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Fate and plasticity of the epidermis in response to congenital activation of BRAF.

Krishnaswami SR, Kumar S, Ordoukhanian P, Yu BD - J. Invest. Dermatol. (2014)

Bottom Line: Germline and somatic mutations in RAS and its downstream effectors are found in several congenital conditions affecting the skin.However, restoration of epidermal differentiation was non-cell autonomous and required dermal tissue to be present in tissue recombination studies.These studies indicate that early activation of the RAF signaling pathway in the ectoderm has effects on specific steps of epidermal differentiation, which may be amenable to treatment with currently available pharmacologic inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Division of Dermatology, Department of Medicine, Institute for Genomic Medicine, Stem Cell Program, University of California, San Diego, La Jolla, California, USA.

ABSTRACT
Germline and somatic mutations in RAS and its downstream effectors are found in several congenital conditions affecting the skin. Here we demonstrate that activation of BRAF in the embryonic mouse ectoderm triggers both craniofacial and skin defects, including hyperproliferation, loss of spinous and granular keratinocyte differentiation, and cleft palate. RNA sequencing of the epidermis confirmed these findings but unexpectedly revealed evidence of continued epidermal maturation, expression of >80% of epidermal differentiation complex genes, and formation of a hydrophobic barrier. Spinous and granular differentiation were restored by pharmacologic inhibition of MAPK/ERK kinase or BRAF. However, restoration of epidermal differentiation was non-cell autonomous and required dermal tissue to be present in tissue recombination studies. These studies indicate that early activation of the RAF signaling pathway in the ectoderm has effects on specific steps of epidermal differentiation, which may be amenable to treatment with currently available pharmacologic inhibitors.

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Expression of intermediate and late stage epidermal differentiation genes in K14-cre; BrafV600E mice(a) Differentiation markers used to identify basal (SB), spinous (SS), granular (SG), and cornified envelope (SC) keratinocytes. (b) Real-time PCR for gene expression at E17.5 skin reveals loss of spinous and granular differentiation markers in K14-cre; BrafV600E embryos and presence of late differentiation markers (Lce3b, Lce3c, Sprr2i, S100a1, S100a10). Error bars represent SD of four animals of each genotype. (c) E17.5 in situ hybridization of basal K5/K14, spinous K1/K10, granular Lor/Flg, involucrin (Ivl), and cornified envelope Lce3b keratinocyte gene expression. These studies show the loss of spinous and granular keratinocyte gene expression and pattern of Ivl and cornified envelope genes in upper layers. Suppl. Fig. S3 further localize gene expression by tyramide signal amplification. Scale bars, 20 µm.
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Figure 3: Expression of intermediate and late stage epidermal differentiation genes in K14-cre; BrafV600E mice(a) Differentiation markers used to identify basal (SB), spinous (SS), granular (SG), and cornified envelope (SC) keratinocytes. (b) Real-time PCR for gene expression at E17.5 skin reveals loss of spinous and granular differentiation markers in K14-cre; BrafV600E embryos and presence of late differentiation markers (Lce3b, Lce3c, Sprr2i, S100a1, S100a10). Error bars represent SD of four animals of each genotype. (c) E17.5 in situ hybridization of basal K5/K14, spinous K1/K10, granular Lor/Flg, involucrin (Ivl), and cornified envelope Lce3b keratinocyte gene expression. These studies show the loss of spinous and granular keratinocyte gene expression and pattern of Ivl and cornified envelope genes in upper layers. Suppl. Fig. S3 further localize gene expression by tyramide signal amplification. Scale bars, 20 µm.

Mentions: To confirm transcriptional changes identified by RNA-seq of pooled samples, real-time quantitative PCR (real-time PCR) for select differentiation markers was performed on individual embryos (Fig. 3 and Suppl. Table S1). K14-cre; BrafV600E consistently demonstrated high levels of basal lineage gene expression (K5, K14), while spinous (K1, K10) and granular (Lor, Flg) specific gene expression was markedly reduced (Fig. 3b). Late epidermal differentiation genes, Lce3b, Lce3c, S100a1, S100a10, and Sprr2i, were still expressed in the K14-cre; BrafV600E embryos and in some cases, expressed at higher levels than in wildtype littermates.


Fate and plasticity of the epidermis in response to congenital activation of BRAF.

Krishnaswami SR, Kumar S, Ordoukhanian P, Yu BD - J. Invest. Dermatol. (2014)

Expression of intermediate and late stage epidermal differentiation genes in K14-cre; BrafV600E mice(a) Differentiation markers used to identify basal (SB), spinous (SS), granular (SG), and cornified envelope (SC) keratinocytes. (b) Real-time PCR for gene expression at E17.5 skin reveals loss of spinous and granular differentiation markers in K14-cre; BrafV600E embryos and presence of late differentiation markers (Lce3b, Lce3c, Sprr2i, S100a1, S100a10). Error bars represent SD of four animals of each genotype. (c) E17.5 in situ hybridization of basal K5/K14, spinous K1/K10, granular Lor/Flg, involucrin (Ivl), and cornified envelope Lce3b keratinocyte gene expression. These studies show the loss of spinous and granular keratinocyte gene expression and pattern of Ivl and cornified envelope genes in upper layers. Suppl. Fig. S3 further localize gene expression by tyramide signal amplification. Scale bars, 20 µm.
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Figure 3: Expression of intermediate and late stage epidermal differentiation genes in K14-cre; BrafV600E mice(a) Differentiation markers used to identify basal (SB), spinous (SS), granular (SG), and cornified envelope (SC) keratinocytes. (b) Real-time PCR for gene expression at E17.5 skin reveals loss of spinous and granular differentiation markers in K14-cre; BrafV600E embryos and presence of late differentiation markers (Lce3b, Lce3c, Sprr2i, S100a1, S100a10). Error bars represent SD of four animals of each genotype. (c) E17.5 in situ hybridization of basal K5/K14, spinous K1/K10, granular Lor/Flg, involucrin (Ivl), and cornified envelope Lce3b keratinocyte gene expression. These studies show the loss of spinous and granular keratinocyte gene expression and pattern of Ivl and cornified envelope genes in upper layers. Suppl. Fig. S3 further localize gene expression by tyramide signal amplification. Scale bars, 20 µm.
Mentions: To confirm transcriptional changes identified by RNA-seq of pooled samples, real-time quantitative PCR (real-time PCR) for select differentiation markers was performed on individual embryos (Fig. 3 and Suppl. Table S1). K14-cre; BrafV600E consistently demonstrated high levels of basal lineage gene expression (K5, K14), while spinous (K1, K10) and granular (Lor, Flg) specific gene expression was markedly reduced (Fig. 3b). Late epidermal differentiation genes, Lce3b, Lce3c, S100a1, S100a10, and Sprr2i, were still expressed in the K14-cre; BrafV600E embryos and in some cases, expressed at higher levels than in wildtype littermates.

Bottom Line: Germline and somatic mutations in RAS and its downstream effectors are found in several congenital conditions affecting the skin.However, restoration of epidermal differentiation was non-cell autonomous and required dermal tissue to be present in tissue recombination studies.These studies indicate that early activation of the RAF signaling pathway in the ectoderm has effects on specific steps of epidermal differentiation, which may be amenable to treatment with currently available pharmacologic inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Division of Dermatology, Department of Medicine, Institute for Genomic Medicine, Stem Cell Program, University of California, San Diego, La Jolla, California, USA.

ABSTRACT
Germline and somatic mutations in RAS and its downstream effectors are found in several congenital conditions affecting the skin. Here we demonstrate that activation of BRAF in the embryonic mouse ectoderm triggers both craniofacial and skin defects, including hyperproliferation, loss of spinous and granular keratinocyte differentiation, and cleft palate. RNA sequencing of the epidermis confirmed these findings but unexpectedly revealed evidence of continued epidermal maturation, expression of >80% of epidermal differentiation complex genes, and formation of a hydrophobic barrier. Spinous and granular differentiation were restored by pharmacologic inhibition of MAPK/ERK kinase or BRAF. However, restoration of epidermal differentiation was non-cell autonomous and required dermal tissue to be present in tissue recombination studies. These studies indicate that early activation of the RAF signaling pathway in the ectoderm has effects on specific steps of epidermal differentiation, which may be amenable to treatment with currently available pharmacologic inhibitors.

Show MeSH
Related in: MedlinePlus