During early development, multiple classes of interneurons are generated in the spinal cord including association interneurons that synapse with motor neurons and regulate their activity. Very little is known about the molecular mechanisms that generate these interneuron cell types, nor is it known how axons from association interneurons are guided toward somatic motor neurons. By targeting the axonal reporter gene τ-lacZ to the En1 locus, we show the cell-type-specific transcription factor Engrailed-1 (EN1) defines a population of association neurons that project locally to somatic motor neurons. These EN1 interneurons are born early and their axons pioneer an ipsilateral longitudinal projection in the ventral spinal cord. The EN1 interneurons extend axons in a stereotypic manner, first ventrally, then rostrally for one to two segments where their axons terminate close to motor neurons. We show that the growth of EN1 axons along a ventrolateral pathway toward motor neurons is dependent on netrin-1 signaling. In addition, we demonstrate that En1 regulates pathfinding and fasciculation during the second phase of EN1 axon growth in the ventrolateral funiculus (VLF); however, En1 is not required for the early specification of ventral interneuron cell types in the embryonic spinal cord.

Reference

Reference
Bastiani
M.
,
Goodman
C. S.
(
1986
)
Guidance of neuronal growth cones in the grasshopper embryo. III. Recognition of specific glial pathways.
J. Neurosci
6
,
3542
3551
Brose
K.
,
Bland
K. S.
,
Wang
K. H.
,
Arnott
D.
,
Henzel
W.
,
Goodman
C. S.
,
Tessier-Lavigne
M.
,
Kidd
T.
(
1999
)
Slit proteins bind robo receptors and have an evolutionarily conserved role in repulsive axon guidance.
Cell
96
,
795
806
Briscoe
J.
,
Sussel
L.
,
Serup
P.
,
Hartigan-O'Conner
D.
,
Jessell
T. M.
,
Rubenstein
J. L. R.
,
Ericson
J.
(
1999
).
Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling.
Nature
398
,
622
627
Callahan
C. A.
,
Thomas
J. B.
(
1994
)
Tau--galactosidase, an axon targeted fusion protein.
Proc. Natl. Acad. Sci. USA
91
,
5972
5976
Caudy
M.
,
Bentley
D.
(
1986
)
Pioneer growth cone steering along a series of neuronal and non-neuronal cues of different affinities.
J. Neurosci
6
,
1781
1795
Condron
B. G.
,
Patel
N. H.
,
Zinn
K.
(
1994
)
engrailed controls glial/neuronal cell fate decisions at the midline of the central nervous system.
Neuron
13
,
541
554
Daston
M. M.
,
Bastmeyer
M.
,
Rutishauser
U.
,
O'Leary
D. D. M.
(
1996
)
Spatially restricted increase in polysialic acid enhances corticospinal axon branching related to target recognition and innervation.
J. Neurosci
16
,
5488
5497
Davis
C. A.
,
Holmyard
D. P.
,
Millen
K. J.
,
Joyner
A. L.
(
1991
)
Examining pattern formation in mouse, chicken and frog embryos with an En-specific antiserum.
Development
111
,
287
298
Easter
S. S.
,
Ross
L. S.
,
Frankfurter
A.
(
1993
)
Initial tract formation in the mouse brain.
J. Neurosci
13
,
285
99
Eccles
J. C.
,
Fatt
P.
,
Koketsu
K.
(
1954
)
Cholinergic and inhibitory synapses in a path from motor-axon collaterals to motorneurones.
J. Physiol
126
,
524
562
Ericson
J.
,
Rashbass
P.
,
Schedl
A.
,
Brenner-Morton
S.
,
Kawakami
A.
,
Van Heyningen
V.
,
Jessell
T. M.
,
Briscoe
J.
(
1997
)
Pax6 controls progenitor cell identity and neuronal fate in response to graded Shh signaling.
Cell
90
,
169
180
Fambrough
D.
,
Goodman
C. S.
(
1996
)
The Drosophila beaten path gene encodes a novel secreted protein that regulates defasciculation at motor axon choice points.
Cell
87
,
1049
1058
Fazeli
A.
,
Dickinson
S. L.
,
Hermiston
M. L.
,
Tighe
R. V.
,
Steen
R. G.
,
Small
C. G.
,
Stoeckli
E. T.
,
Keino-Masu
K.
,
Masu
M.
,
Rayburn
H.
,
Simons
J.
,
Bronson
R. T.
,
Gordon
J. I.
,
Tessier-Lavigne
M.
,
Weinberg
R. A.
(
1997
)
Phenotype of mice lacking functional Deleted in Colorectal Cancer (DCC) gene.
Nature
386
,
796
804
Gutman
C. R.
,
Ajmera
M. K.
,
Hollyday
M.
(
1993
)
Organization of motor pools supplying axial muscles in the chicken.
Brain Research
609
,
129
136
Hanks
M.
,
Wurst
W.
,
Anson-Cartwright
L.
,
Auerbach
A. B.
,
Joyner
A. L.
(
1995
)
Rescue of the En-1 mutant phenotype by replacement of En-1 with En-2.
Science
269
,
679
682
Holley
J. A.
,
Silver
J.
(
1987
)
Growth pattern of pioneering chick spinal cord axons.
Dev. Biol
123
,
375
388
Jankowska
E.
,
Lindstrom
S.
(
1972
)
Morphology of interneurons mediating Ia reciprocal inhibition of motor neurons in the spinal cord of the cat.
J. Physiol
226
,
805
823
Jankowska
E.
,
Roberts
W. J.
(
1972
)
An electrophysiological demonstration of the axonal projectories of single spinal interneurons in the cat.
J. Physiol
226
,
805
823
Kennedy
T. E.
,
Serafini
T.
,
de la Torre
J. R.
,
Tessier-Lavigne
M.
(
1994
)
Netrins are diffusable chemotrophic factors for commissural axons in the embryonic spinal cord.
Cell
78
,
425
435
Kidd
T.
,
Brose
K.
,
Mitchell
K. J.
,
Fetter
R. D.
,
Tessier-Lavigne
M.
,
Goodman
C. S.
,
Tear
G.
(
1998
)
Roundabout controls axon crossing of the CNS midline and defines a novel subfamily of evolutionarily conserved guidance receptors.
Cell
92
,
205
215
Kolodzeij
P. A.
,
Timpe
L. C.
,
Mitchell
K. J.
,
Fried
S. R.
,
Goodman
C. S.
,
Jan
L. Y.
,
Jan
Y. N.
(
1996
)
frazzled encodes a Drosphila member of the DCC immunoglobulin subfamily and is required for CNS and motor axon guidance.
Cell
87
,
197
204
Kuwada
J. Y.
(
1986
)
Cell recognition by neuronal growth cones in a simple vertebrate embryo.
Science
233
,
740
746
Landmesser
L.
(
1978
)
The distribution of motoneurones supplying the chick hindlimb muscles.
J. Physiol
284
,
371
389
Lee
K. J.
,
Mendelsohn
M.
,
Jessell
T. M.
(
1998
)
Neuronal patterning by BMPs: a requirement for GDF7 in the generation of a discrete class of commissural interneurons in the mouse spinal cord.
Genes Dev
12
,
3394
3407
Lin
J. H.
,
Saito
T.
,
Anderson
D. J.
,
Lance-Jones
C.
,
Jessell
T. M.
,
Arber
S.
(
1998
)
Functionally related motor neuron pool and muscle sensory afferent subtypes defined by coordinate ETS gene expression.
Cell
95
,
393
407
Loomis
C. A.
,
Harris
E.
,
Michaud
J.
,
Wurst
W.
,
Hanks
M.
,
Joyner
A. L.
(
1996
)
The mouse Engrailed-1 gene and ventral limb patterning.
Nature
382
,
360
363
Lundell
M. J.
,
Chu-LaGraff
Q.
,
Doe
C. Q.
,
Hirsh
J.
(
1996
)
The engrailed and huckebein genes are essential for development of serotonin neurons in the Drosophila CNS.
Mol. Cell. Neuro
7
,
46
61
McIntire
S. L.
,
Garriga
G.
,
White
J.
,
Jacobson
D.
,
Horvitz
H. R.
(
1992
)
Genes necessary for directed axonal elongation or fasciculation in C. elegans.
Neuron
8
,
307
322
Matise
M. P.
,
Joyner
A. L.
(
1997
)
Expression patterns of developmental control genes in normal and Engrailed-1 mutant mouse spinal cord reveal early diversity in developing interneurons.
J. Neurosci
17
,
7805
7816
Mitchell
K. J.
,
Doyle
J. L.
,
Serafini
T.
,
Kennedy
T. E.
,
Tessier-Lavigne
M.
,
Goodman
C. S.
,
Dickson
B. J.
(
1996
)
Genetic analysis of Netrin genes in Drosophila: Netrins guide CNS commissural axons and peripheral motor axons.
Neuron
17
,
203
215
Pfaff
S. L.
,
Mendelsohn
M.
,
Stewart
C. L.
,
Edlund
T.
,
Jessell
T. M.
(
1996
)
Requirement for LIM homeobox gene Isl1 in motor neuron differentiation reveals a motor-neuron dependent step in interneuron differentiation.
Cell
84
,
309
320
Renshaw
B.
(
1946
)
Central effects of centripetal impulses in axons of spinal ventral roots.
J. Neurophysiol
9
,
191
204
Serafini
T.
,
Kennedy
T. E.
,
Galko
M. J.
,
Mirzayan
C.
,
Jessell
T. M.
,
Tessier-Lavigne
M.
(
1994
).
The netrins define a family of axon outgrowth-promoting proteins homologous to C.elegans UNC-6.
Cell
78
,
409
424
Serafini
T.
,
Colamarino
S. A.
,
Leonardo
E. D.
,
Wang
H.
,
Beddington
R.
,
Skarnes
W. C.
,
Tessier-Lavigne
M.
(
1996
)
Netrin-1 is required for commissural axon guidance in the developing vertebrate nervous system.
Cell
87
,
1001
1014
Sharma
K.
,
Sheng
H. Z.
,
Lettieri
K.
,
Li
H.
,
Karavanov
A.
,
Potter
S.
,
Westphal
H.
,
Pfaff
S. L.
(
1998
)
LIM homeodomain factors Lhx3 and Lhx4 assign subtype identies for motor neurons.
Cell
95
,
817
828
Shirasaki
R.
,
Tamada
A.
,
Katsumata
R.
,
Murakami
F.
(
1995
)
Guidance of cerebellofugal axons in the rat embryo: directed growth toward the floor plate and subsequent elongation along the longitudinal axis.
Neuron
14
,
961
972
Skarnes
W. C.
,
Moss
J. E.
,
Hurtley
S. M.
,
Beddington
R. S. P.
(
1995
)
Capturing genes encoding membrane and secreted proteins important for mouse development.
Proc. Natl. Acad. Sci. USA
92
,
6592
6596
Smith
C. L.
,
Hollyday
M.
(
1983
)
The development and postnatal organization of motor nuclei in the rat thoracic spinal cord.
J Comp. Neur
220
,
16
28
Stoekli
E. T.
,
Landmesser
L. T.
(
1995
)
Axonin-1, Nr-CAM and Ng-CAM play different roles in the in vivo guidance of chick commissural neurons.
Neuron
14
,
1165
1179
Tanabe
Y.
,
Jessell
T. M.
(
1996
)
Diversity and pattern in the developing spinal cord.
Science
274
,
1115
1123
Tanabe
Y.
,
Henderson
C.
,
Jessell
T. M.
(
1998
)
Specification of motor neuron identity by the MNR2 homeodomain protein.
Cell
95
,
67
80
Tuttle
R.
,
Braisted
J. E.
,
Richards
L. J.
,
O'Leary
D. D. M.
(
1998
)
Retinal axon guidance by region-specific cues in the diencephalon.
Development
125
,
791
801
Vaughn
J. E.
,
Grieshaber
J. A.
(
1973
)
A morphological investigation of an early reflex pathway in developing rat spinal cord.
J. Comp. Neur
148
,
177
210
Vaughn
J. E.
,
Sims
T.
,
Nakishima
M.
(
1977
)
A comparison of the early development of axodendritic and axosomatic synapses upon embryonic mouse spinal motor neurons.
J. Comp. Neurol
175
,
79
100
Wightman
B.
,
Clark
S. G.
,
Taskar
A. M.
,
Forrester
W. C.
,
Maricq
A. V.
,
Bargmann
C. I.
,
Garriga
G.
(
1996
)
The C. elegans gene vab-8 guides posteriorly directed axon outgrowth and cell migration.
Development
122
,
671
682
Wurst
W.
,
Auerbach
A. B.
,
Joyner
A. L.
(
1994
)
Multiple developmental defects in Engrailed-1 mutant mice: an early mid-hindbrain deletion and patterning defects in forelimbs and sternum.
Development
120
,
2065
2075
Yaganuma
H.
,
Shiga
T.
,
Homma
S.
,
Ishihara
R.
,
Oppenheim
R. W.
(
1990
)
Identification of early developing axon projections from spinal interneurons in the chick embryo with a neuron specific-tubulin antibody.
Development
108
,
705
716
Zallen
J. A.
,
Yi
B. A.
,
Bargmann
C. I.
(
1998
)
The conserved immunoglobulin superfamily member SAX3/Robo directs multiple aspects of axon guidance in C. elegans.
Cell
92
,
217
227
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