Objective

In this experiment, a region of the neural crest will be transplanted from one stage-fourteen embryo to another (Figure 1). To facilitate the visualization of the grafts and observe the cell fates of the tranplanted region, both albino and wild type embryos will be used. Ten transplants will be attempted in which a region of an albino embryo neural crest will be tranplanted into the regularly pigmented wild-type embryo. In sucessful transplants, a region devoid of pigment should develop at the transplant site along the dorsal side of mature axolotls.

Introduction

Traditionally, amphibians have been one of the most widely studied organisms in the field of developmental biology. Two amphibian species often used in development experiments are the Xenopus laevis frog and the Ambystoma mexicanum, or Axolotl salamander. Axolotls are particularly useful for study of the early stages of development because the eggs are large, easy to harvest year round, and can be arrested at various stages of development by storing at 4°C (Gilbert, 2000).

One of the most commonly used techniques for observing the fate of cells within early gastrulas and neurulas is to transplant regions, either to locations within the same embryo, or form one embryo to another. In the early twentieth century, Hilde Mangold transplanted rgions of the dorsal lip of an amphibian embryo and observed the structural and developmental implications of the transplants (Gilbert, 2000). Hilde Mangold, along with Hans Spemann, used the similar transplantation techniques to identify the Organizer--later termed the dorsal lip-- along the dorsal side of the embryo. This discovery was a seminal work in developmental biology because it demonsrated some of the first evidence of regulative development, and lent significant support to the argument for epigenesis and against te theory of the homonculus (Spemann, 1929). Since then, several other techniques have been developed. In this experiment, the transplant will occur between albino and wild type (regularly pigmented) embryos. Transplantation between pigmented and non-pigmented embryos will provide an easily visible indicator of the fate of the transplaned cels.

In this experiment, the protocols developed by E.S. Wilson and D.L. Lawrence hav been adapted in order to transplant regions of an albino Axolotl embryo and a pigmented embryo (Wilson, 2002, Lawrence, 2000). Because albino embryos are less robust and are more susceptible to being destroyed in the course of the surgery, the albino embryos will serve as the recipient embryo.

Protocol

note: When the embryos are not being manipulated, keep them in a solution of HBSt containing antibiotics in order to keep the cell hydrated and to minimize the chance of infection.

1. Manually remove the jelly coating surrounding a stage fourteen Axolotl embryo using blunt foreceps.

2. Rinse an agarose Petri dish with an alliquot of the HBSt solution.

3. Place a prepared embryo into the operating dish containing HBSt with anitbiotics.

4. Maintain sterile surgical conditions through out the procedure by cleaning all tools in 70% ethanol and following surgical procedure.

5. Remove the membrane around the embryo by gently tearing with fine foreceps. Be careful not to crush embryo, and make sure to keep the embryo under the surface of the HBSt solution while removing the membrane. If done correctly, the embryo should slide out one side of the membrane.

6. Using an eyebrow knife, create a small ventral pocket in the right-hand side of the neural crest region of the recipient (regularly pigmented) embryo (Figure 2a). No removal of cells is necessary in the recipient.

7. Remove a section of the neural fold from the right side of the donor (albino) embryo. Use the eyebrow knife to make a thin, consistent incision in the lightly pigmented epidermis very close to the distal edge of the neural fold (Figure 2a). Continue to cut until the unpigmented cells beneath the surface are exposed.

8. Make an incision parallel to the initial incision along the inner edge of the right neural fold (Figure 2b).

9. Extract the section with two short cuts in order to harvest the rectangular cell sample (Figure 2c). If the excision is sucessful, the sample should include the whitish endodermal cells as well as a layer of the tan-colored ectoderm.

10. Using a surgical loop to manipulate the excised portion, transfer the neural fold segment from the albino donor to the regularly pigmented recipient. The implant should fit snugly within the incisio made in step 6.

11. Allow the embryos to heal. Then transfer both the donor and the host embryos into a sterile dish containing lower concentration HBSt by gradually reducing the concentration until 20% HBSt is acheived.

12. Allow development to continue until full grown axolotls are obtained.

13. Observe whether the grafting procedure was sucessful, and note any difference in the development of the grafted areas, particularly the pigment. Photograph the resulting embryos.

Results

Ten transplants were sucessfully completed using portions of albino embryos inserted into regularly pigmented embryos (Figure 3). Of these ten transplants, nine continued to develop twenty-four hours post surgery. However, after forty eight hours, once the process of diluting the HBSt solution was begun, development of all remaining embryos failed due to developmental abnormalities unrelated to the transfer region of the embryo (Figure 4). Cells lost their cohesiveness and failed to divide within the expected patterns, resulting in fatal delamination and abnormal extensions of mesoderm. These abnormailites resulted in the termination of development.

Discussion

Termination of development was caused by the abnormal cell growth of region of the embryos not directly affected by the transplant. These growths may have been caused by insufficient cadherin expression between cells (Gilbert, 2000), given that the protective and somewhat shape sustaining membrane had been removed. Once the membrane was removed from the embryo, there may not have been sufficient structural constrictions to maintain proper form and direction of growth. Furthermore, the agar plates used as operating dishes in this experiment had depressions in order to facilitate the surgery, and to hold the embryos stable post-surgery. Post surgery, however, the embryos lost their shape and mesodermal and endodermal cells extended into the depressions. The changes in shape most likely were sufficient to destroy the embryos.

However, with the exception of one embryo, all transplants were sucessful up to twenty four hours after surgery (Figure 5). In the failed embryo, the transplant may have been inserted into the transplant incision incorectly, that is, the cells that were fated to become ectoderm were aligned with the endo derm of the recipient embryo. Grafts transplanted in this manner would fail to heal because the ectodermal cells would express different cadherins than would the endodermal cells (Gilbert, 2000). Experimental errors such as this may be avoidable if the transplant was taken from a pigmented donor and grafted to an albino recipient because in pigmented specimens, the ectoderm is easily distinguishable from the endoderm, and inversions of the grafts would not occur.

In the remaining nine embryos, however, the grafts began to heal, indicating that, had other unrelated problems not arisen, the transplants would have resulted in fully developed Axolotl embryos displaying albino regions on their dorsal side. In the future, dilutions of the HBSt solution should be made more gradually, and the embryos should be transferred to a petri dish without depressions as soon after the transplant as possible.

Figures

Figure 1. A pigmented stage fourteen embryo. Stage fourteen embryos are characterized by the closing of the neural tube. The name \"keyhole stage\" has been used for stage fourteen due to the unique shape of the neural crest. Pigmented embryos such as this one were used as recipient embryos in the transplant procedure.

a)

b)

c)

Figure 2. A step-by-step schematic of the incisions necessary to excise a region of the axolotl embryo. a) This initial incision should be made on both the recipient embryo (to become the site of tranplantation) and the donor embryo. b) The second parallel incision to be made alongside the initial incision of the donor embryo. c) These two small cuts should be made perpendicular to the first two incisions in the donor embryo in order to free a region of the neural fold for excision and transplantation.

Figure 3. Sucessful transplants of regions (circa twenty-four hours after transplant) near the neural crest region of the axolotl embryo. The transplanted region orginating from an albino embryo can be easily identifiedin the recipient pigmented embryo as a region devoid of pigment.

Figure 4. Developmental abnormalities leading to the termination of development. The transplanted pigment-free region (indicated by the black arrow) had begun to heal before development failed. However other regions of the embryo failed to develop properly (indicated by the white arrow).

Figure 5. A failed transplant of the neural crest region in an axolotl embryo. The cells of the graft failed to adhere to the surrounding cells in the recipient embryo, possibly because of incorrect alignment of the ectodermal and endodermal cells of the donor and recipient. Development arrested less than forty eight hours after the surgery.

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