BL-CFC potential of Scl-expressing EB populations and Scl expression during blast colony development. (A) Percentage of cells expressing SCL in day-3 EBs. Boxes indicate the populations isolated by cell sorting. (B) Frequency (colonies/10⁵ cells) of blast colonies in isolated populations; PS indicates presort; SCL^-, SCL/lacZ^-; SCL⁺, SCL/lacZ⁺ fractions. Blast colonies (BLASTS; ▪) and secondary EBs (2° EBs; ▦) were scored after 4 days in culture. Data are presented as mean number of blast colonies from 3 dishes. Bars, where visible, represent SEM. (C) Expression analyses of the PS, SCL^-, and SCL⁺ fractions. Each lane represents the amplified products from 1000 single cells deposited directly into lysis buffer. The 3′ cDNA was prepared and analyzed as described.^30,45  (D) Total blast colonies in each fraction. Data are presented as mean number of blast colonies from 3 dishes. Bars, where visible, represent SEM. (E) FLK-1⁺ SCL/lacZ^- cells were isolated from day 3 EBs and replated into hemangioblast cultures. Shown is colony morphology at day 1, the core stage; day 2, the early hematopoietic stage; and day 3, the blast colony stage. Arrow indicates the developing vascular core at the day 2 early hematopoietic stage of blast colony development. Original magnification, × 400. Objective × 40, numerical aperture 0.50. MagnaFire software (Optronics, Goleta, CA) was used to analyze the images. (F) Expression analyses of the starting population and of colonies at the 3 different developmental stages; 10⁵ FLK-1⁺ SCL/lacZ^- (F⁺ S^-) cells, 1000 day 1 and day 2 pooled colonies and 500 day 3 pooled colonies were used for this analysis. Hybridization was carried out with probes designed against the 3′ UTR of the indicated genes. Hybridization to the ribosomal gene L-32 was included to control for the level of cDNA in each lane.