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Fig. 9. Differentiation of Mixl1 heterozygote (GFP/w) and null (GFP/GFP) ES cells in serum-free (SF) media supplemented with BMP4 or activin A. In SF media, activin A (A) was a weaker inducer of GFP expression in Mixl1^GFP/w cells than BMP4 (B). In comparison with serum-induced differentiation (C), the magnitude of the response to SF+BMP4 media was less and GFP induction was delayed. There was no GFP expression in SF media alone. (D) BMP4 increased the percentage of viable cells isolated from day 4 and day 5 cultures of ES cells differentiated in SF media (P<0.01). Error bars represent 1 s.d. and P values were calculated using a two-tailed t-test (n=7). (E) The previously observed defect (see Fig. 5A) in the ability of Mixl1-null ES cells to generate FLK1⁺ cells at day 4 was exacerbated in SF+BMP4 differentiation cultures. At d7, differences in frequency of FLK1-positive cells between the two cell lines had diminished and both cell lines were able to generate CD34-positive cells. (F) Summary of methylcellulose culture experiments showing that Mixl1-deficient ES cells generated fewer haematopoietic CFCs in SF+BMP4 differentiation cultures than their heterozygous counterparts.

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