Supplemental Figure S1. Generation of MrgprB4 knockout mice. (a) UPPER diagram

illustrates the targeting construct; LOWER the genomic structure of the wild-type MrgprB4 locus.

Following homologous recombination (dashed lines), the entire open reading frame (ORF) of

MrgprB4 (large arrow) was replaced by PLAP-pGK-Hyg-loxP. (b) Genomic DNA from wild-type

and MrgprΒ4� PLAP/+ heterozygous mice were digested with the restriction enzyme SphI and probed

with a 32P-labeled 5’ probe. The correctly sized bands (7.1 kb for the wild type allele and 6.4 kb for

the mutant allele) on the Southern blot confirmed the homologous recombination event. The

expression of PLAP in MrgprΒ4� PLAP/+ heterozygous mice was coincident with that of endogenous

MrgprB4 mRNA, as determined by double-label in situ hybridization (Q. Ma, X. D. and D.J.A.,

unpublished). Homozygous MrgprΒ4� PLAP/PLAP mice were viable and fertile, and did not show any

obvious phenotypic abnormalities.

Supplemental Figure S2. Further characterization of MrgprB4+ neurons. (a-c)

Developmental onset of PLAP expression in thoracic DRG at postnatal days 1, 4 and 7. (d-l)

Double-labeling for PLAP (green) and the indicated markers (red) in MrgprB4� PLAP/+ heterozygous

mice. MrgprB4+ neurons co-express IB4 (e, f, arrowheads) and the GDNF co-receptor c-RET (h,

i arrowheads), but not NF200, a marker of myelinated sensory neurons (j-l). (m-o) MrgprB4+

neurons innervate hairy but not glabrous skin. Sections through the glabrous plantar skin of the

hindpaw (m) and hairy skin (n) followed by PLAP histochemistry reveals MrgprB4+ fibers

exlusively in hairy skin. (o) L4 DRG from a heterozygous MrgprB4�PLAP adult mouse, stained for

MrgprB4 (PLAP) after DiI retrograde tracing from the plantar surface of the hind-paw. MrgprB4+

neurons (green) do not overlap with DiI labeled neurons (red).

Supplemental Figure S3. The innervation pattern of MrgprB4 expressing neurons in the

hindlimb. (a) Hindlimb skin from heterozygous MrgprB4�PLAP adult mice stained by whole-mount

PLAP histochemistry. Glabrous skin is outlined by the green line, while hairy skin is outlined by

the red line. The dashed lines indicate the most distal edges of hind paw. The arrowheads point to

MrgprB4+ fibers (blue patches) on the dorsal surface of the distal hindpaw. No epidermal

innervation by MrgprB4+ fibers is found in the glabrous skin. (b) Higher magnification view of

the MrgprB4+ innervation patches indicated by the arrowheads in (A). Note that the innervation

pattern of MrgprB4-PLAP fibers was indistinguishable in MrgprB4� PLAP/+ heterozygous and

MrgprΒ4� PLAP/PLAP homozygous mice, arguing that MrgprB4 does not play an essential role in axon

guidance.

Supplemental Figure S4. MrgprB4 axons terminate in lamina IIo of the dorsal spinal cord,

between the laminae defined by CGRP and PKCγ labeling. Confocal images of thoracic (a and b)

and lumbar (L4-L6; c and d) regions of adult spinal cord from heterozygous MrgprB4�PLAP mice. (a

and c) MrgprB4+ axons centrally project to lamina IIo, ventral to the CGRP+ lamina. (b and d)

MrgprB4+ axons project to lamina IIo, dorsal to the PKCγ+ lamina (IIi). Unlike in thoracic regions

(a and b), MrgprB4+ projection within L4-L6 region is restricted to the lateral part of the dorsal

horn (c and d), while CGRP+ and PKCγ+ laminae occupy the entire medio-lateral extent of the

spinal cord. Panels at the bottom are higher magnification views of boxed regions in (c) and (d),

shown as single and merged color channels.

Note 1: Consistent with our findings in the mouse, the necks of hair follicles in the rat are also

innervated with IB4+ fibers that are nonpeptidergic, which may be the MrgprB4 equivalent in that

species1. However, the necks of the rat follicles also have IB4+ peptidergic fibers, which may

express a different Mrgprd family member. Still other IB4+ fibers innervate the arteries and the

inner conical body of follicle-sinus complexes (FSCs), and contribute to the pilo-neural complexes

of guard hairs. None of the fibers contributing to these locations in the mouse express either

MrgprD or MrgprB4. Although the percentage of MrgprB4 neurons is very small, the fur may be

supplied by at least 20 different varieties of sensory neurons, of which a large percentage, like

MrgprD neurons, may terminate exclusively in the epidermis. The observation that MrgprB4+

neurons correspond to ~5% of IB4+ neurons is, therefore, consistent with this estimate.

REFERENCES

1. Fundin, B. T. et al. J Comp Neurol 385, 185-206 (1997).

Supplemental Table S1: Axonal Projections in MrgprB4PLAP/+ Knockout Mice

SYSTEM Tissue MrgprB4 projections

Peripheral Nervous System

Dorsal root ganglia + Trigeminal ganglia +

Sympathetic ganglia - Enteric neurons -

Central Nervous System

Spinal cord + Brain Spinal Trigeminal Nucleus* +

Amygdala - Brainstem-nucleus caudalis - Brainstem—all other areas - Cerebellum - Cerebral cortex - Hippocampus - Hypothalamus - Meninges - Olfactory bulb - Septum - Striatum - Thalamus -

Skin

Glabrous skin - Hairy skin +

Circular follicle neck endings# + Penetrating follicle neck endings# - Circumferential free nerve endings# - Lanceolate/club endings - Merkel cells and endings - Meissner’s corpuscles -

Blood vessels/vascular endings - Sweat glands -

Digestive System

Tongue - Esophagus - Liver - Stomach - Pancreas - Small intestine - Colon -

Respiratory system

Trachea - Lung -

Urinary system

Kidney - Bladder -

Reproductive system

Prostate gland - Testis - Skin of the genitalia -

Circulatory system

Heart - Blood vessel -

Others

Eye-cornea - Eye-retina - Skeletal muscle - Spleen -

* The brain region that receives central projections from the trigeminal ganglia # Detailed description in Zylka et al, Neuron 45, 17-25 (2005)

SUPPLEMENTAL INFORMATION: MATERIALS AND METHODS and

DISCUSSION

Materials and Methods

Molecular Biology

The genomic sequence of mouse MrgprB4 was obtained from the Mouse Genomic Project

(NCBI). The arms of the MrgprB4 targeting constructs were obtained by PCR-amplification from

a 129/SvJ genomic DNA using Expand High Fidelity PCR System (Roche). The PCR primer

sequences for the 5’ arm are 5’-CAGGCGCGCCTGCTTAGGAATTTTCCACTGG-3’ and 5’-

CTGTACACCATAGTCTCTAGAAAGG-3’. The PCR primer sequences for the 3’ arm are 5’-

CAGGCGCGCCAGTAGTTGAGTGAGTCCCTGG-3’ and 5’-

CAGTTTAAACGATTTACCTGCAAACCTCCTG-3’. The lengths of 5’ and 3’ arms are 4.3 and

3.0 kb, respectively. The entire open reading frame of MrgprB4, which is encoded by a single

exon, was replaced with a genomic fragment of human placental alkaline phosphatase (PLAP).

The PLAP coding sequence was ligated in-frame to NcoI site at the endogenous ATG start codon

of the MrgprB4 coding sequence. Following PLAP is a single loxP site and pGK-hygromycin

resistance gene. Homologous recombination was performed in mouse CJ7 embryonic stem (ES)

cells following standard procedures. 200 �g/ml of hygromycin B (Invitrogen) was used for the

positive selection of homologous recombination at the MrgprB4 locus. Correctly targeted ES cell

clones were identified by PCR the genomic DNA of the clones using primer sets flanking the 5’

and 3’ arms of the targeting construct and were further confirmed by� Southern Blot hybridization

using probe that flanked the 5’ arms of the targeting construct. Chimeric MrgprB4-PLAP mice

were produced by blastocyst injection of positive ES cells. MrgprB4-PLAP heterozygous mice

were generated by mating the chimeric mice to C57Bl/6 mice.

Immunofluorescence

Adult mice (8-12 weeks old) were anesthetized with pentobarbital and perfused with 20 ml 0.1

M phosphate buffer solution (PBS; pH 7.4; 4oC) followed with 25 ml 4% paraformaldehyde in

PBS (4oC). Spinal cord, dorsal root ganglia (DRG) and peripheral tissues (including mystacial

pad) were dissected from the perfused mice. DRG was postfixed in 4% paraformaldehyde at 4oC

for 30 min, and other tissues were fixed for 2 hr.

Body hairy skin and paw glabrous skin were dissected from nonperfused mice and fixed in 2%

paraformaldehyde at 4oC overnight.

Newborn mice were anesthetized in ice cold water until motionless. DRG were dissected

freshly and postfixed in 4% paraformaldehyde for 30 min.

All tissues were cryoprotected in 20% sucrose in PBS at 4oC for 24 hr, and frozen in OCT at –

80oC. Tissues were sectioned at 20 �m with a cryostat. The sections on slides were dried at 37oC

for 30 min, and fixed with 4% paraformaldehyde at room temperature for 10 min. The slides were

washed with PBS containing 0.1% Triton X-100 (PBT) for 3 times and blocked with 10% goat

serum in PBT for 30 min. All sections were incubated with primary antibodies diluted in blocking

solution at 4oC for overnight. The primary antibodies used: mouse anti-human PLAP (clone 8B6;

DAKO; 1:200), rabbit anti-CGRP (T-4239; Peninsula; 1:1000), rabbit anti-NF200 (AB1982;

Chemicon; 1:1000), rabbit anti-P2X3 (AB5895; Chemicon; 1:1000), Armenian hamster anti-c-

RET (Lo and Anderson, 1995) 1:4, rabbit anti-Trpv1 (PC420; Oncogene Research Products;

1:200), rabbit anti-PKCγ (sc-211; Santa Cruz Biotechnology; 1:1000), rabbit anti-GFP (A-11122;

Molecular Probes; 1:1000), Mouse anti-Neuronal nuclei (MAB377; Chemicon; 1:300). Sections

were washed with PBT, and incubated with secondary antibodies at room temperature for 1 hr. The

secondary antibodies used: goat anti-mouse IgG2a (A21134, Alexa 568 conjugated; A21131,

Alexa 488 conjugated; Molecular Probes); goat anti-rabbit (A11011, Alexa 568 conjugated;

A11008, Alexa 488 conjugated; Molecular Probes); goat anti-Armenian hamster (127-165-160,

cy3 conjugated; Jackson Lab); goat anti-mouse IgG1 (A21124, Alexa 568 conjugated; Molecular

Probes). All secondary antibodies were 1:500 diluted in blocking solution. To detected IB4

binging, sections were incubated with 1:200 diluted Griffonia simplicifolia isolectin GS-IB4-Alexa

568 (I-21412; Molecular Probes) during secondary antibody incubations. Sections were washed

with PBT and mounted with glycerol. Images were obtained using Zeiss LSM510 confocal

microscope system.

Quantification

Confocal images from heterozygous and homozygous mice were counted to quantify the

percentage of MrgprB4+ sensory neurons that coexpress a given nociceptive marker. Paired

student’s T test was performed between genotypes.

PLAP histochemistry

Tissues were dissected from perfused adult mice. DRG from newborn mice that were

anesthetized in ice-cold water until motionless, were dissected freshly and postfixed in 4%

paraformaldehyde at 4oC for 30 min.

Tissues were sectioned as described before. Sections were dried at 37oC for 30 min and fixed

in 4% paraformaldehyde in PBS at room temperature for 10 min. Sections were washed with

Hank’s balanced salt solution (HBSS), and incubated in HBSS at 65-68oC for 2 hr. Then sections

were washed 3 times with B1 buffer (0.1 M Tris pH 7.5, 0.15 M NaCl), and subsequently 3 times

with B3 buffer (0.1 M Tris pH 9.5, 0.1 M NaCl, 50 mM MgCl2, 1-5 mM levamisole added

freshly). PLAP activity was then visualized by incubation with 37.5 �g/ml nitro blue tetrazolium,

175 �g/ml –bromo-4-chloro-3-indolyl phosphate in B3 buffer at room temperature until signals

appeared.

PLAP whole mount histochemistry staining

DRG dissected from perfused adult mice and postfixed in 4% paraformaldehyde at 4oC for 30

min. For the hairy skin whole mount staining, the hairy skin was shaved and treated with

commercial Hair Remover to remove the hair. Tape stripping was performed to completely remove

stratum corneum until the skin surface appeared to be glistening. Skin was fixed in 4%

paraformaldehyde at 4oC for 2 hr. Tissues were washed with Hank’s balanced salt solution

(HBSS), and incubated in HBSS at 65-68oC for 2 hr. Then tissues were washed 3 times with B1

buffer (0.1 M Tris pH 7.5, 0.15 M NaCl), and subsequently 3 times with B3 buffer (0.1 M Tris

pH 9.5, 0.1 M NaCl, 50 mM MgCl2, 1-5mM levamisole added freshly). PLAP activity was then

visualized by incubation with 37.5 �g/ml nitro blue tetrazolium, 175 �g/ml –bromo-4-chloro-3-

indolyl phosphate in B3 buffer at room temperature until signals appeared. The tissues were fixed

in 4% paraformaldehyde at 4oC overnight. DRG was cleared in glycerol at 4oC overnight. Skin

was dehydrated with 50%, 75%, 100% ethanol (1 hr at each step), cleared in BABB (Benzyl

Alcohol, sigma Aldrich 402834; Benzyl Benzoate, sigma B-6630; 1:2 mixed together) for 5min-

2hr and mounted in Permount (Fisher).

Whole mount fluorescent immunostaining

Hair was removed from the skin with commercial Hair Remover. Fat and most of the

connective tissue were separated using scissors. The skin was cut into pieces and fixed in 4%

paraformaldehyde at 4oC for 2 hr and washed 3 times with PBS. Skin sheets were treated with

0.5% Triton X-100 in PBS at room temperature for 1 hr, and then blocked with 10% goat serum in

PBT (PBS containing 0.1% Triton X-100) for 1 hr. Sheets were incubated with the primary

antibody at 4oC overnight. The sheets were washed for 2 hr in PBT, and then incubated with the

secondary antibody for 2 hr in blocking solution. After incubation, the sheets were washed with

PBT, mounted in glycerol, and visualized by confocal microscopy.

Electron Microscopy

Homozygous MrgprB4�PLAP mice were anesthetized and perfused with 4% formaldehyde,

0.05% glutaraldehyde, 0.1 M phosphate buffer (pH 7.4). Thoracic nerves were removed, postfixed

for 2 hr at 4°C, and then washed with 0.1 M phosphate buffer [pH 7.4]). The nerves were heated

at 65°C for 2 hr to inactivate endogenous phosphatases, then PLAP activity was visualized by

precipitating electron-dense cerium phosphate, based on the “oCPP” method (Zylka et al., 2005).

After oxidation of Ce(III) to Ce(IV), the nerves were postfixed in 2% glutaraldehyde followed by

osmium tetroxide (1% OsO4, 0.1 M cacodylate, 1.8 mM CaCl2, 0.8 mM MgSO4 [pH 7.4]),

counterstained en bloc with 1% uranyl acetate and 0.4% lead citrate, and embedded in Epon.

Transverse ultrathin sections were cut, mounted on formvar-coated slotted grids, and imaged using

a Philips 420 transmission electron microscope.

Supplemental Discussion

Low-threshold C-fiber tactile afferents have been electrophysiologically characterized in

numerous mammalian species1-7. Several lines of evidence suggest that MrgprB4 may mark such

CT afferents. First, the small diameter and unmyelinated axons of MrgprB4+ neurons support their

classification as C-fibers. Second, like CT low-threshold mechanoreceptors studied in other

rodents8,9, MrgprB4+ neurons express neither CGRP nor the capsaicin-sensitive channel TrpV1.

Third, MrgprB4+ peripheral fibers project exclusively to hairy skin, where CT afferents are

present10-12. Fourth, the size (~0.3-1 mm2) and scattered distribution of MrgprB4+ arborizations in

murine skin are strikingly similar to the size and distribution of touch-sensitive spots within human

CT receptive fields as determined by microneurography (cf. Fig. 2g, h and j-l); moreover the

number of terminals per MrgprB4+ neuron (~1-3) is within the range of that reported for human

CT afferents13. Fifth, the distribution of MrgprB4+ terminals in limb skin is proximally biased, and

it is thought that CT afferents are less frequently encountered in distal limb11, consistent with data

from primates14. CT low threshold afferents, like MrgprB4+ fibers, are also known to be more

abundant in ear skin than on the lower limb15. Finally, MrgprB4+ central afferents project to lamina

II, where CT afferents are known to terminate15-17. Lamina II has been reported to engage dorsal

horn projections to forebrain limbic structures in rodents18, consistent with the finding that, in

humans, CT afferents activate brain regions involved in emotion12.

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2. Douglas, W. W. & Ritchie, J. M. Nonmedullated fibres in the saphenous nerve which signal touch. J Physiol 139, 385-99 (1957).

3. Lynn, B. & Carpenter, S. E. Primary afferent units from the hairy skin of the rat hind limb. Brain Res 238, 29-43 (1982).

4. Shea, V. K. & Perl, E. R. Sensory receptors with unmyelinated (C) fibers innervating the skin of the rabbit's ear. J Neurophysiol 54, 491-501 (1985).

5. Bessou, P. & Perl, E. R. Response of cutaneous sensory units with unmyelinated fibers to noxious stimuli. J Neurophysiol 32, 1025-43 (1969).

6. Iggo, A. Cutaneous mechanoreceptors with afferent C fibres. J Physiol 152, 337-53 (1960). 7. Iggo, A. & Kornhuber, H. H. A quantitative study of C-mechanoreceptors in hairy skin of the

cat. J Physiol 271, 549-65 (1977). 8. Lawson, S. N., Crepps, B. & Perl, E. R. Calcitonin gene-related peptide immunoreactivity and

afferent receptive properties of dorsal root ganglion neurones in guinea-pigs. J Physiol 540, 989-1002 (2002).

9. Seno, N. & Dray, A. Capsaicin-induced activation of fine afferent fibres from rat skin in vitro. Neuroscience 55, 563-9 (1993).

10. Vallbo, A., Olausson, H., Wessberg, J. & Norrsell, U. A system of unmyelinated afferents for innocuous mechanoreception in the human skin. Brain Res 628, 301-4 (1993).

11. Vallbo, A. B., Olausson, H. & Wessberg, J. Unmyelinated afferents constitute a second system coding tactile stimuli of the human hairy skin. J Neurophysiol 81, 2753-63 (1999).

12. Olausson, H. et al. Unmyelinated tactile afferents signal touch and project to insular cortex. Nat Neurosci 5, 900-4 (2002).

13. Wessberg, J., Olausson, H., Fernstrom, K. W. & Vallbo, A. B. Receptive field properties of unmyelinated tactile afferents in the human skin. J Neurophysiol 89, 1567-75 (2003).

14. Kumazawa, T. & Perl, E. R. Primate cutaneous sensory units with unmyelinated (C) afferent fibers. J Neurophysiol 40, 1325-38 (1977).

15. Sugiura, Y., Lee, C. L. & Perl, E. R. Central projections of identified, unmyelinated (C) afferent fibers innervating mammalian skin. Science 234, 358-61 (1986).

16. Kumazawa, T. & Perl, E. R. Primate cutaneous receptors with unmyelinated (C) fibres and their projection to the substantia gelatinosa. J Physiol (Paris) 73, 287-304 (1977).

17. Light, A. R., Trevino, D. L. & Perl, E. R. Morphological features of functionally defined neurons in the marginal zone and substantia gelatinosa of the spinal dorsal horn. J Comp Neurol 186, 151-71 (1979).

18. Braz, J. M., Nassar, M. A., Wood, J. N. & Basbaum, A. I. Parallel "pain" pathways arise from subpopulations of primary afferent nociceptor. Neuron 47, 787-93 (2005).