Facial Transplants in Xenopus laevis Embryos

Summary

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.

Abstract

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.

Introduction

To understand mechanisms underlying craniofacial birth defects^1-2, 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 juxtaposed^3-4. 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 transplante....

Protocol

1. Preparing Reagents

1. 10x MBS: Prepare 1 L of 10x Modified Barth'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.
2. 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.
3. 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 ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Pasteur pipette	VWR	14672-400	Lime Glass
Size 5 3/4 in	Cotton Plugged
Graduated Transfer Pipette	VWR	16001-180	Disposable
#5/45 forceps	Fine Science Tools by Dupont medical	11251-35	Angled 45°
Standard Pattern Forceps	Fine Science Tools	11000-20	Straight; serrated tip; stainless steel
Capillary Tubing (for needles)	FHC	30-30-1	Borosil 1.0 mm OD x 0.5 mm ID/Fiber
Cover slip	VWR	48393 252	24 x 60 mm micro cover glass;
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 3.00 mm square box filaments, 3.0 mm wide.
Model P-80	Flaming / Brown micropipette puller
Stereomicroscope	Zeiss
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: 0-1 mm
Moria Nickel Plated Pin Holder	Fine Science Tools	26016-12	Jaw opening Diameter: 0-1 mm
Tungsten Needles	Fine Science Tools	10130-05	0.125 mm Rod diameter
Van Aken Plastalina	Blick	#33268-2981
Modeling Clay- white, red, or yellow
mMessage mMashine SP6 or T7 Kit	Ambion	AM1340