脊椎动物眼睛发育基因调控要求

1、脊椎动物眼睛发育基因调控要求 眼睛是人体最重要的感觉器官-视觉听觉、嗅觉、味觉等80% 人从外界环境获得的各种信息中，80%以上经由视觉通道输入；视觉的敏锐与否直接影响个人的劳动、学习和生活的能力眼睛是发育过程中最易发生畸变的器官之一管怀庆等, , 1998眼睛是发育过程中最易发生畸变的器官之一- 视觉器官各种遗传性疾病的发病已达2%左右；近视、斜视、散光等等在我国青年中的发病率已达到50%左右；眼睛的老视、白内障几乎是每一个人进入中老年都会遇到的问题。 研究意义基各因种突诱变变 : 因 : 素预防诊断治疗致病机理研究眼睛发育的机制劳动、学习和生活其他器官发育和疾病的机制研究意义- 确定引起各

2、种先天性和后天性眼病的遗传和环境因素，促进对其致病机理研究，提高眼病预防、诊断和治疗水平。 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。国际研究状况美国：University of Texas, the Hermann Eye Centre：Retnet Howard Hughes Medical Institute, Connie CepkoSt Jude Childrens Research Hospital, Guillermo Oliver欧洲：意大利Telethon Institute of Genetics and Medicine (TIGEM)德国马普

3、研究所, Peter Gruss瑞士巴塞尔大学，Gehring WJBiotechnology EEC network - 英国剑桥大学、University College London - 德国马普研究所- 意大利比萨大学- 西班牙（CSIC）Cajal研究所生物研究技术的进步也加快了与眼睛发育有关的基因的发现 222179国内研究现状 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，对眼睛发育的分子机制研究则是近年才开始。 Department of Ophthalmology and Visual Sciences The Chin

4、ese University of Hong Kong, Hong Kong. The Laboratory of Visual Information Processing, Institute of Biophysics, Chinese Academy of Sciences研究目标 分离确定与眼睛发育和疾病发生相关的基因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和环境因素，阐明其致病机理, 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。研究内容 与眼睛发育及疾病相关的基因的

5、筛选(What?) 基因的功能研究 (When and Where?) 基因调控机制 (How?) 研究方法基因筛选- 致病突变的定位- 基因表达的差式分析基因的功能研究- 基因表达模式分析- 基因的超表达和功能失活研究基因调控机制研究- 基因间及与信号通路间的关系- 基因及其表达蛋白的结构和功能 基因的分离鉴定建立cDNA或蛋白质库遗传病家系的建立基因的染色体定位基因的功能及调控机理研究芯片技术、转录/蛋白质组技术病理材料的收集结构及发育阶段特异性组织材料的收集发育机制致病机理基因筛选同源基因的分离疾病的模式动物Dres Expression in eye结构及发育阶段特异性组织材料的收集脑

6、（端、间、中、后）、脊髓、视网膜神经管（前、后）、视杯/视泡神经板（前、后）、脊索基因筛选研究内容及方法基因筛选基因的功能研究 - 基因表达模式分析 (expression pattern analysis) = 可能性 (possibility)- 基因的超表达研究(gain-of-function) = 充分性(sufficiency)- 基因的功能失活研究(loss-of-function) = 必要性(necessity)基因调控机制研究- 基因间及与信号通路间的关系- 基因及其表达蛋白的结构和功能 基因结构分析 (启动子、内含子/外显子分析) 蛋白结构分析（X射线晶体学、NMR） 结

7、构和功能关系分析（点突变，片段缺失） 1The main stages of gene expressionSouthern(DNA)Northern(RNA)Western(protein)- 基因表达模式分析转录水平(mRNA)： RT-PCR, Northern, 原位杂交翻译水平(蛋白)： western blot, immunocytochemistry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） 基因的功能研究1The main stages of gene expressionenhancersilencerknock-out/

8、in expression vector?RNAi?RNAimRNAAntisense RNAsiRNAmiRNAmiRNAmorpholinoproteinantibody?CA pr.DN pr.Functional analysis - Approaches Gain-of-function loss-of-function DNA template DNA (expression vector) knock-out/in knock-in transcription enhancer silencer RNA processing - RNAi transportation - - m

9、RNA mRNA RNAi (siRNA) translation - (Morpholino, miRNA) protein Modification - - Protein Wild-type pr. antibody Targeting constitutively active (CA) pr. dominant negative (DN) pr. - 基因表达模式分析转录水平(mRNA)： RT-PCR, Northern, 原位杂交翻译水平(蛋白)： western blot, immunohistochemistry- 基因的超表达和功能失活研究 显微注射(microinject

10、on) 果蝇 DNA水平 电穿孔(electroporation) 斑马鱼 RNA水平 转染(lipofection) 爪蟾 蛋白水平 体外处理(in vitro treatment) 鸡 小鼠基因的功能研究- 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。基因功能干扰的位置和时间（剂量）控制 ：（1）人工导入的控制（2）特异性启动子的控制：tissue/cell type/gene specific, hsp, etc ( 3 ) 可诱导融合蛋白 显微注射(microinjecton) 果蝇 DNA水平 电穿孔(electroporation)

11、斑马鱼 RNA水平 转染(lipofection) 爪蟾 蛋白水平 体外处理(in vitro treatment) 鸡 小鼠基因的功能分析Wolpert, L, Principle of Development, 2nd, 2002 果蝇 斑马鱼 非洲爪蟾 鸡 小鼠Drosophila Zebrafish Xenopus Chick Mouse模式动物: The vertebrate eyeGiuseppina Barsacchi视觉神经系统Xenopus developmentFertilizationCleavage: blastula blastomereGastrulation:gu

12、strulaNeurulation: neurulaOrganogenesis tailbud tadpoleMetamorphosisMaturation: adultNeural inductionNeurogenesisconnectiongenesisFate Map of Neural PlateEagleson G, et al, J. Neurobiol.,1995 (Nieuwkoop and Faber, 1994)眼睛发育- Morphogenesis/patterningEye field specificationEye field splitpatterning /

13、MorphogenesisVentralDorsalProximalDistalPigmented epitheliumRetinaLensCorneaOptic nerveProsencephalonRetinaLensAPAPEarly neurulaMid-neurulaLate neurulaTailbudTadpoleVertebrate eye developmentEye field specificationEye field splitEye vescicleEye cup(O. Mangold, 1931)patterning / MorphogenesisGiuseppi

14、na Barsacchi眼睛发育- Morphogenesis/patterningProjection眼睛发育的基因调控结构基因： “housekeeping” genes, Crystallin (晶状体球蛋白）调节基因： 转录因子 - homeobox genes: Pax, rx1, vax, Zinc Finger genes: RAR, RXR Helix-Loop Helix genes: Mi, Mash 信号通路分子及受体 - 小有机分子： retinoic acid (RA) 分泌蛋白和膜受体：BMPs, hedgehogs, PKA, A model for Hox re

15、gulation (Garcia-Bellido, 1975)ActivatorSelectorRealizatorActivator genes delimit the 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, cytoskelet

16、on, cell proliferation, apoptosis, .眼睛发育的基因调控Eye Field (EF) specification : EFTF(toy, ET, Pax6, rx1)Eye Field (EF) split : hedgehog (shh) Nodal (cyclops, squint)Polarity Patterning Proximal Distal (P-D): hh,Pax2, Pax6, crystallins Dorsal -Ventral (D-V): BMP, RA, vax2 Anterior-Posterior (A-P): BF1, B

17、F2, SOHo, GH6 Central-Peripheral (C-P) : hh, atonal Visual formation projection, connection, growing, learningPax6 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 Drosoph

18、ila(Halder et al., 1995)(Quiring et al., 1994)Pax6 is sufficient to make the vertebrate eyeChow RL, et al, Development, 1999 ?(Gehring and Ikeo, 1999)眼睛发育的基因调控Eye Field (EF) specification : EFTF(toy, ET, Pax6, rx1)Eye Field (EF) split : hedgehog (shh) Nodal (cyclops, squint)Polarity Patterning Centr

19、al-Peripheral (C-P) : hh, atonal Anterior-Posterior (A-P): BF1, BF2, SOHo, GH6 Proximal Distal (P-D): hh,Pax2, Pax6, crystallins Dorsal -Ventral (D-V): BMP, RA, vax2 Visual formation projection, connection, growing, learning研究内容及方法基因筛选- 致病突变的定位- 基因表达的差式分析基因的功能研究 - 基因表达模式分析 (expression pattern analys

20、is) = 可能性 (possibility)- 基因的超表达研究(gain-of-function) = 充分性(sufficiency)- 基因的功能失活研究(loss-of-function) = 必要性(necessity)基因调控机制研究- 基因间及与信号通路间的关系- 基因及其表达蛋白的结构和功能Relative timing of EFTF expression. Zuber M E et al. Development 2003;130:5155-5167Pax6Otx2Pax 6Rx1Six3Otx2Pax6Six3BF-1Rx1Eye field transcription

21、 factors (EFTFs)Zuber M E et al. Development 2003;130:5155-5167Relative spatial expression of EFTFsstage 15stage 12.5ETLhx2Zuber M E et al. Development 2003;130:5155-5167RNA microinjection.and animal cap assayfor: gain-of-function loss-of-functionGiuseppina Barsacchi-NCNCNCNCNCNCOptx2NC-NCNCtllNCNC-

22、NCNCNCLhx2-NCNCRx1NCNC-NCSix3NC-NCPax6NCNCNC-ETOptx2tllLhx2Rx1Six3Pax6ET*Animal cap expressesEmbryos injected withETRx1Pax6Six3tllLhx2Optx2Epigenetic interactions among the EFTFsZuber M E et al. Development 2003;130:5155-5167Epigenetic interactions among the EFTFsZuber M E et al. Development 2003;13

23、0:5155-5167Evolutionary conserved mechanisms for eye development? Drosophilaey (Pax6)toysoeyadacdppRx1ETPax6EyaDachSix3Optx2LhxTlllens placodeBMP ?VertebrateGiuseppina BarsacchiStem cells make retina in a dishMouse cells have been coaxed into forming a retina, the most complex tissue yet engineered.

24、 Heres lookin at you kid.M. Eiraku and Y.Sasai at RIKEN Center for Developmental Biology 眼睛发育的基因调控Eye Field (EF) specification : EFTF(toy, ET, Pax6, rx1)Eye Field (EF) split : hedgehog (shh) Nodal (cyclops, squint)Patterning Central-Peripheral (C-P) : hh, atonal Anterior-Posterior (A-P): BF1, BF2, S

25、OHo, GH6 Proximal Distal (P-D): hh,Pax2, Pax6, crystallins Dorsal -Ventral (D-V): BMP, RA, vax2 Visual formation projection, connection, growing, learning Cyclopia The phenotypic spectrum of 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,

26、(From Gilbert, 2002.) shh is necessary for the split of eye fieldHedgehog signalling pathway眼睛发育的基因调控Eye Field (EF) specification : EFTF(toy, ET, Pax6, rx1)Eye Field (EF) split : hedgehog (shh) Nodal (cyclops, squint)Polarity Patterning Central-Peripheral (C-P) : hh, atonal Anterior-Posterior (A-P):

27、 BF1, BF2, SOHo, GH6 Proximal Distal (P-D): hh, Pax2, XRALDH3, Pax6 Dorsal -Ventral (D-V): BMP, RA, vax2 Maturation/Visual formation projection, connection, growing, learningGain-of-function of shh in Drosophila phenotype on eyeRorth P, et al, Development, 1998 Polarity PatterningPeters MA, Current

28、Opinion in Neurobiology, 2002molecular markers for retinaDorsal retina:ET, Xvent2Ventral retina:Xvax2, EphB2Optic stalk:Xpax2, XRALDH3Xvax1(b), Xvax2Ventral forebrain:Nkx2.1, Xvax1(b), Xvax2Lupo G, et al (2000), Int. J. Dev. Biol. 44(6); 627-636. Overexpression of Xvax2BarbieriAM, Lupo G, et al, 199

29、9Overexpression of Xvax2 ventralized the eyeBarbieri, A.M., et al, Development, 1999 Schulte, D., et al, Neron, 1999- by lineage tracing and transplantationSpecification or determination ?Xenopus developmentFertilizationCleavage: blastula blastomereGastrulation: gastrulaNeurulation: neurulaOrganogen

30、esis tailbud tadpoleMetamorphosisMaturation: adultNeural inductionNeurogenesisconnectiongenesisMolecular and cellular mechanismsGenetic information + environmental signalDifferential gene expressionParticular protein componentProperties and behaviors of cells Specification, proliferation, migration,

31、 differentiation, deathAxon guidance, synapse formation, information processing Structure and function/behavior: development, aging, diseasesstructure (CNS/PNS): induction, morphogenesis, growth/maturationCell proliferation during Xenopus developmentNeural tube and crest in early developmentCiliary

32、marginal zone (CMZ) of mature retinaSubventricular zone of mature brainApproaches:BrdU incorporationphosphohistone-H3Xvax2 expression colocalizes with proliferative and postmitotic regions of the late embryonic ventral retina and brainLiu, et al., Dev. Dyn. 2008Xvax2/BrdUXvax2/BrdUScale bars = 100 u

33、mXvax2 overexpression inhibits proliferationCyclinD1Liu, et al., Dev. Dyn. 2008antiphosphohistone-H3HoechstBrdU/HoechstMicroinjectionmiR-124 overexpression decreases cell proliferationRed: BrdU-positive cells;Blue: hoechst counterstained cells;Green: gfp-positive coinjectionMolecular and cellular me

34、chanismsGenetic information + environmental signalDifferential gene expressionParticular protein componentProperties and behaviors of cells Specification, proliferation, migration, differentiation, deathAxon guidance, synapse formation, information processing Structure and function/behavior: develop

35、ment, aging, diseasesstructure (CNS/PNS): induction, morphogenesis, growth/maturationNeuronal migrationReferences to the files for “migration of neural crest”Molecular and cellular mechanismsGenetic information + environmental signalDifferential gene expressionParticular protein componentProperties

36、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/maturationCell DifferentiationThe process

37、in which a cell takes on the appearance and characteristics of a neuron is known as cell differentiationCell type identificationMorphology: single cell labelling (staining/imaging)Position: single cell labelling (staining/imaging)molecular marker: RNA/protein detectionXvax2 overexpression inhibits p

38、roliferationLiu, et al., Dev. Dyn. 2008Lipofection: The different cell types distributed in distinct cell layers and can be distinguished by their specific morphologyMolecular and cellular mechanismsGenetic information + environmental signalDifferential gene expressionParticular protein componentPro

39、perties 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/maturation眼睛发育的基因调控Eye Field (EF)

40、specification : EFTF(toy, ET, Pax6, rx1)Eye Field (EF) split : hedgehog (shh) Nodal (cyclops, squint)Polarity Patterning Central-Peripheral (C-P) : hh, atonal Anterior-Posterior (A-P): BF1, BF2, SOHo, GH6 Proximal Distal (P-D): hh,Pax2, Pax6, crystallins Dorsal -Ventral (D-V): BMP, RA, vax2 Maturati

41、on/Visual formation projection, connection, growing, learningProjection Select cells with GFP fluorescence14 - 20 hr rtTurning Assaytwo-cell stageFertilized eggStage 22dissociationcDNA袁小兵Growth cone turning assayNetrinsShhNTsNetrinsSemaphorinsSlits袁小兵袁小兵Growth cone turning assayGrowth cone of retina

42、l ganglion cell axon showing asymmetrical distribution of beta-actin after exposure to a 5 minute gradient of netrin-1 (top right). This 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). Axon Guidan

43、ce in the Developing BrainSingle retinal ganglion cell in the embryonic visual system. The cell is stained with a dye (HRP) to reveal its 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 mot

44、ile growth process, the growth cone, which responds to guidance cues along the pathway and leads the axon to its final destination in the midbrain. 用顺行追踪的方法显示爪蟾正常的视束用HRP显示的st48/49的爪蟾的视束（左侧注射）魏园园,2004ProjectionMolecular control of eye polarityPeters MA, Current Opinion in Neurobiology, 2002The ventra

45、l regionalization of the eye is specified by interactions of Hh, RA and FGFR signaling.Lupo G, Liu Y et al, Development, 2005眼睛发育的基因调控Eye Field (EF) specification : EFTF(toy, ET, Pax6, rx1)Eye Field (EF) split : hedgehog (shh) Nodal (cyclops, squint)Polarity Patterning Central-Peripheral (C-P) : hh, atonal Anterior-Posterior (A-P): BF1, BF2, SOHo, GH6 Proximal Distal (P-D): hh,Pax2, Pax6, crystallins Dorsal -Ventral (D-V): BMP, RA, vax2 Maturation/V