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The Organizer Experiment: Transplantation of the Upper Lip of the Blastopore

 

  • The organizer experiment represents the classical case of a complex induction. Spemann and Mangold discovered that the upper lip of the blastopore of a young urodele gastrula, when transplanted to the flank or ventral region of another young gastrula, they would invaginate, self differentiate into notochord and somites, and, in addition, induce adjacent host tissue to form a second medullary plate and supplementary mesodermal structures. The self differentiating transplant and the induced host structures together formed a well integrated set of secondary axial and paraxial structures, with its own polarity and bilateral symmetry, approaching a complete secondary embryo at the ventral region of the host embryo. This capacity for integrative inductions has earned the upper lip the designation organizer.

    In the organizer action, the following components can be distinguished :

  • a.The transplanted upper lip invaginates. It posses autonomous gastrulation tendencies.
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  • b.It self differentiates into notochord and somites or other mesodermal tissue.
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  • c.The transplant induces adjacent host mesoderm to form mesodermal structures, such as contributions to notochord, somites, lateral plate, etc.
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  • d.The transplant induces overlying host ectoderm to form a secondary neural tube, which is frequently subdivided into brain with optic vesicle and spinal cord.

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  • The most complete secondary embryos can be obtained when large median pieces of the dorsal lip of early gastrulae are implanted in the ventral lip, opposite to the dorsal lip of the host. See the figure on the right. . In this instance, the direction of invagination of the host and of the transplant will be parallel, and the host and donor structures will be in corresponding levels. In this way, the host will only slightly interfere with the formation of the secondary embryo.

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  • Procedure :

  • 1. Select gastrulae that have sickle shaped blastopores. Remove the outer jelly membranes. Wash the embryos in dilute medium. Transfer embryos to an operation dish with a higher concentration.
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  • 2. With a glass needle, make two flat wide groves in the agar bottom, they should be close together, so that both embryos are in the visual field. The grooves should be larger in diameter than the gastrulae, and not too deep. Smooth edges with a ball tip. Place donor and host in the grooves, they will orient themselves with the animal pole upward because they rotate freely in the perivitelline space.
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  • 3. Remove the vitelline membrane from both embryos. The embryos can now be oriented. Using the hair loop, turn them over with very gentle movements, so that the blastopores face upward.
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  • 4. Preparing the host - With the tip of a very fine needle, cut out a square area of the surface layer opposite to the blastopore, in the region where the ventral lip would later appear. Remove a few of the large yolk cells.
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  • 5. Cut out the transplant - Place the donor in such a position that the upper lips points away from you. Cut out a square piece of the upper, slightly larger than in the hole host. Make three cuts and include the upper lip itself as the fourth edge. Remove loose ectoderm cells on the inner surface with the tip of the glass needle. Do not lose the orientation of the transplant.
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  • 6. Implantation – Transfer the transplant to the host on the tip of the needle or with the hair loop. Implant in such away that the dorsal lip of the transplant is opposite to that of the host. Press the transplant gently into position, fit it in tightly, and enlarge the hole if necessary. Push the glass bridge over the transplant, and press it down gently so that the transplant fits in well and is in the desired orientation. Remove the donor embryo to another dish. Healing takes about an hour.
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  • 7. Once healed. Carefully remove the glass bridge. Very carefully, replace the concentrated solution stepwise by dilute medium, using a sterile pipette. Carefully release the fluid.
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  • 8. Over the next few days, try not to disturb the embryo. Whem gastrulation is complete, turn it right side up to allow neural folds to develop.

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  • Figure - Self differentiation of the dorsal blastopore lip tissue. The dorsal lip is transplanted into another gastrula in the region that would normally become the ventral epidermis. A second archenteron forms and then a second embryonic axis. Eventually, a second embryo forms that is joined to the host.