Vol 440|9 March 2006|doi:10.1038/nature04545

LETTERSV1 spinal neurons regulate the speed of vertebrate
locomotor outputs
Simon Gosgnach1*, Guillermo M. Lanuza1*, Simon J. B. Butt3†, Harald Saueressig1, Ying Zhang1,
Tomoko Velasquez1, Dieter Riethmacher4, Edward M. Callaway2, Ole Kiehn3 & Martyn Goulding1

The neuronal networks that generate vertebrate movements suchas walking and swimming are embedded in the spinal cord1–3.
These networks, which are referred to as central pattern generators (CPGs), are ideal systems for determining how ensembles ofneurons generate simple behavioural outputs. In spite of efforts toaddress the organization of the locomotor CPG in walking animals2,4–6, little is known about the identity and function of the
spinal interneuron cell types that contribute to these locomotor
networks. Here we use four complementary genetic approaches
to directly address the function of mouse V1 neurons, a class of
local circuit inhibitory interneurons that selectively express thetranscription factor Engrailed1. Our results show that V1 neurons
shape motor outputs during locomotion and are required forgenerating ‘fast’ motor bursting. These findings outline animportant role for inhibition in regulating the frequency of thelocomotor CPG rhythm, and also suggest that V1 neurons may
have an evolutionarily conserved role in controlling the speed ofvertebrate locomotor movements.
Several genetically defined classes of neurons have been identified
in the developing spinal cord7, including a class of ipsilaterally

projecting inhibitory neurons that innervate motor neurons, theEngrailed1 (En1)-expressing V1 neurons8–11. To assess the function of
V1 neurons in the locomotor CPG, we used two mouse models thathave selective loss of the En1-expressing V1 neuronal population(Fig. 1). This was achieved through the altered specification of V1
neurons in Pax6-knockout (Pax6 2/2) mice and by the selective
ablation of these neurons in En1 Cre; R26-lacZ flox/DTA (En1-DTA;
ref. 12) mice. At embryonic day (E)12.5, a marked reduction in
En1-positive V1 neuron cell numbers was apparent in spinal cords
isolated from Pax6 2/2 and En1-DTA embryos (Fig. 1a, e). This was
confirmed using an En1 Cre lacZ or green fluorescent protein (GFP)
reporter system9 , which also showed that significantly fewer
En1-derived V1 neurons are present at E18.5 (Fig. 1b, f). Both
Pax6 2/2 and En1-DTA mice had normal numbers of lumbar spinal
motor neurons that were appropriately organized into lateral andmedial motor columns. (Fig. 1c, g). Spinal cords from Pax6 2/2 mice
did show an increase in commissural V0 and V3 interneuron cellnumbers, coupled with a slight decrease in ipsilaterally projecting
Chx10-positive V2 neurons13 (Chx10 is a marker of V2 neurons;
Supplementary Fig. S1). However, there were no changes in ventral

Figure 1 | V1 neurons are reduced in Pax6 2/2
and En1-DTA spinal cords. a, b, Cross-sections
through E12.5 (a) and E18.5 (b) wild-type(WT) and Pax6 2/2 spinal cords, showing fewer
En1-positive V1 neurons in the Pax6 2/2 spinal
cord. V1 neurons were marked using
En1 Cre; R26 lacZ reporter alleles. c, Lumbar-level
Hb9 staining of motor neurons is largely
unaffected in Pax6 2/2 embryos.
d, Quantification of V1 neuron loss in the
Pax6 2/2 spinal cord. e, f, En1-DTA embryos
show a selective loss of En1-positive V1 neurons
at E12.5 (e) and E18.5 (f). g, Numbers of
Hb9-positive motor neurons and columnarorganization are normal in the En1-DTA spinal
cord. h, Quantification of V1 neuron loss in the
En1-DTA cord. Error bars indicate s.d.

1
Molecular Neurobiology Laboratory and 2System Neurobiology Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037,
USA. 3Mammalian Locomotor Laboratory, Department of Neuroscience, The Karolinska Institute, Retzius vag 8, Stockholm 17177, Sweden. 4Centre for Molecular Neurobiology,
University of Hamburg, Hamburg 20251, Germany. †Present address: Department of Developmental Genetics, The Skirball Institute, New York University, New York, New York
10016, USA.
*These authors contributed equally to this work.

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Figure 2 | Mice lacking V1 neurons show slow locomotor-like activity.
a–c, Left, electroneurograms from left (lL2) and right (rL2) L2 and left L5(lL5) ventral roots after application of 5 mM NMDA and 10 mM 5-HT,
showing locomotor-like activity in E18.5 spinal cords from wild-type (a),Pax6 2/2 (b) and En1-DTA (c) mice. Right, circular plots showing thecoupling of bursts in lL5 and rL2 with respect to lL2. Each point represents
the vector point for one experiment. d, Mean step-cycle period in wild-type,Pax6 2/2 and En1-DTA spinal cords. e, Plot of step-cycle period versus 5-HT
concentration for Pax6 2/2 (open circles), En1-DTA (open squares) andwild-type/Pax6 þ/2 spinal cords (filled circles). Error bars indicate s.d.

interneuron specification in the En1-DTA mice (Supplementary
Fig. S1).
Limbed walking movements in vertebrates are characterized by therepetitive oscillatory bursting of motor neurons, in which flexor–
extensor and left–right motor activities alternate. These locomotorlike oscillations can be induced in the isolated spinal cord by
excitatory neurotransmitter agonists, and typically show a stepcycle period of 2–4 s (refs 5, 14, 15 and Fig. 2a). We asked whether
V1 neurons regulate the coordination of flexor–extensor motor
neurons during locomotion, because many V1 neurons differentiate
into Renshaw cells and Ia inhibitory interneurons, two inhibitory cell
types that have been proposed to coordinate flexor and extensor
muscle activity around ipsilateral limb joints9,16–19. Spinal cords
lacking V1 neurons showed a normal pattern of alternating flexor(L2) activity and extensor (L5) activity (Fig. 2a–c). Left–rightalternation (that, is lL2 versus rL2) was also normal. However,‘locomoting’ Pax6 2/2 and En1-DTA spinal cords did show a significant lengthening of both the step-cycle period and motor neuron
burst duration (Fig. 2b–e).
Whereas 5 mM NMDA (N-methyl-D -aspartate) in combination
with 5 mM serotonin (5-hydroxytryptamine or 5-HT) typicallyinduces robust locomotor-like activity in wild-type spinal cords15(Fig. 2a), we were only able to elicit a stable pattern of locomotor-like
activity in Pax6 2/2 spinal cords by increasing the concentration of
5-HT to 7.5–20 mM (Fig. 2b, e). In experiments using 10 mM 5-HT,
the step-cycle period (8.4 ^ 2.9 s, mean ^ s.d.) and burst duration(4.4 ^ 1.0 s) in Pax6 2/2 spinal cords (n ¼ 7) were increased significantly (P , 0.001, Student’s t-test) compared to wild-type andheterozygous (Pax6 þ/2) spinal cords (step-cycle period 3.8 ^ 0.8 s,burst duration 1.7 ^ 0.8 s; n ¼ 10). This relative increase in the
step-cycle period was seen over a wide range of 5-HT concentrations(Fig. 2e). Similar lengthening of the step cycle (Fig. 2c, d) and burst
duration was seen in En1-DTA mice (9.3 ^ 2.3 s and 5.4 ^ 1.1 s,
respectively; n ¼ 5 spinal cords; P , 0.001, t-test). V1 neurons are
therefore dispensable for flexor–extensor coordination, but have an
essential role in determining the speed of the locomotor rhythm.
Current-clamp recordings of motor neurons in wild-type andPax6 2/2 spinal cords (Fig. 3a–d) revealed that Pax6 2/2 motor

neurons show prolonged periods of membrane potential depolarization, and that they continue to fire action potentials throughout thisdepolarized phase (Fig. 3c). This is consistent with flexor- andextensor-related CPG neurons remaining active for longer periodsof time. These intracellular recordings also revealed that the alternating excitatory and inhibitory drive that normally underlies
rhythmic changes in motor neuron membrane potential20 is stillpresent in the Pax6 2/2 spinal cord (Fig. 3e). Importantly, Pax6 2/2
motor neurons still receive the phasic inhibitory inputs that are
essential for coordinating alternating flexor and extensor activity(Fig. 3f). The origin of these phasic inhibitory inputs is not known—
although they could in principle be derived from contralateralcommissural interneurons21,22, this seems not to be the case, as
ipsilateral locomotor coordination is normal in the Pax6 2/2 cord
after spinal cord hemisection (data not shown).
We then used En1 tlZ/tlZ null ‘knock-in’ mice (see ref. 8) to address
the function of V1 neurons in the intact animal. Spinal cords isolated
from E18.5 En1 tlZ/tlZ mice show a 3–4-fold slowing of the locomotor
step cycle and lengthening of the burst duration (Fig. 4a, b;
11.2 ^ 2.3 s and 6.1 ^ 1.8 s, respectively; n ¼ 10 spinal cords),
which closely resemble the behavioural changes seen in Pax6 2/2
and En1-DTA spinal cords (compare with Fig. 2). Using a Wnt1 En1transgene23 to rescue the En1 mutant midbrain–hindbrain phenotype, we obtained adult En1 tlZ/tlZ mice in which the V1 neuron
connectivity defects in the spinal cords8,9 are not rescued (H.S.,
unpublished observation). When rotarod tests were performed on
4–6-month-old En1 tlZ/tlZ; Wnt1 En1 mice and age-matched controls,
En1 tlZ/tlZ; Wnt1 En1 mice showed a clear deficit in their ability to walkand maintain their balance at higher rotarod speeds (Fig. 4c; see
Methods). Although the duration (and the speed) for which the
mutant animals walked on the rotarod increased during the trial
period, at no time did they attain walking speeds above 18 r.p.m.
However, these animals did walk on the rotarod for extended periodsof times at idling speed (5 r.p.m., data not shown). In contrast, wildtype animals walked and maintained their balance at much higher
speeds (approximately 22 r.p.m. on day 1 and up to 35 r.p.m. on
day 5). This suggests that adult En1 mutants are impaired in theirability to perform quick stepping movements.

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Figure 3 | Motor neuron firing during locomotion in the Pax6 2/2 spinal
cord is prolonged and shows rhythmic inhibition. a, Schematic of recording
setup, with a suction electrode recording electroneurogram activity fromrL2 ventral root and an intracellular electrode recording from a motor
neuron located in L2. Motor neurons were identified by the presence of anantidromic action potential in response to ventral root simulation (upper
right panel, arrow) and by a small after-depolarization following
orthodromic spikes (lower right panel, arrow), which is unique to rodent
motor neurons. b, c, Recording from motor neurons located in rL2 ofwild-type (b) and Pax6 2/2 (c) mice after application of NMDA and 5-HT.
d, Chart showing the mean number of action potentials (mean ^ s.d.) fired
per depolarization of the motor neuron and the mean duration of thedepolarized phase (LDP) of the motor neuron during locomotion inwild-type and Pax6 2/2. e, f, Intracellular recording from a motor neuron
located in the rL2 segment (upper traces) and electroneurogram recordingfrom the rL2 ventral root (lower traces, rectified and integrated) in a Pax6 2/2
mouse. Grey area indicate the period during which rL2 is inactive. In e,
recordings were performed at resting membrane potential (E m 250 mV)
after application of 5 mM NMDA þ 15 mM 5-HT. The motor neuron is active
in-phase with ventral root activity. In f, the motor neuron is hyperpolarized
to 288 mV, beyond the calculated Cl2 reversal potential, which results in theemergence of out-of-phase depolarization owing to reversed inhibitory
currents.

Next, we used transgenic mice (AlstR192 mice) that conditionallyexpress the Drosophila allatostatin G-protein-coupled receptor(AlstR) to test whether acutely suppressing V1 neuron excitability
causes a similar lengthening of the locomotor step cycle (Fig. 5a).
AlstR couples to endogenous mammalian GIRK channels (inwardrectifying Kþ channels), causing a decrease in cellular input resistance and neuronal excitability24. In control experiments, allatostatin(100 nM–5 mM) had no effect on motor rhythm generation, locomotor coordination or the step-cycle period (Supplementary
Fig. S2a). In contrast, spinal cords from nestin Cre; AlstR192 mice
showed strong allatostatin-dependent depression in rhythmic motor
activity (Supplementary Fig. S2b).
En1 Cre; AlstR192 mice were then used to selectively express AlstR
in V1 neurons (Fig. 5a, b). In spinal cord slices prepared from
En1 Cre; AlstR192 mice, we observed a pattern of GFP reporter
expression that was identical to the normal distribution of V1
neurons (Fig. 5c). Whole-cell recordings from identified GFPpositive V1 neurons (n ¼ 7 cells) revealed a reversible decrease inneuronal excitability in response to current steps (Fig. 5d) and ramps(Fig. 5e) after the addition of allatostatin (10 nM). The decrease inexcitability (2–3-fold), although not as pronounced as in isolated
adult neurons24, was nevertheless substantial, and it was not seen incontrol, AlstR-negative neurons (Supplementary Fig. S2c).
When locomotor-like activity was induced in postnatal day P0–P2
En1 Cre; AlstR192 spinal cords (5 mM NMDA þ 10 mM 5-HT; Fig. 5f),
we observed a step-cycle period of 3–4 s (3.3 ^ 0.8 s, n ¼ 15 spinal
cords), comparable to that seen in wild-type animals of a similar age.
Application of allatostatin (1–5 mM) to these spinal cords resulted inmarked and significant lengthening of the step-cycle period(7.9 ^ 1.8 s, n ¼ 15 spinal cords; P ¼ 0.002, paired t-test), which
was reversed upon washing out the neuropeptide (Fig. 5f). As such,lengthening of the locomotor step cycle that results from the defects
in V1 neuron development can be reproduced when V1 neuronal
activity is suppressed. Two recent studies have provided evidence thatmodifying neuronal activity during development can alter theneurotransmitter phenotype of spinal neurons25 and produce smallchanges in CPG motor outputs26, indicating that activity may have arole in organizing these locomotor networks However, our results
show that the changes in locomotor CPG outputs following the acute

Figure 4 | En1 tlZ/tlZ mice show a slowing of stepping movements. a, Left,electroneurogram recordings from lL2, lL5 and rL2 ventral roots after
application of 5 mM NMDA/7.5 mM 5-HT to an isolated E18.5 En1 tlZ/tlZ
spinal cord. Right, circular plots indicate normal ventral root alternation.
Each small circle corresponds to the vector point for a single experiment.
b, Graph showing the increase in the step-cycle period in En1 tlZ/tlZ mutants
compared with wild-type spinal cords. c, Rotarod testing of mouse motor
behaviours, showing the time and bar speed that age-matched wild-type(open bars) and En1 tlZ/tlZ; Wnt1 En1 mice (filled bars; three animals each)were able to run on an accelerating rotarod. Error bars in b, c show s.d.

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Figure 5 | Acute silencing of V1 neurons via the allatostatin receptor causesa slowing of locomotor-like rhythmicity. a, Schematic showing theallatostatin receptor (Z/AlstR) transgene. Crossing mice that harbour thistransgene with an En1 Cre mouse excises the loxP-flanked b-geo sequences,leading to transcription of AlstR in En1-derived cells. b, Co-expression of
GFP (green) and En1 (red) in an E11.5 En1 Cre; AlstR192 mouse, showing V1cells expressing AlstR. Many newborn V1 neurons in the subventricularzone are AlstR/GFP-negative owing to a delay in En1-dependent Cre
removal of the stop cassette. c, Spinal cord slice from a P0 En1 Cre; AlstR192
mouse carrying the ZnG reporter allele, showing GFP expression in V1

neurons. d, Recording from a V1 neuron expressing AlstR in a P1
En1 Cre; AlstR192; ZnG mouse in response to current steps. e, Recordings
from an AlstR-expressing V1 neuron in response to a current ramp. Before
allatostatin (AL) application, the cell begins to fire upon current injection of
105 pA. After allatostatin application (10 nM), the cell only fires when theinjected current reaches 190 pA. f, Electroneurogram recordings from lL2
and lL5 ventral roots in a P1 En1 Cre; AlstR192 mouse, showing the effects ofallatostatin (5 mM) application and wash-out on locomotor activity in theisolated spinal cord. The lengthening of the step cycle after allatostatin
application was significant (P ¼ 0.02, paired t-test; n ¼ 15 spinal cords).

suppression of V1 neurons compared with the early loss of V1
neurons are highly concordant. This suggests that the absence ofV1 neurons during development does not markedly reconfigure thespinal locomotor CPG network.
Our findings outline an essential role for V1 neurons in mammals
in regulating the duration of the locomotor step cycle, and hence thespeed of the locomotion. This function is specific for V1 neurons, as
we have not seen marked changes in the frequency or duration of thestep cycle when either V0 or V3 neurons are deleted from the spinal
cord5 (M.G., unpublished observations). Our conclusion that V1
neurons are necessary for ‘fast’ locomotor outputs in mice issupported by studies in Xenopus tadpoles showing that aIN neurons,
the homologues of mouse V1 neurons, are the primary source ofearly cycle inhibition to the swimming locomotor CPG10. Although
the role of aIN neurons in regulating the speed of Xenopus swimming
movements was not examined, a strong correlation was seen between
aIN-derived inhibitory inputs to CPG neurons and the frequency ofswimming movements10. This raises the possibility that the Xenopus
homologues of V1 neurons may facilitate fast swimming movements,
in much the same way that their mammalian counterparts are
required for fast ‘walking’ movements.
The exact contribution individual V1 interneuron subtypes maketo regulating the duration of motor neuron bursting during fictive
locomotion remains to be determined. Inhibition of Renshaw cellswith cholinergic blockers causes a small increase in the step-cycle
period26 (S.G. and O.K., unpublished observations), suggesting that agroup of non-Renshaw V1 neurons or the V1 population as a wholeregulates the step-cycle period during fictive locomotion. AlthoughRenshaw cells and Ia inhibitory interneurons have described roles incoordinating flexor–extensor motor neurons during spinal reflexes27,
our study demonstrates that the V1 population as a whole does nothave a primary role in regulating locomotor CPG flexor–extensor
activity. It therefore seems that an additional group of non-V1
interneurons have been incorporated into the walking CPG ofterrestrial vertebrates to secure flexor–extensor alternation.
Our study also demonstrates that the acute silencing of a selectpopulation of neurons using genetic approaches can be used toelucidate their function with respect to a defined behaviour suchas locomotion. Our results highlight the feasibility of using ligandactivated GIRK channels to selectively manipulate neuronal
excitability in the vertebrate nervous system, and the conditional
AlstR192 mice described here can be used to selectively silence
neurons throughout the nervous system. This and similar genetic
approaches now make it feasible to selectively probe the function ofsmall populations of neurons, which should facilitate the mapping ofneural circuits at higher resolutions than was previously possible.

218

METHODS

Animals. The generation and genotyping of the Pax6 lacZ, Sey, En1 Cre, En1 tlZ,
Wnt1 En1, nestin Cre, R26-lacZ flox/DTA, R26 lacZ and R26 GFP alleles in mice has
been previously described8,9,12,23,28. Embryos and tissues were obtained fromtimed matings.
Generation of conditional AlstR mice. The Drosophila AlstR coding sequence,
followed by an IRES-EGFP24, was inserted downstream of a loxP-flanked bgeo/
polyA stop sequence in the conditional Z/EG construct29 to produce the Z/AlstR
expression vector (see Fig. 4a). The linearized Z/AlstR construct was electroporated into the embryonic stem cell line W9.5. G418-resistant clones werescreened for uniform and robust lacZ expression, and for single transgene
integrants. Two clones gave germline transmission (AlstR172 and AlstR192).

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AlstR192 mice that showed lacZ expression throughout the nervous system wereused for all further experiments.
In situ hybridization and immunohistochemistry. Immunostainings were
performed as previously described5. Images were captured using a Zeiss
LSM510 confocal microscope and assembled using Adobe Photoshop. Cell
numbers are indicated as mean ^ s.d. per section per spinal cord side, andwere determined on counts of at least three embryos (five sections each). In situ
hybridization and staining for b-galactosidase activity were performed aspreviously described5,8.
Electrophysiology: electroneurogram recordings. Electrophysiological experiments were performed on E18.5–P2 mice in accordance with the ethical rules
stipulated by NIH and the Swedish government. Animals were anaesthetized anddecapitated, and spinal cords were dissected out in ice-cold Ringers’ solution4.
Recordings were made in Ringers’ solution at 20 8C by placing bipolar suction
electrodes on three of the second and fifth lumbar ventral roots (that is rL2,
lL2, rL5, lL5). Electroneurogram signals were amplified, band-pass filtered(100 Hz–1 kHz), digitized and collected using Axoscope software (Axon Instruments). Rhythmic locomotor activity was induced by adding NMDA (5 mM) and5-HT (5–30 mM) to the Ringers’ solution. Effects of allatostatin (10 nM–5 mM)
were examined by adding the peptide to the perfusion solution.
Electrophysiology: analysis of locomotor activity. Step-cycle period and burst
duration were determined by analysing lL2 or rL2 activity4. Step-cycle period
and burst duration averages were determined from all recorded bursts after the
onset of stable locomotor-like activity. The effects of allatostatin were measured
10 min after application to allow for drug wash-in. All measurements are
given ^ s.d. P values less than 0.05 were considered significant. Circularstatistics19 were used to determine the coupling strength between left and rightventral roots, as well as between flexor-related (L2) and extensor-related (L5)ventral roots on the same side of the spinal cord. L2 bursts were used as a reference.
Vector points representing the phase values between 0 and 1 were plotted for eachexperiment, and show the mean phase as well as the concentration of phase values
around the mean. Appropriate left–right or flexor–extensor alternation isrepresented by phase values around 0.5.
Electrophysiology: intracellular recordings. Whole-cell patch-clamp recordings from motor neurons were made using recording pipettes with a resistance of
4–5 MQ. The microelectrode was driven into the spinal cord through a pial patchin the ventrolateral surface. Cells were patched in the motor neuron area andrecorded using an AxoClamp 2B amplifier (Axon Instruments).
Electrophysiology: acute spinal cord slice recordings. For slice recordings,
spinal cord slices 250–300-mm thick were cut using a Leica VY1000E vibrating
microtome. After a 1-h recovery period, slices were transferred to a recording
chamber, mounted on an Olympus BX51W1 microscope and perfused withoxygenated Ringers’ solution at room temperature. GFP-positive cells werevisualized using a DAGE-MTI IR-1000 CCD camera and patched visuallyusing a Sutter MPC-325 micromanipulator. Recordings were made in currentclamp mode using a MultiClamp 700B amplifier (Axon Instruments).
Behavioural testing. Rotarod test were performed during the light phase of a
12 h:12 h light:dark cycle as previously described30. Mice were placed on a
Rotamex 4/8 (Columbus Instruments) idling at 5 r.p.m. Rotarod speed was set
to increase gradually from 5 to 65 r.p.m. over the course of 3 min. Retention timeon the rod was recorded for three trials per mouse per day for five consecutive
days, using three mutant and three wild-type mice.
Received 15 November; accepted 22 December 2005.
1.
2.

3.

4.

5.

6.

7.

Grillner, S. The motor infrastructure: from ion channels to neuronal networks.
Nature Rev. Neurosci. 4, 573–-586 (2003).
Kiehn, O. & Kjaerulff, O. Distribution of central pattern generators for rhythmic
motor outputs in the spinal cord of limbed vertebrates. Ann. NY Acad. Sci. 860,
110–-129 (1998).
Roberts, A., Soffe, S. R., Wolf, E. S., Yoshida, M. & Zhao, F. Y. Central circuits
controlling locomotion in young frog tadpoles. Ann. NY Acad. Sci. 860, 19–-34(1998).
Butt, S. J., Harris-Warrick, R. M. & Kiehn, O. Firing properties of identified
interneuron populations in the mammalian hindlimb central pattern generator.
J. Neurosci. 22, 9961–-9971 (2002).
Lanuza, G. M., Gosgnach, S., Pierani, A., Jessell, T. M. & Goulding, M. Genetic
identification of spinal interneurons that coordinate left-right locomotor
activity necessary for walking movements. Neuron 42, 375–-386 (2004).
Angel, M. J., Jankowska, E. & McCrea, D. A. Candidate interneurones
mediating group I disynaptic EPSPs in extensor motoneurones during fictive
locomotion in the cat. J. Physiol. (Lond.) 563, 597–-610 (2005).
Jessell, T. M. Neuronal specification in the spinal cord: inductive signals andtranscriptional codes. Nature Rev. Genet. 1, 20–-29 (2000).

8.

Saueressig, H., Burrill, J. & Goulding, M. Engrailed-1 and netrin-1 regulate axon
pathfinding by association interneurons that project to motor neurons.
Development 126, 4201–-4212 (1999).
9. Sapir, T. et al. Pax6 and Engrailed-1 regulate two distinct aspects of Renshaw
cell development. J. Neurosci. 24, 1255–-1264 (2004).
10. Li, W. C., Higashijima, S., Parry, D. M., Roberts, A. & Soffe, S. R. Primitive roles
for inhibitory interneurons in developing frog spinal cord. J. Neurosci. 24,
5840–-5848 (2004).
11. Higashijima, S., Masino, M. A., Mandel, G. & Fetcho, J. R. Engrailed-1
expression marks a primitive class of inhibitory spinal interneuron. J. Neurosci.
24, 5827–-5839 (2004).
12. Brockschnieder, D. et al. Cell depletion due to diphtheria toxin fragment A after
Cre-mediated recombination. Mol. Cell. Biol. 24, 7636–-7642 (2004).
13. Ericson, J. et al. Pax6 controls progenitor cell identity and neuronal fate in
response to graded Shh signaling. Cell 90, 169–-180 (1997).
14. Jiang, Z., Carlin, K. P. & Brownstone, R. M. An in vitro functionally maturemouse spinal cord preparation for the study of spinal motor networks. Brain
Res. 816, 493–-499 (1999).
15. Kullander, K. et al. Role of EphA4 and EphrinB3 in local neuronal circuits that
control walking. Science 299, 1889–-1892 (2003).
16. Wenner, P., O’Donovan, M. J. & Matise, M. P. Topographical and physiologicalcharacterization of interneurons that express engrailed-1 in the embryonicchick spinal cord. J. Neurophysiol. 84, 2651–-2657 (2000).
17. Alvarez, F. J. et al. Postnatal phenotype and localization of V1-derived
interneurons. J. Comp. Neurol. 493, 177–-192 (2005).
18. Jankowska, E., Fu, T. C. & Lundberg, A. Reciprocal Ia inhibition during the latereflexes evoked from the flexor reflex afferents after DOPA. Brain Res. 85,
99–-102 (1975).
19. Kjaerulff, O. & Kiehn, O. Distribution of networks generating and coordinating
locomotor activity in the neonatal rat spinal cord in vitro: a lesion study.
J. Neurosci. 16, 5777–-5794 (1996).
20. Kiehn, O., Hounsgaard, J. & Sillar, K. T. in Neurons, Networks, and Motor
Behaviour (eds Stein, P. S. G., Stuart, D., Selverston, A. & Grillner, S.) 47–-59(MIT Press, Cambridge, Massachusetts, 1997).
21. Kjaerulff, O. & Kiehn, O. Crossed rhythmic synaptic input to motoneurons
during selective activation of the contralateral spinal locomotor network.
J. Neurosci. 17, 9433–-9447 (1997).
22. Butt, S. J. & Kiehn, O. Functional identification of interneurons responsible forleft-right coordination of hindlimbs in mammals. Neuron 38, 953–-963 (2003).
23. Danielian, P. S. & McMahon, A. P. Engrailed-1 as a target of the Wnt-1 signalling
pathway in vertebrate midbrain development. Nature 383, 332–-334 (1996).
24. Lechner, H. A., Lein, E. S. & Callaway, E. M. A genetic method for selective andquickly reversible silencing of mammalian neurons. J. Neurosci. 22, 5287–-5290(2002).
25. Borodinsky, L. N. et al. Activity-dependent homeostatic specification oftransmitter expression in embryonic neurons. Nature 429, 523–-530 (2004).
26. Myers, C. P. et al. Cholinergic input is required during embryonic development
to mediate proper assembly of spinal locomotor circuits. Neuron 46, 37–-49(2005).
27. Burke, R. E. in The Synaptic Organization of the Brain (ed. Shepherd, G. M.)
77–-120 (Oxford Univ. Press, Oxford, 1998).
28. St-Onge, L., Sosa-Pineda, B., Chowdhury, K., Mansouri, A. & Gruss, P. Pax6 is
required for differentiation of glucagon-producing a-cells in mouse pancreas.
Nature 387, 406–-409 (1997).
29. Novak, A., Guo, C., Yang, W., Nagy, A. & Lobe, C. G. Z/EG, a double reportermouse line that expresses enhanced green fluorescent protein uponcre-mediated excision. Genesis 28, 147–-155 (2000).
30. Brandon, E. P. et al. Choline transporter 1 maintains cholinergic function incholine acetyltransferase haploinsufficiency. J. Neurosci. 24, 5459–-5466(2004).

Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank J. Rivier for the allatostatin peptide, P. Gruss and
A. Mansouri for Pax6 mutant mice, P. Soriano for Rosa26 lacZ mice, A. McMahon
for Wnt1 En1 mice, S. Narayan for generating ZnG reporter mice, and M. Gross,
A. Joyner, S. Pfaff and P. Slesinger for materials. We thank G. Lemke, S. Pfaff,
P. Gray and K. Quinlan for their comments on the manuscript. This research was
supported by grants from the National Institutes of Health (M.G., O.K.) and the
Human Frontiers Science Program (O.K. and M.G.). G.M.L. was supported by an
HFSP postdoctoral fellowship.
Author Contributions are provided in the Supplementary Information.
Author Information Reprints and permissions information is available at
npg.nature.com/reprintsandpermissions. The authors declare no competing
financial interests. Correspondence and requests for materials should be
addressed to M.G. (goulding@salk.edu).

© 2006 Nature Publishing Group

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