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.
Although LMC neurons remain segregated from other
columnar subtypes, we observed that the area of the ventral
spinal cord occupied by the LMC was approximately 20%
greater in bDMNg?/?than in control embryos, and the packing
density of FoxP1+LMC neurons was reduced (Figures 2G, 2J,
and 2K). However overall neuronal packing density within the
confines of the LMC, defined by NeuN expression was un-
changed (Figures 2G, 2J, and 2K), implying that other neuronal
classes have encroached the boundaries of the LMC. Con-
sistent with this, we detected an approximately 2.5-fold increase
in the number of En1+V1 interneurons and an approximately
3-fold increase in Chx10+V2a interneurons within the confines
of the LMC in bDMNg?/?embryos (Figures S2C–S2L). Thus, the
erosion of LMC cohesion elicited by loss of b- and g-catenin
activities permits intercalation of ventral interneurons.
b- and g-Catenin Activities Required for LMC Divisional
Segregation and Pool Sorting
We examined whether the segregation of LMC neurons is
affected by the loss of b- and g-catenin activities. In bDMNg?/?
embryos, we observed a marked intermixing of medial and
lateral LMC neurons, evident by e11.5 (Figures 3A and 3B). We
devised a divisional mixing index (Dmi) to monitor the extent to
which Isl1+medial (M) LMC neurons invade the confines of
the HB9+lateral (L) LMC division, and vice versa. In control
e13.5 embryos we observed a relatively low incidence of
Figure 1. b- and g-Catenin Expression in Developing Motor Neurons
(A and B) b-catenin (b-cat) expression in e10.5 lumbar spinal cord.
(C and D) g-catenin (g-cat) expression in e10.5 lumbar spinal cord. Arrow marks blood vessels. Between e15.5 and p0, g-catenin transcript is extinguished from
most spinal motor neurons (see also Figures S1E–S1H).
(E–H) b-cat and g-cat expression in GFP+lumbar motor neurons in e13.5 Hb9::GFP transgenic mice.
(I and K) b-cat expression in e13.5 lumbar spinal cord from control b-catfl/fland bDMNembryos.
a marked upregulation of g-catenin protein (see also Figures S1I–S1L).
(M–P) Absence of g-cat expression in e11.5 lumbar spinal cord of g-cat?/?mice.
RNA expression data in (A), (C), (I), and (K) with positive signal in black.
Cell 147, 641–652, October 28, 2011 ª2011 Elsevier Inc. 643
interdivisional mixing (Dmi: M/L 0.23, L/M 0.30) (Figures 3C,
3E, 3G, and 3H). In bDMNg?/?embryos we detected an approxi-
mately 2.5- to 3-fold increase in Dmivalues (Dmi: M/L 0.70, L/
M 0.71; p < 0.0001, versus control embryos) (Figures 3D, 3F, 3G,
and 3H). Dmivalues in b- and g-catenin single mutants were
similar to those in control embryos (data not shown). Thus, b-
and g-catenin activities promote the divisional segregation of
We next examined whether b- and g-catenin are required for
the clustering and segregation of motor pools. We focused on
motor pool complexes that occupy medial or lateral LMC divi-
sions and are identifiable by expression of homeodomain and
ETS transcription factors (De Marco Garcia and Jessell, 2008).
The adductor/gracilis (A/G) pool complex was identified by co-
expression of Nkx6.1 and Er81; hamstring (H) pools by expres-
sion of Nkx6.1; vasti (V) pools by expression of Er81, and the
rectus femoris/tensor fasciae latae (R/T) pool complex by
expression of Nkx6.2 (Figures 3I and 3K). We detected no
change in the number of motor neurons allocated to these pool
complexes in bDMNg?/?embryos (data not shown).
To provide a quantitative assessment of motor pool organiza-
tion, we used a pool mixing index (Pmi) to document neuronal
sessed the impact of b- and g-catenin inactivation on the clus-
tering of motor pools that reside in different LMC divisions,
analyzing the segregation of lateral R/T from medial H pools. In
control embryos, R/T neurons were clustered in a position lateral
to H neurons and exhibited a low incidence of intermixing (Pmi
[H/R/T]0.32) (Figures 3I, 3K, and 3M). In bDMNg?/?embryos, R/
T neurons were no longer clustered or laterally restricted and
were intermingled with Hneurons, with Pmivalues approximately
3-fold greater than control (Pmi [H/R/T]0.88, p < 0.0001 versus
control) (Figures 3J, 3L, and 3M). A similar analysis of inter-
mixing between R/T and A/G pool complexes revealed an
(Table S4). We did not observe interdivisional mixing of R/T and
H pools in single b- and g-catenin mutants (Table S4). Thus,
neurons in motor pools that normally occupy different LMC divi-
sions are intermingled in catenin mutants, a result consistent
with divisional intermixing.
We also examined whether the clustering and positioning of
motor pools that reside within a single (lateral) LMC division
are affected by the loss of b- and g-catenin activities. In control
embryos neurons in R/T and V pools were tightly clustered
and exhibited little intermixing (Pmi [V/R/T] 0.26) (Figures 3K
and 3M), whereas in bDMNg?/?embryos neurons in these two
pools were more scattered and extensively intermingled (Pmi
[V/R/T]0.66; p < 0.0001 versus control) (Figures 3L and 3M). In-
tradivisional mixing of these pools was not observed in b- and
g-catenin single mutants (Table S4). Thus, the segregation of
motor pools that normally occupy the same LMC division is
also disrupted by the loss of motor neuron b- and g-catenin
activities. In addition, we examined whether there are defects
in motor pool position along the rostrocaudal axis of the spinal
cord. Analysis of the position of molecularly defined motor pools
limits were preserved in bDMNg?/?embryos (Figure S3). Thus, b-
and g-catenin activities control intrasegmental but not rostro-
caudal pool organization.
MotorPoolIdentity Still PredictsMuscle Target in b-and
We next asked whether the disruption in motor pool positioning
that accompanies the loss of b- and g-catenin activities erodes
Figure 2. Motor Neuron Columnar Segregation in
b- and g-Catenin Mutants
(A) Topographic order in motor innervation of the mouse
muscles, hypaxial motor column (HMC) neurons innervate
body wall muscles, lateral motor column (LMC) neurons
innervate limb muscles, and preganglionic motor column
(PGC) neurons innervate sympathetic ganglion (SG)
(B and C) Segregation of Lhx3+MMC and FoxP1+LMC
neurons at lumbar levels of e11.5 control and bDMNg?/?
(D and E) Segregation of Lhx3+, Hb9+, Isl1+MMC, and
Hb9+, Isl1+LMC neurons at lumbar levels of e13.5 control
spinal cord of control (b+/±g+/±) and bDMNg?/?embryos.
Motor neurons/100 mm: mean ± SEM (difference from
control significant for MMC neurons; t test, p < 0.0001).
Control catenin group (b+/±g+/±) includes genotypes:
b+/+g+/+(wt), b+/DMNg+/+, b+/+g+/?, and b+/DMNg+/?. For
additional quantitative data, see also Table S2.
(G) NeuN+and FoxP1+neuronal densities in the LMC of
e13.5 control and bDMNg?/?embryos; mean ± SEM (t test,
p < 0.001 versus control for FoxP1+density).
(H and I) pSMAD+, nNOS+PGC neurons in e13.5 control
(J and K) NeuN+, FoxP1off, and NeuN+, FoxP1+neurons within the LMC in e13.5 of control and bDMNg?/?embryos.
For additional related data regarding motor neuron migration and invasion, see Figure S2.
644 Cell 147, 641–652, October 28, 2011 ª2011 Elsevier Inc.
the predictive link between motor neuron transcriptional identity,
axonal trajectory, and limb muscle innervation.
similar in the hindlimbs of control and bDMNg?/?embryos that
carried an Hb9::GFP allele (Figures S4A and S4B). This finding
permitted us to probe the link between LMC divisional identity
and axonal trajectory. We therefore monitored the transcriptional
status of motor neurons retrogradely labeled after focal rhoda-
mine-dextran (Rh-D) tracer injection into the dorsal or ventral
halves of the hindlimb. After ventral Rh-D injection in control or
neurons expressed Isl1, a medial LMC profile (Figures 4A–4D).
Conversely, after dorsal Rh-D injection in control and bDMNg?/?
embryos, 99% and 96%, respectively, of labeled motor neurons
excluded Isl1 (Figures 4E–4H). Thus, the loss of b- and g-catenin
activities does not perturb the link between the divisional identity
of LMC neurons and the ability of axons to select appropriate
dorsoventral trajectories upon entering the limb.
We next examined whether the link between motor pool iden-
pool scrambling. In control and bDMNg?/?embryos, 82% and
83%, respectively, of LMC neurons labeled after Rh-D injection
into the adductor magnus muscle appropriately coexpressed
Isl1 and Nkx6.1 (Figures 4I–4L), and 96% and 94%, respectively,
of LMC neurons labeled after Rh-D injection into the rectus
femoris muscle appropriately coexpressed Hb9 and Nkx6.2
targets only when they occupy positions coincident with their
normal pool location. As a consequence, we examined
the spatial distribution of retrogradely labeled motor neurons
with reference to the total cohort of motor neurons within medial
or lateral LMC divisions. We found that retrogradely labeled
adductor magnus or rectus femoris motor neurons in bDMNg?/?
embryos were not confined to a localized subdomain, and many
were positioned far from their normal pool epicenter (Figures 4M
mutants does not disturb the predictive link between molecular
identity, axonal trajectory, and target muscle specificity.
We also examined the impact of loss of b- and g-catenin activ-
ities on early stages of motor neuron dendritic development,
focusing on adductor motor neurons, which exhibit a stereotypic
radial dendritic architecture. In control embryos the dendrites
of adductor motor neurons, delineated by muscle Rh-D injec-
tion at e14.5, were elongated and possessed approximately
five primary branch points (Figures S4C, S4D, and S4I–S4K). In
bDMNgDMNembryos, both total dendritic length and primary
ing were not observed in single b- and g-catenin mutant back-
grounds (Figures S4I–S4K). Thus, b- and g-catenin also act
redundantly to control early stages of motor neuron dendritic
Motor Neuron Catenin Phenotypes Do Not Involve Wnt
Catenins have been implicated in Wnt as well as cadherin
signaling (Nelson and Nusse, 2004). Defining which cell surface
Figure 3. Impaired LMC Divisional and Pool Segregation in b- and g-
(A and B) FoxP1+, Lhx1offmedial, and FoxP1+, Lhx1+lateral LMC neurons in
e11.5 control and bDMNg?/?embryos.
(C–F) Isl1+medial and Hb9+lateral LMC neurons at lumbar (L)2 and L4 levels in
e13.5 control and bDMNg?/?embryos.
LMC neurons in e13.5 control (B) and bDMNg?/?(C) embryos; mean ± SEM (t
test, p < 0.0001 for M/L and L/M).
(I–L) LMC pools at L2 and L4 levels in e13.5 control and bDMNg?/?embryos.
Nkx6.1+, Er81+adductor/gracilis (A/G) neurons; Er81+, Nkx6.1offvasti (V)
neurons; Nkx6.2+rectus femoris/tensor fasciae latae (R/T) neurons; and
Nkx6.1+, Er81offhamstring (H) motor neurons.
(M) Pool mixing indices (Pmi) of H/R/T and V/R/T pools in e13.5 control (B)
and bDMNg?/?(C) embryos (c2test, p < 0.0001 for H/R/T and V/R/T). See
also Table S4.
For additional related data regarding b- and g-catenin activity in interseg-
mental pool organization, see Figure S3.
Cell 147, 641–652, October 28, 2011 ª2011 Elsevier Inc. 645