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Our understanding of how human skin cells differ according to anatomical site and tumour formation is limited. To address this, we have created a multiscale spatial atlas of healthy skin and basal cell carcinoma (BCC), incorporating in vivo optical coherence tomography, single-cell RNA sequencing, spatial global transcriptional profiling, and in situ sequencing. Computational spatial deconvolution and projection revealed the localisation of distinct cell populations to specific tissue contexts. Although cell populations were conserved between healthy anatomical sites and in BCC, mesenchymal cell populations including fibroblasts and pericytes retained signatures of developmental origin. Spatial profiling and in silico lineage tracing support a hair follicle origin for BCC and demonstrate that cancer-associated fibroblasts are an expansion of a POSTN+ subpopulation associated with hair follicles in healthy skin. RGS5+ pericytes are also expanded in BCC suggesting a role in vascular remodelling. We propose that the identity of mesenchymal cell populations is regulated by signals emanating from adjacent structures and that these signals are repurposed to promote the expansion of skin cancer stroma. The resource we have created is publicly available in an interactive format for the research community. [ABSTRACT FROM AUTHOR]

Stem cells in organoids self-organize into tissue patterns with unknown mechanisms. Here, we use skin organoids to analyze this process. Cell behavior videos show that the morphological transformation from multiple spheroidal units with morphogenesis competence (CMU) to planar skin is characterized by two abrupt cell motility–increasing events before calming down. The self-organizing processes are controlled by a morphogenetic module composed of molecular sensors, modulators, and executers. Increasing dermal stiffness provides the initial driving force (driver) which activates Yap1 (sensor) in epidermal cysts. Notch signaling (modulator 1) in epidermal cyst tunes the threshold of Yap1 activation. Activated Yap1 induces Wnts and MMPs (epidermal executers) in basal cells to facilitate cellular flows, allowing epidermal cells to protrude out from the CMU. Dermal cell–expressed Rock (dermal executer) generates a stiff force bridge between two CMU and accelerates tissue mixing via activating Laminin and β1-integrin. Thus, this self-organizing coalescence process is controlled by a mechano-chemical circuit. Beyond skin, self-organization in organoids may use similar mechano-chemical circuit structures. [ABSTRACT FROM AUTHOR]

Vertebrate Hox genes are critical for the establishment of structures during the development of the main body axis. Subsequently, they play important roles either in organizing secondary axial structures such as the appendages, or during homeostasis in postnatal stages and adulthood. Here, we set up to analyze their elusive function in the ectodermal compartment, using the mouse limb bud as a model. We report that the HoxC gene cluster was co-opted to be transcribed in the distal limb ectoderm, where it is activated following the rule of temporal colinearity. These ectodermal cells subsequently produce various keratinized organs such as nails or claws. Accordingly, deletion of the HoxC cluster led to mice lacking nails (anonychia), a condition stronger than the previously reported loss of function of Hoxc13, which is the causative gene of the ectodermal dysplasia 9 (ECTD9) in human patients. We further identified two mammalian-specific ectodermal enhancers located upstream of the HoxC gene cluster, which together regulate Hoxc gene expression in the hair and nail ectodermal organs. Deletion of these regulatory elements alone or in combination revealed a strong quantitative component in the regulation of Hoxc genes in the ectoderm, suggesting that these two enhancers may have evolved along with the mammalian taxon to provide the level of HOXC proteins necessary for the full development of hair and nail.

Stiffness and actomyosin contractility are intrinsic mechanical properties of animal cells required for the shaping of tissues. However, whether tissue stem cells (SCs) and progenitors located within SC niche have different mechanical properties that modulate their size and function remains unclear. Here, we show that hair follicle SCs in the bulge are stiff with high actomyosin contractility and resistant to size change, whereas hair germ (HG) progenitors are soft and periodically enlarge and contract during quiescence. During activation of hair follicle growth, HGs reduce contraction and more frequently enlarge, a process that is associated with weakening of the actomyosin network, nuclear YAP accumulation, and cell cycle reentry. Induction of miR-205, a novel regulator of the actomyosin cytoskeleton, reduces actomyosin contractility and activates hair regeneration in young and old mice. This study reveals the control of tissue SC size and activities by spatiotemporally compartmentalized mechanical properties and demonstrates the possibility to stimulate tissue regeneration by fine-tuning cell mechanics. [ABSTRACT FROM AUTHOR]

Low-threshold mechanoreceptors (LTMRs) and their cutaneous end organs convert light mechanical forces acting on the skin into electrical signals that propagate to the central nervous system. In mouse hairy skin, hair follicle-associated longitudinal lanceolate complexes, which are end organs comprising LTMR axonal endings that intimately associate with terminal Schwann cell (TSC) processes, mediate LTMR responses to hair deflection and skin indentation. Here, we characterized developmental steps leading to the formation of Aß rapidly adapting (RA)-LTMR and Ad-LTMR lanceolate complexes. During early postnatal development, Aß RA-LTMRs and Ad-LTMRs extend and prune cutaneous axonal branches in close association with nascent TSC processes. Netrin-G1 is expressed in these developing Aß RA-LTMR and Ad-LTMR lanceolate endings, and Ntng1 ablation experiments indicate that Netrin-G1 functions in sensory neurons to promote lanceolate ending elaboration around hair follicles. The Netrin-G ligand (NGL-1), encoded by Lrrc4c, is expressed in TSCs, and ablation of Lrrc4c partially phenocopied the lanceolate complex deficits observed in Ntng1 mutants. Moreover, NGL-1-Netrin-G1 signaling is a general mediator of LTMR end organ formation across diverse tissue types demonstrated by the fact that Aß RA-LTMR endings associated with Meissner corpuscles and Pacinian corpuscles are also compromised in the Ntng1 and Lrrc4c mutant mice. Thus, axon-glia interactions, mediated in part by NGL-1-Netrin-G1 signaling, promote LTMR end organ formation. [ABSTRACT FROM AUTHOR]

Aging manifests with architectural alteration and functional decline of multiple organs throughout an organism. In mammals, aged skin is accompanied by a marked reduction in hair cycling and appearance of bald patches, leading researchers to propose that hair follicle stem cells (HFSCs) are either lost, differentiate, or change to an epidermal fate during aging. Here, we employed single-cell RNAsequencing to interrogate aging-related changes in the HFSCs. Surprisingly, although numbers declined, aging HFSCs were present, maintained their identity, and showed no overt signs of shifting to an epidermal fate. However, they did exhibit prevalent transcriptional changes particularly in extracellular matrix genes, and this was accompanied by profound structural perturbations in the aging SC niche. Moreover, marked age-related changes occurred in many nonepithelial cell types, including resident immune cells, sensory neurons, and arrector pili muscles. Each of these SC niche components has been shown to influence HF regeneration. When we performed skin injuries that are known to mobilize young HFSCs to exit their niche and regenerate HFs, we discovered that aged skin is defective at doing so. Interestingly, however, in transplantation assays in vivo, aged HFSCs regenerated HFs when supported with young dermis, while young HFSCs failed to regenerate HFs when combined with aged dermis. Together, our findings highlight the importance of SC:niche interactions and favor a model where youthfulness of the niche microenvironment plays a dominant role in dictating the properties of its SCs and tissue health and fitness. [ABSTRACT FROM AUTHOR]

Innocuous mechanical stimuli acting on the skin are detected by sensory neurons, known as low-threshold mechanoreceptors (LTMRs). LTMRs are classified based on their response properties, action potential conduction velocity, rate of adaptation to static indentation of the skin, and terminal anatomy. Here, we report organizational properties of the cutaneous and central axonal projections of the five principal hairy skin LTMR subtypes. We find that axons of neurons within a particular LTMR class are largely nonoverlapping with respect to their cutaneous end organs (e.g., hair follicles), with Aβ rapidly adapting-LTMRs being the sole exception. Individual neurons of each LTMR class are mostly nonoverlapping with respect to their associated hair follicles, with the notable exception of C-LTMRs, which exhibit multiple branches that redundantly innervate individual hair follicles. In the spinal cord, LTMR central projections exhibit rostrocaudal elongation and mediolateral compression, compared with their cutaneous innervation patterns, and these central projections also exhibit a fine degree of homotypic topographic adjacency. These findings thus reveal homotypic tiling of LTMR subtype axonal projections in hairy skin and a remarkable degree of spatial precision of spinal cord axonal termination patterns, suggesting a somatotopically precise tactile encoding capability of the mechanosensory dorsal horn. [ABSTRACT FROM AUTHOR]

Skin tissues, in particular the epidermis, are severely affected by zinc deficiency. However, the zinc-mediated mechanisms that maintain the cells that form the epidermis have not been established. Here, we report that the zinc transporter ZIP10 is highly expressed in the outer root sheath of hair follicles and plays critical roles in epidermal development. We found that ZIP10 marked epidermal progenitor cell subsets and that ablating Zip10 caused significant epidermal hypoplasia accompanied by down-regulation of the transactivation of p63, a master regulator of epidermal progenitor cell proliferation and differentiation. Both ZIP10 and p63 are significantly increased during epidermal development, in which ZIP10-mediated zinc influx promotes p63 transactivation. Collectively, these results indicate that ZIP10 plays important roles in epidermal development via, at least in part, the ZIP10-zinc-p63 signaling axis, thereby highlighting the physiological significance of zinc regulation in the maintenance of skin epidermis. [ABSTRACT FROM AUTHOR]

The complex mechanical properties of biomaterials such as hair, horn, skin, or bone are determined by the architecture of the underlying fibrous bionetworks. Although much is known about the influence of the cytoskeleton on the mechanics of isolated cells, this has been less studied in tridimensional tissues. We used the hair follicle as a model to link changes in the keratin network composition and architecture to the mechanical properties of the nascent hair. We show using atomic force microscopy that the soft keratinocyte matrix at the base of the follicle stiffens by a factor of ∼360, from 30 kPa to 11 MPa along the first millimeter of the follicle. The early mechanical stiffening is concomitant to an increase in diameter of the keratin macrofibrils, their continuous compaction, and increasingly parallel orientation. The related stiffening of the material follows a power law, typical of the mechanics of nonthermal bending-dominated fiber networks. In addition, we used X-ray diffraction to monitor changes in the (supra)molecular organization within the keratin fibers. At later keratinization stages, the inner mechanical properties of the macrofibrils dominate the stiffening due to the progressive setting up of the cystine network. Our findings corroborate existing models on the sequence of biological and structural events during hair keratinization. [ABSTRACT FROM AUTHOR]

How stem cells maintain their identity and potency as tissues change during growth is not well understood. In mammalian hair, it is unclear how hair follicle stem cells can enter an extended period of quiescence during the resting phase but retain stem cell potential and be subsequently activated for growth. Here, we use lineage tracing and gene expression mapping to show that the Wnt target gene Axin2 is constantly expressed throughout the hair cycle quiescent phase in outer bulge stem cells that produce their own Wnt signals. Ablating Wnt signaling in the bulge cells causes them to lose their stem cell potency to contribute to hair growth and undergo premature differentiation instead. Bulge cells express secreted Wnt inhibitors, including Dickkopf (Dkk) and secreted frizzled-related protein 1 (Sfrp1). However, the Dickkopf 3 (Dkk3) protein becomes localized to the Wnt-inactive inner bulge that contains differentiated cells. We find that Axin2 expression remains confined to the outer bulge, whereas Dkk3 continues to be localized to the inner bulge during the hair cycle growth phase. Our data suggest that autocrine Wnt signaling in the outer bulge maintains stem cell potency throughout hair cycle quiescence and growth, whereas paracrine Wnt inhibition of inner bulge cells reinforces differentiation. [ABSTRACT FROM AUTHOR]

Adult tissue stem cells (SCs) reside in niches, which orchestrate SC behavior. SCs are typically used sparingly and exist in quiescence unless activated for tissue growth. Whether parsimonious SC use is essential to conserve long-term tissue-regenerating potential during normal homeostasis remains poorly understood. Here, we examine this issue by conditionally ablating a key transcription factor Forkhead box C1 (FOXC1) expressed in hair follicle SCs (HFSCs). FOXC1-deficient HFSCs spend less time in quiescence, leading to markedly shortened resting periods between hair cycles. The enhanced hair cycling accelerates HFSC expenditure, and impacts hair regeneration in aging mice. Interestingly, although FOXC1-deficient HFs can still form a new bulge that houses HFSCs for the next hair cycle, the older bulge is left unanchored. As the new hair emerges, the entire old bulge, including its reserve HFSCs and SC-inhibitory inner cell layer, is lost. We trace this mechanism first, to a marked increase in cell cycle-associated transcripts upon Foxc1 ablation, and second, to a downstream reduction in E-cadherin-mediated inter-SC adhesion. Finally,we show that when the old bulge is lostwith each hair cycle, overall levels of SC-inhibitory factors are reduced, further lowering the threshold for HFSC activity. Taken together, our findings suggest that HFSCs have restricted potential in vivo, which they conserve by coupling quiescence to adhesion-mediated niche maintenance, thereby achieving long-term tissue homeostasis. [ABSTRACT FROM AUTHOR]

Microscopic mites of the genus Demodex live within the hair follicles of mammals and are ubiquitous symbionts of humans, but little molecular work has been done to understand their genetic diversity or transmission. Here we sampled mite DNA from 70 human hosts of diverse geographic ancestries and analyzed 241 sequences from the mitochondrial genome of the species Demodex folliculorum. Phylogenetic analyses recovered multiple deep lineages including a globally distributed lineage common among hosts of European ancestry and three lineages that primarily include hosts of Asian, African, and Latin American ancestry. To a great extent, the ancestral geography of hosts predicted the lineages of mites found on them; 27% of the total molecular variance segregated according to the regional ancestries of hosts. We found that D. folliculorum populations are stable on an individual over the course of years and that some Asian and African American hosts maintain specific mite lineages over the course of years or generations outside their geographic region of birth or ancestry. D. folliculorum haplotypes were much more likely to be shared within families and between spouses than between unrelated individuals, indicating that transmission requires close contact. Dating analyses indicated that D. folliculorum origins may predate modern humans. Overall, D. folliculorum evolution reflects ancient human population divergences, is consistent with an out-of-Africa dispersal hypothesis, and presents an excellent model system for further understanding the history of human movement. [ABSTRACT FROM AUTHOR]

Among the unique features of humans, one of the most salient is the ability to effectively cool the body during extreme prolonged activity through the evapotranspiration of water on the skin's surface. The evolution of this novel physiological ability required a dramatic increase in the density and distribution of eccrine sweat glands relative to other mammals and a concomitant reduction of body hair cover. Elucidation of the genetic underpinnings for these adaptive changes is confounded by a lack of knowledge about how eccrine gland fate and density are specified during development. Moreover, although reciprocal changes in hair cover and eccrine gland density are required for efficient thermoregulation, it is unclear if these changes are linked by a common genetic regulation. To identify pathways controlling the relative patterning of eccrine glands and hair follicles, we exploited natural variation in the density of these organs between different strains of mice. Quantitative trait locus mapping identified a large region on mouse Chromosome 1 that controls both hair and eccrine gland densities. Differential and allelic expression analysis of the genes within this interval coupled with subsequent functional studies demonstrated that the level of En1 activity directs the relative numbers of eccrine glands and hair follicles. These findings implicate En1 as a newly identified and reciprocal determinant of hair follicle and eccrine gland density and identify a pathway that could have contributed to the evolution of the unique features of human skin. [ABSTRACT FROM AUTHOR]

The generation and functions of human peripheral blood (PB) IgM+ IgD+CD27+ B lymphocytes with somatically mutated IgV genes are controversially discussed. We determined their differential gene expression to naive B cells and to IgM-only and IgG+ memory B cells. This analysis revealed a high similarity of IgM+(IgD+)CD27+ and IgG+ memory B cells but also pointed at distinct functional capacities of both subsets. In vitro analyses revealed a tendency of activated IgM+IgD+CD27+ B cells to migrate to B-cell follicles and undergo germinal center (GC) B-cell differentiation, whereas activated IgG+ memory B cells preferentially showed a plasma cell (PC) fate. This observation was supported by reverse regulation of B-cell lymphoma 6 and PR domain containing 1 and differential BTB and CNC homology 1, basic leucine zipper transcription factor 2 expression. Moreover, IgM+IgD+CD27+ B lymphocytes preferentially responded to neutrophil-derived cytokines. Costimulation with catecholamines, carcinoembryonic antigen cell adhesion molecule 8 (CEACAM8), and IFN-γ caused differentiation of IgM+IgD+ CD27+ B cells into PCs, induced class switching to IgG2, and was reproducible in cocultures with neutrophils. In conclusion, this study substantiates memory B-cell characteristics of human IgM+ IgD+CD27+ B cells in that they share typical memory B-cell transcription patterns with IgG+ post-GC B cells and show a faster and more vigorous restimulation potential, a hallmark of immune memory. Moreover, this work reveals a functional plasticity of human IgM memory B cells by showing their propensity to undergo secondary GC reactions upon reactivation, but also by their special role in early inflammation via interaction with immunomodulatory neutrophils. [ABSTRACT FROM AUTHOR]

Regulation of adult stem cells (SCs) is fundamental for organ maintenance and tissue regeneration. On the body surface, different ectodermal organs exhibit distinctive modes of regeneration and the dynamics of their SC homeostasis remain to be unraveled. A slow cycling characteristic has been used to identify SCs in hair follicles and sweat glands; however, whether a quiescent population exists in continuously growing nails remains unknown. Using an in vivo label retaining cells (LRCs) system, we detected an unreported population of quiescent cells within the basal layer of the nail proximal fold, organized in a ring-like configuration around the nail root. These nail LRCs express the hair stem cell marker, keratin 15 (K15), and lineage tracing show that these K15-derived cells can contribute to both the nail structure and peri-nail epidermis, and more toward the latter. Thus, this stem cell population is bifunctional. Upon nail plucking injury, the homeostasis is tilted with these SCs dominantly delivering progeny to the nail matrix and differentiated nail plate, demonstrating their plasticity to adapt to wounding stimuli. Moreover, in vivo engraftment experiments established that transplanted nail LRCs can actively participate in functional nail regeneration. Transcriptional profiling of isolated nail LRCs revealed bone morphogenetic protein signaling favors nail differentiation over epidermal fate. Taken together, we have found a previously unidentified ring-configured population of bifunctional SCs, located at the interface between the nail appendage organ and adjacent epidermis, which physiologically display coordinated homeostatic dynamics but are capable of rediverting stem cell flow in response to injury. [ABSTRACT FROM AUTHOR]

Reaction-diffusion models have been used as a paradigm for describing the de novo emergence of biological patterns such as stripes and spots. In many organisms, these initial patterns are typically refined and elaborated over the subsequent course of development. Here we study the formation of secondary hair follicle patterns in the skin of developing mouse embryos. We used the expression of sex-determining region Y box 2 to identify and distinguish the primary and secondary hair follicles and to infer the spatiotemporal dynamics of the follicle formation process. Quantitative analysis of the specific follicle patterns observed reveals a simple geometrical rule governing the formation of secondary follicles, and motivates an expansion-induction (EI) model in which new follicle formation is driven by the physical growth of the embryo. The EI model requires only one diffusible morphogen and provides quantitative, accurate predictions on the relative positions and timing of secondary follicle formation, using only the observed configuration of primary follicles as input. The same model accurately describes the positions of additional follicles that emerge from skin explants treated with an activator. Thus, the EI model provides a simple and robust mechanism for predicting secondary space-filling patterns in growing embryos. [ABSTRACT FROM AUTHOR]

De novo organ regeneration has been observed in several lower organisms, as well as rodents; however, demonstrating these regenerative properties in human cells and tissues has been challenging. In the hair follicle, rodent hair follicle-derived dermal cells can interact with local epithelia and induce de novo hair follicles in a variety of hairless recipient skin sites. However, multiple attempts to recapitulate this process in humans using human dermal papilla cells in human skin have failed, suggesting that human dermal papilla cells lose key inductive properties upon culture. Here, we performed global gene expression analysis of human dermal papilla cells in culture and discovered very rapid and profound molecular signature changes linking their transition from a 3D to a 2D environment with early loss of their hair-inducing capacity. We demonstrate that the intact dermal papilla transcriptional signature can be partially restored by growth of papilla cells in 3D spheroid cultures. This signature change translates to a partial restoration of inductive capability, and we show that human dermal papilla cells, when grown as spheroids, are capable of inducing de novo hair follicles in human skin. [ABSTRACT FROM AUTHOR]

The spindle assembly checkpoint (SAC) ensures correct chromosome segregation during mitosis by preventing aneuploidy, an event that is detrimental to the fitness and survival of normal cells but oncogenic in tumor cells. Deletion of SAC genes is incompatible with early mouse development, and RNAi-mediated depletion of SAC components in cultured cells results in rapid death. Here we describe the use of a conditional KO of mouse Mad2, an essential component of the SAC signaling cascade, as a means to selectively induce chromosome instability and aneuploidy in the epidermis of the skin. We observe that SAC inactivation is tolerated by interfollicular epidermal cells but results in depletion of hair follicle bulge stem cells. Eventually, a histologically normal epidermis develops within ∼1 mo after birth, albeit without any hair. Mad2-deficient cells in this epidermis exhibited abnormal transcription of metabolic genes, consistent with aneuploid cell state. Hair follicle bulge stem cells were completely absent, despite the continued presence of rudimentary hair follicles. These data demonstrate that different cell lineages within a single tissue respond differently to chromosome instability: some proliferating cell lineages can survive, but stem cells are highly sensitive. [ABSTRACT FROM AUTHOR]

Smoothened antagonists directly target the genetic basis of human basal cell carcinoma (BCC), the most common of all cancers. These drugs inhibit BCC growth, but they are not curative. Although BCC cells are monomorphic, immunofluorescence microscopy reveals a complex hierarchical pattern of growth with inward differentiation along hair follicle lineages. Most BCC cells express the transcription factor KLF4 and are committed to terminal differentiation. A small CD200+ CD45- BCC subpopulation that represents 1.63 ± 1.11% of all BCC cells resides in small clusters at the tumor periphery. By using reproducible in vivo xenograft growth assays, we determined that tumor initiating cell frequencies approximate one per 1.5 million unsorted BCC cells. The CD200+ CD45- BCC subpopulation recreated BCC tumor growth in vivo with typical histological architecture and expression of sonic hedgehog-regulated genes. Reproducible in vivo BCC growth was achieved with as few as 10,000 CD200+ CD45- cells, representing ∼1,500-fold enrichment. CD200- CD45- BCC cells were unable to form tumors. These findings establish a platform to study the effects of Smoothened antagonists on BCC tumor initiating cell and also suggest that currently available anti-CD200 therapy be considered, either as monotherapy or an adjunct to Smoothened antagonists, in the treatment of inoperable BCC. [ABSTRACT FROM AUTHOR]

Hair follicles facilitate the study of stem cell behavior because stem cells in progressive activation stages, ordered within the follicle architecture, are capable of cyclic regeneration. To study the gene network governing the homeostasis of hair bulge stem cells, we developed a Keratin 15-driven genetic model to directly perturb molecular signaling in the stem cells. We visualize the behavior of these modified stem cells, evaluating their hair-regenerating ability and profile their molecular expression. Bone morphogenetic protein (BMP)-inactivated stem cells exhibit molecular profiles resembling those of hair germs, yet still possess multipotentiality in vivo. These cells also exhibit up-regulation of Wnt7a, Wnt7b, and Wnt16 ligands and Frizzled (Fzd) 10 receptor. We demonstrate direct transcriptional modulation of the Wnt7a promoter. These results highlight a previously unknown intra-stem cell antagonistic competition, between BMP and Wnt signaling, to balance stem cell activity. Reduced BMP signaling and increased Wnt signaling tilts each stem cell toward a hair germ fate and, vice versa, based on a continuous scale dependent on the ratio of BMP/Wnt activity. This work reveals one more hierarchical layer regulating stem cell homeostasis beneath the stem cell-dermal papilla-based epithelial-mesenchymal interaction layer and the hair follicle-intradermal adipocyte-based tissue interaction layer. Although hierarchical layers are all based on BMP/Wnt signaling, the multilayered control ensures that all information is taken into consideration and allows hair stem cells to sum up the total activators/inhibitors involved in making the decision of activation. [ABSTRACT FROM AUTHOR]

Regeneration of peripheral differentiated tissue in mammals is rare, and regulators of this process are largely unknown. We carried out a forward genetic screen in mice using N-ethyl-N-nitrosourea mutagenesis to identify genetic mutations that affect regenerative healing in vivo. More than 400 pedigrees were screened for closure of a through-and-through punch wound in the mouse ear. This led to the identification of a single pedigree with a heritable, fast, and regenerative wound-healing phenotype. Within 5 wk after ear-punch, a threefold decrease in the diameter of the wound was observed in the mutant mice compared with the wild-type mice. At 22 wk, new cartilage, hair follicles, and sebaceous glands were observed in the newly generated tissue. This trait was mapped to a point mutation in a receptor for TGF-β, TGFBR1. Mouse embryonic fibroblasts from the affected mice had increased expression of a subset of TGF-β target genes, suggesting that the mutation caused partial activation of the receptor. Further, bone marrow stromal cells from the mutant mice more readily differentiated to chondrogenic precursors, providing a plausible explanation for the enhanced development of cartilage islands in the regenerated ears. This mutant mouse strain provides a unique model to further explore regeneration in mammals and, in particular, the role of TGFBR1 in chondrogenesis and regenerative wound healing. [ABSTRACT FROM AUTHOR]

Receptor activator of NF-κB (RANK), known for controlling bone mass, has been recognized for its role in epithelial cell activation of the mammary gland. Because bone and the epidermo-pilosebaceous unit of the skin share a lifelong renewal activity where similar molecular players operate, and because mammary glands and hair follicles are both skin appendages, we have addressed the function of RANK in the hair follicle and the epidermis. Here, we show that mice deficient in RANK ligand (RANKL) are unable to initiate a new growth phase of the hair cycle and display arrested epidermal homeostasis. However, transgenic mice overexpressing RANK in the hair follicle or administration of recombinant RANKL both activate the hair cycle and epidermal growth. RANK is expressed by the hair follicle germ and bulge stem cells and the epidermal basal cells, cell types implicated in the renewal of the epidermo-pilosebaceous unit. RANK signaling is dispensable for the formation of the stem cell compartment and the inductive hair follicle mesenchyme, and the hair cycle can be rescued by Rankl knockout skin transplantation onto nude mice. RANKL is actively transcribed by the hair follicle at initiation of its growth phase, providing a mechanism for stem cell RANK engagement and hair-cycle entry. Thus, RANK-RANKL regulates hair renewal and epidermal homeostasis and provides a link between these two activities. [ABSTRACT FROM AUTHOR]

Chronic wounds and acute trauma constitute well-established risk factors for development of epithelial-derived skin tumors, although the underlying mechanisms are largely unknown. Basal cell carcinomas (BCCs) are the most common skin cancers displaying a number of features reminiscent of hair follicle (HF)-derived cells and are dependent on deregulated Hedgehog (Hh)/GLI signaling. Here we show, in a mouse model conditionally expressing GLI1 and in a model with homozygous inactivation of Ptch1, mimicking the situation in human BCCs, that the wound environment accelerates the initiation frequency and growth of BCC-like lesions. Lineage tracing reveals that both oncogene activation and wounding induce emigration of keratinocytes residing in the lower bulge and the nonpermanent part of the HFs toward the interfollicular epidermis (IFE). However, only oncogene activation in combination with a wound environment enables the participation of such cells in the initiation of BCC-like lesions at the HF openings and in the IFE. We conclude that, in addition to the direct enhancement of BCC growth, the tumor-promoting effect of the wound environment is due to recruitment of tumor-initiating cells originating from the neighboring HFs, establishing a link between epidermal wounds and skin cancer risk. [ABSTRACT FROM AUTHOR]

A wide variety of human cancers are associated with injury. Although stem cells participate in tissue regeneration after wounding, it is unclear whether these cells also contribute to epithelial tumors. Human basal cell carcinomas (BCCs) are associated with misactivation of Hedgehog (Hh) signaling, commonly through acquisition of mutations in Smoothened (Smo). We have found that expression of an activated form of Smo by stem cells of the hair-follicle bulge and secondary hair germ does not induce robust Hh signaling or produce BCCs. However, wounding recruits these cells from the follicle to the wound site, where downstream Hh signal transduction is derepressed, giving rise to superficial BCC-like tumors. These findings demonstrate that BCC-like tumors can originate from follicular stem cells and provide an explanation for the association between wounding and tumorigenesis. [ABSTRACT FROM AUTHOR]

In mammals, skin begins as a single-layered epithelium, which, through a series of signals, either stratifies and differentiates to become epidermis or invaginates downward to make hair follicles (HFs). To achieve and maintain proper tissue architecture, keratinocytes must intricately balance growth and differentiation. Here, we uncover a critical and hitherto unappreciated role for Yes-associated protein (YAP), an evolutionarily conserved transcriptional coactivator with potent oncogenic potential. We show that YAP is highly expressed and nuclear in single-layered basal epidermal progenitors. Notably, nuclear YAP progressively declines with age and correlates with proliferative potential of epidermal progenitors. Shortly after initiation of HF morphogenesis, YAP translocates to the cytoplasm of differentiating cells. Through genetic analysis, we demonstrate a role for YAP in maintaining basal epidermal progenitors and regulating HF morphogenesis. YAP overexpression causes hair placodes to evaginate into epidermis rather than invaginate into dermis. YAP also expands basal epidermal progenitors, promotes proliferation, and inhibits terminal differentiation. In vitro gain-and-loss of function studies show that primary mouse keratinocytes (MKs) accelerate proliferation, suppress differentiation, and inhibit apoptosis when YAP is activated and reverse these features when YAP is inhibited. Finally, we identify Cyr61 as a target of YAP in MKs and demonstrate a requirement for TEA domain (TEAD) transcriptional factors to comediate YAP functions in MKs. [ABSTRACT FROM AUTHOR]

The switch between black and yellow pigment is mediated by the interaction between Melanocortin receptor 1 (Mclr) and its antagonist Agouti, but the genetic and developmental mechanisms that modify this interaction to obtain different coat color in distinct environments are poorly understood. Here, the role of Wnt/β-catenin signaling in the regulation of pigment-iype switching was studied. Loss and gain of function of β-catenin in the dermal papilla (DP) of the hair follicle results in yellow and black animals, respectively. β-Catenin activity in the DP suppresses Agouti expression and activates Corin, a negative regulator of Agouti activity. In addition, β-catenin activity in the DP regulates melanocyte activity by a mechanism that is independent of both Agouti and Corin. The coordinate and inverse regulation of Agouti and Corin renders pelage pigmentation sensitive to changes in β-catenin activity in the DP that do not alter pelage structure. As a result, the signals that specify two biologically distinct quantitative traits are partially uncoupled despite their common regulation by the β-catenin pathway in the same cells. [ABSTRACT FROM AUTHOR]

A thorough understanding of the circadian clock requires qualitative evaluation of circadian clock gene expression. Thus far, no simple and effective method for detecting human clock gene expression has become available. This limitation has greatly hampered our understanding of human circadian rhythm. Here we report a convenient, reliable, and less invasive method for detecting human clock gene expression using biopsy samples of hair follicle cells from the head or chin. We show that the circadian phase of clock gene expression in hair follicle cells accurately reflects that of individual behavioral rhythms, demonstrating that this strategy is appropriate for evaluating the human peripheral circadian clock. Furthermore, using this method, we indicate that rotating shift workers suffer from a serious time lag between circadian gene expression rhythms and lifestyle. Qualitative evaluation of clock gene expression in hair follicle cells, therefore, may be an effective approach for studying the human circadian clock in the clinical setting. [ABSTRACT FROM AUTHOR]

Although multiple follicles are present in mammalian ovaries, most of them remain dormant for years or decades. During reproductive life, some follicles are activated for development. Genetically modified mouse models with oocyte-specific deletion of genes in the PTEN-PI3K-Akt-Foxo3 pathway exhibited premature activation of all dormant follicles. Using an inhibitor of the Phosphatase with TENsin homology deleted in chromosome 10 (PTEN) phosphatase and a P13K activating peptide, we found that short-term treatment of neonatal mouse ovaries increased nuclear exclusion of Foxo3 in primordial oocytes. After transplantation under kidney capsules of ovariectomized hosts, treated follicles developed to the preovulatory stage with mature eggs displaying normal epigenetic changes of imprinted genes. After in vitro fertilization and embryo transfer, healthy progeny with proven fertility were delivered. Human ovarian cortical fragments from cancer patients were also treated with the PTEN inhibitor. After xeno-transplantation to immune-deficient mice for 6 months, primordial follicles developed to the preovulatory stage with oocytes capable of undergoing nuclear maturation. Major differences between male and female mammals are unlimited number of sperm and paucity of mature oocytes. Thus. short-term in vitro activation of dormant ovarian follicles after stimulation of the PI3K-Akt pathway allows the generation of a large supply of mature female germ cells for future treatment of infertile women with a diminishing ovarian reserve and for cancer patients with cryo-preserved ovaries. Generation of a large number of human oocytes also facilitates future derivation of embryonic stem cells for regenerative medicine. [ABSTRACT FROM AUTHOR]

Hair is maintained through a cyclic process that includes periodic regeneration of hair follicles in a stem cell-dependent manner. Little is known, however, about the cellular and molecular mechanisms that regulate the layered differentiation of the hair follicle. We have established a mutant mouse with a cyclic alopecia phenotype resulting from the targeted disruption of Sox21, a gene that encodes a HMG-box protein. These mice exhibit progressive hair loss after morphogenesis of the first hair follicle and become completely nude in appearance, but then show hair regrowth. Sox21 is expressed in the cuticle layer and the progenitor cells of the hair shaft in both mouse and human. The lack of this gene results in a loss of the interlocking structures required for anchoring the hair shaft in the hair follicle. Furthermore, the expression of genes encoding the keratins and keratin binding proteins in the hair shaft cuticle are also specifically down-regulated in the Sox21-null mouse. These results indicate that Sox21 is a master regulator of hair shaft cuticle differentiation and shed light on the possible causes of human hair disorders. [ABSTRACT FROM AUTHOR]

During the hair cycle, follicle stem cells (SCs) residing in a specialized niche called the ‘bulge’ undergo bouts of quiescence and activation to cyclically regenerate new hairs. Developmental studies have long implicated the canonical bone morphogenetic protein (BMP) pathway in hair follicle (HF) determination and differentiation, but how BMP signaling functions in the hair follicle SC niche remains unknown. Here, we use loss and gain of function studies to manipulate BMP signaling in the SC niche. We show that when the Bmpr1a gene is conditionally ablated, otherwise quiescent SCs are activated to proliferate, causing an expansion of the niche and loss of slow-cycling cells. Surprisingly, follicle SCs are not lost, however, but rather, they generate long-lived, tumor-like branches that express Sox4, Lhx2, and Sonic Hedgehog but fail to terminally differentiate to make hair. A key component of BMPRIA-deficient SCs is their elevated levels of both Lef1 and β-catenin, which form a bipartite transcription complex required for initiation of the hair cycle. Although β-catenin can be stabilized by Wnt signaling, we show that BMPR1A deficiency enhances β-catenin stabilization in the niche through a pathway involving PTEN inhibition and PI3K/AKT activation. Conversely, sustained BMP signaling in the SC niche blocks activation and promotes premature hair follicle differentiation. Together, these studies reveal the importance of balancing BMP signaling in the SC niche. [ABSTRACT FROM AUTHOR]

Melanoma is one of the most aggressive cancers, and its incidence is increasing. These tumors derive from the melanocyte lineage and remain incurable after metastasis. Here we report that SONIC HEDGEHOG (SHH)-GLI signaling is active in the matrix of human hair follicles, and that it is required for the normal proliferation of human melanocytes in culture. SHH-GLI signaling also regulates the proliferation and survival of human melanomas: the growth, recurrence, and metastasis of melanoma xenografts in mice are prevented by local or systemic interference of HH-GLI function. Moreover, we show that oncogenic RAS-induced melanomas in transgenic mice express Gli1 and require Hh-Gli signaling in vitro and in vivo. Finally, we provide evidence that endogenous RAS-MEK and AKT signaling regulate the nuclear localization and transcriptional activity of GLI1 in melanoma and other cancer cells. Our data uncover an unsuspected role of HH-GLI signaling in melanocytes and melanomas, demonstrate a role for this pathway in RAS-induced tumors, suggest a general integration of the RAS/AKT and HH-GLI pathways, and open a therapeutic approach for human melanomas. [ABSTRACT FROM AUTHOR]

Hairs, feathers, and scales normally exhibit precise orientations with respect to the body axes. In Frizzled6 (Fz6)-/- mice, the global orientation of hair follicles is disrupted, leading to waves, whorls, and tufts, each comprising many hundreds of hairs. By analyzing the orientation of developing hair follicles, we observed that the nearly parallel arrangement of wild-type (WT) hairs arises from fields of imperfectly aligned follicles, and that the Fz6-/- hair patterns arise from fields of grossly misoriented or randomly oriented follicles. Despite their large size, both mutant and WI hair follicles display a remarkable and unexpected plasticity, reorienting on a time scale of days in what seems to be a self-organized refinement process. The essential features of this process can be studied with a simple cellular automata model in which a local consensus "rule" acts iteratively to bias each hair's orientation in favor of the average orientation of its neighbors. These experiments define two systems for hair orientation: a global orienting system that acts early in development and is Fz6-dependent, and a local self-organizing system that acts later and is Fz6 independent. [ABSTRACT FROM AUTHOR]

Hair follicles are spaced apart from one another at regular intervals through the skin. Although follicles are predominantly epidermal structures, classical tissue recombination experiments indicated that the underlying dermis defines their location during development. Although many molecules involved in hair follicle formation have been identified, the molecular interactions that determine the emergent property of pattern formation have remained elusive. We have used embryonic skin cultures to dissect signaling responses and patterning outcomes as the skin spatially organizes itself. We find that ectodysplasin receptor (Edar)-bone morpho- genetic protein (BMP) signaling and transcriptional interactions are central to generation of the primary hair follicle pattern, with restriction of responsiveness, rather than localization of an inducing ligand, being the key driver in this process. The crux of this patterning mechanism is rapid Edar-positive feedback in the epi- dermis coupled with induction of dermal BMP4/7. The BMPs in turn repress epidermal Edar and hence follicle fate. Edar activation also induces connective tissue growth factor, an inhibitor of BMP signaling, allowing BMP action only at a distance from their site of synthesis. Consistent with this model, transgenic hyperactivation of Edar signaling leads to widespread overproduction of hair follicles. This Edar-BMP activation-inhibition mechanism appears to operate alongside a labile prepattern, suggesting that Edar-mediated stabilization of β-catenin active foci is a key event in determining definitive follicle locations. [ABSTRACT FROM AUTHOR]

The hair follicle bulge area is an abundant, easily accessible source of actively growing, pluripotent adult stem cells. Nestin, a protein marker for neural stem cells, also is expressed in follicle stem cells and their immediate, differentiated progeny. The fluorescent protein GFP, whose expression is driven by the nestin regulatory element in transgenic mice, served to mark the follicle cell fate. The pluripotent nestin-driven GFP stem cells are positive for the stem cell marker CD34 but negative for keratinocyte marker keratin 15, suggesting their relatively undifferentiated state. These cells can differentiate into neurons, glia, keratinocytes, smooth muscle cells, and melanocytes in vitro. In vivo studies show the nestin-driven GFP hair follicle stem cells can differentiate into blood vessels and neural tissue after transplantation to the subcutis of nude mice. Equivalent hair follicle stem cells derived from transgenic mice with β-actin-driven GFP implanted into the gap region of a severed sciatic nerve greatly enhance the rate of nerve regeneration and the restoration of nerve function. The follicle cells transdifferentiate largely into Schwann cells, which are known to support neuron regrowth. Function of the rejoined sciatic nerve was measured by contraction of the gastrocnemius muscle upon electrical stimulation. After severing the tibial nerve and subsequent transplantation of hair follicle stem cells, walking print length and intermediate toe spread significantly recovered, indicating that the transplanted mice recovered the ability to walk normally. These results suggest that hair follicle stem cells provide an important, accessible, autologous source of adult stem cells for regenerative medicine. [ABSTRACT FROM AUTHOR]

Adult stem cells are essential for tissue renewal, regeneration, and repair, and their expansion in culture is of paramount importance for regenerative medicine. Using the whisker follicle of the rat as a model system, we demonstrate that (i) clonogenicity is an intrinsic property of the adult stem cells of the hair follicle; (ii) after cultivation for >140 doublings, these stem cells, transplanted to the dermo-epidermal junction of newborn mouse skin, form part or all of the developing follicles; (iii) the stem cells incorporated into follicles are multipotent, because they generate all of the lineages of the hair follicle and sebaceous gland; (iv) thousands of hair follicles can be generated from the progeny of a single cultivated stem cell; (v) cultured stem cells express the self-renewal genes Bmi1 and Zfp 145; (vi) several stem cells participate in the formation of a single hair bulb; and (vii) there are many more stem cells in whisker follicles than could be anticipated from label-retaining experiments. [ABSTRACT FROM AUTHOR]

The mammalian hair cycle involves periodic regeneration of a tiny organ, the hair follicle, through a stem-cell-mediated process. The Hairless (Hr) gene encodes a nuclear receptor corepressor (HR) that is essential for hair follicle regeneration, but its role in this process is unknown. Here, we demonstrate that transgenic expression of HR in progenitor keratinocytes rescues follicle regeneration in Hr-/- mice. We show that expression of Wise, a modulator of Wnt signaling, is repressed by HR in these cells, coincident with the timing of follicle regeneration. This work links HR and Wnt function, providing a model in which HR regulates the precise timing of Wnt signaling required for hair follicle regeneration. [ABSTRACT FROM AUTHOR]

We have recently shown that the expression of nestin, the neural stem cell marker protein, is expressed in bulge-area stem cells of the hair follicle. We used transgenic mice with GFP expression driven by the nestin regulatory element Lnestin-driven GFP (ND- GFP)]. The ND-GFP stem cells give rise to the outer-root sheath of the hair follicle as well as an ND-GFP interfollicular vascular network. ln this study, we demonstrate that ND-GFP stem cells isolated from the hair-follicle bulge area that are negative for the keratinocyte marker keratin 15 can differentiate into neurons, glia, keratinocytes, smooth muscle cells and melanocytes in vitro These pluripotent ND-GFP stem cells are positive for the stem cell marker CD34, as well as keratin 15-negative, suggesting their relatively undifferentiated state. The apparent primitive state of the ND-GFP stem cells is compatible with their pluripotency. Furthermore, we show that cells derived from ND-GFP stem cells can differentiate into neurons after transplantation to the subcutis of nude mice These results suggest that hair-follicle bulge-area ND-GFP stem cells may provide an accessible, autologous source of undifferentiated multipotent stem cells for therapeutic application. [ABSTRACT FROM AUTHOR]

The hair-growth cycle, a complex biological system requiring coordinate alterations in gene expression and cellular behavior, provides a challenging model for investigating the interplay of specific transcriptional regulation events. Here we report that the Barx2 homeodomain factor serves as a regulator of hair follicle remodeling (catagen), and loss of Barx2 in mice causes a defect both in the initiation and progression of catagen, resulting in a protracted first catagen, and later, causing short hair in adult gene-deleted mice. Barx2 negatively regulates its own promoter, and our study highlights the role of Barx2 as a repressor in the skin that can, unexpectedly, functionally interact with two WD40- domain factors distantly related to the yeast corepressor Tup1. These two corepressors, transducin-like enhancer of split and transducin β-like 1, function through distinct and independent interactions with Barx2 for the repression of gene targets, including the Barx2 gene itself, emphasizing the roles of complementary repression strategies in engrailed homology-1 motif-containing homeodomain factors. Together, our data suggest that the hair- remodeling defect of Barx2 mutant mice could be explained, in part, by failure to repress one or more critical target genes. [ABSTRACT FROM AUTHOR]

Besides forming hair shafts, the highly organized, metabolically vigorous hair follicle plays several crucial roles in skin architecture. The follicle contains a distinct population of presumptive follicular stem cells that express nestin, also a marker for neural stem cells. These nestin-expressing follicle cells are located principally in the follicular bulge region. Nestin-driven GFP (ND-GFP), transfected into mice, principally labels cells in the bulge region, which is consistent with the cells' being the stem cells of the hair follicle. We report here that ND-GFP also labels developing skin blood vessels that appear to originate from hair follicles and form a follicle-linking network. This is seen most clearly by transplanting ND-GFP-labeled vibrissa (whisker) hair follicles to unlabeled nude mice. New vessels grow from the transplanted follicle, and these vessels increase when the local recipient skin is wounded. The ND-GFP-expressing structures are blood vessels, because they display the characteristic endothelial-cell-specific markers CD31 and von Willebrand factor. This model displays very early events in skin angiogenesis and can serve for rapid antiangiogenesis drug screening. [ABSTRACT FROM AUTHOR]

Deregulation of the p16[supINK4a] -- cyclin D:cyclin-dependent kinases (cdk) 4/6-retinoblastoma (pRB) pathway is a common paradigm in the oncogenic transformation of human cells and suggests that this pathway functions linearly in malignant transformation. However, it is not understood why p16[supINK4a] and cyclin D:cdk4/6 mutations are disproportionately more common than the rare genetic event of RB inactivation in human malignancies such as melanoma. To better understand how these complexes contribute to altered tissue homeostasis, we blocked cdk4/6 activation and acutely inactivated Rb by conditional mutagenesis during mouse hair follicle cycling. Inhibition of cdk4/6 in the skin by subcutaneous administration of a membrane-transducible TAT. p16[supINK4a] protein completely blocked hair follicle growth and differentiation. In contrast, acute disruption of Rb in the skin of homozygous Rb[supLoxP/LoxP] mice via subcutaneous administration of TAT-Cre recombinase failed to affect hair growth. However, loss of Rb resulted in severe depigmentation of hair follicles. Further analysis of follicular melanocytes in vivo and in primary'cell culture demonstrated that pRB plays a cell-autonomous role in melanocyte survival. Moreover, functional inactivation of all three Rb family members (Rb, p107, and p130) in primary melanocytes by treatment with a transducible TAT-E1A protein did not rescue the apoptotic phenotype. These findings suggest that deregulated cyclin D:cdk4/6 complexes and pRB perform nonoverlapping functions in vivo and provide a cellular mechanism that accounts for the low incidence of RB inactivation in cancers such as melanoma. [ABSTRACT FROM AUTHOR]

The intermediate filament protein, nestin, marks progenitor cells of the CNS. Such CNS stem cells are selectively labeled by placing GFP under the control of the nestin regulatory sequences. During early anagen or growth phase of the hair follicle, nestin-expressing cells, marked by GFP fluorescence in nestin-GFP transgenic mice, appear in the permanent upper hair follicle immediately below the sebaceous glands in the follicle bulge. This is where stem cells for the hair follicle outer-root sheath are thought to be located. The relatively small, ovalshaped, nestin-expressing cells in the bulge area surround the hair shaft and are interconnected by short dendrites. The precise locations of the nestin-expressing cells in the hair follicle vary with the hair cycle. During telogen or resting phase and in early anagen, the GFP-positive cells are mainly in the bulge area. However, in mid- and late anagen, the GFP-expressing cells are located in the upper outer-root sheath as well as in the bulge area but not in the hair matrix bulb. These observations show that the nestin-expressing cells form the outer-root sheath. Results of the immunohistochemical staining showed that nestin, GFP, keratin 5/8, and keratin 15 colocalize in the hair follicle bulge cells, outer-root sheath cells, and basal cells of the sebaceous glands. These data indicate that nestin-expressing cells, marked by GFP, in the hair follicle bulge are indeed progenitors of the follicle outer-root sheath. The expression of the unique protein, nestin, in both neural stem cells and hair follicle stem cells suggests their possible relation. [ABSTRACT FROM AUTHOR]

Investigates the genetic modification of hair follicles and growing hair shafts. Isolation of hair follicles; Application of collagenase treatment; Induction of anagen.

Focuses on the role of mouse homolog of Drosophila ovo on the formation of epidermal appendage differentiation. Observation of the hair follicles; Formation of the bipartite transcription activator; Measurement of the luciferase activity.

Examines the abnormal differentiation of epidermis in transgenic mice constitutively expressing cyclooxygenase-2 (COX-2) in skin. Use of keratin 5 promoter to explore the function of COX-2 in epidermis; Reduction of hair follicle density in heterozygous transgenic mice; Description of adult transgenic mice.

Examines the effect of phorbol 12-myristate 13-acetate (PMA) in vivo and hair plucking, well known experimental anagen initiators to determine whether signal transducer and activator of transcription three is required for anagen induction in response to exogenous stimuli. Finding that PMA treatment or hair plucking activated the telogen follicles.

Explores the role of estrogen and estrogen receptor in the hair follicle cycle. Indication of an estrogen receptor pathway within the dermal papilla; Regulation of the telogen-anagen follicle transition; Influence of the diffusible factors associated with the anagen follicle in the cell proliferation in the epidermis.

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