a, Single-cell injection; RNAs are co-injected with fluorescein-dextran (green) into wild-type embryos at the 128–256-cell stage; embryos are fixed 3 h later at 50% epiboly (left, lateral view; right, animal pole view). ntl, gsc, flh and bik expression is examined by whole-mount RNA in situ hybridization (blue stain); injected cells are stained brown by an anti-fluorescein antibody. b–n, Single-cell injections of RNA; all images are animal pole views. b, 6 pg of lacZ mRNA; no ectopic ntl expression is observed (n = 12/12). c–e, 4 pg of sqt RNA; ntl (c, n = 27/27) and flh (e, n = 18/18) are expressed in a wider domain than gsc (d, n = 34/35); arrowheads in c indicate the domain of ectopic ntl expression, about eight cell diameters from sqt-expressing cells (about 100 µm). f, g, 0.4 pg of sqt RNA; the ectopic ntl domain almost overlaps with the Sqt-producing cells (f, n = 14/15); no ectopic gsc expression is induced (g, n = 13/14). h, 4 pg of cyc–sqt RNA (n = 22/22); the ectopic ntl domain is similar to that induced by sqt RNA (c). i, j and l, 6 pg of cyc RNA; the ectopic ntl and bik domains almost overlap with the Cyc-producing cells (i, n = 18/20; l, n = 11/11) even though the level of Cyc is high enough to induce ectopic gsc (j, n = 16/18). k, 6 pg of sqt–cyc RNA; the ectopic ntl domain is similar to that induced by cyc RNA injection (n = 21/22). m, n, 4 pg of sqt RNA in wild-type (m) and MZoep (n) embryos; ectopic bik expression is induced up to eight cells away from the Sqt-producing cells in wild type (m, n = 20/20), but no ectopic bik expression is induced in MZoep embryo (n, n = 14/14). o–s, bik expression at the blastoderm margin at 50% epiboly in different genetic backgrounds, lateral views; bik expression is dependent on Nodal signalling around the entire margin.