 |
 Previous Article | Next Article 
The Journal of Neuroscience, 2001, 21:RC183:1-4
RAPID COMMUNICATION
Reelin Does Not Directly Influence Axonal Growth
Yves
Jossin and
André M.
Goffinet
Neurobiology Unit, University of Namur School of Medicine, B5000
Namur, Belgium
 |
ABSTRACT |
Reelin is a large extracellular glycoprotein involved in the
development of architectonic patterns, particularly in the cerebral cortex and hippocampus, where it is synthesized primarily by
Cajal-Retzius cells. In the hippocampus, Reelin also regulates the
growth and/or distribution of afferent entorhinal and commissural
axons. To assess further the possible action of Reelin on axonal
growth, we used the three-dimensional collagen gel assay to
measure axonal elongation from reeler cortical explants in the presence
of Reelin. Because Reelin is proteolytically processed in
vivo, normal explants and Reelin-transfected human
embryonic kidney 293T cells were used, respectively, as sources
of processed and full-length protein. The reliability of the assay was
tested by demonstrating a clear repulsive action of semaphorin 3F
(p < 0.0001). However, neither full-length
nor processed Reelin exhibited any significant attraction or repulsion
on cortical axons. Our results suggest that the reported effects of
Reelin on axonal pathways are indirect, secondary to the architectonic
disturbances that result from Reelin deficiency, and that the effects
of Cajal-Retzius cells on connectivity are primarily independent of Reelin.
Key words:
Reelin; axon guidance; collagen gel assay; cortical
explants; hippocampus; Cajal-Retzius cells
| |
INTRODUCTION |
Reelin,
the extracellular glycoprotein defective in reeler mutant mice
(D'Arcangelo et al., 1995 ), plays a key role in architectonic brain
development (for review, see Lambert de Rouvroit and Goffinet, 1998).
In the embryonic cortex and hippocampus, Reelin synthesized by neurons
in the marginal zone, including Cajal-Retzius (CR) cells, acts locally
on end-migration neurons of the cortical plate and instructs their
radial organization. At the surface of target cells, Reelin binds to
two lipoprotein receptors, very-low-density lipoprotein receptor
(VLDLR) and apolipoprotein E receptor 2 (ApoER2), which relay
the signal into the cell via the adapter Dab1 (Bar and Goffinet, 1999;
Cooper and Howell, 1999; Trommsdorf et al., 1999). In addition to their
Reelin-dependent effect on neuronal patterning, hippocampal CR cells
were shown to guide entorhinal axons to the stratum lacunosum
moleculare of the hippocampus, and perturbation experiments with the
Reelin-blocking antibody CR50 suggested that Reelin may play a part in
this guidance (Del Rio et al., 1997). However, observations that
hippocampal afferents successfully reach their target in reeler mice,
although with a significant delay (Borell et al., 1999a,b; Deller et
al., 1999), suggest that other factors produced by CR cells are more
important than Reelin, which could serve to promote collateral
branching in terminal fields rather than guide entorhinal axons.
Whether the developmental delay of hippocampal afferents in reeler mice reflects a direct action of Reelin on axonal growth or is secondary to
the profuse architectonic malformation remains unclear. To study that
question, the action of Reelin on cortical axonal growth was studied
in vitro using the three-dimensional (3D) collagen gel assay. Because Reelin is cleaved in vivo, probably by a
metalloproteinase (Lambert de Rouvroit et al., 1999), and the
physiological consequences of this processing are unknown, the actions
of both the full-length and the processed form of Reelin were analyzed.
Neither form of the protein displayed a significant repulsive or
attractive effect on axonal outgrowth. Although these observations
cannot exclude a direct action of Reelin solely on a subset of axons,
they strongly suggest that the observed disturbances of axonal growth
in Reelin-deficient mice are secondary to the brain malformation in the
mutant, possibly including disturbances of some recently described
projections from CR cells (Ceranik et al., 1999).
| |
MATERIALS AND METHODS |
Explant culture. Normal BALB/c and homozygous reeler
(Reln, Orleans allele) mice were used. Pregnancies were
dated by checking females for the presence of a vaginal plug; the day
of the plug was noted as embryonic day 0 (E0). Mice were killed by
cervical dislocation and the embryonic brains were removed under cold
anesthesia. Experiments were performed in accordance with National and
Institutional Guidelines for animal care and were approved by the
competent Animal Ethics Committee. To obtain cortical explants, the
dorsal tiers of the hemispheres were dissected, meningeal membranes
were pealed off, and the tissue was cut into 300 µm explants with a McIlwain tissue chopper (Campden Instruments, Leicester, UK).
Explants (E14-E18) were cultured in three-dimensional collagen gels as
described previously (Lumsden and Davies, 1983; Toran-Allerand, 1990).
Briefly, explants were embedded in 20 µl of collagen [9 parts
rat-tail collagen, 0.9 parts 10× Eagle's medium with
L-glutamine (Life Technologies, Grand Island, NY),
and 0.1 parts 0.08 mM NaHCO3 in 0.1 M NaOH) in culture dishes. The explants were positioned at
a distance of 300-500 µm from each other or from cell pellets. The
dishes were then placed in an incubator at 37°C for 30 min to gelify
the collagen, before being covered with culture medium [Eagle's
medium with L-glutamine, 0.1% penicillin-streptomycin, Fisher's cocktail (1 mg/ml BSA, 1 mg/ml transferrin, 10 µg/ml aprotinin, 600 nM sodium selenite, and 250 µg/ml
insulin), and 5% horse serum] as described previously (D'Arcangelo
et al., 1997).
Reelin and semaphorin production in human embryonic kidney 293T
cells. Human embryonic kidney 293T cells were transfected with the Reelin cDNA construct pCrl (D'Arcangelo et al., 1997), a
Myc-tagged semaphorin 3A construct, or a Myc-tagged semaphorin 3F construct (provided by M. Tessier-Lavigne, University of California, San Francisco, CA). Cells were seeded at 3 × 105 cells per 35 mm well and transfected
16-20 hr later with 2 µg of Reelin cDNA using Lipofectamine (10 µl
in 1 ml of opti-MEM; Life Technologies). After 5 hr, the supernatant
and cell debris were removed and 2 ml of culture medium was added
(Iscove's modified Dulbecco's medium with 10% heat-inactivated fetal
bovine serum, all from Life Technologies). Pellets of transfected 293T
cells were produced with the hanging-drop method (Métin et al.,
1997). At 12 hr after initiation of transfection, cell layers were
detached with trypsin, washed twice, and suspended in culture medium
with 1% serum (40 µl per 35 mm well). Drops (20 µl) of the cell
suspension were placed on the lids of 35 mm dishes, which were inverted
over dishes containing 2 ml of medium. Hanging-drop cultures were
incubated for 14-16 hr, after which the cell pellets were harvested
into explant culture medium and embedded in collagen.
Quantification of axon growth. Axon growth was quantified by
examination and photography under phase contrast or after
immunostaining with an anti-neurofilament 200 kDa antibody (clone RT97;
Boehringer Mannheim, Mannheim, Germany); analysis of results showed
that both methods were comparable. To avoid the possible effects of endogenous Reelin, target explants were all from reeler mice. The zone
surrounding target explants was divided into quadrants, thus defining a
proximal and a distal quadrant in relation to the source of Reelin
(either cell pellet or normal explant), as schematized in Figure
1. The area covered by the neurites
growing from the target explant was measured in the proximal and distal quadrants using Scion Image software (available from
http://rsb.info.nih.gov/nih-image). The ratios of the proximal and
distal areas were compared using Student's t test after
logarithmic transformation and verification of homogeneity of variance
(Bartlett test).
View larger version (22K):
[in this window]
[in a new window]
|
Figure 1.
Collagen gel assay for analysis of axonal
outgrowth from a test reeler explant in the presence of a source of
Reelin, either a normal explant or transfected cells, or in the
presence of control cells. The ratio of the proximal
(P) versus distal (D) areas
covered with axon outgrowth is used as an index for statistical
analysis.
|
|
| |
RESULTS |
To verify that normal cortical explants and transfected 293T cells
expressed Reelin in the 3D collagen gel culture, supernatants were
tested by Western blot analysis with the G10 monoclonal antibody, directed against the N-terminal region of Reelin (de Bergeyck et al.,
1997). As shown in Figure 2, transfected
293T cells secreted a full-length Reelin of ~400-450 kDa, whereas
normal explants primarily produced processed Reelin, as evidenced by
the demonstration of a predominant 180-200 kDa N-terminal fragment.
Control reeler explants (de Bergeyck et al., 1998) and untransfected
293T cells did not secrete any Reelin. These two situations were used
to study the effect of full-length and processed Reelin on axonal outgrowth. Similarly, the secretion of semaphorin 3A and semaphorin 3F
in the supernatants of transfected cells was verified using anti-Myc
antibodies. Both semaphorin 3A and semaphorin 3F were detected as
full-length (95 kDa) and processed products, namely a 60 kDa
C-terminal fragment for semaphorin 3A and two fragments of 60 kDa and
35 kDa for semaphorin 3F (Adams et al., 1997).
View larger version (11K):
[in this window]
[in a new window]
|
Figure 2.
Reelin protein expression, revealed by Western
blot analysis with antibody G10. Lane 1, Control,
nontransfected cells. Lane 2, Normal cortical explants
(E17) primarily produce processed Reelin, the N-terminal 180 kDa
fragment of which is revealed with G10. Lane 3,
Reelin-transfected 293T cells primarily produce full-length Reelin.
Markers are shown in kilodaltons.
|
|
To assess the effect of Reelin on cortical axons, target reeler
explants were cultured next to normal cortical explants, used as a
source of processed Reelin, or next to a pellet of transfected 293T
cells, used as a source of full-length Reelin (Fig.
3). Recombinant Reelin binds to both
ApoER2 and VLDLR receptors and triggers the phosphorylation of the Dab1
adapter, suggesting that it is biologically active (Cooper and Howell,
1999; Trommsdorf et al., 1999) (data not shown). In cultures, the
control situation consisted of two reeler explants or a reeler explant
cultured next to nontransfected cells. To get a positive control
of the assay, the effects of semaphorin 3A and semaphorin 3F were
tested. After 3 d in vitro, axonal outgrowth from the
reeler explant was analyzed as described above (Fig. 1). The following
situations were analyzed: 29 instances of reeler explants facing
Reelin-transfected 293T cells (Fig. 3A), 36 instances of
reeler versus normal explants (Fig. 3B), 15 control
experiments of reeler-reeler co-explants, 12 experiments with reeler
explants facing nontransfected 293T cells, 26 instances of normal
explants facing semaphorin 3A-transfected cells (Fig. 3C),
and 25 instances of normal explants facing semaphorin 3F-transfected cells (Fig. 3D). The results of the statistical analysis are
shown in Table 1. Whereas a significant
repulsive action of semaphorin 3F on cortical axons was evident
(p < 0.0001), no repulsion was exerted
on cortical axons by semaphorin 3A in this system. The axonal outgrowth
from reeler explants was not statistically different when the explants
faced sources of full-length Reelin (transfected cells), sources of
full-length Reelin (normal explants), or Reelin-deficient sources
(reeler explants or untransfected cells).
View larger version (114K):
[in this window]
[in a new window]
|
Figure 3.
Examples of axonal growth in collagen gel culture
assays. A, A Reeler explant (T)
near a pellet of Reelin-transfected cells as the source
(S). B, A Reeler explant
(T) near a normal explant as the source
(S). C, Normal explant
(T) near a pellet of semaphorin 3A-transfected
cells (S). D, Normal explant
(T) near a pellet of semaphorin 3F-transfected
cells (S). A repulsive action is clearly seen.
Scale bar, 150 µm.
|
|
| |
DISCUSSION |
This study was undertaken following evidence that CR cells and
possibly Reelin play a permissive role during the development of the
entorhinohippocampal pathway (Del Rio et al., 1997). These findings
suggested that, in addition to its well-known role in neuronal
migration and architectonic patterning, Reelin may also regulate
aspects of axonal growth and guidance (Gosh, 1997). The present results
show that the outgrowth of axons from reeler cortical explants was not
significantly influenced by the presence of full-length or
proteolytically processed Reelin; therefore, Reelin does not appear to
have any direct attractive or repulsive action on cortical growth
cones, at least in vitro. The collagen gel assay (Lumsden and Davies, 1983) has been used successfully in various settings and
its sensitivity is considered high (Métin et al., 1997;
Chédotal et al., 1998). That the negative result obtained with
Reelin is not attributable to a lack of sensitivity of the setup
is also demonstrated by the clear-cut axonal repulsion exerted by
semaphorin 3F. The somewhat unexpected absence of repulsion by
semaphorin 3A may be related to the cells used for transfection, as
indicated by the observation of different cleavage products for
semaphorin 3A and semaphorin 3F (Adams et al., 1997).
The absence of detectable effects of Reelin shows that the protein does
not exert any general direct influence on the guidance and growth of
cortical axons. Thus far, Reelin has been shown to affect solely the
distribution of commissural and entorhinal afferent axons to the
hippocampus. Because the explant system examines outgrowth from several
types of axons and thus is not selective, our data cannot rule out a
direct effect of Reelin on a specific set of axons at a given
developmental time. This reservation being made, our results are
generally in agreement with other observations that suggest that the
actions of Reelin on axonal pathways are likely indirect, secondary to
its primary effect on architectonic patterning, and that the
hodological effects of CR cells are primarily independent of Reelin.
Studies in organotypic slice cultures showed that the ablation of CR
cells prevents the ingrowth of entorhinal fibers. Interference with
Reelin using the CR50 antibody has a less drastic effect: it does not
prevent ingrowth of entorhinal fibers but reduces their branching and collateral expansion in the hippocampal target field (Del Rio et al.,
1997; Borrell et al., 1999a). Similarly, entorhinal fibers reach their
hippocampal target in Reelin-deficient mice, although with a
significant delay, but they fail to form a normal contingent of
collaterals and synapses (Borrell et al., 1999b). In addition, hippocampal commissural fibers in reeler mice disperse broadly in the
stratum lacunosum moleculare, but they are strictly segregated in
normal mice. This anomaly is correlated with the different distributions of CR cells and granule neurons in reeler versus normal
mice (Borrell et al., 1999a,b; Deller et al., 1999). The observation that hippocampal CR cells project to the entorhinal cortex
suggests that their processes may play a direct role in guiding
entorhinal fibers to the hippocampal marginal zone. Surely, even if
such actions are dependent on Reelin, they cannot be detected in
collagen gel assays in which anatomical connections between explants
are not present. Together, these studies suggest that the prominent
effects of CR cells on the growth of entorhinal afferents to the
hippocampus are not primarily attributable to Reelin; in addition, the
present observation that Reelin has no direct effect on cortical growth
cones provides a strong argument for this view. The consequences of
Reelin deficiency observed in vivo are likely to be
indirect, secondary to the profuse laminar malformation of the reeler
entorhinal cortex and hippocampus. Given the possible role of the
recently described projections from hippocampal CR cells to the
entorhinal cortex (Ceranik et al., 1999), it would certainly be
interesting to study that projection in reeler mice. Finally, the
action of Reelin on collateral branching should be studied further, for
example using in vitro systems such as slice cultures in
which the architecture of the tissue is better preserved and can be
more accurately assessed than in explant cultures.
| |
FOOTNOTES |
Received April 30, 2001; revised Aug. 27, 2001; accepted Sept. 4, 2001.
This work was supported by Contract 3.4533.95 from the Fonds de la
Recherche Scientifique Médicale, by Contract 94/99-186 from the Association pour la Recherche sur le Cancer, and by the Fondation Médicale Reine Elisabeth. Y.J. is supported by the Fonds de Recherche pour l'Industrie et l'Agriculture. We thank C. Métin and Z. Molnar for advice with the collagen assay, M. Tessier-Lavigne for the gift of the semaphorin plasmids, E. Depierreux for statistics, and M. Frotscher and F. Polleux for discussion.
Correspondence should be addressed to André M. Goffinet at the
above address. E-mail: Andre.Goffinet{at}fundp.ac.be.
This article is published in
The Journal of Neuroscience, Rapid Communications Section,
which publishes brief, peer-reviewed papers online, not in print. Rapid
Communications are posted online approximately one month earlier than
they would appear if printed. They are listed in the Table of Contents
of the next open issue of JNeurosci. Cite this article as:
JNeurosci, 2001, 21:RC183 (1-4). The
publication date is the date of posting online at
www.jneurosci.org.
| |
REFERENCES |
-
Adams RH,
Lohrum M,
Klostermann A,
Betz H,
Püschel AW
(1997)
The chemorepulsive activity of secreted semaphorins is regulated by furin-dependent proteolytic processing.
EMBO J
16:6077-6086.
-
Bar I,
Goffinet AM
(1999)
Decoding the Reelin signal.
Nature
399:645-646.
-
Borrell V,
Del Rio JA,
Alcantara S,
Derer M,
Martinez A,
D'Arcangelo G,
Nakajima K,
Mikoshiba K,
Derer P,
Curran T,
Soriano E
(1999a)
Reelin regulates the development and synaptogenesis of the layer-specific entorhino-hippocampal connections.
J Neurosci
19:1345-1358.
-
Borrell V,
Ruiz M,
Del Rio JA,
Soriano E
(1999b)
Development of commissural connections in the hippocampus of reeler mice: evidence of an influence of Cajal-Retzius cells.
Exp Neurol
156:268-282.
-
Ceranik K,
Deng J,
Heimrich B,
Lubke J,
Zhao S,
Forster E,
Frotscher M
(1999)
Hippocampal Cajal-Retzius cells project to the entorhinal cortex: retrograde tracing and intracellular labelling studies.
Eur J Neurosci
11:4278-4290.
-
Chédotal A,
Del Rio JA,
Ruiz M,
He Z,
Borrell V,
de Castro F,
Ezan F,
Goodman CS,
Tessier-Lavigne M,
Sotelo C,
Soriano E
(1998)
Semaphorins III and IV repel hippocampal axons via two distinct receptors.
Development
125:4313-4323.
-
Cooper JA,
Howell BW
(1999)
Lipoprotein receptors: signaling functions in the brain?
Cell
97:671-674.
-
D'Arcangelo G,
Miao GG,
Chen S,
Soares HD,
Morgan JI,
Curran T
(1995)
A protein related to extracellular matrix proteins deleted in the mouse mutant reeler.
Nature
374:719-723.
-
D'Arcangelo G,
Nakajima K,
Miyata T,
Ogawa M,
Mikoshiba K,
Curran T
(1997)
Reelin is a secreted glycoprotein recognized by the CR-50 monoclonal antibody.
J Neurosci
17:23-31.
-
de Bergeyck V,
Nakajima K,
Lambert de Rouvroit C,
Naerhuyzen B,
Goffinet AM,
Miyata T,
Ogawa M,
Mikoshiba K
(1997)
A truncated Reelin protein is produced but not secreted in the "Orleans" reeler mutation (Reln[rl-Orl]).
Brain Res Mol Brain Res
50:85-90.
-
de Bergeyck V,
Naerhuyzen B,
Goffinet AM,
Lambert de Rouvroit C
(1998)
A panel of monoclonal antibodies against reelin, the extracellular matrix protein defective in reeler mutant mice.
J Neurosci Methods
82:17-24.
-
Deller T,
Drakew A,
Frotscher M
(1999)
Different primary target cells are important for fiber lamination in the fascia dentata: a lesson from reeler mutant mice.
Exp Neurol
156:239-253.
-
Del Rio JA,
Heimrich B,
Borrell V,
Forster E,
Drakew A,
Alcantara S,
Nakajima K,
Miyata T,
Ogawa M,
Mikoshiba K,
Derer P,
Frotscher M,
Soriano E
(1997)
A role for Cajal-Retzius cells and reelin in the development of hippocampal connections.
Nature
385:70-74.
-
Gosh A
(1997)
Axons follow Reelin routes.
Nature
385:23-24.
-
Lambert de Rouvroit C,
Goffinet AM
(1998)
The reeler mouse as a model of brain development.
Adv Anat Embryol Cell Biol
150:1-106.
-
Lambert de Rouvroit C,
de Bergeyck V,
Cortvrindt C,
Bar I,
Eeckhout Y,
Goffinet AM
(1999)
Reelin, the extracellular matrix protein deficient in reeler mutant mice, is processed by a metalloproteinase.
Exp Neurol
156:214-217.
-
Lumsden AG,
Davies AM
(1983)
Earliest sensory nerve fibres are guided to peripheral targets by attractants other than nerve growth factor.
Nature
306:786-788.
-
Métin C,
Deléglise D,
Serafini T,
Kennedy TE,
Tessier-Lavigne M
(1997)
A role for netrin-1 in the guidance of cortical efferents.
Development
124:5063-5074.
-
Toran-Allerand CD
(1990)
Long-term organotypic culture of central nervous system in Maximow assemblies.
In: Methods in neurosciences, Vol 2, Cell culture (Conn PM,
ed), pp 275-296. San Diego: Academic.
-
Trommsdorf M,
Gotthardt M,
Hiesberger T,
Shelton J,
Stockinger W,
Nimpf J,
Hammer RE,
Richardson JA,
Herz J
(1999)
Reeler/disabled-like disruption of neuronal migration in knockout mice lacking the VLDL receptor and ApoE receptor 2.
Cell
97:689-701.
Copyright © Society for Neuroscience 0270-6474//$05.00/0
This article has been cited by other articles:
|
|
|
|
 
T. Matsuki, A. Pramatarova, and B. W. Howell
Reduction of Crk and CrkL expression blocks reelin-induced dendritogenesis
J. Cell Sci.,
June 1, 2008;
121(11):
1869 - 1875.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Alcantara, E. Pozas, C. F. Ibanez, and E. Soriano
BDNF-modulated Spatial Organization of Cajal-Retzius and GABAergic Neurons in the Marginal Zone Plays a Role in the Development of Cortical Organization
Cereb Cortex,
April 1, 2006;
16(4):
487 - 499.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. R. Senechal, C. Thaller, and J. L. Noebels
ADPEAF mutations reduce levels of secreted LGI1, a putative tumor suppressor protein linked to epilepsy
Hum. Mol. Genet.,
June 15, 2005;
14(12):
1613 - 1620.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
B. P. Rubin, R. P. Tucker, M. Brown-Luedi, D. Martin, and R. Chiquet-Ehrismann
Teneurin 2 is expressed by the neurons of the thalamofugal visual system in situ and promotes homophilic cell-cell adhesion in vitro
Development,
March 12, 2003;
129(20):
4697 - 4705.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
.gif?ad=17923&adview=true)
TOP
ABSTRACT
INTRODUCTION
