Figure 7: BMP and MAPK/ERK signalling cooperate to drive iHP reprogramming.

(a) Venn diagram demonstrating the numbers of 4 dpi Lmo2-bound genes that were upregulated in 14 dpi cells. The box demonstrates enrichment of genes associated with various signalling pathways that both Lmo2-bound (4 dpi) transcriptionally upregulated (14 dpi). (b) Mouse genome screenshots demonstrating the binding of 4 dpi Lmo2 onto the promoters of various signalling components (for example, Bmp4, Smad5, BMP4 cascade; Gab1, MAPK-MEK cascade). These are representatives from a single experiment. (c) Addition of signalling pathway modulators during SLRB iHP reprogramming reveals that specific inhibition of either BMP or MEK pathways abrogates iHP formation; SLR factors were delivered in one polycistronic construct. LDN193189 (0.4 μM), DMH1 (1 μM), PD0325901 (0.8 μM) and GSK1120212 (0.4 μM) were added every other day from 1–21 dpi. These data are from four independent experiments. (d) BMP signalling is critical for iHP reprogramming at early stage (from 1–7 dpi), while MEK signalling is required throughout SLRB iHP reprogramming. SLR factors were delivered in one polycistronic construct. Pictures shown exemplify hematopoietic colonies observed at 21 dpi. The effects of various small-molecule inhibitors on hematopoietic colony formation was normalized to DMSO-treated control cultures (21 dpi). Images are representative of four independent experiments. Scale bar: 100 μm. Data shown were mean±s.d. of four independent experiments. Chemicals were added at different time windows (I, II, III, IV and V) as diagrammed in c.