1 Randall Centre, New Hunt's House, Guy's Campus, King's College London, London
SE1 1UL, UK
2 Department of Molecular and Cell Biology, University of California, Berkeley,
CA 94720-3204 USA
3 Western Regional Research Center, Albany CA 94710 USA
4 Department of Biology, University of York, York YO10 5YW, UK
Author for correspondence (e-mail:
simon.hughes{at}kcl.ac.uk)
Accepted 19 March 2004
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SUMMARY |
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Key words: Dermomyotome, Slow muscle, MyoD, Pax3, Myf5, Engrailed
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Introduction |
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In zebrafish, the medial adaxial myogenic cell population arises early in
presomitic mesoderm (PSM) next to the notochord. Adaxial cells have a distinct
cuboidal morphology and express the myogenic basic helix-loop-helix
transcription factors myf5 and myod, followed by slow myosin
heavy chain (MyHC) (Chen et al.,
2001; Coutelle et al.,
2001
; Devoto et al.,
1996
; Weinberg et al.,
1996
). Subsequently, as somite borders form, myf5 and
myod mark a distinct population of cells in the lateral somite
(Chen et al., 2001
;
Coutelle et al., 2001
;
Weinberg et al., 1996
). The
fate of the medial and lateral myogenic cells is known: medial adaxial cells
form slow muscle and most migrate laterally to generate the superficial slow
muscle fibres that lie within the somite under the epidermis
(Blagden et al., 1997
;
Devoto et al., 1996
). At a
slightly later stage of development, lateral somitic cells give rise to the
fast muscle that makes up the bulk of the myotome
(Devoto et al., 1996
).
Subsequently, the myotome grows by the addition of further cell populations at
its dorsal and ventral extremes, a situation reminiscent of the dorsomedial
and ventrolateral dermomyotomal lips of amniotes
(Barresi et al., 2001
;
van Raamsdonk et al., 1982
;
Veggetti et al., 1990
).
Secreted signalling molecules encoded by Hedgehog (Hh) genes, which are
expressed in ventral midline tissues, are required for appropriate medial slow
muscle development (Barresi et al.,
2000; Blagden et al.,
1997
; Coutelle et al.,
2001
; Currie and Ingham,
1996
; Du et al.,
1997
; Lewis et al.,
1999
; Norris et al.,
2000
). Hh is not required for myogenesis of lateral fast muscle or
a second wave of slow fibres formed from the dorsoventral tips of the myotome
(Barresi et al., 2001
;
Blagden et al., 1997
). Thus, in
fish, the distinct contractile fibre type of successive waves of fibres has
permitted elucidation of several modes of fibre formation.
In amniotes, independent regulation of several somitic muscle precursor
populations has also been described although no clear-cut distinction, e.g. on
the basis of fibre type, between the products of different myogenic inductions
has been reported (Hadchouel et al.,
2003; Kahane et al.,
2001
; Sacks et al.,
2003
). In birds and rodents, as in fish, early populations of
fibres express slow MyHC, whereas some later fibres do not. As in fish,
prevention of ventral midline signalling or blocking sonic hedgehog
(shh) in birds eliminates markers of the earliest myogenic cells in
the somite (Borycki et al.,
1998
; Pownall et al.,
1996
), while shh overexpression can enhance expression of
myod and terminal muscle differentiation
(Johnson et al., 1994
;
Kahane et al., 2001
). Mice
with ablated Shh function show reduced epaxial myogenesis at the
dorsomedial lip of the dermomyotome
(Borycki et al., 1999
;
Chiang et al., 1996
).
Elimination of the Shh and indian hedgehog (Ihh) genes or
their receptor Smoothened in the mouse leads to more severe loss of medial
myogenesis (Zhang et al.,
2001
), just as occurs in zebrafish. Lateral myogenesis within the
somite is relatively unaffected by loss of Hh signalling. However, the
parallel role of Hh in induction of sclerotome
(Fan et al., 1995
), which
constitutes a large part of the early somite in amniotes, and conflicting
views on the role of Hh in dermomyotome development makes interpretation
difficult (Borycki et al.,
1999
; Cann et al.,
1999
; Huang et al.,
2003
; Kruger et al.,
2001
; Teillet et al.,
1998
). Actinopterygian teleost species diverged from
sarcopterygian amniote ancestors over 400 Mya. Nevertheless, the similarities
between amniote and fish myogenesis raise the possibility that a common system
has been co-opted to different ends during evolution. To address this issue,
we turned to amphibia, which, although sarcopterygian derived, diverged from
amniotes over 350 Mya.
In Xenopus laevis, the axial musculature begins to differentiate
early (Cary and Klymkowsky,
1994; Chanoine and Hardy,
2003
; Hopwood and Gurdon,
1990
; Hopwood et al.,
1991
). As in zebrafish, axial muscles are functional by 24-26
hours of development (stage 22-24, when the frog has 12-15 somites) and
differentiation progresses in an anterior to posterior direction. Thus, over a
number of developmental stages, anterior myotomes contain muscle cells that
are more advanced than posterior myotomes. There is a major difference in fate
of anterior and posterior somites in Xenopus: anterior (trunk)
somites ultimately generate the complex vertebral and muscular structure of
the metamorphosed adult, whereas posterior (tail) somites are fated to cell
death (Nieuwkoop and Faber,
1967
). Although the differentiation of muscle fibres starts at a
very early stage, differences between muscle fibre types have not been
reported until stage 35 or later (Hughes
et al., 1998
; Kordylewski,
1986
; Schwartz and Kay,
1988
). As in fish, the bulk of the late Xenopus embryo
somite is built of large fibres, whereas, on the lateral surface of the
myotome, there is a monolayer of distinct fibres. In many anurans a cell layer
designated dermatome exists in this lateral location
(Radice et al., 1989
). We
describe the pattern of differentiation of slow and fast muscle fibre types at
early stages and its dependence on anteroposterior position. We show that
manipulation of Hh signalling can affect the decision between fast and slow
muscle formation in a manner similar to that observed in zebrafish. We go on
to characterise later somite myogenesis and suggest a model for evolutionary
adaptation of myogenesis in the transition from fish to tetrapod bodyplan.
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Materials and methods |
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Immunochemistry and histology
Antibodies used in this paper are available from Alexis, ATCC, the DSHB,
Iowa and/or DSG Braunschweig, Germany. Antibody A4.1025 (IgG2a) recognises
many, probably all, sarcomeric MyHCs in species from Drosophila to
human (Dan-Goor et al., 1990).
BA-F8 (IgG2b) was raised against human MyHC and reported to react with slow
and cardiac MyHC in humans and mice, and BA-D5 (IgG2b) was raised against
human MyHC and reported to detect slow MyHCs in rodent, chicken and zebrafish
(Blagden et al., 1997
;
Schiaffino et al., 1989
).
Antibody EB165 (IgG1), raised against chicken fast MyHC, was a gift from Dr
Everett Bandman (Cerny and Bandman,
1986
). The muscle marker 12/101 was a gift from Dr Jeremy Brockes.
Cryosections of staged embryos fixed in Dent or paraformaldehyde fixative were
stained according to (Blagden et al.,
1997
), with the use of class or subclass-specific secondary and
fluorescent tertiary reagents (Jackson ImmunoResearch). After whole-mount in
situ mRNA hybridisation, embryos were cryosectioned and reacted for all MyHC
with A4.1025 or anti-proliferating cell nuclear antigen (PCNA, Sigma).
Analysis and photomicrography was on Zeiss Axiophot or Axiocam. For plastic
sections, devitellinized embryos were fixed in 2% glutaraldehyde in amphibian
Ringer solution, embedded in Epon-Araldite 812 mixture and semi-thin sections
stained with crystal violet and basic fuchsin.
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Results |
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Several muscle fibre populations arise sequentially
We investigated the timing of appearance of the differentiated slow and
fast muscle fibre populations in Xenopus
(Fig. 2). The earliest-formed
muscle is fast, appearing in anterior somites before stage 22
(Fig. 2A,B). As in zebrafish,
fast cells form the majority of medial/deep muscle in the older animal
(Fig. 1F; Fig. 2K-N). By contrast, the
slow fibres arise later in development, being first detected weakly in the
tail tip of stage 27/28 embryos where a group of slow cells appear to span the
somite from adjacent to the notochord towards the lateral somite surface
(Fig. 2F, inset). At this
stage, embryos have about 20 somites, and yet the older trunk somites do not
have slow fibres (data not shown). By stage 35 the posterior half of the
embryo, including somites 18-20, contains superficial slow cells outlining the
lateral border of each somite (Fig.
2I, see also Fig.
5A). In somite 36 at the tail tip, the slow fibre markers span the
somite transversely, as occurred earlier in the 20th somite (compare
Fig. 2F, inset, 2J). As the
functional studies below reveal, slow fibres formed prior to about stage 35
have a similar origin and we designate them `first wave slow fibres'. At later
stages, slow superficial fibres appear in successively more anterior somites
such that, by stage 48, all somites contain an outer layer of slow cells
(Fig. 2K-N). This anterior
extension of slow fibres roughly parallels the retraction of the gut, so that
somites without underlying endodermal tissue contain slow fibres. The
superficial slow fibres are detected preferentially at the dorsal and ventral
extremes of the somite in stage 48 embryos, which is not the case at stage 35
(Fig. 2I-N). Functional studies
below indicate that these later-formed slow fibres have a distinct
embryological origin and we designate them `second wave slow fibres'. Thus,
because anterior (trunk) somites develop first, deep medial `fast' fibres
arise first during development. In more posterior (tail) somites, by contrast,
first wave slow fibres arise first, initially in the medial somite and then
become located more superficially as the bulk of the somite differentiates
into fast muscle. Second wave slow fibres arise later in all somites.
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At early stages, cyclopamine has no effect upon medial XMyf5 expression in trunk regions, where first wave slow muscle does not form, even though Xptc2 expression is suppressed (data not shown). However, cyclopamine downregulates XMyf5 expression in the PSM and nascent somites in the tail creating a `gap' in tailbud expression just where untreated embryos initiate first wave slow myogenesis in the tail (Fig. 4C). Reduction of XMyf5 is specific to the gap region at this stage, as expression in dorsal and ventral myotome of maturing somites is not affected. Strikingly, the missing XMyf5 expression domain is the region where Xptc2 and XMyf5 mRNAs are high in medial cells flanking notochord (see Fig. 4B, Fig. 7G). Moreover, expression of XMyoD is reduced in the tail, though less markedly than XMyf5, but is less affected anteriorly (Fig. 4C). Thus, Hh signalling is required for normal myogenesis in the region of the first slow fibre formation in the Xenopus tail.
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Dermomyotomal myogenesis requires Hh signalling
Trunk somites are not completely insensitive to cyclopamine. Although
second wave slow fibres form normally, cyclopamine treatment reduces
XMyoD expression in dorsal and ventral edges of trunk and anterior
tail somites, whereas XMyf5 expression is normal at these locations
(Fig. 4C). Subsequently, the
somite is reduced dorsoventrally and there is a lack of what appears to be a
transient population of slow fibres at the dorsomedial myotomal edge
(Fig. 5C,D). In addition,
ventral muscle fibres over the belly are aberrant, indicating disruption of
ventral lip myogenesis. Section analysis of unmanipulated embryos reveals that
strong XMyoD expression in the dorsal and ventral somite edges is
associated with the dermomyotomal lips
(Fig. 7E,F). Thus, Hh
signalling is required for some aspects of trunk myogenesis.
Xenopus dermomyotome: a potential source of second wave slow fibres
To investigate the sources of second wave slow fibres formed in
Xenopus somites, we prepared serial plastic sections of embryos at
stages 22, 28 and 35 (Fig.
6A-F). Throughout the period, two epidermal cell layers surround
the embryo as described (Nieuwkoop and
Faber, 1967). At stage 22 and 28, the anterior 18 somites contain
one striking specialisation, a layer of thin cells covering the lateral somite
surface in some regions (Fig.
6A-C) (Blackshaw and Warner,
1976
; Hamilton,
1969
). As no slow fibres have yet formed in trunk somites, this
layer is reminiscent of amniote dermomyotome. At stage 35, a single
superficial monolayer of distinct cells is discerned both in trunk somites
that lack first wave slow fibres and in tail somites that contain superficial
first wave slow fibres (Fig.
6D-H). This distinct superficial cell layer is not slow muscle
because it lacks MyHC and sarcomeric myofibrils
(Fig. 6G,H;
Fig. 7C-F). Pax3, a
dermomyotome marker in amniotes (Bober et
al., 1994
; Goulding et al.,
1994
), is expressed in cells superficial to the differentiated
muscle (Fig. 6I-K). Strikingly,
however, expression is greater in trunk than in tail regions beyond about
somite 12 from stage 29-34 (Fig.
6I,J). Later, at stage 37/38, Pax3 expression increases
in posterior tail somites, possibly in parallel with dermomyotome formation
(Fig. 6E,H,K). Like amniote
Engrailed1 (Davidson et al.,
1988
; Davis et al.,
1991
), Xenopus En1 is also expressed at stage 33, level
with the notochord in the outermost layer of trunk but not tail somites
(Fig. 6L). We conclude that in
trunk and anterior tail somites a cell layer, which hereafter we call
dermomyotome, covers the superficial surface of the somite.
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To further examine the fate of dermomyotomal cells in Xenopus, we
analysed Col1a1 expression, which has been used to mark dermis
(Goto et al., 2000).
Xenopus Col1a1 is expressed in the somitic regions from stage 25 and
widely throughout the dorsal body at later stages when dermomyotome is mature
(Fig. 7K,L). Transverse
sections reveal that the dermomyotomal layer expresses Col1a1, as
does overlying tissue of the epidermis
(Fig. 7M). Thus, the stage 35
Xenopus trunk dermomyotome shares many characteristics with amniote
dermomyotome.
Slow fibre myogenesis and migration
Comparison of MRF and MyHC expression in the tail bud provides insight into
the early events in posterior muscle patterning when first wave slow and fast
fibres types are generated. In Xenopus, the XMyf5 expression
pattern is similar to that in fish (Fig.
4C, Fig. 7A)
(Coutelle et al., 2001). The
most posterior XMyf5 mRNA is abundant in a deep layer of cells
adjacent to the notochord but is not detected in more superficial cells of the
pre-somitic mesoderm. These XMyf5-expressing cells do not express
MyHC (Fig. 7G). In anterior
PSM, XMyf5-expressing cells no longer exclusively cluster around the
notochord: some appear to span the somite, but XMyf5 is still not
detected in the outermost layer of cells
(Fig. 7H). Some
XMyf5-expressing cells with mediolaterally elongated nuclei express
PCNA, which often marks proliferating cells
(Fig. 7I). Cyclopamine prevents
this anterior PSM XMyf5 expression, but not tailbud expression
(Fig. 4C). These data suggest
that Hh signalling is required to maintain XMyf5 expression in medial
cells, which then become orientated mediolaterally, simultaneously losing
XMyf5.
As XMyf5 expression declines, XMyoD mRNA appears. The most posterior XMyoD expression is detected weakly in medial cells but within a few serial sections more anterior, is found exclusively superficially, consistent with loss of XMyf5 and accumulation of XMyoD during lateral migration (Fig. 7J; data not shown). Reduction of this most posterior XMyoD expression is seen in cyclopamine-treated embryos (Fig. 4C). In plastic sections of this region, cells can be seen elongated mediolaterally across the somite, reminiscent of XMyf5-expressing cells and the slow fibres in these somites [compare Fig. 2F (inset) 2J with Fig. 6C,F and Fig. 7H]. Further anterior, XMyoD mRNA is present in newly differentiated superficial muscle beneath the dermomyotome (e.g. Fig. 7F). It seems likely, therefore, that the most superficial layer of cells in the most posterior tail somites contains nascent differentiating slow fibres that express XMyoD, but that shortly thereafter a dermomyotome arises to overlie the slow muscle.
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Discussion |
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Muscle fibre types in Xenopus development
We have used antibody reagents to distinguish several fibre types in
Xenopus. Abundant evidence shows that the superficial fibres are
slow, including oxidative metabolism, early Hh dependence and labelling with
BA-F8, a slow antibody in mammals. However, there is potential for confusion
because BA-D5 and EB165 epitopes show a different spatial pattern in
Xenopus to that observed in zebrafish embryos, where these antibodies
also distinguish slow and fast fibres. In zebrafish, BA-D5 is first expressed
in first wave slow fibres, located medially within the somite
(Blagden et al., 1997).
Subsequently, most of these BA-D5-reactive slow fibres migrate to a lateral
and superficial position (Blagden et al.,
1997
; Devoto et al.,
1996
). In Xenopus, by contrast, the BA-D5 epitope is
present in all/most larval muscle. Thus, the medial muscle that we have
designated `fast' to fit with zebrafish nomenclature, may actually have a slow
contractile rate. In amniotes, all early fibres express some slow MyHC gene,
regardless of their later fate (Page et
al., 1992
). In zebrafish, the medial myotome differentiates into
EB165-reactive fast muscle (Blagden et al.,
1997
). In Xenopus, by contrast, EB165 marks slow fibres.
The simplest explanation is that in Xenopus two epitopes
characteristic of MyHC isoforms have come to be expressed in different cell
populations. These observations emphasise that evolution can rapidly alter
MyHC fibre type, but that MyHC markers are, nevertheless, useful in
conjunction with other functional and molecular data to distinguish cell types
of different developmental origin within one species.
Ancestral pattern of myogenesis: slow, quick, slow
In Xenopus tail somites, slow fibres arise initially in contact
with the medial surface adjacent to the notochord and then become located
superficially, as the bulk of the somite differentiates into fast muscle. This
is what happens throughout the body axis in zebrafish
(Blagden et al., 1997;
Devoto et al., 1996
). Strong
evidence for homology derives from (1) the mediolateral migration of slow
precursors (see below); (2) the Hh-dependence of tailbud XMyf5 and
XMyoD expression and slow fibre formation; and (3) the generation of
extra slow fibres when Hh signalling is increased. Hh dependence is also a
feature of the first wave of slow myogenesis in zebrafish
(Barresi et al., 2000
;
Blagden et al., 1997
;
Coutelle et al., 2001
;
Du et al., 1997
;
Lewis et al., 1999
;
Norris et al., 2000
).
Similarly, in both species, formation of deep fast fibres and a second wave of
superficial slow fibres is Hh independent
(Fig. 8A, tail series)
(Barresi et al., 2001
;
Blagden et al., 1997
;
Du et al., 1997
). The striking
similarities between zebrafish and Xenopus in the formation of slow
and fast muscle at both cellular and molecular levels suggests that the common
ancestor of Xenopus and zebrafish (i.e. of sarcopterygian and
actinopterygian fish) developed muscle in the manner observed in
Xenopus tails and throughout zebrafish. The widespread presence of a
superficial slow muscle layer in agnathans and primitive jawed fish strongly
suggests that primitive vertebrates had this organisation
(Flood et al., 1977
). So the
direct ancestor of amniotes probably generated at least three waves of somite
muscle fibres: early Hh-dependent first wave superficial slow, medial fast and
later Hh-independent second wave slow.
|
First wave slow myogenesis blocked in trunk somites
In Xenopus, first wave (i.e. early Hh dependent) slow muscle is
only formed in the tail. Why is first wave slow muscle not formed in the
trunk? Lack of Hh expression, secretion or responsiveness is unlikely because
expression of Xshh and Xbhh are detected throughout the axis
of stage 22-35 embryos (Ekker et al.,
1995; Mariani et al.,
2001
; Stolow and Shi,
1995
). Moreover, Xptc genes, markers of Hh response, are
upregulated flanking the notochord in trunk PSM dependent on Hh signalling
(Koebernick et al., 2001
;
Takabatake et al., 2000
)
(Fig. 4B). Another possible
reason for the missing first wave is that precocious fast muscle
differentiation in Xenopus mesoderm occurs prior to Hh exposure,
ensuring that the cells exposed to Hh are already committed to fast
differentiation. However, we found that early Hh overexpression did not induce
slow muscle in trunk somites, either at stage 22 or stage 35
(Fig. 3) (A.G., unpublished).
Nor did Shh overexpression ever completely converted tail somites to slow
myogenesis, as can happen in zebrafish
(Blagden et al., 1997
).
Although generation of fast and second wave slow fibres is similar in trunk
and tail somites, it seems there is a block on Hh response that prevents
Xshh and Xbhh from promoting differentiation or survival of
first wave slow fibres in trunk somites
(Fig. 8A, trunk series).
In both fish and Xenopus trunk, myf5 expression is
highest close to the notochord, whereas myod expression persists
further laterally (Chen et al.,
2001; Coutelle et al.,
2001
; Polli and Amaya,
2002
; Pownall et al.,
2002
; Weinberg et al.,
1996
). Notochord ablation prevents medial trunk XMyf5
expression but has little effect on lateral expression of MRFs. This finding
suggests the presence of two distinct XMyf5-expressing myogenic cell
populations in trunk, with the adaxial ones being dependent on signals from
notochord. However, we found that cyclopamine does not modify adaxial trunk
XMyf5 expression (M.E.P., unpublished), even though Hh is active
anteriorly because Xptc genes are upregulated in the medial somite
(Koebernick et al., 2001
;
Takabatake et al., 2000
), and
this Xptc expression is blocked by cyclopamine. So Hh signalling,
although occurring, is not required for initial XMyf5 expression. In
mouse and zebrafish, initiation of myf5 expression is also less
sensitive to Hh removal than is myf5 maintenance
(Asakura and Tapscott, 1998
;
Borycki et al., 1999
;
Coutelle et al., 2001
;
Kruger et al., 2001
;
Teboul et al., 2003
;
Zhang et al., 2001
). In avian
trunk, MRF regulation by Hh depends on other midline/ectoderm signals
(Borycki et al., 2000
). We
suggest, therefore, that the normal function of Hh to maintain MRF expression
in adaxial cells is blocked in Xenopus trunk somites, paralleling
loss of first wave slow fibres. It is unclear whether differences in
myogenesis between amniote trunk and tail are homologous to those in
Xenopus.
Slow fibre migration and somite rotation
Several pieces of evidence suggest that first wave slow cells migrate from
adjacent to the notochord to the superficial somite surface as they terminally
differentiate into slow fibres. First, MRF expression patterns suggest that
medial presomitic mesoderm cells gain XMyoD as they lose XMyf5, move laterally
and undergo terminal differentiation. Second, in tail tip regions slow MyHC
and mediolaterally elongated nuclei are detected in single cells that span the
somite from notochord to lateral surface. More anteriorly, slow fibres are
located on the superficial somite surface and orientated anteroposteriorly, as
are their nuclei. Third, blockade of Hh signalling leads to gap in tailbud
XMyf5 expression followed by decreased XMyoD expression and
absence of first wave slow muscle cells. Based on Xptc1 expression,
Hh signalling acts medially (Koebernick et
al., 2001). Taken together with the known migration of
Hh-dependent first wave slow fibres in zebrafish
(Blagden et al., 1997
;
Devoto et al., 1996
), these
data indicate that first wave slow fibres in Xenopus migrate to the
somite surface around the time of somite formation. However, cell tracking in
vivo would be required to prove this view.
The mediolaterally elongated nascent slow cells are reminiscent of the
cells orientated perpendicular to the notochord in somite I, the next
somite to form (Hamilton,
1969; Keller,
2000
; Youn and Malacinski,
1981a
). First wave slow cells re-orientate parallel to the
notochord as each somite forms. Synchronously, underlying medial somite cells
differentiate into fast muscle fibres and elongate in the same direction.
Thus, the terminal differentiation of two waves of myogenic cells leads to a
change from cells elongated perpendicular to the notochord to fibres
orientated parallel to the notochord (Fig.
8B). Re-orientation of cells in successively older somites has
been interpreted as demonstrating rotation of nascent Xenopus somites
(Hamilton, 1969
). Yet all
cells do not rotate synchronously as a block
(Youn and Malacinski, 1981b
).
Indeed, the morphology of `rotating' Xenopus somite cells and
migrating zebrafish slow muscle is remarkably similar
(Cortes et al., 2003
;
Youn and Malacinski, 1981b
).
Our data, therefore, raise the possibility that a fish-like cell migration
coincident with somite formation accounts for many of the morphological
changes, rather than rotation of the entire somite. Other anuran species do
not show such a somite rotation (Keller,
2000
; Youn and Malacinski,
1981a
). In the light of our finding, we suggest that the previous
interpretation of a wholesale rotation of Xenopus somites should be
regarded with caution until in vivo cell tracking has demonstrated which cells
move where in developing somites.
Xenopus dermomyotome and the evolution of somites
Much has been written concerning the evolution of paired appendages in the
transition from fish to tetrapods, but less attention has been paid to
evolution of the dramatic somitic modifications required for the move to land.
Our findings focus attention on the evolution of the tetrapod trunk,
particularly dermomyotome. Our data show that a superficial layer of slow
muscle is probably the ancestral condition of the common ancestor of teleosts
and anurans. Dermomyotome has not been described in teleosts, instead their
myotomes grow by polarized hyperplasia, a process by which extra muscle fibres
are generated in discrete superficial somitic zones, often at dorsal and
ventral extremes. In zebrafish, these zones give rise to Hh-independent slow
fibres (Barresi et al., 2001)
and pectoral fin musculature (Neyt et al.,
2000
). In this paper, we show that Xenopus trunk somites
do not form the first wave of slow fibres but develop a dermomyotomal layer
shortly after their formation.
First wave slow fibre migration and re-orientation occurs in tail somites.
If we are right that this cell re-orientation accounts for the seeming
`rotation' of tail somites, then similar cell migrations probably explain the
`rotation' of trunk somites (Hamilton,
1969; Youn and Malacinski,
1981b
). Such movements may carry the notochord-dependent adaxial
XMyf5-expressing cells to the lateral somite surface. Loss of contact
with midline-derived signals may explain failure of maintenance of
XMyf5 expression, as occurs in zebrafish notochord mutants
(Coutelle et al., 2001
).
Whereas in the tail Hh drives XMyoD upregulation and slow fibre
formation, in trunk such migratory cells may adopt another fate. Possible
fates include fast muscle, myoblasts or dermomyotome. In chicken, most trunk
myotomal cells arise from the medial half of nascent somites
(Ordahl and Le Douarin, 1992
),
suggesting a medial origin of dermomyotome. If notochord-dependent adaxial
cells form dermomyotome, they may not be committed to myogenesis despite
XMyf5 mRNA expression. In mice, myf5-expressing cells in
brain do not make muscle (Daubas et al.,
2000
). We suggest that at all axial somite levels medial
XMyf5-expressing cells may migrate to lie on the superficial somite
surface, where they may become exposed to other signals influencing their
fate. Perhaps an altered response to Hh was a key evolutionary innovation
permitting these migratory cells to pursue fates other than first wave slow
muscle.
Regardless of its origin, a morphologically distinct cell layer appears on
the superficial surface of Xenopus somites that we believe
constitutes the dermomyotome. This MyHC-negative layer arises shortly after
somitogenesis and expresses Pax3, Col1a1 and, in specific zones,
XMyf5 and XMyoD. These zones correspond well with the
reported sites of myogenesis in the amniote dermomyotome
(Ordahl et al., 2001) and with
the sites of polarized hyperplasia in fish
(Fig. 8A). It is important to
distinguish superficial slow fibres from dermomyotome. Indeed, histology and
electron microscopy (Fig. 6)
suggest that the outer layer of tail somites is initially slow muscle fibres.
These rapidly become covered by a layer of small `spindly' cells, that
coalesce into the dermomyotome overlying the slow fibre layer by stage 35. At
the stages we examined, we found no evidence to support the earlier views that
the dermomyotome is separated from the myotomal portion of the somite, nor
that it forms a dermatome `curtain' draped over the myotomes of more than one
somite (Blackshaw and Warner,
1976
; Hamilton,
1969
). Instead, we concur with the idea that dermomyotome is also
segmented (Youn and Malacinski,
1981b
). As in the mouse
(Davidson et al., 1988
), in
Xenopus trunk somites En1 mRNA is restricted to the medial
region of the dermomyotome. We propose this layer is the evolutionary
homologue of amniote dermomyotome, generating cells for myotome growth.
It is likely that proliferative cells at the dorsal and ventral somitic
lips contribute cells to the dermomyotome, as occurs in birds
(Ordahl et al., 2001)
(Fig. 8A). In addition, the
MRF-expressing lips probably yield the second wave of Hh-independent slow
muscle fibres. In trunk somites, this `second wave' generates the first slow
fibres. During metamorphosis the somites of the trunk increase in size and
form numerous muscles of the back (Ryke,
1953
). Perhaps failure of generation of the earliest slow fibres
is related to the evolution of a pool of cells within the dermomyotome adapted
to building later muscle in tetrapods
(Shimizu-Nishikawa et al.,
2002
).
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: University of Insubria, Department of Structural and
Functional Biology, via J.H. Dunant 3, 21100 Varese, Italy
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