Yap1 is dispensable for self-renewal but required for proper differentiation of mouse embryonic stem (ES) cells

Abstract

Yap1 is a transcriptional co-activator of the Hippo pathway. The importance of Yap1 in early cell fate decision during embryogenesis has been well established, though its role in embryonic stem (ES) cells remains elusive. Here, we report that Yap1 plays crucial roles in normal differentiation rather than self-renewal of ES cells. Yap1-depleted ES cells maintain undifferentiated state with a typical colony morphology as well as robust alkaline phosphatase activity. These cells also retain comparable levels of the core pluripotent factors, such as Pou5f1 and Sox2, to the levels in wild-type ES cells without significant alteration of lineage-specific marker genes. Conversely, overexpression of Yap1 in ES cells promotes nuclear translocation of Yap1, resulting in disruption of self-renewal and triggering differentiation by up-regulating lineage-specific genes. Moreover, Yap1-deficient ES cells show impaired induction of lineage markers during differentiation. Collectively, our data demonstrate that Yap1 is a required factor for proper differentiation of mouse ES cells, while remaining dispensable for self-renewal.

Keywords: Hippo pathway; Yap1; differentiation; embryonic stem cells; self‐renewal.

Figure EV1. Yap1 is dispensable for self‐renewal of J1 mouse ES cells (related to Fig 1)

1. Yap1 mRNA levels measured by RT–qPCR upon shRNA‐based KD. Five different shRNA sequences were tested, and shRNAs 1 and 2 (KD1 and KD2) were used for further studies. Data are represented as mean ± SD.

2. Cell proliferation rates of Yap1 KD cells and control cells.

3. mRNA levels of lineage‐specific marker genes upon KD of Yap1. Differentiating ES cells (dESC) were used as control cells. Data are represented as mean ± SD.

Figure 1. Yap1 is dispensable for self‐renewal of J1 mouse ES cells (see also Figs EV1, EV2, EV3)

1. A

   Colony morphology and alkaline phosphatase (AP) activity of ES cells upon KD of Yap1 and Pou5f1. KD1 and KD2 indicate two different shRNA sequences tested. All the following cell morphology and AP staining pictures were taken two passages (4 days) after lentivirus infection unless otherwise stated.

2. B, C

   mRNA expression levels of Pou5f1, Nanog, Sox2, Esrrb (B), and Yap1 (C) upon KD of Yap1. All the following mRNA samples were harvested 4 days after lentivirus infection while passaged every 2 days unless otherwise stated. Data are represented as mean ± SD.

3. D

   Protein levels of Yap1, Pou5f1, and Nanog upon KD of Yap1. All the following protein samples were harvested 4 days after lentivirus infection while passaged every 2 days unless otherwise stated.

4. E

   Colony morphology and AP activity of mouse embryonic stem cells (ESC) and three Yap1 KO clones (KO1‐KO3).

5. F

   mRNA levels of Pou5f1, Nanog, Sox2, and Esrrb upon KO of Yap1. Data are represented as mean ± SD.

6. G

   Protein levels of Yap1, Pou5f1, and Nanog in Yap1 KO clones.

7. H

   A heatmap showing relative mRNA expression levels of 3,605 genes differentially expressed (> twofold) between ES cells and differentiating ES cells (dESC). Genes were sorted by the fold changes of gene expression between dESC and ES cells. Corresponding gene expression profiles obtained from Yap1 KO1, Yap1 KO2, and Yap1 KD cells are also shown.

8. I

   mRNA expression levels of lineage‐specific marker genes upon KD of Yap1. dESC were used as control cells.

9. J

   A heatmap showing Pearson's correlation coefficients of gene expression profiles obtained from ESC, control virus‐infected ES cells (Control), dESC, Yap1 KD cells, and Yap1 KO cells.

10. K

    Relative average module activities (Core and PRC) in Yap1 KD1 cells, KO cells, and dESC. Module activities were normalized by the data obtained in ES cells. Data are represented as mean ± SEM.

11. L

    Scatter plots showing log₁₀ (FPKM) values of genes in Yap1 KD1 cells and Control (upper left panel), dESC and ESC (bottom left panel), and Yap1 KO cells and ES cells (right two panels). Pearson's correlation coefficients (R ²) are indicated. “FPKM” indicates fragments per kilobase of transcript per million fragments mapped.

Figure EV2. Yap1 is dispensable for self‐renewal of E14 and CJ7 mouse ES cells (related to Fig 1)

1. A–C

   Data from CJ7 ES cells. Colony morphology and AP activity of Yap1 KD cells (A), mRNA levels of Pou5f1, Nanog, Sox2, and Esrrb in Yap1 KD cells (B), and protein expression levels of Yap1, Pou5f1, and Gapdh were measured in Yap1 KD cells (C). mRNA level data are represented as mean ± SD.

2. D–F

   Data from E14 ES cells. Colony morphology and AP activity of Yap1 KD cells (D), mRNA levels of Pou5f1, Nanog, Sox2, and Esrrb in Yap1 KD cells (E), and protein expression levels of Yap1, Pou5f1, and Gapdh were measured in Yap1 KD cells (F). mRNA level data are represented as mean ± SD.

3. G–I

   Data from CJ7 ES cells (ESC). Colony morphology and AP activity of Yap1 KO clones (G), mRNA levels of Pou5f1, Nanog, Sox2, and Esrrb in Yap1 KO clones (H), and protein expression levels of Yap1, Pou5f1, and Gapdh were measured in Yap1 KO clones (I). mRNA level data are represented as mean ± SD.

4. J–L

   Data from E14 ES cells. Colony morphology and AP activity of Yap1 KO clones (J), mRNA levels of Pou5f1, Nanog, Sox2, and Esrrb in Yap1 KO clones (K), and protein expression levels of Yap1, Pou5f1, and Gapdh were measured in Yap1 KO clones (L). mRNA level data are represented as mean ± SD.

Figure EV3. Yap1‐depleted ES cells can maintain self‐renewal for more than a month in culture (related to Fig 1)

1. Colony morphology and AP activity of Yap1 KO clones cultured for more than a month.

2. mRNA levels of Pou5f1, Nanog, Sox2, and Esrrb in Yap1 KO clones shown in (A). Data are represented as mean ± SD.

3. Protein expression levels of Yap1, Pou5f1, and Gapdh were measured in Yap1 KO clones shown in (A).

Figure 2. Taz and Tead family proteins are not required for the self‐renewal of J1 mouse ES cells (see also Fig EV4)

1. A

   Colony morphology and AP activity of ES cells upon KD of Yap1 and Taz.

2. B–D

   mRNA expression levels of Yap1 and Taz (B), Pou5f1, Nanog, Sox2, and Esrrb (C), and lineage‐specific marker genes (D) upon KD of Yap1 and Taz. Data are represented as mean ± SD.

3. E

   Colony morphology and AP activity of ES cells upon KD of Tead1/Tead3/Tead4.

4. F, G

   mRNA expression levels of Tead1, Tead3, and Tead4 (F) and Pou5f1, Nanog, Sox2, and Esrrb (G) upon KD of Tead 1/3/4. Data are represented as mean ± SD.

5. H

   Protein levels of Tead1, Tead3, Tead4, and Pou5f1 upon KD of Tead1/Tead3/Tead4. N.S., non‐specific.

6. I

   mRNA expression levels of lineage‐specific marker genes upon KD of Tead1/Tead3/Tead4. Data are represented as mean ± SD.

Figure EV4. Tead family proteins are not required for the self‐renewal of ES cells (related Fig 2)

1. Colony morphology and AP activity of Tead2 KD ES cells.

2. mRNA levels of Tead2 measured by RT–qPCR upon shRNA‐based KD.

3. mRNA expression levels of Pou5f1, Nanog, Sox2, and Esrrb upon KD of Tead2. Data are represented as mean ± SD.

4. Protein levels of Pou5f1 and Nanog upon KD of Tead2.

5. Colony morphology of three Tead2 KO clones (KO1‐KO3) and control ES cells.

6. mRNA levels of Pou5f1, Nanog, Sox2, and Esrrb upon KO of Tead2. Data are represented as mean ± SD.

7. Protein levels of Pou5f1 and Nanog in Tead2 KO clones.

8. Colony morphology of ES cells upon KO of Tead4 and KD of Tead1/3.

9. mRNA levels of Pou5f1, Nanog, Sox2, and Esrrb upon KO of Tead4 and KD of Tead1/3. Data are represented as mean ± SD.

10. Protein levels of Pou5f1 and Nanog in Tead4 KO and Tead1/3 KD ES cells. N.S., non‐specific bands.

Figure 3. Yap1 is up‐regulated and translocated into nucleus during ES cell differentiation (see also Fig EV5)

1. A

   Relative mRNA levels of Yap1, Pou5f1, and Gata6 during time‐course embryoid body (EB) differentiation. Gene expression data were obtained from GSE3749. Pou5f1 and Gata6 serve as representative ES cell marker and lineage‐specific marker, respectively.

2. B

   Relative Yap1 mRNA levels in ES cells (ESC) and differentiating ES cells (dESC) (LIF withdrawal for 4 days) and data are represented as mean ± SD. To differentiate ES cells, cells were incubated in LIF‐withdrawn medium for 4 days. Both ESC and dESC were passaged every 2 days.

3. C

   Protein levels of Yap1 and Pou5f1 during time‐course differentiation upon LIF withdrawal.

4. D

   Phospho‐Yap1 levels during time‐course differentiation. Samples were normalized by total Yap1 level.

5. E–G

   Immunofluorescence (IF) images depicting localization of Yap1 in J1 (E), CJ7 (F), and E14 (G) mouse ESC (top) and dESC (bottom). The white arrow indicates nucleolus. Bottom panels represent higher magnification of the above panels. Dashed circle indicates nucleus border.

6. H

   A schematic diagram depicting a Yap1‐responsive luciferase reporter (8xGTIIC) construct.

7. I

   Luciferase reporter assay using Yap1‐responsive luciferase reporter (8xGTIIC) upon transient overexpression (OE) Yap1 in ES cells. P‐values were calculated using Student's t‐test. Data are represented as mean ± SD. **P < 0.01. “Control” indicates ES cells infected with control virus not expressing any specific shRNA sequence.

8. J

   Relative activity of Yap1‐responsive luciferase reporter gene in ESC and dESC. P‐values were calculated using Student's t‐test. Data are represented as mean ± SD. **P < 0.01.

9. K

   Relative Yap1 mRNA levels in Control and Pou5f1 KD ES cells. Data are represented as mean ± SD. **P < 0.01. “Control” indicates ES cells infected with control virus not expressing any specific shRNA sequence.

10. L

    IF images depicting localization of Yap1 in Control and Pou5f1 KD ES cells.

11. M

    Relative activity of Yap1‐responsive luciferase reporter gene upon Pou5f1 KD in ES cells. P‐values were calculated using Student's t‐test. Data are represented as mean ± SD. **P < 0.01.

Figure EV5. Yap1 is translocated into the nucleus upon differentiation of ES cells (related to Fig 3)

1. A

   Immunofluorescence (IF) images depicting Yap1 signals in J1 ES cells (ESC) and Yap1 KO clone.

2. B–D

   Quantification of relative Yap1 localization between ESC and differentiating ES cells (dESC) from three different cell lines: J1 (B), CJ7 (C), and E14 (D). See Appendix Supplementary Methods for detailed quantification method. Data are represented as mean ± SD.

Figure 4. Yap1 is required for differentiation of ES cells (see also Fig EV6)

1. A

   Colony morphology and AP activity of Control and Yap1 KD ES cells upon differentiation. Morphology and AP staining pictures were taken 2 days after differentiation.

2. B

   A heatmap showing relative mRNA expression levels of 1,995 genes differentially expressed (> twofold) between Yap1 KD ES cells and Control upon 4 days of differentiation. Genes were sorted by the fold changes of gene expression between Yap1 KD ES cells and Control (first column). Corresponding gene expression changes between ES cells (ESC) and differentiating ES cells (dESC) are shown in the second column.

3. C

   Relative average module activities (Core and PRC modules) between Yap1 KD ES cells and Control cells upon differentiation. Data are represented as mean ± SEM.

4. D

   Colony morphology and AP activity in Yap1 OE cells. Two different Yap1 OE clones (OE1 and OE2) and pool of Yap1 OE (OE pool) were used. Cell morphology and AP staining pictures were taken 3 weeks after electroporation.

5. E

   A heatmap showing relative mRNA expression levels of 2,137 genes differentially expressed (> twofold) between Yap1 OE ES cells and control ES cells. Genes were sorted by the fold changes of gene expression between Yap1 OE ES cells and control ES cells (first column) and corresponding gene expression profiles obtained from dESC are shown.

6. F

   Relative average module activities (Core and PRC modules) between Yap1 OE cells and control cells are shown. Data are represented as mean ± SEM.

7. G

   Genes up‐regulated in Yap1 OE cells were tested using David 6.7. Significantly enriched gene ontology (GO) terms (biological functions) are shown. Developmental process‐related GO terms are highlighted in red.

Figure EV6. Alteration of Yap1 affects differentiation of ES cells (related to Fig 4)

1. A, B

   mRNA levels of ES cell core factors (A) and lineage‐specific markers (B) in Yap1 KD1 cells upon 4 days of differentiation. White bars indicate the levels of genes tested in control virus‐infected ES cells (Control) upon differentiation. Data are represented as mean ± SD.

2. C

   Bar graphs showing significantly enriched gene ontology (GO) terms (biological functions). GO analysis of genes down‐regulated in Yap1 KD cells upon differentiation was performed using David 6.7 tools. Developmental process‐related terms are highlighted in red.

3. D

   IF images showing localization of Yap1 in control and Yap1 OE cells.

4. E

   Quantification of nuclear Yap1 in control and Yap1 OE cells. Data are represented as mean ± SD.

5. F

   mRNA levels of ES cell (ESC) core factors and lineage‐specific marker genes in Yap1 OE cells and Yap1 OE pool. Data are represented as mean ± SD.