Migration of Merkel cells in the labial mucous epithelium of adult rabbits following mental nerve resection

Summary Merkel cells in the lower labial mucosa of adult rabbits were studied electron microscopically, 9, 21, 28, and 50 days after resection of the mental nerves. By day 9, nerve fibers were completely retracted from the epithelial layer of the mucosa. On and after day 21, Merkel cells were located not only in the basal layer but also in the prickle or more superficial cell layers. The ultrastructure of the migrating Merkel cells was unchanged, both as to the amount and location of the specific cored granules in the cytoplasm, until the cells reached the granular cell layer. The position of the migrating Merkel cells differed from cell to cell, and migration continued for at least 50 days. A remarkably large number of immature Merkel cells was observed in the basal and suprabasal cell layers of the denervated epithelium even by day 50. Therefore, the possibility of the reproduction of Merkel cells exists. The migrating Merkel cells, as well as the keratinocytes in the same cell layer, had degenerated drastically in the parakeratinized cell layer. This seems to indicate that the Merkel cells belong to the line of keratinocytes.     

Current considerations about Merkel cells

Since the discovery of Merkel cells by Friedrich S. Merkel in 1875, knowledge of their structure has increased with the progression of new technologies such as electron and laser microscopy, and immunohistochemical techniques. For most vertebrates, Merkel cells are located in the basal layer of the epidermis and characterized by dense-core granules that contain a variety of neuropeptides, plasma membrane spines and cytoskeletal filaments consisting of cytokeratins and desmosomes. The presence of the two latter structures would suggest that Merkel cells originate from the epidermis rather than from the neural crest, even though such a hypothesis is not unanimously accepted. The function of the Merkel cell is also very controversial. For a long time, it has been accepted that Merkel cells with associated nerve terminals act as mechanoreceptors although the transduction mechanism has not yet been elucidated. Merkel cells that do not make contact with nerve terminals have an endocrine function. The present review aims to shed new and comparative light on this field with an attempt to investigate the stimuli that Merkel cells are able to perceive.     

Distribution and ultrastructure of Merkel cell of the fishing bat (Myotis ricketti)

The distribution and ultrastructure of Merkel cells were described in detail in piscivorous bats through immunohistochemistry and transmission electron microscopy techniques. The findings indicated that Merkel cells are commonly found in raised-domes,hair follicles and in the basal epidermis of the skin from their back,abdomen,intercrural membranes,wing membranes and footpads. However,the density of Merkel cells is significantly higher in the footpad than in other places. These results suggested that there ...     

Ultrastructure of Merkel corpuscles and so-called "transitional" cells in the white Leghorn chicken

In the chicken Merkel corpuscles are located in the dermis and consist of specialized Merkel cells, discoid nerve endings and lamellar cells. Merkel cells contain characteristic membrane-bound dense-core granules and bundles of microfilaments. Asymmetric junctions, synapse like, with thickened membranes and clusters of dense-core vesicles were observed between the Merkel cells and the nerve endings. The nerve ending is derived from myelinated nerves and sometimes contains clusters of clear vesicles. A laminar system formed by lamellar cells of the Schwann cell type encloses the Merkel cells and the nerve endings. So called "transitional" cells, showing some of the morphological features of both keratinocytes and Merkel cells, were observed in the basal layer of the epidermis. One was located partly in the epidermis and partly in the dermis. The structure of Merkel corpuscles is compared with that of Merkel cells in other tetrapods. The developmental significance of "transitional" cells and the origin of Merkel cells are discussed.     

Neural crest origin of mammalian Merkel cells

Here, we provide evidence for the neural crest origin of mammalian Merkel cells. Together with nerve terminals, Merkel cells form slowly adapting cutaneous mechanoreceptors that transduce steady indentation in hairy and glabrous skin. We have determined the ontogenetic origin of Merkel cells in Wnt1-cre/R26R compound transgenic mice, in which neural crest cells are marked indelibly. Merkel cells in whiskers and interfollicular locations express the transgene, beta-galactosidase, identifying them as neural crest descendants. We thus conclude that murine Merkel cells originate from the neural crest.     

Merkel-like basal cells in the nasal septal island of dromedaries: Ultrastructure and possible functions

Unlike other Merkel cell types, the morphology and functions of the Merkel-like basal cells remain unclear. The aim of the present study was to investigate the ultrastructural features of Merkel-like basal cells in the nasal septal island (NSI) of dromedaries (Camelus dromedarius) using transmission electron microscopy and to speculate their potential functions. Ten pairs of nasal septal islands obtained from ten heads of dromedary camels were used for the current study. Interestingly, these cells have been identified in the basal layer of the neuroepithelium of the dromedary nasal septal island near the sensory nerve endings. These cells were ovoid to elliptical in shape and rested on the basal lamina. Their surface had spine like cytoplasmic processes which interwined with the adjacent basal cells. Their nuclei were large lobulated with 2–3 deep notches. Moreover, numerous dense-core granules surrounded by electron-lucent halo were aggregated in the basal portion of the cells close to the nerve ending as well as melanin pigments in the apical portion. The ultrastructural characteristics of the Merkel-like basal cells of NSI were typical to those of Merkel cells, but with some morphological differences, including their location, cellular attachments, and connections to other structures. The potential functions were discussed in the light of the cellular context and architecture. The Merkel-like basal cells of the NSI neuroepithelium might play a role in nociception and magnetoreception in dromedaries.     

Merkel cell distribution in human hair follicles of the fetal and adult scalp

The distribution of Merkel cells in fetal and adult terminal hair follicles of human scalp was studied immunohistochemically using cytokeratin (CK) 20 as a specific Merkel cell marker. In hair follicles of adult scalp, abundant Merkel cells were found enriched in two belt-like clusters, one in the deep infundibulum and one in the isthmus region. No Merkel cells were found in the deep follicular portions including the bulb, or in the dermis. In early fetal hair follicles (bulbous peg stage), Merkel cells were only detected in the basal layer of the developing infundibulum but not in deeper follicular areas. In later stages, Merkel cells were also present in the isthmus and bulge. No Merkel cells were seen in the dermis around developing hair follicles. Nerve growth factor receptor was not only present in nerves but was found to be widely distributed within fetal skin. In adult skin, this receptor was localized to the basal cell layers of the outer root sheath of the bulb and the suprabulbar area, but was not detectable in the areas containing Merkel cells. The present study localizing Merkel cells within the permanent hair follicle structures close to their possible stem cells suggests that they have paracrine functions.     

The adult hair follicle: cradle for pluripotent neural crest stem cells

This review focuses on the recent identification of two novel neural crest-derived cells in the adult mammalian hair follicle, pluripotent stem cells, and Merkel cells. Wnt1-cre/R26R compound transgenic mice, which in the periphery express beta-galactosidase in a neural crest-specific manner, were used to trace neural crest cells. Neural crest cells invade the facial epidermis as early as embryonic day 9.5. Neural crest-derived cells are present along the entire extent of the whisker follicle. This includes the bulge area, an epidermal niche for keratinocyte stem cells, as well as the matrix at the base of the hair follicle. We have determined by in vitro clonal analysis that the bulge area of the adult whisker follicle contains pluripotent neural crest stem cells. In culture, beta-galactosidase-positive cells emigrate from bulge explants, identifying them as neural crest-derived cells. When these cells are resuspended and grown in clonal culture, they give rise to colonies that contain multiple differentiated cell types, including neurons, Schwann cells, smooth muscle cells, pigment cells, chondrocytes, and possibly other types of cells. This result provides evidence for the pluripotentiality of the clone-forming cell. Serial cloning showed that bulge-derived neural crest cells undergo self-renewal, which identifies them as stem cells. Pluripotent neural crest cells are also localized in the back skin hair of adult mice. The bulge area of the whisker follicle is surrounded by numerous Merkel cells, which together with innervating nerve endings form slowly adapting mechanoreceptors that transduce steady skin indentation. Merkel cells express beta-galactosidase in double transgenic mice, which confirms their neural crest origin. Taken together, our data indicate that the epidermis of the adult hair follicle contains pluripotent neural crest stem cells, termed epidermal neural crest stem cells (eNCSCs), and one newly identified neural crest derivative, the Merkel cell. The intrinsic high degree of plasticity of eNCSCs and the fact that they are easily accessible in the skin make them attractive candidates for diverse autologous cell therapy strategies.     

Merkel cells and permanent disesthesia in the oral mucosa after soft tissue grafts

Connective tissue grafts are routinely procedures in the treatment of gingival defects. The clinical success of the gingival tissue graft procedures anyway should ensure not only the aesthetic integration between the tissues but also the physiological activity of the graft in terms of sensitivity and immunity because the skin and the mucosae constitute the first natural aspecific borders against pathogens. The aim of this paper was to investigate nervous net recovery after connective graft procedure, in relation with sensorial alteration in the injured area. Results showed that there is a close link among the number of Merkel cells and the alteration of sensations. Merkel cells can be found isolated standing in the basal layer, supposed to have neuroendocrine functions in the epithelia or in larger group not associated with nerves; when found in association with nerves they are named Merkel complexes, acting as slow adapter mechanical receptor. Our data can be explained in two ways: Merkel cells increase as a consequence of tissue injury, a sort of “SOS cells” that secrete neuroendocrine signals to guide tissue healing; as an alternative the presence of the Merkel cells could be read as a derailment of tissue regeneration with the stop of cellular differentiation in the direction of an abnormal proliferation, a sort of mad stem cell. J. Cell. Physiol. 224:205–209, 2010 © 2010 Wiley‐Liss, Inc.     

Ultrastructure of Merkel corpuscles in the tongue of the finch,Lonchura striata

Summary Merkel corpuscles in the lingual mucosa of the finch, Lonchura striata, were examined by means of the argyrophilic reaction and electron microscopy. These corpuscles are composed of 12 to 20 flattened Merkel cells and enclosed nerve terminals. The present study demonstrated for the first time argyrophilia in avian subepithelial Merkel cells with the use of Grimelius silver stain. Electron-microscopically, the Merkel cell was characterized by the presence of numerous densecore granules, approximately 80 to 140 nm in diameter, as well as specialized contacts with nerve terminals. The granules showed a tendency to accumulate in the cytoplasm in close association with both nerve terminals and basal lamina. This study also provided unequivocal evidence for exocytotic discharge of Merkel-cell granules at the plasma membrane facing not only the nerve terminals but also the basal lamina. The exocytotic figures toward the nerve terminals can be regarded as synaptic discharge of Merkel-cell granules, but the possibility also exists that the Merkel-cell granules may exert a trophic effect on the nerve terminals. The exocytotic release of Merkel-cell granules toward the basal lamina with no relation to nerve terminals may suggest an endocrine (paracrine) function for the Merkel cell. The avian subepithelial Merkel cells qualify as paraneurons, but their exact nature and function remain enigmatic as is the case of intraepithelial Merkel cells in other vertebrates.     

Contribution to ontogenesis of Merkel cells

Merkel cells appear in the epidermis of planum nasale of the rat fetuses from the 16th day of i. u. development, namely in the 2nd-3rd layer of epidermal cells. Nerve fibres appear in the subepidermal connective tissue from the 20th day of i.u. development. Long cytoplasmic processes filled in with specific dense core vesicles grow from Merkel cells against them. Intraepidermally, nerve fibres appear in postnatal period (from 3rd day after birth). Granular vesicles of Merkel cells probably have the leading role in the formation and maintenance of contacts between Merkel cell and the nerve ending. The results of studying ontogenetic development of Merkel cells in the rat are favour of hypothesis about the differentiation of Merkel cells in the epidermis, however, the possibility of secondary equipment of epidermis with Merkel cells independently on the development of nerve fibres is not eliminated.     

Response of Merkel cells in the palatal rugae to the continuous mechanical stimulation by palatal plate

The aim of the present study was to investigate the responses of Merkel cells that are numerous in the palatine rugae, due to the continuous mechanical stimulation exerted by the palatal plate. Forty golden hamsters were used in this experiment. The palatal plate was made of adhesive resin and it was set on the palate of the animal. To exert a continuous pressure, a 0.8?mm elevation on the internal surface of the palatal plate was created at the middle portion of the fourth palatine ruga. Thereafter, the number of Merkel cells in the mucosa was calculated by immunohistochemical observation. Morphological changes of Merkel cells were examined by electron microscopy. There was significant difference among the control and any of the treated groups on the number of CK20 positive Merkel cells (p?<?0.05) and that numbers were decreased at the sites where continuous mechanical stimulation was exerted. Degeneration of the cytoplasm mitochondria and nerve endings, and a decrease in both the number of neurosecretory granules and cytoplasmic processes were observed. Furthermore, the presence of nuclear chromatin aggregation and fragmentation was recognized. The continuous mechanical stimulation by the palatal plate affected the responses of Merkel cells and nerve endings, thus inducing a decrease in the number of Merkel cells. A portion of these changes was also associated with the expression of apoptosis.     

Characterization of Merkel cells and mechanosensory axons of the rat by styryl pyridinium dyes

Summary The epidermal Merkel cells and their sensory innervation serve tactile sensation in vertebrates. In this study the fluorescent cationic mitochondrial dye, 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-ASP), which has recently been used as a vital stain for motor and autonomic nerve terminals, was tested for its ability to stain Merkel cells and sensory fibers in the snout of the rat. Brightly-fluorescent structures resembling Merkel cells as well as nerve fibers and their terminations were evident in whole mounts of the vibrissal follicle. Unilateral denervation of the vibrissal follicles soon after birth resulted in a staining pattern remarkably similar to that obtained after labelling of the Merkel cells selectively with the fluorescent marker quinacrine, but all fiber staining was abolished. Likewise, in the separated epidermis of other skin regions, including the hairy and glabrous skin of the nose, the staining pattern revealed by 4-Di-2-ASP was indistinguishable from that obtained by quinacrine fluorescence. These results indicate that certain styryl pyridinium dyes may be used as vital stains for epidermal Merkel cells as well as cutaneous mechanosensory axons.     

Calcitonin gene-related peptide (CGRP) immunoreactivity in the neuroendocrine Merkel cells and nerve fibres of pig and human skin

Summary The presence of calcitonin gene-related peptide (CGRP) in the skin of pig snout and human fingertip was investigated using immunohistochemical techniques. CGRP immunoreactivity was found in Merkel cells and nerve fibres of both species. In pig snout skin, Merkel cells containing CGRP were seen forming clusters at the tips of rete ridge epidermis and in the external root sheath of sinus hair follicles (vibrissae). Human Merkel cells immunostained for CGRP were found isolated or forming small groups in the basal layer of glandular epidermal ridges. In all cases, immunoreactivity was more intense on the side of the Merkel cell facing the associated nerve terminal (which was never positive for CGRP). This part of the Merkel cell has the greatest density of dense-cored granules, suggesting that CGRP must be stored in these granules. Nerve, bundles containing CGRP-immunoreactive fibres were found at dermal and hypodermal level, and blood vessels were often surrounded by CGRP nerve fibres. In pig snout skin some nerve fibres containing CGRP penetrated the epidermis and terminated as free endings, and in the human fingertip a small number of CGRP-immunoreactive nerve fibres were seen in Meissner's corpuscles.     

Long-term staining of live Merkel cells with FM dyes

Live Merkel cells in the skin and hair follicles are known to incorporate a fluorescence dye, quinacrine, which has been utilized to identify and dissect the cells for experiments. Quinacrine fluorescence of the cells is, however, quickly lost and quinacrine-stained Merkel cells soon become difficult to identify in tissue culture. To find dyes that remain in the cells for a long period of time, we tested many fluorescence dyes and found that FM dyes (such as FM1-43) are useful markers for live Merkel cells. In the rat footpad skin, FM1-43 was shown to stain 95% of live Merkel cells that were already stained with quinacrine. FM4-64 stained 98% of quinacrine-stained Merkel cells. Merkel cells in sinus hair follicles were also stained with FM dyes. The fluorescence intensity of FM dyes was stronger than that of quinacrine, and the shape of the cells was more distinct in the FM-dye-stained cells. To test how long FM dyes remain in live cells, FM-dye-stained Merkel cells in hair follicles were embedded in collagen gel and were cultured in a serum-free medium. FM-dye-stained cells were easily identified even after 7 days of culture. During the culture, Merkel cells changed their shape, moved in the preparation and tended to aggregate on the surface. We conclude that FM dyes are powerful tools for tracing live Merkel cells in in vitro experiments. Moreover, the finding that Merkel cells incorporate FM dyes suggests that vesicles in the cells are likely to have mechanisms of recycling in a manner similar to those in neurons and secretory cells.     

Immunohistochemical analyses point to epidermal origin of human Merkel cells

Merkel cells, the neurosecretory cells of skin, are essential for light-touch responses and may probably fulfill additional functions. Whether these cells derive from an epidermal or a neural lineage has been a matter of dispute for a long time. In mice, recent studies have clearly demonstrated an epidermal origin of Merkel cells. Given the differences in Merkel cell distribution between human and murine skin, it is, however, unclear whether the same holds true for human Merkel cells. We therefore attempted to gain insight into the human Merkel cell lineage by co-immunodetection of the Merkel cell marker protein cytokeratin 20 (CK20) with various proteins known to be expressed either in epidermal or in neural stem cells of the skin. Neither Sox10 nor Pax3, both established markers of the neural crest lineage, exhibited any cell co-labeling with CK20. By contrast, β1 integrin, known to be enriched in epidermal stem cells, was found in nearly 70 % of interfollicular epidermal and 25 % of follicular Merkel cells. Moreover, LRIG1, also enriched in epidermal stem cells, displayed significant co-immunolabeling with CK20 as well (approximately 20 % in the interfollicular epidermis and 7 % in the hair follicle, respectively). Further epidermal markers were detected in sporadic Merkel cells. Cells co-expressing CK20 with epidermal markers may represent a transitory state between stem cells and differentiated cells. β1 integrin is probably also synthesized by a large subset of mature Merkel cells. Summarizing, our data suggest that human Merkel cells may originate from epidermal rather than neural progenitors.     

DNA synthesis and content in the nuclei of epidermal cells of mice during their differentiation and specialization

Synthesis and content of DNA in the nuclei of differentiating cells of mouse skin epidermis was studied by using cytomorphometric, autoradiographic and cytophotometric methods. It has been shown that the cells of the keratinoid series divide only in the basal layer and contain 2-4c DNA. Keratinocytes of the thorny layer are mostly tetraploid, 2c cells are lacking. H4c and 8c cells comprise 12% of the population. In the keratinocytes of the granular layer DNA content is somewhat lower due to nuclei break down and conversion of cells into anucleate scale. Part of the melanocytes of the basal layer also contain 4c DNA. Highly specialized element of the basal layer Merkel and Langerhans cells are polyploid. Conclusion is drawn that DNA hyper-replication by multiplication of the whole genome is part of the development program of the population.     

Merkel cells in the stratified squamous nonkeratinized epithelium of the human oesophagus

The aim of our research was to identify Merkel cells in stratified squamous nonkeratinized epithelium of the human oesophagus. We chose the middle and lower part of the oesophagus. For identification of Merkel cells we used antibodies against simple-epithelial cytokeratines, especially anti CK 20, because it is providing the highest degree of specificity. Our investigation showed increased number of CK 20- positive Merkel cells in lower (distal) part of the oesophagus, approximately 60%. In the middle part of the oesophagus there were 30% of CK20-positive Merkel cells.     

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.