Fig. 1. Brachyury expression in Flk1⁺ and Flk1^– cells. (A) At day 2.75 of differentiation, wild-type EBs were harvested, dissociated and cells fractionated by cell sorting based on their level of Flk1 expression. (B) Blast-CFC potential was assayed in the unfractionated population (Presort), the Flk1⁺ and Flk1^– fractions. Blast cell colonies (Blast) and secondary EBs (2nd EBs) were scored following 4 days of culture. Data are presented as the mean number of colonies from three dishes. Bars, where visible, represent s.d. of the mean. (C) Expression analysis of subpopulations positive and negative for Flk1. 3' cDNA from pools of sorted cells (1000 cells per lane) was prepared by RT-PCR and total cDNAs were separated on a 1.5% agarose gel, blotted and probed separately with 3' probes from the indicated genes. (D) Expression analysis of single cells. One-hundred individual cells from the Flk1⁺ sorted fraction were picked using a mouth pipette and deposited directly into the lysis buffer. 3' cDNA was prepared and analyzed as described above. A representative expression pattern for 40 such individual cells is shown here. Hybridization with a 3' probe from the L32 ribosomal protein gene was included to control for amounts of cDNA in each lane.