Sorting Nexin7调节斑马鱼肝脏发育的机制研究
摘要
肝脏是脊椎动物体内最大的内脏器官，同时也是与代谢相关的最重要的器官，而与肝脏相关的各种疾病一直以来都是对人类健康最重大的威胁之一。阐明肝脏的发育过程及其行使生理功能的分子机制，一直都是生物医学研究的重要领域。分选蛋白（Sorting Nexin，SNX）是一类有着共同的PX（Phox）结构域的基因家族，它们多分布在细胞内膜系统的各个位置，广泛参与细胞内蛋白质的运输和分选过程并起着重要的调节作用。在人类的基因组中，迄今已鉴定并命名了33个SNX家族成员，但目前的研究报道仅仅涉及到其中的一小部分。本论文所论述的研究是第一次用斑马鱼动物模型从胚胎整体和器官发育水平对SNX潜在的生理功能进行的研究，我们发现SNX7是一个在肝脏特异表达的SNX家族成员，它的异常表达能够特异性地抑制斑马鱼胚胎肝脏的发育而对其他内脏器官和整体胚胎的发育则没有明显的影响，进一步的研究发现，由morpholino介导的SNX表达的敲降并不影响早期内胚层前体细胞向肝祖细胞的转化，但接下来肝祖细胞进一步的生长增殖和成熟过程则受到了显著的抑制,在分子水平，我们发现一些与凋亡过程相关的关键因子，如Caspase8，bax，p53及其相关的靶标基因的表达水平在SNX7敲降的斑马鱼胚胎中都有着显著地升高，为此我们得出结论：在斑马鱼胚胎早期肝脏发育过程中，SNX7通过某条抑制凋亡的信号通路对肝祖细胞的存活起着不可替代地调控作用。紧接着，在体外试验中，用siRNA转染特异性敲降SNX7表达的方法在HeLa细胞系中能够有效地诱导出凋亡，并最终找到了SNX7在诱导凋亡过程中重要的下游靶分子：c‐FLIPs，体内挽救实验确凿无疑地证明了c‐FLIPs就是联系SNX7和凋亡的关键分子。在本论文阐述的研究中，我们结合体内外的多种技术手段，分析了SNX7对于包括肝脏在内的内胚层各器官发育的影响，重点研究了SNX7调节肝祖细胞生长/分化/凋亡的机制，并对SNX7调节肝脏发育所涉及的上、下游信号通路进行了初步探究，试图在将来从分子、细胞以及器官和胚胎整体水平阐明SNX7的作用机制，从而建立一个包括SNX7在内的调节肝脏发育的信号通路网络。
Liver is the largest and also the most important metabolism‐related internal organ in vertebrate, liver correlative diseases always a substantial threaten of health. To elucidate the liver’s developmental process and how does this organ exercise its physiological fuction, always been a important and intriguing research area of biological and medical science. Sorting Nexin（SNX） is a gene family which shares the Phox（PX） domain, most SNX genes locate on the endomembrane system and play important role in the transport and sorting process of proteins. There are33SNXs which have been discovered and named in human genome, but we still know very little about the physiologic fuction of this conservative and apparently important family. In this paper, we use a model organism:Zebrafish, to study the potential fuction of SNXs from individual and organ level, throμgh our research, we discovered SNX7specifically expressed in the liver cell, low‐expressing level of SNX7causes severe repress liver development and almost with no influence to other internal organ. Further research revealed that knockdown of SNX7by morpholino‐microinjection does’t affect the process that original precursor cells differentiate into hepatoblast, but SNX7do severely inhibit the following growing/proliferating/maturing procedure. At the molecular level, we found that several pro‐apoptotic molecules including Caspase8, bax, p53and its target genes were up‐regulated in SNX7morphants. We concluded that an anti‐apoptotic signaling pathway involving SNX7was indispensible for the survival of hepatoblasts during embryonic liver development. In the in Vitro part,we induced apoptosis in HeLa celline by transfection of SNX7siRNAs,finally,we found the crucial molecular connect SNX7and apoptosis was c‐FLIPS,we then proved it by rescue test.In this research,we combine multiple research methods, both in vivo and in vitro, to explore how SNX7regulate the development of endodermal organs, especially how SNX7regulates the growing/proliferating/maturing process of hepatoblast, we want to find the upstream and downstream signaling pathways of SNX7‐regulating‐liver‐development, our goal is build a network of signaling pathways of SNX7regulating process, to elucidate the mechanism of such process from different researching leve:molecular,cellular,organic and even embryoic and individual.
Liver is the largest and also the most important metabolism‐related internal organ in vertebrate, liver correlative diseases always a substantial threaten of health. To elucidate the liver’s developmental process and how does this organ exercise its physiological fuction, always been a important and intriguing research area of biological and medical science. Sorting Nexin（SNX） is a gene family which shares the Phox（PX） domain, most SNX genes locate on the endomembrane system and play important role in the transport and sorting process of proteins. There are33SNXs which have been discovered and named in human genome, but we still know very little about the physiologic fuction of this conservative and apparently important family. In this paper, we use a model organism:Zebrafish, to study the potential fuction of SNXs from individual and organ level, throμgh our research, we discovered SNX7specifically expressed in the liver cell, low‐expressing level of SNX7causes severe repress liver development and almost with no influence to other internal organ. Further research revealed that knockdown of SNX7by morpholino‐microinjection does’t affect the process that original precursor cells differentiate into hepatoblast, but SNX7do severely inhibit the following growing/proliferating/maturing procedure. At the molecular level, we found that several pro‐apoptotic molecules including Caspase8, bax, p53and its target genes were up‐regulated in SNX7morphants. We concluded that an anti‐apoptotic signaling pathway involving SNX7was indispensible for the survival of hepatoblasts during embryonic liver development. In the in Vitro part,we induced apoptosis in HeLa celline by transfection of SNX7siRNAs,finally,we found the crucial molecular connect SNX7and apoptosis was c‐FLIPS,we then proved it by rescue test.In this research,we combine multiple research methods, both in vivo and in vitro, to explore how SNX7regulate the development of endodermal organs, especially how SNX7regulates the growing/proliferating/maturing process of hepatoblast, we want to find the upstream and downstream signaling pathways of SNX7‐regulating‐liver‐development, our goal is build a network of signaling pathways of SNX7regulating process, to elucidate the mechanism of such process from different researching leve:molecular,cellular,organic and even embryoic and individual.
引文
[1]Chu, J. and K.C. Sadler, New school in liver development: lessons from zebrafish.Hepatology,2009.50(5):1656‐63.
[2]Tao, T. and J. Peng, Liver development in zebrafish (Danio rerio). J GenetGenomics,2009.36(6):325‐34.
[3]Ober, E.A., et al., Mesodermal Wnt2b signalling positively regulates liverspecification. Nature,2006.442(7103):688‐91.
[4]Shin, D., et al., Bmp and Fgf signaling are essential for liver specification inzebrafish. Development,2007.134(11):2041‐50.
[5]Chung, W.S., C.H. Shin, and D.Y. Stainier, Bmp2signaling regulates the hepaticversus pancreatic fate decision. Dev Cell,2008.15(5):738‐48.
[6]Negishi, T., et al., Retinoic acid signaling positively regulates liver specificationby inducing wnt2bb gene expression in medaka. Hepatology,2009.
[7]Field, H.A., et al., Formation of the digestive system in zebrafish. I. Livermorphogenesis. Dev Biol,2003.253(2):279‐90.
[8]Goessling, W., et al., APC mutant zebrafish uncover a changing temporalrequirement for wnt signaling in liver development. Dev Biol,2008.320(1):161‐74.
[9]Chen, J., et al., Loss of function of def selectively up‐regulates Delta113p53expression to arrest expansion growth of digestive organs in zebrafish. GenesDev,2005.19(23):2900‐11.
[10]Chen, J., et al., p53isoform delta113p53is a p53target gene that antagonizesp53apoptotic activity via BclxL activation in zebrafish. Genes Dev,2009.23(3):278‐90.
[11]Sadler, K.C., et al., Liver growth in the embryo and during liver regeneration inzebrafish requires the cell cycle regulator, uhrf1. Proc Natl Acad Sci U S A,2007.104(5):1570‐5.
[12]Farooq, M., et al., Histone deacetylase3(hdac3) is specifically required forliver development in zebrafish. Dev Biol,2008.317(1):336‐53.
[13]Noel, E.S., et al., Organ‐specific requirements for Hdac1in liver and pancreasformation. Dev Biol,2008.322(2):237‐50.
[14]Rai, K., et al., Dnmt2functions in the cytoplasm to promote liver, brain, andretina development in zebrafish. Genes Dev,2007.21(3):261‐6.
[15]Matthews, R.P., et al., The zebrafish onecut gene hnf‐6functions in anevolutionarily conserved genetic pathway that regulates vertebrate biliarydevelopment. Dev Biol,2004.274(2):245‐59.
[16]Matthews, R.P., K. Lorent, and M. Pack, Transcription factor onecut3regulatesintrahepatic biliary development in zebrafish. Dev Dyn,2008.237(1):124‐31.
[17]Lorent, K., et al., Inhibition of Jagged‐mediated Notch signaling disruptszebrafish biliary development and generates multi‐organ defects compatiblewith an Alagille syndrome phenocopy. Development,2004.131(22):5753‐66.
[18]Dong, P.D., et al., Fgf10regulates hepatopancreatic ductal system patterningand differentiation. Nat Genet,2007.39(3):397‐402.
[19]Carlton, J., et al., Sorting nexins‐‐unifying trends and new perspectives. Traffic,2005.6(2):75‐82.
[20]Kurten, R.C., D.L. Cadena, and G.N. Gill, Enhanced degradation of EGFreceptors by a sorting nexin, SNX1. Science,1996.272(5264):1008‐10.
[21]Seet, L.F. and W. Hong, The Phox (PX) domain proteins and membrane traffic.Biochim Biophys Acta,2006.1761(8):878‐96.
[22]Cullen, P.J., Endosomal sorting and signalling: an emerging role for sortingnexins. Nat Rev Mol Cell Biol,2008.9(7):574‐82.
[23]Schwarz, D.G., et al., Genetic analysis of sorting nexins1and2reveals aredundant and essential function in mice. Mol Biol Cell,2002.13(10):3588‐600.
[24]Zheng, B., et al., Essential role of RGS‐PX1/sorting nexin13in mousedevelopment and regulation of endocytosis dynamics. Proc Natl Acad Sci U S A,2006.103(45):16776‐81.
[25]Nasevicius, A. and S.C. Ekker, Effective targeted gene 'knockdown' in zebrafish.Nat Genet,2000.26(2):216‐20.
[26]Yoo, K.W., et al., Snx5, as a Mind bomb‐binding protein, is expressed inhematopoietic and endothelial precursor cells in zebrafish. FEBS Lett,2006.580(18):4409‐16.
[27]Li‐Fong Seet, Wan jin Hong, The Phox(PX) domain proteins and membranetraffic. Biochim Biophys Acta.2006Aμg,1761(8):878‐96.
[28]Jan R.T.van Weering, Paul Verkade, Peter J. Cullen. SNX‐BAR proteins inphosphoinositide‐mediated, tubular‐based endosomal sorting. Semin Cell DevBiol.2010Jun;21(4):371‐80.
[29]R.A.Weinberg著；詹启敏，刘芝华主译.《癌生物学》：科学出版社，2009年4月.
[30]Watson AJ.Apoptosis and colorectal cancer. Gut.2004Nov;53(11):1701‐9.
[31]Nika N Danial,Stanley J Korsmeyer.Cell death: critical control points.Cell.2004;116(2):205‐19.
[32]Hershko, A. Lessons from the discovery of the ubiquitin system. Trends.Biochem. Sci.1996;21:445–449.
[33]Coux, O., Tanaka, K, and Goldberg, A. L. Annu. Rev. Biochem.1996;65:801‐847.
[34]Rechsteiner M. In Ubiquitin and the Biology of the Cell, ed. D Finley, JM Peters.New York/London: Plenum.1997.
[35]Cenci, G., Rawson, R.B., Belloni, G., Castrillon, D.H., Tudor, M., Petrucci, R.,Goldberg, M.L., Wasserman, S.A., Gatti, M.. UbcD1, a Drosophila ubiquitin‐conjugating enzyme required for proper telomere behavior. Genes Dev.1997,11(7):863‐‐875.
[36]Muralidhar MG, Thomas JB. The Drosophila bendless gene encodes a neuralprotein related to ubiquitin‐conjugating enzymes. Neuron.1993;11:253‐66.
[37]Roest HP, van Klaveren J, de Wit J, van Gurp CG, Koken MH, Vermey M, vanRoijen JH, Hoogerbrugge JW, Vreeburg JT, Baarends WM, Bootsma D,Grootegoed JA, Hoeijmakers JH. Inactivation of the HR6B ubiquitin‐conjugatingDNA repair enzyme in mice causes male sterility associated with chromatinmodification. Cell.1996Sep6;86(5):799‐810.
[38]Harbers K, Müller U, Grams A, Li E, Jaenisch R, Franz T.Provirus integration intoa gene encoding a ubiquitin‐conjugating enzyme results in a placental defectand embryonic lethality.Proc Natl Acad Sci U S A.1996Oct29;93(22):12412‐7.
[39]Huibregtse JM, Scheffner M, Beaudenon S, Howley PM. A family of proteinsstructurally and functionally related to the E6‐AP ubiquitin‐protein ligase.ProcNatl Acad Sci U S A.1995Mar28;92(7):2563‐7.
[40]Sudakin V, Ganoth D, Dahan A, Heller H, Hershko J, Luca FC, Ruderman JV,Hershko A. The cyclosome, a large complex containing cyclin‐selectiveubiquitin ligase activity, targets cyclins for destruction at the end ofmitosis.Mol Biol Cell.1995Feb;6(2):185‐97.
[41]King RW, Deshaies RJ, Peters JM, Kirschner MW. How proteolysis drives thecell cycle. Science.1996Dec6;274(5293):1652‐9.
[42]Michael H. Glickman,Aaron Ciechanover. The Ubiquitin‐ProteasomeProteolytic Pathway: Destruction for the Sake of Construction. Physiol Rev.2002;82:373–428.
[43]F Ujimuro M, Takada H, S Aeki Y, T Oh‐E A, T Anaka K, Andyokosawa H.Growth‐dependent change of the26S proteasome in budding yeast. BiochemBiophys Res Commun.1998,251:818‐823.
[44]G Roll M, Ditzel L, L Oewe J, S Tock D, Bochtler M, Bartunik Hd, HUBER R.Structure of20S proteasome from yeast at a2.4Angstrom resolution.Nature.1997,386:463–471.
[45]Dengxw, D Ubiel W, Wein, H Ofmannk, M Undt K, C Olicellij, K Ato Jy, NAumann M, Segal D, S Eeger M, Carra, Glickman Mh,Chamovitzda. Unifiednomenclature for the COP9signalosome: an essential regulator ofdevelopment. Trends Genet Sci.2000,16:202–203.
[46]G Lickman Mh, R Ubin Dm, C Ouxo, Wefes I, P Feifer G, Cjeka Z, Baumeister W,Fried Va, Finley D. A subcomplex of the proteasome regulatory particlerequired for ubiquitin‐conjugate degradation and related to theCOP9/Signalosome and eIF3. Cell.1998,94:615–623.
[47]K Apelarib, B Ech‐Otschir D, Hegerl R, S Chade R, Dumdey R, Dubiel W. Electronmicroscopy and subunit‐subunit interaction studies reveal a first architectureof COP9signalosome. J Mol Biol.2000,300:1169–1178.
[48]Maupin‐F Urlow Ja, W Ilson Hl, Kaczowka Sj, Andou Ms. Proteasomes in thearchaea: from structure to function. Front Biosci.2000,5: D837–D865.
[49]De Mot R, Nagyi, W Alz J, Baumeister W. Proteasomes and otherself‐compartmentalizing proteases in prokaryotes. Trends Microbiol.1999,7:88–92.
[50]Mayer,A.N.and Fishman, M.C. Nilperos encodesa conserved RNA recognitionmotif protein required for morphogenesis and cyto differentiation of digestiveorgans in zebrafish. Development2003.130:3917–3928
[51]Workman, J.L. and Kingston, R.E. Alteration of nucleosome structure as amechanism of transcriptional regulation. Annu Rev Biochem.1998.67:545‐79.
[52]Hansen, J.C. et al. Structure and function of the core histone N‐termini: morethan meets the eye. Biochemistry,1998.37,17637‐41.
[53]Strahl, B.D. and Allis, C.D. The language of covalent histone modifications.Nature,2000.403,41‐5.
[54]Cheung, P. et al. Signaling to chromatin throμgh histone modifications.Cell,2000.103,263‐71.
[55]Bernstein, B.E. and Schreiber, S.L. Global approaches to chromatin. ChemBiol,2002.9,1167‐73.
[56]Jaskelioff, M. and Peterson, C.L. Chromatin and transcription: histonescontinue to make their marks. Nat Cell Biol,2003.5,395‐9.
[57]Thorne, A.W. et al. Patterns of histone acetylation. Eur J Biochem,1990.193,701‐13.
[58]Hendzel, M.J. et al. Mitosis‐specific phosphorylation of histone H3initiatesprimarily within pericentromeric heterochromatin during G2and spreads in anordered fashion coincident with mitotic chromosome condensation.Chromosoma,1997.106,348‐60.
[59]Goto, H. et al. Identification of a novel phosphorylation site on histone H3coupled with mitotic chromosome condensation. J Biol Chem,1999.274,25543‐9.
[60]Preuss, U. et al. Novel mitosis‐specific phosphorylation of histone H3at Thr11mediated by Dlk/ZIP kinase.Nucleic Acids,Res.2003.31,878‐85.
[61]Dai, J. et al. The kinase haspin is required for mitotic histone H3Thr3phosphorylation and normal metaphase chromosome alignment. GenesDev,2005.19,472‐88.
[62]JenniferL.Shepard. et al. A zebrafish bmyb mutation causes genome instabilityand increased cancer susceptibility. Proc Natl Acad Sci U S A.2005. Sep13;102(37):
[63]Curado S, Stainier DY, Anderson RM. Nitroreductase‐mediated cell/tissueablation in zebrafish: a spatially and temporally controlled ablation methodwith applications in developmental and regeneration studies. Nat Protoc.2008;3(6):948‐54.
[64]James Summerton. Morpholino Antisense Oligos:Applications inBiopharmaceutical Research. Innovations in Pharmaceutical Technologyissue.2005.17.
[65]Doyon Y, McCammon J M, Miller J C,FarajiF, NgoC, Katibah G E,etal. Heritabletargeted gene disruption in zebrafish using designed zinc‐fingernucleases.NatBiotech.2008;26:702‐708.
[66]Foley J E, Yeh JR, Maeder ML,Reyon D,Sander JD, Peterson RT,etal. Rapidmutation of endogenous zebrafish genes using zinc finger nucleases made byOligomerized Pool ENgineering(OPEN). PLoSONE.2009;4: e4348.
[67]Field HA, Ober EA, Roeser T, Stainier DY. Formation of the digestive system inzebrafish. I.Liver morphogenesis. DevBiol.2003;253:279‐290.
[68]Farooq M, Sulochana KN, Pan X, To J, Sheng D, Gong Z,etal. Histonedeacetylase3(hdac3) is specifically required for liver development in zebrafish.Dev Biol.2008;317:336‐353.
[69]Korzh S, Pan X, Garcia‐Lecea M, Winata CL,Wohland T, Korzh V,etal.Requirement of vasculogenesis and blood circulation in latest ages oflivergrowth in zebrafish.BMC Dev Biol.2008;8:84.
[70]Sadler, K.C., et al., A genetic screen in zebrafish identifies the mutants vps18,nf2and foie gras as models of liver disease. Development,2005.132(15):3561‐72.
[71]Chen, J., et al., Loss of function of def selectively up‐regulates Delta113p53expression to arrest expansion growth of digestive organs in zebrafish. GenesDev,2005.19(23):2900‐11.
[72]Ober EA,Field HA,Stainier DY. From endoderm formation to liver and pancreasdevelopment in zebrafish.Mech Dev.2003,120:5‐18.
[73]Shin D,Shin CH,Tucker J,Ober EA,Rentzsch F,Poss KD,etal.Bmp and Fgf signalingare essential for liver specification in zebrafish.Development2007,134:2041‐2050.
[74]Sun Z, Hopkins N. vhnf1, the MODY5and familial GCKD‐associated gene,regulates regional specification of the zebrafish gut, pronephros, andhindbrain.Genes Dev.2001,15:3217‐3229.
[75]Holtzinger A, Evans T. Gata4regulates the formation of multipleorgans.Development.2005,132:4005‐4014.
[76]Reiter JF, Alexander J, Rodaway A, Yelon D, Patient R, Holder N,etal.Gata5isrequired for the development of the heart and endoderm in zebrafish.GenesDev.1999;13:2983‐2995.
[77]Reiter JF, Kikuchi Y, Stainier DY. Multiple roles for Gata5in zebrafish endodermformation. Development.2001;128:125‐135.
[78]Deutsch G, Jung J, Zheng M, Lora J, Zaret KS. A bipotential precursorpopulation for pancreas and liver within the embryoniccendoderm.Development.2001;128:871‐881.
[79]Serls AE, Doherty S, Parvatiyar P, Wells JM, Deutsch GH. Different Thresholdsof fibroblast growth factors pattern the ventral foregut intoLiver andlung.Development.2005;132:35‐47.
[80]Monga SP, Monga HK, Tan X, Mule K, Pediaditakis P, MichalopoulosGK.Beta‐catenin antisense studies in embryonic liver cultures: role inproliferation, apoptosis, and lineage specification.Gastroenterology.2003;124:202‐216.
[81] Suksaweang S, Lin CM, Jiang TX, Hμghes MW, Widelitz RB, Chuong CM.Morphogenesis of chicken liver:identification of localized growth zones andthe role of beta‐catenin/Wnt in size regulation. DevBiol.2004;266:109‐122.
[82]Lorent K, Yeo SY, Oda T, Chandrasekharappa S, Chitnis A, Matthews RP, etal.Inhibition of Jagged‐mediated Notch signaling disrupts zebrafish biliarydevelopment and generates multi‐organ defects compatible with an Alagillesyndrome phenocopy. Development.2004;131:5753‐5766.
[83]Oda T, Elkahloun AG, Pike BL,Okajima K, Krantz ID, GeninA, etal.Mutations inthe human Jagged1gene are responsible for Alagille syndrome.NatGenet.1997;16:235‐242.
[84]Li L, Krantz ID, Deng Y, Genin A, Banta AB, Collins CC, etal. Alagille syndrome iscaused by mutations in human Jagged1, which encodes a ligand for Notch1.NatGenet.1997;16:243‐251.
[85]Parviz F, Matullo C, Garrison WD, Savatski L, Adamson JW, Ning G,etal.Hepatocyte nuclear factor4alpha controls the development of a hepaticepithelium and liver morphogenesis. Nat Genet.2003;34:292‐296.
[86]Chiba H, Gotoh T, Kojima T, Satohisa S, Kikuchi K, Osanai M,etal.Hepatocytenuclear factor(HNF)‐4alpha triggers formation of functional tight junctionsand establishment of polarized epithelial morphology in F9embryonalcarcinoma cells. Exp Cell Res.2003;286:288‐297.
[87]Liedtke, C., et al., The human Caspase‐8promoter sustains basal activitythroμgh SP1and ETS‐like transcription factors and can be up‐regulated by ap53‐dependent mechanism. J Biol Chem,2003.278(30):27593‐604.
[88]Liedtke, C., et al., Interferon‐alpha enhances TRAIL‐mediated apoptosis byup‐regulating Caspase‐8transcription in human hepatoma cells. J Hepatol,2006.44(2):342‐9.
[89]Kurita, S., et al., DNMT1and DNMT3b silencing sensitizes human hepatomacells to TRAIL‐mediated apoptosis via up‐regulation of TRAIL‐R2/DR5andCaspase‐8. Cancer Sci,2010.101(6):1431‐1439.
[90]Yamaguchi, Y.,et al., Adenovirus‐mediated transfection of Caspase‐8sensitizeshepatocellular carcinoma to TRAIL‐and chemotherapeutic agent‐induced celldeath. Biochim Biophys Acta,2006,1763(8):844‐53.
[91]http://www.genecards.org/cgi‐bin/carddisp.pl?gene=SNX7&search=snx7
[92]Western印迹法‐Western Blotting‐实验方法技术‐生物在线.http://www.bioon.com.cn/protocol/showarticle.asp?newsid=6946
[2]Tao, T. and J. Peng, Liver development in zebrafish (Danio rerio). J GenetGenomics,2009.36(6):325‐34.
[3]Ober, E.A., et al., Mesodermal Wnt2b signalling positively regulates liverspecification. Nature,2006.442(7103):688‐91.
[4]Shin, D., et al., Bmp and Fgf signaling are essential for liver specification inzebrafish. Development,2007.134(11):2041‐50.
[5]Chung, W.S., C.H. Shin, and D.Y. Stainier, Bmp2signaling regulates the hepaticversus pancreatic fate decision. Dev Cell,2008.15(5):738‐48.
[6]Negishi, T., et al., Retinoic acid signaling positively regulates liver specificationby inducing wnt2bb gene expression in medaka. Hepatology,2009.
[7]Field, H.A., et al., Formation of the digestive system in zebrafish. I. Livermorphogenesis. Dev Biol,2003.253(2):279‐90.
[8]Goessling, W., et al., APC mutant zebrafish uncover a changing temporalrequirement for wnt signaling in liver development. Dev Biol,2008.320(1):161‐74.
[9]Chen, J., et al., Loss of function of def selectively up‐regulates Delta113p53expression to arrest expansion growth of digestive organs in zebrafish. GenesDev,2005.19(23):2900‐11.
[10]Chen, J., et al., p53isoform delta113p53is a p53target gene that antagonizesp53apoptotic activity via BclxL activation in zebrafish. Genes Dev,2009.23(3):278‐90.
[11]Sadler, K.C., et al., Liver growth in the embryo and during liver regeneration inzebrafish requires the cell cycle regulator, uhrf1. Proc Natl Acad Sci U S A,2007.104(5):1570‐5.
[12]Farooq, M., et al., Histone deacetylase3(hdac3) is specifically required forliver development in zebrafish. Dev Biol,2008.317(1):336‐53.
[13]Noel, E.S., et al., Organ‐specific requirements for Hdac1in liver and pancreasformation. Dev Biol,2008.322(2):237‐50.
[14]Rai, K., et al., Dnmt2functions in the cytoplasm to promote liver, brain, andretina development in zebrafish. Genes Dev,2007.21(3):261‐6.
[15]Matthews, R.P., et al., The zebrafish onecut gene hnf‐6functions in anevolutionarily conserved genetic pathway that regulates vertebrate biliarydevelopment. Dev Biol,2004.274(2):245‐59.
[16]Matthews, R.P., K. Lorent, and M. Pack, Transcription factor onecut3regulatesintrahepatic biliary development in zebrafish. Dev Dyn,2008.237(1):124‐31.
[17]Lorent, K., et al., Inhibition of Jagged‐mediated Notch signaling disruptszebrafish biliary development and generates multi‐organ defects compatiblewith an Alagille syndrome phenocopy. Development,2004.131(22):5753‐66.
[18]Dong, P.D., et al., Fgf10regulates hepatopancreatic ductal system patterningand differentiation. Nat Genet,2007.39(3):397‐402.
[19]Carlton, J., et al., Sorting nexins‐‐unifying trends and new perspectives. Traffic,2005.6(2):75‐82.
[20]Kurten, R.C., D.L. Cadena, and G.N. Gill, Enhanced degradation of EGFreceptors by a sorting nexin, SNX1. Science,1996.272(5264):1008‐10.
[21]Seet, L.F. and W. Hong, The Phox (PX) domain proteins and membrane traffic.Biochim Biophys Acta,2006.1761(8):878‐96.
[22]Cullen, P.J., Endosomal sorting and signalling: an emerging role for sortingnexins. Nat Rev Mol Cell Biol,2008.9(7):574‐82.
[23]Schwarz, D.G., et al., Genetic analysis of sorting nexins1and2reveals aredundant and essential function in mice. Mol Biol Cell,2002.13(10):3588‐600.
[24]Zheng, B., et al., Essential role of RGS‐PX1/sorting nexin13in mousedevelopment and regulation of endocytosis dynamics. Proc Natl Acad Sci U S A,2006.103(45):16776‐81.
[25]Nasevicius, A. and S.C. Ekker, Effective targeted gene 'knockdown' in zebrafish.Nat Genet,2000.26(2):216‐20.
[26]Yoo, K.W., et al., Snx5, as a Mind bomb‐binding protein, is expressed inhematopoietic and endothelial precursor cells in zebrafish. FEBS Lett,2006.580(18):4409‐16.
[27]Li‐Fong Seet, Wan jin Hong, The Phox(PX) domain proteins and membranetraffic. Biochim Biophys Acta.2006Aμg,1761(8):878‐96.
[28]Jan R.T.van Weering, Paul Verkade, Peter J. Cullen. SNX‐BAR proteins inphosphoinositide‐mediated, tubular‐based endosomal sorting. Semin Cell DevBiol.2010Jun;21(4):371‐80.
[29]R.A.Weinberg著；詹启敏，刘芝华主译.《癌生物学》：科学出版社，2009年4月.
[30]Watson AJ.Apoptosis and colorectal cancer. Gut.2004Nov;53(11):1701‐9.
[31]Nika N Danial,Stanley J Korsmeyer.Cell death: critical control points.Cell.2004;116(2):205‐19.
[32]Hershko, A. Lessons from the discovery of the ubiquitin system. Trends.Biochem. Sci.1996;21:445–449.
[33]Coux, O., Tanaka, K, and Goldberg, A. L. Annu. Rev. Biochem.1996;65:801‐847.
[34]Rechsteiner M. In Ubiquitin and the Biology of the Cell, ed. D Finley, JM Peters.New York/London: Plenum.1997.
[35]Cenci, G., Rawson, R.B., Belloni, G., Castrillon, D.H., Tudor, M., Petrucci, R.,Goldberg, M.L., Wasserman, S.A., Gatti, M.. UbcD1, a Drosophila ubiquitin‐conjugating enzyme required for proper telomere behavior. Genes Dev.1997,11(7):863‐‐875.
[36]Muralidhar MG, Thomas JB. The Drosophila bendless gene encodes a neuralprotein related to ubiquitin‐conjugating enzymes. Neuron.1993;11:253‐66.
[37]Roest HP, van Klaveren J, de Wit J, van Gurp CG, Koken MH, Vermey M, vanRoijen JH, Hoogerbrugge JW, Vreeburg JT, Baarends WM, Bootsma D,Grootegoed JA, Hoeijmakers JH. Inactivation of the HR6B ubiquitin‐conjugatingDNA repair enzyme in mice causes male sterility associated with chromatinmodification. Cell.1996Sep6;86(5):799‐810.
[38]Harbers K, Müller U, Grams A, Li E, Jaenisch R, Franz T.Provirus integration intoa gene encoding a ubiquitin‐conjugating enzyme results in a placental defectand embryonic lethality.Proc Natl Acad Sci U S A.1996Oct29;93(22):12412‐7.
[39]Huibregtse JM, Scheffner M, Beaudenon S, Howley PM. A family of proteinsstructurally and functionally related to the E6‐AP ubiquitin‐protein ligase.ProcNatl Acad Sci U S A.1995Mar28;92(7):2563‐7.
[40]Sudakin V, Ganoth D, Dahan A, Heller H, Hershko J, Luca FC, Ruderman JV,Hershko A. The cyclosome, a large complex containing cyclin‐selectiveubiquitin ligase activity, targets cyclins for destruction at the end ofmitosis.Mol Biol Cell.1995Feb;6(2):185‐97.
[41]King RW, Deshaies RJ, Peters JM, Kirschner MW. How proteolysis drives thecell cycle. Science.1996Dec6;274(5293):1652‐9.
[42]Michael H. Glickman,Aaron Ciechanover. The Ubiquitin‐ProteasomeProteolytic Pathway: Destruction for the Sake of Construction. Physiol Rev.2002;82:373–428.
[43]F Ujimuro M, Takada H, S Aeki Y, T Oh‐E A, T Anaka K, Andyokosawa H.Growth‐dependent change of the26S proteasome in budding yeast. BiochemBiophys Res Commun.1998,251:818‐823.
[44]G Roll M, Ditzel L, L Oewe J, S Tock D, Bochtler M, Bartunik Hd, HUBER R.Structure of20S proteasome from yeast at a2.4Angstrom resolution.Nature.1997,386:463–471.
[45]Dengxw, D Ubiel W, Wein, H Ofmannk, M Undt K, C Olicellij, K Ato Jy, NAumann M, Segal D, S Eeger M, Carra, Glickman Mh,Chamovitzda. Unifiednomenclature for the COP9signalosome: an essential regulator ofdevelopment. Trends Genet Sci.2000,16:202–203.
[46]G Lickman Mh, R Ubin Dm, C Ouxo, Wefes I, P Feifer G, Cjeka Z, Baumeister W,Fried Va, Finley D. A subcomplex of the proteasome regulatory particlerequired for ubiquitin‐conjugate degradation and related to theCOP9/Signalosome and eIF3. Cell.1998,94:615–623.
[47]K Apelarib, B Ech‐Otschir D, Hegerl R, S Chade R, Dumdey R, Dubiel W. Electronmicroscopy and subunit‐subunit interaction studies reveal a first architectureof COP9signalosome. J Mol Biol.2000,300:1169–1178.
[48]Maupin‐F Urlow Ja, W Ilson Hl, Kaczowka Sj, Andou Ms. Proteasomes in thearchaea: from structure to function. Front Biosci.2000,5: D837–D865.
[49]De Mot R, Nagyi, W Alz J, Baumeister W. Proteasomes and otherself‐compartmentalizing proteases in prokaryotes. Trends Microbiol.1999,7:88–92.
[50]Mayer,A.N.and Fishman, M.C. Nilperos encodesa conserved RNA recognitionmotif protein required for morphogenesis and cyto differentiation of digestiveorgans in zebrafish. Development2003.130:3917–3928
[51]Workman, J.L. and Kingston, R.E. Alteration of nucleosome structure as amechanism of transcriptional regulation. Annu Rev Biochem.1998.67:545‐79.
[52]Hansen, J.C. et al. Structure and function of the core histone N‐termini: morethan meets the eye. Biochemistry,1998.37,17637‐41.
[53]Strahl, B.D. and Allis, C.D. The language of covalent histone modifications.Nature,2000.403,41‐5.
[54]Cheung, P. et al. Signaling to chromatin throμgh histone modifications.Cell,2000.103,263‐71.
[55]Bernstein, B.E. and Schreiber, S.L. Global approaches to chromatin. ChemBiol,2002.9,1167‐73.
[56]Jaskelioff, M. and Peterson, C.L. Chromatin and transcription: histonescontinue to make their marks. Nat Cell Biol,2003.5,395‐9.
[57]Thorne, A.W. et al. Patterns of histone acetylation. Eur J Biochem,1990.193,701‐13.
[58]Hendzel, M.J. et al. Mitosis‐specific phosphorylation of histone H3initiatesprimarily within pericentromeric heterochromatin during G2and spreads in anordered fashion coincident with mitotic chromosome condensation.Chromosoma,1997.106,348‐60.
[59]Goto, H. et al. Identification of a novel phosphorylation site on histone H3coupled with mitotic chromosome condensation. J Biol Chem,1999.274,25543‐9.
[60]Preuss, U. et al. Novel mitosis‐specific phosphorylation of histone H3at Thr11mediated by Dlk/ZIP kinase.Nucleic Acids,Res.2003.31,878‐85.
[61]Dai, J. et al. The kinase haspin is required for mitotic histone H3Thr3phosphorylation and normal metaphase chromosome alignment. GenesDev,2005.19,472‐88.
[62]JenniferL.Shepard. et al. A zebrafish bmyb mutation causes genome instabilityand increased cancer susceptibility. Proc Natl Acad Sci U S A.2005. Sep13;102(37):
[63]Curado S, Stainier DY, Anderson RM. Nitroreductase‐mediated cell/tissueablation in zebrafish: a spatially and temporally controlled ablation methodwith applications in developmental and regeneration studies. Nat Protoc.2008;3(6):948‐54.
[64]James Summerton. Morpholino Antisense Oligos:Applications inBiopharmaceutical Research. Innovations in Pharmaceutical Technologyissue.2005.17.
[65]Doyon Y, McCammon J M, Miller J C,FarajiF, NgoC, Katibah G E,etal. Heritabletargeted gene disruption in zebrafish using designed zinc‐fingernucleases.NatBiotech.2008;26:702‐708.
[66]Foley J E, Yeh JR, Maeder ML,Reyon D,Sander JD, Peterson RT,etal. Rapidmutation of endogenous zebrafish genes using zinc finger nucleases made byOligomerized Pool ENgineering(OPEN). PLoSONE.2009;4: e4348.
[67]Field HA, Ober EA, Roeser T, Stainier DY. Formation of the digestive system inzebrafish. I.Liver morphogenesis. DevBiol.2003;253:279‐290.
[68]Farooq M, Sulochana KN, Pan X, To J, Sheng D, Gong Z,etal. Histonedeacetylase3(hdac3) is specifically required for liver development in zebrafish.Dev Biol.2008;317:336‐353.
[69]Korzh S, Pan X, Garcia‐Lecea M, Winata CL,Wohland T, Korzh V,etal.Requirement of vasculogenesis and blood circulation in latest ages oflivergrowth in zebrafish.BMC Dev Biol.2008;8:84.
[70]Sadler, K.C., et al., A genetic screen in zebrafish identifies the mutants vps18,nf2and foie gras as models of liver disease. Development,2005.132(15):3561‐72.
[71]Chen, J., et al., Loss of function of def selectively up‐regulates Delta113p53expression to arrest expansion growth of digestive organs in zebrafish. GenesDev,2005.19(23):2900‐11.
[72]Ober EA,Field HA,Stainier DY. From endoderm formation to liver and pancreasdevelopment in zebrafish.Mech Dev.2003,120:5‐18.
[73]Shin D,Shin CH,Tucker J,Ober EA,Rentzsch F,Poss KD,etal.Bmp and Fgf signalingare essential for liver specification in zebrafish.Development2007,134:2041‐2050.
[74]Sun Z, Hopkins N. vhnf1, the MODY5and familial GCKD‐associated gene,regulates regional specification of the zebrafish gut, pronephros, andhindbrain.Genes Dev.2001,15:3217‐3229.
[75]Holtzinger A, Evans T. Gata4regulates the formation of multipleorgans.Development.2005,132:4005‐4014.
[76]Reiter JF, Alexander J, Rodaway A, Yelon D, Patient R, Holder N,etal.Gata5isrequired for the development of the heart and endoderm in zebrafish.GenesDev.1999;13:2983‐2995.
[77]Reiter JF, Kikuchi Y, Stainier DY. Multiple roles for Gata5in zebrafish endodermformation. Development.2001;128:125‐135.
[78]Deutsch G, Jung J, Zheng M, Lora J, Zaret KS. A bipotential precursorpopulation for pancreas and liver within the embryoniccendoderm.Development.2001;128:871‐881.
[79]Serls AE, Doherty S, Parvatiyar P, Wells JM, Deutsch GH. Different Thresholdsof fibroblast growth factors pattern the ventral foregut intoLiver andlung.Development.2005;132:35‐47.
[80]Monga SP, Monga HK, Tan X, Mule K, Pediaditakis P, MichalopoulosGK.Beta‐catenin antisense studies in embryonic liver cultures: role inproliferation, apoptosis, and lineage specification.Gastroenterology.2003;124:202‐216.
[81] Suksaweang S, Lin CM, Jiang TX, Hμghes MW, Widelitz RB, Chuong CM.Morphogenesis of chicken liver:identification of localized growth zones andthe role of beta‐catenin/Wnt in size regulation. DevBiol.2004;266:109‐122.
[82]Lorent K, Yeo SY, Oda T, Chandrasekharappa S, Chitnis A, Matthews RP, etal.Inhibition of Jagged‐mediated Notch signaling disrupts zebrafish biliarydevelopment and generates multi‐organ defects compatible with an Alagillesyndrome phenocopy. Development.2004;131:5753‐5766.
[83]Oda T, Elkahloun AG, Pike BL,Okajima K, Krantz ID, GeninA, etal.Mutations inthe human Jagged1gene are responsible for Alagille syndrome.NatGenet.1997;16:235‐242.
[84]Li L, Krantz ID, Deng Y, Genin A, Banta AB, Collins CC, etal. Alagille syndrome iscaused by mutations in human Jagged1, which encodes a ligand for Notch1.NatGenet.1997;16:243‐251.
[85]Parviz F, Matullo C, Garrison WD, Savatski L, Adamson JW, Ning G,etal.Hepatocyte nuclear factor4alpha controls the development of a hepaticepithelium and liver morphogenesis. Nat Genet.2003;34:292‐296.
[86]Chiba H, Gotoh T, Kojima T, Satohisa S, Kikuchi K, Osanai M,etal.Hepatocytenuclear factor(HNF)‐4alpha triggers formation of functional tight junctionsand establishment of polarized epithelial morphology in F9embryonalcarcinoma cells. Exp Cell Res.2003;286:288‐297.
[87]Liedtke, C., et al., The human Caspase‐8promoter sustains basal activitythroμgh SP1and ETS‐like transcription factors and can be up‐regulated by ap53‐dependent mechanism. J Biol Chem,2003.278(30):27593‐604.
[88]Liedtke, C., et al., Interferon‐alpha enhances TRAIL‐mediated apoptosis byup‐regulating Caspase‐8transcription in human hepatoma cells. J Hepatol,2006.44(2):342‐9.
[89]Kurita, S., et al., DNMT1and DNMT3b silencing sensitizes human hepatomacells to TRAIL‐mediated apoptosis via up‐regulation of TRAIL‐R2/DR5andCaspase‐8. Cancer Sci,2010.101(6):1431‐1439.
[90]Yamaguchi, Y.,et al., Adenovirus‐mediated transfection of Caspase‐8sensitizeshepatocellular carcinoma to TRAIL‐and chemotherapeutic agent‐induced celldeath. Biochim Biophys Acta,2006,1763(8):844‐53.
[91]http://www.genecards.org/cgi‐bin/carddisp.pl?gene=SNX7&search=snx7
[92]Western印迹法‐Western Blotting‐实验方法技术‐生物在线.http://www.bioon.com.cn/protocol/showarticle.asp?newsid=6946