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1、Chapter 18Gene Regulation during DevelopmentuuThree Strategies by which Cells Are Instructed to Express Specific Sets of Genes during DevelopmentuExamples of the Three Strategies for Establishing Differential Gene ExpressionuThe Molecular Biology of Drosophila EmbryogenesisThree Strategies:n nmRNA l
2、ocalizationn nCell-to-cell contactn nSignaling through the diffusion of secreted signaling moleculesmRNA localizationmRNAs serve as a critical regulatory moleculeThey are transported along elements of the cytoskeleton which have intrinsic polarityThe transportation is realized by an “adapter protein
3、”“Adapter” proteinsContaining two domainsOne recognizing the 3 UTR of the mRNAThe other associates with the components of the cytoskeletonThereby it crawls along the actin filamentCell-to-Cell Contact and Secreted Cell Signaling MoleculesnThree steps:1.The synthesized signaling molecules are deposit
4、ed in the membrane or secreted into the extracellular matrix.2.They are recognized by the receptor on the surface of recipient cells3.The changes in gene expression in the recipient cell is achieved through the signal transduction pathwaysSignal Transduction Pathwaysa. a.Ligand-receptor interaction
5、induces kinase cascade that modifies regulatory proteins present in nucleus.b.Activated receptor cause the release of DNA-binding protein so it can enter the nucleus, regulate gene transcription.c.The intracytoplasmic domain of the activated receptor is cleaved to enter the nucleus and interact with
6、 DNA-binding protein.Gradient of Secreted Signaling Molecules Two concepts:nPositional information: a cells development is influenced by its location within the developing embryo.nMorphogens: signaling molecules that control position information a. a.extracellular gradientextracellular gradientb.Cel
7、ls located near the source of morphogen receive high concentration of the signaling molecule, experience peak activation of receptorsc.determine most regulatory protein enter the nucleus;d. The different levels of the regulatory factor lead to the expression of different sets of gene.Part Two: Examp
8、les of the Three Strategies1.The Localized Ash1 Repressor Controls Mating Type in Yeast by Silencing the HO gene2.A Localized mRNA Initiates Muscle Differentiation in the Sea Squirt Embryo3.Cell-to-Cell Contact Elicits Differential Gene Expression in the Sporulating Bacterium4.A Skin-Nerve Regulator
9、y Switch Is Controlled by Notch Signaling in the Insect CNS5.A Gradient of the Sonic Hedgehog Morphogen Controls the Formation of Different Neurons in the Vertebrate Neural Tube1.mRNA localization2.Cell-to-cell contact3.Signaling through the diffusion of secreted signaling molecules1. The Localized
10、Ash1 Repressor Controls Mating Type in Yeast by Silencing the HO geneYeast lifecycle1.Budding 2.Conjugation 3.Spore The The HOHO gene gene is involved in the budding of yeast. is involved in the budding of yeast.It has two It has two activatorsactivators : : SWI5SWI5 and and SBFSBF. .alter the nucle
11、osomeActive only at correct stage of cell cycle1. Mating Type in Yeast by Silencing the HO genenA haploid yeast cell budding to produce a mother cell and a smaller daughter cell.nThe daughter cell cant switch due to localized Ash1 repressor,it cant express HO which initiate switching.nThe mother cel
12、l can switch: it lacks Ash1,and is able to express HO.The The ash1ash1 gene is transcribed in the mother cell during gene is transcribed in the mother cell during budding.The encoded mRNA localized within the daughter cell budding.The encoded mRNA localized within the daughter cell by sliding along
13、polarized by sliding along polarized actinactin filaments, depends on She2 and filaments, depends on She2 and Sh3 adapter proteins that bind the 3UTR and myosin. Sh3 adapter proteins that bind the 3UTR and myosin. 2.A Localized mRNA initiates Muscle Differentiation in the Sea Squirt Embryon nMacho-1
14、Macho-1 regulatory protein is a major determinant to regulatory protein is a major determinant to form muscle .form muscle .n nThe The Macho-1Macho-1 mRNA encodes a zinc finger DNA- mRNA encodes a zinc finger DNA-binding protein that is believed to activate the binding protein that is believed to ac
15、tivate the transcription of muscle-specific genes,such as transcription of muscle-specific genes,such as actinactin and and myosin.myosin.n nThese genes are expressed only in muscles because These genes are expressed only in muscles because Macho-1Macho-1 is made only in those cells. is made only in
16、 those cells. nMacho-1 mRNA is ninitially distributed nthroughout the cytoplasm of unfertilized eggs but becomes localized to the vegetal(bottom)region shortly after fertilization, ultimately inherited by two cells of the eight-cell embryos,thus the two cells go on to form the tail muscles.3. Cell-t
17、o-Cell Contact Elicit Differential Gene Expression in the Sporulating Bacterium, B.subtilisn nB.subtilisB.subtilis spore formation:a septum form at an spore formation:a septum form at an asymmetric location within the sporangium, asymmetric location within the sporangium, produce two cells remain at
18、tached through produce two cells remain attached through abutting membranes.The smaller cell is abutting membranes.The smaller cell is foresporeforespore, , it ultimately forms the spore.The larger cell is the it ultimately forms the spore.The larger cell is the mother cell,it aids the development o
19、f the spore.mother cell,it aids the development of the spore.n nThe The foresporeforespore influences the expression of genes influences the expression of genes in the neighboring mother cell.in the neighboring mother cell.1.1.芽孢形成过程芽孢形成过程2.2.形成过程中基因活性的调控形成过程中基因活性的调控: : 特异特异亚基亚基控制基因组活性控制基因组活性 a. Spo
20、OA 对外界刺激产生应答对外界刺激产生应答决定芽孢是否决定芽孢是否/何时形成何时形成 b.前孢子和母细胞中的前孢子和母细胞中的亚基级联亚基级联时间依赖的变化时间依赖的变化 c. 细胞细胞-细胞间的信号传导：细胞间的信号传导： 位于隔上的受体位于隔上的受体前孢子前孢子和和母细胞中事件的协调母细胞中事件的协调 3. Cell-to-Cell Contact Elicit Differential Gene Expression in the Sporulating Bacterium, B.subtilisA A 和和H H亚基亚基E E 和和F F亚基亚基 胞胞胞胞外外外外信号信号信号信号 蛋白
21、激酶蛋白激酶蛋白激酶蛋白激酶 SpoOASpoOASpoOASpoOA SpoOASpoOAPPFigure 12.20 Role of SpoOA in Bacilius sporulation激活激活E E 和和F F 的表达基因的表达基因 A A：F F 激活激活结果：结果： E E 是是母细胞特异的母细胞特异的 F F 是是前孢子特异的前孢子特异的Activation of Activation of k k during during BacilusBacilus sporulationsporulation前孢子中的前孢子中的F F 和和母细胞中母细胞中E E 被被激活激活亚基级联
22、亚基级联F F G G ：识别芽孢分化后期识别芽孢分化后期要转录的基因（要转录的基因（SpoBSpoB）SpoFSpoF K K： 指导母细胞后期指导母细胞后期要转录的基因要转录的基因n n F factor in F factor in foresporeforespore activated the activated the spoIIRspoIIR gene. gene.n nThe encoded The encoded SpoIIRSpoIIR protein is secreted and protein is secreted and associate with the se
23、ptum where it triggers the associate with the septum where it triggers the proteolyticproteolytic processing of an inactive form of processing of an inactive form of E(pro-E(pro- E)in the mother cell.The pro-E)in the mother cell.The pro- E protein E protein contains an N-terminal inhibitory domain t
24、hat contains an N-terminal inhibitory domain that blocks blocks E activity and tethers the protein to the E activity and tethers the protein to the membrane of the mother cell.membrane of the mother cell.n nAfter the cleavage of the N-terminal peptide, the After the cleavage of the N-terminal peptid
25、e, the activated activated E protein leads to the transcription of E protein leads to the transcription of target genes.target genes.n nSpoIIR functions as a signaling molecule that acts at the interface between the forespore and the mother cell,elicits differential gene expressions.n nInduction req
26、uires cell-to-cell contact because the forespore produces small quantities of SpoIIR which are insufficient to elicit the processing of E in the other cells except the abutting mother cell.4.Delta-Notch Signaling control skin-nerve regulatory in the Insect CNSn nIn insect embryo, Neurons In insect e
27、mbryo, Neurons of the ventral nerve cord of the ventral nerve cord arises from arises from neurogenicneurogenic ectoderm, the other cell ectoderm, the other cell population is ventral skin.population is ventral skin.n nSignaling between the two Signaling between the two populations decide which popu
28、lations decide which to become skin or neuron.to become skin or neuron.n nThe developing neurons The developing neurons contain a signaling molecule contain a signaling molecule DeltaDelta on their surface,which on their surface,which binds to and activates the binds to and activates the NotchNotch
29、receptor on the skin receptor on the skin cells.cells.n nActivation causes the Activation causes the intracytoplasmicintracytoplasmic domain of domain of Notch (Notch (NotchICNotchIC) to be ) to be released from the cell released from the cell membrane and enter the membrane and enter the nuclei,the
30、n it associates with nuclei,then it associates with the DNA-binding protein the DNA-binding protein Su(H).Su(H).n nNotchIC displaces the repressor proteins in complex with Su(H),turning Su(H) into a activator instead. n nThe Su(H)-NotchIC complex activates genes that encodes transcriptional represso
31、rs which block the development of neurons.5. A Gradient of the Shh Morphogen Controls the Formation of Different Neurons in the Vertebrate Neural TubenIn all vertebrate embryos, there is a stage when cells located along the dorsal ectoderm move toward internal regions of the embryo and form the neur
32、al tube.nCells located in the ventralmost region of the neutral tube form floorplate, where the secreted signaling molecule Sonic Hedgehog(Shh) is expressed. Shh functions as a gradient morphogen. n nShhShh is secreted from the is secreted from the floorplatefloorplate and diffuses through the and d
33、iffuses through the extracellular matrix of the neutral tube.extracellular matrix of the neutral tube.n nthe high concentrations of the high concentrations of ShhShh high number of high number of ShhShh receptors receptors activated activated n nThe graded distribution of the The graded distribution
34、 of the ShhShh protein leads to the formation of protein leads to the formation of distinct neuronal cell types in the ventral half of the neural tube. distinct neuronal cell types in the ventral half of the neural tube. n nHigh and intermediate levels lead to the development of the V3 High and inte
35、rmediate levels lead to the development of the V3 neurons and neurons and motorneurons,respectively.Lowmotorneurons,respectively.Low and lower levels lead to and lower levels lead to the development of the V2 and V1 the development of the V2 and V1 interneuronsinterneurons. .uuThree Strategies by wh
36、ich Cells Are Instructed to Express Specific Sets of Genes during DevelopmentuExamples of the Three Strategies for Establishing Differential Gene ExpressionuThe Molecular Biology of Drosophila Embryogenesisn nLocalized determinants and cell signaling Localized determinants and cell signaling pathway
37、s are both used to establish pathways are both used to establish positional information that result in gradients of regulatory that result in gradients of regulatory proteins that pattern the anterior-posterior (head-proteins that pattern the anterior-posterior (head-tail) and dorsal-ventral (back-b
38、elly) body axes.tail) and dorsal-ventral (back-belly) body axes.n nA recurring theme is the use of complex A recurring theme is the use of complex regulatory regulatory DNAsDNAs to bring transcriptional to bring transcriptional activators and repressors to genes whose product activators and represso
39、rs to genes whose product define different regions of the embryo. define different regions of the embryo. The Molecular Biology of Drosophila EmbryogenesisOocyte n. 卵母细胞卵母细胞 metamorphosis n.变形变形Larval adj.幼虫的幼虫的 Larva n.幼虫幼虫 pupa n.昆昆蛹蛹Molt n. 换毛期换毛期 Thorax n. 胸腔胸腔An Overview of Drosophila Embrogene
40、sisThe Molecular Biology of Drosophila EmbryogenesisOocyte n. 卵母细胞卵母细胞 metamorphosis n.变形变形Larval adj.幼虫的幼虫的 Larva n.幼虫幼虫 pupa n.昆昆蛹蛹Molt n. 换毛期换毛期 Thorax n. 胸腔胸腔A Morphogen Gradient Controls Dorsal-Ventral Patterning of the Drosophila Embryon nThe dorsal-ventral patterning of the early Drosophila e
41、mbryo is The dorsal-ventral patterning of the early Drosophila embryo is controlled by a regulatory protein called controlled by a regulatory protein called DorsalDorsal. .n nRegulated nuclear transport of the Dorsal protein is controlled by Regulated nuclear transport of the Dorsal protein is contr
42、olled by the cell signaling molecule the cell signaling molecule SpSp tzletzle, , which is distributed in a which is distributed in a ventral-to-dorsal gradient within the extracellular matrix.ventral-to-dorsal gradient within the extracellular matrix.nAfter fertilization, Sptzle binds to the cell s
43、urface Toll receptor.Depending on the concentration of Sptzle,Toll is activated to greater or lesser extent.Peak activation of Toll is in ventral regions, where the Sptzle concentration is highest.nToll signaling causes the degration of a cytoplasmic inhibitor Cactus,and the release of Dorsal from t
44、he cytoplasm into nuclei.nThe Dorsal gradient specifies three major thresholds of gene expression across the dorsal-ventral axis of embryos undergoing cellularization.twist generhomboid genesog gene The highest levels of the Dorsal gradient activate the The highest levels of the Dorsal gradient acti
45、vate the expression of the expression of the twist genetwist gene in the in the ventralmostventralmost 18cells that 18cells that forms the mesoderm. The twist 5 regulatory DNA contains forms the mesoderm. The twist 5 regulatory DNA contains two low-affinity Dorsal binding sites, so peak levels of th
46、e two low-affinity Dorsal binding sites, so peak levels of the Dorsal gradient are required for the efficient occupancy of Dorsal gradient are required for the efficient occupancy of these sites.these sites. The rhomboid geneThe rhomboid gene is activated by intermediate levels of the is activated b
47、y intermediate levels of the Dorsal protein in the ventral Dorsal protein in the ventral neurogenicneurogenic ectoderm.The ectoderm.The enhancer in it has a cluster of mostly low-affinity Dorsal enhancer in it has a cluster of mostly low-affinity Dorsal binding sites but one high-affinity site. Thus
48、, the rhomboid binding sites but one high-affinity site. Thus, the rhomboid enhancer can be activated by both the high and the enhancer can be activated by both the high and the intermediate levels of Dorsal protein.intermediate levels of Dorsal protein. The lowest levels of the Dorsal protein are s
49、ufficient to activate The lowest levels of the Dorsal protein are sufficient to activate the the sogsog gene gene in both the ventral and the dorsal in both the ventral and the dorsal neurogenicneurogenic ectoderm. The enhancer of the gene contains four high-affinity ectoderm. The enhancer of the ge
50、ne contains four high-affinity Dorsal binding sites. Dorsal binding sites. Both rhomboid and sog gene are kept off by the transcriptional repressor Snail in the mesoderm for they have binding sites for it . Thus the Snail repressor and the affinities of the Dorsal binding sites together determine sp
51、ecific gene expression. twistSegmentation Is Initiated by Localized RNAs at the Anterior and Posterior Poles of the Unfertilized Eggn nIn fertilization, Drosophila egg contains two localized mRNAs. One, the bicoid mRNA, is located at the anterior pole, while the other,the oskar mRNA at the posterior
52、 pole.n nThe oskar mRNA is first deposited at the anterior end of the oocyte, then transported from anterior to posterior regions. n nthe the oskaroskar mRNA interacts mRNA interacts with adapter proteins which with adapter proteins which associate with the associate with the growing + growing + end
53、s of the microtubulesends of the microtubules depends on specific sequences depends on specific sequences within the 3UTR region, within the 3UTR region, thereby transported into the thereby transported into the posterior posterior plasmplasm. .n nAfter fertilization the After fertilization the cell
54、scells that inherit the localized that inherit the localized oskaroskar mRNA form the pole cells.mRNA form the pole cells.n nThe localization of the The localization of the bicoidbicoid mRNA in mRNA in anterior regions also anterior regions also depends on sequences depends on sequences contained wi
55、thin its 3 contained within its 3 UTR. Therefore, the UTR. Therefore, the 3UTR is important in 3UTR is important in determine where each determine where each mRNA becomes mRNA becomes localized.localized.n nIf the 3UTR from the If the 3UTR from the oskaroskar mRNA is replaced with that mRNA is repla
56、ced with that from from biciodbiciod, the hybrid , the hybrid oskaroskar mRNA is located to anterior mRNA is located to anterior regions (just as regions (just as biciodbiciod normally is). normally is). The Bicoid Gradient Regulates the Expression of Segmentation Genes in a Concentration-Dependent
57、Fashion n nThe Bicoid regulatory protein diffuses away from its source of synthesis at the anterior pole and simply distributed across the syncitial embryo.n nThere are peak levels of the Bicoid protein in anterior regions, intermediate levels in the central regions and low levels in posterior regio
58、ns.nOnly high concentrations of Bicoid activate the expression of orthodenticle, while both high and intermediate concentrations are sufficient to activate hunchback.nThis differential regulation of orthodenticle and hunchback depends on the binding affinities of Biciod recognition. nThe Bicoid prot
59、ein binds to DNA as a monomer, Bicoid monomers interact with each other to foster the cooperative occupancy of adjacent sites.Hunchback Expression Is also Regulated at the level of Translationn nThe hunchback gene is actually transcribed from two promoters:one activated by the Bicoid gradient, the o
60、ther is maternal promoter. n nNanos is an RNA-binding protein which block the translation of the maternal transcript in posterior regions.This dual regulation of hunchback expression produces a steep Hunchback protein gradient with the highest concentrations located in the anterior half of the embry
61、o and sharply diminishing levels in the posterior half.AP轴线形成模式Hunchback：母体mRNA在卵中均匀分布，受精后前区高浓度的Bicoid蛋白激活合子hunchback基因的表达，从而帮助形成hunchback蛋白浓度梯度。Caudal: 母体mRNA在卵中均匀分布，受精后bicoid蛋白抑制其在前区的表达，因而Caudal蛋白形成类似于nanos的浓度梯度。The Gradient of Hunchback Repressor Establishes Different Limits of Gap Gene Expressio
62、nn nHunchback functions as a transcriptional repressor to establish different limits of expression of the gap genes, Krppel, knirps and giant.n nHigh levels of the Hunchback protein repress the transcription of Krppel, whereas intermediate and low levels of the protein repress the expression of the
63、knirps and giant, respectively.n nNot the binding affinities but the number of Hunchback repressor sites may be more critical for distinct patterns of Kr ppel, knirps and giant expression.一定浓度的hunchback蛋白激活kruppel基因的表达，而高浓度的hunchback则对后者起抑制作用。Hunchback and Gap proteins produce Segmentation Stripes o
64、f Gene Expression n nThe eve gene is expressed in a series of seven alternating or pair-rule stripes that extend along the length of the embryo.n nThe eve protein coding sequence is less than 2kb in length while the 12kb of the regulatory DNA contains five separate enhancers that together produce th
65、e seven different stripes of eve expression. We will consider the expression of eve stripe 2 for example.n nEve stripe 2 contains binding sites for four different regulatory proteins: Bicoid, Hunchback, Giant, and Krppel.n nIn principle, Bicoid and Hunchback can activate the stripe 2 enhancer in the
66、 entire anterior half of the embryo where they both present, Giant and Krppel function as repressors that form the anterior and posterior borders, respectively.Pair-rulePair-rule基因的每个表达横纹由一组基因的每个表达横纹由一组GapGap转录因子控制转录因子控制Pair-rule基因表达的间隔性重复，无法通过单一浓度梯度来控制，而是由多个转录因子来控制。例如， Even-skipped在第三类体节中的表达受bicoid
67、和和hunch-back的激活，而受giant和和kruppel的抑制。n One is competition. Two of the three Krppel binding sites directly overlap Boicoid activator sites,precludes the activator to bind. KrppelKrppel mediates transcriptional repression through mediates transcriptional repression through two distinct mechanismstwo di
68、stinct mechanisms. .n The other is quenching. The third Krppel is able to inhibit the action of the Bicoid activator bound nearby. It depends on the recruitment of the transcriptional repressor CtBP, which contains a enzymatic activity that impairs the function of neighboring activatorsGap Repressor
69、 Gradients Produce many Stripes of Gene Expressionn nThe same basic mechanism of how eve stripe 2 is formed applies to the regulation of the other eve enhancers as well. The stripe borders are defined by localized gap repressors:Hunchback establishes the anterior border, while Knirps specifies the p
70、osterior border.n nHowever,the differential regulation of the the two enhancers by the repressor gradient produces distinct anterior borders for the eve stripes. The The eve eve stripe 3 enhancer is repressedstripe 3 enhancer is repressed by high levels of the by high levels of the Hunchback gradien
71、t but low levels of the Hunchback gradient but low levels of the KnirpsKnirps gradient, gradient, while the stripe 4 enhancer is just the opposite, this while the stripe 4 enhancer is just the opposite, this differences are due to the number of repressor binding sites. differences are due to the num
72、ber of repressor binding sites. 黑腹果蝇发育调控机制n n母体基因建立了果蝇胚胎中母体基因建立了果蝇胚胎中母体基因建立了果蝇胚胎中母体基因建立了果蝇胚胎中的蛋白梯度的蛋白梯度的蛋白梯度的蛋白梯度 n n基因表达级联将位置信息转基因表达级联将位置信息转基因表达级联将位置信息转基因表达级联将位置信息转化为分节模式化为分节模式化为分节模式化为分节模式n n分节特征由同源异形选择基分节特征由同源异形选择基分节特征由同源异形选择基分节特征由同源异形选择基因决定因决定因决定因决定 Translation of a localized Translation of a loc
73、alized mRNA generates a gradient of mRNA generates a gradient of protein as the products protein as the products diffuses away from the site of diffuses away from the site of synthesis.synthesis.Bicoid 和和Nanos做为转录调节因子激活做为转录调节因子激活Hunchback 基因的表达，抑制基因的表达，抑制 caudal的表达的表达 Hunchback和和 caudal形成浓度梯度形成浓度梯度B
74、icoid mRNA和和Nanos mRNA 翻译，在合胞体中扩散，形成浓度梯度翻译，在合胞体中扩散，形成浓度梯度BicoidBicoid mRNA mRNA定位于卵的前部，定位于卵的前部，定位于卵的前部，定位于卵的前部，NanosNanos mRNA mRNA 被被被被运至卵细胞的后端运至卵细胞的后端运至卵细胞的后端运至卵细胞的后端Hunchback Hunchback 和和和和 caudal caudal 均匀分布均匀分布均匀分布均匀分布母体效应基因母体效应基因建立了果蝇胚胎中的蛋白梯度建立了果蝇胚胎中的蛋白梯度 缺口基因缺口基因 位置信息更加精确位置信息更加精确成对基因成对基因分布模式分
75、布模式体节极性基因体节极性基因精确定位（幼虫体节的大小和位置）精确定位（幼虫体节的大小和位置）激活激活 The role of the gap The role of the gap gene productsgene productsThe The antennapediaantennapedia and and bithoraxbithorax gene gene complexes of complexes of melanogastermelanogaster 体节极性基因产物分布的体节极性基因产物分布的位置信息位置信息 同源异形选择基因同源异形选择基因 表达转录因子（表达转录因子（HTHHTH结构域）结构域） + +共激活物共激活物 开启特定体节发育起始的一套基因开启特定体节发育起始的一套基因uuThree Strategies by which Cells Are Instructed to Express Specific Sets of Genes during DevelopmentuExamples of the Three Strategies for Establishing Differential Gene ExpressionuThe Molecular Biology of Drosophila Embryogenesis
