Epidermal Merkel cells are mechanosensory cells that tune mammalian touch receptors

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Abstract

Touch submodalities, such as flutter and pressure, are mediated by somatosensory afferents whose terminal specializations extract tactile features and encode them as action potential trains with unique activity patterns1. Whether non-neuronal cells tune touch receptors through active or passive mechanisms is debated. Terminal specializations are thought to function as passive mechanical filters analogous to the cochlea’s basilar membrane, which deconstructs complex sounds into tones that are transduced by mechanosensory hair cells. The model that cutaneous specializations are merely passive has been recently challenged because epidermal cells express sensory ion channels and neurotransmitters2,3; however, direct evidence that epidermal cells excite tactile afferents is lacking. Epidermal Merkel cells display features of sensory receptor cells4,5 and make ‘synapse-like’ contacts5,6 with slowly adapting type I (SAI) afferents7,8,9. These complexes, which encode spatial features such as edges and texture1, localize to skin regions with high tactile acuity, including whisker follicles, fingertips and touch domes. Here we show that Merkel cells actively participate in touch reception in mice. Merkel cells display fast, touch-evoked mechanotransduction currents. Optogenetic approaches in intact skin show that Merkel cells are both necessary and sufficient for sustained action-potential firing in tactile afferents. Recordings from touch-dome afferents lacking Merkel cells demonstrate that Merkel cells confer high-frequency responses to dynamic stimuli and enable sustained firing. These data are the first, to our knowledge, to directly demonstrate a functional, excitatory connection between epidermal cells and sensory neurons. Together, these findings indicate that Merkel cells actively tune mechanosensory responses to facilitate high spatio-temporal acuity. Moreover, our results indicate a division of labour in the Merkel cell–neurite complex: Merkel cells signal static stimuli, such as pressure, whereas sensory afferents transduce dynamic stimuli, such as moving gratings. Thus, the Merkel cell–neurite complex is an unique sensory structure composed of two different receptor cell types specialized for distinct elements of discriminative touch.

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Figure 1: Merkel cells exhibit touch-evoked ionic currents and preferentially express Piezo2.
Figure 2: Merkel cells are necessary and sufficient to elicit sustained action-potential trains in touch-dome afferents.
Figure 3: Atoh1CKO and Piezo2CKO mice show intermediately adapting responses.
Figure 4: Model of active Merkel-cell inputs in touch reception.

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Acknowledgements

Thanks to R. Axel, A. MacDermott and the Lumpkin laboratory for helpful discussions and to D. Florez and R. Piskorowski for advice on whole-cell recordings. Funding was provided by NIH/NIAMS grants R01AR051219 and R21AR062307 (to E.A.L.), R01DE022358 (to A.P.) and fellowships to S.M. (5T32HL087745-05 and NIH/NINDS F32NS080544), M.N. (JSPS Research Fellowships for Young Scientists 24-7585), and A.M.N. (McNair Foundation). Microscopy and flow cytometry was performed with core support from the Columbia SDRC (P30AR044535) and Cancer Center (P30CA013696). Initial studies were performed at Baylor College of Medicine with assistance from Flow Cytometry and Genetically Engineered Mouse Shared Resources (P30CA125123).

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S.M. screened transgenic mouse lines, and performed and analysed all ex vivo optogenetic experiments (Fig. 2 and Extended Data Figs 36). M.N. performed and analysed all whole-cell recordings (Fig. 1, Extended Data Fig. 1a, b and Extended Data Table 1). Y.B. performed and analysed recordings from Atoh1 and Piezo2 strains (Fig. 3, Extended Data Fig. 8 and Extended Data Table 2). A.M.N. performed qRT-PCR (Fig. 1i) and calcium imaging (Extended Data Fig. 1c–i). K.L.M. performed immunohistochemistry in Atoh1 strains (Extended Data Fig. 7) and assisted in preparation of all figures. S.A.W. and E.A.L. conceived optogenetic strategies. P.F. generated initial ChR2 transgenic mouse lines. E.A.L. conceived and supervised the project. During this manuscript’s peer-review process, we entered into a collaboration with S.H.W., S.R. and A.P., to analyse unpublished Piezo2CKO mice. S.R. generated Piezo2flox/flox mice and S.-H.W. generated and validated Krt14Cre;Piezo2flox/flox mice in the laboratory of A.P. The manuscript was written by S.M., M.N., Y.B. and E.A.L. and edited by A.M.N., K.L.M., S.A.W., P.F., S.-H.W. and A.P.

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Correspondence to Ellen A. Lumpkin.

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Extended data figures and tables

Extended Data Figure 1 Mechanically activated responses in Merkel cells.

a, Representative trace of mechanically evoked current induced by 1-µm mechanical displacement. Application of Ruthenium red (RR, 100 µM) attenuated mechanically activated current. b, Peak currents (Ipeak) were estimated from 250 µs around peak and steady-state currents (Iss) were estimated from the last 5 ms (black bar in 1a) of mechanical displacements. Data were normalized by Ipeak for each cell. With Ruthenium red, Ipeak was reduced to 38 ± 7% of control condition. Steady state currents were also reduced by Ruthenium red (n = 4; control: 9 ± 1% of Ipeak; RR: 2 ± 1% of Ipeak). **P < 0.01; paired Student’s t-test (two-tailed). c–i, Merkel cells display reversible Ca2+ responses to focal displacements applied to somata. c, Representative pseudocolour images of fura-2 ratios (340:380) of a Merkel cell at rest. d, A Merkel cell activated by depolarizing (high-K+) solution. e, A brightfield image showing the position of the stimulus probe. f–h, Peak responses corresponding to each displacement. ‘Fold Δ’ is the fold change in fluorescence ratio from baseline. Scale bar, 10 µm. i, Representative time course of mean fura-2 ratios during the touch stimuli shown above. Stimulus onset in f–h is indicated by arrows. Calcium responses were stimulus-dependent. Similar responses were observed from 11 Merkel cells.

Extended Data Figure 2 ChR2+ Merkel cells display light-activated inward currents.

a, Light-activated currents were recorded with whole-cell, tight-seal voltage clamp methods. b, Fluorescent image of a ChR2-tdTomato expressing Merkel cell. Scale bar, 10 µm. c, Representative trace for light-activated inward currents at a holding potential of −70 mV. Inactivation kinetics were measured by fitting a single exponential curve (red).

Extended Data Figure 3 Immunostaining of ChR2-expressing touch domes.

a–e, Whole-mount staining and confocal axial projection of the touch dome shown in Fig. 2d. a, Merged image. b–d, Expression of ChR2-tdTomato was present in Merkel cells (Krt8), but absent from sensory terminals (neurofilament heavy, NFH). e, Some terminal Schwann cells (Nestin)38 also expressed ChR2 (arrowheads in b and e). f–i, Immunostaining of skin cryosections. f, Merged image. g–i, ChR2-tdTomato was present in some S100+ Schwann cells that also expressed Nestin, a marker for type II terminal Schwann cells38 (arrowheads in f–i). Scale bars, 20 µm.

Extended Data Figure 4 Light-evoked activity is specific to touch-dome illumination.

a, f, Responses to light stimuli centred on a touch dome. b–e, When the light stimulus was positioned around the touch dome, no light-evoked activity was observed. Illuminating a cluster of ChR2+ dermal cells did not evoke any responses (c). f, To confirm that the absence of light-evoked activity was not due to the loss of Merkel cells and/or neuronal fibres, the experiment ended by re-positioning the light stimulus over the touch dome to re-elicit light-evoked activity. Images have been thresholded for clarity. Scale bars, 200 µm.

Extended Data Figure 5 K14Cre;ChR2loxP/+ mice exhibit light-evoked SAI activity.

a, Confocal image of a touch dome illustrating ChR2-tdTomato expression driven by K14Cre. ChR2-tdTomato expressed much stronger in Merkel cells than in neighbouring keratinocytes. b, Light-evoked responses from the touch dome shown in a to seven light intensities as indicated. Spike sorting and clustering analysis were used to identify the unit that fired in phase with light (lower trace with spike positions and their amplitudes). c, Mean IFFs for light with varying illumination intensities on a log-intensity scale (n = 3 recordings). Scale bar, 20 µm.

Extended Data Figure 6 Confocal axial projection of a touch dome shows selective ArchT–EGFP expression in Merkel cells driven by CckCre.

ArchT–EGFP expression was not observed in touch-dome afferents. Scale bar, 20 µm.

Extended Data Figure 7 Structure of touch-dome afferents in Atoh1CKO mice.

Immunostaining of skin cryosections from Atoh1CKO and control genotypes are shown. Antibodies labelling myelinated afferents (NFH; cyan), Merkel cells (Krt8; yellow), nodes of Ranvier (βIV spectrin; magenta) show that the general structure of touch-dome afferents, including myelinated branches and Nodes of Ranvier (arrowheads), appears normal even in the absence of Merkel cells. Scale bar, 20 µm.

Extended Data Figure 8 Comparison of ISI distributions in Atoh1CKO, Piezo2CKO and control genotypes.

a, Histogram of ISI distribution during saturating responses in Atoh1CKO (mean ± s.d., 43.4 ± 59.2 ms, median: 29.8 ms; n = 466 intervals from n = 6 units) and control genotypes (mean ± s.d., 16.5 ± 12.9 ms, median: 13.8 ms; n = 1,412 intervals from n = 5 units). Inset on the left illustrates all ISIs, including those > 150 ms, which were excluded from the main histograms (14/466 intervals in Atoh1CKO and 1/1,412 in control genotypes). At right, bar graphs show the minimum ISIs during dynamic and static phases. Minimum ISIs were longer in Atoh1CKO than control mice for both phases, indicating a loss of high-frequency firing during dynamic stimuli and static displacement (**P < 0.02, ***P < 0.01; Student’s t-test). Mann–Whitney tests indicated that median values were also significantly different (P < 0.001). b, Histogram of ISI distribution for Piezo2CKO (Mean ± s.d., 41.9 ± 32.3 ms, median: 23.4 ms; n = 792 intervals from N = 6 units) and control genotypes (mean ± s.d., 13.9 ± 1.4 ms, median: 11.8 ms; n = 1,845 intervals from n = 5 units). Main histograms excluded long intervals (>150 ms; 4/792 intervals in Piezo2CKO and 2/1,845 in control mice.) Minimum ISIs were not significantly different in the dynamic phase (P ≥ 0.76; Student’s t-test and Mann–Whitney test); indicating that high-frequency firing is preserved in touch-dome afferents in these mice. For static phase firing, the means were not significantly different (P = 0.095; Student’s t-test); however, non-parametric analysis indicated that medians differed between genotypes (P = 0.0043; Mann–Whitney test).

Extended Data Table 1 Properties of mechanically and light-evoked currents in Merkel cells
Extended Data Table 2 Summary of touch-dome responses from Atoh1CKO and control mice

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Maksimovic, S., Nakatani, M., Baba, Y. et al. Epidermal Merkel cells are mechanosensory cells that tune mammalian touch receptors. Nature 509, 617–621 (2014). https://doi.org/10.1038/nature13250

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