发育生物学——体轴与胚层教案资料.ppt

哺乳动物胚轴形成与胚层分化,VertebrateembryosshowdifferencesinformbeforegastrulationAftergastrulation,rathersimilaratPhylotypicstage(种系型期):headstructure,neuraltube,notochord,somites,Lifecycleofthemouse,9weeksfertilizationintheoviduct,meioisIIcompletedclevagemorula,卵裂,蛙的早期卵裂。A第一次卵裂，B第二次卵裂，C第四次卵裂，动物极和植物极细胞出现差异。,卵裂期是指受精卵开始有丝分裂并产生由较小的细胞构成的囊胚(blastula)的过程。主要特点包括：分裂周期短;分裂球的体积下降：海胆胚胎的质/核比由550降至6;早期卵裂中合子基因大多处于休眠状态；卵裂常经历由均等裂向不均等裂变化。,3.hatching,paction,2.blastocystcavityformation,geneexpressionstarts,128cell,Day7-humanDay5.5-mouse,哺乳动物的早期卵裂发生在输卵管中,Mousepre-implantationstages:cleavage,compaction,blastocystcavityformation,Compactionatthelate8-cellstage,Withapical-basalpolarizationofcells,128-cellblastocyst,compaction,128-cellblastocyst,紧密化(Compaction):8细胞分裂后不久，卵裂球突然挤在一起，卵裂球之间的接触面增大，形成一个紧密的细胞球体。,哺乳动物卵裂球的compaction:发生第三次卵裂后不久,小鼠8细胞胚胎compaction前后,小鼠桑椹胚的压缩现象8细胞时期，小鼠细胞表面光滑，微绒毛均匀分布，压缩后微绒毛仅分布于细胞的外表面，细胞之间的联系加强了。,未压缩的和压缩的8细胞小鼠胚胎的比较,Compaction的机制:8细胞胚胎的外层胞间形成致密连接(tightjunctions)，可将球内部的细胞与外环境隔绝，起稳定细胞球的作用。而内层胞间形成缝隙连接(gapjunction)，可以交换小分子和离子。,注射E-Cadherin抗体的胚胎不能够致密化。在4细胞期激活蛋白激酶C导致compaction发生。因此，Compaction可能始于蛋白激酶C的活化，它引起细胞骨架的重排，在膜上均匀分布的E-Cadherin重新定位在胞间相交处。,哺乳动物囊胚细胞命运的早期分化位置决定论16细胞期桑椹胚(morula):位于内部的少数细胞产生的子细胞将组成内细胞团(innercellmass)，而位于外部的细胞产生的子细胞大多构成滋养外胚层(trophoblast)。32细胞期胚泡(blastocyst):位于囊胚腔一端的内细胞团(约10个细胞)将发育胚胎的本体及与其相连的卵黄囊、尿囊和羊膜；外层的滋胚层生成绒毛膜，为胎儿从母体摄取营养物质和氧，并产生激素以避免母体的排斥反应。,内细胞团的产生是哺乳动物早期发育的关键步骤之一。通过对活体胚胎的观察研究，发现这种重要决定作用仅仅依赖于细胞于某一正确时间出现在某一正确的位置。压缩后位于外层的细胞将形成滋养层细胞，而内部的细胞将发育成胚胎。一个细胞是否成为胚胎或滋养层细胞，完全取决于压缩作用后细胞所处的位置是位于外周还是内部。,哺乳动物胚泡的着床胚泡在向子宫移动过程中体积增大，是由定位于滋胚层细胞膜囊胚腔一侧的NaK泵将外部Na+泵入腔中，最后通过渗透作用吸水使囊胚腔增大。,随着囊胚腔内离子浓度的增加，水分就通过渗透作用进入进入囊胚腔内。囊胚腔内液体的积累对囊胚腔壁造成一个向外的压力，这种静水压是参与形成和维持囊胚为球形的一个重要作用力。,哺乳动物胚泡的着床胚胎外的透明带阻止了胚泡与输卵管壁的粘连-异位妊娠。胚泡到达子宫时，胚胎细胞分泌Strypsin（一种类胰蛋白酶)，它使透明带穿孔，胚泡从孔中“孵化”(hatch)出与子宫壁接触，通过一系列反应而着床。,人类的同卵双生的发生(占出生总数的0.25%),分离发生在滋胚层形成前(约受精后5天前)的分割，有独立的绒毛膜和羊膜。占同卵双生的33%。,发生在滋胚层形成后但羊膜形成前(约受精后59天)的分割，共用绒毛膜，有独立的羊膜。占同卵双生的66%,发生在羊膜形成后(约受精9天后)的分割，共用绒毛膜和羊膜，易出现连体儿。,人类的连体婴儿,嵌合胚：早期卵裂球有同等的发育潜力。自然人群中也出现过同时有XX型和XY型细胞的人，异卵双生的两个胚胎融合形成的单一混合个体。胚胎干细胞：保持了分化为胚胎本体的潜能的、可在体外增殖的胚胎细胞。在基因功能研究和疾病治疗方面有重要的作用。,哺乳动物中的嵌合胚、胚胎干细胞,人工受精：如主要用于治疗不育症、保存和运输优良个体。胚胎切割：在24细胞期分割胚胎细胞，用于繁殖优良家畜个体。,哺乳动物中的人工受精和胚胎切割,原肠作用Gastrulation,原肠作用是指囊胚细胞有规则的移动，使细胞重新排列，用来形成内胚层和中胚层器官的细胞迁入胚胎内部，而要形成外胚层的细胞铺展在胚胎表面。原肠作用期的胚胎叫原肠胚(gastrula)。,通过原肠作用，胚胎首先建立起三个胚层，即外胚层、中胚层和内胚层；其次为重新占有新位置的胚胎细胞之间的相互作用奠定了基础。因此，原肠作用是从尚未分化到分化为三个胚层和器官原基决定的关键时期。动物身体的主轴（包括前后轴、背腹轴和左右轴）也会在卵裂和原肠作用期间建立起来，胚胎各部分的细胞会获得各自的发育潜能。,人类的原肠作用,上胚层和下胚层的形成：在原肠作用开始时，内细胞团分裂为两层。与囊胚腔接触的一层为下胚层，将用于形成yolksac；另一层为上胚层。上胚层细胞间的缝隙将合并、扩大成为羊膜腔，腔中的液体可防止胚胎脱水和保护胚胎受振荡。上胚层将发育为胚胎的本体。,原肠作用前的胚泡，示内细胞团和滋养层细胞。,下胚层细胞从内细胞团中分离出来形成卵黄囊，滋养层细胞分裂形成细胞滋养层和合胞体滋养层；细胞滋养层形成绒毛，合胞体滋养层融入子宫组织。,上胚层分裂形成羊膜外胚层（包绕羊膜腔）和胚胎上胚层，哺乳类胚胎由胚胎上胚层形成。,胚外内胚层形成卵黄囊,人体胚胎三胚层的建立及其命运,人类原肠作用过程中，羊膜结构的形成和细胞的运动。,Note:thehumanepiblastisaflatdisc.Themouseepiblastiscup-shaped-hardertovisualize.,40天妊娠后人类的胚胎被羊膜包裹，外面再被绒毛膜包被。血管已深入到绒毛膜内，绒毛膜上的微绒毛最大限度地与母体血液接触。,人类胚胎与母体间的物质交流,人体胚胎细胞的分化路线,Earlypost-implantationdevelopmentofthemouseembryo,Innercellmass:primitiveendoderm(extra-embryonicmembrane)andprimitiveectodermorepiblast(embryoproperandsomeextra-embryoniccomponents)Polartrophectoderm:theectoplacentalconeandtheplacentaMuraltrophectoderm:trophoblastgiantcells,Earlypost-implantationdevelopmentofthemouseembryo,Eggcylinder:aninternalcavity(proaminoticcavity)forms,U-shapedOnsetofgastrulation:theappearanceofprimitivestreak,小鼠胚胎上胚层细胞的程序性死亡导致囊胚腔的形成。,Gastrulationinthemouseembryo,6.5d:beginningofgastrulation,theappearanceofprimitivestreak;antero-posterioraxis7.5d:primitivestreakelongatesuntilthebottomofthecup,formingnotochord(headprocess),Gastrulationandneurulationinthemouseembryo,7d:thenodeformation,anteriorectodermneurectoderm(brainandspinalcord)Theanteriorpartoftheembryogrowsinsizeandheadfoldappears,definitiveendodermreplacevisceralendodermtoformanouterlayerontheventralsurfaceoftheembryo.Thenotochordbeginstoform.,Gastrulationandneurulationinthemouseembryo,8d:headisdistinct,neuralfoldshaveformed,guthaveclosed,somitesbeginstoformoneithersideofthenotochord.10.5d:thecompleteofgastrulationandneurulationafterturning.,Tuninginthemouseembryo,Turning:adevelopmentalquirkpeculiartorodents:humanembryosaresurroundedbytheirextra-embryonicmembranesfromthebeginning.8.5-9.5d:themouseembryobecomesentirelyenclosedintheprotectiveamnionandamnioticfluid.Thevisceralyolksac,amajorsourceofnutrition,surroundstheaminon,theumbilicalcordconnectstotheplacenta.,胚轴形成,胚胎细胞形成不同组织、器官，构成有序空间结构的过程称为图式形成（patternformation）。在动物胚胎发育中，最初的图式形成主要涉及胚轴（embryonicaxes）形成及其一系列相关的细胞分化过程。胚轴指胚胎的前后轴（anterior-posterioraxis）和背腹轴（dorsal-ventralaxis）。胚轴的形成是在一系列基因的多层次、网络性调控下完成的。,Bodyaxesformation：小鼠胚胎发育的早期无法辨认体轴,Thespecificationoftheinnercellmassofamouseembryodependsontherelativepositionofthecellswithrespecttotheinsideandoutsideoftheembryos.Transcriptionfactors:Cdx2(滋养层细胞的分化)andOct/4(内细胞团的分化),Theaxesoftheearlymouseembryo,Blastocyststage:4d:innercellmassisconfinedtoembryonicregionandthisdefinesanembryonic-abembryonicaxis(geometrically,notcellfate)5.5d:theantero-posterioraxisvisible,withtheformationofprimitivestreakattheposteriorend.,Thesymmetry-breakingeventisthespecificationoftheanteriorvisceralendoderm(前端内脏内胚层,AVE),5.5d:Asmallregionofvisceralendodermatthedistal-mostendofthecupisspecifiesasAVEinducedbyNodalsignalingfromtheepiblastandbeginstoproliferateandextendtoonesideofepiblast.Theanteriorfateisrestrictedtothedistalregionbyinhibitorysignalsfromtheextra-embryonicectodermtotheproximalvisceralendoderm;inducesanteriorectoderm,Thesymmetry-breakingeventisthespecificationoftheanteriorvisceralendoderm(前端内脏内胚层,AVE),6.5d:primitivestreakbeginstoformontheoppositeside,markingtheposteriorendoftheaxis,RoleoftheAVEinestablishingtheanterior-posterioraxisofthemouseembryo,CylindricalSymmetry,BilateralSymmetry,A,P,Cerberus(anti-Nodal)Dkk(anti-Wnt)Lefty(anti-Nodal),Otx2Anteriorneuralectoderm,Nodal,Cripto(co-receptorForNodal)Endo-mesoderminduction,Day5.25Day5.5Day6.25,orhypoblast,Endo-mesoderminduction,Primitivestreakwnt,brachyury,fgfexpressed,Epiblastectoderm,Theleft-rightasymmetry,Whilethevertebratebodyisoutwardlysymmetric,mostinternalorgansareinfactasymmetricwithrespecttotheleftandrightsides,calledleft-rightasymmetry,e.g.,theheart,therightlunghasmorelobesthantheleft.1/10,000inhumans,situsinversusmutationintheivgenecauses50%ofmicehaveheartsthatlooptotheright.Keyproteinsinestablishingleftness:extracellelarsignalproteinNodalandtranscriptionfactorPitx2,Determinationofleft-rightasymmetryinthechick,left-rightsymmetryisbrokenatanearlystagebytheasymmetricactivityofaproton-potassiumpump(H+/K+-ATPase)ThereleaseofCa2+fromcellsintheleftsideaspartofthesymmetry-breakingprocess.Pitx2:leftness;Lefty(Nodalantagonist)providemidlinebarriertopreventNodalcrossingtotheright-handside;ActivininhibitShhactivity,Left-rightasymmetryinthemouseembryo,Cilia(纤毛)-directedflowgeneratesleft-rightasymmetryDirectedleftwardflowofextracellularfluidatthenodeataroundthelatehead-foldstageLeftwardflowoffluidpropagatesthereleaseofintracellularCa2+toupregulateNodalexpressionontheleftside,胚层的来源和特化(specification),Fatemap:telluswhichtissuesthedifferentregionsoftheembryogiverisetoThefatemapsandthegenesinvolvedingerm-layerspecificationaremoresimilarinallvertebratemodelsthanareinearlyaxisspecification根据被标记细胞的命运，构建两栖类早期囊胚的命运图：labeledbyinjectionofhigh-molecular-weightmoleculessuchasrhodamine(cannotpassthroughcellmembranes),asrhodaminefluorescesredinUVlight,sodetectedunderaUVmicroscope.Thefluorescentproteingreenfluorescentprotein(GFP)isnowwidelyused.,胚层的来源和特化(specification),FatemappingoftheearlyXenopusembryo:C3islabeledwithfluorescein(green)-tailbudstage,thecellsgiverisetomesodermcells,胚层的来源和特化(specification),FatemapofalateXenopusblastulaEctoderm:epidermis,nervoussystemAlongthedorso-ventralaxisthemesoderm:notochord,somites,heart,kidneyandbloodEndodermoverlyingthemesoderminthemarginalzone,Fatemapofamouseatthelategastrulastage,Node:exclusivelynotochordThemiddlepartofthestreak:mainlylateral-platemesodermTheposteriorpartofthestreak:tailbud,extra-embryonicmesodermthatformstheextra-embryonicmembranes-theamnion,visceralyolksac,allantois.,不同脊椎动物的fatemap基于一个图式的改变,Similarities:thefuturenotochordmesodermoccupiesacentraldorsalposition;theneuralectodermliesadjacenttothenotochord,withtherestoftheectodermanteriortoit.Differences:mainlytotheyolkinessofthedifferenteggs,whichdeterminesthepatternsofcleavageandinfluencestheshapeoftheearlyembryo.Endodermoverlyingthemesoderminthemarginalzone,Fatemapsofvertebrateembryosatalateblastulaorearlygastrula,早期脊椎动物的胚胎细胞的命运并未决定，是可以调整的,Fusionofmouseembryosgiverisetoachimera(micethataremosaicsofcellswith2differentgeneticconstitutions-byfusing2embryos):8-cellstageembryoofanunpigmentedstrainofmouseisfusedwithapigmentedstrainwillgiverisetoachimericanimal,Graftingexperiments:transplantingthecellsorregionstoahostembryo,ifdetermined,theywilldevelopaccordingtotheiroriginalposition;ifnotyetdetermined,developinlinewiththenewpositionEarlymouseembryoscanregulatetoachievethecorrectsize:retainsconsiderablecapacitytoregulateuntillateingastrulation.Innercellmassremainpluripotentupto4.5daysafterfertilization-theycangiverisetomanycelltypes.CellsinICMcanbeculturedtoproduceembryonicstemcells(EScells),小鼠发育相关突变的产生,Studyingtheroleofaparticulargeneindevelopment,tostudytheeffectsofamutationinthatgeneMice:transgenictechniques,overexpressorknock-outagivengeneIntroducingembryonicstemcells(EScells)carryingthemutationintotheblastocyst,小鼠的中胚层诱导和模式建成发生在原条形成时期,6days:theAVEinduceanteriorcharacterintheunderlyingepiblastandearlymarkersofthefutureprimitivestreak(PS)arerestrictedtotheproximalepiblast6.5days:BMPactivatestheexpressionofmesodermmarkersandtheposteriormovementofcellsexpressingPSmarkersresultsinPSformation,小鼠的中胚层诱导和模式建成发生在原条形成时期,7days:extra-embryonicmesodermisproducedfromtheposteriorendofPSwhilethenodeformsattheanteriorend,whichwillgiverisetoaxialmesendoderm(formdoralmesodermandgutendoderm),小鼠的中胚层诱导和模式建成发生在原条形成时期,Themolecularinteractionswithinthestreakat6.5daysBMPactivatestheexpressionofmesodermmarkers,NodalandWntexpre