Single-cell transcriptomics defines keratinocyte differentiation in avian scutate scales

The 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.

The Epidermis: Redox Governor of Health and Diseases

A functional epithelial barrier necessitates protection against dehydration, and ichthyoses are caused by defects in maintaining the permeability barrier in the stratum corneum (SC), the uppermost protective layer composed of dead cells and secretory materials from the living layer stratum granulosum (SG). We have found that loricrin (LOR) is an essential effector of cornification that occurs in the uppermost layer of SG (SG1). LOR promotes the maturation of corneocytes and extracellular adhesion structure through organizing disulfide cross-linkages, albeit being dispensable for the SC permeability barrier. This review takes psoriasis and AD as the prototype of impaired cornification. Despite exhibiting immunological traits that oppose each other, both conditions share the epidermal differentiation complex as a susceptible locus. We also review recent mechanistic insights on skin diseases, focusing on the Kelch-like erythroid cell-derived protein with the cap “n” collar homology-associated protein 1/NFE2-related factor 2 signaling pathway, as they coordinate the epidermis-intrinsic xenobiotic metabolism. Finally, we refine the theoretical framework of thiol-mediated crosstalk between keratinocytes and leukocytes in the epidermis that was put forward earlier.

NFAT5 Controls the Integrity of Epidermis

The skin protects the human body against dehydration and harmful challenges. Keratinocytes (KCs) are the most abundant epidermal cells, and it is anticipated that KC-mediated transport of Na+ ions creates a physiological barrier of high osmolality against the external environment. Here, we studied the role of NFAT5, a transcription factor whose activity is controlled by osmotic stress in KCs. Cultured KCs from adult mice were found to secrete more than 300 proteins, and upon NFAT5 ablation, the secretion of several matrix proteinases, including metalloproteinase-3 (Mmp3) and kallikrein-related peptidase 7 (Klk7), was markedly enhanced. An increase in Mmp3 and Klk7 RNA levels was also detected in transcriptomes of Nfat5-/- KCs, along with increases of numerous members of the ‘Epidermal Differentiation Complex’ (EDC), such as small proline-rich (Sprr) and S100 proteins. NFAT5 and Mmp3 as well as NFAT5 and Klk7 are co-expressed in the basal KCs of fetal and adult epidermis but not in basal KCs of newborn (NB) mice. The poor NFAT5 expression in NB KCs is correlated with a strong increase in Mmp3 and Klk7 expression in KCs of NB mice. These data suggests that, along with the fragile epidermis of adult Nfat5-/- mice, NFAT5 keeps in check the expression of matrix proteases in epidermis. The NFAT5-mediated control of matrix proteases in epidermis contributes to the manifold changes in skin development in embryos before and during birth, and to the integrity of epidermis in adults.

Reporter System Controlled by the Involucrin Promoter as a Tool to Follow the Epidermal Differentiation

Different approaches have been explored to study skin biology, including the use of stem cells. Mesenchymal stem cells (MSC) from umbilical cord can be safely and easily obtained, however a simple strategy to monitor their differentiation is essential. Involucrin is a marker of keratinocyte terminal differentiation, and its promoter (pINV) directs stratum-specific expression of this protein. We designed a reporter system containing EGFP under control of pINV to assess MSC differentiation into keratinocytes. The functional sequence of pINV was inserted into a lentiviral vector, originating LeGO-GpINV. MSC were transduced with the LeGO-GpINV and induced to differentiate into keratinocytes upon cultivation with Keratinocyte Serum Free Medium supplemented. MSC differentiation was confirmed by morphological changes and by the expression of epidermal markers, by flow cytometry, quantitative PCR and western blot. The activity of kallikreins 5, 6 and 7 was detected using fluorogenic substrates. After 14 days of differentiation, MSC transduced with LeGO-GpINV showed an increase in EGFP fluorescence and expressed CK10, CK14, involucrin and filaggrin. There was also an increase in the kallikrein activity. This reporter system allowed to temporally assess the epidermal differentiation, simultaneously with involucrin expression, opening perspectives for the in vivo study of skin biology and in regenerative medicine.

Rice leaves undergo a rapid metabolic reconfiguration during a specific stage of primordium development

Leaf development is crucial to establish the photosynthetic competency of plants. It is a process that requires coordinated changes in cell number, cell differentiation, transcriptomes, metabolomes and physiology. However, despite the importance of leaf formation for our major crops, early developmental processes for rice have not been comprehensively described. Here we detail the temporal developmental trajectory of early rice leaf development and connect morphological changes to metabolism. In particular, a developmental index based on the patterning of epidermal differentiation visualised by electron microscopy enabled high resolution staging of early growth for single primordium metabolite profiling. These data demonstrate that a switch in the constellation of tricarboxylic acid (TCA) cycle metabolites defines a narrow window towards the end of the P3 stage of leaf development. Taken in the context of other data in the literature, our results substantiate that this phase of rice leaf growth, equivalent to a change of primordium length from around 5 to 7.5 mm, defines a major shift in rice leaf determination towards a photosynthetically defined structure. We speculate that efforts to engineer rice leaf structure should focus on the developmental window prior to these determining events.

Combined Metallomics/Transcriptomics Profiling Reveals a Major Role for Metals in Wound Repair

Endogenous metals are required for all life, orchestrating the action of diverse cellular processes that are crucial for tissue function. The dynamic wound healing response is underpinned by a plethora of such cellular behaviours, occurring in a time-dependent manner. However, the importance of endogenous metals for cutaneous repair remains largely unexplored. Here we combine ICP-MS with tissue-level RNA-sequencing to reveal profound changes in a number of metals, and corresponding metal-regulated genes, across temporal healing in mice. Wound calcium, magnesium, iron, copper and manganese are elevated at 7 days post-wounding, while magnesium, iron, aluminium, manganese and cobalt increase at 14 days post-wounding. At the level of transcription, wound-induced pathways are independently highly enriched for metal-regulated genes, and vice versa. Moreover, specific metals are linked to distinct wound-induced biological processes and converge on key transcriptional regulators in mice and humans. Finally, we reveal a potential role for one newly identified transcriptional regulator, TNF, in calcium-induced epidermal differentiation. Together, these data highlight potential new and diverse roles for metals in cutaneous wound repair, paving the way for further studies to elucidate the contribution of metals to cellular processes in the repair of skin and other tissues.

Genome-Wide Selective Signatures Reveal Candidate Genes Associated with Hair Follicle Development and Wool Shedding in Sheep

Hair follicle development and wool shedding in sheep are poorly understood. This study investigated the population structures and genetic differences between sheep with different wool types to identify candidate genes related to these traits. We used Illumina ovine SNP 50K chip genotyping data of 795 sheep populations comprising 27 breeds with two wool types, measuring the population differentiation index (Fst), nucleotide diversity (θπ ratio), and extended haplotype homozygosity among populations (XP-EHH) to detect the selective signatures of hair sheep and fine-wool sheep. The top 5% of the Fst and θπ ratio values, and values of XP-EHH < −2 were considered strongly selected SNP sites. Annotation showed that the PRX, SOX18, TGM3, and TCF3 genes related to hair follicle development and wool shedding were strongly selected. Our results indicated that these methods identified important genes related to hair follicle formation, epidermal differentiation, and hair follicle stem cell development, and provide a meaningful reference for further study on the molecular mechanisms of economically important traits in sheep.

START domain mediates Arabidopsis GLABRA2 transcription factor dimerization and turnover independently of homeodomain DNA binding

Class IV homeodomain leucine-zipper transcription factors (HD-Zip IV TFs) are key regulators of epidermal differentiation that are characterized by a DNA-binding homeodomain (HD) in conjunction with lipid sensor domain termed START (Steroidogenic Acute Regulatory (StAR)-related lipid Transfer). Previous work demonstrated that the START domain of GLABRA2 (GL2), a HD-Zip IV member from Arabidopsis, is required for transcription factor activity. Here, we address the functions and possible interactions of START and the HD in DNA binding, dimerization, and protein turnover. Deletion analysis of the HD and missense mutations of a conserved lysine (K146) result in phenotypic defects in leaf trichomes, root hairs and seed mucilage, similar to those observed for START mutants, despite nuclear localization of the mutant proteins. Gel shift and ChIP-seq experiments demonstrate that while HD mutations impair binding to target DNA, the START domain is dispensable for DNA binding. Vice versa, yeast two-hybrid assays reveal impaired GL2 dimerization for START domain mutants, but not HD mutants. Using in vivo cycloheximide chase experiments, we provide evidence for the role of START, but not HD, in maintaining protein stability. This work advances our fundamental understanding of HD-Zip TFs as multidomain regulators of epidermal development in plants.

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

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.