Method

Here the host embryo is operated in ovo, which is only suitable with ease for embryos older than about 36-48 h incubation. As an example, the operation described here consists of a graft of presomitic mesoderm (segmental plate) with a quail embryo as the donor and a chick embryo as the host. An identical method can be followed to transplant paraxial mesoderm that has already formed somites, but it should be remembered that this is much easier when it involves the youngest (most caudal) 5-6 pairs of somites at stages 10-14. Somites situated more anteriorly in the embryo have already separated into dermomyotome and sclerotome (Fig. 1) and special care is needed both to keep these components together during grafting and to separate them cleanly. Whatever the operation to be performed, it is generally a good idea to fix for histo-logical analysis some of the pieces like those to be transplanted, as well as some embryos immediately after the operation, to confirm that the graft involves the tissues of interest and no contaminating cells.

2.2.1. Preparation of Donor Quail Embryo

1. Remove quail eggs from incubator. With the scissors, gently tap near the blunt end of an egg in order to penetrate the shell. Use the tip of the scissors to cut off a small cap of shell near this end, carefully to avoid damaging the yolk.

2. Allow egg white to pour into waste bucket, assisted by the scissors, taking care to avoid damage to the yolk. You may need occasionally to cut through the rather thick albumen using the scissors.

3. Once most of the albumen has been poured off, make sure the embryo is uppermost; if not, turn the yolk by stroking it very gently with the sides of the scissors.

4. Use the scissors to make four cuts into the vitelline membrane around the embryo. If the embryo does not lie exactly in the center of the egg, make the first cut on the side of the embryo nearest the shell, and proceed in this way until all four cuts have been made. Make sure all the cuts meet each other.

5. Pick up the square of embryo/membrane by grasping one corner with fine forceps and transfer it immediately to the Sylgard-coated 35-mm dish containing about 5 mL CMF-Tyrode's solution.

6. Using two pairs of fine forceps, separate the vitelline membrane from the embryo, but leave the extraembryonic membranes attached to the embryo.

7. Stretch out the embryo (either side up) by placing 4-6 entomological pins (size A1 or D1) through the corners of extraembryonic membranes, into the Sylgard rubber. The embryo should be under some tension. Put the dish aside while preparing the host.

2.2.2. Preparation of Chick Host Embryo

The procedure described here differs from that in the chapters on grafting of the notochord and neural tube, AER/ZPA and neural crest (see Chapters 18-

20). In the method described here, a small window is cut into the shell and the embryo floated up to the level of this window for the operation. The advantages of this technique are:

a. The embryo lies very close to the operator's hands.

b. It is completely submerged in liquid at all times (which avoids drying out and simplifies some of the manipulations).

c. The liquid above the embryo can be changed several times, for example to wash the embryo.

d. The light illuminating it can be shone tangentially to its surface (the bubble of liquid above acts as a lens that greatly enhances the optical clarity).

e. The tension generated by floating the embryo seems to aid the action of the trypsin, such that tissues almost appear to dissect themselves.

f. It is very easy to notice if the endoderm has been punctured accidentally because ink will fountain out very quickly.

g. After the operation the small hole can be closed very tightly with plastic (PVC, or electrical) tape, which allows the egg to be incubated with the window downwards and turned—both of these greatly enhance survival.

1. Shape plasticine (modeling clay) into a ring about 2 in. (5 cm) in diameter and place it on the stage of the microscope. Place a hens' egg (host) onto the plasticine ring, being careful that it does not rotate with respect to its resting position.

2. Using the 5-mL syringe with 21-gage needle, held nearly vertical, insert needle into blunt end of egg until the shell is felt at the bottom surface. Withdraw 0.5-1 mL egg albumen, which should come up easily.

3. Score a shallow 1 x 1 cm square on the top of the shell with the scalpel and lift up the square of shell.

4. With a pair of watchmakers' forceps, pierce and remove the underlying shell membrane, after wetting it with CMF. Avoid damage to the embryo underneath: before air is allowed into the egg, the embryo will lie very close to the membrane.

5. Fill the cavity with CMF so that the embryo floats up to the level of the window.

6. After ensuring that there are no air bubbles in the syringe with Indian ink or in the needle, insert the needle under the vitelline membrane, tangentially, at a position as far away from the embryo proper as possible. Point toward and slightly below the embryo, and inject about 20-50 pL. It is important to minimize movement of the needle after penetrating the vitelline membrane, or the hole will be very large and yolk/ink will leak out. Introduce and withdraw the needle with one clean, decisive movement and do not stir the needle inside the yolk; only one attempt per egg!

7. Draw a shallow, continuous border of silicon grease around the window. This will contain a standing drop in which the operation will be done. Now fill this chamber with CMF saline until there is a standing drop, and adjust the fibre optic light to shine tangentially to the surface of the egg so that the embryo can be seen very clearly with minimal light intensity from the light source.

2.2.3. Grafting Procedure

1. Break the vitelline membrane just over the region to be operated with a needle. The hole should be as small as possible. The segmental plates are the rod-like structures lying on either side of the neural tube at the tail end of the embryo, just behind the last somite. The portion to be rotated in this example is the most anterior half of the plate.

2. Replace the bubble of CMF with trypsin/CMF. Increase the magnification of the microscope as much as possible.

3. Operating in the drop of trypsin, use the micro-knife to make initially very shallow cuts in the ectoderm next to the neural tube in the region of the operation.

4. Gradually deepen the cuts using the knife blade more as a spatula, allowing penetration of the trypsin, than as a sharp cutting edge. Once the ectoderm has been penetrated with the tip of the blade, the trypsin does the rest. Find the lateral border of the segmental plate, and do the same there: a shallow cut in the ectoderm first, then separate the tissue gradually. In both cases, make sure you do not penetrate the endoderm (which is 1-cell thick) or ink will pour out. Finally, free the posterior end of the piece and loosen the graft.

5. Remove the piece of segmental plate with a Gilson micropipet set to 1-2 |L. Replace the bubble over the embryo with fresh CMF twice to remove the trypsin solution. Make a new bubble of CMF while obtaining the graft from the donor.

6. Turn to the donor embryo in the Sylgard dish. Replace almost all of the CMF in which it was submerged by trypsin solution. Repeat the trypsin wash and perform the dissection in this solution at room temperature.

7. Cut out an equivalent piece of segmental plate as the one removed from the host, using the same technique.

8. Pick up the graft with the Gilson. With the other hand, place the host under the microscope and, observing under low magnification, carefully place the graft into the CMF bubble over the embryo.

9. Use the knife and work at low magnification to manipulate the graft into the gap made by removal of the host piece of segmental plate.

10. When the graft is in position (approximately), very carefully remove the most of the fluid from above the embryo with a Pasteur pipet while watching under low power. If necessary, reposition the graft with a mounted needle.

11. Insert the 5-mL syringe with 21-gage needle into the original hole in the blunt part of the eggshell, vertically, and carefully withdraw 2.5-3 mL thin egg albumen. This will lower the operated embryo back to its original position. Be careful when you insert the needle, since the pressure could make the graft come out of its site.

12. Add 2-3 drops of antibiotic/antimycotic concentrate (away from the graft site).

13. Wipe the edges of the shell with tissue paper moistened lightly in 70% alcohol to remove the silicon grease.

14. Cut a piece of PVC tape about 6 cm long. Stretch it slightly, and then let it relax. Place it over the window, smoothing out any unevenness carefully to avoid breaking the shell or applying too much pressure on the window.

15. Keeping the egg on its side, place it (window down!) into an egg tray in a humidified incubator at 38°C. If you are worried about the graft falling out, it is a good idea to incubate the embryo with the window upwards until the next day, and then to turn it.

16. Incubate 1-3 d. Embryo survival 2 d after this operation should be 80-100%.

Was this article helpful?

0 0

Post a comment