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First published online 1 November 2006
doi: 10.1242/dev.02665
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1 Laboratory for Developmental Gene Regulation, Brain Science Institute, The
Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako,
Saitama 351-0198, Japan.
2 Core Research for Evolutional Science and Technology (CREST), Japan Science
and Technology Corporation (JST), 4-1-8 Honcho, Kawaguchi, Saitama, 332-0012,
Japan.
* Author for correspondence (e-mail: hitoshi{at}brain.riken.jp)
Accepted 28 September 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Zebrafish, frizzled, celsr, Facial motor neuron, Neuroepithelium
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Hatten,
2002). The mechanisms by which neurons select specific layers as
their migrating pathway remain largely unknown.
The zebrafish is a good model to address this issue. In the developing
zebrafish hindbrain, the facial (nVII) motor neurons originate in rhombomere
(r)4 and migrate caudally to r6, where they form the facial motor nucleus
(Chandrasekhar et al., 1997;
Higashijima et al., 2000).
These neurons migrate near the pial surface of the hindbrain
(Wada et al., 2005). Several
molecules that regulate the migration of nVII motor neurons have been
identified in our, and other, laboratories. The trilobite/strabismus
(tri/stbm; stbm is also known as vangl2 - Zebrafish
Information Network) and prickle1 (pk1) genes, originally
identified in Drosophila as planar cell polarity (PCP) genes
(reviewed by Tree et al.,
2002; Klein and Mlodzik,
2005), regulate caudal migration of the nVII motor neurons
(Bingham et al., 2002;
Jessen et al., 2002;
Carreira-Barbosa et al., 2003).
We have previously shown that a cytoplasmic protein Landlocked/Scribble1
(Llk/Scrb1) is required for migration of the nVII motor neurons and that it
genetically interacts with Tri/Stbm during convergent extension (CE) movements
(Wada et al., 2005). A recent
study has shown that the tri/stbm gene controls the
anterior-posterior polarity of the neuroepithelial cells to regulate their
reintegration in the zebrafish spinal cord after cell division
(Ciruna et al., 2006). Thus,
the neuroepithelial cells may regulate migration of nVII motor neurons through
their polarized activity mediated by these gene products. However, the
mechanisms by which these molecules regulate neuronal migration have not been
investigated.
Here, we demonstrate that expression of the off-limits/frizzled3a (olt/fz3a) and off-road/celsr2 (ord/celsr2) genes in neuroepithelium maintains the nVII motor neurons near the pial surface during their caudal migration in the zebrafish hindbrain. In the absence of olt/fz3a expression in the neuroepithelium, the nVII motor neurons failed to migrate caudally; instead, they migrated radially into the dorsomedial part of the hindbrain by extending aberrant radial processes. Mosaic analyses showed that expression of the olt/fz3a gene in the surrounding neuroepithelial cells prevented integration of the nVII motor neurons into the neuroepithelial layer.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
The zebrafish Isl1-GFP transgenic line (registered as
Tg(CM-isl1:GFP)rw0 in the Zebrafish
National BioResource Project of Japan,
http://www.shigen.nig.ac.jp/zebra/index_en.html)
(Higashijima et al., 2000) was
derived from the RIKEN-Wako wild-type strain. The WIK strain was used for
genetic mapping (Shimoda et al.,
1999). Mutagenesis using N-ethyl-N-nitrosourea
(ENU) has been described previously (Wada
et al., 2005). One allele for the olt locus
(oltrw689) and four alleles for the ord locus
(ordrw71, ordrw135, ordrw166 and
ordrw380) were identified and used in this study. All
embryos used in this study carried the Isl1-GFP transgene. Images were
captured using a fluorescent dissecting microscope (Leica MZFLIII) with a CCD
camera (Hamamatsu C5810). All strains are available from the Zebrafish
National BioResource Project of Japan.
Mapping of the mutant loci
Genetic mapping of the mutant loci was carried out as described previously
(Wada et al., 2005). In total,
463 olt and 1027 ord homozygous embryos were used to assign
the locus to a linkage group. Expressed sequence tags (ESTs) and genomic
sequences were obtained from the T51 radiation hybrid panel (Research
Genetics,
http://www.resgen.com/)
and the Sanger Centre genome database
(http://www.ensembl.org/index.html).
The SSLP markers mrck1 and wz12343 were generated based on the sequences of a
putative gene annotated as myotonin-related Cdc42-binding kinase
(mrck)
and an EST (wz12343), respectively
(Table 1).
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Immunohistochemistry, in situ hybridization and retrograde cell labeling
Immunohistochemistry was performed according to standard protocols
(Westerfield, 2000), using
anti-acetylated -tubulin antibody (Sigma, diluted 1:1000),
zn-5-antibody (Oregon Monoclonal Bank, diluted 1:100)
(Trevarrow et al., 1990),
anti-ß-catenin antibody (Sigma, diluted 1:500) and a secondary antibody
conjugated to Alexa-533 (Santa Cruz Biotechnology, diluted 1:500). The samples
were viewed under confocal microscopy (Zeiss LSM 510). Isl1-GFP signals were
captured simultaneously in the samples. In situ hybridization using RNA probes
was carried out as described (Westerfield,
2000). To monitor hindbrain differentiation, we used RNA probes
for tag1 (also known as cntn2 - Zebrafish Information
Network) (Warren, Jr et al.,
1999), hoxb1a (Prince
et al., 1998), krox20
(Oxtoby and Jowett, 1993) and
val/mafb (Moens et al.,
1998). Images were captured using a differential interference
contrast microscope (Zeiss Axioplan2) with a CCD camera (Olympus DP50).
Retrograde labeling of the reticulospinal neurons and the octavolateralis
(OLe) neurons was carried out as described previously
(Wada et al., 2005). At least
ten embryos were monitored in each experiment.
Gene knockdown
Antisense morpholino oligonucleotides (MO) to target the first exon/intron
boundary of fz3a, and the second exon/intron boundary of celsr2,
celsr1a and celsr1b were designed by Gene Tools (see
Table 1 and Fig. S2D,I in the
supplementary material). Approximately 1 nl of MO (5 mg/ml) was injected into
one-cell-stage embryos (5 ng per embryo), as described
(Nasevicius and Ekker, 2000).
RT-PCR assay was carried out to confirm the splicing defects of the MOs, as
described (see Fig. S2E,J in the supplementary material)
(Goutel et al., 2000).
Injection of mRNA and detection of the expressed proteins
The wild-type fz3a, the mutated fz3arw689 and
the truncated fz3a-
C (1515 bp, which lacks the cytoplasmic
region) cDNAs were amplified by RT-PCR and subcloned into pCS2 expression
vectors. Sense-capped mRNA was synthesized using a mMessage mMachine kit
(Ambion). Approximately 1 nl of mRNA (0.5 mg/ml) was injected into
one-cell-stage embryos (0.5 ng per embryo). To monitor the subcellular
localization of the expressed proteins, Venus-fused cDNAs (fz3a-venus
and fz3arw689-venus) were generated
(Nagai et al., 2002) and the
corresponding mRNA was injected into one-cell-stage embryos, as described. To
monitor the subcellular localization of Deshevelled (Dsh), mRNA encoding
Xdsh:GFP (Rothbacher et al.,
2000) was co-injected with fz3a or
fz3arw689 mRNAs. For each construct, five embryos were
monitored under confocal microscopy at 10-12 hpf.
Cell transplantation
Cell transplantation was performed as described
(Wada et al., 2005;
Moens et al., 1996).
MZ-oltrw689 and MZ-ordrw71 embryos
were produced by homozygous crosses. Live mosaic embryos were analyzed at
36-48 hpf. To confirm that the transplanted donor cells developed into nVII
motor neurons, we monitored peripheral axons from donor cells labeled with
rhodamine, as described previously (Wada
et al., 2005). We used IMARIS 4.2 software (Bitplane) to visualize
mosaicism and optical sections in the neuroepithelium.
Time-lapse observations
The procedures for time-lapse observations were essentially the same as
described (Langenberg et al.,
2003; Bingham et al.,
2005) with modifications (H.T. and H.O., unpublished). Briefly,
the hindbrain regions were excised with a fine blade from embryos at 18 hpf.
The resulting hindbrain explants were embedded in 1.5% agarose in a small
chamber and covered with L-15 medium (Gibco BRL) supplemented with
penicillin/streptomycin cocktail (Gibco BRL). The chamber was sealed with a
cover slip and observed under confocal microscopy every 10 minutes at 18-33
hpf (lateral views) or 18-22 hpf (cross sections). Five samples were analyzed
in each experiment. Aberrant radial processes of the nVII motor neurons were
counted in live embryos at 20-22 hpf.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), to
identify novel mutants with perturbed caudal migration of the nVII motor
neurons (Wada et al., 2005).
The olt and ord mutations were further characterized, and
one allele for the olt locus (oltrw689) and four
alleles for the ord locus (ordrw71,
ordrw135, ordrw166 and
ordrw380) were used in this study.
In wild-type embryos, the nVII motor neurons begin to express GFP in r4 at
16 hours post-fertilization (hpf), after which they start migrating caudally
towards r6 (Fig. 1D,G)
(Chandrasekhar et al., 1997;
Higashijima et al., 2000;
Wada et al., 2005). The
olt and ord embryos display impairment in migration of the
nVII motor neurons, albeit differently. In the olt embryos, none of
the nVII motor neurons migrated caudally, but they all migrated dorsally into
r4 (Fig. 1E,H). By contrast, in
the ord embryos, some of the nVII motor neurons also migrated into
the r5 region (Fig. 1F,I).
Embryos homozygous for each of the four alleles of ord showed similar
impairment in migration of the nVII motor neurons. The other morphological
features of the olt and ord embryos were normal as compared
to those of wild-type embryos (Fig.
1A-C), and the adult mutants were viable and fertile. We obtained
maternal-and-zygotic (MZ) embryos by mating homozygous fish. Because the
MZ-oltrw689 and MZ-ordrw71 embryos
were also viable and displayed the same defects in neuronal migration, we used
them in the subsequent analyses. In the following experiments, we showed that
the ord and olt genes are barely expressed maternally (see
below and Fig. S2C,G in the supplementary material). These results suggest
that these genes are not required in the early stages of development,
supporting the viability of the MZ-oltrw689 and
MZ-ordrw71 embryos.
Despite their ectopic localization, nVII motor neurons in the mutant embryos expressed tag-1 mRNA and extended peripheral axons normally to the correct target muscles (see Fig. S1A-F in the supplementary material). Moreover, the expression patterns of the rhombomere-specific genes and the formation of hindbrain neurons appeared unaffected in the mutant embryos (see Fig. S1G-U in the supplementary material). These results suggest that the overall patterning and differentiation of the hindbrain neurons were unaffected by the mutation, except for the aberrant migration of the nVII motor neurons.
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). Because
another frizzled3 homolog was identified
(Fig. 2D), we termed the gene
associated with the olt locus zebrafish frizzled3a
(fz3a) and the other homolog frizzled3b (fz3b).
Sequence analyses showed that the allele oltrw689 carries
a mis-sense mutation resulting in substitution of the second conserved
cysteine of the extracellular cysteine-rich domain (CRD) of Fz3a (C38S)
(Fig. 2C; see also Fig. S2A in
the supplementary material). This amino acid substitution may disrupt proper
folding, and thereby the function, of the Frizzled family proteins, because
the second and fourth conserved cysteines of the CRD form a disulfide bond
(Dann et al., 2001). Indeed, in
Drosophila, the mutated Dfz2C3, which carries a
substitution of the fourth conserved cysteine of the CRD to serine (C118S),
shows a reduced capacity to reach the cell surface
(Chen et al., 2004).
Ectopically expressed Frizzled family proteins recruit the downstream effector
Dsh to the plasma membrane (Axelrod et al.,
1998), and recruitment of Dsh to the plasma membrane is essential
for downstream signaling (Park et al.,
2005). Thus, to determine whether the amino acid substitution
resulting from the mutation in the ordrw689 allele leads
to functional disruption of the Fz3a protein, we examined the subcellular
localization of Fz3a and mutated Fz3arw689, and of Xenopus
Dsh (Xdsh), when Fz3a or Fz3arw689 is overexpressed. We found that
the mutated Fz3arw689 protein was not associated with the plasma
membrane and that it lost its capacity to recruit Xdsh to the plasma membrane
(see Fig. S2B in the supplementary material). To confirm that the loss of function of the fz3a gene is responsible for the olt phenotype, we used an antisense MO (fz3a-MO) (see Fig. S2D,E in the supplementary material). The resulting morphant phenotype was identical to that of the olt mutant embryos (Fig. 2F-H), with impaired migration of the nVII motor neurons (100% of MO-injected embryos; n=47). Control MO (fz3a-MO-5mis) did not impair migration of the nVII motor neurons in the injected embryos (n=30). There was little expression of fz3a mRNA up to the gastrula stages (see Fig. S2C in the supplementary material), but fz3a mRNA was expressed throughout the hindbrain at 24 hpf, at the time when the nVII motor neurons are migrating (Fig. 2E,E').
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C) lacking the C-terminal intracellular domain, a
significant proportion of embryos showed loss of nVII motor-neuron migration,
without early pattern formations or CE movements being affected
(Fig. 2I,I'; 36% of
injected embryos, n=55). These results demonstrated that
Fz3a-C acts dominant-negatively in a highly specific manner, confirming
the specific role of the fz3a gene in the migration of nVII motor
neurons.
ord encodes zebrafish Celsr2
The ord locus was genetically mapped to linkage group 22
(Fig. 3A). We showed that this
genomic region contains a gene coding for Celsr2 (for cadherin, EGF-like,
LAG-like and seven-pass receptor), which is a vertebrate homolog of
Drosophila Flamingo (Fmi, also known as Stan - Flybase)
(Fig. 3B-D)
(Usui et al., 1999;
Formstone and Little, 2001;
Shima et al., 2002). Sequence
analyses showed that each of the four alleles of the ord locus
carries a point mutation in the celsr2 gene. The alleles
ordrw71, ordrw166 and
ordrw380 carry a mis-sense mutation resulting in amino
acid substitution (C1835S, E582K and V1168D, respectively) in the
extracellular domain, and the ordrw135 allele carries a
premature stop codon (L288Stop) in the extracellular domain
(Fig. 3C, see also Fig. S2F in
the supplementary material).
To confirm that loss of function of the celsr2 gene is responsible for the ord phenotype, we used an antisense MO (celsr2-MO) (see Fig. S2I,J in the supplementary material). The resulting morphant phenotype was identical to that of the ord mutant embryos (Fig. 3H-J), with impaired migration of the nVII motor neurons (95% of MO-injected embryos; n=99), confirming that the ord gene encodes Celsr2. Migration of the nVII motor neurons was not impaired in 74 embryos injected with control MO (celsr2-MO-5mis). The celsr2 mRNA was expressed throughout the embryo at the gastrula stages, and became restricted to the CNS thereafter (see Fig. S2G in the supplementary material). The celsr2 mRNA was expressed throughout the brain at 24 hpf, when the nVII motor neurons were migrating (Fig. 3E,E').
celsr1a and celsr1b function redundantly with ord/celsr2 in regulating migration of the nVII motor neurons
The celsr1, celsr2 and celsr3 mRNAs are differentially
expressed in developing mouse CNS
(Formstone and Little, 2001;
Shima et al., 2002). To
understand the function of zebrafish celsr homologs in migration of the nVII
motor neurons, we identified celsr family genes. A BLAST homology search and
phylogenetic tree analyses revealed that two celsr1 orthologs and one
celsr3 ortholog exists in the zebrafish genome
(Fig. 3D). The celsr1
orthologs have been described and referred to previously as flamingo
(fmi)1a and fmi1b
(Formstone and Mason, 2005).
To abide by the rule of the Zebrafish Nomenclature Committee
(http://zfin.org/zf_info/nomen.html)
to follow the mammalian terminology, we refer to these genes as
celsr1a (equivalent to fmi1a) and celsr1b
(equivalent to fmi1b) in this study. We demonstrated that
celsr1a and celsr1b mRNAs, but not celsr3 mRNA,
were also expressed in the developing hindbrain
(Fig. 3F,F',G,G'
and Fig. S2H in the supplementary material). We therefore analyzed the
function of the celsr1a and celsr1b genes.
We designed celsr1a-MO and celsr1b-MO (see Fig. S2I,J in the supplementary material). Injection of celsr1a-MO or celsr1b-MO alone into wild-type embryos did not cause any defect in neuronal migration (Fig. 3L). However, co-injection of celsr1a-MO and celsr1b-MO gave rise to a phenotype similar to that of the ord embryos, showing impaired migration of the nVII motor neurons (34% of injected embryos, n=82; Fig. 3L). Interestingly, injection of celsr1a-MO or celsr1b-MO into the MZ-ord embryos enhanced the severity of neuronal migration defects. In most of the resulting embryos, the nVII motor neurons did not migrate at all (79% of embryos injected with celsr1a-MO, n=85; 85% of embryos injected with celsr1b-MO, n=72; Fig. 3L). Moreover, co-injection of celsr1a-MO and celsr1b-MO resulted in complete loss of neuronal migration in all the MZ-ord embryos, as observed in the olt embryos (97%, n=151; Fig. 3K,L,). Control MOs did not enhance the severity of neuronal migration defects in the MZ-ord embryos (none of the 71 embryos injected with celsr1a-MO-5mis, or of the 60 embryos injected with celsr1b-MO-5mis). These results suggest that celsr1a and celsr1b act in concert with ord/celsr2 to regulate migration of the nVII motor neurons.
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-tubulin
antibody (red), and the Isl1-GFP signals (green) were captured simultaneously
(Fig. 4A-K). The peripheral
axons of the nVII motor neurons and the medial longitudinal fascicles (MLF)
were immunoreactive to anti-acetylated -tubulin antibody
(Fig. 4A-K). In the wild-type
embryos, the migrating motor neurons were localized close to the pial surface
of the hindbrain and were connected to the MLF
(Fig. 4A,D,G). Some of the
migrating nVII motor neurons appeared to be in direct contact with the MLF;
others, which were located a short distance away from the MLF, had axons
projecting to the MLF tracts (Fig.
4J), showing that migrating motor neurons also extend axons. A
time-lapse study using hindbrain explants
(Bingham et al., 2005) (see
Materials and methods) confirmed that the motor neurons were always located
near the pial surface of the hindbrain during migration
(Fig. 5A, see also Movie S1 in
the supplementary material).
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nVII motor neurons migrate in aberrant directions by extending radial processes in the mutant embryos
In the wild-type embryos, the migrating nVII motor neurons showed biased
caudal protrusive activity (see Movie S1 in the supplementary material)
(Jessen et al., 2002). Our
time-lapse observations revealed that the nVII motor neurons migrated dorsally
in the hindbrain explants from the olt embryos (arrow in
Fig. 5B, see Movie S2 in the
supplementary material). Moreover, the motor neurons migrated aberrantly by
extending long processes towards the ventricle at r4
(Fig. 5C, Movie S5 in the
supplementary material). On average, 1.7 processes per embryo were observed at
a single time point (Fig. 4M,O;
27 olt embryos were observed). A large proportion (47%;
n=47) of these aberrant radial processes reached the ventricular
surface (Fig. 4M). By contrast,
radial processes were seldom observed in the wild-type embryos
(Fig. 4L,O, see Movie S4 in the
supplementary material). Only two aberrant processes were detected in 39
wild-type embryos and they did not reach the ventricular surface. The
ord embryos injected with celsr1a-MO and celsr1b-MO
showed an identical phenotype to that of the olt embryos
(Fig. 4O; on average, 1.7
processes per embryo were observed in 24 morphants). These results suggest
that the fz3a and celsr genes function in the same
manner.
Next, we quantified the direction of the aberrant processes in the olt embryos. Most of the nVII motor neurons extended aberrant processes at right angles to the ventricular surface (Fig. 4N,P). Mismigrating motor neurons sometimes reached the ventricles (arrowhead in Fig. 4H), indicating that these aberrant processes could steer the migrating motor neurons in the wrong direction.
Incomplete migration of nVII motor neurons in ord embryos
Next, we performed time-lapse observations of hindbrain explants from
ord embryos. As described above, some of the nVII motor neurons
migrated into r5 in the ord embryos because of the functional
redundancy of celsr1a and celsr1b
(Fig. 3H-L). These incompletely
migrated neurons were not associated with the pial surface of the hindbrain
(Fig. 4K).
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We previously traced cell movements in wild-type embryos and showed that
nVII motor neurons migrate independently of other neuroepithelial structures
(Wada et al., 2005). To
compare the behavior of nVII motor neurons with neighboring neuroepithelial
cells, we observed mosaic embryos in which several neuroepithelial cells were
randomly labeled with rhodamine-conjugated dextran (see Fig. S3A,B in the
supplementary material). We confirmed that, in the ord embryos, some
of the nVII motor neurons became detached from the pial surface of the brain
and migrated caudally relative to the neighboring cells (see Fig. S3B in the
supplementary material). These results demonstrated that the nVII motor
neurons can migrate caudally to some extent without keeping direct contact
with the pial structure of the hindbrain in the ord embryos. However,
we can not exclude the possibility that some component of the pial structures,
such as the MLF, may have a long-distance influence.
fz3a and celsr2 genes mainly act in a non-cell-autonomous manner during migration of nVII motor neurons
During the development of zebrafish embryos, the hindbrain everts, and its
ventricle opens at 18-22 hpf and begins to expand at 24 hpf
(Lowery and Sive, 2005). The
olt embryos also showed rapid expansion of the ventricle (as observed
in the wild-type embryos) when they were stained with anti-ß-catenin
antibody at 24 hpf and 33 hpf (Fig.
6A,B) (Lowery and Sive,
2005). As a consequence, the orientation of the neuroepithelial
cells became more parallel to the midline, and the nVII motor-neuron clusters
changed their positions to be relatively further away from the ventricle and
the neurons were located laterally in the olt embryos
(Fig. 6B).
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fz3a is required in the neuroepithelial cells to restrict the nVII motor neurons near the pial surface of the hindbrain
We then investigated the cell type responsible for regulating the migration
of the nVII motor neurons in the developing hindbrain. Because fz3a
and celsr2 mRNAs are expressed widely in the hindbrain
(Fig. 2E,E';
Fig. 3E,E'), the
neuroepithelial cells surrounding the nVII motor neurons are good candidates
as regulators of nVII motor-neuron migration. In the mutant embryos, the nVII
motor neurons migrated into the neuroepithelial layer at r4
(Fig. 4E,F). A function of
wild-type neuroepithelium may, therefore, be to prevent integration of the
nVII motor neurons into the neuroepithelium and restrict them near the pial
surface of the brain.
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Next, we observed the behavior of nVII motor neurons in olt embryos in which the neuroepithelium contained wild-type-derived cells (Fig. 6H,J-J''). In this case, the wild-type-derived cells (red) were incorporated into the neuroepithelium of the MZ-olt host embryos at r4 and r5 (Fig. 6H). In the intact side (left side of the embryo shown in Fig. 6H), the nVII motor neurons (green) mismigrated dorsally in the olt mutant neuroepithelium, as described (Fig. 4B,E). However, in the mosaic region (right side of the embryo shown in Fig. 6H), the MZ-olt-derived nVII motor neurons failed to invade the wild-type cell cluster (Fig. 6H,J,J'; the example shown is representative of ten mosaic embryos; also see Fig. S6 in the supplementary material for observations of other mosaic embryos). These results showed that the wild-type neuroepithelial cells prevented the nVII motor neurons from invading the neuroepithelial layer (Fig. 6J''). In Fig. 6H, it is apparent that several nVII motor neurons in the mosaic regions migrated slightly caudally in comparison with the intact side, suggesting that the caudal migration may be rescued by the associated wild-type neuroepithelium. However, we could not introduce wild-type large-cell clusters encompassing the r4 and r5 regions in the other mosaic embryos. Therefore, we could not make sufficient observations to provide statistical support for this possibility.
Together, the above results demonstrate that the fz3a gene acts in the neuroepithelium to regulate the direction of neuronal migration by preventing integration of nVII motor neurons into the neuroepithelium and by restricting them to a region near the pial surface of the brain (Fig. 7).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). These
may include Reelin (Ohshima et al.,
2002; Rossel et al.,
2005) and Sdf-1 (Sapede et
al., 2005) signaling. In the present study, we have clearly shown
that the neuroepithelial cells function in preventing integration of the nVII
motor neurons into the neuroepithelial layer of the hindbrain, and that this
radial exclusion is essential for normal migration of the nVII motor neurons.
The mosaic experiments using tri/stbm
(Jessen et al., 2002),
llk/scrb1 (Wada et al.,
2005), olt/fz3a and ord/celsr2 (present study)
genes showed that the wild-type-derived nVII motor neurons completely failed
to migrate caudally in the mutant host embryos. We showed that the nVII motor
neurons of MZ-olt host embryos did not invade the wild-type
neuroepithelial cell cluster but that, conversely, the nVII motor neurons of
wild-type host embryos did invade the mutant neuroepithelial cell cluster
(Fig. 6G-J). These findings
indicate that normal migration requires a functional olt/fz3a gene
mainly in the surrounding neuroepithelial cells. However, as a non-negligible
proportion of the mutant embryo-derived nVII motor neurons also failed to
migrate in the wild-type host embryos (Fig.
6C), we can not exclude the possibility that these genes may also
act in the migrating nVII motor neurons themselves. We observed that some of the early-born nVII motor neurons mismigrated radially away from the pial surface in the olt embryos (Fig. 5C). However, we can still not exclude the possibility that some other late-born neurons may have stalled during migration on their way to the pial surface, before they started expressing GFP.
Neuroepithelial cells play a contrasting role for the re-integration of newly divided neuroepithelial cells
It has been shown that the tri/stbm gene in neuroepithelial cells
regulates the re-integration of daughter cells into the neuroepithelial layer
after cell division (Ciruna et al.,
2006). By contrast, neuroepithelial cells prevent integration of
nVII motor neurons into the neuroepithelial layer, as shown in the present
study. Hence, we propose opposite roles for the neuroepithelial cells
depending on the cell type that they act upon - integration of neuroepithelial
daughter cells into the neuroepithelial layer and exclusion of nVII motor
neurons from the neuroepithelial layer. Fz3a and Celsr2 together with Stbm may
control cell-adhesion or cell-repulsion molecules that are specifically
required for the integration or exclusion of cells from the
neuroepithelium.
Radial exclusion and caudal migration of the nVII motor neurons are interdependent mechanisms
We showed that in the olt embryos, the mismigrated nVII motor
neurons extended aberrant processes towards the ventricle at right angles to
the anterior-posterior axis (Fig.
4N,P). These observations suggest that the nVII motor neurons in
the olt embryos lose their ability to migrate caudally. There was
some, albeit incomplete, migration of the nVII motor neurons in the
ord embryos, apparently because of functional redundancy with the
celsr1a and celsr1b genes. However, the migration of the
nVII motor neurons in the ord embryos was by no means normal in
nature, and these neurons stopped migrating at r5 and failed to reach r6. In
the ord embryos, the nVII motor neurons also invaded the
neuroepithelial layer. These results suggest that radial exclusion and caudal
migration are interdependent mechanisms of normal nVII motor neuron migration.
A recent study has shown that the tri/stbm gene is essential for
localization of Prickle to the anterior membrane of neuroepithelial cells
(Ciruna et al., 2006),
suggesting that these genes may control the anterior-posterior polarity of
these cells (Ciruna et al.,
2006). This might provide some basis for caudal migration of the
nVII motor neurons.
In a previous study, we showed that the r4-derived nVII motor neurons are
composed of the branchiomotor neurons and the OLe neurons
(Higashijima et al., 2000;
Wada et al., 2005), and that
the OLe neurons are possibly among the population to first migrate out of r4
(Fig. 5D). However, by labeling
the OLe neurons with DiI retrogradely, we demonstrated that the OLe neurons
failed to migrate caudally and remained in r4 in the ord embryos
(n=4, see Fig. S7 in the supplementary material).
Possible roles of the fz3a and celsr genes in the neuroepithelium
We showed that maternal and zygotic impairment of Fz3a, or overexpression
of Fz3a-C, specifically disrupted nVII motor-neuron migration without
affecting the early pattern formations or CE movements during gastrulation
(Fig. 1B;
Fig. 2I). These results suggest
that Fz3a interacts with only a specific ligand to regulate the
neuroepithelial functions. Conversely, Silberblick (Slb)/Wnt11 and Pipetail
(Ppt)/Wnt5a regulate CE movements but do not regulate nVII motor-neuron
migration (Bingham et al.,
2002; Jessen et al.,
2002). Our data, taken together with these results, suggest that
the genetic cascades regulating the neuroepithelial functions adopt a
different ligand-receptor system to that used for the regulation of CE
movements.
It remains unclear as to how the other downstream effectors of Fz3a,
including Dsh, are involved in the regulation of neuroepithelial functions. We
showed that simultaneous overexpression of Fz3a and Xenopus Dsh
(Xdsh) led to the recruitment of Xdsh to the plasma membrane at the blastula
stages (see Fig. S2B in the supplementary material). These results suggest
that Fz3a can interact with Xdsh. By contrast, overexpression of a mutant form
of Xdsh, Xdd1, which lacks a PDZ domain disrupts CE movements in a
dominant-negative manner but does not impair migration of the nVII motor
neurons (Jessen et al., 2002).
Therefore, there may be functional specialization among different Dsh proteins
in zebrafish, as described above for Wnts and Fzs, and
Xenopus-derived Xdd1 may antagonize a special Dsh that is required
for CE movements only. Alternatively, the domains of Dsh required for the
neuroepithelial functions may be different from those regulating CE movements,
and Xdd1 may still be able to transmit signals that normally regulate
neuroepithelial functions. We also do not exclude the possibility that Fz3a
may act independently of Dsh in its regulation of neuroepithelial
functions.
It has been suggested that homophilic interaction of Fmi through the
extracellular cadherin-repeat domain plays an important role at cell-cell
boundaries in epithelial planar polarity
(Usui et al., 1999). Thus, it
is possible that the Celsr proteins also regulate cell adhesion between
adjacent neuroepithelial cells to restrict invasion of the nVII motor neurons
into the neuroepithelium.
The Eph receptors and their ligands, the ephrins, are other good candidate
mediators of cell repulsion (for reviews, see
Wilkinson, 2001;
Kullander and Klein, 2002). In
the mouse and chick hindbrain, Eph receptors and ephrin ligands are expressed
in the nVII motor neurons and neuroepithelial cells, respectively
(Cowan et al., 2000;
Kury et al., 2000). Recent
studies have shown that Dsh forms a complex with EphrinB to mediate cell
repulsion in Xenopus embryos
(Tanaka et al., 2003;
Lee et al., 2006). Thus, it is
possible that Fz and Celsrs may regulate the activity of cell-repulsive
molecules, such as ephrins, on the surface of the neuroepithelial cells.
Novel roles of the frizzled and celsr family genes in brain development
Our present finding that neuroepithelial cells are involved in positioning
specific neurons near the pial surface suggests a fundamental role for the
neuroepithelium in brain development. In the mammalian cortex, neurons are
generated in ventricular germinal zones and migrate radially towards the pial
surface to form architectural layered structures. In mouse embryos, Reelin
signaling regulates the positioning of neurons during layer formation of the
cerebrum (reviewed by Tissir and Goffinet,
2003), and is essential for radial migration of the nVII motor
neurons (Ohshima et al., 2002;
Rossel et al., 2005). These
data suggest that similar mechanisms regulate the proper positioning of both
the hindbrain motor neurons and the cortical layer neurons.
In the mouse cerebral cortex, many wnt and frizzled family genes are
expressed in gene-specific regional and lamina patterns
(Shimogori et al., 2004). Such
patterned expression suggests the possibility that these genes are involved in
other aspects of brain development. Recent studies have shown that functional
fzd3 and celsr3 genes are required for the development of
the anterior commissure, and the cortico-subcortical, thalamocortical and
corticospinal tracts (Wang et al.,
2002; Tissir et al.,
2005). It is possible that the mouse fzd3 and
celsr3 genes regulate neuroepithelial cells to guide these axonal
tracts to the proper region in a similar manner to that by which the zebrafish
fz3a and celsr genes act in neuroepithelial cells to
restrict the migrating nVII motor neurons near the pial surface of the
hindbrain. Our demonstration of a role for neuroepithelial cells in preventing
integration of differentiated neurons into the neuroepithelial layer may
provide new insights into the general mechanisms underlying the formation of
layered structures in the mammalian brain, such as in the cerebral cortex.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/23/4749/DC1
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ACKNOWLEDGMENTS |
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