ASTM A790 2507 / 2205 1.4462 / 1.4410 Tuubbada Welded ee Qaybta Kiimikada Warshadaha, yaraanta SPECC1L waxay keenaysaa xasilloonida kordhaysa ee kala-goysyada kala-goysyada iyo hoos u dhaca daadinta unugyada unugyada neerfaha ee cranial.

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ASTM A790 2507 / 2205 1.4462 / 1.4410 Duplex Tube Welded ee Warshadaha Kiimikada

Liaocheng Sihe SS Material Co., Ltd.waa shirkad hormood ah oo ku takhasustay tuubooyinka birta ah ee aan sinnayn, tuubooyinka daloola ee dhalaalaya, tuubooyinka duuban ee aan xidhnayn iwm.Si loo fududeeyo macaamiisha , waxaanu sidoo kale haysanaa tubooyinka alxanka iyo tuubooyinka .Liaocheng Sihe SS Material Co., Ltd.wuxuu haystaa qalabka wax soo saarka iyo tijaabinta ugu horumarsan .Waxaan si buuxda u buuxin karnaa shuruudahaaga.Marka loo eego heerka aad u adag, tuubooyinka aan soo saarno had iyo jeer waxay leeyihiin dulqaad OD iyo WT sax ah.Xakamaynta dulqaadku waxay si adag u waafaqsan tahay heerarka soo saarista.Alaabtayadu had iyo jeer waxay ku qanacsan yihiin macaamiisha .Macaamiishu waxay iibsadeen alaabtayada waxay abuureen faa'iido badan .
a) OD ( Dhexroorka Dibadda): 3.18mm ilaa 101.6mm
b) WT ( Dhumucda gidaarka): 0.5mm ilaa 20mm
c) Dhererka: Marka loo eego shuruudaha macmiilka
d) Heerarka: ASTM A312;ASTM A269;ASTM A789;ASTM A790 iwm
e) Habka Habka: ERW, EFW iwm

Sliders oo muujinaya saddex maqaal sawirkiiba.Isticmaal badhamada dambe iyo kuwa xiga si aad ugu dhex gudubto boggaga, ama badhamada kantaroolaha slide ee dhamaadka si aad ugu gudubto sawir kasta.
Unugyada ceerfaha neerfaha ee cranial (CNCC) ayaa ka leexiya laalaabka neerfaha ee uurjiifka oo u haajiraan dabada farnjiga, kuwaas oo ka samaysan inta badan qaab-dhismeedka wejiga dhexe.Cilad la'aanta CNCC waxay door muhiim ah ka ciyaartaa etiology ee dillaaca orofacial, cillad la'aanta dhalmada ee caadiga ah.Isbeddellada Heterozygous SPECC1L ayaa laga helay bukaannada leh dildilaaca aan caadiga ahayn iyo syndromic.Halkan, waxaan ku soo wargelinaynaa wasakhaynta la xoojiyey ee qaybaha isku xidhka xabagta ee canonical (AJ), β-catenin iyo E-cadherin ee ku jira unugyada garaaca SPECC1L, iyo micrographs elektarooniga ayaa muujinaya faafinta apical-basal ee AJ.Si loo fahmo doorka SPECC1L ee morphogenesis craniofacial, waxaan abuurnay moodal jiirka oo yaraada Specc1l.Mutants-ka Homozygousku waa dilaa uurjiif ah oo muujiya xidhitaanka tuubada neerfaha ee daciifka ah iyo CNCC lamination.Midabaynta borotiinka AJ waxay ku korodhay laalaabka neerfaha ee mutant.Ciladdan AJ waxay la socotaa cilad ku jirta delamination CNCC, oo u baahan kala dirid AJ.Intaa waxaa dheer, mutants Specc11 ayaa hoos u dhigay calaamadaha PI3K-AKT iyo korodhka apoptosis.In vitro, xannibaadda khafiifka ah ee calaamadaynta PI3K-AKT ee unugyada nooca duurjoogta ah ayaa ku filnaa inay keento isbeddelada AJ.Muhiimad ahaan, isbeddelada AJ ee ay keentay garaacista SPECC1L waxa lagu beddeli karaa hawlgelinta dariiqa PI3K-AKT.Isku soo wada duuboo, xogtani waxay soo jeedinaysaa in SPECC1L, oo ​​ah nidaamiye cusub oo calaamadaynta PI3K-AKT iyo bayoolaji AJ, loo baahan yahay xidhitaanka tuubada neerfaha iyo CNCC stratification.
Unugyada Cranial neural Crest (CNCCs) waxay u soo baxaan neuroectoderm dorsal waxayna ka go'aan neuroepithelium ee laalaabka neerfaha ee soo koraya iyada oo loo marayo habka ku lug leh kala-guurka epithelial-mesenchymal (EMT) 1,2,3.CNCC-yada epithelial-ka hore u soo baxay waxay carqaladeeyaan isgoysyada unugyada dhexdooda waxayna noqdaan guuritaan mesenchymal CNCCs kuwaas oo buuxiya qanjidhada koowaad iyo labaad oo sameeya inta badan carjawda craniofacial.Sidaa darteed, hiddo-wadaha xakameynaya shaqada CNCC ayaa badanaa lagu carqaladeeyaa etiology ee cilladaha ku dhasha craniofacial sida jeexjeexyada orofacial, inta badan waxay saameeyaan 1/800 dhalashada Maraykanka oo keliya.Mid ka mid ah cilladaha lagu dhasho8.
Kala saarista CNCC waxay ku beegan tahay xidhitaanka tuubada neerfaha hore inta u dhaxaysa 8.5 iyo 9.5 maalmood ee koritaanka embriyaha ee jiirarka.Mutants tiro ka mid ah hiddo-wadaha-jiirka-jidhka-jidhka-jiidka ah ayaa sidoo kale muujinaya nooc ka mid ah cilladaha tuubada neerfaha, oo ay ku jiraan Irf69,10, Ghrl310, Cfl111, iyo Pdgfrα12.Si kastaba ha ahaatee, hababka xidhitaanka tuubada neerfaha iyo CNCC stratification ayaa loo tixgelin karaa madax banaan, sida Splotch mutant mouse (Pax3) waxay muujinaysaa cilladaha xiritaanka tuubada neerfaha iyada oo aan wax saameyn ah ku yeelanayn CNCC stratification ama socdaalka 13,14.Moodooyinka dheeraadka ah ee mouse-ka ee leh cilladaha kala-goynta CNCC iyo xidhitaanka tuubada neerfaha ayaa kaa caawin doona in la xaddido saldhigga molecular ee labadan habraac.
Go'doominta CNCC ee unugyada neuroepithelial waxay u baahan tahay kala dirida isgoysyada dhejiska (AJs), kuwaas oo ka kooban isku-dhafka borotiinka oo ay ku jiraan, iyo kuwa kale, E-cadherin, β-catenin, α-E-catenin, iyo α-actin oo la xidhiidha actin filaments 2 Daraasadaha xad dhaafka ah ee E-cadherin ee laalaabka neerfayaasha ayaa muujiyay hoos u dhac ama dib u dhac ku yimaada delamination CNCC.Taa beddelkeeda, xakamaynta E-cadherin natiijooyinka hore ee stratification15,16.Qaar badan oo ka mid ah qodobbada dhexdhexaadiya EMT inta lagu jiro isbarbardhigga CNCC waa qodobbada qoraalka (AP2a, Id2, FOXD3, SNAIL, TWIST, SOX10) iyo borotiinnada dib-u-qaabaynta (ECM) ee borotiinnada dib-u-qaabaynta sida matrix metalloproteinases (MMPs), si kastaba ha ahaatee CNCC-yadu waa kuwa si toos ah u xakameynaya cytoskeletal AJ. weli lama oga.Dariiqa PI3K-AKT waxaa loo yaqaanaa inay ka soo horjeedaan heerarka E-cadherin, inta badan cilmi baarista kansarka17.Daraasadihii ugu dambeeyay waxay muujiyeen in luminta PDGFa-ku-saleysan PI3K-AKT calaamadeynta jiirarka ay keento cillado aan caadi ahayn, oo ay ku jiraan jeexjeexyada iyo cilladaha tuubada neerfaha12.Si kastaba ha ahaatee, xidhiidhka ka dhexeeya dariiqa PI3K-AKT iyo xasiloonida AJ ee ku salaysnaanta CNCC ma cadda.
Waxaan hore u aqoonsannay SPECC1L inuu yahay hiddo-wadaha mutant ee ugu horreeya ee laba qof oo leh dildilaac daran oo ka soo baxa afka ilaa isha, oo loo yaqaan dildilaaca oblique (ObFC) ama dillaaca Tessier IV18.Isbeddellada SPECC1L ayaa lagu aqoonsaday laba qoys oo kala duwan oo leh autosomal Dominant Opitz G/BBB syndrome (OMIM #145410), kaas oo shakhsiyaadka ay saameeyeen ay muujiyeen hyperdistance iyo dillaac / dillaacsanaan / palate19, iyo hal qoys oo qaba xanuunka Tibi ee dheeraadka ah (OMIM #145420)20 .in ka badan kala badh kiisaska Opitz G/BBB syndrome waa X-ku xidhan (OMIM #300000) waxaana sababa isbeddellada hidda-wadaha MID1, kaas oo soo koobaya borotiinka 22 ee qalfoofka unugyada la xidhiidha microtubule.Waxaan qiyaasaynaa in SPECC1L, oo ​​sidoo kale ah borotiinka la xidhiidha microtubules iyo actin cytoskeleton, waxay dhexdhexaadin kartaa calaamadaynta loo baahan yahay dib-u-qaabaynta cytoskeleton actin inta lagu jiro adhesion cell iyo socdaalka 18.Iyada oo loo marayo daraasadaha gudaha iyo vivo, waxaan hadda ku qeexnay SPECC1L inay tahay nidaamiye cusub oo xasilloonida AJ iyada oo loo marayo calaamadaynta PI3K-AKT.Heerka gacanta, yaraanta SPECC1L waxay keentay hoos u dhac ku yimid heerka borotiinka pan-AKT iyo kororka faafinta apical-basal ee AJ, kaas oo lagu baabi'iyay kicinta kiimikaad ee waddada AKT.In vivo, uurjiifka Specc11-yar ayaa muujinaya xiritaanka tuubada neerfaha oo daciif ah iyo kala-goynta CNCC oo yaraaday.Sidaa darteed, SPECC1L waxay u shaqeysaa calaamad muujinaysa ku-xidhnaanta unugga ee nidaamsan ee loo baahan yahay shaqada caadiga ah ee CNCC inta lagu jiro morphogenesis wajiga.
Si loo qeexo doorka SPECC1L ee heerka gacanta, waxaanu isticmaalnay khadka unugga osteosarcoma ee xasiloon ee hore ee U2OS ee SPECC1L18.Unugyadan xasilloon ee U2OS ee leh SPECC1L (kd) garaacid waxay lahaayeen dhexdhexaad (60-70%) hoos u dhac ku yimid heerarka qoraallada SPECC1L iyo borotiinada, oo ay weheliyaan cilladaha socdaalka iyo dib-u-habaynta cytoskeleton actin 18. Taas bedelkeeda, hoos u dhac ku-meel-gaar ah oo daran SPECC1L ayaa la tusay inay horseeddo cilladaha mitotic-ka 23.Markaynu sii tilmaamno, waxaanu ogaanay in unugyadeena SPECC1L-kd ee deggan ay beddeleen qaab-dhismeedka qaab-dhismeedka heer aad u sarreeya (Jaantuska 1).Unugyada kantaroolka ee shakhsi ahaaneed iyo unugyada kd ee isku dhafka hooseeya waxay u ekaayeen isku mid (Jaantuska 1A,D).24 saacadood ka dib isku-dhafka, unugyada kantaroolku waxay xajisteen qaabkooda cuboidal (Jaantus. 1B, E), halka unugyada SPECC1L-kd ay dheereeyeen (Jaantus. 1C, F).Baaxadda isbedelkan qaabka unugga waxaa qabtay in vivo sawirka noolaha ee unugyada kantaroolka iyo unugyada kd (filimka 1).Si loo go'aamiyo doorka SPECC1L ee unugyada isku dhafan, waxaan marka hore baarnay muujinteeda.Waxaan ogaanay in heerarka borotiinka ee SPECC1L ay kordheen marka la isku daro (Jaantuska 1G), halka SPECC1L heerarka qoraalku aanay kordhin (Jaantuska 1H).Intaa waxaa dheer, sida cufnaanta unuggu korodhay, borotiinka SPECC1L wuxuu ku ururay xuduudaha intercellular (Jaantus. 2A-E), oo leh qaab isku dhafan oo leh β-catenin xuub-xiran (Jaantus 2A'-E').Marka la eego xiriirka SPECC1L ee leh actin cytoskeleton 18,23 waxaan qiyaasnay ​​in SPECC1L ay la falgasho isgoysyada dhejiska ku saleysan actin (AJ).
(AF) Unugyada garaacista SPECC1L (DF) waxay ku dheeraanayaan isku dhafka sare (F) marka la barbardhigo unugyada U2OS (AC).Halkan waxaa ku yaal saddex ka mid ah lixda dhibcood ee wakhtiga (T1, T3, T6) ee aan u dooranay cufnaanta unugyada kala duwan.(G) Falanqaynta xuubka Galbeedka oo muujinaya in borotiinka SPECC1L uu ku xasilay heer aad u sarreeya marka loo eego heerka hoose ee isku dhafka unugyada kantaroolka.Galbeedka SPECC1L waxay tusinaysaa guutada 120 kDa ee la filayo iyo koox miisaankeedu sarreeyo, oo suurtogal ah in dib loo turjumo (*).Falanqaynta xuubka galbeedka ayaa lagu sameeyay isla shuruudo isku mid ah oo hooseeya iyo mid sare.Sawirada muujinaya SPECC1L meel hoose iyo mid sare ayaa laga soo qaaday isla hal dhibic.Isla bartii ayaa laga saaray oo dib loogu baaray β-actin antibody.(H) Falanqaynta tirada RT-PCR ma muujin isbeddello la taaban karo oo ku yimid heerarka qoraalka SPECC1L.Baararka khaladku waxay ka dhigan yihiin SEM-yada afar tijaabo oo madaxbannaan.
(AE) Waxaanu dooranay lix dhibcood oo wakhti ah (T1-T6) oo ka dhigan cufnaanta unugyada kala duwan si loo caadiyeeyo falanqaynta qaabka unugyada iyo isbeddelada AJ ee unugyada U2OS ee leh SPECC1L garaacid (kd).Shanta ugu horreysa ee dhibcaha waqtigan waxaa ka mid ah unugyo keliya (T1), 50-70% isku-dhafka unugyada unugyada yaryar (T2), fiyuus aan dib-u-qaabayn unugyada kd (T3), dib-u-qaabaynta unugyada kd (T4), iyo 24 saacadood oo isbeddel ah.qaabka dambe ee unugyada kd (T5).Barootiinka SPECC1L ayaa inta badan ku kala firdhiyey cytoplasm ee T1 (A), laakiin ururintiisa ayaa lagu arkay xuduudaha intercellular ee waqtiyada xiga (B–E, fallaadho).(FJ) β-catenin waxay muujinaysaa isku-ururin la mid ah xudduudaha intercellular ee la xidhiidha dhismaha AJ.(A'-E') SPECC1L iyo β-catenin waxay muujinayaan wasakhaynta isku dhafan ee xudduudaha unugyada cufnaanta unugyada sare (Falaaraha).(F'-J') Unugyada SPECC1L-kd, midabaynta β-catenin waxay u muuqataa mid caadi ah cufnaanta unugyada hoose (F'-H'), laakiin waxay sii fidaysaa marka qaabka unuggu isbeddelo (I', J'; fallaadho), taasoo muujinaysa in AJ ayaa isbedelay.Baararka = 10 µm.
Waxaan markaa isku daynay inaan go'aan ka gaarno saameynta ay ku leedahay yaraanta SPECC1L ee AJ.Waxaan isticmaalnay dhowr calaamadood oo xiriir la leh AJ, oo ay ku jiraan qaybaha canonical F-actin, myosin IIb, β-catenin, iyo E-cadherin24,25,26,27.Fiilooyinka cadaadiska Actin ayaa kordhay unugyada SPECC1L-kd sida hore loogu sharraxay (Jaantus 3A, B) 18.Myosin IIb ee la xidhiidha actin filaments waxay muujisay koror la mid ah unugyada SPECC1L-kd ee vitro (Jaantus. 3C,D).AJ-ku-xiran β-catenin waxay ku xidhan tahay cadhrin ee xuubka unugyada, oo muujinaya qaabka caadiga ah ee "shirka malabka" ee xakamaynta cubocytes (Jaantus. 3E, G).Waxa xiiso leh, sawirada fidsan ee isticmaalaya microscopy confocal, β-catenin (Sawir 3E,F) iyo E-cadherin (Jaantus. 3G, H) oo ku dhejinaya xuubka unugyada unugyada isku-darka ah ee SPECC1L waxay muujiyeen qaabab caan ah oo midabaynta fidsan.Balaadhintan AJ-ku-xiran β-catenin midabaynta unugyada kd waxay ahayd mid aad loogu dhawaaqo marka la isku daro, laakiin waxay u muuqatay inay ka horeyso isbeddelada qaabka unugyada (Jaantus. 2F-J, F'-J').Si loo go'aamiyo dabeecadda jireed ee midabaynta AJ ee la fidiyay, waxaanu baarnay xuduudaha unugyada ee dusha sare ee apical-basal ee unugyada SPECC1L-kd U2OS anagoo adeegsanayna microscopy elektarooniga ah (TEM) (Jaantuska 3I,J).Si ka duwan unugyada kantaroolka (Jaantus. 3I), kuwaas oo lahaa gobollo cufan oo elektaroonig ah oo kala duwan oo tilmaamaya AJ (falaadhaha), unugyada kd (Jaantus. 3J) waxay muujiyeen gobollo waaweyn oo isku xiran oo ah cufnaanta elektaroonigga sare ee AJ oo ay weheliyaan diyaaradda apicobasal..Intaa waxaa dheer, qaybaha transverse, waxaan ku aragnay xuubka unugyada ballaaran ee unugyada kd (Sawir S1A, B), taas oo sharxaysa qaabka fidsan ee β-catenin iyo E-cadherin midabaynta (Jaantus 3F, H).Taageerada doorka SPECC1L ee AJ-yada, β-catenin ayaa la wadaagay difaaca jirka ee SPECC1L ee lysates ee unugyada U2OS ee isku dhafan (Jaantus. 3K).Iyada oo ay weheliso difaac-ilaalinta fidsan ee calaamadaha AJ, falanqaynta TEM waxay la socotaa mala-awaalkeena ah in yaraanta SPECC1L ay kordhiso cufnaanta AJ apical-basal iyo kala duwanaanshiyaha.
(AH) Kordhinta midabaynta F-actin ee unugyada kd 48 saacadood ka dib-fudud (T6; A, B).Wasakhda la beddelay ee myosin IIb ee la xidhiidha F-actin (C, D).Qaabka siman ee β-catenin iyo E-cadherin xuubka xuubka ee unugyada kantaroolka (E, G) ayaa lagu xoojiyay unugyada SPECC1L-kd (F, H).Baararka = 10 µm.(I-J) Mikrogarafyada korantada ee eegaya isgoysyada apical-basal intercellular.Unugyada kantaroolku waxay muujinayaan gobolo cufan oo kala duwan oo elektaroonik ah oo muujinaya isgoysyo dhegdheg leh (I, fallaadho).Taas bedelkeeda, dhammaan isku xidhka apical-basal ee unugyada SPECC1L-kd waxay u muuqdeen cufan elektaroonig ah (J, fallaadho), taasoo muujinaysa cufnaanta korodhay iyo kala firdhisanaanta isgoysyada koollada.(K) β-catenin waxaa si wadajir ah looga tallaalay SPECC1L gudaha unugyada U2OS ee isku dhafan.Sawir laga soo qaaday hal meel oo ka dhigan mid ka mid ah afar tijaabo oo madaxbannaan.
Si loo fahmo doorka SPECC1L ee morphogenesis craniofacial, waxaan abuurnay moodal jiirka yar oo Specc1l ah anagoo adeegsanayna laba xariiqo unug dabin ah oo madax banaan, DTM096 iyo RRH048 (BayGenomics, CA), kuwaas oo u taagan intron 1 iyo Specc1l qoraallada lagu qabtay 15 (Sawir 1) .4A, sawirka S2).Meesha genomic-ga ee galinta vector-ka sakhraansan waxaa lagu go'aamiyay isku xigxiga guud ee genome-ga waxaana xaqiijiyay PCR (Jaantus. S2).Labada naqshadood ee dabinka hidde-sidaha ayaa sidoo kale oggolaaday isku-dhafka gudaha ee suxufiyiinta Specc11-lacZ markii la qabto.Sidaa darteed, muujinta lacZ ee lagu go'aamiyay midabaynta X-gal ayaa loo adeegsaday tilmaame muujinta Specc11.Labada noocba waxay muujiyeen qaababka muujinta lacZ ee isku midka ah, oo leh dabinka hidda-wadaha DTM096 ee gudaha 1 oo muujinaya muujin ka xoog badan RRH048 gudaha gudaha 15 (aan la muujin).Si kastaba ha ahaatee, Specc1l si ballaaran ayaa loo muujiyay, iyadoo si gaar ah u muujinta xooggan ee laalaabka neerfaha ee E8.5 (Jaantuska 4B), ee tuubada neerfaha iyo hababka wejiga ee E9.5 iyo E10.5 (Jaantus 4C, D), iyo horumarinta addimada ee E10.5 iyo indhaha (Jaantuska 4D).Waxaan horey u soo sheegnay in muujinta SPECC1L ee qaansada hore ee pharyngeal ee E10.5 ay ku jirtay epithelium iyo mesenchyme18 hoosta hoose, oo la socota CNCC.Si loo tijaabiyo muujinta SPECC1L ee CNCC, waxaanu samaynay E8.5 laalaabka neerfaha (Jaantuska 4E-J) iyo E9.5 qaybaha dhakada (Jaantuska 4K-).Markay tahay E8.5, SPECC1L waxay si xoog leh u laallaabtay neerfaha (Jaantus. 4E, H), oo ay ku jiraan unugyo leh sumadaha NCC (Jaantus. 4G, J).Markay tahay E9.5, SPECC1L (Jaantus. 4K, N) si xoog leh u walaaqday guuritaanka CNCC oo leh AP2A (Jaantus. 4L, M) ama SOX10 (Jaantus. 4O, P).
(A) Qaabka qaabaynta hiddo-wadaha Specc11 ee muujinaya galinta vector-ka-soo-saarka ee ES DTM096 (intron 1) iyo RRH048 (intron 15) clones unug.(BD) midabaynta lacZ ee heterozygous Specc1lDTM096 embriyaha oo ka dhigan Specc1l muujinta E8.5 ilaa E10.5.NE = neuroectoderm, NF = laabta neerfaha, PA1 = qanjidhada koowaad ee pharyngeal.(EP) SPECC1L is-difaacid oo leh calaamadaha NCC AP2A iyo SOX10 ee E8.5 (NF; EJ) laalaabka neerfaha iyo E9.5 (KP) qaybaha madaxa.Midabaynta SPECC1L ayaa si weyn loogu arkay laalaabyada neerfaha E8.5 (E, H; fallaaro madaxyada), oo ay ku jiraan unugyo lagu calaamadeeyay AP2A (F, G; falaaraha) iyo SOX10 (I, J; fallaadho).Markay tahay E9.5, SPECC1L waxay si adag u wasakhaysay guuritaanka CNCCs (K, N; fallaadho) ku suntan AP2A (L, M; fallaadho) iyo SOX10 (O, P; fallaadho).
Ka gudubka inta u dhaxaysa heterozygous Specc1lDTM096/+ iyo Specc1lRRH048/+ jiirarka waxay tusinaysaa in labada dabin hiddo-sideyaasha aanay ahayn kuwo is dhammaystira iyo in isku-xidhka heterozygotes iyo homozygotes-ka embriyaha ee labada hidde-dhabarka ay yihiin kuwo dilaa ah embriyaha (Shaxda S1).Saamiyada Mendelian waxay muujiyeen hoos u dhac ku yimid heerka badbaadada heterozygotes xilliga dhalashada (la filayo 1.34 vs. 2.0).Waxaan ogaanay dhimashada dhalmada oo hooseysa ee heterozygotes, qaar ayaa lahaa cillad maskaxeed (Jaantus. S3).Si kastaba ha ahaatee, gelitaanka hoose ee dabeecadaha 'craniofacial phenotypes' ee dhalmada ayaa ka dhigaysa mid adag in la barto hababka jirkooda ee hoose.Sidaa darteed, waxaanu diirada saarnay nooca embriyaha dilaaga ah ee mutaant-yada homozygous Specc11.
Inta badan embriyaha heterozygous ama homozygous Specc1lDTM096/RRH048 mutant embriyaha ma kobcin ka dib E9.5-10.5 (Sawir 5A-D), iyo tuubada neural ma xirmin hore (Sawir. 5B, D) iyo mararka qaarkood xiran gadaal (lama muujin) ..Ciladdan xiritaanka tuubada neerfaha ee cranial waxay la xiriirtay inta badan CNCC ee calaamadisay DLX2 ee ku haray laalaabka neerfayaasha ee E10.5, taasoo muujineysa inaan kala goyn (Jaantuska 5A'-D').Si loo go'aamiyo haddii cabbirka guud ee CNCC sidoo kale la dhimay, waxaan ku dhejinay khadadka CNCC GFP ee khadadka dabinka hidda-wadaha Wnt1-Cre iyo ROSAmTmG.Waxa aanu kala soocnay GFP+ NCC iyo GFP-(RFP+) aan NCC ahayn ee uurjiifka oo dhan.Markay tahay E9.5, saamiga qulqulka-sooc-soocidda ee GFP-ku calaamadeeyay CNCC-yada si weyn isuma beddelin WT iyo embriyaha mutant (aan la muujin), taasoo muujinaysa qeexitaanka CNCC ee caadiga ah.Sidaa darteed, waxaan qiyaasnay ​​in haraaga Wnt1-Cre iyo DLX2 ee ku jira laallaabyada neerfaha ee bannaan (Jaantuska 5B') ay sabab u tahay lakabka CNCC ee cilladaysan, oo ay suurtogal tahay inay ugu wacan tahay cufnaanta korodhay ama kala firdhisanaanta unugyada AJ, sida lagu arkay unugyada SPECC1L-kd.Waxaan isticmaalnay calaamadaha NCC ee SOX10, AP2A, iyo DLX2 si aan u xaqiijino joogitaanka CNCC ee labka neerfaha (Jaantus 5E-R).Markay tahay E8.5, midabaynta neerfaha ee saddexda calaamadood ee NCC ayaa lagu arkay qaybaha WT (Jaantus. 5E, G, I) iyo Specc1l mutant (Jaantus. 5F, H, J).Markay tahay E9.5, halka calaamadaha NCC ay dhaliyeen guuritaanka NCC ee qaybaha WT (Jaantus. 5M, O, Q), midabaynta NCC ee hadhaaga ah ayaa lagu arkay laallaabyada neerfaha ee embriyaha ee Specc1l mutant (Jaantus. 5N, P, R).Sababtoo ah SOX10 iyo DLX2 waxay calaamadeeyaan guuritaanka CNCCs, natiijadani waxay soo jeedinaysaa in CNCC-yaraanta SPECC1L ay gaadhaan qeexida socdaalka ka dib laakiin waxay ku guul daraysteen inay ka haajiraan laalaabka neerfaha.
Yaraanta Specc11 waxay keentaa xidhitaanka tuubada neerfaha ee cilladaysan, delamination unugyada crest neural neural iyo AJs.
(A, B') E9.5 WT (A) embriyaha sidda unugyada crest neural neural ee guuraya (CNCC) oo ku suntan Wnt1-Cre (A').Taas bedelkeeda, embriyaha mutant Specc11 waxay muujinayaan laalaabka neerfaha ee furan (B), madax falaarta) iyo CNCC-yada aan guurin (B', fallaadho).(C, D') Sawirada goobta dhalaalaya (C, D') iyo difaaca jirka (C', D') ee calaamadeeyaha CNCC DLX2 ee E10.5 WT embriyaha (C, C') iyo Specc1l (D, D').embriyaha WT E10.5, DLX2-positive CNCC waxay gumaysataa qaansooyinka gill (C', fallaadho), halka mutants-ka, wasakhaynta muuqata ay ku sii jirto laalaabka neerfaha ee furan (D', fallaadho) iyo qaansooyinka pharyngeal ee ugu horreeya (D', fallaadho).) oo leh qaar ka mid ah wasakhaynta (Falaaraha) oo muujinaya delamination liidata iyo socdaalka CNCC.ER) Qaybaha WT iyo Specc1l embriyaha mutant ee heerarka E8.5 (E-L) iyo E9.5 (M–R) ayaa lagu calaamadeeyay calaamadaha NCC SOX10 (E, F, M, N), AP2A (G, H, O, P) iyo DLX2 (I, J, Q, R).Markay ahayd E8.5, midabaynta NCC ayaa lagu arkay laalaabka neerfaha ee duurjoogta ah (NF) iyo qaybaha mutant.Isku-dhafka SOX10 iyo β-catenin ee E8.5 WT (K) iyo mutant (L) ayaa muujiyay kororka β-catenin ee xuduudaha unugyada ee laalaabka neerfaha.Markay tahay E9.5, midabaynta nooca duurjoogta ah ee CNCCs (M, O, Q) ayaa lagu arkay, halka isku-dhafan, CNCC-yada aan la aqoonsanayn ay wasakheeyeen laallaabyada neerfaha ee furan (N, P, R).(S–Z) In vivo AJ falanqaynta summaynta ee qaybaha coronal ee WT iyo Specc11DTM096/RRH048 uurjiifka leh E9.5 mutation.Diyaarad qaybeed qiyaas ah ayaa lagu muujiyay geeska sare ee midig.Qaybaha unugyada mutant, kor u kaca midabaynta F-actin (S, T) iyo myosin IIb (U, V) ayaa lagu arkay.Si la mid ah natiijooyinka in vitro ee Jaantuska 3, ee uurjiifka mutant, midabaynta xuubka ee β-catenin (W, X) iyo E-cadherin (Y, Z) ayaa la arkay.(AA-BB) Mikrogaraafka elektarooniga ah ee qayb ka mid ah embriyaha nooca duurjoogta ah ee eegaya meel ka baxsan xudduudda unugga apical-basal waxay muujinaysaa gobol cufan oo elektaroonig ah oo gaar ah oo tilmaamaya isku-goysyada kobacyada (AA, fallaaro).Taas bedelkeeda, qaybaha Specc11 uurjiifka mutant (BB, fallaadho), dhammaan isgoysyada apicobasal waa cufan elektaroonig ah, taas oo muujinaysa cufnaanta korodhay iyo kala firdhinta isgoysyada koollada.
Si aan u tijaabino mala-awaalkeena ah in lakabka hoos u dhaca ay sabab u tahay AJ oo la beddelay, waxaan baarnay calaamadeynta AJ ee laalaabka neerfaha ee qaawan ee uurjiifka mutant Specc1l (Jaantus. 5S-Z).Waxaan aragnay kororka fiilooyinka cadaadiska actin (Jaantus. 5S, T) iyo isku-dhafan oo kordhay ee myosin IIB midabaynta fiilooyinka actin (Jaantus. 5U, V).Muhiimad ahaan, waxaanu aragnay midabaynta β-catenin oo kordhay (Jaantus. 5W, X) iyo E-cadherin (Jaantus. 5Y,Z) ee xuduudaha intercellular.Waxaan sidoo kale baaray β-catenin midabaynta NCC ee laalaabka neerfaha ee E8.5 embriyaha (Jaantus. 5K, L).Midabaynta β-catenin waxay u muuqatay inay ku xoog badan tahay Specc1l laalaabka neerfaha ee mutant (Jaantus. 5L iyo K), oo soo jeedinaysa in isbeddellada AJ ay bilaabeen.Mikrogaraafka elektarooniga ah ee qaybaha dhakada E9.5 embriyaha, waxaan mar kale ku aragnay wasakhaynta korantada-cufan ee kororka ee embriyaha mutant Specc1l marka la barbar dhigo WT (Jaantus. 5AA, BB iyo S1E-H).Isku soo wada duuboo, natiijooyinkani waxay taageerayaan natiijooyinkayaga in vitro-ga ee unugyada SPECC1L-kd U2OS waxayna soo jeedinayaan in wasakhaynta AJ-ka-soo-baxa ahi ay ka horreyso shaandhaynta CNCC ee embriyahayada mutant.
Marka la eego xidhiidhka ka soo horjeeda ee la yaqaan ee ka dhexeeya dhaqdhaqaaqa AKT iyo xasilloonida E-cadherin,17,28 waxaanu qiyaasnay ​​ku lug lahaanshaha calaamadaynta PI3K-AKT.Intaa waxa dheer, waxaanu ku aragnay finan ka hooseeya uurjiifkayaga ka soo baxsaday dhimashada (<5%) E9.5-10.5 beddelkeedana degay agagaarka E13.5 (Jaantus. S3).Xididdada hoose waa calaamad muujinaysa hoos u dhigista PI3K-AKT ee ku salaysan PDGFRα12.Fantauzzo iyo al.(2014) ayaa sheegay in carqaladaynta PDGFRa-ku-salaysan PI3K firfircoonida ee PdgfraPI3K/PI3K embriyaha mutant ay keenayso xuubka subpidermal, cilladaha tuubada neerfaha, iyo phenotypes dillaacsan.Runtii, heerarka pan-AKT iyo fosforyaalka firfircoon ee Ser473-AKT ayaa lagu dhimay vivo gudaha unugyada mutant Specc1l ilaa E9.5 xiritaanka uurjiifka (Jaantus. 6A-D).Hoos u dhaca heerarka fosfooraska Ser473-AKT waxaa laga yaabaa inay gebi ahaanba sabab u tahay hoos u dhaca heerarka pan-AKT ee vivo (FIG. 6E) iyo in vitro (FIG. 6F).Hoos u dhaca vitro ayaa la arkay kaliya marka unugyada U2OS ay si xooggan ugu biireen isbeddellada qaabka unugga iyo cufnaanta AJ (Jaantuska 6D).Sidaa darteed, xogtayadu waxay soo jeedinaysaa in SPECC1L ay tahay nidaamiye togan oo cusub oo PI3K-AKT ah oo tilmaamaya morphogenesis craniofacial.
(A–E) E8.5 (A,B) iyo E9.5 (C,D) qaybaha dhakada ama E9.5 lysates ka Specc1l mutant embryos (E) oo muujinaya heerarka fosfooraska firfircoon ee S473-AKT iyo pan-AKT Protein dhimis , marka la barbar dhigo kantaroolka WT.Burburinta reer galbeedka ayaa lagu sameeyay lysates-ka duurjoogta ah iyo lysates-ka mutant-ka ah ee hoos yimaada xaalado isku mid ah.Sawirada lagu muujiyay SPECC1L waxaa laga soo qaatay hal dhibic.Isla bartii ayaa laga saaray oo dib loogu baaray anti-pan-ACT iyo β-actin.Heerarka Pan-AKT ee E8.5 laalaabka neerfayaasha (A, B) iyo heerarka fosfooraska S473-AKT ee qaybaha E9.5 ayaa si weyn hoos loogu dhigay.(F) Heerarka Pan-AKT si la mid ah ayaa loo dhimay lysates ee unugyada SPECC1L-kd U2OS ee laga soo goostay isku-xidhnaan sare.Baararka khaladku waxay ka dhigan yihiin SEM-yada laga soo qaatay saddex qiyaasood oo madax-bannaan oo Galbeedka ah.(GJ) Qaybaha uurjiifka WT ee E9.5 oo leh KI67 iyo jeexjeexan 3, siday u kala horreeyaan, oo muujinaya kororka unugyada (G, G') iyo hawl yar oo apoptotic ah (H, H').Specc11 embriyaha mutant waxay muujinayaan kororka unugyada isbarbardhigga (I), laakiin tirada unugyada ku jira apoptosis ayaa si weyn u kordhay (J).
Waxaan markaa baarnay calaamadaha faafinta iyo apoptosis.Ma aanan arkin wax farqi ah oo u dhexeeya fidinta E9.5 embriyaha (Jaantus. 6E, G marka la barbardhigo I) oo leh index fidinta 82.5% ee mutants WT iyo 86.5% ee Specc1l mutants oo lagu qiyaasay KI67 midabaynta (p <0.56, Fisher's imtixaan sax ah).Sidoo kale, ma aanan arkin wax farqi ah oo apoptosis ah oo lagu cabbiray midabaynta 3 ee laalaabka neerfaha ee E8.5 ilaa embriyaha la xiro (lama muujin) (lama muujin).Taas bedelkeeda, apoptosis ayaa si aad ah u kordhay dhammaan E9.5 embriyaha mutant (Jaantus. 6F, H iyo J).Korodhkan guud ee apoptosis wuxuu la socdaa hoos u dhigista PI3K-AKT iyo dhimashada uurjiifka hore29,30,31.
Marka xigta, si loo xaqiijiyo doorka sababa ee calaamadaynta PI3K-AKT ee isbeddelada AJ ee unugyadeena kd, waxaanu si kiimiko ah u bedelnay dariiqa xakamaynta iyo unugyada kd (Jaantuska 7A-F).Waxaan u isticmaalnay calaamad ahaan qaabka unugyada isbeddelka phenotype ee lagu arkay unugyada isku dhafan ee SPECC1L-kd, kaas oo aan ku qiyaasnay ​​annagoo adeegsanayna saamiga cabbirka ugu dheer (dhererka) cabbirka toosan (ballaca).Saamiga 1 ayaa laga filayaa unugyada wareega ama kuboidalka ah (Jaantuska 7G).Marka lagu daro qaabka unugyada, waxaan sidoo kale xaqiijinay saameynta AJ ee β-catenin midabaynta (Jaantus. 7A'-F').Joojinta dariiqa PI3K-AKT iyadoo la adeegsanayo wortmannin ayaa ku filnayd inay bedesho qaabka unugyada unugyada kantaroolka (Jaantuska 7A,C) iyo AJ (Jaantus 7A').PI3K-AKT activator SC-79 ma saameynin qaabka unugyada (FIG. 7A, E) ama ballaarinta AJ (FIG. 7A') ee unugyada kantaroolka.Unugyada SPECC1L-kd, xakamaynta dheeraadka ah ee dariiqa PI3K-AKT waxay keentay kororka apoptosis (Jaantus. 7B,D) iyo korodhka calaamadsan ee β-catenin midabaynta (Jaantus. 7B'), oo ku habboon inteena in vivo mutants culus.Muhiimad ahaan, dhaqaajinta dariiqa PI3K-AKT ayaa si weyn u wanaajiyay qaabka unugyada (Jaantuska 7B,F) iyo AJ phenotypes (Jaantuska 7B”).Isbeddellada qaabka unugga waxaa lagu qiyaasaa saamiga wareega unugga (CCR) waxaana marka la barbar dhigo muhiimada sida kor lagu sheegay (FIG. 7G).Runtii, unugyada kantaroolka (Sawir 7G, CCR = 1.56), daawaynta wortmannin waxay ku filnayd inay si weyn u bedesho qaabka unugyada ee SPECC1L.-kd unugyada (Jaantus. 7G, CCR = 3.46).Daaweynta Wortmannin ee unugyada SPECC1L-kd (Sawir 7G, CCR = 3.60, dayacan) ma ahayn mid ka muhiimsan unugyada kd ee aan la daweyn (Jaantus. 7G, CCR = 3.46, dayacan) ama unugyada xakamaynta wortmannin-la daweeyay (Jaantus. 7G)., CCR = 3.46, dayacan) ayaa sidoo kale saameynaya ballaarinta unugyada (7G, CCR = 3.61, dayacan).Tan ugu muhiimsan, SC-79 AKT activator ayaa dib u soo celiyay phenotype-dheer ee unugyada SPECC1L-kd (Jaantus. 7G, CCR = 1.74, p <6.2 × 10-12).Natiijooyinkani waxay xaqiijinayaan in SPECC1L ay maamusho calaamadaha PI3K-AKT waxayna soo jeedinayaan in hoos u dhaca dhexdhexaadka ah ee SPECC1L uu saameeyo adhesion unug, halka hoos u dhac xoog leh uu keeno apoptosis (Jaantus. 8).
(A-F') Xakamaynta (A, C, E) iyo SPECC1L-kd (B, D, F) unugyada lagu daaweeyay PI3K-AKT dariiqa inhibitor wortmannin (C, D) ama SC-79 activator (E, F) Unugyada kantaroolka ee aan la daweynin waa kuboidal (A) oo leh midabaynta unugyada β-cat ee caadiga ah (A'), halka unugyada kd ay dheer yihiin (B) oo leh kororka β-cat (B').Ka dib markii la xakameeyey dariiqa PI3K-AKT, unugyada kantaroolku way dheereeyeen (C) oo leh balaadhinta β-cat (C'), halka unugyada kd ay bilaabeen inay maraan apoptosis (D), oo la mid ah embriyahayada aadka isu beddelay oo muujinaya β-cat oo aad loo xoojiyay.midabaynta (D').Ka dib markii la hawlgeliyay dariiqa PI3K-AKT, unugyada kantaroolku waxay noqdeen cuboidal (E) waxayna lahaayeen β-cat (E') midabaynta caadiga ah, halka unugyada kd ay muujiyeen inay si weyn u wanaajiyeen qaabka unugyada (F) iyo β-cat (F'), taasoo muujinaysa (G) Heerka isbeddelka qaabka unugga ee (AF) waxaa lagu qiyaasey iyadoo la isticmaalayo saamiga wareega unugga (CCR) ee cabbirka ugu dheer (dhererka) iyo cabbirka toosan ee u dhigma (ballaadhka) iyadoo la isticmaalayo software MetaMorph.Unugyada aan la daaweynin (NT) SPECC1L-kd (CCR = 3.46) ayaa aad uga dheeraa unugyada kantaroolka (CCR = 1.56, p <6.1 × 10-13).Joojinta Wort ee dariiqa PI3K-AKT ee unugyada kantaroolka ayaa ku filnaa inay keento koror la mid ah qaabka unugyada (CCR=3.61, p<2.4 × 10-9).Sidoo kale, firfircoonida AKT ee SC-79 ee unugyada SPECC1L-kd waxay soo celisay ballaarinta unugyada si loo xakameeyo heerarka (CCR = 1.74, p <6.2 × 10-12).Daaweynta Wortmannin ee unugyada SPECC1L-kd waxay keentay kororka apoptosis laakiin ma kordhin wax dheeraad ah oo ku saabsan isbeddelka qaabka unugyada (CCR = 3.60) marka la barbardhigo kd aan la daweyn (CCR = 3.46, ns) ama unugyada xakamaynta wortmannin-la daaweeyay (3.61) lagu arkay.ns = dhib ma leh.+/- Cabbirrada SEM ee 50 unug ayaa la muujiyey.Kala duwanaanshaha tirakoobka ayaa la xisaabiyay iyadoo la isticmaalayo t-tijaabka Ardayga.
(A) Matalaadda qaabaynta xannibaadda iyo hawlgelinta waddada PI3K-AKT taasoo keentay isbeddellada AJ iyo samatabbixinta, siday u kala horreeyaan.(B) Qaabka la soo jeediyay ee xasilinta borotiinka AKT ee SPECC1L.
CNCC-yada hordhaca ah waxay u baahan yihiin lysis AJ si ay uga soocaan unugyada neerfaha hore ee neerfaha neuroepithelial1,15,32.Wasakhaynta kordhay ee qaybaha AJ iyo luminta qaybinta apical-basal AJ asymmetric ee unugyada SPECC1L-la'aanta ah ee labadaba gudaha vitro iyo in vivo, oo ay weheliso u dhawaanshaha jireed ee SPECC1L ilaa β-catenin, waxay soo jeedinayaan in SPECC1L u shaqeyso si ay si sax ah u ilaaliso xasilloonida gudaha AJ murqaha ururka.actin cytoskeleton.Ururka SPECC1L oo leh actin cytoskeleton iyo β-catenin iyo kororka tirada fiilooyinka actin-ka ee la'aanta SPECC1L waxay la socotaa kororka la arkay ee cufnaanta AJ.Suurtagalnimada kale ayaa ah in tirada sii kordheysa ee fiilooyinka actin ee unugyada SPECC1L-yar ay horseeddo isbeddel ku yimaada xiisadda unugyada dhexdooda.Sababtoo ah walbahaarka gacanta wuxuu saameeyaa dhaqdhaqaaqa AJ 33, isbeddelada tamarta waxay keeni kartaa in badan oo faafin AJ 34 .Markaa isbedel kastaa wuxuu saamayn doonaa lakabyada CNCC.
Wnt1 waxa lagu muujiyaa laalaabka neerfaha hore ee keena unugyada crest neural.Markaa, raadraaca abtirsiinta Wnt1-cre waxay calaamad u tahay ka hor iyo guuritaanka NCC35.Si kastaba ha noqotee, Wnt1 waxay sidoo kale calaamadisaa xirmooyinka unugyada maskaxda ee dhabarka sidoo kale waxay ka soo jeedaan laalaabka neerfaha hore ee 35,36, taasoo ka dhigaysa inay u badan tahay in midabkeena E9.5 mutants ee calaamadaha Wnt1 ee laalaabka neerfaha ee furan maaha CNCC.Wasakhayntayada togan ee calaamadaha NCC AP2A iyo SOX10 waxay xaqiijiyeen in laallaabyada neerfaha ee qaawan ee uurjiifka mutant Specc11 ay runtii ku jiraan CNCC.Intaa waxaa dheer, maadaama AP2A iyo SOX10 ay yihiin calaamadaha socdaalka hore ee NCC, midabaynta togan ayaa muujisay in unugyadani ay yihiin CNCC-guurka ka dib oo aan lagu hagaajin karin E9.5.
Xogtayadu waxay soo jeedinaysaa in nidaamka molecular ee AJ ee SPECC1L ay dhexdhexaadiso calaamadaynta PI3K-AKT.Calaamadaynta AKT waa lagu dhimay unugyada iyo unugyada ka maqan SPECC1L.Natiijooyinka Fantauzzo et al.taageer doorka tooska ah ee calaamadaynta PI3K-AKT ee morphogenesis craniofacial.(2014) waxay muujisay in la'aanta firfircoonida PDGFRa-ku-saleysan PI3K-AKT calaamad u horseedayso phenotype dillaacsan.Waxaan sidoo kale tuseynaa in xannibaadda dariiqa PI3K-AKT ay ku filan tahay in la beddelo AJ iyo qaabka unugyada unugyada U2OS.Si waafaqsan natiijooyinkayaga, Kayn et al.37 waxay muujisay in hoos u dhigista qaybta PI3K α110 ee unugyada endothelial ay keento koror isku mid ah oo ku yimaada midabaynta pericellular β-catenin, oo loo tixraacayo kororka "tusaha isku xirnaanta".Si kastaba ha noqotee, unugyada endothelial kuwaas oo fiilooyinka actin-ka horeyba si heer sare ah loo abaabulay, xakamaynta dariiqa PI3K-AKT waxay keenaysaa qaab unug dabacsan.Taas bedelkeeda, unugyada SPECC1L-kd U2OS waxay muujiyeen qaab unug dheer.Farqigani waxa uu noqon karaa nooca unugyada gaarka ah.Iyadoo la xakameynayo calaamadaha PI3K-AKT ay si joogto ah u saameeyaan cytoskeleton actin, saameynta qaabka unugyada waxaa lagu go'aamiyaa isbeddelada xiisadda oo ay keento isbeddelada cufnaanta iyo abaabulka fiilooyinka actin dhexe.Unugyada U2OS, waxaan u isticmaalnay kaliya isbeddellada qaabka unugga si ay u calaamadiyaan isbeddelka iyo soo kabashada AJ ee SPECC1L.Gebogebadii, waxaanu qiyaasaynaa in joojinta dariiqa AKT ee yaraanta SPECC1L ay kordhiso xasilloonida AJ oo ay yarayso delamination CNCC.
Waxa xiiso leh, heerarka pan-AKT waa la dhimay gudaha vitro iyo in vivo marka lagu daro heerarka fosfooraska 473-AKT ee maqnaanshaha SPECC1L, soo jeedinta nidaaminta PI3K-AKT calaamadaynta heerka xasilloonida borotiinka AKT ama wareejinta.Hidde-sideyaasha SPECC1L iyo MID1, labaduba waxay la xiriiraan cudurka Opitz/GBBB, waxay dejiyaan borotiinnada xasiliya microtubules 18,22.Habka ay SPECC1L iyo MID1 ku dhexdhexaadiyaan xasilinta microtubule si buuxda looma fahmin.Xaaladda SPECC1L, xasilintan waxaa ku jira acetylation la xoojiyay ee qayb ka mid ah microtubules 18.Waxaa suurtogal ah in SPECC1L ay isticmaasho hab la mid ah si ay u dejiso borotiinnada kale sida AKT.Waxaa la muujiyay in acetylation ee hadhaaga lysine ee borotiinka AKT ay keento hoos u dhac ku yimaada meelaynta xuubka iyo fosfooraska38.Intaa waxaa dheer, meelaynta silsiladda K63 ee isla hadhaaga lysine ee AKT ayaa looga baahan yahay meelaynta xuubka iyo firfircoonida39,40.Waxaa ka mid ah dhowr arrimood oo la falgalaya borotiinnada SPECC1L oo lagu aqoonsaday khamiirka sare ee kala duwan ee labada shaashad ee isku-dhafan, afar - CCDC841, ECM2942, APC iyo UBE2I43 - ayaa lagu lug yeeshay beddelka borotiinka ama xasilloonida iyada oo loo marayo meel-ka-soo-bax ama sumoylation.SPECC1L waxa laga yaabaa inay ku lug yeelato wax ka beddelka turjumaadda dambe ee hadhaaga AKT lysine, taasoo saamaynaysa xasilloonida AKT.Si kastaba ha ahaatee, doorka muhiimka ah ee SPECC1L ee meelaynta iyo xasiloonida borotiinka AKT ayaa weli ah in la caddeeyo.
Cilladaha ba'an ee muujinta SPECC1L ee vivo waxay keentay kororka sumaynta AJ iyo daboolka cilladaysan ee CNCC, iyo sidoo kale kororka apoptosis iyo dhimashada embriyaha hore.Warbixinadii hore waxay muujiyeen in jiirka jiirka ee leh heerarka korodhay ee apoptosis ay la xiriiraan cilladaha tuubada neerfaha 44,45,46,47 iyo cilladaha craniofacial48.Waxaa la soo jeediyay in dhimashada unugyada xad-dhaafka ah ee laalaabka neerfaha ama qanjidhada pharyngeal ay keeni karaan tiro aan ku filnayn unugyada loo baahan yahay dhaqdhaqaaqa morphogenetic habboon 48,49,50.Taas bedelkeeda, xariiqayada unugyada ee SPECC1L oo si dhexdhexaad ah loo dhimay muujinta SPECC1L waxay muujisay kaliya isbeddelada AJ iyada oo aan caddayn dhimashada unugyada korodhay.Si kastaba ha ahaatee, xakameynta kiimikada ee dariiqa PI3K-AKT ee unugyadan Kd waxay keentay kororka apoptosis.Markaa, hoos u dhac dhexdhexaad ah oo ku yimaadda muujinta ama shaqada SPECC1L waxay hubisaa badbaadada unugyada.Tani waxay la socotaa fiirsashada in uurjiifyada naadirka ah ee Specc11 ee ka baxsanaya xadhiga st.E9.5-laga yaabee inay sabab u tahay hoos u dhaca waxtarka qabsashada hidda-wadaha-waxay awoodaan inay xiraan tuubooyinkooda neerfaha oo ay joojiyaan goor dambe ee horumarka, inta badan cilladaha craniofacial (Jaantus. S3).Waxa kale oo la socota tan waa dhacdo naadir ah oo ah embriyaha heterozygous Specc1l oo leh cillado craniofacial-malaha sababtoo ah korodhka waxtarka qabsashada hidda-wadaha - iyo sidoo kale helitaanka zebrafish kaas oo mid ka mid ah labada SPECC1L orthologues (specc1lb) uu keeno ifafaale dambe oo uurjiif ah, oo ay ku jiraan luminta embriyaha. daanka hoose iyo dildilaaca laba geesoodka ah51.Sidaa daraadeed, heterozygous SPECC1L isbeddelada shaqada ee lumay ee lagu aqoonsaday bukaanka bini'aadamka waxay keeni kartaa naafo yar oo ku jirta shaqada SPECC1L inta lagu jiro morphogenesis craniofacial, oo ku filan inay sharaxdo jeexjeexyada orofacial.Nidaaminta ku saleysan SPECC1L ee xiriirada intercellular ayaa sidoo kale laga yaabaa inay door ka ciyaaraan palatogenesis iyo fusion of arches pharyngeal.Daraasado dheeraad ah oo ku saabsan shaqada SPECC1L waxay gacan ka geysan doontaa in la caddeeyo doorka xiriirka ku-meel-gaarka ah ee ku-meel-gaarka ah ee CNCC inta lagu jiro xiritaanka tuubada neerfaha ee dhaqdhaqaaqa unugyada neuroepithelial iyo morphogenesis craniofacial.
U2OS osteosarcoma kantaroolka iyo unugyada SPECC1L-kd ayaa horay loogu sifeeyay (Saadi et al., 2011).Ka-hortagga unugyada ka-hortagga SPECC1L ayaa sidoo kale hore loogu sifeeyay (Saadi et al., 2011).Ka-hortagga-β-catenin-ka-hortagga (bakayle; 1:1000; Santa Cruz, Dallas, TX) (jiirka; 1: 1000; Tignoolajiyada Calaamadaynta Unugyada, 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), fosfo-Ser473-AKT (1:1000; Tignoolajiyada Calaamadaynta Unugyada, Danvers, MA), pan-AKT (1:1000; ThermoFisher Scientific, Waltham, MA) ) , KI67 (1:1000; Tignoolajiyada Calaamadaynta Unugyada, Danvers, MA), 3 (1:1000; Tignoolajiyada Calaamadaynta Unugyada, Danvers, MA) iyo β-actin (1:2500; Sigma-Aldrich, St. Louis, MO) ayaa loo adeegsaday sida lagu tilmaamay..Fiilooyinka Actin waxaa lagu sumeeyay Acti-stain rhodamine phalloidin (Cytoskeleton, Denver, Colorado).
Unugyada kontoroolka U2OS iyo unugyada SPECC1L-kd waxaa lagu soo barbaariyey gulukoos heer sare ah oo DMEM ah oo lagu kabay 10% serum bovine ee uurjiifka (Technology Life, Carlsbad, CA).Isbedelada AJ, 2 x 105 unugyo ayaa lagu beeray dhalada lagu daaweeyay 0.1% jelatin porcine (Sigma-Aldrich, St. Louis, MO) waxaana lagu arkay isbedelada qaabka unugga.Unugyada waxaa lagu soo ururiyay waqtiyo kala duwan oo la tilmaamay: 4 saacadood ka dib abuurka (t = 1), 24 saacadood ka dib abuuritaanka (t = 2), isku darka iyada oo aan isbeddelin qaabka unugyada (t = 3), isbeddelka qaabka unugyada (t = 4) , 24 h ka dib isbedelka qaabka unugga (t = 5) iyo 48 h ka dib isbeddelka qaabka unugyada (t = 6) (Jaantus 1, 2, 3).Si wax looga beddelo dariiqa PI3K-AKT, unugyadu waxaa lagu soo barbaariyey inta la tilmaamay ee PI3K-AKT inhibitor wortmannin (TOCRIS Biosciences, Minneapolis, Minnesota) ama SC-79 activator (TOCRIS Biosciences, Minneapolis Adams, Minnesota).Dhexdhexaadka ay ku jiraan kiimikooyinka ayaa la beddelay maalin kasta.
Duubabka qaab-dhismeedka ayaa lagu sameeyay xakamaynta tooska ah iyo unugyada KD ee xaaladaha dhaqanka caadiga ah, iyo sawirada isbarbardhigga wajiga ayaa la ururiyay 10kii daqiiqoba 7 maalmood.Sawirada waxaa lagu helay iyada oo la isticmaalayo kombuyuutar lagu maamulo Leica DM IRB mikroskoob rogan oo ku qalabaysan marxalad farsamo iyo ujeedo 10 × N-PLAN ah oo ku xidhan kamarada QImaging Retiga-SRVInta lagu jiro sawir-qaadista, dhaqamada unugga waxaa lagu ilaalinayay 37°C jawi qoyan oo leh 5% CO2.
Laba hidde-side dabin ES unug ee DTM096 iyo RRH048 ee Xarunta Kheyraadka Mutant Mouse ee Gobolka (UC Davis, CA) ayaa loo adeegsaday in lagu dhaliyo khadadka jiirka ee Specc11, loo qoondeeyay Specc1lgtDTM096 iyo Specc1lgtRRH046.Si kooban, 129/unugyada REJ ES ayaa lagu duray blastocysts C57BL6.Natiijadii ka soo baxday jiirarka lab ee chimeric waxaa lagu saray dhedig C57BL6 jiirarka si loo aqoonsado faraca leh midabka jaakada agouti.Joogitaanka galinta dabinka hidde-sideyaasha ayaa loo isticmaalay in lagu aqoonsado heterozygotes.Jiirarka waxaa lagu hayaa asal isku dhafan oo ah 129/REJ;C57BL6.Meesha la geliyo galka dabinka hidde-sideyaasha waxaa xaqiijiyay RT-PCR, isku-xiridda genome-ka, iyo dhammaystirka hidde-sidaha (Jaantuska Dheeraadka ah 1).Si loo raadiyo lineage CNCC ee jiirarka heterozygous Specc1lGT double, ROSAmTmG (# 007576) iyo Wnt1-Cre (# 003829) jiirarka (Jackson Laboratory, Bar Harbor, ME) ayaa laga gudbay si loo soo saaro ROSAmTmG iyo Wnt1-Cre allele in Speccyol.Dhammaan tijaabooyinka jiirarka waxaa la sameeyay si waafaqsan borotokoollada ay ansixiyeen Hay'adda Xannaanada Xoolaha iyo Guddiga Isticmaalka ee Jaamacadda Kansas Medical Center.
Uurjiifka waxaa lagu hagaajiyay (1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2, 0.02% NP-40, 5 mM EGTA) 60 min heerkulka qolka.Ka dib markii lagu hagaajiyo X-gal wasakheynta (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, 1 mg / ml X-gal) Horumarinta wasakhda ayaa lagu sameeyay 37 ° C .°C 1-6 saacadood gudahood.Uurjiifka waxaa lagu dhejiyay 4% PFA oo la arkay.
Burburinta reer galbeedka, unugyadu waxa lagu saleeyay lysis dadban (Promega, Fitchburg, WI) oo lagu kabay isku-dar ah HAT protease inhibitors (Sigma-Aldrich, St. Louis, MO).Lysates waxaa lagu farsameeyay 12% polyacrylamide Mini-PROTEAN TGX jeel diyaarsan (Bio-Rad, Hercules, CA) waxaana loo wareejiyay xuubabka Immobilon PVDF (EMD Millipore, Billerica, MA).Xuubabka waxaa lagu xannibay 5% caanaha PBS oo ka kooban 0.1% Tween.Unugyada difaaca jirka waxaa lagu duray habeenkii 4°C ama hal saac heerkulka qolka.Femto SuperSignal West ECL reagent (Thermo Scientific, Waltham, MA) ayaa loo isticmaalay soo saarista calaamadaha.Difaaca difaaca jirka, embriyaha waxaa lagu hagaajiyay habeen 4% PFA/PBS waana la xafiday.Unugyada cryosections ayaa lagu xannibay PBS oo ay ku jiraan 1% serum riyaha caadiga ah (Thermo Scientific, Waltham, MA) iyo 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) ka dibna lagu beeray 4°C gudaha kubatarka xilliga habeen.leh anti-antibody iyo fluorescent secondary antibody (1:1000) 1 saac at 4°C.Qaybaha wasakhaysan ayaa lagu meeleeyay dhexdhexaadka dahabiga ah ee ProLong (Thermo Scientific, Waltham MA) iyo sawirro fidsan ayaa la helay iyadoo la adeegsanayo mikroskoob-koobeedka Leica TCS SPE.Is-difaacid kasta waxa loo sameeyay sidii saddex tijaabo oo madax-bannaan oo ku saabsan xuubka xuubka ugu yaraan laba uurjiif ah.Tijaabo wakiil ah ayaa la muujiyay.
Unugyada waxaa lagu dhex daray RIPA kaydka la beddelay (20 mM Tris-HCl, pH 8.0, 1% NP-40, 130 mM NaCl, 10% glycerol, 2 mM EDTA, iyo HALT protease inhibitor (Sigma-Aldrich, St. Louis, MO) Si kooban, lysates waxaa lagu sii sifeeyay borotiinka G magnetic beads (Life Technologies, Carlsbad, CA) ka dibna lagu daray habeenkii oo dhan 4 ° C. oo leh anti-SPECC1L ama borotiinka borotiinka IgG ayaa loo adeegsaday si loo soo saaro SPECC1L -β-catenin antibody-ka kor lagu sifeeyay Tijaabooyinka wada-shaqaynta IP-ga ee la muujiyay waxay matalaan afar tijaabo oo madaxbannaan.
Unugyo dhaqan oo go'an ama unugyo embriyaha jiirka ayaa la siiyay xarunta mikroskoobyada elektarooniga ah ee Jaamacadda Kansas Medical Center.Si kooban, muunado ayaa lagu dhex daray EMbed 812 resin (Electron Microscope Sciences, Fort Washington, PA), polymerized habeenkii 60°C, waxaana lagu qaybiyay 80 nm iyadoo la isticmaalayo Leica UC7 ultramicrotome oo ku qalabaysan daab dheeman ah.Qaybaha waxaa lagu sawiray iyadoo la isticmaalayo JEOL JEM-1400 mikroskoob elektaroonik ah oo gudbinta oo ku qalabaysan 100 kV Lab6 qori.
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