Dorsal Lip Transplant in Amphibian Embryos
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Introduction
In early stages of development in amphibian embryos, very few cells' fates are rigidly determined. However, previous experiments show that cells in one area of the developing embryo are determined before gastrulation. This area in the amphibian embryo has since been termed the organizer for its role in cell fate determination. In 1924 Hans Spemann and Hilde Mangold showed that cells transplanted from this "organizer" region of the blastopore lip of one embryo will induce gastrulation in any kind of tissue. They used this information to induce the growth of a second neural tube on an already developing embryo. This experiment attempts to repeat Spemann and Mangold's experiment in Xenopus embryos.
Detailed protocol
1. The operation should be performed under aseptic conditions. All phases of the operation should be performed in Steinberg's solution to promote healing. After healing, however, embryos should be transferred to spring water or 10% amphibian ringers solution.
2. Dip the tip of the eyebrow knife in 90% alcohol and then passed through asmall beaker containing the operating medium. Make sure all instruments are sterile.
3. Transfer the Xenopus embryo to the agar dish containing operating medium. Removejelly coats and vitelline membrane, handle the denuded embryo CAREFULLY.
4. With a ball tipped glass needle or watchmakers forceps make a shallow depression in the agar in which the embryo can sit comfortably, dorsal side up. Using an eyebrow knife, carefully remove part of the dorsal lip. With a single motion, bottom to top, make a thin tear in the epidermis. Follow the first longitudinal cut with a second longitudinal cut. The subsequent two cuts are transverse cuts, to complete a rectangular area of dorsal lip tissue.
5. Life the entire rectangular area of tissue away from the embryo with the tip of the glass needle.
6. Prepare a small pocket in the ventral surface of the host embryo. Do not attempt to cut a rectangular section, all that is needed is a mere slit, or slight hole. The pocket can then be enlarged to receive the transplant by carefully removing some of the mesoderm cells.
7. Transfer the excised piece of tissue to the slit in the host. Gently, yet firmly, push the whole mass of cells through the narrow incision, it may be necessary to enlarge the slit to accommodate the transplant, but a tight-fitting transplant is desirable.
8. Once the transplant has been pushed into the slit, quickly cover it with the flat surface of a thin coverslip. The weight of the cover slip should hold the tissue in place.
9. After and hour, the piece of coverslip should be carefully removed. If the transplant has not healed, place it again under pressure, with a piece of coverslip, for an additional half hour. After healing is complete, transfer the embryo to spring water or ringers solution.
10. Observe to see if the transplanted tissue forms a second neural fold.
Results/Discussion
The Xenopus embryos were very fragile and easily injured during the transplant surgery. They were also reluctant to heal well. However, there were a few successful transplants, with minimal injury (figure 1A). Several of the embryos developed visible secondary axis'. The original axis is discernible by what will form the eye and mouth and the secondary axis is characterized by a less well developed bud (figure 1 B,C). None of the embryos survived to form adult Xenopus.
