Slow muscle regulates the pattern of trunk neural crest migration in zebrafish
Yasuko Honjo* and
Judith S. Eisen
Institute of Neuroscience, 1254 University of Oregon, Eugene, OR 97403,
USA
*
Author for correspondence (e-mail:
yhonjo{at}uoneuro.uoregon.edu)
Accepted 4 August 2005
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SUMMARY
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In avians and mice, trunk neural crest migration is restricted to the
anterior half of each somite. Sclerotome has been shown to play an essential
role in this restriction; the potential role of other somite components in
specifying neural crest migration is currently unclear. By contrast, in
zebrafish trunk neural crest, migration on the medial pathway is restricted to
the middle of the medial surface of each somite. Sclerotome comprises only a
minor part of zebrafish somites, and the pattern of neural crest migration is
established before crest cells contact sclerotome cells, suggesting other
somite components regulate the pattern of zebrafish neural crest migration.
Here, we use mutants to investigate which components regulate the pattern of
zebrafish trunk neural crest migration on the medial pathway. The pattern of
trunk neural crest migration is aberrant in spadetail mutants that
have very reduced somitic mesoderm, in no tail mutants injected with
spadetail morpholino antisense oligonucleotides that entirely lack
somitic mesoderm and in somite segmentation mutants that have normal somite
components but disrupted segment borders. Fast muscle cells appear dispensable
for patterning trunk neural crest migration. However, migration is abnormal in
Hedgehog signaling mutants that lack slow muscle cells, providing evidence
that slow muscle cells regulate the pattern of trunk neural crest migration.
Consistent with this idea, surgical removal of adaxial cells, which are slow
muscle precursors, results in abnormal patterning of neural crest migration;
normal patterning can be restored by replacing the ablated adaxial cells with
ones transplanted from wild-type embryos.
Key words: Myotome, Danio rerio, spadetail, beamter, fused somites, sonic hedgehog, sonic-you, slow muscle omitted, Smoothened, unplugged, you too, after eight, deadly seven, Notch pathway mutants
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Introduction
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Although all vertebrates have a similar body plan, the early cell movements
that establish that body plan can differ considerably in different vertebrate
subgroups. For example, neural crest cells, a defining feature of vertebrates,
migrate from their origin in the region of the dorsal neural tube throughout
the body and differentiate into a well-characterized set of derivatives
(Le Douarin and Kalcheim,
1999
; Kalcheim,
2000
; Eisen and Weston,
1993
). The pathways of neural crest migration are highly
regulated, but differ between amniotes, such as avians and mice, and
anamniotes, such as zebrafish. The neural crest medial migration pathway in
zebrafish is restricted to the middle of the medial surface of each somite
(Fig. 1)
(Raible et al., 1992
); thus,
trunk neural crest cells migrate in a pattern of `streams' in which there is a
single stream medial to the middle of each somite. By contrast, the equivalent
migration pathway in avians and mice is restricted to the anterior half of
each somite. Despite these differences in migration path in amniotes and
anamniotes, early-migrating crest cells in both groups generate similar
neuronal derivatives (Le Douarin and
Kalcheim, 1999
; Kalcheim,
2000
; Christiansen et al.,
2000
).
The pattern of trunk crest migration is likely to be established by
different somite components in amniotes and anamniotes. In avian and mammalian
embryos, trunk neural crest migration is regulated by environmental cues
thought to be derived from somites, and especially from sclerotome. Many
studies provide evidence that neural crest migrates through the anterior half
sclerotome of each somite, and several molecules expressed in posterior half
sclerotome repel crest migration (Newgreen
et al., 1990
) (reviewed by Le
Douarin and Kalcheim, 1999
;
Krull, 2001
;
Kalcheim, 2000
;
Kuan et al., 2004
). In
contrast to amniote embryos, the majority of cells in the somites of
anamniotes such as zebrafish and frogs are myotomal, rather than sclerotomal
(Fig. 1)
(Morin-Kensicki and Eisen,
1997
; Keller,
2000
). In zebrafish, sclerotome precursors are first seen at the
most ventromedial region of the somite. These cells migrate dorsally and
eventually meet neural crest cells, but not until after the segmental pattern
of neural crest migration streams on the medial pathway is already
established. Thus, the mechanisms that initiate the segmental pattern of
neural crest migration streams in zebrafish are currently unknown.
Myotomal cells are present at the right time and place to establish the
segmental pattern of migration of zebrafish trunk neural crest on the medial
pathway. Studies of zebrafish sclerotome showed that, in contrast to avian
embryos, sclerotome was not required for formation of dorsal root ganglia or
for pathfinding by motor axons, leading Morin-Kensicki and Eisen
(Morin-Kensicki and Eisen,
1997
) to suggest that zebrafish myotome contains the patterning
information attributed to avian sclerotome. This idea is supported by the
observation that as neural crest cells begin to migrate along the medial
pathway, myotome cells are the first cells they encounter, and the onset of
neural crest migration coincides with contact between the two cell types
(Raible et al., 1992
). The
myotomal precursors of zebrafish slow and fast muscle fibers can be recognized
very early in development. Slow muscle precursors, called adaxial cells, can
be distinguished before segmentation occurs as a sheet of cuboidal cells in
the segmental plate adjacent to the notochord
(Devoto et al., 1996
). Shortly
after somite formation, adaxial cells start to elongate on the medial myotome
surface as a monolayer of muscle fibers. These cells then migrate as elongated
muscle fibers through the myotome to the lateral surface, where they form a
monolayer of slow muscle fibers (Devoto et
al., 1996
; Cortés et
al., 2003
). Adaxial cell migration away from the medial myotome
surface begins shortly after neural crest cells enter the medial migration
pathway [compare time courses of adaxial cell migration in Devoto et al.
(Devoto et al., 1996
) and
neural crest migration onset in Raible et al.
(Raible et al., 1992
)]. A
subset of adaxial cells becomes muscle pioneer cells that extend through the
myotome from the notochord to the lateral surface and define the position of
the horizontal myoseptum that separates dorsal and ventral myotome regions
(Halpern et al., 1993
). Fast
muscle precursors are initially lateral to adaxial cells but later become
medial muscle fibers, after adaxial cell migration
(Fig. 1)
(Devoto et al., 1996
;
Blagden et al., 1997
) (reviewed
by Stickney et al., 2000
).

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Fig. 1. Neural crest migration and somite development in zebrafish. Myotome is the
major component of zebrafish somites. Slow muscle precursors (red), called
adaxial cells, are located adjacent to notochord before segmentation (left).
During segmentation stages they become a single-layered sheet of fully
extended muscle cells along the anteroposterior axis and then migrate through
the somite to the lateral surface (right). A subset of these cells, called
muscle pioneers, remain medial and define the horizontal myoseptum (yellow).
The major part of each somite is composed of fast muscle cells (orange).
Sclerotome cells (blue) arise in ventromedial somite and migrate dorsally.
Neural crest migrates on a medial pathway in the middle of the medial aspect
of each somite (arrow).
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In this paper, we explore how somites affect the restricted migration of
trunk neural crest cells in streams along the middle region of the medial
surface of each zebrafish somite, with an emphasis on how this pattern is
initially established. Analysis of two kinds of somite mutants, ones with
reduced somitic mesoderm and ones with segmentation defects, shows that proper
somite patterning is required for normal patterning of neural crest migration.
Neural crest forms normal migration streams in embryos that lack fast muscle,
showing that this somite component is not required to pattern these streams.
By contrast, neural crest migration is disrupted in Hedgehog (Hh) signaling
mutants that have normal somite boundaries but reduced numbers of slow muscle
cells (van Eeden et al., 1996
;
Stickney et al., 2000
),
suggesting that slow muscle regulates the pattern of neural crest migration on
the medial pathway. We tested this hypothesis by examining neural crest
migration in embryos after surgical removal and replacement of adaxial cells.
The pattern of neural crest migration streams is abnormal in somites that lack
slow muscle; normal patterning can be restored by transplantation of wild-type
slow muscle cells. These data support the hypothesis that slow muscle
regulates the pattern of neural crest migration on the medial pathway in
zebrafish.
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Materials and methods
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Animals
Embryos were obtained from natural spawnings of a wild-type colony (AB) or
crosses of identified carriers heterozygous for specific mutations. Fish were
maintained in the University of Oregon Zebrafish Facility on a 14 hour
light/10 hour dark cycle at 28.5°C and embryos staged according to Kimmel
et al. (Kimmel et al., 1995
)
by number of somites or hours post fertilization (hpf) at 28.5°C. Mutant
embryos were generated by crossing two heterozygous adult carriers. The
following mutant alleles were used in this study; sptb104
(Griffin et al., 1998
),
ntlb195
(Schulte-Merker et al., 1994
;
Amacher et al., 2002
),
beatm98, fsste314
(van Eeden et al., 1996
;
van Eeden et al., 1998
;
Nikaido et al., 2002
),
syut4 (Schauerte et
al., 1998
), smub641
(Varga et al., 2001
),
aeitm223, deste322b
(Holley et al., 2000
;
Holley et al., 2002
) and
yotty17 (Karlstrom et
al., 1999
). At least 30 embryos were examined for each
mutation.
RNA in situ hybridization and immunohistochemistry
RNA in situ hybridization was performed as described previously
(Appel and Eisen, 1998
). The
crestin antisense riboprobe was detected by Fast Fast Red (Sigma) or
NBT/BCIP (Roche). DIG-labeled or fluorescein-labeled antisense RNA probes
(Roche Diagnostics) for RNA in situ hybridization were generated from plasmids
as follows: crestin plasmid (Luo
et al., 2001
) was cut with EcoRI and transcribed with T7
polymerase; and myod plasmid
(Weinberg et al., 1996
) was
cut with XbaI and transcribed with T7 polymerase. F59 monoclonal
antibody (Crow and Stockdale,
1986
) was used at a 1:10 dilution to detect slow muscle cells
(Devoto et al., 1996
); EB165
monoclonal antibody (Blagden et al.,
1997
) was used at 1:5000 dilution to detect fast muscle cells; and
anti-HuC antibody (16A11) was used at 1:1000 dilution
(Marusich et al., 1994
;
Henion et al., 1996
). Antibody
staining was carried out after RNA in situ hybridization. Alexa-488- or
Alexa-546-conjugated goat anti-rabbit polyclonal antibody was used as
secondary antibody. Fluorescence was visualized using a confocal laser
scanning microscope (Biorad Radiance 2100). Fluorescent images shown in Figs
2,
3,
4,
5,
6 represent
z-projections.
mRNA and morpholino microinjection
Embryos were injected with 2-5 nl diluted RNA solution (50 ng/ml) or
morpholino antisense oligonucleotides (MOs) into the yolk at the one-cell
stage. spt MO mix [spt MO 1 1.2 mg/ml; spt MO 2 0.5
mg/ml; MO sequences reported by Lewis and Eisen
(Lewis and Eisen, 2004
)] were
injected into ntlb195 mutant embryos. mRNA was transcribed
as described by Lewis and Eisen (Lewis and
Eisen, 2001
). Pipettes were pulled on a Sutter Instruments
Micropipette puller (Model P-2000). Injections were performed with an air
injection apparatus (ASI).
Removal and transplantation of adaxial cells
Adaxial cell removal
At the one- to three-somite stage,
20 adaxial cells were removed from
wild-type embryos by gentle suction with a micropipette, as described by Eisen
and Pike (Eisen and Pike,
1991
). Briefly, embryos were embedded in 5% methylcellulose, a
micropipette inserted approximately five somite widths posterior to the
most-recently formed somite and
20 adaxial cells removed from a two- to
three-somite wide region by gentle suction. We carried out these experiments
under Nomarski optics, which allows adaxial cells to be readily discerned from
neighboring non-adaxial cells (Hirsinger
et al., 2004
), and later confirmed by staining with F59 antibody,
which labels slow muscle fibers, that adaxial cells were removed. Only embryos
whose adaxial cells were removed were fixed at 21-24 hpf to examine slow
muscle and neural crest; embryos in which adaxial cells were still present
were discarded. Adaxial cells were removed from 39 embryos. In 20 cases, slow
muscle appeared normal; it is unclear whether the ablation did not
successfully remove all adaxial cells or whether adaxial cells were replaced
as we have seen in previous muscle ablation experiments (J.S.E., unpublished).
These animals served as controls; neural crest migration was normal in 17/20.
In 19 cases, slow muscle was reduced or absent from the target somites. We
examined these embryos to learn whether neural crest migration was
affected.
One- to three-somite stage adaxial cell transplantation
For transplantation, wild-type donor embryos were labeled by injection of a
mixture of 2.5% rhodamine dextran (lysinated, 3x103
Mr; Molecular Probes) and 2.5% fluorescein dextran
(lysinated, 3x103 Mr; Molecular Probes)
in 0.2 M KCl into the yolk cell at the one-cell stage. Adaxial cells were
removed as described above from donor and host embryos. Adaxial cells from
dye-labeled, wild-type donors were transplanted into unlabeled
smub641 mutant hosts. smu mutants with
transplanted adaxial cells were fixed at 21-24 hpf. The neural crest was
visualized by in situ hybridization with crestin probe, and the
transplanted cells were visualized with anti-fluorescein antibody. Later,
these embryos were stained with F59 antibody to confirm that transplanted
cells were slow muscle cells. Adaxial cells were transplanted in 63 embryos.
Of these, 47 were +/+ or smu/+; we did not score these. In 17
cases, hosts were smu/; 11 of these had transplanted
wild-type slow muscle cells.
Shield stage adaxial cell transplantation
Transplantation was performed as described by Maves et al.
(Maves et al., 2002
). In
brief, wild-type donors were labeled at the one-cell stage as described above.
Cells were removed by using a pulled glass micropipette as a knife and donor
tissue (about 50-100 cells) was excised and then inserted into an unlabeled
smub641 mutant host. Cells were transplanted
40°
from the shield, 10-15 cells from the edge and from the deepest layer of
cells. smu mutants with transplanted adaxial cells were fixed at
21-24 hpf and processed as described above.
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Results
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Somites play an essential role in patterning zebrafish neural crest migration
Somites are necessary to pattern trunk neural crest migration in amniote
vertebrates (Bronner-Fraser and Stern,
1991
; Tosney,
1988
) (reviewed in Kuan et
al., 2004
). To learn whether this is also the case in zebrafish,
we examined neural crest migration in two kinds of somite defect mutants: (1)
mutants in T-box genes that have little or no somitic mesoderm; and (2)
mutants that have segmentation defects leading to improperly formed somites.
We visualized migrating neural crest cells by RNA in situ hybridization with a
riboprobe for crestin, a gene expressed in almost all zebrafish trunk
neural crest cells (Luo et al.,
2001
), and slow muscle by staining with F59 antibody that
recognizes slow muscles and also reveals somite boundaries
(Devoto et al., 1996
).
Double-labeling revealed that in wild-type embryos, neural crest migration on
the medial pathway is restricted to a single stream of cells in the middle of
the medial surface of each somite (Fig.
2). Slow muscle fibers are initially located on the medial surface
of the somite. They then migrate through the somite and, by the stage shown in
Fig. 2, they form a monolayer
of slow muscle fibers on the lateral surface of each somite
(Devoto et al., 1996
).

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Fig. 2. Neural crest migration in wild-type embryos. Whole-mount staining of 21 hpf
wild-type embryo with crestin riboprobe (red) and F59 antibody
(green) to reveal trunk neural crest and slow muscle cells. (A) Neural crest
alone, (B) slow muscle alone, (C) the merged image, (D) a Nomarski
differential interference contrast (DIC) image. Neural crest migrates in the
middle of the medial aspect of the myotome in one- to two-cell wide streams.
Slow muscle fibers extend throughout the anteroposterior axis of each somite.
At this stage of development, midtrunk level neural crest cells are in a more
medial focal plane than slow muscle fibers, but they are seen together in this
z-projection. Similar z-projections are shown in subsequent
figures. Lateral view, anterior toward the left. Scale bar: 20 µm.
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T-box mutants
spadetail (spt) encodes zebrafish Tbx16
(Griffin et al., 1998
) (see
http://zfin.org
for nomenclature). In spt mutants, somitic mesoderm is reduced or
absent (Molven et al., 1990
;
Ho and Kane, 1990
) and there
are very few slow muscle cells, as shown by F59 staining
(Fig. 3). Neural crest migrates
in spt mutants, but migration is entirely unrestricted and there is
no pattern of segmental streams (Fig.
3). Because muscle fibers do form in some spt mutants, we
decided to examine embryos lacking function of both spt and another
T-box gene, no tail (ntl), by injecting spt MOs
into ntl mutants. These embryos entirely lack trunk mesoderm and
never make any muscle fibers (Lewis and
Eisen, 2004
; Amacher et al.,
2002
), as shown by lack of expression of myod
(Fig. 3). As in spt
mutants, in ntl mutants injected with spt MOs, trunk neural
crest cells migrated, but their migration was entirely unrestricted and there
was no segmental pattern (Fig.
3). Thus, we conclude that somites are unnecessary for neural
crest motility, but they are necessary to restrict neural crest migration into
coherent, segmental streams.
Segmentation mutants
The lack of segmentally patterned trunk neural crest migration in
spt mutants and ntl mutants injected with spt MOs
suggested that normal somite segmentation might be required to establish a
normal pattern of crest migration on the medial pathway. To test this
hypothesis, we examined neural crest migration in embryos with mutations in
genes required for proper somite segmentation, including beamter
(bea) (van Eeden et al.,
1996
), fused somites [fss (tbx24
Zebrafish Information Network)]
(Nikaido et al., 2002
),
after eight [aei (deltaD Zebrafish
Information Network)] (Holley et al.,
2000
) and deadly seven [des (notch1a
Zebrafish Information Network)]
(Holley et al., 2002
). In
bea mutants, normal somite boundaries form only for the most anterior
5-7 somites; no normal somite boundaries form at all in fss mutants.
Although there is an overall segmental pattern to neural crest migration on
the medial pathway in these mutants, the pattern is abnormal (Figs
4 and
5). Streams of neural crest
cells are closer together or farther apart than in wild types, in correlation
with the abnormal size and shape of somites in these mutants. In most cases,
the streams were appropriately located in the middle of the medial aspect of
the myotome, consistent with our hypothesis that somites pattern trunk neural
crest migration. However, in some cases the streams of neural crest cells
branched and neural crest cells migrated in abnormal positions. These
patterning defects occurred only where somite segmentation was disturbed, i.e.
posterior to somite 7 in bea mutants and from the first somite in
fss mutants (Fig. 5).
Thus, the neural crest migration patterning defect is precisely correlated
with the region of defective segmentation. We observed the same phenotype in
aei and des mutants (data not shown). Together these
experiments show that where somites are absent or their patterning is
disrupted, trunk neural crest cells migrate aberrantly on the medial pathway,
supporting the idea that somite-derived signals regulate neural crest
migration.

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Fig. 3. Neural crest cells migrate in an unrestricted pattern in spt
mutants and ntl mutants injected with spt MOs. (A-D)
Whole-mount staining of 21 hpf spt mutant with crestin
riboprobe (red) and F59 antibody (green). (A) Neural crest alone, (B) slow
muscle alone, (C) the merged image, (D) a DIC image. F59 antibody staining
reveals that spt mutants have few slow muscle cells. Neural crest
cells migrate in spt mutants, but migration is not restricted to a
specific pathway, thus there are no migration streams. (E-H) Whole-mount
staining of 21 hpf ntl mutant injected with spt MOs. (E)
Neural crest alone, (F) slow muscle alone, (G) the merged image, (H) a DIC
image. F59 antibody staining reveals that ntl mutants injected with
spt MOs have no muscle. As in spt mutants, neural crest
migrates in these embryos, but migration is not restricted to a specific
pathway and there are no migration streams in ntl mutants injected
with spt MOs that have no muscle cells, as shown by absence of
myod expression (H). Scale bar: 20 µm.
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Fig. 4. Disruption of neural crest migration is correlated with segmentation
defects. Whole-mount staining with crestin riboprobe (red) and F59
antibody (green) of bea (A-D; posterior of somite 9) and fss
mutants (E-H) at 21 hpf. Streams of neural crest cells (red, A,C,E,G) are
present, but less regular than in wild types and the streams show some
branching, consistent with abnormal somite shape and size. However, neural
crest still migrates generally in the middle of the medial aspect of the
myotome in both mutants. (D,H) DIC images of bea and fss
mutants, respectively. Scale bar: 20 µm.
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Fig. 5. In segmentation mutants, neural crest migration is disrupted in the same
somites in which segmentation is disrupted. Whole-mount staining with a
crestin riboprobe of wild-type (A) fss (B), bea (C)
and smu mutants (D) at 21 hpf to reveal neural crest cells. (A) In
wild-type embryos, neural crest cells migrate in segmental streams along the
entire AP axis. (B) Neural crest migration is abnormal from the first somite
in fss mutants. (C) In bea mutants, neural crest migration
is disrupted posterior to somite 6. (D) In smu mutants there is no
clear segmental pattern of neural crest migration; in addition, neural crest
cells stall at the level of the dorsal aspect of the notochord. (E,F) EB165
antibody staining reveals that in smu mutants (F) somites are smaller
along the DV axis, but a significant amount of fast muscle is still present,
although there may be less than in wild types (E). Scale bar: 60 µm in A-D;
25 µm in E,F.
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Fast muscle cells are not required for formation of normal neural crest migration streams
To learn which somite components are involved in patterning trunk neural
crest migration, we first considered which components are in close proximity
to neural crest cells as they begin to migrate on the medial pathway. Although
many studies in amniote embryos have implicated sclerotome in patterning
neural crest migration (Newgreen et al.,
1990
) (reviewed by Le Douarin
and Kalcheim, 1999
; Krull,
2001
; Kalcheim,
2000
), in zebrafish, neural crest cells do not encounter
sclerotome cells until the segmental pattern of neural crest migration streams
is already established (Morin-Kensicki and
Eisen, 1997
). Thus, it is unlikely that sclerotome participates in
the initial patterning of neural crest migration, although we cannot rule out
a role of sclerotome in later stages of neural crest migration, particularly
migration ventral of the horizontal myoseptum. Previous studies showed that
the first cells encountered by zebrafish neural crest cells are myotomal and
that the onset of crest migration coincides with contact between the two cell
types (Raible et al., 1992
).
This suggests that fast or slow muscle cells, or their precursors, establish
the initial pattern of neural crest migration streams. To test whether fast
muscle cells are important for patterning neural crest migration, we injected
sonic hedgehog (shh) mRNA into one- to two-cell stage
embryos. Overexpression of shh mRNA results in expansion of slow
muscle cells at the expense of fast muscle cells
(Du et al., 1997
;
Barresi et al., 2000
); thus,
shh mRNA-injected embryos have more slow muscle cells but few or no
fast muscle cells. In shh mRNA-injected embryos, somite shapes were
disrupted so that there were spaces between adjacent somites dorsal of the
horizontal myoseptum (Fig. 6).
Some neural crest migrated in these spaces. As we showed earlier for
spt mutants and ntl mutants injected with spt MOs,
trunk neural crest cells are motile in embryos lacking somitic muscle;
however, the migration of those cells is not segmentally patterned. Thus, we
interpret the migration of trunk neural crest cells in the spaces between
adjacent dorsal somites to be unpatterned migration because of lack of somitic
mesoderm. In addition to this unpatterned migration, neural crest cells also
migrated in normal streams along the middle of the medial aspect of each
somite (Fig. 6). This suggests
that fast muscle cells are necessary for adjacent somites to remain in contact
along their boundaries. However, fast muscle cells are not essential for
patterning the streams of neural crest migration on the medial pathway. This
result raises the possibility that this patterning role falls to slow muscle
cells or their precursors.

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Fig. 6. Fast muscle is not essential for neural crest migration on the medial
pathway. (A-D) Whole-mount staining of shh mRNA-injected embryo at 21
hpf. (A) crestin RNA expression (red) in neural crest cells. (B) F59
protein localization (green) in slow muscle. (C) Merged image. (D) DIC image.
In the absence of fast muscle, the somites do not adhere to one another,
creating a somite-free space in the intersomitic cleft. Neural crest migrates
in this region, presumably because neural crest cells are motile in the
absence of somites. However, neural crest also migrates in normal streams
medial to the middle of somites (arrows in C). (E,F) Cross-section of
shh mRNA-injected embryo. The somite on the left side of this embryo
has no fast muscle and excess slow muscle (all the muscle is slow, as
indicated by F59 staining in F); however, neural crest cells (crestin
riboprobe, purple in E) have migrated to the same extent as on the right side,
where fast muscle is present and slow muscle has migrated to the myotome
periphery, as in wild type. Scale bar: 20 µm.
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Neural crest migration is disrupted in mutants that lack slow muscle cells
To determine whether slow muscle cells regulate the pattern of trunk neural
crest migration on the medial pathway, we examined mutants in which slow
muscle cell formation was disrupted. Hedgehog (Hh) signaling is required for
specification of slow muscle cells; thus, we focused on mutants affecting the
Hh pathway. These mutants have normal somite boundaries but lack muscle
pioneer cells and have few or no slow muscle cells; fast muscle cells are
unaffected (Fig. 5E,F)
(Hirsinger et al., 2004
;
van Eeden et al., 1996
)
(reviewed by Stickney et al.,
2000
). We investigated neural crest migration in embryos with
mutations in three different genes in the Hh signaling pathway: sonic
you (syu), which encodes Sonic hedgehog; slow muscle
omitted (smu), which encodes Smoothend, an essential component
of the Hh signaling pathway; and you-too (yot), which
encodes Gli2, a downstream effector of Hh signaling. syu mutants have
a significant number of slow muscle cells in most somites
(Fig. 7B,C). Consistent with
this, the general pattern of trunk neural crest migration on the medial
pathway was normal, although the streams of cells were much less regular than
in wild types (Fig. 7A-C).
smu mutants essentially lack slow muscle cells
(Fig. 7F) (van Eeden, 1996)
because of an autonomous requirement for Smoothened activity for their
formation (Barresi et al.,
2000
) and neural crest migration was very abnormal
(Fig. 7E-G) (see also
Ungos et al., 2003
). Although
a significant number of neural crest cells migrated, they did not migrate
along the middle of the medial aspect of each somite, nor did they migrate in
coherent streams. Instead, many of these cells migrated under the overlying
somite boundaries (Fig. 8F). In
addition, in these mutants, neural crest migration did not extend ventral of
the dorsal aspect of the notochord (Fig.
7E-G); thus, neural crest cells tended to pile up at the level of
the notochord. This is in contrast to wild-type embryos, in which trunk neural
crest cells migrate much farther ventrally
(Fig. 5A,
Fig. 8E). yot mutants
showed a phenotype similar to smu mutants (data not shown). All of
these Hh signaling mutants have normal somite segmentation, suggesting that
normal segmentation is not sufficient to define the normal pattern of trunk
neural crest migration. Together, these data suggest that slow muscle cells
are required to regulate the segmental streams of neural crest migration on
the medial pathway. In addition, slow muscle cells may also be necessary for
later aspects of migration along the pathway.

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Fig. 7. Neural crest migration is disrupted in Hedgehog signaling mutants.
Whole-mount staining (A-C,E-G), and DIC image (D,H) of syu (A-D) and
smu mutants (E-H) at 21 hpf with crestin riboprobe and F59
antibody. syu mutants have a significant number of slow muscle cells.
Consistent with this, neural crest migration is fairly normal, although the
streams are less regular than in wild types. By contrast, smu mutants
entirely lack slow muscle and neural crest cells are abnormally patterned and
stall at the level of the dorsal aspect of the notochord (arrowhead in E).
Somite boundaries are shown as broken lines in G. Scale bar: 20 µm.
|
|
Wild-type adaxial cells can restore normal neural crest migration patterning in smu mutants
To further test the hypothesis that slow muscle patterns trunk neural crest
migration, we carried out two types of experiment. In the first experiment, we
surgically removed adaxial cells from wild-type embryos at the one- to
three-somite stage and examined neural crest migration on the medial pathway
by in situ hybridization with a crestin riboprobe between the 22-26
somite stages. In control experiments, we first removed adaxial cells then
immediately replaced them. This had no effect on the pattern of neural crest
migration on the medial pathway (data not shown). In 20 out of 39 removal
experiments, slow muscle still formed, although it was not entirely clear
weather the number of slow muscle cells was decreased relative to
unmanipulated embryos. At the 22-26 somite stage, neural crest migration
appeared normal in these embryos (Fig.
8D). By contrast, in 19 out of 39 removal experiments, slow muscle
was absent (Fig. 8A,B). In the
somites lacking slow muscle cells, the streams of migrating neural crest were
abnormal. In some cases, the streams were not restricted to the middle of the
medial aspect of the somite (Fig.
8A,B; n=7); in other cases, there were two streams in one
somite (n=4; data not shown); and in some cases, streams were
branched (n=1; data not shown). In some cases (n=7), neural
crest streams both migrated in an inappropriate position and were branched, or
there were two streams in one somite and they both branched.

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Fig. 8. Slow muscle regulates neural crest migration. (A-C) Removal of adaxial
cells (black box in A; absence of orange stain) results in disruption of
neural crest cell (purple) migration. F59 staining reveals slow muscle cells
(orange). (B,C) Higher magnification views of where adaxial cells were removed
(B, black box in A) and normal slow muscle cells are present (C, blue box in
A). (D) Control experiment in which adaxial cells were removed, but slow
muscle cells are present at 21 hpf and neural crest migration is essentially
normal. (E) Nomarski DIC image of wild-type embryo shows neural crest migrates
in the middle of somite. (F-H) Transplantation of adaxial cells in
smu mutants. (F,G) Nomarski DIC images show neural crest migration
(crestin riboprobe; purple) and transplanted slow muscle cells (red).
Somite boundaries are shown as broken lines. (H) F59 staining (green) of the
same embryo shows wild-type adaxial cells. (G) Higher magnification of F.
Wild-type adaxial cells transplanted dorsal of the notochord partially
restored neural crest migration in the middle of the somite, whereas neural
crest migrates on the boundaries of somites without slow muscle. Scale bar: 85
µm in A; 75 µm in B,C; 70 µm in D-F; 40 µm in G,H.
|
|
In the second experiment, we transplanted wild-type adaxial cells into
smu mutants. Adaxial cells were transplanted from shield stage or 1-3
somite stage, dye-labeled, wild-type donor embryos into unlabeled smu
mutant hosts of the same developmental stage. Host embryos were fixed between
the 22- and 26-somite stages, and examined by in situ hybridization with a
crestin riboprobe. Transplantation of adaxial cells into the somite
dorsal of the level of the notochord partially restored the normal pattern of
neural crest migration (Fig.
8F-H). In these somites, neural crest cells migrated as a coherent
stream that tended to be in the middle of the medial aspect of the somite
(Table 1). By contrast,
transplantation of adaxial cells into the somite ventral of the level of the
notochord did not restore a normal pattern of neural crest migration
(Table 1). Together these
results provide strong support for the hypothesis that slow muscle cells or
their precursors pattern trunk neural crest migration into coherent streams on
the medial pathway.
The migration pathway is crucial for proper development of some neural crest derivatives
To test the role of the migration pathway in formation of neural crest
derivatives, we examined dorsal root ganglion (DRG) neurons by HuC antibody
staining in embryos whose adaxial cells were surgically removed. At 72 hpf,
clusters of DRG neurons are aligned along the neural tube, typically in a
single cluster per somite (Fig.
9A,B,E). By contrast, following adaxial cell removal, DRG neurons
are often found in two clusters per somite that are spatially segregated along
the dorsoventral axis (Fig.
9C,D,F), suggesting that proper migration of neural crest cells is
essential for proper development of at least some derivatives.
 |
Discussion
|
---|
Our key finding is that slow muscle cells or their precursor adaxial cells
establish the segmental pattern of trunk neural crest migration on the medial
pathway in zebrafish and that this is important for proper formation of at
least some derivatives. Although adaxial cells are initially located medially
within each somite, they elongate and move laterally through the myotome at
approximately the same time that neural crest cells are migrating on the
medial pathway (Raible et al.,
1992
; Devoto et al.,
1996
; Cortés et al.,
2003
). How might adaxial or slow muscle cells regulate the pattern
of neural crest migration on the medial pathway? From their studies of motor
axon pathfinding, Zhang and Granato (Zhang
and Granato, 2000
) proposed that slow muscle might express a
guidance molecule and leave it behind on the medial myotome surface as it
migrates through the myotome to the lateral surface. unplugged and
diwanka mutants have defects in motor axon pathfinding along this
pathway; these defects can be rescued by transplantation of a small number of
wild-type slow muscle cells just dorsal to the muscle pioneers that form the
horizontal myoseptum, even though slow muscle cells are already migrating
toward the lateral myotome surface by the time motor axons extend out of the
spinal cord (Zeller and Granato,
1999
; Zhang and Granato,
2000
). It is possible that similar mechanisms restrict neural
crest migration.
Myotome may establish the initial pattern of migration of neural crest in
avian embryos. There is considerable evidence that sclerotome plays a role in
regulating neural crest migration in avian and mammalian embryos
(Bronner-Fraser, 1986
;
Loring and Erickson, 1987
;
Teillet et al., 1987
;
Newgreen et al., 1990
);
however, the role of myotome in initial patterning of neural crest migration
has received less attention. Many studies document neural crest migration on
the anterior half-sclerotome of each somite, and several molecules expressed
in posterior half-sclerotome have been shown to repel neural crest migration
(Newgreen et al., 1990
)
(reviewed by Le Douarin and Kalcheim,
1999
; Krull, 2001
;
Kalcheim, 2000
). However,
Tosney et al. (Tosney et al.,
1994
) provided evidence that neural crest cells prefer to migrate
on the myotome basal lamina rather than on sclerotome. They found that neural
crest cells invade sclerotome only when they fail to contact the myotome basal
lamina. Moreover, during the onset of neural crest dispersal from the
migration staging area in chick, it appears that neural crest cells contact
epithelial somite cells that will become myotome
(Loring and Erickson, 1987
;
Teillet et al., 1987
;
Kahane et al., 1998
;
Kiefer and Hauschka, 2001
).
This is similar to the situation in zebrafish, in which neural crest migration
does not begin until neural crest cells contact myotomal cells
(Raible et al., 1992
). In
avians, neural crest cells do not appear to contact sclerotome until later
(Loring and Erickson, 1987
;
Teillet et al., 1987
). This
suggests that in avian embryos, as in zebrafish embryos, myotome is
responsible for the initial pattern of neural crest migration.
Sclerotome is not present at the right place and time to pattern the
initial migration of zebrafish neural crest. We have previously shown that
neural crest cells and sclerotome migrate in opposite directions along the
same pathway (Morin-Kensicki and Eisen,
1997
). However, these two cell types only encounter one another
when they both reach the level of the horizontal myoseptum. Thus, the initial
segmentally restricted pattern of migration of both cell types is already
established before the two cell types meet, raising the possibility that the
myotome is responsible for the migration pattern of both neural crest and
sclerotome. However, it remains likely that interactions with sclerotome cells
regulate aspects of zebrafish neural crest migration along more ventral
regions of the migration pathway.

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Fig. 9. Slow muscle removal results in aberrant DRGs. (A-D) Views of a single 72
hpf embryo: (A,B) control side; (C,D) experimental side. All figures are
printed with anterior toward the left and dorsal toward the top for ease of
comparison. (A,C) Anti-HuC antibody staining (red) reveals clusters of DRG
neurons adjacent to the neural tube. DRGs are indicated only in the first
segment in A (arrow). Enteric neurons are indicated with asterisks. (B,D) F59
staining reveals slow muscle cells (green). (A,B) There is a single DRG per
somite in most segments, although one segment (open arrowhead) has two DRG
neuron clusters. (C,D) Adaxial cells were removed from the experimental side
and F59 staining shows less slow muscle. In many segments there are two DRG
neuron clusters per somite; the second cluster is often much further ventral
than the normal DRG position (arrowheads in C). (E,F) Schematic showing DRG
formation in somites 3-10 of five embryos from which adaxial cells were
removed from somites on one side; each color represents a different embryo.
(E) Control side. (F) Experimental side; the colored lines represent the
somites with reduced or missing slow muscle. Scale bar: 20 µm.
|
|
Are the molecular mechanisms that regulate neural crest migration conserved
between zebrafish, avian and mammalian embryos? In avian and mammalian
embryos, neural crest migrates on the anterior half sclerotome of each somite.
Interactions between Ephrins and their Eph receptors have been implicated in
patterning neural crest migration in amniotes
(Le Douarin and Kalcheim,
1999
; Krull, 2001
;
Kalcheim, 2000
;
Halloran and Berndt, 2003
).
Ephrin B proteins are expressed in posterior half sclerotome and repel neural
crest migration, presumably through interactions with Eph proteins expressed
in neural crest cells (Krull et al.,
1997
; Wang and Anderson,
1997
). Interestingly, the same Ephrin B proteins expressed in the
posterior half of the sclerotome are also expressed in dermamyotome
(Wang and Anderson, 1997
) and
thus might also mediate interactions between neural crest and dermamyotome. In
zebrafish, slow muscle, not sclerotome, establishes the initial pattern of
neural crest migration. Thus, molecules involved in patterning neural crest
migration should be expressed in slow muscle. Zebrafish Eph family members,
such as epha4a, ephrin-A-L1 and ephrin B genes are segmentally
expressed early in development, and have been implicated in somite formation
(Cooke et al., 1997
;
Durhin et al., 1998
;
Chan et al., 2001
). However,
they are diffusely expressed in somites after they differentiate, but the
pattern does not resemble that described for amniotes
(Cooke et al., 1997
;
Durhin et al., 1998
;
Chan et al., 2001
). In
addition, no Eph family members have been reported to be expressed in
zebrafish trunk neural crest cells. Thus, if Eph family members regulate
neural crest migration patterning in zebrafish, the molecular mechanisms are
likely to be distinct from those of avian and mammalian embryos, because in
zebrafish neural crest migration occurs in the middle of the medial myotome
surface, rather than through the anterior half somite. In addition to Eph
family members, several other proteins have been implicated in patterning
neural crest migration in amniotes, including tenascin C, laminins and
F-spondin (Le Douarin and Kalcheim,
1999
; Krull, 2001
;
Kalcheim, 2000
). In zebrafish,
tenascin C proteins are expressed in somite boundaries intensely, throughout
somites weakly and also within neural crest cells
(Tongiorgi, 1999
). Because
tenascin C is expressed both in the region of the neural crest migration
pathway and in the region between adjacent neural crest migration pathways,
its expression pattern is not consistent with a role in patterning neural
crest migration on the medial pathway. laminin
1
(lamc1 Zebrafish Information Network) mRNA is expressed
throughout somites and laminin ß1 (lamb1
Zebrafish Information Network) mRNA is expressed throughout embryos
weakly in zebrafish (Parsons et al.,
2002
). The gene encoding F-spondin and the related genes
mindin 1 (spon2a Zebrafish Information Network) and
mindin 2 (spon2b Zebrafish Information Network) are
also not expressed in slow muscle
(Higashijima et al., 1997
). So
far in zebrafish, no molecules have expression patterns that make them good
candidates for establishing the neural crest migration pattern on the medial
pathway. Thus the molecular mechanisms that regulate patterning of neural
crest migration in zebrafish remain to be resolved.
 |
ACKNOWLEDGMENTS
|
---|
We thank Frank Stockdale for kindly providing F59 antibody, Estelle
Hirsinger for helpful technical advice, Jon Muyskens for spt MOs, Jim
Weston, Chuck Kimmel, Julie Kuhlman and Stephan Schneider for comments on
earlier drafts of the manuscript, and the staff of the University of Oregon
Zebrafish Facility for fish husbandry. Supported by Sankyo Life Science
Foundation, Uehara Foundation of Life Science and NIH Grant HD22486.
 |
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