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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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Time course of relative mRNA expression of several p63-regulated genes.(A) Real-time RT-PCR analysis for p63-targets genes at 3 differentiation time points, normalized to untreated control ES cultures. The data represents the average of three independent experiments±sd. (B) Relative mRNA expression for different p63-regulated genes. (A) Real-time RT-PCR analysis for p63-target genes at 3 differentiation time points, normalized to undifferentiated ES cells at day 0. (B) Real-time RT-PCR analysis for p63-target genes in control and sh-p63 cl2 ES cells at day 14 normalized to undifferentiated WT ES cells at day. The data represents the average of three experiments±sd.
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pone-0003441-g004: Time course of relative mRNA expression of several p63-regulated genes.(A) Real-time RT-PCR analysis for p63-targets genes at 3 differentiation time points, normalized to untreated control ES cultures. The data represents the average of three independent experiments±sd. (B) Relative mRNA expression for different p63-regulated genes. (A) Real-time RT-PCR analysis for p63-target genes at 3 differentiation time points, normalized to undifferentiated ES cells at day 0. (B) Real-time RT-PCR analysis for p63-target genes in control and sh-p63 cl2 ES cells at day 14 normalized to undifferentiated WT ES cells at day. The data represents the average of three experiments±sd.

Mentions: Recently, several genes dependent on p63 expression and associated with the murine early embryonic epidermal morphogenesis have been identified [16]–[18]. Among them, BMP-7, FGFR2b, Notch1 and Jagged1 transcripts have been shown to be absent from the developing epidermis of p63-deficient mice [18]. IKKα and GATA3 are two other genes regulated by p63 and associated with epidermal development [16], [19], [20]. Finally, Perp, a protein promoting the stable assembly of desmosomal adhesive complexes, was one of the first described p63 regulated proteins implicated in epidermal development [17]. We thus monitored their expression during ES epidermal differentiation as compared to undifferentiated ES cells. Upon BMP-4 treatment, in parallel to the induction of ΔNp63 gene expression, all the transcripts were detected by qRT-PCR (Fig. 4A). The relatively low activation of some of them (2–5 fold) as compared to the very pronounced induction of ΔNp63 (100 fold) reflected that ΔNp63 was not expressed at all before BMP-4 treatment (>35 cycles of qPCR) while mRNA of these genes were already detected before (22–25 cycles). To demonstrate that p63 is directly responsible for at least part of this modulation, the expression of potential p63-target genes were tested at day 14 in sh-63-cl2 ES cells as compared to control ES cells. Absence of p63 drastically reduced the expression of each gene during epidermal commitment, with the exception of BMP-7, which could be activated by an alternative mechanism, independent of the epidermal commitment (Fig. 4B). Similar results were obtained with the second sh-p63-cl1 (data not shown). We can conclude that the present model of ES-derived stratified epithelium commitment faithfully recapitulates molecular events known to be essential in epidermal morphogenesis.


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)

Time course of relative mRNA expression of several p63-regulated genes.(A) Real-time RT-PCR analysis for p63-targets genes at 3 differentiation time points, normalized to untreated control ES cultures. The data represents the average of three independent experiments±sd. (B) Relative mRNA expression for different p63-regulated genes. (A) Real-time RT-PCR analysis for p63-target genes at 3 differentiation time points, normalized to undifferentiated ES cells at day 0. (B) Real-time RT-PCR analysis for p63-target genes in control and sh-p63 cl2 ES cells at day 14 normalized to undifferentiated WT ES cells at day. The data represents the average of three experiments±sd.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2562986&req=5

pone-0003441-g004: Time course of relative mRNA expression of several p63-regulated genes.(A) Real-time RT-PCR analysis for p63-targets genes at 3 differentiation time points, normalized to untreated control ES cultures. The data represents the average of three independent experiments±sd. (B) Relative mRNA expression for different p63-regulated genes. (A) Real-time RT-PCR analysis for p63-target genes at 3 differentiation time points, normalized to undifferentiated ES cells at day 0. (B) Real-time RT-PCR analysis for p63-target genes in control and sh-p63 cl2 ES cells at day 14 normalized to undifferentiated WT ES cells at day. The data represents the average of three experiments±sd.
Mentions: Recently, several genes dependent on p63 expression and associated with the murine early embryonic epidermal morphogenesis have been identified [16]–[18]. Among them, BMP-7, FGFR2b, Notch1 and Jagged1 transcripts have been shown to be absent from the developing epidermis of p63-deficient mice [18]. IKKα and GATA3 are two other genes regulated by p63 and associated with epidermal development [16], [19], [20]. Finally, Perp, a protein promoting the stable assembly of desmosomal adhesive complexes, was one of the first described p63 regulated proteins implicated in epidermal development [17]. We thus monitored their expression during ES epidermal differentiation as compared to undifferentiated ES cells. Upon BMP-4 treatment, in parallel to the induction of ΔNp63 gene expression, all the transcripts were detected by qRT-PCR (Fig. 4A). The relatively low activation of some of them (2–5 fold) as compared to the very pronounced induction of ΔNp63 (100 fold) reflected that ΔNp63 was not expressed at all before BMP-4 treatment (>35 cycles of qPCR) while mRNA of these genes were already detected before (22–25 cycles). To demonstrate that p63 is directly responsible for at least part of this modulation, the expression of potential p63-target genes were tested at day 14 in sh-63-cl2 ES cells as compared to control ES cells. Absence of p63 drastically reduced the expression of each gene during epidermal commitment, with the exception of BMP-7, which could be activated by an alternative mechanism, independent of the epidermal commitment (Fig. 4B). Similar results were obtained with the second sh-p63-cl1 (data not shown). We can conclude that the present model of ES-derived stratified epithelium commitment faithfully recapitulates molecular events known to be essential in epidermal morphogenesis.

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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