1 Department of Anatomy and Developmental Biology, University College London,
London WC1E 6BT, UK
2 Department of Endocrinology, Centre of Excellence on Neurodegenerative
Diseases, University of Milan, 20133 Milan, Italy
3 Developmental Neurobiology Unit, University of Louvain Medical School, B1200
Brussels, Belgium
* Author for correspondence (e-mail: j.parnavelas{at}ucl.ac.uk)
Accepted 9 August 2005
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SUMMARY |
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Key words: GnRH neurons, Migration, Reelin
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Introduction |
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GnRH neurons originate in the nasal compartment at the level of the medial
olfactory placode where they are identified by the production of GnRH mRNA or
the peptide early in embryonic life (E10-11 in mouse; E12-13 in rat)
(Schwanzel-Fukuda and Pfaff,
1989; Wray et al.,
1989a
; Wray et al., 1998b;
Tobet et al., 2001
). They
migrate in association with olfactory/vomeronasal nerves (VNN) until they pass
the cribriform plate. The fascicles of VNN split at this level, with the
majority entering the main and accessory olfactory bulbs; the remaining axons,
the so-called caudal VNN (cVNN), take a caudal and ventral turn and enter the
basal forebrain (Schwarting et al.,
2004
). These pioneer axons can be labelled with the intermediate
filament marker peripherin (Wray et al.,
1994
). Migrating GnRH neurons follow the cVNN into the basal
forebrain where they detach from the guiding fibres and find their positions
in the hypothalamic region by the time of birth in rodents. Only a small
number of these neurons proceeds dorsally and enter the cerebral cortex
(Yoshida et al., 1995
). In the
human, failure of GnRH neurons to migrate normally results in reproductive
dysfunction and delayed or absent pubertal maturation. Kallmann's syndrome
(KS) is a genetic developmental disorder characterized by anosmia and
hypogonadotropic hypogonadism (Seminara et
al., 1998
). In the X-linked form of the disease, the VNN and GnRH
neurons fail to cross the cribriform plate and remain clustered in this area
(Schwanzel-Fukuda et al.,
1989
).
What are the molecular mechanisms that guide GnRH neurons during their long
and tortuous journey from the nasal compartment to the forebrain? A number of
molecules have been shown to affect their migration (reviewed by
Wray, 2001;
MacColl et al., 2002
;
Pimpinelli and Maggi, 2004
;
Wierman et al., 2004
). Amongst
them are: adhesion molecules (NCAM and its polysialylated form PSA-NCAM,
peripherin, TAG-1 and nasal embryonic LHRH factor), secreted molecules (GABA,
netrin-1, HGF), the transcription factor Ebf2
(Corradi et al., 2003
), and the
gene product responsible for the X-linked KS, anosmin-1
(Cariboni et al., 2004
). Most
of these molecules affect the migration of GnRH neurons indirectly by altering
the underlying migratory pathway and nearly all appear to act at the early
stages of their migration within the nasal compartment. The cues that instruct
GnRH neurons to avoid the olfactory bulb and migrate caudally to the basal
forebrain remain largely unknown.
Reelin is an extracellular protein extensively studied for its function in
neuronal migration and lamination in the cerebral and cerebellar cortices
(D'Arcangelo et al., 1995;
Ogawa et al., 1995
;
D'Arcangelo and Curran, 1998
).
It has also been found to have important roles in a number of developmental
events in the hippocampus (Del Rio et al.,
1997
; Niu et al.,
2004
; Zhao et al.,
2004
) and other areas of the CNS
(Lambert de Rouvroit and Goffinet,
1998
), and has been implicated in the migration of autonomic
neurons in the spinal cord (Yip et al.,
2000
). Reelin is highly expressed in the olfactory
system, including the olfactory bulb, vomeronasal organ and VNN
(Ikeda and Terashima, 1997
;
Alcantara et al., 1998
;
Teillon et al., 2003
), and
investigators have recently queried its role in this system. Here, we tested
the hypothesis that reelin acts as a guidance signal for the migration of GnRH
neurons and their disposition in the forebrain. Using immortalized GnRH
neurons as well as in vitro and in vivo experiments in rodents, we found that
reelin does affect the migratory activity and distribution of GnRH-releasing
neurons. Analysis of embryonic and postnatal reeler mouse brains of
both sexes showed a reduction in GnRH neurons in the hypothalamus, providing
an explanation for the observed reduced fertility in these animals
(Caviness et al., 1972
;
Green, 1989
). Finally,
examination of animals mutants for ApoER2/Vldlr and Dab1,
the components of reelin signalling, showed a normal complement of GnRH
neurons, suggesting that this pathway is not involved in mediating the effects
of reelin in their migration to the hypothalamus.
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Materials and methods |
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Brains of embryos, removed from pregnant Sprague Dawley albino rats at
different stages during the last week of gestation (E1, day vaginal plug was
found), were used for the preparation of slice cultures and for
immunofluorescence experiments. Reeler, scrambler and double
Vldlr/ApoER2 mutant brains (provided by A.G.) were fixed by
intracardiac perfusion with 4% paraformaldehyde in PBS and postfixed in the
same fixative solution for 24 hours. Reeler mutant mice (Orleans
allele) were bred by crossing homozygous mutant animals on a mixed,
predominantly CD1 background (Goffinet,
1984; D'Arcangelo et al.,
1997
). Scrambler mutants with a spontaneous inactivation
of Dab1 (Sheldon et al., 1997
;
Ware et al., 1997
) were also
maintained by crossing homozygous animals on a predominant CD1 background. The
Vldlr/ApoER2 mutant mice were previously generated by targeting in ES
cells (Trommsdorff et al.,
1999
) and were bred on mixed 129 and CD1 backgrounds. The testes
of adult (3 months) reeler and wild-type animals were fixed by
intracardiac perfusion with Bouin's fluid and postfixed in the same solution
for 24 hours.
Immunohistochemistry
Embryonic or adult fixed brains were embedded in 3.5% agarose and sectioned
at 60-80 µm with a Vibroslice (Campden Instruments, UK). Sections were
blocked with 5% normal goat serum (Gibco, NY, USA) in PBS + 0.3% Triton X-100
for 30 minutes, and incubated in primary antibody (see list below) diluted in
PBS + 0.3% Triton X-100 for 36 hours. After washes in PBS, sections were
incubated with Alexa FITC- or TRITC-conjugated isotype-specific secondary
antibodies at a dilution of 1:400 for 2 hours at room temperature, washed and
mounted with Citifluor anti-fading solution (Agar, Essex, UK). Preparations
was then examined with a confocal microscope (Leica). Images were
reconstructed using MetaMorph imaging software (Universal Imaging, West
Chester, PA, USA).
For immunoperoxidase experiments, sections were incubated with an anti-rabbit biotinylated secondary antibody and processed using an ABC kit (Vector Laboratories, Burlingame, CA) with 3,3'-diaminobenzidine (0.015%; Sigma) as a chromogen. Sections were then washed in PBS, mounted, dehydrated and coverslips placed on top. Images were taken using black and white camera (Quantix Photometrics, Princeton Instruments, UK) and edited using Adobe Photoshop.
The primary antibodies used were: anti-GnRH (rabbit polyclonal, 1:400 for
immunofluorescence and 1:4000 for immunoperoxidase; Immunostar Inc.,
Wisconsin), anti-peripherin (rabbit polyclonal, 1:4000, Chemicon Int.,
Temecula), anti-reelin (mouse monoclonal, clone G10, 1:500), anti-ApoER2
(mouse monoclonal, clone 2H8, 1:50), anti-Dab1 (mouse monoclonal, clone L2,
1:500), anti-3 integrin (mouse monoclonal, clone MAB1952Z, 1:500;
Chemicon Int.), anti-EGFP (rabbit polyclonal, 1:400; Molecular Probes, Eugene,
OR) and anti-beta III tubulin (mouse monoclonal, 1:500; Sigma, UK).
Histology of testes
The testes of reeler and wild-type mice were embedded in paraffin
wax, and serially sectioned at 10 µm. Sections were deparaffinized,
dehydrated and stained with Haematoxylin and Eosin. The number of seminiferous
tubules was assessed by counting three random 1 mm2 fields of
testes derived from two reeler and two wild-type animals.
Preparation of slice cultures and application of fluorescent tracers
Pregnant Sprague Dawley albino rats at E18 (n=3) were killed by
cervical dislocation. The foetuses were rapidly removed and placed in Gey's
balanced salt solution medium supplemented with glucose (6.5 mg/ml) at
4°C. Brain slices were prepared as described previously (Nadarajah et al.,
2002). Briefly, brains embedded in 3% low-melting point agarose (Sigma) were
sectioned in the sagittal plane in ice-cold oxygenated artificial
cerebrospinal fluid (ACSF), pH 7.4, at 300 µm using a Vibroslice (Campden
Instruments). Slices, proceeding from lateral to medial forebrain, were
mounted onto porous nitrocellulose filters (0.45 µm; Millipore, London, UK)
and transferred to 24-well culture plates.
To label the population of neurons arising or passing through the olfactory
bulb, tungsten particles coated with a fluorescent tracer, 4-chloromethyl
benzoyl amino tetramethyl rhodamine (CMTMR; Molecular Probes) were applied
adjacent to the olfactory bulb (Fig.
2A) using micropipettes as described by Alifragis et al.
(Alifragis et al., 2002).
Following application of particles, slices were placed in an incubator for 48
hours in culture medium containing DMEM (Sigma), 5% N-2 (Gibco), 100 µM
heat-inactivated foetal bovine serum (Gibco), 1x µM L-glutamine, 2.4
g/l D-glucose and penicillin/streptomycin (1:1000, Sigma). For the
reelin-blocking experiments, monoclonal anti-reelin antibody (clone CR-50;
kindly provided by Kazunori Nakajima, Keio University, Japan) was added to the
culture medium every 6 hours at a dilution of 1:100
(Ogawa et al., 1995
;
Miyata et al., 1997
;
Nakajima et al., 1997
). After
incubation, sections were fixed in 4% paraformaldehyde and mounted on slides
with Citifluor. Images of CMTMR-labelled neurons were examined using a
confocal microscope (Leica) and edited using Photoshop imaging software.
Cell lines
GN11 cells, generously given to R.M. by S. Radovick (University of Chicago,
Chicago, IL, USA), and COS-7 cells (American Type Culture Collection, ATCC,
Manassas, VA, USA) were grown as a monolayer at 37°C in a humidified
CO2 incubator in Dulbecco's MEM containing 1 mM sodium pyruvate,
100 mg/ml streptomycin, 100 U/ml penicillin and 10 mg/l of phenol red
(Biochrom KG, Berlin, Germany) and supplemented with 10% foetal bovine serum
(FBS; Gibco, Grand Island, NY, USA). The medium was replaced at 2-day
intervals. Subconfluent cells were harvested by trypsinization and cultured in
57 cm2 dishes (at a density of 1x105 and
2.5x105 for GN11 and COS-7 cells, respectively). Cells within
six passages were used in all experiments. A stable line of GN11 cells
expressing EGFP (GN11-EGFP) was obtained by G418 selection following
transfection with the pEGFP-N2 plasmid (ClonTech, La Jolla, CA,
USA) (Cariboni et al.,
2004).
Collagen gel co-cultures
Cell aggregates of GN-EGFP cells were prepared by the `hanging drop'
technique as described previously (Kennedy
et al., 1994). Rat tail collagen solution was prepared as
described by Guthrie and Lumsden (Guthrie
and Lumsden, 1994
). Collagen gels were generated by adding 10%
(v/v) of 10x concentrated DMEM (without phenol red) and 0.8 M sodium
bicarbonate to an aliquot of collagen stock solution. Pairs of olfactory bulbs
were dissected from E18 rats brains and cut into pieces (explants) of 100-300
µm in diameter; explants and GN11-EGFP cell aggregates were subsequently
plated on four-well dishes coated with poly-L-lysine and overlaid with 80
µl of collagen solution. Following polymerization (30 minutes), the
preparation was covered with 500 µl of serum-free medium containing B-27
supplement (Gibco) and cultured in a humidified, 5% CO2, 37°C
incubator. After 48 hours in vitro, olfactory bulb explants and GN11 cell
aggregates were fixed in 4% paraformaldehyde and immunostained with a
monoclonal anti-beta III tubulin antibody (Sigma) and a polyclonal anti-EGFP
antibody, respectively. Cell displacement away from the aggregate was studied
by estimating the difference in the percentage of migrating cells present in
the proximal (P) and distal (D) quadrants as illustrated in
Fig. 2B.
For the reelin-blocking experiments, 1 µl of CR-50 antibody (100 µg/ml, diluted in culture medium) was added 3 times a day during the 48 hours incubation period.
This analysis was performed blindly with the aid of the Metamorph image analysis system (Universal Imaging Corporation). Statistical analysis was performed using the Prism4 program (GraphPad Software Inc., San Diego, CA, USA).
Production of reelin-enriched conditioned media
Full-length reelin cDNA plasmid (pCRl) was kindly provided by
Gabriella D'Arcangelo (Houston, TX, USA) and Tom Curran (Memphis, TN, USA);
pCDNA3 plasmid was chosen as a control. For transfection, COS-7 cells (at 80%
confluence) were grown in culture plates in complete culture medium for 24
hours and incubated for 3 hours with the selected expression vector (1
µg/ml) in the presence of Lipofectamine-2000 (Life Technologies, MD, USA)
according to the manufacturer's instructions. The expression of the different
constructs was verified by immunofluorescence. Conditioned media from pCRl
(reelin-CM) or pCDNA3 (control-CM) transfected COS-7 cells were
obtained as described by Hack et al. (Hack
et al., 2002). Briefly, transfected COS-7 cells were left in
culture for 48 hours in complete medium; this was then replaced with
serum-free medium and the cells incubated for 24 hours. Cell supernatant was
centrifuged at 3000 g for 5 minutes and immediately used for
microchemotaxis assays. Secretion of reelin was confirmed by
immunohistochemistry and western blot analysis (data not shown).
Chemomigration assays
In order to quantify the migratory activity of GnRH neurons in the presence
of reelin, we performed chemomigration assays on GN11 cells
(Maggi et al., 2000) using a
48-well Boyden's chamber according to the manufacturer's instructions
(Neuroprobe, Cabin John, MD, USA). Briefly, subconfluent cells were suspended
(105 cells/50 µl) in serum-free medium and placed in the
open-bottom wells of the upper compartment of the chamber. These wells were
separated from facing wells of the lower compartment by a
polyvinylpirrolidone-free polycarbonate porous membrane (8 µm pores)
precoated with gelatin (0.2 mg/ml in PBS). To measure chemotaxis (the directed
migration of cells towards a concentration gradient of chemotactic factors),
control-CM and reelin-CM were placed into the lower compartment of the
chamber. Haptotaxis (the directed movement up to a gradient of
substratum-bound chemoattractant) was measured by coating the lower surface of
the gelatin treated-porous membrane with control- or reelin-CM at 4°C for
24 hours.
The chamber was kept in an incubator at 37°C for 3 hours, which is the
minimum time required to attain significant migratory activity of GN11 neurons
(Pimpinelli and Maggi, 2004).
The cells migrated through the pores during incubation, and adhered to the
underside of the membrane. They were subsequently fixed and stained using the
Diff-Quick stain kit (Biomap, Milan, Italy) and mounted onto glass slides. For
quantitative analysis, the membranes were observed using an Olympus light
microscope with a 20x objective. Three random fields of stained cells
were counted for each well, and the mean number of migrating
cells/mm2 for each experimental condition was calculated.
Statistical analysis was performed using the Prism4 program.
RT-PCR
Total RNA was isolated by extraction with TRIzol (Life Technologies,
Inc.).
Single-strand cDNA was synthesized by AMV reverse transcriptase (Promega) and random hexamers (Promega). PCR was carried out using 0.5 µl cDNA and the appropriate oligonucleotides (0.6-6 µM) in 25 µl reaction mix using Taq DNA polymerase (Qiagen) and the following conditions: 35 cycles of denaturing for 60 seconds at 94°C, annealing for 60 seconds at 60°C and extension for 120 seconds at 72°C. In order to verify quality and quantity of RNA retrotranscribed into cDNA, GADPH amplification was carried out under the same conditions (data not shown). PCR products were analysed by electrophoresis on a 2% agarose gel, and bands visualized under UV illumination following ethidium bromide staining. The primers used are listed in Table 1.
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Results |
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Reelin affects the migration of GnRH neurons
In vivo studies of GnRH neurons are hindered by their small number and
widespread distribution in the basal forebrain
(Merchenthaler et al., 1984).
Furthermore, attempts to maintain a pure population in culture have been
unsuccessful (Terasawa et al.,
1993
; Kusano et al.,
1995
). However, a number of cell lines have been obtained by
genetically targeted tumourigenesis of GnRH neurons in mice
(Mellon et al., 1990
;
Radovick et al., 1991
). These
cells express neuronal markers and retain many of the features of
GnRH-secreting neurons (Liposits et al.,
1991
; Wetsel,
1995
; Gore and Roberts,
1997
). One of these lines, GN11, shows a strong chemomigratory
response in vitro and is thought to represent a good model to study the
molecular mechanisms of GnRH neuronal migration
(Maggi et al., 2000
;
Giacobini et al., 2002
;
Pimpinelli et al., 2003
). We
used GN11 cells to study the effects of reelin on the migratory activity of
GnRH neurons.
We first performed co-culture experiments in three-dimensional collagen
gels containing rat E18 olfactory bulb explants, as a source of reelin
(Hack et al., 2002), and
aggregates of GN11-EGFP cells. The co-cultures were maintained for 48 hours
and subsequently fixed, stained and analysed with a confocal microscope
(Fig. 2A). The percentages of
cells that had moved away from the aggregate were estimated for the proximal
and distal quadrants as illustrated in Fig.
2B. Analysis of 24 samples showed that roughly twice as many
GN11-EGFP cells had moved into the distant quadrant, away from the olfactory
bulb explant, than in the proximal quadrant
(Fig. 2C). However, when this
experiment was performed in the presence of reelin-blocking antibody (CR-50),
no difference in the percentage of GN11-EGFP neurons migrating away from the
aggregate was observed (Fig.
2D,E).
To confirm the repulsive activity of reelin in the migration of GN11 cells, we performed chemomigration assays using a 48-well Boyden's chamber, a method that provides a sensitive and quantitative measure of cellular responses to specific chemotropic signals. In these experiments, the effects of reelin on GN11 cells were analysed in terms of their chemotactic and haptotactic responses (see Materials and methods). The chemotactic response of GN11 cells was no different to a gradient of soluble reelin, presented by placing the extracellular protein-enriched conditioned media (CM) into the wells of the lower compartment of the chamber, than to control-CM (Fig. 3A). However, when reelin-enriched CM was adsorbed to the gelatin-coated lower surface of the porous membrane, the haptotactic response of GN11 cells was significantly reduced compared to control-CM (Fig. 3B). These results indicate that reelin exerts an inhibitory effect on the migration of immortalized GnRH neurons, but only when it is bound to the substratum.
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GnRH neurons in reeler, scrambler and reelin receptor mutant mice
Reelin deficiency results in the reeler phenotype. Characteristics
of the mutant include abnormal lamination of cerebral, cerebellar and
hippocampal cortices and neuronal ectopias in other brain areas
(Lambert de Rouvroit and Goffinet,
1998). In mice, inactivating mutations of Dab1
(scrambler mice), and double mutations of ApoER2 and
Vldlr generate reeler-like phenotypes
(Tissir and Goffinet, 2003
).
To confirm the importance of reelin in the migration of GnRH neurons, we
examined brains of developing and adult reeler mice, as well as of
scrambler and double lipoprotein receptor (ApoER2 and
Vldlr) mutants at the adult stage.
|
Analysis of serial sections taken through entire brains of postnatal day (P) seven reeler mice (two males and one female) also revealed a marked reduction of GnRH immunoreactive neurons in the hypothalamus compared to wild-type controls (reeler 304±24.6; wt 528±23.3; n=3; P<0.01; Student's t-test), with very few labelled cells present in the cortex at this stage. By the adult stage, reeler brains contained a paucity of faintly labelled GnRH neurons scattered in the hypothalamic region (Fig. 7A,B,B'). In the reeler median eminence, where the long processes of GnRH neurons normally converge to access the pituitary portal vessels, labelled processes were very few compared to the profuse array of such processes observed in the wild-type animals (Fig. 7C,D). This picture was consistent for all six (3 males and 3 females) adult reeler brains examined. To investigate whether the reduction in the number of GnRH neurons is reflected in alterations in the gonads, we analysed the testes of adult male reeler and wild-type mice (n=2 for each type). There was no gross difference in the weight, volume or appearance of testes between the wild-type and reeler mice. However, histological examination revealed a significant reduction in the density of seminiferous tubules (mean count/mm2 ±s.d.; wt 21.33±5.84; reeler 11.00±2.28; P<0.01; Student's t-test) and dilation of these tubules in reeler animals. Spermatogenesis appeared complete in the seminiferous tubules of both groups of animals and Sertoli cells were present in all samples. In addition, there was no hyperplasia of the Leydig cells, but an apparent decrease in the interstitial tissue in reeler testes (data not shown).
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Discussion |
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Reelin and GnRH neuronal migration
Reelin, an extracellular protein, is present in the nervous system of all
vertebrates and its expression is widespread. Numerous studies have focused on
its role in the developing brain (reviewed by
Curran and D'Arcangelo, 1998;
Rice and Curran, 2001
;
Tissir and Goffinet, 2003
),
particularly in providing positional cues to radially migrating neurons in the
cerebral cortex (D'Arcangelo and Curran,
1998
). Reelin is highly expressed in the olfactory system and
especially in the main (mitral cell layer) and accessory olfactory bulbs
(Alcantara et al., 1998
;
Teillon et al., 2004). In this area, migrating GnRH neurons appear to lose
their association with the olfactory and VNN fibres, change their course and
move caudally along the cVNN to enter the basal forebrain. Furthermore, recent
reports have documented the expression of ApoER2 in the olfactory bulb region
(Perez-Garcia et al., 2004
).
These observations prompted us to hypothesize that reelin acts as a repellent
during the migration of GnRH neurons, guiding them away from the olfactory
bulb and into the basal forebrain.
Functional studies of GnRH-secreting neurons have been facilitated by the
availability of two cell lines of immortalized mouse GnRH-secreting neurons:
GT1 cells [which includes GT-1, -3 and -7 subclones
(Mellon et al., 1990)] and GN
cells [which includes GN10, GN11 and NLT subclones
(Radovick et al., 1991
)].
These cell lines may be considered as postmigratory and migratory GnRH
neurons, respectively. In particular, GN11 cells retain features of
GnRH-secreting neurons, including strong chemomigratory activity in vitro
(Maggi et al., 2000
) and, as
such, have been used to investigate mechanisms involved in GnRH neuron
migration (Giacobini et al.,
2002
; Cariboni et al.,
2004
).
Using collagen gels and microchemotaxis assays, we found that reelin inhibits the migration of GN11 cells. In the collagen gel assay, we used embryonic olfactory bulb explants as a source of reelin and observed that the migration of immortalized GnRH neurons is inhibited. However, when the action of reelin was blocked by CR-50 antibody, no differences were observed in GN11-EGFP cell displacement from the aggregate, supporting the hypothesis for a role of this protein in the migration of GnRH neurons.
More direct evidence was provided by the chemomigration assays that showed the repulsive activity of substrate-bound reelin on GN11 cells. The results of both migration assays are in accordance with the purported nature of reelin as a secreted protein bound to extracellular matrix represented here by the collagen gel matrix or by the gelatin-coated surface of the porous membrane in the Boyden's chamber. Notably, the inhibitory effect on GN11 cells was observed in the haptotaxis but not the chemotaxis assays, pointing to the requirement of a physical substrate for the action of reelin and not to the presence of a soluble gradient of concentration.
The results of these migration assays are consistent with our observations
in cultured embryonic rat brain slices. When the activity of reelin is blocked
with CR-50 antibody in these slices, the migratory path of the majority of
cells labelled with CMTMR at their point of entry into the brain is diverted
to the cerebral cortex instead of the hypothalamic region. It is known that
CR-50 is directed against an N-terminal epitope, interferes with the
aggregation of reelin, and blocks its function in vitro and in vivo
(Ogawa et al., 1995;
Miyata et al., 1997
;
Nakajima et al., 1997
;
D'Arcangelo et al., 1999
;
Kubo et al., 2002
). Although
it was not technically feasible to double label cells for CMTMR and GnRH, it
may be assumed that at least some of the cells labelled with the fluorescent
tracer were indeed neurosecretory neurons. The fate of these neurons in the
cortex, similar to the small number of GnRH neurons that enter the cortical
anlage in normal animals, is presently unknown.
Reception of the classical reelin signal requires the presence of at least
one of two lipoprotein receptors, ApoER2 and Vldlr, as well as the
intracellular adaptor protein Dab1. Using RT-PCR, we found that GN11 cells
express both receptor mRNAs, but in immunohistochemically stained sections of
embryonic brains, a small proportion of GnRH neurons express only ApoER2. This
may be due to the presence of the receptor in one of the different
subpopulations of GnRH neurons (Tobet et
al., 1996) or, alternatively, ApoER2 may be transiently expressed
during migration. Furthermore, we observed that both GN11 cells and GnRH
neurons lacked Dab1. These observations indicate that the repulsive activity
of reelin on migrating GnRH neurons is independent of Dab1 and unlikely to be
mediated by the conventional reelin signalling pathway. Other molecules have
been suggested to bind reelin. Among them, the protocadherin CNR1 has been
proposed to act as a co-receptor for reelin
(Senzaki et al., 1999
), but
this proposal has recently been disproved
(Jossin et al., 2004
). Reelin
has been found to inhibit radial neuronal migration by binding to
3ß1 integrin receptors (Anton
et al., 1999
; Dulabon et al.,
2000
; Schmid et al.,
2005
). However, our results of migration assays using GN11 cells
together with immunohistochemical observations of brain sections suggest that
this integrin is not involved in the reception of the reelin signal by GnRH
neurons.
Mutants of the reelin signalling pathway
Here we demonstrated that the inhibitory activity of reelin is important
for guiding the migration of the GnRH neurons to the basal forebrain. Our
observations in developing and adult reeler mice confirmed the
importance of reelin in the development of these neuroendocrine neurons in the
hypothalamus. We first noted a defect in their migration during embryonic
life, with a significant increase in cells misrouted to the cerebral cortex
instead of the hypothalamus. This resulted in a marked reduction of cell
number in postnatal animals, with adults showing only sparse and faintly
stained GnRH-secreting neurons in the hypothalamus and few processes
projecting to the median eminence. This reduction in hypothalamic GnRH neurons
is likely the cause of the consistently observed delayed pubertal maturation
and low fertility of reeler mice, which was thought to be due to
their ataxic behaviour (Caviness et al.,
1972; Goffinet,
1984
; Green,
1989
). However, our histological analysis of adult reeler
testes showed a clear reduction in the density of seminiferous tubules, but no
hyperplasia of Leydig cells. The lack of hyperplasia of Leydig cells is
indicative of hypogonadotropic hypogonadism rather than an intrinsic defect of
the testes (Mason et al.,
1986
; Corradi et al.,
2003
).
Our in vitro experiments suggest that the repulsive activity of reelin on
the migration of GnRH neurons is not mediated by the classical reelin
signalling pathway. The presence of a normal complement of GnRH-secreting
neurons in the hypothalamus of mutants lacking both lipoprotein receptors and
in scrambler mice lacking Dab1 is consistent with the in vitro
findings. Although we are at present unable to identify a mechanism that
mediates the action of reelin in this neurendocrine system, it is worth noting
that effects of reelin, independent of the conventional reelin signalling
pathway, have recently been described
(Rossel et al., 2005) in the
developing mouse hindbrain. These authors identified a ventral
reelin-dependent migration that is independent of the receptors, ApoER2 and
Vldlr.
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ACKNOWLEDGMENTS |
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