Supplementary Figure 1

Characterization of mechanically activated currents and Piezo2 expression in proprioceptive neurons.

a, Pvalb immunostaining with tdTomato epifluorescence in lumbar DRG from adult Pvalb-Cre;Ai9 mice. Scale bar: 100 μm. b, Current (I)-voltage (V) relationship of one mechanically activated IA current found in tdTomato+ neurons from adult Pvalb-Cre;Ai9 DRG. Voltage clamp experiments conducted identically to those depicted in Fig. 1b and c. Mechanically activated current responses to threshold plus 4-5 µm were used for I-V measurements.

Nature Neuroscience: doi:10.1038/nn.4162

Supplementary Figure 2

Characterization of GFP expression in Piezo2GFP lumbar DRG and skeletal muscles from hind leg.

a, Immunofluorescence for GFP and Pvalb in adult Piezo2GFP lumbar DRG. Circles mark cell bodies that co-express GFP and Pvalb. b, Immunofluorescence for GFP and Vglut1 in MSs and GTOs in various skeletalmuscles from P8 Piezo2GFP hind leg. c, Immunofluorescence for GFP and αBTX (a marker of neuromuscularjunction) in P6 Piezo2GFP hind leg muscle. Scale bars: a, 100 μm; b, 50 μm; c, 20 μm.

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Supplementary Figure 3

Characterization of HoxB8-Cre;Ai9 DRG, skeletal muscles, and dorsal skin.

a, Based on caudal bias in the HoxB8-Cre activity, we assessed tdTomato epifluorescence in cervical/upperthoracic (left), lower thoracic (middle), and lumbar (right) DRG. tdTomato expression was sparse in neuronalcell bodies of cervical/upper thoracic (Th1-3) DRG stained with Piezo2 antibody (left). Strong tdTomatoexpression was detected in a majority of neuronal cell bodies in lower thoracic (middle) and lumbar (right)DRG stained with Piezo2 and Pvalb antibodies. White arrowheads mark tdTomato cell bodies that express Piezo2 and Pvalb. This indicates that 1.5% (12/806) of total DRG neurons do not express HoxB8-Cre. b, tdTomato epifluorescence with Vglut1 immunostaining in proprioceptor endings in forelimb (left) and hindlimb (right) muscles of adult HoxB8-Cre;Ai9 reporter. tdTomato was detected in MS endings and skeletal

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muscles of hind limbs, while it was absent in forelimb muscles and associated MSs. c, tdTomato epifluorescence with Nefh and DAPI staining in lanceolate endings (left, hair follicle) and with Nefh staining in Merkel cell-neurite complexes (right, touch dome) in adult HoxB8-Cre;Ai9 dorsal skin. Dotted line on left indicates the hair follicle, and the bracket marks lanceolate endings. Dotted circles on right mark Merkel cells, and the dotted line marks the epidermal-dermal junction. HS, hair shaft; TD, touch dome; GH, guard hair. Scalebars: a, 100 μm; b, 50 μm; c, 20 μm.

Nature Neuroscience: doi:10.1038/nn.4162

Supplementary Figure 4

Characterization of Pvalb-Cre;Piezo2cKO mice.

a, b, Piezo2 (green puncta) and Pvalb (red puncta) dual in situ hybridization in lumbar DRG of adult Piezo2fl/+

(WT, a) and Pvalb-Cre;Piezo2cKO mice (b). Left panels show entire DRG. Middle panels are the magnifiedview of dotted insets in the left panels. Smaller panels on right show the magnified view of the area markedwith orange lines. White arrowheads in WT DRG (a) indicate Pvalb+ cell bodies with intact Piezo2. White circles in cKO DRG (b) indicate Pvalb+ cell bodies without Piezo2.White asterisk indicates one Pvalb+ cell

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body with intact Piezo2. 11.2% of Pvalb+ DRG neurons (10/89) still retained Piezo2 in Pvalb-Cre;Piezo2cKO

mice. c, Quantification of MSs found in EDL (extensor digitorum longus) and soleus muscles from P10-11 WT and Pvalb-Cre;Piezo2cKO mice. Bars represent mean ± s.e.m. ns, not statistically significant. Unpaired t-test with Welch’s correction. d, Example images of Vglut1-stained MSs found in P30 WT and Pvalb-Cre;Piezo2cKO

mice. e, Central projection of tdTomato+ proprioceptors in lumbar spinal cord of P5 Pvalb-Cre;Ai9 (WT) and Pvalb-Cre;Ai9;Piezo2cKO mice. Scale bars: a, b, e, 100 μm; d, 20 μm.

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Supplementary Figure 5

Characterization of HoxB8-Cre;Piezo2cKO mice.

a, Piezo2 remaining (%) in cervical/upper thoracic (left) and lumbar (right) DRG from adult Piezo2fl/+ (WT) and HoxB8-Cre;Piezo2cKO mice by qRT-PCR. Note that HoxB8-Cre;Piezo2cKO mice refer to HoxB8-Cre;Piezo2fl/– (only one allele of Piezo2 is present prior to Cre recombination). Bars represent mean ± s.e.m. **P < 0.01; ****P < 0.0001. Unpaired t-test with Welch’s correction. b, Piezo2 (green puncta) in situ hybridization in lumbar DRG of adult Piezo2fl/+ (WT, top) and HoxB8-Cre;Piezo2cKO mice (bottom). Left panels show entire DRG outlined by solid lines. Right panels show the magnified view of dotted insets in the left panels. White asterisk (bottom, right) indicates one cell body with intact Piezo2. 1.2% of lumbar DRG neurons (2/165) still retained Piezo2 in HoxB8-Cre;Piezo2cKO mice, and this likely suggests that 1.2% of lumbar DRG neurons do not express HoxB8-Cre. c, Central projection of proprioceptors via Pvalb staining in lumbar spinal cord of P5 Piezo2fl/+ (WT) and HoxB8-Cre;Piezo2cKO mice. Scale bars: b, c, 100 μm.

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Supplementary Figure 6

Maximal current responses observed in tdTomato+ neurons from adult Pvalb-Cre;Ai9 (WT) and Pvalb-Cre;Ai9;Piezo2cKO DRG.

Whole-cell voltage-clamp experiments from a holding potential of –60 mV. Mechanical stimulation was applied as described in Figures. 1 and 3. a, Individual data plot of maximal current densities of RA current responses. b, Individual data plot of maximal current densities of IA current responses.

Nature Neuroscience: doi:10.1038/nn.4162

Supplementary Figure 7

Activation of putative group III/IV acid-sensitive sensory neurons by pH 6.0 lactic acid in adult WT andPiezo2 cKO muscles.

Note that both Pvalb-Cre;Piezo2cKO and HoxB8-Cre;Piezo2cKO lines responded similarly. a, A typical WT response to pH 6.0 lactic acid with baseline firing activity. b, Lactic acid response in Piezo2 cKO mouse with no baseline firing activity.

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AP current threshold (pA)

PvalbCre;Ai9 (WT)

Resting potential (mV)

PvalbCre;Ai9;Piezo2cKO

AP voltagethreshold (mV)

Input resistance (MΩ)

-62.83 ± 0.93 -35.61 ± 1.01 0.728 ± 0.05 78.2 ± 16.82 741.66 ± 202.68

-61.31 ± 0.98 -29.48 ± 2.88 0.922 ± 0.074 182.89 ± 23.09 411.25 ± 86.68

AP halfwidth (ms)

Supplementary Table 1. Electrical properties of cultured tdTomato+ DRG neurons from PvalbCre;Ai9 (WT) and PvalbCre;Ai9;Piezo2cKO mice.

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PvalbCre;Piezo2cKO

65 ± 8 62 ± 4

24.6 ± 1.9 21.8 ± 2.9

Measurement

Age (days)

Body weight (g)

Muscle weight (mg)

Lo (mm)

Muscle cross sectional area (cm2)

Maximal tetanic contractile force (N/cm2)

10.2 ± 0.7 10.1 ± 0.5

11.8 ± 0.7 11.9 ± 0.8

8.2 ± 0.4 8.4 ± 0.8

17.4 ± 3.5 16.4 ± 4.5

Piezo2fl/+ (WT) HoxB8Cre;Piezo2cKO

88 ± 11 80 ± 12

25.6 ± 1.2 23.1 ± 0.8*

Measurement

Age (days)

Body weight (g)

Muscle weight (mg)

Lo (mm)

Muscle cross sectional area (cm2)

Maximal tetanic contractile force (N/cm2)

9.5 ± 1.1 10.7 ± 0.8*

12.2 ± 0.8 11.3 ± 1.2

7.4 ± 1.2 9.0 ± 1.2*

18.9 ± 7.8 18.6 ± 6.3

a

b

Piezo2fl/+ (WT)

Supplementary Table 2. Properties of PvalbCre;Piezo2cKO and HoxB8Cre;Piezo2cKO mice used for ex vivo muscle-nerve recordings.a, No values are significantly different between groups with Independent t-test. Mouse numbers = 4 PvalbCre;Piezo2cKO and 3 WT animals; Muscle numbers = 8 PvalbCre;Piezo2cKO and 5 WT muscles. b, * denotes p < 0.05 from WT from Independent t-test. Mouse numbers = 4 HoxB8Cre;Piezo2cKO and 5 WT animals; Muscle numbers = 8 HoxB8Cre;Piezo2cKO and 10 WT muscles.

Nature Neuroscience: doi:10.1038/nn.4162