Mechanisms and molecules in motorneuron specification and axonpathfindingJohn Jacob, Adam Hacker and Sarah Guthrie*

SummaryThe vertebrate nervous system performs the mostcomplex functions of any organ system. This feat is medi-ated by dedicated assemblies of neurons that must beprecisely connected to one another and to peripheraltissues during embryonic development. Motor neurons,which innervate muscle and regulate autonomic func-tions, form an integral part of this neural circuitry. Thefirst part of this review describes the remarkable progressin our understanding of motor neuron differentiation,which is arguably the best understood model of neuronaldifferentiation to date. During development, the coordi-nate actions of inductive signals from adjacent non-neural tissues initiate the differentiation of distinct motorneuron subclasses, with specific projection patterns, atstereotypical locations within the neural tube. Underlyingthis specialisation is the expression of specific home-odomain proteins, which act combinatorially to confermotor neurons with both their generic and subtype-specific properties. Ensuring that specific motor neuronsubtypes innervate the correct target structure, however,requires precise motor axon guidance mechanisms. Thesecond half of this review focuses on how distinct motorneuron subtypes pursue highly specific projection pat-terns by responding differentially to spatially discreteattractive and repulsive molecular cues. The tight linkbetween motor neuron specification and axon pathfind-ing appears to be established by the dominant role ofhomeodomain proteins in dictating the ways that navi-gating motor axons interpret the plethora of guidancecues impinging on growth cones. BioEssays 23:582±595, 2001. ß 2001 John Wiley & Sons, Inc.

Introduction

The pioneering neuroanatomical studies of the Spanish

neuroscientist, Ramon y Cajal revealed the remarkably

intricate, yet stereotypical patterns of neural connections

in the nervous system. How are these connections estab-

lished? The last decade has seen dramatic progress in our

understanding of the molecular basis of neural connectivity,(1)

building on earlier descriptions of the formation of neural

projections at embryonic stages.(2) Among developing neu-

rons, motor neurons are unique in sending axon projections

out of the central nervous system into the periphery. The

projections of motor neurons to their targets are specific and

accurate from the outset,(2) which implies the operation of a

highly coordinated process. The various mechanisms that

guide motor axons must be robust enough to compensate for

the rapid morphogenetic changes that result in axons seeking

outÐquite literallyÐa moving target. Moreover, the motor

axon growth cone must be able to discriminate the appropriate

target structure from amongst a wide array of alternative

structures.

The division of the central nervous system into cranial and

spinal components is paralleled by differences between motor

neurons at these axial levels in terms of their organisation,

identity, axon trajectories and target specificities. These

distinctions between cranial and spinal motor neurons are

rooted in their distinct ontogenies and, in particular, in their

combinatorial profiles of homeodomain protein expression.

This review encompasses the full spectrum of developmental

events, beginning with motor neuron determination and

the acquistion of subtype identity, to the mechanisms that

enable motor axons to navigate to their targets with precision.

These events are deeply interrelated at the molecular level

and, as we shall see, the development of spinal and cranial

motor neurons offers an intriguing mix of similarities and

differences. Readers unaccustomed to embryological termi-

nology will find a glossary of the terms used in this review

provided on the next page.

Specification of motor neurons

Motor neurons form subpopulationsat distinct axial levelsThe central nervous system is first recognisable in vertebrate

embryos as a flat sheet of cells called the neural plate, which

subsequently folds to form the neural tube. Its early cyclindrical

configuration is soon modified by local expansions along the

longitudinal axis. Within this cylinder, neuronal types develop

at specific locations in the dorsoventral and rostrocaudal axes.

All motor neurons differentiate exclusively in ventral regions,

582 BioEssays 23.7 BioEssays 23:582±595, ß 2001 John Wiley & Sons, Inc.

MRC Centre for Developmental Neurobiology, King's College,

London.

Funding agencies: JJ was a Wellcome Trust Clinical Training Fellow.�Correspondence to: Dr. Sarah Guthrie, MRC Centre for Develop-

mental Neurobiology, King's College, Guy's Campus, 4th Floor, New

Hunt's House, London SE1 1UL. E-mail: sarah.c.guthrie@kcl.ac.uk

Review articles

but once they are post-mitotic may later migrate to occupy

more dorsal positions on the ipsilateral side. In other cases, for

example in the midbrain, motor neurons may translocate

contralaterally, crossing the floor plate as they do so.(3) As

development progresses, motor neurons also start to assume

distinct identities. Three generic classes can be identified,

based on their dorsoventral (DV) position, axon pathway and

synaptic target (Table 1). The cranial region contains somatic

motor (SM), visceral motor (VM) and branchiomotor (BM)

neurons, whilst the spinal region contains SM and VM neurons

only. SM neurons and spinal VM neurons project their axons

ventrally out of the neural tube to supply skeletal muscle and

sympathetic ganglia, respectively. Cranial VM neurons in-

nervate parasympathetic ganglia, whilst BM neurons inner-

vate branchial arch muscles. The latter two subtypes both

exhibit dorsal axon trajectories and dorsal patterns of cell body

migration.

Induction of motor neuron fatein neural progenitorsMotor neurons arise from initially uncommitted, mitotically

active ventral progenitors in the ventricular layer of the neural

tube. The molecular basis of motor neuron differentiation has

been most extensively investigated in chick and mouse

embryos, hence further discussion on this subject will be

confined to studies in these species. Commitment to motor

neuron identity involves a progressive restriction of progenitor

fate imposed by a ventral-to-dorsal gradient of an extrinsic

signal, sonic hedgehog (shh), which is produced by the sub-

jacent notochord and the floor plate.(2) Graded sonic signalling

establishes multiple progenitor domains within the ventral

neural tube. Each domain in turn gives rise in turn gives rise to

distinct neuronal classes, by activating or repressing the

expression of a key set of DNA-binding, homeodomain

proteins at different concentration thresholds (Fig. 1A).(5)

In the hindbrain, the progenitors of BM and VM neurons are

delineated by coexpression of the homeodomain proteins

Nkx2.2 and Nkx6.1. Nkx6.1 expression extends further dor-

sally to encompass the SM neuron progenitor domain, which

abuts the BM/VM progenitor domain at its ventral border.(6)

At spinal levels, the progenitors of all motor neuron subtypes

are marked by an Nkx6.1 expression domain whilst the Nkx2.2

domain gives rise to a separate ventral interneuron popula-

tion.(7) Consistent with a crucial role in motor neuron fate

determination, misexpression of Nkx6.1 in the dorsal spinal

cord of the chick leads to the ectopic generation of motor

neurons.(5) Conversely, targeted deletion of Nkx6.1 largely

prevents the formation of all subclasses of spinal motor

neurons and hindbrain SM neurons but spares cranial BM/VM

neuron generation.(6) Homeodomain proteins that specify

distinct progenitor domains and share a common boundary

negatively regulate each other's expression by recruiting

members of the Groucho class of transcriptional repres-

sors.(5,8) Consequently, the absence of Nkx6.1 leads to a

ventral expansion of the dorsally adjacent Dbx2 expression

domain. Dbx2 specifies a distinct population of ventral

interneurons which are present in greater numbers in Nkx6.1

null mutant mice (Fig. 1B) .(6) That the loss of Nkx6.1 function

has no discernible effect on cranial BM/VM neuron formation is

thought to be due to the activity of Nkx2.2 and the highly related

Glossary of key terms

Branchial arches Bars of mesenchymal tissue which

will contribute to the formation of

the head and neck

Chemoattraction Permissive and/or attractive effect

on growth cones by a diffusible

molecule

Chemorepulsion Inhibitory and/or repulsive effect

on growth cones by a diffusible

molecule

Cranial paraxial Non-segmented head mesoderm

mesoderm lying adjacent to the midline

Dermamyotome Dorsolateral part of the somite

which gives rise to the dermis

and back musculature

Fasciculation Bundling together of nerve fibres

Floor plate Non-neural cells found at the ventral

midline of the neural tube

Growth cone The growing tip of an axon that has a

key role in axon pathfinding and

later in synapse formation

Isthmic region Portion of the neural tube lying

between midbrain and hindbrain,

which is a source of inductive

signals that pattern the adjacent

midbrain and rostral hindbrain

Limb bud Primordium of the future limb

Nucleus Aggregation of nerve cell bodies

within the central nervous system

that have a common function

Otic vesicle Primordium of inner ear structures

Paraxial Mesoderm lying adjacent to the

mesoderm midline

Plexus Present at fore- and hind-limb

levels; demar cates the points

at which axon pathways are re-

arranged

Sclerotome Ventromedial compartment of the

somite, which will form the ver-

tebrae and ribs

Somite Segmental paraxial mesoderm of

the trunk

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gene, Nkx2.9, in BM/VM progenitors. In mice lacking Nkx2.2

function there is no detectable abnormality of cranial BM/VM

neuron development, suggesting that Nkx2.2 acts in redun-

dant fashion with Nkx2.9 to control BM/VM progenitor fate.(7)

Motor neuron differentiation in the spinal cordOnce the motor neuron progenitor domain is established,

committed precursors initiate an autonomous, homeodomain

protein-mediated program of differentiation, marked by inde-

pendence from sonic signalling.(9) In the chick, the molecular

pathway of SM neuron differentiation is triggered by auto-

activation of MNR2, acting as a selector gene, in motor neuron

precursors during their terminal mitotic cycle.(10) Misexpres-

sion of MNR2 in the dorsal spinal cord is sufficient to induce

ectopic somatic motor neuron differentiation in the absence of

any detectable change in progenitor identity.(10) In the mouse,

the closely related gene, HB9, drives motor neuron progeni-

tors to a somatic motor neuron fate. Targeted inactivation of

HB9 results in inappropriate expression of interneuron mark-

ers by prospective motor neurons, with associated disruption

Table 1. Major distinguishing features of motor neuron subtypes

Spatial locationSubtype identity Longitudinal/DV Axon trajectory Target tissue

SM Cranial/ventral Ventral Extraocular/tongue muscles

SM Spinal/ventral Ventral Trunk/limb muscles

VM Cranial/dorsal Dorsal Parasympathetic ganglia

VM Spinal/dorsal Ventral Sympathetic ganglia

BM Cranial/dorsal Dorsal Branchial arches

Figure 1. Mechanism of ventral cell fate determination bygraded sonic hedgehog (shh) signalling A: A concentration

gradient of shh in the ventral neural tube establishes ventral

expression domains of key transcription factors within ventral

neural progenitors. Shh either represses (class I, ÿ ) or induces(class II, �) homeodomain protein expression at different

concentration thresholds, leading to the formation of bound-

aries, which are refined by negative cross-regulatory interac-

tions between those proteins that share the same boundary (forexample Dbx2 and Nkx6.1) Five neuronal subtypes, comprising

motor neurons and distinct interneuron subtypes, arise from the

five progenitor domains established by graded shh signalling.The p3 domain gives rise to BM/VM neurons in the hindbrain

and to V3 interneurons at spinal cord level. Although Nkx6.1 is

also expressed ventral to the pMN domain, motor neurons do

not develop in this region due to the dominant suppressiveeffect of Nkx2.2 on motor neuron differentiation. B: In Nkx6.1mutants, there is a ventral expansion of Dbx2 expression which

is associated with an increase in V1 interneuron generation,

and a large reduction in numbers of motor neurons and V2interneurons. C: Horizontal section of the spinal cord of the

chick embryo at limb level showing the motor columns in the

ventral spinal cord and their expression of LIM domain proteins.D, dorsal; v, ventral; Isll, Islet-1; Isl2, Islet-2; LMC (m), medial

subdivision of lateral motor column; LMC(1), lateral subdivision

of lateral motor column; MMC, median motor column (Figure

adapted from(9)).

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584 BioEssays 23.7

of motor neuron development and axon pathfinding.(11,12)

HB9 therefore actively suppresses interneuron differentiation

programs in developing motor neurons. Not surprisingly, HB9

misexpression in the chick spinal cord mimics the effect of

MNR2. However, since its endogenous activation occurs post-

mitotically, following that of MNR2, HB9 does not act as a

selector gene in vivo in the chick.

Which transcription factors lie downstream of MNR2 and

what role do they play in motor neuron differentiation? Four

LIM domain proteins, namely Islet-1, Islet-2, Lim1 and Lim3,

(Lhx3), are combinatorially expressed by distinct motor neuron

subtypes, which are distinguished by their peripheral axon

trajectories and settling position within medial or lateral

subdivisions of two major longitudinal columns in the ventral

spinal cord.(13) Median motor column (MMC) neurons inner-

vate trunk muscles, whereas neurons in the lateral motor

column (LMC), which is only present at limb levels, project to

limb muscles (Fig. 1C). An elegant series of misexpression

studies in the chick showed that MNR2 induces Islet-1

expression and cooperates with the latter protein in activating

Islet-2; MNR2 also lies upstream of Lhx3 which in turn acti-

vates HB9 coordinately with Islet-1.(10)

Islet-1 is expressed by all motor neurons as soon as they

are born.(14) In mutant mice lacking Islet-1 function, there is a

block in the differentiation of all classes of motor neurons which

die as soon as they become post-mitotic.(15) Therefore, Islet-1

activation represents an early critical point of convergence in

the cascade of molecular events that promote motor neuron

diversification and survival. Misexpression of Islet-1 in the

spinal cord does not lead to the ectopic generation of motor

neurons, suggesting that Islet-1 is necessary but not sufficient

to instruct motor neuron differentiation.(10) Lhx3 and the highly

related gene, Lhx4 are initially expressed during the final

progenitor division by precursors of both LMC, MMC and

spinal VM neurons, but their expression is rapidly extinguished

in all except medial subdivision neurons (MMCM) of the

MMC.(16) Both genes coordinately determine two important

phenotypic characteristics, namely cell body migration pattern

and ventral axon trajectory. Combined inactivation of Lhx3

and Lhx4 results in the acquisition of an inappropriate identity

by LMC, MMC and spinal VM neurons, reflected in dorsal cell

body migration and dorsal axon trajectories, which are nor-

mally displayed only by BM/VM motor neurons of the hindbrain

and a distinct subpopulation of motor neurons in the rostral

spinal cord.(16) In keeping with a key role in determining

columnar identity, the singular activity of Lhx3 is sufficient to

instruct binary choices between MMCM and all other spinal

motor neuron identities. Forced maintenance of Lhx3 expres-

sion in spinal VM, LMC and lateral MMC (MMCL) motor

neurons endows them with an MMCM-like identity, with con-

comitant rerouting of motor axons to axial muscles.(17) By

contrast, Lim-1, which is activated in post-mitotic lateral

subdivision (LMCL) neurons of the LMC as they down-regulate

Islet-1, is not necessary for the specification of LMCL identity

and its function is confined to regulating axon pathfinding

decisions in the periphery (see later also).(18) Finally, Islet-2 is

expressed by MMCL and LMC neurons, but its role in motor

neuron differentiation has yet to be elucidated by loss- and

gain-of function studies. In summary, the differential activation

of homeodomain transcription factors is causally linked to the

progressive refinement of motor neuron identity. Thus, dis-

ruption of the transcriptional program of differentiation early in

development affects motor neuron specification or columnar

identity, whereas later disruption affects more restricted fea-

tures of motor neuron phenotype.

The presence of multiple motor neuron subtypes at the

same axial level that are derived from progenitors with identical

dorsoventral locations raises the question of how this diversity

is generated. LMCL neurons migrate past early-born medial

LMC (LMCM) neurons which are a source of retinoic acid

(RA). RA induces differentiation of LMCL neurons in vitro,

down-regulating Islet-1 and up-regulating Lim-1 that distin-

guishes LMCL from LMCM neurons.(19) Local signalling

interactions between post-mitotic motor neurons therefore

represent an additional modality in the acquisition of motor

neuron subtype identity. At a finer level of resolution, further

distinctions in motor neuron subtype identity are evident.

Within individual columns, motor neurons innervating a single

muscle are organised into discrete pools consisting of some

hundreds of neurons,(20) which can be defined by their

combinatorial expression of LIM domain proteins and mem-

bers of the ETS(21) and forkhead(22) classes of transcription

factors.

Differentiation of cranial motor neuronsIn the hindbrain and midbrain, motor neurons are organised in

twelve discrete clusters, or nuclei, each containing one or

more SM, BM or VM types of motor neuron (Fig. 2). SM

neurons in the hindbrain have a discontinuous distribution and

their development is thought to be closely similar to that of their

counterparts in the spinal cord.(23,24) However, SM neurons

that form the trochlear and oculomotor nuclei in the isthmic

region and midbrain, respectively, have a distinct ontogeny

characterised at the molecular level by sequential expr-

ession of the paired-like homeodomain transcription factors

Phox2a(25) and the highly related protein, Phox2b.(26) In mice

with a null mutation in the Phox2a gene, both nuclei are

absent.(26) In contrast, Phox2a expression is conspicuously

absent in hindbrain SM nuclei, and they develop normally in

Phox2a null mutant mice.(27)

The early development of BM and VM neurons is closely

similar and identical transcriptional determinants are

expressed by progenitors of both generic subtypes and

post-mitotic motor neurons.(7,26,28) During BM/VM neuron

development, the sequence of Phox2 gene activation is

reversed so that Phox2b is expressed before Phox2a.(26) In

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BioEssays 23.7 585

the absence of Phox2b function, there is a block in the

differentiation of all BM/VM neurons in the hindbrain,

accompanied by a failure to express generic neuronal

markers, indicating that Phox2b controls subtype-specific

and generic neuronal properties.(29,30) In addition, Phox2b

activity has been implicated in the promotion of cell cycle exit of

BM/VM progenitors, a role that has not so far been ascribed to

any transcriptional determinant of SM and spinal VM

neurons.(31) Therefore, cranial motor neuron subpopulations

have complementary expression profiles of Phox2 genes and

the temporal order of expression predicts the relative

importance of Phox2a and Phox2b in their specification.

Figure 2. Organisation of cranial motor neurons A: Diagram of a flat-mounted chick embryo brainstem showing the rhombomericorganisation of BM/VM (red) and SM (blue, yellow and green) neurons in the different cranial motor nuclei. Rhombomeres (r1-r8), the

cranial motor nuclei and the sensory ganglia are shown. The oculomotor (III) nucleus also contains a contingent of VM neurons. In

addition to facial motor neurons, which project axons into the periphery, rhombomere 4 (r4) also contains a population of contralaterally-

migrating vestibuloacoustic (cva) neurons, which also send axon projections out of the hindbrain. The accessory abducens (aVI) nucleusin r5 splits from the main abducens nucleus during development. The dashed line represents the midbrain-hindbrain boundary (adapted

from (28)). B: A transverse section of the embryo in (A) taken at the level of r5. Branchiomotor (BM) and visceral motor (VM) neurons

(red) send their axons dorsally towards dorsal exit points. Their cell bodies translocate laterally (arrow) during development. Somaticmotor (SM) neurons (blue and green) have ventral axon projections, with the exception of trochlear (IV) neurons, which project dorsally.

Colour coding is the same as in (A). Rhombomeres are numbered r1-r8. III, oculomotor nucleus; IV, trochlear nucleus; V, trigeminal

nucleus; mVI, abducens nucleus; VII, facial nucleus; IX, glossopharyngeal nucleus; X/XI, vagus/cranial accessory nerve; XII,

hypoglossal nucleus; gV-gX, cranial sensory ganglia; ov, otic vesicle.

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586 BioEssays 23.7

Cranial motor nuclei also express different combinations of

LIM genes. BM and VM neurons with dorsal axon pathways

express only Islet-1 (red in Fig. 2B), whereas hindbrain SM

neurons that project ventrally express Islet-1 and Islet-2 and/or

Lhx3 (blue in Fig. 2B) .(28) The specification of somatic motor

neuron identity by Lhx3 is conserved at cranial levels of the

neuraxis. When Lhx3 is misexpressed in the caudal hindbrain

of the chick, there is an expansion of ventrally projecting

hypoglossal SM neurons, at the expense of dorsally projecting

vagal motor neurons.(16) The differential effects of specific

homeobox genes on cranial and spinal motor neuron sub-

populations are summarised in Table 2.

Identifying the molecular `codes' that distinguish cranial BM

from VM neurons has thus far proved elusive. Both generic

subtypes share the same LIM code, which suggests that the

expression of other genes must distinguish between them.

Possible candidates are the Phox2 genes. Phox2a is expres-

sed initially by both subtypes, but is later downregulated in

facial BM neurons and maintained in facial VM neurons at

the stage when their axons are growing to their respective

targets.(32,33) Interestingly, there is a complementary down-

regulation of Phox2b expression in developing facial VM neu-

rons and maintenance of expression in facial BM neurons

(Guthrie, and Studer unpublished observations). To determine

the importance of dynamic regulation of Phox2 gene expres-

sion for the differentiation of motor neuron subtypes, Phox2a

and Phox2b expression could be maintained at high levels

using a transgenic approach in developing facial BM and VM

neurons, respectively. This might reveal whether a quantita-

tive difference in levels of expression of these proteins is the

cause or, simply, a consequence of the differences between

these subtypes.

Axial differences in motor neuron patterningAside from the obvious differences in the arrangement of

motor neurons in the head and trunk, and even between the

hindbrain and spinal cord, there are further axial differences

in motor neuron patterning. What are the mechanisms that

generate these differences? In the hindbrain, motor neurons

develop in specific segments, or rhombomeres (r), which have

unique identities reflected by the phenotypes of their consti-

tuent neurons.(34) At an early stage in embryonic development,

well in advance of the birth of motor neurons, the Hox family of

genes are differentially expressed in nested patterns which

pre-figure hindbrain segments.(35) There is now considerable

evidence that rhombomeres are patterned by specific Hox

genes. For example, mutation of Hoxb1, which is strongly

expressed in r4 leads to aberrant migration of the facial BM

neuron cell bodies therein and agenesis of the facial motor

nucleus.(36,37) Conversely, misexpression of Hoxb1 in r2 resu-

lts in the ectopic generation of facial-like motor neurons, at the

expense of the normal resident trigeminal motor neuron

population.(38) Ectopic motor neurons with facial and trig-

eminal characteristics are induced even in regions normally

devoid of motor neurons such as r1, following misexpression

of Hoxb1 and Hoxa2, respectively.(39) These findings demon-

strate that individual Hox genes can impose specific fates

upon hindbrain neural progenitors and, under certain condi-

tions, are able to override endogenous segmental differentia-

tion programs.

There is evidence also for the role of non-cell autonomous

factors in determining the characteristics of motor neuron

populations along the rostrocaudal axis of the neural tube.

When limb level paraxial mesoderm in the chick is transplanted

to thoracic levels, ectopic formation of the LMCL is induced.(40)

Presumably, this is mediated by an inductive signal(s) from the

adjacent mesoderm that directly or indirectly instructs the

columnar identity of spinal motor neurons. Cranial paraxial

mesoderm that lies caudal to the otic vesicle (post-otic meso-

derm) may also possess inductive activity that specifies the

phenotype of motor neurons. Following the transposition of

presumptive pre-otic rhombomeres to post-otic levels the

transposed segment generates motor neurons appropriate

to the new environment and alters its Hox `code' accord-

ingly.(41,42) However, converse post-otic to pre-otic rhombo-

mere transposition, or transposition in rostral or caudal

directions within the pre-otic region does not alter motor

neuron specification.(42±44) One possible explanation for this

difference is that following early exposure to this `caudalising'

signal progenitor fate becomes irreversibly determined and is

not subject to change. Thus, inductive factors from adjacent

non-neural tissues contribute to the diversification of motor

neuron subtypes along the rostrocaudal and dorsoventral

axes.

Motor axon pathfinding

During development, motor neurons acquire distinct identities

that are reflected in their choice of specific axon pathways and

synaptic targets. Motor axon pathfinding occurs in a step-wise

manner that is dependent on the differential action of guidance

cues, which are serially deployed at discrete locations along

the pathway to the target.(41,42) Table 3 provides an overview

of the tissues and molecules implicated in cranial and spinal

motor axon guidance, as discussed in detail below.

Motor axons are repelled by the floor plateMotor axons grow away from the floor plate and penetrate

the neuroepithelium at specific exit points to grow into the

periphery. Collagen gel co-culture experiments have demon-

strated that all classes of motor axons are repelled when

placed adjacent to floor plate cells in culture(46) (Fig. 3A).

Furthermore, in the same study, in vivo transplantation of floor

plate tissue to ectopic locations in the chick hindbrain caused

motor axons to be deflected in such a way as to avoid the graft.

The floor plate therefore deflects motor axons away from the

midline by providing chemorepulsive cues.

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Molecular candidates for the repellent effect of the ventral

midline/floor plate include the axon guidance molecules,

semaphorins, netrins and slit proteins.(47,48) Cell aggregates

secreting the vertebrate semaphorin, SEMA3A, chemorepel

all classes of motor axons in culture.(49,50) Netrin-1 repels only

dorsally projecting axons and not ventrally projecting ax-

ons.(50) Thus, signals controlling the trajectory of ventrally

projecting neurons may be distinct from those that trigger

dorsal projection. Evidence for the involvement of additional

midline chemorepellents comes from the observation that

midbrain oculomotor (SM) neurons are also repelled by

the floor plate, but fail to respond to either netrin-1 or

SEMA3A.(50) A role for Slit proteins in midline chemorepulsion

first came from the identification of Drosophila slit as a ligand

for the repulsive guidance receptor, robo1.(51) Human Slit2

(hSlit2), one of three mammalian slit proteins, repels motor

axons from ventral spinal cord explants cultured at a distance,

or in contact with hSlit2 expressing cell aggregates.(52)

The unexpected finding that Slit2 and Slit3 are also

expressed in the ventral motor column(52) suggests that Slit

proteins produced by motor neurons may have a cell

autonomous/local signalling effect that might act in conjunc-

tion with a non-cell autonomous effect of Slit proteins produced

by the floor plate. There is no evidence, thus far, that Slit

proteins chemorepel cranial motor axons, however, and it

would be of particular interest to determine whether they can

repel oculomotor axons, for which as yet there are no

candidate chemorepellents.

Exit points are specific conduitsfor motor axon outgrowthAlthough all classes of motor neurons appear to be repelled by

the midline, other mechanisms must segregate motor axons

along their distinct dorsal and ventral routes. Spinal SM and

VM axons, and cranial SM axons forge a path into the ventral

mesenchyme close to the midline. By contrast, cranial BM

and VM axons grow dorsally for some distance within the

neuroepithelium before coalescing in groups towards large

single dorsal exit points within even-numbered rhombomeres.

Both dorsal and ventral exit points contain a cellular com-

ponent that is neural crest-derived.(53) An important distinction

Table 2. Summary table comparing effects of specific homeobox genes on the birth and differentiation of spinal

and cranial motor neurons

Loss of function Misexpression

Homeobox gene Expressed in... Spinal cord Brainstem Spinal cord Brainstem

Nkx6.1 Progenitors Loss of all Loss of Ectopic spinal

of all spinal motor neurons hindbrain motor neurons

motor neurons SM neurons

and cranial

SM neurons

MNR2 Progenitors of Ectopic SM neurons

SM neurons

HB9 Progenitors Interneuron Hindbrain Ectopic SM neurons

of SM neurons markers activated SM nuclei disrupted;

in motor neurons; axons misrouted

axons misrouted

Lhx3/4 Progenitors of SM Motor neurons Switch to Swtich to Swtich to cranial

neurons and migrate and BM/VM-like MMCM-like SM-like identity;

spinal VM neurons project dorsally identity; loss of identity loss of BM/VM

hindbrain neurons

SM neurons

Islet-1 All motor Early death Early death No effect

neurons of all motor of all motor

neurons neurons

Phox2a Midbrain/isthmic No effect Loss of

SM and midbrain

cranial BM/VM and isthmic

neurons SM neurons

Phox2b Midbrain/isthmic No effect Loss of cranial

SM and cranial BM/VM

MB/VM neurons neurons

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588 BioEssays 23.7

is that while ventral exit points contain only motor axons, dorsal

exit points provide common conduits for both outgoing motor

and incoming sensory axons which grow into the CNS from the

adjacent ganglia (Fig. 2). Is the dual passage of sensory and

motor axons important for the guidance of either axonal

population? In the chick embryo, trigeminal sensory ganglion

cells send axons into the hindbrain a few hours before the

lateral migration of BM neurons of the trigeminal nerve. Using

micromanipulation techniques, motor axonal outgrowth and

somatic translocation were demonstrated to be contingent on

the in-growth of sensory axons.(54) Perhaps then, at least one

mechanism of dorsal exit point facilitation of motor axon

outgrowth might operate indirectly, by bringing motor axons

into close proximity with sensory axons on which they rely for

their correct navigation. A more direct influence of dorsal exit

points on motor axon pathfinding is suggested by the rostral

growth of motor axons in r3 and r5 towards their appropriate

exit point following 180� rhombomere reversal in the chick.(55)

This suggests that exit points may also be the source of

diffusible guidance cues for navigating BM and VM growth

cones and, conceivably, might even confer specificity to axon

pathfinding at particular axial levels. Exit points might also be

the origin of contact-mediated guidance cues, for example the

cadherin, cad7, is expressed by the neural crest-derived cells

at the exit points.(56) In the case of the trochlear nerve, exit from

the neuroepithelium in the isthmic region appears to be

dependent on the neuropilin-2 receptor, since in mice mutant

for neuropilin-2, the trochlear nerve fails to exit the neu-

roepithelium correctly.(57±58) In vitro, trochlear aons were

found to be repelled by SEMA3F, which is a ligand for

Table 3. The response of motor axons to pathway tissues in the head and spinal cord

Head Spinal cord

Candidate CandidateMotor axons... Tissue molecules Tissues molecules

Are repelled by... Floor plate Netrin-1, Floor plate Netrin-1, SEMA3A,

SEMA3A slits

Grow towards... Exit points Exit points

Are segmented... Prior to leaving In the somite

the hindbrain

Grow through... Cranial paraxial Rostral sclerotome CSPG, PNA-

mesoderm binding proteins,

T-cadherin,

Ephrin- B1, B2, B4

Are sorted in the... Cranial sensory Paraxial mesoderm EphA7, NCAM

ganglia and and limb plexus

branchial arches

Grow into... Branchial arches HGF, SEMA3A Limbs HGF, EphA4

Figure 3. Developing hindbrain motor axons are chemor-

epelled by the floor plate and chemoattracted by the branchial

arches or HGF loaded beads A: An explant of unilateralhindbrain motor column has been cultured together with a

floor plate explant (FP) in a collagen gel and the axonal

outgrowth is shown in black immunostained using a pan-

neuronal antibody. Axons grow preferentially from the sidefacing away from the floor plate, reflecting repulsion. B: A

similar explant to that in (A) has been cultured alone, and

shows radial outgrowth. C: Axons from a bilateral ventral

hindbrain explant show enhanced and oriented outgrowthtowards branchial arch mesenchyme in cultrue. D: HGF

loaded beads mimic the outgrowth-promoting and orienting

effects of branchial arch tissue on axons emerging from abilateral ventral hindbrain explant.

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BioEssays 23.7 589

neuropilin-2, and is expressed in domains on either side of the

trochlear nerve's normal exit site.(57) The exit of other cranial

notor axons from the neuroepithelium was normal in the

neuropilin-2 mutants, although the oculomotor nerve showed

defasciculation.(57±58)

Guidance of motor axons within mesodermAt spinal levels, the adjacent mesoderm is divided into a series

of segmented tissue blocks, the somites, which become

partitioned into sclerotome and dermamyotome components.

Motor axons emerging from the spinal cord traverse the

sclerotomal component of the somite, which permits the

passage of axons only within the rostral half of this tissue

(Fig. 4A). Repulsive and attractive activities derived from

rostral and caudal halves of the sclerotome, respectively,

impose the periodic arrangement of motor nerves exiting from

the spinal cord.(59)

Multiple redundant mechanisms are responsible for the

repulsive activity. Peanut agglutinin binding glycoproteins

(PNA's), chondroitin sulphate proteoglycans and the Ca2�-

dependent glycoprotein, T-cadherin, are discretely expressed

in caudal sclerotome only.(60±62) The same studies also

showed that when these molecules are presented as sub-

strates to motor and sensory neurons in vitro, they cause

growth cone collapse. Specific function-blocking antibodies

abolish this activity, suggesting that inhibitory molecules

could prevent motor axon invasion of the caudal sclerotome

in vivo. The ephrin family of transmembrane and GPI-

anchored molecules have recently been implicated in barrier

repellent functions. In general, ephrin-A ligands bind EphA

receptors, whilst ephrin-B ligands bind EphB receptors. An

exception to this rule is EphA4 which binds ephrin-A in addition

to ephrin-B2 and ephrin-B3 ligands.(63) Members of this

family are involved in a number of developmental events,

including axon guidance.(63) Ephrin-B1 and ephrin-B4 in

the chick and ephrin-B2 in the mouse are restricted to the

caudal half of the sclerotome and can repel spinal motor

axons when these ligands are coated in stripes that

alternate with a permissive substratum such as laminin.(64)

The caudal sclerotome also prevents spinal nerves that

innervate the back muscles from crossing the dorsal midline.

SEMA3A produced by the sclerotome mediates this barrier

function. In mice lacking the SEMA3A gene, spinal nerves

cross the midline, breaching the sclerotome boundary.(65)

Thus, the available evidence suggests that repulsive cues

shape the segmental array of spinal motor axons. In vitro

studies, however, have also shown that the sclerotome

promotes the outgrowth of spinal motor axons, suggesting a

Figure 4. Schematic diagram of spinal and cranial motoraxon pathways in the chick embryo A. Spinal motor axon

pathways into the limb (adapted after [Tosney, 1991 #726]

Motor axons originating from different motor pools within the

cord grow through the rostral, and avoid the caudal (shadedgrey) part of the sclerotome, to form the spinal nerves. Sorting

occurs within the plexus region whereupon motor axons

segregate into either the dorsal (D) or ventral (V) nerve trunks

as they enter the limb. B. Lateral view showing projections ofBM/VM and SM cranial motor axons. Only the motor

projections (blue) to the branchial arches are shown. Branchial

nerves project in a segmental pattern to the corresponding

branchial arch (b1-4). The ventral pathways of the somaticmotor nerves (except the IVth nerve) are shown in red. Rostral

spinal nerves are shown in green. Abbreviations: t, telence-

phalon; d, diencephalon, m, mesencephalon, b1-b4, branchialarches 1 to 4.

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590 BioEssays 23.7

role for chemoattraction in the guidance of motor axons.(66)

Part of this in vitro activity is due to hepatocyte growth factor

(HGF), but the relevance of these findings is in some doubt

since HGF is not detectably expressed by the sclerotome in

vivo.(66) Further work is necessary to determine whether

attractant cues from rostral sclerotome are involved in the

guidance of developing motor axons. Leaving aside the

possible involvement of attractive cues, what is the signifi-

cance of multiple repulsive cues in the caudal sclerotome?

These different cues might act synergistically on motor axons

or multiple repulsive cues may be necessary to overcome an

intrinsic attractive activity within caudal sclerotome. Some

evidence for the latter comes from in vitro studies which have

shown that posterior sclerotome can chemoattract spinal

motor axons, although this activity appears to be masked

in vivo.(66)

In the head, the mesoderm adjacent to the neural tube is not

segmented rostral to the otic vesicle (Fig. 4B). The periodic

arrangement of the dorsally projecting branchial nerves arises

because of the segmented arrangement of rhombomeres and

their associated neural crest, which contribute to the formation

of cranial nerve roots.(67) Thus, mechanisms intrinsic to the

cranial neural tube dictate the patterning of branchial nerves,

in contrast to the spinal cord where mesoderm imposes

segmental motor nerve organisation.

Spinal motor axons grow along a system of`highways' into the limb but make pathwaychoices in specific `decision' regionsMany studies have used experimental manipulations to force

spinal motor axons into foreign territory and so challenge

axons to make pathfinding choices. The results of these

experiments broadly support a general scheme in which motor

axons are able to project along common `highways' yet can

respond to specific cues at `decision' regions.(67) If motor

axons are displaced too far from highways, they cannot find

the correct pathways.(69) Highway boundaries are demarcated

by the repulsive molecule SEMA3A which `hems in' the growth

of spinal nerves along stereotyped trajectories. Disruption of

SEMA3A function in transgenic mice results in the ectopic

growth and branching of spinal nerves into inappropriate

territory such as cartilaginous structures.(65)

In the chick embryo, LMC motor axons destined for the limb

express ephrin-A5, and converge at the limb base, which

expresses the cognate receptor EphA7.(70) It is here that motor

axons belonging to a common motor pool are brought together

in a region termed the plexus.(71) Extinguishing EphA7 exp-

ression blocks the convergence of axons at the plexus.(70)

Within the plexus region axon trajectories are highly indivi-

dualistic, with many abrupt turns, perhaps reflecting a process

of active sorting.(72) The axon defasciculation and refascicula-

tion that must occur to permit the rearrangement of motor

axons in the plexus, depends on the biochemical modification

of NCAM on motor axons, by the addition or removal of

polysialic acid residues.(73)

Cranial sensory ganglia are important decisionpoints in cranial motor axon pathfindingCranial sensory ganglia are positioned along the course of the

dorsally-projecting branchial cranial nerves (Fig. 2). Here too,

as in the limb plexus region, a process of motor axon sorting

occurs, with axons segregating into distinct branchial or

visceral motor pathways distal to the ganglia. In vivo place-

ment of impermeable barriers between the trigeminal ganglion

and the exit point, before motor axon outgrowth, results in

trigeminal motor axons tracing aberrant pathways around the

barrier to reach the ganglion. Moreover, only those axons that

make contact with the ganglion are able subsequently to reach

their target branchial arch.(74) Rhombomere transplantation

experiments have shown that ectopically displaced motor

neuron subpopulations are able to reroute their axons towards

the appropriate sensory ganglion, suggesting that specific

ganglion derived cues are differentially recognised by distinct

cranial motor neuron subtypes.(38,75) However, in vitro, cranial

motor axons with different axial origins show enhanced growth

towards the trigeminal ganglion, although the magnitude of

the attractive response is modest.(76±77) To date, candidate

chemoattractants that might mediate the effect of the cranial

sensory ganglia on cranial motor axon growth have not been

identified.

Guidance of motor axons into their target zonesis mediated by growth factors which act aschemoattractantsA considerable body of evidence points to the involvement of

limb-derived diffusible factors in the guidance of spinal motor

axons to their eventual targets within the limb in mammals, or

the fin in fish. Ectopic transplantation of fin buds to a more

posterior axial level results in the rerouting of axon trajectories

to the base of the ectopic fin. Moreover, grafting additional fins

attracts motor axons into both the ectopic fin and the host

fin.(78) Explants of the spinal cord of tadpoles show directed

outgrowth to limb mesenchymal tissue, which varies inversely

with increasing separation of the two tissues.(79) Finally, the

latter study also showed that limb-conditioned medium signi-

ficantly enhances outgrowth from tadpole spinal cord explants.

The chemoattractive activity of limb tissue is mediated by

hepatocyte growth factor (HGF) and can be neutralised by

antibodies against HGF. In addition, motor axons express the

HGF receptor, c-met when they invade the limb and HGF

is present in the tissue environment at these stages.(66)

Recently, HGF produced by the branchial arches was found to

be growth-promoting and chemoattractive for cranial motor

axons (Fig. 3C,D).(77) Therefore, a single chemoattractant,

HGF is implicated in the guidance of multiple different motor

neuron subtypes along the neuraxis. Is HGF in fact necessary

Review articles

BioEssays 23.7 591

for motor axon guidance? Interestingly, the formation of major

nerve branches in the limbs of HGF knockout mice are intact,

although certain muscle branches are missing.(66) Likewise, in

the cranial region, the innervation of the branchial arches is

preserved, but the somatic motor nerve supply to the tongue is

truncated.(77) A plethora of other growth factors have been

shown to promote the survival and growth of motor neurons,(80)

raising the possibility that some of these factors might also

function as chemoattractants, either fully or partially compen-

sating for the lack of HGF. Trigeminal sensory axons are

chemoattracted by NT-3 and BDNF, which are expressed by

the branchial arches,(81) but the effects of these molecules on

developing cranial motor axons has not been tested. The

branchial arches are not only a source of chemoattractants, but

also produce at least one chemorepellent mentioned earlier,

SEMA3A, which is needed to channel the growth of branchial

nerves into their termination zone. Targeted disruption of

SEMA3A function in mice leads to defasciculation of motor

axons which become spread over a wider area, but the axons

nevertheless terminate in broadly correct target regions.(65)

Role of the target in the axon pathfindingof VM neuronsRecent investigations are beginning to shed light on the cues

responsible for VM axon pathfinding. Cranial parasympathetic

axons exit the neural tube dorsally and project, via cranial

sensory ganglia, to their parasympathetic ganglion targets,

whereas spinal sympathetic axons exit the cord ventrally and

project to sympathetic ganglia. A number of observations

suggest that cranial VM neuron populations require the

presence of the synaptic target for their navigation. First, in

transgenic mice which lack the target of facial VM axons,

namely the sphenopalatine ganglion, these axons fail to

project correctly showing a ``stalling phenotype.'' Second,

facial VM neurons transposed to ectopic locations pathfind to

the sphenopalatine ganglion via novel routes.(75) Finally, in

preliminary experiments, motor axons from cultured explants

of hindbrain neuroepithelium containing VM neurons grow

preferentially towards parasympathetic ganglion tissue, thus

implicating chemoattraction as a possible guidance mechan-

ism (Jacob and Guthrie, unpublished data). By contrast, spinal

VM axons, which share a common functional identity with

cranial VM axons, do not appear to require the presence of the

target to navigate to their eventual destination, instead relying

on cues from the somites for their guidance.(82,83)

Pathway selection at decision points isinfluenced by cell autonomous andnon-cell autonomous factorsThe development of proper patterns of neuromuscular

connectivity critically depends on motor axons choosing

the appropriate pathways at decision regions. On emerging

from the plexus, motor axons of the LMCM and LMCL

segregate at the limb base to form distinct ventral and dorsal

nerve trunks, respectively (Fig. 5). In transgenic mice with

targeted inactivation of the LMCL marker gene, Lim1, motor

axons make a random choice between dorsal and ventral

pathways in the limb.(18) A similar phenotype is obtained

upon inactivation of another LIM domain gene, Lmx1b,

which is normally expressed by dorsal mesenchyme cells in

the limb.(18) These findings in vertebrates complement the

earlier discovery in Drosophila that certain LIM genes direct

motor axon trajectories, presumably by regulating the cellular

signalling events involved in the differential recognition of and

response to specific axon guidance cues.(84)

Dorsoventral axon trajectories in the limb are also affected

in transgenic mice with a null mutation of the EphA4 gene

(Fig. 5). EphA4 is strongly expressed by dorsally-projecting

LMCL axons and its absence leads to the misprojection of all

LMCL motor axons into the ventral pathway.(85) The aberrant

innervation pattern may be due to the loss of normal repulsive

interactions between EphA4 and its cognate ligands, ex-

pressed in the ventral compartment, which could normally

prevent ingrowth by LMCL axons.(86) The more interesting

implication, recognised by Helmbacher et al., is the existence

of ventral cues in the limb that are potentially attractive for all

LMC axons. Nevertheless, spatially co-extensive repulsive

Eph-ephrin forces normally prevail, to keep LMCL axons out of

the ventral limb compartment. An obvious question is whether

EphA4 expression in LMCL axons is positively regulated

by Lim-1. The answer could be provided by examining the

expression of EphA4 in LMC neurons of Lim1ÿ /ÿ mice. Such

regulation, however, would not be sufficient to explain the

randomisation of dorsoventral projections that takes place

in the absence of Lim1. More likely, as Helmbacher et al.

postulate, Lim-1 regulates growth cone responsiveness to

both ventrally and dorsally channeling cues.

Once individual motor axons are in the vicinity of their target

muscle, they must not only distinguish their target from other

nearby muscles but must also synapse with the appropri-

ate muscle fibre. Motor neurons that innervate muscle are

matched with their target in such a way as to generate a

precise, topographic map.(87) This feat of precision guidance

relies again on Eph-ephrin interactions, as at least one of the

mechanisms. Ephrin-A subfamily members are differentially

expressed on muscles along the A-P axis during develop-

ment,(88) and subsets of motor axons express at least three

cognate Eph receptors.(89,90) Selective activation or inactiva-

tion of members of this group of ephrins through genetic gain-

of-function and loss-of-function experiments results in disrup-

tion of the topographic patterns of muscle innervation at the

synaptic level.(88)

Conclusions

A wealth of data has accumulated on the specification and

guidance mechanisms of motor axons, which began with the

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592 BioEssays 23.7

systematic characterisation of motor neuron subtypes and

their projection patterns. Neural progenitors at varying

positions along the rostrocaudal and dorsoventral axes

detect graded inductive signals from midline and adjacent

non-neural tissues at distinct concentration thresholds.

Spatially distinct progenitor populations interpret such

graded signals by all-or-none activation or repression of

specific patterning genes, which define future neuronal

subtypes including motor neurons. The consolidation and

subsequent refinement of motor neuron identity depends

on the cell autonomous and/or non-cell autonomous

regulation of transcription factor activity in committed motor

neuron precursors. At cranial levels, Hox genes are instru-

mental in diversifying segmentally repeated motor neuron

subsets that are necessary to subserve highly specialised

functions.

Post-mitotic, differentiated motor neurons pathfind to their

targets by relying on attractive and repulsive short- or long-

range guidance cues which are discretely and serially de-

ployed in tissues along the pathway to the target. The guidance

of both cranial and spinal motor axon populations to muscle

targets share certain features. First, motor neurons at all levels

of the neuraxis are responsive to midline chemorepellents.

Second, signals from exit points may be important for motor

axon growth out of the neural tube. Third, the same target-

derived chemoattractant, HGF is involved in the guidance of

spinal and cranial motor axons. Important differences be-

tween these two populations include the mechanisms used

to segment spinal and cranial motor nerves and the chemo-

attractive guidance of cranial, but not spinal motor axon sub-

populations by intermediate ganglionic targets, the cranial

sensory ganglia.

Altogether, recent investigations of motor neuronal speci-

fication and motor axon navigation have converged on the

involvement of specific transcription factors in the regulation of

both these processes. These discoveries concerning tran-

scriptional regulation of motor axon pathfinding open an excit-

ing new chapter in motor axon pathfinding mechanisms. An

important next step will be the identification of the actual

downstream targets of homeodomain transcription factors.

These may include novel axon guidance receptors and signal

transduction pathways.

Figure 5. Regulation of motor axon pathfinding within the limb Axon pathways of motor neurons in the LMCM (shown in green) and

LMCL (shown in blue) in wild-type mice, LIM1 mutants, Lmx1b mutants and EphA4 mutants. Lmx1b is expressed selectively by

mesenchyme cells in the dorsal part of the limb bud. In wild-type animals, LMCM axons project into the ventral motor nerve branch,

whereas LMCL neurons send their axons into the dorsal branch. In the absence of LIM1, motor axons of the LMCL appear to projectrandomly into either the dorsal or ventral motor nerve branches within the limb bud. Subsequently, LMCL axons retract from the limb

(shown as dashed blue line). The LMCM axon projection is unaffected in LIM1 mutants. In Lmx1b mutants, both LMCL and LMCM axons

extend into dorsal and ventral nerve branches. EphA4 mutants lack the dorsal nerve trunk and all LMCL axons grow into the ventral

compartment of the limb.

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BioEssays 23.7 593

Acknowledgments

We are grateful to J.F. Brunet, Y. Zhu and A. Varela-

EchavarrõÂa for comments on the manuscript and helpful

discussions.

References1. Mueller BK. Growth cone guidance: first steps towards a deeper

understanding. Annu Rev Neurosci 1999;22:351±388.

2. Landmesser L. The development of motor projection patterns in the

chick hind limb. J Physiol (Lond) 1978;284:391±414.

3. Puelles-Lobez L, Malagon-Cobos F, Genis-Galvez JM. The migration of

oculomotor neuroblasts across the midline in the chick embryo. Exp

Neurol 1975;47:459±469.

4. Ericson J, Briscoe J, Rashbass P, van Heyningen V, Jessell TM. Graded

sonic hedgehog signaling and the specification of cell fate in the ventral

neural tube. Cold Spring Harb Symp Quant Biol 1997;62:451±466.

5. Briscoe J, Pierani A, Jessell TM, Ericson J. A homeodomain protein code

specifies progenitor cell identity and neuronal fate in the ventral neural

tube. Cell 2000;101:435±445.

6. Sander M, Paydar S, Ericson J, Briscoe J, Berber E, German M, Jessell

TM, Rubenstein JL. Ventral neural patterning by Nkx homeobox genes:

Nkx6.1 controls somatic motor neuron and ventral interneuron fates.

Genes Dev 2000;14:2134±2139.

7. Briscoe J, Sussel L, Serup P, Hartigan-O'Connor D, Jessell TM,

Rubenstein JL, Ericson J. Homeobox gene Nkx2.2 and specification of

neuronal identity by graded Sonic hedgehog signalling. Nature 1999;

398:622±627.

8. Muhr J, Andersson E, Persson M, Jessell T, Ericson J. Groucho-

mediated transcriptional repression establishes progenitor cell pattern

and neuronal fate in the ventral neural tube. Cell 2001;104:861±873.

9. Jessell TM. Neuronal specification in the spinal cord: inductive signals

and transcriptional codes. Nature Rev Genet 2000;1:20±29.

10. Tanabe Y, William C, Jessell TM. Specification of motor neuron identity

by the MNR2 homeodomain protein. Cell 1998;95:67±80.

11. Arber S, Han B, Mendelsohn M, Smith M, Jessell TM, Sockanathan S.

Requirement for the homeobox gene Hb9 in the consolidation of motor

neuron identity. Neuron 1999;23:659±674.

12. Thaler J, Harrison K, Sharma K, Lettieri K, Kehrl J, Pfaff SL. Active

suppression of interneuron programs within developing motor neurons

revealed by analysis of homeodomain factor HB9 [see comments].

Neuron 1999;23:675±687.

13. Tsuchida T, Ensini M, Morton SB, Baldassare M, Edlund T, Jessell TM,

Pfaff SL. Topographic organisation of embryonic motor neurons defined

by expression of LIM homeobox genes. Cell 1994;79:957±970.

14. Ericson J, Thor S, Edlund T, Jessell TM, Yamada T. Early stages of motor

neuron differentiation revealed by expression of homeobox gene Islet-1.

Science 1992;256:1555±1560.

15. Pfaff SL, Mendelsohn M, Stewart CL, Edlund T, Jessell TM. Requirement

for LIM homeobox gene Isl-1 in motor neuron generation reveals a

motor neuron-dependent step in interneuron diffentiation. Cell 1996;84:

309±320.

16. Sharma K, Sheng HZ, Lettieri K, Li H, Karavanov A, Potter S, Westphal H,

Pfaff SL. LIM homeodomain factors Lhx3 and Lhx4 assign subtype

identities for motor neurons. Cell 1998;95:817±828.

17. Sharma K, Leonard AE, Lettieri K, Pfaff SL. Genetic and epigenetic

mechanisms contribute to motor neuron pathfinding. Nature 2000;406:

515±519.

18. Kania A, Johnson RL, Jessell TM. Coordinate roles for LIM homeobox

genes in directing the dorsoventral trajectory of motor axons in the

vertebrate limb [In Process Citation]. Cell 2000;102:161±173.

19. Sockanathan S, Jessell TM. Motor-neuron derived retinoid signalling

specifies the subtype identity of spinal motor neurons. Cell 1998;94:503±

514.

20. Landmesser L. The distribution of motoneurones supplying chick hind

limb muscles. J Physiol (Lond) 1978;284:371±389.

21. Lin JH, Saito T, Anderson DJ, Lance-Jones C, Jessell TM, Arber S.

Functionally related motor neuron pool and muscle sensory afferent

subtypes defined by coordinate ETS gene expression [see comments].

Cell 1998;95:393±407.

22. Dou C, Ye X, Stewart C, Lai E, Li SC. TWH regulates the development of

subsets of spinal cord neurons. Neuron 1997;18:539±551.

23. Ericson J, Rashbass P, Schedl A, Brenner-Morton S, Kawakami A, van

HV, Jessell TM, Briscoe J. Pax 6 controls progenitor cell identity and

neuronal fate in response to graded Shh signaling. Cell 1997;90:169±

180.

24. Osumi N, Hirota A, Ohuchi H, Nakafuku M, Tadahiro I, Kuratani S,

Fujiwara M, Noji S, Kazuhiro E. Pax-6 is involved in the specification of

hindbrain motor neuron subtype. Development 1997;124:2961±2972.

25. Valarche I, Tissier-Seta JP, Hirsch MR, Martinez S, Goridis Cm,

Brunet JF. The mouse homeodomain protein Phox2 regulates Ncam

promoter activity in concert with Cux/CDP and is a putative determin-

ant of neurotransmitter phenotype. Development 1993;119:881±896.

26. Pattyn A, Morin X, Cremer H, Goridis C, Brunet JF. Expression and

interactions of the two closely related homeobox genes Phox2a and

Phox2b during neurogenesis. Development 1997;124:4065±4075.

27. Morin X, Cremer H, Hirsch MR, Kapur RP, Goridis C, Brunet JF. Defects

in sensory and autonomic ganglia and absence of locus coeruleus in

mice deficient for the homeobox gene Phox2a. Neuron 1997;18:411±

423.

28. Varela-Echavarria A, Pfaff SL, Guthrie S. Differential expression of LIM

homeobox genes among motor neuron subpopulations in the developing

chick brain stem. Mol Cell Neuroscience 1996;8:242±257.

29. Pattyn A, Morin X, Cremer H, Goridis C, Brunet JF. The homeobox gene

Phox2b is essential for the development of autonomic neural crest

derivatives. Nature 1999;399:366±370.

30. Pattyn A, Hirsch M, Goridis C, Brunet JF. Control of hindbrain motor

neuron differentiation by the homeobox gene Phox2b. Development

2000;127:1349±1358.

31. Dubreuil V, Hirsch M, Pattyn A, Brunet J, Goridis C. The Phox2b

transcription factor coordinately regulates neuronal cell cycle exit and

identity. Development 2000;127:5191±5201.

32. Tiveron MC, Hirsch MR, Brunet JF. The expression pattern of the

transcription factor Phox2 delineates synaptic pathways of the auto-

nomic nervous system. J Neurosci 1996;16:7649±7660.

33. Jacob J, Tiveron MC, Brunet JF, Guthrie S. Role of the target in the

pathfinding of facial visceral motor axons. Mol Cell Neurosci 2000;16:

14±26.

34. Lumsden A, Keynes R. Segmental patterns of neuronal development in

the chick hindbrain. Nature 1989;337:424±428.

35. Keynes R, Krumlauf R. Hox genes and regionalization of the nervous

system. Ann Rev Neurosci 1994;17:109±132.

36. Goddard JM, Rossel M, Manley NR, Capecchi MR Mice with targeted

disruption of hoxb-1 fail to form the motor nucleus of the VIIth nerve.

Development 1996;122:3217±3228.

37. Studer M, Lumsden A, Ariza-McNaughton L, Bradley A, Krumlauf, R.

Altered segmental identity and abnormal migration of motor neurons in

mice lacking Hoxb-1. Nature 1996;384:630±634.

38. Bell E, Wingate RJ, Lumsden A. Homeotic transformation of rhombomere

identity after localized Hoxb1 misexpression. Science 1999;284:2168±

2171.

39. Jungbluth S, Bell E, Lumsden A. Specification of distinct motor neuron

identities by the singular activities of individual Hox genes. Development

1999;126:2751±2758.

40. Ensini M, Tsuchida TN, Belting HG, Jessell TM. The control of rostro-

caudal pattern in the developing spinal cord: specification of motor

neuron subtype identity is initiated by signals from paraxial mesoderm.

Development 1998;125:969±982.

41. Grapin-Botton A, Bonnin MA, McNaughton LA, Krumlauf R, Le Douarin

NM. Plasticity of transposed rhombomeres: Hox gene induction is corre-

lated with phenotypic modifications. Development 1995;121:2707±2721.

42. Itasaki N, Sharpe J, Morrison A, Krumlauf R. Reprogramming Hox

expression in the vertebrate hindbrain: influence of paraxial mesoderm

and rhombomere transposition. Neuron 1996;16:487±500.

43. Guthrie S, Muchamore I, Kuroiwa A, Marshall H, Krumlauf R, Lumsden A.

Neuroectodermal autonomy of Hox-2.9 expression revealed by rhombo-

mere transpositions. Nature 1992;356:157±159.

44. Simon H, Hornbruch A, Lumsden A. Independent assignment of antero-

posterior and dorso-ventral positional values in the developing chick

hindbrain. Curr Biol 1995;5:205±214.

Review articles

594 BioEssays 23.7

45. Tessier-Lavigne M, Goodman CS. The molecular biology of axon

guidance. Science 1996;274:1123±1133.

46. Guthrie S, Pini A. Chemorepulsion of developing motor axons by the floor

plate. Neuron 1995;14:1117±1130.

47. Varela-Echavarria A, Guthrie S. Molecules making waves in axon

guidance. Genes Dev 1997;11:545±557.

48. Brose K, Tessier-Lavigne M. Slit proteins: key regulators of axon

guidance, axonal branching, and cell migration. Curr Opin Neurobiol

2000;10:95±102.

49. Colamarino SA, Tessier-Lavigne M. The axonal chemoattractant netrin-1

is also a chemorepellent for trochlear motor axons. Cell 1995;81:621±

629.

50. Varela-Echavarria A, Tucker A, P&schel AW, Guthrie S. Motor axon

subpopulations respond differentially to the chemorepellents netrin-1

and semaphorin D. Neuron 1997;18:193±207.

51. Kidd T, Bland KS, Goodman CS, Slit is the midline repellent for the robo

receptor in Drosophila. Cell 1999;96:785±794.

52. Brose K, Bland KS, Wang KH, Arnott D, Henzel W, Goodman CS,

Tessier-Lavigne M, Kidd T. Slit proteins bind Robo receptors and have

an evolutionarily conserved role in repulsive axon guidance. Cell

1999;96:795±806.

53. Niederlander C, Lumsden C. Late emigrating neural crest cells migrate

specifically to the exit points of cranial branchiomotor nerves. Develop-

ment 1996;122:2367±2374.

54. Moody SA, Heaton MB. Developmental relationships between trigeminal

ganglia and trigeminal motoneurons in chick embryos. II. Ganglion axon

ingrowth guides motoneuron migration. J Comp Neurol 1983;213:344±

349.

55. Guthrie S, Lumsden A. Motor neuron pathfinding following rhombomere

reversals in the chick embryo hindbrain. Development 1992;114:663±

673.

56. Nakagawa S, Takeichi M. Neural crest cell-cell adhesion controlled by

sequential and subpopulation-specific expression of novel cadherins.

Development 1995;121:1321±1332.

57. Giger RJ, Cloutier J-F, Sahay A, Prinjha RK, Levengood DV, Moore SE,

Pickering S, Simmons D, Rastan S, Walsh FS, Kolodkin AL, Ginty DD,

Geppert M. Neuropilin-2 is required in vivo for selective axon guidance

responses to secreted semaphorins. Neuron 2000;25:29±41.

58. Chen H, Bagri A, Zupcich JA, Zou Y, Stoeckli E, Pleasure SJ, Lowenstein

DH, Skarnes WC, CheÂdotal A, Tessier-Lavigne M. Neuropilin-2 regulates

the development of select cranial and sensory nerves and hippocampal

mossy fibre projections. Neuron 2000;25:43±56.

59. Keynes RJ, Stern CD. Segmentation in the vertebrate nervous system.

Nature 1984;310:786±789.

60. Davies JA, Cook GM, Stern CD, Keynes RJ. Isolation from chick somites

of a glycoprotein fraction that causes collapse of dorsal root ganglion

growth cones. Neuron 1990;4:11±20.

61. Oakley RA, Tosney KW. Contact-mediated mechanisms of motor axon

segmentation. The Journal of Neuroscience 1993;13:3773±3792.

62. Fredette BJ, Miller J, Ranscht B. Inhibition of motor axon growth by

T-cadherin substrata. Development 1996;122:3163±3171.

63. Flanagan JG, Vanderhaeghen P. The ephrins and Eph receptors in

neural development. Annu Rev Neurosci 1998;21:309±345.

64. Wang HU, Anderson DJ. Eph family transmembrane ligands can

mediate repulsive guidance of trunk neural crest migration and motor

axon outgrowth. Neuron 1997;18:383±396.

65. Taniguchi M, Yuasa S, Fujisawa H, Naruse I, Saga S, Mishina M, Yagi T.

Disruption of semaphorin III/D gene causes severe abnormality in

peripheral nerve projection. Neuron 1997;19:519±530.

66. Ebens A, Brose K, Leonardo ED, Gartz HJM, Bladt F, Birchmeier C,

Barns BA, Tessier-Lavigne M. Hepatocyte Growth Factor/Scatter Factor

is an axonal chemoattractant and neurotrophic factor for spinal motor

neurons. Neuron 1996;17:1157±1172.

67. Kuratani SC, Eichele G. Rhombomere transplantation repatterns the

segmental organisation of cranial nerves and reveals cell-autonomous

expression of a homeodomain protein. Development 1993;117:105±117.

68. Landmesser L. The development of specific motor pathways in the chick

embryo. Trends Neurosci 1984;7:336±339.

69. Hollyday M. Rules of motor innervation in chick embryos with super-

numary limbs. Journal of Comparative Neurology 1981;202:439±

465.

70. Araujo M, Piedra ME, Herrera MT, Ros MA, Nieto MA. The expression

and regulation of chick EphA7 suggests roles in limb patterning and

innervation. Development 1998;125:4195±4204.

71. Lance-Jones C, Landmesser L. Pathway selection by embryonic chick

motoneurons in an experimentally altered environment. Proc R Soc Lond

B Biol Sci 1981;214:19±52.

72. Tosney KW, Landmesser LT. Growth cone morphology and trajectory in

the lumbosacral region of the chick embryo. J Neurosci 1985;5:2345±

2358.

73. Tang J, Landmesser L, Rutishauser U. Polysialic acid influences specific

pathfinding by avian motoneurons. Neuron 1992;8:1031±1044.

74. Moody SA, Heaton MB. Developmental relationships between trigeminal

ganglia and trigeminal motoneurons in chick embryos. III. Ganglion

perikarya direct motor axon growth in the periphery. J Comp Neurol

1983;213:350±364.

75. Jacob J, Guthrie S. Facial visceral motor neurons display specific

rhombomere origin and axon pathfinding behavior in the chick.

J Neurosci 2000;20:7664±7671.

76. Tucker A, Lumsden A, Guthrie S. Cranial motor axons respond differently

to the floor plate and sensory ganglia in collagen gel co-cultures. Eur J

Neurosci 1996;8:906±916.

77. Caton A, Hacker A, Naeem A, Livet J, Maina F, Bladt F, Klein R,

Birchmeier C, Guthrie S. The branchial arches and HGF are growth-

promoting and chemoattractant for cranial motor axons. Development

2000;127:1751±1766.

78. Okamoto H, Kuwada JY. Alteration of pectoral fin nerves following

ablation of fin buds and by ectopic fin buds in the Japanese medaka

fish. Dev Biol 1991;146:62±71.

79. Pollack ED, Muhlach WL, Liebig V. Neurotropic influence of mesench-

ymal limb target tissue on spinal cord neurite growth in vitro. J Comp

Neurol 1981;200:393±405.

80. Henderson CE. Role of neurotrophic factors in neuronal development.

Curr Opin Neurobiol 1996;6:64±70.

81. O'Connor R, Tessier-Lavigne M. Identification of maxillary factor, a

maxillary process-derived chemoattractant for developing trigeminal

sensory axons. Neuron 1999;24:165±178.

82. Yip JW. Target cues are not required for the guidance of sympathetic

preganglionic axons. Brain Res 1987;429:155±159.

83. Yip JW. Specificity of sympathetic preganglionic projections in the chick

is influenced by the somitic mesoderm. J Neurosci 1996;16:612±

620.

84. Thor S, Andersson SG, Tomlinson A, Thomas JB. A LIM-homeodomain

combinatorial code for motor-neuron pathway selection. Nature 1999;

397:76±80.

85. Helmbacher F, Schneider-Maunoury S, Topilko P, Tiret L, Charnay P.

Targeting of the EphA4 tyrosine kinase receptor affects dorsal/

ventral pathfinding of limb motor axons. Development 2000;127:3313±

3324.

86. Ohta K, Iwamasa H, Drescher U, Terasaki H, Tanaka H. The inhibitory

effect on neurite outgrowth of motoneurons exerted by the ligands ELF-1

and RAGS. Mech Dev 1997;64:127±135.

87. Laskowski MB, Sanes JR. Topographic mapping of motor pools onto

skeletal muscles. J Neurosci 1987;7:252±260.

88. Feng G, Laskowski MB, Feldheim DA, Wang H, Lewis R, Frisen J,

Flanagan JG, Sanes JR. Roles for ephrins in positionally selective

synaptogenesis between motor neurons and muscle fibers. Neuron

2000;25:295±306.

89. Kilpatrick TJ, Brown A, Lai C, Gassmann M, Goulding M, Lemke G.

Expression of the Tyro4/Mek4/Cek4 gene specifically marks a subset of

embryonic motor neurons and their muscle targets. Mol Cell Neurosci

1996;7:62±74.

90. Ohta K, Nakamura M, Hirokawa K, Tanaka S, Iwama A, Suda T, Ando M,

Tanaka H. The receptor tyrosine kinase, Cek8, is transiently expressed

on subtypes of motoneurons in the spinal cord during development.

Mech Dev 1996;54:59±69.

Review articles

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