ARTICLE |
Correspondence to: Kuniaki Toyoshima, Dept. of Oral Anatomy and Neurobiology, Kyushu Dental College, Manazuru, Kokurakita-ku, Kitakyushu 803-8580, Japan..
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Merkel cells represent a population of epithelial cells in the skin and oral mucosa. Although Merkel cells are reliably distinguishable from other epithelial cells at the ultrastructural level, these cells are usually not discernible by standard light microscopy and need special techniques for their identification. Villin is an actin-crosslinking protein that is associated with the actin filament cores of brush border microvilli. In this study we show that an antibody against villin is an excellent marker of Merkel cells and their microvilli even at the light microscopic level. The surrounding keratinocytes and subepithelial connective tissue cells do not show any significant affinity for the antibody against villin. Confocal laser micrographs reconstructed from serial images 0.5 µm thick of Merkel cells that were immunostained with villin clearly reveal the three-dimensional morphology of Merkel cells and their microvilli. The presence of villin in Merkel cell microvilli lends support to the idea that these cells might have a mechanoreceptor function. (J Histochem Cytochem 46:13291334, 1998)
Key Words: Merkel cell, villin, microvilli, immunohistochemistry, confocal laser microscopy
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MERKEL CELLS represent a population of specialized intraepithelial cells in the skin and oral mucosa and have been qualified as a representative of paraneurons (
The function(s) of Merkel cells is(are) still controversial. The widely held belief that the Merkel cell is a mechanoreceptor cell is probably based on the presence of microvilli at the cell surface and the accumulation of dense-cored granules in the cytoplasm facing the nerve terminals. These microvilli may act as "feelers" sensitive to mechanical distortion, which may cause the release of transmitters from the Merkel cell. The present study was undertaken to identify Merkel cells by the use of an antibody specific for villin, the actin-crosslinking protein, which is associated with the actin filament cores of microvilli. In the present report, we show that the antibody against villin is an excellent marker of Merkel cells and their characteristic microvilli for both light and confocal laser microscopy.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Rabbits were sacrificed by IV injection of an overdose of sodium pentobarbital. The hard palate membrane was removed and immediately fixed in 4% paraformaldehyde in 0.1 M phosphate buffer for 4 hr. After rinsing overnight with the same buffer containing 30% sucrose, the tissues were quickly frozen to the temperature of a dry iceisopentane mixture and cut at 8 or 20 µm thickness in a freezing microtome. The sections were then either processed for diaminobenzidine (DAB) immunohistochemistry (8-µm sections) or immunofluorescence histochemistry (20-µm sections). DAB immunohistochemistry was done with the avidinbiotinperoxidase complex (ABC) method (
For double immunolabeling, the sections were incubated in an equal part mixture of a 1:100 dilution of monoclonal antibody against villin and a 1:20 dilution of monoclonal antibody against CK 20 (Progen Biotechnik; Heidelberg, Germany). Reaction products were sequentially labeled with FITC-conjugated goat anti-mouse IgG1 (Southern Biotechnology Associates) and tetrarhodamine isothiocyanate (TRITC)-conjugated goat anti-mouse IgG2a (Southern Biotechnology Associates) by incubation for 90 min at RT for each labeled antibody, and were observed under a confocal laser scanning microscope.
For electron microscopic immunohistochemistry, the preembedding technique was employed on cryostat sections of rabbit hard palate that was fixed in 4% paraformaldehyde. Sections were incubated with the villin antibody and then treated with the ABC method as described above. The sections were postfixed for 2 hr in 2% osmium tetroxide, dehydrated in ethanol, and embedded in Epon 812. Ultrathin sections were obtained from selected areas containing immunopositive Merkel cells, as assessed by light microscopic evaluation of semithin sections, and were observed without further staining. A paired adjacent ultrathin section was stained routinely with uranyl acetate followed by lead. Both types of sections were examined in a JEM-1200 EX electron microscope.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Merkel cells were clustered in the basal cell layer at the base of epithelial rete pegs of every palate raphe. Ultrastructurally, Merkel cells were characterized by the following features: (a) many dense-cored granules 80120 nm in diameter; (b) Golgi complexes in the cytoplasm opposite the major aggregation of dense-cored granules; (c) many microvilli extending randomly from the cell surface; (d) desmosomal attachments to neighboring epithelial cells; and (e) frequent association with nerve terminals (Figure 1A). The core of each microvillus contained a bundle of parallel actin filaments extending from its tip into the cell body (Figure 1B and Figure 1C).
|
In cryosections of hard palate, the monoclonal antibody specific for villin gave intense labeling of Merkel cells scattered in the basal layer of the epithelium (Figure 2A and Figure 2B). In contrast to Merkel cells, the surrounding keratinocytes and subepithelial connective tissue cells did not show any significant affinity for the antibody against villin. The villin antibody displayed a strong affinity both for microvilli and for a narrow cytoplasmic band beneath the Merkel cell membrane. In addition, diffuse or reticular staining of variable density was detected in the cell body. In favorable planes of tangential sections, the cross-sectioned microvilli appeared as densely labeled dots. Confocal laser microscopy of Merkel cells labeled for villin showed that the microvillar processes were more brightly fluorescent than the cell bodies (Figure 2C).
|
A particular advantage of using confocal microscopy reconstructions from 20 serial images at steps of 0.5 µm was the identification of the three-dimensional extent of characteristic microvilli of Merkel cells (Figure 2D and Figure 2E). There were at least 50 microvilli in each Merkel cell. These microvilli measured about 0.30.5 µm in width and 24 µm in length, and usually had a straight configuration. However, bent or bifurcated microvilli also were occasionally observed. In double-labeling experiments with antibodies against CK20 (Figure 2F) and villin (Figure 2G), the CK20-positive Merkel cell population also could be labeled specifically with villin.
Electron microscopic immunostaining for villin clearly revealed that immunoreactions were exclusively confined to the Merkel cells (Figure 3A). Immunoperoxidase label was found mainly in association with microvilli of Merkel cells. When a paired adjacent ultrathin section was stained with uranyl acetate followed by lead, dense-cored granules and microvilli characteristics of Merkel cells could be recognized in the same cell (Figure 3B).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
This study shows that a specific marker, villin, allows reliable identification of Merkel cells and their characteristic microvilli by immunohistochemistry. Villin, a calcium-regulated actin binding protein associated with the axial microfilament bundle, was originally isolated from intestinal microvilli (
It is known that villin plays an important role in the organization of the brush border cytoskeleton (
The function of Merkel cells is still under debate. Since
However, controversy continued as to whether or not the Merkel cell is a mechanoreceptor (
If the Merkel cell is not a mechanoreceptor, then the significance of microvilli at the cell surface remains unexplained. The presence of villin in Merkel cell microvilli lends support to the notion that these cells engage in mechanoreception.
![]() |
Acknowledgments |
---|
We thank Dr B. Tandler for critical reading of the manuscript and M. Ueda and T. Tanaka for assistance with the CLMS.
Received for publication January 20, 1998; accepted July 14, 1998.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bretscher A, Weber K (1979) Villin: the major microfilament-associated protein of the intestinal microvillus. Proc Natl Acad Sci USA 76:2321-2325[Abstract]
Crowe R, Whitear M (1978) Quinacrine fluorescence of Merkel cells in Xenopus laevis. Cell Tissue Res 190:273-283[Medline]
Dell DA, Munger BL (1986) The early embryogenesis of papillary (sweat duct) ridges in primate glabrous skin: the dermatotrophic map of cutaneous mechanoreceptors and dermatoglyphics. J Comp Neurol 244:511-532[Medline]
Diamond J, Mills LR, Mearow KM (1988) Evidence that the Merkel cell is not the transducer in the mechanosensory Merkel cell-neurite complex. Prog Brain Res 74:51-56[Medline]
Drenckhahn D, Dermietzel R (1988) Organization of the actin filament cytoskeleton in the intestinal brush border: a quantitative and qualitative immunoelectron microscope study. J Cell Biol 107:1037-1048[Abstract]
Friederich E, Huet C, Arpin M, Louvard D (1989) Villin induces microvilli growth and actin redistribution in transfected fibroblasts. Cell 59:461-475[Medline]
Fujita T, Kanno T, Kobayashi S (1988) The Paraneuron. Tokyo, Berlin, Heidelberg, New York, London, Paris, Springer-Verlag
Garant PR, Feldman J, Cho MI, Cullen MR (1980) Ultrastructure of Merkel cells in the hard palate of the squirrel monkey (Saimiri sciureus). Am J Anat 157:155-167[Medline]
Hartschuh W, Weihe E (1988) Multiple messenger candidates and marker substances in the mammalian Merkel cell-axon complex: a light and electron microscopic immunohistochemical study. Prog Brain Res 74:181-187[Medline]
Hartschuh W, Weihe E, Reinecke M (1986) The Merkel cell. In BereiterHahn AGE, Matoltsy AG, Richards KS, eds. Biology of the Integument 2. Berlin, Springer-Verlag, 605-620
Hartschuh W, Weihe E, Yanaihara N (1989) Immunohistochemical analysis of chromogranin A and multiple peptides in the mammalian Merkel cell: further evidence for its paraneuronal function? Arch Histol Cytol 52(suppl):423-431[Medline]
Höfer D, Drenckhahn D (1992) Identification of brush cells in the alimentary and respiratory systems by antibodies to villin and fimbrin. Histochemistry 98:237-242[Medline]
Höfer D, Drenckhahn D (1996) Cytoskeletal markers allowing discrimination between brush cells and other epithelial cells of the gut including enteroendocrine cells. Histochem Cell Biol 105:405-412[Medline]
Horvat B, Osborn M, Damjanov I (1990) Expression of villin in the mouse oviduct and seminiferous ducts. Histochemistry 93:661-663[Medline]
Hsu SM, Raine L, Fanger H (1981) Use of avidinbiotinperoxidase complex (ABC) in immunoperoxidase techniques. A comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577-580[Abstract]
Kasper M, Höfer D, WoodcockMitchel J, Migheli A, Attanosio A, Rudolf T, Müller M, Drenckhahn D (1994) Colocalization of cytokeratin 18 and villin in type III alveolar cells (brush cells) of the rat lung. Histochemistry 101:57-62[Medline]
Luciano L, Reale E (1979) A new morphological aspect of the brush cells of the mouse gallbladder epithelium. Cell Tissue Res 201:37-44[Medline]
Luciano L, Reale E (1990) Brush cells of the mouse gallbladder. Cell Tissue Res 262:339-349[Medline]
Matsudaira PT, Burgess DR (1979) Identification and organization of the components in the isolated microvillus cytoskeleton. J Cell Biol 83:667-673[Abstract]
Merkel F (1875) Tastzellen und Tastkörperchen bei den Haustieren und beim Menschen. Arch Mikrosk Anat 11:636-652
Mills LM, Diamond J (1995) Merkel cells are not the mechanosensory transducers in the touch dome of the rat. J Neurocytol 24:117-134[Medline]
Moll I, Kuhn C, Moll R (1995) Cytokeratin 20 is a general marker of cutaneous Merkel cells while certain neuronal proteins are absent. J Invest Dermatol 104:910-915[Abstract]
Moll R, Moll I, Franke WW (1984) Identification of Merkel cells in human skin by specific cytokeratin antibodies: changes of cell density and distribution in fetal and adult plantar epidermis. Differentiation 28:136-154[Medline]
Mooseker MS, Graves TA, Wharton KA, Falco N, Home CL (1980) Regulation of microvillus structure: calcium-dependent solution and cross-linking of actin filaments in the microvilli of intestinal cells. J Cell Biol 87:809-822[Abstract]
Narisawa Y, Hashimoto K (1991) Immunohistochemical demonstration of nerveMerkel cell complex in fetal human skin. J Dermatol Sci 2:361-370[Medline]
Ogawa H (1996) The Merkel cell as a possible mechanoreceptor cell. Prog Neurobiol 49:317-334[Medline]
Pasche F, Mérot Y, Carraux P, Saurat JH (1990) Relationship between Merkel cells and nerve endings during embryogenesis in the mouse epidermis. J Invest Dermatol 95:247-251[Abstract]
Robine S, Huet C, Moll R, SahuquiilloMerino C, Coudrier E, Zweibaum A, Louvard D (1985) Can villin be used to identify malignant and undifferentiated normal digestive epithelial cells? Proc Natl Acad Sci USA 82:8488-8492[Abstract]
Rodman JS, Mooseker M, Farquhar MG (1986) Cytoskeletal proteins of the rat kidney proximal tubule brush border. Eur J Cell Biol 42:319-327[Medline]
Scott SA, Cooper E, Diamond J (1981) Merkel cells as targets of the mechanosensory nerves in salamander skin. Proc R Soc Lond B 211:455-470[Medline]
Tachibana T (1995) The Merkel cell: recent findings and unresolved problems. Arch Histol Cytol 58:379-396[Medline]
Whitear M (1989) Merkel cells in lower vertebrates. Arch Histol Cytol 52(suppl):415-422[Medline]
Yamashita Y, Toida K, Ogawa H (1993) Observation of Merkel cells with scanning electron microscopy. Neurosci Lett 159:155-158[Medline]
Zelená J (1994) Nerves and Mechanoreceptors: the Role of Innervation in the Development and Maintenance of Mammalian Mechanoreceptors. London, Glasgow, Weinheim, New York, Melbourne, Madras, Chapman & Hall