刘缨-眼睛发育的基因调控2015

1、刘刘 缨缨 副研究员副研究员中国科学院生物物理研究所中国科学院生物物理研究所视觉视觉听觉、嗅觉、味觉等听觉、嗅觉、味觉等80%管怀庆等, , 1998 视觉器官各种遗传性视觉器官各种遗传性疾病的发病已达疾病的发病已达2%2%左右；左右；近视、斜视、散光等等在近视、斜视、散光等等在我国青年中的发病率已达我国青年中的发病率已达到到50%50%左右；眼睛的老视、左右；眼睛的老视、白内障几乎是每一个人进白内障几乎是每一个人进入中老年都会遇到的入中老年都会遇到的问题问题。 基各基各因种因种突诱突诱变变变变 : 因因 : 素素l 确定引起各种先天性和后天性眼病的遗确定引起各种先天性和后天性眼病的遗传和环境

2、因素，促进对其致病机理研究，提传和环境因素，促进对其致病机理研究，提高眼病预防、诊断和治疗水平。高眼病预防、诊断和治疗水平。l 揭开脊椎动物眼睛发育的秘密，并为其揭开脊椎动物眼睛发育的秘密，并为其它器官和系统发育机制的研究提供线索。它器官和系统发育机制的研究提供线索。222179 各大医院的眼科同行在临床防治视觉器官疾病方面做了大各大医院的眼科同行在临床防治视觉器官疾病方面做了大量的工作，但是从基因水平来进行研究的工作还非常有限，量的工作，但是从基因水平来进行研究的工作还非常有限，对眼睛发育的分子机制研究则是近年才开始。对眼睛发育的分子机制研究则是近年才开始。 Department of Op

3、hthalmology and Visual Sciences The Chinese University of Hong Kong, Hong Kong. The Laboratory of Visual Information Processing, Institute of Biophysics, Chinese Academy of Sciences1.分离确定与眼睛发育和疾病发生相关的基因分离确定与眼睛发育和疾病发生相关的基因2.阐明上述基因的功能和调控机制阐明上述基因的功能和调控机制阐明脊椎动物眼睛发育基因调控机制，为其它阐明脊椎动物眼睛发育基因调控机制，为其它器官和中枢神经系统

4、发育机制的研究提供线索器官和中枢神经系统发育机制的研究提供线索由此确定引起各种先天性和后天性眼病的遗传由此确定引起各种先天性和后天性眼病的遗传和环境因素，阐明其致病机理和环境因素，阐明其致病机理, 最终为建立相关最终为建立相关眼病的预防、诊断和临床治疗方法提供依据眼病的预防、诊断和临床治疗方法提供依据。l基因筛选基因筛选l- 致病突变的定位致病突变的定位l- 基因表达的差式分析基因表达的差式分析l基因的功能研究基因的功能研究l- 基因表达模式分析基因表达模式分析l- 基因的超表达和功能失活研究基因的超表达和功能失活研究l基因调控机制研究基因调控机制研究l- 基因间及与信号通路间的关系基因间及与

5、信号通路间的关系l- 基因及其表达蛋白的结构和功能基因及其表达蛋白的结构和功能 基因的分离鉴定基因的分离鉴定建立建立cDNA或蛋白质库或蛋白质库遗传病家系的建立遗传病家系的建立基因的染色体定位基因的染色体定位基因的功能及调控机理研究基因的功能及调控机理研究芯片技术、转录芯片技术、转录/蛋白质组技术蛋白质组技术病理材料的收集病理材料的收集结构及发育阶段特异性结构及发育阶段特异性组织材料的收集组织材料的收集发育机制发育机制致病机理致病机理同源基因的分离同源基因的分离疾病的模式动物疾病的模式动物l脑（端、间、中、后）、脊髓、视网膜l神经管（前、后）、视杯/视泡l神经板（前、后）、脊索l基因筛选基因筛

6、选l基因的功能研究基因的功能研究 l- 基因表达模式分析基因表达模式分析 (expression pattern analysis) = 可能性可能性 (possibility)l- 基因的超表达研究基因的超表达研究(gain-of-function) = 充分性充分性(sufficiency)l- 基因的功能失活研究基因的功能失活研究(loss-of-function) = 必要性必要性(necessity)l基因调控机制研究基因调控机制研究l- 基因间及与信号通路间的关系基因间及与信号通路间的关系l- 基因及其表达蛋白的结构和功能基因及其表达蛋白的结构和功能l 基因结构分析基因结构分析 (

7、启动子、内含子启动子、内含子/外显子分析外显子分析)l 蛋白结构分析（蛋白结构分析（X射线晶体学、射线晶体学、NMR）l 结构和功能关系分析（点突变，片段缺失结构和功能关系分析（点突变，片段缺失） 1Southern(DNA)Northern(RNA)Western(protein)- - 基因表达模式分析基因表达模式分析l转录水平转录水平(mRNA)(mRNA)： RT-PCR, Northern, RT-PCR, Northern, 原位杂交原位杂交l翻译水平翻译水平( (蛋白蛋白) )： western blot, immunocytochemistrywestern blot, imm

8、unocytochemistry基因的表达模式决定基因功能干扰分析的位置和时间。基因的表达模式决定基因功能干扰分析的位置和时间。- - 基因表达和功能的干扰（超表达和功能失活研究）基因表达和功能的干扰（超表达和功能失活研究） 1enhancersilencerknock-out/in expression vector?RNAi?RNAimRNAAntisense RNAsiRNAmiRNAmiRNAmorpholinoproteinantibody?CA pr.DN pr. Gain-of-function loss-of-function DNA template DNA (express

9、ion vector) knock-out/in knock-in transcription enhancer silencer RNA processing - RNAi transportation - - mRNA mRNA RNAi (siRNA) translation - (Morpholino, miRNA) protein Modification - - Protein Wild-type pr. antibody Targeting constitutively active (CA) pr. dominant negative (DN) pr. - - 基因表达模式分析

10、基因表达模式分析l转录水平转录水平(mRNA)(mRNA)： RT-PCR, Northern, RT-PCR, Northern, 原位杂交原位杂交l翻译水平翻译水平( (蛋白蛋白) )： western blot, immunohistochemistrywestern blot, immunohistochemistry- - 基因的超表达和功能失活研究基因的超表达和功能失活研究 显微注射显微注射(microinjecton) 果蝇果蝇 DNA水平水平 电穿孔电穿孔(electroporation) 斑马鱼斑马鱼 RNA水平水平 转染转染(lipofection) 爪蟾爪蟾 蛋白水平蛋白

11、水平 体外处理体外处理(in vitro treatment) 鸡鸡 小鼠小鼠- - 基因表达和功能的干扰（超表达和功能失活研究）基因表达和功能的干扰（超表达和功能失活研究）基因的表达模式决定基因功能干扰分析的位置和时间基因的表达模式决定基因功能干扰分析的位置和时间。基因功能干扰的位置和时间（剂量）控制基因功能干扰的位置和时间（剂量）控制 ：（1）人工导入的控制）人工导入的控制（2）特异性启动子的控制：）特异性启动子的控制：tissue/cell type/gene specific, hsp, etc ( 3 ) 可诱导融合蛋白可诱导融合蛋白 显微注射显微注射(microinjecton)

12、果蝇果蝇 DNA水平水平 电穿孔电穿孔(electroporation) 斑马鱼斑马鱼 RNA水平水平 转染转染(lipofection) 爪蟾爪蟾 蛋白水平蛋白水平 体外处理体外处理(in vitro treatment) 鸡鸡 小鼠小鼠Wolpert, L, Principle of Development, 2nd, 2002: : The vertebrate eyeGiuseppina BarsacchiFertilizationCleavage: blastula blastomereGastrulation:gustrulaNeurulation: neurulaOrganoge

13、nesis tailbud tadpoleMetamorphosisMaturation: adultNeural inductionNeurogenesisconnectiongenesisEagleson G, et al, J. Neurobiol.,1995 (Nieuwkoop and Faber, 1994)Eye field specificationEye field splitpatterning / MorphogenesisVentralDorsalProximalDistalPigmented epitheliumRetinaLensCorneaOptic nerveP

14、rosencephalonRetinaLensAPAPEarly neurulaMid-neurulaLate neurulaTailbudTadpoleVertebrate eye developmentEye field specificationEye field splitEye vescicleEye cup(O. Mangold, 1931)patterning / MorphogenesisGiuseppina Barsacchil结构基因：结构基因： “housekeeping” genes, Crystallin (晶状体球蛋白）晶状体球蛋白）l调节基因：调节基因： 转录因子

15、转录因子 - homeobox genes: Pax, rx1, vax, Brn3.0 Zinc Finger genes: RAR, RXR Helix-Loop Helix genes: Mi, Mash 信号通路分子及受体信号通路分子及受体 - 小有机分子：小有机分子： retinoic acid (RA) 分泌蛋白和膜受体：分泌蛋白和膜受体：BMPs, hedgehogs, PKA, A model for Hox regulation (Garcia-Bellido, 1975)ActivatorSelectorRealizatorActivator genes delimit t

16、he realm of expression of Selector genes. Maternal signalSelectors confer different segment identity along AP axis by controlling Realizator genes. Hox genesThe ultimate genes who do the job: cell adhesion, cytoskeleton, cell proliferation, apoptosis, .EFTF(toy, ET, Pax6, rx1)hedgehog (shh) Nodal (c

17、yclops, squint) hh,Pax2, Pax6, crystallins BMP, RA, vax2 BF1, BF2, SOHo, GH6 hh, atonal Pax6 is necessary to make the vertebrate eyeXenopus (Chow et al., 1999)Mouse (Hill et al., 1991)Homo(Glaser et al., 1992)eyeless/Pax6 is necessary and sufficient for eye formation in Drosophila(Halder et al., 199

18、5)(Quiring et al., 1994)Chow RL, et al, Development, 1999 ? (Gehring and Ikeo, 1999)EFTF(toy, ET, Pax6, rx1)hedgehog (shh) Nodal (cyclops, squint) hh, atonal BF1, BF2, SOHo, GH6 hh,Pax2, Pax6, crystallins BMP, RA, vax2 l基因筛选基因筛选l- 致病突变的定位致病突变的定位l- 基因表达的差式分析基因表达的差式分析l基因的功能研究基因的功能研究 l- 基因表达模式分析基因表达模式分

19、析 (expression pattern analysis) = 可能性可能性 (possibility)l- 基因的超表达研究基因的超表达研究(gain-of-function) = 充分性充分性(sufficiency)l- 基因的功能失活研究基因的功能失活研究(loss-of-function) = 必要性必要性(necessity)l基因调控机制研究基因调控机制研究l- 基因间及与信号通路间的关系基因间及与信号通路间的关系l- 基因及其表达蛋白的结构和功能基因及其表达蛋白的结构和功能Zuber M E et al. Development 2003;130:5155-5167Pax6

20、Otx2Pax 6Rx1Six3Otx2Pax6Six3BF-1Rx1Eye field transcription factors (EFTFs)Zuber M E et al. Development 2003;130:5155-5167Zuber M E et al. Development 2003;130:5155-5167RNA microinjection.and animal cap assayfor: gain-of-function loss-of-functionGiuseppina Barsacchi*Animal cap expressesZuber M E et a

21、l. Development 2003;130:5155-5167Zuber M E et al. Development 2003;130:5155-5167 Drosophilaey (Pax6)toysoeyadacdppRx1ETPax6EyaDachSix3Optx2LhxTlllens placodeBMP ?VertebrateGiuseppina BarsacchiMouse cells have been coaxed into forming a retina, the most complex tissue yet engineered.Published online

22、6 April 2011 | Nature | doi:10.1038/news.2011.215 Heres lookin at you kid.M. Eiraku and Y.Sasai at RIKEN Center for Developmental Biology EFTF(toy, ET, Pax6, rx1)hedgehog (shh) Nodal (cyclops, squint) hh, atonal BF1, BF2, SOHo, GH6 hh,Pax2, Pax6, crystallins BMP, RA, vax2 The phenotypic spectrum of

23、holoprosencephaly. (From Muenke, 1995.)美国国家地理网站 2011十大奇异动物：独眼鲨上榜 Cyclopia of lamb and mouse by loss of shh, (From Gilbert, 2002.)shh is necessary for the split of eye fieldEFTF(toy, ET, Pax6, rx1)hedgehog (shh) Nodal (cyclops, squint) hh, atonal BF1, BF2, SOHo, GH6 hh, Pax2, XRALDH3, Pax6 BMP, RA, v

24、ax2 Rorth P, et al, Development, 1998 Peters MA, Current Opinion in Neurobiology, 2002Lupo G, et al (2000), Int. J. Dev. Biol. 44(6); 627-636. BarbieriAM, Lupo G, et al, 1999Barbieri, A.M., et al, Development, 1999 Schulte, D., et al, Neron, 1999- by lineage tracing and transplantationSpecification

25、or determination ?FertilizationCleavage: blastula blastomereGastrulation: gastrulaNeurulation: neurulaOrganogenesis tailbud tadpoleMetamorphosisMaturation: adultNeural inductionNeurogenesisconnectiongenesisGenetic information + environmental signalDifferential gene expressionParticular protein compo

26、nentProperties and behaviors of cells Specification, proliferation, migration, differentiation, deathAxon guidance, synapse formation, information processing Structure and function/behavior: development, aging, diseasesstructure (CNS/PNS): induction, morphogenesis, growth/maturationlNeural tube and

27、crest in early developmentlCiliary marginal zone (CMZ) of mature retinalSubventricular zone of mature brainApproaches:lBrdU incorporationlphosphohistone-H3Xvax2 expression colocalizes with proliferative and postmitotic regions of the late embryonic ventral retina and brainLiu, et al., Dev. Dyn. 2008

28、Xvax2/BrdUXvax2/BrdUScale bars = 100 umXvax2 overexpression inhibits proliferationCyclinD1Liu, et al., Dev. Dyn. 2008antiphosphohistone-H3HoechstBrdU/HoechstMicroinjectionRed: BrdU-positive cells;Blue: hoechst counterstained cells;Green: gfp-positive coinjectionGenetic information + environmental si

29、gnalDifferential gene expressionParticular protein componentProperties and behaviors of cells Specification, proliferation, migration, differentiation, deathAxon guidance, synapse formation, information processing Structure and function/behavior: development, aging, diseasesstructure (CNS/PNS): indu

30、ction, morphogenesis, growth/maturationReferences to the files for “migration of neural crest”Genetic information + environmental signalDifferential gene expressionParticular protein componentProperties and behaviors of cells Specification, proliferation, migration, differentiation, deathAxon guidan

31、ce, synapse formation, information processing Structure and function/behavior: development, aging, diseasesstructure (CNS/PNS): induction, morphogenesis, growth/maturationThe process in which a cell takes on the appearance and characteristics of a neuron is known as cell differentiationMorphology: s

32、ingle cell labelling (staining/imaging)Position: single cell labelling (staining/imaging)molecular marker: RNA/protein detectionXvax2 overexpression inhibits proliferationLiu, et al., Dev. Dyn. 2008Lipofection: The different cell types distributed in distinct cell layers and can be distinguished by

33、their specific morphologyGenetic information + environmental signalDifferential gene expressionParticular protein componentProperties and behaviors of cells Specification, proliferation, migration, differentiation, deathAxon guidance, synapse formation, information processing Structure and function/

34、behavior: development, aging, diseasesstructure (CNS/PNS): induction, morphogenesis, growth/maturationEFTF(toy, ET, Pax6, rx1)hedgehog (shh) Nodal (cyclops, squint) hh, atonal BF1, BF2, SOHo, GH6 hh,Pax2, Pax6, crystallins BMP, RA, vax2 Select cells with GFP fluorescence14 - 20 hr rtTurning Assaytwo

35、-cell stageFertilized eggStage 22dissociationcDNA袁小兵Growth cone turning assayNetrinsShhNTsNetrinsSemaphorinsSlits袁小兵袁小兵Growth cone turning assayGrowth cone of retinal ganglion cell axon showing asymmetrical distribution of beta-actin after exposure to a 5 minute gradient of netrin-1 (top right). Thi

36、s spatial asymmetry is generated by local translation of beta-actin mRNA and is critical for attractive turning towards netrin-1 (see Leung et al, 2006). http:/www.pdn.cam.ac.uk/staff/holt/Single retinal ganglion cell in the embryonic visual system. The cell is stained with a dye (HRP) to reveal its

37、 soma and developing dendrites in the eye and its long axon extending across the midline (optic chiasm) into the contralateral optic tract. It is tipped with a motile growth process, the growth cone, which responds to guidance cues along the pathway and leads the axon to its final destination in the

38、 midbrain. 魏园园,2004Peters MA, Current Opinion in Neurobiology, 2002The ventral regionalization of the eye is specified by interactions of Hh, RA and FGFR signaling.Lupo G, Liu Y et al, Development, 2005EFTF(toy, ET, Pax6, rx1)hedgehog (shh) Nodal (cyclops, squint) hh, atonal BF1, BF2, SOHo, GH6 hh,P

39、ax2, Pax6, crystallins BMP, RA, vax2 DsRed2 and GFP-synaptobrevin plasmids were co-lipofected with 1:1 DOTAP into the eye primordia of stage 22-24 Xenopus embryos.中科大，胡兵中科大，胡兵中科大，胡兵中科大，胡兵0.2-1.0 nanoliters of anti-BDNF (330 g/ml), APV (50 M), MK801 (20 M), recombinant BDNF (200 ng/ l), or vehicle solution (50% Niu Twitty) was injected into the optic tectum soon after the first image was taken.中科大，胡兵Genetic in