Preparation of Donor Tissues

1. Remove donor embryos from eggs. This may be done by cracking eggs into a Petri dish or by windowing them (see Chapter 14). If you are dealing with embryos of stage 9 or younger, it is more reliable to window eggs to avoid losing embryos by accidentally puncturing the blastoderm.

2. Remove donor embryos by cutting through the vitelline membrane and the blastoderm and around the embryo using spring scissors.

3. Slip a small spatula under the embryo and transfer into a dish of Ringer solution.

4. Dissect away extraneous tissues to isolate the region of interest. In the case of rhombomere transplants, for example, the entire hindbrain from E2 embryos is dissected away from spinal cord, midbrain, and forebrain, and the heart and gut removed (Fig. 1).

5. Place the isolated regions in a small volume of Dispase (1 mL) in a 35-mm Petri dish, and leave for 5 min.

6. After this time, use tungsten needles to test whether adjacent tissues are dissociating from the neural tubes. If so, transfer immediately to a fresh dish of HBSS. If

Fig. 1. This figure illustrates diagrammatically the procedure for grafting a rhombomere unilaterally from a donor stage 10 chick embryo into an isochronic host embryo. Once the embryo has been removed from the egg, the entire hindbrain region is dissected free of adjacent tissues, and the adhering mesenchyme cells are removed by exposure to Dispase. Then individual rhombomeres are separated from each other, and at the desired axial level (rhombomere 3 in this case), the right and left halves are separated from each other. Meanwhile the region that will receive the graft in the host embryo is prepared by removing the host rhombomere 5 using 4 cuts in the numbered sequence. The graft tissue can then be transferred into the egg and put in place in the ectopic location. The right-hand side piece of the rhombomere has been grafted such that the anteroposterior and dorsoventral polarity have been maintained.

Fig. 1. This figure illustrates diagrammatically the procedure for grafting a rhombomere unilaterally from a donor stage 10 chick embryo into an isochronic host embryo. Once the embryo has been removed from the egg, the entire hindbrain region is dissected free of adjacent tissues, and the adhering mesenchyme cells are removed by exposure to Dispase. Then individual rhombomeres are separated from each other, and at the desired axial level (rhombomere 3 in this case), the right and left halves are separated from each other. Meanwhile the region that will receive the graft in the host embryo is prepared by removing the host rhombomere 5 using 4 cuts in the numbered sequence. The graft tissue can then be transferred into the egg and put in place in the ectopic location. The right-hand side piece of the rhombomere has been grafted such that the anteroposterior and dorsoventral polarity have been maintained.

not, leave for a minute or so longer—the exact time will depend on the age of embryo and region to be dissected. In any case, it is crucial that embryos are not exposed to Dispase for too long. Otherwise, neural tissue will become irreversibly damaged.

7. Adjacent mesodermal tissues can be teased away from the neural tubes using tungsten needles and then clean neural tubes transferred into fresh HBSS. Cut away undesired tissues by pressing down on the plastic Petri dish with the tungsten needle. When pulling away mesenchymal or other tissues, avoid contacting the neural tissue directly with the needles.

8. From now on, keep tissues as far as possible on ice, since this prevents deterioration.

9. Transfer individual neural tubes from the dish on ice to the microscope for further subdissection, e.g., dissection of individual rhombomeres (Fig. 1A), but keep the rest cold. At this stage or previous stages of the dissection, a few Carmine particles can be brushed against the surface of the neural tissue using a needle in order to label its polarity, if this is required.

10. Different tissue pieces may be stored in individual wells in multiwell plates, and chick tissues can be kept on ice for up to 8 h without detriment. For rhombomere transplantation in which unilateral grafts are to be performed, the bilateral rhombomere fragments can be kept intact until just before grafting and then separated into right and left halves (Fig. 1).

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