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Mechanisms of Development 121 (2004) 1103–1115
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Review
The generation and diversification of spinal motor
neurons: signals and responses
Stephen R. Pricea,*, James Briscoeb,*
a
Department of Anatomy and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK
b
Developmental Neurobiology, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK
Received 6 April 2004; received in revised form 26 April 2004; accepted 26 April 2004
Abstract
Motor neurons are probably the best characterised neuronal class in the vertebrate central nervous system and have become a paradigm for
understanding the mechanisms that control the development of vertebrate neurons. For many investigators working on this problem the chick
embryo is the model system of choice and from these studies a picture of the steps involved in motor neuron generation has begun to emerge.
These findings suggest that motor neuron generation is shaped by extracellular signals that regulate intrinsic, cell-autonomous determinants
at sequential steps during development. The chick embryo has played a prominent role in identifying the sources of these signals, defining
their molecular identities and determining the cell intrinsic programs they regulate.
q 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Motor neurons; Chick; Extrinsic factors; Intrinsic factors; Spinal cord
1. Introduction
Systematic studies on the development of the spinal cord
of the chick can be traced back at least to the late 1850s
(Clarke, 1862; Bidder and Kupffer, 1857; Kolliker, 1861
quoted in Clarke, 1862). Throughout the 20th century, the
experimentally tractable nature of chick embryos (Lillie,
1908 quoted in Cowan, 2001) and the detailed and extensive
description of the morphological stages in its development
(Hamburger and Hamilton, 1951) resulted in it remaining
the model system of choice for many investigators. These
studies have included seminal work such as the classic
embryological neuroanatomical studies of Santiago Ramon
y Cajal (1894, 1906, 1911) and the ground breaking work of
Rita Levi-Montalcini and Viktor Hamburger which led to
the discovery of Nerve Growth Factor as a peripheral signal
critical to the survival of motor neurons (Hamburger and
Levi-Montalcini, 1949 and see references within Cowan,
2001). In this review, we focus on the generation
and diversification of motor neuron subtype identity,
* Corresponding authors. Tel.: þ 44-20-7679-3884; fax: þ 44-207679-7349 (S.R. Price), Tel.: þ44-20-8816-2559; fax: þ 44-20-88162523 (J. Briscoe).
E-mail addresses: [email protected] (S.R. Price), james.briscoe@
nimr.mrc.ac.uk (J. Briscoe).
0925-4773/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mod.2004.04.019
emphasising the contributions that studies of the chick
embryo have made in these endeavours. We attempt to put
the results of the past decade on the elucidation of the
identity of extracellular signals and cell intrinsic programs
that regulate motor neuron development in the context of the
seminal contributions made previously. However, for any
failures to do this adequately we must apologise at the
outset.
Spinal somatic motor neurons are easily identifiable, as
one of only a few neuronal types that project axons out of
the CNS and their activity is readily recorded through their
output—the contraction of a muscle. Consequently, motor
neurons represent probably the best functionally and
molecularly characterized neuronal subtype in vertebrates.
The process of specifying motor neuron identity can be
thought of as encompassing three stages (Jessell, 2000).
First, the specification of generic motor neuron identity
leads to the generation of motor neurons in the appropriate
position in the spinal cord. Second, the soma of functionally
related groups of motor neurons that are destined to share
common projection targets settle in longitudinally oriented
columns in the spinal cord and their axons project towards
their target regions. Third, the cell bodies of motor neurons
that innervate the same muscle form clusters known as
motor pools and both pre- and post-synaptic connections are
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made. Each of these steps involves extracellular signals that
regulate intrinsic, cell-autonomous determinants of motor
neuron identity, which together define and shape motor
neuron development. Identifying the sources of these
signals, defining their molecular identities and determining
the cell intrinsic programs they regulate has been the major
focus of research over the last century and the chick embryo
has played a key, if not dominant, role in these studies.
2. Specification of generic motor neuron identity
Neurogenesis in the spinal cord proceeds with bilateral
symmetry generating distinct cell types at different positions
along the dorsal ventral axis of the neural tube (Clarke,
1862). Motor neurons are produced in a ventrally restricted
region of the spinal cord (see Ramón y Cajal, 1906) and are
generated earlier in development than more dorsal neuronal
subtypes resulting in an emerging reduction in the number
of ventral neuroblasts. Consequently the ventricular zone is
thicker at the dorsal midline than at the ventral midline.
These observations suggested that the dorsal – ventral
position of a progenitor cell in the neural tube determined
the progeny it generates and raised the question of what
signals controlled the emergence of this pattern. A series of
studies focused attention on the notochord—the rod of axial
mesodermal cells that underlies the neural tube—and the
floor plate—a group of specialised glial cells occupying the
ventral midline of the neural tube in close proximity to MNs
(reviewed in Placzek et al., 1991). It was noted, in a chick
which had a spontaneously duplicated notochord positioned
next to a single spinal cord, that floor plate like structures
developed next to each notochord (Watterson et al., 1955).
This observation was consistent with previous experimental
analyses of amphibian development which indicated that
mesodermal structures adjacent to the neural tube influenced neural development. Subsequently, embryological
manipulations of chick tissue in vivo provided support for
this idea: grafting an ectopic notochord next to the neural
tube resulted in the induction of floor plate cells adjacent to
the graft (van Straaten et al., 1985a,b) while the floor plate
appeared to be lost in embryos in which the notochord had
been removed (van Straaten et al., 1987). These data
indicated that the notochord was capable of inducing floor
plate identity, however, further progress was hampered by
the reliance on cell morphology to distinguish distinct cell
types. It was the characterisation of antigens and molecular
markers selectively expressed by floor plate cells and/or
motor neurons that removed this limitation and provided the
opportunity to examine in detail the role of the notochord
and floor plate in the control of ventral neural tube
patterning (Placzek et al., 1991; Yamada et al., 1991).
These studies indicated that motor neuron differentiation
was abrogated by removal of the notochord and floor plate,
conversely, grafting a supernumerary notochord or floor
plate lateral to the neural tube resulted in the generation of
ectopic floor plate cells and motor neurons in place of the
normal cell differentiation program. Moreover, careful
analysis demonstrated that ectopic floor plate cells differentiated immediately adjacent to the grafted tissue while
motor neurons were located at a discrete distance, separated
from floor plate by an intervening strip of cells (Yamada
et al., 1991). Together, these experiments indicated that the
notochord was not only sufficient to induce floor plate
differentiation but also had instructive roles in establishing
the identity and position of motor neuron generation.
Whether the floor plate is normally induced by signals
emanating from the notochord has become a matter of some
controversy as Teillet et al. (1998) using chick-quail
chimeras provided evidence that floor plate cells are derived
from axial mesodermal cells and inserted into the neural
tube as Hensen’s node regresses. This suggested that floor
plate cells have a shared lineage with notochord and raised
the possibility that notochord signals were not necessary for
floor plate induction. Arguments for and against each of
these models have been discussed elsewhere (Le Douarin
and Halpern, 2000; Placzek et al., 2000).
In order to define more clearly the signals necessary for
motor neuron generation an in vitro explant culture system
was developed in which a region of naı̈ve neural plate
equidistant from the ventral and dorsal midlines was
removed and cultured in vitro for a number of days
(Yamada et al., 1993). This technique became an important
tool for the characterisation of patterning signals and
illustrates a number of the strengths of the chick system
such as the accessibility of chick embryos, the relative ease
of its micro-dissection and the ability to grow naı̈ve neural
tissue in vitro in serum-free defined medium. With this
approach, Yamada et al. (1993) confirmed that floor plate
and motor neuron differentiation could be induced by a
factor derived from the notochord. While floor plate
induction appeared to require direct contact between the
explant and notochord, a diffusible signal was sufficient to
promote motor neuron production. Together with the in vivo
data, these findings led to a model in which a signal from the
notochord induced floor plate differentiation. Once induced,
the floor plate cells shared all patterning activities of the
notochord and collectively these two structures acted as the
source of the signal to control motor neuron differentiation
(Yamada et al., 1993; Placzek et al., 1993). Moreover, the
constant spatial relationship between the position of the
grafted notochord and induced floor plate and motor
neurons raised the possibility that the signal acted in a
graded manner to control ventral neural tube development.
The hunt for the molecular identity of the notochord
derived motor neuron inducing signal culminated with the
publication of a series of studies in 1993 –1994 proposing
that the factor corresponded to Sonic Hedgehog (Shh), a
secreted protein and vertebrate orthologue of the Drosophila
segment polarity gene hedgehog (Echlard et al., 1993;
Krauss et al., 1993; Riddle et al., 1993; Chang et al., 1994;
Roelink et al., 1994). Expression of Shh by the notochord
S.R. Price, J. Briscoe / Mechanisms of Development 121 (2004) 1103–1115
and floor plate was observed at the stages when these two
structures exhibited their patterning activities (Fig. 1) and
direct evidence for the activity of Shh came from the
demonstration that its ectopic expression in vivo and in vitro
induced the differentiation of ventral cell types. Most
notably, cells transfected with a Shh expression vector,
placed in contact with neural plate explants, were able to
induce the differentiation of floor plate and motor neurons
(Roelink et al., 1994).
Elegant studies of Drosophila Hedgehog (Hh) from the
Beachy lab demonstrated that Hh protein is synthesised as a
precursor which is cleaved via an autocatalytic activity of
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the carboxyl-terminal domain to generate amino-terminal
and carboxyl-terminal fragments (Lee et al., 1994).
Vertebrate proteins are also processed in a similar manner,
(Porter et al., 1995; Bumcrot et al., 1995) raising the
question of whether a single molecule induced the
differentiation of both floor plate and motor neurons or
whether the two products of the Shh protein provided
distinct inductive activities. The advantages of the chick
neural plate explant system were exploited to address this
issue. Assays using the two isoforms of Shh led to the
conclusion that all the inducing activities attributed to Shh
resided in the amino-terminal product (Roelink et al., 1995;
Fig. 1. Motor neurons are generated from a specific domain of ventral neural progenitors of the spinal cord. Progenitor domains are defined by graded Shh
signalling regulating the combinatorial expression of transcription factors. (A) Shh is expressed by the notochord and floor plate. Five distinct ventral neuronal
subtypes arise at distinct positions along the dorsal ventral axis of the neural tube. Progressively more dorsal progenitor domains are exposed to a decreasing
concentration of Shh protein and the concentration of Shh determines the neuronal subtype generated. (B) The concentration gradient of Shh regulates the
ventral expression domains of a series of transcription factors in ventral progenitor cells. Shh either represses (class I) or induces (class II) expression at
different concentration thresholds. Progenitor gene expression domains are refined and maintained by negative cross-regulatory interactions between those
proteins that share the same boundary. The combinatorial expression of homeodomain proteins by distinct progenitor domains determines the neuronal subtype
that arises from each domain. FP, floor plate; MN, motor neuron; V, ventral interneuron; Shh, Sonic Hedgehog; N, notochord.
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Marti et al., 1995). Moreover, these experiments indicated
that the induction of different cell types was controlled by
different concentrations of amino terminal Shh—higher
concentrations of Shh being necessary for the induction of
floor plate cells, than for motor neuron differentiation
(Roelink et al., 1995). The importance of Shh in the
generation of the floor plate and motor neurons has been
confirmed by the loss of ventral neural tube cell fates in
embryos in which Shh signalling had been eliminated using
either antibody blockade in vitro or by gene targetting in
mouse (Ericson et al., 1996; Chaing et al., 1996).
Subsequently, the identification of molecular markers for
additional ventral neuronal subtypes extended these observations and it has become apparent that incremental 2 – 3
fold changes in Shh concentration generate at least five
molecularly distinct classes of ventral neurons in addition to
floor plate cells (Fig. 1; Ericson et al., 1997a). The
concentration of Shh necessary to induce each of these
neuronal classes in vitro corresponds to its distance from the
source of Shh in vivo (reviewed in Ericson et al., 1997b).
Thus, neurons generated in progressively more ventral
regions of the neural tube require correspondingly higher
Shh concentrations for their induction. Therefore, Shh
appears to function in a graded manner to pattern the ventral
neural tube, directing the position of generation and subtype
identity of the neurons at defined concentration thresholds.
Is there evidence for a gradient of Shh activity in vivo?
Although the ability of Shh to organize the pattern of
neurogenesis in the ventral neural tube in a concentration
dependent manner suggests that Shh acts directly at long
range to control gene expression, no direct observation of a
gradient of Shh protein in vivo had been made. Additionally, the long-range wing patterning activity of Hh in
Drosophila is exerted indirectly by the induction of the
secreted morphogen Decapentaplegic (Zecca et al., 1995).
Thus, it was possible that Shh exerted its long-range effect
by inducing a series of intermediary signals that relay
positional information to adjacent cells. In this view, Shh
signalling would act only at short range to initiate the relay
signal(s) that would act to pattern the neural tube. To test the
range of Shh signalling in the neural tube a method of
blocking the response of individual cells to Shh was devised
by constructing a mutated form of the Hh receptor, Patched
(Ptc). This mutant Ptc acted as a dominant inhibitor of Shh
signalling even in the presence of Shh ligand (Briscoe et al.,
2001). In ovo electroporation of the neural tube was used to
produce mosaic unilateral expression of mutant Ptc resulting in the blockade of Shh signalling in clusters of cells in
the ventral neural tube. Analysis of the transfected neural
tubes demonstrated inhibiting Shh signalling led to the cellautonomous inhibition of the generation of motor neurons
and other ventral neuronal classes arguing against signal
relay models and indicating that Shh functions directly, at
long-range to control ventral neural tube patterning.
Although these findings supported the idea that a
gradient of Shh signalling activity controls neuronal subtype
identity from progenitor cells, they posed the problem of
how cells respond to and interpret graded Shh signals.
Studies of both mouse and chick spinal cord suggested that
homeodomain transcription factors expressed by ventral
progenitor cells act as intermediaries in the interpretation of
graded Shh signalling (Fig. 1). On the basis of their mode of
regulation by Shh signalling, these transcription factors can
be subdivided into two groups, termed class I and II proteins
(Briscoe et al., 2000). The expression of each class I protein
is repressed at distinct thresholds of Shh activity, consequently, their ventral limits of expression are determined by
Shh signalling. Conversely, neural expression of the class II
proteins depends on Shh signalling, so their dorsal
boundaries of expression are defined by graded Shh
signalling (Ericson et al., 1997a; Briscoe et al., 1999,
2000; Vallstedt et al., 2001).
The use of the chick in vitro explant culture system
defined the threshold responses of the class I and class II
proteins to graded Shh signalling, however, it raised the
question of how the strikingly sharp boundaries of
expression could be generated by small changes in the
concentration of extracellular Shh. Gain- and loss- of
function studies in chick and mouse, respectively, implicated selective cross-repressive interactions between complementary pairs of class I and class II proteins expressed in
adjacent, abutting domains in the establishment of boundaries (Ericson et al., 1997a; Briscoe et al., 2000; Vallstedt
et al., 2001). These studies suggested that the crossrepressive interactions establish the dorsoventral domains
of expression of class I and class II proteins and generate
sharp boundaries between domains (Fig. 1). Together these
characteristics provide a mechanism to convert a gradient of
Shh signalling into discrete all or none changes in gene
expression and the cross-repressive interactions may serve
to consolidate progenitor domain identity relieving a
requirement for a prolonged period of graded Shh signalling. The principles of this model resemble mechanisms
involved in other developing tissues, such as the anterioposterior patterning of the Drosophila embryo (Small and
Levine, 1991), thus, this strategy may represent a general
mechanism for the regional allocation of cell fate in
response to graded inductive signals.
The combined expression profiles of the class I and
class II proteins defines 5 domains of progenitor cells within
the ventral neural tube and this subdivision is an initial
requirement for the generation of distinct neurons. The
profile of homeodomain protein expression appears to
specify the identity of neurons generated from each
progenitor domain such that the five progenitor domains
correspond to the position of generation of the five
molecularly identified classes of ventral interneuron
(Fig. 1). Consistent with this, the forced expression of a
class I or class II protein in the neural tube changed the fate
and position of generation of individual neuronal subtypes
in a manner predicted by the normal profile of homeodomain protein expression (Briscoe et al., 2000).
S.R. Price, J. Briscoe / Mechanisms of Development 121 (2004) 1103–1115
Conversely, the targeted inactivation, in mice, of individual
class I or class II proteins resulted in predictable switches of
neuronal fate (Ericson et al., 1997; Briscoe et al., 1999;
Vallstedt et al., 2001). Gain of function studies revealed that
the majority of the class I and class II proteins act as
transcriptional repressors via the groucho family of
co-repressors emphasizing the importance of transcription
repression in the regulation of dorsal –ventral patterning in
the neural tube (Muhr et al., 2001). Together these studies
provided insight into the molecular basis of the control of
neuronal subtype identity in the ventral neural tube and
allowed the construction of models of the genetic network
involved. For MNs, the combinatorial action of three
homeodomain proteins—Nkx2.2, Nkx6.1 and Irx3—
restricts the generation of motor neurons to the appropriate
region of the neural tube (Briscoe et al., 2000). Ventrally
Nkx2.2 and dorsally Irx3 ensure that motor neuron
induction is not initiated outside of this region. Within the
motor neuron progenitor domain, Nkx6.1 activity induces
the expression of domain restricted transcription factors that
are essential for motor neuron specification, notable among
these are Olig2 and MNR2 (Tanabe et al., 1998; Novitch
et al., 2001). Olig2, a bHLH protein, appears to co-ordinate
the acquisition of pan-neuronal properties and subtype
characteristics of differentiating motor neurons (Novitch
et al., 2001). Specifically, forced expression of Olig2 was
sufficient to repress Irx3, thus maintaining the motor neuron
potential of progenitors, and induce the expression of the
pan-neuronal gene Ngn2. MNR2, on the other hand, is a
homeodomain protein that acts as a dedicated determinant
of motor neuron identity. It is first expressed in motor
neuron progenitors during their final cell division and
ectopic expression of MNR2 is sufficient to elicit the ectopic
generation of motor neurons without affecting the
expression of class I and class II proteins (Tanabe et al.,
1998).
The inductive signals that direct the differentiation of
MNs and other spinal neurons have to be co-ordinated with
the regulatory pathways that specify general neuronal traits.
Recent studies have begun to shed light on this aspect of
MN generation (Diez del Corral et al., 2003; Novitch et al.,
2003). FGF emanating from the regressing node into
posterior neural tissue appears to act as a general repressor
of neuronal differentiation, while RA synthesized in
condensing paraxial mesoderm attenuates FGF signalling
and is required for neuronal differentiation and expression
of key ventral neural patterning genes, most notably Olig2.
Within differentiating progenitors the onset of neuronal
differentiation is at least in part co-ordinated by homeodomain proteins transcriptionally regulating the expression
of bHLH genes that control the acquisition of pan-neuronal
properties (Scardigli et al., 2003). However, evidence has
also emerged of direct association between bHLH transcription factors and the homeodomain proteins that
determine MN identity (Lee and Pfaff, 2003). This
integration converges on the promoter of a gene induced
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in differentiating MNs and is mediated by a dedicated
adapter protein (Lee and Pfaff, 2003). These mechanisms
synchronize neuronal subtype specification with neurogenesis, providing temporal and spatial control over MN
generation.
A major unresolved issue is the pathway through which
graded Shh signalling initially regulates class I and class II
protein expression. The Gli class of zinc finger transcription
factors appear to be key mediators of Shh signalling as
repression of all Gli mediated transcription inhibits ventral
neural tube patterning (Persson et al., 2002; Meyer and
Roelink, 2003). Gli proteins can act as both transcriptional
repressors and activators and an attractive model suggests
that Shh signalling inhibits the transcriptional repressor
activity and potentiates transcriptional activation (reviewed
in Jacob and Briscoe, 2003). Whether Shh regulation of Gli
activity is sufficient to transduce the graded aspect of Shh
signalling and how small differences in the extracellular
concentration of Shh are transmitted to nucleus to
differentially regulate gene expression remains to be
determined. Moreover the analysis of mouse mutants
lacking both Shh and Gli3 has uncovered a Shh independent
patterning mechanism (Litingtung and Chiang, 2000), the
nature of this signal is currently unclear. In this context, it is
interesting to note that the response of ventral neural
progenitors to Shh seems to be influenced by BMP
signalling. Exposure of neural plate explants to a fixed
concentration of Shh in the presence of BMPs results in a
ventral-to-dorsal shift in progenitor and neuronal subtype
identity (Liem et al., 2000). Conversely, BMP inhibitory
proteins ventralize the response of neural plate cells to a set
Shh concentration (Liem et al., 2000). BMP signalling could
therefore play a role in establishing a dorsal ventral
patterning and may represent the source of positional
information revealed in the Shh/Gli3 double mutant mice.
3. Subtype identity of motor neurons: the motor columns
of the spinal cord
Following the initial generation of generic motor neuron
identity, further refinement of subtype identity divides
motor neurons into subclasses that participate in distinct
circuits in the central nervous system and innervate different
target muscles in the periphery. As with the studies on the
generation of generic motor neuron identity, dissecting this
step in motor neuron development proceeded from ground
breaking anatomical and descriptive studies exploiting the
accessibility and malleability of chick embryos through to
the identification of the molecular components involved and
the manipulation of these components to reveal the
underlying genetic networks. Diversification of motor
neuron subtype identity was initially recognised by the
characteristic settling patterns of the motor neuron soma in
the spinal cord and by the distinct pathways selected by
motor axons in the periphery (Fig. 2A; Landmesser, 1980).
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Fig. 2. (A) Schematic showing the initial trajectories of the MMCm, LMCm and LMCl motor axons and the expression of LIM-Homeodomain transcription
factor within each of the columns. (B,C) Schematic of motor neuron soma movements during development. (B) LMCl motor neurons are generated later than
LMCm motor neurons and migrate through the LMCm to acquire their lateral position. (C) Overlapping with the ‘inside out’ migration of neurons that will
populate the LMCm and LMCl motor columns, motor pool formation occurs, F represents the Femorotibialis motor pool and A represents the Adductor motor
pool. (D) Schematic showing the motor pool specific expression of LIM-Homeodomain and ETS family transcription factors at Stage 35 chick lumbosacral
level 2. A, eF, HR, S, and ITR represent the Adductor, external Femorotibialis, Hip Retractor, Sartorius and Iliotrochanterici motor pools, respectively.
Orthograde and retrograde tracing of motor axons provided
the means to link the segregation of motor neurons into
discrete columns in the spinal cord with their axonal
projections (Landmesser, 1978a,b, 1980; Hollyday, 1980;
Gutman et al., 1993).
After motor axons project out of the spinal cord they
make a number of binary choices along a path to the target
muscle they will innervate. On leaving the spinal cord,
motor axons project either dorsally, towards axial muscles
or ventrally towards body wall muscles or limb muscles.
MNs innervating these distinct muscle types are positioned
into longitudinal columns. Axial muscles are innervated by
neurons located in a medial sub column called the MMCm
that is present throughout the rostro-caudal extent of the
spinal cord. Found progressively more laterally in the
spinal cord are the sub columns that project to body wall
muscles (the MMCl) and those that project to limb muscles
(LMC). Along the anterior – posterior axis of the spinal
cord, LMC motor neurons are present only at limb levels,
and MMCl motor neurons present only at thoracic levels
(Fig. 2A). Additionally, a group of preganglionic autonomic motor neurons, the Column of Terni, is present only
at thoracic levels (Prasad and Hollyday, 1991). Subsequent
to this initial axonal choice point, LMC axons face a
second binary choice at the base of the limb where they
project to either dorsally or ventrally derived limb muscles
(Landmesser, 2001). The lateral LMC (LMCl) sub column
motor neurons project to dorsally derived (largely extensor) muscles and the medial sub column (LMCm) motor
neurons project to ventrally derived (largely flexor)
muscles. Thus, within a column, sub-columnar identity
on the basis of the location within the limb of the muscle
that is innervated can be defined. Of key importance to our
understanding of the specification of motor neuron
columnar identity was the finding that each of these subcolumns may be identified by their combinatorial
expression of members of the LIM homeodomain family
of transcription factors, Isl-2, Isl-1, Lim-1and Lim-3 prior
to the innervation of muscle (Fig. 2A; Tsuchida et al.,
1994). Isl-1 expression demarcates the Column of Terni,
the combination of Isl2- and Lim-1 identifies the LMCl,
Isl-2 and Isl-1 defines LMCm and Isl-1, Isl-2 and Lim-3
labels the MMCm. Moreover, members of the Hox-c
homeodomain protein cluster were shown to be expressed
by motor neurons generated at distinct rostrocaudal levels
(Liu et al., 2001; Dasen et al., 2003). Experiments utilising
the ease of manipulation of the chick embryo, both in vivo
and in vitro, coupled to the ability to identify motor
columns through their expression of transcription factors
have yielded considerable insight into the nature of
extrinsic signals and intrinsic gene expression that specify
these different motor neuron subtypes.
S.R. Price, J. Briscoe / Mechanisms of Development 121 (2004) 1103–1115
4. Specification of the spatial extent of the motor columns
“The embryologist is confronted with the problem of
whether a causal relation exists during embryonic
development between the differentiation of the limb
musculature and that of the motor columns…. This
problem is a special aspect of a larger problem: The
definition of the roles of extrinsic and intrinsic factors in
the development of the central nervous system” Hamburger and Keefe (1944).
What determines that the lateral motor column lies in
register with the limb field, whereas the MMCm column is
present at all spinal levels? The use of LIM-homeodomain
and Hox-c protein expression as markers of columnar
identity (Ensini et al., 1998) has helped in the identification
of the source of the extrinsic signals that control the
establishment of the columnar organisation. Transplanting
segments of a quail neural tube from limb level to the
thoracic level of a chick host soon after neural tube closure
resulted in the appropriate respecification of MN columnar
identity so that MNs in the transplanted segment expressed
markers and acquired characteristics of thoracic MNs
(Ensini et al., 1998). Respecification is also possible in
grafts of thoracic quail neural tube to brachial chick hosts.
Moreover, the transposition of paraxial mesoderm to
different rostrocaudal positions resulted in the appropriate
transformation in the columnar identity of spinal MNs, thus
the specification signal(s) derive at least in part from the
paraxial mesoderm. This re-specification of motor column
identity is possible only in a short time-window from neural
tube closure, up to stage 15 (Hamburger and Hamilton,
1951), before the appearance of the first postmitotic motor
neurons (Ensini et al., 1998). Transplants that occur at stage
15 result in Hox and Lim homeodomain protein expression
in motor columns characteristic of the original anterior –
posterior position of the graft. Together these studies
suggested that signals derived from paraxial mesoderm
control columnar subtype identity and indicated that this
specification is an early event, occurring soon after the
specification of motor neuron progenitors.
By culturing chick neural tube explants in defined
conditions, and assaying the expression of Hox-c genes as
markers of rostrocaudal identity of motor neuron columns,
Liu et al. (2001) identified fibroblast growth factors, Gdf11
and retinoids as candidate signals responsible for establishing columnar identity. Further analysis indicated that the
neural Hox-c expression and columnar identity could be
established by signals from Hensen’s node in addition to
those from paraxial mesoderm (Liu et al., 2001; Dasen et al.,
2003). This suggested that the paraxial mesoderm signal
acts to refine rostrocaudal patterning initiated prior to neural
tube closure by signals emanating from the node and
notochord. Dasen et al. (2003) subsequently provided
evidence that FGFs emmanating from the Node mediated
this activity. Thus, FGFs may be involved in co-ordinating
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both motor column identity as well as the identity in the
early limb mesenchyme. However, the complete expression
profile required retinoid signaling at cervical levels while at
caudal thoracic and lumbar levels the BMP family member,
Gdf11 appeared to refine the Hox-c expression profile.
Moreover, analysis of the role of Hox-c genes indicated
that these proteins act at sequential stages of motor neuron
specification. In chick, forced expression in the brachial
neural tube of a Hox-c gene normally restricted to thoracic
levels generated motor neurons with thoracic columnar
identity, while a brachial Hox-c gene was sufficient to
reprogramme thoracic levels to produce features of brachial
columnar identity (Dasen et al., 2003). Restricting the
expression of Hox proteins to post-mitotic neurons, using a
promoter element selectively expressed in differentiating
motor neurons, provided evidence that the brachial LMC
identity can be specified solely through the actions of Hox-c
genes in post-mitotic motor neurons. Additionally, Sockanathan et al. (2003) have provided evidence that retinoid
signalling to postmitotic motor neurons can also influence
the columnar identity of brachial LMC neurons versus
thoracic motor neurons. Thus, multiple extrinsic signalling
events can provide checkpoints in the specification of motor
neuron subtype identity, a recurrent theme in the further
specification of motor neuron identity. Together with the
studies described above, these data have begun to establish
the molecular links between the imposition of rostrocaudal
identity and the emergence of motor neurons with distinct
topographic projections.
Further complexity in MN diversification is evident from
the generation, at the same rostrocaudal level, of more than
one motor column and by the subdivision within columns of
motor neurons with different axonal projection patterns.
Limb projecting motor neurons are subdivided into medially
placed LMC neurons projecting to ventrally derived limb
muscles and laterally placed LMC neurons, which project to
dorsally derived limb muscles (Landmesser, 1978a,b;
Tosney and Landmesser, 1985a,b). Both groups of neurons
are generated from progenitors that occupy the same
rostrocaudal and dorsoventral positions within the spinal
cord, however, they can be distinguished by differences in
the time at which they are generated. The ontogeny of
generation of postmitotic motor neurons from progenitor
cells (or neuroblasts) was studied first by Cajal in sheep,
rodents, pigs, ox, man and chick. However, these studies
were limited by the difficulty of morphologically distinguishing motor neuroblasts from precursors of interneurons and glia. Subsequently, Fujita (1964), followed by
Langman and Haden (1970) and Hollyday and Hamburger
(1977) undertook an autoradiographic analysis of the
birthdate and migratory pattern of motor neuron differentiation. These studies provided evidence that motor neuron
columnar differentiation occurs in an inside out fashion
(Fig. 2B). Earlier born motor neurons reside in the medial
part of the lateral motor column whereas later born motor
neurons must migrate past these to lie laterally.
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Thus, LMCm neurons are born earlier than LMCl neurons.
The timing of the formation of each of these sub-columns
within the LMC and their ‘inside-out migration’ has a
critical effect on their identity. The expression of two LIM
homeodomain proteins, Isl1 and Lim1 (Tsuchida et al.,
1994) distinguishes medial and lateral LMC: Isl1 is initially
expressed by all LMC neurons but is rapidly downregulated
from lateral LMC neurons as expression of Lim1 is induced
(Tsuchida et al., 1994); Using these as molecular markers,
Sockanathan and Jessell provided evidence that the switch
in LMC subtype depends on retinoid signals provided by
early-born LMC neurons. LMC neurons express the retinoid
synthesizing enzyme RALDH2 and exposure of LMC
neurons to retinoids represses Isl1 and promotes Lim1
expression (Sockanathan and Jessell, 1998). Similar to the
cross-repressive functions of class 1 and class 2 genes in
spinal progenitor domain specification, Lim1 and Isl1 have
a mutual cross-repressive interaction. Thus, forced
expression of Lim1 downregulates Isl1 expression and
promotes a lateral settling position in the spinal cord and
vice versa for forced expression of Isl1 (Kania and Jessell,
2003). Together, these data imply that, like the development
of many tissues, the specification of MN subtype and
columnar identity depends on the integrated, co-ordinated
action of multiple extrinsic signalling pathways.
5. Specification of motor axon pathfinding
Does the molecular specification of these different
columnar subtypes of motor neurons allow motor axons to
choose the correct pathway in the periphery? Manipulations
of motor neuronal positioning, through inversions or rostrocaudal shifts of the neural tube (Lance-Jones and Landmesser, 1980, 1981b; O’Brien and Oppenheim, 1989), or of
limb targets, through transplant of supernumerary limbs
(Hollyday et al., 1977; Whitelaw and Hollyday, 1983a),
limb bud rotations (Ferns and Hollyday, 1993; Whitelaw
and Hollyday, 1983b) and limb bud ablations (Shorrey,
1909; Barron, 1948; Hamburger and O’Keefe, 1944;
Whitelaw and Hollyday, 1983c; Tosney and Landmesser,
1984), have revealed that there are both permissive and
instructive signals in the periphery that motor axons use to
be guided to their correct targets. These experiments have
yielded the model that local guidepost signals within the
limb mesenchyme are used by motor axons to navigate. One
crucial guidepost occurs at the base of the limb, where LMC
motor axons are faced with their first choice of projections
(Lance-Jones and Landmesser, 1981a,b). LMCl axons
project dorsally at this choice-point, whilst LMCm
axons project ventrally. Inversions of the limb-bud distal
to this choice-point result in LMC axons making projections
dorsally or ventrally appropriate to the choice point (which
had not been inverted) (Whitelaw and Hollyday, 1983b).
These axons are then faced with inappropriate targets, due to
the distal inversion of the limb, and thus fail to make
connections with the correct muscle targets. Conversely,
rotations of the limb bud that include the choice-point result
in LMCl axons projecting ventrally (into dorsal tissue) and
vice versa for LMCm axons, thus, appropriate guidance
decisions are made towards the correct muscle targets
(Ferns and Hollyday, 1993). However, the gross morphology of nerve branches within the limb is maintained,
indicating that there are permissive pathways within the
limb mesenchyme that allow motor axon growth. The
concept that motor axons respond to guidance cues located
within the limb field is illustrated by an experiment
performed by Wang and Scott (2000) replacing limb buds
with those of various different developmental ages. Once
LMC motor axons reach the choice point at the base of the
limb, they pause for around one day during the so-called
waiting period. Are motor axons waiting for a change of
developmental profile of themselves, or of the limb
mesenchyme? It appears that the latter is the case, grafting
older limb buds causes motor axons to reduce the length of
the waiting period. These experiments elegantly demonstrate that motor axons respond to local cues positioned both
in time and space in the limb.
Rotations and rostro-caudal shifts of the neural tube that
result in motor axons being faced with novel territory yield
similar interpretations. As long as a given motor axon is not
shifted dramatically away from its appropriate entry point in
the limb, it is able to make appropriate choices to innervate
the correct target muscles (Lance-Jones and Landmesser,
1980, 1981b). However, in extreme cases where motor
axons are confronted with a novel choice-point (in
experiments utilising hind limb rotations where there are
two possible choice points that correspond to the two plexi
(the sciatic plexus and the crural plexus)), motor axons are
unable to project appropriately (Lance-Jones and Landmesser, 1981b).
What molecules control motor axon guidance? Experiments using both mouse and chick model systems have
implicated a role for the LIM homeodomain transcription
factors, the expression of which defines motor neuron
subcolumns of the LMC, in specifying the choices these
motor axons make at the plexus. Genetic manipulation in
the mouse of Lhx3 (Lim3) and Lhx4 (Gsh4) expression
indicated that these genes have selective roles in specifying
MMC motor neuron identity (Sharma et al., 1998, 2000).
Removal of Lim-1 expression in mouse results in LMCl
axons projecting both dorsally and ventrally with no
apparent preference, whereas LMCm axons project correctly to ventral limb mesenchyme (Kania et al., 2000).
Conversely, forced expression of either Lim1 or Isl-1 in
chick LMC motor neurons promotes the selection of a
dorsal or ventral trajectory appropriate for the LIM homeodomain that was misexpressed (Kania and Jessell, 2003).
What are candidate molecules downstream of this transcription cascade that direct appropriate axon trajectory?
Possible cell-surface effectors that direct the projection of
LMC axons to the correct limb domain are the EphA, class
S.R. Price, J. Briscoe / Mechanisms of Development 121 (2004) 1103–1115
of receptor tyrosine kinases and their ligands the ephrinAs
(Eberhart et al., 2002; Kania and Jessell, 2003). Misexpression of EphA4, which is normally expressed in LMCl
neurons, in LMCm neurons results in their axons being
redirected into the dorsal limb. This correlates with the
expression of the ephrinA ligands, which are preferentially
expressed in the ventral limb. Thus, a putative repulsive
signal mediated by EphA – ephrin-A interaction causes
LMCl motor axons to preferentially invade the dorsal
limb mesenchyme. This model is consistent with the earlier
Lim-1 knockout data (Kania et al., 2000) where LMCl
motor axons project with equal avidity to both dorsal and
ventral limb mesenchyme. Loss of Lim-1 in motor neurons
results in a downregulation of EphA4 in those motor
neurons which makes them less susceptible to the repulsive
effects of ephrin A ligands within the ventral limb
mesenchyme.
Despite the advances in our understanding of how the
binary choice between dorsal and ventral derived limb
musculature is determined by limb and motor neuron
intrinsic factors, little is known of how motor axons destined
to innervate a single muscle are guided to their correct
target. However, considerable progress has been made in
identifying factors expressed both peripherally and centrally
that define these subpopulations of motor neurons that will
innervate a single muscle, the so-called motor pools.
6. Further subdivisions in motor identity: motor neuron
pool formation
Motor pools are clusters of functionally related motor
neurons located within each of the sub-columns of the LMC
(Elliott, 1942; Romanes, 1942, 1964; Hollyday, 1980;
Landmesser, 2001). All of the neurons within a single
motor pool project to a single muscle in the periphery,
receive the same proprioceptive input from neurons located
in the dorsal root ganglia and are electrically coupled
together (Brenowitz et al., 1983), presumably by gapjunctions (Chang and Balice-Gordon, 2000). Each motor
pool has a rostrocaudal extent that is shorter than that of the
LMC. The organisation of these motor pools resembles a
complicated topographical map with no simple relationship
between motor pool position and the muscle it innervates
(Hollyday, 1980). For example, large motor pools that
innervate the large thigh muscles are present at the same
segmental level as small motor pools that innervate calf and
foot muscles. However, a careful analysis of motor pool
position in the developing and adult chick spinal cords by
retrograde tracing of motor neuron cell bodies by peripheral
injection of horse radish peroxidase (HRP) in defined
muscles, has revealed that motor pool position is related to
the muscle precursor positions on the original sheets of the
developing muscle mass (Hollyday, 1980). Nonetheless,
within a single species the positions of individual motor
pools are highly stereotyped and conserved between
1111
animals and provide a topographic map, albeit a complex
one, upon which motor output is coordinated. Motor pool
maps have been extensively characterised (Lance-Jones and
Landmesser, 1981a; Landmesser, 1978a,b; Hollyday, 1980)
and the electrophysiological and anatomical analysis of
innervation patterns in spinal motor circuit formation are
aided by the ease of manipulation of the chick embryo and
the large size of chick limbs and motor pools. Although the
signals that control the positioning of motor neuron pools in
the rostrocaudal axis are currently ill-defined, three lines of
evidence suggest that, similar to motor columns, different
motor neuron pools are specified early during spinal cord
development, prior to muscle innervation. First, autoradiographic studies of motor pool formation indicate that each
motor pool may have a characteristic birthdate (Whitelaw
and Hollyday, 1983c). Second, motor neuron pools that
project to extensor versus flexor muscles have characteristically different patterns of spontaneous activity identifiable long before their axons have reached their muscle
targets (Milner and Landmesser, 1999). Third, work from
the labs of David Anderson, Cynthia Lance Jones and
Thomas Jessell have found that members of the ETS family
of transcription factors, Er81 and PEA3, are expressed in a
motor pool restricted fashion with the initiation of ETS
protein expression occurring before muscle innervation (Lin
et al., 1998). Together, these data suggest that motor neuron
pools are specified centrally prior to muscle innervation.
Morphologically, individual motor neuron pools can
only be unambiguously identified once they have innervated
their target muscle. However, the identification of the motor
pool restricted expression of ETS genes provided a
watershed in the study of motor neuron pool formation.
On the basis of the combinatorial expression of the ETS
genes (as motor pool markers) and the LIM homeodomain
genes (as sub-columnar markers) it is possible to identify
individual motor neuron pools at time points earlier than the
final innervation of individual muscles (Fig. 2D; Lin et al.,
1998). It thus became clear that motor pools become
clustered during development in a phase that overlaps with
the inside out migration of LMCl through LMCm, a finding
that was almost impossible to make were it not for the
developmental profile of ETS gene expression (Fig. 2C, D).
Subsequent to the discovery of the motor pool specific
expression of Er81 and PEA3, many other genes, including
Eph’s (Iwamasa et al., 1999), cadherins (Fredette and
Ranscht, 1994; Price et al., 2002), semaphorins (Feiner et al.,
1997) and Lim-only proteins (Chen et al., 2002), have been
found that are expressed in restricted numbers of motor
pools. For example, similar to the ETS-LIM code for motor
pool identity, the combinatorial expression of members of
the cadherin family of cell adhesion molecules (Nollet et al.,
2000) distinguishes different motor pools. Together, these
findings allowed an investigation of motor neuron pool
formation, in terms of factors involved in the specification of
motor pool identity and the formation of motor pools as
discrete clusters in the spinal cord.
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How is motor pool identity acquired? Embryological
inversions of the spinal cord during development (Matise
and Lance-Jones, 1996) reveal the same critical period in
the specification of motor neuron pool identity (as defined
by retrograde tracing of motor neuron cell bodies by
peripheral injection of HRP) as that found in the specification of motor column identity. Consistent with these
findings, inversions of the lumbar spinal segments LS1-3
up to stage 15 resulted in an appropriate rostro-caudal
pattern of expression of Er81 (Lin et al., 1998). After stage
15, however, this rostro-caudal pattern of Er81 expression
was inverted, indicating that the identity of the motor pools,
assessed through Er81 expression, had already been
specified before the time of inversion. However, signals
from the limb are crucial for the induction of ETS protein
expression within motor pools. Limb ablation results in the
loss of ETS protein expression in motor neurons prior to the
period of naturally occurring cell death (Lin et al., 1998).
Together, these findings suggest that although motor pool
identity may be specified at early time points in motor
neuron development, prior to axonal outgrowth, signals
within the limb mesenchyme are important in reinforcing
this motor pool identity. Consistent with this view, the
expression of one member of the cadherin superfamily, MNcadherin, has an initial expression which is independent of
the limb, but a later, motor pool specific expression, is
dependent on limb signals (Price et al., 2002). These
findings thus suggested the possibility that peripheral
signals induce the expression of Ets genes, which then
refine the expression of cadherin genes. Consistent with this
model, misexpression of Er81 is capable of deregulating
expression of MN-cadherin (Price et al., 2002). Further, the
expression of cadherin-8 in the lattisiumus dorsai motor
pool is lost in the PEA3 knockout whereas cadherin-7, the
mouse homologue of MN-cadherin, becomes ectopically
expressed in that mutant mouse (Livet et al., 2002). The
nature of the peripheral signals that induce the expression of
ETS proteins in the central nervous system appears to
involve members of the neurotrophic factor family. Loss of
the neurotrophin GDNF (expressed in the periphery), or one
of its cognate receptors GFRa-1 (which is expressed in the
central nervous system) causes a dramatic loss of PEA3
expression in motor neurons (Haase et al., 2002). Additionally, explants of neural tube induce PEA3 in an appropriate
pattern dependent on the presence of GDNF. These data
provide evidence that neurotrophic signals play a permissive rather than instructive role in the induction of ETS
genes in a motor pool restricted pattern. The nature of the
signals, either peripherally or centrally that define motor
pools at the earliest timepoint is currently an unknown.
The formation of motor neuron pools into discrete
clusters is a common theme in motor organisation of all
limbed vertebrates that have been studied. The correct
juxtapositioning of motor neuron soma into these nuclei is
presumably critical to the co-ordination of motor output, a
theory supported by the fact that motor neurons within an
individual pool (but not between different pools) are
electrically and dye coupled by gap junctions (Chang and
Balice-Gordon, 2000). How is it that motor neurons
destined to populate an individual motor pool become
clustered and exclude those neurons of different pools?
Recent evidence has suggested that members of the cadherin
gene family, the expression of which depends on ETS
protein expression, play a crucial role in the formation of
motor neuron pools (Price et al., 2002). Perturbation of
cadherin expression using a dominant negative construct
perturbs general motor neuron organisation and cadherin
gene expression is required in the formation of individual
motor pools as discrete clusters. For example, the Adductor
(A) motor pool is distinguishable from the Femorotibialis
(F) motor pool by its expression of MN-cad; T-cadherin,
cadherin-6b and cadherin-8 expression being shared
between the two motor pools. Normalisation of cadherin
gene expression between these two pools, through electroporation in ovo of MN-cad in the F motor pool or a
dominant negative version of MN-cad in the A motor pool,
results in a cell autonomous perturbation of the sorting of A
and F motor pools. These findings provide strong evidence
that the combinatorial of cadherin protein expression directs
the specificity of interaction of neurons destined to populate
a given motor pool. Consistent with these data from chick
embryos, motor neuron pool formation is perturbed in the
PEA3 knockout mouse, a finding that correlates with the
deregulation of cadherin gene expression in those motor
pools (Livet et al., 2002). Taken together, these data provide
evidence that the induction of ETS transcription factor
expression by peripheral signals is important for the shaping
of cadherin gene expression in motor neuron pools. This
cadherin expression, in turn, shapes the morphology of
motor pools as clusters. However, whether motor neurons
destined to cluster into motor pools remain desegregated in
the absence of these peripheral signals and the consequences
to motor neurons of remaining desegregated are currently
unknown. Cadherin gene expression on its own, however, is
insufficient to explain all the specificity with which motor
pools cluster. How are motor pools restricted in the
anteriorposterior extent? How is the topographic organisation of motor pool positioning within the LMC established? Recent evidence from the knockout of the EphA4
gene suggests that it plays a role in the rostro-causal
positioning of certain motor pools within the spinal cord
(Coonan et al., 2003). Whether this rostro-caudal positioning is dependent on the induction of EphA4 expression in
motor neurons by extrinsic signals must await further
investigation.
7. Concluding remarks
In this review we have attempted to emphasize the
important place the chick embryo occupies in the study of
motor neuron development, from initial generation of motor
S.R. Price, J. Briscoe / Mechanisms of Development 121 (2004) 1103–1115
neuroblasts to formation of the spinal circuits that coordinate movement. These studies have provided an
indispensable framework around which exploitation of
genome sequence information can be laid and it is clear
from the continuing interest and enthusiasm for the chick
model that future directions—dissecting the molecular
mechanisms that regulate aspects of MN identity and
function—will continue to rely on the chick embryo.
Acknowledgements
We thank Artur Kania, Ben Novitch and Kate Storey for
their insightful comments. We apologise to those authors
whose work we have been unable to cite because of space
constraints. SRP is supported by grants from the BBSRC,
Wellcome Trust and Royal Society. JB is supported by the
MRC.
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