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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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RNA-sequencing identifies the fate of epidermis in BrafV600E mutant mice and persistence of EDC gene expression(a) Functional classification of genes differentially expressed in E17.5 K14-cre; BrafV600E epidermis, demonstrating number of genes and statistical significance of their association. (b) Read coverage of wildtype vs. K14-cre; BrafV600E transcripts across the mouse Krt14 and Krt10 loci. (c) Intermediate and late differentiation genes clustered in the EDC locus and close proximity of closely related paralogs. (d) Volcano plots of relative expression of all EDC group genes from pooled wildtype vs. K14-cre; BrafV600E embryos. Specific groups of EDC genes, LCE-like (green), SPRR-like (red), and S100 family genes (blue), are colored and shown as balloons in respective cluster III, IV and V. The levels of gene expression in RNA-seq are estimated from (b) and have associated log2 q-values or FDRs (vertical axis) for this estimate. Note: this q-value does not reflect sample variance since specimens have been pooled.
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Figure 2: RNA-sequencing identifies the fate of epidermis in BrafV600E mutant mice and persistence of EDC gene expression(a) Functional classification of genes differentially expressed in E17.5 K14-cre; BrafV600E epidermis, demonstrating number of genes and statistical significance of their association. (b) Read coverage of wildtype vs. K14-cre; BrafV600E transcripts across the mouse Krt14 and Krt10 loci. (c) Intermediate and late differentiation genes clustered in the EDC locus and close proximity of closely related paralogs. (d) Volcano plots of relative expression of all EDC group genes from pooled wildtype vs. K14-cre; BrafV600E embryos. Specific groups of EDC genes, LCE-like (green), SPRR-like (red), and S100 family genes (blue), are colored and shown as balloons in respective cluster III, IV and V. The levels of gene expression in RNA-seq are estimated from (b) and have associated log2 q-values or FDRs (vertical axis) for this estimate. Note: this q-value does not reflect sample variance since specimens have been pooled.

Mentions: To characterize differentiation and fate of the K14-cre; BrafV600E epidermis, high-throughput sequencing of transcripts was performed from the E17.5 epidermis, when the skin was phenotypically abnormal but lacked extensive signs of cytolysis seen at later stages. Pooled total RNA from four control littermate and mutant E17.5 epidermis were used to generate 48.4 and 56.3 million read libraries, respectively, and unique reads were aligned to the genome and annotated (Fig. 2). 2,189 coding genes were differentially expressed in the K14-cre; BrafV600E epidermis, of which many participate in epidermal differentiation and keratinization (Fig. 2a). Due to the heterogeneity of epidermal tissue, gene expression data may also reflect the presence of other cell types and follicular tissues. This data was used to study the activity of genes representing specific epidermal lineages (Fig. 2b; Suppl. Fig. S1), including late steps in differentiation, which involve activation of >70 epidermal differentiation complex (EDC) genes (de Guzman Strong et al., 2010). Read coverage within the 3.3 MB interval of conserved gene cluster was analyzed and identified 63 transcriptional units, which can be categorized into five physical groups (Fig. 2c). Loss of EDC gene expression was most prominent in the LCE-like group III, where the majority of these genes were decreased by 2-fold; however, four outliers in this group representing Lce3 paralogs were upregulated (Fig. 2d). In the remaining four EDC groups, >85% (41 genes) were expressed at normal or higher levels in K14-cre; BrafV600E epidermis (Suppl. Fig. S2). These findings confirm that despite the loss of early and intermediate gene differentiation, the vast majority of transcriptional features of late differentiation remain active.


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)

RNA-sequencing identifies the fate of epidermis in BrafV600E mutant mice and persistence of EDC gene expression(a) Functional classification of genes differentially expressed in E17.5 K14-cre; BrafV600E epidermis, demonstrating number of genes and statistical significance of their association. (b) Read coverage of wildtype vs. K14-cre; BrafV600E transcripts across the mouse Krt14 and Krt10 loci. (c) Intermediate and late differentiation genes clustered in the EDC locus and close proximity of closely related paralogs. (d) Volcano plots of relative expression of all EDC group genes from pooled wildtype vs. K14-cre; BrafV600E embryos. Specific groups of EDC genes, LCE-like (green), SPRR-like (red), and S100 family genes (blue), are colored and shown as balloons in respective cluster III, IV and V. The levels of gene expression in RNA-seq are estimated from (b) and have associated log2 q-values or FDRs (vertical axis) for this estimate. Note: this q-value does not reflect sample variance since specimens have been pooled.
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Related In: Results  -  Collection

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Figure 2: RNA-sequencing identifies the fate of epidermis in BrafV600E mutant mice and persistence of EDC gene expression(a) Functional classification of genes differentially expressed in E17.5 K14-cre; BrafV600E epidermis, demonstrating number of genes and statistical significance of their association. (b) Read coverage of wildtype vs. K14-cre; BrafV600E transcripts across the mouse Krt14 and Krt10 loci. (c) Intermediate and late differentiation genes clustered in the EDC locus and close proximity of closely related paralogs. (d) Volcano plots of relative expression of all EDC group genes from pooled wildtype vs. K14-cre; BrafV600E embryos. Specific groups of EDC genes, LCE-like (green), SPRR-like (red), and S100 family genes (blue), are colored and shown as balloons in respective cluster III, IV and V. The levels of gene expression in RNA-seq are estimated from (b) and have associated log2 q-values or FDRs (vertical axis) for this estimate. Note: this q-value does not reflect sample variance since specimens have been pooled.
Mentions: To characterize differentiation and fate of the K14-cre; BrafV600E epidermis, high-throughput sequencing of transcripts was performed from the E17.5 epidermis, when the skin was phenotypically abnormal but lacked extensive signs of cytolysis seen at later stages. Pooled total RNA from four control littermate and mutant E17.5 epidermis were used to generate 48.4 and 56.3 million read libraries, respectively, and unique reads were aligned to the genome and annotated (Fig. 2). 2,189 coding genes were differentially expressed in the K14-cre; BrafV600E epidermis, of which many participate in epidermal differentiation and keratinization (Fig. 2a). Due to the heterogeneity of epidermal tissue, gene expression data may also reflect the presence of other cell types and follicular tissues. This data was used to study the activity of genes representing specific epidermal lineages (Fig. 2b; Suppl. Fig. S1), including late steps in differentiation, which involve activation of >70 epidermal differentiation complex (EDC) genes (de Guzman Strong et al., 2010). Read coverage within the 3.3 MB interval of conserved gene cluster was analyzed and identified 63 transcriptional units, which can be categorized into five physical groups (Fig. 2c). Loss of EDC gene expression was most prominent in the LCE-like group III, where the majority of these genes were decreased by 2-fold; however, four outliers in this group representing Lce3 paralogs were upregulated (Fig. 2d). In the remaining four EDC groups, >85% (41 genes) were expressed at normal or higher levels in K14-cre; BrafV600E epidermis (Suppl. Fig. S2). These findings confirm that despite the loss of early and intermediate gene differentiation, the vast majority of transcriptional features of late differentiation remain active.

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