Has Vertical Signaling Already Occurred Use of Skewered Sandwiches

One must also be certain that vertical signals have not occurred prior to testing the effect of planar signals alone. As pointed out above, the prospective nervous system is very wide and very short, and Xenopus shows early, cryptic movement of the leading edge mesoderm inside. This means that the distance the leading edge of the organizer must move to come beneath a good part of the prospective nervous system, and the time it takes to do so are very short. If staging is not absolutely precise, or too late a stage is used, some opportunity for vertical signaling will already have occurred prior to experimental manipulations used to study solely planar signals (see refs. 52,80).

We use the following method to determine how far beneath the prospective neural ectoderm the Organizer has reached at the time of making explants. Explants are made at stages 10- through 10+. Also, as the dorsal sector is dissected, the highest point of contact of any involuted mesoderm/endoderm is marked by piercing the prospective neural ectoderm with an eyebrow hair (Fig. 8A, A'). Two similarly marked explants are selected and sandwiched together. A small glass bead (glass beads, acid-washed, 106 ^ and finer, US Sive 140, cat. no. G4649, Sigma Chemical Co.) is inserted into the hole made by the eyebrow hair (Fig. 8B, B'), marking the upper limit of mesodermal/endoder-mal contact. The two halves are then held together beneath a glass coverslip for healing. After healing, the coverslip and glass bead are removed and the explant is skewered to a bed of 2% agarose, with a hair pushed through the hole left by the bead. The hair permanently marks the site of mesodermal/endodermal contact (Fig. 8C, C'). Video microscopy shows convergent extension is not affected by the presence of the bead and hair. Convergent extension occurs, moving the explant with respect to the anchor point provided by the skewer. Using this method, we have found that expression of the homeobox gene Hoxb-1 is affected by the early vertical contact of PM with neural tissue, whereas

Fig. 8. The skewer method of marking the anterior limit of contact of the involuting mesoderm with the overlying neural ectoderm is shown in diagrams in cases of no contact (A-C) and substantial, anterior contact (A'-C'). When the AC is cut open, in preparation for making the explant, an eyebrow hair knife is used to puncture the outer gastrula wall at the point of inflection of the VE to gastrula wall at early stages (A), or at the top edge of contact of the involuted material with the gastrula wall (A'). Two explants with holes in matched or nearly matched positions are then sandwiched and the holes marked with small glass beads (B, B') before pressing them together under a coverslip. After the coverslip is removed, the beads are removed and the explants skewered to the bottom of a dish covered with 2% agarose. As the explants converge and extend, the position of the original contact is marked by the eyebrow hairs (C, C').

Fig. 8. The skewer method of marking the anterior limit of contact of the involuting mesoderm with the overlying neural ectoderm is shown in diagrams in cases of no contact (A-C) and substantial, anterior contact (A'-C'). When the AC is cut open, in preparation for making the explant, an eyebrow hair knife is used to puncture the outer gastrula wall at the point of inflection of the VE to gastrula wall at early stages (A), or at the top edge of contact of the involuted material with the gastrula wall (A'). Two explants with holes in matched or nearly matched positions are then sandwiched and the holes marked with small glass beads (B, B') before pressing them together under a coverslip. After the coverslip is removed, the beads are removed and the explants skewered to the bottom of a dish covered with 2% agarose. As the explants converge and extend, the position of the original contact is marked by the eyebrow hairs (C, C').

convergent extension is not (80). Expression of other neural properties and genes may or may not be affected by these early, transient signals.

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