LETTER. Photoentrainment and pupillary light reflex are mediated by distinct populations of iprgcs

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1 LETTER doi: /nture10206 Photoentrinment nd pupillry light reflex re medited y distinct popultions of iprgcs S.-K. Chen 1, T. C. Bde 2 & S. Httr 1,3 Intrinsiclly photosensitive retinl gnglion cells (iprgcs) express the photopigment melnopsin nd regulte wide rry of lightdependent physiologicl processes Genetic ltion of iprgcs elimintes circdin photoentrinment nd severely disrupts the pupillry light reflex (PLR) 12,13. Here we show tht iprgcs consist of distinct supopultions tht differentilly express the Brn3 trnscription fctor, nd cn e functionlly distinguished. Brn3-negtive M1 iprgcs innervte the suprchismtic nucleus (SCN) of the hypothlmus, wheres Brn3-positive iprgcs innervte ll other known rin trgets, including the olivry pretectl nucleus. Consistent with these innervtion ptterns, selective ltion of Brn3-positive iprgcs severely disrupts the PLR, ut does not impir circdin photoentrinment. Thus, we find tht moleculrly distinct supopultions of M1 iprgcs, which re morphologiclly nd electrophysiologiclly similr, innervte different rin regions to execute specific light-induced functions. In ddition to rod nd cone photoreceptors, the retin contins smll suset of iprgcs tht express the photopigment melnopsin 1,9. iprgcs project to the suprchismtic nucleus (SCN) nd the olivry pretectl nucleus (OPN), regions in the rin tht control circdin rhythms nd the pupillry light reflex (PLR), respectively. In the sence of the melnopsin protein (Opn4), iprgcs lose their intrinsic photosensitivity 7, ut still innervte the correct rin regions 7 nd convey rod/cone input 7,14,15 to drive non-imge-forming visul functions 7,16. Recent studies hve shown tht iprgcs re not uniform nd cn e further sudivided into distinct sutypes sed on their morphology, electrophysiology nd discrete rin trgets 2,17. M1 iprgcs cn e redily distinguished from other iprgc sutypes (herein referred to s non-m1 iprgcs) ecuse they re the only sutype with exclusive dendritic strtifiction in the OFF sulmin of the inner plexiform lyer (IPL) in the retin 18,19. The previling view is tht M1 iprgcs re homogeneous popultion tht send collterl xonl rnches to two rely nuclei, the SCN nd OPN, to drive circdin photoentrinment nd PLR 20. Genetic ltion of iprgcs y diphtheri toxin (in Opn4 DTA mice) elimintes circdin photoentrinment nd disrupts PLR 12. Here we surprisingly found tht M1 iprgcs re not uniform popultion, ut consist of functionlly distinct supopultions defined y their expression of the POU domin trnscription fctor, Brn3. Previously, we showed tht Brn3 (lso known s Pou4f2) mutnt mice, which lck 80% of RGCs, hve pronounced deficits in PLR, ut re still cple of wek photoentrinment 21. These findings rise the possiility tht the remining Brn3-negtive M1 iprgcs selectively medite photoentrinment. To determine the extent of Brn3 expression in the M1 iprgc popultion, we performed nti-brn3 immunohistochemistry on retins from Opn4 tu-lcz mice, together with X-gl (5-romo-4-chloro-3-indolyl--D-glctoside) stining tht lels only M1 iprgcs 21. A frction of -glctosidse-positive RGCs stined for Brn3 in the dult retin (Fig. 1). To determine the projections of Brn3-positive iprgcs, we mted mice in which n inducile Cre recominse is driven y the melnopsin promoter (Opn4 CreERT2/1 ) to mice hving either uiquitous Credependent lkline phosphtse (AP) reporter (Ros26-IAP) 22 or conditionl Brn3 knock-in (Brn3 CKOAP/1 ) 21 in which Cre recomintion cuses the lkline phosphtse coding region to e expressed y the Brn3 promoter (Supplementry Fig. 1). Tmoxifen injections in Opn4 CreERT2/1 ;R26 IAP/1 nimls result in lelling of M1 nd non-m1 iprgcs y lkline phosphtse histochemistry (Fig. 1c), ut only of Brn3-expressing iprgcs in Opn4 CreERT2/1 ;Brn3 CKOAP/1 nimls (Fig. 1 nd Supplementry Tle 1). Alkline phosphtse lelling of Brn3-positive iprgcs llowed us to nlyse the dendritic roriztions nd centrl projections of these cells, independently of Brn3negtive iprgcs (Fig. 1, d f). Mny Brn3-positive iprgcs hd dendrites rorizing in the ON sulmin of the IPL similr to previous oservtions for non-m1 iprgcs 2,18,20,23. This indictes tht Brn3 expression is not just restricted to M1 iprgcs, ut is lso expressed in non-m1 iprgcs (Fig. 1). Compring the lelling of Brn3positive M1 nd non-m1 iprgcs with ll iprgc sutypes, we find tht most rin trgets of iprgcs show similr ptterns of innervtion (Fig. 1d i). In prticulr, the OPN is innervted fully in oth cses (Fig. 1f, i). However, notle difference is found in the SCN; in the Opn4 CreERT2/1 ;R26 IAP/1 mice, the SCN ws completely innervted y lkline-phosphtse-positive iprgc fires (Fig. 1g) similrly to previous studies 2. In contrst, in the Opn4 CreERT2/1 ;Brn3 CKOAP/1 mice, Opn4 lcz + Anti-Brn3 c Opn4 CreERT2 ; Brn3 CKOAP/+ Opn4 CreERT2 ; R26 IAP/+ d e f OPN Opn4 CreERT2 ; Brn3 CKOAP/+ dlgn IGL vlgn g h i Opn4 CreERT2 ; R26 IAP/+ Figure 1 Co-expression of melnopsin nd Brn3 defines specific set of iprgcs., Retinl flt mounts from Opn4 tu-lcz/1 mice stined with nti- Brn3 ntiody (rown) nd X-gl stining (lue) show Brn3-positive (rrowheds; 140 Brn3-positive iprgcs from 988 lcz1 cells, n 5 5) nd Brn3-negtive (rrows), M1 iprgcs. i, Alkline phosphtse histochemistry of retin ( nd c) nd coronl rin sections (d i) from Opn4 CreERT2/1 ;Brn3 CKOAP/1 mice (, d f), or from Opn4 CreERT2/1 ;R26 IAP/1 mice (c, g i). Suprchismtic region shows prtil innervtion in Opn4 CreERT2/1 ;Brn3 CKOAP/1 mice (d), compred to full innervtion of the SCN in Opn4 CreERT2/1 ;R26 IAP/1 mice (g). Both mouse lines show significnt iprgc projections to the IGL nd vlgn, nd sprse innervtion to the dlgn (e nd h) nd intense lelling of the OPN (f nd i). Scle rs re 25 mm (), 1 mm ( nd c), nd 400 mm (d i). SCN 1 Deprtment of Biology, Johns Hopkins University, Bltimore, Mrylnd 21218, USA. 2 Retinl Circuit Development & Genetics Unit, N-NRL/NEI/NIH, Bethesd, Mrylnd 20892, USA. 3 Deprtment of Neuroscience, Johns Hopkins University-School of Medicine, Bltimore, Mrylnd 21218, USA. 92 NATURE VOL AUGUST Mcmilln Pulishers Limited. All rights reserved

2 LETTER the SCN ws sprsely innervted y Brn3-positive iprgcs (Fig. 1d), with the medil regions of the SCN completely devoid of innervtion (Fig. 1d). We further confirmed tht the diminished SCN innervtion is not due to the use of the inducile Opn4 CreERT2 line, ecuse crossing the Opn4 Cre line 2 with the Brn3 knock-in llele (Opn4 Cre ; Brn3 CKOAP/1 nimls) lso results in reduced SCN innervtion (Supplementry Fig. 2). Thus, iprgcs cn e seprted into two supopultions sed on their Brn3 expression nd connectivity. To lel the centrl projection of Brn3-negtive iprgcs nd determine the physiologicl functions of oth Brn3-negtive nd Brn3positive iprgcs, we specificlly eliminted cells tht co-express melnopsin nd Brn3 y crossing Opn4 Cre nd Brn3 Z-dt lines (Supplementry Tle 1). The Brn3 Z-dt knock-in line 24 expresses diphtheri toxin A suunit (DTA) from the endogenous Brn3 gene promoter (Supplementry Fig. 1) only in the presence of Cre 25. Thus, in Opn4 Cre/1 ; Brn3 Z-dt/1 mice, Brn3-expressing iprgcs re lted, wheres Brn3-negtive iprgcs nd conventionl (melnopsin negtive) RGCs re left intct (Supplementry Fig. 3). Using melnopsin immunofluorescence tht only revels M1 iprgcs in Opn4 Cre/1 ; Brn3 Z-dt/1 retins, we oserved less thn 200 surviving M1 iprgcs (Fig. 2, ). To determine the extent of ltion of ll iprgcs in the Opn4 Cre/1 ; Brn3 Z-dt/1 mice, we generted triple heterozygous Opn4 Cre/1 ; Brn3 Z-dt/1 ; Z/AP mice (Supplementry Tle 1). Alkline phosphtse lelling in the Opn4 Cre/1 ; Z/AP mice revels ll M1 nd non-m1 iprgcs (,2,000 cells) 2. Using lkline phosphtse histochemistry in the triple heterozygous mice, we oserved similr numers of surviving iprgcs s with melnopsin immunofluorescence (Fig. 2). These results show tht ll non-m1 cells re lted nd tht the surviving 10% (200 out of 2,000) of totl iprgcs re Brn3-negtive nd predominntly elong to the M1 sutype. To ssess the centrl projections of these surviving M1 Brn3negtive iprgcs, we crossed Opn4 Cre/1 ; Brn3 Z-dt/1 mice with the either the Opn4 tu-lcz reporter 19,20 or Z/AP reporter 26. Although only 200 M1 iprgcs remined in the Opn4 Cre/tu-LcZ ; Brn3 Z-dt/1 or Opn4 Cre/1 ; Brn3 Z-dt/1 ; Z/AP mice, we oserved tht their fires completely innervted the SCN t levels comprle to those oserved in the control groups (Fig. 2c). However, innervtion of the intergeniculte leflet (IGL) ws highly ttenuted (Fig. 2d) nd OPN projections were completely olished (Fig. 2e). Interestingly, the shell of the OPN showed no fires in the Opn4 Cre/tu-LcZ ; Brn3 Z-dt/1 compred to control mice (Fig. 2e). Given tht oth the SCN nd OPN shell re innervted y M1 iprgcs 5,20, differentil lelling of these iprgc trgets in Opn4 Cre/tu-LcZ ; Brn3 Z-dt/1 mice shows tht the M1 sutype of iprgcs is not uniform popultion. To ensure tht RGCs tht re not intrinsiclly photosensitive re intct in the Opn4 Cre/1 ; Brn3 Z-dt/1 mice, we used choler toxin injection in the eye to lel ll RGC fires in the rin nterogrdely. RGCs innervted the dorsl nd ventrl lterl geniculte nuclei (dlgn nd vlgn) normlly in these mice (Fig. 2f). This is further supported y the similr visul cuity mesured in Opn4 Cre/1 ; Brn3 Z-dt/1 nd wild-type mice (Fig. 2g). Given tht iprgc projections to the OPN re lost, ut SCN projections re lrgely intct, the Opn4 Cre/1 ; Brn3 Z-dt/1 mice llow the reltive contriutions of Brn3-negtive iprgcs to the pupillry light reflex (PLR) nd circdin light responses to e determined. We first mesured PLR in Opn4 Cre/1 ; Brn3 Z-dt/1 mice t two light intensities in the middle of the dy (zeitgeer time 8, ZT8). The pupil of wild-type mice is 95.61% constricted under high light intensity nd 79.47% under low light intensity (Fig. 3, c). In contrst, Opn4 Cre/1 ; Brn3 Z-dt/1 mice showed highly ttenuted PLR t ZT8 under high nd low light intensities (Fig. 3, c). This phenotype is remrkly similr to the PLR deficits oserved in Opn4 DTA/DTA homozygous nimls 12. We further investigted the PLR in the middle of the night (ZT20), nd found tht Opn4 Cre/1 ; Brn3 Z-dt/1 nimls hve no pupillry constriction to high or low light stimultions (Supplementry Fig. 4 nd Supplementry Text). The residul PLR response t ZT8 c d e f SCN LGN OPN LGN Brn3 Z-dt/+ Brn3 +/+ Opn4 tu-lcz/+ Brn3 Z-dt/+ Opn4 tu-lcz/cre Brn3 Z-dt/+ CTB CTB 594 Opn4 +/+ Brn3 Z-dt/+ Brn3 Z-dt/+ Cell numer per retin Brn3 +/+ ; Z/AP Brn3 +/+ Brn3 Z-dt/+ ; Z/AP Brn3 Z-dt/+ in Opn4 DTA/DTA nd Opn4 Cre/1 ; Brn3 Z-dt/1 mice suggests tht other melnopsin-negtive RGCs contriute to this reflex. One cndidte popultion could e Brn3-positive RGCs, which project to the OPN 21. We then sked whether the surviving M1 iprgcs tht innervte the SCN in Opn4 Cre/1 ; Brn3 Z-dt/1 mice re sufficient to drive circdin photoentrinment. Strikingly, we found tht Opn4 Cre/1 ; Brn3 Z-dt/1 mice re le to photoentrin s well s controls under norml 24-h light drk cycles or skeleton photoperiods (Fig. 4, c, Supplementry Figs 5 nd 6 nd Supplementry Text). In ddition, they cn redjust to jet lg light-drk cycle prdigm with dvnced nd delyed drk onsets (Fig. 4). We lso oserved no difference in ctivity during the drk phse of the ultrdin :3.5 light/drk (LD) cycle (Fig. 4, d). Moreover, 15-min light pulse presented erly during the ctive phse g Visul cuity Brn3 +/+ Brn3 Z-dt/+ Figure 2 Genetic ltion of Brn3-positive iprgcs does not impir trgeting to the SCN., Melnopsin immunofluorescence revels reduction in iprgc numers in Opn4 Cre/1 ;Brn3 Z-dt/1 retin compred to control (Opn4 Cre/1 ;Brn3 1/1 )., Quntifiction of surviving melnopsin-positive cells in Opn4 Cre/1 ;Brn3 Z-dt/1 ( cells per retin; n 5 4) nd control ( cells per retin; n 5 4) mice. c e, Coronl rin sections of Opn4 tu-lcz/1 ;Brn3 Z-dt/1 nd Opn4 Cre/tu-LcZ ;Brn3 Z-dt/1 (c e, left two pnels), nd Opn4 Cre/1 ;Brn3 1/1 ;Z/AP nd Opn4 Cre/1 ;Brn3 Z-dt/1 ;Z/AP (c e, right two pnels) mice using X-gl (c e, left two pnels) or lkline phosphtse histochemistry (c e, right two pnels). Sections show SCN (c), LGN (d) nd OPN (e). f, Lelling of ll RGCs with Alex Fluor 594- nd 488-conjugted choler toxin B (CTB 488 nd CTB 594, respectively) in the left (red) nd right eye (green), respectively, shows norml rin trgeting to imge forming regions. g, Visul cuity ws the sme etween Opn4 1/1 ;Brn3 Z-dt/1 (n 5 5) nd Opn4 Cre/1 ;Brn3 Z-dt/1 mice (n 5 6). Scle rs re 100 mm. Error rs represent s.e.m Mcmilln Pulishers Limited. All rights reserved 4 AUGUST 2011 VOL 476 NATURE 93

3 LETTER Brn3 Z-dt/+ of mice mintined under constnt conditions generted similr phse shifts ( nd min for control nd Opn4 cre/1 ; Brn3 Z-dt/1 nimls, respectively; Fig. 4e). Together, these results indicte tht Brn3-negtive iprgcs, comprising only 10% of ll identified iprgc sutypes, re sufficient for circdin photoentrinment. c f Ultrdin LL DD LD Skeleton DD Period (hour) Time (h) ; Brn3 Z-dt/+ Time (h) Drk Low light High light DD LL Brn3 Z-dt/+ g Brn3 + iprgcs M1 iprgcs d Percent ctivity in drk (ultrdin) e Phse shift (min) Trget OPN core, vlgn nd dlgn Brn3 Z-dt/+ Brn3 Z-dt/+ Trget OPN shell nd most of the IGL Trget the SCN nd prt of the IGL Figure 4 Opn4 Cre/1 ;Brn3 Z-dt/1 mice show norml circdin photoentrinment. c, Representtive ctogrms from control nd Opn4 Cre/1 ;Brn3 Z-dt/1 nimls under series of lighting prdigms:, 12:12 h LD cycle, jet lg, constnt drkness (DD), nd constnt light (LL);, ultrdin 3.5:3.5 h cycles; c, skeleton photoperiod. The grey ckground indictes drkness nd the yellow dot indictes the 15-min light pulse t CT16 (circdin time 16). Opn4 Cre/1 ;Brn3 Z-dt/1 nimls hve similr photoentrinment to controls with minor deficits in period lengthening. d, Percent ctivity in the drk portion of the ultrdin cycle shows no significnt difference etween the genotypes. e, Quntifiction of phse shifts shows no significnt differences etween the two groups. f, Quntifiction of circdin period from the two groups under constnt drk nd constnt light conditions. Both groups of nimls show significnt period lengthening under constnt light. g, Venn digrm showing Brn3-positive iprgcs in yellow nd Brn3 negtive iprgcs in green (full description is provided in Supplementry Tle 1). indictes P, 0.01, indictes P, 0.05 using Student s t-test. Error rs represent s.e.m. c Pupil constriction (%) Low High Low High Brn3 Z-dt/+ Figure 3 Opn4 Cre/1 ;Brn3 Z-dt/1 mice show severe deficits in the pupillry light reflex (PLR).,, Representtive imges of PLR from control nd Opn4 Cre/1 ;Brn3 Z-dt/1 mice. Left pnels show pupils under drk conditions, middle pnels show pupils under low light intensity (22 mwcm 22 ) nd right pnels show pupils under high light intensity (5.66 mw cm 22 ). c, Quntifiction of PLR dt from control (n 5 5) nd Opn4 Cre/1 ;Brn3 Z-dt/1 (n 5 6) nimls. indictes P, 0.01 with one-wy ANOVA. Error rs represent s.e.m. 0 However, Opn4 cre/1 ; Brn3 Z-dt/1 mice show minor deficit in period lengthening under constnt light conditions (Fig. 4f). Becuse period lengthening is positively correlted with light intensity, ttenuted projections to the IGL in Opn4 Cre/1 ; Brn3 Z-dt/1 mice could underlie this difference. Here we show tht, lthough M1 iprgcs hve homogeneous morphologicl nd electrophysiologicl chrcteristics, they consist of t lest two different supopultions, which cn e discriminted y expression of the Brn3 trnscription fctor (Fig. 4g). The two M1 supopultions hve distinct rin trgets nd re involved in different non-imge forming visul functions. Using precise moleculr genetic tools to lte Brn3-expressing iprgcs, we disrupted the pupillry light reflex ut not circdin photoentrinment. Thus, iprgcs hve prllel pthwys for controlling non-imge forming functions, nlogous to the specilized properties of RGCs tht medite imgeforming functions 27. METHODS SUMMARY Animls. All experiments were conducted in ccordnce with NIH guidelines nd pproved institutionl niml cre nd use committees of the Johns Hopkins University. Behviourl nlyses. We used previously descried ehviourl tests 12 tht mesure visul cuity (optomotor), pupil constriction (PLR), the period of the circdin oscilltor (wheel running ctivity), the djustment of the circdin clock to different light stimultions (circdin photoentrinment, jet lg prdigms, phse shifting, nd skeleton photoperiod) nd direct light effects on ctivity (constnt light nd ultrdin). Histology. X-gl nd lkline phosphtse histochemistry were performed s descried previously 2,5,28. Full Methods nd ny ssocited references re ville in the online version of the pper t Received 22 Mrch; ccepted 18 My Pulished online 17 July Berson, D. M., Dunn, F. A. & Tko, M. Phototrnsduction y retinl gnglion cells tht set the circdin clock. Science 295, (2002). 2. Ecker, J. L. et l. Melnopsin-expressing retinl gnglion-cell photoreceptors: cellulr diversity nd role in pttern vision. Neuron 67, (2010). 3. Gooley, J. J., Lu, J., Fischer, D. & Sper, C. B. A rod role for melnopsin in nonvisul photoreception. J. Neurosci. 23, (2003). 4. Hnnil, J. & Fhrenkrug, J. Melnopsin contining retinl gnglion cells re light responsive from irth. Neuroreport 15, (2004). 5. Httr, S., Lio, H. W., Tko, M., Berson, D. M. & Yu, K. W. Melnopsin-contining retinl gnglion cells: rchitecture, projections, nd intrinsic photosensitivity. Science 295, (2002). 6. Httr, S. et l. Melnopsin nd rod-cone photoreceptive systems ccount for ll mjor ccessory visul functions in mice. Nture 424, (2003). 7. Lucs, R. J. et l. Diminished pupillry light reflex t high irrdinces in melnopsin-knockout mice. Science 299, (2003). 8. Pnd, S. et l. Melnopsin (Opn4) requirement for normllight-induced circdin phse shifting. Science 298, (2002). 9. Provencio, I., Rollg, M. D. & Cstrucci, A. M. Photoreceptive net in the mmmlin retin. Nture 415, 493 (2002). 10. Ruy, N. F. et l. Role of melnopsin in circdin responses to light. Science 298, (2002). 11. Tu, D. C. et l. Physiologic diversity nd development of intrinsiclly photosensitive retinl gnglion cells. Neuron 48, (2005). 12. Güler, A. D. et l. Melnopsin cells re the principl conduits for rod-cone input to non-imge-forming vision. Nture 453, (2008). 13. Htori, M. et l. Inducile ltion of melnopsin-expressing retinl gnglion cells revels their centrl role in non-imge forming visul responses. PLoS ONE 3, e2451 (2008). 14. Schmidt, T. M., Tniguchi, K. & Kofuji, P. Intrinsic nd extrinsic light responses in melnopsin-expressing gnglion cells during mouse development. J. Neurophysiol. 100, (2008). 15. Wong, K. Y., Dunn, F. A., Grhm, D. M. & Berson, D. M. Synptic influences on rt gnglion-cell photoreceptors. J. Physiol. (Lond.) 582, (2007). 16. Mrosovsky, N. & Httr, S. Impired msking responses to light in melnopsinknockout mice. Chronoiol. Int. 20, (2003). 17. Brown, T. M. et l. Melnopsin contriutions to irrdince coding in the thlmocorticl visul system. PLoS Biol. 8, e (2010). 18. Berson, D. M., Cstrucci, A. M. & Provencio, I. Morphology nd mosics of melnopsin-expressing retinl gnglion cell types in mice. J. Comp. Neurol. 518, (2010). 19. Httr, S. et l. Centrl projections of melnopsin-expressing retinl gnglion cells in the mouse. J. Comp. Neurol. 497, (2006). 94 NATURE VOL AUGUST Mcmilln Pulishers Limited. All rights reserved

4 LETTER 20. Bver, S. B., Pickrd, G. E. & Sollrs, P. J. Two types of melnopsin retinl gnglion cell differentilly innervte the hypothlmic suprchismtic nucleus nd the olivry pretectl nucleus. Eur. J. Neurosci. 27, (2008). 21. Bde, T. C., Chill, H., Ecker, J., Httr, S. & Nthns, J. Distinct roles of trnscription fctors Brn3 nd Brn3 in controlling the development, morphology, nd function of retinl gnglion cells. Neuron 61, (2009). 22. Bde, T. C. et l. New mouse lines for the nlysis of neuronl morphology using CreER(T)/loxP-directed sprse leling. PLoS ONE 4, e7859 (2009). 23. Schmidt, T. M. & Kofuji, P. Functionl nd morphologicl differences mong intrinsiclly photosensitive retinl gnglion cells. J. Neurosci. 29, (2009). 24. Mu, X. et l. Gnglion cells re required for norml progenitor- cell prolifertion ut not cell-fte determintion or ptterning in the developing mouse retin. Curr. Biol. 15, (2005). 25. Gn, L., Wng, S. W., Hung, Z. & Klein, W. H. POU domin fctor Brn-3 is essentil for retinl gnglion cell differentition nd survivl ut not for initil cell fte specifiction. Dev. Biol. 210, (1999). 26. Loe, C. G. et l. Z/AP, doule reporter for Cre-medited recomintion. Dev. Biol. 208, (1999). 27. Wässle, H. Prllel processing in the mmmlin retin. Nture Rev. Neurosci. 5, (2004). 28. Bde, T. C., Wng, Y. & Nthns, J. A noninvsive genetic/phrmcologic strtegy for visulizing cell morphology nd clonl reltionships in the mouse. J. Neurosci. 23, (2003). Supplementry Informtion is linked to the online version of the pper t Acknowledgements We thnk J. Nthns for providing severl niml lines (Brn3 CKOAP, R26 IAP nd Z/AP) tht were crucil for the completion of this study. We thnk J. L. Ecker, who creted the inducile cre line (Opn4 CreERT2 )weusedinthisstudy. We thnk Z. Yng in D. Zck s lortory for providing the Brn3 Z-dt mouse line, which ws generously provided y the originl lortory tht creted this line: W. Klein. We lso thnk R. Kuruvill, H. Zho, M. Hlpern, A. P. Smpth nd T. Schmidt for their creful reding of the mnuscript nd helpful suggestions nd the Johns Hopkins University Mouse Tri-L for support. This work ws supported y the Ntionl Institutes of Helth grnt GM (S.H.), the Dvid nd Lucile Pckrd Foundtion (S.H.), nd the Alfred P. Slon Foundtion (S.H.). Author Contriutions S.-K.C., T.C.B. nd S.H. performed ll experiments nd wrote the pper. Author Informtion Reprints nd permissions informtion is ville t The uthors declre no competing finncil interests. Reders re welcome to comment on the online version of this rticle t Correspondence nd requests for mterils should e ddressed to S.H. (shttr@jhu.edu) or T.C.B. (tudor.de@nih.gov) Mcmilln Pulishers Limited. All rights reserved 4 AUGUST 2011 VOL 476 NATURE 95

5 LETTER METHODS Mice. All mice were of mixed ckground (BL/6;129SvJ). Littermte mle nimls tht were used in the ehviourl nlyses were ged etween 4 nd 12 months. Animls were housed nd treted in ccordnce with NIH nd IACUC guidelines, nd used protocols pproved y the Johns Hopkins University Animl Cre nd Use Committees. Genertion of Opn4 CreERT2 line. To generte Opn4 CreERT2 mice, we used the trgeting rms nd generl strtegy detiled in ref. 2. The only difference is tht the constructcontined rit-gloinintron, CreERT2recominsendnIRESLcZ cssette immeditely downstrem of the strt codon for mouse melnopsin. Immunohistochemistry. Mouse retin ws fixed s whole eyecup for t lest 30 min in 4% prformldehyde (PFA) nd cryoprotected in 30% sucrose overnight. Retin sections (40 mm) were otined y cryostt nd incuted with locking solution (0.3% Triton X-100 nd 5% norml got serum in PBS) for 1 h efore stining with primry ntiody overnight t 4 uc. Sections were wshed in 13 PBS three times for 30 min nd incuted with secondry ntiody t room temperture for 2 h efore mounting in vector-shield mounting solution. Imges were tken with n Olympus microscope y epifluorescence. The dilution for melnopsin ntiody (Advnced Trgeting Systems) is 1:1,000. Tmoxifen injections. The intensity of lelling depends on the mount of tmoxifen injected into nimls s well s the efficiency of excision from loxp regions in the reporter mice. Therefore, ll intrperitonel injections of tmoxifen were stndrdized to lel ll the identified iprgc sutypes (M1 M5). In fct, iprgcs with morphologies chrcteristic for ll identified iprgc sutypes re oserved in the flt mount retins in Fig. 1, c. Retin shown in Fig. 1 ws from n niml injected with 500 mg tmoxifen t P14 (postntl dy 14). Brins shown in Fig. 1d f were from n niml injected with 250 mg tmoxifen t P5. For Fig. 1c, g i, imges re from n niml injected with 1 mg tmoxifen t emryonic dy 17 (E17). There is no prticulr reson to injecting tmoxifen t different developmentl times. We simply used the lkline phosphtse stining s trcing method to revel iprgc trgets in the rin. Histology. X-gl stining. Mice were perfused with 15 ml of 4% PFA, the rin ws dissected out, cryoprotected in 30% sucrose for 2 dys nd 50 mm coronl sections were otined y cryostt. Brin sections were incuted in stining solution with 1mgml 21 X-gl for 2 dys t room temperture, post-fixed in 4% PFA for 1 h nd mounted with glycerol 5. Alkline phosphtse (AP) stining. Mice were perfused with 45 ml of 4% PFA, the rin nd retin were dissected out. Whole-mount retin ws post-fixed for 30 min nd 200 mm coronl rin sections were otined y virotome. Both retin nd rin sections were het-inctivted t 65 uc for 90 min nd incuted in lkline phosphtse stining solution. After stining, the sections nd retin whole-mount were post-fixed in 4% PFA for overnight nd wshed with ethnol series efore mounting 2, 28. Choler toxin injections in the eye. Mice were nesthetized with vertin. Eyes were injected intrvitrelly with 2 ml of choler toxin B suunit conjugted with Alex Fluor 488 (CTB-488) or Alex Fluor 594 (CTB 594) (Invitrogen). Three dys fter injection, rins were isolted, sectioned nd mounted. Visul cuity. A virtul cylinder OptoMotry (Cererl Mechnics) ws used to determine visul cuity y mesuring the imge-trcking reflex of mice. A sinewve grting ws projected on the screen rotting in virtul cylinder. The niml ws ssessed for trcking response on stimultion for out 5 s. All cuity thresholds were determined y using the stircse method with 100% contrst. Pupillry light reflex. All nimls were kept under 12:12 LD cycle efore testing PLR. Before ech experiment, ll nimls were drk-dpted for t lest 1 h. While one eye received light stimultion with specific intensity descried in the min text from 470-nm light-emitting-diode light source (Super Bright LEDs), digitl cmcorder (DCRHC96; Sony) ws used to record from the other eye (for 30 s) t 30 frmes per second under 940-nm light (LDP). The percentge pupil constriction ws clculted s the percentge of pupil re t 30 s fter initition of the stimulus (stedy stte) reltive to the dilted pupil size (right efore light stimultion). The sme group of nimls were used for wheel-running ctivity. The control nimls re littermtes to the experimentl nimls (Opn4 Cre/1 ; Brn3 Z-dt/1 ) with either Opn4 Cre/1 ; Brn3 1/1 or Opn4 1/1 ; Brn3 Z-dt/1 genotypes. Wheel-running ctivity. Mice were plced in cges with 4.5-inch running wheel, nd their ctivity ws monitored with VitlView softwre (MiniMitter). The period ws clculted with ClockL (Actimetrics). Mice were initilly plced under 12:12 LD cycle for 2 weeks. Animls were then exposed to two jet lg light prdigms: 10 dys of 6-h dvnce followed y 10 dys of 6-h dely. After the jet lg prdigms, mice were kept under constnt drkness for 2 weeks followed y 10 dys of constnt light. Phse-shifting experiments were crried out on the 7th dy of constnt drkness where ech niml ws exposed to 15 min light pulse t CT16 (1,500 lx). Animls were re-entrined to 12:12 LD cycle for 2 weeks efore exposing them to ultrdin 3.5:3.5 light/drk cycles. The intensity of light for ll the light drk cycle were,1,000 lx. Another set of mice ws tested using skeleton photoperiod, where two 1-h light pulses (800 lx) seprted y 10 h of drk were dministered Mcmilln Pulishers Limited. All rights reserved

6 doi: /nture10206 Supplementry text Pupillry light reflex shows diurnl rhythm We mesured PLR in ;Brn3 Z-dt/+ mice t two light intensities in the middle of the dy (ZT 8) nd the middle of the night (ZT 20). The PLR in wild type mice shows diurnl rhythm with higher constriction during the dytime (t ZT 8 pupil constriction is 95.61% under high light intensity [5.66 mw/cm 2 ]) nd 79.47% under low light intensity [22 µw/cm 2 ]) compred to night time (t ZT 20 pupil constriction is 82.61% under high light intensity nd 42.44% under the low light intensity) (Figure 3 nd supplementry Figure 4). In contrst, ;Brn3 Z-dt/+ mice showed highly ttenuted PLR t ZT 8 under oth high light nd low light intensities, nd no detectle PLR t ZT20 even under the high light intensity (Figure 3 nd supplementry Figure 4). This phenotype is remrkly similr to the PLR deficits oserved in the Opn4 DTA/DTA homozygous nimls, lthough the ;Brn3 Z-dt/+ nimls still hve single functionl copy of the melnopsin gene. Heterozygous Opn4 DTA/+ nimls show mixed responses with PLR, especilly t high light intensities 1. Circdin nd msking studies on ;Brn3 Z-dt/+ mice To study circdin photoentrinment nd msking in the ;Brn3 Z-dt/+ mice, we crried out the following procedures: 1- We first plced the nimls under 12h:12h light drk cycle. All ;Brn3 Z-dt/+ mice photoentrined to the light drk cycle y confining their ctivity to the drk, showing stle phse reltionship with the light drk cycle nd producing n exct 24-hour period length. 2- In the second LD cycle, the drk ws dvnced y 6 hours to mesure re-entrinment. Both the experimentl nd the control groups show the sme ility for re-entrinment. The reentrinment ility of mice to 6-hr dvnce in the drk cycle depends on two fctors: the phse of the clock (circdin) nd direct drk ctivtion of ctivity (msking). These experiments show tht oth experimentl nd control nimls re cple of redjusting their ctivity to the new imposed light drk cycle. 3- We then delyed the drk onset in the LD cycle y 6 hours. Agin, oth the experimentl nd control nimls pper to directly entrin. However, this is ctully n rtifct due to the fct tht mice do not like to run under right light conditions (msking). To see the speed of re- 1

7 entrinment, we hve to oserve the time t which mice cese their ctivity (indicted y the red line in the ctogrms showing negtive slope), which shows tht ll nimls (control nd experimentl) require on verge six dys to re-entrin. 4- To mesure if the circdin oscilltor is functionl, nimls re plced in constnt drkness. We oserve little vrition in the oscilltor s period length etween control nd experimentl group. Also, ll nimls (control nd experimentl) respond to rief right light pulse y shifting their onset in drk stly for severl dys fter the dministrtion of the light pulse. 5- To further mesure the influence of light on these nimls, we crried out constnt light exposure experiments. These studies reveled deficits in light responsiveness of the experimentl group under LL conditions. Note tht in the control group, some nimls rely run on the wheel in LL nd re nerly rrhythmic (nimls 1 nd 3). This is n indiction of strong effect of light on their ehvior. In the experimentl group, severl prmeters highlight their ttenuted light responses in LL: - lck of complete rrhythmicity, - higher ctivity levels, c- weker lengthening of the period nd d- n ppernce of two period lengths (niml 1 of the experimentl group). Thus lthough nimls with Brn3-ipRGCs deleted re completely cple of photoentrinment, phse-shifting, nd msking responses under the ultrdin cycle (see point 7), they do show deficits in light-responsiveness under LL conditions. This indictes tht rin regions distinct from the SCN, such s the IGL, which receives weker innervtion ptterns in the Brn3-ipRGCs deleted nimls, my medite LL responsiveness. Further experiments re needed to completely prove this possiility. 6- Even fter prolonged LL, ll mice re le to re-entrin to LD including nimls tht show little ctivity in LL (nimls 1 nd 3 of the control group). 7- All nimls respond similrly to n ultrdin cycle composed of 3.5 hours of light nd 3.5 hours of drk tht mesures msking responses to light. 8- We lso mesured the ility of ;Brn3 Z-dt/+ mice to entrin to skeleton photoperiod nd show tht oth control nd experimentl group entrin to the skeleton photoperiod. 2

8 Brn3 CKOAP Brn3 locus rn3 AP Brn3 Z-dt Brn3 locus LcZ Stop DTA Opn4 Cre opn4 locus NLS Cre Opn4 CreERT2 opn4 locus CreERT2 IRES LcZ Opn4 tu-lcz opn4 locus tu-lcz Z/AP CAG promoter geo(lcz+neo) Stop AP R26 IAP Ros26 Locus AP exon1 3 UTR AP exon 2 3 UTR Supplementry Figure 1. Schemtic representtion of the mouse genetic lines Schemtic representtion of ll the genetic mouse lines tht we utilized in this pper, yellow tringles indicte the LoxP site. Brn3 CKOAP, which ws previously vlidted 2 hs 2 LoxP sites flnking the Brn3 open reding nd upon Cre excision, n lkline phosphtse gene ecomes in frme with the Brn3 promoter. Therefore, the expression of AP will e restricted to Brn3 positive cells only upon Cre expression. Brn3 Z-dt ws pulished previously 3 nd hs 2 LoxP sites flnking the βgeo cssette (LcZ-neo), which prevents the downstrem DTA from eing expressed in the sence of Cre. Therefore, DTA will only e expressed upon the Cre dependent excision of the intervening cssette. Opn4 Cre ws used in our previous study to revel the diversity of iprgcs nd their trgets in the rin 4, wheres Opn4 tu-lcz mice were pulished severl times nd show leling of only M1 iprgcs 5-7. Opn4 CreERT2/+ is recently generted niml tht ws vlidted in this study upon mting with previously pulished niml R26 IAP mice 8 which in the presence of Cre cuses n inversion tht restores the functionl open reding frme of AP. Finlly, Z/AP mice 9 is possily one of the most widely used Cre-dependent reporter line. We used R26 IAP insted of Z/AP in our conditionl Cre nlysis, since we oserved higher rte of recomintion with this niml upon tmoxifen injection. 3

9 ; Brn3 KOAP/+ OPN LGN SCN Supplementry Figure 2. Brin innervtion pttern in ; Brn3 CKOAP/+ line Representtive imges using histochemicl stining with AP in coronl sections from the rin of n ; Brn3 CKOAP/+ mouse. Note the intense leling of the LGN nd the OPN in greement with the conditionl stining (Figure 1h nd i). Higher leling intensity is expected since we used the conventionl Cre line, which lels ll Brn3-positive iprgcs. Interestingly, despite the intense leling in the LGN nd the OPN, the stining of SCN is still restricted to the lterl edges of the nucleus. Scle r is 400 µm. 4

10 opn4 +/+ ; Brn3 Z-dt/+ opn4 Cre/+ ; Brn3 Z-dt/+ Supplementry Figure 3. Totl numer of RGCs is similr in control nd experimentl group X-gl histochemicl stining of β-glctosidse from:. Opn4 +/+ ; Brn3 Z-dt/+ nd. ; Brn3 Z-dt/+ mouse retins. The deletion of Brn3-positive iprgcs does not impct the totl numer of RGCs in the retin in greement with the visul cuity test (Figure 2g). Scle r is 200 µm. 5

11 Drk Low High Brn3 Z-dt/+ c 100% Pupil Constriction 75% 50% 25% 0% Low High Low High ;Brn3 Z-dt/+ Supplementry Figure 4. PLR shows highly ttenuted responses in ; Brn3 Z-dt/+ mice nd. Representtive imges of pupillry light reflex from control () nd ; Brn3 Z- dt/+ mice () t ZT 20. The dsh circles mrk the edge of pupil. The left pnels show pupils under drk, the middle pnels show pupils under low light intensity (22 µw/cm 2 ) nd the right pnels show pupils under high light intensity (5.66 mw/cm 2 ). Ech row represents imges from the sme niml. There is lmost no detectle PLR in the ; Brn3 Z-dt/+ mice even t high light intensity. Note tht oth control nd ; Brn3 Z-dt/+ groups show less pupil constriction during the night compred to the dy (Figure 3). c. Quntifiction of PLR dt from control (n=5) nd ; Brn3 Z-dt/+ (n=6) nimls. indictes p<0.01 with 1-wy ANOVA. Error rs represent SEMs. 6

12 LD 0 Ultr LD LD LL DD Opn4Cre/+ ; Brn3Z-dt/+ LL DD Ultr LD LL DD LD Ultr LD Ultr LD Littermte LL DD LD 0 12 Supplementry Figure 5. All ctogrms of experimentl nd control groups nd, Actogrms from ll Opn4Cre/+ ; Brn3Z-dt/+ mice () nd control mice (). The yellow indictes 15 min light pulse for shifting the circdin oscilltor. Red line shows re-entrinment fter the shift in the LD cycle. Note tht ll the tretments re exctly the sme s those explined in Figure 4. W W W. N A T U R E. C O M / N A T U R E 7

13 Cre/+;Brn3 Z-dt/ DD Skeleton LD DD Skeleton LD Supplementry Figure 6. All ctogrms of experimentl nd control groups under skeleton photoperiod nd, Actogrms from ll ; Brn3 Z-dt/+ mice () nd control mice () under skeleton photoperiod. Both groups show norml circdin photoentrinment s reveled y the onset of their free running ctivity in constnt drk conditions. 8

14 Brn3(+) iprgcs Supplementry Tle 1 Mjor Trgets: OPN core, vlgn nd dlgn Mjor Trgets: OPN shell nd IGL Mjor Trgets: SCN nd prt of the IGL M1 iprgcs Opn4 CreERT2/+ ; Brn3 CKOAP/+ : Lels ll Brn3+ iprgcs sutypes, including ll non-m1 nd suset of M1 iprgcs. lels ll known iprgc rin trgets with miniml innervtion of the SCN Opn4 CreERT2/+ ; R26 IAP/+ : Lels ll iprgcs sutypes lels ll known iprgc rin trgets Opn4 Cre/tu-LcZ ;Brn3 Z-dt/+ : ; Brn3 Z-dt/+ ; Z/AP : Elimintes ll Brn3+ iprgcs, which includes ll non-m1 nd suset of M1 iprgcs Elimintes ll Brn3+ iprgcs, which includes ll non-m1 nd suset of M1 iprgcs Leling with X-gl stining is only oserved in SCN nd prtilly in the IGL Leling with AP stining is only oserved in SCN nd prtilly in the IGL (non-m1 not leled) ; Brn3 Z-dt/+ : Elimintes ll Brn3+ iprgcs, which This line ws used for ehvior: includes ll non-m1 nd suset pupillry light reflex deficit nd of M1 iprgcs norml photoentrinment Opn4 DTA/DTA (from Guler 2008 Nture) SCN nd shell of OPN re not leled Pupillry light reflex nd circdin photoentrinment deficits only in homozygotes Supplementry Tle 1 Summry tle with Venn digrm explining the rtionle for the use of the genetic mouse lines. A supopultion of iprgcs re M1 iprgcs (Green Circle). Yellow circle represents ll 9

15 Brn3-positive iprgcs tht re M1 or non-m1. Yellow color (not merged with green) represents non-m1 iprgcs tht re Brn3 positive (nerly 100% re Brn3 positive). Blue (yellow nd green circles merge) represents the M1 iprgcs tht re Brn3-positive nd project to the IGL nd the shell of the OPN, wheres crescent green represents M1 iprgcs tht predominntly trget the SCN nd re Brn3 negtive. It is noteworthy to mention tht the re of the circles correspond to the percentge of the different sutypes of iprgcs. Note tht no dt ws included in this study from the Opn4 DTA/DTA nimls, which were previously pulished. Reference: 1 Guler, A.D. et l., Melnopsin cells re the principl conduits for rod cone input to non imge forming vision. Nture 453 (7191), (2008). 2 Bde, T.C., Chill, H., Ecker, J., Httr, S., & Nthns, J., Distinct roles of trnscription fctors rn3 nd rn3 in controlling the development, morphology, nd function of retinl gnglion cells. Neuron 61 (6), (2009). 3 Mu, X. et l., Gnglion cells re required for norml progenitor cell prolifertion ut not cell fte determintion or ptterning in the developing mouse retin. Curr Biol 15 (6), (2005). 4 Ecker, J.L. et l., Melnopsin expressing retinl gnglion cell photoreceptors: cellulr diversity nd role in pttern vision. Neuron 67 (1), (2010). 5 Bver, S.B., Pickrd, G.E., & Sollrs, P.J., Two types of melnopsin retinl gnglion cell differentilly innervte the hypothlmic suprchismtic nucleus nd the olivry pretectl nucleus. Eur J Neurosci 27 (7), (2008). 6 Httr, S. et l., Centrl projections of melnopsin expressing retinl gnglion cells in the mouse. J Comp Neurol 497 (3), (2006). 7 Httr, S., Lio, H.W., Tko, M., Berson, D.M., & Yu, K.W., Melnopsin contining retinl gnglion cells: rchitecture, projections, nd intrinsic photosensitivity. Science 295 (5557), (2002). 8 Bde, T.C. et l., New mouse lines for the nlysis of neuronl morphology using CreER(T)/loxP directed sprse leling. PLoS ONE 4 (11), e7859 (2009). 9 Loe, C.G. et l., Z/AP, doule reporter for cre medited recomintion. Dev Biol 208 (2), (1999). 10