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1、Major Developmental signaling pathways1.RTK Pathways: FGF, EGF, Jak-Stat 2.TGF-beta3.Wnt4.Hh5.Notch 6.10 Structure and function of a receptor tyrosine kinase6.12 The widely used RTK signal transduction pathway6.13 Activation of the Mitf transcription factor through the binding of stem cell factor by
2、 the Kit RTK protein (Part 2) Copyright 2013 by W. H. Freeman and CompanyMolecular Cell Biology, 7th EditionLodish et al.Figure 16.4 Ligand-induced dimerization of HER1, a human receptor for epidermal growth factor (EGF). Copyright 2013 by W. H. Freeman and CompanyMolecular Cell Biology, 7th Edition
3、Lodish et al.Figure 16.5 Structure of the fibroblast growth factor (FGF) receptor, stabilized by heparan sulfate. Copyright 2013 by W. H. Freeman and CompanyMolecular Cell Biology, 7th EditionLodish et al.Figure 16.6 Activation of EGF receptor by EGF results in the formation of an asymmetric kinase
4、domain dimer. Copyright 2013 by W. H. Freeman and CompanyMolecular Cell Biology, 7th EditionLodish et al.Figure 16.7 The HER family of receptors and their ligands. Copyright 2013 by W. H. Freeman and CompanyMolecular Cell Biology, 7th EditionLodish et al.Figure 16.8 Erythropoietin and formation of r
5、ed blood cells (erythrocytes).6.14 A STAT pathway: the casein gene activation pathway activated by prolactin Copyright 2013 by W. H. Freeman and CompanyMolecular Cell Biology, 7th EditionLodish et al.Figure 16.27 Structure of TGF-b b superfamily of signaling molecules.6.21 Relationships among member
6、s of the TGF- superfamily6.21 Relationships among members of the TGF- superfamily (Part 1)6.21 Relationships among members of the TGF- superfamily (Part 2)6.23 The Smad pathway activated by TGF- superfamily ligands (Part 1)6.23 The Smad pathway activated by TGF- superfamily ligands (Part 2) Copyrigh
7、t 2013 by W. H. Freeman and CompanyMolecular Cell Biology, 7th EditionLodish et al.Figure 16.29 Model of Ski-mediated down-regulation of Smad transcription-activating function.Genetic control of Drosophila embryogenesis Edward B. Lewis Christiane Nuesslein- Volhard Eric Wieschaus Wg:GreenHh: RedSegm
8、ent Polarity mutantswingless (wg)hedgehog (hh) Copyright 2013 by W. H. Freeman and CompanyMolecular Cell Biology, 7th EditionLodish et al.Figure 16.30 Wnt signaling pathway.6.20 The Wnt signal transduction pathways (Part 1)6.20 The Wnt signal transduction pathways (Part 2)6.20 The Wnt signal transdu
9、ction pathways (Part 3) Copyright 2013 by W. H. Freeman and CompanyMolecular Cell Biology, 7th EditionLodish et al.Figure 16.31 Processing of Hedgehog (Hh) precursor protein. Copyright 2013 by W. H. Freeman and CompanyMolecular Cell Biology, 7th EditionLodish et al.Figure 16.32 Hedgehog signaling in
10、 flies. Copyright 2013 by W. H. Freeman and CompanyMolecular Cell Biology, 7th EditionLodish et al.Figure 16.33 Hedgehog signaling in vertebrates. Copyright 2013 by W. H. Freeman and CompanyMolecular Cell Biology, 7th EditionLodish et al.Figure 16.34 (a) Activation of the NF- B signaling pathway. Co
11、pyright 2013 by W. H. Freeman and CompanyMolecular Cell Biology, 7th EditionLodish et al.Figure 16.34 (b) Activation of the NF- B signaling pathway.6.26 Mechanism of Notch activityFigure 16.35 Notch/Delta signaling pathway.Figure 16.36 Proteolytic cleavage of APP and Alzheimers disease.Figure 16.40
12、Multiple signal transduction pathways interact to regulate adipocyte differentiation.Figure 15.6 GTPase switch proteins cycle between active and inactive forms.Figure 15.7 Switching mechanism of G proteins.Figure 15.15 General structure of G proteincoupled receptors.Figure 15.17 General mechanism of
13、 the activation of effector proteins associated with G proteincoupled receptors.Experimental Figure 15.18 Activation of G proteins occurs within seconds of ligand binding in amoeba cells.Figure 15.20 Activation of the muscarinic acetylcholine receptor and its effector K+ channel in heart muscle.Figu
14、re 15.32 Activation of CREB transcription factor following ligand binding to Gs proteincoupled receptors.Figure 15.34 Role of b b-arrestin in GPCR desensitization and signal transduction.Phenotypes of Mutants in the Wingless Sigaling Pathway Wild Type Loss of Wg Signaling Constitutive Wg SignalingWi
15、ldtypewinglesssulfateless(sfl)sugarless (sgl)Phenotypes of sulfateless and sugarless Biosynthesis of Heparan Sulfate ProteoglycanH2COHCOOH2COHH2COHH2COHHNAcUDP-D-glucoseUDP-D-glucuronic acidUDP-D-glucose dehydrogenaseSugarless HS N-deacetylase/N-sulfotransferasesulfatelessUronosyl C5-epimerization2-
16、O-sulfation6-O-sulfationCOOCOOCOOH2COHHNAcHNAcHNAcH2COHH2COHH2COHH2COHCOOCOOCOOCOOHNSO3HNSO3HNSO3HNAcH2COSO3H2COSO3H2COSO3H2COHHNSO3HNSO3HNAcHNSO3Syndecan Glypican PerlecanStructure of Heparan Sulfate ProteoglycansThe Gal-4/UAS techniqueEnhancer Trap-Gal4UAS-Gene XXTranscriptional Activation of Gene
17、 XTussue Specific Expression of Gal-4UAS Genomic EnhancerBrand and Perrimon: Development (1993)Gene XActivated Arm protein can rescue the cuticle defects of sfl and sgl Control PrdGAL4 / UAS ArmActsflsglOverexpression of Wg in sfl and sgl can rescue Wg signaling defect in a dose dependent mannerCont
18、rol16CPrd GAL4 / UAS Wg 25CsflsglPrd GAL4 / UAS WgDFZ2DFZ2HSPGWg expressing cellDFZ2DFZ2HSPGWg expressing cellWild typesgl or sfl mutantsnGlypican in Wnt signaling nHaecker et al (1997) DevelopmentnLin and Perrimon (1999) Nature nLin et al (2001) Development nHan et al (2004) DevelopmentnHan et al (
19、2005) Development nTao et al. (2005) CellnYan et al. (2009) Dev Cell nCK-1a in Wnt signaling nLiu, et al (2002) CellnPygopus (Pygo) in Wnt signaling:nBelenkaya et al (2002) Development nSchwab et al (2007) BMC BiologynSong et al (2007) Development Wnt信号通路成员的鉴定信号通路成员的鉴定53深层分析Morphogen (形态发生素形态发生素) :
20、An important concept in BiologyTuring, 1952Morgan, 1897Target genes Cell fates54深层分析发育过程中关键的形态发生素发育过程中关键的形态发生素nWnt / WinglessnHedgehog (Hh)nTGF- / BMPnFGF特性特性1. 进化上保守进化上保守2. 对胚胎发育至关重要对胚胎发育至关重要3. 功能异常与多种人类疾病密切相关功能异常与多种人类疾病密切相关55深层分析Cell proliferation and differentiation.(Driesch 1891, 1908)When he se
21、parated two sea urchin blastomeres, they produced twohalf-sized blastula, showing that cells are potentially independent,but function together to form a whole organism (Driesch 1891, 1908).(Morgan 1901).Morgan noted the polarity of organisms and that regeneration inworms occurs with different rates
22、at different positions. This ledhim to postulate that regeneration phenomena are influenced bygradients of “formative substances”Morphogen 56深层分析Morphogen “ A form generating substance that diffuses through a tissue, its distribution dictating the development of cells in the tissue”Alan Turing (1952
23、). The chemical basis of morphogenesis. Philos. Trans. R. Soc. Lond. B Biol. Sci. 237, 37-7257深层分析Growth Factor MorphogensSignaling molecules produced in a restricted regions within a tissue. Morphogens emanate from their source to form a long-range concentration gradient.Receiving cells interpret m
24、orphogen gradient by activating target gene expression at discrete concentration thresholds thereby acquiring positional information.Two criteria: 1. Concentration dependence. 2. Direct action at a distanceWnt, Hh, and TGF- are morphogens.58深层分析形态发生素功能失调相关的人类疾病形态发生素功能失调相关的人类疾病前脑无裂畸形前脑无裂畸形(HPE)(HPE)
25、骨发育不良骨发育不良神经管发育缺陷神经管发育缺陷肾母细胞瘤肾母细胞瘤 结肠癌结肠癌 家族性渗出性玻璃家族性渗出性玻璃体视网膜病变体视网膜病变 骨质疏松骨质疏松- -假性神经胶质瘤假性神经胶质瘤综合症综合症(OPS) (OPS) 裂足症裂足症先天性四肢切断症先天性四肢切断症59深层分析Vertebrates: SHh Wnt BMPHh, Wg, Dpp: essential morphogens in wing disc60深层分析 Key questions for morphogen gradient formation (1) how do they secreted (2) how d
26、o they move(3) how do they establish their activity gradients61深层分析Models for morphogen MovementTwo main modelsDiffusionTranscytosisOther modelsCytonemesArgosomes62深层分析Dpp is a morphogen in wing patterning Dpp Sal Omb P-MADUAS-GFP-Dpp Dpp-Gal463深层分析Models on Dpp movement A prevailing view is that Dp
27、p moves across cells by planar transcytosis initiated by dynamin-mediated endocytosis. (Entchev, E. V., Schwabedissen, A., and Gonzalez-Gaitan, M. (2000). Cell 103, 981-991)However, mathematical modeling suggested that diffusive mechanism(s) is much more plausible than non-diffusive mechanisms. (Lan
28、der, A. D., Nie, Q., and Wan, F. Y. (2002). Dev Cell 2, 785-796)Question: Which mechanism controls Dpp movement?64深层分析Extracellular Dpp gradient coincides with Dpp activity gradient in wing disc65深层分析Dynamin-mediated endocytosis66深层分析Dpp signaling is reduced cell-autonomously in dynamin-defective ce
29、lls, but is normal in cells behind the dynamin-defective cells67深层分析Extracellular Dpp can move through dynamin-defective cells68深层分析Extracellular Dpp can move through dynamin-defective cells69深层分析Dynamin-mediated endocytosisIs required for Dpp signalingDown-regulates extracellular Dpp levelsIs not e
30、ssential for Dpp movement70深层分析Structure of heparan sulfate proteoglycansSyndecanGlypicanPerlecanGene Number in Drosophila 12Dally Dally-like 171深层分析72深层分析Dpp fails to move across cells mutant for sflsfl mutant clones73深层分析Dpp fails to move across cells mutant for or dally-dlydally-dly mutant clones
31、74深层分析Dpp fails to move across cells mutant for sfl or dally-dlyGFP-Dpp Extracellular Dpp Mergesfl dally-dly75深层分析The extracellular Dpp gradient represents Dpp activity gradient. Dynamin-mediated endocytosis is not required for Dpp movement, but is involved in Dpp signaling.The HSPGs Dally and Dly a
32、re required for Dpp movement.We propose that Dpp moves along the cell surface by attachment to the HSPGs Dally and Dly.76深层分析A model for Dpp movement across cells77深层分析形态发生素形态发生素（Wnt、Hh、BMP）梯度形成机制梯度形成机制限制扩散机制限制扩散机制Wnt morpohgen: Beag et al. (2001) Development Han et al. (2004) Development. Han et al
33、. (2005) Development Tao et al. (2005) Cell Yan et al (2009) Developmental Cell Hh morphogen: Han et al (2004) Development Yan et al (2010) Development BMP morphogen: Belenkaya et al (2004) Cell78深层分析Wg acts as a morphogen in the wing discDllSensMergeExtracellular Wg 79深层分析Wnt Secretion Model in 200
34、6 Wntless/Evi/SrtK. Balser lab (2006) CellM. Boutros lab (2006) CellE. Selva Lab (2007) Development 80深层分析What is Retromer complex?81深层分析What does retromer do? 82深层分析1. Retrieval of receptors from endosomes to Golgi- Lysosomal hydrolases in mammalian cells - Vacuolar hydrolases in yeast2. Promoting
35、of polymeric immunoglobulin receptor transcytosisKnown functions of retromer complex83深层分析Role of retrormer complex in Wingless signalingPrevious views:Retromer is required for Wnt signaling. Retromer controls Wnt signaling in its producing cells. Disruption of retromer affects Wnt gradient formatio
36、nRetromer may control Wnt modifications or association with lipoproteins.Coudreuse et al. Science, 2006; Prasad et al. Development, 2006.Questions:How does retromer control Wnt signaling activity in Wnt producing cells?84深层分析Generation of dVps35 null mutant and RNAi construct85深层分析Wg is accumulated
37、in dVps35 mutant cellsdVps35 clonesUAS-dVps35 Ri /En-G486深层分析Depletion of dVps35 decreases Wg secretion in S2 cells87深层分析Retromer is not required for Hh and Dpp functionsHh distribution and signaling Dpp signaling 88深层分析In the absence of dVps35 activity:Wg protein is accumulated inside Wg-producing
38、cells, but reduced on the cell surface of Wg-producing cells and in surrounding Wg-recieving cells.Wg protein levels are reduced in conditioned medium. Conclusion: Retromer is required for Wg secretion.Question: How Does Retromer regulate Wg secretion ?89深层分析Similarity between dvps35 and Wls mutants
39、BAWgGFPE-cadmergeBAWgGFPE-cadmergeBAWgGFPE-cadmergeBAWgGFPE-cadmergeGFP Wg MergewlsHypothesis: Retromer may control Wg/Wnt secretion by regulating Wls activity90深层分析dVps35 interacts with Wls91深层分析Question: What would happen with Wls in the absence of retromer?92深层分析Wls GFP Merge Wls protein is reduc
40、ed in the vps35 mutant cells 93深层分析Retromer controls Wls stability6hrs8hrsWls-V5 Hh-G4/vps35 RNAi Wls-V5 Hh-G4/vps35 RNAi1hr3hrs94深层分析QuestionsWhat is the cellular distribution of Wls ?What is the role of retromer in regulating Wls activity?95深层分析Wls is distributed on the cell surface 96深层分析Localiza
41、tion of Wls on cell membraneWls-V5CD8-mRFPmerge97深层分析Wls particles co-localize with endosome markersRab5-GFP Wls Merge GFP-2xFYVE Wls Merge 98深层分析Dynamin-mediated endocytosis99深层分析Wls is elevated on the plasma membrane of shi cellsshits1Rab5(RNAi)hrsd28Wls DE-cad Merge 100深层分析Dynamin-mediated endocy
42、tosis101深层分析Enhanced Wls levels in rab5 and hrs mutantshrs cloneWls Rab5 MergeRab5 RNAiHh-Gal4Wls DE-cad Merge 102深层分析Question: Does Retromer regulate Wnt secretion using similar mechanism(s) in vertebrate cells?103深层分析Depletion of Vps35 reduced Wnt3A and Wnt5A secretion104深层分析hWls forms a complex w
43、ith Vps35105深层分析Human retromer complex controls the stability of hWls protein 106深层分析Belenkaya et al. Developmental Cell 2008 Port et al. Nature Cell Biology 2008Yang et al. Developmental Cell 2008 Franch-Marro et al. Nature Cell Biology 2008Pan et al. Developmental Cell 2008Wnt Secretion Model in 2
44、008107深层分析Question 1: Whats role of retromer in Wnt signalingQuestion 2: Mechanisms of Retrmer in Wnt secretion: Role of Snx3 in Wnt secretionQuestion 3: Roles of Retromer in other developmental patterning: Apical-Basal Polarity108深层分析 What does Retromer do? 109深层分析Homology between Human sorting nex
45、ins and Drosophila SNXs110深层分析 Null allels of Drosophila SNX homologues111深层分析Generation of Dsnx3 Null mutants by P-element hopping 112深层分析Dsnx3 is required for Wg secretion and signaling113深层分析 Wg expression monitored by wg-lacZ is not alteredExtracellular Wg is reduced in the Dsnx3 mutant cells 11
46、4深层分析Wg (conditioned media)Wg (lysate)Beta-actinControl RNAi1 RNAi2 Dsnx3 is essential for Wg secretion in cultured S2 cells115深层分析Dsnx3 is required for Wg secretion and signalingUAS-Dsnx12RNAi116深层分析Dsnx3 function is not essential for Hh and Dpp signaling117深层分析 HypothesisDsnx3 influences Wnt secre
47、tion by disrupting Wls recycling from endosomes to the Trans-Golgi Network.118深层分析Wls levels were markedly reduced in Dsnx3 clones Wg secretion defect can be rescued by Wls overexpression 119深层分析 Dsnx3 can colocalize with hWls in early Endosomes of Hela cells 120深层分析Dsnx3 interacts with Dwls in S2 c
48、ells121深层分析 Mutiple alignment of Snx3 homologue genes PtdIns3P binding motif 122深层分析PtdIns3P binding site is essential for Dsnx3 functionUAS-SNX12-EGFPMerge 123深层分析 Dsnx3Wnt Secretion model in 2011124深层分析Casali & Batlle, 2009, Cell Stem Cell 4:124-127.哺乳动物结肠隐窝哺乳动物结肠隐窝果蝇中肠果蝇中肠ISC: Intestinal Stem Cel
49、ls ISC: Intestinal Stem Cells 肠干细胞肠干细胞TA: Transient Amplifying TA: Transient Amplifying 短暂扩充细胞短暂扩充细胞EB: Enteroblast cells EB: Enteroblast cells 成肠细胞成肠细胞EC: Eterocyte cells EC: Eterocyte cells 肠上皮细胞肠上皮细胞EE: Enteroendocrine cells EE: Enteroendocrine cells 内分泌细胞内分泌细胞果蝇中肠是研究果蝇中肠是研究稳态（稳态（homeostasis) )维持
50、和维持和干细胞调控干细胞调控的理想系统的理想系统 关键科学问题：关键科学问题：形态发生素信号如何调控干细胞和组织稳态维持？形态发生素信号如何调控干细胞和组织稳态维持？125深层分析BMPBMP信号通过气管与肠道两种不同器官之间的对话来实现信号通过气管与肠道两种不同器官之间的对话来实现调控肠道干细胞和组织内稳态调控肠道干细胞和组织内稳态Li et al., Developmental Cell 2013Highlighted by F1000BMPBMP信号通过气管与肠道两种不同器官之信号通过气管与肠道两种不同器官之间的对话来实现调控肠道干细胞和组织内间的对话来实现调控肠道干细胞和组织内稳态；提
51、出器官稳态；提出器官- -器官之间建立信号通讯器官之间建立信号通讯并协同调控稳态维持的概念并协同调控稳态维持的概念126深层分析HhHh信号调控肠干细胞活性和稳态维持信号调控肠干细胞活性和稳态维持Li et al., Li et al., Stem Cell Reports 2014Stem Cell Reports 2014HhHh信号通过激活信号通过激活JNKJNK和和JAK/STATJAK/STAT调调控稳态维持控稳态维持127深层分析Zorn AM and Wells JM. Annu. Rev. Cell Cev Biol. 2009哺乳动物内胚层器官的形成及谱系建立哺乳动物内胚层器官的形成及谱系建立128深层分析Model of early A-P patterningMesodermEndodermZorn and Wells(2009) Annu Rev Cell Dev Biol. Wnt signaling and endoderm patterning?repress foregut identity promote hindgut fate129深层分析
