Claudio D Stern 1 Introduction

Soon after Spemann and 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, and 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 100 pm 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 h incubation, Hamburger and Hamilton (5) [HH] stages 35) are most easily performed in whole embryo culture, as described by New (6) (see Chapter 15). 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 easily achieved by 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 Dil (see refs. 7 and 8). In a recent study, using these techniques in combination with tissue- and region-specific markers, Storey et al. (9) were able to determine that Hensen's node is at the peak of its inducing

From: Methods in Molecular Biology, Vol. 97: Molecular Embryology: Methods and Protocols Edited by: P. T. Sharpe and I. Mason © Humana Press Inc., Totowa, NJ

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 (9,10). 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 (9-11).

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 (12). 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 h, followed by a period (3-5 h) at 30°C before placing it at 38°C. 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

Fig. 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.

contact with one another, and the result may therefore be different than when these cells are prevented from interacting.

In the following sections, I 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. (9) and rotation of the node about its rostrocaudal axis in situ, to demonstrate embryonic regulation as done by Abercrombie (13).

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