Fig. 1. Formation of early primitive ectoderm-like cells in suspension culture. (A-D). 7 µm sections of EBM⁴ (A,B) and EB⁴ (C,D) stained with Haemotoxylin and Eosin (A,C) and Hoechst 22358 (B,D) and viewed using brightfield (A,C) and fluorescent (UV2A filter; B,D) microscopy. Scale bar: 170 µm. (E) RT-PCR analysis for the presence of AFP and actin transcripts in EB⁴ and EB⁹, two independent populations of EBM⁴ and EBM⁹, and 8.5 d.p.c. mouse embryos. A control reaction in which reverse transcriptase was omitted is included (no RT). (F) Northern blot analysis of 20 µg of RNA isolated from EB^2-5 and EBM^2-5 probed for Oct4 (1.55 kb), Fgf 5 (2.7 kb), brachyury (2.1 kb) and mGAP (1.5 kb). (G-L). Whole-mount in situ hybridisation analysis of EBM⁴ (G,H,K) and EB⁴ (I,J,L) probed with digoxigenin-labelled antisense probes to Oct4 (G,I), Fgf5 (H,J) and brachyury (K,L). Scale bar: 85 µm.

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