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1 DOI: 1.18/n71 In the formt provie y the uthors n uneite. Ault GFP/ K GFP/ K GFP - Lgr + vs Lgr (1 genes up; 6 genes own) Lgr + vs Luminl ( genes up; 187 genes own) Lgr vs Luminl (17 genes up; 1619 genes own) Top 1 own genes in the ontrst of Lgr + vs Lgr sl ells Lgr + sl Lgr sl Luminl 9H1Rik Uts Ag Spok Crl1 Dn Col91 Tnfrsf19 Gni1 Ror Col1 Igfp Lrrn1 Dlx A6H16Rik Krt8 Mo Psk6 Phyhip Sps Gr1 Wisp1 Alh11 Htr1 Pst1 Vw1 Hgf Sors1 Vllr Arhgp Tx Tpl1 Dlk Tn Hpl Phgh Sem Ey1 Knk Hsst1 Fmo Pmp 1719N1Rik Se Slmp Nte Apo1 Up1 Hfy Egln Cs Upk1 Hsst1 Bnf Ch1 Alh17 Mtmr Hs6st 67O6Rik Pgf Fmnl Eif Aif1l Ak Lptm Hif1 Plsr D8KRik Ak Etv um Itm Gnp1 Sl1 AI11878 Rok Mtn Rrg Esm1 Lm1 Vn Guy1 Knm1 Slf Srm Pmt1 Col1 AK7767 D11Bwg17e C Cln1 Tmem18 BC6 Sl61 Str 11NRik Cp6 Cnjl Arsj Ce GO Enrihment (Up in Lgr + sl ells) GO Enrihment (Down in Lgr + sl ells) Stem ell ifferentition Negtive regultion of ell tivtion Woun heling Estlishment of pil/sl ell polrity Integrl omponent of memrne Regultion of emryoni evelopment Negtive regultion of non-nonil Wnt signling pthwy Cell hesion Cilium movement Cell surfe reeptor signling pthwy Estlishment of monopolr ell polrity Metlloenopeptise tivity Regultion of mirotuule polymeriztion or epolymeriztion Integrl omponent of plsm memrne Extrellulr mtrix Ativtion of NF-kppB-inuing kinse tivity Cytoskeletl protein ining Mirotuule unle formtion Cell migrtion Cell ifferentition Cell morphogenesis Plsm memrne - - log1 (p vlue) Cell ivision Crohyrte erivtive iosyntheti proess Glyosyl ompoun iosyntheti proess Nuleosie iosyntheti proess Regultion of RNA spliing Regultion of mitohonril trnsltion Positive regultion of GMP iosyntheti proess Sugr trnsmemrne trnsporter tivity Orgni sustne trnsport Orgni i metoli proess Enoplsmi retiulum Polysome Mitohonril memrne Crohyrte erivtive metoli proess L-serine iosyntheti proess Glyoprotein metoli proess Regultion of mrna proessing Positive regultion of trnsltion Smll moleule metoli proess Extrellulr mtrix Extrellulr vesiulr exosome Extrellulr region prt log1 (p vlue) -8 Supplementry Figure 1 Gene expression profiling of Lgr + ells in the ult mmmry gln () Whole-mount D onfol imge n optil setion of utl portion lote in the istl prt of the mmmry gln from n ult (9 week-ol) Lgr-GFP-IRES-reER T femle (representtive of mie, inepenent experiments) immunostine for K (lue). Lgr + ells only pper in the sl popultion of the ult mmmry glns. Sle rs, µm (whole-mount); 1 µm (optil setions). () Het mp showing ll the DE genes in ny pirwise omprison mongst the luminl, Lgr-GFP + (Lgr + ) n Lgr-GFP (Lgr ) sl popultions. Expression vlues re on log sle n re men-orrete for eh gene. Three iologil replites were sorte from 9 week-ol Lgr-GFP-IRES-CreER T femles n their trnsriptomes nlyse y mirorry. The utoff for DE ws FDR<. n solute fol-hnge (7 mie were nlyse in totl of n= inepenent experiments). () Het mp for the top 1 upregulte genes for Lgr + vs Lgr sl ells in (). () GO enrihment nlysis of DE genes etween Lgr + versus Lgr sl ells in () Mmilln Pulishers Limite, prt of Springer Nture. All rights reserve.
2 1 Luminl Primry outgrowths in virgin reipients Lgr + hi Lgr hi 1 Bsl.6 CD 1 1 Strom CD Lgr + Lgr C7Bl/6 CD 1 Luminl Bsl e Primry outgrowths in pregnnt reipients Lgr + hi Lgr hi 1 Strom CD Lgr + Lgr FVB/N Elf-rtTA-GFP CD Elf-GFP f Seonry outgrowths in virgin reipients Lgr + hi Lgr hi CD g 1 luminl ells S/G/M S/G/M Lgr + Lgr 7-AAD..1 G G py.1.1 G G Supplementry Figure FACS nlysis of -expressing ells in the ult mmmry gln n representtive outgrowth generte y ifferent sl supopultions () Representtive FACS plots showing -expressing ells in the mmmry glns of 9 week-ol C7BL/6 femles (1 mie, inepenent experiments). () Representtive FACS plots showing -expressing ells in the mmmry glns of 9 week-ol FVB/N femles (1 mie, inepenent experiments). () Representtive FACS plots showing overlp of Elf-GFP + n + ells in the luminl popultion from the mmmry glns of 9 week-ol Elf-rtTA-GFP femles ( mie, inepenent experiments). () Trnsplnttion of the ifferent supopultions ( ells) into the lere ft ps of -week ol reipient femle mie. Glns were ollete 1 weeks post-trnsplnttion. Representtive whole-mount imges re shown (6 mie per popultion). Sle r, mm. (e) Trnsplnttion of the ifferent supopultions ( ells) into the lere ft ps of reipient femles. Femles were mte 9-1 weeks fter trnsplnttion n mmmry glns were ollete t 18. ys of pregnny. Representtive wholemounts of outgrowths re shown (6 mie per popultion). Sle r, mm. (f) Representtive whole-mount imges of seonry outgrowths. Bsl ells were sorte from primry outgrowths generte from the inite susets efine y Lgr n expression, n trnsplnte into the lere ft ps of seonry reipient femles. The seonry outgrowths were ollete 1 weeks post-trnsplnttion. Sle r, mm (-6 mie for eh popultion per experiment, inepenent experiments). (g) Representtive FACS plots showing the DNA n RNA ontent of the luminl omprtment isolte on the sis of expression (ll Lin CD9 lo CD + ). Sorte ells from the mmmry glns of 9 week-ol Lgr-GFP-IRES-reER T femles were fixe n stine with 7-AAD n pyroniny (py). G is efine s G1 ells with low RNA ontent ( mie, inepenent experiments) Mmilln Pulishers Limite, prt of Springer Nture. All rights reserve.
3 Gene Lgr Lgr hi Lgr+ Lgr + hi At Krt Krt Krt Krt Trp Col Atg Myh Sni I Cytokine proution Response to ytokine Response to wouning Cellulr response to stimulus Cell ommunition Cell migrtion Cell surfe reeptor signling pthwy Cell hesion Integrl omponent of memrne Extrellulr region Plsm memrne GO Enrihment (up in Lgr + hi vs Lgr + ) GO Enrihment (own in Lgr + hi vs Lgr + ) Cell yle phse trnsition Mitoti ytokinesis Cytoskeleton DNA mge response Chromosome onenstion Chromosome segregtion Orgnelle fission Nuler ivision Cell ivision Cell yle proess Mitoti ell yle proess log1 (p vlue) -6 log1 (p vlue) Col61 Col6 LOC Clip Mu1 Sfrp Atp11 Ni Col71 D1 Sng Amts Amts1 Emp1 Serping1 Crip1 Fn1 Slpi Gfr Sfrp1 Enh Pygl Emp My Esyt Oln Ptpr Snhg11 Sstr1 Nrm Fox1 C1 Tnfip6 Ntn1 Dpt Gr1 Lox Ifit1 Bmp Fp Iigp1 Th Rr Anx8 Il6 Col1 Cxl1 Col1 Sprl1 Cpe Prex Lrr Mmrn C17G19Rik Ly61 Cyp1 C Tgtp1 Nrp1 C6 Rgs16 Ly6 Lrg1 Ifit C Lpl Fox1 Erg H A Cxl9 Islr Cm Emn Fxy Rmp Gp Mnl AI6787 Ifi Il AW111 Meox Tgtp Semg Pi16 Loxl1 Cle Ogn Nrxn1 Wf18 Mmp Frmp Mfp Mn Ifi Ifi H 1 Ptpr Ighm 1 1 Lm1 Reln Ppp Col6 Ereg Frem Amts17 Gr1 Col111 Cpn6 Hs6st Roo Atg Mox1 Pk Tt9 Fn Ankr6 St8si6 Crlf1 Knm1 As Igfp Tx Hpl Sl76 Chr Sors1 Cmk1 Sem Wisp1 Np Ryr Upk1 Hsst1 Trpm Knm1 Coro6 Gm1961 Mkx Col91 Dio Zfhx Ptprt Fgfr Kn6 Coh Am11 Sv Bt1 Xylt1 Gpx Serpin1 Knk Gm6 Cmkv Wnt6 Ey1 Gp Serpinh Stox1 Bves Dlx Lep Ch6 F91NRik 1719DRik Ltr Tf Tnfrsf19 118D6Rik Ism DGRik Rpsn Ryr1 Serpin Spok Up1 Ag Tmem1 Lhfpl1 Spok1 Uts Egfem1 A89NRik Fgf1 Cyp1 Dsg1 Lrfn 9H1Rik Gl1l Nrp Cyr Lrp LOC1866 Gm6 Knip1 Lrr 67ORik Am8 e Signture sores Lgr + T8 hi vs AllOthers Cluin low Lum A Lum B HER Bsl Signture sores Cluin low Lgr T8 hi vs Lgr T8 Lum A Lum B HER Bsl f Lgr + hi Lgr hi Enrihment Enrihment 1..6 Up 1.6 Lgr +. Lgr 1.9 Lgr + hi Lgr hi Lgr + Lgr Cluin-low vs sl rest ner p vlue = sttistis 11.6 Down Supplementry Figure Gene expression signtures of quiesent mmmry stem ells n omprison with the mjor sutypes of humn rest ner () Reltive expression (log pm vlues) of known sl mrkers ross the four ifferent myoepithelil/sl supopultions s etermine y RNA-seq nlysis. (, ) Het mps showing expression of the top 1 DE genes on omprison of Lgr + hi ells versus the verge of the other three susets, either upregulte () or ownregulte (). Two iologil replites were sorte y flow ytometry from the mmmry glns of 9 week-ol Lgr-GFP- IRES-reER T femles n their trnsriptomes were etermine y RNA-seq (9 mie were nlyse in totl of n= inepenent experiments) () GO enrihment nlysis of DE genes for Lgr + hi versus Lgr + ells (9 mie were nlyse in totl of n= inepenent experiments). (e) Box plots of quiesent MSC signture sores y tumour sutype. The signture sores for the Lgr + hi su-popultion ompre to ny other popultion re strongly orrelte with the luin-low sutype of rest ner. Boxes show %, % n 7% perentiles. Whiskers exten to minimum n mximum vlues. Vlues more thn 1. interqurtile rnges from the ox re shown s iniviul points. Two RNA smples per mmmry supopultion were use to interrogte the rest ner tse: Cluin-low: n = 19; LumA: n = 7; LumB: n = 7; HER: n = ; Bsl: n = tumours. (f) Broe plot epiting the strongly ssoite gene expression signtures of quiesent MSCs n luin-low tumours ompre to the sl-like sutype. Genes re orere from right to left s most upregulte to most ownregulte in luin-low ner. The re lines esignte upregulte genes in quiesent MSCs (Lgr + hi vs All Others), wheres lue lines esignte ownregulte genes. The utoff for DE is FDR<. n solute fol-hnge (9 mie were nlyse in totl of n= inepenent experiments). P vlues mesuring the overll orreltion were erive from the rost funtion of the limm softwre pkge Mmilln Pulishers Limite, prt of Springer Nture. All rights reserve.
4 luminl weeks 9 weeks 6 months LN ft p proximl re proximl istl istl re CD Prous (x, pregnny) 1 1 LN ft p proximl re proximl istl istl re CD CD CD month-ol virgin LN mile ft p istl istl re mile re re CD Lgr-GFP CD9 Supplementry Figure Lolistion of hi sl ells in the ult mmmry gln () Representtive FACS plots showing + luminl ells in the proximl n istl res of mmmry glns isolte from Lgr- GFP-IRES-reER T femles t weeks, 9 weeks or 6 months of ge (1 mie were nlyse for eh ge in totl of inepenent experiments). () Representtive FACS plots showing hi sl ells in the proximl n istl res of mmmry glns from C7BL/6 femles fter ompletion of three pregnny yles ( mie, inepenent experiments). () Representtive FACS plots showing the sl supopultions efine y expression of Lgr n in the, mile n istl res of mmmry glns from the Lgr-GFP-IRES-reER T femles t the ge of 9 months ( mie, inepenent experiments) Mmilln Pulishers Limite, prt of Springer Nture. All rights reserve.
5 S U P P L E M E N TA R Y I N F O R M AT I O N GFP/K/K8 GFP/K/K8 ololistion hnnel K/K8 f e E18. % of GFP+ ells 1 GFP/K/K Per ent CD trunk CD Ault MSCs Fetl MSCs 1 Lgr-GFP Fetl skin i 11. rnhes Lgr-GFP Fetl mmmry ruiments Lgr-GFP-IRES-CreERT 1 * h E18. ns GFP/K/K8 g K/K8 K K8 **** **** Lgr-GFP Supplementry Figure Chrteristion of Lgr- n -positive ells in fetl mmmry ells () Whole-mount D onfol imge of n entire mmmry ruiment from Lgr-GFP-IRES-reERT emryo t E18. immunostine for GFP (green), Kertin (re) n K8/K18 (lue) (representtive of emryos). () Cololistion hnnel (white) uilt in the Imris softwre showing the ololistion pttern for K (sl) n K8/K18 (luminl). Note tht ololistion minly ours in the growing uts. () Enlrgement from () showing the trunk portion of the emryoni mmmry gln, where the epithelil lyers re more efine n there re rre oule-positive ells (for K n K8/K18). (, e) Enlrgement from () showing eveloping rnhes. (e) shows the ololistion hnnel (white). Most ells re oule-positive for K n K8/K18 ( emryos, inepenent experiments). Sle rs, 1 µm (whole-mounts); µm (optil setions). (f) Br hrt showing the perentge of Lgr-GFP+ K/K8+, K+ n K8+ ells in the growing uts (us) n the trunk (n = emryos). K8 in -f esigntes the omine K8/K18 Trom ntioy (n= emryos, inepenent experiments). Error rs represent men ± SEM; * p <.; ****p <.1, Stuent s t-test. (g) Representtive imge of mmmry ruiment from Lgr-GFP-IRESSupplementry Figure Sle rs, reert femle emryo t E18. (representtive of emryos). µm. (h) Representtive FACS plot showing Lgr n expression in fetl mmmry glns n skin. Emryos were hrveste t E18. from pregnnt Lgr-GFP-IRES-reERT femles. Mmmry glns from femle emryos were issete uner fluoresene mirosope n GFP+ mmmry ruiments from 8-1 emryos were poole for the preprtion of single ell suspensions for FACS nlysis (8-1 femle emryos for eh experiment, inepenent experiments). (i) Comprison of the expression levels of LgrGFP in the fetl n ult MSC-enrihe popultions ( mie or 16 femle emryos, inepenent experiments) Mmilln Pulishers Limite, prt of Springer Nture. All rights reserve.
6 S U P P L E M E N TA R Y I N F O R M AT I O N E18. /p6 /p6/dapi E17., TAM nlysis irth 11 week Lgr-GFP-IRES-reERT/ R6R-tTomto 1 t ttomto Luminl 1 Luminl 1 1 MSC/ sl CD MSC/sl 6. 1 CD CD 1 / ttomto EU E1. E18. nlysis 11 week P I I II II Figure S8 GFP/EU/E Supplementry Figure 6 Contriution of fetl Lgr+ ells n emryoni origin of Lgr+hi ells in the ult mmmry gln () Representtive onfol imges of n E18. femle mmmry utl tree stine with (re), p6 (green) n DAPI (lue) ( emryos, inepenent experiments). () Emryoni Lgr-expressing ells ontriute to the luminl n sl lineges in the ult mmmry gln. 1, Whole-mount imge of n entire mmmry gln from Lgr-GFP-IRES-reERT/R6R-tTomto mie 11 weeks fter tmoxifen injetion t 17. ys of pregnny (representtive of mie, inepenent experiments). Sle rs: 1 m., Whole-mount D imge of utl portion lelle for E-herin (lue). Inset, optil setion from the enlrgement showing Tomto+ luminl n myoepithelil ells lelle in the ut. Sle rs, 1 µm (whole-mount); µm (optil setion). () Representtive FACS plots emonstrting the Supplementry mmmry repopulting Figure tivity of emryoni Lgr-ex6 pressing ells. Lgr-GFP-IRES-reERT/R6R-tTomto mie were nlyse t 11 weeks fter tmoxifen injetion t 17. ys of pregnny ( mie, inepenent experiments). () Whole-mount D onfol imge of the proximl portion of utl tree from Lgr-GFP-IRES-reERT mouse. EU ws IP injete twie per y from E1. to E18. n then hse for 11 weeks. The whole-mount ws lelle for GFP (green), EU (re) n E-herin (lue). I, Optil setion from the whole-mount imge showing utl portion emnting from the re, with no EU retention. II, Optil setion from the whole-mount imge showing utl portion in the re isplying EU retention (representtive of mie, inepenent experiments). Sle rs, µm (whole-mount); µm (optil setions) Mmilln Pulishers Limite, prt of Springer Nture. All rights reserve. 6
7 S U P P L E M E N TA R Y I N F O R M AT I O N Vehile I II Distl region Proximl region II I MPA+E Proximl region f Distl region e g Luminl MSC/sl Vehile E + MPA Lgr hi EU Supplementry Figure 7 Quiesent mmmry stem ells n e tivte y syntheti hormonl ues (-) Whole-mount D onfol imges n optil setions of the proximl () n istl () prts of mmmry gln from Lgr-GFP-IRES-reERT/R6R-tTomto mouse two weeks fter tmoxifen injetion t 9 weeks n immunolelle for E-herin (lue). Mie were trete with vehile. (I, II) Enlrge res showing sprsely istriute ttomto+ myoepithelil n luminl ells in vehile-trete mie (). Representtive imges re shown ( mie, inepenent experiments). (f) Whole-mount D onfol imges n optil setions of the proximl (, ) n istl (e, f) prts of mmmry gln from Lgr-GFP-IRESreERT/R6R-tTomto mouse two weeks fter tmoxifen injetion t Lgr+hi Lgr CD Lgr Supplementry Figure 7 weeks, uring ulthoo n immunolelle for E-herin (lue). Mie were trete with the syntheti progestin meroxyprogesterone ette (MPA) plus estrogen (E + MPA). () Enlrgement from () epiting rnhing u with luminl n sl Tomto+ ells erive from Lgr+ ells in mie trete with E + MPA. Right hn pnel, optil setion of the lveolr u. (f) Enlrgement from (e) showing utl rnh in the istl re omprising only Tomto+ sl ells. Representtive of mie, inepenent experiments. Sle rs: µm (whole-mounts), µm (optil setions). (g) FACS plots showing EU+ ells in the luminl n sl popultions isolte from LgrGFP-IRES-reERT mie trete with vehile or E + MPA. Representtive plots re shown (1 mie, inepenent experiments) Mmilln Pulishers Limite, prt of Springer Nture. All rights reserve. 7
8 S U P P L E M E N TA R Y I N F O R M AT I O N weeks Involution Proximl region Distl region Supplementry Figure 8 Quiesent mmmry stem ells n e tivte uring involution (-) Whole-mount D onfol imges n optil setions from enlrgements of the proximl (, ) n istl (, ) prts of mmmry gln from Lgr-GFP-IRES-reERT/R6R-tTomto mouse two weeks uring involution fter tmoxifen injetion t 9 weeks of ge. Glns were immunolelle with E-herin (lue). Expnsion n e visulise in oth regions of the gln. Sle rs: µm (whole-mounts), µm (optil setions). Representtive imges re shown ( mie, inepenent experiments). Supplementry Figure Mmilln Pulishers Limite, prt of Springer Nture. All rights reserve. 8
9 Supplementry Vieo Legens Supplementry Vieo 1 Movie epiting D reonstrution of mmmry sprout t E18. (represente in Figure 6A1). This movie shows Lgr + (GFP, green) n K + (lue) ells. Representtive vieo of emryos, inepenent experiments Mmilln Pulishers Limite, prt of Springer Nture. All rights reserve.
Supplementary figure 1
Supplementry figure 1 Igf 1 mrna 5 15 1 5 Arg1 mrna 16 1 8 Col11 mrna 5 3 1 Mmp 13 mrna 15 1 5 Tslp mrna 3 1 Il5 mrna 3 1-1 Il33 mrna 6 Figure 1s. Kinetis of woun heling ftors n erly Th-type response ytokines
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DOI: 1.138/n24 Reltive let7i expression Reltive let7i expression CAL27 Twist1 shsr shtwist1 shbmi1 Reltive let7i expression 6. 5. 4. 3. pflagcmv pflagtwist1 Cse 1 Cse 2 BMI1 Eherin Reltive migrtory ility
supplementary information
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DOI:.38/n3343 DIC/Ht Ht DAPI Anphse/Telophse (% of ells) 6 1 18 Time from HU relese (min) untrete Nsg1-GFP Myo1-mCherry -4-4 6 8 untrete HU pulse 5 15 Onset of ring ontrtion to NE segregtion (min) untrete
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doi:1.1/nture1 NF-κB/RE-GFP TNF-α pdna CA CA + DN d e NF-κB/RE-GFP GFP+DAPI NF-κB/RE-GFP GFP+DAPI NF-κB/RE-GFP GFP+DAPI V V DAPI DAPI NF-κB RE/GFP V V Mediosl hypothlmus Thlmus Cortex Suppl. Figure 1.
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VOLUME: ARTICLE NUMBER: 3 In the formt provided y the uthors nd unedited. Developmentl constrints shpe the evolution of the nemtode mid-developmentl trnsition Hrel Zlts nd Iti Yni,3 Fculty of Biology,
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DOI: 1.138/n2131 Protein levels (% of ) e Full-length protein remining (%) 1 5 1 5 1 1 5 5 Hs7 Syt1 Syt2 β-atin CSP +tsyn Hs7 Syt1 Syt2 P4 rin.1.1.1 1 [Trypsin] (g/l) f +tsyn SNARE-omplexes remining (%)
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doi:1.138/nture11813 NK cells NKp46 + RORγt - 5.4 RORγt + ILCs SSC-A FSC-H Linege 22.4 98.2 28.8 NKp46 1.2 15.4 NKp46 NKp46 + RORγt + ILCs NKp46 - RORγt + ILCs FSC-A FSC-A CD45 RORγt RORγt CCR6 + T-et
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DOI: 1.138/nc2975 GM13 / DNA F-ctin α shrna CLASP1 shrna shrna shrna CLASP1 shrna shrna e Tuulin CLASP1 CLASP1 shrna #32 shrna #33 #55 #58 Tuulin c Tuulin ppmlc E-Cdherin shrna CLASP1 shrna shrna f Golgi
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SUPPLEMENTARY INFORMATION DOI:./n Supplementry Figure Stroml ell senesene in mie with mesenhyml (RBP-Jk) eletion. () Skin o neworn (P) mouse with eletion o the (RBP-Jk) gene (KO), ierent rom the one shown
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doi:.38/nture8499 doi:.38/nture8499 5 6 5 4.5 Firing rte (Hz) -67-65 -66-6 -58 V m (mv) -7-67 -68-66 -64 c Thet power (mv ) -73-69 -7-7 -7.5.8 3....9.9.4.6.6. 9 8 9 8 9 8 9 8 9 8 Supplementry Figure Firing
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Supplementry Figure (nesthetized) (wke) Normlized mplitude.5 Pek width (ms).6.4.2 4 2 2 x 3 Wveform slope Normlized mplitude.5 Pek width (ms).6.4.2 x 3 3 2 Wveform slope c (nesthetized) d (wke) Normlized
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SUPPLEMENTARY INFORMATION oi:1.138/nture12343 Myogenin M-Cherin E9.5 E11.5 Thymus Spleen White ipose tissue Tongue Lim Musle Hert Kiney Brin Liver Lung Bler Stomh 28s Fol expression (Reltive to Thymus)
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