Fig. 1. Induction and monitoring of Gsc expression. (A) Our strategy for inserting GFP gene into Gsc allele. Southern blots demonstrate correct insertion of Gsc-gfp transgene and removal of Neo cassette. (B-F) Gsc induction under various culture conditions. Cultures were basically performed on collagen IV-coated dishes, except embryoid body (EB) formation experiments (right panels in B, part i and F). Gsc^gfp/+ES cells were cultured for 4 days (D4) or 6 days (D6). (B, part i) Day 4 FACS analysis of cells cultured in serum containing medium (SCM). (B, part ii) RT-PCR expression analysis of GFP⁺ and GFP^- population induced in SCM for 4 days. The specific molecules for the organizer were only expressed in GFP⁺ population. (B, part iii) GFP expression in tetraploid embryo. GFP expression was strongly detected in the organizer region of mid-streak stage embryo. (C-F) SF-O3 serum-free media (SFM) were used in Gsc induction. (C, part i) Selective induction of Gsc-GFP. Ninety-three percent of cells expressed Gsc-GFP on day 6 in the monolayer culture with activin. (C, part ii) RT-PCR analysis of GFP⁺ and GFP^- population on day 4. (C, part iii) Expression pattern of visceral endoderm markers. Upper panel shows RT-PCR analysis of total cells differentiated in SFM with activin for 4 days. Lower panel shows that of EBs differentiated in SCM for 4 days. (C, part iv) Immunostaining showing that almost all of cells on day 6 in SFM plus activin expressed brachyury protein. (D) Effect of Nodal on the generation of Gsc⁺ cells. Nodal showed a similar activity to induce Gsc⁺ cells. (E,F) Bmp4 and serum inhibited the generation of Gsc⁺ cells even in the presence of a large amount of activin. The culture with EB formation is less efficient for the generation of Gsc⁺ cells even in SFM (F), than the monolayer culture (C). The units displayed for activin (Act), nodal and Bmp4 are ng/ml.