Discovery of novel determinants of endothelial lineage using chimeric heterokaryons
- Houston Methodist Research Institute, United Staes
- University of Iowa, United Staes
- Stanford University School of Medicine, United States
Figures
Figure 1
Heterokaryon recapitulates gene expression of endothelial ontogeny.
(a) Scheme for heterokaryon generation. GFP-labeled murine ESCs (mESCs) were fused with Cell Tracker Red labeled human ECs (hECs) by HVJ-enveloped fusagen to induce multinucleated heterokaryons. (b) …
Figure 2
Heterokaryon bi-species RNA-seq reveals transcriptome reprogramming during differentiation of pluripotent stem cells into endothelial lineages.
(a) Heat map to show expression level of genes that are up- or down-regulated either uniquely in the Heterokaryon (Het) at 6 hr after fusion or commonly in all het samples relative to the mESC …
Figure 3
Role of Pou3f2 as a novel transcription factor in endothelial differentiation from murine embryonic stem cells.
(a) Gene expression pattern of Pou3f2 in heterokaryons consisting of mESC and hEC compared to Co-culture control. (b) Validation of expression of Pou3f2 during differentiation of mESC into …
Figure 4 with 3 supplements
Pou3f2 knockdown in the tg(fli1:EGFP)y1 zebrafish embryo.
(a) Bright-field images of embryos injected with caged morpholino against Pou3f2 translation start site in the absence of photoactivation (control), or with photoactivation with UV light at 6 or 24 …
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Figure 4—source data 1
Intersegmental vessel analysis in zebrafish embryos following Pou3f2 knockdown.
- https://doi.org/10.7554/eLife.23588.007
Figure 4—figure supplement 1
In-situ hybridization for Pou3f2 in zebrafish embryos.
Sense and Pou3f2-specific antisense RNA probe shows high expression of Pou3f2 in the head region, including the eye, hindbrain, midbrain and forebrain. Sense RNA probe was used as negative control.
Figure 4—figure supplement 2
Pou3f2 gene expression in endothelial and hematopoietic lineages.
Endothelial (A) and hematopoietic (B) cells were FACS purified from Tg(Fli1:EGFP cells) and Tg(C-myb:EGFP) larvae at 48 and 96 hpf, respectively. Total RNA was extracted and real-time PCR performed …
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Figure 4—figure supplement 2—source data 1
Real time PCR analysis of Pou3f2 in zebrafish embryo Fli1+ and Cmyb+ cells.
- https://doi.org/10.7554/eLife.23588.010
Figure 4—figure supplement 3
Gene expression of endothelial markers following Pou3f2 knockdown.
Total RNA was isolated from whole embryos injected with Pou3f2-targeted morpholino (Pou3f2-Mo) or a mismatch (Ctrl-Mo). Real-time PCR showed that Pou3f2 KD impaired significantly the expression of …
Figure 5
Role of Pou3f2 as a novel transcription factor in endothelial differentiation from human-induced pluripotent stem cells.
(a) Gene expression pattern of Pou3f2 in heterokaryons consisting of hiPSC and mEC compared to co-culture control. (b) Validation of expression of Pou3f2 during differentiation of hiPSC into …
Figure 6
Functional assays further reveal the importance of Pou3f2 during differentiation of human iPSC into endothelial phenotype.
(a–c) Representative FACS plots and summarized diagram showing Pou3f2 KD reduced iPSC-EC generation compared to scrambled control. (d) Representative immunofluorescence images revealed lower …
Figure 7
Chromatin immunoprecipitation (ChIP) and qPCR analysis reveal Pou3f2 binds to the promoters of endothelial-related transcription factors.
(a–d) Binding of Pou3f2 to the promoters of endothelial-related transcription factors including Ets1, Lmo2, Hey1 and Hey2 was significantly inhibited in Pou3f2 KD cells compared to scrambled control …
