| Method |
Operations were of two types, and were performed synchronously with the onset of blockage of the cell cycle. In one, a second stage 10 organizer was grafted into the marginal zone of a stage 10+ or 10.5 gastrula, at an angle of about 100° as described in Paper I. Typical results of such operation are described in Paper II (Cooke, 1972b). In the other, the head organizer region was simply excised, as from the donor in the previous operation, but from a stage 10+ gastrula. Control embryos at the time of blockage, and control and blocked, operated embryos at stages 14-15 and stages 22-24, were disaggregated in pairs in Ca2+- and Mg2+-free Holtfreter at pH 8-2, containing 150 mg/1 EDTA. After dissecting free the endodermal mass of large fragile and yolky cells from gastrulae, or their well-known derivatives from later stages, the remaining cell sheets, incorporating neurectoderm, mesoderm and a little head endoderm were transferred rapidly to 0-5 ml of the disaggregation solution per pair. After 10 min they were pipetted gently to a single-cell suspension with a Spemann pipette, and counted immediately. |