If one maintains the host embryo with its ventral side down during gastrulation, gravity will help maintain the grafted tissue in the ventral part of the blastocoel cavity, resulting in more uniform results across transplanted embryos Others may end up on a more lateral side, resulting in a smaller secondary embryo or a secondary embryo lacking a head. Some explants may end up on the anterior-most part of the embryo and therefore result in a well-formed head in the secondary embryo. The results may also vary depending on where the grafted tissue ends up at the conclusion of gastrulation. This difference is caused by a change in the host tissue’s competency to respond to the signals secreted by the Spemann–Mangold organizer. If the embryo is already at the gastrula stage, the secondary axis may only consist of trunk and tail tissues (although we obtained full head-twinning when grafting CNC at stage 10+). If the host is at blastula stage, the grafted tissue will be able to induce a full secondary embryo attached to the ventral or lateral side of the primary embryo. The quality of the secondary axis induced depends on two variables: the age of the host embryos at the time of transplantation and the position of the explant after the host gastrulation is complete. In the case of the gastrula blastopore lip, a stage 10+ explant will be able to induce a secondary embryo with a fully formed head while an explant taken later (stage 10.5 or 11) will induce more posterior structures such as trunk and tail ( Saxén and Toivonen 1962). The nature and extent of the induced axis will vary depending on the type of tissue or amount of growth factor used. Students that failed to induce secondary anterior structures with the Spemann–Mangold organizer transplants were usually able to do so using the Einsteck technique. This particular protocol has been used successfully in a classroom environment.
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