Three-Dimensional Morphology of Touch Domes in Human Hairy Skin by Correlative Light and Scanning Electron Microscopy

Abstract

keratin 20 light microscopy scanning electron microscopy three-dimensional The presence of tactile structures in human hairy skin was first reported by Pinkus in 1902. He discovered distinct epidermal disc-like structures with nerves and “Tastzellen” (i.e., Merkel cells) at the base of the epidermis, and named these structures “Haarscheiben” because of their close association with hair follicles. These structures were revisited by later investigators (Kamide, 1955Kamide J. On the findings of skin nerves supravitally stained with methylene blue, especially on the Haarscheibe of the human skin.Jap J Derm. 1955; 65: 339-355Google Scholar; Kawamura et al., 1964Kawamura T. Nishiyama S. Ikeda S. et al.The human haarscheibe, its structure and function.J Invest Dermatol. 1964; 42: 87-90Abstract Full Text PDF PubMed Scopus (15) Google Scholar). Recent researchers have also reported the histology of the human “Haarscheiben” (Moll et al., 2005Moll I. Roessler M. Brandner J.M. et al.Human Merkel cells--aspects of cell biology, distribution and functions.Eur J Cell Biol. 2005; 84: 259-271Crossref PubMed Scopus (110) Google Scholar; Reinisch and Tschachler, 2005Reinisch C.M. Tschachler E. The touch dome in human skin is supplied by different types of nerve fibers.Ann Neurol. 2005; 58: 88-95Crossref PubMed Scopus (43) Google Scholar), whereas some other investigators showed that Merkel cells arise from the epidermal progenitor cells under control of the Atoh1 transcription factor (Morrison et al., 2009Morrison K.M. Miesegaes G.R. Lumpkin E.A. et al.Mammalian Merkel cells are descended from the epidermal lineage.Dev Biol. 2009; 336: 76-83Crossref PubMed Scopus (142) Google Scholar; Van Keymeulen et al., 2009Van Keymeulen A. Mascre G. Youseff K.K. et al.Epidermal progenitors give rise to Merkel cells during embryonic development and adult homeostasis.J Cell Biol. 2009; 187: 91-100Crossref PubMed Scopus (181) Google Scholar). In the present study, we aimed at investigating the three-dimensional (3D) morphology of the human “Haarscheiben”, or touch domes, by the combined use of light microscopy (LM) and scanning electron microscopy (SEM). Touch domes observed by LM in hematoxylin–eosin-stained sections were characterized by their slightly thickened epidermis with clear cells, delineated by thick epidermal ridges in the periphery (Figure 1a and b). Immunohistochemical study for keratin 20 (K20), showing Merkel cells, and protein gene product 9.5, which stains nerve fibers, revealed the presence of numerous Merkel cell-neurite complexes in the base of the touch dome epidermis (Figure 1c). 3D reconstruction of K20-immunostained serial sections clearly showed the overall shape of the touch dome (Figure 1d and e). When viewed from the dermal side, the dome was an oval concave area with Merkel cells in the epidermal base, bordered by a circumferential deep epidermal ridge. These results corresponded well with those obtained by previous investigators (e.g., Pinkus, 1902Pinkus F. Ueber einen bisher unbekannten Nebenapparat am Haarsystem des Menschen: Haarscheiben.Dermatologische Zeitschrift. 1902; 9: 465-469Crossref Google Scholar). KOH treatment was then used to separate the epidermis from the dermis. The localization of Merkel cells in the touch dome was determined by direct comparison of LM and SEM images of KOH-treated/K20-immunostained tissues (Figure 2a and b). The base of Merkel cells appeared to be covered by neuronal components because of their continuity to the branch of unmyelinated fibers (Figure 2c and d). The number of Merkel cells ranged from 60 to 265 Merkel cells per touch dome (n=6, the 65-year-old cadaver), depending on the dome size. The density of Merkel cells in the touch dome was 608±142 Merkel cells per mm2 (mean±SD). Touch domes were further studied by SEM observation of the dermal side after KOH-collagenase treatment (Figure 2e–h and Supplementary Figure S1 online). Domes were determined as concave areas bordered by a thick epidermal ridge, and they were characterized by the accumulation of unmyelinated nerve fibers in the dermal region. The shape of the touch domes varied from round to irregular (Supplementary Figure S1 online). The epidermal ridges in the touch domes were generally shallow, but they sometimes appeared thicker in certain areas, as if dividing the domes into two or more subcompartments (Figure 2e). The sizes of the touch domes were 0.036±0.035mm2 (mean±SD), ranging from 0.002 to 0.130mm2 in our SEM images; the smallest dome was estimated to be 50 × 40μm2 and the largest 580 × 290μm2 (n=26). Although hair follicles were sometimes associated with touch domes, they were not in the center of the domes but were rather beside them (Figure 2e and Supplementary Figure S1 online). Some touch domes appeared completely independent from hair follicles (Figure 2f and Supplementary Figure S1 online). In the 26 touch domes (four individuals) analyzed in our SEM studies, 13 touch domes were associated with hair follicles and the other 13 domes were independent from hair follicles. Download .pdf (8.16 MB) Help with pdf files Supplementary Information Our SEM analysis also revealed the arrangement of Merkel cell-nerve complexes in the touch domes (Figure 2g and h). Schwann cell bodies were seen as spherical swellings around nerve branches. Unmyelinated nerve terminals extended into the base of Merkel cells and often innervated more than two Merkel cells with one terminal. Some nerve terminals also ended in the epidermis after innervating one or two Merkel cells (Figure 2g). Smith, 1970Smith Jr, K.R. The ultrastructure of the human Haarscheibe and Merkel cell.J Invest Dermatol. 1970; 54: 150-159Crossref PubMed Scopus (80) Google Scholar used transmission electron microscopy to show that axon terminals attaching to Merkel cells did not connect directly with other Merkel cells. However, our SEM findings clearly demonstrated that nerve branches often innervated more than two Merkel cells with one terminal. A similar finding was recently reported in the touch dome in cat hairy skin (Ebara et al., 2008Ebara S. Kumamoto K. Baumann K.I. et al.Three-dimensional analyses of touch domes in the hairy skin of the cat paw reveal morphological substrates for complex sensory processing.Neurosci Res. 2008; 61: 159-171Crossref PubMed Scopus (16) Google Scholar) using confocal laser scanning microscopy. Thus, it is probable that terminal unmyelinated branches form some territories in the human touch domes by innervating multiple Merkel cells with en-passant endings. The function of the touch dome has been studied by previous investigators. Iggo and colleagues carried out electron microscopic studies in combination with physiological recordings from touch domes in cats and primates, and demonstrated that the touch domes generated a localized, highly sensitive, and slowly adapting discharge by vertical surface pressure (Iggo and Kenshalo, 1968Iggo A. Electrophysiological and histological studies of cutaneous mechanoreceptors.in: Kenshalo D.R. The Skin Senses: Proceedings. Thomas, Springfield, IL1968: 84-105Google Scholar; Iggo and Muir, 1969Iggo A. Muir A.R. The structure and function of a slowly adapting touch corpuscle in hairy skin.J Physiol. 1969; 200: 763-796Crossref PubMed Scopus (568) Google Scholar). Recent experimental studies have also shown that Merkel cells are important for the slowly adapting response to sustained mechanical stimuli (Cahusac et al., 2005Cahusac P.M. Senok S.S. Hitchcock I.S. et al.Are unconventional NMDA receptors involved in slowly adapting type I mechanoreceptor responses?.Neuroscience. 2005; 133: 763-773Crossref PubMed Scopus (35) Google Scholar; Lumpkin et al., 2010Lumpkin E.A. Marshall K.L. Nelson A.M. The cell biology of touch.J Cell Biol. 2010; 191: 237-248Crossref PubMed Scopus (136) Google Scholar). These findings imply that the touch dome has an important role in mechanoreception in hairy skin. Previous studies showed that a tylotrich follicle is present in the center of the touch dome in mice, rats, and guinea pigs, suggesting that the dome detects pressure upon the guard hair in those animals (Smith, 1967Smith Jr, K.R. The structure and function of the Haarscheibe.J Comp Neurol. 1967; 131: 459-474Crossref PubMed Scopus (96) Google Scholar). The present study, however, revealed that the human touch dome was rather independent from hair follicles, although they were sometimes located beside the touch dome. This finding suggests that human touch domes function as touch spots independent from the structure for detecting the pressure upon the hair. The general consensus is that Merkel cells are connected by terminals from a single myelinated Aβ fiber (Delmas et al., 2011Delmas P. Hao J. Rodat-Despoix L. Molecular mechanisms of mechanotransduction in mammlian sensory neurons.Nat Rev Neurosci. 2011; 12: 139-153Crossref PubMed Scopus (321) Google Scholar), whereas Reinisch and Tschachler, 2005Reinisch C.M. Tschachler E. The touch dome in human skin is supplied by different types of nerve fibers.Ann Neurol. 2005; 58: 88-95Crossref PubMed Scopus (43) Google Scholar demonstrated the presence of Aδ and C-fibers as well as Aβ fibers in the human touch dome. We also showed that nerve fibers innervating the touch dome mostly ended as Merkel cell-neurite complexes, but partly ended on the base of keratinocytes in this area. It is probable that the human touch dome exceeds simple mechanosensation and has an important role in complex sensation as a touch spot in human hairy skin. We thank the members in the Division of Microscopic Anatomy, Niigata University Graduate School of Medical and Dental Sciences, for their technical assistance and valuable discussions throughout the study. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid