ASTM A790 2507 / 2205 1.4462 / 1.4410 Duplex Welded Tube Bakeng sa Karolo ea lik'hemik'hale ea Indasteri ea Lik'hemik'hale, Khaello ea SPECC1L e lebisa ho botsitso bo eketsehileng ba manonyeletso a spliced ​​le ho fokotseha ho fokolang ha lisele tsa cranial neural crest.

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ASTM A790 2507 / 2205 1.4462 / 1.4410 Duplex Welded Tube Bakeng sa Indasteri ea Lik'hemik'hale

Liaocheng Sihe SS Material Co., Ltd.ke moetsi ea ka sehloohong ea ikhethang ka liphaephe tse se nang seamless tsa tšepe, li-tubes tse khanyang tse nang le li-annealed, li-tubing tse seamless tse kopantsoeng joalo-joalo.E le ho nolofatsa bareki , re boetse re na le liphaephe tse cheselitsoeng le liphaephe.Liaocheng Sihe SS Material Co., Ltd.e na le lisebelisoa tse tsoetseng pele ka ho fetisisa tsa tlhahiso le tlhahlobo.Re ka khotsofatsa tlhoko ea hau ka botlalo.Ho latela maemo a thata haholo, li-tubes tse hlahisoang ke rona li lula li na le mamello e nepahetseng ea OD le WT.Taolo ea mamello e lumellana ka thata le ho hlahisa litekanyetso .Lihlahisoa tsa rona li lula li khotsofetse ke bareki.Bareki ba rekile lihlahisoa tsa rona ba entse phaello e eketsehileng.
a) OD ( Kantle Diameter) : 3.18mm ho 101.6mm
b) WT (Botenya ba Lerako): 0.5mm ho isa ho 20mm
c) Length : Ho ea ka tlhokahalo ea moreki
d) Maemo : ASTM A312;ASTM A269;ASTM A789;ASTM A790 joalo-joalo
e) Mokhoa oa Ts'ebetso: ERW, EFW joalo-joalo

Li-slide tse bonts'ang lingoloa tse tharo selaeteng se seng le se seng.Sebelisa likonopo tse ka morao le tse latelang ho tsamaea ka har'a li-slide, kapa likonopo tsa selaoli se qetellong ho tsamaea selaeteng ka seng.
Lisele tsa cranial neural crest cell (CNCC) li thella mameno a embryonic neural 'me li fallela ho li-arch tsa pharyngeal, tse etsang boholo ba libopeho tse bohareng.Ho se sebetse hantle ha CNCC ho phetha karolo ea bohlokoa ho etiology ea orofacial cleft, phoso e tloaelehileng ea tsoalo.Liphetoho tsa Heterozygous SPECC1L li fumanoe ho bakuli ba nang le mapheo a sa tloaelehang le a syndromic.Mona, re tlaleha ho silafala ho ntlafalitsoeng ha likaroloana tsa canonical adhesive junction (AJ), β-catenin le E-cadherin ka liseleng tse holisitsoeng tsa SPECC1L, le li-micrograph tsa elektronike li bonts'a phallo ea apical-basal ea AJ.Ho utloisisa karolo ea SPECC1L ho craniofacial morphogenesis, re thehile mofuta oa mouse o haellang oa Specc1l.Homozygous mutants ke embryonic lethal mme e bonts'a ho koaloa ha methapo ea methapo e sa sebetseng hantle le CNCC lamination.AJ protheine staining e eketsehile ka mutant neural folds.Phoso ena ea AJ e tsamaisana le sekoli ho CNCC delamination, e hlokang hore AJ e hlahisoe.Ho feta moo, liphetoho tsa Specc11 li fokolitse pontšo ea PI3K-AKT le ho eketseha ha apoptosis.In vitro, thibelo e bonolo ea pontšo ea PI3K-AKT ka liseleng tsa mofuta o hlaha e ne e lekane ho susumetsa liphetoho tsa AJ.Habohlokoa, liphetoho tsa AJ tse bakiloeng ke ho kokota ha SPECC1L li ka khutlisoa ka ts'ebetso ea tsela ea PI3K-AKT.Ha li kopantsoe hammoho, lintlha tsena li fana ka maikutlo a hore SPECC1L, joalo ka molaoli o mocha oa pontšo ea PI3K-AKT le biology ea AJ, ea hlokahala bakeng sa ho koaloa ha methapo ea methapo le stratification ea CNCC.
Lisele tsa Cranial neural crest cell (CNCCs) li lula sebakeng sa dorsal neuroectoderm 'me li itokolla ho neuroepithelium ea methapo e ntseng e hōla ea methapo ka mokhoa o amanang le phetoho ea epithelial-mesenchymal (EMT)1,2,3.Li-CNCC tsa pele tsa li-epithelial li senya li-intercellular junctions 'me li fetoha li-CNCC tsa mesenchymal tse fallang tse tlatsang li-arches tsa pele le tsa bobeli tsa pharyngeal le ho etsa boholo ba lefufuru la craniofacial.Ka hona, liphatsa tsa lefutso tse laolang ts'ebetso ea CNCC hangata li sitisoa ho etiology ea craniofacial congenital anomalies joalo ka orofacial clefts, hangata e amang bana ba 1/800 US feela.E 'ngoe ea bokooa ba congenital8.
Delamination ea CNCC e tsamaisana le ho koaloa ha "neural tube ea anterior" pakeng tsa matsatsi a 8.5 le 9.5 a nts'etsopele ea embryonic ho litoeba.Liphetoho tsa mefuta e mengata e amanang le mouse orofacial cleft-associated le tsona li bontša mofuta o itseng oa neural tube defect, ho akarelletsa le Irf69,10, Ghrl310, Cfl111, le Pdgfrα12.Leha ho le joalo, lits'ebetso tsa ho koaloa ha methapo ea methapo le CNCC stratification li ka nkuoa li ikemetse, joalo ka ha Splotch mutant mouse (Pax3) e bonts'a bofokoli ho koaloa ha methapo ea methapo ntle le phello ho CNCC stratification kapa ho falla 13,14.Mefuta e meng ea litoeba e nang le mefokolo ho CNCC dissection le neural tube closure e tla thusa ho hlalosa motheo o tloaelehileng oa limolek'hule oa lits'ebetso tsena tse peli.
Ho itšehla thajana ha CNCC ho tloha liseleng tsa neuroepithelial ho hloka ho felisoa ha lihlopha tse khomarelang (AJs), tse entsoeng ka li-protein complexes tse nang le, har'a tse ling, E-cadherin, β-catenin, α-E-catenin, le α-actinin e amanang le actin filaments 2. Liphuputso tse feteletseng tsa E-cadherin ka har'a methapo ea methapo li bontšitse ho fokotseha kapa ho lieha ha CNCC delamination.Ka lehlakoreng le leng, khatello ea E-cadherin e fella ka stratification ea pele15,16.Lintho tse ngata tse kenyelletsang EMT nakong ea CNCC stratification ke lintlha tse ngotsoeng (AP2α, Id2, FOXD3, SNAIL, TWIST, SOX10) le extracellular matrix (ECM) liprotheine tse nchafatsang tse kang matrix metalloproteinases (MMPs), leha ho le joalo CNCC ke li-cytoskeletal AJ regulators. ha e so tsejoe.Tsela ea PI3K-AKT e tsejoa ka ho hanyetsa maemo a E-cadherin, haholo-holo ho tsoa lipatlisisong tsa mofets'e17.Liphuputso tsa morao-rao li bontšitse hore tahlehelo ea PI3K-AKT e thehiloeng ho PDGFα-based ho litoeba e lebisa ho sa tloaelehang ha craniofacial, ho kenyelletsa le cleft palate le neural tube defects12.Leha ho le joalo, kamano pakeng tsa tsela ea PI3K-AKT le botsitso ba AJ holim'a stratification ea CNCC ha e hlake.
Re kile ra tsebahatsa SPECC1L e le mofuta oa pele oa phetoho ho batho ba babeli ba nang le lekhalo le tebileng le tlohang molomong ho ea leihlong, le tsejoang ka hore ke oblique cleft (ObFC) kapa Tessier IV18 cleft.Liphetoho tsa SPECC1L li khethiloe malapeng a mabeli a mefuta-futa a nang le lefu la autosomal dominant Opitz G/BBB (OMIM #145410), moo batho ba amehileng ba bonts'itseng hyperdistance le cleft lip/palate19, le lelapeng le leng le nang le Tibi overdistance syndrome (OMIM #145420)20 .ho feta halofo ea linyeoe tsa lefu la Opitz G/BBB ke X-linked (OMIM #300000) 'me li bakoa ke liphetoho tsa lefutso la MID1, le kenyang protheine ea 22 ea skeleton ea sele e amanang le microtubule.Re nahana hore SPECC1L, hape e leng protheine e amanang le microtubules le actin cytoskeleton, e ka 'na ea e-ba lipakeng tsa pontšo e hlokahalang bakeng sa ho lokisoa ha actin cytoskeleton nakong ea ho khomarela lisele le ho falla 18.Ka lithuto tsa in vitro le in vivo, joale re hlalosa SPECC1L e le molaoli o mocha oa botsitso ba AJ ka pontšo ea PI3K-AKT.Boemong ba lisele tsa cellular, khaello ea SPECC1L e ile ea fella ka ho fokotseha ha boemo ba protheine ea pan-AKT le ho eketseha ha apical-basal dispersion ea AJ, e ileng ea felisoa ke ts'ebetso ea lik'hemik'hale ea tsela ea AKT.Ka vivo, mahe a senyehileng a Specc11 a bonts'a ho koaloa ha methapo ea methapo e sa sebetseng hantle le ho fokotsa sekhahla sa CNCC.Ka hona, SPECC1L e sebetsa ka pontšo e laoloang haholo ea ho khomarela lisele tse hlokahalang bakeng sa ts'ebetso e tloaelehileng ea CNCC nakong ea morphogenesis ea sefahleho.
Ho hlahisa karolo ea SPECC1L boemong ba liselefouno, re sebelisitse mohala o tsitsitseng oa osteosarcoma cell line U2OS e haelloang ke SPECC1L18.Lisele tsena tse tsitsitseng tsa U2OS tse nang le SPECC1L (kd) knockdown li bile le ho fokotseha ho itekanetseng (60-70%) maemong a SPECC1L transcripts le liprotheine, hammoho le mefokolo ea ho falla le ho hlophisoa bocha ha actin cytoskeleton 18. Ka lehlakoreng le leng, ho fokotseha ho matla ha nakoana SPECC1L e bontšitsoe hore e lebisa ho bokooa ba mitotic 23.Ha re ntse re hlalosa ka ho eketsehileng, re fumane hore lisele tsa rona tse tsitsitseng tsa SPECC1L-kd li fetotse morpholoji ka tekanyo e phahameng haholo ea confluence (Setšoantšo sa 1).Lisele tsa taolo ea motho ka mong le lisele tsa kd ka har'a confluence e tlase li ne li shebahala li tšoana (Setšoantšo sa 1A, D).Lihora tsa 24 ka mor'a ho kopanya, lisele tsa taolo li ile tsa boloka sebōpeho sa tsona sa cuboidal (setšoantšo sa 1B, E), ha lisele tsa SPECC1L-kd li le telele (setšoantšo sa 1C, F).Tekanyo ea phetoho ena ea sebopeho sa sele e hapiloe ke in vivo live imaging ea lisele tsa taolo le lisele tsa kd (filimi 1).Ho tseba karolo ea SPECC1L liseleng tse kopaneng, re ile ra qala ho hlahloba polelo ea eona.Re fumane hore litekanyetso tsa protheine tsa SPECC1L li eketsehile ha li kopana (Setšoantšo sa 1G), athe litekanyetso tsa SPECC1L ha lia ka tsa eketseha (Setšoantšo sa 1H).Ho phaella moo, ha selefouno e ntse e eketseha, protheine ea SPECC1L e bokelloa meeling ea li-intercellular (setšoantšo sa 2A-E), se nang le mokhoa o kopantsoeng le oa membrane-associated β-catenin (Fig. 2A'-E ').Ka lebaka la ho kopana ha SPECC1L le actin cytoskeleton 18,23 re ile ra nahana hore SPECC1L e sebelisana le li-adhesive junctions tse thehiloeng ho actin (AJ).
(AF) SPECC1L knockdown (DF) lisele tse hole ka phahameng confluence (F) bapisoa le taolo U2OS lisele tse (AC).Mona ho bonts'itsoe lintlha tse tharo ho tse ts'eletseng (T1, T3, T6) tseo re li khethileng bakeng sa density ea lisele tse fapaneng.(G) Tlhahlobo ea blot ea Bophirimela e bontšang hore protheine ea SPECC1L e tsitsitse ka tekanyo e phahameng ea ho kopana ha e bapisoa le tekanyo e tlaase ea ho kopana ha lisele tsa taolo.Western blot ea SPECC1L e bonts'a sehlopha se lebelletsoeng sa 120 kDa le sehlopha se phahameng sa boima ba limolek'hule, mohlomong se fetotsoeng ka morao ho phetolelo (*).Tlhahlobo ea blot ea Bophirimela e ile ea etsoa tlas'a maemo a tšoanang bakeng sa confluence e tlaase le e phahameng.Litšoantšo tse bonts'ang SPECC1L ka kopanelo e tlase le e phahameng li nkuoe sebakeng se le seng.Letlapa le tšoanang le ile la tlosoa 'me la hlahlobjoa hape ka antibody ea β-actin.(H) Tlhahlobo ea palo ea RT-PCR e bontšitse hore ha ho liphetoho tse kholo ho maemo a ngotsoeng a SPECC1L.Li-barbar tsa liphoso li emela li-SEM ho tsoa litekong tse 'ne tse ikemetseng.
(AE) Re khethile lintlha tse tšeletseng tsa nako (T1-T6) tse emelang mefuta e mengata ea lisele ho etsa hore tlhahlobo ea sebopeho sa sele e tloaelehe le liphetoho tsa AJ liseleng tsa U2OS ka SPECC1L knockdown (kd).Lintlha tsa pele tse hlano tsa nako ena li ne li kenyelletsa lisele tse le 'ngoe (T1), 50-70% fusion ea lihlopha tse nyenyane tsa lisele (T2), fusion ntle le ho fetola lisele tsa kd (T3), ho fetola lisele tsa kd (T4), le liphetoho tsa lihora tse 24.ka mokhoa o ka morao oa lisele tsa kd (T5).Protheine ea SPECC1L e ne e hasane ka ho fetisisa ho cytoplasm ho T1 (A), empa ho bokellana ha eona ho ile ha hlokomeloa meeling ea li-intercellular ka nako e latelang (B-E, metsu).(FJ) β-catenin e bontša ho bokellana ho tšoanang meeling ea li-intercellular e amanang le motsoako oa AJ.(A'-E') SPECC1L le β-catenin li bonts'a litšila tse fetang moeling oa lisele ka bongata bo boholo ba lisele (metsu).(F'-J') Liseleng tsa SPECC1L-kd, β-catenin staining e bonahala e tloaelehile ho low cell density (F'-H'), empa ea atoloha ha sebopeho sa sele se fetoha (I', J'; metsu), ho bontša hore AJ li fetohile.Litšepe = 10 µm.
Eaba re leka ho fumana phello ea khaello ea SPECC1L ho AJ.Re sebelisitse matšoao a 'maloa a amanang le AJ, ho kenyelletsa le likarolo tsa canonical F-actin, myosin IIb, β-catenin, le E-cadherin24,25,26,27.Li-fibers tsa khatello ea actin li eketsehile ka lisele tsa SPECC1L-kd joalokaha ho hlalositsoe pele (Setšoantšo sa 3A, B) 18.Myosin IIb e amanang le actin filaments e bontšitse keketseho e tšoanang ea lisele tsa SPECC1L-kd in vitro (Fig. 3C, D).β-catenin e amanang le AJ e tlama ho cadherin ka lera la sele, e bontšang mokhoa o tloaelehileng oa polelo ea "honeycomb" ho laola li-cubocyte (Fig. 3E, G).Hoa thahasellisa hore litšoantšong tse bataletseng li sebelisa confocal microscopy, β-catenin (Fig. 3E, F) le E-cadherin (Fig. 3G, H) e silafatsang lesela la lisele tsa lisele tse haelloang ke SPECC1L tse kopantsoeng li bontšitse mekhoa e hlaheletseng ea litšila tse atolositsoeng.Katoloso ena ea litšila tse amanang le AJ-e amanang le β-catenin liseleng tsa kd e ne e tsebahala haholo nakong ea ho kopana, empa e bonahala e etella pele liphetoho tsa sebopeho sa sele (Setšoantšo sa 2F-J, F'-J').Ho fumana sebopeho sa 'mele oa letheba lena le atolositsoeng la AJ, re ile ra hlahloba meeli ea lisele sebakeng sa apical-basal sa lisele tsa SPECC1L-kd U2OS ka transmission electron microscopy (TEM) (Figure 3I,J).Ho fapana le lisele tse laolang (setšoantšo sa 3I), se neng se e-na le libaka tse arohaneng tsa elektronike tse teteaneng tse bontšang AJ (metsu), lisele tsa kd (setšoantšo sa 3J) se bontšitse libaka tse kholo, tse nang le libaka tse phahameng tsa elektronike tse bontšang AJ hammoho le sefofane sa apicobasal..Ho phaella moo, likarolong tse pota-potileng, re ile ra bona li-membrane tsa lisele tse pharaletseng ka lisele tsa kd (Fig. S1A, B), e hlalosang mokhoa o atolositsoeng oa lihlopha tse silafatsang tsa β-catenin le E-cadherin (Fig. 3F, H).E le ho tšehetsa karolo ea SPECC1L ho AJs, β-catenin e ne e kopantsoe le SPECC1L ka li-lysates tsa lisele tsa U2OS tse nang le confluent (Fig. 3K).Hammoho le immunostaining e atolositsoeng bakeng sa matšoao a AJ, tlhahlobo ea TEM e ne e lumellana le maikutlo a rona a hore khaello ea SPECC1L e eketsa bongata ba AJ apical-basal le phapang.
(AH) Ho eketsa litšila tsa F-actin liseleng tsa kd ka lihora tse 48 ka mor'a fusion (T6; A, B).Lebala le fetotsoeng la myosin IIb le amanang le F-actin (C, D).Mokhoa o boreleli oa membrane ea β-catenin le E-cadherin e silafatsa liseleng tsa taolo (E, G) e ile ea ntlafatsoa ka lisele tsa SPECC1L-kd (F, H).Litšepe = 10 µm.(I-J) Li-micrograph tsa elektrone tse shebileng mateano a apical-basal intercellular.Lisele tsa taolo li bonts'a libaka tse fapaneng tse teteaneng tsa elektrone tse bonts'ang likhohlano tse khomarelang (I, metsu).Ka lehlakoreng le leng, motsoako oohle oa apical-basal ka liseleng tsa SPECC1L-kd o ile oa hlaha e le "electron dense" (J, metsu), e bontšang ho eketseha ha sekhahla le ho hasana ha li-junctions tsa sekhomaretsi.(K) β-catenin e ne e kopantsoe le SPECC1L ka li-lysates tsa U2OS tse kopanetsoeng.Setšoantšo se nkiloeng sebakeng se le seng se emelang e 'ngoe ea liteko tse' nè tse ikemetseng.
Ho utloisisa karolo ea SPECC1L ho craniofacial morphogenesis, re thehile mohlala oa Specc1l o haelloang ke mouse ho sebelisa mela e 'meli e ikemetseng ea ES trap cell, DTM096 le RRH048 (BayGenomics, CA), e emelang intron 1 le Specc1l transcripts e hapuoe ho 15 (Setšoantšo sa 1) .4A, setšoantšo S2).Sebaka sa genomic sa ho kenya vector ea decoy se ile sa khethoa ke tatellano ea genome eohle 'me e tiisitsoe ke PCR (Fig. S2).Meralo ka bobeli ea sefi sa liphatsa tsa lefutso le eona e lumelletse ho kopanngoa ha foreimi ea baqolotsi ba litaba ba Specc11-lacZ ha ba tšoaroa.Ka hona, polelo ea lacZ e khethiloeng ke mabala a X-gal e sebelisitsoe e le sesupo sa polelo ea Specc11.Li-alleles ka bobeli li bonts'itse mekhoa e tšoanang ea polelo ea lacZ, le DTM096 gene trap in intron 1 e bonts'a polelo e matla ho feta RRH048 intron 15 (e sa bonts'itsoeng).Leha ho le joalo, Specc1l e hlalosoa ka ho pharaletseng, ka polelo e matla ka ho khetheha ho li-neural folds ho E8.5 (Setšoantšo sa 4B), ka har'a methapo ea methapo le mekhoa ea sefahleho ho E9.5 le E10.5 (Setšoantšo sa 4C, D), le ho nts'etsopele ea maoto le matsoho. ho E10.5 le mahlo (Setšoantšo sa 4D).Re kile ra tlaleha hore polelo ea SPECC1L karolong ea pele ea pharyngeal e E10.5 e ne e le teng ka har'a epithelium le mesenchyme18 e ka tlaase, e lumellanang le leloko la CNCC.Ho leka polelo ea SPECC1L ho CNCC, re entse E8.5 neural folds (Setšoantšo sa 4E-J) le likarolo tsa lehata tsa E9.5 (Setšoantšo sa 4K-).Ho E8.5, SPECC1L e silafalitse li-neural folds ka matla (Fig. 4E, H), ho kenyelletsa le lisele tse silafalitsoeng ka matšoao a NCC (Fig. 4G, J).Ho E9.5, SPECC1L (setšoantšo sa 4K, N) se silafetseng ka matla se fallang CNCC se kopantsoeng le AP2A (setšoantšo sa 4L, M) kapa SOX10 (setšoantšo sa 4O, P).
(A) Kemiso ea moralo ea mofuta oa mouse ea Specc11 e bontšang ho kenngoa ha vector ea decoy ho ES DTM096 (intron 1) le RRH048 (intron 15) li-cell clones.(BD) lacZ staining ea heterozygous Specc1lDTM096 embryos e emelang Specc1l polelo ho tloha E8.5 ho E10.5.NE = neuroectoderm, NF = neural fold, PA1 = pele pharyngeal arch.(EP) SPECC1L immunostaining e nang le matšoao a NCC AP2A le SOX10 ho E8.5 (NF; EJ) methapo ea methapo le likarolo tsa lehata tsa E9.5 (KP).SPECC1L staining e ne e hlokomeloa haholo ka li-neural folds E8.5 (E, H; li-arrowheads), ho kenyelletsa le lisele tse ngotsoeng ka AP2A (F, G; arrowheads) le SOX10 (I, J; arrowheads).Ho E9.5, SPECC1L li-CNCC tse fallang tse silafetseng ka matla (K, N; metsu) tse ngotsoeng AP2A (L, M; metsu) le SOX10 (O, P; metsu).
Ho tšela pakeng tsa heterozygous Specc1lDTM096/+ le Specc1lRRH048/+ litoeba li bontša hore alleles tse peli tsa gene trap ha li tlatsane le hore motsoako oa li-heterozygote le li-embryonic homozygote bakeng sa mofuta ofe kapa ofe oa trap allele ke lefu le bolaeang embryonic (Letlapa la S1).Likarolo tsa Mendelian li bontšitse ho fokotseha ha lebelo la ho phela ha li-heterozygote nakong ea tsoalo (e lebeletsoeng 1.34 vs. 2.0).Re hlokometse lefu le tlaase la perinatal har'a li-heterozygotes, tse ling li ne li e-na le li-anomalies tsa craniofacial (Fig. S3).Leha ho le joalo, ho kenella ho tlase ha li-perinatal craniofacial phenotypes ho etsa hore ho be thata ho ithuta mekhoa ea bona ea motheo ea pathophysiological.Ka hona, re tsepamisitse maikutlo ho embryonic lethal phenotype ea homozygous Specc11 mutants.
Boholo ba metsoako ea heterozygous kapa homozygous Specc1lDTM096/RRH048 e emolisitsoeng e fetohileng ha ea ka ea ntlafala ka mor'a E9.5-10.5 (Figs. 5A-D), 'me neural tube ha ea ka ea koala ka ntle (Figs. 5B, D) 'me ka linako tse ling e koetsoe ka morao (e sa bontšoang). ..Sekoli sena sa cranial neural tube closure se ne se amahanngoa le boholo ba CNCC e tšoailoeng DLX2 e setseng ho neural folds ho E10.5, e bontšang hore ha ho na dissection (Figure 5A'-D').Ho fumana hore na boholo ba kakaretso ba CNCC le bona bo fokotsehile, re ile ra tšoaea mela ea CNCC le GFP mekhoeng ea rona ea liphatsa tsa lefutso le Wnt1-Cre le ROSAmTmG.Re tsamaisa GFP+ NCC e hlophisitsoeng le GFP- (RFP+) e seng NCC ho tsoa ho mahe a emolisitsoeng.Ho E9.5, karolo ea li-CNCC tse ngotsoeng ka phallo ea GFP ha ea ka ea fetoha haholo pakeng tsa WT le mahe a emolisitsoeng a fetohileng (ha a bontšoe), a bontšang tlhaloso e tloaelehileng ea CNCC.Ka hona, re ile ra nahana hore masala a Wnt1-Cre le DLX2 masala a li-neural folds (Figure 5B') a bakiloe ke CNCC layering e nang le bokooa, mohlomong ka lebaka la ho eketseha kapa ho hasana ha lisele tsa AJ, joalo ka ha ho bonoa liseleng tsa SPECC1L-kd.Re sebelisitse matšoao a NCC SOX10, AP2A, le DLX2 ho netefatsa boteng ba CNCC ka har'a neural fold (Setšoantšo sa 5E-R).Ho E8.5, neural fold staining bakeng sa matšoao a mararo a NCC a ile a bonoa likarolong tsa WT (Fig. 5E, G, I) le Specc1l mutant (Fig. 5F, H, J).Ho E9.5, ha matšoao a NCC a ne a silafatsa NCC e fallang likarolong tsa WT (Fig. 5M, O, Q), masala a NCC a ne a hlokomeloa ka mameno a neural a pepenene a Specc1l mutant embryos (Fig. 5N, P, R).Hobane SOX10 le DLX2 li tšoaea li-CNCC tse fallang, sephetho sena se fana ka maikutlo a hore li-CNCC tse haelloang ke SPECC1L li finyella litlhaloso tsa ka mor'a ho falla empa li hlōleha ho falla ho tloha ho neural folds.
Ho haella ha Specc11 ho lebisa ho koaleheng ha methapo ea methapo e sa sebetseng hantle, delamination ea lisele tsa cranial neural crest le AJs.
(A, B') E9.5 WT (A) Lehe le jereng lisele tsa cranial neural crest cell (CNCC) tse ngotsoeng ka Wnt1-Cre (A').Ka lehlakoreng le leng, Specc11 e emolisitsoeng e fetohileng e bontša mameno a neural a bulehileng (B), lihlooho tsa metsu) le li-CNCC tse so falle (B', lihlooho tsa metsu).(C, D') Litšoantšo tse khanyang (C, D') le immunostaining (C', D') ea CNCC marker DLX2 ea E10.5 WT embryos (C, C') le Specc1l (D, D').Ho li-embryos tsa WT E10.5, DLX2-positive CNCC e bokella li-gill archs (C', metsu), ha li ntse li fetoha, matheba a hlahelletseng a ntse a tsoela pele ka har'a mameno a neural a bulehileng (D', metsu) le lithapong tsa pele tsa pharyngeal (D', metsu).) e nang le matheba a mang (metsu) e bontšang delamination e mpe le ho falla ha CNCC.E. O, P ) le DLX2 (I, J, Q, R).Ho E8.5, ho silafala ha NCC ho ile ha hlokomeloa karolong e hlaha ea neural fold (NF) le likarolo tse fetohileng.Co-staining ea SOX10 le β-catenin ho E8.5 WT (K) le mutant (L) e senotse ho eketseha ha β-catenin ho silafatsa meeli ea sele ka har'a methapo ea methapo.Ho E9.5, ho ile ha hlokomeloa hore ho na le litšila tse hlaha tsa li-CNCC tse fallang (M, O, Q), ha li ntse li fetoha, li-CNCC tse sa tsitsang li ne li silafatsa mameno a neural a bulehileng (N, P, R).(S–Z) In vivo AJ tlhahlobo ea ho ngola likarolong tsa coronal tsa WT le Specc11DTM096/RRH048 embryos ka phetoho ea E9.5.Ho na le sefofane se lekanyelitsoeng se hlahang hukung e kaholimo ho le letona.Likarolong tsa lisele tse fetohileng, ho eketseha ha litšila tsa F-actin (S, T) le myosin IIb (U, V) ho ile ha hlokomeloa.Ho tšoana le liphello tsa in vitro setšoantšong sa 3, li-embryos tse feto-fetohang, ho ntlafala ha membrane bakeng sa β-catenin (W, X) le E-cadherin (Y, Z) ho ile ha hlokomeloa.(AA-BB) Elektrone micrograph ea karolo ea embryo ea mofuta o hlaha e shebileng ka nqane ho moeli oa sele ea apical-basal e bonts'a sebaka se ikhethileng sa elektronike se nang le li-junctions tsa sekhomaretsi (AA, metsu).Ka lehlakoreng le leng, likarolong tsa Specc11 li-embryos tse feto-fetohang (BB, metsu), motsoako oohle oa apicobasal ke electron dense, e bontšang ho eketseha ha sekhahla le ho hasana ha lihlopha tsa sekhomaretsi.
Ho leka maikutlo a rona a hore ho fokotseha ha layering ho bakoa ke AJ e fetotsoeng, re ile ra hlahloba AJ labeling ka mameno a neural a pepenene a Specc1l mutant embryos (Fig. 5S-Z).Re bone ho eketseha ha likhoele tsa khatello ea actin (setšoantšo sa 5S, T) le ho eketseha ha sebaka sa myosin IIB ho silafatsa li-fiber tsa actin (Fig. 5U, V).Habohlokoa, re bone ho eketseha ha litšila tsa β-catenin (setšoantšo sa 5W, X) le E-cadherin (setšoantšo sa 5Y, Z) meeling ea li-intercellular.Re ile ra boela ra hlahloba β-catenin staining ea NCC ka har'a li-neural folds tsa E8.5 embryos (Fig. 5K, L).β-catenin staining e bonahala e le matla ho Specc1l mutant neural folds (Fig. 5L le K), e bontšang hore liphetoho tsa AJ li qalile.Ka electron micrographs ea likarolo tsa lehata tsa E9.5 embryos, re ile ra boela ra bona ho eketseha ha litšila tsa elektronike tse teteaneng ka har'a mahe a Specc1l a fetohileng ha a bapisoa le WT (Fig. 5AA, BB le S1E-H).Ha li kopane, liphetho tsena li tšehetsa liphetho tsa rona tsa in vitro liseleng tsa SPECC1L-kd U2OS 'me li fana ka maikutlo a hore litšila tsa AJ tse sa sebetseng li tla pele ho stratification ea CNCC maheeng a rona a fetohileng.
Ka lebaka la kamano e tsebahalang e hanyetsanang pakeng tsa mosebetsi oa AKT le botsitso ba E-cadherin, 17,28 re ile ra nahana ka ho kenya letsoho ha pontšo ea PI3K-AKT.Ho phaella moo, re ile ra bona ho phatloha ha subepidermal ho tse ling tsa mahe a emolisitsoeng a fetohileng a ileng a phonyoha lefu (<5%) ho E9.5-10.5 'me ho e-na le hoo a lula ho pota E13.5 (setšoantšo sa S3).Li-vesicles tsa Subepidermal ke letšoao la ho fokotseha ha pontšo ea PI3K-AKT e thehiloeng ho PDGFRα12.Fantauzzo et al.(2014) e tlalehile hore ho sitisoa ha PDGFRα-based PI3K activation ho PdgfraPI3K/PI3K embryos mutant ho fella ka li-subepidermal vesicles, neural tube defects, le cleft palate phenotypes.Ka sebele, maemo a pan-AKT le Ser473-AKT e sebetsang ea phosphorylated e fokotsoe ka vivo ho Specc1l mutant tissues ho E9.5 embryonic arrest (Fig. 6A-D).Ho fokotseha ha maemo a phosphorylated Ser473-AKT e ka ba ka ho feletseng ka lebaka la ho fokotseha ha maemo a pan-AKT ka vivo (FIG. 6E) le in vitro (FIG. 6F).Ho fokotseha ha in vitro ho ile ha hlokomeloa feela ha lisele tsa U2OS li ne li kopana ka matla le liphetoho tsa sebopeho sa sele le AJ density (Figure 6D).Ka hona, lintlha tsa rona li fana ka maikutlo a hore SPECC1L ke molaoli o mocha oa pontšo ea PI3K-AKT ho craniofacial morphogenesis.
(A–E) E8.5 (A,B) le E9.5 (C,D) likarolo tsa lehata kapa E9.5 lysates ho tloha Specc1l mutant embryos (E) e bontšang maemo a phosphorylated S473-AKT e sebetsang le phokotso ea protheine ea pan-AKT , ha e bapisoa le taolo ea WT.Ho senya Bophirimela ho ne ho etsoa ka li-lysates tsa mofuta o hlaha le li-mutant lysates tlas'a maemo a tšoanang.Litšoantšo tse bonts'itsoeng bakeng sa SPECC1L li nkuoe letlapeng le le leng.Letlapa le tšoanang le ile la tlosoa 'me la hlahlojoa hape ka li-anti-pan-ACT le β-actin antibodies.Maemo a pan-AKT ho E8.5 neural folds (A, B) le maemo a phosphorylated S473-AKT likarolong tsa lehata tsa E9.5 a fokotsehile haholo.(F) Maemo a Pan-AKT a ile a fokotsoa ka mokhoa o ts'oanang ho li-lysate tsa lisele tsa SPECC1L-kd U2OS tse kotuloang ka confluence e phahameng.Mehala ea liphoso e emela li-SEM ho tsoa ho lipalo tse tharo tse ikemetseng tsa Western blot.(GJ) Likarolo tsa li-embryo tsa WT ho E9.5 tse silafalitsoeng ka KI67 le caspase 3 e phunyeletsoeng, ka ho latellana, e bontšang ho ata ha lisele (G, G') le mosebetsi o monyenyane oa apoptotic (H, H').Li-embryos tsa Specc11 tse fetotsoeng li bonts'a ho ata ha sele (I), empa palo ea lisele tse fetang apoptosis e eketsehile haholo (J).
Eaba re hlahloba matšoao a ho ata le apoptosis.Ha rea ​​ka ra bona phapang leha e le efe ea ho ata ha li-embryos tsa E9.5 (Setšoantšo sa 6E, G ha se bapisoa le I) ka index ea ho ata ea 82.5% bakeng sa liphetoho tsa WT le 86.5% bakeng sa liphetoho tsa Specc1l tse lekantsoeng ke staining ea KI67 (p <0.56, Fisher's tekolo hantle).Ka mokhoa o ts'oanang, ha rea ​​ka ra bona phapang leha e le efe ea apoptosis e lekantsoeng ka ho silafatsa caspase 3 ka mameno a neural ho E8.5 ho fihlela ho tšoaroa ha embryo (e sa bontšoang) (e sa bontšoang).Ka lehlakoreng le leng, apoptosis e ne e eketsehile haholo ho li-embryo tsohle tsa E9.5 tse fetotsoeng (setšoantšo sa 6F, H le J).Keketseho ena ka kakaretso ea apoptosis e lumellana le pontšo e fokotsehileng ea PI3K-AKT le lefu la pele la embryonic29,30,31.
Ka mor'a moo, ho netefatsa karolo ea sesosa bakeng sa pontšo ea PI3K-AKT ho liphetoho tsa AJ liseleng tsa rona tsa kd, re ile ra fetola lik'hemik'hale tsela ea taolo le lisele tsa kd (Setšoantšo sa 7A-F).Re sebelisitse e le lesupa phetoho ea sebopeho sa sele e bonoang ka har'a lisele tse kopaneng tsa SPECC1L-kd, tseo re ileng ra li lekanya ho sebelisa karo-karolelano ea boholo bo bolelele (bolelele) ho ea boemong bo otlolohileng (bophara).Karolelano ea 1 e lebeletsoe bakeng sa lisele tse batlang li pota-potile kapa tsa cuboidal (Setšoantšo sa 7G).Ntle le sebopeho sa sele, re boetse re netefalitse phello ea AJ ka β-catenin staining (Fig. 7A'-F ').Ho thibela tsela ea PI3K-AKT ho sebelisa wortmannin ho ne ho lekane ho fetola sebopeho sa sele liseleng tsa taolo (Setšoantšo sa 7A, C) le AJ (Setšoantšo sa 7A ').PI3K-AKT activator SC-79 ha e ea ama sebopeho sa sele (FIG. 7A, E) kapa katoloso ea AJ (FIG. 7A') liseleng tsa taolo.Liseleng tsa SPECC1L-kd, ho hatelloa ho eketsehileng ha tsela ea PI3K-AKT ho ile ha fella ka ho eketseha ha apoptosis (setšoantšo sa 7B, D) le ho eketseha ho hoholo ha β-catenin staining (Fig. 7B '), e lumellanang le liphetoho tsa rona tse boima tsa vivo.Habohlokoa, ts'ebetso ea tsela ea PI3K-AKT e ntlafalitse haholo sebopeho sa sele (Setšoantšo sa 7B, F) le AJ phenotypes (Setšoantšo sa 7B").Liphetoho tsa sebopeho sa sele li ne li lekantsoe e le karo-karolelano ea selefouno (CCR) 'me e bapisoa le bohlokoa bo hlalositsoeng ka holimo (FIG. 7G).Ka sebele, liseleng tsa taolo (setšoantšo sa 7G, CCR = 1.56), phekolo ea wortmannin e ne e lekane ho fetola haholo sebōpeho sa sele (setšoantšo sa 7G, CCR = 3.61, p <2.4 × 10-9) ho isa tekanyong e tšoanang le e hlokometsoeng. ho SPECC1L.-kd lisele (setšoantšo sa 7G, CCR = 3.46).Kalafo ea Wortmannin ea lisele tsa SPECC1L-kd (setšoantšo sa 7G, CCR = 3.60, se sa tsotelleng) e ne e se sa bohlokoa ho feta lisele tsa kd tse sa tšoaroeng (setšoantšo sa 7G, CCR = 3.46, se sa tsotelleng) kapa lisele tse laolang li-wortmannin (setšoantšo sa 7G)., CCR = 3.46, ha e na thuso) ho phaella moo e ama selelele e telele (7G, CCR = 3.61, e hlokomoloha).Habohlokoa ka ho fetisisa, SC-79 AKT activator e tsosolositse phenotype e telele ea lisele tsa SPECC1L-kd (Fig. 7G, CCR = 1.74, p <6.2 × 10-12).Liphetho tsena li tiisa hore SPECC1L e laola pontšo ea PI3K-AKT 'me e fana ka maikutlo a hore ho fokotseha ho itekanetseng ho SPECC1L ho ama ho khomarela lisele, ha ho fokotseha ho matla ho lebisa ho apoptosis (setšoantšo sa 8).
(A–F') Control (A, C, E) le SPECC1L-kd (B, D, F) lisele tse tšoaroang ka PI3K-AKT pathway inhibitor wortmannin (C, D) kapa SC-79 activator (E, F) Treatment .Lisele tsa taolo tse sa phekoloeng li na le li-cuboidal (A) tse nang le litšila tse tloaelehileng tsa β-cat cellular (A'), ha lisele tsa kd li lelefalitsoe (B) ka ho eketseha ha β-cat staining (B').Kamora ho hatelloa ha tsela ea PI3K-AKT, lisele tsa taolo li lelefalitsoe (C) ka katoloso ea β-cat (C'), ha lisele tsa kd li qala ho ba le apoptosis (D), joalo ka mahe a emolisitsoeng a fetohileng haholo 'me a bonts'a β-cat e ntlafalitsoeng haholo.litšila (D').Ka mor'a ts'ebetso ea tsela ea PI3K-AKT, lisele tsa taolo li ile tsa lula li le cuboidal (E) 'me li na le litšila tse tloaelehileng tsa β-cat (E'), ha lisele tsa kd li ne li bontša ho ntlafatsa haholo sebopeho sa sele (F) le β-cat (F'), se bontšang (G) Tekanyo ea phetoho ea sebopeho sa sele ho (AF) e ile ea lekanyetsoa ho sebelisoa tekanyo ea sele e pota-potileng (CCR) ea tekanyo e telele ka ho fetisisa (bolelele) le tekanyo e lumellanang e emeng (bophara) ho sebelisoa software ea MetaMorph.Lisele tse sa phekoloeng (NT) SPECC1L-kd (CCR = 3.46) li ne li le telele haholo ho feta lisele tse laolang (CCR = 1.56, p <6.1 × 10-13).Thibelo ea Wort ea tsela ea PI3K-AKT liseleng tsa taolo e ne e lekane ho baka bolelele bo tšoanang ka sebopeho sa sele (CCR = 3.61, p<2.4 × 10-9).Ka mokhoa o ts'oanang, ts'ebetso ea AKT ke SC-79 liseleng tsa SPECC1L-kd e tsosolositse selelele ea sele ho ea maemong a taolo (CCR = 1.74, p <6.2 × 10-12).Kalafo ea Wortmannin ea lisele tsa SPECC1L-kd e ile ea fella ka ho eketseha ha apoptosis empa ha ho na keketseho e 'ngoe ea phetoho ea sebōpeho sa sele (CCR = 3.60) ha e bapisoa le kd e sa sebetsoeng (CCR = 3.46, ns) kapa lisele tse laolang phekolo ea wortmannin (3.61) e hlokometsoeng ka .ns = ha ho tsotellehe.+/- Litekanyo tsa SEM bakeng sa lisele tse 50 lia bontšoa.Liphapano tsa lipalo li baloa ho sebelisoa tlhahlobo ea t ea Seithuti.
(A) Boemeli ba moralo oa ho thibela le ho kenya tšebetsong tsela ea PI3K-AKT e bakang liphetoho tsa AJ le pholoso, ka ho latellana.(B) Mohlala o reriloeng bakeng sa botsitso ba protheine ea AKT ka SPECC1L.
Li-CNCC tsa pele li hloka hore AJ lysis e arohane le lisele tse ka hare tsa neural fold neuroepithelial cell1,15,32.Ho eketseha ha litšila tsa likarolo tsa AJ le tahlehelo ea apical-basal AJ asymmetric distribution ka lisele tse haelloang ke SPECC1L ka bobeli ka hare ho vitro le ka vivo, hammoho le kamano e haufi ea SPECC1L ho β-catenin, e fana ka maikutlo a hore SPECC1L e sebetsa ho boloka botsitso ba sebaka sa AJ hantle bakeng sa mesifa ea mokhatlo.actin cytoskeleton.Mokhatlo oa SPECC1L le actin cytoskeleton le β-catenin le keketseho ea palo ea condensed actin filaments ka ho ba sieo ha SPECC1L e lumellana le keketseho e hlokometsoeng ea AJ density.Monyetla o mong ke hore palo e ntseng e eketseha ea li-actin fibers liseleng tse haelloang ke SPECC1L li lebisa phetohong ea tsitsipano ea intercellular.Hobane khatello ea cellular e ama matla a AJ 33, liphetoho tsa motlakase li ka fella ka ho ata ha AJ 34 haholoanyane.Kahoo liphetoho leha e le life li tla ama likarolo tsa CNCC.
Wnt1 e hlalosoa ka mameno a pele a methapo a hlahisang lisele tsa neural crest.Kahoo, Wnt1-cre track tracking e tšoaea NCC35 pele le ho falla.Leha ho le joalo, Wnt1 e boetse e tšoaea li-clones tsa lisele tsa boko ba mokokotlo le tsona tse nkiloeng ho li-neural folds tsa pele 35,36, e leng se etsang hore ho be le monyetla oa hore ho silafala ha rona ka liphetoho tsa E9.5 bakeng sa matšoao a Wnt1 mamenong a neural a bulehileng ha se CNCC.Matheba a rona a matle bakeng sa matshwao a NCC AP2A le SOX10 a netefalitse hore mameno a neural a pepeneneng a Specc11 mutant embryos ehlile a na le CNCC.Ho phaella moo, kaha AP2A le SOX10 ke matšoao a NCC ea pele e falla, litšila tse ntle li bontšitse hore lisele tsena ke CNCC ea ka mor'a ho falla e ke keng ea aroloa ke E9.5.
Lintlha tsa rona li fana ka maikutlo a hore taolo ea limolek'hule ea AJ ka SPECC1L e fetisoa ke pontšo ea PI3K-AKT.Letšoao la AKT le fokotsehile ka lisele tse haellang tsa SPECC1L le lisele.Liphuputso ka Fantauzzo et al.tšehetsa karolo e tobileng bakeng sa pontšo ea PI3K-AKT ho craniofacial morphogenesis.(2014) e bontšitse hore ho haella ha ts'ebetso ea pontšo ea PDGFRα-based PI3K-AKT e lebisa ho phenotype e hlakileng ea palate.Re boetse re bontša hore ho thibela tsela ea PI3K-AKT ho lekane ho fetola AJ le sebopeho sa sele ka lisele tsa U2OS.Tumellanong le seo re se fumaneng, Kaine le ba bang.37 e bontšitse hore ho fokotseha ha karoloana ea PI3K α110 liseleng tsa endothelial ho fella ka keketseho e tšoanang ea pericellular β-catenin staining, eo ho thoeng ke keketseho ea "index ea khokahanyo".Leha ho le joalo, liseleng tsa endothelial tseo li-filaments tsa actin li seng li hlophisitsoe haholo, ho hatelloa ha tsela ea PI3K-AKT ho fella ka sebopeho sa sele se hlephileng.Ka lehlakoreng le leng, lisele tsa SPECC1L-kd U2OS li bonts'itse sebopeho se selelele sa sele.Phapang ena e ka ba mofuta o ikhethileng oa lisele.Le hoja ho hatelloa ha pontšo ea PI3K-AKT ho ama ka ho sa feleng actin cytoskeleton, phello ea sebopeho sa sele e khethoa ke liphetoho tsa tsitsipano e bakoang ke liphetoho tsa letsoalo le mokhatlo oa li-fiber tsa actin tse bohareng.Ka liseleng tsa U2OS, re sebelisitse feela liphetoho tsa sebopeho sa sele e le letšoao la phetoho le ho hlaphoheloa ha SPECC1L e haelloang ke AJ.Qetellong, re nahana hore ho thibela tsela ea AKT ka khaello ea SPECC1L ho eketsa botsitso ba AJ le ho fokotsa delamination ho CNCC.
Hoa thahasellisa hore maemo a pan-AKT a fokotsoe ka in vitro le ka vivo ho phaella ho phosphorylated 473-AKT maemo a ho ba sieo ha SPECC1L, ho fana ka maikutlo a ho laola pontšo ea PI3K-AKT boemong ba AKT protheine botsitso kapa phetoho.Liphatsa tsa lefutso tsa SPECC1L le MID1, tse peli tse amanang le lefu la Opitz / GBBB, li kenyelletsa liprotheine tse tsitsitseng microtubules 18,22.Mokhoa oo SPECC1L le MID1 ba kopanyang botsitso ba microtubule ha bo utloisisoe ka botlalo.Tabeng ea SPECC1L, ho tsitsisa hona ho kenyelletsa ho ntlafatsa acetylation ea subset ea microtubules 18.Ho ka etsahala hore SPECC1L e sebelise mokhoa o ts'oanang oa ho tsitsisa liprotheine tse ling tse kang AKT.Ho bontšitsoe hore acetylation ea masala a lysine ka protheine ea AKT e lebisa ho fokotseha ha membrane localization le phosphorylation38.Ho feta moo, ho hlokahala hore ho behoe hohle ha ketane ea K63 sebakeng se tšoanang sa lysine ho AKT bakeng sa sebaka sa eona sa membrane le ho kenya tšebetsong39,40.Har'a lintlha tse 'maloa tse sebetsanang le liprotheine tsa SPECC1L tse khetholloang ka mefuta e sa tšoaneng ea tomoso ea li-skrini tse peli tse nyalisitsoeng, tse' nè - CCDC841, ECM2942, APC le UBE2I43 - li kenyelelitsoe phetohong ea liprotheine kapa botsitso ka hohle kapa ho sumoylation.SPECC1L e ka 'na ea ameha ho fetoleng ka morao ho phetolelo ea masala a AKT lysine, e amang botsitso ba AKT.Leha ho le joalo, karolo ea bohlokoa ea SPECC1L sebakeng sa sebaka le botsitso ba protheine ea AKT e ntse e lokela ho hlakisoa.
Bofokoli bo matla ho polelo ea SPECC1L ka vivo bo bakile ho eketseha ha lets'oao la AJ le ho koaheloa ka phoso ho CNCC, hammoho le ho eketseha ha apoptosis le lefu la pele la embryonic.Litlaleho tse fetileng li bontšitse hore liphetoho tsa mouse tse nang le maemo a eketsehileng a apoptosis li amahanngoa le bokooa ba neural tube 44,45,46,47 le craniofacial defects48.Ho 'nile ha boleloa hore lefu le feteletseng la sele ka har'a li-neural folds kapa li-pharyngeal arches li ka fella ka palo e sa lekaneng ea lisele tse hlokahalang bakeng sa motsamao o nepahetseng oa morphogenetic 48,49,50.Ka lehlakoreng le leng, lisele tsa rona tsa SPECC1L tse haelloang ke lisele tse nang le polelo e fokolang ea SPECC1L li bontšitse feela liphetoho tsa AJ ntle le bopaki ba ho eketseha ha lisele tsa lefu.Leha ho le joalo, ho thibela lik'hemik'hale tsa tsela ea PI3K-AKT liseleng tsena tsa Kd ho ile ha fella ka ho eketseha ha apoptosis.Ka hona, ho fokotseha ho itekanetseng ha polelo kapa ts'ebetso ea SPECC1L ho netefatsa hore lisele li phela.Sena se lumellana le tlhokomeliso ea hore mahe a sa tloaelehang a Specc11 a fetotsoeng a phonyohang ho tšoaroa St.E9.5-mohlomong ka lebaka la ho fokotsa matla a ho tšoara liphatsa tsa lefutso-ba khona ho koala methapo ea bona ea methapo le ho emisa hamorao tsoelo-pele, hangata ba e-na le bokooa ba craniofacial (Fig. S3).Hape ho lumellana le sena ke ketsahalo e sa tloaelehang ea mahe a mahe a bitsoang "heterozygous Specc1l" a nang le lintho tse sa tloaelehang tsa craniofacial-mohlomong ka lebaka la ho eketseha ho matla ha liphatsa tsa lefutso-hammoho le ho fumanoa ho zebrafish moo e 'ngoe ea li-orthologues tse peli tsa SPECC1L (specc1lb) e bakang morao embryonic phenotypes, ho akarelletsa le tahlehelo ea mehlahare e ka tlaase le maphao a mahlakore a mabeli51.Ka hona, liphetoho tsa ho lahleheloa ke ts'ebetso ea heterozygous SPECC1L tse khetholloang ho bakuli ba batho li ka 'na tsa baka tšenyo e nyenyane mosebetsing oa SPECC1L nakong ea craniofacial morphogenesis, e lekaneng ho hlalosa liphahlo tsa bona tsa orofacial.Taolo e thehiloeng ho SPECC1L ea li-intercellular contacts e ka boela ea phetha karolo ea palatogenesis le fusion ea li-arches tsa pharyngeal.Lithuto tse ling tsa ts'ebetso ea SPECC1L li tla thusa ho hlakisa karolo ea likhokahano tsa nakoana tsa li-intercellular ho CNCC nakong ea ho koaloa ha methapo ea methapo ho neuroepithelial cell motility le craniofacial morphogenesis.
Taolo ea U2OS osteosarcoma le lisele tsa SPECC1L-kd li hlalositsoe pejana (Saadi et al., 2011).Li-antibodies khahlanong le SPECC1L le tsona li 'nile tsa tsejoa pele (Saadi et al., 2011).Anti-β-catenin antibodies (mmutla; 1: 1000; Santa Cruz, Dallas, TX) (toeba; 1: 1000; Cell Signaling Technology, Danvers, MA), myosin IIb (1: 1000; Sigma-Aldrich, St. Louis) ) , MO) ), E-cadherin (1:1000; Abkam, Cambridge, MA), AP2A (1:1000; Novus Biologicals, Littleton, Colo.), SOX10 (1:1000; 1000; Aviva Systems Biology, San Diego , California), DLX2 (1:1000; Abcam, Cambridge, MA), phospho-Ser473-AKT (1:1000; Cell Signaling Technology, Danvers, MA), pan-AKT (1:1000; ThermoFisher Scientific, Waltham, MA ), KI67 (1: 1000; Cell Signaling Technology, Danvers, MA), e phunyeletse caspase 3 (1: 1000; Cell Signaling Technology, Danvers, MA) le β-actin (1: 2500; Sigma-Aldrich, St. Louis, MO ) e sebelisitsoe joalo ka ha ho hlalositsoe..Li-filaments tsa Actin li ne li silafalitsoe ke Acti-stain rhodamine phalloidin (Cytoskeleton, Denver, Colorado).
Lisele tsa taolo tsa U2OS le lisele tsa SPECC1L-kd li ile tsa hlahisoa ka DMEM ea tsoekere e phahameng e tlatsitsoeng ka 10% ea fetal bovine serum (Life Technologies, Carlsbad, CA).Bakeng sa liphetoho tsa AJ, lisele tsa 2 x 105 li ile tsa kenngoa ka khalase e tšoaroang ka 0.1% porcine gelatin (Sigma-Aldrich, St. Louis, MO) 'me e hlokometsoe bakeng sa liphetoho tsa sebōpeho sa sele.Lisele li ile tsa bokelloa ka linako tse fapaneng tse bontšitsoeng: lihora tsa 4 ka mor'a ho jala peo (t = 1), lihora tse 24 ka mor'a peo (t = 2), confluence ntle le phetoho ea sebopeho sa sele (t = 3), phetoho ea sebopeho sa sele (t = 4) , 24 h ka mor'a phetoho ea sebōpeho sa sele (t = 5) le 48 h ka mor'a phetoho ea sebopeho sa sele (t = 6) (setšoantšo sa 1, 2, 3).Ho fetola tsela ea PI3K-AKT, lisele li ile tsa hlahisoa ka mokhoa o bontšitsoeng ka PI3K-AKT inhibitor wortmannin (TOCRIS Biosciences, Minneapolis, Minnesota) kapa SC-79 activator (TOCRIS Biosciences, Minneapolis Adams, Minnesota).Mocheso o nang le lik'hemik'hale o ne o fetoloa letsatsi le letsatsi.
Lirekoto tsa foreimi li entsoe ka taolo e phelang le lisele tsa KD tlas'a maemo a tloaelehileng a setso, 'me litšoantšo tse fapaneng tsa mekhahlelo li ne li bokelloa metsotso e meng le e meng ea 10 bakeng sa matsatsi a 7.Litšoantšo li ile tsa fumanoa ho sebelisoa microscope e laoloang ke k'homphieutha ea Leica DM IRB e nang le sethala sa mechine le sepheo sa 10 × N-PLAN se amanang le khamera ea QImaging Retiga-SRV.Nakong ea litšoantšo, litloaelo tsa lisele li ne li bolokiloe ho 37 ° C sebakeng se mongobo se nang le 5% CO2.
Mehala e 'meli ea liphatsa tsa lefutso tsa ES cell line DTM096 le RRH048 ho tloha Regional Mutant Mouse Resource Center (UC Davis, CA) li ile tsa sebelisoa ho hlahisa mela ea mouse e haellang ea Specc11, e khethiloeng Specc1lgtDTM096 le Specc1lgtRRH046.Ka bokhutšoanyane, lisele tse 129/REJ ES li ile tsa kenngoa ka har'a li-blastocysts tsa C57BL6.Litoeba tse tona tsa chimeric tse hlahisitsoeng li ile tsa hlahisoa ka litoeba tsa C57BL6 tse tšehali ho tsebahatsa bana ba nang le 'mala oa seaparo sa agouti.Ho ba teng ha liphatsa tsa lefutso tsa liphatsa tsa lefutso ho ne ho sebelisetsoa ho khetholla li-heterozygotes.Litoeba li ne li bolokiloe mokokotlong o tsoakiloeng oa 129 / REJ; C57BL6.Sebaka sa sebaka sa ho kenngoa ha mochine oa liphatsa tsa lefutso se tiisitsoe ke RT-PCR, tatellano ea liphatsa tsa lefutso, le ho tlatsana ha liphatsa tsa lefutso (Setšoantšo sa Tlatsetso 1).Ho sala morao moloko oa CNCC oa litoeba tse habeli tsa heterozygous Specc1lGT, ROSAmTmG (#007576) le Wnt1-Cre (#003829) litoeba (Jackson Laboratory, Bar Harbor, ME) li ile tsa tšeloa ho hlahisa ROSAmTmG le Wnt1-Cre allele ho Specc1l mutant embryos.Liteko tsohle tsa litoeba li entsoe ho latela melaoana e amohetsoeng ke Komiti ea Setheo ea Tlhokomelo ea Liphoofolo le Tšebeliso ea Setsi sa Bongaka sa Univesithi ea Kansas.
Li-embryo li ne li tsitsitse (1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2, 0.02% NP-40, 5 mM EGTA) bakeng sa 60 min mocheso oa kamore.Ka mor'a ho lokisa motsoako oa X-gal staining (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, 1 mg/ml X-gal) Tsoelo-pele ea letheba e entsoe ka 37°C. .°C nakong ea lihora tse 1-6.Li-embryo li ile tsa lokisoa ka morao ho 4% PFA 'me tsa bonoa.
Bakeng sa ho thibela Bophirimela, lisele li ne li lysed ka passive lysis buffer (Promega, Fitchburg, WI) e tlatsitsoeng ka motsoako oa HALT protease inhibitors (Sigma-Aldrich, St. Louis, MO).Li-lysates li ne li sebelisoa ka 12% polyacrylamide Mini-PROTEAN TGX li-gel tse itokiselitseng (Bio-Rad, Hercules, CA) 'me li fetisetsoa ho Immobilon PVDF membranes (EMD Millipore, Billerica, MA).Li-membrane li ne li koetsoe ka lebese la 5% ho PBS e nang le 0.1% Tween.Lithibela-mafu li ile tsa kenngoa bosiung ba 4°C kapa hora e le ’ngoe mochesong oa kamore.Femto SuperSignal West ECL reagent (Thermo Scientific, Waltham, MA) e ne e sebelisetsoa ho hlahisa lipontšo.Bakeng sa immunostaining, mahe a emolisitsoeng a ile a ts'oaroa bosiu bo le bong ho 4% PFA/PBS mme a bolokoa.Li-tissue cryosections li ne li koetsoe ka PBS e nang le 1% ea serum ea pōli e tloaelehileng (Thermo Scientific, Waltham, MA) le 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) 'me ea kenngoa ka 4°C ka har'a incubator nakong ea bosiu.e nang le anti-anti-antibody le anti-antibody le fluorescent secondary antibody (1:1000) bakeng sa hora e le 1 ho 4°C.Likarolo tse silafalitsoeng li ile tsa behoa ka har'a proLong khauta medium (Thermo Scientific, Waltham MA) 'me litšoantšo tse bataletseng li ile tsa fumanoa ho sebelisoa microscope ea Leica TCS SPE confocal.Thibelo e 'ngoe le e 'ngoe ea ho itšireletsa mafung e ile ea etsoa e le liteko tse tharo tse ikemetseng mabapi le li-cirossections tsa bonyane mahe a mabeli a fetohileng.Ho bontšoa teko ea moemeli.
Lisele li ne li kentsoe ka har'a buffer e fetotsoeng ea RIPA (20 mM Tris-HCl, pH 8.0, 1% NP-40, 130 mM NaCl, 10% glycerol, 2 mM EDTA, le HALT protease inhibitor (Sigma-Aldrich, St. Louis, MO) Ka bokhutšoanyane, li-lysate li ne li hloekisitsoe ka lifaha tsa magnetic G (Life Technologies, Carlsbad, CA) 'me tsa kenngoa bosiu bo le bong ka 4 ° C. ka li-anti-SPECC1L kapa IgG protheine G lifaha li ile tsa sebelisoa ho ntša SPECC1L le Western blotting e ne e etsoa ho sebelisoa anti-SPECC1L kapa IgG protein G. -β-catenin antibody e hlalositsoeng ka holimo Liteko tsa co-IP tse bontšitsoeng li emela liteko tse 'nè tse ikemetseng.
Lisele tse tsitsitseng kapa li-embryonic tissues li ile tsa fanoa setsing sa electron microscopy se Univesithing ea Kansas Medical Center.Ka bokhutšoane, lisampole li ile tsa kenngoa ho EMbed 812 resin (Electron Microscopy Sciences, Fort Washington, PA), e entsoeng ka polymer bosiung ba 60 ° C, 'me e arotsoe ka 80 nm ho sebelisa Leica UC7 ultramicrotome e nang le lehare la daemane.Likarolo li ile tsa bonoa ho sebelisoa microscope ea elektronike ea JEOL JEM-1400 e nang le sethunya sa 100 kV Lab6.
Mokhoa oa ho qotsa sengoloa sena: Wilson, NR et al.Khaello ea SPECC1L e lebisa ho botsitso bo eketsehileng ba manonyeletso a kopantsoeng le ho fokotsa delamination ea lisele tsa cranial neural crest.saense.6, 17735;doi:10.1038/srep17735 (2016).
Saint-Jeanne, J.-P.Induction le phapang ea neural crest.(Springer Science + Business Media; Landes Bioscience/Eurekah.com, 2006).
Cordero, DR et al.Lisele tsa Cranial neural crest tse tsamaeang: karolo ea tsona ho nts'etsopele ea craniofacial.American Journal of Medical Genetics.Karolo ea A 155A, 270-279, doi:10.1002/ajmg.a.33702 (2011).
Boland, RP Neurocristopathia: kholo le nts'etsopele ea eona ho feta lilemo tse 20.Ngaka ea bana.pathology.laboratori.moriana.17, 1–25 (1997).
Mangold E., Ludwig KU le Noten MM Katleho ho liphatsa tsa lefutso tsa maphao a orofacial.Mekhoa ea Meriana ea Limolek'hule 17, 725-733, doi: 10.1016 / j.molmed.2011.07.007 (2011).
Minu, M. le Riley, Mekhoa ea limolek'hule ea FM ea ho falla ha lisele tsa cranial neural crest le mohlala nakong ea nts'etsopele ea craniofacial.Ntlafatso ea 137, 2605-2621, doi: 10.1242 / dev.040048 (2010).
Dixon, MJ, Marazita, ML, Beaty, TH le Murray, JK Cleft molomo le mahalapa: ho utloisisa tšusumetso ea liphatsa tsa lefutso le tikoloho.maikutlo a tlhaho.Genetics 12, 167-178, doi: 10.1038/nrg2933 (2011).
Ingram, CR le ba bang.Morphogenesis e sa tloaelehang ea letlalo, maoto le matsoho, le sebaka sa craniofacial ho interferon-regulating factor-6 (Irf6) -e haelloang ke litoeba.Genette ea Sechaba.38, 1335–1340, doi: 10.1038/ng1903 (2006).
Peyrard-Janvid, M. et al.Liphetoho tse kholo ho GRHL3 li baka lefu la van der Waord le ho sitisa nts'etsopele ea periderm ea molomo.Am J Hum Genet 94, 23–32, doi: 10.1016/j.ajhg.2013.11.009 (2014).
Harris, MJ le Juriloff, DM Ntlafatsa lenane la liphetoho tsa litoeba tse nang le bokooa ho koaloa ha methapo ea methapo le tsoelo-pele e lebisang kutloisisong e felletseng ea liphatsa tsa lefutso tsa ho koaloa ha methapo ea methapo.Patlisiso ea bokooa ba tsoalo.Karolo ea A, Teratology ea Clinical le Molecular 88, 653-669, doi: 10.1002 / bdra.20676 (2010).
Fantauzzo, KA & Soriano, P. PI3K-mediated PDGFRalpha signaling e laola ho pholoha le ho ata ha nts'etsopele ea masapo ka tsela ea p53 e itšetlehileng ka intracellular.Gene Development 28, 1005-1017, doi: 10.1101 / gad.238709.114 (2014).
Kopp, AJ, Green, ND le Murdoch, JN Disheveled: Kamano ea ho atolosoa ha converrgent ho koala neural tube.Mekhoa ea methapo ea kutlo.26, 453-455, doi: 10.1016 / S0166-2236 (03) 00212-1 (2003).