During development of the cerebellum, Sonic hedgehog (SHH) is expressed in migrating and settled Purkinje neurons and is directly responsible for proliferation of granule cell precursors in the external germinal layer. We have previously demonstrated that SHH interacts with vitronectin in the differentiation of spinal motor neurons. Here, we analysed whether similar interactions between SHH and extracellular matrix glycoproteins regulate subsequent steps of granule cell development. Laminins and their integrin receptor subunit alpha6 accumulate in the outer most external germinal layer where proliferation of granule cell precursors is maximal. Consistent with this expression pattern, laminin significantly increases SHH-induced proliferation in primary cultures of cerebellar granule cells. Vitronectin and its integrin receptor subunits alpha(v) are expressed in the inner part of the external germinal layer where granule cell precursors exit the cell cycle and commence differentiation. In cultures, vitronectin is able to overcome SHH-induced proliferation, thus allowing granule cell differentiation. Our studies indicate that the pathway in granule cell precursors responsible for the conversion of a proliferative SHH-mediated response to a differentiation signal depends on CREB. Vitronectin stimulates phosphorylation of cyclic-AMP responsive element-binding protein (CREB), and over-expression of CREB is sufficient to induce granule cell differentiation in the presence of SHH. Taken together, these data suggest that granule neuron differentiation is regulated by the vitronectin-induced phosphorylation of CREB, a critical event that terminates SHH-mediated proliferation and permits the differentiation program to proceed in these cells.

Reference

Coppolino
M. G.
,
Woodside
M. J.
,
Demaurex
N.
,
Grinsten
S.
,
St-Arnaud
R.
,
Dedhar
S.
(
1997
)
Calreticulin is essential for integrin mediated calcium signalling and cell adhesion.
Nature
386
,
343
347
Cowley
S.
,
Paterson
H.
,
Kemp
P.
,
Marshall
C. J.
(
1994
)
Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of HIH 3T3 cells.
Cell
77
,
841
852
Crossley
P. H.
,
Martínez
S.
,
Martin
G. R.
(
1996
)
Midbrain development induced by FGF8 in the chick embryo.
Nature
380
,
66
68
Dahmane
N.
,
Ruiz i Altaba
A.
(
1999
)
Sonic hedgehog regulates the growth and patterning of the cerebellum.
Development
126
,
3089
3100
De Curtis
I.
,
Reichardt
L.
(
1993
)
Function and spatial distribution in developing chick retina of the laminin receptor6 1 and its isoforms.
Development
118
,
377
388
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 signalling molecules, is implicated in regulation of CNS polarity.
Cell
75
,
1417
1430
Epstein
D. J.
,
Martí
E.
,
Scott
M. P.
,
McMahon
A. P.
(
1996
)
Antagonizing cAMP-dependent protein kinase A in the dorsal CNS activates a conserved Sonic hedgehog signal.
Development
122
,
2885
2894
García-Segura
L. M.
,
Baetens
D.
,
Roth
J.
,
Norman
A. W.
,
Orci
L.
(
1984
)
Immunohistochemical mapping of calcium-binding protein immunoreactivity in the rat central nervous system.
Brain Res
296
,
75
68
Gonzales
G. A.
,
Montminy
M. R.
(
1989
)
Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at serine 133.
Cell
59
,
675
680
Hallonet
M.
,
LeDouarin
N. M.
(
1993
)
Tracing neuroepithelial cells of the mesencephalic and metencephalic alar plates during cerebellar ontogeny in quail-chick chimeras.
Eur. J. Neurosci
5
,
1145
1155
Hamburger
V.
,
Hamilton
H. L.
(
1951
)
A series of normal stages in the development of chick embryo.
J. Morphol
88
,
49
92
Hammerschimdt
M.
,
Brook
A.
,
McMahon
A. P.
(
1997
)
The world according to hedgehog.
Trends Genet
13
,
14
21
Hammerschimdt
M.
,
McMahon
A. P.
(
1998
)
The effect of pertussis toxin on zebrafish development: a possible role for inhibitory G-proteins in hedgehog signalling.
Dev. Biol
194
,
166
171
Hatten
M. E.
(
1999
)
Central nervous system neuronal migration.
Annu. Rev. Neurosci
22
,
511
539
Hatten
M. E.
,
Heintz
N.
(
1995
)
Mechanisms of neural patterning and specification in the developing cerebellum.
Annu. Rev. Neurosci
18
,
385
408
Howe
A.
,
Alpin
A. E.
,
Alahari
S. K.
,
Juliano
R. L.
(
1998
)
Integrin signalling and growth control.
Curr. Opin. Cell Biol
10
,
220
231
Hynes
M.
,
Stone
D. M.
,
Dowd
M.
,
Pitts-Meek
S.
,
Goddard
A.
,
Gurney
A.
,
Rosenthal
A.
(
1997
)
Control of cell pattern in the neural tube bythe zinc finger trancription factor and oncogene Gli-1.
Neuron
19
,
15
26
Hynes
R. O.
(
1992
)
Integrins: versatility, modulation and signalling in cell adhesion.
Cell
69
,
11
25
Ingham
P. W.
(
1998
)
Transducing Hedgehog: the story so far.
EMBO J
17
,
3505
3511
Joyner
A. L.
(
1996
)
Engrailed, wnt and pax genes regulate midbrain-hindbrain development.
Trends Genet
12
,
15
20
Lumsden
A.
,
Krumlauf
R.
(
1996
)
Patterning the vertebrate neuraxis.
Science
274
,
1109
1115
McMahon
A. P.
,
Bradley
A.
(
1990
)
The Wnt-1 (int-1) proto-oncogene is required for development of a large region of the mouse brain.
Cell
62
,
1073
1085
Martí
E.
,
Bumcrot
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
Martí
E.
,
Takada
R.
,
Bumcrot
D. A.
,
McMahon
A. P.
(
1995
)
Distribution of Sonic hedgehog peptides in the developing chick and mouse embryo.
Development
121
,
2537
2547
Martínez
S.
,
Alvarado-Mallart
R. M.
(
1989
)
Rostral cerebellum originates from the caudal portion of the so-called ‘mesencephalic’ vesicle: a study using chick-quail chimeras.
Eur. J. Neurosci
1
,
549
560
Martínez-Morales
J. R.
,
Martí
E.
,
Frade
J. M.
,
Rodríguez-Tebar
A.
(
1995
)
Developmentally regulated vitronectin influences cell differentiation, neuron survival and process outgrowth in the developing chicken retina.
Neuroscience
68
,
245
253
Martínez-Morales
J. R.
,
Barbas
J. A.
,
Martí
E.
,
Bovolenta
P.
,
Edgar
D.
,
Rodríguez-Tebar
A.
(
1997
)
Vitronectin is expressed in the ventral region of the neural tube and promotes the differentiation of motor neurones.
Development
124
,
5139
5147
Meyer-Franke
A.
,
Kaplan
M. R.
,
Pfrieger
F. W.
,
Barres
B. A.
(
1995
)
Characterisation of the signalling interactions that promote the survival and growth of developing retinal ganglion cells in culture.
Neuron
15
,
805
819
Murase
S.
,
Hayashi
Y.
(
1998
)
Concomitant expression of genes encoding integrin alpha v beta 5 heterodimer and vitronectin in growing parallel fibers of postnatal rat cerebellum: a possible role as mediators of parallel fiber elongation.
J. Comp. Neurol
397
,
199
212
Nagano
Y.
,
Hamano
T.
,
Nakashima
N.
,
Ishiwaka
M.
,
Miyazaki
K.
,
Hayashi
M.
(
1992
)
Yolk Vitronectin: Purification and differences from its blood homologue in molecular size, heparin binding, collagen binding and bound carbohydrate.
J. Biol. Chem
267
,
24863
24870
Perrimon
N.
,
Bernfield
M.
(
2000
)
Specificities of heparan sulphate proteoglycans in developmental processes.
Nature
404
,
725
728
Pons
S.
,
Martí
E.
(
2000
)
Sonic hedgehog synergizes with the extracellular matrix protein Vitronectin, to induce spinal motor neuron differentiation.
Development
127
,
333
342
Powel
S. K.
,
Williams
C. C.
,
Nomizu
M.
,
Yamada
Y.
,
Kleinman
H. K.
(
1998
)
Laminin-like proteins are differentially regulated during cerebellar development and stimulate granule cell neurite outgrowth in vitro.
J. Neurosci. Res
54
,
233
247
Riddle
R. D.
,
Jonson
R. L.
,
Laufer
E.
,
Tabin
C.
(
1993
)
Sonic hedgehog mediates the polarising activity of the ZPA.
Cell
75
,
1401
1416
Rowitch
D. H.
,
St-Jacques
B.
,
Lee
S. M. K.
,
Flax
J. D.
,
Snyder
E. Y.
,
McMahon
A. P.
(
1999
)
Sonic hedgehog regulates proliferation and inhibits differentiation of CNS precursors.
J. Neurosci
19
,
8954
8965
Ruiz i Altaba
A.
(
1999
)
Gli proteins and hedgehog signalling.
Trends Genet
15
,
418
425
Selleck
S. B.
(
2000
)
Proteoglycans and pattern formation.
Trends Genet
16
,
206
212
Shaywitz
A. J.
,
Greenberg
M. E.
(
1999
)
CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals.
Annu. Rev. Biochem
68
,
821
861
The
I.
,
Bellaiche
Y.
,
Perrimon
N.
(
1999
)
Hedgehog movement is regulated through tout velu-dependent sysnthesis of a heparan sulfate proteoglycan.
Mol. Cell
4
,
633
639
Thomas
K. R.
,
Capecci
M. R.
(
1990
)
Targeted disruption of the murine int-1 proto-oncogene resulting in severe abnormalities in midbrain and cerebellar development.
Nature
346
,
847
850
Traiffort
E.
,
Charytoniuk
D.
,
Watroba
L.
,
Faure
H.
,
Ruat
M.
(
1999
)
Discrete localisation of hedgehog signalling components in the developing and adult rat nervous system.
Eur. J. Neurosci
11
,
3199
3214
Wallace
V. A.
(
1999
)
Purkinje-cell-derived Sonic hedgehog regulates granule neurone precursor cell proliferation in the developing mouse cerebellum.
Curr. Biol
9
,
445
448
Wechsler-Reya
R. J.
,
Scott
M. P.
(
1999
)
Control of neuronal precursor proliferation in the cerebellum by sonic hedgehog.
Neuron
22
,
103
114
Zheng
X.
,
Saunders
T. L.
,
Camper
S. A.
,
Samuelson
L. C.
,
Ginsburg
D.
(
1995
)
Vitronectin is not essential for normal mammalian development and fertility.
Proc. Natl. Acad. Sci. USA
92
,
12426
12430
This content is only available via PDF.