Department of Anatomy and Cell Biology, Hebrew University-Hadassah Medical School, Jerusalem 91120-PO Box 12272, Israel
* Author for correspondence (e-mail: kalcheim{at}nn-shum.cc.huji.ac.il)
Accepted 8 December 2004
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SUMMARY |
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Key words: Avian embryo, Cell dissociation, Desmin, Epithelial-mesenchymal conversion, Myotome, PAX3, PAX7, Satellite cells, Somite
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Introduction |
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The DM consists of a central epithelial sheet and four contiguous curved
edges: the dorsomedial and ventrolateral lips (DML and VLL), which are in
apposition to the neural tube and intermediate/lateral plate mesoderm,
respectively; and the rostral and caudal lips, abutting successive
intersomitic spaces. Lineage tracing of distinct regions within the DM
revealed that, following establishment of the initial myotomal scaffold formed
of pioneer cells (Kahane et al.,
1998a; Kahane et al.,
2002
), subsequent myofibers arise from progenitors residing in all
four lips of the DM (Kahane et al.,
1998b
; Kahane et al.,
2002
; Cinnamon et al.,
1999
; Cinnamon et al.,
2001
; Huang and Christ,
2000
; Venters et al.,
1999
; Gros et al.,
2004
) [see other references for a primary contribution by the DML
and VLL only (Denetclaw et al.,
1997
; Denetclaw and Ordahl,
2000
; Ordahl et al.,
2001
; Venters and Ordahl,
2002
)].
Because the precedent progenitors exit the cell cycle upon myotome
colonization, continuous growth and muscle differentiation are ensured by
subsequent cellular contributions. Starting at embryonic day (E) 2.5,
mitotically active progenitors enter the myotome while expressing the FGF
receptor FREK but not MyoD, Myf5 or FGF4
(Marcelle et al., 1995;
Sechrist and Marcelle, 1996
;
Kahane et al., 2001
), yet they
do not undergo muscle differentiation until E6
(Kahane et al., 2001
). Using a
combination of iontophoretic injections of CM-DiI with BrdU labeling, we found
that they originate from both rostral and caudal DM edges at a time when these
lips still supply myofibers to the myotome and the DM is still fully
epithelial (Kahane et al.,
2001
). Notably, within 2 days from their first appearance, these
mitotically competent progenitors become the predominant myotomal population
(85% of total nuclei approximately), suggesting that they might also arise
from additional, yet unexplored, sources.
The central portion of the DM in between the marginal lips has been
classically referred to as dermatome, as it contributes to the dermis of the
back (Brent and Tabin, 2002,
Scaal and Christ, 2004
). By
E3.5 in the avian embryo, this central sheet begins dissociating, leaving the
epithelial lips intact for a few days yet. We found that the resulting dorsal
dermis derives from dissociating progenitors residing along the entire medial
to lateral extent of epithelial somites. As these project onto corresponding
regions of the DM sheet, the developing dermis is generated in a regionally
restricted fashion (Ben-Yair et al.,
2003
). These results contrast with previous findings suggesting
that the entire dorsal dermis derives from medial somitic halves exclusively
(Olivera-Martinez et al.,
2000
). Altogether, lineage tracing studies and direct measurements
of growth patterns led us to conclude that the dorsal somite, the DM and the
resulting myotome and dermis expand by a coherent and proportional mechanism
whereby all derivatives exhibit a direct topographical relationship with their
predecessors (Kahane et al.,
2002
; Ben-Yair et al.,
2003
). A direct spatial relationship between myocytes in the
myotome and their progenitors in the DM was also observed using the LaacZ
method of clonal analysis in mouse embryos
(Eloy-Trinquet and Nicolas,
2002
).
In addition, available fate mapping results suggest that dermal and muscle
fates are spatially segregated to distinct regions of the DM, with muscle
progenitors arising from the DM edges and dermis from the central DM sheet. In
the present study we analyzed the fate of the central DM and found that, in
addition to dermis, it also furnished cells that colonized the underlying
myotome. Further characterization revealed that these cells are proliferative
myotome progenitors (PMPs) that maintain expression of PAX7 and PAX3, two DM
markers that are downregulated in the nascent dermis. These cells also express
FREK, similar to previously characterized proliferative progenitors emanating
from the extreme DM lips. Hence, the mitotically competent wave of myotome
colonization arises sequentially from two sources, first from the rostral and
caudal DM lips (Kahane et al.,
2001) and slightly later from the dissociating DM sheet.
Our finding that the central DM develops into PMPs as well as dermis raised the question whether this epithelium is composed of differentially specified progenitors, or alternatively, whether both cell types arise from single DM cells. To resolve this question, we performed clonal analysis in ovo. We found that single DM precursors give rise to both derivatives. We also identified the presence of intermediate progenitor types, suggesting that fate segregation occurs progressively in association with a sequential conversion of the DM from epithelium into mesenchyme. Notably, these events are associated with a change in the plane of cell division, which, in the young epithelium, is parallel to the apico-basal orientation of the cells and then, starting prior to dissociation, progressively shifts to become mostly perpendicular to their apico-basal axis. Our results support the notion that the DM sheet is composed of (at least) bipotent progenitors able to give rise both to myogenic and dermogenic lineages.
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Materials and methods |
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CM-DiI labeling combined with Pax7 immunostaining
The central region of flank-level DMs of stage 16/17 embryos was labeled
midway between adjacent intersomitic clefts with CM-DiI as previously
described (Kahane et al.,
1998a; Cinnamon et al.,
2001
). Embryos were re-incubated until E4 (stage 20), fixed in 4%
formaldehyde and processed for cryostat sectioning. Sections were then
counterstained with PAX7 antibodies as described below.
Electroporation
An expression construct encoding an enhanced version of GFP, the pCAGGS-AFP
(4 µg/µl) (Momose et al.,
1999) was microinjected into flank-level DMs of HH16/17 quail
embryos. The central sheet of a single DM was microinjected with a minimal
amount of DNA using a micropipette mounted on an FST MM-33 micromanipulator
and positioned approximately 45° to the longitudinal axis of the embryo.
For electroporation, the negative L-shape tungsten electrode was placed
underneath the blastoderm with the tip just ventral to the medial part of the
DM and the positive electrode was located in a dorsal position with respect to
the DM. A square wave electroporator (BTX, San Diego, CA) was used to deliver
a single pulse of current at 20 volts, 10 milliseconds long. Embryos were
re-incubated for 6 hours to monitor localization of fluorescent protein
following initial expression of the transgene and then re-incubated until E5.
Embryos were fixed in 4% formaldehyde, processed for paraffin wax embedding
and sectioning and then immunolabeled with antibodies to GFP and desmin in
combination with in-situ hybridization for chick-specific Dermo1
(Scaal et al., 2001
).
Transfection of GFP-encoding DNA into single DM progenitors
The GFP-encoding construct was microinjected unilaterally into the central
DM sheet of stage 18 embryos. Six consecutive flank-level DMs were injected
per embryo. Injection was carried out using a micromanipulator-mounted
micropipette as described above. Micropipettes had a tip diameter ranging
between 8 and 10 µm. The epithelium of each DM was pierced once through the
ectoderm with the micropipette and a minimal volume of DNA mixed with Fast
Green was pressure injected. GFP fluorescence became evident as early as 2
hours after transfection. However, in order to ensure robust and uniform
levels of transgene expression, the re-incubation period for the
calibration\control series was 6 hours. Additional embryos were grown for 24
or 48 hours, fixed in 4% formaldehyde, processed for paraffin-wax embedding
and immunolabeled with antibodies to GFP and desmin.
Calibration series
To achieve transfections of single DM cells, different construct
concentrations were examined by counting the number of GFP-expressing cells in
serial sections of treated embryos 6 hours following transfection. An optimal
DNA concentration ranging between 0.05 and 0.1 µg/µl was determined,
which produced an acceptable success rate (see below).
Control series
Once an optimal DNA concentration was established, 46 embryos were injected
as described above. In these experiments, six consecutive DMs were injected on
both sides of each embryo. Serial section analysis revealed the presence of 33
GFP-expressing cells per 552 injected DMs. In 29/31 successful injections, a
single GFP-positive cell was identified per DM 6 hours after injection. In
2/31 such injections, two GFP-expressing cells per DM were detected. The
percent of successful injections resulting in a single GFP-expressing cell is,
therefore, assessed to be higher than 90% at the specified DNA
concentration.
Experimental series
Thirty eight and 41 successful transfections resulting from injection of
600 and 498 DMs in 100 and 83 embryos, were analyzed in serial transverse
sections 24 and 48 hours after treatment, respectively. The clonal progeny of
single transfected cells was classified according to location in either the
myotome, the intermediate domain (ID) and/or the dermal layer, based on
position relative to the desmin-immunostained myotome and on stage-specific
landmarks. The latter included the compact PAX7-positive ID at 24 hours, and
the PAX7-negative loose dermal mesenchyme at 48 hours (see Results).
Successful cases were classified as producing exclusively dermal cells,
myotomal precursors, ID cells or any combination of the three derivatives.
Segments containing labeled cells in either sclerotome or ectoderm were not
considered.
BrdU incorporation
BrdU (50 µl of a 10 mmol/l solution; Sigma) was applied for 1 hour to E4
embryos, followed by fixation in 4% formaldehyde and processing for cryostat
sectioning. Embryos were sectioned at 10 µm, and immunolabeled with
anti-BrdU and anti-PAX7 antibodies as described below.
Embryo processing and sectioning
Embryos were fixed with either 4% formaldehyde or Fornoy (the latter for
in-situ hybridization on sections) and processed for paraffin-wax embedding as
previously described (Cinnamon et al.,
1999). Serial 10 µm transverse sections were mounted on
Superfrost/Plus slides. For immunohistochemistry with PAX7 and PAX3
antibodies, embryos were processed for cryosectioning as previously described
(Ben-Yair et al., 2003
).
In-situ hybridization
Embryos were subjected to in-situ hybridization on sections
(Kahane et al., 2001) with
avian-specific probes for Frek
(Marcelle et al., 1994
),
Dermo1 (Scaal et al.,
2001
), Pax7 and Pax3 (gifts from Peter Gruss).
While the original antisense PAX3 probe was prepared by BamH1
digestion at the 5' end of the insert, a new, shorter probe containing
100 bp upstream of the original sequence was prepared by PvuII
digestion, ensuring no PAX7 was recognized. Hybridized sections were further
subjected to immunohistochemistry with anti-desmin, anti-PAX 7 or anti-GFP
antibodies.
Immunohistochemistry
Immunostaining was carried out with polyclonal and monoclonal antibodies to
desmin (ICN, 1:100 and Sigma, 1:20, respectively), to GFP (Molecular Probes,
1:200), polyclonal anti-BrdU antibodies (Abcam, 1:150), and monoclonal
antibodies to PAX3 and PAX7 (Hybridoma Bank, 1:10). Specificity of the latter
antibodies to their respective antigens was confirmed by Western blot analysis
(not shown). Secondary antibodies coupled either to FITC, Rhodamine or
horseradish peroxidase were used.
Determination of the plane of cell division
Embryos at HH16/17, 18 and 20 were fixed in 4% formaldehyde. Whole embryos
were immunolabeled with -tubulin antibodies (Sigma, T3559, diluted
1:500) to label the centrosomes and further processed for paraffin-wax
embedding. Serial 10 µm transverse sections were then subjected to Hoechst
nuclear staining. Individual mitoses were identified and respective
-tubulin and Hoechst stainings were separately photographed with a
magnification objective of x100 using an Olympus DP70 digital camera.
Since the two centrosomes belonging to individual mitoses were sometimes found
at slightly different focal depths, each mitosis was documented by capturing
2-3 consecutive images. The angles of the mitotic spindles relative to the
mediolateral plane of the DM epithelium were measured from the combined
(
-tubulin +Hoechst) images using Adobe Photoshop 7.
Statistical analysis
Significance of the results was determined by the 2 test
(P<0.0001). The power of the test was calculated using the SAS
Macro for sample size analysis.
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Results |
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Next, we asked whether these PAX7-positive cells are mitotically active. To this end, E4 embryos were pulsed with Brdu for 1 hour, and fixed and processed for immunostaining with antibodies to Brdu and PAX7. As shown in Fig. 2F-I, double-labeled cells were found throughout the myotome (n=4). As expected, only a subset of PAX7-expressing cells co-expressed BrdU, as only a fraction of the proliferative cells in the myotome are encountered in the S-phase of the cycle during a 1 hour period. These results demonstrate that the myotomal progenitors of central DM origin that express PAX7 are mitotically active.
Since the DM is characterized by expression of both PAX7 and PAX3, we
determined whether these myotomal progenitors also retained PAX3.
Fig. 3A shows the presence of
PAX3-positive cells in the myotome (white arrowheads). These cells do not
overlap with the desmin-expressing myofibers, suggesting they belong to the
mitotically active category. In previous studies, proliferative progenitors in
the myotome were shown to express FREK
(Kahane et al., 2001;
Marcelle et al., 1995
;
Sechrist and Marcelle, 1996
).
We then examined whether PAX7-expressing myotomal progenitors co-express FREK.
Virtually all PAX7-positive cells also expressed frek mRNA
(Fig. 3B). Such double-labeled
cells were detected throughout the myotome, suggesting that all proliferative
progenitors, whether derived from the rostral and caudal lips as originally
demonstrated (Kahane et al.,
2001
) or later from the DM sheet
(Fig. 2), express both markers.
Hence, the central DM epithelium produces mitotic cells that colonize the
myotome and co-express PAX7 and FREK. Because the desmin-negative myotomal
cells also express PAX3, the data suggest that the PMPs co-express all three
markers.
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GFP-encoding DNA was introduced into single DM progenitors as described in the Materials and methods. Of 276 somites similarly injected (time zero controls), labeled cells were found in only 31 cases (11%). Of these 31 somites, in 29 cases a single GFP-expressing epithelial progenitor was detected 6 hours following transfection, the earliest time point in which a strong and unambiguous GFP signal became evident (Fig. 5A). In the two remaining somites, two adjacent epithelial cells were found that might have resulted from a double transfection event, or alternatively from a single transfected cell that divided within the 6 hour interval until fixation. These results show that, although being highly ineffective, the conditions we used are suitable for labeling single progenitor cells.
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Analysis of the composition of single clones at 24 hours post-transfection revealed that in 38.2% of cases, the progeny of a single injected cell were still confined to the DM-derived ID (Fig. 6C,E; see Table 1 in the supplementary material). One day later, no labeled cells could be detected any longer in only the ID. Instead, in 48.8% of injected somites, the progeny of labeled founders was found in both the PMP and the dermis (Fig. 5E1-E5; Fig. 6D,E; see Table 1 in the supplementary material). In 50% of the latter clones, labeled progeny were also detected in the residual ID, but for simplicity this triple clonal combination was pooled along with the PMP+D category. These results demonstrate that the DM contains single cells that give rise to both myogenic as well as dermogenic lineages. Since the proportion of clones containing labeled progeny in the ID (ID alone, PMP+ID and dermis+ID) at 24 hours is significantly reduced a day later (79.3% compared with 19.5%, respectively; Fig. 6E, P<0.0001) we infer that the mixed clones containing both PMP and dermis are likely to derive from the disappearing ID population between 24 and 48 hours post-transfection (E4 and E5, respectively).
Along with mixed PMP+dermis clones, others containing labeled cells in the myotome or in dermis exclusively (21.9 and 9.7%, respectively at E5) (Fig. 5F,G; and Fig. 6E) were also observed. These cells might have originated from a population of early-specified progenitors that coexist along with at least `bipotent precursors' in the DM. Alternatively, they might have derived from similar bipotent progenitors that were nevertheless stimulated by local cues to express only one of their potential fates (see Discussion). In 19.5% of total cases (14% approximately of total cells at E5), we detected the presence of mixed clones containing cells in either the PMP+ID or dermis+ID (Fig. 6E). Whereas cells in PMP and dermis are likely to further propagate their own lineages, cells in the ID could give rise either to PMPs, to dermis or to both phenotypes. The latter possibility seems the most likely one based on the observation that the disappearance of the ID is compensated for by appearance of mixed PMP+dermis clones between E4 and E5. Analysis for longer time periods would be required to follow the final fates of ID progenitors still remaining at E5. This is, however, complicated by the transient lifespan of the GFP transgene, which we found to be strongly transcribed for 48 hours and then to progressively decline.
Clonal analysis clearly reveals the existence of bipotent DM cells fated to become both PMPs and dermis. Furthermore, it also uncovers the presence and dynamics of intermediate progenitors along these lineages (PMP+ID, dermis+ID, ID only), and of clones with single phenotypes (see Fig. 9).
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Discussion |
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Regional fate map of DM derivatives
Three main derivatives directly arise from the DM: postmitotic
mononucleated myofibers, mitotically competent PMPs and the dorsal dermis. The
present findings show that the central portion of the DM gives rise both to
PMPs and to dermis, further complementing previous data concerning fates of
the DM lips. An outline of derivatives of the various DM regions is
illustrated in Fig. 8. Using
DiI labeling and pulse-chase experiments with thymidine, we reported that
myofibers originate from all four lips of the overlying DM
(Kahane et al., 1998b;
Kahane et al., 2002
;
Cinnamon et al., 1999
;
Cinnamon et al., 2001
). The
origin of myofibers from the four edges was further confirmed using the quail
marker technique and GFP electroporation
(Huang and Christ, 2000
;
Gros et al., 2004
).
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The question remained of the fate of the central DM sheet. Contrary to
previous findings (Olivera-Martinez et
al., 2000), we observed that the entire mediolateral aspect of the
DM (and of the earlier epithelial somite), contributed to the dorsal dermis,
with dermal cells demonstrating a direct spatial relationship vis-à-vis
their epithelial predecessors (Ben-Yair et
al., 2003
). This is consistent with the dorsal dermis developing
when the whole DM is still epaxially located before its hypaxial domain
expands into the somatopleura (Christ et
al., 1983
). Surprisingly, in initial studies we observed that
labeling the central domain of the DM with CM-DiI also produced cells that
colonize the myotome but are mesenchymal rather than fibers
(Ben-Yair et al., 2003
). The
timing of myotome colonization by these progenitors (from E3.5) and their
characterization as mitotically active, PAX3/7/FREK-positive cells (see
Results), led us to conclude that epithelial progenitors from the non-lip
regions of the DM are late contributors of the third wave category of
mitotically active progenitors, which initially invade the myotome from the
rostral and caudal lips (Kahane et al.,
2001
). Hence, by the time of dissociation, the DM epithelium
produces mesenchymal cells that sort out in two opposed directions; both
superficially to colonize the subectodermal space and give rise to dermis, and
also toward the myotome to further contribute to its growth
(Fig. 8). Thus, the immediate
fates of DM cells appear to be regionalized, with myofiber generation limited
to the DM lips, PMP production to the rostral and caudal lips followed by the
DM sheet, and dermis exclusively from the DM sheet. Although dermis is likely
to be a major derivative, a subset of DM progenitors at flank levels of the
axis also contributes to the scapula blade
(Huang et al., 2000a
), though
the precise location of these precursors in the DM remains to be mapped, and
to endothelial cells (Huang et al.,
2000b
). Likewise, the final fate of the PMPs may be varied.
Although most progenitors are likely to withdraw from the cell cycle at
progressive stages to undergo myogenesis, some may also contribute to muscle
fibroblasts and others may remain as undifferentiated muscle satellite cells.
A limitation of the present study is that due to the transient lifespan of
transfected GFP, daughter cells cannot be followed for long enough to
determine their final fates.
Our study showing a unique behavior of the DM sheet would suggest that this
area follows a developmental molecular program that is, at least partly,
different from that of the DM lips. Consistent with such a possibility is the
expression of several transcription factors such as EN1 and
SIM1 to different mediolateral subdomains of the central DM sheet in
both avian and mouse embryos (Cheng et al.,
2004; Sporle,
2001
). Notably, following DM dissociation, these markers remain
expressed in both myotomal and dermal domains at corresponding mediolateral
locations (Cheng et al., 2004
),
consistent with results of our lineage studies (this paper) (see also
Ben-Yair et al., 2003
). Another
gene that characterizes the central DM region is Alx4, which later
becomes restricted to the dermis (Cheng et
al., 2004
). Reciprocally, we show here that PAX 3 and 7, initially
widespread throughout the entire avian DM, get restricted to the PMPs in the
myotome and disappear from the nascent dermis. Several studies also identified
genes specifically localized to a central region of the myotome. In mice,
Myf5, Fzd9 and connexin 40 are transiently expressed in this
subdomain [Sporle (Sporle,
2001
) and references therein]. Regulatory sequences of the
Myf5 gene directing expression of nlacZ were detected in an
intercalated subregion of the myotome in transgenic mice
(Hadchouel et al., 2000
;
Hadchouel et al., 2003
).
Xmyf5 was detected in the presumptive DM of Xenopus somites
(Grimaldi et al., 2004
).
Altogether, these results would suggest the existence of myogenic regions in
the DM proper other than the lips. It remains to be clarified whether the
various myotomal markers are expressed in myofibers, in PMPs or
indistinguishably in both cell types.
Lineage segregation of the DM
As the DM is a structure that produces several derivatives, one of the
critical questions related its development concerns the timing and mechanisms
of segregation of the different lineages from the primary pseudostratified
epithelium. In-vivo lineage tracing permits the fate of individual progenitors
to be assessed under normal conditions because the labeled cells remain
subjected to the instructive/selective environments of the embryo. Given that
DM fates are regionally restricted (see previous section), and that specific
DM domains generate more than one cell type, the question of the state of
specification of individual progenitors is an important one. For instance,
rostral and caudal lips of the DM contain heterogeneous subsets of
MyoD-positive and -negative cells, which correlate with their outcome as
myofibers versus PMPs, respectively
(Kahane et al., 2001).
Although not yet formally demonstrated, these data would suggest an early fate
restriction among epithelial precursors residing in the extreme DM lips.
In this study, we focused our initial analysis on the central sheet of the
DM, which produces both PMPs and dermis. We uncovered the existence of
individual DM progenitors that generate both cell types, others that produce
exclusively PMPs or dermis, and intermediate stages containing combinations of
ID cells and PMPs or ID cells and dermis. Clearly, the different outcomes
could be temporally separated, with 80% of clones 1 day after labeling still
containing cells in the ID, with PMPs segregated slightly before dermis, and
clones containing both PMPs and dermis appearing between 1 and 2 days
post-transfection. These results stress a time-dependent generation of
restricted progeny from the early DM (Fig.
9). Such an outcome could either be explained as stochastic
differentiation of a homogenous population of initially multipotent cells or
by the presence of both multipotent and fate-restricted precursor cells in the
early DM. Our data generally favor the first mechanism, for several reasons.
First, expression of PAX3 and PAX7, as well as the weak transcription of
frek mRNA, are homogeneous in the DM sheet and DM-derived ID with no
apparent segregation to cell subsets, unlike that observed for MyoD in the
extreme DM lips (Kahane et al.,
2001). Second, all mixed clones containing both PMPs and dermis
become apparent only 48 hours post-transfection
(Fig. 9) but not a day earlier.
This strengthens the notion that even in the ID, a significant proportion of
progenitors still remain bipotent. Third, 12 hours after transfection we
already observed clones containing cells in ID+PMPs at a time that precedes
formation of a mesenchymal dermis (R.B.-Y. and C.K., unpublished); the time
gap between these two cell types is further emphasized by the higher
proportion of clones containing PMPs compared with dermis at 24 hours. This
earlier segregation of PMPs does not necessarily need to result from the
existence in the DM epithelium of distinct progenitors. Instead, it is likely
to occur because the well-developed myotome, which exists prior to the initial
dermis, stimulates or at least permits the entry of stem-like cells that
differentiate further to myotome colonization. Fourth, the orientation of cell
divisions in the DM is mostly parallel to the mediolateral plane of the
epithelium, consistent with a distribution of the resulting progeny along the
direction of DM growth. This favors the notion that the central DM is composed
of a homogeneous population of self-renewing epithelial progenitors (see next
section) rather than a mixture of cells with different states of
commitment.
The existence of single progenitors that generate more than one cell type
has recently been documented in a specific somite domain using a retroviral
approach (Kardon et al.,
2002). In this study, 26% of clones derived from the lateral
portion of hindlimb-level somites of chick embryos were shown to contain both
muscle cells and endothelial cells that colonized the limb. Furthermore,
within the muscle lineage, they were not restricted in their ability to
develop into fast versus slow fibers, or to populate specific muscles in the
limb. Likewise, the mitotically active PAX7/3-positive PMPs that we identified
in the myotome might not necessarily be restricted to give rise to myofibers
and could also generate endothelial cells, muscle satellite cells etc. An
additional similarity between our study and that of Kardon et al.
(Kardon et al., 2002
) is that
both revealed the existence of clones containing single cell types along with
clones containing mixed progeny. In both studies, the presence of mixed clones
was, in general, positively associated with clonal size, in further support of
a stochastic model of lineage segregation.
A shift in the plane of cell division precedes fate segregation of individual ID progenitors
In the young pseudostratified DM epithelium, mitoses occur at the apical
pole (Fig. 7) and DNA synthesis
in the basal half of the epithelium
(Ben-Yair et al., 2003). At
this stage, the orientation of the vast majority of cell divisions is parallel
to the plane of the epithelium. We observed that in the mature DM, a
progressive shift in the plane of cell division becomes apparent; and with
initial dissociation of the DM into the aggregated ID, the orientation of
71.7% of the cells changes to a perpendicular direction. This value resembles
the proportion of mixed clones obtained at E5 (68.3%). These results raise the
intriguing question of the significance of this shift to lineage segregation
of DM cells into its derivatives. In the nervous system, it has been
hypothesized that parallel divisions serve to expand the progenitor pool,
whereas the perpendicular type of mitoses produce two different daughter
cells, one that remains in the ventricular zone as a progenitor and one that
migrates away to differentiate (e.g.
Kornac and Rakic, 1995
;
Chenn and McConnell, 1995
;
Doe, 1996
). Along the same
line, we propose that the early DM contains a pool of self-renewing
progenitors (Fig. 9) that, by
dividing in a parallel orientation, contribute to mediolateral expansion of
the DM, a process found to occur uniformly along this structure
(Ben-Yair et al., 2003
). Also
similar to the neuroepithelium, we find that the orientation of cell divisions
changes in the DM prior to dissociating into PMPs and dermis. Unlike the
neuroepithelium, however, the central portion of the DM dissociates in its
entirety, giving rise to the aggregated ID, which in itself lasts for 1.5 days
approximately. A subset of ID cells are also likely to self-renew because
progeny in this domain are found at both 24 and 48 hours following
transfection. Nevertheless, at this stage, a major outcome of the ID is the
production of mixed clones containing both PMPs and dermis. By analogy to the
neuroepithelium, it is tempting to speculate that this fate segregation
results from the change in orientation of cell divisions. Direct demonstration
that this is the case is still lacking due to the technical difficulty of
tracing in ovo the final fates of individual cells that specifically divide
perpendicular to the epithelium.
Supportive evidence for such a mechanism could potentially derive from
asymmetric allocation of cell-fate-determinants to one of two daughter cells
dividing in the perpendicular orientation, as demonstrated in invertebrates
(e.g. Rhyu et al., 1994;
Knoblich, 2001
;
Lu et al., 2000
). NUMB is one
such protein shown to inhibit NOTCH signaling and also to divide
asymmetrically in the nervous system of vertebrates
(Chenn and McConnell, 1995
;
Zhong et al., 1996
;
Zhong et al., 1997
;
Cayouette et al., 2001
;
Cayouette and Raff, 2003
;
Wakamatsu et al., 1999
).
However, we have not observed any asymmetric allocation of NUMB to the central
DM sheet or to ID cells (R.B.-Y. and C.K., unpublished).
The integrity of intercellular junctions could also play a role in
maintaining the orientation of mitoses. N-cadherin is expressed in the
epithelial DM with adherens junctions located apically
(Duband et al., 1987;
Duband et al., 1988
). The
observed shift in mitotic orientation could be secondary to, and perhaps also
result from, a loss of N-cadherin-associated signaling that occurs upon
dissociation of the epithelium. Consistent with such a possibility, the
stereotypic orientation of mitoses in the zebrafish neuroepithelium was found
to be partially perturbed by loss of function of specific junctional
components (Geldmacher-Voss et al.,
2003
). Factors emanating from the adjacent ectoderm or myotome
could also influence the observed shift in planes of cell divisions in the ID.
Consistently, the pigmented epithelium was reported to control the plane of
cell division in the adjacent retina
(Cayouette et al., 2001
). In
spite of environmental modulation, it appears that the orientation of cell
divisions is intrinsic to the epithelium, as cultured dissociated cells keep
their original orientations ex vivo
(Cayouette et al., 2001
;
Estvill-Torrus et al., 2002
).
It will be interesting to elucidate whether similar general mechanisms operate
during DM ontogeny and to determine their precise molecular identity.
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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/132/4/689/DC1
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