Plzf regulates limb and axial skeletal patterning

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1 rtile Plzf regultes lim n xil skeletl ptterning Mri Brn 1, Niol Hwe 1, Lee Niswner 2 & Pier Polo Pnolfi 1 The promyeloyti leukemi zin finger (Plzf) protein (enoe y the gene Zfp145) elongs to the POZ/zin-finger fmily of trnsription ftors. Here we generte Zfp145 / mie n show tht Plzf is essentil for ptterning of the lim n xil skeleton. Plzf intivtion results in ptterning efets ffeting ll skeletl strutures of the lim, inluing homeoti trnsformtions of nterior skeletl elements into posterior strutures. We emonstrte tht Plzf ts s growth-inhiitory n pro-poptoti ftor in the lim u. The expression of memers of the ominl (A) Hox gene omplex, s well s genes enoing one morphogeneti proteins (Bmps), is ltere in the eveloping lim of Zfp145 / mie. Plzf regultes the expression of these genes in the sene of errnt polrizing tivity n inepenently of known ptterning genes. Zfp145 / mie lso exhiit nterior-irete homeoti trnsformtion throughout the xil skeleton with ssoite ltertions in Hox gene expression. Plzf is therefore meitor of nterior-to-posterior (AP) ptterning in oth the xil n ppeniulr skeleton n ts s regultor of Hox gene expression. Introution The verterte lim evelops from smll us tht rise from the lterl plte mesoerm. Cells t the tip of the lim u rpily proliferte, wheres mesenhyme ells t the se of the u egin to terminlly ifferentite. A funtionlly efine zone of polrizing tivity (ZPA) is lote in the posterior lim u mesenhyme n is mrke y the presene of Shh (ref. 1). This region is importnt in meiting AP ptterning of the eveloping lim u, ut the signls ting ownstrem or inepenently of Shh signlling tht re responsile for iretly regulting the growth n ptterning of the lim re not well hrterize, lthough the Hox genes hve een implite. In the xil strutures, the Hox genes re expresse long the AP xis n re responsile for the speifition of segmentl ientity of verterl strutures 2. Gin-of-funtion n loss-offuntion experiments hve emonstrte tht misexpression of given Hox gene results in morphologil trnsformtion of the tissue into struture tht woul hve normlly expresse this gene 2. In ition, etopi expression of Hox genes uses ltertions in oth lim n xil skeletl ptterning. The gene ZFP145 (enoing PLZF) ws ientifie y virtue of hromosoml trnslotions involving RARA (enoing retinoi i reeptor-α) leing to ute promyeloyti leukemi 3 (APL). PLZF is nuler phosphoprotein, ontining t the roxy terminus nine Krüppel-type zin-finger omins, whih in iretly to DNA (refs 3 5). At the mino terminus, PLZF ontins BTB/POZ omin tht meites self-ssoition n trnsriptionl repression through reruitment of nuler orepressors 6 8. Here we show tht Plzf is essentil for ptterning of the lim n xil skeleton n ts s min upstrem regultor of Hox gene expression. Results Trgete isruption of Zfp145 in emryoni stem ells By homologous reomintion, we sustitute exon 2 of Zfp145, whih ontins the 5 region spnning the region enoing the POZ omin n Plzf zin fingers 1 n 2, with neomyin-resistne gene ssette (Fig. 1,). We emonstrte intivtion of Plzf y the sene of ny etetle protein from ells from Zfp145 / mie y western-lot nlysis (Fig. 1, n t not shown). Lim homeosis n ptterning efets in Zfp145 / mie The verterte hinlim is ivie into three regions: stylopo (femur), zeugopo (tii n fiul) n utopo (trsls, mettrsls n phlnges). Zfp145 / mie isply morphologil efets ffeting ll the skeletl strutures of the hinlim, wheres skeletl efets in the forelim our t lower frequeny. In the hinlim utopo of these nimls, igit I is homeotilly trnsforme into igit II (92%), s inite y the presene of n itionl phlnx (Fig. 2,). In suset of ses (40%), Zfp145 / mie possess n extr igit prexilly, whih morphologilly resemles igit V in the hinlim, or showe omplete sene of igit I (Fig. 2, n t not shown). The tlus, whih normlly lies proximl to igits I, II n III, is lso homeotilly trnsforme into lneus (90%; Fig. 2,). Morphologil efets in the forelim of Zfp145 / mie our t lower frequeny (5%) n re lrgely restrite to the utopo. As with their hinlim, Zfp145 / mie isply homeoti trnsformtion of igit I into igit II in the forelim n n itionl igit is present prexilly, whih morphologilly resemles igit V (Fig. 2,e). In the mettrsl region, homeoti trnsformtions hnge the nterior C1 one into neighouring nviulre one (38%) or into the most posterior struture, the uoi one (42%; Fig. 2f,g, 1 Deprtment of Humn Genetis n Moleulr Biology Progrm, 2 Howr Hughes Meil Institute n Moleulr Biology Progrm, Memoril Slon- Kettering Cner Center, Slon-Kettering Division, Grute Shool of Meil Sienes, Cornell University, New York, New York, USA. Corresponene shoul e resse to P.P.P. (e-mil: p-pnolfi@ski.msk.org). 166 nture genetis volume 25 june 2000

2 rtile 1 k Fig. 1 Trgete isruption of Zfp145., Shemti representtion n prtil restrition mp of mouse Zfp145. Mile, the trgeting vetor, in whih exon 1 (whih enoes the strt oon, POZ omin n Zfp145 zin fingers 1 n 2) is reple y neo. Bottom, the enogenous Zfp145 genomi region following orret integrtion of the trgeting onstrut vi homologous integrtion., Southern-lot nlysis of EoRI-igeste genomi DNA from ES ell lones proe with the 3 externl proe BB, initing n 18-k wiltype Zfp145 llele n 3-k n resulting from the reomine llele. This n is present in the lnes orresponing to the two reomine lones, 137 n 159. Further hrteriztion of the reomine lones use 5 externl proe XB6 n internl proe NEO. The reomine llele reognize y these proes gives rise to n of 14 k. The wil-type n of 18 k is lso etete., Intivtion of Plzf ws verifie y western-lot nlysis with n ntioy (R94) rise ginst the C terminus of Plzf, whih etets 78-kD n in wil-type, ut not in Zfp145 /, mie. 18 k 14 k 3 k proe XB proe BB proe NEO n t not shown). In some ses (15%), the entire mettrsl region is fuse, forming lrge ony mss lking ny morphologil ientity (Fig. 2f,h). Zfp145 / mie isply ony outgrowths in the mettrsl region (69%) whih re not present in wil-type mie (Fig. 2f h). In ition, nterior strutures suh s the tiile re sent from Zfp145 / hinlims (98%; Fig. 2f h). In the zeugopo of Zfp145 / mie, the tii n fiul re seprte n fil to fuse (96%), n the fiul is thikene (32%; Fig. 3,). The tii of Zfp145 / mie is often trnsforme into fiullike struture (38%), giving rise to two zeugopo elements tht re of equl length n lk rtiultions (Fig. 3,). In ertin ses (36%), shortening of the zeugopo region ours in Zfp145 / mie, whih results in the sene of isernle strutures from this region or the omplete sene of fiul struture (16%; Fig. 3,, n t not shown). Ptterning efets in more proximl strutures re lso oserve, inluing uplition (s mny s seven) or sene of the ptell (52%), s well s the sene of the menisus (28%; Fig. 3e,f). The meil sesmoi one is either sent from Zfp145 / mie or trnsforme into lterl sesmoilike struture (70%; Fig. 3e,f, n t not shown). In the stylopo of Zfp145 / mie, the mjor n minor trohnters ( prominene t the upper prt of the femur serving s tthment of musles) of the femur re similr in size n shpe (Fig. 3). 117 kd 75 kd 42 kd 31 kd 17 kd Plzf β-tin Thus, Zfp145 intivtion ffets ptterning of ll skeletl strutures of the lim. This hnge in skeletl ptterning is etete uring formtion of the rtilge templte, s Alin lue n Alizrin re stining of Zfp145 / emryos t 14.5 n 17.5 ys post oitum (.p..) isloses skeletl ltertions inluing mettrsl n igit trnsformtions (Fig. 4, n t not shown). Plzf regultes poptosis n prolifertion in the lim u Preise ontrol of prolifertion uring evelopment is essentil for the norml growth of the lim. Furthermore, interigitl progrmme ell eth is neessry prerequisite for the seprtion of igits. Zfp145 / mie n e ientifie s erly s 12.5.p.., s the hinlim igitl plte is wier thn tht of wil type (Fig. 4). At 13.5.p.., Zfp145 / mie isply greter quntity of unifferentite mesenhyme etween prospetive nterior igits (Fig. 4) thn o wil-type mie. At 14.5.p.., in ition to n etopi igitl onenstion present etween nterior igits in suset of f Fig. 2 Autopo skeleton., A representtive exmple of wil-type hinlim utopo. The igits re numere in romn numerls, igit I eing the most nterior n igit V the most posterior. The lelle mer lui rwing of the istl mettrsl region inites the lneus (C), tlus (T) n tiile (TB)., In the hinlim of Zfp145 / mie, igit I is homeotilly trnsforme into igit II (sterisk; note the presene of three phlnges, P1 3). The mer lui rwing illustrtes tht the tlus is homeotilly trnsforme into lneus., Presene of n etopi igit with three phlnges (P1 3, rrow) lote nteriorly to homeotilly trnsforme igit I (II*) in the Zfp145 / hinlim utopo., A representtive exmple of wil-type forelim utopo. e, In the forelim utopo of Zfp145 / mie, igit I is homeotilly trnsforme into igit II (sterisk; note the presene of three phlnges (P1 3), whih re omprle in length to those of wil-type igit II). An etopi igit with three phlnges (P1 3, rrow) is lote nteriorly to homeotilly trnsforme igit I (II*). f, Wil-type premettrsl region n lelle mer lui rwing of this region illustrting the ones tht re present proximl to the igits: uoi (CU), uneiforme 3 (C3), nviulre (N) n uneiforme 1 (C1). g, Zfp145 / premettrsl region n lelle mer lui rwing show homeoti trnsformtion of the C1 one to uoi struture. h, Zfp145 / premettrsl region n lelle mer lui rwing showing the fusion of the entire region, with no istinguishle one ientities. Note the presene of ony outgrowths (sterisks) whih projet out of the pge, n tht the tiile one (TB) is sent from lmost ll Zfp145 / mie. e g h nture genetis volume 25 june

3 rtile Zfp145 / mie (30%), ll Zfp145- mutnt hinlims show elye regression of interigitl mesenhyme (Fig. 4). We onfirme tht poptosis of interigitl regions ws reue in Zfp145 / mie y TUNEL n Nile lue stining. Cell eth is not etete etween igits I n II of Zfp145 / hinlims t 13.5.p.. n 14.5.p.. (Fig. 4, n t not shown). Furthermore, ult Zfp145 / mie isply interigitl weing in hinlim (60%) n forelim (5%), lrgely etween igits I n II (Fig. 4). Zfp145 / mie lso isply hnges in ell prolifertion. Flow ytometri nlysis revele n inrese in the perentge of ells tht inorporte BrU, in utero, in the hinlim utopo of Zfp145 / mie t 13.5.p.. n 14.5.p.. (Fig. 4). No ifferenes in prolifertion re pprent in Zfp145 / mie t 15.5.p.. (t not shown), when ptterning of the igit primori is omplete. Immunohistohemistry of hinlim tissue setions with the Ki-67 ntioy revele n inrese perentge of proliferting ells in the interigitl regions of Zfp145 / mie t 13.5.p.. (t not shown). Thus, Plzf inhiits prolifertion n funtions s pro-poptoti ftor in the lim. Expression of Zfp145 in eveloping lim Morphologil ltertions in Zfp145 / hinlim orrelte with the regions of Zfp145 expression in the lim. Onset of Zfp145 Fig. 3 Zeugopo skeleton., Wil-type hinlim zeugopo region with normlly fuse tii (T) n fiul (F)., A Zfp145 / hinlim zeugopo in whih the fiul is thikene n the tii hs een homeotilly trnsforme into fiul-like struture., Wil-type hinlim., Zfp145 / hinlim. The rkets n rrows highlight the zeugopo. Only ruimentry tii n fiul strutures re present in the Zfp145 / zeugopo. Note the equl size n shpe of the mjor n minor trohnter of the femur (Fe) s inite y n sterisk. e,f, Knee region of the hinlim of wil-type n Zfp145 / mouse, respetively. Note in Zfp145 / lims the presene of two ptells (P), whih re shifte proximlly, n the sene of the menisus (me) n meil sesmoi (ms) one (rrows). expression is onomitnt with lim-u formtion, n Zfp145 is expresse t 10.5.p.. n 11.5.p.. throughout the lim mesenhyme. At 12.5 n 13.5.p.., Zfp145 expression is highest in the interigitl regions surrouning the perihonrium of prospetive igit nlges (Fig. 5). Homeoti trnsformtions re not use y etopi polrizing tivity The gene Shh is involve in pttern formtion long the AP lim xis 9,10. Shh n omponents of the Shh signlling pthwy, suh s Pth n Gli, re expresse normlly in Zfp145 / lims (Fig. 6, n t not shown). In ition, Fgf4 n Fgf8 trnsripts re not nteriorize in Zfp145 / lims (Fig. 6,n t not shown). Trnsplnttion of nterior mesoerm from mouse mutnts tht possess n etopi polrizing region to the nterior mrgin of host hik lim u results in igit uplitions 9. Intivtion of Plzf oes not result in etopi polrizing tivity, s norml igit pttern is oserve following grft of nterior mesenhyme from Zfp145 / mie (n=7) or wil-type littermtes (n=38) to hik lim u (t not shown). These results suggest tht Plzf ts ownstrem or inepenently of Shh to regulte lim ptterning. e f Fig. 4 Inrese prolifertion n erese poptosis in the hinlim of Zfp145 / mie., Alin lue n Alizrin re stining of p.. Zfp145 / emryos revele skeletl ltertions inluing the trnsformtion of igit I into igit II ientity n etopi skeletl onenstions (rrow)., Snning eletron mirosopy of the hinlim of wil-type n Zfp145 / emryos. Romn numerls inite igit onenstions., Nile lue stining in the hinlim of Zfp145 / n ontrol mie t 13.5.p.. The rrow inites the lk of Nile lue stining etween igits I n II of the Zfp145 / hinlim. Bottom, n ult Zfp145 / hinlim with interigitl weing etween igits I n II (rrow)., Flow ytometri nlysis of lim tissues, s ssesse y BrU uptke. Zfp145 / mie h sttistilly signifint inrese (P<0.002) in the numer of BrU-positive ells in the utopo of the hinlim t 13.5.p.. n 14.5.p nture genetis volume 25 june 2000

4 rtile Altere expression of the A Hox omplex n Bmp genes in Zfp145 / lims The Hox genes re importnt regultors of lim morphogenesis, n gin-of-funtion experiments hve revele tht ltertions of Hox gene expression within the lim n result in homeoti trnsformtions s well s morphologil efets In Zfp145 / mie, the expression of ll 5 Hox genes t 11.5.p.. is nteriorize so tht trnsripts re present throughout the utopo. In norml mie t 12.5.p.., Hox gene expression egins to e restrite to the perihonrium. Zfp145 / mie, however, show high level of Hox expression throughout the utopo n zeugopo regions t this time (Fig. 6). Memers of the 5 Hox omplex hve een shown to e importnt for lim evelopment No ltertions re oserve in Hox11 expression in Zfp145 / emryos (t not shown), ut Hox13 trnsripts re nteriorize n expresse in more proximl regions of the lim spnning the zeugopo (Fig. 6). Fig. 5 Expression of Zfp145 in the hinlim. Whole-mount RNA in situ hyriiztion of Zfp145 uring lim evelopment. Bmps re expresse in the eveloping lim, where they funtion in ontrolling ell prolifertion n progrmme ell eth 18. Zfp145 / mie isply reution in Bmp7 expression in the lim mesenhyme t 10.5.p.. (Fig. 6), ut its expression in the pil etoerml rige (AER) is not ltere. At 11.5.p.., Zfp145 / mie show erese in expression of oth Bmp2 n Bmp7 in the mesenhyme (Fig. 6, n t not shown). We i not etet ltertions in the expression of Bmp genes t 12.5.p.. in Zfp145 / mie (t not shown). Therefore, Plzf is require for proper expression of Hox n Bmp genes in the eveloping lim. Axil homeoti trnsformtions n ptterning efets in Zfp145 / mie Zfp145 / mie possess xil skeletl normlities tht onsist of homeoti trnsformtions of verterl segments into morphology typil of more nterior segments. Wil-type mie hve 13 thori vertere, wheres Zfp145 / mie (70%) hve 14 (Fig. 7). This itionl thori segment in Zfp145 / mie is ssoite with n extr pir of ris, whih re tthe to the sternum, thus resulting in n pprent trnsformtion of T8 n L1 into more nterior n posterior thori segments, respetively (Fig. 7). In ition, the xiphoi proess of Zfp145 / mie is mlforme n isplys the presene of fissures or holes tht re sent from wil-type mie (Fig. 7). In Zfp145 / mie (70%) there is shift of the trnsitionl vertere from T10 to T11, suh tht T10 is nteriorize to resemle T9 (Fig. 7). Zfp145 / mie (80%) lso show the presene of homeoti trnsformtions within the srl region of the verterl olumn. These inlue n nterioriztion of the most srl vertere, S4, to resemle S3 n fusion of the trnsverse proesses to those of S3, feture tht is not oserve in wil-type mie (Fig. 7). A suset of Zfp145 / mie show trnsformtion of the seon ul vertere S2 into n S1 morphology, in whih the rtiulr surfe of the ilium is extene to the seon srl vertere inste of eing limite to S1 s seen in wil-type mie (Fig. 7e). In ition, the Fig. 6 Altere Hox n Bmp gene expression in the hinlim of Zfp145 / mie., Whole-mount in situ hyriiztion of ll 5 Hox genes t 11.5.p.. n 12.5.p.., n Hox13 t 11.5.p.., in Zfp145 / emryos n wil-type ontrols. All 5 Hox genes, s well s Hox13, show eregulte pttern of expression in Zfp145 / lims. The top right-hn orner n ottom left-hn orner enote the nterior n posterior regions of the lim, respetively, in ll pnels., Whole-mount in situ hyriiztion of Shh n Fgf4. Their expression is not ltere in Zfp145 / lims., Whole-mount in situ hyriiztion of Bmp7 n 10.5.p.. n Bmp2 t 11.5.p.. The expression of these genes is reue in the mesenhyme of Zfp145 / lims. nture genetis volume 25 june

5 rtile e Fig. 7 Homeoti trnsformtions n ptterning efets in the xil skeleton of Zfp145 / mie., Verterl olumn of wil-type n Zfp145 / mie. The totl numer of thori vertere n ris re inrese in Zfp145 / mie., Left, sternum with ssoite tthe ris in wil-type n Zfp145 / mie. Note the presene of n itionl pir of tthe ris in Zfp145 / mie. Right, lose-up of the xiphoi proess in wil-type n Zfp145 / mie. The rrowhe inites the presene of hole or fissure in the xiphoi proess of Zfp145 / mie., Trnsitionl rtiulr proesses (rrow) of thori vertere in wil-type n Zfp145 / mie. Note the shift of the trnsitionl vertere from T10 in wil type to T11 in Zfp145 / mie (sterisk)., Alizrin re stining of ult srl vertere in wil-type n Zfp145 / mie. In Zfp145 / mie, the morphology of the fourth srl vertere (S3*) is ltere s its trnsverse proesses re ompletely fuse to those of S3 (fille rrowhe). e, Left, Alizrin re stining of ult srl vertere in Zfp145 / mie in whih the seon srl vertere exhiits morphology hrteristi of wil-type S1. Right, trnsitionl proesses of srl vertere seen in the left pnel. Note the trnsformtion of the trnsitionl proess (rrow) of the seon srl vertere (S1*) in Zfp145 / mie to tht of wil-type S1. f, Til morphology of wil-type n Zfp145 / mie. Note tht Zfp145 / mie show trunte n kinke til. trnsverse proess of the seon srl vertere of Zfp145 / mie resemles tht of n S1 vertere in wil-type mie (Fig. 7e). Furthermore, Zfp145 / mie show loss in the numer of ul vertere, resulting in shortening of the til, n fusion of rtilge etween vertere, using kinke til (Fig. 7f). Plzf intivtion therefore ffets ptterning of the xil skeleton. Altertions in Hox gene expression in the AP xis Loss-of-funtion n overexpression experiments of Hox genes in the mouse result in trnsformtions of xil skeletl strutures 19,20. The prxil expression ptterns of oth Hox6 n Hox8 re expne posteriorly in Zfp145 / emryos, s the posterior ounries of expression of oth genes re shifte y t lest two prevertere (Fig. 8). Other Hox genes (suh s Hox2) re expresse normlly in the xil skeleton n lims of Zfp145 / emryos (Fig. 8, n t not shown). Altertions in the expression ptterns of Hox genes re ssoite with regions of Zfp145 expression, s Zfp145 trnsripts re present in oth spinl or n prevertere 21. Zfp145 tivity therefore regultes the expression of seletive Hox genes in oth xil n ppeniulr skeletons. Disussion Role of Zfp145 in ontrol of lim n xil skeletl ptterning. Zfp145 ontrols AP ptterning in the lim in oth istl n proximl strutures. This my e meite, t lest in prt, through regultion of Hox genes, whih re nteriorize n etopilly expresse in Zfp145 / mie. It hs een previously suggeste tht Hox genes my meite ptterning of the lim long the AP xis; however, Hox loss-of-funtion mutnts lrgely result in truntion of speifi skeletl elements n not AP ptterning efets. In ontrst, gin-of-funtion stuies of iniviul Hox genes result in AP ptterning hnges, lrgely restrite to one or few elements. Zfp145 / mie isply efets in lim ptterning, ffeting skeletl elements long the entire proximoistl xis. The more extensive AP phenotype oserve in f Zfp145 / mie my e ue to the onomitnt ltertions in expression of the 5 Hox n Hox genes. In the xil skeleton, Zfp145 / mie lso exhiit nterior-irete homeoti trnsformtion. The thori trnsformtions re similr to those ssoite with misexpression of Hox6 n Hox8, n in Zfp145 / emryos the posterior ounries of expression of oth genes re misexpresse. Plzf n therefore regulte Hox gene expression in oth the xil n ppeniulr skeleton. In this regr, we hve ientifie Zfp145 ining sites within the A Hox gene lous (unpulishe t) n it my e tht Plzf ts s trnsriptionl repressor to iretly regulte Hox gene expression. Plzf regultes poptosis n prolifertion in the eveloping lim. Plzf lso influenes the expression of Bmp genes, whih is Fig. 8 Expression of Hox genes within prevertere (Pv). Whole-mount in situ hyriiztion is shown of Hox6, Hox8 n Hox2 in wil-type n Zfp145 / emryos t 12.5.p.. Note the posterior expnsion in Hox6 n Hox8 expression omins in Zfp145 / emryos. No ifferenes in Hox2 expression were oserve. 170 nture genetis volume 25 june 2000

6 rtile reue in Zfp145 / mie. Bmp7-null mie exhiit nterior lim ptterning efets, inluing the presene of n extr prexil igit 22,23. The iminishe expression of Bmp genes in Zfp145 / emryos my therefore lso ontriute to the lim skeletl ptterning efets oserve in these mie. In ition, Bmps my meite the pro-poptoti n growth inhiitory funtion of Zfp145. The misexpression of Hox genes within the interigits of Zfp145 / emryos t 12.5.p.. my lso result in erese progrmme ell eth, onsistent with the retrovirl overexpression of Hox11 in the hik, whih results in weing of the igits 11. Morphologil ltertions in the forelim of Zfp145 / mie re present t lower frequeny (5%) thn in the hinlim (100%). In this respet, it is notle tht two homologues of Zfp145 hve reently een ientifie 24, one of whih is highly expresse in forelim 25. Therefore, the loss of Zfp145 funtion in the forelim my e ompenste y the presene of Zfp145 homologues. The ility of Zfp145 to regulte Hox n Bmp expression ppers to e ownstrem or inepenent of Shh. In ontrst, other mouse mutnts tht exhiit posterior trnsformtions of nterior skeletl elements isply etopi polrizing tivity, ue to either iret tivtion of Shh signlling or loss of repressor of Shh tivity. Plzf n therefore meite ptterning of the lim inepenently or ownstrem of polrizing signl. Implitions for leukemogenesis. APL is hrterize y the lonl expnsion of mlignnt myeloi ells loke t the promyeloyti stge of hemopoieti evelopment. As onsequene of trnslotions etween hromosomes 11 n 17, two reiprol fusion genes re generte tht enoe PLZF RARA n RARA PLZF fusion proteins, whih re o-expresse in the leukemi lst 26. Both proteins n t s ominnt-negtive prouts on PLZF funtion 26. Our finings therefore hve implitions for the pthogenesis of APL, in whih errnt PLZF funtion might len seletive growth n survivl vntge to the leukemi ells through eregulte expression of PLZF trget genes. In this regr, HOX genes n BMPs hve een implite in the ontrol of prolifertion n ifferentition of primitive hemopoieti ells 27,28. Aerrnt regultion of HOX genes in APL my e exerte y the ft tht PLZF RARA n onomitntly interfere with the trnsriptionl funtion of RARA, whih hs lso een implite in the regultion of HOX gene expression. Methos Genertion n ientifition of Zfp145 / mie. We isolte Zfp145 genomi lones y sreening 129 Sv mouse genomi lirry (Strtgene) with humn ZFP145 proe. To generte the trgeting onstrut, we sulone 10-k BmHI frgment into pbluesript vetor, in whih the KpnI n XhoI sites h een elete from the multiple loning site. This filitte removl of 3.5-k KpnI/BspEI frgment n replement with the neomyin ssette, therey eleting the 5 Plzf oing region. This ws then sulone into the PYTK vetor, whih ontins the TK gene 29. The trgeting onstrut ws eletroporte into CJ7 ES ells. We selete trnsfetnts in G418 (350 µg/ml) n gnylovir (2 µm) n expne them for Southern-lot nlysis. Chimeri mie were proue y miroinjetion of two inepenently generte, trgete ES ell lones with norml kryotypes into 3.5-.p.. C57BL6/J lstoysts (Jkson Lortories). We verifie germline trnsmission of the mutte llele y Southern-lot nlysis. Genertion of Plzf ntioy n western-lot nlysis. We rrie out immunolot nlysis using the ECL western-lot kit (Amershm) following the mnufturer s protool. Whole-ell extrts from p.. Zfp145 / n wil-type emryos were use for western-lot nlysis. The filters were loke with 10% nonft ry milk in TBST for 1 h, inute with nti-plzf ntioies for 1 2 h t RT, wshe 3 times in TBST n inute with pproprite seonry ntioies onjugte to horserish peroxise. The western-lot signls were evelope using regents supplie y the mnufturer. The filters were strippe with Tris-HCl (62.5 mm, ph 6.8), 2% SDS n β-merptoethnol (100 mm) t 50 C for 30 min, n re-hyriize with n nti-β-tin ntioy (1:3,000). We use two ntioies for western-lot nlysis: NPGP reognize the Plzf POZ omin 30 ; n R94 reognize peptie (KPEEIPPDWRIEKTY) lote t the very C terminus of the Plzf protein ( ). Skeletl nlysis of Zfp145 / mie. For skeletl nlysis, skin n internl viser of 26 Zfp145 / nimls were remove. We then fixe the nimls overnight in ethnol n rrie out Alin lue n Alizrin re stining proeures s esrie 31. The lering step ws moifie to 2% potssium hyroxie tretment overnight for ult mie. Snning eletron mirosopy. Emryos t ifferent evelopmentl stges were issete in PBS n fixe overnight in 4% prformlehye n 1% glutrlehye in oyli uffer (0.1 M). After postfixtion with 1% OsO 4 in oylte uffer (0.1 M; 1 h t 4 C), the emryos were ehyrte y n sening ethnol series n rie y the ritil point metho, sputtere-ote with pllium-gol n exmine with snning eletron mirosope. Nile lue stining. We issete n p.. emryos in PBS, ple them into solution of 1.5% Nile lue (Sigm) in DMEM (Gio) for 45 min t RT, then ple them in PBS. The lims were remove n photogrphe from orsl perspetive. Flow ytometry. We injete BrU lelling solution (Amershm) intrperitonelly into pregnnt femle mie t 13.5, 14.5 n 15.5 ys of gesttion (1 ml/100 g oy weight). One hour fter injetion, the mie were kille n emryos issete in PBS. A single-ell suspension of lim tissue ws otine y tissue grining. Cells were wshe twie with PBS n fixe in 70% ethnol for 30 min, n wshe gin with PBS. A solution of 2N HCl/0.5% Triton X-100 ws e slowly while ells were gently vortexe n inute for 30 min. We e solution of soium orte (0.1 M) to neutrlize the i n resuspene ells in PBS/1% BSA/0.5% Tween-20 for 30 min efore stining. Cells were then inute with FITConjugte nti-bru ntioy for 30 min, vortexe, resuspene in PBS n lelle ells sorte on FACS mhine. Polrizing tivity ssy. A piee of nterior or posterior mesenhyme ( 150 µm ue) from Zfp145 / or wil-type littermte emryos ( p..) ws implnte uner the AER on the nterior sie of stge hik lim u. We fixe host emryos t y 8 10 of inution n stine the skeleton with Alin lue. Digit uplitions (pttern rnge from 2234 to ) were oserve following posterior mesenhyme grft from Zfp145 / or wil-type lims (n=15; genotype i not ffet the extent of uplition). Whole-mount in situ hyriiztion. We rrie out whole-mount in situ hyriiztion using igoxygenin-lelle ntisense RNA proes s esrie 32. We etete the lel using n lkline phosphtse-onjugte nti-igoxygenin ntioy (Boehringer). Aknowlegements We thnk D. Ruggero n S. Pizette for isussions; M. Brnfor n C. Kuro for help with experiments; J.H. Dong n V. Sores for help with the genertion n the mngement of the Zfp145 mutnts; N. Lmpin n B. Guminer for help n vie with the eletron snning mirosopy nlysis; D. Duoule n P. Gruss for Hox n Hox proes, respetively; M. Sott n A. Joyner for Pth n Gli proes, respetively; M. Cpehi n A. Boulet for Hox6 n Hox8 proes; n P.G. Pelii n M. Ruthrt for PLZF ntioy n proe. P.P.P. is Sholr of the Leukemi n Lymphom Soiety (formerly known s the Leukemi Soiety of Ameri). L.N. is n Assistnt Investigtor of the Howr Hughes Meil Institute. This work ws supporte y the NIH (CA n CA wre to Slon-Kettering Institute n P.P.P.). Reeive 6 Mrh; epte 7 April nture genetis volume 25 june

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