Figure 3.

(A) flk-1^−/− ES cells give rise to EryP colonies in vitro. EryP progenitors were quantified from day 4 flk-1^−/−, +/− and +/+ EBs using methylcellulose colony assay. R1, wild-type R1 ES cells; 8145, a flk-1^+/− line; 47/48, two independent flk-1^−/− ES cell lines. Results obtained from three independent replatings were similar. An average number from one such replating, obtained from triplicate plates, is shown. Error bars indicate standard deviations. The difference in the number of EryP colonies between 47 and 48 could reflect clonal variations. (B) flk-1^−/− ES cells give rise to definitive erythroid and myeloid progenitors. Types of day 9 colonies produced: MAC, macrophage; ERY^D, definitive erythroid; Ery/MAC, mixed erythroid/macrophage; GM, granulocyte/macrophage; Mast, mast cell; Mix, mixed myeloid. Results obtained from three independent replatings were similar. An average number from one such replating, obtained from triplicate plates, is shown. Error bars indicate standard deviations. In all cases, while clonal variation is observed among cell lines used, overall, the hematopoietic potential of flk-1^−/− cells was indistinguishable from that of their +/− and +/+ counterparts. (C) Appearance of endothelial marker genes during differentiation of flk-1^−/−, +/−, +/+ ES cells. RT-PCR was performed with total RNA prepared from timed EBs, but with primers specific to a series of endothelial marker genes. The appearance of endothelial markers in flk-1^−/− day 3–day 7 EBs is indistinguishable from that of their +/− and +/+ counterparts. Marker gene expression was not detectable on day 0 (not shown). Transcripts detected are indicated on the right; d3–d7, days of differentiation; −/−, flk-1^−/−; +/−, flk-1^+/−; +/+, flk-1^+/+.