Fig. 3. Expression pattern of kringelchen in buds as detected by in situ hybridization with a digoxigenin-labelled antisense probe. Staging of buds is according to Otto and Campbell (Otto and Campbell, 1977). (A,B) Stage 1 (ectoderm thickening and early evagination); (C) stage 3 (about 5 hours after first signs of evagination); (D) stage 4; (E-H) stage 5 (the tentacle in G,H belongs to the parent); (I-K) stage 7 (tentacle buds form); (L) stage 10 (shortly before detachment). (M-O) Budding region of the parent polyp after detachment: (M) immediately after detachment, (N) 30 minutes afterwards, (O) 60 minutes afterwards. (P) Semi-thin section of a stage 5 bud. (Q) Close-up of a stage 2 bud tip, in which about 60 ectodermal cells are distinguishable. (R,S) Single cell preparations of tissue derived from the budding region with kringelchen-positive epithelial cells. (R) Phase contrast imaging, allowing the evaluation of non labelled cell types; (S) a group of epithelial cells from the budding region shown by light microscopy.