SUPPLEMENTARY INFORMATION

Transcription

1 doi: 1.138/nture9142 BglII EoRI EoRV Chr. 17 p2 p1 p7 p3 p4 p5 p k 9 k 3 k p1 p2 p3 p4 p5 p6 p7 p p p p4 96 p5 p p mino id d p1 p2 p3 p4 p5 p6 p7 nuleotide Figure S1. Mouse V2Rp gene fmily () Southern lot nlysis of mouse V2Rp genes. Genomi DNA ws digested with BglII, EoRI nd EoRV, nd hyridized with pn-v2rp DNA proe. () Genomi orgniztion of the mouse V2Rp genes lustered on hromosome 17. Blue oxes represent individul V2Rp genes nd red rrows indite the orienttion of trnsription. () Nuleotide nd mino id sequene identities mong the V2Rp fmily of genes. (d) RT-PCR nlysis of V2Rp mrna expression in the mouse VNO. High levels of V2Rp2, 4 nd 5 mrnas nd low levels of V2Rp1 nd 6 mrnas re expressed in the VNO, while V2Rp3 nd 7 mrnas were not detetle. 1

2 doi: 1.138/nture9142 V2Rp5 mrna V2Rp5 protein merge Figure S2. Chrteriztion of the nti-v2rp5 ntiody () Immunostining of VNO setion leled with nti-v2rp5 polylonl ntiody. The nti-v2rp5 polylonl ntiody ws rised ginst syntheti peptide orresponding to the C-terminl mino-id sequene of the mouse V2Rp5 protein. The dendrites nd som of smll suset of VSNs were strongly leled with the V2Rp5 ntiody. The lower pnel shows the enlrged view of the re indited y the dshed retngle in the upper pnel. Sle r, 1 µm. () Two-olor fluoresent stining of V2Rp5 mrna (mgent) nd V2Rp5 protein (green). All of the V2Rp5-immunoretive VSNs were lso positive for V2Rp5 mrna nd vie vers. Sle r, 1 µm 2

3 doi: 1.138/nture9142 V2Rp5 protein -Fos protein merge ESP % of -Fos / V2Rp5 1 VNO rostrl udl Figure S3. Ativtion of V2Rp5-expressing VSNs y, ut not ESP4, nd the grdul derese in -indued -Fos-positive ells long the rostro-udl xis of the VNO () Doule-immunostining of VNO setions olleted from - nd ESP4-stimulted mie leled with nti-v2rp5 (green) nd nti--fos (gry) ntiodies. Fos-expressing VSNs re indited y the yellow rrowheds. All -indued, -Fos-positive neurons were leled with V2Rp5 ntiody, while ESP4- indued, -Fos-positive neurons were not leled. Sle r, 2 µm. () Perentge of -indued -Fos-positive VSNs per V2Rp5-expressing VSNs in the representtive VNO setions (upper pnel) loted t the lue dshed lines in the lower pnel (n=3). The perentge of - Fos-expressing VSNs mong V2Rp5-positive VSNs vried signifintly long the rostro-udl xis of the VNO, nd rnged from 75% in the rostrl setions to 33% in the udl setions. The grdul derese towrds the udl region of the VNO is most likely due to the onentrtion grdient of tken up into the vomeronsl vity y vsulr pumping mehnisms, resulting in n verge of 5% expression. Error rs represent SEM. Sle r, 5 µm. 3

4 doi: 1.138/nture9142 IRES-tWGA/DsRed V2Rp5 ATG 15 k TAA 17 k 12 k DsRed V2Rp5 merge DsRed V2Rp5 merge d Figure S4. DsRed fluoresene in the VNO of V2Rp5-DsRed mie () Shemti digrm illustrting the struture of the V2Rp5-DsRed BAC trnsgene. The V2Rp5 gene onsists of six exons (gry oxes) nd five introns. The IRES-tWGA/DsRed (red ox) ssette ws inserted immeditely downstrem of the V2Rp5 stop odon. () Whole-mount lterl view of the VNO from V2Rp5-DsRed mouse. Bright DsRed fluoresene ws oserved in VSNs expressing twga/dsred. We otined two BAC trnsgeni mouse lines (V2Rp5DsRed lines #1 nd #2) tht showed strong DsRed fluoresene in smll popultion of VSNs. Sle r, 5 µm. () Coronl setion of the VNO from V2Rp5-DsRed mouse. Mgent nd green represent DsRed fluoresene nd V2Rp5 immunoretivity, respetively. All of the DsRed-positive VSNs displyed V2Rp5 immunoretivity, suggesting tht V2Rp5 protein ws expressed in onjuntion with DsRed from the trnsgeni llele. Among the V2Rp5-positive VSNs, pproximtely the sme numer of DsRedpositive nd DsRed-negtive VSNs were oserved in oth lines (line #1, 51%; #2, 57%), whih likely reflets V2Rp5 expression from the trnsgeni nd endogenous lleles. Sle r, 1 µm. (d) Higher mgnifition of the re outlined in the dshed retngle of (). Three DsRed-fluoresent VSNs with V2Rp5 immunoretivity were oserved, while one V2Rp5-immunoretive VSN ws devoid of DeRed fluoresene. Sle r, 25 µm. 4

5 doi: 1.138/nture9142 (1) (2) (3) (4) 1 k V2Rp5 +/+ V2Rp5 -/- +/+ +/- -/- V2Rp5 exon ires-gp-venus lox p self-ex Neo DT-A 1.8 k 1.2 k Figure S5. Genertion of V2Rp5 knokout mie () Shemti digrms illustrting the struture of the trgeting vetor for V2Rp5 (1), the orgniztion of the V2Rp5 gene lous (2), the trgeted V2Rp5 lous fter homologous reomintion in n ES ell where tndem ssette onsisting of n ires-gp-venus nd self-ex Neo ws inserted into exon 6 (3), nd the V2Rp5 knok-out lous fter self-exision of the neomyin ssette in the germ line (4). DT-A, diphtheri toxin A suunit; Self-ex Neo, self-exision neomyin-resistnt gene. () PCR genotyping of wild-type (+/+), heterozygous (+/-) nd homozygous (-/-) V2Rp5-defiient mie. The 1.8-k nd 1.2-k nds represent the PCR produts from the V2Rp5 knokout nd V2Rp5 intt llele, respetively. () V2Rp5 immunostining of VNO setions from V2Rp5+/+ nd V2Rp5-/- mie. V2Rp5-positive VSNs ompletely disppered in the V2Rp5-defiient mie. Sle r, 1 µm. 5

6 doi: 1.138/nture9142 TRPC2+/- TRPC2-/- -Fos : V2Rp5 -Fos V2Rp5-Red; TRPC2+/+ V2Rp5-Red; TRPC2-/- DsRed V2Rp5 -Fos merge DsRed V2Rp5 -Fos merge V2Rp5-Red; TRPC2+/+ pil rostrl udl sl V2Rp5-Red; TRPC2-/- d Numer of Mount mount lordosis intromission TRPC2+/+ TRPC2-/ Perent of Lordosis Perent of Intromission Figure S6. -indued neurl tivtion nd sexul ehvior requires TRPC2 () Doule immunostining of VNO setions from -stimulted TRPC2+/- nd TRPC2-/-. Gry nd green represent the VSNs leled with nti--fos ntiody nd nti-v2rp5 ntiody, respetively. -Fospositive VSNs disppered in the -stimulted TRPC2-/- mie. The perentge of -Fos-positive VSNs mong the V2Rp5-expressing VSNs were 9% (13/15) in TRPC2+/- nd % (/33) in TRPC2-/-. Sle r, 1 µm. () Triple leling of VNO setions from -stimulted V2Rp5-DsRed; TRPC2+/+ nd V2Rp5-DsRed; TRPC2-/- mie. Mgent, green nd gry represent the VSNs leled with DsRed fluoresene, nti-v2rp5 ntiody nd nti--fos-ntiody, respetively. -indued, -Fos-positive VSNs re sent in the BAC trnsgeni V2Rp5- nd DsRed-expressing VSNs on the TRPC2-defiient kground. The perentges of - Fos-positive VSNs mong the DsRed-fluoresent VSNs were 16% (7/44) in V2Rp5-DsRed;TRPC2+/+ nd % (/94) in V2Rp5-DsRed;TRPC2-/- mie. Sle r, 1 µm. () -Fos immunostining of the sgittl setions of the AOB from V2Rp5-DsRed; TRPC2+/+ nd V2Rp5- DsRed; TRPC2-/- mie (upper pnel). The lower pnel shows the high mgnifition view of the re indited y the dshed retngle in the upper pnel. Blue, green nd red represent the DAPI-stined nulei, -Fos-immunoretive neurons nd DsRed-expressing glomerulus, respetively. Sle r, 1 µm. (d) Left: Numer of mounts of the stud mle towrd -exposed TRPC2+/+ nd TRPC2 -/- femle mie (n = 3). The numer of mounts ws not ffeted y the TRPC2 genotype of the femle. Middle: Perentge of lordosis response per totl mounts of -exposed TRPC2+/+ nd TRPC2-/- femle mie (n = 3). - exposed TRPC2-/- femle mie displyed more thn 2-fold derese in lordosis ehvior ompred to -exposed TRPC2+/+ femle mie. Right: Perentge of suessful mle intromissions per totl mounts towrd -exposed TRPC2+/+ nd TRPC2-/- femle mie (n = 3). The perentge of suessful intromissions of mles towrd -exposed TRPC2-/- femle mie were lower thn tht towrd - exposed TRPC2+/+ femle mie. Error rs represent SEM. **P <.1 6

7 doi: 1.138/nture9142 -Fos -Fos : V2Rp5 pil rostrl udl sl Figure S7. -indued neurl tivtion does not require β2-mirogloulin () Doule immunostining of VNO setions from -stimulted β2-mirogloulin-/- mie. Gry nd green represent the VSNs leled with nti--fos ntiody nd nti-v2rp5 ntiody, respetively. -Fospositive VSNs were intt in the -stimulted β2-mirogloulin-/- mie. The perentge of -Fospositive VSNs mong the V2Rp5-expressing VSNs ws 28% (131/476) in β2-mirogloulin-/- mie. Sle r, 1 µm. () -Fos immunostining of the sgittl setions of the AOB from -exposed β2-mirogloulin-/- mie. -indued, -Fos-positive neurons were lso intt in the AOB of β2-mirogloulin-/- mie. Sle r, 1 µm. () The enlrged view of the re indited y the dshed retngle in (). Sle r, 5 µm. 7

8 doi: 1.138/nture9142 MOB xon undle rostrl AOB left AOB udl right AOB rin rostrl udl pil rostrl udl sl Figure S8. Glomerulr innervtion ptterns of V2Rp5-expressing VSNs () Representtive whole-mount dorsl views of the AOB from femle nd mle V2Rp5-DsRed trnsgeni mie (line #1). Mgent nd green represent the xon terminus of the DsRed-fluoresent V2Rp5- expressing VSNs nd ll VSN xons projeting to the AOB leled with fluoresein-onjugted soyen gglutinin, respetively. The DsRed-positive VSNs projeted xons onto 3-5 glomeruli in the udl zone of the AOB, nd these fluoresent glomeruli formed line long the medio-lterl xis of the AOB. MOB: min olftory ul. Sle r, 1 mm. () Higher mgnifition of the AOB from the femle indited in the re within the dshed retngles in prt (left pnel). Sle r, 5 µm. () Immunostining of sgittl setion of the AOB from V2Rp5-DsRed mouse. Mgent nd green represent DsRed-positive glomerulus nd leling with nti-gαi ntiody whih is mrker for vomeronsl xons tht innervte the rostrl zone of the AOB, respetively. DsRed-positive, V2Rp5- expressing glomeruli were lwys loted in the middle portion of the udl zone. This projetion pttern ws onsistent regrdless of trnsgeni line, sex or individul smple exmined. Sle r, 1 µm. 8

9 doi: 1.138/nture9142 mouse1 mouse2 mouse3 left right left right left right Gαi zone rostrl udl Gαo zone Figure S9. Comprtmentl loliztion of -tivted neurons in the AOB Shemti digrm of the distriution of DsRed glomeruli (red) nd -Fos-positive mitrl/tufted ells (green) in the AOB of three different V2Rp5-DsRed mie exposed to. 9

10 doi: 1.138/nture9142 -Fos-positive ells/setion ter fluid nsolriml dut VNO C57BL/6 BALB/ Mle Femle Mle Femle C57BL/6 BALB/ d nsolriml dut eye ELG BALB/ mle nose Figure S1. Strin nd sex differenes in the response of VSNs nd seretion into the ter fluids () Men numer of -Fos-positive VSNs per VNO setion from C57BL/6 nd BALB/ mie stimulted with or (n = 3). In mle nd femle C57BL/6 mie stimulted with, -Fos indution ws oserved in similr fshion. In ontrst, no -Fos indution ws oserved in the BALB/ mle mie. Error rs represent SEM. **P <.1 () Western lotting of ter fluids nd nsolriml dut nd VNO extrts otined from BALB/ or C57BL/6 mie using ffinity-purified nti- ntiody. is oserved in the ters, nsolriml dut nd VNO extrts of BALB/ mle mie, ut not of BALB/ femles or C57BL/6 mles nd femles. () immunostining of setion of the BALB/ mle nsl vity. Tringles show immunoretivesignls. Sle r, 1 mm. (d) Shemti illustrtion of the seretion pthwy from the extroritl lriml glnd (ELG) to the vomeronsl orgn (VNO) vi the nsolriml dut, suggesting tht BALB/ mle mie re onstntly exposed to their own, nd therefore, the -Fos signling pthwy in the VSNs is desensitized. 1

11 doi: 1.138/nture9142 stimulus exposure for 3 min housing in the test ge for 24 hr (+) (-) exmining ourtship for 6 min exmining ourtship for 6 min Figure S11. Shemti illustrtion of the ehviorl ssy method () Sexully nive C57BL/6 femle mie were ovrietomized (OVX) nd treted with estrogen nd progesterone to indue estrous onditions. Sujet femles were exposed to either, ESP4, mixture of ESP6/16/31 (ESP mix), mle urine or odornt-inding protein 1 (OBP1) for 3 min nd then pired with stud Sl:ICR mles nd ourtship ehvior ws exmined for 6 min. This method ws used for the experiments presented in Figure 4-, S6d, S12, S13 nd S14. () Sexully experiened BALB/ or C57BL/6 mle mie were individully housed in the test ge 24 h prior to ourtship testing in order to deposit their own seretions into the edding, nd then pired with estrogen- nd progesterone-treted OVX femle mie nd ourtship ehvior ws exmined for 6 min. This method ws used for the experiments presented in Figure 4d. 11

12 doi: 1.138/nture nogenitl sniffing 8 grooming 3 USV Sniffing Durtion (se) ESP4 ESPmix Urine Grooming Durtion (se) 4 ESP4 ESPmix Urine Numer of USV 2 1 Figure S12. No hnges were oserved in mle sniffing or grooming ehvior or in the numer of ultrsoni voliztion events towrd vrious stimuli-exposed femles () Totl durtion of mle nogenitl sniffing towrd -, -, ESP4-, ESP mix- or urine-exposed C57BL/6 femle mie (n = 6-9) in the 6-min ourtship trils. Anogenitl sniffing ws evluted sed on the oservtion of mle sniffing of the nogenitl re of the femle mie. Error rs represent SEM. () Totl durtion of mle grooming ehvior in the presene of -, -, ESP4-, ESP mix- or urine-exposed C57BL/6 femle mie (n = 6-9) in the 6-min ourtship trils. Grooming ws evluted sed on the oservtion of mle mie self-grooming the ody inluding the fe, nogenitl re nd ody trunk. Error rs represent SEM. () Totl numer of mle ultrsoni voliztions (USVs) towrd - or -exposed C57BL/6 femle mie (n = 7) during the 6-min ourtship trils. The numer of USVs rnging from 3-14 khz were sored. Error rs represent SEM. 12

13 doi: 1.138/nture pproh/sniffing 4 grooming Sniffing Durtion (se) Grooming Durtion (se) 2 ESP4 ESPmix Urine ESP4 ESPmix Urine Figure S13. No hnges were oserved in femle pproh nd sniffing or grooming ehviors following exposure () Totl durtion of pproh nd sniffing ehviors towrds -, -, ESP4-, ESP mix- or urineexposed C57BL/6 femle mie (n = 6-9) in the 6-min ourtship trils. Approh nd sniffing ehvior ws sored using the riterion of strethed-out pproh nd sniffing towrd the mle stud. Error rs represent SEM. () Totl durtion of grooming ehvior towrds -, -, ESP4-, ESP mix- or urine-exposed C57BL/6 femle mie (n = 6-9) during the 6-min ourtship trils. Grooming ws evluted using the riterion of self-grooming the ody inluding the fe, nogenitl re nd ody trunk. Error rs represent SEM. 13

14 doi: 1.138/nture9142 Rering Durtion (se) rering ESP4 ESPmix Urine Durtion of Loomotion (se) loomotion exposure time (min) Figure S14. No hnges were oserved in femle rering or loomotion ehviors following exposure () The totl durtion of rering ehvior towrds -, -, ESP4-, ESP mix- or urine-exposed C57BL/6 femle mie in the presene of mle mie (n = 6-9) during the 6-min ourtship trils. Rering ws evluted using the riterion tht oth forepws were in the ir nd the k ws stright nd strethed. Error rs represent SEM. () Durtion of loomotion towrds - or -exposed C57BL/6 femle mie in the sene of mle mie (n = 12). Green nd lue plots indite the loomotive tivity of - nd -exposed femle mie, respetively. Eh point represents the totl loomotion time during eh 5-min period of the 6-min ourtship trils. Stimulus-ontining otton ws pplied t the time indited y the dshed line. Loomotion ws sored when the hind pws were moved for more thn two steps. The inresed loomotion in the first 1-15 min ws due to explortory ehvior towrds the stimulus-ontining otton. Error rs represent SEM. () Durtion of femle loomotion during the ourtship trils without pre-exposure to stimuli. Femle mie were pired with mle mie immeditely fter touhing - or -ontining otton (n=4-6). Green nd lue plots indite the loomotive tivity of - nd -exposed femles, respetively. Eh point represents the totl loomotion time during eh 5-min period of the 6-min ourtship trils. The numer of mle mounts (left), the perentge of lordosis response (middle) nd the perentge of intromission (right) in these trils re shown in the inset. Error rs represent SEM. **P <.1 Durtion of Loomotion (se) loomotion Numer of Mount 1 % of Lordosis time (min) ** % of Intromission 5 ** 14