Stle reprogrmmed heterokryons form spontneously in Purkinje neurons fter one mrrow trnsplnt Jmes M. Weimnn 1,2,Cls B. Johnsson 1,Angeli Trejo 1 nd Helen M. Blu 1,2 Heterokryons re the produt of ell fusion without susequent nuler or hromosome loss. Dedes of reserh using Sendivirus or polyethylene glyol (PEG)-medited fusion in tissue ulture showed tht the terminlly differentited stte of ell ould e ltered. But whether stle non-dividing heterokryons ould our in nimls hs remined unler. Here, we show tht green fluoresent protein (GFP)-positive one-mrrow-derived ells (BMDCs) ontriute to dult mouse Purkinje neurons through ell fusion. The formtion of heterokryons inreses in liner mnner over 1.5 yers nd seems to e stle. The dominnt Purkinje neurons used the BMDC nulei within the resulting heterokryons to enlrge, exhiit dispersed hromtin nd tivte Purkinje neuron-speifi trnsgene, L7-GFP. The oserved reprogrmmed heterokryons tht form in rin my provide insights into gene regultion ssoited with ell-fte plstiity. The formtion of stle inulete ells, heterokryons, through the fored fusion of disprte ell types helped eluidte fundmentl priniples regrding gene expression efore the dvent of trnsfetion tehnologies. Evidene tht ftors funtioned in trns to tivte or repress gene expression ws first otined through fusion of different ell types to form hyrid ells 1 5.These studies were extended y the disovery tht heterokryons ould e formed, in whih ellulr division nd susequent hromosome loss ws voided 6 9.Suh stle fusion produts demonstrted tht the phenotype of ell ould e ltered. Indeed, the differentited stte of ell ws shown to require ontinuous regultion nd to e determined lrgely y the lne of ftors within tht ell t ny given time. In reent yers, severl reports hve demonstrted tht BMDCs n ontriute to the regenertion of diverse dult tissues, suh s epithelium, musle, liver, hert nd rin, in oth mie nd humns 11,12.Two mehnisms hve een suggested to ount for the ontriution of BMDCs to dult tissue regenertion: first, hnges in gene expression through de novo genertion of tissue-speifi ells from BMDCs in response to the tissue miro-environment 13 17 ;seond, hnges in gene expression fter fusion of BMDCs with pre-existing ells nd exposure to n ltered intrellulr milieu 18 20. Purkinje neurons re mononulete diploid ells tht re generted only during gesttion nd not repled fter loss through trum or geneti disese 21 26.The omplexity nd importne of the Purkinje neuron is undersored y the ft tht the xons of the Purkinje neurons re the only efferent from the ereellum to other rin regions, nd in humns eh Purkinje neuron n reeive over 1 million inputs from other neurons. Indeed, these lrge, highly speilized, Purkinje neurons of the ereellum re ritil to lne nd fine motor ontrol, nd defets in these ells result in txis 27. Previously, we showed tht BMDCs ontriuted to Purkinje neurons in the rins of dult women who reeived one mrrow from mle donors for the tretment of hemtologil mlignnies; however the underlying mehnism remined unler 28.From these dt, it ws pprent tht in humns, s in mie, donor-derived one mrrow ells ould ross the lood rin rrier nd ontriute to the Purkinje neuron pool. However, euse of tehnil limittions, we ould not disern whether this ourred through de novo genertion of Purkinje neurons, s suggested in mie 17,or through fusion of BMDCs to preexisting Purkinje ells. As result, we undertook n in-depth nlysis in the mouse. Here, we provide unexpeted evidene tht the mehnism wherey BMDCs ontriute to Purkinje neurons in dult mie is not merely fusion, ut the formtion of stle euploid heterokryons. The previously unreognized finding tht inulete, hromosomlly lned heterokryons re produed in vivo in tissues suh s rin is remrkle, s stle heterokryons were only thought to our rtifiilly in tissue ulture 6 9,29,30.In these in vivo heterokryons, the neurons were dominnt over the BMDCs, s no mitosis ws evident nd the morphology ws typil of funtionl Purkinje neurons, with omplex dendriti trees nd xons. Moreover, ytoplsmi ftors within the Purkinje neurons reprogrmmed the fused BMDC nulei resulting in nuler swelling, deondensed hromtin nd tivtion of Purkinje neuronspeifi trnsgene, L7-GFP. We hypothesize tht the reprogrmmed 1 Bxter Lortory in Geneti Phrmology, Deprtment of Miroiology nd Immunology, Deprtment of Moleulr Phrmology, Stnford University Shool of Mediine, Stnford, CA 94305, USA. 2 Correspondene should e ddressed to J.M.W. nd H.M.B. (e-mil: jweimnn@stnford.edu nd hlu@stnford.edu). Pulished online: 15 Otoer 2003; DOI:.38/n53 NATURE CELL BIOLOGY VOLUME 5 NUMBER 11 NOVEMBER 2003 959
Of Ctx Th GCL heterokryons oserved here, whih inrese in frequeny with ge, my provide insights into hromtin re-rrngement nd gene regultion. RESULTS Bone-mrrow-derived Purkinje neurons Sine our previous studies in humn rins showed tht BMDCs ontriute to Purkinje neurons, we deided to exmine mie to investigte ML PCL Figure 1 GFP-positive Purkinje neurons in the ereellum. () A shemti representtion of mouse rin, showing the nterior olftory ul (Of), ererl ortex (Ctx), thlmus (Th) nd the udlly loted ereellum (C). (). In thik setions (45 µm) ut from the ereellum of post-onemrrow-trnsplnted mouse, individul donor-derived GFP-positive Purkinje neurons re evident in the Purkinje ell lyer (PCL). The dendrites from these ells extend into the ell-sprse moleulr lyer (ML), wheres their xon projets through the grnulr ell lyer (GCL) nd is the only output onnetion from the ereellum to the rest of the rin. Three loes of the ereellum in the ox in n e seen in. Note the mny one-mrrowderived (GFP-positive) ells in the prenhym. () A high-power lsersnning onfol imge of this ell shows its mny synpti spines nd single output xon (rrow). The two GFP-positive BMDC ells re proly mirogli or mrophges in PCL nd ML (rrowheds). Sle rs represent 2 mm in, 0 µm in nd 50 µm in. C whether the underlying mehnism is de novo genertion or fusion. The one mrrow from trnsgeni mie uiquitously expressing GFP ws hrvested nd trnsplnted y til-vein injetion into lethlly irrdited syngenei reipient mie. Severl months lter, Purkinje neurons expressing GFP were deteted in the ereell of reipient nimls (Fig. 1). These GFP-positive Purkinje ells were indistinguishle from norml Purkinje neurons, with their som in the Purkinje ell lyer (PCL) nd lrge, pil nd highly rnhed dendriti tree tht extended into the ell-sprse moleulr lyer (ML; Fig. 1). The single xon from the Purkinje neuron extended through the grnulr ell lyer (GCL) into the white mtter nd ws the only output xon from this neuron to other rin regions. An imge of one-mrrow-derived Purkinje neuron t low mgnifition shows this ell in the ontext of ereellr loe (Fig. 1). At higher mgnifition, lser-snning onfol mirosopy revels prt of the desending xon (Fig. 1, rrow) nd mny smll synpti spines on the extensive dendriti tree. Other GFPpositive BMDCs, suh s mirogli nd mrophges, were redily pprent throughout the rin (Fig. 1); two suh ells re mrked with rrowheds in Fig. 1. The rhiteture nd struture of the dendriti tree of the GFP-positive Purkinje ell with its mny synpti spines re indistinguishle from typil Purkinje neurons nd re hrteristi of helthy funtioning neurons. We investigted whether GFP-positive Purkinje neurons expressed genes tht re typilly found in Purkinje ells, one mrrow ells, or oth. When nlysed y immunofluoresene mirosopy, ll of the GFP-positive Purkinje neurons strongly expressed the lium-inding protein, lindin, hllmrk of the Purkinje ell type (Fig. 2, ). No other ell type expressed lindin in the ereellum. To ssess whether they still expressed mrkers typil of one mrrow ells, GFP-positive Purkinje neurons were ssyed for hemtopoieti mrkers. Setions ontining BMDC Purkinje neurons were stined with ntiodies ginst CD45 ( pn-hemtopoieti mrker), CD11 ( mrophge/mirogli mrker), F4/80 nd I1 (miroglil mrkers; Fig. 2 j). The GFP-positive Purkinje neurons were negtive for ll four of these hemtopoieti mrkers, suggesting tht the genes enoding these produts were either intivted or never expressed in the BMDCs tht resulted in the GFP-positive Purkinje neurons in the rin. The BMDCs lso yield other ell types in the ereellr prenhym, inluding GFP-positive mirogli nd mrophge ells. As expeted, these GFP-positive BMDCs expressed hemtopoieti mrkers (insets in Fig. 2, d; rrowheds in Fig. 2e j). Thus, o-expression of Purkinje neuron gene mrkers nd hemtopoieti mrkers ws not oserved. We then determined the time ourse of BMDC ontriution to the Purkinje ell pool. Mie were trnsplnted with one mrrow t two months of ge nd the numer of GFP-positive Purkinje neurons deteted in 20 mie under non-seletive onditions ws sored over period spnning 1.5 yers, pproximtely 75% of the verge mouse lifespn (Fig. 3). GFP-positive neurons were not pprent until severl months fter trnsplnttion, nd the mximum numer oserved under these non-seletive onditions ws 60 neurons in one niml fter 1.5 yers. A liner inrese in GFP-positive neurons ws oserved tht orrelted with ge, pttern tht ws sttistilly signifint up to 16 months fter trnsplnttion (see Fig. 3 legend). GFP-positive BMDCs fuse to mture Purkinje neurons We nlysed the nuler omposition of the GFP-positive Purkinje neurons to determine whether they rose de novo from BMDCs or through fusion to endogenous Purkinje neurons. Using lser-snning onfol mirosope, seril 1-µm optil setions were otined through the entire ell ody of GFP-positive Purkinje ells. Seril 960 NATURE CELL BIOLOGY VOLUME 5 NUMBER 11 NOVEMBER 2003
reonstrution of these ells reveled tht in the more thn 300 ses where it ws possile to imge the full extent of the som, two nulei were lwys deteted. A typil GFP-positive Purkinje neuron with n xon exiting the som from the top right nd primry dendrite with severl seondry nd tertiry dendrites is shown (Fig. 3, ). As with ll of the GFP-positive Purkinje neurons, numerous smll synpti spines (the post-synpti speiliztions of tive synpses) were redily pprent on the dendrites. The endogenous Purkinje nuleus of the reipient ws enlrged, with dispersed hromtin nd prominent nuleolus (Fig. 3, ; middle rrow), similr to other neighouring Purkinje nulei evident in this field of view (Fig. 3, ; left nd right rrows). By ontrst, the puttive one-mrrow-derived nuleus tht fused into the host Purkinje neuron ontined ompt highly ondensed hromtin (Fig. 3, rrowhed). These results suggest tht BMDCs ontriute to the Purkinje neurons y fusion nd not y de novo neurogenesis. To determine definitively the ellulr origin of eh nuleus within GFP-positive Purkinje neurons, we trnsplnted one mrrow from mle donor mie into femle reipient mie using the sme experimentl prdigm desried ove. The presene of mle nuleus ws ssyed using Texs-Red-lelled DNA proe speifi to the Y hromosome nd exmintion y fluoresene in situ hyridiztion (FISH). One yer fter trnsplnttion, rins were setioned t 12 µm nd seril ereellr setions were stined for GFP nd ounterstined with the nuler stin To-Pro3 to visulize nuler DNA. Seril 1-µm optil setions were otined to determine the numer of nulei in GFP-positive ells. A motorized stge ws used to reord the x, y nd z oordintes, ensuring the preise relotion of GFP-positive ells fter the proteinse K digestion nd FISH stining protool, whih removes most of the GFP stining. Representtive exmples of GFP-positive Purkinje neurons with two distint To-Pro3-lelled nulei re shown (Fig. 4, ). After FISH, red Y-hromosome ws deteted in one of the two nulei in eh ell (Fig. 4, d; rrowhed). The two sets of pnels in this figure show ells in the sme lotions efore nd fter the extensive proteinse K digestion of the tissue tht is neessry for FISH. The other nuleus within the GFP-positive som (Fig. 4, rrow) is the endogenous ell nuleus of the Purkinje neuron tht does not ontin Y-hromosome. In the exmple shown in Fig. 4, d, the GFP-positive som ws found in two djent setions. The hromtin in the donorderived Y-hromosome-positive nuleus of this ell ws s dispersed s the host nuleus, with prominent nuleolus; struture not seen in the ompt hromtin hrteristi of mrrow-derived ells. Donorderived miroglil ells were evident in the host tissue, nd these ells lso ontined Y hromosome (dt not shown). Despite the hnge in nuler morphology, there ws no evidene of ytokinesis or kryokinesis in ny of the ells nlysed. Indeed, the fused ells seemed to e stle heterokryons tht persisted over time. Furthermore, there ws no evidene of GFP-positive Purkinje neuron deth, suh s leing or memrne frgmenttion, mong the more thn 300 GFP-positive neurons exmined. These dt indite tht the GFP-positive Purkinje neurons found in the host ereellum re the result of fusion etween host femle Purkinje ell nd mle BMDC. Chromtin reorgniztion nd gene tivtion in fused BMDCs During the ourse of our nlysis, we oserved tht the struture of the two nulei differed mrkedly mong the GFP-positive Purkinje ells. Approximtely 50% of the GFP-positive ells ontined one lrge Purkinje-like nuleus, with dispersed hromtin, nd one smll one-mrrow-like nuleus, with ompt hromtin. In the other ells sored, oth nulei ppered Purkinje-like. The rnge of nuler morphologies tht were oserved is shown in Fig. 5. A time ourse of e g i d f h j ClB CD45 CD11 F4/80 I1 50 µm Figure 2 Mrker expression in GFP-positive Purkinje neurons. Immunohistohemistry with speifi ntiodies demonstrtes the presene of Purkinje-speifi mrkers, ut not mrrow mrkers. (, ) Clindin, lium-inding protein, stins the dendrites nd somt of Purkinje ells. All of the GFP-positive Purkinje neurons were lso lindin-positive (rrow; 20/20 ells), wheres none of the mirogli or other ell types in the ereellum were lindin-positive. (, d) GFP-positive Purkinje ells were negtive for the pn-hemtopoieti mrker CD45 (rrow; 0/15 ells), wheres donor nd host mirogli were positive (insets). (e h) These GFPpositive Purkinje ells were lso negtive for the mrophge/mirogli mrkers CD11 (rrow; 0/8 ells) nd F4/80 (rrow; 0/12 ells). Note tht other BMDCs remin positive for these mrkers (rrowheds). (i, j). The mirogli lium-inding protein I1 did not stin ny of the GFP-positive neuronl som or dendrites (0/7 ells; rrow) ut strongly lelled djent mirogli (rrowheds). Sle r represents 50 µm in ll pnels. NATURE CELL BIOLOGY VOLUME 5 NUMBER 11 NOVEMBER 2003 961
Numer of GFP-positive purkinje neurons 30 25 20 15 5 0 0 2 4 6 8 12 14 16 Months d Figure 4 Fusion of mle BMDC to femle Purkinje neuron. () A Texs- Red-lelled Y hromosome proe ws used to detet donor-derived mle nulei y FISH. A single 1-mm onfol optil setion through 12-mm setion of GFP-positive Purkinje ell ontining two nulei. () After protese digestion nd hyridiztion, it is evident tht the top nuleus (rrowhed) hs Y hromosome nd is donor-derived. Note tht some displement of the nulei ours through digestion nd in situ proessing. () Another doule-nulei GFP-positive ell. The host nuleus in d is eneth the mle donor-derived nuleus shown in (rrowhed). The host nuleus visile in d does not possess Y hromosome. Sle r represents 20 µm. Figure 3 Time ourse of GFP-positive Purkinje neuron pperne. () Mie were one-mrrow-trnsplnted t two months of ge nd ereell were olleted nd nlysed t vrious times. The numer of GFP-positive Purkinje ells inresed in liner mnner over time, with liner regression of 0.92. One niml nlysed t 18 months hd higher thn expeted numer of ells (n = 60) tht my reflet some spet of the geing proess. This one dt point ws not inluded in the grph. (, ) All of the GFP-positive Purkinje ells oserved ontined two nulei. This Purkinje ell hs distintive dendriti tree with mny synpti spines nd n xon exiting the som t the left. One of the two nulei in the ell is ompt (rrowhed) nd is the puttive BMD nuleus. The other nuleus hs dispersed hromtin similr to other Purkinje neurons (rrows). In 752 ontrol Purkinje neurons from trnsplnted nd norml mie, no inuleted ells were oserved. Sle r represents 20 µm. hromtin ltertion demonstrtes tht the rtio of nulei with dispersed-to-ompt hromtin in the ereell of individul mie inresed over time (Fig. 5i). These dt suggest tht one BMDC with ompt nuleus fuses to Purkinje neuron (Fig. 5, e), the one-mrrow-derived nuleus eomes less ompt nd dense (Fig. 5, f) nd finlly ssumes the morphology of the Purkinje nuleus to whih it fused (Figs 4 nd 5, d, g, h). This inresing trend towrds dispersed hromtin in the fused BMDC nuleus over time suggests tht the fusion events re stle. The tivtion of previously silent genes y intrellulr signls generted y one of the two nulei in heterokryon hs een well estlished in vitro 31.To determine whether the hnges in hromtin struture oserved in the fused BMDC nulei orrelte with reprogrmming nd tivtion of Purkinje genes, trnsgeni mouse tht expresses GFP under the ontrol of the Purkinje-speifi promoter, L7- pp-2 ws used s one-mrrow donor 32.We onfirmed the previously desried expression pttern of the L7-GFP promoter y nlysing setions from the rin of these trnsgeni mie. In the rin, the only GFP-positive ells deteted were the Purkinje neurons (dt not shown). Flow ytometry nlysis of the L7-GFP one mrrow showed tht these trnsgeni mie do not express GFP in their one mrrow. Indeed the fluoresene-tivted ell sorting (FACS) plots of the L7-GFP trnsgeni ells were indistinguishle from those of wildtype mrrow nd were three orders of mgnitude lower thn the GFP fluoresene otined from GFP-positive one mrrow (Fig. 6). Thus, the L7-GFP trnsgeni promoter is intive in the one mrrow of this mouse line. Four mie were srified five months fter reeiving one mrrow trnsplnt from the L7-GFP mouse nd then nlysed. L7-GFP-positive Purkinje neurons were found in the ereell of ll four mie, nd on verge 2 3 fully mture GFP-positive neurons were oserved in eh mouse (Fig. 7), orrelting with our erlier predition for five months fter trnsplnttion (Fig. 3). All of the L7-GFP-positive Purkinje ells ontined two nulei (Fig. 7, ). In the ells shown in Fig. 7, d, one nuleus ws evident in the onfol imge, wheres the 962 NATURE CELL BIOLOGY VOLUME 5 NUMBER 11 NOVEMBER 2003
d e f g h Perentge of dispersed/ompt nulei i 90 80 70 60 50 40 30 20 0 0 2 4 6 8 12 14 16 Months fter trnsplnt Figure 5 Chnges in nuler morphology within heterokryons over time. In some ses one of the two nulei hs ompt hromtin (, e), in others the hromtin is eginning to look dispersed (, f), wheres in others the two nulei pper identil (, g). In some ses, oth pper s norml Purkinje nulei with highly dispersed hromtin nd prominent nuleolus (d, h). Arrowheds indite donor-derived one mrrow nuleus nd rrows point to Purkinje-like dispersed nulei. (i) The nuler pperne of oth nulei in the fused ells ws mesured. Nulei tht were 20% the dimeter of norml Purkinje nulei were onsidered ompt, wheres nulei tht were 60% or greter in dimeter were onsidered s dispersed. The rtio of dispersed-to-ompt nulei per niml is plotted over period of 16 months post-trnsplnt. Sle r represents 20 µm in pnels h. other ws in different plne of fous. Donor-derived hemtopoieti ells suh s mirogli nd mrophge ells re known to e present in the rin prenhym fter one mrrow trnsplnt (see Fig. 1, nd ref. 33), ut these donor-derived ells did not express GFP (Fig. 7), further indition for the speifiity of the L7-pp-2 promoter. These results demonstrte tht under physiologil onditions, trnsplnted BMDCs not only fuse to pre-existing Purkinje neurons, ut n lso tivte the Purkinje neuron-speifi promoter, L7-pp-2. Thus, in these ells the BMDC nuleus ws reprogrmmed fter it fused to the Purkinje ell, enling expression of the Purkinje-speifi promoter L7-pp-2.These results show tht gene tivtion, only otined previously in vitro in heterokryons, n our spontneously in vivo. The results strongly suggest tht the one-mrrow-derived nulei re not only ltered morphologilly, ut lso reprogrmmed in the dult Purkinje ell, s shown y the expression of the reporter gene GFP under the ontrol of the L7-pp-2 promoter. DISCUSSION Two mehnisms hve een proposed for BMDCs to ontriute to speilized dult tissues suh s rin: first, de novo genertion of tissuespeifi ells; seond, fusion with pre-existing ells within tissues. Despite their differenes, oth mehnisms ultimtely involve hnges in gene expression; tht is, nuler reprogrmming, providing in vivo evidene tht the differentited stte n e ltered 31.In the first se, BMDCs undergo hnge in funtion in response to signls in the lol environment nd differentite ordingly 13 15,34. De novo genertion of tissue-speifi ells hs een reported in endothelium, the entrl nervous system nd liver 14 17.In ddition, in musle, physiologil progression ws demonstrted in whih BMDCs first quired hrteristis of heritly diploid musle stem ells (stellite ells) nd lter ontriuted to mture myofires of musle tissue 13.Tken together, these dt suggest tht one-mrrow-derived stem ells n undergo ell-fte hnge s diploid mononulete ells nd, one reprogrmmed, ontriute to vrious tissues. In the seond se, BMDCs undergo hnge in ell funtion s result of signls within the ells to whih they fuse 19,20.Although evidene for oth mehnisms hs een reported, fusion hs not een shown to our nturlly in vivo in the sene of strong seletive pressure. Moreover, the previously demonstrted exmples of fusion re in liver nd re the result of prolifertion of polyploid or neuploid ells. By ontrst, we show here tht ells from the mrrow n fuse spontneously with highly omplex Purkinje neurons of the NATURE CELL BIOLOGY VOLUME 5 NUMBER 11 NOVEMBER 2003 963
,000 Wild type L7-GFP Control-GFP,000,000 00 00 00 Sidestter 0 0 0 1 1 0 1,000,000 1 1 0 1,000,000 1 1 0 1,000,000 GFP GFP GFP Figure 6 Flow ytometry of one mrrow from the L7-GFP mouse. FACS nlysis, showing tht one mrrow ells do not express the Purkinje neuron-speifi trnsgene, L7-pp2-GFP. Bone mrrow ws dissoited from two wild-type mie, two GFP trnsgeni mie nd four L7-GFP trnsgeni mie. Comprison of the FACS profiles shows tht the wild-type nd L7-GFP mie disply no fluoresene when ompred with the GFP one mrrow. Gting of PI-lelled ells (ded ells) exluded them from the ontour profiles. ereellum to form stle, non-dividing, inulete, hromosomlly lned heterokryons. Previously we showed tht, in the ereell of women trnsplnted with mle one mrrow, Y hromosomes were deteted in Purkinje neurons 28.These dt demonstrted lerly tht BMDCs ould ross the lood rin rrier nd ontriute to the Purkinje ell popultion in dult humn rins. However, the mehnism y whih this ourred ould not e determined in these humn utopsy smples. Here, we show lerly in mie tht fusion is the underlying mehnism y whih BMDCs ontriute to Purkinje neurons. Fusion ours spontneously nd physiologilly to generte stle heterokryons in the sene of seletive pressure through geneti defets or drug tretment. The frequeny inreses over time, even though the lood rin rrier opens only trnsiently 35.After one mrrow trnsplnt from trnsgeni mouse uiquitously expressing GFP, numerous GFP-positive ells were found to e inulete Purkinje/BMDC heterokryons in whih the nulei remined intt nd distint. Suh heterokryons inresed in frequeny with inresing ge of the mouse. The morphology of the more thn 300 GFP-positive ells nlysed ws typil of funtionl thriving Purkinje ells, with xons nd full omplex dendriti trees from whih synpti spines projeted. Fusion of BMDC ws speifi to these ells, s no other neurons in this prt of the rin expressed GFP fter trnsplnt. This finding is of prtiulr interest, s Purkinje neurons re the most omplex nd elorte in the ereellum nd hve ritil funtion in lne nd movement 25,26. Although erlier studies with su-optiml ssys for ssessing DNA ontent one suggested tht Purkinje ells were tetrploid 36, using improved ssys others hve now lerly shown tht Purkinje ells re normlly diploid in oth mouse nd humn 21 23. Thus, the heterokryons formed hd n equl hromosoml ontriution from oth ell types. Definitive proof tht the inulete ells resulted from fusion ws otined fter trnsplnttion of mle one mrrow into femle mie nd detetion of Y hromosome in one of the two nulei per heterokryon. The BMDC nulei inside Purkinje heterokryons were reprogrmmed. BMDC nulei re generlly ompt, wheres Purkinje neurons re not. The proportion of Purkinje neurons with two enlrged nulei with dispersed hromtin inresed with the ge of the mouse. On the sis of these hnges in nuler morphology over time, we hypothesized tht in its new intrellulr milieu, the BMDC nuleus ws progressively reprogrmmed. In support of this hypothesis, trnsplnttion of one mrrow from trnsgeni mouse hrouring silent Purkinjespeifi trnsgene, L7-GFP, resulted in its tivtion nd GFP expression in Purkinje-BMDC heterokryons. Tken together with the typil Purkinje neuron morphology in GFP-expressing heterokryons nd the lk of expression of three distint lood ell mrkers, CD11, F4/80 nd CD45, these results suggest tht the Purkinje ell is dominnt in these inulete heterokryons. To dte, the only other exmple of BMDC fusion to tissue-speifi ells in vivo is in the liver 19,20.The results of these studies re distint from those reported here in severl respets: first, lthough the initil frequeny of fusion grees with the dt in this report (1/50,000), the heptoyte/bmdc fusion produt is not stle heterokryon. Insted, it prolifertes extensively, resulting in millions of highly neuploid progeny; seond, the liver studies used strong seletive pressure, the survivl of mouse with lethl geneti disorder, tyrosinemi, s well s repeted drug dministrtion. For survivl, expnsion of the rre BMDC/heptoyte fusion events ws solutely neessry. The resulting kryotypi instility ws presumly well tolerted euse dult heptoytes re typilly multinuler, polyploid nd even neuploid 37 39.As 6% of donor one-mrrow-derived heptoytes were diploid 20, the possiility remins tht ell-fte hnge from BMDC to heptoyte ourred efore fusion, rther thn fter fusion with host heptoytes. Thus, it is unler whih me first, ell fte hnge or ell fusion. Cell fusion studies hve hd long history in tissue ulture, providing the first evidene of trns-ting repressors nd tivtors of gene expression 2,3,5,40. Gene repression ws most ommonly oserved in hyrid ells, ut whether this ws used y loss of the gene of interest or the gene enoding the tivtor ws diffiult to disern, s the proliferting fusion produts were kryotypilly unstle 3,41. Gene tivtion ws first oserved in synkryons, hyrids etween heptoytes nd other ell types, nd ws ttriuted to the inresed gene dosge provided y the polyploid liver ells reltive to their fusion prtners 1,4.Gene tivtion ws generlly trnsient in these ses euse of the loss of nuler hromosomes fter the kryokinesis nd ytokinesis typil of hyrids (synkryons). Heterokryons, in ontrst, do not undergo mitosis nd stly retin ll nuler nd ytoplsmi omponents of the ell types fused in distint intt nulei 5.Using heterokryons in vitro, the expression of previously silent genes in disprte nulei ould e stly indued, prtiulrly if the nuler rtio of the two fused ell types ws 964 NATURE CELL BIOLOGY VOLUME 5 NUMBER 11 NOVEMBER 2003
d into sterile ulture dish. Mrrow frgments were dissoited y triturting through the 25-guge needle, nd the resulting suspension ws filtered through sterile 70-µm nitex mesh (BD-Flon, Frnklin Lkes, NJ). The filtrte ws ooled on ie, spun for 5 min t 250g, nd the pellet ws resuspended in ie-old HBSS with 2.5% FCS to 8 7 nuleted ells per ml. Simultneously, 8 - week-old C57BL/6 mie (Stnford) were lethlly irrdited with two doses of 4.8 Gy 3h prt. Eh irrdited reipient reeived 125 µl of the unfrtionted mrrow ell suspension y til-vein injetion within 1 h of the seond irrdition dose. Hrvesting of rins. Mie were srified t vrious times fter one mrrow trnsplnttion. The mie reeived lethl injetion of pentoritl (Sleepwy, Fort Dodge Animl Helth, Fort Dodge, IA) nd were immeditely perfused with ie-old phosphte uffer (PB) followed y 4% prformldehyde in PB. The rins were then removed nd ryoproteted in 20% surose/pb solution overnight. Thik tissue setions (35 50 µm) for ntiody stining, enumertion of donor derived ell numer nd nuler ontent were otined on sliding mirotome (SM2000R; Lei, Bnnokurn, IL). Thin setions for FISH were mde on ryostt (CM3050S; Lei) t 12 µm nd mounted on geltin-oted slides (Goldsel, Portsmouth, NH). Figure 7 Evidene for reprogrmming of BMDCs fter fusion to Purkinje neurons. () Low-power imge of L7-GFP one-mrrow-derived Purkinje neuron with the som in the PCL nd dendrite extending into the ML. ( d) High-power imges of three L7-GFP one-mrrow-derived Purkinje neurons. All eight of the L7-GFP neurons hd doule nulei. In ll imges, Green represents GFP nd Red represents To-Pro3. Sle r represents 50 µm in nd 20 µm in d. unequl, demonstrting tht gene dosge or the lne of ellspeifi ytoplsmi regultors ws ritil 6,30. In summry, the findings reported here re highly unexpeted nd signifint for severl resons: heterokryons formed spontneously in vivo through the fusion of two disprte ell types, resulting in stly inulete ells with equivlent hromosoml input. These dt demonstrte tht ell fusion in Purkinje neurons of the mouse rin n our under physiologil onditions without ongoing seletive pressure. The result of this fusion is heterokryon ontining reprogrmmed one mrrow nuleus, presumly through the inresed dosge of regultory proteins in the muh lrger Purkinje ell ytoplsm. Eh GFP-positive fusion produt, of the hundreds exmined, ws inulete, nd the frequeny of this event inresed with ge. Future studies will fous on the eluidtion of the moleulr sis of fusion nd possile mens to enhne its frequeny. These studies will lso ontriute to eluidtion of the mehnisms underlying ell-fte plstiity. METHODS Bone mrrow trnsplnttion. Mrrow ws isolted under sterile onditions from 8 -week-old C57BL/6 trnsgeni mie tht uiquitously expressed enhned green fluoresent protein (GFP) 42.Donor mie were killed y ervil dislotion, riefly immersed in 70% ethnol nd their skin peeled k from midline, irumferentil, inision. After the femurs, tiis nd humeri were removed, ll musle ws srped wy with rzor lde nd the ones were pled in ml of lium nd mgnesium-free, Hnk s lned slt solution (HBSS, Invitrogen, Crlsd, CA) with 2.5% foetl lf serum (FCS; SH30072.03, HyClone, Logn, UT) on ie for up to 90 min. The tips of the ones were removed nd 25-guge needle ontining 1 ml of ie-old HBSS with 2.5% FCS ws inserted into the mrrow vity nd used to wsh the mrrow out Antiody Stining. Antiodies ginst GFP (mouse 1:00; #A-11120; rit 1:2000; A-11122, Moleulr Proes, Eugene, OR), Clindin (1:00; C9848, Sigm, St Louis, MO), MAP2 (M2376; 1:0, Sigm), CD11 (1:0; #553308, BD Biosienes PhrMingen, Sn Diego, CA), CD45 (1:200; #553076BD Biosienes PhrMingen), F4/80 (#RM2900; 1:50, Cltg, Burlingme, CA), I 1 (1:00, gift from Y. Imi, Ntionl Institute of Neurosienes, Tokyo Jpn), were pplied for 12 h t 4 C to the floting setions fter pre-inution in loking solution for 2 h. When mouse or rit primry ntiodies were used, nti-cd16/cd32 (1:200) ws lso inluded (#553142; BD Biosienes PhrMingen). The setions were then inuted in pproprite seondry ntiodies overnight t 4 C. The loking solution ontined 5% got serum, 3% BSA nd 0.3% Triton X-0. FISH nlysis. Thin setions (12 µm) of the ereell from GFP-trnsplnted mie were proessed for GFP using stndrd immunohistohemistry. The nulei were then ounterstined with To-Pro3. These setions were then viewed for the presene of GFP-positive Purkinje neurons nd snned t 1-µm optil setion using snning onfol mirosope (LSM5; Zeiss, Thornwood, NY). The x- nd y-position of the GFP-positive ell odies were reorded with respet to the orners of the slide, to relote the ext position fter FISH. The FISH protool ws modified from ref. 43 nd protools from Applied Spetrl Imging (Crlsd, CA). Briefly, setions were then dehydrted, treted with proteinse K t 45 C for 7 15 min, rinsed in 2 SSC nd dentured in 70% formmide in 2 SSC t 68 C for 5 min. The slides were then dehydrted nd wrmed to 50 C. The X nd Y hromosome proes were dentured nd pplied s direted (see CmBio nd Applied Spetrl Imging wesite). After 36 h t 37 C, the proe ws wshed off in 2 SCC, efore inution in 2 SSC/0.1% NP40 t 50 C for 2 min nd mounted with Vysis DAPI mounting solution with 1:3000 To-Pro3. Flow ytometry nd FACS Anlysis. Bone mrrow ws prepred s desried ove, with the exeption tht erythroytes were lysed in lysis uffer (0.15 M NH 4 Cl, 1.0 mm KHCO 3 nd 0.1 mm N 2 EDTA t ph 7.4) for 5 min on ie efore inution with propidium iodine (PI; finl onentrtion 0 µg ml 1 ) to exlude ded ells. Totl unfrtionted one mrrow (0 µl) from five L7/GFP-Pp-2 trnsgeni, one GFP-trnsgeni nd three wild-type mie, respetively, were used to quire dt to determine whether one mrrow ells (one million ells) expressed GFP, using FACSCliur (BD Biosienes, Sn Diego, CA). These experiments were repeted in triplite. Dt were nlysed nd presented with FlowJo v.4.3 softwre (Tree Str, In., Ashlnd, CA), displyed s ontour plot t 5% proility, s funtion of side sttered versus GFP fluoresene. All nimls were proessed simultneously. ACKNOWLEDGMENTS We thnk K. Kolekr for her exellent tehnil support nd R. Doyonns, M. LBrge, A. Okd, F. Rossi nd M. Springer for useful omments. We lso thnk M. Yuzki for the generous gift of the L7-pp-2 trnsgeni mie. This work ws supported y fellowship from the Wenner-Gren Foundtion, Sweden, to C.B.J. NATURE CELL BIOLOGY VOLUME 5 NUMBER 11 NOVEMBER 2003 965
nd Ntionl Institutes of Helth grnt AG20961 to J.M.W. nd H.M.B. nd NIH grnts AG09521, HL65572, HD18179, Ellison Medil Foundtion grnt AG-33-0817, the MKnight Endowment Fund nd the Bxter Foundtion to H.M.B. COMPETING FINANCIAL INTERESTS The uthors delre tht they hve no ompeting finnil interests. Reeived 14 July 2003; epted 30 Septemer 2003; Pulished online t http://www.nture.om/ntureelliology 1. Drlington, G. J., Bernrd, H. P. & Ruddle, F. H. Humn serum lumin phenotype tivtion in mouse heptom humn leukoyte ell hyrids. Siene 185, 859 862 (1974). 2. Ephrussi, B. & Weiss, M. C. Interspeifi hyridiztion of somti ells. Pro. Ntl Ad. Si. USA 53, 40 42 (1965). 3. Hrris, H. Cell fusion: the Dunhm letures (Hrvrd University Press, Cmridge, 1970). 4. Peterson, J. A. & Weiss, M. C. Expression of differentited funtions in heptom ell hyrids: indution of mouse lumin prodution in rt heptom mouse firolst hyrids. Pro. Ntl Ad. Si. USA 69, 571 575 (1972). 5. Ringertz, N. & Svge, R. E. 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