ASTM A790 2507 / 2205 1.4462 / 1.4410 Duplex Welded Tube No ka ʻenehana ʻenehana ʻenehana, ʻo ka hemahema o SPECC1L e alakaʻi i ka hoʻonui ʻia o nā hui spliced ​​a me ka hoʻohaʻahaʻa ʻana i ka hoʻoheheʻe ʻana o nā cell neural crest cranial.

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ASTM A790 2507 / 2205 1.4462 / 1.4410 Duplex Welded Tube No ka ʻOihana Kemika

Liaocheng Sihe SS Material Co., Ltd.He mea hana koʻikoʻi ia i nā paipu hili ʻole kila, nā paipu annealed ʻālohilohi, nā paipu wili liʻiliʻi a me nā mea ʻē aʻe.I mea e maʻalahi ai nā mea kūʻai aku, ua wili pū mākou i nā paipu a me nā paipu.Liaocheng Sihe SS Material Co., Ltd.loaʻa i nā lako hana ʻoi loa a hoʻāʻo.Hiki iā mākou ke hoʻokō pono i kāu koi.E like me ke kūlana koʻikoʻi, ʻo nā paipu i hana ʻia e mākou e loaʻa mau i ka OD a me ka WT hoʻomanawanui.ʻO ka mana hoʻomanawanui e pili pono i ka hana ʻana i nā kūlana.Māʻona mau kā mākou huahana me nā mea kūʻai aku.Ua kūʻai nā mea kūʻai aku i kā mākou huahana i loaʻa nā waiwai hou aʻe.
a) OD (Outer Diameter): 3.18mm i 101.6mm
b) WT (Ka Mānoanoa Wall): 0.5mm a 20mm
c) Length: E like me ka makemake o ka mea kūʻai aku
d) Nā Kūlana : ASTM A312;ASTM A269;ASTM A789;ASTM A790 etc
e) Ke Kaʻina Hana: ERW, EFW etc

ʻO nā slider e hōʻike ana i ʻekolu ʻatikala ma kēlā me kēia paheʻe.E hoʻohana i nā pihi hope a i hope no ka neʻe ʻana i nā paheʻe, a i ʻole nā ​​pihi hoʻokele paheʻe ma ka hopena e neʻe i kēlā me kēia paheʻe.
Hoʻopau ka cranial neural crest cell (CNCC) i nā ʻāpana neural embryonic a neʻe i nā ʻāʻī pharyngeal, ka mea i hana i ka hapa nui o nā hale waena.He mea koʻikoʻi ko CNCC dysfunction i ka etiology o orofacial cleft, he malformation congenital maʻamau.Ua loaʻa nā hoʻololi Heterozygous SPECC1L i nā poʻe maʻi me nā maʻi atypical a syndromic clefts.Maʻaneʻi, hōʻike mākou i ka hoʻonui ʻia ʻana o nā ʻāpana canonical adhesive junction (AJ), β-catenin a me E-cadherin i loko o nā pūnaewele knockdown SPECC1L moʻomeheu, a hōʻike nā micrographs electron i ka hoʻopuehu apical-basal o AJ.No ka hoʻomaopopo ʻana i ke kuleana o SPECC1L i ka craniofacial morphogenesis, ua hana mākou i kahi kiʻi ʻiole hemahema Specc1l.Homozygous mutants he embryonic lethal a hōʻike i ka impaired neural tube pani a me CNCC lamination.Hoʻonui ʻia ka hoʻopaʻa ʻana o ka protein AJ i nā ʻāpana neural mutant.Kūlike kēia hemahema AJ me kahi kīnā i ka delamination CNCC, e koi ana i ka hoʻoheheʻe AJ.Eia kekahi, ua hōʻemi ʻo Specc11 mutants i ka hōʻailona PI3K-AKT a hoʻonui i ka apoptosis.I loko o ka vitro, ua lawa ka pale ʻana o ka PI3K-AKT hōʻailona ma nā ʻano ʻano hihiu e hoʻohuli i nā loli AJ.ʻO ka mea nui, hiki ke hoʻohuli ʻia nā hoʻololi AJ i hoʻoiho ʻia e SPECC1L knockdown e ka hoʻāla ʻana o ke ala PI3K-AKT.Hoʻohui pū ʻia, hōʻike kēia mau ʻikepili e koi ʻia ʻo SPECC1L, ma ke ʻano he mea hoʻoponopono hou o ka hōʻailona PI3K-AKT a me ka biology AJ, no ka pani ʻana o ka neural tube a me ka stratification CNCC.
ʻO nā pūnaewele neural crest cranial (CNCCs) localize i ka neuroectoderm dorsal a hoʻokaʻawale mai ka neuroepithelium o ka ulu ʻana o nā neural folds ma o ke kaʻina hana e pili ana i ka epithelial-mesenchymal transition (EMT)1,2,3.Hoʻopilikia ka epithelial CNCCs i nā hui intercellular a lilo i CNCC mesenchymal mesenchymal e hoʻopiha ana i nā arches pharyngeal mua a me ka lua a hana i ka hapa nui o ka cartilage craniofacial.No laila, hoʻopilikia pinepine ʻia nā genes e hoʻoponopono i ka hana CNCC i ka etiology o nā anomalies congenital craniofacial e like me nā ʻāʻī orofacial, ka mea maʻamau e pili ana i ka 1/800 hānau ma US wale nō.ʻO kekahi o nā hemahema o ka hānau ʻana8.
Hoʻopili ka Delamination o ka CNCC me ka pani ʻana o ka paipu neural mua ma waena o 8.5 a me 9.5 mau lā o ka ulu ʻana o ka embryonic i nā ʻiole.Hōʻike pū kekahi mau ʻano o nā ʻano ʻano o ka neural tube defect, me Irf69,10, Ghrl310, Cfl111, a me Pdgfrα12.Eia naʻe, hiki ke noʻonoʻo ʻia nā kaʻina hana o ka neural tube closure a me CNCC stratification, no ka mea, ʻo ka Splotch mutant mouse (Pax3) e hōʻike ana i nā hemahema i ka pani ʻana o ka neural tube me ka ʻole o ka hopena i ka stratification CNCC a i ʻole neʻe 13,14.ʻO nā kumu hoʻohālike ʻiole hou me nā hemahema i ka wehe ʻana i ka CNCC a me ka pani ʻana o ka neural tube e kōkua i ka wehewehe ʻana i ke kumu molekala maʻamau o kēia mau kaʻina hana ʻelua.
ʻO ka hoʻokaʻawale ʻana o CNCC mai nā cell neuroepithelial e pono ai ka hoʻopau ʻana o nā junction adhesive (AJs), i haku ʻia me nā paʻakikī protein i loaʻa, i waena o nā mea ʻē aʻe, E-cadherin, β-catenin, α-E-catenin, a me α-actinin pili me nā filament actin 2 Ua hōʻike ʻia nā haʻawina overexpression E-cadherin i loko o nā neural folds i ka hōʻemi a i ʻole ka lohi ʻana i ka delamination CNCC.ʻO ka mea ʻē aʻe, ʻo ka hoʻopau ʻana i ka E-cadherin ka hopena i ka stratification mua15,16.ʻO ka nui o nā mea e hoʻopili ai i ka EMT i ka wā CNCC stratification he mau mea transcription (AP2α, Id2, FOXD3, SNAIL, TWIST, SOX10) a me ka extracellular matrix (ECM) remodeling proteins e like me matrix metalloproteinases (MMPs), akā naʻe he pololei nā CNCCs cytoskeletal AJ regulators. aole i ikeia.ʻIke ʻia ke ala PI3K-AKT e kūʻē i nā pae E-cadherin, ʻoi loa mai ka noiʻi maʻi maʻi17.Ua hōʻike ʻia nā haʻawina hou i ka nalo ʻana o ka PDGFα-based PI3K-AKT hōʻailona i nā ʻiole e alakaʻi i nā mea ʻino craniofacial, e komo pū me ka palate a me nā neural tube defects12.Eia nō naʻe, ʻaʻole maopopo ka pilina ma waena o ke ala PI3K-AKT a me ka kūpaʻa AJ ma ka stratification CNCC.
Ua ʻike mua mākou ʻo SPECC1L ʻo ia ka gene mutant mua i loko o nā kānaka ʻelua me kahi māwae koʻikoʻi mai ka waha a hiki i ka maka, i kapa ʻia ʻo oblique cleft (ObFC) a i ʻole Tessier IV18 cleft.Ua ʻike ʻia nā hoʻololi ʻana o SPECC1L i loko o ʻelua mau ʻohana multigenerational me ka autosomal dominant Opitz G/BBB syndrome (OMIM #145410), kahi i hōʻike ʻia ai nā poʻe i pili i ka hyperdistance a me nā lehelehe māwae / palate19, a i loko o hoʻokahi ʻohana me Tibi overdistance syndrome (OMIM #145420)20 .ʻoi aku ma mua o ka hapalua o nā hihia o Opitz G / BBB syndrome he X-linked (OMIM #300000) a ke kumu ʻia e nā hoʻololi ʻana i ka MID1 gene, kahi e hoʻopili ai i ka protein 22 o ka ʻiʻo o ka microtubule-associated cell skeleton.Manaʻo mākou ʻo SPECC1L, he pūmua hoʻi e pili ana me nā microtubules a me ka cytoskeleton actin, hiki ke hoʻopili i ka hōʻailona e pono ai no ka hoʻoponopono hou ʻana o ka cytoskeleton actin i ka wā o ka hoʻopili ʻana o ka cell a me ka neʻe 18.Ma o nā haʻawina in vitro a me in vivo, wehewehe mākou iā SPECC1L ma ke ʻano he mea hoʻoponopono hou o ka paʻa AJ ma o ka hōʻailona PI3K-AKT.Ma ka cellular level, SPECC1L deficiency i ka emi ʻana o ke kiʻekiʻe o ka protein pan-AKT a me ka hoʻonui ʻana i ka hoʻopuehu apical-basal o AJ, i hoʻopau ʻia e ka hoʻoulu ʻana kemika o ke ala AKT.I loko o vivo, hōʻike nā embryos deficient Specc11 i ka pani ʻana o ka paipu neural a hōʻemi i ka wehe ʻana o CNCC.No laila, hana ʻo SPECC1L i ka hoʻopaʻa ʻia ʻana o ka cell adhesion-based signaling i koi ʻia no ka hana CNCC maʻamau i ka wā o ka morphogenesis facial.
No ka ʻike ʻana i ke kuleana o SPECC1L ma ka pae kelepona, ua hoʻohana mākou i ka laina kelepona osteosarcoma paʻa i hōʻike mua ʻia ʻo U2OS hemahema ma SPECC1L18.ʻO kēia mau pūnaewele U2OS paʻa me SPECC1L (kd) knockdown he haʻahaʻa haʻahaʻa (60-70%) i nā pae o nā transcripts SPECC1L a me nā protein, me nā hemahema i ka neʻe ʻana a me ka hoʻonohonoho hou ʻana o ka cytoskeleton actin 18. Ua hōʻike ʻia ʻo SPECC1L e alakaʻi i nā hemahema mitotic 23 .Ma ka hōʻike hou ʻana, ʻike mākou ua hoʻololi kā mākou SPECC1L-kd paʻa i ka morphology ma kahi kiʻekiʻe kiʻekiʻe o ka hui ʻana (Figure 1).Ua like ke ano like o na keena hoomalu kanaka a me na keena kd ma ka hui haahaa (Figure 1A,D).24 mau hola ma hope o ka hui ʻana, mālama ʻia nā pūnaewele hoʻomalu i ko lākou ʻano cuboidal (Fig. 1B, E), aʻo SPECC1L-kd cell elongated (Fig. 1C, F).Ua hopu ʻia ka nui o kēia hoʻololi ʻana i ke ʻano o ke kelepona e ka in vivo imaging ola o nā cell control and cell kd (kiʻiʻoniʻoni 1).No ka hoʻoholo ʻana i ke kuleana o SPECC1L i nā cell confluent, ua nānā mua mākou i kāna ʻōlelo.Ua ʻike mākou ua hoʻonui ʻia nā pae protein SPECC1L ma luna o ka fusion (Figure 1G), akā ʻaʻole i hoʻonui nā pae transcript SPECC1L (Figure 1H).Eia kekahi, i ka hoʻonui ʻia ʻana o ka nui o ka cell, ua hōʻiliʻili ʻia ka protein SPECC1L ma nā palena intercellular (Fig. 2A-E), me kahi kumu hoʻohālike me ka β-catenin pili i ka membrane (Fig. 2A'-E').Hāʻawi ʻia i ka hui ʻana o SPECC1L me ka actin cytoskeleton 18,23 ua manaʻo mākou e pili ana ka SPECC1L me nā junction adhesive adhesive (AJ).
(AF) SPECC1L knockdown (DF) pūnaewele elongate i ka hui kiʻekiʻe (F) i hoʻohālikelike ʻia me ka mālama ʻana i nā pūnaewele U2OS (AC).Hōʻike ʻia ma ʻaneʻi ʻekolu o nā kiko manawa ʻeono (T1, T3, T6) a mākou i koho ai no nā density cell like ʻole.(G) Hōʻike ʻia ʻo Western blot analysis e hoʻokūpaʻa ʻia ka protein SPECC1L ma kahi kiʻekiʻe o ka hui ʻana i hoʻohālikelike ʻia me kahi haʻahaʻa haʻahaʻa o ka hui ʻana i nā cell control.Hōʻike ka blot Western o SPECC1L i ka pūʻulu 120 kDa i manaʻo ʻia a me kahi pūna kaumaha molekala kiʻekiʻe, i hoʻololi ʻia ma hope o ka unuhi ʻana (*).Ua hana ʻia ka loiloi Western blot ma lalo o nā kūlana like no ka hui haʻahaʻa a kiʻekiʻe.Ua lawe ʻia nā kiʻi e hōʻike ana i ka SPECC1L ma ka hui haʻahaʻa a me ke kiʻekiʻe mai ka blot hoʻokahi.Ua wehe ʻia ka blot like a nānā hou ʻia me ka antibody β-actin.(H) ʻAʻohe hoʻololi koʻikoʻi o ka helu RT-PCR nui i nā pae transcript SPECC1L.Hōʻike nā pahu hewa i nā SEM mai nā hoʻokolohua kūʻokoʻa ʻehā.
(AE) Ua koho mākou i ʻeono mau helu manawa (T1-T6) e hōʻike ana i kahi ʻano o nā density cell e hoʻomaʻamaʻa i ka nānā ʻana i ke ʻano o ke kelepona a me nā loli AJ i nā cell U2OS me SPECC1L knockdown (kd).ʻO ka ʻelima mua o kēia mau helu manawa, ʻo ia ka pūnaewele hoʻokahi (T1), 50-70% ka hui ʻana o nā puʻupuʻu liʻiliʻi liʻiliʻi (T2), ka hui ʻana me ka ʻole o ka hoʻololi ʻana i nā pūnaewele kd (T3), ka hoʻololi hou ʻana i nā cell kd (T4), a me nā hoʻololi 24 hola.ma ke ano hope o kd (T5).Ua hoʻopuehu nui ʻia ka protein SPECC1L i loko o ka cytoplasm ma T1 (A), akā ʻike ʻia kona hōʻiliʻili ʻana ma nā palena intercellular ma nā wahi manawa hope (B-E, nā pua).(FJ) β-catenin hōʻike i ka hōʻiliʻili like ma nā palena intercellular e pili ana me ka complex AJ.(A'-E') SPECC1L a me β-catenin e hōʻike ana i ka hoʻopaʻa ʻia ʻana ma nā palena cell ma ke kiʻekiʻe o ka cell density (nā pua).(F'-J') I loko o nā pūnaewele SPECC1L-kd, ʻike ʻia ka staining β-catenin ma ke ʻano maʻamau i ka haʻahaʻa cell density (F'-H'), akā e hoʻonui ʻia ke hoʻololi ʻia ke ʻano o ke kelepona (I', J'; nā pua), e hōʻike ana i ka AJ. ua loli.Nā pā = 10 µm.
A laila hoʻāʻo mākou e hoʻoholo i ka hopena o ka hemahema SPECC1L ma AJ.Ua hoʻohana mākou i kekahi mau māka pili AJ, me nā mea canonical F-actin, myosin IIb, β-catenin, a me E-cadherin24,25,26,27.Ua hoʻonui ʻia nā fiber stress Actin i loko o nā pūnaewele SPECC1L-kd e like me ka mea i wehewehe ʻia ma mua (Fig. 3A, B) 18.ʻO ka Myosin IIb e pili ana me nā filament actin i hōʻike i ka hoʻonui like i nā pūnaewele SPECC1L-kd in vitro (Fig. 3C, D).Hoʻopili ka β-catenin pili i ka AJ i ka cadherin ma ka membrane cell, e hōʻike ana i kahi hiʻohiʻona "honeycomb" maʻamau i nā cubocytes control (Fig. 3E, G).ʻO ka mea e mahalo ai, ma nā kiʻi palahalaha e hoʻohana ana i ka microscopy confocal, β-catenin (Fig. 3E, F) a me E-cadherin (Fig. 3G,H) i hoʻopaʻa ʻia ma ka membrane cell o nā pūnaewele hemahema SPECC1L i hōʻike ʻia i nā hiʻohiʻona koʻikoʻi o ka hoʻonui ʻia.ʻO kēia hoʻonui ʻia ʻana o ka β-catenin pili i ka AJ i loko o nā pūnaewele kd i ʻōlelo nui ʻia ma ka hui ʻana, akā ua ʻike ʻia ma mua o ka hoʻololi ʻana i ke ʻano cell (Fig. 2F-J, F'-J').No ka hoʻoholo ʻana i ke ʻano kino o kēia hoʻopaʻa ʻana AJ lōʻihi, ua nānā mākou i nā palena cell ma ka ʻili apical-basal o SPECC1L-kd U2OS pūnaewele ma o ka lawe ʻana i ka microscopy electron (TEM) (Figure 3I,J).Ma ka hoʻohālikelike ʻana i nā pūnaewele hoʻomalu (Fig. 3I), i loaʻa nā ʻāpana electron dense ʻokoʻa e hōʻike ana i ka AJ (nā pua), ua hōʻike ʻo kd cell (Fig. 3J) i nā ʻāpana nui a pili o ke kiʻekiʻe electron density indicative o AJ ma ka mokulele apicobasal..Eia kekahi, ma nā ʻāpana transverse, ua ʻike mākou i nā ʻāpana membrane cell nui i loko o nā pūnaewele kd (Fig. S1A,B), e wehewehe ana i ke ʻano lōʻihi o ka β-catenin a me ka E-cadherin staining bands (Fig. 3F,H).I ke kākoʻo ʻana i ke kuleana o SPECC1L i nā AJs, ua hui pū ʻia ʻo β-catenin me SPECC1L i nā lysates o nā pūnana U2OS hui pū (Fig. 3K).Me ka immunostaining lōʻihi no nā māka AJ, ua kūlike ka loiloi TEM me kā mākou kuhiakau e hoʻonui ka hemahema SPECC1L i ka AJ apical-basal density a me ka ʻokoʻa.
(AH) Hoʻonui ka F-actin staining ma kd cell ma nā hola 48 post-fusion (T6; A, B).Hoʻololi ʻia ke kala ʻana o ka myosin IIb pili me F-actin (C, D).Ua hoʻonuiʻia keʻano maʻalahi o ka β-catenin a me ka E-cadherin membrane i loko o nā pūnaewele mana (E, G) i nā pūnaewele SPECC1L-kd (F, H).Nā pā = 10 µm.(I-J) Electron micrographs e nānā ana i ka ʻāpana apical-basal intercellular junction.Hōʻike nā pūnaewele mana i nā ʻāpana electron-dense ʻokoʻa e hōʻike ana i nā hui pili (I, nā pua).ʻO ka ʻokoʻa, ʻo ka hui apical-basal holoʻokoʻa i loko o nā pūnaewele SPECC1L-kd ua ʻike ʻia ka electron dense (J, nā pua), e hōʻike ana i ka hoʻonui ʻia a me ka hoʻopuehu ʻana o nā hui adhesive.(K) β-catenin i hui pū ʻia me SPECC1L i nā lysates cell U2OS.Kiʻi ʻia mai kekahi wahi e hōʻike ana i kekahi o nā hoʻokolohua kūʻokoʻa ʻehā.
No ka hoʻomaopopo ʻana i ke kuleana o SPECC1L i ka craniofacial morphogenesis, ua hana mākou i kahi hiʻohiʻona ʻiole deficient Specc1l me ka hoʻohana ʻana i ʻelua mau laina kelepona pahele ES kūʻokoʻa, DTM096 a me RRH048 (BayGenomics, CA), e hōʻike ana i nā transcripts intron 1 a me Specc1l i hopu ʻia ma 15 (Fig. 1) .4A, huahelu S2).Ua hoʻoholo ʻia ka wahi genomic o ka hoʻokomo vector decoy e ka hoʻonohonoho genome holoʻokoʻa a hoʻopaʻa ʻia e PCR (Fig. S2).Ua ʻae ʻia nā hoʻolālā gene trap ʻelua i ka hui ʻana i loko o ka pahu o nā mea hoʻolaha Specc11-lacZ i ka hopu ʻana.No laila, ua hoʻohana ʻia ka ʻōlelo lacZ i hoʻoholo ʻia e X-gal staining ma ke ʻano he hōʻailona o ka ʻōlelo Specc11.Ua hōʻike nā allele ʻelua i nā hiʻohiʻona hōʻike lacZ like, me ka DTM096 gene trap i loko o ka intron 1 e hōʻike ana i ka manaʻo ikaika ma mua o RRH048 i ka intron 15 (ʻaʻole i hōʻike ʻia).Eia nō naʻe, ua hōʻike nui ʻia ʻo Specc1l, me ka ʻōlelo ikaika loa i nā neural folds ma E8.5 (Figure 4B), i ka neural tube a me nā kaʻina hana ma E9.5 a me E10.5 (Figure 4C,D), a i ka hoʻomohala ʻana i nā lālā. ma E10.5 a me nā maka (Figure 4D).Ua hōʻike mua mākou i ka hōʻike SPECC1L i ka pharyngeal arch mua ma E10.5 aia i loko o ka epithelium a me ka mesenchyme18 lalo, e kūlike me ka laina CNCC.No ka ho'āʻoʻana i ka'ōlelo SPECC1L ma CNCC, ua hana mākou i ka E8.5 neural folds (Figure 4E-J) a me E9.5 skull sections (Figure 4K-).Ma E8.5, SPECC1L stained neural folds ikaika loa (Fig. 4E, H), me nā pūnaewele i hoʻopaʻa ʻia me nā kaha NCC (Fig. 4G, J).Ma E9.5, SPECC1L (Fig. 4K, N) ikaika stained migrating CNCC co-stained with AP2A (Fig. 4L, M) or SOX10 (Fig. 4O, P).
(A) Hōʻike hoʻohālike o ka ʻiole Specc11 gene e hōʻike ana i ka hoʻokomo ʻana i ka vector decoy ma ES DTM096 (intron 1) a me RRH048 (intron 15) cell clones.(BD) lacZ staining o heterozygous Specc1lDTM096 embryos e hoike ana i ka hoakaka Specc1l mai E8.5 a i E10.5.NE = neuroectoderm, NF = neural fold, PA1 = mua pharyngeal arch.(EP) SPECC1L immunostaining me NCC markers AP2A a me SOX10 ma E8.5 (NF; EJ) neural folds a me E9.5 (KP) pauku po'o.Ua ʻike nui ʻia ka hoʻopaʻa ʻana o SPECC1L i loko o nā neural folds E8.5 (E, H; arrowheads), me nā cell i kapa ʻia me AP2A (F, G; arrowheads) a me SOX10 (I, J; arrowheads).Ma E9.5, SPECC1L i hoʻopaʻa ʻia i nā CNCC e neʻe ana (K, N; nā pua) i kapa ʻia ʻo AP2A (L, M; nā pua) a me SOX10 (O, P; nā pua).
ʻO ka hele ʻana ma waena o nā ʻiole heterozygous Specc1lDTM096/+ a me Specc1lRRH048/+ e hōʻike ana ʻaʻole i hoʻohui ʻia nā allele gene trap ʻelua a ʻo kēlā hui heterozygotes a me nā homozygotes embryonic no kēlā me kēia gene trap allele he embryonic lethal (Papa S1).Ua hōʻike ʻia nā lakio Mendelian i ka emi ʻana o ke ola o nā heterozygotes i ka hānau ʻana (manaʻo ʻia 1.34 vs. 2.0).Ua ʻike mākou i ka make perinatal haʻahaʻa ma waena o nā heterozygotes, aia kekahi i nā anomalies craniofacial (Fig. S3).Eia nō naʻe, ʻo ka haʻahaʻa haʻahaʻa o kēia perinatal craniofacial phenotypes he mea paʻakikī ke aʻo ʻana i kā lākou mau kumu pathophysiological.No laila, ua nānā mākou i ka embryonic lethal phenotype o homozygous Specc11 mutants.
ʻAʻole i ulu ka hapa nui o ka heterozygous a i ʻole homozygous Specc1lDTM096/RRH048 mutant embryos ma hope o E9.5–10.5 (Fig. 5A–D), a ʻaʻole i pani mua ka paipu neural (Fig. 5B, D) a i kekahi manawa pani ʻia ma hope (ʻaʻole i hōʻike ʻia) ..Hoʻopili ʻia kēia hemahema panina neural tube me ka hapa nui o ka CNCC i kaha ʻia ʻo DLX2 i koe i loko o nā neural folds ma E10.5, e hōʻike ana ʻaʻohe dissection (Figure 5A'-D').No ka hoʻoholo ʻana inā ua hoʻemi ʻia ka nui holoʻokoʻa o CNCC, ua kau mākou i nā laina CNCC me GFP i kā mākou laina pahele gene me Wnt1-Cre a me ROSAmTmG.Hoʻokahe mākou i ka GFP+ NCC a me GFP- (RFP+) non-NCC mai nā embryos holoʻokoʻa.Ma E9.5, ʻaʻole i hoʻololi nui ʻia ka hapa o nā CNCC i hoʻokaʻawale ʻia i ka GFP ma waena o WT a me nā embryos mutant (ʻaʻole i hōʻike ʻia), e hōʻike ana i ka kikoʻī CNCC maʻamau.No laila, ua manaʻo mākou ʻo ke koena o Wnt1-Cre a me DLX2 ka waihoʻoluʻu ʻana i loko o nā ʻāpana neural i hōʻike ʻia (Figure 5B') ma muli o ka hoʻoheheʻe ʻana o CNCC, ma muli paha o ka hoʻonui ʻana a i ʻole ka hoʻopuehu ʻana o nā pūnaewele AJ, e like me ka ʻike ʻia ma nā pūnaewele SPECC1L-kd.Ua hoʻohana mākou i nā māka NCC SOX10, AP2A, a me DLX2 e hōʻoia i ka hele ʻana o CNCC i ka neural fold (Figure 5E-R).Ma E8.5, ua ʻike ʻia ka ʻili ʻana o ka neural fold no nā māka NCC ʻekolu i nā ʻāpana o WT (Fig. 5E, G, I) a me Specc1l mutant (Fig. 5F, H, J).Ma ka E9.5, ʻoiai nā kaha NCC i hoʻopaʻa ʻia i ka neʻe ʻana i ka NCC ma nā ʻāpana WT (Fig. 5M, O, Q), ua ʻike ʻia ke koena NCC staining i nā ʻāpana neural i hōʻike ʻia o Specc1l mutant embryos (Fig. 5N, P, R).Ma muli o ka hōʻailona ʻana o SOX10 a me DLX2 i ka neʻe ʻana i nā CNCC, hōʻike kēia hopena i ka loaʻa ʻana o nā CNCC hemahema SPECC1L i ka kikoʻī post-migratory akā ʻaʻole hiki ke neʻe mai nā neural folds.
ʻO ka hemahema o Specc11 ke alakaʻi nei i ka pani ʻana o ka paipu neural hemahema, ka hoʻopau ʻana i nā pūnaewele neural crest cranial a me nā AJ.
(A, B') E9.5 WT (A) Embryo e lawe ana i ka neʻe ʻana i nā pūnaʻi neural neural crest (CNCC) i hōʻailona ʻia me Wnt1-Cre (A').ʻO ka ʻokoʻa, hōʻike nā embryos mutant Specc11 i nā ʻāpana neural hāmama (B), nā pana pua) a me nā CNCC i neʻe ʻole (B', nā pana pua).(C, D') Nā kiʻi kiʻi mālamalama (C, D') a me ka immunostaining (C', D') o ka CNCC marker DLX2 o E10.5 WT embryos (C, C') a me Specc1l (D, D').Ma WT E10.5 embryos, DLX2-maikaʻi CNCC colonize na gill arches (C', arrows), oiai ma mutants, conspicuous staining mau i loko o ka hamama neural folds (D', arrows) a me ka mua pharyngeal arches (D', nā pua).) me kekahi mau staining (nā pua) e hōʻike ana i ka delamination maikaʻi ʻole a me ka neʻe ʻana o CNCC.ER) Ua hōʻailona ʻia nā ʻāpana o WT a me Specc1l mutant embryos ma nā pae E8.5 (E–L) a me E9.5 (M–R) me nā māka NCC SOX10 (E, F, M, N), AP2A (G, H, O, P ) a me DLX2 (I, J, Q, R).Ma E8.5, ʻike ʻia ka ʻili ʻana o ka NCC ma nā ʻāpana neural fold (NF) a me nā ʻāpana mutant.ʻO ka co-staining o SOX10 a me ka β-catenin ma E8.5 WT (K) a me ka mutant (L) i hōʻike i ka hoʻonui ʻia ʻana o ka β-catenin staining ma nā palena cell i nā neural folds.Ma E9.5, ʻike ʻia ke ʻano ʻāhiu o nā CNCC e neʻe nei (M, O, Q), ʻoiai ma nā mutants, unstratified CNCCs stained open neural folds (N, P, R).(S–Z) In vivo AJ hōʻailona hōʻailona ma nā ʻāpana coronal o WT a me Specc11DTM096/RRH048 embryos me ka mutation E9.5.Hōʻike ʻia kahi ʻāpana ʻāpana ma ke kihi ʻākau i luna.Ma nā ʻāpana o nā ʻiʻo mutant, ua ʻike ʻia ka hoʻonui ʻia ʻana o F-actin (S, T) a me myosin IIb (U, V).E like me nā hopena in vitro ma Fig. 3, i loko o nā embryos mutant, hoʻonui ʻia ka pale ʻana o ka membrane no β-catenin (W, X) a me E-cadherin (Y, Z).(AA-BB) ʻO kahi micrograph electron o kahi ʻāpana o kahi ʻano ʻano ʻāhiu e nānā ana ma waho o ka palena o ke kelepona apical-basal e hōʻike ana i kahi ʻāpana electron-dense ʻokoʻa e hōʻike ana i nā hui pipili (AA, nā pua).Ma ka ʻokoʻa, ma nā ʻāpana o Specc11 mutant embryos (BB, nā pua), ʻo ka hui apicobasal holoʻokoʻa he electron dense, e hōʻike ana i ka hoʻonui ʻana a me ka hoʻopuehu ʻana o nā junction adhesive.
No ka hoʻāʻo ʻana i kā mākou kuhiakau no ka hoʻololi ʻana i ka AJ, ua nānā mākou i ka lepili AJ ma nā ʻāpana neural i hōʻike ʻia o Specc1l mutant embryos (Fig. 5S-Z).Ua ʻike mākou i ka piʻi ʻana o nā fibers stress actin (Fig. 5S, T) a me ka hoʻonui ʻana i ka localization o ka myosin IIB staining ma nā fibers actin (Fig. 5U, V).ʻO ka mea nui, uaʻike mākou i ka hoʻonuiʻia o ka staining o β-catenin (Fig. 5W, X) a me E-cadherin (Fig. 5Y, Z) ma nā palena intercellular.Ua nānā pū mākou i ka β-catenin staining o NCC i nā neural folds o E8.5 embryos (Fig. 5K, L).Ua ʻoi aku ka ikaika o ka staining β-catenin ma Specc1l mutant neural folds (Fig. 5L a me K), e hōʻike ana ua hoʻomaka nā loli AJ.Ma nā micrographs electron o nā ʻāpana iwi poʻo o E9.5 embryos, ua ʻike hou mākou i ka hoʻonui ʻia ʻana o ka diffuse electron-dense staining ma Specc1l mutant embryos i hoʻohālikelike ʻia me WT (Fig. 5AA, BB a me S1E-H).Hoʻohui pū ʻia, kākoʻo kēia mau hopena i kā mākou hopena in vitro i nā SPECC1L-kd U2OS cell a manaʻo ʻia ʻo ka ʻokoʻa AJ aberrant ma mua o ka stratification CNCC i kā mākou embryos mutant.
Hāʻawi ʻia i ka pilina antagonistic i ʻike ʻia ma waena o ka hana AKT a me ka paʻa E-cadherin, 17,28 ua kuhi mākou i ke komo ʻana o ka hōʻailona PI3K-AKT.Eia kekahi, ua ʻike mākou i ka ʻōpū o ka subepidermal i loko o kekahi o kā mākou embryos mutant i pakele i ka lethality (<5%) ma E9.5-10.5 a noho ma kahi o E13.5 (Fig. S3).ʻO nā vesicles subepidermal kahi hōʻailona o ka hōʻemi ʻana i ka hōʻailona PI3K-AKT ma muli o PDGFRα12.ʻO Fantauzzo et al.(2014) hōʻike i ka hoʻohaunaele o PDGFRα-based PI3K activation ma PdgfraPI3K/PI3K mutant embryos ka hopena i nā vesicles subepidermal, neural tube defects, and cleft palate phenotypes.ʻOiaʻiʻo, ua hoʻemi ʻia nā pae o ka pan-AKT a me ka Ser473-AKT phosphorylated ikaika i loko o ka vivo i loko o Specc1l mutant tissues i ka hopu ʻana i ka embryonic E9.5 (Fig. 6A-D).ʻO ka emi ʻana o nā pae o ka phosphorylated Ser473-AKT ma muli o ka emi ʻana o nā pae o ka pan-AKT in vivo (FIG. 6E) a me in vitro (FIG. 6F).Ua ʻike ʻia ka emi ʻana o ka in vitro i ka wā i hui pū ʻia ai nā pūnaewele U2OS me nā loli i ke ʻano o ke kelepona a me ka nui AJ (Figure 6D).No laila, hōʻike kā mākou ʻikepili ʻo SPECC1L kahi mea hoʻoponopono maikaʻi o ka PI3K-AKT hōʻailona i ka craniofacial morphogenesis.
(A–E) E8.5 (A,B) a me E9.5 (C,D) ʻāpana poʻo a i ʻole E9.5 lysates mai Specc1l mutant embryos (E) e hōʻike ana i nā pae o ka phosphorylated ikaika S473-AKT a me ka pan-AKT Protein hoʻemi. , hoʻohālikelike i ka mana WT.Ua hana ʻia ka blotting Western ma nā lysates ʻano hihiu a me nā lysates mutant ma lalo o nā kūlana like.Ua lawe ʻia nā kiʻi i hōʻike ʻia no SPECC1L mai hoʻokahi blot.Ua wehe ʻia ka blot like a nānā hou ʻia me nā anti-pan-ACT a me β-actin antibodies.ʻO nā pae Pan-AKT ma E8.5 neural folds (A, B) a me nā pae o ka phosphorylated S473-AKT i nā ʻāpana iwi poʻo E9.5 ua hoʻemi nui ʻia.(F) Ua hoʻemi like ʻia nā pae Pan-AKT i nā lysates o nā pūnaewele SPECC1L-kd U2OS i ʻohi ʻia ma ka hui kiʻekiʻe.Hōʻike nā pahu hewa i nā SEM mai ʻekolu mau helu kūʻokoʻa Western blot.(GJ) ʻO nā ʻāpana o WT embryos ma E9.5 i hoʻopaʻa ʻia me KI67 a me ka caspase 3 i hoʻokaʻawale ʻia, e hōʻike ana i ka hoʻonui ʻana o ke kelepona (G, G ') a me ka hana apoptotic liʻiliʻi (H, H ').Hōʻike ka specc11 mutant embryos e like me ka hoʻonui ʻia ʻana o ka cell cell proliferation (I), akā ua hoʻonui nui ʻia ka helu o nā cell e loaʻa ana i ka apoptosis (J).
A laila ua nānā mākou i nā hōʻailona o ka proliferation a me ka apoptosis.ʻAʻole mākou i ʻike i kekahi ʻokoʻa i ka hoʻonui ʻia ʻana o nā embryos E9.5 (Fig. 6E, G i hoʻohālikelike ʻia me I) me kahi helu hoʻonui o 82.5% no nā mutants WT a me 86.5% no nā mutants Specc1l i ana ʻia e KI67 staining (p <0.56, Fisher's. hōʻoia pololei).Pēlā nō, ʻaʻole mākou i ʻike i kekahi ʻokoʻa o ka apoptosis i ana ʻia e ka ʻili ʻana no ka caspase 3 i hoʻopaʻa ʻia i nā neural folds ma E8.5 a hiki i ka hopu ʻana o ka embryo (ʻaʻole i hōʻike ʻia) (ʻaʻole i hōʻike ʻia).ʻO ka hoʻohālikelike, ua hoʻonui nui ʻia ka apoptosis i nā embryos mutant E9.5 a pau (Fig. 6F, H a me J).ʻO kēia piʻi nui o ka apoptosis e kūlike me ka hōʻemi ʻana i ka hōʻailona PI3K-AKT a me ka lethality embryonic mua29,30,31.
A laila, e hōʻoia i kahi kumu kumu no ka hōʻailona PI3K-AKT i nā hoʻololi AJ i kā mākou kd cell, ua hoʻololi mākou i ke ala i ka mana a me nā cell kd (Figure 7A-F).Ua hoʻohana mākou ma ke ʻano he hōʻailona i ke ʻano o ka hoʻololi ʻana o ke ʻano o ka cell i ʻike ʻia i loko o nā pūnaewele SPECC1L-kd hui pū ʻia, a mākou i helu ai me ka hoʻohana ʻana i ka ratio o ka anana lōʻihi loa (lōʻihi) i ke ana kū pololei (ākea).Manaʻo ʻia ka lakio o 1 no nā pūnaewele puni a i ʻole cuboidal (Figure 7G).Ma waho aʻe o ke ʻano o ka cell, ua hōʻoia pū mākou i ka hopena ma ka AJ e ka staining β-catenin (Fig. 7A'-F').Ua lawa ka pale ʻana i ke ala PI3K-AKT me ka wortmannin no ka hoʻololi ʻana i ke ʻano o ka cell i loko o nā keʻena mana (Figure 7A, C) a me AJ (Figure 7A ').PI3K-AKT activator SC-79 'aʻole i hoʻopili i ke kinona pūnaewele (FIG. 7A, E) a i ʻole AJ hoʻonui (FIG. 7A') i loko o nā keena hoʻomalu.Ma nā pūnaewele SPECC1L-kd, hoʻopau hou i ke ala PI3K-AKT i hoʻonui i ka apoptosis (Fig. 7B, D) a me ka hoʻonui nui ʻana i ka staining β-catenin (Fig. 7B '), e like me kā mākou in vivo heavy mutants.ʻO ka mea nui, ʻo ka hoʻoulu ʻana o ke ala PI3K-AKT i hoʻomaikaʻi nui i ke ʻano o ka cell (Figure 7B, F) a me AJ phenotypes (Figure 7B").Ua helu ʻia nā hoʻololi ʻana i ke ʻano o ke kelepona ma ke ʻano he lākiō pōʻai o ke kelepona (CCR) a hoʻohālikelike ʻia no ke ʻano nui e like me ka mea i wehewehe ʻia ma luna (FIG. 7G).ʻOiaʻiʻo, i loko o nā pūnaewele mana (Fig. 7G, CCR = 1.56), ua lawa ka mālamaʻana i ka wortmannin e hoʻololi nui i keʻano o ke kelepona (Fig. 7G, CCR = 3.61, p <2.4 × 10-9) i keʻano like me ka mea iʻikeʻia. ma SPECC1L.-kd pūnaewele (Fig. 7G, CCR = 3.46).ʻO ka mālamaʻana i ka Wortmannin o nā pūnaewele SPECC1L-kd (Fig. 7G, CCR = 3.60, negligible)ʻaʻole iʻoi aku ka nui ma mua o nā pūnaewele kd i mālamaʻoleʻia (Fig. 7G, CCR = 3.46, negligible) aiʻole nā ​​pūnaewele mana wortmannin (Fig 7G)., CCR = 3.46, negligible) pili hou i ka elongation cell (7G, CCR = 3.61, negligible).ʻO ka mea nui loa, ua hoʻihoʻi ʻo SC-79 AKT activator i ka phenotype elongated o SPECC1L-kd cell (Fig. 7G, CCR = 1.74, p <6.2 × 10-12).Hōʻoia kēia mau hopena i ka hoʻoponopono ʻana o SPECC1L i ka hōʻailona PI3K-AKT a manaʻo ʻo ka emi ʻana o ka SPECC1L e pili ana i ka pili o ka cell, aʻo ka emi ʻana o ka ikaika e alakaʻi i ka apoptosis (Fig. 8).
(A–F') Manaʻo (A, C, E) a me SPECC1L-kd (B, D, F) i mālama ʻia me ka PI3K-AKT alahele inhibitor wortmannin (C, D) a i ʻole SC-79 activator (E, F) Lapaʻau. .He kuboidal (A) nā pūnae hoʻomalu i hoʻoponopono ʻole ʻia me ka β-cat cellular staining maʻamau (A'), ʻoiai ua hoʻolōʻihi ʻia nā pūnaewele kd (B) me ka hoʻonui ʻia o ka β-cat staining (B').Ma hope o ka hoʻopau ʻana i ke ala PI3K-AKT, ua hoʻolōʻihi ʻia nā cell control (C) me ka hoʻonui ʻana i ka β-cat (C'), ʻoiai ua hoʻomaka nā cell kd e hana i ka apoptosis (D), e like me kā mākou embryos mutated a hōʻike i ka β-cat i hoʻonui ʻia.hoʻopaʻa ʻia (D').Ma hope o ka ho'āla ʻia ʻana o ke ala PI3K-AKT, noho mau nā cell control cuboidal (E) a loaʻa ka β-cat (E') maʻamau, ʻoiai ʻo kd cell i hōʻike i ka hoʻomaikaʻi nui ʻana i ke ʻano o ka cell (F) a me ka β-cat (F') staining, e hōʻike ana. (G) Ua helu ʻia ke degere o ka hoʻololi ʻana o ke ʻano o ke kelepona ma (AF) me ka hoʻohana ʻana i ka lākiō pōʻai cell (CCR) o ka ana lōʻihi loa (lōʻihi) a me ke ana kū pololei (ākea) me ka hoʻohana ʻana i ka polokalamu MetaMorph.ʻO nā pūnaewele SPECC1L-kd i mālamaʻoleʻia (NT) (CCR = 3.46) iʻoi aku ka lōʻihi ma mua o nā pūnaewele mana (CCR = 1.56, p <6.1 × 10-13).Ua lawa ka pale ʻana o Wort i ke ala PI3K-AKT i loko o nā keʻena hoʻomalu e hana i kahi elongation like i ke ʻano cell (CCR=3.61, p<2.4 × 10-9).Pēlā nō, hoʻihoʻi hou ʻia ka hoʻonui ʻana o ka cell cell AKT e SC-79 i nā cell SPECC1L-kd e hoʻokele i nā pae (CCR = 1.74, p <6.2 × 10-12).ʻO ka mālamaʻana i ka Wortmannin o nā pūnaewele SPECC1L-kd ka hopena i ka hoʻonuiʻana i ka apoptosis akāʻaʻole i hoʻonui houʻia i ka hoʻololiʻana i keʻano o ke kelepona (CCR=3.60) i hoʻohālikelikeʻia me ka kd i mālamaʻoleʻia (CCR=3.46, ns) a iʻole nā ​​pūnaewele mana i mālamaʻia i ka wortmannin (3.61) iʻikeʻia ma .ns = ʻaʻole pili.+/- Hōʻike ʻia nā ana SEM no 50 mau kelepona.Ua helu ʻia nā ʻokoʻa ʻokoʻa me ka hoʻohana ʻana i ka ho'āʻo-t a Student.
(A) Hōʻike hoʻolālā o ka inhibition a me ka hoʻoulu ʻana o ke ala PI3K-AKT e hopena i nā loli AJ a me ka hoʻopakele, kēlā me kēia.(B) Hoʻohālike i manaʻo ʻia no ka hoʻopaʻa ʻana i ka protein AKT e SPECC1L.
Pono nā CNCC premigratory i ka AJ lysis e hoʻokaʻawale mai nā neural fold neuroepithelial cells1,15,32.ʻO ka hoʻonui ʻia ʻana o nā ʻāpana AJ a me ka nalo ʻana o ka puʻupuʻu asymmetric apical-basal AJ i loko o nā pūnaewele hemahema SPECC1L i loko o ka vitro a i loko o ka vivo, i hui pū ʻia me ka pili kino o SPECC1L i β-catenin, e manaʻo e hana ʻo SPECC1L i ka mālama pono ʻana i ka paʻa kūloko o AJ. nā ʻiʻo hui.actin cytoskeleton.ʻO ka huiʻana o SPECC1L me ka actin cytoskeleton a me ka β-catenin a me ka hoʻonuiʻana i ka helu o nā filaments actin condensed i ka nele o SPECC1L e kūlike me ka piʻiʻana o ka nui o AJ.ʻO kekahi mea ʻē aʻe, ʻo ka hoʻonui ʻana i ka nui o nā fibers actin i nā cell SPECC1L-deficient e alakaʻi i kahi hoʻololi i ka ʻāʻī intercellular.No ka mea, pili ke koʻikoʻi kelepona i ka AJ 33 dynamics, hiki i nā hoʻololi uila ke hopena i ka AJ 34 diffuse.No laila, e pili ana nā hoʻololi i nā papa CNCC.
Hōʻike ʻia ʻo Wnt1 i nā ʻāpana neural mua e hoʻāla ai i nā pūnaewele neural crest.No laila, ʻo Wnt1-cre laina laina e hōʻailona ana ma mua o ka neʻe ʻana iā NCC35.Eia nō naʻe, hōʻailona ʻo Wnt1 i nā clones dorsal brain tissue i loaʻa mai i nā neural folds mua 35,36, ʻo ia ka mea ʻaʻole CNCC kā mākou staining o E9.5 mutants no Wnt1 markers ma nā neural folds.Ua hōʻoia ko mākou ʻili maikaʻi no nā māka NCC AP2A a me SOX10 ua loaʻa maoli i nā ʻāpana neural o Specc11 mutant embryos i loko o CNCC.Eia kekahi, ʻoiai ʻo AP2A a me SOX10 nā mea hōʻailona o ka neʻe mua ʻana o ka NCC, ua hōʻike ʻia ka staining maikaʻi i kēia mau cell post-migratory CNCC ʻaʻole hiki ke stratified e E9.5.
Hōʻike kā mākou ʻikepili e hoʻoponopono ʻia ka hoʻoponopono molecular o AJ e SPECC1L e ka hōʻailona PI3K-AKT.Hoʻemi ʻia ka hōʻailona AKT i loko o nā pūnaewele hemahema SPECC1L a me nā ʻiʻo.Nā ʻike a Fantauzzo et al.kākoʻo i kahi kuleana kūpono no ka hōʻailona PI3K-AKT i ka morphogenesis craniofacial.(2014) hōʻike i ka nele o ka hoʻāla ʻana o ka hōʻailona PI3K-AKT e pili ana i ka PDGFRα e alakaʻi i kahi phenotype palate cleft.Hōʻike pū mākou ua lawa ka pale ʻana i ke ala PI3K-AKT e hoʻololi i ka AJ a me ke ʻano o ke kelepona i nā ʻāpana U2OS.Kūlike me kā mākou ʻike, ʻo Kain et al.Ua hōʻike ʻo 37 i ka hoʻohaʻahaʻa ʻana o ka subunit PI3K α110 i loko o nā cell endothelial e hopena i ka hoʻonui like ʻana o ka pericellular β-catenin staining, i kapa ʻia ʻo ka hoʻonui ʻana i ka "index connectivity".Eia nō naʻe, i loko o nā pūnaewele endothelial nona nā filament actin i hoʻonohonoho nui ʻia, hoʻopau ʻia ke ala PI3K-AKT i ke ʻano o ke ʻano o ke kelepona.I ka hoʻokaʻawale ʻana, ua hōʻike ʻo SPECC1L-kd U2OS i kahi ʻano kelepona elongated.ʻO kēia ʻokoʻa paha ke ʻano cell type.ʻOiai ʻo ka hoʻopau ʻana i ka hōʻailona PI3K-AKT e pili mau ana i ka cytoskeleton actin, ua hoʻoholo ʻia ka hopena ma ke ʻano o ke kelepona e nā loli i ka ʻāʻī i hoʻololi ʻia e nā loli i ka nui a me ka hoʻonohonoho ʻana o nā fibers actin waena.Ma nā pūnaewele U2OS, ua hoʻohana wale mākou i nā hoʻololi ʻano kelepona ma ke ʻano he hōʻailona o ka hoʻololi a me ka hoʻihoʻi hou ʻana o SPECC1L-deficient AJ.I ka hopena, manaʻo mākou ʻo ka pale ʻana i ke ala AKT i ka hemahema SPECC1L e hoʻonui i ka paʻa AJ a hoʻemi i ka delamination ma CNCC.
ʻO ka mea e mahalo ai, ua hoʻemi ʻia nā pae pan-AKT i loko o ka vitro a i loko o ka vivo i hoʻohui ʻia i nā pae phosphorylated 473-AKT i ka nele o SPECC1L, e hōʻike ana i ka hoʻoponopono ʻana o ka hōʻailona PI3K-AKT ma ke kiʻekiʻe o ke kūpaʻa o ka protein AKT a i ʻole ka huli ʻana.ʻO nā genes SPECC1L a me MID1, pili pū me Opitz/GBBB syndrome, hoʻopili i nā protein e hoʻopaʻa i nā microtubules 18,22.ʻAʻole maopopo loa ke ʻano hana a SPECC1L a me MID1 i hoʻopaʻa ʻia i ka microtubule stabilization.Ma ka hihia o SPECC1L, ua hoʻonui ʻia kēia hoʻopaʻa ʻana i ka acetylation o kahi ʻāpana o nā microtubules 18.Hiki ke hoʻohana ʻo SPECC1L i kahi ʻano hana like e hoʻopaʻa i nā protein ʻē aʻe e like me AKT.Ua hōʻike ʻia ʻo ka acetylation o nā koena lysine i ka protein AKT e alakaʻi i ka emi ʻana o ka localization membrane a me ka phosphorylation38.Eia kekahi, pono ka ubiquitination o ke kaulahao K63 ma ke koena lysine like ma AKT no kona localization a me ka hoʻoulu ʻana39,40.Ma waena o nā mea e pili ana me nā protein SPECC1L i ʻike ʻia i loko o nā ʻano kiʻekiʻe throughput yeast ʻelua-hybrid pale, ʻehā - CCDC841, ECM2942, APC a me UBE2I43 - ua hoʻopili ʻia i ka huli ʻana o ka protein a i ʻole ke kūpaʻa ma o ka ubiquitination a i ʻole sumoylation.Hiki ke komo ʻo SPECC1L i ka hoʻololi ʻana ma hope o ka unuhi ʻana o nā koena lysine AKT, e pili ana i ka kūpaʻa AKT.Eia nō naʻe, ʻo ka hana koʻikoʻi o SPECC1L i ka localization a me ka paʻa o ka protein AKT e hoʻomau ʻia e elucidated.
ʻO nā hemahema koʻikoʻi ma ka hōʻike SPECC1L i vivo i hoʻonui ʻia i ka hoʻonui ʻia ʻana o ka marker AJ a me ka overlay CNCC hemahema, a me ka hoʻonui ʻana i ka apoptosis a me ka lethality embryonic mua.Ua hōʻike mua nā hōʻike e pili ana nā mutants ʻiole me nā kiʻekiʻe o ka apoptosis me nā neural tube defects 44,45,46,47 a me craniofacial defects48.Ua manaʻo ʻia ʻo ka make nui o ka cell i loko o nā neural folds a i ʻole pharyngeal arches hiki ke hopena i ka lawa ʻole o nā cell i koi ʻia no ka neʻe morphogenetic kūpono 48,49,50.Ma ka ʻokoʻa, ʻo kā mākou SPECC1L nā laina kelepona hemahema me ka hōʻike SPECC1L hōʻemi haʻahaʻa i hōʻike ʻia i nā loli AJ wale nō me ka ʻole o ka hōʻike o ka piʻi ʻana o ka make cell.Eia nō naʻe, ʻo ka hoʻopaʻa ʻana i nā kemika o ke ala PI3K-AKT i kēia mau keena Kd i hoʻonui i ka apoptosis.No laila, ʻo ka hoʻohaʻahaʻa haʻahaʻa o ka ʻōlelo SPECC1L a i ʻole ka hana e hōʻoia i ke ola o ka cell.Ua kūlike kēia me ka ʻike ʻana i nā embryos mutant Specc11 i pakele i ka hopu ʻia ma st.E9.5—ma muli paha o ka emi ʻana o ka hana hopu gene—hiki iā ia ke pani i ko lākou mau ʻōpū neural a hoʻōki ma hope o ka hoʻomohala ʻana, pinepine me nā hemahema craniofacial (Fig. S3).E like pū me kēia, ʻo ka loaʻa ʻana o nā embryos Specc1l heterozygous me nā mea ʻino o ka craniofacial-ma muli paha o ka hoʻonui ʻana i ka hana hopu gene-a me ka loaʻa ʻana i ka iʻa zebra i kahi o nā orthologues SPECC1L ʻelua (specc1lb) ke kumu o nā phenotypes embryonic hope loa, me ka nalowale o ka nā āwae lalo a me nā māwae lua51.No laila, ʻo ka heterozygous SPECC1L nalowale-o-hana hoʻololi i ʻike ʻia i loko o nā maʻi kanaka hiki ke hoʻopilikia liʻiliʻi i ka hana SPECC1L i ka wā o ka craniofacial morphogenesis, lawa e wehewehe i ko lākou mau ʻāpana orofacial.Hiki i ka SPECC1L ke hoʻoponopono i nā pilina intercellular ke hana i ka palatogenesis a me ka hui ʻana o nā arches pharyngeal.ʻO nā haʻawina hou aʻe o ka hana SPECC1L e kōkua i ka hoʻomaʻamaʻa ʻana i ka hana o nā pilina intercellular pōkole ma CNCC i ka wā o ka pani ʻana o ka neural tube i ka neuroepithelial cell motility a me ka craniofacial morphogenesis.
Ua wehewehe mua ʻia ka mana o ka osteosarcoma U2OS a me nā cell SPECC1L-kd (Saadi et al., 2011).Ua hōʻike mua ʻia nā antibody kūʻē iā SPECC1L (Saadi et al., 2011).Anti-β-catenin antibodies (rabbit; 1: 1000; Santa Cruz, Dallas, TX) (mouse; 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 , Kaleponi), 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) a me β-actin (1:2500; Sigma-Aldrich, St. Louis, MO ) ua hoʻohana ʻia e like me ka wehewehe ʻana..Ua hoʻopaʻa ʻia nā filament Actin me Acti-stain rhodamine phalloidin (Cytoskeleton, Denver, Colorado).
Ua hoʻomaʻamaʻa ʻia nā pūnana mana U2OS a me SPECC1L-kd i ka DMEM glucose kiʻekiʻe maʻamau i hoʻohui ʻia me 10% fetal bovine serum (Life Technologies, Carlsbad, CA).No nā hoʻololi AJ, 2 x 105 mau pūnaewele i hua ʻia ma ke aniani i mālama ʻia me 0.1% porcine gelatin (Sigma-Aldrich, St. Louis, MO) a nānā ʻia no nā loli i ke ʻano o ke kelepona.Ua hōʻiliʻili ʻia nā kelepona i nā manawa manawa like ʻole: 4 mau hola ma hope o ke kanu ʻana (t = 1), 24 mau hola ma hope o ke kanu ʻana (t = 2), hui pū me ka ʻole o ka hoʻololi ʻana i ke ʻano cell (t = 3), hoʻololi i ke ʻano cell (t = 4) , 24 mau hola ma hope o ka hoʻololi ʻana o ke ʻano o ke kelepona (t = 5) a me 48 mau hola ma hope o ka hoʻololi ʻana o ke ʻano o ka cell (t = 6) (Fig. 1, 2, 3).No ka hoʻololi ʻana i ke ala PI3K-AKT, ua hoʻomaʻamaʻa ʻia nā cell i nā manaʻo i hōʻike ʻia me ka PI3K-AKT inhibitor wortmannin (TOCRIS Biosciences, Minneapolis, Minnesota) a i ʻole SC-79 activator (TOCRIS Biosciences, Minneapolis Adams, Minnesota).Ua hoʻololi ʻia ke ʻano me nā kemika i kēlā me kēia lā.
Ua hoʻopaʻa ʻia nā hoʻopaʻa paʻi paʻi ma ke kiʻi ma ka mana ola a me nā pūnaewele KD ma lalo o nā kūlana moʻomeheu maʻamau, a ua hōʻiliʻili ʻia nā kiʻi ʻokoʻa i kēlā me kēia 10 minuke no 7 lā.Ua kiʻi ʻia nā kiʻi me ka hoʻohana ʻana i ka microscope inverted Leica DM IRB i hoʻopaʻa ʻia me ka pae mechanical a me kahi pahuhopu 10 × N-PLAN i hoʻopili ʻia i kahi kāmela QImaging Retiga-SRV.I ka wā o ke kiʻi kiʻi, mālama ʻia nā moʻomeheu cell ma 37 ° C i kahi ea haʻahaʻa me 5% CO2.
Ua hoʻohana ʻia ʻelua mau gene trap ES cell line DTM096 a me RRH048 mai ka Regional Mutant Mouse Resource Center (UC Davis, CA) no ka hana ʻana i nā laina ʻiole hemahema Specc11, i koho ʻia ʻo Specc1lgtDTM096 a me Specc1lgtRRH046.ʻO ka pōkole, ua hoʻokomo ʻia nā cell 129 / REJ ES i nā blastocysts C57BL6.Ua hānai ʻia nā ʻiole kāne chimeric me nā ʻiole wahine C57BL6 e ʻike ai i nā keiki me ka waihoʻoluʻu ʻaʻahu agouti.Ua hoʻohana ʻia ka loaʻa ʻana o nā mea hoʻokomo hoʻokomo ʻana i nā mea hoʻokomo i nā heterozygotes.Ua mālama ʻia nā ʻiole ma kahi ʻano huikau o 129/REJ;C57BL6.Ua hōʻoia ʻia ka wahi o ka wahi hoʻokomo o ka genetic trap vector e RT-PCR, genome sequencing, a me genetic complementation (Supplementary Figure 1).No ka ʻimi ʻana i ka ʻiole CNCC o nā ʻiole Specc1lGT heterozygous pālua, ROSAmTmG (#007576) a me Wnt1-Cre (#003829) ʻiole (Jackson Laboratory, Bar Harbor, ME) ua hele ʻia e hana i ka ROSAmTmG a me Wnt1-Cre allele i Specc1osl muce.Ua hana ʻia nā hoʻokolohua āpau i nā ʻiole e like me nā protocols i ʻae ʻia e ka Institutional Animal Care and Use Committee o ke Kulanui o Kansas Medical Center.
Hoʻopaʻa ʻia nā embryos i (1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2, 0.02% NP-40, 5 mM EGTA) no 60 min ma ka lumi wela.Ma hope o ka hoʻopaʻa ʻana i ka solution staining X-gal (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, 1 mg/ml X-gal) Ua hana ʻia ka hoʻomohala ʻana i ka stain ma 37 ° C .°C i loko o 1-6 hola.Ua hoʻopaʻa ʻia nā Embryos ma 4% PFA a ʻike ʻia.
No ka Western blotting, ua lysed nā pūnaewele i loko o ka passive lysis buffer (Promega, Fitchburg, WI) i hoʻohui ʻia me ka hui ʻana o ka HALT protease inhibitors (Sigma-Aldrich, St. Louis, MO).Ua hana ʻia nā Lysates ma 12% polyacrylamide Mini-PROTEAN TGX i hoʻomākaukau ʻia i nā gels (Bio-Rad, Hercules, CA) a hoʻoneʻe ʻia i nā membranes Immobilon PVDF (EMD Millipore, Billerica, MA).Ua ālai ʻia nā membranes i ka waiū 5% ma PBS i loaʻa ka 0.1% Tween.Hoʻokomo ʻia nā antibody i ka pō ma 4 ° C a i ʻole hoʻokahi hola ma ka lumi wela.Ua hoʻohana ʻia ʻo Femto SuperSignal West ECL reagent (Thermo Scientific, Waltham, MA) no ka hana hōʻailona.No ka immunostaining, ua hoʻopaʻa ʻia nā embryos i ka pō ma 4% PFA/PBS a cryopreserved.Hoʻopaʻa ʻia nā cryosections tissue i loko o ka PBS i loaʻa ka 1% maʻamau kao serum (Thermo Scientific, Waltham, MA) a me 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) a laila incubated ma 4 ° C i loko o kahi incubator i ka wā o ka pō.me ka anti-antibody a me ka fluorescent secondary antibody (1:1000) no 1 hola ma 4°C.Ua hoʻokomo ʻia nā ʻāpana i hoʻopaʻa ʻia i loko o ka medium gula ProLong (Thermo Scientific, Waltham MA) a ua loaʻa nā kiʻi pālahalaha me ka hoʻohana ʻana i kahi microscope confocal Leica TCS SPE.Ua hana ʻia kēlā me kēia immunostaining e like me ʻekolu mau hoʻokolohua kūʻokoʻa ma nā cirossections o ʻelua mau embryos mutant.Hōʻike ʻia kahi hoʻokolohua hōʻike.
Hoʻopiliʻia nā pūnaewele i loko o ka RIPA buffer i hoʻololiʻia (20 mM Tris-HCl, pH 8.0, 1% NP-40, 130 mM NaCl, 10% glycerol, 2 mM EDTA, a me HALT protease inhibitor (Sigma-Aldrich, St. Louis, MO) ʻO ka pōkole, ua hoʻomaʻemaʻe mua ʻia nā lysates me ka protein G magnetic beads (Life Technologies, Carlsbad, CA) a laila hoʻomoʻi ʻia i ka pō ma 4 ° C. me ka anti-SPECC1L a i ʻole IgG protein G protein beads i hoʻohana ʻia e unuhi i ka SPECC1L a ua hana ʻia ka blotting Western me ka hoʻohana ʻana i ka anti-SPECC1L a i ʻole IgG protein G. -β-catenin antibody i wehewehe ʻia ma luna ʻO nā hoʻokolohua co-IP i hōʻike ʻia he hōʻailona o nā hoʻokolohua kūʻokoʻa ʻehā.
Ua hāʻawi ʻia nā cell moʻomeheu paʻa a i ʻole nā ​​ʻiʻo embryonic ʻiole i ke kikowaena microscopy electron ma ke Kulanui o Kansas Medical Center.ʻO ka pōkole, ua hoʻokomo ʻia nā laʻana i loko o EMbed 812 resin (Electron Microscopy Sciences, Fort Washington, PA), polymerized i ka pō ma 60 ° C, a ʻāpana ʻia ma 80 nm me ka hoʻohana ʻana i kahi ultramicrotome Leica UC7 i lako me ka pahi daimana.Ua ʻike ʻia nā ʻāpana me ka JEOL JEM-1400 transmission electron microscope i lako me kahi pū 100 kV Lab6.
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