mir-124 acts through CoREST to control onset of Sema3A sensitivity in navigating retinal growth cones

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1 cts through CoREST to control onset of Sem3A sensitivity in nvigting retinl growth cones Mrie-Lure Budet 1, Krishn H Zivrj 1, Cei Areu-Goodger 2, Alistir Muldl 1, Jvier Armisen 3, Cherie Blenkiron 3, Leonrd D Goldstein 3, Eric A Misk 3 & Christine E Holt Nture Americ, Inc. All rights reserved. During xon pthfinding, growth cones commonly show chnges in sensitivity to guidnce cues tht follow cell-intrinsic timetle. The cellulr timer mechnisms tht regulte such chnges re, however, poorly understood. Here we hve investigted micrornas (mirnas) in the timing control of sensitivity to the semphorin Sem3A in Xenopus levis retinl gnglion cell (RGC) growth cones. A developmentl profiling screen identified s cndidte timer. Loss of delyed the onset of Sem3A sensitivity nd concomitnt neuropilin-1 (NRP1) receptor expression nd cused cell-utonomous pthfinding errors. CoREST, cofctor of NRP1 repressor, ws newly identified s trget nd meditor of for this highly specific temporl spect of RGC growth cone responsiveness. Our findings indicte tht is importnt in regulting the intrinsic temporl chnges in RGC growth cone sensitivity nd suggest tht mirnas my ct rodly s liner timers in verterte neuronl development. Axons nvigte in complex nd chnging environment to estlish connections with their trgets. Chemotropic cues in this environment ttrct nd repel growing xons 1,2, nd growth cones must modulte their responsiveness en route to void stlling t ttrctive intermedite trgets or invding non-trgets. Growth cones of commissurl neurons in the verterte spinl cord, for exmple, re initilly ttrcted to Netrin-1 nd unresponsive to Slits, ut, fter crossing the midline (n intermedite trget), they ecome unresponsive to Netrin-1 nd repelled y Slits 3,4. Similrly, RGC xons chnge their responsiveness to severl cues s they dvnce long the pthwy 5 7 with growth cones initilly showing ttrction to Netrin-1 nd neutrl responses to repellents (Sem3A nd Slit2) nd lter gining sensitivity to repellents nd switching the polrity of their response to Netrin-1. This developmentl regultion helps to mtch growth cone sensitivity ppropritely with pthwy stimuli nd therey ensures correct nvigtion. The timetle of chnges in growth cone sensitivity cn e remrkly precise, much like other spects of neuronl differentition, rising the question of how it is controlled. Wheres evidence in spinl cord commissurl neurons fvors extrinsic midline-medited control of growth cone sensitivity 3,4,8, in RGC xons, the developmentl chnges re primrily under intrinsic control. Pthwy-nive RGC growth cones in vitro, for exmple, show the sme progrm of chnges in cue sensitivity, polrity switching nd receptor expression nd with similr timetle s pthwy-experienced growth cones in vivo 5,6. These findings point to the involvement of n intrinsic moleculr clock, or liner timer. Such timers regulte the liner progression of seril events in these postmitotic neurons nd could serve to synchronize the sensitivity of dvncing growth cones with their chnging environment. Wheres much is known out the moleculr sis of cyclicl clocks tht control repetitive processes such s the cell cycle nd somite segmenttion 9,1, little is known out liner timers tht control the sequentil progrm of cell differentition. mirnas re emerging s key molecules regulting the liner timing of developmentl events in Cenorhditis elegns nd vertertes 14. mirnas re smll, noncoding RNAs of ~22 nucleotides tht pir to the 3 untrnslted region (UTR) of trget mrnas to repress their expression y inducing either their degrdtion or trnsltionl repression 15. Of interest, severl mirnas hve een reported to e specificlly distriuted within the nervous system, including in differentiting neurons 16,17. Thus, mirnas re prime cndidte regultors of growth cone ge-relted chnges. We hve therefore investigted whether mirnas contriute to the intrinsic temporl regultion of growth cone responsiveness to Sem3A. We identify s strong cndidte in RGCs, on the sis of developmentl mirna profiling of different ge retins nd in situ hyridiztion (ISH). Using loss-of-function pproch, we demonstrte tht regultes the onset of growth cone responsiveness to Sem3A. Moreover, we show tht cts y trgeting the mrna encoding CoREST (lterntive nme, rcor1), known cofctor of REST (repressor element 1 silencing trnscription fctor), which in turn represses trnscription of NRP1, encoding Sem3A receptor. We propose tht in norml development cuses the upregultion of NRP1 nd the onset of sensitivity of growth cones to Sem3A, which is essentil for norml xon guidnce. RESULTS mirna profiling identifies s cndidte timer To determine which mirna(s) might e involved in RGC growth cone ging, we identified the repertoire of mirnas in Xenopus RGCs. 1 Deprtment of Physiology, Development & Neuroscience, University of Cmridge, Cmridge, UK. 2 Ntionl Lortory of Genomics for Biodiversity (Lngeio), CINVESTAV, Irputo, Gunjuto, México. 3 Wellcome Trust/Cncer Reserch UK Gurdon Institute, University of Cmridge, Cmridge, UK. Correspondence should e ddressed to C.E.H. (ceh@mole.io.cm.c.uk). Received 8 August; ccepted 5 Octoer; pulished online 4 Decemer 211; doi:1.138/nn.2979 nture NEUROSCIENCE VOLUME 15 NUMBER 1 JANUARY

2 212 Nture Americ, Inc. All rights reserved. Figure 1 Expression profiling of mirnas in the developing retin. () Het mp representing the log 2 of the normlized numer of reds for the 51 mirnas profiled in stge 4 retin (neurl retin nd retinl pigmented epithelium) using Illumin sequencing. The nmes of mirnas with >1, reds re underlined. () Retinl distriution of the most undnt mirnas t stge 4 y ISH. Only nd mir-13 re distriuted in RGCs, long with other retinl cells, wheres mir-182 nd mir-183 re locted in the developing photoreceptor lyers. (c) Hierrchicl clustering of the 35 mirnas detected with >5 reds t ny of the stges studied. The het mp shows the reltive stndrdized mirna expression (Z-score) indicted in the color key. There were 1, 22 nd 3 mirnas tht ppered, respectively, to decrese over time, increse over time or pek t stge 32. (d,e) ISH on whole mounts (d) or tissue sections (e) showing detection in the spinl cord, rin nd retin of Xenopus emryos, including in differentiting RGCs (rrows in insets). Lines in d indicte pproximte plnes of section shown elow in e; oxed regions in e re mgnified in insets. mir- 124 ws sent or low in proliferting cells nd in the ciliry mrginl zone (CMZ). F, forerin; GCL, gnglion cell lyer; H, hindrin; INL, inner nucler lyer; IPL, inner plexiform lyer; L, outer plexiform lyer; PR, photoreceptor lyer; NR, neurl retin; pnr, presumptive NR (dshed outline) within optic vesicles (solid outline); RPE, retinl pigmented epithelium. Scle rs, 1 µm (,e); 25 µm (d). We first profiled mirnas in stge 4 retins, e when most RGCs hve lredy een orn, using Illumin sequencing, which detected 51 mirnas t stge 4 (Fig. 1). Previously F identified timer mirnas, including orthologs of mirnas shown to regulte developmentl timing in C. elegns 18 (let-7-i, mir-98, mirpnr 125 nd mir-125), were either sent or were not mong the most undnt mirnas in the retin (Fig. 1). ISH nlysis of the distriution of the most undnt (>1, reds) Optic vesicles mirnas showed, mir-13, mir-182 nd mir-183 in the neurl retin (Fig. 1), wheres mir-184 nd mir-1 were detected solely in extr-retinl tissues (Supplementry Fig. 1). nd mir-13 were the only two to e detected in RGCs, lthough they were lso present in other retinl cells (Fig. 1). mir-13, however, ws found to e uiquitously distriuted in ll cells of the Xenopus hed, wheres ws present exclusively in postmitotic neurons (Fig. 1), in greement with mmmlin studies 19,2. These initil results, therefore, identified the neuronspecific s cndidte of interest. We resoned tht liner timer would e expected to show progressive chnges in expression with time, s shown for mirnas involved in developmentl timing 11 13,18. Therefore, we next quntified expression t three developmentl stges (24, 32 nd 4) tht spn the period of cell prolifertion nd RGC differentition, including xon initition, elongtion nd guidnce. Pioneer RGC xons rech the optic chism t stge 32, rrive t the tectum t stge 37/38 nd hve rorized y stge 4 to form functionl visul connections mir-184 mir-182 mir-183 mir-1 mir-13 mir-9* mir-143 mir-1 mir-31 let-7f let-7 mir-24 mir-146 mir-26 mir-96 mir-13c mir-122 mir-1c mir-181 mir-363-3p mir-9 mir-9 mir-181-1* mir-1 mir-128 mir-26 mir-365 mir-7 mir-222 mir-11 mir-16c mir-3c mir-181 mir-13 mir-199* mir-93 mir-93 mir-22 mir-21 mir-3e mir-19 mir-16 mir-19 mir-3d mir-25 mir-92 mir-214 mir-17 mir-148 mir-181-2* d Eye 8 19 c RPE CMZ Lens PR L INL IPL GCL Stge 24 Brin NR Retin mir Stge 24 Stge 32 Stge 4 Stge 32 Stge 4/41 F mir-182 mir-183 Control CMZ Lens Retin As determined y Illumin sequencing nlysis, we found tht mir- 124 incresed over the three stges nlyzed (Fig. 1c) y fctor of 1.33 etween stges 24 nd 32, nd y fctor of 1.64 etween stges 32 nd 4. ISH confirmed this temporl regultion (Fig. 1d,e). A mir- 124-ssocited signl ws fintly present t stge 24 in the presumptive neurl retin, rin nd spinl cord, nd the intensity of the signl in these tissues incresed t stge 32, then further t stge 4 (Fig. 1d,e). This progressive increse ws prticulrly evident in the neurl retin, including RGCs (Fig. 1e). expression did not seem to chnge fter stge 4, when xons hve strted to rorize (dt not shown). knockdown does not lter RGC differentition We next investigted whether is implicted in RGC growth cone ge-relted chnges, using loss-of-function pproch. is highly conserved mirna expressed y only one gene in Xenopus tropiclis nd is the sole memer of its fmily (mirbse relese 16) 22. We therefore designed morpholino ntisense oligonucleotide, F H mir-96 mir-143 mir-1 mir-183 mir-182 mir-24 mir-181 let-7 mir-181 mir-222 mir-122 mir-365 mir-1c mir-1 mir-31 mir-26 mir-9* mir-1 mir-7 mir-181-1* let-7f mir-9 mir-9 mir-184 mir-16c mir-11 mir-363-3p mir-146 mir-13 mir-13c mir-3c mir-26 mir-13 mir VOLUME 15 NUMBER 1 JANUARY 212 nture neuroscience

3 212 Nture Americ, Inc. All rights reserved. Figure 2 knockdown does not ffect the timing of RGC genesis nd differentition. () Morpholino-medited knockdown of endogenous ws confirmed t stge 4 y whole-mount ISH. No signl ws detected in emryos injected with, unlike in controls (uninjected or injected). () Representtive stge 4 retin stined with Islet-1 (green), Sox2 (red) nd DAPI (lue). The white outline delinetes the developing RGC lyer. A cell ws considered RGC when it ws locted in the innermost prt of the retin, positive for Islet-1 nd negtive for Sox2. dac, displced mcrine cell. (c) Illustrtive retins stined for Islet-1 nd Sox2 from stge 32 4 emryos microinjected with or. (d) Numers of RGCs per retinl section in stge (st.) 32 4 retin of emryos injected with or. (e,f) Proportion of Brn3-positive cells (e) nd EdU-positive, Sox2-negtive cells (f) per GCL of emryos injected with or - MO. All smples pssed Kolmogorov-Smirnov normlity test. (d) Anlysis of vrince (ANOVA) followed y Bonferroni post-test. (e,f) Unpired Student s t-test. Vlues re men ± s.e.m., not significnt. Numers of retins nlyzed re indicted in rs. Up to ten 12-µm sections were nlyzed per retin; out 25, (d), 2, (e) nd 8, (f) cells were counted in totl. Scle rs, 25 µm (); 1 µm (, left), 1 µm (, right); 5 µm (c)., to lock the function of endogenous. ws injected t the eight-cell stge into oth dorsl lstomeres fted to give rise to the centrl nervous system, including the neurl retin. In injected emryos, endogenous ws sent t stge 4 (pproximtely 72 h post-fertiliztion (hpf)), wheres strong ISH signl ws present in the retin, rin nd spinl cord in uninjected or control morpholino ()-injected emryos (Figs. 1d,e nd 2). This indictes ISH Stge 32 tht morpholino-medited knockdown ws oth effective nd long-lsting. Morpholinos ct y preventing the mturtion of mirna 23, nd ecuse ws introduced efore mir- 124 ws expressed, the sence of n endogenous signl likely reflected the sence of mture. Although is undnt nd highly specific to postmitotic neurons 16,17,2, we detected no gross morphologicl phenotype in the morphnt (Supplementry Fig. 2). We first exmined whether influences the generl progression of RGC differentition, which in turn might ffect the intrinsic mturtion of growth cones. Indeed, the exct role of in differentition is not cler 24. We therefore counted differentited RGCs t four developmentl stges, from erly RGC differentition (stge 32) to mturtion (stge 4), to determine whether knockdown ltered cell numers. RGCs were identified s Islet-1 positive, Sox2 negtive. The RGC lyer is composed of RGCs nd displced mcrine cells, oth of which re positive for Islet-1 (ref. 25). Displced mcrine cells re lso positive for Sox2 (ref. 25) (Fig. 2). At ll stges studied, the numer of Islet-1 + Sox2 RGCs ws similr in morphnt nd control retins, indicting tht knockdown does not c RGCs per retinl section St No MO Lens 8 7 GCL St. 33/34 St. 37/38 St Percentge Brn3 + cells per GCL Percentge EdU + Sox2 cells per GCL influence the generl timetle of RGC differentition (Fig. 2c,d). We further ssessed the timing of RGC cell differentition using different RGC mker, Brn3 (ref. 26), nd found no difference etween the numer of Brn3-positive cells in the RGC lyer t stge 4 in morphnts nd controls (Fig. 2e nd Supplementry Fig. 3). Finlly, we investigted whether the timing of RGC neurogenesis ws impired. We incuted stge emryos with irthdting gent, 5-ethynyl-2 -deoxyuridine (EdU), when out hlf of the RGCs hd een orn nd mesured the numer of EdU-positive, Sox2- negtive cells in the gnglion cell lyer (GCL) t stge 4 (Fig. 2f nd Supplementry Fig. 3). Agin, no significnt difference ws found in the percentge of leled RGCs in morphnt nd control retins. Although these results do not gree with reported evidence tht promotes neuronl differentition in vivo 27,28, they re consistent with severl other reports showing tht the loss of does not ffect neuronl differentition 29,3. knockdown delys onset of Sem3A sensitivity RGC xonl growth cones show intrinsic temporl regultion of cue sensitivity exemplified y the switching on of Sem3A-induced 1 RGC Stge 33/34 Stge 37/38 Stge 4 d e f St. 4 St dac Sox2 Islet nture NEUROSCIENCE VOLUME 15 NUMBER 1 JANUARY

4 212 Nture Americ, Inc. All rights reserved. Figure 3 Responsiveness of growth cones to Sem3A is delyed in morphnts. () Mesure of the numer of collpsed growth cones in stge 24 retinl explnts grown in culture for h in the presence (+) or sence ( ) of Sem3A. The onset of retinl growth cone responsiveness to Sem3A occurred etween 28 nd 32 h in culture y collpse ssy. () Schemtic representtion of the experiment; 125 µm or mir- 124-MO were injected. Collpsed growth cones (ox) hve retrcted lmellipodi nd hve less thn two filopodi. (c) Numer of collpsed growth cones in stge 24 retinl explnts dissected from emryos injected with either or nd grown in culture for h. Growth cone responsiveness to Sem3A ws delyed in morphnts compred to control. All smples pssed Kolmogorov-Smirnov normlity test. ANOVA followed y Bonferroni post-test. Vlues re men ± s.e.m.;, not significnt; P <.1. Numer of growth cones nlyzed re indicted in rs. Stge 24 emryo imge in modified from ref. 46, copyright 1994 P.D. Nieuwkoop nd J. Fer. Reproduced y permission of Tylor nd Frncis Group, LLC, division of Inform plc. collpse responses nd NRP1 expression severl hours fter xon initition in neurons without pthwy experience 5,6. We investigted whether the temporl regultion of this sucellulr spect of differentition (growth cone cue sensitivity) ws influenced y. We resoned tht if cts s timer, disrupting its function would induce either precocious or retrded cue responses. First we determined the norml time of onset of Sem3A sensitivity. We explnted emryonic retinl tissue t stge 24, efore xonogenesis 21,31, to ensure de novo xon growth nd tested the responsiveness of growth cones to Sem3A t vrious times using collpse ssys. A significnt increse in Sem3A-induced collpse response occurred within 4 h time frme etween 28 h (36% ± 3, s.e.m. collpse, equivlent to ckground collpse) nd 32 h (65% ± 5) fter plting. At 32 h full collpse response ws reched, s it did not significntly differ from tht oserved t lter time points (4 h nd 48 h) (Fig. 3). The gin in Sem3A sensitivity thus occurred ruptly etween 28 h nd 32 h. We next ssessed whether the onset of Sem3A responsiveness ws ltered in retinl explnts from morphnts (Fig. 3). At 36 h, full collpse response ws oserved in control cultures (Fig. 3c), ut y contrst, morphnt retinl growth cones did not respond to Sem3A. At 48 h, morphnt RGC growth cones responded significntly (P <.1) to Sem3A (52% ± 9) compred to BSA control (21% ± 5), ut the collpse response ws significntly (P <.1) lower thn tht of control () growth cones (79% ± 4; Fig. 3c). At 64 h, full Sem3A-medited collpse response ws oserved in morphnts (72% ± 3) tht did not differ significntly (P >.5) from tht oserved in controls (73% ± 3) (Fig. 3c). The full collpse response ws thus oserved 32 h lter thn norml. Becuse is sent until t lest 72 hpf, when the delyed response egins, this indictes tht ffects the timing rther thn simple repression of responsiveness. To test the specificity of the phenotype, we performed rescue experiments using duplex, mimic of endogenous doule-strnded mture. We electroported the duplex into stge 24 eyes of emryos tht hd een injected with mir- 124-MO t the eight-cell stge (Fig. 4). Sectioned mteril verified tht the duplex-electroported regions of retin positive for CAAXmCherry, construct encoding the red fluorescent protein mcherry trgeted to the plsm memrne y the CAAX ox, showed strong ISH stining for in n otherwise -negtive morphnt ckground (Fig. 4). This indictes tht the electroported duplex restored mture to sustntil degree fter morpholinomedited knockdown. Retinl explnts cultured from eyes tht were electroported with high efficiency (ssessed y mcherry fluorescence) with oth the duplex nd Retinl growth cone collpse (%) Sem3A Sem3A + 24 h or Eight-cell stge emryo c Retinl growth cone collpse (%) Stge 24 emryo showed high Sem3A-induced collpse (64% ± 7) t 4 h in culture, similr to uninjected control growth cones t this time (Fig. 4c). This response ws significntly higher thn tht oserved in morphnt growth cones, whether electroported with control duplex (32% ± 2) or not (41% ± 5). The duplex thus rescued the loss of Sem3A responsiveness oserved in morphnts. To determine whether controls the onset of sensitivity to other cues, we exmined two other repulsive cues, Slit2 nd lysophosphtidic cid (LPA). Like Sem3A, collpse ssys show tht RGC growth cones gin responsiveness to Slit2 (refs. 6,7) nd LPA etween 24 h nd 48 h in culture (Supplementry Fig. 4). We oserved full collpse with LPA nd Slit2 in morphnt growth cones t 48 h (Supplementry Fig. 4,c). This contrsts with Sem3A, which induced significntly less collpse t 48 h in growth cones (see ove), nd indictes tht does not regulte the onset of ll repellent cue sensitivity. knockdown induces dely in expression of NRP1 NRP1 is Sem3A receptor, nd its precocious overexpression in young Xenopus RGC growth cones, normlly unresponsive to Sem3A, confers Sem3A sensitivity 6. NRP1 is thus sufficient to induce Sem3A responsiveness. Therefore, we resoned tht might lter Sem3A responsiveness y regulting the temporl expression of NRP1. To ssess this, we compred NRP1 immunorectivity in RGC growth cones using quntittive immunofluorescence. The NRP1 signl intensity (men pixel intensity per unit re) ws significntly lower on verge in stge 24 morphnt explnts grown for 36 h nd 48 h in culture thn in controls (Fig. 5,). Although most individul morphnt growth cones showed wek NRP1 stining, we noted some vriility, with occsionl growth cones displying high levels of NRP1 (Fig. 5c). By 64 h, however, NRP1 hd incresed significntly (P <.1) compred to 36 h nd 48 h, eing on verge lower thn ut not significntly different from controls (Fig. 5). Incresing the morpholino concentrtion y + 28 h + 32 h + 4 h + 48 h h + 48 h + 64 h Cultured retinl explnt 1 + Sem3A Sem3A Sem3A h + 48 h + 64 h 32 VOLUME 15 NUMBER 1 JANUARY 212 nture neuroscience

5 212 Nture Americ, Inc. All rights reserved. Figure 4 Electroported duplex rescues norml onset of Sem3A response. () Schemtic representtion of the experiment nd illustrtive collpsed growth cone (ox). Positive nd negtive electrodes re shown. We electroported 5 µm or control duplexes, long with 4 ng µl 1 CAAXmCherry to ssess the electroportion success. () ISH of stge 37/38 Xenopus hed sections. Exogenous duplex, ut not control duplex, ws detected in electroported retinl res (delineted y solid lck (rightfield imge) or white (fluorescence imges) line) in morphnt ckground. The decrese in fluorescent DAPI signl is likely due to msking effect of the ISH BM Purple precipitte. (c) Numer of collpsed growth cones in stge 24 retinl explnts grown in culture for 4 h. Growth cone responsiveness to Sem3A ws restored to control levels in morphnts electroported with duplex ut not with control duplex. All smples pssed Kolmogorov-Smirnov normlity test. ANOVA followed y Bonferroni post-test. Vlues re men ± s.e.m.;, not significnt; **P <.1. Numer of growth cones nlyzed re indicted in rs. Scle rs, 1 µm. Stge 24 emryo imges in modified from ref. 46, copyright 1994 P.D. Nieuwkoop nd J. Fer. Reproduced y permission of Tylor nd Frncis Group, LLC, division of Inform plc. fctor of two to four did not lter the chnge in NRP1 expression oserved in morphnts (Supplementry Fig. 5), indicting tht the effect ws not simply due to morpholino titrtion over time. This is consistent with the possiility tht regultes the temporl expression of NRP1 in growth cones. In ddition, the dt showed tht NRP1 expression in morphnts reched control levels t similr time to the onset of Sem3A responsiveness. The dely in NRP1 expression in morphnt growth cones is thus likely to underlie the concomitnt dely in Sem3A sensitivity. Eight-cell stge emryo mcherry + or control + duplex Stge 24 emryo Stge 24 emryo Cultured retinl explnt + Sem3A + 4 h + control duplex mir124-mo + duplex mcherry c 1 Retinl growth cone collpse (%) Control + Control duplex duplex DAPI morphnt RGC xons mke trgeting errors in vivo We next exmined whether the dely of Sem3A responsiveness shown in vitro in growth cones lcking could lso led to errors in pthfinding in vivo. Sem3A is encountered in discrete domins in the distl prts of the optic pthwy, including the ** ** + Sem3A Sem3A + 36 h + 48 h + 64 h c NRP1 NRP1 fluorescence intensity (% control) h + 48 h + 64 h Numer of growth cones (% totl) Fluorescence (AU) >55 Figure 5 Expression of NRP1 in growth cones is delyed in morphnts. () Illustrtive growth cones stined for NRP1; the white lines delinete the oundries of the growth cone nd the distl xon shft. Contrst nd rightness were oth incresed for illustrtion purposes. Scle r, 1 µm. () Fluorescence intensity ssocited with NRP1 immunostining mesured in growth cones from stge 24 explnts grown in culture for h. The fluorescence intensity ws lower in retinl growth cones from morphnts thn in controls fter 36 nd 48 h in culture ut not fter 64 h. Kruskl-Wllis test followed y Dunn post-test. Vlues re men ± s.e.m.;, not significnt; P <.1. Numers of growth cones nlyzed re indicted in rs. (c) Distriution of growth cone NRP1 fluorescence intensity from stge 24 explnts grown in culture for 48 h. AU, ritrry units. nture NEUROSCIENCE VOLUME 15 NUMBER 1 JANUARY

6 Dil or HRP Ipsi c d or Eight-cell stge emryo Stge 4 emryo Dissected contrlterl rin P-vlue Numer of emryos nlyzed Numer of optic projections with >5 ectopic xons <.5 Numer of ectopic xons per optic projection (% control ± s.e.m.) 1% ± % ± 48.9 <.1 c Devition ngle of ventrl xons (± s.e.m.) d 26.8 ± ± 1.5 Sem3A 212 Nture Americ, Inc. All rights reserved. Figure 6 RGC xons re misrouted in the ventrl tectum in morphnts. () Schemtic representtion of the experiment. We microinjected 125 µm or. () DiI-leled RGC xons visulized in the contrlterl rin (lterl view). The dotted lue line represents the nterior oundry of the tectum. (c) HRP-leled RGC xons (rown) visulized long with Sem3A mrna (purple). The lck dotted line delinetes the ventrl tectl oundry of the optic projection. In rins from morphnt emryos (,c, ottom), suset of ventrl xons (rrowheds) djcent to tectl site of Sem3A production filed to stop nd ppered misrouted towrd the ventrl prt of tectum. (d) Quntittive nlysis of the phenotype. Fisher exct test. A conservtive cut-off vlue (>5 errntly Sem3A trgeting xons) ws chosen to determine whether given ws considered norml. Vlues were normlized to the width of the t the nterior oundry of the tectum to ccount for vrition in DiI filling. c Unpired Mnn- Whitney U test. d Devition ngles formed y the 5 8 most ventrl xons () nd ventrl ectopic xons () to the medin xon of the (see lso Supplementry Fig. 6). We counted 213 ectopic xons in totl, nd the ngles formed y 337 xons were mesured in totl. Imges were djusted for rightness nd contrst. HRP, horserdish peroxidse; Ipsi, ipsilterl;, optic projection;, tectum. Scle rs, 5 µm (,c); inset (c), 1 µm. Stge 4 emryo imge in modified from ref. 46, copyright 1994 P.D. Nieuwkoop nd J. Fer. Reproduced y permission of Tylor nd Frncis Group, LLC, division of Inform plc. mid-optic trct, the ventrl order of the optic tectum (Supplementry Fig. 6) nd the cudl midrin 6. We rised morphnt emryos until stge 4, when pioneer retinl xons hve reched nd terminted in the tectum 31, nd trced RGC xons y nterogrde filling using the lipophilic dye DiI (Fig. 6). We found tht suset of RGC xons consistently filed to terminte in the tectum nd, insted, continued to project ventro-cudlly, often exiting the tectum t its ventrl order (Fig. 6) nd errntly trespssing into the Sem3A domin (Fig. 6c). This phenotype occurred in 67% of morphnt rins nlyzed, ut rrely (1%) in controls, (P <.5) nd ws chrcterized y significnt (P <.1) 3.5-fold increse in the numer of ectopic xons (Fig. 6d). Mesurement of the men ngle of devition from the optic projection medin showed tht the mistrgeting xons devited y 9.1, compred with 26.8 in control xons in the ventrl tectl re (Fig. 6d nd Supplementry Fig. 6). Long-rnge guidnce through the optic trct did not seem to e ffected. morphnt RGC xons thus showed errors t the ventrl tectl order close to where Sem3A is expressed, suggesting filure to respond to Sem3A in this re. Consistent with this, we found tht NRP1 expression ws significntly (P <.1) reduced y 5.2% ± 6.1 in morphnt RGC xons in vivo (Supplementry Fig. 7,c). To ddress whether this is cell-utonomous phenotype, we knocked down exclusively in retinl cells y electroporting ntisense oligonucleotides (ASOs) (either morpholino or LNA knockdown proes) in one eye of stge 26 emryos, when the first RGCs strt to differentite 21,31 (Fig. 7). We judged the knockdown of to e effective, s ASO electroported retinl cells were devoid of signl y ISH t stge 4 (Fig. 7 nd Supplementry Fig. 8). Pthfinding of ASO-electroported RGC xons ws nlyzed t stge 4. Agin, retinotectl xons misprojected t the ventrl tectl order (Fig. 7c f) like those in morphnts. The percentge of ectopic xons ws pproximtely three times tht in controls: 15% ± 2.9 ASO-electroported RGC xons errntly exited the ventrl tectum, compred to 5% ± 1.4 in controls (Fig. 7g). Time-lpse recording of elongting pioneer xons t stge 39/4 cptured few xons entering the tectum nd then misprojecting eyond its ventrl oundry (Fig. 7h). Although non-utonomous function of tectl mirna-124 cnnot e completely excluded, these dt collectively suggest tht cts intrinsiclly in the retin to promote ccurte trgeting of RGC xons to the tectum. regultes responsiveness to Sem3A vi CoREST As knockdown leds to the downregultion of NRP1 in the growth cone nd s mirnas generlly repress the expression of their trget mrnas, is likely to regulte NRP1 indirectly. We entertined the hypothesis tht such regultion might occur through silencing of repressive effector. Although little is known out the molecules regulting NRP1 expression, recent study reported tht the trnscriptionl repressor REST represses the expression of NRP1 in humn kertinocyte cell line, leding to loss of sensitivity to Sem3A 32. Of note, the 3 UTR of REST s primry cofctor, CoREST, ut not tht of REST itself, hs predicted inding site for (7mer-A1) tht is highly conserved from Xenopus to humn (Fig. 8). We tested, therefore, whether the predicted -inding site in the CoREST 3 UTR ws sufficient for medited inhiition of expression, using dul Renill:firefly luciferse reporter ssy (Fig. 8)., ut not the control duplex, induced 43% ± 2.6 decrese in the rtio of Renill to firefly luciferse ctivity. By contrst, hd no significnt effect (P >.5) on luciferse ctivity compred to control duplex when the seed of the inding site in the CoREST 3 UTR ws disrupted y muttion to give 3-nt mismtch (Fig. 8c). Tken together, these results demonstrte tht CoREST 3 UTR driven trnsltion or stility is negtively regulted y. We next ssessed the developmentl expression pttern of CoREST in the retin. If CoREST is one fide trget in vivo, it would e predicted to decline s increses. Indeed, ISH nlysis 34 VOLUME 15 NUMBER 1 JANUARY 212 nture neuroscience

7 or control ASO + Electroported stge 26 emryo Stge 4 emryo Ispi Dissected contrlterl rin Control ASO ASO DAPI FITC-ASO ISH g ASO Control ASO c e d f Percentge ectopic xons Control ASO ASO 19 Control ASO 17 ASO h Nture Americ, Inc. All rights reserved. Figure 7 cts utonomously to cuse retinl xon misrouting () Schemtic representtion of the experiment. ASOs (1 µm morpholino or µm LNA knockdown proe) were electroported. () ISH performed on emryos electroported with fluorescein isothiocynte (FITC)- leled morpholino nd fixed t stge 4. Cells electroported with ASO were devoid of endogenous ssocited signl, wheres controls showed strong ssocited signl throughout the retin, even in electroported cells. (c f) Fluorescing RGC xons projecting to the contrlterl rin (c,e), with corresponding zoomed imges (d,f). A supopultion of xons from RGCs, electroported with ASO, did not stop in the tectum ut misprojected ventrlly (e,f, rrow), wheres RGC xons ll stopped in the tectum in controls (c,d). (g) Numer of ectopic xons normlized to the numer electroported RGC xons in the tectum. Unpired Mnn-Whitney U test. Vlues re men ± s.e.m., P <.1. Numers of optic projections exmined re indicted in rs; 556 xons were nlyzed in totl. (h) Time-lpse imging of xons misprojecting in the ventrl tectum. After reching the tectum, n xon took shrp, 9 turn nd ws misrouted in distl-ventrl direction, insted of stopping nd rnching within the tectum (rrow). Time is shown in minutes. Imges were djusted for rightness nd contrst. Ipsi, ipsilterl;, optic projection;, tectum. Scle rs, 5 µm (); 1 µm (c,e); 5 µm (d,f); 2 µm (h). Emryo imges in modified from ref. 46, copyright 1994 P.D. Nieuwkoop nd J. Fer. Reproduced y permission of Tylor nd Frncis Group, LLC, division of Inform plc. reveled strong CoREST signl t stge 32 tht decresed progressively to rely detectle levels in the RGC lyer t stge 4, lthough wek signl persisted in the inner nucler lyer (Fig. 8d,e). Similr results were otined with two rioproes complementry to different regions of the CoREST coding sequence (dt not shown). Moreover, significnt (P <.1) CoREST signl persisted in the RGC lyer in morphnt retins t stge 4 compred to tht in controls (Fig. 8e,f), indicting tht regultes CoREST mrna stility in RGCs. High CoREST ctivity in RGCs when their xons enter the tectum would e predicted to repress NRP1. CoREST might thus ct downstrem of to determine the time of onset of growth cone sensitivity to Sem3A. We therefore next exmined whether CoREST, s trget, is responsile for the ltered Sem3A responsiveness seen in morphnts. If this is the cse, knocking down CoREST in morphnt should restore growth cone responsiveness to Sem3A. We thus knocked down oth CoREST nd y morpholino microinjection nd oserved tht this doule knockdown led to the rescue of Sem3A responsiveness in stge 24 explnts t 4 h (Fig. 8g). Finlly, we tested whether the CoREST-MO induced rescue of Sem3A sensitivity in morphnts ws due to chnge in NRP1 undnce. Upon microinjection of CoREST-MO, the NRP1 signl intensity ws significntly (P <.1) higher in growth cones thn in growth cones in stge 24 explnts t 4 h (Supplementry Fig. 9). Together the findings support model in which medited silencing of CoREST progressively leds to incresed NRP1 expression nd the concomitnt induction of growth cone sensitivity to Sem3A (Fig. 8h,i). DISCUSSION Growth cones chnge their responsiveness to cues en route to their trget 3,5,33, nd, in Xenopus RGCs, this chnge seems to e regulted y n intrinsic clock of unknown mechnism 5. Using loss-of-function pproch, we hve demonstrted tht controls the timing of expression of NRP1 nd therey confers growth cone sensitivity to Sem3A t the right time nd plce during nvigtion. Our dt lso reveled tht promotes proper trgeting to the tectum, consistent with medited regultion of retinl growth cone sensitivity to this repulsive guidnce cue. We identified CoREST s trget nd showed tht it medites s influence on growth cone ging. Tken together, these results indicte tht intrinsiclly regultes specific ge-relted chnges in the ehvior of growth cones nd thus promotes ccurte pthfinding. Growth cone ging in Xenopus RGCs is chrcterized y intrinsic chnges in the response to vrious cues nd in the undnce of their cognte receptors over time 5,6. We hve shown here tht ffects the temporl chnge in growth cone responsiveness to Sem3A, ut not to LPA or Slit2. This suggests tht does not ct s mster timer regulting growth cone ging overll ut rther s n uxiliry timer regulting only some spects of the progrm of growth cone chnges. Other molecules, perhps other mirnas, might ct in concert with to regulte other spects of growth cone ging. nture NEUROSCIENCE VOLUME 15 NUMBER 1 JANUARY

8 212 Nture Americ, Inc. All rights reserved. X. levis X. tropiclis Gllus gllus Mus musculus Homo spiens * * * * * * * * * * * * * * * * * * * * d Stge 32 Stge 37/38 Stge 4 d r r r 3 - L L 7mer-A1 site 5 xl- e GCL f CoREST mrna-relted intensity in GCL (% control) Figure 8 CoREST medites ction on Sem3A responsiveness. () Sequence lignment of the 3 UTR of L MO g Retinl growth cone collpse (%) WT 3 UTR P Renill luc. CoREST 3 UTR P Firefly luc. MUT 3 UTR P Renill luc. CoREST 3 UTR * * Control h Young RGC: CoREST Repressor NRP1 No response to Sem3A co -MO Old RGC: CoREST Repressor Response to Sem3A CoREST from vrious species. () Schemtic representtion of Xenopus CoREST 3 UTR, with wild-type (WT) or mutted (MUT) inding site, sucloned downstrem of dul Renill:firefly luciferse (luc.) reporter. (c) Quntifiction of reporter ctivity fter trnsfection of NRP1 i Concentrtion Concentrtion P Firefly luc. + CoREST-MO Wild type corest mir-124 NRP1 + Sem3A Sem3A St. 24 Onset of St. 4 Sem3A responsiveness morphnt St. 24 c Renill:firefly luciferse ctivity (% control) No duplex Control duplex duplex WT 3 UTR St. 4 Threshold MUT 3 UTR Threshold Time Time Onset of Sem3A responsiveness Dely 1 5 pmol Xenopus or control duplex in HEK (humn emryonic kidney)-293t cells. (d) Oculr distriution of CoREST mrna detected y ISH. Solid lck line delinetes the outer oundry of the retin nd the dshed line demrctes the peripherl re devoid of stining. (e) Retinl CoREST mrna distriution in stge 4 emryos injected with 125 µm or. (f) Quntifiction of CoREST mrna signl intensity in the GCL. (g) Numer of collpsed growth cones in stge 24 retinl explnts grown in culture for 4 h fter microinjection of 25 µm (1.1 ng) CoREST-MO, stndrd commercilly ville control morpholino (co -MO) nd/or 125 µm. (h,i) Model of -medited regultion of growth cone ging (see text for explntion). Sttistics: (c,g) All smples pssed Kolmogorov-Smirnov normlity test. ANOVA followed y Bonferroni post-test. (f) Unpired Mnn-Whitney U test. Vlues re men ± s.e.m. throughout figure;, not significnt; *P <.5, P <.1. Numers in rs indicte numer of retins exmined (f; up to seven 18-µm sections were nlyzed per retin) or numer of growth cones nlyzed (g)., nterior; d, dorsl; L, lens; P, promoter; r, retin; xl, Xenopus levis. Scle rs, 1 µm (d), 25 µm (e). It is of interest tht other mirnas profiled in the Xenopus retin, such s mir-9, re lso predicted to trget guidnce receptors tht chnge over time. In ddition, the temporlly coordinted ction of set of mirnas hs een reported to control progenitor cell fte y silencing multiple trgets in vertertes 14. Individul mirnas my thus hve more restricted role in regulting developmentl timing in vertertes, s previously proposed 18, thn in Cenorhditis elegns. Our results reveled tht knockdown of CoREST rescues the reduced Sem3A responsiveness of morphnt growth cones through prtil restortion of NRP1 to the growth cone. Therefore, it seems resonle to infer tht CoREST medites s effect on the regultion of NRP1 nd ssocited growth cone responsiveness to Sem3A. Of note, other vlidted trgets re present in RGCs 34 (Supplementry Tle 1), suggesting tht might ffect dditionl pthwys. Although none of these trgets hs een shown to e iologiclly linked to neuropilin-1 or CoREST function in neurons, we cnnot rule out cross-tlk etween these potentil regulted pthwys nd CoREST-medited regultion of NRP1 nd growth cone sensitivity to Sem3A. The loss of did not led to the sustined loss of NRP1 ut to its delyed expression. ws sent from emryos until t lest 72 hpf similr to oservtions in zerfish, where - MO induces complete knockdown of endogenous until t lest hpf (ref. 23). This period spns the time frme for which we oserved the resumption of growth cone sensitivity to Sem3A. In ddition, the oserved phenotype ws not secondry to the morpholino eing clered from the RGCs. Thus NRP1 expression nd Sem3A sensitivity re eventully switched on in the sence of, rguing for n effect on timing rther thn simple recovery from repression through return of endogenous. Our results showed tht CoREST nd hd opposite expression profiles in the retin over the sme time period, such tht 36 VOLUME 15 NUMBER 1 JANUARY 212 nture neuroscience

9 212 Nture Americ, Inc. All rights reserved. CoREST decresed while incresed. In ddition, knockdown delyed the developmentl decline in CoREST mrna expression in the GCL. The medited post-trnscriptionl silencing of CoREST tht we report here might thus e due to ccelerted clernce of the remining CoREST mrnas in RGCs. We therefore suggest tht, rther thn cting s simple off switch like C. elegns heterochronic mirnas 11 13, shrpens the window over which CoREST mrna clernce occurs. Such interction hs een descried for other developmentl processes 15, such s the mir-43 medited ccelerted degrdtion of mternl mrnas in zerfish 35. Along these lines, we propose model wherey the temporl increse of over the period of RGC differentition leds to the ccelerted decy of CoREST expression, promoting, in turn, rpid NRP1 expression in growth cones, synchronizing the onset of Sem3A sensitivity with Sem3A encounters. Overll, the increse in determines the time t which NRP1 is expressed ove the level tht is necessry for growth cones to show sensitivity to Sem3A in res where Sem3A is expressed, such s the ventrl tectl order. In sence of, this level would e reched lter in development, desynchronizing growth cone sensitivity from exposure to Sem3A in the pthwy (Fig. 8h,i). Previous studies hve demonstrted function for cue-induced locl trnsltion in xons nd growth cones in guidnce, with the min clss of trnslted proteins identified s cytoskeletl regultors such s RhoA nd β-ctin Of interest, CoREST mrna is present in RGC somt ut not in growth cones throughout the period of pthfinding 34. Similrly here, ppered enriched in the cell som nd ws only wekly detected in growth cones y ISH (dt not shown). The increse in NRP1 t the growth cone my thus e direct consequence of trnscriptionl de-repression nd susequent trnsport of the protein to this comprtment. This possiility is supported y the recent findings tht NRP1 mrna is sent from RGC growth cones 34. This is lso consistent with other studies tht show trnscriptionl control of guidnce receptors in xons; for instnce, Roo 39. mrnas encoding receptors for guidnce cues such s neuropilin-2 ( receptor for Sem3F nd Sem3C 4 ), ctivin nd Eph receptors re detected in RGC growth cones 34, nd EphA2 hs een shown to e trnslted in commissurl xons 41. This suggests receptor nd context-specific trnsltionl regultion in growth cones. Overll, this implies tht induced growth cone ging my e centrlly nd not loclly regulted, nd this centrl regultion might led to progressive chnges in the moleculr mkeup of the growth cone over time. knockdown resulted in the filure of suset of RGC xons to stll t the ventrl order of the tectum. The sutlety of this in vivo phenotype contrsted with the roust in vitro defect nd likely reflects moleculr redundncy. Severl different guidnce mechnisms comine to ccurtely guide xons to their trgets in vivo, nd retinl xons re guided y multiple cues to the tectum 42. For instnce, doule ut not single knockdown of Sem3A nd Slit leds to strong guidnce phenotype of the retinotectl projection 43. Similrly, in studies of retinotectl topogrphy, multiple-knockdown pproch is needed to fully impir even single mpping xis (for exmple, ref. 44). As ffects the responsiveness to Sem3A ut not to the repulsive tectl cue Slit or LPA, repulsive ction of other cues likely contriutes to the sutle phenotype reported in our study. The xons tht project erroneously out of the tectum originted from xons positioned very close to the Sem3A domin tht orders the ventrl tectum, nd these xons my e prticulrly vulnerle ecuse they my rely more hevily on Sem3A thn other cues t this point in the pthwy. Despite its sutlety, the in vivo trgeting defect is consistent with the possiility tht morphnt RGC growth cones re not ppropritely repelled y Sem3A owing to delyed NRP1 expression. This is supported y our findings tht upregultion of NRP1, nd concomitnt growth cone responsiveness to Sem3A, ws delyed nd NRP1 undnce ws significntly lower in most morphnt RGC growth cones thn in controls, t n in vitro stge corresponding to stge 4 in vivo, nd in stge 4 RGC xons in vivo. Overll, might ensure tht RGC growth cones express sufficient NRP1 to enle n dequte repulsive response to tectl Sem3A t the right time, nd trgeting of xons in the tectum. would thus regulte xon guidnce. A previous report 45 reveled tht mirnas, s clss of molecule, re involved in xon guidnce in the developing visul system. However, the mirna(s) responsile for this phenotype were not identified. In ddition, the study did not prove tht the phenotype ws directly cused y lck of mirnas in RGCs. The present work is thus, to our knowledge, the first report demonstrting tht specific mirna directly regultes xon pthfinding. In summry, we provide evidence tht, through the direct repression of CoREST, specificlly nd intrinsiclly regultes the timing of NRP1 expression, ensuring tht the onset of growth cone responsiveness to its cognte lignd, Sem3A, occurs t the right time in the development of the optic pthwy. thus contriutes to the intrinsic regultion of Xenopus RGC growth cone ging nd therey ensures proper xonl trgeting to the tectum. Our findings further suggest tht, y cting s cellulr timer in RGCs, is importnt in synchronizing the responsiveness of RGC growth cones to relevnt cues in their locl microenvironment. METHODS Methods nd ny ssocited references re ville in the online version of the pper t Accession codes. Gene Expression Omnius: GSE Note: Supplementry informtion is ville on the Nture Neuroscience wesite. Acknowledgments The uthors wish to thnk J. Woolford for her contriution to the nlysis of high-throughput sequencing dt, H. Lynn for help on cloning nd M. Wood for prticiption in collpse nlysis. We lso thnk A. Dwivedy, L. Strochlic, J. Flk, K. Leung, M. Agthocleous, A. Lin, S. McFrlne nd A. Almeid for technicl ssistnce. We thnk W. Hrris, A.C. Lee, J. Yoon nd M. Agthocleous for criticlly reding the mnuscript, s well s ll the memers of the Hrris/Holt lortory for their input. This work ws supported y postdoctorl fellowships from the Ntionl Reserch Council of Cnd (M.-L.B.) nd Alert Heritge Foundtion for Medicl Reserch (M.-L.B.), y Wellcome Trust Progrmme nd UK Medicl Reserch Council grnts (C.E.H.) nd y Europen Frmework 6 grnt (SIROCCO) (E.A.M.). AUTHOR CONTRIBUTIO C.E.H. nd M.-L.B. conceived the project. M.-L.B. performed most of the experiments nd dt nlysis. M.-L.B., K.H.Z., C.A.-G., J.A., C.B. nd L.D.G. prticipted in profiling experiments nd nlyses. K.H.Z. contriuted to Slit2 nd LPA collpse ssys. C.A.-G. determined the puttive trgets of. A.M. performed ISH on cryosections of wild-type Xenopus. E.A.M. designed nd discussed profiling experiments. M.-L.B. nd C.E.H. wrote the mnuscript. competing FINANCIAL INTERESTS The uthors declre no competing finncil interests. Pulished online t Reprints nd permissions informtion is ville online t reprints/index.html. 1. Tessier-Lvigne, M. & Goodmn, C.S. The moleculr iology of xon guidnce. Science 274, (1996). 2. Dickson, B.J. Moleculr mechnisms of xon guidnce. Science 298, (22). nture NEUROSCIENCE VOLUME 15 NUMBER 1 JANUARY

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