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.
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.
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 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.
1. Preparing Reagents
2. Preparing Glass Operating Tools
3. Preparing for the Embryo Operation
4. Preoperation Embryo Preparation
5. Performing the Face Transplant Surgery
6. Face Transplant Post-operation Recovery
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 rotated relative to its position in the donor body, as shown in Figure 3D. After several hours, the transplanted tissue and surrounding face should heal, and by the next day, the embryo should appear as the example shown in Figures 4A and 4A’. One can observe, under fluorescence, that the transplant remains in place in Figure 4B. At stage 41, the transplanted control tissue will contribute to the mouth, and will remain florescent green, seen in Figure 4B’. Wild type EAD tissue transplanted into wild type hosts should give rise to normal faces, when compared with unperturbed wild type embryos. However, with LOF or GOF donor tissue, faces should heal and remain in the head, but these may or may not give rise to normal craniofacial structures, as shown in Figure 3 of Dickinson and Sive5.
Reagent | Ingredients | Instructions |
1 M CaCl2 Solution | 111 g of CaCl2 per liter | Autoclave and store in 1 ml aliquots at -20 or 4 °C. |
10x Modified Barth’s Saline (MBS) Solution | 880 mM NaCl, 51.4 g | Adjust volume up to 1 L with distilled water. Adjust final pH to 7.8 with NaOH and then autoclave. |
10 mM KCl, 745.5 mg | ||
10 mM MgSO4, 1.2 g | ||
50 mM HEPES (pH 7.8), 11.9 g | ||
25 mM NaHCO3, 2.1 g | ||
1x MBS Solution | Final Concentrations: | Prepare 1x MBS solution by mixing 100 ml of 10x MBS salts solution with 0.7 ml of 1M CaCl2 solution. Adjust volume up to 1 L with distilled water. Dilute this solution to make 0.5x MBS and 0.1x MBS. |
88 mM NaCl | ||
1 mM KCl | ||
0.7 mM CaCl2 | ||
1 mM MgSO4 | ||
5 mM HEPES (pH 7.8) | ||
2.5 mM NaHCO3 |
Table 1. Reagents, ingredients, and instructions.
Heat | Pull | Vel. | Time |
800 | 70 | 40 | 50 |
Table 2. Needle Puller Settings*.
*Needle puller settings vary from machine to machine so each lab will probably need to optimize their own needle puller settings.
Figure 1. Tools used. A) Shows an intact, unbroken needle and a broken needle after the flexible tip has been removed. B) Depicts two pipette tools with their ends fully sealed and rounded. C) Shows three sample glass bridges. Scale bars = 1,000 μm.
Figure 2. Summary of face transplant method. A) General Transplant Scheme: Schematic of the face transplant method. a. Embryos are injected with a fluorescent agent and an antisense oligonucleotide morpholino at the 1 cell stage. The fluorescent agent can either be GFP mRNA, FITC-tagged morpholino, or fluorescent dextran. b. Presumptive mouth is removed at stage 22 from a host wild type embryo and a donor morphant embryo. The transplanted tissue can also be enlarged to include the presumptive nose, which lies directly above the mouth region. The donor morphant tissue is transplanted to the wild type host, and then is secured in place with a glass bridge. Grey arch: hatching gland. c. Facial development is scored at stages 40-41. B) Experimental Considerations. a. Summary of incisions used to remove the face from a donor embryo. For surgical excision of the face, make incisions 1 through 4 in order. This is the preferred order of cuts, but the order of incisions can vary. b. The resulting donor is shown with the ectoderm and endoderm removed from the face, such that an opening exists from the outside of the embryo to the foregut. c. The diagram shows ideal placement of the glass bridge. The glass contacts both the transplant and host face, pressing the EAD tissue into the head to oppose extrusive forces from wound contraction during healing. Please click here to view a larger version of this figure.
Figure 3. Schematic of embryos shortly after transplantation. Frontal views are shown. The transplanted tissue is outlined in red dots. A and A’ depict an ideal outcome at stage 22. The tissue is fully inserted and correctly positioned in the head. B and B’ show an incorrect transplant, with the tissue partially inserted into the head. C and C’ show an incorrect transplant with most of the tissue inserted into the head, but the excess region is protruding from the healing site. This tissue will necrose, and inhibit healing of the surrounding, properly inserted regions. D and D’ show an improper transplant, where the face has been rotated in the host, relative to its position in the donor. The tissue will heal into the head, but the face will not develop normally. Please click here to view a larger version of this figure.
Figure 4. Older embryos with ideal outcomes. Frontal views are shown. cg: cement gland; mo: mouth. A) Displays an embryo one day after transplantation, showing a properly healed transplant at stage 32. A’) Shows this same embryo with a fluorescent overlay, confirming that the transplanted tissue remains in place. B) Shows the same embryo at stage 42 which had a control morpholino, GFP+ transplant several days prior. The face has developed properly. B’) Shows the same embryo with a fluorescent overlay, confirming that the transplanted tissue remains in the head and has contributed normally to facial structures. Please click here to view a larger version of this figure.
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 not heal as well and may need to be reduced in size to fit into the host’s facial hole. The transplant’s size and shape must roughly match the size and shape of the recipient’s facial cavity, to allow successful insertion.
Face transplants are most successful when performed between stages 19-22, with embryos that are matched by stage. Older face transplants, between stages 22-26, are possible, but are more challenging and may disrupt neural crest migration into the sides of the face since the crest-free midline region becomes significantly narrower from stage 22-26.
Both the recipient and donor must be at similar stages for the transplant to work optimally. Ideally, they should be the same stage, and minimally have to be within a few hours of each other. Embryos injected with antisense-RNA morpholino oligonucleotides ("morphants") sometimes develop more slowly than wild type or control morphant embryos, requiring that morphants be grown at a higher temperature to match the wild type embryos’ stages.
The donor face tissue should not be rotated in the host body relative to its original position in the donor embryo, otherwise the face will not develop normally. The cement gland does not need to be included in the face transplant for the procedure to work; the face develops normally without it. However, the cement gland is often included in the extirpated facial tissue because it serves as a distinct marker to indicate and position the bottom of the transplant. This marker will help avoid accidently rotating the tissue during the transfer of the face tissue to the recipient. If the transplant has been inserted incorrectly in the host face, the donor tissue can be removed and discarded. One can attempt to insert a new transplant into the same host, if the opening has not begun to constrict. However, if the host opening has noticeably shrunk, then discard the host and begin anew.
It is crucial to place the glass bridge directly on the transplanted face, so that the transplant is held in the host’s head during healing. If the transplant is not held in place, the transplant can be extruded from host’s face. Transplants that are not fully inserted into the face or that pop out during healing will undergo necrosis. Even in perfect transplants, small amounts of tissue death may occur around the edges of the transplant. Usually this does not cause adverse effects and a completely normal face can still be formed. In successful controls we have not observed any malformation after four weeks of development suggesting that the cartilages formed normally and that rejection of the tissue is rare.
Finally, if a morpholino perturbs a protein required for normal wound healing, this technique may not work, as the transplanted LOF tissue may not be incorporated into the host head.
Possible Modifications and Trouble Shooting: Modifications can be made to the procedure. With practice, the researcher can learn to transplant smaller regions, for example, half of the EAD. Shallow incisions allow ectodermal transplants, leaving the deeper endoderm undisturbed. Other regions of embryonic tissue can be transplanted, using similar approaches.
If the transplanted tissue dies after insertion, there are a couple of possible causes. Transplanted tissue that is not completely inserted into the host or appropriately held in place with a glass bridge, will die and impede proper healing. Make sure to fully insert the transplanted tissue and secure it in place with a glass bridge. If the transplant is derived from a morpholino or RNA injected donor, then the tissue may die due to toxicity from injected agents. Similarly, if the host embryos are morphants or RNA-injected and frequently die, then the amount of morpholino or RNA will need to be reduced. To resolve these issues, titrate the amount of injected RNA or morpholino and determine a safe amount for successful face transplants. Finally, increasing the salt concentration of the MBS solution above 0.1x can also assist healing.
Significance: The face transplant technique described allows analysis of the local requirements and activity of gene products during craniofacial development. This approach can clarify signaling between the developing noncrest, neural crest, and surrounding structures. It allows one to examine LOF or GOF (using any strategy) in all EAD-derived tissues, which is not possible with any single promoter driven construct. Though there is a long history of tissue transplantation in developmental biology this is the first application of transplantation to the study of craniofacial development in frogs and is crucial for mechanistic studies7. Thus the technique can help to unravel the complex mechanisms controlling patterning and formation of the vertebrate face and to clarify causes of craniofacial developmental defects.
The authors have nothing to disclose.
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.
Pasteur pipette | VWR | 14672-400 | Lime Glass |
Size 5 3/4’’ | Cotton Plugged | ||
Disposable | |||
Graduated Transfer Pipette | VWR | 16001-180 | Disposable |
Polyethylene | |||
#5/45 forceps | Fine Science Tools by Dupont medical | 11251-35 | Angled 45 degrees |
Standard Pattern Forceps | Fine Science Tools | 11000-20 | Straight; serrated tip |
Stainless Steel; | |||
20cm long | |||
Capillary Tubing (for needles) | FHC | 30-30-1 | Borosil 1.0mm OD x 0.5mm ID/Fiber |
100mm each | |||
Cover slip | VWR | 48393 252 | 24x60mm |
micro cover glass | or | or | |
(for glass bridges) | 48393 230 | 24x40mm | |
No.1.5 | |||
Ficoll 400 | Sigma-Aldrich | F9378 | |
Needle Puller | Sutter Instrument Co | Needle Puller: discontinued Filament: FB300B | The most similar, currently available needle puller is the P-97. For filaments, use Sutter 3.00mm square box filaments, 3.0mm wide. |
Model P-80 | Flaming / Brown micropipette puller | ||
(discontinued) | |||
Stereomicroscope | Zeiss | ||
Zeiss Stemi 1000 | |||
Stereomicroscope Lighting by Fostec | Fostec | Use a light box with 2 fiberoptic arms. | |
Nickel Plated Pin Holder | Fine Science Tools | 26018-17 | Jaw Opening Diameter: 0 to 1mm |
Length: 17cm | |||
Moria Nickel Plated Pin Holder | Fine Science Tools | 26016-12 | Jaw opening Diameter: 0 to 1mm |
Length: 12cm | |||
Tungsten Needles | Fine Science Tools | 10130-05 | 0.125mm Rod diameter |
Van Aken Plastalina | Blick | #33268-2981 | |
Modeling Clay- white, red or yellow | |||
mMessage mMashine SP6 or T7 Kit | Ambion | AM1340 |