Epidermal club cells in the cardinal tetra (Paracheirodon axelrodi): Presence, distribution, and relationship to antipredator behavior.

Epidermal club cells (ECCs) are present in many species of teleost fish. In an attempt to justify their presence in the epidermis of fish, they have been associated with numerous functions. One proposed function is communication with conspecifics during a predation event, as these cells may passively release substances upon rupture, which may occur during predation. We identified the presence and distribution of ECCs in the body skin of adult cardinal tetra, Paracheirodon axelrodi (Schultz, 1956) and analyzed the animal's behavioral response to conspecific skin extract in a laboratory setting. The identification and distribution of ECCs in the epidermis of the animals were confirmed by conventional histology and immunohistochemistry. Our results demonstrated that: ECCs are present in the skin of the entire body; a high density is observed in the dorsal side from head to tail, in the insertion of the fins and in the epidermis covering them; and ventral distribution is less extensive and more dispersed than dorsal distribution. Treatment of P. axelrodi specimens with skin preparations of conspecifics resulted in behavioral changes in the animals: they showed erratic swimming movements, they showed avoidance of the area of stimulus application and they decreased the time spent moving. Overall, these results allow us to conclude that P. axelrodi possesses ECCs throughout the body, with a greater presence in areas of high exposure to predation events (dorsal area and fins). Animals exposed to conspecific skin extract showed a significant increase in behaviors described as anti-predatory in other species. This supports the hypothesis that ECCs may be the origin of chemical alarm cues that are passively released when skin damage occurs, alerting the rest of the group to the risk of predation.

[Research advances on the role and mechanism of epidermal stem cells in skin wound repair].

Epidermal stem cells play an pivotal role in skin self-renewal, wound repair, and re-epithelialization process. The emergence of new technologies and concepts such as single-cell sequencing and gene knockout further revealed a new mechanism of epidermal stem cells in epidermal self-renewal and wound repair, providing new ideas for wound repair. In this review, the mechanisms of proliferation, differentiation, and migration of epidermal stem cells are discussed. Combined with the analysis of researches on stem cell heterogeneity and cell plasticity, the physiological function of epidermal stem cells can be further understood. The application advances of epidermal stem cells in wound repair is also summarized, which would provide some advice for workers engaged in clinical and basic research on wound repair.

The Skin's Barrier: A Cryo-EM Based Overview of its Architecture and Stepwise Formation.

A major role of the skin is to serve as a barrier toward the environment. The skin's permeability barrier consists of a lipid structure positioned in the stratum corneum. Recent progress in high-resolution cryo-electron microscopy (cryo-EM) has allowed for elucidation of the architecture of the skin's barrier and its stepwise formation process representing the final stage of epidermal differentiation. In this review, we present an overview of the skin's barrier structure and its formation process, as evidenced by cryo-EM.

Stem cells expand potency and alter tissue fitness by accumulating diverse epigenetic memories.

Immune and tissue stem cells retain an epigenetic memory of inflammation that intensifies sensitivity to future encounters. We investigated whether and to what consequence stem cells possess and accumulate memories of diverse experiences. Monitoring a choreographed response to wounds, we found that as hair follicle stem cells leave their niche, migrate to repair damaged epidermis, and take up long-term foreign residence there, they accumulate long-lasting epigenetic memories of each experience, culminating in post-repair epigenetic adaptations that sustain the epidermal transcriptional program and surface barrier. Each memory is distinct, separable, and has its own physiological impact, collectively endowing these stem cells with heightened regenerative ability to heal wounds and broadening their tissue-regenerating tasks relative to their naïve counterparts.

Cyclic growth of dermal papilla and regeneration of follicular mesenchymal components during feather cycling.

How dermis maintains tissue homeostasis in cyclic growth and wounding is a fundamental unsolved question. Here, we study how dermal components of feather follicles undergo physiological (molting) and plucking injury-induced regeneration in chickens. Proliferation analyses reveal quiescent, transient-amplifying (TA) and long-term label-retaining dermal cell (LRDC) states. During the growth phase, LRDCs are activated to make new dermal components with distinct cellular flows. Dermal TA cells, enriched in the proximal follicle, generate both peripheral pulp, which extends distally to expand the epithelial-mesenchymal interactive interface for barb patterning, and central pulp, which provides nutrition. Entering the resting phase, LRDCs, accompanying collar bulge epidermal label-retaining cells, descend to the apical dermal papilla. In the next cycle, these apical dermal papilla LRDCs are re-activated to become new pulp progenitor TA cells. In the growth phase, lower dermal sheath can generate dermal papilla and pulp. Transcriptome analyses identify marker genes and highlight molecular signaling associated with dermal specification. We compare the cyclic topological changes with those of the hair follicle, a convergently evolved follicle configuration. This work presents a model for analyzing homeostasis and tissue remodeling of mesenchymal progenitors.

New mouse models for high resolution and live imaging of planar cell polarity proteins in vivo.

The collective polarization of cellular structures and behaviors across a tissue plane is a near universal feature of epithelia known as planar cell polarity (PCP). This property is controlled by the core PCP pathway, which consists of highly conserved membrane-associated protein complexes that localize asymmetrically at cell junctions. Here, we introduce three new mouse models for investigating the localization and dynamics of transmembrane PCP proteins: Celsr1, Fz6 and Vangl2. Using the skin epidermis as a model, we characterize and verify the expression, localization and function of endogenously tagged Celsr1-3xGFP, Fz6-3xGFP and tdTomato-Vangl2 fusion proteins. Live imaging of Fz6-3xGFP in basal epidermal progenitors reveals that the polarity of the tissue is not fixed through time. Rather, asymmetry dynamically shifts during cell rearrangements and divisions, while global, average polarity of the tissue is preserved. We show using super-resolution STED imaging that Fz6-3xGFP and tdTomato-Vangl2 can be resolved, enabling us to observe their complex localization along junctions. We further explore PCP fusion protein localization in the trachea and neural tube, and discover new patterns of PCP expression and localization throughout the mouse embryo.

Whole-body clonal mapping identifies giant dominant clones in zebrafish skin epidermis.

Skin expansion during development is predominantly driven by growth of basal epithelial cell (BEC)-derived clonal populations, which often display varied sizes and shapes. However, little is known about the causes of clonal heterogeneity and the maximum size to which a single clone can grow. Here, we created a zebrafish model, basebow, for capturing clonal growth behavior in the BEC population on a whole-body, centimeter scale. By tracking 222 BECs over the course of a 28-fold expansion of body surface area, we determined that most BECs survive and grow clonal populations with an average size of 0.013 mm2. An extensive survey of 742 sparsely labeled BECs further revealed that giant dominant clones occasionally arise on specific body regions, covering up to 0.6% of the surface area. Additionally, a growth-induced extracellular matrix component, Lamb1a, mediates clonal growth in a cell-autonomous manner. Altogether, our findings demonstrate how clonal heterogeneity and clonal dominance may emerge to enable post-embryonic growth of a vertebrate organ, highlighting key cellular mechanisms that may only become evident when visualizing single cell behavior at the whole-animal level.

Cryptic genetic variation in a heat shock protein modifies the outcome of a mutation affecting epidermal stem cell development in C. elegans.

A fundamental question in medical genetics is how the genetic background modifies the phenotypic outcome of mutations. We address this question by focusing on the seam cells, which display stem cell properties in the epidermis of Caenorhabditis elegans. We demonstrate that a putative null mutation in the GATA transcription factor egl-18, which is involved in seam cell fate maintenance, is more tolerated in the CB4856 isolate from Hawaii than the lab reference strain N2 from Bristol. We identify multiple quantitative trait loci (QTLs) underlying the difference in phenotype expressivity between the two isolates. These QTLs reveal cryptic genetic variation that reinforces seam cell fate through potentiating Wnt signalling. Within one QTL region, a single amino acid deletion in the heat shock protein HSP-110 in CB4856 is sufficient to modify Wnt signalling and seam cell development, highlighting that natural variation in conserved heat shock proteins can shape phenotype expressivity.

Basal epidermis collective migration and local Sonic hedgehog signaling promote skeletal branching morphogenesis in zebrafish fins.

Teleost fish fins, like all vertebrate limbs, comprise a series of bones laid out in characteristic pattern. Each fin's distal bony rays typically branch to elaborate skeletal networks providing form and function. Zebrafish caudal fin regeneration studies suggest basal epidermal-expressed Sonic hedgehog (Shh) promotes ray branching by partitioning pools of adjacent pre-osteoblasts. This Shh role is distinct from its well-studied Zone of Polarizing Activity role establishing paired limb positional information. Therefore, we investigated if and how Shh signaling similarly functions during developmental ray branching of both paired and unpaired fins while resolving cellular dynamics of branching by live imaging. We found shha is expressed uniquely by basal epidermal cells overlying pre-osteoblast pools at the distal aspect of outgrowing juvenile fins. Lateral splitting of each shha-expressing epidermal domain followed by the pre-osteoblast pools precedes overt ray branching. We use ptch2:Kaede fish and Kaede photoconversion to identify short stretches of shha+basal epidermis and juxtaposed pre-osteoblasts as the Shh/Smoothened (Smo) active zone. Basal epidermal distal collective movements continuously replenish each shha+domain with individual cells transiently expressing and responding to Shh. In contrast, pre-osteoblasts maintain Shh/Smo activity until differentiating. The Smo inhibitor BMS-833923 prevents branching in all fins, paired and unpaired, with surprisingly minimal effects on caudal fin initial skeletal patterning, ray outgrowth or bone differentiation. Staggered BMS-833923 addition indicates Shh/Smo signaling acts throughout the branching process. We use live cell tracking to find Shh/Smo restrains the distal movement of basal epidermal cells by apparent 'tethering' to pre-osteoblasts. We propose short-range Shh/Smo signaling promotes these heterotypic associations to couple instructive basal epidermal collective movements to pre-osteoblast repositioning as a unique mode of branching morphogenesis.

The Potential of Nail Mini-Organ Stem Cells in Skin, Nail and Digit Tips Regeneration.

Nails are highly keratinized skin appendages that exhibit continuous growth under physiological conditions and full regeneration upon removal. These mini-organs are maintained by two autonomous populations of skin stem cells. The fast-cycling, highly proliferative stem cells of the nail matrix (nail stem cells (NSCs)) predominantly replenish the nail plate. Furthermore, the slow-cycling population of the nail proximal fold (nail proximal fold stem cells (NPFSCs)) displays bifunctional properties by contributing to the peri-nail epidermis under the normal homeostasis and the nail structure upon injury. Here, we discuss nail mini-organ stem cells' location and their role in skin and nail homeostasis and regeneration, emphasizing their importance to orchestrate the whole digit tip regeneration. Such endogenous regeneration capabilities are observed in rodents and primates. However, they are limited to the region adjacent to the nail's proximal area, indicating the crucial role of nail mini-organ stem cells in digit restoration. Further, we explore the molecular characteristics of nail mini-organ stem cells and the critical role of the bone morphogenetic protein (BMP) and Wnt signaling pathways in homeostatic nail growth and digit restoration. Finally, we investigate the latest accomplishments in stimulating regenerative responses in regeneration-incompetent injuries. These pioneer results might open up new opportunities to overcome amputated mammalian digits and limbs' regenerative failures in the future.

The Scf/Kit pathway implements self-organized epithelial patterning.

How global patterns emerge from individual cell behaviors is poorly understood. In the Xenopus embryonic epidermis, multiciliated cells (MCCs) are born in a random pattern within an inner mesenchymal layer and subsequently intercalate at regular intervals into an outer epithelial layer. Using video microscopy and mathematical modeling, we found that regular pattern emergence involves mutual repulsion among motile immature MCCs and affinity toward outer-layer intercellular junctions. Consistently, Arp2/3-mediated actin remodeling is required for MCC patterning. Mechanistically, we show that the Kit tyrosine kinase receptor, expressed in MCCs, and its ligand Scf, expressed in outer-layer cells, are both required for regular MCC distribution. Membrane-associated Scf behaves as a potent adhesive cue for MCCs, while its soluble form promotes their mutual repulsion. Finally, Kit expression is sufficient to confer order to a disordered heterologous cell population. This work reveals how a single signaling system can implement self-organized large-scale patterning.

N6 -methyladenosine modification of lncRNA Pvt1 governs epidermal stemness.

Dynamic chemical modifications of RNA represent novel and fundamental mechanisms that regulate stemness and tissue homeostasis. Rejuvenation and wound repair of mammalian skin are sustained by epidermal progenitor cells, which are localized within the basal layer of the skin epidermis. N6 -methyladenosine (m6 A) is one of the most abundant modifications found in eukaryotic mRNA and lncRNA (long noncoding RNA). In this report, we survey changes of m6 A RNA methylomes upon epidermal differentiation and identify Pvt1, a lncRNA whose m6 A modification is critically involved in sustaining stemness of epidermal progenitor cells. With genome-editing and a mouse genetics approach, we show that ablation of m6 A methyltransferase or Pvt1 impairs the self-renewal and wound healing capability of skin. Mechanistically, methylation of Pvt1 transcripts enhances its interaction with MYC and stabilizes the MYC protein in epidermal progenitor cells. Our study presents a global view of epitranscriptomic dynamics that occur during epidermal differentiation and identifies the m6 A modification of Pvt1 as a key signaling event involved in skin tissue homeostasis and wound repair.

Cell Volume Regulation in the Epidermis.

In order to cope with external stressors such as changes in humidity and temperature or irritating substances, the epidermis as the outermost skin layer forms a continuously renewing and ideally intact protective barrier. Under certain circumstances, this barrier can be impaired and epidermal cells have to counteract cell swelling or shrinkage induced by osmotic stress via regulatory volume decrease (RVD) or increase (RVI). Here, we will review the current knowledge regarding the molecular machinery underlying RVD and RVI in the epidermis. Furthermore, we will discuss the current understanding how cell volume changes and its regulators are associated with epidermal renewal and barrier formation.

Interplay of opposing fate choices stalls oncogenic growth in murine skin epithelium.

Skin epithelium can accumulate a high burden of oncogenic mutations without morphological or functional consequences. To investigate the mechanism of oncogenic tolerance, we induced HrasG12V in single murine epidermal cells and followed them long term. We observed that HrasG12V promotes an early and transient clonal expansion driven by increased progenitor renewal that is replaced with an increase in progenitor differentiation leading to reduced growth. We attribute this dynamic effect to emergence of two populations within oncogenic clones: renewing progenitors along the edge and differentiating ones within the central core. As clone expansion is accompanied by progressive enlargement of the core and diminishment of the edge compartment, the intraclonal competition between the two populations results in stabilized oncogenic growth. To identify the molecular mechanism of HrasG12V-driven differentiation, we screened known Ras-effector in vivo and identified Rassf5 as a novel regulator of progenitor fate choice that is necessary and sufficient for oncogene-specific differentiation.

Hair Follicle Transplantation for Wound Repair.

Significance: Hair follicles are complex miniorgans that reside in the dermal layer of the skin. When the skin is wounded, epidermal stem cells in the hair follicle activate and start migrating into the wound site, differentiating into epidermal cells. and contributing to the reepithelialization of the wound. The hair follicles represent the deepest epidermal elements in the skin, which are extremely beneficial in partial-thickness burns and abrasions where the skin can regenerate from the hair follicles. Recent Advances: Advanced animal models have demonstrated that the contribution of epidermal stem cells in the hair follicle bulge and isthmus regions is important for wound healing. In addition, several clinical studies have shown successful harvesting and transplantation of hair follicles as a treatment modality to accelerate wound healing. Critical Issues: Deep and large wounds require hospitalization and, without exception, surgical treatment. Harvesting and direct transplantation of hair follicles could provide a great source of autologous epidermal stem cells for wound healing. The procedure can be done in an outpatient setting, quickly and without creating a large donor site wound. Future Directions: Transplantation of hair follicles in a combination with novel biomaterials could provide advantageous treatment possibilities for both chronic wounds and burns. There is a substantial amount of molecular signaling data available on the role of hair follicles during wound repair, but almost all the data are derived from rodent models, and thus, more information from large animals and most importantly from humans would be beneficial and help to advance this promising treatment further.

Establishment and characterization of proliferating primary cultures of equine epidermal keratinocytes.

Skin-derived tissue cultures are a useful model to study molecular mechanisms of skin renewal and pathogenesis of dermal diseases. Horses often suffer from skin diseases, skin trauma and problems with proper wound healing, which could be improved by in vitro grown keratinocyte grafts. Herein we describe establishment and characterization of equine skin-derived primary cell cultures, using enzymatic and explant methods. The established cell lines of primary equine keratinocytes (peK) maintained high proliferative capacity for over five passages and expressed different epithelial/keratinocyte-specific markers. Characterization of the primary culture was performed in parallel with localization studies of the markers in the skin histological sections, using commercially available antibodies. Relative expression of typical differentiation stage-specific markers was determined in the established cell lines, using RT-qPCR. Basal (proliferating) keratinocytes were the predominant cell type in the established cell lines, but low expression of post-mitotic keratinocyte markers was also detected. Differences in marker expression were observed neither between the peK originating from two different animals nor between the peK established with two different methods (enzymatically or by explanting). The described methods in combination with the suggested characterization and differentiation markers are suitable for establishment of proliferating peK and evaluation of their differentiation status.

Cellular heterogeneity and microenvironmental control of skin cancer.

Healthy tissues harbour a surprisingly high number of cells that carry well-known cancer-causing mutations without impacting their physiological function. In recent years, strong evidence accumulated that the immediate environment of mutant cells profoundly impact their prospect of malignant progression. In this review, focusing on the skin, we investigate potential key mechanisms that ensure tissue homeostasis despite the presence of mutant cells, as well as critical factors that may nudge the balance from homeostasis to tumour formation. Functional in vivo studies and single-cell transcriptome analyses have revealed a tremendous cellular heterogeneity and plasticity within epidermal (stem) cells and their respective niches, revealing for example wild-type epithelial cells, fibroblasts or immune-cell subsets as critical in preventing cancer formation and malignant progression. It's the same cells, however, that can drive carcinogenesis. Therefore, understanding the abundance and molecular variation of cell types in health and disease, and how they interact and modulate the local signalling environment will thus be key for new therapeutic avenues in our battle against cancer.

Identification of epidermal differentiation genes of the tuatara provides insights into the early evolution of lepidosaurian skin.

The tuatara (Sphenodon punctatus) is the phylogenetically closest relative of squamates (including lizards and snakes) from which it diverged around 250 million years ago. Together, they constitute the clade Lepidosauria. Fully terrestrial vertebrates (amniotes) form their skin barrier to the environment under the control of a gene cluster, termed the epidermal differentiation complex (EDC). Here we identified EDC genes in the genome of the tuatara and compared them to those of other amniotes. The organization of the EDC and proteins encoded by EDC genes are most similar in the tuatara and squamates. A subcluster of lepidosaurian EDC genes encodes corneous beta-proteins (CBPs) of which three different types are conserved in the tuatara. Small proline-rich proteins have undergone independent expansions in the tuatara and some, but not all subgroups of squamates. Two genes encoding S100 filaggrin-type proteins (SFTPs) are expressed during embryonic skin development of the tuatara whereas SFTP numbers vary between 1 and 3 in squamates. Our comparative analysis of the EDC in the tuatara genome suggests that many molecular features of the skin that were previously identified in squamates have evolved prior to their divergence from the lineage leading to the tuatara.

Phenotypic suppression of acral peeling skin syndrome in a patient with autosomal recessive congenital ichthyosis.

Autosomal recessive congenital ichthyosis (ARCI) manifests with generalized scaling often associated with generalized erythema. Mutations in at least 13 different genes have been reported to cause ARCI. Acral peeling skin syndrome (APSS) is a rare autosomal recessive disorder manifesting with peeling over the distal limbs and dorsal surfaces of hands and feet. APSS is mostly due to mutations in TGM5, encoding transglutaminase 5. Both ARCI and APSS are fully penetrant genetic traits. Here, we describe a consanguineous family in which one patient with mild ARCI was found to carry a homozygous mutation in ALOXE3 (c.1238G > A; p.Gly413Asp). The patient was also found to carry a known pathogenic homozygous mutation in TGM5 (c.1335G > C; p.Lys445Asn) but did not display acral peeling skin. Her uncle carried the same homozygous mutation in TGM5 but carried the ALOXE3 mutation in a heterozygous state and showed clinical features typical of APSS. Taken collectively, these observations suggested that the ALOXE3 mutation suppresses the clinical expression of the TGM5 variant. We hypothesized that ALOXE3 deficiency may affect the expression of a protein capable of compensating for the lack of TGM5 expression. Downregulation of ALOXE3 in primary human keratinocytes resulted in increased levels of corneodesmosin, which plays a critical role in the maintenance of cell-cell adhesion in the upper epidermal layers. Accordingly, ectopic corneodesmosin expression rescued the cell-cell adhesion defect caused by TGM5 deficiency in keratinocytes as ascertained by the dispase dissociation assay. The present data thus provide evidence for phenotypic suppression in a human hereditary skin disorder.