Mechanical tension mobilizes Lgr6+ epidermal stem cells to drive skin growth.

Uniquely among mammalian organs, skin is capable of marked size change in adults, yet the mechanisms underlying this notable capacity are unclear. Here, we use a system of controlled tissue expansion in mice to uncover cellular and molecular determinants of skin growth. Through machine learning-guided three-dimensional tissue reconstruction, we capture morphometric changes in growing skin. We find that most growth is driven by the proliferation of the epidermis in response to mechanical tension, with more limited changes in dermal and subdermal compartments. Epidermal growth is achieved through preferential activation and differentiation of Lgr6+ stem cells of the epidermis, driven in part by the Hippo pathway. By single-cell RNA sequencing, we uncover further changes in mechanosensitive and metabolic pathways underlying growth control in the skin. These studies point to therapeutic strategies to enhance skin growth and establish a platform for understanding organ size dynamics in adult mammals.

Collagen XVII deficiency alters epidermal patterning.

Vertebrates exhibit patterned epidermis, exemplified by scales/interscales in mice tails and grooves/ridges on the human skin surface (microtopography). Although the role of spatiotemporal regulation of stem cells (SCs) has been implicated in this process, the mechanism underlying the development of such epidermal patterns is poorly understood. Here, we show that collagen XVII (COL17), a niche for epidermal SCs, helps stabilize epidermal patterns. Gene knockout and rescue experiments revealed that COL17 maintains the width of the murine tail scale epidermis independently of epidermal cell polarity. Skin regeneration after wounding was associated with slender scale epidermis, which was alleviated by overexpression of human COL17. COL17-negative skin in human junctional epidermolysis bullosa showed a distinct epidermal pattern from COL17-positive skin that resulted from revertant mosaicism. These results demonstrate that COL17 contributes to defining mouse tail scale shapes and human skin microtopography. Our study sheds light on the role of the SC niche in tissue pattern formation.

Choline transporter-like protein 2 interacts with chitin synthase 1 and is involved in insect cuticle development.

Chitin is an aminopolysaccharide present in insects as a major structural component of the cuticle. However, current knowledge on the chitin biosynthetic machinery, especially its constituents and mechanism, is limited. Using three independent binding assays, including co-immunoprecipitation, split-ubiquitin membrane yeast two-hybrid assay, and pull-down assay, we demonstrate that choline transporter-like protein 2 (Ctl2) interacts with krotzkopf verkehrt (kkv) in Drosophila melanogaster. The global knockdown of Ctl2 by RNA interference (RNAi) induced lethality at the larval stage. Tissue-specific RNAi to silence Ctl2 in the tracheal system and in the epidermis of the flies resulted in lethality at the first larval instar. The knockdown of Ctl2 in wings led to shrunken wings containing accumulated fluid. Calcofluor White staining demonstrated reduced chitin content in the first longitudinal vein of Ctl2 knockdown wings. The pro-cuticle, which was thinner compared to wildtype, exhibited a reduced number of chitin laminar layers. Phylogenetic analyses revealed orthologues of Ctl2 in different insect orders with highly conserved domains. Our findings provide new insights into cuticle formation, wherein Ctl2 plays an important role as a chitin-synthase interacting protein.

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.

Tissue-Engineered Skin Regenerative Units for Epidermal Keratinocytes Expansion and Wound Healing.

Chronic wounds have become an increasing medical and economic problem of aging societies because they are difficult to manage. Tissue engineering provides new perspectives for the clinically applicable skin substitutes. Epidermal keratinocytes play an important role in wound epithelization and construction of tissue-engineered skin substitutes. How to obtain a large number of autologous epidermal keratinocytes in a short time is the main problem that limits the application of tissue-engineered skin and epidermal cell membranes. Developing an appropriate method for reproducing the biological potential of cell-cell interactions and simulating the three-dimensional structure between cells has great significance for epidermal keratinocytes expansion and full-thickness skin regeneration. In this article, we propose the concept of tissue-engineered skin regeneration units (TESRUs) as the smallest unit with complete full-thickness skin regeneration ability. First, autologous dermal fibroblasts are cultured in biodegradable macroporous microcarriers to provide the mesenchyme support. Second, autologous epidermal keratinocytes and autologous melanocytes are incubated with the fibroblasts-loaded microcarriers and expand in vitro. Incorporating the above co-culture method into the macroporous microcarriers is reasonable for maintaining cell-cell interactions in spatial and temporal context and providing a suitable growth niche for epidermal keratinocytes. Moreover, TESRUs are composed of fibroblasts, keratinocytes, and melanocytes and have complete full-thickness skin regeneration ability. We suggest that TESRUs could be a promising strategy to repair full-thickness skin defects for clinical applications if the hypothesis proves to be practical.

Delta/Jagged-mediated Notch signaling induces the differentiation of agr2-positive epidermal mucous cells in zebrafish embryos.

Teleosts live in aquatic habitats, where they encounter ionic and acid-base fluctuations as well as infectious pathogens. To protect from these external challenges, the teleost epidermis is composed of living cells, including keratinocytes and ionocytes that maintain body fluid ionic homeostasis, and mucous cells that secret mucus. While ionocyte progenitors are known to be specified by Delta-Notch-mediated lateral inhibition during late gastrulation and early segmentation, it remains unclear how epidermal mucous cells (EMCs) are differentiated and maintained. Here, we show that Delta/Jagged-mediated activation of Notch signaling induces the differentiation of agr2-positive (agr2+) EMCs in zebrafish embryos during segmentation. We demonstrated that agr2+ EMCs contain cytoplasmic secretory granules and express muc5.1 and muc5.2. Reductions in agr2+ EMC number were observed in mib mutants and notch3 MOs-injected notch1a mutants, while increases in agr2+ cell number were detected in notch1a- and X-Su(H)/ANK-overexpressing embryos. Treatment with γ-secretase inhibitors further revealed that Notch signaling is required during bud to 15 hpf for the differentiation of agr2+ EMCs. Increased agr2+ EMC numbers were also observed in jag1a-, jag1b-, jag2a- and dlc-overexpressing, but not jag2b-overexpressing embryos. Meanwhile, reductions in agr2+ EMC numbers were detected in jag1a morphants, jag1b mutants, jag2a mutants and dlc morphants, but not jag2b mutants. Reduced numbers of pvalb8-positive epidermal cells were also observed in mib or jag2a mutants and jag1a or jag1b morphants, while increased pvalb8-positive epidermal cell numbers were detected in notch1a-overexpressing, but not dlc-overexpressing embryos. BrdU labeling further revealed that the agr2+ EMC population is maintained by proliferation. Cell lineage experiments showed that agr2+ EMCs are derived from the same ectodermal precursors as keratinocytes or ionocytes. Together, our results indicate that specification of agr2+ EMCs in zebrafish embryos is induced by DeltaC/Jagged-dependent activation of Notch1a/3 signaling, and the cell population is maintained by proliferation.

Developmental genetics of color pattern establishment in cats.

Intricate color patterns are a defining aspect of morphological diversity in the Felidae. We applied morphological and single-cell gene expression analysis to fetal skin of domestic cats to identify when, where, and how, during fetal development, felid color patterns are established. Early in development, we identify stripe-like alterations in epidermal thickness preceded by a gene expression pre-pattern. The secreted Wnt inhibitor encoded by Dickkopf 4 plays a central role in this process, and is mutated in cats with the Ticked pattern type. Our results bring molecular understanding to how the leopard got its spots, suggest that similar mechanisms underlie periodic color pattern and periodic hair follicle spacing, and identify targets for diverse pattern variation in other mammals.

Regulation of integrin and extracellular matrix genes by HNRNPL is necessary for epidermal renewal.

Stratified epithelia such as the epidermis require coordinated regulation of stem and progenitor cell proliferation, survival, and differentiation to maintain homeostasis. Integrin-mediated anchorage of the basal layer stem cells of the epidermis to the underlying dermis through extracellular matrix (ECM) proteins is crucial for this process. It is currently unknown how the expression of these integrins and ECM genes are regulated. Here, we show that the RNA-binding protein (RBP) heterogeneous nuclear ribonucleoprotein L (HNRNPL) binds to these genes on chromatin to promote their expression. HNRNPL recruits RNA polymerase II (Pol II) to integrin/ECM genes and is required for stabilizing Pol II transcription through those genes. In the absence of HNRNPL, the basal layer of the epidermis where the stem cells reside prematurely differentiates and detaches from the underlying dermis due to diminished integrin/ECM expression. Our results demonstrate a critical role for RBPs on chromatin to maintain stem and progenitor cell fate by dictating the expression of specific classes of genes.

Regional Specific Differentiation of Integumentary Organs: Regulation of Gene Clusters within the Avian Epidermal Differentiation Complex and Impacts of SATB2 Overexpression.

The epidermal differentiation complex (EDC) encodes a group of unique proteins expressed in late epidermal differentiation. The EDC gave integuments new physicochemical properties and is critical in evolution. Recently, we showed ß-keratins, members of the EDC, undergo gene cluster switching with overexpression of SATB2 (Special AT-rich binding protein-2), considered a chromatin regulator. We wondered whether this unique regulatory mechanism is specific to ß-keratins or may be derived from and common to EDC members. Here we explore (1) the systematic expression patterns of non-ß-keratin EDC genes and their preferential expression in different skin appendages during development, (2) whether the expression of non-ß-keratin EDC sub-clusters are also regulated in clusters by SATB2. We analyzed bulk RNA-seq and ChIP-seq data and also evaluated the disrupted expression patterns caused by overexpressing SATB2. The results show that the expression of whole EDDA and EDQM sub-clusters are possibly mediated by enhancers in E14-feathers. Overexpressing SATB2 down-regulates the enriched EDCRP sub-cluster in feathers and the EDCH sub-cluster in beaks. These results reveal the potential of complex epigenetic regulation activities within the avian EDC, implying transcriptional regulation of EDC members acting at the gene and/or gene cluster level in a temporal and skin regional-specific fashion, which may contribute to the evolution of diverse avian integuments.

Gene expression profiling of epidermal cell types in C. elegans using Targeted DamID.

The epidermis of Caenorhabditis elegans is an essential tissue for survival because it contributes to the formation of the cuticle barrier as well as facilitating developmental progression and animal growth. Most of the epidermis consists of the hyp7 hypodermal syncytium, the nuclei of which are largely generated by the seam cells, which exhibit stem cell-like behaviour during development. How seam cell progenitors differ transcriptionally from the differentiated hypodermis is poorly understood. Here, we introduce Targeted DamID (TaDa) in C. elegans as a method for identifying genes expressed within a tissue of interest without cell isolation. We show that TaDa signal enrichment profiles can be used to identify genes transcribed in the epidermis and use this method to resolve differences in gene expression between the seam cells and the hypodermis. Finally, we predict and functionally validate new transcription and chromatin factors acting in seam cell development. These findings provide insights into cell type-specific gene expression profiles likely associated with epidermal cell fate patterning.

Multitargeted Approach for the Optimization of Morphogenesis and Barrier Formation in Human Skin Equivalents.

In vitro skin tissue engineering is challenging due to the manifold differences between the in vivo and in vitro conditions. Yet, three-dimensional (3D) human skin equivalents (HSEs) are able to mimic native human skin in many fundamental aspects. However, the epidermal lipid barrier formation, which is essential for the functionality of the skin barrier, remains compromised. Recently, HSEs with an improved lipid barrier formation were generated by (i) incorporating chitosan in the dermal collagen matrix, (ii) reducing the external oxygen level to 3%, and (iii) inhibiting the liver X receptor (LXR). In this study, we aimed to determine the synergic effects in full-thickness models (FTMs) with combinations of these factors as single-, double-, and triple-targeted optimization approaches. The collagen-chitosan FTM supplemented with the LXR inhibitor showed improved epidermal morphogenesis, an enhanced lipid composition, and a better lipid organization. Importantly, barrier functionality was improved in the corresponding approach. In conclusion, our leading optimization approach substantially improved the epidermal morphogenesis, barrier formation, and functionality in the FTM, which therefore better resembled native human skin.

Gene duplications and gene loss in the epidermal differentiation complex during the evolutionary land-to-water transition of cetaceans.

Major protein components of the mammalian skin barrier are encoded by genes clustered in the Epidermal Differentiation Complex (EDC). The skin of cetaceans, i.e. whales, porpoises and dolphins, differs histologically from that of terrestrial mammals. However, the genetic regulation of their epidermal barrier is only incompletely known. Here, we investigated the EDC of cetaceans by comparative genomics. We found that important epidermal cornification proteins, such as loricrin and involucrin are conserved and subtypes of small proline-rich proteins (SPRRs) are even expanded in numbers in cetaceans. By contrast, keratinocyte proline rich protein (KPRP), skin-specific protein 32 (XP32) and late-cornified envelope (LCE) genes with the notable exception of LCE7A have been lost in cetaceans. Genes encoding proline rich 9 (PRR9) and late cornified envelope like proline rich 1 (LELP1) have degenerated in subgroups of cetaceans. These data suggest that the evolution of an aquatic lifestyle was accompanied by amplification of SPRR genes and loss of specific other epidermal differentiation genes in the phylogenetic lineage leading to cetaceans.

High proliferation and delamination during skin epidermal stratification.

The development of complex stratified epithelial barriers in mammals is initiated from single-layered epithelia. How stratification is initiated and fueled are still open questions. Previous studies on skin epidermal stratification suggested a central role for perpendicular/asymmetric cell division orientation of the basal keratinocyte progenitors. Here, we use centrosomes, that organize the mitotic spindle, to test whether cell division orientation and stratification are linked. Genetically ablating centrosomes from the developing epidermis leads to the activation of the p53-, 53BP1- and USP28-dependent mitotic surveillance pathway causing a thinner epidermis and hair follicle arrest. The centrosome/p53-double mutant keratinocyte progenitors significantly alter their division orientation in the later stages without majorly affecting epidermal differentiation. Together with time-lapse imaging and tissue growth dynamics measurements, the data suggest that the first and major phase of epidermal development is boosted by high proliferation rates in both basal and suprabasally-committed keratinocytes as well as cell delamination, whereas the second phase maybe uncoupled from the division orientation of the basal progenitors. The data provide insights for tissue homeostasis and hyperproliferative diseases that may recapitulate developmental programs.

Exploration of novel candidate genes involved in epidermal keratinocyte differentiation and skin barrier repair in man.

A proper skin barrier function requires constant formation of stratum corneum, i.e. the outermost layer of epidermis composed of terminally differentiated keratinocytes. The complex process of converting proliferative basal keratinocytes into corneocytes relies on programmed changes in the activity of many well-established genes. Much remains however to be investigated about this process, e.g. in conjunction with epidermal barrier defects due to genetic errors as in ichthyosis. To this end, we re-analyzed two sets of microarray-data comparing altered gene expression in differentiated vs. proliferating keratinocytes and in the skin of patients with autosomal recessive congenital ichthyosis (ARCI) vs. healthy controls, respectively. We thus identified 24 genes to be upregulated in both sets of array and not previously associated with keratinocyte differentiation. For 10 of these genes (AKR1B10, BLNK, ENDOU, GCNT4, GLTP, RHCG, SLC15A1, TMEM45B, TMEM86A and VSNL1), qPCR analysis confirmed the array results and subsequent immunostainings of normal epidermis showed superficial expression of several of the proteins. Furthermore, induction of keratinocyte differentiation using phorbol esters (PMA) resulted in increased expression of eight of the genes, whereas siRNA silencing of PPARδ, a transcription factor supporting differentiation, had the opposite effect. In summary, our results identify ten new candidate genes seemingly involved in human epidermal keratinocyte differentiation and possibly important for epidermal repair in a genetic skin disease characterized by barrier failure.

Getting under the skin of Polycomb-dependent gene regulation.

The Polycomb repressive system functions through chromatin to regulate gene expression and development. In this issue of Genes & Development, Cohen and colleagues (pp. 354-366) use the developing mouse epidermis as a model system to show that the two central Polycomb repressive complexes, PRC1 and PRC2, have autonomous yet overlapping functions in repressing Polycomb target genes. They show that this cooperation enables the stable repression of nonepidermal transcription factors that would otherwise compromise epidermal cell identity and disrupt normal skin development.

Ultrastructural observations on the oncomiracidium epidermis and adult tegument of Discocotyle sagittata, a monogenean gill parasite of salmonids.

During their different life stages, parasites undergo remarkable morphological, physiological, and behavioral "metamorphoses" to meet the needs of their changing habitats. This is even true for ectoparasites, such as the monogeneans, which typically have a free-swimming larval stage (oncomiracidium) that seeks out and attaches to the external surfaces of fish where they mature. Before any obvious changes occur, there are ultrastructural differences in the oncomiracidium's outer surface that prepare it for a parasitic existence. The present findings suggest a distinct variation in timing of the switch from oncomiracidia epidermis to the syncytial structure of the adult tegument and so, to date, there are three such categories within the Monogenea: (1) Nuclei of both ciliated cells and interciliary cytoplasm are shed from the surface layer and the epidermis becomes a syncytial layer during the later stages of embryogenesis; (2) nuclei of both ciliated cells and interciliary syncytium remain distinct and the switch occurs later after the oncomiracidia hatch (as in the present study); and (3) the nuclei remain distinct in the ciliated epidermis but those of the interciliary epidermis are lost during embryonic development. Here we describe how the epidermis of the oncomiracidium of Discocotyle sagittata is differentiated into two regions, a ciliated cell layer and an interciliary, syncytial cytoplasm, both of which are nucleated. The interciliary syncytium extends in-between and underneath the ciliated cells and sometimes covers part of their apical surfaces, possibly the start of their shedding process. The presence of membranous whorls and pyknotic nuclei over the surface are indicative of membrane turnover suggesting that the switch in epidermis morphology is already initiated at this stage. The body tegument and associated putative sensory receptors of subadult and adult D. sagittata are similar to those in other monogeneans.

The dynamics of epidermal stratification during post-larval development in zebrafish.

BACKGROUND: The epidermis, as a defensive barrier, is a consistent trait throughout animal evolution. During post-larval development, the zebrafish epidermis thickens by stratification or addition of new cell layers. Epidermal basal stem cells, expressing the transcription factor p63, are known to be involved in this process. Zebrafish post-larval epidermal stratification is a tractable system to study how stem cells participate in organ growth. METHODS: We used immunohistochemistry, in combination with EdU cell proliferation detection, to study zebrafish epidermal stratification. For this procedure, we selected a window of post-larval stages (5-8 mm of standard length or SL, which normalizes age by size). Simultaneously, we used markers for asymmetric cell division and the Notch signaling pathway. RESULTS: We found that epidermal stratification is the consequence of several events, including changes in cell shape, active cell proliferation and asymmetrical cell divisions. We identified a subset of highly proliferative epidermal cells with reduced levels of p63, which differed from the basal stem cells with high levels of p63. Additionally, we described different mechanisms that participate in the stratification process, including the phosphorylation of p63, asymmetric cell division regulated by the Par3 and LGN proteins, and expression of Notch genes.

Investigating Epidermal Interactions Through an In Vivo Cutaneous Wound-Healing Assay.

Cutaneous wound healing is an intricate and multifaceted process. Despite these complexities, the distinct phases of wound healing provide a unique opportunity to evaluate the roles of different targets in these coordinated responses. This protocol details an in vivo wound healing assay to study the intersection of cellular, molecular, and systemic effector pathways. The role of certain proteins in the wound healing process can be efficiently explored in vivo through the generation of tissue-specific deficient mice. This approach, although optimized for use with animal models displaying epithelial deficiencies, can be used for other tissue-specific deficiencies, and utilizes simple and cost-effective methods, allowing investigators to precisely devise their experimental design. The coordination of immunological, epithelial, vascular, and microenvironmental factors in wound healing makes this technique a valuable tool for investigators across fields.

Parallel evolution of direct development in frogs - Skin and thyroid gland development in African Squeaker Frogs (Anura: Arthroleptidae: Arthroleptis).

BACKGROUND: Cases of parallel evolution offer the possibility to identify adaptive traits and to uncover developmental constraints on the evolutionary trajectories of these traits. The independent evolution of direct development from the ancestral biphasic life history in frogs is such a case of parallel evolution. In frogs, aquatic larvae (tadpoles) differ profoundly from their adult forms and exhibit a stunning diversity regarding their habitats, morphology and feeding behaviors. The transition from the tadpole to the adult is a climactic, thyroid hormone (TH)-dependent process of profound and fast morphological rearrangement called metamorphosis. One of the organ systems that experiences the most comprehensive metamorphic rearrangements is the skin. Direct-developing frogs lack a free-swimming tadpole and hatch from terrestrial eggs as fully formed froglets. In the few species examined, development is characterized by the condensed and transient formation of some tadpole-specific features and the early formation of adult-specific features during a "cryptic" metamorphosis. RESULTS: We show that skin in direct-developing African squeaker frogs (Arthroleptis) is also repatterned from a tadpole-like to an adult-like histology during a cryptic metamorphosis. This repatterning correlates with histological thyroid gland maturation. A comparison with data from the Puerto Rican coqui (Eleutherodactylus coqui) reveals that the evolution of direct development in these frogs is associated with a comparable heterochronic shift of thyroid gland maturation. CONCLUSION: This suggests that the development of many adult features is still dependent on, and possibly constrained by, the ancestral dependency on thyroid hormone signaling.

Accelerated human epidermal turnover driven by increased hyaluronan production.

BACKGROUND: Hyaluronan (HA) is an essential component of extracellular matrix in the skin, but its functions in the epidermis remain elusive. OBJECTIVE: We examined the interaction of increased HA production mediated by 1-ethyl-ß-N-acetylglucosaminide (ß-NAG2), a newly developed highly selective inducer of HA production which is intracellularly converted to UDP-N-acetylglucosamine, a substrate of HA, with epidermal proliferation and differentiation. METHODS: The amount, molecular size and epidermal tissue distribution of HA and expression of CD44, a cell surface receptor for HA, were analyzed in ß-NAG2-treated organ cultured human skin, reconstructed human skin equivalents or cultured human skin keratinocytes. The relationship between HA and epidermal proliferation or differentiation was examined. RESULTS: ß-NAG2 significantly increased HA production in the epidermis of skin explants or skin equivalents without affecting molecular size of HA (>2000 kDa) or CD44 mRNA expression. Histochemical experiments revealed that ß-NAG2 enhances HA signals in the basal to granular layers of the epidermis of skin equivalents, accompanying increased epidermal stratification. Immunohistochemical experiments demonstrated that signals of Ki67, transglutaminase 1 and filaggrin are increased in ß-NAG2-treated skin equivalents, and these observations were confirmed by the data showing that mRNA expression of PCNA, transglutaminase 1 (TGM1) and filaggrin (FLG) is significantly up-regulated by ß-NAG2 in skin equivalents. Importantly, blockade of HA production by inhibiting conversion of ß-NAG2 to UDP-NAG abolished ß-NAG2-mediated up-regulation of PCNA, TGM1 and FLG mRNA expression in cultured keratinocytes. CONCLUSION: These results suggest that increased epidermal HA production plays a key role in epidermal morphogenesis and homeostasis by accelerating keratinocyte proliferation and differentiation.