li>
		<li>Post-hybridization Washes:
		<ol>
			<li>Add 1 ml of 50% FA/2x SSC onto the coverslip on the slide to loosen it; remove the coverslip carefully from the slide (taking care not to scrape the cells) and place it cell-side up into a 4-well plate containing 50% FA/2x SSC. For sections on slide, remove the coverslip in a similar manner and place the slide in a Coplin jar containing 50% FA/2x SSC.</li>
			<li>Perform 3 washes, each for 7 min with pre-warmed 50% FA/2x SSC at 42 &#176;C or 44 &#176;C for coverslip or slide, respectively.</li>
			<li>Perform 3 washes with pre-warmed 2x SSC for 5 min each at 42 &#176;C or 44 &#176;C for coverslip or slide, respectively.</li>
		</ol>
		</li>
		<li>Counterstain nuclei by washing in 2x SSC with 1 &#181;g/ml DAPI for 3 min at RT. Rinse twice with 2x SSC.</li>
	</ol>
	</li>
	<li>Mounting of Coverslips and Slides
	<ol>
		<li>For small coverslip from cultured TGCs, apply 5 &#181;l mounting medium on a slide. Place the coverslip cell-side down on top of the drop.</li>
		<li>For slide with sections, apply 15 &#181;l mounting medium on the slide. Place a 22 x 22 mm2 coverslip on top of the drop.</li>
		<li>Avoid bubbles. Wipe off excess mounting solution. Seal the coverslip with a small amount of nail polish. If possible, image slides immediately or store for up to several months at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
4. Microscopy and Analysis
<ol>
	<li>Acquire 3D sequential z axis images in 0.3 &#181;m using a fluorescence microscope (63X objective) and perform the analysis of 3D image stacks using the imageJ software14.</li>
</ol>

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The authors have nothing to disclose.

Nascent RNA FISH represents an easy and sensitive method for the single cell analysis of transcriptional activity in embryonic tissues at different developmental stages. The power of this approach is the capacity to identify different embryonic lineages at any particular stage according to morphological criteria. However this also requires that minimal background fluorescence is present. Any such background renders the identification of different embryonic regions and cell types challenging. To ensure minimal background, there are two critical steps in this protocol. The first is the quality of the cryosection and the second is the efficiency (signal to noise ratio) of the RNA FISH, which depends on the level of gene expression and the quality of the probe. For the latter, probes are therefore always tested on cultured cells on coverslips (embryonic stem cells or somatic cells) prior to use on sections.
In this protocol, we provide the techniques required to perform RNA FISH analysis on TGCs from two different types of sample preparation (cryostat sections and primary cultures of embryonic explants). Such single cell analysis enables dynamic changes in gene expression at different post-implantation stages to be assessed.
The methods we describe to generate secondary TGCs (at E7-7.5 post-implantation stages) were used in our lab to study the X-chromosome inactivation patterns of an extra-embryonic lineage which is constituted of TGCs at post-implantation stages. Extra-embryonic development in rodents depends on the differentiation of TGCs. Indeed, TGCs are essential for placental and thus embryonic development. Defects in TGC differentiation cause embryonic lethality (for review see reference15). Transcriptional activity of TGCs using RNA FISH allowed us to demonstrate an unusual X chromosome inactivation status of such cell type during mouse development3.
The methods presented here to obtain and study secondary TGCs can be applied to study molecular pathways in development of the important extra-embryonic tissues they are part of, both in normal and mutant mice. These approaches could be adapted to investigate TGC development in other mammals. Our analysis involved the use of wild type mouse embryos allowing us to assess the transcriptional activity of various genes in in vivo TGCs and in in vitro TGCs derived from EPC explant. This method could be extended to the analysis of transgenic mice and/or by the addition of inhibitor molecules in the culture medium. We have successfully used this method of RNA FISH on another type of TGCs, such as primary TGCs which appear at an earlier stage of mouse development, E3.0 blastocyts, which can be individually cultured for 4-5 days during which developed outgrowth, the ICM being surrounded by large primary TGCs16,17. Nascent transcripts for different genes as well as Xist could be visualized and quantified using the same RNA FISH approach3.
Combined immunofluorescence and RNA FISH can also be performed on cryostat sections as well as in vitro cultured TGCs3. This demonstrates that the method is quite robust, as primary transcripts are highly susceptible to degradation and there is an absolute requirement of RNase free compounds. IF/RNA FISH provides additional information about transcription levels, cellular localization and protein expression, simultaneously in a given cell. Although, sections of paraffin-embedded tissue have previously been used to detect nascent RNA in human tumors while maintaining tissue morphology18, in our hands, cryostat sections are more appropriate for preserving both the nascent RNA and the epitopes required for detection by antibodies during immunofluorescence.
In addition to RNA FISH, following immunofluorescence, chromosomal DNA FISH can also be performed on secondary TGCs using probes labeled with fluorochromes, similar to those used for RNA FISH3. Fluorescent probes can be either plasmids/fosmids or BACs, labeled with fluorescent dUTPs as used here, and described in Chaumeil et al.,8 . Alternatively, fluorescently labeled oligonucleotides can be used19. Since DNA FISH does not require strand-specificity, oligonucleotide probes can be designed to target either of the two complementary strands in the target region. Branched DNA probes, where signal amplification is achieved by two sequential rounds of specific and amplifier probes can also be used to enhance the signal-to-noise ratio of FISH signals20. Alternatively, RNA probes such as riboprobes can be used although in our DNA-based probes guarantee a better tradeoff between signal quality, specificity and ease of use.
Finally, 3D image acquisition is essential in order to obtain the required spatial information in these large cells, and can be performed using a variety of fluorescence microscopes, such as the Apotome microscope, or epifluorescence microscopes with deconvolution such as the Deltavision (GEH), or other microscopes suitable for imaging tissue sections, and large (&#62;20 &#181;m) TGCs. It should be noted that for single copy DNA loci, the punctate signal detected by nascent RNA FISH or DNA FISH may not be readily detected by confocal microscopy.
In conclusion, the methods we describe here should be useful both for the detailed analysis of TGS in a developmental context, but also in other disease situations. Many genes that are involved in TGC development and function in rodents are conserved between rodents and humans, such as transcription factors, proteases and cell adhesion molecules21. Mouse TGCs are a cell model for studying genes that regulate placental development and therefore give insights into human placental diseases. Furthermore, due to the fact that these cells are endoreplicating and since some cancer cells engage endocycle programs, in addition to gene amplification processes, the methods we describe should also be helpful in single cell investigations required to explore mechanisms leading to genome instability in cancer cells.

Mammalian trophoblast giant cells (TGCs) form a barrier between maternal and embryonic tissues. They mediate implantation and invasion of the conceptus into the uterus and play critical roles in development for the formation of the placenta. They produce several growth factors (cytokines) and hormones of the Prolactin/Placental Lactogen family and steroids, necessary for embryonic growth and survival. TGCs are large, mononucleated and polyploid cells with a cell cycle, the endocycle, that consists of alternating S and G phases. Indeed, TGCs are endoreplicating cells, able to undergo multiple rounds of DNA synthesis without any division2. In order to investigate the transcriptional status of genes in vivo, in TGCs compared to other embryonic and extraembryonic cell types, nascent RNA FISH can be performed in vivo on cryostat sections3 at specific post-implantation stages. TGCs are easily recognizable on sections due to their large size and their transcriptional gene activity can be recorded but their number at early post-implantation stages is low. Paucity of TGCs on E7 embryonic sections, led us to perform short-term cultures of embryonic trophectodermal tissues to obtain differentiated TGCs in order to study the regulation of gene expression during trophectoderm development. Moreover, in order to remain as physiological as possible, established cell lines ie trophectoderm stem cells (TS) are not always suitable to investigate developmental mechanisms Generation of secondary TGCs provide a valuable tool to study pathological pregnancies associated with defects in TGCs due to abnormal gene regulation in mice.
Trophoblast cells of the ectoplacental cone (EPC) are precursors of secondary TGCs4. Spontaneous differentiation of cultured EPCs to secondary TGCs has previously been reported5. However, in contrast to primary TGCs, studies on secondary TGC differentiation have remained limited, presumablydue to the difficulties of isolating EPC explants free of any maternal or embryonic tissues. We adapted these methods, in order to perform RNA FISH on secondary TGCs derived from individual embryo at E7, a developmental post-implantation stage where TGCs are very few but could be generated from EPC precursors. RNA FISH to analyze nuclear primary transcripts has never been done at the level of the single cell level on secondary TGCs. This allows precise analysis of transcription and was used to show the epigenetic instability of TGCs at post-implantation stages3.
A classic example of epigenetics in mammals, the X-chromosome inactivation (XCI) is studied in the Heard lab6. In this process one of the 2 X chromosomes in female is inactivated. The non-coding Xist transcript coats the X chromosome from which it is expressed in female cells and triggers silencing of most genes. Using RNA FISH, nascent transcripts of X-linked genes can be investigated as can the accumulation of Xist RNA on the inactive X chromosome (Xi). We describe here a procedure to perform RNA FISH on sections of post-implantation embryos and on short-term cultures of EPC. This protocol adapted from those that were used to study XCI in differentiating female embryonic stem cells and preimplantation embryos7-11. We provide examples of XCI in female embryos in vivo, as well as in vitro TGCs.

<table><tbody><tr><td>Stereomicroscope</td><td>Nikon</td><td>SMZ 1500</td><td></td></tr><tr><td>Stereomicroscope</td><td>Zeiss</td><td>Stemi SV6</td><td></td></tr><tr><td>Scissors Pascheff-Wolff</td><td>Moria</td><td>MC19</td><td></td></tr><tr><td>Dumont #5 forceps</td><td>Roth</td><td>PK78.1</td><td></td></tr><tr><td>4-well tissue culture dishes&amp;nbsp;</td><td>Nunc</td><td>176740</td><td></td></tr><tr><td>60 mm Petri dishes Falcon</td><td>Dutsher, France</td><td>353004</td><td></td></tr><tr><td>100 mm Petri dishes Falcon</td><td>Dutsher, France</td><td>353003</td><td></td></tr><tr><td>Coverslips 18 mm x 18 mm&amp;nbsp;</td><td>VWR</td><td>631-1331</td><td></td></tr><tr><td>Coverslips 22 mm x 22 mm</td><td>VWR</td><td>631-0125</td><td></td></tr><tr><td>12 mm glass round coverslips&amp;nbsp;</td><td>Harvard apparatus</td><td>64-0712</td><td></td></tr><tr><td>Slides Superfrost plus</td><td>VWR</td><td>631-9483</td><td></td></tr><tr><td>4-slide Transport&amp;nbsp; box&amp;nbsp; Lockmailer</td><td>Dutsher, France</td><td>40684</td><td></td></tr><tr><td>Cryotubes 1.8 ml Corning&amp;nbsp;</td><td>Fisher Science</td><td>10418571</td><td></td></tr><tr><td>Glass Coplin staining jars</td><td>Fischer Scientific</td><td>W1561L</td><td></td></tr><tr><td>TissueTek O.C.T compound</td><td>VWR</td><td>4583</td><td></td></tr><tr><td>RPMI 1640 medium</td><td>Invitrogen</td><td>61870</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>Sigma-Aldrich</td><td>D1408</td><td>10x&amp;nbsp; is used</td></tr><tr><td>Water&amp;nbsp;</td><td>Sigma-Aldrich</td><td>W3500</td><td></td></tr><tr><td>Paraformaldehyde&amp;nbsp;</td><td>Panreac Quimica, Spain</td><td>141451</td><td>3% in PBS</td></tr><tr><td>Triton-X-100&amp;nbsp;&amp;nbsp;</td><td>Euromedex</td><td>&amp;nbsp;2000-A</td><td>0.5% final</td></tr><tr><td>Vanadyl ribonucleoside complex (VRC)&amp;nbsp;</td><td>New England Biolabs, USA</td><td>S1402S</td><td></td></tr><tr><td>Sodium dextran sulfate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D8906</td><td></td></tr><tr><td>Bovine serum albumin&amp;nbsp; (BSA)</td><td>New England Biolabs, USA</td><td>B9001S</td><td></td></tr><tr><td>Formamide</td><td>Sigma-Aldrich</td><td>47671-1L-F</td><td>&amp;nbsp;aliquots kept at -20 &amp;deg;C</td></tr><tr><td>Illustra TempliPhi&amp;nbsp; Kit Construct (Kit MDA)</td><td>Dutsher, France</td><td>25-6400-80</td><td></td></tr><tr><td>Nick translation kit</td><td>Abbott, USA</td><td>07J00-001</td><td></td></tr><tr><td>20x SSC buffer concentrate</td><td>Sigma-Aldrich</td><td>&amp;nbsp;S6639</td><td></td></tr><tr><td>Spectrum green dUTP&amp;nbsp;</td><td>Abbott, USA</td><td>02N32-050</td><td></td></tr><tr><td>Spectrum red dUTP&amp;nbsp;</td><td>Abbott, USA</td><td>02N34-050</td><td></td></tr><tr><td>Cy-5 dUTP&amp;nbsp;</td><td>Dutsher, France</td><td>PA55022</td><td></td></tr><tr><td>Mouse Cot-1 DNA&amp;nbsp;</td><td>Invitrogen</td><td>18440016</td><td></td></tr><tr><td>DNA, MB grade</td><td>Invitrogen</td><td>Roche</td><td>DNA from fish sperm</td></tr><tr><td>4&amp;prime;,6-diamidino-2-phenylindole dihydrochloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D9564</td><td>DAPI</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G9012</td><td></td></tr><tr><td>p-phenylenediamine</td><td>Sigma-Aldrich</td><td>695106</td><td></td></tr><tr><td>Centrifuge 5417R</td><td>Eppendorf, Germany</td><td></td><td>molecular biology grade</td></tr><tr><td>Eppendorf concentrator plus</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Eppendorf Thermomixer comfort</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>Liquiport Liquid pump</td><td>KNF Neuberger, Trenton, USA</td><td></td><td></td></tr><tr><td>Shake&#039;N&#039;Bake Hybridization oven</td><td>Boekel Scientific, USA</td><td></td><td></td></tr><tr><td>Cryostat&amp;nbsp;</td><td>Leica</td><td>CM3050</td><td></td></tr></tbody></table>

transcriptional analysis by nascent rna fish of in vivo trophoblast giant cells or in vitro short-term cultures of ectoplacental cone explants

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells differentiate into primary trophoblast giant cells (TGCs) while the polar trophectoderm overlying the inner cell mass continues to proliferate later differentiating into secondary TGCs. TGCs play a key role in developing placenta and are essential for a successful pregnancy. Investigation of transcriptional regulation of specific genes during post-implantation development can give insights into TGCs development. Cells of the ectoplacental cone (EPC) from embryos at 7-7.5 days of gestation (E7-7.5), derived from the polar trophectoderm, differentiate into secondary TGCs1. TGCs can be studied in situ, on cryostat sections of embryos at E7 although the number of TGCs is very low at this stage. An alternative means of analyzing secondary TGCs is to use short-term cultures of individual EPCs from E7 embryos. We propose a technique to investigate the transcriptional status of genes of interest both in vivo and in vitro at the single-cell level using fluorescent in situ hybridization (RNA FISH) to visualize nascent transcripts. This technique provides a direct readout of gene expression and enables assessment of the chromosomal status of TGCs, which are large endoreplicating cells. Indeed, a key feature of terminal differentiation of TGCs is that they exit the cell cycle and undergo multiple rounds of endoreplication.This approach can be applied to detect expression of any gene expressed from autosomes and/or sex chromosomes and can provide important information into developmental mechanisms as well as placental diseases.

TGCs can be identified on E7 sections upon DAPI staining due to their localization in the conceptus and their large size. This is illustrated in Figure 1A on a longitudinal section. RNA FISH was performed on such embryonic sections in order to study X chromosome inactivation in this extra-embryonic lineage.
Several slides or coverslips can be processed at the same time. By using different dyes to label RNA of different genes, one can detect different primary transcripts in the same nucleus. At least 2 probes can be mixed together, for instance Xist coupled to SG (green signal) and Atrx coupled to SR (red signal). An example of a female TGC is shown in Figure 1B where 2 other lineages are represented, the embryo proper (E) and the visceral endoderm (VE). A TGC nucleus is shown with Xist RNA covering the X chromosome which is inactivated (Xi) while the other X chromosome which is active (Xa) displays a few pinpoints. X-linked gene Atrx primary transcripts are expressed from the active X chromosome (not decorated by Xist) in several copies due to endoreplication. This illustrates monoallelic expression e.g., inactivation of Atrx on one X chromosome.
Since TGCs are heterogeneous in size as illustrated in Figure 2B, only the largest ones are recorded. Three probes can be also used as illustrated in Figure 2C for secondary TGCs. In this case, Xist-SG (green), two X-linked genes: G6pd-SR (red) and Huwe1-Cy (magenta) were analyzed at the same time in the same nucleus. While G6pd is monoallelically expressed as shown here (and previously shown in secondary TGCs3) Huwe1 is biallelically expressed demonstrating its property to escape XCI. Endoreplication, with several pinpoints i.e., nascent transcripts copies, is also evident.
<img alt="Figure 1" src="/files/ftp_upload/54386/54386fig1.jpg" /> 
Figure 1. Primary transcript expression in TGCs from E7 female embryonic section. (A) Longitudinal section of the E7 conceptus stained with DAPI. The embryo and extra-embryonic tissues are surrounded by the mother tissue, the decidua. The localization of the different lineages is done upon DAPI staining with 5X objective. TGCs are identified due to their large size. Ch, chorion, E, embryo, EPC, ectoplacental cone, VE, visceral endoderm, TGC, trophoblast giant cells. Scale bar = 100 &#181;m. (B) Higher magnification of the boxed-in area in A (63X objective) RNA FISH. Atrx primary transcript in red and Xist RNA in green are visualized on the 3 lineages = E, VE, TGC. Scale bar = 10 &#181;m. Example of an in vivo TGC where Atrx is monoallelically expressed (Xa, arrowhead) and silent on the Xist-coated X chromosome (Xi); several Atrx signals seen on the Xa are due to endoreplication. Atrx = BAC clone RP23-260I15. <a href="https://www.jove.com/files/ftp_upload/54386/54386fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54386/54386fig2.jpg" /> 
Figure 2. Primary transcript expression in secondary TGCs derived from EPC. (A) General view of growing secondary TGCs (x5 objective). Scale bar = 100 &#181;m. (B) Asterisk indicate example of TGCs according to their size (10X objective). Scale bar = 60 &#181;m. (C) Example of a secondary female TGC RNA FISH with the use of 3 probes (Xist domain in green, covering the inactivated X chromosome: Xi) showing endoreplication of G6pd and Huwe1 primary transcripts (several pinpoints, in red and magenta). While G6pd is expressed monoallelically, Huwe1 expression is biallelic in this nucleus. Huwe1 = BAC clone RP24-157H12 ; G6pd = BAC clone RP23-13D21. Xi = arrow; Xa = arrowhead (63X objective). Scale bar = 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/54386/54386fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Transcriptional  Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants at JoVE.com

Transcriptional  Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

Trophoblast giant cells (TGCs) play a key role in the placenta to ensure a healthy pregnancy. We present a protocol for assessing the transcriptional status of genes in TGCs by nascent fluorescent in situ hybridization on cryostat sections of post-implantation embryos or short-term cultures of embryonic day 7 ectoplacental cones.

Transcriptional Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

The placenta derives from one extra-embryonic lineage, the trophectoderm. In the peri-implantation murine blastocyst, mural trophectoderm cells ...

transcriptional-analysis-nascent-rna-fish-vivo-trophoblast-giant

Research

JoVE Journal

Developmental Biology

7.8K Views.  Institut Curie, PSL Research University, CNRS UMR 3215, INSERM U934. Trophoblast giant cells (TGCs) play a key role in the placenta to ensure a healthy pregnancy. We present a protocol for assessing the transcriptional status of genes in TGCs by nascent fluorescent in situ hybridization on cryostat sections of post-implantation embryos or short-term cultures of embryonic day 7 ectoplacental cones. 

Video: Transcriptional  Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

Genetics

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density centrifugation, media gradient isolation and immunomagnetic purification. As observed in vivo, mononucleated villous cytotrophoblast cells in primary culture differentiate into multinucleated syncytiotrophoblast cells after 72 hr. Compared to normoxia (8% O2), villous cytotrophoblast cells that undergo hypoxia/reoxygenation (0.5% / 8% O2) undergo increased oxidative stress and intrinsic apoptosis, similar to that observed in vivo in pregnancy complications such as preeclampsia, preterm birth, and intrauterine growth restriction. In this context, primary villous trophoblasts cultured under hypoxia/reoxygenation conditions represent a unique experimental system to better understand the mechanisms and signalling pathways that are altered in human placenta and facilitate the search for effective drugs that protect against certain pregnancy disorders. Human villous trophoblasts produce melatonin and express its synthesizing enzymes and receptors. Melatonin has been suggested as a treatment for preeclampsia and intrauterine growth restriction because of its protective antioxidant effects. In the primary villous cytotrophoblast cell model described in this paper, melatonin has no effect on trophoblast cells in normoxic state but restores the redox balance of syncytiotrophoblast cells disrupted by hypoxia/reoxygenation. Thus, human villous trophoblast cells in primary culture are an excellent approach to study the mechanisms behind the protective effects of melatonin on placental function during hypoxia/reoxygenation.

This manuscript presents a unique in vitro model of immunopurified human villous cytotrophoblast cells cultured under hypoxia/reoxygenation. This model is suitable to study the protective effects of promising treatments, such as melatonin, on pregnancy complications associated with increased oxidative stress and altered placental function.

This protocol describes how villous cytotrophoblast cells are isolated from placentas at term by successive enzymatic digestions, followed by density ...

Protocol for the Direct Conversion of Murine Embryonic Fibroblasts into Trophoblast Stem Cells

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining the ability to self-renew and to differentiate into all subtypes of the trophoblast lineage, equivalent to the in vivo stem cell population giving rise to the fetal portion of the placenta. Therefore, TSCs offer a unique model to study placental development and embryonic versus extra-embryonic cell fate decision in vitro. From the blastocyst stage onwards, a distinct epigenetic barrier consisting of DNA methylation and histone modifications tightly separates both lineages. Here, we describe a protocol to fully overcome this lineage barrier by transient over-expression of trophoblast key regulators Tfap2c, Gata3, Eomes and Ets2 in murine embryonic fibroblasts. The induced trophoblast stem cells are able to self-renew and are almost identical to blastocyst derived trophoblast stem cells in terms of morphology, marker gene expression and methylation pattern. Functional in vitro and in vivo assays confirm that these cells are able to differentiate along the trophoblast lineage generating polyploid trophoblast giant cells and chimerizing the placenta when injected into blastocysts. The induction of trophoblast stem cells from somatic tissue opens new avenues to study genetic and epigenetic characteristics of this extra-embryonic lineage and offers the possibility to generate trophoblast stem cell lines without destroying the respective embryo.

Here we present a protocol for the direct conversion of murine embryonic fibroblasts into fully functional and stable trophoblast stem cells by ten day over-expression of Tfap2c, Gata3, Eomes and Ets2.

Trophoblast stem cells (TSCs) arise as a consequence of the first cell fate decision in mammalian development. They can be cultured in vitro, retaining ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Mouse In Vivo Placental Targeted CRISPR Manipulation

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients and waste between the mother and fetus and the production and delivery of growth factors and hormones. Placental genetic manipulations in mice are critical for understanding the placenta's specific role in prenatal development. Placental-specific Cre-expressing transgenic mice have varying effectiveness, and other methods for placental gene manipulation can be useful alternatives. This paper describes a technique to directly alter placental gene expression using CRISPR gene manipulation, which can be used to modify the expression of targeted genes. Using a relatively advanced surgical approach, pregnant dams undergo a laparotomy on embryonic day 12.5 (E12.5), and a CRISPR plasmid is delivered by a glass micropipette into the individual placentas. The plasmid is immediately electroporated after each injection. After dam recovery, the placentas and embryos can continue development until assessment at a later time point. The evaluation of the placenta and offspring after the use of this technique can determine the role of time-specific placental function in development. This type of manipulation will allow for a better understanding of how placental genetics and function impact fetal growth and development in multiple disease contexts.

Mouse In Vivo Placental Targeted CRISPR Manipulation

Here we describe a time-specific method to effectively manipulate critical developmental pathways in the mouse placenta in vivo. This is performed through the injection and electroporation of CRISPR plasmids into the placentas of pregnant dams on embryonic day 12.5.

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Biochemistry

Analyzing Angiogenesis: In Vitro Assay for Tube Formation on BM Matrix

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of mesenchymal to endothelial-like transition. Traditionally, this process is evaluated by an in vitro tube-formation assay, where the cells organize themselves into tube-like structures when seeded over a polymerized basement membrane preparation. Although several structural features can be measured in photomicrographs of the structures, to assess the real commitment of EVT to the endothelial-type phenotype, biochemical analysis of cell extracts is required. Scraping the cells from the culture dish to obtain RNA and/or protein extracts is not an alternative since the tube-like structures are severely contaminated by the bulk of proteins from the polymerized basement membrane. Thus, a strategy to separate the cells from the basement membrane proteins prior to the preparation of cell extracts is needed. Here, a simple, fast, and cost-effective method to recover the tube-like structures from the in vitro tube-formation assay and the subsequent analysis by biochemical techniques is presented. Tube-like structures formed by HTR8/SVneo cells, an EVT cell line, were liberated from the polymerized basement membrane by a short incubation with PBS supplemented with ethylenediaminetetraacetic acid (EDTA). After serial washes, a ready-to-use pellet of purified tube-like structures can be obtained. This pellet can be subsequently processed to obtain RNA and protein extracts. qPCR analysis evidenced the induced expression of VE-cadherin and alphav-integrin, two endothelial cell markers, in EVT-derived tube-like structures compared to control cells, which was consistent with the induction of the endothelial cell marker, CD31, evaluated by immunofluorescence. Western blot analysis of the tube-like structures' protein extracts revealed the overexpression of RECK in transfected HTR8/SVneo cells. Thus, this simple method allows to obtain cell extracts from the in vitro tube-formation assay for the subsequent analysis of RNAs and protein expression.

The present protocol allows the recovery of tube-like structures formed in the classical in vitro angiogenesis-tube formation assay where cells, such as trophoblast cells, organize in capillary-like structures over a layer of extracellular basement membrane matrix. The recovered structures are used for RNA and protein isolation for various biochemical analyses.

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Biology

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Transcriptional Analysis by Nascent RNA FISH of In Vivo Trophoblast Giant Cells or In Vitro Short-term Cultures of Ectoplacental Cone Explants

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