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. 2014 Jun 14;3:e02224. doi: 10.7554/eLife.02224

Figure 1. Pou3f1 is essential for ESC neural differentiation.

(A) Schematic expression profiles of Pou3f1 and of several key marker genes during ESC neural differentiation in serum-free medium. Rex1, ESC marker; Fgf5, EpiSC marker; Sox1, NPC marker; Tuj1, neuron marker. Detection of Pou3f1 protein expression during ESC neural differentiation by Western blotting. (B) Gene expression levels in control-ESCs (Ctrl) and in Pou3f1-knockdown ESCs (Pou3f1-KD1, Pou3f1-KD3) at neural differentiation day 4 were determined by Q-PCR. Three independent experiments were performed. (C) Immunocytochemical assays of Sox/Oct4, Pax6, and Tuj1 in day 4 EBs described in B. DNA is stained with DAPI. Scale bars: 50 μm. (D) Statistical analysis of Sox+/Oct4−, Pax6+, and Tuj1+ cells in C. (E) Gene expression levels in control-ESCs and inducible Pou3f1-overexpressing (Pou3f1-OE) ESCs at unbiased differentiation (10%FBS) day 8 were determined by Q-PCR. Dox (2 μg/ml) was added for 8 days. (F) Immunocytochemical assays of Sox/Oct4, Pax6, Nestin, and of Tuj1 in day 8 EBs described in E. Scale bars, 50 μm. (G) Statistical analysis of Sox+/Oct4−, Pax6+, and Tuj1+ cells in F. (H) Pou3f1-knockdown ESCs were transfected with control or with Pou3f1-overexpressing lentiviruses. Gene expression levels at neural differentiation day 4 were determined by Q-PCR. The values represent the mean ± SD for B, D, E, G, and for H. (*p<0.05; **p<0.01).

DOI: http://dx.doi.org/10.7554/eLife.02224.003

Figure 1.

Figure 1—figure supplement 1. Pou3f1-knockdown ESCs could differentiate into non-neural cell lineages.

Figure 1—figure supplement 1.

(A) Expression profiling of Pou3f1 and of several key marker genes during ESC neural differentiation in serum-free medium, was determined by Q-PCR. (B) a, Knockdown efficiency of Pou3f1 with control-shRNA and Pou3f1-KD1/2/3 lentivirus-transfected ESCs was determined by Q-PCR. b, Knockdown of Pou3f1 protein by Pou3f1-shRNAs. (C) Gene expression levels in control and Pou3f1-knockdown ESCs were determined by Q-PCR. (D) Expression levels of germ layer genes in control and Pou3f1-knockdown ESCs at unbiased differentiation day 8 were determined by Q-PCR. The values represent the mean ± SD. (*p<0.05; **p<0.01).
DOI: http://dx.doi.org/10.7554/eLife.02224.004
Figure 1—figure supplement 2. Brn2 could compensate for the Pou3f1 depletion during ESC neural fate commitment.

Figure 1—figure supplement 2.

(A) Expression levels of the POU III family members Pou3f1, Brn1, and Brn2 during ESC neural differentiation in serum-free medium. (B) Brn1 and Brn2 expression levels in control and Pou3f1-knockdown ESCs were determined by Q-PCR. (C) Expression levels of POUIII family members in control, Pou3f1-knockdown and Pou3f1/Brn2-knockdown ESCs undergoing differentiation for 4 days in serum-free medium. (D) Expression levels of neural marker genes in control, Pou3f1-knockdown, and Pou3f1/Brn2-knockdown ESCs undergoing differentiation for 4 days in serum-free medium.
DOI: http://dx.doi.org/10.7554/eLife.02224.005
Figure 1—figure supplement 3. Overexpression of Pou3f1 accelerates ESC neural differentiation in serum-free condition.

Figure 1—figure supplement 3.

(A) Expression levels of neural marker genes in control and Pou3f1-stable-overexpression ESCs differentiated in serum-free medium from days 0 to 6. (B) Immunocytochemical assays of Sox/Oct4, Nestin, and Tuj1 in day 4 EBs described in A. Cells in day 4 EBs were replated in N2 medium for 2 days. Immunostaining of Tuj1 (red) was performed (g and h). DNA is stained with DAPI. Scale bars: 50 μm. (C) Statistical analysis of Sox+/Oct4−, Nestin+, and Tuj1+ cells in EBs and percentages of Tuj1+ cells in adherent culture during neural differentiation from days 0 to 6 in A.
DOI: http://dx.doi.org/10.7554/eLife.02224.006
Figure 1—figure supplement 4. Pou3f1 promotes neural differentiation in a cell-autonomous manner.

Figure 1—figure supplement 4.

(A) a, Q-PCR and b, Western blotting analysis of induced Pou3f1 overexpression. ESCs in adherent cultures were treated with Dox for 48 hr. (B) a, Immunocytochemical assays for Tuj1 (red) and GFP (green) using the co-cultured EBs. Wt ESCs (GFP−) were co-cultured with control ESCs (GFP+) or with Pou3f1-overexpressing ESCs (GFP+) in serum-free medium for 6 days. b, Cells in ‘a’ were immunostained by the Tuj1 antibody and then analyzed by fluorescence-activated cell sorting. The values represent the mean ± SD. (*p<0.05; **p<0.01).
DOI: http://dx.doi.org/10.7554/eLife.02224.007