Modulating embryonic signaling pathways paves the way for regeneration in wound healing.

Epithelial tissues, including the skin, are highly proliferative tissues with the capability to constant renewal and regeneration, a feature that is essential for survival as the skin forms a protective barrier against external insults and water loss. In adult mammalian skin, every injury will lead to a scar. The scar tissue that is produced to seal the wound efficiently is usually rigid and lacks elasticity and the skin's original resilience to external impacts, but also secondary appendages such as hair follicles and sebaceous glands. While it was long thought that hair follicles develop solely during embryogenesis, it is becoming increasingly clear that hair follicles can also regenerate within a wound. The ability of the skin to induce hair neogenesis following injury however declines with age. As fetal and neonatal skin have the remarkable capacity to heal without scarring, the recapitulation of a neonatal state has been a primary target of recent regenerative research. In this review we highlight how modulating dermal signaling or the abundance of specific fibroblast subsets could be utilized to induce de novo hair follicles within the wound bed, and thus to shift wound repair with a scar to scarless regeneration.

Molecular and spatial landmarks of early mouse skin development.

A wealth of specialized cell populations within the skin facilitates its hair-producing, protective, sensory, and thermoregulatory functions. How the vast cell-type diversity and tissue architecture develops is largely unexplored. Here, with single-cell transcriptomics, spatial cell-type assignment, and cell-lineage tracing, we deconstruct early embryonic mouse skin during the key transitions from seemingly uniform developmental precursor states to a multilayered, multilineage epithelium, and complex dermal identity. We identify the spatiotemporal emergence of hair-follicle-inducing, muscle-supportive, and fascia-forming fibroblasts. We also demonstrate the formation of the panniculus carnosus muscle (PCM), sprouting blood vessels without pericyte coverage, and the earliest residence of mast and dendritic immune cells in skin. Finally, we identify an unexpected epithelial heterogeneity within the early single-layered epidermis and a signaling-rich periderm layer. Overall, this cellular and molecular blueprint of early skin development-which can be explored at https://kasperlab.org/tools-establishes histological landmarks and highlights unprecedented dynamic interactions among skin cells.

A Roadmap for a Consensus Human Skin Cell Atlas and Single-Cell Data Standardization.

Single-cell technologies have become essential to driving discovery in both basic and translational investigative dermatology. Despite the multitude of available datasets, a central reference atlas of normal human skin, which can serve as a reference resource for skin cell types, cell states, and their molecular signatures, is still lacking. For any such atlas to receive broad acceptance, participation by many investigators during atlas construction is an essential prerequisite. As part of the Human Cell Atlas project, we have assembled a Skin Biological Network to build a consensus Human Skin Cell Atlas and outline a roadmap toward that goal. We define the drivers of skin diversity to be considered when selecting sequencing datasets for the atlas and list practical hurdles during skin sampling that can result in data gaps and impede comprehensive representation and technical considerations for tissue processing and computational analysis, the accounting for which should minimize biases in cell type enrichments and exclusions and decrease batch effects. By outlining our goals for Atlas 1.0, we discuss how it will uncover new aspects of skin biology.

Myofibroblast stroma differentiation in infiltrative basal cell carcinoma is accompanied by regulatory T-cells.

INTRODUCTION: The implications of infiltrative compared to non-infiltrative growth of cutaneous basal cell carcinoma (BCC) on the tumor stroma and immune cell landscape are unknown. This is of clinical importance, because infiltrative BCCs, in contrast to other BCC subtypes, are more likely to relapse after surgery and radiotherapy. MATERIALS AND METHODS: This descriptive cross-sectional study analyzed 38 BCCs collected from 2018 to 2021. In the first cohort (n = 28), immune cells were characterized by immunohistochemistry and multiplex immunofluorescence staining for CD3, CD8, CD68, Foxp3, and α-SMA protein expression. In the second cohort (n = 10) with matched characteristics (age, sex, location, and BCC subtype), inflammatory parameters, including TGF-ß1, TGF-ß2, ACTA2, IL-10, IL-12A, and Foxp3, were quantified via RT-qPCR after isolating mRNA from BCC tissue samples and perilesional skin. RESULTS: Infiltrative BCCs showed significantly increased levels of α-SMA expression in fibroblasts (p = 0.0001) and higher levels of Foxp3+ (p = 0.0023) and CD3+ (p = 0.0443) T-cells compared to non-infiltrative BCCs. CD3+ (p = 0.0171) and regulatory T-cells (p = 0.0026) were significantly increased in α-SMA-positive tumor stroma, whereas CD8+ T-cells (p = 0.1329) and CD68+ myeloid cells (p = 0.2337) were not affected. TGF-ß1 and TGF-ß2 correlated significantly with ACTA2/α-SMA mRNA expression (p = 0.020, p = 0.005). CONCLUSION: Infiltrative growth of BCCs shows a myofibroblastic stroma differentiation and is accompanied by an immunosuppressive tumor microenvironment.

Hedgehog Signaling in Papillary Fibroblasts Is Essential for Hair Follicle Regeneration during Wound Healing.

Patients suffering from large scars such as burn victims not only encounter aesthetic challenges but also ongoing itching or pain that substantially deteriorates their quality of life. Skin appendages such as hair follicles rarely regenerate within the healing wound. Because they are crucial for skin homeostasis and the lack thereof constitutes one of the main limitations to scarless wound healing, their regeneration represents a major objective for regenerative medicine. Fibroblasts, the main resident cell type of the skin dermis, mediate embryonic hair follicle morphogenesis and are particularly involved in wound healing because they orchestrate extracellular matrix remodeling and collagen deposition in the wound bed. Importantly, dermal fibroblasts originate from two distinct developmental lineages with unique functions that differently mediate the response to epidermal signals such as Hedgehog signaling. In this study, we show that Hedgehog signaling in the reticular fibroblast lineage promotes the initial phase of wound repair, possibly by modulating angiogenesis and fibroblast proliferation, whereas Hedgehog signaling in papillary fibroblasts is essential to induce de novo hair follicle formation within the healing wound.

Mus musculus papillomavirus 1 is a key driver of skin cancer development upon immunosuppression.

Epidemiological and experimental data implicate cutaneous human papillomavirus infection as co-factor in the development of cutaneous squamous cell carcinomas (cSCCs), particularly in immunocompromised organ transplant recipients (OTRs). Herein, we established and characterized a skin cancer model, in which Mus musculus papillomavirus 1 (MmuPV1) infection caused cSCCs in cyclosporine A (CsA)-treated mice, even in the absence of UV light. Development of cSCCs and their precursors were observed in 70% of MmuPV1-infected, CsA-treated mice on back as well as on tail skin. Immunosuppression by systemic CsA, but not UV-B irradiation, was a prerequisite, as immunocompetent or UV-B-irradiated mice did not develop skin malignancies after infection. In the virus-driven cSCCs the MmuPV1-E6/E7 oncogenes were abundantly expressed, and transcriptional activity and productive infection demonstrated. MmuPV1 infection induced the expression of phosphorylated H2AX, but not degradation of proapoptotic BAK in the cSCCs. Transfer of primary cells, established from a MmuPV1-induced cSCC from back skin, into athymic nude mice gave rise to secondary cSCCs, which lacked viral DNA, demonstrating that maintenance of the malignant phenotype was virus independent. This papillomavirus-induced skin cancer model opens future investigations into viral involvement, pathogenesis, and cancer surveillance, aiming at understanding and controlling the high incidence of skin cancer in OTRs.

Deciphering the functional heterogeneity of skin fibroblasts using single-cell RNA sequencing.

Though skin fibroblasts (FB) are the main cell population within the dermis, the different skin FB subsets are not well characterized and the traditional classification into reticular and papillary FBs has little functional relevance. To fill the gap of knowledge on FB diversity in human skin, we performed single-cell RNA sequencing. Investigation of marker genes for the different skin cell subtypes revealed a heterogeneous picture of FBs. When mapping reticular and papillary FB markers, we could not detect cluster specificity, suggesting that these two populations show a higher transcriptional heterogeneity than expected. This finding was further confirmed by in situ hybridization, showing that DPP4 was expressed in both dermal layers. Our analysis identified six FB clusters with distinct transcriptional signatures. Importantly, we could demonstrate that in human skin DPP4+ FBs are the main producers of factors involved in extracellular matrix (ECM) assembly. In conclusion, we provide evidence that hitherto considered FB markers are not ideal to characterize skin FB subpopulations in single-cell sequencing analyses. The identification of DPP4+ FBs as the main ECM-producing cells in human skin will foster the development of anti-fibrotic treatments for the skin and other organs.

Inhibition of p38/MK2 Signaling Prevents Vascular Invasion of Melanoma.

Melanoma cells can switch between distinct gene expression profiles, resulting in proliferative or invasive phenotypes. Signaling pathways involved in this switch were analyzed by gene expression profiling of a cohort of 22 patient-derived melanoma cell lines. CDH1 negativity was identified as a surrogate marker for the invasive phenotype. CDH1 expression could be turned on and off by modulating activity of p38 or its downstream target MK2, suggesting that this pathway controls melanoma progression. Mechanistically, MK2 inhibition prevented melanoma-induced vascular barrier disruption, reduced the expression of PODXL and DEL-1, and prevented vascular dissemination in vivo. PODXL and DEL-1 expression in patients with melanoma were associated with poor survival and thus can be used as prognostic markers. Downstream targets of MK2 may thus serve as candidate therapeutics.

Hair eruption initiates and commensal skin microbiota aggravate adverse events of anti-EGFR therapy.

Epidermal growth factor receptor (EGFR)-targeted anticancer therapy induces stigmatizing skin toxicities affecting patients' quality of life and therapy adherence. The lack of mechanistic details underlying these adverse events hampers their management. We found that EGFR/ERK signaling is required in LRIG1-positive stem cells during de novo hair eruption to secure barrier integrity and prevent the invasion of commensal microbiota and inflammatory skin disease. EGFR-deficient epidermis is permissive for microbiota outgrowth and displays an atopic-like TH2-dominated signature. The opening of the follicular ostia during hair eruption allows invasion of commensal microbiota into the hair follicle, initiating an additional TH1 and TH17 response culminating in chronic folliculitis. Restoration of epidermal ERK signaling via prophylactic FGF7 treatment or transgenic SOS expression rescues the barrier defect in the absence of EGFR, highlighting a therapeutic anchor point. These data reveal that commensal skin microbiota provoke atopic-like inflammatory skin diseases by invading into the follicular opening of erupting hair.

EGFR Controls Hair Shaft Differentiation in a p53-Independent Manner.

Epidermal growth factor receptor (EGFR) signaling controls skin development and homeostasis in mice and humans, and its deficiency causes severe skin inflammation, which might affect epidermal stem cell behavior. Here, we describe the inflammation-independent effects of EGFR deficiency during skin morphogenesis and in adult hair follicle stem cells. Expression and alternative splicing analysis of RNA sequencing data from interfollicular epidermis and outer root sheath indicate that EGFR controls genes involved in epidermal differentiation and also in centrosome function, DNA damage, cell cycle, and apoptosis. Genetic experiments employing p53 deletion in EGFR-deficient epidermis reveal that EGFR signaling exhibits p53-dependent functions in proliferative epidermal compartments, as well as p53-independent functions in differentiated hair shaft keratinocytes. Loss of EGFR leads to absence of LEF1 protein specifically in the innermost epithelial hair layers, resulting in disorganization of medulla cells. Thus, our results uncover important spatial and temporal features of cell-autonomous EGFR functions in the epidermis.

Isolation of Papillary and Reticular Fibroblasts from Human Skin by Fluorescence-activated Cell Sorting.

Fibroblasts are a highly heterogeneous cell population implicated in the pathogenesis of many human diseases. In human skin dermis, fibroblasts have traditionally been attributed to the superficial papillary or lower reticular dermis according to their histological localization. In mouse dermis, papillary and reticular fibroblasts originate from two different lineages with diverging functions regarding physiological and pathological processes and a distinct cell surface marker expression profile by which they can be distinguished. Importantly, evidence from explant cultures from superficial and lower dermal layers suggest that at least two functionally distinct dermal fibroblasts lineages exist in human skin dermis as well. However, unlike for mouse skin, cell surface markers enabling the discrimination of different fibroblast subsets have not yet been established for human skin. We developed a novel protocol for the isolation of human papillary and reticular fibroblast populations via fluorescence-activated cell sorting (FACS) using the two cell surface markers Fibroblast Activation Protein (FAP) and Thymocyte antigen 1 (Thy1)/CD90. This method enables the isolation of pure fibroblast subsets without in vitro manipulation, which was shown to affect gene expression, thus permitting accurate functional analysis of human dermal fibroblast subsets in regard to tissue homeostasis or disease pathology.

CXCL5 as Regulator of Neutrophil Function in Cutaneous Melanoma.

Chemokines mold the tumor microenvironment by recruiting distinct immune cell populations, thereby strongly influencing disease progression. Previously, we showed that CXCL5 expression is upregulated in advanced stages of primary melanomas, which correlates with the presence of neutrophils in the tumor. The analysis of neutrophil populations in various tissues revealed a distinct phenotype of tumor-associated neutrophils. Tumor-associated neutrophils expressed PD-L1, CXCR4, CCR5, Adam17, and Nos2 and were immunosuppressive in a T-cell proliferation assay. To investigate the impact of CXCL5 and neutrophils in vivo, we established a syngeneic mouse tumor transplantation model using CXCL5-overexpressing and control melanoma cell lines. Growth behavior or vascularization of primary tumors was not affected by CXCL5 expression and neutrophils alone. However, in combination with Poly(I:C), tumor-associated neutrophils were able to attenuate induced antitumoral T-cell responses. CXCL5-overexpressing tumors had reduced lung metastasis compared with control tumors. Neutrophil depletion reversed this effect. In vitro, unstimulated lung-derived neutrophils had higher levels of reactive oxygen species compared with tumor-associated neutrophils, and CXCL5 stimulation further increased reactive oxygen species levels. In summary, in melanoma, neutrophils play a context-dependent role that is influenced by local or systemic factors, and interfere with therapies activating the acquired immune system. Actively switching neutrophils into antitumorigenic mode might be a new therapeutic strategy.

Lineage Identity and Location within the Dermis Determine the Function of Papillary and Reticular Fibroblasts in Human Skin.

Human skin dermis is composed of the superficial papillary dermis and the reticular dermis in the lower layers, which can easily be distinguished histologically. In vitro analyses of fibroblasts from explant cultures from superficial and lower dermal layers suggest that human skin comprises at least two fibroblast lineages with distinct morphology, expression profiles, and functions. However, while for mouse skin cell surface markers have been identified, allowing the isolation of pure populations of one lineage or the other via FACS, this has not been achieved for human skin fibroblasts. We have now discovered two cell surface markers that discriminate between papillary and reticular fibroblasts. While FAP+CD90- cells display increased proliferative potential, express PDPN and NTN1, and cannot be differentiated into adipocytes, FAP-CD90+ fibroblasts express high levels of ACTA2, MGP, PPARγ, and CD36 and readily undergo adipogenic differentiation, a hallmark of reticular fibroblasts. Flow cytometric analysis of fibroblasts isolated from superficial and lower layers of human dermis showed that FAP+CD90- cells are enriched in the papillary dermis. Altogether, functional analysis and expression profiling confirms that FAP+CD90- cells represent papillary fibroblasts, whereas FAP-CD90+ fibroblasts derive from the reticular lineage. Although papillary and reticular fibroblasts are enriched in the upper or lower dermis, respectively, they are not spatially restricted, and the microenvironment seems to affect their function.

An evolutionarily conserved ribosome-rescue pathway maintains epidermal homeostasis.

Ribosome-associated mRNA quality control mechanisms ensure the fidelity of protein translation1,2. Although these mechanisms have been extensively studied in yeast, little is known about their role in mammalian tissues, despite emerging evidence that stem cell fate is controlled by translational mechanisms3,4. One evolutionarily conserved component of the quality control machinery, Dom34 (in higher eukaryotes known as Pelota (Pelo)), rescues stalled ribosomes 5 . Here we show that Pelo is required for mammalian epidermal homeostasis. Conditional deletion of Pelo in mouse epidermal stem cells that express Lrig1 results in hyperproliferation and abnormal differentiation of these cells. By contrast, deletion of Pelo in Lgr5-expressing stem cells has no effect and deletion in Lgr6-expressing stem cells induces only a mild phenotype. Loss of Pelo results in accumulation of short ribosome footprints and global upregulation of translation, rather than affecting the expression of specific genes. Translational inhibition by rapamycin-mediated downregulation of mTOR (mechanistic target of rapamycin kinase) rescues the epidermal phenotype. Our study reveals that the ribosome-rescue machinery is important for mammalian tissue homeostasis and that it has specific effects on different stem cell populations.

Dermal Blimp1 Acts Downstream of Epidermal TGFß and Wnt/ß-Catenin to Regulate Hair Follicle Formation and Growth.

B-lymphocyte-induced maturation protein 1 (Blimp1) is a transcriptional repressor that regulates cell growth and differentiation in multiple tissues, including skin. Although in the epidermis Blimp1 is important for keratinocyte and sebocyte differentiation, its role in dermal fibroblasts is unclear. Here we show that Blimp1 is dynamically regulated in dermal papilla cells during hair follicle (HF) morphogenesis and the postnatal hair cycle, preceding dermal Wnt/ß-catenin activation. Blimp1 ablation in E12.5 mouse dermal fibroblasts delayed HF morphogenesis and growth and prevented new HF formation after wounding. By combining targeted quantitative PCR screens with bioinformatic analysis and experimental validation we demonstrated that Blimp1 is both a target and a mediator of key dermal papilla inductive signaling pathways including transforming growth factor-ß and Wnt/ß-catenin. Epidermal overexpression of stabilized ß-catenin was able to override the HF defects in Blimp1 mutant mice, underlining the close reciprocal relationship between the dermal papilla and adjacent HF epithelial cells. Overall, our study reveals the functional role of Blimp1 in promoting the dermal papilla inductive signaling cascade that initiates HF growth.

A genome-wide screen identifies YAP/WBP2 interplay conferring growth advantage on human epidermal stem cells.

Individual human epidermal cells differ in their self-renewal ability. To uncover the molecular basis for this heterogeneity, we performed genome-wide pooled RNA interference screens and identified genes conferring a clonal growth advantage on normal and neoplastic (cutaneous squamous cell carcinoma, cSCC) human epidermal cells. The Hippo effector YAP was amongst the top positive growth regulators in both screens. By integrating the Hippo network interactome with our data sets, we identify WW-binding protein 2 (WBP2) as an important co-factor of YAP that enhances YAP/TEAD-mediated gene transcription. YAP and WPB2 are upregulated in actively proliferating cells of mouse and human epidermis and cSCC, and downregulated during terminal differentiation. WBP2 deletion in mouse skin results in reduced proliferation in neonatal and wounded adult epidermis. In reconstituted epidermis YAP/WBP2 activity is controlled by intercellular adhesion rather than canonical Hippo signalling. We propose that defective intercellular adhesion contributes to uncontrolled cSCC growth by preventing inhibition of YAP/WBP2.