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First published online 21 February 2007
doi: 10.1242/dev.000067
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Research Report |
Department of Biology, Wesleyan University, Middletown, CT 06459, USA.
Author for correspondence (e-mail:
sdevoto{at}wesleyan.edu)
Accepted 1 February 2007
SUMMARY
The myogenic precursors responsible for muscle growth in amniotes develop from the dermomyotome, an epithelium at the external surface of the somite. In teleosts, the myogenic precursors responsible for growth have not been identified. We have used single cell lineage labeling in zebrafish to show that anterior border cells of epithelial somites are myogenic precursors responsible for zebrafish myotome growth. These cells move to the external surface of the embryonic myotome and express the transcription factor Pax7. Some remain on the external surface and some incorporate into the fast myotome, apparently by moving between differentiated slow fibres. The posterior cells of the somite, by contrast, elongate into medial muscle fibres. The surprising movement of the anterior somite cells to the external somite surface transforms a segmentally repeated arrangement of myogenic precursors into a medio-lateral arrangement similar to that seen in amniotes.
Key words: Myotome, Anterior border cells, Cell fate, Paraxial mesoderm, Somite, Dermomyotome, Zebrafish, Fast muscle, Pax7, Myogenesis, Teleost
INTRODUCTION
Vertebrate axial skeletal muscle first develops in a compartment of the
somite called the myotome. In amniotes the myotome grows from a supply of
myogenic precursors in the dermomyotome, an epithelium external to the myotome
(Scaal and Christ, 2004
). The
transcription factors Pax3 and Pax7 are expressed in mitotically active muscle
progenitors in the dermomyotome (Relaix et
al., 2004). As these cells differentiate into muscle they express
myogenic regulatory factors (MRFs) such as MyoD, Myf5 and myogenin
(Relaix, 2006).
Recently, we and others proposed that cells expressing Pax3 and Pax7 on the
surface of the teleost myotome are myogenic precursors
(Devoto et al., 2006;
Groves et al., 2005;
Steinbacher et al., 2006).
Here, we combine lineage tracing and gene expression analysis to test whether
these Pax3- and Pax7-expressing cells are myogenic precursors and to identify
their origin in zebrafish. We show that cells along the anterior border of
epithelial somites move to the external surface of the developing myotome and
express Pax7. A subset of these later differentiates into muscle fibres of the
lateral myotome. We discuss the similarities and differences between the
cellular basis for myotome growth in amniotes and in zebrafish.
MATERIALS AND METHODS
Embryos, immunohistochemistry, birthdating and morphometrics
Zebrafish (danio rerio) were raised according to standard procedures.
MF20 and Pax7 antibodies were obtained from Developmental Studies Hybridoma
Bank (DSHB) (Bader et al.,
1982; Kawakami et al.,
1997). Prox1 antibodies were from Chemicon, whereas MEF2, Myf5
[which recognises MyoD and not Myf5 in zebrafish
(Hammond et al., 2007)] and
myogenin were from Santa Cruz Biotechnology. Antibody labeling and BrdU
birthdating was performed as previously described
(Barresi et al., 2001;
Barresi et al., 2000;
Devoto et al., 2006;
Feng et al., 2006). For
phalloidin labeling, dehydration was omitted and Triton was substituted for
Tween. Labeled embryos were visualised and photographed using a Zeiss LSM
confocal microscope. Morphometric analysis of fast fibre cross-sectional area
was done as previously described (Johnston
et al., 2003); slow fibres were omitted from this analysis.
Time-lapse and cell labeling
Fluorescent images of six-somite embryos carrying the histone2A.F/Z:GFP
transgene (Pauls et al., 2001)
were captured every 2 minutes for 2 hours. For lineage tracing, cells were
injected in 12-somite embryos as previously described
(Devoto et al., 1996). Embryos
were viewed and photographed using a Zeiss Axioplan compound microscope and a
Zeiss LSM 510 confocal microscope.
RESULTS AND DISCUSSION
Cell rearrangements in the epithelial somite
The first indication of myogenic specification in zebrafish is in the
segmental plate, when adaxial cells along the notochord express MRF genes
(Fig. 1A)
(Weinberg et al., 1996).
Somites in zebrafish form as epithelial balls surrounding a core of
mesenchymal cells (Fig. 1D,E).
After somites form, only posterior epithelial cells express MyoD
(Fig. 1A)
(Coutelle et al., 2001). This
suggests that posterior cells have committed to myogenesis. By contrast, Pax7
mRNA is first detectable in anterior cells approximately 2 hours after
segmentation (Feng et al.,
2006; Hammond et al.,
2007; Seo et al.,
1998). Three hours after segmentation, Pax7 and myogenin
immunoreactivity is strongest in non-overlapping subsets of somite cells, with
Pax7-positive cells restricted to lateral and anterior domains of the somite
and myogenin-positive cells restricted to medial and posterior domains
(Fig. 1B). Some
myogenin-positive nuclei appear to be in elongating cells, which are in the
medial posterior region of the somite just lateral to elongated adaxial cells
that span the antero-posterior length of the somite
(Fig. 1B). At the end of the
segmentation period (24 hours), Pax7-expressing cells are restricted to the
external somite surface (Fig.
1C) (Devoto et al.,
2006; Feng et al.,
2006; Groves et al.,
2005; Hammond et al.,
2007).
The change in Pax7 distribution suggests that cells move from the anterior
to the external surface of the somite. In the midtrunk (somites 9 to 14), the
anterior of the second to last formed somite (ss II)
(Ordahl, 1993) consists of a
readily identifiable row of 7 to 9 cells along the medio-lateral axis, from
the anterior-most adaxial cell to the lateral border of the somite
(Fig. 1E). We call these
anterior border cells (ABCs). We used embryos expressing nuclear localised
green fluorescent protein (GFP) (Pauls et
al., 2001) to follow groups of ABCs during the time when the
distribution of Pax7-positive cells changes. In all embryos, ABCs moved
laterally during the first few hours after segmentation, and those that
reached the lateral surface moved posteriorly
(Fig. 1F-K). During this time,
medial and posterior somite cells elongated into muscle fibres
(Fig. 1K). We conclude that
during the first few hours after becoming incorporated into a somite, the
posterior cells begin to elongate into muscle fibres, whereas ABCs instead
remain as undifferentiated cells that shift from the anterior to the lateral
somite surface.
|
).
|
). Our data indicate that the germinal layer derives from ABCs
that move laterally to become external cells, and then back medially to become
muscle fibres. In contrast to ABCs, posterior cells differentiated very early into elongated muscle fibres. Five hours after injection posterior cells had not moved laterally, and instead had elongated medially within the myotome (Fig. 2L). Differences in timing of elongation were not related to initial medio-lateral position of the injected cells - ABCs and posterior cells at similar distances from the midline elongated at quite different times (data not shown). Unlike ABCs, all injected posterior cells had elongated, and/or fused with other cells to span the anterior-posterior length of the somite before 24 hours (n=6/6). Elongated cells derived from posterior cells were always deep within the myotome (Fig. 2M,O, Fig. 3A), and expressed MyHC (n=6/6; Fig. 2N,O). Thus, posterior cells and ABCs give rise to separate waves of myogenesis and generate muscle fibres in distinct myotome locations.
We next wished to determine whether ABCs develop into Pax7-expressing
cells. Seven to 12 hours after injection, half of injected ABCs developed into
Pax7-positive cells on the external surface (n=14/27;
Fig. 2D,E). One injected ABC
gave rise to both a Pax7-positive cell and a muscle fibre. Like Waterman's
external cells (Waterman,
1969), Pax7-positive injected cells are extremely flattened and
occupy the external surface of the myotome in close apposition to superficial
muscle fibres (Fig. 2E). Most
of the Pax7-negative cells derived from ABCs were elongated muscle fibres
(n=9/12) that may have already downregulated Pax7 expression. A few
Pax7-negative cells had a morphology similar to external cells
(n=3/12). We suspect that these non-elongated, Pax7-negative cells
would have later expressed Pax7, but it is possible that a minority of ABCs
develop into cells on the external surface of the myotome that never express
Pax7. We observed no strict correlation between the medio-lateral position of
an ABC and the likelihood of it developing into a Pax7-positive precursor
(data not shown).
The zebrafish myotome expands during larval stages as new muscle fibres are
added (Rowlerson and Veggetti,
2001). Pax7-positive cells remain on the surface of the myotome
into the larval period and beyond (Fig.
2R,S and data not shown). To determine whether ABCs also
contribute to these late Pax7-positive potential myogenic precursors, we
allowed some embryos with rhodamine-injected ABCs to develop into the early
larval period. Some injected ABCs remained as undifferentiated cells on the
external surface of the myotome as long as we observed. After approximately 48
hours, these external cells assume a more protracted morphology, and are found
primarily at the dorsal and ventral extremes of myotome, as well as at the
horizontal myoseptum. They continue to express Pax7
(Fig. 2T,U).
|
; Relaix et al.,
2005). Later myogenic precursors from the centre of the
dermomyotome enter the myotome directly, retaining Pax7 expression for a
prolonged time. These cells are responsible for fetal muscle growth and the
generation of muscle satellite cells. We charted the position of Pax7-positive
nuclei within the myotome to provide an indication of where external cells
enter the myotome in zebrafish. We found that Pax7-positive nuclei within the
myotome were excluded from rostral and caudal myotome ends, and were instead
found scattered along the central region of the myotome
(Fig. 3B,C). This suggests
myogenic precursors enter the myotome directly, by moving between superficial
slow muscle fibres along their length, but not around their ends. These
Pax7-positive cells within the myotome are usually positive for MRFs (data not
shown) (Devoto et al., 2006).
We suspect that these cells differentiate soon after entering the myotome, and
are not equivalent to late myogenic precursors derived from the centre of the
dermomyotome in amniotes.
Medial to lateral expansion of the myotome
The above gene expression and fate map data indicate that
early-differentiating posterior cells give rise to medial fast fibres, whereas
later-differentiating ABCs give rise to lateral fast fibres. Consistent with
these observations, medial fast fibres became post-mitotic prior to lateral
fast fibres. Early BrdU exposure led to BrdU-positive muscle fibre nuclei
throughout the entire medio-lateral extent of the myotome
(Fig. 3D). By contrast, late
BrdU exposure led to BrdU-positive nuclei only along the lateral edge of the
fast myotome, just medial to slow muscle fibres
(Fig. 3E).
To confirm these results we used fibre cross-sectional area measurements to
map the distribution of new fast fibres [very few slow fibres are added
(Barresi et al., 2001)]. At
48-hour, 72-hour and 96-hour stages, smaller diameter [presumptive new
(Veggetti et al., 1993)] fast
fibres were in the lateral myotome, whereas larger diameter fast fibres were
in the medial myotome (Fig.
3F-H).
CONCLUSIONS AND PERSPECTIVE
In sum, zebrafish myogenesis proceeds in distinct waves. First, posterior cells of the epithelial somite and adaxial cells generate an embryonic myotome, while ABCs form a layer of undifferentiated myogenic precursors external to this myotome (Fig. 3I). Second, some of these external cells generate new fast fibres lateral to the earlier formed, deep, fast fibres, probably by moving between and not around pre-existing slow fibres (Fig. 3J).
|
; Feng et al.,
2006; Hammond et al.,
2007; Johnson et al.,
1994). We do not yet know if later zebrafish external cells also
share features with later amniote dermomyotome cells, which give rise to
dermis, endothelial cells, cells that migrate into limbs, and cells that enter
the myotome as mitotically active, Pax7-positive myogenic precursors
(Yusuf and Brand-Saberi,
2006). The zebrafish dermis is acellular until 4 weeks of life
(Le Guellec et al., 2004); it
remains unknown whether external cells in zebrafish contribute to dermis or
other cell types later in larval life.
Unlike the amniote dermomyotome, zebrafish external cells derive from the
anterior border of the epithelial somite. Interestingly, in Xenopus,
anterior epithelial somite cells also move to the lateral surface, during the
process of somite rotation (Afonin et al.,
2006). Whether these cells become external cells
(Grimaldi et al., 2004) is
unknown. However, in other amphibians
(Keller, 2000), as well as in
chick (Selleck and Stern,
1991; Stern and Canning,
1990) and in mouse
(Eloy-Trinquet and Nicolas,
2002), it is clear that the dermomyotome (external cells) do not
derive from the anterior border of the epithelial somite. Our results
demonstrate that although the cellular movements underlying the initial
development of external cells in teleosts and the dermomyotome in amniotes may
have diverged, the presence of an external myogenic precursor population is
conserved among vertebrate lineages.
ACKNOWLEDGMENTS
We thank all members of our laboratory, Laura Grabel, Ann Burke, Peter Steinbacher and Walter Stoiber for helpful comments on the manuscript. A Donaghue Investigator Award, and NIH grants HD37509 and HD044929 to S.H.D. supported our work.
Footnotes
* Current address: CADIC-CONICET, Bernardo Houssay 200, (9410) Ushuaia,
Tierra del Fuego, Argentina 
Current address: The Jackson Laboratory, 600 Main Street, Bar Harbor, ME
04609, USA
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