![]() 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. (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). In a recent study, using these techniques in combination with tissue- and region-specific markers, Storey et al. 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). QCPN) or using species-specific riboprobes in in situ hybridization analysis. 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. 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. In the avian embryo, operations involving Hensen's node at the primitive streak stage (10-20 hours' incubation, Hamburger & Hamilton (5) stages 3-5) are most easily performed in whole embryo culture, as described by New (6) (see 7). At this point, the three germ layers of the embryo are in very close apposition. 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. 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). 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. In: Molecular Embryology: Methods and Protocols. ![]()
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