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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.

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Affiliation: Fondazione IRCCS SDN, Napoli, Italy.

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Reduced progenitor marker expression in p63+/L514F and Fgfr2b−/− mice and in skin of AEC patientsImmunostaining reveals reduced Krt15 expression (green) in the epidermis and in the hair follicle of p63+/L514F and Fgfr2b−/− compared to +/+ control. Nuclei are stained with DAPI. Scale bar: 50 µm.Immunoblotting for Krt15 confirms decreased protein expression in p63+/L514F epidermis compared to +/+. Tubulin was used to normalize samples.Immunostaining for Krt15 (green) of skin biopsies from AEC patients (AEC1 and AEC2) and a human unaffected control (CTR) demonstrates reduced Krt15 expression in AEC skin. Scale bar: 50 µm.Real-time RT-PCR for Krt15 mRNA in human skin isolated from AEC patients or controls (CTR) reveals a quantitative reduction of Krt15 mRNA levels in the skin of AEC patients compared to wild-type. Data are represented as mean ± SD (p-value = 0.026).Immunofluorescence for Sox9 on neonatal mouse skin reveals reduced Sox9 expression in p63+/L514F and Fgfr2b−/− compared to +/+ control. Scale bar: 50 µm.Levels of Sox9 mRNA in human skin isolated from AEC patients or controls (CTR) were assessed by real-time RT-PCR. Data are represented as mean ± SD (p-value = 3.85 × 10−5).
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fig07: Reduced progenitor marker expression in p63+/L514F and Fgfr2b−/− mice and in skin of AEC patientsImmunostaining reveals reduced Krt15 expression (green) in the epidermis and in the hair follicle of p63+/L514F and Fgfr2b−/− compared to +/+ control. Nuclei are stained with DAPI. Scale bar: 50 µm.Immunoblotting for Krt15 confirms decreased protein expression in p63+/L514F epidermis compared to +/+. Tubulin was used to normalize samples.Immunostaining for Krt15 (green) of skin biopsies from AEC patients (AEC1 and AEC2) and a human unaffected control (CTR) demonstrates reduced Krt15 expression in AEC skin. Scale bar: 50 µm.Real-time RT-PCR for Krt15 mRNA in human skin isolated from AEC patients or controls (CTR) reveals a quantitative reduction of Krt15 mRNA levels in the skin of AEC patients compared to wild-type. Data are represented as mean ± SD (p-value = 0.026).Immunofluorescence for Sox9 on neonatal mouse skin reveals reduced Sox9 expression in p63+/L514F and Fgfr2b−/− compared to +/+ control. Scale bar: 50 µm.Levels of Sox9 mRNA in human skin isolated from AEC patients or controls (CTR) were assessed by real-time RT-PCR. Data are represented as mean ± SD (p-value = 3.85 × 10−5).

Mentions: To further evaluate the presence of cells with high-proliferative potential in mutant skin, we immunostained neonatal skin for markers that have been shown to correlate with these cells, such as Krt15 (Liu et al, 2003; Stachelscheid et al, 2008) and Sox9 (Nowak et al, 2008; Vidal et al, 2005). Krt15 expression was dramatically reduced in p63+/L514F and in Fgfr2b−/− neonatal epidermis and hair follicle as assessed by immunofluorescence and, for p63+/L514F epidermis, also by immunoblotting (Fig 7A and B). Importantly, a similar strong downregulation of Krt15 was also observed in AEC patient skin by immunofluorescence and by real-time RT-PCR (Fig 7C and D). Similarly, Sox9 was strongly reduced in p63+/L514F and Fgfr2b−/− hair follicles even in the rare well-formed follicles containing a visible bulge region (Fig 7E). A significant reduction of Sox9 mRNA was also detected in skin samples derived from AEC patients compared to wild-type controls (Fig 7F).


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)

Reduced progenitor marker expression in p63+/L514F and Fgfr2b−/− mice and in skin of AEC patientsImmunostaining reveals reduced Krt15 expression (green) in the epidermis and in the hair follicle of p63+/L514F and Fgfr2b−/− compared to +/+ control. Nuclei are stained with DAPI. Scale bar: 50 µm.Immunoblotting for Krt15 confirms decreased protein expression in p63+/L514F epidermis compared to +/+. Tubulin was used to normalize samples.Immunostaining for Krt15 (green) of skin biopsies from AEC patients (AEC1 and AEC2) and a human unaffected control (CTR) demonstrates reduced Krt15 expression in AEC skin. Scale bar: 50 µm.Real-time RT-PCR for Krt15 mRNA in human skin isolated from AEC patients or controls (CTR) reveals a quantitative reduction of Krt15 mRNA levels in the skin of AEC patients compared to wild-type. Data are represented as mean ± SD (p-value = 0.026).Immunofluorescence for Sox9 on neonatal mouse skin reveals reduced Sox9 expression in p63+/L514F and Fgfr2b−/− compared to +/+ control. Scale bar: 50 µm.Levels of Sox9 mRNA in human skin isolated from AEC patients or controls (CTR) were assessed by real-time RT-PCR. Data are represented as mean ± SD (p-value = 3.85 × 10−5).
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fig07: Reduced progenitor marker expression in p63+/L514F and Fgfr2b−/− mice and in skin of AEC patientsImmunostaining reveals reduced Krt15 expression (green) in the epidermis and in the hair follicle of p63+/L514F and Fgfr2b−/− compared to +/+ control. Nuclei are stained with DAPI. Scale bar: 50 µm.Immunoblotting for Krt15 confirms decreased protein expression in p63+/L514F epidermis compared to +/+. Tubulin was used to normalize samples.Immunostaining for Krt15 (green) of skin biopsies from AEC patients (AEC1 and AEC2) and a human unaffected control (CTR) demonstrates reduced Krt15 expression in AEC skin. Scale bar: 50 µm.Real-time RT-PCR for Krt15 mRNA in human skin isolated from AEC patients or controls (CTR) reveals a quantitative reduction of Krt15 mRNA levels in the skin of AEC patients compared to wild-type. Data are represented as mean ± SD (p-value = 0.026).Immunofluorescence for Sox9 on neonatal mouse skin reveals reduced Sox9 expression in p63+/L514F and Fgfr2b−/− compared to +/+ control. Scale bar: 50 µm.Levels of Sox9 mRNA in human skin isolated from AEC patients or controls (CTR) were assessed by real-time RT-PCR. Data are represented as mean ± SD (p-value = 3.85 × 10−5).
Mentions: To further evaluate the presence of cells with high-proliferative potential in mutant skin, we immunostained neonatal skin for markers that have been shown to correlate with these cells, such as Krt15 (Liu et al, 2003; Stachelscheid et al, 2008) and Sox9 (Nowak et al, 2008; Vidal et al, 2005). Krt15 expression was dramatically reduced in p63+/L514F and in Fgfr2b−/− neonatal epidermis and hair follicle as assessed by immunofluorescence and, for p63+/L514F epidermis, also by immunoblotting (Fig 7A and B). Importantly, a similar strong downregulation of Krt15 was also observed in AEC patient skin by immunofluorescence and by real-time RT-PCR (Fig 7C and D). Similarly, Sox9 was strongly reduced in p63+/L514F and Fgfr2b−/− hair follicles even in the rare well-formed follicles containing a visible bulge region (Fig 7E). A significant reduction of Sox9 mRNA was also detected in skin samples derived from AEC patients compared to wild-type controls (Fig 7F).

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