Single-cell transcriptomics defines keratinocyte differentiation in avian scutate scales

AbstractThe growth of skin appendages, such as hair, feathers and scales, depends on terminal differentiation of epidermal keratinocytes. Here, we investigated keratinocyte differentiation in avian scutate scales. Cells were isolated from the skin on the legs of 1-day old chicks and subjected to single-cell transcriptomics. We identified two distinct populations of differentiated keratinocytes. The first population was characterized by mRNAs encoding cysteine-rich keratins and corneous beta-proteins (CBPs), also known as beta-keratins, of the scale type, indicating that these cells form hard scales. The second population of differentiated keratinocytes contained mRNAs encoding cysteine-poor keratins and keratinocyte-type CBPs, suggesting that these cells form the soft interscale epidermis. We raised an antibody against keratin 9-like cysteine-rich 2 (KRT9LC2), which is encoded by an mRNA enriched in the first keratinocyte population. Immunostaining confirmed expression of KRT9LC2 in the suprabasal epidermal layers of scutate scales but not in interscale epidermis. Keratinocyte differentiation in chicken leg skin resembled that in human skin with regard to the transcriptional upregulation of epidermal differentiation complex genes and genes involved in lipid metabolism and transport. In conclusion, this study defines gene expression programs that build scutate scales and interscale epidermis of birds and reveals evolutionarily conserved keratinocyte differentiation genes.

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Terminal differentiation in keratinocytes involves positive as well as negative regulation by retinoic acid receptors and retinoid X receptors at retinoid response elements

Molecular and Cellular Biology ◽

10.1128/mcb.12.11.4862-4871.1992 ◽

1992 ◽

Vol 12 (11) ◽

pp. 4862-4871

Author(s):

B J Aneskievich ◽

E Fuchs

Keyword(s):

Retinoic Acid ◽

Terminal Differentiation ◽

Cell Types ◽

Epidermal Differentiation ◽

Retinoic Acid Receptors ◽

Keratinocyte Differentiation ◽

Epidermal Keratinocytes ◽

Retinoid X Receptors ◽

Response Elements ◽

X Receptors

Terminal differentiation of epidermal keratinocytes is inhibited by 1 microM retinoic acid, a concentration which induces differentiation in a number of cell types, including F9 teratocarcinoma cells. The molecular basis for these opposing retinoid responses is unknown, although retinoic acid receptors (RARs) and retinoid X receptors (RXRs) have been detected in both cell types. When F9 cells are stably transfected with a truncated RAR alpha lacking the E/F domain necessary for ligand binding and RAR/RXR dimerization, action at retinoid response elements is suppressed and cells produce a retinoic acid-resistant phenotype; i.e., they are blocked in differentiation (A. S. Espeseth, S. P. Murphy, and E. Linney, Genes Dev. 3:1647-1656, 1989). If retinoid receptors influence epidermal differentiation only in a negative fashion, then suppression of transactivation at retinoid response elements would be expected to enhance, rather than block, keratinocyte differentiation. In this study, we show that surprisingly, even though constitutive expression of an analogous truncated RAR gamma in keratinocytes specifically suppressed transactivation at retinoid response elements, keratinocytes were blocked, rather than enhanced, in their ability to undergo morphological and biochemical features of differentiation. These findings demonstrate a direct and hitherto unrecognized role for RARs and RXRs in positively as well as negatively regulating epidermal differentiation. Additionally, our studies extend those of Espeseth et al. (Genes Dev. 3:1647-1656, 1989), indicating a novel RAR function independent of the E/F domain.

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The role of cathepsin E in terminal differentiation of keratinocytes

Biological Chemistry ◽

10.1515/bc.2011.060 ◽

2011 ◽

Vol 392 (6) ◽

Cited By ~ 8

Author(s):

Tomoyo Kawakubo ◽

Atsushi Yasukochi ◽

Kuniaki Okamoto ◽

Yoshiko Okamoto ◽

Seiji Nakamura ◽

...

Keyword(s):

Hair Follicle ◽

Primary Cultures ◽

Terminal Differentiation ◽

Epidermal Differentiation ◽

Keratinocyte Differentiation ◽

Epidermal Keratinocytes ◽

Marker Genes ◽

Differentiation Markers ◽

Cathepsin E

Abstract Cathepsin E (CatE) is predominantly expressed in the rapidly regenerating gastric mucosal cells and epidermal keratinocytes, in addition to the immune system cells. However, the role of CatE in these cells remains unclear. Here we report a crucial role of CatE in keratinocyte terminal differentiation. CatE deficiency in mice induces abnormal keratinocyte differentiation in the epidermis and hair follicle, characterized by the significant expansion of corium and the reduction of subcutaneous tissue and hair follicle. In a model of skin papillomas formed in three different genotypes of syngeneic mice, CatE deficiency results in significantly reduced expression and altered localization of the keratinocyte differentiation induced proteins, keratin 1 and loricrin. Involvement of CatE in the regulation of the expression of epidermal differentiation specific proteins was corroborated by in vitro studies with primary cultures of keratinocytes from the three different genotypes of mice. In wild-type keratinocytes after differentiation inducing stimuli, the CatE expression profile was compatible to those of the terminal differentiation marker genes tested. Overexpression of CatE in mice enhances the keratinocyte terminal differentiation process, whereas CatE deficiency results in delayed differentiation accompanying the reduced expression or the ectopic localization of the differentiation markers. Our findings suggest that in keratinocytes CatE is functionally linked to the expression of terminal differentiation markers, thereby regulating epidermis formation and homeostasis.

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Terminal differentiation in keratinocytes involves positive as well as negative regulation by retinoic acid receptors and retinoid X receptors at retinoid response elements.

Molecular and Cellular Biology ◽

10.1128/mcb.12.11.4862 ◽

1992 ◽

Vol 12 (11) ◽

pp. 4862-4871 ◽

Cited By ~ 32

Author(s):

B J Aneskievich ◽

E Fuchs

Keyword(s):

Retinoic Acid ◽

Terminal Differentiation ◽

Cell Types ◽

Epidermal Differentiation ◽

Retinoic Acid Receptors ◽

Keratinocyte Differentiation ◽

Epidermal Keratinocytes ◽

Retinoid X Receptors ◽

Response Elements ◽

X Receptors

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PML Regulates the Epidermal Differentiation Complex and Skin Morphogenesis during Mouse Embryogenesis

Genes ◽

10.3390/genes11101130 ◽

2020 ◽

Vol 11 (10) ◽

pp. 1130

Author(s):

Anna Połeć ◽

Alexander D. Rowe ◽

Pernille Blicher ◽

Rajikala Suganthan ◽

Magnar Bjørås ◽

...

Keyword(s):

Gene Expression ◽

Developmental Stages ◽

Expression Patterns ◽

Growth Control ◽

Wild Type Mouse ◽

Global Gene Expression ◽

Mouse Embryos ◽

Epidermal Differentiation ◽

Envelope Gene ◽

Epidermal Differentiation Complex

The promyelocytic leukemia (PML) protein is an essential component of nuclear compartments called PML bodies. This protein participates in several cellular processes, including growth control, senescence, apoptosis, and differentiation. Previous studies have suggested that PML regulates gene expression at a subset of loci through a function in chromatin remodeling. Here we have studied global gene expression patterns in mouse embryonic skin derived from Pml depleted and wild type mouse embryos. Differential gene expression analysis at different developmental stages revealed a key role of PML in regulating genes involved in epidermal stratification. In particular, we observed dysregulation of the late cornified envelope gene cluster, which is a sub-region of the epidermal differentiation complex. In agreement with these data, PML body numbers are elevated in basal keratinocytes during embryogenesis, and we observed reduced epidermal thickness and defective hair follicle development in PML depleted mouse embryos.

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Immunolocalization of corneous beta proteins of the Epidermal Differentiation Complex in the developing claw of the alligator

Annals of Anatomy - Anatomischer Anzeiger ◽

10.1016/j.aanat.2020.151513 ◽

2020 ◽

Vol 231 ◽

pp. 151513

Author(s):

Lorenzo Alibardi

Keyword(s):

Epidermal Differentiation ◽

Epidermal Differentiation Complex ◽

Beta Proteins

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The Effect of Single CpG Demethylation on the Pattern of DNA-Protein Binding

International Journal of Molecular Sciences ◽

10.3390/ijms20040914 ◽

2019 ◽

Vol 20 (4) ◽

pp. 914 ◽

Cited By ~ 1

Author(s):

Barbara Sobiak ◽

Wiesława Leśniak

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Epidermal Differentiation ◽

Keratinocyte Differentiation ◽

Splicing Factors ◽

Targeted Next Generation Sequencing ◽

Factors Analysis ◽

Epidermal Differentiation Complex ◽

Complex Process ◽

Next Generation Sequencing Ngs

Epidermal differentiation is a complex process and its regulation may involve epigenetic factors. Analysis of DNA methylation in 20 selected regions within the epidermal differentiation complex (EDC) gene cluster by targeted next-generation sequencing (NGS) detected no or only minor changes in methylation, mostly slight demethylation, occurring during the course of keratinocyte differentiation. However, a single CpG pair within the exon of the PGLYRP3 gene underwent a pronounced demethylation concomitant with an increase in PGLYRP3 expression. We have employed a DNA-affinity precipitation assay (DAPA) and mass spectrometry to examine changes in the composition of proteins that bind to DNA containing either methylated or unmethylated CpG. We found that the unmethylated probe attracted mostly RNA binding proteins, including splicing factors, which suggests that demethylation of this particular CpG may facilitate PGLYRP3 transcription and/or pre-mRNA splicing.

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Effect of SUV39H1 Histone Methyltransferase Knockout on Expression of Differentiation-Associated Genes in HaCaT Keratinocytes

Cells ◽

10.3390/cells9122628 ◽

2020 ◽

Vol 9 (12) ◽

pp. 2628

Author(s):

Barbara Sobiak ◽

Wiesława Leśniak

Keyword(s):

Gene Expression ◽

Skin Diseases ◽

Human Keratinocytes ◽

Histone Methyltransferase ◽

Epidermal Differentiation ◽

Gene Encoding ◽

Epidermal Differentiation Complex ◽

Expression Of Genes ◽

State Dependent ◽

Genomic Locations

Keratinocytes undergo a complex differentiation process, coupled with extensive changes in gene expression through which they acquire distinctive features indispensable for cells that form the external body barrier—epidermis. Disturbed epidermal differentiation gives rise to multiple skin diseases. The involvement of epigenetic factors, such as DNA methylation or histone modifications, in the regulation of epidermal gene expression and differentiation has not been fully recognized yet. In this work we performed a CRISPR/Cas9-mediated knockout of SUV39H1, a gene-encoding H3K9 histone methyltransferase, in HaCaT cells that originate from spontaneously immortalized human keratinocytes and examined changes in the expression of selected differentiation-specific genes located in the epidermal differentiation complex (EDC) and other genomic locations by RT-qPCR. The studied genes revealed a diverse differentiation state-dependent or -independent response to a lower level of H3K9 methylation. We also show, by means of chromatin immunoprecipitation, that the expression of genes in the LCE1 subcluster of EDC was regulated by the extent of trimethylation of lysine 9 in histone H3 bound to their promoters. Changes in gene expression were accompanied by changes in HaCaT cell morphology and adhesion.

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Immunolocalization of the EDWM-Protein Indicates a Matrix Role in Cornification of Lizard Epidermis

Russian Journal of Herpetology ◽

10.30906/1026-2296-2021-28-5-267-274 ◽

2021 ◽

Vol 28 (5) ◽

pp. 267-274

Author(s):

Lorenzo Alibardi

Keyword(s):

Amino Acids ◽

Matrix Protein ◽

Light And Electron Microscopy ◽

Basal Layer ◽

Anolis Carolinensis ◽

Epidermal Differentiation ◽

Alpha Cells ◽

Chemical Bonds ◽

Epidermal Differentiation Complex ◽

Beta Proteins

During epidermal differentiation in the scales of lizards and snakes, from the basal layer beta- and later alpha-keratinocytes are generated to form beta-and alpha-corneous layers. In the lizard Anolis carolinensis, minor proteins derived from the EDC (Epidermal Differentiation Complex) are added to the main constituent proteins, IFKs (Intermediate Filament Keratins) and CBPs (Corneous Beta Proteins, formerly indicated as beta keratins). One of these proteins that previous studies showed to be exclusively expressed in the skin, EDWM (EDC protein containing high GSRC amino acids) is rich in cysteine and arginine, amino acids that form numerous –S–S– and electro-static chemical bonds in the corneous material. Light and electron microscopy immunolbeling for EDWM show a diffuse localization in differentiating beta-cells and in some alpha-cells, in particular those of the clear-layer, involved in epidermal shedding. The study suggests that EDWM may function as a matrix protein that binds to IFKs and CBPs, contributing to the formation of the specific corneous material present in beta- and alpha-corneous layers. In particular, its higher immunolocalization in the maturing clear layer indicates that this protein is important for its differentiation and epidermal shedding in A. carolinensis and likely also in other lepidosaurian reptiles.

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Oct-6: a regulator of keratinocyte gene expression in stratified squamous epithelia

Molecular and Cellular Biology ◽

10.1128/mcb.14.5.3263-3275.1994 ◽

1994 ◽

Vol 14 (5) ◽

pp. 3263-3275

Author(s):

I Faus ◽

H J Hsu ◽

E Fuchs

Keyword(s):

Gene Expression ◽

Immunoblot Analysis ◽

Negative Influence ◽

Epidermal Differentiation ◽

Epidermal Keratinocytes ◽

Rnase Protection ◽

Pou Domain ◽

Octamer Motif ◽

Repressive Effect ◽

Squamous Epithelia

POU domain proteins have been implicated as regulators of differentiation and development, particularly in early embryogenesis and in neural morphogenesis. Given that neural and epidermal lineages originate from a common precursor (ectodermal) cell, we explored the possibility that POU proteins are involved in epidermal differentiation. Using reverse transcription-PCR and degenerate oligonucleotides, we generated several POU domain cDNAs from cultured human epidermal mRNAs. One of these encoded a sequence identical to the rodent Tst-1/SCIP/Oct-6 POU domain. Subsequently, we isolated a cDNA encoding a 45.3-kDa protein with 98% sequence identity to rat Tst-1/SCIP and 94% identity to mouse Oct-6. This protein bound specifically to the canonical octamer motif, warranting its designation as human Oct-6. By RNase protection assays, by PCR, and by immunoblot analysis, Oct-6 was expressed in cultured epidermal keratinocytes. By in situ hybridization, Oct-6 mRNA was detected not only in epidermis but also a variety of other stratified squamous epithelia and with greater signals than testis, the tissue in which this POU protein was originally discovered. Moreover, Oct-6 exerted a marked and specific negative influence on expression of the K5 and K14 genes, abundantly expressed in most dividing stratified squamous epithelial cells and downregulated as cells commit to terminally differentiate. The repressive effect was complex, but it was not observed with Oct-1, nor was it seen with a truncated Oct-6 missing the POU domain. Taken together, our studies suggest that Oct-6 may play an important role in controlling gene expression in stratified squamous epithelia, including epidermis.

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Platelet-Released Growth Factors Induce Differentiation of Primary Keratinocytes

Mediators of Inflammation ◽

10.1155/2017/5671615 ◽

2017 ◽

Vol 2017 ◽

pp. 1-12 ◽

Cited By ~ 3

Author(s):

Andreas Bayer ◽

Mersedeh Tohidnezhad ◽

Justus Lammel ◽

Sebastian Lippross ◽

Peter Behrendt ◽

...

Keyword(s):

Gene Expression ◽

Growth Factors ◽

Terminal Differentiation ◽

Human Keratinocytes ◽

Keratinocyte Differentiation ◽

Dependent Manner ◽

Primary Human Keratinocytes ◽

Transglutaminase 1 ◽

Keratin 1

Autologous thrombocyte concentrate lysates, for example, platelet-released growth factors, (PRGFs) or their clinically related formulations (e.g., Vivostat PRF®) came recently into the physicians’ focus as they revealed promising effects in regenerative and reparative medicine such as the support of healing of chronic wounds. To elucidate the underlying mechanisms, we analyzed the influence of PRGF and Vivostat PRF on human keratinocyte differentiation in vitro and on epidermal differentiation status of skin wounds in vivo. Therefore, we investigated the expression of early (keratin 1 and keratin 10) and late (transglutaminase-1 and involucrin) differentiation markers. PRGF treatment of primary human keratinocytes decreased keratin 1 and keratin 10 gene expression but induced involucrin and transglutaminase-1 gene expression in an epidermal growth factor receptor- (EGFR-) dependent manner. In concordance with these results, microscopic analyses revealed that PRGF-treated human keratinocytes displayed morphological features typical of keratinocytes undergoing terminal differentiation. In vivo treatment of artificial human wounds with Vivostat PRF revealed a significant induction of involucrin and transglutaminase-1 gene expression. Together, our results indicate that PRGF and Vivostat PRF induce terminal differentiation of primary human keratinocytes. This potential mechanism may contribute to the observed beneficial effects in the treatment of hard-to-heal wounds with autologous thrombocyte concentrate lysates in vivo.

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