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First published online 3 July 2008
doi: 10.1242/dev.022178
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Institute of Neuroscience, 1254 University of Oregon, Eugene, OR 97403, USA.
* Author for correspondence (e-mail: eisen{at}uoneuro.uoregon.edu)
Accepted 11 June 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: ErbB2, ErbB3, Neuregulin, Dorsal root ganglion, Neural crest migration, Zebrafish
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). DRGs
are located adjacent to the spinal cord, and their component neurons and glia
arise from trunk neural crest (NC) cells
(Le Douarin and Kalcheim,
1999; Kalcheim,
2000; Eisen and Weston,
1993). Neural crest is a transient, embryonic cell population that
emigrates from the dorsal neuroepithelium, migrates along specific pathways
and generates a variety of progeny, including neurons and glia of the
peripheral nervous system, pigment cells and craniofacial cartilages. Trunk NC
cells migrate on two pathways: a ventral (also called medial) pathway between
the somites and neural tube; and a dorsolateral (also called lateral) pathway
between the somites and overlying ectodermal epithelium
(Weston and Butler, 1966;
Serbedzija et al., 1989;
Serbedzija et al., 1990;
Erickson et al., 1992;
Erickson and Weston, 1983;
Raible et al., 1992). The
ventral or medial migration pathway is restricted to a particular region of
the somites (Le Douarin and Kalcheim,
1999; Kalcheim,
2000); in zebrafish, this pathway is restricted to the middle of
the medial surface of each somite, half way between adjacent somite boundaries
(Honjo and Eisen, 2005;
Raible et al., 1992). Thus,
zebrafish trunk NC cells migrate in a pattern of `streams'; a single stream
underlies the middle of each somite. Zebrafish DRG neurons are derived from
among the earliest NC cells to migrate on the medial pathway
(Raible and Eisen, 1994).
However, whether DRGs arise from a specified NC subpopulation, how the fates
of individual NC cells are regulated during migration, and how NC cells
recognize specific target locations appropriate for their eventual
differentiation are currently not well understood.
In amniote embryos, ErbB3 is one factor that has been implicated in NC
migration. ErbB3 is a receptor tyrosine kinase proto-oncogene member of the
epidermal growth factor (EGF) receptor family. Although ErbB3 can form
homodimers, active receptors require heterodimerization with ErbB2
(Guy et al., 1994;
Tzahar et al., 1996;
Graus-Porta et al., 1997;
Kim et al., 1998;
Olayioye et al., 2000;
Holbro et al., 2003).
ErbB2/ErbB3 heterodimers bind to neuregulin (Nrg) 1 or Nrg2, members of the
EGF-like polypeptide growth factor superfamily
(Riese et al., 1995;
Busfield et al., 1997;
Carraway et al., 1997;
Chang et al., 1997;
Olayioye et al., 2000;
Holbro et al., 2003). Nrg1
signaling via ErbB3 is important for several developmental processes,
including glial migration and development
(Riethmacher et al., 1997;
Erickson et al., 1997;
Garratt et al., 2000;
Lyons et al., 2005),
development of myelination (Chen et al.,
2006), muscle acetylcholine receptor expression
(Morris et al., 1999;
Woldeyesus et al., 1999;
Lin et al., 2000; Fall, 2003),
and neural migration (Anton et al.,
1997; Rio et al.,
1997; Olayioye et al.,
2000).
The initial suggestion of a role for ErbB3 in NC migration came from mice
with targeted mutations in Erbb2, Erbb3 or Nrg1
(Britsch et al., 1998). These
mice lack sympathetic ganglia, presumably because without ErbB
receptor-mediated neuregulin signaling, NC cells cannot migrate to the region
lateral of the dorsal aorta, where sympathetic neuron differentiation occurs
(Britsch et al., 1998). By
contrast, DRGs, the progenitors of which do not have to migrate any
significant distance after emigrating from the neural folds
(Teillet et al., 1987),
initially appear normal in Erbb3 mutant mice
(Britsch et al., 1998).
However, although NC cells form an ectopic cluster around the DRGs in
Erbb3 mutant mice, these ectopic NC cells do not differentiate into
DRG neurons or glia (Britsch et al.,
1998); later, most DRG neurons reportedly die
(Riethmacher et al., 1997).
Erbb2 mutant mice die of cardiac defects too early to determine
whether ErbB2 is required for DRG formation
(Morris et al., 1999;
Woldeyesus et al., 1999).
Thus, the role of ErbB signaling in DRG formation remains unresolved.
Here, we show that zebrafish mutants lacking function of either ErbB3b or
ErbB2 do not form trunk NC-derived DRG or sympathetic neurons. By contrast,
cranial NC-derived enteric neurons appear normal. Previous studies have shown
that zebrafish erbb3b is expressed in NC cells and that
erbb3b mutants have defects in glial migration and in myelinating
Schwann cell terminal differentiation
(Lyons et al., 2005), similar
to mouse Erbb3 mutants (Lee et
al., 1995; Meyer and
Birchmeier, 1995; Erickson et
al., 1997; Meyer et al.,
1997; Riethmacher et al.,
1997). We provide evidence that in zebrafish erbb3b
mutants, migrating trunk NC cells do not pause in the position where DRGs
normally form. Our results suggest that ErbB receptor signaling is required
during specific periods when NC-derived cells normally pause at the location
where DRGs form. Thus, we suggest that the absence of DRGs in erbb3b
mutants is a consequence of inappropriate trunk NC cell migration resulting
from the inability of mutant NC cells to recognize a specific target location.
We further hypothesize that the absence of sympathetic ganglia also results
from failure of correct trunk NC cell migration. By contrast, the apparently
normal formation of enteric neurons in erbb3b mutants suggests that
at least some cranial NC migrates appropriately in the absence of ErbB
receptor signaling. To learn which ligands affect trunk NC migration, we
isolated genes encoding three different zebrafish neuregulins. We knocked
these down using morpholino antisense oligonucleotides (MOs) and found that
two of the neuregulins act in concert to regulate trunk NC migration and DRG
formation.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Fish
were maintained in the University of Oregon Zebrafish Facility on a 14-hour
light/10-hour dark cycle at 28.5°C. Embryos and larvae were staged
according to Kimmel et al. (Kimmel et al.,
1995) by number of somites, by hours post fertilization at
28.5°C (hpf), or by days post fertilization at 28.5°C (dpf). Mutant
embryos were generated by crossing two heterozygous adult carriers. We also
used the Tg(-4.9sox10:EGFP)ba2 transgenic line [hereafter
referred to as Tg(sox10:GFP)] in which GFP is driven by the
sox10 promoter (Wada et al.,
2005; Carney et al.,
2006). Homozygous Tg(sox10:GFP) fish have a mutant
phenotype; therefore, to obtain erbb3b mutants carrying this
transgene, heterozygous erbb3b mutant adults were crossed with
erbb3b; Tg(sox10:GFP) fish.
RNA in situ hybridization and immunohistochemistry
RNA in situ hybridization was performed as described previously
(Appel and Eisen, 1998).
Antisense riboprobes were detected by NBT/BCIP (Roche Diagnostics).
DIG-labeled antisense RNA probes (Roche Diagnostics) for RNA in situ
hybridization were generated from plasmids as follows: neurogenin 1
(neurog1) plasmid (Blader et al.,
1997) was cut with BamHI and transcribed with T7
polymerase; neurod plasmid (Blader
et al., 1997) was cut with NotI and transcribed with T3;
crestin plasmid (Luo et al.,
2001) was cut with EcoRI and transcribed with T7;
sox10 plasmid (Dutton et al.,
2001) was cut with SalI and transcribed with T7.
Anti-Elavl antibody (16A11; previously known as anti-HuC/D) was used at 1:1000
(Marusich et al., 1994;
Henion et al., 1996) and
anti-tyrosine hydroxylase (TH) antibody (Pel-Freeze) was used at 1:100
(An et al., 2002). Alexa-488
conjugated goat anti-rabbit polyclonal antibody was used as secondary
antibody. Frozen sectioning was performed after RNA in situ hybridization.
Time-lapse confocal microscopy
Migration of NC expressing GFP was visualized using a confocal laser
scanning microscope (Zeiss LSM5 Pascal). Embryos were mounted in 0.2% agar
containing dilute tricaine. Images were taken from around 24-72 hpf. For
identifying mutants, embryos were fixed after imaging and genotyped by PCR as
described by Lyons et al. (Lyons et al.,
2005).
AG1478 treatment
AG1478 [4-(3-chloroanilino)-6,7-dimethoxyquinazoline; Calbiochem] was used
at 4 µM in 0.4% DMSO as described by Lyons et al.
(Lyons et al., 2005). As a
control, we used 0.4% DMSO alone. All embryos were dechorionated before
treatment. DRG axial level was scored at 4 dpf in AG1478-treated embryos by
counting somite number at this stage from the most posterior Elavl-positive
cranial ganglia (ganglia of vagus and posterior lateral line nerves)
(Raible and Kruse, 2000). The
most anterior somite, which is the one closest to these ganglia, was
designated as somite one, consistent with our previous studies showing that
the most anterior five somites remain present through at least 12 dpf
(Morin-Kensicki et al., 2002).
We counted somites, rather than DRGs, because we found that DRGs were variably
present medial to the first two somites in 4 dpf wild-type embryos.
neurogenin 1 and neurogenin 2 gene cloning
Primers for nrg1 cloning were designed based on EST clones found
by using murine Nrg1 sequence to search the zebrafish Ensemble
database
(http://www.ensembl.org/Danio_rerio/index.html).
For nrg2, the Fugu Ensemble database
(http://www.ensembl.org/Takifugu_rubripes/index.html)
was first searched by using murine sequences and then the zebrafish database
was searched using Fugu sequence. Two high homology sequences were found on
chromosome (Chr) 14 and Chr 21. Primers were designed based on EST clones and
amplified by PCR. 5'- or 3'-RACE were performed using SMART RACE
cDNA Amplification kit (Clontech). Although we found an EST clone (clone
ID810694; BC139893) that is nrg2a by sequence comparison, our clone
from RT-PCR and 5', 3' RACE is different in the 3' region
(see Fig. S2 in the supplementary material). We have never been able to
isolate exactly the same clone as the EST described above. This might be
because of background strain or tissue sample differences.
RT-PCR
Splice-blocking and mismatch control MO-injected embryos were collected and
RNA extracted with TRI reagent (MRC). RT-PCR was performed with 2 or 3 µg
of each RNA using SuperscriptIII (Invitrogen). For MO confirmation,
gene-specific primers were used; an oligo dT primer was used at each stage to
examine gene expression. Subsequently, fragments were amplified by PCR.
Primers sequences used for RT-PCR are as follows: nrg1 (MO) forward
primer, 5'-ctgttgatacggaaatccac-3'; nrg1 (MO) reverse
primer, 5'-tcgctctcgtaaactctgcc-3'; nrg1 (type I) forward
primer, 5'-atggctgaggtgaaagcaggcaa-3'; nrg1 (type II)
forward primer, 5'-atggcgattctgccaggacgca-3'; nrg1 (type
III) forward primer, 5'-gagatgaagtcggaggcggcgga-3', nrg1
(all types) reverse primer, 5'-cagtattccttctcgctctcgttg-3';
nrg2a forward primer, 5'-ggcggcgactgttactacat-3';
nrg2a reverse primer, 5'-cccattggccaggttgcgatt-3'; nrg2b
forward primer, 5'-ctacacctgtgtggtggagaa-3'; nrg2b reverse primer,
5'-tgcctccatacacgccgggctactgc-3'; β-actin forward primer,
5'-tggcatcacaccttctac-3'; β-actin reverse primer,
5'-agaccatcaccagagttc-3'.
Morpholino microinjection
Embryos were injected with 2-5 nl of MOs into the yolk at the one-cell
stage. nrg1 MO [100 µM-1 mM; MO sequences reported by Milan et al.
(Milan et al., 2006)],
nrg2a MO (75 µM for single MO injections, 100 µM for
co-injection with nrg1 MO;
5'-tgacagaggagaaactcacttgcag-3') and nrg2b MO (100 µM;
5'-ttgacaatgtgtaacttacttgcag-3') were injected into wild-type
embryos. Mismatch MOs were used as a control for each MO. Pipettes were pulled
on a Sutter Instruments Micropipette puller (Model P-2000). Injections were
performed with an air injection apparatus (ASI). The efficacy of the
nrg1 MO varied from injection to injection. Therefore, we performed
an RT-PCR for each injection and compared fish that had similar levels of
mis-spliced transcript, rather than comparing fish that received similar MO
concentrations.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Henion et al.,
1996; Honjo and Eisen,
2005), sympathetic (An et al.,
2002; Stewart et al.,
2004) and enteric (Kelsh and
Eisen, 2000; Kuhlman and
Eisen, 2007) neurons. At 7 dpf, wild-type embryos have
Elavl-positive DRG neurons in every segment, except they are occasionally
absent from the first segment. By contrast, erbb3b mutants lack DRG
neurons (Fig. 1). To determine
whether DRG neurons formed earlier, we examined expression of neurod
and neurog1, the earliest-known markers of nascent DRG neurons
(Cornell and Eisen, 2002;
Ungos et al., 2003). These
markers were never expressed in the DRG region of erbb3b mutants
(Fig. 1), suggesting that they
never form DRG neurons. DRG neurons are derived from trunk NC, which also
generates sympathetic neurons (Raible and
Eisen, 1994); erbb3b mutants lack sympathetic neurons at
7 dpf (Fig. 1). In contrast to
DRG and sympathetic neurons, enteric neurons are derived from cranial NC
(Tobin et al., 2008;
Reichenbach et al., 2008);
enteric neurons appeared normal in erbb3b mutants
(Fig. 1).
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ErbB2 and ErbB3 are required for migrating trunk neural crest cells to pause at the location where DRGs normally form
The absence of DRG and sympathetic neurons in erbb3b and
erbb2 mutants might result from trunk NC depletion. To test this
possibility, we labeled erbb3b
(Fig. 2) and erbb2
(not shown) mutants with riboprobes for two genes expressed in migrating NC
cells, crestin (Luo et al.,
2001) and sox10
(Dutton et al., 2001); the
number of NC cells and their migratory pattern appeared normal at 24 hpf. At
27 hpf, a subset of NC cells normally clusters at the dorsal aspect of the
notochord, where DRGs form in wild type. A subset of NC cells remains
clustered at the dorsal and ventral aspects of the notochord at 30 hpf, when
many NC cells have already migrated to a position ventral of the notochord. NC
cell clustering at the dorsal aspect of the notochord was never seen in
erbb3b and erbb2 mutants, and the region ventral of the
notochord appeared depleted of NC cells
(Fig. 2 and data not shown).
This diminution in ventral NC cells did not result from delayed migration,
because the region dorsal of the notochord had approximately the same number
of NC cells in wild type, erbb3b and erbb2 mutants
(Fig. 2), suggesting some other
alteration of NC migration in the mutants.
To understand migration of NC cells, we performed time-lapse confocal
microscopy using the Tg(sox10:GFP) transgenic line
(Wada et al., 2005;
Carney et al., 2006). In wild
type, as NC cells migrate along the medial pathway, some of them pause at the
dorsal and ventral aspects of the notochord, often for more than 24 hours
(Fig. 3; average pause=27
hours; n=3 segments in each of three embryos). Interestingly, these
NC cells contact one another, as previously described for migrating NC cells
(Fig. 3)
(Krull et al., 1995;
Kasemeier-Kulesa et al.,
2005). Later, between about 50-70 hpf, these paused NC cells
become undetectable at mid-trunk levels, presumably because sox10 is
downregulated. In erbb3b mutants, very few NC cells paused at the
dorsal and ventral aspects of the notochord, and instead they continuously
migrated away (Fig. 3). The
small number of NC cells paused at the dorsal aspect of the notochord in
erbb3b mutants did so only briefly compared with wild type (average
pause=4.8 hours; n=3 segments in each of three embryos). These data
are consistent with results from fixed embryos, and together suggest that
ErbB3b is required for migrating NC cells to pause just dorsal of the
notochord, in the location where DRGs normally form.
|
), there are no detailed studies linking trunk NC migration
and trunk sympathetic ganglion formation in zebrafish. Zebrafish trunk
sympathetic ganglia are not segmentally organized for at least the first week
of development (Fig. 1; Y.H.
and J.S.E., unpublished). This is consistent with studies from other species
showing that sympathetic ganglion segmentation appears to arise by rostral or
caudal migration of cells, rather than being imposed by the segmental
migration of trunk NC (Young et al.,
2004; Kasemeier-Kulesa et al.,
2005). Thus, to learn how ErbB2/ErbB3 signaling affects NC
migration and formation of trunk ganglia, we focused on DRG formation.
To test directly when ErbB signaling is required for migrating NC cells in
DRG neuron formation, we blocked ErbB signaling conditionally with a
pharmacological inhibitor, AG1478. Previous studies have shown that AG1478
inhibits signaling through all ErbB receptors
(Busse et al., 2000;
Levitzki and Gazit, 1995);
Lyons et al. (Lyons et al.,
2005) showed that in zebrafish, AG1478 treatment replicates the
phenotype of erbb3b mutants. We treated zebrafish embryos with 4
µM AG1478 starting at 20 hpf and followed migrating NC cells by time-lapse
confocal microscopy, as we had done with wild type and erbb3b mutants
(see above). The NC migration phenotype of AG1478-treated embryos was
essentially the same as the NC migration phenotype of erbb3b mutants;
NC cells failed to pause at the dorsal and ventral aspects of the notochord,
and instead migrated away (Fig.
3; average pause=7.65 hours; n=3 segments in each of
three embryos).
After showing that AG1478 treatment mimics the erbb3b mutant phenotype, we treated embryos at various time periods to learn when ErbB receptors are required for DRG formation (Fig. 4). We performed two series of experiments. In one series we treated embryos from 8 hpf until various later time points. In the other series we treated embryos from various early time points until 30 hpf. DRGs formed medial to the anterior-most five somites even with the earliest treatment, which started at 8 hpf; thus, we only analyzed DRGs in segments posterior of the fifth somite. When we treated embryos from 8 to 14 or 18 hpf, most DRG neurons were missing from segments 6-18, but many DRG neurons formed in more posterior segments (Fig. 4A,B,G). Embryos treated from 8 to 30 hpf lacked DRG neurons from segments 6-30; embryos treated from 8 hpf to times between 18 and 30 hpf had intermediate phenotypes in which the axial level at which DRGs did not form was progressively more posterior for later treatments (Fig. 4G). Embryos treated beginning at 8, 14 or 18 hpf through 30 hpf lacked DRG neurons from segments 6-30 (Fig. 4H). Embryos treated beginning at 22, 24, or 26 hpf to 30 hpf formed DRGs neurons in anterior segments, but not in more posterior ones. Based on these results, we conclude that ErbB signaling is required between 18-30 hpf for DRG neuron formation in segments 6-30, consistent with the period when migrating NC cells pause at the location where DRGs normally form.
neuregulin 1 alone is not required for DRG neuron formation
Mice with targeted mutations in Erbb3 or Erbb2 have NC
cell migration defects (Britsch et al.,
1998). Mice with a targeted mutation in the ErbB2/ErbB3 ligand
Nrg1 show the same NC cell defects as Erbb3 and
Erbb2 mutant mice, suggesting that in mouse Nrg1 is required for
normal NC cell migration. To determine whether Nrg1 affects NC cell migration
or DRG formation in zebrafish, we isolated zebrafish nrg1 and found
that there are five splice variants that conform to the three splice variant
types described in mouse and human (see Fig. S1 in the supplementary material)
(Falls, 2003). To determine
the temporal expression pattern of each nrg1 isoform, we performed
RT-PCR at various stages from 50% epiboly to 72 hpf, and in adult brain
(Fig. 5). The type I isoform is
expressed throughout almost all developmental stages, except 36 hpf, and is
only very weakly expressed in adult brain. The type II isoform is strongly
expressed during epiboly, but is then downregulated and upregulated at several
developmental stages. It is more strongly expressed in adult brain than is the
type I isoform. The type III isoform is strongly expressed at all stages, with
the exception of 36 hpf. We then designed isoform-specific riboprobes
(Fig. 5). A type I-specific
riboprobe showed weak expression in the somites along the NC medial migration
pathway. A type II-specific riboprobe showed similar somite expression, and
also showed expression in clusters of spinal cord cells in the position of
motoneurons. A type III-specific riboprobe showed no somite expression, but
similar to type II, was expressed in clusters of ventral spinal cord cells.
Considering that somites are important for NC cell migration
(Honjo and Eisen, 2005) and
that ErbB3 signaling is required from 18-30 hpf for DRG neuron formation, the
type I isoform is the most likely candidate ErbB2/ErbB3 receptor ligand for
DRG neuron formation.
|
) that
targeted the EGF-like domain and knocked down all Nrg1 isoforms. Knockdown of
Nrg1 resulted in occasional mislocalization or loss of DRG neurons
(Fig. 5;
Table 1). However, the
phenotype was not as severe as erbb3b or erbb2 mutants,
suggesting that other ErbB3 ligands are also important for DRG formation.
|
;
Busfield et al., 1997;
Carraway et al., 1997;
Chang et al., 1997;
Olayioye et al., 2000;
Holbro et al., 2003;
Lin and Winer, 2004). To
examine whether Nrg2 is important for zebrafish DRG neuron development, we
isolated nrg2 and found that zebrafish has two nrg2 genes
(nrg2a and nrg2b; see Fig. S2 in the supplementary
material). nrg2a is located on chromosome 21 and nrg2b is
located on chromosome 14. Both of these genes have the typical neuregulin gene
structure (see Fig. S2 in the supplementary material). nrg2a has four splice variants, whereas nrg2b has only a single splice variant. We performed RT-PCR to examine expression of the two nrg2 genes during development and in adult brain. nrg2a is first expressed during epiboly and continues to be expressed until 72 hpf; it is also expressed in adult brain. By contrast, nrg2b is first expressed at 16 hpf. Like nrg1 type II, nrg2b is downregulated and upregulated during development; it is also expressed in adult brain. We designed riboprobes specific to either nrg2a or nrg2b and found that nrg2a is expressed in somites, whereas nrg2b appears to be expressed primarily in ventral spinal cord at 24 hpf (Fig. 6). The distinct expression patterns of these genes suggest that they have different roles in zebrafish development.
Neuregulin 2a is important for DRG development
To test whether either of the two Nrg2 proteins affects DRG development, we
knocked them down individually using splice-blocking MOs designed against the
EGF-encoding domain of each gene. Like Nrg1, the EGF domain is crucial for
binding ErbB receptors and thus for Nrg2 function
(Jones et al., 1999;
Falls, 2003). Embryos injected
with nrg2a MO lacked most DRG neurons at 2 dpf, however, many DRG
neurons were present at 4 dpf (Fig.
7), although some were mislocalized
(Table 1). This recovery might
occur because MO efficacy decreased over time; thus, we performed RT-PCR to
examine nrg2a splicing from 1-4 dpf. RT-PCR showed that the MOs
affected EGF-like domain splicing through the first 2 days of development.
However, at later stages, both full-length and incorrectly spliced mRNA were
present (Fig. 7). As we
described above, our data from pharmacological inhibition revealed that the
crucial period of ErbB signaling for DRG formation is between 18-30 hpf
(Fig. 4). Thus, we would not
expect diminution of MO efficacy at 3 or 4 dpf to affect DRG neurons.
Therefore, our results suggest that Nrg2a plays a role in DRG neuron
development in zebrafish, but that other factors must also be involved.
|
Our results showed a partial requirement for both Nrg1 and Nrg2a in DRG neuron development. To learn whether DRGs are present in the absence of both of these neuregulins, we knocked them down together by injecting nrg1 and nrg2a MOs simultaneously and confirmed by RT-PCR that both genes were incorrectly spliced through 2 dpf. Embryos injected with nrg1 plus nrg2a MOs lacked DRG neurons at 4 dpf (Fig. 8; Table1), suggesting that Nrg1 and Nrg2a both act during DRG neuron formation.
To learn whether the absence of DRG neurons in nrg1 plus nrg2a MO-injected embryos resulted from abnormal NC migration, as it did in erbb3b or erbb2 mutants, we labeled double MO-injected embryos with either crestin or sox10 riboprobe. As in the mutants, the number of NC cells and their initial migration appeared normal at 24 hpf (Fig. 8). However, at 27 and 30 hpf there were fewer NC cells ventral of the notochord than in wild type (Fig. 8), consistent with the idea that Nrg1 and Nrg2a both act as ligands for ErbB2/ErbB3 receptors in DRG neuron formation and are important for migrating NC cells to pause at the location where DRGs normally form.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ErbB2/ErbB3 signaling is required during a specific time window of trunk neural crest migration for DRG neuron formation
In the absence of ErbB2/ErbB3 signaling in zebrafish, migrating NC cells
fail to pause at the location where DRGs normally form, and DRG neurons are
not produced. Our time-lapse observations of Tg(sox10:GFP) fish
suggest that DRG neurons do not form in the mutants because their progenitors
continue migrating ventrally rather than pausing. Consistent with this idea,
our experiments using the AG1478 inhibitor show that ErbB signaling is
required between 18 and 30 hpf, precisely when DRG progenitors reach the
dorsal aspect of notochord and pause. For example, trunk NC starts to migrate
around 16 hpf at the level of somite 6 and around 19 hpf at the level of
somite 17 (Raible et al.,
1992), and it takes about 3-5 hours of migration for the first
cell to reach the dorsal aspect of the notochord (Y.H. and J.S.E.,
unpublished). Consistent with this timing, starting inhibitor treatment at 18
hpf prevented DRG neuron formation beginning just posterior of somite 6, and
starting treatment at 24 hpf prevented DRG neuron formation just posterior of
somite 20. Interestingly, earlier treatment, from 8-14 hpf inhibited DRG
neuron formation anteriorly, possibly because the inhibitor takes some time to
wash out. However, consistent with the idea that ErbB signaling is required
around the time NC reaches the position where it normally pauses, DRG neurons
formed posteriorly in this treatment protocol. Together, these observations
argue that ErbB signaling is required for DRG progenitors to pause in a
location where they receive a DRG neuron differentiation-promoting signaling,
and that in the absence of ErbB signaling, the progenitors migrate through
this location, thus they fail to receive the signal and cannot generate DRG
neurons.
Mice with targeted mutations in the Erbb3 and Erbb2 genes
also have defects in trunk NC migration
(Britsch et al., 1998). Our
observations and those of Budi and colleagues
(Budi et al., 2008) provide
evidence that zebrafish trunk NC cells migrate past their normal targets in
erbb3b mutants. By contrast, in mouse ErbB3, ErbB2 and
Nrg1 mutants, trunk NC cells stop migrating immediately after
emigrating from the neural tube. This apparent difference between zebrafish
and mouse might result from differences in the migration pathway of DRG
progenitors. In zebrafish, DRG progenitors migrate ventrally until they reach
the dorsal aspect of notochord. By contrast, in mouse, DRGs are formed very
close to the region where trunk NC cells emigrate from the neural tube. Thus,
DRG progenitors do not migrate any significant distance in mouse, whereas in
zebrafish they need both to migrate a significant distance and to recognize a
specific location at which to stop migrating. ErbB receptors have previously
been shown to regulate expression of adhesion molecules
(D'Souza and Taylor-Papadimitriou,
1994; Sweeney et al.,
2001; Bertucci et al.,
2004; Amin et al.,
2005; Way and Lin,
2005; Yasmeen et al.,
2006). We predict that there will be species-specific differences
in this regulation, based on the different distances that DRG progenitors
migrate in mouse and zebrafish.
|
);
Erbb2 mutant mice die of cardiac defects too early to assay DRGs
(Morris et al., 1999;
Woldeyesus et al., 1999). Most
DRG neurons die in Erbb3 mutant mice
(Riethmacher et al., 1997) and
rescue of the cardiac defects in Erbb2 mutant mice reveals that, as
for Erbb3 mutant mice, there is a severe loss of DRG neurons in the
absence of Erbb2 (Morris et al.,
1999; Woldeyesus et al.,
1999). ErbB3 is thought to function autonomously in glial cell
development and non-autonomously in survival of DRG neurons in Erbb3
mutant mice (Riethmacher et al.,
1997). However, as in cardiac defect-rescued Erbb2 mutant
mice, in Erbb3 mutant mice the loss of DRG neurons occurs very early,
almost simultaneously with the loss of glia
(Riethmacher et al., 1997),
raising the possibility that the same mechanisms could be affecting both cell
types autonomously. This possibility is consistent with the observation that
ErbB3 is expressed in at least some mouse DRG neurons
(Riethmacher et al., 1997;
Meyer et al., 1997) and
virtually all rat DRG neurons are ErbB2 and ErbB3 immunoreactive
(Pearson and Carroll, 2004).
Whether ErbB2/ErbB3 signaling is required autonomously in DRG neurons has not
yet been tested directly.
Zebrafish has multiple neuregulins, two of which participate in DRG neuron formation
We isolated one nrg1 gene and two nrg2 genes in
zebrafish. MO-mediated knockdown showed that Nrg1 and Nrg2a work together in
zebrafish DRG neuron development, whereas Nrg2b appears to be uninvolved in
this process. These results are consistent with the expression patterns of
these genes, nrg1 type I and type II and nrg2a are expressed
in somites, whereas nrb2b is only expressed in ventral spinal cord
neurons and is expressed more strongly after 48 hpf. In mouse, only
Nrg1 has been shown to be required for trunk NC cell migration. By
contrast, nrg1 and nrg2a both act during trunk NC cell
migration in zebrafish. DRG development was not examined in mice with a
targeted Nrg2 deletion (Britto et
al., 2004), so whether DRGs were normal is still an unanswered
question. Furthermore, there have been no reports about DRG development in
mice with targeted deletions of both Nrg1 and Nrg2. Thus,
whether both Nrg1 and Nrg2 are required for DRG neuron development in mammals
remains unknown.
|
),
and we report here that this is also the case in zebrafish. Development of
cervical and trunk sympathetic neurons has been described in zebrafish
(An et al., 2002;
Stewart et al., 2004).
However, virtually nothing is known about the NC migratory processes during
formation of zebrafish trunk sympathetic ganglia, because early markers such
as sox10 are downregulated several days before later markers such as
Elavl and TH are expressed, leaving a window during which sympathetic ganglion
formation cannot be followed. We observed that NC cells failed to pause at the
ventral aspect of the notochord in erbb3b mutants and we hypothesize
that the NC cells that normally pause in this region in wild-type embryos are
sympathetic ganglion precursors. Thus, we propose that, as for DRG precursors,
ErbB2/ErbB3 signaling is also required for sympathetic ganglion precursors to
pause at a specific location. Sympathetic ganglion formation appears much less
regular than DRG formation in zebrafish. For example, DRGs are segmentally
arranged and develop in a strict rostral-to-caudal sequence. By contrast,
sympathetic ganglia initially are not segmentally arranged and it is unclear
whether they develop in a strict rostral-to-caudal sequence
(An et al., 2002). Studies in
avians suggest that sympathetic ganglion progenitors migrate rostrally and
caudally during formation of sympathetic ganglia
(Young et al., 2004;
Kasemeier-Kulesa et al.,
2005). These studies also suggest that the segmental arrangement
of sympathetic ganglia arises late and is a consequence of this rostral and
caudal migration, rather than of the earlier segmental NC migration that
results in segmentally arranged DRGs
(Young et al., 2004). Thus, we
are unable to draw conclusions about when ErbB2/ErbB3 signaling is required
for sympathetic neuron formation. However, sympathetic neurons were present in
the AG1478 treatments in which DRGs were absent, suggesting that ErbB2/ErbB3
signaling is required either over a longer time period or over an entirely
different time period for sympathetic neuron formation than for DRG
formation.
ErbB2/ErbB3 signaling is not a general requirement for formation of peripheral neurons
ErbB2/ErbB3 signaling is required for both DRG and sympathetic neuron
formation, raising the possibility of a general requirement for ErbB signaling
in NC-derived peripheral neurons. However, this is apparently not the case,
because ErbB2/ErbB3 signaling is unnecessary for zebrafish enteric neuron
formation. Previous work suggested a role for ErbB2/ErbB3 signaling in
formation of mouse enteric neurons
(Erickson et al., 1997).
However, studies of conditional Erbb2 mutant mice revealed that ErbB2
is unnecessary for initial enteric neuron formation, but is required cell
non-autonomously for enteric neuron survival, consistent with expression of
ErbB2 in the colonic epithelium (Crone et
al., 2003). Thus, in mouse
(Crone et al., 2003), as in
zebrafish, the cranial NC cells that generate enteric neurons are able to
migrate normally into the intestine and to generate enteric neurons in the
absence of ErbB2. Together these studies suggest that ErbB2/ErbB3 signaling
may play distinct roles in migration of cranial and trunk NC. This is
consistent with other reports suggesting that, although cranial ganglia are
defective in Nrg1, Erbb2 and Erbb3 mutant mice, this likely
arises not because of an effect on cranial NC migration, but because
Nrg1-mediated ErbB2/ErbB3 signaling is required for survival of cranial
NC-derived neurons (Meyer and Birchmeier,
1995; Erickson et al.,
1997).
Although we argue that ErbB2/ErbB3 signaling is required for migrating trunk NC cells to pause in locations where they receive differentiation signals, there are other possibilities that we cannot yet rule out. For example, it is possible that ErbB2/ErbB3 signaling makes cells competent to receive differentiation signals and that failure of trunk NC cells to migrate properly in erbb2 and erbb3b mutants is a secondary consequence of failure to perceive a differentiation signal. Furthermore, the failure of DRG progenitors to pause at the right place in erbb2 and erbb3b mutants could cause defects in other progenitors, for example sympathetic ganglion progenitors. The lack of multiple trunk NC neuronal derivatives in erbb2 and erbb3b mutants raises the possibility that ErbB2/ErbB3 signaling acts early in trunk NC cells, perhaps in specific subpopulations. Although we cannot completely rule out this possibility, our inhibitor treatment experiments show that loss of ErbB2/ErbB3 signaling later, during trunk NC migration, is sufficient to cause the DRG neuron defect. In addition, early inhibitor treatment had no effect on sympathetic neuron formation. These results suggest that lack of DRG and sympathetic neurons in erbb2 and erbb3b mutants does not result from absence of specific subsets of trunk NC cells, but rather results from effects that occur to those cells during their migration.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/15/2615/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Amin, D. N., Tuck, D. and Stern, D. F. (2005). Neuregulin-regulated gene expression in mammary carcinoma cells. Exp. Cell Res. 309,12 -23.[CrossRef][Medline]
An, M., Luo, R. and Henion, P. D. (2002). Differentiation and maturation of zebrafish dorsal root and sympathetic ganglion neurons. J. Comp. Neurol. 446,267 -275.[CrossRef][Medline]
Anton, E. S., Marchionni, M. A., Lee, K. F. and Rakic, P. (1997). Role of GGF/neuregulin signaling in interactions between migrating neurons and radial glia in the developing cerebral cortex. Development 124,3501 -3510.[Abstract]
Appel, B. and Eisen, J. S. (1998). Regulation of neuronal specification in the zebrafish spinal cord by Delta function. Development 125,371 -380.[Abstract]
Bertucci, F., Borie, N., Ginestier, C., Groulet, A., Charafe-Jauffret, E., Adelaide, J., Geneix, J., Bachelart, L., Finetti, P., Koki, A. et al. (2004). Identification and validation of an ERBB2 gene expression signature in breast cancers. Oncogene 23,2564 -2575.[CrossRef][Medline]
Blader, P., Fischer, N., Gradwohl, G., Guillemot, F. and Strahle, U. (1997). The activity of neurogenin1 is controlled by local cues in the zebrafish embryo. Development 124,4557 -4569.[Abstract]
Britsch, S., Li, L., Kirchhoff, S., Theuring, F., Brinkmann, V.,
Birchmeier, C. and Riethmacher, D. (1998). The ErbB2 and
ErbB3 receptors and their ligand, neuregulin-1, are essential for development
of the sympathetic nervous system. Genes Dev.
12,1825
-1836.
Britto, J. M., Lukehurst, S., Weller, R., Fraser, C., Qiu, Y.,
Hertzog, P. and Busfield, S. J. (2004). Generation and
characterization of neuregulin-2-deficient mice. Mol. Cell.
Biol. 24,8221
-8226.
Budi, E. H., Patterson, L. B. and Parichy, D. M.
(2008). Embryonic requirements for ErbB signalling in neural
crest development and adult pigment pattern formation.
Development 135,2603
-2614.
Busfield, S. J., Michnick, D. A., Chickering, T. W., Revett, T. L., Ma, J., Woolf, E. A., Comrack, C. A., Dussault, B. J., Woolf, J., Goodearl, A. D. et al. (1997). Characterization of a neuregulin-related gene, Don-1, that is highly expressed in restricted regions of the cerebellum and hippocampus. Mol. Cell. Biol. 17,4007 -4014.[Abstract]
Busse, D., Doughty, R. S. and Arteaga, C. L. (2000). HER-2/neu (erbB-2) and the cell cycle. Semin. Oncol. 27,3 -8.[Medline]
Carney, T. J., Dutton, K. A., Greenhill, E., Delfino-Machin, M.,
Dufourcq, P., Blader, P. and Kelsh, R. N. (2006). A direct
role for Sox10 in specification of neural crest-derived sensory neurons.
Development 133,4619
-4630.
Carraway, K. L. 3rd, Weber, J. L., Unger, M. J., Ledesma, J., Yu, N., Gassmann, M. and Lai, C. (1997). Neuregulin-2, a new ligand of ErbB3/ErbB4-receptor tyrosine kinases. Nature 387,512 -516.[CrossRef][Medline]
Chang, H., Riese, D. J., 2nd, Gilbert, W., Stern, D. F. and McMahan, U. J. (1997). Ligands for ErbB-family receptors encoded by a neuregulin-like gene. Nature 387,509 -512.[CrossRef][Medline]
Chen, S., Velardez, M. O., Warot, X., Yu, Z. X., Miller, S. J.,
Cros, D. and Corfas, G. (2006). Neuregulin 1-erbB signaling
is necessary for normal myelination and sensory function. J.
Neurosci. 26,3079
-3086.
Cornell, R. A. and Eisen, J. S. (2002).
Delta/Notch signaling promotes formation of zebrafish neural crest by
repressing Neurogenin 1 function. Development
129,2639
-2648.
Crone, S. A., Negro, A., Trumpp, A., Giovannini, M. and Lee, K. F. (2003). Colonic epithelial expression of ErbB2 is required for postnatal maintenance of the enteric nervous system. Neuron 37,29 -40.[CrossRef][Medline]
D'Souza, B. and Taylor-Papadimitriou, J.
(1994). Overexpression of ERBB2 in human mammary epithelial cells
signals inhibition of transcription of the E-cadherin gene. Proc.
Natl. Acad. Sci. USA 91,7202
-7206.
Dutton, K. A., Pauliny, A., Lopes, S. S., Elworthy, S., Carney,
T. J., Rauch, J., Geisler, R., Haffter, P. and Kelsh, R. N.
(2001). Zebrafish colourless encodes sox10 and specifies
non-ectomesenchymal neural crest fates. Development
128,4113
-4125.
Eisen, J. S. and Weston, J. A. (1993). Development of the neural crest in the zebrafish. Dev. Biol. 159,50 -59.[CrossRef][Medline]
Erickson, C. A. and Weston, J. A. (1983). An SEM analysis of neural crest migration in the mouse. J. Embryol. Exp. Morphol. 74,97 -118.[Medline]
Erickson, C. A., Duong, T. D. and Tosney, K. W. (1992). Descriptive and experimental analysis of the dispersion of neural crest cells along the dorsolateral path and their entry into ectoderm in the chick embryo. Dev. Biol. 151,251 -272.[CrossRef][Medline]
Erickson, S. L., O'Shea, K. S., Ghaboosi, N., Loverro, L., Frantz, G., Bauer, M., Lu, L. H. and Moore, M. W. (1997). ErbB3 is required for normal cerebellar and cardiac development: a comparison with ErbB2-and heregulin-deficient mice. Development 124,4999 -5011.[Abstract]
Falls, D. L. (2003). Neuregulins: functions, forms, and signaling strategies. Exp. Cell Res. 284, 14-30.[CrossRef][Medline]
Garratt, A. N., Britsch, S. and Birchmeier, C. (2000). Neuregulin, a factor with many functions in the life of a schwann cell. BioEssays 22,987 -996.[CrossRef][Medline]
Graus-Porta, D., Beerli, R. R., Daly, J. M. and Hynes, N. E. (1997). ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling. EMBO J. 16,1647 -1655.[CrossRef][Medline]
Guy, P. M., Platko, J. V., Cantley, L. C., Cerione, R. A. and
Carraway, K. L., 3rd (1994). Insect cell-expressed p180erbB3
possesses an impaired tyrosine kinase activity. Proc. Natl. Acad.
Sci. USA 91,8132
-8136.
Henion, P. D., Raible, D. W., Beattie, C. E., Stoesser, K. L., Weston, J. A. and Eisen, J. S. (1996). Screen for mutations affecting development of Zebrafish neural crest. Dev. Genet. 18,11 -17.[CrossRef][Medline]
Holbro, T., Civenni, G. and Hynes, N. E. (2003). The ErbB receptors and their role in cancer progression. Exp. Cell Res. 284,99 -110.[CrossRef][Medline]
Honjo, Y. and Eisen, J. S. (2005). Slow muscle
regulates the pattern of trunk neural crest migration in zebrafish.
Development 132,4461
-4470.
Jones, J. T., Akita, R. W. and Sliwkowski, M. X. (1999). Binding specificities and affinities of egf domains for ErbB receptors. FEBS Lett. 447,227 -231.[CrossRef][Medline]
Kalcheim, C. (2000). Mechanisms of early neural crest development: from cell specification to migration. Int. Rev. Cytol. 200,143 -196.[CrossRef][Medline]
Kasemeier-Kulesa, J. C., Kulesa, P. M. and Lefcort, F.
(2005). Imaging neural crest cell dynamics during formation of
dorsal root ganglia and sympathetic ganglia.
Development 132,235
-245.
Kelsh, R. N. and Eisen, J. S. (2000). The zebrafish colourless gene regulates development of non-ectomesenchymal neural crest derivatives. Development 127,515 -525.[Abstract]
Kim, H. H., Vijapurkar, U., Hellyer, N. J., Bravo, D. and Koland, J. G. (1998). Signal transduction by epidermal growth factor and heregulin via the kinase-deficient ErbB3 protein. Biochem. J. 334,189 -195.[Medline]
Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B. and Schilling, T. F. (1995). Stages of embryonic development of the zebrafish. Dev. Dyn. 203,253 -310.[Medline]
Krull, C. E., Collazo, A., Fraser, S. E. and Bronner-Fraser, M. (1995). Segmental migration of trunk neural crest: time-lapse analysis reveals a role for PNA-binding molecules. Development 121,3733 -3743.[Abstract]
Kuhlman, J. and Eisen, J. S. (2007). Genetic screen for mutations affecting development and function of the enteric nervous system. Dev. Dyn. 236,118 -127.[CrossRef][Medline]
Le Douarin, N. M. and Kalcheim, C. (1999). The Neural Crest. Cambridge, UK: Cambridge University Press.
Lee, K. F., Simon, H., Chen, H., Bates, B., Hung, M. C. and Hauser, C. (1995). Requirement for neuregulin receptor erbB2 in neural and cardiac development. Nature 378,394 -398.[CrossRef][Medline]
Levitzki, A. and Gazit, A. (1995). Tyrosine
kinase inhibition: an approach to drug development.
Science 267,1782
-1788.
Lin, N. U. and Winer, E. P. (2004). New targets for therapy in breast cancer: small molecule tyrosine kinase inhibitors. Breast Cancer Res. 6,204 -210.[CrossRef][Medline]
Lin, W., Sanchez, H. B., Deerinck, T., Morris, J. K., Ellisman,
M. and Lee, K. F. (2000). Aberrant development of motor axons
and neuromuscular synapses in erbB2-deficient mice. Proc. Natl.
Acad. Sci. USA 97,1299
-1304.
Luo, R., An, M., Arduini, B. L. and Henion, P. D. (2001). Specific pan-neural crest expression of zebrafish Crestin throughout embryonic development. Dev. Dyn. 220,169 -174.[CrossRef][Medline]
Lyons, D. A., Pogoda, H. M., Voas, M. G., Woods, I. G., Diamond, B., Nix, R., Arana, N., Jacobs, J. and Talbot, W. S. (2005). erbb3 and erbb2 are essential for schwann cell migration and myelination in zebrafish. Curr. Biol. 15,513 -524.[CrossRef][Medline]
Marusich, M. F., Furneaux, H. M., Henion, P. D. and Weston, J. A. (1994). Hu neuronal proteins are expressed in proliferating neurogenic cells. J. Neurobiol. 25,143 -155.[CrossRef][Medline]
Meyer, D. and Birchmeier, C. (1995). Multiple essential functions of neuregulin in development. Nature 378,386 -390.[CrossRef][Medline]
Meyer, D., Yamaai, T., Garratt, A., Riethmacher-Sonnenberg, E., Kane, D., Theill, L. E. and Birchmeier, C. (1997). Isoform-specific expression and function of neuregulin. Development 124,3575 -3586.[Abstract]
Milan, D. J., Giokas, A. C., Serluca, F. C., Peterson, R. T. and
MacRae, C. A. (2006). Notch1b and neuregulin are required for
specification of central cardiac conduction tissue.
Development 133,1125
-1132.
Morin-Kensicki, E. M., Melancon, E. and Eisen, J. S. (2002). Segmental relationship between somites and vertebral column in zebrafish. Development 129,3851 -3860.[Medline]
Morris, J. K., Lin, W., Hauser, C., Marchuk, Y., Getman, D. and Lee, K. F. (1999). Rescue of the cardiac defect in ErbB2 mutant mice reveals essential roles of ErbB2 in peripheral nervous system development. Neuron 23,273 -283.[CrossRef][Medline]
Olayioye, M. A., Neve, R. M., Lane, H. A. and Hynes, N. E. (2000). The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J. 19,3159 -3167.[CrossRef][Medline]
Pearson, R. J., Jr and Carroll, S. L. (2004
