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 ...

<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. 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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. 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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 vistas.  Harvard University.  Una técnica para el trasplante "Extreme anterior Dominio" tejido facial entre Xenopus laevis embriones se ha desarrollado. Los tejidos pueden moverse de un fondo de la expresión génica en otro, lo que permite el estudio de las necesidades locales para el desarrollo craneofacial y de señalización interacciones entre las regiones faciales. 