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Mutant p63 causes defective expansion of ectodermal progenitor cells and impaired FGF signalling in AEC syndrome.

Ferone G, Thomason HA, Antonini D, De Rosa L, Hu B, Gemei M, Zhou H, Ambrosio R, Rice DP, Acampora D, van Bokhoven H, Del Vecchio L, Koster MI, Tadini G, Spencer-Dene B, Dixon M, Dixon J, Missero C - EMBO Mol Med (2012)

Bottom Line: The AEC mutation exerts a selective dominant-negative function on wild-type p63 by affecting progenitor cell expansion during ectodermal development leading to a defective epidermal stem cell compartment.These phenotypes are associated with impairment of fibroblast growth factor (FGF) signalling resulting from reduced expression of Fgfr2 and Fgfr3, direct p63 target genes.Restoring Fgfr2b expression in p63(+/L514F) epithelial cells by treatment with FGF7 reactivates downstream mitogen-activated protein kinase signalling and cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Fondazione IRCCS SDN, Napoli, Italy.

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The epidermal self-renewing compartment is reduced in p63+/L514F miceClonogenicity assay performed with freshly isolated epidermal cells cultured for 3 weeks reveals a reduced clonogenic potential of p63+/L514F keratinocytes.Left panel: Percentage of total number of p63+/L514F compared to +/+ clones. The difference is not statistically significant. Right panel: A reduction in clones larger than 2 mm is observed in p63+/L514F mice compared to +/+. Data are represented as mean ± SD (*p-value = 0.0076; n = 6).Clonogenicity assay performed with freshly isolated Fgfr2b−/− and +/+ keratinocytes cultured for 3 weeks. Clonogenicity assay reveals a reduction in the number of clones with a high-clonogenic potential (>2 mm) in Fgfr2b−/− mice compared to wild-type. Data are represented as mean ± SD (*p-value = 0.035; n = 6).Secondary and tertiary replating of clones reveals that p63+/L514F epidermal cells can self-renew similarly to +/+ in tertiary cultures. The percentage of secondary clones that formed tertiary clones is indicated (+/+ n = 37; p63+/L514F = 27; Fgfr2−/−n = 19).
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fig06: The epidermal self-renewing compartment is reduced in p63+/L514F miceClonogenicity assay performed with freshly isolated epidermal cells cultured for 3 weeks reveals a reduced clonogenic potential of p63+/L514F keratinocytes.Left panel: Percentage of total number of p63+/L514F compared to +/+ clones. The difference is not statistically significant. Right panel: A reduction in clones larger than 2 mm is observed in p63+/L514F mice compared to +/+. Data are represented as mean ± SD (*p-value = 0.0076; n = 6).Clonogenicity assay performed with freshly isolated Fgfr2b−/− and +/+ keratinocytes cultured for 3 weeks. Clonogenicity assay reveals a reduction in the number of clones with a high-clonogenic potential (>2 mm) in Fgfr2b−/− mice compared to wild-type. Data are represented as mean ± SD (*p-value = 0.035; n = 6).Secondary and tertiary replating of clones reveals that p63+/L514F epidermal cells can self-renew similarly to +/+ in tertiary cultures. The percentage of secondary clones that formed tertiary clones is indicated (+/+ n = 37; p63+/L514F = 27; Fgfr2−/−n = 19).

Mentions: Loss of p63 has been reported to affect the proliferative potential of stratified epithelial stem cells, including those of the skin (Senoo et al, 2007). To explore the possibility that mutant p63 may affect the pool of epidermal self-renewing cells, we first tested whether p63+/L514F cells have an altered clonogenic potential by plating freshly isolated newborn keratinocytes at clonogenic density and analyzing their ability to form clones over time. Initial plating efficiency was similar between mutant and wild-type cells. However, after 3 weeks in culture the number of large clones (>2 mm) generated by p63+/L514F cells was significantly lower than their wild-type counterparts (Fig 6A). Under clonogenic conditions, the overall mutant population was less proliferative (Supporting Information Fig S10A) consistent with a reduced number of clonogenic cells or with a reduced ability to proliferate under clonogenic conditions. However, no differences in cell proliferation were detected between p63+/L514F mutant and wild-type large clones 3 weeks after plating in spite of a maintained reduction in Fgfr2 protein expression (Supporting Information Fig S10B-C). Accordingly, Fgfr2b−/− keratinocytes had a very similar reduction in the number of large clones (>2 mm) compared to wild-type controls in spite of a similar initial plating efficiency (Fig 6B).


Mutant p63 causes defective expansion of ectodermal progenitor cells and impaired FGF signalling in AEC syndrome.

Ferone G, Thomason HA, Antonini D, De Rosa L, Hu B, Gemei M, Zhou H, Ambrosio R, Rice DP, Acampora D, van Bokhoven H, Del Vecchio L, Koster MI, Tadini G, Spencer-Dene B, Dixon M, Dixon J, Missero C - EMBO Mol Med (2012)

The epidermal self-renewing compartment is reduced in p63+/L514F miceClonogenicity assay performed with freshly isolated epidermal cells cultured for 3 weeks reveals a reduced clonogenic potential of p63+/L514F keratinocytes.Left panel: Percentage of total number of p63+/L514F compared to +/+ clones. The difference is not statistically significant. Right panel: A reduction in clones larger than 2 mm is observed in p63+/L514F mice compared to +/+. Data are represented as mean ± SD (*p-value = 0.0076; n = 6).Clonogenicity assay performed with freshly isolated Fgfr2b−/− and +/+ keratinocytes cultured for 3 weeks. Clonogenicity assay reveals a reduction in the number of clones with a high-clonogenic potential (>2 mm) in Fgfr2b−/− mice compared to wild-type. Data are represented as mean ± SD (*p-value = 0.035; n = 6).Secondary and tertiary replating of clones reveals that p63+/L514F epidermal cells can self-renew similarly to +/+ in tertiary cultures. The percentage of secondary clones that formed tertiary clones is indicated (+/+ n = 37; p63+/L514F = 27; Fgfr2−/−n = 19).
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Related In: Results  -  Collection

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fig06: The epidermal self-renewing compartment is reduced in p63+/L514F miceClonogenicity assay performed with freshly isolated epidermal cells cultured for 3 weeks reveals a reduced clonogenic potential of p63+/L514F keratinocytes.Left panel: Percentage of total number of p63+/L514F compared to +/+ clones. The difference is not statistically significant. Right panel: A reduction in clones larger than 2 mm is observed in p63+/L514F mice compared to +/+. Data are represented as mean ± SD (*p-value = 0.0076; n = 6).Clonogenicity assay performed with freshly isolated Fgfr2b−/− and +/+ keratinocytes cultured for 3 weeks. Clonogenicity assay reveals a reduction in the number of clones with a high-clonogenic potential (>2 mm) in Fgfr2b−/− mice compared to wild-type. Data are represented as mean ± SD (*p-value = 0.035; n = 6).Secondary and tertiary replating of clones reveals that p63+/L514F epidermal cells can self-renew similarly to +/+ in tertiary cultures. The percentage of secondary clones that formed tertiary clones is indicated (+/+ n = 37; p63+/L514F = 27; Fgfr2−/−n = 19).
Mentions: Loss of p63 has been reported to affect the proliferative potential of stratified epithelial stem cells, including those of the skin (Senoo et al, 2007). To explore the possibility that mutant p63 may affect the pool of epidermal self-renewing cells, we first tested whether p63+/L514F cells have an altered clonogenic potential by plating freshly isolated newborn keratinocytes at clonogenic density and analyzing their ability to form clones over time. Initial plating efficiency was similar between mutant and wild-type cells. However, after 3 weeks in culture the number of large clones (>2 mm) generated by p63+/L514F cells was significantly lower than their wild-type counterparts (Fig 6A). Under clonogenic conditions, the overall mutant population was less proliferative (Supporting Information Fig S10A) consistent with a reduced number of clonogenic cells or with a reduced ability to proliferate under clonogenic conditions. However, no differences in cell proliferation were detected between p63+/L514F mutant and wild-type large clones 3 weeks after plating in spite of a maintained reduction in Fgfr2 protein expression (Supporting Information Fig S10B-C). Accordingly, Fgfr2b−/− keratinocytes had a very similar reduction in the number of large clones (>2 mm) compared to wild-type controls in spite of a similar initial plating efficiency (Fig 6B).

Bottom Line: The AEC mutation exerts a selective dominant-negative function on wild-type p63 by affecting progenitor cell expansion during ectodermal development leading to a defective epidermal stem cell compartment.These phenotypes are associated with impairment of fibroblast growth factor (FGF) signalling resulting from reduced expression of Fgfr2 and Fgfr3, direct p63 target genes.Restoring Fgfr2b expression in p63(+/L514F) epithelial cells by treatment with FGF7 reactivates downstream mitogen-activated protein kinase signalling and cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Fondazione IRCCS SDN, Napoli, Italy.

Show MeSH
Related in: MedlinePlus