The transcription factor Pou3f1 promotes neural fate commitment via activation of neural lineage genes and inhibition of external signaling pathways
- Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China
- West China Hospital, Sichuan University, China
- CAS-MPG Partner Institute for Computational Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China
- Institute of Life Science, Nanchang University, China
Figures
Figure 1 with 4 supplements
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, …
Figure 1—figure supplement 1
Pou3f1-knockdown ESCs could differentiate into non-neural cell lineages.
(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 …
Figure 1—figure supplement 2
Brn2 could compensate for the Pou3f1 depletion during ESC neural fate commitment.
(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 …
Figure 1—figure supplement 3
Overexpression of Pou3f1 accelerates ESC neural differentiation in serum-free condition.
(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, …
Figure 1—figure supplement 4
Pou3f1 promotes neural differentiation in a cell-autonomous manner.
(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 …
Figure 2
Pou3f1 promotes the neural differentiation from EpiSCs to NPCs.
(A) Inducible Pou3f1-overexpressing ESCs were cultured as EBs for 2 days in the medium with or without Dox and then subjected to the ESD-EpiSC colony formation assay for 6 days in Dox-free CDM/AF …
Figure 3 with 1 supplement
Pou3f1 promotes neural fate commitment in vivo.
(A) Whole-mount in situ hybridization of Pou3f1 in early mouse embryos (E5.5–E8.0). The arrowhead marks the position-plane of the transverse section of the corresponding embryo below. Scale bars, …
Figure 3—figure supplement 1
Information of chimeric mice generated from Pou3f1-overexpressing or knockdown ESCs.
https://doi.org/10.7554/eLife.02224.010
Figure 4 with 1 supplement
RNA-seq and ChIP-seq analysis of Pou3f1 downstream targets.
(A) RNA-seq gene expression heat map of control and of inducible Pou3f1-overexpressing ESCs with Dox-treatment for 6 days. Heat-map colors (red, up-regulation; blue, down-regulation) indicate gene …
Figure 4—figure supplement 1
Pou3f1 is enriched in the loci of multiple downstream target genes.
(A) Correlation between Q-PCR and RNA-Seq data. Approximately, 30 genes were chosen to confirm the RNA-Seq results. (B) ChIP-qPCR verification of the ChIP-seq data represented in Figure 4E. Pou3f1 …
Figure 5 with 1 supplement
Pou3f1 increases neural lineage-specifier expression.
(A) Gene expression levels in control and in Pou3f1-knockdown ESCs differentiated in serum-free medium for 4 days. (B) Gene expression levels in control and in inducible Pou3f1-overexpressing ESCs …
Figure 5—figure supplement 1
HOMEO domain is essential for the neural-promoting effect of Pou3f1.
(A) Schematic structure of full-length and domain-deleted mutant Pou3f1 proteins. Row 1, full-length Pou3f1; Row 2, Pou3f1 without the N-terminus (ΔN, missing 1–244 amino acids); Row 3, Pou3f1 …
Figure 6 with 1 supplement
Pou3f1 represses BMP and Wnt signaling at the transcriptional level.
(A) Expression levels of BMP signaling target genes in control and Pou3f1-knockdown ESCs differentiated in serum-free medium. (B) Expression levels of BMP signaling target genes in control and …
Figure 6—figure supplement 1
Pou3f1 interferes with BMP and Wnt signaling pathways at the transcriptional level.
(A) In situ hybridization of cId1 expression (blue) in chick embryos that were electroporated with IRES-GFP (control vector, a and a′) or with Pou3f1-IRES-GFP (b and b′). GFP expression (brown) was …
Figure 7
Pou3f1 alleviates the inhibitory effects of BMP4 and Wnt3a on neural fate commitment.
(A) Inducible Pou3f1-overexpressing ESCs were cultured as EBs in serum-free medium for 4 days with or without BMP4/Dox treatment from day 2 to day 4. Gene expression levels were detected by Q-PCR. (B…
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Additional files
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Supplementary file 1
Primer list for PCR analysis. (A) The PCR primers used to clone Pou3f1 into the lentiviral expression vector pFUGW-IRES-EGFP. (B) Oligo sequences used for Pou3f1 RNAi. (C) Primers used for Real-time Q-PCR analysis. (D) Primers used for ChIP-qPCR.
- https://doi.org/10.7554/eLife.02224.018
