Name: Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme (Video) | JoVE Journal
Uploaded: 2013-04-12T13:00:00.000+00:00
Duration: 8 min 36 s
Description: Learn to separate mouse embryonic facial ectoderm and mesenchyme using Dispase II treatment. This protocol enables detailed studies of facial development and congenital disorder mechanisms.

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5 minutes

The authors would like to thank Irene Choi for illustrating embryo heads in Fig 1 and the other members of the laboratory for help and discussion. This work is supported by NIH grant DE012728 (T.W.)

1. Prepare Dispase II
<ol>
	<li>Prepare fresh Hepes-buffered Saline (HBS) (50 mM Hepes/KOH pH 7.4, 150 mM NaCl). Add 2 ml 0.5 M Hepes/KOH pH 7.4 and 1.2 ml 2.5 M NaCl into 16.8 ml Ultrapure H2O.</li>
	<li>Make 10 mg/ml Dispase II. Weigh 0.2 g Dispase II (Roche, Cat# 4942078001) and dissolve in 20 ml HBS. Prepare 1.0 ml aliquots in individual Eppendorf tubes and store at -20 &deg;C for long-term storage.</li>
	<li>Dilute 10 mg/ml Dispase II 1:10 in PBS before use. Put the diluted Dispase II on ice.</li>
</ol>
2. Dissect CD-1 E10.5 Embryos from Pregnant Female
All animal experiments were performed in accordance with protocols approved by the University of Colorado Denver (UCD) Animal Care and Usage Committee. CD-1 (ICR) mice were obtained from Harlan Laboratories (Indianapolis, IN).
<ol>
	<li>Clean the dissection area and tools with 70% ethanol.</li>
	<li>Euthanize pregnant CD-1 female using approved protocols.</li>
	<li>Spray the abdomen with 70% ethanol then open the abdomen. Use one set of forceps to pull the uterus and another to tear away the mesometrium from the uterus. Remove the uterus from the body. Repeat for uterine horn on other side of the abdomen.</li>
	<li>Briefly rinse the uterus with ice cold PBS in a 10-cm Petri dish to wash out blood.</li>
	<li>Transfer the uterus into a new Petri dish with ice cold PBS. Separate into one-embryo fragments by cutting between implantation sites.</li>
	<li>Transfer one embryo into ice-cold PBS under a stereomicroscope. Use fine forceps to tear off the muscular wall of uterus starting at one of the cut sites to expose the embryo with the yolk sac. Peel off the yolk sac and amnion. Transfer dissected embryo to a 6-cm Petri dish with PBS on ice. Proceed until all embryos have been collected in this way.</li>
</ol>
3. Dispase II Treatment
<ol>
	<li>Wash all the embryos with PBS in a 6-cm Petri dish. Replace PBS with 10 ml diluted Dispase II in PBS. Cover the Petri dish and incubate at 37 &deg;C for 25 min - a bacterial incubator or warm room should suffice - we have not used a CO2 tissue culture incubator for this step.</li>
	<li>Take the Petri dish out and observe the embryo under a stereomicroscope. The ectoderm should have become loose, typified by a clear gap between this tissue layer and the underlying mesenchyme. If not, incubate for an additional 5 min at 37 &deg;C. Then put the Petri dish on ice.</li>
</ol>
4. Dissect out the Intact Facial Prominences
<ol>
	<li>Use a disposable glass pipette (VWR, Cat# 14672-380) to transfer one Dispase II treated embryo into ice-cold PBS in a 6-cm Petri dish under a stereomicroscope.</li>
	<li>Use one pair of forceps to hold the embryo. Use another pair of fine forceps carefully to cut the boundaries of the facial prominences attached to head (Figure 1 shows the boundaries of the facial prominences). Start from one side of the MnP, then the MxP, and last the FNP. Do the same to the other side to dissect the facial prominences out intact (Figure 2A).</li>
</ol>
5. Separate Facial Ectoderm from Mesenchyme
<ol>
	<li>Observe the facial prominences under a stereomicroscope with 3x working distance. Use a long glass Pasteur pipette to gently pipette the facial prominences up and down 5 times. After this, the ectoderm is easy to peel off.</li>
	<li>Use one pair of forceps to hold the facial prominences. Use the other pair of forceps to peel off the ectoderm very slowly. Figure 2B shows the separated sheets of facial ectoderm.</li>
	<li>Use a new long glass Pasteur pipette to transfer the facial ectoderm into a 1.5 ml Eppendorf tube with PBS on ice. Transfer the mesenchyme to another 1.5 ml Eppendorf tube with PBS on ice.</li>
	<li>Use new ice cold PBS for each dissection. Collect samples.</li>
	<li>Wash the samples with PBS. Centrifuge at 4 &deg;C, 500 x g for 3 min.</li>
	<li>Aspirate the PBS using long glass Pasteur pipettes. If the samples are to be used for ChIP assay, proceed to step 6.1. For RNA extraction, proceed to step 6.2. For Protein extraction, proceed to step 6.3.</li>
</ol>
6. Process Samples for Further Experimentation
<ol>
	<li>Samples for ChIP assay
	<ol>
		<li>Add 1 ml PBS with 1% formaldehyde to crosslink. Homogenize the samples.</li>
		<li>Incubate at room temperature for 10 min with rotation.</li>
		<li>Add 110 &mu;l of 1.25 M Glycine to each tube to quench unreacted formaldehyde.</li>
		<li>Mix and incubate at room temperature for 5 min.</li>
		<li>Spin at 500 x g at 4 &deg;C for 3 min. Remove supernatant.</li>
		<li>Add 1 ml cold PBS to wash the tissues.</li>
		<li>Spin at 500 x g at 4 &deg;C for 3 min.</li>
		<li>Remove PBS and repeat PBS wash once.</li>
		<li>Remove supernatant, Snap freeze the tissues in liquid nitrogen. Store the samples at -80 &deg;C for subsequent pooling and processing.</li>
	</ol>
	</li>
	<li>Samples for RNA extraction
	<ol>
		<li>Place the facial ectoderm and mesenchyme in tubes containing RNAlater (Ambion).</li>
		<li>Incubate the samples in RNAlater Solution overnight at 4 &deg;C to allow thorough penetration of the tissue.</li>
		<li>Store at -20 &deg;C for subsequent pooling and processing.</li>
	</ol>
	</li>
	<li>Samples for protein extraction</li>
</ol>
Add lysis buffer to the sample for subsequent processing. Or snap freeze the tissues in liquid nitrogen. Store the samples at -80 &deg;C for subsequent pooling and processing.
Notes
<ol>
	<li>Time constraints. Plan on 4-6 hr from the start of dissection through to the beginning of section 6.</li>
	<li>When proficient, it takes ~10-15 min per embryo to perform sections 4 and 5 (prominence dissection and ectoderm separation).</li>
	<li>If the samples are for RNA isolation, every solution should be treated with Diethylpyrocarbonate first. Also clean the forceps and the working area with RNase Away.</li>
	<li>Keep the samples on ice at all times except for the Dispase II treatment and dissections. Change to new ice cold PBS for each dissection to make sure the PBS used for dissection is cold.</li>
	<li>Sharp forceps are critical to obtain precise dissection. Clean the forceps completely to prevent any contamination.</li>
	<li>For collecting samples from different prominences, dissect each prominence separately after Dispase II treatment. Then separate the ectoderm and mesenchyme.</li>
	<li>After treatment with Dispase II, the facial ectoderm is easy to peel off. Pay special attention to contamination from other ectoderm. Discard all potentially contaminated tissue.</li>
	<li>Once you start, don't stop until the samples reach the appropriate point in Section 6. Finish separation of facial ectoderm and mesenchyme for all the embryos as quickly as you can.</li>
	<li>The enzyme activities of Dispase II might be different between batches and brands. It is necessary to adjust the treatment time for different brands and batches of Dispase II.</li>
</ol>

<ol><li>Wilkie, A. O., Morriss-Kay, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Genetics+of+craniofacial+development+and+malformation%5BTitle%5D)+AND+%22Nat.+Rev.+Genet%22%5BJournal%5D)">Genetics of craniofacial development and malformation.</a> Nat. Rev. Genet. 2, 458-468 (2001).</li>
<li>Gritli-Linde, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chapter+2+-+The+Etiopathogenesis+of+cleft+lip+and+cleft+palate%3A+usefulness+and+caveats+of+mouse+models%5BTitle%5D)+AND+%22Current+Topics+in+Developmental+Biology%22%5BJournal%5D)">Chapter 2 - The Etiopathogenesis of cleft lip and cleft palate: usefulness and caveats of mouse models.</a> Current Topics in Developmental Biology. 84, 37-138 (2008).</li>
<li>Schutte, B. C., Murray, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+many+faces+and+factors+of+orofacial+clefts%5BTitle%5D)+AND+%22Human+Molecular+Genetics%22%5BJournal%5D)">The many faces and factors of orofacial clefts.</a> Human Molecular Genetics. 8, 1-7 (1999).</li>
<li>Chai, Y., Maxson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+advances+in+craniofacial+morphogenesis%5BTitle%5D)+AND+%22Dev.+Dyn%22%5BJournal%5D)">Recent advances in craniofacial morphogenesis.</a> Dev. Dyn. 235, 2353-2375 (2006).</li>
<li>Jiang, R., Bush, J. O., Lidral, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+the+upper+lip%3A+Morphogenetic+and+molecular+mechanisms%5BTitle%5D)+AND+%22Dev.+Dyn%22%5BJournal%5D)">Development of the upper lip: Morphogenetic and molecular mechanisms.</a> Dev. Dyn. 235, 1152-1166 (2006).</li>
<li>Reid, B. S., Yang, H., Melvin, V. S., Taketo, M. M., Williams, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ectodermal+WNT%2F%CE%B2-catenin+signaling+shapes+the+mouse+face%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Ectodermal WNT/β-catenin signaling shapes the mouse face.</a> Developmental Biology. 349, 261-269 (2011).</li>
<li>Feng, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spatial+and+temporal+analysis+of+gene+expression+during+growth+and+fusion+of+the+mouse+facial+prominences%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Spatial and temporal analysis of gene expression during growth and fusion of the mouse facial prominences.</a> PLoS ONE. 4, e8066(2009).</li>
<li>Chia, R., Achilli, F., Festing, M. F., Fisher, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+origins+and+uses+of+mouse+outbred+stocks%5BTitle%5D)+AND+%22Nat.+Genet%22%5BJournal%5D)">The origins and uses of mouse outbred stocks.</a> Nat. Genet. 37 (11), 1181-1186 (2005).</li>
<li>Nagai, T., Aruga, J., Takada, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+expression+of+the+mouse+Zic1%2C+Zic2%2C+and+Zic3+gene+suggests+an+essential+role+for+Zic+genes+in+body+pattern+formation%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">The expression of the mouse Zic1, Zic2, and Zic3 gene suggests an essential role for Zic genes in body pattern formation.</a> Developmental Biology. 182, 299-313 (1997).</li>
<li>Britsch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+transcription+factor+Sox10+is+a+key+regulator+of+peripheral+glial+development%5BTitle%5D)+AND+%22Genes+Dev%22%5BJournal%5D)">The transcription factor Sox10 is a key regulator of peripheral glial development.</a> Genes Dev. 15 (1), 66-78 (2001).</li>
</ol>

The authors have no competing interests or other conflicts associated with the contents of this article.

This protocol provides a straightforward method to separate embryonic mouse facial ectoderm and mesenchyme based on an initial Dispase II treatment step. In previous studies, we have performed dissection of the facial prominences prior to Dispase II treatment, but we have consistently found that the mesenchyme becomes "sticky" and more difficult to manipulate. Our new protocol avoids this problem. Combination Dispase II treatment with gentle physical force by pipetting up and down makes separation of ectoderm and mesench...

<table><tbody><tr><td>Dispase II (neutral protease, grade II)</td><td>Roche</td><td>4942078001</td><td>Make 10 mg/ml Dispase II stock solution in HBS. Aliquot and store at -20 &amp;deg;C . </td></tr><tr><td>RNAlater</td><td>Ambion</td><td>AM7021</td><td></td></tr></tbody></table>

separation of mouse embryonic facial ectoderm and mesenchyme

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Orofacial clefts are the most frequent craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal facial development3. In human and mouse, initial growth and patterning of the face relies on several small buds of tissue, the facial prominences4,5. The face is derived from six main prominences: paired frontal nasal processes (FNP), maxillary prominences (MxP) and mandibular prominences (MdP). These prominences consist of swellings of mesenchyme that are encased in an overlying epithelium. Studies in multiple species have shown that signaling crosstalk between facial ectoderm and mesenchyme is critical for shaping the face6. Yet, mechanistic details concerning the genes involved in these signaling relays are lacking. One way to gain a comprehensive understanding of gene expression, transcription factor binding, and chromatin marks associated with the developing facial ectoderm and mesenchyme is to isolate and characterize the separated tissue compartments.
Here we present a method for separating facial ectoderm and mesenchyme at embryonic day (E) 10.5, a critical developmental stage in mouse facial formation that precedes fusion of the prominences. Our method is adapted from the approach we have previously used for dissecting facial prominences7. In this earlier study we had employed inbred C57BL/6 mice as this strain has become a standard for genetics, genomics and facial morphology8. Here, though, due to the more limited quantities of tissue available, we have utilized the outbred CD-1 strain that is cheaper to purchase, more robust for husbandry, and tending to produce more embryos (12-18) per litter than any inbred mouse strain8. Following embryo isolation, neutral protease Dispase II was used to treat the whole embryo. Then, the facial prominences were dissected out, and the facial ectoderm was separated from the mesenchyme. This method keeps both the facial ectoderm and mesenchyme intact. The samples obtained using this methodology can be used for techniques including protein detection, chromatin immunoprecipitation (ChIP) assay, microarray studies, and RNA-seq.

After Dispase II treatment, the ectoderm of the embryo tends to be loose. The facial prominences should be intact after dissection (Figure 2A). The isolated facial ectoderm is clear and free of mesenchyme tissue (Figure 2B). To determine the efficacy of the protocol and to detect cross contamination we assayed for forebrain expressed Zic39, ectodermal specific cdh15 and mesenchymal specific sox1010 using reverse transcriptase PCR. Our studies confirmed th...

A protocol for separation of embryo facial ectoderm and mesenchyme is described. We use Dispase II to treat whole embryos first, dissect whole facial prominences out, and then separate the facial ectoderm and mesenchyme.

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

The overall goal of this procedure is to separate mouse embryonic, facial ectoderm, and mechy. This is accomplished by first isolating CD one E 10.5 embryos. The second step of the procedure is to treat the whole embryos with dys.Phase two. After the enzymatic treatment, the facial prominences are isolated and the final step is to carefully separate the facial derm from mesenchyme. The isolated facial derm and mesenchyme are then ready for further experimentation.The main advantage of this technique over existing methods like dis space treatment of dissected facial prominence, is that treatment of the whole embryo with DYS space keeps both the facial accident and intact. This method can help answer key questions in the mammalian developmental biology field, such as how differential gene expression, epigenetic modification, and transcription factor binding can regulate the molecular crosstalk between facial derm and mesenchyme. During embryogenesis Plan on four to six hours between now and the isolation of the tissues, clean the dissection area and tools for 70%ethanol.And if the samples are for RNA isolation, use RNAs away. It is critical that the forceps are sharp. Additionally, for RNA isolation, the solution should be treated with ethyl percarbonate.Now euthanize a pregnant CD one female using approved protocols to collect E 10.5 embryos. Spray the abdomen with 70%ethanol, open the abdomen. Use one set of forceps to pull the uterus and another to tear the meso nutrium away from the uterus.Remove the uterus from the body and repeat the process on the opposite uterine horn. Briefly rinse the uterus with ice cold PBS in a 10 centimeter Petri dish to wash out the blood. Transfer the uterus into a new dish with ice cold PBS.Separate it into one embryo fragments by cutting it between the implantation sites. Then transfer one embryo into ice cold PBS under a stereo microscope. Starting at one of the cut sites.Use the fine forceps to tear off the muscular wall of the uterus to expose the embryo with the yolk sack. Then peel off the yolk sack and amnion. Transfer the dissected embryo to a six centimeter Petri dish with PBS on ice, and repeat the procedure until all embryos have been collected.Before treating the embryos for dys paste two, wash them with ice cold PBS in a six centimeter Petri dish. Replace the PBS with 10 milliliters of ice cold diluted dys Pastes two in PBS. Cover the dish and incubate it at 37 degrees Celsius for about 25 minutes.Adjust the time according to the enzymatic activity of the dys phase two, which can vary to confirm the digestion look under a stereo microscope and check that the derm has become loose. This is typified by a clear gap between this tissue layer and the underlying mechy. If it is not loose, incubate the tissue for another five minutes.When the digestion is complete, put the samples on ice. Keep them chilled from now onward, except during dissections after the treatment with dys. Phase two, the facial derm is easy to peel off.Use a disposable glass pipette to transfer a treated embryo into ice cold PBS in a six centimeter dish under a stereo microscope. Use forceps to hold the embryo and find forceps to carefully cut the boundaries of the facial prominences attached to the head. Pay special attention to contamination from other ectoderm, discard, or potentially contaminated tissue to dissect the facial prominences out intact.Start the dissection from one side First, dissect under the MNP. Then proceed with the MXP and the FNP. Repeat this process from the other side to complete the isolation at this point if needed, separate and isolate the three facial prominences with all the tissues readied under three x magnification.Separate the facial derm and mechy as quickly as possible. First, use a long glass pasta pipette to gently pipette the facial prominences up and down five times. The ectoderm is now easily removed.Use one forceps to hold the facial prominences and a second forceps to peel them off the ectoderm very slowly. This is the most difficult step of this procedure. Makes certain that the forceps are very sharp.And when gently peeling the odum from the magic climb, do it very slowly. Also, always adjust your position as necessary. Using a new long glass pasta pipette.Transfer the facial derm into a 1.5 milliliter tube with fresh PBS on ice. Transfer the mesenchyme to another 1.5 milliliter tube with fresh PBS on ice. Now wash the samples with PBS next centrifuge.The samples of four degrees Celsius of 500 G and for three minutes aspirate the PBS using long glass pasta pipettes. The samples can now be processed for a chip assay or RNA extraction or protein extraction. After the dys phases two treatment, the ectoderm of the embryo tends to be loose.The facial prominences should remain intact after the dissection. The isolated facial ectoderm should be clear and free of mechy tissue to determine the efficacy of the protocol. Cross-contamination was tested for on the molecular level.RT PCR was performed on each tissue for the following, for brain express sick. Three, ODEM specific CD CDH one and Mechy specific. So 10, the results confirmed that the tissues expressed the expected genes and were free of cross-contamination.While attempting these procedure, it's important to remember to keep the samples on eyes at all times except for the DASE two treatment and dissections After its development. This technique paved the way for researchers in the field of craniofacial development to explore differential gene expression, molecular signal, crosstalk, and downstream target identification in embryonic facial derm and mechy with relevance to facial development and or facial clefting. Following this procedure are the methods like chipsy arming as ASIC and protein detection can be performed in order to answer additional questions like how gene expression differences in the differential transcription factor binding distinguishes the odum and amazing climb.

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Learn to separate mouse embryonic facial ectoderm and mesenchyme using Dispase II treatment. This protocol enables detailed studies of facial development and congenital disorder mechanisms.

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

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