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First published online 31 January 2007
doi: 10.1242/dev.02789
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1 Centre for Regenerative Medicine and Developmental Biology Programme,
Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY,
UK.
2 Victor Chang, Cardiac Research Institute, 384 Victoria Street, Darlinghurst,
Sydney 2010, Australia.
3 MRC Centre for Developmental and Biomedical Genetics, University of Sheffield,
Firth Court, Western Bank, Sheffield S10 2TN, UK.
4 Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2
3DY, UK.
5 Institute of Neuroscience, University of Oregon, Eugene, OR 97403, USA.
Author for correspondence (e-mail:
bssrnk{at}bath.ac.uk)
Accepted 18 December 2006
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Neural crest, Migration, Patterning, Pigment pattern formation, Melanophore, Melanocyte, Xanthophore, Chromatophore, Slow muscle, Fast muscle, Horizontal myoseptum, cho, you-type, Sdf1a (Cxcl12a), Zebrafish
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Krull,
2001). The somites have particular importance in patterning NC
migration, as is perhaps best characterised in developing chick dorsal root
ganglia (Lallier and Bronner-Fraser,
1988). Here, NC migrates on a ventral pathway (between somites and
neural tube; called the medial pathway in zebrafish) only through the rostral
portion of each sclerotome (Rickmann et
al., 1985). This patterned migration results from differential
expression of migration-promoting molecules such as thrombospondin, and
migration-inhibiting molecules such as Fspondin and ephrin ligands, in rostral
and caudal sclerotome, respectively
(Davies et al., 1990).
Mechanisms regulating migration on the dorsolateral (lateral pathway in
zebrafish) pathway, between the skin and dermomyotome, are incompletely
understood. Timelapse studies in zebrafish reveal that premigratory NC cells
extend numerous protrusions that actively explore the lateral migration
pathway. Before lateral pathway migration begins, these protrusions undergo
rapid collapse, apparently because of somite-associated inhibitory activity
(Jesuthasan, 1996). Later, the
frequency of protrusion collapse decreases, allowing migration over the
somite. Similarly, lateral pathway `maturation' is indicated by heterologous
extracellular matrix (ECM) transplantation studies in axolotl, with precocious
NC migration initiated by ECM from older embryos
(Löfberg et al., 1985;
Löfberg et al., 1989).
Two signals controlling the time when migration begins have been identified.
Kit ligand attracts melanoblasts onto the dorsolateral pathway in mouse
(Wehrle-Haller and Weston,
1995) and, in chick, ephrinB acting via EphB receptors regulates
which neural crest cell types migrate on the dorsolateral pathway
(Santiago and Erickson, 2002).
In mouse and chick, only melanocytes utilise the dorsolateral pathway, and
these become distributed ubiquitously, whereas in fish, several distinct types
of chromatophores use this pathway and pigment pattern formation results from
controlling their migration and final positioning
(Kelsh, 2004).
In zebrafish embryos, pigment patterns form from three chromatophore types.
Black melanophores (equivalent to mammalian melanocytes) are principally
arranged in four longitudinal stripes, including dorsal and ventral stripes
extending from the head to the tip of the tail and the lateral stripe along
the horizontal myoseptum of somites 6-26
(Kelsh et al., 1996).
Iridescent iridophores are found within some melanophore stripes, whereas
yellow xanthophores lie scattered throughout the embryo flank. The prominence
and reproducibility of their pigment patterns make zebrafish ideally suited
for mutant screens to identify NC patterning cues. Numerous loci necessary for
correct development of embryonic (Kelsh et
al., 1996; Odenthal et al.,
1996) and adult (Haffter et
al., 1996; Parichy et al.,
2000a) zebrafish pigment cells have been identified. As with the
pigment mutant collections in mice (Lyon
and Searle, 1989), zebrafish pigmentation mutants exhibit a
changed appearance to the body pigmentation and are commonly referred to as
`pigment pattern mutants', although only a small subclass of the pigment
pattern mutants actually has altered pigment cell positions. Studies of fish
pigment pattern mutants (sensu stricto) have focused to date on the
adult pattern (Asai et al.,
1999; Johnson et al.,
1995; Rawls and Johnson,
2001; Parichy and Turner,
2003; Quigley et al.,
2004), but this is likely to be under very different control from
the embryonic pigment pattern.
Stromal cell-derived factor 1 (SDF1; CXCL12 - Human Gene Nomenclature
Database) in humans is the ligand for CXCR4, a Gprotein-coupled receptor known
for its role in both leukocyte and neural stem cell migration and as a
co-receptor for HIV-1 (Feng et al.,
1996; Imitola et al.,
2004; Loetscher et al.,
1994; Oberlin et al.,
1996). Zebrafish have two SDF1 and two CXCR4 orthologues
(Chong et al., 2001;
David et al., 2002;
Doitsidou et al., 2002;
Knaut et al., 2003;
Li et al., 2004;
Li et al., 2005). Zebrafish
mutant and morphant studies demonstrate that Cxcr4b-Sdf1a (Cxcl12a - Zebrafish
Information Network) signalling is necessary for correct migration of
primordial germ cells, the posterior lateral line (PLL) primordium, retinal
ganglion cell axons and hindbrain neuron cell bodies
(David et al., 2002;
Doitsidou et al., 2002;
Gilmour et al., 2004;
Knaut et al., 2003;
Li et al., 2004). Further
study in zebrafish germ cells has dissected specific roles for distinct
intracellular signalling pathways in driving the motility and directionality
of response to Sdf1a signalling (Dumstrei
et al., 2004). cxcr4b is expressed in the migrating
cells, whereas sdf1a expression neatly delineates the migration
routes. The PLL primordium migration route coincides with the position of the
lateral stripe melanophores; whether or not Sdf1a activity contributes to
their patterning is currently unknown.
The choker (cho) mutant was identified in the
Tübingen 1996 genetic screen and was classified as a `you-type'
(you is also known as scube2 - Zebrafish Information
Network) muscle mutant, so named because of the U-shaped appearance of their
somites [V-shaped in wild type (WT)], a phenotype thought to be due to a lack
of structural integrity resulting from loss of the horizontal myoseptum
(van Eeden et al., 1996).
Zebrafish myotomes consist of two types of slow-twitch muscle fibres and
fast-twitch muscle fibres. Most somite cells develop into fast-twitch muscles,
whereas slow muscle fibres derive from adaxial cells developing adjacent to
the notochord. Most adaxial cells migrate laterally to form a subcutaneous
layer of surface slow muscle fibres by 24 hours post-fertilisation (hpf)
(Devoto et al., 1996), whilst
a few develop into muscle pioneer fibres located at the position of the future
horizontal myoseptum. Hedgehog signalling is necessary for specification of
slow muscle cells (Currie and Ingham,
1996; Blagden et al.,
1997), and most you-type mutants affect the production or
transduction of the Hedgehog signals
(Schauerte et al., 1998;
Karlstrom et al., 1999;
Barresi et al., 2000;
Kawakami et al., 2005;
Nakano et al., 2004;
Woods and Talbot, 2005). PLL
primordium migration is aberrant in you-type mutants owing to the
loss of sdf1a expression from the horizontal myoseptal region
(David et al., 2002;
Li et al., 2004). In addition,
they all show a pigment pattern defect: lateral stripe melanophores are
completely absent (Kelsh et al.,
1996). Uniquely, cho mutants also have an ectopic
melanophore band (collar) spanning the anterior-most five somites and bridging
the dorsal and ventral stripes. Despite the absence of the lateral stripe, and
in contrast to the other you-type mutants, muscle pioneer cells
differentiate normally in choker mutant embryos. We find, however,
that both slow- and fast-twitch muscle fibres are patterned aberrantly in the
anterior-most somites and there is a general disorganisation of myotomal
architecture in this region. We show by somite transplantation experiments
that the pigment defects occur as secondary consequences of abnormal somite
development.
We have investigated the timing of melanophore accumulation and the mechanism of ectopic collar formation. Collar formation corresponds to a post-migratory phase for NC cells in WT and defects are restricted to two pigment cell types, both of which utilise the lateral migratory pathway. Our results suggest that the two defects are independent and show that the ectopic collar forms by aberrant invasion of melanophores from both dorsal and ventral stripes. Observing abnormal sdf1a expression in cho mutant somites, we uncover a role for Sdf1a in melanophore patterning in both lateral stripe generation in WT and melanophore collar formation in cho mutants.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and
reared in Embryo Medium (Kimmel and Warga,
1987) at 28.5°C. The following mutant alleles were used:
chotm26 (Kelsh et al.,
1996; van Eeden et al.,
1996) and nacrew2
(Lister et al., 1999). Double
mutants were created by crossing identified carriers for the single mutant
alleles; offspring were reared to adulthood and carriers for both alleles were
identified in the expected mendelian ratios.
Whole-mount RNA in situ hybridisation, immunocytochemistry and Alcian Blue staining
RNA in situ hybridisation was performed according to Kelsh et al.
(Kelsh et al., 2000b). Probes
used were as follows: dopachrome tautomerase (dct)
(Kelsh et al., 2000b),
nacre (mitfa) (Lister et
al., 1999), GTP cyclohydrolase 1 (gch)
(Parichy et al., 2000b),
sdf1a (David et al.,
2002), engrailed
(Ekker et al., 1992) and
smbp (Neyt et al.,
2000). To partially inhibit melanisation, embryos were placed in
0.003% phenyl-thio-urea (PTU) in Embryo Medium and reared normally.
Antibody staining was performed using anti-Hu mAb 16A11
(Marusich et al., 1994) and
anti-acetylated tubulin (Sigma) primary antibodies and detected with Alexa
fluor secondary antibodies (Molecular Probes). The anti-Engrailed mAb 4D9 was
detected with DAB (Vector Laboratories). F59
(Crow and Stockdale, 1986)
antibody staining was performed using peroxidase-antiperoxidase (Sternberger
Monoclonals). Antibody staining with fast muscle-specific EB165 and slow
muscle-specific BAD5 was performed as described in Lewis et al.
(Lewis et al., 1999).
Pan-myosin antibody MF20 was detected using a TcsSP confocal microscope
(Leica).
Alcian Blue staining of craniofacial cartilage was performed according to
Kelsh and Eisen (Kelsh and Eisen,
2000).
For microscopy, live embryos were anaesthetised with 0.003% tricaine (Sigma), examined under an Eclipse E800 compound microscope (Nikon) and photographed with a U-III (Nikon) or a Spot (Diagnostic Instruments) camera.
Melanophore counts
Embryos were reared to the required developmental stages, then
anaesthetised in 0.003% tricaine in Embryo Medium and fixed in 4%
paraformaldehyde. Melanophores were counted on the lateral pathway over the
first five somites on both sides of each embryo on an Eclipse E800 microscope;
cells with 25% or more of their area on the lateral pathway were included.
Melanophore counts in sdf1a morphant studies were counted after
fixation in 4% paraformaldehyde on an Olympus IX70 inverted microscope.
Timelapse microscopy
Embryos were dechorionated and anaesthetised in 0.003% tricaine in Embryo
Medium and immobilised in 0.5% agarose 0.003% tricaine in Embryo Medium on a
circular glass coverslip, which was placed into a metal chamber. The chamber
was filled with Embryo Medium and a small amount of perfluorodecalin (F2
Chemicals) that had been saturated with oxygen by bubbling prior to addition
to the chamber. The chamber was sealed with another glass coverslip and
observed under an Eclipse E1000 (Nikon) microscope, using a 20x dry
objective. The microscope stage was surrounded by a heated chamber maintained
at approximately 30°C. Images were collected for up to 30 hours with a
Hamamatsu C4880 CCD camera, and processed using Metamorph software (Universal
Imaging, USA). Development appeared normal, although somewhat slowed.
Timelapses were started at either 30 or 48 hpf, with WT and cho
mutant embryos mounted together and observed in parallel.
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) except
that the antibody was detected fluorescently. Labelled embryos were mounted in
glycerol under a coverslip and imaged with the Zeiss LSM510 confocal system.
Confocal sections were rendered and animated using the LSM510 system
software.
Somite transplantation and bead implantation
Somite transplantation was performed as previously described
(Haines et al., 2004). Bead
implantation utilised a novel method of agarose adhesion to avoid puncture of
the epidermis. AffiGel Blue Gel beads (Biorad) were rinsed several times,
soaked in a 1 µg/µl solution of recombinant human SDF1 (Chemicon
International, CA) in water, and then rinsed and stored in water until use,
usually within 5 hours. Control beads were rinsed in water before agarose
mounting. Embryos were embedded in 0.7% agarose on coverslips. Agarose was
dissected away and an SDF1-soaked bead was placed next to the skin. More
agarose was added to hold the bead in place and, where possible, agarose was
dissected away from the head, heart region and tail. Embryos were incubated at
28.5°C for the desired period.
Morpholino injection
Morpholino oligonucleotide sdf1a-Mo (5'-ATCACTTTGAGATC
CAT-GTTTGCA-3'; translation start site is indicated in bold) was
obtained from GeneTools (Philomath, OR) and was injected at 1.0 mM. This
morpholino is of identical sequence to that used previously
(David et al., 2002), and our
injected embryos had consistent misrouting of the PLL nerve as described by
these authors.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Timelapse microscopy revealed melanophore behaviour during collar formation. We studied cho mutant and WT embryos from 24 hpf, when collar formation was initially not obvious, and from 48 hpf, when cho mutants were readily sorted by their melanophore collars. Timelapses of 24 hpf embryos for over 15 hours showed indistinguishable melanophore behaviour in this early phase. Both WT and cho mutant melanophores were highly motile, frequently migrating on the lateral pathway in both dorsoventral and ventrodorsal directions; frequent changes of direction and temporary cessation of migration induced by contact with another melanophore were also observed (data not shown). Towards the end of these early timelapses, melanophores in the WT evacuated the collar region, whilst in cho mutants one or two melanophores remained in the collar region. That this behaviour represented the beginning of collar formation was confirmed by timelapses starting at 48 hpf, which showed dramatic differences in melanophore behaviour between WT and cho mutants (Fig. 2 and see Movies 1 and 2 in the supplementary material). The anterior trunk of WT embryos usually remained entirely devoid of melanophores (Fig. 2A-D). Although melanophores in both dorsal and ventral stripes were highly protrusive and frequently extended processes into the surrounding region, they remained in the dorsal and ventral stripes (Fig. 2I-L). Occasional melanophores were observed in the anterior trunk lateral pathway of a WT at the beginning of the timelapse, but these always migrated rapidly and joined either the ventral or dorsal stripe. Thus, from 48 hpf, WT melanophores exhibited exploratory behaviour but did not migrate through the collar region. By 48 hpf, cho mutants had a few melanophores in the nascent collar. These cells were highly active, extending numerous processes, but usually remained stationary and displayed the characteristic radially-symmetric stellate shape of non-migratory melanophores. As in WT siblings, melanophores from both dorsal and ventral stripes of cho mutants showed extensive protrusive behaviour exploring the lateral pathway, but unlike WT, these cells frequently migrated into the collar region (Fig. 2E-H,M-P). Thus, cho mutant melanophores migrated into the lateral pathway at times when WT melanophores were restricted to one of the pigment stripes. Such behaviour was observed only in the collar region of cho mutants.
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), but Alcian Blue staining revealed no differences in
jaw cartilages in 3- and 5-day post-fertilisation (dpf) cho mutants
and their WT siblings (Fig.
3A,B; data not shown). Likewise, this region generates cardiac NC
(Li et al., 2003;
Sato and Yost, 2003), but
simple observation revealed normal heart morphology, regular heart beat and
good blood flow (data not shown).
NC cells migrating on the medial pathway generate neurons and glia of the
sensory and sympathetic ganglia, plus melanophores and iridophores. Using
incident lighting to detect iridophores, we observed no abnormalities in
iridophore number, size or distribution in cho mutants (data not
shown). Likewise, foxd3 expression
(Kelsh et al., 2000a) revealed
no change in glia on the PLL nerve (data not shown), although projection of
the nerve itself was aberrant (see below). Immunofluorescent studies using the
anti-Hu antibody (Kelsh and Eisen,
2000) revealed that enteric neurons were normal in cho
mutants (Fig. 3C,D). Similarly,
sensory neurons were largely unaffected; the only difference being a slight
increase in the number of DRG neurons in the anterior trunk of cho
mutants at 5 dpf (Fig. 3E-H;
Table 1).
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) is an
early and persistent marker of the xanthoblast lineage. A gch
riboprobe in situ hybridisation revealed normally distributed,
gch-positive cells in 20-hpf cho mutants. A gradual clearing
of xanthoblasts from the region of the collar, but not from more anterior or
posterior regions, was observed in cho mutants at subsequent stages
beginning at 24 hpf (Fig.
3I-N). By 48 hpf, the collar region was essentially devoid of
xanthophores. Taken together with the prominent melanophore collar, it is
clear that both NC derivatives utilising the lateral migration pathway display
severe defects restricted to the collar region of cho mutants.
Collar xanthophore defects in cho mutants are causally independent of melanophore defects
In view of the remarkable correlation between the xanthophore and
melanophore defects in the collar, we asked whether these pigment cell defects
might be causally related. Although our studies revealed melanophore
accumulation beginning after xanthoblast loss, unpigmented precursor cells of
the melanophore lineage migrating through this collar region could conceivably
drive xanthophore loss. We therefore asked whether xanthophore loss occurred
in the absence of melanophores or their precursors using the nacre
(nac) mutant to eliminate the melanophore lineage
(Lister et al., 1999). Mutant
embryos doubly homozygous for nac and cho exhibited an
additive combination of the loss of gch-expressing cells from the
collar region as in cho mutants, and the absence of all melanophores
as in nac mutants (Fig.
3Q,R). We conclude that xanthophore loss in cho mutants
is independent of the melanophore lineage.
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).
Transverse sections of the collar region identified a defect in dorsal
extension of the myotome causing this dorsoventral flattening
(Fig. 4A-D).
Immunohistochemistry using F59 revealed disorganisation of slow muscle fibres
in the anterior somites (Fig.
4I-L). Transverse sections showed that these anterior-most
cho mutant somites had abnormally positioned slow fibres, with these
fibres located medially in the myotome
(Fig. 4F). By contrast, slow
muscle was found peripherally in posterior trunk somites of cho
mutants and at all axial levels in WT siblings
(Fig. 4E,G,H). To address the
time of onset of slow muscle defects, we examined stage-specific markers of
slow muscle differentiation. Slow myosin binding protein (Smbp; P.D.C.,
unpublished), is absent from migratory slow fibres, but expressed within
differentiated muscle pioneer cells (MPs)
(Ekker et al., 1992) and
post-migratory surface slow muscle. RNA in situ hybridisation with
smbp confirmed the defect in radial migration of slow muscle cells
(Fig. 4M,N), and indicated that
slow muscle cells had differentiated on schedule. In situ hybridisation of
16-18 hpf cho mutants and WT with a specific marker for pre-migratory
slow muscle precursor cells (prox1)
(Glasgow and Tomarev, 1998)
showed that cho mutants were indistinguishable from WT prior to
migration of slow muscle progenitors (data not shown). Furthermore, there was
no alteration in the expression of adaxial cell markers such as ptc1
and myod, suggesting that the initial Hedgehog-dependent
specification of these cells was unaltered in cho mutant homozygotes.
In situ hybridisation with an engrailed probe, as well as the
anti-Engrailed antibody 4D9, to identify MPs showed that cho mutants
possess MPs at 24 hpf and 30 hpf. cho mutant MPs were morphologically
indistinguishable from those of WT at 24 hpf, but showed reduced labelling by
30 hpf, consistent with the general reduction in all fibre types at this stage
(data not shown). Collectively, these analyses suggest that the initial
specification and differentiation of muscle cells is unaltered within the
anterior myotomes of cho mutants, whereas the subsequent
morphogenesis of these cells is altered within mutants. As expected, given the
aberrant distribution of slow muscle, fast muscle fibres were also abnormally
organised, but in addition were clearly reduced in number
(Fig. 4O,P). Counts of both
fast and slow muscle fibres labelled with anti-MyHc (myosin heavy chain)
antibody in transverse sections showed that the number of both types of fibres
was reduced in anterior myotomes of cho mutants to less than 50% of
those present in WT siblings (data not shown). Examination of embryos labelled
with anti-MyHc antibody showed progressive fibre loss through the embryonic
and larval period (see Fig. S1 and Movies 3 and 4 in the supplementary
material). In contrast to the migration defect, this quantitative aspect of
the slow muscle phenotype extended beyond the anterior-most myotomes, but was
strongest anteriorly and absent in the posteriormost trunk
(Fig. 5). Confocal 3D
reconstruction revealed that individual muscle fibres frequently failed to
span the entirety of the myotome (see Movie 5 in the supplementary material).
Therefore, the decreased fibre number evident in transverse sections is likely
to result from fibre loss through fibre detachment as well as reduction in
overall initial fibre number.
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), the
activation and maintenance of the alpha actin-GFP transgene is a sensitive
indicator of muscle integrity. Thus, muscle differentiation appeared normal
within the transplant. Further analysis of the integrity of muscle within the
transplant was monitored by differential interference contrast (DIC) optics,
to assay the presence of intact sarcomeric arrays within fibres. Sarcomeric
assembly appeared normal in the transplanted somites, suggesting that muscle
differentiated and was maintained normally within transplanted tissue.
Melanophores freely traversed the transplanted tissue at early developmental
time points both in WT 
WT (n=17) and WT cho
mutant (n=6) transplants (Fig.
6D-F). However, by 72 hpf, melanophores were excluded from the
regions lying directly above transplanted WT somites in cho mutants
(6 out of 6). By contrast, they collected freely at other locations lacking WT
tissue, such as within the collar region adjacent to the transplant, as well
as on the contralateral side of the embryo
(Fig. 6A-C). No defect in
melanophore migration or patterning was seen within control WT WT
transplants (17 of 17). This shows that the cho mutant somites cause
the mispatterning of melanophores to form the collar.
Abnormally distributed sdf1a correlates with pigment cell pattern defects in cho mutants
As previously reported (David et al.,
2002; Li et al.,
2004), sdf1a is expressed at the horizontal myoseptum in
WT embryos but not in you-type mutants. cho mutants lack
sdf1a expression along the horizontal myoseptum but have ectopic
sdf1a over the anterior somites in the collar region
(Fig. 7A-C,E). This pattern of
expression initiates at 24 hpf and persists through to at least 55 hpf,
coinciding with the time of melanophore collar formation. Expression of
sdf1a is localised to the lateral surface of the somite, adjacent to
the lateral NC migration pathway, in a subpopulation of external cells
(Devoto et al., 2006;
Groves et al., 2005;
Waterman, 1969). Thus, in
embryos labelled both for sdf1a and with MF20 antibody, the
sdf1a-expressing cells were clearly seen to lie immediately lateral
to the MF20-staining myotomal populations
(Fig. 7D). In the
you-type mutants smu (smo - Zebrafish Information
Network) and con (disp1 - Zebrafish Information Network),
absence of sdf1a in the horizontal myoseptum results in misrouting of
the PLL nerve to the ventral domain of sdf1a expression
(David et al., 2002;
Li et al., 2004). By contrast,
in cho mutants, the PLL primordium was found to migrate dorsally over
the ectopic sdf1a-expressing collar region, as shown using acetylated
alpha tubulin as a PLL nerve marker at 5 dpf
(Fig. 8A,B). or the PLL
primordium marker cxcr4b at 36 hpf
(Fig. 8C,D).
Sdf1 is a chemoattractant for melanophores
The altered pattern of sdf1a expression in cho mutants
corresponds to both melanophore patterning defects. To test whether Sdf1 is a
chemoattractant for migrating melanophores. We placed beads soaked in
recombinant human SDF1 protein against the epidermis of WT embryos, and
analysed their effect on melanophore localisation. In cases in which
SDF1-soaked beads remained attached to embryos for at least 16 hours during
melanophore migration, melanophores accumulated at the location adjacent to
the bead (7 of 9) (Fig. 9). By
contrast, melanophores never accumulated next to control beads (7 out of 7;
data not shown). We note that beads placed on the posterior trunk, at least as
posterior as the vicinity of the thirteenth somite, readily attracted
melanophores (Fig. 9C-E). The
integrity of muscle adjacent to the transplant was checked by serial
sectioning of bead-implanted embryos and staining with anti-MyHc antibody to
determine if muscle differentiation and maintenance were affected. In all
cases, muscle adjacent to the bead appeared normal (n=5); a
representative section containing ectopic melanophores immunolabelled for MyHc
is illustrated (Fig. 9E).
Implanted animals were also monitored using DIC optics to analyse fibre
integrity, which also appeared normal. We then placed SDF1-soaked beads on
cho mutant embryos at various locations in the trunk and tail. As in
WT embryos, ectopic melanophores accumulated adjacent to the bead (10 of 14)
(Fig. 9F). Thus, in both WT and
cho mutant embryos, ectopic SDF1 can act as a chemoattractant for
melanophores throughout the entire trunk.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The latter
study indicated that NC cell invasion of the lateral pathway is delayed by an
inability of the substrate to support cell adhesion and traction. Here, we
have shown that a similar incompatibility of substrate and pigment cells is
reasserted around 36 hpf. Interestingly, this constraint is overcome in
cho mutants, at least in part owing to the ectopic expression of
Sdf1a.
We have demonstrated a role for Sdf1a in cho mutant collar and
normal lateral stripe formation. Notably, sdf1a expression is not
disrupted in anterior somites in other you-type mutants examined
(David et al., 2002) (data not
shown), consistent with this being a key feature driving the unique collar
pigmentation of cho mutants. In the case of germ cell and PLL
primordium migration, Sdf1a expression acts as a `roadway' constraining the
migrating cells to a specific route
(Doitsidou et al., 2002;
Knaut et al., 2003;
Schier, 2003). Nevertheless,
in both systems, ectopic expression of the ligand has established that
redirection of both germ cell and PLL primordium migration can occur at a
distance (Doitsidou et al.,
2002; Knaut et al.,
2003; Li et al.,
2004). Which of these mechanisms is most important for lateral
stripe formation is unclear, as the migration route taken by lateral stripe
melanoblasts is still poorly defined. Although our data suggest a role for
Sdf1a signalling in lateral stripe formation, other aspects of the WT
melanophore pattern do not show a correlation with sdf1a expression
and are likely to be independently controlled. Consistent with this, both
dorsal and ventral stripe melanophores are unaffected in sdf1a
morphants (data not shown).
|
). However, initial studies of cxcr4a morphants
do not reveal any melanophore patterning defect (T.R.C. and R.N.K.,
unpublished). Moreover, we have been unable to identify any other
cxcr4-like genes in the zebrafish genome. One possibility is that the
effects of Sdf1a are mediated by a novel Sdf1 receptor in pigment cells.
Consistent with this proposition is the demonstration that an orphan receptor,
RDC1 (CXCR7 - Human Gene Nomenclature Database), is responsible for SDF1
signalling in human T lymphocytes
(Balabanian et al., 2005).
Finally, Sdf1a might act only indirectly in melanophore patterning via its
effects on another cell type, as shown in other contexts. For example,
migration of the PLL nerve sensory afferent growth cones along the horizontal
myoseptum is dependent upon Cxcr4b-Sdf1a signalling, yet elegant analyses of
genetic chimaeras demonstrate that the guidance cues act on the PLL primordium
which migrates ahead of these growth cones and tows them along
(Gilmour et al., 2004).
Further work to identify the precise mechanism whereby Sdf1a influences
melanophores will now be paramount.
It is notable that whilst cho mutants have a profound effect on
lateral pathway migration, the effects on the medial pathway are at most
minor. The identification of ectopic sdf1a expression as an
underlying cause of the collar phenotype helps clarify this, as the ectopic
sdf1a expression is localised only to the lateral face of the
developing somite. It is therefore likely that NC cells utilising the medial
pathway are largely shielded from this influence. Furthermore, a recent study
identified cells of the slow muscle lineage as a key guidance cue for medial
pathway NC migration (Honjo and Eisen,
2005). Since slow muscle fibres initially form normally and show
regular organisation in the majority of somites in cho mutants, it is
perhaps not unexpected that medial pathway NC migration is normal.
cho mutants show more severe somite defects anteriorly than posteriorly
Some aspects of the cho mutant phenotype affect somites along the
whole anterior-posterior axis, including the U-shaped appearance and
detachment of many muscle fibres from vertical myosepta. By contrast, some
defects, such as ectopic sdf1a expression, lack of somite dorsal
extension and slow muscle migration, only affect the anterior-most five
somites. These defects are likely to be an autonomous property of anterior
somitic cells of cho mutants, as WT tissue transplanted into
cho mutants does not develop similar structural defects as the
surrounding cho mutant tissue.
There are other examples of anterior somites behaving differently to
more-posterior somites, including mutants in which only more-posterior somites
are affected (van Eeden et al.,
1996). Indeed, adaxial cell specification might be regulated by
distinct signalling pathways in anterior and posterior somites; for instance,
pertussis toxinmediated manipulation of intracellular signalling pathways is
reported to result in defects restricted to posterior somites
(Hammerschmidt and McMahon,
1998). Furthermore, recent observations suggest that cell adhesion
systems may also be differentially deployed in anterior and posterior somites,
as mutations in integrin alpha 5 alter segmentation of only the first
seven somites (Julich et al.,
2005). These observations are particularly intriguing in relation
to the cho mutant muscle detachment phenotype.
cho mutants differ from other you-type mutants
Although traditionally grouped with the you-type mutants owing to
its U-shaped somites and weak myoseptal phenotype, cho has been
proposed to act downstream of the other you-type genes in the somite
development pathway (van Eeden et al.,
1996). Our characterisation of the cho mutant muscle
phenotype reveals extensive differences from the you-type group, e.g.
general myotome disruption including both slow and fast muscle and retention
of muscle pioneer cells. We propose that cho does not strictly belong
to the you-type gene class and that the shared somite shape phenotype
may be caused by different mechanisms. Molecular characterisation of the
cho locus may illuminate this. The cho mutation has been
mapped to a crucial region but is not yet identified. Interestingly, we have
been unable to identify genes encoding known components of the SDF signaling
pathway in this crucial region (G.E.H. and P.D.C., unpublished).
In summary, the studies reported here demonstrate the nature of the myotomal and pigment patterning defects evident in the cho mutant, defining a novel phenotype distinct from the you-type mutant class to which cho was initially assigned. Analysis of this phenotype has revealed that misregulation of the chemokine Sdf1a within the myotome is a key causative factor in the melanophore patterning defects of cho homozygotes. This is significant because, for the first time, it implicates the Sdf1-mediated signal transduction pathway in pigment pattern formation. It will be of interest now to determine how widely this patterning mechanism is used to control the patterning of NC derivatives throughout the vertebrate clades.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/5/1011/DC1
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ACKNOWLEDGMENTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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