小鼠胚胎发育过程中Brachyury对Wnt信号通路的作用研究
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摘要
Brachyury对调控小鼠胚胎发育起着至关重要的作用,缺乏Brachyury蛋白的小鼠胚胎不能正常发育。Wnt信号通路在小鼠胚胎发育中可控制胚胎的轴向发育等重要的生理过程,Brachyury可能通过与Wnt信号通路的相互作用导致短尾表型的产生。为了揭示Brachyury与Wnt信号通路相互作用关系,本研究制作了Brachyury突变小鼠,通过提取不同时期的胚胎并提取总RNA,经反转录进行qPCR检测Brachyury与Wnt信号通路相关成分的表达关系。结果显示,Brachyury、Axin2、Dkk1及Wnt3a的表达在突变胚胎和野生胚胎中的表达有显著差异。因此,Brachyury作为转录因子对上述Wnt信号通路成分的表达有调节作用,它们形成一个调控网络调控小鼠胚胎的正常发育。本研究为小鼠胚胎发育期间Brachyury (T)的功能作用提供了理论基础。
Brachyury plays a crucial role in the regulation of mouse embryonic development, lacking of Brachyury in mouse embryos can cause the deviant development. Wnt signaling pathway can control the important physiological processes such as the axial elongation of embryos in mouse embryonic development, and Brachyury may cause short-tail phenotype through the interaction with Wnt signaling pathway. In order to reveal the interaction between Brachyury and Wnt signaling pathway, we built Brachyury mutant mice. By extracting embryos from different periods and extracting total RNA, qPCR was used to detect the relative expression relation between Brachyury and Wnt signaling components. The results showed that Brachyury, Axin2, Dkk1, and Wnt3 a expression in mutant embryos and wild embryos were significantly different. In conclusion, Brachyury, as a transcription factor, regulates the expression of Wnt signaling pathway components mentioned above, and they form a regulatory network regulating the normal development of mouse embryos. Thus, this experiment provide a theoretical basis for the functional role of Brachyury(T) during mouse embryo development.
Brachyury plays a crucial role in the regulation of mouse embryonic development, lacking of Brachyury in mouse embryos can cause the deviant development. Wnt signaling pathway can control the important physiological processes such as the axial elongation of embryos in mouse embryonic development, and Brachyury may cause short-tail phenotype through the interaction with Wnt signaling pathway. In order to reveal the interaction between Brachyury and Wnt signaling pathway, we built Brachyury mutant mice. By extracting embryos from different periods and extracting total RNA, qPCR was used to detect the relative expression relation between Brachyury and Wnt signaling components. The results showed that Brachyury, Axin2, Dkk1, and Wnt3 a expression in mutant embryos and wild embryos were significantly different. In conclusion, Brachyury, as a transcription factor, regulates the expression of Wnt signaling pathway components mentioned above, and they form a regulatory network regulating the normal development of mouse embryos. Thus, this experiment provide a theoretical basis for the functional role of Brachyury(T) during mouse embryo development.
引文
Ar nold S.J.,Stappert J.,Bauer A.,Kispert A.,Herrmann B.G.,and Kemler R.,2000,Brachyury is a target gene of the wnt/β-catenin signaling pathway,Mech.Develop.,91(1):249-258
Clevers H.,2006,Wnt/β-catenin signaling in development and disease,Cell,127(3):469-480
Colas A.R.,McKeithan W.L.,Cunningham T.J.,Bushway P.J.,Garmire L.X.,Duester G.,Subramaniam S.,and Mercola M.,2012,Whole-genome microrna screening identifies let-7and mir-18 as regulators of germ layer formation during early embryogenesis,Genes Dev.,26(23):2567-2579
Evans A.L.,Faial T.,Gilchrist M.J.,Down T.,Vallier L.,Pedersen,R.A.,Wardle F.C.,and Smith J.C.,2012,Genomic targets of brachyury(T)in differentiating mouse embryonic stem cells,PLoS One,7(3):33346
Galceran J.,Hsu S.C.,and Grosschedl R.,2001,Rescue of a wnt mutation by an activated form of lef-1:regulation of maintenance but not initiation of brachyury expression,PNAS,98(15):8668-8673
Guder C.,Philipp I.,Lengfeld T.,Watanabe H.,Hobmayer B.,and Holstein T.W.,2006,The wnt code:cnidarians signal the way,Oncogene,25(57):7450-7460
Jambhekar N.A.,Rekhi B.,Thorat K.,Dikshit R.,Agrawal M.,and Puri A.,2010,Revisiting chordoma with brachyury,a“new age”marker:analysis of a validation study on 51 cases,Arch.Pathol.Lab.Med.,134(8):1181-1187
Kavka A.I.,and Green J.B.A.,1997,Tales of tails:brachyury and the t-box genes,BBA,1333(2):73-84
Lako M.,Lindsay S.,Lincoln J.,Cairns P.M.,Armstrong L.,and Hole N.,2001,Characterisation of wnt gene expression during the differentiation of murine embryonic stem cells in vitro:role of wnt3 in enhancing haematopoietic differentiation,Mech.Develop.,103(1-2):49-59
Liu F.,and Tang X.D.,2015,Research progress of T-box transcription factor Brachyury in tumor,Shengming Kexue(Chinese Bulletin of Life Sciences),27(2):135-142(刘飞,唐旭东,2015,T-box转录因子Brachyury在肿瘤相关研究中的进展,生命科学,27(2):135-142)
Nentwich O.,Dingwell K.S.,Nordheim A.,and Smith J.C.,2009,Downstream of FGF during mesoderm formation in Xenopus:the roles of elk-1 and egr-1,Dev.Biol.,336(2):313-326
Pennimpede T.,Proske J.,Konig A.,Vidigal J.A.,Morkel M.,Bramsen J.B.,Herrmann B.G.,and Wittler L.,2012,In vivo knockdown of brachyury results in skeletal defects and urorectal malformations resembling caudal regression syndrome,Dev.Biol.,372(1):55-67
Shah S.R.,David J.M.,Tippens N.D.,Mohyeldin A.,Martinez-Gutierrez J.C.,Ganaha S.,Schiapparelli P.,Hamilton D.H.,Palena C.,Levchenko A,and Qui觡ones-Hinojosa A.,2017,Br achyury-YAP regulatory axis drives stemness and growth in cancer,Cell Rep.,21(2):495-507
Shao Y.X.,Chen B.,Zhang C.X.,Chen C.B.,Xue Z.F.,and Li H.D.,2005,Generating a tail short mouse using ENU mutagenesis and mapping of the mutant gene,Dongwuxue Zazhi(Chinese Journal of Zoology),40(1):8-13(邵义祥,陈兵,张成香,陈春波,薛整风,李厚达,2005,ENU诱导获得一种短尾小鼠及其突变基因的初步定位,动物学杂志,40(1):8-13)
Showell C.,Binder O.,and Conlon F.L.,2004,T-box genes in early embryogenesis,Dev.Dyn.,229(1):201-218
Stott D.,Kispert A.,and Herrmann B.G.,1993,Rescue of the tail defect of brachyury mice,Gene.Dev.,7(2):197-203
Tada M.,and Smith J.C.,2001,T-targets:clues to understanding the functions of t-box proteins,Dev.Growth Differ.,43(1):1-11
Tortelote G.G.,Hernández-Hernández J.M.,Quaresma A.J.C.,Nickerson J.A.,Imbalzano A.N.,and Rivera-Pérez J.A.,2013,Wnt3 function in the epiblast is required for the maintenance but not the initiation of gastrulation in mice,Dev.Biol.,374(1):164-173
Wilkinson D.G.,Bhatt S.,and Herrmann B.G.,1990,Expression pattern of the mouse T gene and its role in mesoderm formation,Nature,343(6259):657-659
Wu B.,Shao Y.,Chen B.,Liu C.,Xue Z.F.,Wu P.,and Li H.,2010,Identification of a novel mouse brachyury(T)allele causing a short tail mutation in mice,Cell Biochem.Biophys.,58(3):129-135
Yamaguchi T.P.,Takada S.,Yoshikawa Y.,Wu N.,and Mcmahon A.P.,1999,T(brachyury)is a direct target of wnt3a during paraxial mesoderm specification,Gene.Dev.,13(24):3185-3190
Zhang F.T.,Zhou S.B.,Zhang C.,Chen H.,Cai T.C.,Zhuang W.J.,2014,Cloning and characterization of the Arogenate dehydrogenase gene from Arachis hypogaca L.,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),12(3):478-484(张福婷,周双彪,张冲,陈华,蔡铁城,庄伟建,2014,花生预苯胺酸脱氢酶基因AhADH的克隆及分子特性分析,分子植物育种,12(3):478-484)
Clevers H.,2006,Wnt/β-catenin signaling in development and disease,Cell,127(3):469-480
Colas A.R.,McKeithan W.L.,Cunningham T.J.,Bushway P.J.,Garmire L.X.,Duester G.,Subramaniam S.,and Mercola M.,2012,Whole-genome microrna screening identifies let-7and mir-18 as regulators of germ layer formation during early embryogenesis,Genes Dev.,26(23):2567-2579
Evans A.L.,Faial T.,Gilchrist M.J.,Down T.,Vallier L.,Pedersen,R.A.,Wardle F.C.,and Smith J.C.,2012,Genomic targets of brachyury(T)in differentiating mouse embryonic stem cells,PLoS One,7(3):33346
Galceran J.,Hsu S.C.,and Grosschedl R.,2001,Rescue of a wnt mutation by an activated form of lef-1:regulation of maintenance but not initiation of brachyury expression,PNAS,98(15):8668-8673
Guder C.,Philipp I.,Lengfeld T.,Watanabe H.,Hobmayer B.,and Holstein T.W.,2006,The wnt code:cnidarians signal the way,Oncogene,25(57):7450-7460
Jambhekar N.A.,Rekhi B.,Thorat K.,Dikshit R.,Agrawal M.,and Puri A.,2010,Revisiting chordoma with brachyury,a“new age”marker:analysis of a validation study on 51 cases,Arch.Pathol.Lab.Med.,134(8):1181-1187
Kavka A.I.,and Green J.B.A.,1997,Tales of tails:brachyury and the t-box genes,BBA,1333(2):73-84
Lako M.,Lindsay S.,Lincoln J.,Cairns P.M.,Armstrong L.,and Hole N.,2001,Characterisation of wnt gene expression during the differentiation of murine embryonic stem cells in vitro:role of wnt3 in enhancing haematopoietic differentiation,Mech.Develop.,103(1-2):49-59
Liu F.,and Tang X.D.,2015,Research progress of T-box transcription factor Brachyury in tumor,Shengming Kexue(Chinese Bulletin of Life Sciences),27(2):135-142(刘飞,唐旭东,2015,T-box转录因子Brachyury在肿瘤相关研究中的进展,生命科学,27(2):135-142)
Nentwich O.,Dingwell K.S.,Nordheim A.,and Smith J.C.,2009,Downstream of FGF during mesoderm formation in Xenopus:the roles of elk-1 and egr-1,Dev.Biol.,336(2):313-326
Pennimpede T.,Proske J.,Konig A.,Vidigal J.A.,Morkel M.,Bramsen J.B.,Herrmann B.G.,and Wittler L.,2012,In vivo knockdown of brachyury results in skeletal defects and urorectal malformations resembling caudal regression syndrome,Dev.Biol.,372(1):55-67
Shah S.R.,David J.M.,Tippens N.D.,Mohyeldin A.,Martinez-Gutierrez J.C.,Ganaha S.,Schiapparelli P.,Hamilton D.H.,Palena C.,Levchenko A,and Qui觡ones-Hinojosa A.,2017,Br achyury-YAP regulatory axis drives stemness and growth in cancer,Cell Rep.,21(2):495-507
Shao Y.X.,Chen B.,Zhang C.X.,Chen C.B.,Xue Z.F.,and Li H.D.,2005,Generating a tail short mouse using ENU mutagenesis and mapping of the mutant gene,Dongwuxue Zazhi(Chinese Journal of Zoology),40(1):8-13(邵义祥,陈兵,张成香,陈春波,薛整风,李厚达,2005,ENU诱导获得一种短尾小鼠及其突变基因的初步定位,动物学杂志,40(1):8-13)
Showell C.,Binder O.,and Conlon F.L.,2004,T-box genes in early embryogenesis,Dev.Dyn.,229(1):201-218
Stott D.,Kispert A.,and Herrmann B.G.,1993,Rescue of the tail defect of brachyury mice,Gene.Dev.,7(2):197-203
Tada M.,and Smith J.C.,2001,T-targets:clues to understanding the functions of t-box proteins,Dev.Growth Differ.,43(1):1-11
Tortelote G.G.,Hernández-Hernández J.M.,Quaresma A.J.C.,Nickerson J.A.,Imbalzano A.N.,and Rivera-Pérez J.A.,2013,Wnt3 function in the epiblast is required for the maintenance but not the initiation of gastrulation in mice,Dev.Biol.,374(1):164-173
Wilkinson D.G.,Bhatt S.,and Herrmann B.G.,1990,Expression pattern of the mouse T gene and its role in mesoderm formation,Nature,343(6259):657-659
Wu B.,Shao Y.,Chen B.,Liu C.,Xue Z.F.,Wu P.,and Li H.,2010,Identification of a novel mouse brachyury(T)allele causing a short tail mutation in mice,Cell Biochem.Biophys.,58(3):129-135
Yamaguchi T.P.,Takada S.,Yoshikawa Y.,Wu N.,and Mcmahon A.P.,1999,T(brachyury)is a direct target of wnt3a during paraxial mesoderm specification,Gene.Dev.,13(24):3185-3190
Zhang F.T.,Zhou S.B.,Zhang C.,Chen H.,Cai T.C.,Zhuang W.J.,2014,Cloning and characterization of the Arogenate dehydrogenase gene from Arachis hypogaca L.,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),12(3):478-484(张福婷,周双彪,张冲,陈华,蔡铁城,庄伟建,2014,花生预苯胺酸脱氢酶基因AhADH的克隆及分子特性分析,分子植物育种,12(3):478-484)