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Fig. 1. Blocking vegetal signaling produces permanent blastulae with an expanded animal plate that differentiates as unpatterned neural tissue. (A-D,F) Embryos injected with -cadherin RNA. (A) DIC image of embryo (72 hour) injected with -cadherin RNA. (B) Anti-serotonin immunoreactive cells in a -cadherin-injected embryo. Confocal stack of the animal pole. (C) Anti-synaptotagmin immunoreactivity (SpSyn B) is principally in neurites of the serotonergic cells. (D) Merged image of B,C. (E) Merged image of control embryo (72 hours); serotonin (green) and synB (magenta) co-localize in apical organ neurons and synB identifies ciliary band neurons. (F) Detail of serotonergic and non-serotonergic neurons of the animal plate of a -cadherin RNA injected embryo (120 hours). (G) Anti-serotonin immunoreactive cells in embryo expressing Gsk. Inset is DIC image. (H) Permanent blastula from animal cap blastomeres with abundant unpatterned anti-serotonin immunoreactive cells. Inset is DIC image. (I) Uninjected embryo (72 hours) expressing SpHnf6 (green) in ciliary band and SpNk2.1 protein (magenta). Only the animal plate co-expresses these transcription factors. (J) A single confocal, optical section at the midline showing a lateral view of the region co-expressing SpHnf6 and SpNk2.1 (bracket). Enlargement of the region shown with asterisk in inset. o, oral ectoderm; ab, aboral ectoderm. (K-M) Recombined confocal image of -cadherin RNA injected embryo from animal pole. All of the thickened epithelial cells express SpHnf6 (K) and SpNk2.1 (L). (M) Merged image of (K,L). (N) Lateral view of a -cadherin RNA-injected embryo labeled with in situ RNA hybridization of SpHnf6 and immunolocalization of SpNk2.1. Arrowheads indicate the border between thick and thin epithelium. (O) Single confocal optical section of -cadherin RNA-injected embryo. Serotonergic cells (green) express SpNk2.1 (magenta), but SpNk2.1 is cytoplasmic rather than nuclear in these cells (arrows). Scale bar: 20 µm.

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