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Guanino nukleotidų mainų faktorius net1 palengvina nugaros ląstelių likimo nustatymą zebrafish embrionuose, skatinant motinos β-katenino aktyvaciją | ląstelių tyrimai
 Embriogenezė
 Fosforilinimas
 Signalizavimas Wnt / β-kateninu yra būtinas norint inicijuoti nugaros ir ventralinį modeliavimą stuburinių embriogenezės metu. Motinos β-kateninas kaupiasi nugariniuose kraštiniuose branduoliuose skilimo etapuose, tačiau kritiniai jo tiksliniai genai, būtini dorsalizacijai, yra tylūs iki blastulos vidurio perėjimo (MBT). Čia mes pastebime , kad zebrafish net1 , guanino nukleotidų mainų faktorius, yra specialiai išreikštas nugaros kraštiniuose blastomeruose po MBT ir veikia kaip zigotinis faktorius, skatinantis nugaros ląstelių likimų specifikaciją. Funkcijos praradimo ir padidėjimo eksperimentai rodo, kad norint aktyvuoti Wnt / β-katenino signalizaciją zebrafish embrionuose ir žinduolių ląstelėse, reikalingas Net1 GEF aktyvumas. „Net1“ išskiria ir suaktyvina PAK1 dimerus, o PAK1 kinazės aktyvinimas sukelia β-katenino S675 fosforilinimą po MBT, o tai galiausiai lemia pasroviui taikomų genų transkripciją. Apibendrinant, mūsų rezultatai atskleidžia, kad Net1 reguliuojamas β-katenino aktyvinimas vaidina lemiamą vaidmenį nugarinės ašies formavimosi metu plėtojant zebrafish.
 Išsaugotas visų stuburinių embrionų bruožas yra pagrindinio kūno plano sudarymas, į kurį įeina ir priekinės, ir užpakalinės, ir dorsalinės bei ventrinės ašys, 1, 2 . Ankstesniais tyrimais nustatyta, kad motinos Wnt / β-katenino signalizacija yra reikalinga norint, kad besivystantys embrionai darytų dorsalinį ir ventrinį šabloną. 3, 4, 5, 6, 7, 8 . β-kateninas yra pagrindinis kanoninio Wnt signalizacijos kelio veiksnys, o jo stabilumas ir aktyvumas lemia paskesnių taikinių genų ekspresiją. Kai Wnt signalo nėra, β-kateninas skaidomas naikinimo kompleksu, kurį sudaro aksinas, adenomatozinis polipozės coli baltymas, kazeino kinazė 1α (CK1α) ir glikogeno sintazės kinazės-3β (GSK-3β), kuris fosforilina β-kateniną ant N- galiniai serino ir treonino likučiai ubikvitino sukeltam skilimui 9, 10, 11 . Wnt ligando buvimas slopina skilimo kompleksą ir sukelia β-katenino stabilizavimąsi bei vėlesnį perkėlimą į branduolį, kur jis suaktyvina taikinio geno transkripciją 12, 13 . Anamniotuose po apvaisinimo mikrotubulų judėjimas lemia asimetrinę motinos Wnt ligandų lokalizaciją, kuri skatina β-katenino kaupimąsi būsimos nugarinės pusės blastomeruose skilimo etapuose 4, 14 . Nors šis asimetriškas β-katenino kaupimasis suaktyvina nedidelio skaičiaus genų transkripciją prieš vidurinio blastulės perėjimą (MBT) 15, kritiniai taikiniai, kurie slopina ventralizuojančių veiksnių, įskaitant bozozoką ( boz ) ir chordiną ( chd ), veikimą , yra neišreikšta tik po MBT 1, 2, 16, 17, 18, 19 . Šie stebėjimai rodo, kad branduolinio β-katenino transkripcinis aktyvumas gali būti ribotas prieš MBT. Tolesnis ląstelių modifikacijos išaiškinimas ir tolesnė β-katenino aktyvacija yra labai svarbūs norint suprasti šiuos vystymosi procesus.
 Fosforilinimas β-kateninas ne tik sukelia jo skilimą, bet taip pat buvo įrodyta, kad jis sukelia kitokį poveikį jo ląstelių funkcijai 20 . Be fosforilinimo, gauto N-gale naudojant GSK-3β ir CK1α, β-kateninas taip pat gali būti fosforilinamas S552, Y654, S663 ir S675, todėl padidėja baltymo stabilumas arba transkripcinis aktyvumas 20, 21, 22, 23, 24 . Tačiau šių β-katenino C-galinių modifikacijų vystymosi vaidmuo formuojant stuburo stuburo stuburo ašį dar nėra išaiškintas.
 Kaupiami įrodymai rodo, kad Rho GTPazės sukeliamas β-katenino fosforilinimas vaidina gyvybiškai svarbų vaidmenį reguliuojant Wnt signalizaciją. „RhoA“, „Rac1“ ir „Cdc42“ priklauso mažų GTPazių Rho šeimai ir veikia kaip molekuliniai jungikliai, besisukantys tarp neaktyvios, su BVP susijusios būsenos, ir aktyvios, su GTP susijusios, būsenos. Rho GTPazių aktyvaciją griežtai kontroliuoja specifiniai guanino nukleotidų mainų veiksniai (GEF), kurie stimuliuoja GTP mainus BVP, kuris yra svarbus daugeliui nuo aktino priklausomų procesų, tokių kaip ląstelių migracija, adhezija ir morfogenezė 25, 26 . Įrodyta, kad Rac1 aktyvina JNK2, kuris savo ruožtu fosforilina β-kateniną prie Ser191 ir Ser605 ir kontroliuoja β-katenino branduolio translokaciją pelės kaulų čiulpų gautoje ST2 ląstelių linijoje 27 . Rac1 taip pat aktyvina p21 aktyvuotą kinazę 1 (PAK1), kad skatintų β-katenino S675 fosforilinimą ir aktyvaciją storosios žarnos vėžio ląstelėse 23 . Daugybė mažų GTPazių Rho šeimos GEF taip pat vaidina gyvybiškai svarbų vaidmenį reguliuojant Wnt / β-katenino signalą. Kolorektalinio vėžio ląstelėse Rac1 mainų faktorius Tiam1 susijęs su Wnt reaguojančiais promotoriais, reaguodamas į Wnt ligando stimuliaciją, ir aktyvuoja branduolinį Rac1, kad padidintų Wnt taikinio geno transkripciją 28 . Storosios žarnos vėžyje ir HEK293 ląstelėse DOCK4 ir β 1 Pix, kiti du Rac GEF, taip pat gali skatinti kanoninį Wnt signalizavimą, padidindami β-katenino 29, 30 stabilumą arba transkripcinį aktyvumą. Be to, neseniai atliktas tyrimas parodė, kad „Rho GEF Pebble“ / ECT2 vaidina konservuotą vaidmenį slopindamas Wnt aktyvumą β-katenino stabilizavimo pasroviui metu ir kad jo praradimas ir funkcijos padidėjimas Drosophila embrionuose sukelia modeliavimo defektus 31 . Ankstesnis mūsų tyrimas atskleidė, kad viena tokia GEF, neuroepitelinė ląstelė, transformuojanti 1 ( net1 ), yra labai išreikšta priešdorsaliniame organizme , kai prasideda gastrologija zebrafish embrione 32 . Atsižvelgiant į tai, kad net1 yra erdviniu ir laiko atžvilgiu išreikštas zebrafish embriono regione, kuriame, kaip žinoma, Wnt signalizacija vaidina svarbų vaidmenį vystymosi metu, atrodo įtikėtina, kad Net1 greičiausiai dalyvaus Wnt / β-katenino kelyje.
 „Net1“ yra RhoA specifinis GEF, iš pradžių išskirtas iš neuroepitheliomos ląstelių kaip naujas onkogenas 33 . „Net1“ baltyme yra katalitinis Dbl homologijos (DH) domenas ir gretimas plekstrino homologijos (PH) domenas, apklijuotas N- ir C-galo plėtiniais 34 . DH ir PH domenai yra būtini prisijungiant prie GTPazės ir stimuliuojant nukleotidų mainų aktyvumą 34 . Kadangi „Net1“ savo N gale turi dvi branduolio lokalizacijos signalo (NLS) sekas, laukinio tipo „Net1“ daugiausia yra 35 branduolyje, tačiau „Net1“ gali patekti į citoplazmą, o tik citoplazminė „Net1“ suaktyvina RhoA ir skatina streso skaidulų susidarymą 36 . Mutacija NLS arba N-galo ištrynimas sąlygojo dalinį Net1 persiskirstymą į citozolį 35, 36 . Todėl „Net1“ branduolinė lokalizacija suteikia potencialų mechanizmą, kaip atskirti GEF nuo RhoA 35 . Tačiau branduolinis „Net1“ taip pat egzistuoja aktyvios formos ir, kaip buvo pranešta, padidina branduolinio RhoB aktyvumą gydant DNR kenkėjais 37, tačiau jo fiziologiniai vaidmenys nėra tiksliai apibrėžti.
 Įrodyta, kad „Net1“ ir „RhoA“ vaidina svarbų vaidmenį įvairiais stuburinių embrionų vystymosi ir organogenezės aspektais. „ Xenopus“ programoje „ Net1“ asocijuojasi su „Disheveled“ ir aktyvina „RhoA“, kad sureguliuotų 38 skrandžio veiklą. Embrionuose, auginančiuose viščiukus, mažinant Net1 arba RhoA ekspresiją epiblastinėse ląstelėse prieš epitelio pasikeitimą į mezenchiminį perėjimą (EMT), susidaro įėjimo ir migracijos defektai 39 . Pelių embrionuose net1 turinčios pieno liaukos pasižymi sumažėjusiu miozino lengvosios grandinės ir miozino lengvosios grandinės fosfatazės reguliuojamųjų subvienetų RhoA sukeltu fosforilinimu, dėl kurio sumažėja ir neorganizuotas latakų išsišakojimas 40 . Be to, nustatyta, kad zebrafish, RhoA kontroliuoja konvergenciją ir pailgėjimą (CE) skrandžio metu 41 . Tačiau Net1 vystymosi vaidmuo niekada nebuvo įvertintas zebrafish embriono vystymuisi. Nors mūsų ankstesnis tyrimas parodė, kad net1 yra išreikštas prieš-dorsaliniu zebrafish embriono 32 organizatoriumi, vis dar reikia išsiaiškinti, kokiu būdu Net1 pasireiškia pasroviui.
 Čia parodome, kad „Net1“ reguliuoja β-katenino fosforilinimą ties S675, o tai yra būtina β-katenino transkripcijos, sukeliančios nugaros ląstelių liktelius, indukcijai pasroviui. „Net1“ veikia prieš PAK1, kad skatintų β-katenino fosforilinimąsi ankstyvojo embriono vystymosi metu. Tiksliau, mes parodome, kad „Net1“ per neidentifikuotą GTPazę disocijuoja ir aktyvuoja PAK1 dimerus, kurie savo ruožtu fosforilina β-kateniną S675 vietoje. Todėl mes pateikiame tiesioginį norminės kaskados, susidedančios iš „Net1-GTPase-PAK1“, kontroliuojančios kanoninį Wnt signalizavimą, įrodymą ir parodome, kad β-katenino C-terminis fosforilinimas yra kritinis zebrafish embrionų stuburo vystymosi reikalavimas.
 „Zebrafish net1“ yra būtinas organizatoriaus formavimuisi ir nugarinės likimo specifikacijai
 Buvo nustatyta, kad žinduolių net1 yra specifinis RhoA GEF, kuris yra padidintas daugelyje karcinomų, kad padidintų ląstelių migraciją ir invaziją. 42, 43, 44, 45, 46, tačiau jo funkcija embriono vystymosi metu nėra iki galo aprašyta. „ Zebrafish net1“ , kuris koduoja baltymą, turintį panašią domeno struktūrą ir turinčią didelę seką su savo žmogaus ir pelės ortologų (atitinkamai 57, 17% ir 57, 66%), pirmą kartą buvo identifikuotas kaip dorsaliai išreikštas genas, skrandžio pradžioje 32 . Viso RNR hibridizavimas in situ atskleidė, kad zebrafish net1 transkripcijos motinos stadijose nebuvo ir pirmą kartą buvo aptiktas dorsoventraliniame gradiente blastodermos krašte, esant didžiausiam jo nugariniam kraštui, esant 30% epibolijos stadijai (1A pav.). Skydo etape „ net1“ išraiška tapo labiau apribota nugaros organizatoriumi ir išlaikė žemesnį lygį šoninėje paraštėje (1A pav.). Remiantis organizuotojo išraiška, net1 ryškiausiai buvo išreikštas ašinėje mezodermoje vidurinės gastrikacijos stadijoje (75% epibolija; papildoma informacija, S1A pav.). Įdomu tai, kad segmentuojant somitogenezę, net1 išraiškos domenas pereina į presomitinę mezodermą (papildoma informacija, S1A pav.). Be to, Western blot analizė atskleidė, kad Net1 baltymo motinos stadijoje nebuvo, tačiau jis buvo pradėtas ekspresuoti prieš skrandį (1B pav.). Naudodami jautresnę aptikimo sistemą, pusiau kiekybinę RT-PGR, mes nustatėme, kad zebrafish embrionai pradėjo ekspresuoti net1 transkriptą aplink MBT (1K ląstelių stadija; 1C pav.). Šie rezultatai rodo, kad net1 yra ankstyvasis zigotinis genas, kuris gali turėti reikšmės organizmo formavime ir nugarinės lemties specifikacijoje.
 „Zebrafish Net1“ yra teigiamas nugaros ašies formavimosi moduliatorius. (A) „ Net1“ išraiškos šoniniai vaizdai nurodytuose etapuose. (B) Net1 baltymo laikino išraiškos profilio viso embriono lizatuose Western blot analizė. Tubulinas buvo naudojamas kaip įkrovos kontrolė. (C) Pusiau kiekybinė „ net1“ transkripto profilio RT-PGR analizė esant kaltinamoms stadijoms. Β-aktino ekspresija buvo naudojama kaip vidinė kontrolė. (D ir E) Dorsalinių žymenų genų boz , chd ir gsc ekspresija cMO ir net1 MO1 injekuotuose embrionuose sferos ir skydo stadijose. (D) Šoniniai vaizdai su gyvūnų stulpu viršuje. (E) Dorsaliniai vaizdai, kurių viršuje yra gyvūnų stulpas. Nurodyti paveiktų embrionų santykiai. (F) Dorsalinio neuroektoderminio žymeklio geno sox3 ir ventralinio neneralinio ektoderminio žymeklio geno gata2 raiškos analizė 75% epibolijos stadijoje. Šoniniai vaizdai, nugarinė pusė nukreipta į dešinę. (G) Neuroninio ektoderminio žymeklio geno bmp2b raiškos modeliai net1 morfotuose skydo stadijoje. Šoniniai vaizdai su nugarine puse į dešinę viršutinėse plokštėse; gyvūnų vaizdai su nugarine puse į dešinę apatinėse plokštėse. (H) Tinklo 1 ekspresijos zebra žuvyse schema. Embrionams buvo įšvirkšta 200 pg „ Flag-net1“ mRNR ir 1 ng „AS-Flag-photo-MO“ („ net1 2 mix“) vienos ląstelės stadijoje, po to veikiami ultravioletiniu spinduliu 64 ląstelių stadijoje 10 min. (I) boz ir chd išraiška (gyvūnų vaizdas su nugarine puse į dešinę) sferos stadijoje embrionuose, įpuršktuose net1 2mix. (J) Per didelis „ net1“ ekspresija išgelbėja „ net1“ morfolių nugarinius defektus. Iš viso į embrionus buvo įšvirkšta 4 ng net1 MO1 kartu su 200 pg Flag-net1 mRNR ir 1 ng AS-Flag-photo-MO ( net1 3 mix ), o grupė net1 3 mix įšvirkštų embrionų buvo veikiami UV. Boz ir chd raiška buvo ištirta hibridizacijos in situ srityje sferoje. (D ', E', I ' ir J') Nurodytų žymenų genų ekspresijos lygiai buvo atskirai tiriami qRT-PGR. Β-aktino ekspresijos lygiai buvo naudojami kaip atskaitos taškas normalizuojant mRNR kiekį kiekviename mėginyje. Žvaigždutės rodo statistinį skirtumo reikšmingumą (* P <0, 05, ** P <0, 01, ** P <0, 001; n = 3). Klaidų juostos nurodytos SD.
 Kadangi net1 yra vienas iš anksčiau nustatytų Nodal / Smad2 taikinių genų prieš pradedant skrandį 32, mes toliau tyrėme , ar net1 išraišką ties nugaros blastodermos kraštu reguliuoja Nodal signalizacija. Kaip parodyta papildomoje informacijoje, S1B paveiksle, net1 ekspresija buvo panaikinta mazgais neturinčiuose MZ oep mutantuose 30 %epibolijos ir skydo stadijose. Priešingai, „ net1“ nugaros raiškos sritis buvo smarkiai išplėsta Nodal ligando skint ( mq ) mRNR sušvirkštuose embrionuose (papildoma informacija, S1C pav.). Be to, akivaizdžiai sumažėjo net1 ekspresija MZ oep mutantuose , kuriuos atskleidė pusiau kiekybinis RT-PGR rutulio stadijoje, tai yra būtinas laikotarpis, per kurį nustatomos nugaros ląstelių likimo savybės (papildoma informacija, S1D pav.). Šie rezultatai rodo, kad mazgo signalas suaktyvina net1 išraišką ikikariniame organizme .
 Norėdami ištirti net1 vystymosi funkciją, pirmiausia panaudojome morfolino pagrindu sukurtą genų numušimo technologiją, kad sutrikdyčiau net1 ekspresiją laukinio tipo zebrafish embrionuose. Suprojektuoti ir susintetinti du sujungimus blokuojantys morfolinai (MO1 ir MO2), nukreipti į skirtingas net1 pre-mRNR eksonono ir introno ribas. Palyginus su neatitinkančiomis kontrolinės morfolino (cMO) injekcijomis, 4 ng arba MO1, arba MO2 injekcija efektyviai blokavo endogeninės net1 mRNR ir baltymo ekspresiją, o morfotuose buvo aptikti tik trukdantys mRNR produktai (papildoma informacija, S2A ir S2B paveikslai). ). Šie rezultatai rodo „ net1 MO“ specifiškumą ir efektyvumą. Embrionai, įpurškiami skirtingais kiekiais MO1, turėjo ryškių nugaros defektų, priklausomų nuo dozės, kūno plano formavimo metu, įskaitant mažesnį organizmą skydo stadijoje, kintamą sumažintą galvos dydį ir sutrumpintą kūno ašį esant 24 AG / h (valandos po apvaisinimo; papildoma informacija, S2C pav.). Taip pat MO2 įpurškimas sąlygojo panašius nugaros ašies formavimosi defektus (papildoma informacija, S2D pav.).
 Be morfologinių net1 morfologinių defektų, rutulio ir skydo stadijose reikšmingai sumažėjo nugaros žymeklių, tokių kaip boz , chd ir goosecoid ( gsc ), išraiška (1D-1E paveikslas ir papildoma informacija, S2E paveikslas). Tačiau daugelio ankstyvųjų zigotinių genų, įskaitant BI891768 , apoeb , lrwd1 ir sox11a , kurie yra plačiai ekspresuojami visoje blastodermoje po MBT 47, raiška akivaizdžiai nepasikeitė net1- MO1 įšvirkštuose embrionuose (papildoma informacija, S2F pav.), siūlantys esminę ir specifinę net1 funkciją zebrafish nugaros likimo specifikacijose. Be to, „ net1“ morfomanai parodė daug mažesnę nugaros neuroektodermą (kaip rodo „ Sox3“ išraiška), kurią paprastai sukelia organizatoriaus signalai, taip pat išsiplėtusi veninė neneurinė ektoderma (kaip rodo „ gata2“ ir „ bmp2b“ išraiška; 1F ir 1G pav.) ). Norėdami atmesti galimybę, kad sumažėjęs nugaros žymeklio genų ekspresija net1 morfotuose atsirado dėl MO injekcijos sukeltų globalių ląstelių proliferacijos ar išgyvenimo defektų, mes ištyrėme, ar tarp cMO ir MO1 įšvirkštų embrionų yra skirtumų tarp ląstelių proliferacijos ar apoptozės. Imuninis dažymas fosforilintam H3 histonui parodė panašų ryškų ląstelių proliferaciją net1 morfotuose, palyginti su kontroliniais embrionais skrandžio metu (papildoma informacija, S3A pav.). Nepaisant akivaizdaus TUNEL tyrimo metu aptiktos ląstelių apoptozės, kai net1 morfofitai buvo 24 AG / ff, nė vienas akivaizdus apoptozės signalas nebuvo pastebėtas nei cMO, nei MO1 įšvirkštuose embrionuose gastrulės stadijose (papildoma informacija, S3B pav.). Nuosekliai, p53 mutantiniai embrionai, įpurškiami MO1, turėjo aiškių morfologinių nugaros defektų ir sumažėjusią nugaros žymeklio išraišką, toliau pašalindami galimą painų morfolino sukeltos p53 aktyvacijos ir vėlesnės ląstelių žūties indėlį (papildoma informacija, S3C ir S3D paveikslai). Be to, su „dorsal“ susijusių audinių žymenų genų, įskaitant prechordalinį plokštelinį žymeklį gsc , priekinio smegenų žymeklį six3 ir notochord žymeklį ntl, ekspresija smarkiai sumažėjo net1 morfotuose pumpurų ir segmentacijos etapuose (papildoma informacija, S4A ir S4B paveikslai), leidžiant manyti, kad šie nugaros defektus sukelia ne MO injekcijos sukeltas vystymosi uždelsimas. Siekiant pašalinti galimą MO injekcijos poveikį ne pagal tikslą, buvo tiriama siRNR pagrindu sukurta genų nutildymo strategija, kuri, kaip įrodyta, yra alternatyvi specifinio genų ekspresijos numušimo zebrafish 48, 49, 50 metodika . net1 . Sukūrėme ir susintetinome dvi siRNR (siRNR1 ir siRNR2), nukreipdami skirtingas zebrafish net1 mRNR sekas. Įšvirkštus siRNA1 arba siRNA2 į embrionus, dramatiškai sumažėjo endogeninės net1 ekspresija ir atsirado nugaros defektai esant 24 AG / h (papildoma informacija, S5A ir S5B paveikslai). Tolesnės analizės atskleidė, kad net1 siRNR sušvirkštuose embrionuose buvo smarkiai sutrikdytas nugaros likimas (papildoma informacija, S5C pav.). Šie stebėjimai rodo, kad net1 reikalingas organizmo formavimui ir dorsalinių ląstelių likimo nustatymui zebrafish embrionuose.
 Toliau paklausėme, ar per didelis tinklo1 ekspresija gali paskatinti nugaros ašies formavimąsi. Kadangi labai mažo net1 mRNR (10 pg) kiekio sušvirkštimas į 1 ląstelių stadijos embrionus sukėlė didelius blastodermos apsigimimus ir labai ankstyvą embriono mirtingumą, mes panaudojome antisense nuotrauka skaidomą morfoliną, nukreiptą į N-galo vėliavos seką. „Flag-net1“ mRNR (AS-Flag-photo-MO), kad būtų užblokuotas jo ankstyvasis vertimas. Embrionai buvo įšvirkšti mišiniu, kuriame yra Flag-net1 mRNR ir AS-Flag-photo-MO ( net1 2 mix) 1 ląstelių stadijoje, veikiami UV 64 ląstelių stadijoje, po to surinkti sferos stadijoje fenotipo analizei atlikti (pav. 1H). Embrionai, ekstensyviai eksploatuojantys net1, parodė padidėjusias nugaros žymeklių boz ir chd ekspresijos sritis, palyginti su laukinio tipo kontrolinėmis medžiagomis arba embrionais, kuriems buvo įšvirkštas net1 2 mix be UV spindulių poveikio (1I ir 1I paveikslai), ir tai rodo, kad net1 vaidina teigiamą vaidmenį formuojant nugaros ašį. Svarbu tai, kad net1 morfolių nugarinės likimo specifikacijos trūkumus buvo galima ištaisyti pervertinant net1 (1J ir 1J paveikslai), dar labiau nurodant net1 MO specifiškumą. Panašiai, sumažėjus prechordalinio plokštelinio žymeklio gsc ir priekinių smegenų žymeklio šeštojo3 raiška pumpurų stadijos morfomanams, taip pat buvo atstatyta naudojant net1 perraišką (papildoma informacija, S6 pav.). Visi šie rezultatai rodo, kad net1 veikia kaip zigotinis faktorius, skatinantis ankstyvojo dorsalinio signalo centro formavimąsi ir dorsalinės ašies sukūrimą zebrafish embriono vystymosi metu.
 „Net1“ pagerina Wnt / β-katenino signalizaciją
 Kanoninis Wnt signalizavimas yra būtinas, norint suformuoti nugaros organizatorius ir nustatyti stuburo ir ventralinės stuburo dalies vidurius 2, 4, 14 . Faktas, kad net1 yra būtinas norint nustatyti nugaros ląstelių likimus ir išreikšti boz - tiesioginį motinos Wnt / β-katenino signalizacijos 51 tikslą, rodo, kad jis gali būti svarbus Wnt / β-katenino signalo reguliatorius. Norėdami patikrinti savo hipotezę, pirmiausia paklausėme, ar tbx6 ir cdx4 , dviejų tiesioginių zigotinio Wnt / β- katenino 52, 53, 54 taikinių, išraišką taip pat reguliuoja net1 . Kaip parodyta 2A paveiksle, skydo stadijoje tbx6 ir cdx4 yra specifiškai ekspresuojami ventrolateraliniame mezodermoje, kur net1 yra silpnai išreikštas, o jų ekspresija buvo žymiai sumažinta įpurškiant net1 MO1. Remiantis šiais rezultatais, net1 panaikinimas nutraukė Wnt sukeltą gfp reporterio ekspresiją TOPdGFP transgeninių embrionų 55 dorsaliniame organizme ir ventrolateriniame mezoderme (2B paveikslas), rodantis, kad net1 yra būtinas tiek motinos, tiek zigotiniam Wnt / β-katenino signalizavimui. veikla. Be to, sumažėjusi nugaros žymeklio genų boz ir chd raiška net1 morfotuose buvo atkurta į normalų lygį, kartu sušvirkščiant wnt8a arba ΔN - β-katenino mRNR (2C pav.). Šie duomenys rodo, kad zebrafish net1 vaidina pagrindinį vaidmenį motinos Wnt / β-katenino signalo perdavime ankstyvojo embriono vystymosi metu. Be to, kadangi zygotinis Wnt / β-katenino signalas reikalingas ventralinių audinių 56 vystymuisi, kartu sukeliantis zigotinio Wnt / β-katenino signalo aktyvumo sumažėjimą ventrolaterinėje mezodermoje net1 morfotuose greičiausiai lėmė tai, kad nėra per daug uodegos audinių, kurie dažnai stebimi embrionuose, turinčiuose nugaros defektus (papildoma informacija, S2C ir S2D paveikslai).
 „Net1“ skatina Wnt / β-katenino signalizaciją zebrafish embrionuose ir žinduolių ląstelėse. (A) Zigotinio Wnt / β- katenino tikslinio geno tbx6 ir cdx4 raiška 4 ng cMO arba net1 MO1 injekuotuose embrionuose skydo stadijoje. Šoniniai vaizdai su gyvūnų stulpu viršuje. (B) Visavertis hibridizavimas in situ parodo sumažintą GFP raišką net1 MO1 injekuotų TOPdGFP reporterių transgeninių zebrafish embrionų embrionuose. Embrionams buvo įšvirkšta 4 ng cMO arba net1 MO1 vienos ląstelės stadijoje ir buvo paimti skydo stadijoje hibridizavimui in situ . Gyvūnų vaizdai su nugarine puse į dešinę. (C) Per didelis wnt8a arba ΔN-β- katenino ekspresija išgelbėja stuburo vystymosi defektus net1 morfotuose. Embrionai buvo įšvirkšti nurodytais MO arba mRNR vienos ląstelės stadijoje ir surinkti skydo stadijoje, kad būtų galima hibridizuoti in situ su boz ir chd zondais. Injekcijos dozės: cMO, 4 ng; net1 MO1, 4 ng; wnt8a mRNR, 10 pg; ΔN-β-katenino mRNR, 100 psl. (D) Net1 ekspresija HEK293T ląstelėse padidina super-TOPFlash luciferazės aktyvumą, reaguojant į Wnt3a. HEK293T ląstelės, kartu transfekuotos su super-TOPFlash luciferazės reporteriu, ir vis didesnis net1 ekspresijos plazmidžių kiekis 12 valandų buvo gydomos Wnt3a CM arba be jo, po to surinktos luciferazės tyrimams. ** P <0, 01; *** P <0, 001, studento t- testas. (E) net1 shRNR konstrukcijos efektyviai numuša endogeninę žmogaus net1 raišką. HEK293T ląstelės buvo transfekuotos nurodytomis shRNR plazmidėmis ir surinktos 48 valandas po transfekcijos, kad būtų galima atlikti Western blot analizę. (F ir G) net1 numušimas HEK293T (F) ir MCF-7 ląstelėse (G) sumažina Wnt3a sukeltą super-TOPFlash luciferazės aktyvumą. ** P <0, 01; *** P <0, 001, studento t- testas. (H) net1 numušimas HEK293T ląstelėse slopina Axin2, c-myc ir LEF1 raišką. Nurodytomis plazmidėmis transfekuotos HEK293T ląstelės 12 valandų buvo gydomos Wnt3a CM arba be jo, ir surinktos qRT-PGR analizei. * P <0, 05; ** P <0, 01, studento t- testas.
 Norint nustatyti, ar net1 yra grįžtamojo ryšio modifikatorius Wnt / β-katenino signalizavimui, buvo ištirta zebrafish net1 raiška embrionuose, turinčiuose nepakankamą arba per didelį Wnt kiekį. Kaip parodyta papildomoje informacijoje, S7A pav., Wnt8a arba ΔN - β - katenino mRNR (koduojančios konstituciškai aktyvią β- katenino formą) perdėta ekspresija buvo pakankama, kad sukeltų negimdinę chd išraišką , o Wnt / β-katenino signalo slopinimas injekcijomis ΔN - tcf3 mRNR (koduojanti dominuojančią neigiamą Tcf3 formą) sumažino chd ekspresiją. Keista, bet net1 raiškos nepakeitė nei Wnt / β-katenino signalizacijos suaktyvėjimas ar slopinimas zebrafish embrionuose (papildoma informacija, S7B pav.).
 Toliau mes ištyrėme Net1 vaidmenį reguliuojant kanoninį Wnt kelią žinduolių ląstelėse. Per didelis Net1 ekspresija HEK293T ląstelėse žymiai padidino Wnt3a sukeltą super-TOPFlash luciferazės reporterio aktyvumą priklausomai nuo dozės (2D paveikslas). Norėdami išsamiau ištirti endogeninio žinduolio Net1 funkciją, išanalizavome net1 numušimo įtaką β-katenino / TCF priklausomai transkripcijai. Mes sukūrėme dvi trumpų plaukų smeigtukų RNR (shRNR, pavadintos net1 shRNA1 ir net1 shRNA2) prieš du skirtingus žmogaus net1 regionus , kurie abu veiksmingai slopino endogeninio Net1 baltymo ekspresiją (2E pav.). HEK293T ir MCF-7 ląstelių transfekcija šiomis SHRNR sąlygojo reikšmingą luciferazės aktyvumo sumažėjimą, palyginti su kontrole (2F ir 2G paveikslai). Šis poveikis buvo dar kartą patvirtintas atliekant qRT-PGR analizę, kuri atskleidė žymiai sumažintą Wnt / β- katenino tikslinių genų Axin2 , c-myc ir LEF1 raišką net1-shRNR transfekuotose HEK293T ląstelėse (2H pav.). Visi šie rezultatai rodo, kad „Net1“ turi išsaugotą funkciją stiprinant Wnt / β-katenino aktyvumą tarp skirtingų stuburinių rūšių.
 „Net1“ reikalauja GEF aktyvumo, kad būtų reguliuojamas Wnt / β-katenino signalizavimas ir nugaros ašies formavimasis
 „Net1“ yra sudėtingas baltymas, turintis trumpą C-galinį domeną, be katalizinio DH domeno ir gretimo PH domeno, turinčio bendrą PDZ jungimosi motyvą. Buvo sukonstruota daugybė Net1 delecijų ir taškų mutacijų, siekiant nustatyti, kurie domenai buvo svarbūs jo tarpląstelinei lokalizacijai ir GEF aktyvumui 34, 36, 57 . Norėdami nustatyti, kurie Net1 domenai yra svarbūs Wnt / β-catenino signalizavimui, sukūrėme zebrafish delecijos mutantą, pavadintą Net1-ΔC4, kuriame buvo išbrauktos keturios paskutinės aminorūgštys, turinčios PDZ-surišimo motyvą 57, taip pat du nuostoliai. -funkciniai mutantai, žymimi Net1-L266E ir Net1-W437L, kuriuose yra taškinės mutacijos atitinkamai DH ir PH domenuose, dėl ko prarandamas GEF aktyvumas 34 . HEK293T ląstelės buvo transfekuotos šiomis konstrukcijomis, esant arba be Wnt3a, siekiant nustatyti poveikį Wnt signalizacijai naudojant super-TOPFlash luciferazės reporterį. Mes nustatėme, kad luciferazės aktyvumas smarkiai padidėjo dėl tiek Net1-ΔC4, tiek laukinio tipo Net1 ekspresijos. Tai rodo, kad Net1 PDZ rišantis motyvas nėra būtinas Wnt / β-katenino signalizacijos aktyvavimui (3A pav.). Priešingai, GEF mutantai Net1-L266E ir Net1-W437L buvo ekspresuojami panašiu lygiu kaip laukinio tipo Net1, tačiau nebegalėjo stimuliuoti Wnt3a sukeltos luciferazės aktyvumo (3B pav.). Be to, reporterio aktyvumą net1 shRNA1 transfekuotose HEK293T ląstelėse išgelbėjo perdėta laukinio tipo zebrafish Net1 ekspresija, tačiau nei GEF mutanto L266E, nei W437L ekspresija neturėjo (3C pav.). Sutikus su šiais rezultatais, net1 MO1 injekcija kartu su net1-L266E arba net1-W437L mRNR, palyginti su laukinio tipo net1 mRNR, negalėjo išgelbėti sumažėjusios nugaros žymeklių boz ir chd ekspresijos net1 morfotuose (3D paveikslas). . Be to, per didelis laukinio tipo net1, bet ne net1 GEF mutantų ekspresija išplėtė chd ir boz raiškos sritis (papildoma informacija, S8 pav.). Remdamiesi šiais stebėjimais, mes padarėme išvadą, kad GEF veikla yra būtina Net1 tarpininkaujant reguliuoti kanoninę Wnt signalizaciją ir nugarinę likimo specifikaciją.
 Tiek citoplazminė, tiek branduolinė Net1 savo GEF aktyvumu reguliuoja kanoninį Wnt signalizavimą. (AC) Laukinio tipo zebrafish Net1 arba Net1-CC4, bet ne jo mutantų, kuriuose nėra GEF aktyvumo, per didelis ekspresija padidina Wnt / β-katenino signalo perdavimą (A ir B) ir atkuria santykinį super-TOPFlash reporterių luciferazės aktyvumą tinkle1 - išeikvotos HEK293T ląstelės (C) . HEK293T ląstelės, kotransfekuotos super-TOPFlash, ir nurodytos plazmidės 12 valandų buvo gydomos Wnt3a CM arba be jo, ir surinktos luciferazės tyrimams. Laukinio tipo Net1 ir įvairių jo mutantų ekspresijos lygis buvo tiriamas Western blot'ais, kurie buvo parodyti apatinėse A ir B lentelėse. ** P <0, 01; *** P <0, 001, studento t- testas. (D) Zebrafish embrionai buvo sušvirkšti kartu su nurodytais MO ir mRNR vienos ląstelės stadijoje ir, kaip nurodyta, veikiami UV spinduliais. Visi embrionai buvo surinkti sferos stadijoje, kad būtų galima hibridizuoti in situ . net1 3mix , 4 ng net1 MO1 kartu su 200 pg Flag-net1 mRNR ir 1 ng AS-Flag-photo-MO; L266E 3 mix , 4 ng net1 MO1 kartu su 200 pg Flag-net1-L266E mRNR ir 1 ng AS-Flag-photo-MO; „W437L 3mix“ , 4 ng net1 MO1 kartu su 200 pg Flag-net1-W437L mRNR ir 1 ng AS-Flag-photo-MO. Atminkite, kad laukinio tipo Net1 ekspresija, veikiama ultravioletiniais spinduliais, bet ne dėl to, kad ji praranda funkciją, galėtų atkurti nugaros žymeklio genų ekspresiją net1 morfotuose. (E) Hela ląstelės buvo transfekuotos naudojant Flag-NES-ΔN-Net1 arba Flag-NLS-Net1. Ląstelės buvo fiksuotos, imuniniu būdu pritvirtintos anti-Flag antikūnais ir priešpriešiais padengtos DAPI, kad būtų parodyta šių Net1 mutantų ląstelių lokalinė vieta. Svarstyklės, 10 μm. (FI) HEK293T ląstelės buvo transfekuotos nurodytomis plazmidėmis ir apdorotos Wnt3a CM arba be jo prieš imant derlių luciferazės tyrimams. Atminkite, kad super-TOPFlash reporterio Wnt3a sukeltą atsaką sustiprino ir citoplazma, ir branduolinis Net1 (F) . Citoplazminio ir branduolinio Net1 L266E ir W437L mutantai, kuriems trūksta GEF aktyvumo, prarado gebėjimą skatinti Wnt / β-katenino aktyvumą (G ir H) . Bet koks citoplazminis ar branduolinis Net1 gali atkurti santykinį luciferazės reporterio aktyvumą net1 ląstelėse, kurių išeikvotos (I) . * P <0, 05; ** P <0, 01; *** P <0, 001, studento t- testas. (J) Net1 morfolių nugaros raidos defektus blokuoja citoplazminė ar branduolinė Net1 ekspresija. Embrionuose , įpuršktuose nurodytais MO ir mRNR, sferos stadijoje buvo tiriama bozo ir chd raiška. NES-ΔN 3 mišinys , 4 ng net1 MO1 kartu su 200 pg vėliava-NES-ΔN-net1 mRNR ir 1 ng AS-vėliava-nuotrauka-MO; NLS 3 mix , 4 ng net1 MO1 kartu su 200 pg Flag-NLS-net1 mRNR ir 1 ng AS-Flag-photo-MO.
 Atsižvelgiant į tai, kad Net1 baltymas daugiausia lokalizuojasi branduolyje ir kad Net1 perkėlimas į citoplazmą yra labai svarbus jo funkcijai, aktyvinant RhoA, siekiant skatinti streso pluošto susidarymą 35, 58, tada mes ištyrėme, ar reikalingas Net1 citoplazminis pasiskirstymas norint reguliuoti Wnt / β -katenino signalizacijos. Mes sukūrėme zebrafish Net1 konstrukcijas, kuriose branduolinio eksporto signalo (NES) seka MAPKK buvo pridėta prie onkogeninės Net1 versijos N galo (ΔN-Net1, kuriai trūko pirmųjų 60 aminorūgščių) ir NLS seka SV40 dideliam T -antigenas buvo pridėtas prie laukinio tipo Net1 N galo. Kaip ir tikėtasi, NES-ΔN-Net1 buvo lokalizuotas išskirtinai citoplazmoje, tuo tarpu NLS-Net1 buvo apribotas branduoliu (3E pav.). Be to, GEF trūkumų turinčios NES-ΔN-Net1 ir NLS-Net1 mutacijos nepakeitė jų tarpląstelinės lokalizacijos (duomenys nepateikti). Įdomu tai, kad nei NES-ΔN-Net1, nei NLS-Net1 reikšmingai padidino Wnt3a sukeltą luciferazės reporterio aktyvumą (3F pav.), O jų giminingi GEF mutantai (L266E ir W437L) neturėjo (3G ir 3H paveikslai). Furthermore, overexpression of NES-ΔN-Net1 or NLS-Net1 recovered the ability of net1 -depleted cells to respond to Wnt3a stimulation (Figure 3I). Consistent with the results in mammalian cells, defects in dorsal axis formation in net1 morphants were corrected by overexpression of NES-ΔN-net1 or NLS-net1 (Figure 3J). Taken together, these data suggest that both cytoplasmic and nuclear Net1 can promote canonical Wnt signaling and dorsal axis formation in a GEF activity-dependent manner.
 Net1 controls phosphorylation of β-catenin at serine residue 675
 To elucidate the mechanisms underlying net1 function in dorsal development, we performed several experiments to map the position of Net1 activity along the canonical Wnt signaling pathway. We have confirmed that Net1 is a key modulator of Wnt ligand-triggered signal activation (Figure 2F and 2G). We also found that shRNA knockdown of net1 in HEK293T cells significantly attenuated expression of the super-TOPFlash reporter induced by GSK-3β-selective inhibitors, LiCl and 6-bro-moindirubin-3-oxime (BIO) 59 (Supplementary information, Figure S9A and S9B). Moreover, Wnt3a-induced β-catenin nuclear accumulation was not changed in response to Net1 overexpression or depletion in mammalian cells (Supplementary information, Figure S9C-S9E). Likewise, knockdown of net1 in embryos did not result in obvious effects on the cellular distribution of endogenous β-catenin (Supplementary information, Figure S9F). These findings together suggest that Net1 acts in parallel with or downstream of β-catenin.
 Phosphorylation of β-catenin at the C-terminal tyrosine and serine residues has been shown to increase its biological activity 21, 22, 23, 24 . Specifically, the phosphorylation of S675 effectively modulates transcriptional activity of β-catenin in a variety of cells and tissues, but its effect on β-catenin stability is cell type-dependent 22, 23, 60 . We aimed to investigate whether β-catenin S675 phosphorylation is regulated by Net1 expression. In support of this hypothesis, overexpression of Net1 in HEK293T cells enhanced S675 phosphorylation in the presence of Wnt3a (Figure 4A). This increase in S675 phosphorylation relies on the presence of Wnt3a, as overexpression of Net1 alone could not induce S675 phosphorylation (Figure 4A). In addition, Wnt3a stimulation significantly promoted wild-type β-catenin phosphorylation which could not be detected in S675A mutant–the unphosphorylated form of β-catenin (Supplementary information, Figure S10A). These data demonstrate the specificity of the anti-phospho-β-catenin (Ser675) antibody we used in this study and further confirms the requirement of Wnt signal activation in β-catenin S675 phosphorylation.
 Net1-regulated β-catenin S675 phosphorylation is required for zebrafish dorsal axis formation. (A and B) Overexpression of wild-type Net1 (A) but not Net1 loss-of-function mutants (B) results in an increase in β-catenin S675 phosphorylation. HEK293T cells transfected with plasmids encoding Flag-Net1, Flag-Net1-L266E or Flag-Net1-W437L were treated with or without Wnt3a CM. Lysates were analyzed by western blot using the indicated antibodies. (C) Knockdown of net1 leads to a significant decrease in β-catenin S675 phosphorylation. HEK293T cells were transfected with net1 shRNA expressing plasmids and treated with or without Wnt3a CM before immunoblotting. (D) HEK293T cells were co-transfected net1 shRNA and Net1 plasmids, then treated with Wnt3a CM. Total cell lysates were prepared and immunoblotted with the indicated antibodies. Note that only wild-type Net1 can rescue the relative decrease in β-catenin S675 phosphorylation in net1 -depleted cells. (E and F) Western blots of total lysates from wild-type embryos, 4 ng cMO or net1 MO1-injected embryos, and net1 morphants co-injected with 200 pg mRNAs encoding wild-type net1 or net1 mutants. The phosphorylation level of β-catenin S675 is upregulated around MBT (1 K-cell stage) in wild-type embryos (E, left panels), while this relative increase in β-catenin S675 phosphorylation is abolished in net1 morphants (E, right panels). β-cat, β-catenin. Only overexpression of wild-type Net1 can rescue the decrease in β-catenin S675 phosphorylation in net1 morphants (F) . In panel AF, quantification is the relative density of phospho-specific signals to corresponding total protein signals (mean ± SD, three independent biological repeats). (G) The phosphorylation of β-catenin S675 is essential for dorsal gene expression. The expression of boz and chd was examined at the sphere stage in embryos injected with the indicated MOs and mRNAs. Injection dose: β - catenin S675D mRNA, 150 pg; β - catenin S675A mRNA, 400 pg; net1 MO1, 4 ng. (H) Phosphorylated β-catenin has stronger ability to enhance dorsal gene expression. The expression of boz and chd was examined at the sphere stage in embryos injected with the 150 pg indicated mRNAs. (I) Phosphorylated β-catenin induces much earlier expression of boz and chd. The expression of boz and chd was examined at the 512-cells stage and High stage, respectively, in embryos injected with 150 pg indicated mRNAs.
 Consistent with the requirement of Net1 GEF activity in regulating canonical Wnt signal, overexpression of Net1-L266E or Net1-W437L mutants did not increase the phosphorylation level of β-catenin S675 in the presence of Wnt3a (Figure 4B). We further tested the role of endogenous Net1 in regulating β-catenin phosphorylation. Knockdown of net1 expression in HEK293T cells resulted in a significant decrease of basal and Wnt3a-induced phosphorylation levels of β-catenin S675 (Figure 4C). Reintroduction of wild-type Net1, but not its GEF defective mutants, into net1 -depleted cells reversed the reduction of β-catenin S675 phosphorylation (Figure 4D). Thus, Net1 contributes to β-catenin S675 phosphorylation.
 We subsequently addressed the biological functions of Net1-regulated β-catenin S675 phosphorylation during zebrafish dorsal axis formation. Endogenous β-catenin in zebrafish embryos was phosphorylated at S675 at a very low level before the 1K-cell stage, but this phosphorylation significantly increased during the MBT (Figure 4E). Phosphorylation of β-catenin S675 was almost abolished in net1 morphants compared with wild-type control (Figure 4E), suggesting that net1 is the major positive modulator of β-catenin S675 phosphorylation during MBT. Injection of 200 pg mRNA of wild-type net1 , but not its loss-of-function mutants, could restore phosphorylation level of β-catenin at S675 in net1 MO1-injected embryos (Figure 4F), indicating the requirement for Net1 GEF activity for β-catenin S675 phosphorylation. To verify the functional relevance of S675 phosphorylation in dorsal axis formation, we overexpressed β-catenin S675D and S675A mutants, which respectively mimic phosphorylated and unphosphorylated forms of the protein and retain the same cellular localization as the wild-type 23 . In situ hybridization assays revealed that only injection of β-catenin S675D mRNA could significantly expand the expression domain of boz and chd in wild-type embryos (Figure 4G). Furthermore, β-catenin S675D mRNA also recovered the expression defects of these dorsal marker genes in net1 morphants (Figure 4G). Injection of β-catenin S675D mRNA induced much more expanded expression domains of boz and chd compared with wild-type β-catenin overexpression, and most importantly, resulted in a much earlier expression of these dorsal marker genes at 512-cell stage or high stage when their transcripts could not be detected in wild-type embryos or wild-type β-catenin mRNA-injected embryos (Figure 4H and 4I). Taken together, β-catenin S675 phosphorylation is required for dorsal gene expression, and net1 reinforces dorsal development by increasing the phosphorylation of β-catenin at residue S675.
 Since Nodal signal is require for the pre-dorsal organizer expression of net1 , we next examined whether the β-catenin S675 phosphorylation is decreased in Nodal-deficient embryos. Interestingly, MZ oep mutants exhibited similar phosphorylation level of β-catenin S675 as is in wild-type embryos at sphere and 30% epiboly stages (Supplementary information, Figure S10B). As Smad4-deficient HCT116 cells show elevated PAK1 expression 61, we speculate that the expression of some Nodal-suppressed genes might be upregulated to maintain β-catenin phosphorylation in MZ oep mutants.
 Net1 acts upstream of PAK1 to regulate β-catenin phosphorylation
 Since S675 of β-catenin has been shown to be a major target of PAK1 and protein kinase A (PKA) 22, 23, we first examined the potential involvement of these two kinases during dorsal axis formation in zebrafish. Embryos injected with the mRNA encoding a specific inhibitory protein for PKA (PKI) 22, 62 or treated with the PKA selective chemical inhibitor H89 63 exhibited normal expression of the dorsal markers and the phospho-β-catenin (S675; Supplementary information, Figure S11A and S11B), thus excluding a major role for PKA in zebrafish dorsalization. These results are in good agreement with previous reports that the level of PKA activity in the dorsal region is lower than that in the ventral region from late blastula to gastrula stages, and PKA modulates morphogenetic movement but not cell fate specification during Xenopus gastrulation 64 . However, when embryos were treated with IPA-3, a selective non-ATP competitive PAK1 inhibitor, or injected with pak1 K299A mRNA, a dominant negative PAK1 mutant ( dnpak1 ) 23, expression of dorsal markers and phospho-β-catenin (S675) was dramatically reduced (Figure 5A and Supplementary information, Figure S11C). Furthermore, overexpression of pak1 T423E , a constitutively active form of PAK1 ( capak1 ) 23, expanded the dorsal marker expression domains in wild-type embryos, and also rescued the phenotypes resulting from net1 -depletion in morphants (Figure 5A and 5B). To explore whether the activity of PAK1 is differentially activated in the dorsal and ventral regions of the embryos after MBT, gsc:GFP transgenic fish embryos were injected with pak1 mRNA (encoding Flag-PAK1) at 1-cell stage, and cut into dorsal and ventral halves with a microblade at 30% epiboly stage. Expression of GFP, and phosphorylation of PAK1 Thr423 which occurs in activated PAK1 65, were only detected in the dorsal regions of zebrafish embryos, indicating that embryos were correctly dissected into ventral and dorsal halves and PAK1 is primarily activated in the dorsal blastomeres (Figure 5C). Importantly, net1 MO1 injection strongly repressed PAK1 phosphorylation in zebrafish embryos (Figure 5C). Likewise, injection of the mRNA of capak1 , but not dnpak1 , into net1 morphants derepressed the phosphorylation of β-catenin S675 (Figure 5D). Thus, PAK1 activity appears to be required for dorsal axis formation, and Net1 likely promotes the C-terminal phosphorylation of β-catenin by activating PAK1 in the dorsal blastomeres.
 Net1 controls β-catenin S675 phosphorylation via PAK1. (A) Expression patterns of boz and chd in sphere stage embryos treated with 10 μM IPA-3 or embryos injected with 225 pg dnpak1 or 300 pg capak1 mRNA. (B) Expression patterns of boz and chd in sphere stage embryos co-injected with 4 ng net1 MO1 and 300 pg capak1 mRNA. Note that overexpression of capak1 restored the expression of dorsal marker genes in net1 morphants. (C) net1 is essential for PAK1 activition in dorsal region. gsc:GFP transgenic embryos injected with 200 pg Flag-PAK1 mRNA and indicated MOs were cut into dorsal and ventral halves under fluorescence microscope. The dissected tissues (left panel) or whole embryos (right panel) were lysed with TNE at 30% epiboly stage. Then lysates were subjected to immunoprecipitation and western blot analysis with the indicated antibodies. (D) Western blots of total lysates from sphere-stage embryos injected with the indicated MOs and mRNAs. net1 MO1, 4 ng; capak1 mRNA, 300 pg; dnpak1 mRNA, 225 pg. (EG) HEK293T cells were transfected with the indicated plasmids and treated with or without Wnt3a CM before being harvested for luciferase assays. The Wnt3a-induced response of the super-TOPFlash reporter was enhanced by CAPAK1 and inhibited by DNPAK1 (E) . Forced expression of DNPAK1 inhibited the ability of net1 to enhance Wnt3a-induced luciferase activity (F), but overexpression of CAPAK1 significantly increased Wnt/β-catenin signal transduction in net1 -depleted cells (G) . NS, non significant; * P <0, 05; ** P <0, 01; *** P < 0.001, Student's t -test. (H) Western blot analysis showing reduced p-PAK1 in HEK293T cells transfected with plasmids expressing net1 shRNA1 or shRNA2 for 48 h. (IK) Cell lysates from HEK293T cells co-transfected with the indicated plasmids were subjected to immunoprecipitation with anti-Flag M2 agarose beads. Total lysates and IPs were subjected to western blot analysis with the indicated antibodies. Note that overexpression of wild-type Net1, but not its loss-of-function mutants, strongly blocked the interaction between β-catenin and HDAC1 (I) . In D and I, quantification is the relative density of phospho-specific signals to corresponding total protein signals, while in H, quantification is the mean relative ratio of co-immunoprecipitated signals over lysate (mean±SD, three independent biological repeats).
 We next examined the epistatic relationship between Net1 and PAK1 in modulating canonical Wnt signaling. Overexpression of CAPAK1 increased the luciferase activity of the super-TOPFlash reporter by approximately threefold over the control in response to Wnt3a stimulation, whereas DNPAK1 caused an approximately 50% decrease in reporter activity (Figure 5E), suggesting that PAK1 is a positive regulator of canonical Wnt signaling as previously reported 23 . Importantly, co-expression of CAPAK1 with Net1 further increased luciferase reporter activity, while disruption of endogenous PAK1 by forced expression of DNPAK1 inhibited the ability of Net1 to enhance Wnt3a-induced luciferase activity (Figure 5F). However, overexpression of CAPAK1, but not DNPAK1, still dramatically induced Wnt/β-catenin signal transduction in net1 -depleted cells (Figure 5G). In addition, the expression of phosphorylated PAK1 (Thr423) was abolished in net1 shRNA transfected cells (Figure 5H). PAK1 has been reported to promote β-catenin transcriptional activity by inhibiting the association of β-catenin and its general transcriptional repressor HDAC1 23, 66 . Consistent with these previous reports, wild-type Net1, but not its GEF mutants, could strongly block β-catenin/HDAC1 interaction, but had no effects on β-catenin/LEF1/TCF4 association (Figure 5I-5K). These results suggest that Net1 acts upstream of PAK1 to regulate β-catenin phosphorylation and ultimately Wnt-responsive gene transcription.
 Net1 dissociates and activates PAK1 dimers to promote β-catenin phosphorylation
 After establishing the functional significance of the Net1-PAK1 cascade in promoting β-catenin activation during zebrafish dorsal development, we aimed to elucidate the mechanism by which Net1 enhances PAK1 activation in mammalian cells and zebrafish embryos. Co-immunoprecipitation experiments were first performed to examine relative binding affinity. The results indicated that Myc-tagged or endogenous PAK1 was specifically co-precipitated with overexpressed Flag-Net1 in HEK293T cells (Figure 6A and 6B). The amount of Myc-PAK1 co-immunoprecipitated with GEF defective mutants, Flag-Net1-L266E or W437L, was significantly decreased compared with wild-type control (Figure 6C), suggesting that their association requires Net1 GEF activity. Moreover, in contrast with wild-type PAK1, the H83L/H86L mutant containing an inactive GTPase binding domain (GBD), which cannot bind small GTPases such as Cdc42 and Rac1 65, showed a much lower affinity for Net1 (Figure 6D). Thus, Net1 is a binding partner of PAK1, and the interaction of these two proteins requires Net1 GEF activity.
 Effect of Net1 on PAK1 dimerization. (AD) Flag-tagged Net1 interacts with overexpressed (A) or endogenous (B) PAK1. HEK293T cells were transfected as indicated with expression plasmids encoding Myc-tagged PAK1 and Flag-tagged Net1 or Net1 mutants, then harvested for immunoprecipitation with anti-Flag M2 agarose beads. Note that compared with the wild-type control, Flag-Net1-L266E or W437L has a much lower binding affinity for Myc-PAK1 (C) . Conversely, Myc-tagged PAK1 H83L/H86L mutant (which cannot bind Rho family small G proteins such as Cdc42 and Rac1) loses the ability to associate with wild-type Net1 (D) . (E and F) Wild-type Net1 (E) but not its L266E or W437L mutant (F) disrupts dimerization of PAK1. HEK293T cells were co-transfected with plasmids expressing Flag- or Myc-tagged PAK1 and HA-tagged wild-type Net1 or Net1 mutants as indicated. Lysates were immunoprecipitated with anti-Flag M2 agarose beads and immunoblotted as indicated. (G) Net1 disrupts the dimerization of DNPAK1, the kinase-inactive mutant. HEK293T cells were co-transfected with plasmids expressing Flag- or Myc-tagged PAK1 K299A mutant proteins and HA-tagged Net1. Lysates were subjected to immunoprecipitations and immunoblots. (H) net1 -depletion enhances PAK1 dimerization. HEK293T cells transfected with net1 shRNA1 or shRNA2 were harvested for immunoprecipitation and immunoblotting. (I and J) Wnt3a CM treatment enhances the interaction between PAK1 and Net1, and disassociates the dimerization of PAK1. HEK293T cells co-transfected with indicated plasmids were treated with or without Wnt3a CM for 12 h before harvest, and then lysed for immunoprecipitations. (K and L) Net1 enhances the binding affinity of PAK1 for β-catenin (K) and promotes PAK1-induced phosphorylation of β-catenin S675 (L) in the presence of Wnt3a. HEK293T cells transfected with the indicated constructs were treated with or without Wnt3a CM for 12 h before harvesting for immunoprecipitation and western blot analyses. In CK, quantification is the mean relative ratio of co-immunoprecipitated signals over lysate, while in L, quantification is the relative density of phospho-specific signals to corresponding total protein signals (mean ± SD, three independent biological repeats).
 Previous studies have shown that majority of PAK1 exists as inactive homodimers by the interaction between the N-terminal inhibitory switch domain and the C-terminal kinase domain 67 . GTP-bound Cdc42 or Rac1 binds to GBD of PAK1 disrupting its dimerization, resulting in fully autophosphorylated and activated monomers 65 . Both the decreased phosphorylation level of PAK1 Thr423 in net1 -depleted cells (Figure 5H) and the physical association between these two proteins prompted us to examine whether Net1 controls PAK1 dimerization. HEK293T cells were transfected with plasmids expressing Flag-PAK1 and Myc-PAK1. Myc-PAK1 could be specifically detected in anti-Flag immunoprecipitates, suggesting the overexpressed PAK1 is in dimerized form (Figure 6E). As expected, PAK1 homodimers were dissociated upon co-expression of wild-type Net1 but not its mutants without GEF activity (Figure 6E and 6F). Interestingly, dimers of DNPAK1, the kinase-inactive mutant K299A, was similarly disrupted by Net1 overexpression (Figure 6G). This finding is consistent with the previous conclusions that PAK1 kinase-dead mutant, which lost its ability to autophosphorylate the crucial Thr423 residue, efficiently forms homodimers and its dimerization is strongly dissociated upon GTPase binding 65 . These data also suggest that the disruption of PAK1 homodimer by Net1 is not a consequence of autophosphorylation of key residues, including Thr423. In contrast, the association of Flag-PAK1 with Myc-tagged PAK1 was moderately enhanced in net1 shRNAs transfected cells (Figure 6H). These results reveal that Net1 and its GEF activity are essential for the dissociation of PAK1 homodimers.
 In addition, Wnt3a stimulation strengthened the association of Net1 with PAK1 and promoted the dissociation of PAK1 homodimers (Figure 6I and 6J). Co-expression of Net1 with PAK1 enhanced the affinity of PAK1 kinase for its substrate β-catenin in the presence of Wnt3a (Figure 6K). Consistently, PAK1-induced phosphorylation of β-catenin S675 was increased in cells transfected with plasmids expressing wild-type Net1 but not Net1 mutants (Figure 6L). These results therefore support the idea that Net1 promotes β-catenin phosphorylation via disrupting PAK1 dimerization.
 Net1 activates an unidentified Rho family GTPase to regulate Wnt signaling and dorsal development
 The requirements of Net1 GEF activity during Net1-regulated PAK1 activation and β-catenin phosphorylation suggest the involvement of GTPases in these processes. Although mammalian Net1 specifically activates RhoA but not Rac1 or Cdc42 34, 68, whether Net1 functions on zebrafish RhoA, Rac1 and Cdc42 activation has not been investigated. Previous work demonstrated that the GBD of PAK1 (PBD) binds the activated GTP-bound forms of Rac1 and Cdc42, but not RhoA, while the GBD of Rhotekin (RBD) selectively binds the activated GTP-bound form of RhoA, but not Rac1 or Cdc42 69, 70, 71 . GST-PBD and GST-RBD fusion proteins were generated and used as probes in pull-down assays to test the activation of these GTPases by Net1 overexpression in zebrafish embryos. Consistent with their mammalian homologous proteins, zebrafish Rac1, Cdc42 and RhoA were able to bind GST-PBD and GST-RBD, respectively (Supplementary information, Figure S12A). Overexpression of zebrafish Net1 significantly increased the binding of GST-RBD and zebrafish RhoA, but had no effect on the binding of GST-PBD and zebrafish Rac1 or Cdc42, suggesting Net1 selectively activates zebrafish RhoA (Supplementary information, Figure S12A). RhoA has context-dependent functions either enhancing or restraining Wnt/β-catenin signal 72, 73 ; we then examined whether RhoA was involved in dorsal development. Injection of RhoA-specific inhibitor C3 or overexpression of the dominant-negative rhoA mutant ( rhoA-N19 ) in zebrafish embryos resulted in significant morphological changes including reduced body size and shortened anterior-posterior body axis at 24 hpf (Supplementary information, Figure S12B and S12C). Consistently, in rhoA-N19 mRNA injected embryos, the adaxial mosodermal tissues exhibited shorter anterior-posterior axis with more distant bilateral columns (as indicated by myod1 expression), and the notochord became wider and shorter (as indicated by ntl expression; Supplementary information, Figure S12D). These phenotypes resembled the CE movement defects observed in rhoA morphants 41 . The inhibition of RhoA activity did not cause obvious dorsal fate defects during early embryonic development (Supplementary information, Figure S12E and S12F). Thus, RhoA is not necessary for dorsal fate specification.
 To confirm the involvement of GTPases in Net1-induced β-catenin activation and dorsal development, we used compactin, a pan Rho family GTPase inhibitor, to reduce endogenous GTPase activity in mammalian cells and zebrafish embryos 74 . In HEK293T cells, compactin treatment dramatically reduced the association between Net1 and PAK1 (Figure 7A), and further inhibited the ability of Net1 to disrupt PAK1 dimerization (Figure 7B). As expected, ectopic Net1 expression stimulated super-TOPFlash reporter activity (Figure 7C). However, the Net1-induced promotion of reporter expression was significantly repressed by compactin treatment (Figure 7C). Importantly, compactin treatment decreased the expression of dorsal marker genes in wild-type embryos, and erased the rescue effects of net1 mRNA on dorsal axis formation in net1 morphants (Figure 7D and 7E). These data provide evidence that an unidentified Rho family small GTPase is required for Net1-regulated PAK1 homodimer dissociation, canonical Wnt signal transduction and dorsal axis formation. Overall, these results support a model consisting of the GEF Net1, which activates an unidentified GTPase but not RhoA to bind to the GBD domain of PAK1. Net1 then dissociates PAK1 homodimers and relieves the inhibition of its kinase activity to enhance canonical Wnt signal transduction by phosphorylating β-catenin at S675 during dorsal development of ebrafish embryo (Figure 7F).
 An unidentified Rho family GTPase is necessary for Net1-regulated canonical Wnt signal transduction and dorsal axis formation. (AC) Compactin treatment inhibits the interaction between Net1 and PAK1 (A), the Net1-induced dissociation of PAK1 homodimers (B) and Net1-promoted increase in super-TOPFlash reporter luciferase activity (C) . HEK293T cells were treated with or without 10 μM compactin for 6 h then transfected with the indicated plasmids. After transfection, cells were subjected to compactin treatment for another 24 h before harvesting for immunoprecipitation and western blot analyses (A and B) or luciferase assays (C) . *** P < 0.001, Student's t -test. In A and B, quantification is the mean relative ratio of co-immunoprecipitated signals over lysate (mean ± SD, three independent biological repeats). (D) Compactin treatment disrupts the dorsal development of wild-type embryos. Wild-type embryos were treated with 10 μM compactin from 16-cell stage until sphere stage, before harvesting for in situ hybridization. (E) Compactin treatment suppresses the rescue effects of net1 mRNA on dorsal axis formation in net1 morphants. Embryos were co-injected with the indicated MOs and mRNAs at the one cell stage. net1 3mix injected-embryos were subjected to compactin treatment and UV exposure as indicated. All embryos were harvested at sphere stage for in situ hybridization. net1 3mix: 4 ng net1 MO1 together with 200 pg Flag-net1 mRNA and 1 ng AS-Flag-photo-MO. (F) An integrated model for the activation of canonical Wnt signaling by Net1. U-GDP, GDP-bound form of unidentified GTPase; U-GTP, GTP-bound form of unidentified GTPase. (G and H ) A number of Rho family GTPase signal transduction-related genes are upregulated in net1 mutants compared with wild-type embryos and net1 morphants. (G) Heat map of 16 Rho family GTPase signal transduction-related genes which are differentially expressed in wild-type embryos, net1 morphants and net1Δ53 mutants. The transcript abundance was quantified from RNA-Seq data. Induced genes are represented in red, while suppressed genes are represented in green. (H) The expression differences of these 16 genes in wild-type embryos, net1 morphants and net1Δ53 mutants were validated by qRT-PCR analyses. * P <0, 05; ** P <0, 01; *** P < 0.001, Student's t -test.
 To further confirm the function of net1 in dorsal axis formation, we generated net1 mutants using Cas9/gRNA system targeting exon 2, which encodes a unique amino acid sequence upstream of the DH domain 75 . We obtained two deletion mutants named net1Δ20 (20 bp deletion with 1 bp insertion) and net1Δ53 (53 bp deletion) (Supplementary information, Figure S13A). Both of these two net1 mutations led to a shift of the open-reading frame with a premature stop codon. The results of in situ hybridization experiments with net1 probe and western blot analyses using an anti-Net1 antibody showed that the expression of net1 transcript and protein was totally abolished in net1Δ20 and net1Δ53 homozygous embryos (Supplementary information, Figure S13B and S13C), suggesting that these two mutants are null alleles of the net1 gene. At first glance, net1Δ20 and net1Δ53 homozygotes showed no obvious morphological defects compared with wild-type control (Supplementary information, Figure S13D). Further analyses revealed that the dorsal marker genes were normally expressed at early stages during embryo development (Supplementary information, Figure S13E). Thus, unlike the severe dorsal developmental defects observed in net1 morphants, no obvious phenotypes are found in net1 mutants.
 The phenotypic differences between mutants and morphants may be due to the activation of a compensatory network in mutants 76, 77 . Actually, net1 MO1 or siRNA1 injection caused dorsal fate defects in wild-type embryos, but did not influence the dorsal development of mutants (Supplementary information, Figure S13F and S13G), indicating the specificity of the MOs and siRNAs used to knock-down zebrafish net1 in our study and the insensitiveness of the net1 mutants to net1 MOs and siRNAs. To identify the molecules with a compensatory role in net1 mutants, we performed RNA sequencing analyses in wild-type embryos, net1 morphants and net1Δ53 homozygotes at sphere stage. We identified a set of genes encoding GEFs, GTPases, and GTPase effectors that were upregulated in net1Δ53 mutants but not in wild-type embryos and net1 morphants (Figure 7G). Quantitative real-time PCR experiments using a number of primers listed in Supplementary information, Data S1, further confirmed the specific activation of most of these genes in net1Δ53 mutants (Figure 7H). Furthermore, net1Δ53 mutants treated with compactin or injected with dnpak1 mRNA exhibited clearly decreased expression of dorsal markers, suggesting that the upregulation of these GTPase-mediated signal transduction-related genes in net1 mutants compensates for the loss of net1 (Supplementary information, Figure S13H and S13I). In addition, these results provide valuable evidence from another perspective to support the important function of Net1 and its GEF activity in activating Wnt/β-catenin signaling during dorsal development.
 The neuroepithelial cell transforming gene net1 encodes a RhoA-specific GEF that regulates cell migration in different embryonic stages of various animal species 38, 39, 40 . In this study, we found that zebrafish net1 is prominently expressed in the dorsal side of the embryo after MBT. Loss-of-function and gain-of-function experiments in zebrafish embryos and mammalian cells revealed that Net1 acts as a zygotic factor to trigger dorsal regionalization. Mechanistically, Net1 disrupts PAK1 dimerization by activating an unknown GTPase, and subsequently promotes PAK1-mediated phosphorylation of β-catenin at S675 to fully activate Wnt/β-catenin signaling during the dorsal development of zebrafish embryos. To our knowledge, this is the first genetic evidence to demonstrate that Net1 contributes to dorsal axis formation by modulating maternal Wnt signaling.
 Previous studies have shown that the expression of net1 is rapidly induced by TGF-β/Smad signaling in several human and mouse cell lines 58, 78 . Our previous work also indicated that zebrafish net1 is a direct downstream target of Nodal/Smad2 signaling at the onset of gastrulation 32 . In current study, we found that Nodal signal activates the expression of net1 in the pre-dorsal organizer from sphere stage to shield stage. In the early zebrafish embryo, a dorsal to ventral gradient of Nodal activity is established 79 . As net1 exhibits highest expression level in the dorsal side but much weaker expression level in the lateral margin, we hypothesize that net1 is a high-threshold-activated gene in response to Nodal signaling. Interestingly, Nodal-deficient embryos exhibit a normal phosphorylation level of β-catenin S675. It is likely that some Nodal-suppressed genes might be upregulated to maintain β-catenin phosphorylation in Nodal-deficient embryos. In agreement with these findings, the dorsal axis still forms in embryos lacking Nodal signaling 79, 80, 81, 82 .
 Overexpression of net1 can rescue the dorsal fate defects in net1 morphants and result in markedly enlarged expression domains of dorsal marker genes in wild-type embryos. Furthermore, inactivation of zebrafish net1 by an alternative technique, siRNA-based gene silencing, induces similar dorsal fate defects to those observed in net1 morphants. In addition, knockdown of endogenous net1 expression by shRNAs in mammalian cells causes a significant decrease in Wnt/β-catenin signaling activity and target gene expression. Because these different knockdown strategies lead to similar effects in zebrafish embryos and mammalian cells, it is likely that any potential off-target effects in net1 knockdown experiments are minimal.
 We also generated net1 null mutants using the Cas9/gRNA system. Surprisingly, net1 mutants display no obvious developmental defects; and are also, importantly, insensitive to the injection of net1 MOs or siRNAs. Previous biochemical and genetic studies as well as clinical reports indicate that compensatory changes may allow organisms to adapt to genetic mutations 77, 83, 84 . Such properties have also been described in zebrafish genetic studies. Embryos injected with egfl7 morpholinos exhibit severe vascular defects, while egfl7 mutants do not show any obvious phenotypes, as a set of genes are upregulated to compensate for the induced genetic alteration 76 . Interestingly, we also observed a clear upregulation of a subset of genes encoding GEFs, GTPases and GTPase effectors in net1 mutants. These net1 mutants are still sensitive to compactin treatment or PAK1 activity inhibition, strongly suggesting that upregulation of these GTPase-mediated signal transduction-related genes compensates for the loss of Net1 function during embryonic development. Thus, our findings provide another example of compensatory gene regulation in response to an altered genetic environment.
 Mammalian Net1 exists in two isoforms, Net1 and Net1A, which are produced by alternative splicing and differ only in their N-terminus. Both isoforms are mainly localized in the nucleus in quiescent cells, thereby preventing them from activating RhoA 35, 36, and their presence in the cytoplasm is tightly controlled. TGF-β/Smad signaling has been reported to induce not only Net1 expression, but also its cytoplasmic localization, which is required for TGF-β-induced EMT of human retinal pigment epithelial cells 58 . Furthermore, active Rac1 stimulates Net1A re-localization to the cytoplasm by promoting acetylation on the N-terminal sites 85 . Although our study clearly demonstrates that both cytoplasmic and nuclear Net1 can efficiently enhance canonical Wnt signaling and dorsal axis formation, it remains to be seen if zebrafish Net1 has alternatively spliced isoforms and whether these isoforms are localized to the cytoplasm during embryonic development.
 Our study suggests a role of Net1 in regulating the transcriptional activity of β-catenin. During vertebrate embryonic development, maternal β-catenin protein, the transcriptional effector of canonical Wnt signaling, is stabilized in dorsal blastomeres by asymmetrically localized Wnt ligands to initiate the dorsal-ventral axis 1, 2 . In zebrafish, the maternal wnt8a mRNA is transported from the vegetal pole to the future dorsal margin by a microtubule-dependent mechanism, which then leads to β-catenin nuclear translocation 14 . Zebrafish maternal β-catenin accumulates in the nuclei of dorsal marginal blastomeres as early as the 128-cell stage (2.25 hpf) 81, 86, but activates the transcription of dorsal genes including boz and chd only after the MBT (about 3 hpf) to inhibit the action of ventralizing Bmp signals 2 . The mechanism by which the transcriptional activity of maternal β-catenin is inhibited before MBT remains to be elucidated. Recent evidence suggests that alterations in DNA methylation and histone modifications around the time of MBT are important for zygotic gene activation 47, 87, 88, 89, 90 . Similarly, β-catenin recruits the arginine methyltransferase Prmt2 to target promoters, thereby establishing poised chromatin architecture and priming organizer gene expression 91 . Whether the transcriptional activity of maternal β-catenin is controlled by internal self-regulating protein modifications remains unknown.
 Our findings suggest that Net1 is essential for β-catenin activation to pattern the zebrafish embryonic body plan. Interestingly, we found that the relative phosphorylation level of β-catenin S675 is very low before MBT, but becomes significantly increased during and after MBT. The expression of net1 results in the phosphorylation at S675, but not protein stability of β-catenin. Importantly, overexpression of β-catenin S675D but not wild-type β-catenin could lead to a much earlier expression of the dorsal marker genes. These observations suggest that β-catenin S675 phosphorylation, which is regulated by Net1, is a key requirement for the transcriptional activity of maternal β-catenin to trigger target gene expression and establish dorsal axis in zebrafish embryos. Whether the C-terminal phosphorylation of β-catenin at other sites plays additional roles in embryonic patterning remains to be determined.
 Multiple serine residues within the C terminus of β-catenin can be phosphorylated by various protein kinases. In particular, the S675 residue is a common target of PAK1 and PKA 22, 23, 60 . Our biochemical and genetic studies in zebrafish embryos and mammalian cells demonstrate that Net1 acts upstream of PAK1, but not PKA, to control β-catenin phosphorylation and dorsal axis formation. This premise is further supported because Net1 requires its GEF activity to promote β-catenin phosphorylation, and the kinase activity of PAK1 is regulated by GTP-loaded GTPases 65 . Interestingly, our results suggest that Net1 dissociates PAK1 homodimers thereby initiating PAK1 kinase activity to phosphorylate β-catenin in a GEF-dependent manner. Net1 specifically interacts with both GTP- and GDP-bound RhoA and catalyzes GDP exchange on RhoA, but not Rac1 or Cdc42 34, 68 . Moreover, our results, as well as those from previous genetic studies, indicate that RhoA mediates cell movements during zebrafish gastrulation but not cell fate determination 41, 92, 93 . Conversely, intracellular GTP-Rac1 and GTP-cdc42, but not activated RhoA, are known to disrupt PAK1 dimerization, thereby controlling β-catenin S675 phosphorylation and Wnt signal transduction 23, 65 . Because Net1 GEF activity is required for regulating PAK1 homodimer dissociation, Wnt/β-catenin signal transduction and zebrafish dorsal axis formation, we propose that a previously unidentified GTPase exists and is activated by Net1 to regulate downstream Wnt/β-catenin signaling and dorsal patterning. Compactin, a pan Rho family GTPase inhibitor, was then used to reduce endogenous GTPase activity in mammalian cells and zebrafish embryos. Compactin treatment dramatically reduced the association between Net1 and PAK1, inhibited the ability of Net1 to disrupt PAK1 dimerization, suppressed Net1-induced Wnt/β-catenin signal activity, and erased the rescue effect of net1 mRNA on dorsal axis formation in net1 morphants. These data confirm the requirement of Rho family GTPase activity in Net1-regulated canonical Wnt signal transduction and dorsal fate specification. We have tried pull-down experiments and yeast two-hybrid screening using full-length zebrafish Net1 as bait to identify Net1-regulated GTPases, but did not find any useful clue. Interestingly, a recent study found that PAK1 phosphorylates Net1 on three serine residues and negatively regulates its GEF activity 94, suggesting a negative-feedback loop in Net1-regulated Wnt signal activation.
 In summary, our results reveal a conserved regulatory cascade of Net1-GTPase-PAK1 that controls canonical Wnt signaling in zebrafish embryos and mammalian cells. Future studies may determine whether this regulatory cascade similarly functions in the dorsal axis formation of other vertebrate animals. In addition, because upregulated net1 expression and aberrant Wnt/β-catenin signaling are widely implicated in numerous cancers 12, 42, 43, 44, 45, 46, it will be important to investigate the functional interaction of Net1 and Wnt/β-catenin signaling in cancer cell proliferation, motility and invasiveness.
 Zebrafish kamienai
 Embryos and adult fish were raised and maintained under standard laboratory conditions. Embryos were staged by morphology as previously described 95 . Homozygous p53(M214K) mutant embryos (abbreviated as p53 −/− ) carrying a loss-of-function p53 point mutation were employed as controls to account for any non-specific cell apoptosis induced by morpholino injection. TOPdGFP transgenic embryos were used to confirm the endogenous activity of Wnt/β-catenin signaling during embryonic development. Our studies involving zebrafish embryo collection and analyses were in full compliance with the Regulations for the Care and Use of Laboratory Animals by the Ministry of Science and Technology of China, and with the Institute of Zoology's Guidelines for the Care and Use of Laboratory Animals. The experimental protocol was approved by the Animal Care and Use Committee at the Institute of Zoology, Chinese Academy of Sciences (Permission Number: IOZ-13048).
 HEK293T, MCF-7, Hela and L cell lines (American Tissue Culture Collection, ATCC, USA) were cultured in DMEM medium supplemented with 10% FBS in a 37 °C humidified incubator in a 5% CO 2 environment. The Wnt3a-expressing L cell line was grown under similar conditions, but supplemented with 0.4 mg/ml G-418 to select for Wnt3a-expressing cells. The Wnt3a conditioned medium (Wnt3a CM) was generated following the protocol of ATCC. For Wnt stimulation experiments, Wnt3a CM was mixed with normal culture medium at the volume ratio 1:1. Cell transfection was performed using Lipofectamine 2000 (11668019, Invitrogen) following the manufacturer's instructions.
 RNA and morpholino microinjection
 Capped mRNAs for zebrafish net1 , wnt8a , ΔN-β-catenin , β-catenin S675D , β-S675A , dnpak1 , capak1 and rhoA N19 were synthesized in vitro from corresponding linearized plasmids using the mMessage mMachine kit (Ambion). The following morpholinos were synthesized by Gene Tools and re-suspended in nuclease free water: net1 MO1 (5′-CTTGCTCCGGCTGTACTCACCTCTT-3′), net1 MO2 (5′-AAAGAATGTACAAACCTTTCGGGCT-3′), control MO (mis-MO1) (5′-CTTCCTGCGCCTGTAGTCACGTCTT-3′) and AS-Flag-photo-MO (5′-TCATCGTCGTpCTTGTAGTCCAT-3′). Microinjection was performed as previously described. For net1 mRNA overexpression experiments, embryos were co-injected with pre-mixed Flag-net1 mRNA and AS-Flag-photo-MO (200 pg Flag-net1 mRNA and 1 ng AS-Flag-photo-MO per embryo) at 1-cell stage, then treated with 365 nm UV 10 min by Lightbox (Gene Tools, LLC) at 64-cell stage, and collected at sphere stage. All of these processes were conducted under constant dark conditions.
 Viso montavimo in situ hibridizacija
 Digoxigenin-UTP-labeled antisense RNA probes were transcribed using MEGAscript Kit (Ambion) according to the manufacturer's instructions. Whole-mount in situ hybridizations (WISH) were performed according to previously published methods 32 .
 To block RhoA activity, embryos were injected with 1 nl of 20 μM C. botulinum toxin C3 transferase (C3) (A8724, Sigma) at the 1-cell stage, then collected at sphere stage for WISH. To disrupt Ras family GTPase activity, embryos were treated with 10 μM compactin (M2537, Sigma) at the 16-cell stage, then harvested at the sphere stage for subsequent experiments. To inhibit PAK1 activity, embryos were treated with 10 μM IPA-3 (I2285, Sigma) at the 1-cell stage, and then subjected to in situ hybridization at sphere stage. To repress PKA activity, embryos were treated with 10 μM H89 dihydrochloride hydrate (B1427, Sigma) at the 16-cell stage, and collected at sphere stage for WISH.
 Dviejų liuciferazių reporterių testai
 For the luciferase reporter assays, either HEK293T or MCF-7 cells were transfected with super-TOPFlash and the indicated plasmids. The Renilla luciferase reporter was used as an internal control. Cells were stimulated with or without Wnt3a CM for 12 h before harvesting for luciferase activity assays. Each luciferase reporter assay was repeated at least three times, and the average activity was calculated.
 For immunoprecipitation assays, HEK293T cells were transfected with the indicated plasmids. Cells were harvested 48 h after transfection and lysed with TNE lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA and 0.5% Nonidet P-40) containing a protease inhibitor cocktail. Lysates were subjected to immunoprecipitation assay as previously described 32 .
 For immunoblotting, we used affinity-purified anti-Flag (F2555, Sigma), anti-HA (CW0092A, CW), anti-Myc (M047-3, MBL), anti-β-catenin (M24002, Abmart), anti-Phospho-β-catenin (Ser675) (4176, Cell Signaling Technology), anti-PAK1 (2602, Cell Signaling Technology), anti-Phospho-PAK1 (Thr423) (2601, Cell Signaling Technology), anti-Net1(AI13372, Abgent), anti-Phospho-Histone H3 (3377, Cell Signaling Technology), anti-GFP (A11120, Thermo Fisher) and anti-GST (M20007, Abmart) antibodies.
 Imunofluorescencinė analizė
 Cells on coverslips were fixed in 4% paraformaldehyde/PBS for 10 min at room temperature then permeabilized with 0.2% Triton X-100/PBS for 10 min. After 30 min blocking in 1.5% FBS/PBS, cells were incubated with the indicated antibodies for 1 h at room temperature or overnight at 4 °C. 4′, 6-Diamidine-2′-phenylindole dihydrochloride (DAPI, 10236276001, Sigma) was used to counterstain nuclei. All immunofluoresence images within each experiment were captured using Nikon A1R+ confocal microscope under the same settings.
 Student's t -test was performed to analyze all data sets (Microsoft Excel software). At a minimum, experiments were performed in triplicate. Results were considered statistically significant at P < 0.05.
 Autoriaus įnašai
 SW and QW conceived and designed the experiments and wrote the manuscript. SW performed most of the experiments and analysis. MD and ZL performed experiments presented in Figure 2F-2H. YM constructed net1 shRNA vectors. HS and YC verified the functions of RhoA in zebrafish embryo development. QW supervised the entire project. All authors reviewed and commented on the manuscript.
 Konkurencingi finansiniai interesai
 The expression of net1 at the dorsal blastoderm margin is regulated by Nodal signal.
 Knockdown of net1 by MOinjection blockszebrafish dorsal axis formation.
 The dorsal imperfections in net1 morphants is not resulted from MO injection-induced global defects in cell proliferation or survival.
 The dorsal imperfections in net1 morphants are not resulted from MO injection-induced developmental delay.
 Knockdown of net1 by siRNA injection results in severe dorsal development defects.
 Overexpression of net1 rescues the dorsal defects in net1 morphants.
 Papildoma informacija, S7 pav
 Zebrafish net1 expression is not regulated by Wnt/β-catenin signaling.
 Papildoma informacija, S8 pav
 The GEF activity of net1 is essential to induce dorsal gene expression.
 Papildoma informacija, S9 pav
 Net1 functions in parallel with or downstream of β-catenin to regulate canonical Wnt signaling.
 Papildoma informacija, S10 pav
 Nodal signal is dispensable for β-Catenin S675 phosphorylation in zebrafish embryos.
 Disruption of endogenous PKA protein activity has no effect on dorsal axis formation and β-Catenin S675 phosphorylation.
 RhoA is not necessary for dorsal fate specification.
 net1 null alleles exhibit no obvious developmental defects.
 Supplementary information Data S1
 ( Papildoma informacija yra susieta su internetine straipsnio versija ląstelių tyrimų svetainėje.)
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type: contracts

jurisdiction: EU

text:
 19.12.2013
 HU
 Az Európai Unió Hivatalos Lapja
 L 345/13
 AZ EGT VEGYES BIZOTTSÁG 144/2013 HATÁROZATA
 (2013. július 15.)
 az Európai Gazdasági Térségről szóló megállapodás XIII. mellékletének (Közlekedés) módosításáról
 AZ EGT VEGYES BIZOTTSÁG,
 tekintettel az Európai Gazdasági Térségről szóló megállapodásra (a továbbiakban: az EGT-megállapodás) és különösen annak 98. cikkére,
 mivel:
 (1)
 Az adathordozót (mikrocsipet) tartalmazó vezetői engedélyekre vonatkozó műszaki követelmények megállapításáról szóló, 2012. május 4-i 383/2012/EU bizottsági rendeletet (1) bele kell foglalni az EGT-megállapodásba.
 (2)
 Az EGT-megállapodás XIII. mellékletét ezért ennek megfelelően módosítani kell,
 ELFOGADTA EZT A HATÁROZATOT:
 1. cikk
 Az EGT-megállapodás XIII. melléklete a 24f. pont (2006/126/EK európai parlamenti és tanácsi irányelv) után a következő szöveggel egészül ki:
 „24fa.
 32012 R 0383: a Bizottság 2012. május 4-i 383/2012/EU rendelete az adathordozót (mikrocsipet) tartalmazó vezetői engedélyekre vonatkozó műszaki követelmények megállapításáról (HL L 120., 2012.5.5., 1. o.).
 E megállapodás alkalmazásában a rendelet rendelkezéseit a következő kiigazítással kell értelmezni:
 A III. melléklet III.4.2. pontjának a) alpontja a következőkkel egészül ki a megkülönböztető számok tekintetében:
 »—
 14 Izland
 —
 15 Liechtenstein
 —
 16 Norvégia« ”.
 2. cikk
 A 383/2012/EU rendeletnek az Európai Unió Hivatalos Lapja EGT-kiegészítésében közzéteendő izlandi és norvég nyelvű szövege hiteles.
 3. cikk
 Ez a határozat 2013. július 16-án lép hatályba, feltéve, hogy az EGT-megállapodás 103. cikkének (1) bekezdésében előírt összes bejelentést megtették (2).
 4. cikk
 Ezt a határozatot az Európai Unió Hivatalos Lapjának EGT-re vonatkozó részében és EGT-kiegészítésében ki kell hirdetni.
 Kelt Brüsszelben, 2013. július 15-én.
 az EGT Vegyes Bizottság részéről
 az elnök
 Thórir IBSEN
 (1) HL L 120., 2012.5.5., 1. o.
 (2) Alkotmányos követelmények fennállását nem jelezték.


type: other

text:
Portaria 228/2016, 2016-08-25 - DRE
 Portaria n.o 228/2016
 Páginas:2900 - 2923
 ELI:https://data.dre.pt/eli/port/228/2016/08/25/p/dre/pt/html
 Decorreram mais de cinco anos desde a data em que o EUROMILHÕES foi alvo da última revisão.
 Através da presente portaria, visa-se adequar o jogo nacional de exploração coordenada de maior sucesso na Europa, em exploração há doze anos, à realidade atual e às expectativas dos apostadores.
 Assim, através do aumento do preço da aposta em (euro) 0,20, valor que não era objeto de qualquer alteração desde 2004, aquando do lançamento do jogo, bem como do aumento das percentagens da receita destinadas ao 1.o prémio e ao fundo de reserva, que agora passa a poder ser utilizado para incrementar o valor de todas as categorias de prémios, é possível oferecer jackpots mínimos garantidos de valor superior aos atuais, assim como uma diversificação dos sorteios promocionais, contemplando com prémios um maior número de apostadores.
 É igualmente revisto o número máximo de sorteios consecutivos em que o 1.o prémio pode oferecer o valor de 190 milhões de euros, passando dos atuais dois para cinco sorteios. Deste modo, com a manutenção da regra de que o remanescente do valor acumulado na primeira categoria de prémios, após atingir o seu montante máximo, reverte para o 2.o prémio, procura-se também estimular a importância da segunda categoria de prémios.
 Ao abrigo do n.o 2 do artigo 2.o do Decreto-Lei n.o 210/2004, de 20 de agosto, alterado e republicado pelo Decreto-Lei n.o 44/2011, de 24 de março, e Decreto-Lei n.o 43/2016, de 16 de agosto, e dos artigos 2.o e 27.o, n.o 3, alínea i) dos Estatutos da Santa Casa da Misericórdia de Lisboa, aprovados pelo artigo 1.o do Decreto-Lei n.o 235/2008, de 3 de dezembro, alterado pelo Decreto-Lei n.o 114/2011, de 30 de novembro, e pelo Decreto-Lei n.o 67/2015, de 29 de abril, manda o Governo, pelo Ministro do Trabalho, Solidariedade e Segurança Social, o seguinte:
 1 - Os artigos 3.o, 4.o, 7.o, 8.o, 10.o, 13.o, 15.o, 16.o, 17.o, 18.o, 19.o e 23.o do Regulamento do EUROMILHÕES, aprovado pela Portaria n.o 1267/2004, de 1 de outubro, e publicado em anexo à mesma, dela fazendo parte integrante, alterado pelas Portarias n.os 1528/2004, de 31 de dezembro, 147/2006, de 20 de fevereiro, 867/2006, de 28 de agosto, 8-A/2007, de 3 de janeiro, 93/2009, de 28 de janeiro, 699/2009, de 2 de julho, 65/2011, de 4 de fevereiro, 127/2011, de 31 de março, 320-F/2011, de 30 de dezembro, 113/2013, de 21 de março, e 15/2014, de 23 de janeiro, passam a ter a seguinte redação:
 1 - O preço de cada aposta no jogo EUROMILHÕES é de (euro) 2,20.
 2 - Sem prejuízo do pagamento do preço referido no número anterior, a participação no jogo EUROMILHÕES implica o pagamento adicional do preço de (euro) 0,30 por cada aposta realizada, habilitando-se os apostadores a participar no jogo «Totosorteio», cujo regime jurídico foi aprovado pelo Decreto-Lei n.o 43/2016, de 16 de agosto.
 5 - (Anterior n.o 3):
 c) [Anterior alínea c)];
 d) [Anterior alínea d)];
 h) [Anterior alínea h)];
 i) [Anterior alínea i)];
 j) [Anterior alínea j)];
 m) [Anterior alínea n)].
 12 - Na situação prevista no número anterior, caso não sejam escrutinadas apostas com direito ao 1.o prémio após cinco concursos consecutivos, o respetivo montante acresce ao valor do 2.o prémio ou, caso este não seja atribuído, ao valor do prémio da categoria imediatamente inferior em que haja, pelo menos, uma aposta premiada.
 13 - Na situação prevista na parte final dos n.os 11 e 12, quando não forem escrutinadas apostas premiadas em qualquer categoria de prémios, o montante total acumulado acresce ao valor do 1.o prémio do concurso imediatamente seguinte, aplicando-se o disposto nos n.os 7 e 11.
 14 - O montante indicado nos n.os 7 e 11 pode ser objeto de revisão, a publicitar pelo departamento de jogos, antes do início da aceitação das apostas para o concurso em que o novo montante se aplique.
 15 - Sem prejuízo do disposto nos n.os 7, 11, 12, 13 e 14, podem realizar-se concursos, com base no fundo de reserva a que se refere a alínea a) do n.o 2, nos quais o montante do 1.o prémio, caso não haja vencedores nessa categoria, acresce ao montante do 2.o prémio ou, caso este não seja atribuído, ao montante do prémio da categoria imediatamente inferior em que haja, pelo menos, uma aposta premiada, a publicitar pelo departamento de jogos antes do início da aceitação das apostas para esses concursos.
 16 - Podem, também, realizar-se concursos, com base no fundo de reserva a que se refere a alínea a) do n.o 2, nos quais o valor do primeiro prémio pode ser superior ao valor acumulado durante o ciclo de jackpots, a publicitar pelo departamento de jogos antes do início da aceitação das apostas para esses concursos, aplicando-se o disposto nos n.os 7, 11, 12, 13 e 14.
 17 - Cumulativamente com o EUROMILHÕES podem também ser organizados sorteios adicionais de prémios, com base no fundo de reserva a que se refere a alínea a) do n.o 2, de quantidade e montante a publicitar pelo departamento de jogos antes do início da aceitação das apostas para os respetivos concursos, nos termos seguintes:
 f) A data do sorteio é a do dia em que o mesmo se realizar.
 6 - As apostas podem ser anuladas no terminal onde foram registadas nos vinte minutos posteriores ao registo ou até à hora de encerramento da aceitação de apostas para o concurso a que respeitam, conforme o que ocorrer primeiro, implicando a anulação das apostas no jogo EUROMILHÕES a consequente anulação das apostas que lhe estão associadas e a que se referem o n.o 1 do artigo 3.o e o n.o 17 do artigo 10.o
 b) A cópia de segurança dos ditos suportes tenha sido enviada pelo órgão de fiscalização denominado LOI (lottery operator independent), a que se refere o artigo 14.o do presente regulamento, e a mesma tenha sido rececionada e se encontre à guarda do auditor independente previsto no artigo 6.o, n.o 2 do Decreto-Lei n.o 210/2004, de 20 de agosto, antes da hora do começo do(s) sorteio(s), encontrando-se a mesma arquivada sob custódia do referido LOI.
 São publicadas as tabelas constantes dos anexos I, II e III, relativas, respetivamente, aos sistemas de apostas múltiplas, à distribuição da percentagem da importância destinada a prémios para o 1.o prémio e o fundo de reserva e aos prémios em apostas múltiplas, as quais fazem parte integrante do presente regulamento.»
 2 - É republicado em anexo o Regulamento do EUROMILHÕES, aprovado pela Portaria n.o 1267/2004, de 1 de outubro, alterado pelas Portarias n.os 1528/2004, de 31 de dezembro, 147/2006, de 20 de fevereiro, 867/2006, de 28 de agosto, 8-A/2007, de 3 de janeiro, 93/2009, de 28 de janeiro, 699/2009, de 2 de julho, 65/2011, de 4 de fevereiro, 127/2011, de 31 de março, 320-F/2011, de 30 de dezembro, 113/2013, de 21 de março, e 15/2014, de 23 de janeiro, e pela presente portaria.
 3 - A presente portaria produz efeitos para as apostas registadas a partir do dia 24 de setembro de 2016.
 6 - Para participar no EUROMILHÕES apenas podem ser utilizados os suportes autorizados pelo Departamento de Jogos, nos termos do presente Regulamento, sem prejuízo do disposto no Decreto-Lei n.o 282/2003, de 8 de novembro.
 1 - Os prognósticos fazem-se pela marcação de cruzes (X), cujos pontos de interseção devem estar dentro de cada um dos retângulos das grelhas dos conjuntos existentes no bilhete.
 2 - Os prognósticos podem também ser gerados aleatoriamente ou ser escolhidos pelos jogadores, mediante solicitação de digitação e impressão no terminal de jogo por mediador dos jogos sociais do Estado, através do sítio da Internet www.jogossantacasa. pt ou noutros canais, nos termos regulados pelo Departamento de Jogos, cujo acesso é disponibilizado através da sua plataforma de acesso multicanal.
 2 - Os prognósticos efetuados, nos termos do Decreto-Lei n.o 282/2003, de 8 de novembro, em outros suportes distintos do bilhete físico de apostas devem obedecer às regras constantes do número anterior.
 1 - Da receita de cada concurso, constituída pelo montante total das apostas admitidas e das apostas anuladas sem direito a restituição, é destinada a prémios a importância correspondente a 50 %, conforme previsto no n.o 2 do artigo 8.o do Decreto-Lei n.o 210/2004, de 20 de agosto.
 k) Ao 11.o, as que tenham prognosticado um dos cinco números extraídos no 1.o sorteio e os dois números extraídos no 2.o sorteio;
 l) Ao 12.o, as que tenham prognosticado dois dos cinco números extraídos no 1.o sorteio e um dos dois números extraídos no 2.o sorteio;
 m) Ao 13.o, as que tenham prognosticado apenas dois dos cinco números extraídos no 1.o sorteio.
 9 - Quando não forem escrutinadas apostas com direito ao 13.o prémio, o montante a ele destinado acresce ao montante do 1.o prémio do concurso imediatamente seguinte.
 10 - A importância de cada prémio é repartida em quinhões iguais pelas apostas premiadas de cada uma das categorias de prémios referidas no n.o 2, arredondados para a quantia em cêntimos imediatamente inferior.
 3 - O mediador é responsável perante o Departamento de Jogos pelo pagamento do preço de todas as apostas registadas nos termos do n.o 2 do artigo 9.o e que não tenham sido anuladas, nos termos do regulamento respetivo.
 d) A utilização do cartão de jogador nos outros canais da plataforma de acesso multicanal do Departamento de Jogos, nomeadamente o sítio da Internet www.jogossantacasa.pt, nos termos do Decreto-Lei n.o 282/2003, de 8 de novembro.
 5 - Quando, por qualquer motivo, o jogador não pague imediatamente as apostas efetuadas, as mesmas são anuladas pelo mediador, através da reintrodução do recibo no terminal que imprimirá na frente a palavra «ANULADO» ou «CANCELADO», o valor da aposta, data e hora, o qual será enviado ao Departamento de Jogos pelo mediador dos jogos sociais do Estado, não podendo em caso algum ser entregue ao jogador.
 7 - O sistema central anula igualmente as apostas registadas e validadas através do sistema de registo e validação informático quando se verificar que as mesmas foram efetuadas com violação do disposto no artigo 3.o, n.o 3, tendo o jogador direito à devolução do preço das apostas pagas.
 1 - Sem prejuízo dos órgãos de controlo e fiscalização estabelecidos pelos diversos exploradores de jogos participantes no EUROMILHÕES, nomeadamente o LOI português, órgão independente constituído por um representante da Inspeção-Geral de Finanças, ao júri dos concursos, com a constituição fixada no artigo 30.o dos Estatutos da Santa Casa da Misericórdia de Lisboa, aprovados pelo Decreto-Lei n.o 235/2008, de 3 de dezembro, alterado pelo Decreto-Lei n.o 114/2011, de 30 de novembro, e pelo Decreto-Lei n.o 67/2015, de 29 de abril, compete também:
 a) A receção e a guarda em segurança da cópia dos registos de apostas efetuadas através do sistema de registo e validação informático, previstas no artigo 13.o, n.o 10, alínea b), cuja entrega é feita pelo LOI;
 1 - O 1.o sorteio de números de cada concurso do EUROMILHÕES, denominado «Sorteio A», efetua-se mediante a extração de 5 bolas, de uma esfera contendo 50 bolas homogéneas, iguais em material, volume e peso, numeradas de 1 a 50.
 6 - O sorteio complementar limita-se à extração do número de bolas necessário para completar o total de cinco bolas para o 1.o sorteio (A) e de duas bolas para o 2.o sorteio (B) de cada concurso.
 5 - O controlo dos prémios relativos a apostas efetuadas no sistema de registo e validação informático é efetuado pelo júri dos concursos, por comparação com a cópia de segurança prevista no artigo 13.o, n.o 10, alínea b), prevalecendo esta em caso de dúvida.
 2 - Quando haja alteração dos resultados provisórios, o número definitivo das apostas premiadas bem como o valor dos respetivos quinhões são tornados públicos através do cartaz referido no número anterior, após o julgamento das reclamações nos termos do artigo 19.o
 b) No caso de o recibo apresentar um prémio igual ou inferior a (euro) 150, após confirmação por parte do jogador premiado de que pretende receber o seu prémio, é impresso pelo terminal na frente do recibo a palavra «PAGO», o valor do prémio, data e hora, e o mediador ou o Departamento de Jogos procedem ao respetivo pagamento;
 c) No caso de o recibo apresentar um prémio de valor superior a (euro) 150 e inferior a (euro) 5.000, o pagamento é efetuado através de transferência para a conta bancária indicada pelo portador do título premiado;
 e) Os prémios de valor igual ou superior a (euro) 5.000 são pagos junto do Departamento de Jogos da Santa Casa da Misericórdia de Lisboa, mediante transferência para conta bancária do portador do título premiado, após a respetiva identificação pessoal, nos termos da legislação que regula as medidas de natureza preventiva e repressiva de combate ao branqueamento de vantagens de proveniência ilícita e de financiamento do terrorismo;
 a) Os prémios de valor igual ou inferior a (euro) 150 são transferidos automaticamente para o cartão de jogador;
 10 - O jogador é exclusivamente responsável pela correta e atempada realização dos atos necessários ao recebimento do prémio, responsabilizando-se o Departamento de Jogos pelo pagamento dos prémios antes do decurso do prazo de caducidade, sem prejuízo do disposto nos artigos 19.o e 20.o
 1 - As reclamações são julgadas por um júri constituído nos termos do artigo 35.o dos Estatutos da Santa Casa da Misericórdia de Lisboa, aprovados pelo Decreto-Lei n.o 235/2008, de 3 de dezembro.


text:
 29.11.2018
 SL
 Uradni list Evropske unije
 L 305/15
 SKLEP SKUPNEGA ODBORA EGP
 št. 51/2017
 z dne 17. marca 2017
 o spremembi Priloge I (Veterinarske in fitosanitarne zadeve) in Priloge II (Tehnični predpisi, standardi, preskušanje in certificiranje) k Sporazumu EGP [2018/1815]
 SKUPNI ODBOR EGP JE –
 ob upoštevanju Sporazuma o Evropskem gospodarskem prostoru (v nadaljnjem besedilu: Sporazum EGP) in zlasti člena 98 Sporazuma EGP,
 ob upoštevanju naslednjega:
 (1)
 Uredbo Komisije (EU) 2016/1822 z dne 13. oktobra 2016 o spremembi prilog II, III in V k Uredbi Evropskega parlamenta in Sveta (ES) št. 396/2005 glede mejnih vrednosti ostankov za aklonifen, deltametrin, fluazinam, metomil, sulkotrion in tiodikarb v ali na nekaterih proizvodih (1) je treba vključiti v Sporazum EGP.
 (2)
 Uredbo Komisije (EU) 2016/1866 z dne 17. oktobra 2016 o spremembi prilog II, III in V k Uredbi Evropskega parlamenta in Sveta (ES) št. 396/2005 glede mejnih vrednosti ostankov za 3-decen-2-on, acibenzolar-S-metil in heksaklorobenzen v ali na nekaterih proizvodih (2) je treba vključiti v Sporazum EGP.
 (3)
 Ta sklep se navezuje na zakonodajo v zvezi s krmo in živili. Zakonodaja v zvezi s krmo in živili se ne uporablja za Lihtenštajn, dokler se Sporazum med Evropsko skupnostjo in Švicarsko konfederacijo o trgovini s kmetijskimi proizvodi uporablja tudi za Lihtenštajn, kakor je podrobno določeno v sektorskih prilagoditvah Priloge I in v uvodu k poglavju XII Priloge II k Sporazumu EGP. Ta sklep se zato ne uporablja za Lihtenštajn.
 (4)
 Prilogi I in II k Sporazumu EGP bi bilo zato treba ustrezno spremeniti –
 SPREJEL NASLEDNJI SKLEP:
 Člen 1
 V točki 40 (Uredba Evropskega parlamenta in Sveta (ES) št. 396/2005) poglavja II Priloge I k Sporazumu EGP se dodata naslednji alinei:
 „—
 32016 R 1822: Uredba Komisije (EU) 2016/1822 z dne 13. oktobra 2016 (UL L 281, 18.10.2016, str. 1),
 —
 32016 R 1866: Uredba Komisije (EU) 2016/1866 z dne 17. oktobra 2016 (UL L 286, 21.10.2016, str. 4).“
 Člen 2
 V točki 54zzy (Uredba Evropskega parlamenta in Sveta (ES) št. 396/2005) poglavja XII Priloge II k Sporazumu EGP se dodata naslednji alinei:
 „—
 32016 R 1822: Uredba Komisije (EU) 2016/1822 z dne 13. oktobra 2016 (UL L 281, 18.10.2016, str. 1),
 —
 32016 R 1866: Uredba Komisije (EU) 2016/1866 z dne 17. oktobra 2016 (UL L 286, 21.10.2016, str. 4).“
 Člen 3
 Besedili uredb (EU) 2016/1822 in (EU) 2016/1866 v islandskem in norveškem jeziku, ki se objavita v Dopolnilu EGP k Uradnemu listu Evropske unije, sta verodostojni.
 Člen 4
 Ta sklep začne veljati 18. marca 2017 pod pogojem, da so bila predložena vsa uradna obvestila v skladu s členom 103(1) Sporazuma EGP (*1).
 Člen 5
 Ta sklep se objavi v sklopu EGP Uradnega lista Evropske unije in Dopolnilu EGP k Uradnemu listu Evropske unije.
 V Bruslju, 17. marca 2017
 Za Skupni odbor EGP
 Predsednik
 Claude MAERTEN
 (1) UL L 281, 18.10.2016, str. 1.
 (2) UL L 286, 21.10.2016, str. 4.
 (*1) Navedena ni nobena ustavna zahteva.