Chick midbrain comprises two major components along the dorsoventral axis, the tectum and the tegmentum. The alar plate differentiates into the optic tectum, while the basal plate gives rise to the tegmentum. It is largely unknown how the differences between these two structures are molecularly controlled during the midbrain development. The secreted protein Sonic hedgehog (Shh) produced in the notochord and floor plate induces differentiation of ventral cell types of the central nervous system. To evaluate the role of Shh in the establishment of dorsoventral polarity in the developing midbrain, we have ectopically expressed Shh unilaterally in the brain vesicles including whole midbrain of E1.5 chick embryos in ovo. Ectopic Shh repressed normal growth of the tectum, producing dorsally enlarged tegmentum region. In addition, the expression of several genes crucial for tectum formation was strongly suppressed in the midbrain and isthmus. Markers for midbrain roof plate were inhibited, indicating that the roof plate was not fully generated. After E5, the tectum territory of Shh-transfected side was significantly reduced and was fused with that of untransfected side. Moreover, ectopic Shh induced a considerable number of SC1-positive motor neurons, overlapping markers such as HNF-3(beta) (floor plate), Isl-1 (postmitotic motor neuron) and Lim1/2. Dopaminergic and serotonergic neurons were also generated in the dorsally extended region. These changes indicate that ectopic Shh changed the fate of the mesencephalic alar plate to that of the basal plate, suppressing the massive cell proliferation that normally occurs in the developing tectum. Taken together our results suggest that Shh signaling restricts the tectum territory by controlling the molecular cascade for tectum formation along dorsoventral axis and by regulating neuronal cell diversity in the ventral midbrain.

REFERENCES

Acampora
D.
,
Mazan
S.
,
Lallemand
Y.
,
Avantaggiato
V.
,
Maury
M.
,
Simeone
A.
,
Brulet
P.
(
1995
)
Forebrain and midbrain regions are deleted in Otx-2 /mutants due to a defective anterior neuroectoderm specification during gastrulation.
Development
121
,
3279
3290
Acampora
D.
,
Avantaggiato
V.
,
Tuorto
F.
,
Simeone
A.
(
1997
)
Genetic control of brain morphogenesis through Otx gene dosage requirement.
Development
124
,
3639
3650
Ang
S. L.
,
Jin
O.
,
Rhinn
M.
,
Daigle
N.
,
Stevenson
L.
,
Rossant
J.
(
1996
)
A targeted mouse Otx-2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to delete of rostral brain.
Development
122
,
243
252
Araki
I.
,
Nakamura
H.
(
1999
)
Engrailed defines the position of dorsal diencephalic boundary by repressing diencephalic fate.
Development
126
,
5127
5135
Brand
M.
,
Heisenberg
C. P.
,
Jiang
Y. J.
,
Beuchle
D.
,
Lun
K.
,
Furutani-Seiki
M.
,
Granato
M.
,
Haffter
P.
,
Hammerschmidt
M.
,
Kane
D. A.
,
Mullins
M. C.
,
Odenthal
J.
,
van Eeden
F. J.
,
Nusslein-Volhard
C.
(
1996
)
Mutations in zebrafish genes affecting the formation of the boundary between midbrain and hindbrain.
Development
123
,
179
190
Broccoli
V.
,
Boncinelli
E.
,
Wurst
W.
(
1999
)
The caudal limit of Otx2 expression positions the isthmic organizer.
Nature
401
,
164
168
Crossely
P. H.
,
Martinez
S.
,
Martin
G.R.
(
1996
)
Midbrain development induced by FGF8 in the chick embryo.
Nature
380
,
66
68
Echelard
Y.
,
Epstein
D. J.
,
St-Jacques
B.
,
Shen
L.
,
Mohler
J.
,
McMahon
J. A.
,
McMahon
A. P.
(
1993
)
Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity.
Cell
75
,
1417
1430
Ekker
S. C.
,
Ungar
A. R.
,
Greenstein
P.
,
von Kessler
D. P.
,
Porter
J. A.
,
Moon
R. T.
,
Beachy
P. A.
(
1995
)
Patterning activities of vertebrate hedgehog proteins in the developing eye and brain.
Curr. Biol
5
,
944
955
Ericson
J.
,
Thor
S.
,
Edlund
T.
,
Jessell
T. M.
,
Yamada
T.
(
1992
)
Early stages of motor neuron differentiation revealed by expression of homeobox gene Islet-1.
Science
256
,
1555
1560
Ericson
J.
,
Muhr
J.
,
Placzek
M.
,
Lints
T.
,
Jessell
T. M.
,
Edlund
T.
(
1995
)
Sonic hedgehog induces the differentiation of ventral forebrain neurons: a common signal for ventral patterning within the neural tube.
Cell
81
,
747
756
Ericson
J.
,
Morton
S.
,
Kawakami
A.
,
Roelink
H.
,
Jessell
T. M.
(
1996
)
Two crucial periods of sonic hedgehog signaling required for the specification of motor neuron identity.
Cell
87
,
661
673
Fan
C. M.
,
Tessier-Lavigne
M.
(
1994
)
Patterning of mammalian somites by surface ectoderm and notochord: evidence for sclerotome induction by a hedgehog homolog.
Cell
79
,
1175
1186
Fan
C. M.
,
Porter
J. A.
,
Chiang
C.
,
Chang
D. T.
,
Beachy
P. A.
,
Tessier-Lavigne
M.
(
1995
)
Long-range sclerotome induction by Sonic hedgehog: direct role of the amino-terminal cleavage product and modulation by the cyclic AMP signaling pathway.
Cell
81
,
457
465
Funahashi
J.-i.
,
Okafuji
T.
,
Ohuchi
H.
,
Noji
S.
,
Tanaka
H.
,
Nakamura
H.
(
1999
)
Role of Pax-5 in the regulation of a mid-hindbrain organizer's activity.
Dev. Growth Differ
41
,
59
72
Goldberg
S.
(
1974
)
Studies on the mechanics of development of visual pathways in the chick embryo.
Dev. Biol
36
,
24
43
Goodrich
L. V.
,
Johnson
R. L.
,
Milenkovic
L.
,
McMahon
J. A.
,
Scott
M. P.
(
1996
)
Conservation of the hedgehog/patched signaling pathway from flies to mice: induction of a mouse patched gene by Hedgehog.
Genes Dev
10
,
301
312
Hamburger
V.
,
Hamilton
H. L.
(
1951
)
A series of normal stages in the development of the chick embryo.
J. Morphol
88
,
49
92
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
Holliday
M.
,
McMahon
J. A.
,
McMahon
A. P.
(
1995
)
Wnt expression patterns in chick embryo nervous system.
Mech. Dev
52
,
9
25
Hynes
M.
,
Porter
J. A.
,
Chiang
C.
,
Chang
D.
,
Tessier-Lavigne
M.
,
Beachy
P. A.
,
Rosenthal
A.
(
1995
)
Induction of midbrain dopaminergic neurons by Sonic hedgehog.
Neuron
15
,
35
44
Itasaki
N.
,
Nakamura
H.
(
1992
)
Rostrocaudal polarity of the tectum in birds: correlation of en gradient and topographic order in retinotectal projection.
Neuron
8
,
787
798
Itasaki
N.
,
Nakamura
H.
(
1996
)
A role for gradient en expression in positional specification on the optic tectum.
Neuron
16
,
55
62
Kawakami
A.
,
Kimura-Kawakami
M.
,
Nomura
T.
,
Fujisawa
H.
(
1997
)
Distributions of PAX6 and PAX7 proteins suggest their involvement in both early and late phases of chick brain development.
Mech. Dev
66
,
119
130
Krauss
S.
,
Maden
M.
,
Holder
N.
,
Wilson
S. W.
(
1992
)
Zebrafish pax[b] is involved in the formation of the midbrain-hindbrain boundary.
Nature
360
,
87
89
Logan
C.
,
Wizenmann
A.
,
Drescher
U.
,
Monschau
B.
,
Bonhoeffer
F.
,
Lumsden
A.
(
1996
)
Rostral optic tectum acquires caudal characteristics following ectopic engrailed expression.
Curr. Biol
6
,
1006
1014
Lumsden
A.
,
Krumlauf
R.
(
1996
)
Patterning thevertebrate neuroaxis.
Science
274
,
1109
1115
Marigo
V.
,
Tabin
C. J.
(
1996
)
Regulation of Patched by Sonic hedgehog in the developing neural tube.
Proc. Natl. Acad. Sci. USA
93
,
9346
9351
Martí
E.
,
Buncrot
D. A.
,
Takada
R.
,
McMahon
A. P.
(
1995
)
Requirement of 19K form of Sonic hedgehog for induction of distinct ventral cell types in CNS explants.
Nature
375
,
322
325
Marusich
M. F.
,
Furneaux
H. M.
,
Henion
P. D.
,
Weston
J. A.
(
1994
)
Hu neuronal proteins are expressed in proliferating neurogenic cells.
J. Neurobiol
25
,
143
155
Matsuo
I.
,
Kuratani
S.
,
Kimura
C.
,
Takeda
N.
,
Aizawa
S.
(
1995
)
Mouse Otx2 functions in the formation and patterning of rostral head.
Genes Dev
9
,
2646
2658
Millet
S.
,
Bloch-Gallego
E.
,
Simeone
A.
,
Alvarado-Mallart
R.-M.
(
1996
)
The caudal limit of Otx2 gene expression as a marker of the midbrain/hindbrain boundary: a study using in situ hybridization and chick/quail homotopic grafts.
Development
122
,
3785
97
Muramatsu
T.
,
Mizutani
Y.
,
Ohmori
Y.
,
Okumura
J.
(
1997
)
Comparison of three nonviral transfection methods for foreign gene expression in early chick embryo in ovo.
Biochem. Biophys. Res. Commun
230
,
376
380
Nomura
T.
,
Kawakami
A.
,
Fujisawa
H.
(
1998
)
Correlation between tectum formation and expression of two PAX family genes, PAX7 and PAX6, in avian brains.
Dev. Growth Differ
40
,
485
495
Okafuji
T.
,
Funahashi
J.-i.
,
Nakamura
H.
(
1999
)
Roles of Pax-2 in initiation of the chick tectal development.
Dev. Brain Res
116
,
41
49
Patel
N. H.
,
Martin-Blanco
E.
,
Coleman
K. G.
,
Poole
S. J.
,
Ellis
M. C.
,
Kornberg
T. B.
,
Goodman
C. S.
(
1989
)
Expression of engrailed proteins in arthropods, annelids, and chordates.
Cell
58
,
955
968
Platt
K. A.
,
Michaud
J.
,
Joyner
A. L.
(
1997
)
Expression of the mouse Gli and Ptc genes is adjacent to embryonic sources of hedgehog signals suggesting a conservation of pathways between flies and mice.
Mech. Dev
62
,
121
135
Porter
J. A.
,
von Kessler
D. P.
,
Ekker
S. C.
,
Young
K. E.
,
Lee
J. J.
,
Moses
K.
,
Beachy
P. A.
(
1995
)
The product of hedgehog autoproteolytic cleavage active in local and long-range signaling.
Nature
374
,
363
366
Riddle
R. D.
,
Johnson
R. L.
,
Laufer
E.
,
Tabin
C.
(
1993
)
Sonic hedgehog mediates the polarizing activity of the ZPA.
Cell
75
,
1401
1416
Roelink
H.
,
Augsburger
A.
,
Heemskerk
J.
,
Korzh
V.
,
Norlin
S.
,
Ruiz i Altaba
A.
,
Tanabe
Y.
,
Placzek
M.
,
Edlund
T.
,
Jessell
T. M.
,
Dodd
J.
(
1994
)
Floor plate and motor neuron induction by vhh-1, a vertebrate homolog of hedgehog expressed by the notochord.
Cell
76
,
761
775
Roelink
H.
,
Porter
J. A.
,
Chiang
C.
,
Tanabe
Y.
,
Chang
D. T.
,
Beachy
P. A.
,
Jessell
T. M.
(
1995
)
Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of Sonic hedgehog autoproteolysis.
Cell,
81
,
445
455
Ruiz i Altaba
A.
,
Placzek
M.
,
Baldassare
M.
,
Dodd
J.
,
Jessell
T. M.
(
1995
)
Early stages of notochord and floor plate development in the chick embryo defined by normal and induced expression of HNF-3.
Dev. Biol
170
,
299
313
Senut
M. C.
,
Alvarado-Mallart
R.-M.
(
1987
)
Cytodifferentiation of quail tectum primodium transplanted homotopically into the chick embryo.
Dev. Brain. Res
32
,
187
205
Shamim
H.
,
Mahmood
R.
,
Logan
C.
,
Doherty
P.
,
Lumsden
A.
,
Mason
I.
(
1999
)
Sequential roles for Fgf4, En1 and Fgf8 in specification and regionalisation of the midbrain.
Development
126
,
945
959
Shigetani
Y.
,
Funahashi
J.-i.
,
Nakamura
H.
(
1997
)
En-2 regulates the expression of the ligands for Eph type tyrosine kinases in chick embryonic tectum.
Neurosci. Res
27
,
211
217
Streit
P.
,
Reubi
J. C.
(
1977
)
A new and sensitive staining method for axonally transported horseradish peroxidase (HRP) in the pigeon visual system.
Brain Res
126
,
530
537
Suemori
H.
,
Kadokawa
Y.
,
Goto
K.
,
Araki
I.
,
Kondoh
H.
,
Nakatsuji
N.
(
1990
)
A mouse embryonic stem cell line showing pluripotency of differentiation in early embryos and ubiquitous beta-galactosidase expression.
Cell Differ. Dev
29
,
181
186
Sugiyama
S.
,
Funahashi
J.
,
Kitajewski
J.
,
Nakamura
H.
(
1998
)
Crossregulation between En-2 and Wnt-1 in chick tectal development.
Dev. Growth Differ
40
,
157
166
Suzuki
H. R.
,
Padanilam
B. J.
,
Vitale
E.
,
Ramirez
F.
,
Solursh
A.
(
1991
)
Repeating developmental expression of G-Hox7, a novel homeobox-containing gene in the chicken.
Dev. Biol
148
,
375
388
Tanabe
Y.
,
Jessell
T. M.
(
1996
)
Diversity and pattern in the developing spinal cord.
Science
274
,
1115
1123
Tanabe
Y.
,
William
C.
,
Jessell
T. M.
(
1998
)
Specification of motor neuron identity by MNR2 homeodomain protein.
Cell
95
,
67
80
Tanaka
H.
,
Obata
K.
(
1984
)
Developmental changes in unique cell surface antigens of chick embryo spinal motor neurons and ganglion cells.
Dev. Biol
106
,
26
37
Urbánek
P.
,
Wang
Z. Q.
,
Fetka
I.
,
Wagner
E. F.
,
Busslinger
M.
(
1994
)
Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5/BSAP.
Cell
79
,
901
912
Wakamatsu
Y.
,
Watanabe
Y.
,
Nakamura
H.
,
Kondoh
H.
(
1997
)
Regulation of the neural crest cell fate by N-myc: promotion of ventral migration and neuronal differentiation.
Development
124
,
1953
1962
Wakamatsu
Y.
,
Weston
J. A.
(
1997
)
Sequential expression and role of Hu RNA-binding proteins during neurogenesis.
Development
124
,
3449
3460
Wang
M. Z.
,
Jin
P.
,
Bumcrot
D. A.
,
Marigo
V.
,
McMahon
A. P.
,
Wang
E. A.
,
Woolf
T.
,
Pang
K.
(
1995
)
Induction of dopaminergic neuron phenotype in the midbrain by Sonic hedgehog protein.
Nat. Med
1
,
1184
1188
Wurst
W.
,
Auerbach
A.
,
Joyner
A.
(
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
Ye
W.
,
Shimamura
K.
,
Rubenstein
J. L. R.
,
Hynes
M.
,
Rosenthal
A.
(
1998
)
FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate.
Cell
93
,
755
766
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