(a) Double in situ hybridization for Zic1/Foxi1c, Foxi1c/LPGDS and Foxi1c/RALDH2 showing that Zic1, LPGDS and RALDH2 are expressed at a distance from the PPR (Foxi1c-expressing cells). Frontal views, dorsal to top. Scale bars, 200 μm. (b) Animal explants dissected from embryos injected in one blastomere at the 2-cell stage with Zic1GR and GFP mRNAs were cultured for 8 hours in dexamethasone and analyzed by In situ hybridization. (c) In situ hybridization (ISH) for Foxi1c, Six1, LPGDS and RALDH2 on sections of animal explants derived from embryos injected with Zic1GR and GFP mRNA in one blastomere at the 2-cell stage (left panels). In each case the Zic1GR-expressing cells (GFP-positive) are shown (middle panels). Merge of fluorescence and in situ hybridization images (right panels). Three independent experiments were performed with similar results for each probe as pictured (Foxi1c, n=14; Six1, n=13; LPGDS, n=13 and RALDH2, n=11). Scale bars, 100 μm.