Emanuel Rognoni

Wounding induces dedifferentiation of epidermal Gata6 + cells and acquisition of stem cell properties

The epidermis is maintained by multiple stem cell populations whose progeny differentiate along diverse, and spatially distinct, lineages. Here we show that the transcription factor Gata6 controls the identity of the previously uncharacterized sebaceous duct (SD) lineage and identify the Gata6 downstream transcription factor network that specifies a lineage switch between sebocytes and SD cells. During wound healing differentiated Gata6+ cells migrate from the SD into the interfollicular epidermis and dedifferentiate, acquiring the ability to undergo long-term self-renewal and differentiate into a much wider range of epidermal lineages than in undamaged tissue. Our data not only demonstrate that the structural and functional complexity of the junctional zone is regulated by Gata6, but also reveal that dedifferentiation is a previously unrecognized property of post-mitotic, terminally differentiated cells that have lost contact with the basement membrane. This resolves the long-standing debate about the contribution of terminally differentiated cells to epidermal wound repair.

Inhibition of β-catenin signalling in dermal fibroblasts enhances hair follicle regeneration during wound healing

New hair follicles (HFs) do not form in adult mammalian skin unless epidermal Wnt signalling is activated genetically or within large wounds. To understand the postnatal loss of hair forming ability we monitored HF formation at small circular (2 mm) wound sites. At P2, new HFs formed in back skin, but HF formation was markedly decreased by P21. Neonatal tail also formed wound-associated HFs, albeit in smaller numbers. Postnatal loss of HF neogenesis did not correlate with wound closure rate but with a reduction in Lrig1-positive papillary fibroblasts in wounds. Comparative gene expression profiling of back and tail dermis at P1 and dorsal fibroblasts at P2 and P50 showed a correlation between loss of HF formation and decreased expression of genes associated with proliferation and Wnt/β-catenin activity. Between P2 and P50, fibroblast density declined throughout the dermis and clones of fibroblasts became more dispersed. This correlated with a decline in fibroblasts expressing a TOPGFP reporter of Wnt activation. Surprisingly, between P2 and P50 there was no difference in fibroblast proliferation at the wound site but Wnt signalling was highly upregulated in healing dermis of P21 compared with P2 mice. Postnatal β-catenin ablation in fibroblasts promoted HF regeneration in neonatal and adult mouse wounds, whereas β-catenin activation reduced HF regeneration in neonatal wounds. Our data support a model whereby postnatal loss of hair forming ability in wounds reflects elevated dermal Wnt/β-catenin activation in the wound bed, increasing the abundance of fibroblasts that are unable to induce HF formation. Summary: Postnatal mouse skin exhibits a decline in its ability to regenerate hair follicles in the wound bed and this can be partially reversed by inhibiting dermal β-catenin activation.

Fate of Prominin-1 Expressing Dermal Papilla Cells During Homeostasis, Wound Healing and Wnt Activation

Prominin-1/CD133 (Prom1) is expressed by fibroblasts in the dermal papilla (DP) of the hair follicle (HF). By examining endogenous Prom1 expression and expression of LacZ in the skin of Prom1CreERLacZ (Prom1C-L) mice, in which a CreERT2-IRES-nuclear LacZ cassette is knocked into the first ATG codon of Prom1, we confirmed that Prom1 is expressed in the DP of all developing HFs and also by postnatal anagen follicles. To analyze the fate of Prom1+ DP cells, we crossed Prom1C-L mice with Rosa26-CAG flox/stop/flox tdTomato reporter mice and applied 4-hydroxytamoxifen (4OHT) to back skin at postnatal day (P) 1 and P2. We detected tdTomato+ cells in ~50% of DPs. The proportion of labeled cells per DP increased between P5 and P63, while the total number of cells per DP declined. Following full thickness wounding, there was no migration of tdTomato-labeled cells out of the DP. When β-catenin was activated in Prom1+ DP cells there was an increase in the size of anagen and telogen DP, but the proportion of tdTomato-labeled cells did not increase. We conclude that Prom1+ DP cells do not contribute to dermal repair but are nevertheless capable of regulating DP size via β-catenin-mediated intercellular communication.

Dermal Blimp1 Acts Downstream of Epidermal TGFβ and Wnt/β-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/β-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-β and Wnt/β-catenin. Epidermal overexpression of stabilized β-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.

Skin Cell Heterogeneity in Development, Wound Healing, and Cancer

Skin architecture and function depend on diverse populations of epidermal cells and dermal fibroblasts. Reciprocal communication between the epidermis and dermis plays a key role in skin development, homeostasis and repair. While several stem cell populations have been identified in the epidermis with distinct locations and functions, it is now recognised that there is additional heterogeneity within the mesenchymal cells of the dermis. Here, we discuss recent insights into how these distinct cell populations are maintained and coordinated during development, homeostasis, and wound healing. We highlight the importance of the local environment, or niche, in cellular plasticity. We also discuss new mechanisms that have been identified as influencing wound repair and cancer progression.

Loxl2 is dispensable for dermal development, homeostasis and tumour stroma formation

Lysyl oxidase-like 2 (LOXL2) is a copper-dependent monoamine oxidase that contributes to the remodelling of the extracellular matrix (ECM) by cross linkage of collagen and elastin fibres and has emerged as a potential therapeutic target in cancer and fibrosis. In the skin, LOXL2 is essential for epidermal cell polarity and differentiation. However, its role in the dermis has not been evaluated. We found that Loxl2 is dispensable for mouse dermal development, maturation and homeostasis, yet affects dermal stiffness. Neither loss of Loxl2 nor increased Loxl2 expression affected dermal architecture following treatment with the phorbol ester TPA. Furthermore, Loxl2 expression did not alter the stroma of DMBA-TPA-induced tumours. We conclude that, although Loxl2 is expressed in both dermis and epidermis, its function appears largely confined to the epidermis.

Fibroblast state switching orchestrates dermal maturation and wound healing

Murine dermis contains functionally and spatially distinct fibroblast lineages that cease to proliferate in early postnatal life. Here, we propose a model in which a negative feedback loop between extracellular matrix (ECM) deposition and fibroblast proliferation determines dermal architecture. Virtual‐tissue simulations of our model faithfully recapitulate dermal maturation, predicting a loss of spatial segregation of fibroblast lineages and dictating that fibroblast migration is only required for wound healing. To test this, we performed in vivo live imaging of dermal fibroblasts, which revealed that homeostatic tissue architecture is achieved without active cell migration. In contrast, both fibroblast proliferation and migration are key determinants of tissue repair following wounding. The results show that tissue‐scale coordination is driven by the interdependence of cell proliferation and ECM deposition, paving the way for identifying new therapeutic strategies to enhance skin regeneration.

Reply to Chi et al.

TO THE EDITOR We are grateful to Bruce Morgan and his colleagues for engaging in a dialog over our own article (Kaushal et al., 2015). We believe that open discussion about conflicting observations is important in moving forward research—far too often results that are at odds with one another are simply ignored. There are certainly aspects of the Chi et al. (2017) study that are superior to ours. In particular, we agree that there are advantages of studying the dermal papilla (DP) in unpigmented skin, and we note that the quantitation by Chi et al. is based on a larger number of hair follicles, and mice, than in our study. Chi et al. (2017) suggest that our results are due to misidentification of hair follicle types. We agree that DP size alone cannot be used to identify hair follicle type. We analyzed skin of P65 mice in which the hair follicle cycle was asynchronous so that we could report on anagen (stage IV; early anagen of the second hair follicle cycle, according to the classification of Müller-Röver et al. [2001]) and telogen follicles from the same mice. We believe that Chi et al. are looking at a later anagen stage. Given the low frequency of non-zigzag hairs in our samples, it is unlikely that they would skew our data. The tissue in our article (Kaushal et al., 2015) was not flash frozen but fixed with 4% paraformaldehyde before being cryopreserved in Optimal Cutting Temperature medium. Tissue preservation under these conditions is excellent (Driskell et al., 2012, 2013). The movies in the Supplemental Material of Kaushal et al. show that the 60-umethick horizontal whole mounts

Letter in reply to Chi et al

TO THE EDITOR We are grateful to Bruce Morgan and his colleagues for engaging in a dialog over our own article (Kaushal et al., 2015). We believe that open discussion about conflicting observations is important in moving forward research—far too often results that are at odds with one another are simply ignored. There are certainly aspects of the Chi et al. (2017) study that are superior to ours. In particular, we agree that there are advantages of studying the dermal papilla (DP) in unpigmented skin, and we note that the quantitation by Chi et al. is based on a larger number of hair follicles, and mice, than in our study. Chi et al. (2017) suggest that our results are due to misidentification of hair follicle types. We agree that DP size alone cannot be used to identify hair follicle type. We analyzed skin of P65 mice in which the hair follicle cycle was asynchronous so that we could report on anagen (stage IV; early anagen of the second hair follicle cycle, according to the classification of Müller-Röver et al. [2001]) and telogen follicles from the same mice. We believe that Chi et al. are looking at a later anagen stage. Given the low frequency of non-zigzag hairs in our samples, it is unlikely that they would skew our data. The tissue in our article (Kaushal et al., 2015) was not flash frozen but fixed with 4% paraformaldehyde before being cryopreserved in Optimal Cutting Temperature medium. Tissue preservation under these conditions is excellent (Driskell et al., 2012, 2013). The movies in the Supplemental Material of Kaushal et al. show that the 60-umethick horizontal whole mounts