Establishment of Axon-Dendrite Polarity in Developing Neurons

ANRV379-NE32-15 ARI 13 May 2009 8:40

Establishment ofAxon-Dendrite Polarityin Developing NeuronsAnthony P. Barnes1 and Franck Polleux2

1Pediatric Neuroscience Research Program, Department of Pediatrics, Oregon Healthand Science University, Portland, Oregon 97239-3098; email: [email protected] Center, Department of Pharmacology, University of North Carolina,Chapel Hill, North Carolina 27599-7250; email: [email protected]

Annu. Rev. Neurosci. 2009. 32:347–81

First published online as a Review in Advance onMarch 24, 2009

The Annual Review of Neuroscience is online atneuro.annualreviews.org

This article’s doi:10.1146/annurev.neuro.31.060407.125536

Copyright c© 2009 by Annual Reviews.All rights reserved

0147-006X/09/0721-0347$20.00

Key Words

neuronal migration, cortex, signaling, LKB1, Par complex

AbstractNeurons are among the most highly polarized cell types in the body, andthe polarization of axon and dendrites underlies the ability of neuronsto integrate and transmit information in the brain. Significant progresshas been made in the identification of the cellular and molecular mecha-nisms underlying the establishment of neuronal polarity using primarilyin vitro approaches such as dissociated culture of rodent hippocam-pal and cortical neurons. This model has led to the predominant viewsuggesting that neuronal polarization is specified largely by stochastic,asymmetric activation of intracellular signaling pathways. Recent evi-dence shows that extracellular cues can play an instructive role duringneuronal polarization in vitro and in vivo. In this review, we synthesizethe recent data supporting an integrative model whereby extracellularcues orchestrate the intracellular signaling underlying the initial breakof neuronal symmetry leading to axon-dendrite polarization.

347

Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 348EMERGENCE OF NEURONAL

POLARITY IN VIVO . . . . . . . . . . . . . 349COMPARISON OF NEURONAL

POLARIZATION IN VITROAND IN VIVO. . . . . . . . . . . . . . . . . . . . 349

SIGNALING MECHANISMSUNDERLYINGESTABLISHMENT OFAXON-DENDRITE POLARITY . 351PI3-Kinase and Potential Effectors . 351PTEN . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353AKT/Protein Kinase B . . . . . . . . . . . . . 353Glycogen Synthase Kinase 3 . . . . . . . . 354LKB1 and SAD-A/B and MARK

Kinases, the MammalianOrthologs of Par4 and Par1 . . . . . 355

Ras- and Rho-family of SmallGTPases . . . . . . . . . . . . . . . . . . . . . . . 358

PAR3-PAR6-aPKC . . . . . . . . . . . . . . . . 362GLOBAL CELLULAR

MECHANISMS OF NEURONALMORPHOGENESIS . . . . . . . . . . . . . . 365Local Protein Degradation . . . . . . . . . 365Cytoskeletal Dynamics . . . . . . . . . . . . . 366

Cytoplasmic Flow and DirectedMembrane Trafficking . . . . . . . . . . 367

Molecular Motors . . . . . . . . . . . . . . . . . . 367Diffusional Barrier . . . . . . . . . . . . . . . . . 368

ROLE OF EXTRACELLULARCUES IN ORCHESTRATINGINTRACELLULAR SIGNALINGDURING NEURONALPOLARIZATION . . . . . . . . . . . . . . . . . 368In Vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . 368In Vivo . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369

POTENTIAL RELATIONSHIPBETWEENNEUROEPITHELIAL CELLPOLARITY ANDPOSTMITOTIC NEURONPOLARITY . . . . . . . . . . . . . . . . . . . . . . . 370

SPECIFICATION OF DENDRITICIDENTITY . . . . . . . . . . . . . . . . . . . . . . . 371

INTERPLAY BETWEENEXTRACELLULAR-INTRACELLULARREGULATORS OF NEURONALPOLARITY: INSIGHTS FROMCAENORHABDITIS ELEGANS . . . . 371

CONCLUSION . . . . . . . . . . . . . . . . . . . . . 372

INTRODUCTION

Cell polarity lies at the center of manybiological processes including epithelial mor-phogenesis, cell migration, and chemotaxis.Its disruption is thought to underlie severalpathological states including cell transforma-tion and metastasis. Neurons are among themost polarized cell types in the body and arecompartmentalized into two molecularly andfunctionally distinct domains: the axon and thedendrites. Neurons typically form a single axonand multiple dendrites, which underlie thedirectional flow of information transfer in thecentral nervous system. Dendrites integratesynaptic inputs, triggering the generation of

action potentials at the level of the soma, whichpropagate along the axon, making presynapticcontacts onto target cells. How are the axonaland dendritic compartments generated duringdevelopment? This question has received a lotof attention at both the cellular and the molecu-lar levels over the past three decades. A seminalreview published by Craig & Banker (1994)fifteen years ago in this journal observed that, atthe time, “we [knew] almost nothing about thecellular mechanisms responsible for the com-partmentation in neurons” (p. 278). On the ba-sis of existing data, Craig & Banker provided aconceptual framework for the experiments that,over the past decade and a half, have improvedour understanding of how neuronal polarity is

348 Barnes · Polleux

Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

Gustavo

Highlight

ANRV379-NE32-15 ARI 13 May 2009 8:40

established during development. This reviewprovides an updated and contextualized modelsynthesizing the body of recent work suggestingthat in vivo, neuronal polarity is most probablythe result of a complex interaction betweenextracellular cues and intrinsic cell polaritypathways.

EMERGENCE OF NEURONALPOLARITY IN VIVO

Neuronal polarization can be divided intoseveral specific steps. Upon cell cycle exit,mammalian neurons usually migrate over a longdistance before reaching their final destination.In vivo, most neurons undergo axon-dendritepolarization during migration. While migrat-ing, postmitotic neurons form a leading processand a trailing process, each becoming the axonor the dendrite depending on the cell type(Figure 1). Careful examination of themorphological transition between neural pro-genitors and postmitotic neurons reveals thatneurons can inherit their axon and dendritepolarity directly from the apico-basal polarityof their progenitors. This is the case for retinalganglion cells and bipolar cells in the develop-ing vertebrate retina (Figure 1a–b) (Hinds &Hinds 1978, Morgan et al. 2006, Zolessi et al.2006). The morphology of other neurons,however, undergoes extensive stereotypicalchanges, leading to polarized outgrowth oftheir axon and dendrites (Figure 1c–d ). This isthe case for cerebellar granule neurons (CGN)as well as cortical and hippocampal pyramidalneurons (PN), three of the best-studied modelsof neuronal polarization both in vitro andin vivo (Gao & Hatten 1993; Hatanaka &Murakami 2002; Komuro et al. 2001; Noctoret al. 2004; Rakic 1971, 1972; Shoukimas &Hinds 1978). Both CGN and PN acquiretheir axon-dendrite polarity from the polarizedemergence of the trailing-leading processesduring migration. Therefore, in these twoneuronal cell types, an important functionalrelationship exists between the molecularmechanisms underlying polarized migrationand the final axon-dendrite polarity.

COMPARISON OF NEURONALPOLARIZATION IN VITROAND IN VIVO

Historically, the advent of in vitro dissoci-ated neuronal cultures provided an experimen-tal template for improving our understandingof the cell biology of neuronal polarity. Pio-neering work using these cultures establisheda paradigm in which isolated neurons in cul-ture can adopt spatially and functionally distinctdendritic and axonal domains (Craig & Banker1994, Goslin & Banker 1989). Careful analysisof these cultures led to the observation that cul-tured hippocampal neurons transition throughseveral stages, from freshly plated stage 1cells bearing immature neurites to stage 5 cellsthat exhibit mature axons, dendrites, dendriticspines, and functional synapses (Craig & Banker1994, Dotti et al. 1988) (Figure 2). It should benoted that in the classical E18 rat hippocampalcultures, most plated cells were polarized post-mitotic neurons prior to dissociation; therefore,neuronal polarization using this in vitro modellikely corresponds to the repolarization of pre-viously polarized neurons in vivo. It is there-fore important to keep in mind that molecu-lar manipulations in this in vitro model act onpreviously polarized neurons that may retainsome aspects of polarization, which can be criti-cal for interpreting the results. Recent advancesin the techniques allowing the manipulation ofgene expression more specifically in neural pro-genitors such as in utero or ex utero corticalelectroporation (Hand et al. 2005, Hatanaka &Murakami 2002, Saito & Nakatsuji 2001,Tabata & Nakajima 2001) provide a paradigmto (a) manipulate gene expression in progen-itors, i.e., before neuronal polarization occursupon cell cycle exit, and (b) visualize the earlieststages of neuronal polarization in a contextualcellular environment, i.e., in organotypic slicesor intact embryonic brain (Barnes et al. 2007,Calderon de Anda et al. 2008, Hand et al. 2005).

Most studies reviewed in this article wereperformed primarily using in vitro approaches.The classic paradigm to confirm a regulatoryrole for a gene in neuronal polarity is to

www.annualreviews.org • Axon-Dendrite Polarity 349

Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

Gustavo

Highlight

Gustavo

Highlight

Gustavo

Underline

someterse

Gustavo

Highlight

Gustavo

Underline

llegada

ANRV379-NE32-15 ARI 13 May 2009 8:40

Mousebipolarretinal

cells

EGL

IGL

PCL

ML

b

P1 P5 P10 P20

Granule cells in early postnatal rodent cerebellum

GCL

ML

VZ

Zebrafishand mouseretinalganglion cells

Apical

Basal

OPL

IPL

GCL

ML

c

a

CP

VZ

SVZ

IZ

MZ

6

5

CP

MZ

IZ

E11–E17

P1– P7

SVZ

Rodent and primate neocortex d

Apical

Basal

LP

TP2

2

2

2

4

4

44

5

5

5

6

6

6

7

8

9

10

1

1

1

1

3

3

3

3


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

show that downregulation of its expressionusing shRNA technology or gene knockouttechnology is required for axon formationusing both staining with axon-specific markersand measurement of neurite length becausethe axon usually grows 5–10 times faster thando neurites becoming dendrites. However,this type of evidence is usually not sufficientto distinguish unambiguously an effector ofneuronal polarity from a molecule simplyregulating axon growth ( Jiang & Rao 2005).Conversely, showing that overexpression oroveractivation of a candidate molecule leads tothe emergence of multiple axons is generallyused to suggest that this molecule is sufficientto confer axon identity to immature neurites.However, this approach is limited by the factthat it relies on an overexpression phenotype,which could be complicated by ectopic activa-tion of a pathway normally not involved in axonspecification or neuronal polarity. Given thesetechnical advances, a more biologically rele-vant validation should include the test of the

requirement of a candidate gene for neuronalpolarity in vivo or ex vivo using gene knockoutor shRNA-mediated knockdown technologies.

SIGNALING MECHANISMSUNDERLYING ESTABLISHMENTOF AXON-DENDRITE POLARITY

PI3-Kinase and Potential Effectors

The lipid kinase phosphatidylinositol 3-kinase(PI3K) lies downstream of Ras during signaltransduction and generates localized sites of themembrane enriched for phosphatidylinositol(3,4,5)-triphosphate (PIP3). Work from severalgroups has implicated PI3K in axon specifica-tion on the basis of the fact that pharmacologicinhibition of PI3K activity using LY294002 orWortmannin prevents axon formation ( Jianget al. 2005, Menager et al. 2004, Shi et al. 2003,Yoshimura et al. 2006). Conversely, overexpres-sion of the constitutively active catalytic sub-unit of PI3K (p110α) leads to the formation of

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 1Cell-type specific patterns of neuronal polarization in vivo. Examples of the sequence of events leading to the polarized emergence ofaxon and dendrites in four distinct vertebrate neuronal cell types in vivo. Throughout these figures, the nascent axon is depicted inpurple and the somatodendritic domain in green. (a) In vivo polarization of retinal ganglion cells in zebrafish (Danio rerio) and mouse(Mus musculus). Neuroepithelial progenitors characterized by an apical and a basal attachment undergo asymmetrical cell division at theapical surface (a1–a3). Upon cell cycle exit, the nucleus undergoes basal translocation (a4) and specifically loses its apical attachmentwhile its basal process starts growing along the basal membrane (a5). The axon ( purple) develops from the basal process and thedendrite from the apical process (a6). (b) Polarization of mouse bipolar cells in the mouse retina. Neuroepithelial progenitors(b1) transform into bipolar cells by first losing their basal attachment, which starts branching in the inner plexiform layer (IPL) whilethe apical process starts branching in the prospective outer plexiform layer (OPL) before (b2) losing its apical attachment (b3). The axonarises from the basal process ( purple) and the dendrite emerges from the apical process (b4). (c) Polarization of granule cells in themammalian cerebellum. Granule cell progenitors divide rapidly in the external plexiform layer (EGL; c1) and, upon cell cycle exit, startto adopt a bipolar morphology (c2) before migrating tangentially with a leading and a trailing process (c3). Another process emergesorthogonally from the cell body (c4) and becomes the leading process, directing its migration toward the inner granule layer (IGL; c5).The trailing processes form a characteristic T-shaped axon ( purple in c6), whereas the leading process gives rise to the dendritic domain( green). (d ) Polarization of radially migrating pyramidal neurons in the mammalian neocortex. Neurons are generated between E11 andE17 by radial glial progenitors in the ventricular zone (VZ) of the mouse neocortex. These cells have a long basal (radial) processattached to the basal membrane at the pial surface and a short apical process on the ventricle side (d1; see detail in Figure 5). Upon cellcycle exit through asymmetric cell division (d2), the postmitotic neuron (blue) goes through a multipolar transition where multipleneurites emerge rapidly from the cell body (d3) before one major process forms in the radial direction (d4) and becomes the leadingprocess (LP). At this point, the neuron initiates radial translocation along a radial glial process (d5) and leaves behind a trailing process,which elongates tangentially in the intermediate zone (IZ) ( purple). The cell body continues to translocate toward its final destination(the top of the cortical plate; CP) while the axon rapidly elongates (d6). The leading process gives rise to the apical dendrite (green ind7), which initiates local branching in the marginal zone (MZ) while over the first postnatal week (until radial migration ends) the cellbody will translocate ventrally (d8–d9) as neurons born at later stages (orange in d10) bypass their predecessors (inside-out accumulationpattern). Adapted from Hinds & Hinds (1978), Zolessi et al. (2006), Morgan et al. (2006), Gao & Hatten (1993), Hatanaka &Murakami (2002), Komuro et al. (2001), Noctor et al. (2004), Rakic (1971, 1972), and Shoukimas & Hinds (1978).


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

Gustavo

Underline

inversamente

ANRV379-NE32-15 ARI 13 May 2009 8:40

a

CP

VZ

SVZ

IZ

MZ

E11 E17

Polarization of cortical neurons in vivo

1

2/3

4

5

6

WM

P1–21

LP

TP

Axon initiation segment

Somato-dendritic domain

Axon

Spine

Polarization of cortical neurons in vitrob

Lamellipodial

and filopodial

protrusion

Stage 1

E14 + 0div

Multiple immature

neurite extension

Stage 2

E14 + 1–2div

Stage 3

E14 + 2–4div

Breaking of

symmetry: axon

specification Axon and dendrite

outgrowth, branching

Stage 4

E14 + 4–15div

Spine morphogenesis

Synapse formation

Stage 5

E14 + 15–25div

Figure 2Parallel between neuronal polarization in vitro and in vivo. Comparison of the sequence of events leading to the polarization of corticalpyramidal neurons in vivo and in vitro. (a) As depicted in Figure 1d, the axon-dendrite polarity of pyramidal neurons is derived fromthe polarized emergence of the trailing (TP) and leading processes (LP), respectively. In vivo, pyramidal neurons acquire other keyfeatures of their terminal polarity such as the axon initiation segment (AIS; yellow cartridge) and dendritic spines ( gray protrusions) duringthe first postnatal weeks of development. (b) In dissociated cultures, postmitotic cortical neurons display specific transitions as classicallydescribed for hippocampal neurons by Dotti et al. (1988). At stage 1, immature postmitotic neurons display intense lamellipodial andfilopodial protrusive activity, which leads to the emergence of multiple immature neurites, stage 2. Stage 3 represents a critical stepwhen neuronal symmetry breaks and a single neurite grows rapidly to become the axon ( purple) while other neurites acquire dendriticidentity. Stage 4 is characterized by rapid axon and dendritic outgrowth. Finally, stage 5 neurons are terminally differentiated pyramidalneurons harboring dendritic spines and the AIS.

PH: pleckstrinhomology

PIP3:phosphatidylinositol(3,4,5)-triphosphate

multiple axons (Yoshimura et al. 2006), sug-gesting that PI3K activation is both requiredand sufficient for axon specification. Using thepleckstrin homology (PH) domain of AKTfused to GFP (PHAKT-GFP) as a biosensor for

PIP3 formation, Menager et al. (2004) haveshown that PIP3 accumulates selectively withina single neurite following local application oflaminin in a single neurite of stage 2 hippocam-pal neurons. Future investigations will need to


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

address when and where PI3K activation occursin vivo during neuronal polarization and whatclass of PI3K is required (class 1, 2 or 3) usinggenetic loss-of-function approaches.

Two PI3K-interacting proteins, Shootin1(Toriyama et al. 2006) and Singar1/2 (Moriet al. 2007), were recently identified as po-tential regulators of axon formation using amass-spectrometry approach. Overexpressionof Shootin1 leads to the generation of super-numerary axons, and RNAi knockdown in-hibits axon formation. Shootin1 is transportedvia a myosin-dependent mechanism to axonalgrowth cones, and its overexpression leadsto aberrant accumulation of PI3K in minorneurites, likely leading to the observed alter-ation of axon specification. Shootin1 colocal-izes with active pools of PI3K, and inhibitionof PI3K activity significantly reduces the abil-ity of Shootin1 to induce multiple axons. Thesedata suggest a role for Shootin1 in regulat-ing PI3K activity, and its selective transportto the nascent axon is likely critical for es-tablishing axonal identity. Singar exists as atleast two splice forms, Singar1 and Singar2,and both are expressed in developing neurons.RNAi against both forms causes cultured neu-rons to form multiple axons, and this effect isprevented when PI3K activity is inhibited. Un-like Shootin1, overexpression of Singar is notsufficient to affect axon formation. When co-expressed, Singar1, but not Singar2, can reducethe multiple axon phenotype of Shootin1 over-expression. This result suggests an antagonis-tic relationship between Shootin1 and Singar 1and that Singar proteins may inhibit PI3K ac-tivity. Both Singar proteins contain a RUN do-main, a motif known to be a site of interactionwith small GTPases including Rap2. Singar in-teracts with Rap2 and not with the closely re-lated Rap1 (Kukimoto-Niino et al. 2006). Workfrom the immune system indicates that Rap2can inhibit PI3K activity (Christian et al. 2003).However, the exact mechanism by which ei-ther Shootin1 or Singar1/2 exert their effectson PI3K awaits further elucidation. An addi-tional source of PIP3 may come in the formof PIP3-containing vesicles transported to the

PTEN: phosphataseand tensin homologdeleted onchromosome 10

forming axon. Guanylate kinase associated ki-nesin (GAKIN) is important for neuronal po-larity because it binds to PIP3-binding protein(Horiguchi et al. 2006). In studies using over-expression of wild-type and dominant-negative(motor domain deleted) forms of GAKIN, theobserved effect was similar to that describedfor other proteins that modulate PIP3 levels(Horiguchi et al. 2006). This result suggeststhat, in addition to locally derived PIP3, a trans-ported pool may be required to maintain axonalidentity.

PTEN

PTEN (phosphatase and tensin homologdeleted on chromosome 10) is a lipid and pro-tein phosphatase that acts in direct oppositionto PI3K activity as PTEN dephosphorylatesPIP3 into PIP2 and thus limits PIP3 signal-ing both spatially and temporally. Increasinglevels of PTEN expression lead to a loss ofaxon formation ( Jiang et al. 2005, Shi et al.2003), whereas reduction of PTEN expressionvia RNAi-mediated knockdown leads to a mul-tiple axon phenotype ( Jiang et al. 2005). Thiseffect is consistent with the gain-of-functionmutation of PI3K described above and high-lights the critical need to maintain the deli-cate balance of phospholipid composition at themembrane to ensure proper neuronal polariza-tion and axon formation.

The identification of a complex betweenPTEN and the PAR3/6 polarity complex (seecorresponding section below) represents an im-portant convergence between two previouslyindependent pathways (Feng et al. 2008). Asis true for other signaling cascades, the closeproximity of positive (PAR3/6) and negative(PTEN) regulators might allow strict spatialand temporal control of the activation and de-activation of a given pathway.

AKT/Protein Kinase B

Several proteins are recruited via their PIP3-specific PH domains to sites of PIP3-enrichedmembranes created by PI3K activity. The


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

GSK3: glycogensynthase

protein kinase AKT also called protein kinaseB (PKB) undergoes such a translocation tothe membrane via its PH domain, a step re-quired for its dual phosphorylation on T308and S473 and activation by membrane-targetedprotein kinase PKD1 and PKD2, respectively.This activated form of AKT is enriched ingrowth cones of polarized neurons (Shi et al.2003). When a myristoylation site is added torecombinant AKT (myr-AKT), it is constitu-tively targeted to the membrane, independentof PI3K, and therefore acts as a constitutivelyactive form. When overexpressed in neurons,this form of AKT is sufficient for multipleaxon formation (Yoshimura et al. 2006), con-sistent with a unified pathway in which AKTacts downstream of PI3K in regulating axonformation. In addition to PKD, another PI3Kregulated kinase, ILK (integrin linked kinase),increases AKT activity via S473 phosphory-lation (Delcommenne et al. 1998). Similar toother regulators of AKT, hyperactived ILK(S343D) increases multiple axon formation incultured neurons, and RNAi reduction or phar-macologic inhibition leads to failure of axonformation without affecting the adoption of adendritic fate (Guo et al. 2007). These experi-ments point to an important but not exclusiverole of ILK in regulating AKT. A common tar-get protein of both ILK and AKT is GSK3β

(glycogen synthase kinase β) (Delcommenneet al. 1998), and phosphorylation by either pro-tein is capable of inactivating GSK3 kinase ac-tivity. In addition, Oinuma et al. (2007) recentlydemonstrated that the small GTPase R-Ras actsupstream to regulate this ILK-GSK pathway.The results of several groups point to GSK3β

as the most important target of these proteinsin the neuronal polarity cascade. In fact, thecotransfection of a nonphosphorylatable formof GSK3β (S9A) with ILK-S343D (constitu-tively active) or a CAAX box containing subunitof PI3K (constitutively active) and ILK-S343A(dominant negative) is sufficient to suppress atleast partially the generation of multiple axons,whereas Myr-AKT is unaffected by ILK-S343A(Guo et al. 2007), indicating the central impor-tance of GSK3β in this polarity pathway.

Glycogen Synthase Kinase 3

GSK3 is a well-studied serine/threonine pro-tein kinase initially identified for its role in reg-ulating glycogen synthesis. Two genes encod-ing GSK3 (α and β) perform essentially re-dundant functions. GSK3 has the fairly uniqueproperty of being constitutively active, a statethat is reversed following phosphorylation atSer9 in GSK3β or Ser21 in GSK3α by multiplekinases including AKT, ILK, and atypical pro-tein kinase C (aPKC) (Etienne-Manneville &Hall 2003). Recent work has implicated GSKβ

as a critical regulator of neuronal polarity. Ex-periments using several types of GSK3 in-hibitors indicate that GSK3α/β act as nega-tive regulators of axon formation because theylead to formation of multiple axons ( Jiang et al.2005, Yoshimura et al. 2005) and can evenconvert dendritic processes into axons, demon-strating that dendrites retain the potential tobecome axons and that this action is normallyprevented by dendritic GSK3 activity, a resultpreviously hinted at by axon-severing experi-ments in culture (Bradke & Dotti 2000, Dotti& Banker 1987). This effect mimics constitu-tively active AKT/ILK-mediated phosphoryla-tion and inactivation of GSK3β and suggestsa model for local activation of these kinases,leading to GSK3 inhibition and axon forma-tion. This model is bolstered by experimentsshowing that GSK3β S9A mutants can suppressmultiple axon phenotypes in cases of constitu-tively active ILK (Guo et al. 2007), expressionof membrane-targeted (myr-)Akt ( Jiang et al.2005), or PTEN knockdown ( Jiang et al. 2005).

A recent paper by Gartner et al. (2006) sug-gests that the situation may be somewhat morecomplex in vivo. Using double knockin-micebearing single point mutations in GSK3β (S9A)and GSK3α (S21A), Gartner et al. reportedno obvious deficits in neuronal morphogene-sis in vivo and in vitro (Gartner et al. 2006). Infact, these mice are viable and do not show anyobvious developmental phenotype in the cen-tral nervous system. However, using inhibitorsof GSK3α/β such as lithium chloride or,more specifically, SB-415286, SB-216763, and


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

AR-A014418, Gartner et al. were able to repli-cate the multiple axon phenotype obtained byothers (Garrido et al. 2007, Jiang et al. 2005).These results indicate that although the exactrole of Ser9/Ser21 phosphorylation in GSK3inactivation remains to be understood or mayinvolve an alternate site (Thornton et al. 2008),it is clear that the catalytic activity of GSK3 isa critical regulator of neuronal polarity.

Several downstream targets of GSK3 arepotential effectors of neuronal polarity, andmany involve regulation of the cytoskele-ton. Collapsin-response mediator protein-2(CRMP-2) is one such microtubule-bindingprotein that is enriched in tips of the nascentaxon and is regulated by GSK3β such that phos-phorylated CRMP-2 displays a decreased bind-ing affinity for tubulin heterodimers (Inagakiet al. 2001, Yoshimura et al. 2005; reviewedin Arimura et al. 2004). As seen for other po-larity regulators, overexpression of CRMP-2 issufficient to induce the formation of multipleaxons, and truncated forms of CRMP-2 canimpair axon formation (Inagaki et al. 2001).Although the ability of CRMP-2 to facilitatemicrotubule assembly is important in regulat-ing axon formation, CRMP-2 is also known toassociate with several other factors, such as theactin polymerization-regulating Sra-1/WAVE1complex, which might contribute to its functionin axogenesis. Recently, Kawano et al. (2005)showed that CRMP-2 links the Sra-1/WAVE1complex with the microtubule-based motorprotein Kinesin 1, and Sra1/WAVE expressionis likely required for CRMP-2’s induction ofmultiple axons.

APC (adenomatous polyposis coli) is an-other well-established effector of GSK3 thatis enriched in the neurite that will becomethe axon early in neuronal polarization (Shiet al. 2004). Phosphorylation of APC byGSK3β blocks its ability to bind the plus endsof microtubules, leading to increased micro-tubule stability, and inhibition of GSK3β leadsto an accumulation of APC in multiple neu-rites (Shi et al. 2004). Expression of truncatedforms of APC is sufficient to inhibit axon for-mation (Shi et al. 2004, Zhou et al. 2004).

MAP: microtubuleassociated protein

Recent work suggests that the APC/GSK dyadregulates targeting of another polarity proteinPAR3 as overexpression of full-length or trun-cated APC disrupts neuronal polarization, in-hibition of GSKβ reduces the pool of APC inthe nascent axon (Shi et al. 2004, Zhou et al.2004), and growth factor–triggered inactiva-tion of GSK3β by PI3K signaling acts throughAPC to control axogenesis (Zhou et al. 2004).Investigators have observed similar results fortwo other GSK targets, the microtubule as-sociated proteins (MAPs) MAP1b (Gonzalez-Billault et al. 2004) and Tau (Sperber et al.1995), that when phosphorylated by GSK, altermicrotubule dynamics. These results empha-size a key principle that underlies much of whatis known about neuronal polarization, namelythat the cytoskeleton is a major endpoint forpolarity regulators. Interestingly, PTEN wasalso recently identified as a GSK3β substrate(Maccario et al. 2007), which may represent anegative feedback loop for AKT signaling fol-lowing activation via stabilization of PTEN.

LKB1 and SAD-A/B and MARKKinases, the Mammalian Orthologsof Par4 and Par1

A pioneering genetic screen performed byKemphues and colleagues in the late eightiesidentified six Par genes encoding distinct pro-tein families. Many studies have since demon-strated that invertebrate and vertebrate Pargenes play critical roles in epithelial cell po-larity during development as well as in thecontext of cell transformation and metastasis(Goldstein & Macara 2007, Kemphues et al.1988). Although this pathway is critical to po-larity in many species, the signaling linking thispathway to extracellular cues has remained elu-sive. The furthest upstream component knownin this cascade is an evolutionarily conserved ki-nase named LKB1 or PAR4. LKB1 translocatesfrom the nucleus and is activated by hetero-dimerization with one of two related pseu-dokinases known as Stradα and -β (Dorfman& Macara 2008). In addition to bindingStrad, LKB1 function in neuronal polarity


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

RAS Akt/PKBPI3K

PKA

Microtubules

MAPs

PAR6

aPKC

PAR3Tiam1/Stef

PAK1

DOCK7

Rac

p90RSK

Raf

MEK

ERK

MKK

JNK—JIP

?

Shootin1

Singar1/2Dvl1

Stathmin/Op18

GAKIN

GSK3β

GSK3β

ILK

Dvl1

Microtubules

Growth factorreceptor

GPCR

Cell–cell or ECM–cell receptors

LIMK

CRMP2 APC

Sra1/WAVE1

Cofilin

PTEN

PIP3Activation

Inhibition

MARK 1– 4

PAK5

F-actin F-actin

LKB1

SAD-A/B

cdc42

Figure 3Signaling pathways involved in mammalian axon specification during neuron polarization. See text for details. GPCR, Gprotein–coupled receptor; ECM: extracellular matrix. Red arrows indicate negative regulation, whereas blue arrows indicate activation.

requires its phosphorylation at S431, a tar-get of both protein kinase A and p90RSK ki-nases (Collins et al. 2000, Sapkota et al. 2001),and this phosphorylation can be triggeredby extracellular cues such as BDNF (brain-derived neurotrophic factor) (Shelly et al. 2007)(Figure 3). This event might be mediated partlyby cues providing chemotactic attraction ofradially migrating neurons toward the corti-cal plate such as Sema3A (Chen et al. 2008,Polleux et al. 1998) or by other extracellu-lar cues including neurotrophins (NTs) such asBDNF/NT4/NT3 (Shelly et al. 2007), Netrin(Adler et al. 2006), FGFs, or any other cues thatcan activate cAMP-dependent protein kinase(PKA) or p90 RSK (RSK1-3) or another un-characterized serine/threonine protein kinaseable to phosphorylate S431 on LKB1 (see later

section on extracellular cues; Figures 4 and5). Future investigations need to identify therelevant extracellular cues and the correspond-ing signaling pathways triggering phosphoryla-tion of LKB1 in position S431, thereby speci-fying the axon in developing cortical pyramidalneurons.

Once LKB1 is activated by binding toits necessary coactivator Strad and S431-phosphorylation (which occurs only in the neu-rite becoming the axon), LKB1 phosphorylatesSAD-A/B kinases (and probably microtubuleaffinity-regulated kinases, MARK1-4), whichare required for axon specification partly byphosphorylating microtubule-associated pro-teins such as Tau. On the basis of the functionof SAD kinases in presynaptic vesicular clus-tering in C. elegans (Crump et al. 2001), we


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

can hypothesize that SAD-A/B kinases mightalso specify axon identity by directing vesiculartrafficking in the neurite becoming the axon.Most important, genetic deletion of LKB1 incortical pyramidal neurons prevents axon for-mation, whereas overexpression of LKB1 andits coactivator Strad in neural progenitors orLKB1 alone in postmitotic cells is sufficient tolead to the formation of multiple axons (Asadaet al. 2007, Barnes et al. 2007, Shelly et al. 2007).

Experiments in Xenopus laevis suggestedthat LKB1 may regulate aPKC inactivation ofGSK3β (Ossipova et al. 2003), two proteins in-volved in neuronal polarity (see below). How-ever, at this point, the exact contribution ofLKB1 in APC/GSK3β function in neuronalpolarity is poorly understood. LKB1 also phos-phorylates and activates a family of 13 pro-tein kinases related to the C. elegans PAR1protein (Lizcano et al. 2004). To date, threeof these have been implicated in regulatingaxon formation: SAD-A and SAD-B kinases aswell as MARK-2 (microtubule affinity regulat-ing kinase-2). RNAi knockdown of SAD ki-nases partially abrogates the ability of LKB1over-activation to induce multiple axon forma-tion in cortical neurons, indicating that LKB1’sfunction in promoting axogenesis largely (butmaybe not completely) derives from activationof SAD-A/B kinases (Barnes et al. 2007). Dou-ble knockout mice for SAD-A and SAD-B resultin neurons that cannot form axons in vivo (Kishiet al. 2005), and overexpression of SAD-A/B in-duces a modest, but significant increase in mul-tiple axon formation (Choi et al. 2008). SADand MARK kinases target several MAPs includ-ing MAP2, MAP4, and Tau by phosphorylat-ing three K-X-G-S motifs within each protein,which reduces their microtubule binding affin-ity, thus destabilizing microtubules (Dreweset al. 1997, Illenberger et al. 1996). Little isknown about SAD kinase regulation; however,a recent study suggested that the protein phos-phatase PP2 might downregulate SAD catalyticactivity by reversing LKB1-mediated phospho-rylation (Bright et al. 2008). Another study hasrecently implicated the tuberous sclerosis com-plex (TSC) genes TSC1/2 in regulating SAD

protein abundance (Choi et al. 2008). The mi-crotubule regulatory scheme is the same for thefour members of MARK kinase family, but atthis point, only MARK2 has been implicatedin neuronal polarity (Biernat et al. 2002, Chenet al. 2006b). Because RNAi-mediated knock-down of MARK2 induces supernumerary axonsand overexpression of MARK2 inhibits axonformation, it is tempting to hypothesize thatMARK2 is a negative regulator of axogene-sis (Chen et al. 2006b). Intriguingly, MARK2can interact with the serine/threonine kinasePAK5, and this interaction is thought to inhibitMARK2 kinase activity while simultaneouslydestabilizing actin cytoskeleton (Matenia et al.2005). Thus the MARK2/PAK5 dyad might co-ordinate actin and microtubule cytoskeletal dy-namics during the establishment and/or main-tenance of neuronal polarity (discussed later inthis review).

Recent work has revealed that other po-tential regulators of neuronal polarity act byregulating MARK2. GSK3β can inactivateMARK2 catalytic activity through phosphory-lation (Timm et al. 2008), and similarly aPKCcan inhibit MARK2 activity through T595phosphorylation (Timm et al. 2008). The pla-nar cell polarity signaling molecules Dishev-elled1 (Dvl1) and Wnt5a have been added intothe MARK2/aPKC pathway of neuronal po-larization (Zhang et al. 2007). In this scenario,Wnt5a activation of its receptor Frizzled (Fzl)leads to stabilization of aPKC through its directinteraction with Dvl1. This increase in aPKCthen leads to an increase in the inhibitory phos-phorylation of MARK2. Consistent with thismodel, increased Dvl1 expression leads to mul-tiple axons, and RNAi knockdown inhibits axonformation (Zhang et al. 2007). Furthermore,the combination of RNAi against MARK2 andDvl1 actually results in normal axon forma-tion. c-Jun N-terminal kinase ( JNK) is anotherpotential target for Dvl1 signaling (Ciani &Salinas 2007) and plays a role in neuronal po-larization. Inhibition of JNK blocks neuronalpolarization in a reversible manner (Oliva et al.2006). The JNK binding partner JIP is also re-quired for axon initiation (Dajas-Bailador et al.


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

GEF: guanine-nucleotide exchangefactor

2008), a function that might require phospho-rylation by the c-Abl tyrosine kinase. It is un-clear whether this effect is due to mislocaliza-tion of JNK, JIP’s role in linking vesicle cargoesto motor proteins (Verhey et al. 2001), its inter-action with p190RhoGEF (Meyer et al. 1999),or a combination of these factors.

Ras- and Rho-family of Small GTPases

Small GTPases are critical regulators of cy-toskeletal and membrane dynamics underly-ing cell motility, cell polarity, and cell growth.Small GTPase proteins are molecular switchesthat constitute a critical component of cellular

PLL-

lam

inin

Ng

CA

MN

gC

AM

t = 8 h t = 13 h t = 13.5 h

Ng

CA

MPL

L-la

min

inPL

L-la

min

inPL

LPL

L-B

DN

FPL

L-B

DN

F

PLL

PLL-

BSA

PLL-

BSA

PLL

PLL-

BD

NF

PLL-

BD

NF

Expressio

n o

f LKB

1S431A

a b

c

f g

d e

Slice overlay assay to determine if extracellular cues can polarize axon emergence in cortical neurons [Sema3A]

Label dissociatedcortical neurons

with DilPlate over

cortical slices

Examine axon outgrowthby video microscopy

or?

Scenario 1

Scenario 2

Pial

Ventricular


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

homeostasis and generally act on downstreameffectors when bound to guanosine triphos-phate (GTP) and are inactive when thisGTP is hydrolyzed to guanosine diphosphate(GDP). Rho-GTPases possess relatively slowintrinsic GTP hydrolysis activity, and theircatalytic activity is regulated by GTPase-activating proteins (GAP; 53 predicted in thehuman genome). GAPs therefore act as nega-tive regulators of GTPase activity by promotingthe GDP bound (inactive) state. Activationof small GTPases by exchanging GDP forGTP is controlled by guanine nucleotide ex-change factors (GEFs; 69 predicted in thehuman genome).

Several members of the Ras-family of smallGTPases have been shown to regulate neuronalpolarity including H-Ras, R-Ras, K-Ras, andN-Ras. Overexpressing either wild-type or aconstitutively active mutant (V12 or Q61L) ofthe H-Ras or the related protein R-Ras (Q87L)leads to the production of multiple axons (Fivazet al. 2008, Oinuma et al. 2007, Yoshimura et al.2006). Ras proteins regulate both the MAPkinase and phosphoinositide-3 kinase (PI3K)pathways, and pharmacologic inhibition of ei-ther pathway was sufficient to inhibit the pro-duction of additional axons, but surprisinglyit did not impact axon formation in general

(Yoshimura et al. 2006). Ras activation is cou-pled to many cell surface receptors includinggrowth factor receptors, and an EGFR tyrosinekinase inhibitor, AG1478, can inhibit axon for-mation (Shi et al. 2003). Recently, elegant workusing a fluorescent reporter of Ras activationdemonstrates the restricted nature of Ras sig-naling and its recruitment during axon determi-nation to contribute to a positive feedback loopwith PI3K (Fivaz et al. 2008). Additional workremains to identify which upstream activatorsmay act through Ras during neuronal symme-try breaking to fate the nascent axon; we discusssome potential candidates later in this review.

The best studied of all mammalian Rho-family small GTPases (22 total) are Cdc42,RhoA, and Rac1. Expression of dominant-negative (locked in GDP-bound state) or con-stitutively active (locked in GTP-bound state)mutants of each of these small GTPases in po-larizing neurons, or treatment with the Rho-GTPase inhibitor toxin B (Bradke & Dotti1999), indicates a critical role for both cdc42and Rac1 both in vitro in rodent neurons(Nishimura et al. 2005, Schwamborn & Puschel2004) and in Drosophila in vivo (Luo et al.1994). Specifically, expression of Cdc42L28, acdc42 mutant which autonomously cycles be-tween a GDP- and GTP-bound state, leads

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 4Experimental evidence for the instructive effects of extracellular cues on neuronal polarization in vitro. (a, b) When hippocampalneurons are plated on stripes coated with alternating substrates such as the cell-adhesion molecule NgCAM (blue) and Poly-l-Lysine(PLL) plus laminin ( yellow), the first neurite contacting the new substrate (arrowhead in a and b) is specified to become the axon( purple). Note that this axon specification event can occur regardless of the type of substrate interface (see a and b), suggesting that inthese conditions, axon specification can occur when unspecified neurites detect a relative change in the substrate composition ratherthan a specific substrate. Adapted from Esch et al. (1999). (c–e) Hippocampal neurons plated on control stripes coated with PLL ( green)or PLL plus bovine serum albumin (BSA, gray) show no trend for axon specification when neurites encounter a stripe boundary (c),whereas an immature neurite encountering a stripe containing brain-derived neurotrophic factor (BDNF) becomes an axon (d ). Thiseffect is abrogated if neurons express a nonphosphorylatable form of LKB1 (LKB1S431A; e). Adapted from Shelly et al. (2007). ( f–g)The slice overlay assay was developed to test if the cortical wall contains extracellular cues that could polarize axon emergence towardthe ventricle. In this assay, immature E18 rat cortical neurons are dissociated and fluorescently labeled with DiI before being platedonto isochronic E18 or heterochronic (P3) cortical slices. Only three hours after plating, most neurons have a short neurite becomingthe axon, allowing investigators to test between two hypotheses: In scenario 1, polarized axon emergence is the sole result of intrinsicpolarization inherited, for example, from neuroepithelial cell progenitors (see Figures 5 and 6). In this case, the plated neurons shouldshow a randomized direction of axon emergence. According to scenario 2, graded extracellular cues can polarize the direction of axonemergence; therefore, neurons plated on the slice should show a directed axon outgrowth toward the ventricle. Polleux et al. (1998)demonstrated that scenario 2 is the most likely one because the overwhelming majority of pyramidal neurons present in the corticalplate show directed axon outgrowth in this assay only a few hours after plating [arrowheads in Figure 4g (Polleux et al. 1998)].


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

Polarized axon initiation in HSN neuron of C. elegans

Dorsal muscle

12 h/early L2

PML axon

Ventral nerve cord

HSN

UNC-6 (netrin)

Ventral muscle

12 h/early L2

10 min/early L1

25 h/mid-L3 30 h/L4

Wild type Unc-6

a

UNC-40::GFP

or MIG-10::GFPUNC-40::GFP

Apical complex(aPKC, α-catenin, F-actin)

Centrosome

Par3

Migration and axon polarization of zebrafish retinal ganglion cells

Basal lamina

Basal lamina

Apical

Basal

GCL

OFL

RPE

b

Axon

Dendrite

2

2

1

1

3

3

4BasalApical dendrite

Leading processUnspecified

neurites

c

Cell cycleexit

Neuronalpolarization

Apical

Trailing process

Axon

PKA or p90RSK?

LKB1S431

phosophorylation

SAD-A/B kinasephosphorylation/activation

Vesiculartrafficking?

Phosphorylation of MAPs

Axon specification

Other effectors?(MARK1-4?)

Stradbinding

+

?Mig

ratio

n

Stage 1 Stage 2 Stage 3

Polarized extracellular cues?(NTs, FGF, Sema3A, WNT, others?)

Po

lari

zed

ext

race

llula

r cu

es?

Par4

Par1

essocess

atio

na

tion

StradαLKB1Phospho-S431-LKB1


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

to the formation of multiple axons in rodentneurons [interestingly, a constitutively activemutant leads to a failure of neurite formation(Schwamborn & Puschel 2004)]. The loss ofcdc42 expression, either through siRNA knock-down (Schwamborn & Puschel 2004) or geneticablation (Garvalov et al. 2007), leads to a strongaxon specification defect. In the case of cdc42conditional knockout mice, the axon phenotypemay be due to increased levels of phosphory-lated (inactive) cofilin, a regulator of actin dy-namics enriched in developing axons (Garvalovet al. 2007). This phosphorylation is achievedby LIM kinase, an activity stimulated by a cdc42effector kinase, Pak1. Paradoxically, Pak1 activ-ity is greatly reduced in cdc42-null mice, sug-gesting that the deregulation of another path-way regulating cofilin occurs in the absenceof cdc42, most likely the RhoA-regulated ki-nase ROCK (Maekawa et al. 1999). The loss ofPak1 itself also inhibits neuronal polarization,and conversely, constitutively active Pak1 in-duces multiple tau1-positive processes ( Jacobset al. 2007). The latter effect can be partiallymitigated by coexpression of either an unphos-phorylatable form of cofilin or a GDP-lockedRac1, suggesting that Rac1 may act down-

stream of Pak1 activation. Taken together, theseresults demonstrate a role for activated cdc42in neuronal polarization beyond its associationwith the PAR3/6 complex described later in thisreview.

RhoA is another small GTPase, and it istypically associated with destabilization of theactin cytoskeleton and myosin-based contrac-tility. Experiments using a constitutively ac-tive form of RhoA show that it inhibits neu-ritogenesis (Bito et al. 2000, Schwamborn &Puschel 2004), whereas a dominant-negativeform of RhoA enhances neurite outgrowth(Schwamborn & Puschel 2004). This findingis consistent with the regulatory role proposedabove for p190RhoGAP as well as the ef-fect of inhibiting the RhoA-activated kinase,ROCK, on axogenesis (Bito et al. 2000). An-other pathway implicated in ROCK inhibitionduring axon formation potentially involves thelocalization of ganglioside-converting enzymePMGS (plasma membrane ganglioside siali-dase) (Da Silva et al. 2005). The overexpres-sion of PMGS leads to the generation of multi-ple axons, and inactivation of ROCK observedfollowing PMGS overexpression might involveenhancement of neurotrophic receptor TrkA

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 5Experimental evidence for instructive effects of extracellular cues on neuronal polarization in vivo. (a) In C. elegans, the immature HSNneurons undergo a series of morphological changes leading to the polarized outgrowth of their axon ventrally at larval stage 4 (L4) (a1).Most noticeably, the polarized lamellipodial outgrowth observed at stage early L2 correlates with a polarized distribution of theattractive Netrin receptor (Unc40) and the intracellular cytoskeletal effector Lamellipodin (MIG-10) (a2) and also requires thepresence of Netrin (UNC-6) secreted from the ventral part of the embryo because the Unc6 mutant shows nondirectional processoutgrowth at early L2 accompanied by nonasymmetrical distribution of UNC-40 (a3). Adapted from Adler et al. (2006) (b) Axonpolarization during the migration of Zebrafish retinal ganglion cells. As described in Figure 1a, retinal ganglion cells inherit theiraxon-dendrite polarity as their cell bodies translocate basally to the ganglion cell layer. In these cells, the basal process of the dividingprogenitor gives rise to the leading process of the migrating RGC, which becomes the axon ( purple). Using live cell imaging, Zolessiet al. demonstrated that the centrosome and the polarity complex protein PAR3 are localized to the apical side of the RGC duringtranslocation. The apical membrane containing atypical protein kinase C (aPKC), α-catenin, and F-actin is also localized apically in thetranslocating RGC in the trailing process. Therefore, in RGC, the PAR3/apical polarity complex is localized in the trailing process,which becomes the dendritic domain; on the basal side, the leading process becomes the axon, which grows rapidly along the basalmembrane. Adapted from Zolessi et al. (2006). (c) A cellular and molecular model of the function of LKB1 and SAD kinases in thepolarization of pyramidal cortical neurons. (c1) Upon asymmetric cell division of radial glial progenitors, early unpolarized postmitoticneurons show a transient phase of nondirected neurite outgrowth in the subventricular zone (c2) before adopting a bipolar morphologyin the intermediate zone (c3) where they engage radial migration with a leading process directed toward the pial surface and a trailingprocess directed toward the ventricle. (c4) On the basis of recent reports (Barnes et al. 2007, Shelly et al. 2007), we propose that in vivo,the trailing process is specified to become the axon in response to putative extracellular cues that preferentially induce phosphorylationof LKB1 on Serine 431. Modified from Barnes et al. (2008).


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

signaling, resulting in signaling that requiresboth PI3K and Rac1. Much work is neededto identify the requirement for PGMS signal-ing for axogenesis in vivo as well as to definemore clearly the molecular mechanisms under-lying the function of RhoA in axon specificationversus axon outgrowth.

The examination of Rac1’s role in neu-ronal polarization has led to some confound-ing results. In Drosophila, expression of eitherdominant-negative (GDP-locked) Rac (Luoet al. 1994) or loss of Rac expression (Hakeda-Suzuki et al. 2002, Ng et al. 2002, Ng &Luo 2004) affects outgrowth but not polar-ity. Similarly, siRNA knockdown of mammalianRac1 typically does not affect axon identity(Gualdoni et al. 2007), although some reportsdetected unpolarized neurons following expres-sion of the dominant-negative form of Rac1(Nishimura et al. 2005). In cultured neurons, aconstitutively active version of Rac1 does not af-fect axon specification (Schwamborn & Puschel2004). These results, while mixed, do hint ata more complex regulation of Rac1 in neu-ronal polarization. This fact becomes clearerlater in this review because the only GEF pro-teins shown to be crucial for axon formationappear to control Rac1. This observation’s ap-parent disjunction with the lack of strong phe-notype may reflect the importance of subcellu-lar localization of activated pools of Rac1 andcompensation by related small GTPases.

Small GTPases have a plethora of effectorswithin cells, and proper activation of theseeffectors, both spatially and temporally, re-quires exquisite control of both activation andinactivation by GEFs and GAPs, respectively.Apart from p190RhoGAP (discussed later inthis review), most studies have so far focused onthe function of GEFs in neuronal polarity. Thisincludes the two GEFs Tiam1 and STEF, de-scribed later, and the GEF DOCK7, recentlyreported to be a regulator of axon specificationby activating Rac1 triggering phosphorylationof Stathmin/Op18, a microtubule-destabilizingfactor critical for axogenesis (Watabe-Uchidaet al. 2006). Another axonally enriched, un-

conventional Rac1 regulatory protein is thecytoplasmic dynein light chain Tctex-1(Chuang et al. 2005). Increased levels of Tctex-1 result in increases in GTP-loaded Rac1 anda drop in GTP-Rac1 levels following Tctex-1siRNA treatment. Multiple axons result fromoverexpression, and this effect is preservedusing a mutant form (T94E) that cannot binddynein heavy chain. Consistent with a rolein controlling Rac1, the supranumerary axonphenotype is suppressed by constitutivelyactive RhoA or dominant negative Rac1.

Rap1b, a member of the Ras superfamily ofGTPases, is also required for proper neuronalpolarity (Schwamborn & Puschel 2004). It isfound at the tip of the nascent axon, and its over-expression leads to hippocampal neurons bear-ing multiple axons. The loss of Rap1b followingsiRNA knockdown abrogates axon formation,and expression of auto-cycling cdc42 can rescuethe phenotype. Expression of a constitutivelyactive Rap1b fails to reverse the loss of axons ob-served following a loss of cdc42, indicating thatRap1b lies upstream of cdc42 in this pathway ofneuron polarization. Similarly, suppressing ax-ogenesis via pharmacological inhibition of PI3-kinase can be reversed by auto-cycling eithercdc42 or constitutively active Rap1b, placingboth of these small GTPases downstream ofPI3K signaling during axon specification. In ad-dition to its place in one of the canonical polar-ity pathways, studies on Rap1b have explored anovel mechanism for protein localization dur-ing neuronal polarity, namely selective pro-tein degradation (Schwamborn et al. 2007b).This means of controlling protein activity ap-pears to apply to several polarity-regulatingproteins and is expanded upon later in thisreview.

PAR3-PAR6-aPKC

The core components of the other ma-jor polarity complex identified in C.elegans are the scaffolding proteins PAR-3and PAR-6. Many binding partners for thiscomplex have been implicated in regulating the


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

polarity of epithelial cells, and neuroepithelialradial glia express the PAR3/6 complexat their analogous apical domain along theventricular wall (Costa et al. 2008, Manabe et al.2002). Proteins reported to exist in complexwith PAR3/6 include atypical forms of proteinkinase C (aPKC: PKC λ and ζ) ( Joberty etal. 2000, Lin et al. 2000, Qiu et al. 2000), thesmall GTPase cdc42 ( Joberty et al. 2000, Linet al. 2000, Qiu et al. 2000), the kinesin motorprotein KIF3A (Nishimura et al. 2004), theguanine exchange factor Tiam1/STEF (Chen& Macara 2005, Nishimura et al. 2005), thelipid and protein phosphatase PTEN (Fenget al. 2008, von Stein et al. 2005), the GT-Pase activating protein (GAP) p190RhoGAP(Zhang & Macara 2008), the tumor suppressorlethal giant larvae (lgl) (Plant et al. 2003), thescaffold protein inscuteable (Schober et al.1999), the ubiquitin ligases Smurf1 (Ozdamaret al. 2005) and Smurf2 (Schwamborn et al.2007a), and the transforming growth factorreceptor 1 (TGFβR1) (Ozdamar et al. 2005).Each of these proteins has also been implicatedin controlling polarity in nonneuronal cells aspart of the PAR3/6 complex.

We consider the potential function of someof these PAR3/6 interacting proteins (Lgl andSmurf1/2) later in this review in the contextof progenitor polarity and protein stability, re-spectively. PAR3/6 are enriched in the nascentaxon in stage 3 hippocampal neurons, and over-expression of wild-type and truncated forms ofeither PAR3 or PAR6 perturb the formationof a single axonal process in hippocampal neu-rons (Shi et al. 2003). In Drosophila, orthologsof PAR3 (bazooka), PAR6, or aPKC do not ap-pear to be required for proper axon-dendritespecification (Rolls & Doe 2004). This couldmean that PAR3/6 have acquired a function inneuronal polarity late during evolution in thevertebrate radiation. Alternatively, there is sofar no genetic loss-of-function evidence in ver-tebrates (especially in mammals) demonstratingthat Par3 and Par6 are required for axon speci-fication. This evidence is clearly more challeng-ing to obtain than in Drosophila because thereare four Par6 genes and two Par3-like genes in

mammalian genomes (Barnes et al. 2008, Gold-stein & Macara 2007).

The role of aPKC in neuronal polarity istightly linked to its ability to associate to thePAR3-PAR6 complex. The activity of aPKCis greatly reduced when associated with PAR6(Yamanaka et al. 2001), and this partnershipprovides a regulatory scheme that requires ad-ditional signaling events to produce a veryspatially limited pool of activated aPKC asGTP-bound cdc42 binding relieves this inhibi-tion. Phosphorylation targets of aPKC includethe PAR3/6-binding partner Lethal giant larvae(Lgl ), and this posttranslational modificationis thought to play a crucial role in regulatingthe subcellular localization of Lgl during polar-ization in several cellular contexts (Betschingeret al. 2003, Plant et al. 2003, Yamanaka et al.2003). As mentioned previously, aPKC can alsophosphorylate the PAR1 ortholog MARK2 onThreonine 595 (Hurov et al. 2004, Suzuki et al.2004), in this case inhibiting its kinase activity.Global inhibition of aPKC activity in polarizingneurons clearly indicates a role for this kinase inthe establishment of neuronal polarity at least invitro (Shi et al. 2003), consistent with observedenrichment of activated aPKC in the nascentaxon (Schwamborn & Puschel 2004, Zhanget al. 2007). PAR3/6 axonal localization likelyoccurs via PAR3 interaction with the micro-tubule plus-end-directed kinesin KIF3A be-cause interference with KIF3A function leads tothe delocalization of PAR3 and aPKC from thenascent axon tip (Nishimura et al. 2004). As dis-cussed previously, inhibition of GSK3 or per-turbation of APC localization also eliminatesPAR3 targeting from the tip of the nascent axon(Shi et al. 2004), indicating a potential hierar-chical scheme that enriches the Par3/6 complexin the developing axon (Figure 6).

The Rac1-GEF Tiam1 also interacts withPAR3/6, which is of particular interest becausethis protein regulates axon formation in vitro.Overexpression of Tiam1 leads to multiple tau1positive processes, and reducing its expressionlevel is sufficient to block neurons in the unpo-larized stage 2 (Kunda et al. 2001, Nishimuraet al. 2005). Current models suggest that PAR3


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

may limit the distribution of Tiam1, thus lim-iting the activation of the small GTPase Rac1(Chen & Macara 2005, Nishimura et al. 2005,Zhang & Macara 2006). This Rac1 regulatoryrole for PAR3/Tiam1 requires the associationof another GTPase known as cdc42 and its as-sociation with PAR6 (Nishimura et al. 2005).The tight regulation of this pathway is likelyrequired for specificity at early stages of po-

larization following the cell’s encounter with asymmetry-breaking cue.

The recent report of an interaction be-tween the RhoA-specific GAP, p190RhoGAP,and aPKC-PAR3/6 pathways (Zhang & Macara2008) opens the possibility that PAR3/6 mayserve as a scaffolding hub for signaling involvingthe most well-studied small GTPases (RhoA,Rac, and cdc42). In this scenario, p190RhoGAP

AJ

TJAJ

n

a

b

Apical membrane

Baso-lateralmembrane

Apicalmembrane

Baso-lateralmembrane?

Neuroepithelial celland radial glia cell polarityEpithelial cell polarity

???

?

?

Neuroepithelialprogenitors

Unpolarized(premigratory)

postmitotic neuron

Polarized (migrating)postmitotic neuron

?Trailing process ➞ axon

Leading process➞ apical dendrite

Cep120

TACC1–3

Centrosome

1

2

3

N– and E–cadherins

α– and β–catenins

Numb

Atypical PKC

Cdc42

Par3 (+Par6?)

Microtubules

Prominin

Nucleusn

n

n

F–actin

Axonspecification?

n

2

1


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

would locally inactivate RhoA while the associ-ated Tiam1/STEF would increase active Rac1levels, thus promoting neurite outgrowth/axonformation. Several observations potentiallystrengthen this possibility. First, p190RhoGAPactivity is reduced by GSK3β phosphorylationso that the requisite GSK3β inactivation in ax-ogenesis would lead to decreased levels of ac-tivated RhoA and increased process extension( Jiang et al. 2008). Second, mice lacking ei-ther of the two p190RhoGAP (A and B) genesdemonstrate significant decrease in axon tracts(Brouns et al. 2000, 2001; Matheson et al. 2006),which is compatible with an axon specificationor an axon growth defect. Last, p190RhoGAPactivity can be stimulated by activating insulin-like growth factor 1 (IGF-1) signaling (Sordellaet al. 2003), which stimulates axon formation incultured neurons (Sosa et al. 2006) and axonoutgrowth in vivo (Ozdinler & Macklis 2006).Returning to the theme of balanced antago-nism, a very recent study suggests that RhoAactivity may inhibit the interaction of PAR3and PAR6 by activating Rho-kinase (Nakayamaet al. 2008), likely disrupting or altering sig-naling from the PAR3/6 complex. In this way,RhoA activity might be part of a negative feed-back loop negatively regulating PAR3/6 sig-naling in neurites becoming dendrites. Clearly,much work needs to be done to test this model,which is based mostly on evidence in nonneu-ronal systems.

GLOBAL CELLULARMECHANISMS OF NEURONALMORPHOGENESIS

Local Protein Degradation

The spatial regulation of protein expressionby selective degradation has been describedin various contexts during neuronal differ-entiation, including axonal pruning duringdevelopment (Watts et al. 2004) and vari-ous aspects of axon guidance (Bloom et al.2007, Campbell & Holt 2001, DiAntonioet al. 2001, Lewcock et al. 2007), synapse for-mation (DiAntonio et al. 2001, Nakata et al.2005), synapse maintenance (Aravamudan &Broadie 2003, DiAntonio et al. 2001, Ehlers2003, Speese et al. 2003), and synapse elimina-tion (Ding et al. 2007) (reviewed in DiAntonio& Hicke 2004). Acute treatment with the pro-teosome inhibitor lactacystin blocks axogene-sis in dorsal root ganglion cells (Klimaschewskiet al. 2006). Furthermore, more prolonged in-hibition of protein degradation with lactacystinleads to formation of multiple axons (Yan et al.2006). The protein kinase AKT that we pre-viously described as critical for neuronal po-larity may undergo selective degradation (Yanet al. 2006). In fact, this degradation selectivelytargets the inactive pool of AKT in neurites, re-sulting in a net enrichment of AKT in a singleprocess that contains that most active AKT, thenascent axon. This phenomenon is consistent

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 6Relationship between epithelial cell polarity, neuroepithelial progenitor polarity, and postmitotic neuron polarity. (a) Parallel betweenepithelial cell polarity and neuroepithelial cell polarity. Epithelial cells have two main cell membrane compartments: the apicalmembrane and the baso-lateral membrane separated by tight junctions (TJ) and adherens junctions (AJ), which act both as cell adhesionsites and as strong diffusion barriers preventing direct exchange between the apical and the baso-lateral domains. Neuroepithelialprogenitors form a pseudostratified epithelium in vivo, which is characterized by a basal attachment through their radial process to thepial surface and an apical domain separated by Cadherin-based adherens junctions. (b) Potential model for the molecular control ofapical polarity in cortical neuroepithelial progenitors and its potential relationship with postmitotic neuron polarity. Several proteincomplexes recently involved in the apical polarity of cortical progenitors include the centrosomal proteins Cep120-TACC1–3,microtubules, Numb-regulated adherens junctions composed of cadherins and catenins, and atypical protein kinase C(aPKC)-cdc42-Par3/6 (see text for details). Question marks point to unresolved issues regarding the functional interactions betweenspecific molecular components of the apical polarity complex. During cell cycle exit, the centrosome might play a role in axonspecification (de Anda et al. 2005) by leaving an apical trace transiently localizing to the base of multipolar neurons, which might play arole in specifying the position of the trailing process (future axon) before translocating to the base of the leading process (future apicaldendrite) when neurons initiate radial translocation (Tsai et al. 2007).


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

with the negative feedback signal model pro-posed by Kaibuchi and colleagues (Arimura &Kaibuchi 2007) to explain axon specificationof a single axon during neuronal polarization.Schwamborn et al. (2007b) recently showedthat the small GTPase Rap1b encounters a reg-ulatory scheme very similar to that of AKTbecause the active form Rap1b is spared fromdegradation and ultimately enriched in theaxon. In this case, the ubiquitin ligase acting onRap1b is specifically Smurf-2, whereas the re-lated Smurf1 appears to affect only neurite out-growth. Additional work has demonstrated thatan interaction between Smurf2 and the polar-ity scaffold PAR3 must exist for proper neuronpolarization (Schwamborn et al. 2007a). Thisfinding appears to be related to PAR3 target-ing of Smurf2 to the axon because perturba-tion of the interaction of either PAR3-Smurf2or PAR3-KIF3A results in Rap1b increase inall neurites. The converse situation exists forLIM kinase (LIMK) because levels of the pro-tein must be reduced for axon initiation (Tursunet al. 2005). The protein Rnf6 catalyzes theubiquitination of LIMK, and blocking Rnf6expression leads to increased levels of axonalLIMK similar to those observed following pro-teasome inhibition. Although this phenomenonrepresents a powerful mechanism to regulateprotein enrichment, complete understandingwill require identification of both upstream reg-ulatory signals responsible for its spatially lim-ited activity and the cohort of proteins targetedfor degradation.

Cytoskeletal Dynamics

Regulation of the actin and microtubule cy-toskeleton has been the focus of intense stud-ies because of their role in neuronal polariza-tion. Experiments using the actin-destabilizingagents lactrunculin B and cytochalasin D indi-cate that remodeling the actin-based cytoskele-ton is an important regulatory step in axonformation (Bradke & Dotti 1999). Specifically,the actin-depolymerization localized to a sin-gle neurite in unpolarized stage 2 hippocampalneurons is sufficient to confer axonal identity.

In this way, the loose actin filaments wouldpermit the egress of microtubules and lead torapid elongation of a given neurite, perhapsoutpacing the transport of negative regulatorsof axonal identity. The idea of cellular asym-metries being reinforced by localized micro-tubule stabilization and invasion proposed byKirschner & Mitchison (1986) was elegantlydemonstrated using a photoactivatable form ofthe tubulin-stabilizing compound taxol, whichcan direct axonal specification to a single im-mature neurite (Witte et al. 2008). This workdirectly confirms a hypothesis about the roleof tubulin in axon-dendrite polarity that hasbeen developed as a result of the observationsby many groups regarding posttranslation mod-ifications of tubulin and microtubule-bindingproteins.

Early experiments showed that severing theputative axon of cultured neuron results inthe assignment of an alternate process as theaxon (Dotti & Banker 1987), which may alsobe related to changes in microtubule dynam-ics. Work from several groups indicated thatthe distance from the cell body dictates ifthe axon will regenerate from the same neu-rite or if a dendrite will adopt the axonal fateupon axotomy (Bradke & Dotti 1997, 2000;Goslin & Banker 1989; Takahashi et al. 2007).More recent work indicates that this regen-erative phenomenon can occur in relativelymature neurons integrated in establishedsynaptic networks (Gomis-Ruth et al. 2008).This work also predicts that the presence ofa stable pool of microtubules in the lesionedneurite may dictate if axon regeneration or ax-onal respecification will occur. The existence ofan intrinsic bipolar polarity axis has also beendescribed in hippocampal neurons with axo-tomized cells regrowing their axons from an an-tipodal neurite (Calderon de Anda et al. 2008).This work also demonstrates that following ax-otomy, the microtubule-organizing center, thecentrosome, does not reorient toward the newlyassigned axon, in contrast to a previous sugges-tion of a role for the centrosome in directingthe destiny of the axonal neurite (de Anda et al.2005). This discrepancy may reflect the fact


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

that at later stages of neuronal differentiation,the centrosome is no longer required for axonrespecification. This and other observations(Calderon de Anda et al. 2008) suggest that dur-ing respecification of the axonal process, unlikethe centrosome, membrane flow does respondby reorienting. The requirement of centrosomeand Golgi positioning in relation to axon initi-ation still awaits in vivo confirmation (see alsoFigures 4 and 5).

Cytoplasmic Flow and DirectedMembrane Trafficking

Subtle changes in membrane trafficking, as in-dicated by neurite growth cone size and phase-contrast tracking of organelles using time-lapsemicroscopy, are often predictive of axon specifi-cation (Bradke & Dotti 1997). These results in-dicated that mitochondria, membrane vesicles,and other cytoplasmic components are prefer-entially directed toward the nascent axon andare consistent with earlier analysis of polarizingneurons using electron microscopy (Deitch &Banker 1993). The components of cytoplasmicflow are likely to include some of the cytoplas-mic molecules described earlier as regulators ofaxogenesis and, as indicated, the anterogradetrafficking of vesicles. Proteins with an estab-lished role in synapse function are targeted tothe nascent axon (Fletcher et al. 1991); how-ever, very few of the regulatory molecules con-trolling this activity have been identified. Earlywork showed that using antisense oligonu-cleotide knockdown of Rab8, a critical regula-tor of vesicular trafficking, can block neuronalpolarization (Huber et al. 1995). Similarly,a dominant-negative form of the tetanusneurotoxin-insensitive vesicle-associated mem-brane protein (TI-VAMP) blocks neuronal po-larization (Martinez-Arca et al. 2001). A corol-lary to selective axonal transport comes fromwork exploring the asymmetric genesis of theapical dendrite. In this work, the polarized po-sition of the Golgi network leads to preferen-tial membrane deposition in a single neuritethat becomes the apical dendrite (Horton et al.

2005), a step likely to follow shortly after axonspecification.

A recent Drosophila screen for dendritic ar-bor reduction (dar) mutants made the intriguingdiscovery that perturbing some secretory path-way specifically affects dendrite and not ax-onal development, a result also observed inmammalian neurons along with the existenceof Golgi outposts (Ye et al. 2007). Polar-ity mutants have been identified that disruptthe polarized sorting of cargoes in the nema-tode C. elegans, as well. These genes includethe kinases LRK-1 (Park-8) (Levy-Strumpf &Culotti 2007) and SAD1 (Crump et al. 2001.Hung et al. 2007) (as described previously, alsorequired for polarity in mammalian neurons),the SAD kinase binding partner Nab-1 (Neura-bin) (Hung et al. 2007), and the UNC-76(FEZ1)/UNC-69 complex (Hung et al. 2007).The RNAi-mediated knockdown of the mam-malian ortholog, FEZ1, inhibits axon forma-tion in part owing to regulating mitochondrialmotility (Ikuta et al. 2007) and likely by regu-lating the engagement of kinesin-1 in coopera-tion with the JNK-scaffold protein JIP1 (Blasiuset al. 2007). Taken together, these insights fromC. elegans buttress the high degree of conserva-tion of some of the pathways regulating neu-ronal polarity beyond the core group of PARproteins.

Molecular Motors

Investigators have long postulated that molec-ular motor proteins play a role in neuronal po-larity. Intriguing evidence indicates that a fewof the microtubule plus-end-directed kinesinproteins are the best candidates. As mentionedabove, the kinesin-like protein GAKIN likelyhas a crucial role in transporting PIP3, andlikely other cargoes, to the developing axon(Horiguchi et al. 2006). Similarly, the KIF3Amotor is important for PAR3 protein local-ization (Nishimura et al. 2004. Schwambornet al. 2007a). Work with kinesin-1/KIF5C-EGFP fusion protein presents a particularlyfascinating picture of motor dynamics dur-ing axon specification ( Jacobson et al. 2006).


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

Time-lapse video microscopy of isolated hip-pocampal neurons in dissociated culture at stage2 shows that KIF5C-EGFP transiently exploressingle immature neurites but makes a prolongedinvasion of the neurite becoming the axon intransition to stage 3 ( Jacobson et al. 2006).More work needs to be done to (a) testif this mechanism occurs during neuronalpolarization in vivo and (b) test specifi-cally when after cell cycle exit this prefer-ential kinesin-based motor transport occursduring axon specification when neurons engagemigration. Finally, it will be of great interest touse this visualizing tool to test how extracellu-lar cues and their intracellular effectors directmicrotubule-based transport activity duringaxon specification. Given the large number ofmotor proteins (Hirokawa & Takemura 2004.2005), these data likely represent only the be-ginning of a complex story regarding the selec-tive transport of proteins, membrane vesicles,and organelles during neuronal polarization.

Diffusional Barrier

A straightforward scenario for the subcellulartargeting of neuronal proteins envisions polar-ized microtubule-based transport coupled withdocking of specific cargoes at their destination.This situation is likely true for many cytoplas-mic and some membrane-bound proteins, butexperiments utilizing fluorescent lipids indicatethat a barrier to communication between ax-onal and somatic membranes exists (Kobayashiet al. 1992) and would be required to main-tain asymmetries established during early neu-ronal polarization events. A continual refine-ment of our understanding of the nature ofthis barrier has led to an appreciation that thebarrier is dependent on the actin cytoskeletonand represents a significant physical barrier tolateral diffusion (Winckler & Mellman 1999).Later experiments have shown that this barrierforms over time in cultured neurons (Nakada etal. 2003), suggesting that vectorial traffickingand anchoring/stablization contribute to theearly phase of polarization and that the barrier

contributes to later maintenance of polarity.The consensus that a molecular fence existswithin the axon initial segment (AIS) is fairlysolid, but the molecular mechanisms under-lying the assembly of the barrier during de-velopment remain to be elucidated. It is notclear if the morphological structure that is en-riched in components such as sodium channelsand ankyrin-G is identical to the diffusion bar-rier, but both are contemporaneous develop-mentally (Nakada et al. 2003). One recentlyidentified component of the axon initial seg-ment is the phorphorylated form of IκBα, aninhibitor of NFκB (Sanchez-Ponce et al. 2008).In this study, inhibition of the IκB kinasesinhibited axon formation through a currentlyunknown mechanism. Two papers disruptingknown components of the AIS have shown crit-ical roles for the scaffold protein, ankyrin G, aswell as NF-186 and its linkage to the brevican-containing extracellular matrix (Hedstromet al. 2007, Yang et al. 2007). Very recently,Hedstrom et al. have shown a loss of es-tablished neuronal polarity via knockdown ofankyrin-G which included the development ofPSD-95 positive spines in the former axon(Hedstrom et al. 2008). In the coming years,similar work in this area will further expandour understanding of the function of the AIS inspecification and/or maintenance of neuronalpolarity.

ROLE OF EXTRACELLULARCUES IN ORCHESTRATINGINTRACELLULAR SIGNALINGDURING NEURONALPOLARIZATION

In Vitro

Several lines of evidence suggest that extracel-lular cues can direct the polarized emergenceof the axon and the dendrites both in vitro andin vivo. First, dissociated cortical or hippocam-pal pyramidal neurons plated on striped sub-strates coated with two different cell adhesionmolecules (Laminin and NgCAM, for example)


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

can play an instructive role for axon specifica-tion (Figure 4a–b). The first immature neu-rite of E18 hippocampal neurons that contactsthe boundary between two stripes systemati-cally becomes the axon. This situation occursregardless of the fact that the initial outgrowthof immature neurites occurred on laminin orNgCAM, suggesting that immature neuritescan detect changes in the nature of the extracel-lular substrate rather than the absolute natureof the novel substrate they are encountering(Esch et al. 1999). Using a similar approach,Shelly and colleagues showed that neurites ofimmature hippocampal neurons growing on apatterned substrate can detect the presence ofBDNF, which plays an instructive role in axonspecification because the first neurite contact-ing a BDNF stripe systematically becomes theaxon (Shelly et al. 2007) (Figure 4c–d ). Theeffect of BDNF on axon specification requirescAMP-dependent protein kinase (PKA) activa-tion and phosphorylation of LKB1 in position431 by PKA (Shelly et al. 2007) (Figure 4e),suggesting that LKB1 phosphorylation on S431acts as a detector of neuronal symmetry break-ing by extracellular cues such as BDNF in thisin vitro context.

The overlay assay is an in vitro assay de-veloped to detect the existence of putative ex-tracellular cues playing a role in cortical axonguidance and neuron polarization. As depictedin Figure 4e, this rather simple assay revolvesaround plating fluorescently labeled dissociatedcortical neurons onto cortical slices to test ifpolarized axon emergence in vivo is mainly theresult of asymmetric activation of intracellulareffectors (maybe inherited by progenitors) or ifextracellular cues can play a role in axon speci-fication (scenario 2 in Figure 4e). Polleux et al.(1998) have demonstrated that scenario 2 is themost likely because only a couple of hours af-ter plating, the vast majority of cortical neuronsdisplayed a single, short axon directed ventrallytoward the ventricle as found in vivo. These au-thors went on to demonstrate that the class 3 se-creted semaphorin, Sema3A, which is enrichedin the most superficial part of the cortical wall

(the top of the cortical plate), plays a role inrepulsing axon initiation ventrally toward theventricle (Polleux et al. 1998). This work sug-gests that the polarized emergence of a singleaxon is controlled at least in part by extracel-lular cues expressed in a graded manner alongtheir migratory path.

In Vivo

Is there any in vivo evidence for the role ofextracellular cues in the specification of neu-ronal polarity? In C. elegans, elegant workhas demonstrated that the polarized emer-gence of a single axon in HSN neurons re-quires Netrin (UNC6)-induced localization ofNetrin-attractive receptor UNC-40 (DCC) aswell as a cytoskeletal effector called MIG-10(lamellipodin) on the ventral part of the neuronat the early L2 stage (Figure 5a) (Adler et al.2006).

Careful live imaging experiments of Xeno-pus retinal ganglion cell polarization revealedthat polarized axon outgrowth requires someunidentified extracellular cues present in thebasal lamina (Zolessi et al. 2006) (Figure 5b).The axon of developing RGCs normally growson the basal side of the neuron. In a mutantcalled Nok, characterized by the absence of reti-nal pigmented epithelium (RPE), some postmi-totic RGC neurons show a defective polarizedoutgrowth of their axon on the apical side alongthe now-exposed basal lamina. In this context,the polarized emergence of the axon on thebasal side of the RGC is correlated with the po-sition of the centrosome, Par3, and the apicalcomplex (containing at least aPKC, α-catenin,and F-Actin) on the apical side of the cell wherethe dendrite will emerge (Figure 5b). Taken to-gether, this work strongly suggests that (a) thebasal lamina contains some important extracel-lular cues playing a role in the polarized emer-gence of the axon of RGC neurons and that(b) RGC neurons inherit the intrinsic apico-basal polarity of their progenitor at least withregard to the Par3/aPKC components of thepolarity complex. Important questions remain


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

to be addressed: Which extracellular compo-nent of the basal lamina controls the polarizedemergence of the axon? Which signaling path-way is involved in this axon specification? Howis this pathway linking the extracellular cuespolarizing axon outgrowth to the apical polar-ity pathway? Future investigations will addresssome of these questions using this unique invivo paradigm.

To date, the only signaling cascade linkedto extracelluar cues is the recently identifiedLKB1-SAD kinase pathway (Barnes et al. 2007).Recent studies using either RNAi knockdownor genetic loss of function have explored thispathway in the developing cerebral cortex.Conditional LKB1 deletion in cortical progen-itors leads to a severe loss of axon initiation incortical neurons but does not impact migration.Structure/function analysis indicates that phos-phorylation of LKB1 at Serine 431 is requiredfor its function in axon specification (Barneset al. 2007), and Shelly et al. (2007) link thisphosphorylation to the ability of BNDF appli-cation to stimulate PKA-dependent phospho-rylation of S431 in the nascent axon.

POTENTIAL RELATIONSHIPBETWEEN NEUROEPITHELIALCELL POLARITY ANDPOSTMITOTIC NEURONPOLARITY

One of the important questions emerging fromthe work reviewed in Figures 1 and 5 revolvesaround determining if there is a systematic re-lationship between neural progenitor polarityand postmitotic neuron polarity in mammals.Clearly, in several cell types, such as retinal gan-glion cells and bipolar cells, postmitotic neu-rons directly inherit the intrinsic apico-basalpolarity of progenitors, which is transformedinto axon-dendrite polarity upon cell cycle exit(Hinds & Hinds 1978, Morgan et al. 2006,Zolessi et al. 2006). However, this relationshipbetween progenitor polarity and postmitoticneuron polarity is not so clear at first glancefor other neuronal subpopulations such as

cerebellar granule cells and pyramidal corticaland hippocampal neurons.

Dividing neuroepithelial progenitors inthe neocortex and elsewhere in the nervoussystem presents a strong apico-basal polaritywhere the apical and baso-lateral membranesare separated by cadherin-based adherensjunctions (AJ) but not by tight junction as ob-served in epithelial cells (Figure 6a) (reviewedby Gotz & Huttner 2005). Recent resultshave allowed investigators to determine themolecular composition of the apical membraneof cortical neuroepithelial progenitors, whichrevealed that the apical membrane containsthe core components of the apical polaritycomplex (aPKC/Par3/Par6) as well as cdc42.Interference with Par6 or cdc42 expression incortical progenitors alters the balance betweenasymmetric versus symmetric cell divisions(Cappello et al. 2006, Chen et al. 2006a, Costaet al. 2008). The apical domain of corticalprogenitors is also closely associated with thecadherin-based AJ and the centrosome, mi-crotubule organizing center (MTOC) (Chennet al. 1998, Rasin et al. 2007, Xie et al. 2007). Itis not clear at this point whether any of the pro-teins or organelles present in the apical polaritycomplex of progenitors are inherited by postmi-totic neurons upon cell-cycle exit (Figure 6b).Recent analysis suggested that the centrosomeposition upon the terminal mitosis predictedthe axon initiation site in postmitotic neurons atleast in vitro (de Anda et al. 2005). Although thiswork awaits in vivo confirmation, it is temptingto hypothesize that there is a functionalrelationship between (a) the apical position ofthe Par3/Par6/aPKC/cdc42 polarity complex,(b) the centrosome position, and (c) the axoninitiation site during the multipolar to bipolartransition (Figure 6b). Based on the fact thatcdc42 is required for axon specification invivo and that interference with Par3/Par6and aPKC signaling also has an impact onneuronal polarization at least in vitro, thishypothesis of a functional relationship betweenthe apico-basal polarity of neural progenitorsand the axon-dendrite polarity of postmitotic


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

neurons represents an exciting avenue forfuture investigations.

SPECIFICATION OF DENDRITICIDENTITY

Axon specification is for many neurons, bothin vitro and in vivo, the earliest symmetry-breaking event. Our review has focused onthis event because most recent studies haveidentified molecular regulators of axon spec-ification, but very few studies have identifiedgenes involved in specification of dendriticidentity. On the other hand, the molecu-lar mechanisms involved in dendritic growth,guidance, and branching are intensely studied,but we refer the reader to recent, exhaustive re-views on this topic (Grueber et al. 2005, Scott& Luo 2001, Whitford et al. 2002a).

As described earlier, motor proteins canaffect neurite identity, and CHO1/MKLP1 (re-named kinesin member 23 or Kif23) is requiredfor dendrite initiation (Sharp et al. 1997) andmaintenance (Yu et al. 2000). As mentionedearlier, the polarized orientation of the Golgiappears to be another event during dendriteoutgrowth (Horton et al. 2005). Work in cellsfrom the superior cervical ganglion, which donot form dendrites when cultured at low den-sity, demonstrates an astounding dendritogen-esis in response to application of soluble ligandbone morphogenetic protein 7 (BMP7/OP-1)(Lein et al. 1995). This effect requires Smad1signaling, and inhibition of proteasome func-tion can disrupt this activity (Guo et al. 2001).In addition to Smad1 signaling in dendriteformation, the activated BMP receptor 2 alsobinds and facilitates the activation of the ki-nase LIMK1 (Lee-Hoeflich et al. 2004), whichwould lead to increased cofilin phosphoryla-tion, a situation previously shown to inhibitaxon formation (Garvalov et al. 2007), and maybias neurites to become dendrites. The smallGTPase Rit has proved to be antagonistic toBMP-stimulated dendrite formation by stim-ulating ERK1/2 activation (Lein et al. 2007).The related ligand TGF-β has a similar effect

on dendrite initiation in Xenopus retinal gan-glion cells (Hocking et al. 2008).

These results present a picture of dendriteformation utilizing the same pathway or onerelated to those seen previously for axogen-esis. This finding is consistent with observa-tions made for a secreted cue, Semaphorin3A, that can regulate the site of axonal out-growth through its repulsive function (Polleuxet al. 1998) and, simultaneously, the orientationand outgrowth of the leading process/apicaldendrite through its attractive function (Chenet al. 2008, Polleux et al. 2000). The out-growth of Drosophila neurons also providesstrong evidence for extracellular cues in reg-ulating dendritogenesis. Two cues implicatedin dendrite formation and orientation are theSlit-Robo (Furrer et al. 2007, Godenschwegeet al. 2002, Whitford et al. 2002b) and netrin-frazzled (DCC ortholog) (Furrer et al. 2003;reviewed in Kim & Chiba 2004). Similar mech-anisms are described below with regard to axonformation in the nematode.

INTERPLAY BETWEENEXTRACELLULAR-INTRACELLULAR REGULATORSOF NEURONAL POLARITY:INSIGHTS FROMCAENORHABDITIS ELEGANS

Although many results described above havebeen established using Drosophila and mam-malian neurons, important progress in ourunderstanding of the molecular and cellularmechanisms specifying neuronal polarity hasalso been made using the C. elegans model. Inparticular, this work has resulted in significantprogress in how extracellular cues instructaxon specification in vivo. The neurons ofthe nematode have a stereotyped morphology,e.g., specific projections along the anterior-posterior body axis. Two studies have identifiedthe diffusible signal Wnt and its receptor ascritical regulators of neuronal polarity (Hilliard& Bargmann 2006, Prasad & Clark 2006).In addition to identifying loss of function for


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

Lin-44 (Wnt) and its receptor Lin-17(Frizzled), one of the screens identified VPS-35, a component of retromer complex thatregulates vesicular traffic and is required forproper Wnt secretion (Prasad & Clark 2006).Further experiments have identified anotherextracellular cue, UNC-6 (netrin), along withits receptor UNC-40 (DCC), as a criticalgene orchestrating axon specification in vivo(Adler et al. 2006) (Figure 5). This work alsoidentified downstream proteins in this pathway(mammalian orthologs are shown in paren-thesis when established), including AGE-1(PI3K), DAF-18 (PTEN), UNC-34 (Enabled),CED-10 (Rac), UNC-115/AbLIM, and MIG-10/Lamellipodin (mammalian orthologs areshown in parenthesis when known). The cur-rent model for the relationship of these genesto the signaling of UNC-6 involves DAF-18’slimitation of AGE-1 activity following UNC-40 stimulation and the asymmetric recruitmentof MIG-10 to the plasma membrane (seeFigure 5). Very recent work has shown thatthis recruitment requires activated CED-10directly binding to MIG-10 and the involve-ment of the PAK-like kinase, Pak-1 (Adleret al. 2006). The involvement of a kinase incytoskeletal rearrangement is consistent withthe similar role of MIG-10 and the likelymechanism under which it operates oncerecruited to the plasma membrane to stimulatedirected neurite outgrowth. Another regulatorthought to act in concert to drive filopodial for-mation with MIG-10 is the Enabled homologmentioned above, UNC-34 (Chang et al.2006). SLT-1 (Slit) is another extracellular cuethat likely acts through MIG-10 recruitment(Chang et al. 2006) to control neuronal polar-ity. Recent work links GSK3β and aPKC toSlit-mediated repolarization in migratingmammalian neurons (Higginbotham et al.2006).

Subsequent genetic screens have identifiedmore genes that underlie this polarity pheno-type in C. elegans. These include a component ofthe AP-2 adaptor complex (dpy-23); in the samestudy, both dpy-23 and the VPS-35 mutants actby perturbing MIG-14 (Wntless) and its role in

UNC-6 secretion (Pan et al. 2008). Four othergenes have been involved in this signaling: theSLT-1 receptor Sax-3 (robo), the kinesin-likeprotein VAB-8, the RacGEF UNC-73 (trio),and the small GTPase MIG-2 (Levy-Strumpf& Culotti 2007, Watari-Goshima et al. 2007).Analysis of these genes reinforces the roles ofUNC-6 and SLT-1, and somewhat surprisinglygiven their expected role as receptor effectors,VAB-8, UNC-73, and MIG-2 act upstream ofSax-3 and UNC-40 to control the localiza-tion and trafficking of these receptors. Thisobservation suggests a positive reinforcementmechanism to enrich receptors in the locale ofpreviously activated ones. A link between extra-cellular cues and the transition from initial neu-ron polarization to the maintainance of the po-larized state has been observed using mutants ofthe Netrin and Wnt signaling pathways. Poonet al. observed that loss of either pathway in thenematode results in mislocalization of presy-naptic components into the dendrite (Poonet al. 2008). Genetic evidence from Vab-7(even-skipped) mutants also indicates thatstereotyped responses to cues can result aspart of a larger transcription factor–controlledprogram of cell fate and polarity orientation(Esmaeili et al. 2002). It will be important totest if the mammalian orthologs of any of thesegenes play a role in the specification of neuronalpolarity in higher vertebrates.

CONCLUSION

The polarization of axon and dendrites under-lies the ability of neurons to integrate and trans-mit information in the brain. Neuronal polar-ization has been addressed using a broad rangeof techniques and model systems. The adventof genetic labeling and time-lapse imaging nowallows the observation of the earliest aspects ofneuronal polarization in a contextual cellularenvironment. The picture that emerges fromthese more recent approaches shows that axon-dendrite polarization is specified when neuronsengage migration in vivo, and genetic per-turbation studies have demonstrated the im-portance of conserved polarity pathways [e.g.


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

LKB1 and SAD kinases (Par4/Par1 dyad)]. Itis now clear that extracellular cues play an in-structive role during neuronal polarization bothin vitro and in vivo. Here, we have reviewedsome of these recent results and highlightedfuture challenges in the field. Outstanding the-matic questions include understanding the re-lationship of neuroepithelial cell polarization

and postmitotic neuronal polarity, identifyingthe extracellular cues controlling neuronal po-larization in vivo and how they regulate thesignaling networks involved in axon and den-drite specification during polarization in vivo,and dissecting the interplay between the cy-toskeletal dynamics underlying migration andpolarization.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.

ACKNOWLEDGEMENTS

This work was supported by K01MH080259-01 (A.P.B.), R01AG031524 (F.P.), and a Pew ScholarAward in Biomedical Sciences (F.P.).

LITERATURE CITED

Adler CE, Fetter RD, Bargmann CI. 2006. UNC-6/Netrin induces neuronal asymmetry and defines the siteof axon formation. Nat. Neurosci. 9:511–18

Aravamudan B, Broadie K. 2003. Synaptic Drosophila UNC-13 is regulated by antagonistic G-protein path-ways via a proteasome-dependent degradation mechanism. J. Neurobiol. 54:417–38

Arimura N, Kaibuchi K. 2007. Neuronal polarity: from extracellular signals to intracellular mechanisms.Nat. Rev. Neurosci. 8:194–205

Arimura N, Menager C, Fukata Y, Kaibuchi K. 2004. Role of CRMP-2 in neuronal polarity. J. Neurobiol.58:34–47

Asada N, Sanada K, Fukada Y. 2007. LKB1 regulates neuronal migration and neuronal differentiation in thedeveloping neocortex through centrosomal positioning. J. Neurosci. 27:11769–75

Barnes AP, Lilley BN, Pan YA, Plummer LJ, Powell AW, et al. 2007. LKB1 and SAD kinases define a pathwayrequired for the polarization of cortical neurons. Cell 129:549–63

Barnes AP, Solecki D, Polleux F. 2008. New insights into the molecular mechanisms specifying neuronalpolarity in vivo. Curr. Opin. Neurobiol. 18:44–52

Betschinger J, Mechtler K, Knoblich JA. 2003. The Par complex directs asymmetric cell division by phospho-rylating the cytoskeletal protein Lgl. Nature 422:326–30

Biernat J, Wu YZ, Timm T, Zheng-Fischhofer Q, Mandelkow E, et al. 2002. Protein kinase MARK/PAR-1is required for neurite outgrowth and establishment of neuronal polarity. Mol. Biol. Cell 13:4013–28

Bito H, Furuyashiki T, Ishihara H, Shibasaki Y, Ohashi K, et al. 2000. A critical role for a Rho-associatedkinase, p160ROCK, in determining axon outgrowth in mammalian CNS neurons. Neuron 26:431–41

Blasius TL, Cai D, Jih GT, Toret CP, Verhey KJ. 2007. Two binding partners cooperate to activate themolecular motor Kinesin-1. J. Cell Biol. 176:11–17

Bloom AJ, Miller BR, Sanes JR, DiAntonio A. 2007. The requirement for Phr1 in CNS axon tract formationreveals the corticostriatal boundary as a choice point for cortical axons. Genes Dev. 21:2593–606

Bradke F, Dotti CG. 1997. Neuronal polarity: vectorial cytoplasmic flow precedes axon formation. Neuron19:1175–86

Bradke F, Dotti CG. 1999. The role of local actin instability in axon formation. Science 283:1931–34Bradke F, Dotti CG. 2000. Differentiated neurons retain the capacity to generate axons from dendrites.

Curr. Biol. 10:1467–70


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

Bright NJ, Carling D, Thornton C. 2008. Investigating the regulation of brain-specific kinases 1 and 2 byphosphorylation. J. Biol. Chem. 283:14946–54

Brouns MR, Matheson SF, Hu KQ, Delalle I, Caviness VS, et al. 2000. The adhesion signaling molecule p190RhoGAP is required for morphogenetic processes in neural development. Development 127:4891–903

Brouns MR, Matheson SF, Settleman J. 2001. p190 RhoGAP is the principal Src substrate in brain andregulates axon outgrowth, guidance and fasciculation. Nat. Cell Biol. 3:361–67

Calderon de Anda F, Gartner A, Tsai LH, Dotti CG. 2008. Pyramidal neuron polarity axis is defined at thebipolar stage. J. Cell Sci. 121:178–85

Campbell DS, Holt CE. 2001. Chemotropic responses of retinal growth cones mediated by rapid local proteinsynthesis and degradation. Neuron 32:1013–26

Cappello S, Attardo A, Wu X, Iwasato T, Itohara S, et al. 2006. The Rho-GTPase cdc42 regulates neuralprogenitor fate at the apical surface. Nat. Neurosci. 9:1099–107

Chang C, Adler CE, Krause M, Clark SG, Gertler FB, et al. 2006. MIG-10/lamellipodin and AGE-1/PI3Kpromote axon guidance and outgrowth in response to slit and netrin. Curr. Biol. 16:854–62

Chen G, Sima J, Jin M, Wang KY, Xue XJ, et al. 2008. Semaphorin-3A guides radial migration of corticalneurons during development. Nat. Neurosci. 11:36–44

Chen L, Liao G, Yang L, Campbell K, Nakafuku M, et al. 2006a. Cdc42 deficiency causes Sonic hedgehog-independent holoprosencephaly. Proc. Natl. Acad. Sci. USA 103:16520–25

Chen X, Macara IG. 2005. Par-3 controls tight junction assembly through the Rac exchange factor Tiam1.Nat. Cell Biol. 7:262–69

Chen YM, Wang QJ, Hu HS, Yu PC, Zhu J, et al. 2006b. Microtubule affinity-regulating kinase 2 functionsdownstream of the PAR-3/PAR-6/atypical PKC complex in regulating hippocampal neuronal polarity.Proc. Natl. Acad. Sci. USA 103:8534–39

Chenn A, Zhang YA, Chang BT, McConnell SK. 1998. Intrinsic polarity of mammalian neuroepithelial cells.Mol. Cell Neurosci. 11:183–93

Christian SL, Lee RL, McLeod SJ, Burgess AE, Li AH, et al. 2003. Activation of the Rap GTPases in Blymphocytes modulates B cell antigen receptor-induced activation of Akt but has no effect on MAPKactivation. J. Biol. Chem. 278:41756–67

Choi YJ, Di Nardo A, Kramvis I, Meikle L, Kwiatkowski DJ, et al. Tuberous sclerosis complex proteins controlaxon formation. Genes Dev. 22:2447–53

Chuang JZ, Yeh TY, Bollati F, Conde C, Canavosio F, et al. 2005. The dynein light chain Tctex-1 has adynein-independent role in actin remodeling during neurite outgrowth. Dev. Cell 9:75–86

Ciani L, Salinas PC. 2007. c-Jun N-terminal kinase ( JNK) cooperates with Gsk3beta to regulate Dishevelled-mediated microtubule stability. BMC Cell Biol. 8:27

Collins SP, Reoma JL, Gamm DM, Uhler MD. 2000. LKB1, a novel serine/threonine protein kinase andpotential tumour suppressor, is phosphorylated by cAMP-dependent protein kinase (PKA) and prenylatedin vivo. Biochem. J. 345(Pt. 3):673–80

Costa MR, Wen G, Lepier A, Schroeder T, Gotz M. 2008. Par-complex proteins promote proliferativeprogenitor divisions in the developing mouse cerebral cortex. Development 135:11–22

Craig AM, Banker G. 1994. Neuronal polarity. Annu. Rev. Neurosci. 17:267–310Crump JG, Zhen M, Jin Y, Bargmann CI. 2001. The SAD-1 kinase regulates presynaptic vesicle clustering

and axon termination. Neuron 29:115–29Dajas-Bailador F, Jones EV, Whitmarsh AJ. 2008. The JIP1 scaffold protein regulates axonal development in

cortical neurons. Curr. Biol. 18:221–26Da Silva JS, Hasegawa T, Miyagi T, Dotti CG, Abad-Rodriguez J. 2005. Asymmetric membrane ganglioside

sialidase activity specifies axonal fate. Nat. Neurosci. 8:606–15de Anda FC, Pollarolo G, Da Silva JS, Camoletto PG, Feiguin F, Dotti CG. 2005. Centrosome localization

determines neuronal polarity. Nature 436:704–8Deitch JS, Banker GA. 1993. An electron microscopic analysis of hippocampal neurons developing in culture:

early stages in the emergence of polarity. J. Neurosci. 13:4301–15Delcommenne M, Tan C, Gray V, Rue L, Woodgett J, Dedhar S. 1998. Phosphoinositide-3-OH kinase-

dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linkedkinase. Proc. Natl. Acad. Sci. USA 95:11211–16


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

DiAntonio A, Haghighi AP, Portman SL, Lee JD, Amaranto AM, Goodman CS. 2001. Ubiquitination-dependent mechanisms regulate synaptic growth and function. Nature 412:449–52

DiAntonio A, Hicke L. 2004. Ubiquitin-dependent regulation of the synapse. Annu. Rev. Neurosci. 27:223–46

Ding M, Chao D, Wang G, Shen K. 2007. Spatial regulation of an E3 ubiquitin ligase directs selective synapseelimination. Science 317:947–51

Dorfman J, Macara IG. 2008. STRAD{alpha} regulates LKB1 localization by blocking access to Importin-{alpha}, and by association with Crm1 and Exportin-7. Mol. Biol. Cell 19:1614–26

Dotti CG, Banker GA. 1987. Experimentally induced alteration in the polarity of developing neurons. Nature330:254–56

Dotti CG, Sullivan CA, Banker GA. 1988. The establishment of polarity by hippocampal neurons in culture.J. Neurosci. 8:1454–68

Drewes G, Ebneth A, Preuss U, Mandelkow EM, Mandelkow E. 1997. MARK, a novel family of protein kinasesthat phosphorylate microtubule-associated proteins and trigger microtubule disruption. Cell 89:297–308

Ehlers MD. 2003. Activity level controls postsynaptic composition and signaling via the ubiquitin-proteasomesystem. Nat. Neurosci. 6:231–42

Esch T, Lemmon V, Banker G. 1999. Local presentation of substrate molecules directs axon specification bycultured hippocampal neurons. J. Neurosci. 19:6417–26

Esmaeili B, Ross JM, Neades C, Miller DM 3rd, Ahringer J. 2002. The C. elegans even-skipped homologue,vab-7, specifies DB motoneurone identity and axon trajectory. Development 129:853–62

Etienne-Manneville S, Hall A. 2003. Cdc42 regulates GSK-3beta and adenomatous polyposis coli to controlcell polarity. Nature 421:753–56

Feng W, Wu H, Chan LN, Zhang M. 2008. Par-3-mediated junctional localization of the lipid phosphatasePTEN is required for cell polarity establishment. J. Biol. Chem. 283;23440–49

Fivaz M, Bandara S, Inoue T, Meyer T. 2008. Robust neuronal symmetry breaking by Ras-triggered localpositive feedback. Curr. Biol. 18:44–50

Fletcher TL, Cameron P, De Camilli P, Banker G. 1991. The distribution of synapsin I and synaptophysinin hippocampal neurons developing in culture. J. Neurosci. 11:1617–26

Furrer MP, Kim S, Wolf B, Chiba A. 2003. Robo and Frazzled/DCC mediate dendritic guidance at the CNSmidline. Nat. Neurosci. 6:223–30

Furrer MP, Vasenkova I, Kamiyama D, Rosado Y, Chiba A. 2007. Slit and Robo control the development ofdendrites in Drosophila CNS. Development 134:3795–804

Gao WQ, Hatten ME. 1993. Neuronal differentiation rescued by implantation of Weaver granule cell pre-cursors into wild-type cerebellar cortex. Science 260:367–69

Garrido JJ, Simon D, Varea O, Wandosell F. 2007. GSK3 alpha and GSK3 beta are necessary for axonformation. FEBS Lett. 581:1579–86

Gartner A, Huang X, Hall A. 2006. Neuronal polarity is regulated by glycogen synthase kinase-3(GSK-3beta) independently of Akt/PKB serine phosphorylation. J. Cell Sci. 119:3927–34

Garvalov BK, Flynn KC, Neukirchen D, Meyn L, Teusch N, et al. 2007. Cdc42 regulates cofilin during theestablishment of neuronal polarity. J. Neurosci. 27:13117–29

Godenschwege TA, Simpson JH, Shan X, Bashaw GJ, Goodman CS, Murphey RK. 2002. Ectopic expressionin the giant fiber system of Drosophila reveals distinct roles for roundabout (Robo), Robo2, and Robo3in dendritic guidance and synaptic connectivity. J. Neurosci. 22:3117–29

Goldstein B, Macara IG. 2007. The PAR proteins: fundamental players in animal cell polarization. Dev. Cell13:609–22

Gomis-Ruth S, Wierenga CJ, Bradke F. 2008. Plasticity of polarization: changing dendrites into axons inneurons integrated in neuronal circuits. Curr. Biol. 18(13):992–1000

Gonzalez-Billault C, Jimenez-Mateos EM, Caceres A, Diaz-Nido J, Wandosell F, Avila J. 2004. Microtubule-associated protein 1B function during normal development, regeneration, and pathological conditions inthe nervous system. J. Neurobiol. 58:48–59

Goslin K, Banker G. 1989. Experimental observations on the development of polarity by hippocampal neuronsin culture. J. Cell Biol. 108:1507–16


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

Gotz M, Huttner WB. 2005. The cell biology of neurogenesis. Nat. Rev. Mol. Cell Biol. 6:777–88Grueber WB, Yang CH, Ye B, Jan YN. 2005. The development of neuronal morphology in insects. Curr. Biol.

15:R730–38Gualdoni S, Albertinazzi C, Corbetta S, Valtorta F, de Curtis I. 2007. Normal levels of Rac1 are important

for dendritic but not axonal development in hippocampal neurons. Biol. Cell 99:455–64Guo W, Jiang H, Gray V, Dedhar S, Rao Y. 2007. Role of the integrin-linked kinase (ILK) in determining

neuronal polarity. Dev. Biol. 306:457–68Guo X, Lin Y, Horbinski C, Drahushuk KM, Kim IJ, et al. 2001. Dendritic growth induced by BMP-7 requires

Smad1 and proteasome activity. J. Neurobiol. 48:120–30Hakeda-Suzuki S, Ng J, Tzu J, Dietzl G, Sun Y, et al. 2002. Rac function and regulation during Drosophila

development. Nature 416:438–42Hand R, Bortone D, Mattar P, Nguyen L, Heng JI, et al. 2005. Phosphorylation of Neurogenin2 specifies

the migration properties and the dendritic morphology of pyramidal neurons in the neocortex. Neuron48:45–62

Hatanaka Y, Murakami F. 2002. In vitro analysis of the origin, migratory behavior, and maturation of corticalpyramidal cells. J. Comp. Neurol. 454:1–14

Hedstrom KL, Ogawa Y, Rasband MN. 2008. AnkyrinG is required for maintenance of the axon initialsegment and neuronal polarity. J. Cell Biol. 183:635–40

Hedstrom KL, Xu X, Ogawa Y, Frischknecht R, Seidenbecher CI, et al. 2007. Neurofascin assembles aspecialized extracellular matrix at the axon initial segment. J. Cell Biol. 178:875–86

Higginbotham H, Tanaka T, Brinkman BC, Gleeson JG. 2006. GSK3beta and PKCzeta function in cen-trosome localization and process stabilization during Slit-mediated neuronal repolarization. Mol. CellNeurosci. 32:118–32

Hilliard MA, Bargmann CI. 2006. Wnt signals and frizzled activity orient anterior-posterior axon outgrowthin C. elegans. Dev. Cell 10:379–90

Hinds JW, Hinds PL. 1978. Early development of amacrine cells in the mouse retina: an electron microscopic,serial section analysis. J. Comp. Neurol. 179:277–300

Hirokawa N, Takemura R. 2004. Molecular motors in neuronal development, intracellular transport anddiseases. Curr. Opin. Neurobiol. 14:564–73

Hirokawa N, Takemura R. 2005. Molecular motors and mechanisms of directional transport in neurons.Nat. Rev. Neurosci. 6:201–14

Hocking JC, Hehr CL, Chang RY, Johnston J, McFarlane S. 2008. TGFbeta ligands promote the initiationof retinal ganglion cell dendrites in vitro and in vivo. Mol. Cell Neurosci. 37:247–60

Horiguchi K, Hanada T, Fukui Y, Chishti AH. 2006. Transport of PIP3 by GAKIN, a kinesin-3 family protein,regulates neuronal cell polarity. J. Cell Biol. 174:425–36

Horton AC, Racz B, Monson EE, Lin AL, Weinberg RJ, Ehlers MD. 2005. Polarized secretory traffickingdirects cargo for asymmetric dendrite growth and morphogenesis. Neuron 48:757–71

Huber LA, Dupree P, Dotti CG. 1995. A deficiency of the small GTPase rab8 inhibits membrane traffic indeveloping neurons. Mol. Cell Biol. 15:918–24

Hung W, Hwang C, Po MD, Zhen M. 2007. Neuronal polarity is regulated by a direct interaction betweena scaffolding protein, Neurabin, and a presynaptic SAD-1 kinase in Caenorhabditis elegans. Development134:237–49

Hurov JB, Watkins JL, Piwnica-Worms H. 2004. Atypical PKC phosphorylates PAR-1 kinases to regulatelocalization and activity. Curr. Biol. 14:736–41

Ikuta J, Maturana A, Fujita T, Okajima T, Tatematsu K, et al. 2007. Fasciculation and elongation proteinzeta-1 (FEZ1) participates in the polarization of hippocampal neuron by controlling the mitochondrialmotility. Biochem. Biophys. Res. Commun. 353:127–32

Illenberger S, Drewes G, Trinczek B, Biernat J, Meyer HE, et al. 1996. Phosphorylation of microtubule-associated proteins MAP2 and MAP4 by the protein kinase p110mark. Phosphorylation sites and regu-lation of microtubule dynamics. J. Biol. Chem. 271:10834–43

Inagaki N, Chihara K, Arimura N, Menager C, Kawano Y, et al. 2001. CRMP-2 induces axons in culturedhippocampal neurons. Nat. Neurosci. 4:781–82


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

Jacobs T, Causeret F, Nishimura YV, Terao M, Norman A, et al. 2007. Localized activation of p21-activatedkinase controls neuronal polarity and morphology. J. Neurosci. 27:8604–15

Jacobson C, Schnapp B, Banker GA. 2006. A change in the selective translocation of the Kinesin-1 motordomain marks the initial specification of the axon. Neuron 49:797–804

Jiang H, Guo W, Liang X, Rao Y. 2005. Both the establishment and the maintenance of neuronal polarityrequire active mechanisms: critical roles of GSK-3beta and its upstream regulators. Cell 120:123–35

Jiang H, Rao Y. 2005. Axon formation: fate versus growth. Nat. Neurosci. 8:544–46Jiang W, Betson M, Mulloy R, Foster R, Levay M, et al. 2008. P190A RHOGAP is a glycogen synthase

kinase-3beta substrate required for polarized cell migration. J. Biol. Chem. 283:20978–88Joberty G, Petersen C, Gao L, Macara IG. 2000. The cell-polarity protein Par6 links Par3 and atypical protein

kinase C to Cdc42. Nat. Cell Biol. 2:531–39Kawano Y, Yoshimura T, Tsuboi D, Kawabata S, Kaneko-Kawano T, et al. 2005. CRMP-2 is involved in

kinesin-1-dependent transport of the Sra-1/WAVE1 complex and axon formation. Mol. Cell Biol. 25:9920–35

Kemphues KJ, Priess JR, Morton DG, Cheng NS. 1988. Identification of genes required for cytoplasmiclocalization in early C. elegans embryos. Cell 52:311–20

Kim S, Chiba A. 2004. Dendritic guidance. Trends Neurosci. 27:194–202Kirschner M, Mitchison T. 1986. Beyond self-assembly: from microtubules to morphogenesis. Cell 45:329–42Kishi M, Pan YA, Crump JG, Sanes JR. 2005. Mammalian SAD kinases are required for neuronal polarization.

Science 307:929–32Klimaschewski L, Hausott B, Ingorokva S, Pfaller K. 2006. Constitutively expressed catalytic proteasomal

subunits are up-regulated during neuronal differentiation and required for axon initiation, elongationand maintenance. J. Neurochem. 96:1708–17

Kobayashi T, Storrie B, Simons K, Dotti CG. 1992. A functional barrier to movement of lipids in polarizedneurons. Nature 359:647–50

Komuro H, Yacubova E, Rakic P. 2001. Mode and tempo of tangential cell migration in the cerebellar externalgranular layer. J. Neurosci. 21:527–40

Kukimoto-Niino M, Takagi T, Akasaka R, Murayama K, Uchikubo-Kamo T, et al. 2006. Crystal structure ofthe RUN domain of the RAP2-interacting protein x. J. Biol. Chem. 281:31843–53

Kunda P, Paglini G, Quiroga S, Kosik K, Caceres A. 2001. Evidence for the involvement of Tiam1 in axonformation. J. Neurosci. 21:2361–72

Lee-Hoeflich ST, Causing CG, Podkowa M, Zhao X, Wrana JL, Attisano L. 2004. Activation of LIMK1 bybinding to the BMP receptor, BMPRII, regulates BMP-dependent dendritogenesis. EMBO J. 23:4792–801

Lein P, Johnson M, Guo X, Rueger D, Higgins D. 1995. Osteogenic protein-1 induces dendritic growth inrat sympathetic neurons. Neuron 15:597–605

Lein PJ, Guo X, Shi GX, Moholt-Siebert M, Bruun D, Andres DA. 2007. The novel GTPase Rit differentiallyregulates axonal and dendritic growth. J. Neurosci. 27:4725–36

Levy-Strumpf N, Culotti JG. 2007. VAB-8, UNC-73 and MIG-2 regulate axon polarity and cell migrationfunctions of UNC-40 in C. elegans. Nat. Neurosci. 10:161–68

Lewcock JW, Genoud N, Lettieri K, Pfaff SL. 2007. The ubiquitin ligase Phr1 regulates axon outgrowththrough modulation of microtubule dynamics. Neuron 56:604–20

Lin D, Edwards AS, Fawcett JP, Mbamalu G, Scott JD, Pawson T. 2000. A mammalian PAR-3-PAR-6 compleximplicated in Cdc42/Rac1 and aPKC signalling and cell polarity. Nat. Cell Biol. 2:540–47

Lizcano JM, Goransson O, Toth R, Deak M, Morrice NA, et al. 2004. LKB1 is a master kinase that activates13 kinases of the AMPK subfamily, including MARK/PAR-1. EMBO J. 23:833–43

Luo L, Liao YJ, Jan LY, Jan YN. 1994. Distinct morphogenetic functions of similar small GTPases: DrosophilaDrac1 is involved in axonal outgrowth and myoblast fusion. Genes Dev. 8:1787–802

Maccario H, Perera NM, Davidson L, Downes CP, Leslie NR. 2007. PTEN is destabilized by phosphorylationon Thr366. Biochem. J. 405:439–44

Maekawa M, Ishizaki T, Boku S, Watanabe N, Fujita A, et al. 1999. Signaling from Rho to the actin cytoskeletonthrough protein kinases ROCK and LIM-kinase. Science 285:895–98


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

Manabe N, Hirai S, Imai F, Nakanishi H, Takai Y, Ohno S. 2002. Association of ASIP/mPAR-3 with adherensjunctions of mouse neuroepithelial cells. Dev. Dyn. 225:61–69

Martinez-Arca S, Coco S, Mainguy G, Schenk U, Alberts P, et al. 2001. A common exocytotic mechanismmediates axonal and dendritic outgrowth. J. Neurosci. 21:3830–38

Matenia D, Griesshaber B, Li XY, Thiessen A, Johne C, et al. 2005. PAK5 kinase is an inhibitor of MARK/Par-1, which leads to stable microtubules and dynamic actin. Mol. Biol. Cell 16:4410–22

Matheson SF, Hu KQ, Brouns MR, Sordella R, VanderHeide JD, Settleman J. 2006. Distinct but overlappingfunctions for the closely related p190 RhoGAPs in neural development. Dev. Neurosci. 28:538–50

Menager C, Arimura N, Fukata Y, Kaibuchi K. 2004. PIP3 is involved in neuronal polarization and axonformation. J. Neurochem. 89:109–18

Meyer D, Liu A, Margolis B. 1999. Interaction of c-Jun amino-terminal kinase interacting protein-1 withp190 rhoGEF and its localization in differentiated neurons. J. Biol. Chem. 274:35113–18

Morgan JL, Dhingra A, Vardi N, Wong RO. 2006. Axons and dendrites originate from neuroepithelial-likeprocesses of retinal bipolar cells. Nat. Neurosci. 9:85–92

Mori T, Wada T, Suzuki T, Kubota Y, Inagaki N. 2007. Singar1, a novel RUN domain-containing protein,suppresses formation of surplus axons for neuronal polarity. J. Biol. Chem. 282:19884–93

Nakada C, Ritchie K, Oba Y, Nakamura M, Hotta Y, et al. 2003. Accumulation of anchored proteins formsmembrane diffusion barriers during neuronal polarization. Nat. Cell Biol. 5:626–32

Nakata K, Abrams B, Grill B, Goncharov A, Huang X, et al. 2005. Regulation of a DLK-1 and p38 MAPkinase pathway by the ubiquitin ligase RPM-1 is required for presynaptic development. Cell 120:407–20

Nakayama M, Goto TM, Sugimoto M, Nishimura T, Shinagawa T, et al. 2008. Rho-kinase phosphorylatesPAR-3 and disrupts PAR complex formation. Dev. Cell 14:205–15

Ng J, Luo L. 2004. Rho GTPases regulate axon growth through convergent and divergent signaling pathways.Neuron 44:779–93

Ng J, Nardine T, Harms M, Tzu J, Goldstein A, et al. 2002. Rac GTPases control axon growth, guidance andbranching. Nature 416:442–47

Nishimura T, Kato K, Yamaguchi T, Fukata Y, Ohno S, Kaibuchi K. 2004. Role of the PAR-3-KIF3 complexin the establishment of neuronal polarity. Nat. Cell Biol. 6:328–34

Nishimura T, Yamaguchi T, Kato K, Yoshizawa M, Nabeshima Y, et al. 2005. PAR-6-PAR-3 mediates Cdc42-induced Rac activation through the Rac GEFs STEF/Tiam1. Nat. Cell Biol. 7:270–77

Noctor SC, Martinez-Cerdeno V, Ivic L, Kriegstein AR. 2004. Cortical neurons arise in symmetric andasymmetric division zones and migrate through specific phases. Nat. Neurosci. 7:136–44

Oinuma I, Katoh H, Negishi M. 2007. R-Ras controls axon specification upstream of glycogen synthasekinase-3beta through integrin-linked kinase. J. Biol. Chem. 282:303–18

Oliva AA Jr, Atkins CM, Copenagle L, Banker GA. 2006. Activated c-Jun N-terminal kinase is required foraxon formation. J. Neurosci. 26:9462–70

Ossipova O, Bardeesy N, DePinho RA, Green JB. 2003. LKB1 (XEEK1) regulates Wnt signalling in vertebratedevelopment. Nat. Cell Biol. 5:889–94

Ozdamar B, Bose R, Barrios-Rodiles M, Wang HR, Zhang Y, Wrana JL. 2005. Regulation of the polarityprotein Par6 by TGFbeta receptors controls epithelial cell plasticity. Science 307:1603–9

Ozdinler PH, Macklis JD. 2006. IGF-I specifically enhances axon outgrowth of corticospinal motor neurons.Nat. Neurosci. 9:1371–81

Pan CL, Baum PD, Gu M, Jorgensen EM, Clark SG, Garriga G. 2008. C. elegans AP-2 and retromer controlWnt signaling by regulating mig-14/Wntless. Dev. Cell 14:132–39

Plant PJ, Fawcett JP, Lin DC, Holdorf AD, Binns K, et al. 2003. A polarity complex of mPar-6 and atypicalPKC binds, phosphorylates and regulates mammalian Lgl. Nat. Cell Biol. 5:301–8

Polleux F, Giger RJ, Ginty DD, Kolodkin AL, Ghosh A. 1998. Patterning of cortical efferent projections bysemaphorin-neuropilin interactions. Science 282:1904–6

Polleux F, Morrow T, Ghosh A. 2000. Semaphorin 3A is a chemoattractant for cortical apical dendrites. Nature404:567–73

Poon VY, Klassen MP, Shen K. 2008. UNC-6/netrin and its receptor UNC-5 locally exclude presynapticcomponents from dendrites. Nature 455:669–73


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

Prasad BC, Clark SG. 2006. Wnt signaling establishes anteroposterior neuronal polarity and requires retromerin C. elegans. Development 133:1757–66

Qiu RG, Abo A, Steven Martin G. 2000. A human homolog of the C. elegans polarity determinant Par-6 linksRac and Cdc42 to PKCzeta signaling and cell transformation. Curr. Biol. 10:697–707

Rakic P. 1971. Neuron-glia relationship during granule cell migration in developing cerebellar cortex. A Golgiand electronmicroscopic study in Macacus rhesus. J. Comp. Neurol. 141:283–312

Rakic P. 1972. Mode of cell migration to the superficial layers of fetal monkey neocortex. J. Comp. Neurol.145:61–83

Rasin MR, Gazula VR, Breunig JJ, Kwan KY, Johnson MB, et al. 2007. Numb and Numbl are requiredfor maintenance of cadherin-based adhesion and polarity of neural progenitors. Nat. Neurosci. 10:819–27

Rolls MM, Doe CQ. 2004. Baz, Par-6 and aPKC are not required for axon or dendrite specification inDrosophila. Nat. Neurosci. 7:1293–95

Saito T, Nakatsuji N. 2001. Efficient gene transfer into the embryonic mouse brain using in vivo electropo-ration. Dev. Biol. 240:237–46

Sanchez-Ponce D, Tapia M, Munoz A, Garrido JJ. 2008. New role of IKK alpha/beta phosphorylated IkappaBalpha in axon outgrowth and axon initial segment development. Mol. Cell Neurosci. 37:832–44

Sapkota GP, Kieloch A, Lizcano JM, Lain S, Arthur JS, et al. 2001. Phosphorylation of the protein kinasemutated in Peutz-Jeghers cancer syndrome, LKB1/STK11, at Ser431 by p90(RSK) and cAMP-dependentprotein kinase, but not its farnesylation at Cys(433), is essential for LKB1 to suppress cell vrowth. J. Biol.Chem. 276:19469–82

Schober M, Schaefer M, Knoblich JA. 1999. Bazooka recruits Inscuteable to orient asymmetric cell divisionsin Drosophila neuroblasts. Nature 402:548–51

Schwamborn JC, Khazaei MR, Puschel AW. 2007a. The interaction of mPar3 with the ubiquitin ligase Smurf2is required for the establishment of neuronal polarity. J. Biol. Chem. 282:35259–68

Schwamborn JC, Muller M, Becker AH, Puschel AW. 2007b. Ubiquitination of the GTPase Rap1B by theubiquitin ligase Smurf2 is required for the establishment of neuronal polarity. EMBO J. 26:1410–22

Schwamborn JC, Puschel AW. 2004. The sequential activity of the GTPases Rap1B and Cdc42 determinesneuronal polarity. Nat. Neurosci. 7:923–29

Scott EK, Luo L. 2001. How do dendrites take their shape? Nat. Neurosci. 4:359–65Sharp DJ, Yu W, Ferhat L, Kuriyama R, Rueger DC, Baas PW. 1997. Identification of a microtubule-associated

motor protein essential for dendritic differentiation. J. Cell Biol. 138:833–43Shelly M, Cancedda L, Heilshorn S, Sumbre G, Poo MM. 2007. LKB1/STRAD promotes axon initiation

during neuronal polarization. Cell 129:565–77Shi SH, Cheng T, Jan LY, Jan YN. 2004. APC and GSK-3beta are involved in mPar3 targeting to the nascent

axon and establishment of neuronal polarity. Curr. Biol. 14:2025–32Shi SH, Jan LY, Jan YN. 2003. Hippocampal neuronal polarity specified by spatially localized mPar3/mPar6

and PI 3-kinase activity. Cell 112:63–75Shoukimas GM, Hinds JW. 1978. The development of the cerebral cortex in the embryonic mouse: an electron

microscopic serial section analysis. J. Comp. Neurol. 179:795–830Sordella R, Jiang W, Chen GC, Curto M, Settleman J. 2003. Modulation of Rho GTPase signaling regulates

a switch between adipogenesis and myogenesis. Cell 113:147–58Sosa L, Dupraz S, Laurino L, Bollati F, Bisbal M, et al. 2006. IGF-1 receptor is essential for the establishment

of hippocampal neuronal polarity. Nat. Neurosci. 9:993–95Speese SD, Trotta N, Rodesch CK, Aravamudan B, Broadie K. 2003. The ubiquitin proteasome system acutely

regulates presynaptic protein turnover and synaptic efficacy. Curr. Biol. 13:899–910Sperber BR, Leight S, Goedert M, Lee VM. 1995. Glycogen synthase kinase-3 beta phosphorylates tau protein

at multiple sites in intact cells. Neurosci. Lett. 197:149–53Suzuki A, Hirata M, Kamimura K, Maniwa R, Yamanaka T, et al. 2004. aPKC acts upstream of PAR-1b in

both the establishment and maintenance of mammalian epithelial polarity. Curr. Biol. 14:1425–35Tabata H, Nakajima K. 2001. Efficient in utero gene transfer system to the developing mouse brain using

electroporation: visualization of neuronal migration in the developing cortex. Neuroscience 103:865–72


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

Takahashi D, Yu W, Baas PW, Kawai-Hirai R, Hayashi K. 2007. Rearrangement of microtubule polarityorientation during conversion of dendrites to axons in cultured pyramidal neurons. Cell Motil. Cytoskeleton64:347–59

Thornton TM, Pedraza-Alva G, Deng B, Wood CD, Aronshtam A, et al. 2008. Phosphorylation by p38MAPK as an alternative pathway for GSK3beta inactivation. Science 320:667–70

Timm T, Balusamy K, Li X, Biernat J, Mandelkow E, Mandelkow EM. 2008. Glycogen synthase kinase(GSK) 3-beta directly phosphorylates serine 212 in the regulatory loop and inhibits microtubule affinityregulating kinase (MARK) 2. J. Biol. Chem. 283(27):18873–82

Toriyama M, Shimada T, Kim KB, Mitsuba M, Nomura E, et al. 2006. Shootin1: a protein involved in theorganization of an asymmetric signal for neuronal polarization. J. Cell Biol. 175:147–57

Tsai JW, Bremner KH, Vallee RB. 2007. Dual subcellular roles for LIS1 and dynein in radial neuronal migrationin live brain tissue. Nat. Neurosci. 10:970–79

Tursun B, Schluter A, Peters MA, Viehweger B, Ostendorff HP, et al. 2005. The ubiquitin ligase Rnf6 regulateslocal LIM kinase 1 levels in axonal growth cones. Genes Dev. 19:2307–19

Verhey KJ, Meyer D, Deehan R, Blenis J, Schnapp BJ, et al. 2001. Cargo of kinesin identified as JIP scaffoldingproteins and associated signaling molecules. J. Cell Biol. 152:959–70

von Stein W, Ramrath A, Grimm A, Muller-Borg M, Wodarz A. 2005. Direct association of Bazooka/PAR-3with the lipid phosphatase PTEN reveals a link between the PAR/aPKC complex and phosphoinositidesignaling. Development 132:1675–86

Watabe-Uchida M, John KA, Janas JA, Newey SE, Van Aelst L. 2006. The Rac activator DOCK7 regulatesneuronal polarity through local phosphorylation of stathmin/Op18. Neuron 51:727–39

Watari-Goshima N, Ogura K, Wolf FW, Goshima Y, Garriga G. 2007. C. elegans VAB-8 and UNC-73 regulatethe SAX-3 receptor to direct cell and growth-cone migrations. Nat. Neurosci. 10:169–76

Watts RJ, Schuldiner O, Perrino J, Larsen C, Luo L. 2004. Glia engulf degenerating axons during develop-mental axon pruning. Curr. Biol. 14:678–84

Whitford KL, Dijkhuizen P, Polleux F, Ghosh A. 2002a. Molecular control of cortical dendrite development.Annu. Rev. Neurosci. 25:127–49

Whitford KL, Marillat V, Stein E, Goodman CS, Tessier-Lavigne M, et al. 2002b. Regulation of corticaldendrite development by Slit-Robo interactions. Neuron 33:47–61

Winckler B, Mellman I. 1999. Neuronal polarity: controlling the sorting and diffusion of membrane compo-nents. Neuron 23:637–40

Witte H, Neukirchen D, Bradke F. 2008. Microtubule stabilization specifies initial neuronal polarization.J. Cell Biol. 180:619–32

Xie Z, Moy LY, Sanada K, Zhou Y, Buchman JJ, Tsai LH. 2007. Cep120 and TACCs control interkineticnuclear migration and the neural progenitor pool. Neuron 56:79–93

Yamanaka T, Horikoshi Y, Sugiyama Y, Ishiyama C, Suzuki A, et al. 2003. Mammalian Lgl forms aprotein complex with PAR-6 and aPKC independently of PAR-3 to regulate epithelial cell polarity.Curr. Biol. 13:734–43

Yamanaka T, Horikoshi Y, Suzuki A, Sugiyama Y, Kitamura K, et al. 2001. PAR-6 regulates aPKC activity ina novel way and mediates cell-cell contact-induced formation of the epithelial junctional complex. GenesCells 6:721–31

Yan D, Guo L, Wang Y. 2006. Requirement of dendritic Akt degradation by the ubiquitin-proteasome systemfor neuronal polarity. J. Cell Biol. 174:415–24

Yang Y, Ogawa Y, Hedstrom KL, Rasband MN. 2007. betaIV spectrin is recruited to axon initial segmentsand nodes of Ranvier by ankyrinG. J. Cell Biol. 176:509–19

Ye B, Zhang Y, Song W, Younger SH, Jan LY, Jan YN. 2007. Growing dendrites and axons differ in theirreliance on the secretory pathway. Cell 130:717–29

Yoshimura T, Arimura N, Kawano Y, Kawabata S, Wang S, Kaibuchi K. 2006. Ras regulates neuronal polarityvia the PI3-kinase/Akt/GSK-3beta/CRMP-2 pathway. Biochem. Biophys. Res. Commun. 340:62–68

Yoshimura T, Kawano Y, Arimura N, Kawabata S, Kikuchi A, Kaibuchi K. 2005. GSK-3beta regulates phos-phorylation of CRMP-2 and neuronal polarity. Cell 120:137–49

Yu W, Cook C, Sauter C, Kuriyama R, Kaplan PL, Baas PW. 2000. Depletion of a microtubule-associatedmotor protein induces the loss of dendritic identity. J. Neurosci. 20:5782–91


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

ANRV379-NE32-15 ARI 13 May 2009 8:40

Zhang H, Macara IG. 2006. The polarity protein PAR-3 and TIAM1 cooperate in dendritic spine morpho-genesis. Nat. Cell Biol. 8:227–37

Zhang H, Macara IG. 2008. The PAR-6 polarity protein regulates dendritic spine morphogenesis throughp190 RhoGAP and the Rho GTPase. Dev. Cell 14:216–26

Zhang X, Zhu J, Yang GY, Wang QJ, Qian L, et al. 2007. Dishevelled promotes axon differentiation byregulating atypical protein kinase C. Nat. Cell Biol. 9:743–54

Zhou FQ, Zhou J, Dedhar S, Wu YH, Snider WD. 2004. NGF-induced axon growth is mediated by localizedinactivation of GSK-3beta and functions of the microtubule plus end binding protein APC. Neuron42:897–912

Zolessi FR, Poggi L, Wilkinson CJ, Chien CB, Harris WA. 2006. Polarization and orientation of retinalganglion cells in vivo. Neural. Develop. 1:2


Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

AR379-FM ARI 20 May 2009 14:8

Annual Review ofNeuroscience

Volume 32, 2009Contents

Neuropathic Pain: A Maladaptive Response of the NervousSystem to DamageMichael Costigan, Joachim Scholz, and Clifford J. Woolf � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Synaptic Mechanisms for Plasticity in NeocortexDaniel E. Feldman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �33

Neurocognitive Mechanisms in Depression: Implications forTreatmentLuke Clark, Samuel R. Chamberlain, and Barbara J. Sahakian � � � � � � � � � � � � � � � � � � � � � � � � � �57

Using Diffusion Imaging to Study Human Connectional AnatomyHeidi Johansen-Berg and Matthew F.S. Rushworth � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �75

Serotonin in Affective ControlPeter Dayan and Quentin J.M. Huys � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �95

Physiology and Pharmacology of Striatal NeuronsAnatol C. Kreitzer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 127

The Glial Nature of Embryonic and Adult Neural Stem CellsArnold Kriegstein and Arturo Alvarez-Buylla � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 149

Representation of Number in the BrainAndreas Nieder and Stanislas Dehaene � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 185

Neuronal Gamma-Band Synchronization as a Fundamental Processin Cortical ComputationPascal Fries � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 209

The Neurobiology of Individual Differences in ComplexBehavioral TraitsAhmad R. Hariri � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 225

The Science of Neural Interface SystemsNicholas G. Hatsopoulos and John P. Donoghue � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 249

The Neuropsychopharmacology of Fronto-Executive Function:Monoaminergic ModulationT.W. Robbins and A.F.T. Arnsten � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 267

v

Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

AR379-FM ARI 20 May 2009 14:8

The Influence of Stress Hormones on Fear CircuitrySarina M. Rodrigues, Joseph E. LeDoux, and Robert M. Sapolsky � � � � � � � � � � � � � � � � � � � � � � � 289

The Primate Cortical Auditory System and Neural Representation ofConspecific VocalizationsLizabeth M. Romanski and Bruno B. Averbeck � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 315

Establishment of Axon-Dendrite Polarity in Developing NeuronsAnthony P. Barnes and Franck Polleux � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 347

Axon Growth and Guidance: Receptor Regulationand Signal TransductionMichael O’Donnell, Rebecca K. Chance, and Greg J. Bashaw � � � � � � � � � � � � � � � � � � � � � � � � � � � � 383

Cerebellum and Nonmotor FunctionPeter L. Strick, Richard P. Dum, and Julie A. Fiez � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 413

Advances in Light Microscopy for NeuroscienceBrian A. Wilt, Laurie D. Burns, Eric Tatt Wei Ho, Kunal K. Ghosh,Eran A. Mukamel, and Mark J. Schnitzer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 435

Indexes

Cumulative Index of Contributing Authors, Volumes 23–32 � � � � � � � � � � � � � � � � � � � � � � � � � � � 507

Cumulative Index of Chapter Titles, Volumes 23–32 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 511

Errata

An online log of corrections to Annual Review of Neuroscience articles may be found athttp://neuro.annualreviews.org/

vi Contents

Ann

u. R

ev. N

euro

sci.

2009

.32:

347-

381.

Dow

nloa

ded

from

arj

ourn

als.

annu

alre

view

s.or

gby

Uni

vers

idad

de

Con

cepc

ion

on 1

2/08

/09.

For

per

sona

l use

onl

y.

Documents

Establishment of Axon-Dendrite Polarity in Developing Neurons