SUPPLEMENTARY INFORMATION

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Clarissa Lester
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DOI:.38/n2816 Primr pir C lox P lox P Primr pir B Primr pir A - WT CM KO WT ES -50 kd -37 kd flox/flox X ZP3 Cr/Cr flox/+ -ZP3 Cr/+ flox/flox -ZP3 Cr/+ flox/flox -ZP3 Cr/+ X X flox/flox flox/flox

1 DOI:.38/n2816 Primr pir C lox P lox P Primr pir B Primr pir A - WT CM KO WT ES -50 kd -37 kd flox/flox X ZP3 Cr/Cr flox/+ -ZP3 Cr/+ flox/flox -ZP3 Cr/+ flox/flox -ZP3 Cr/+ X X flox/flox flox/flox Ooyts without mtrnl -Flox -Wil-typ -Cr Gn xprssion rltiv to Hprt Ctr1 Singl ooyt Ctr2 Ctr3 Ctr4 KO1 KO2 KO3 KO4 Fgf4 Tx1 Utf1 Nnog Sox2 Sll4 Stll Brg1 T4 Cx2 Eoms Gt4 Hprt1 f g Littr siz of flox/flox -ZP3 Cr/+ fml mi Fml Ml p -250 p Offspring of flox/flox ZP3 Cr/+ fml mi WT -Ctr Figur S1 Elimintion of mtrnl A i not show ny fft on th ooyt s vlopmntl omptn. () Shmti rprsnttion of th A trgting DNA onstrut n position of th gnotyping primr sts moifi from Khlr t l. (2004). Fill rrowhs: lox P sits; Ovl ox: promotr; fill rtngls: xons 1 5. () Mting strtgy to gnrt mtrnl A-null ooyts. Th flox/flox mi wr mt with ZP3 Cr/Cr mi to prou offspring with th flox/+ /ZP3 Cr/+ gnotyp. Thn th flox/+ /ZP3 Cr/+ ml mi wr kross with flox/ flox fml mi to otin flox/flox /ZP3 Cr/+ mi. Th ml mi of this gnotyp wr kross with flox/flox mi gin to otin flox/ flox /ZP3 Cr/+ fml mi tht woul prou A-null ooyts. () Th tils of offspring wr ut n gnotyp with primr pir B to tt for th prsn of th flox lll (449 p), Wt lll (415 p), n primr pir for Cr (373 p). Th lst ln ws us s ngtiv ontrol without ing ny DNA. () Vlition of limintion of mtrnl A t th protin lvl y Wstrn lot nlysis. Ooyt smpls ompris xtrts from 400 or mor GV ooyts. A monlonl A ntioy tt wk n of protin in wil-typ ooyts (WT) n vry strong n in th ES ll smpl (ES) of pproximtly 45 kd, ut not in th -knokout ooyts (KO) n umulus lls (CM). () Gn xprssion of singl ooyts t th GV stg, nlyz y Fluiigm qrt-pcr using th Biomrk Dynmi Arry systm (Fluiigm) furthr onfirm th limintion of th A trnsript without signifint impt on th xprssion of ooytn ling-spifi gns xmin. Th numr (1, 2, 3, or 4) right ftr th rvition (Ctr, for wil-typ ontrol, or KO, for knokout) rfrs to th iologil rplits. (f) A-knokout ooyts n support stlishmnt of totipotny, whih is nssry for full-trm vlopmnt, s shown y th norml littr siz from th rossing of flox/flox /ZP3 Cr/+ fml mi with CD1 wil-typ ml mi. (g) PCR gnotyping of th offspring from th ov rossing onfirm tht th A lll h n lt. Th rs rprsnt th mns from 3 thnil rplits, rsult rprsnttiv of h iologil rplit in. Th unropp vrsion of is shown in Supplmntry Fig. 5 n sour t for r shown in Supplmntry Tl

WT- - Gn xprssion rltiv to Control 1-1 -2 Iniviul lstomrs from iopsy Iniviul 8-ll mryos -Ctr -362 p -245 p Nnog Sox2 Fgf4 Utf1 Cx2 Eoms Tx1 Tp Ctr KO M-Z KO Mtrnl KO Zygoti KO WT M-Z KO Control Nnog

54 Nnog Nnog+Cx2 Cx2 Figur S2 Phnotyp of A-null mryos () Gnotyping with nst PCR on singl iopsi lstomrs from mryos otin y rossing flox/flox /ZP3 Cr/+ fml mi with +/- ml mi.

2 WT- - Gn xprssion rltiv to Control Iniviul lstomrs from iopsy Iniviul 8-ll mryos -Ctr -362 p -245 p Nnog Sox2 Fgf4 Utf1 Cx2 Eoms Tx1 Tp Ctr KO M-Z KO Mtrnl KO Zygoti KO WT M-Z KO Control Nnog Mrg Trom-1 Mrg Numrs of positiv lls E4.5 mryos Ctr KO Nnog Nnog+Cx2 Cx2 Figur S2 Phnotyp of A-null mryos () Gnotyping with nst PCR on singl iopsi lstomrs from mryos otin y rossing flox/flox /ZP3 Cr/+ fml mi with +/- ml mi. () Quntittiv RT-PCR on singl gnotyp 8-ll mryos with triplits shows tht limintion os not ly tivtion of Nnog gn trnsription. Ctr: A +/, KO: mtrnl n zygoti Aknokout. () Immunoytohmistry of E2.5 mryos for Nnog (grn) n (r), M-Z KO: mtrnl n zygoti A knokout. () Immunoytohmistry of E3.5 lstoysts for Trom-1 (r), nothr TE mrkr, n (grn) loliz th protin to th TE, whih furthr onfirm th ling sprtion of ICM/TE in A-null mryos. () Avrg ll numrs of Nnog- n Cx2-positiv lls pr E4.5 mryo wr ount on onfol immgs of immnostin mryos. KO: A-knokout; M-Z KO: mtrnl n zygoti A knokout. Th sl rs rprsnt 25 mm in n. Vlu rprsnts mn±s.d. of 3 iologil rplits in n mn±s.d. of 61 n 41 mryo smpls for wiltyp n A KO, rsptivly in. Sour t for r shown in Supplmntry Tl

Flox- WT- - -379 p -345 p -245 p 1 2 3 4 5 6 7 8 9 11 12 13 14 -Ctr Fluorsn intnsity of -GFP 60 E2.5 n=8 50 n=16 40 30 n=12 n=12 20 Ngtiv Control sigfp sisll4 160 140 120 0 80 60 40 20 E3.

5 Blstoysts Utf1 siutf1 Figur S3 -GFP xprssion is tivt in A-null mryos. () At th n of th tim-lps osrvtion on -GFP xprssion, h iniviul mryo ws mrk y numr with its gnotyp (mtrnl/zygoti) s trmin y.

3 Flox- WT p -345 p -245 p Ctr Fluorsn intnsity of -GFP 60 E2.5 n=8 50 n= n=12 n=12 20 Ngtiv Control sigfp sisll E3.5 n= n=8 n=12 n=13 0 Ngtiv Control sigfp sisll4 Exprssion rltiv to E3.5 Blstoysts Tpt1 sitpt E3.5 Blstoysts Zsn4 sizsn4 Exprssion rltiv to E3.5 Blstoysts Esrr siesrr E3.5 Blstoysts Utf1 siutf1 Figur S3 -GFP xprssion is tivt in A-null mryos. () At th n of th tim-lps osrvtion on -GFP xprssion, h iniviul mryo ws mrk y numr with its gnotyp (mtrnl/zygoti) s trmin y. () Gnrt y rossing flox/flox /ZP3 Cr/+ fml mi with OG2-GFP +/- - +/ ml mi, GFP-xprssing E4.5 mryos wr slt s shown. Gnotyping of ths mryos rvl tht hlf (17/36) wr mtrnl/zygoti knokout n suggst tht OG2-GFP ws still tivt in A-null mryos. () Gnotyp ws trmin y nst PCR with orrsponing numrs to. () -GFP xprssion ws not fft in E2.5 (lft) n E3.5 (right) mryos following injtion of sisll4 into zygots otin y rossing flox/flox /ZP3 Cr/+ fml mi with GOF18-GFP ml mi. Emryos without th -GFP trnsgn wr us s ngtiv ontrol n sirna trgting GFP (sigfp) ws us s positiv ontrol. Fluorsn intnsity ws quntifi y ImgJ softwr. () Effiint knokown of Tpt1, Zsn4, Esrr n Utf1 t th mrna lvls y injtion of sirna uplxs s ssss y rl-tim RT-PCR. No signifint fft on xprssion ws osrv. Exprssion lvls of Hprt1 wr us s th intrnl ontrol to normliz th t n sigfp-injt mryos wr us s lirtors. Sl rs rprsnt 50 mm in n. Th rror rs rprsnt mn±s.e.m. of 8-16 iologil rplits in n mn±s.d. of 3 iologil rplits in. Th unropp vrsion of is shown in Supplmntry Fig. 5 n sour t for r shown in Supplmntry Tl

OtA4KO ooyt NT WT ooyt NT AKO ooyt PA WT ooyt PA Exprssion lvl rltiv to ESCs Nnog Mrg Gn xprssion rltiv to ES 3 2 1-1 -2-3 NT Blstoysts Fgf4 Utf1 Nnog Sox2 Sll4 Stll Dzl Cx2Eoms Gt4 ES Ctr1 Ctr2 Ctr3

0-NT lstoysts show tivtion of Nnog n xprssion y A-knokout (A KO) ooyts. WT: wil typ; NT: Nulr trnsfrr; PA: prthnogni.

4 OtA4KO ooyt NT WT ooyt NT AKO ooyt PA WT ooyt PA Exprssion lvl rltiv to ESCs Nnog Mrg Gn xprssion rltiv to ES NT Blstoysts Fgf4 Utf1 Nnog Sox2 Sll4 Stll Dzl Cx2Eoms Gt4 ES Ctr1 Ctr2 Ctr3 KO1 KO2 KO3 PA1 PA2 PA3 DIC CAG-mRFP -GFP * * Figur S4 A-null ooyts rprogrmm somti ll nuli to pluripotnt sttus. () Immunoytohmistry of E4.0-NT lstoysts show tivtion of Nnog n xprssion y A-knokout (A KO) ooyts. WT: wil typ; NT: Nulr trnsfrr; PA: prthnogni. () Gn xprssion profiling of NT lstoysts rvl tivtion of xprssion of th pluripotnt gns n Nnog without mtrnl A xprssion. Th gn xprssion lvls wr otin with pools of 3 lstoysts with triplits n prsnt in omprison with ES lls (ES). Ctr: NT mryos using wil-typ ooyts; KO: NT mryos using A-knokout ooyts; PA: prthnogni mryos using -knokout ooyts. Th numr (1, 2, or 3) right ftr th rvition (Ctr n KO) rfrs to th iologil rplits. () Morphology of NT-ES lls grown on MEFs xprssing CAG-mRFP n -GFP. () Histology of trtom from NT-ES ll lin RG6 4 wks ftr injtion into SCID mi s ssss y hmtoxylin n osin stining. Th trtom ontin lls of ll 3 mryoni grm lyrs. Uppr lft pnl: krtiniz strtifi squmous pithlil lls (torml); uppr right pnl: nurl rostts (torml); lowr lft pnl: strit musl (msorml); lowr right: ilit olumnr pithlil lls jnt to pnrti inr lls (oth norml). () A littr of nontl NT-ES ll riv pups livr y srn stion on E19.0. In this prtiulr littr, 8 pups show norml full-trm vlopmnt, of whih on ws n on fil to initit rthing (*). Th sl rs rprsnt 30 mm in, 0 mm in n 50 mm in. Vlus rprsnt mn±s.d. of 3 iologil rplits. Sour t for r shown in Supplmntry Tl

5 Figur S5 Unropp figurs for Fig. 1, 2, 2, 4 n Supplmntry Fig. 1 n

6 Supplmntry Tl lgns Supplmntry Tl 1 Squning rsults of RT-PCR mplion using primrs spnning xon 2 n xon 3 of th gn in A-null ooyts n mryos. Supplmntry Tl 2 Squning rsults of RT-PCR mplion using primrs spnning xon 3 n xon 4 of gn in A-null ooyts n mryos. Supplmntry Tl 3 Primrs for gn xprssion stuy. Supplmntry Tl 4 sirna trgt squns Supplmntry Tl 5 Sour t fil. This fil inlus th originl sour t for rl-tim RT-PCR ssy. Lgns to Supplmntry Vios Supplmntry Vio 1 Tim-lps roring of in vitro vlopmnt of A-null 8-ll mryo A iopsi n gnotyp morul with mtrnl/zygoti A-null ws ultur on MEFs in ES ll mium n osrv on th stg of mirosop with n inution hmr (TOKAI HIT, Jpn) fill with 5% CO 2 in ir n mintin t 37 C. Brightfil piturs wr tkn vry 5 min for 4 ys n wr ompil into movi with 24 frms pr son. Th vio monstrts tht A-null mryos initit vittion n form grossly norml-looking lstoysts with istint ICM. Howvr, immunostining of th outgrowth (Fig. 3) show ytoplsmi loliztion of Nnog s wll s frgmnttion of nuli. Supplmntry Vio 2 Tim-lps onfol roring rvl tivtion of -GFP xprssion in mtrnl-knokout n mtrnl/zygoti-knokout mryos Twlv 2-ll mryos from th mting of flox/flox /ZP3 Cr/+ fml mi with A +/ /-GFP +/+ ml mi n 4 mryos (#1, 3, 4 n 8) from th mting of flox/flox fml mi with A +/ /-GFP +/+ ml mi wr pl in KSOM AA in glss ottom ish with th sm onition s Supplmntry Vio 1 for onfol xmintion with 488 nm lsr. A onfol pitur h n tkn vry min for 3 ys n ws ompil into movi with 24 frms pr son. Th vio monstrt tht rgrlss of th gnotyp, ll mryos tivt -GFP t roun E2.5 in timly fshion, s i wil-typ mryos. Th gnotyp of h mryo is shown in Fig. S3. Supplmntry Vio 3 Brightfil tim-lps roring of th sm mryos t th sm tim point s Supplmntry Vio 2 This vio ws us to monitor th vlopmntl stg of th mryos n to tr th position of iniviul mryos for gnotyp trmintion. 6

Above. Below H&E. Above CD4. CD11b. Fluoromyelin BDA BDA BDA. Cerebral cortex. Focal lesion. Thoracic cord. Lumbar cord. Hindlimb placing score

Above. Below H&E. Above CD4. CD11b. Fluoromyelin BDA BDA BDA. Cerebral cortex. Focal lesion. Thoracic cord. Lumbar cord. Hindlimb placing score 1. 1. 1 1 3 Tim tr lsion inution () wks wks 3 1 1 1 3 Tim tr lsion inution () i Ltrl sor. Mil Aov Blow.. 1 wk On sgmnt ov g 3. Lsion Fluoromylin Fluoromylin h CD11 Hinlim pling sor CD Aov Blow Fol lsion