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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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Phenotype of K14-cre; BrafV600E neonatal and perinatal mice(a) Appearance of normal neonatal (upper row) and K14-cre; BrafV600E (bottom row) littermates. The K14-cre; BrafV600E skin appeared flaky and fissured overlying areas of translucent skin. (b) Cleft palate defects in K14-cre; BrafV600E whole mount preparations were counterstained with toluidine blue. (c) Histologic and immunofluorescent analysis of K14-cre; BrafV600E (BrafV600E) neonatal skin. Hematoxylin and eosin (HE) stained sections demonstrate thickened epidermis, separation of individual cells, and severe reduction in cytoplasmic keratohyalin granules. Immunofluorescent staining for K10, BrdU, loricrin (LOR), filaggrin (FLG) and K6 are shown for perinatal wildtype littermate and K14-cre; BrafV600E E18.5-P0 mouse skin. Note the absence of K10pos-spinous keratinocytes and LOR/FLGpos granular keratinocytes in the K14-cre; BrafV600E mouse skin. K6, a marker for hyperproliferative skin, is increased in the K14-cre; BrafV600E mouse skin. Scale bars, 20 µm.
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Figure 1: Phenotype of K14-cre; BrafV600E neonatal and perinatal mice(a) Appearance of normal neonatal (upper row) and K14-cre; BrafV600E (bottom row) littermates. The K14-cre; BrafV600E skin appeared flaky and fissured overlying areas of translucent skin. (b) Cleft palate defects in K14-cre; BrafV600E whole mount preparations were counterstained with toluidine blue. (c) Histologic and immunofluorescent analysis of K14-cre; BrafV600E (BrafV600E) neonatal skin. Hematoxylin and eosin (HE) stained sections demonstrate thickened epidermis, separation of individual cells, and severe reduction in cytoplasmic keratohyalin granules. Immunofluorescent staining for K10, BrdU, loricrin (LOR), filaggrin (FLG) and K6 are shown for perinatal wildtype littermate and K14-cre; BrafV600E E18.5-P0 mouse skin. Note the absence of K10pos-spinous keratinocytes and LOR/FLGpos granular keratinocytes in the K14-cre; BrafV600E mouse skin. K6, a marker for hyperproliferative skin, is increased in the K14-cre; BrafV600E mouse skin. Scale bars, 20 µm.

Mentions: To activate BRAF in the ectoderm, we utilized a mouse model, where expression of a mutant allele (BrafV600E) remains under the control of its endogenous locus and produces mutant product following Cre-mediated deletion of a transcription stop cassette (Dankort et al., 2007). BrafV600E floxed females were bred to Keratin 14 (K14)-cre) transgenic males, which express Cre in the epidermis at embryonic day (E) 14.5 (Vasioukhin et al., 1999). K14-cre-positive, BrafV600E-positive (K14-cre; BrafV600E) offspring were produced at near expected Mendelian frequency when assessed prior to birth (25.6% observed; 25% expected from 167 late stage embryos). Postnatally, the majority of K14-cre; BrafV600E newborns were cannibalized by adults, and at the time of weaning, only 3 K14-cre; BrafV600E mice out of > 20 litters were detected at the time of weaning. In litters observed at the moment of birth, K14-cre; BrafV600E newborns showed severe ectodermal defects, including thick, fissured scale overlying translucent edematous skin and displayed rhythmic ventilation and pink oxygenation. Further examination of K14-cre; BrafV600E newborns also revealed lack of ingested milk in their stomachs and cleft palate defects in >84% (Fig. 1b). The latter defect may result from Cre expression in the palate epithelium of K14-cre animals (Okubo et al., 2009). Histologic analysis of the skin revealed a thickened epidermis, basaloid cells, cytolysis and loss of keratohyalin granules (Fig. 1c). Although a stratified epithelium was present in K14-cre; BrafV600E mice, immunofluorescent analysis revealed loss of K10+ spinous and LOR/FLG+ granular keratinocytes (Fig. 1c). The K14-cre; BrafV600E epidermis was hyperproliferative as evidenced by increased BrdU-staining and the overexpression of K6 protein.


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)

Phenotype of K14-cre; BrafV600E neonatal and perinatal mice(a) Appearance of normal neonatal (upper row) and K14-cre; BrafV600E (bottom row) littermates. The K14-cre; BrafV600E skin appeared flaky and fissured overlying areas of translucent skin. (b) Cleft palate defects in K14-cre; BrafV600E whole mount preparations were counterstained with toluidine blue. (c) Histologic and immunofluorescent analysis of K14-cre; BrafV600E (BrafV600E) neonatal skin. Hematoxylin and eosin (HE) stained sections demonstrate thickened epidermis, separation of individual cells, and severe reduction in cytoplasmic keratohyalin granules. Immunofluorescent staining for K10, BrdU, loricrin (LOR), filaggrin (FLG) and K6 are shown for perinatal wildtype littermate and K14-cre; BrafV600E E18.5-P0 mouse skin. Note the absence of K10pos-spinous keratinocytes and LOR/FLGpos granular keratinocytes in the K14-cre; BrafV600E mouse skin. K6, a marker for hyperproliferative skin, is increased in the K14-cre; BrafV600E mouse skin. Scale bars, 20 µm.
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Related In: Results  -  Collection

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Figure 1: Phenotype of K14-cre; BrafV600E neonatal and perinatal mice(a) Appearance of normal neonatal (upper row) and K14-cre; BrafV600E (bottom row) littermates. The K14-cre; BrafV600E skin appeared flaky and fissured overlying areas of translucent skin. (b) Cleft palate defects in K14-cre; BrafV600E whole mount preparations were counterstained with toluidine blue. (c) Histologic and immunofluorescent analysis of K14-cre; BrafV600E (BrafV600E) neonatal skin. Hematoxylin and eosin (HE) stained sections demonstrate thickened epidermis, separation of individual cells, and severe reduction in cytoplasmic keratohyalin granules. Immunofluorescent staining for K10, BrdU, loricrin (LOR), filaggrin (FLG) and K6 are shown for perinatal wildtype littermate and K14-cre; BrafV600E E18.5-P0 mouse skin. Note the absence of K10pos-spinous keratinocytes and LOR/FLGpos granular keratinocytes in the K14-cre; BrafV600E mouse skin. K6, a marker for hyperproliferative skin, is increased in the K14-cre; BrafV600E mouse skin. Scale bars, 20 µm.
Mentions: To activate BRAF in the ectoderm, we utilized a mouse model, where expression of a mutant allele (BrafV600E) remains under the control of its endogenous locus and produces mutant product following Cre-mediated deletion of a transcription stop cassette (Dankort et al., 2007). BrafV600E floxed females were bred to Keratin 14 (K14)-cre) transgenic males, which express Cre in the epidermis at embryonic day (E) 14.5 (Vasioukhin et al., 1999). K14-cre-positive, BrafV600E-positive (K14-cre; BrafV600E) offspring were produced at near expected Mendelian frequency when assessed prior to birth (25.6% observed; 25% expected from 167 late stage embryos). Postnatally, the majority of K14-cre; BrafV600E newborns were cannibalized by adults, and at the time of weaning, only 3 K14-cre; BrafV600E mice out of > 20 litters were detected at the time of weaning. In litters observed at the moment of birth, K14-cre; BrafV600E newborns showed severe ectodermal defects, including thick, fissured scale overlying translucent edematous skin and displayed rhythmic ventilation and pink oxygenation. Further examination of K14-cre; BrafV600E newborns also revealed lack of ingested milk in their stomachs and cleft palate defects in >84% (Fig. 1b). The latter defect may result from Cre expression in the palate epithelium of K14-cre animals (Okubo et al., 2009). Histologic analysis of the skin revealed a thickened epidermis, basaloid cells, cytolysis and loss of keratohyalin granules (Fig. 1c). Although a stratified epithelium was present in K14-cre; BrafV600E mice, immunofluorescent analysis revealed loss of K10+ spinous and LOR/FLG+ granular keratinocytes (Fig. 1c). The K14-cre; BrafV600E epidermis was hyperproliferative as evidenced by increased BrdU-staining and the overexpression of K6 protein.

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