1 MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4
2XU, UK
2 The Victor Chang Cardiac Research Institute, 384 Victoria Street, Darlinghurst
2010, NSW, Australia
* Author for correspondence (e-mail: p.currie{at}victorchang.unsw.edu.au)
Accepted 15 July 2004
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SUMMARY |
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Key words: Hypaxial muscle, Met, Zebrafish
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Introduction |
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Until recently it was believed that the specification of limb muscle
precursors was entirely dependent on limb-derived environmental cues. Flank
somites grafted into the limb region result in the grafted tissue contributing
to the forming limb musculature, despite their non-limb level origin
(Chevallier et al., 1977;
Christ et al., 1977
;
Hayashi and Ozawa, 1995
).
Furthermore, when ectopic limbs are induced adjacent to flank somites, these
somites are re-specified to generate migratory muscle precursors that develop
into normal limb muscles. However, Alvares et al.
(Alvares et al., 2003
) have
recently shown that limb muscle precursors are not naïve, and the ability
of somites to produce limb muscle precursors depends on the axial level from
which individual somites derive. Somites transplanted from limb level to the
flank retain the ability to initiate limb myoblast specific gene expression.
Furthermore, altering the positional identity of somites, through the
manipulation of Hox gene expression, again changes the ability of cells to
express limb myoblast-specific marker genes. However, despite an underlying
reliance on positional information, the positional identity of somites can be
completely over-ridden by signals emanating from the limb environment, as
flank somites can contribute to limb muscle formation when grafted adjacent to
the limb environment. Thus, the specification of limb muscle precursors is
influenced by two different layers of regulatory control: one reliant on the
position of an individual somite; the second dependent on signals produced by
the limb environment, which are sufficient, in themselves, for limb myoblast
induction.
Several genes have been identified that are required for the formation of
the limb musculature. Perhaps the best understood of the cohort of limb muscle
control genes, from a mechanistic point of view, is the receptor tyrosine
kinase Met and its ligand hepatocyte growth factor (Hgf) (reviewed by
Birchmeier et al., 2003). The
EMT of limb myoblasts is mediated by Met and Hgf, a process shown to be
necessary for limb myoblast migration
(Yang et al., 1996
;
Heymann et al., 1996
;
Brand-Saberi et al., 1996
).
During limb formation, met is expressed in the medial and lateral
lips of the dermomyotome at all axial levels
(Yang et al., 1996
). Localized
activation of Met occurs through the restricted expression of hgf,
which is present within the mesenchyme of the limb bud, during limb muscle
migration. Targeted inactivation of either met or hgf within
the mouse embryo results in a lack of appendicular muscle of the limbs, as
well as other hypaxially derived migratory muscles such as the diaphragm and
tongue, despite these cells being initially specified normally
(Bladt et al., 1995
;
Schmidt et al., 1995
). Limb
muscle precursors, however, are unable to migrate from the somite into the
limb bud in met and hgf mutants, as indicated by the
continued expression of lbx1 in the dorsolateral lip of somites at
limb levels (Dietrich et al.,
1999
). In addition, implanting Hgf-soaked beads at inter-limb
level, where Hgf is not normally expressed, can result in cells undergoing an
EMT before delaminating from the somites in this region
(Heymann et al., 1996
;
Brand-Saberi et al., 1996
).
Thus, the formation of appendicular muscles of the amniote limb is
characterized by a Met/Hgf dependent EMT and consequent long-range migration
of limb muscle precursors to their site of differentiation within the dorsal
and ventral muscle masses of the limbs.
Analyses in fish species, however, have revealed the existence of two
phylogenetically distinct processes operating to generate the appendicular
muscles of the fin. The first mechanism, which is thought to be analogous to
that present in amniote embryos, has been shown to operate in zebrafish
(Neyt et al., 2000). However,
a second primitive mode of appendicular muscle formation was found to occur
within chondricthyan species such as the spotted dogfish shark
(Scyliorhinus canicula). An examination of fin muscle formation at a
number of different developmental stages revealed the existence of direct
myotomal extensions headed by epithelial buds within the developing fin bud,
which lay down differentiating myocytes in a proximal-to-distal manner as the
epithelial bud extends within the fin
(Dohrn, 1884
;
Braus, 1899
;
Goodrich, 1958
;
Neyt et al., 2000
) (reviewed
by Galis, 2001
). Furthermore,
S. canicula somite extensions do not express genes that mark
migrating limb/fin myoblasts in other species, reinforcing the primitive
nature of the control Selachian fin muscle formation.
These studies raise a number of important issues about the molecular nature of fin muscle formation in zebrafish. Are fin muscle precursor cells influenced by the local fin adjacent environment, or do they possess an intrinsic ability to form fin musculature based on anterior posterior positioning within the embryo? Is the Met-dependent EMT evident within amniote species a true component of the derived mode of fin and limb muscle formation, or is it a synapomorphy that was generated in response to the differing architecture of the tetrapod body plan? To answer these questions, we have investigated the competence of zebrafish somites positioned at different anteroposterior levels to contribute to pectoral fin muscle formation and examined the role that Met-mediated signaling plays in the formation of hypaxial and specifically appendicular muscle formation in zebrafish.
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Materials and methods |
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Time-lapse analysis of muscle migration
Embryos transgenic for the actin GFP transgene were anaesthetized
(0.17 mg/ml tricane, Sigma) at 36 hpf, embedded in 0.7% agarose/0.17 mg/ml
tricaine, and orientated obliquely on a large coverslip such that the anterior
somites and yolk were positioned directly against the glass. The embedded
embryo was inverted into a deep well depression glass slide into which embryo
media (Westerfield, 1994
) plus
0.17 mg/ml tricaine had been placed. The edge of the depression was coated
with small amounts of petroleum jelly so that when the coverslip was inverted
onto the depression slide, an airtight seal was produced, and the sample did
not dry out during time-lapse. Cell movements were visualized on an Axioskop
FS (Zeiss, Germany) by taking near simultaneous DIC and fluorescent images
every 5 minutes using a Hamamatsu ORCA digital camera driven by IPLab software
(Scanalytic, Fairfax, VA, USA).
Cloning of zebrafish met and hgf
A full description of the cloning of met and hgf
sequences can be found in the legend for Fig. S1 in the supplementary
material.
Morpholino injections, in situ hybridization, antibody stains and histological methods
Two antisense morpholino oligos were designed against the met
sequence (Genetools, OR). The initial morpholino (CM1
ATAGTGAATTGTCATCTTTGTTCCT) was designed to anneal against ATG-containing
sequences, and the second was targeted against the 5' untranslated
region of met (CM2 CTGTAAAATAAAGACACCTGTCGGA). A control morpholino
was also injected that contained a 4 bp mismatch to CM1 (CM1mm,
ATAATGGATTGTCATCCTTGCTCCT). Morpholinos were injected into actin GFP
transgenic embryos at the two-cell stage, at concentrations of 0.5 and 0.75
mM. In situ hybridization was carried out as described previously
(Jowett and Lettice, 1994
)
with some minor modifications. Cryosectioning of somite transplants were
performed after overnight fixation in 4% PFA, using standard procedures
(Westerfield, 1994
). Wax
sections of embryos processed for in situ hybridization or
immunohistochemistry were performed using standard techniques
(Nusslein-Volhard and Dahm,
2002
).
Bead implants and antibody ablation
Anti-human HGF antibody (R&D Systems, UK) and a control antibody,
anti-human slow MyHC (Chemicon, USA), were both diluted to 500 µg/ml in PBS
and injected into -actin GFP embryos at either 16-20 somites or 28 hpf
stages of development. Embryos were incubated at 28.5°C in system water
with Methylene Blue, until they reached 48 hpf and analyzed for defects in
hypaxial muscle formation. Recombinant Hgf protein (R&D Systems, UK) was
diluted in PBS to 50 µg/ml and incubated with shards of Affi Blue beads
(BioRad, USA) that had been trimmed with 30 gauge needles to an appropriate
diameter, for 1-2 hours at room temperature in a moist chamber. Beads were
then back loaded into an injection pipette, under control of a hydraulic
microdrive (Narashige, Japan), and injected sub-epidermally at the appropriate
positions.
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Results |
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Three different types of transplantation experiment were performed. Initially, we randomly chose somites from any rostral/caudal level and transplanted into the region of somite 4 within the host. Contribution to the fin musculature by donor tissue could be detected by the presence of GFP-positive cells, derived from the transgenically marked donor tissue transplanted into somite 4, within the developing fin. Surprisingly, our analyses revealed that only 5% of successful transplants (n=22) were able to contribute to the fin musculature. This suggested that the somites already possessed, at the 13- to 17-somite stage, some restriction in competence for provision of fin muscles.
To determine how somites are restricted in their ability to contribute to
fin musculature, they were isolated from actin GFP donors and
dissected into two groups. The first series of transplants used tissue derived
from the six rostral-most somites and the second group of dissections used
somites caudal to somite 9 at the 13-17 somite stage. This combination of
dissections was aimed at minimizing errors in dissection level leading to an
overlap in the rostrocaudal level from which donor somites were derived.
Individual somites from each group were transplanted ventrally into the host
somite at the approximate level of somite 4, and the contribution of donor
tissue to the fin musculature monitored by the presence or absence of
GFP-positive myoblasts within the host fin. Rostral transplants gave rise to
fin myoblasts 45% of the time (Fig.
1, n=11), while transplants caudal to somite 9 gave rise
to fin myoblasts in only one instance (n=18), which we attribute to
an experimental error in correctly assigning dissected somites to either the
rostral or caudal donor class. We could also show that the failure to give
rise to fin myoblasts did not result from a difference in developmental age
between the transplanted tissue of posterior and anterior somites at 13-17
somites. Transplants of donor somites performed at 24 hpf, a time at which
somites posterior to somite 9 would have been of roughly similar developmental
age as the anterior somites within 13- to 17-somite stage embryo, also failed
to give rise to fin myoblasts (n=16). Collectively, we have
interpreted these results to mean that somites transplanted in this manner
possess an anteroposterior fate restriction at a very early stage of their
development that cannot be over-ridden by local inductive signals.
|
|
|
met expression is restricted to somite levels that contribute to the hypaxial musculature and is also expressed in the posterior lateral line primordia
The results of our transplantation studies revealed a surprisingly early
competence restriction to the anterior somites for the ability to produce
hypaxial musculature. We wished to determine if molecular correlates of this
competence were similarly restricted. In order to address this issue, we
identified zebrafish orthologs of met and hgf, and examined
their expression during fin myoblast migration
(Fig. 4). A single open reading
frame was identified within the zebrafish genome that encoded the Met receptor
(Fig. 4A, see Fig. S1 in the
supplementary material). We find that, in contrast to amniote embryos,
met somitic expression is specifically restricted to the ventral
lateral margin of anterior somites, which we have shown generates hypaxial
muscle in zebrafish and cannot be detected within other somites positioned at
more posterior levels (Fig.
4C-E). During fin myoblast and PHM migration, expression can be
detected in migrating myoblast cells, but in the case of the PHM, expression
is restricted only to cells exiting the somite
(Fig. 4H-K). By contrast, at 48
hpf, a stage at which the majority of fin myoblast migration is thought to
have been completed (Neyt et al.,
2000), met expression remains high in the post migratory
dorsal and ventral muscle masses of the fin with expression within the PHM no
longer detectable at this stage, despite its continued migration towards its
attachment point at the cleithrum (Fig.
4L, Fig. 2).
|
Expression of Hgf during zebrafish pectoral fin and lateral line development
Given that it is the localized expression of the Met ligand, Hgf, which
restricts the activation of the Met receptor in amniote embryos, it was of
interest to determine how hgf was expressed during fin myoblast
migration. We also isolated fragments of two genes encoding Hgf, which we have
termed hgf1 and hgf2. The two different genes gave identical
expression profiles during development and hence will collectively be called
hgf for the purposes of this study. hgf initiates expression
broadly and at low level within somites at all axial levels at 22 hpf, with
stronger expression evident at somite boundaries
(Fig. 4M,N). Expression is also
evident within the caudal aspect of the notochord at this stage, with
widespread expression also evident in the neural tube by 24 hpf
(Fig. 4N). By 30 hpf,
hgf transcripts can be detected throughout the fin bud mesenchyme
with expression in the fin increasing at 36 and 48 hpf
(Fig. 4O-Q). At 48 hpf,
hgf transcripts can no longer be detected within the trunk of the
embryo.
Met morphants lack hypaxial muscles and possess defects in PLLP-derived neuromast deposition
To investigate the function of Met in the tissues in which it is expressed,
we `knocked-down' Met activity, using two different antisense morpholino
oligonucleotides. One morpholino was targeted to overlie the sequences
encoding the ATG (CM1) and the other to sequences 5' of the ATG, within
the 5' untranslated region of the met gene (CM2).
Microinjection of either of these morpholinos (0.5 mM) into embryos derived
from the actin GFP transgenic strain resulted in a similar, severe,
reduction in the amount of GFP-positive cells in the dorsal and ventral muscle
masses of the pectoral fin as well as a reduction in cells of the PHM (89%,
n=269, Fig. 5A-J). By
contrast, no defect in fin muscle and PHM migration was evident in embryos
injected with a control morpholino oligonucleotide (CM1mm, n=118, 0.5
mM), containing a 4 bp mismatch to the met ATG spanning morpholino
oligonucleotide CM1.
|
As a second prominent region of expression of met was within
another migratory cell type, the PLLP, we were interested to determine if
there was a similar requirement for Met in directing either the migration of,
or the exit of, neuromast clusters from, the PLLP. The vital dye DASPEI
(Whitfield et al., 1996) marks
the regularly spaced neuromast-derived hair cells present on both sides of the
trunk of the embryo (Fig. 6A),
and identification of fluorescent hair cell clusters therefore can be used to
monitor neuromast deposition. Microinjection of either morpholino CM1 or CM2
(0.5 mM) resulted in a similar, severe, reduction in the number of deposited
neuromast clusters as monitored by DASPEI staining
(Fig. 6A-C). By contrast, no
defect in neuromast deposition was evident in embryos injected with a control
morpholino oligonucleotide (CM1mm, n=148, 0.5 mM), containing a 4 bp
mismatch to the met ATG-spanning morpholino oligonucleotide, CM1. On
average, CM1/CM2 injected embryos possessed 2.87±1.31 (n=114)
clusters per side, as defined by the presence or absence of fluorescent hair
cells at 48 hpf. Uninjected embryos possess 6.0±0.58 (n=12)
per side at this stage of development. Furthermore, when neuromast-derived
hair cell clusters are deposited in met morpholino-injected embryos,
the number of hair cells within individual clusters is drastically reduced
(Fig. 6D,E), possessing fewer
than 50% (n=11) of the number of differentiated hair cells present in
wild-type clusters. Spacing of hair cell clusters, when deposited, was also
altered in met morphants, with larger than usual gaps evident between
clusters, spanning two to three times the number of somites present between
neuromasts in wild-type embryos (Fig.
6C). Furthermore, differentiated clusters appear to be randomly
deposited within a somite, instead of possessing the stereotypical location at
somite boundaries. The non-neural component of the lateral line, the
supporting cells, are believed to be marked by the expression of the
follistatin gene (Fig.
6F,G) (Mowbray et al.,
2001
). An analysis of follistatin expression within
met morpholino injected embryos, reveals a similar lack of support
cells to that evident for the hair cells
(Fig. 6H,I), suggesting all
PLLP-derived fates are affected by the injection of met
morpholinos.
|
Gain or loss of function of the Met ligand Hgf perturbs hypaxial muscle formation
In order to determine if ectopic activation of the Met/Hgf signaling
pathway could perturb formation of migratory myoblasts, we developed
techniques for implanting beads soaked in Hgf protein adjacent to anterior
somites. Using such techniques, we hoped to determine if application of
ectopic Hgf could produce delamination of ectopic muscle cells from adjacent
somites, as had been shown in the chick embryo, and whether there was any
restriction in the ability of somites at different anteroposterior levels to
do this, as suggested by the restricted nature of met expression.
These experiments demonstrated that if a bead was implanted lateral to the fin
at 20 hpf, by 36 hpf ectopic muscle fibers could be detected in two
stereotypical locations that were never present in control embryos. We
observed a spur of muscle from somite 4 and elongating muscle fibers on the
yolk adjacent to this somite (Fig.
7A,B,D, n=12). On the control side, where no bead or a
control bead soaked in PBS (n=14) was implanted, no ectopic fibers
were detected (Fig. 7C, data
not shown). In addition, implantation of Hgf-soaked beads at other positions
within the embryo did not result in ectopic muscle fiber development from
other somites or induction of met expression within adjacent somites
(n=5, data not shown). However, implanting a bead in the neural tube
of the embryo did result in the ectopic expression of met around the
bead (n=2, data not shown). These results reveal that the ability of
Hgf to stimulate exit of somitic cells is restricted to anterior, and
specifically, fin adjacent somites. It also reveals that the initiation of
hypaxial myoblast differentiation is triggered when precociously delaminating
cells emerge from the somite environment, rather than by the deployment of
some intrinsic cell-autonomous timing of the myoblasts themselves.
|
Injection of the antibody at 16- to 20-somite stage resulted in a reduction to complete absence of GFP-positive cells in the dorsoventral muscle masses of the fin and PHM in 49% of injected embryos (Fig. 7H, n=47). Fin bud formation was not affected in these embryos, as evidenced by its normal development, viewed by DIC optics (Fig. 7I). However, if the anti-HGF antibody was injected at 28 hpf, a reduction, but never a complete absence, in the number of GFP-positive cells in the dorsoventral muscle masses was seen within the fin, suggesting that cells that had already exited the somites could migrate normally to contribute to the fin musculature, but those that had yet to exit could not (Fig. 7J, 50% n=26). Strikingly, injection of the anti-HGF antibody at 28 hpf also resulted in a gap forming within the migrating PHM (Fig. 7J). The anterior most cells of the PHM on the injected side migrate to the same anterior position as the PHM cells on the contralateral uninjected side, revealing that Hgf function was not required for the onward migration of these cells. However, the presence of a posterior gap within the PHM suggests that Hgf is required for the initial delamination of PHM precursors from the somite, and once the antibody is removed or degraded from the extracellular space, PHM precursor cells are able to exit from anterior somites. This observation also reinforces the unique nature of PHM morphogenesis, which results from the ordered addition of myoblasts distal to the migrating primordia from specific somites, and the retention of this addition order throughout the migration of the PHM to its attachment to the cleithrum. Control injections with an identically prepared antibody raised against an unrelated epitope resulted in no phenotype at any stage at which it was injected (n=20).
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Discussion |
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Why this should be the case remains to be fully resolved, but one clue
could come from the stereotypical position that the pectoral fin occupies in
all fish species, both extinct and extant. The pectoral fin is always tightly
physically linked to the bones of the skull through a series of fish-specific
bones such as the cleithrum, and consequently its relative rostrocaudal
positioning may not be easily altered. Within tetrapods, the pectoral girdle
is structurally and functionally detached from the skull and this lack of
physical association is believed to have allowed the fore-limb repositioning
or `posteriorization' evident within early and extant tetrapods to have
occurred (Goodrich, 1958).
This would have required the evolution of a mechanism to uncouple the
positioning of paired appendages from a particular somite level and must have
necessarily included within it the ability to generate appendicular muscle
from any somite level at which the limb became juxtaposed. In early tetrapods,
for example, limbs may have needed to be placed at more posterior positions to
support the extra body weight that would be evident in evolving modes of
aquatic habitation or terrestrial environs. Once such a mechanism evolved, it
would have allowed greater freedom in the formation of different tetrapod body
plans, resulting in the marked alteration in the number of pre- and
post-forelimb vertebrae (a proxy for somite positioning in this context)
evident in different vertebrate species
(Burke et al., 1995
). Fish,
with their highly evolutionarily successful design, never required any
mechanism other than the specification of fin myoblasts by rostrocaudal
identity (presumably imparted by the Hox code) as the positioning of the
pectoral fin bud always occurred at the same somitic level regardless of the
species involved. Although a detailed phylogenetic survey of the relationship
of pectoral fin position and somite number has yet to be performed to
definitively support such an argument, an analysis of the existing literature
(Killeen et al., 1999
;
Okamoto and Kuwada, 1991
;
Ballard and Needham, 1964
;
Detlaff et al., 1993
),
together with our direct observations (embryos examined, zebrafish, salmon,
trout, carp, stickleback, paddlefish, sturgeon and lungfish; N.J.C. and
P.D.C., unpublished) reveal that in every bony fish species for which we could
obtain data, the pectoral fin always arises adjacent to the first six somites.
Hence, the two layers of regulation of limb myoblast formation elegantly
demonstrated by Alvares et al. (Alvares et
al., 2003
) may in fact represent the primitive `Hox-imparted' mode
of specification, which is present in all vertebrates with paired appendages,
and the derived `local induction' mode that may have arisen to accommodate the
designs of the tetrapod body plan.
Identification of a new hypaxially derived muscle in zebrafish
Fate mapping and somite transplantation have defined a hypaxial muscle with
a distinct origin and morphogenesis to that of the fin musculature. Although
the continuous nature of the extension of the PHM from its origin is
reminiscent of inter-limb level hypaxial muscle formation in amniotes and
hypaxial muscle formation in general within cartilaginous fish (the primitive
mode of hypaxial muscle morphogenesis), we believe that the two processes are
not related. First, the PHM highly expresses markers such as lbx
(Neyt et al., 2000) and
met (this study), which specifically mark limb/fin and anterior
migratory myoblasts in this and other contexts. Furthermore, inter-limb level
somites exhibit the primitive epithelial morphogenesis of hypaxial muscle
formation and the PHM does not migrate as an epithelium. However, we believe
the existence of the PHM and the fin associated migration that it undergoes
may underlie a number of previous reports, based on comparative morphology,
that suggest the pectoral fin muscle of a number of teleost species are
produced via the primitive mode of epithelial extension (reviewed by
Galis, 2001
). We also believe
that the PHM is not likely to have a directly analogous muscle in amniote
species, given its attachment to the cleithrum, which is a bone of dermal
origin found predominantly in fish and primitive tetrapods.
Control of hypaxial muscle by Met and Hgf signaling
We have demonstrated that the restricted competence of anterior zebrafish
somites to produce hypaxial and appendicular muscle is mirrored by a
restricted expression of met. In contrast to the postulated function
of Met in stimulating active migration of amniote limb myoblasts
(Birchmeier et al., 2003), we
can only implicate Met-mediated signaling in the control of the initial
delamination of hypaxial muscle precursors from zebrafish somites. However, we
note that little genetic evidence exists to definitively show that Met
signaling is required during the migratory process in amniotes, as conditional
knockout mice have yet to be generated that remove Met or Hgf function solely
during the migratory period. Furthermore it should be noted that our results
cannot rule out a role for Hgf in mediating cell proliferation and
differentiation of post-migratory myoblasts within in the fin environment as
has been postulated previously to occur for post-migratory limb myoblasts in
the chick (Scaal et al.,
l999
).
Further models of Met and Hgf function developed from results of in vitro
studies suggest a chemotactic role for Hgf in guiding migration of stimulated
cells (Lee et al., 1999).
Again, our results do not support such a role, as beads implanted with Hgf
protein do not alter the path of migration of either fin myoblasts or cells of
the PHM, instead resulting in a precocious delamination of cells from
Met-expressing somites. These results are in line with previous studies in
chick and mice that similarly discount a chemotactic role for Hgf in limb
myoblast migration (Heymann et al.,
1996
; Dietrich et al.,
1999
).
Met/Hgf function in control of the lateral line primordia
How might Met signal activation control proneuromast deposition from the
PLLP? Our results suggest that whatever this mechanism may be, it is
independent from the process that directs PLLP migration, which has been shown
to be controlled by the chemokine SDF1 and its receptor CXCR4
(David et al., 2002), as PLLP
migration proceeds normally in Met-deficient embryos.
One clue may come from the fact that the PLLP, despite its migratory
morphogenesis, expresses a number of markers of epithelial identity at high
levels. We have shown that a number of these markers, such as prox1
and met itself are specifically downregulated in the trailing edge of
the PLLP before proneuromast deposition occurs. One possible model, which is
consistent with the available data, is that migration of the PLLP beyond
discrete regions of Hgf expression, present at somite boundaries, could
generate pulses of Met-mediated de-epithelization within a subset of
predetermined cells of the PLLP (Itoh and
Chitnis, 2001). Once sufficient somite boundaries have been
traversed by the PLLP and trailing edge cells have downregulated epithelial
markers sufficiently, they loose contact with the remainder of the primordium
and are released. This model explains a number of puzzling features of
neuromast deposition, including its attenuated periodicity of approximately
every five somites and the association of differentiated neuromasts with
somite boundaries. Furthermore, the model suggests a novel mechanism of
`de-epithelialization thresholding', which is controlled by the Met receptor,
may act as a developmental timekeeper for cellular morphogenesis.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/131/19/4857/DC1
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