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Stern, C.D. (1999) Grafting Hensen's node. In: Molecular Embryology: Methods and Protocols. (P.T. Sharpe and I. Mason, eds.). New Jersey: Humana Press. pp. 245-253. (Meth. Mol. Biol. 97, 245-253).
Soon after Spemann & Mangold's (1) famous demonstration in 1924 that the dorsal lip of the blastopore of the gastrulating amphibian embryo has the unique ability to induce a second axis when grafted into an ectopic site in a host embryo, Waddington (2,3) showed that Hensen's node is its equivalent in amniotes. After transplanting this region into an ectopic site in interspecific combinations of rabbit, duck or chick embryos, he found that a second axis developed, where the nervous system was derived from the host ectoderm (4). Hensen's node is situated at the anterior (cranial) tip of the primitive streak during gastrulation, and in chick embryos appears as a bulbous thickening, some 100m in diameter, centered around a depression, the primitive pit. At this point, the three germ layers of the embryo are in very close apposition.
In the avian embryo, operations involving Hensen's node at the primitive streak stage (10-20 hours' incubation, Hamburger & Hamilton (5) [HH] stages 3-5) are most easily performed in whole embryo culture, as described by New (6) (see 7). For assays of induction, it is essential to be able to distinguish donor from host cells because the change of fate of the host cells is central to the definition of induction. This can be achieved most easily using interspecies chimaeras, for example quail donors and chick hosts, whose cells can be distinguished by either the Feulgen-Rossenbeck technique or using anti-quail cell antibodies (e.g. QCPN) or using species-specific riboprobes in in situ hybridization analysis. Another way to trace the fate of the grafted cells is to label the transplanted node with a cell autonomous vital dye, such as the carbocyanine dye DiI (see 8, 9). In a recent study, using these techniques in combination with tissue- and region-specific markers, Storey et al. (10) were able to determine that Hensen's node is at the peak of its inducing ability at the primitive streak stage but that this ability quickly declines as soon as the head process begins to emerge (HH stage 5).
One problem with the way in which Waddington originally performed his grafting experiments (2, 3) is that he placed the transplanted node into a region now known to be fated to form neural plate, and therefore, although he demonstrated that this was able to initiate the formation of a second axis in the host, it is impossible to conclude from his experiment that the host cells underwent a change in fate. One way to overcome this is to place the grafts into a peripheral ring of the avian blastoderm, the inner third of the area opaca (Fig. 1). During normal development, this region only contributes to extraembryonic tissues, but is nevertheless able to respond to a graft of Hensen's node by generating a complete embryonic axis, where the host epiblast changes its fate from extraembryonic ectoderm to neural tissue (10, 11). The competence of this region to respond to such a graft declines rapidly, such that by HH stage 5 it is no longer able to respond to grafts of nodes derived from donors of any stage (10-12).
Manipulating Hensen's node in its normal position in the embryo is technically very difficult, as are most other microsurgical operations on chick embryos at the primitive streak stage. This is particularly true when the manipulation involves all three germ layers (ectoderm, mesoderm, endoderm), because cutting through the whole thickness of the embryo often leads to holes that expand greatly and eventually destroy the embryo. The main reason for this is that at these stages, the embryo only develops well when it is under some tension. This tension is maintained by the migration of cells at the peripheral edge of the area opaca on the vitelline membrane, to which they are attached. There are several ways to overcome this problem, at least in part. One is to remove the embryo from its vitelline membrane and to culture it, epiblast side down, on the surface of agar-albumen or agar-egg extract, as described by Spratt (13). But under these conditions growth of the embryo is stunted and abnormalities of the development of the axis are the rule rather than the exception. Another way is to excise the most peripheral edge of the area opaca but leaving the embryo on its vitelline membrane. The excised cells slowly appear to regenerate, while the hole has time to heal, and the embryo gradually develops tension once again in time for normal axial development to occur. In my experience, this is a very successful way to proceed. A third way to prevent large holes from expanding is to keep the newly-operated embryo at room temperature for 2-3 hours, followed by a period (3-5 hours) at 30C before placing it at 38C. The low temperature appears to slow down expansion of the area opaca while allowing healing to occur. This is also a successful approach. Whatever the course of action chosen, it is important to consider that the extent of the healing process will probably determine the outcome of the experiment. Healing after excision of a large portion of the embryo will bring new cells into contact with one another, and the result may therefore be different than when these cells are prevented from interacting.
In the following sections, I will consider two examples of operations on Hensen's node: excision from a donor quail embryo and transplantation to the inner ring of the area opaca of a host chick embryo to demonstrate embryonic induction, as done by Storey et al. (10) and rotation of the node about its rostrocaudal axis in situ, to demonstrate embryonic regulation as done by Abercrombie (14).
2. DEMONSTRATION OF EMBRYONIC INDUCTION BY TRANSPLANTATION OF HENSEN'S NODE
1. Dissecting microscope, preferrably one with trasmitted light base
2. Pannett-Compton saline:
solution A: 121 g NaCl, 15.5 g KCl, 10.42 g CaCl2.2H2O, 12.7 g MgCl2.6H2O, H2O to 1 litre;
solution B: 2.365 g Na2HPO4.2H2O, 0.188 g NaH2PO4.2H2O, H2O to 1 litre;
before use, mix (in order): 120 ml A, 2700 ml H2O and 180 ml B.
3. Two pairs of watchmakers' forceps, number 4 or 5
4. One pair of coarse forceps, about 15 cm (6") long
5. One pair of small, fine scissors, with straight blades about 2 cm (3/4") long.
6. a spoon/spatula or teaspoon
7. Petri dish (about 10-15cm diameter) to collect embryos
8. Container for egg waste
9. Pasteur pipette with the end cut off at the shoulder, stump flamed to remove sharp edges, and rubber teat
10. Pasteur pipette (short form), end lightly flamed to remove sharp edges; rubber teat
11. Pyrex baking dish about 2" (5 cm) deep, 2 litre capacity
12. 35 mm plastic dishes with lids
13. watch glasses, about 5-7 cm diameter
14. rings cut from glass tubing, approx. 27 mm outer diameter, 24 mm inner diameter, 3-4 mm deep
15. very fine needles (e.g. entomological size A1 or D1) or sharpened tungsten wire mounted by melting the fine end of a Pasteur pipette (to act as a handle) or into a metal needle holder
16. 1 small beaker (50-100 ml)
17. plastic lunch box with lid for incubating culture dishes
18. 38C incubator
19. hens' eggs incubated 12-18 h (depending on stage needed)
20. quails' eggs incubated 12-18 h (depending on stage needed)
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 so as to penetrate the shell. Use the tip of the scissors to cut off a small cap of shell near this end, carefully so as 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 4 cuts into the vitelline membrane around the embryo with scissors. If the embryo does not lie exactly in the centre of the egg, make the first cut on the side of the embryo nearest the shell, and proceed in this way until all 4 cuts have been made. Make sure all the cuts meet each other.
5. Pick up the square of embryo/membrane with the spoon/spatula, trying to collect only a minimal amount of yolk.
6. Transfer the yolk/embryo/membrane with the spoon into the large Petri dish with Pannett-Compton saline under a dissecting microscope. With fine forceps, turn the square of yolk/membrane/embryo so that the embryo is uppermost.
7. After the desired number of donor embryos have been placed into the Petri dish, use two pairs of forceps to separate the embryo from adhering yolk. Working at low magnification; pick up a corner of the square of vitelline membrane with one pair of forceps and slowly but steadily fold it back, steadying the yolk with the other pair of forceps. During the whole procedure the membrane and embryo should remain totally submerged in saline. The embryo should be attached to the membrane. If not, peel the membrane completely and then use foceps gently to remove the embryo from the underlying yolk.
8. Pick up the embryo, with or without adhering membrane, with the wide-mouth Pasteur pipette, and transfer it to a 35mm dish with clean saline for final cleaning and dissection. The edges of the extraembryonic membranes will be perfectly circular, provided that the embryo has not been damaged during the explantation procedure. Put this aside while preparing the host embryo.
2.2.2. Preparation of host (chick) embryo
The following description is for preparing cultures based on the method of New (6) but with some modifications as described in Stern & Ireland (15). The procedure has been adapted from (16, 17). You may also follow the method described in the previous chapter (7). The main difference between these methods and that originally described by New (6) is the use of rings cut from glass tubing, rather than bent from a glass rod with circular cross-section. The advantage of these rings, with rectangular profile, is that they grip the vitelline membrane tightly and therefore allow transfer of the assembly to a flat plastic dish. Above, I have recommended rings of 27 mm outer diameter, because it is easier to wrap the membrane around these for a novice. However, if larger (c. 30 mm diameter) rings are used, the embryos will develop up to 6-9 hours longer. The longevity of the cultured embryo appears to depend both on the amount of thin albumen under the ring and on the length of time for which it can be cultured before the edges of the area opaca reach the ring.
1. Fill the large Pyrex dish about 3/4 full with saline (about 1.5 litres).
2. Open an incubated hen's egg by tapping the blunt egg with coarse forceps, and carefully removing pieces of shell. Tip the egg gently to collect the thin albumen in the small beaker (this is required for culturing), and discard the thicker albumen, assisted with the coarse forceps. Try to remove as much albumen as possible, which will simplify the later steps.
3. When yolk is clean and free from adhering albumen, carefully tip it into the saline container, taking care not to damage the vitelline membrane on the edges of the broken shell. The blastoderm should face upwards. If not, carefully turn the yolk with the side of the coarse forceps.
4. Make a cut into the vitelline membrane enveloping the yolk just below the equator. Continue to cut all the way around the circumference of the yolk.
5. With two pairs of fine forceps, slowly but steadily 'peel' the North Pole of the vitelline membrane, all the way off the yolk. Do not stop during this process. The embryo should come off with the membrane. Let the membrane rest on the bottom of the dish, inner face (containing the embryo) pointing upwards.
6. Lower a watch glass and a glass ring into the container. Slide the vitelline membrane, preserving its orientation, onto the watch glass, and arrange the ring over it so that membrane protrudes around the ring. Pull out the assembly from the saline.
7. With fine forceps, work carefully to fold the cut edges of the vitelline membrane over the edge of the ring, all the way around its circumference. Do not pull too tightly but ensure that the bottom of the membrane is smooth and free from wrinkles as you work around the circumference.
8. Place the watch glass over a black surface. Suck off as much fluid as possible from the outside of the ring with the flamed Pasteur pipette. If there is much yolk remaining over and/or around the embryo, wash it carefully with clean saline. Discard any embryos in which the vitelline membrane has been damaged. Leave the host submerged in saline for the operation.
2.2.3. Grafting and incubation
1. Having prepared both donor quail and host chick embryos, you are ready for the operation. First bring the dish with the donor quail embryo under the microscope and arrange it so that its ventral (endoderm) surface is uppermost. Using two fine needles, carefully cut out the very tip of the primitive streak, cutting through the whole thickness of the embryo but making sure that you do not cut through the vitelline membrane if this is still attached.
2. Lower the magnification of the microscope, keeping track of the excised node, and pick this up with a Gilson P20 fitted with a yellow tip, set to 1-2 l.
3. Move the dish with donor embryos away and place the watch glass with the host chick embryo under the microscope. While looking down the microscope, insert the tip of the Gilson under the saline covering the host embryo and gently expel the quail node onto its surface, keeping track of it at all times.
4. Use fine needles to manipulate the donor node to close to the desired grafting site (Fig. 1). Now carefully lift up a portion of the flap of yolky cells (germ wall margin) that covers the inner margin of the area opaca, working outwards from the area pellucida and taking care not to penetrate the ectoderm underneath, which is only 1 cell thick. This will produce a pocket into which the graft can be inserted.
5. Slide the quail node into the pocket, pushing it as deep as possible so that when the flap of germ wall margin is replaced it will cover the graft completely.
6. Working under the microscope, carefully remove any remaining saline, both inside and outside the ring. During this process, keep watching the graft to make sure that it does not become dislodged. It is important that the embryo and the inside of the ring remain completely dry during incubation.
7. Now pour some thin albumen (about 2-3 mm thick layer) on the bottom of a 35 mm plastic dish. Slide the ring with vitelline membrane off the watch glass, and transfer it to the dish, over the pool of egg albumen. Press lightly on the ring with two forceps to allow it to adhere to the dish.
8. If the level of albumen comes close to the edge of the ring, remove the excess. Also aspirate any remaining fluid from inside the ring. It is best if the vitelline membrane bulges upwards, above a good pool of albumen. This will also help to drain off further fluid accumulated during culture to the edges of the ring.
9. Wet the lid of the plastic dish with albumen. Discard the excess, and seal.
10. Place the dish in a plastic box containing a piece of tissue paper or cotton wool wetted in distilled water, seal the box, and place it in an incubator at 38C.
After the desired period of incubation, fix the embryo by flooding with methanol (for most immunological detection procedures), Zenker's fixative (for Feulgen-Rossenbeck staining) or in 4% formaldehyde in PBS (for in situ hybridization and most other procedures). In the case of methanol or Zenker's, which are "rapid" fixatives, it is advantageous first to submerge the cultured embryo in saline, then to detach it from the vitelline membrane and to transfer it to a clean dish with saline prior to fixation. Otherwise the embryo will adhere permanently to the membrane and the fixative will denature the albumen, generating threads of protein that will tend to stick to the embryo. In the case of formaldehyde, this can be poured directly onto the embryo provided that the embryo is then detached from the membrane within a few minutes.
Whatever the fixative and subsequent method of processing chosen, it is advantageous to ensure that at the time of fixation the embryo is as flat as possible. If it has been detached from the membrane prior to fixation, place it in a small drop of saline on a plastic surface, then suck off most of the saline with a fine Pasteur pipette so that the embryo flattens on the plastic, and then place the first drop of fixative directly onto the surface of the embryo, taking care not to break it. After this it is safe simply to submerge the embryo in fixative, perhaps transferring it to a glass vial. Embryos fixed in this way usually remain flat through all subsequent manipulations.
Depending on the purpose of the experiment to be performed, embryos operated and cultured as described can be subjected to histochemistry, immunological procedures (as whole mounts) or whole-mount in situ hybridization. In many cases it is possible to combine two or more of these methods. For example, it is possible to fix the embryos in formaldehyde, process them as whole mounts for in situ hybridization, postfix in formaldehyde and then perform whole-mount immunoperoxidase histochemistry with QCPN antibody to detect the quail cells. After this they can be embedded in wax and sectioned. Methods for this have been published elsewhere in some detail (18).
The procedure outlined below is "generic", and similar manipulations can be done to operate on smaller or larger portions of embryo. For example, whole large sections of the primitive streak may be rotated as done by Abercrombie (14) and others, or very small sub-sectors of Hensen's node transplanted as described by Selleck & Stern (19) and Storey et al. (9).
The materials required are the same as listed in section 2.1. In addition you will need fine capillary glass connected to an aspirator (mouth) tube. The former can be manufactured by pulling the thin end of a Pasteur pipette over a very hot bunsen flame and pulling rapidly. The latter can be purchased from Sigma (A5177).
1. Follow steps 1-8 of the method in section 2.2.2 above to place a chick embryo in modified New (1955) culture as if to receive a graft.
2. Use fine needles to cut out the node, involving the whole thickness of the embryo but being very careful to avoid damaging the vitelline membrane underneath. Even a small hole will cause leakage of albumen and prevent healing, or even displace the graft. It is best to work in steps: first make a very superficial cut of the shape required. Then deepen the cuts, a little at a time, until the node is finally freed all around.
3. It is of advantage to mark one edge of the node with fine carbon (e.g. pencil lead shavings) or carmine (Sigma C1022) particles, using a fine needle. This allows the orientation of the node to be controlled through subsequent manipulations.
4. Manoeuvre the excised node, using the fine needles, to the desired orientation, again taking care not to damage the vitelline membrane that is now exposed.
5. Still observing under the microscope, carefully and slowly withdraw as much saline as possible from inside and outside the ring, as described for grafting Hensen's nodes above. In this experiment, where the manipulated node is not secured under a flap of tissue, it is much easier to lose it while sucking off the fluid. If necessary, replace it in position as required, using the fine needles.
6. Once all the fluid has been removed, use the fine capillary and mouth tube (if necessary) to remove all remaining fluid from the site of the graft. It is likely that the excised piece will appear to have shrunk. Sucking the fluid off in this way will close the gap and "knit" the pieces together, if performed with care.
7. Now slide the ring from the watch glass and set up the culture as described in steps 7-10, section 2.2.3. above. Finally, if necessary, suck off some more fluid with the mouth capillary assembly to ensure that the graft site is totally dry and appears closed.
8. Before placing in the incubator at 38C, it is advantageous to keep the operated embryo at room temperature (in the sealed Petri dish) for 2-3 hours. After this, place it at 30C for 3-5 hours. Finally transfer the dishes to an incubator at 38C. These periods at lower temperature will help the healing process as described above.
Operated embryos may be analyzed by histology, whole-mount immunohistochemistry and/or in situ hybridization with appropriate probes, as described for Hensen's node grafts.
1. Spemann, H. and Mangold, H. (1924). Über Induktion von Embryonanlagen durch Implantation artfremder Organisatoren. Wilh. Roux Arch. EntwMech. Organ. 100, 599-638.
2. Waddington, C. H. (1932). Experiments on the development of chick and duck embryos, cultivated in vitro. Phil. Trans. R. Soc. Lond. B 221, 179-230.
3. Waddington, C. H. (1933). Induction by the primitive streak and its derivatives in the chick. J. Exp. Biol. 10, 38-46.
4. Streit, A., Théry, C. and Stern, C.D. (1994) Of mice and frogs. Trends Genet. 10, 181-183.
5. Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morph. 88, 49-92.
6. New, D. A. T. (1955). A new technique for the cultivation of the chick embryo in vitro. J. Embryol. Exp. Morph. 3, 326-331.
7. Hornbruch, A. (1996). New culture. (this volume)
8. Selleck, M.A.J. & Stern, C.D. (1991) Fate mapping and cell lineage analysis of Hensen's node in the chick embryo. Development 112, 615-626.
9. Storey, K.G., Selleck, M.A.J. & Stern, C.D. (1995) Induction by different subpopulations of cells in Hensen's node. Development 121, 417-428
10. Storey, K.G., Crossley, J.M., De Robertis, E.M., Norris, W.E. & Stern, C.D. (1992) Neural induction and regionalisation in the chick embryo. Development 114, 729-741.
11. Stern, C.D. (1994) The avian embryo: a powerful model system for studying neural induction. FASEB J. 8, 687-691
12. Streit, A., Stern, C.D., Théry, C., Ireland, G.W., Aparicio, S., Sharpe, M. & Gherardi, E. (1995) A role for HGF/SF in neural induction and its expression in Hensen's node during gastrulation. Development 121, 813-824
13. Spratt, N.T. Jr. (1947) A simple method for explanting and cultivating early chick embryos in vitro. Science 106, 452
14. Abercrombie, M. (1950) The effects of antero-posterior reversal of lengths of the primitive streak in the chick. Phil. Trans. Roy. Soc. Lond. 234, 317-338
15. Stern, C. D. and Ireland, G. W. (1981). An integrated experimental study of endoderm formation in avian embryos. Anat. Embryol. 163, 245-263.
16. Stern, C.D. (1993a). Avian embryos. In: Essential Developmental Biology: A Practical Approach. (C.D. Stern and P.W.H. Holland, eds.) IRL Press at Oxford University Press, Oxford. pp. 45-54.
17. Stern, C.D. (1993b). Transplantation in avian embryos. In: Essential Developmental Biology: A Practical Approach. (C.D. Stern and P.W.H. Holland, eds.) IRL Press at Oxford University Press, Oxford. pp. 111-117.
18. Stern, C.D. & Holland, P.W.H. (eds.) (1993). Essential Developmental Biology: A Practical Approach. IRL Press at Oxford University Press, Oxford. 333pp.
19. Selleck, M.A.J. & Stern, C.D. (1992) Commitment of mesoderm cells in Hensen's node of the chick embryo to notochord and somite. Development 114, 403-415.
Figure 1. Diagram illustrating the operation of grafting a quail Hensen's node into a chick host to demonstrate embryonic induction in the inner part of the area opaca of the host.
Grafting Hensen's node
Claudio D. Stern
Department of Genetics and Development
College of Physicians & Surgeons of Columbia University
701 West 168th Street #1602, New York, NY 10032, U.S.A.
1. INTRODUCTION
2.1. Materials
2.2. Method
2.3. Analysis of results
3. ROTATION OF THE NODE IN SITU
3.1. Materials
3.2. Method
3.3. Analysis of results
References
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