Preliminary evidence that Merkel cells exert chemosensory functions in human epidermis.

The mechanotransduction of light-touch sensory stimuli is considered to be the main physiological function of epidermal Merkel cells (MCs). Recently, however, MCs have been demonstrated to be also thermo-sensitive, suggesting that their role in skin physiologically extends well beyond mechanosensation. Here, we demonstrate that in healthy human skin epidermal MCs express functional olfactory receptors, namely OR2AT4, just like neighbouring keratinocytes. Selective stimulation of OR2AT4 by topical application of the synthetic odorant, Sandalore®, significantly increased Piccolo protein expression in MCs, as assessed by quantitative immunohistomorphometry, indicating increased vesicle trafficking and recycling, and significantly reduced nerve growth factor (NGF) immunoreactivity within MCs, possibly indicating increased neurotrophin release upon OR2AT4 activation. Live-cell imaging showed that Sandalore® rapidly induces a loss of FFN206-dependent fluorescence in MCs, suggesting OR2AT4-dependent MC depolarization and subsequent vesicle secretion. Yet, in contrast to keratinocytes, OR2AT4 stimulation by Sandalore® altered neither the number nor the proliferation status of MCs. These preliminary ex vivo findings demonstrate that epidermal MCs also exert OR-dependent chemosensory functions in human skin, and invite one to explore whether these newly identified properties are dysregulated in selected skin disorders, for example, in pruritic dermatoses, and if these novel MC functions can be therapeutically targeted to maintain/promote skin health.

Do Merkel complexes initiate mechanical itch?

Itch is a common sensation which is amenable to disabling patients' life under pathological and chronic conditions. Shared assertion easily limits itch to chemical itch, without considering mechanical itch and alloknesis, its pathological counterpart. However, in recent years, our understanding of the mechanical itch pathway, particularly in the central nervous system, has been enhanced. In addition, Merkel complexes, conventionally considered as tactile end organs only responsible for light touch perception due to Piezo2 expressed by both Merkel cells and SA1 Aß-fibres - low threshold mechanical receptors (LTMRs) -, have recently been identified as modulators of mechanical itch. However, the tactile end organs responsible for initiating mechanical itch remain unexplored. The consensus is that some LTMRs, either SA1 Aß- or A∂- and C-, are cutaneous initiators of mechanical itch, even though they are not self-sufficient to finely detect and encode light mechanical stimuli into sensory perceptions, which depend on the entire hosting tactile end organ. Consequently, to enlighten our understanding of mechanical itch initiation, this article discusses the opportunity to consider Merkel complexes as potential tactile end organs responsible for initiating mechanical itch, under both healthy and pathological conditions. Their unsuspected modulatory abilities indeed show that they are tuned to detect and encode light mechanical stimuli leading to mechanical itch, especially as they host not only SA1 Aß-LTMRs but also A∂- and C-fibres.

Ex Vivo Analysis of Mechanically Activated Ca2+ Transients in Urothelial Cells.

Mechanically activated ion channels are biological transducers that convert mechanical stimuli such as stretch or shear forces into electrical and biochemical signals. In mammals, mechanically activated channels are essential for the detection of external and internal stimuli in processes as diverse as touch sensation, hearing, red blood cell volume regulation, basal blood pressure regulation, and the sensation of urinary bladder fullness. While the function of mechanically activated ion channels has been extensively studied in the in vitro setting using the patch-clamp technique, assessing their function in their native environment remains a difficult task, often because of limited access to the sites of expression of these channels (e.g., afferent terminals, Merkel cells, baroreceptors, and kidney tubules) or difficulties applying the patch-clamp technique (e.g., the apical surfaces of urothelial umbrella cells). This protocol describes a procedure to assess mechanically evoked Ca2+ transients using the fluorescent sensor GCaMP5G in an ex vivo urothelial preparation, a technique that could be readily adapted for the study of mechanically evoked Ca2+ events in other native tissue preparations.

Miswiring of Merkel cell and pruriceptive C fiber drives the itch-scratch cycle.

Itch sensation provokes the scratch reflex to protect us from harmful stimuli in the skin. Although scratching transiently relieves acute itch through activation of mechanoreceptors, it propagates the vicious itch-scratch cycle in chronic itch by further aggravating itch over time. Although well recognized clinically, the peripheral mechanisms underlying the itch-scratch cycle remain poorly understood. Here, we show that mechanical stimulation of the skin results in activation of the Piezo2 channels on Merkel cells that pathologically promotes spontaneous itch in experimental dry skin. Three-dimensional reconstruction and immunoelectron microscopy revealed structural alteration of MRGPRA3+ pruriceptor nerve endings directed toward Merkel cells in the setting of dry skin. Our results uncover a functional miswiring mechanism under pathologic conditions, resulting in touch receptors triggering the firing of pruriceptors in the skin to drive the itch-scratch cycle.

Merkel cell polyomavirus large T antigen binding to pRb promotes skin hyperplasia and tumor development.

Clear evidence supports a causal link between Merkel cell polyomavirus (MCPyV) and the highly aggressive human skin cancer called Merkel cell carcinoma (MCC). Integration of viral DNA into the human genome facilitates continued expression of the MCPyV small tumor (ST) and large tumor (LT) antigens in virus-positive MCCs. In MCC tumors, MCPyV LT is truncated in a manner that renders the virus unable to replicate yet preserves the LXCXE motif that facilitates its binding to and inactivation of the retinoblastoma tumor suppressor protein (pRb). We previously developed a MCPyV transgenic mouse model in which MCC tumor-derived ST and truncated LT expression were targeted to the stratified epithelium of the skin, causing epithelial hyperplasia, increased proliferation, and spontaneous tumorigenesis. We sought to determine if any of these phenotypes required the association between the truncated MCPyV LT and pRb. Mice were generated in which K14-driven MCPyV ST/LT were expressed in the context of a homozygous RbΔLXCXE knock-in allele that attenuates LT-pRb interactions through LT's LXCXE motif. We found that many of the phenotypes including tumorigenesis that develop in the K14-driven MCPyV transgenic mice were dependent upon LT's LXCXE-dependent interaction with pRb. These findings highlight the importance of the MCPyV LT-pRb interaction in an in vivo model for MCPyV-induced tumorigenesis.

POU4F3 pioneer activity enables ATOH1 to drive diverse mechanoreceptor differentiation through a feed-forward epigenetic mechanism.

During embryonic development, hierarchical cascades of transcription factors interact with lineage-specific chromatin structures to control the sequential steps in the differentiation of specialized cell types. While examples of transcription factor cascades have been well documented, the mechanisms underlying developmental changes in accessibility of cell type-specific enhancers remain poorly understood. Here, we show that the transcriptional "master regulator" ATOH1-which is necessary for the differentiation of two distinct mechanoreceptor cell types, hair cells in the inner ear and Merkel cells of the epidermis-is unable to access much of its target enhancer network in the progenitor populations of either cell type when it first appears, imposing a block to further differentiation. This block is overcome by a feed-forward mechanism in which ATOH1 first stimulates expression of POU4F3, which subsequently acts as a pioneer factor to provide access to closed ATOH1 enhancers, allowing hair cell and Merkel cell differentiation to proceed. Our analysis also indicates the presence of both shared and divergent ATOH1/POU4F3-dependent enhancer networks in hair cells and Merkel cells. These cells share a deep developmental lineage relationship, deriving from their common epidermal origin, and suggesting that this feed-forward mechanism preceded the evolutionary divergence of these very different mechanoreceptive cell types.

Epigenetic regulation and signalling pathways in Merkel cell development.

Merkel cells are specialized epithelial cells connected to afferent nerve endings responsible for light-touch sensations, formed at specific locations in touch-sensitive regions of the mammalian skin. Although Merkel cells are descendants of the epidermal lineage, little is known about the mechanisms responsible for the development of these unique mechanosensory cells. Recent studies have highlighted that the Polycomb group (PcG) of proteins play a significant role in spatiotemporal regulation of Merkel cell formation. In addition, several of the major signalling pathways involved in skin development have been shown to regulate Merkel cell development as well. Here, we summarize the current understandings of the role of developmental regulators in Merkel cell formation, including the interplay between the epigenetic machinery and key signalling pathways, and the lineage-specific transcription factors involved in the regulation of Merkel cell development.

Disruption of tph1 genes demonstrates the importance of serotonin in regulating ventilation in larval zebrafish (Danio rerio).

Serotonergic neuroepithelial cells (NECs) in larval zebrafish are believed to be O2 chemoreceptors. Serotonin (5-HT) within these NECs has been implicated as a neurotransmitter mediating the hypoxic ventilatory response (HVR). Here, we use knockout approaches to discern the role of 5-HT in regulating the HVR by targeting the rate limiting enzyme for 5-HT synthesis, tryptophan hydroxylase (Tph). Using transgenic lines, we determined that Tph1a is expressed in skin and pharyngeal arch NECs, as well as in pharyngeal arch Merkel-like cells (MLCs), whereas Tph1b is expressed predominately in MLCs. Knocking out the two tph1 paralogs resulted in similar changes in detectable serotonergic cell density between the two mutants, yet their responses to hypoxia (35 mmHg) were different. Larvae lacking Tph1a (tph1a-/- mutants) displayed a higher ventilation rate when exposed to hypoxia compared to wild-types, whereas tph1b-/- mutants exhibited a lower ventilation rate suggesting that 5-HT located in locations other than NECs, may play a dominant role in regulating the HVR.

Merkel cells of human oral mucosa express the pluripotent stem cell transcription factor Sox2.

Merkel cells are neuroendocrine cells associated to a neural sensitive ending and localized primarily in the epidermis, although they are also found in oral mucosa. Sox2 or SRY-box2 is a key transcription factor important in the maintenance of embryonic neural crest stem cell pluripotency. Sox2 has been described in Merkel cells of skin and in Merkel cell carcinomas, but not specifically in oral Merkel cells. The aims of the present study were to analyze the density of Merkel cells in human oral mucosa and to study the expression of Sox2 in these cells. For these purposes, immunohistochemical analyses for Sox2 and CK20 (the best marker for Merkel cells) were automatically performed on sections of normal human oral mucosa. Double immunofluorescence for Sox2 and CK20 was also performed. To analyze the density of Merkel cells, CK20 positive cells were counted in each sample and the length of the epithelial apical edge was measured (cells/mm). Merkel cells, demonstrated by CK20 immunoreactivity, were found in 95% of oral mucosa specimens studied (n=21). Mean density of Merkel cells in oral mucosa was 1.71±2.34 cells/mm. Sox2 immunoreactivity was found in the nuclei of scattered cells located at the basal layer. Serial sections immunostained for Sox2 and CK20 showed that Sox2-positive cells of oral mucosa coexpressed CK20, confirming that they were Merkel cells. Immunofluorescence for Sox2 and CK20 showed colocalization of both markers, demonstrating that virtually all oral Merkel cells expressed Sox2. This transcription factor could play a role in Merkel cell maturation and maintenance.

Conversion of Sox2-dependent Merkel cell carcinoma to a differentiated neuron-like phenotype by T antigen inhibition.

Viral cancers show oncogene addiction to viral oncoproteins, which are required for survival and proliferation of the dedifferentiated cancer cell. Human Merkel cell carcinomas (MCCs) that harbor a clonally integrated Merkel cell polyomavirus (MCV) genome have low mutation burden and require viral T antigen expression for tumor growth. Here, we showed that MCV+ MCC cells cocultured with keratinocytes undergo neuron-like differentiation with neurite outgrowth, secretory vesicle accumulation, and the generation of sodium-dependent action potentials, hallmarks of a neuronal cell lineage. Cocultured keratinocytes are essential for induction of the neuronal phenotype. Keratinocyte-conditioned medium was insufficient to induce this phenotype. Single-cell RNA sequencing revealed that T antigen knockdown inhibited cell cycle gene expression and reduced expression of key Merkel cell lineage/MCC marker genes, including HES6, SOX2, ATOH1, and KRT20 Of these, T antigen knockdown directly inhibited Sox2 and Atoh1 expression. MCV large T up-regulated Sox2 through its retinoblastoma protein-inhibition domain, which in turn activated Atoh1 expression. The knockdown of Sox2 in MCV+ MCCs mimicked T antigen knockdown by inducing MCC cell growth arrest and neuron-like differentiation. These results show Sox2-dependent conversion of an undifferentiated, aggressive cancer cell to a differentiated neuron-like phenotype and suggest that the ontology of MCC arises from a neuronal cell precursor.

A bioinspired hydrogen bond-triggered ultrasensitive ionic mechanoreceptor skin.

Biological cellular structures have inspired many scientific disciplines to design synthetic structures that can mimic their functions. Here, we closely emulate biological cellular structures in a rationally designed synthetic multicellular hybrid ion pump, composed of hydrogen-bonded [EMIM+][TFSI-] ion pairs on the surface of silica microstructures (artificial mechanoreceptor cells) embedded into thermoplastic polyurethane elastomeric matrix (artificial extracellular matrix), to fabricate ionic mechanoreceptor skins. Ionic mechanoreceptors engage in hydrogen bond-triggered reversible pumping of ions under external stimulus. Our ionic mechanoreceptor skin is ultrasensitive (48.1-5.77 kPa-1) over a wide spectrum of pressures (0-135 kPa) at an ultra-low voltage (1 mV) and demonstrates the ability to surpass pressure-sensing capabilities of various natural skin mechanoreceptors (i.e., Merkel cells, Meissner's corpuscles, Pacinian corpuscles). We demonstrate a wearable drone microcontroller by integrating our ionic skin sensor array and flexible printed circuit board, which can control directions and speed simultaneously and selectively in aerial drone flight.

Effects of norepinephrine and ß2 receptor antagonist ICI 118,551 on whisker hair follicle mechanoreceptors dissatisfy Merkel discs being adrenergic synapses.

Merkel discs, located in skin touch domes and whisker hair follicles, are tactile end organs essential for environmental exploration, social interaction, and tactile discrimination. Recent studies from our group and two others have shown that mechanical stimulation excites Merkel cells via Piezo2 channel activation to subsequently activate sensory neural pathways. We have further shown that mechanical stimulation leads to the release of 5-HT from Merkel cells to synaptically transmit tactile signals to whisker afferent nerves. However, a more recent study using skin touch domes has raised the possibility that Merkel discs are adrenergic synapses. It was proposed that norepinephrine is released from Merkel cells upon mechanical stimulation to subsequently activate ß2 adrenergic receptors on Merkel disc nerve endings leading to nerve impulses. In the present study, we examined effects of norepinephrine and ß2 adrenergic receptor antagonist ICI 118,551 on Merkel disc mechanoreceptors in mouse whisker hair follicles. We show that norepinephrine did not directly induce impulses from Merkel disc mechanoreceptors. Furthermore, we found that ICI 118,551 at 50 µM inhibited voltage-gated Na+ channels and suppressed impulses of Merkel disc mechanoreceptors, but ICI 118,551 at 1 µM had no effects on the impulse. These findings challenge the hypothesis of Merkel discs being adrenergic synapses.

Merkel cells immunohistochemical study in striped dolphin (Stenella coeruleoalba) skin.

Cetacean mechanical senses, such as hearing, echolocation, active touch and the perception of water movements, are essential for their survival. Dolphins skin possesses dense packing of dermal papillae associated with the cutaneous ridges that suggests a sensory function, furthermore they are well innervated and very sensitive to touch. This is mediated by mechanoreceptors, abundant in the region of the head and in the dorsal part of the body. Most odontocetes possess vibrissae (i.e., sensory hair) that have been well described in literature and present a microanatomy similar to that of terrestrial mammals. The aim of this study was to characterize Merkel cell through use of specific antibodies: Substance P, Anti-calbindin DK28, Anti-5HT, Leu- enkephalin, Protein Gene Product 9.5 (PGP9.5) and Anti-Human Neuronal Protein, for the first time. Merkel cells (MCs) in the dolphin skin are specialized skin receptors, characterized by their particular location and close association with nerve terminals. The presence of neuroendocrine markers and different neuropeptides confirms that MCs play also neuroendocrine function and are considered as part of the diffuse neuroendocrine system. Furthermore, the presence of Leu-enkephalin in Merkel cells could involve these cells in inflammatory responses in the skin.

Study on the Role of Histochemical Stains in Identifying Merkel Cells in Dogs.

Merkel cells (MCs) are neuroendocrine cells involved with tactile sense, growth, differentiation, and homeostasis of the skin as well as in different cutaneous diseases. Specific staining techniques are required for their identification because they are not easily visible in paraffin sections stained with hematoxylin and eosin. The present study assess the histochemical features of the MCs in dogs comparing with those described for other mammals in the literature and with the use of immunohistochemistry. A systematic study of samples from MCs-rich areas from healthy dogs was carried out by use of several histologic stains, including metachromatic staining, silver stains, methylene blue, periodic acid-Schiff stain, and osmium-based staining method. MCs were detected by the Grimelius argyrophilic stain in 86.7% of the specimens. The staining was showed as dark-brown granular cytoplasmic and consistently polarized to the basal cell cytoplasm matching with the cellular distribution of the characteristic neurosecretory granules. Some modifications in the standard staining protocol, including rinsing, silver reimpregnation, and counterstain dye, enhanced the MCs identification in stratified squamous epithelium. When compared with Cytokeratin 20-immunolabeled serial sections several MCs appeared nonstained with the argyrophilic method. These differences in MC numbers between stains were statistically significant. Other histologic stains failed to identify MCs in the specimens. The results of this study indicate that Grimelius argyrophilic stain is a suitable method for demonstration of MCs in the stratified squamous epithelium of skin and mucosa. Discussion on its utility when compared with immunohistochemistry and a review of the scientific literature is also presented. Anat Rec, 302:1458-1464, 2019. © 2018 Wiley Periodicals, Inc.

FGF signalling controls the specification of hair placode-derived SOX9 positive progenitors to Merkel cells.

Merkel cells are innervated mechanosensory cells responsible for light-touch sensations. In murine dorsal skin, Merkel cells are located in touch domes and found in the epidermis around primary hairs. While it has been shown that Merkel cells are skin epithelial cells, the progenitor cell population that gives rise to these cells is unknown. Here, we show that during embryogenesis, SOX9-positive (+) cells inside hair follicles, which were previously known to give rise to hair follicle stem cells (HFSCs) and cells of the hair follicle lineage, can also give rise to Merkel Cells. Interestingly, while SOX9 is critical for HFSC specification, it is dispensable for Merkel cell formation. Conversely, FGFR2 is required for Merkel cell formation but is dispensable for HFSCs. Together, our studies uncover SOX9(+) cells as precursors of Merkel cells and show the requirement for FGFR2-mediated epithelial signalling in Merkel cell specification.

In vitro formation of the Merkel cell-neurite complex in embryonic mouse whiskers using organotypic co-cultures.

A Merkel cell-neurite complex is a touch receptor composed of specialized epithelial cells named Merkel cells and peripheral sensory nerves in the skin. Merkel cells are found in touch-sensitive skin components including whisker follicles. The nerve fibers that innervate Merkel cells of a whisker follicle extend from the maxillary branch of the trigeminal ganglion. Whiskers as a sensory organ attribute to the complicated architecture of the Merkel cell-neurite complex, and therefore it is intriguing how the structure is formed. However, observing the dynamic process of the formation of a Merkel cell-neurite complex in whiskers during embryonic development is still difficult. In this study, we tried to develop an organotypic co-culture method of a whisker pad and a trigeminal ganglion explant to form the Merkel cell-neurite complex in vitro. We initially developed two distinct culture methods of a single whisker row and a trigeminal ganglion explant, and then combined them. By dissecting and cultivating a single row from a whisker pad, the morphogenesis of whisker follicles could be observed under a microscope. After the co-cultivation of the whisker row with a trigeminal ganglion explant, a Merkel cell-neurite complex composed of Merkel cells, which were positive for both cytokeratin 8 and SOX2, Neurofilament-H-positive trigeminal nerve fibers and Schwann cells expressing Nestin, SOX2 and SOX10 was observed via immunohistochemical analyses. These results suggest that the process for the formation of a Merkel cell-neurite complex can be observed under a microscope using our organotypic co-culture method.

Notch pathway signaling in the skin antagonizes Merkel cell development.

Merkel cells are mechanosensitive skin cells derived from the epidermal lineage whose development requires expression of the basic helix-loop-helix transcription factor Atoh1. The genes and pathways involved in regulating Merkel cell development during embryogenesis are poorly understood. Notch pathway signaling antagonizes Atoh1 expression in many developing body regions, so we hypothesized that Notch signaling might inhibit Merkel cell development. We found that conditional, constitutive overexpression of the Notch intracellular domain (NICD) in mouse epidermis significantly decreased Merkel cell numbers in whisker follicles and touch domes of hairy skin. Conversely, conditional deletion of the obligate NICD binding partner RBPj in the epidermis significantly increased Merkel cell numbers in whisker follicles, led to the development of ectopic Merkel cells outside of touch domes in hairy skin epidermis, and altered the distribution of Merkel cells in touch domes. Deletion of the downstream Notch effector gene Hes1 also significantly increased Merkel cell numbers in whisker follicles. Together, these data demonstrate that Notch signaling regulates Merkel cell production and patterning.

Merkel Cells Sense Cooling with TRPM8 Channels.

In the skin, Merkel cells connect with keratinocytes and Aß nerve fibers to form a touch receptor that functions as a slow adapting mechanoreceptor (slow adapting type 1). In human and mouse Merkel cells, we observed an increased concentration of intracellular Ca2+ ions in response to cold temperature and transient receptor potential melastatine 8 (TRPM8) ion channel agonists. A reduction in the response to cooling and TRPM8 agonists occurred after the addition of TRPM8 antagonists, as well as in TRPM8 knockout mice. Cold temperature and TRPM8 agonists also induced a current that was inhibited by a TRPM8 antagonist. Our results indicate that Merkel cells sense cooling through TRPM8 channels. We hypothesized that cooling modulates the slow adapting type 1 receptor response. Cooling mouse skin to 22°C reduced the slow adapting type 1 receptor discharge frequency. Interestingly, we observed no such reduction in TRPM8 knockout mice. Similarly, in human skin, a temperature of 22°C applied to the slow adapting type 1 receptive field reduced the spiking discharge. Altogether, our results indicate that Merkel cells are polymodal sensory cells that respond to mild cold stimuli through the activation of TRPM8 channels. Thermal activation of Merkel cells, and possibly other TRPM8-expressing non-neuronal cells, such as keratinocytes, potentially adapts the discharge of slow adapting type 1 receptors during cooling.

Merkel cells and Meissner's corpuscles in human digital skin display Piezo2 immunoreactivity.

The transformation of mechanical energy into electrical signals is the first step in mechanotransduction in the peripheral sensory nervous system and relies on the presence of mechanically gated ion channels within specialized sensory organs called mechanoreceptors. Piezo2 is a vertebrate stretch-gated ion channel necessary for mechanosensitive channels in mammalian cells. Functionally, it is related to light touch, which has been detected in murine cutaneous Merkel cell-neurite complexes, Meissner-like corpuscles and lanceolate nerve endings. To the best of our knowledge, the occurrence of Piezo2 in human cutaneous mechanoreceptors has never been investigated. Here, we used simple and double immunohistochemistry to investigate the occurrence of Piezo2 in human digital glabrous skin. Piezo2 immunoreactivity was detected in approximately 80% of morphologically and immunohistochemically characterized (cytokeratin 20+ , chromogranin A+ and synaptophisin+ ) Merkel cells. Most of them were in close contact with Piezo2- nerve fibre profiles. Moreover, the axon, but not the lamellar cells, of Meissner's corpuscles was also Piezo2+ , but other mechanoreceptors, i.e. Pacinian or Ruffini's corpuscles, were devoid of immunoreactivity. Piezo2 was also observed in non-nervous tissue, especially the basal keratinocytes, endothelial cells and sweat glands. The present results demonstrate the occurrence of Piezo2 in cutaneous sensory nerve formations that functionally work as slowly adapting (Merkel cells) and rapidly adapting (Meissner's corpuscles) low-threshold mechanoreceptors and are related to fine and discriminative touch but not to vibration or hard touch. These data offer additional insight into the molecular basis of mechanosensing in humans.