The Giant Sandwich

In the giant explant, the entire AC, NIMZ, and IMZ are excised and sandwiched (Fig. 10). The vegetal region of the embryo is turned uppermost, and single-stroke cut is made (Fig. 4A), from the center of vegetal region toward the midventral line, through the entire embryo all the way to the AC (cut #1, Fig. 10A). The embryo is then turned over, and the AC, NIMZ and IMZ are pulled away from any mesoderm/endoderm that has involuted (Fig. 10A; see Subheading 3.2. and Fig. 4C). These tissues are separated from the involuted tissue, as well as the VE by a second cut at the boundary of the VE (cut #2, Fig. 10A). The explant is then laid out, inner surface uppermost. Any adherent, postinvolution cells are removed from its inner surface (see Subheading 3.3.

Fig. 10. To test the results of planar signaling in the context of the entire AC, a giant sandwich is used. A giant sandwich is made by cutting through the entire gastrula, from the ventral pole animally, along a line that extends from the center of the vegetal pole along the ventral midline (step #1, A). The embryo is then turned over, the entire gastrula wall peeled off from any involuting material, and the entire involuting marginal zone and AC cut away from the VE (step #2, A). This large piece, spanning the entire dorsoventral extent of the embryo, is then trimmed at the animal end to form a rectangle and sandwiched with a similar piece from another embryo to form a giant explant (B). As is the case with the standard sandwich explant, this explant shows convergent extension of both the neural and mesodermal regions. It differentiates neural and epidermal regions in the former, and notochord and somitic mesoderm in the latter (C). Prospective areas or the corresponding differentiated tissues are shown, including notochord (N), somitic mesoderm (S), epidermis (Ep), forebrain/midbrain (F), RH, SC, and neural tissue (Ne). The mesodermal prospective fates are shown as they would appear if the overlying epithelial endoderm were removed. The arrows in A and B show the anterior to posterior polarity of the neural and mesodermal tissues.

and Fig. 4D), and it is trimmed at the animal end to make a clean rectangle. The explant consists of the entire array of prospective tissues of the gastrula, its free edges at the ventral midline (Fig. 10B). Two of these giant explants are sandwiched together and allowed to develop. Both the neural and mesodermal/ endodermal regions of the explant converge and extend (Figs. 10C, 11A,B). Moreover, the cells of the neural ectoderm columnarize, wedge, and attempt to roll the neural plate into a neural tube. Generally, a neural groove, rather than a tube, forms on both sides of the explant (pointers, Fig. 11B). Patterning in such explants can be visualized by RNA whole-mount in situ hybridization according to the method of Harland (7). Otx2, a marker for forebrain and head meso-

Fig. 11. Giant sandwiches are shown shortly after construction (A) and at stage 27 (B). The vegetal end at the bottom and the AC is at the top of all figures. The neural region has converged, extended, and rolled into a neural trough (pointers, B), whereas the lateral regions form epidermis. The mesodermal/endodermal region converges and extends in the opposite direction, and consists of notochord (N) centrally and somitic mesoderm (S) on both sides (B). A whole-mount RNA in situ hybridization (C) shows an explant stained for Otx-2, expressed in the forebrain region of the giant explant (top of explant) and in the prechordal mesoderm (bottom of explant). An explant stained with the antibody tor 70 shows the notochord in the deep region of the mesodermal/ endodermal component of the explant (D).

Fig. 11. Giant sandwiches are shown shortly after construction (A) and at stage 27 (B). The vegetal end at the bottom and the AC is at the top of all figures. The neural region has converged, extended, and rolled into a neural trough (pointers, B), whereas the lateral regions form epidermis. The mesodermal/endodermal region converges and extends in the opposite direction, and consists of notochord (N) centrally and somitic mesoderm (S) on both sides (B). A whole-mount RNA in situ hybridization (C) shows an explant stained for Otx-2, expressed in the forebrain region of the giant explant (top of explant) and in the prechordal mesoderm (bottom of explant). An explant stained with the antibody tor 70 shows the notochord in the deep region of the mesodermal/ endodermal component of the explant (D).

derm (Fig. 11C), and staining with tor 70 antibody, a marker for notochord (Fig. 11D), shows the locations of these tissues in the giant sandwich explant.

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