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by Senok, Silas Solomon. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 194-217). / ACKNOWLEDGEMENT --- p.ii / ABSTRACT --- p.v / Chapter 1 --- INTRODUCTION AND OBJECTIVES --- p.1 / Chapter 2 --- LITERATURE REVIEW --- p.8 / Chapter 2.1 --- Classification of Cutaneous Mechanoreceptors --- p.8 / Chapter 2.2 --- Characteristics of Some Mammalian Cutaneous Mechanoreceptors --- p.21 / Chapter 2.3 --- Vibrissae --- p.30 / Chapter 2.4 --- Mechanosensory Transduction --- p.40 / Chapter 2.5 --- The Merkel Cell Controversy --- p.44 / Chapter 2.6 --- Intracellular Calcium Mobilisation --- p.53 / Chapter 3 --- METHODS --- p.59 / Chapter 3.1 --- The Isolated Vibrissal Preparation --- p.59 / Chapter 3.2 --- Experimental Protocol --- p.77 / Chapter 3.3 --- Data Analysis --- p.79 / Chapter 4 --- RESULTS --- p.82 / Chapter 4.1 --- Characterisation of The Receptors --- p.82 / Chapter 4.2 --- Stability And Viability of The Preparation --- p.94 / Chapter 4.3 --- Pharmacologic Studies --- p.103 / Chapter 5 --- DISCUSSION --- p.160 / Chapter 5.1 --- Outcome of Project --- p.160 / Chapter 5 2 --- The Isolated Vibrissal Preparation --- p.162 / Chapter 5.3 --- The Vibrissal Slowly Adapting Mechanoreceptors --- p.165 / Chapter 5 4 --- Mechanism of Chloroquine Inhibtion of Merkel Cell Receptors --- p.170 / Chapter 5.5 --- Evidence For CICR In Merkel Cell Endings --- p.175 / Chapter 5.6 --- Mechano-Electric Transduction In Merkel Cell Receptors --- p.182 / Chapter 5.7 --- Transduction in St II Nerve Terminals --- p.190 / Chapter 5.8 --- What Next? --- p.192 / Chapter 5.9 --- Conclusion --- p.193 / REFERENCES --- p.194

Touch sensation is initiated by diverse mechanosensory neurons that innervate distinct skin structures; however, little is known about how touch receptors are patterned during mammalian skin development. During the course of my PhD training, I analyzed embryonic and neonatal development of mouse touch domes, which contain Merkel cell-neurite complexes that encode pressure and object features. I found that developing touch domes share three key features with canonical sensory placodes: discrete patches of specialized epithelial, co-clustered mesenchymal cells capable of engaging in molecular crosstalk with the epithelium, and selective recruitment of sensory neurons. During embryogenesis, molecularly distinct patches of epithelial Merkel cells and keratinocytes clustered with a previously unsuspected population of BMP4-expressing dermal fibroblasts in nascent touch domes. Concurrently, two populations of sensory neurons preferentially targeted touch domes compared with other skin regions. Surprisingly, only one neuronal population persisted in mature touch domes. Overexpression of Noggin, a BMP antagonist, in epidermis at embryonic age 14.5 resulted in fewer touch domes, a loss of Merkel cells, and decreased innervation density in skin areas where touch domes are typically found. Thus, touch domes bear hallmarks of placode-derived sensory epithelia that require BMP signaling for proper specification.

The skin epidermis ensures critical vital functions: it represents the first line of defense against external aggressions.<p>The Epidermal Proliferative Unit (EPU) and the stochastic model are two opposite theories that have been proposed to explain the homeostasis of the interfollicular epidermis (IFE). During my thesis, we addressed this question. We used two inducible CREER transgenic mouse models (K14CREER and InvCREER) to labeled basal cells of the IFE using the RosaYFP reporter system, allowing us to follow their fate. Using confocal microscopy to analyze the basal YFP positive clones induced, we demonstrated the existence of two distinct basal progenitors. The mathematical modeling of our data set confirmed this observation and clearly demonstrated the existence of a quiescent stem cell (SC) population (targeted by the K14 promoter), which eventually gives rise to a more rapidly proliferating cells, the committed progenitors (CPs, targeted by the Inv promoter) that participate actively to replace the IFE cells lost during the normal turnover of the tissue. By using an independent genetic mouse model (K5tTA tetO-H2BGFP) and by studying the transcriptional profiling of the two basal populations, we confirmed our hypothesis, and thus reconciling the two seemingly opposite EPU and stochastic models. Finally, we challenged the function of the K14 SCs and Inv CPs during IFE wound healing. Conversely to what happens during homeostasis, we observed that SCs are massively proliferating and participating to the repair and maintenance of the damaged area, while CPs are poorly recruited to the wound.<p>The skin is also a highly sensitive organ, which contains many different receptors specialized in a precise sensation, and richly innervated by somatosensory neurons.<p>The Merkel Cells (MCs) are mechanoreceptors present in the basal layer of the IFE that have features of both neurons and keratinocytes, and might be implicated in the development of a rare but very aggressive skin tumor. Since their discovery in 1875, many studies claimed the neural crest cells (NCCs) or the epidermal progenitors are at the origin of MC, but no clear evidence has been proposed. <p>During my thesis, we were also interested in the embryonic origin of the MC. We used different transgenic mouse models to assess the NCC (Wnt1CRE and Pax3CRE RosaYFP) and the epidermal (K14CRE RosaYFP) hypotheses and we finally demonstrated that MC arise from embryonic epidermal progenitors. Using induction of adult epidermis transgenic mouse (K14CREER RosaYFP, K15CREPR RosaYFP), we showed that the epidermis also participates to the renewing of adult MC. Finally, we deleted the Math1 gene in all the MC progenitors (K14CRE Mathfl/fl) of mouse embryos, resulting in the absence of MCs in adult mice, demonstrating the importance of this transcription factor for MCs specification during embryogenesis.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

by Chan Eliza. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 183-196). / ACKNOWLEDGEMENTS / ABSTRACT --- p.i / Chapter CHAPTER ONE: --- INTRODUCTION --- p.1 / Chapter CHAPTER TWO: --- LITERATURE REVIEW / Chapter Section 1: --- History of Merkel cells --- p.3 / Chapter Section 2: --- Morphology and characteristic response of Merkel cell receptors in the skin --- p.5 / Chapter Section 3: --- Merkel cells and other mechanoreceptors in the mammalian sinus hair --- p.16 / Chapter Section 4: --- Functions of Merkel cells --- p.29 / Chapter Section 5: --- Review of technical approaches in the study of Merkel cell physiology --- p.39 / Chapter Section 6: --- Monitoring intracellular Ca2+ with the microfluorimetric technique --- p.42 / Chapter Section 7: --- Properties of voltage-gated and ligand-operated Ca2+ channels --- p.52 / Chapter CHAPTER THREE: --- METHODS / Chapter Section 1: --- Isolation of the rat vibrissal follicles --- p.60 / Chapter Section 2: --- Procedures for fluorimetric studies --- p.63 / Chapter Section 3: --- Procedures for electrophysiological study --- p.72 / Chapter Section 4: --- Chemicals --- p.82 / Chapter CHAPTER FOUR: --- RESULTS / Chapter Section 1: --- Electrophysiological studies in an isolated sinus hair preparation --- p.89 / Chapter Section 2: --- Electrophysiological studies on slowly adapting type I (SA I) mechanoreceptors in an isolated skin-nervein vitro preparation --- p.109 / Chapter Section 3: --- Microfluorimetric studies of Merkel cells in the isolated sinus hair preparation --- p.117

Somatosensory neurons densely innervate skin, our largest sensory organ. Adult skin continually remodels throughout the lifespan to maintain a protective barrier for our bodies. How sensory neurons maintain their peripheral endings in the face of continual turnover of their target tissue is not well understood. To address this gap in knowledge, I analyzed the temporal dynamics and mechanisms of structural plasticity of touch receptors in healthy adult skin. My studies focused on the terminals of Merkel-cell afferents in mouse touch domes. These two-part touch receptors comprise epithelial Merkel cells innervated by branching axons of fast-conducting sensory neurons. I show that Merkel cells and their afferents are structurally plastic over the course of hair growth in adults. These two components simplify during active hair growth, with fewer terminal neurites and fewer Merkel cells per touch dome at this stage compared with other phases of hair growth. Merkel-cell removal was observed with multiple molecular markers. Additionally, mice showed diminished touch-evoked behavior during hair growth compared with follicle quiescence. Next, I showed that Sarm1, a key effector of Wallerian degeneration, is not required for structural plasticity of Merkel cell-neurite complexes in young adulthood. Finally, I developed a technique to perform time-lapse in vivo imaging of identified Merkel cells and afferent terminals over the course of a month. These structures were highly plastic, with afferent terminals undergoing frequent growth and regression, as well as both Merkel cells and terminal branches being added or removed. Together, these studies reveal that peripheral nerve terminals undergo a previously unsuspected amount of structural plasticity in healthy tissue.

La détection de la température externe par la peau est le point de départ de nombreuses adaptations cellulaires et comportementales permettant de maintenir notre température interne constante. Selon ce concept, les fibres sensorielles cutanées sont les seuls récepteurs sensoriels de la peau pour la détection de la température. Plusieurs canaux ioniques activés directement par des températures chaudes ou froides ont été identifiés, ce sont les canaux TRPs. Le froid peut-il modifier le fonctionnement des organes du toucher?Nous montrons chez l’homme et la souris que les cellules de Merkel (CMs), qui sont les cellules tactiles des complexes de Merkel, peuvent être activées par le froid. Chez les souris dépourvues du canal TRPM8 (KO M8) la réponse au froid des CMs diminue. Le BCTC et le M8B, 2 bloqueurs du canal TRPM8, diminuent également la réponse au froid des CMs. Pour déterminer l’impact de cette sensibilité au froid sur la performance tactile, nous avons enregistré les variations de l’activité nerveuse des récepteurs de Merkel chez les souris WT et KO M8. Un froid modéré (20°C) appliqué sur la peau diminue le train de potentiels d’action issu d’un récepteur de Merkel stimulé mécaniquement. A 20°C ni le seuil de déclenchement des potentiels d’action, ni le train de potentiels d’action en réponse à une stimulation électrique ne sont modifiés. En revanche chez les souris KO M8 cette réponse mécanique tactile n’est plus diminuée. Ce résultat montre pour la première fois qu’une cellule non nerveuse de la peau, la cellule de Merkel, contient un récepteur au froid, le canal TRPM8, qui ajuste l’activité des récepteurs de Merkel lors d’une stimulation tactile. / In the skin, Merkel cells (Mcs) are connected to keratinocytes and A sensory nerve fibers and the complexes works as a slow adaptive mechanoreceptor (SA1 receptor). We observe that cooling human and mouse Merkel cells to 15°C increases intracellular Ca2+ ions concentration. The TRPM8 agonist’s provoke intracellular Ca2+ increases. The responses to cooling and TRPM8 agonist’s are reduced in absence of extracellular Ca2+ ions, by the TRPM8 antagonist’s and in KO M8 mouse. These results show that MCs sense cooling through TRPM8 channels. We hypothesize that cooling sensitivity modulate mechano-transduction and we investigate the modulation of SA1 response using the skin nerve and microneurography techniques in mouse and human, respectively. In mouse, cooling the skin at 22°C reduces the frequency of the SA1 discharge, without modifying the nerve conduction. This reduction disappeared in KO M8 mouse. These results suggest that MCs activity reduced the discharge of SA1 receptor at mild fresh temperature, anticipating effect of lower temperature on A nerve fiber excitability.This study is the first report about the sensitivity of MCs to cold temperature and its consequences on the SA1 receptor activity in mouse and human. We conclude that cold sensitivity of Merkel cells mediated by TRPM8 regulates the SA1 mechanical response, particularly at mild fresh temperature, when the nerve conduction is not significantly modified by cold. This is the first description of an active inhibitory process, driven by a TRP channel, during sensory transduction in the skin.

L’opossum nait dans un état très immature, mais rampe avec ses membres antérieurs (MA) de l’orifice urogénital de la mère à une tétine, où il s’attache pour poursuivre son développement. Des informations sensorielles sont nécessaires pour guider le nouveau-né vers une tétine et déclencher son attachement. Des expériences précédentes ont montré que le système du trijumeau, dont dépend l’innervation somesthésique du museau, influence les mouvements précoces des MA. Le présent projet vise à déterminer si les mécanorécepteurs faciaux sont fonctionnels et exercent une influence sur les MA. On s’intéresse particulièrement aux cellules de Merkel, un mécanorécepteur épidermique innervé par des fibres à adaptation lente de type I (SA I). Ces cellules ont été localisées sur le pourtour du museau de l’opossum nouveau-né en utilisant un traceur cellulaire, l’AM1-43. Nous avons analysé les réponses musculaires des MA consécutives à l’application de forces calibrées au museau sur des préparations in vitro. Ces réponses sont bilatérales et simultanées, très variables, et leur intensité augmente avec la force de la stimulation. Lors de stimulations répétitives pendant 60 min, les réponses diminuent avec le temps. Le retrait de la peau faciale abolit presque ces réponses. De plus, l’application d’un antagoniste des récepteurs métabotropiques du glutamate, qui affecte l’activité des fibres SA I, ou d’un antagoniste des récepteurs purinergiques les diminue fortement, suggérant une participation des cellules de Merkel. Ces résultats soutiennent que le sens du toucher facial relayé par le système du trijumeau est fonctionnel chez l’opossum nouveau-né et qu’il pourrait influencer les mouvements des MA. / The opossum, Monodelphis domestica, is born very immature but crawls, unaided, with its forelimbs (FL) from the mother's birth canal to a nipple where it attaches to pursue its development. Sensory clues are needed to guide the newborn to the nipple and trigger its attachment to it. We postulated that the trigeminal system, responsible for sensory innervation of the face, is involved. Indeed, light pressure applied on the snout evokes FL movements in vivo, low intensity electrical stimulation of the trigeminal ganglion induces motor responses of the FL in vitro, and trigeminal fibers is distributed in the facial dermis and basal epidermis of the newborn. Also, slowly adapting mechanosensory receptors Merkel cells (AM1-43 positive) are present in the face epidermis. To determine if Merkel cells exert an influence on locomotion of newborn opossums, we analyzed the FL muscles responses following application of calibrated forces on the snout in in vitro preparations. Pressure applied to the face induced bilateral and simultaneous FL motor responses, which intensity is proportional to stimulation force. Following consecutive stimulations during 60 min, the responses tended to decrease. Removing the facial skin nearly abolished the responses. Bath applications of the glutamate metabotropic receptor antagonist YM298198, and of the purinergic receptors (P2) antagonist PPADS, decreased the muscles responses. These results support that touch sensitivity of the snout relayed by the trigeminal system is functional in newborn opossums and may influence FL movement, possibly contributing to guiding the animal to the nipple.

La peau est organisée en niche de cellules souches/progénitrices qui contribuent au maintien des lignées épidermiques pendant l’homéostasie permettant ainsi de conserver l’intégrité du tissue. Les différentes cascades de signalisation qui régulent cet équilibre sont essentielles et la perturbation de ces voix peuvent amener à une différentiation anormale des kératinocytes, pouvant engendrer des cancers de la peau. Le but de cette thèse était d’identifier et de caractériser la population de progéniteurs responsables de la maintenance d’une niche épidermique spécialisée dans la mechanotransduction du toucher léger appelée les cellules de Merkel. Mon étude a porté sur le rôle des progéniteurs épithéliaux localisés dans le dôme du toucher (DT) de l’épiderme dans des conditions d’homéostasie ainsi que sur le développement du carcinome des cellules de Merkel. Basé sur l’analyse de données de microarray, j’ai identifié une nouvelle population de progéniteurs qui expriment de manière spécifique Kératine 17 (K17). Des expériences de traçage de lignées cellulaires démontrent que ces cellules donnent naissance aux cellules de Merkel (CM) ainsi qu’aux cellules squameuses. De plus, l’ablation génétique sélective des progéniteurs des CMs dans le DT a montré que cette niche est isolée et indépendante du reste de l’épiderme. Ces résultats établissent les CMs comme la quatrième lignée cellulaire de la peau. Cette découverte a permis l’établissement de nouveaux outils pour l’étude de conditions pathologiques associées à la lignée des cellules de Merkel, telles que les carcinomes des cellules de Merkel et le déclin du toucher léger avec l’âge. / The skin is organized in highly regionalized stem or progenitor cell niches that are in charge of maintaining all epidermal lineages during homeostasis. Disruption of molecular pathways that tightly regulate this balance leads to abnormal specification and differentiation of keratinocytes, eventually causing skin cancer. The goal of this thesis was to identify and characterize the progenitor population responsible for the maintenance of an epidermal niche specialized for mechanosensory signaling: the Merkel cell lineage. This work focused on the role of the epithelial progenitors located in the touch dome (TD) of hairy skin under homeostatic conditions and in a Merkel cell carcinoma (MCC) context. Based on previous microarray data, I first identified a distinct population of the interfollicular epidermis uniquely expressing Keratin 17 (K17). By lineage tracing analysis, I demonstrated that these cells give rise to the Merkel cell (MC) and squamous lineage. More importantly, selective genetic ablation of K17+ TD keratinocytes (TDKC) showed that the TD is a self-autonomous niche defining it as the 4th lineage of the skin. Interestingly, TDKCs may be involved in maintaining innervation of the Merkel cell-neurite complex. These critical results have established a new plateform for the field to allow studies of pathological skin conditions such as Merkel cell carcinoma and the loss of tactile acuity with age.