Motor Neuron Position
and Topographic Order Imposed
by b- and g-Catenin Activities
Elena Y. Demireva,1Lawrence S. Shapiro,2Thomas M. Jessell,1,2,* and Niccolo ` Zampieri1
1Department of Neuroscience
2Department of Biochemistry and Molecular Biophysics
Howard Hughes Medical Institute, Kavli Institute for Brain Science, Columbia University, New York, NY 10032, USA
Neurons typically settle at positions that match the
location of their synaptic targets, creating topo-
graphic maps. In the spinal cord, the organization
of motor neurons into discrete clusters is linked to
the location of their muscle targets, establishing a
topographic map of punctate design. To define the
significance of motor pool organization for neuro-
muscular map formation, we assessed the role of
cadherin-catenin signaling in motor neuron posi-
tioning and limb muscle innervation. We find that
joint inactivation of b- and g-catenin scrambles
motor neuron settling position in the spinal cord but
fails to erode the predictive link between motor
neuron transcriptional identity and muscle target.
Inactivation of N-cadherin perturbs pool positioning
in similar ways, albeit with reduced penetrance.
These findings reveal that cadherin-catenin signaling
directs motor pool patterning and imposes topo-
graphic order on an underlying identity-based neu-
In many regions of the central nervous system (CNS), the
dained plan in which the identity and/or location of an individual
neuron is inexorably linked to the position of its synaptic target,
creating a neural map. Highly ordered maps, typified by retinal
projections in the visual system, exhibit a continuously graded
topography that links the position of neuronal cell bodies and
synaptic targets along linear axes (Sperry, 1963; Luo and Flana-
gan, 2007). A second class of topographic maps, exemplified by
the divergent projections of nuclear subgroups in the amygdala
(Pitka ¨nen et al., 1997), assigns neuronal cell body position in
punctate rather than linear coordinates, yet preserves the pre-
dictive link between neuronal position and innervation target.
For a third class of maps, notably those found in olfactory
systems, topography is cast aside and molecular identity rather
than position binds sensory neurons to their target structures
(Imai et al., 2010). For topographic maps, however, it remains
unclear if the precise positioning of neurons is a critical element
in the formation of target connections, or is merely an incidental
byproduct of circuit assembly.
The spatial order of CNS neurons is readily apparent in the
spinal cord, where motor neurons serving different biomechan-
ical functions are organized into circumscribed groups, each
occupying an invariant location. The precision of motor neuron
positioning is at its most intricate for circuits that control limb
movement: each of the 50 or so muscles used in locomotion
and object manipulation receives input from a dedicated set of
motor neurons whose cell bodies are clustered into stereotypic
pools (Romanes, 1964; Vanderhorst and Holstege, 1997). The
constancy of motor pool positioning has potential implications
for the assembly of sensory-motor circuits; motor neuron posi-
tion is predictive of sensory input specificity (Ladle et al.,
2007), as well as the pattern of muscle target innervation (Land-
messer, 1978). Thus, scrambling motor neuron position could
uncouple the link between identity and muscle target or simply
downgrade neuromuscular mapping to a state in which identity
but not position is aligned with target connectivity. To date,
however, it has not been possible to manipulate the position of
motor neurons in a manner subtle enough to permit analysis of
consequent alterations in patterns of target innervation.
Manipulating cell surface recognition systems that control the
segregation and settling of motor neurons could help to define
how neuronal position influences motor circuit assembly. Clas-
sical cadherins represent a prominent family of adhesion pro-
teins expressed by spinal motor neurons (Price and Briscoe,
2004). Manipulation of type II cadherin expression profiles in
chick embryos disrupts the normal settling pattern of motor
pools (Price et al., 2002; Patel et al., 2006). Nevertheless, mice
in which individual type II cadherins have been eliminated genet-
ically exhibit no obvious defects in motor neuron positioning
(E.Y.D., S. Price, N.Z., and T.M.J., unpublished data). Given
the diversity of type II cadherins expressed by motor neurons
(Price et al., 2002; this study), it is possible that residual profiles
of cadherin expression are sufficient to maintain molecular and
spatial distinctions between motor neuron subsets. Moreover,
type I cadherins expressed by spinal motor neurons (Zelano
et al., 2006) could also participate in motor neuron sorting.
Cell 147, 641–652, October 28, 2011 ª2011 Elsevier Inc. 641
To circumvent the cadherin diversity problem, we turned our
attention to catenins, the primary intracellular transducers of
classical cadherin-mediated adhesion and recognition (Kemler,
1993; Nelson, 2008). Within the catenin family, the activities of
b- and g-catenin can be distinguished from those of a- and
d-catenin on the basis of different modes of interaction with
the cytoplasmic domain of cadherins (Huber and Weis, 2001).
b-catenin is the most widely studied mediator of classical cad-
nonneural cell types (Butz et al., 1992; Zhurinsky et al., 2000).
But there has been no systematic exploration of the respective
roles of b- and g-catenin as mediators of cadherin signaling in
the developing CNS, nor of their potential role in motor neuron
positioning and target connectivity.
In this study, we inactivated the cadherin-catenin signaling
pathway in spinal motor neurons to assess the significance of
motor neuron position in motor circuit assembly. Genetic inacti-
vation of both b- and g-catenin, or of N-cadherin, disrupts motor
neuron positioning. This degradation of positional order fails to
undermine the predictive link between transcriptional identity,
axonal trajectory, and muscle target selectivity. Thus, the clus-
tering and positioning of cell bodies is not required for motor
neuron subtypes to innervate appropriate target muscles. Nev-
ertheless, the emergence of neuromuscular maps of topo-
graphic rather than identity-based design is dependent on cad-
Overlapping Expression of b- and g-Catenin in Spinal
To assess the involvement of catenins in motor neuron posi-
tioning and target topography, we examined b- and g-catenin
expression profiles in mouse spinal cord between embryonic
day (e) 9.5 and postnatal day (p) 0.
Broad expression of b-catenin mRNA and protein was de-
tected from e9.5 (Figures 1A and 1B and Figures S1A and S1B
available online). In embryonic motor neurons marked by GFP
expression in Hb9::GFP transgenic mice (Wichterle et al.,
2002), b-catenin protein was expressed at high levels on the
cell surface, but at negligible levels in the cytoplasm (Figures
1E and 1F). From e10.5 to e11.5, expression of g-catenin was
also expressed by neurons in other domains of the spinal cord
(Figures S1C and S1D). In motor neurons, g-catenin protein
with lower levels in the cytoplasm (Figures 1G and 1H). Thus, b-
and g-catenin are coexpressed by spinal motor neurons as
they settle in distinct positions and establish axonal trajectories.
Eliminating b- and g-Catenin from Spinal Motor Neurons
We evaluated the impact of inactivating b- and g-catenin genes
from motor neurons. In mouse embryos, b-catenin activity is re-
quired at early developmental stages and constitutive b-catenin
mutants die before motor neuron generation (Haegel et al.,
1995). To bypass this early requirement, we crossed mice
carrying two copies of a floxed b-catenin (b-catflox/flox) allele
(Brault et al., 2001) with an Olig2::Cre driver line that directs
recombination in motor neuron progenitors (Dessaud et al.,
2007), to generate bDMNmice. We found that over 90% of motor
neurons in bDMNmice lacked b-catenin transcript at e13.5, with
no obvious loss from other spinal cells (Figures 1I–1L), and
mutant mice died within 24 hr of birth (Table S1). Constitutive
g-catenin (g?/?) mutant mice (Ruiz et al., 1996) died between
e10.5 and e15.5 from heart defects (Table S1), and revealed
a complete loss of g-catenin mRNA and protein from embryonic
motor neurons and other spinal cord cells (Figures 1M–1P).
We generated b- and g-catenin double-mutant (bDMNg?/?)
embryos by introducing constitutive g-catenin mutant alleles
into an Olig2::Cre+/?; b-cat
bDMNg?/?embryos were detected at lower than predicted
(1:16) Mendelian frequencies: approximately 1:30 at e10.5 and
approximately 1:44 at e13.5. We attempted to extend the
viability of catenin double-mutant embryos through the genera-
tion and use of a conditional g-catenin mutant allele (g-catflox;
Figures S1M–S1O). Crossing Olig2::Cre and g-catflox/floxmice
produced embryos in which g-catenin protein was eliminated
preferentially from motor neurons (Figures S1P–S1S). But re-
combined Olig2::Cre+/?; g-cat-/flox; b-catflox/flox
double conditional mutants survived only until e14.5 (Table S1),
limiting analysis of motor neuron differentiation to defects evi-
dent by this developmental stage.
We first assessed the impact of catenin activity on motor neu-
ron differentiation through analysis of phenotypes in b- and
g-catenin single, and bDMNg?/?double-mutant embryos. Motor
neuron columnar classes (Figure 2A) were identified by tran-
scription factor expression profiles (Dasen et al., 2008). At
lumbar levels, lateral motor column (LMC) neurons express
FoxP1, whereas median motor column (MMC) neurons express
Lhx3. At thoracic levels, hypaxial motor column (HMC) neurons
coexpress Hb9 and Isl1/2 but not FoxP1 or Lhx3, and pregangli-
onic column (PGC) neurons coexpress nNOS and pSMAD.
to e13.5 was similar in control, b- and g-catenin single-mutant,
and bDMNg?/?double-mutant embryos (Table S2). Although the
number of LMC, HMC, and PGC neurons was similar in
bDMNg?/?and control embryos, we detected an approximately
25% reduction in the number of MMC neurons in bDMNg?/?, as
well as in single b-catenin mutants (Figure 2F and Table S2). In
addition, we observed that in bDMNg?/?embryos approximately
15% of motor neurons, primarily MMC neurons, failed to migrate
away from the ventricular zone (Figures 2C and S2A and Table
S3). Nevertheless, the number of postmigratory LMC, HMC,
and PGC neurons was similar in bDMNg?/?and control embryos
(Figure 2F). Moreover, LMC and HMC neurons remained segre-
gated from MMC neurons at lumbar and thoracic levels, respec-
tively (Figures 2B–2E; data not shown).In contrast, PGC neurons
were scattered in ectopic ventral positions in e13.5 bDMNg?/?
embryos (Figures 2H and 2I; Figure S2B). Thus, despite the
disruption in MMC and PGC organization, the specification,
lateral migration, and segregation of LMC neurons are little
affected by the loss of b- and g-catenin activities.
642 Cell 147, 641–652, October 28, 2011 ª2011 Elsevier Inc.
Analysis of Motor Neuron Clustering and Mixing
Neuronal mixing indices for segregation of LMC neurons are described in
Extended Experimental Procedures.
Retrograde Labeling of Motor Neurons
Embryos (e11.5 to e14.5) were injected with a 10% rhodamine-dextran (3K
MW) solution into individual hindlimb muscles and incubated for 3–5 hr at
27?C before processing.
Motor Neurite Outgrowth
Motor neurons were derived from e10.5 embryos carrying an Hb9::GFP allele,
and plated on a confluent monolayer of naive or N-cad-transfected CHO
cells, and cultured for 16–20 hr in medium supplemented with trophic factors.
GFP+neurite length was determined using MetaMorph software (Molecular
figures,and four tablesand can befound withthisarticle online atdoi:10.1016/
The authors are indebted to Stephen Price for early contributions to this
project. The authors thank Qiaolian Liu, BarbaraHan and IraSchieren for tech-
nical help, Susan Brenner-Morton for antibody generation, Monica Mendel-
sohn, Jennifer Kirkland, Barbara Han, and Susan Kales for help in the genera-
tion of conditional g-catenin mutant mice, Laskaro Zagoraiou and Apostolos
Klinakis for DNA constructs, Corey Washington for statistical analysis, Kendall
Doerr for valuable perspective, and Ira Schieren and Kathy MacArthur for help
in preparing the manuscript. We are grateful to Rolf Kemler, Patricia Ruiz,
Masatoshi Takeichi, Barbara Ranscht, Glenn Radice, and Makoto Taketo for
mouse lines. Natalia de Marco Garcia, Sebastian Poliak, Joriene de Nooij,
Jeremy Dasen, Carol Mason, Christopher Henderson, and Barbara Ranscht
provided advice and helpful comments on the manuscript. E.Y.D. was sup-
ported by NINDS RO1 NS033245; N.Z. was supported by HHMI; L.S.P. was
supported by NIH R01GM062270; T.M.J. was supported by grants from the
NINDS RO1 NS033245, the Wellcome Trust, the G. Harold and Leila Y. Math-
ers Foundation, and Project A.L.S. and is an Investigator of the Howard
Hughes Medical Institute.
Received: March 18, 2011
Revised: July 14, 2011
Accepted: September 26, 2011
Published: October 27, 2011
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