The Edgewise Planar Apposition of Sandwiched Tissues

The best way to demonstrate planar induction is to place the Organizer in edgewise apposition to a responding tissue that normally does not undergo neural development. We have done such an experiment to demonstrate the planar induction of convergent extension (52) by grafting a labeled piece of Organizer to the edge of a piece of dorsal ectoderm, taken from far above the blastoporal lip, so that it did not include the original converging and extending neural tissue (Fig. 9A). In another experiment, a second Organizer was abutted to the animal end of the dorsal sector of the gastrula, inducing a second extension of polarity opposite that of the original (Fig. 9B). These explants can be made as sandwiches of two full thickness of tissue, grafted with their inner surfaces together, as shown above (Fig. 6). In this case, the explant consists of the outer epithelium, the apposed deep layers of each, and the outer epithelium of the second.

A somewhat easier way to make these explants is to excise one-half of such an explant from one embryo and graft to its inner, deep surface the corresponding epithelium from a second embryo, as shown in Fig. 9A,B. This provides a complete epithelial covering, maintaining the advantage of a controlled internal environment. In addition, these thinner explants are somewhat easier to abut edgewise than their thicker counterparts.

Multistep preparations such as these are easier done if several rules are followed. They should be done fast. Otherwise, problems will arise, such as rolling of the epithelial sheets, migration of the epithelium over the cut edges to be apposed, and "bowing" of the tissues, which results in tissues "saddling" crosswise to one another when the sandwich is made. Also, the preparations should be done in stages, at least until one develops expertise. For example, this experiment is easier if two sandwiches, one of the Organizer and one of the responding ectoderm, are made first. Each of these sandwiches consist of an explant and the corresponding epithelium from another embryo (steps #1 and #2, Fig. 9). When these sandwiches are slightly healed, they are removed from beneath their coverslips and abutted to one another edgewise (step #3, Fig. 9). A fresh wound surface should be made at the surfaces to be mated so that they will join and heal.

In any case, these "blunt-end ligations" of tissue are difficult. Epithelial sheets abhor a free edge, and immediately after being cut, their margins will migrate across the exposed deep cells at the ends of the explants, until they meet another epithelium. If the edges of the explants do not match precisely and a gap appears where the deep cells are abutted, the epithelial sheets will not span the breach. Rather, each epithelium will migrate down along its own deep cells to the bottom of the gap, where it will meet and heal with its counterpart. At this point, the continuous epithelium will pop up out of the groove and

Fig. 9. A method for edgewise apposition of inducing and responding tissue is shown. In the first case, Organizer tissue is placed in edgewise (planar) contact with AC ectoderm at the early gastrula (A). In the second case, a second organizer is grafted in planar apposition to the opposite end of the AC ectoderm, whereas the original Organizer is left in place (B). In both cases, the inducing and responding tissues are excised and sandwiched with an epithelial layer from the corresponding region of another embryo (steps #1 and #2, A and B), to provide a protective covering. The inducing and responding tissues are then abutted edgewise (step #3, A and B). A method of making a slight lap joint, which will aid in abutment and healing of tissues joined edgewise, is shown (C). One explant is cut such that flaps of epithelial tissue are exposed (#1, C), and the other is cut such that the deep nonepithelial cells are exposed (#2, C). The two are laid facing one another (#3, C) and then pushed together and held there with a coverslip until healed (# 4, C).

Fig. 9. A method for edgewise apposition of inducing and responding tissue is shown. In the first case, Organizer tissue is placed in edgewise (planar) contact with AC ectoderm at the early gastrula (A). In the second case, a second organizer is grafted in planar apposition to the opposite end of the AC ectoderm, whereas the original Organizer is left in place (B). In both cases, the inducing and responding tissues are excised and sandwiched with an epithelial layer from the corresponding region of another embryo (steps #1 and #2, A and B), to provide a protective covering. The inducing and responding tissues are then abutted edgewise (step #3, A and B). A method of making a slight lap joint, which will aid in abutment and healing of tissues joined edgewise, is shown (C). One explant is cut such that flaps of epithelial tissue are exposed (#1, C), and the other is cut such that the deep nonepithelial cells are exposed (#2, C). The two are laid facing one another (#3, C) and then pushed together and held there with a coverslip until healed (# 4, C).

form a smooth surface. This takes some time, and such slow or incomplete healing could block any induction that might have occurred.

To avoid these problems, we often make a slight lap joint, or "sticky-end ligation", in which the cut edges of the deep cells and epithelial cells do not coincide. One end of an explant is cut off squarely, and several rows of epithelial cells are lifted off the deep region at the edge, with the eyebrow hair (#1, Fig. 9C). This operation is repeated on the opposite side. A cut is then made downward, through most of the deep layer; the operation is repeated from the opposite side, removing a short section of deep layer (#3, Fig. 9C). On the other explant, the opposite operation is performed; an eyebrow hair is pushed back under the epithelium a couple of cells, and a hairloop is rubbed across the top, cutting off a short piece of epithelium (#2, Fig. 9C); the operation is repeated on the other side, making a notched explant (#3, Fig. 9C). If one works slowly, it is best to reverse the order of these operations, since the free epithelial sheets in the second type of explant will curl faster than their counterparts will advance across the deep cells in the first type of explant. Next, the edges of the free epithelial sheets are teased outward and the exposed deep cells of the other explant are stuffed in between them (#4, Fig. 9C). Finally, the entire preparation is pressed lightly between a coverslip and the bottom of the dish for the duration of healing, about 10-15 min.

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