Patterning of the vertebrate neural tube depends on intercellular signals emanating from sources such as the notochord and the floor plate. The secreted protein Sonic hedgehog and the extracellular matrix protein Vitronectin are both expressed in these signalling centres and have both been implicated in the generation of ventral neurons. The proteolytic processing of Sonic hedgehog is fundamental for its signalling properties. This processing generates two secreted peptides with all the inducing activity of Shh residing in the highly conserved 19 kDa amino-terminal peptide (N-Shh). Here we show that Vitronectin is also proteolitically processed in the embryonic chick notochord, floor plate and ventral neural tube and that this processing is spatiotemporally correlated with the generation of motor neurons. The processing of Vitronectin produces two fragments of 54 kDa and 45 kDa, as previously described for Vitronectin isolated from chick yolk. The 45 kDa fragment lacks the heparin-binding domain and the integrin-binding domain, RGD, present in the non-processed Vitronectin glycoprotein. Here we show that N-Shh binds to the three forms of Vitronectin (70, 54 and 45 kDa) isolated from embryonic tissue, although is preferentially associated with the 45 kDa form. Furthermore, in cultures of dissociated neuroepithelial cells, the combined addition of N-Shh and Vitronectin significantly increases the extent of motor neuron differentiation, as compared to the low or absent inducing capabilities of either N-Shh or Vitronectin alone. Thus, we conclude that the differentiation of motor neurons is enhanced by the synergistic action of N-Shh and Vitronectin, and that Vitronectin may be necessary for the proper presentation of the morphogen N-Shh to one of its target cells, the differentiating motor neurons.

Averbuch-Heller
L.
,
Pruginin
M.
,
Kahane
N.
,
Tsoulfas
P.
,
Parada
L.
,
Rosenthal
A.
,
Kalcheim
C.
(
1994
)
Neurotrophin 3 stimulates the differentiation of motorneurons from avian neural tube progenitors.
Proc. Natl. Acad. Sci. USA
91
,
3247
3251
Bellaiche
Y.
,
The
I.
,
Perrimon
N.
(
1998
)
Tout-velu is a Drosophila homologue of the putative tumour suppresor EXT-1 and is needed for Hh diffusion.
Nature
394
,
85
88
Bumcrot
D. A.
,
Takada
R.
,
McMahon
A. P.
(
1995
)
Proteolytic processing yields two secreted forms of sonic hedgehog.
Mol. Cell Biol
15
,
2294
2303
Chiang
C.
,
Litingung
E.
,
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
Cohen
J.
,
Burne
J.F.
,
Winter
J.
,
Barlett
P.
(
1986
)
Retinal ganglion cells lose response to laminin with maturation.
Nature
322
,
465
467
Dutton
R.
,
Yamada
T.
,
Turnley
A.
,
Bartlett
P. F.
,
Murphy
M.
(
1999
)
Sonic hedgehog promotes neuronal differentiation of murine spinal cord precursors and collaborates with Neurotrophin 3 to induce islet-1.
J. Neurosc
19
,
2601
2608
Ericson
J.
,
Muhr
J.
,
Placzek
M.
,
Lints
T.
,
Jessell
T. M.
,
Edlund
T.
(
1995
)
Sonic hedgehog induces differentiation of ventral forebrain neurones: 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 critical periods of Sonic hedgehog signalling required for the specification of motor neuron identity.
Cell
87
,
661
673
Fariñas
I.
,
Jones
K. R.
,
Backus
C.
,
Wang
X. Y.
,
Reichardt
L. F.
(
1994
)
Severe sensory and sympathetic deficits in mice lacking neurotrophin-3.
Nature
369
,
658
661
Frade
J. M.
,
Martínez-Morales
J. M.
,
Rodríguez-Tebar
A.
(
1996
)
Laminin-1 selectively stimulates neuron generation from cultured retinal neuroepithelial cells.
Exp. Cell Res
222
,
140
149
Gotz
R.
,
Kilbek
R.
,
Lottspeich
F.
,
Barde
Y.-A.
(
1992
)
Production and characterisation of recombinant mouse neurotrophin-3.
Eur. J. Biochem
204
,
745
749
Gotz
W.
,
Osmers
R.
,
Herken
R.
(
1995
)
Localisation of extracellular matrix components in the embryonic human notochord and axial mesenchyme.
J. Anat
186
,
111
121
Hall
T. M.
,
Porter
J. A.
,
Beachy
P. A.
,
Leahy
D. J.
(
1995
).
A potential catalytic site revealed by the 1.7-A crystal structure of the amino-terminal signalling domain of Sonic hedgehog.
Nature
378
,
212
216
Hall.
T. M.
,
Porter
J. A.
,
Young
K. E.
,
Koonin
E. V.
,
Beachy
P. A.
,
Leahy
D. J.
(
1997
)
Crystal structure of a Hedgehog autoprocessing domain: homology between Hedgehog and self splicing proteins.
Cell
91
,
85
97
Hamburger
V.
,
Hamilton
H. L.
(
1951
)
A series of normal stages in the development of chick embryo.
J. Morph
88
,
49
92
Hammerschimdt
M.
,
Brook
A.
,
McMahon
A. P.
(
1997
)
The world according to hedgehog.
Trends Genet
13
,
14
21
Higashijima
S.
,
Nose
A.
,
Eguchi
G.
,
Hotta
Y.
,
Okamoto
H.
(
1997
)
Mindin/F-Spondin family: novel ECM proteins expressed in the zebrafish embryonic axis.
Dev. Biol
192
,
211
227
Hynes
M. A.
,
Porter
J. A.
,
Chiang
C.
,
Chang
D.
,
Tessier-Lavigne
M.
,
Beachy
P. A.
,
Rosenthal
A.
(
1995
)
Induction of midbrain dopaminergic neurones by Sonic hedgehog.
Neuron
15
,
35
44
Kalyani
A.
,
Hobson
K.
,
Rao
M. S.
(
1997
)
Neuroepithelial stem cells from the embryonic spinal cord: isolation, characterisation, and clonal analysis.
Dev. Biol
186
,
202
223
Kalyani
A. J.
,
Piper
D.
,
Mutjaba
T.
,
Lucero
M. T.
,
Rao
M. S.
(
1998
)
Spinal cord neuronal precursors generate multiple phenotypes in culture.
J. Neurosc
18
,
7856
7868
Kinoshita
Y.
,
Kinoshita
C.
,
Heurer
J. G.
,
Bothwell
M.
(
1993
)
Basic fibroblast growth factor promotes interactions of neuroepithelial cells from chick neural tube with extracellular matrix proteins in culture.
Development
119
,
943
956
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
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
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
Nagano
Y.
,
Hamano
T.
,
Nakashima
N.
,
Ishikawa
M.
,
Miyazaki
K.
,
Hayashi
M.
(
1992
)
Purification and differences from its blood homologue in molecular size, heparin binding, collagen binding, and bound carbohydrate.
J. Biol. Chem
267
,
24863
24870
Placzek
M.
(
1995
)
The role of the notochord and floor plate in inductive interactions.
Curr. Opin. Genet. Dev
5
,
499
506
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 signalling.
Nature
374
,
363
366
Porter
J. A.
,
Young
K.
,
Beachy
P. A.
(
1996
)
Cholesterol modification of hedgehog signalling proteins in animal development.
Science
274
,
255
259
Preissner
K. T.
(
1991
)
Structure and biological role of Vitronectin.
Ann.Rev. Cell Biol
7
,
275
310
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
Seiffer
D.
,
Poenninger
J.
,
Binder
B. R.
(
1993
)
Organisation of the gene encoding mouse vitronectin.
Gene
134
,
303
304
Seiffert
D.
,
Iruela-Arispe
M. L.
,
Sage
E. H.
,
Loskutoff
D. J.
(
1995
)
Distribution of Vitronectin mRNA during murine development.
Dev. Dyn
203
,
71
79
Tanabe
Y.
,
Roelink
H.
,
Jessell
T. M.
(
1995
)
Induction of motor neurons by Sonic hedgehog is independent of floor plate differentiation.
Curr. Biol
5
,
651
658
The
I.
,
Bellaiche
Y.
,
Perrimon
N.
(
1999
)
Hedgehog movement is regulated through tout velu-dependent synthesis of a heparon sulfate proteoglycan.
Mol. Cell
4
,
633
639
Thor
S.
,
Ericson
J.
,
Brannstrom
T.
,
Edlund
T.
(
1991
)
The homeodomain LIM protein Isl-1 is expressed in subsets of neurones and endocrine cells in the adult rat.
Neuron
7
,
881
889
Tucker
R. P.
,
Hagios
C.
,
Chiquet-Ehrismann
R.
,
Lawler
J.
(
1997
)
In situ localization of thrombospondin-1 and thrombospondin-3 transcripts in the avian embryo.
Dev. Dyn
208
,
326
337
Urry
L. A.
,
Whittaker
C. A.
,
Duquette
M.
,
Lawler
J.
,
DeSimeone
D. W.
(
1998
)
Thrombospondins in early Xenopus embryo: dynamic patterns of expression suggest diverse roles in nervous system, notochord and muscle development.
Dev. Dyn
211
,
390
407
Werb
Z.
(
1998
)
ECM and cell surface proteolysis: regulating cellular ecology.
Cell
91
,
439
442
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.