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DeltaNp63 is essential for epidermal commitment of embryonic stem cells.

Medawar A, Virolle T, Rostagno P, de la Forest-Divonne S, Gambaro K, Rouleau M, Aberdam D - PLoS ONE (2008)

Bottom Line: DeltaNp63 gene expression remains high during epithelial development.Interestingly, other epithelial cell fates are not affected, allowing the production of K5/K18-positive epithelial cells.Therefore, our results demonstrate that DeltaNp63 may be dispensable for some epithelial differentiation, but is necessary for the commitment of ES cells into K5/K14-positive squamous stratified epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U898, Nice, France.

ABSTRACT
In vivo studies have demonstrated that p63 plays complex and pivotal roles in pluristratified squamous epithelial development, but its precise function and the nature of the isoform involved remain controversial. Here, we investigate the role of p63 in epithelial differentiation, using an in vitro ES cell model that mimics the early embryonic steps of epidermal development. We show that the DeltaNp63 isoform is activated soon after treatment with BMP-4, a morphogen required to commit differentiating ES cells from a neuroectodermal to an ectodermal cell fate. DeltaNp63 gene expression remains high during epithelial development. P63 loss of function drastically prevents ectodermal cells to commit to the K5/K14-positive stratified epithelial pathway while gain of function experiments show that DeltaNp63 allows this commitment. Interestingly, other epithelial cell fates are not affected, allowing the production of K5/K18-positive epithelial cells. Therefore, our results demonstrate that DeltaNp63 may be dispensable for some epithelial differentiation, but is necessary for the commitment of ES cells into K5/K14-positive squamous stratified epithelial cells.

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Ectopic expression of ΔNp63 does not induce efficient stratified epithelial commitment of ES cells.(A) Differentiating ES cells were either treated with BMP-4 (at day 3) or transduced (at day 4) with lentiviruses expressing either ΔNp63 or irrelevant proteins (red tomato or Pax6) as negative controls. Real-time RT-PCR analysis for K5, K14 and laminin-5 gene expression was performed at days 5, 8 and 14 of ES cell differentiation. The value for each gene was normalized to either untreated control cultures for BMP-4-treated ES cells or red tomato transduced control cultures for p63-transduced ES cells. The values represent an average of three determinations±sd. (B) Wild type ES or ES cells stably expressing ΔNp63 were induced to differentiate with and without BMP-4. Real-time RT-PCR analysis for K5, K14 and p63 gene expression was performed at day 14. The value for each gene was normalized to untreated wild type ES cells at day 14. The values represent an average of three determinations±sd.
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pone-0003441-g005: Ectopic expression of ΔNp63 does not induce efficient stratified epithelial commitment of ES cells.(A) Differentiating ES cells were either treated with BMP-4 (at day 3) or transduced (at day 4) with lentiviruses expressing either ΔNp63 or irrelevant proteins (red tomato or Pax6) as negative controls. Real-time RT-PCR analysis for K5, K14 and laminin-5 gene expression was performed at days 5, 8 and 14 of ES cell differentiation. The value for each gene was normalized to either untreated control cultures for BMP-4-treated ES cells or red tomato transduced control cultures for p63-transduced ES cells. The values represent an average of three determinations±sd. (B) Wild type ES or ES cells stably expressing ΔNp63 were induced to differentiate with and without BMP-4. Real-time RT-PCR analysis for K5, K14 and p63 gene expression was performed at day 14. The value for each gene was normalized to untreated wild type ES cells at day 14. The values represent an average of three determinations±sd.

Mentions: Since ΔNp63 expression was detected at day 7, before the appearance of K14-positive cells, we evaluated whether exogenous expression of ΔNp63 could, in the absence of a BMP-4 dependent signalling pathway, induce keratinocyte commitment. ES cells were transduced, at day 4 of differentiation, with a lentiviral vector expressing ΔNp63. At days 8 and 14, expression of keratinocyte-specific gene was barely detected after infection with ΔNp63-lentivirus, as compared to cells cultivated in the presence of BMP-4 and serum (Fig. 5A). Careful systematic counting of large areas under the microscope detected very few K14+ cells (data not shown). Thus, ΔNp63 is not able to induce keratinocyte differentiation in absence of BMP-4. To define if ΔNp63 can synergize the effect of BMP-4, a ΔNp63-expressing construct was stably inserted into ES cells which were induced to differentiate with and without BMP-4. As illustrated in Fig. 5B, no enhancement of K14+ or K5+ cell production was detected by qRT-PCR when the ES cells were treated with both BMP-4 and ΔNp63 as compared to BMP-4 alone. Furthermore, infection of ES cells with a lentivirus expressing ΔNp63 at day 4 of differentiation did not enhance the effect of BMP-4 on keratinocyte production (data not shown). Since we previously have shown that BMP-4 efficiently induces ES cell to differentiate into ectodermal K8+/K18+ cells ([13] and Fig. S2-A), it suggested that ΔNp63 can induce epidermal commitment only in ES-derived cells that already have received and integrated ectodermal signals delivered by BMP-4.


DeltaNp63 is essential for epidermal commitment of embryonic stem cells.

Medawar A, Virolle T, Rostagno P, de la Forest-Divonne S, Gambaro K, Rouleau M, Aberdam D - PLoS ONE (2008)

Ectopic expression of ΔNp63 does not induce efficient stratified epithelial commitment of ES cells.(A) Differentiating ES cells were either treated with BMP-4 (at day 3) or transduced (at day 4) with lentiviruses expressing either ΔNp63 or irrelevant proteins (red tomato or Pax6) as negative controls. Real-time RT-PCR analysis for K5, K14 and laminin-5 gene expression was performed at days 5, 8 and 14 of ES cell differentiation. The value for each gene was normalized to either untreated control cultures for BMP-4-treated ES cells or red tomato transduced control cultures for p63-transduced ES cells. The values represent an average of three determinations±sd. (B) Wild type ES or ES cells stably expressing ΔNp63 were induced to differentiate with and without BMP-4. Real-time RT-PCR analysis for K5, K14 and p63 gene expression was performed at day 14. The value for each gene was normalized to untreated wild type ES cells at day 14. The values represent an average of three determinations±sd.
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Related In: Results  -  Collection

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pone-0003441-g005: Ectopic expression of ΔNp63 does not induce efficient stratified epithelial commitment of ES cells.(A) Differentiating ES cells were either treated with BMP-4 (at day 3) or transduced (at day 4) with lentiviruses expressing either ΔNp63 or irrelevant proteins (red tomato or Pax6) as negative controls. Real-time RT-PCR analysis for K5, K14 and laminin-5 gene expression was performed at days 5, 8 and 14 of ES cell differentiation. The value for each gene was normalized to either untreated control cultures for BMP-4-treated ES cells or red tomato transduced control cultures for p63-transduced ES cells. The values represent an average of three determinations±sd. (B) Wild type ES or ES cells stably expressing ΔNp63 were induced to differentiate with and without BMP-4. Real-time RT-PCR analysis for K5, K14 and p63 gene expression was performed at day 14. The value for each gene was normalized to untreated wild type ES cells at day 14. The values represent an average of three determinations±sd.
Mentions: Since ΔNp63 expression was detected at day 7, before the appearance of K14-positive cells, we evaluated whether exogenous expression of ΔNp63 could, in the absence of a BMP-4 dependent signalling pathway, induce keratinocyte commitment. ES cells were transduced, at day 4 of differentiation, with a lentiviral vector expressing ΔNp63. At days 8 and 14, expression of keratinocyte-specific gene was barely detected after infection with ΔNp63-lentivirus, as compared to cells cultivated in the presence of BMP-4 and serum (Fig. 5A). Careful systematic counting of large areas under the microscope detected very few K14+ cells (data not shown). Thus, ΔNp63 is not able to induce keratinocyte differentiation in absence of BMP-4. To define if ΔNp63 can synergize the effect of BMP-4, a ΔNp63-expressing construct was stably inserted into ES cells which were induced to differentiate with and without BMP-4. As illustrated in Fig. 5B, no enhancement of K14+ or K5+ cell production was detected by qRT-PCR when the ES cells were treated with both BMP-4 and ΔNp63 as compared to BMP-4 alone. Furthermore, infection of ES cells with a lentivirus expressing ΔNp63 at day 4 of differentiation did not enhance the effect of BMP-4 on keratinocyte production (data not shown). Since we previously have shown that BMP-4 efficiently induces ES cell to differentiate into ectodermal K8+/K18+ cells ([13] and Fig. S2-A), it suggested that ΔNp63 can induce epidermal commitment only in ES-derived cells that already have received and integrated ectodermal signals delivered by BMP-4.

Bottom Line: DeltaNp63 gene expression remains high during epithelial development.Interestingly, other epithelial cell fates are not affected, allowing the production of K5/K18-positive epithelial cells.Therefore, our results demonstrate that DeltaNp63 may be dispensable for some epithelial differentiation, but is necessary for the commitment of ES cells into K5/K14-positive squamous stratified epithelial cells.

View Article: PubMed Central - PubMed

Affiliation: INSERM, U898, Nice, France.

ABSTRACT
In vivo studies have demonstrated that p63 plays complex and pivotal roles in pluristratified squamous epithelial development, but its precise function and the nature of the isoform involved remain controversial. Here, we investigate the role of p63 in epithelial differentiation, using an in vitro ES cell model that mimics the early embryonic steps of epidermal development. We show that the DeltaNp63 isoform is activated soon after treatment with BMP-4, a morphogen required to commit differentiating ES cells from a neuroectodermal to an ectodermal cell fate. DeltaNp63 gene expression remains high during epithelial development. P63 loss of function drastically prevents ectodermal cells to commit to the K5/K14-positive stratified epithelial pathway while gain of function experiments show that DeltaNp63 allows this commitment. Interestingly, other epithelial cell fates are not affected, allowing the production of K5/K18-positive epithelial cells. Therefore, our results demonstrate that DeltaNp63 may be dispensable for some epithelial differentiation, but is necessary for the commitment of ES cells into K5/K14-positive squamous stratified epithelial cells.

Show MeSH
Related in: MedlinePlus