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First published online 15 February 2006
doi: 10.1242/dev.02291
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Department of Anatomy and Cell Biology, Hebrew University-Hadassah Medical School, PO Box 12272, Jerusalem 91120, Israel.
* Author for correspondence (e-mail: kalcheim{at}nn-shum.cc.huji.ac.il)
Accepted 18 January 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Adherens junctions, Asymmetric cell division, Avian embryo, Cell adhesion, Dermis, Dermomyotome, Desmin, Epithelial to mesenchymal transition, Muscle, Myod, Myf5, Pax3, Pax7, Somite
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Buckingham,
2001; Hollway and Currie,
2003; Kalcheim and Ben-Yair,
2005). Studies in avian embryos showed that the dorsomedial lip
(DML) of the dermomyotome (DM) and, later, the ventrolateral lip (VLL)
contribute to myotome growth in medial and lateral orientations, respectively
(Denetclaw et al., 2001;
Denetclaw et al., 1997;
Denetclaw and Ordahl, 2000;
Ordahl et al., 2001). Our
studies distinguished at least two separable waves that account for the
formation of the postmitotic myotome. A first wave of pioneer myoblasts
constitutes the medial region of the epithelial somite with cells expressing
Myod and Myf5. Upon sclerotome dissociation, pioneers
delaminate, migrate towards the rostral somitic domain, and, from this region,
generate myofibers in rostrocaudal and mediolateral directions
(Kahane et al., 1998b;
Kahane et al., 2002).
Following the establishment of this primary structure, the advent of a second
wave of myoblasts was shown, which emanates from all four lips of the DM, the
contribution of the DML and VLL being restricted to the epaxial and hypaxial
domains, respectively, and that of the rostral and caudal lips spreading along
the entire mediolateral myotome. This unique mechanism leads to an overall
intercalatory mode of myotome expansion
(Cinnamon et al., 2001;
Cinnamon et al., 1999;
Kahane et al., 1998a;
Kahane et al., 2002). The
contributions of the four DM lips were further confirmed by quail-chick
analysis (Huang and Christ,
2000) and by GFP electroporation
(Gros et al., 2004).
Subsequent growth of the myotome occurs by addition of cells that remain
transiently proliferative. We reported that the first mitotically active
muscle progenitors, termed the `third wave', originate in the rostral and
caudal lips of the DM (Kahane et al.,
2001). Somewhat later, the central DM sheet dissociates and its
progeny, composed of at least bipotent cells, colonizes both the subectodermal
space to generate dermis, and the myotome to give rise to the majority of
mitotic muscle progenitors, a population that maintains the expression of DM
markers, such as PAX3, PAX7 and FREK
(Ben-Yair and Kalcheim, 2005;
Ben-Yair et al., 2003). The
final fate of these progenitors is varied. Most are likely to withdraw from
the cell cycle at progressive stages and undergo myogenesis, some may also
contribute to muscle fibroblasts, endothelial cells and muscle satellite cells
(Gros et al., 2005;
Kassar-Duchossoy et al., 2005;
Relaix et al., 2005;
Scaal and Christ, 2004).
Altogether, an emerging fate map suggests that the entire DM epithelium has
myogenic potential that is, nevertheless, regionally biased to the immediate
production of myofibers and/or of mitotic muscle progenitors.
The existence of these discrete somitic microenvironments endowed with different and multiple fates raises fundamental questions about the mechanisms of lineage segregation in the various subdomains. Based on these findings, a functional analysis of genes expressed in somites can now be undertaken in the perspective of a clearer cellular picture underlying morphogenesis. In the present study, we investigate the precise expression and role of N-cadherin in each of the described waves of somitic myogenesis.
N-cadherin belongs to a family of Ca2+-dependent cell adhesion
molecules (Hatta et al., 1988;
Hatta et al., 1987;
Hatta and Takeichi, 1986;
Volk and Geiger, 1984;
Volk and Geiger, 1986a;
Volk and Geiger, 1986b) and is
crucial for a diversity of steps during embryonic development
(Gumbiner, 2000;
Nelson and Nusse, 2004). It is
characterized by five extracellular cadherin-binding domains that are
separated by Ca2+-binding pockets, a transmembrane domain and an
intracellular ß-catenin-binding domain
(Tepass et al., 2000). Among
numerous other systems, N-cadherin is also dynamically expressed in the
paraxial mesoderm (Duband et al.,
1987; Duband et al.,
1988). In vitro studies implicated N-cadherin in the regulation of
the specification and differentiation of muscle progenitors
(George-Weinstein et al.,
1997; Goichberg and Geiger,
1998; Holt et al.,
1994; Knudsen et al.,
1990), but knowledge of its role(s) in vivo is still very limited.
A pivotal role for N-cadherin in somite morphogenesis was demonstrated
(Horikawa et al., 1999;
Linask et al., 1998;
Radice et al., 1997). However,
this early requirement during somite formation, and the premature death of
embryos carrying a mutation in N-cadherin, precluded phenotypic
analysis at muscle-forming stages, thus, genetic evidence awaits the analysis
of conditional knockouts. In avians, adenovirus-mediated missexpression of
N-cadherin mutants revealed a role in myofiber arrangement, yet this technique
allowed analysis of only an early subset of myofibers
(Horikawa and Takeichi, 2001).
In zebrafish, an interplay between N- and M-cadherins was reported to control
the migration of slow muscle cells from their paranotochordal region of origin
to the most lateral surface of the myotome
(Cortes et al., 2003).
In a previous study, we determined that the dual generation of dermal and
mitotic muscle progenitors from the central DM is associated with a sharp
change in the plane of cell division from the young epithelium, in which
symmetrical divisions occur parallel to the mediolateral plane of the DM, to
the dissociating DM, in which cell divisions become mostly perpendicular with
one daughter cell facing the dermis and the other positioned adjacent to the
myotome (Ben-Yair and Kalcheim,
2005). Here, we report that during the growth phase of the DM
sheet, when the orientation of mitotic spindles is parallel to the
mediolateral extent of the epithelium, N-cadherin is inherited by both
daughter cells. Prior to DM dissociation into dermis and muscle progenitors,
when mitoses become perpendicularly oriented, N-cadherin remains associated
with only the apical cell located in apposition to the myotome, and is absent
from the basally oriented cell, thus generating molecular asymmetry between
basal and apical progeny. In line with this differential segregation, local
gene misexpression confirms that N-cadherin-mediated adhesion is sufficient
for promoting myotome colonization, whereas interfering with the above process
prematurely drives the cells towards the dermal domain with no PAX-positive
progenitors colonizing the myotome. Hence, N-cadherin-mediated adhesion is
both necessary and sufficient for the differential allocation of DM
progenitors into the myotome. Notably, cells overexpressing N-cadherin that
translocate into the myotome differentiate into myofibers, rather than
remaining as mitotically active PAX+ progenitors, as has been
reported for their normal counterparts, suggesting that N-cadherin is also
involved in muscle specification. Likewise, interference with the activity of
N-cadherin-mediated cell adhesion in the bordering lips (DML, rostral and
caudal) caused the rapid dissociation of the DM epithelium. Consequently, no
cells entered the myotome and no fibers differentiated. Notably, in the
rostral and caudal lips of the DM, early electroporation prevented the onset
of Myod transcription and myofiber generation, further implying that
N-cadherin-mediated adhesion is required for proper myogenic specification. In
contrast to DM lip-derived fibers, differentiation of the pioneer fibers was
not affected. This finding provides the first molecular evidence that the
pioneer population is unique and distinct from the later myofibers deriving
from the DM and, in particular, from the DML.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Expression vectors and electroporation
Four different expression vectors were employed: pCAGGS-AFP, which served
as control (Momose et al.,
1999); full-length chicken N-cadherin; a dominant-negative version
of N-cadherin lacking part of the extracellular domain (cN390
); and
N-cadherin lacking its intracellular ß-catenin-binding domain (CBR-)
(Fujimori and Takeichi, 1993;
Nakagawa and Takeichi, 1998).
The latter three were subcloned into the pCAGGS vector and fused in frame to a
GFP-encoding sequence.
Electroporations were performed under a dissecting microscope. DNA (4 µg/µl) was microinjected into the center of flank-level epithelial or dissociating somites at specific stages as depicted in the figure legends. Five different types of electroporation were performed: to the medial epithelial somite, to the DM sheet, to the DML and to the rostral and caudal lips (Fig. 1). To this end, tungsten electrodes were mounted on two separate micromanipulators and placed to reach each somitic domain as described in Fig. 1. The direction of the electric field is indicated by the black arrow. A four parameter PulseAgile square wave electroporator (PA-4000, Cyto Pulse Sciences) was used to deliver three groups of sequential pulses as follows: 3x30 V, 20 mseconds each; 1x38 V, 5 mseconds; 3x30 V, 20 mseconds each.
The number of embryos showing a given phenotype out of the total number of transfected embryos for each treatment is presented in the corresponding figure legends.
Tissue processing, immunocytochemistry and in situ hybridization
Embryos were fixed with 4% formaldehyde in PBS, embedded in paraffin wax
and sectioned at 8 µm. Immunostaining for desmin was as described
(Kahane et al., 2001). Rabbit
anti-GFP (Molecular Probes) was used at a dilution of 1:500, alone, in
combination with desmin immunolabeling, or with in situ hybridization for
Myod, Alx4 or Pax7
(Cinnamon et al., 2001).
Monoclonal anti-N-cadherin antibodies were from Zymed and were used at a 1:100
dilution in PBS containing 5% fetal calf serum and 0.1% Triton X-100. PAX7
immunolabeling, staining of centrosomes with 
-tubulin antibodies and
Hoechst nuclear staining were as described by Ben-Yair and Kalcheim
(Ben-Yair and Kalcheim, 2005).
Whole-mount embryo preparations and sections were photographed using a DP70
(Olympus) cooled CCD digital camera mounted on a BX51 microscope
(Olympus).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Kahane et al., 1998b).
N-cadherin is homogeneously distributed throughout the epithelium, including
the medial domain, with predominant staining in the apical surfaces and weaker
expression in the somitocoele (Fig.
2A). Upon somite dissociation, pioneer myoblasts bend underneath
the forming DM, dissociate into mesenchymal cells and engage in caudorostral
migration (Kahane et al.,
1998b; Kahane et al.,
2002) (see also Fig.
2F and Fig. 8C).
Slightly before this process begins, pioneer myoblasts that express desmin
downregulate N-cadherin to a basal level (arrowheads in
Fig. 2B-G), similar to the
behavior of dissociating sclerotomal cells
(Fig. 2B,C)
(Duband et al., 1987). The
young DM retains strong apical N-cadherin expression
(Fig. 2B-I). Following
polarized migration, the first myotomal fibers are generated in rostrocaudal
and mediolateral directions (Kahane et
al., 1998a; Kahane et al.,
1998b; Kahane et al.,
2002), a stage at which they regain N-cadherin protein on their
surface (Fig. 2H,I). Hence,
pioneer myoblasts are positive for N-cadherin at the epithelial stage, lose
the protein during dissociation and migration, and re-express it during
differentiation into myofibers.
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;
Kahane et al., 1998a) (see
also Fig. 7), and express
N-cadherin primarily in the apical aspect of the epithelial cell, as revealed
by N-cadherin staining of cells transfected with a GFP-encoding DNA
(Fig. 3D-G).
The DML and VLL are also N-cadherin positive
(Fig. 3H-M). We have previously
shown that these lips contribute indirectly to myofiber generation, by
releasing intermediate progenitors that localize underneath these lips in a
region we termed the sub-lip domain (SLD). Similar to the DML and VLL, the SLD
is also negative for desmin but, unlike the DML and VLL, it is positive for
Myod, Myf5 and FREK
(Cinnamon et al., 2001), and
also for N-cadherin, even though these cells are no longer epithelial
(Fig. 3H-J).
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).
This part of the epithelium also reveals N-cadherin immunoreactivity enriched
in the apical pole of the cells (arrowheads in
Fig. 3A-C). N-cadherin protein
is, however, downregulated in the emerging dermis
(Fig. 3K-M), but is stably
maintained in desmin-expressing myofibers that, contrary to the DM, are
decorated in a homogeneous pericellular pattern (Figs
3,
4).
Differential segregation of N-cadherin to the progeny of DM sheet cells during mitosis
Following dissociation, many individual DM sheet progenitors generate both
mitotic myotomal progenitors as well as dermis. This is associated with a
shift in the orientation of cell divisions which, in the young DM epithelium,
are parallel to the mediolateral axis of the DM and prior to DM dissociation
progressively change to become perpendicularly oriented
(Ben-Yair and Kalcheim, 2005).
To begin approaching the relationship between mitotic orientations,
dissociation of the DM epithelium and fate segregation, we determined the
localization of N-cadherin protein during mitosis
(Fig. 4). As in other types of
epithelia, the mitotic phase of the cell cycle in the DM occurs at the apical
pole where N-cadherin expression is predominant
(Fig. 4A). In all parallel cell
divisions, daughter cells remain side by side, associated with the apical
surface of the epithelium, and N-cadherin is symmetrically inherited by both
cells (Fig. 4B). In
perpendicular mitoses, one daughter cell points towards the dermis and the
other towards the myotome. In all such cases, N-cadherin remains associated
only with the apical cell located in apposition to the myotome and is lost
from the basally positioned cell (Fig.
4C,D), thus generating a molecular asymmetry between the basal and
apical progeny. Notably, N-cadherin in oblique mitoses was similarly
restricted to the apical progeny, similar to what was observed in the
perpendicular cell divisions (Fig.
4E), further validating by molecular means the classification
previously employed to distinguish between parallel and perpendicular mitoses
(Ben-Yair and Kalcheim, 2005;
Cayouette et al., 2001).
Effects of N-cadherin-mediated adhesion on cell translocation and fate segregation of DM sheet progenitors
The observation that myotomal cells are N-cadherin positive whereas dermal
progenitors downregulate the protein (Figs
3,
4), together with the finding
that N-cadherin becomes differentially segregated to the apical daughter cells
during mitosis (Fig. 4), suggest that, of the single cells previously shown to generate both muscle and
dermal progenitors, the N-cadherin-positive DM daughter cell translocates into
the N-cadherin-expressing myotome as a result of homophilic interactions,
whereas the cell lacking N-cadherin generates dermis. To examine this
possibility, the nascent DM sheet was focally electroporated (see
Fig. 1B), with control GFP,
wtN-cadherin-GFP or N-cadherin bearing a deletion in the extracellular domain
(cN390-GFP) (Fig. 5).
This mutation was documented to interfere with activity of endogenous cadherin
by competing for interactions with cytoskeletal components, resulting in the
abrogation of cadherin-mediated cell adhesion
(Fujimori and Takeichi, 1993).
The transfection method used did not reach the bordering lips and was confined
to the center of the DM sheet. Up to 18 hours after transfection, the
GFP-labeled DM was still epithelial in control-treated segments, but in cells
expressing cN390-GFP the beginning of de-epithelialization could be
detected as early as 5 hours following electroporation; this was also revealed
by a gradual loss of basement membrane-associated laminin immunoreactivity,
which is observed in the basal surface of the DM (see Fig. S1 in the
supplementary material, data not shown). By 30 hours, the DM sheet had already
dissociated in control segments, and GFP-labeled cells localized in both
dermis and myotome (Fig. 5A)
(Ben-Yair and Kalcheim, 2005).
In clear contrast, the cells expressing cN390-GFP failed to colonize
the myotome and localized instead to the prospective dermal domain. There,
they sorted out from the unlabeled progenitors to become aggregated
subectodermally (Fig. 5B).
These data suggest that N-cadherin-mediated adhesion is necessary for myotome
colonization. To examine whether it is also sufficient, wtN-cadherin-GFP was
similarly delivered. In contrast to control GFP and to cN390-GFP, all
cells overexpressing N-cadherin translocated into the myotome with no
contribution to dermis (Fig.
5C). This effect was mimicked by overexpression of a mutant of
N-cadherin in which the intracellular ß-catenin-binding domain (CBR-) was
deleted (Fig. 5D). Altogether,
these results suggest that the extracellular domain of N-cadherin that
mediates homophilic cell adhesion is both necessary and sufficient `in vivo'
to drive cell translocation into the myotome.
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-GFP. As previously documented, control GFP-treated cells
remained as mesenchymal Pax7-positive muscle progenitors in the myotome
(Fig. 5E) (Ben-Yair and Kalcheim, 2005).
By contrast, N-cadherin (wt or CBR-)-treated cells entered the myotome as
Pax-positive progenitors, gradually downregulated Pax by 24-30 hours (data not
shown) and then differentiated into fibers by 48 hours after transfection
(Fig. 5F). Thus, N-cadherin is
not only necessary and sufficient for cell translocation into the myotome, it
also triggers myogenic specification followed by differentiation in cells that
otherwise remain as proliferative myotomal progenitors.
We then examined the phenotype of cells that received cN390-GFP and
localized subectodermally to where dermis develops
(Fig. 5B). Similar to control
GFP-treated cells, these cells remained mitotically active (see Fig. S2 in the
supplementary material). Like normal dermis progenitors, they maintained
expression of Alx4, a marker of the central DM region that is later
expressed in dermis (Cheng et al.,
2004) (Fig. 5G,H),
and they rapidly downregulated Pax7 mRNA expression
(Fig. 5I), while still
exhibiting a low level of Pax7 protein; the protein had completely disappeared
by 20 hours after transfection (see Fig. S2 in the supplementary material, data not shown). Thus, cN390-GFP-treated cells display features of
early dermal progenitors (maintenance of Alx4 and downregulation of
Pax genes). Because of a relatively rapid loss of the transfected signal, we
were unable to trace them after E5, when more mature markers of dermis
development, such as Dermo1, or differentiation features of mature
dermis become apparent.
Effects of N-cadherin-mediated adhesion on the ontogeny of the myotome
The dynamic expression of N-cadherin to pioneer muscle progenitors and to
DM lips, as well as its effects on cells originating in the DM sheet, prompted
us to further examine its involvement in the development of the first two
myogenic waves that generate myofibers.
Effect of N-cadherin-mediated adhesion on the DML
In previous studies, we subdivided the contribution of the medial somite
into early pioneer myoblasts and later DML-derived fibers. Here, we wished to
determine whether these two populations can be further distinguished by a
differential sensitivity to N-cadherin (see Figs
6,
8). To examine its effect on
DML progenitors, the nascent DML was electroporated with control GFP or with
cN390-GFP (Fig. 1C). In
control-treated segments, the DML remained both epithelial and labeled for the
entire duration of the experiments (Fig.
6A-F), further suggesting that this domain acts as a continuous
source for myofibers (Ordahl et al.,
2001); this is different from pioneers, which are a limited cell
subset leaving no residual labeled cells after myogenesis (see
Fig. 8). Approximately 20 hours
after electroporation, no DML-derived fibers were yet apparent (data not
shown); this was again different from pioneers, many of which by this time had
already differentiated into myofibers (see
Fig. 8D). Thirty hours
following electroporation, the first labeled fibers became apparent in the
desmin-positive myotome, being restricted to its medial region
(Fig. 6E). In striking
contrast, shortly after electroporation of the mutant cadherin, some labeled
cells had already adopted a round morphology and become localized
subectodermally (Fig. 6G-J).
This phenotype became stronger by 30 hours, when, in addition, the labeled
cells completely failed to enter the myotome
(Fig. 6K). This effect does not
reflect a delay in myotome colonization because a similar picture was observed
42 hours after transfection, at stages following DM dissociation and dermis
formation (Fig. 6L, compare with
6F).
Effect of N-cadherin-mediated adhesion on the rostral and caudal DM lips
When dissociating somites were electroporated with control GFP at their
rostral and caudal edges (Fig.
1D), expression of the transgene appeared shortly afterwards in
the rostral and caudal lips of the DM, respectively (not shown). A day later,
fibers emanating from all along the corresponding lips elongated toward the
opposite direction (Fig. 7A,C,
see also Fig. S2A in the supplementary material), and Pax7/GFP-immunoreactive
cells were also apparent in the myotome (Fig. S2 in the supplementary
material, arrows in A and B). By contrast, overexpression of cN390-GFP
at this stage caused the dissociation of the lip cells into round progenitors
apparent in the intersomitic region, and no myofibers or Pax7-positive
progenitors colonized the myotome (Fig.
7B,D, and Fig. S2D-F in the supplementary material). By contrast,
overexpression of wtN-cadherin robustly generated myofibers
(Fig. 7E).
To begin to understand the mechanism underlying the failure of myofiber
differentiation observed in cN390-GFP-treated lips, control and
experimental embryos were in situ hybridized to detect Myod. In
previous studies, we have shown that Myod mRNA appears in the extreme
DM lips in a scattered fashion following establishment of the pioneer myotome,
about a day after epithelial somites had formed
(Kahane et al., 2001;
Kahane et al., 1998a). By this
time, control GFP-positive epithelial cells in both lips co-expressed
Myod (Fig. 7F,H,J).
However, cN390-GFP-treated cells were Myod negative
(Fig. 7G,I,K). These results
suggest that the cN390-induced early dissociation of the somite
epithelium prevented the onset of Myod transcription and the
subsequent formation of myotomal fibers. Together with our finding that forced
expression of wtN-cadherin or N-cadherin CBR-stimulate myofiber
differentiation in otherwise mitotic progenitors, these data support the
notion that, in vivo, N-cadherin triggers a cascade of events leading to
muscle specification and differentiation.
Loss or gain of N-Cadherin function does not affect the generation of early pioneer myofibers
The origin, migration and pattern of differentiation of pioneer myoblasts
was previously documented using lineage tracing with the lipophilic dye DiI,
pulse-chase experiments with tritiated thymidine, and expression of Myod,
Myf5 and desmin (Kahane et al.,
2002). To interfere with N-cadherin activity in pioneers, we first
determined conditions to specifically transfect DNA into this cell population.
A GFP-encoding DNA was electroporated into the medial somite as shown in
Fig. 1A; this method
differentiates between the pioneer population and the prospective DML and DM
sheet (Fig. 1B,C). The position
of fluorescent cells observed 5 hours after transfection
(Fig. 8A,B, see also 8E)
confirms the specificity of labeling. Consistent with previous results, by 16
hours after transfection, labeled cells have mesenchymalized and are
predominantly localized to the rostral half of the segment, revealing a
general triangular pattern (Fig.
8C) that, as directly demonstrated elsewhere, reflects
caudorostral cell movement (Kahane et al.,
1998b; Kahane et al.,
2002). In addition, the first fibers to arise are located adjacent
to the medial edge and are anchored to the rostral lip of the segment
(Fig. 8C, arrow), further
substantiating the medial-to-lateral and rostral-to-caudal directions of fiber
generation (Kaehn et al.,
1988) [see also figure 1B,C in Kahane et al.
(Kahane et al., 2002)]. By 24
hours, full-length myofibers are already present that span a significant
fraction of the mediolateral extent of the somite
(Fig. 8D).
Next, we examined the function of N-cadherin on the formation of the
pioneer fibers by electroporating the medial epithelial somite with
cN390-GFP (Fig. 8E,F).
Electroporation of 23-25ss embryos revealed the formation of a normal myotome
(Fig. 8G), similar to control
GFP-transfected somites. Similar electroporations performed on younger embryos
aged 15-16ss also resulted in the formation of normal myofibers. which were
already apparent by 20 hours (Fig.
8H,I). To further test whether preventing the normal
downregulation of N-cadherin that takes place upon cell dissociation (see
Fig. 2B-E) has any effect on
the development of the pioneer fibers, full-length N-cadherin-GFP was
similarly delivered. N-cadherin overexpression did not prevent the
dissociation of medial epithelial progenitors (data not shown). Furthermore,
no effect on the differentiation of pioneer fibers was detected, with the
earliest full-length fibers being generated medially and the lateral somite
still containing mesenchymal cells 20 hours after transfection
(Fig. 8J,K). Thus,
N-cadherin-mediated adhesion is not necessary for the development of the first
wave of myotomal fibers. Moreover, forced expression of N-cadherin does not
impair either their dissociation or their ability to generate fibers.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) reflects a change to an asymmetric mode of cell
division, and is therefore of functional significance for fate segregation, as
shown during the development of several invertebrate and vertebrate systems
(Betschinger and Knoblich,
2004).
Differential sensitivity of pioneers and DML-derived fibers to N-cadherin further emphasizes the uniqueness of these two medial somitic populations
Previous studies postulated that the contribution of the medial somite to
the formation of the myotome occurs via a single mechanism whereby stem cells
constituting the DML directly translocate into the myotome and differentiate
without migration (Denetclaw et al.,
2001; Gros et al.,
2004). We found instead that the medial contribution is subdivided
into two components: first, the pioneer wave; and, later, fibers from the DML.
This distinction is based on several findings. Pioneer myoblasts arise in the
medial wall of the still epithelial somite, defining a region different from
the future DML. This region expresses Myod and Myf5 and is
characterized by a significantly lower rate of cell proliferation, including
the presence of post-mitotic cells (Kahane
et al., 1998b), suggesting that this is a limited myogenic
subpopulation. Hence, it differs from the later DML, which in avians lacks
expression of Myod or Myf5, and reveals a continuously high
rate of cell proliferation (Ben-Yair et
al., 2003). This is consistent with the suggestion that DML cells
behave as a continuous source of myoblasts
(Ordahl et al., 2001;
Venters and Ordahl, 2002). The
distinction between fibers derived from pioneers relative to DML was further
confirmed in this study, in which GFP-DNA was differentially electroporated
into the medial versus the dorsomedial regions of the somite. In the first
case, following dissociation of the epithelial pioneers and fiber formation,
no residual GFP-labeled cells remained in the epithelium (indicative of a
finite cell subset), whereas a similar transfection to the DML always revealed
residual labeling of the lip (suggesting a stem-like mechanism). Moreover,
pioneer myoblasts generated a myotome with a significant amount of full-length
fibers present already by 20 hours after GFP transfection, whereas DML-derived
fibers were still absent after 24 hours.
Another unique feature of pioneer myoblasts is that they undergo a
polarized migration towards the rostral edge of the DM, from which initial
myofiber generation proceeds in a rostral to caudal direction
(Kahane et al., 2002). This
particular migratory behavior was further confirmed in this study by tracing
the cells after focal GFP electroporation (see
Fig. 8). The results of our
lineage analyses are consistent with the observed expression patterns of
Myod, Myf5, desmin (Kahane et
al., 2002; Kalcheim et al.,
1999), and the flamingo homologue c-fmi
(Formstone and Mason, 2005) in
the developing myotome.
This mode of initial myotome formation clearly differs from the
contribution of the DML that generates fibers through an intermediate SLD
(Cinnamon et al., 2001) (see
also Denetclaw et al., 2001;
Gros et al., 2004). The medial
cells involved in generation of the second myogenic wave (the DML itself and
the SLD) and the resulting fibers, express N-cadherin throughout the entire
process. Consistent with this continuous expression, cN390-GFP caused
the rapid dissociation of the DML, as well as that of the DM sheet, with cells
that sorted out to become localized subectodermally, and consequent failure of
myotome colonization. These data suggest that, in addition to keeping the
epithelial integrity of the somite, N-cadherin-mediated homophilic attraction
between DM progenitors and the underlying pre-existing myofibers is necessary
for progenitor translocation into the myotomal domain.
By contrast, we report that N-cadherin-mediated adhesion is not necessary
for establishment of the first wave of pioneer fibers. This might be related
to the early downregulation of the protein that is apparent immediately before
the dissociation and rostralward migration of the pioneers. It is, however,
unlikely to depend upon the state of specification of these progenitors, which
already express Myod and Myf5 at epithelial stages, as a
similar electroporation of cN390-GFP to epithelial somites of younger
embryos (aged 15ss), prior to significant medial expression of the myogenic
genes and to their withdrawal from the cell cycle (N. Kahane and C.K.,
unpublished), also had no effect on myofiber development.
Overexpression of wtN-cadherin at this stage was also without effect,
strongly suggesting that N-cadherin is neither necessary nor sufficient for
the earliest progenitors to migrate and generate a myotome. This further
highlights the molecular differences between pioneers and second wave
progenitors, and suggests that mechanisms other than, or additional to,
N-cadherin operate to regulate the formation of this cell population. Unlike
the avian pioneers, differential cell adhesion mediated by N- and M-cadherins
in the zebrafish myotome drives the migration of adaxial cells
(Cortes et al., 2003). Adaxial
cells, like the avian pioneers, originate in a medial position in the somite,
where they express Myod and Myf5, and migrate laterally to
localize superficially in the myotome
(Devoto et al., 1996).
Furthermore, on their way, zebrafish adaxial cells also exhibit an
intermediate phase of rostralward migration
(Cortes et al., 2003).
Nevertheless, whereas zebrafish adaxials continuously express N- and
M-cadherins from their site of origin and throughout migration
(Cortes et al., 2003), avian
pioneers lose N-cadherin expression prior to or concomitant with the onset of
delamination from the medial somite, migrating as cadherin-negative
progenitors. Hence, the molecular mechanisms underlying the migration of the
avian pioneers seem to differ from those of the zebrafish adaxial cells.
Our findings are consistent with results in which the infection of the
chick segmental plate with adenoviruses expressing either full-length
N-cadherin or cN390 had no effect on early myofiber generation
(Horikawa and Takeichi, 2001).
Collectively, a wealth of independent evidence, including lineage tracing with
DiI and GFP (this study), patterns of marker expression, distinct
proliferative behavior, and also functional data on the differential
sensitivity to N-cadherin-mediated adhesion (this study), fully validates the
notion that pioneers and DML cells are distinct myogenic populations.
N-cadherin mediated adhesion and its significance to myogenic specification and differentiation
During myogenesis, the onset of the second wave of muscle colonization
generally follows the formation of the pioneer myotome
(Kahane et al., 1998a;
Kahane et al., 1998b). The
production of myofibers from the extreme lips is associated with the local
upregulation of Myod, with transcripts appearing scattered to
subpopulations of lip cells (Kahane et
al., 2001; Kahane et al.,
1998b). The factors inducing the transcription of Myod in
these lips had remained unknown. Here, we show that the disruption of
N-cadherin-mediated adhesion in the nascent lips prevents the synthesis of
Myod and, consequently, the generation of myofibers. Hence,
N-cadherin acts by mediating specific adhesive interactions among neighboring
lip cells, and/or between lips cells and the pre-existing pioneer myofibers
that attach to the lips. These interactions in turn promote signaling events
that trigger myogenic specification.
This notion is further supported by the finding that, in the DM sheet,
overexpression of N-cadherin not only drives cell entry into the myotome at
the expense of dermis, but also causes myofiber formation rather than
maintenance of mitotic myotomal progenitors, as is observed under normal
conditions. The reason for this phenotypic shift is unclear. One possibility
is that in normal development, Pax3/Pax7-positive mitotic cells that enter the
myotome as N-cadherin-expressing cells subsequently lose the protein in order
to remain in a progenitor state. An additional possibility is that the
relatively high amount of protein in N-cadherin-transfected cells is not
compatible with maintenance of a mitotic state, whereas physiological amounts
are. As this phenotype was mimicked by N-cadherin (CBR-), we favor the
interpretation that N-cadherin-mediated adhesion rather than
N-cadherin-mediated signaling is responsible both for cell translocation and
differentiation. This is further strengthened by the observations that
N-cadherin delivered to the central DM sheet, which is normally devoid of
Myod, was not sufficient to upregulate Myod in the epithelium (Y.C.,
unpublished), and that the N-cadherin-overexpressing cells entered the myotome
as Pax3/Pax7-positive progenitors, which generated fibers only 24 hours after
transfection into the DM sheet. Therefore, N-cadherin is likely to indirectly
affect muscle-specific gene transcription and myogenesis by mediating
homophilic cell interactions that enable cell translocation into an
appropriate environment. Our results are consistent with the notion proposed
on the basis of in vitro paradigms, that local cell-cell interactions are
required for myogenesis (Cossu et al.,
1995; Gurdon et al.,
1993), and that N-cadherin is capable of mediating such
interactions by inducing first the assembly of adherens-type junctions and
then the expression of muscle-specific markers
(Goichberg and Geiger, 1998;
Holt et al., 1994). Also, in
vitro, activation of N-cadherin was found to stimulate Rho GTPase activity,
which in turn activates the serum response factor that enhances
muscle-specific transcription and promotes myogenesis (reviewed by
Krauss et al., 2005). This
molecular cascade couples changes in cell adhesion and morphology with
patterns of gene expression and cell differentiation.
|
ACKNOWLEDGMENTS |
|---|
-GFP and
N-cadherin (CBR-)-GFP. We also thank J. Yisraeli for critical reading of the
manuscript. We are indebted to M. Takeichi for the N-cadherin DNAs. This work
was supported by grants from the Israel Science Foundation (ISF), the EEU 6th
Framework program Network of Excellence MYORES, the March of Dimes, ICRF and
DFG (SFB 488) to C.K. |
Footnotes |
|---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/6/1101/DC1
|
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