Neural Development Notes PDF
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2024
Serena Bovetti
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These notes cover neural development, focusing on topics including neural induction, body pattern formation, neurogenesis, and cell migration. The document is for the academic year 2024-2025, providing insights into different developmental stages and underlying mechanisms.
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NEURAL DEVELOPMENT – Prof.sse S. Bovetti – A.A 2024-2025 – anno1 semestre1 – Margherita Bosso Serena Bovetti AA 2024 2025 ANNO 1 - SEMESTRE...
NEURAL DEVELOPMENT – Prof.sse S. Bovetti – A.A 2024-2025 – anno1 semestre1 – Margherita Bosso Serena Bovetti AA 2024 2025 ANNO 1 - SEMESTRE 1 1 NEURAL DEVELOPMENT – Prof.sse S. Bovetti – A.A 2024-2025 – anno1 semestre1 – Margherita Bosso Sommario Lesson1-07.11(Prof.ssaBovetti).....................................................................................................................................................5 PROGRAM:.......................................................................................................................................................................5 NEURALINDUCTION...............................................................................................................................................................................................5 Evolutionaryoriginsofmetazoans.....................................................................................................................................................................5 Anhistoricalpointofview..........................................................................................................................................................................5 C.Elegansdevelopment............................................................................................................................................................................6 ExperimentonC.eleganscelldivision:......................................................................................................................................7 Amphibianembryonicdevelopment...........................................................................................................................................................8 Mammalianembryonicdevelopment.........................................................................................................................................................8 Neuraltubedefects..................................................................................................................................................................10 Demonstratingself-regulationinembryos...............................................................................................................................................10 1891-Driesch..........................................................................................................................................................................11 1903-Spemann......................................................................................................................................................................11 1903-Weismann.....................................................................................................................................................................11 Spemann(opticcup)................................................................................................................................................................11 Anchorpoint(SpemannandMangold)....................................................................................................................................11 Identifyingmoleculesthatmediateneuralinduction................................................................................................................................12 Signalsfromtheorganizer.......................................................................................................................................................12 Whatorganizestheorganizer?................................................................................................................................................13 MechanismsonBMPworking.................................................................................................................................................13 Summary-lesson1..................................................................................................................................................................................14 Lesson2-14.11(Prof.ssaBovetti)...................................................................................................................................................14 DEVELOPMENTOFABODYPATTERN...............................................................................................................................................................14 PATTERNINGOFTHENEUROECTODERM..................................................................................................................................................14 DevelopmentinDrosophila......................................................................................................................................................14 Patternsofgeneexpressionareset-upbymorphogens.........................................................................................................................15 Establishingpolarityintheegg................................................................................................................................................................16 PatterningintheAPaxisofthevertebrateCNS..............................................................................................................................................17 Bicoid.......................................................................................................................................................................................17 Thecascadeofgeneregulationinfruitflies............................................................................................................................................18 Hoxgenes................................................................................................................................................................................................19 Evolutionarytreeofvertebrates...............................................................................................................................................20 Developmentofvertebratenervoussystem............................................................................................................................................21 PatterningintheDVaxisofthevertebrateCNS..............................................................................................................................................22 SignalsgradientsthatdriveDVpatterninginvertebrates........................................................................................................22 Summary-lesson2..................................................................................................................................................................................23 Lesson3-21.11(Prof.ssaBovetti)...................................................................................................................................................24 NEUROGENESISANDCELLMIGRATION............................................................................................................................................................24 Neurogenesis...................................................................................................................................................................................................24 Symmetricalandasymmetricalneuraldivisions......................................................................................................................24 Thedevelopingcerebralcortex................................................................................................................................................................25 SymmetricandAsymmetriccelldivisions................................................................................................................................................26 AsymmetriccelldivisioninDrosophila.....................................................................................................................................26 Asymmetriccelldivisioninvertebrates....................................................................................................................................27 Postmitoticneuronaldifferentiation..........................................................................................................................................................27 ProneuralgenesinDrosophila-Mutuallateralinhibition........................................................................................................27 Proneuralgenesinvertebrates-Mutuallateralinhibition........................................................................................................28 Neurogenesisinadultbrain.............................................................................................................................................................................28 Adultneurogenesis:theolfactoryepithelium...........................................................................................................................................28 Adultneurogenesis:TheSVZandOlfactoryBulb(OB)neurogenesis....................................................................................................29 Neuronalmigration...........................................................................................................................................................................................29 Directvisualization...................................................................................................................................................................29 Indirectvisualization................................................................................................................................................................30 Majormodesofmigration........................................................................................................................................................................31 1-Individualmigration:............................................................................................................................................................31 2-Chainmigration:..................................................................................................................................................................32 3-Scaffoldmigration:...............................................................................................................................................................32 Defectinneuronalmigration:GnRHneurons-GnRHcellsmigrationandKallmannSyndrome............................................33 Summary-lesson3..................................................................................................................................................................................34 Lesson4-28.11(Prof.ssaBovetti)...................................................................................................................................................34 MATURATIONOFFUNCTIONALPROPERTIES...................................................................................................................................................34 2 NEURAL DEVELOPMENT – Prof.sse S. Bovetti – A.A 2024-2025 – anno1 semestre1 – Margherita Bosso Howneuronsdeveloptheirshapes.................................................................................................................................................34 Thecytoskeletoninmatureaxonsanddendrites....................................................................................................................................35 Thegrowingneurite.................................................................................................................................................................................35 Stagesofneuriteoutgrowth.....................................................................................................................................................................36 Regulationofdendritemorphology..........................................................................................................................................................37 1.InteractionbetweenSISTERDENDRITESofthesameneuron..................................................................................................38 2.BetweendendritesofNEIGHBORINGNEURONSOFDIFFERENTTYPE................................................................................39 3.BetweendendritesofNEIGHBORINGNEURONSOFTHESAMETYPE..................................................................................40 SYNAPTOGENESISANDSPINOGENESIS...........................................................................................................................................................40 Precursorstosynapses...........................................................................................................................................................40 Evolutionofsynapses..............................................................................................................................................................................41 Thestructureofa(excitatory)synapse....................................................................................................................................................42 Stagesofsynaptogenesis........................................................................................................................................................................42 1.Synapticspecificationandinduction....................................................................................................................................43 2.Synapseformation...............................................................................................................................................................44 3.Synapseselectionandstabilization.....................................................................................................................................44 Theneuromuscularjunctions...................................................................................................................................................................45 Spinogenesis...........................................................................................................................................................................................46 Molecularregulatorsofspinedevelopment.............................................................................................................................................47 Dendriticspinescompeteforsurvival......................................................................................................................................47 FragileXsyndromesuggeststherecanbetoomuchofagoodthing.....................................................................................................48 InVivoOpticalImagingofFXSModelMice.....................................................................................................................................48 AlteredStructuralandFunctionalSynapticPlasticitywithMotorSkillLearninginaMouseModelofFragileXSyndrome............49 DendriticSpinesinEarlyPostnatalFragileXMiceAreInsensitivetoNovelSensoryExperience..................................................49 HypersensitivityinresponsetosensorystimuliandneocorticalhyperexcitabilityareprominentfeaturesofFragileXSyndrome.50 Lesson5-2.12(Prof.ssaBovetti)...................................................................................................................................................51 ADVANCEDOPTICALIMAGINGTECHNIQUESFORNEURALDEVELOPMENT..............................................................................................51 Analyzingstructureandfunction..............................................................................................................................................................51 Lightscatteringinbiologicaltissue..........................................................................................................................................................52 Propertiesoflight.............................................................................................................................................................................52 Principlesoffluorescence................................................................................................................................................................52 Widefieldillumination......................................................................................................................................................................53 Confocalillumination........................................................................................................................................................................53 Principlesoftwo-photonexcitation..........................................................................................................................................................54 Whynon-linearismorethanlinear?................................................................................................................................................55 Multiphotonsystem..................................................................................................................................................................................55 NikonA1two-photonmicroscope@NICO.....................................................................................................................................56 Fluorescenttoolsfor2Pimaging.............................................................................................................................................................56 Fluorescentreportersofcellactivity........................................................................................................................................................57 TheCalciumIonasanIndirectReporterofNeuronalActivity.................................................................................................57 Invivotwophotoncalciumimaging.........................................................................................................................................................58 Lightscatteringinbiologicaltissue..........................................................................................................................................................58 Imagethewholemousebrain:theproblemoftissuescattering......................................................................................................59 Light-sheetfluorescencemicroscopy..............................................................................................................................................59 Confocalimaging.....................................................................................................................................................................60 Summary-lesson5.................................................................................................................................................................................60 Lesson6-05.12(StefanoZucca-seminar)............................................................................................................................................60 Basicconceptsinbiophysics...........................................................................................................................................................................61 Neurons...................................................................................................................................................................................................61 Cellpotential............................................................................................................................................................................................61 Ions’flowacrossthemembrane..............................................................................................................................................61 Howisionflowregulated?.......................................................................................................................................................61 Ionchannels............................................................................................................................................................................62 Ions’permeability.....................................................................................................................................................................62 Recordingneuronalelectricalproperties.........................................................................................................................................................63 Patchclamp.............................................................................................................................................................................................63 SchemePatch-ClampRecordings...........................................................................................................................................63 Patch-Clamp:Voltage-ClampvsCurrent-Clamp....................................................................................................................64 Intrinsicneuronalproperties.............................................................................................................................................................................65 BiophysicsoftheActionPotential............................................................................................................................................................66 DevelopmentalchangesofActionPotentialproperties...........................................................................................................67 RoleofCa++............................................................................................................................................................................67 Neuronalchemicalcommunicationduringdevelopment.................................................................................................................................68 3 NEURAL DEVELOPMENT – Prof.sse S. Bovetti – A.A 2024-2025 – anno1 semestre1 – Margherita Bosso GABAreceptors.......................................................................................................................................................................68 Summary-Intrinsicneuronalproperties..................................................................................................................................................69 Microbialopsins.......................................................................................................................................................................................69 LightControlofProteinActivity................................................................................................................................................................72 Summary-Optogenetics.........................................................................................................................................................................72 Applicationof2Pinvivoimaging-SEELESSON8................................................................................................................................72 Lesson7-12.12(Prof.Bovetti).......................................................................................................................................................74 ACTIVITY-GUIDEDNEURALDEVELOPMENT-MAPFORMATION....................................................................................................................74 Coarsemaps....................................................................................................................................................................................................75 Finemaps:topographicmaps..........................................................................................................................................................................75 Finemaps:featuremaps.................................................................................................................................................................................76 Principlesofmapformation.............................................................................................................................................................................76 Developmentoftopographicmaps..................................................................................................................................................................76 Developmentoftopographicmaps:thechemoaffinityhypothesis...........................................................................................78 Neuronalactivity:tectalexpansionorcompressionrearrangements......................................................................................79 Map-formation:theroleofneuronalactivity.....................................................................................................................................................79 Map-formation:theroleofneuronalactivity(segregationinhumans).....................................................................................79 EXPERIMENT1-discoveryofsegregation.............................................................................................................................80 EXPERIMENT2-inductionofsegregation,TheThree-EyedFrogsexperiment.....................................................................80 Spontaneousactivityrole.........................................................................................................................................................................82 Spontaneousactivityandformationofsegregation.................................................................................................................82 Paper.......................................................................................................................................................................................................82 SpontaneousWavesofRetinalActivityFormOcularDominanceBandsintheLG................................................................83 Hebbiansynapse-Hebbianprocess.......................................................................................................................................................83 Long-termpotentiation(LTP)confirmstheexistenceofHebbiansynapses............................................................................83 Paper-LTP..............................................................................................................................................................................................84 Summary-lesson“MAPFORMATION”..................................................................................................................................................84 DEVELOPMENTOFFEATUREMAPS...................................................................................................................................................................85 EXPERIMENT:DavidHubel,TorstenWiesel-finemaps........................................................................................................85 Developmentoforientationanddirectionmapsintheferretvisualcortex..............................................................................................85 Visualcortexofferretgrownupindarkenvironment..............................................................................................................86 Lesson8-19.12(Prof.ssaBovetti).................................................................................................................................................86 EXPERIENCE-DEPENDENTNEURALDEVELOPMENT......................................................................................................................................86 Effectofexperienceonvisualsystemdevelopment........................................................................................................................................87 Experiment:Effectofexperienceonvisualsystemdevelopment............................................................................................................87 (DavidHubelandTorstenWiesel)...........................................................................................................................................87 Effectsofstrabismusonphysiologicalresponsesofneuronsinthevisualcortex...........................................................................88 Physiologicalchangesinoculardominancepriortoanatomicalchanges.......................................................................................89 Physiologicalchangesinoculardominancepriortoanatomicalchanges...............................................................................................90 Mammalsrequirevisualexperienceduringasensitiveperiodtodevelopfunctionalvision............................................................................90 CRITICALPERIODS(CP)...............................................................................................................................................................................91 InhibitorycircuitsofGABAsignallingandthesensitiveperiod................................................................................................................92 Exampleofmultimodalintegration:..........................................................................................................................................93 SOCIALLYGUIDEDNEURALDEVELOPMENT....................................................................................................................................................93 PAPER:Developmentalencodingofnaturalsoundsinthemouseauditorycortex.........................................................................................93 AtwhichagemicestarttohearUSVsandhowUSVsprocessingchangesacrossdevelopment?........................................94 Invivo2Pfunctionalimagingofsound-evokedactivity...........................................................................................................94 L2/3cellstunedforthesamestimulusdisplayedhighlycorrelatedspontaneousactivity.......................................................96 CONCLUSIONS......................................................................................................................................................................96 HowtoReadandUnderstandaScientificPaper................................................................................................................................................97 4 NEURAL DEVELOPMENT – Prof.sse S. Bovetti – A.A 2024-2025 – anno1 semestre1 – Margherita Bosso NEURAL DEVELOPMENT esson1 - 07.11 (Prof.ssa Bovetti) L PROGRAM: Neural induction Development of a body pattern Patterning of the neuroectoderm Neurogenesis and cell migration Maturation of functional properties Synaptogenesis and spinogenesis Activity-guided neural development Experience-dependent neural development Socially guided neural development NEURAL INDUCTION Evolutionary origins of metazoans t some point natural selection A starts favoring individuals whose cells stayed together to form a multicellular body. In sponges there are specialized cells but no real tissues, no symmetry or regularity in the body shape. 600 million years ago: first metazoans multicellular animals with a body composed of more than one type of cell like cnidaria. In jellyfish and similar organisms have a basic NS composed by a network of neurons of the same type. Jellyfishes have a digestive system, but not a circulatory system,sothey’reabletorespond tostimulisuchaslight,objectand so they can move in a specific direction: reflexive responses to ncountering prey or other objects and e specialized sensory cells and connections that coordinate moving toward or away from light, diving in response to rough water, and avoiding rock walls brought to the cellular differentiation and coordination of cell types so we can identify a BODY PLAN: organization of different cell types to survive and reproduce. The NS is part of the body sootherpartsof the body are connected with it like heart, eyes, muscles, ecc and the NS is also responsible for coordination of all these parts. An historical point of view A ristotle(384–322bce)addressedthequestionofhowtypesofcellsarearrangedinpartsofthebodyby examining hens’ eggs. ○ Epigenesis: the body gradually changes shape, acquiring new structures and growing more complex, with time. 5 NEURAL DEVELOPMENT – Prof.sse S. Bovetti – A.A 2024-2025 – anno1 semestre1 – Margherita Bosso E nlightenment (17th and 18th centuries): initial use of the microscope. Robert Hooke called“c ells”the different compartments of the cork that he observed with the microscope. Jan Swammerdam (1637–1680) used his microscope to find, tucked inside a caterpillar, the wings and bodyofthebutterfly.Heconcludedthattheadultformhad been tucked inside the juvenile’s body all along. ○ Preformationism:developmentoforganismsconsistsofasimpleenlargement of a body plan. Early 1800s, scientists began reporting that fertilized eggs from hens and from amphibians divided repeatedly to form clusters of cells each having the same shape. ○ Ontogeny: the process of individual development; growing up and growing old 1900: several scientists independently rediscovered Gregor Mendel’s 1866 paper demonstrating that pea plants inherit traits as discrete units. The fusion of Darwin’s theory of evolution bynaturalselectionandMendel’slawsof discrete inheritance became known as the modern synthesis of evolution, which underlies evolutionary biology today. Scientists would propose the word gene to describethediscretehereditaryunitthat Mendel referred to as a “factor.” It would be many years before we realized that genes consist of stretches of DNA. Scientists domesticated a simple worm to address the question of cell differentiation: Caenorhabditiselegans.1963:SydneyBrennerdecidedtoestablishC.elegansasamodel system, and used it to explore gene function. It isvery useful because: Simple anatomy Limited number of cells Transparent embryos Genome fully sequenced Easy to maintain in the lab Easily manipulated: we can easily induce mutations Readily induce mutations Hermaphrodites: both male and female organs Life cycle: form egg to adult: 3 days (low impact on lab costs) Life span: 2-3 weeks (low impact on lab costs) Discoveries 1963: Sydney Brenner decided to establish C. elegans as a modelsystem,andusedittoexploregene function. 1976:JohnSulston,publishedacompletecelllineageofC.elegans.Hefollowedthedescentofeverycell as it divided and differentiated and found that first five cell divisions produce six founder cells that differentiate to ultimately give rise to all of the different tissues in the organism. 1986: Robert Horvitz published his pioneering work on the discovery of "death genes“. 2002: Sydney Brenner, John Sulston and Robert Horvitz shared the Nobel Prize in Physiology and Medicine for their seminal work done in C. elegans. 2006: Andrew Fire and Craig Mello shared the Nobel Prize in Physiology and Medicine for their groundbreaking work on RNA interference, or RNAi, a process that results in silencing of genes via degradation of specific mRNA molecules. 2008:MartinChalfiereceivedtheNobelPrizeinChemistryforshowingthattheGreenFluorescentProtein (or GFP)could be expressed in C. elegans and usedas a fluorescent reporter. C. Elegans development he problem of cell differentiation, seems T moremanageableinC.elegansbecausethe entire worm, with a body about1mmlong, consists of just under 1,000 cells, all well known by scientists. itotic lineage reveals cell fate in the M wormC.elegans.Theearliestcelldivisions inC.elegansareasymmetrical,soscientists could name the daughter cellsproducedby eachroundofmitosis,keepingtrackoftheir 6 NEURAL DEVELOPMENT – Prof.sse S. Bovetti – A.A 2024-2025 – anno1 semestre1 – Margherita Bosso rder and “lineage,” as they descended from the fertilized egg. o Thereisastrictorderofcelldivisionsthathappensaboutthesame way in every individual worm. You canmakea“map”thatshows every celldivisionthattakesplaceinthetransitionfromzygoteto adult (mitotic map), and so the position of every single cell. The pattern of mitosis perfectly matches every cell’s fate. First cells: P1 = most posterior part → EMS + P2 AB = most anterior part → ABa + ABp Experiment onC.eleganscell division: he2daughtercellsofthezygoteweredivided:ABweredividedbyP1andtheysawthatthedevelopmentdidnot T change, so if the cells are dividedtheygoonontheirownpath:P1dividedintoP2andEMScellswhileAbcell divided into ABa and ABp cells, so an entire organism would not develop anymore. So,everycellfollowsitsparticulardestinynomatterwhatitsneighboringcellsareuptodo:thisbehavioriscalled mosaicspecificationofcellfate→theinteractionofcellsisnotimportantbecausetheguideisinsidethecells themself. ThishappensbecauseP1andABhavethesamemothercellbutprobablywhentheydivideandthecycleendsthe symmetrical division is not very symmetrical: different TF are released in one of the 2 parts. Notmanyfactorsdifferfromthe2cells,butmanyimportantaredifferent,for exampleskin1(SKN1).ThisSKN1factorisreleasedtotheP1cellandso, there is anasymmetricaldistributionofthisfactoralreadyinthemother cell: SKN1 is more concentrated into theposteriorpart.ThenalsoinP1 cellsSKN1ismoreconcentratedintheposteriorpartsoboththedaughter cells would not receive SKN1, but only the posterior one, that is EMS cell. As further divisions ensue, each cell receives a unique mixture of transcription factors varying in their relative concentrations as they are parceled out among descendants. The particular composition of transcription factors each cell receives may be a resultofwhere,exactly, that cell falls in the mitotic lineage.Thatparticularmixtureoftranscription factors direct gene expression in the cell and differentiationofthatcellto takeonitsfate.IfthemotherhasnofunctionalcopiesofSKN-1geneand cannotlaydowntheproteinintheposteriorpart,noneofheroffspringwill form a pharynx embryos of most species do not develop in that way!!! Removing SKN1 the 2 daughter cells developequally.Nowweknowthat therearefewTFactivatedsequentiallyanddifferentgradientsactivatedifferentTF,sothecodeofTFdetermines the complexity of each cell type. Most of the organisms has not a mosaic specification of cells in development. 7 NEURAL DEVELOPMENT – Prof.sse S. Bovetti – A.A 2024-2025 – anno1 semestre1 – Margherita Bosso Amphibian embryonic development In virtually all animals the first several divisions of the zygote result in a more or less spherical cluster of cells leaving a fluid filled hollow space in the center. At this stage the embryo is calledblastula. Adimplecalledblastoporeappearsonthesurfaceoftheblastulaandinvadestheinterioroftheembryo.Nowthe embryoisdesignedasgastrula.Inamphibiangastrulationstartsfromtheinvaginationoftheblastulaformingthe blastoporeandcellsstarttomoveintheblastoporethroughcellmigrationanddivide,andanewcavityformsand will rise to the digestive system. The nervous system derives from specialized parts of ectoderm called neuroectoderm - ectoderm (blue)- most external - mesoderm (pink) - endoderm (yellow) - most internal The NS forms under the effect of notochord (in Chordates)thatisastructureofthemesodermandinducethe ectoderm to become neuroectoderm and then NS. Mammalian embryonic development heblastulationandgastrulationaredifferentfromtheamphibianones.Theblastulaisimplantedintheuterusand T surroundedbytheplacenta.Mitosisandmigrationintheblastulaformstheinnercellmasswhichwillgiveriseto the whole body from theinner cell mass ICM,thatgives rise to the entire embryo when implanted. Aprimitivestreakthatisthemidlineoftheorganisms:fromtheICM(grey)aninvaginationoccursinbothsidesand cells migrate into the streak bymovingintheinternalpart andthe3layersareformed. At the end in the most internal part we’ll have the endoderm, mesorìdem and ectodem. The primitive streak would be crossed by cells that enter into the structure and go down forming the endoderm. The first cohort of cells will give rise to endoderm. The later arriving cellswillfillthe surface between the outer layer and the endoderm formingmesoderm. Gastrulation begins when the inner cell mass form a disc two cell layers thick inside the embryo Cells on the upper surface migrate toward the center forming a crease called primitive streak (midline of the animal) Invertebrateembryos,theprimitivestreakmarksthemidlineposition,wherethenervoussystemwilldevelopinthe ectoderm.Thecreaseformedbytheprimitivestreakismorepronouncedatoneend,calledthenodeanditmarks where the animal’s brain and head will form. 8 NEURAL DEVELOPMENT – Prof.sse S. Bovetti – A.A 2024-2025 – anno1 semestre1 – Margherita Bosso henervoussystembeginsasanelongatedlayer T of ectodermal cells on the surface, called the neural plate. As cell divisions and migrations continue, the sides oftheneuralplateriseuptoformthewalls ofaneuralgroove.Thesewallsmeetandfuseat the midline to form aneural tube=neurulation. The rest of the nervous system will arise from a group of ectodermal cells that were atthepeaks of the two sides of the neural groove thatwhere not included into the neural tube, called neural crest cells, that will migrate in different parts of thebodyinveryimportantstructuresandwillgive rise to the peripheral nervous system. NEURULATION: process by which a gastrula transforms into a neurula ←see video The neural plate and neural tube 9 NEURAL DEVELOPMENT – Prof.sse S. Bovetti – A.A 2024-2025 – anno1 semestre1 – Margherita Bosso sdevelopmentproceedstherewillbetheformationofvesicles:theprimaryvesicleduringdevelopmentwillgive A rise to secondary vesicles.Primary vesiclesare: - the FOREBRAIN (PROSENCEPHALON) - most anterior, - the MIDBRAIN (MESENCEPHALON) and - the HINDBRAIN (RHOMBENCEPHALON) and thesecondary vesiclesare: - the TELENCEPHALON and DIENCEPHALON (from the forebrain), - the MESENCEPHALON (from the midbrain) - the METENCEPHALON and the MYELENCEPHALON (from the hindbrain) As the system is mature these structures will give rise to correspondent adult derivatives (see uppertable) Neural tube defects nencephaly:cranialneuralfoldsdonotfuseinthedevelopingembryoin A the anterior part and most or all of the brain is missing (usually not compatible with life) Spina bifida: failure of the neural tube to fuse at its posterior end, resultingineitheranopenlesiononthespine,withsignificantdamageto the nerves and spinal cord. Ectodermal contributions to the body: Demonstrating self-regulation in embryos Tounderstandthemechanismsofneurulation,weneedtoreturntotheworkofearlyembryologistsandunderstand the concept of “self-regulation” 10 NEURAL DEVELOPMENT – Prof.sse S. Bovetti – A.A 2024-2025 – anno1 semestre1 – Margherita Bosso 1891 - Driesch eusedahydra(simpleorganism)tostudydevelopment:heseparatedthe4cellsthatnormallywouldmakeone H larva.Fromeachcells4almostcompletehydradeveloped,eveniftheyweresmalleranddifferentinsizefromthe “original” one. Entirely give rise to whole individuals so are 4 totipotent stem cells. 1903 - Spemann Heusedanamphibianandtookfromtheembryodividedtheblastulain2parts:if1nucleusmigratesintooneof the two portions, it brought to the formation of 2 entire organisms. 1903 - Weismann He removedapartoftheblastulaandsawthattheorganismdeveloponrmallybecausetheblastulacanrecover the damage and give rise to an entire organism. heseexperimentsbroughttotheconceptofSelf-regulation:processbywhichembryosmanagetocompensate T for missing or damaged cellsandneverthelessproduceanentireindividual→conditionalspecificationofcell fate, because this process depends on cell-cell interaction C. Elegans → Mosaic specification of cell fate → Fate predestined by its mitotic lineage, no matter what eighboring cells do - NO INDUCTION PROCESS n Cell fate depends on environmental conditions. Otheranimals→Fatepredestinedbyitsmitoticlineage,nomatterwhatneighboringcellsdo→Cellstakeonafate that is appropriate for their location in the body, which is determined by thetypeofcellsthatsurroundthem.In other words, cellular differentiation is guided by cell-cell interactions, the communication and influence between developing cells: INDUCTION Spemann (optic cup) he optic cup induces the epithelium to form a lens: T Induction: The processbywhichonegroupofcells directs the differentiation of other, nearby cells. HansSpemann(1900)notedthatthevertebrateeye develops when the neural tube extends two optic cups, out to the epithelium. The optic cup will developtheretina,whiletheoverlyingepitheliumwill produce the transparent lens and cornea. Hetriedtoimplanttheopticcupinadifferentlocation and a correspondent retina developed, but this not happenedwhenthesiteoftransplantationoftheopticcupwastoofarawayfromthenormalpointofformation.He discovered that the graft of lens can send signals soitisanINDUCTIVEPROCESS,sothereisafactorthatis releasedandhastoreachareceptortohaveaconsequence,soalsoCOMPETENCEisimportant.Intheposterior part the graft of the optic cup does not have consequences because here there are not receptors. Anchor point (Spemann and Mangold) ansSpemannandHildeMangold(1924)begantransplantingportionsof H one blastula into other blastulas. IN one of their experiments they transplantedaspecificpartthatisthedorsallipandinthiscaseitnotonly induced the formation of some structures, it seemed to induce the formation of an entirely new individual, with a headandcentralnervous system: thedorsal lip is the ORGANIZER. Cells in the early embryo that would eventually form the skin can be induced to change their fates. These cells must retain the ability, or competence, to adopt other fates. In another experiment the dorsal lip of the blastopore was transplanted from a donor blastula to a receiving blastula and a new NS developed: they transplanted a specific region that istheORGANIZERthatinduces the development of NS despite epithelium. The transplanted dorsal lip not only induced the formation of some structures,itseemedtoinducetheformationofanentirelynewindividual, withaheadandcentralnervoussystem.Itwouldbeover50yearsbefore any techniques would be available to isolate and identify the particular 11 NEURAL DEVELOPMENT – Prof.sse S. Bovetti – A.A 2024-2025 – anno1 semestre1 – Margherita Bosso molecule(s) from the dorsal lip of the blastopore that organizes a new individual. Identifying molecules that mediate neural induction he discovery of thedefault model: 2 experiments were performed: T 1- Animal caps dissected and cultured intact without dissociation → epidermis. 2- Animal caps cells are separated from each other → neural cells Dissociation allowed molecules that prevent neural induction to escapefrom around the animal cap cells into the surrounding fluid, thereby reducing the concentration of those blocking molecules and allowing the cells to adopt a neural fate by default. The molecules preventing neural induction are called members of Bone morphogeneticproteinsfamily(BMP2,BMP4,BMP7)andwerefoundinthe animal cap of the blastula: to confirm thatthesemoleculeswereresponsible for the inhibition of neural development dissociated cell were cultured with BMP and they brought to the formation of epidermis, as in case1. The organizer might promote neural induction because it produces molecules that inhibit BMPs in the animal cap, preventing its cells from acquiring an epidermal fate. What are the active molecules produced by the organizer to induce neural tissue? Signals from the organizer hen the experiment was repeated in amphibians T 2 models: - the exposure to LiCl will give rise to an amphibian with an enormous head. Here they supposed to have a major concentration of BMP - the exposure to UV will give rise to an amphibian without head SotheytookanamphibianblastulaandexposedittoLiCltoincrease the concentration of BMPs near the blastopore near theportionthat willformtheNSandthentheyextractedmRNAfromthesecellsandinjecteditintoablastulathatwasirradiated with UV. They saw that doing this there was a recovery of the UV-irradiated embryo that developed a nervous system. (A) hen they took the normal amphibian embryo and extracted mRNA from the dorsal lip of the blastopore, T sequenceditcreatingasequencingcDNAlibraryandinjectedsinglemRNAsinmanyUV-irradiatedblastulas.They discovered that by injecting isolatedNoggingmRNAthe phenotype was entirely rescued. o, some mRNA was able to recover, such as Nogging and other mRNAs. S They also discovered that concentration is fondamental: by injecting no Noggin or just a few there is no recovery and injecting too much Nogging thereisnorecoverysotheexactconcentrationisimportant.Nogginworksin a concentration dependent manner. 12 NEURAL DEVELOPMENT – Prof.sse S. Bovetti – A.A 2024-2025 – anno1 semestre1 – Margherita Bosso fterNogginothermoleculeswereisolated,suchasChordinandFollistatin,thatwerefoundtobeexpressedin A the dorsal lip of the blastopore. These molecules were discovered by knockout experiments. Homologues of these genes were promptly found in mammals. All use the same basic mechanism to induce ectoderm to form the nervous system What organizes the organizer? How did the dorsal lip of the blastopore come to express the rganizer signals? o 1. Endoderm induces the cells in the dorsal lip of the blastopore (the presumptive chordo-mesorderm) to start expressing organizer signal genes 2. The protein that endodermal cells secrete to induce the dorsal lip of the blastopore to start making organizers is calledβ-catenin. The mother strategically placed mRNAs and proteins in the egg cytoplasm so that β-catenin would be concentrated in the right place in the endoderm. β-catenins released from themothercell so mRNAs are asymmetrically organized in the cells during development, inducing the ventral or dorsal fate. Chain of inductive events that are needed to make a really complicated metazoan. One advantage of having many different inductive steps is that if a chunk of embryo is removed or damaged, the embryo will self-regulate in compensation, and you’ll still end up with a complete individual. Mechanisms on BMP working he cell expressing the receptor for T BMPs, and when BMP binds the receptor,thatcellbecomesanepidermal cell. Having Nogging, Chordin or Follistatin,thereceptorisblockedbythis antagonist, so BMP can’t ligate the receptor and this cell will become neuron. BMPs prevent the induction of the neural fate inducing the non-neural development.IfBMPscan’tbind,thecell goes through neuronal development. Other signals are released from the notochordandareveryimportantforthe development of the dorso-ventral axis! (next lesson…) 13 NEURAL DEVELOPMENT – Prof.sse S. Bovetti – A.A 2024-2025 – anno1 semestre1 – Margherita Bosso Summary - lesson1 InC.elegans,celldivisionallocatesthetranscriptionfactorsthatwereunevenlydistributedintheeggand consequentlyeachresultingcellhasauniquemixoftranscriptionfactors:mosaicspecificationofcellfate, according to which every cell follows its particular destiny no matter what its neighboring cells are; Inotherspecies,thismosaicspecificationofcellfatehasbeensupplantedbyconditionalspecificationof cell fatethroughcell-cell interactionsand signalsreleased by the neighbouring cells; In vertebrates, maternal factors trigger a chain of events to form the nervous system: ○ accumulation of β-catenin in blastomeres that will become the endoderm; ○ Theβ-catenincausesthecellsthatwillformthedorsallipoftheblastoporetosecreteorganizing proteins such as noggin and chordin; ○ These mesodermal organizers block BMPsignalingin the overlying ectoderm to induce those cells to switch fates from epidermal cells to neural cells, forming the neural tube. The organizer cells migrate to become mesoderm and, eventually, the notochord. Lesson2 - 14.11 (Prof.ssa Bovetti) DEVELOPMENT OF A BODY PATTERN ncetheneuroectodermhasbeenestablished,thenextstepinneuraldevelopmentistodivideitupintoareasthat O willlaythebasisforregionalspecializationsinthestructureofthematurenervoussystem.Thisprocessisknown aspatterning. PATTERNING OF THE NEUROECTODERM In both Drosophila and vertebrates, patterningthenervoussysteminvolvessubdivisionsinthetwo xes of the neuroectoderm: anteroposterior and dorsoventral axis(see picture). a In the dorsal part of the gastrula the neural ectoderm and neural plate forms under the releasing of specific moleculesbythenotochord.Themidlineisimportantbecauseitisthefirstindicatorofthebodyplan:inthemidline theneuraltubeforms.Thenwecandistinguishtheanteroposterioraxisandwhentheneuraltubecloseswecan identify a dorso-ventral orientation. The dorsal portion would be in the opposite part from the midline. In the dorsal part the braindevelopsandtherewillbedifferent structures according to the expression of different genes. As development proceed morphological changes became evident: from anterior to posterior there will be the forebrain, the midbrain, the hindbrain ad finally the spinal chord. Intheimageweseetheformationoftheneurosystemininsects (D rosophila). Development in Drosophila he Drosophila gene decapentaplegic (dpp) encodes a homologue of vertebrate BMP2 and BMP4. The gene T encoding vertebrate chordin is homologous to the Drosophila gene short gastrulation (sog) Neuroectoderm → ventral portion 14 NEURAL DEVELOPMENT – Prof.sse S. Bovetti – A.A 2024-2025 – anno1 semestre1 – Margherita Bosso hedarkgreyisventralpositionedandlightgreyisdorsalpositionandrepresentsthegradientoftheexpressionof T Dorsal.Dotsare different cells. DorsalactivateSnailgene(ventral)andinthedorsalpartactivateDPP,whileinthemedialpartactivatesSogso depending on the concentration of dorsal different genes are activated. The most dorsal portion and thelateral portion are separated because of the antagonist mechanism between DPP and Sog. Thesegenesinteractwitheachother:SnailrepresstheactionofSogandsogrepresstheactionofDPPandvice versa.Thisrepressionmakesthatthesegenesareexpressedinsomeportions.Themostventralpart(mesoderm in violet). Themesodermgoesinsideandthe2piecesoftheectodermjoininthemiddlepartandgoinginsidethe2lateral portions start totoucheachotherandthesewillbecametheneuroectoderm(inDrosophilatheneuraltubeisnot present). Thentherewillbethedelaminationprocessinwhichproliferationofcellsbringstotheformationofdifferentlayers: different layers of cells will give rise to a specific cells type of nervous system. Patterns of gene expression are set-up by morphogens atterning involves long‐range signalling that provides cells with P informationabouttheirlocationwithintheneuralepithelium:POSITIONAL information. Blue cells are sense high concentrations of the signal while red cells sense lower concentration of the signal. Organizer:thesignallingsource(signallingcentre),releasethesignalling molecules forming a gradient Morphogen: A diffusible signal that provokes more than one cellular response depending on its concentration around the cell The response of a cell reflects its distance from the source and its response according to a specific concentration of the morphogen. The cellular response to a morphogen is usually nottheacquisitionofaspecificcellfatebuttheexpressionof specific transcription factorsin domains within the epithelium. TFs influence cell behaviour or fates through affecting the expression of other genes. This process is very long andgradually gives rise to the specific cell type. Whicharethemechanismsthattypicallyorganizeabodyplanincludingbrainregions?Ourbestunderstandingof subsequent events comes from work in fruit flies (Drosophila Melanogaster), because: - Easy to grow → advanced genetics and molecular tools - Life cycle: about 10 days at 25°C (from fertilization to adult age) - embryo development about 24 hours 3 instar - stages sexual maturity reached in 12 hours - Life Span: 60 - 80 days - 17000 genes (many of which are named) - all genome is known - 50% have mammalian homologous - 75% of human disease-associated genes have fly homologues - Olfactory memory and behavioral control Many mutant lines → Our best understanding of subsequent events comes from work in fruit flies ★ 1910:ThomasHuntMorgandiscoveredawhite-eyedflyamongacollectionofred-eyedflies.Heobserved thebandingpatternsofchromosomes,andsawthatthesamepatternwasalwaysobservedinwhite-eyed flies.Withtheseexperimentsheestablishedthechromosomaltheoryofinheritanceforwhichhewonthe Nobel Prize in 1933. ★ 1927: Hermann Muller, discovered X-rays can induce genetic mutations. Muller won the Nobel Prize in 1946 for his discovery. 15 NEURAL DEVELOPMENT – Prof.sse S. Bovetti – A.A 2024-2025 – anno1 semestre1 – Margherita Bosso ★ ’70s and ’80s: Ed Lewis, Christiane Nusslein-Volhard, and Eric Wieschaus identifiedsomeofthegenes thatestablishthedorsal-ventralandanterior-posterioraxesoftheembryo,aswellasthegenesinvolvedin segmentation, which specify the body plan. They won the Nobel Prize in 1995. http://nobelprize.org/nobel_prizes/medicine/laureates/1995/index.html http://superstarsofscience.com/scientists ★ 1990’s:JulesHoffmannusedDrosophilaforresearchoninnateimmunity.HediscoveredTollreceptorsand demonstratedtheirimportanceforsensinganddefendingagainstpathogens.HoffmanwontheNobelPrize in2011forhisworkontheDrosophilainnateimmunesystem,andsharedtheprizewithBruceBeutlerand Ralph Steinman for their work on innate immunity in mammals. Establishing polarity in the egg fter fecondation the cellgoesthroughcellulardivisionwithout A formation of the membrane. In 2 hours: 13 mitotic cycles and 6000 nuclei in a unique cell. Fruit fly embryogenesis differs from that of vertebrates. In insects, the early embryo does not divide into separate cells; rather, the nucleus alone divides repeatedly until the egg consists of a single cell, which we call asyncytium,withone continuous cytoplasm containing many nuclei. imultaneous multi-view imaging of mitotic cycles 10-13inthe S Drosophila syncytial blastoderm (His2Av-GFPS65T transgenic stock). The entire embryowasrecordedin25-secondintervals using light sheet fluorescence microscopy (Tomer et al., 2012), with which we can keep the sample in vivo underthemicroscopeandmarkingcellswithfluorophores (transgenicDrosophilaexpressingGFPundercontrolofa promoter - inducible GFP). Drosophila will be set transparent. Only at that point cells can begin become different from eachother.Allnucleicandetectwhatishappeninginthe entire structure, while cellularization, the formation of membranes will isolate the cells. So there is a passage from uniform behavior to distinct behavior. https://digital-embryo.janelia.org/ ll the proteins and RNAs are supplied by theembryo’s A mother: the embryo hasbeendoingsomethingrepetitive using only maternally supplied genes products. Zygotic RNAsandproteins:thesearerequiredtodonewthings(presentfromstage13).Already5minuteslatercellsno longerhavethesameshape,andacephalicfoldappearswheretheheadwillform.Theformationofacephalicfold is followed by a process of cell movement and invagination (no proliferation). At one point in the anterior part something starts to change in morphology and shape in cellsandthisinduces movementsofcellsintheposteriorpartwi