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.

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			<td height="21" style="height:21px;">Cryotubes 1.8 ml Corning&#160;</td>
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			<td>10418571</td>
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			<td>Invitrogen</td>
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			<td>Sigma-Aldrich</td>
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			<td>10x&#160; is used</td>
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			<td>W3500</td>
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			<td height="21" style="height:21px;">Paraformaldehyde&#160;</td>
			<td>Panreac Quimica, Spain</td>
			<td style="width:80px;">141451</td>
			<td>3% in PBS</td>
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			<td height="21" style="height:21px;">Triton-X-100&#160;&#160;</td>
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			<td>0.5% final</td>
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			<td height="21" style="height:21px;">Vanadyl ribonucleoside complex (VRC)&#160;</td>
			<td>New England Biolabs, USA</td>
			<td>S1402S</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Sodium dextran sulfate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D8906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin&#160; (BSA)</td>
			<td>New England Biolabs, USA</td>
			<td>B9001S</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Formamide</td>
			<td>Sigma-Aldrich</td>
			<td>47671-1L-F</td>
			<td>&#160;aliquots kept at -20&#176;C</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Illustra TempliPhi&#160; Kit Construct (Kit MDA)</td>
			<td>Dutsher, France</td>
			<td>25-6400-80</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Nick translation kit</td>
			<td style="width:195px;">Abbott, USA</td>
			<td>07J00-001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">20x SSC buffer concentrate</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;S6639</td>
			<td></td>
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			<td height="21" style="height:21px;">Spectrum green dUTP&#160;</td>
			<td>Abbott, USA</td>
			<td>02N32-050</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Spectrum red dUTP&#160;</td>
			<td>Abbott, USA</td>
			<td>02N34-050</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Cy-5 dUTP&#160;</td>
			<td>Dutsher, France</td>
			<td>PA55022</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Mouse Cot-1 DNA&#160;</td>
			<td>Invitrogen</td>
			<td>18440016</td>
			<td></td>
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			<td height="21" style="height:21px;width:297px;">DNA, MB grade</td>
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			<td>Roche</td>
			<td>DNA from fish sperm</td>
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			<td height="21" style="height:21px;">4&#8242;,6-diamidino-2-phenylindole dihydrochloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>D9564</td>
			<td>DAPI</td>
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			<td height="21" style="height:21px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G9012</td>
			<td></td>
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			<td height="21" style="height:21px;">p-phenylenediamine</td>
			<td>Sigma-Aldrich</td>
			<td>695106</td>
			<td></td>
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			<td height="21" style="height:21px;">Centrifuge 5417R</td>
			<td>Eppendorf, Germany</td>
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			<td>molecular biology grade</td>
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			<td height="21" style="height:21px;">Eppendorf concentrator plus</td>
			<td>Eppendorf</td>
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			<td height="21" style="height:21px;">Eppendorf Thermomixer comfort</td>
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			<td height="21" style="height:21px;">Liquiport Liquid pump</td>
			<td colspan="2">KNF Neuberger, Trenton, USA</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Shake&#39;N&#39;Bake Hybridization oven</td>
			<td>Boekel Scientific, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Cryostat&#160;</td>
			<td style="width:195px;">Leica</td>
			<td style="width:80px;">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

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the maternal circulation. The maternal uterine vasculature needs to adapt to this temporary demand and the success of this arterial remodeling process has implications for fetal growth. Cells of the maternal immune system, especially natural killer (NK) cells, play a critical role in this process. Here we describe a method to assess the degree of remodeling of maternal spiral arteries during mouse pregnancy. Hematoxylin and eosin-stained tissue sections are scanned and the size of the vessels analysed. As a complementary validation method, we also present a qualitative assessment for the success of the remodeling process by immunohistochemical detection of smooth muscle actin (SMA), which normally disappears from within the arterial vascular media at mid-gestation. Together, these methods enable determination of an important parameter of the pregnancy phenotype. These results can be combined with other endpoints of mouse pregnancy to provide insight into the mechanisms underlying pregnancy-related complications.

This protocol describes a technique to assess changes in the maternal vasculature during pregnancy in mice. Using stereological methods, remodeling of the decidual spiral arteries is assessed quantitatively and the results confirmed qualitatively using immunohistochemistry.

The placenta mediates the exchange of factors such as gases and nutrients between mother and fetus and has specific demands for supply of blood from the ...

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.

Spatial and Temporal Analysis of Active ERK in the C. elegans Germline

We present an immunofluorescence&#160;imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.

The evolutionarily conserved&#160;extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

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 ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We quantitatively assessed follicle survival, measured follicle and oocyte diameters, and examined ultrastructure of the oocytes produced from the system. We observed decreased follicle survival, downregulation of expressions of proliferating cell nuclear antigen and growth differentiation factor 9, as indicators for the development of granulosa cells and oocytes, respectively, and oocyte ultrastructural abnormalities under the simulated microgravity condition. The simulated microgravity experimental setup needs to be optimized to provide a model for investigation of the mechanisms involved in the oocyte/follicle in vitro development.

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

A highly promising technique to generate tissue constructs without using matrix is to culture cells in a simulated microgravity condition. Using a rotary culture system, we examined ovarian follicle growth and oocyte maturation in terms of follicle survival, morphology, growth, and oocyte function under the simulated microgravity condition.

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...