ASTM A790 2507 / 2205 1.4462 / 1.4410 Duplex Welded Tube Rau Kev Lag Luam Kev Lag Luam chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemical Chemistry

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ASTM A790 2507 / 2205 1.4462 / 1.4410 Duplex Welded Tube Rau Kev Lag Luam Tshuaj

Liaocheng Sihe SS Material Co., Ltd.yog cov chaw tsim khoom lag luam uas tshwj xeeb hauv stainless hlau seamless kav, ci annealed hlab, seamless coiled tubing thiab lwm yam.Txhawm rau pab txhawb cov neeg siv khoom, peb muaj cov kav dej thiab cov kav dej.Liaocheng Sihe SS Material Co., Ltd.muaj cov cuab yeej ua tau zoo tshaj plaws thiab kev sim.Peb tuaj yeem ua raws li koj qhov yuav tsum tau ua.Raws li tus qauv nruj heev, cov raj uas tsim los ntawm peb ib txwm muaj qhov tseeb OD thiab WT kam rau ua.Kev tswj kev kam rau siab yog nruj me ntsis raws li cov qauv tsim.Peb cov khoom yeej ib txwm txaus siab rau cov neeg siv khoom.Cov neeg muas zaub yuav peb cov khoom tsim tau nyiaj ntau dua.
a) OD (On Diameter): 3.18mm txog 101.6mm
b) WT (Wall Thickness): 0.5mm txog 20mm
c) Ntev: Raws li cov neeg siv khoom xav tau
d) Cov Qauv: ASTM A312;ASTM A269;ASTM A789;ASTM A790 thiab lwm yam
e) Txheej Txheem: ERW, EFW thiab lwm yam

Sliders qhia peb kab lus rau ib tus swb.Siv cov nyees khawm rov qab thiab tom ntej kom txav mus los ntawm cov slides, lossis cov khawm tswj swb thaum kawg kom txav mus los ntawm txhua tus swb.
Cranial neural crest cells (CNCC) slough tawm ntawm embryonic neural folds thiab tsiv mus rau pharyngeal arches, uas tsim feem ntau ntawm cov qauv midface.CNCC dysfunction plays lub luag haujlwm tseem ceeb hauv etiology ntawm orofacial cleft, ib qho kev tsis sib haum xeeb hauv lub cev.Heterozygous SPECC1L kev hloov pauv tau pom nyob rau hauv cov neeg mob atypical thiab syndromic clefts.Ntawm no, peb tshaj tawm kev txhim kho staining ntawm canonical adhesive junction (AJ) Cheebtsam, β-catenin thiab E-cadherin hauv kab lis kev cai SPECC1L knockdown cells, thiab electron micrographs qhia apical-basal diffusion ntawm AJ.Txhawm rau nkag siab lub luag haujlwm ntawm SPECC1L hauv craniofacial morphogenesis, peb tsim Specc1l tus qauv tsis muaj nas.Homozygous mutants yog embryonic tuag thiab nthuav tawm cov hlab ntsha tsis zoo kaw thiab CNCC lamination.AJ protein staining yog nce nyob rau hauv mutant neural folds.Qhov kev tsis zoo AJ no zoo ib yam nrog rau qhov tsis xws luag hauv CNCC delamination, xav tau AJ dissolution.Tsis tas li ntawd, Specc11 mutants tau txo PI3K-AKT signaling thiab nce apoptosis.Hauv vitro, me me inhibition ntawm PI3K-AKT signaling nyob rau hauv cov tsiaj qus hom yog txaus los ntxias AJ hloov.Qhov tseem ceeb, AJ hloov pauv los ntawm SPECC1L knockdown tuaj yeem thim rov qab los ntawm kev ua kom txoj hauv kev PI3K-AKT.Ua ke, cov ntaub ntawv no qhia tias SPECC1L, raws li tus tshiab regulator ntawm PI3K-AKT signaling thiab AJ biology, yuav tsum tau rau neural raj kaw thiab CNCC stratification.
Cranial neural crest cells (CNCCs) nyob rau hauv dorsal neuroectoderm thiab tshem tawm los ntawm cov neuroepithelium ntawm kev txhim kho neural folds los ntawm cov txheej txheem cuam tshuam nrog kev hloov pauv ntawm epithelial-mesenchymal (EMT) 1,2,3.Premigrating epithelial CNCCs cuam tshuam intercellular junctions thiab ua migrating mesenchymal CNCCs uas sau thawj thiab thib ob pharyngeal arches thiab tsim feem ntau ntawm cov pob txha mos craniofacial.Yog li, cov noob uas tswj CNCC muaj nuj nqi feem ntau cuam tshuam rau hauv etiology ntawm craniofacial congenital anomalies xws li orofacial clefts, feem ntau cuam tshuam rau 1/800 yug hauv Teb Chaws Asmeskas ib leeg.Ib tug ntawm congenital deformities8.
Delamination ntawm CNCC coincides nrog kaw lub anterior neural raj ntawm 8.5 thiab 9.5 hnub ntawm embryonic kev loj hlob nyob rau hauv nas.Mutants ntawm ib tug xov tooj ntawm nas orofacial cleft-associated genes kuj pom ib co daim ntawv ntawm neural raj tsis xws luag, xws li Irf69,10, Ghrl310, Cfl111, thiab Pdgfrα12.Txawm li cas los xij, cov txheej txheem ntawm neural tube kaw thiab CNCC stratification tuaj yeem suav tias yog kev ywj pheej, raws li Splotch mutant nas (Pax3) nthuav tawm qhov tsis xws luag hauv neural raj kaw yam tsis muaj kev cuam tshuam rau CNCC stratification lossis tsiv teb tsaws 13,14.Cov qauv nas ntxiv nrog qhov tsis xws luag hauv CNCC kev txiav tawm thiab kaw lub raj neural yuav pab qhia txog cov qauv molecular ntawm ob txheej txheem no.
Kev rho tawm ntawm CNCC los ntawm cov hlwb neuroepithelial yuav tsum muaj kev sib cais ntawm cov nplaum nplaum (AJs), uas yog tsim los ntawm cov protein ntau uas muaj, ntawm lwm tus, E-cadherin, β-catenin, α-E-catenin, thiab α-actinin txuam nrog actin filaments 2. Kev tshawb fawb ntau dhau ntawm E-cadherin hauv neural folds pom tias txo qis lossis qeeb hauv CNCC delamination.Hloov pauv, kev tawm tsam ntawm E-cadherin ua rau thaum ntxov stratification15,16.Ntau yam uas kho EMT thaum lub sij hawm CNCC stratification yog transcription yam (AP2α, Id2, FOXD3, SNAIL, TWIST, SOX10) thiab extracellular matrix (ECM) remodeling proteins xws li matrix metalloproteinases (MMPs), txawm li cas los CNCCs yog ncaj qha cytoskeletal AJ. tseem tsis tau paub.Txoj hauv kev PI3K-AKT paub txog kev tawm tsam E-cadherin qib, feem ntau los ntawm kev tshawb fawb qog noj ntshav17.Cov kev tshawb fawb tsis ntev los no tau pom tias poob ntawm PDGFα-raws li PI3K-AKT signaling nyob rau hauv nas ua rau craniofacial txawv txav, nrog rau cleft palate thiab neural tube defects12.Txawm li cas los xij, kev sib raug zoo ntawm PI3K-AKT txoj hauv kev thiab AJ kev ruaj ntseg ntawm CNCC stratification tsis meej.
Peb yav dhau los tau txheeb xyuas SPECC1L yog thawj cov noob caj noob ces hauv ob tus neeg uas muaj qhov sib cais loj heev uas nthuav tawm ntawm lub qhov ncauj mus rau qhov muag, hu ua oblique cleft (ObFC) lossis Tessier IV18 cleft.SPECC1L kev hloov pauv tau raug txheeb xyuas nyob rau hauv ob tsev neeg ntau yam nrog autosomal dominant Opitz G / BBB syndrome (OMIM #145410), uas cuam tshuam rau cov tib neeg tau pom qhov ua siab ntev thiab cleft di ncauj / palate19, thiab hauv ib tsev neeg nrog Tibi overdistance syndrome (OMIM #145420) .Ntau tshaj li ib nrab ntawm cov neeg mob ntawm Opitz G / BBB syndrome yog X-txuas (OMIM # 300000) thiab yog tshwm sim los ntawm kev hloov hauv MID1 noob, uas encodes protein 22 ntawm microtubule-txuas nrog cell skeleton.Peb xav tias SPECC1L, kuj yog cov protein txuam nrog microtubules thiab cov actin cytoskeleton, tuaj yeem kho qhov teeb meem xav tau rau kev hloov kho actin cytoskeleton thaum lub sijhawm cell adhesion thiab tsiv teb tsaws 18.Los ntawm kev tshawb fawb hauv vitro thiab hauv vivo, peb tam sim no piav qhia SPECC1L ua tus tswj hwm tshiab ntawm AJ kev ruaj ntseg los ntawm PI3K-AKT signaling.Ntawm qib cellular, SPECC1L tsis txaus ua rau txo qis hauv qib ntawm pan-AKT protein thiab nce hauv apical-basal dispersion ntawm AJ, uas tau tshem tawm los ntawm kev ua kom muaj tshuaj lom neeg ntawm AKT txoj hauv kev.Hauv vivo, Specc11-tsis muaj embryos qhia qhov tsis zoo ntawm lub raj kaw thiab txo CNCC kev txiav tawm.Yog li, SPECC1L ua haujlwm nyob rau hauv cov txheej txheem tswj hwm ntawm tes adhesion-raws li cov teeb liab yuav tsum tau ua rau CNCC muaj nuj nqi thaum lub ntsej muag morphogenesis.
Txhawm rau qhia lub luag haujlwm ntawm SPECC1L ntawm qib cellular, peb tau siv cov lus piav qhia yav dhau los ruaj khov osteosarcoma cell kab U2OS tsis txaus hauv SPECC1L18.Cov hlwb U2OS ruaj khov nrog SPECC1L (kd) knockdown muaj qhov nruab nrab (60-70%) txo qis hauv cov qib ntawm SPECC1L cov ntawv sau thiab cov proteins, nrog rau cov kev tsis zoo hauv kev tsiv teb tsaws thiab rov tsim kho ntawm actin cytoskeleton 18. Hauv qhov sib piv, qhov kev hloov pauv loj zuj zus hauv SPECC1L tau pom tias ua rau mitotic defects 23 .Raws li tus cwj pwm ntxiv, peb pom tias peb cov hlwb SPECC1L-kd ruaj khov hloov morphology ntawm qhov sib txuam siab heev (Daim duab 1).Tus kheej tswj cov hlwb thiab kd hlwb ntawm qhov tsis sib haum xeeb zoo sib xws (Daim duab 1A,D).24 teev tom qab fusion, tswj cov hlwb khaws lawv cov duab cuboidal (Fig. 1B, E), thaum SPECC1L-kd hlwb elongated (Fig. 1C, F).Qhov luaj li cas ntawm qhov kev hloov pauv ntawm lub xovtooj ntawm tes tau raug ntes los ntawm vivo nyob imaging ntawm tswj hlwb thiab kd hlwb (movie 1).Txhawm rau txiav txim siab lub luag haujlwm ntawm SPECC1L hauv cov hlwb sib txuas, peb thawj zaug tshuaj xyuas nws cov lus qhia.Peb pom tias SPECC1L cov protein ntau tau nce thaum fusion (Daim duab 1G), thaum SPECC1L cov ntawv teev lus tsis tau nce (Daim duab 1H).Tsis tas li ntawd, raws li cell ceev nce, SPECC1L protein ntau nyob rau ntawm intercellular ciam teb (Daim duab. 2A-E), nrog ib tug qauv overlapping nrog cov membrane-cuam tshuam β-catenin (Fig. 2A'-E').Muab lub koom haum ntawm SPECC1L nrog cov actin cytoskeleton 18,23 peb xav tias SPECC1L cuam tshuam nrog actin-based adhesive junctions (AJ).
(AF) SPECC1L knockdown (DF) hlwb elongate ntawm siab confluence (F) piv rau tswj U2OS hlwb (AC).Qhia ntawm no yog peb ntawm rau lub sijhawm cov ntsiab lus (T1, T3, T6) uas peb xaiv rau qhov sib txawv ntawm tes.(G) Western blot tsom xam qhia tias SPECC1L protein nyob ruaj khov ntawm qhov siab ntawm confluence piv rau qib qis ntawm confluence hauv cov hlwb tswj.Western blot ntawm SPECC1L qhia qhov xav tau 120 kDa band thiab ntau dua molecular luj band, tejzaum nws tom qab hloov pauv hloov (*).Western blot tsom xam tau ua nyob rau hauv tib yam kev mob rau qis thiab siab confluence.Cov duab uas qhia SPECC1L ntawm qhov qis thiab qhov sib txuam siab tau raug coj los ntawm tib lub blot.Tib lub blot raug tshem tawm thiab rov tshuaj xyuas nrog β-actin antibody.(H) Quantitative RT-PCR tsom xam pom tsis muaj kev hloov pauv tseem ceeb hauv SPECC1L cov ntawv sau tseg.Cov kab yuam kev sawv cev SEMs los ntawm plaub qhov kev sim ywj pheej.
(AE) Peb tau xaiv rau lub sij hawm cov ntsiab lus (T1-T6) sawv cev rau ntau yam ntawm cov xov tooj ntawm tes kom normalize cell duab tsom xam thiab AJ hloov hauv U2OS hlwb nrog SPECC1L knockdown (kd).Thawj tsib lub sij hawm cov ntsiab lus no suav nrog tib lub hlwb (T1), 50-70% fusion ntawm cov cell me (T2), fusion yam tsis muaj reshaping kd hlwb (T3), reshaping kd hlwb (T4), thiab 24 teev hloov.nyob rau hauv daim ntawv posterior ntawm kd (T5) hlwb.Cov protein SPECC1L feem ntau tau tawg mus rau hauv cytoplasm ntawm T1 (A), tab sis nws cov tsub zuj zuj tau pom ntawm thaj tsam intercellular ntawm lub sijhawm tom ntej (B-E, xub).(FJ) β-catenin qhia tau hais tias zoo sib xws ntawm cov ciam teb intercellular txuam nrog AJ complex.(A'-E') SPECC1L thiab β-catenin qhia kev sib tshooj ntawm cov ciam teb ntawm tes ntawm cov xov tooj ntawm tes siab (cov xub).(F'-J') Hauv SPECC1L-kd hlwb, β-catenin staining zoo li qub ntawm cov xov tooj ntawm tes qis (F'-H'), tab sis nthuav dav raws li kev hloov pauv ntawm tes (I', J'; xub), qhia tias AJ tau hloov.Luas = 10 µm.
Peb mam li sim txiav txim siab qhov cuam tshuam ntawm SPECC1L deficiency ntawm AJ.Peb tau siv ob peb AJ-kawg cim, suav nrog cov canonical Cheebtsam F-actin, myosin IIb, β-catenin, thiab E-cadherin24,25,26,27.Actin stress fibers tau nce hauv SPECC1L-kd hlwb raws li tau piav ua ntej (Fig. 3A,B) 18 .Myosin IIb cuam tshuam nrog actin filaments pom qhov zoo sib xws hauv SPECC1L-kd hlwb hauv vitro (Fig. 3C,D).AJ-associated β-catenin khi rau cadherin ntawm lub cell membrane, qhia ib txwm "honeycomb" qhia qauv hauv kev tswj cubocytes (Fig. 3E, G).Interestingly, nyob rau hauv cov duab tiaj tus uas siv confocal microscopy, β-catenin (Fig. 3E, F) thiab E-cadherin (Fig. 3G, H) staining ntawm lub cell membrane ntawm confluent SPECC1L-tsis muaj hlwb pom cov qauv tseem ceeb ntawm kev staining txuas ntxiv.Qhov kev nthuav dav ntawm AJ-koom nrog β-catenin staining hauv kd hlwb tau tshaj tawm ntawm qhov sib txuam, tab sis tau tshwm sim ua ntej hloov pauv ntawm cov duab ntawm tes (Fig. 2F-J, F'-J').Txhawm rau txiav txim siab lub cev ntawm qhov txuas ntxiv AJ staining, peb tau tshuaj xyuas cov ciam teb ntawm tes ntawm apical-basal nto ntawm SPECC1L-kd U2OS hlwb los ntawm kev xa hluav taws xob microscopy (TEM) (Daim duab 3I,J).Nyob rau hauv sib piv rau cov tswj hlwb (Fig. 3I), uas muaj cais electron ntom cheeb tsam qhia ntawm AJ ( xub xub), kd hlwb (Fig. 3J) pom loj, contiguous cheeb tsam ntawm high electron ceev qhia AJ raws lub apicobasal dav hlau..Tsis tas li ntawd, nyob rau ntawm qhov chaw hla, peb tau pom ntau cov cell membrane folds hauv kd hlwb (Fig. S1A, B), uas piav qhia txog cov qauv txuas ntxiv ntawm β-catenin thiab E-cadherin staining bands (Fig. 3F, H).Hauv kev txhawb nqa lub luag haujlwm ntawm SPECC1L hauv AJs, β-catenin tau co-immunoprecipitated nrog SPECC1L hauv lysates ntawm confluent U2OS hlwb (Fig. 3K).Nrog rau kev tiv thaiv txuas ntxiv rau AJ cov cim, TEM tsom xam tau ua raws li peb qhov kev xav tias SPECC1L tsis muaj peev xwm nce AJ apical-basal ntom thiab sib txawv.
(AH) Nce F-actin staining hauv kd hlwb ntawm 48 teev tom qab fusion (T6; A, B).Kev hloov pauv ntawm myosin IIb cuam tshuam nrog F-actin (C, D).Tus qauv du ntawm β-catenin thiab E-cadherin membrane staining hauv cov hlwb tswj (E, G) tau txhim kho hauv SPECC1L-kd (F, H) hlwb.Luas = 10 µm.(I-J) Electron micrographs saib lub apical-basal intercellular junction.Tswj cov hlwb qhia cov cheeb tsam electron-dense uas qhia cov junctions nplaum (I, xub).Nyob rau hauv sib piv, tag nrho cov apical-basal hlws ris nyob rau hauv SPECC1L-kd hlwb tshwm electron ntom (J, xub), qhia tau hais tias muaj zog ceev thiab dispersion ntawm nplaum junctions.(K) β-catenin yog co-immunoprecipitated nrog SPECC1L nyob rau hauv confluent U2OS cell lysates.Duab coj los ntawm ib qho chaw sawv cev rau ib qho ntawm plaub qhov kev sim ywj pheej.
Txhawm rau nkag siab txog lub luag haujlwm ntawm SPECC1L hauv craniofacial morphogenesis, peb tau tsim Specc1l tus qauv tsis muaj nas siv ob txoj kab ES ntxiab ntawm tes, DTM096 thiab RRH048 (BayGenomics, CA), uas sawv cev rau intron 1 thiab Specc1l cov ntawv sau tau raug ntes ntawm 15 (daim duab 1) .4A, duab S2).Qhov chaw genomic ntawm decoy vector ntxig tau txiav txim siab los ntawm tag nrho cov genome sequencing thiab paub tseeb los ntawm PCR (Fig. S2).Ob qho kev tsim cov cuab yeej cuab tam kuj tau tso cai nyob rau hauv-nruab nrab fusion ntawm Specc11-lacZ reporters thaum ntes.Yog li, lacZ qhia tau txiav txim siab los ntawm X-gal staining tau siv los ua qhov taw qhia ntawm Specc11 qhia.Ob leeg alleles pom cov qauv lacZ zoo sib xws, nrog DTM096 gene ntxiab hauv intron 1 qhia kev qhia muaj zog dua li RRH048 hauv intron 15 (tsis qhia).Txawm li cas los xij, Specc1l tau nthuav dav dav, nrog kev qhia tshwj xeeb hauv cov pob txha neural ntawm E8.5 (Daim duab 4B), hauv cov hlab ntsha neural thiab cov txheej txheem ntawm lub ntsej muag ntawm E9.5 thiab E10.5 (Daim duab 4C, D), thiab hauv kev tsim cov ceg tawv. hauv E10.5 thiab ob lub qhov muag (Daim duab 4D).Peb yav dhau los tau tshaj tawm tias SPECC1L kev qhia nyob rau hauv thawj pharyngeal koov ntawm E10.5 yog tam sim no nyob rau hauv lub epithelium thiab hauv qab mesenchyme18, raws li CNCC kab.Txhawm rau kuaj SPECC1L qhia hauv CNCC, peb tau ua E8.5 neural folds (Daim duab 4E-J) thiab E9.5 pob txha taub hau seem (Daim duab 4K-).Ntawm E8.5, SPECC1L stained neural folds intensely (Fig. 4E, H), nrog rau cov hlwb stained nrog NCC cov cim (Fig. 4G, J).Ntawm E9.5, SPECC1L (Fig. 4K, N) muaj zog stained migrating CNCC co-stained nrog AP2A (Fig. 4L, M) los yog SOX10 (Fig. 4O, P).
(A) Schematic sawv cev ntawm nas Specc11 noob uas qhia decoy vector insertion hauv ES DTM096 (intron 1) thiab RRH048 (intron 15) cell clones.(BD) lacZ staining ntawm heterozygous Specc1lDTM096 embryos sawv cev Specc1l qhia los ntawm E8.5 txog E10.5.NE = neuroectoderm, NF = neural fold, PA1 = first pharyngeal arch.(EP) SPECC1L immunostaining nrog NCC cov cim AP2A thiab SOX10 hauv E8.5 (NF; EJ) neural folds thiab E9.5 (KP) pob txha taub hau.SPECC1L staining tau dav pom nyob rau hauv neural folds E8.5 (E, H; arrowheads), nrog rau cov hlwb sau nrog AP2A (F, G; arrowheads) thiab SOX10 (I, J; arrowheads).Ntawm E9.5, SPECC1L muaj zog stained migrating CNCCs (K, N; xub) sau npe AP2A (L, M; xub) thiab SOX10 (O, P; xub).
Hla ntawm heterozygous Specc1lDTM096/+ thiab Specc1lRRH048/+ nas qhia tau hais tias ob lub gene ntxiab alleles tsis complementary thiab cov compound heterozygotes thiab embryonic homozygotes rau ob hom gene trap allele yog embryonic tuag (Table S1).Mendelian piv tau qhia txog qhov txo qis ntawm kev muaj sia nyob ntawm heterozygotes thaum yug (xav tias 1.34 vs. 2.0).Peb tau sau tseg tias tsis muaj kev tuag perinatal ntawm cov heterozygotes, qee qhov muaj craniofacial anomalies (Fig. S3).Txawm li cas los xij, qhov qis qis ntawm cov perinatal craniofacial phenotypes ua rau nws nyuaj rau kev kawm lawv cov txheej txheem pathophysiological.Yog li ntawd, peb tsom mus rau lub embryonic lethal phenotype ntawm homozygous Specc11 mutants.
Feem ntau cov tshuaj heterozygous lossis homozygous Specc1lDTM096/RRH048 mutant embryos tsis tsim tom qab E9.5–10.5 (Figs. 5A–D), thiab cov hlab ntsha neural tsis kaw anteriorly (Figs. 5B, D) thiab qee zaum kaw tom qab (tsis qhia) ..Qhov no cranial neural raj kaw qhov tsis xws luag yog txuam nrog feem ntau ntawm CNCC cim DLX2 nyob rau hauv lub neural folds ntawm E10.5, qhia tias tsis muaj dissection (Daim duab 5A'-D').Txhawm rau txiav txim siab seb qhov loj tag nrho ntawm CNCC kuj raug txo, peb tau sau CNCC kab nrog GFP hauv peb cov kab kab nrog Wnt1-Cre thiab ROSAmTmG.Peb kwj txheeb GFP + NCC thiab GFP- (RFP +) tsis yog NCC los ntawm tag nrho embryos.Ntawm E9.5, qhov kev faib ua feem ntawm cov dej-sorted GFP-labeled CNCCs tsis hloov pauv ntawm WT thiab mutant embryos (tsis pom), qhia txog CNCC cov lus qhia.Yog li ntawd, peb xav tias qhov seem ntawm Wnt1-Cre thiab DLX2 staining nyob rau hauv cov ntaub so ntswg neural folds (Daim duab 5B') yog vim CNCC txheej txheej tsis zoo, tejzaum nws yog vim muaj zog ntau los yog dispersion ntawm AJ hlwb, raws li pom nyob rau hauv SPECC1L-kd hlwb.Peb tau siv NCC cov cim SOX10, AP2A, thiab DLX2 kom paub meej tias muaj CNCC nyob rau hauv lub qhov quav neural (Daim duab 5E-R).Ntawm E8.5, neural fold staining rau tag nrho peb NCC cov cim tau pom nyob rau hauv ntu ntawm WT (Fig. 5E, G, I) thiab Specc1l mutant (Fig. 5F, H, J).Ntawm E9.5, thaum NCC cov cim stained migrating NCC nyob rau hauv WT seem (Fig. 5M, O, Q), residual NCC staining tau pom nyob rau hauv exposed neural folds ntawm Specc1l mutant embryos (Fig. 5N, P, R).Vim hais tias SOX10 thiab DLX2 cim migrating CNCCs, qhov tshwm sim no qhia tias SPECC1L-tsis muaj CNCCs ua tiav cov ntsiab lus tom qab tsiv teb tsaws tab sis ua tsis tiav los ntawm neural folds.
Specc11 deficiency ua rau muaj qhov tsis zoo ntawm cov hlab ntsha kaw, delamination ntawm cranial neural crest hlwb thiab AJs.
(A, B') E9.5 WT (A) Embryo nqa migrating cranial neural crest cells (CNCC) sau nrog Wnt1-Cre (A').Nyob rau hauv sib piv, Specc11 mutant embryos qhia qhib neural folds (B), arrowheads) thiab CNCCs uas tsis tau tsiv teb tsaws (B', arrowheads).(C, D') Bright field images (C, D') thiab immunostaining (C', D') ntawm CNCC marker DLX2 ntawm E10.5 WT embryos (C, C') thiab Specc1l (D, D').Hauv WT E10.5 embryos, DLX2-zoo CNCC colonize lub gill arches (C', xub), thaum nyob rau hauv mutants, conspicuous staining persists nyob rau hauv lub qhib neural folds (D', xub) thiab nyob rau hauv thawj pharyngeal arches (D', xub).) nrog qee qhov staining ( xub) qhia tias tsis zoo delamination thiab tsiv teb tsaws ntawm CNCC.ER) Ntu ntawm WT thiab Specc1l mutant embryos ntawm theem E8.5 (E–L) thiab E9.5 (M–R) tau sau nrog NCC cov cim SOX10 (E, F, M, N), AP2A (G, H, O, P) thiab DLX2 (I, J, Q, R).Ntawm E8.5, NCC staining tau pom nyob rau hauv qus-hom neural fold (NF) thiab mutant seem.Co-staining ntawm SOX10 thiab β-catenin hauv E8.5 WT (K) thiab mutant (L) tau nthuav tawm ntau dua β-catenin staining ntawm cell ciam teb nyob rau hauv lub neural folds.Ntawm E9.5, cov tsiaj qus staining ntawm migrating CNCCs (M, O, Q) tau pom, thaum nyob rau hauv mutants, unstratified CNCCs stained qhib neural folds (N, P, R).(S–Z) Nyob rau hauv vivo AJ sau npe tsom xam hauv coronal seem ntawm WT thiab Specc11DTM096/RRH048 embryos nrog E9.5 kev hloov pauv.Ib qho kwv yees seem dav hlau tau pom nyob rau sab xis sab xis.Hauv seem ntawm cov ntaub so ntswg mutant, nce staining ntawm F-actin (S, T) thiab myosin IIb (U, V) tau pom.Zoo ib yam li cov txiaj ntsig hauv vitro hauv daim duab 3, hauv mutant embryos, txhim kho daim nyias nyias staining rau β-catenin (W, X) thiab E-cadherin (Y, Z) tau pom.(AA-BB) Ib qho hluav taws xob micrograph ntawm ib ntu ntawm cov tsiaj qus-hom embryo saib dhau ntawm ciam teb ntawm apical-basal cell qhia tau hais tias ib cheeb tsam electron-dense uas qhia txog cov nplaum junctions (AA, xub).Nyob rau hauv sib piv, nyob rau hauv seem ntawm Specc11 mutant embryos (BB, xub), tag nrho cov apicobasal hlws ris yog electron ntom, qhia ib tug nce ceev thiab dispersion ntawm nplaum junctions.
Txhawm rau kuaj peb qhov kev xav tias kev txo qis yog vim hloov pauv AJ, peb tau tshuaj xyuas AJ daim ntawv lo rau hauv cov kab mob neural folds ntawm Specc1l mutant embryos (Fig. 5S-Z).Peb tau pom qhov nce hauv actin kev ntxhov siab fibers (Daim duab 5S, T) thiab ib qho kev nce hauv zos ntawm myosin IIB staining ntawm actin fibers (Fig. 5U, V).Ib qho tseem ceeb, peb tau pom qhov ua kom muaj xim ntau ntxiv ntawm β-catenin (Fig. 5W, X) thiab E-cadherin (Fig. 5Y, Z) ntawm thaj tsam intercellular.Peb kuj tau tshuaj xyuas β-catenin staining ntawm NCC nyob rau hauv neural folds ntawm E8.5 embryos (Fig. 5K, L).β-catenin staining tshwm sim muaj zog hauv Specc1l mutant neural folds (Fig. 5L thiab K), qhia tias AJ hloov pauv tau pib.Nyob rau hauv electron micrographs ntawm pob txha taub hau ntawm E9.5 embryos, peb rov pom muaj zog diffuse electron-dense staining nyob rau hauv Specc1l mutant embryos piv rau WT (Fig. 5AA, BB thiab S1E-H).Ua ke, cov txiaj ntsig no txhawb nqa peb cov txiaj ntsig hauv vitro hauv SPECC1L-kd U2OS hlwb thiab qhia tias aberrant AJ staining precedes CNCC stratification hauv peb mutant embryos.
Muab qhov paub txog kev sib raug zoo ntawm AKT kev ua haujlwm thiab E-cadherin stability, 17,28 peb xav txog kev koom tes ntawm PI3K-AKT signaling.Tsis tas li ntawd, peb pom cov kab mob subepidermal blistering nyob rau hauv ib co ntawm peb mutant embryos uas dim lethality (<5%) ntawm E9.5-10.5 thiab es tsis txhob nyob ib ncig ntawm E13.5 (Fig. S3).Subepidermal vesicles yog lub cim ntawm kev txo qis PI3K-AKT signaling raws li PDGFRα12.Fantauzzo et al.(2014) tau tshaj tawm tias kev cuam tshuam ntawm PDGFRα-raws li PI3K ua kom muaj zog hauv PdgfraPI3K / PI3K mutant embryos ua rau subepidermal vesicles, neural tube defects, thiab cleft palate phenotypes.Tseeb, theem ntawm pan-AKT thiab nquag phosphorylated Ser473-AKT raug txo nyob rau hauv vivo hauv Specc1l mutant cov ntaub so ntswg rau E9.5 embryonic ntes (Fig. 6A-D).Kev txo qis hauv qib ntawm phosphorylated Ser473-AKT tuaj yeem yog vim qhov txo qis hauv qib ntawm pan-AKT hauv vivo (Fig. 6E) thiab hauv vitro (Daim duab 6F).Kev txo qis hauv vitro tau pom tsuas yog thaum U2OS hlwb tau sib haum xeeb nrog kev hloov pauv ntawm tes thiab AJ ntom ntom (Daim duab 6D).Yog li, peb cov ntaub ntawv qhia tias SPECC1L yog ib qho tshiab zoo regulator ntawm PI3K-AKT signaling nyob rau hauv craniofacial morphogenesis.
(A–E) E8.5 (A, B) thiab E9.5 (C, D) pob txha taub hau los yog E9.5 lysates los ntawm Specc1l mutant embryos (E) qhia qib ntawm active phosphorylated S473-AKT thiab pan-AKT Protein txo. , piv rau tswj WT.Western blotting tau ua rau cov tsiaj qus lysates thiab mutant lysates nyob rau hauv tib yam mob.Cov duab qhia rau SPECC1L tau coj los ntawm ib qho blot.Tib lub blot raug tshem tawm thiab rov tshuaj xyuas nrog cov tshuaj tiv thaiv-yias-ACT thiab β-actin antibodies.Pan-AKT qib hauv E8.5 neural folds (A, B) thiab qib phosphorylated S473-AKT hauv E9.5 pob txha taub hau tau txo qis.(F) Pan-AKT qib tau zoo ib yam txo qis hauv lysates ntawm SPECC1L-kd U2OS hlwb tau sau los ntawm kev sib txuam siab.Cov kab yuam kev sawv cev rau SEMs los ntawm peb qhov kev ywj pheej ntawm Western blot quantifications.(GJ) Ntu ntawm WT embryos ntawm E9.5 stained nrog KI67 thiab cleaved caspase 3, raws li, qhia cell proliferation (G, G') thiab me ntsis apoptotic kev ua si (H, H').Specc11 mutant embryos qhia qhov sib piv ntawm cov cell proliferation (I), tab sis tus naj npawb ntawm cov hlwb uas raug apoptosis tau nce ntau (J).
Tom qab ntawd peb soj ntsuam cov cim ntawm kev loj hlob thiab apoptosis.Peb tsis tau pom qhov sib txawv ntawm qhov kev loj hlob ntawm E9.5 embryos (Daim duab 6E, G piv rau kuv) nrog qhov kev loj hlob ntawm 82.5% rau WT mutants thiab 86.5% rau Specc1l mutants ntsuas los ntawm KI67 staining (p <0.56, Fisher's xeem xeem).Ib yam li ntawd, peb tsis tau pom qhov txawv ntawm apoptosis ntsuas los ntawm staining rau cleaved caspase 3 nyob rau hauv neural folds ntawm E8.5 kom txog rau thaum embryo ntes (tsis pom) (tsis pom).Hauv qhov sib piv, apoptosis tau nce ntxiv hauv tag nrho E9.5 mutant embryos (Fig. 6F, H thiab J).Qhov kev nce ntxiv hauv apoptosis no zoo ib yam nrog txo PI3K-AKT signaling thiab ntxov embryonic lethality29,30,31.
Tom ntej no, kom paub meej tias lub luag haujlwm tseem ceeb rau PI3K-AKT signaling hauv AJ hloov pauv hauv peb cov hlwb kd, peb tau hloov kho txoj hauv kev tswj thiab kd hlwb (Daim duab 7A-F).Peb siv los ua tus cim lub xov tooj ntawm tes hloov phenotype pom nyob rau hauv confluent SPECC1L-kd hlwb, uas peb quantified siv qhov piv ntawm qhov ntev tshaj plaws dimension (ntev) mus rau qhov sib thooj ntsug dimension (dav).Ib qho piv txwv ntawm 1 yog xav tau rau cov kab mob sib npaug lossis cuboidal (Daim duab 7G).Ntxiv rau cov duab ntawm tes, peb kuj tau lees paub qhov cuam tshuam ntawm AJ los ntawm β-catenin staining (Fig. 7A'-F').Inhibition ntawm PI3K-AKT txoj kev siv wortmannin yog txaus los hloov cov duab ntawm tes hauv cov hlwb tswj (Daim duab 7A, C) thiab AJ (Daim duab 7A').PI3K-AKT activator SC-79 tsis cuam tshuam rau cov duab ntawm tes (FIG. 7A, E) lossis AJ nthuav dav (Daim duab 7A') hauv cov hlwb tswj.Nyob rau hauv SPECC1L-kd hlwb, ntxiv kev tawm tsam ntawm PI3K-AKT txoj hauv kev ua rau muaj kev nce ntxiv ntawm apoptosis (Fig. 7B,D) thiab muaj kev nce ntxiv hauv β-catenin staining (Fig. 7B'), zoo ib yam nrog peb hauv vivo hnyav mutants.Qhov tseem ceeb, kev ua kom txoj hauv kev PI3K-AKT tau txhim kho cov duab ntawm tes (Daim duab 7B, F) thiab AJ phenotypes (Daim duab 7B").Kev hloov pauv ntawm cov duab ntawm tes tau suav tias yog qhov sib piv ntawm cell roundness ratio (CCR) thiab muab piv rau qhov tseem ceeb raws li tau piav qhia saum toj no (Daim duab 7G).Tseeb, nyob rau hauv kev tswj hlwb (Fig. 7G, CCR = 1.56), wortmannin kev kho mob yog txaus los hloov lub cell zoo (Daim duab. 7G, CCR = 3.61, p < 2.4 × 10-9) rau qhov zoo ib yam li qhov pom. hauv SPECC1L.-kd cells (Fig. 7G, CCR = 3.46).Wortmannin kev kho mob ntawm SPECC1L-kd hlwb (Fig. 7G, CCR = 3.60, negligible) yog tsis muaj qhov tseem ceeb tshaj li untreated kd hlwb (Fig. 7G, CCR = 3.46, negligible) los yog wortmannin-kho hlwb (Fig. 7G)., CCR = 3.46, negligible) kuj cuam tshuam rau cell elongation (7G, CCR = 3.61, negligible).Qhov tseem ceeb tshaj plaws, SC-79 AKT activator tau kho cov elongated phenotype ntawm SPECC1L-kd hlwb (Fig. 7G, CCR = 1.74, p < 6.2 × 10-12).Cov txiaj ntsig no tau lees paub tias SPECC1L tswj hwm PI3K-AKT cov cim qhia thiab qhia tias qhov kev txo qis hauv SPECC1L cuam tshuam rau cell adhesion, thaum lub zog qis ua rau apoptosis (Fig. 8).
(A–F') Tswj (A, C, E) thiab SPECC1L-kd (B, D, F) hlwb kho nrog PI3K-AKT pathway inhibitor wortmannin (C, D) lossis SC-79 activator (E, F) Kev Kho Mob .Untreated tswj hlwb yog cuboidal (A) nrog ib txwm β-cat cellular staining (A'), thaum kd hlwb yog elongated (B) nrog nce β-cat staining (B').Tom qab kev tawm tsam ntawm PI3K-AKT txoj hauv kev, tswj cov hlwb elongated (C) nrog β-cat nthuav dav (C'), thaum kd hlwb pib mus rau apoptosis (D), zoo ib yam li peb cov embryos hloov pauv thiab qhia tau zoo heev β-cat.staining (D').Tom qab ua haujlwm ntawm PI3K-AKT txoj hauv kev, tswj cov hlwb tseem nyob hauv cuboidal (E) thiab muaj ib txwm β-cat (E') staining, thaum kd hlwb pom tau zoo ntawm cov cell zoo (F) thiab β-cat (F') staining, qhia. (G) Lub degree ntawm kev hloov pauv ntawm tes nyob rau hauv (AF) tau suav hais tias siv lub cell roundness ratio (CCR) ntawm qhov ntev tshaj plaws (ntev) thiab qhov sib thooj ntsug qhov ntev (dav) siv MetaMorph software.Untreated (NT) SPECC1L-kd hlwb (CCR = 3.46) tau ntev dua li cov hlwb tswj (CCR = 1.56, p < 6.1 × 10–13).Wort's inhibition ntawm PI3K-AKT txoj hauv kev tswj cov hlwb tau txaus los ua kom zoo sib xws hauv cov duab ntawm tes (CCR = 3.61, p <2.4 × 10-9).Ib yam li ntawd, kev ua kom AKT los ntawm SC-79 hauv SPECC1L-kd hlwb rov qab los ntawm cell elongation los tswj qib (CCR = 1.74, p < 6.2 × 10–12).Wortmannin kev kho mob ntawm SPECC1L-kd hlwb ua rau muaj kev nce apoptosis tab sis tsis muaj kev nce ntxiv ntawm cov cell hloov pauv (CCR = 3.60) piv rau cov tsis kho kd (CCR = 3.46, ns) lossis wortmannin-kho hlwb (3.61) pom hauv .ns = tsis muaj teeb meem.+/- SEM ntsuas rau 50 lub hlwb tau pom.Qhov sib txawv ntawm kev txheeb cais tau suav nrog Cov Tub Ntxhais Kawm T-test.
(A) Schematic sawv cev ntawm inhibition thiab activation ntawm PI3K-AKT txoj kev uas ua rau AJ hloov thiab cawm, raws li.(B) Npaj qauv rau AKT protein stabilization los ntawm SPECC1L.
Premigratory CNCCs xav kom AJ lysis cais los ntawm anterior neural fold neuroepithelial cells1,15,32.Ua kom muaj xim ntawm AJ cov khoom thiab poob ntawm apical-basal AJ asymmetric tis nyob rau hauv SPECC1L-tsis muaj hlwb ob leeg hauv vitro thiab hauv vivo, ua ke nrog lub cev sib thooj ntawm SPECC1L rau β-catenin, qhia tias SPECC1L ua haujlwm kom tswj tau AJ kev ruaj ntseg hauv zos rau koom haum cov leeg.actin cytoskeleton ua.Lub koom haum ntawm SPECC1L nrog rau actin cytoskeleton thiab β-catenin thiab qhov nce ntawm cov condensed actin filaments nyob rau hauv qhov tsis muaj SPECC1L yog zoo ib yam nrog rau kev soj ntsuam nce nyob rau hauv AJ ntom.Lwm qhov ua tau yog tias muaj ntau cov actin fibers hauv SPECC1L-tsis muaj hlwb ua rau muaj kev hloov pauv hauv kev sib zog ntawm cov cell.Vim tias kev ntxhov siab ntawm tes cuam tshuam rau AJ 33 kev hloov pauv, qhov hloov pauv hluav taws xob tuaj yeem ua rau ntau qhov sib txawv ntawm AJ 34.Yog li txhua yam kev hloov pauv yuav cuam tshuam rau CNCC txheej.
Wnt1 yog qhia nyob rau hauv thaum ntxov neural folds uas ua rau nce mus rau neural crest hlwb.Yog li, Wnt1-cre kab tracing cim ob qho tib si ua ntej thiab tsiv NCC35.Txawm li cas los xij, Wnt1 kuj tseem cim dorsal hlwb cov ntaub so ntswg clones kuj muab tau los ntawm thaum ntxov neural folds 35,36, ua rau nws zoo li peb staining ntawm E9.5 mutants rau Wnt1 cov cim nyob rau hauv qhib neural folds tsis CNCC.Peb qhov zoo staining rau NCC cov cim AP2A thiab SOX10 tau lees paub tias qhov cuam tshuam neural folds ntawm Specc11 mutant embryos tau muaj tseeb muaj CNCC.Tsis tas li ntawd, txij li AP2A thiab SOX10 yog cov cim ntawm kev tsiv teb tsaws NCC thaum ntxov, qhov zoo staining qhia tias cov hlwb no yog CNCC tom qab tsiv teb tsaws chaw uas tsis tuaj yeem stratified los ntawm E9.5.
Peb cov ntaub ntawv qhia tias kev tswj hwm molecular ntawm AJ los ntawm SPECC1L yog kho los ntawm PI3K-AKT signaling.AKT signaling yog txo nyob rau hauv SPECC1L tsis muaj hlwb thiab cov ntaub so ntswg.Nrhiav los ntawm Fantauzzo et al.txhawb nqa lub luag haujlwm ncaj qha rau PI3K-AKT signaling hauv craniofacial morphogenesis.(2014) tau pom tias qhov tsis muaj kev ua kom muaj zog ntawm PDGFRα-raws li PI3K-AKT signaling ua rau cleft palate phenotype.Peb kuj qhia tau hais tias inhibition ntawm PI3K-AKT txoj kev yog txaus los hloov AJ thiab cell zoo nyob rau hauv U2OS hlwb.Raws li peb qhov kev tshawb pom, Cain li al.37 tau qhia tias kev txo qis ntawm PI3K α110 subunit hauv cov hlwb endothelial ua rau muaj kev nce ntxiv hauv pericellular β-catenin staining, hu ua qhov nce hauv "kev sib txuas index".Txawm li cas los xij, nyob rau hauv cov hlwb endothelial uas nws cov actin filaments twb tau koom nrog, kev tawm tsam ntawm PI3K-AKT txoj hauv kev ua rau lub cev xoob.Nyob rau hauv sib piv, SPECC1L-kd U2OS hlwb pom ib tug elongated cell duab.Qhov sib txawv no yuav yog hom cell tshwj xeeb.Thaum kev tawm tsam ntawm PI3K-AKT teeb liab cuam tshuam rau cov actin cytoskeleton mus tas li, cov nyhuv ntawm cov duab ntawm tes yog txiav txim siab los ntawm kev hloov ntawm qhov nro los ntawm kev hloov ntawm qhov ntom thiab lub koom haum ntawm central actin fibers.Hauv U2OS hlwb, peb tsuas yog siv cov xovtooj ntawm tes hloov pauv raws li tus cim ntawm SPECC1L-tsis muaj AJ hloov pauv thiab rov qab los.Hauv kev xaus, peb xav tias inhibition ntawm AKT txoj hauv kev hauv SPECC1L deficiency ua rau AJ ruaj khov thiab txo qis delamination hauv CNCC.
Interestingly, pan-AKT qib tau txo qis hauv vitro thiab hauv vivo ntxiv rau phosphorylated 473-AKT qib thaum tsis muaj SPECC1L, qhia txog kev tswj hwm ntawm PI3K-AKT signaling ntawm qib AKT protein stability lossis kev hloov pauv.Cov noob SPECC1L thiab MID1, ob qho tib si cuam tshuam nrog Opitz / GBBB syndrome, encode cov proteins uas ruaj khov microtubules 18,22.Lub tshuab uas SPECC1L thiab MID1 kho microtubule stabilization tsis nkag siab tag nrho.Nyob rau hauv rooj plaub ntawm SPECC1L, qhov stabilization no suav nrog kev txhim kho acetylation ntawm ib pawg ntawm microtubules 18 .Nws yog qhov ua tau tias SPECC1L siv cov txheej txheem zoo sib xws kom ruaj khov rau lwm cov proteins xws li AKT.Nws tau raug pom tias acetylation ntawm lysine residues hauv AKT protein ua rau txo qis hauv daim nyias nyias hauv zos thiab phosphorylation38.Tsis tas li ntawd, ubiquitination ntawm K63 saw ntawm tib lysine residue ntawm AKT yog xav tau rau nws cov membrane localization thiab activation39,40.Ntawm ntau yam cuam tshuam nrog SPECC1L cov proteins uas tau txheeb xyuas nyob rau hauv ntau qhov sib txawv ntawm cov poov xab ob-hybrid cov ntxaij vab tshaus, plaub - CCDC841, ECM2942, APC thiab UBE2I43 - tau cuam tshuam rau cov protein hloov pauv lossis ruaj khov ntawm ubiquitination lossis sumoylation.SPECC1L tuaj yeem koom nrog hauv kev hloov pauv tom qab hloov pauv ntawm AKT lysine residues, cuam tshuam rau AKT stability.Txawm li cas los xij, lub luag haujlwm tseem ceeb ntawm SPECC1L hauv thaj chaw thiab kev ruaj ntseg ntawm AKT protein tseem yuav tsum tau piav qhia.
Kev mob hnyav hauv SPECC1L kev qhia hauv vivo ua rau nce AJ cim staining thiab tsis zoo CNCC overlay, nrog rau nce apoptosis thiab ntxov embryonic lethality.Cov ntaub ntawv dhau los tau qhia tias nas mutants nrog nce qib ntawm apoptosis yog txuam nrog neural tube defects 44,45,46,47 thiab craniofacial defects48.Nws tau raug qhia tias kev tuag ntawm tes ntau dhau ntawm cov pob txha hauv neural lossis pharyngeal arches tuaj yeem ua rau muaj cov hlwb tsis txaus uas yuav tsum tau ua rau kev txav morphogenetic 48,49,50.Hauv qhov sib piv, peb cov SPECC1L cov kab tsis muaj cell nrog kev txo qis SPECC1L qhia pom tsuas yog AJ hloov pauv yam tsis muaj pov thawj ntawm kev tuag ntawm tes.Txawm li cas los xij, chemical inhibition ntawm PI3K-AKT txoj hauv kev hauv cov Kd hlwb no ua rau muaj kev nce apoptosis.Yog li, qhov txo qis hauv SPECC1L kev qhia lossis kev ua haujlwm kom muaj sia nyob ntawm tes.Qhov no yog ua raws li qhov kev soj ntsuam uas tsis tshua muaj Specc11 mutant embryos uas khiav dim ntawm st.E9.5-tej zaum vim yog txo qis kev ntes cov noob tau zoo-muaj peev xwm kaw lawv cov hlab ntsha neural thiab nres tom qab hauv txoj kev loj hlob, feem ntau muaj cov kab mob craniofacial (Fig. S3).Tsis tas li ntawd zoo ib yam nrog qhov no yog qhov tsis tshua muaj tshwm sim ntawm heterozygous Specc1l embryos nrog craniofacial abnormalities-tej zaum yog vim muaj ntau cov noob capture efficiency-nrog rau kev tshawb pom nyob rau hauv zebrafish nyob rau hauv uas ib tug ntawm ob lub SPECC1L orthologues (specc1lb) ua rau lig embryonic phenotypes, nrog rau kev poob. sab puab tsaig thiab ob sab clefts51.Yog li, heterozygous SPECC1L poob-ntawm-function mutations pom nyob rau hauv tib neeg cov neeg mob yuav ua rau me me impairments nyob rau hauv SPECC1L muaj nuj nqi thaum lub sij hawm craniofacial morphogenesis, txaus los piav lawv orofacial clefts.SPECC1L-raws li kev cai ntawm intercellular hu kuj tseem tuaj yeem ua lub luag haujlwm hauv palatogenesis thiab fusion ntawm pharyngeal arches.Cov kev tshawb fawb ntxiv ntawm SPECC1L muaj nuj nqi yuav pab qhia txog lub luag haujlwm ntawm kev sib cuag ntawm lub cev ib ntus hauv CNCC thaum lub sij hawm kaw lub raj hauv neuroepithelial cell motility thiab craniofacial morphogenesis.
U2OS osteosarcoma tswj thiab SPECC1L-kd hlwb tau piav qhia yav dhau los (Saadi li al., 2011).Antibodies tiv thaiv SPECC1L kuj tau ua tus yam ntxwv yav dhau los (Saadi li al., 2011).Anti-β-catenin antibodies (Lavbit; 1:1000; Santa Cruz, Dallas, TX) (nas; 1:1000; Cell Signaling Technology, Danvers, MA), myosin IIb (1:1000; Sigma-Aldrich, St. ), 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), cleaved caspase 3 (1:1000; Cell Signaling Technology, Danvers, MA) thiab β-actin (1:2500; Sigma-Aldrich, St. MO) tau siv raws li tau piav qhia..Actin filaments tau stained nrog Acti-stain rhodamine phalloidin (Cytoskeleton, Denver, Colorado).
U2OS tswj cov hlwb thiab SPECC1L-kd hlwb tau coj mus rau hauv cov qauv siab qabzib DMEM ntxiv nrog 10% fetal bovine serum (Life Technologies, Carlsbad, CA).Rau AJ kev hloov pauv, 2 x 105 hlwb tau cog rau ntawm iav kho nrog 0.1% porcine gelatin (Sigma-Aldrich, St. Louis, MO) thiab tau pom zoo rau kev hloov pauv ntawm cov duab ntawm tes.Cells tau sau los ntawm cov ntsiab lus sib txawv ntawm lub sijhawm: 4 teev tom qab cog (t = 1), 24 teev tom qab cog (t = 2), confluence yam tsis hloov pauv ntawm cov duab cell (t = 3), hloov hauv cell duab (t = 4) , 24 h tom qab cell zoo hloov (t = 5) thiab 48 h tom qab cell hloov (t = 6) (Fig. 1, 2, 3).Txhawm rau hloov kho txoj hauv kev PI3K-AKT, cov hlwb raug coj los ntawm cov ntsiab lus qhia nrog PI3K-AKT inhibitor wortmannin (TOCRIS Biosciences, Minneapolis, Minnesota) lossis SC-79 activator (TOCRIS Biosciences, Minneapolis Adams, Minnesota).Qhov nruab nrab uas muaj cov tshuaj tau hloov txhua hnub.
Cov ntaub ntawv kaw lus-los ntawm-ncej tau tsim los ntawm kev tswj hwm nyob thiab KD hlwb nyob rau hauv cov kab lis kev cai ib txwm muaj, thiab cov duab sib piv tau sau txhua 10 feeb rau 7 hnub.Cov duab tau txais los ntawm kev siv lub khoos phis tawj tswj hwm Leica DM IRB inverted microscope nruab nrog lub tshuab theem thiab lub hom phiaj 10 × N-PLAN txuas nrog QImaging Retiga-SRV lub koob yees duab.Thaum kuaj pom, cov kab lis kev cai ntawm tes tau khaws cia ntawm 37 ° C hauv qhov chaw ntub nrog 5% CO2.
Ob lub noob ntxiab ES kab ntawm tes DTM096 thiab RRH048 los ntawm Lub Chaw Pabcuam Hauv Cheeb Tsam Mutant Mouse (UC Davis, CA) tau siv los tsim cov kab Specc11 tsis txaus nas, xaiv Specc1lgtDTM096 thiab Specc1lgtRRH046.Luv luv, 129/REJ ES hlwb raug txhaj rau hauv C57BL6 blastocysts.Qhov tshwm sim chimeric txiv neej nas tau bred nrog poj niam C57BL6 nas los txheeb xyuas cov xeeb ntxwv nrog agouti lub tsho tiv no xim.Lub xub ntiag ntawm gene trap vector inserts tau siv los txheeb xyuas cov heterozygotes.Cov nas tau khaws cia rau ntawm keeb kwm sib xyaw ntawm 129 / REJ; C57BL6.Qhov chaw ntawm qhov chaw ntxig ntawm cov cuab yeej cuab tam vector tau lees paub los ntawm RT-PCR, genome sequencing, thiab genetic complementation (Cov duab ntxiv 1).Txhawm rau taug qab CNCC kab ntawm ob lub heterozygous Specc1lGT nas, ROSAmTmG (#007576) thiab Wnt1-Cre (#003829) nas (Jackson Laboratory, Bar Harbor, ME) tau hla los tsim ROSAmTmG thiab Wnt1-Cre allele mullele hauv Specc.Txhua qhov kev sim hauv nas tau ua raws li cov txheej txheem pom zoo los ntawm Pawg Saib Xyuas Tsiaj thiab Siv Khoom ntawm University of Kansas Medical Center.
Embryos tau tsau rau hauv (1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2, 0.02% NP-40, 5 mM EGTA) rau 60 min ntawm chav tsev kub.Tom qab kho hauv X-gal staining tov (5 mM potassium ferricyanide, 5 mM poov tshuaj ferrocyanide, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, 1 mg / ml X-gal) Stain kev loj hlob ntawm 37 ° C. .° C hauv 1-6 teev.Embryos tau tom qab kho hauv 4% PFA thiab pom pom.
Rau Western blotting, hlwb tau lysed nyob rau hauv passive lysis tsis (Promega, Fitchburg, WI) supplemented nrog ib tug sib tov ntawm HALT protease inhibitors (Sigma-Aldrich, St. Louis, MO).Lysates tau ua tiav ntawm 12% polyacrylamide Mini-PROTEAN TGX npaj ua gels (Bio-Rad, Hercules, CA) thiab xa mus rau Immobilon PVDF daim nyias nyias (EMD Millipore, Billerica, MA).Cov membranes raug thaiv hauv 5% mis nyuj hauv PBS uas muaj 0.1% Tween.Cov tshuaj tiv thaiv tau muab tso rau hmo ntuj ntawm 4 ° C lossis rau ib teev ntawm chav tsev kub.Femto SuperSignal West ECL reagent (Thermo Scientific, Waltham, MA) tau siv rau kev tsim teeb liab.Rau kev tiv thaiv kab mob, embryos tau kho thaum hmo ntuj hauv 4% PFA / PBS thiab cryopreserved.Cov ntaub so ntswg cryosections raug thaiv nyob rau hauv PBS uas muaj 1% ib txwm tshis serum (Thermo Scientific, Waltham, MA) thiab 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) thiab ces incubated ntawm 4 ° C nyob rau hauv lub incubator thaum lub sij hawm lub hmo.nrog cov tshuaj tiv thaiv kab mob thiab cov tshuaj fluorescent Secondary antibody (1:1000) rau 1 teev ntawm 4 ° C.Stained seem tau muab tso rau hauv ProLong kub nruab nrab (Thermo Scientific, Waltham MA) thiab cov duab tiaj tus tau txais los ntawm Leica TCS SPE confocal microscope.Txhua qhov kev tiv thaiv kab mob tau ua raws li peb qhov kev sim ywj pheej ntawm kev sib tw ntawm tsawg kawg ob lub embryos mutant.Ib qho kev sim sawv cev tau qhia.
Cells raug incubated nyob rau hauv hloov kho RIPA buffer (20 mM Tris-HCl, pH 8.0, 1% NP-40, 130 mM NaCl, 10% glycerol, 2 mM EDTA, thiab HALT protease inhibitor (Sigma-Aldrich, St. Louis, MO) Luv luv, lysates tau prepurified nrog protein G magnetic hlaws dai (Lub neej Technologies, Carlsbad, CA) thiab ces incubated thaum hmo ntuj ntawm 4 ° C. nrog anti-SPECC1L los yog IgG protein G protein hlaws tau siv los rho tawm SPECC1L thiab Western blotting tau ua siv cov tshuaj tiv thaiv. -β-catenin antibody tau piav qhia saum toj no Cov kev sim co-IP tau pom yog tus sawv cev ntawm plaub qhov kev sim ywj pheej.
Tsau kab lis kev cai lossis nas embryonic cov ntaub so ntswg tau muab rau lub chaw electron microscopy ntawm University of Kansas Medical Center.Luv luv, cov qauv tau muab tso rau hauv EMbed 812 resin (Electron Microscopy Sciences, Fort Washington, PA), polymerized thaum hmo ntuj ntawm 60 ° C, thiab ntu ntawm 80 nm siv Leica UC7 ultramicrotome nruab nrog lub pob zeb diamond hniav.Cov seem tau pom pom siv JEOL JEM-1400 kis tau tus mob electron microscope nruab nrog 100 kV Lab6 phom.
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