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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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Fate of pulse-labeled basal keratinocytes in wildtype and BrafV600E explantsTo determine the origin of spinous and granular layer revertants in wildtype and BrafV600E explants, proliferating basal keratinocytes were labeled in utero with BrdU and after embryo harvest and explant culture for 24 hrs in the presence of vehicle or PLX4720, BrdU was localized. (a) Double staining for K10 and pulse-chase BrdU demonstrate the labeling at time 0 hr and retention in post-mitotic cells 24 hrs after vehicle vs. PLX4720 treatment. (b) Double staining for loricrin and pulse-chase BrdU demonstrate lack of BrdU-labeled cells in loricrin-positive cells. In all panels, dotted lines indicate epidermal junction with dermis. Scale bars, 20 µm.
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Figure 5: Fate of pulse-labeled basal keratinocytes in wildtype and BrafV600E explantsTo determine the origin of spinous and granular layer revertants in wildtype and BrafV600E explants, proliferating basal keratinocytes were labeled in utero with BrdU and after embryo harvest and explant culture for 24 hrs in the presence of vehicle or PLX4720, BrdU was localized. (a) Double staining for K10 and pulse-chase BrdU demonstrate the labeling at time 0 hr and retention in post-mitotic cells 24 hrs after vehicle vs. PLX4720 treatment. (b) Double staining for loricrin and pulse-chase BrdU demonstrate lack of BrdU-labeled cells in loricrin-positive cells. In all panels, dotted lines indicate epidermal junction with dermis. Scale bars, 20 µm.

Mentions: Plasticity in the above studies could arise from basal keratinocytes responding to BRAF/MEK inhibition or keratinocytes already residing in distal layers. To label these populations, we performed a pulse-chase labeling to follow the fate of proliferating basal keratinocytes after BRAF inhibition (Fig. 5 and Suppl. Fig. S8). BrdU was introduced in utero to pregnant females, and embryos and skin explants were isolated 2 hrs later to identify pulse-labeled cells. In K14-cre; BrafV600E explants, 47.3 ± 4.4% of basal keratinocytes vs. 29.8 ± 4.7% in wildtype basal keratinocytes were BrdU-labeled at t=2 hours (Fig. 5a). After a 24-hour chase in BrdU-free culture, we found that the majority of K10- and LOR-positive keratinocytes in PLX4720-treated explants do not arise from label-retaining keratinocytes cells. These observations suggest that PLX4720 inhibition triggers post-mitotic layers to express K10 and Lor gene expression.


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)

Fate of pulse-labeled basal keratinocytes in wildtype and BrafV600E explantsTo determine the origin of spinous and granular layer revertants in wildtype and BrafV600E explants, proliferating basal keratinocytes were labeled in utero with BrdU and after embryo harvest and explant culture for 24 hrs in the presence of vehicle or PLX4720, BrdU was localized. (a) Double staining for K10 and pulse-chase BrdU demonstrate the labeling at time 0 hr and retention in post-mitotic cells 24 hrs after vehicle vs. PLX4720 treatment. (b) Double staining for loricrin and pulse-chase BrdU demonstrate lack of BrdU-labeled cells in loricrin-positive cells. In all panels, dotted lines indicate epidermal junction with dermis. Scale bars, 20 µm.
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Related In: Results  -  Collection

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Figure 5: Fate of pulse-labeled basal keratinocytes in wildtype and BrafV600E explantsTo determine the origin of spinous and granular layer revertants in wildtype and BrafV600E explants, proliferating basal keratinocytes were labeled in utero with BrdU and after embryo harvest and explant culture for 24 hrs in the presence of vehicle or PLX4720, BrdU was localized. (a) Double staining for K10 and pulse-chase BrdU demonstrate the labeling at time 0 hr and retention in post-mitotic cells 24 hrs after vehicle vs. PLX4720 treatment. (b) Double staining for loricrin and pulse-chase BrdU demonstrate lack of BrdU-labeled cells in loricrin-positive cells. In all panels, dotted lines indicate epidermal junction with dermis. Scale bars, 20 µm.
Mentions: Plasticity in the above studies could arise from basal keratinocytes responding to BRAF/MEK inhibition or keratinocytes already residing in distal layers. To label these populations, we performed a pulse-chase labeling to follow the fate of proliferating basal keratinocytes after BRAF inhibition (Fig. 5 and Suppl. Fig. S8). BrdU was introduced in utero to pregnant females, and embryos and skin explants were isolated 2 hrs later to identify pulse-labeled cells. In K14-cre; BrafV600E explants, 47.3 ± 4.4% of basal keratinocytes vs. 29.8 ± 4.7% in wildtype basal keratinocytes were BrdU-labeled at t=2 hours (Fig. 5a). After a 24-hour chase in BrdU-free culture, we found that the majority of K10- and LOR-positive keratinocytes in PLX4720-treated explants do not arise from label-retaining keratinocytes cells. These observations suggest that PLX4720 inhibition triggers post-mitotic layers to express K10 and Lor gene expression.

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