ReVieWs

Roles of axon guidance molecules

in neuronal wiring in the developing
spinal cord
Alain Chédotal   
Abstract | The spinal cord receives, relays and processes sensory information from the periphery
and integrates this information with descending inputs from supraspinal centres to elicit precise
and appropriate behavioural responses and orchestrate body movements. Understanding how
the spinal cord circuits that achieve this integration are wired during development is the focus
of much research interest. Several families of proteins have well-​established roles in guiding
developing spinal cord axons, and recent findings have identified new axon guidance molecules.
Nevertheless, an integrated view of spinal cord network development is lacking, and many
current models have neglected the cellular and functional diversity of spinal cord circuits.
Recent advances challenge the existing spinal cord axon guidance dogmas and have provided
a more complex, but more faithful, picture of the ontogenesis of vertebrate spinal cord circuits.

Propriospinal neurons
Interneurons distributed along
the spinal cord that connect
multiple spinal cord segments
and project to the brain. They
play a role in the coordination
of locomotion and
proprioception.
Ventricular zone
(VZ). The cellular layer lining
the CNS ventricles that
contains proliferating neural
progenitors (radial glia).
Sorbonne Université, INSERM,
CNRS, Institut de la Vision,
Paris, France.
e-​mail: alain.chedotal@
inserm.fr
https://doi.org/10.1038/
s41583-019-0168-7
In mammals, the spinal cord is the main point at which
the somatosensory system enters the CNS, receiving
inputs from all the sensory neurons localized in the
30 (human) to 34 (mouse) dorsal root ganglia (DRG)
located on each side of the spinal cord. These neurons
convey somatosensory information, information about
noxious and non-​noxious stimuli and propriocep-
tive information. This sensory information is directly
transmitted to supraspinal brain centres; however, it is
also processed and coded locally by microcircuits of
propriospinal neurons1 (Box 1). The spinal cord also con-
tains motor neurons, which are responsible for loco-
motion and for autonomic nervous system function.
The precise pattern and sequences of motor neuron
activation during movement are controlled by networks
of spinal cord neurons known as central pattern gene­
rators (CPGs)2–4. Spinal cord neurons and circuits also
receive extensive descending inputs from the motor
cortex and other supraspinal neurons, which modulate
skilled motor behaviour5,6 and the sensation of touch7
(Box 2). In addition to increasing our knowledge of these
key physiological functions, understanding how spinal
cord circuits form is essential for the development of
new therapeutic strategies for the treatment of spinal
cord injury8–11 and pain and may help to delineate the
aetiological bases of congenital movement disorders12.
The cellular and molecular mechanisms that control
the wiring of spinal cord circuits during development
have been studied for several decades. A particular focus
has been to understand the mechanisms of spinal cord
axon laterality4,13–15 — the decision made by neurons to
extend their axon on the same side (ipsilateral) or the
opposite side (contralateral) of the spinal cord with
respect to their cell body. A number of protein fami-
lies have been assigned now well-​established roles in
this process (Table 1). However, recent observations
have demonstrated that a large spectrum of molecules,
including lipids, sugars and peptides, work together
with these canonical families of axon guidance mol­
ecules (Table 1) to guide axon wiring. The palette of cells
that are known to provide cues to spinal cord axons
has also been extended (Table 1). Recent studies have
also highlighted the singularity of spinal cord network
ontogenesis in comparison with other regions of the
nervous system; as described above, local spinal cir-
cuits integrate inputs from peripheral sensory afferents
with descending tracts from the brain and contribute
to (motor) efferent pathways that leave the CNS, mean-
ing that developing axons need to cross boundaries and
extend over longer distances than other axons to reach
their targets. In this Review, I aim to highlight the main
principles and signalling pathways involved in the devel-
opment of mammalian spinal circuits. I will primarily
focus on studies in the mouse, which is currently the
best-​u nderstood mammalian model and the most
amenable to genetic manipulations.
How motor axons escape the spinal cord
In the mouse spinal cord, the first motor neurons are
generated around embryonic day 9.5 (E9.5) from pro-
genitors in a ventral domain of the ventricular zone
(VZ)16–18. Their axons extend away from the floor plate

Nature Reviews | Neuroscience

Reviews
Box 1 | classification and organization of spinal cord neurons
the adult spinal cord grey matter is classically divided into ten superposed domains
(laminas i–iX and area X) that are defined by the density, dendritic morphology and
electrophysiological properties of their neurons1,209. However, genetic mouse models
and single-​cell profiling have recently revealed a much larger diversity of spinal cord
neuron subtypes.
understanding this diversity is essential to understanding the spinal cord wiring
diagram and how it develops. it is known that spinal cord neurons are generated in
11 distinct ventricular zone progenitor domains, defined by the expression of a
particular combination of transcription factors and organized along the dorsoventral
axis of the spinal cord210–212. However, with a few exceptions2,212, the lineage of adult
spinal cord neurons has not been reconstructed.
the overall pattern and diversity of neuronal classes appears to be conserved along
the rostro-​caudal axis of the spinal cord, although there is variation in the relative
proportions of each class75,213.
the ventral horn contains two types of motor neuron, α-​motor neurons and γ-​motor
neurons, which project to extrafusal and intrafusal muscle fibres, respectively.
Motor neurons innervating the same muscle (which may be up to 60 for limb muscles)
are clustered in pools within longitudinal columns occupying precise locations along
the dorsoventral, mediolateral and longitudinal axes of the spinal cord. The medial
motor column exists at all spinal cord levels, whereas the lateral motor column is found
only at limb levels and the hypaxial and preganglionic motor columns are present only
at thoracic levels214.
Other spinal cord neurons integrate sensory and supraspinal inputs and modulate
motor neuron activation. there are multiple and intertwined classifications of these
neurons that are based on their cell lineage (resulting in dI1–dI6, dL1A, dL1B and
V0–V3c classes), their influence on their postsynaptic partners (excitatory versus
inhibitory) and the neurotransmitters that they release (GABAergic, glycinergic,
cholinergic or glutamatergic), their sensory inputs (proprioceptive or cutaneous, for
example), their axon laterality (ipsilateral, contralateral or bilateral), their axonal length
(segmental or intersegmental), the longitudinal orientation and branching pattern of
their axons (ascending, descending or bifurcating), their connections with supraspinal
neurons (projection neurons versus local propriospinal neurons), their synaptic partners
(premotor versus last-​order interneurons; directly connected (or not) to motor neurons)
and their dendritic architecture1,42,83,154.
Recent studies have further extended our understanding of spinal cord neuron
diversity. single-​cell profiling and cluster analysis of adult mouse spinal cord neurons
has identified between 28 (ref.215) and 42 (ref.216) distinct neuronal types in addition to
motor neurons. Another study reported at least 30 genetically distinct neuronal types
in the dorsal horn alone217, half of which were inhibitory and half excitatory. Although
these neuronal classes tend to be organized in specific patterns (including distinct
dorsoventral layers for glutamatergic neurons217), neurons sharing similar molecular
properties are found in diverse parts of the spinal cord and their distribution has no
obvious overlap with the classic cytoarchitectonic laminas. in other studies, 11 classes
of cutaneous axon recipient neurons have been characterized using transgenic mice47,
and progenitors from the v1 domain alone have been estimated to produce 50 different
neuronal types expressing different combinations of 19 transcription factors75,218.
Cross analysis of these data will be required to determine how many different
neuronal classes exist and their function in spinal cord microcircuits. Similar cell
classification studies will also need to be conducted in the developing spinal cord to
reconstruct spinal cord cell lineages.
to reach a ventral exit point, where they break through
the basal lamina and leave the CNS19. By contrast, motor
neuron cell bodies are prevented from leaving the spinal
cord by so-​called boundary cap cells, which surround
nerve roots and express repulsive factors such as sema­
phorin 6A (SEMA6A)20,21. The cellular and molecu-
lar mechanisms that allow motor axons to breach the
CNS–peripheral nervous system (PNS) boundary, which
is composed of the glia limitans (the endfeet of radial
glia and/or neuronal progenitors) and the abutting
basal lamina, are largely unknown19; however, there is
evidence from studies in Xenopus laevis embryos that
this process requires matrix degradation and that motor
neuron growth cones form invadosomes (cellular struc-
tures essential for normal and pathological cell invasion)
to exit the spinal cord22. Most motor axons leave the CNS
ventrally; however, at cervical (C) levels (comprising
spinal nerves C1–C4 in rodents), a subset of motor neu-
rons, the spinal accessory motor neurons (SACMNs),
project via a dorsolateral exit point to innervate two
neck and back muscles23 (Fig. 1). How motor axons nav-
igate outside the CNS to their muscle targets has been
extensively reviewed and is not discussed here4,15.
Newly born motor neurons express the netrin 1
receptors deleted in colorectal cancer (DCC)24,25, neo-
genin26, UNC5A and UNC5C26–28 and the slit homo-
logue (SLIT) protein receptors roundabout homologue 1
(ROBO1) and ROBO2 (refs 25,29,30) (Table  1) . These
ligand–receptor pairs control motor axon pathfinding
within the spinal cord, acting at multiple levels and in an
intertwined manner. In commissural axons, the interac-
tion between netrin 1 and DCC is known to guide axons
towards the midline31,32. Unexpectedly, despite also
expressing DCC, motor neuron axons initially extend
away from the floor plate, suggesting that they are, at
least at this stage, unresponsive to netrin 1. An explana-
tion for this paradox has been provided by the findings
of a genetic screen in mice25. This screen revealed that,
in mice lacking expression of both Robo1 and Robo2,
some motor axons are unable to leave the CNS and
many extend across the floor plate25 (Fig. 1). As motor
neurons are known to express SLIT2 and SLIT3 (ref.33)
in addition to ROBO1 and ROBO2, it was proposed that
SLIT proteins released by motor neurons trigger — in
an autocrine and/or paracrine manner — the silencing
of DCC by ROBO1 and/or ROBO2, thereby prevent-
ing motor axons from extending towards the midline25.
Support for this idea comes from studies of presenilin 1
mutant embryos, in which the cleavage of the intracyto­
plasmic domain of DCC by presenilin 1 is absent and
DCC stubs that are unable to interact with ROBO1
or ROBO2 accumulate at the cell membrane. In these
embryos, ROBO1 and/or ROBO2 are therefore unable
to silence DCC, causing abnormal attraction of motor
axons to the midline25,34.
Interestingly, at later stages, motor neuron axons pro-
jecting both dorsally and ventrally respond to netrin 1
and SLIT proteins; however, their responses differ.
In Dcc and Ntn1 knockout mice, the exit point of the
axons of ventral motor neurons (vMNs) is shifted dor-
sally35, suggesting that they are normally guided ven-
trally by netrin 1, whereas axons from SACMNs extend
towards the floor plate, indicating that they are repelled
dorsally by netrin 1 (ref.24). Likewise, vMN axons exit
the spinal cord more ventrally in Slit1 and Slit2 or Robo1
and Robo2 double knockout mice35, suggesting that SLIT
proteins drive repulsion from the midline, whereas
SACMN axons appear to be attracted by SLIT proteins
expressed around their dorsal exit point36 (Fig. 1). What
could explain this differential behaviour? First, mouse
SACMNs show higher levels of expression of the UNC5C
receptor, which seems to mediate repulsive responses to
netrin 1 (ref.28). Second, the G protein-​coupled receptor
(GPCR) CXC-​chemokine receptor 4 (CXCR4) is tran-
siently expressed by newborn vMNs and its ligand, the
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Box 2 | organization of spinal cord longitudinal tracts
some spinal cord neurons are connected to target neurons within the same or adjacent
spinal cord segments; however, many project their axons along tracts running along the
longitudinal axis of the spinal cord in a caudal or, most often, anterior direction,
sometimes reaching supraspinal levels. These longitudinal tracts contain primary axon
branches from sensory neurons, axons from ipsilateral and contralateral spinal cord
projection neurons and descending axons from the brain.
ipsilateral projections
The dorsal funiculus (DF), also known as the dorsal or posterior column, is a mixed tract
containing several types of axon. These include dorsal root ganglion (DRG) axons from
proprioceptive and Aβ rapidly adapting low-​threshold mechanoreceptor (LTMR)
neurons, which form the direct dorsal column (DDC) pathway and project to the dorsal
spinal cord and hindbrain in a somatotopic manner. Axons coming from DRGs below
the thoracic nerve 6 (T6) are positioned medially in the gracilis fasciculus, whereas
those from neurons above T6 occupy a more lateral position in the cuneatus fasciculus.
In each fascicle, ascending Aβ and proprioceptive axons are segregated, with LTMR
axons extending for a long distance in a rostral direction and occupying a medial
position and proprioceptive axons occupying a lateral position and extending only over
a few segments219. As a result, only proprioceptive axons from above T6 reach the
hindbrain. The DF also contains axons from dorsal spinal projection neurons (most
originating from lamina iv). these form the postsynaptic dorsal column (PsDC)
pathway, which also projects to the gracilis and cuneatus hindbrain nuclei. in the DF,
PSDC axons are positioned and grow under DDC axons182,183,219.
the dorsal spinocerebellar tract occupies the dorsolateral part of the lateral funiculus
and contains axons originating from Clarke’s column neurons in the medial part of
lamina VII of the spinal cord, at the thoracic and upper lumbar level (T1–L3). Clarke’s
column neurons directly relay proprioceptive information from the hindlimbs and the
lower part of the body to the anterior cerebellum. they also receive major input from
the corticospinal tract (Cst)220–222.
At more rostral levels, the cuneocerebellar tract relays proprioceptive information
from the forelimbs to the cerebellum.
Decussations and contralateral projections
Longitudinal tracts also contain axons from ventrally projecting commissural neurons.
For example, the contralateral ventral spinocerebellar tract (vSCT) originates from
neurons within laminae vi to viii of the spinal cord and mostly conveys proprioceptive
information from the lower limbs and lower trunk. the vsCt projects to the cerebellum,
where it re-​crosses the midline220.
The lateral spinothalamic tract (also known as the anterolateral tract) contains axons
that carry pain and temperature information and that predominantly originate from
contralateral projection neurons in lamina i of the spinal cord223. these neurons project
anteriorly for 1–2 segments in Lissauer’s tract and then decussate in the ventral
commissure to ascend to the ventral posterolateral thalamic nucleus. the anterior
spinothalamic tract, on the other hand, conveys information about light touch and
extends more ventrally. Its neurons reside in lamina III–V of the spinal cord and project
to the contralateral thalamus via the ventrolateral funiculus32,46.
Descending axons from the brain and supraspinal inputs
The CST occupies the most ventral part of the DF and contains axons from layer V
neurons of the contralateral primary (M1) and secondary (M2) motor cortices and from
the somatosensory cortex (S1). A subset of corticospinal axons also project medially
and anteriorly, including some with cell bodies residing in the ipsilateral cortex189.
M1 and M2 neurons connect with premotor neurons and Clarke’s column neurons221,
whereas S1-derived CST axons target neurons in laminas III–V of the spinal cord that
are also innervated by LTMRs7,47.
Other descending axons originate from the raphe nucleus, hypothalamus, vestibular
and spinal trigeminal nuclei, pons and red nucleus, among others224–226. How these
axons are guided within the spinal cord is unknown.
CXC-​chemokine ligand 12 (CXCL12), is present in the
mesenchyme and meninges surrounding the ventral
spinal cord37,38 (Fig. 1). In Cxcr4 and Cxcl12 knockout
Marginal zone mice, vMN axons are misrouted dorsally, suggesting that
The cell-​poor superficial region
of the developing CNS that is
CXCR4–CXCL12 signalling normally inactivates a ven-
located under the basal tral repulsive signal, inactivates a dorsal attractive sig-
lamina. nal or attracts motor axons to spinal exit points (Fig. 1).
Nature Reviews | Neuroscience
Reviews
Although the underlying mechanisms remain elusive,
CXCR4 has been shown to interact with ROBO139, and
there is evidence suggesting that CXCL12 antagonizes
SLIT protein-​mediated repulsion39,40. In addition, signal
transducing adaptor molecule 1 (STAM1), a compo-
nent of an endosomal sorting complex that is required
for intracellular protein transport, plays a role in vMN
guidance by regulating the endosomal targeting of
ubiquitinylated CXCR4 (ref.41).
Connecting with the periphery
Two main categories of sensory axons innervate the spi-
nal cord. Proprioceptive DRG neurons convey input to
motors and belong to three main classes. Type Ib DRG
neurons innervate the tendons, whereas type Ia and
II neurons innervate limb muscles and connect directly
with the 50–200 motor neurons that innervate the same
muscles4,42. In addition, the spinal cord is innervated
by DRG cutaneous neurons (comprising about 80% of
the DRG neurons43), which have a peripheral axonal
branch innervating the inner organs or skin and a cen-
tral branch projecting into the spinal cord in a somato-
topic manner44. These neurons are the entry point of the
somatosensory system and convey sensory modalities
such as pain, temperature and light touch45. Cutaneous
axons are connected to interneurons and projection
neurons in the dorsal spinal cord and some, such as
Aβ-​low-threshold mechanoreceptors (Aβ-​LTMRs), also
project directly to brainstem nuclei (Box 2). Cutaneous
axons terminate at different levels of the spinal cord in
a pattern that is specific to each modality46,47. A major
challenge has been to understand how these sensory
axons find their targets within the spinal cord.
Sensory axon bifurcation and the waiting period.
Mouse sensory axons reach the spinal cord dorsal root
entry zone (DREZ) around E10.5 but remain confined
to the marginal zone for a waiting period of several
days, forming the primordium of the dorsal funiculus
(also known as the oval bundle of His)48. Proprioceptive
and cutaneous afferents enter the cord at different dorso­
ventral positions, with proprioceptive afferents invad-
ing a more dorsomedial area of the dorsal horn, above
cutaneous axons49 (Fig. 2). How sensory axons find the
DREZ is largely unknown. However, a few studies sug-
gest that the initial patterning of sensory axons might
be shaped by guidance factors that are differentially dis-
tributed along the dorsoventral axis of the spinal cord49.
For example, the high level of netrin 1 in the ventral
spinal cord is thought to repel sensory axons, causing
them to extend in a dorsal direction50. In addition, the
glycerophospholipid lyso-​phosphatidyl-β-​d-glucoside
(LysoPtdGlc) is selectively released by dorsal spinal cord
progenitors and repels cutaneous nociceptive axons but
not proprioceptive axons49 (Fig. 2). LysoPtdGlc repul-
sion is mediated by the GPCR GPR55, and inhibition of
GPR55 signalling drives cutaneous axons to invade the
dorsal spinal cord in chick and mouse embryos49.
After entering the marginal zone, sensory axons
progressively arborize along the longitudinal axis
across adjacent segments (some covering up to eight
segments at E13.5) without leaving the presumptive
Reviews

dorsal funiculus and Lissauer’s tract48. However, dif-
ferent classes of sensory axons exhibit distinct branch-
ing profiles. In most cases, sensory axon growth cones
split51 into a caudal and a rostral branch, adopting a
T-​shaped or Y-​shaped morphology (Fig. 2). By contrast,
A∂-LTMRs and C-​LTMRs do not divide but simply
turn in a rostral direction44. What underlies these dis-
tinct branching behaviours is unknown; however, the
molecular mechanisms controlling growth cone split-
ting and the appearance of the first branch point are

Table 1 | Axon guidance molecules and their function in the development of spinal cord circuits
Axon guidance Location and cell type receptors and Function in spinal cord circuit development
molecules expression pattern co-receptors
Canonical axon guidance molecules
Netrin 1 Floor plate, VZ progenitors and the DCC34 CA confinement to the ventral spinal cord31,32 and CNS88,89,
(refs94,95) basal lamina72,90,94 sensory axon repulsion and50,58 motor axon midline repulsion25,35
UNC5C27 Sensory axon repulsion during waiting period58
Neogenin230,231 CA guidance in ventral spinal cord31,230
DSCAM , 232
Proposed role in CA guidance (mostly in vitro evidence235)
ADORA2B233 and APP234
ROBO3 (refs141,146) CA guidance in ventral spinal cord129,141
SLIT proteins33,236 Floor plate (SLIT1, SLIT2 and SLIT3) ROBO1 and ROBO2 Post-​crossing CA repulsion125,128–130, sensory neuron axon
and motor neurons (SLIT2 and (refs33,129,236) branching56 and motor neuron axon repulsion33,35
SLIT3)30,33,130
Plexin A1 (ref.132) Post-​crossing CA repulsion132
α-​Dystroglycan131 SLIT protein localization and CA repulsion131
Semaphorins
SEMA3A59 Ventral spinal cord, VZ and Neuropilin 1 and Cutaneous axon targeting60,62,63
astrocytes59,60 plexin A4 (ref.229)
SEMA3B125,126 Floor plate and VZ125,126,237 Neuropilin 2 (ref.238) Midline switch and post-​crossing CA repulsion125,126,133
and plexin A1 (ref.239)
SEMA3E240 Motor neurons76,78,79 Plexin D1 (refs77,239) Proprioceptive axon targeting78,79
SEMA6D240 Oligodendrocytes and the dorsal Plexin A1 (ref.239) Sensory axon pathfinding64,65 and CST axon pruning189
spinal cord64,65
Morphogens
SHH107 Floor plate107 BOC109 Pre-​crossing CA guidance109
PTCH1 and/or SMO Post-​crossing CA guidance
GDF7 and/or Dorsal spinal cord BMPR1A 116
CA dorsal repulsion113,115
BMP7 (refs113,115)
WNT proteins Floor plate FZD3 (ref.161) Longitudinal CA guidance161,163,165
RYK 190
CST axon guidance190
Other axon guidance cues
Draxin114 Dorsal spinal cord VZ114 DCC and netrin 1 CA dorsal repulsion (?), CA fasciculation (?) and netrin 1
(refs117–119) antagonism (?)114
LysoPtdGlc49 Dorsal spinal cord VZ49 GPR55 (ref.49) Sensory axon sorting49
Ephrin B3 (ref. ) Spinal cord midline glia
175 175,183
EPHA4 (ref. ) 175
Midline crossing (dorsal and ventral)175,176,183
NELL2 (ref.91) Motor neurons91 ROBO3 (ref.91) CA guidance in the ventral spinal cord91
VEGF 108
Floor plate 108
KDR 108
CA guidance in the ventral spinal cord108
CXCL12 (ref.37) Meninges and VZ37,38 CXCR4 (ref.37) Motor axon guidance37
GDNF 133
Floor plate 133
NCAM1 and/or GFRA1 CA guidance and midline switch133
(ref.133)
CNP52 Dorsal spinal cord52 NPR2 (ref.51) Sensory axon branching51,54
NT3 (ref. ) 70
Ventral spinal cord and/or muscle 43,70
NTRK3 (ref. ) 70
Sensory axon targeting70,71
IGF1 (ref.192) Spinal cord IGF1R CST axon guidance192 and sensory axon guidance182
ADORA2B, adenosine A2b receptor ; APP, amyloid precursor protein; BMP7 , bone morphogenetic protein 7; BMPR1A , bone morphogenetic protein receptor
type 1 A ; BOC, brother of CDO; CA , commissural axon; CNP, C-​type natriuretic peptide; CST, corticospinal tract; CXCL12, CXC-​chemokine ligand 12; CXCR4,
CXC-chemokine receptor 4; DCC, deleted in colorectal cancer ; draxin, dorsal inhibitory axon guidance protein; DSCAM, down syndrome cell adhesion molecule;
EPHA4, ephrin type A receptor 4; FZD3, frizzled 3; GDF7 , growth differentiation factor 7; GDNF, glial cell line-​derived neurotrophic factor ; GFRA1, GDNF family
receptor-​α1; GPR55, G protein-​coupled receptor 55; IGF1, insulin growth factor 1; IGFR1, insulin growth factor receptor 1; KDR , kinase insert domain receptor ;
LysoPtdGlc, lyso-​phosphatidyl-β-​d-glucoside; NCAM1, neural cell adhesion molecule 1; NELL2, neural epithelial growth factor-​like-like 2; NPR2, cGMP-​producing
atrial natriuretic peptide receptor 2; NT3, neurotrophin 3; NTRK3, NT-3 growth factor receptor ; PTCH1, patched; ROBO, roundabout homologue; SEMA ,
semaphorin; SHH, sonic hedgehog; SLIT, slit homologue; SMO, smoothened; VEGF, vascular endothelial growth factor ; VZ, ventricular zone.

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Wild-type mice Slit1, Slit2 or Robo2 KO mice Cxcr4 or Cxcl12 KO mice

VZ progenitor
processes
RP DREZ
Spinal +
cord +
+
Meninges + DRG
–
–
– –
– – – – – – –
– – – – – – – –
– –– –
– – – – – – – –

FP
Mesenchyme
Ventral
Netrin 1 vMNs SACMNs +– SLIT proteins – CXCL12
nerve root

Fig. 1 | Guidance of motor axons to their exit points. In wild-​type mice (left panel), ventral motor neurons (vMNs) project
their axons to exit the spinal cord at a ventral nerve root; however, at cervical levels, a separate population of motor neurons,
spinal accessory motor neurons (SACMNs), exit the spinal cord dorsally at the dorsal root entry zone (DREZ)23. The position of
these motor neuron axon exit points is established by a balance of attractive and repulsive forces involving various guidance
molecules. Netrin 1 is expressed by the floor plate (FP) and by ventricular zone (VZ) progenitors, which extend processes to
the pial surface72,94,227. Although netrin 1 is a secreted protein, recent studies show that its diffusion is limited; it accumulates
at the basal lamina (left panel, arrowheads) and primarily acts as a short-​range and/or haptotactic cue72,90. Motor neurons
express the netrin 1 receptor deleted in colorectal cancer (DCC)25,35. Slit homologue (SLIT) proteins are found at the FP33 and
close to the DREZ36, where they respectively exhibit repulsive (indicated by –) or attractive (indicated by + ) activities. vMNs
also express SLIT proteins25,33, and all motor neurons express the SLIT receptors roundabout homologue 1 (ROBO1) and
ROBO2 (not shown)25,33. In the absence of SLIT–ROBO signalling (in Slit1, Slit2 or Robo2 knockout (KO) mice, for example),
vMN axons are misguided; the ventral root is shifted ventrally (middle panel, dashed arrow)35, and some motor axons project
across the ventral midline (middle panel, arrowhead)25, whereas a subset of SACMNs fail to reach their dorsal exit point
(middle panel, arrow)36. Ventrally , the mesenchyme and meninges (left panel, dashed line) surrounding the spinal cord also
secrete the CXC-​chemokine ligand 12 (CXCL12), whose receptor CXC-​chemokine receptor 4 (CXCR4) is expressed by
vMNs37. In Cxcr4 and Cxcl12 KO mice, some vMNs aberrantly follow an SACMN trajectory (right panel, arrowhead) or project
dorsally after exiting the CNS (right panel, arrow)37. DRG, dorsal root ganglia; RP, roof plate.

better understood. C-​type natriuretic peptide (CNP) is
enriched in the dorsal spinal cord and plays a key role
in this process. In Cnp mutant embryos, sensory axons
fail to split and grow either rostrally or caudally52 (Fig. 2).
The contribution of CNP to branching involves the
cGMP-​producing atrial natriuretic peptide receptor 2
(NPR2)51. NPR2 activation generates cGMP, which
activates the α-​isoform of cGMP-​dependent protein
kinase I (PRKG1α) and downstream factors (possibly
glycogen synthase kinase 3β (GSK3β)) that ultimately
trigger growth cone bifurcation53. Accordingly, sensory
axons fail to form first-​order branches in Npr2 and Prkg1
mutant mice51,53, although collaterals still bud from the
axon shaft (albeit prematurely) and both cutaneous and
proprioceptive axons follow a normal pathway to their
spinal cord targets54.
Although in vitro experiments had first suggested
that SLIT proteins, which are expressed near the
DREZ, promote sensory axon branching55, first-​order
branches develop normally in Slit1 and Slit2 double
knockout mice and Robo1 and Robo2 double knock-
out mice56 (Fig. 2). However, in these mice, one of the
sister branches deviates from the dorsal funiculus to
penetrate the dorsal spinal cord56. Why a single branch
is misguided in these mutants is unclear, but these
findings suggest that, after bifurcation, the two sister
branches might differentially respond to longitudinal
guidance cues.
Development of collateral branches and penetration
of the cord. What are the mechanisms controlling the
formation of collateral, or side, branches from axons in
the dorsal funiculus? As mentioned above, the sensory
axon growth remains confined to the oval bundle of
His during a waiting period of at least 2 days in mice.
Proprioceptive axons start arborizing second-​order
branches first, around E13.5, followed by cutaneous
afferents from about E14.5 to E15.5 (refs48,55). This
observation suggests that repulsive signals might initially
restrict the sprouting of second-​order collateral branches
from the longitudinal axon shaft into the grey matter.
One candidate repulsive signal is netrin 1, which is
expressed at the DREZ around E11.5 (refs57,58). Netrin 1
has been proposed to block the development of axonal
collaterals via UNC5C because sensory axon collater-
als form prematurely in Ntn1 and Unc5c mutant mice58.
However, the extension of sensory axons along the
rostro-​caudal axis is also altered in these mice, suggest-
ing that netrin 1 influences the growth and bifurcation
of first-​order branches rather than collateral branches.
During the next phase of axon growth (between
E13.5 and E18.5), sensory axons navigate across the
spinal cord to connect with their neuronal targets. As
discussed above, cutaneous and proprioceptive axons
are pre-​patterned in the dorsal funiculus, and the axon
collaterals respect this initial topography with cutane-
ous axons growing directly to the dorsal horn laminas

Nature Reviews | Neuroscience

Reviews

Wild-type mice Gpr55 KO mice Npr2, Prkg1 or Cnp KO mice, or

Anterior Slit1 and Slit2 double KO mice
Dorsal
spinal
Posterior cord

Dorsal RP

DREZ

Cutaneous
neuron
Proprioceptive
DRG neuron
Ventral FP
LysoPtdGlc CNP SLIT proteins SEMA3A

Fig. 2 | The development of sensory projections. The dorsal root ganglia (DRG) on both sides of the spinal cord
contain proprioceptive and cutaneous neurons, whose axons enter the spinal cord at the dorsal root entry zone (DREZ).
Both types of axon bifurcate upon entering the cord and elongate two branches extending in opposite directions along
the anteroposterior axis of the spinal cord. The left panel illustrates the expression pattern of the main guidance cues
involved in DRG axon guidance. Cutaneous axons are positioned below proprioceptive axons, outside a domain
containing the soluble lipid lyso-​phosphatidyl-β-​d-glucoside (LysoPtdGlc), which selectively repels these axons49.
The repulsive activity of LysoPtdGlc is mediated by the GPR55 receptor, expressed by cutaneous neurons. In Gpr55
knockout (KO) mice (middle panel), the position of the cutaneous axons is shifted dorsally (arrow)49. The bifurcation of
sensory axons is triggered by C-type natriuretic peptide (CNP), which is expressed in the dorsal spinal cord52 and is
mediated by the cGMP-​producing atrial natriuretic peptide receptor 2 (NPR2) and the α-​isoform of cGMP-​dependent
protein kinase I (PRKG1α), expressed by sensory neurons51,54,228. In mice lacking any one of these proteins, sensory axons
fail to bifurcate (right panel) and extend in only one direction51,52,54,228. Slit homologue (SLIT) protein repellents are present
near the DREZ, and DRG axons express roundabout homologue (ROBO) family receptors55,56. Both SLIT proteins and
ROBO receptors also influence the guidance of primary sensory axon branches in the dorsal funiculus56. Other guidance
cues are present in the embryonic spinal cord, such as the secreted semaphorin 3 A (SEMA3A), which forms a high
ventral–low dorsal gradient and repels cutaneous axons (Fig. 3). FP, floor plate; RP, roof plate.

and proprioceptive axons growing more medially before
turning towards the ventral spinal cord48 (Fig. 3a). This
patterning prompted a search for inhibitory factors that
prevent cutaneous axons from invading the ventral cord
and proprioceptive axons from arborizing the dorsal
cord. In retrospect, it is unsurprising that semaphorins,
the largest family of repulsive molecules, turn out to be
involved in both processes (Table 1).
At the time that sensory axons invade the spinal
cord59, the secreted semaphorin, SEMA3A, is present
only in the ventral half of the cord and is enriched in
astrocytes and VZ progenitors60. In vitro assays showed
that, at this stage, SEMA3A is a potent chemorepellent
for cutaneous, but not proprioceptive, axons59. Indeed,
only cutaneous axons express the SEMA3A recep-
tor neuropilin 1. This finding suggested that SEMA3A–
neuropilin 1 repulsion sculpts the basic dorsoventral
topography of sensory projections. The organization
of cutaneous projections in Sema3a and Nrp1 mutant
mice was either reported to be normal61 or to exhibit a
limited ventral ingrowth of nociceptive axons62; however,
the conditional ablation of SEMA3A from astrocytes
resulted in a significant ventral ingrowth of cutaneous
axons60 (Fig. 3a). Likewise, in Nrp1Sema- mutants, which
express a neuropilin 1 receptor that is unable to bind
SEMA3A63, a subset of cutaneous axons invade the
ventral cord. Whether the misrouted axons belong to a
specific subclass of cutaneous axons was not investigated.
Proprioceptive axons also express plexin A1, a recep-
tor of the transmembrane semaphorins SEMA6C and
SEMA6D (Table 1), which are both present throughout
the dorsal horn with the exception of a medial ‘corri-
dor’ that is followed by proprioceptive axons64,65. In mice
lacking either Plxna1 or Sema6d, proprioceptive axon
collaterals colonize the rest of the dorsal horn (Fig. 3a).
Interestingly, the arrangement of cutaneous axons in
these mice is also disorganized by the presence of ectopic
oligodendrocytes, which accompany and myelinate the
misguided proprioceptive axons. Ablating these ectopic
oligodendrocytes can restore a normal patterning of
cutaneous axons65. These studies therefore emphasize
the varied roles played by glial cells in the development
of sensory projections.
Finding target neurons in the spinal cord ‘haystack’.
After sensory axons have been channelled to their dorsal
or ventral domains, they need to identify and connect
with their proper targets. How cutaneous axons select
second-​order neurons is largely unknown, in line with
our poor understanding of the organization of dorsal
spinal cord microcircuits (the complexity and diversity
of which has just started to be apprehended; Box 1).
A recent study showed that each axon of the 5 known
classes of LTMRs contacts at least 4–11 types of dor-
sal spinal cord interneuron and that each interneuron
receives inputs from at least 2 different types of LTMRs47.
In addition, layer V of the dorsal horn was shown to
contain a small population of neurons expressing DNA-​
binding protein SATB2 that receive direct inputs from
proprioceptive neurons and the motor cortex as well
as indirect nociceptive inputs from dorsal spinal cord
interneurons66,67. Interestingly, in the absence of SATB2,

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 Reviews

a
Wild-type mice Astrocytic Sema3a KO mice or Nrp1Sema– mice Sema6d or Plxna1 KO mice

Cutaneous
neuron

Proprioceptive
neuron

b c
Brachial spinal cord Lumbar spinal cord
Wild-type mice Sema3e or Plxnd1 KO mice Wild-type mice Sema3e or Plxnd1 KO mice
Non-plexin
D1-expressing
neuron

Plexin D1-
expressing neuron
Extensor motor neurons Flexor motor neurons SEMA3E-expressing neurons

Fig. 3 | semaphorins control sensory axon guidance. a | Cutaneous axons arborize in the dorsal horn of the spinal cord,
whereas proprioceptive axons bypass the dorsal horn and follow a medial pathway before projecting towards the ventral
cord (left panel)48. Cutaneous axons express the semaphorin 3A (SEMA3A) receptor neuropilin 1 (NRP1)229 and are kept
away from the ventral spinal cord where SEMA3A is localized (Fig. 2). In mice in which SEMA3A–NRP1 signalling is
inactivated by knocking out Sema3a expression in astrocytes or knocking in a variant of NRP1 that is unable to bind
SEMA3A , cutaneous axons invade the ventral cord (middle panel)60,62,63. Semaphorins also shape the proprioceptive
axon trajectory. SEMA6D is found in the dorsal spinal cord and repels proprioceptive axons, which express its receptor
plexin A1 (PLXNA1)64,65. Proprioceptive axons aberrantly cross the dorsal spinal cord laminas in Sema6d and Plxna1
knockout (KO) mice and displace cutaneous axons (right panel). b,c | SEMA3E repulsion controls the development of
monosynaptic inputs to motor neurons. In the brachial spinal cord (part b), a subset of proprioceptive axons expressing
PLXND1 does not usually establish direct contacts with a motor neuron pool expressing SEMA3E78. In absence of SEMA3E
or PLXND1, this subset of proprioceptive axons forms abnormal synapses on these motor neurons (arrow)78. In the lumbar
spinal cord (part c), SEMA3E is selectively expressed by a pool of extensor motor neurons but is absent from an adjacent
pool of flexor motor neurons innervated by PLXND1-expressing proprioceptive axons79. In Sema3e and Plxnd1 KO mice,
these proprioceptive axons establish ectopic connections (arrow) with extensor motor neurons79.

these neurons are shifted laterally and their proprio-
ceptive inputs are significantly reduced, suggesting that
sensory axon connectivity could be regulated by the
position of second-​order neurons and layer-​specific or
neuron-​specific guidance cues68,69.
How proprioceptive axons select their spinal cord tar-
gets is better understood. After leaving the dorsal spinal
cord, the different categories of proprioceptive axons
make distinct pathfinding decisions and reach different
territories: type Ia axons preferentially connect with ‘self ’
motor neurons, which innervate a common or syner-
gistic extrafusal muscle and interneurons projecting
to ‘non-​self ’ motor neurons innervating antagonistic
muscles, whereas type Ib axons synapse on premotor
interneurons42. The growth programme controlling
the arborization of proprioceptive axons in the ventral
cord appears to be initiated by neurotrophin 3 via SAD
(synapses of amphids defective) kinases70,71.
The pattern of proprioceptive axons mirrors and
respects the topography of pools of motor neurons,
suggesting that proprioceptive axon wiring could be
instructed by motor neurons themselves. This hypo­
thesis was tested in forkhead box protein P1 (Foxp1)
knockout mice, in which motor neuron position is

Nature Reviews | Neuroscience

Reviews
randomized. In these mice, proprioceptive axons termi-
nated at the proper dorsoventral location and therefore
synapsed with motor neurons of the wrong identity69,
implying that unidentified non-​motor neuron-​derived
cues shape this sensory map. Interestingly, the hetero-
geneous dorsoventral expression of guidance factors
such as netrin 1 (ref.72), LysoPtdGlc49, SEMA3A60, reelin
and SLIT1 (ref.73) in radial glia and spinal cord astro-
cytes suggests that these cells could be major players in
sensory axon pathfinding.
Although motor neurons do not seem to control the
dorsoventral patterning of proprioceptive axons, they do
influence their anteroposterior targeting. In tetrapods,
the number of motor neuron pools is higher and their
3D organization is more complex at limb levels (lumbar
and brachial) than it is in the thoracic spinal cord.
Moreover, cervical proprioceptive axons do not contact
motor neurons in thoracic segments and vice versa74.
The homeobox protein HOXC9, a transcription factor,
is instrumental for this longitudinal patterning. In Hoxc9
knockout mice, in which thoracic motor neurons adopt
the characteristic features and molecular identity of limb
column motor neurons, cervical proprioceptive axons
extend more caudally to colonize thoracic levels, where
they coexist with properly targeted thoracic axons74.
Therefore, motor neurons provide some topographical
information to proprioceptive axons. However, in Hoxc9
loss-​of-function experiments, there are many spinal neu-
rons whose identity is changed74,75; therefore, it is impor-
tant to be careful about ascribing all the phenotypes only
to the effects of motor neurons.
Motor neurons could also play a role in the selection
of motor neuron pool and of self and non-​self motor
neurons by proprioceptive axons. Once more, sema-
phorins and repulsive mechanisms are involved in this
process (Fig. 3b,c). During development, motor neuron
pools can be distinguished by their combinatorial expres-
sion of secreted semaphorins76. For instance, SEMA3F is
enriched in all lumbar motor neurons, whereas SEMA3E
is expressed in discrete motor pools at cervical, lumbar
and brachial levels. SEMA3E is unusual among secreted
semaphorins because it binds plexin D1 rather than
neuropilin 1 or neuropilin 2 (refs77,78). SEMA3E there-
fore restrains the monosynaptic connection of plexin
D1-expressing proprioceptive axons to a pool of brachial
motor neurons that innervate the cutaneous maximus78
(Fig. 3b). At the lumbar level, SEMA3E is expressed in
a motor neuron pool that innervates an extensor mus-
cle but is absent from a neighbouring pool connected
to a flexor muscle79. Proprioceptive axons innervating
the two motor pools differentially express plexin D1,
with the lowest level of expression in axons innervating
the SEMA3E-​expressing pool. In the absence of plexin
D1 or SEMA3E, the proprioceptive axons that normally
innervate the flexor muscle aberrantly innervate both
pools of motor neurons79 (Fig. 3c).
Together, these data suggest that, to a large extent,
sensory axon guidance within the spinal cord primarily
relies on repulsive forces that exclude axons from specific
spinal cord territories or motor neuron pools. Therefore,
and unexpectedly, there is currently no direct evidence
for chemoaffinity-​based processes in the wiring of spinal
cord circuits, unlike in other parts of the CNS (such as
the visual system80).
Bridging the left and the right
Commissural neurons project to targets on the con-
tralateral side of the CNS; however, they do not form a
homogeneous population and their trajectories depend
on their site of origin and birthdate81–83. Some cross
the midline ventrally (others dorsally), some project
to both sides of the spinal cord and some turn ros-
trally after crossing (others ventrally). In addition, they
originate from multiple progenitor domains along the
dorsoventral axis of the spinal cord VZ83 (Boxes 1,2).
Interestingly, although axon laterality in these neurons
appears to be genetically encoded, it is also influenced
by exogenous factors; the proportion of premotor com-
missural neuron inputs (originating from premotor spi-
nal interneurons in the deep dorsal and ventral horns)
received by motor neurons depends on their identity and
position along the spinal cord74. Recent data also sug-
gest that commissural axons influence the positioning of
both ipsilaterally and contralaterally projecting interneu-
rons in the ventral spinal cord by providing a substrate
for their migration84.
Very early in development (around E9.5 in the
mouse) spinal cord neurons start extending their axons
and immediately face a major dilemma: to cross or not to
cross the midline. Until about E14, spinal cord commis-
sural axons primarily cross the midline ventrally, at the
floor plate; however, at later stages, crossing also occurs
dorsally, above the central canal85. The mechanisms
gover­ning such midline crossing have been the subject of
intensive study for many years. However, the findings
of recent studies have questioned our understanding of
this developmental process.
Guiding commissural axons to the ventral midline.
The axons of the first commissural neurons grow at a
superficial position86, underneath the pial surface and
glial endfeet, but remain in the CNS. The co-​culture
of dorsal spinal cord explants with tissue from the
meninges showed that they secrete factors repelling
commissural axons87. This observation suggested that
repulsive factors secreted by meningeal cells could con-
fine commissural axons to the spinal cord87. However,
recent findings suggest that growth-​promoting factors,
in particular netrin 1, are also essential to keep commis-
sural axons inside the spinal cord88,89. In Ntn1 mutant
mice, a signi­ficant fraction of commissural axons trans-
gress the CNS–PNS border to extend into the periphery
(Fig. 4). They leave preferentially through the DREZ, but
some also follow the ventral motor roots. A milder ver-
sion of this phenotype is also observed in Dcc knock-
outs, suggesting that this receptor is involved in this
process88,89. Netrin 1 is produced by VZ progenitors and
accumulates near their basal processes72,90. Thus, one
current model suggests that the preferential binding
and hapto­tactic extension of commissural axons on a
netrin 1-expressing substrate is the force that confines
them to the spinal cord (Fig. 4a).
After this initial phase of development, ventral com-
missural axons seem to lose their preference for pial
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Axonal adhesion
The process that allows axons
to adhere preferentially to
some cells or substrates
containing cell adhesion
molecules that exert adhesive
forces on the axon.
Fasciculation
The mechanism through which
growing axons extend along
other axons and adhere to
each other forming tracts and
bundles.
Nature Reviews | Neuroscience
netrin 1 and dive towards the floor plate. On their way,
they avoid the motor columns, suggesting that they
might be repelled from motor neurons; indeed, a can-
didate motor-​neuron-derived repellent, the secreted
glycoprotein neural epithelial growth factor-​like-like 2
(NELL2), has been recently characterized91. However,
most commissural axons still grow above the motor
columns in Nell2 knockout mice, suggesting either
that other factors act redundantly with NELL2 or that
NELL2 influences only a subset of commissural axons91.
Importantly, many commissural axons from neurons
born at later stages (E13 and older) do extend across
the motor column before entering the midline92, indi-
cating that these late-​born commissural axons are not
responsive to NELL2 repulsion (Fig. 4b).
Why do commissural axons grow ventrally to the
midline? The remarkable ventral growth of commis-
sural axons led Ramon y Cajal93 to postulate that they
follow gradients of chemoattractive factors secreted by
the floor plate. Almost one century later, netrin 1 was
identified94–96 and fulfilled all of the criteria expected
from a bona fide commissural axon chemoattractant,
particularly the ability to attract axons when presented in
a gradient. Moreover, midline crossing was found to be
severely reduced in mice lacking netrin 1 or its receptors
DCC and neogenin31,32,57,97–100. These results (together
with similar observations made in other bilateria) laid
the basis for a well-​accepted model in which floor plate-​
derived netrin 1 is a long-​range diffusible cue for spinal
cord commissural axons13,101,102.
However, other observations suggest that we need to
reconsider this model. There was evidence from in vitro
assays and experiments in Drosophila melanogaster that
substrate-​bound netrin 1 might be as efficient (or even
more efficient) at guiding axons as soluble netrin 1 and
that netrin 1 could also act at short range to promote
axonal adhesion or fasciculation103–105. More recent genetic
studies in mice have shown that commissural axons
reach and cross the midline in mice selectively depleted
of floor plate netrin 1, further questioning its ability to
act at a long distance72,90,92,106.
In the spinal cord (but not in the hindbrain), floor
plate netrin 1 seems to influence some aspects of com-
missural guidance, such as axonal fasciculation or the
timing of midline crossing92. However, VZ progenitors
have now been identified as a major source of netrin 1
(refs 72,90,92) , as was previously envisaged 94 (Fig.  4a) .
Interestingly, midline crossing defects are mild in
the spinal cord of VZ netrin 1-depleted or floor plate
netrin 1-depleted mutant mice72,90,92, but crossing is
almost absent in the spinal cord of mice depleted of
netrin 1 from both sources92. This finding shows that
the two netrin 1 sources cooperate to guide spinal
cord commissural axons. A second, and non-​exclusive,
hypothesis is that additional floor plate chemoattractants
act redundantly with netrin 1. Sonic hedgehog (SHH)107
and vascular endothelial growth factor (VEGF)108 were
both shown to influence spinal cord commissural axon
guidance through receptors brother of CDO (BOC)109
and kinase insert domain receptor (KDR; also known
as VEGFR2 or FLK1)108, respectively. There is also evi-
dence from a microfluidic-​based assay for synergistic
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chemoattractive activities of SHH and netrin 1 on
commissural axons110 (Fig. 4a). Accordingly, some com-
missural axons are misrouted and extend across the
motor columns before reaching the midline in floor
plate netrin 1-depleted mutant mice and in netrin 1 full
knockout mice, and these guidance errors are more fre-
quent if BOC is simultaneously deleted92,106. The various
attractive signals, acting in the last 150–250 micrometres
before axons cross the floor plate106, could be integrated
by downstream effectors, such as SRC kinases, which are
common members of both SHH and netrin 1 signalling
pathways110,111.
Whether chemotactic gradients play a role in com-
missural axon guidance in the spinal cord therefore
remains to be established 112. Importantly, there is
evidence that commissural axons are expelled from the
dorsal spinal cord (rather than attracted ventrally) by two
categories of chemorepellents: bone morphogenetic pro-
teins (BMPs, such as BMP7 and growth/differentiation
factor 7 (GDF7) heterodimers)113 and the dorsal repul-
sive axon guidance protein draxin114. BMP7 and GDF7
are produced by the roof plate and repel commissural
axons that express their receptors BMP receptor type 1B
(BMPR1B) and/or BMPR1A 113,115,116. Commissural
axons are misrouted dorsally in Bmp7, Gdf7, Bmpr1a
or Bmpr1b knockouts115,116. Draxin is restricted to the
dorsal spinal cord at E10.5 and repels cultured commis-
sural axons114. However, these axons still extend ven-
trally in Draxin knockout mice, where they primarily
exhibit a defasciculation phenotype114. Current models
infer that draxin binds DCC117,118 and netrin 1 (refs118,119)
through two distinct domains and could thus antago-
nize the binding of netrin 1 to DCC, thereby blocking its
growth-​promoting activity119. Alternatively, draxin could
promote the fasciculation of DCC-​expressing axons by
inducing the trans-​interaction of draxin-​bound DCC
receptors with netrin 1-bound receptors118.
A midline switch to leave the floor plate. After they
have reached the floor plate, commissural axons quickly
change their behaviour; having entered the midline, they
then rapidly leave it to extend on the contralateral side
of the spinal cord. How this switch in responsiveness is
orchestrated remains a burning question despite years of
research120–122. The current consensus is that commissural
axons become unresponsive to floor plate attractants
and gain responsiveness to SLIT proteins and SEMA3B
repellents123–125 (Fig. 3a). Although this model probably
needs some adjustment in light of the findings that have
given us new information on the role of floor plate-​
derived netrin 1 (refs72,90), in vitro and in vivo data sup-
port SEMA3B and SLIT function in midline repulsion.
Commissural axon crossing defects were found in mice
lacking SEMA3B126, its receptor neuropilin 2 (refs125,127)
or its co-​receptor plexin A1 (ref.126) (Fig. 4b). Likewise,
crossing is affected in Robo1 or Robo2 knockout mice
and in Slit1, Slit2 and Slit3 triple knockout mice128,129.
Midline crossing defects are also detectable in mice with
a floor plate-​specific loss of expression of all three SLIT
proteins130. SLIT proteins are cleaved by unknown pro-
teases55 to produce a short carboxy-​terminal fragment
(SLIT-​C) and a long amino-​terminal fragment (SLIT-​N),
Reviews

a b Ntn1–/–, Ntn1∆FP+∆VZ
Wild-type mice or Dcc–/– mice Ntn1∆FP mice Ntn1∆VZ mice

VZ progenitor RP
processes

Spinal
cord
Commissural
neuron

–
+ +
– –
– –+
FP Slit triple KO, Robo1 and
Nell2–/–;Robo3+/–, Boc–/–, Robo3–/– or Dcc–/–;Neo1–/– Robo2 double KO, Plxna1–/–,
Pre-crossing receptors Post-crossing receptors Kdr–/– or Vegfa–/– mice mice Nrp2–/– or Sema3b–/– mice
• ROBO3 • ROBO1 and ROBO2
• DCC • Plexin A1
• BOC • NRP2
• KDR • FZD3
• BMPR1A • PTCH1/SMO
Netrin 1 + Growth-promoting cues
Motor neuron (SHH and VEGFA)
repellents Dorsal repellents (draxin, BMP7
GDNF and GDF7) – –
– Inhibitory FP cues (SLIT1, SLIT2, – –
– – – –
SLIT3, SEMA3B, SHH and – –– – ––
WNT proteins)

Robo1 and Robo2 double

c Wild-type mice Epha4 or Efnb3 KO mice Fzd3 KO, PCP KO or
KO or Slit triple KO mice
FP VF/LF Smo KO mice

Anterior
Posterior Dorsal
RP

Ventral
Ventral

FP

WNT proteins
SHH
Anterior
Ephrin B3
Commissural neuron
Ipsilateral neuron
CST axon
Posterior

d Ryk or Wnt loss-of- Robo1 and Robo2 double

Wild-type or Robo3 KO mice Epha4 or Efnb3 KO mice
function mutant mice KO mice
Dorsal

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◀ Fig. 4 | The development of contralateral and ipsilateral circuits. a | Commissural axons
(CAs) are guided towards the ventral midline by netrin 1 from the ventricular zone (VZ)
and floor plate (FP)57,72,227 and by sonic hedgehog (SHH)107 and vascular endothelial growth
factor (VEGFA)108, both expressed at the FP. They are repelled from the dorsal spinal cord by
dorsal inhibitory axon guidance protein draxin114 and the roof plate (RP) morphogens bone
morphogenetic protein 7 (BMP7) and growth/differentiation factor 7 (GDF7)113,115 and
from the motor columns by the secreted glycoprotein neural epithelial growth factor-​
like-like 2 (NELL2)91. Pre-​crossing CAs express the receptors deleted in colorectal cancer
(DCC)34, roundabout homologue 3 (ROBO3)141, brother of CDO (BOC)109, kinase insert
domain receptor (KDR)108 and BMP receptor type 1 A (BMPR1A)116. Upon reaching the FP,
a molecular switch triggered by glial cell line-​derived neurotrophic factor (GDNF)133 and
SHH134 produced by the FP activates repulsive signalling pathways. This activation involves
the expression by CAs of receptors including plexin A1, neuropilin 2 (NRP2)125,126,132,133,
patched (PTCH1), smoothened (SMO)134,160 and frizzled 3 (FZD3)161,163,165,166 and their
interaction with their ligands semaphorin 3B (SEMA3B), SHH and WNT proteins, which
are present in the FP. Slit homologue (SLIT) protein–ROBO receptor-​mediated repulsion is
also activated125. These repulsive forces push CAs away from the midline and prevent
re-crossing. b | Schematic illustration of the guidance errors induced by the inactivation
of each axon guidance pathway. In the netrin 1 full knockout (KO) mouse (Ntn1−/−)57,98,99,230,
a conditional KO mouse that lacks netrin 1 expression at the FP and VZ (Ntn1∆FP+∆VZ)92,
and the Dcc KO mouse (Dcc−/−)31,230, most CAs fail to cross the midline and some exit the
CNS to invade the peripheral nervous system88,89. CAs still cross the midline in mutants
selectively depleted of FP netrin 1 (Ntn1ΔFP), but some invade the motor column and
crossing is delayed92,106. The ablation of netrin 1 from VZ progenitors (Ntn1ΔVZ) perturbs
CA guidance with axons invading the VZ90,92,106. In Boc109, Kdr or Vegfa108 KO mice or in
Nell2−/−;Robo3+/− compound mutants91, midline guidance is perturbed with CAs extending
across the motor columns. CAs fail to cross the FP in Robo3 KO mice128 and in mice in which
Dcc and neogenin (Neo1) are knocked out230. Midline crossing is also perturbed in Slit1, Slit2
and Slit3 triple KO mice (Slit triple KO)128,130, Robo1 and Robo2 double KO mice129,144, Sema3b
KO mice, Plxna1 KO mice and Nrp2 KO mice125–127, with axons stalling at the midline or
re-crossing it. c | Longitudinal guidance of ipsilateral and contralateral axons in the spinal
cord. In the ventral spinal cord, WNT proteins and SHH are expressed along the midline in
two opposite gradients160,161. Post-​crossing commissural axons extend anteriorly in
response to the combined action of SHH repulsion and WNT attraction, mediated by SMO
and FZD3, part of the planar cell polarity (PCP) pathway , respectively160,161,163–166. Most
post-crossing axons transiently follow the FP before deviating laterally to enter the ventral
and lateral funiculi (VF/LF)82. SLIT–ROBO signalling prevents commissural axon re-​crossing
but also controls their mediolateral positioning158. Ipsilateral axons, which express ephrin
type A receptor 4 (EPHA4), are driven away from the FP by ephrin B3 and invade the
midline when this pathway is inactivated174,175,177,183. The corresponding phenotypes of Fzd3
(or other PCP pathway gene), Smo, Robo, Slit, Efnb3 and EphA4 mutant mice are shown.
d | In the dorsal spinal cord, WNT proteins are also expressed along the midline in an
anterior (high expression) to posterior (low expression) gradient190. Corticospinal tract
(CST) axons express the non-​canonical WNT receptor RYK and grow caudally in the dorsal
funiculus down the WNT gradient190. CST axons express EPHA4 and are prevented from
re-crossing the dorsal midline by ephrin B3 (refs6,174,175). Ephrin B3–EPHA4 repulsion
also repels ascending ipsilateral axons from the dorsal midline183,184. However, several
populations of commissural neurons project across the dorsal midline147. Dorsal crossing
involves SLIT–ROBO repulsion but, unlike ventral crossing, is independent of ROBO3
(ref.150). The corresponding phenotypes of Wnt, Ryk, Efnb3, Epha4 and Robo mutant mice
are shown.
which have different properties and functions. The glyco­
sylated α-​subunit of dystroglycan 1 (DAG1), an extra-
cellular matrix protein, selectively anchors SLIT-​C at the
ventral midline, and Dag1 mutant mice partially pheno-
copy Slit and Robo mutants131. SLIT-N binds to ROBO1
and/or ROBO2 receptors, whereas SLIT-​C binds to
plexin A1 (see below)33,132.
How does the floor plate become repulsive for com-
missural axons? Netrin 1, SLIT proteins and SEMA3B
are all present at the floor plate before commissural
axon crossing, indicating that the regulation of commis-
sural axon responsiveness probably occurs at the level
of their receptors or downstream signalling partners.
It also implies that preventing a premature repulsion of
Nature Reviews | Neuroscience
Reviews
pre-​crossing axons is necessary in order for commissural
axons to enter the floor plate. For example, one model
proposes that neuropilin 2 is transiently expressed by
floor plate cells and ‘traps’ SEMA3B, thereby preventing
it from repelling pre-​crossing axons127.
Several parallel mechanisms involving translational
and post-​translational modifications of plexin A1 and
ROBO receptors seem to ensure that post-​crossing
commissural axons escape and do not re-​cross the
midline120–122. A series of studies have shown that in
pre-​crossing commissural axons, plexin A1 cleavage
by calpain silences SEMA3B-​mediated repulsion126,133.
Once these axons reach the midline, floor plate cells
secrete glial cell line-​d erived neurotrophic factor
(GDNF), which binds to a neural cell adhesion mol­
ecule (NCAM) family member present on commis-
sural axon growth cones, inactivates calpain and allows
plexin A1 to reach the membrane to trigger SEMA3B
repulsion126,133. Plexin A1 upregulation has been shown
to be indispensable for SEMA3B repulsive activity126,133;
however, SHH also participates in this process via a non-​
canonical signalling pathway in which SHH decreases
cAMP production and the activity of protein kinase A134.
This switch mechanism is reminiscent of a mecha-
nism that takes place in D. melanogaster, in which the
endocytic trafficking of Robo by protein commissure-
less 1 (Comm) leads to Robo degradation, protecting
pre-​crossing axons from protein slit (Sli) repulsion135–137.
A vertebrate homologue of Comm does not seem to
exist, suggesting that other mechanisms regulate ROBO
receptor responsiveness in mammalian commissural
axons138,139. Nevertheless, it was recently proposed that
the short transmembrane protein proline-​r ich and
γ-carboxyglutamic acid (Gla) domain 4 (PRRG4), which
contains the PPxY and Gla motifs found in D. melano-
gaster Comm, could be its functional homologue140.
However, PRRG4 function was tested only in hetero­
logous systems (in D. melanogaster and in cell lines),
and its role in vertebrate commissural guidance has not
yet been assessed.
Earlier work implicated the ROBO3 receptor, which
is expressed by vertebrate spinal cord commissural axons
and is downregulated after midline crossing141–143. The
ventral spinal cord commissure is completely absent in
Robo3 knockout mice141. In mice, two different splice
isoforms of ROBO3 — ROBO3.1 and ROBO3.2 — are
differentially expressed in pre-​crossing and post-​crossing
commissural axons144,145. Selectively re-​expressing each
isoform in commissural axons of Robo3 knockout
mice showed that only ROBO3.1 can restore midline
crossing144. Commissural axon crossing is also par-
tially restored in mice lacking Robo1, Robo2 and Robo3
(ref.129), providing genetic evidence for an interaction of
ROBO3 with ROBO1 and/or ROBO2 and supporting
a model in which ROBO3.1 suppresses ROBO1 and
ROBO2 repulsion before crossing, whereas ROBO3.2
cooperates with ROBO1 and ROBO2 to repel com-
missural axons away from the midline after crossing.
In crossing axons, uncharacterized floor plate signals
induce the translation, in commissural axon growth
cones, of ROBO3.2 transcripts, the levels of which are
tightly regulated by nonsense-​mediated decay145.
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Although this model is appealing, there is no
evidence that ROBO3 and ROBO1 and/or ROBO2
receptors heterodimerize. In addition, ROBO1 and
ROBO2 expression appears low or absent in pre-​
crossing axons129,141, and, in mammals, ROBO3.1 does
not bind SLIT proteins but instead interacts with DCC
and participates in the netrin 1 response146. Moreover,
the genetic evidence linking Robo3 and Robo1 and/or
Robo2 was obtained using full knockout mice and
should be interpreted cautiously as Robo1 and Robo2 are
broadly expressed in the CNS, and these crosses do not
show that the receptors interact in commissural neurons.
It has been suggested that ROBO3.1 might also mediate
NELL2 repulsion from the motor column91. In addition,
ROBO3.2 transcripts are highly divergent in vertebrates,
suggesting that the proposed regulatory mechanism
might be specific to mice143. Interestingly, plexin A1
also contributes to SLIT-​mediated repulsion at the mid-
line132 because SLIT-​C binds to plexin A1 and collapses
commissural axons. This observation suggests that the
upregulation of plexin A1 expression at the midline
switches on both semaphorin and SLIT signalling.
Crossing dorsally. In rodents, less-​well-character-
ized neuronal populations cross the midline dorsally,
between the ventral tip of the dorsal funiculus and the
central canal147. These neurons start crossing around
E15.5, after the fusion of the dorsal neural tube and
the disappearance of the roof plate148. Some dorsal
horn commissural axons originating in layer IV of the
dorsal horn appear to establish reciprocal commissural
connections with contralateral layer IV neurons85,149,150.
A subset of commissural axons might also originate
from lamina I neurons151. The VZ origin of these axons
and their genetic characterization is still elusive, but
recently, a small population of GABAergic inhibitory
neurons deriving from pancreas transcription factor 1α
(PTF1A)-expressing progenitors and projecting across
the dorsal midline, was characterized150 (Fig. 4c). These
axons still cross in the Dreher mouse mutant152, which
lacks a roof plate, indicating that this midline structure
is dispensable for crossing. Although they fail to decus-
sate in Robo1 and Robo2 double mutant mice, crossing
is normal in Robo3 knockout mice, suggesting that the
molecular mechanisms controlling dorsal and ventral
crossing differ. Finally, a fraction of sensory fibres also
cross the dorsal midline at the thoracic level to terminate
in the medial and lateral spinal cord48,153. Interestingly,
these sensory axons fail to cross in the absence of serine/
threonine (SAD) kinases71. Understanding the guidance
mechanisms of dorsal spinal cord commissural axons
will first require us to better trace their origin and
genetic identity.
Guiding axons longitudinally
Post-​crossing commissural axons. Immediately after
crossing the floor plate, most commissural axons turn
longitudinally to extend along the midline (Fig.  4c).
Tracing experiments in rodents and chick embryos
showed that the reorientation of post-​crossing commis-
sural axons differs between neuronal classes; although
most grow in an anterior direction, some extend caudally
or bifurcate to send longitudinal branches in both direc-
tions82,154–156. Moreover, a large fraction of commissural
axons progressively deviate laterally to continue growing
in the ventral and lateral funiculi where they fasciculate
with the axons of ipsilaterally projecting neurons155,157.
Post-​crossing commissural axons are laterally shifted in
Slit1, Slit2 and Slit3 triple knockout mice and in Robo1
and Robo2 single or double knockout mice, suggesting
that their lateral positioning is at least partly mediated
by SLIT protein-​dependent repulsion128,129,158. Evidence
from studies in chick embryos also suggests that motor
neurons and the DRG provide guidance cues for some
post-​crossing axons159. The molecular mechanisms
underlying these heterogeneous and contrasting path-
finding choices are for the most part poorly charac-
terized. However, we do have some insight into the
mechanisms of rostral turning, which involves WNT
proteins and SHH.
In the mouse floor plate, SHH is expressed at a
higher level at caudal spinal cord levels than at anterior
levels160. After crossing, the responsiveness of commis-
sural axons to SHH switches so that the morphogen now
initiates a repulsive response and constrains the axons
to progress rostrally, down the SHH gradient. This
repulsion involves the transmembrane receptor protein
patched (PTCH1)134, its effector (the GPCR smooth-
ened (SMO)) and the adaptor protein 14-3-3 (ref.160),
but not BOC109,134.
Although it might be expected that commissural
axons would simply grow away from the floor plate in
response to SHH, this is often not the case owing to
the counteracting attractive activity of WNT proteins.
At least five WNT proteins are expressed by midline
cells (floor plate and ventral VZ progenitors) at the
stage at which post-​crossing axons turn rostrally161–163
and display a rostral–caudal expression gradient along
the spinal cord161 (Fig. 4c). Antagonizing WNT proteins
through the application of members of the secreted
frizzled-​related protein (SFRP) family to spinal cord
explants161 or by genetically blocking WNT protein
secretion from the floor plate in vivo163 severely per-
turbs post-​crossing axon guidance. In CAs, WNT pro-
tein activity is mediated by frizzled 3 (FZD3)161, which
belongs to the so-​called non-​canonical planar cell polar-
ity (PCP) pathway. Strikingly, genetic and biochemical
evidence has demonstrated that all components of the
PCP pathway are required for rostral turning161,164–166
(Fig. 4c). The activation of rostral turning is triggered by
a cascade of events that ensure that FZD3 becomes active
only after crossing. A key regulator of this mechanism is
the transmembrane protein, SHISA2, which binds FZD3
and blocks its glycosylation and transport to the plasma
membrane of pre-​crossing growth cones163. At the mid-
line, SHH acts via SMO to block SHISA2 expression,
thereby allowing FZD3 to reach the membrane and
activate WNT attraction163.
These data suggest that SHH controls commissural
axon rostral turning and midline crossing via two dis-
tinct pathways, both of which require SMO. How attrac-
tive and repulsive SHH signals are integrated by the
post-​crossing growth cone remains an enigma. In addi-
tion, whether WNT proteins and SHH have the same
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Pyramidal decussation
The region at the junction of
the brainstem and spinal cord
at which axons of the
corticospinal tract deviate
dorsally and cross the midline.
Nature Reviews | Neuroscience
effects on ipsilaterally projecting axons is unknown.
Unexpectedly, and despite all the changes that occur in
commissural axons at the midline, crossing per se is not
required for axons to change direction. In Robo3 knock-
out mice, axons that fail to cross the midline still turn
longitudinally and extend along the proper pathways
to reach their appropriate targets (albeit on the wrong
(ipsilateral) side of the brain)167–169. This observation
suggests that the longitudinal switch and the ability to
recognize anteroposterior cues might not require floor
plate contact.
Ephrin B family members are transmembrane pro-
teins that can act as either ligands or receptors of EPH
receptors (Table 1) and act on post-​crossing axons. The
expression of ephrin type B receptors by post-​crossing
commissural axons (see below) and of their ligands,
ephrin B1 and ephrin B2 in the dorsolateral part of
the spinal cord, suggested that these repulsive mol­
ecules could act as a barrier and constrain post-​crossing
commissural axons to the ventral half of the spinal
cord170,171. However, this does not seem to be the case
as the position of post-​crossing axons is not altered in
Efnb1 or Efnb2 mutant mice or in mice lacking ephrin
type B receptors.
Development of ipsilateral circuits. Many populations
of spinal cord interneurons and projection neurons
extend their axons only on the ipsilateral side (Box 2)
and develop concomitantly to commissural neurons
(Fig. 4c). Although the development of ipsilateral circuits
has been less well studied than that of commissural neu-
rons, there is evidence for a key role of ephrin repellents
in this process.
In the developing spinal cord, ephrin B3 is expressed
at the ventral and dorsal midline in floor plate and roof
plate cells, respectively. At later stages, ephrin B3 is still
present in radial glia cells at the dorsal midline172, where
it acts as a repulsive midline barrier for many classes
of ipsilateral axons expressing ephrin type A receptor 4
(EPHA4). Epha4 and Efnb3 knockout mice exhibit a
so-​called ‘hopping’ gait173–176 that physiological, genetic
and neuroanatomical studies have shown is explained
by the abnormal ventral crossing of EPHA4-expressing
excitatory axons originating from neurons that normally
innervate ipsilateral motor neurons177,178 (Fig. 4c). This
phenotype is also observed in mice deficient in EPHA4
downstream partners, such as the RAC-​GTPase activat-
ing protein N-​chimaerin179,180. Importantly, a hopping
gait was present both in mice selectively lacking EPHA4
in most spinal cord neurons176 and in those in which it
was deficient only in dorsal neurons181.
Recent studies show that ephrins also guide ipsi-
lateral axons in the dorsal spinal cord. The dorsal
funiculus (Box 2) starts to develop around E14.5 after
the dorsal midline has emerged from the roof plate182.
It contains at least three categories of axons: third-​order
branches of sensory axons and axons from dorsal spi-
nal cord neurons projecting in the postsynaptic dorsal
column (PSDC) pathway (Box 2). PSDC neurons have
multiple origins and comprise late-​born interneuron
class B (dILB) glutamatergic neurons183. In the spinal
cord, dILB neurons selectively express the transcription
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factor ZIC2 (ref.184), which is a direct activator of EPHA4.
Downregulating ZIC2 in dILB neurons allows some
of them to extend axons across the dorsal midline184.
Likewise, the conditional ablation of EPHA4 in ZIC2-
expressing neurons is also followed by a partial con-
tralateral re-​routing of their axons, suggesting that they
are normally repelled from the midline by ephrin B3
(ref.183) (Fig. 4c). Interestingly, a subset of dorsal commis-
sural neurons (dI1c) is rewired ipsilaterally following
the ectopic expression of ZIC2, and this is accompa-
nied by the induction of EPHA4 expression and the
downregulation of ROBO3 expression. These results
suggest that ipsilateral growth requires the activation of
repulsive pathways and the silencing of attractive ones.
Recent data also suggest that the rostral extension of
PSDC axons is promoted by midline radial glia cells182
expressing various growth factors.
Guiding corticospinal tract axons in the spinal cord.
In the mouse, the corticospinal tract (Box 2) reaches the
spinal cord around birth to extend in the most ventral
part of the dorsal funiculus185. Corticospinal tract axons
are ipsilateral and ventral in the brainstem, but most are
rerouted at the pyramidal decussation. They cross the dor-
sal midline, enter the dorsal funiculus and descend into
the spinal cord, reaching the lumbar spinal cord around
postnatal day 9 in mice185,186. Interestingly, the developing
corticospinal tract contains axons from the motor and
sensory cortices (Box 2) that are still present in the adult
but also axons from the visual cortex that are pruned
postnatally5,187. This developmental pruning involves
plexin A3 and neuropilin 2 signalling188. Recent results
further illustrate the importance of axon remodelling
in corticospinal tract development; viral tracing exper-
iments showed that, during the first 2 postnatal weeks,
corticospinal tract axons establish functional synapses
on motor neurons that are later eliminated and replaced
by connections to premotor neurons189. Although the
exact mechanism involved is unclear, the elimination
of corticospinal tract–motor neuron contacts requires
plexin A1 (expressed by corticospinal tract axons) and
its ligand SEMA6D189.
The mechanisms controlling the formation of the
pyramidal decussation have started to be elucidated
and involve netrin 1 and semaphorin signalling186, but
how corticospinal tract axons are guided along the
spinal cord is poorly understood. Corticospinal tract
axons express the WNT receptor RYK and are repelled
by WNT proteins190. Interfering with RYK function per-
turbs the caudal extension of the axons, suggesting that
they are normally driven away from the rostral spinal
cord where the expression of WNT proteins is highest
(Fig. 4c). Direct genetic evidence supporting the role of
RYK and WNT proteins in corticospinal tract guidance
is still lacking, although a Ryk conditional knockout
mouse now exists191. In addition, corticospinal tract
axons express the insulin-​like growth factor 1 (IGF1)
receptor (IGFR), and their invasion of the spinal cord is
promoted by IGF1 (ref.192).
The dorsal midline has an essential role in preventing
corticospinal tract axons from entering the contralat-
eral spinal cord after their initial crossing. A subset
Reviews

of corticospinal tract axons abnormally re-​cross the
midline dorsally in Epha4 and Efnb3 knockout mice,
suggesting that ephrin B3 keeps the axons away from
the midline6,178,183,193. These midline crossing errors
are recapitulated in mice selectively lacking EPHA4
or N-​chimaerin expression194 in either corticospinal
tract neurons or spinal cord neurons6,183. This non-​cell
autonomous defect is attributed to the invasion of the
dorsal midline by dorsal spinal cord neurons that open
‘gaps’ allowing corticospinal tract axons to re-​cross
the midline183,184. What controls the arborization of
corticospinal tract axonal branches at specific levels
of the spinal cord and guides them to their targets is
currently unknown.
Conclusion and perspectives
Significant progress has been made in our understand-
ing of spinal cord wiring. Recently, new guidance factors
expressed by spinal cord neurons, glia and their progen-
itors have been discovered, opening exciting avenues
for research. However, our knowledge is still far from
reflecting the paramount complexity and diversity of spi-
nal cord circuits. For instance, the combined expression
of DCC and ROBO3 cannot account for the diversity of
trajectories followed by commissural axons. Likewise,
the mechanisms that allow LTMRs to connect with their
target neurons in the dorsal spinal cord are completely
unknown. Single-​cell profiling and transcriptomic
analyses of spinal cord neurons should help to identify
specific guidance pathways; however, such studies have
thus far been conducted only in postnatal mice.
What have mice carrying mutations in axon guid-
ance genes told us about spinal circuits more generally?
First, they have provided direct evidence supporting
the pivotal role of spinal cord interneurons in motor
control. Although abnormal corticospinal tract decus-
sation and midline re-​crossing were initially thought
to be accountable for the hopping-​gait defects in Dcc
and EphA4 knockout mice, the development of con-
ditional alleles has demonstrated that defects in spinal
cord neurons are the main culprits183,195,196. This finding
suggests that this could also be the case in patients with
congenital mirror movement disorders, who also carry
mutations in axon guidance genes197–199. Second, these
studies have identified potential drug targets for pain68
and for promoting axonal regeneration after injury200.
It will now be important to determine whether axon
guidance pathways can be reactivated in the injured
adult spinal cord in order to repair lesioned circuits or
to trigger the de novo formation of parallel circuits8.
For example, there is evidence indicating that WNT pro-
teins inhibit axonal regeneration in the adult spinal cord
and that blocking their function improves the recovery
of locomotor function201,202.
Getting a better understanding of spinal cord wiring
mechanisms in mice also brings new tools and models
to study the evolution of locomotor circuits in verte-
brates. Although the basic building blocks of locomotor
circuits appear to have been largely conserved for over
400 million years203, there is now mounting evidence for
a little anticipated diversification of guidance mecha-
nisms across evolution101,102. For instance, mammalian
ROBO3 receptors became unable to bind SLIT proteins
and gained the ability to mediate netrin 1 (ref.146) signal-
ling. In birds, the Dcc gene was lost (in some species)
and the molecular components of the midline switch
diverged from those of mice204–207. Likewise, changes in
developmental programmes dependent on homeobox
protein HOXD1 might explain the differential organiza-
tion of spinal cord nociceptive projections between mice
and birds208. Finally, the existence of a direct connection
between corticospinal tract axons and spinal cord motor
neurons in humans and primates could also be linked to
a modification of semaphorin and/or plexin function189.
These few examples illustrate the need for more in-​depth
comparative analysis of the expression of axon guidance
molecules and their receptors during the development
of spinal cord circuits.
Published online xx xx xxxx

1. Abraira, V. E. & Ginty, D. D. The sensory neurons of
touch. Neuron 79, 618–639 (2013).
This article provides a comprehensive review on the
classification of LTMRs and their molecular and
physiological properties.
2. Goulding, M. Circuits controlling vertebrate locomotion:
moving in a new direction. Nat. Rev. Neurosci. 10,
507–518 (2009).
3. Kiehn, O. Decoding the organization of spinal circuits
that control locomotion. Nat. Rev. Neurosci. 17,
224–238 (2016).
4. Catela, C., Shin, M. M. & Dasen, J. S. Assembly and
function of spinal circuits for motor control. Annu. Rev.
Cell Dev. Biol. 31, 669–698 (2015).
5. Wang, X. et al. Deconstruction of corticospinal circuits for
goal-​directed motor skills. Cell 171, 440–455 (2017).
6. Serradj, N. et al. EphA4-mediated ipsilateral
corticospinal tract misprojections are necessary for
bilateral voluntary movements but not bilateral
stereotypic locomotion. J. Neurosci. 34, 5211–5221
(2014).
7. Liu, Y. et al. Touch and tactile neuropathic pain
sensitivity are set by corticospinal projections. Nature
561, 547–550 (2018).
8. Chen, B. et al. Reactivation of dormant relay pathways
in injured spinal cord by KCC2 manipulations. Cell
174, 521–535 (2018).
This remarkable study shows that drugs targeting
spinal cord interneurons can restore locomotion in
mice with spinal cord injury.
9. Courtine, G. et al. Recovery of supraspinal control of
stepping via indirect propriospinal relay connections
after spinal cord injury. Nat. Med. 14, 69–74
(2008).
10. Asboth, L. et al. Cortico–reticulo–spinal circuit
reorganization enables functional recovery after
severe spinal cord contusion. Nat. Neurosci. 21,
576–588 (2018).
11. Hollis, E. R. et al. Remodelling of spared proprioceptive
circuit involving a small number of neurons supports
functional recovery. Nat. Commun. 6, 6079 (2015).
12. Meneret, A., Welniarz, Q., Trouillard, O. & Roze, E.
Congenital mirror movements: from piano player to
opera singer. Neurology 84, 860–860 (2015).
13. Tessier-​Lavigne, M. & Goodman, C. S. The molecular
biology of axon guidance. Science 274, 1123–1133
(1996).
14. Pignata, A., Ducuing, H. & Castellani, V. Commissural
axon navigation: control of midline crossing in the
vertebrate spinal cord by the semaphorin 3B
signaling. Cell Adh. Migr. 10, 604–617 (2016).
15. Bonanomi, D. Axon pathfinding for locomotion.
Semin. Cell Dev. Biol. 85, 26–35 (2019).
16. Nornes, H. O. & Carry, M. Neurogenesis in spinal cord
of mouse: an autoradiographic analysis. Brain Res.
159, 1–16 (1978).
17. Ericson, J., Thor, S., Edlund, T., Jessell, T. M. &
Yamada, T. Early stages of motor neuron
differentiation revealed by expression of homeobox
gene Islet-1. Science 256, 1555–1560 (1992).
18. Jessell, T. M. Neuronal specification in the spinal cord:
inductive signals and transcriptional codes. Nat. Rev.
Genet. 1, 20–29 (2000).
19. Fraher, J. P., Dockery, P., O’Donoghue, O., Riedewald, B.
& O’Leary, D. Initial motor axon outgrowth from the
developing central nervous system. J. Anat. 211,
600–611 (2007).
20. Vermeren, M. et al. Integrity of developing spinal
motor columns is regulated by neural crest
derivatives at motor exit points. Neuron 37,
403–415 (2003).
21. Bron, R. et al. Boundary cap cells constrain spinal
motor neuron somal migration at motor exit points by
a semaphorin-​plexin mechanism. Neural Dev. 2, 21
(2007).
22. Santiago-​Medina, M., Gregus, K. A., Nichol, R. H.,
O’Toole, S. M. & Gomez, T. M. Regulation of ECM
degradation and axon guidance by growth cone
invadosomes. Development 142, 486–496 (2015).
23. Snider, W. D. & Palavali, V. Early axon and dendritic
outgrowth of spinal accessory motor neurons studied
with dii in fixed tissues. J. Comp. Neurol. 297,
227–238 (1990).
24. Dillon, A. K. et al. Molecular control of spinal
accessory motor neuron/axon development in the
mouse spinal cord. J. Neurosci. 25, 10119–10130
(2005).
25. Bai, G. et al. Presenilin-​dependent receptor processing
is required for axon guidance. Cell 144, 106–118
(2011).

www.nature.com/nrn

26. Poliak, S. et al. Synergistic integration of Netrin and
ephrin axon guidance signals by spinal motor neurons.
eLife 4, 7250–7257 (2015).
27. Leonardo, E. D. et al. Vertebrate homologues of
C.elegans UNC-5 are candidate netrin receptors.
Nature 386, 833–838 (1997).
28. Dillon, A. K. et al. UNC5C is required for spinal
accessory motor neuron development. Mol. Cell.
Neurosci. 35, 482–489 (2007).
29. Kidd, T. et al. Roundabout controls axon crossing of
the CNS midline and defines a novel subfamily of
evolutionarily conserved guidance receptors. Cell 92,
205–215 (1998).
30. Jaworski, A. & Tessier-​Lavigne, M. Autocrine/
juxtaparacrine regulation of axon fasciculation by
Slit-Robo signaling. Nat. Neurosci. 15, 367–369
(2012).
31. Fazeli, A. et al. Phenotype of mice lacking functional
Deleted in colorectal cancer (Dcc) gene. Nature 386,
796–804 (1997).
32. da Silva, R. V. et al. DCC is required for the development
of nociceptive topognosis in mice and humans. Cell Rep.
22, 1105–1114 (2018).
33. Brose, K. et al. Slit proteins bind Robo receptors and
have an evolutionarily conserved role in repulsive axon
guidance. Cell 96, 795–806 (1999).
34. Keino-​Masu, K. et al. Deleted in colorectal cancer
(DCC) encodes a Netrin receptor. Cell 87, 175–185
(1996).
35. Kim, M., Fontelonga, T. M., Lee, C. H., Barnum, S. J. &
Mastick, G. S. Motor axons are guided to exit points
in the spinal cord by Slit and Netrin signals. Dev. Biol.
432, 178–191 (2017).
36. Bravo-​Ambrosio, A., Mastick, G. & Kaprielian, Z.
Motor axon exit from the mammalian spinal cord is
controlled by the homeodomain protein Nkx2.9 via
Robo-​Slit signaling. Development 1446, 1435–1446
(2012).
37. Lieberam, I., Agalliu, D., Nagasawa, T., Ericson, J. &
Jessell, T. M. A. Cxcl12-CXCR4 chemokine signaling
pathway defines the initial trajectory of mammalian
motor axons. Neuron 47, 667–679 (2005).
38. Zhu, Y., Matsumoto, T., Nagasawa, T., Mackay, F. &
Murakami, F. Chemokine signaling controls integrity of
radial glial scaffold in developing spinal cord and
consequential proper position of boundary cap cells.
J. Neurosci. 35, 9211–9224 (2015).
39. Wu, J. Y. et al. The neuronal repellent Slit inhibits
leukocyte chemotaxis induced by chemotactic factors.
Nature 410, 948–952 (2001).
40. Chalasani, S. H., Sabelko, K. A., Sunshine, M. J.,
Littman, D. R. & Raper, J. A. A chemokine, SDF-1,
reduces the effectiveness of multiple axonal
repellents and is required for normal axon pathfinding.
J. Neurosci. 23, 1360–1371 (2003).
41. Nam, H. & Lee, S. Identification of STAM1 as a novel
effector of ventral projection of spinal motor neurons.
Development 143, 2334–2343 (2016).
42. Arber, S. Motor circuits in action: specification,
connectivity, and function. Neuron 74, 975–989
(2012).
43. Patel, T. D. et al. Peripheral NT3 signaling is required
for ETS protein expression and central patterning of
proprioceptive sensory afferents. Neuron 38,
403–416 (2003).
44. Li, L. et al. The functional organization of cutaneous
low-​threshold mechanosensory neurons. Cell 147,
1615–1627 (2011).
45. Zimmerman, A., Bai, L. & Ginty, D. D. The gentle touch
receptors of mammalian skin. Science 346, 950–955
(2014).
46. Todd, A. J. Identifying functional populations among
the interneurons in laminae I-​III of the spinal dorsal
horn. Mol. Pain 13, 1–19 (2017).
47. Abraira, V. E. et al. The cellular and synaptic architecture
of the mechanosensory dorsal horn. Cell 168, 295–310
(2017).
48. Ozaki, S. & Snider, W. D. Initial trajectories of sensory
axons toward laminar targets in the developing
mouse spinal cord. J. Comp. Neurol. 380, 215–229
(1997).
49. Guy, A. T. et al. Glycerophospholipid regulation of
modality-​specific sensory axon guidance in the spinal
cord. Science 349, 974–977 (2015).
This very interesting study uncovers a role for lipids
in sensory axon guidance.
50. Masuda, T. et al. Netrin-1 acts as a repulsive guidance
cue for sensory axonal projections toward the spinal
cord. J. Neurosci. 28, 10380–10385 (2008).
51. Schmidt, H. et al. The receptor guanylyl cyclase Npr2
is essential for sensory axon bifurcation within the
spinal cord. J. Cell Biol. 179, 331–340 (2007).
Nature Reviews | Neuroscience
52. Schmidt, H. et al. C-​Type natriuretic peptide (CNP) is a
bifurcation factor for sensory neurons. Proc. Natl
Acad. Sci. USA 106, 16847–16852 (2009).
53. Zhao, Z. et al. Regulate axon branching by the cyclic
GMP pathway via inhibition of glycogen synthase
kinase 3 in dorsal root ganglion sensory neurons.
J. Neurosci. 29, 1350–1360 (2009).
54. Schmidt, H. et al. cGMP-​mediated signaling via cGKIα
is required for the guidance and connectivity of
sensory axons. J. Cell Biol. 159, 489–498 (2002).
55. Wang, K. H. et al. Biochemical purification of a
mammalian Slit protein as a positive regulator of
sensory axon elongation and branching. Cell 96,
771–784 (1999).
56. Ma, L. & Tessier-​Lavigne, M. Dual branch-​promoting
and branch-​repelling actions of Slit/Robo signaling on
peripheral and central branches of developing sensory
axons. J. Neurosci. 27, 6843–6851 (2007).
57. Serafini, T. et al. Netrin-1 is required for commissural
axon guidance in the developing vertebrate nervous
system. Cell 87, 1001–1014 (1996).
58. Watanabe, K. et al. Dorsally derived netrin 1 provides
an inhibitory cue and elaborates the ‘waiting period’
for primary sensory axons in the developing spinal
cord. Development 133, 1379–1387 (2006).
59. Messersmith, E. K. et al. Semaphorin III can function
as a selective chemorepellent to pattern sensory
projections in the spinal cord. Neuron 14, 949–959
(1995).
60. Molofsky, A. V. et al. Astrocyte-​encoded positional
cues maintain sensorimotor circuit integrity. Nature
509, 189–194 (2014).
61. Kitsukawa, T. et al. Neuropilin-​semaphorin III/D-​
mediated chemorepulsive signals play a crucial role in
peripheral nerve projection in mice. Neuron 19,
995–1005 (1997).
62. Behar, O., Golden, J. A., Mashimo, H., Schoen, F. J. &
Fishman, M. C. Semaphorin III is needed for normal
patterning and growth of nerves, bones and heart.
Nature 383, 525–528 (1996).
63. Gu, C. et al. Neuropilin-1 conveys semaphorin and
VEGF signaling during neural and cardiovascular
development. Dev. Cell 5, 45–57 (2003).
64. Yoshida, Y., Han, B., Mendelsohn, M. & Jessell, T. M.
PlexinA1 signaling directs the segregation of
proprioceptive sensory axons in the developing spinal
cord. Neuron 52, 775–788 (2006).
65. Leslie, J. R. et al. Ectopic myelinating oligodendrocytes
in the dorsal spinal cord as a consequence of altered
semaphorin 6D signaling inhibit synapse formation.
Development 138, 4085–4095 (2011).
66. Levine, A. J. et al. Identification of a cellular node for
motor control pathways. Nat. Neurosci. 17, 586–593
(2014).
67. Hilde, K. L. et al. Satb2 is required for the
development of a spinal exteroceptive microcircuit
that modulates limb position. Neuron 91, 763–776
(2016).
This work identifies the transcription factor SATB2
as a key genetic determinant of a small population
of relay interneurons in lamina V that are involved
in specific sensorimotor behaviours.
68. Hayano, Y. et al. Dorsal horn interneuron-​derived
Netrin-4 contributes to spinal sensitization in chronic
pain via Unc5B. J. Exp. Med. 213, 2949–2966
(2016).
69. Sürmeli, G., Akay, T., Ippolito, G. C., Tucker, P. W. &
Jessell, T. M. Patterns of spinal sensory-​motor
connectivity prescribed by a dorsoventral positional
template. Cell 147, 653–665 (2011).
70. Usui, N. et al. Role of motoneuron-​derived
neurotrophin 3 in survival and axonal projection of
sensory neurons during neural circuit formation.
Development 139, 1125–1132 (2012).
71. Lilley, B. N., Pan, Y. A. & Sanes, J. R. SAD kinases
sculpt axonal arbors of sensory neurons through long-
and short-​term responses to neurotrophin signals.
Neuron 79, 39–53 (2013).
72. Dominici, C. et al. Floor-​plate-derived netrin-1 is
dispensable for commissural axon guidance. Nature
545, 350–354 (2017).
73. Hochstim, C., Deneen, B., Lukaszewicz, A., Zhou, Q. &
Anderson, D. J. Identification of positionally distinct
astrocyte subtypes whose identities are specified by a
homeodomain code. Cell 133, 510–522 (2008).
74. Baek, M., Pivetta, C., Liu, J. P., Arber, S. & Dasen, J. S.
Columnar-​intrinsic cues shape premotor input
specificity in locomotor circuits. Cell Rep. 21,
867–877 (2017).
75. Sweeney, L. B. et al. Origin and segmental diversity of
spinal inhibitory interneurons. Neuron 97, 341–355
(2018).
Reviews
This study shows that at least 50 types of spinal
cord V1 interneurons with segment specificity can
be identified on the basis of their combinatorial
expression of 19 transcription factors.
76. Cohen, S. et al. A semaphorin code defines
subpopulations of spinal motor neurons during mouse
development. Eur. J. Neurosci. 21, 1767–1776
(2005).
77. Gu, C. et al. Semaphorin 3E and plexin-​D1 control
vascular pattern independently of neuropilins. Science
307, 265–268 (2005).
78. Pecho-​Vrieseling, E., Sigrist, M., Yoshida, Y., Jessell, T. M.
& Arber, S. Specificity of sensory–motor connections
encoded by Sema3e–Plxnd1 recognition. Nature 459,
842–846 (2009).
79. Fukuhara, K. et al. Specificity of monosynaptic
sensory-​motor connections imposed by repellent
Sema3E-​PlexinD1 signaling. Cell Rep. 5, 748–758
(2013).
80. Zipursky, S. L. & Sanes, J. R. Chemoaffinity revisited:
dscams, protocadherins, and neural circuit assembly.
Cell 143, 343–353 (2010).
81. Eide, A. L., Glover, J., Kjaerulff, O. & Kiehn, O.
Characterization of commissural interneurons in the
lumbar region of the neonatal rat spinal cord. J. Comp.
Neurol. 403, 332–345 (1999).
82. Kadison, S. R. & Kaprielian, Z. Diversity of contralateral
commissural projections in the embryonic rodent spinal
cord. J. Comp. Neurol. 472, 411–422 (2004).
83. Chédotal, A. Development and plasticity of
commissural circuits: from locomotion to brain repair.
Trends Neurosci. 37, 551–562 (2014).
84. Laumonnerie, C., Tong, Y. G., Alstermark, H. &
Wilson, S. I. Commissural axonal corridors instruct
neuronal migration in the mouse spinal cord.
Nat. Commun. 6, 7028 (2015).
85. Orlino, J., Wong, C. M. & Phelps, P. E. L1 and GAD65
are expressed on dorsal commissural axons in
embryonic rat spinal cord. Dev. Brain Res. 125,
117–130 (2000).
86. Wentworth, L. E. The development of the cervical
spinal cord of the mouse embryo. II. A Golgi analysis
of sensory, commissural, and association cell
differentiation. J. Comp. Neurol. 222, 96–115
(1984).
87. Suter, T. A. C. S., DeLoughery, Z. J. & Jaworski, A.
Meninges-​derived cues control axon guidance.
Dev. Biol. 430, 1–10 (2017).
88. Laumonnerie, C., Da Silva, R. V., Kania, A. &
Wilson, S. I. Netrin 1 and Dcc signalling are
required for confinement of central axons within the
central nervous system. Development 141, 594–603
(2014).
89. Moreno-​Bravo, J. A. et al. Commissural neurons
transgress the CNS/PNS boundary in absence of
ventricular zone-​derived Netrin-1. Development 145,
dev159400 (2018).
90. Varadarajan, S. G. et al. Netrin1 produced by neural
progenitors, not floor plate cells, is required for axon
guidance in the spinal cord. Neuron 94, 790–799
(2017).
91. Jaworski, A. et al. Operational redundancy in axon
guidance through the multifunctional receptor Robo3
and its ligand NELL2. Science 350, 961–965
(2015).
92. Moreno-​Bravo, J. A., Roig Puiggros, S., Mehlen, P. &
Chédotal, A. Synergistic activity of floor-​plate- and
ventricular-​zone-derived Netrin-1 in spinal cord
commissural axon guidance. Neuron 101, 625–634
(2019).
This article presents one of the recent studies
showing that netrin 1 secreted by VZ progenitors
promotes the ventral extension of commissural
axons.
93. Ramón y Cajal, S. La rétine des vertébrés [French].
Cellule 9, 119–257 (1892).
94. Kennedy, T. E., Serafini, T., de la Torre, J. &
Tessier-Lavigne, M. Netrins are diffusible chemotropic
factors for commissural axons in the embryonic spinal
cord. Cell 78, 425–435 (1994).
95. Serafini, T. et al. The netrins define a family of axon
outgrowth-​promoting proteins homologous to
C. elegans UNC-6. Cell 78, 409–424 (1994).
96. Tessier-​Lavigne, M., Placzek, M., Lumsden, A. G.,
Dodd, J. & Jessell, T. M. Chemotropic guidance of
developing axons in the mammalian central nervous
system. Nature 336, 775–778 (1988).
97. Xu, X. et al. An integrative approach to understanding
bird origins. Science 346, 1253293 (2014).
98. Yung, A. R., Nishitani, A. M. & Goodrich, L. V.
Phenotypic analysis of mice completely lacking
netrin 1. Development 142, 3686–3691 (2015).
Reviews
99. Bin, J. M. et al. Complete loss of Netrin-1 results in
embryonic lethality and severe axon guidance defects
without increased neural cell death. Cell Rep. 12,
1099–1106 (2015).
100. Rabe, N., Gezelius, H., Vallstedt, A., Memic, F. &
Kullander, K. Netrin-1-dependent spinal interneuron
subtypes are required for the formation of left-​right
alternating locomotor circuitry. J. Neurosci. 29,
15642–15649 (2009).
101. Goodman, C. S. The likeness of being: phylogenetically
conserved molecular mechanisms of growth cone
guidance. Cell 78, 353–356 (1994).
102. Dickson, B. J. Molecular mechanisms of axon
guidance. Science 298, 1959–1964 (2002).
103. Keleman, K. & Dickson, B. J. Short- and long-​range
repulsion by the Drosophila Unc5 netrin receptor.
Neuron 32, 605–617 (2001).
104. Brankatschk, M. & Dickson, B. J. Netrins guide
Drosophila commissural axons at short range.
Nat. Neurosci. 9, 188–194 (2006).
105. Moore, S. W., Biais, N. & Sheetz, M. P. Traction on
immobilized Netrin-1 is sufficient to reorient axons.
Science 325, 166–166 (2009).
106. Wu, Z. et al. Long-​range guidance of spinal
commissural axons by Netrin1 and sonic hedgehog
from midline floor plate cells. Neuron 101, 635–647
(2019).
107. Charron, F., Stein, E., Jeong, J., McMahon, A. P. &
Tessier-​Lavigne, M. The morphogen sonic hedgehog is
an axonal chemoattractant that collaborates with
netrin-1 in midline axon guidance. Cell 113, 11–23
(2003).
108. Ruiz de Almodovar, C. et al. VEGF mediates
commissural axon chemoattraction through its
receptor Flk1. Neuron 70, 966–978 (2011).
109. Okada, A. et al. Boc is a receptor for sonic hedgehog
in the guidance of commissural axons. Nature 444,
369–373 (2006).
110. Sloan, T. F. W., Qasaimeh, M. A., Juncker, D., Yam, P. T.
& Charron, F. Integration of shallow gradients of Shh
and Netrin-1 guides commissural axons. PLOS Biol.
13, e1002119 (2015).
111. Yam, P. T., Langlois, S. D., Morin, S. & Charron, F.
Sonic hedgehog guides axons through a noncanonical,
Src-​family-kinase-​dependent signaling pathway.
Neuron 62, 349–362 (2009).
112. Goodhill, G. J. Can molecular gradients wire the brain?
Trends Neurosci. 39, 202–211 (2016).
113. Augsburger, A., Schuchardt, A., Hoskins, S., Dodd, J. &
Butler, S. BMPs as mediators of roof plate repulsion of
commissural neurons. Neuron 24, 127–141 (1999).
114. Islam, S. M. et al. Draxin, a repulsive guidance protein
for spinal cord and forebrain commissures. Science
323, 388–393 (2009).
115. Butler, S. J. & Dodd, J. A role for BMP heterodimers in
roof plate-​mediated repulsion of commissural axons.
Neuron 38, 389–401 (2003).
116. Yamauchi, K., Phan, K. D. & Butler, S. J. BMP type I
receptor complexes have distinct activities mediating
cell fate and axon guidance decisions. Development
135, 1119–1128 (2008).
117. Ahmed, G. et al. Draxin inhibits axonal outgrowth
through the Netrin receptor DCC. J. Neurosci. 31,
14018–14023 (2011).
118. Liu, Y. et al. Structural basis for draxin-​modulated
axon guidance and fasciculation by Netrin-1 through
DCC. Neuron 97, 1261–1267 (2018).
119. Gao, X. et al. A floor-​plate extracellular protein-​protein
interaction screen identifies draxin as a secreted
Netrin-1 antagonist. Cell Rep. 12, 694–708 (2015).
120. Ducuing, H., Gardette, T., Pignata, A., Tauszig-​
Delamasure, S. & Castellani, V. Commissural axon
navigation in the spinal cord: a repertoire of repulsive
forces is in command. Semin. Cell Dev. Biol. 85, 3–12
(2018).
121. Neuhaus-​Follini, A. & Bashaw, G. J. Crossing the
embryonic midline: molecular mechanisms regulating
axon responsiveness at an intermediate target.
Wiley Interdiscip. Rev. Dev. Biol. 4, 377–389 (2015).
122. Chédotal, A. Further tales of the midline. Curr. Opin.
Neurobiol. 21, 68–75 (2011).
123. Stein, E. & Tessier-​Lavigne, M. Hierarchical organization
of guidance receptors: silencing of netrin attraction by
slit through a Robo/DCC receptor complex. Science
291, 1928–1938 (2001).
124. Shirasaki, R., Katsumata, R. & Murakami, F. Change in
chemoattractant responsiveness of developing axons
at an intermediate target. Science 279, 105–107
(1998).
This landmark paper, together with that of Zou
et al. (2000), provides the first direct experimental
evidence for the ‘midline switch’ model.
125. Zou, Y., Stoeckli, E., Chen, H. & Tessier-​Lavigne, M.
Squeezing axons out of the gray matter: a role for slit
and semaphorin proteins from midline and ventral
spinal cord. Cell 102, 363–375 (2000).
126. Nawabi, H. et al. A midline switch of receptor
processing regulates commissural axon guidance in
vertebrates. Genes Dev. 24, 396–410 (2010).
127. Hernandez-​Enriquez, B. et al. Floor plate-​derived
neuropilin-2 functions as a secreted semaphorin sink
to facilitate commissural axon midline crossing.
Genes Dev. 29, 2617–2632 (2015).
128. Long, H. et al. Conserved roles for Slit and Robo
proteins in midline commissural axon guidance.
Neuron 42, 213–223 (2004).
129. Jaworski, A., Long, H. & Tessier-​Lavigne, M.
Collaborative and specialized functions of Robo1
and Robo2 in spinal commissural axon guidance.
J. Neurosci. 30, 9445–9453 (2010).
130. Dominici, C., Rappeneau, Q., Zelina, P., Fouquet, S.
& Chédotal, A. Non-​cell autonomous control of
precerebellar neuron migration by Slit and Robo
proteins. Development 145, dev150375 (2018).
131. Wright, K. M. et al. Dystroglycan organizes axon
guidance cue localization and axonal pathfinding.
Neuron 76, 931–944 (2012).
132. Delloye-​Bourgeois, C. et al. PlexinA1 is a new Slit
receptor and mediates axon guidance function of Slit
C-​terminal fragments. Nat. Neurosci. 18, 36–45
(2015).
133. Charoy, C. et al. gdnf activates midline repulsion by
Semaphorin3B via NCAM during commissural axon
guidance. Neuron 75, 1051–1066 (2012).
134. Parra, L. M. & Zou, Y. Sonic hedgehog induces
response of commissural axons to Semaphorin
repulsion during midline crossing. Nat. Neurosci. 13,
29–35 (2009).
135. Kidd, T., Russell, C., Goodman, C. S. & Tear, G.
Dosage-​sensitive and complementary functions of
roudabout and commissureless control axon crossing
of the CNS midline. Neuron 20, 25–33 (1998).
136. Keleman, K., Ribeiro, C. & Dickson, B. J. Comm function
in commissural axon guidance: cell-autonomous sorting
of Robo in vivo. Nat. Neurosci. 8, 156–163 (2005).
137. Keleman, K. et al. Comm sorts robo to control axon
guidance at the Drosophila midline. Cell 110,
415–427 (2002).
138. Evans, Ta & Bashaw, G. J. Slit/Robo-​mediated axon
guidance in Tribolium and Drosophila: divergent
genetic programs build insect nervous systems.
Dev. Biol. 363, 266–278 (2012).
139. Tear, G. et al. Commissureless controls growth cone
guidance across the CNS midline in Drosophila and
encodes a novel membrane protein. Neuron 16,
501–514 (1996).
140. Justice, E. D., Barnum, S. J. & Kidd, T. The WAGR
syndrome gene PRRG4 is a functional homologue of
the commissureless axon guidance gene. PLOS Genet.
13, e1006865 (2017).
141. Sabatier, C. et al. The divergent Robo family protein
Rig-1/Robo3 is a negative regulator of Slit
responsiveness required for midline crossing by
commissural axons. Cell 117, 157–169 (2004).
142. Jen, J. C. et al. Mutations in a human ROBO gene
disrupt hindbrain axon pathway crossing and
morphogenesis. Science 304, 1509–1513 (2004).
143. Friocourt, F. & Chédotal, A. The Robo3 receptor, a key
player in the development, evolution, and function of
commissural systems. Dev. Neurobiol. 77, 876–890
(2017).
144. Chen, Z., Gore, B. B., Long, H., Ma, L. &
Tessier-Lavigne, M. Alternative splicing of the Robo3
axon guidance receptor governs the midline switch
from attraction to repulsion. Neuron 58, 325–332
(2008).
145. Colak, D., Ji, S.-J., Porse, B. T. & Jaffrey, S. R. Regulation
of axon guidance by compartmentalized nonsense-​
mediated mRNA decay. Cell 153, 1252–1265 (2013).
146. Zelina, P. et al. Signaling switch of the axon guidance
receptor Robo3 during vertebrate evolution. Neuron
84, 1258–1272 (2014).
147. Ramón y Cajal, S. in Atlas der Pathologischen
Histologie des Nerven Systems [German] (ed. Babes, V.)
3–35 (Verlag van August Hirschwald, 1895).
148. Sturrock, R. R. An electron microscopic study of the
development of the ependyma of the central canal of
the mouse spinal cord. J. Anat. 132, 119–136
(1981).
149. Petkó, M., Veress, G., Vereb, G., Storm-​Mathisen, J. &
Antal, M. Commissural propriospinal connections
between the lateral aspects of laminae III-​IV in the
lumbar spinal cord of rats. J. Comp. Neurol. 480,
364–377 (2004).
150. Comer, J. D. et al. Sensory and spinal inhibitory dorsal
midline crossing is independent of Robo3. Front. Neural
Circuits 9, 36 (2015).
This interesting work provides some novel insights
about midline crossing in the dorsal spinal cord.
151. Todd, A. J. Neuronal circuitry for pain processing in
the dorsal horn. Nat. Rev. Neurosci. 11, 823–836
(2010).
152. Millonig, J. H., Millen, K. J. & Hatten, M. E. The mouse
Dreher gene Lmx1a controls formation of the roof
plate in the vertebrate CNS. Nature 403, 764–769
(2000).
153. Smith, C. L. Sensory neurons supplying touch domes
near the body midlines project bilaterally in the
thoracic spinal cord of rats. J. Comp. Neurol. 245,
541–552 (1986).
154. Sakai, N. & Kaprielian, Z. Guidance of longitudinally
projecting axons in the developing central nervous
system. Front. Mol. Neurosci. 5, 59 (2012).
155. Avraham, O. et al. Transcriptional control of axonal
guidance and sorting in dorsal interneurons by the
Lim-​HD proteins Lhx9 and Lhx1. Neural Dev. 4, 21
(2009).
156. Kadison, S. R., Murakami, F., Matise, M. P. &
Kaprielian, Z. The role of floor plate contact in the
elaboration of contralateral commissural projections
within the embryonic mouse spinal cord. Dev. Biol.
296, 499–513 (2006).
157. Saueressig, H., Burrill, J. & Goulding, M. Engrailed-1
and netrin-1 regulate axon pathfinding by association
interneurons that project to motor neurons.
Development 126, 4201–4212 (1999).
158. Reeber, S. L. et al. Manipulating Robo expression
in vivo perturbs commissural axon pathfinding in the
chick spinal cord. J. Neurosci. 28, 8698–8708
(2008).
159. Avraham, O. et al. Motor and dorsal root ganglion
axons serve as choice points for the ipsilateral turning
of dI3 axons. J. Neurosci. 30, 15546–15557 (2010).
160. Yam, P. T. et al. 14-3-3 proteins regulate a cell-​intrinsic
switch from Sonic Hedgehog-​mediated commissural
axon attraction to repulsion after midline crossing.
Neuron 76, 735–749 (2012).
161. Lyuksyutova, A. I. et al. Anterior-​posterior guidance of
commissural axons by Wnt-​frizzled signaling. Science
302, 1984–1988 (2003).
162. Agalliu, D., Takada, S., Agalliu, I., McMahon, A. P. &
Jessell, T. M. Motor neurons with axial muscle
projections specified by Wnt4/5 signaling. Neuron 61,
708–720 (2009).
163. Onishi, K. & Zou, Y. Sonic Hedgehog switches on
Wnt/planar cell polarity signaling in commissural axon
growth cones by reducing levels of Shisa2. eLife 6,
e25269 (2017).
164. Onishi, K. et al. Antagonistic functions of Dishevelleds
regulate Frizzled3 endocytosis via filopodia tips in
Wnt-​mediated growth cone guidance. J. Neurosci. 33,
19071–19085 (2013).
165. Shafer, B., Onishi, K., Lo, C., Colakoglu, G. & Zou, Y.
Vangl2 promotes Wnt/planar cell polarity-​like
signaling by antagonizing Dvl1-mediated feedback
inhibition in growth cone guidance. Dev. Cell 20,
177–191 (2011).
166. Wolf, A. M. et al. Phosphatidylinositol-3-kinase-​
atypical protein kinase C signaling is required for Wnt
attraction and anterior-​posterior axon guidance.
J. Neurosci. 28, 3456–3467 (2008).
167. Renier, N. et al. Genetic dissection of the function of
hindbrain axonal commissures. PLOS Biol. 8,
e1000325 (2010).
168. Wilson, S. I., Shafer, B., Lee, K. J. & Dodd, J. A.
Molecular program for contralateral trajectory: Rig-1
control by LIM homeodomain transcription factors.
Neuron 59, 413–424 (2008).
169. Michalski, N. et al. Robo3-driven axon midline
crossing conditions functional maturation of a large
commissural synapse. Neuron 78, 855–868 (2013).
170. Imondi, R. & Kaprielian, Z. Commissural axon
pathfinding on the contralateral side of the floor plate:
a role for B-​class ephrins in specifying the dorsoventral
position of longitudinally projecting commissural
axons. Development 128, 4859–4871 (2001).
171. Jevince, A. R., Kadison, S. R., Pittman, A. J., Chien, C.-B.
& Kaprielian, Z. Distribution of EphB receptors and
ephrin-​B1 in the developing vertebrate spinal cord.
J. Comp. Neurol. 497, 734–750 (2006).
172. Imondi, R., Wideman, C. & Kaprielian, Z.
Complementary expression of transmembrane ephrins
and their receptors in the mouse spinal cord: a
possible role in constraining the orientation of
longitudinally projecting axons. Development 127,
1397–1410 (2000).
www.nature.com/nrn

173. Yokoyama, N. et al. Forward signaling mediated by
ephrin-​B3 prevents contralateral corticospinal axons
from recrossing the spinal cord midline. Neuron 29,
85–97 (2001).
174. Kullander, K. Ephrin-​B3 is the midline barrier that
prevents corticospinal tract axons from recrossing,
allowing for unilateral motor control. Genes Dev. 15,
877–888 (2001).
175. Kullander, K. Role of EphA4 and EphrinB3 in local
neuronal circuits that control walking. Science 299,
1889–1892 (2003).
176. Borgius, L. et al. Spinal glutamatergic neurons defined
by EphA4 signaling are essential components of normal
locomotor circuits. J. Neurosci. 34, 3841–3853
(2014).
177. Butt, S. J. B., Lundfald, L. & Kiehn, O. EphA4 defines a
class of excitatory locomotor-​related interneurons.
Proc. Natl Acad. Sci. USA 102, 14098–14103
(2005).
178. Restrepo, C. E. et al. Change in the balance of
excitatory and inhibitory midline fiber crossing as an
explanation for the hopping phenotype in EphA4
knockout mice. Eur. J. Neurosci. 34, 1102–1112
(2011).
179. Iwasato, T. et al. Rac-​GAP alpha-​chimerin regulates
motor-​circuit formation as a key mediator of EphrinB3/
EphA4 forward signaling. Cell 130, 742–753 (2007).
180. Wegmeyer, H. et al. EphA4-dependent axon guidance
is mediated by the RacGAP α2-chimaerin. Neuron 55,
756–767 (2007).
181. Vallstedt, A. & Kullander, K. Dorsally derived spinal
interneurons in locomotor circuits. Ann. NY Acad. Sci.
1279, 32–42 (2013).
182. Kridsada, K. et al. Roof plate-​derived radial glial-​like
cells support developmental growth of rapidly
adapting mechanoreceptor ascending axons. Cell Rep.
23, 2928–2941 (2018).
183. Paixão, S. et al. EphrinB3/EphA4-mediated guidance
of ascending and descending spinal tracts. Neuron 80,
1407–1420 (2013).
184. Escalante, A., Murillo, B., Morenilla-​Palao, C., Klar, A.
& Herrera, E. Zic2-dependent axon midline avoidance
controls the formation of major ipsilateral tracts in the
CNS. Neuron 80, 1392–1406 (2013).
This paper shows that the transcription factor ZIC2
controls axon laterality in the spinal cord and not
only in the visual system.
185. Canty, A. J. & Murphy, M. Molecular mechanisms of
axon guidance in the developing corticospinal tract.
Prog. Neurobiol. 85, 214–235 (2008).
186. Welniarz, Q., Dusart, I. & Roze, E. The corticospinal
tract: evolution, development, and human disorders.
Dev. Neurobiol. 77, 810–829 (2017).
187. Stanfield, B. B., O’Leary, D. D. M. & Fricks, C. Selective
collateral elimination in early postnatal development
restricts cortical distribution of rat pyramidal tract
neurones. Nature 298, 371–373 (1982).
188. Low, L. K., Liu, X.-B., Faulkner, R. L., Coble, J. &
Cheng, H.-J. Plexin signaling selectively regulates the
stereotyped pruning of corticospinal axons from visual
cortex. Proc. Natl Acad. Sci. USA 105, 8136–8141
(2008).
189. Gu, Z. et al. Control of species-​dependent cortico-​
motoneuronal connections underlying manual
dexterity. Science 357, 400–404 (2017).
This beautiful work shows how species-​specific
expression of the transcription factor FEZF2 and
the plexin A1 receptor might explain the different
targeting of corticospinal axons between rodents
and higher primates.
190. Liu, Y. et al. Ryk-​mediated Wnt repulsion regulates
posterior-​directed growth of corticospinal tract.
Nat. Neurosci. 8, 1151–1159 (2005).
191. Hollis, E. R. et al. Ryk controls remapping of motor
cortex during functional recovery after spinal cord
injury. Nat. Neurosci. 19, 697–705 (2016).
192. Özdinler, P. H. & Macklis, J. D. IGF-​I specifically
enhances axon outgrowth of corticospinal motor
neurons. Nat. Neurosci. 9, 1371–1381 (2006).
193. Kullander, K. et al. Kinase-​dependent and kinase-​
independent functions of EphA4 receptors in major
axon tract formation in vivo. Neuron 29, 73–84
(2001).
194. Katori, S., Noguchi-​Katori, Y., Itohara, S. & Iwasato, T.
Spinal RacGAP α-​chimaerin is required to establish the
midline barrier for proper corticospinal axon guidance.
J. Neurosci. 37, 7682–7699 (2017).
Nature Reviews | Neuroscience
195. Peng, J. et al. Loss of Dcc in the spinal cord is
sufficient to cause a deficit in lateralized motor control
and the switch to a hopping gait. Dev. Dyn. 247,
620–629 (2018).
196. Satoh, D., Pudenz, C. & Arber, S. Context-​dependent
gait choice elicited by EphA4 mutation in Lbx1 spinal
interneurons. Neuron 89, 1046–1058 (2016).
197. Méneret, A. et al. Mutations in the netrin-1 gene
cause congenital mirror movements. J. Clin. Invest.
127, 3923–3936 (2017).
198. Srour, M. et al. Mutations in DCC cause congenital
mirror movements. Science 328, 592 (2010).
199. Gallea, C. et al. RAD51 deficiency disrupts the
corticospinal lateralization of motor control. Brain
136, 3333–3346 (2013).
200. Hollis, E. R. Axon guidance molecules and neural circuit
remodeling after spinal cord injury. Neurotherapeutics
13, 360–369 (2016).
201. Liu, Y. et al. Repulsive Wnt signaling inhibits axon
regeneration after CNS injury. J. Neurosci. 28,
8376–8382 (2008).
202. Hollis, E. R. & Zou, Y. Reinduced Wnt signaling limits
regenerative potential of sensory axons in the spinal
cord following conditioning lesion. Proc. Natl Acad.
Sci. USA 109, 14663–14668 (2012).
203. Jung, H. et al. The ancient origins of neural substrates
for land walking. Cell 172, 667–682 (2018).
204. Friocourt, F. et al. Recurrent DCC gene losses during
bird evolution. Sci. Rep. 7, 37569 (2017).
205. Patthey, C., Tong, Y. G., Tait, C. M. & Wilson, S. I.
Evolution of the functionally conserved DCC gene in
birds. Sci. Rep. 7, 42029 (2017).
206. Avilés, E. C. & Stoeckli, E. T. Canonical wnt signaling
is required for commissural axon guidance.
Dev. Neurobiol. 76, 190–208 (2016).
207. Stoeckli, E. T. Understanding axon guidance: are we
nearly there yet? Development 145, dev151415
(2018).
208. Guo, T. et al. An evolving NGF-​Hoxd1 signaling
pathway mediates development of divergent neural
circuits in vertebrates. Nat. Neurosci. 14, 31–36
(2011).
209. Rexed, B. A cytoarchitectonic atlas of the spinal
cord in the cat. J. Comp. Neurol. 100, 297–379
(1954).
210. Lai, H. C., Seal, R. P. & Johnson, J. E. Making sense
out of spinal cord somatosensory development.
Development 143, 3434–3448 (2016).
211. Lu, D. C., Niu, T. & Alaynick, W. A. Molecular and
cellular development of spinal cord locomotor circuitry.
Front. Mol. Neurosci. 8, 25 (2015).
212. Alaynick, W. A., Jessell, T. M. & Pfaff, S. L. SnapShot:
spinal cord development. Cell 146, 178–178
(2011).
213. Hayashi, M. et al. Graded arrays of spinal and
supraspinal V2a interneuron subtypes underlie
forelimb and hindlimb motor control. Neuron 97,
869–884 (2018).
214. Dasen, J. S. & Jessell, T. M. Hox networks and the
origins of motor neuron diversity. Curr. Top. Dev. Biol.
88, 169–200 (2009).
215. Rosenberg, A. B. et al. Single-​cell profiling of the
developing mouse brain and spinal cord with split-​pool
barcoding. Science 360, 176–182 (2018).
This article presents one of the recent cell profiling
and RNA sequencing studies that illustrate the
unknown diversity of spinal cord interneurons.
216. Sathyamurthy, A. et al. Massively parallel single
nucleus transcriptional profiling defines spinal cord
neurons and their activity during behavior. Cell Rep.
22, 2094–2106 (2018).
217. Häring, M. et al. Neuronal atlas of the dorsal horn
defines its architecture and links sensory input to
transcriptional cell types. Nat. Neurosci. 21,
869–880 (2018).
218. Bikoff, J. B. et al. Spinal inhibitory interneuron
diversity delineates variant motor microcircuits. Cell
165, 207–219 (2016).
219. Niu, J. et al. Modality-​based organization of ascending
somatosensory axons in the direct dorsal column
pathway. J. Neurosci. 33, 17691–17709 (2013).
220. Bermingham, N. A. et al. Proprioceptor pathway
development is dependent on Math1. Neuron 30,
411–422 (2001).
221. Hantman, A. W. & Jessell, T. M. Clarke’s column
neurons as the focus of a corticospinal corollary
circuit. Nat. Neurosci. 13, 1233–1239 (2010).
Reviews
222. Yuengert, R. et al. Origin of a non-​clarke’s column
division of the dorsal spinocerebellar tract and the role
of caudal proprioceptive neurons in motor function.
Cell Rep. 13, 1258–1271 (2015).
223. Tracey, D. P. in The Rat Nervous System 3rd edn
(ed. Paxinos, G.) 149–164 (Academic Press, 2004).
224. Caggiano, V. et al. Midbrain circuits that set locomotor
speed and gait selection. Nature 553, 455–460
(2018).
This work identifies two populations of supraspinal
glutamatergic neurons in the midbrain that control
two different locomotor behaviours.
225. Esposito, M. S., Capelli, P. & Arber, S. Brainstem
nucleus MdV mediates skilled forelimb motor tasks.
Nature 508, 351–356 (2014).
226. Hippenmeyer, S. et al. A developmental switch in the
response of DRG neurons to ETS transcription factor
signaling. PLOS Biol. 3, e159 (2005).
227. Varadarajan, S. G. & Butler, S. J. Netrin1 establishes
multiple boundaries for axon growth in the developing
spinal cord. Dev. Biol. 430, 177–187 (2017).
228. Ter-​Avetisyan, G., Rathjen, F. G. & Schmidt, H.
Bifurcation of axons from cranial sensory neurons is
disabled in the absence of Npr2-induced cGMP
signaling. J. Neurosci. 34, 737–747 (2014).
229. He, Z. & Tessier-​Lavigne, M. Neuropilin is a receptor
for the axonal chemorepellent Semaphorin III. Cell 90,
739–751 (1997).
230. Xu, K. et al. Structures of netrin-1 bound to two
receptors provide insight into its axon guidance
mechanism. Science 344, 1275–1279 (2014).
231. Wang, H., Copeland, N. G., Gilbert, D. J., Jenkins, N. A.
& Tessier-​Lavigne, M. Netrin-3, a mouse homolog of
human NTN2L, is highly expressed in sensory ganglia
and shows differential binding to netrin receptors.
J. Neurosci. 19, 4938–4947 (1999).
232. Ly, A. et al. DSCAM is a netrin receptor that
collaborates with DCC in mediating turning responses
to Netrin-1. Cell 133, 1241–1254 (2008).
233. Corset, V. et al. Netrin-1-mediated axon outgrowth
and cAMP production requires interaction with
adenosine A2b receptor. Nature 407, 747–750
(2000).
234. Rama, N. et al. Amyloid precursor protein regulates
Netrin-1-mediated commissural axon outgrowth.
J. Biol. Chem. 287, 30014–30023 (2012).
235. Thiry, L., Lemieux, M., Laflamme, D. O. & Bretzner, F.
Role of DSCAM in the development of the spinal
locomotor and sensorimotor circuits. J. Neurophysiol.
115, 1338–1354 (2016).
236. Li, H. S. et al. Vertebrate slit, a secreted ligand for the
transmembrane protein roundabout, is a repellent for
olfactory bulb axons. Cell 96, 807–818 (1999).
237. Arbeille, E. et al. Cerebrospinal fluid-​derived
Semaphorin3B orients neuroepithelial cell divisions in
the apicobasal axis. Nat. Commun. 6, 6366 (2015).
238. Chen, H., Chédotal, A., He, Z., Goodman, C. S. &
Tessier-​Lavigne, M. Neuropilin-2, a novel member of
the neuropilin family, is a high affinity receptor for the
semaphorins Sema E and Sema IV but not Sema III.
Neuron 19, 547–559 (1997).
239. Tamagnone, L. et al. Plexins are a large family of
receptors for transmembrane, secreted, and
GPI-anchored semaphorins in vertebrates. Cell 99,
71–80 (1999).
240. Seiradake, E., Jones, E. Y. & Klein, R. Structural
perspectives on axon guidance. Annu. Rev. Cell
Dev. Biol. 32, 577–608 (2016).
Acknowledgements
A.C. is supported by grants from the Agence Nationale de la
Recherche (ANR; ANR-14-CE13-0004-01), the LABEX
LIFESENSES (reference ANR-10-LABX-65) and French state
funds managed by the ANR within the Investissements d’Avenir
programme under reference ANR-11-IDEX-0004-02.
Competing interests
The author declares no competing interests.
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Reviewer information
Nature Reviews Neuroscience thanks G. Bashaw, D.
Bonanomi, Y. Zou and other anonymous reviewer(s) for their
contribution to the peer review of this work.