Neural Plate Explants

The neural plate can be excised cleanly from the underlying dorsal meso-derm as diagrammed in Fig. 14. The tip of the eyebrow hair should be pushed only through the neural plate, which consists of an epithelium and one layer of deep cells at these stages (stages 11-12.5), using the method described above (Fig. 4B). The depth of the neural plate is determined by experience, judging at what depth the eyebrow hair is two cells deep, staying on the shallow side. The eyebrow hair tip is used to tease away the edges of the two layers of the neural plate, searching for the easily separable interface that lies between the neural ectoderm and the underlying mesoderm. If only one layer comes up, the cut is too shallow. If the edge of the patch is lifted and more than two layers come up, the vertical cut likely was too deep, traversing into the mesoderm. In this case, the underlying mesoderm on the neural plate side of the cut will come

Fig. 14. The neural plate can be removed from later-stage gastrulae by first cutting through the two layers of the neural plate (NP) completely surrounding the area to be removed, using method diagrammed in Fig. 4B. A separation is then begun at the interface of the neural plate and underlying mesoderm, using the tip of the eyebrow hair. We prefer to begin at the posterior, right corner (asterisk). Once a sufficiently large area is lifted, the hairloop is used to deflect the neural plate animally, and the tip of the eyebrow hair is run along the interface, separating it from the underlying meso-derm. From stage 11.5 onward, the notochordal mesoderm (N) can be distinguished from somitic mesoderm (S) lying on both sides. The neural plate and underlying dorsal mesoderm can be removed together at late gastrula stages (B), by cutting (heavy dashed lines) through the body wall, into the archenteron (AR, outlined with light dashed lines), from its lateral aspect. A cross-section (C-C') shows the line of cut (dashed lines) through the body wall, into the archenteron (AR).

Fig. 14. The neural plate can be removed from later-stage gastrulae by first cutting through the two layers of the neural plate (NP) completely surrounding the area to be removed, using method diagrammed in Fig. 4B. A separation is then begun at the interface of the neural plate and underlying mesoderm, using the tip of the eyebrow hair. We prefer to begin at the posterior, right corner (asterisk). Once a sufficiently large area is lifted, the hairloop is used to deflect the neural plate animally, and the tip of the eyebrow hair is run along the interface, separating it from the underlying meso-derm. From stage 11.5 onward, the notochordal mesoderm (N) can be distinguished from somitic mesoderm (S) lying on both sides. The neural plate and underlying dorsal mesoderm can be removed together at late gastrula stages (B), by cutting (heavy dashed lines) through the body wall, into the archenteron (AR, outlined with light dashed lines), from its lateral aspect. A cross-section (C-C') shows the line of cut (dashed lines) through the body wall, into the archenteron (AR).

along with the neural plate. If this occurs, one should use the tip of the eyebrow hair to start a cleft at the interface of neural and mesodermal tissues. The neural cells are usually smaller and grayer than those of the mesoderm. However, once the integrity of the mesoderm has been violated, the forces of peeling off the neural plate may tear it further; if so, one must start over with a new embryo.

This procedure is easy to do at stages 10.5-11.5, but at stage 11.5 onward, the region lying above the notochord, the notoplate (87), progressively adheres more tightly to the underlying notochord. As one peels off the neural plate, the notoplate cells will tend to stay with the notochord, or vice versa. When the undersurface of the notoplate is inspected, gaps will appear, reflecting notoplate cells that adhered to the notochord. Conversely, cells sitting above the general surface of the notoplate are notochord cells that have adhered to the notoplate and were pulled out of the notochord. These cells must be stripped off with an eyebrow hair, one by one (see Fig. 4D) to assure a pure neural plate cell population. It is best to check the explant after fixation with tor 70 staining for notochord cells, since just a very few notochord cells can affect development of the neural plate (see ref. 80). Laterally, contamination of the neural plate with the underlying somitic cells is less of a problem, since these cells adhere weakly to the neural plate.

Another difficulty is controlling whether or not mesoderm is left in planar apposition with the posterior neural plate. This requires knowing precisely where the posterior boundary of the neural plate lies. If one desires mesoderm to be present, the explant should be cut at the blastoporal lip. If not, one must err on the neural side of the boundary to be assured that no mesoderm will be included. This presents a dilemma. Although considerable convergent extension has occurred at the mid- to late-gas-trula stages, the posterior neural tissue is still relatively short and wide. Cutting a little too far animally will remove all the prospective posterior neural tissue that would have converged, extended, and made the spinal cord. Likewise, lateral epidermis can be included in the explant, or not, depending on where the lateral cuts isolating the explant are made with respect to the boundaries shown in the fate maps. This type of explant has been used to study the effect of the lateral epidermis on morphogenesis of the neural tube in the newt and the axolotl (59).

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