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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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p63 is required for epithelial differentiation of ES cells.Stable ES cell clones in which ΔNp63 gene expression has been inhibited with shRNA, were produced and induced to differentiate into the epidermal fate. (A) Analysis of ES clones transfected with a control pSuper sh-RNA (ctrl) or a pSuper sh-RNA specific for p63 (clones sh-1, sh-2). Wild type ES cells and stable clones were transiently transfected with a ΔNp63-expressing vector and analyzed 48 h later by Western blot with anti-ΔNp63 and anti-Erk2 (as loading control) antibodies. The two p63 sh-RNA clones displayed a strong inhibition of the ectopic ΔNp63 expression while the pSuper control was unable to regulate it. (B) Immunofluorescence staining for the indicated proteins in control and sh-p63 ES cell clones at day 14 of differentiation. Dapi staining is shown in blue. Bars = 50 µm. (C) Percentages of K14- or K5-positive cells differentiating from wt-ES cells, control-sh or p63-specific sh ES clones at day 14 determined by flow cytofluorometric analysis. (D) Real-time RT-PCR analysis at day 14 of differentiation for ΔNp63, K5, K14 and Lama3 (left panel) and for K1, K10, filaggrin and involucrin (right panel) genes. ES cell clones expressing either a control- or p63-specific shRNA were tested.
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pone-0003441-g003: p63 is required for epithelial differentiation of ES cells.Stable ES cell clones in which ΔNp63 gene expression has been inhibited with shRNA, were produced and induced to differentiate into the epidermal fate. (A) Analysis of ES clones transfected with a control pSuper sh-RNA (ctrl) or a pSuper sh-RNA specific for p63 (clones sh-1, sh-2). Wild type ES cells and stable clones were transiently transfected with a ΔNp63-expressing vector and analyzed 48 h later by Western blot with anti-ΔNp63 and anti-Erk2 (as loading control) antibodies. The two p63 sh-RNA clones displayed a strong inhibition of the ectopic ΔNp63 expression while the pSuper control was unable to regulate it. (B) Immunofluorescence staining for the indicated proteins in control and sh-p63 ES cell clones at day 14 of differentiation. Dapi staining is shown in blue. Bars = 50 µm. (C) Percentages of K14- or K5-positive cells differentiating from wt-ES cells, control-sh or p63-specific sh ES clones at day 14 determined by flow cytofluorometric analysis. (D) Real-time RT-PCR analysis at day 14 of differentiation for ΔNp63, K5, K14 and Lama3 (left panel) and for K1, K10, filaggrin and involucrin (right panel) genes. ES cell clones expressing either a control- or p63-specific shRNA were tested.

Mentions: To evaluate whether ΔNp63 was required for stratified epithelial commitment, stable ES cell clones that express a small hairpin (sh) RNA inhibiting p63 expression were produced. As shown in Fig. 3A, two independent ES cell clones (sh-p63-cl1 and sh-p63-cl2) displayed, by Western-blot analysis, a strong inhibition of exogenously expressed ΔNp63, which was not observed with the sh-control clone. Upon differentiation with BMP-4 and serum, the sh-p63 clones produced, at day 14, a number of ectodermal K18-positive cells similar to the wild type control cells (Fig. S3-A, B). It confirmed that the ectodermal differentiation of ES cells was independent of the expression of ΔNp63. However, while large patches of K14-positive cells were observed within the sh-control clone (Fig. 3-B), very few isolated K14-positive cells were detected with sh-p63-cl1 and sh-p63-cl2 clones at day 14. Analysis by FACS confirmed that the absence of ΔNp63 resulted in a strong reduction of K14 and K5-expressing cells at day 14 (Fig. 3-C). At the transcriptional level, we further confirmed the strong inhibition of the K14 mRNA level in differentiating sh-p63-ES clones (Fig. 3-D). Accordingly, lama3 gene expression, as well as terminal differentiation markers such as K1, K10, involucrin and filaggrin were completely repressed when p63 was knocked down (Fig. 3E). The two other genes encoding for laminin-5 (lamb3 and lamc2) were repressed as well in sh-p63 ES cells (data not shown). Interestingly, while K14 expression was drastically reduced, the expression of K5 mRNA was not modulated by the absence of ΔNp63 and a significant percentage of K5-positive cells was still observed by FACS analysis (Fig. 3-C) with the two sh-p63-ES clones (5.6% and 5.7%, respectively), as compared to sh-control and wt-ES cells (17.4% and 18.0%, respectively). In the absence of K14, K5-positive cells were not able to produce keratinocytes and accordingly we could no longer observe the patches of K14/K5-positive cells obtained during the differentiation of control ES cells (Fig. 3-B). Instead, the K5-positive cells, orphans of K14, were systematically detected within structures of rounded flat clones still positive for the K18 ectodermal marker (Fig. 3-B). Actually, this particular structure was also detectable within wt and sh-control ES cell differentiation along with the large K14/K5-positive patches. Since they were detected as early as day 11 and still present at the end of the culture (up to day 17), they may represent alternative epithelial cells.


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)

p63 is required for epithelial differentiation of ES cells.Stable ES cell clones in which ΔNp63 gene expression has been inhibited with shRNA, were produced and induced to differentiate into the epidermal fate. (A) Analysis of ES clones transfected with a control pSuper sh-RNA (ctrl) or a pSuper sh-RNA specific for p63 (clones sh-1, sh-2). Wild type ES cells and stable clones were transiently transfected with a ΔNp63-expressing vector and analyzed 48 h later by Western blot with anti-ΔNp63 and anti-Erk2 (as loading control) antibodies. The two p63 sh-RNA clones displayed a strong inhibition of the ectopic ΔNp63 expression while the pSuper control was unable to regulate it. (B) Immunofluorescence staining for the indicated proteins in control and sh-p63 ES cell clones at day 14 of differentiation. Dapi staining is shown in blue. Bars = 50 µm. (C) Percentages of K14- or K5-positive cells differentiating from wt-ES cells, control-sh or p63-specific sh ES clones at day 14 determined by flow cytofluorometric analysis. (D) Real-time RT-PCR analysis at day 14 of differentiation for ΔNp63, K5, K14 and Lama3 (left panel) and for K1, K10, filaggrin and involucrin (right panel) genes. ES cell clones expressing either a control- or p63-specific shRNA were tested.
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Related In: Results  -  Collection

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pone-0003441-g003: p63 is required for epithelial differentiation of ES cells.Stable ES cell clones in which ΔNp63 gene expression has been inhibited with shRNA, were produced and induced to differentiate into the epidermal fate. (A) Analysis of ES clones transfected with a control pSuper sh-RNA (ctrl) or a pSuper sh-RNA specific for p63 (clones sh-1, sh-2). Wild type ES cells and stable clones were transiently transfected with a ΔNp63-expressing vector and analyzed 48 h later by Western blot with anti-ΔNp63 and anti-Erk2 (as loading control) antibodies. The two p63 sh-RNA clones displayed a strong inhibition of the ectopic ΔNp63 expression while the pSuper control was unable to regulate it. (B) Immunofluorescence staining for the indicated proteins in control and sh-p63 ES cell clones at day 14 of differentiation. Dapi staining is shown in blue. Bars = 50 µm. (C) Percentages of K14- or K5-positive cells differentiating from wt-ES cells, control-sh or p63-specific sh ES clones at day 14 determined by flow cytofluorometric analysis. (D) Real-time RT-PCR analysis at day 14 of differentiation for ΔNp63, K5, K14 and Lama3 (left panel) and for K1, K10, filaggrin and involucrin (right panel) genes. ES cell clones expressing either a control- or p63-specific shRNA were tested.
Mentions: To evaluate whether ΔNp63 was required for stratified epithelial commitment, stable ES cell clones that express a small hairpin (sh) RNA inhibiting p63 expression were produced. As shown in Fig. 3A, two independent ES cell clones (sh-p63-cl1 and sh-p63-cl2) displayed, by Western-blot analysis, a strong inhibition of exogenously expressed ΔNp63, which was not observed with the sh-control clone. Upon differentiation with BMP-4 and serum, the sh-p63 clones produced, at day 14, a number of ectodermal K18-positive cells similar to the wild type control cells (Fig. S3-A, B). It confirmed that the ectodermal differentiation of ES cells was independent of the expression of ΔNp63. However, while large patches of K14-positive cells were observed within the sh-control clone (Fig. 3-B), very few isolated K14-positive cells were detected with sh-p63-cl1 and sh-p63-cl2 clones at day 14. Analysis by FACS confirmed that the absence of ΔNp63 resulted in a strong reduction of K14 and K5-expressing cells at day 14 (Fig. 3-C). At the transcriptional level, we further confirmed the strong inhibition of the K14 mRNA level in differentiating sh-p63-ES clones (Fig. 3-D). Accordingly, lama3 gene expression, as well as terminal differentiation markers such as K1, K10, involucrin and filaggrin were completely repressed when p63 was knocked down (Fig. 3E). The two other genes encoding for laminin-5 (lamb3 and lamc2) were repressed as well in sh-p63 ES cells (data not shown). Interestingly, while K14 expression was drastically reduced, the expression of K5 mRNA was not modulated by the absence of ΔNp63 and a significant percentage of K5-positive cells was still observed by FACS analysis (Fig. 3-C) with the two sh-p63-ES clones (5.6% and 5.7%, respectively), as compared to sh-control and wt-ES cells (17.4% and 18.0%, respectively). In the absence of K14, K5-positive cells were not able to produce keratinocytes and accordingly we could no longer observe the patches of K14/K5-positive cells obtained during the differentiation of control ES cells (Fig. 3-B). Instead, the K5-positive cells, orphans of K14, were systematically detected within structures of rounded flat clones still positive for the K18 ectodermal marker (Fig. 3-B). Actually, this particular structure was also detectable within wt and sh-control ES cell differentiation along with the large K14/K5-positive patches. Since they were detected as early as day 11 and still present at the end of the culture (up to day 17), they may represent alternative epithelial cells.

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