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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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Epidermal differentiation of murine ES cells.(A) Appearance of K5+/K14+ and involucrin-positive epidermal cells from mouse ES cells treated with BMP-4 and serum. The differentiated cells were immunostained for cytokeratin K14, K5, K10 or involucrin at day 14 (panels a–e). Scale bars = 50 µm. Confocal analysis of differentiated cells (at day 14) (panel f). (B) Flow cytofluorimetry analysis of K5 and K14 on differentiated cells at day 14 of culture. The graphs represent the fluorescence intensity for the indicated proteins plotted against the cell size (forward scatter, FSC). The percentages of positive cells among the gated populations are indicated on graphs. (C) Gene expression was analyzed by real-time RT-PCR, for K18, K5, and K14 at the indicated differentiation time points (panel a), for laminin-5 chains at day 14 (panel b), and for K1, K10, filaggrin and involucrin at day 14 (panel c). The value for each gene was normalized to untreated control ES cultures, and represents the average of three independent experiments±sd.
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pone-0003441-g001: Epidermal differentiation of murine ES cells.(A) Appearance of K5+/K14+ and involucrin-positive epidermal cells from mouse ES cells treated with BMP-4 and serum. The differentiated cells were immunostained for cytokeratin K14, K5, K10 or involucrin at day 14 (panels a–e). Scale bars = 50 µm. Confocal analysis of differentiated cells (at day 14) (panel f). (B) Flow cytofluorimetry analysis of K5 and K14 on differentiated cells at day 14 of culture. The graphs represent the fluorescence intensity for the indicated proteins plotted against the cell size (forward scatter, FSC). The percentages of positive cells among the gated populations are indicated on graphs. (C) Gene expression was analyzed by real-time RT-PCR, for K18, K5, and K14 at the indicated differentiation time points (panel a), for laminin-5 chains at day 14 (panel b), and for K1, K10, filaggrin and involucrin at day 14 (panel c). The value for each gene was normalized to untreated control ES cultures, and represents the average of three independent experiments±sd.

Mentions: During ES cell differentiation, K5/K14-positive epithelial cells were detected by day 10 /11 and further proliferated into large patches by day 14 (Fig. 1-A panels a–c). FACS analysis indicated that, among the differentiated cells, about 10% became epidermal cells at day 14 (Fig. 1-B). The terminal differentiation events occured as K10-positive cells as well as involucrin-positive cells were also detected by immunofluorescence analysis (Fig. 1-A, panels d,e). These cells were in close contact to patches of K5/K14-positive cells (Fig. 1-A, panels d,e) suggesting that the stratification program was efficiently initiated during ES differentiation. Confocal analysis clearly confirmed that the differentiated K10-positive cells were different from the proliferating K14-positive cells (Fig. 1-A, panel f). Real-time Q-PCR (qRT-PCR) analysis was performed at different time points: day 5 (48 h after BMP-4 treatment), day 8 (24 h before serum addition) and day 14 (end of the culture) (Fig. 1-C). Transcription of the K18, K14 and K5 genes was induced after BMP-4 treatment (days 5 and 8). Following serum addition (day 14), K18 gene expression was reduced while transcription of K5 and K14 genes were enhanced (Fig. 1-C, panel a). A detailed kinetic experiment for K18 and K14 gene expression confirmed the progression along the epithelial developmental process (Fig. S2-A). The transcription of the three genes encoding laminin-5, the major adhesion ligand of keratinocytes, was also clearly induced during this epithelial differentiation process (Fig. 1-C, panel b). At day 14, specific markers of terminal differentiation of stratified epithelia (K1, K10, involucrin and filaggrin) were also detected (Fig. 1-C, panel c). These results indicate that this ES cell model recapitulates in vitro the stepwise appearance of stratified epidermal cells and is therefore suitable to study the function of p63 isoforms during embryonic epithelial cell differentiation.


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)

Epidermal differentiation of murine ES cells.(A) Appearance of K5+/K14+ and involucrin-positive epidermal cells from mouse ES cells treated with BMP-4 and serum. The differentiated cells were immunostained for cytokeratin K14, K5, K10 or involucrin at day 14 (panels a–e). Scale bars = 50 µm. Confocal analysis of differentiated cells (at day 14) (panel f). (B) Flow cytofluorimetry analysis of K5 and K14 on differentiated cells at day 14 of culture. The graphs represent the fluorescence intensity for the indicated proteins plotted against the cell size (forward scatter, FSC). The percentages of positive cells among the gated populations are indicated on graphs. (C) Gene expression was analyzed by real-time RT-PCR, for K18, K5, and K14 at the indicated differentiation time points (panel a), for laminin-5 chains at day 14 (panel b), and for K1, K10, filaggrin and involucrin at day 14 (panel c). The value for each gene was normalized to untreated control ES cultures, and represents the average of three independent experiments±sd.
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Related In: Results  -  Collection

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pone-0003441-g001: Epidermal differentiation of murine ES cells.(A) Appearance of K5+/K14+ and involucrin-positive epidermal cells from mouse ES cells treated with BMP-4 and serum. The differentiated cells were immunostained for cytokeratin K14, K5, K10 or involucrin at day 14 (panels a–e). Scale bars = 50 µm. Confocal analysis of differentiated cells (at day 14) (panel f). (B) Flow cytofluorimetry analysis of K5 and K14 on differentiated cells at day 14 of culture. The graphs represent the fluorescence intensity for the indicated proteins plotted against the cell size (forward scatter, FSC). The percentages of positive cells among the gated populations are indicated on graphs. (C) Gene expression was analyzed by real-time RT-PCR, for K18, K5, and K14 at the indicated differentiation time points (panel a), for laminin-5 chains at day 14 (panel b), and for K1, K10, filaggrin and involucrin at day 14 (panel c). The value for each gene was normalized to untreated control ES cultures, and represents the average of three independent experiments±sd.
Mentions: During ES cell differentiation, K5/K14-positive epithelial cells were detected by day 10 /11 and further proliferated into large patches by day 14 (Fig. 1-A panels a–c). FACS analysis indicated that, among the differentiated cells, about 10% became epidermal cells at day 14 (Fig. 1-B). The terminal differentiation events occured as K10-positive cells as well as involucrin-positive cells were also detected by immunofluorescence analysis (Fig. 1-A, panels d,e). These cells were in close contact to patches of K5/K14-positive cells (Fig. 1-A, panels d,e) suggesting that the stratification program was efficiently initiated during ES differentiation. Confocal analysis clearly confirmed that the differentiated K10-positive cells were different from the proliferating K14-positive cells (Fig. 1-A, panel f). Real-time Q-PCR (qRT-PCR) analysis was performed at different time points: day 5 (48 h after BMP-4 treatment), day 8 (24 h before serum addition) and day 14 (end of the culture) (Fig. 1-C). Transcription of the K18, K14 and K5 genes was induced after BMP-4 treatment (days 5 and 8). Following serum addition (day 14), K18 gene expression was reduced while transcription of K5 and K14 genes were enhanced (Fig. 1-C, panel a). A detailed kinetic experiment for K18 and K14 gene expression confirmed the progression along the epithelial developmental process (Fig. S2-A). The transcription of the three genes encoding laminin-5, the major adhesion ligand of keratinocytes, was also clearly induced during this epithelial differentiation process (Fig. 1-C, panel b). At day 14, specific markers of terminal differentiation of stratified epithelia (K1, K10, involucrin and filaggrin) were also detected (Fig. 1-C, panel c). These results indicate that this ES cell model recapitulates in vitro the stepwise appearance of stratified epidermal cells and is therefore suitable to study the function of p63 isoforms during embryonic epithelial cell differentiation.

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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