Oligodendrocyte progenitors balance growth with self-repulsion to achieve homeostasis in the adult brain

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1 Oligonroyt prognitors ln growth with slf-rpulsion to hiv homostsis in th ult rin Ethn G Hughs 1,3, Shin H Kng 1 3, Mshiro Fuky 1 3 & Dwight E Brgls 1 npg 213 Ntur Amri, In. All rights rsrv. Th ult CNS ontins n unnt popultion of oligonroyt prursor lls (NG2 + lls) tht gnrt oligonroyts n rpir mylin, ut how ths uiquitous prognitors mintin thir nsity is unknown. W gnrt NG2-mEGFP mi n us in vivo two-photon imging to stuy thir ynmis in th ult rin. Tim-lps imging rvl tht NG2 + lls in th ortx wr highly ynmi; thy survy thir lol nvironmnt with motil filopoi, xtn growth ons n ontinuously migrt. Thy mintin uniqu trritoris though slf-voin, n NG2 + ll loss though th, iffrntition or ltion triggr rpi migrtion n prolifrtion of jnt lls to rstor thir nsity. NG2 + lls rruit to sits of fol CNS injury wr similrly rpl y prolifrtiv urst surrouning th injury sit. Thus, homostti ontrol of NG2 + ll nsity through ln of tiv growth n slf-rpulsion nsurs tht ths prognitors r vill to rpl oligonroyts n prtiipt in tissu rpir. Homostti ontrol of ll nsity is n ssntil ftur of tissu n orgn mintnn, llowing ll rplmnt n rgnrtion to offst ll loss rsulting from injury, iss or g-pnnt gnrtion 1,2. Tight ontrol ovr ll prolifrtion is spilly ritil in th ult CNS, whih hs limit pity to ommot growth s rsult of its omplx llulr rhittur n its nsmnt in on. In ontrst with nurons, whih, prt from rstrit popultions in th hippompus n olftory ul, r not rpl vn in th ontxt of injury n iss 3, mny glil lls xhiit rmrkl pity for slf-rnwl 4,5. Howvr, it is not known how th nsity n istriution of iffrnt lsss of glil lls r mintin in th ult CNS. Glil prognitor lls tht xprss th honroitin sulft protoglyn NG2, trm NG2 + lls (or oligonroyt prursor lls), ompris th mjority of prolifrting lls in th ult CNS 6. During vlopmnt, ths glil lls migrt from grminl zons, prolifrt n iffrntit into mylinting oligonroyts 7 9. Although mylint trts r form rly in lif, NG2 + lls r rtin throughout th ult CNS, whr thy r orgniz in gri-lik or til mnnr, with iniviul lls oupying non-ovrlpping omins 1. In vivo gnti ft tring stuis init tht NG2 + lls ontinu to iffrntit into oligonroyts in ults 7,11 13 n r rpily moiliz to rpl oligonroyts in niml mols of ut n hroni mylintion 4,14,15, suggsting tht thy r importnt for oth norml oligonroyt homostsis n th rgnrtion of mylin. Although ontinul rnwl of ths prognitors is likly to ruil for ffiint oligonrognsis, th mhnisms tht ontrol thir uniform istriution n high nsity in th ult CNS rmin unknown, in prt us thir ynmis hv not n xmin in th intt ult CNS 9,16,17. NG2 + ll prolifrtion is nhn following mylintion 15, trumti injury to th CNS 18 n in hroni nurognrtiv iss 7,19 ; howvr, th rltionship twn prolifrtion of ths prognitors n th gnrtion of nw oligonroyts rmins unrtin 2. Morovr, unontroll growth of ths prognitors ls to tumor formtion 21, n rnt stuis hv suggst tht NG2 + lls r likly to ll of origin for rtin forms of gliom 22,23, highlighting th importn of unrstning how th prolifrtion of ths lls is ontroll in vivo. To rss ths qustions, w vlop lin of trnsgni mi tht xprss mmrn-nhor form of EGFP unr th ontrol of th NG2 (Cspg4) promotr (NG2-mEGFP-H mi) n prform in vivo two-photon imging of NG2 + lls in th mous somtosnsory ortx. W foun tht NG2 + lls wr highly ynmi in th ult rin; thy xtn motil filopoi, rorgniz thir prosss n ontinuously mov through th prnhym. Although thir position ws not fix, NG2 + lls mintin inpnnt omins through slf-rpulsion, n loss of lls through th, iffrntition or xprimntl ltion triggr rpi migrtion n prolifrtion of jnt NG2 + lls to prsrv thir nsity. Long-trm imging rvl tht NG2 + lls irtly iffrntit into oligonroyts without prolifrtion, initing tht ivision of ths prognitors is homostti rspons to ll rmovl, rthr thn th gnrtion of oligonroyts through symmtri ivision. Although ult NG2 + lls n srv s oligonroyt prognitors, thy lso migrt to sits of fol injury to hlp form glil sr n wr similrly rpl through prolifrtion of nighoring NG2 + lls. By lning tiv growth with slf-rpulsion, NG2 + lls mintin onstnt nsity in th CNS, nsuring tht thy r vill to prtiipt in rgnrtion n rpir of th CNS throughout lif. 1 Th Solomon H. Snyr Dprtmnt of Nurosin, Johns Hopkins Univrsity Shool of Miin, Bltimor, Mryln, USA. 2 Prsnt rsss: Cntr for Nurl Rpir n Rhilittion, Tmpl Univrsity Shool of Miin, Phillphi, Pnnsylvni, USA (S.H.K.) n Dprtmnt of Antomy, Kitsto Univrsity Shool of Miin, Sgmihr, Jpn (M.F). 3 Ths uthors ontriut qully to this work. Corrsponn shoul rss to D.E.B. (rgls@jhmi.u). Riv 16 Jnury; pt 28 Mrh; pulish onlin 28 April 213; orrt onlin 6 My 213; oi:1.138/nn VOLUME 16 NUMBER 6 JUNE 213 ntur NEUROSCIENCE

2 npg 213 Ntur Amri, In. All rights rsrv. RESULTS Ault NG2 + lls xtn prosss with ynmi filopoi To fin th hvior of NG2 + lls in th ult rin, w gnrt BAC trnsgni mi tht xprss mmrn-nhor form of EGFP 24 unr th ontrol of th NG2 promotr (NG2-mEGFP-H mi; Supplmntry Fig. 1), whih llow visuliztion of th full xtnt of thir omplx morphologis (Supplmntry Fig. 2). In vivo two-photon imging through rnil winow rvl tht fluorsnt NG2 + lls wr istriut in highly orr mnnr throughout th uppr lyrs of th ortx of ths mi (Supplmntry Movi 1), with lls oupying non-ovrlpping omins. NG2 + lls xtn highly rmifi prosss stu with numrous filopoi-lik protrusions into th surrouning nuropil (Fig. 1). In tim-lps rorings, ths thin protrusions wr highly ynmi, ontinully xtning, rtrting, rnhing n ltring thir trjtory (Fig. 1 n Supplmntry Movis 2 n 3) with n vrg sp of.3 ±.1 µm min 1 (185 protrusions on 1 lls in 5 mi). Motil filopoi wr prsnt long th lngth of h pross n vn xtn from th som (Fig. 1,,), initing tht NG2 + lls tivly survy thir lol nvironmnt. This ynmi hvior is similr to tht xhiit y miroglil lls 25,26, lthough NG2 + ll filopoi lk th ulous nings tht r hrtristi of miroglil prosss n mov mor slowly (mirogli pross sp = 1.47 ±.1 µm min 1 ; from rf. 26). Dspit th tiv growth of NG2 + ll prosss, ths lls mintin ril morphology n ontts twn prosss wr rrly osrv. Prvious stuis foun Figur 1 NG2 + lls xtn ynmi filopoi n xhiit slf-rpulsion in th ult ortx. () Mximum-intnsity projtion of n iniviul NG2 + ll uring 1 h of tim-lps imging. Stl rgions r shown in lk n ynmi rgions r shown in mgnt. () Iniviul NG2 + ll filopoi xtn (top), rnh (mil) n ltr thir trjtory (ottom) in minuts. () Plots showing th hng in lngth ovr tim of six NG2 + ll filopoi. () Mximum-intnsity projtion of on NG2 + ll pross, olor o for tim (intrvls low = 4 min). Stl rs r rprsnt in whit. () Grph showing th istriution of ynmi filopoi long NG2 + ll prosss (n = 13 rnhs on 7 lls in 5 mi, n.s. inits not signifint (P >.5), on-wy ANOVA with Tuky post ho tst). Error rs rprsnt s..m. (f) In vivo timlps imgs of two psuo-olor NG2 + ll prosss. Contt l to filopoil rtrtion. (g) Plot of th hng in lngth of NG2 + ll filopoi for n ftr ontting nothr NG2 + ll prosss (n = 35 filopoi on 1 lls in 5 mi). Avrg hng in lngth for ll prosss is shown in lk. (h) In vivo timlps imging of vning (top) n rtrting (ottom) NG2 + ll prosss. Montgs t fr right ( 6 min) r mximum-intnsity projtion imgs showing two NG2 + ll prosss olor o for tim (intrvls low = 3 min). Stl rs r rprsnt in whit. Not th prsn of highly motil filopoi t th tip of th vning pross n th sn of filopoi on th rtrting pross. tht ontt-mit inhiition n hlp stlish ril morphology n ontrol ll sping through homotypi rpulsion 27,28. In tim-lps imging, growing filopoi of NG2 + lls lwys rtrt whn thy ontt pross of th sm or n jnt NG2 + ll (Fig. 1f,g n Supplmntry Movi 3), initing tht ths prognitors ngg in ontinul slf-voin through onttmit inhiition. Prvious stuis hv shown tht rmovl of th skull n implnttion of hroni rnil winow n inu rtiv hngs in glil lls n nhn th ynmis of nriti spins on nurons 29. To trmin whthr th ynmi hvior of NG2 + lls ws rtiv rspons to injury, w prpr NG2-mEGFP-H mi with thinn-skull rnil winows 3 n prform tim-lps imging. NG2 + lls img through th skull h omplx morphologis n highly ynmi filopoi wr osrv long thir prosss, oth of whih wr omprl to thos osrv in mi implnt with hroni rnil winows (Supplmntry Fig. 3 n Supplmntry Movi 4). Morovr, mi implnt with hroni rnil winows i not show rtiv hngs in mirogli or inrss in th numr of prolifrting lls rltiv to ontrols whn in vivo imging ws initit (Supplmntry Fig. 4). Ths rsults init tht Stl Dynmi (1h) Filopoil ynmis 6 min f h 5 µm 1 µm 1 min Avning pross Rtrting pross µm µm g Lngth (µm) Numr of ynmi filopoi Contt min 33 min min n.s. Distn from ll oy (µm) 2 µm Tim (min) 2 µm ntur NEUROSCIENCE VOLUME 16 NUMBER 6 JUNE

3 Figur 2 NG2 + lls ontinully hng thir position in th ult ortx. () Img of NG2 + lls in on rgion of ortx on y (lft) n 32 ltr (right, montg of two tim points). Stl priyts r shown in yllow. () Montg showing th hng in morphology n position of thr NG2 + lls ovr 4. () Histogrm showing th istns tht NG2 + lls (grn rs) n privsulr lls (r rs) mov ovr 2 wks (318 NG2 + lls, 53 privsulr lls in 5 mi, P <.5, Mnn-Whitny tst). () Histogrm of th sp tht NG2 + lls n priyts mov ovr 2 wks (318 NG2 + lls, 53 privsulr lls in 5 mi, P <.5, Mnn-Whitny tst). () Thr-imnsionl grphs showing th movmnts of thr NG2 + lls (lft) n thr privsulr lls (right) in th somtosnsory ortx ovr 2-wk prio. (f) Grph showing th isplmnt of somt ovr tim for fiv NG2 + lls n fiv priyts. (g) Vtor plots of th irtion n isplmnt of 95 NG2 + ll movmnts in th X-Y pln (lft, P =.578, Moor-Ryligh tst) n th Z pln (right) for lls >9 µm low th pi mtr. Frquny Priyts 5 µm 8 NG2 + lls Priyts 6 Frquny µm npg 213 Ntur Amri, In. All rights rsrv. th xtnsion of ynmi filopoi from NG2 + ll prosss is norml ftur of ths prognitors in th uninjur rin, rthr thn rtiv rspons to rnil winow implnttion. NG2 + lls migrt ontinuously through th ult ortx Th tips of vning NG2 + ll prosss isply numrous motil filopoi, rminisnt of nuronl growth ons, whrs rtrting prosss lk filopoi (Fig. 1h), suggsting tht ths ynmis my nl ll migrtion. To trmin whthr pross motility is ompni y somti trnslotion, w r-img NG2 + lls in th sm volum of tissu 1 month ltr. Although th position of vsulr-ssoit priyts in ths rgions ws unhng, th istriution of NG2 + ll somt n th orinttion of thir prosss wr mrkly ltr, n it ws not possil to unmiguously intify th sm NG2 + lls from th prvious imging sssion (Fig. 2). Mor frqunt imging rvl tht NG2 + lls ontinully rorint thir prosss n mov n vrg istn of 2.1 ±.1 µm 1 (318 NG2 + lls in 5 mi; Fig. 2 n Supplmntry Movis 5 n 6), in ontrst with priyts, whih rmin lrgly sttionry (Fig. 2,f). Iniviul NG2 + lls xhiit htrognous hviors, with som lls moving t ontinuous rt uring th 2-wk imging prio, whrs othrs intrrupt prios of rltiv stility with lrg movmnts of >3 µm ovr prio of fw ys (Fig. 2f) f Distn (µm) Z (µm) Totl istn (µm pr 2 wks) Sp (µm pr ) Cll 1 Cll 2 Cll X (µm) NG2 + lls Priyts 4 6 Dys NG2 + lls g Displmnt (µm) Y (µm) Cll 1 Cll 2 Cll 3 X-Y pln Z pln Vtor sum Long-rng us gui th migrtion of NG2 + lls uring vlopmnt 31, rising th possiility tht similr grints influn thir movmnt in th ult CNS. Howvr, nlysis of th trjtoris of ll NG2 + lls in.6-mm 3 ortil volum rvl tht thr ws no irtionl is in thir movmnt (Fig. 2g), suggsting tht thir hvior is ontroll y lol intrtions rthr thn lrg-sl grints of ttrtiv or rpulsiv us Z (µm) Priyts X (µm) Displmnt (µm) Y (µm) Prnt of lls pr y Prolifrtion Diffrntition Dth Dth Diffrntition Prolifrtion Stl In Out Unsur Cll ount gin µm Dys Numr of NG2 + lls Figur 3 NG2 + ll nsity is mintin spit prolifrtion, iffrntition, n th. ( ) Squntil imgs from in vivo timlps rorings illustrting iniviul NG2 + lls unrgoing ivision (), th () n iffrntition (). Th img intnsity t 9 in ws inrs thrfol to illustrt th trnsition to n oligonroyt morphology. EGFP intnsity rs with iffrntition s rsult of ownrgultion of th NG2 promotr 1. () Grph illustrting th proportion of NG2 + lls ngg in iffrnt hviors on h y (1,118 NG2 + lls in 5 mi). () Comin plot showing th numr of NG2 + lls unrgoing prolifrtion, iffrntition n th (olor rs) n th totl NG2 + ll nsity (lu lin) in.6-mm 3 volum of ult somtosnsory ortx ovr 4- prio. 67 VOLUME 16 NUMBER 6 JUNE 213 ntur nuroscience

4 Diffrntition Dth gin Figur 4 NG2 + ll nsity is mintin through lol prolifrtion. (,) Imgs from tim-lps imgs showing tht NG2 + ll iffrntition (grn ll, ) or th (r ll, ) is ssoit with prolifrtion (yllow rrowhs) of nighoring NG2 + ll (yn). Th intnsity of th iffrntiting ll t y 4 in hs n inrs thrfol to highlight th morphologil hng. () Prntg of iffrntiting, ying 5 µm Prntg with lol prolifrtion Prntg with lol prolifrtion 1 ** ** Diffrntition 8 6 Dth Control ** * Diffrntition Dth Control n stl NG2 + lls tht wr ssoit with prolifrtion of nighoring NG2 + ll (72 iffrntiting, 54 ying, 187 stl NG2 + lls in 5 mi; **P <.5 on-wy ANOVA with Tuky post ho tst). () Tim ours of lol prolifrtion rltiv to th onst of ll loss through th or iffrntition (*P <.3, **P <.5, on-wy ANOVA with Tuky post ho tst). Error rs rprsnt s..m. npg 213 Ntur Amri, In. All rights rsrv. Constnt turnovr of NG2 + lls in th ult rin In ition to th onstnt rorgniztion of NG2 + ll position in th ortx, thr wr ynmi hngs in th ovrll popultion rsulting from th prolifrtion, iffrntition n th of iniviul NG2 + lls (Fig. 3 ). Cll th ws hrtriz y hyprtrophy, rtrtion of prosss n rupt ispprn of th ll (Supplmntry Fig. 5), whrs iffrntition into oligonroyts ws ompni y grul rs in fluorsn, trnsformtion of thir highly rmifi prosss to long, unrnh prosss n xprssion of mylin protins (Supplmntry Fig. 6). NG2 + lls i not iffrntit into stroyts or nurons in this Diffrntition 2 4 rgion, proviing furthr vin tht ths glil lls srv s lingrstrit prognitors in th norml CNS 7 ; howvr, if EGFP is gr mor rpily uring trnsiffrntition, option of ths ltrnt fts woul iffiult to ssss using this pproh. In ontrst with th sgrgtion of prinipl nurons into istint ortil lmin, NG2 + lls wr vnly istriut mong ortil lyrs, n th or hviors (prolifrtion, iffrntition n th) wr not is to prtiulr lmin (Supplmntry Fig. 7). On h y, 3% of NG2 + lls wr ngg in ths ynmi hviors (Fig. 3), whih xpn to inlu 36% of th popultion ovr 2 wks. Notly, ll loss through th or iffrntition µm Ar of inving prosss (µm) 2, * 1,5 1, 5 Avrg 2 2 Diffrntition Dth Figur 5 Nighoring NG2 + ll prosss Invsion 25 µm inv th trritory of iffrntiting, ut not ying, NG2 + lls. () In vivo tim-lps imgs of n iniviul NG2 + ll (psuoolor grn) tht iffrntit into n oligonroyt uring th 8- imging prio. Not tht iffrntition rsult in prosss xtnsion n prolifrtion (yllow rrowhs) Dth of th nighoring NG2 + ll to th lft of th iffrntiting ll. () Montg showing th trritory of th iffrntiting ll (yllow) ovrli with th prosss of nighoring NG2 + lls (whit) tht ntr th trritory of th iffrntiting ll. Thr ws xtnsiv invsion of ll trritory vry rly in th iffrntition 25 µm pross. () In vivo tim-lps imgs of n iniviul NG2 + ll (psuo-olor r) tht i uring th 4- imging prio. () Montg showing th trritory of th ying ll (yllow) ovrli with th prosss of nighoring NG2 + lls (whit) tht ntr th trritory of th ying ll. Sl r pplis to oth n. () Ar of prosss tht inv th trritory of iffrntiting or ying NG2 + lls (1 iffrntiting, 1 ying NG2 + lls in 5 mi, *P <.5, pir two-til Stunt s t tst). Error rs rprsnt s..m. ntur NEUROSCIENCE VOLUME 16 NUMBER 6 JUNE

5 Figur 6 NG2 + ll ltion triggrs trritory invsion n ivision of nighoring NG2 + ll. () In vivo tim-lps imgs of n iniviul NG2 + ll ovr 1. Th trritory of th ll is outlin in yllow. Prosss of nighoring NG2 + lls hv n psuo-olor grn. Not th lk of trritory invsion ovr 1 (fr right, invsion). () Singl Z pln imgs of n iniviul NG2 + ll for n ftr rif xposur of th ll oy to th fous lsr m to inu ltion. Th r rrow highlights th inrs in utofluorsn of th nulus ftr illumintion. Pnl with = 17 µm. () Mximum intnsity projtion img of th NG2 + ll shown in for ltion. Th trritory of th ll is outlin in yllow. () Montg of th sm volum of tissu in 1 following ll ltion. Prosss of nighoring NG2 + lls hv n psuo-olor grn. Sl r pplis to, n. () In vivo timlps imgs of thr nighoring NG2 + lls (psuo-olor grn, yllow n mgnt) from thinn-skull prprtion. Two NG2 + lls (shown in yllow n mgnt) wr rmov y lsr-mit ltion on y 1. On y 4, nighoring NG2 + ll (grn) ivi (yllow rrowhs). (f) Mp of th som position of NG2 + lls ovr 4 in ontrol. Nwly gnrt NG2 + lls r rprsnt in r. (g) Snpshot of NG2 + lls istriution for ll ltion. Clls to trgt for ltion r irl in lu. (h) Mp of th som position of NG2 + lls ovr 4 following ltion of four NG2 + lls (shown in g). Nwly gnrt NG2 + lls r rprsnt in r. Bfor Control Bfor min 1 min 1 h Cll ltion 1 Bfor 1 1 invsion 1 invsion 25 µm 4 npg 213 Ntur Amri, In. All rights rsrv. (1.2 ±.1% 1 ) ws ln y ll ition through prolifrtion (1.5 ±.1% 1, P =.9), llowing th nsity of NG2 + lls to rmin onstnt (, 162 NG2 + lls; 14, 159 NG2 + lls; 5 mi, P =.9, unpir two-til Stunt s t tst; Fig. 3), initing tht this prognitor pool is onstntly rorgniz y th ontinul loss n ition of nw lls. NG2 + ll nsity is mintin through lol prolifrtion If homostti ontrol of NG2 + ll nsity is govrn y lol intrtions, thn iffrntition or th shoul ssoit with ivision of nighoring ll. W osrv tht 98% of iffrntiting Frquny 1, Numr of ll ivisions Frquny µm Cll yl lngth () NG2 + ll Oligonroyt Cll ltion 5 µm Prolifrtion Bfor -4 f g h Control 5 µm n 76% of ying NG2 + lls wr ssoit with th prolifrtion of nighoring NG2 + ll (<5 µm wy), whrs only 43% of stl NG2 + lls wr ssoit with lol prolifrtion (P <.5 for oth th n iffrntition; Fig. 4 ). Morovr, th proility of lol prolifrtion in instns of th or iffrntition ws highst t th tim of ll loss, ut i not vry with tim for ontrol NG2 + lls (Fig. 4). Nighoring NG2 + lls rpily inv th trritory of iffrntiting lls (Fig. 5,,), suggsting tht th homotypi rpulsiv us tht prvnt ovrgrowth r ownrgult with iffrntition. In ontrst, ying NG2 + lls mintin istint, lit progrssivly smllr, trritoris until rmovl (Fig. 5 ). Th grul rs in th trritory of ying lls (Supplmntry Fig. 5) osionlly rsult in rpositioning of nighoring NG2 + lls without ll prolifrtion, ounting for th somwht lowr inin of nighor prolifrtion in ss of th. To furthr ssss whthr ll loss is suffiint to inu lol prolifrtion, w lt iniviul NG2 + lls in vivo using fol lsr illumintion n xmin th rspons of nighoring NG2 + lls. Following ll rmovl, nighoring NG2 + lls rorint thir prosss n inv th formr trritory of th lt ll, proviing Stl Diffrntition Dth Out of fil Dys Dys Figur 7 NG2 + lls irtly iffrntit into oligonroyts without symmtri ivision. () Squntil imgs from in vivo tim-lps rorings of on NG2 + ll tht ivi twi ovr 26. Nwly gnrt lls r shown in yn n yllow. () Histogrm of th istriution of prolifrtion frqunis of NG2 + lls in th somtosnsory ortx (828 NG2 + lls in thr mi img for 4 ). () Histogrm of NG2 + ll yl lngth (97 NG2 + ll ivisions in 3 mi). () Ling trs of highly prolifrtiv NG2 + lls illustrting th rng of fts of sistr lls. () Ling trs of NG2 + lls tht iffrntit into oligonroyts (17 NG2 + lls in 5 mi). 672 VOLUME 16 NUMBER 6 JUNE 213 ntur nuroscience

6 Bfor Lsion h 1 h 24 h Rspons inx Control Lsion * npg 213 Ntur Amri, In. All rights rsrv. Lsion Lsion Bfor h 2 h 4 h 6 h 8 h 1 h µm f Displmnt (µm) wks for lsion 3 wks ftr lsion Lsion sit Vtor sum furthr vin tht NG2 + ll omins r mintin y slfvoin (prnt trritory invsion: ontrol, 6 ± 1%, n = 1 lls; ll ltion, 52 ± 1%, n = 12 lls; P <.1, Stunt s t tst; Fig. 6 ). Morovr, rmovl of svrl NG2 + lls in th imging fil triggr rorinttion, ll migrtion n prolifrtion of NG2 + lls in th viinity, rsulting in th rpi rstortion of thir nsity (numr of prolifrting lls: ontrol, 1 ±.4 lls; ll ltion, 5 ±.3 lls; volum nlyz =.2 mm 3, from four ontrol n thr ltion mi; *P <.1, Stunt s t tst; Fig. 6 h). Ths t init tht NG2 + lls mintin onstnt nsity using lol homostti mhnism in whih ll loss rsults in rls from growth inhiition n rpi rplmnt through ivision of nighoring ll. NG2 + lls irtly iffrntit into oligonroyts NG2 + lls in th ult CNS n gnrt from SVZ prognitors 32, whih my hv grtr pity for prolifrtion n iffrntition thn lls with longr rsin tim. To trmin whthr rpopultion is omplish y sust of highly prolifrtiv, slf-rnwing NG2 + lls 33, w follow th hvior of prolifrting NG2 + lls y rptly imging th sm ortil volum for mny wks. During this prio, th mjority of prolifrting NG2 + lls ivi g 5 µm 25 µm Lsion Prolifrtion 25 µm Chng in lngth (µm) h Cll ount Tim (h) 1 5 only on, n, for lls tht ivi multipl tims, ll yl lngth ws vril (vrg ll yl, 18.5 ± 1, n = 97 lls; Fig. 7 ), s woul xpt if xtrinsi ftors (tht is, th or iffrntition of nighor) trmin whn NG2 + lls ivi. In, lls tht unrwnt multipl ivisions wr lot in rgions in whih thr ws sustntil turnovr or iffrntition. Morovr, ling trs of highly prolifrtiv lls rvl instns in whih oth sistr lls unrwnt multipl ivisions, s wll s instns in whih on sistr ll ithr i or iffrntit n th othr rmin stl (Fig. 7). Ths finings, n th onsistnt prolifrtion of immit nighors following ll loss, suggst tht homostsis is hiv y nowing ll NG2 + lls with th pity to ivi 7, rthr thn y sing th rin with sust of highly prolifrtiv lls. Nw nurons r form in th CNS through symmtri or symmtri ivision of prognitors 34 ; howvr, it is unlr whthr oligonroyts r form through similr mhnisms or y irt iffrntition of NG2 + lls 2. Ling tring through in vivo imging rvl tht iffrntition ws rrly pr y ll ivision (7 of 17 iffrntition vnts; Fig. 7), initing tht oligonroyts in th ult CNS r primrily form through irt 5 * ** *** Tim (h) 4 Prolifrtion 25 Lost Totl Ling pross Triling pross * ** *** Tim () Figur 8 NG2 + lls surroun rs of CNS mg n prolifrt to mintin thir nsity. () Tim-lps imging of NG2 + ll rsponss to fol lsr injury (sit of lsion shown in yllow). () Grph showing umultion of EGFP + NG2 + ll prosss within 75 µm of th lsion ovr tim. Conntri irls on th 24-h tim point in highlight th rs msur to trmin th rspons inx (n = 5 mi pr onition, *P <.5, two-wy ANOVA). () Squntil imgs showing th rspons of NG2 + ll to fol lsr injury (yllow spot). () Quntifition of xtnsion n rtrtion of ling n triling prosss of NG2 + lls rltiv to th lsion (n = 2 rnhs on 1 lls in 4 mi; *P <.5, **P <.5, ***P <.5, on-wy ANOVA with Tuky post ho tst). () Montg of thr imgs of on NG2 + ll ollt on iffrnt ys showing th migrtion of th ll towr th lsion (yllow). (f) Vtor plots showing th irtion n isplmnt of NG2 + lls within 75 µm of th lsion sit 3 wks for n ftr lsion inution (73 lls in 4 mi; for, P =.73; ftr, P = , Moor-Ryligh tst). Lin irtion rprsnts th ngl of th isplmnt rltiv to th lsion sit. R lin is th vtor sum of ll isplmnts. (g) Mximum-intnsity projtion of 15 µm ov n low fol lsr injury (yllow) 2 ftr lsion inution. Two prolifrting NG2 + lls (grn rrowhs) jnt to th lsion sit r highlight in orng. (h) Comin plot showing th numr of NG2 + lls unrgoing prolifrtion n th n/or iffrntition (omin) n th totl NG2 + ll nsity (lu lin) within 75 µm of th lsion. Fol lsr injury ws inu on y (n = 4 mi). Error rs rprsnt s..m. 5 Numr of NG2 + lls ntur NEUROSCIENCE VOLUME 16 NUMBER 6 JUNE

7 npg 213 Ntur Amri, In. All rights rsrv. iffrntition of ths prognitors. Thus, prolifrtion of NG2 + lls in vivo rflts homostti rspons to ll pltion rthr thn oligonrognsis. NG2 + lls prtiipt in glil sr formtion NG2 + ll prolifrtion is oftn nhn ftr injury n th NG2 protoglyn umults t lsion sits, whr it n inhiit xon outgrowth 18,35. Howvr, th rson for th nhn prolifrtion of ths lls is unknown n th prtiiption of NG2 + lls in sr formtion hs not n stlish, s NG2 is lso xprss y mrophgs n priyts in injur tissu 36,37. To ssss th hvior of NG2 + glil lls to CNS injury, w inu fol lsr lsions 25 in th ortx of ult NG2-mEGFP-H mi. Immitly following injury, NG2 + lls jnt to th lsion xtn prosss towr th lsion sit n vntully surroun th sit of injury (Fig. 8 n Supplmntry Movi 7). Although thir rspons to fol injury ws similr to mirogli 25, NG2 + lls xtn thir prosss mor slowly thn mirogli to th lsion sit (Fig. 8). As onsqun, srs quir lyr strutur onsisting of miroglil ll prosss surroun y NG2 + ll prosss (Supplmntry Fig. 8). Rorinttion n xtnsion of NG2 + ll prosss ftr injury ws follow y migrtion of ths lls to th lsion sit in svrl wks, n vntul rmovl of ths lls s th sr rsolv (Fig. 8,f n Supplmntry Movi 8). As prit y thir homostti hvior to ll loss, pltion of NG2 + lls from th r surrouning th lsion ws ompni y prolifrtion of nighoring NG2 + lls (Fig. 8g), with th numr of prolifrting lls mthing th numr lost (for lsion, 18 ± 2 NG2 + lls; ftr lsion, 16 ± 1 NG2 + lls; n = 4 mi, P =.5; Fig. 8h). Ths finings init tht NG2 + lls ontriut to glil sr formtion n provi n xplntion for th nhn prolifrtion of NG2 + lls tht ours following mny typs of CNS trum. DISCUSSION NG2 + glil lls ompris th most unnt popultion of prolifrtiv lls in th ult CNS. Ths prognitors rtin th pity to iffrntit into oligonroyts n ontriut to rgnrtion of mylin following injury n iss. Homostti ontrol of thir nsity n istriution llows not only ffiint rplmnt of oligonroyts, ut lso nsurs tht ll growth os not pro unhk n rsult in tumor formtion. To trmin how NG2 + ll nsity is mintin in th ult CNS, w vlop mi tht xprss mmrn-nhor EGFP in NG2 + lls (NG2-mEGFP-H mi) n xmin th hvior of ths uiquitous prognitors in th somtosnsory ortx of ult mi using in vivo two-photon imging. W foun tht NG2 + lls r highly ynmi in th rsting rin; thy tivly survy thir lol nvironmnt with motil filopoi n ontinully migrt through th prnhym, mintining xlusiv trritoris through slf-rpulsion. NG2 + lls rmov from th popultion y iffrntition or th wr rpily rpl through prolifrtion of n immit nighor (Supplmntry Fig. 9). By lning tiv growth with slf-rpulsion, NG2 + lls nsur tht prolifrtion is oupl oth sptilly n tmporlly to ll loss, prsrving high nsity of ths prognitors throughout th CNS to ffiintly gnrt oligonroyts n prtiipt in tissu rpir. NG2 + glil lls r istriut in gri-lik or til mnnr in th ult CNS, with iniviul lls oupying non-ovrlpping omins. As this gri is onstntly rorgniz, mhnisms must xist to tivly limit th growth of ths prognitors. In th mmmlin rtin, th mosi sping of nurons is rgult y homotypi rpulsiv intrtions through trnsmmrn rptors (for xmpl, MEGFs n DSCAM) 27,38. Similr rpulsiv intrtions r likly to involv in oth stlishing n mintining th nsity of NG2 + lls, s sltiv ltion of iniviul NG2 + lls ws suffiint to triggr rpi invsion of th trritory of th rmov ll, n ontt twn NG2 + ll prosss ws lwys follow y rtrtion (Figs. 1 n 6). Homotypi rpulsion woul lso provi th mns to stlish n mintin th ril orinttion of thir prosss 28. Altrntivly, prolifrtion of ths prognitors my triggr y nhn ss to mitogns, suh s pltlt-riv growth ftor (PDGF), s ovrxprssion of PDGF inrss th numr of oligonroyt prognitors in th vloping spinl or 39 n inus tumors from ths lls in ults 4. Howvr, oligonroyt prognitors xhiit nsity-pnnt rution in prolifrtion in sturting PDGF in vitro 41, suggsting tht irt intrtions n limit thir growth. Although guin us, suh s ntrin n smphorins, hv n shown to influn th isprsion n iffrntition of oligonroyt prognitors uring vlopmnt 31, th moluls tht mintin thir nsity in th ult CNS rmin unknown. As NG2 + ll prosss ovrlp xtnsivly with othr glil lls tht xhiit tiling, suh s stroyts n mirogli, th molulr pthwys tht ontrol th istriution n nsity of iffrnt glil ll typs r likly to istint. Long-trm tim-lps imging of NG2 + lls in th ortx rvl tht th ntir ntwork of NG2 + lls is onstntly rorgniz s rsult of th ontinul loss of lls n thir intrinsi prssur to tivly survy thir lol nvironmnt. Th migrtion of iniviul lls ws inu y lol vnts th iffrntition, th or isplmnt of nighoring lls rthr thn lrg-sl ttrtiv or rpulsiv grints (Fig. 2). In som instns, multipl lls in th img volum i or iffrntit, rting lrgr vois tht rsult in grtr rorgniztion through migrtion of nry NG2 + lls. Howvr, lrg-sl migrtion of iniviul lls through th prnhym ws not osrv n mintnn of th popultion ws hiv through lol prolifrtion of nighoring lls, rthr thn through supopultion of highly migrtory, prolifrtiv NG2 + lls (Fig. 7), onsistnt with th mrk slf-voin xhiit y ths lls. Dynmi filopoi wr istriut long th lngth of NG2 + lls prosss n vn projt from thir somt. This onstnt xplortion of th lol nvironmnt y NG2 + lls my llow thm to not only ssss thir nsity, ut lso th viility of oligonroyts n th stt of mylintion of nry xons 4. If lol intrtions r rquir to stimult oligonrognsis, it woul xplin why high nsity of ths prognitors is mintin throughout th ult CNS. In multipl slrosis, NG2 + ll nsity is mrkly ru in mny hronilly mylint whit mttr lsions 42. This impirmnt of NG2 + ll homostsis my prvnt ttion of mylint xons n limit oligonroyt rgnrtion, rising th possiility tht fol rstortion of NG2 + ll numrs in hroni lsions my thrputilly nfiil in multipl slrosis. NG2 + lls form funtionl synpss with glutmtrgi nurons in ll of th rgions of th CNS tht hv n xmin Th high motility of NG2 + ll prosss n onstnt movmnt of ths lls through th prnhym ontrst with th mrk stility of glutmtrgi xons in th ortx osrv through similr in vivo imging, whih, prt from th rfinmnt of som outons, o not hng thir position ovr svrl months 46. Th xtnt of NG2 + ll movmnt suggsts tht synpti onntions twn xons n NG2 + lls r ontinully rmol on tim sl of ys to wks. 674 VOLUME 16 NUMBER 6 JUNE 213 ntur nuroscience

8 npg 213 Ntur Amri, In. All rights rsrv. Th trnsint ntur of ths synpss my xplin why NG2 + lls in th sm rin rgion xhiit wily vrying lvls of synpti onntivity 45. Although th rol of this rpi form of nuron-glil ll ommunition hs not n stlish in vivo, glutmtrgi signling influns th prolifrtion n iffrntition of ths prognitors in vitro 47. As NG2 + lls oupy non-ovrlpping omins, th ontinul rorgniztion of thir prosss my nl ths prognitors to smpl th tivity of grtr proportion of xons to nl lrning-inu hngs in mylintion 48, n nsur tht oligonroyts tht gnrt s rsult of injury, iss or norml ging r rpily rpl. In vivo gnti ft tring stuis hv suggst tht NG2 + lls ontinu to gnrt oligonroyts in th ult CNS, lthough lss frquntly thn uring rly postntl lif 7, Consistnt with ths finings, our tim-lps imging rvl tht NG2 + lls in th ortx of 5-month-ol mi ontinu to iffrntit into oligonroyts t low rt (Fig. 3). In vitro stuis init tht oligonroyts r gnrt through symmtri ivision of ths prognitors 2 ; howvr, th stps ling to oligonrognsis in vivo rmin unrtin. Using long-trm imging, w isovr tht th mjority of NG2 + lls in th ult ortx irtly iffrntit into oligonroyts without unrgoing symmtri ivision. Thus, prolifrtion of NG2 + lls in vivo is not irt rfltion of oligonrognsis, ut rthr homostti rspons to rpl prognitors tht hv iffrntit or i. Our nlysis of NG2 + lls fous xlusivly on lyrs 1 n 2/3 of th ortx s rsult of th limittions of two-photon imging t grtr pths. Although h ortil lyr hs istint ytorhittur, NG2 + ll hvior ws similr throughout ths uppr lyrs of ortx (Supplmntry Fig. 7), onsistnt with th ility of ths lls to prolifrt n gnrt nw oligonroyts throughout th CNS 7,13. Although not onsir tritionl whit mttr rgion, lyr 1 of th ortx ontins rltivly high nsity of mylint xons tht ours horizontlly through th nriti tufts of pyrmil nurons. Nvrthlss, it is possil tht NG2 + ll hviors my iffr twn gry n whit mttr in th ult CNS, prtiulrly if ths rgions vry with rgr to ll turnovr or oligonrognsis. Th vlopmnt of nw pprohs for high-rsolution imging in p rin struturs suh s th orpus llosum or long-trm imging of orsl olumn whit mttr in th spinl or will rquir to xmin NG2 + ll hvior in ult whit mttr. Injury to th CNS, whthr inu utly or through hroni iss, is oftn ssoit with gliosis, hrtriz y nhn prolifrtion n umultion of gli roun lsions to form glil srs. This rruitmnt of gli to sits of trum is thought to ruil for limiting furthr injury n promoting rpir. In ition to stroyts, mirogli n mrophgs, lls tht xprss th NG2 protoglyn umult t lsions, with thir numrs pking 1 wk ftr injury n thn lining slowly ovr n xtn prio 37,49. Dtrmining th sour of NG2 hs n prolmti, s NG2 is lso xprss y priyts n sust of mirogli n mrophgs following injury 36,37 n n rls into th xtrllulr sp through nzymti lvg y mtrix mtlloprotinss 5. Tim-lps imging of iniviul NG2 + lls rvl tht ths prognitors r ttrt to th sit of injury in mnnr similr to mirogli 25 ; thy rorint n xtn thir prosss towr th lsion n vntully migrt to th pri-lsion r to prtiipt in th formtion of th sr. NG2 + lls rruit to th lsion no longr xhiit slf-rpulsion, suggsting tht injury my trnsform ths lls into istint sr-forming lls with uniqu hrtristis. Aoringly, loss of lls to th sr triggr homostti prolifrtion of surrouning NG2 + lls to rstor thir nsity. Although NG2 + lls gn to rorint thir prosss within minuts of injury, thy rriv t th lsion svrl hours ltr thn mirogli (xtnsion rt: NG2 + lls, 2.7 ±.4 µm h 1, Fig. 8; mirogli, 1.25 ±.6 µm min 1, from rf. 25). Th squntil pprn of ths iffrnt typs of glil lls t th lsion suggsts tht thy hv istint rols in th rspons to ut injury. Long-trm tim-lps imging rvl tht umultion of NG2 + ll prosss rh its pk 1 wk following injury inution (Supplmntry Movi 8) n wr vntully rmov s th sr rsolv. Th prtiiption of NG2 + lls in th formtion n rsolution of glil srs suggsts tht tht ths uiquitous prognitors not only srv s rsrvoir to gnrt oligonroyts, ut lso s survilln ntwork to tt CNS injury n promot tissu rpir. Mthos Mthos n ny ssoit rfrns r vill in th onlin vrsion of th ppr. Not: Supplmntry informtion is vill in th onlin vrsion of th ppr. Aknowlgmnts W thnk M. Puk, N. Y n T. L for thnil ssistn, B. Cumor (Johns Hopkins Univrsity) n S. Wng (Printon Univrsity) for vi on rnil winow implnttion, W.-B. Gn (Nw York Univrsity) for vi on prpring thinn skull winows, n mmrs of th Brgls lortory for isussions. E.G.H. ws support y Kirshstin Ntionl Rsrh Srvi Awr grnt from th US Ntionl Instituts of Hlth (F32NS7698). Funing ws provi y grnts from th US Ntionl Instituts of Hlth (NS5159, NS5274) n th Brin Sin Institut t Johns Hopkins Univrsity. AUTHOR CONTRIBUTIONS E.G.H., M.F., S.H.K. n D.E.B. sign th xprimnts. E.G.H. sign, xut n nlyz th xprimnts sri in th figurs, movis n txt. M.F. m sminl osrvtions of NG2 + ll ynmis n thir rspons to lsr-inu lsions in thinn skull prprtions, n gnrt t for Supplmntry Figur 8. S.H.K. gnrt n hrtriz th NG2-mEGFP-H n NG2-mEGFP-L mous lins n rt Supplmntry Figur 1. E.G.H. n D.E.B. wrot th mnusript. COMPETING FINANCIAL INTERESTS Th uthors lr no ompting finnil intrsts. Rprints n prmissions informtion is vill onlin t rprints/inx.html. 1. Bitu, B., Hohmuth, C.E. & Jspr, H. Mintining tissu homostsis: ynmi ontrol of somti stm ll tivity. Cll Stm Cll 9, (211). 2. Simons, B.D. & Clvrs, H. Strtgis for homostti stm ll slf-rnwl in ult tissus. Cll 145, (211). 3. Zho, C., Dng, W. & Gg, F.H. Mhnisms n funtionl implitions of ult nurognsis. Cll 132, (28). 4. Frnklin, R.J., Gilson, J.M. & Blkmor, W.F. Lol rruitmnt of rmylinting lls in th rpir of mylintion in th ntrl nrvous systm. J. Nurosi. Rs. 5, (1997). 5. Ajmi, B., Bnntt, J.L., Krigr, C., Ttzlff, W. & Rossi, F.M. Lol slf-rnwl n sustin CNS mirogli mintnn n funtion throughout ult lif. Nt. Nurosi. 1, (27). 6. Dwson, M.R., Polito, A., Lvin, J.M. & Rynols, R. NG2-xprssing glil prognitor lls: n unnt n wispr popultion of yling lls in th ult rt CNS. Mol. Cll Nurosi. 24, (23). 7. Kng, S.H., Fuky, M., Yng, J.K., Rothstin, J.D. & Brgls, D.E. NG2 + CNS glil prognitors rmin ommitt to th oligonroyt ling in postntl lif n following nurognrtion. Nuron 68, (21). 8. Kssris, N. t l. Compting wvs of oligonroyts in th forrin n postntl limintion of n mryoni ling. Nt. Nurosi. 9, (26). 9. Kiry, B.B. t l. In vivo tim-lps imging shows ynmi oligonroyt prognitor hvior uring zrfish vlopmnt. Nt. Nurosi. 9, (26). ntur NEUROSCIENCE VOLUME 16 NUMBER 6 JUNE

9 npg 213 Ntur Amri, In. All rights rsrv. 1. Nishiym, A., Komitov, M., Suzuki, R. & Zhu, X. Polynroyts (NG2 lls): multifuntionl lls with ling plstiity. Nt. Rv. Nurosi. 1, 9 22 (29). 11. Rivrs, L.E. t l. PDGFRA/NG2 gli gnrt mylinting oligonroyts n piriform projtion nurons in ult mi. Nt. Nurosi. 11, (28). 12. Dimou, L., Simon, C., Kirhhoff, F., Tkyshi, H. & Gotz, M. Progny of Olig2-xprssing prognitors in th gry n whit mttr of th ult mous rrl ortx. J. Nurosi. 28, (28). 13. Young, K.M. t l. Oligonroyt ynmis in th hlthy ult CNS: vin for mylin rmoling. Nuron 77, (213). 14. Tripthi, R.B., Rivrs, L.E., Young, K.M., Jmn, F. & Rihrson, W.D. NG2 gli gnrt nw oligonroyts, ut fw stroyts, in murin xprimntl utoimmun nphlomylitis mol of mylinting iss. J. Nurosi. 3, (21). 15. Lvin, J.M. & Rynols, R. Ativtion n prolifrtion of nognous oligonroyt prursor lls uring thiium romi-inu mylintion. Exp. Nurol. 16, (1999). 16. Hr, M., Vutrin, S., Fry, E.J. & Muri, K.K. Sutyp-spifi oligonroyt ynmis in orgnotypi ultur. Gli 57, (29). 17. Hrlnt, C. t l. Gry mttr NG2 lls isply multipl C 2+ -signling pthwys n highly motil prosss. PLoS ONE 6, (211). 18. MTigu, D.M., Wi, P. & Stoks, B.T. Prolifrtion of NG2-positiv lls n ltr oligonroyt numrs in th ontus rt spinl or. J. Nurosi. 21, (21). 19. Mgnus, T. t l. Ault glil prursor prolifrtion in mutnt SOD1G93A mi. Gli 56, 2 28 (28). 2. Sugirto, S. t l. Asymmtry-ftiv oligonroyt prognitors r gliom prursors. Cnr Cll 2, (211). 21. Ivkovi, S., Cnoll, P. & Golmn, J.E. Constitutiv EGFR signling in oligonroyt prognitors ls to iffus hyprplsi in postntl whit mttr. J. Nurosi. 28, (28). 22. Liu, C. t l. Mosi nlysis with oul mrkrs rvls tumor ll of origin in gliom. Cll 146, (211). 23. Prsson, A.I. t l. Non-stm ll origin for oligonrogliom. Cnr Cll 18, (21). 24. Bniktsson, A.M., Shhtl, S.J., Grn, S.H. & Dily, M.E. Bllisti lling n ynmi imging of stroyts in orgnotypi hippompl sli ulturs. J. Nurosi. Mthos 141, (25). 25. Dvlos, D. t l. ATP mits rpi miroglil rspons to lol rin injury in vivo. Nt. Nurosi. 8, (25). 26. Nimmrjhn, A., Kirhhoff, F. & Hlmhn, F. Rsting miroglil lls r highly ynmi survillnts of rin prnhym in vivo. Sin 38, (25). 27. Ky, J.N., Chu, M.W. & Sns, J.R. MEGF1 n MEGF11 mit homotypi intrtions rquir for mosi sping of rtinl nurons. Ntur 483, (212). 28. Lfvr, J.L., Kostinov, D., Chn, W.V., Mnitis, T. & Sns, J.R. Protohrins mit nriti slf-voin in th mmmlin nrvous systm. Ntur 488, (212). 29. Xu, H.T., Pn, F., Yng, G. & Gn, W.B. Choi of rnil winow typ for in vivo imging ffts nriti spin turnovr in th ortx. Nt. Nurosi. 1, (27). 3. Yng, G., Pn, F., Prkhurst, C.N., Grutznlr, J. & Gn, W.B. Thinn-skull rnil winow thniqu for long-trm imging of th ortx in liv mi. Nt. Proto. 5, (21). 31. Spssky, N. t l. Dirtionl guin of oligonroglil migrtion y lss 3 smphorins n ntrin-1. J. Nurosi. 22, (22). 32. Mnn, B. t l. Origin of oligonroyts in th suvntriulr zon of th ult rin. J. Nurosi. 26, (26). 33. Pshouli, K., Jmn, F., Young, K.M. & Rihrson, W.D. Cll yl ynmis of NG2 lls in th postntl n ging rin. Nuron Gli Biol. 5, (29). 34. Krigstin, A. & Alvrz-Buyll, A. Th glil ntur of mryoni n ult nurl stm lls. Annu. Rv. Nurosi. 32, (29). 35. Bush, S.A. & Silvr, J. Th rol of xtrllulr mtrix in CNS rgnrtion. Curr. Opin. Nuroiol. 17, (27). 36. Göritz, C. t l. A priyt origin of spinl or sr tissu. Sin 333, (211). 37. Jons, L.L., Ymguhi, Y., Stllup, W.B. & Tuszynski, M.H. NG2 is mjor honroitin sulft protoglyn prou ftr spinl or injury n is xprss y mrophgs n oligonroyt prognitors. J. Nurosi. 22, (22). 38. Furst, P.G., Koizumi, A., Msln, R.H. & Burgss, R.W. Nurit roriztion n mosi sping in th mous rtin rquir DSCAM. Ntur 451, (28). 39. Clvr, A.R. t l. Oligonroyt popultion ynmis n th rol of PDGF in vivo. Nuron 2, (1998). 4. Assnh, M.C. t l. PDGF stimults th mssiv xpnsion of glil prognitors in th nontl forrin. Gli 57, (29). 41. Zhng, H. & Millr, R.H. Dnsity-pnnt fk inhiition of oligonroyt prursor xpnsion. J. Nurosi. 16, (1996). 42. Chng, A., Nishiym, A., Ptrson, J., Prins, J. & Trpp, B.D. NG2-positiv oligonroyt prognitor lls in ult humn rin n multipl slrosis lsions. J. Nurosi. 2, (2). 43. Chittjllu, R., Aguirr, A. & Gllo, V. NG2-positiv lls in th mous whit n gry mttr isply istint physiologil proprtis. J. Physiol. (Lon.) 561, (24). 44. Brgls, D.E., Rorts, J.D., Somogyi, P. & Jhr, C.E. Glutmtrgi synpss on oligonroyt prursor lls in th hippompus. Ntur 45, (2). 45. D Bis, L.M., Nishiym, A. & Brgls, D.E. Exitility n synpti ommunition within th oligonroyt ling. J. Nurosi. 3, (21). 46. D Pol, V. t l. Cll typ spifi struturl plstiity of xonl rnhs n outons in th ult noortx. Nuron 49, (26). 47. Gllo, V. t l. Oligonroyt prognitor ll prolifrtion n ling progrssion r rgult y glutmt rptor mit K + hnnl lok. J. Nurosi. 16, (1996). 48. Bngtsson, S.L. t l. Extnsiv pino prtiing hs rgionlly spifi ffts on whit mttr vlopmnt. Nt. Nurosi. 8, (25). 49. Lvin, J.M. Inrs xprssion of th NG2 honroitin-sulft protoglyn ftr rin injury. J. Nurosi. 14, (1994). 5. Lrsn, P.H., Wlls, J.E., Stllup, W.B., Opnkkr, G. & Yong, V.W. Mtrix mtlloprotins-9 filitts rmylintion in prt y prossing th inhiitory NG2 protoglyn. J. Nurosi. 23, (23). 676 VOLUME 16 NUMBER 6 JUNE 213 ntur nuroscience

10 npg 213 Ntur Amri, In. All rights rsrv. ONLINE METHODS Gnrtion of NG2-mEGFP trnsgni mi. A mous tril rtifiil hromosom (BAC) lon ontining th NG2 (Cspg4) gn (RP23-39G21) ws purhs from BACPAC Rsours Cntr n moifi y homologous romintion 51. To trgt EGFP to th plsm mmrn, th first 26 mino is of Lk, Sr fmily tyrosin kins tht ontins two plmitoyltion omins n myristoyltion omin (gift from M. Dily n S. Grn, Univrsity of Iow) 24, ws fus to th N trminus of EGFP in pegfp-n1 (Clonth). A DNA noing this mmrn-nhor EGFP (megfp) n susqunt rit β-gloin polya signl wr pl t th trnsltionl inititor (ATG) of th NG2 (Cspg4) gn with two flnking homology rms (5 p for h rm). Th moifi BAC ws linriz y NotI igstion, injt into th pronulus of mous mryos in th John Hopkins Univrsity Trnsgni Cor n implnt into psuoprgnnt fmls. Of th thr lins of NG2-mEGFP mi gnrt, two lins wr slt for furthr stuis. In vivo two-photon mirosopy. All xprimnts wr prform in strit orn with protools pprov y th Animl Cr n Us Committ t Johns Hopkins Univrsity. To prpr rnil winows, NG2-mEGFP-H mi (2 3 months ol) wr nsthtiz y intrpritonl injtion of ktmin (1 mg pr kg of oy wight) n xylzin (1 mg pr kg); oy tmprtur ws mintin t 37 C with thrmostt-ontroll hting plt. Th skin ovr th right rrl hmisphr ws rtrt n th skull ln. A mtl plt with ntr hol ws tth to th skull with ntl mnt (C&B Mton) to llow for h stiliztion. A ~4-µm-imtr rgion of skull ovr somtosnsory ortx (.5 to 2 mm post rgm n 1 to 3.5 mm ltrl) ws ithr thinn (~2 µm thiknss) or rmov using high-sp ntl rill 3,52. For rnil winows, pi of ovr glss (VWR, No. 1) ws pl in th rniotomy n sl with ntl mnt. In vivo imging sssions gn immitly ftr surgry (thinn-skull prprtion) or ftr minimum of 3 wks (hroni rnil winow). Although rniotomy n inu gliosis 29, ths smll rnil winows i not inu rtiv hngs in miroglil lls or NG2 + lls 3 wks following implnttion (Supplmntry Fig. 4). Mi wr nsthtiz with isoflurn (1. 1.5%, mix with.5 l min 1 O 2 ) uring imging. Imgs wr ollt using Ziss LSM 71 mirosop quipp with GAsP ttor using molok Ti:spphir lsr (Cohrnt Ultr II) tun to 92 nm. Th vrg powr t th smpl uring imging ws <3 mw. Vsulr lnmrks (EGFP + priyts) wr us to intify th sm imging r on susqunt ys. Two-photon lsr-inu lsions n iniviul ll ltion. Smll lsions wr inu in ortil gry mttr y illuminting with th Ti:spphir lsr for svrl sons (78 nm, ~15 mw, 1 3 s) 25. This protool inu ~2-µm r of mg visil s n utofluorsnt sphr. To lt iniviul NG2 + lls in mi with thinn-skull winows, ll somt wr sujt to rpt, short-urtion lsr irrition (78 nm, ~15 mw, 1 5 ms, 1 6 rptitions) 53. Sussful ltions wr pr y slight swlling of th som tht ws follow y ll ispprn within 1 8 h. Lsr powr, puls urtion n puls rptition wr vri pning on th pth of th trgt ll n thiknss of th ovrlying thinn-skull. Img prossing n nlysis. Img stks n tims sris wr nlyz using Fiji 54. All nlysis ws prform on unpross imgs. For prsnttion in figurs, img rightnss n ontrst lvls wr just for lrity. Filopoil imgs wr itionlly pross with Gussin filtr (rius = 2 pixls) to rmov ttor nois. For psuo-olor isply of iniviul lls, th thrimnsionl ll volum ws fin pln-y-pln for h tim point n ustom Fiji sript ws us to sgmnt n/or oloriz th ll. Filopoil nlysis. Filopoi on NG2 + ll prosss ( 5 µm in lngth) wr nlyz frm y frm using ustom Fiji sript to quntify filopoil lngth, motility, nsity n liftim. Filopoi wr onsir to ynmi if th lngth vri mor thn 2 µm uring th imging prio. For nlysis of filopoil nsity in fix tissu, protrusions with lngth t lst twi th with n minimum lngth of 2 µm wr lssifi s filopoi. oorints n tgorizing llulr hvior (for xmpl, prolifrtion, iffrntition, t.). NG2 + lls wr lssifi s prolifrting if two lls ppr in th sm lotion whr thr ws on ll t th prvious tim point, th somt wr sprt y lss thn 5 µm, th prosss of rntly ivi lls wr orint in opposing irtions n th somt lk mjor ll prosss t th point losst to th sistr ll. Th thr-imnsionl volum roun h prolifrting ll ws xmin to xlu nighoring NG2 + lls tht migrt into th fil. NG2 + lls wr lssifi s iffrntiting if th lls xhiit progrssiv rs in EGFP fluorsn, th lls hng from hving ril, highly rnh prosss to hving long, unrnh prosss hrtristi of oligonroyts, n th prosss of nighoring NG2 + lls inv th trritory of th iffrntiting ll. NG2 + lls wr lssifi s ying if lls xhiit shortning n/or ling of prosss, rution in trritory siz, n inrs in rightnss of th ll som n omplt ispprn of EGFP fluorsn in <3 without movmnt from th imging r. NG2 + lls tht lost EGFP fluorsn, ut oul not finitivly intifi s iffrntit or ying, wr lssifi s unsur. NG2 + ll trritory invsion. To lult trritory invsion following th or iffrntition, w omput onvx hull pln y pln for h tim point roun th iffrntiting n/or ying ll, th ll msk n EGFP + prosss ontin in th onvx hull utothrshol using th Fiji IsoDt lgorithm. A mximum projtion of thr-imnsionl volum fin y th onvx hull ws gnrt n th r of prosss for h tim point trmin. To lult trritory invsion following NG2 + ll ltion, th thr-imnsionl volum of th lt ll ws mrg with th img of th following tim point to intify inving prosss. For ontrol lls, th ll in th son tim point ws msk n th rgion of th thr-imnsionl volum of th ll in th first tim point ws ovrli onto th son tim point. A mximum projtion of thr-imnsionl volum of th inving prosss ws utothrshol using th Fiji IsoDt lgorithm n th xtnt of trritory invsion lult y normlizing th r oupi y inving prosss t th lst tim point to th r oupi y th ll t th first tim point. Anlysis of lol prolifrtion. NG2 + lls tht unrwnt prolifrtion, iffrntition or th wr intifi uring th imging prio. For h iffrntiting ll, th tim point t whih th ll isply prmylinting oligonroyt morphology 55, n nighoring lls h gun inving tht ll s trritory, ws mrk s y. For h ll tht i, th tim point tht th ll isply ltr morphology (inrs rightnss of ll som n ru trritory siz), immitly pring ispprn, ws mrk s y. Aftr intifition of NG2 + ll tht unrwnt iffrntition/th, immitly jnt NG2 + lls wr xmin for ll ivision pring n following th vnt. Th y of prolifrtion ws mrk s th tim point t whih th two sistr lls h unrgon ytokinsis n h no joining prosss. Clls wr xlu from nlysis if th ntir iffrntition/th pross i not our uring th imging prio or if not ll jnt lls wr lot within th fil of viw. For ontrols, w nlyz lls tht i not unrgo prolifrtion, iffrntition or ll th for th ntir imging prio n i not hv mor thn on ll ivision within 1 µm. Anlysis of th rspons to fol injury. To follow pprn of NG2 + lls t lsions, EGFP + prosss ntring 75-µm zon surrouning lsion sits (r X) from lrgr 15-µm zon (r Y) wr msur s funtion of tim (Fig. 4) 25. To lult th r surrouning th lsion oupi y NG2 + ll prosss, imgs wr utothrshol using th Fiji IsoDt lgorithm n th numr of pixls ws ssss. Th hng in pixl numr ovr tim (R x (t)) ompr to th initil vlu of pixls (R x ()) rprsnt NG2 + ll prosss umultion. To ontrol for vritions in th numr NG2 + lls surrouning th lsion sit in iffrnt xprimnts, umultion of NG2 + ll prosss ws lult rltiv to th numr of prosss in th outr r Y immitly following inution of th lsion (R y ()). Th NG2 + ll rspons inx t ny tim point, (R(t)), is givn y th qution Cll trking. NG2 + lls wr trk in thr imnsions using ustom Fiji sripts y fining som position t h tim point, roring xyz Rx( t) Rx( ) R( t) = Ry( ) (1) oi:1.138/nn.339 ntur NEUROSCIENCE

11 npg 213 Ntur Amri, In. All rights rsrv. To quntify rspons sp, w msur th lngth of NG2 + ll prosss within 15 µm of th lsion sit ovr tim. To trmin th ling n triling prosss of NG2 + lls, th oorint systm ws rott so tht th y xis pss through th lsion sit t th top n th som of th ll ws lot t th origin (lsion sit = 9 from th ll of intrst). Th ngl t whih prosss xtn from th som trmin whthr thy wr on th ling g ( 18 ) or triling g ( ) of th NG2 + ll. To trmin th mgnitu n irtion of NG2 + ll movmnts with rspt to th lsion sit, th oorint systm ws rott suh tht th x xis pss through th lsion sit t th right n th strt point of th ll ws lot t th origin (lsion sit = from th strt point). Nxt, th ngl θ twn th isplmnt vtor of NG2 + ll movmnt n th x xis (isplmnt vtor twn th strt point n th lsion sit) ws trmin using q i L i = ros i L whr i is th isplmnt vtor for th ll, i n L r th isplmnt vtors twn th strt point of th ll n th lsion sit, rsptivly, i L is th ot prout twn th two vtors, n =. Th sign of th ngl ws trmin y lulting th trminnt of th two vtors using x y z x y z x y z = xyz + yzx + zxy zyx xz y yx z whr th Crtsin oorints of th lsion sit, th strt position n th finl position of th NG2 + ll of intrst r fin s, n, rsptivly. (2) (3) Immunohistohmistry. Mi wr ministr n ovros of nsthsi (pntoritl, 1 mg pr kg, intrpritonl), prfus trnsrilly with 4% formlhy (vol/vol, in.1 M phospht uffr, ph 7.4), n thir rins wr xtrt n postfix in 4% formlhy for 4 h or ovrnight t 4 C, thn ryoprott in 3% suros solution (wt/vol, in phospht-uffr slin, ph 7.4) t 4 C for up to 36 h. Brins wr frozn in TissuTk, stion (oronl, rgm.2 to 1.9 mm) t 3 5 µm thik n inut fr-floting for 1 2 h t 2 3 C in loking solution (5% norml onky srum (vol/vol),.3% Triton X-1 (vol/vol) in phospht-uffr slin, ph 7.4). Stions wr inut with primry ntiois (Supplmntry Tl 1) suspn in loking solution ovrnight t 4 C on n oritl shkr. For Olig2 immunostining, rin stions wr inut in LAB solution (Polysins) for 1 min for loking. For BrU immunostining, stions wr trt with 2 N HCl t 37 C for 3 min, n thn nutrliz with.1 M soium ort uffr (ph 8.5) for inution with primry ntiois. Aftr wshing in loking solution, stions wr inut with fluorsntly onjugt sonry ntiois (Supplmntry Tl 2) for 2 h t 2 3 C n thn mount on slis with Aqu Poly/Mount (Polysins). Imgs wr quir using ithr n pifluorsn mirosop (Ziss Axio-imgr M1) n Axiovision softwr (Ziss) or onfol lsr-snning mirosop (Ziss LSM 51 Mt). For ll prolifrtion nlysis, mi wr provi with BrU-ontining rinking wtr (1 mg ml 1 supplmnt with 1% suros) n riv two injtions of BrU (5 mg pr kg, intrpritonl) pr y, t lst 8 h prt, for 1 wk for prfusion. To nlyz th ffts of rnil winow implnttion, urr hols wr rill <1 mm outsi th riniotomy r n 1 µl of xtrn-onjugt rhomin ws injt into th rin to ll th position of th rnil winow just for th mous ws kill. Sttistil nlysis. Sttistil nlyss wr prform in OriginPro (OriginL), Exl (Mirosoft) or Fiji. Smpl sizs wr hosn oring to th stnr prti in th fil. Normlity of th t ws tst with th Shpiro-Wilk tst. Signifin ws trmin using th Mnn-Whitny tst, unpir or pir two-til Stunt s t tst, or on-wy ANOVA with Tuky post ho tst, s not. All irtion sttistis wr prform using moifition to th Ryligh s tst 56, implmnt in Chmotxis n Migrtion Tool (ImgJ plug-in, Iii). 51. Yng, X.W., Mol, P. & Hintz, N. Homologous romintion s moifition in Eshrihi oli n grmlin trnsmission in trnsgni mi of tril rtifiil hromosom. Nt. Biothnol. 15, (1997). 52. Holtmt, A. t l. Long-trm, high-rsolution imging in th mous noortx through hroni rnil winow. Nt. Proto. 4, (29). 53. Chung, S.H. & Mzur, E. Fmtoson lsr ltion of nurons in C. lgns for hviorl stuis. Appl. Phys. A Mtr. Si. Pross. 96, (29). 54. Shinlin, J. t l. Fiji: n opn-sour pltform for iologil-img nlysis. Nt. Mthos 9, (212). 55. Trpp, B.D., Nishiym, A., Chng, D. & Mklin, W. Diffrntition n th of prmylinting oligonroyts in vloping ront rin. J. Cll Biol. 137, (1997). 56. Moor, B.R. A moifition of th Ryligh tst for vtor t. Biomtrik 67, (198). ntur NEUROSCIENCE oi:1.138/nn.339