Enforcement of developmental lineage specificity by transcription factor Oct1
- University of Utah School of Medicine, United States
- The Broad Institute of MIT and Harvard, United States
- Howard Hughes Medical Institute, Massachusetts Institute of Technology, United States
Figures
Figure 1 with 1 supplement
Abnormal developmental gene induction in ESCs lacking Oct1.
(A) Phase microscopy images of four ESC lines (two Oct1 deficient, two WT littermate controls) derived from Pou2f1-/+ intercrosses. Passage 5 ESCs on feeder fibroblasts are shown. (B) Immunoblot …
Figure 1—figure supplement 1
Abnormal morphology in differentiating Oct1-deficient cells manifests by day 5 of EB formation.
(A) Phase microscopy images of WT and Oct1-deficient ESCs aggregating into EBs. Top: 1 day of culture. Bottom: day 2. (B) Day 5. EBs are in low attachment dishes.
Figure 2 with 1 supplement
Effect of Oct1 loss on RA-mediated differentiation and neurogenesis.
(A) Quantitative RT-PCR results are shown for Pou5f1 (Oct4), Sox2, and Pou2f1 (Oct1) mRNA relative to a GAPDH standard. Average of three biological replicates ±standard deviation is shown. (B) …
Figure 2—figure supplement 1
β-tubulin III staining of neuralizing WT and Oct1-deficient EBs.
(A) WT cells. (B) Oct1-deficient cells. Two day-old EBs were used.
Figure 3
Defective cardiomyocyte differentiation in ESCs lacking Oct1.
(A) Cardiomyocytes were generated from individual EBs using 24-well dishes with gelatin. Functionality (±beating) was assessed for each well (16 per genotype) and plotted. (B) The wells assessed in …
Figure 4 with 2 supplements
Gene expression defects upon differentiation of Oct1 inducible-conditional ESCs.
(A) YFP-epifluorescence and phase microscopy images of inducible-conditional ESCs. Top: parent Pou2f1fl/fl cells were treated with 500 nM 4-OHT for 24 hr. A colony with good morphology and …
Figure 4—figure supplement 1
Steady-state metabolite levels in parental WT and 4-OHT-treated Pou2f1Δ/Δ ESCs as determined by GC-MS.
(A) Identified metabolites with total ion current (TIC) levels between 0.25 and 2.5. Normalization were achieved by setting the total ion current area under curve for all metabolites equal to each …
Figure 4—figure supplement 2
Images of parental WT and 4-OHT-treated KO ESCs differentiating in the presence of RA.
Left: phase microscopy images. Right: epifluorescence images taken using a YFP filter.
Figure 5 with 1 supplement
Smaller, less differentiated teratomas and lack of contribution to adult mouse tissues in Pou2f1Δ/Δ ESCs.
(A) 1 × 106 ESCs were injected into flanks (left flank: control Pou2f1fl/fl parent cells, right flank: derived Pou2f1Δ/Δ ESCs) of NCr Nude mice. Images are shown at 4 weeks. (B) Images of dissected …
Figure 5—figure supplement 1
Pluripotent phenotype of ESCs immediately prior to blastocyst injection.
Microscopy images of parental WT and two derived 4-OHT-treated, YFP-expressing, Pou2f1Δ/Δ ESC lines cultured in the absence of feeder fibroblasts. Images were taken by the University of Utah …
Figure 6 with 2 supplements
Genome-wide changes in developmental gene expression following differentiation of Oct1 conditional-inducible deficient ESCs.
(A) For each gene, averaged RNAseq FKPM (aligned Fragments Per Kilobase per Million aligned reads) values from three undifferentiated or 14-day RA-differentiated parent and KO ESCs were plotted on a …
Figure 6—figure supplement 1
Differences in gene expression in differentiated Oct1 deficient cells revealed by RNAseq.
(A) Heat map of gene expression differences across all biological replicates generated using unsupervised hierarchical clustering. 10,500 genes that vary at least fourfold across all samples were …
Figure 6—figure supplement 2
Differences in gene expression in differentiated Oct1 deficient cells revealed by RNAseq.
(A) Genome tracks of additional genes showing decreased expression in the differentiated state identified by RNAseq. (B) Genome tracks of elevated genes.
Figure 7 with 1 supplement
Unique and common Oct1 and Oct4 targets in ESCs.
(A) Venn diagram illustrating Oct1 and Oct4 target gene profile and intersection with RNAseq gene set. (B) Motif analysis for peaks occupied uniquely by Oct1 or Oct4, and for peaks occupied by both …
Figure 7—figure supplement 1
Oct1 and Oct4 ChIPseq read density at example co-bound genes.
(A) Genome tracks of Oct1 and Oct4 enrichment at Ell in ESCs. (B) Ahcy. (C) Rras2.
Figure 8
Oct1/Oct4 co-binding to MOREs in ESCs, and inducible Oct1 binding to poised targets upon differentiation.
(A) Conserved MOREs at two genes (Ahcy and Polr2a) co-bound by Oct1 and Oct4 in ESCs. The top co-bound de novo motif from Figure 7B is shown at top. The MORE sequence (Reményi et al., 2001; Tomilin …
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Videos
Video 1
Example WT ESC line cardiomyocyte differentiation 1.
https://doi.org/10.7554/eLife.20937.008
Video 2
Example WT ESC line cardiomyocyte differentiation 2.
https://doi.org/10.7554/eLife.20937.009
Video 3
Example WT ESC line cardiomyocyte differentiation 3.
https://doi.org/10.7554/eLife.20937.010
Video 4
Example WT ESC line cardiomyocyte differentiation 4.
https://doi.org/10.7554/eLife.20937.011
Video 5
Example WT ESC line cardiomyocyte differentiation 5.
https://doi.org/10.7554/eLife.20937.012
Video 6
Example WT ESC line cardiomyocyte differentiation 6.
https://doi.org/10.7554/eLife.20937.013
Video 7
Example WT ESC line cardiomyocyte differentiation 1.
https://doi.org/10.7554/eLife.20937.014
Video 8
Example WT ESC line cardiomyocyte differentiation 2.
https://doi.org/10.7554/eLife.20937.015
Video 9
Example WT ESC line cardiomyocyte differentiation 3.
https://doi.org/10.7554/eLife.20937.016
Video 10
Example WT ESC line cardiomyocyte differentiation 4.
https://doi.org/10.7554/eLife.20937.017
Video 11
Example WT ESC line cardiomyocyte differentiation 5.
https://doi.org/10.7554/eLife.20937.018
Video 12
Example WT ESC line cardiomyocyte differentiation 6.
https://doi.org/10.7554/eLife.20937.019Additional files
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Supplementary file 1
Normalized RNAseq gene expression changes.
- https://doi.org/10.7554/eLife.20937.031
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Supplementary file 2
Genes identified by ChIPseq in comparison to RNAseq data.
- https://doi.org/10.7554/eLife.20937.032
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Supplementary file 3
Oligonucleotides for RT-qPCR and ChIP.
- https://doi.org/10.7554/eLife.20937.033
