Varying amounts of Organizer tissue (prospective PM and pharyngeal endoderm) can be included in the giant, pita, and standard sandwich explants. The amount of Organizer tissue included is determined by the age of the embryos from which the explants are made and where, with respect to the blas-toporal lip, the anterior end of the explant is trimmed. For example, when explants are made from stage 10 or 10+ embryos, trimming off the vegetal end of the explant just above the VE, at the level of the BC, will leave behind with the embryo most of the prospective PM, which has already involuted (Fig. 13A). In order to include prechordal mesoderm in explants at this stage, the following method should be used. The wall of the gastrula (the preinvolution IMZ-NIMZ-AC) should be peeled outward, as described above, and the postinvolution prospective PM cut away from the central endoderm of the gas-trula (Fig. 13B). The explant should be bent at the point of involution such that the AC-NIMZ-preinvolution IMZ and postinvolution IMZ material are spread open and apart, like the leaves of a strap hinge (Fig. 13C). Finally, two such explants are flattened and sandwiched together. The PM or head mesoderm will form a ball of tissue that does not converge and extend at the anterior end of the IMZ portion of the explant. It should stain with PM markers, such as Otx2 (Fig. 11C).
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