ASTM A790 2507 / 2205 1.4462 / 1.4410 Duplex Welded Tube Yemakemikari Indasitiri chikamu chemakemikari, Kushaikwa kweSPECC1L kunotungamirira kukuwedzera kugadzikana kwemajoini akatsemuka uye kuderedzwa kudeurwa kwecranial neural crest masero.

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ASTM A790 2507 / 2205 1.4462 / 1.4410 Duplex Welded Tube For Chemical Industry

Liaocheng Sihe SS Material Co., Ltd.mugadziri anotungamira ane hunyanzvi mune simbi isina musono pombi, anopenya annealed chubhu, isina musono coiled tubing etc.Kuitira kurerutsa vatengi , tine welded pombi uye machubhu zvakare .Liaocheng Sihe SS Material Co., Ltd.ine michina yepamusoro yekugadzira uye yekuedza.Tinogona kugutsa zvachose zvaunoda.Zvinoenderana neyakajairwa zvakanyanya, machubhu anogadzirwa nesu anogara aine chaiyo OD uye WT kushivirira.Iyo tolerance control inonyatso kuenderana kugadzira zviyero.Zvigadzirwa zvedu zvinogara zvichigutsikana nevatengi.Vatengi vakatenga zvigadzirwa zvedu vakagadzira purofiti yakawanda.
a) OD (Outer Diameter): 3.18mm kusvika 101.6mm
b) WT (Wall Ukobvu): 0.5mm kusvika 20mm
c) Kureba: Zvinoenderana nezvinodiwa nemutengi
d) Maitiro : ASTM A312;ASTM A269;ASTM A789;ASTM A790 nezvimwe
e) Maitiro ekuita: ERW, EFW nezvimwe

Masiraidhi anoratidza zvinyorwa zvitatu pane siraidhi.Shandisa mabhatani ekumashure neanotevera kufamba nemumasiraidhi, kana mabhatani ekudzora masiraidhi ari kumagumo kuti ufambe nemumasiraidhi ega ega.
Iwo cranial neural crest masero (CNCC) anodonhedza kubva paembryonic neural folds uye anotamira kune pharyngeal arches, ayo anoumba mazhinji epakati pechiso zvimiro.CNCC dysfunction inoita basa rakakosha mune etiology yeorofacial cleft, yakajairika congenital malformation.Heterozygous SPECC1L shanduko dzakawanikwa muvarwere vane atypical uye syndromic clefts.Pano, tinoshuma kusvibiswa kwakasimbiswa kwecanonical adhesive junction (AJ) zvikamu, β-catenin uye E-cadherin mumaseru eSPECC1L ekugogodza, uye maelectron micrographs anoratidza apical-basal diffusion yeAJ.Kuti tinzwisise basa re SPECC1L mu craniofacial morphogenesis, takagadzira Specc1l inoshomeka mbeva modhi.Homozygous mutants inouraya embryonic uye inoratidzira kukanganisa neural chubhu kuvharwa uye CNCC lamination.AJ protein staining inowedzera mune mutant neural folds.Ichi chikanganiso cheAJ chinopindirana nehurema muCNCC delamination, inoda kubviswa kweAJ.Mukuwedzera, Specc11 mutants yakaderedza PI3K-AKT kusaina uye kuwedzera apoptosis.In vitro, inhibition yakapfava yePI3K-AKT kusaina mumasero emhando yemusango yaive yakakwana kuti iite shanduko yeAJ.Zvakakosha, shanduko yeAJ yakakonzerwa ne SPECC1L kugogodza inogona kudzoserwa nekuitwa kwePI3K-AKT nzira.Takatorwa pamwechete, idzi data dzinoratidza kuti SPECC1L, semugadziri wenyaya wePI3K-AKT kusaina uye AJ biology, inodiwa kuti neural tube kuvharwa uye CNCC stratification.
Cranial neural crest masero (CNCCs) anogara kune dorsal neuroectoderm uye anobvisa kubva kune neuroepithelium yekuvandudza neural folds kuburikidza nemaitiro anosanganisira epithelial-mesenchymal transition (EMT)1,2,3.Premigrating epithelial CNCCs inokanganisa intercellular junctions uye inotamira mesenchymal CNCCs inozadza yekutanga uye yechipiri pharyngeal arches uye inoumba yakawanda ye craniofacial cartilage.Saka, majini anodzora basa reCNCC anowanzo vhiringwa mune etiology yecraniofacial congenital anomalies senge orofacial clefts, kazhinji inokanganisa 1 / 800 vanozvarwa muUS chete.Imwe yehurema hwekuzvarwa8.
Delamination yeCNCC inopindirana nekuvharwa kweanterior neural tube pakati pe8.5 uye 9.5 mazuva ekukura kwema embryonic mumakonzo.Mutants ehuwandu hwemakonzo orofacial cleft-akabatanidzwa majini anoratidzawo imwe fomu yeneural tube defect, kusanganisira Irf69,10, Ghrl310, Cfl111, uye Pdgfrα12.Nekudaro, maitiro ekuvhara neural chubhu uye CNCC stratification anogona kunzi akazvimirira, sezvo Splotch mutant mbeva (Pax3) inoratidzira hurema mukuvharwa neural chubhu pasina mhedzisiro paCNCC stratification kana kutama 13,14.Kuwedzera mbeva modhi ine hurema muCNCC dissection uye neural chubhu kuvharwa kuchabatsira kutsanangura iyo yakajairika mamorekuru maitiro maviri aya.
Kuparadzaniswa kweCNCC kubva kumasero euroepithelial kunoda kuparara kwezvibatanidza zvinonamatira (AJs), izvo zvinoumbwa neprotein complexes ine, pakati pevamwe, E-cadherin, β-catenin, α-E-catenin, uye α-actinin inobatanidza ne actin filaments 2. .Kunyanya kuwedzeredza zvidzidzo E-cadherin mune neural folds yakaratidza kuderedzwa kana kunonoka muCNCC delamination.Ukuwo, kudzvinyirirwa kweE-cadherin kunoguma nekutanga stratification15,16.Zvizhinji zvezvinhu zvinomiririra EMT panguva yeCNCC stratification zvinhu zvekunyora (AP2α, Id2, FOXD3, SNAIL, TWIST, SOX10) uye extracellular matrix (ECM) inogadzirisa mapuroteni akadai sematrix metalloproteinases (MMPs), zvisinei CNCCs yakananga cytoskeletal AJ regulators. hazvisati zvazivikanwa.Iyo PI3K-AKT nzira inozivikanwa kupikisa E-cadherin mazinga, kunyanya kubva mukutsvaga kenza17.Zvidzidzo zvenguva pfupi zvakaratidza kuti kurasikirwa kwePDGFα-based PI3K-AKT kusaina mumakonzo kunotungamirira kune craniofacial abnormalities, kusanganisira cleft palate uye neural tube defects12.Nekudaro, hukama pakati pePI3K-AKT nzira uye AJ kugadzikana pane CNCC stratification haina kujeka.
Isu takamboona SPECC1L seyekutanga mutant gene muvanhu vaviri vane mukaha wakakomba unobva pamuromo uchienda kuziso, unozivikanwa se oblique cleft (ObFC) kana Tessier IV18 cleft.SPECC1L shanduko dzakaonekwa mumhuri mbiri dzakasiyana-siyana dzine autosomal dominant Opitz G/BBB syndrome (OMIM #145410), umo vanhu vakakanganisika vakaratidza hyperdistance uye cleft lip/palate19, uye mune imwe mhuri ine Tibi overdistance syndrome (OMIM #145420)20 .inopfuura hafu yezviitiko zveOpitz G/BBB syndrome ndeye X-yakabatana (OMIM #300000) uye inokonzerwa nekushanduka kweMID1 gene, iyo inokodha protein 22 ye microtubule-associated cell skeleton.Isu tinofungidzira kuti SPECC1L, uyewo puroteni inobatanidza ne microtubules uye actin cytoskeleton, inogona kupindirana chiratidzo chinodiwa kuti actin cytoskeleton remodeling panguva yekunamatira sero uye kutama 18.Kuburikidza ne in vitro uye mu vivo zvidzidzo, isu ikozvino tinotsanangura SPECC1L seanoveli regulator yeAJ kugadzikana kuburikidza nePI3K-AKT kusaina.Panzvimbo yemasero, kushayikwa kweSPECC1L kwakaguma nekuderera kwehuwandu hwepan-AKT protein uye kuwedzera kweapical-basal dispersion yeAJ, iyo yakabviswa nemakemikari activation yeAKT nzira.Mu vivo, Specc11-inoshaya embryos inoratidza kusagadzikana neural chubhu kuvharwa uye kuderedzwa kweCNCC dissection.Saka, SPECC1L inoshanda mune yakanyanya kurongeka sero adhesion-based siginecha inodiwa kune yakajairika CNCC basa panguva yechiso morphogenesis.
Kuratidzira basa reSPECC1L padanho remaserura, takashandisa yakatsanangurwa yakagadzika osteosarcoma cell line U2OS inoshomeka muSPECC1L18.Aya masero akagadzikana eU2OS ane SPECC1L (kd) knockdown aiva nepakati (60-70%) kuderera mumazinga e SPECC1L zvinyorwa uye mapuroteni, pamwe nekukanganisa kwekufamba uye kurongeka kwe actin cytoskeleton 18. Kusiyana neizvi, kuderera kwakanyanya kwenguva pfupi mu SPECC1L yakaratidzwa kutungamirira kune mitotic defects 23.Pamusoro pekuwedzera maitiro, takaona kuti masero edu akagadzikana SPECC1L-kd akachinja morphology pane yakanyanya dhigirii yekusangana (Mufananidzo 1).Ega ega ekudzora maseru uye kd maseru payakaderera confluence aitaridzika akafanana (Mufananidzo 1A, D).Maawa 24 mushure mekusanganiswa, masero ekudzora akachengetedza chimiro chavo che cuboidal (Fig. 1B, E), nepo SPECC1L-kd masero akareba (Fig. 1C, F).Hukuru hweshanduko iyi muchimiro chesero yakabatwa ne in vivo live imaging yemasero ekudzora uye kd masero (bhaisikopo 1).Kuti tione basa re SPECC1L mumaseru akaungana, takatanga taongorora kutaura kwayo.Takaona kuti SPECC1L mapuroteni mazinga akawedzera pane fusion (Mufananidzo 1G), asi SPECC1L zvinyorwa zvinyorwa hazvina kuwedzera (Mufananidzo 1H).Mukuwedzera, sezvo masero emasero akawedzera, SPECC1L protein yakaunganidzwa pamiganhu ye intercellular (Fig. 2A-E), ine muenzaniso inopindirana neye membrane-yakabatanidzwa β-catenin (Fig. 2A'-E ').Tichifunga nezvekubatana kwe SPECC1L ne actin cytoskeleton 18,23 isu takafungidzira kuti SPECC1L inopindirana ne actin-based adhesive junctions (AJ).
(AF) SPECC1L knockdown (DF) maseru anorebesa pakusangana kwakakwira (F) zvichienzaniswa nekutonga U2OS masero (AC).Anoratidzwa pano matatu emapoinzi matanhatu (T1, T3, T6) atakasarudza kune akasiyana masero density.(G) Kuongorora kweWestern blot kunoratidza kuti SPECC1L protein yakagadzikana pahupamhi hwepamusoro kana ichienzaniswa nehuwandu hwehuwandu hwekugadzirisa masero.Western blot ye SPECC1L inoratidza inotarisirwa 120 kDa bhendi uye yepamusoro molecular uremu bhendi, pamwe post-translationally modified (*).Kuongororwa kweWestern blot kwakaitwa pasi pemamiriro akafanana kune yakaderera uye yakakwirira confluence.Mifananidzo inoratidza SPECC1L pazasi uye yakakwira confluence yakatorwa kubva kune imwechete blot.Iyo blot imwechete yakabviswa uye yakaongororwazve ne β-actin antibody.(H) Quantitative RT-PCR kuongororwa kwakaratidza kusina kuchinja kukuru mu SPECC1L zvinyorwa zvinyorwa.Mabhawa ekukanganisa anomiririra maSEM kubva kuzviyedzo zvina zvakazvimirira.
(AE) Takasarudza zvitanhatu nguva mapoinzi (T1-T6) anomiririra huwandu hwemasero density kuti ive yakajairika kuongororwa kwesero chimiro uye AJ shanduko mumaseru eU2OS ane SPECC1L knockdown (kd).Zvishanu zvekutanga zvenguva idzi zvaisanganisira masero ega (T1), 50-70% fusion yemasumbu madiki emasero (T2), fusion pasina kuumbazve kd masero (T3), reshaping kd masero (T4), uye 24 maawa shanduko.mumashure maitiro e kd (T5) masero.Iyo SPECC1L protein yainyanya kuparadzirwa mucytoplasm paT1 (A), asi kuunganidza kwayo kwakaonekwa pamiganhu yeintercellular panguva dzinotevera (B-E, miseve).(FJ) β-catenin inoratidza kuunganidza kwakafanana pamiganhu ye intercellular inobatanidza neAJ yakaoma.(A'-E') SPECC1L uye β-catenin inoratidza kupindirana kwemavara pamiganhu yesero pakakwirira sero density (miseve).(F'-J') MuSPECC1L-kd masero, β-catenin staining inoratidzika seyakajairika pane yakaderera cell density (F'-H'), asi inowedzera sezvo sero chimiro chinoshanduka (I', J'; miseve), zvichiratidza kuti AJ zvachinja.Mabhawa = 10 µm.
Takazoedza kuona mhedzisiro yekushomeka kwe SPECC1L paAJ.Isu takashandisa akati wandei AJ-akabatanidzwa mamakisi, kusanganisira canonical zvikamu F-actin, myosin IIb, β-catenin, uye E-cadherin24,25,26,27.Actin stress fibers yakawedzera mu SPECC1L-kd masero sezvakatsanangurwa kare (Fig. 3A, B) 18.Myosin IIb yakabatana ne actin filaments yakaratidza kuwedzera kwakafanana kweSPECC1L-kd masero mu vitro (Fig. 3C, D).AJ-yakabatana β-catenin inosunga ku cadherin pa membrane yesero, ichiratidza yakajairika "uchi" kutaura muenzaniso mukutonga cubocytes (Fig. 3E, G).Sezvineiwo, mumifananidzo yakatsetseka uchishandisa confocal microscopy, β-catenin (Fig. 3E, F) uye E-cadherin (Fig. 3G, H) inosvibisa pa cell membrane ye confluent SPECC1L-yakashaya masero yakaratidza maitiro akakurumbira ekusvibiswa kwakawedzerwa.Uku kuwedzera kweAJ-inosanganiswa ne β-catenin kusvibisa mu kd masero kwainyanya kutaurwa pakusangana, asi kwakaratidzika kutangira shanduko muchimiro chesero (Fig. 2F-J, F'-J').Kuti titarise chimiro chechimiro cheiyi AJ staining yakawedzerwa, takaongorora miganhu yesero pane apical-basal pamusoro peSPECC1L-kd U2OS masero nekutapurirana electron microscopy (TEM) (Mufananidzo 3I, J).Kusiyana nekudzora maseru (Fig. 3I), iyo yaive neakaparadzana erekitironi dense matunhu anoratidza AJ (miseve), kd masero (Fig. 3J) airatidza makuru, contiguous nzvimbo dzepamusoro maerekitironi density inoratidza AJ pamwe apicobasal ndege..Mukuwedzera, pazvikamu zvakachinjika, takaona yakakura sero membrane folds mu kd masero (Fig. S1A, B), iyo inotsanangura nzira yakawedzerwa ye β-catenin uye E-cadherin staining bands (Fig. 3F, H).Mukutsigira basa re SPECC1L muAJs, β-catenin yakanga yakabatanidzwa ne SPECC1L mu lysates ye confluent U2OS masero (Fig. 3K).Pamwe chete nekuwedzera immunostaining yeAJ mamaki, kuongororwa kweTEM kwaienderana nefungidziro yedu yekuti kushomeka kweSPECC1L kunowedzera AJ apical-basal density uye musiyano.
(AH) Kuwedzera F-actin kusvibisa mu kd masero pamaawa 48 mushure mekusanganiswa (T6; A, B).Yakashandurwa kusvibiswa kwemyosin IIb yakabatana neF-actin (C, D).Nzira yakatsetseka ye β-catenin uye E-cadherin membrane inosvibisa mumasero ekutonga (E, G) yakasimbiswa mumasero eSPECC1L-kd (F, H).Mabhawa = 10 µm.(I-J) Electron micrographs inotarisa apical-basal intercellular junction.Maseru ekudzora anoratidza akasiyana maerekitironi-dense matunhu anoratidza anonamira makutano (I, miseve).Kusiyana neizvi, iyo yese apical-basal junction muSPECC1L-kd masero akaoneka erekitironi dense (J, miseve), zvichiratidza kuwedzera kuwanda uye kupararira kweanonamira makutano.(K) β-catenin yaive co-immunoprecipitated ne SPECC1L mune confluent U2OS cell lysates.Mufananidzo wakatorwa kubva panzvimbo imwe unomiririra chimwe chezviedzo zvina zvakazvimirira.
Kuti tinzwisise basa re SPECC1L mu craniofacial morphogenesis, takagadzira Specc1l inoshaya mbeva modhi tichishandisa maviri akazvimirira ES trap cell mitsara, DTM096 uye RRH048 (BayGenomics, CA), inomiririra intron 1 uye Specc1l zvinyorwa zvakatorwa pa15 (Fig. 1) .4A, mufananidzo S2).Nzvimbo ye genomic ye decoy vector insert yakagadziriswa neyese genome sequencing uye yakasimbiswa nePCR (Fig. S2).Magadzirirwo ese ari maviri ejini musungo akabvumirawo mu-frame fusion yeSpecc11-lacZ vatori venhau pavakabatwa.Naizvozvo, kutaura kwelacZ kwakatemwa neX-gal staining kwakashandiswa sechiratidzo cheSpecc11 kutaura.Ose ma alleles airatidza akafanana lacZ kutaura mapatani, neiyo DTM096 gene trap in intron 1 inoratidza kutaura kwakasimba kupfuura RRH048 intron 15 (isina kuratidzwa).Zvisinei, Specc1l inoratidzwa zvakanyanya, iine kutaura kwakasimba zvakanyanya muNeural folds paE8.5 (Mufananidzo 4B), muNeural tube uye maitiro echiso paE9.5 uye E10.5 (Mufananidzo 4C, D), uye mukukura kwemakumbo. pa e10.5 uye maziso (Mufananidzo 4D).Isu takambotaura kuti SPECC1L kutaura mune yekutanga pharyngeal arch paE10.5 yaivepo muepithelium uye yakadzika mesenchyme18, inopindirana neCNCC mutsara.Kuti uedze kutaura kwe SPECC1L muCNCC, takaita E8.5 neural folds (Mufananidzo 4E-J) uye E9.5 skull zvikamu (Mufananidzo 4K-).PaE8.5, SPECC1L yakasvibiswa neural folds zvakanyanya (Fig. 4E, H), kusanganisira masero akasvibiswa neNCC markers (Fig. 4G, J).PaE9.5, SPECC1L (Fig. 4K, N) yakasvibiswa zvakasimba ichitama CNCC yakasvibiswa neAP2A (Fig. 4L, M) kana SOX10 (Fig. 4O, P).
(A) Schematic inomiririra yembeva Specc11 gene inoratidza decoy vector kuiswa muES DTM096 (intron 1) uye RRH048 (intron 15) cell clones.(BD) lacZ tsvina yeheterozygous Specc1lDTM096 embryos inomiririra Specc1l kutaura kubva E8.5 kusvika E10.5.NE = neuroectoderm, NF = neural fold, PA1 = yekutanga pharyngeal arch.(EP) SPECC1L immunostaining neNCC markers AP2A uye SOX10 muE8.5 (NF; EJ) neural folds uye E9.5 (KP) zvikamu zvedehenya.SPECC1L staining yaionekwa zvakanyanya muNeural folds E8.5 (E, H; arrowheads), kusanganisira masero akanyorwa neAP2A (F, G; arrowheads) uye SOX10 (I, J; miseve).PaE9.5, SPECC1L yakasvibiswa zvakasimba inotama CNCCs (K, N; miseve) yakanyorwa AP2A (L, M; miseve) uye SOX10 (O, P; miseve).
Kuyambuka nepakati peheterozygous Specc1lDTM096/+ uye Specc1lRRH048/+ mbeva zvinoratidza kuti magene maviri etepi alleles haawirirane uye kuti mukomboni heterozygotes uye embryonic homozygotes kune chero gene trap allele inouraya embryonic (Table S1).Mendelian ratios yakaratidza kuderera kwehuwandu hwehupenyu hweheterozygotes pakuberekwa (inotarisirwa 1.34 vs. 2.0).Takacherechedza pasi perinatal kufa pakati peheterozygotes, vamwe vaiva necraniofacial anomalies (Fig. S3).Nekudaro, iyo yakaderera kupinda kweiyi perinatal craniofacial phenotypes inoita kuti zviome kudzidza maitiro avo epathophysiological.Nokudaro, takatarisa pane embryonic lethal phenotype ye homozygous Specc11 mutants.
Yakawanda inosanganiswa heterozygous kana homozygous Specc1lDTM096/RRH048 mutant embryos haina kukura mushure meE9.5-10.5 (Fig. 5A-D), uye neural tube haina kuvhara mberi (Fig. 5B, D) uye dzimwe nguva yakavharwa kumashure (isina kuratidzwa). ..Iyi cranial neural chubhu yekuvhara defect yakabatanidzwa nehuwandu hweCNCC yakanyorwa DLX2 yakasara muNeural folds paE10.5, ichiratidza kuti hapana dissection (Mufananidzo 5A'-D').Kuti tione kana hukuru hwese hweCNCC hwakaderedzwawo, takaisa CNCC mitsara neGFP mumitsara yedu yejeni neWnt1-Cre uye ROSAmTmG.Isu tinoyerera yakarongedzwa GFP+ NCC uye GFP- (RFP+) isiri-NCC kubva kumazamu ese.PaE9.5, chikamu chekuyerera-yakarongedzwa GFP-yakanyorwa CNCCs haina kushanduka zvakanyanya pakati peWT uye mutant embryos (isina kuratidzwa), zvichiratidza zvakajairika CNCC kutsanangurwa.Nokudaro, takafungidzira kuti zvakasara zveWnt1-Cre uye DLX2 kusvibisa mumaneural folds (Mufananidzo 5B') zvakakonzerwa nekusagadzikana kweCNCC layering, zvichida nekuda kwekuwedzera kuwanda kana kupararira kwemasero eAJ, sezvinoonekwa mumaseru SPECC1L-kd.Isu takashandisa NCC mamaki SOX10, AP2A, uye DLX2 kusimbisa kuvepo kweCNCC muNeural fold (Mufananidzo 5E-R).PaE8.5, neural fold staining kune ese matatu NCC mamaki akaonekwa muzvikamu zveWT (Fig. 5E, G, I) uye Specc1l mutant (Fig. 5F, H, J).PaE9.5, nepo NCC mamaki akashatisa kutama kweNCC muzvikamu zveWT (Fig. 5M, O, Q), kusvibiswa kweNCC kwakasara kwakaonekwa mumapendekete akaiswa pachena eSpecc1l mutant embryos (Fig. 5N, P, R).Nekuda kwekuti SOX10 uye DLX2 maki ari kutama maCNCC, mhedzisiro iyi inoratidza kuti SPECC1L-inoshomeka CNCCs inowana post-migratory specification asi inotadza kutama kubva neural folds.
Kushaikwa kweSpecc11 kunotungamira kuvharika neural chubhu kuvharwa, delamination yecranial neural crest masero uye maAJs.
(A, B') E9.5 WT (A) Embryo yakatakura inotama cranial neural crest cells (CNCC) yakanyorwa kuti Wnt1-Cre (A').Mukupesana, Specc11 mutant embryos anoratidza akavhurika neural folds (B), arrowheads) uye CNCC asina kutama (B', arrowheads).(C, D') Bright field images (C, D') uye immunostaining (C', D') yeCNCC marker DLX2 yeE10.5 WT embryos (C, C') uye Specc1l (D, D').MuWT E10.5 embryos, DLX2-positive CNCC inogadzirisa gill arches (C', miseve), nepo mumachinjika, kusviba kunooneka kunorambira mune yakavhurika neural folds (D', miseve) uye mune yekutanga pharyngeal arches (D', miseve).) ine mamwe mavara (miseve) inoratidza kushata delamination uye kutama kweCNCC.ER) Zvikamu zveWT uye Specc1l mutant embryos pamatanho E8.5 (E-L) uye E9.5 (M-R) zvakanyorwa neNCC mamaki SOX10 (E, F, M, N), AP2A (G, H, O, P ) uye DLX2 (I, J, Q, R).PaE8.5, kusvibiswa kweNCC kwakaonekwa mumusango-mhando neural fold (NF) uye zvikamu zvinoshanduka.Co-staining yeSOX10 uye β-catenin muE8.5 WT (K) uye mutant (L) yakaratidza yakawedzera β-catenin tsvina pamiganhu yesero mumaneural folds.PaE9.5, kusvibiswa kwemhando dzesango dzeCNCC dzinotama (M, O, Q) dzakaonekwa, apo mune mutants, unstratified CNCCs yakasvibiswa neural folds yakazaruka (N, P, R).(S-Z) In vivo AJ yekunyorwa kwekuongorora mu coronal zvikamu zveWT uye Specc11DTM096/RRH048 embryos ine E9.5 mutation.Ndege inokwana kuita chikamu inoratidzwa mukona yepamusoro yekurudyi.Muzvikamu zvehutumbi hunoshanduka, kuwedzera kusvibiswa kweF-actin (S, T) uye myosin IIb (U, V) yakaonekwa.Zvakafanana nemigumisiro ye in vitro muFig. 3, mu mutant embryos, yakasimbiswa membrane staining ye β-catenin (W, X) uye E-cadherin (Y, Z) yakaonekwa.(AA-BB) Erekitironi micrograph yechikamu chemusango-embryo yakatarisa mhiri kwemuganhu weapical-basal cell inoratidza yakasarudzika electron-dense dunhu rinoratidza makutano ekunamira (AA, miseve).Kusiyana neizvi, muzvikamu zveSpecc11 mutant embryos (BB, miseve), iyo yese apicobasal junction ndeye electron dense, zvichiratidza kuwanda kwekuwedzera uye kupararira kweanonamatira junctions.
Kuti tiedze fungidziro yedu yekuti yakaderedzwa layering imhaka yekushandurwa kweAJ, takaongorora AJ kunyora muakavhurika neural folds eSpecc1l mutant embryos (Fig. 5S-Z).Takaona kuwedzera kwe actin stress fibers (Fig. 5S, T) uye kuwirirana kwakawedzera nzvimbo ye myosin IIB inosvibisa pa actin fibers (Fig. 5U, V).Zvinonyanya kukosha, takaona kuwedzera kusvibiswa kwe β-catenin (Fig. 5W, X) uye E-cadherin (Fig. 5Y, Z) pamiganhu ye intercellular.Takaongororawo β-catenin tsvina yeNCC mumakumbo emagetsi eE8.5 embryos (Fig. 5K, L).β-catenin staining yakaratidzika kuva yakasimba muSpecc1l mutant neural folds (Fig. 5L uye K), zvichiratidza kuti kuchinja kweAJ kwakatanga.Mumaelectron micrographs ezvikamu zvedehenya zveE9.5 embryos, takaona zvakare kuwedzera diffuse electron-dense staining muSpecc1l mutant embryos zvichienzaniswa neWT (Fig. 5AA, BB uye S1E-H).Takatorwa pamwechete, idzi mhedzisiro dzinotsigira yedu in vitro mhedzisiro muSPECC1L-kd U2OS maseru uye inokurudzira kuti aberrant AJ staining inotangira CNCC stratification mumatant embryos edu.
Tichifunga nezvehukama hunopesana hunozivikanwa pakati pebasa reAKT uye E-cadherin kugadzikana, 17,28 isu takafungidzira kubatanidzwa kwePI3K-AKT kusaina.Mukuwedzera, takaona subepidermal blistering mune mamwe embryos edu mutant akapukunyuka kuuraya (<5%) paE9.5-10.5 uye panzvimbo payo akagara kumativi E13.5 (Fig. S3).Subepidermal vesicles chiratidzo chekuderedzwa kwePI3K-AKT kusaina kwakavakirwa paPDGFRα12.Fantauzzo et al.(2014) yakashuma kuti kukanganisa kwePDGFRα-based PI3K activation muPdgfraPI3K/PI3K mutant embryos inoguma ne subepidermal vesicles, neural tube defects, uye cleft palate phenotypes.Zvechokwadi, mazinga epan-AKT uye inoshanda phosphorylated Ser473-AKT yakaderedzwa mu vivo muSpecc1l mutant tissues kusvika kuE9.5 embryonic arrest (Fig. 6A-D).Kuderera kwemazinga ephosphorylated Ser473-AKT inogona kunge yakakonzerwa zvachose nekuderera kwemazinga epan-AKT mu vivo (FIG. 6E) uye in vitro (FIG. 6F).Kuderera kwemu vitro kwakaonekwa chete apo masero eU2OS ainyatsoenderana nekuchinja kwesero chimiro uye AJ density (Mufananidzo 6D).Saka, data redu rinoratidza kuti SPECC1L ibhuku rakanaka regulator yePI3K-AKT kusaina mu craniofacial morphogenesis.
(A-E) E8.5 (A,B) uye E9.5 (C,D) zvikamu zvedehenya kana E9.5 lysates kubva kuSpecc1l mutant embryos (E) inoratidza mazinga ekushanda phosphorylated S473-AKT uye pan-AKT Protein kuderedza. , zvichienzaniswa nekudzora WT.Western blotting yakaitwa pamusango-mhando lysates uye mutant lysates pasi pemamiriro akafanana.Mifananidzo yakaratidzwa ye SPECC1L yakatorwa kubva kune rimwe blot.Iyo blot imwechete yakabviswa uye yakaongororwa zvakare ne-anti-pan-ACT uye β-actin antibodies.Pan-AKT mazinga muE8.5 neural folds (A, B) uye mazinga e phosphorylated S473-AKT muE9.5 skull zvikamu zvakaderedzwa zvakanyanya.(F) Pan-AKT mazinga akaderedzwa zvakafanana mu lysates ye SPECC1L-kd U2OS masero akakohwa pakusangana kukuru.Mabhawa ekukanganisa anomiririra maSEM kubva kune matatu akazvimiririra eWestern blot quantifications.(GJ) Zvikamu zveWT embryos paE9.5 yakasvibiswa neKI67 uye yakatsemuka caspase 3, zvichiteerana, ichiratidza kuwedzera kwesero (G, G') uye zvishoma apoptotic chiitiko (H, H').Specc11 mutant embryos anoratidza kuenzaniswa kwesero kuwedzera (I), asi huwandu hwemasero ari kuita apoptosis hunowedzera zvakanyanya (J).
Takazoongorora mamaki ekuwedzera uye apoptosis.Hatina kucherechedza chero mutsauko mukupararira kweE9.5 embryos (Fig. 6E, G ichienzaniswa neI) ine proliferation index ye82.5% yeWT mutants uye 86.5% yeSpecc1l mutants yakayerwa neKI67 staining (p <0.56, Fisher's bvunzo chaiyo).Saizvozvo, isu hatina kucherechedza chero mutsauko muapoptosis yakayerwa nekusvibisa kune yakatsemuka caspase 3 mumaneural folds paE8.5 kusvika embryo kusungwa (isina kuratidzwa) (isina kuratidzwa).Kusiyana neizvi, apoptosis yakawedzera zvakanyanya muE9.5 mutant embryos (Fig. 6F, H uye J).Uku kuwedzera kwese kweapoptosis kunoenderana nekuderedzwa kwePI3K-AKT kusaina uye yekutanga embryonic lethality29,30,31.
Tevere, kusimbisa basa rinokonzera rePI3K-AKT kusaina muAJ shanduko mumaseru edu kd, isu takashandura kemikari nzira yekutonga uye kd masero (Mufananidzo 7A-F).Isu takashandisa sechiratidzo chekuchinja kwesero shanduko phenotype inocherechedzwa mune confluent SPECC1L-kd masero, ayo isu takaita tichishandisa reshiyo yehurefu hwakareba (kureba) kune inoenderana vertical dimension (hupamhi).Chiyero che1 chinotarisirwa kune akatenderera kana cuboidal maseru (Mufananidzo 7G).Mukuwedzera kune chimiro chesero, takasimbisawo maitiro paAJ ne β-catenin staining (Fig. 7A'-F ').Kuvharidzirwa kwePI3K-AKT nzira uchishandisa wortmannin yakanga yakakwana kushandura chimiro chesero mumasero ekudzora (Mufananidzo 7A, C) uye AJ (Mufananidzo 7A ').PI3K-AKT activator SC-79 haina kukanganisa sero chimiro (FIG. 7A, E) kana AJ kuwedzera (FIG. 7A') mukudzora maseru.MuSPECC1L-kd masero, kuwedzera kudzvinyirirwa kwePI3K-AKT nzira yakaguma nekuwedzera apoptosis (Fig. 7B, D) uye kuwedzera kwakajeka mu β-catenin staining (Fig. 7B '), inopindirana neyedu in vivo heavy mutants.Zvakakosha, kushandiswa kwePI3K-AKT nzira yakagadziridza zvakanyanya sero chimiro (Mufananidzo 7B, F) uye AJ phenotypes (Mufananidzo 7B").Kuchinja kwechimiro chesero kwakaverengerwa sereshiyo yekutenderera kwesero (CCR) uye kuenzaniswa nekukosha sekutsanangurwa pamusoro apa (FIG. 7G).Zvechokwadi, mumasero ekutonga (Fig. 7G, CCR = 1.56), kurapwa kwewortmannin kwakanga kwakakwana kuchinja zvakanyanya chimiro chesero (Fig. 7G, CCR = 3.61, p <2.4 × 10-9) kusvika pamwero wakafanana newakaonekwa. mu SPECC1L.-kd masero (Fig. 7G, CCR = 3.46).Wortmannin kurapwa kweSPECC1L-kd masero (Fig. 7G, CCR = 3.60, isingakoshi) yakanga isinganyanyi kukosha kupfuura isina kubatwa kd masero (Fig. 7G, CCR = 3.46, isina hanya) kana wortmannin-treated control cells (Fig. 7G)., CCR = 3.46, isina hanya) inokanganisa kuwedzera kwesero (7G, CCR = 3.61, isingakoshi).Chinonyanya kukosha, SC-79 AKT activator yakadzorera elongated phenotype ye SPECC1L-kd masero (Fig. 7G, CCR = 1.74, p <6.2 × 10-12).Migumisiro iyi inosimbisa kuti SPECC1L inodzora PI3K-AKT chiratidzo uye inoratidza kuti kuderera kwepakati mu SPECC1L kunokanganisa kusungirirwa kwesero, asi kuderera kwakasimba kunotungamirira kuapoptosis (Fig. 8).
(A-F') Kudzora (A, C, E) uye SPECC1L-kd (B, D, F) masero anobatwa nePI3K-AKT pathway inhibitor wortmannin (C, D) kana SC-79 activator (E, F) Kurapa. .Masero asingatarisirwe asina kubatwa ari cuboidal (A) ane yakajairika β-cat cellular staining (A'), nepo kd masero akareba (B) nekuwedzera β-cat staining (B').Mushure mekudzvinyirirwa kwePI3K-AKT nzira, kudzora masero akareba (C) ne β-cat kuwedzera (C'), apo kd masero akatanga kutarisana neapoptosis (D), yakafanana nemaembryo edu akashandurwa zvakanyanya uye achiratidza zvakanyanya kuwedzeredzwa β-katsi.kusviba (D').Mushure mekushandiswa kwePI3K-AKT nzira, masero ekudzora akaramba ari cuboidal (E) uye aive neyakajairwa β-cat (E') tsvina, nepo kd masero airatidza zvakanyanya kuvandudzwa sero chimiro (F) uye β-cat (F') kusvibisa, zvichiratidza. (G) Iyo dhigirii rekuchinja kwechimiro chesero mu (AF) yakaverengerwa pachishandiswa sero kutenderera reshiyo (CCR) yeyakareba dimension (kureba) uye inoenderana vertical dimension (hupamhi) uchishandisa MetaMorph software.Kusagadziriswa (NT) SPECC1L-kd masero (CCR = 3.46) akanga akareba zvakanyanya kudarika masero ekudzora (CCR = 1.56, p <6.1 × 10-13).Wort's inhibition yePI3K-AKT nzira mukudzora maseru yakanga yakakwana kukonzera kuenzana kwakafanana musero chimiro (CCR = 3.61, p <2.4 × 10-9).Saizvozvo, AKT activation neSC-79 muSPECC1L-kd masero akadzoreredza sero elongation kusvika kudzora mazinga (CCR = 1.74, p <6.2 × 10-12).Wortmannin kurapwa kweSPECC1L-kd masero kwakaguma nekuwedzera apoptosis asi hapana kuwedzera kwesero shanduko yekuchinja (CCR = 3.60) kana ichienzaniswa neasina kubatwa kd (CCR = 3.46, ns) kana wortmannin-yakagadziriswa masero ekudzora (3.61) akaonekwa mu.ns = hazvina basa.+/- SEM zviyero zve50 masero zvinoratidzwa.Misiyano yenhamba yakaverengerwa pachishandiswa t-test yeMudzidzi.
(A) Schematic inomiririra ye inhibition uye activation yePI3K-AKT nzira inoguma nekuchinja kweAJ nekununura, zvichiteerana.(B) Yakarongwa modhi yeAKT protein kugadzikana ne SPECC1L.
Premigratory CNCCs inoda AJ lysis kuti iparadzanise kubva kune anterior neural fold neuroepithelial cells1,15,32.Kuwedzera kusvibiswa kwezvikamu zveAJ uye kurasikirwa kweapical-basal AJ asymmetric distribution mu SPECC1L-yakashaya masero zvose mu vitro uye in vivo, pamwe chete nehukama hwepedyo hwe SPECC1L kusvika ku β-catenin, inoratidza kuti SPECC1L inoshanda kuchengetedza zvakakodzera AJ kugadzikana kwenzvimbo. tsandanyama dzesangano.actin cytoskeleton.Kubatana kwe SPECC1L ne actin cytoskeleton uye β-catenin uye kuwedzera kwenhamba ye condensed actin filaments mukusavapo kwe SPECC1L inopindirana nekuwedzera kunoonekwa muAJ density.Imwe mukana ndeyokuti kuwedzera kwenhamba ye actin fibers mu SPECC1L-inoshaya masero inotungamirira kukuchinja kwe intercellular tension.Nemhaka yokuti kushungurudzika kwemasero kunokanganisa AJ 33 dynamics, kuchinja kwemagetsi kunogona kukonzera kupararira kweAJ 34.Saka chero shanduko ichakanganisa CNCC layer.
Wnt1 inoratidzwa mukutanga neural folds izvo zvinopa kusimuka kweneural crest masero.Saka, Wnt1-cre mutsara wekutsvagira mamakisi ese ari maviri-ekutanga uye anofamba NCC35.Zvisinei, Wnt1 inoratidzawo dorsal brain tissue clones zvakare yakatorwa kubva mukutanga neural folds 35,36, zvichiita kuti kusvibiswa kwedu kweE9.5 mutants yeWnt1 marker mumagetsi akazaruka neural folds haisi CNCC.Kusviba kwedu kwakanaka kweNCC mamaki AP2A uye SOX10 yakasimbisa kuti yakafumurwa neural folds yeSpecc11 mutant embryos zvechokwadi yaive neCNCC.Mukuwedzera, sezvo AP2A neSOX10 zviri zviratidzo zvekutanga kutama kweNCC, kusvibiswa kwakanaka kwakaratidza kuti masero aya ari post-migratory CNCC iyo isingagoni kugadziriswa neE9.5.
Yedu data inoratidza kuti molecular regulation yeAJ ne SPECC1L inopindirana nePI3K-AKT kusaina.Kuratidzira kweAKT kunoderedzwa muSPECC1L masero asina kukwana uye matishu.Zvakawanikwa naFantauzzo et al.tsigira basa rakananga rePI3K-AKT kusaina mu craniofacial morphogenesis.(2014) yakaratidza kuti kushayikwa kwekushandiswa kwePDGFRα-based PI3K-AKT kusaina kunotungamirira kune cleft palate phenotype.Isu tinoratidza zvakare kuti inhibition yePI3K-AKT nzira yakakwana kushandura AJ uye sero chimiro muU2OS masero.Zvinoenderana nezvatakawana, Kaini et al.37 yakaratidza kuti kuderedzwa kwePI3K α110 subunit mumasero ekupedzisira kunoguma nekuwedzera kwakafanana kwepericellular β-catenin staining, inonzi kuwedzera kwe "connectivity index".Nekudaro, mumasero ekupedzisira ane actin filaments atove akarongeka zvakanyanya, kudzvanywa kwePI3K-AKT nzira inoguma mune yakasununguka sero chimiro.Mukupesana, SPECC1L-kd U2OS masero airatidza akareba sero chimiro.Musiyano uyu unogona kunge uri werudzi rwesero.Nepo kudzvinyirirwa kwePI3K-AKT kusaina kunokanganisa zvachose actin cytoskeleton, mhedzisiro pane sero chimiro inotarwa nekuchinja kwekunetsana kunokonzerwa nekuchinja kwehudhindi uye kurongeka kwepakati actin fibers.Mumasero eU2OS, isu takashandisa chete sero chimiro shanduko sechiratidzo che SPECC1L-inoshaya AJ shanduko uye kupora.Mukupedzisa, isu tinofungidzira kuti inhibition yeAKT nzira mu SPECC1L kushomeka kunowedzera AJ kugadzikana uye kunoderedza delamination muCNCC.
Sezvineiwo, pan-AKT mazinga akaderedzwa mu vitro uye mu vivo mukuwedzera kune phosphorylated 473-AKT mazinga mukusavapo kweSPECC1L, zvichiratidza kudzorwa kwePI3K-AKT kusaina padanho reAKT protein kugadzikana kana kudzoka.Iyo SPECC1L uye MID1 genes, zvose zvakabatana neOpitz / GBBB syndrome, encode mapuroteni anogadzirisa microtubules 18,22.Iyo nzira iyo SPECC1L uye MID1 inopindirana microtubule kugadzikana haina kunyatsonzwisiswa.Munyaya ye SPECC1L, kugadzikana uku kunosanganisira kuwedzerwa acetylation ye subset ye microtubules 18.Zvinogoneka kuti SPECC1L inoshandisa nzira yakafanana yekudzikamisa mamwe mapuroteni akadai seAKT.Yakave yakaratidzwa kuti acetylation ye lysine yakasara muAKT protein inotungamirira kuderera kwe membrane localization uye phosphorylation38.Pamusoro pezvo, kuzara kweketani yeK63 pane imwechete lysine yakasara paAKT inodiwa kune iyo membrane localization uye activation39,40.Pakati pezvinhu zvakati wandei zvinodyidzana neSPECC1L mapuroteni akaonekwa mune akasiyana-siyana akakwira mbiriso mbiri-yakasanganiswa skrini, ina - CCDC841, ECM2942, APC uye UBE2I43 - zvakabatanidzwa mukuchinja kweprotein kana kugadzikana kuburikidza neubiquitination kana sumoylation.SPECC1L inogona kubatanidzwa mukushandurwa kwemashure kwekushandura kweAKT lysine zvakasara, zvichikanganisa kugadzikana kweAKT.Zvisinei, basa rakakosha re SPECC1L munzvimbo uye kugadzikana kweprotein yeAKT inoramba ichinyatsojekeswa.
Kukanganisa kwakanyanya mu SPECC1L kutaura mu vivo kwakakonzera kuwedzera AJ marker staining uye yakaremara CNCC overlay, pamwe nekuwedzera apoptosis uye yekutanga embryonic lethality.Mishumo yapfuura yakaratidza kuti mbeva inochinja nekuwedzera mazinga eapoptosis inosanganiswa neural tube defects 44,45,46,47 uye craniofacial defects48.Izvo zvave zvichiratidzwa kuti zvakanyanya kufa kwesero mumaneural folds kana pharyngeal arches zvinogona kukonzera nhamba isina kukwana yemasero anodiwa kune yakakodzera morphogenetic kufamba 48,49,50.Kusiyana neizvi, yedu SPECC1L yakashomeka mitsara yesero ine zvine mwero yakaderedzwa SPECC1L kutaura kwakaratidza chete AJ shanduko pasina humbowo hwekuwedzera kwesero kufa.Nekudaro, kemikari inhibition yePI3K-AKT nzira mumasero aya eKd yakaguma nekuwedzera apoptosis.Saka, kuderera zvine mwero mukutaura kwe SPECC1L kana basa kunoita kuti masero ararame.Izvi zvinoenderana nekuona kuti zvisingawanzo Specc11 mutant embryos anotiza kusungwa paSt.E9.5-zvichida nekuda kwekuderedzwa kwemajini ekutora maitiro-vanokwanisa kuvhara neural tubes uye kumira gare gare mukuvandudzika, kazhinji necraniofacial defects (Fig. S3).Zvakare zvinopindirana neizvi ndiko kusawanzoitika kweheterozygous Specc1l embryos ine craniofacial abnormalities-zvichida nekuda kwekuwedzera gene kubatwa zvakanaka-pamwe nekuwanikwa muzebrafish umo imwe yeviri SPECC1L orthologues (specc1lb) inokonzera kunonoka embryonic phenotypes, kusanganisira kurasikirwa kwe shaya dzezasi nekutsemuka kwemativi maviri51.Nokudaro, heterozygous SPECC1L kurasikirwa-kwe-kushanda kuchinja kunowanikwa muvarwere vevanhu kunogona kukonzera kukanganisa kuduku mu SPECC1L basa panguva yecraniofacial morphogenesis, yakakwana kutsanangura maorofacial clefts.SPECC1L-based regulation ye intercellular contacts inogonawo kuita basa mu palatogenesis uye fusion ye pharyngeal arches.Zvimwe zvidzidzo zve SPECC1L basa zvichabatsira kujekesa basa renguva pfupi intercellular contacts muCNCC panguva yekuvhara neural tube mu neuroepithelial cell motility uye craniofacial morphogenesis.
U2OS osteosarcoma control uye SPECC1L-kd masero akatsanangurwa kare (Saadi et al., 2011).Mishonga inorwisa SPECC1L yakave yakambozivikanwa kare (Saadi et al., 2011).Anti-β-catenin antibodies (tsuro; 1: 1000; Santa Cruz, Dallas, TX) (mbeva; 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), yakatsemuka caspase 3 (1: 1000; Cell Signaling Technology, Danvers, MA) uye β-actin (1: 2500; Sigma-Aldrich, St. Louis, MO ) yakashandiswa sezvakatsanangurwa..Actin filaments yakasvibiswa neActi-stain rhodamine phalloidin (Cytoskeleton, Denver, Colorado).
U2OS kudzora maseru uye SPECC1L-kd masero akagadzirwa mune yakajairwa yakakwira glucose DMEM yakawedzerwa ne10% fetal bovine serum (Life Technologies, Carlsbad, CA).Nokuda kwekuchinja kweAJ, 2 x 105 masero akadyarwa pagirazi akabatwa ne 0.1% porcine gelatin (Sigma-Aldrich, St. Louis, MO) uye akacherechedza kuchinja kwechimiro chemasero.Masero akaunganidzwa panguva dzakasiyana dzakaratidzwa: maawa mana mushure mekudyara (t = 1), maawa makumi maviri nemana mushure mekudyara (t = 2), confluence pasina shanduko musero chimiro (t = 3), shanduko musero chimiro (t = 4) , 24 h mushure mekuchinja kwesero (t = 5) uye 48 h mushure mekuchinja kwesero (t = 6) (Fig. 1, 2, 3).Kugadzirisa iyo PI3K-AKT nzira, maseru akagadzirwa panzvimbo dzakaratidzwa nePI3K-AKT inhibitor wortmannin (TOCRIS Biosciences, Minneapolis, Minnesota) kana SC-79 activator (TOCRIS Biosciences, Minneapolis Adams, Minnesota).Hurukuro ine makemikari yaichinjwa zuva nezuva.
Marekodhi-ne-frame marekodhi akaitwa pakurarama kwekutonga uye masero eKD pasi peyakajairika tsika mamiriro, uye chikamu chekusiyanisa mifananidzo yakaunganidzwa yega maminetsi gumi kwemazuva manomwe.Mifananidzo yakatorwa pachishandiswa komputa-inodzorwa Leica DM IRB inverted maikorosikopu yakashongedzerwa ine mechanical stage uye 10 × N-PLAN chinangwa chakabatana neQImaging Retiga-SRV kamera.Munguva yekufungidzira, tsika dzemasero dzakachengetwa pa 37 ° C mumhepo ine hunyoro ine 5% CO2.
Mitsetse miviri yemajini trap ES cell lines DTM096 uye RRH048 kubva kuRegional Mutant Mouse Resource Center (UC Davis, CA) yakashandiswa kugadzira Specc11 mitsara isina kukwana mbeva, yakatarwa Specc1lgtDTM096 uye Specc1lgtRRH046.Muchidimbu, masero 129/REJ ES akaiswa muC57BL6 blastocysts.Iwo akakonzeresa chimeric mbeva dzechirume dzakazvarwa nevakadzi C57BL6 mbeva kuti vaone vana vane agouti jasi coloration.Kuvapo kweiyo gene trap vector kuisa kwakashandiswa kuratidza heterozygotes.Mbeva dzakachengetwa pane yakasanganiswa kumashure kwe129/REJ;C57BL6.Nzvimbo yekuisa nzvimbo ye genetic trap vector yakasimbiswa neRT-PCR, genome sequencing, uye genetic complementation (Supplementary Figure 1).Kutsvaga iyo CNCC mutsara wembiri heterozygous Specc1lGT mbeva, ROSAmTmG (#007576) uye Wnt1-Cre (#003829) mbeva (Jackson Laboratory, Bar Harbor, ME) akayambuka kugadzira iyo ROSAmTmG neWnt1-Cre allele muSpecc1l mutant embryos.Zvese zviedzo mumakonzo zvakaitwa zvinoenderana nemaprotocol akabvumidzwa neInstitutional Animal Care uye Use Committee yeYunivhesiti yeKansas Medical Center.
Embryos yakagadziriswa mu (1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2, 0.02% NP-40, 5 mM EGTA) kwemaminetsi makumi matanhatu pakupisa kwekamuri.Mushure mekugadzirisa muX-gal staining solution (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, 1 mg/ml X-gal) Stain development yakaitwa pa37°C. .°C mukati maawa 1-6.Embryos yakagadziriswa-yakagadziriswa mu4% PFA uye yakaonekwa.
Nokuda kweWestern blotting, masero akaiswa lysed mu passive lysis buffer (Promega, Fitchburg, WI) yakawedzerwa nemusanganiswa weHALT protease inhibitors (Sigma-Aldrich, St. Louis, MO).Lysates akagadziriswa pa12% polyacrylamide Mini-PROTEAN TGX akagadzirira-akagadzirwa gels (Bio-Rad, Hercules, CA) uye akaendeswa kune Immobilon PVDF membranes (EMD Millipore, Billerica, MA).Iyo membrane yakavharwa mu5% mukaka muPBS ine 0.1% Pakati.Masoja ekudzivirira zvirwere akaiswa usiku hwose pa4°C kana kuti kweawa imwe patembiricha.Femto SuperSignal West ECL reagent (Thermo Scientific, Waltham, MA) yakashandiswa kugadzira chiratidzo.Nezve immunostaining, embryo dzakagadziriswa husiku humwe mu4% PFA/PBS uye cryopreserved.Matishu akavharwa muPBS aine 1% serum yembudzi yakajairwa (Thermo Scientific, Waltham, MA) uye 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) ndokuzoiswa pa4°C muincubator panguva ye husiku.ine anti-antibody uye fluorescent yechipiri masoja ekudzivirira chirwere (1:1000) kweawa imwe pa4°C.Zvikamu zvakasvibiswa zvakaiswa muProLong yegoridhe yepakati (Thermo Scientific, Waltham MA) uye mifananidzo yakatsetseka yakawanikwa uchishandisa Leica TCS SPE confocal microscope.Imwe neimwe immunostaining yakaitwa sezviyedzo zvitatu zvakazvimiririra pazvisungo zvemazamu maviri anoshanduka.Muedzo wekumiririra unoratidzwa.
Masero akaiswa mumodified RIPA buffer (20 mM Tris-HCl, pH 8.0, 1% NP-40, 130 mM NaCl, 10% glycerol, 2 mM EDTA, uye HALT protease inhibitor (Sigma-Aldrich, St. Louis, MO) Muchidimbu, malysates akafanocheneswa neprotein G magnetic beads (Life Technologies, Carlsbad, CA) ndokuzoiswa usiku hwose pa4° C. ne anti-SPECC1L kana IgG protein G protein beads dzakashandiswa kubvisa SPECC1L uye Western blotting yakaitwa pachishandiswa anti -β-catenin antibody inotsanangurwa pamusoro apa Co-IP miedzo inoratidzwa inomiririra zviedzo zvina zvakazvimirira.
Maseru akasimirirwa kana mbeva embryonic tissues akapihwa eelectron microscopy centre paUniversity yeKansas Medical Center.Nenguva pfupi, samples dzakaiswa muEMbed 812 resin (Electron Microscopy Sayenzi, Fort Washington, PA), yakagadziriswa husiku pa60 ° C, uye yakakamurwa pa80 nm uchishandisa Leica UC7 ultramicrotome ine blade yedhaimani.Zvikamu zvakaonekwa pachishandiswa JEOL JEM-1400 transmission electron microscope ine 100 kV Lab6 pfuti.
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