Fig. 5

Eomes functions downstream of Foxh1 in FLK1⁺ cell generation. a TGET ΔFoxh1 ES cells (BFP positive) were differentiated either by themselves (left) or with wild-type A2lox ES cells (BFP negative, right). Subsequently, BFP-negative (wild type) or BFP-positive (ΔFoxh1) cells were analyzed for FLK1, T-GFP, and Etv2-tdTomato expression on D4. b iFoxh1-∆Foxh1 EB cells were treated with 500 ng/mL DOX for different time span as indicated and analyzed by flow cytometry on D3.5. c RT-qPCR analysis of indicated genes in D3.5 iFoxh1-∆Foxh1 EB cells treated with 500 ng/mL DOX for different time span. Frzb and Smad1, two signaling factors important for mesoderm development were included in the list. d RT-qPCR analysis of Etv2 was performed using iFoxh1-∆Foxh1 ES cells differentiated with or without DOX (500 ng/mL, D2.5–3.5), and then for additional 1 day without DOX. e Eomes mRNA levels in indicated populations sorted from D3.5 TGET EBs and undifferentiated ES cells (“d0”) are shown. f Eomes mRNA levels are shown in indicated populations sorted from E7.5 embryos. g Flow cytometry analysis of D3.5 iEomes-∆Foxh1 EB cells treated with or without 50 ng/mL DOX for 24 h. h RT-qPCR analysis of Etv2 was performed using iEomes-∆Foxh1 ES cells differentiated with or without DOX (50 ng/mL, D2.5–3.5), and then for additional 1 day without DOX. *P value <0.05 in Student’s t test, **P < 0.01, ***P < 0.001, and n = 3. Error bars are s.d