Neurobiology, Stem Cell Biology, and Immunology: An Emerging Triad for Understanding Tissue Homeostasis and Repair.

The peripheral nervous system (PNS) endows animals with the remarkable ability to sense and respond to a dynamic world. Emerging evidence shows the PNS also participates in tissue homeostasis and repair by integrating local changes with organismal and environmental changes. Here, we provide an in-depth summary of findings delineating the diverse roles of peripheral nerves in modulating stem cell behaviors and immune responses under steady-state conditions and in response to injury and duress, with a specific focus on the skin and the hematopoietic system. These examples showcase how elucidating neuro-stem cell and neuro-immune cell interactions provides a conceptual framework that connects tissue biology and local immunity with systemic bodily changes to meet varying demands. They also demonstrate how changes in these interactions can manifest in stress, aging, cancer, and inflammation, as well as how these findings can be harnessed to guide the development of new therapeutics.

Chromatin Potential Identified by Shared Single-Cell Profiling of RNA and Chromatin.

Cell differentiation and function are regulated across multiple layers of gene regulation, including modulation of gene expression by changes in chromatin accessibility. However, differentiation is an asynchronous process precluding a temporal understanding of regulatory events leading to cell fate commitment. Here we developed simultaneous high-throughput ATAC and RNA expression with sequencing (SHARE-seq), a highly scalable approach for measurement of chromatin accessibility and gene expression in the same single cell, applicable to different tissues. Using 34,774 joint profiles from mouse skin, we develop a computational strategy to identify cis-regulatory interactions and define domains of regulatory chromatin (DORCs) that significantly overlap with super-enhancers. During lineage commitment, chromatin accessibility at DORCs precedes gene expression, suggesting that changes in chromatin accessibility may prime cells for lineage commitment. We computationally infer chromatin potential as a quantitative measure of chromatin lineage-priming and use it to predict cell fate outcomes. SHARE-seq is an extensible platform to study regulatory circuitry across diverse cells in tissues.

Cell Types Promoting Goosebumps Form a Niche to Regulate Hair Follicle Stem Cells.

Piloerection (goosebumps) requires concerted actions of the hair follicle, the arrector pili muscle (APM), and the sympathetic nerve, providing a model to study interactions across epithelium, mesenchyme, and nerves. Here, we show that APMs and sympathetic nerves form a dual-component niche to modulate hair follicle stem cell (HFSC) activity. Sympathetic nerves form synapse-like structures with HFSCs and regulate HFSCs through norepinephrine, whereas APMs maintain sympathetic innervation to HFSCs. Without norepinephrine signaling, HFSCs enter deep quiescence by down-regulating the cell cycle and metabolism while up-regulating quiescence regulators Foxp1 and Fgf18. During development, HFSC progeny secretes Sonic Hedgehog (SHH) to direct the formation of this APM-sympathetic nerve niche, which in turn controls hair follicle regeneration in adults. Our results reveal a reciprocal interdependence between a regenerative tissue and its niche at different stages and demonstrate sympathetic nerves can modulate stem cells through synapse-like connections and neurotransmitters to couple tissue production with demands.

Transit-amplifying cells orchestrate stem cell activity and tissue regeneration.

Transit-amplifying cells (TACs) are an early intermediate in tissue regeneration. Here, using hair follicles (HFs) as a paradigm, we show that emerging TACs constitute a signaling center that orchestrates tissue growth. Whereas primed stem cells (SCs) generate TACs, quiescent SCs only proliferate after TACs form and begin expressing Sonic Hedgehog (SHH). TAC generation is independent of autocrine SHH, but the TAC pool wanes if they can't produce SHH. We trace this paradox to two direct actions of SHH: promoting quiescent-SC proliferation and regulating dermal factors that stoke TAC expansion. Ingrained within quiescent SCs' special sensitivity to SHH signaling is their high expression of GAS1. Without sufficient input from quiescent SCs, replenishment of primed SCs for the next hair cycle is compromised, delaying regeneration and eventually leading to regeneration failure. Our findings unveil TACs as transient but indispensable integrators of SC niche components and reveal an intriguing interdependency of primed and quiescent SC populations on tissue regeneration.

Corticosterone inhibits GAS6 to govern hair follicle stem-cell quiescence.

Chronic, sustained exposure to stressors can profoundly affect tissue homeostasis, although the mechanisms by which these changes occur are largely unknown. Here we report that the stress hormone corticosterone-which is derived from the adrenal gland and is the rodent equivalent of cortisol in humans-regulates hair follicle stem cell (HFSC) quiescence and hair growth in mice. In the absence of systemic corticosterone, HFSCs enter substantially more rounds of the regeneration cycle throughout life. Conversely, under chronic stress, increased levels of corticosterone prolong HFSC quiescence and maintain hair follicles in an extended resting phase. Mechanistically, corticosterone acts on the dermal papillae to suppress the expression of Gas6, a gene that encodes the secreted factor growth arrest specific 6. Restoring Gas6 expression overcomes the stress-induced inhibition of HFSC activation and hair growth. Our work identifies corticosterone as a systemic inhibitor of HFSC activity through its effect on the niche, and demonstrates that the removal of such inhibition drives HFSCs into frequent regeneration cycles, with no observable defects in the long-term.

Dynamics between stem cells, niche, and progeny in the hair follicle.

Here, we exploit the hair follicle to define the point at which stem cells (SCs) become irreversibly committed along a differentiation lineage. Employing histone and nucleotide double-pulse-chase and lineage tracing, we show that the early SC descendents en route to becoming transit-amplifying cells retain stemness and slow-cycling properties and home back to the bulge niche when hair growth stops. These become the primary SCs for the next hair cycle, whereas initial bulge SCs become reserves for injury. Proliferating descendents further en route irreversibly lose their stemness, although they retain many SC markers and survive, unlike their transit-amplifying progeny. Remarkably, these progeny also home back to the bulge. Combining purification and gene expression analysis with differential ablation and functional experiments, we define critical functions for these non-SC niche residents and unveil the intriguing concept that an irreversibly committed cell in an SC lineage can become an essential contributor to the niche microenvironment.

Hyperactivation of sympathetic nerves drives depletion of melanocyte stem cells.

Empirical and anecdotal evidence has associated stress with accelerated hair greying (formation of unpigmented hairs)1,2, but so far there has been little scientific validation of this link. Here we report that, in mice, acute stress leads to hair greying through the fast depletion of melanocyte stem cells. Using a combination of adrenalectomy, denervation, chemogenetics3,4, cell ablation and knockout of the adrenergic receptor specifically in melanocyte stem cells, we find that the stress-induced loss of melanocyte stem cells is independent of immune attack or adrenal stress hormones. Instead, hair greying results from activation of the sympathetic nerves that innervate the melanocyte stem-cell niche. Under conditions of stress, the activation of these sympathetic nerves leads to burst release of the neurotransmitter noradrenaline (also known as norepinephrine). This causes quiescent melanocyte stem cells to proliferate rapidly, and is followed by their differentiation, migration and permanent depletion from the niche. Transient suppression of the proliferation of melanocyte stem cells prevents stress-induced hair greying. Our study demonstrates that neuronal activity that is induced by acute stress can drive a rapid and permanent loss of somatic stem cells, and illustrates an example in which the maintenance of somatic stem cells is directly influenced by the overall physiological state of the organism.

Treg cells require Izumo1R to regulate γδT cell-driven inflammation in the skin.

Izumo1R is a pseudo-folate receptor with an essential role in mediating tight oocyte/spermatozoa contacts during fertilization. Intriguingly, it is also expressed in CD4+ T lymphocytes, in particular Treg cells under the control of Foxp3. To understand Izumo1R function in Treg cells, we analyzed mice with Treg-specific Izumo1r deficiency (Iz1rTrKO). Treg differentiation and homeostasis were largely normal, with no overt autoimmunity and only marginal increases in PD1+ and CD44hi Treg phenotypes. pTreg differentiation was also unaffected. Iz1rTrKO mice proved uniquely susceptible to imiquimod-induced, γδT cell-dependent, skin disease, contrasting with normal responses to several inflammatory or tumor challenges, including other models of skin inflammation. Analysis of Iz1rTrKO skin revealed a subclinical inflammation that presaged IMQ-induced changes, with an imbalance of Rorγ+ γδT cells. Immunostaining of normal mouse skin revealed the expression of Izumo1, the ligand for Izumo1R, electively in dermal γδT cells. We propose that Izumo1R on Tregs enables tight contacts with γδT cells, thereby controlling a particular path of skin inflammation.

A family business: stem cell progeny join the niche to regulate homeostasis.

Stem cell niches, the discrete microenvironments in which the stem cells reside, play a dominant part in regulating stem cell activity and behaviours. Recent studies suggest that committed stem cell progeny become indispensable components of the niche in a wide range of stem cell systems. These unexpected niche inhabitants provide versatile feedback signals to their stem cell parents. Together with other heterologous cell types that constitute the niche, they contribute to the dynamics of the microenvironment. As progeny are often located in close proximity to stem cell niches, similar feedback regulations may be the underlying principles shared by different stem cell systems.

Hair follicles' transit-amplifying cells govern concurrent dermal adipocyte production through Sonic Hedgehog.

Growth and regeneration of one tissue within an organ compels accommodative changes in the surrounding tissues. However, the molecular nature and operating logic governing these concurrent changes remain poorly defined. The dermal adipose layer expands concomitantly with hair follicle downgrowth, providing a paradigm for studying coordinated changes of surrounding lineages with a regenerating tissue. Here, we discover that hair follicle transit-amplifying cells (HF-TACs) play an essential role in orchestrating dermal adipogenesis through secreting Sonic Hedgehog (SHH). Depletion of Shh from HF-TACs abrogates both dermal adipogenesis and hair follicle growth. Using cell type-specific deletion of Smo, a gene required in SHH-receiving cells, we found that SHH does not act on hair follicles, adipocytes, endothelial cells, and hematopoietic cells for adipogenesis. Instead, SHH acts directly on adipocyte precursors, promoting their proliferation and their expression of a key adipogenic gene, peroxisome proliferator-activated receptor γ (Pparg), to induce dermal adipogenesis. Our study therefore uncovers a critical role for TACs in orchestrating the generation of both their own progeny and a neighboring lineage to achieve concomitant tissue production across lineages.

Inhibition of pyruvate oxidation as a versatile stimulator of the hair cycle in models of alopecia.

Hair follicle stem cells (HFSCs) are known to be responsible for the initiation of a new hair cycle, but typically remain quiescent for very long periods. In alopecia, or hair loss disorders, follicles can be refractory to activation for years or even permanently. Alopecia can be triggered by autoimmunity, age, chemotherapeutic treatment, stress, disrupted circadian rhythm or other environmental insults. We previously showed that hair follicle stem cells and the hair cycle can be manipulated by regulation of pyruvate entry into mitochondria for subsequent oxidation to fuel the TCA cycle in normal adult mice with typical hair cycling. Here, we present new data from our efforts to develop murine models of alopecia based on environmental triggers that have been shown to do the same in human skin. We found that inhibition of pyruvate transport into mitochondria can accelerate the hair cycle even during refractory hair cycling due to age, repeated chemotherapeutic treatment and stress. Hair cycle acceleration in these alopecia models led to the formation of histologically normal hair follicles within 30-40 days of treatment without any overt signs of toxicity or deleterious effects. Therefore, we propose inhibition of pyruvate entry into mitochondria as a versatile treatment strategy for alopecia in humans.

Melanocortin 1 receptor is dispensable for acute stress induced hair graying in mice.

Stress is a risk factor for many skin conditions, but the cellular and molecular mechanisms of its impacts have only begun to be revealed. In mice, acute stress induces loss of melanocyte stem cells (MeSCs) and premature hair greying. Our previous work demonstrated that the loss of MeSCs upon acute stress is caused by the hyperactivation of the sympathetic nervous system. Stress also induces the secretion of stress hormones from the hypothalamic-pituitary-adrenal (HPA) axis; however, whether stress hormones are involved in the hair greying process has not been fully examined. In particular, the adrenocorticotropic hormone (ACTH) is released from the pituitary glands upon stress. ACTH is a ligand for the melanocortin 1 receptor (MC1R), which plays critical roles in regulating MeSC migration and skin pigmentation. We investigated whether the MC1R pathway is required for the stress-induced hair greying. We confirmed that MC1R is the major melanocortin receptor expressed in MeSCs. However, induction of acute stress via resiniferatoxin (RTX) injection still leads to hair greying in Mc1r mutant mice, suggesting that the ACTH-MC1R pathway is not a major contributor in acute stress-induced premature hair greying.

Stress-associated ectopic differentiation of melanocyte stem cells and ORS amelanotic melanocytes in an ex vivo human hair follicle model.

Hair greying depends on the altered presence and functionality of hair follicle melanocytes. Melanocyte stem cells (MelSCs) reside in the bulge of hair follicles and give rise to migrating and differentiating progeny during the anagen phase. Ageing, genotoxic stress, redox stress and multiple behaviour-associated acute stressors have been seen to induce hair greying by depleting the MelSC pool, a phenomenon which is accompanied by ectopic pigmentation of these cells, followed by their depletion from the stem cell niche. This aberrant differentiation produces a state from which a return to stem cell-like quiescence appears to be lost. The cellular features of stress-induced hair greying have been extensively studied in murine models. Here, we describe a method to assess and quantify human hair follicle MelSC differentiation by measuring ectopically pigmented MelSCs in isolated human hair follicles exposed to specific stress signal mediators. Ionizing radiation, hydrogen peroxide and noradrenaline have been shown to cause hair greying in mice. We demonstrate here that isolated, ex vivo cultured human hair follicles exposed to these treatments display similar ectopic pigmentation within the bulge area which is accompanied by induction of differentiated melanocytic markers. This study suggests that as in murine models, stress signalling induces closely matching phenotypic changes in human hair follicles which can be monitored and studied as a surrogate model for early steps in human hair greying.

Anagen hair follicle repair: Timely regenerative attempts from plastic extra-bulge epithelial cells.

Anagen hair follicle repair (AHFR) is the regenerative scheme activated to restore the structure and hair growth following injuries to anagen hair follicles. Compared with telogen-to-anagen regeneration and hair follicle neogenesis, AHFR is a clinically important, yet relatively unexplored regenerative feature of hair follicles. Due to their highly proliferative character, germinative cells and matrix cells within hair bulbs are highly susceptible to injuries, such as chemotherapy and radiotherapy. Clinical and experimental observations suggest that damaged anagen hair follicles are able to repair themselves to resume anagen growth, bypassing premature catagen/telogen entry. Mechanistically, extra-bulge epithelial cells in the outer root sheath and the lower proximal cup are quickly mobilized for regeneration. These cells acquire stem cell-like properties, exhibiting high plasticity by breaking lineage restriction to regenerate all cell types in the lower segment of anagen hair follicles. Facilitating extra-bulge epithelial cells' mobilization ameliorates hair loss from chemo- and radiotherapy. On the other hand, quiescent bulge stem cells can also be activated, but only after more severe injuries and with slower activation dynamics. They show limited plasticity and regenerate part of the outer root sheath only. The dysrhythmic activation might render bulge stem cells susceptible to concomitant injuries due to their exit from quiescence.

Polycomb-Mediated Repression and Sonic Hedgehog Signaling Interact to Regulate Merkel Cell Specification during Skin Development.

An increasing amount of evidence indicates that developmental programs are tightly regulated by the complex interplay between signaling pathways, as well as transcriptional and epigenetic processes. Here, we have uncovered coordination between transcriptional and morphogen cues to specify Merkel cells, poorly understood skin cells that mediate light touch sensations. In murine dorsal skin, Merkel cells are part of touch domes, which are skin structures consisting of specialized keratinocytes, Merkel cells, and afferent neurons, and are located exclusively around primary hair follicles. We show that the developing primary hair follicle functions as a niche required for Merkel cell specification. We find that intraepidermal Sonic hedgehog (Shh) signaling, initiated by the production of Shh ligand in the developing hair follicles, is required for Merkel cell specification. The importance of Shh for Merkel cell formation is further reinforced by the fact that Shh overexpression in embryonic epidermal progenitors leads to ectopic Merkel cells. Interestingly, Shh signaling is common to primary, secondary, and tertiary hair follicles, raising the possibility that there are restrictive mechanisms that regulate Merkel cell specification exclusively around primary hair follicles. Indeed, we find that loss of Polycomb repressive complex 2 (PRC2) in the epidermis results in the formation of ectopic Merkel cells that are associated with all hair types. We show that PRC2 loss expands the field of epidermal cells competent to differentiate into Merkel cells through the upregulation of key Merkel-differentiation genes, which are known PRC2 targets. Importantly, PRC2-mediated repression of the Merkel cell differentiation program requires inductive Shh signaling to form mature Merkel cells. Our study exemplifies how the interplay between epigenetic and morphogen cues regulates the complex patterning and formation of the mammalian skin structures.

Theory and Practice of Lineage Tracing.

Lineage tracing is a method that delineates all progeny produced by a single cell or a group of cells. The possibility of performing lineage tracing initiated the field of Developmental Biology and continues to revolutionize Stem Cell Biology. Here, I introduce the principles behind a successful lineage-tracing experiment. In addition, I summarize and compare different methods for conducting lineage tracing and provide examples of how these strategies can be implemented to answer fundamental questions in development and regeneration. The advantages and limitations of each method are also discussed.

Drosophila TCTP is essential for growth and proliferation through regulation of dRheb GTPase.

Cellular growth and proliferation are coordinated during organogenesis. Misregulation of these processes leads to pathological conditions such as cancer. Tuberous sclerosis (TSC) is a benign tumour syndrome caused by mutations in either TSC1 or TSC2 tumour suppressor genes. Studies in Drosophila and other organisms have identified TSC signalling as a conserved pathway for growth control. Activation of the TSC pathway is mediated by Rheb (Ras homologue enriched in brain), a Ras superfamily GTPase. Rheb is a direct target of TSC2 and is negatively regulated by its GTPase-activating protein activity. However, molecules required for positive regulation of Rheb have not been identified. Here we show that a conserved protein, translationally controlled tumour protein (TCTP), is an essential new component of the TSC-Rheb pathway. Reducing Drosophila TCTP (dTCTP) levels reduces cell size, cell number and organ size, which mimics Drosophila Rheb (dRheb) mutant phenotypes. dTCTP is genetically epistatic to Tsc1 and dRheb, but acts upstream of dS6k, a downstream target of dRheb. dTCTP directly associates with dRheb and displays guanine nucleotide exchange activity with it in vivo and in vitro. Human TCTP (hTCTP) shows similar biochemical properties compared to dTCTP and can rescue dTCTP mutant phenotypes, suggesting that the function of TCTP in the TSC pathway is evolutionarily conserved. Our studies identify TCTP as a direct regulator of Rheb and a potential therapeutic target for TSC disease.

Building and Maintaining the Skin.

The skin forms a crucial, dynamic barrier between an animal and the external world. In mammals, three stem cell populations possess robust regenerative potential to maintain and repair the body's protective surface: epidermal stem cells, which maintain the stratified epidermis; hair follicle stem cells, which power the cyclic growth of the hair follicle; and melanocyte stem cells, which regenerate pigment-producing melanocytes to color the skin and hair. These stem cells reside in complex microenvironments ("niches") comprising diverse cellular repertoires that enable stem cells to rejuvenate tissues during homeostasis and regenerate them upon injury. Beyond their niches, skin stem cells can also sense and respond to fluctuations in organismal health or changes outside the body. Here, we review these diverse cellular interactions and highlight how far-reaching signals can be transmitted at the local level to enable skin stem cells to tailor their actions to suit the particular occasion and optimize fitness.

Defining a Role for G-Protein Coupled Receptor/cAMP/CRE-Binding Protein Signaling in Hair Follicle Stem Cell Activation.

Manipulation of adrenergic signaling has been shown experimentally and clinically to affect hair follicle growth. In this study, we provide direct evidence that canonical cAMP/CRE-binding protein signaling through adrenergic receptors can regulate hair follicle stem cell (HFSC) activation and hair cycle. We found that CRE-binding protein activation is regulated through the hair cycle and coincides with HFSC activation. Both isoproterenol and procaterol, agonists of adrenergic receptors, show the capacity to activate the hair cycle in mice. Furthermore, deletion of ADRB2 receptor, which is thought to mediate sympathetic nervous system regulation of HFSCs, was sufficient to block HFSC activation. Downstream, stimulation of adenylyl cyclase with forskolin or inhibition of phosphodiesterase to increase cAMP accumulation or direct application of cAMP was each sufficient to promote HFSC activation and accelerate initiation of hair cycle. Genetic induction of a Designer Receptors Exclusively Activated by Designer Drug allele showed that G-protein coupled receptor/GαS stimulation, specifically in HFSCs, promoted the activation of the hair cycle. Finally, we provide evidence that G-protein coupled receptor/CRE-binding protein signaling can potentially act on HFSCs by promoting glycolytic metabolism, which was previously shown to stimulate HFSC activation. Together, these data provide mechanistic insights into the role of sympathetic innervation on HFSC function.