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Patterning and axon guidance of
cranial motor neurons
Sarah Guthrie
Abstract | The cranial motor nerves control muscles involved in eye, head and neck
movements, feeding, speech and facial expression. The generic and specific properties of
cranial motor neurons depend on a matrix of rostrocaudal and dorsoventral patterning
information. Repertoires of transcription factors, including Hox genes, confer generic and
specific properties on motor neurons, and endow subpopulations at various axial levels with
the ability to navigate to their targets. Cranial motor axon projections are guided by
diffusible cues and aided by guideposts, such as nerve exit points, glial cells and muscle
primordia. The recent identification of genes that are mutated in human cranial
dysinnervation disorders is now shedding light on the functional consequences of
perturbations of cranial motor neuron development.
Neural tube
The primordium of the nervous
system.
Floor plate
The ventral midline structure of
the CNS. It has a role in
patterning and axon guidance.
Branchial arches
Repeated bars of
mesenchymal tissue that
contribute to the lower jaw and
neck; each contains a
cartilaginous component, a
muscular component, a nerve
and an artery.
MRC Centre for
Developmental Neurobiology,
King’s College, Guy’s Campus,
London, SE1 1UL, UK.
e‑mail:
[email protected]
doi:10.1038/nrn2254
In humans, the cell bodies of cranial motor neurons lie
in the brainstem, and their axons extend through the
cranial nerves to control muscles in the head and neck.
Other vertebrates (including fish, chicks and mice) show
a high degree of conservation in both the arrangement
of brainstem motor neurons and the muscles they innervate. Developing motor axons perform a spectacular feat,
navigating over long distances from the CNS to their
targets in the periphery.
Early in development, the neural tube acquires a series of
swellings at its rostral end, presaging the development
of the forebrain, the midbrain and the hindbrain.
Caudally, the neural tube remains narrow and elongates
to form the spinal cord. Motor neurons differentiate
ventrally, on either side of a midline structure, the floor
plate. Cranial motor neurons reside in the midbrain and
the hindbrain (which together constitute the brainstem),
where they are partitioned into a series of nuclei. By
contrast, spinal motor neurons form a number of discontinuous columns along the length of the cord (for
reviews, see refs 1,2). Cranial motor axons follow dorsal
or ventral pathways from the brainstem; the axial position of this site of exit in turn dictates their peripheral
paths to muscles of the eye, tongue, branchial arches or to
parasympathetic ganglia3 (FIG. 1; TABLE 1).
Surprisingly, despite the functional significance of
cranial motor nerves, an understanding of the molecular
mechanisms that underlie their development is only just
starting to emerge4,5. Exciting progress has been made in
understanding cranial motor neuron development, particularly from gene gain-of-function and loss-of-function
nature reviews | neuroscience
experiments. The specificity of cranial motor neuron
projections is governed by rostrocaudal and dorsoventral patterning mechanisms that produce a diversity of
motor neuron subpopulations with distinct differentiation programmes. Some of the guidance molecules that
are involved in elaborating axon projections have also
been characterized. However, many important questions remain. The unique features of the differentiation
programmes of each of the cranial nerves are only partly
characterized. In particular, we know little about how
patterning genes dictate the repertoires of receptors on
axons, or how these receptors determine axon pathfinding behaviour to particular muscle targets. Deciphering
these molecular mechanisms is a major challenge in
developmental neurobiology.
In this context, it is fascinating that cranial dysinnervation disorders, which reflect abnormalities of one or
more cranial nerves, are starting to be genetically characterized in humans6. Clinical studies, together with studies
in animal models, are now providing fresh impetus to
understand how normal and abnormal cranial nerve wiring develops. In this Review, I describe the latest findings
in cranial motor neuron patterning and axon guidance,
focusing mainly on mouse and chick studies (as zebrafish
studies have been reviewed elsewhere: see REF. 5).
Motor nuclei form at distinct axial levels
Cranial motor neurons comprise three subsets: branchiomotor (BM), visceral motor (VM) and somatic
motor (SM) neurons (FIG. 2; TABLE 1). Early in development, these neurons arise in longitudinal progenitor
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MB
FB
III
IV
HB
V
VII/VII
BAs
OV
VI
XII
IX
X/XI
Figure 1 | Cranial nerves in the chick embryo. A lateral
view of cranial nerves in the chick
embryo
at embryonic
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| Neuroscience
day four, showing the pathways from the hindbrain (HB), on
the right, into the branchial arches (BAs) and other head
structures (the midbrain (MB) and the forebrain (FB)), on the
left. Roman numerals denote the nerves: III, oculomotor; IV,
trochlear; V, trigeminal; VI, abducens; VII/VIII, facial/
vestibuloacoustic; IX, glossopharyngeal; X, vagus; XI,
cranial accessory; XII, hypoglossal. OV, otic vesicle. Figure
modified, with permission, from ref. 3  (1990) Wiley-Liss.
Basal plate
The ventral half of the
neuroepithelium.
Alar plate
The dorsal half of the
neuroepithelium.
Neuroepithelium
A part of the early nervous
system that consists of dividing
progenitors arranged in a
columnar epithelium.
Homeobox
A conserved 180 base pair
sequence that encodes
homeodomain regions of
proteins that are involved in
binding to DNA and regulating
transcription.
domains in the hindbrain basal plate : BM and VM
neuronal somata migrate dorsally into the alar plate,
whereas SM somata remain ventral (in the basal
plate). BM and VM axons extend dorsally through the
neuroepithelium to large common exit points, whereas
SM axons leave the neuroepithelium ventrally in small
groups (FIG. 2c), with the exception of trochlear SM
axons, which grow dorsally and cross the dorsal midline at the midbrain–hindbrain boundary to project
contralaterally.
Individual motor nuclei can contain one or more
of BM, VM and SM neuron subsets. The oculomotor
nucleus (nucleus III), which contains SM and VM
neurons, lies most rostrally in the midbrain. Along the
rostrocaudal axis, the hindbrain is divided into rhombomeres, segmental entities that contain repeating sets
of neurons with distinct differentiation programmes
at different axial levels7–9. Motor nuclei differentiate
in individual rhombomeres or pairs of rhombomeres.
Rostral rhombomere one (r1) contains the trochlear
nucleus (nucleus IV), which contains SM neurons. The
trigeminal nucleus (nucleus V; BM neurons) occupies
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r1, r2 and r3 (in mice) or r2 and r3 (in chicks), the facial
nucleus (nucleus VII; BM and VM neurons) lies in r4
and r5, the glossopharyngeal nucleus (nucleus IX; BM
and VM neurons) lies in r6 (in mice) or r6 and r7 (in
chicks), and the vagus nucleus (nucleus X; BM and
VM neurons) and cranial accessory nucleus (XI; BM
neurons) occupy r7 and r8 (REF.10) (FIG. 2). In the caudal hindbrain, the abducens nucleus (nucleus VI, SM
neurons) occupies r5 in mice and r5 and r6 in chicks,
with the extended hypoglossal nucleus (nucleus XII,
SM neurons) found in r8. In both mice and chicks, the
facial motor neurons of the BM subtype are segregated
in r4, and those of the VM subtype are segregated in r5
(REFS 11,12). In all except avian species, the facial branchiomotor (FBM) neurons are born in r4 and then
undertake a striking caudal migration to r6 (REFS 10,13),
unlike most BM and VM neuron somata, which migrate
dorsally14. Rhombomere 4 also contains a population of
vestibuloacoustic neurons, which are efferent to the hair
cells of the inner ear; a subset of these neurons (contralateral vestibuloacoustic neurons) translocate their cell
bodies across the midline15.
Following their exit into the periphery, cranial
motor axons converge to form components of the
cranial nerves (FIG. 1). BM axons travel, through the
trigeminal, facial, glossopharyngeal, vagus and cranial
accessory nerves, towards branchial arches 1, 2, 3, 4
and 6, respectively, where they innervate muscles of
the jaw and muscles that control facial expression, as
well as the pharynx and the larynx. VM axons project
towards parasympathetic ganglia, the neurons of
which supply salivary and lacrimal glands, smooth
muscle and visceral organs. Oculomotor, trochlear
and abducens SM neurons innervate the six eye muscles, with an additional oculomotor VM component
synapsing at the ciliary ganglion. Hypoglossal neurons
project rostrally through the floor of the pharynx to
the tongue muscles. Cranial motor nuclei conform to a
theme, sharing common features, such as morphology
and initial axon trajectory, but nevertheless possessing distinct positional identity, synaptic targets and
functions.
Rostrocaudal patterning of the brainstem
The midbrain is divided into a series of ‘arcs’, which
have been proposed to underlie the differentiation of
nuclei and are distinguished by the expression of various homeobox genes and other molecular markers16–18.
The most medial arc contains oculomotor neurons,
and fibroblast growth factor 8 (FGF8), produced by
the midbrain–hindbrain boundary, has been proposed
to dictate the rostrocaudal position of the oculomotor nucleus, because misexpression of FGF8 shifts the
nucleus rostrally17. Differentiating oculomotor neurons
express the homeobox gene paired-like homeobox 2a
(Phox2a)16, which is an important determinant of oculomotor identity, as oculomotor neurons (as well as
trochlear motor neurons) are absent in Phox2a-mutant
mice19. Further details of the transcriptional hierarchy
that underlies oculomotor neuron determination remain
to be discovered.
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Table 1 | Motor components of the cranial nerves and their targets in humans
Nerve
Subtype
Nucleus
Target muscles or ganglia
III
Somatic motor
Oculomotor
Superior, inferior and medial recti muscles;
inferior oblique, levator palpebrae superioris
Visceral motor
Edinger-Westphal
Ciliary ganglion
IV
Somatic motor
Trochlear
Superior oblique
V
Branchiomotor
Trigeminal motor
Muscles of mastication, tensor tympani,
anterior belly of digastric, others
VI
Somatic motor
Abducens
Lateral rectus muscle
VII
Branchiomotor
Facial motor
Muscles of facial expression, stapedius,
posterior belly of digastric
Visceral motor
Superior salivatory
Pterygopalatine/sphenopalatine ganglion,
submandibular ganglion
Branchiomotor
Nucleus ambiguus
Stylopharyngeus muscle
Visceral motor
Inferior salivatory
Otic ganglion
Branchiomotor
Nucleus ambiguus
Laryngeal and pharyngeal muscles
Visceral motor
Dorsal motor
Non-striated muscle of thoracic and abdominal
viscera
Cranial XI
Branchiomotor
Nucleus ambiguus
Laryngeal and pharyngeal muscles
Spinal XI
Branchiomotor
Accessory nucleus,
cervical spinal cord
Sternocleidomastoid and trapezius muscles
XII
Somatic motor
Hypoglossal
Tongue muscles
IX
X
In the hindbrain, a large number of transcription
factors and other genes pattern rhombomere territories
through their segmental expression9,13,20. Many of these
genes regulate motor neuron development, either directly
or indirectly. For example, the zinc finger transcription
factor early growth response 2 (EGR2; also known as
KROX20) is expressed early in r3 and r5 in the mouse21
and determines many of the features of odd-numbered
rhombomeres. In Egr2-mutant mice, r3 and r5 are
largely missing, depleting the motor neurons at these
levels22. The transcription factor MAFB (also known as
Kreisler) is similarly expressed early and regulates r5 and
r6 development23,24; in Mafb-mutant mice, r5 and r6 are
lost, causing the deletion of the r5 VM facial and SM
abducens neurons25.
Both Egr2 and Mafb lie upstream of, and activate
the transcription of, Hox genes, which contain an
Antennapedia-class homeobox sequence and have a preeminent role in hindbrain patterning. In vertebrates there
are four Hox-gene clusters (named a–d) on four separate
chromosomes26. There are 13 paralogue groups, (although
no individual cluster contains all 13 genes). Paralogous
genes (for example, Hoxa3 and Hoxb3) often exhibit overlapping functions; indeed, dosage-dependent effects have
been shown in the case of Hoxa3 and Hoxd3 (Ref. 27). Hox
genes show nested domains of expression in the hindbrain
(FIG. 3), with each paralogue group expressed from the spinal cord rostrally to particular rhombomere boundaries.
Thus, group 2, 3 and 4 gene expression domains end at the
r2–r3, r4–r5 and r6–r7 boundaries, respectively (although
Hoxa2 expression terminates at the r1–r2 boundary).
Group 1 genes show an anomalous rostral restriction at
the r3–r4 boundary, and Hoxb1 is expressed at high levels
in r4. The combination of Hox genes that are expressed
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in a particular rhombomere, as well as the timing of the
onset of the expression and the expression level, dictates
segmentation and segment identity at that axial level.
The patterns of Hox gene expression in the hindbrain
are established, at least in part, by the diffusible action
of FGF8 and retinoic acid (RA) at the rostral and caudal
ends of the hindbrain, respectively28,29. Rostrally, FGF8
that is produced by the midbrain–hindbrain boundary
sets the rostral boundary of Hoxa2 expression30. RA that
is derived from the mesodermal somites, which flank the
caudal hindbrain, is thought to specify the caudal hindbrain (r5 to r8) through the induction of Hox genes, in
a dose-dependent manner28. Experimental depletion of
RA in mice and chicks supports this idea: it causes caudal
rhombomeres to assume more rostral identities (either
r3 or r4)31,32. In some cases, the expression of caudal
Hox genes is induced through upstream Retinoic Acid
Response Elements (RAREs)33,34. RA is implicated in the
production of SM neurons in the spinal cord35,36, raising
the possibility that it might also generate SM neurons in
r5 to r8 (the abducens and hypoglossal nuclei). Somatic
motor neurons differentiate throughout the rostrocaudal
extent of chick hindbrain explants following application of
RA37, whereas a reduction in RA signalling in the zebrafish
produces a loss of hindbrain cranial motor neurons38. The
absence of SM neurons in the rostral hindbrain might be
maintained by the action of the RA‑degrading enzyme
CYP26, as inhibition of CYP26 caused the differentiation
of SM neurons throughout hindbrain explants37. Thus,
FGF8 and RA, through their role in patterning Hox genes,
and possibly through independent inductive mechanisms,
pattern motor neurons such that BM and VM neurons
differentiate throughout the hindbrain, whereas SM
neurons are restricted to caudal rhombomeres.
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a Chick at embryonic day 4
b Mouse at embryonic day 11.5
MB
MB
III
HB
III
HB
IV
r1
gV
r1
FP
r2
gV
V
r3
r5 aVI
gVIII
OV
VII/VIII
V
r6
IX
X, XI
gIX
OV
VII/VIII
VE
VI
VI
r6
IX
FP
G
r7
X, XI
gIX
r8
c Transverse section
of chick branchial region
r4 CVA
r5 aVI
gVII
gVIII
VI
VI
r7
gX
FP
r2
r3
r4 CVA
gVII
IV
r8
gX
XII
PX
XII
BA
Figure 2 | Motor neuron organization in the vertebrate brainstem. a | The organization of motor neurons in a flatReviews | Neuroscience
mounted chick brainstem at embryonic day four (E4). b | The organization of motor neurons inNature
a flat-mounted
mouse
brainstem at E11.5. In parts a and b, rhombomere (r) levels are indicated; branchiomotor and visceral motor neurons are
shown in red; somatic motor neurons are shown in blue. Roman numbers alone denote the nerves: III, oculomotor; IV,
trochlear; V, trigeminal; VI, accessory abducens; VII/VIII, facial/vestibuloacoustic; IX, glossopharyngeal; X, vagus; XI, cranial
accessory; XII, hypoglossal. Roman numerals with the letter g denote the cranial ganglia: gV, trigeminal ganglion; gVII,
geniculate ganglion; gVIII, vestibuloacoustic ganglion; gIX, petrosal ganglion; gX, nodose ganglion. c | Cranial motor axon
pathways in a transverse section of the branchial region in the chick embryo at E4 (for details, see REF. 10). aVI, accessory
abducens nucleus; BA, branchial arch; CVA, contralateral vestibuloacoustic neurons; FP, floor plate; G, cranial sensory
ganglion; OV, otic vesicle; PX, pharynx; VE, ventricle.
Hox genes control motor neuron identity
As mentioned above, Hox genes are key controllers of
rostrocaudal patterning in the head, including hindbrain segmentation and rhombomere identity 4,39. As
well as being expressed in neuroepithelial domains
(FIG. 3), Hox genes are expressed in neural crest cells,
which emigrate predominantly from even-numbered
rhombomeres into the branchial arches (FIG. 4), generating skeletal tissues and cranial ganglia40–42. As a result,
several Hox-mutant mice show defects in the patterning of branchial arch-derived structures 39. There is
positional registration, that is, motor neurons project
into peripheral territory that expresses the same Hox
repertoire as their rhombomere of origin. One expectation of this pattern is that Hox genes dictate the expression of matching axon guidance receptors and ligands,
in neurons and their targets, respectively. But, hitherto,
there is little data on this issue. Nevertheless, a number
of studies with Hox-mutant mice have shown specific
defects in the generation and patterning of cranial
motor neurons. Loss of rostrally expressed Hox genes
in paralogue groups 1 and 2 leads to patterning defects
of the trigeminal and facial BM and VM nuclei, which
occupy rostral rhombomeres, whereas SM neurons in
caudal rhombomeres are affected by the loss of group 3
paralogues.
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Hoxa2 regulates trigeminal motor neuron differentiation.
Hoxa2 is expressed up to the r1–r2 boundary and is
the only Hox gene to be expressed in r2, whereas in
r3 it is co-expressed with Hoxb2 (Refs 39,43). In both
mice and chicks, only r4 second branchial arch, not r2,
(first branchial arch) neural crest cells express Hoxa2,
and loss of Hoxa2 in mice produces a striking transformation of the second branchial arch into a first-arch
phenotype44–46. In Hoxa2-mutant mice, presumptive
trigeminal motor neurons in r3, and a subset in r2,
project through the r4 exit point46. This suggests that
trigeminal motor neurons misroute to the transformed
second arch, either because of a change in motor neuron
identity and/or because of a change in axon guidance
cues. The r4-derived facial nerve is also reduced in
size later in development, possibly owing to a loss of
second branchial arch character and arch-derived factors46. When Hoxa2 is ectopically expressed in chick r1,
which normally lacks motor neurons, trigeminal
neurons are generated 47, supporting the idea that
Hoxa2 specifies trigeminal motor neurons. In Hoxb2mutant mice there is also a mis-specification of some
trigeminal motor neurons in r3, which project through
the r4 exit point, but the effect on trigeminal axon
projections is less striking than with Hoxa2 mutation (Ref. 48). It is likely, therefore, that r2 trigeminal
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MB
Mafb
r5
Mafb
r6
Hoxd4
Egr2
Hoxb4
r4
Hoxa4
Hoxb1
Hoxd3
r3
Hoxb3
Egr2
Hoxb2
r2
Hoxa3
Hoxa1
r1
Hoxa2
HB
r7
r8
Figure 3 | Expression patterns of Hox genes in the vertebrate hindbrain. The
domains of mRNA expression of the Hox genes Egr2 and Mafb
in the
hindbrain
of the
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| Neuroscience
mouse and chick embryo, at embryonic day 11.5 (E11.5) and E4, respectively. The bars
labelled with different Hox genes show the genes’ expression domains, which extend
from the caudal hindbrain up to particular rhombomere (r) boundaries4,168. Darker
shading indicates higher levels of expression. HB, hindbrain; MB, midbrain.
neurons are patterned by Hoxa2, whereas those in r3
are patterned by a combination of Hoxa2 and Hoxb2.
Hoxb1 is a key regulator of FBM neurons in r4. Extensive
insight has been gained into the genetic hierarchy that
underlies FBM neuron development in r4. Hoxa1 and
Hoxb1 are expressed up to the r3–r4 boundary in dividing progenitors, but the onset of Hoxa1 expression comes
earlier (at embryonic day eight), and after neuronal differentiation, only Hoxb1 is maintained at high levels in r4
and in the postmitotic FBM neurons40. In Hoxa1-mutant
mice, rhombomeric segmentation of r3 to r8 is disrupted,
the r3–r4 boundary does not form correctly, r4 is reduced
in size and r5 is absent49–51. By embryonic day 18.5, this
early role of Hoxa1 in rhombomere segmentation and
identity leads to loss of the abducens nerve and reduction
or loss of the facial nerve. By contrast, Hoxb1 has a later
role in FBM neuron specification, reflected in the observation that in Hoxb1-mutant mice, segmentation occurs
normally but there is a striking absence of FBM neurons
(which fail to migrate caudally to r6 and eventually die);
CVA neurons are also missing52,53. Motor neurons that do
differentiate in r4 project axons to the first branchial arch,
suggesting that they default to a trigeminal-like identity.
The instructive role of Hoxb1 was demonstrated in chicks
by overexpressing Hoxb1 in r2. This converts trigeminal
motor neurons to an FBM fate, resulting in anomalous
facial-like axon projections to the second branchial arch54.
However, when Hoxb1 is misexpressed in both r2 and the
first branchial arch, ectopic FBM axons navigate from r2
nature reviews | neuroscience
into the first arch54. Global Hoxb1 overexpression appears
to cause the coordinated transformation of motor neurons
and the first arch, such that they develop an FBM-like
second arch identity. The molecular significance of this
positional registration in Hox gene expression between
BM neurons and their innervation territory was unclear
for some time. However, recent studies show that Hoxb1positive, r4-derived neural crest cells preferentially give
rise to Schwann cells, which might provide the guidance cues and/or survival factors that are needed for the
projection and maintenance of the facial motor nerve55.
Conditional deletion of Hoxb1 only in the neural crest
results in a significant proportion of animals showing
facial paralysis, indicative of a loss of facial motor neurons
(as in Hoxb1-null mice)56. The facial nerve fails to branch
correctly and eventually the facial motor neurons die56.
FBM axons might require an interaction with Schwann
cells for their guidance and survival, perhaps through the
production by the Schwann cells of neurotrophic factors.
An intriguing piece of evidence in favour of this idea is
the prolonged survival of mis-specified FBM neurons in
Hoxb1-mutant mice that are also mutant for Bax (a gene
that is required in cell death pathways that are associated
with neurotrophic factor deprivation)57.
As with the pairing of Hoxa2 and Hoxb2 in trigeminal
neuron specification, Hoxa1 and Hoxb1 appear to
act synergistically to pattern facial motor neurons.
Hoxa1;Hoxb1 double-mutants reflect this fact, and
show more extensive FBM neuron defects than either
of the single mutants58,59. Hoxb2 also functions in FBM
specification, possibly because it is a downstream target
of Hoxb1 (Refs 48,60). The network of regulatory interactions that has been revealed so far for r4 provides a
glimpse of how daunting it will be to achieve a thorough
understanding of hindbrain motor neuron patterning.
Following the activation of Hoxa1 and Hoxb1 by RA,
Hoxa1 transactivates Hoxb1, Hoxb1 auto-regulates its
own expression and activates Hoxb2 and Hoxa2 (Ref. 61).
Mutation studies have also shown that the genes Gata2,
Gata3 and T-box 20 (Tbx20) are among the downstream
targets of Hoxb1 in FBM specification62,63.
One outstanding question is how the specificity of Hox
proteins in binding to particular DNA target regions is
conferred. An answer might lie in the fact that Hox geneencoded homeoproteins form complexes with specific
cofactors, possibly restricting the proteins’ binding specificity. These cofactors are the Pbx and Meis homeoproteins, which form a tripartite complex with Hox proteins
to regulate downstream transcription64. In pbx4-mutant
zebrafish, FBM neuron development is defective, and
the resultant phenotype is identical to that which results
from a deficiency in hoxb1a, the zebrafish Hoxb1 homologue65,66. Complete elimination of maternal and zygotic
Pbx function produces a hindbrain r1 ‘ground state’ in
r2 to r7, reflected by a lack of characteristic molecular
markers and neuronal types, especially BM and VM neurons67. The specificity of the interactions between Pbx
proteins and Hox proteins remains to be characterized,
but HOXB1–Pbx-protein complexes have been shown to
bind to specific regulatory regions of the Hoxb1 and Hoxb2
genes to regulate their expression in r4 (Refs 60,68).
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MB
r1
r2
HB
FB
r3
r4
r5
r6
OV
r7
Md
BA2 Mx
BA3 BA1
BA4
Hox 1
Hox 2
Hox 3
Hox 4
BA4
BA3
BA2
BA1
Figure 4 | Patterns of neural crest migration and
branchial arch Hox gene expression
in chick
and
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| Neuroscience
mouse embryos. A schematic diagram of a chick head at
embryonic day two, showing pathways of neural crest
migration in the chick and mouse embryo and patterns of
Hox gene expression in the branchial arches (BAs)42,102,169,170.
FB, forebrain; HB, hindbrain; MB, midbrain; Md, mandibular
part of BA1; Mx, maxillary part of BA1; OV, otic vesicle;
r, rhombomere.
Hox3 genes regulate SM neuron differentiation. Hox3
paralogues are good candidates for genes that regulate
the production of SM neurons in r5 to r8. Hoxb3, for
example, is expressed at high levels in r5 and r6 in
chicks, but only in r5 in mice69,70, corresponding with
the location of abducens neurons in these two species.
Knockout studies have shown that abducens neurons are
lost in Hoxa3;Hoxb3 mouse double-mutants, but can be
ectopically induced by rostral expression of Hoxa3 in
chicks71,72. Future studies might reveal whether Hox4
paralogues, which are expressed in a caudal domain that
extends up to the r6–r7 boundary, specify hypoglossal
motor neurons in this region.
Dorsoventral patterning of Hox
Combinations of other homeobox-containing transcription factors specify progenitor domains along the
dorsoventral axis of the brainstem1,73. Sonic hedgehog
protein (SHH) has been proposed to form a ventral-todorsal gradient that induces dose-dependent neuronal
differentiation. Although this model is based largely on
experiments on the spinal cord, it is thought to apply to
the hindbrain as well, and both cranial and spinal motor
neurons are missing in Shh–/– mouse mutants74. SHH also
controls the differentiation of the midbrain arc, which
contains oculomotor neurons16. The model proposes
that graded SHH signalling produces the graded activity of Gli transcription factors75, which in turn activate
or repress the expression of homeodomain proteins in
864 | november 2007 | volume 8
specific dorsoventral progenitor domains76. The expression domains of these transcription factors show crossrepressive interactions at their boundaries, consolidating
their identity and generating groups of postmitotic neurons that express repertoires of transcription factors that
are involved in their further specification77. This model
has given insight into the mechanism of differentiation
of cranial motor neurons, the progenitors of which
occupy distinct dorsoventral domains.
In the hindbrain, the progenitor domain that flanks
the floor plate (known as the p3 domain) gives rise to
BM and VM neurons78,79, whereas the dorsally adjoining progenitor domain (known as the pMN domain)
generates SM neurons (FIG. 5a). P3 and pMN progenitors
express distinct ‘codes’ of transcription factors (FIG. 5),
most of which act as transcriptional repressors to control
neuronal identity80–83. The key regulators of BM and VM
neuron fate are suspected to be Nkx2.2 and Nkx2.9, but
mutation of either gene on its own leaves these neurons
intact, probably because of redundancy78,84. Nkx6.1 and
Nkx6.2 are not required to specify BM and VM neurons
per se, but they act to repress alternative interneuron
fates, and in double mutants, BM and VM neurons
express some interneuron markers79. Loss-of-function
of Nkx6.1 alone or with Nkx6.2 also results in aberrant
BM and VM neuron migration and axon pathfinding79,85.
In the pMN domain, loss-of-function of Nkx6.1 and/or
Nkx6.2, or of paired box gene 6 (Pax6), deletes abducens
and hypoglossal SM neurons80,82,83,86. Combined Nkx
gene activity in turn induces the expression of the basic
helix-loop-helix transcription-factor-encoding gene
oligodendrocyte transcription factor 2 (Olig2), which
coordinates generic neuronal differentiation with motor
neuron subtype87,88 and induces the homeobox-containing
gene MNR2.
As a result of these early specification events, postmitotic SM neurons express a combination of the homeo­
box-containing genes MNR2 (which is chick-specific),
motor neuron and pancreas homeobox 1 (MNX1; also
known as HB9) and the LIM-homeobox genes Islet 1
(Isl1), Isl2, Lhx3 and Lhx4 refs 73,87,89,90 (FIG. 5b).
Interestingly, hindbrain SM neurons show variations
on this pattern, for example, subsets of abducens SM
neurons express different combinations of these genes91.
MNR2 and MNX1 are involved in specifying SM neuron
fate and repressing interneuron fates, respectively90,92,93.
Lhx3 and Lhx4 are determinants of ventral pathway
choice; SM neurons, including abducens and hypoglossal subpopulations, are absent in Lhx3;Lhx4 double
knockouts, and BM and VM neurons that misexpress
these transcription factors extend axons ventrally rather
than dorsally94. Like SM neurons, postmitotic BM and
VM neurons express Isl1 but, unlike SMs, they also
express Tbx20 (Ref. 95), Phox2a and Phox2b, with Phox2b
expressed first19 (FIG. 5b). Phox2b is a key gene in BM and
VM neuron generation96, and in Phox2b-mutant mice,
all BM and VM neurons are absent97. Conversely, in mice
that lack Phox2a, which is expressed before Phox2b in
the oculomotor and trochlear nuclei, both of these nuclei
are missing19. The generation of knock-in mouse lines,
in which Phox2b was replaced by the Phox2a locus, or
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a
b
pMN
Pax6, Olig2,
Nkx6.1, NKx6.2.
SM neurons
MNX1, MNR2,
Islet-1, Islet-2,
Lhx3, Lhx4.
p3
Nkx2.2, Nkx2.9,
Nkx6.1, Nkx6.2.
SM neurons
BM and VM neurons
SHH
pMN
p3
BM/VM neurons
Islet-1, Phox2b,
Phox2a, Tbx20.
FP
FP
N
N
Figure 5 | Dorsoventral patterning of cranial motor neurons. a | A schematic diagram of a transverse section
through the hindbrain, showing pMN and p3 progenitor domains, which give rise to branchiomotor (BM) and visceral
motor (VM) neurons, and somatic motor (SM) neurons, respectively. The arrows extending from the floor plate (FP) and
Nature Reviews | Neuroscience
the notochord (N) show the presumed diffusion of Sonic hedghog protein (SHH) during motor neuron differentiation.
For the pMN and p3 domains, the repertoires of transcription factors that are expressed by the progenitors are listed.
b | A schematic diagram of a transverse section through the hindbrain, showing the location of postmitotic cranial motor
neurons following dorsal migration by BM and VM neurons. The repertoires of transcription factors that are expressed by
the neurons are listed. Lhx, LIM homeobox protein; MNX1, motor neuron and pancreas homeobox 1; Olig2,
oligodendrocyte transcription factor 2; Pax6, paired box gene 6; Phox2b, paired-like homeobox 2b; Tbx20, T-box 20.
vice versa, has revealed that the two genes are not functionally equivalent98. Oculomotor and trochlear motor
neurons are only partially rescued by the substitution
of Phox2b for Phox2a, and in the Phox2a into Phox2b
knock-in line, FBM neurons differentiate but fail to
migrate correctly 98.
Phox2b is regulated by converging streams of rostrocaudal and dorsoventral patterning information, which
are dependent on Hox gene function and Nkx2.2 and
Nkx6 gene function, respectively. Loss-of-function studies reveal that Nkx genes cooperate with Hoxb1 to maintain Phox2b expression in r4 and favour an FBM over
a serotonergic neuronal fate99. Hoxb1, Hoxb2 or Hoxa2
misexpression can all activate Phox2b ectopically in
ventral regions of the hindbrain, but co-electroporation
of Hoxb1 or Hoxa2 with Nkx2.2 is required to generate
ectopic motor neurons in dorsal regions100. An upstream
enhancer region that can be transactivated by Hoxb1,
Hoxb2 or Hoxa2 has recently been identified in the
Phox2b gene; this region contains conserved Pbx and
Meis protein binding sites, and its transcriptional activity
can be enhanced by Pbx and Meis cofactors99.
Cranial paraxial mesoderm
The population of mesoderm
cells that originates adjacent to
the brainstem and gives rise
to many head muscles.
Sphenopalatine ganglion
A parasympathetic ganglion
that is innervated by VM
neurons of the facial nerve.
Diffusible cues guide cranial motor axons
These early genetic programmes specify cranial motor
neurons and dictate their responses to local guidance
cues in the developing head (TABLE 2). The first pathway
choice made by cranial motor axons is whether to project
ventrally in small groups into the perinotochordal mesenchyme (in the case of SM neurons) or dorsally (in the
case of BM and VM neurons) through large common exit
points. SM axons then innervate eye muscles derived from
the prechordal mesoderm and cranial paraxial mesoderm
(CPM), or tongue muscles derived from somites 1–4
(Ref. 10). VM axons supply parasympathetic ganglia, such
as the ciliary, sphenopalatine and otic ganglia, which are
neural crest-derived structures101,102. BM axons project
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into the branchial arch muscles, along paths that were
previously followed by CPM cells; these CPM cells
migrate dorsoventrally to form the branchial muscle
plates103, which later split up into a characteristic set of
muscles104.
Motor axons are repelled from the midline. The initial
phase of cranial motor axon extension involves repulsion of all subtypes of cranial motor axons by the floor
plate105,106. Candidates for the mediation of this effect
are the axon guidance molecules netrin 1, the Slit proteins and SEMA3A, all of which can repel BM and VM
axons in vitro107–109. Cranial motor neurons express the
UNC5A receptor (which mediates the repellent effect
of netrin 1), the Slit receptors ROBO1 and ROBO2,
and the SEMA3A receptor neuropilin 1 (Refs 109–112).
However, only netrin 1 and the Slit proteins are highly
expressed in the brainstem floor plate at the time of
axon extension109,110,113–115, suggesting that these are the
relevant repellents in vivo. In particular, attenuation of
Slit–ROBO signalling in chicks or mice in vivo leads to
BM and VM axon pathfinding defects, suggesting an
in vivo role for the Slit proteins109. A third Robo receptor,
ROBO3 (also known as Rig1), has been shown to have
a role in midline crossing of some neuronal types116, but
might not be expressed in motor neurons.
There is as yet no clear evidence for a role for netrin 1
motor neuron repulsion in vivo, although trochlear motor
neuron cell bodies enter the floor plate in netrin 1
mutants, suggesting a loss of repulsion from the midline117. Motor axon pathways have not been investigated
in Unc5a mutants, but there are peripheral motor axon
guidance defects in mutants for a related Unc5 family
member, Unc5c118,119. Unc5c is also prematurely expressed
in FBM neurons that lack Nkx6.1 and fail to migrate85,
suggesting that regulation of Unc5 family members
might be important in aspects of FBM guidance and
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Table 2 | Molecules involved in cranial motor axon guidance
Molecule
Effect on cranial motor axon guidance
HOXB1
In Hoxb1 mutants, facial branchiomotor neurons project aberrantly to the first
branchial arch. When Hoxb1 is ablated in the neural crest only, the facial nerve fails
to branch correctly. Ectopic expression of Hoxb1 in r2 converts trigeminal motor
neurons into facial motor neurons
52–54,56
HOXA2
In Hoxa2 mutants, trigeminal motor axons misroute into the second branchial arch
46
HOXB2
In Hoxb2 mutants, a subset of trigeminal motor axons misroute into the second
branchial arch
48
NKX6.1, NKX6.2
In Nkx6.1 mutants, branchiomotor axons mistarget exit points and pathfind
aberrantly
LHX3, LHX4
Direct cranial motor axons along ventral trajectories
PHOX2A
In Phox2a mutants, visceral motor axons fail to project due to an absence of
ganglionic targets
Netrin 1
Repels branchiomotor axons in vitro
UNC5C
In Unc5c mutants, trochlear motor axons project aberrantly
118
Slits
Repel branchiomotor axons in vitro. In Slit1;Slit2 double mutants, branchiomotor
axons aberrantly enter the midline
109
Robos
In Robo1 and Robo2 mutants, or in chick embryos that express dominant-negative
Robo receptors, branchiomotor axons enter the midline and fail to reach exit
points
109
SEMA3A
Repels branchiomotor, visceral motor and somatic motor axons in vitro. Sema3Amutant mice show defasciculation of cranial motor nerves
Neuropilin 1
Neuropilin 1 mutants show defasciculation of cranial motor nerves
SEMA3F
SEMA3F repels trochlear motor axons in vitro or in the chick embryo in vivo. In
Sema3F mutants, the trochlear nerve fails to exit and the oculomotor nerve is
defasciculated
Neuropilin 2
In Neuropilin 2 mutants, the trochlear nerve fails to exit and the oculomotor nerve
is defasciculated
FGF8
FGF8 attracts trochlear axons, causing them to exit the brain
148
Ephrin As
Overexpression of ephrin As in branchial muscle causes trigeminal motor axon
branching defects
143
EPHAs
Trigeminal motor neurons that express dominant-negative EPHAs show axon
branching defects
143
SDF‑1/CXCL12
In SDF‑1 mutants, abducens and hypoglossal axons project dorsally rather than
ventrally
124
CXCR4
The phenotype of CXCR4 mutants is identical to that of SDF‑1 mutants
124
migration. In some contexts, Unc5a can also bind to the
DCC (deleted in colorectal carcinoma) receptor, which
normally mediates netrin 1 attraction, to mediate repulsion120. In the fly embryo, UNC5A and DCC–UNC5A
mediate short-range and long-range repulsion of dorsally
projecting motor neurons, respectively121,122. A similar
possible role of netrin 1 in the rat embryo is implied by
the finding that BM and VM neurons express Unc5a
during early axon projection but co-express Dcc during
later axon extension110; however, this requires functional
confirmation.
It is currently unknown what molecules mediate
the floor plate repulsion of SM axons110, because only
BM and VM axons are repelled by netrin 1 and Slit proteins108,109. SM neurons do respond to SEMA3A, which is
expressed by the notochord and might have a function in
spacing apart the exiting abducens and hypoglossal SM
axons108,123 (FIG. 2c). Cranial SM axon exit from the hindbrain depends on the chemokine SDF‑1 (also known as
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References
79,85
94
138
107,108
108,134
135
149,151,152
149,150
CXCL12), which is expressed in the mesenchyme that
underlies the neuroepithelium, at least in the trunk124.
The receptor for SDF‑1, CXCR4, is expressed by spinal
and cranial SM neurons, but not BM or VM neurons, and
in CXCR4 mutants, spinal, abducens and hypoglossal SM
axons fail to project ventrally and instead extend across
the floor plate or project dorsally124. In other neuronal
types, SDF‑1 has been shown to attenuate responses
to repellents, such as Semaphorins or Slits, and so in
motor axons, this molecule might attenuate the effects of
Semaphorins or of unidentified SM floor plate-derived
chemorepellents124,125.
The exit point is a key guidepost. The next step in pathfinding is projection to the exit point. For BM and VM
axons, exit from the hindbrain appears to require the
presence of cranial sensory ganglia, which are apposed to
the large dorsal exit points. Ablation of these structures
leads to a reduction in peripheral axon projections126.
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The most proximal cells of the ganglion are specialized
boundary cap cells which, in chicks, arise from a lateemigrating population of cadherin‑7-expressing neural
crest cells127,128. Transplantation experiments in chick
embryos demonstrated that, when an odd-numbered
rhombomere is rotated 180o, most BM axons reorientate to navigate rostrally to their correct exit points129.
However, evidence for an exit point-derived chemoattractant has not been forthcoming, and the alar plate
itself is chemorepellent 130, possibly due to a narrow
stripe of Slit2 expression at the rhombic lip109. Thus, BM
and VM axon tracts might be hemmed in by floor plate
repellents medially and alar plate repellents dorsally.
The sensory ganglia exert only a weak chemoattractant influence on BM axons in vitro130, and the role of
boundary cap cells has yet to be explored, but this role
is likely to be pivotal, judging by the intriguing finding
that these cells limit spinal motor neuron emigration
from the neural tube131.
Rhombic lip
The structure at the dorsal
extreme of the hindbrain.
A balance of attraction and repulsion in the periphery.
Cranial motor axon behaviour in the periphery depends
on a balance of positive and negative influences. The
perinotochordal mesenchyme, which is derived from the
neural crest and differentiates into cartilage, repels spinal
motor axons132. Although this role has not been directly
demonstrated for cranial SM axons, motor axons avoid
the forming cartilage of the branchial arches, and the
parachordal cartilages, by channelling into the developing muscle plate103. Branchial arch neural crest cells also
express Semaphorins, including SEMA3A, and in mouse
mutants that lack Sema3A or neuropilin 1, cranial motor
axons become defasciculated and invade inappropriate
regions of the periphery133–135. The oculomotor nerve is
normal in Sema3A mutants, consistent with the observation that oculomotor axons are not repelled by SEMA3A
in vitro108,134. Skeletogenic cells that express Semaphorins
therefore influence peripheral axon pathways, and might
interact with growing nerves to influence the positioning of skull foramina, which are the mature pathways
of the cranial nerves. Secreted Semaphorins, especially
SEMA3A and SEMA3C, are expressed in particular
subsets of cranial motor neurons, where their function
remains to be explored111,112.
Chemoattraction by the branchial arches can also
strongly orient the trajectories of BM and VM axons130.
Hepatocyte growth factor (HGF) is expressed by the
branchial muscle plate at the time of axon extension and
accounts for most of this activity, as HGF presented on
beads is chemoattractant, and HGF-blocking antibodies
can eliminate a large portion of the arch-mediated chemoattraction. However, in HGF-mutant mice, BM and
VM axon trajectories are normal, and only SM hypoglossal pathways are affected, suggesting that there are other
branchial arch-derived chemoattractants130. Indeed, at
least three other neurotrophic factors are capable of
promoting cranial motor neuron outgrowth before they
become crucial factors for survival136. Brain-derived
neurotrophic factor (BDNF) has a strong effect on motor
axon outgrowth136, is expressed in the branchial arches
during early axon pathfinding, and has been shown to
nature reviews | neuroscience
chemoattract trigeminal sensory axons137. Therefore,
other neurotrophic factors are likely candidates in BM
and VM axon guidance.
The divergence of VM axons from their BM neighbours, for example, in the facial nerve, points to the
existence of distinct VM guidance cues. Indeed, r5derived VM axons navigate to their parasympathetic
ganglion targets by following chemoattractant signals;
genetic removal of the sphenopalatine ganglion in
Phox2a mutants results in the specific loss of the relevant
branch of the facial nerve138. The relevant chemoattractant molecules have not been identified, but the glial cell
line-derived neurotrophic factor (GDNF) family of
neurotrophic factors, which is implicated in the guidance and arborization of autonomic neurons, might be
involved139.
Motor pools and their target muscles
Little is known about how cranial motor axons recognize their target structures at appropriate axial levels.
For the spinal motor axon–limb system it has been proposed that major guidance information comes from the
lateral plate mesenchyme140. In the head, embryological
evidence strongly suggests that the neural crest from
a particular axial level patterns muscles, the myogenic
progenitors of which migrate from the same level102.
Fate mapping has shown that neural crest cells form
connective tissue sheaths and skeletal attachment points
around muscles derived from the same axial level42. The
neural crest might thus provide specific guidance information for BM axons and/or induce branchial muscles
to produce such guidance signals. However, the nature
of these guidance signals is currently unknown.
Following the rostrocaudal inversion of an oddnumbered rhombomere, trigeminal BM axons that
project to incorrect target muscles are eliminated, suggesting a specific recognition between BM neurons and
their targets141. Studies in fish and chicks suggest that
r2- and r3-derived regions of the trigeminal nucleus
contribute to separate subnuclei with different synaptic
targets142,143. Rhombomere 2- and r3-derived trigeminal
motor neurons express high and low levels of ephrin
A receptors, respectively, whereas r3 target muscle
showed patterned ephrin A expression143,144. Expression
of dominant-negative ephrin A receptors in r3 trigeminal motor neurons, or overexpression of ephrin As in
their target, led to aberrant axon branching patterns,
suggesting a role for ephrins in this topographic axon
targeting143.
Extraocular muscle innervation
The six eye muscles that rotate the eyeball and provide fine control of visual tracking movements are
innervated by three nerves. This is a promising system in which to study the mechanism that controls
nerve–muscle targeting, as well as a model that is of
clinical relevance to humans. The abducens nerve
innervates the lateral rectus muscle, the trochlear
nerve innervates the superior oblique (the dorsal
oblique in chicks) and the oculomotor nerve innervates the remaining four muscles, the medial, dorsal
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Congenital fibromatosis of
the extraocular muscles
(CFEOM). A group of
congenital syndromes that
involve cranial nerve miswiring
and paralysis or paresis of the
extraocular muscles, often
associated with drooping of the
upper eyelid.
Duane syndrome
A congenital eye movement
disorder characterized by
impeded horizontal eye
movements that resut from
miswiring of the eye muscles.
Horizontal gaze palsy with
progressive scoliosis
(HGPPS). A rare congenital
syndrome that is characterized
by the absence of conjugate
horizontal eye movements and
by deformities in the spine.
Möbius syndrome
A rare congenital disorder
caused by abnormal
development of the cranial
nerves, which results in paresis
or paralysis of the facial
muscles and, in some cases,
other abnormalities.
Marcus Gunn syndrome
Also known as jaw-winking
syndrome. It consists of an
elevation or depression of the
eyelid on chewing and/or
suckling, and is thought to be
caused by aberrant innervation
of branches of the trigeminal
and oculomotor nerves.
and ventral recti, and the inferior oblique. In chicks,
the lateral rectus primordium differentiates precociously relative to the other extraocular muscles, in a
position ventral to r2 and r3 (Ref. 145). From embryonic day three onwards, the abducens nerve extends
rostrally from its origin in r5 and r6, contacting the
lateral rectus primordium by embryonic day four146.
A double immunohistochemical study contrasted the
early abducens outgrowth with the delayed outgrowth
of the oculomotor nerve, which contacts its first and
most distant muscle target, the ventral oblique (VO),
by embryonic day five147. After the oculomotor nerve
reaches the VO, branches appear along the oculomotor
nerve to its other targets (the medial, dorsal and
ventral recti). It is not known whether this occurs
by interstitial branching of pre-existing axons or by
de novo growth of neurons. However, FGF8 and the
Semaphorins have been shown to function in trochlear
and oculomotor initial projections. The dorsal exit of
trochlear axons from the neuroepithelium depends
on attraction by the midbrain–hindbrain boundary
and FGF8 (Ref. 148). SEMA3F is expressed rostral and
caudal to the midbrain–hindbrain boundary and acts
through its receptor neuropilin 2 to guide the exit and
initial trajectory of trochlear and oculomotor nerves in
both mice and chicks149–152. SEMA3F repels trochlear
axons in vitro, and in neuropilin‑2- or Sema3F-mutant
mice, trochlear axons fail to exit the brain correctly
and the oculomotor nerve is defasciculated. The role
of SEMA3F in extraocular innervation thus provides
an interesting counterpart to the role of SEMA3A in
guiding the branchiomotor nerves. Guidance cues for
later growth and branching and for targeting of the
nerves III, IV and VI to specific extraocular muscles
remain to be characterized.
Dysinnervation disorders in humans
It will be vital to unravel in more detail the principles
that underlie extraocular muscle wiring, in order
to understand a group of human syndromes termed
cranial dysinnervation disorders (CDDs6,153). These
disorders are characterized most notably by deficits
of horizontal eye movements (complex strabismus),
and include defects such as congenital fibromatosis
of the extraocular muscles (CFEOM) , Duane syndrome ,
horizontal gaze palsy with progressive scoliosis (HGPPS),
Möbius syndrome and Marcus Gunn syndrome. In some
cases these disorders are congenital, and it has been
proposed that they are caused primarily by defects in
cranial motor neuron development and axon navigation. Strabismus (squinting) often results from an
imbalance in the function of the lateral (innervated by
the abducens) and medial (innervated by oculomotor)
recti muscles, which respectively abduct and adduct
the eyeball. Familial studies, including linkage screens,
and mapping to candidate genetic loci have implicated
five different genes in five separate syndromes. These
are HOXA1, PHOX2A, SALL4, KIF21A and ROBO3
(also known as RIG1) 154–158. Three of these genes
– HOXA1, PHOX2A and SALL4 – are transcription
factors, one is involved in axonal transport (KIF21A)
868 | november 2007 | volume 8
and one is an axon guidance molecule (ROBO3),
supporting the notion of a developmental basis for
congenital CDDs.
Mutations in HOXA1 have been shown to result in
two overlapping syndromes, Bosley-Salih-Alorainy syndrome (BSAS) and Athabascan brainstem dysgenesis
syndrome (ABDS)154. These disorders are characterized by impaired horizontal eye movements, ear and
vascular defects and, in some cases, autism or mental
retardation. In Hoxa1-knockout mice, the abducens
nerve is lacking50, and so it is likely that abducens development and consequent innervation of the lateral rectus
muscle is aberrant in BSAS and ABDS patients. Clinical
characterization of a loss of ocular motility, especially
abduction (which is mediated by the abducens nerve),
broadly agrees with this interpretation159. Another
congenital CDD, Duane syndrome type 2 (DURS2),
is also characterized by an absence or reduction of the
abducens nerve, whereas in some cases the oculomotor nerve branches aberrantly into the lateral rectus
muscle160,161. This innervation pattern bears a striking
resemblance to the pattern that is seen in Hoxa3- and
Hoxb3-mutant mice, which lack the abducens nerve
and manifest aberrant innervation of the lateral rectus
through another nerve of uncertain origin71. However,
there is no published genetic evidence to link Hox3 or
other genes with DURS2.
CFEOM2, a CFEOM variant which involves fibrosis of the extraocular muscles and severe loss of ocular
motility, has been linked to mutations in PHOX2A155.
Neuroimaging data show that patients lack the oculomotor and trochlear nerves 162, a phenotype that
is identical to that of Phox2a-mouse mutants19. This
suggests that the primary defect is neural, and that the
extraocular muscle atrophy that occurs in CFEOM
might occur secondarily to the lack of innervation.
Similarly, patients with CFEOM1 were found to have
hypoplasia of the oculomotor nerve, and in some cases
of the abducens nerve163. CFEOM1 has been shown to
result from mutations in the kinesin motor protein
KIF21A 156, which has a role in anterograde axonal
transport164.
The rare syndrome HGPPS involves the absence of
the conjugate horizontal eye movements that are mediated by the abducens and oculomotor nerves, as well as
scoliosis, and is caused by mutations in ROBO3 (Ref. 158).
ROBO3 is required for axons to cross the midline and
form commissures116, which are absent in the brainstems
of HGPPS patients. It is likely that horizontal eye movement disturbances result from the failure of supranuclear
tracts, such as the paramedian pontine reticular formation, to cross the midline and innervate the abducens
and oculomotor nuclei158.
A combination of clinical genetics and developmental neurobiology might in future further illuminate
the causes of CDDs in humans. In some CDDs, such
as Möbius syndrome, an association with autism has
been proposed165, suggesting that disorders of cranial
motor neuron development might have far-reaching
significance for understanding human disorders of
brain wiring.
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REVIEWS
Future prospects and challenges
Great progress been made in understanding how rostrocaudal and dorsoventral patterning processes regulate
cranial motor neuron specification. However, there is a
sizeable gap in our knowledge that concerns how combinations of transcription factors govern the expression of
axon guidance receptors and motor axon pathway decisions. There are few characterized head-specific guidance cues, which stems from the lack of basic knowledge
about tissue patterning in the head. Glial cells are likely
to have a key role in cranial motor axon guidance, but
the idea that they might be molecularly heterogeneous
has not been addressed. Similarly, the specific character of the extraocular versus the tongue and branchial
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Acknowledgements
Thanks to A. Lumsden and R. Knight for critical comments on
the manuscript.
DATABASES
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query.
fcgi?db=gene
BDNF | cadherin‑7 | CXCR4 | CYP26 | DCC | EGR2 | FGF8 |
Gata2 | Gata3 | HGF | Hoxa1 | HOXA1 | Hoxa2 | Hoxa3 | Hoxb1 |
hoxb1a | Hoxb2 | Hoxb3 | Hoxd3 | Isl1 | Isl2 | KIF21A | Lhx3 | Lhx4
| MAFB | MNR2 | MNX1 | netrin 1 | neuropilin 1 | neuropilin 2 |
Nkx2.2 | Nkx2.9 | Nkx6.1 | Nkx6.2 | Olig2 | Pax6 | pbx4 | Phox2a |
PHOX2A | ROBO1 | ROBO2 | ROBO3 | ROBO3 | SALL4 | SDF‑1
| SEMA3A | SEMA3C | SEMA3F | Slit2 | SHH | Tbx20 | UNC5A |
Unc5c
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