Neuroepithelial and radial glial cells span between the ventricular and the pial surfaces of the neural tube and express two intermediate filaments (IFs), nestin and vimentin, which form a filamentous network throughout the length of the cells. In this report we study the polymerization characteristics of nestin and examine how mutations affect the assembly and localization of the nestin protein in cultured cells and in the developing CNS of transgenic mice. A wild-type rat nestin gene transfected into the IF-free SW13 cell line failed to assemble into a filamentous network but was incorporated into the existing IF network of a subclone expressing vimentin, demonstrating that nestin requires vimentin for proper assembly. In transgenic mice, rat nestin formed a network indistinguishable from that formed by endogenous nestin and vimentin, but a mutant form lacking five amino acids at the carboxy terminus of the rod domain was largely restricted to the pial endfeet. Since nestin mRNA is localized to the pial endfoot region we propose that both transgenes are translated there, but that the wild-type protein is preferentially incorporated into the IF network. These observations provide evidence for hierarchical assembly and a complex organization of the IF network along the ventricular-pial axis in the early CNS.

REFERENCES

Albers
K.
,
Fuchs
E.
(
1987
).
The expression of mutant epidermal keratin cDNAs transfected in simple epithelial and squamous cell carcinoma lines.
J. Cell Biol
105
,
791
806
Albers
K.
,
Fuchs
E.
(
1989
).
Expression of mutant keratin cDNAs in epithelial cells reveals posible mechanisms for initiation and assembly of intermediate filaments.
J. Cell Biol
108
,
1477
1493
Almazan
G.
,
Afar
D.
,
Bell
J.
(
1993
).
Phosphorylation and disruption of intermediate filament proteins in oligodendrocyte precursor cultures treated with Calyculin A.
J. Neurosci. Res
36
,
163
172
Andreoli
J.
,
Trevor
K.
(
1994
).
Fate of a headless vimentin protein in stable cell cultures: soluble and cytoskeletal forms.
Exp. Cell Res
214
,
177
188
Brittis
P.
,
Silver
J.
(
1994
).
Exogenous glycosaminoglycans induce complete inversion of retinal ganglion cell bodies and their axons within the retinal neuroepithelium.
Proc. Nat. Acad. Sci. USA
91
,
7539
7542
Chen
C.
,
Okayama
H.
(
1987
).
High-efficiency transformation of mammalian cells by plasmid DNA.
Mol. Cell Biol
7
,
2745
2752
Chin
S.
,
Macioce
P.
,
Liem
R.
(
1991
).
Effects of truncated neurofilament proteins on the endogenous intermediate filaments in transfected fibroblasts.
J. Cell Sci
99
,
335
350
Ching
G.
,
Liem
R.
(
1993
).
Assembly of Type IV neuronal intermediate filaments in nonneuronal cells in the absence of preexisting cytoplasmic intermediate filaments.
J. Cell Biol
122
,
1323
1335
Chou
Y.-H.
,
Bischoff
J.
,
Beach
D.
,
Goldman
R.
(
1990
).
Intermediate filament reorganization during mitosis is mediated by p34cdc2phosphorylation of vimentin.
Cell
62
,
1063
1071
Chou
Y.-H.
,
Skalli
O.
,
Goldman
R. D.
(
1997
).
Intermediate filaments and cytoplasmic networking: new connections and more functions.
Curr. Opin. Cell Biol
9
,
49
53
Colluci-Guyon
E.
,
Portier
M.-M.
,
Dunia
I.
,
Paulin
D.
,
Pournin
S.
,
Babinet
C.
(
1994
).
Mice lacking vimentin develop and reproduce without an obvious phenotype.
Cell
79
,
679
694
Cote
F.
,
Collard
J.-F.
,
Julien
J.-P.
(
1993
).
Progressive neuronopathy in transgenic mice expressing the human neurofilament heavy gene: a mouse model of amyotrophic lateral sclerosis.
Cell
73
,
35
46
Coulombe
P.
,
Chan
Y.-M.
,
Albers
K.
,
Fuchs
E.
(
1990
).
Deletions in epidermal keratins leading to alteration in filament organization in vivo and in intermediate filament assembly in vitro.
J. Cell Biol
111
,
3049
3064
Coulombe
P.
,
Fuchs
E.
(
1990
).
Elucidating the early stages of keratin filament assembly.
J. Cell Biol
111
,
153
169
Dahlstrand
J.
,
Collins
V.
,
Lendahl
U.
(
1992
).
Expression of the Class VI intermediate filament nestin in human central nervous system tumors.
Cancer Res
52
,
5334
5341
Dahlstrand
J.
,
Lardelli
M.
,
Lendahl
U.
(
1995
).
Nestin mRNA expression correlates with the CNS progenitor cell state in many, but not all, regions of the CNS.
Dev. Brain Res
84
,
109
129
Eyer
J.
,
Peterson
A.
(
1994
).
Neurofilament-deficient axons and perikaryal aggregates in viable transgenic mice expressing a neurofilament—galactosidase fusion protein.
Neuron
12
,
389
405
Frederiksen
K.
,
McKay
R.
(
1988
).
Proliferation and differentiation of rat neuroepithelial precursor cells in vivo.
J. Neurosci
8
,
1144
1151
Frisen
J.
,
Johansson
C.
,
Torok
C.
,
Risling
M.
,
Lendahl
U.
(
1995
).
Rapid, widespread and long lasting induction of nestin contributes to thegeneration of glial scar tissue after CNS injury.
J. Cell Biol
131
,
453
64
Fuchs
E.
(
1994
).
Intermediate filaments and disease: mutations that cripple cell strength.
J. Cell Biol
125
,
511
516
Galou
M.
,
Colluci-Guyon
E.
,
Ensergueix
D.
,
Ridet
J.-L.
,
Giminez y Ribotta
M.
,
Privat
A.
,
Babinet
C.
,
Dupouey
P.
(
1996
).
Disrupted glial fibrillary acidic protein network in astrocytes from vimentin knockout mice.
J. Cell Biol
133
,
853
863
Gill
S.
,
Wong
P.
,
Monteiro
M.
,
Cleveland
D.
(
1990
).
Assembly properties of dominant and recessive mutations in the small mouse nuerofilament (NF-L) subunit.
J. Cell Biol
111
,
2005
2019
Gomi
H.
,
Yokoyama
T.
,
Fujimoto
K.
,
Ikeda
T.
,
Katoh
A.
,
Itoh
T.
,
Itohara
S.
(
1995
).
Mice devoid of the glial fibrillary acidic protein develop normally and are susceptible to scrapie prions.
Neuron
14
,
29
41
Hatzfeld
M.
,
Weber
K.
(
1992
).
A synthetic peptide representing the consensus sequence motif at the carboxy-terminal end of the rod domain inhibits intermediate filament assembly and disassembles preformed filaments.
J. Cell Biol
116
,
157
166
Hedberg
K.
,
Chen
L.
(
1986
).
Absence of intermediate filaments in a human adrenal cortex cercinoma-derived cell line.
Exp. Cell Res
163
,
509
517
Heins
S.
,
Wong
P.
,
Muller
S.
,
Goldie
K.
,
Cleveland
D.
,
Aebi
U.
(
1993
).
The rod domain of NF-L determines neurofilament architecture whereas the end domains specify filament assembly and network formation.
J. Cell Biol
123
,
1517
1533
Herrmann
H.
,
Hoffmann
I.
,
Franke
W.
(
1992
).
Identification of a nonapeptide motif in the vimentin head domain involved in intermediate filament assembly.
J. Mol. Biol
223
,
637
650
Herman
J.-P.
,
Victor
J.
,
Sanes
J.
(
1993
).
Developmentally regulated and spatially restricted antivens of radial glial cells.
Dev. Dynam
197
,
307
318
Hockfield
S.
,
McKay
R.
(
1985
).
Identification of major cell classes inthe developing mammalian nervous system.
J. Neurosci
5
,
3310
3328
Hoffmann
I.
,
Herrmann
H.
(
1992
).
Interference in vimentin assembly in vitro by synthetic peptides derived from the vimentin head.
J. Cell Sci
101
,
687
700
Holmin
S.
,
Almqvist
P.
,
Lendahl
U.
,
Mathiesen
T.
(
1997
).
Adult CNS stem cells differentiate to astrocytes in response to brain injury.
Eur. J. Neurosci
9
,
65
75
Kachinsky
A.
,
Dominov
J.
,
Miller
J.
(
1994
).
Myogenesis and the intermediate filament protein nestin.
Dev. Biol
165
,
216
228
Kolodziej
P.
,
Young
R.
(
1991
).
Epitope tagging and protein surveillance.
Meth. Enzymol
194
,
508
519
Kouklis
P.
,
Traub
P.
,
Georgatos
S.
(
1992
).
Involvement of the consensus sequence motif at coil 2b in the assembly and stability of vimentin filaments.
J. Cell Sci
102
,
31
41
Laird
P.
,
Zijderveld
A.
,
Linders
K.
,
Rudniki
M.
,
Jaenisch
R.
,
Berns
A.
(
1991
).
Simplified mammalian DNA isolation procedure.
Nucl. Acids Res
19
,
4293
4294
Lee
M.
,
Xu
Z.
,
Wong
P.
,
Cleveland
D.
(
1993
).
Neurofilaments are obligate heteropolymers in vivo.
J. Cell Biol
122
,
1337
1350
Lendahl
U.
,
Zimmerman
L.
,
McKay
R.
(
1990
).
CNS stem cells express a new class of intermediate filament protein.
Cell
60
,
585
595
Letai
A.
,
Coulombe
P.
,
Fuchs
E.
(
1992
).
Do the ends justify the mean?Proline mutations at the ends of the keratin coiled-coil rod segment are more disruptive than internal mutations.
J. Cell Biol
116
,
1181
1195
Li
Z.
,
Colucci-Guyon
E.
,
Pincon-Raymond
M.
,
Mericskay
M.
,
Pournin
S.
,
Paulin
D.
,
Babinet
C.
(
1996
).
Cardiovascular lesions and skeletal myopathy in mice lacking desmin.
Dev. Biol
175
,
362
366
Liem
R.
(
1993
).
Molecular biology of neuronal intermediate filaments.
Curr. Opin. Cell Biol
5
,
12
16
Milner
D. J.
,
Weitzer
G.
,
Tran
D.
,
Bradley
A.
,
Capetanaki
Y.
(
1996
).
Disruption of muscle architecture and myocardial degeneration in mice lacking desmin.
J. Cell Biol
134
,
1255
1270
Ngai
J.
,
Coleman
T.
,
Lazarides
E.
(
1990
).
Localization of newly synthesized vimentin subunits reveals a novel mechanism of intermediate filament assembly.
Cell
60
,
415
427
Ohara
O.
,
Gahara
Y.
,
Miyake
T.
,
Teraoke
H.
,
Kitamura
T.
(
1993
).
Neurofilament deficiency in quail caused by nonsense mutation in neurofilament-L gene.
J. Cell Biol
121
,
387
395
Pekny
M.
,
Leveen
P.
,
Pekna
M.
,
Eliasson
C.
,
Berthold
C. H.
,
Westermark
B.
,
Betsholtz
C.
(
1995
).
Mice lacking glial fibrillary acidic protein display astrocytes devoid of intermediate filaments but develop and reproduce normally.
EMBO J
14
,
1590
1598
Raats
J.
,
Gerards
W.
,
Schreuder
M.
,
Grund
C.
,
Henderik
J.
,
Hendriks
I.
,
Famaekers
F.
,
Bloemendal
H.
(
1992
).
Biochemical and structural aspects of transiently and stably expressed mutant desmin in vimentin-free and vimentin containing cells.
Eur. J. Cell Biol
58
,
108
127
Raats
J.
,
Pieper
F.
,
Vree Egberts
W.
,
Verrijp
K.
,
Ramaekers
F.
,
Bloemendal
H.
(
1990
).
Assembly of amino-terminally deleted desmin in vimentin-free cells.
J. Cell Biol
111
,
1971
1975
Rakic
P.
(
1971
).
Neuron-glia relationship during granule cell migration in developing cerebellar cortex.
J. Comp. Neurol
141
,
283
312
Reynolds
B.
,
Weiss
S.
(
1992
).
Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.
Science
255
,
1707
1710
Sarria
A.
,
Nordeen
S.
,
Evans
R.
(
1990
).
Regulated expression of vimentin cDNA in cells in the presence and absence of a preexisting vimentin filament network.
J. Cell Biol
111
,
553
565
Sarria
A.
,
Panini
A.
,
Evans
R.
(
1992
).
A functional role for vimentin intermediate filaments in the metabolism of lipoprotein-derived cholesterol in human SW13 cells.
J. Biol. Chem
267
,
19455
19463
Sauer
F.
(
1935
).
Mitosis in the neural tube.
J. Comp. Neurol
62
,
377
405
Sejersen
T.
,
Lendahl
U.
(
1993
).
Transient expression of the intermediate filament nestin during skeletal muscle differentiation.
J. Cell Sci
106
,
1291
1300
Shea
T.
,
Beermann
M.
,
Fischer
I.
(
1993
).
Transient requirement for vimentin in neuritogenesis: intracellular delivery of anti-vimentin antibodies and antisense oligonucleotieds inhibit neurite initiation but not elongation of existing neurites in neuroblastoma.
J. Neurosci Res
36
,
66
76
Sjöberg
G.
,
Jiang
W.-Q.
,
Ringertz
N. R.
,
Lendahl
U.
,
Sejersen
T.
(
1994
).
Colocalization of nestin and vimentin/desmin demonstrated by computer-aided double immunofluorescence analysis.
Exp. Cell Res
214
,
447
458
Steinert
P.
(
1993
).
Structure, function and dynamics of keratin intermediate filaments.
J. Invest. Dermatol
100
,
729
734
Steinert
P.
,
Roop
D.
(
1988
).
Molecular and cellular biology of intermediate filaments.
Annu. Rev. Biochem
57
,
593
625
Stewart
M.
(
1993
).
Intermediate filament structure and assembly.
Curr. Opin. Cell Biol
5
,
3
11
Thomas
T.
,
Dziadek
M.
(
1993
).
Genes coding for basement membrane glycoportiens laminin, nidogen and collagen IV are differentially expressed in the nervous system and by epithelial, endothelial and mesenchymal cells.
Exp. Cell Res
208
,
54
67
Tohyama
T.
,
Lee
V. M.-Y.
,
Rorke
L.
,
Marvin
M.
,
McKay
R.
,
Trojanowski
J.
(
1992
).
Nestin expression in embryonic human neuroepithelium and in human neuroepithlial tumor cells.
Lab. Invest
66
,
303
313
Valtz
N.
,
Hayes
T.
,
Norregard
T.
,
Liu
S.
,
McKay
R.
(
1991
).
An embryonic origin for medulloblastoma.
New Biol
3
,
364
371
van den Heuvel
R.
,
van Eys
G.
,
Ramaekers
F.
,
Quax
W.
,
Vree Egberts
W.
,
Schaart
G.
,
Cuypers
H.
(
1987
).
Intermediate filament formation after transfection with modified hamster vimentin and desmin genes.
J. Cell Sci
88
,
475
482
Vikstrom
K.
,
Borisy
G.
,
Goldman
R.
(
1989
).
Dynamic aspects of intermediate filament networks in BHK-21 cells.
Proc. Nat. Acad. Sci. USA
86
,
549
553
Weber
K.
,
Riemer
D.
,
Dodemont
H.
(
1991
).
Aspects of evolution of the lamin/intermediate filament protein family: a current analysis of invertebrate intermediate filament proteins.
Biochem. Soc. Trans
19
,
1021
1023
Weinstein
D.
,
Shelanski
M.
,
Liem
R.
(
1991
).
Suppression by antisense mRNA demonstrates a requirement for the glial fibrillary acidic protein in the formation fo stable astrocytic processes in response to neurons.
J. Cell Biol
112
,
1205
1213
Weitzer
G.
,
Milner
D.
,
Kim
J.
,
Bradley
A.
,
Capetanaki
Y.
(
1995
).
Cytoskeletal control of myogenesis: A desmin null mutation blocks the myogenic pathway during embryonic stem cell differentiation.
Dev. Biol
172
,
422
439
Wilson
A. K.
,
Coulombe
P. A.
,
Fuchs
E.
(
1992
).
The role of K5 and K14 head, tail, and R/KLLEGE domains in keratin filament assembly in vitro.
J. Cell Biol
119
,
401
414
Wong
P. C.
,
Cleveland
D. W.
(
1990
).
Characterization of dominant and recessive assembly-defective mutation in mouse neurofilament NF-M.
J. Cell Biol
111
,
1987
2003
Wroblewski
J.
,
Engström
M.
,
Edwall-Arvidsson
C.
,
Sjöberg
G.
,
Sejersen
T.
,
Lendahl
U.
(
1997
).
Distribution of nestin in the developing limb bud in vivo and in micro-mass culutres of cells isolated from limb buds.
Differentiation
61
,
151
159
Xu
Z.
,
Cork
L.
,
Griffin
J.
,
Cleveland
D.
(
1993
).
Increased expression of neurofilament subunit NF-L produces morphological alterations that resemble the pathology of human motor neuron disease.
Cell
73
,
23
33
Yang
H.-Y.
,
Lieska
N.
,
Shao
D.
,
Kriho
V.
,
Pappas
G.
(
1993
).
Immunotyping of radial glia and their glial derivatives during development of the rat spinal cord.
J. Neurocytol
22
,
558
571
Zimmerman
L.
,
Lendahl
U.
,
Cunningham
M.
,
McKay
R.
,
Parr
B.
,
Gavin
B.
,
Mann
J.
,
Vassileva
G.
,
McMahon
A.
(
1994
).
Independent regulatory elements in the nestin gene directs transgene expression to neural stem cells or muscle precursors.
Neuron
12
,
11
24
This content is only available via PDF.