The secreted protein sonic hedgehog (Shh) is required to establish patterns of cellular growth and differentiation within ventral regions of the developing CNS. The expression of Shh in the two tissue sources responsible for this activity, the axial mesoderm and the ventral midline of the neural tube, is regulated along the anteroposterior neuraxis. Separate cis-acting regulatory sequences have been identified which direct Shh expression to distinct regions of the neural tube, supporting the view that multiple genes are involved in activating Shh transcription along the length of the CNS. We show here that the activity of one Shh enhancer, which directs reporter expression to portions of the ventral midbrain and diencephalon, overlaps both temporally and spatially with the expression of Sim2. Sim2 encodes a basic helix-loop-helix (bHLH-PAS) PAS domain containing transcriptional regulator whose Drosophila homolog, single-minded, is a master regulator of ventral midline development. Both vertebrate and invertebrate Sim family members were found sufficient for the activation of the Shh reporter as well as endogenous Shh mRNA. Although Shh expression is maintained in Sim2(−)(/)(−) embryos, it was determined to be absent from the rostral midbrain and caudal diencephalon of embryos carrying a dominant-negative transgene that disrupts the function of bHLH-PAS proteins. Together, these results suggest that bHLH-PAS family members are required for the regulation of Shh transcription within aspects of the ventral midbrain and diencephalon.

REFERENCES

Anderson
M. G.
,
Perkins
G. L.
,
Chittick
P.
,
Shrigley
R. J.
,
Johnson
W. A.
(
1995
)
drifter, a Drosophila POU-domain transcription factor, is required for correct differentiation and migration of tracheal cells and midline glia.
Genes Dev
9
,
123
137
Arendt
D.
,
Nubler-Jung
K.
(
1999
)
Comparison of early nerve cord development in insects and vertebrates.
Development
126
,
2309
2325
Aza-Blanc
P.
,
Kornberg
T. B.
(
1999
)
Ci: a complex transducer of the hedgehog signal.
Trends Genet
15
,
458
462
Brunskill
E. W.
,
Witte
D. P.
,
Shreiner
A. B.
,
Potter
S. S.
(
1999
)
Characterization of npas3, a novel basic helix-loop-helix PAS gene expressed in the developing mouse nervous system.
Mech Dev
88
,
237
241
Chiang
C.
,
Litingtung
Y.
,
Lee
E.
,
Young
K. E.
,
Corden
J. L.
,
Westphal
H.
,
Beachy
P. A.
(
1996
)
Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function.
Nature
383
,
407
413
Crews
S. T.
(
1998
)
Control of cell lineage-specific development and transcription by bHLH-PAS proteins.
Genes Dev
12
,
607
620
Crews
S. T.
,
Fan
C.-M.
(
1999
)
Remembrance of things PAS: regulation of development by bHLH-PAS proteins.
Curr. Opin. Genet. Dev
9
,
580
587
Dahmane
N.
,
Charron
G.
,
Lopes
C.
,
Yaspo
M. L.
,
Maunoury
C.
,
Decorte
L.
,
Sinet
P. M.
,
Bloch
B.
,
Delabar
J. M.
(
1995
)
Down syndrome-critical region contains a gene homologous to Drosophila sim expressed during rat and human central nervous system development.
Proc. Natl. Acad. Sci. USA
92
,
9191
9195
Dale
J. K.
,
Vesque
C.
,
Lints
T. J.
,
Sampath
T. K.
,
Furley
A.
,
Dodd
J.
,
Placzek
M.
(
1997
)
Cooperation of BMP7 and SHH in the induction of forebrain ventral midline cells by prechordal plate.
Cell
90
,
257
269
Ding
Q.
,
Motoyama
J.
,
Gasca
S.
,
Mo
R.
,
Sasaki
H.
,
Rossant
J.
,
Hui
C.-C.
(
1998
)
Diminished Sonic Hedgehog signaling and lack of floor plate differentiation in Gli2 mutant mice.
Development
125
,
2533
2543
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 and limb polarity.
Cell
75
,
1417
1430
Echelard
Y.
,
Vassileva
G.
,
McMahon
A. P.
(
1994
)
Cis -acting regulatory sequences governing Wnt-1 expression in the developing mouse CNS.
Development
120
,
2213
2224
Ema
M.
,
Morita
M.
,
Ikawa
S.
,
Tanaka
M.
,
Matsuda
Y.
,
Gotoh
O.
,
Saijoh
Y.
,
Fujii
H.
,
Hamada
H.
,
Kikuchi
Y.
,
Fujii-Kuriyama
Y.
(
1996
)
Two new members of the murine Sim gene family are transcriptionalrepressors and show different expression patterns during mouse embryogenesis.
Mol. Cell Biol
16
,
5865
5875
Epstein
D. J.
,
Marti
E.
,
Scott
M. P.
,
McMahon
A. P.
(
1996
)
Antagonizing cAMP-dependent protein kinase A in the dorsal CNS activates a conserved Sonic hedgehog signaling pathway.
Development
122
,
2885
2894
Epstein
D. J.
,
McMahon
A. P.
,
Joyner
A. L.
(
1999
)
Regionalization of Sonic hedgehog transcription along the anteroposterior axis of the mouse central nervous system is regulated by Hnf3-dependent and-independent mechanisms.
Development
126
,
281
292
Ericson
J.
,
Morton
S.
,
Kawakami
A.
,
Roelink
H.
,
Jessell
T. M.
(
1996
)
Two critical periods of sonic hedgehog signaling required for the specification of motor neuron identity.
Cell
87
,
661
673
Ericson
J.
,
Rashbass
P.
,
Schedl
A.
,
Brenner-Morton
S.
,
Kawakami
A.
,
van Heynigen
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
Fan
C. M.
,
Kuwana
E.
,
Bulfone
A.
,
Fletcher
C. F.
,
Copeland
N. G.
,
Jenkins
N. A.
,
Crews
S.
,
Martinez
S.
,
Puelles
L.
,
Rubenstein
J. L.
,
Tessier-Lavigne
M.
(
1996
)
Expression patterns of two murine homologs of Drosophila single-minded suggest possible roles in embryonic patterning and in the pathogenesis of Down syndrome.
Mol. Cell Neurosci
7
,
1
16
Goodrich
L. V.
,
Jung
D.
,
Higgins
K. M.
,
Scott
M. P.
(
1999
)
Overexpression of ptc1 inhibits induction of Shh target genes and prevents normal patterning in the neural tube.
Dev. Biol
211
,
323
334
Hui
C. C.
,
Joyner
A. L.
(
1992
)
A mouse model of greig cephalopolysyndactyly syndrome: the extra-toesJ mutation contains an intragenic deletion of the Gli3 gene.
Nat Genet
3
,
241
246
Hui
C. C.
,
Slusarski
D.
,
Platt
K. A.
,
Holmgren
R.
,
Joyner
A. L.
(
1994
)
Expression of three mouse homologs of the Drosophila segment polarity gene cubitus interruptus, Gli, Gli-2, and Gli-3, in ectoderm-and mesoderm-derived tissues suggests multiple roles during postimplantation development.
Dev. Biol
162
,
402
413
Jain
S.
,
Maltepe
E.
,
Lu
M. M.
,
Simon
C.
,
Bradfield
C. A.
(
1998
)
Expression of ARNT, ARNT2, HIF1, HIF2 and Ah receptor mRNAs in the developing mouse.
Mech Dev
73
,
117
123
Kim
S. H.
,
Crews
S. T.
(
1993
)
Influence of Drosophila ventral epidermal development by the CNS midline cells and spitz class genes.
Development
118
,
893
901
Marti
E.
,
Bumcrot
D. A.
,
Takada
R.
,
McMahon
A. P.
(
1995
)
Requirement of 19K forms of Sonic hedgehog for induction of distinct ventral cell types in CNS explants.
Nature
375
,
322
325
Matise
M. P.
,
Epstein
D. J.
,
Park
H. L.
,
Platt
K. P.
,
Joyner
A. L.
(
1998
)
Gli2 is required for induction of floor plate but not most ventral neurons in the mouse central nervous system.
Development
125
,
2759
2770
Mo
R.
,
Freer
A. M.
,
Zinyk
D. L.
,
Crackower
M. A.
,
Michaud
J.
,
Heng
H. H.-Q.
,
Chik
K. W.
,
Shi
X.-M.
,
Tsui
L.-C.
,
Cheng
S. H.
,
Joyner
A. L.
,
Hui
C.-C.
(
1997
)
Specific and redundant functions of Gli2 and Gli3 zinc finger genes in skeletal patterning and development.
Development
124
,
113
123
Moffett
P.
,
Reece
M.
,
Pelletier
J.
(
1997
)
The murine Sim-2 gene product inhibits transcription by active repression and functional interference.
Mol. Cell Biol
17
,
4933
4947
Moffett
P.
,
Pelletier
J.
(
2000
)
Different transcriptional properties of mSim-1 and mSim-2.
FEBS Lett
466
,
80
86
Nambu
J. R.
,
Franks
R. G.
,
Hu
S.
,
Crews
S. T.
(
1990
)
The single-minded gene of Drosophila is required for the expression of genes important for the development of CNS midline cells.
Cell
63
,
63
75
Nambu
J. R.
,
Lewis
J. O.
,
Wharton
K. A.
Jr
,
Crews
S. T.
(
1991
)
The Drosophila single-minded gene encodes a helix-loop-helix protein that acts as a master regulator of CNS midline development.
Cell
67
,
1157
1167
Park
H. L.
,
Bai
C.
,
Platt
K. A.
,
Matise
M. P.
,
Beeghly
A.
,
Hui
C. C.
,
Nakashima
M.
,
Joyner
A. L.
(
2000
)
Mouse Gli1 mutants are viable but have defects in SHH signaling in combination with a Gli2 mutatiion.
Development
127
,
1593
1605
Pierani
A.
,
Brenner-Morton
S.
,
Chiang
C.
,
Jessell
T. M.
(
1999
)
A sonic hedgehog-independent, retinoid-activated pathway of neurogenesis in the ventral spinal cord.
Cell
97
,
903
915
Probst
M. R.
,
Fan
C. M.
,
Tessier-Lavigne
M.
,
Hankinson
O.
(
1997
)
Two murine homologs of the Drosophila single-minded protein that interact with the mouse aryl hydrocarbon receptor nuclear translocator protein.
J. Biol. Chem
272
,
4451
4457
Reisz-Porszasz
S.
,
Probst
M. R.
,
Fukunaga
B. N.
,
Hankinson
O.
(
1994
)
Identification of functional domains of the aryl hydrocarbon receptor nuclear translocator protein (ARNT).
Mol. Cell Biol
14
,
6075
86
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.
(
1998
)
Combinatorial Gli gene function in floor plate and neuronal inductions by Sonic hedgehog.
Development
125
,
2203
2212
Sasaki
H.
,
Hui
C.-c.
,
Nakafuku
M.
,
Kondoh
H.
(
1997
)
A binding site for Gli proteins is essential for HNF-3 floor plate enhancer activity in transgenics and can respond to Shh in vitro.
Development
124
,
1313
1322
Schweitzer
R.
,
Shaharabany
M.
,
Seger
R.
,
Shilo
B. Z.
(
1995
)
Secreted Spitz triggers the DER signaling pathway and is a limiting component in embryonic ventral ectoderm determination.
Genes Dev
9
,
1518
1529
Skeath
J. B.
(
1998
)
The Drosophila EGF receptor controls the formation and specification of neuroblasts along the dorsal-ventral axis of the Drosophila embryo.
Development
125
,
3301
3312
Sussel
L.
,
Marin
O.
,
Kimura
S.
,
Rubenstein
J. L.
(
1998
).
Loss of Nkx2.1 homeobox gene function results in a ventral to dorsal molecular respecification within the basal telencephalon: evidence for a transformation of the pallidum into the striatum.
Development
126
,
3359
3370
Taylor
A. M.
,
Nakano
Y.
,
Mohler
J.
,
Ingham
P. W.
(
1993
)
Contrasting distributions of patched and hedgehog proteins in the Drosophila embryo.
Mech. Dev
42
,
89
96
Teillet
M. A.
,
Lapointe
F.
,
Le Douarin
N. M.
(
1998
)
The relationships between notochord and floor plate in vertebrate development revisited.
Proc. Natl. Acad. Sci. USA
95
,
11733
11738
Tole
S.
,
Ragsdale
C. W.
,
Grove
E. A.
(
2000
)
Dorsoventral patterning of the telencephalon is disrupted in the mouse mutant extra-toes(J).
Dev. Biol
217
,
254
265
Weiss
J. B.
,
Von Ohlen
T.
,
Mellerick
D. M.
,
Dressler
G.
,
Doe
C. Q.
,
Scott
M. P.
(
1998
)
Dorsoventral patterning in the Drosophila central nervous system: the intermediate neuroblasts defective homeobox gene specifies intermediate column identity.
Genes Dev
12
,
3591
3602
Wharton
K. A.
Jr.
,
Franks
R. G.
,
Kasai
Y.
,
Crews
S. T.
(
1994
)
Control of CNS midline transcription by asymmetric E-box-like elements: similarity to xenobiotic responsive regulation.
Development
120
,
3563
3569
Ye
W.
,
Shimamura
K.
,
Rubenstein
J. L. R.
,
Hynes
M. A.
,
Rosenthal
A.
(
1998
)
FGF and Shh signals control dopaminergic and serotonergic cell fate in the anterior neural plate.
Cell
93
,
755
766
Zhou
Y. D.
,
Barnard
M.
,
Tian
H.
,
Li
X.
,
Ring
H. Z.
,
Francke
U.
,
Shelton
J.
,
Richardson
J.
,
Russell
D. W.
,
McKnight
S. L.
(
1997
)
Molecular characterization of two mammalian bHLH-PAS domain proteins selectively expressed in the central nervous system.
Proc. Natl. Acad. Sci. USA
94
,
713
718
Zinn
K.
,
Sun
Q.
(
1999
)
Slit branches out: a secreted protein mediates both attractive and repulsive axon guidance.
Cell
97
,
1
4
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