We thank Radek Sindelka for his help, and Cas Bresilla for assisting with frog husbandry and embryo preparation. This work was funded by the NIH via the grant R01DE021109 to H.L.S. Laura Jacox was funded by the Herschel Smith Graduate Fellowship at Harvard University and an F30 individual fellowship grant F30DE022989-01 through the NIDCR.

1. Preparing Reagents
<ol>
	<li>10x MBS: Prepare 1 L of 10x Modified Barth&#39;s Saline (MBS) solution17. Refer to Table 1, Reagents, ingredients, and instructions. Use distilled water for all solutions. Mix in a beaker, using a stir bar, until full dissolution. All solutions should be made at room temperature.</li>
	<li>1x MBS: Dilute 100 ml of 10x MBS solution in 900 ml of distilled water to make 1 L of 1x MBS. Add 0.7 ml of 1 M CaCl2 solution.</li>
	<li>0.1x MBS: Dilute 1x MBS to prepare 1 L of 0.5x MBS solution and 2 L of 0.1x MBS solution. To 1 L of 0.1x MBS solution, add 1 ml of 10 mg/ml gentamycin solution. The 0.1x MBS solution with gentamycin will be used for long-term embryo culture.</li>
	<li>Ficoll/MBS: Add 15 grams of Ficoll 400 to 500 ml of 0.5x MBS solution. Mix vigorously. Add a stir bar and mix until Ficoll is fully dissolved (several hours).</li>
	<li>70% ethanol: Dilute 100% ethanol to 70% ethanol using distilled water.</li>
</ol>
2. Preparing Glass Operating Tools
<ol>
	<li>Needle preparation: Load capillary tubing into a needle puller.
	<ol>
		<li>Pull the needles according to the settings shown in Table 2: Needle puller settings. The settings are for a Sutter Instrument Co. Model P-80/PC micropipette puller and capillary tubings, as described in the Table of specific reagents and equipment. However, these settings are specific to the Sutter needle puller, and will need to be adjusted for other machines. Settings can be determined using a ramp test, as specified by the machine&#39;s manufacturer.</li>
		<li>Pull 4-6 needles in preparation for the procedure. The needles should be broken such that the flexible, hair-like portion of the glass tip is fully removed, which is typically 2-3 mm long. The tip must be relatively rigid, but still narrow enough to be used as a cutting tool. See photo of an ideal needle in Figure 1A.</li>
		<li>Store the needles in a Petri dish with a strip of clay down the center. Press the shaft of each needle into the clay, to hold it in place and to keep the fragile sharp tip away from the bottom and sides.</li>
	</ol>
	</li>
	<li>For additional tools, obtain glass cover slips, a pair of long standard pattern forceps, a Bunsen burner, and 3-4 glass Pasteur pipettes (Size 5 &#190; in).</li>
	<li>Pipette tool: To make a pipette tool, light the Bunsen burner and place the tip of a glass Pasteur pipette into the blue part of the flame while rotating it, such that the tip melts and the hole completely seals, forming a closed, rounded end. The rounded, sealed tip is later used to make depressions in the clay dish that holds the embryos during the operation. See Figure 1B.</li>
	<li>Glass bridges: To make glass bridges, use long standard pattern forceps to carefully break off 3 mm by 3 mm chunks of cover slip glass. While holding the piece of cover slip with tweezers, place the glass in the flame until all four edges soften and curve downward, forming a tiny glass dome. The edges should no longer be sharp. See Figure 1C.</li>
	<li>Store the glass cover slip bridges by inserting them, edges down, into modeling clay, lining the bottom of a Petri dish. Insert the bridges such that the top of the bridge remains above the clay surface. Do not push too hard such that the bridge breaks, and do not fully embed the bridge into the clay, because it can be difficult to remove. After sterilization with a flame or 70% ethanol, the bridges can be reused from experiment to experiment.</li>
</ol>
3. Preparing for the Embryo Operation
<ol>
	<li>Line a small 60 mm plastic Petri dish with modeling clay. Between uses, the dish and clay surface is thoroughly washed with distilled water and then with 70% ethanol. 
	NOTE: Use red, white or yellow, Van Aken Plastalina modeling clay, which can be bought at a local toy or art store. Black clay is not recommended because it releases a residue.</li>
	<li>Fill the dish with 3% Ficoll 0.5x MBS solution. The higher salt concentration prevents tissue dissociation, and the polysaccharide Ficoll helps to thicken the solution, which assists in holding the face in position.</li>
	<li>Use the flamed Pasteur pipette tool (see Figure 1) to make shallow, 2-3 mm depressions in the clay, about the depth of a stage 20 embryo body. Make 20-30 depressions, 1-2 mm apart, on each side of the dish, so there is a total of 40-60 depressions. Label one side LOF/GOF, and the other side wild type, by carving initials into the clay with forceps.</li>
</ol>
4. Preoperation Embryo Preparation
<ol>
	<li>Obtain and culture Xenopus laevis embryos using standard methods17. For a detailed description of frog husbandry, please see Sive et al.17
	<ol>
		<li>Forty-eight hours prior to the experiment obtain eggs from female frogs and perform in vitro fertilization.</li>
		<li>Inject 0.5-1 ng of capped membrane GFP mRNA, plus any desired mRNA or anti-sense morpholino-modified antisense oligonucleotides (&#34;morpholinos&#34;) at the one cell stage or into 2 cells at the 2 cell stage. The one cell stage lasts approximately 70-90 min. Inject a total volume of 1-3 nl. In place of RNA coding for a fluorescent protein (such as GFP or RFP RNA), one can inject FITC labeled morpholino or fluorescently labeled dextran. The fluorescence is important for determining whether the transplanted tissue heals and remains in the head.</li>
		<li>Capped mRNA can be prepared from a linearized plasmid using a mMessage mMachine SP6 or T7 kit. Morpholinos can be designed and ordered through Gene Tools LLC. The amount of morpholino required for a desired effect must be determined for each gene18.</li>
		<li>Store the injected embryos at 15 &#176;C for 48 hr, until they reach stage 19-20. (For all subsequent steps, stage embryos according to the Normal table of Xenopus laevis by Nieuwkoop and Faber19.) 
		NOTE: On the day of the surgery, both recipient and donor embryos must be within one stage of each other for the transplants to work optimally. However, embryos injected with morpholinos (&#34;morphants&#34;) sometimes develop more slowly than wild type or control morphant embryos, making it necessary to coordinate experiments so both morphant and wild type embryos are at the same stage. Morphants may need to be maintained at a higher temperature for 12-24 hr prior to the procedure. To increase the likelihood that embryos can be found at matching stages, embryos can be maintained at several temperatures. Twenty-four hours prior to the experiment, one should divide the embryos into a several dishes, and place morphants at 18-20 &#176;C and wild type embryos at 15-18 &#176;C.</li>
	</ol>
	</li>
	<li>On the day of the transplantation experiment, remove the embryos from the15 &#176;C incubator and stage them according to Nieuwkoop and Faber19. If they are younger than late neurula (stage 19), leave them at room temperature for 1-2 hr, until they reach stage 19.</li>
	<li>Screen the injected embryos under a fluorescent microscope. Select embryos that show uniform, bright fluorescence for the experiment.</li>
	<li>Remove clutter and possible contaminants near the operating area. Wipe down the operating surface, stereomicroscope and all tools with 70% ethanol.</li>
	<li>Once the embryos are at stage 19, remove the vitelline membrane using #5/45 Dumont forceps under a stereomicroscope.
	<ol>
		<li>Remove the vitelline membrane of 20-30 of each of donor and host embryos.</li>
		<li>For the first several face transplant experiments, one should practice with a few embryos for each condition. The method is challenging and requires careful practice before it can be used on a larger scale, quickly and successfully. One can work up to doing 20 transplants/experiment.</li>
	</ol>
	</li>
	<li>Move host embryos into the operating dish using a plastic graduated transfer pipette with the tip cut off such that the opening is much wider than an embryo (at least a few millimeters). Be careful to avoid touching embryos to bubbles or the water surface, as embryos will explode in the surface tension.</li>
	<li>Use #5/45 forceps to insert the embryos into the clay depressions, with the posterior of the embryo in the clay. Gently close the clay around the bases of the embryo using the forceps, leaving the top quarter of the embryo, the head, protruding from the depression.</li>
	<li>Once all host embryos are secured in their depressions, move to the other side of the plate and begin to insert the donor embryos into their depressions. Repeat the process of securing the embryos in their wells.</li>
</ol>
5. Performing the Face Transplant Surgery
<ol>
	<li>Cutting knife: As a cutting knife for removal of donor EAD tissue, use an appropriately broken glass capillary needle (see step 2.3 and Figure 1). 
	NOTE: One can directly hold the capillary between fingers (this works well for people with small hands) or one can mount the needle in an insect pin holder. Electrosharpened tungsten needles17 loaded into a pin holder may be used instead of a capillary tube needle. Please see suggested pin holders and tungsten needles in the Table of Specific Reagents and Equipment.</li>
	<li>Under a stereomicroscope, insert the needle into the head of the embryo to the left of the cement gland. The needle should be inserted deeply, so that it passes from outside the embryo, through the head, and into the foregut. To transplant the entire EAD, cuts should extend from the outside of the embryo to the foregut. For ectoderm-only transplants, cuts should be shallower and extend only through the ectoderm.
	<ol>
		<li>Flick the needle from the left to the right side of the head, across the entire width of the cement gland. The flicking is important as the motion gives a clean cut. Refer to Figure 2A for a summary of the technique and Figure 2B for a demonstration of the cuts. The cement gland and eyes are important landmarks for the cuts. The order of the cuts does not affect the outcome and can vary based on the user&#39;s preference or handedness.</li>
	</ol>
	</li>
	<li>Place the needle at the start of the previous cut, at the left border of the cement gland, and flick the needle upward until it reaches the bottom of the left eye. This will create a vertical cut from the left border of the cement gland to the bottom of the left eye.</li>
	<li>Place the needle at the right border of the cement gland, and flick the needle upward until it reaches the bottom of the right eye. This will create a vertical cut from the right border of the cement gland to the bottom of the right eye.</li>
	<li>To fully excise the tissue, flick the needle from the bottom of the left eye to the bottom of the right eye, creating a horizontal cut that will free the tissue. Cuts can extend from the outside of the embryo to the foregut, including both ectoderm and endoderm in the EAD explant. Alternately, shallow cuts can be used for ectoderm-only EAD transplants. Once the EAD tissue is removed, there should be a rectangular hole from the outside of the embryo into the foregut, stretching from the cement gland to just below the eyes (top to bottom). The hole should extend from the inside border of the left eye to the inside border of the right eye (side to side). See Figure 2Bb.</li>
	<li>Gently push the excised tissue on the tip of the needle, and lift it through the buffer to the part of the dish containing host embryos. Do not expose the tissue to the surface air or it will become damaged.</li>
	<li>Excise the same tissue from the host embryo, as for the donor. Discard the host EAD explant or save it to insert into the donor face, for reciprocal transplants.</li>
	<li>Insert the donor explant into the resulting host hole using #5/45 forceps.</li>
	<li>Once the donor tissue is correctly positioned and fully inserted, carefully place a glass bridge (see Figure 1 and Figure 2Bc) over the embryo&#39;s face to hold the transplant in place. The ends of the bridge should insert into the clay, holding it in place. The bridge should gently apply pressure to the transplanted tissue, such that the transplant lies flush with the host head, without it sticking out of the head or lying deep within the head. The head can be slightly flattened by the cover slip, but be careful not to damage the embryo with too much pressure. Transplants should be performed within 5 minutes. 
	NOTE: An experienced investigator can perform around twenty transplants per experiment, over 2-3 hr. During this period the embryos will progress from stage 20-22. Completing the face transplants at stage 21 or 22 does not affect outcomes. Later transplants (at stages 22-26) can be done but are more difficult as the crest-free EAD midline region is narrowed as cranial neural crest moves into the face. Consistency across transplants is critical.</li>
</ol>
6. Face Transplant Post-operation Recovery
<ol>
	<li>Healing typically takes 2-3 hr. Leave the embryos at room temperature undisturbed in their clay depressions with the glass bridges holding the donor tissue in place.</li>
	<li>Once the transplants have healed, carefully remove the glass bridges, remove the clay from around the base of the embryos using forceps, and use a plastic graduated transfer pipette to gently vacuum the embryos out of their depressions.</li>
	<li>Put the embryos into an appropriately labeled Petri dish, half filled with clean 0.1x MBS with gentamycin.</li>
	<li>Grow the embryos at 15 &#176;C or 18 &#176;C for several days until they reach feeding tadpole stage at stage 40, when facial phenotypes can be scored.
	<ol>
		<li>The 0.1x MBS solution with gentamycin should be changed daily, and any dead embryos should be removed promptly to prevent contamination and death of other embryos.</li>
		<li>The mouth opens at stage 40. At stage 40-41, one must check that the transplanted tissue remains healed in place by viewing its fluorescence. Transplants occasionally fall out, so one must ensure that all scored embryos have the donor tissue in place.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Gorlin, R. J., Cohen, M., Levin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Syndromes of the head and neck. , (1990).</li><li>Trainor, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Craniofacial+birth+defects:+The+role+of+neural+crest+cells+in+the+etiology+and+pathogenesis+of+Treacher+Collins+syndrome+and+the+potential+for+prevention.">Craniofacial birth defects: The role of neural crest cells in the etiology and pathogenesis of Treacher Collins syndrome and the potential for prevention.</a> Am. J. Med. Gen. A. 152, 2984-2994 (2010).</li><li>Dickinson, A. J., Sive, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+primary+mouth+in+Xenopus+laevis.">Development of the primary mouth in Xenopus laevis.</a> Dev. Bio. 295, 700-713 (2006).</li><li>Dickinson, A. J., Sive, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positioning+the+extreme+anterior+in+Xenopus:+cement+gland,+primary+mouth+and+anterior+pituitary.">Positioning the extreme anterior in Xenopus: cement gland, primary mouth and anterior pituitary.</a> Sem. Cell Dev. Bio. 18, 525-533 (2007).</li><li>Dickinson, A. J., Sive, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Wnt+antagonists+Frzb-1+and+Crescent+locally+regulate+basement+membrane+dissolution+in+the+developing+primary+mouth.">The Wnt antagonists Frzb-1 and Crescent locally regulate basement membrane dissolution in the developing primary mouth.</a> Dev. 136, 1071-1081 (2009).</li><li>Gilbert, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Developmental Biology. , (2010).</li><li>Borchers, A., Epperlein, H. H., Wedlich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assay+system+to+study+migratory+behavior+of+cranial+neural+crest+cells+in+Xenopus.">An assay system to study migratory behavior of cranial neural crest cells in Xenopus.</a> Dev. Genes Evol. 210, 217-222 (2000).</li><li>Grunz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homoiogenetic+neural+inducing+activity+of+the+presumptive+neural+plate+of+Xenopus+laevis.">Homoiogenetic neural inducing activity of the presumptive neural plate of Xenopus laevis.</a> Dev. Growth Differ. 32, 583-589 (1990).</li><li>Servetnick, M., Grainger, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+neural+and+lens+competence+in+Xenopus+ectoderm:+evidence+for+an+autonomous+developmental+timer.">Changes in neural and lens competence in Xenopus ectoderm: evidence for an autonomous developmental timer.</a> Dev. Bio. 112, 177-188 (1991).</li><li>Servetnick, M., Grainger, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeogenetic+neural+induction+in+Xenopus.">Homeogenetic neural induction in Xenopus.</a> Dev. Bio. 147, 73-82 (1991).</li><li>Couly, G., Coltey, P., Le Douarin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+triple+origin+of+skull+in+higher+vertebrates:+a+study+in+quail-chick+chimeras.">The triple origin of skull in higher vertebrates: a study in quail-chick chimeras.</a> Dev. 117, 409-429 (1993).</li><li>Couly, G. F., Le Douarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+the+early+neural+primordium+in+quail-chick+chimeras+:+I.">Mapping of the early neural primordium in quail-chick chimeras : I.</a> Developmental relationships between placodes, facial ectoderm. 110, 422-439 (1985).</li><li>Couly, G. F., Le Douarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+the+early+neural+primordium+in+quail-chick+chimeras+II.+The+prosencephalic+neural+plate+and+neural+folds:+Implications+for+the+genesis+of+cephalic+human+congenital+abnormalities.">Mapping of the early neural primordium in quail-chick chimeras II. The prosencephalic neural plate and neural folds: Implications for the genesis of cephalic human congenital abnormalities.</a> Dev. Bio. 120, 198-214 (1987).</li><li>Lievre, A. L., Le Douarin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+early+development+of+cranial+sensory+ganglia+and+the+potentialities+of+their+component+cells+studied+in+quail-chick+chimeras.">The early development of cranial sensory ganglia and the potentialities of their component cells studied in quail-chick chimeras.</a> Dev. Bio. 94, 291-310 (1982).</li><li>Kennedy, A., Dickinson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Median+facial+clefts+in+Xenopus+laevis:+roles+of+retinoic+acid+signaling+and+homeobox+genes.">Median facial clefts in Xenopus laevis: roles of retinoic acid signaling and homeobox genes.</a> Dev. Bio. 365, 229-240 (2012).</li><li>Trainor, P., Tam, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+paraxial+mesoderm+and+neural+crest+of+the+mouse+embryo-+codistribution+in+the+craniofacial+mesenchyme+but+distinct+segregation+in+the+branchial+arches.">Cranial paraxial mesoderm and neural crest of the mouse embryo- codistribution in the craniofacial mesenchyme but distinct segregation in the branchial arches.</a> Dev. 121, 2569-2582 (1995).</li><li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Early Development of Xenopus laevis. , (2000).</li><li>Tandon, P., Showell, C., Christine, K., Conlon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morpholino+injection+in+Xenopus.">Morpholino injection in Xenopus.</a> Methods Mol. Biol. 843, 29-46 (2012).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal Table of Xenopus laevis (Daudin). , (1994).</li></ol>

The authors have nothing to disclose.

Critical Steps and Limitations: The EAD face transplant procedure is time and work intensive. It requires practice, steady hands, and dexterity to perfect. The face transplant protocol relies on the researcher's ability to efficiently remove and transplant tissue. If one takes too long to insert the transplant into the host's face, the host face will begin to contract and heal. Forceps can be used to delicately expand the facial region. However, if significant wound contraction has occurred, the transplant will no...

To understand mechanisms underlying craniofacial birth defects1-2, important tissues and their signaling contributions during craniofacial development must be identified. In the frog Xenopus laevis, part of the face, including the mouth and nostrils form from the "Extreme Anterior Domain" (EAD), where ectoderm and endoderm are directly juxtaposed3-4. The EAD also acts as a signaling center to influence surrounding tissues, including the cranial neural crest, which forms the jaws and other facial regions5. To identify genes that contribute to EAD function, a 'face transplant' technique was developed, where tissue is transplanted from a donor into a host embryo, after removing the corresponding host region. Following the transplant, resulting facial development is assessed. Thus, the effects of loss of function (LOF) or gain of function (GOF) for a specific gene&#160;in the EAD are analyzed locally, where the rest of the head and body is composed of wild type tissue. The reciprocal transplant can be performed, where wild type tissue is transplanted into embryos with global LOF or GOF in specific genes. Transplantation has been frequently used in Xenopus and chick studies6. For example, Xenopus transplantation has addressed homogenetic neural induction, lens and neural competence, and neural crest migration7-10. Quail-chick chimeric grafting has analyzed development of the anterior neural plate, anterior neural ridge, neural crest, and cranial bones11-14. This is the first transplant technique for study of craniofacial development in Xenopus. This technique has demonstrated a novel role for the Wnt inhibitors Frzb1 and Crescent in regulating basement membrane formation in the presumptive mouth5. Xenopus laevis is an ideal model for study of craniofacial development as embryos are large, develop externally, and the face is readily visible, allowing micromanipulation and imaging of development. Mechanisms underlying facial development appear conserved, indicating that findings made in the frog provide insight into human development4,15-16.

<table border="0" cellpadding="0" cellspacing="0" width="559">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Pasteur pipette</td>
			<td rowspan="3" style="width:101px;">VWR</td>
			<td rowspan="3" style="width:136px;">14672-400</td>
			<td style="width:128px;">Lime Glass&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Size 5 3/4&#8217;&#8217;</td>
			<td style="width:128px;">Cotton Plugged</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;"></td>
			<td style="width:128px;">Disposable</td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:195px;">Graduated Transfer Pipette</td>
			<td rowspan="2" style="width:101px;">VWR</td>
			<td rowspan="2" style="width:136px;">16001-180</td>
			<td style="width:128px;">Disposable&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Polyethylene</td>
		</tr>
		<tr height="80">
			<td height="100" rowspan="2" style="height:100px;width:195px;">#5/45 forceps</td>
			<td rowspan="2" style="width:101px;">Fine Science Tools by Dupont medical</td>
			<td rowspan="2" style="width:136px;">11251-35</td>
			<td rowspan="2" style="width:128px;">Angled 45 degrees</td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="40">
			<td height="81" rowspan="3" style="height:81px;width:195px;">Standard Pattern Forceps</td>
			<td rowspan="3" style="width:101px;">Fine Science Tools</td>
			<td rowspan="3" style="width:136px;">11000-20</td>
			<td style="width:128px;">Straight; serrated tip</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Stainless Steel;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">20cm long</td>
		</tr>
		<tr height="60">
			<td height="81" rowspan="2" style="height:81px;width:195px;">Capillary Tubing (for needles)</td>
			<td rowspan="2" style="width:101px;">FHC</td>
			<td rowspan="2" style="width:136px;">30-30-1</td>
			<td style="width:128px;">Borosil 1.0mm OD x 0.5mm ID/Fiber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">100mm each</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Cover slip&#160;</td>
			<td rowspan="4" style="width:101px;">VWR</td>
			<td style="width:136px;">48393 252&#160;</td>
			<td style="width:128px;">24x60mm&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">micro cover glass</td>
			<td style="width:136px;">or&#160;</td>
			<td style="width:128px;">or&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">(for glass bridges)</td>
			<td style="width:136px;">48393 230</td>
			<td style="width:128px;">24x40mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">No.1.5</td>
			<td style="width:136px;"></td>
			<td style="width:128px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:195px;">Ficoll 400</td>
			<td style="width:101px;">Sigma-Aldrich</td>
			<td style="width:136px;">F9378</td>
			<td style="width:128px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Needle Puller&#160;</td>
			<td style="width:101px;">Sutter Instrument Co</td>
			<td rowspan="3" style="width:136px;">Needle Puller: discontinued Filament: FB300B</td>
			<td rowspan="3" style="width:128px;">The most similar, currently available&#160; needle puller is the P-97. For filaments, use Sutter 3.00mm square box filaments, 3.0mm wide.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:195px;">Model P-80</td>
			<td style="width:101px;">Flaming / Brown micropipette puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">(discontinued)</td>
			<td style="width:101px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Stereomicroscope</td>
			<td rowspan="2" style="width:101px;">Zeiss</td>
			<td rowspan="2" style="width:136px;"></td>
			<td rowspan="2" style="width:128px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Zeiss Stemi 1000</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:195px;">Stereomicroscope Lighting by Fostec</td>
			<td style="width:101px;">Fostec</td>
			<td style="width:136px;"></td>
			<td style="width:128px;">Use a light box with 2 fiberoptic arms.&#160;&#160;</td>
		</tr>
		<tr height="60">
			<td height="81" rowspan="2" style="height:81px;width:195px;">Nickel Plated Pin Holder</td>
			<td rowspan="2" style="width:101px;">Fine Science Tools</td>
			<td rowspan="2" style="width:136px;">26018-17</td>
			<td style="width:128px;">Jaw Opening Diameter: 0 to 1mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Length: 17cm</td>
		</tr>
		<tr height="60">
			<td height="101" rowspan="3" style="height:101px;width:195px;">Moria Nickel Plated Pin Holder</td>
			<td rowspan="3" style="width:101px;">Fine Science Tools</td>
			<td rowspan="3" style="width:136px;">26016-12</td>
			<td style="width:128px;">Jaw opening Diameter: 0 to 1mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Length: 12cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;"></td>
		</tr>
		<tr height="40">
			<td height="60" rowspan="2" style="height:60px;width:195px;">Tungsten Needles</td>
			<td rowspan="2" style="width:101px;">Fine Science Tools</td>
			<td rowspan="2" style="width:136px;">10130-05</td>
			<td rowspan="2" style="width:128px;">0.125mm Rod diameter</td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Van Aken Plastalina</td>
			<td rowspan="2" style="width:101px;">Blick&#160;</td>
			<td rowspan="2" style="width:136px;">#33268-2981</td>
			<td rowspan="2" style="width:128px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:195px;">Modeling Clay- white, red or yellow</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:195px;">mMessage mMashine SP6 or T7 Kit</td>
			<td style="width:101px;">Ambion</td>
			<td style="width:136px;">AM1340</td>
			<td style="width:128px;"></td>
		</tr>
	</tbody>
</table>

facial transplants in xenopus laevis embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Transplanted tissue should be fully inserted into the host head after transplantation as shown in Figure 3A, and have a glass bridge appropriately placed on the embryo's face, as shown in Figure 2Bc. The transplanted donor tissue must be correctly sized for the host opening, for the transplant to be successful. The EAD tissue should not protrude from the head, in any way, as seen in Figures 3B and 3C. Additionally, the face transplant should not be rotat...

Watch this Scientific Journal Video about Facial Transplants in Xenopus laevis Embryos at JoVE.com

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

facial-transplants-in-xenopus-laevis-embryos

Research

JoVE Journal

Biology

10.1K Views.  Harvard University. A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Video: Facial Transplants in Xenopus laevis Embryos

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and widely-utilized +TIPs for analyzing MT dynamics is the End-Binding protein, EB1, which binds all growing MT plus-ends, and thus, is a marker for MT polymerization1. Many studies of EB1 behavior within growth cones have used time-consuming and biased computer-assisted, hand-tracking methods to analyze individual MTs1-3. Our approach is to quantify global parameters of MT dynamics using the software package, plusTipTracker4, following the acquisition of high-resolution, live images of tagged EB1 in cultured embryonic growth cones5. This software is a MATLAB-based, open-source, user-friendly package that combines automated detection, tracking, visualization, and analysis for movies of fluorescently-labeled +TIPs. Here, we present the protocol for using plusTipTracker for the analysis of fluorescently-labeled +TIP comets in cultured Xenopus laevis growth cones. However, this software can also be used to characterize MT dynamics in various cell types6-8.

Using plusTipTracker Software to Measure Microtubule Dynamics in Xenopus laevis Growth Cones

The MATLAB-based, open source software package, plusTipTracker, can be used to analyze image series of fluorescently-labeled +TIPs to quantify microtubule dynamics.

Microtubule (MT) plus-end-tracking proteins (+TIPs) localize to the growing plus-ends of MTs and regulate MT dynamics1,2. One of the most well-known and ...

Methods Article

Facial Transplants in Xenopus laevis Embryos