劉纓-眼睛發(fā)育的基因調控2015

1、劉劉 纓纓 副研究員副研究員中國科學院生物物理研究所中國科學院生物物理研究所視覺視覺聽覺、嗅覺、味覺等聽覺、嗅覺、味覺等80%管懷慶等, , 1998 視覺器官各種遺傳性視覺器官各種遺傳性疾病的發(fā)病已達疾病的發(fā)病已達2%2%左右；左右；近視、斜視、散光等等在近視、斜視、散光等等在我國青年中的發(fā)病率已達我國青年中的發(fā)病率已達到到50%50%左右；眼睛的老視、左右；眼睛的老視、白內障幾乎是每一個人進白內障幾乎是每一個人進入中老年都會遇到的入中老年都會遇到的問題問題。 基各基各因種因種突誘突誘變變變變 : 因因 : 素素l 確定引起各種先天性和后天性眼病的遺確定引起各種先天性和后天性眼病的遺傳和環(huán)境

2、因素，促進對其致病機理研究，提傳和環(huán)境因素，促進對其致病機理研究，提高眼病預防、診斷和治療水平。高眼病預防、診斷和治療水平。l 揭開脊椎動物眼睛發(fā)育的秘密，并為其揭開脊椎動物眼睛發(fā)育的秘密，并為其它器官和系統(tǒng)發(fā)育機制的研究提供線索。它器官和系統(tǒng)發(fā)育機制的研究提供線索。222179 各大醫(yī)院的眼科同行在臨床防治視覺器官疾病方面做了大各大醫(yī)院的眼科同行在臨床防治視覺器官疾病方面做了大量的工作，但是從基因水平來進行研究的工作還非常有限，量的工作，但是從基因水平來進行研究的工作還非常有限，對眼睛發(fā)育的分子機制研究則是近年才開始。對眼睛發(fā)育的分子機制研究則是近年才開始。 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.分離確定與眼睛發(fā)育和疾病發(fā)生相關的基因分離確定與眼睛發(fā)育和疾病發(fā)生相關的基因2.闡明上述基因的功能和調控機制闡明上述基因的功能和調控機制闡明脊椎動物眼睛發(fā)育基因調控機制，為其它闡明脊椎動物眼睛發(fā)育基因調控機制，為其它器官和中樞神經(jīng)系統(tǒng)

4、發(fā)育機制的研究提供線索器官和中樞神經(jīng)系統(tǒng)發(fā)育機制的研究提供線索由此確定引起各種先天性和后天性眼病的遺傳由此確定引起各種先天性和后天性眼病的遺傳和環(huán)境因素，闡明其致病機理和環(huán)境因素，闡明其致病機理, 最終為建立相關最終為建立相關眼病的預防、診斷和臨床治療方法提供依據(jù)眼病的預防、診斷和臨床治療方法提供依據(jù)。l基因篩選基因篩選l- 致病突變的定位致病突變的定位l- 基因表達的差式分析基因表達的差式分析l基因的功能研究基因的功能研究l- 基因表達模式分析基因表達模式分析l- 基因的超表達和功能失活研究基因的超表達和功能失活研究l基因調控機制研究基因調控機制研究l- 基因間及與信號通路間的關系基因間及與

5、信號通路間的關系l- 基因及其表達蛋白的結構和功能基因及其表達蛋白的結構和功能 基因的分離鑒定基因的分離鑒定建立建立cDNA或蛋白質庫或蛋白質庫遺傳病家系的建立遺傳病家系的建立基因的染色體定位基因的染色體定位基因的功能及調控機理研究基因的功能及調控機理研究芯片技術、轉錄芯片技術、轉錄/蛋白質組技術蛋白質組技術病理材料的收集病理材料的收集結構及發(fā)育階段特異性結構及發(fā)育階段特異性組織材料的收集組織材料的收集發(fā)育機制發(fā)育機制致病機理致病機理同源基因的分離同源基因的分離疾病的模式動物疾病的模式動物l腦（端、間、中、后）、脊髓、視網(wǎng)膜l神經(jīng)管（前、后）、視杯/視泡l神經(jīng)板（前、后）、脊索l基因篩選基因篩

6、選l基因的功能研究基因的功能研究 l- 基因表達模式分析基因表達模式分析 (expression pattern analysis) = 可能性可能性 (possibility)l- 基因的超表達研究基因的超表達研究(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基因調控機制研究基因調控機制研究l- 基因間及與信號通路間的關系基因間及與信號通路間的關系l- 基因及其表達蛋白的結構和功能基因及其表達蛋白的結構和功能l 基因結構分析基因結構分析 (

7、啟動子、內含子啟動子、內含子/外顯子分析外顯子分析)l 蛋白結構分析（蛋白結構分析（X射線晶體學、射線晶體學、NMR）l 結構和功能關系分析（點突變，片段缺失結構和功能關系分析（點突變，片段缺失） 1Southern(DNA)Northern(RNA)Western(protein)- - 基因表達模式分析基因表達模式分析l轉錄水平轉錄水平(mRNA)(mRNA)： RT-PCR, Northern, RT-PCR, Northern, 原位雜交原位雜交l翻譯水平翻譯水平( (蛋白蛋白) )： western blot, immunocytochemistrywestern blot, imm

8、unocytochemistry基因的表達模式?jīng)Q定基因功能干擾分析的位置和時間?；虻谋磉_模式?jīng)Q定基因功能干擾分析的位置和時間。- - 基因表達和功能的干擾（超表達和功能失活研究）基因表達和功能的干擾（超表達和功能失活研究） 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. - - 基因表達模式分析

10、基因表達模式分析l轉錄水平轉錄水平(mRNA)(mRNA)： RT-PCR, Northern, RT-PCR, Northern, 原位雜交原位雜交l翻譯水平翻譯水平( (蛋白蛋白) )： western blot, immunohistochemistrywestern blot, immunohistochemistry- - 基因的超表達和功能失活研究基因的超表達和功能失活研究 顯微注射顯微注射(microinjecton) 果蠅果蠅 DNA水平水平 電穿孔電穿孔(electroporation) 斑馬魚斑馬魚 RNA水平水平 轉染轉染(lipofection) 爪蟾爪蟾 蛋白水平蛋白

11、水平 體外處理體外處理(in vitro treatment) 雞雞 小鼠小鼠- - 基因表達和功能的干擾（超表達和功能失活研究）基因表達和功能的干擾（超表達和功能失活研究）基因的表達模式?jīng)Q定基因功能干擾分析的位置和時間基因的表達模式?jīng)Q定基因功能干擾分析的位置和時間。基因功能干擾的位置和時間（劑量）控制基因功能干擾的位置和時間（劑量）控制 ：（1）人工導入的控制）人工導入的控制（2）特異性啟動子的控制：）特異性啟動子的控制：tissue/cell type/gene specific, hsp, etc ( 3 ) 可誘導融合蛋白可誘導融合蛋白 顯微注射顯微注射(microinjecton)

12、果蠅果蠅 DNA水平水平 電穿孔電穿孔(electroporation) 斑馬魚斑馬魚 RNA水平水平 轉染轉染(lipofection) 爪蟾爪蟾 蛋白水平蛋白水平 體外處理體外處理(in vitro treatment) 雞雞 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結構基因：結構基因： “housekeeping” genes, Crystallin (晶狀體球蛋白）晶狀體球蛋白）l調節(jié)基因：調節(jié)基因： 轉錄因子

15、轉錄因子 - homeobox genes: Pax, rx1, vax, Brn3.0 Zinc Finger genes: RAR, RXR Helix-Loop Helix genes: Mi, Mash 信號通路分子及受體信號通路分子及受體 - 小有機分子：小有機分子： retinoic acid (RA) 分泌蛋白和膜受體：分泌蛋白和膜受體：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基因篩選基因篩選l- 致病突變的定位致病突變的定位l- 基因表達的差式分析基因表達的差式分析l基因的功能研究基因的功能研究 l- 基因表達模式分析基因表達模式分

19、析 (expression pattern analysis) = 可能性可能性 (possibility)l- 基因的超表達研究基因的超表達研究(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基因調控機制研究基因調控機制研究l- 基因間及與信號通路間的關系基因間及與信號通路間的關系l- 基因及其表達蛋白的結構和功能基因及其表達蛋白的結構和功能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.)美國國家地理網(wǎng)站 2011十大奇異動物：獨眼鯊上榜 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. 魏園園,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