Pax-6 is a member of the vertebrate Pax gene family, which is structurally related to the Drosophila pair-rule gene, paired. In mammals, Pax-6 is expressed in several discrete domains of the developing CNS and has been implicated in neural development, although its precise role remains elusive. We found a novel Small eye rat strain (rSey2) with phenotypes similar to mouse and rat Small eye. Analyses of the Pax-6 gene revealed one base (C) insertion in an exon encoding the region downstream of the paired box of the Pax-6 gene, resulting in generation of truncated protein due to the frame shift. To explore the roles of Pax-6 in neural development, we searched for abnormalities in the nervous system in rSey2 homozygous embryos. rSey2/rSey2 exhibited abnormal development of motor neurons in the hindbrain. The Islet-1-positive motor neurons were generated just ventral to the Pax-6-expressing domain both in the wild-type and mutant embryos. However, two somatic motor (SM) nerves, the abducent and hypoglossal nerves, were missing in homozygous embryos. By retrograde and anterograde labeling, we found no SM-type axonogenesis (ventrally growing) in the mutant postotic hindbrain, though branchiomotor and visceral motor (BM/VM)-type axons (dorsally growing) were observed within the neural tube. To discover whether the identity of these motor neuron subtypes was changed in the mutant, we examined expression of LIM homeobox genes, Islet-1, Islet-2 and Lim-3. At the postotic levels of the hindbrain, SM neurons expressed all the three LIM genes, whereas BM/VM-type neurons were marked by Islet-1 only. In the Pax-6 mutant hindbrain, Islet-2 expression was specifically missing, which resulted in the loss of the cells harboring the postotic hindbrain SM-type LIM code (Islet-1 + Islet-2 + Lim-3). Furthermore, we found that expression of Wnt-7b, which overlapped with Pax-6 in the ventrolateral domain of the neural tube, was also specifically missing in the mutant hindbrain, while it remained intact in the dorsal non-overlapping domain. These results strongly suggest that Pax-6 is involved in the specification of subtypes of hindbrain motor neurons, presumably through the regulation of Islet-2 and Wnt-7b expression.

REFERENCES

Appel
B.
,
Korzh
V.
,
Glasgow
E.
,
Thor
S.
,
Edlund
T.
,
Dawid
I. B.
,
Eisen
J. S.
(
1995
)
Motoneuron fate specification revealed by patterned LIM homeobox gene expression in embryonic zebrafish.
Development
121
,
4117
4125
Bourgouin
C.
,
Lundgren
S. E.
,
Thomas
J. B.
(
1992
)
Apterous is a Drosophila LIM domain gene required for the development of a subset of embryonic muscles.
Neuron
9
,
549
561
Cohen
B.
,
McGuffin
M. E.
,
Pfeifle
C.
,
Segal
D.
,
Cohen
S. M.
(
1992
)
apterous, a gene required for imaginal disc development in Drosophila encodes a member of the LIM family of developmental regulatory proteins.
Genes Dev
6
,
715
729
Colamarino
S. A.
,
Tessier-Lavigne
M.
(
1995
)
The axonal chemoattractant netrin-1 is also a chemorepellent for trochlear motor axons.
Cell
81
,
621
629
Davis
J. A.
,
Reed
R. R.
(
1996
)
Role of Olf-1 and Pax-6 transcription factors in neurodevelopment.
J. Neurosci
16
,
5082
5094
Ericson
J.
,
Thor
S.
,
Jessell
T. M.
,
Edlund
T.
,
Yamada
T.
(
1992
)
Early stages of motor neuron differentiation revealed by expression of homeobox gene Islet-1.
Science
256
,
1555
1560
Freyd
G.
,
Kim
S. K.
,
Horvitz
H. R.
(
1990
)
Novel cysteine-rich motif and homeodomain in the product of the Caenorhabditis elegans cell lineage gene lin-11.
Nature
344
,
876
879
Fritzsch
B.
,
Northcutt
R. G.
(
1993
)
Cranial and spinal nerve organization in amphioxus and lampreys: evidence for an ancestral cranial pattern.
Act. Anat
148
,
96
109
Fujiwara
M.
,
Uchida
T.
,
Osumi-Yamashita
N.
,
Eto
K.
(
1994
)
Uchida rat (rSey): a new mutant rat with craniofacial abnormalities resembling those of the mouse Sey mutant.
Differentiation
57
,
31
38
Gavin
B. J.
,
McMahon
J. A.
,
McMahon
A. P.
(
1990
)
Expression of multiple novel Wnt-1/int-1 -related genes during fetal and adult mouse development.
Genes Dev
4
,
2319
2332
Goulding
M. D.
,
Chalepakis
G.
,
Deutsch
U.
,
Erselius
J. R.
,
Gruss
P.
(
1991
)
Pax-3, a novel murine DNA binding protein expressed during early neurogenesis.
EMBO J
10
,
1135
1147
Goulding
M. D.
,
Lumsden
A.
,
Gruss
P.
(
1993
)
Signals from the notochord and floor plate regulate the region-specific expression of two Pax genes in the developing spinal cord.
Development
117
,
1001
1016
Guthrie
S.
,
Pini
A.
(
1995
)
Chemorepulsion of developing motor axons by the floor plate.
Neuron
14
,
1117
1130
Hanson
I.
,
Van Heyningen
V.
(
1995
)
Pax-6: more than meets the eye.
Trends Genet
11
,
268
272
Hill
R. E.
,
Favor
J.
,
Hogan
B. L. M.
,
Ton
C. C. T.
,
Saunders
G. F.
,
Hanson
I. M.
,
Prosser
J.
,
Jordan
T.
,
Hastie
N. D.
,
Heyningen
V. V.
(
1991
)
Mouse Small eye results from mutation in a paired-like homeobox-containing gene.
Nature
354
,
522
–.
Hollyday
M.
,
McMahon
J. A.
,
McMahon
A. P.
(
1995
)
Wnt expression patterns in chick embryo nervous system.
Mech. Dev
52
,
9
25
Kennedy
T. E.
,
Serafini
T.
,
de la Torre
J. R.
,
Tessier-Lavigne
M.
(
1994
)
Netrins are diffusible chemotropic factors for commissural axons in the embryonic spinal cord.
Cell
78
,
425
435
Krauss
S.
,
Maden
M.
,
Holder
N.
,
Wilson
S.W.
(
1991
)
Zebrafish pax[b] is involved in the formation of the midbrain-hindbrain boundary.
Nature
360
,
87
89
Lee
Y. M.
,
Osumi-Yamashita
N.
,
Ninomiya
Y.
,
Moon
C. K.
,
Eriksson
U.
,
Eto
K.
(
1995
)
Retinoic acid stage-dependently alters the migrationpattern and identity of hindbrain neural crest cells.
Development
121
,
825
837
Lumsden
A.
(
1995
)
A ‘LIM code’ for motor neurons?.
Curr. Biol
5
,
491
495
Lumsden
A.
,
Krumlauf
R.
(
1996
)
Patterning the vertebrate neuraxis.
Science
274
,
1109
1115
Lundgren
S. E.
,
Callahan
C. A.
,
Thor
S.
,
Thomas
J. B.
(
1995
)
Control of neuronal pathway selection by the Drosophila LIM homeodomain gene apterous.
Development
121
,
1769
1773
Macdonald
R.
,
Barth
A.
,
Xu
Q.
,
Holder
N.
,
Mikkola
I.
,
Wilson
S. W.
(
1995
)
Midline signalling is required for Pax gene regulation and patterning of the eyes.
Development
121
,
3267
3278
Mansouri
A.S.
,
Hallonet
M.
,
Gruss
P.
(
1996
)
Pax genes and their roles in cell differentiation and development.
Curr. Biol
8
,
851
857
Matsuo
T.
,
Osumi-Yamashita
N.
,
Noji
S.
,
Ohuchi
H.
,
Koyama
E.
,
Myokai
F.
,
Matsuo
N.
,
Taniguchi
S.
,
Doi
H.
,
Iseki
S.
,
Ninomiya
Y.
,
Fujiwara
M.
,
Watanabe
T.
,
Eto
K.
(
1993
)
A mutation in the Pax-6 gene in rat small eye is associated with impaired migration of midbrain crest cells.
Nature Genetics
3
,
299
304
McMahon
A. P.
(
1993
)
Cell signalling in induction and anterior-posterior patterning of the vertebrate central nervous system.
Curr. Opin. Neurobiol
3
,
4
7
Nakagawa
Y.
,
Kaneko
T.
,
Ogura
T.
,
Suzuki
T.
,
Torii
M.
,
Kaibuchi
K.
,
Arai
K.-I.
,
Nakamura
S.
,
Nakafuku
M.
(
1996
)
Roles of cell-autonomous mechanisms for differential expression of region-specific transcription factors in neuroepithelial cells.
Development
122
,
2449
2464
Nusse
R.
,
Varmus
H. E.
(
1992
)
Wnt genes.
Cell
69
,
1073
1083
Osumi-Yamashita
N.
,
Kuratani
S.
,
Ninomiya
Y.
,
Aoki
K.
,
Iseki
S.
,
Chareonvit
S.
,
Doi
H.
,
Fujiwara
M.
,
Watanabe
T.
,
Eto
K.
(
1997
)
Cranial anomaly of homozygous rSey rat is associated with a defect in the migration pathway of midbrain crest cells.
Devel. Growth Differ
39
,
53
67
Parr
B. A.
,
McMahon
A. P.
(
1993
)
Dorsalizing signal Wnt-7a required for normal polarity of D-V and A-P axes of mouse limb.
Nature
374
,
350
353
Parr
B. A.
,
Shea
M. J.
,
Vassileva
G.
,
McMahon
A. P.
(
1993
)
Mouse Wnt genes exhibit discrete domains of expression in the early embryonic CNS and limb buds.
Development
119
,
247
261
Perrimon
N.
(
1994
)
The genetic basis of patterned baldness in Drosophila.
Cell
76
,
781
784
Riddle
R. D.
,
Ensini
M.
,
Nelson
C.
,
Tsuchida
T.
,
Jessell
T. M.
,
Tabin
C.
(
1995
)
Induction of the LIM homeobox gene Lmx1 by WNT7a establishes dorsoventral pattern in the vertebrate limb.
Cell
83
,
631
640
Rowitch
D. H.
,
McMahon
A. P.
(
1995
)
Pax-2 expression in the murine neural plate precedes and encompasses the expression domains of Wnt-1 and En-1.
Mech Devel
52
,
3
8
Rubenstein
J. L.
,
Martinez
S.
,
Shimamura
K.
,
Puelles
L.
(
1994
)
The embryonic vertebrate forebrain: the prosomeric model.
Science
266
,
578
580
Schmahl
W.
,
Knoedlseder
M.
,
Favor
J.
,
Davidson
D.
(
1993
)
Defects of neuronal migration and the pathogenesis of cortical malformations are associated with small eye (sey) in the mouse, a point mutation at the Pax-6 locus.
Acta Neuropathol
86
,
126
135
Smith
J. C.
(
1993
)
Dorso-ventral patterning in the neural tube.
Curr. Biol
3
,
582
585
Stoykova
A.
,
Gruss
P.
(
1994
)
Roles of Pax-genes in developing and adult brain as suggested by expression patterns.
J. Neurosci
14
,
1395
1412
Stoykova
A.
,
Walther
C.
,
Gruss
P.
(
1996
)
Forebrain patterning defects in Small eye mutant mice.
Development
122
,
3453
3465
Tamada
A.
,
Shirasaki
R.
,
Murakami
F.
(
1995
)
Floor plate chemoattracts crossed axons and chemorepels uncrossed axons in the vertebrate brain.
Neuron
14
,
1083
1093
Tanabe
Y.
,
Jessell
T. M.
(
1996
)
Diversity and pattern in the developing spinal cord.
Science
274
,
1115
1123
Tessier-Lavigne
M.
(
1994
)
Axon guidance by diffusible repellants and attractants.
Curr. Opin. Genet. Dev
4
,
596
601
Tessier-Lavigne
M.
,
Goodman
C. S.
(
1996
)
The molecular biology of axon guidance.
Science
274
,
1123
1133
Tokumoto
M.
,
Gong
Z.
,
Tsubokawa
T.
,
Hew
C. L.
,
Uyemura
K.
,
Hotta
Y.H.
,
Okamoto
H.
(
1995
)
Molecular heterogeneity among primary motoneurons and within myotomes revealed by the differential mRNA expression of novel islet-1 homologs in embryonic zebrafish.
Dev. Biol
171
,
578
589
Tremblay
P.
,
Pitello
F.
,
Gruss
P.
(
1996
)
In hibition of floor plate differentiation by Pax −3: evidence from ectopic expression in transgenic mice.
Development
122
,
2555
2567
Tsuchida
T.
,
Ensini
M.
,
Morton
S. B.
,
Baldassare
M.
,
Edlund
T.
,
Jessell
T. M.
,
Pfaff
S. L.
(
1994
)
Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes.
Cell
79
,
957
970
Tucker
A.
,
Lumsden
A.
,
Guthrie
S.
(
1996
)
Cranial motor axons respond differently to the floor plate and sensory ganglia in colagen gel co-cultures.
Eur. J. Neurosci
8
,
906
916
Varela-Echavarria
A.
,
Pfaff
S. L.
,
Guthrie
S.
(
1997
)
Differential expression of LIM homeobox genes among motor neuron subpopulations in the developing chick brain stem.
Mol. Cell Neurosci
8
,
242
257
Varela-Echavarria
A.
,
Tucker
A.
,
Puschel
A. W.
,
Guthrie
S.
(
1997
)
Motor axon subpopulations respond differentially to the chemorepellents netrin-1 and semaphorin D.
Neuron
18
,
193
207
Vogel
A.
,
Rodriguez
C.
,
Warnken
W.
,
Izpisua-Belmonte
J. C.
(
1995
)
Dorsal cell fate specified by chick Lmx1 during vertebrate limb development.
Nature
378
,
716
720
Way
J. C.
,
Chalfie
M.
(
1988
)
mec-3, a homeobox-containing gene that specifies differentiation of the touch receptor neurons in C. elegans.
Cell
54
,
5
16
Wilson
S. W.
,
Placzek
M.
,
Furley
A.
(
1993
)
Border disputes: Do boundaries play a role in growth cone guidance?.
Trends Neurosci
16
,
316
322
Yamada
T.
,
Pfaff
S. L.
,
Edudlund
T.
,
Jessell
T. M.
(
1993
)
Control of cell pattern in the neural tube: motor neuron induction by diffusible fuctors from notochord and floor plate.
Cell
73
,
673
686
This content is only available via PDF.