Download The transcription factors Nkx2.2 and Nkx2.9 play a

RESEARCH ARTICLE 4249
Development 137, 4249-4260 (2010) doi:10.1242/dev.053819
© 2010. Published by The Company of Biologists Ltd
The transcription factors Nkx2.2 and Nkx2.9 play a novel role
in floor plate development and commissural axon guidance
Andreas Holz1, Heike Kollmus1,*, Jesper Ryge2, Vera Niederkofler3, Jose Dias4, Johan Ericson4,
Esther T. Stoeckli3, Ole Kiehn2 and Hans-Henning Arnold1,†
SUMMARY
The transcription factors Nkx2.2 and Nkx2.9 have been proposed to execute partially overlapping functions in neuronal
patterning of the ventral spinal cord in response to graded sonic hedgehog signaling. The present report shows that in mice
lacking both Nkx2 proteins, the presumptive progenitor cells in the p3 domain of the neural tube convert to motor neurons (MN)
and never acquire the fate of V3 interneurons. This result supports the concept that Nkx2 transcription factors are required to
establish V3 progenitor cells by repressing the early MN lineage-specific program, including genes like Olig2. Nkx2.2 and Nkx2.9
proteins also perform an additional, hitherto unknown, function in the development of non-neuronal floor plate cells. Here, we
demonstrate that loss of both Nkx2 genes results in an anatomically smaller and functionally impaired floor plate causing severe
defects in axonal pathfinding of commissural neurons. Defective floor plates were also seen in Nkx2.2+/–;Nkx2.9–/– compound
mutants and even in single Nkx2.9–/– mutants, suggesting that floor plate development is sensitive to dose and/or timing of Nkx2
expression. Interestingly, adult Nkx2.2+/–;Nkx2.9–/– compound-mutant mice exhibit abnormal locomotion, including a permanent
or intermittent hopping gait. Drug-induced locomotor-like activity in spinal cords of mutant neonates is also affected,
demonstrating increased variability of left-right and flexor-extensor coordination. Our data argue that the Nkx2.2 and Nkx2.9
transcription factors contribute crucially to the formation of neuronal networks that function as central pattern generators for
locomotor activity in the spinal cord. As both factors affect floor plate development, control of commissural axon trajectories
might be the underlying mechanism.
INTRODUCTION
The spinal cord contains anatomically distinct classes of neurons
that contribute to sensory and motor tasks. Neurons that exert and
regulate motor control reside in the ventral spinal cord, and their
development in the mammalian embryo is largely controlled by
genetic programs (for reviews, see Briscoe and Novitch, 2008;
Jessell, 2000; Shirasaki and Pfaff, 2002) that specify at least five
classes of neuronal progenitor cells. These progenitors give rise to
motor neurons (MNs) and four cardinal classes of ventral
interneurons, referred to as V0 to V3 neurons (Ericson et al.,
1997a; Pierani et al., 1999). The different progenitor cell types are
generated in response to the graded activity of the signaling protein
sonic hedgehog (Shh), which is first produced by the notochord
and subsequently by the overlying floor plate, the most ventral
structure of the neural tube (Chiang et al., 1996; Ericson et al.,
1996; Marti et al., 1995; Roelink et al., 1995). The concentration
gradient of Shh along the dorsoventral (DV) axis of the neural tube
leads to regional activation or repression of homeodomain (HD)
transcription factors, generating a transcriptional code for distinct
1
Cell and Molecular Biology, University of Braunschweig, Spielmannstraße 7, 38106
Braunschweig, Germany. 2Mammalian Locomotor Laboratory, Department of
Neuroscience, Karolinska Institute, 17177 Stockholm, Sweden. 3Institute of
Molecular Life Sciences, University of Zurich, 8057 Zurich, Switzerland. 4Institute of
Cell and Molecular Biology, Karolinska Institute, 17177 Stockholm, Sweden.
*Present address: Department of Infection Genetics, Helmholtz Centre for Infection
Research, Inhoffenstraße 7, 38124 Braunschweig, Germany
†
Author for correspondence ([email protected])
Accepted 11 October 2010
progenitor cell populations (Briscoe and Ericson, 2001; Briscoe et
al., 2000; Briscoe et al., 1999; Ericson et al., 1997a; Ericson et al.,
1997b; Novitch et al., 2001).
The most ventral neuronal population in the spinal cord,
referred to as Sim1-expressing (Sim1+) V3 interneurons, is
derived from progenitor cells expressing Nkx2.2 and Nkx2.9 HD
transcription factors. This cell population is located immediately
dorsal to the floor plate and encounters high concentrations of
Shh signal. Both Nkx2 genes are structurally, and presumably
functionally, related and exhibit very similar spatiotemporal
expression patterns in the central nervous system (CNS) in a
Shh-dependent manner (Briscoe et al., 1999; Pabst et al., 1998;
Pabst et al., 2000). In Nkx2.2-deficient mice, the domain (p3) of
early V3 progenitor cells forms and is indistinguishable from
wild type between the ventral floor plate and dorsal Pax6expressing cells. However, p3 progenitors in the mutant mouse
undergo a ventral-to-dorsal transformation and generate MNs in
place of Sim1+ V3 interneurons (Briscoe et al., 1999; Ericson et
al., 1997a). This indicates that Nkx2.2 is essential for the correct
differentiation of V3 neurons but not for establishing the p3
progenitor domain. Nkx2.9, which is initially co-expressed with
Nkx2.2, might substitute for the missing Nkx2.2 function.
Slightly later, when expression of Nkx2.9 stops in the neural
tube at embryonic day (E) 10.5 (Briscoe et al., 1999), the
essential requirement of Nkx2.2 to establish V3 neuronal fate
becomes apparent. Alternatively, Nkx2.2 might play no role in
setting up p3 progenitor cells and entirely different factors could
be responsible. Targeted disruption of the Nkx2.9 gene in mouse
causes no obvious neuronal phenotype in spinal cord, consistent
with the idea that Nkx2.2, which is expressed for a longer time
DEVELOPMENT
KEY WORDS: Nkx2.2, Nkx2.9, Floor plate development, Central pattern generators in spinal cord, Mouse
4250 RESEARCH ARTICLE
mutant embryos appear to be transformed into MN progenitors
leading to considerably reduced expression of various floor platespecific markers. Moreover, we observe axonal pathfinding defects
of commissural axons that might be related to the loss of floor
plate integrity in mutants. Finally, we demonstrate that
Nkx2.2+/–;Nkx2.9–/– compound mutants exhibit locomotor defects
in vivo and during fictive locomotor-like activity in vitro,
indicating that both HD transcription factors contribute to the
development and function of CPG circuits in the spinal cord.
MATERIALS AND METHODS
Generation and genotyping of the Nkx2.2;Nkx2.9 double-mutant
mouse
Double-homozygous mice were obtained by crossing Nkx2.2- (Sussel et
al., 1998) and Nkx2.9- (Pabst et al., 2003) deficient mutant mice. Doublenull mutants showed perinatal lethality, as already reported for the
Nkx2.2–/– mouse (Sussel et al., 1998); however, Nkx2.2+/–;Nkx2.9–/–
mutants were viable. Genotyping for Nkx2.9 was performed by PCR as
described previously (Pabst et al., 2003). Nkx2.2 genotyping was done by
Southern blotting as described (Sussel et al., 1998) or by PCR using the
following primers: Nkx2.2 #6 sense, 5⬘-TTCCAAAGGCACCACAAATCGC-3⬘; Nkx2.2 #7 antisense, 5⬘-GGTCTTGGGAGTCAAGTGGATGAAG-3⬘; Nkx2.2 mut#1 antisense, 5⬘-TGAAGAACGAGATCAGCAGCCTCT-3⬘. Embryos were staged by counting the morning of vaginal plug
detection as E0.5.
Immunohistochemistry
Immunohistochemical detection of proteins was performed on fixed tissue
sections of mouse embryos as described previously (Pabst et al., 2003).
Primary mouse-specific monoclonal antibodies (obtained from the
Developmental Studies Hybridoma Bank, University of Iowa) used were:
FoxA2 (Foxa2 – Mouse Genome Informatics) clone 4C7 (1:800), Nkx2.2
clone 74.5A5 (1:1000), Nkx6.1 clone F55A10 (1:600), Pax6 (1:600),
HB9/MNR2 (Mnx1 – Mouse Genome Informatics) clone 81.5C10 (1:300),
Islet1 (Isl1 – Mouse Genome Informatics) clone 39.4D5 (1:500), Lim3
(Lhx3 – Mouse Genome Informatics) clone 67.4E12 (1:500) and Tag1
(Cntn2 – Mouse Genome Informatics) clone 4D7 (1:15). Other primary
antibodies used were: rabbit anti-Olig2 (IBL; 1:400), anti-BrdU (Santa
Cruz; 1:400) and rabbit anti-Rig1 (a gift from Dr Fujio Murakami, Osaka
University, Japan; 1:600) (Tamada et al., 2008). Secondary antibodies used
were: anti-mouse IgG-Cy2, anti-mouse IgG1-Cy2, anti-mouse IgG2b-Cy3
and anti-rabbit IgG-Cy5 (all from Jackson ImmunoResearch; 1:600). The
slides were counterstained with DAPI (Molecular Probes) and documented
on a Zeiss Apotome or Zeiss LSM 510 confocal microscope. Galactosidase activity was detected on cryosections (14-16 m) as
described (Pabst et al., 2003) and documented with a digital color camera
mounted onto a Leica DM RBE microscope.
For quantification, specifically marked cells on sections (16 m) of
neural tube were photographed on the Zeiss Axioplan 2 fluorescent
microscope and counted using Adobe Photoshop software. For each
genotype, a minimum of five sections from at least three individual animals
was analyzed. Statistical analysis (t-test) and graph preparation was carried
out using GraphPad software (San Diego).
In situ hybridization and riboprobes
Strand-specific digoxigenin-labeled riboprobes were used for in situ
hybridization as described previously (Pabst et al., 2003). The following
gene fragments from mouse were subcloned into pSP70/71 vectors: sonic
hedgehog, nucleotides 1650–2668 (accession number AK077688); slit,
ClaI-SmaI restriction fragment of IMAGE clone IRAVp968B10159
(ImaGenes, Berlin); Dbx1, SmaI-HindIII fragment of IMAGE clone
IMAGp998C241002 (ImaGenes, Berlin); Chx10 (Vsx2 – Mouse Genome
Informatics), HindIII-XhoI Chx10 3⬘UTR fragment of IMAGE clone
IRAVp968a0893 (ImaGenes, Berlin). IMAGE clone IRAKp961D0140Q
(ImaGenes, Berlin) was used to generate netrin 3⬘ untranslated region
(UTR) riboprobes, the FoxA2 riboprobe was described previously (Ang et
al., 1993) and a 5⬘ probe of engrailed 1 cDNA was obtained from F. Vauti
(University of Braunschweig, Germany) (Wurst et al., 1994).
DEVELOPMENT
period, provides redundant functions and thereby rescues V3
neurons (Pabst et al., 2003). Thus, the individual phenotypes of
single Nkx2.2 and Nkx2.9 mutant mice suggest that the two
genes have overlapping roles in neuronal patterning of the
ventral spinal cord. In order to clarify the extent of shared
functions between the two transcription factors, we examined
double-mutant mice lacking both Nkx2.2 and Nkx2.9 proteins
with particular focus on their individual and combined roles in
early cell lineage decisions in the spinal cord.
Although floor plates were reported to be anatomically normal
in Nkx2.2 and Nkx2.9 single-mutant mice (Briscoe et al., 1999;
Pabst et al., 2003), early expression of both regulators in the floor
plate area suggests that they might have a function in
development and/or maintenance of this important organizing and
signaling center. Nkx2.2 expression in the ventral spinal cord of
zebrafish and chicken embryos has been shown to extend beyond
the p3 domain into the lateral floor plate (Chapman et al., 2002;
Strahle et al., 2004). In the mouse, expression of Nkx2.2 in early
floor plate has also been described (Jeong and McMahon, 2005)
and preliminary evidence showed that the Nkx2.9 knock-in allele
directs lacZ expression in the most ventral part of the neural tube
(Pabst et al., 2003). The floor plate is not composed of a single
population of uniform cells but consists of different cell groups,
most notably the medial and the lateral floor plate cells, which
can be distinguished by molecular markers including Nkx2.2
(Placzek and Briscoe, 2005; Strahle et al., 2004). To investigate
the hypothesis that both HD transcription factors could play a role
in floor plate development, we utilized Nkx2.2;Nkx2.9 doublemutant embryos to analyze the histological and functional
consequences of the combined null mutations on floor plate
development.
The neuronal circuits that generate locomotor behavior in
mammals are embedded in the ventral half of the spinal cord
(Kiehn, 2006; Kiehn and Kjaerulff, 1998). These networks are
commonly called central pattern generators (CPGs), which are
composed of excitatory and inhibitory neurons belonging to the
four main classes of V0, V1, V2 and V3 interneurons (Briscoe et
al., 2000; Goulding and Pfaff, 2005; Jessell, 2000; Kiehn, 2006)
with further subtypes within each class (Al-Mosawie et al., 2007;
Lanuza et al., 2004; Lundfald et al., 2007). Individual contributions
of the various types of interneurons to locomotor CPGs have been
investigated by genetic disruption of early cell type-specific
transcription factors, selective cell ablation experiments and
permanent or acute blockage of synaptic transmission (for reviews,
see Goulding, 2009; Kiehn, 2006; Kiehn et al., 2010; Stepien and
Arber, 2008). The role of V3 neurons in the CPG was probed by
functional interference in two ways. First, a Sim1-Cre-mediated
conditionally expressed tetanus toxin light chain permanently
blocked synaptic release from V3 cells; secondly, an allostatin
receptor-based approach acutely attenuated the activity of V3
neurons in the adult mouse. Both experimental interventions
resulted in disruption of the reliable rhythmicity of locomotor
activity in vitro without a major effect on left-right alternation
(Zhang et al., 2008). Nkx2.2 single- and Nkx2.2;Nkx2.9 doublemutant mice fail to form Sim1+ V3 interneurons thereby providing
an alternative model to study the role of V3 neurons in the spinal
locomotor network.
Here, we report the detailed phenotypic analysis of
Nkx2.2;Nkx2.9 double-null embryos. We show that neuronal
progenitor cells within the p3 domain are not specified correctly
and that putative V3 progenitors are entirely replaced by MN
precursors. Likewise, cells of the lateral floor plate in double-
Development 137 (24)
Nkx2 transcription factors regulate floor plate development
RESEARCH ARTICLE 4251
Anterograde labeling of commissural axons
Embryos were collected at E12.5, fixed in 4% paraformaldehyde in PBS
for 45 minutes then washed in PBS. Subsequently, spinal cords were
dissected carefully, dorsal root ganglia were removed and the tubes were
cut open at the dorsal midline (roof plate) to obtain ‘open-book’
preparations, as described previously (Perrin and Stoeckli, 2000).
Following a second fixation step, DiI solution (5 mg/ml in methanol;
Molecular Probes) was injected with finely drawn-out glass capillaries into
the soma of dorsal commissural neurons (dl1 neurons) (Zou et al., 2000).
DiI was allowed to penetrate axon trajectories in cold PBS for 40-48 hours.
Analysis and documentation was performed on a standard fluorescence or
confocal microscope.
Fictive locomotion in isolated spinal cord preparations
RESULTS
V3 neuronal progenitor cells are replaced by
Olig2+ MN precursors in the ventral neural tube of
Nkx2.2–/–;Nkx2.9–/– double-mutant embryos
In order to investigate potentially overlapping or redundant roles
of the structurally related HD transcription factors Nkx2.2 and
Nkx2.9, we generated double-mutant mice lacking both proteins.
Crossing the single-knockout mutants (Pabst et al., 2003; Sussel
et al., 1998) yielded the expected combinatorial genotypes
including the Nkx2.2–/–;Nkx2.9–/– double-null mutants, which were
born with no apparent anatomical defects. However, double-null
mutants died shortly after birth, possibly as a result of respiratory
problems or a lack of pancreatic islet cells, as described previously
for the Nkx2.2–/– mutant mouse (Sussel et al., 1998). Because
double-null mutant embryos developed to term, neither
transcription factor, individually or in combination, can be vitally
important during prenatal mouse development. Interestingly, a
single Nkx2.2 wild-type allele in Nkx2.2+/–;Nkx2.9–/– compound
Fig. 1. Early patterning defect in the ventral neural tube of
Nkx2.2–/–;Nkx2.9–/– double-mutant mouse embryos: loss of the p3
progenitor domain. (A-L)Immunohistochemistry for neuronal
progenitor cells in the ventral spinal cord of E9.5 (A-D) and E10.5 (E-L)
mouse embryos at forelimb (E-H) and hindlimb (I-L) levels. Transverse
sections of wild type (A,E,I,G,K), Nkx2.2–/– (B), Nkx2.9–/– (C) and
Nkx2.2–/–;Nkx2.9–/– mutant (D,F,H,J,L) animals reveal unaltered
expression of Pax6 (green in A-F,I,J) and Nkx6.1 (green in G,H,K,L) in
single and double mutants whereas Olig2+ MN progenitor cells (blue)
expand more ventrally into the p3 domain in the double mutant
(D,F,H,J,L). Olig2+ MN precursors initially occupy the entire width of the
neural tube and become restricted to the ventricular zone later. The
ventral expansion of the MN progenitor domain in rostral sections of
mutant embryos is highlighted by vertical lines (E-H). Scale bars: 50m.
mutants was sufficient for postnatal survival. However, most of
these animals displayed abnormal locomotion that manifested as
a hopping gait.
To test the hypothesis that functional redundancy exists between
both genes, we analyzed the neuronal patterning in spinal cords of
double-homozygous Nkx2.2–/–;Nkx2.9–/– mouse embryos using
diagnostic markers, such as the transcription factors Olig2, Pax6
and Nkx6.1, which mark all neuronal progenitors ventral to the p2
domain (Sander et al., 2000). We found that at E9.5 the p3 domain
in Nkx2.2 and Nkx2.9 single-knockout mutant mice was located
ventral to MN progenitor cells forming the correct dorsal boundary
with Olig2-expressing (Olig2+) cells as observed in wild-type
embryos (Fig. 1A-C) (Mizuguchi et al., 2001; Novitch et al., 2001;
Sun et al., 2001). By contrast, Olig2+ progenitors in double-null
Nkx2.2–/–;Nkx2.9–/– embryos at E9.5 expanded into the ventral
neural tube, including the p3 domain and, surprisingly, also a major
part of the floor plate (Fig. 1D; Fig. 4M). This phenotype persisted
in double-mutant embryos at E10.5 when Olig2 expression began
to retract from the medial floor plate area (Fig. 1F,H). The ventral
border of Pax6-expressing cells and the expression domain of
DEVELOPMENT
For in vitro recordings, neonatal pups from postnatal day (P) 0 to P2
were used except for one initial litter of E18.5 Nkx2.2 embryos.
Nkx2.9–/–;Nkx2.2–/– double-knockout animals die at birth and were not
included in this study. All electrophysiological experiments were carried
out as described in detail elsewhere (Quinlan and Kiehn, 2007). Fictive
locomotion was induced with a mixture of N-methyl-D-aspartic acid
(NMDA; 3-7.5 M) and 5-hydroxytryptamine (5-HT; 3-20 M). In a few
cases (n5) dopamine (20-50 M) was also added. For blocked inhibition
experiments, 10 M picrotoxin and 0.3 M strychnine was applied. The
left and right activities of the L2 and L5 ventral roots were recorded with
suction electrodes. To estimate the level of coordination, ventral root
recordings were analyzed from stable locomotor activity, taken at least 10
minutes after the initial burst of drug-induced activity using circular
statistics as described previously (Kjaerulff and Kiehn, 1996; Tresch and
Kiehn, 1999). In brief, raw traces of the ventral root activity were rectified
and smoothed (moving average) using custom written applications in
R software (http://www.r-project.org/). Cycle period onsets were
automatically calculated based on the intersection between the smoothed
tracing with a horizontal line running through the center of this trace. The
cycle periods of the reference trace (LL2) were each normalized to a scale
of 360 degrees, with the onset of a locomotor burst in the reference ventral
root corresponding to a phase value of zero and the onset of the next burst
corresponding to a phase value of 360. The phase delay of the period cycle
onset with respect to the reference was calculated for test traces (LL5, RL2
or RL5). Based on these values, an estimate of ventral root coordination
was made and locomotor cycles were selected randomly. From these
values, a vector representing the level of individual animals was calculated.
The angle of the vector represents the average phase delay with respect to
the reference root, and the length is proportional to the clustering around
this value. Raleigh’s test was used to determine the degree of clustering
with statistical significance (P<0.05; illustrated as a blue circle). Of the 58
animals tested, only 32 were included in the final circular statistics because
of poorly defined signals in the remaining preparations.
4252 RESEARCH ARTICLE
Development 137 (24)
Fig. 2. Development of V0, V1 and V2 neurons is unaffected in
the ventral spinal cord of Nkx2.2–/–;Nkx2.9–/–and
Nkx2.2+/–;Nkx2.9–/–mutant mouse embryos. (A,B)In situ
hybridization using probes for Dbx1, En1, Chx10 and Sim1 on transverse
sections of E11.5 wild-type, Nkx2.2+/–;Nkx2.9–/– and Nkx2.2–/–;Nkx2.9–/–
mutant embryos. No major differences are observed in V0 (Dbx1), V1
(En1) and V2a (Chx10) neurons (A). Notably, Sim1-expressing V3
neurons are present in Nkx2.2+/–;Nkx2.9–/– mutant embryos of the
presumptive hopping phenotype (B). (C)Immunofluorescent analysis
using Lim3 (green) and Islet1 (red) antibodies on embryos at E12.5
illustrates an increase in the number of somatic motor neurons (yellow)
within the medial motor column (MMC) of Nkx2.2+/–;Nkx2.9–/– and
Nkx2.2–/–;Nkx2.9–/– mutants. Islet1-positive MN cells of the lateral motor
column (LMC) and Lim3+ V2 progenitors (green in encircled area) appear
unaffected in double-mutant embryos. DRG, dorsal root ganglia.
(D) Quantification of V2 progenitor, MMC and LMC cells demonstrates a
significant increase of motor neurons in the MMC in compound and
double-null mutants versus wild type. Seven or more independent
sections were counted at forelimb level from wild type (n3) and
Nkx2.2–/–;Nkx2.9–/– (Nkx2ko; n3) mutant embryos. Data are expressed
as mean ± s.d. Scale bar: 50m.
Nkx2.9 is activated in the p3 domain, including
the floor plate territory, of Nkx2.2–/–;Nkx2.9–/–
double-null embryos
The model for neuronal patterning in the ventral spinal cord
proposes that progenitor cells in the p3 domain enter the lineage of
V3 neurons driven by a high concentration of Shh signal and
subsequent induction of the transcription factors Nkx2.2 and Nkx2.9
(Dessaud et al., 2007; Ericson et al., 1997b). No other specific
markers have been described for these early cells. To confirm that
DEVELOPMENT
Nkx6.1 were unaffected in double-mutant embryos suggesting that
both Nkx2.2 and Nkx2.9 were not essential for control of the
expression pattern of those transcription factors (Fig. 1). Thus,
molecular markers in the embryonic spinal cord of the
Nkx2.2–/–;Nkx2.9–/– double mutant clearly indicated that V3
progenitor cells were absent and replaced entirely by MN precursor
cells. This is in contrast to Nkx2.2 and Nkx2.9 single-mutant
embryos, which contained V3 progenitor cells in the p3 domain, as
observed in wild type (Fig. 1B,C) (Briscoe et al., 1999; Pabst et al.,
2003). Taken together, these observations support the idea that
Nkx2.2 and Nkx2.9 play redundant roles and that one or both
factors is required and sufficient for the formation of the early p3
progenitor domain.
In order to confirm that simultaneous depletion of Nkx2.2 and
Nkx2.9 did not cause indirect or compensatory effects on the
development of the other neuronal cell populations in ventral spinal
cord, we performed in situ hybridization with specific probes for
Dbx1, En1, Chx10 and Sim1 to identify V0, V1, V2a and V3
neurons, respectively. Our results indicated that the normal
dorsoventral distribution, and presumably numbers, of V0, V1 and
V2a interneurons were maintained in Nkx2.2–/–;Nkx2.9–/– doublenull embryos at E11.5 (Fig. 2A). Counts of Lim3+ and Islet1expressing cells in Nkx2.2–/–;Nkx2.9–/– embryos at E12.5 confirmed
that motor neurons in the medial motor column (MMC) were
specifically expanded at the expense of V3 cells (Fig. 2C,D). By
contrast, numbers of Lim3+ and Islet1– V2 progenitor cells
representing the next dorsal class of interneurons, and Islet1+
neurons of the lateral motor column (LMC) appeared to be
unaffected in mutant embryos (Fig. 2C,D). This latter result
confirms and expands previously published observations in the
Nkx2.2–/– mutant, which demonstrated that ectopic MN progenitors
in p3 contribute to the MMC only and not to the LMC (Agalliu et
al., 2009). Notably, Nkx2.2+/–;Nkx2.9–/– compound-mutant embryos,
most of which developed the hopping gait, also showed a moderate
increase of MMC motor neurons (Fig. 2C,D) and a reduction, but
not a complete loss, of mature Sim1+ V3 interneurons (Fig. 2B).
This decrease was in line with fewer Nkx2.2-expressing V3
progenitor cells, which we identified by immunostaining in the
Nkx2.2+/–;Nkx2.9–/– compound mutant (Fig. 4F,L). All other
cardinal classes of interneurons in the ventral spinal cord of
Nkx2.2+/–;Nkx2.9–/– mutant embryos were indistinguishable from
those observed in wild-type animals (Fig. 2A).
Fig. 3. The Nkx2.9-lacZ knock-in allele is expressed in the floor
plate of E10.5 Nkx2.2;Nkx2.9-deficient embryos. -Galactosidase
activity (blue) in whole-mount embryos (left panels) reflecting Nkx2.9
activity in the CNS and in transverse sections at the rostral trunk level
(right panels) illustrating that Nkx2.9 is activated in the most ventral
neural tube, including the floor plate. This expression domain is
established independently of both Nkx2.2 and Nkx2.9 proteins.
prospective V3 progenitors were actually generated but take another
fate in Nkx2.2–/–;Nkx2.9–/– double-mutant embryos, we utilized the
lacZ reporter gene inserted into the Nkx2.9 mutant allele (Pabst et
al., 2003). In E10.5 Nkx2.9-deficient embryos, -galactosidase
activity was observed throughout the normal Nkx2.9 expression
domain regardless of whether or not Nkx2.2 was also present (Fig.
3). This finding suggests that initial Nkx2.9 gene activation is
probably directly controlled by the Shh signal and is not subject to
feedback control, such as auto- or cross-regulation by Nkx2.2 or
Nkx2.9. More interestingly, -galactosidase-positive cells were not
only found in the p3 domain but also more ventrally within the floor
plate region, suggesting that Nkx2.9-expressing cells initially
include prospective progenitors of neuronal cell lineages as well as
non-neuronal floor plate cells.
Typical floor plate markers are reduced in mutant
embryos lacking Nkx2.2 and Nkx2.9
In an attempt to assess a function for Nkx2.2 and Nkx2.9
transcription factors in the development of the floor plate we first
determined the expression of the forkhead transcription factor
FoxA2, a key regulator for floor plate cells in the embryonic spinal
cord (Ruiz i Altaba et al., 1995). Immunohistochemistry with a
FoxA2-specific antibody on rostral (forelimb) and caudal (hindlimb)
transverse sections of the spinal cord from E10.5 mouse embryos
revealed almost normal expression in Nkx2.2–/– mutants, and a
moderately smaller expression domain particularly on caudal
sections of Nkx2.9–/– single mutants (Fig. 4G-J). In the
Nkx2.2–/–;Nkx2.9–/– double knockout (Fig. 4C,D) and also in
Nkx2.2+/–;Nkx2.9–/– compound-mutant mice (Fig. 4E,F), FoxA2expressing cells and/or the accumulation of FoxA2 protein were
significantly reduced (Fig. 4A,B) and replaced by Olig2+ MN
progenitor cells that were already present at E9.5 (Fig. 4K-M). Some
cells in the ventral midline, however, retained FoxA2 suggesting that
RESEARCH ARTICLE 4253
only the lateral and not the medial floor plate acquired an MN fate
in the double-null mutant. A similar reduction of floor plate territory
was also observed for Shh (data not shown). Incorporation of BrdU
and immunostaining for active caspase 3 did not provide evidence
for major changes in proliferation or enhanced apoptosis in ventral
spinal cords of mutant embryos, supporting the idea of cell-fate
conversion (see Fig. S1 in the supplementary material). Counting
immunostained cells on multiple sections of the various mutant and
wild-type embryos confirmed that floor plates in double-null and
Nkx2.2+/–;Nkx2.9–/– compound mutants were significantly smaller
than normal (Fig. 4K). Floor plates in both mutant genotypes
appeared to be more affected at caudal than rostral levels. To provide
further evidence of the mutant floor plate phenotype, we performed
in situ hybridization with additional markers and extended the
analysis to older embryos (E11.5). At E10.5 and E11.5 hybridization
signals for Shh, netrin 1, FoxA2 and Slit1 transcripts were drastically
diminished in double-null Nkx2.2–/–;Nkx2.9–/– (Fig. 4W-Z,II-LL) and
compound Nkx2.2+/–;Nkx2.9–/– (Fig. 4S-V,EE-HH) mutant embryos
in comparison with wild type (Fig. 4O-R,AA-DD). Taken together,
these results provide compelling evidence that many cells,
particularly in the lateral floor plate, were transformed into MN
progenitors in double-null and compound Nkx2.2+/–;Nkx2.9–/– mutant
embryos.
Aberrant pathfinding of commissural axons in
spinal cord of Nkx2.2;Nkx2.9 mutant embryos
Besides its role as signaling center for dorsoventral patterning of
the neural tube (Jessell, 2000), the floor plate also controls
pathfinding of commissural axons (Charron et al., 2003; Matise et
al., 1999; Serafini et al., 1994). The massive changes in floor plate
morphology and/or specific gene expression in Nkx2.2;Nkx2.9
mutant embryos prompted us to monitor axon guidance by
anterograde labeling of neurons in open-book preparations of the
neural tube from E12.5 embryos, as described previously (Bourikas
et al., 2005). DiI injection into dorsally located somata in spinal
cord preparations from wild-type embryos revealed straight and
parallel bundles of commissural axons that projected towards the
floor plate, crossed the midline and turned rostrally, as expected
(Fig. 5A). Although commissural axons in spinal cords of most
Nkx2.2–/– and Nkx2.9–/– single-knockout embryos displayed normal
projections, 10-20% of these mutants appeared to form less tightly
packed axon bundles that often stalled at the midline without
reaching the contralateral side (Fig. 5B,C). The most frequent
pathfinding errors of commissural axons were detected in spinal
cords isolated from Nkx2.2–/–;Nkx2.9–/– double-null mutants and
Nkx2.2+/–;Nkx2.9–/– mutants that develop the hopping phenotype
(Fig. 5D-F). Approximately 50% of DiI injection sites in doublehomozygous and compound mutants revealed highly abnormal
axons with premature ipsilateral turning, random rostral and caudal
turning after crossing the floor plate, and increased density of
growth cones indicating stalling at the midline (Fig. 5E). Generally,
deviations in axon projections appeared to be most complex in
double-null mutants, although the numbers of injection sites
displaying abnormal axon projections were similar in compound
Nkx2.2+/–;Nkx2.9–/– mutants (Fig. 5G). Potential defects in DV
pathfinding or fasciculation of axons prior to reaching the floor
plate were analyzed on transverse sections of DiI-injected spinal
cord preparations. The results confirmed aberrant axonal navigation
at the ventral midline and showed that the vast majority of axons
reached the ipsilateral floor plate border correctly, even in mice
lacking both Nkx2.2 and Nkx2.9 (Fig. 5K,L). This observation
was supported by immunofluorescent labeling of transverse
DEVELOPMENT
Nkx2 transcription factors regulate floor plate development
4254 RESEARCH ARTICLE
Development 137 (24)
spinal cord sections from wild-type, Nkx2.2+/–;Nkx2.9–/– and
Nkx2.2–/–;Nkx2.9–/– embryos using Rig1- (Tamada et al., 2008) and
Tag1-specific (Dodd et al., 1988) antibodies (Fig. 5H-J). We found
no evidence for any major deviations of dorsoventral axon
trajectories before midline crossing.
In summary, our data demonstrate severe defects in
pathfinding of commissural axons at the ventral midline of the
spinal cord in mice that lack one or both of the transcription
factors Nkx2.2 and Nkx2.9. The aberrant axonal navigation and
the reduced expression of the axonal guidance molecules Shh,
netrin 1 and Slit1 in mutant floor plates support the model that
correct formation of the ventral commissure requires Nkx.2.2
and/or Nkx2.9 activity.
The adult Nkx2.2+/–;Nkx2.9–/– mutant mouse
exhibits abnormal locomotor activity and
coordination defects during walking
Nkx2.2+/–;Nkx2.9–/– mutant mice with one wild-type Nkx2.2 gene
copy were viable but ~90% of these compound mutants displayed
abnormal patterns of locomotor coordination (Fig. 6A-D). The gait
DEVELOPMENT
Fig. 4. Reduction of the floor plate in Nkx2.2+/–;Nkx2.9–/– and Nkx2.2–/–;Nkx2.9–/– double-null embryos. (A-J)Immunofluorescence for
Nkx2.2 (red) and the floor plate marker FoxA2 (green) on rostral (forelimb; A,C,E,G,I) and caudal (hindlimb; B,D,F,H,J) transverse sections of E10.5
mouse embryos reveals a significantly smaller FoxA2-positive floor plate in Nkx2.2–/–;Nkx2.9–/– (C,D) and Nkx2.2+/–;Nkx2.9–/– (E,F) mutant embryos
compared with wild type (A,B) and Nkx2.9–/– (G,H) and Nkx2.2–/– (I,J) single mutants. (K-M)Triple immunofluorescence at E9.5 demonstrates that all
of the p3 domain and a major portion of the floor plate were replaced by Olig2-positive (blue) MN progenitors in the Nkx2.2–/–;Nkx2.9–/– mutant.
(N)FoxA2-expressing nuclei were counted on 57 (wild type), 14 (Nkx2.2–/–), 30 (Nkx2.9–/–), 23 (Nkx2.2+/–;Nkx2.9–/–) and 43 (Nkx2.2–/–;Nkx2.9–/–)
individual sections of three to five embryos per genotype. Note the stronger effect in the caudal spinal cord. Data are expressed as mean ± s.d.
(O-LL) In situ hybridization using specific probes for Shh, netrin 1, FoxA2 and Slit1 demonstrate the dramatic loss of these markers in
Nkx2.2+/–;Nkx2.9–/– and Nkx2.2–/–;Nkx2.9–/– mouse embryos at E10.5 (O-Z) and E11.5 (AA-LL). Scale bars: 50m.
Nkx2 transcription factors regulate floor plate development
RESEARCH ARTICLE 4255
Fig. 6. Adult Nkx2.2+/–;Nkx2.9–/– animals display a hopping gait.
(A-C)The hopping phenotype of adult mutant mice is illustrated for
two animals with either strong (A) or weak (B) deviations from normal
locomotor behavior (C). To demonstrate foot prints on a sheet of paper,
fore and hind paws were painted with black and red ink, respectively.
Tracings are depicted by foot symbols of matching color. Synchronous
activation of the hind limbs is highlighted by the blue bars. Locomotor
coordination appears to be affected more strongly in hind than fore
limbs, and animals can change between normal alternating and
hopping locomotion. Occasionally, extra forelimb footsteps disrupt the
regular step pattern (arrowhead in A). (D)Percentage of animals with
strong and weak gait phenotypes for the available mutant genotypes at
the age of 3-6 months. Nkx2.9+/–, n35; Nkx2.2+/–;Nkx2.9+/–, n50;
Nkx2.9–/–, n121; Nkx2.2+/–;Nkx2.9–/–, n171.
Locomotor-like activity in isolated spinal cords
from newborn Nkx2.2+/–;Nkx2.9–/– mutant mice
The abnormal motor behavior of adult Nkx2.2+/–;Nkx2.9–/– mutant
mice might reflect defects in spinal neuronal networks, the CPGs
generating locomotion. The elements of the mammalian CPGs are
embedded in the ventral half of the spinal cord where Nkx2.2 and
Nkx2.9 transcription factors are expressed and constitute key
developmental regulators for V3 neurons (Briscoe et al., 1999; Pabst
DEVELOPMENT
Fig. 5. Pathfinding of commissural axons in spinal cords of
Nkx2.2 and Nkx2.9 mutant mice is significantly impaired at the
ventral midline. (A-F)Confocal images of DiI-labeled commissural
axons in open-book explants of wild type (WT; A), Nkx2.2–/– (B) and
Nkx2.9–/– (C) single mutants, Nkx2.2+/–;Nkx2.9–/– (D), and
Nkx2.2–/–;Nkx2.9–/– (E,F) double mutants. In all panels the anterior
end of the spinal cord is at the top. Dotted lines demarcate the floor
plate. The axons in the wild type cross the floor plate and turn
anteriorly (A), whereas axons in mutants frequently stall at the floor
plate, turn randomly before or after the midline, or fail to turn at all
(B-F). (G)Quantification of aberrant injection sites for each mutant
genotype relative to wild type. DiI injection sites were scored as
showing aberrant pathfinding when at least 25% of the labeled
axons did not cross the midline, when more than 50% of the axons
did not turn into and extend along the longitudinal axis of the spinal
cord, or when axons turned into the longitudinal axis prematurely in
the floor plate. Data are expressed as mean ± s.d. (H-J)Normal DV
trajectories of commissural axons in double mutants is illustrated by
immunofluorescence with Rig1 (green) and Tag1 (red) antibodies.
(K,L)Transverse sections of DiI-injected spinal cords illustrate regular
DV projections until axons reach the ipsilateral floor plate border in
Nkx2.2+/–;Nkx2.9–/– (K) and Nkx2.2–/–;Nkx2.9–/– (L) mutant mice.
Horizontal and vertical dashed lines indicate the midline and ventral
border of the spinal cord, respectively. Insets show lower
magnification of DiI-injected sites. ML, midline.
abnormalities were observed predominantly for hindlimbs as
continuous hopping with no alternating activity of left and right
legs, or as less frequent, intermittent hopping. To illustrate the
locomotor phenotype, fore- and hindpaws were painted with black
and red ink, respectively, and color-coded footprints were recorded
on a sheet of paper. More than 60% of the Nkx2.2+/–;Nkx2.9–/–
mutants showed severe locomotion defects of hindlimbs and less
stereotypic movements of forelimbs, including extra steps between
the normal step cycle (Fig. 6A). Approximately 30% of mutants
displayed occasional hopping with only a few interspersed episodes
of abnormal leg movements (Fig. 6B). These locomotor
phenotypes were confirmed for a large number of mice (more
than 70 animals for each genotype) and were never observed in
wild type or heterozygous single and double mutants. However, in
~5% and 20% of homozygous Nkx2.9 single mutants we also
observed the strong and weak hopping phenotypes, respectively
(Fig. 6D).
4256 RESEARCH ARTICLE
Development 137 (24)
et al., 1998; Pabst et al., 2003) and floor plate, as shown here. In
order to determine the functional contribution of Nkx2.2 and Nkx2.9
to the development of the locomotor CPG in addition to the
maturation of the V3 population, we performed in vitro recordings
in spinal cord preparations from newborn mutant mice carrying
various allelic combinations of Nkx2.2 and Nkx2.9 targeted gene
disruptions. Locomotor-like activity was induced by bath application
of NMDA and 5-HT. Because it was generally difficult to induce
fictive locomotion in spinal cords from Nkx2.9-deficient animals,
dopamine (20-50 M) was added in some of these experiments
(n5). Electrical activities of ventral roots on both sides of the spinal
cord were recorded at lumbar level L2 to evaluate left-right
coordination. Activities of the ipsilateral roots at L2 and the more
caudal L5 were also measured to assess the pattern of motor activity
for flexor (L2) and extensor (L5) muscles. Examples of ventral root
recordings from Nkx2.2–/– and Nkx2.2+/–;Nkx2.9–/– compoundmutant spinal cords are shown in Fig. 7A and Fig. 7B, respectively.
Average circular plots of left-right (LL2-RL2) and flexor-extensor
(iL2-iL5) coordination for homozygous and heterozygous Nkx2.2
mutant animals with normal Nkx2.9 wild-type genes revealed no
systematic change in the drug-induced locomotor-like activity (Fig.
7C) compared with wild-type animals (Fig. 7A; data not shown).
These results suggest that blocking the formation of V3 neurons
during embryonic mouse development causes no significant effect
on left-right and flexor-extensor coordination. By contrast, Nkx2.9–/–
animals and, most prominently, the Nkx2.2+/–;Nkx2.9–/– compound
mutants showed markedly reduced coordination of drug-induced
locomotor-like activity with increased variability in both left-right
and flexor-extensor coordination, as illustrated by the circular plots
(Fig. 7D). Unfortunately, double-homozygous Nkx2.2–/–;Nkx2.9–/–
null mutants could not be included in the analysis because of their
early postpartum lethality. The observed increase in variance of
locomotor-like activity was mainly attributable to two phenomena.
First, left-right coordination in these animals often drifted in phase
DEVELOPMENT
Fig. 7. Nkx2.9-deficient mice display defects of left-right and flexor-extensor coordination during locomotor activity in the isolated
spinal cord. (A,B)Representative raw traces of ventral root recordings during drug-induced locomotor activity in spinal cords of Nkx2.2–/– (A) and
Nkx2.2+/–;Nkx2.9–/– (B) newborn mice. Lower panel in B shows traces from the boxed area with higher resolution of the time axis. Activities were
recorded in ventral roots from left (L) and right (R) lumbar segments L2 (LL2, RL2, respectively; flexor-dominated), and the left lumbar segment L5
(LL5; extensor-dominated). Each locomotor cycle is indicated in reference to LL2 by alternating grey and white background. The onset of locomotor
cycles at RL2 and LL5 are shown as raster plots above the corresponding recordings. The superimposed thin red line shows the rectified smoothed
ventral root activity. (C,D)Circular phase diagrams of left-right (L-R) and intersegmental flexor-extensor (iL2-iL5) coordination show no significant
uncoupling in Nkx2.2+/– (green arrow in C; n5) and Nkx2.2–/– (blue arrow in C; n2) pups. By contrast, a number of Nkx2.9-deficient animals, both
Nkx2.9–/– single mutants (red arrow in D; n6) and Nkx2.2+/–;Nkx2.9–/– compound mutants (black arrow in D; n11), exhibited insignificant coupling
between left-right and flexor-extensor ventral root activities (dots falling within the blue circle). One Nkx2.2+/–;Nkx2.9–/– double mutant showed
hopping with phase values around zero. Each dot in the graphs represents the average phase value calculated from 30 locomotor cycles for each
individual animal. The blue circle marks the significance level with P<0.05 outside the blue circle. (E)Representative recording from an isolated
spinal cord of an Nkx2.2+/–;Nkx2.9–/– pup. Fast GABAergic and glycinergic synaptic transmission was blocked with picrotoxin (10M) and strychnine
(0.3M), respectively. Spontaneous slow synchronous bursts of activity occur in all ventral roots.
RESEARCH ARTICLE 4257
between the locomotor cycles on each side of the spinal cord leading
to a relaxed coordination that lacked clear segmental alternation.
Second, a double burst activity was sometimes seen in L5 (seen as
two bursts per L2 cycle, one in and one out of phase with L2; Fig.
7B). Thus, it seemed that the regular out-of-phase flexor-extensor
activity was maintained but complemented with additional activation
of the L5 ventral root in synchrony with the ipsilateral rostral L2 root
in these mutants. In summary, Nkx2.9–/– mice and, even more
significantly, the Nkx2.2+/–;Nkx2.9–/– mutants displayed a high
degree of uncoupling of left and right side activity and aberrant
flexor-related bursting in the otherwise extensor-dominated L5 root.
Seventy-five percent of V3 cells form a population of excitatory
commissural neurons (CINs) (Zhang et al., 2008). This suggests
that crossed excitatory drive might be reduced in
Nkx2.2+/–;Nkx2.9–/– compound mutants. Therefore, we examined
whether these animals were still able to coordinate left-right
activities using the remaining excitatory CINs. To test this, we
recorded locomotor-like activity in the presence of picrotoxin and
strychnine, which block the two main classes of inhibitory
receptors, -aminobutyric acid (GABA) and glycine receptors,
respectively. In spinal cords of wild-type mice, these experimental
conditions led to synchronous activities in all ventral roots
(Hinckley et al., 2005). The same result was obtained with spinal
cords from the Nkx2.2+/–;Nkx2.9–/– mutant (Fig. 7E). Taken
together, the electrophysiological data indicate that developmental
loss of V3 neurons alone probably has little impact on druginduced locomotor-like activity, whereas complete disruption of the
Nkx2.9 gene and partial loss of Nkx2.2 results in substantial
locomotor deficits, both in vitro and in vivo.
shown that Nkx2.2 is able to repress the expression of Olig2
(Novitch et al., 2001). The ectopic expression of Olig2 in Nkx2.2
and Nkx2.9-depleted cells supports the idea of transcriptional
repression of MN lineage-specific genes, such as Olig2. In this
scenario, formation of motor neurons would be driven by Shh
signaling through the direct induction of Olig2 (Dessaud et al.,
2007). Support for this view also comes from the temporal
sequence of marker gene expression in the early ventral neural
tube, in which the basic progenitor organization is probably already
set by E8.5-9.0 by Shh signaling from the notochord (Ericson et al.,
1996; Jeong and McMahon, 2005). Olig2 expression is first seen
in the most ventral part of the early neural tube and precedes that
of Nkx2.2 in an area that soon after will acquire floor plate identity.
Olig2 expression is probably repressed by Nkx2.2 and/or Nkx2.9
in early p3 progenitors and, as we show here, in prospective floor
plate cells.
Nkx2.2 has also been suggested to repress the expression of
Pax6 in p3 cells (Briscoe et al., 2000). However, we find no
evidence for a ventrally expanded Pax6 domain in the spinal cord
of double-null mutant embryos, in agreement with previous
observations for the homozygous Nkx2.2 mutant (Briscoe et al.,
1999). In conclusion, we propose a model in which Nkx2.2 and
Nkx2.9 genes collaborate to serve a dual function during
development of V3 interneurons. First, the repressive activity of
Nkx2.9 and/or Nkx2.2 leads to the determination of the early V3
progenitor cell lineage by preventing Olig2 expression, as shown
here. Second, continued activity of, presumably, Nkx2.2 is essential
to mediate differentiation of progenitors into Sim1+ V3
interneurons (Briscoe et al., 1999).
DISCUSSION
Individual targeted disruptions of the genes encoding the related
HD transcription factors Nkx2.2 (Briscoe et al., 1999; Sussel et al.,
1998) and Nkx2.9 (Pabst et al., 2003) suggested that both proteins
play essential and partially redundant roles for the development of
distinct neuronal populations in hindbrain and ventral spinal cord.
Here, we report three novel findings in double-mutant mice. First,
V3 neural progenitors cells are converted to the MN cell lineage in
the spinal cord of mouse embryos that lack both transcription
factors. Second, development of a structurally and functionally
intact floor plate is impaired in the absence of Nkx2.2 and Nkx2.9,
which might cause the observed severe axonal pathfinding defects
of commissural neurons. Third, the majority of adult
Nkx2.2+/–;Nkx2.9–/– mutant mice exhibits abnormal locomotor
behavior, such as a hopping gait and increased variability of leftright and flexor-extensor coordination in drug-induced locomotorlike activity of isolated spinal cords.
The transcription factors Nkx2.2 and Nkx2.9 are
essential for normal locomotor behavior
Unexpectedly, we observed that the majority of adult compound
Nkx2.2+/–;Nkx2.9–/– mutant mice exhibited a strong walking
impairment. This phenotype was supplemented by the finding that
neonatal mutant mice of the Nkx2.9-null genotype showed
substantially abnormal drug-induced locomotor-like activity, which
was even more pronounced in Nkx2.2+/–;Nkx2.9–/– mutant animals.
Our data therefore indicate the importance of Nkx2.9 and Nkx2.2
factors for a fully functional CPG network. In vitro locomotor
experiments revealed that compound Nkx2.2+/–;Nkx2.9–/– mutant
mice possibly have more than one defect in locomotion, as leftright and flexor-extensor motor activity patterns were disturbed.
Mutant animals displayed alterations of the interplay between L2
and L5 ventral roots. Although the normal out-of-phase activity of
the ipsilateral L2 and L5 segments seemed to be preserved in
mutant animals, it was complemented by an additional activation
of L5 in synchrony with the ipsilateral L2 activation. This extensorrelated double burst presents a CPG phenotype not previously
described for genetic perturbation experiments in any of the V0-V3
groups.
In normal animals, the alternation of motor bursts on both sides
of the spinal cord is coordinated through the activity of CINs.
Proper left-right alternation is fundamental for coordinated walking
behavior in mammals, which is thought to be mediated through
inhibitory and excitatory pathways (Butt and Kiehn, 2003;
Jankowska, 2008; Kiehn, 2006; Quinlan and Kiehn, 2007). Support
for this view was obtained by the analysis of a mouse mutant in
which the V0 class was perturbed by genetically ablated Dbx1, the
fate-determining transcription factor in V0 neurons. This gene
mutation caused disturbances in left-right coordination in the spinal
cord (Lanuza et al., 2004). Rhythmic coupling of left-right motor
Cell fate determination of neuronal progenitors in
the p3 domain requires either Nkx2.2 or Nkx2.9
The present study provides evidence that neuronal progenitor cells
in the p3 domain acquire a more dorsal fate in the absence of
Nkx2.2 and Nkx2.9. This observation, together with previous
studies, clearly indicates that at least one of the two transcription
factors is required and sufficient for correct cell lineage
determination in this part of the spinal cord, supporting the model
of functional redundancy between both genes. In the double-null
mutant, but not in single-mutant or wild-type embryos, cells in the
p3 region express Olig2, which identifies them as prospective MN
precursors that replace the normal V3 progenitors. Recent lineage
tracing studies demonstrated that Nkx2.2 and Olig2 cell lineages
share a common origin (Dessaud et al., 2007). Furthermore, it was
DEVELOPMENT
Nkx2 transcription factors regulate floor plate development
bursts seems to be coordinated by ipsilaterally projecting
interneurons, as genetic depletion for excitatory Chx10-positive V2a
neurons also disrupted left-right alternation (Crone et al., 2008;
Crone et al., 2009). V2a interneurons synapse (among other targets)
on V0 commissural interneurons (Crone et al., 2008) emphasizing
the complexity of the neuronal network that is required to drive the
left-right alternation.
Mice with a conditional block of neurotransmission in all V3derived interneurons have been generated recently by selective
expression of the tetanus toxin light chain subunit (Zhang et al.,
2008). The regularity and robustness of the locomotor rhythm in
these mutant mice was severely perturbed, particularly the duration
of individual motor bursts, and the entire step cycle period of
fictive locomotion appeared to be highly variable (Zhang et al.,
2008). This is in contrast to wild-type mice and even mutants with
complete loss of left-right alternation, in which the length of motor
bursts per segment on either side of the spinal cord were identical
and well coordinated (Fawcett et al., 2007; Kullander et al., 2003;
Wegmeyer et al., 2007). Current knowledge suggests that V3derived neurons ensure normal walking gait by controlling and
balancing the rhythmic motor outputs of independent locomotor
centers on each side of the spinal cord as well as flexor-extensorrelated motor bursts. However, the Sim1+ neurons seem to play a
minor role in the general left-right alternation of the locomotor
cycle and flexor-extensor coordination (Zhang et al., 2008).
Interestingly, in the present study fictive locomotion in spinal
cords from Nkx2.2-deficient neonates that lack V3 neurons was not
significantly different from wild type, suggesting that
compensatory mechanisms during prenatal development of the
CPG circuitry might obscure the phenotype. Because Nkx2.2deficient mice also contain higher numbers of MNs, it seems
unlikely that an increased pool of MNs contributes significantly to
the locomotor phenotype.
Development of the floor plate and formation of
the spinal commissure are defective in
Nkx2.2;Nkx2.9 mutant mice
Nkx2.2+/–;Nkx2.9–/– double-mutant and to a lesser extent Nkx2.9
single-mutant mice show clear defects in locomotor activity both in
vitro and in vivo, although they generate all cardinal classes of
neurons in the ventral neural tube, including some Sim1+ V3 neurons
(Fig. 2) (Pabst et al., 2003). How then can the abnormal locomotor
phenotype be explained? Nkx2.2 and Nkx2.9 are transiently
expressed in cells that will contribute to the floor plate (Chamberlain
et al., 2008; Jeong and McMahon, 2005). We demonstrate here that
targeted disruption of both genes has a major impact on floor plate
development most profoundly in Nkx2.2–/–;Nkx2.9–/– mutants but
also in Nkx2.2+/–;Nkx2.9–/– animals (Fig. 4). Several known guidance
molecules for commissural axons (Evans and Bashaw, 2010) showed
reduced expression in mutants. Specifically, chemoattractants and
repellents, like Shh (Charron et al., 2003), netrin (Kennedy et al.,
1994; Kennedy et al., 2006; Serafini et al., 1994), and Slit
(Hammond et al., 2005; Long et al., 2004) were significantly
diminished in the floor plate of double mutants. Tracing of the
trajectories of dorsal commissural neurons (presumably dl1 neurons)
in open-book preparations of mutant spinal cords provided evidence
of disturbed navigation of axons (Fig. 5), which normally extend
towards the ventral midline attracted by guidance cues derived from
the floor plate (Salinas, 2003). As axons contact the floor plate, they
become unresponsive to these attractive signals and gain sensitivity
to repellent molecules, which allows complete crossing of the
midline and subsequent turning in an anterior direction (Stoeckli,
Development 137 (24)
2006). Shh in the floor plate serves a dual function by first attracting
commissural axons and then inducing the switch to repulsion that is
important to permit entry into the contralateral hemisphere of the
neural tube (Parra and Zou, 2010). Shh is reduced in the floor plate
of the Nkx2 mutants, which might partly explain the aberrant
pathfinding behavior of commissural axons observed in this study.
As defects in axonal guidance and the locomotor phenotype appear
to be correlated in Nkx2.2+/–;Nkx2.9–/– mutant mice, it is not
unreasonable to propose that altered guidance cues for commissural
axons could lead to wiring problems in the CPG circuitry and cause
the abnormal locomotor behavior of these mutants. Although we
have not yet tested which type of commissural neurons might be
affected in the hopping mutant, V0 and dl6 interneurons are good
candidates (Dougherty and Kiehn, 2010; Goulding, 2009; Kiehn et
al., 2010). The reduction of V3 interneurons alone does not provide
a sufficient explanation for the locomotor phenotype, as they are not
required for left-right alternation (Zhang et al., 2008). Nevertheless,
it cannot be ruled out entirely that subtle changes in the function of
mutant V3 neurons contribute to the defective gait.
Acknowledgements
We thank Iris Linde for expert technical assistance, R. Hänsch for advice on
confocal microscopy, S. Schildt and K. Sawinski for experimental help, and Dr
Fujio Murakami for the Rig1 antibody. This work was supported by a grant of
the Deutsche Forschungsgemeinschaft to H.H.A. and A.H., and grants from
the Swedish Research Council and NIH (R01NS0407909) to O.K. Deposited in
PMC for release after 12 months.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.053819/-/DC1
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