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First published online 15 April 2009
doi: 10.1242/dev.033290
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1 Laboratory for Developmental Gene Regulation, RIKEN Brain Science Institute
(BSI), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
2 Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of
Agricultural and Life Sciences, the University of Tokyo, Bunkyo-ku, Tokyo
113-8657, Japan.
3 Department of Life Sciences, Graduate School of Arts and Sciences, the
University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.
4 Research Resource Center, RIKEN BSI, 2-1 Hirosawa, Wako, Saitama 351-0198,
Japan.
Author for correspondence (e-mail:
hitoshi{at}brain.riken.jp)
Accepted 4 March 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Fucosylation, GMDS, Neuroepithelial cells, Neural migration, Vagus motor neurons, Zebrafish
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Many studies have identified molecules involved in the control of neural
migration. These studies have revealed that proper neural migration requires
various genes that function not only in migrating neurons, but also in the
cells that support them. For example, we have reported that the planar cell
polarity genes frizzled3a and celsr2 function in
neuroepithelial cells to guide the caudal migration of facial motor neurons
(Wada et al., 2006). Despite
this, there is still very little known about the molecular mechanisms that
regulate termination of neural migration and the accumulation of neural
progenitors to form the proper layers or nuclei.
The vagus nerve is the tenth cranial nerve, located in rhombomere (r) 8 in
the caudal hindbrain. It controls various body functions, including heart beat
and gastrointestinal movement (Gilland and
Baker, 2005; Taylor et al.,
1999). Correct arrangement of vagus motor nuclei following neural
migration and accumulation may be crucial for the normal functioning of vagus
motor neurons. For example, although sudden infant death syndrome (SIDS), the
most common cause of postnatal infant death, is a complex and multifactorial
disorder (Moon et al., 2007),
it has been suggested that the reduced number of neurons in vagus motor nuclei
in victims of SIDS is due to a failure of neuronal migration
(Macchi et al., 2002). Thus,
there is a need to understand how vagus motor nuclei are formed by neural
migration, and the molecular mechanisms that govern this process.
In the present study, we used vagus motor nuclei as a new model system in
which to study the molecular mechanisms underlying neural migration.
Time-lapse observations of hindbrain explants from the transgenic zebrafish
line Tg(CM-isl1:GFP)rw0, hereafter referred to as
isl1:GFP, which expresses green fluorescent protein (GFP) in motor
neurons, including vagus motor neurons
(Higashijima et al., 2000),
demonstrated that vagus motor neuron progenitors are born near the ventral
midline, migrate in a dorsolateral direction and accumulate to form the vagus
motor nuclei. An N-ethyl-N-nitrosourea (ENU)-based mutant
screen using the isl1:GFP transgenic zebrafish isolated
towhead (twdrw685) mutant embryos, in which the
vagus motor neuron progenitors migrate beyond their proper position to the
dorsal roof of the hindbrain. The twdrw685 locus encodes
GMDS, a key enzyme in the fucosylation pathway
(Staudacher et al., 1999).
Fucosylated glycans are involved in a variety of biological and
pathological processes, including cell migration
(Ma et al., 2006). For
example, sialyl Lewis X (sLex), a fucosylated carbohydrate
structure, is the core recognition epitope that mediates lymphocyte homing and
initial leukocyte-endothelial cell adhesion
(Ma et al., 2006). The donor
substrate for fucosylation, GDP-fucose, is synthesized in the cytoplasm mainly
through a de novo synthesis pathway, which includes an enzymatic reaction
catalyzed by GDP-mannose 4,6-dehydrase (GMDS)
(Fig. 1). Subsequently, a
GDP-fucose transporter at the Golgi membrane transports GDP-fucose from the
cytoplasm to the Golgi lumen, and fucosyltransferases (FUTs) transfer
GDP-fucose to the acceptor molecules. Leukocyte adhesion deficiency II (LAD
II), also known as congenital disorder of glycosylation IIc (CDG IIc), results
in mental retardation, short limbs and stature, and a flat face with a broad,
depressed nasal bridge (Freeze,
2001). These features are caused by a defect in the
Golgi-localized GDP-fucose transporter, resulting in impaired expression of
the fucosylated glycans (Lubke et al.,
2001; Lubke et al.,
1999; Luhn et al.,
2001). Although individuals with LAD II/CDG IIc exhibit
neurological abnormalities, how fucosylated glycans regulate neural
development is not yet understood. The findings of the present study reveal a
novel molecular mechanism in the establishment of the structure of the vagus
motor nuclei following neural migration, whereby fucosylation of the substrate
plays a key role in preventing vagus motor neuron progenitors from migrating
along an aberrant pathway.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Wada et al., 2005;
Wada et al., 2006;
Westerfield, 2007). The simple
sequence length polymorphism (SSLP) marker BX510653-1 was generated based on
the sequence of the eighth intron of the gmds gene. The
isl1:GFP fish and the twdrw685 fish are available
from the National BioResource Project of Japan
(http://www.shigen.nig.ac.jp/zebra/index_en.html).
Time-lapse imaging of hindbrain explant culture and cell transplantation
The procedures for time-lapse imaging of the hindbrain were essentially the
same as those described previously (Tanaka
et al., 2007). The hindbrain explant in the culture chamber was
observed under a confocal microscope (LSM510; Carl Zeiss) every 15 minutes
from 24 to 48 hours post-fertilization (hpf). Labeling of wild-type cells by
rhodamine-dextran and transplantation were performed according to standard
protocols (Westerfield,
2007).
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). Details
of primers and the antisense morpholino oligonucleotides (MOs; Gene Tools)
used in this study can be provided on request. For the injection of mRNA or MO
solution, approximately 1 nl of the solution at the concentrations indicated
in Table 1 was injected into
one-cell stage embryos. The GenBank Accession Numbers for zebrafish
L-gmds and S-gmds are AB290320 and AB290319, respectively.
Amino acid sequence similarity was analyzed using the CLASTAL W program. The
following amino acid sequences of GMDS were used for sequence comparisons:
Homo sapiens (GenBank accession number NM_001500), Mus
musclus (BC093502), Xenopus laevis (BC111472), Drosophila
melanogaster (NM_135044), Caenorhabditis elegans (NM_069162) and
Escherichia coli (NZ_AAJT01000120). Embryo genotypes were identified
by direct sequencing of the mutation site.
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;
Shepard et al., 2004;
Wada et al., 2005;
Westerfield, 2007). Details of
primary antibodies and biotinylated lectins used in the present study can be
provided on request. These probes were detected by fluorescein-conjugated
secondary antibodies (Invitrogen; 1:500 dilution for each) or avidin
(Chemicon; 1:500 dilution). For immunoblotting, 25 embryos of wild-type and
the twdrw685 mutants were pooled and lysed in Laemmli
sample buffer. The remaining steps in the immunoblotting procedure were as
described previously (Ohata et al.,
2009). |
RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Like mammals (Gilland and Baker,
2005; Taylor et al.,
1999), zebrafish form two vagus motor nuclei in r8, the most
caudal part of the hindbrain. One is the dorsolateral motor nucleus of the
vagus (dlX), whereas the other is the medial motor nucleus of the vagus (mmX)
(Fig. 2A, parts a,b). In the
present study, we used time-lapse imaging of hindbrain explants to observe the
process of vagus motor nuclei formation
(Fig. 2B). The vagus motor
neuron progenitors were born near the ventral midline from 24 to 48 hpf
(Fig. 2B; see Movie 1 in the
supplementary material). The dlX was formed first by the dorsolateral
migration and accumulation of vagus motor neuron progenitors from 24 to 36 hpf
(Fig. 2B). Then, mmX was formed
in the ventromedial region of r8 by 48 hpf
(Fig. 2B).
Isolation of the twdrw685 mutants showing defects in the cessation of migration of vagus motor neuron progenitors
To dissect genetically the process of the formation of vagus motor nuclei,
we performed ENU-based mutant screening and isolated the
twdrw685 mutant. In the twdrw685
embryos, the vagus motor nuclei were fused across the midline
(Fig. 3A). In addition, the
distribution of anterior and posterior trigeminal (Va and Vp) and facial (VII)
motor neurons was perturbed in the twdrw685 mutant (33/33
(100%) (Fig. 3A), presumably
because of defects in the lateral migration of the trigeminal motor neurons
and the posterior migration of the facial motor neurons. The morphology of the
twdrw685 embryos appeared to be normal at 2 days
post-fertilization (dpf), except for their `curled up' tails
(Fig. 3B). The
twdrw685 mutants died between 6 and 7 dpf.
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To distinguish between dlX and mmX neurons, we took advantage of the differences in time when these neurons are born. Specifically, wild-type and twdrw685 embryos were labeled with BrdU for 30 minutes at 38 hpf and were fixed at 3 dpf for staining with anti-BrdU and anti-GFP antibodies. In wild-type embryos, most dlX neurons were BrdU negative (Fig. 3D, part a), whereas all mmX neurons were BrdU positive. Most of the ectopic neurons in twdrw685 embryos were also BrdU negative (Fig. 3D, part b), indicating a common origin with dlX neurons. In addition, most BrdU and GFP double-positive cells from isl1:GFP transgenic embryos were localized ectopically in twdrw685 mutants. This indicates that the twdrw685 mutation may affect the migration of both dlX and mmX neurons.
To clarify the effect of the twdrw685 mutation on the migration of dlX progenitors, we observed the embryos using time-lapse imaging. In wild-type embryos, the dlX progenitors migrate in the dorsolateral direction soon after they are born (Fig. 3E; see Movie 1 in the supplementary material). However, in twdrw685 embryos, the dlX progenitors migrate in more dorsal directions, straying away from the normal migratory pathway (Fig. 3E; see Movie 2 in the supplementary material).
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). The formation of zn-5-immunoreactive axons appeared normal
in twdrw685 embryos
(Fig. 4A,B). Furthermore,
labeling of reticulospinal neurons by injection of a tracer dye into the
spinal cord revealed that the anterior-posterior patterning of the
reticulospinal neurons in twdrw685 embryos was identical
to that in wild-type embryos (Fig.
4C,D). The LIM homeodomain proteins Lhx2 and Lhx9, as well as
glutamate decarboxylases Gad1 and Gad2, are segmentally expressed in the
hindbrain (Ando et al., 2005;
Sassa et al., 2007). In the
present study, the location of both Lhx2/9- and Gad1/2-immunoreactive neurons
was almost normal in twdrw685 embryos
(Fig. 4E-H). These results
suggest that the overall patterning and differentiation of neurons other than
the vagus motor neurons in the posterior hindbrain were unaffected by the
twdrw685 mutation.
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The twdrw685 locus encodes GMDS
The twdrw685 locus was mapped between SSLP markers
z14995 and z14614 on linkage group 20 (LG20) and no recombination was detected
at the gene encoding GMDS in 702 meioses
(Fig. 5A). The gmds
gene consists of 12 exons and 11 introns
(Fig. 5B). cDNA cloning of
wild-type embryos identified two splicing variant mRNAs, depending on whether
the fourth exon, which consists of 21 bases, is included (L-gmds) or
skipped (S-gmds) in the mature mRNA. These two variants share an
identical amino acid sequence, except for the seven residues encoded by the
fourth exon (Fig. 5B,C). A
mutation from T to A was detected in the seventh exon of the gmds
gene from twdrw685 embryos
(Fig. 5B-D), resulting in amino
acid substitution from Trp193 in L-GMDS (or Trp186 in
S-GMDS) to Arg. Trp193 (or Trp186 in S-GMDS) is
conserved in the GMDS from various species, ranging from human to bacteria
(Fig. 5C).
To confirm that gmds is the gene responsible for the twdrw685 phenotype, we performed loss-of-function and gain-of-function analyses of gmds. Injection of an MO designed to inhibit splicing of premature gmds mRNA into one-cell stage wild-type embryos effectively blocked the maturation of gmds mRNA (Fig. 6A). Although injection of the 5-mis-pair control MO had no effect on motor neurons in morphants (0/113; 0%) (Fig. 6B, part a), injection of the splice-blocking MO against gmds produced a fused vagus motor nuclei phenotype (130/133; 98%) (Fig. 6B, part b) that was identical to that observed in the twdrw685 mutant (Fig. 3A). In addition, the distribution of trigeminal and facial motor neurons was perturbed in gmds morphant embryos (41/41; 100%) (Fig. 6Bb), as in the twdrw685 mutant embryos (Fig. 3A).
Further experiments were performed in which the effects of injection of either of the in vitro-synthesized sense-capped S- or L-gmds mRNAs (40 or 80 µg/ml) into one-cell stage embryos derived from heterozygous twdrw685 parents were investigated. Of the twdrw685 embryos injected with 80 µg/ml L-gmds mRNA, 88% (21/24) exhibited a completely rescued phenotype (Table 1; Fig. 6B, part c) and 13% exhibited a partially rescued phenotype, in which some, but not all, vagus motor neurons were found in abnormal locations (Fig. 6B, part c', arrowheads). When the lower concentration of L-gmds mRNA (40 µg/ml) was injected, there was a concomitant decrease in the number of twdrw685 embryos rescued [14/27 (52%) and 8/27 (30%) embryos completely and partially rescued, respectively] and 19% (5/27) of mutant embryos still showed the twdrw685 phenotype (Table 1). Injection of 80 µg/ml S-gmds mRNA rescued twdrw685 embryos with a similar efficiency as that seen following injection of the same concentration of L-gmds mRNA [22/30 (73%) and 8/30 (27%) embryos completely and partially rescued, respectively] (Table 1; Fig. 6B, part d). By contrast, injection of the mutant-type mRNA did not result in the rescue of any twdrw685 mutants (Table 1). Overexpression of L- or S-gmds mRNA in wild-type embryos had no effect on the differentiation and migration of vagus motor neurons (Fig. 6B, part e).
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Reduced levels of fucosylated glycans in twdrw685 embryos are rescued by injection of gmds mRNA at the one-cell stage
The expression profile of fucosylated glycans in the hindbrain of zebrafish
embryos was examined in the present study using biotinylated lectins, because
GMDS is a key enzyme in the fucosylation pathway
(Sullivan et al., 1998). In
wild-type embryos, N-linked fucosylated glycans recognized by
Aleuria aurantia lectin (AAL) and Lens culinaris agglutinin
(LCA) were expressed ubiquitously throughout the hindbrain
(Fig. 7A; see Fig. S1A in the
supplementary material) (Varki et al.,
1999). However, in twdrw685 embryos, glycan
levels were markedly reduced (Fig.
7B; see Fig. S1B in the supplementary material). The expression of
other types of N-linked glycans, namely those recognized by
concanavalin A (ConA), wheat-germ agglutinin (WGA) and Ricinus
communis agglutinin I (RCA-I), as well as the expression of HNK-1 antigen
detected using zn12 antibody (Metcalfe et
al., 1990), appeared to be normal in mutant embryos
(Fig. 7D,E; see Fig. S1C-H in
the supplementary material). In wild-type embryos, the fucosylated
tetrasaccharide structure sLex was expressed in the ventricular
zone, but expression of sLex was markedly reduced in
twdrw685 mutant embryos
(Fig. 7F,G). This reduction in
the expression of sLex was confirmed by immunoblotting
(Fig. 7H). O-Linked
glycans recognized by Ulex europaeus agglutinin I (UEA-I) were not
detectable in the hindbrain of either wild-type or
twdrw685 embryos (see Fig. S1I,J in the supplementary
material). In mutant embryos rescued by injection of either L- or
S-gmds variant mRNA, the expression of N-linked fucosylated
glycans recognized by AAL was recovered
(Fig. 7C). By contrast, levels
of AAL-reactive glycans in twdrw685 mutant embryos were
not rescued by injection of either mutant-type L- or S-gmds
variant mRNA (data not shown). These results indicate that fucosylated glycans
are specifically affected by the mutation in the gmds gene.
Repression of FUT7-9, FT1, FT2, POFUT1 and POFUT2 does not phenocopy twdrw685 mutant
FUTs are classified according to the site of fucose addition as follows:
1,2 (encoded by FUT1 and FUT2); 1,3/4 (encoded
by FUT3); 1,3 (encoded by FUT4-7 and FUT9-11
and ft1-2); and 1,6 (encoded by FUT8)
(Kageyama et al., 1999;
Ma et al., 2006;
Mollicone et al., 2008).
Protein O-fucosyl transferases (POFUTs) are encoded by
pofut1 and pofut2. Of the FUTs, pofut1, pofut2 and
FUT8 have already been cloned in zebrafish. In mammals, these genes
encode the enzymes catalyzing O-fucosylation in the consensus
sequence of the epidermal growth factor (EGF)-like repeat (POFUT1)
(Fig. 8A, part a),
O-fucosylation in the consensus sequence of the thrombospondin type-1
repeat (POFUT2) (Fig. 8A, part
b) and 1,6-fucosylation to the innermost N-acetylglucosamine
(GlcNAc) moiety of the core N-linked glycans (FUT8; core
1,6-fucosylation) (Fig.
8A, part c). Therefore, we knocked down these genes to investigate
the importance of O- and core 1,6-fucosylation in the
formation of the vagus motor nuclei. Although MOs against pofut1,
pofut2 and FUT8 effectively knocked down the translation of
EYFP-tagged mRNAs (Fig. 8B,
parts a-f), these MOs did not induce fusion of the bilateral vagus motor
nuclei (Fig. 8C, parts a-c).
Instead, some of the morphants showed aberrant positioning of the vagus motor
neurons lateral to the dlX (Fig.
8C, parts a-c), whereas embryos injected with the standard control
MO were identical to the wild-type embryos (data not shown). These results
indicate that O- and core 1,6-fucosylation do not contribute
to the cessation of migration of vagus motor neurons.
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1,3-fucosylation consists of core 1,3-fucosylation to the
innermost GlcNAc moiety of the core N-linked glycans and terminal
fucosylation, where GDP-fucose is linked to the oligosaccharide chains as a
terminal modification that is not elongated further
(Fig. 8A, part c)
(Ma et al., 2006). Of the
terminal 1,3-FUTs, ft1, ft2, FUT7 and FUT9 have been
identified. However, knocking down of those genes did not induce fusion of the
bilateral vagus motor nuclei [1/30 (3%), 0/26 (0%), 0/38 (0%), 0/33 (0%),
respectively, data not shown]. Next, we knocked down FUT10, which
encodes a core 1,3-FUT. MO against FUT10 effectively knocked
down the translation of EYFP-tagged mRNA
(Fig. 8B, parts g,h), and
caused fusion of the bilateral vagus motor nuclei (22/35; 63%),
(Fig. 8C, part d). However,
unlike twdrw685 mutants, FUT10 morphants did not show the
overshooting of the vagus motor neurons, but showed disruption of
neuroepithelial apicobasal polarity and adherens junctions (data not shown).
These results indicate that 1,3-fucosylation catalyzed by FUT10 affects
neural development in a different manner from that of GMDS.
Notch activity is not reduced in twdrw685 embryos
Fucosylation has been shown to regulate Notch signaling, and genetic
inactivation of the Notch activator 
-Secretase causes mis-migration of
the facial motor neurons (Ishikawa et al.,
2005; Louvi et al.,
2004; Ma et al.,
2006; Sasamura et al.,
2007). Although O-fucosylation has an important role in
Notch signaling, the expression of the Notch target gene her4
(Takke et al., 1999) was not
affected in twdrw685 mutants in the present study
(Fig. 9A-C). Because Notch
signaling has an antineurogenic effect on neural stem cells, we observed
primary motor neurons and Rohan-Beard sensory neurons in the spinal cord
(Inoue et al., 1994;
Tokumoto et al., 1995). There
were no significant differences detected in the spinal cord between wild-type
and twdrw685 mutant embryos
(Fig. 9D-G). Because Notch
activation is also required for the segregation of the rhombomere boundary
(Cheng et al., 2004), we
examined the expression of genetic markers for hindbrain segmentation. The
expression pattern of radical fringe and wnt1 in
twdrw685 mutant embryos was identical to that in wild-type
embryos (Fig. 9H-K), indicating
that there is no significant defect in Notch activity in
twdrw685 mutant embryos.
Surrounding neuroepithelial cells regulate the migration and accumulation of vagus motor neurons
To determine which cells require gmds for the correct formation of
vagus motor nuclei, we performed mosaic analysis by transplanting
rhodamine-dextran-labeled wild-type cells at the blastoderm stage into
gmds morphant host embryos at the shield stage. The progeny of the
donor cells that differentiated into the motor neurons could be identified by
their GFP and rhodamine signals. The wild-type dlX progenitors in the
hindbrain of wild-type localized normally
(Fig. 10A, part a), but those
in the hindbrain of gmds morphant embryos did not stop at the correct
position and were positioned ectopically, close to the midline, similar to the
positioning of dlX progenitors in twdrw685 mutants
(Fig. 10A, parts b,c; C, parts
a,b) (n=3). Similar results were obtained following transplantation
of wild-type dlX progenitors into twdrw685 mutants (data
not shown; n=3). These results indicate that gmds expression
in vagus motor neuron progenitors is not essential for correct migration of
these progenitor cells.
Neuroepithelial cells are likely to be regulators of the migration of vagus
motor neuron progenitors, because they have been shown to support the caudal
migration of facial motor neuron progenitors by preventing the integration of
migrating facial motor neuron progenitors into the neuroepithelial layer
(Wada et al., 2006) and
gmds mRNA was expressed ubiquitously (data not shown). In the present
study, when wild-type neuroepithelial cells were placed into the dorsomedial
region of the hindbrain of gmds morphants, the vagus motor
progenitors were found to migrate to normal positions (line 2 in
Fig. 10B, parts a,f; C, part
c) (n=5). Of note, morphant vagus motor neuron progenitors entered
the dorsomedial region and positioned more dorsally if wild-type cells were
not distributed in the dorsomedial region of the hindbrain (line 1 in
Fig. 10B, parts a,e). These
results suggest that the recovery of fucosylation in neuroepithelial cells in
the dorsomedial region of the hindbrain is sufficient to restore normal
migration of mutant vagus motor progenitors.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The
twdrw685 mutation occurs in the gmds gene, which
encodes a key enzyme in the de novo synthesis pathway (Figs
1 and
5). As a result, expression of
fucosylated glycans recognized by AAL and LCA (core-fucosylation), as well as
the anti-sLex antibody (terminal fucosylation), was markedly and
specifically reduced. Injection of normal gmds mRNA was able to
restore the expression of these glycans
(Fig. 7; see Fig. S1 in the
supplementary material). These results indicate that core and terminal
fucosylation greatly depend on the de novo synthesis pathway.
The structure of GMDS is highly conserved among organisms; in bacteria and
Arabidopsis thaliana, GMDS works as an oligomer
(Mulichak et al., 2002;
Somoza et al., 2000;
Webb et al., 2004). The
twdrw685 mutation changes the conserved hydrophobic
residue Trp193 in L-GMDS (Trp186 in S-GMDS) into a basic
residue, Arg, in the region involved in oligomer formation
(Fig. 5C). Therefore, this
mutation may disrupt oligomerization of GMDS, which may be crucial for its
enzymatic activity. However, we cannot rule out the possibility that the
twdrw685 mutation affects the enzymatic activity directly
and/or the stability of the GMDS protein.
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1,2-, 1,3/4-, 1,3- or core 1,3-fucosylation may be involved in the formation of the vagus motor nuclei1,2-(catalyzed by FUT1 and FUT2),
1,3/4-(catalyzed by FUT3) and 1,3-(catalyzed by FUT4-7 and FUT9,
and FT1-2) fucosylation (Ma et al.,
2006). As a representative structure of terminal fucosylation,
sLex is the core recognition epitope for members of the Selectin
family, which mediate lymphocyte homing and initial leukocyte-endothelial cell
adhesion (Ma et al., 2006).
Core fucosylation includes core 1,3-(catalyzed by FUT10 and 11) and
core 1,6-fucosylation (catalyzed by FUT8), and transfers GDP-fucose to
the reducing terminal of the core structure of an asparagine-linked
oligosaccharide (Ma et al.,
2006). Core 1,6-fucosylation has been reported to be
essential for both transforming growth factor-β1 (TGF-β1) and EGF
receptor signaling (Wang et al.,
2006; Wang et al.,
2005). In O-fucosylation, GDP-fucose is linked directly
to Ser or Thr residues in the consensus sequence of the EGF-like repeat and in
the thrombospondin type-1 repeat by POFUT1 and POFUT2, respectively
(Ma et al., 2006). The Notch
signaling pathway, one of the pathways in which O-fucosylation is
involved, is well characterized (Ma et
al., 2006). In the present study, we knocked down FUT8,
pofut1 and pofut2 genes to investigate the contribution of core
1,6- and O-fucosylation in the migration of vagus motor neuron
progenitors, but did not observe any morphant with bilaterally fused vagus
motor nuclei that were seen in twdrw685 mutants. Our data
suggest that some other glycans than core 1,6- or
O-fucosylated glycans are involved in the signal that stops the
migration of vagus motor neuron progenitors in the right location. Therefore,
fucosylated adhesion molecules, such as Selectin ligands or soluble guidance
molecules may be fucosylation targets involved in the regulation of vagus
motor neuron migration (Ma et al.,
2006). Reelin is a candidate soluble guidance molecule, because it
is fucosylated (Botella-Lopez et al.,
2006), and repression of Reelin cause mis-formation of the vagus
motor nuclei (S. Kikkawa, personal communication).
In the present study, we further knocked down terminal 1,3-FUTs
(FT1, FT2, FUT7 and FUT9) and a core 1,3-FUT (FUT10), which have been
identified in zebrafish. However, those morphants did not show the same
phenotype as the twdrw685 mutants. Therefore, cloning and
knocking down of other FUTs, such as terminal
1,2-FUTs (FUT1, FUT2), a terminal
1,3/4-FUT (FUT3), terminal 1,3-FUTs
(FUT4-6) and a core 1,2-FUT (FUT11)
will be required for the future study.
Effect of the twdrw685 mutation on the Notch signaling pathway
The Notch signaling pathway has been shown to be a key regulator of many
developmental processes, including somitogenesis, vasculogenesis and
neurogenesis (Ma et al.,
2006). Notch ligands trigger Notch signaling by binding to the
extracellular domain of Notch, and this binding depends on the
O-fucosylation of the EGF-like repeats in Notch. However, there was
no prominent decrease in Notch signaling observed in
twdrw685 mutants, as determined by the expression level of
the Notch target gene her4, hindbrain segmentation and the number of
neurons (Fig. 9). These results
suggest that O-fucosylation is less sensitive to the loss of GMDS
compared with core and terminal fucosylation, which are significantly reduced
in the twdrw685 mutant
(Fig. 7; see Fig. S1 in the
supplementary material). This difference in sensitivity may be due to a
smaller quantitative demand for O-fucosylation or to differences in
the properties of POFUT1 and POFUT2 compared with other FUTs.
One possible reason why the Notch signal was not affected in
twdrw685 mutants is that the maternally supplied GMDS is
sufficient for normal activation of Notch during the earliest stages of
neurogenesis. In a preliminary study, we found that injection of an MO that
inhibits the translation of maternal/zygotic gmds mRNA causes severe
embryonic malformations and cell death at 24 hpf. Therefore, maternally
supplied GMDS may have a crucial role in early development. Another
possibility is that the GDP-fucose produced by the salvage pathway is
sufficient for the O-fucosylation of Notch. In mammals, this pathway
uses free cytosolic fucose as a substrate, which is derived from an
extracellular source or from lysosomal degradation, converting it to
GDP-fucose via fucokinase and GDP-fucopyrophosphorylase
(Fig. 1). Conversely, there are
no sequence-encoding enzymes involved in the salvage pathway in the
Drosophila genome (Roos et al.,
2002). Although it is unclear whether the teleost contains the
salvage pathway, a candidate gene that encodes zebrafish fucokinase
is registered in GenBank (XM_001344236)
(Fig. 1). Therefore, zebrafish
may have the salvage pathway, which could account for the
O-fucosylation of the Notch EGF-like repeat in the
twdrw685 mutant. However, in our preliminary study, the
twdrw685 mutants were not rescued after the injection of
fucose. This suggests that even if the salvage pathway does exist in
zebrafish, it is not sufficient to rescue the twdrw685
mutant because less than 10% of GDP-fucose is derived from this salvage
pathway (Ma et al., 2006).
Human congenital disorders of fucosylation
The molecular basis of the immunodeficiency in LAD II/CDG IIc can be
explained by the function of fucosylated glycans in cell adhesion, mediated by
an interaction between fucosylated oligosaccharides, sLex and the
Selectin family (Becker and Lowe,
2003; Ma et al.,
2006). However, it remains unclear why individuals with LAD II/CDG
IIc exhibit other defects, including mental retardation. The mouse and fly
knock-out strains with global depression of fucosylation have been reported
previously (Ishikawa et al.,
2005; Smith et al.,
2002). The FX knockout mouse is embryonic lethal and shows reduced
expression of sialyl Lewis X antigens. Severe Notch-deficiency phenotypes were
observed when GDP-fucose transporter was impaired by mutation in
Drosophila. However, these studies have not explained all the
mechanisms underlying the neuropathology of LAD II/CDG IIc. The
twdrw685 mutant is the first vertebrate model in which the
gmds gene is affected and in which a neural defect can be
demonstrated to result from aberrant migration of vagus motor neuron
progenitors. This mutant provides us with an ideal model system in which to
study the effects of fucosylation on neural migration. Using this model,
neural migration can be visualized in vivo to evaluate the effects of
gain-of-function and loss-of-function following injection of DNA constructs,
mRNA or MOs, or in response to pharmacological treatment. Further analysis of
the twdrw685 mutant, the FX knock out mouse and the
GDP-fucose transporter knockout fly may contribute to a greater understanding
of the mechanisms responsible for the neurological disorders in individuals
with LAD II/CDG IIc.
|
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/136/10/1653/DC1
The authors thank Dr T. M. Jessell (Columbia University) for the anti-Lhx2/9 antibody, Dr H. Kakinuma for providing pofut1 cDNA, Dr S. Kurisu for providing the pCS2-EYFP vector, Dr S. Oka (Kyoto University) for technical comments, Dr S. Kikkawa (Kobe University) for the personal communication, Dr A. Thomson for critical reading of the manuscript, the BSI Research Resources Center for performing the DNA sequence analysis, and the other members of the Okamoto laboratory for their technical assistance, fish care and helpful discussions. This work was supported, in part, by a Grant-in-Aid from the Ministry of Education, Culture, Sport, Science and Technology of Japan, and by grants for Core Research for Evolutional Science and Technology from the Japan Science and Technology Agency.
* These authors contributed equally to this work 
Present address: Department of Life Science and Medical Bio-Science, School
of Advanced Science and Engineering, Waseda University, 2-2 Wakamatu-cho,
Shinjuku-ku, Tokyo 162-8480, Japan
Present address: Center for Transdisciplinary Research, Niigata University,
Ikarashi-2, Nishi-ku, Niigata 950-2181, Japan
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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