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First published online 12 April 2006
doi: 10.1242/dev.02354
1 Division of Behavior and Neurobiology, National Institute for Basic Biology,
Myodaijicho, Okazaki 444-8585, Japan.
2 CREST, Japan Science and Technology, Japan.
3 Laboratory of Neuroscience, Graduate School of Frontier Biosciences, Osaka
University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
Author for correspondence (e-mail:
murakami{at}fbs.osaka-u.ac.jp)
Accepted 9 March 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Tangential migration, Precerebellar neurons, LRN, ECN, Cadherin, Electroporation, Rat, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Marín and Rubenstein,
2003). Many studies at molecular and cellular levels have
identified several mechanisms that control the direction, final position and
motility of the migrating neurons and proposed two major mechanisms,
contact-dependent and contact-independent regulations (reviewed by
Hatten, 1999;
Marín and Rubenstein,
2003). The latter is mediated by attractive or repulsive
activities of diffusible molecules, which have been shown to regulate the
direction of neuronal migration (Alcantara
et al., 2000; Causeret et al.,
2002; de Diego et al.,
2002; Lu et al.,
2001; Marín et al.,
2001; Taniguchi et al.,
2002; Yee et al.,
1999; Zhu et al.,
1999). The former can be mediated by cell-adhesion molecules
(CAMs), which have been shown to positively control neuronal migration by
enhancing neuronal motility (Marín
and Rubenstein, 2003). NCAM, a neural cell-adhesion molecule that
belongs to immunoglobulin superfamily (IgSF), for example, regulates the
migration of olfactory interneurons via its conjugated polysialic acids
(Hu et al., 1996;
Ono et al., 1994); TAG1, an
axonal glycoprotein that is also a member of IgSF, also positively regulates
tangential migration of cortical GABAergic interneurons and a subset of
precerebellar neurons in the caudal hindbrain
(Denaxa et al., 2001;
Kyriakopoulou et al., 2002).
However, our knowledge on the roles of CAMs in neuronal migration is still
limited.
Classic cadherins, Ca2+-dependent homophilic CAMs (called as
cadherins hereafter unless otherwise noted), are potential candidates to be
tested for their involvement in contact-dependent neuronal migration, as they
are expressed throughout the developing nervous system and have been shown to
play important roles in several processes of neuronal development in both
vertebrates and invertebrates (Inoue et
al., 1997; Matsunami and
Takeichi, 1995; Redies and
Takeichi, 1996; Iwai et al.,
2002). In the fruit fly, for example, Drosophila
N-cadherin mediates the axonal fasciculation of the ventral nerve cord and the
target recognition between retinal axons and optic medulla
(Iwai et al., 2002;
Lee et al., 2001). In the
vertebrate, cadherins regulate the formation and maintenance of brain
compartments (Inoue et al.,
2001), axonal elongation
(Matsunaga et al., 1988;
Riehl et al., 1996) and
synapse formation (Togashi et al.,
2002). Their potential roles in neuronal migration were tested in
the neural crest cells, which derive from the dorsal neural tube and migrate
towards their peripheral targets (Nakagawa
and Takeichi, 1998), and it was suggested that cadherins control
the timing of neural crest cells emergence from their origin rather than the
maintenance of migration. Nevertheless, there still remains the possibility
that cadherins are involved in the neuronal migration in the brain.
In this study, we have examined the role of cadherins in the neuronal
migration using the lateral reticular nucleus (LRN) and external cuneate
nucleus (ECN) neurons that relay information to the cerebellum as a model
system. Precerebellar neurons, including the LRN/ECN neurons, originate from
the embryonic lower rhombic lip located at the most dorsal site of the caudal
hindbrain and migrate towards the ventral midline floor plate (FP) along the
marginal or submarginal pathway (Altman and
Bayer, 1987a; Altman and Bayer,
1987b; Altman and Bayer,
1987c; Altman and Bayer,
1987d; Bourrat and Sotelo,
1988; Bourrat and Sotelo,
1990). We first characterize the expression pattern of mRNAs for
cadherins in the migrating LRN/ECN neurons in vivo and then show that
expression of DN forms of cadherins in these neurons perturbs their migration
both in vitro and in vivo. The perturbation of migration is not caused by
altered TAG1 expression, which has been shown previously to be involved in the
migration of these neurons (Kyriakopoulou
et al., 2002), or by the change in responsiveness to the guidance
molecules from the FP that attract them. These findings provide the first
evidence that cadherins positively control the neuronal migration in the
CNS.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) with
minor modifications. Briefly, the myelencephalon taken from an embryonic day
14 (E14) Wistar rat was cut along the dorsal midline and opened onto membrane
filters (Millipore, pore size, 10 µm). Explants were embedded with collagen
gels and cultured on the membranes floating on the culture medium at 37°C
in a 5% CO2 and 95% humidity incubator. Collagen gel co-culture was
performed as described before (Taniguchi
et al., 2002). Images were taken by an epifluorescent microscope
equipped with a camera or a confocal microscope (LSM510, Zeiss).
Immunohistochemistry
Cultured explants and hindbrains from embryos manipulated with exo utero
electroporation were fixed overnight with 4% paraformaldehyde (PFA) in 0.12 M
phosphate buffer (PB) (pH 7.4) at 4°C. The fixed whole hindbrains, in
which enhanced green fluorescent protein (EGFP)-NCAD(t) was transferred by exo
utero electroporation, were then blocked with 10% normal goat serum in PBS
containing 0.3% Triton X-100, followed by the incubation with a rabbit
polyclonal antibody against GFP (1:1000, Molecular Probes) and an Alexa
488-conjugated anti-rabbit IgG antibody (Molecular Probes). Cryostat sections
(20 µm) were made from the cultured explants or whole hindbrains that had
been fixed and immersed overnight in 30% sucrose/4% PFA at 4°C. The
sections were placed on poly-L-lysine-coated slides and blocked with 10%
normal goat serum in PBS containing 0.1% Triton X-100, followed by the
incubation with primary and secondary antibodies. Primary antibodies used were
a rabbit polyclonal antibody against GFP (1:1000, Molecular Probes), a mouse
monoclonal antibody (mAb) against pan-cadherin (CH-19; 1:500; Sigma-Aldrich),
a mouse mAb against FLAG (M2; 1:750; Sigma) and a mouse mAb against TAG1 (4D7;
1:30; Developmental Studies Hybridoma Bank). Secondary antibodies used were a
Cy3-conjugated goat anti-mouse IgG (1:300; Jackson), a Cy3-conjugated goat
anti-mouse IgM (1:300; Jackson) and an Alexa 488-conjugated anti-rabbit IgG
antibody (Molecular Probes).
In situ hybridization
To obtain cDNA fragments for Ecad, VEcad, Rcad, BRcad, Cad8, Cad11,
Mcad and Cad6, we performed PCR using cDNAs from E15 rat brains
or E14 whole rat embryos as templates. Following primers were used:
Ecad, (forward) 5'-ggagggttttcactggttgttgg-3' and
(reverse) 5'-catctgcctaagaatcaggtgtgc-3'; VEcad,
(forward) 5'-cgccagaatgctaagtatgtgcta-3' and (reverse)
5'-gattgagtaaagacggggaagttg-3'; Rcad, (forward)
5'-cagcacactctgaacacaagaaagg-3' and (reverse)
5'-tgatgaactcgggacggttg-3'; BRcad, (forward)
5'-cctgcttctctgggttctgtttg-3' and (reverse)
5'-ctggcactgtttccataggtcg-3'; Cad8, (forward)
5'-acgcttttggacctctggactc-3' and (reverse)
5'-gggcattgtcgttgatgtcttg-3'; Cad11, (forward)
5'-cattctctcaggtgaaggagcg-3' and (reverse)
5'-actttggtgggttgtcgttgac-3'; Mcad, (forward)
5'-cctaagaccaatgagggtgtgc-3' and (reverse)
5'-caaaaagggtgaagcagggc-3'; Cad6, (forward)
5'-tcttgctgctcttttgggtcg-3' and (reverse)
5'-catcggcatcagttgctgtgac-3'. All PCR products were subcloned into
the pGEM-T Easy vector (Promega). A SacI/EcoRI fragment of
rat Ncad (a kind gift from Dr K. Asai) was subcloned into the
pBluescriptSK-. Sense or antisense cRNA probes labeled with digoxigenin
(DIG)-11-UTP were prepared by in vitro transcriptions with SP6 RNA polymerase,
T7 RNA polymerase or T3 RNA polymerase.
Brains were dissected out from E15 Wistar rat embryos and fixed overnight with 4% PFA in 0.12 M PB, followed by immersion in 30% sucrose/4% PFA at 4°C overnight. They were embedded in OCT Compound (Tissue-Tek), frozen in a cryostat at -20°C and cut into 20 µm sections. The sections were collected on poly-L-lysine-coated slides and dried for 3 hours. After rehydration in PBS, they were postfixed with 4% PFA in 0.12 M PB for 10 minutes (min) and washed with KPBS [20 mM KH2PO4, 150 mM NaCl (pH 7.3)]. They were then treated with 10 µg/ml proteinase K in 100 mM Tris-Cl (pH 8.0), 50 mM EDTA at 37°C for 5 minutes and fixed again for 10 minutes, followed by a wash with KPBS for 20 minutes. After brief washes with distilled water and then 0.1 M triethanolamine (pH 8.0), they were acetylated with 0.25% acetic anhydride, 0.1 M triethanolamine for 10 minutes. They were next washed with 2xSSC [1xSSC; 150 mM NaCl, 15 mM Na citrate (pH 7.0)] for 4 minutes, dehydrated with 50%, 70%, 95% and 100% ethanol sequentially for 3 minutes each, and dried in the air. After preincubation with hybridization buffer [50% formamide, 10% dextran sulfate, 300 mM NaCl, 1xDenhardt's solution, 10 mM Tris-Cl (pH 8.0), 1 mM EDTA, 500 µg/ml yeast tRNA, 0.5% SDS] at 56°C for 1 hour, they were hybridized with cRNA probes labeled with DIG at 56°C for 16 hours. After hybridization they were washed with 4xSSC three times for 20 minutes each and treated with 20 µg/ml RNase in 1 M NaCl, 10 mM Tris-Cl (pH 8.0), 1 mM EDTA at 37°C for 30 minutes, followed by two washes with 2xSSC each for 5 minutes, 1xSSC for 5 minutes, 0.5xSSC for 5 minutes, 0.05xSSC at 70°C for 30 minutes and 0.05xSSC for 3 minutes. They were then washed with solution 1 [100 mM Tris-Cl (pH 7.5), 150 mM NaCl], blocked with solution 2 [1% blocking reagent (Roche) in solution 1] for 1 hour and incubated with an anti-DIG antibody conjugated with alkaline phosphatase (1:1000; Roche) at 4°C overnight. After extensive washes with solution 1, they were briefly washed with solution 3 [100 mM Tris-Cl (pH 9.5), 100 mM NaCl, 50 mM MgCl2, 1 mM levamisole] and then incubated with detection solution [2% NBT/BCIP stock solution (Roche), 10% polyvinyl alcohol (70-100kDa) in solution 3]. The detection was stopped by incubation with Tris-EDTA [100 mM Tris-Cl (pH 8.0), 50 mM EDTA]. Finally the sections were briefly washed with distilled water. All steps were performed at room temperature unless otherwise noted. Images were taken by an epifluorescent microscope equipped with a camera.
Expression vectors
The whole coding regions for the FLAG-tagged chicken NCAD (NCAD-FLAG),
FLAG-tagged mouse CAD11 (CAD11-FLAG), FLAG tagged dominant negative chicken
NCAD (cN390
-FLAG) (all FLAG-tagged cadherin cDNAs are kind gifts from
Dr M. Takeichi) were cloned into the pCAGGS vector (a kind gift from J.
Miyazaki), yielding pCAGGS-NCAD-FLAG, pCAGGS-CAD11-FLAG and
pCAGGS-cN390-FLAG. The cytoplasmic form of DN-NCAD
[pCAGGS-EGFP-NCAD(t)] was constructed by fusing the intracellular domain of
rat NCAD with EGFP (Clontech). The coding sequences for EGFP, DsRed (Clontech)
and Venus YFP (a kind gift from Dr A. Miyawaki) were cloned into the pCAGGS
vector to yield pCAGGS-EGFP, pCAGGS-DsRed and pCAGGS-Venus YFP,
respectively.
Gene transfer by electroporation in the flat whole-mount culture
The gene transfer based on electroporation in rat embryos was carried out
as described previously with minor changes
(Taniguchi et al., 2002). In
brief, pCAGGS-EGFP and one of the cadherin constructs used in the present
study were mixed at 2.5 mg/ml each in PBS with 0.02% Trypan Blue. In the
experiments to confirm co-expression, pCAGGS-DsRed and pCAGGS-EGFP-NCAD(t)
were mixed. The cDNA solution was injected into the fourth ventricle of rat
embryos using a capillary glass pipette. Electric pulses (voltage: 40 V;
duration: 50 mseconds; 12 pulses) were applied in PBS by using forceps-shaped
electrodes. Although we found that one side (right side) of the lower rhombic
lips was predominantly labeled with EGFP, the other side (left side)
occasionally showed weak GFP labeling. To avoid mixing of the labeled neurons
derived from both sides, the left rhombic lip was removed before culture.
Exo utero electroporation
Exo utero electroporation was performed as described previously
(Kawauchi et al., 2006). In
brief, either pCAGGS-Venus YFP or pCAGGS-EGFP-NCAD(t) was injected into the
fourth ventricle of E12.5 mouse embryos that had been exposed by cutting the
uterine wall. The DNA solution was mixed with 0.01% Fast Green or Indigo
Carmine for visualization of the injection. Electric pulses (20 V, 50
mseconds, three pulses) were applied by using an electroporator. Embryos were
taken out of pregnant mice at E15.5 or E18.5. Embryos that have the
unilaterally labeled lower rhombic lip were selected for analysis and their
whole hindbrains were imaged using an epifluorescent microscope (VB-G25,
KEYENCE) after dissection and fixation.
Quantification
The fluorescent and bright images of the same area were taken with a CCD
camera (C4880-40-26A, Hamamatsu Photonics) for the analysis. Bright-field
illumination was used to identify the position of the ventral midline (VM).
For quantification we first determined a 150 µm x 300 µm
rectangular area that is the farthest from the VM but includes more than 10
migrating neurons, and then measured the distance between the VM and the
forefront of the cells which reside within the rectangle.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Overexpression of cadherin constructs in the flat whole-mount culture by electroporation
Previously, we developed a flat whole-mount culture preparation of the rat
medulla, in which migrating LRN/ECN neurons were almost exclusively labeled
with EGFP by introducing an Egfp cDNA into the lower rhombic lip by
electroporation (Fig. 2A)
(Taniguchi et al., 2002). In
this culture, LRN/ECN neurons migrated just beneath the pial surface towards
the contralateral side over a distance of 2 mm in 3 days in vitro (div). We
thought that this in vitro system would enable us to study the role of
cadherins in the neuronal migration if cadherin constructs could be
co-expressed with EGFP in the neurons. First, we introduced EGFP into the flat
whole-mount preparation and after 2 div it was immunostained with an antibody
for pan cadherin to examine expression of endogenous cadherins in LRN/ECN
neurons. LRN/ECN neurons migrating just beneath the pial surface expressed
cadherin proteins (Fig.
2B-D).
We next examined if EGFP and the cadherin constructs prepared here could be
co-expressed in LRN/ECN neurons. Full-length NCAD, CAD11 and the
membrane-bound form of DN cad (cN390) were tagged with FLAG to detect
their expression. EGFP was used as a tag to detect the cytoplasmic form of DN
cad [EGFP-NCAD(t)]. Each tagged cadherin construct was introduced into the
flat whole-mount preparations together with EGFP, except for EGFP-NCAD(t),
which was co-expressed with DsRed. After 2 div, many LRN/ECN neurons expressed
FLAG-tagged constructs and EGFP (Fig.
2E,F). EGFP-NCAD(t) was also well co-expressed with DsRed in the
migrating neurons (Fig. 2G). As
early as the first day in vitro, LRN/ECN neurons that have just left the
rhombic lip expressed these cadherins and fluorescent markers (data not
shown). Thus, cadherin constructs can be co-expressed with fluorescent
proteins in migrating LRN/ECN neurons, which endogenously express
cadherins.
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-FLAG, which should
suppress the function of all classic cadherins by sequestering endogenous
ß-catenins, appeared to slow migration of LRN/ECN neurons down
(Fig. 3D,
Fig. 4, compare with
Fig. 3A-C). A similar but
stronger phenotype was observed when EGFP-NCAD(t), a cytoplasmic form of a DN
construct that should have the same function as cN390-FLAG, was used
(Fig. 3E,
Fig. 4). One might argue that
the phenotype resulted from disruption of local interactions between LRN/ECN
neurons and the FP, which play an important role in their midline crossing
(Taniguchi et al., 2002),
rather than slowed migration. If this is the case, it should be expected that
the phenotype occurs only when they cross the ventral midline. However, the
delayed migration of neurons expressing EGFP-NCAD(t) occurred before they
reached the ventral midline, refuting this possibility (compare
Fig. 5C,D with
Fig. 5A,B). Taken together,
these results indicate that classic cadherins are involved in the initiation
and maintenance of migration of LRN/ECN neurons in the medulla.
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). In these experiments, inhibition of the TAG1 function
disrupted proper migration of LRN/ECN neurons in vitro. To test if TAG1
expression could be influenced by cadherin function, EGFP-NCAD(t) introduced
preparations were fixed at 2 div and immunostained for TAG1. We found no
difference in the expression of TAG1 between EGFP control neurons
(Fig. 8A-C) and EGFP-NCAD(t)
LRN/ECN neurons (Fig. 8D-F).
Thus, suppression of cadherin function in LRN/ECN neurons does not cause
changes in the TAG1 expression.
The FP promotes migration of precerebellar neurons by secreting guidance
factors, including netrin 1 (Alcantara et
al., 2000; de Diego et al.,
2002; Taniguchi et al.,
2002; Yee et al.,
1999). LRN/ECN neurons can change their responsiveness to FP cues
during tangential migration of these neurons
(Taniguchi et al., 2002).
Therefore, one might speculate that disruption of cadherin function might
change their responsiveness to FP-derived guidance molecules. To examine this
possibility, an explant from the E14 rat dorsal medulla electroporated with
either EGFP or a mixture of EGFP and EGFP-NCAD(t) was cultured with an FP
explant for 2 days in collagen gels. In this co-culture most of EGFP labeled
neurons emigrating from the dorsal medulla explant have been shown to be TAG1-
and DCC-positive precerebellar neurons
(Taniguchi et al., 2002).
EGFP-NCAD(t) expressing neurons emigrated from the dorsal medulla explant
toward the FP, similarly to those expressing EGFP alone
(Fig. 9), indicating that their
responsiveness to the FP is unaltered. Taken together, these results suggest
that cadherins regulate LRN/ECN neuron migration by directly modulating cell
adhesion, rather than via changes of their TAG1 expression or responsiveness
to FP attractive cues.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Marín and Rubenstein,
2003). Each group of migrating neurons in different areas of CNS
seems to be guided by both mechanisms (reviewed by
Hatten, 1999;
Marín and Rubenstein,
2003). In the hindbrain, LRN/ECN neurons are attracted by the FP
in the vicinity of the ventral midline
(Alcantara et al., 2000;
de Diego et al., 2002;
Taniguchi et al., 2002).
Several lines of evidence also suggest that contact-dependent mechanisms might
also play an important role in the control of their migration. First,
migrating LRN/ECN neurons form a chain-like structure as if they use
themselves as a migration substrate
(Alcantara et al., 2000;
Taniguchi et al., 2002).
Second, it has been suggested that the axons running just beneath the
migratory stream of LRN/ECN neurons confer the substrate on the migrating
neurons (Kyriakopoulou et al.,
2002; Ono and Kawamura,
1989). Third, blocking the TAG1 function perturbs the migration of
LRN/ECN neurons (Kyriakopoulou et al.,
2002). However, molecular mechanisms for the contact-dependent
regulation have remained elusive. In the present study, we showed that mRNA
for CAD6, CAD8, CAD11 and NCAD were expressed in the migratory streams of
LRN/ECN neurons. The expression of cadherin mRNAs together with their
homophilic adhesive property prompted us to hypothesize that they regulate the
migration of LRN/ECN neurons.
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). This
method enabled us to visualize the entire morphology of individual neurons,
and thus to clarify the effect of cadherin constructs on their migration.
Consistent with the proposed hypothesis, overexpression of two different types
of DN cadherin constructs in the lower rhombic lip of the caudal hindbrain
perturbed the migration of LRN/ECN neurons. This phenotype appeared to be due
to a defect not only in initiation but also in maintenance of migration,
because the difference in the migration distance between control cells and
those expressing DN-constructs became more evident after 3 div compared with 1
div. Importantly, a similar phenotype was also observed in vivo when a
cytoplasmic form of a DN cadherin or an shRNA for NCAD was expressed in the
LRN/ECN neurons using exo utero electroporation. These results provide the
first compelling evidence suggesting that classic cadherins control initiation
and maintenance of neuronal migration.
What is the mechanism by which overexpression of DN constructs influenced
the migration of LRN/ECN neurons? One possibility is that the cytoplasmic
domain of the cadherin constructs might disrupt the Wnt signaling pathway by
sequestering ß-catenin, which is one of its canonical components. This is
unlikely because DN constructs but not full-length constructs caused a
phenotype in our experiments, despite the fact that they have the same
cytoplasmic sequence and similar activity to suppress the transactivation by
ß-catenins (Sadot et al.,
1998).
We have found that a cytoplasmic form of a DN construct, EGFP-NCAD(t),
caused more pronounced phenotype than a membrane-bound form of a DN construct,
cN390. One possible explanation could be that EGFP-NCAD(t) affected the
ß-catenins signaling pathway in addition to homophilic interactions
mediated by cadherins, because a cytoplasmic form suppresses the
transactivation by ß-catenins more strongly than a membrane-bound form
(Sadot et al., 1998). Another
could be that EGFP-NCAD(t) is able to sequester ß-catenins more
efficiently than cN390 localized in the plasma membrane as it is
expected to be localized diffusely in the cytoplasm, thereby interfering with
cadherin interactions more potently.
One might argue that overexpression of DN constructs might have changed the
TAG1 expression, which has been implicated in the migration of LRN/ECN neurons
(Kyriakopoulou et al., 2002).
This, however, is unlikely, because overexpression of DN constructs did not
alter TAG1 expression in LRN/ECN neurons.
The migration of LRN/ECN neurons from the dorsal medulla explants elicited by an FP explant in collagen gel co-cultures was not blocked by overexpression of DN constructs. Furthermore, in our flat whole-mount culture, LRN/ECN neurons expressing DN constructs still reached the ventral midline, with some delay, after 3 div and had ability to turn toward the ectopic FP placed laterally to their migratory stream (data not shown). These results negate the possibility that DN constructs altered the responsiveness of LRN/ECN neurons to FP cues. Therefore, the most plausible mechanism for the effect of DN constructs on the migration of LRN/ECN neurons would be disruption of homophilic interactions through classic cadherins between migrating neurons and/or they and accompanying axonal fibers.
It has been shown that switching a class of cadherins from NCAD and CAD6B
to CAD7 plays a crucial role in the emigration of neural crest cells from the
dorsal neural tube: when full-length NCAD or CAD7 were overexpressed in the
neural tube, they failed to emigrate from neuroepithelial cells
(Nakagawa and Takeichi, 1998).
These results contrasts with our findings that overexpression of full-length
cadherins was insufficient to affect the migration of LRN/ECN neurons
implicating the failure to form additional strong adhesion complexes. It is
also of note that appearance of chain-like structures composed of migrating
LRN/ECN neurons was apparently unaltered when DN cadherins or full-length
cadherins were expressed. Our results suggest that classic cadherins promote
the motility of migrating LRN/ECN cells rather than stabilizing them despite
their adhesive property. What, then, is the rational explanation for this
difference? It has been proposed that their conformational changes and
interactions with intracellular proteins such as ß-catenins can modulate
strength of cadherin-mediated adhesion
(Tanaka et al., 2000;
Barth et al., 1997). These
mechanisms could work in migrating LRN/ECN neurons so that weak rather than
strong adhesive interactions are triggered, promoting their gliding along a
cadherin-adhesive path.
The technique of exo utero electroporation also allowed us to analyze the
effect of the perturbation of cadherin functions in the formation of nuclei in
vivo. We showed that inhibition of cadherin functions leads to the ipsilateral
formation of LRN/ECN, which normally occurs contralaterally. It is likely that
the ipsilateral formation of the nuclei is a consequence of slowed migration,
because: (1) LRN/ECN neurons acquire the ability to respond to cues for their
entry into prospective nuclear regions in a developmental time-dependent
manner (see Kawauchi et al.,
2006); and (2) early emigrating EGFP-NCAD(t) neurons do not form
nuclei on the ipsilateral side (D.K. and F.M., unpublished). The regulation of
migration speed therefore may be important for nucleogenesis at a proper
site.
In conclusion, our results demonstrate that classic cadherins play an
essential role in contact-dependent regulation for the tangential migration of
LRN/ECN neurons. We propose that the regulated adhesion and signaling mediated
through the homophilic interaction of cadherins provide the migrating neurons
with forces to move forwards. Interestingly, it has been reported that the
migration of Purkinje cells is disorganized in 
-Ncatenin knockout mice,
suggesting the importance of cadherin function
(Togashi et al., 2002). Thus,
classic cadherins might have a general role as substrates not only in the
tangential migration as presented in this study but also the radial migration,
as seen in the cerebrum and cerebellum.
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ACKNOWLEDGMENTS |
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|
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/10/1923/DC1
* Present address: Cold Spring Harbor Laboratory, Beckman 220, One Bungtown
Road, Cold Spring Harbor, NY, 11724, USA 
Department of Developmental Biology, Graduate School of Medicine, Chiba
University, Chiba 260-8670, Japan
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