Fates-shifted is an F box protein that targets Bicoid for degradation and regulates developmental fate determination in Drosophila embryos

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1 ARTICLES Ftes-shifted is n F ox protein tht trgets Bioid for degrdtion nd regultes developmentl fte determintion in Drosophil emryos Juno Liu 1 nd Jun M 1,2,3 Bioid (Bd) is morphogeneti protein tht instruts ptterning long the nterior posterior (A P) xis in Drosophil melnogster emryos. Despite extensive studies, wht ontrols the formtion of norml onentrtion grdient of Bd remins n unresolved nd ontroversil question. Here, we show tht Bd protein degrdtion is medited y the uiquitin-protesome pthwy. We hve identified n F ox protein, enoded y ftes-shifted (fsd), tht hs n importnt role in Bd protein degrdtion y trgeting it for uiquityltion. Emryos from femles lking fsd hve n ltered Bd grdient profile, resulting in shift of the ftemp long the A P xis. Our study is n experimentl demonstrtion tht, ontrry to n lterntive hypothesis, Bd protein degrdtion is required for norml grdient formtion nd developmentl fte determintion. Morphogens re moleules tht form onentrtion grdients in developing emryo or tissue to provide positionl informtion to ells nd instrut their developmentl ftes 1 5. The Drosophil morphogeneti protein Bioid (Bd) forms onentrtion grdient long the nterior posterior (A P) xis of the emryo nd instruts ptterning y tivting its downstrem trget genes in onentrtion-dependent mnner 6 9. An importnt question out morphogens reltes to the moleulr mehnisms tht ontrol the formtion of norml grdient. Diffusion nd degrdtion of morphogen moleules re two physil properties ritil to oth the proess of grdient formtion nd the finl outome of the grdient 1,5,1,11. Although the diffusion onstnt for Bd moleules in emryos remins highly ontroversil 12 14, wht ontrols Bd degrdtion remins virtully unknown t this time. In this report, we investigte the moleulr mehnisms of Bd protein degrdtion. We present evidene tht Bd degrdtion is required for the formtion of norml onentrtion grdient in emryos nd for instruting ells to dopt their norml developmentl ftes. Results Bd degrdtion in Drosophil ells is sensitive to protesome inhiitors We generted Drosophil S2 ells tht stly express hemgglutinin (HA) tgged wild-type Bd protein from the tin promoter. Western lotting nlysis onfirmed the expression of the full-length Bd protein in these ells (Supplementry Informtion, Fig. S1). This HA Bd protein n tivte trnsription of Bd-dependent reporter gene in S2 ells 15, demonstrting tht it is funtionlly tive. To investigte mehnisms nd pthwys of Bd protein degrdtion, we treted the ells stly expressing HA Bd with vrious inhiitors. The protesome inhiitor MG132 inresed the totl mount of Bd (Fig. 1, lne 4 nd Supplementry Informtion, Fig. S1). Neither Z LL H, whih is n MG132 nlogue nd lpin inhiitor, nor hloroquine diphosphte, whih is lysosome inhiitor, hd suh n effet (Fig. 1, lnes 2 nd 3). Two dditionl protesome inhiitors, ltystin nd epoxomiin, whih inhiit protesome tivities through distint mehnisms 16,17, lso inresed the totl mount of Bd (Fig. 1, lnes 5 nd 6). These results suggest tht the protesome-dependent pthwy is importnt for Bd protein degrdtion. Bd degrdtion in Drosophil emryoni extrts revels role for uiquitin To further investigte Bd degrdtion mehnisms, we generted Drosophil emryoni extrts from emryos t 3 h. An HA Bd fusion protein ws effiiently degrded in these extrts (Fig. 1, lnes 1 6), ut suh degrdtion ws signifintly inhiited y MG132 (Fig. 1, lnes 7 12) or epoxomiin (Supplementry Informtion, Fig. S1, d). Bd degrdtion profiles in the presene or sene of MG132 oth fitted well with the first order dey funtion 18 (Fig. 1), with respetive Adjusted R 2 vlue of.999 nd.981. MG132 extends the estimted hlf-life of Bd y 13.1-fold (± 1.8; n = 3 from experiments performed s in Fig. 1, ). These results further support our onlusion tht Bd is degrded in protesome-dependent mnner. 1 Division of Biomedil Informtis, Cininnti Children s Reserh Foundtion, 3333 Burnet Avenue, Cininnti, OH 45229, USA. 2 Division of Developmentl Biology, Cininnti Children s Reserh Foundtion, 3333 Burnet Avenue, Cininnti, OH 45229, USA. 3 Correspondene should e ddressed to J.M. (e mil: jun.m@hm.org) Reeived 3 April 1; epted 17 Novemer 1; pulished online 19 Deemer 1; DOI: 1.138/n nture ell iology VOLUME 13 NUMBER 1 JANUARY 11

2 ARTICLES Proteins destined for protesome-medited degrdtion re often polyuiquitylted Unlike protein degrdtion in the lysosome, uiquityltion is proess tht requires energy from ATP hydrolysis 22. We therefore investigted whether Bd degrdtion in emryoni extrts is dependent on ATP. Addition of ATP into the emryoni extrts enhned Bd degrdtion, with 61% redution in the estimted hlf-life of Bd (Fig. 2, ). In ddition, depletion of ATP from the extrts through the use of hexokinse nd gluose led to the ttenution of Bd degrdtion (Fig. 2, d), extending the estimted hlf-life of Bd y 2.3-fold. To diretly investigte the role of uiquitin in Bd degrdtion, we used mutnt uiquitin, UM NK, whih hs ll of its seven lysine residues mutted to rginines. This mutnt uiquitin n prevent polyuiquityltion nd inhiit uiquitin-dependent protein degrdtion 23. If Bd degrdtion is indeed dependent on polyuiquityltion, we would expet this mutnt uiquitin to inhiit Bd degrdtion. Our results show tht when UM NK ws dded to the emryoni extrts Bd degrdtion ws ttenuted (Fig. 2e, f), extending its estimted hlf-life y 1.93-fold (s.d. ±.35, n = 3 experiments performed s in Fig. 2e, f). Together, these results suggest tht Bd is degrded in protesome- nd uiquitin-dependent mnner. Bd is uiquitylted To determine whether Bd is uiquitylted, we performed n in vivo uiquityltion ssy in humn emryoni kidney (HEK) 293T ells, whih hve n effiient uiquityltion system 24. To inrese the mount of uiquitinmodified produts, uiquitin-expressing plsmid ws o-trnsfeted into these ells, whih were then treted with MG132 efore hrvest. As shown in Figure 2g, uiquitin-modified Bd protein speies (lne 4) were deteted in Bd- nd MG132-dependent mnner (see other lnes for ontrols). These results support the hypothesis tht Bd is uiquitylted. Identifying n F ox protein involved in Bd degrdtion Uiquityltion is tlysed y three stepwise enzymti retions, with the uiquitin ligse E3 providing sustrte speifiity One group of E3 ligses, the SCF (Skp1 Cul1/Cd53 F-ox protein)-type E3 ligses, hve role in the turnover of mny trnsription ftors 3. The sustrte speifiity in these multi-protein omplexes is onferred y F ox proteins,31,32. We performed doule-strnded RNA interferene (dsrnai) sreen for 38 potentil Drosophil F ox proteins in the S2 ells stly expressing HA Bd. The ddition of dsrna trgeting the ftes-shifted (fsd, CG12765) gene used onsistent inrese in the totl mount of Bd (Supplementry Informtion, Fig. S2, lne 3; see lnes 1 nd 2 for speifiity). Next, we used yloheximide (CHX) to lok trnsltion in the HA Bd-expressing stle ells (Fig. 3) nd found tht fsd dsrna tretment inresed the estimted hlf-life of Bd y 1.5-fold (Fig. 3). In ddition, overexpressing Fsd in S2 ells redued the totl mount of Bd (42%; Fig. 3, lne 2; see lnes 1 nd 3 for ontrols). These results indite tht Fsd hs n importnt role in regulting Bd protein stility in Drosophil ells. Fsd is n F ox protein tht diretly interts with oth Skp1 nd Bd Bsed on the lignment of the 234 sequenes used to rete the F ox profile in the Pfm dtse, F-ox motifs re highly degenerte, lking strit primry onsensus sequene 31. Our lignment of ll potentil F ox domins in Drosophil derived from the Interpro dtse further onfirms the degenerte nture of the primry sequenes of the F ox Perentge Bd remining Time (min): HA Bd Anti-HA Anti- -tin DMSO DMSO Chloroquine Ltystin Epoxomiin HA Bd -tin motif (Supplementry Informtion, Fig. S3). A hllmrk of genuine F ox protein is its ility to intert with Skp1, nother omponent of the SCF omplex,31,32. To determine if Fsd hs this property, we performed o-immunopreipittion experiments using HEK 293T ells. HA Skp1 ws speifilly o-preipitted with Flg Fsd (Fig. 4, lne 6; see other lnes for ontrols), demonstrting tht Fsd nd Skp1 n physilly intert with eh other in ells. To further evlute the role of Skp1 in Bd degrdtion, we mnipulted Skp1 protein levels in the HA Bd-expressing stle ells. The totl mount of Bd in these ells ws deresed when dditionl Skp1 (Flg Skp1) ws expressed (Supplementry Informtion, Fig. S2, lne 3; see lne 1 for ontrol). As expeted, MG132, whih inhiits the downstrem step of protein degrdtion, inresed the totl mount of Bd to similr level regrdless of the sttus of dditionl Skp1 expression (Supplementry Informtion, Fig. S2, lnes 1 4). In ddition, when ells were treted with skpa dsrna, the estimted hlf-life of Bd ws extended y 2.5-fold (Supplementry Z-LL-H MG132 MG132 DMSO MG132 Figure 1 Bd is degrded through the protesome-dependent pthwy. () Effets of protese inhiitors on the totl mount of Bd in Drosophil S2 ells. Cells stly expressing HA Bd were treted with the indited inhiitors nd western lotting ws performed using the HA ntiody to determine the totl mount of HA Bd (top). β tin (ottom) represents the loding ontrol. () Time ourse of Bd degrdtion in emryoni extrts. HA Bd, synthesized in vitro, ws nlysed in emryoni extrts in the presene of MG132 or DMSO (s ontrol). Aliquots were tken from retion tues t the indited times, nd HA Bd ws deteted y western lotting. () Bd degrdtion kinetis. Intensity of the HA Bd nds in were quntified nd lulted s perentge of the intensity of the nd t time zero for eh tretment. The lines represent exponentil fitting to the experimentl dt. Throughout this study, ll kineti experiments shown were performed through side-y-side omprison of western lots (s shown in Fig. 1, ) nd only suh side-y-side results within single pnel n e ompred. Unropped imges of lots re shown in Supplementry Informtion, Fig. S9. nture ell iology VOLUME 13 NUMBER 1 JANUARY 11 23

3 ARTICLES Time (min): HA Bd ATP + ATP hexokinse + hexokinse Time (min): HA Bd e Time (min): HA Bd f Perentge Bd remining Perentge Bd remining Time (min) UM-NK + UM-NK Time (min) ATP + ATP 3 4 UM-NK + UM-NK 3 4 d g Perentge Bd remining Time (min) HA Bd: + + FLAG U: MG132: + + M r (K) hexokinse + hexokinse 3 4 U-modified Bd Figure 2 Bd is uiquitylted. ( f) Time ourse of Bd degrdtion in emryoni extrts with or without ATP ddition (, ), ATP depletion (, d) or UM NK ddition (e, f). Hexokinse (together with gluose) ws used to deplete ATP. For eh experiment, liquots were tken from the retion tues t the indited times. (,, e) HA Bd ws deteted y western lotting. (, d, f) Frtion of remining Bd protein (quntified from intensity of the nds in the western lots) plotted ginst retion time. In eh se the lines represent exponentil fitting to the experimentl dt. (g) Bd uiquityltion deteted in Informtion, Fig. S2, d). These results suggest tht oth Fsd nd Skp1 prtiipte in the Bd degrdtion proess, presumly working together in novel Fsd-ontining SCF (SCF Fsd ) E3 ligse. F-ox proteins trget their sustrtes for degrdtion y reruiting them, through diret protein protein intertions, to the SCF E3 ligse omplexes for uiquityltion,31,32. To determine whether Bd is sustrte of the proposed SCF Fsd E3 ligse, we performed nother o-immunopreipittion experiment. Our results indite tht HA Bd ws speifilly o-preipitted y Flg Fsd (Fig. 4, lne 6; see other lnes for ontrols), demonstrting tht Fsd n physilly intert with Bd. Therefore Fsd seems to e on fide F ox protein tht n diretly intert with oth omponent (Skp1) within the proposed SCF Fsd omplex nd its sustrte Bd. Emryos lking Fsd exhiit posterior shifts in morphologil nd moleulr mrkers Sequene nlysis of n fsd DNA lone (NM_17.3) predited protein of 312 mino ids, with two potentil F ox domins (Fig. 5). Fsd does not seem to ontin ny other onserved domins. fsd trnsripts re uniformly distriuted in oth prelstoderm nd gstrulted emryos (Supplementry Informtion, Fig. S4, ), suggesting oth mternl nd zygoti expression. We otined pulilly ville Drosophil line with ells. HEK 293T ells were trnsfeted with plsmids expressing HA Bd nd were treted with MG132 s indited. HEK 293T ells were o-trnsfeted with plsmids enoding Flg-Uiquitin to inrese uiquitylted produts. Whole-ell extrts were immunopreipitted using n nti-ha ntiody nd nlysed y western lotting using the nti-uiquitin ntiody. Uiquitinmodified Bd produts re indited, nd moleulr weight stndrds re shown on the left. Unropped imges of lots re shown in Supplementry Informtion, Fig. S9. P element insertion tht disrupts the fsd gene, referred to s fsd KG2393. We onfirmed tht the insertion is 15 p downstrem of ATG, whih effetively disrupts the entire open reding frme (Fig. 5). The fsd KG2393 flies re homozygous vile, ut exhiit temperture-dependent hthing defet. Although the hthing rtes of emryos from wild-type nd fsd KG2393 flies were omprle t C (91.3% versus 81.7%, Supplementry Informtion, Fig. S4), emryos from mutnt flies hd signifintly redued hthing rte t 18 C (48.6%, ompred with 92% for wild-type). At 29 C, emryos from fsd KG2393 femles (referred to s fsd emryos from now on) hd hthing rte of %. Cutile exmintion deteted mutnt emryos with vrile A P ptterning defets when inuted t 29 C, inluding missing/fused dentile nds (Supplementry Informtion, Fig. S5 d). Whole-mount in situ hyridiztion nlysis lso deteted mutnt emryos with norml even-skipped (eve) expression ptterns with missing or fused stripes (Supplementry Informtion, Fig. S5f, g). These results suggest tht the hthing defet oserved t 29 C is onsequene of A P ptterning defets used y the fsd muttion. The hthing defet t 29 C is stritly ssoited with the mternl mutnt genotype (dt not shown) nd, furthermore, mutnt flies t non-optiml tempertures (18 C) do not exhiit signifint redution in survivl rtes from lrve to pupe or from pupe to dults (Supplementry Informtion, Fig. S4). 24 nture ell iology VOLUME 13 NUMBER 1 JANUARY 11

4 ARTICLES Time (h): Control fsd dsrna Perentge Bd remining Anti-HA Anti- -tin Control fsd dsrna Time (h) Flg Fsd: HA Skp1: Anti-HA Anti-Flg Flg Fsd: HA Bd: Anti-HA Anti-Flg Input + + Input Anti-Flg IP Anti-Flg IP HA Skp1 Flg Fsd HA Bd Flg Fsd Plsmid: Anti-HA Reltive Bd mount: Control vetor pa-fsd pa-cg1242 HA Bd Figure 4 Fsd interts with oth Skp1 nd Bd. () A o-immunopreipittion experiment deteting the intertion etween Fsd nd Skp1. HEK 293T ells were o-trnsfeted with the indited omintions of plsmids expressing Flg Fsd nd HA Skp1. Anti-Flg ntiody ws used to immunopreipitte (IP) Flg Fsd from whole-ell extrts, followed y western lotting nlyses using nti-ha (top) nd nti-flg (ottom) ntiodies to detet HA Skp1 nd Flg Fsd, respetively. () Experiments were performed s in, exept with the use of plsmid expressing HA Bd (in ple of HA Skp1) to detet the intertion etween Bd nd Fsd. Unropped imges of lots re shown in Supplementry Informtion, Fig. S9. Figure 3 Fsd hs role in Bd protein degrdtion. () CHX ssy using ells stly expressing HA Bd. Cells were treted with CHX, nd hrvested t indited times to detet the totl mount of Bd in ells y western lotting using nti-ha (left). Western lotting of β tin (right) ws used s loding ontrol. Top, ontrol ells without dsrna tretment; ottom ells inuted with fsd dsrna efore CHX. () Frtion of remining Bd protein in ontrol ells nd ells inuted with fsd dsrna (quntified from intensity of nds in nd normlized to β tin intensities) plotted ginst time fter CHX ddition. The lines represent exponentil fitting to the experimentl dt. () Drosophil S2 ells were trnsiently trnsfeted with plsmid enoding HA Bd. Cells were o-trnsfeted with ontrol vetor (pa5.1; vetor used for protein expression), plsmid enoding Fsd (pa-fsd), or ontrol F-ox protein (pa-cg1242). For eh lne, the loding mount ws djusted ording to the tivity of gltosidse expressed from lz ontrol plsmid trnsfeted t the sme time s the indited plsmids (to mesure trnsfetion effiieny). Reltive intensity of the nds is indited t the ottom. Unropped imges of lots re shown in Supplementry Informtion, Fig. S9. These results suggest tht the primry funtion of fsd is onferred mternlly to emryos. Ating s morphogen instruting A P ptterning, the mount of Bd diretly ontrols the ftemp of the entire emryo long the A P xis If Fsd hs role in promoting Bd protein degrdtion in emryos s oserved in our in vitro ssys, fsd emryos my exhiit posterior shift of the ftemp refletive of n ltered mount of Bd. To diretly evlute this possiility, we mesured the position of the ephli furrow, morphologil mrker long the A P xis nd strong inditor of Bd protein levels in emryos 13,34,36. For experiments desried from now on, ll emryos were olleted t C (unless stted otherwise) to void ny temperture influene on Bd grdient formtion nd emryoni ptterning. Figure 5, indites tht the ephli furrow position (in frtionl emryo length, ) ws shifted from.343 ±.5 (n = 1) in wild-type emryos to.2 ±.12 (n = 9) in fsd emryos (P = , Student s t-test). To evlute the effet of the fsd gene muttion on emryoni ptterning t the moleulr level, we mesured the expression pttern of eve using quntittive fluoresene in situ hyridiztion (FISH). To ensure diret omprison, experiments nd dt proessing for wildtype nd mutnt emryos were performed side-y-side. Figure 5d demonstrtes the men expression profiles of eve in wild-type nd mutnt emryos, reveling posterior shift for ll eve stripes in fsd emryos (see Supplementry Informtion Fig. S6, for rw dt). For exmple, the first eve stripe is shifted from.331 ±.14 (n = 7) in wild-type emryos to.353 ±.9 (n = 8) in fsd emryos (P = , Fig. 5d). To tre the origin of the oserved ftemp shift in mutnt emryos, we mesured expression ptterns of the gp genes hunhk (h) nd knirps (kni), oth of whih re diret trgets of Bd 8, Agin, we onduted side-y-side experiments in wild-type nd mutnt emryos for eh gene in our quntittive FISH experiments. As shown in Figure 5e, f, the expression oundries of oth gp genes exhiited signifint posterior shift in fsd emryos (see Supplementry Informtion, Fig. 6 f for rw dt). Speifilly, the h expression oundry hd posterior shift of pproximtely 3% of the emryo length, from of.4 ±.8 (n = 8) in wild-type emryos to.479 ±.18 (n = 7) in fsd emryos (P = , Fig. 5e). The kni expression oundry ws shifted towrd the posterior y pproximtely 2% of the emryo length, from.671 ±.1 (n = 9) in wild-type emryos to.689 ±.17 (n = 8) in fsd emryos (P = , Fig. 5f). These results demonstrte tht mternl loss of fsd funtion results in posterior shift of oth morphologil nd moleulr mrkers long the A P xis. fsd nd d intert genetilly The oserved ftemp shift in fsd emryos is supportive of diret role of Fsd in trgeting Bd for degrdtion in emryos. To further test this hypothesis, we onduted geneti intertion nlysis in emryos from d E1 /+ femles tht re either wild-type or mutnt for fsd. Our nture ell iology VOLUME 13 NUMBER 1 JANUARY 11

5 ARTICLES CG4663 KG2393 F-ox F-ox fsd (CG12765) CG467 e Reltive h intensity WT fsd WT (n = 8) fsd (n = 7) d Reltive kni intensity Reltive eve intensity f WT (n = 9) fsd (n = 8) WT (n = 7) fsd (n = 8) Figure 5 Posterior shift of ftemp long the A P xis in fsd emryos. () Shemti representtion of the fsd gene (not to sle). The rrows under the genes show the diretion of trnsription nd the oxes represent nnotted exons. The lue setions of the oxes represent the nnotted fsd oding sequenes, wheres the unfilled setions represent untrnslted regions. The position of the KG2393 insertion nd the two F ox domins re mrked. Prts of the two nnotted neighouring genes re lso shown. (, ) Midsgittl imges of living emryos from w 1118 () nd fsd KG2393 () flies results show tht the nterior ftemp shift used y mternl d dosge redution is resued prtilly y eliminting fsd mternlly (Supplementry Informtion, Fig. S7 ). These results demonstrte n intertion, genetilly, etween fsd nd d in regulting developmentl fte speifition long the A P xis. fsd emryos hve n ltered Bd grdient profile To diretly determine whether Fsd hs role in Bd grdient formtion, we onduted quntittive immunostining nlysis using nti- Bd ntiodies on whole-mount wild-type nd mutnt emryos 45. For this nlysis we used rw, unproessed Bd fluoresene intensity dt within liner rnge, with kground intensities diretly mesured under identil experimentl onditions. Figure 6, show Bd intensity (B) dt extrted from individul wild-type nd fsd emryos tht were stined, imged nd nlysed side-y-side. Also shown in these figures re the mesured kground intensities. To evlute the shpe of the Bd grdient profiles, we lulted nd ompred the length onstnt (λ) vlues of either the verge Bd profiles from wild-type nd mutnt emryos or vlues from individul emryos. In simple diffusion model 1,5,46, λ of n exponentil profile is funtion of the effetive diffusion oeffiient (D) nd the effetive degrdtion rte of the morphogen moleules (ω): λ 2 = D/ω. As Bd protein itself hs not een ltered in fsd emryos, D is expeted to remin unffeted y fsd muttion, whih llows us to estimte the reltive ω vlues in wild-type nd mutnt emryos sed on the mesured λ vlues. Figure 6 shows ln (B/B mx ) plot s funtion of A P position. Both B nd B mx re from verge Bd profiles of wild-type nd fsd t C, imged under hloron oil. Arrowheds represent the ephli furrow positions. Sle r, μm. (d f) Averge normlized fluoresene intensities from FISH on whole-mount emryos, deteting the trnsripts of eve (d), h (e) nd kni (f) in wild-type (lue) nd fsd (red) emryos. See Supplementry Informtion, Fig. S6 for rw dt, extrted from individul emryos. The eve expression stripes 1 to 7 hve the following posterior shifts in their respetive posterior oundry positions in fsd emryos: 2.2%, 1.9%, 2.9%, 2.8%, 3.4%, 1.5% nd 1.1% of the emryo length. emryos nd re kground sutrted (s neessry) without ny further djustments. The ln trnsformtion onverts n exponentil funtion to liner funtion 42. Here, we perform liner fit within the rnge of.1 to.5, where the Bd intensity dt re lest sensitive to experimentl nd kground mesurement errors 45,46. We lso exlude dt from the most nterior prt of the emryo where Bd profiles re known to devite from n exponentil funtion 1,42,45. The liner fitting (Fig. 6) hd smller slope in fsd emryos thn in wildtype emryos, suggesting lrger λ vlue in fsd emryos. To further ompre Bd profiles, we lulted λ vlues for individul wild-type nd mutnt emryos (Fig. 6d). The lulted λ vlues inresed from ± μm (n = ) in wild-type emryos to ± 14.3 μm (n = 28) in fsd emryos (P = , Fig. 6d). These results (nd Fig. 5 nd Supplementry Informtion, Fig. S6) provide in vivo evidene tht Fsd hs role in regulting Bd protein stility nd determining developmentl ftes long the A P xis. Disussion Advning the morphogen onept requires n understnding of not only how ells respond to the positionl informtion enoded y the morphogen grdients, ut lso how suh grdients re estlished. Aording to widely epted view 1, the formtion of onentrtion grdient requires lolized soure of morphogen prodution, oupled with diffusion nd degrdtion of the morphogen moleules. However, reent studies question whether the formtion of norml onentrtion grdient of Bd in emryos requires either diffusion or dey of Bd moleules 47,48. Before our urrent work, it ws not known how Bd moleules re degrded, 26 nture ell iology VOLUME 13 NUMBER 1 JANUARY 11

6 ARTICLES WT (n = ) fsd (n = 28) 6 B 4 3 B In (B/B mx ) WT (n = ) fsd (n = 28).8 1. d λ (µm) P = WT fsd e B Figure 6 Bd grdient profiles in wild-type nd fsd emryos. (, ) Wholemount wild-type () nd fsd () emryos were immunostined with nti-bd ntiodies, imged y immunofluoresene mirosopy, nd the fluoresene intensity of Bd ws quntified long the A P xis. Eh olour represents dt from n individul emryo. The line t the ottom represents kground intensities. Dt re mens ± s.d. B, Bd intensity. () ln (B/B mx ) plotted ginst for verge Bd profiles from wild-type () nd fsd () emryos. Both B nd B mx re kground-sutrted s neessry, without ny further djustments. The solid lines represent liner fits for Bd profiles from wildtype emryos (y = 7.2x +.61, Adjusted R 2 =.997) nd fsd emryos mking it diffiult to evlute the role of Bd protein degrdtion in grdient formtion in vivo. We report for the first time tht Bd is degrded through the uiquitin-protesome pthwy. The identifition of novel F ox gene, fsd, mkes it possile to experimentlly pertur Bd degrdtion in vivo nd investigte its role on Bd grdient formtion nd developmentl fte determintion. The Bd grdient profile in fsd emryos hs lrger λ vlue thn in wildtype emryos, demonstrting tht, ontrry to reent proposl 47, Bd degrdtion is required for norml grdient formtion. Our oserved differene in λ vlues orresponds to, in simple diffusion model, n pproximtely 48% inrese in Bd stility in fsd emryos, differene suffiient to use signifint shifts in morphologil nd moleulr mrkers (Fig. 5). We note tht, unlike λ, B mx in wild-type nd fsd emryos did not exhiit signifint differene (44.32 ± 9.1 nd ± 9.19, respetively, P =.63, Student s t-test). Like λ, the stedy-stte mount of morphogen moleules t the soure is lso funtion of D nd ω in simple diffusion model, ut this mount is dditionlly dependent on the morphogen prodution rte J 1,5,46. As the Bd prodution site (tht is, d mrna lotion) is not restrited to single point s ssumed in the idelized simple diffusion model 1, λ should represent more relile prmeter (thn, for exmple, B mx ) in evluting the reltive effetive degrdtion rtes of Bd. (y = 6.31x +.45, Adjusted R 2 =.998). (d) λ vlues lulted from Bd intensity profiles of individul wild-type nd fsd emryos from nd. Individul dt points (unfilled irles) nd mens ± s.d. (oxes with error rs) re indited, with P vlue from Student s t-test indited t the top. (e) The verge intensity of Bd long the A P xis ws quntified for wildtype nd fsd emryos shown in nd. The positions t whih the Bd intensities (without djustments) from the wild-type nd fsd emryos ross onentrtion threshold re shown. The mesured verge h oundry positions in these emryos re mrked with solid rrowheds. Dt from wildtype emryos re shown in lue, nd fsd emryos in red. The h expression oundry in fsd emryos exhiits shift towrd the posterior y pproximtely 3% of the emryo length. This is onsistent with the shift of the positionl informtion enoded y the Bd grdient in fsd emryos (Fig. 6e). These results, together with those presented elsewhere 45,46,49, demonstrte tht the h expression oundry is primrily determined y the positionl informtion provided y the Bd grdient. Although gene regultory networks represent importnt mehnisms for mintining or refining gene expression ptterns 11,46,,51, our results suggest tht erly deisions in A P ptterning, suh s h expression, re ontrolled primrily y the positionl informtion enoded y the Bd grdient. The h oundry shift of pproximtely 3% of the emryo length in fsd emryos mthes the oserved ephli furrow shift (pproximtely 3% of the emryo length), inditing tht erly deisions in gene expression re fithfully pssed down to the morphologil level. We note tht the h oundry shift in fsd emryos, though seemingly modest, is signifint when ompred with the oserved shift (pproximtely 7% of the emryo length) used y the douling of the mternl d gene dose to four opies 42. A shift of pproximtely 3% of the emryo length, when expressed in solute length, is omprle to the differene etween our mesured λ vlues in wild-type nd mutnt emryos. We lso note tht the ftemp shift in nture ell iology VOLUME 13 NUMBER 1 JANUARY 11 27

7 ARTICLES fsd emryos ws deteted t C. The hthing defet of fsd emryos t 29 C represents mnifesttion of more severe A P ptterning defets (Supplementry Informtion, Fig. S5), proly refletive of the omplex effets of temperture on oth Bd grdient formtion (tht is, degrdtion nd diffusion) nd other relevnt proesses. Finlly, we note tht the fsd KG2393 llele used throughout our work is proly null llele euse it is indistinguishle from n llele, fsd 5 7, tht hs the entire fsd oding sequene deleted (Supplementry Informtion, Fig. S8). SCF E3 ligses regulte mny iologil proesses, suh s DNA replition, trnsription, signl trnsdution, ell prolifertion nd deth, nd tissue growth nd ptterning,26,31,32. Our study identifies n F ox protein (enoded y fsd) tht hs role in Bd grdient formtion nd developmentl fte speifition. Our study thus expnds the list of iologil proesses tht SCF E3 ligses regulte. Fsd diretly interts with oth its sustrte Bd nd n SCF omponent Skp1, suggesting tht Bd is trgeted for degrdtion y n Fsd-ontining SCF omplex, SCF Fsd, tht funtions s n E3 uiquitin ligse. We urrently do not know whether Fsd my hve other sustrtes. Identifition of dditionl sustrtes of Fsd, if ny, will provide more omprehensive understnding of the iologil funtions of this F ox protein. How norml Bd onentrtion grdient is formed in erly emryos remins n open question urrently under intense dete 1,13,14,47 49,52,53. One prtiulr spet of the dete fouses on the roles of the nuleus. Although some studies suggest tht nulei hve importnt roles in Bd degrdtion 7 nd proper grdient formtion 13,54, others suggest tht the shping of norml Bd grdient is independent of nulei 55. Fsd is rodly distriuted in ells with primry loliztion to the ytoplsm (Supplementry Informtion, Figs. S4d f), onsistent with the notion tht ytoplsm hs role in Bd degrdtion. However, our reent studies hve shown tht dcbp, Bd-interting trnsription oftor 56,57, ffets Bd grdient profile in emryos 46, suggesting tht nulei lso ontriute to Bd degrdtion nd grdient formtion. Although effiient Bd degrdtion requires Fsd oth in vitro nd in vivo, we onsider it unlikely tht SCF Fsd is the only E3 ligse for Bd. Identifying dditionl E3 ligses, prtiulrly those with primry loliztion to the nuleus, should help settle the ongoing ontroversy regrding the role of nulei in Bd grdient formtion. METHODS Methods nd ny ssoited referenes re ville in the online version of the pper t Note: Supplementry Informtion is ville on the Nture Cell Biology wesite Aknowledgements We thnk memers of our groups t CCHMC, in prtiulr F. He, D. Cheung, W. Dui, nd J. Deng, for disussion nd ssistne, nd we thnk Xinhu Lin s l for some of the primers used in our dsrnai sreening. This work ws supported in prt y grnts from NIH nd NSF (to J.M.). Author Contriutions J.L. nd J.M. oneived nd designed the study. J.L. performed ll experiments nd nlysis. J.L. nd J.M. interpreted the dt, J.L. generted ll figures nd J.L. nd J.M. wrote the pper. COMPETING FINANCIAL INTERESTS The uthors delre tht they hve no ompeting finnil interests. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/ 1. Wolpert, L. Positionl informtion nd the sptil pttern of ellulr differentition. J. Theor. Biol, 1 47 (1969). 2. Kerszerg, M. & Wolpert, L. Speifying positionl informtion in the emryo: looking eyond morphogens. Cell 13, 5 9 (7). 3. Lnder, A. D. Morpheus unound: reimgining the morphogen grdient. Cell 128, (7). 4. Mrtinez Aris, A. & Hywrd, P. Filtering trnsriptionl noise during development: onepts nd mehnisms. Nt. Rev. Genet. 7, (6). 5. Wrtlik, O., Kihev, A. & Gonzlez-Gitn, M. Morphogen grdient formtion. Cold Spring Hr. Perspet. Biol. 1, 15 (9). 6. Ephrussi, A. & St. Johnston, D. Seeing is elieving. The ioid morphogen grdient mtures. Cell 116, (4). 7. 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The F ox protein Skp2 prtiiptes in My proteosoml degrdtion nd ts s oftor for My regulted trnsription. Mol. Cell 11, (3). 29. Kornitzer, D., Roy, B., Kulk, R. G. & Fink, G. R. Regulted degrdtion of the trnsription ftor Gn4. EMBO J. 13, (1994). 3. Jing, J. & Struhl, G. Regultion of the Hedgehog nd Wingless signlling pthwys y the F ox/wd4-repet protein Slim. Nture 391, (1998). 31. Kipreos, E. T. & Pgno, M. The F ox protein fmily. Genome Biol 1, reviews32 reviews32.7 (). 32. Ho, M. S., Tsi, P. I. & Chien, C. T. F ox proteins: the key to protein degrdtion. J. Biomed. Si. 13, (6). 33. Frohnhöfer, H. G. & Nüsslein-Volhrd, C. Orgniztion of nterior pttern in the Drosophil emryo y the mternl gene ioid. Nture 324, 1 1 (1986). 34. Berleth, T. et l. The role of loliztion of ioid RNA in orgnizing the nterior pttern of the Drosophil emryo. EMBO J. 7, (1988). 35. Driever, W., Siegel, V. & Nüsslein-Volhrd, C. Autonomous determintion of nterior strutures in the erly Drosophil emryo y the ioid morphogen. Development 19, (199). 36. Driever, W. & Nüsslein-Volhrd, C. The ioid protein determines position in the Drosophil emryo in onentrtion dependent mnner. Cell 54, (1988). 28 nture ell iology VOLUME 13 NUMBER 1 JANUARY 11

8 ARTICLES. Smll, S., Krut, R., Hoey, T., Wrrior, R. & Levine, M. Trnsriptionl regultion of pir-rule stripe in Drosophil. Genes & Dev. 5, (1991). 38. RiverA Pomr, R. & Jkle, H. From grdients to stripes in Drosophil emryogenesis: filling in the gps. Trends Genet. 12, (1996). 39. Driever, W. & Nüsslein-Volhrd, C. Bioid protein is positive regultor of hunhk trnsription in the erly Drosophil emryo. Nture 3, (1989). 4. Perkins, T. J., Jeger, J., Reinitz, J. & Glss, L. Reverse engineering the gp gene network of Drosophil melnogster. PLoS Comput. Biol. 2, e51 (6). 41. Sheffer, V., Jnody, F., Loss, C., Despln, C. & Wimmer, E. A. Bioid funtions without its TATA-inding protein-ssoited ftor intertion domins. Pro. Ntl Ad. Si. USA 96, (1999). 42. Houhmndzdeh, B., Wieshus, E. & Leiler, S. Estlishment of developmentl preision nd proportions in the erly Drosophil emryo. Nture 415, (2). 43. Cruk, O. & Dosttni, N. Bioid determines shrp nd preise trget gene expression in the Drosophil emryo. Curr. Biol. 15, (5). 44. River-Pomr, R., Lu, X., Tuert, H., Perrimon, N. & Jkle, H. Ativtion of posterior gp gene expression in the Drosophil lstoderm. Nture 6, 3 6 (1995). 45. He, F. et l. Proing intrinsi properties of roust morphogen grdient in Drosophil. Dev. Cell 15, (8). 46. He, F. et l. Shping morphogen grdient for positionl preision. Biophys. J. 99, (1). 47. Coppey, M., Berezhkovskii, A. M., Kim, Y., Boettiger, A. N. & Shvrtsmn, S. Y. Modeling the ioid grdient: diffusion nd reversile nuler trpping of stle protein. Dev. Biol. 312, (7). 48. Spirov, A. et l. Formtion of the ioid morphogen grdient: n mrna grdient dittes the protein grdient. Development 136, (9). 49. He, F. et l. Distne mesurements vi the morphogen grdient of Bioid in Drosophil emryos. BMC Dev. Biol. 1, 8 (1).. Jeger, J. et l. Dynmi ontrol of positionl informtion in the erly Drosophil emryo. Nture 43, (4). 51. Mnu et l. Cnliztion of gene expression in the Drosophil lstoderm y gp gene ross regultion. PLoS iology 7, e49 (9). 52. Luhett, E. M., Vinent, M. E. & Ismgilov, R. F. A preise Bioid grdient is nonessentil during yles for preise ptterning in the Drosophil lstoderm. PLoS One 3, e3651 (8). 53. Heht, I., Rppel, W. J. & Levine, H. Determining the sle of the Bioid morphogen grdient. Pro. Ntl Ad. Si. USA 16, (9). 54. Gregor, T., MGregor, A. P. & Wieshus, E. F. Shpe nd funtion of the Bioid morphogen grdient in diptern speies with different sized emryos. Dev. Biol. 316, (8). 55. Grimm, O. & Wieshus, E. The Bioid grdient is shped independently of nulei. Development 1, (1). 56. Fu, D. & M, J. Interply etween positive nd negtive tivities tht influene the role of Bioid in trnsription. Nulei ids reserh 33, (5). 57. Fu, D., Wen, Y. & M, J. The o-tivtor CREB-inding protein prtiiptes in enhner-dependent tivities of Bioid. J. Biol. Chem. 279, (4). nture ell iology VOLUME 13 NUMBER 1 JANUARY 11 29

9 METHODS DOI: 1.138/n2141 Methods Fly lines, plsmids nd ells. The fsd KG2393 flies were otined from the Bloomington Drosophil Stok Center (stok numer 12983). The following vetors were used in this study. pgem3 (Promeg) ws used s templte for in vitro trnsription/trnsltion. The pa5.1/v5-his C vetor (Invitrogen) ontining the Drosophil tin 5C promoter ws used to onstitutively express proteins in Drosophil S2 ells. The pdna3 vetor (Invitrogen) ontining the humn Cytomeglovirus immedite-erly (CMV) promoter ws used for over-expressing proteins in HEK 293T ells. All plsmids used in this study were generted y stndrd loning methods. Drosophil S2 nd HEK 293T ells were ultured in Shneider s Drosophil medium (Invitrogen) nd Duleo s modifition of Egle s medium (Cellgro), respetively, oth ontining 1% (v/v) fetl lf serum (Invitrogen) nd 1 ntiioti-ntimyoti (Invitrogen). Trnsfetion in oth ells ws performed using the FuGENE HD trnsfetion regent (Rohe) ording to the mnufturer s instrutions. Stle Drosophil S2 ells nd protein stility ssy. Drosophil S2 ells tht stly express HA Bd were generted s desried previously 58. The following is list of inhiitors (soures; finl onentrtions) for treting ells: hloroquine diphosphte (Sigm; μm), Z LL H (Peptides Interntionl; μm), MG132 (Boston Biohem; μm), ltystin (Cliohem; 1 μm), epoxomiin (Cliohem; 3 nm), nd CHX (Sigm; μg ml 1 ). For trnsient ssys, lz reporter ws used to normlize trnsfetion effiieny 15. Protein degrdtion ssy in Drosophil emryoni extrts. Drosophil emryoni extrts (extrted from w1118 emryos t 3 h) were prepred s desried previously 59, diluted to finl onentrtion of 1 mg ml 1 nd stored t 8 C efore use. HA Bd protein used in protein degrdtion ssys ws trnslted in vitro using the T N T quik oupled trnsription/trnsltion system (Promeg). Degrdtion retions were performed t 3 C, with or without the following (t given finl onentrtions): MG132 ( μm), epoxomiin ( μm), Mg ATP (Boston Biohem; 5 mm), UM NK (Boston Biohem,.67 μg μl 1 ), gluose (Sigm; 1 mm) nd hexokinse (Sigm;.3 U μl 1 ). We hve notied tht the protein degrdtion tivities of the stored emryoni extrts vried somewht. Thus, ll kineti experiments with speifi tretments inluded side-y-side (t ll steps) no-tretment ontrol. Only omprisons etween side-y-side ssys re meningful nd, thus, no omprisons were mde (or should e mde) etween experiments. Despite this vriility, the effets of different tretments on Bd stility re highly reproduile etween independent experiments. Western lot nd protein quntifitions. For deteting totl protein mounts in ells, ells were diretly oiled in 1 SDS PAGE (sodium dodeyl sulfte polyrylmide gel eletrophoresis) loding uffer ( mm Tris-HCl t ph 6.8, mm dithiothreitol, 2% SDS, 1% glyerol nd.1% romophenol lue) for 8 min, with rief vortexing every 2 min. Proteins were seprted y SDS PAGE nd trnsferred to Immun-Blot PVDF (polyvinylidene fluoride) memrne (Bio-Rd) for western lotting using pproprite primry ntiodies nd HRP (horse rdish peroxidse)- onjugted seondry ntiodies. Western lotting signls were visulized y ECL (enhned hemiluminsesene) plus Western lotting detetion regents (GE Helthre). HA tgged nd Flg-tgged proteins were deteted y nti-ha (Covne) nd nti-flg (Sigm) primry ntiodies, respetively; nti β-tin ntiody (Am) ws used to detet β tin s loding ontrol whenever neessry. nti-ha (1:1,), nti-flg (1:1,) nd nti-β-tin (1:2,). The protein nds deteted in western lotting were quntified s follows. X ry films were snned with the HP Snjet 47 Photosmrt snner, followed y nlysis using the Sion imge softwre. A retngulr ox of the sme size ws hosen to over the individul nds of interest, with men intensities determined y the softwre. Bkground intensity ws otined y pling the sme-sized retngulr ox in lnk lne t the sme position s in experimentl lnes. To estimte the hlf-life of Bd in the emryoni extrts, we plotted the Bd remining mount (frtion of Bd intensity t time zero) ginst the retion time. The dt were then fitted to the exponentil dey eqution (y = e x ) using the Mtl softwre. The resulting ln2/ vlues represent the estimted hlf-life of Bd. In vivo uiquityltion nd o-immunopreipittion ssys. For uiquityltion ssy, the HA Bd-expressing plsmid ws trnsiently trnsfeted into HEK 293T ells, long with seond plsmid expressing Flg-tgged uiquitin. Cell lystes were prepred nd inuted with nti-ha monolonl ntiody (Rohe) nd protein G Sephrose 4 fst flow eds (GE Helthre). Immunopreipittes were resolved y SDS PAGE nd western lotting ws performed using nti-uiquitin ntiody (Zymed 1:). For o-immunopreipittion ssys, HEK 293T ells were o-trnsfeted with plsmids expressing Flg Fsd nd HA Skp1 (or HA Bd). Cell lystes were inuted with nti-flg ntiody (Sigm) nd protein G Sephrose 4 fst flow eds, followed y western lotting using indited ntiodies. dsrnai genertion nd sreening in the HA Bd-expressing stle S2 ells. For dsrnai sreening 6, 38 potentil Drosophil F ox proteins were determined from the Interpro dtse from the Europen Bioinformtis Institute. Primers used for PCR mplifition of eh gene frgment were hosen from the mplion dtse of the Drosophil RNAi Sreening Center t Hrvrd Medil Shool. To trnsrie dsrnas in vitro from the PCR frgments, eh primer ontined T7 RNA polymerse inding site (5ʹ-TAATACGACTCACTATAGGG-3ʹ) t 5ʹ. The dsrnas were generted from the MEGASCRIPT T7 trnsription kit (Amion) nd purified y the RNesy mini kit (Qigen). To minimize the effet of plte-to-plte vritions nd inrese the reliility of ssys during sreening, fold hnge in Bd levels for eh smple ws lulted y dividing the intensity of smple y the men of ll of the 12 smples on given plte, with ll experimentl nd quntifition steps performed stritly on side-y-side sis for ll smples on single plte. Emryo stining, imging nd intensity mesurement. Emryos were olleted t 4 h t the stted tempertures, fixed nd stined for mrna using digoxigenin (Rohe)-lelled ntisense RNA proe s previously desried 61,62 with the following modifitions: emryos were post-fixed with 1% (v/v) formldehyde (Fisher Sientifi) nd hyridiztion ws performed in uffer ontining.3% SDS (4 h inution) without protese K pre-tretment. Cy3-AffiniPure Donkey nti-mouse immunogloulin G (IgG; Jkson ImmunoReserh) ws used s the seondry ntiody. Imging nd quntifition were desried previously 45, with ll imges ptured within liner rnge under identil settings in single imge yle (for oth wild-type nd mutnt emryos tht were stined for gene of interest) to minimize mesurement errors. To mesure the FISH intensities in the ytoplsm, irulr window 61 pixels in size ws used to slide long the emryo edge immeditely outside of the nuler lyer (slly for h nd kni; pilly for eve) during the utomted imge proessing. It hs een shown tht gp gene nd segmenttion gene expression ptterns evolve during nuler yle 14 (refs, 63). To minimize suh effets nd llow urte omprison etween wild-type nd mutnt emryos, we seleted emryos within short developmentl windows with pek expression level for eh gene nlysed. Speifilly, we seleted emryos t erly nuler yle 14 with nuler height:width rtio of 1.3:1 to 1.7:1 for h nd kni expression ptterns, s desried previously 45. For h expression, emryos were further seleted to hve posterior h expression t normlized level etween. nd.. For kni expression, emryos were further seleted to ensure no detetle expression stripe t the position of pproximtely.. The segmenttion gene eve rehes its pek expression t lter time thn the gp genes, nd we seleted emryos t stge (efore the onset of gstrultion) shown to hve highly preise eve pttern 63. To further inrese the preision of developmentl time for eve nlysis, we seleted emryos tht hd the seventh stripe level > % of the first stripe level. The mesured oundry positions for ll three genes were highly preise within eh group of emryos, with stndrd devition tht is less thn tht of the oserved posterior shifts etween wild-type nd fsd emryos in eh se. Emryo stining, imging nd intensity mesurements with nti-bd ntiodies (Snt Cruz Biotehnology) were s desried previously 45. To ompre λ vl- ues of Bd profiles from wild-type nd fsd emryos, we first nlysed their men Bd intensity profiles s funtion of. Bd intensities used in this nlysis were kground-sutrted without further djustments. The liner fit of ln (B/B mx ) ws onduted using the Mtl softwre. The λ vlues in individul emryos were lulted s desried 45. In n lterntive fitting, we used the eqution of B = B mx e x/λ + C nd otined onsistent results; the men λ vlues lulted from Bd profiles of individul wild-type nd fsd emryos re.8 ± 9.9 μm nd 94. ± μm (P = ), respetively. Immunoytohemistry. Drosophil S2 ells were ultured on L-Tek II hmer slide nd trnsfeted with the HA Fsd-expressing plsmid. Cells were fixed, permeilized, inuted with nti-ha ntiody (1: dilution) nd Alex nture ell iology

10 ARTICLES Fluor 488 got nti-mouse IgG (1:4 dilution), nd imged using onfol mirosopy. TO PRO 3 dye (1 μm) ws used to stin nulei. Sttistil nlysis. All relevnt experimentl vlues represent men vlues nd stndrd devitions, with n representing the numer of independent smples. P vlues were lulted y the Student s t-test funtion (two-tiled) using the Mtl softwre (MthWorks). Exponentil nd liner fitting ws done through the urve fitting funtion of the softwre nd the Adjusted R 2 vlues 64 were lulted to determine how well the dt fit to the pplied model ( perfet fit hs vlue of 1). 58. Forler, D. et l. An effiient protein omplex purifition method for funtionl proteomis in higher eukryotes. Nt. Biotehnol. 21, (3). 59. Crevel, G. & Cotterill, S. DNA replition in ell-free extrts from Drosophil melnogster. EMBO J. 1, (1991). 6. Clemens, J. C. et l. Use of doule-strnded RNA interferene in Drosophil ell lines to disset signl trnsdution pthwys. Pro. Ntl Ad. Si. USA 97, (). 61. Tutz, D. & Pfeifle, C. A non-rdiotive in situ hyridiztion method for the loliztion of speifi RNAs in Drosophil emryos revels trnsltionl ontrol of the segmenttion gene hunhk. Chromosom 98, (1989). 62. Kosmn, D. et l. Multiplex detetion of RNA expression in Drosophil emryos. Siene 35, 846 (4). 63. Surkov, S. et l. Chrteriztion of the Drosophil segment determintion morphome. Developmentl iology 313, (8). 64. Lttin, J., Crroll, J. D. & Green, P. E. Anlyzing multivrite dt. (Thompson Books/ Cole, 3). nture ell iology VOLUME 13 NUMBER 1 JANUARY 11 31

11 supplementry informtion DOI: 1.138/n2141 HA-Bd 2 1 M r (K) Time (min): HA-Bd DMSO epoxomiin d HA-Bd WB: nti-β-tin β-tin 1 2 Figure S1 Bd degrdtion is sensitive to protesome inhiitors. () Fulllength HA-Bd protein deteted in the HA-Bd-expressing stle ells. Moleulr weight stndrds re shown. Lne 1 represents dt from ells with vetor lone. () Totl mount of HA-Bd in Drosophil S2 ells trnsiently trnsfeted with the HA-Bd expressing plsmid, with or without MG132 tretment. ( & d) HA-Bd degrdtion ws inhiited y protesome inhiitor epoxomiin in emryoni extrts. See Figs. 1 nd legends for further detils. 1

12 supplementry informtion dsrna: FLAG-Skp1: WB: nti- β-tin Reltive Bd mount: HA-Bd β-tin WB: nti-β-tin WB: nti-flag HA-Bd β-tin FLAG-Skp1 d WB: nti-ha nti-β-tin Time (hr): Control skpa dsrna Control skpa dsrna skpa Figure S2 Skp1 prtiiptes in Bd degrdtion in Drosophil ells. () The totl mount of Bd in HA-Bd-expressing stle ells is inresed y dsrna trgeting fsd (lne 3), ut not y dsrnas trgeting two F-ox ontrol genes (lnes 1 nd 2). () Over-expression of Skp1 promotes Bd degrdtion. In this experiment, HA-Bd-expressing stle ells were trnsfeted with either the plsmid expressing FLAG-Skp1 or the vetor lone, nd the totl mount of HA-Bd ws nlyzed in the presene or sene of MG132. () CHX ssy in the HA-Bd-expressing stle ells in the presene or sene of skpa dsrna. See legend to Fig. 3 for further detils. (d) Stter plot of the experimentl dt from pnel ; see legend to Fig. 3 for further detils. 2

13 supplementry informtion Consensus: Figure S3 Alignment of F-ox domins in Drosophil. F-ox domins were derived from the Interpro dtse nd ligned using the Clustl W method. The two puttive F-ox domins of Fsd re inluded in this lignment. A loose onsensus is shown t the top of lignment, whih does not ontin ny invrile residues mong the sequenes nlyzed; the positions with the highest rte of identil residues re: 77% leuine t positions 3 nd 15, nd 72% isoleuine t position 11. Figur 3

14 supplementry informtion 18 C C 29 C w 1118 fsd KG2393 w 1118 fsd KG2393 w 1118 fsd KG2393 egg lrv lrv pup 23/ (92%) 21/21 (%) 17/35 (48.6%) 17/17 (%) 21/23 (91.3%) 15/15 (%) 49/6 (81.7%) 42/45 (93.3%) 7/34 (.5%) 4/7 (57.1%) /45 (%) N/A pup dult 21/21 (%) 16/17 (94.1%) 15/15 (%) 39/42 (92.9%) 3/4 (.%) N/A d e f Figure S4 Chrteriztions of fsd gene. ( & ) fsd trnsripts in wt emryos deteted y whole mount in situ hyridiztion using n fsd-speifi proe t pre-lstoderm () stge-7 () emryos. Sle r: μm. () Comprison of survivl rtes etween w 1118 nd fsd KG2393 flies t different tempertures nd stges. (d-f) Fsd is primrily lolized to the ytoplsm. S2 ells trnsfeted with the HA-Fsd expressing plsmid were fixed y formldehyde nd stined with To-pro-3 (d) nd nti-ha ntiody (e). Pnel f is merged imge of d nd e. Sle r: 1 μm. 4

15 supplementry informtion d e f g Figure S5 Ptterning defets in fsd emryos t 29 o C. (-d) Cutile ptterns of wt emryo () nd fsd emryos tht exhiit vrile A-P ptterning defets (-d). Cutiles re oriented with the nterior towrds the left nd dorsl upwrds. (e-g) Ptterns of eve expression in wt emryo (e) nd fsd emryos tht exhiit fused/missing eve expression stripes (f nd g). Sle r: μm. 5

16 s u p p l e m e n t r y i n f o r m t i o n wt (n = 7) eve eve fsd (n = 8) wt (n = 8) h d fsd (n = 7) h e wt (n = 9) kni f fsd (n = 8) kni Figure S6 Figure S6 Normlized FISH intensity dt extrted from individul emryos. Dt for eve ( nd ), h ( nd d) nd kni (e nd f) re shown seprtely for wt nd fsd emryos. Eh olor represents dt from n individul emryo. The men intensity nd stndrd devition re lso shown. 6

17 supplementry informtion d E1 /+ (n = 9) fsd KG2393 ;d E1 /+ (n = 9) h h d E1 /+ fsd KG2393 ;d E1 /+ h Figure S7 Geneti intertion etween d nd fsd. ( nd ) Normlized FISH h intensity dt extrted from individul emryos from d E1 /+ () nd fsd KG2393 ; d E1 /+ () femles. () Averge h intensity profiles from pnels nd. The nteriorly shifted h oundry position in d E1 /+ emryos (.368 ±.13, n = 9) is resued, prtilly, y mternl depletion of fsd (.39 ±.12 n = 9; p = ). The nteriorly shifted CF position in d E1 /+ emryos (.277 ±.15, n = 7) is lso prtilly resued y mternl depletion of fsd (.3 ±.12, n = 8; p = ). 7

18 supplementry informtion CG4663 KG2393 F-ox F-ox fsd (CG12765) CG467 (133p deletion) fsd 5-7 : wt (n = 9) fsd 5-7 (n = 1) wt fsd 5-7 d h h h Figure S8 Anlysis of null llele of fsd. () A shemti representtion of the fsd 5-7 llele generted y P-element medited exision. Green oxes represent the nnotted oding sequenes. Like fsd KG2393 flies, fsd 5-7 flies re homozygous vile t C ut emryos fil to hth t 29 C. The mesured CF positions for wt emryos nd emryos from fsd 5-7 femles (referred to fsd 5-7 emryos; ll results shown in this figure were otined t C) re.347 ±.9 (n = 9) nd.6 ±.13 (n = 13), respetively (p = ). ( nd ) Normlized FISH intensity dt for h extrted from individul wt () nd fsd 5-7 () emryos. (d) Averge h intensity profiles from nd. The men h oundry positions in wt nd fsd 5-7 emryos re.447 ±.15 (n = 9) nd.4 ±.11 (n = 1), respetively (p = ). 8

19 supplementry informtion Fig.1 Fig.1 WB: nti-β-tin Fig.2 Fig Fig.2e Fig.2g WB: nti-uiquitin Figure S9 Unropped Western lot dt. 9

20 supplementry informtion Fig.3 WB: nti-β-tin WB: nti-β-tin Fig.3 Fig.4 WB: nti-flag Fig.4 WB: nti-flag Figure S9 ontinued 1