commissural axons

YADDLE
Precisely
pointed paths
Dr Valérie Castellani describes the work of her group in
furthering understanding of the intricate system of multiple cues
that guide the integration of the central nervous system
circuits, connect other neurons of the spinal
cord and also convey information to higher
brain centres.
Why have you monitored growth cone
responsiveness?
Could you explain the context from which
your work emerged?
The first ‘axon guidance molecule’ was
discovered in the 1990s. Since then,
accumulating evidence has indicated that the
mechanism of action of axon guidance cues
is much more complex than initially thought.
In particular, many molecules interact and
novel properties can emerge from cues acting
together, rather than individually. Our current
goals are thus to characterise the molecular
crosstalks between the different protein
families and to understand what particular
information they deliver to growing axons.
How are dorsal interneurons integral to
the nervous system?
Our experimental model is the navigation
of the commissural axons in the spinal
cord. Commissures are projections that
interconnect the two sides of the central
nervous system and ensure coordination
and integration of motor and sensory
commands. A crucial step in their navigation
is the crossing of the midline dividing the
nervous system, which is controlled by
multiple axon guidance cues. In the spinal
cord, the commissures are formed by
interneurons. The commissural projections of
interneurons residing in the dorsal part of the
spinal cord participate in various neuronal
To build neuronal circuits, neurons extend
an axon which elongates and navigates
towards its target cell. The axon is tipped
by a highly motile and enlarged structure
– the growth cone – composed of filopodia
and lamellipodia, which explores the local
environment like a sensor. The growth
cone expresses receptors, allowing it to
perceive and respond to corresponding
guidance cues, and to guide the axon in
the appropriate direction. Thus, to explore
the mechanisms of axon guidance cues, we
monitor the behaviour of the growth cone
under various experimental conditions.
Why are molecular crosstalks of
particular interest in axon guidance?
Axon navigation proceeds by steps, and in
some of them, crucial guidance decisions
have to be made. Crossing or not crossing
the midline is one such decision.
Axon guidance cues must segregate the
axons that have to cross from those that do
not, attract the crossing axons towards the
midline and ensure that they exit the midline
to continue towards their final targets. So it’s
a sort of paradox, because the midline must
be firstly attractive and secondly repulsive.
This switch is accomplished via molecular
crosstalks and modulation of growth cone
responsiveness to midline guidance cues.
The midline co-expresses both attractant
and repellent cues, plus cues that enable the
axons to first perceive the attractants and
then the repellents.
Can you describe the approaches adopted
in your research?
We studied genetically modified mouse
models in vivo. We also manipulated genes
of interest by electroporation of the spinal
cord in chick embryos. Such an approach
allows the rapid investigation of molecular
adjustments, and is therefore a very
powerful alternative to the mouse model.
For example, we engineered a fluorescent
tool to monitor the dynamics of a
guidance receptor during midline crossing
in our chick model. We also set up various
neuronal and tissue cultures, using an
approach that we designed several years
ago which supports the investigation of
signal transduction in fresh commissural
tissue. This confers the great advantage
of enabling us to study neurons in their
physiological environment.
Could you explain how your findings
advance knowledge in this field?
At the molecular level, our findings
revealed the existence of cooperation
between axon guidance cues and
neurotrophic factors. More generally, ours
was the first report to show that regulated
processing of a guidance receptor can
control the competence of the growth
cone to respond or not to a guidance cue.
This molecular strategy might operate
for other guidance receptors and in other
biological contexts whose characterisation
would be of great interest.
Do you work closely with any other
researchers?
The axon guidance community has built
collaborative networks over the years,
which share many molecular tools and
animal models. We have benefited
greatly from such networks, and were
able to transfer mouse colonies from
many different labs in the US and Europe
to our institute. We also regularly
exchange constructs of receptors and
axon guidance cues as a basis for the
generation of additional versions of
modified proteins, which we then share.
Collaboration greatly facilitates the
advancement of our research.
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YADDLE
The complex choreography
of commissural axons
Led by researchers from the University of Lyon, the YADDLE project has deciphered a finely tuned series of
molecular crosstalks that guides the formation of neuronal networks across the central nervous system midline
AMONG THE TASKS carried out by the many
billions of neurons in the human central nervous
system (CNS), left/right motor coordination,
integration of brain function processing and
synchronisation of sensory information are
established by the commissural neurons. The
commissural neurons engineer interconnecting
neuronal networks across the two sides of the
CNS in both the brain and spinal cord during
foetal development, by extending their axons
across the midline of the CNS. In the course
of this, the axons often have to navigate long
distances through diverse environments; as
a consequence, they have to find their way
through multiple pathways.
If the wiring of any of the commissural circuits
is insufficiently established before birth,
communication, tolerance of pain, information
processing or motor coordination may be
affected and, in some cases, severe physical
or mental disability may result. Damage to
the circuits can also arise in adults, through
neurodegenerative diseases, such as multiple
sclerosis and Parkinson’s, or brain or spinal cord
injury. Unfortunately, at present it is impossible
to regenerate damaged axons or to reintegrate
disrupted neuronal circuits.
AXON GUIDANCE
Dr Valérie Castellani is Team Leader in the Centre
de Génétique et de Physiologie Moléculaire et
Cellulaire at the University of Lyon. Her research
focus encompasses the molecular processes in
neural development and neuronal signalling,
with particular emphasis on axon guidance over
recent years. Castellani’s work has contributed
a number of important insights into axon
guidance mechanisms, paving the way to the
discovery of crosstalks between some receptor
molecules and members of the semaphorin
family of proteins, and revealing for the first
time signalling pathways that are both repulsive
84INTERNATIONAL INNOVATION
and attractive to axons in a commissure linking
the hemispheres of the brain.
In a recent project – YADDLE – Castellani’s
group has explored the molecular mechanisms
of the protein interactions that constitute cues
for directing the passage of commissural axons
across the midline of the spinal cord, then
guide them to select their targets to establish
appropriate connections with neurons in the
spine and higher brain centres.
commissural axons towards the CNS midline.
Castellani and her group found that a molecular
pathway controls the commissural axon gain
of response to a member of the semaphorin
family – Sema3B – and that at the midline,
Sema3B then exerts a repulsive effect on the
axons. From this, the team has deduced that
the responsiveness of commissural axons to
Sema3B must first be silenced to allow them to
enter into crossing, and only activated to expel
them once they have passed the midline.
MOLECULES IN CONCERT
CROSSING THE MIDLINE: IN DEPTH
The spinal commissural axons cross the midline
in the floor plate, a structure of specialised glial
cells that extends in the embryonic precursor
of the spine – the neural tube – from the
midbrain to its end. In the process, the tip of
the axon – the growth cone – leads the way. The
key question that Castellani’s study sought to
answer was how a limited number of guidance
cues could direct the growth cones, and thus
the axons, to accomplish the highly complex
task of building neuronal networks. Combining
in vivo and ex vivo approaches, Castellani and
her group therefore investigated the means by
which the growth cone becomes appropriately
sensitised to different navigation cues at each
stage of the crossing to ensure that the axons
respond, follow the right pathway precisely
and only once, and ultimately connect to their
specific target cells.
Castellani and her colleagues have established
that, before crossing, the commissural neurons
synthesise the Sema3B receptors Nrp2 and
PlexinA1 to ensure that the commissural axons
are sensitised to the Sema3B residing at the
midline. The floor plate then acts as an axon
guidance cue activation/deactivation switch,
initiating the post-crossing step by expressing
a growth factor, glial cell-derived neurotrophic
factor (GDNF), which is expressed in multiple
The semaphorin family of proteins and their
corresponding receptors – the plexins – play
an important role in guiding axons during
development. They regulate actin dynamics,
and are highly expressed in scar tissue in lesions
of the CNS. They also control cell motility in
many different organs and altered semaphorin
signalling is associated with cancer metastasis.
In the formation of neuronal connections,
their role is to guide the growth cone tips of
Immunolabelling in three colours showing the expression
of three guidance receptors in a commissural growth
cone, delineated by the white line.
INTELLIGENCE
YADDLE – MOLECULAR CROSSTALKS
DURING COMMISSURAL AXON
GUIDANCE IN THE DEVELOPING
SPINAL CORD
OBJECTIVES
To provide a molecular characterisation of
crosstalks playing important roles during
axon navigation; more specifically:
• To characterise receptors and signal
transduction pathways mediating the
different guidance effects of the Sema3s
• To identify new mechanisms controlling axon
responsiveness to Sema3s and to define in
vivo contexts in which they are acting
KEY TEAM MEMBERS
Actin (red), nucleus (blue) and neuropilin
receptor (green) labelling in cultures of
dissociated spinal commissural neurons.
tissues in the human body, from the heart
and liver to the lungs and testicles. During
foetal development, GDNF regulates neuronal
cell differentiation, proliferation and survival
and participates in cell migration: “We know
now that when commissural axons reach the
midline, PlexinA1 is not available at the growth
cone surface because it is processed by calpains.
This explains why the growth cone is insensitive
to Sema3B,” reveals Castellani. “Upon crossing,
the growth cones become exposed to the GDNF
expressed by the floor plate. The GDNF signal
is transmitted to the growth cone via a neural
cell adhesion molecule receptor; it suppresses
calpain activity, subsequently restores PlexinA1,
and thus switches on the responsiveness of the
commissural growth cones to Sema3B.”
The sequencing of steps in the signalling
process therefore indicates cooperative activity
between the glial cells in the floor plate and the
axons; multiple signalling exchanges explain
the conundrum of how neuronal growth cone
behaviour can be managed via a limited number
of cues – growth cone responses to the same cue
vary at different stages in the crossing process.
The ‘decision’ to cross the midline results from
the integration of successive and intricate
regulatory steps controlling the distribution of
guidance receptors at the growth cone surface:
“A complex and very precisely orchestrated
molecular programme controls the crucial step
of midline crossing,” summarises Castellani.
POSSIBILITIES FOR FINE-TUNED THERAPY
It is its diverse properties and actions in
the adult brain, ranging from regulation
of dopaminergic neurons to conferring
neurotrophic and neuroprotective effects, that
make GDNF an ideal candidate for targeted
drugs for neurodegenerative diseases. Because
of this, it is already under examination as a
possible means of treating Parkinson’s. Now,
the new Sema3B signalling pathway regulation
role that Castellani and her group have found
that GDNF plays in the development of
neuronal networks raises the possibility that
other neurotrophic factors may have similar
properties and also that its regulation of
PlexinA1 may be mirrored with other plexin
receptors. If this is found to be the case, it
would greatly expand the range of interactions
between GDNF and the plexin family and so
widen the scope for finding new therapeutic
targets for a range of maladies.
Sema3B and related family members exert
inhibitory effects that preclude regeneration of
damaged axons. Hence, greater understanding
of their signalling pathways also raises the
prospect of modifying their receptors to
manipulate their behaviour: “Understanding
the mechanisms of action of guidance cues
and identifying their regulators is an obligatory
step towards the design of efficient therapeutic
strategies,” Castellani asserts.
Castellani’s group has explored
the molecular mechanisms of the
protein interactions that constitute
cues for directing the passage
of commissural axons across
the midline of the spinal cord,
then guide them to select their
targets to establish appropriate
connections with neurons in the
spine and higher brain centres
Dr Frédéric Moret • Dr Julien Falk • Dr
Edmund Derrington • Dr Arnaud Jacquier •
Dr Céline Delloye • Elise Arbeille • Camille
Charoy • Leila Boubakar • Muriel Bozon •
Karine Kindbeiter • Karine Thinet •
Florie Reynaud
FUNDING
European Research Council
Labex DevWeCan
Labex CORTEX
CNRS
ANR
CONTACT
Dr Valérie Castellani
Team Leader
University of Lyon 1
Campus de la Doua
Building Gregor Mendel
16 Rue Dubois – 69622
Villeurbanne Cedex
France
T +33 472 43 26 91
E [email protected]
DR VALÉRIE CASTELLANI gained an
MSc in Genetics and obtained her PhD in
Neuroscience in 1998. After postdoctoral
training in the Institute of Developmental
Biology of Marseille Luminy, she obtained
a CNRS research position. In 2003, she was
funded by the CNRS (ATIP programme)
to set up her own group, and moved to
the University of Lyon to become part of
the CNRS research centre, the Centre de
Génétique et de Physiologie Moléculaire et
Cellulaire (CGphiMC).
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