Notes

1. If possible, select a rapidly growing strain of chickens in which early stages of development will be in phase with the ones of quail embryos. A nonpigmented strain of chickens can be chosen in order to use the heavily pigmented quail melanocytes as a second marker. Freshly laid eggs are stored no more than 1 wk at 15°C.

2. Ideal conditions for chick and quail incubation are 38°C, 45% humidity (first two-thirds of incubation time), 75% (last one-third of incubation time and hatching). Small incubators equipped with time programmers can be placed in a 15°C room in order to obtain very precise stages of develoment.

3. Multiple wire tongs, individual egg holders, and hollowed-out wooden slats can be used for preincubation, microsurgical experiment, and postincubation, respectivley, of the chicken eggs.

4. The operations described here are performed at embryonic d 2 (E2) when somites, which can be easily counted, can serve to stage the embryos.

5. In order to perturb the gas exchanges at the level of the shell as little as possible, avoid using too large pieces of tape, and stick it carefully against the shell without folds, which would cause air entry and consequently progressive drying of the egg content.

6. PBS and Tyrode solutions supplemented with antibiotics (10-20 IU/mL) directly deposited on the blastoderm are commonly used to humidify it at any time during the experiment.

7. Pancreatin (Gibco) is diluted one-third to one-sixth with PBS or Tyrode solution. In this way, the tissue dissociation can be easily controlled. Titer and temperature are adapted to the stage of development of the tissues. The younger the tissues, the lower the titer and temperature; for example, tissues from 10-somite stage embryos will be treated with 20% pancreatin in Tyrode on ice, whereas tissues from 20-somite stage embryos will be treated with 30% pancreatin at room temperature. Tyrode solution supplemented with bovine serum will serve to stop enzyme action.

8. Glass micropipets hand-drawn from Pasteur pipets are curved and calibrated according to use: injection of liquids or transfer of pieces of tissues. Calibration of the micropipet according to the size of the rudiment to be transplanted (for instance, neural tube vs brain vesicle) is an important requirement.

9. The Rhodorsyl base is now preferred over the paraffin base. It can be either black or perfectly transparent depending on whether animal carbon is added to the commercial preparation. Moreover, it can be sterilized as often as necessary in dry oven and supports insect pins without damage.

10. Indian ink has to be tested for toxicity before use and must be used without excess.

11. Microscalpels have to be perfectly adapted to each use. Microscalpels, manufactured by stropping and honing steel needles (sewing needles) on an Arkansas oil stone, are the most convenient for excising fragments of neural tube or brain vesicles, because they can be both extremely thin and resistant. For dissociating tissues after enzymatic treatment, they must have a smooth tip. Tungsten microscalpels (43) or microscalpels made up from insect pins are also useful for dissecting very small pieces of tissues. They are quicker to prepare, but more fragile.

12. Formerly used conventional light bulbs with a condensor tend to radiate heat and cause traumatic drying to the embryos during the surgery, so that defects in amnios formation and subsequent death are often observed.

13. Zenker fluids like Carnoy fluid are good fixatives for Feulgen and Rossenbeck staining (13) but they do not allow immunohistochemistry or in situ hybridization to be made.

14. A modification of the Feulgen and Rossenbeck classical protocol consists of performing the hydrolysis in 5 N HCl for 20-30 min at room temperature after Carnoy fixation, instead of 1 N HCl for 4-8 min at 60°C as previously recommended in Gabe (13).

15. Dorso-ventral and rostro-caudal orientations of the graft are recognized either by morphological characters or by various labelings, for instance, a minute precisely localized slit.

16. Differences in stage and caudo-rostral level implicate difference of size. If the fragment of neural tube to be grafted is much bigger than the one that has been removed, it should be ressected before grafting.

17. Transplantations of brain vesicles are made at the 12- to 14-somite stages, which are favorable for the following reasons: Brain vesicles, still uncovered by the amnion, are clearly demarcated by constrictions in the absence of brain curvature; the notochord is no longer strongly adherent to the ventral part of the neural epithelium at this level; the neuroepithelium is not yet vascularized. Some neural crest cells and cephalic mesoderm are transferred along with the brain vesicles. Their presence does not interfere with the development of the brain, and presence of mel-anocytes in the head feathers of the chimera indicates the level of the brain graft.

18. A good adhesion of the graft to the host tissues is favored by sucking out with a micropipet the excess of physiological liquid added during the operation.

19. Daily gentle manual rocking of the operated eggs can enhance embryo survival. Incubator humidity must grow from 45-75% on d 18 of incubation if hatching of the operated embryos is expected.

20. E3-E4 chimeric embryos are fixed as a whole, for 1-3 h at room temperature. Older embryos have to be fixed as fragments and maintained in vacuum, during a growing time according to the stage and the size of the tissue pieces or organs. The same conditions will be applied for dehydratation and paraffin impregnation of the samples.

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