I-ASTM A790 2507 / 2205 1.4462 / 1.4410 I-Duplex Welded Tube Yengxenye yamakhemikhali Yemboni Yamakhemikhali, Ukuntuleka kwe-SPECC1L kuholela ekukhuleni kokuqina kwamalunga ahlukene kanye nokuncishiswa kokuchithwa kwamaseli e-cranial neural crest.

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I-ASTM A790 2507 / 2205 1.4462 / 1.4410 Duplex Welded Tube Yemboni Yamakhemikhali

I-Liaocheng Sihe SS Material Co., Ltd.ingumkhiqizi ohamba phambili ogxile kumapayipi ensimbi engenamthungo, amashubhu acwebile afakwe emanzini, amashubhu ahlanganisiwe angenamthungo njll.Ukuze kube lula amakhasimende , sinamapayipi ashiselwe namashubhu futhi .I-Liaocheng Sihe SS Material Co., Ltd.inemishini yokukhiqiza nokuhlola ethuthuke kakhulu.Singakwazi ukwanelisa ngokuphelele imfuneko yakho.Ngokwezinga eliqinile , amashubhu akhiqizwa yithi ahlala enokubekezelelana okulungile kwe-OD ne-WT .Ukulawulwa kokubekezelelana kuhambisana ngokuqinile nokukhiqiza amazinga.Imikhiqizo yethu ihlale inelisekile ngamakhasimende.Amakhasimende athenge imikhiqizo yethu adale inzuzo eyengeziwe.
a) OD ( Ububanzi Ongaphandle ): 3.18mm kuya ku-101.6mm
b) I-WT (Ukuqina Kodonga): 0.5mm kuya ku-20mm
c) Ubude : Ngokuvumelana nemfuneko yekhasimende
d) Amazinga : ASTM A312;I-ASTM A269;I-ASTM A789;I-ASTM A790 njll
e) Indlela Yokucubungula: ERW, EFW njll

Izilayidi ezibonisa izindatshana ezintathu zesilayidi ngasinye.Sebenzisa izinkinobho ezingemuva nezilandelayo ukuhamba phakathi kwamaslayidi, noma izinkinobho zokulawula ama-slide ekugcineni ukuze uhambe kuslayidi ngasinye.
Amaseli e-cranial neural crest cell (CNCC) ayashesha ukugoqa imizwa ye-embryonic futhi athuthele kuma-pharyngeal arches, akha iningi lezakhiwo ezimaphakathi.Ukungasebenzi kahle kwe-CNCC kudlala indima ebalulekile ku-etiology ye-orofacial cleft, ukukhubazeka okuvamile kokuzalwa.Ukuguqulwa kwe-Heterozygous SPECC1L kutholwe ezigulini ezinemifantu engabonakali kanye ne-syndromic.Lapha, sibika ukungcoliswa okuthuthukisiwe kwezingxenye ze-canonical adhesive junction (AJ), i-β-catenin ne-E-cadherin kumaseli akhulisiwe we-SPECC1L, kanye nama-electron micrographs abonisa ukusabalalisa kwe-apical-basal ye-AJ.Ukuze siqonde indima ye-SPECC1L ku-craniofacial morphogenesis, sidale imodeli yegundane entulekayo ye-Specc1l.Ama-Homozygous mutants ayingozi e-embryonic futhi abonisa ukuvalwa kwe-neural tube ekhubazekile kanye ne-CNCC lamination.I-AJ protein staining iyanda kuma-mutant neural folds.Lokhu kushiyeka kwe-AJ kuhambisana nenkinga ku-CNCC delamination, edinga ukuqedwa kwe-AJ.Ngaphezu kwalokho, izinguquko eziguquguqukayo ze-Specc11 zehlise ukusayinda kwe-PI3K-AKT futhi zanda i-apoptosis.I-In vitro, ukuvinjwa okuncane kokusayina kwe-PI3K-AKT kumaseli ohlobo lwasendle kwakwanele ukunxenxa izinguquko ze-AJ.Okubalulekile, izinguquko ze-AJ ezidalwe ukudilizwa kwe-SPECC1L zingahlehliswa ngokwenza kusebenze indlela ye-PI3K-AKT.Ihlanganiswe ndawonye, ​​le datha iphakamisa ukuthi i-SPECC1L, njengesilawuli esinoveli sokusayinda i-PI3K-AKT kanye ne-AJ biology, iyadingeka ekuvaleni kwe-neural tube kanye ne-CNCC stratification.
Amaseli e-Cranial neural crest cell (CNCCs) ahlala endaweni ye-dorsal neuroectoderm futhi asuse ku-neuroepithelium ye-neural folds ekhulayo ngenqubo ehilela ukuguqulwa kwe-epithelial-mesenchymal (EMT)1,2,3.Ama-CNCC e-epithelial afudukayo aphazamisa ukuhlangana kwe-intercellular futhi abe ama-CNCC afudukayo e-mesenchymal agcwalisa amakholomu e-pharyngeal okuqala futhi akhe ingxenye enkulu ye-craniofacial cartilage.Ngakho-ke, izakhi zofuzo ezilawula ukusebenza kwe-CNCC zivame ukuphazamiseka ku-etiology ye-craniofacial congenital anomalies efana nokuqhekeka kwe-orofacial, okuvamise ukuthikameza ukuzalwa okungu-1/800 e-US kuphela.Okunye kokukhubazeka kokuzalwa8.
I-Delamination ye-CNCC ihambisana nokuvalwa kwe-anterior neural tube phakathi kwezinsuku ezingu-8.5 neziyi-9.5 zokuthuthukiswa kombungu kumagundane.Izinguquko zenani lezinhlobo zofuzo ezihlotshaniswa negundane le-orofacial cleft nazo zibonisa uhlobo oluthile lwe-neural tube defect, okuhlanganisa i-Irf69,10, Ghrl310, Cfl111, ne-Pdgfrα12.Kodwa-ke, izinqubo zokuvalwa kwe-neural tube kanye ne-CNCC stratification zingabhekwa njengezizimele, njengoba igundane le-Splotch mutant (Pax3) libonisa amaphutha ekuvaleni kwe-neural tube ngaphandle kwanoma yimuphi umthelela ku-CNCC stratification noma ukufuduka kwe-13,14.Amamodeli engeziwe wamagundane anokukhubazeka ku-CNCC dissection kanye nokuvalwa kweshubhu le-neural azosiza ukucacisa isisekelo samangqamuzana avamile alezi zinqubo ezimbili.
Ukuhlukaniswa kwe-CNCC kumangqamuzana e-neuroepithelial kudinga ukuqedwa kwama-adhesive junctions (AJs), akhiwe ngama-protein complexes aqukethe, phakathi kwabanye, i-E-cadherin, i-β-catenin, i-α-E-catenin, ne-α-actinin ehlotshaniswa ne-actin filaments 2 Ucwaningo lwe-Overexpression E-cadherin kuma-neural folds lubonise ukuncipha noma ukubambezeleka kwe-CNCC delamination.Ngokuphambene, ukucindezelwa kwe-E-cadherin kubangela ukuhlukaniswa kwangaphambi kwesikhathi15,16.Izinto eziningi ezilamula i-EMT ngesikhathi se-CNCC stratification yizici zokuloba (AP2α, Id2, FOXD3, SNAIL, TWIST, SOX10) kanye namaprotheni avuselelayo we-extracellular matrix (ECM) afana ne-matrix metalloproteinases (MMPs), nokho ama-CNCC angama-cytoskeletal AJ regulator. akukaziwa.Indlela ye-PI3K-AKT yaziwa ngokuphikisana namazinga e-E-cadherin, ikakhulukazi ocwaningweni lomdlavuza17.Ucwaningo lwakamuva lubonise ukuthi ukulahlekelwa kokusayinwa kwe-PDGFα-based PI3K-AKT kumagundane kuholela ekuziphatheni okungavamile kwe-craniofacial, okuhlanganisa i-cleft palate kanye neural tube defects12.Nokho, ubudlelwano phakathi kwendlela ye-PI3K-AKT nokuzinza kwe-AJ ekufakweni kohlu kwe-CNCC akucacile.
Ngaphambilini sihlonze i-SPECC1L njengofuzo lokuqala oluguquguqukayo kubantu ababili abanomfantu oqinile osuka emlonyeni uye esweni, owaziwa ngokuthi yi-oblique cleft (ObFC) noma i-Tessier IV18 cleft.Ukuguqulwa kwe-SPECC1L kukhonjwe emindenini emibili enezizukulwane eziningi ene-autosomal dominant Opitz G/BBB syndrome (OMIM #145410), lapho abantu abathintekile babonise i-hyperdistance kanye ne-cleft lip/palate19, futhi emndenini owodwa one-Tibi overdistance syndrome (OMIM #145420)20 .ngaphezu kwesigamu sezigameko ze-Opitz G/BBB syndrome zixhunywe ku-X (OMIM #300000) futhi zibangelwa ukuguquka kofuzo ku-MID1 gene, ehlanganisa amaprotheni angu-22 ohlaka lwamathambo eseli ahlobene ne-microtubule.Sicabanga ukuthi i-SPECC1L, nayo iphrotheni ehlotshaniswa nama-microtubules kanye ne-actin cytoskeleton, ingase ilamule ukusayina okudingekayo ukuze kulungiswe kabusha i-actin cytoskeleton ngesikhathi sokunamathela kweseli nokufuduka kwe-18.Ngezifundo ze-in vitro ne-vivo, manje sesichaza i-SPECC1L njengesilawuli esinoveli sokuqina kwe-AJ ngokusayina kwe-PI3K-AKT.Ezingeni lamaselula, ukuntuleka kwe-SPECC1L kubangele ukwehla kwezinga leprotheyini ye-pan-AKT kanye nokwanda kwe-apical-basal dispersion ye-AJ, eyaqedwa ngokusebenza kwamakhemikhali kwendlela ye-AKT.Ku-vivo, imibungu entula i-Specc11 ibonisa ukuvalwa kwe-neural tube ekhubazekile kanye nokuncishiswa kwe-CNCC dissection.Ngakho-ke, i-SPECC1L isebenza ekuboniseni okusekelwe kakhulu kokunamathela kweseli okudingekayo ekusebenzeni okujwayelekile kwe-CNCC phakathi ne-morphogenesis yobuso.
Ukuze siveze indima ye-SPECC1L ezingeni lamaselula, sisebenzise ulayini weseli we-osteosarcoma ochazwe ngaphambilini ozinzile we-U2OS ongenawo ku-SPECC1L18.Lawa maseli e-U2OS azinzile ane-SPECC1L (kd) ehlehlisiwe abe nokwehla okumaphakathi (60–70%) kumazinga emibhalo ebhaliwe ye-SPECC1L namaprotheni, kanye namaphutha ekufudukeni nasekuhleleni kabusha kwe-actin cytoskeleton 18. Ngokuphambene, ukwehla okukhulu kwesikhashana I-SPECC1L ikhonjiswe ukuthi iholela ekukhubazekeni kwe-mitotic 23.Emuva kokucaciswa okwengeziwe, sithole ukuthi amaseli ethu azinzile we-SPECC1L-kd ashintshe i-morphology ngezinga eliphezulu kakhulu lokuhlangana (Umfanekiso 1).Amaseli okulawula angawodwana namaseli e-kd ekuhlanganeni okuphansi abukeka afana (Umfanekiso 1A, D).Amahora angu-24 ngemva kokuhlanganiswa, amaseli okulawula agcina umumo wawo we-cuboidal (Fig. 1B, E), kuyilapho amaseli e-SPECC1L-kd enwetshiwe (Fig. 1C, F).Izinga lalolu shintsho ekubunjweni kweseli lithwetshulwe izithombe ezibukhoma ze-vivo zamaseli okulawula namaseli e-kd (ifilimu 1).Ukuze sinqume indima ye-SPECC1L kumaseli ahlanganayo, siqale sahlola ukusho kwayo.Sithole ukuthi amazinga we-SPECC1L amaprotheni anyukile ekuhlanganisweni (Umfanekiso 1G), kanti amazinga okuloba we-SPECC1L awazange anyuke (Umfanekiso 1H).Ukwengeza, njengoba ukuminyana kwamaseli kwanda, amaprotheni e-SPECC1L aqoqwe emingceleni ye-intercellular (Fig. 2A-E), enephethini ehambisana naleyo ye-β-catenin ehlotshaniswa ne-membrane (Fig. 2A'-E').Njengoba kunikezwe ukuhlotshaniswa kwe-SPECC1L ne-actin cytoskeleton 18,23 sacabanga ukuthi i-SPECC1L isebenzisana namajunction anamathelayo asuselwa ku-actin (AJ).
(AF) SPECC1L amaseli e- knockdown (DF) anda lapho ehlangana kakhulu (F) uma kuqhathaniswa nokulawula amaseli e-U2OS (AC).Aboniswa lapha amaphuzu esikhathi amathathu kwayisithupha (T1, T3, T6) esiwakhethele ukuminyana kwamaseli ahlukene.(G) Ukuhlaziywa kwe-blot yaseNtshonalanga okubonisa ukuthi iphrotheni ye-SPECC1L izinzile ngezinga eliphezulu lokuhlangana uma kuqhathaniswa nezinga eliphansi lokuhlangana kumaseli okulawula.I-Western blot ye-SPECC1L ibonisa ibhendi engu-120 kDa elindelekile kanye nebhendi yesisindo semolekyuli ephakeme, okungenzeka ishintshwe ngemva kokuhumusha (*).Ukuhlaziywa kwe-blot yaseNtshonalanga kwenziwa ngaphansi kwezimo ezifanayo zokuhlangana okuphansi nokuphezulu.Izithombe ezibonisa i-SPECC1L ekuhlanganeni okuphansi nokuphezulu zithathwe endaweni efanayo.I-blot efanayo yasuswa futhi yahlolwa kabusha nge-β-actin antibody.(H) Ukuhlaziywa komthamo kwe-RT-PCR akuzange kubonise izinguquko ezibalulekile kumazinga wokuloba we-SPECC1L.Amabha wamaphutha amelela ama-SEM avela ekuhlolweni okune okuzimele.
(AE) Sikhethe amaphoyinti esikhathi ayisithupha (T1-T6) amele uhla lokuminyana kwamaseli ukuze kwenziwe ukuhlaziya komumo weseli kuvame kanye nezinguquko ze-AJ kumaseli e-U2OS nge-SPECC1L knockdown (kd).Amaphuzu esikhathi amahlanu okuqala ahlanganisa amaseli angawodwa (T1), 50-70% ukuhlanganiswa kwamaqoqo amaseli amancane (T2), ukuhlanganisa ngaphandle kokubunjwa kabusha kwamaseli e-kd (T3), ukulungisa kabusha amaseli e-kd (T4), nezinguquko zamahora angu-24.ngesimo sangemuva samaseli we-kd (T5).Iphrotheni ye-SPECC1L yahlakazeka kakhulu ku-cytoplasm ku-T1 (A), kodwa ukunqwabelana kwayo kwabonwa emingceleni ye-intercellular kumaphoyinti esikhathi alandelayo (B-E, imicibisholo).(FJ) i-β-catenin ibonisa ukuqoqwa okufanayo emingceleni ye-intercellular ehlotshaniswa ne-AJ complex.(A'-E') SPECC1L kanye ne-β-catenin zibonisa amabala agqagqene emingceleni yamaseli ekuminyana kwamaseli aphezulu (imicibisholo).(F'-J') Kumaseli e-SPECC1L-kd, ukungcoliswa kwe-β-catenin kubonakala kujwayelekile kumaseli aphansi (F'-H'), kodwa kuyanwebeka njengoba isimo seseli sishintsha (I', J'; imicibisholo), okubonisa ukuthi i-AJ zishintshile.Amabha = 10 µm.
Sibe sesizama ukuthola umphumela wokushoda kwe-SPECC1L ku-AJ.Sisebenzise izimpawu ezimbalwa ezihambisana ne-AJ, okuhlanganisa izingxenye ze-canonical F-actin, myosin IIb, β-catenin, ne-E-cadherin24,25,26,27.Izintambo zokucindezeleka ze-Actin zanda kumaseli we-SPECC1L-kd njengoba kuchazwe ngaphambilini (Umfanekiso 3A, B) 18.I-Myosin IIb ehlotshaniswa ne-actin filaments ibonise ukwanda okufanayo kwamaseli e-SPECC1L-kd ku-vitro (Fig. 3C, D).I-β-catenin ehlotshaniswa ne-AJ ibophezela ku-cadherin kulwelwesi lweseli, okubonisa iphethini evamile yokusho “yekhekheba lezinyosi” ekulawuleni ama-cubocyte (Umfanekiso 3E, G).Kuyathakazelisa ukuthi ezithombeni eziyisicaba zisebenzisa i-confocal microscopy, i-β-catenin (I-Fig. 3E, F) ne-E-cadherin (I-Fig. 3G, H) ingcolisa ulwelwesi lweseli lwamaseli angenayo i-SPECC1L ahlangene abonisa amaphethini avelele ebala elinwetshiwe.Lokhu kwandiswa kokungcoliswa kwe-β-catenin okuhlotshaniswa ne-AJ kumaseli e-kd kugqame kakhulu ekuhlanganeni, kodwa kubonakala kwandulela izinguquko ekubunjweni kweseli (Fig. 2F-J, F'-J').Ukuze sinqume isimo somzimba salokhu kungcoliswa kwe-AJ okunwetshiwe, sihlole imingcele yeseli endaweni ye-apical-basal yamaseli e-SPECC1L-kd U2OS nge-transmission electron microscopy (TEM) (Figure 3I,J).Ngokuphambene namaseli okulawula (Fig. 3I), ayenezifunda ezihlukene zama-electron aminyene abonisa i-AJ (imicibisholo), amaseli e-kd (Fig. 3J) abonise izifunda ezinkulu, ezihlangene zokuminyana kwama-electron aphezulu okubonisa i-AJ eduze kwendiza ye-apicobasal..Ukwengeza, ezigabeni eziphambeneyo, sabona ukugoqa okubanzi kwe-membrane yeseli kumaseli e-kd (Fig. S1A, B), echaza iphethini enwetshiwe ye-β-catenin ne-E-cadherin staining bands (Fig. 3F, H).Ekusekeleni indima ye-SPECC1L kuma-AJs, i-β-catenin yayihlanganiswe ne-SPECC1L kuma-lysates amaseli e-U2OS ahlangene (Fig. 3K).Kanye nokunwetshwa kwe-immunostaining kumamaki e-AJ, ukuhlaziywa kwe-TEM bekuhambisana nenkolelo-ze yethu yokuthi ukushoda kwe-SPECC1L kukhulisa ukuminyana kwe-AJ apical-basal kanye nokuhluka.
(AH) Ukwenyuka kwamabala e-F-actin kumaseli e-kd emahoreni angu-48 ngemva kokuhlanganisa (T6; A, B).Ukushintshwa kwamabala we-myosin IIb okuhlotshaniswa ne-F-actin (C, D).Iphethini ebushelelezi ye-β-catenin ne-E-cadherin membrane staining kumaseli okulawula (E, G) yathuthukiswa kumaseli e-SPECC1L-kd (F, H).Amabha = 10 µm.(I–J) Ama-micrographs e-electron abheka ukuhlangana kwe-apical-basal intercellular.Amaseli okulawula abonisa izifunda eziminyene ze-electron ezibonisa ukuhlangana okunamathelayo (I, imicibisholo).Ngokuphambene, yonke i-apical-basal junction kumaseli e-SPECC1L-kd ivele i-electron dense (J, imicibisholo), okubonisa ukuminyana okukhulayo nokuhlakazeka kokuhlangana okunamathelayo.(K) i-β-catenin yayihlanganiswe ne-SPECC1L kuma-lysates e-U2OS cell.Isithombe esithathwe endaweni eyodwa simele ukuhlola okune okuzimele.
Ukuze siqonde indima ye-SPECC1L ku-craniofacial morphogenesis, sidale imodeli yegundane entula i-Specc1l sisebenzisa imigqa yeseli ye-ES trap ezimbili ezimele, i-DTM096 ne-RRH048 (i-BayGenomics, CA), emele imibhalo ye-intron 1 ne-Specc1l ithathwe ku-15 (Fig. 1) .4A, umfanekiso S2).Indawo ye-genomic yokufaka i-decoy vector yanqunywa ukulandelana kwe-genome yonke futhi yaqinisekiswa yi-PCR (Fig. S2).Yomibili imiklamo ye-gene trap iphinde yavumela ukuhlanganiswa kohlaka lwezintatheli ze-Specc11-lacZ lapho zibanjwa.Ngakho-ke, isisho se-lacZ esinqunywe yi-X-gal staining sisetshenziswe njengenkomba yenkulumo ye-Specc11.Womabili ama-alleles abonise amaphethini wokuvezwa kwe-lacZ afanayo, ne-DTM096 gene trap ku-intron 1 ebonisa inkulumo enamandla kune-RRH048 ku-intron 15 (engaboniswa).Kodwa-ke, i-Specc1l ivezwa kabanzi, ngenkulumo eqinile ikakhulukazi kuma-neural folds ku-E8.5 (Umfanekiso 4B), ku-neural tube kanye nezinqubo zobuso ku-E9.5 no-E10.5 (Umfanekiso 4C, D), nasekuthuthukiseni izitho. kwe e10.5 namehlo (Umfanekiso 4D).Sibike ngaphambilini ukuthi inkulumo ye-SPECC1L ku-arch yokuqala ye-pharyngeal ku-E10.5 yayikhona ku-epithelium kanye ne-mesenchyme18 engaphansi, ehambisana nohlu lozalo lwe-CNCC.Ukuhlola ukusho kwe-SPECC1L ku-CNCC, senze i-E8.5 neural folds (Umfanekiso 4E-J) kanye nezigaba zogebhezi lwe-E9.5 (Umfanekiso 4K-).Ku-E8.5, i-SPECC1L ifake ama-neural folds kakhulu (Fig. 4E, H), kuhlanganise namaseli angcoliswe omaka be-NCC (Fig. 4G, J).Ku-E9.5, i-SPECC1L (I-Fig. 4K, N) i-CNCC enamabala aqinile efudukayo ehlanganiswe ne-AP2A (Fig. 4L, M) noma i-SOX10 (Fig. 4O, P).
(A) Ukumelwa okuhleliwe kwegundane lofuzo lwe-Specc11 okubonisa ukufakwa kwevekhtha ye-decoy kuma-clones weseli e-ES DTM096 (intron 1) kanye ne-RRH048 (intron 15).(BD) i-lacZ staining ye-heterozygous Specc1lDTM096 imibungu emelela inkulumo ye-Specc1l ukusuka ku-E8.5 ukuya ku-E10.5.I-NE = i-neuroectoderm, i-NF = i-neural fold, i-PA1 = i-arch yokuqala ye-pharyngeal.(EP) I-SPECC1L i-immunostaining enezimpawu ze-NCC i-AP2A ne-SOX10 ku-E8.5 (NF; EJ) i-neural folds kanye nezigaba zogebhezi lwe-E9.5 (KP).I-SPECC1L staining yaqashelwa kabanzi kuma-neural folds E8.5 (E, H; imicibisholo), okuhlanganisa namaseli anelebulwe ngokuthi AP2A (F, G; imicibisholo) kanye ne-SOX10 (I, J; imicibisholo).Ku-E9.5, SPECC1L ama-CNCC afudukayo anebala elinamandla (K, N; imicibisholo) anelebulwe ukuthi AP2A (L, M; imicibisholo) kanye ne-SOX10 (O, P; imicibisholo).
Ukweqa phakathi kwe-heterozygous Specc1lDTM096/+ kanye ne-Specc1lRRH048/+ amagundane kubonisa ukuthi ama-allele amabili ogibe lwezakhi zofuzo awaphelelisi nokuthi ama-heterozygote ahlanganisiwe nama-embryonic homozygote kunoma yisiphi i-gene trap allele ayabulala umbungu (Ithebula S1).Izilinganiso ze-Mendelian zibonise ukwehla kwezinga lokusinda lama-heterozygote ngesikhathi sokuzalwa (okulindelekile ku-1.34 vs. 2.0).Siphawule ukufa okuphansi kokubeletha phakathi kwama-heterozygote, amanye ayenokungaqondakali kwe-craniofacial (Fig. S3).Kodwa-ke, ukungena okuphansi kwalezi phenotypes ze-perinatal craniofacial kwenza kube nzima ukutadisha izindlela zabo eziyisisekelo ze-pathophysiological.Ngakho-ke, sigxile ku-embryonic ebulalayo phenotype ye-homozygous Specc11 mutants.
I-heterozygous eminingi ehlanganisiwe noma i-homozygous i-Specc1lDTM096/RRH048 imibungu eguquguqukayo ayizange ithuthuke ngemva kwe-E9.5–10.5 (Amakhiwane 5A-D), futhi ishubhu le-neural alizange livaleke ngaphambili (Amakhiwane 5B, D) futhi ngezinye izikhathi avale ngemuva (akuboniswanga) ..Lesi sici sokuvala ishubhu le-cranial neural tube sihlotshaniswa neningi le-CNCC elimakwe ngokuthi i-DLX2 esele kuma-neural folds ku-E10.5, okubonisa ukungahlukanisi (Umfanekiso 5A'-D').Ukuze sinqume ukuthi ingabe usayizi uwonke we-CNCC naye wehlisiwe, simake imigqa ye-CNCC ne-GFP emigqeni yethu yogibe lofuzo nge-Wnt1-Cre kanye ne-ROSAmTmG.Sisakaza i-GFP+ NCC ehlungiwe kanye ne-GFP- (RFP+) okungeyona i-NCC kusukela kuma-embryo wonke.Ku-E9.5, ingxenye yama-CNCC afakwe ilebula ye-GFP ehlelwe ngokugeleza ayizange ishintshe kakhulu phakathi kwe-WT nemibungu eguquguqukayo (engabonisiwe), okubonisa ukucaciswa okuvamile kwe-CNCC.Ngakho-ke, sacabanga ukuthi ukungcola kwe-Wnt1-Cre ne-DLX2 okusele kuma-neural folds (Umfanekiso 5B') kwakungenxa yokwendlaleka kwe-CNCC okunesici, okungenzeka kube ngenxa yokuminyana okwengeziwe noma ukuhlakazwa kwamaseli e-AJ, njengoba kubonakala kumaseli e-SPECC1L-kd.Sisebenzise omaka be-NCC SOX10, AP2A, kanye ne-DLX2 ukuze siqinisekise ubukhona be-CNCC ku-neural fold (Figure 5E-R).Ku-E8.5, ukugoqa kwe-neural staining kubo bonke omaka be-NCC abathathu kubonwe ezigabeni ze-WT (Fig. 5E, G, I) kanye ne-Specc1l mutant (Fig. 5F, H, J).Ku-E9.5, ngenkathi omaka be-NCC bengcolisa i-NCC efudukayo ezigabeni ze-WT (Fig. 5M, O, Q), insalela ye-NCC staining ibonwe ekugoqekeni kwe-neural okuveziwe kwe-Specc1l imibungu eguquguqukayo (Fig. 5N, P, R).Ngenxa yokuthi i-SOX10 kanye ne-DLX2 imaka ama-CNCC afudukayo, lo mphumela uphakamisa ukuthi ama-CNCC angenayo i-SPECC1L afinyelela ukucaciswa kwangemva kokufuduka kodwa ahluleke ukufuduka esuka kuma-neural folds.
Ukushoda kwe-Specc11 kuholela ekuvaleni kwe-neural tube enesici, i-delamination yamaseli e-cranial neural crest kanye nama-AJs.
(A, B') E9.5 WT (A) Umbungu othwele amaseli afudukayo e-cranial neural crest (CNCC) anelebuli ethi Wnt1-Cre (A').Ngokuphambene, ama-embryo e-Specc11 aguqukayo abonisa ukugoqa kwemizwa evulekile (B), imicibisholo) kanye nama-CNCC angakathutha (B', imicibisholo).(C, D') Izithombe zenkambu ekhanyayo (C, D') kanye ne-immunostaining (C', D') ye-CNCC marker DLX2 ye-E10.5 WT imibungu (C, C') kanye ne-Specc1l (D, D').Kuma-embryo e-WT E10.5, i-DLX2-positive CNCC ihlanganisa ama-gill arches (C', imicibisholo), kuyilapho eziguquguqukayo, amabala agqamayo aphikelela kuma-neural folds (D', imicibisholo) kanye namakhothamo okuqala e-pharyngeal (D', imicibisholo).) enamabala athile (imicibisholo) ekhombisa ukudelela nokufuduka kwe-CNCC.ER) Izigaba ze-WT kanye ne-Specc1l imibungu eguquguqukayo ezigabeni E8.5 (E–L) kanye ne-E9.5 (M–R) zilebulwe ngomaka be-NCC SOX10 (E, F, M, N), AP2A (G, H, O, P ) kanye ne-DLX2 (I, J, Q, R).Ku-E8.5, ukungcoliswa kwe-NCC kwabonwa ku-wild-type neural fold (NF) kanye nezigaba eziguqukayo.I-Co-staining ye-SOX10 ne-β-catenin ku-E8.5 WT (K) kanye ne-mutant (L) yembule ukwanda kwe-β-catenin staining emingceleni yamaseli kuma-neural folds.Ku-E9.5, ukungcoliswa kohlobo lwasendle lwama-CNCC afudukayo (M, O, Q) kwabonwa, kuyilapho eziguquguqukayo, ama-CNCC angahluziwe angcolisa ukugoqa kwemizwa evulekile (N, P, R).(S–Z) Ukuhlaziywa kokulebula kwe-vivo AJ ezigabeni ze-coronal ze-WT kanye nemibungu ye-Specc11DTM096/RRH048 enokuguqulwa kwe-E9.5.Indiza yesigaba esilinganiselwe iboniswa ekhoneni eliphezulu kwesokudla.Ezingxenyeni zezicubu eziguqukayo, ukwanda kokungcoliswa kwe-F-actin (S, T) kanye ne-myosin IIb (U, V) kwabonwa.Ngokufana nemiphumela ye-in vitro ku-Fig. 3, emibungu eguquguqukayo, ukungcoliswa kwe-membrane okuthuthukisiwe kwe-β-catenin (W, X) ne-E-cadherin (Y, Z) kwabonwa.(AA-BB) I-electron micrograph yengxenye yombungu wohlobo lwasendle ebheke ngale komngcele weseli ye-apical-basal ibonisa isifunda esiminyene se-electron esibonisa ukuhlangana okunamathelayo (AA, imicibisholo).Ngokuphambene, ezigabeni ze-Specc11 imibungu eguquguqukayo (BB, imicibisholo), yonke indawo yokuhlangana ye-apicobasal iminyene ye-electron, okubonisa ukuminyana okukhuphukile nokuhlakazeka kokuhlangana okunamathelayo.
Ukuhlola umbono wethu wokuthi ukubekwa kwezingqimba okuncishisiwe kungenxa ye-AJ eshintshiwe, sihlole ukulebula kwe-AJ kuma-neural folds e-Specc1l emibungu eguquguqukayo (Fig. 5S-Z).Sibone ukwanda kwezintambo zokucindezeleka kwe-actin (I-Fig. 5S, T) kanye nokukhuphuka okuhambisanayo kwendawo ye-myosin IIB staining ku-actin fibers (Fig. 5U, V).Okubalulekile, sibone ukwanda kwebala le-β-catenin (Fig. 5W, X) kanye ne-E-cadherin (Fig. 5Y, Z) emingceleni ye-intercellular.Siphinde sahlola i-β-catenin staining ye-NCC kuma-neural folds we-E8.5 embryos (Fig. 5K, L).I-β-catenin staining ibonakala inamandla ku-Specc1l mutant neural folds (Fig. 5L no-K), okuphakamisa ukuthi izinguquko ze-AJ zase ziqalile.Kuma-electron micrographs wezingxenye zogebhezi lwemibungu engu-E9.5, siphinde sabona ukwanda kokungcola kwe-electron-dense emibungu eguquguqukayo ye-Specc1l uma kuqhathaniswa ne-WT (Fig. 5AA, BB kanye ne-S1E-H).Ihlanganiswe ndawonye, ​​le miphumela isekela imiphumela yethu ye-in vitro kumaseli e-SPECC1L-kd U2OS futhi iphakamisa ukuthi ukungcola kwe-AJ okuphambukayo kwandulele ukuhlukaniswa kwe-CNCC kumibungu yethu eguquguqukayo.
Njengoba kunikezwe ubudlelwano obaziwayo obuphikisanayo phakathi komsebenzi we-AKT nokuzinza kwe-E-cadherin,17,28 sicabangele ukubandakanyeka kokusayina kwe-PI3K-AKT.Ngaphezu kwalokho, sabona amabhamuza e-subepidermal kweminye yemibungu yethu eguquguqukayo ephunyuke ebulalayo (<5%) ku-E9.5-10.5 futhi esikhundleni salokho yazinza cishe ku-E13.5 (Fig. S3).Ama-Subepidermal vesicles awuphawu lokubonisa okuncishisiwe kwe-PI3K-AKT okusekelwe ku-PDGFRα12.Fantauzzo et al.(I-2014) ibike ukuthi ukuphazamiseka kokusebenza kwe-PDGFRα-based PI3K ku-PdgfraPI3K/PI3K imibungu eguquguqukayo kubangela ama-subepidermal vesicles, neural tube defects, kanye ne-cleft palate phenotypes.Ngempela, amazinga e-pan-AKT kanye ne-Phosphorylated Ser473-AKT esebenzayo ancishiswa ku-vivo kumathishu aguquguqukayo e-Specc1l kuya ku-E9.5 ukuboshwa kwe-embryonic (Fig. 6A-D).Ukuncipha kwamazinga e-phosphorylated Ser473-AKT kungase kube ngokuphelele ngenxa yokuncipha kwamazinga e-pan-AKT ku-vivo (FIG. 6E) kanye ne-in vitro (FIG. 6F).Ukuncipha kwe-in vitro kwaqashelwa kuphela lapho amaseli e-U2OS ehlangene kakhulu nezinguquko ekubunjweni kweseli nokuminyana kwe-AJ (Umfanekiso 6D).Ngakho-ke, idatha yethu iphakamisa ukuthi i-SPECC1L iyinoveli yokulawula okuhle kokusayina kwe-PI3K-AKT ku-craniofacial morphogenesis.
(A–E) E8.5 (A,B) kanye ne-E9.5 (C,D) izigaba zogebhezi noma ama-E9.5 ama-lysates asuka ku-Specc1l imibungu eguquguqukayo (E) ebonisa amazinga we-phosphorylated esebenzayo S473-AKT kanye nokunciphisa amaprotheni e-pan-AKT , uma kuqhathaniswa nokulawula i-WT.Ukubhula kwaseNtshonalanga kwenziwa kuma-lysates ohlobo lwasendle nama-mutant lysates ngaphansi kwezimo ezifanayo.Izithombe ezibonisiwe ze-SPECC1L zithathwe endaweni eyodwa.I-blot efanayo yasuswa futhi yahlolwa kabusha ngama-anti-pan-ACT kanye ne-β-actin antibodies.Amazinga e-Pan-AKT ku-E8.5 neural folds (A, B) kanye namazinga we-phosphorylated S473-AKT ezigabeni zogebhezi lwe-E9.5 aye ancipha kakhulu.(F) Amaleveli e-Pan-AKT ancishiswa ngokufanayo kuma-lysates amaseli e-SPECC1L-kd U2OS avunwe ngokuhlangana okuphezulu.Amabha wamaphutha amelela ama-SEM avela kuzibalo ezintathu ezimele ze-Western blot.(GJ) Izigaba zemibungu ye-WT ku-E9.5 engcoliswe nge-KI67 futhi iklebhuke i-caspase 3, ngokulandelana, ebonisa ukwanda kwamaseli (G, G') kanye nomsebenzi omncane we-apoptotic (H, H').I-Specc11 imibungu eguquguqukayo ikhombisa ukwanda kwamaseli (I), kodwa inani lamaseli athola i-apoptosis lenyuke kakhulu (J).
Sibe sesihlola izimpawu zokwanda kanye ne-apoptosis.Asibonanga noma yimuphi umehluko ekwandeni kwemibungu engu-E9.5 (I-Fig. 6E, G uma iqhathaniswa ne-I) enenkomba yokwanda engu-82.5% yezinguquko ze-WT kanye no-86.5% we-Specc1l eziguquguqukayo ezilinganiswa nge-KI67 staining (p <0.56, i-Fisher's ukuhlolwa okuqondile).Ngokufanayo, asibonanga noma yimuphi umehluko ku-apoptosis elinganiswa ngokungcolisa i-caspase 3 eqhekeziwe kuma-neural folds ku-E8.5 kuze kuboshwe umbungu (akuboniswanga) (akuboniswanga).Ngokuphambene, i-apoptosis yanda kakhulu kuyo yonke imibungu eguquguqukayo ye-E9.5 (Umfanekiso 6F, H no-J).Lokhu kwanda sekukonke kwe-apoptosis kuhambisana nokuncipha kokusayina kwe-PI3K-AKT kanye nokufa kwe-embryonic29,30,31.
Okulandelayo, ukuze kuqinisekiswe indima eyimbangela yokusayina kwe-PI3K-AKT ekushintsheni kwe-AJ kumaseli ethu e-kd, siguqule ngamakhemikhali indlela yokulawula namaseli e-kd (Umfanekiso 7A-F).Sisebenzise njengomaka i-phenotype yokushintsha komumo weseli ebonwa kumaseli ahlanganayo we-SPECC1L-kd, esiwalinganise sisebenzisa isilinganiso sobukhulu (ubude) obuhambisanayo nobukhulu obuqondile (ububanzi).Isilinganiso esingu-1 silindeleke kumaseli ayindilinga noma ayi-cuboidal (Umfanekiso 7G).Ngaphezu kokuma kweseli, siphinde saqinisekisa umthelela ku-AJ nge-β-catenin staining (Fig. 7A'-F').Ukuvinjelwa kwendlela ye-PI3K-AKT usebenzisa i-wortmannin kwakwanele ukushintsha ukuma kweseli kumaseli okulawula (Umfanekiso 7A, C) kanye ne-AJ (Umfanekiso 7A').I-activator ye-PI3K-AKT i-SC-79 ayizange ithinte umumo weseli (FIG. 7A, E) noma ukunwetshwa kwe-AJ (FIG. 7A') kumaseli okulawula.Kumaseli e-SPECC1L-kd, ukucindezelwa okuqhubekayo kwendlela ye-PI3K-AKT kubangele ukwanda kwe-apoptosis (I-Fig. 7B, D) kanye nokwanda okuphawulekayo kwe-β-catenin staining (Fig. 7B'), ehambisana nezinguquko zethu ezinzima ze-vivo.Okubalulekile, ukwenziwa kusebenze kwendlela ye-PI3K-AKT kuthuthukise kakhulu ukuma kweseli (Umfanekiso 7B, F) kanye nama-phenotypes e-AJ (Umfanekiso 7B”).Izinguquko zomumo weseli zilinganiswa njengesilinganiso sokujikeleza kweseli (CCR) futhi zaqhathaniswa nokubaluleka njengoba kuchazwe ngenhla (FIG. 7G).Ngempela, kumaseli okulawula (I-Fig. 7G, CCR = 1.56), ukwelashwa kwe-wortmannin kwakwanele ukuguqula ngokuphawulekayo ukuma kweseli (I-Fig. 7G, CCR = 3.61, p <2.4 × 10-9) ngezinga elifana nelo elibonwayo. ku-SPECC1L.-kd amaseli (Fig. 7G, CCR = 3.46).Ukwelashwa kwe-Wortmannin kwamaseli e-SPECC1L-kd (I-Fig. 7G, CCR = 3.60, ayinaki) kwakungeyona into ebaluleke kakhulu kunamaseli e-kd angalashwa (Fig. 7G, CCR = 3.46, ayinaki) noma amaseli okulawula aphethwe yi-wortmannin (Fig. 7G)., i-CCR = 3.46, ayinaki) ngaphezu kwalokho ithinta ukwelulwa kweseli (7G, CCR = 3.61, akunakwa).Okubaluleke kakhulu, i-activator ye-SC-79 AKT ibuyisele i-phenotype emide yamaseli e-SPECC1L-kd (Fig. 7G, CCR = 1.74, p <6.2 × 10-12).Le miphumela iqinisekisa ukuthi i-SPECC1L ilawula ukusayinwa kwe-PI3K-AKT futhi iphakamisa ukuthi ukwehla okulinganiselwe ku-SPECC1L kuthinta ukunamathela kweseli, kanti ukwehla okunamandla kuholela ku-apoptosis (Fig. 8).
(A–F') Ukulawula (A, C, E) kanye namaseli e-SPECC1L-kd (B, D, F) aphathwe nge-PI3K-AKT pathway inhibitor wortmannin (C, D) noma i-SC-79 activator (E, F) Ukwelashwa .Amaseli okulawula angalashiwe ayi-cuboidal (A) ane-β-cat cell staining evamile (A'), kuyilapho amaseli e-kd elulwa (B) ngokunyuka kwamabala okuthi β-cat (B').Ngemva kokucindezelwa kwendlela ye-PI3K-AKT, amaseli okulawula anwetshwa (C) ngokunwetshwa kwe-β-cat (C'), kuyilapho amaseli e-kd eqala ukuba ne-apoptosis (D), efana nemibungu yethu eguquguquke kakhulu futhi ebonisa i-β-cat ethuthukisiwe kakhulu.ibala (D').Ngemuva kokusebenza kwendlela ye-PI3K-AKT, amaseli okulawula ahlala e-cuboidal (E) futhi anebala elivamile le-β-cat (E'), kuyilapho amaseli e-kd abonisa ukumila kweseli okuthuthuke kakhulu (F) kanye ne-β-cat (F'), okubonisa ukuthi (G) Izinga lokushintsha komumo weseli ku-(AF) lalinganiswa kusetshenziswa isilinganiso sokujikeleza kweseli (CCR) sohlangothi olude kakhulu (ubude) kanye nobukhulu obuhambisanayo obuqondile (ububanzi) kusetshenziswa isofthiwe ye-MetaMorph.Amaseli angalashiwe (NT) SPECC1L-kd (CCR = 3.46) ayemade kakhulu kunamaseli okulawula (CCR = 1.56, p <6.1 × 10–13).Ukuvinjelwa kwe-Wort kwendlela ye-PI3K-AKT kumaseli okulawula kwakwanele ukubangela ukunwetshwa okufanayo komumo weseli (CCR=3.61, p<2.4×10-9).Ngokufanayo, ukwenziwa kusebenze kwe-AKT nge-SC-79 kumaseli e-SPECC1L-kd kubuyisele ukwelulwa kweseli kumazinga okulawula (CCR = 1.74, p <6.2 × 10–12).Ukwelashwa kwe-Wortmannin kwamaseli e-SPECC1L-kd kuholele ekwenyukeni kwe-apoptosis kodwa akukho okunye ukwanda ekushintsheni komumo weseli (CCR=3.60) uma kuqhathaniswa ne-kd engelashiwe (CCR=3.46, ns) noma amaseli okulawula aphethwe yi-wortmannin (3.61) abonwe ku-.ns = akunandaba.+/- Izilinganiso ze-SEM zamaseli angu-50 ziyaboniswa.Umehluko wezibalo ubalwe kusetshenziswa i-t-test Yomfundi.
(A) Ukumelwa okuhleliwe kokuvimbela nokusebenzisa indlela ye-PI3K-AKT okuholela ekushintsheni kwe-AJ nokuhlenga, ngokulandelanayo.(B) Imodeli ehlongozwayo yokuqiniswa kwamaprotheni e-AKT nge-SPECC1L.
I-Premigratory CNCCs idinga i-AJ lysis ukuze ihlukane ne-anterior neural fold neuroepithelial cells1,15,32.Ukwenyuka kwamabala ezingxenyeni ze-AJ kanye nokulahlekelwa kwe-apical-basal AJ ukusatshalaliswa kwe-asymmetric kumaseli ashodayo we-SPECC1L kokubili ku-vitro kanye ne-vivo, kuhlanganiswe nokuba seduze ngokomzimba kwe-SPECC1L kuya ku-β-catenin, kuphakamisa ukuthi i-SPECC1L isebenza ukuze kugcinwe kahle ukuzinza kwendawo ye-AJ imisipha yenhlangano.i-actin cytoskeleton.Ukuhlotshaniswa kwe-SPECC1L ne-actin cytoskeleton kanye ne-β-catenin kanye nokwanda kwenani le-actin filaments efingqiwe lapho ingekho i-SPECC1L ihambisana nokukhuphuka okuphawulwe kokuminyana kwe-AJ.Okunye okungenzeka ukuthi inani elikhulayo lama-actin fibers kumaseli angenayo i-SPECC1L kuholela ekushintsheni kokungezwani kwe-intercellular.Ngenxa yokuthi ukucindezeleka kwamaselula kuthinta i-AJ 33 dynamics, izinguquko ze-voltage zingabangela ukusabalalisa okuningi kwe-AJ 34.Ngakho noma yiziphi izinguquko zizothinta izendlalelo ze-CNCC.
I-Wnt1 iboniswa kuma-neural folds asekuqaleni abangela amaseli e-neural crest.Ngakho-ke, i-Wnt1-cre yokulandelela yomugqa iphawula kokubili ngaphambi naphakathi kwe-NCC35 efudukayo.Kodwa-ke, i-Wnt1 iphinda imaka ama-clones ezicubu zobuchopho bomgogodla nawo asuselwa ekugoqekeni kwe-neural yokuqala engu-35,36, okwenza kube nokwenzeka ukuthi ukungcola kwethu kwe-E9.5 mutants kumaka we-Wnt1 kuma-neural folds avulekile akuyona i-CNCC.Ibala lethu elihle lezimpawu ze-NCC ze-AP2A ne-SOX10 ziqinisekise ukuthi ukugoqa kwemizwa okuveziwe kwama-embryo e-Specc11 aguquguqukayo ngempela bekuqukethe i-CNCC.Ukwengeza, njengoba i-AP2A ne-SOX10 kuyizimpawu ze-NCC efudukayo ngaphambi kwesikhathi, ukungcola okuhle kubonise ukuthi lawa maseli ayi-CNCC yangemva kokufuduka engakwazi ukuhlukaniswa yi-E9.5.
Idatha yethu iphakamisa ukuthi ukulawulwa kwamangqamuzana e-AJ nge-SPECC1L kulamula ukusayina kwe-PI3K-AKT.Ukusayina kwe-AKT kuncishisiwe kumaseli nezicubu ezintula i-SPECC1L.Okutholakele nguFantauzzo et al.isekela indima eqondile yokusayina kwe-PI3K-AKT ku-craniofacial morphogenesis.(I-2014) ibonise ukuthi ukuntuleka kokusebenza kokusayinwa kwe-PDGFRα-based PI3K-AKT kuholela ku-phenotype ye-cleft palate.Siphinde sibonisa ukuthi ukuvinjwa kwendlela ye-PI3K-AKT kwanele ukushintsha i-AJ nokuma kweseli kumaseli e-U2OS.Ngokuvumelana nokutholakele kwethu, uKayini et al.I-37 ibonise ukuthi ukwehliswa kwe-subunit ye-PI3K α110 kumaseli e-endothelial kubangela ukwanda okufanayo kwe-pericellular β-catenin staining, okubizwa ngokuthi ukwanda "kwenkomba yokuxhuma".Kodwa-ke, kumaseli e-endothelial ama-actin filaments asevele ehlelwe kakhulu, ukucindezelwa kwendlela ye-PI3K-AKT kuholela ekumeni kweseli okuxekethile.Ngokuphambene, amaseli e-SPECC1L-kd U2OS abonise umumo weseli omude.Lo mehluko ungahle uqonde uhlobo lweseli.Nakuba ukucindezelwa kokusayina kwe-PI3K-AKT kuthinta unomphela i-actin cytoskeleton, umthelela ekubunjweni kweseli kunqunywa izinguquko ekucindezelekeni okubangelwa izinguquko ekuminyana nasekuhleleni amafayili e-actin amaphakathi.Kumaseli e-U2OS, sisebenzise izinguquko zomumo weseli kuphela njengomaka wokushintsha nokululama kwe-AJ engena-SPECC1L.Sengiphetha, sicabanga ukuthi ukuvinjwa kwendlela ye-AKT ekuntulekeni kwe-SPECC1L kukhulisa ukuzinza kwe-AJ futhi kunciphisa i-delamination ku-CNCC.
Kuyathakazelisa ukuthi amazinga e-pan-AKT ancishiswa ku-vitro naku-vivo ngaphezu kwamazinga we-phosphorylated 473-AKT lapho ingekho i-SPECC1L, ephakamisa ukulawulwa kokusayina kwe-PI3K-AKT ezingeni le-AKT lokuzinza kwamaprotheni noma inzuzo.Izakhi zofuzo ze-SPECC1L ne-MID1, zombili ezihlotshaniswa ne-Opitz/GBBB syndrome, zihlanganisa amaprotheni azinzisa ama-microtubules 18,22.Indlela i-SPECC1L ne-MID1 elamula ngayo ukuqiniswa kwe-microtubule ayiqondwa ngokugcwele.Endabeni ye-SPECC1L, lokhu kuzinzisa kuhlanganisa i-acetylation ethuthukisiwe ye-subset yama-microtubules 18.Kungenzeka ukuthi i-SPECC1L isebenzisa indlela efanayo ukuze izinze amanye amaprotheni afana ne-AKT.Kuye kwaboniswa ukuthi i-acetylation yezinsalela ze-lysine ku-protein ye-AKT iholela ekunciphiseni kwe-membrane yendawo kanye ne-phosphorylation38.Ngaphezu kwalokho, ukutholakala kwendawo yonke kweketango le-K63 endaweni efanayo yensalela ye-lysine ku-AKT kuyadingeka ukuze kwenziwe ulwelwesi lwalo lwasendaweni kanye nokusebenza39,40.Phakathi kwezici eziningana ezisebenzisana namaprotheni e-SPECC1L ahlonzwe kumvubelo ephezulu yezikrini ezimbili ezixubile, ezine - CCDC841, ECM2942, APC kanye ne-UBE2I43 - ziye zathinteka ekwenziweni kwamaprotheni noma ukuzinza ngokusebenzisa indawo yonke noma i-sumoylation.I-SPECC1L ingase ibandakanyeke ekuguqulweni kwangemuva kokuhumusha kwezinsalela ze-AKT lysine, okuthinta ukuzinza kwe-AKT.Kodwa-ke, indima ebalulekile ye-SPECC1L ekwenzeni indawo kanye nokuzinza kwephrotheni ye-AKT isazocaciswa.
Ukukhubazeka okukhulu ekukhulumeni kwe-SPECC1L ku-vivo kuphumele ekukhuleni komaka kwe-AJ kanye nembondela ye-CNCC enesici, kanye nokwanda kwe-apoptosis kanye nobungozi bombungu bangaphambi kwesikhathi.Imibiko yangaphambilini ibonise ukuthi izinguquko zegundane ezinamazinga akhulayo we-apoptosis zihlotshaniswa nokukhubazeka kwe-neural tube 44,45,46,47 kanye nokukhubazeka kwe-craniofacial48.Kuye kwaphakanyiswa ukuthi ukufa kweseli ngokweqile kuma-neural folds noma ama-pharyngeal arches kungase kubangele inani elinganele lamaseli adingekayo ukuze kuhambisane ne-morphogenetic efanele i-48,49,50.Ngokuphambene, imigqa yethu yeseli entula ye-SPECC1L enenkulumo eyehliswe ngokumaphakathi ye-SPECC1L ibonise izinguquko ze-AJ kuphela ngaphandle kobufakazi bokwanda kokufa kweseli.Kodwa-ke, ukuvinjwa kwamakhemikhali kwendlela ye-PI3K-AKT kulawa maseli e-Kd kubangele ukwanda kwe-apoptosis.Ngakho, ukwehla okumaphakathi kwenkulumo noma umsebenzi we-SPECC1L kuqinisekisa ukusinda kweseli.Lokhu kuhambisana nokuqaphela ukuthi imibungu eguquguqukayo eyivelakancane ye-Specc11 ebalekela ukuboshwa e-st.I-E9.5-mhlawumbe ngenxa yokunciphisa ukusebenza kahle kwezakhi zofuzo-iyakwazi ukuvala amashubhu e-neural futhi ayeke kamuva ekuthuthukisweni, ngokuvamile ngokukhubazeka kwe-craniofacial (Fig. S3).Okunye okuhambisana nalokhu ukuvela okungajwayelekile kwemibungu eyi-heterozygous Specc1l enokungajwayelekile kwe-craniofacial—mhlawumbe ngenxa yokwanda kokuthwebula izakhi zofuzo—kanye nokutholakala ku-zebrafish lapho enye yama-orthologues amabili e-SPECC1L (specc1lb) idala ama-embryonic phenotypes sekwephuzile, okuhlanganisa nokulahlekelwa imihlathi engezansi kanye nokuqhekeka kwamazwe amabili51.Ngakho-ke, ukuguqulwa kwe-heterozygous SPECC1L kokulahlekelwa komsebenzi okuhlonzwe ezigulini ezingabantu kungase kubangele ukukhubazeka okuncane ekusebenzeni kwe-SPECC1L ngesikhathi se-craniofacial morphogenesis, okwanele ukuchaza ukuqhekeka kwe-orofacial.Ukulawulwa okusekelwe ku-SPECC1L koxhumana nabo be-intercellular kungase futhi kubambe iqhaza ku-palatogenesis kanye nokuhlanganiswa kwama-pharyngeal arches.Ucwaningo olwengeziwe lomsebenzi we-SPECC1L luzosiza ukucacisa indima yokuxhumana kwesikhashana kwe-intercellular ku-CNCC ngesikhathi sokuvalwa kwe-neural tube ku-neuroepithelial cell motility kanye ne-craniofacial morphogenesis.
Ukulawulwa kwe-osteosarcoma ye-U2OS namaseli e-SPECC1L-kd achazwe ngaphambilini (Saadi et al., 2011).Ama-antibodies ngokumelene ne-SPECC1L nawo abonakaliswe ngaphambilini (Saadi et al., 2011).Ama-anti-β-catenin antibodies (unogwaja; 1: 1000; Santa Cruz, Dallas, TX) (igundane; 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 esisikiwe (1:1000; Cell Signaling Technology, Danvers, MA) kanye ne-β-actin (1:2500; Sigma-Aldrich, St. Louis, MO ) isetshenziswe njengoba kuchaziwe..I-Actin filaments yayingcoliswe nge-Acti-stain rhodamine phalloidin (Cytoskeleton, Denver, Colorado).
Amaseli okulawula e-U2OS kanye namaseli e-SPECC1L-kd akhuliswe ku-DMEM yeglucose evamile ehambisana no-10% we-fetal bovine serum (Life Technologies, Carlsbad, CA).Ngezinguquko ze-AJ, amaseli angu-2 x 105 ahlwanyelwe engilazini ephathwe nge-gelatin ye-porcine engu-0.1% (Sigma-Aldrich, St. Louis, MO) futhi abhekwa ngokushintsha kwesimo seseli.Amaseli aqoqwe ngezikhathi ezihlukile ezibonisiwe: amahora angu-4 ngemva kokuhlwanyela (t = 1), amahora angu-24 ngemva kokuhlwanyela (t = 2), ukuhlangana ngaphandle koshintsho ekubunjweni kweseli (t = 3), ukuguqulwa kwesimo seseli (t = 4) , Amahora angu-24 ngemva kokuguqulwa komumo weseli (t = 5) kanye namahora angu-48 ngemva kokushintsha komumo weseli (t = 6) (Fig. 1, 2, 3).Ukuze kulungiswe indlela ye-PI3K-AKT, amaseli akhuliswe ekugxiliseni okubonisiwe nge-PI3K-AKT inhibitor wortmannin (TOCRIS Biosciences, Minneapolis, Minnesota) noma i-SC-79 activator (TOCRIS Biosciences, Minneapolis Adams, Minnesota).Indlela equkethe amakhemikhali yayishintshwa nsuku zonke.
Ukurekhodwa kohlaka ngohlaka kwenziwa ekulawuleni okubukhoma namaseli e-KD ngaphansi kwezimo ezijwayelekile zesiko, futhi izithombe zokuqhathanisa nezigaba zaziqoqwa njalo emizuzwini eyi-10 izinsuku eziyi-7.Izithombe zitholwe kusetshenziswa isibonakhulu esilawulwa ikhompuyutha i-Leica DM IRB ehlanekezelwe ngesiteji somshini kanye nenjongo engu-10 × N-PLAN exhunywe kukhamera ye-QImaging Retiga-SRV.Ngesikhathi sokuthwebula izithombe, amasiko amaseli agcinwa ku-37°C endaweni enomswakama ene-5% CO2.
Imigqa yeseli yeseli ye-ES ye-gene trap ye-ES DTM096 kanye ne-RRH048 evela ku-Regional Mutant Mouse Resource Center (UC Davis, CA) yasetshenziselwa ukukhiqiza imigqa yegundane entula i-Specc11, eqokiwe i-Specc1lgtDTM096 kanye ne-Specc1lgtRRH046.Kafushane, amaseli angu-129/REJ ES ajovwe kuma-blastocyst e-C57BL6.Amagundane abesilisa e-chimeric azalaniswa namagundane esifazane i-C57BL6 ukuhlonza inzalo enombala wejazi le-agouti.Ukuba khona kokufakwa kwe-gene trap vector kwasetshenziselwa ukukhomba ama-heterozygotes.Amagundane agcinwe kusizinda esixubile esingu-129/REJ;C57BL6.Indawo yesayithi yokufakwa kwe-genetic trap vector yaqinisekiswa yi-RT-PCR, ukulandelana kofuzo, kanye nokugcwalisa ufuzo (Umfanekiso Owengeziwe 1).Ukulandelela uhlu lwe-CNCC lwamagundane e-heterozygous e-Specc1lGT ephindwe kabili, i-ROSAmTmG (#007576) kanye ne-Wnt1-Cre (#003829) yamagundane (i-Jackson Laboratory, i-Bar Harbor, ME) yawela ukuze kukhiqizwe i-ROSAmTmG kanye ne-Wnt1-Cre allele ku-Speccbryol mutant.Zonke izivivinyo kumagundane zenziwe ngokuvumelana nezinqubo ezigunyazwe iKomidi Lesikhungo Sokunakekelwa Kwezilwane Nokusetshenziswa kwe-University of Kansas Medical Center.
Imibungu ifakwe ku-(1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2, 0.02% NP-40, 5 mM EGTA) imizuzu engama-60 ekamelweni lokushisa.Ngemva kokulungiswa kusixazululo se-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) Ukuthuthukiswa kwamabala kwenziwa ku-37°C .°C phakathi namahora angu-1-6.Imibungu yalungiswa ngemuva ku-4% PFA futhi yabonwa.
E-Western blotting, amaseli aye-lysed ku-passive lysis buffer (i-Promega, i-Fitchburg, i-WI) ehlanganiswe nengxube ye-HALT protease inhibitors (Sigma-Aldrich, St. Louis, MO).Ama-Lysates acutshungulwa ku-12% we-polyacrylamide Mini-PROTEAN TGX amajeli enziwe ngomumo (i-Bio-Rad, i-Hercules, i-CA) futhi adluliselwa kulwelwesi lwe-Immobilon PVDF (EMD Millipore, Billerica, MA).Ama-membrane ayevinjwe kubisi lwe-5% ku-PBS equkethe i-0.1% Tween.Amasosha omzimba ayefukanyelwa ebusuku ku-4°C noma ihora elilodwa ekamelweni lokushisa.I-Femto SuperSignal West ECL reagent (Thermo Scientific, Waltham, MA) isetshenziselwe ukukhiqiza isignali.Ukuze kuvinjwe amasosha omzimba, imibungu yalungiswa ngobusuku obubodwa ku-4% PFA/PBS futhi i-cryopreserved.Ama-tissue cryosections avinjiwe ku-PBS equkethe u-1% we-serum yembuzi evamile (Thermo Scientific, Waltham, MA) kanye no-0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) futhi yafakwa ku-4°C ku-incubator ngesikhathi ebusuku.nge-anti-antibody kanye ne-fluorescent yesibili amasosha omzimba (1:1000) ihora elingu-1 ku-4°C.Izingxenye ezinebala zafakwa ku-ProLong gold medium (Thermo Scientific, Waltham MA) futhi izithombe eziyisicaba zatholwa kusetshenziswa isibonakhulu se-Leica TCS SPE confocal.I-immunostaining ngayinye yenziwa njengokuhlola okuthathu okuzimele kokuguquguquka okungenani kwemibungu emibili eguqukile.Kuboniswa ummeleli wokuhlola.
Amaseli afakwe kubhafa ye-RIPA eshintshiwe (20 mM Tris-HCl, pH 8.0, 1% NP-40, 130 mM NaCl, 10% glycerol, 2 mM EDTA, kanye ne-HALT protease inhibitor (Sigma-Aldrich, St. Louis, MO) Kafushane, ama-lysates ahlanjululwa kusengaphambili nge-protein G yobuhlalu kazibuthe (i-Life Technologies, Carlsbad, CA) abese efukanyelwa ngobusuku obungu-4° C. nge-anti-SPECC1L noma i-IgG protein G ubuhlalu basetshenziselwe ukukhipha i-SPECC1L futhi ukuvinjelwa kwaseNtshonalanga kwenziwa kusetshenziswa i-anti. -β-catenin antibody echazwe ngenhla Ukuhlolwa kwe-co-IP okubonisiwe kumelela ukuhlola okune okuzimele.
Amaseli agxilile amisiwe noma izicubu ze-embryonic zegundane zinikezwe isikhungo se-electron microscopy e-University of Kansas Medical Center.Kafushane, amasampula ashumekwe ku-EMbed 812 resin (Electron Microscopy Sciences, Fort Washington, PA), enziwe i-polymer ngobusuku obungu-60°C, futhi ahlukaniswa ku-80 nm kusetshenziswa i-Leica UC7 ultramicrotome ehlome ngeblade yedayimane.Izigaba zibonwe ngeso lengqondo kusetshenziswa isibonakhulu se-electron yokudlulisa i-JEOL JEM-1400 efakwe isibhamu se-100 kV Lab6.
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