BILD 2: Multicellular LifeLECTURE #26Website: http://www biology ucsd edu/classes/bild2 WI11 1Website: http://www.biology.ucsd.edu/classes/bild2.WI11.1Instructor: Darwin K. Berg ([email protected])ANNOUNCEMENTS:1)Final Review Session: Wed, Mar 16, 5-8 pm, 3500 Pacific Hall. ),,p,Building entrance code that day: 701181.2)Final Exam: Fri, Mar 18, 8-10:50 am, WLH 2001.2)Final Exam: Fri, Mar 18, 810:50 am, WLH 2001.3) Relevant Material for the Final Exam: a) A third devoted exclusively to the last third of the course.)yb) The remaining two-thirds: comprehensive (entire course).Fig. 47-11: Gastrulation in a Chick EmbryoDorsalFertilized eggAnteriorRightLeftEmbryoPrimitive streakVentralPosteriorPi iti t kYolkEpiblastPrimitive streakFutureectodermEndodermMi tiBlastocoelEndodermMigratingcells(mesoderm)HypoblastYOLKFig. 47-13: Organogenesis in a Chick EmbryoNeural tubeEyeEddNeural tubeCoelomNotochordHeartSomiteArchenteronForebrainEndodermMesodermEctodermLateral foldSomitesBloodvesselsThese layersform extraembryonicmembranesYOLKNeural tubeYolk stalkYolk sac(a) Early organogenesisYOLK(b) Late organogenesisFig. 47-7: Body Axes and Their Establishment in an AmphibianDorsalRightPosteriorAnteriorLeftPosteriorAnterior(a) The three axes of the fully developed embryoPi t dFirstVentralAnimal polePigmentedcortexFirstcleavageFuturedorsalAnimal poleAnimalhemispherePoint ofspermnucleusentryGraycrescentdorsalsideVegetalhemisphere(b) Establishing the axesVegetal poleEXPERIMENTFig. 47-23: Role of the Gray CrescentExperimental egg(side view)Control egg(dorsal view)GraycrescentGraycrescentTight thread forces cell division in way that puts all of theThreadFertilized control egg allowed to divide normally, yielding gray crescent evenly divided between the that puts all of the gray crescent in only one of the two blastomeres two blastomeresConclude: Developmental potential of the two RESULTSblastomeres depends on their getting “cytoplasmic determinants” from the gray crescent NormalBelly pieceNormalFig. 47-21: Fate Mapping for Two ChordatesEpidermisEpidermisCentralnervoussystemNotochord64-cell embryosNltbtMesodermEndodermLBlastomeresinjected with dye(b) Cell lineage analysis in a tunicate(a) Fate map of a frog embryoBlastulaNeural tube stage(transverse section)LarvaeFig. 47-24: Induction of Blastopore by Dorsal LipEXPERIMENTRESULTSDorsal lip ofblastoporePrimary embryoPrimary structures:Secondary(induced) embryoPigmented gastrula(donor embryo)Nonpigmented gastrulaPrimary structures:Neural tubeNotochordNonpigmented gastrula(recipient embryo)Secondary structures:Notochord (pigmented cells)Neural tube (mostly nonpigmented cells)...if time permits...BILD 2 SeminarNicotinic Control of GlutamateSynapse FormationDarwin K. BergDivision of Biological SciencesUniversity of California San DiegoUniversity of California, San DiegoNicotinic Cholinergic SignalingThe transmitter acetylcholine (ACh) activates cation-selectiveHeteromeric Homomeric *CCThe transmitter acetylcholine (ACh) activates cation-selective ligand-gated ion channels (nAChRs).Two major nicotinic receptor species in the CNS:2*-nAChRs 7-nAChRsthe CNS:During Development:High levels of nicotinic receptorsblock w DHE block w Bgt, MLAHigh levels of nicotinic receptors.Spontaneous waves of nicotinic excitation.And:Glutamate synapses are rapidly formingAnd:Glutamate synapses are rapidly forming.EM Quantification of Synapses in Vivo in the CA1 at P8in the CA1 at P8***er m20.20***Synapses pe***0.100.150.05WTS2KO7KO 7/2-dKO0Removal of the 7-nAChR gene causes fewer synapses to be present in P8 CA1.E d i ti i h li i ti it tl h lEndogenous nicotinic cholinergic activity apparently helps promote glutamate synapse formation during development.Immunostaining Hippocampal Slices Provides Quantification of Glutamatergic Synapses.WTVGluT PSD-95 MergedThresholded7KO5 mColocalVGluTPSD9500 m2)Co-localVGluTPSD-9510080120uncta (per 40*********6040***Pu******020Fewer total glutamate synapses in 7KOs than WTs at P12.Reduced mEPSC Frequency in 7KOs at P11/12qyWT2KOC Amp (pA)102015C Freq (Hz)***0.20.40.3α7KO25 pA mEPSC05mEPSC00.1Fewer spontaneous mEPSCs in CA1 pyramidal neurons of 250 msppy7KOs vs WTs at P11/12 recorded in TTX & gabazine.Results consistent with fewer functional glutamate synapse in 7KOs.Spine Numbers Depend on2*nAChRs In VivoSpine Numbers Depend on 2*-nAChRs In Vivo Labeled with Sindbis-GFP In Vivo OvernightP4P40P4WTP415**P400 m6**P42KO10510**ines per 124*****10 mWT2KO7/2-dKO7KOSpi02KO7KO7/2-dKOWTConstitutive knockout of the 2-nAChR gene reduces spine numbers in early postnates. The deficit persists.Knockout of 7-nAChR has no effect on spine number.Spine vs Shaft Synapses in 2KOs & 7KOs In Vivo WT2KO7KO[Lbld ithSidbiGFP i i ON th i t i d f PSD95 ( d) d VGl T (bl )]WT2KO7KO10 mCo-localVGluT PSD-95[Labeled with Sindbis-GFP in vivo ON, then immunostained for PSD-95 (red) and VGluT (blue)]----------- Spines -------------Co-local------------- Shafts ------------PSD-95VGluT**er 10 m)64*******Puncta (pe2********************0************Constitutive 2KOs have fewer glutamate synapses on spines but a compensatory increase in synapses on dendritic shafts. Constitutive 7KOs have fewer of both (spine & shaft synapses).SummarySummary1) Activity through7-nAChRs directly recruits components1) Activity through 7-nAChRs directly recruits components required for functional maturation of glutamate synapses.2) Activity through 2*-nAChRs increases dendritic spine bi lltdinumber in a cell-autonomous manner during development.3) Changing the ratio of2*and7nAChR activation3) Changing the ratio of 2*-and 7-nAChR activation changes the location/type of glutamate synapses.Altering nicotinic activity during development mayAltering nicotinic activity during development mayproduce long-lasting changes in neural networks andthe computations they can perform.The People!Zhaoping Liu Rob Neff Nolan Campbell Catarina FernandesAndrew Halff (Campbell, Fernandes, Halff, & Berg, J Neurosci 2010)(Liu, Neff, & Berg; Science 2006)Adrian LozadaKerri MasseyZhaoping Liu NataliaGunko Jingjing DuanAndrew Halff (Lozada, Gunko, Massey, Duan, Liu, Halff, & Berg, in prep)BILD 2: Multicellular LifeLECTURE #26LECTURE #26Website:
View Full Document
Unlocking...