Cellular Migration and Formation of Axons and Dendrites: Comprehensive Developmental Neuroscience PDF
Preview Cellular Migration and Formation of Axons and Dendrites: Comprehensive Developmental Neuroscience
Cellular Migration and Formation of Axons and Dendrites Comprehensive Developmental Neuroscience Second Edition Senior Editors-in-Chief John Rubenstein Department of Psychiatry & Weill Institute for Neurosciences Universityof California, San Francisco, San Francisco, CA, United States Pasko Rakic Department of Neuroscience & Kavli Institute for Neuroscience Yale School of Medicine, New Haven, CT, United States Editors-in-Chief Bin Chen Department of Molecular, Cell & Developmental Biology Universityof California, Santa Cruz, Santa Cruz, CA, United States Kenneth Y. Kwan Michigan Neuroscience Institute & Department of Human Genetics Universityof Michigan, Ann Arbor, MI, United States Section Editors Alex Kolodkin Solomon H. Snyder Department of Neuroscience Johns Hopkins University School of Medicine, Baltimore, MD, United States Eva Anton UNC Neuroscience Center & Department of Cell and Molecular Physiology Universityof North Carolina School of Medicine, Chapel Hill, NC, United States Academic PressisanimprintofElsevier 125London Wall,LondonEC2Y5AS,UnitedKingdom 525BStreet,Suite1650,SanDiego,CA92101,UnitedStates 50HampshireStreet,5thFloor,Cambridge,MA02139,UnitedStates TheBoulevard,Langford Lane,Kidlington,OxfordOX5 1GB,UnitedKingdom Copyright©2020ElsevierInc.Allrightsreserved. Nopart ofthispublicationmay bereproduced ortransmitted inanyform orbyanymeans, electronicor mechanical,including photocopying, recording,oranyinformation storageandretrieval system,withoutpermission inwritingfromthepublisher. 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Tothefullestextentofthelaw,neither thePublishernortheauthors,contributors, oreditors, assumeany liabilityforany injuryand/ordamagetopersonsorpropertyasamatterofproductsliability,negligence orotherwise,or fromanyuseor operation ofanymethods,products, instructions,or ideascontainedinthematerialherein. LibraryofCongressCataloging-in-Publication Data Acatalogrecord forthisbook isavailablefromtheLibrary ofCongress BritishLibraryCataloguing-in-Publication Data Acataloguerecord forthisbook isavailablefromtheBritishLibrary ISBN:978-0-12-814407-7 Forinformation onallAcademic Presspublications visitourwebsite at https://www.elsevier.com/books-and-journals Publisher:NikkiLevy Acquisitions Editor: NatalieFarra EditorialProjectManager:AndraeAkeh ProductionProjectManager:SuryaNarayanan Jayachandran CoverDesigner: Mark Rogers CoverImage:JasonKeil TypesetbyTNQTechnologies Contributors François Beaubien, Montreal Neurological Institute, R.J. Giger, The University of Michigan, Medical School, McGill University, Department of Neurology and Ann Arbor, MI, United States Neurosurgery, Montréal, QC, Canada WesleyB.Grueber,ColumbiaUniversity,NewYork,NY, FranckBielle,InstitutduCerveauetdelaMoelleEpinière, United States Paris, France K. Hayashi, Keio University School of Medicine, Tokyo, Frank Bradke, Laboratory for Axon Growth and Regen- Japan eration,GermanCenterforNeurodegenerativeDiseases Zhigang He, Kirby Center of Neuroscience, Children’s (DZNE), Bonn, Germany HospitalBoston,HarvardMedicalSchool,Boston,MA, Frédéric Charron, Institut de Recherches Cliniques de United States Montréal (IRCM), Montreal, QC, Canada; Integrated Holden Higginbotham, Department of Biology, Brigham PrograminNeuroscience,McGillUniversity,Montreal, Young University, Rexburg, ID, United States QC,Canada;DepartmentofAnatomyandCellBiology, Katrine Iversen, Montreal Neurological Institute, McGill Department of Biology, McGill University, Montreal, University, Department of Neurology and Neuro- QC, Canada; Department of Medicine, University of surgery, Montréal, QC, Canada Montreal, Montreal, QC, Canada; Division of Exper- imental Medicine, McGill University, Montreal, QC, Artur Kania, Neural Circuit Development Laboratory, Canada Institut de Recherches Cliniques de Montréal (IRCM), Montreal, QC, Canada; Integrated Program in Neuro- AlainChédotal,InstitutdelaVision,SorbonneUniversité, science, McGill University, Montreal, QC, Canada; INSERM, CNRS, Paris, France Department of Anatomy and Cell Biology, Division of Jean-François Cloutier, Montreal Neurological Institute, Experimental Medicine, McGill University, Montreal, McGill University, Department of Neurology and QC, Canada Neurosurgery, Montréal, QC, Canada EyalKarzbrun,KavliInstituteofTheoreticalPhysicsand Christopher L. Cunningham, Solomon H. Snyder Department of Physics, University of California, Santa DepartmentofNeuroscience,JohnsHopkinsUniversity Barbara, CA, United States School of Medicine, Baltimore, MD, United States Arnold R. Kriegstein, Department of Neurology, Uni- Fernando de Castro, Instituto Cajal-CSIC, Spanish versityofCalifornia,SanFrancisco,CA,UnitedStates; Research Council/Consejo Superior de Investigaciones The Eli and Edythe Broad Center of Regeneration Científicas-CSIC, Madrid, Spain Medicine and Stem Cell Research, University of Cal- Kevin C. Flynn, Stem Cell and Gene Therapy, Bio- ifornia, San Francisco, CA, United States Techne, Minneapolis, MN, United States Zeljka Krsnik, Croatian Institute for Brain Research, Fernando García-Moreno, Achucarro Basque Center for School of Medicine, University of Zagreb, Zagreb, Neuroscience, Parque Científico UPV/EHU Edif. Sede, Croatia Leioa, Spain; Ikerbasque Foundation, Bilbao, Spain Christophe Laumonnerie, Department of Developmental Sonia Garel, Institut de Biologie de l’École Normale Neurobiology, St. Jude Children’s Research Hospital, Supérieure (IBENS), PSL Université, Paris, France; Memphis, TN, United States Institut National de la Santé et de la Recherche Médi- Julie L. Lefebvre, The Hospital for Sick Children, Tor- cale (INSERM) U1024, Paris, France; Centre National onto, ON, Canada; Department of Molecular Genetics, delaRechercheScientifique(CNRS)UMR8197,Paris, University of Toronto, Toronto, Canada France xvii xviii Contributors Fanny Lepiemme, GIGA-Stem Cells / GIGA-Neuro- Masato Sawada, Department of Developmental and sciences, University of Liège, Liège, Belgium Regenerative Neurobiology, Institute of Brain Science, Nagoya City University Graduate School of Medical Guangnan Li, Department of Neurology, University of Sciences, Nagoya, Japan California, San Francisco, CA, United States JosephJ.LoTurco,UniversityofConnecticut,Mansfield, Kazunobu Sawamoto, Department of Developmental and Regenerative Neurobiology, Institute of Brain Science, CT, United States Nagoya City University Graduate School of Medical Le Ma, Thomas Jefferson University, Philadelphia, PA, Sciences, Nagoya, Japan; Division of Neural Develop- United States ment and Regeneration, National Institute for Physio- Jean-Bernard Manent, Inmed Inserm, Marseille, France logical Sciences, Okazaki, Japan Julie Marocha, The Hospital for Sick Children, Toronto, K. Sekine, Keio University School of Medicine, Tokyo, ON, Canada; Department of Molecular Genetics, Uni- Japan versity of Toronto, Toronto, Canada Carla Silva G., GIGA-Stem Cells / GIGA-Neurosciences, Zoltán Molnár, Department of Physiology, Anatomy and University of Liège, Liège, Belgium Genetics, University of Oxford, Oxford, United David J. Solecki, Department of Developmental Neuro- Kingdom biology, St. Jude Children’s Research Hospital, Mem- K.Nakajima,KeioUniversitySchoolofMedicine,Tokyo, phis, TN, United States Japan Constantino Sotelo, Institut de la Vision, Sorbonne Uni- Laurent Nguyen, GIGA-Stem Cells / GIGA-Neuro- versité, INSERM, CNRS, Paris, France sciences, University of Liège, Liège, Belgium H. Tabata, Keio University School of Medicine, Tokyo, Stephen C. Noctor, Department of Psychiatry and Japan Behavioral Sciences, UC Davis MIND Institute, Sac- MarcTessier-Lavigne,StanfordUniversity,Stanford,CA, ramento, CA, United States United States Hirofumi Noguchi, Department of Neurology, University Stephen R. Tymanskyj, Thomas Jefferson University, of California, San Francisco, CA, United States Philadelphia, PA, United States R. Jeroen Pasterkamp, Department of Translational Marieke G. Verhagen, Department of Translational Neu- Neuroscience, UMC Utrecht Brain Center, University roscience, UMC Utrecht Brain Center, University Medical Center Utrecht, Utrecht University, Utrecht, Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands The Netherlands SamuelJ.Pleasure,DepartmentofNeurology,University Fan Wang, Department of Neurobiology, Duke Uni- of California, San Francisco, CA, United States versity, Durham, NC, United States F. Polleux, Columbia University, New York, NY, United Franco Weth, Karlsruhe Institute of Technology, Zoo- States logicalInstitute,DepartmentofCellandNeurobiology, Tatiana Popovitchenko, Departmentof Neuroscience and Karlsruhe, Germany Cell Biology, Rutgers University, Robert Wood John- Patricia T. Yam, Institut de Recherches Cliniques de sonMedicalSchool,NewBrunswick,NJ,UnitedStates Montréal (IRCM), Montreal, QC, Canada Janet E.A. Prince, Montreal Neurological Institute, Jing Yang, School of Life Sciences, Peking University, McGill University, Department of Neurology and Beijing, China Neurosurgery, Montréal, QC, Canada Bing Ye, University of Michigan, Ann Arbor, MI, United Mladen-Roko Rasin, Department of Neuroscience and States Cell Biology, Rutgers University, Robert Wood John- Bernard Zalc, Université Pierre & Marie Curie-Paris 6, sonMedicalSchool,NewBrunswick,NJ,UnitedStates Centre de Recherche de l’Institut du Cerveau et de la Orly Reiner, Department of Molecular Genetics, The Moelle épinière, UMR_S 975, Inserm U 975, CNRS Weizmann Institute of Science, Rehovot, Israel UMR 7225, Hôpital de la Salpêtrière, Paris, France Chapter 1 Development of neuronal polarity in vivo F. Polleux ColumbiaUniversity,NewYork,NY,UnitedStates Chapter outline 1.1. Introduction 3 1.5.1. Roleofproteindegradationandlocaltranslationin 1.2. Axoninitiationinvitroversusinvivo 4 axonspecificationandaxongrowth 8 1.2.1. Axoninitiationinvitro 4 1.5.2. Roleofcytoskeletaldynamicsinaxoninitiationand 1.2.2. Axoninitiationinvivo 5 growth 9 1.3. Distinctionbetweencuesregulatingaxonspecification 1.5.3. Majorsignalingpathwaysinvolvedinaxoninitiation versusaxongrowth 5 andgrowth 10 1.4. Extracellularcuesregulatingneuronalpolarizationand 1.5.3.1. LKB1anditsdownstreamkinasesSAD-A/B axoninitiation 6 andMARK1-4 10 1.4.1. Netrin-1andWntcontrolaxoninitiationin 1.5.3.2. PAR3ePAR6eAPKC 11 Caenorhabditiselegans 6 1.5.3.3. Ras-andRho-familyofsmallGTPases 12 1.4.2. Polarizedemergenceoftheaxoninretinalganglion 1.5.3.4. PI3KandPTENsignalingduringaxon cellsofXenopus 6 specification 13 1.4.3. Extracellularcuesunderlyingtheemergenceofaxon 1.5.3.5. AKT/proteinkinaseB 14 anddendritesinmammalianneurons 7 1.5.3.6. GSK3andaxonspecification 14 1.5. Intracellularpathwaysunderlyingneuronalpolarization 8 1.6. Conclusionandfuturedirections 15 References 16 1.1 Introduction Theabilityofneuronstoformasingleaxonandmultipledendritesunderliesthedirectionalflowofinformationtransferin the central nervous system (CNS). Dendrites and axons are fundamentally distinct domains of neurons at the molecular, ultrastructural, and functional levels. Dendrites integrate synaptic inputs, triggering the generation of action potentials at the level of the soma and the axon initiation segment (AIS). Action potentials then propagate along the axon and trigger neurotransmitter release at presynaptic contacts onto their postsynaptic partners. This chapter reviews what is currently knownabout thecellularandmolecularmechanisms underlyingtheabilityofneurons topolarize andformasingle axon and multiple dendrites during development. This question has received much attention over the past threedecades using mainly in vitro approaches following some of the pioneering work of Gary Banker and colleagues on dissociated hip- pocampalneuroncultures(CraigandBanker,1994;GoslinandBanker,1989).Intheseassays,immaturehippocampalor cortical pyramidal neurons (PNs) are dissociated and maintained in culture for variable periods of time (typically 1e 3 weeks). Remarkably, in these conditions, PNs can repolarize to form a single axon and multiple dendrites and later establish functional synaptic contacts in these reductionist conditions. This approach became, and remains, the dominant modeltostudymultipleaspectsofneuronalpolarization,includingaxonspecificationaswellasaxonanddendritegrowth, andithasyieldedtheidentificationofmanymoleculesthatregulateaxonformationinvitro.However,thecentralquestion for the field has been to determine if the cellular and molecular mechanisms identified in these in vitro conditions are conservedinvivo.Atpresent,onlyafewofthegenesidentifiedusinginvitroapproacheshavebeenshowntoberequired foraxoninitiationandoutgrowthinvivo.Invitro,axoninitiationandelongationisthoughttoreflecttheintrinsicabilityof neuronstopolarizeintheabsenceofrelevantextracellularcues.However,extracellularcueshavebeenshowntoplayan 3 CellularMigrationandFormationofAxonsandDendrites.https://doi.org/10.1016/B978-0-12-814407-7.00001-8 Copyright©2020ElsevierInc.Allrightsreserved. 4 PART | I Formationofaxonsanddendrites importantroleduringneuronalpolarizationinvivo.Here,wefocusonourcurrentunderstandingofthecomplexinterplay between extracellular cues and intracellular signaling pathways underlying the emergence of axons and dendrites during neuronal polarization in vivo. 1.2 Axon initiation in vitro versus in vivo 1.2.1 Axon initiation in vitro Historically, the advent of in vitroedissociated neuronal cultures provided an experimental template for improving our understanding of the cell biology of neuronal polarity, including the specification of axons and dendrites. Pioneering work usingtheseculturesestablishedaparadigminwhichisolatedneuronsinculturecanadoptspatiallyandfunctionallydistinct dendriticandaxonaldomains(CraigandBanker,1994;GoslinandBanker,1989).Carefulanalysisoftheseculturesledto theobservationthatculturedhippocampalneuronstransitionthroughseveralstages:fromfreshlyplatedstage1cellsbearing immatureneuritestostage5cellsthatexhibitmatureaxons,dendrites,dendriticspines,andfunctionalsynapses(Fig.1.1A) (CraigandBanker,1994;Dottietal.,1988).Itshouldbenotedthat,intheclassicalE18rathippocampalcultures,mostplated FIGURE1.1 Parallelbetweenneuronalpolarizationinvitroandinvivo.Comparisonofthesequenceofeventsleadingtothepolarizationofcortical pyramidalneuronsinvivoandinvitro.(A)Indissociatedcultures,postmitoticcorticalneuronsdisplayspecifictransitionsasclassicallydescribedfor hippocampalneuronsbyDottietal.(1988).Atstage1,immaturepostmitoticneuronsdisplayintenselamellipodialandfilopodialprotrusiveactivity, whichleadstotheemergenceofmultipleimmatureneurites,stage2.Stage3representsacriticalstepwhenneuronalsymmetrybreaksandasingleneurite growsrapidlytobecometheaxon(purple)whileotherneuritesacquiredendriticidentity.Stage4ischaracterizedbyrapidaxonanddendriticoutgrowth. Finally, stage 5 neurons are terminally differentiated pyramidal neurons harboring dendritic spines and the axon initiation segment (AIS). (B) The axonedendrite polarity of pyramidal neurons is derived from the polarized emergence of the trailing (TP) and leading processes (LP), respectively. Invivo,pyramidalneuronsacquireotherkeyfeaturesoftheirterminalpolaritysuchastheAIS(yellowcartridge)anddendriticspines(grayprotrusions) duringthefirstpostnatalweeksofdevelopment. Developmentofneuronalpolarityinvivo Chapter | 1 5 cellswerepolarizedpostmitoticneuronsbeforedissociation;therefore,neuronalpolarizationusingthisinvitromodellikely corresponds to the “repolarization” of previously polarized neurons in vivo. It is therefore important to keep in mind that molecular manipulations in this in vitro model act on previously polarized neurons that may retain some aspects of polar- ization, and this can be critical for interpreting results from such manipulations. Recent advances in techniques that allow manipulationofgeneexpressionmorespecificallyinneuralprogenitors,suchasinuteroorexuterocorticalelectroporation (Hand et al., 2005; Hatanaka and Murakami, 2002; Saito and Nakatsuji, 2001; Tabata and Nakajima, 2001), provide a paradigmto(1)manipulategeneexpressioninprogenitors,beforeneuronalpolarizationoccursuponcellcycleexit;and(2) visualizetheearlieststagesofneuronalpolarizationina contextualcellularenvironment,including inorganotypicslicesor intact embryonic brain (Barnes et al., 2007; Calderon de Anda et al., 2008; Hand et al., 2005). 1.2.2 Axon initiation in vivo Neuronalpolarizationcanbedividedintoseveralspecificstepsinvivo.Uponcellcycleexit,mammalianneuronsusually migrate over long distances before reaching their final destinations. In vivo, most neurons undergo axonedendrite po- larizationduringmigration.Whileinitiatingradialmigration,neocorticalPNsformaleadingprocessandatrailingprocess, each becoming the axon or the dendrite, respectively (Fig. 1.1B). Careful examination of the morphological transition betweenneuralprogenitorandpostmitoticneuronrevealsthatneuronscaninherittheiraxonanddendritepolaritydirectly fromtheapicobasalpolarityoftheirprogenitors.Thisisthecaseforretinalganglioncells(RGCs)andbipolarcellsinthe developing vertebrate retina (Hinds and Hinds, 1978; Morgan et al., 2006; Zolessi et al., 2006, reviewed in Barnes and Polleux, 2009). In other neuronal subpopulations undergoing long-range migration, neuronal morphogenesis involves extensivestereotypicalchanges,leadingtopolarizedoutgrowthoftheiraxonsanddendrites.Thisisthecaseforcerebellar granuleneurons(CGNs)aswellascorticalandhippocampalPNs,twoofthebest-studiedmodelsofneuronalpolarization (Gao and Hatten, 1993; Hatanaka and Murakami, 2002; Komuro et al., 2001; Noctor et al., 2004; Rakic, 1971, 1972; Shoukimas and Hinds, 1978). Both CGNs and PNs acquire their axonedendrite polarity from the polarized emergence of their trailing and leading processes, respectively, during migration (reviewed in Barnes and Polleux, 2009). Precise examination of the process dynamicsoccurringshortlyaftercellcycleexitindorsaltelencephalicprogenitorssuggeststhatthereisoftenaslightdelay betweenformationoftrailing(axoninitiation)andleading(dendriteelaboration)processes,withtrailingprocessformation frequently preceding leading process formation (Calderon de Anda et al., 2008; Hand and Polleux, 2011; Namba et al., 2014). However, one thing is clear in both PNs and CGNs: Axon formation starts before or during radial migration. Interestingly,different neuronalpopulationsdisplaydistinct modesofaxonformation,reflectingtheirmodeofmigration, lineage, and type of axon projection. For example, cortical interneurons, which will form axons projecting only locally withinthecortex,originatefromtheventraltelencephalonandmustmigrateoververylongdistancesbeforeinitiatingtheir axon upon reaching their final destination in the cortex (Bortone and Polleux, 2009; Cobos et al., 2007; Yamasaki et al., 2010). The precise mechanisms underlying axon emergence in cortical interneurons are largelyunknown. However,they are strikingly different from those employed by radially migrating pyramidal cortical neurons, which initiate axon for- mation during migration. This leads to the hypothesis that the ability of interneurons to form an axon is inhibited during tangentialmigrationandthataxoninitiationincorticalinterneuronsmaydependuponfactor(s)presentonlyintheirtarget environment, the cortex. As discussed later in the chapter, an emerging concept from recent work done primarily in Caenorhabditis elegans suggeststhat,invivo,the“symmetry-breaking”eventsthatleadtotheemergenceofthedendriteandtheaxonrequirethe ability of postmitotic neurons to sense gradients of extracellular cues, leading to the asymmetric activation of signaling pathways underlying the emergence of the axon. These data are supported by recent evidence in mammals showing that extracellularcuessuchasdiffusibleTGFbormembrane-boundcelladhesionmoleculessuchasTAG-1playrolesinaxon specification in vivo by triggering specific signaling pathways in the neurite’s trailing process that becomes the axon (Yi et al., 2010; Namba et al., 2014). 1.3 Distinction between cues regulating axon specification versus axon growth Moststudiespublishedoverthepasttwodecadesinthisfieldhavebeenperformedusinginvitroapproaches.Theclassic paradigmforconfirmingtheregulatoryroleofageneinneuronalpolarityistoshowthatdownregulationofitsexpression using shRNA technology or gene knockout technology is required for axon formation. These experiments are typically doneusingstainingwithaxon-specificmakersandmeasurementofneuritelengthsincetheaxonusuallygrows5e10times faster than neuritis, which will eventually become dendrites. However, this type of evidence may not be sufficient to 6 PART | I Formationofaxonsanddendrites distinguishunambiguouslyaneffectorofaxonspecificationfromamoleculesimplyrequiredforaxongrowth(Jiangetal., 2005). Conversely, showing that overexpression or overactivation of a candidate molecule leads to the emergence of multipleneuritesthatdisplaythemolecularidentityofanaxonisgenerallyusedtosuggestthatthismoleculeissufficient to confer axon identity. However, this approach is limited by the fact that it relies on overexpression, which can be confoundedbyabnormalactivationofapathwaynormallynotinvolvedinaxonspecificationorneuronalpolarity.Recent technical advances allow for the manipulation of gene expression in vivo and include in utero cortical electroporation in rodentcortexorcerebellum(Famulskietal.,2010;SaitoandNakatsuji,2001)andalsotransgenicapproachesinXenopus (Zolessi et al., 2006). Therefore, more biologically relevant validation of candidate gene function during neuronal po- larizationoftenincludestestingitsrequirementusing geneknockoutorshRNA-mediatedknockdowntechnologies orthe analysis of conventional or conditional knockout in combination with in utero electroporation allowing single cell reso- lutionanalysisofaxonformation(Barnesetal.,2007;Shellyetal.,2007;Yietal.,2010;Nambaetal.,2014).Finally,one importantcaveatoftheinvivoapproachesmentionedinthischaptertostudymammalianaxonspecificationisthatseveral ofthe molecules identified byloss-of-functionanalysisnotonly affect axon/dendrite (trailing/leading process)generation but also often when compromised lead to abnormal initiation of radial migration. This effect on neuronal migration can complicate the interpretation of experimental results since defects in axon specification could be the direct result of the molecule’sfunctioninaxonspecification,orthesedefectscouldresultfromsecondaryeffectsthatarearesultofpreventing the postmitotic neuron from responding to extracellular cues that are required for neuronal polarization. 1.4 Extracellular cues regulating neuronal polarization and axon initiation 1.4.1 Netrin-1 and Wnt control axon initiation in Caenorhabditis elegans Isthereanyinvivoevidencefortheroleofextracellularcuesinthespecificationofneuronalpolarity?Significantprogress inourunderstandingofthemolecularandcellularmechanismsspecifyingaxoninitiationduringneuronalpolarizationhas been made using C. elegans as a model. This pioneering work has markedly enhanced our understanding of how extra- cellular cues instruct axon initiation in vivo (reviewed by Yogev and Shen, 2017). The neurons of the nematode have a stereotypedmorphology,ascanbeseenwithrespecttospecificprojectionsalongthedorsoventralandanterioreposterior bodyaxes.Elegantexperimentsinvolvinggeneticscreenshaveidentifiedanextracellularcue,UNC-6(Netrin),alongwith itsreceptorUNC-40 (DCC), ascritical genesfororchestratingaxoninitiation invivo(Adleretal.,2006).Thisworkalso identified downstream proteins inthispathway,including (mammalian orthologs are shownin parenthesis,when known) AGE-1(phosphoinositide-3kinase[PI3K]),DAF-18(PTEN),UNC-34(Enabled),CED-10(Rac),UNC-115/AbLIM,and MIG-10/lamellipodin. The current model for the relationship among these genes and UNC-6/netrin signaling involves DAF-18’s limitation of AGE-1 activity following UNC-40/DCC stimulation and the asymmetric recruitment of MIG-10/ lamellipodin to the plasma membrane. This recruitment requires activated CED-10/Rac binding directly to MIG-10/ lamellipodin and the involvement of the PAK-like kinase, Pak-1 (Adler et al., 2006). The function of a kinase in the regulation of cytoskeletal rearrangement is consistent with a similar role played by MIG-10/lamellipodin and the likely mechanismbywhichitoperates oncerecruited totheplasmamembrane tostimulate directedneuriteoutgrowth. Another regulatorthoughttoactinconcertwithMIG-10/lamellipodintodrivefilopodialformationistheEnabledhomolog,UNC- 34 (Chang et al., 2006). Finally, SLT-1 (Slit) is another extracellular cue that likely acts through MIG-10 recruitment to control neuronal polarization (Chang et al., 2006). Two other studies have identified the diffusible signal Wnt and its receptor as critical regulators of axon specification andneuronalpolarity(HilliardandBargmann,2006;PrasadandClark,2006).Inadditiontocharacterizinglossoffunction mutants inthegenesencodingLin-44 (Wnt)anditsreceptorLin-17 (Frizzled[Fzl]),ageneticscreen hasidentifiedVPS- 35, a component of retromer complex that regulates vesicular traffic and is required for proper Wnt secretion, as an important regulator of neuronal polarity (Pan et al., 2008; Prasad and Clark, 2006). 1.4.2 Polarized emergence of the axon in retinal ganglion cells of Xenopus Detailed live imaging experiments of Xenopus RGC polarization revealed that polarized axon outgrowth requires un- identifiedextracellularcuespresentinthebasallamina(Randlettetal.,2011;Zolessietal.,2006).Theaxonofdeveloping RGCs normally grows on the basal side of the neuron. In a mutant called Nok, characterized by the absence of retinal pigmented epithelium, some postmitotic RGC neurons show defective polarized axon outgrowth on the apical side along the now-exposed basal lamina. In this context, the polarized emergence of the axon on the basal side of the RGC is correlated with the position of the centrosome, Par3, and the apical complex (containing the atypical protein kinase C Developmentofneuronalpolarityinvivo Chapter | 1 7 [aPKC], b-catenin, and F-actin) on the apical side of the cell where the dendrite will emerge. Taken together, this work stronglysuggeststhat(1)thebasallaminacontainsimportantextracellularcuesthatplayaroleinorganizingthepolarized emergenceoftheRGCaxonand(2)RGCneuronsinherittheintrinsicapicobasalpolarityoftheirprogenitoratleastwith respect to the Par3/aPKC components of the polarity complex. Recently, asignalingcascade hasbeenlinkedtopotentialextracellularcuesregulatingaxoninitiation invivo(Barnes and Polleux, 2009; Barnes et al., 2008). Conditional deletion of LKB1 in pyramidal cortical neurons (also called Par4 or STK11) demonstrated that LKB1 is required for axon initiation in cortical neurons but does not impact their radial migration (Barnes et al., 2007). Structure/function analysis indicates that phosphorylation of LKB1 at Serine 431 is required for its function in axon specification (Barnes et al., 2007). This phosphorylation event has been linked to the ability of extracellular cues such as brain-derived neurotrophic factor (BDNF) to stimulate cAMP production and protein kinase A (PKA)edependent phosphorylation of LKB1 on S431 in the nascent axon (see below for details; Shelly et al., 2007, 2010, 2011). 1.4.3 Extracellular cues underlying the emergence of axon and dendrites in mammalian neurons Severallinesofevidencesuggestthatextracellularcuescandirectthepolarizedemergenceoftheaxonanddendritesboth in vitro and in vivo. One paradigm involves dissociated cortical or hippocampal PNs plated on striped substrates coated with two different cell adhesion molecules (e.g., laminin and NgCAM, reviewed in Barnes and Polleux, 2009). The first immature neurite of E18 hippocampal neurons contacting the boundary between two stripes systematically becomes the axon.ThisoccursdespiteinitialoutgrowthofimmatureneuritesoccursonlamininorNgCAM,suggestingthatimmature neuritescandetectchangesinthenatureoftheextracellularsubstrateratherthantheabsolutenatureofthenovelsubstrate they are encountering (Esch et al., 1999). Using a similar approach, Shelly and colleagues showed that neurites of immature hippocampal neurons growing on a patterned substrate can detect the presence of BDNF, which plays an instructive role in axon specification because the first neurite contacting a BDNF stripe systematically becomes the axon (Shelly et al., 2007). The effect of BDNF on axon specification requires cAMP-dependent PKA activation and phos- phorylationofLKB1atposition431byPKA(Shellyetal.,2007,2010),suggestingthatLKB1phosphorylationonS431 actsasadetectorthatbreaksneuronalsymmetryfollowingencounterswithextracellularcuessuchasBDNFinthisinvitro context. To detect the existence of extracellular cues that play a role in cortical axon guidance and neuron polarization, an overlay in vitro assay was developed. This rather simple assay involves plating fluorescently labeled dissociated cortical neuronsontocorticalslicestotestwhetherpolarizedaxonemergenceinvivoismainlytheresultofasymmetricactivation ofintracellulareffectors(perhapsinheritedbyprogenitors)orextracellularcuesthatdirectaxonspecification.Polleuxetal. demonstrated that the second scenario is most likely because only a couple of hours after plating, the vast majority of corticalneuronsdisplayedasingle,shortaxondirectedventrallytowardtheventricle(Polleuxetal.,1998),asobservedfor radially migrating neurons in vivo. These authors went on to demonstrate that the class 3 secreted semaphorin, Sema3A, which is enriched in the most superficial part of the cortical wall (the top of the cortical plate), plays a role in repulsing axoninitiationventrallytowardtheventricle(Polleuxetal.,1998).Morerecently,Sema3Awasshowntoalsoregulatethe polarized emergence of the leading process/apical dendrite both in the overlay assay, that is, independently of radial migration where it requires cGMP production and PKG activation (Polleux et al., 2000), and also in vivo during radial migration(Chenetal.,2008).Interestingly,Sema3Acanplayaroleinthespecificationofdendriticidentitybyactivatinga cGMP-dependent pathway involving activation of cGMP-gated calcium channels (Nishiyama et al., 2011) and also by repressing axonal identity in a LKB1-dependent manner (Shelly et al., 2007, 2010, 2011; see Fig. 1.2). Recently,TGFbsignalingwasshowntoberequiredforthepolarizedemergenceoftheaxonofradiallymigratingPN invivo(Yietal.,2010,Fig.1.3).TGFbligandsareexpressedinthegerminalzoneofthecortex,wheretheycouldactasan instructive “ventral”cuefor thepolarized emergenceoftheaxoninmultipolar neurons before engagingradialmigration. InvitroexperimentsdemonstratedthatlocalapplicationofTGFbonasingleneuriteinimmaturestage1corticalneuronsis sufficient to trigger fast axonal extension (Yi et al., 2010). Importantly, conditional genetic deletion of TGFb receptor 2 expression leads to the production of neurons without trailing process/axon in vivo. One noticeable difference compared with the conditional ablation of LKB1 (Barnes et al., 2007), which also leads to the absence of trailing process/axon formation but not radial migration defects, is that TGFb receptor 2 conditional deletion leads to retardation of radial migration in a subset of cortical neurons (Yi et al., 2010) (Fig. 1.2). TGFb receptor function during axon specification also requires the phosphorylation of Par6 on S435, which was previouslyshowntomediatetheepithelial-to-mesenchymaltransition(EMT;Ozdamaretal.,2005).Asdiscussedlaterin this chapter, this “noncanonical” TGFb receptoredependent signaling represents an attractive in vivo signaling pathway
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