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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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ES-derived ectodermal cells differentiate into K14/K5-positive epidermal cells upon ΔNp63 ectopic expression.An ectodermal K8+/K18+ cell line was derived by serial clonal dilutions of differentiating ES cells after BMP-4 treatment. (A) Immunofluorescence staining of the ES-derived ectodermal cell line 48 h after infection with a lentivirus expressing c-myc-tagged-ΔNp63. Control cells were transduced with a control c-myc-tagged-pax6 lentivirus. Nuclei are stained with DAPI. Bars = 50 µm. (B) Percentages of K14-positive cells amongst transduced cells 48 h after infection, as determined by immunofluorescence staining of c-myc and K14-positive cells. (C) Immunofluorescence staining of the ES-derived ectodermal cell line 48 h after cotransfection of c-myc-tagged-ΔNp63 and a p63-responsive reporter luciferase construct. Positive cells were detected with c-myc and luciferase antibodies. Nuclei are stained with DAPI. Bars = 50 µm.
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pone-0003441-g006: ES-derived ectodermal cells differentiate into K14/K5-positive epidermal cells upon ΔNp63 ectopic expression.An ectodermal K8+/K18+ cell line was derived by serial clonal dilutions of differentiating ES cells after BMP-4 treatment. (A) Immunofluorescence staining of the ES-derived ectodermal cell line 48 h after infection with a lentivirus expressing c-myc-tagged-ΔNp63. Control cells were transduced with a control c-myc-tagged-pax6 lentivirus. Nuclei are stained with DAPI. Bars = 50 µm. (B) Percentages of K14-positive cells amongst transduced cells 48 h after infection, as determined by immunofluorescence staining of c-myc and K14-positive cells. (C) Immunofluorescence staining of the ES-derived ectodermal cell line 48 h after cotransfection of c-myc-tagged-ΔNp63 and a p63-responsive reporter luciferase construct. Positive cells were detected with c-myc and luciferase antibodies. Nuclei are stained with DAPI. Bars = 50 µm.

Mentions: To further demonstrate that ΔNp63 is potent to induce ectodermal precursors to become keratinocytes, we isolated by serial dilution of BMP-4-treated ES cells at day 7, a homogenous ectodermal cell line positive for K8/K18 cytokeratins [25]. Exogenous expression of ΔNp63 in this ectodermal cell line indeed induced the differentiation of ΔNp63-transduced cells into K14-positive cells (Fig. 6-A and B). None of the control-transduced cells expressed the keratinocyte-specific genes (Fig. 6-B). Since not all (about 25–40%) of the ectodermal cells expressing de novo ΔNp63 became K14+-cells, we hypothesized that the ES-derived ectodermal K18+ cell population could be heterogeneous in its response to p63. To test if p63 still remains able to transactivate in these unresponsive cells, the ES-derived ectodermal cell line was cotransfected with ΔNp63 and a p63-responsive reporter construct [26]. 48 h after transfection, cells were analyzed by immunofluorescence staining for tagged-p63, luciferase and K14 gene expression (Fig. 6C). Interestingly, although not all the p63-transfected cells became K14+ keratinocytes, they all expressed luciferase, strongly suggesting that the K18+ ectodermal cells unable to become K14+ cells are locked by an unknown mechanism. Further studies will be necessary to identify the molecular differences between cells belonging to this apparent homogenous ectodermal cell population. Finally, p63-target genes were also induced upon ectopic expression of ΔNp63 (not shown, Rostagno et al, in preparation). Altogether, these results indicate that ΔNp63 is necessary to switch-on the differentiation program of pre-committed ectodermal cells into K14-positive 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)

ES-derived ectodermal cells differentiate into K14/K5-positive epidermal cells upon ΔNp63 ectopic expression.An ectodermal K8+/K18+ cell line was derived by serial clonal dilutions of differentiating ES cells after BMP-4 treatment. (A) Immunofluorescence staining of the ES-derived ectodermal cell line 48 h after infection with a lentivirus expressing c-myc-tagged-ΔNp63. Control cells were transduced with a control c-myc-tagged-pax6 lentivirus. Nuclei are stained with DAPI. Bars = 50 µm. (B) Percentages of K14-positive cells amongst transduced cells 48 h after infection, as determined by immunofluorescence staining of c-myc and K14-positive cells. (C) Immunofluorescence staining of the ES-derived ectodermal cell line 48 h after cotransfection of c-myc-tagged-ΔNp63 and a p63-responsive reporter luciferase construct. Positive cells were detected with c-myc and luciferase antibodies. Nuclei are stained with DAPI. Bars = 50 µm.
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Related In: Results  -  Collection

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pone-0003441-g006: ES-derived ectodermal cells differentiate into K14/K5-positive epidermal cells upon ΔNp63 ectopic expression.An ectodermal K8+/K18+ cell line was derived by serial clonal dilutions of differentiating ES cells after BMP-4 treatment. (A) Immunofluorescence staining of the ES-derived ectodermal cell line 48 h after infection with a lentivirus expressing c-myc-tagged-ΔNp63. Control cells were transduced with a control c-myc-tagged-pax6 lentivirus. Nuclei are stained with DAPI. Bars = 50 µm. (B) Percentages of K14-positive cells amongst transduced cells 48 h after infection, as determined by immunofluorescence staining of c-myc and K14-positive cells. (C) Immunofluorescence staining of the ES-derived ectodermal cell line 48 h after cotransfection of c-myc-tagged-ΔNp63 and a p63-responsive reporter luciferase construct. Positive cells were detected with c-myc and luciferase antibodies. Nuclei are stained with DAPI. Bars = 50 µm.
Mentions: To further demonstrate that ΔNp63 is potent to induce ectodermal precursors to become keratinocytes, we isolated by serial dilution of BMP-4-treated ES cells at day 7, a homogenous ectodermal cell line positive for K8/K18 cytokeratins [25]. Exogenous expression of ΔNp63 in this ectodermal cell line indeed induced the differentiation of ΔNp63-transduced cells into K14-positive cells (Fig. 6-A and B). None of the control-transduced cells expressed the keratinocyte-specific genes (Fig. 6-B). Since not all (about 25–40%) of the ectodermal cells expressing de novo ΔNp63 became K14+-cells, we hypothesized that the ES-derived ectodermal K18+ cell population could be heterogeneous in its response to p63. To test if p63 still remains able to transactivate in these unresponsive cells, the ES-derived ectodermal cell line was cotransfected with ΔNp63 and a p63-responsive reporter construct [26]. 48 h after transfection, cells were analyzed by immunofluorescence staining for tagged-p63, luciferase and K14 gene expression (Fig. 6C). Interestingly, although not all the p63-transfected cells became K14+ keratinocytes, they all expressed luciferase, strongly suggesting that the K18+ ectodermal cells unable to become K14+ cells are locked by an unknown mechanism. Further studies will be necessary to identify the molecular differences between cells belonging to this apparent homogenous ectodermal cell population. Finally, p63-target genes were also induced upon ectopic expression of ΔNp63 (not shown, Rostagno et al, in preparation). Altogether, these results indicate that ΔNp63 is necessary to switch-on the differentiation program of pre-committed ectodermal cells into K14-positive 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