I-ASTM A790 2507 / 2205 1.4462 / 1.4410 I-Duplex Welded Tube yecandelo lemichiza yoShishino lweMichiza, Ukunqongophala kwe-SPECC1L kukhokelela ekuzinzeni okwandisiweyo kwamalungu adityanisiweyo kunye nokuncipha kokuchithwa kweeseli ze-neural crest.

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I-ASTM A790 2507 / 2205 1.4462 / 1.4410 Duplex Welded Tube yoShishino lweMichiza

ILiaocheng Sihe SS Material Co., Ltd.ngumvelisi ohamba phambili okhethekileyo kwimibhobho yentsimbi engenamthungo , iityhubhu ezikhanyayo eziqholiweyo , iityhubhu eziphothiweyo ezingenamthungo njl njl .Ukuququzelela abathengi , sinemibhobho edityanisiweyo kunye netyhubhu.ILiaocheng Sihe SS Material Co., Ltd.inezona zixhobo ziphambili zokuvelisa nokuvavanya .Sinokwanelisa ngokupheleleyo imfuno yakho.Ngokomgangatho ongqongqo kakhulu , iityhubhu eziveliswe sithi zihlala zine-OD echanekileyo kunye nokunyamezela i-WT .Ulawulo lokunyamezelana luhambelana ngokungqongqo ukuvelisa imigangatho.Iimveliso zethu zihlala zanelisekile ngabathengi.Abathengi bathenge iimveliso zethu benze inzuzo eninzi .
a) OD ( Ububanzi obungaphandle ) : 3.18mm ukuya kwi-101.6mm
b) WT (Ukutyeba kweendonga): 0.5mm ukuya kwi-20mm
c) Ubude : Ngokwemfuno yomthengi
d) Imigangatho : ASTM A312;I-ASTM A269;I-ASTM A789;I-ASTM A790 njl
e) Indlela yeNkqubo : ERW, EFW njl

Izilayidi ezibonisa amanqaku amathathu kwisilayidi ngasinye.Sebenzisa amaqhosha angasemva nalandelayo ukuhamba kwizilayidi, okanye amaqhosha okulawula isilayidi ekupheleni ukuya kwisilayidi ngasinye.
Iiseli ze-cranial neural crest cells (CNCC) ziyacotha kwi-embryonic neural folds kwaye zifudukela kwi-pharyngeal arches, eyenza uninzi lwezakhiwo eziphakathi.Ukungasebenzi kakuhle kwe-CNCC kudlala indima ebalulekileyo kwi-etiology ye-orofacial cleft, ukukhubazeka okuqhelekileyo kokuzalwa.Iinguqu ze-Heterozygous SPECC1L zifunyenwe kwizigulane ezine-atypical kunye ne-syndromic clefts.Apha, sixela ukungcoliswa okuphuculweyo kwe-canonical adhesive junction (AJ) amacandelo, i-β-catenin kunye ne-E-cadherin kwiiseli ezikhulisiwe ze-SPECC1L, kunye ne-electron micrographs zibonisa i-apical-basal diffusion ye-AJ.Ukuqonda indima ye-SPECC1L kwi-craniofacial morphogenesis, senze imodeli yempuku enqongopheleyo yeSpecc1l.I-Homozygous mutants i-embryonic lethal kwaye ibonisa ukuvalwa kwe-neural tube ephazamisekileyo kunye ne-CNCC lamination.I-AJ protein staining yonyuka kwi-mutant neural folds.Esi siphene se-AJ sihambelana nesiphene kwi-CNCC delamination, efuna ukuchithwa kwe-AJ.Ukongeza, i-Specc11 mutants inciphise ukubonakaliswa kwe-PI3K-AKT kunye nokunyuka kwe-apoptosis.I-in vitro, inhibition ethambileyo ye-PI3K-AKT yokubonisa kwiiseli zohlobo lwasendle yayanele ukwenza utshintsho lwe-AJ.Okubalulekileyo, utshintsho lwe-AJ olunyanzeliswa yi-SPECC1L yokunkqonkqoza lunokubuyiselwa umva ngokusebenza kwendlela ye-PI3K-AKT.Kuthatyathwe kunye, ezi datha zibonisa ukuba i-SPECC1L, njengomlawuli wenoveli ye-PI3K-AKT yokubonisa kunye ne-AJ biology, iyadingeka ukuze kuvalwe i-neural tube kunye ne-CNCC stratification.
Iiseli ze-Cranial neural crest (CNCCs) zihlala kwi-dorsal neuroectoderm kwaye zikhuphe kwi-neuroepithelium ye-neural folds ekhulayo ngokusebenzisa inkqubo ebandakanya i-epithelial-mesenchymal transition (EMT)1,2,3.I-CNCC ye-epithelial ehamba phambili iphazamisa i-intercellular junctions kwaye ibe yi-CNCC efudukayo ye-mesenchymal egcwalisa i-arches yokuqala neyesibini ye-pharyngeal kwaye yenza ininzi ye-craniofacial cartilage.Ke, iijini ezilawula umsebenzi we-CNCC zihlala ziphazamiseka kwi-etiology ye-craniofacial congenital anomalies efana ne-orofacial clefts, edla ngokuchaphazela ukuzalwa kwe-1/800 e-US kuphela.Enye yeziphene zokuzalwa8.
I-Delamination ye-CNCC ihambelana nokuvalwa kwe-neural tube yangaphambili phakathi kwe-8.5 kunye ne-9.5 iintsuku zokuphuhliswa kwe-embryonic kwiigundane.Iziguqulo zenani lemouse ye-orofacial cleft-ayanyaniswa nohlobo lwegenes ekwabonisa uhlobo oluthile lwesiphene se-neural tube, kuquka i-Irf69,10, Ghrl310, Cfl111, kunye ne-Pdgfrα12.Nangona kunjalo, iinkqubo zokuvalwa kwe-neural tube kunye ne-CNCC stratification zingathathwa njengezizimeleyo, njengoko i-Splotch mutant mouse (i-Pax3) ibonisa iziphene ekuvalweni kwe-neural tube ngaphandle kwempembelelo kwi-CNCC stratification okanye ukufuduka kwe-13,14.Iimodeli ezongezelelweyo zemouse ezineziphene kwi-CNCC dissection kunye nokuvalwa kwe-neural tube iya kunceda ukucacisa isiseko seemolekyuli eziqhelekileyo zezi nkqubo zimbini.
Ukuhlukaniswa kwe-CNCC kwiiseli ze-neuroepithelial kufuna ukuchithwa kweendlela zokubambelela (AJs), eziqulethwe kwiiprotheyini zeprotheyini eziqulethe, phakathi kwezinye, i-E-cadherin, i-β-catenin, i-α-E-catenin, kunye ne-α-actinin ehambelana ne-actin filaments 2 Uphononongo olugqithisileyo lwe-E-cadherin kwi-neural folds lubonise ukunciphisa okanye ukulibaziseka kwi-CNCC delamination.Ngakolunye uhlangothi, ukunyanzeliswa kwe-E-cadherin kubangela ukucwangciswa kwangaphambili15,16.Uninzi lwezinto ezidibanisa i-EMT ngexesha le-CNCC stratification zizinto ezibhaliweyo (AP2α, Id2, FOXD3, SNAIL, TWIST, SOX10) kunye ne-extracellular matrix (ECM) yokulungisa iiprotheni ezifana ne-matrix metalloproteinases (MMPs), nangona kunjalo i-CNCC i-cytoskeletal AJ regulators ngqo. ayikaziwa.Indlela ye-PI3K-AKT iyaziwa ngokuchasa amanqanaba e-E-cadherin, ikakhulu kuphando lomhlaza17.Uphononongo lwakutsha nje lubonise ukuba ilahleko ye-PDGFα-esekelwe kwi-PI3K-AKT yokubonisa kwiimpuku kukhokelela kwizinto ezingaqhelekanga ze-craniofacial, kubandakanywa i-cleft palate kunye ne-neural tube defects12.Nangona kunjalo, ubudlelwane phakathi kwendlela ye-PI3K-AKT kunye nokuzinza kwe-AJ kwi-CNCC stratification ayicacanga.
Ngaphambili sichonge i-SPECC1L njengejini lokuqala eliguqukayo kubantu ababini abanomngxuma onzulu osuka emlonyeni uye esweni, owaziwa ngokuba yi-oblique cleft (ObFC) okanye iTessier IV18 cleft.Ukuguqulwa kwe-SPECC1L kuchongiwe kwiintsapho ezimbini zezizwe ngezizwe ezine-autosomal dominant Opitz G/BBB syndrome (OMIM #145410), apho abantu abachaphazelekayo babonisa i-hyperdistance kunye ne-cleft lip / palate19, kunye nentsapho enye ene-Tibi overdistance syndrome (OMIM #145420)20 .ngaphezu kwesiqingatha seemeko ze-Opitz G / BBB syndrome zi-X-linked (OMIM #300000) kwaye zibangelwa ukuguqulwa kwe-MID1 gene, efaka i-protein ye-22 ye-microtubule-associated cell skeleton.Sicinga ukuba i-SPECC1L, nayo iprotheni ehambelana ne-microtubules kunye ne-actin cytoskeleton, inokudibanisa ukubonakaliswa okufunekayo kwi-actin cytoskeleton remodeling ngexesha lokubambelela kweeseli kunye nokufuduka kwe-18.Ngezifundo ze-in vitro kunye ne-vivo, ngoku sichaza i-SPECC1L njengomlawuli wenoveli wozinzo lwe-AJ ngokusebenzisa umqondiso we-PI3K-AKT.Kwinqanaba leselula, ukusilela kwe-SPECC1L kubangele ukuhla kwinqanaba leprotheni ye-pan-AKT kunye nokunyuka kwe-apical-basal dispersion ye-AJ, eyaye yapheliswa ngokusebenza kweekhemikhali kwindlela ye-AKT.Kwi-vivo, i-Specc11-i-embryos engafanelekanga ibonisa ukuvalwa kwe-neural tube ephazamisekileyo kunye nokunciphisa i-CNCC dissection.Ngaloo ndlela, imisebenzi ye-SPECC1L kwi-cell adhesion-based signaling elawulwa kakhulu efunekayo kumsebenzi oqhelekileyo we-CNCC ngexesha le-morphogenesis yobuso.
Ukubonakalisa indima ye-SPECC1L kwinqanaba leselula, sisebenzise umgca weseli ye-osteosarcoma echazwe ngaphambili ezinzileyo ye-U2OS esweleyo kwi-SPECC1L18.Ezi ziseli ze-U2OS ezizinzile kunye ne-SPECC1L (kd) i-knockdown ine-moderate (60-70%) yehla kumanqanaba e-SPECC1L yemibhalo kunye neeprotheni, kunye neziphene ekufudukeni kunye nokulungiswa kwakhona kwe-actin cytoskeleton 18. Ngokwahlukileyo, ukuncipha okukhulu kwexeshana I-SPECC1L ibonakaliswe ukukhokelela kwiziphene ze-mitotic 23.Ngaphezulu kolunye uphawu, sifumene ukuba iiseli zethu ezizinzile ze-SPECC1L-kd zatshintsha i-morphology kwizinga eliphezulu kakhulu lokudibanisa (Umfanekiso 1).Iiseli zolawulo lomntu ngamnye kunye neeseli ze-kd kwi-confluence ephantsi zibukeka zifana (Umfanekiso 1A, D).Iiyure ze-24 emva kokudibanisa, iiseli zokulawula zigcina imilo yazo ye-cuboidal (Umfanekiso 1B, E), ngelixa iiseli ze-SPECC1L-kd zide (Umfanekiso 1C, F).Ubungakanani bolu tshintsho kwimilo yeseli yabanjwa yi-vivo live imaging yeeseli zolawulo kunye neeseli ze-kd (imovie 1).Ukumisela indima ye-SPECC1L kwiiseli ezidibeneyo, siqale savavanya imbonakalo yayo.Sifumene ukuba amanqanaba eprotheyini ye-SPECC1L anyuswe kwi-fusion (Figure 1G), kanti i-SPECC1L amanqanaba e-transcript awazange anyuke (Figure 1H).Ukongezelela, njengoko i-cell density yanda, i-protein ye-SPECC1L eqokelelwe kwimida ye-intercellular (Umfanekiso 2A-E), kunye nephethini edibeneyo kunye ne-membrane ehambelana ne-β-catenin (Umfanekiso 2A'-E ').Ukunikezelwa kwentlangano ye-SPECC1L kunye ne-actin cytoskeleton 18,23 sicinge ukuba i-SPECC1L isebenzisana ne-actin-based adhesive junctions (AJ).
(AF) SPECC1L i-knockdown (DF) iiseli zinwebeka kwi-confluence ephezulu (F) xa kuthelekiswa nolawulo lweeseli ze-U2OS (AC).Apha aboniswe ngamanqaku amathathu kwathandathu (T1, T3, T6) esiwakhethele ukuxinana kweeseli ezahlukeneyo.(G) Uhlalutyo lwe-Western blot olubonisa ukuba iprotheni ye-SPECC1L izinzile kwiqondo eliphezulu lokudibanisa xa kuthelekiswa neqondo eliphantsi lokudibanisa kwiiseli zokulawula.Iblothi yaseNtshona ye-SPECC1L ibonisa ibhendi ye-120 kDa elindelekileyo kunye nebhendi ephezulu yobunzima bemolekyuli, ekunokwenzeka ukuba iguqulwe emva kokuguqulelwa (*).Uhlalutyo lwe-blot lwaseNtshona lwenziwa phantsi kweemeko ezifanayo zokudibanisa okuphantsi kunye nokuphezulu.Imifanekiso ebonisa i-SPECC1L kwi-confluence ephantsi kunye nephezulu ithathwe kwi-blot efanayo.Ela blot linye laye lasuswa laze lavavanywa kwakhona nge-β-actin antibody.(H) Uhlalutyo lobungakanani be-RT-PCR alubonakalisi lutshintsho olubalulekileyo kumanqanaba ombhalo we-SPECC1L.Imivalo yemposiso imele iiSEM ukusuka kwimifuniselo emine ezimeleyo.
(AE) Sakhetha amaxesha amathandathu (T1-T6) amele uluhlu lweeseli ezixineneyo ukwenza uhlalutyo lwemilo yeseli luqheleke kunye notshintsho lwe-AJ kwiiseli ze-U2OS nge-SPECC1L knockdown (kd).Ezintlanu zokuqala zala manqaku exesha ziquka iiseli enye (T1), i-50-70% yokudibanisa amaqoqo amancinci amancinci (T2), ukudibanisa ngaphandle kokuhlaziya iiseli ze-kd (T3), ukubuyisela iiseli ze-kd (T4), kunye neenguqu zeyure ezingama-24.kwimo yangasemva ye kd (T5) iiseli.Iprotheyini ye-SPECC1L yachithwa kakhulu kwi-cytoplasm kwi-T1 (A), kodwa ukuqokelela kwayo kwabonwa kwimida ye-intercellular kumanqaku exesha elilandelayo (B-E, iintolo).(FJ) i-β-catenin ibonisa ukuqokelela okufanayo kwimida ye-intercellular ehambelana ne-AJ complex.(A'-E') SPECC1L kunye ne-β-catenin zibonisa ukudityaniswa okuthe kratya kwimida yeeseli kuxinaniso lweeseli eziphezulu (iintolo).(F'-J') Kwiiseli ze-SPECC1L-kd, i-β-catenin staining ibonakala iqhelekile kwi-low cell density (F'-H'), kodwa iyanda njengoko iiseli zitshintsha (I', J'; arrows), ebonisa ukuba i-AJ zitshintshile.Imivalo = 10 µm.
Emva koko sazama ukufumanisa umphumo we-SPECC1L yokusilela kwi-AJ.Sasebenzisa amanqaku amaninzi ahambelana ne-AJ, kubandakanywa amacandelo e-canonical F-actin, myosin IIb, β-catenin, kunye ne-E-cadherin24,25,26,27.Iifayibha zoxinzelelo ze-Actin zanda kwiiseli ze-SPECC1L-kd njengoko kuchazwe ngaphambili (Umfanekiso 3A, B) 18.I-Myosin IIb ehambelana ne-actin filaments ibonise ukunyuka okufanayo kwiiseli ze-SPECC1L-kd kwi-vitro (Umfanekiso 3C, D).I-AJ ehambelana ne-β-catenin ibophelela kwi-cadherin kwi-membrane yeseli, ibonisa indlela eqhelekileyo yokubonisa "i-honeycomb" kwi-cubocytes yokulawula (Umfanekiso 3E, G).Kuyathakazelisa ukuba kwimifanekiso ecwecwe usebenzisa i-microscopy ye-confocal, i-β-catenin (umzobo 3E, F) kunye ne-E-cadherin (i-Fig. 3G, H) i-staining kwi-cell membrane ye-confluent SPECC1L-cell-deficient cells ibonise iipatheni ezibalaseleyo zokungcola okwandisiweyo.Oku kwandiswa kwe-AJ-enxulumene ne-β-catenin staining kwiiseli ze-kd kwakubonakaliswe kakhulu kwi-confluence, kodwa kubonakala ngathi kwandulela utshintsho kwimilo yeseli (Fig. 2F-J, F'-J').Ukumisela ubume bomzimba wale ndawo ye-AJ eyandisiweyo, sivavanye imida yeeseli kumphezulu we-apical-basal we-SPECC1L-kd U2OS iiseli ngokusasaza i-electron microscopy (TEM) (Figure 3I,J).Ngokwahlukileyo kwiiseli zokulawula (umzobo we-3I), owawunemimandla eyahlukeneyo ye-electron eshinyeneyo ebonisa i-AJ (iintolo), iiseli ze-kd (umzobo 3J) zibonise imimandla emikhulu, edibeneyo ye-electron ephezulu ebonisa i-AJ ecaleni kwendiza ye-apicobasal..Ukongezelela, kumacandelo aguquguqukayo, sabona i-folded cell membranes ebanzi kwiiseli ze-kd (Umfanekiso we-S1A, B), ochaza umzekelo owandisiweyo we-β-catenin kunye ne-E-cadherin staining bands (Fig. 3F, H).Ukuxhasa indima ye-SPECC1L kwi-AJs, i-β-catenin yayiyi-immunoprecipitated kunye ne-SPECC1L kwi-lysates yeeseli ze-U2OS ezidibeneyo (umzobo 3K).Kunye nokwandiswa kwe-immunostaining kumanqaku e-AJ, uhlalutyo lwe-TEM lwaluhambelana ne-hypothesis yethu yokuba ukusilela kwe-SPECC1L kwandisa ubuninzi be-AJ apical-basal kunye nokwahluka.
(AH) Ukunyuswa kwe-F-actin staining kwiiseli ze-kd kwiiyure ze-48 emva kokuxutywa (T6; A, B).Ukutshintshwa kwebala le-myosin IIb ehambelana ne-F-actin (C, D).Ipateni epholileyo ye-β-catenin kunye ne-E-cadherin membrane staining kwiiseli zokulawula (E, G) zandiswa kwiiseli ze-SPECC1L-kd (F, H).Imivalo = 10 µm.(I-J) Iimicrographs ze-electron eziqwalasela i-apical-basal intercellular junction.Iiseli zolawulo zibonisa imimandla eshinyeneyo ye-electron eyahlukileyo ebonisa iindawo ezincamathelayo (I, iintolo).Ngokwahlukileyo, yonke i-apical-basal junction kwiiseli ze-SPECC1L-kd zavela i-electron dense (J, iintolo), ebonisa ukwanda koxinano kunye nokusasazwa kokudibanisa okuncamathelayo.(K) i-β-catenin yayiyi-immunoprecipitated kunye ne-SPECC1L kwi-confluent U2OS cell lysates.Umfanekiso othathwe kwindawo enye emele enye yemifuniselo emine ezimeleyo.
Ukuqonda indima ye-SPECC1L kwi-craniofacial morphogenesis, senze imodeli ye-mouse elahlekileyo ye-Specc1l sisebenzisa imigca emibini ye-ES trap cell ezimeleyo, i-DTM096 kunye ne-RRH048 (i-BayGenomics, i-CA), emele i-intron 1 kunye ne-Specc1l imibhalo yabanjwa kwi-15 (Umfanekiso 1) .4A, umfanekiso S2).Indawo ye-genomic yokufaka i-decoy vector inqunywe ngokulandelelana kwe-genome yonke kwaye iqinisekiswe yi-PCR (Fig. S2).Zombini ziyilo lomgibe wofuzo zikwavumele ukudityaniswa kwesakhelo seentatheli zeSpecc11-lacZ xa zibanjiwe.Ke ngoko, intetho ye-lacZ egqitywe yi-X-gal staining isetyenziswe njengesalathisi sentetho yeSpecc11.Zombini ii-alleles zibonise iipateni ezifanayo zokubonisa i-lacZ, kunye ne-DTM096 i-gene trap kwi-intron 1 ibonisa ukubonakaliswa okunamandla kune-RRH048 kwi-intron 15 (engaboniswanga).Nangona kunjalo, i-Specc1l ibonakaliswe ngokubanzi, kunye nokubonakaliswa okunamandla kwi-neural folds kwi-E8.5 (Umfanekiso 4B), kwi-tube ye-neural kunye neenkqubo zobuso kwi-E9.5 kunye ne-E10.5 (Umfanekiso 4C, D), kunye nokuphuhlisa amalungu. kwi E10.5 kunye namehlo (Figure 4D).Ngaphambili saxela ukuba i-SPECC1L ibonakaliso kwi-arch yokuqala ye-pharyngeal kwi-E10.5 yayikhona kwi-epithelium kunye ne-mesenchyme18 ephantsi, ehambelana nomgca we-CNCC.Ukuvavanya ukubonakaliswa kwe-SPECC1L kwi-CNCC, senze i-E8.5 neural folds (Umfanekiso 4E-J) kunye ne-E9.5 amacandelo e-skull (Umfanekiso 4K-).Kwi-E8.5, i-SPECC1L igxininise i-neural folds kakhulu (Fig. 4E, H), kubandakanywa neeseli ezihlanjululwe ngabamakishi be-NCC (Fig. 4G, J).Kwi-E9.5, i-SPECC1L (Umfanekiso we-4K, N) ugxininise ngamandla ukufuduka kwe-CNCC edibene ne-AP2A (Umfanekiso 4L, M) okanye i-SOX10 (Umfanekiso 4O, P).
(A) Ukubonakaliswa kweskimu semouse ye-Specc11 gene ebonisa ukufakwa kwe-decoy vector kwi-ES DTM096 (intron 1) kunye ne-RRH048 (intron 15) ii-clones zeseli.(BD) i-lacZ staining ye-heterozygous Specc1lDTM096 embryos emele i-Specc1l intetho esuka kwi-E8.5 ukuya kwi-E10.5.NE = neuroectoderm, NF = i-neural fold, PA1 = i-arch yokuqala ye-pharyngeal.(EP) I-SPECC1L i-immunostaining kunye nabamakishi be-NCC AP2A kunye ne-SOX10 kwi-E8.5 (NF; EJ) i-neural folds kunye ne-E9.5 (KP) amacandelo okakayi.I-SPECC1L i-staining yabonwa ngokubanzi kwii-neural folds E8.5 (E, H; ii-arrowheads), kubandakanywa iiseli ezibhalwe nge-AP2A (F, G; iintloko zotolo) kunye ne-SOX10 (I, J; iintolo).Kwi-E9.5, i-SPECC1L enebala elinamandla le-CNCCs ezifudukayo (K, N; iintolo) ezibhalwe AP2A (L, M; iintolo) kunye ne-SOX10 (O, P; iintolo).
Ukuwela phakathi kwe-heterozygous Specc1lDTM096/+ kunye ne-Specc1lRRH048/+ iimpuku zibonisa ukuba i-alleles ezimbini ze-gene trap azihambelani kwaye i-heterozygotes ehlanganisiweyo kunye ne-embryonic homozygotes nokuba yeyiphi i-allele ye-gene trap i-embryonic ebulalayo (Itheyibhile S1).Umlinganiselo we-Mendelian ubonise ukuhla kwezinga lokusinda kwe-heterozygotes ekuzalweni (kulindelwe i-1.34 vs. 2.0).Siye saqaphela ukufa okuphantsi kwe-perinatal phakathi kwe-heterozygotes, ezinye zine-craniofacial anomalies (Fig. S3).Nangona kunjalo, ukungena okuphantsi kwezi phenotypes ze-perinatal craniofacial kwenza kube nzima ukufunda iindlela zabo ezisisiseko ze-pathophysiological.Ke ngoko, sigxile kwi-embryonic lethal phenotype ye-homozygous Specc11 mutants.
Uninzi lwe-heterozygous edibeneyo okanye i-homozygous i-Specc1lDTM096 / RRH048 i-embryos eguqukileyo ayizange ikhule emva kwe-E9.5-10.5 (Amakhiwane 5A-D), kunye ne-neural tube ayizange ivale ngaphambili (Imifanekiso 5B, D) kwaye ngamanye amaxesha ivalwe ngasemva (engaboniswanga) ..Esi siphene sokuvalwa kwetyhubhu ye-cranial idibaniswe noninzi lwe-CNCC ephawulwe i-DLX2 esele kwi-neural folds kwi-E10.5, ebonisa ukuba akukho dissection (Figure 5A'-D').Ukufumanisa ukuba ubungakanani obupheleleyo be-CNCC buye bancitshiswa, sibhale imigca ye-CNCC kunye ne-GFP kwimizila yethu ye-gene trap kunye ne-Wnt1-Cre kunye ne-ROSAmTmG.Sisasaza i-GFP+ NCC ehleliweyo kunye ne-GFP- (RFP+) engeyiyo i-NCC ukusuka kwiimbumba ezipheleleyo.Kwi-E9.5, umlinganiselo we-GFP-ebhalwe nge-GFP ebhalwe nge-CNCC ayizange itshintshe kakhulu phakathi kwe-WT kunye ne-embryos eguqukayo (engaboniswanga), ebonisa ukucaciswa kwe-CNCC eqhelekileyo.Ngoko ke, siye saqikelela ukuba intsalela ye-Wnt1-Cre kunye ne-DLX2 ingcolisa kwii-neural folds eziveziweyo (Figure 5B') kwaba ngenxa yokusilela kwe-CNCC umaleko, okunokwenzeka ngenxa yokwanda koxinzelelo okanye ukusasazwa kweeseli ze-AJ, njengoko kubonwa kwiiseli ze-SPECC1L-kd.Sasebenzisa iimpawu ze-NCC ze-SOX10, i-AP2A, kunye ne-DLX2 ukuqinisekisa ubukho be-CNCC kwi-neural fold (Umfanekiso 5E-R).Kwi-E8.5, i-neural fold staining kubo bonke abathathu abamakishi be-NCC yabonwa kumacandelo e-WT (Fig. 5E, G, I) kunye ne-Specc1l mutant (Fig. 5F, H, J).Kwi-E9.5, ngelixa iimpawu ze-NCC zingcolise i-NCC efudukayo kumacandelo e-WT (Umfanekiso we-5M, O, Q), i-NCC eshiyekileyo yabonwa kwii-neural folds ze-Specc1l eziguquguqukayo ze-embryos (Fig. 5N, P, R).Ngenxa yokuba i-SOX10 kunye ne-DLX2 iphawula ii-CNCC ezifudukayo, esi siphumo sibonisa ukuba i-SPECC1L-engenayo i-CNCCs iphumelele ukucaciswa kokuhamba emva kokufuduka kodwa iyasilela ukufuduka kwi-neural folds.
Ukusilela kwe-Specc11 kukhokelela ekuvalweni kwe-neural tube enesiphako, i-delamination yeeseli ze-cranial neural crest kunye nee-AJs.
(A, B') E9.5 WT (A) Imbumba ethwele iiseli ze-cranial neural crest ezifudukayo (CNCC) ezibhalwe nge-Wnt1-Cre (A').Ngokuchaseneyo, iimbumba eziguqukayo zeSpecc11 zibonisa i-neural folds evulekileyo (B), iintloko zotolo) kunye ne-CNCC ezingekafuduki (B', iintloko zotolo).(C, D') Imifanekiso yentsimi eqaqambileyo (C, D') kunye ne-immunostaining (C', D') ye-CNCC marker DLX2 ye-E10.5 WT embryos (C, C') kunye ne-Specc1l (D, D').Kwi-WT E10.5 embryos, i-DLX2-positive CNCC igcina i-arches yegill (C', iintolo), ngelixa i-mutants, i-staining staining iqhubeka kwi-neural folds evulekileyo (D', iintolo) nakwi-arches yokuqala ye-pharyngeal (D', iintolo).) kunye nenye ibala (iintolo) ezibonisa ukusilela kunye nokufuduka kwe-CNCC.ER) Amacandelo e-WT kunye ne-Specc1l iimbumba eziguqukayo kwizigaba ze-E8.5 (E-L) kunye ne-E9.5 (M–R) zibhalwe ngamagama e-NCC SOX10 (E, F, M, N), AP2A (G, H, O, P ) kunye ne-DLX2 (I, J, Q, R).Kwi-E8.5, i-NCC staining yabonwa kwi-wild-type neural fold (NF) kunye namacandelo aguqukayo.I-Co-staining ye-SOX10 kunye ne-β-catenin kwi-E8.5 WT (K) kunye ne-mutant (L) ibonakalise ukunyuka kwe-β-catenin kwimida yeeseli kwi-neural folds.Kwi-E9.5, i-wild-style staining ye-CNCCs efudukayo (M, O, Q) yabonwa, ngelixa i-mutants, i-CNCC engabonakaliyo igxininise i-neural folds evulekile (N, P, R).(S-Z) In vivo AJ uhlalutyo lokuleyibhela kumacandelo e-coronal ye-WT kunye ne-Specc11DTM096/RRH048 iimbumba ezinotshintsho lwe-E9.5.Inqwelomoya yecandelo eqikelelweyo iboniswe kwikona ephezulu ngasekunene.Kumacandelo ezicubu eziguqukayo, ukunyuka kwe-F-actin (S, T) kunye ne-myosin IIb (U, V) kwabonwa.Ngokufana neziphumo ze-in vitro kwi-Fig. 3, kwi-embryos eguquguqukayo, i-membrane ephuculweyo yokucoca i-β-catenin (W, X) kunye ne-E-cadherin (Y, Z) yabonwa.(AA-BB) I-electron micrograph yecandelo le-embryo yasendle ejonge ngaphaya komda weseli ye-apical-basal ibonisa ummandla ocacileyo we-electron-dense obonisa ukudibanisa okunamathelayo (AA, iintolo).Ngokwahlukileyo, kumacandelo e-Specc11 e-embryos eguquguqukayo (BB, iintolo), yonke i-apicobasal junction i-electron dense, ebonisa ukuxinana okwandisiweyo kunye nokusabalalisa kweendlela zokubambelela.
Ukuvavanya i-hypothesis yethu yokuba ukwehliswa kokuncipha kubangelwa yi-AJ etshintshiweyo, siye savavanya ukuleyibheli kwe-AJ kwii-neural folds eziveziweyo ze-Specc1l iimbumba eziguqukileyo (Fig. 5S-Z).Siye sabona ukwanda kwe-actin imicu yoxinzelelo (umzobo 5S, T) kunye ne-concomitant yokwandisa indawo ye-myosin IIB staining kwi-actin fibers (Fig. 5U, V).Okubalulekileyo, siye sabona ukunyuka kwe-β-catenin (umzobo 5W, X) kunye ne-E-cadherin (umzobo 5Y, Z) kwimida ye-intercellular.Siphinde sihlolisise i-β-catenin staining ye-NCC kwi-neural folds ye-E8.5 embryos (Umfanekiso 5K, L).I-β-catenin staining ibonakala yomelele kwi-Specc1l i-mutant neural folds (Fig. 5L kunye ne-K), ebonisa ukuba utshintsho lwe-AJ luqalile.Kwimikrografu ye-electron yamacandelo ekhanda leembumba ze-E9.5, siphinde sabona ukwanda kwe-electron-dense staining kwi-Specc1l embryos eguqukileyo xa kuthelekiswa ne-WT (Fig. 5AA, BB kunye ne-S1E-H).Zithathiwe kunye, ezi ziphumo zixhasa iziphumo zethu ze-in vitro kwiiseli ze-SPECC1L-kd U2OS kwaye zicebisa ukuba ukungcola kwe-AJ okungahambiyo kwandulela ukucwangciswa kwe-CNCC kwiimbumba zethu eziguqukayo.
Ukunikezelwa kobudlelwane obuchasayo obaziwayo phakathi komsebenzi we-AKT kunye nokuzinza kwe-E-cadherin, i-17,28 sicingela ukubandakanyeka kokubonakaliswa kwe-PI3K-AKT.Ukongezelela, siye sabona i-subepidermal blistering kwezinye zeembumba zethu eziguqukayo ezibalekele ukufa (<5%) kwi-E9.5-10.5 kwaye endaweni yoko zahlala malunga ne-E13.5 (Fig. S3).I-Subepidermal vesicles luphawu lokunciphisa umqondiso we-PI3K-AKT ngokusekelwe kwi-PDGFRα12.Fantauzzo et al.(I-2014) ibike ukuba ukuphazamiseka kwe-PDGFRα-based based PI3K activation kwi-PdgfraPI3K / PI3K i-embryos ye-mutant iphumela kwi-subepidermal vesicles, i-neural tube defects, kunye ne-cleft palate phenotypes.Enyanisweni, amanqanaba e-pan-AKT kunye ne-Ser473-AKT esebenzayo ye-phosphorylated yancitshiswa kwi-vivo kwi-Specc1l izicubu eziguqukayo kwi-E9.5 yokubanjwa kwe-embryonic (Umfanekiso 6A-D).Ukuncipha kwamanqanaba e-phosphorylated Ser473-AKT inokuba ngokupheleleyo ngenxa yokuncipha kwamanqanaba e-pan-AKT kwi-vivo (FIG. 6E) kunye ne-vitro (FIG. 6F).Ukuncipha kwe-in vitro kwabonwa kuphela xa iiseli ze-U2OS zidibene kakhulu kunye notshintsho kwimilo yeseli kunye nobuninzi be-AJ (Umfanekiso 6D).Ke, idatha yethu icebisa ukuba i-SPECC1L iyinoveli yolawulo olulungileyo lwe-PI3K-AKT yokubonisa kwi-craniofacial morphogenesis.
(A-E) E8.5 (A,B) kunye ne-E9.5 (C,D) amacandelo ekhakhayi okanye i-E9.5 i-lysates esuka kwi-Specc1l i-mutant embryos (E) ibonisa amanqanaba e-phosphorylated esebenzayo S473-AKT kunye ne-pan-AKT yokunciphisa iProtheyini , xa kuthelekiswa nolawulo lwe-WT.I-Western blotting yenziwa kwi-wild-type lysates kunye ne-mutant lysates phantsi kweemeko ezifanayo.Imifanekiso ebonisiweyo ye-SPECC1L ithathwe kwiblothi enye.I-blot efanayo yasuswa kwaye yaphononongwa kwakhona nge-anti-pan-ACT kunye ne-β-actin antibodies.Amanqanaba e-Pan-AKT kwi-E8.5 neural folds (A, B) kunye namanqanaba e-phosphorylated S473-AKT kumacandelo e-skull E9.5 ancitshiswe kakhulu.(F) Amanqanaba e-Pan-AKT ancitshiswe ngokufanayo kwii-lysates ze-SPECC1L-kd U2OS iiseli ezivunwe kwi-confluence ephezulu.Imivalo yemposiso imele ii-SEMs ezivela kwi-quantifications ze-blot ezintathu ezizimeleyo zaseNtshona.(GJ) Amacandelo e-WT embryos kwi-E9.5 ehlanjululwe nge-KI67 kunye ne-caspase eqhekezayo 3, ngokulandelanayo, ebonisa ukwanda kweeseli (G, G ') kunye nomsebenzi omncinci we-apoptotic (H, H').Iimbumba eziguqukayo zeSpecc11 zibonisa ukwanda kweeseli ezithelekisekayo (I), kodwa inani leeseli ezifumana i-apoptosis landa kakhulu (J).
Emva koko siye savavanya iimpawu zokwanda kunye ne-apoptosis.Asizange siqaphele naluphi na umehluko ekwandeni kwee-embryo ze-E9.5 (umzobo 6E, G xa kuthelekiswa no-I) kunye ne-index indexation ye-82.5% ye-WT mutants kunye ne-86.5% ye-Specc1l mutants elinganiswe yi-KI67 staining (p <0.56, i-Fisher's uvavanyo ngqo).Ngokufanayo, asizange sigcine naluphi na umahluko kwi-apoptosis elinganiswe ngokungcolisa i-caspase ecatshulweyo 3 kwi-neural folds kwi-E8.5 de kube kubanjwe umbungu (engaboniswanga) (engaboniswanga).Ngokwahlukileyo, i-apoptosis yanda kakhulu kuzo zonke ii-embryo ze-E9.5 eziguqukayo (umzobo 6F, H kunye noJ).Oku kunyuka ngokubanzi kwe-apoptosis kuhambelana nokuncitshiswa kwe-PI3K-AKT ukubonakaliswa kunye nokubulawa kwangaphambili kwe-embryonic29,30,31.
Emva koko, ukuqinisekiswa kwendima ye-causal ye-PI3K-AKT yokubonisa kwiinguqu ze-AJ kwiiseli zethu ze-kd, siguqule ikhemikhali indlela yokulawula kunye neeseli ze-kd (Umfanekiso 7A-F).Sisebenzise njengesiphawuli utshintsho lwemilo yeseli kwiphenotype ebonwe kwiiseli ezidityanisiweyo ze-SPECC1L-kd, esithe sazilinganisa sisebenzisa umlinganiselo wowona mlinganiso omde (ubude) ukuya kumlinganiselo othe nkqo ohambelanayo (ububanzi).Umlinganiselo we-1 ulindeleke kwiiseli ezingqukuva okanye ezi-cuboidal (Figure 7G).Ukongeza kwimilo yeseli, siye saqinisekisa umphumo kwi-AJ nge-β-catenin staining (Fig. 7A'-F ').Ukuvinjelwa kwendlela ye-PI3K-AKT usebenzisa i-wortmannin yayanele ukutshintsha ukuma kweeseli kwiiseli zokulawula (Umfanekiso 7A, C) kunye ne-AJ (Umfanekiso 7A ').I-activator ye-PI3K-AKT i-SC-79 ayizange ichaphazele imilo yeseli (FIG. 7A, E) okanye ukwandiswa kwe-AJ (FIG. 7A') kwiiseli zokulawula.Kwiiseli ze-SPECC1L-kd, ukunyanzeliswa okuqhubekayo kwendlela ye-PI3K-AKT kubangele ukwanda kwe-apoptosis (umzobo 7B, D) kunye nokunyuka okuphawulekayo kwi-β-catenin staining (Fig. 7B '), ehambelana ne-vivo yethu eguquguqukayo enzima.Okubalulekileyo, ukusetyenziswa kwendlela ye-PI3K-AKT kuphuculwe kakhulu ukumila kweeseli (Umfanekiso 7B, F) kunye ne-AJ phenotypes (Umfanekiso 7B").Utshintsho kwimilo yeseli lucatshangelwe njengomlinganiselo wokujikeleza kweeseli (CCR) kwaye kuthelekiswa ngokubaluleka njengoko kuchazwe ngasentla (FIG. 7G).Enyanisweni, kwiiseli zokulawula (umzobo 7G, i-CCR = 1.56), unyango lwe-wortmannin lwalukwanele ukuguqula ngokuphawulekayo ukuma kweeseli (Umfanekiso we-7G, i-CCR = 3.61, p <2.4 × 10-9) ukuya kwinqanaba elifana nelo libonwayo. kwi-SPECC1L.-kd iiseli (Umfanekiso 7G, CCR = 3.46).Unyango lwe-Wortmannin lweeseli ze-SPECC1L-kd (umzobo 7G, i-CCR = 3.60, ayinanto) yayingabalulekanga ngaphezu kweeseli ze-kd ezingaphendulwanga (Umfanekiso we-7G, i-CCR = 3.46, ingenamsebenzi) okanye iiseli zokulawula unyango lwe-wortmannin (Umfanekiso 7G)., I-CCR = 3.46, ayinanto) iphinda ichaphazele i-cell elongation (7G, CCR = 3.61, ayinanto).Okubaluleke kakhulu, i-activator ye-SC-79 AKT ibuyisele i-phenotype emide yeeseli ze-SPECC1L-kd (Umfanekiso 7G, CCR = 1.74, p <6.2 × 10-12).Ezi ziphumo ziqinisekisa ukuba i-SPECC1L ilawula ukubonakaliswa kwe-PI3K-AKT kwaye iphakamisa ukuba ukuhla okuphakathi kwe-SPECC1L kuchaphazela ukunamathela kweseli, ngelixa ukuhla okunamandla kukhokelela kwi-apoptosis (Umfanekiso 8).
(A-F') Ulawulo (A, C, E) kunye ne-SPECC1L-kd (B, D, F) iiseli eziphathwa nge-PI3K-AKT pathway inhibitor wortmannin (C, D) okanye SC-79 activator (E, F) Unyango .Iiseli ezingaphathwanga zolawulo ziyi-cuboidal (A) kunye ne-β-cat ye-cellular staining (A'), ngelixa iiseli ze-kd zandiswa (B) kunye nokunyuka kwe-β-cat staining (B').Emva kokucinezelwa kwendlela ye-PI3K-AKT, iiseli zokulawula zide (C) kunye nokwandiswa kwe-β-cat (C'), ngelixa iiseli ze-kd zaqala ukudlula i-apoptosis (D), efana neembumba zethu eziguqulwe kakhulu kwaye zibonisa i-β-cat ephuculweyo kakhulu.ukungcola (D').Emva kokusebenza kwendlela ye-PI3K-AKT, iiseli zokulawula zahlala zi-cuboidal (E) kwaye zine-beta-cat (E') eqhelekileyo, ngelixa iiseli ze-kd zibonise ukuphuculwa kakhulu kwe-cell shape (F) kunye ne-β-cat (F') staining, ebonisa (G) Iqondo lotshintsho kwimilo yeseli kwi- (AF) yalinganiswa kusetyenziswa umlinganiselo weseli wokujikeleza (CCR) wowona mlinganiselo mde (ubude) kunye nomlinganiselo othe nkqo ohambelanayo (ububanzi) kusetyenziswa isoftware yeMetaMorph.Iiseli ezingaphathwanga (NT) SPECC1L-kd (CCR = 3.46) zazide kakhulu kuneeseli zokulawula (CCR = 1.56, p <6.1 × 10-13).I-Wort's inhibition ye-PI3K-AKT indlela kwiiseli zokulawula yayanele ukuba ibangele ukunyuka okufanayo kwi-cell shape (CCR = 3.61, p <2.4 × 10-9).Ngokufanayo, ukusebenza kwe-AKT nge-SC-79 kwiiseli ze-SPECC1L-kd zibuyisele ukunyuswa kweeseli kumanqanaba okulawula (CCR = 1.74, p <6.2 × 10-12).Unyango lwe-Wortmannin lweeseli ze-SPECC1L-kd lubangele ukwanda kwe-apoptosis kodwa akukho kwandiso ngakumbi kwinguqu ye-cell shape (CCR = 3.60) xa kuthelekiswa ne-kd engaphathwanga (CCR = 3.46, ns) okanye iiseli zokulawula unyango lwe-wortmannin (3.61) ezibonwe kwi.ns = ayinamsebenzi.+/- Imilinganiselo ye-SEM yeeseli ze-50 ibonisiwe.Umahluko ngokwamanani ubalwe kusetyenziswa uvavanyo luka-t loMfundi.
(A) Ukubonakaliswa kweSchematic of inhibition kunye nokusebenza kwendlela ye-PI3K-AKT ebangela ukuba i-AJ iguqule kunye nokuhlangula, ngokulandelanayo.(B) Imodeli ecetywayo ye-AKT yokuzinzisa iprotheni nge-SPECC1L.
Ii-CNCC ze-Premigratory zifuna i-AJ lysis ukuba ihlukane ne-neural fold neuroepithelial cells1,15,32.Ukunyuka kwamacandelo e-AJ kunye nokulahlekelwa kwe-apical-basal AJ yokusabalalisa i-asymmetric kwi-SPECC1L-iseli ezinqongopheleyo zombini kwi-vitro kunye ne-vivo, edityaniswe nokusondela ngokomzimba kwe-SPECC1L ukuya kwi-β-catenin, iphakamisa ukuba imisebenzi ye-SPECC1L ukugcina ngokufanelekileyo i-AJ ukuzinza kwendawo izihlunu zombutho.actin cytoskeleton.Umbutho we-SPECC1L kunye ne-actin cytoskeleton kunye ne-β-catenin kunye nokunyuka kwenani le-actin filaments edibeneyo ngokungabikho kwe-SPECC1L ihambelana nokunyuka okubonakalayo kwi-AJ density.Enye into enokwenzeka kukuba inani elongezelelweyo le-actin fibers kwiiseli ezingenayo i-SPECC1L zikhokelela ekutshintsheni ukuxhatshazwa kwe-intercellular.Ngenxa yokuba uxinzelelo lweselula luchaphazela i-AJ 33 dynamics, utshintsho lwe-voltage lunokubangela ukwanda kwe-AJ 34.Ngoko naluphi na utshintsho luya kuchaphazela iileya ze-CNCC.
I-Wnt1 ibonakaliswe kwi-neural folds yokuqala evelisa iiseli ze-neural crest.Ngoko ke, i-Wnt1-cre yokulandelela umkhondo womgca wamanqaku amabini ngaphambili kunye ne-NCC35 efudukayo.Nangona kunjalo, i-Wnt1 iphinda iphawule ii-clones zeethishu ze-dorsal nazo ziphuma kwi-neural folds yokuqala 35,36, okwenza ukuba kube lula ukuba ukungcola kwethu kwee-E9.5 mutants kwiimpawu ze-Wnt1 kwi-neural folds evulekileyo ayiyo-CNCC.Ibala lethu elihle leempawu ze-NCC ze-AP2A kunye ne-SOX10 ziqinisekisile ukuba i-neural folds eveziweyo ye-Specc11 embryos eguqukileyo ngokwenene yayiqulathe i-CNCC.Ukongeza, ekubeni i-AP2A kunye ne-SOX10 ziziphawuli ze-NCC efudukayo kwangoko, i-staining positive ibonise ukuba ezi seli ziyi-CNCC zasemva kokufuduka ezingenako i-stratified yi-E9.5.
Idatha yethu ibonisa ukuba ukulawulwa kwe-molecular ye-AJ nge-SPECC1L idibene ne-PI3K-AKT yokubonisa.Ukubonakaliswa kwe-AKT kuncitshiswe kwiiseli ezinqongopheleyo ze-SPECC1L kunye nezicubu.Iziphumo zikaFantauzzo et al.xhasa indima ethe ngqo ye-PI3K-AKT yokubonisa kwi-craniofacial morphogenesis.(I-2014) ibonise ukuba ukungabikho kokusebenza kwe-PDGFRα-based based PI3K-AKT ukubonakaliswa kukhokelela kwi-phenotype ye-cleft palate.Kwakhona sibonisa ukuba ukuvinjelwa kwendlela ye-PI3K-AKT yanele ukutshintsha i-AJ kunye nokuma kweeseli kwiiseli ze-U2OS.Ngokuvumelana neziphumo zethu, uKayin et al.I-37 ibonise ukuba ukuthotywa kwe-PI3K α110 subunit kwiiseli ze-endothelial kubangela ukunyuka okufanayo kwi-pericellular β-catenin staining, ekubhekiselwa kuyo njengokunyuka "kwisalathisi sokuxhuma".Nangona kunjalo, kwiiseli ze-endothelial ezine-actin filaments sele zilungelelaniswe kakhulu, ukunyanzeliswa kwendlela ye-PI3K-AKT kuphumela kwimilo yeseli ekhululekile.Ngokwahlukileyo, iiseli ze-SPECC1L-kd U2OS zibonise ukumila kweeseli ezinde.Lo mahluko usenokuba luhlobo oluthile lweeseli.Ngelixa ukunyanzeliswa kwe-PI3K-AKT ukubonakaliswa kuchaphazela ngokusisigxina i-actin cytoskeleton, umphumo kwimilo yeseli inqunywe ngotshintsho kwixinzelelo olubangelwa utshintsho kubuninzi kunye nombutho we-actin fibers.Kwiiseli ze-U2OS, sisebenzise kuphela utshintsho lwemilo yeseli njengesiphawuli se-SPECC1L-esilelayo ye-AJ yokutshintsha kunye nokubuyisela.Ekugqibeleni, sicinga ukuba ukuvinjelwa kwendlela ye-AKT kwi-SPECC1L yokusilela kwandisa ukuzinza kwe-AJ kunye nokunciphisa i-delamination kwi-CNCC.
Okuthakazelisayo kukuba, amanqanaba e-pan-AKT ancitshiswe kwi-vitro kunye ne-vivo ngaphezu kwe-phosphorylated 473-AKT amanqanaba ngokungabikho kwe-SPECC1L, ebonisa ukulawulwa kwe-PI3K-AKT yokubonisa kwinqanaba le-AKT iprotheyini yokuzinza okanye ukuguqulwa.I-SPECC1L kunye ne-MID1 izakhi zofuzo, zombini ezinxulumene ne-Opitz / GBBB syndrome, zifake iiprotheni ezizinzisa i-microtubules 18,22.Indlela apho i-SPECC1L kunye ne-MID1 idibanisa ukuzinza kwe-microtubule ayiqondwa ngokupheleleyo.Kwimeko ye-SPECC1L, oku kuzinziswa kubandakanya i-acetylation ephuculweyo ye-subset ye-microtubules 18.Kungenzeka ukuba i-SPECC1L isebenzisa indlela efanayo ukuzinzisa ezinye iiprotheni ezifana ne-AKT.Kuye kwaboniswa ukuba i-acetylation yeentsalela ze-lysine kwiprotheni ye-AKT ikhokelela ekunciphiseni kwi-membrane yendawo kunye ne-phosphorylation38.Ukongeza, u-biquitination yekhonkco ye-K63 kwi-residu ye-lysine efanayo kwi-AKT iyadingeka kwi-membrane yayo yendawo kunye nokusebenza39,40.Phakathi kwezinto ezininzi ezisebenzisana neeprotheni ze-SPECC1L ezichongiweyo kwi-yeast ephezulu ye-throughput izikrini ezimbini ze-hybrid, ezine-CCDC841, ECM2942, i-APC kunye ne-UBE2I43 - ziye zabandakanyeka kwi-protein turnover okanye ukuzinza ngokusebenzisa indawo yonke okanye i-sumoylation.I-SPECC1L inokubandakanyeka ekuguquleni emva kokuguqulwa kweentsalela ze-AKT lysine, ezichaphazela ukuzinza kwe-AKT.Nangona kunjalo, indima ebalulekileyo ye-SPECC1L kwindawo kunye nokuzinza kweprotheni ye-AKT ihlala icacisiwe.
Iziphene ezimandundu kwinkcazo ye-SPECC1L kwi-vivo ibangele ukonyuka kwe-AJ marker staining kunye ne-CNCC enesiphene yokwaleka, kunye nokunyuka kwe-apoptosis kunye nokubulawa kwe-embryonic kwangaphambili.Iingxelo zangaphambili zibonise ukuba utshintsho lwemouse kunye namanqanaba anyukileyo e-apoptosis adibaniswa ne-neural tube defects 44,45,46,47 kunye ne-craniofacial defects48.Kuye kwacetyiswa ukuba ukufa kweeseli ezigqithisileyo kwii-neural folds okanye i-pharyngeal arches kunokubangela inani elaneleyo leeseli ezifunekayo kwintshukumo efanelekileyo ye-morphogenetic 48,49,50.Ngokwahlukileyo, i-SPECC1L yethu imigca yeeseli ezinqongopheleyo kunye nokuncitshiswa ngokulinganayo kwe-SPECC1L ibonakaliso ibonise kuphela utshintsho lwe-AJ ngaphandle kobungqina bokunyuka kokufa kweeseli.Nangona kunjalo, ukuvinjwa kweekhemikhali kwendlela ye-PI3K-AKT kwezi seli ze-Kd kubangele ukwanda kwe-apoptosis.Ngaloo ndlela, ukuhla okuphakathi kwintetho ye-SPECC1L okanye umsebenzi uqinisekisa ukusinda kweeseli.Oku kuhambelana noqwalaselo lokuba iimbumba zeSpecc11 eziguqukileyo ezinqabileyo ezibaleka ukubanjwa eSt.I-E9.5-mhlawumbi ngenxa yokunciphisa i-gene yokubamba i-gene-iyakwazi ukuvala i-neural tubes kunye nokuyeka kamva ekuphuhlisweni, ngokuphindaphindiweyo kunye neziphene ze-craniofacial (Fig. S3).Kwakhona okuhambelana noku kukwenzeka okungaqhelekanga kweembumba ze-heterozygous Specc1l ezine-craniofacial abnormalities-mhlawumbi ngenxa yokwanda kokubanjwa kwe-gene-kunye nokufunyaniswa kwe-zebrafish apho enye yee-orthologues ezimbini ze-SPECC1L (specc1lb) ibangela i-embryonic phenotypes kade, kubandakanywa nokulahlekelwa kwe-embryonic phenotypes. imihlathi engezantsi kunye nokuqhekeka kwamacala amabini51.Ngaloo ndlela, i-heterozygous SPECC1L ilahleko-ye-function yenguquko echongiweyo kwizigulane zabantu inokubangela ukuphazamiseka okuncinci kwi-SPECC1L umsebenzi ngexesha le-craniofacial morphogenesis, eyaneleyo ukucacisa i-orofacial clefts.Ulawulo olusekelwe kwi-SPECC1L yoqhagamshelwano lwe-intercellular lungaphinda ludlale indima kwi-palatogenesis kunye nokudibanisa kwee-arches pharyngeal.Uphononongo olongezelelweyo lomsebenzi we-SPECC1L luya kunceda ukucacisa indima yoqhagamshelwano lwesikhashana lwe-intercellular kwi-CNCC ngexesha lokuvalwa kwe-neural tube kwi-neuroepithelial cell motility kunye ne-craniofacial morphogenesis.
Ulawulo lwe-U2OS osteosarcoma kunye neeseli ze-SPECC1L-kd zichazwe ngaphambili (Saadi et al., 2011).Ii-antibodies ezichasene ne-SPECC1L nazo zibonakaliswe ngaphambili (Saadi et al., 2011).I-anti-β-catenin antibodies (umvundla; 1: 1000; Santa Cruz, Dallas, TX) (mouse; 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), caspase 3 (1: 1000; Cell Signaling Technology, Danvers, MA) kunye ne-β-actin (1: 2500; Sigma-Aldrich, St. Louis, MO ) isetyenziswe njengoko kuchaziwe..I-Actin filaments yangcoliswa nge-Acti-stain rhodamine phalloidin (Cytoskeleton, Denver, Colorado).
Iiseli zolawulo ze-U2OS kunye neeseli ze-SPECC1L-kd zakhuliswa kumgangatho ophezulu we-glucose ye-DMEM eyongezwa nge-10% ye-fetal bovine serum (Life Technologies, Carlsbad, CA).Kwiinguqu ze-AJ, iiseli ze-2 x 105 zahlwayelwa kwiglasi ephathwe nge-0.1% ye-porcine gelatin (Sigma-Aldrich, St. Louis, MO) kwaye yaqaphela utshintsho kwimilo yeseli.Iiseli zaqokelelwa ngamaxesha ahlukeneyo abonakalisiweyo: iiyure ze-4 emva kokuhlwayela (t = 1), iiyure ze-24 emva kokuhlwayela (t = 2), ukudibanisa ngaphandle kokutshintsha kwimilo yeseli (t = 3), utshintsho kwimilo yeseli (t = 4) , 24 h emva kokutshintsha kobume beseli (t = 5) kunye ne-48 h emva kokutshintsha kobume beseli (t = 6) (Umfanekiso 1, 2, 3).Ukumodareyitha indlela ye-PI3K-AKT, iiseli zikhuliswe kwiindawo ezibonakalisiweyo kunye ne-PI3K-AKT inhibitor wortmannin (TOCRIS Biosciences, Minneapolis, Minnesota) okanye i-SC-79 activator (TOCRIS Biosciences, Minneapolis Adams, Minnesota).Indlela equlethe imichiza yayitshintshwa yonke imihla.
Ukurekhodwa kwesakhelo-nge-frame kwenziwa kulawulo oluphilayo kunye neeseli ze-KD phantsi kweemeko eziqhelekileyo zenkcubeko, kunye nemifanekiso ehlukeneyo yesigaba yaqokelelwa yonke imizuzu ye-10 kwiintsuku ze-7.Imifanekiso yafunyanwa kusetyenziswa ikhomputha elawulwa yiLeica DM IRB i-microscope eguquliweyo exhotywe ngesiteji somatshini kunye nenjongo ye-10 × N-PLAN exhunywe kwikhamera ye-QImaging Retiga-SRV.Ngexesha lokucinga, iinkcubeko zeeseli zigcinwe kwi-37 ° C kwindawo efumileyo kunye ne-5% CO2.
Imizila emibini ye-gene trap ES celllines DTM096 kunye ne-RRH048 evela kwi-Regional Mutant Mouse Resource Centre (UC Davis, CA) yasetyenziselwa ukuvelisa i-Specc11 imigca yegundane enqongopheleyo, ekhethiweyo i-Specc1lgtDTM096 kunye ne-Specc1lgtRRH046.Ngokufutshane, iiseli ze-129/REJ ES zafakwa kwi-C57BL6 blastocysts.Iimpuku ze-chimeric eziyindoda zaye zakhuliswa kunye neempuku ze-C57BL6 zasetyhini ukuchonga inzala enombala wengubo ye-agouti.Ubukho bokufakelwa kwe-gene trap vector kwakusetyenziselwa ukuchonga i-heterozygotes.Iigundane zigcinwe kwimvelaphi edibeneyo ye-129 / REJ; C57BL6.Indawo yokufakwa kwendawo ye-genetic trap vector yaqinisekiswa yi-RT-PCR, ulandelelwano lwe-genome, kunye ne-genetic complementation (Umfanekiso owongezelelweyo 1).Ukulandelela umnombo we-CNCC weempuku ze-heterozygous eziphindwe kabini ze-Specc1lGT, i-ROSAmTmG (#007576) kunye ne-Wnt1-Cre (#003829) yeempuku (i-Jackson Laboratory, i-Bar Harbour, ME) zawelwa ukuvelisa i-ROSAmTmG kunye ne-Wnt1-Cre allele kwi-Specc1l embryos.Zonke iimvavanyo kwiimpuku zenziwa ngokwemigaqo evunyiweyo yiKomiti yeZiko loNyango lweZilwanyana kunye nokuSetyenziswa kweZiko lezoNyango leYunivesithi yaseKansas.
Ii-embryo zilungiswe (1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2, 0.02% NP-40, 5 mM EGTA) kwi-60 min kwiqondo lokushisa.Emva kokulungiswa kwisisombululo se-X-gal staining (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, 0.01% ye-sodium deoxycholate, 0.02% NP-40, 1 mg / ml X-gal) Uphuhliso lwe-stain lwenziwa kwi-37 ° C. .°C kwiiyure ezi-1-6.Iimbumba zilungiswe emva kwe-4% ye-PFA kwaye zabonwa.
Kwi-Western blotting, iiseli ze-lysed kwi-passive lysis buffer (i-Promega, i-Fitchburg, i-WI) yongezwa ngomxube we-HALT protease inhibitors (Sigma-Aldrich, St. Louis, MO).I-Lysates yacutshungulwa kwi-12% ye-polyacrylamide Mini-PROTEAN TGX i-gel esele ilungile (i-Bio-Rad, i-Hercules, i-CA) kwaye idluliselwe kwi-Immobilon PVDF membranes (EMD Millipore, Billerica, MA).Iimbumba zavalwa kwi-5% yobisi kwi-PBS equkethe i-0.1% Tween.Ii-antibody zafakwa ngobusuku kwi-4°C okanye iyure enye kubushushu begumbi.I-Femto SuperSignal West ECL reagent (i-Thermo Scientific, Waltham, MA) yayisetyenziselwa ukuvelisa umqondiso.Kwi-immunostaining, ii-embryo zalungiswa ngobusuku kwi-4% PFA / PBS kunye ne-cryopreserved.I-tissue cryosections ivaliwe kwi-PBS equlethe i-1% ye-serum yebhokhwe eqhelekileyo (i-Thermo Scientific, Waltham, MA) kunye ne-0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) kwaye emva koko ifakwe kwi-4 ° C kwi-incubator ngexesha busuku.kunye ne-anti-antibody kunye ne-fluorescent yesibini ye-antibody (1:1000) iyure enye kwi-4°C.Amacandelo anebala abekwe kwi-ProLong medium yegolide (i-Thermo Scientific, i-Waltham MA) kunye nemifanekiso eflethi yafunyanwa kusetyenziswa i-microscope ye-Leica TCS SPE confocal.I-immunostaining nganye yenziwa njengemifuniselo emithathu ezimeleyo kwi-cirossections ubuncinane iimbumba ezimbini eziguqukayo.Umlingelo omele uyaboniswa.
Iiseli zafakwa kwi-buffer ye-RIPA elungisiweyo (20 mM Tris-HCl, pH 8.0, 1% NP-40, 130 mM NaCl, 10% glycerol, 2 mM EDTA, kunye ne-HALT protease inhibitor (Sigma-Aldrich, St. Louis, MO) Ngokufutshane, iilysates zaye zahlanjululwa ngeprotheyini ye-G yamaso kazibuthe (i-Life Technologies, Carlsbad, CA) kwaye emva koko yafukanyelwa ngobusuku kwi-4 ° C. kunye ne-anti-SPECC1L okanye i-IgG yeprotheyini ye-G amaso yayisetyenziselwa ukukhupha i-SPECC1L kunye nokuchithwa kwe-Western kwenziwa kusetyenziswa i-anti. -β-catenin antibody echazwe ngasentla Imifuniselo ye-co-IP ebonisiweyo imele imifuniselo emine ezimeleyo.
Iiseli ezizinzileyo ezicwangcisiweyo okanye izihlunu zempuku embryonic zanikezelwa kwiziko le-electron microscopy kwiYunivesithi yaseKansas Medical Centre.Ngokufutshane, iisampulu zifakwe kwi-EMbed 812 resin (i-Electron Microscopy Sciences, i-Fort Washington, PA), i-polymerized ngobusuku kwi-60 ° C, kwaye yahlula kwi-80 nm usebenzisa i-Leica UC7 ultramicrotome exhotywe nge-diamond blade.Amacandelo aye abonwa kusetyenziswa i-JEOL JEM-1400 yokuhanjiswa kwe-electron microscope exhotyiswe ngompu we-100 kV Lab6.
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