FIG. 10.

RA modulates the frequency of GATA-2-dependent hematopoietic colony formation by ES cells. (A) RT-PCR analysis of GATA-2 expression in an ES cell clone containing a TET-regulatable GATA-2 expression vector. The first lane (day 5) is from an ES culture grown for 5 days on OP9 stroma in the presence of added TET. The remaining lanes are samples taken at further 1-day intervals (days 6 to 9) after the initial day 5 culture had been trypsinized and reseeded onto OP9 stroma in the presence or absence of TET. (B and C) When grown on OP9 stroma, ES cells give rise to immature, definitive, multipotent, hematopoietic progenitors which begin to emerge at day 5 of culture. The effects of (i) induction of exogenous GATA-2 expression, (ii) addition of RA, and (iii) both were assessed at this time point by withdrawal of TET and/or addition of 1 μM RA or control diluent. Colonies were examined 2 days later (day 7). Photomicrographs of representative colonies are shown in panel B, and the arrowhead indicates a colony that has undergone pseudoemperipolesis. A summary of the number of colonies produced is presented in panel C; the results presented represent the average of six cultures analyzed. Panel D shows colony frequency data obtained in a similar series of experiments (n = 6) conducted with an ES cell line expressing a GATA-2-ER chimera. In this case the activity of the exogenous GATA-2-ER was regulated by addition of 1 μM β-estradiol.