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 9 June 2003
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SUMMARY |
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Key words: Avian embryo, Cell delamination, Dermis, Dermomyotome, Desmin, Epithelial-mesenchymal conversion, Myotome, Somite
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INTRODUCTION |
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A wealth of information is available on early patterning of somite
derivatives that involves opposite and mutually inhibitory gradients of factor
activity (reviewed by Brent and Tabin,
2002). Yet, the mechanisms by which these basic gradients are
translated into morphogenetic processes remain incompletely understood. Ordahl
and colleagues (Denetclaw et al.,
1997
; Denetclaw and Ordahl,
2000
) have proposed that growth of both the myotome and DM is
driven by two stem cell systems restricted to the dorsomedial and
ventrolateral lips (DML and VLL, respectively) of the DM. The DML was
suggested to drive medial growth of the myotome and the VLL to be responsible
for expansion in a ventral direction
(Denetclaw et al., 1997
;
Denetclaw and Ordahl, 2000
;
Ordahl et al., 2001
).
Consequently, both myotome and DM expand incrementally in opposite medial and
lateral directions, with the youngest cells approaching the medial and lateral
extremes and a central domain which would correspond to the oldest fibers.
This latter intervening space was suggested to remain quiescent in terms of
cell growth and contribution to the myotome
(Denetclaw and Ordahl, 2000
).
At variance with this view, we have found that the myotome grows evenly in the
mediolateral orientation owing to progressive and simultaneous intercalation
of myoblasts from all four lips of the overlying DM among pre-existing pioneer
myofibers (Kahane et al.,
1998a
; Kahane et al.,
1998b
; Kahane et al.,
2001
; Cinnamon et al.,
1999
; Cinnamon et al.,
2001
). Hence, both young and old fibers evenly spread all along
the mediolateral extent and the growth of the myotome follows an overall
uniform pattern (Kahane et al.,
2002
). Altogether, these findings demonstrated that progenitor
cells for muscle growth are not restricted to the DML and VLL but also prevail
along the entire mediolateral extent of both rostral and caudal DM lips. The
origin of the myotome from all four DM lips was now further substantiated
(Huang and Christ, 2000
;
Venters et al., 1999
), and the
existence of intercalatory mechanisms accounting for coherent myotome growth
was independently shown in the mouse embryo using the LaacZ method
for clonal labeling (Eloy-Trinquet and
Nicolas, 2002
).
Based on our results inferring a coherent and uniform mode of overall
myotome expansion, we asked whether development of the DM epithelium follows a
similar pattern, or is, alternatively, controlled by restricted pools of stem
cells driving directional growth. Such a question was also relevant for the
formation of the DM-derived dermis, which was suggested, based on grafting of
half-somites from quail donors into chick hosts, to arise entirely from the
medial half of the somite
(Olivera-Martinez et al.,
2000). To this end, we studied the cellular events that govern DM
development from epithelial somites and the regional origin of the dermis.
Measurements of cell proliferation, nuclear density and cellular
rearrangements revealed that the developing DM can be subdivided in the
transverse plane into three distinct and dynamic regions: medial, central and
lateral, rather than simply into epaxial and hypaxial domains. To understand
how these temporally and spatially restricted changes affect overall DM
growth, lineage tracing with CM-DiI was performed. A proportional pattern of
growth was measured along the entire DM. These results suggest a model of
coherent mediolateral growth of the DM as opposed to a stem cell view implying
focal and inversely oriented sources of growth at the medial and lateral
edges. In line with the uniform mediolateral growth of the dorsal epithelial
somite and the DM, lineage tracing experiments showed that the dorsal dermis
derives from progenitors residing along the entire medial to lateral extent of
epithelial somites which contribute to dermis in a regionally restricted
fashion. Taken together, our results support the view that all derivatives of
the dorsal somite exhibit a direct topographical relationship with their
ascendants.
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MATERIALS AND METHODS |
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Quantification of DM cell proliferation, nuclear density and nuclear
layers
Embryos at stages T0, T1 and T2 received a single pulse of 10 mM BrdU and
were reincubated for 1 hour, fixed in 4% formaldehyde and processed for
paraffin wax embedding. Serial sections (5 µm) were prepared, immunolabeled
with a BrdU-specific antibody and counterstained with Hoechst to reveal total
nuclei (Burstyn-Cohen and Kalcheim,
2002). Cell proliferation was measured as the proportion of
BrdU-positive of total nuclei, the number of nuclear layers per thickness of
the pseudostratified epithelium was counted and the actual thickness of the DM
was directly measured and presented as arbitrary units. All measurements were
performed along the entire mediolateral extent of the dorsal epithelial somite
(at T0) or the DM (for T1 and T2) and were conducted in the central domain of
each segment midway between the rostral and caudal edges. To this end, the
dorsal epithelium was subdivided mediolaterally into equal fields, 625
µm2 each. The entire mediolateral extent of the dorsal
epithelium at T0 was included into three such fields, and the DM at T1 and T2
comprised five and ten contiguous microscopic fields, respectively. To compare
between stages, the whole mediolateral length of the epithelium was normalized
to 100%, hence 0% represents the medialmost point in the epithelium and 100%
the lateral edge. For each parameter, 10-20 alternate transverse sections were
measured in two somites per embryo. Results represent the mean±s.d. of
three or four independent embryos.
CM-DiI labeling
The medial, central and/or lateral aspects of dorsal epithelial somites or
DM were labeled midway between adjacent intersomitic clefts with CM-DiI or
with a combination of DiI and DiO as previously described
(Kahane et al., 1998a;
Cinnamon et al., 2001
).
Experiments were performed in interlimb-level segments of embryos aged T0 or
T1. Embryos were reincubated until T2 or T3, fixed in 4% formaldehyde and
processed either for paraffin-wax embedding (CM-DiI-treated embryos) or
cryostat sectioning (DiI/DiO) as previously described
(Cinnamon et al., 1999
). CM-DiI
labeling of the sclerotome was performed at T1 after initial dissociation of
the somite. To avoid labeling of the DM, the latter was separated
microsurgically from the sclerotome, lifted and pulled temporarily aside.
Selected sections were counterstained with desmin antibodies
(Cinnamon et al., 2001
) to
delineate the myotome.
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 epithelial somites of 25
somite-stage quail embryos. The lateral domain of two or three successive
segments was microinjected using a micropipette positioned parallel to the
longitudinal axis of the embryo. To electroporate the dorsolateral half of the
epithelium, the negative L-shape tungsten electrode was placed underneath the
blastoderm with the tip just ventral to the medial part of somites and the
positive electrode was located in a dorsolateral position with respect to the
somites. A square wave electroporator (BTX, San Diego, CA) was used to deliver
four pulses of current at 20 V, 20 mseconds each. Embryos were reincubated for
7-8 hours to monitor localization of fluorescent protein following initial
expression of the transgene and then reincubated till E4. Embryos were fixed
in 4% formaldehyde, processed for paraffin wax embedding, sectioned at 10
µm, and immunolabeled with anti GFP
(Burstyn-Cohen and Kalcheim,
2002
) and desmin antibodies.
Laser-scanning confocal microscopy
Laser-scanning confocal microscopy and digital imaging were performed as
already described (Cinnamon et al.,
2001). Frozen sections (50 µm) of embryos whose somites were
double-labeled with DiI and DiO were scanned at 2 µm increments through the
z-axis and sequential images were collected. The confocal images represent
cumulative scans of single sections that include, in each case, all
fluorescently stained cells.
Measurement of DM growth by DiI labeling
The localization of dye-labeled cells relative to the mediolateral aspect
of the epithelium was measured in epithelial somites (T0) of living embryos
shortly after injection. Embryos were further incubated until T2, fixed and
sectioned, and the position of dye-labeled cells measured in consecutive
serial sections. In each case, the localization of the medial and lateral
borders of the dye-labeled domain was monitored. The midpoint between the two
values was calculated and expressed as a function of total mediolateral length
of the corresponding segment. The localization of the midpoint values at T2
relative to T0 was plotted for each injected somite (see
Fig. 5).
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RESULTS |
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Spatially and temporally distinct morphogenetic traits characterize
development of the DM
Cell proliferation in the dorsal somite and developing DM
The dorsal aspect of the epithelial somite which corresponds to the
prospective DM displays a homogeneous pattern of cell proliferation along its
entire mediolateral extent with 45-50% of total nuclei having incorporated
BrdU following a 1 hour pulse (Fig.
2A,B,E, upper panel). Notably, in the medial aspect of the somite
(Fig. 2A,B, asterisk) a subset
of BrdU-negative nuclei is apparent that corresponds to the earliest
post-mitotic progenitors that will develop into pioneer myofibers, as
previously documented (Kahane et al.,
1998a). Changes in the even pattern of proliferation of dorsal
cells appeared upon somite dissociation. Quantification of the data revealed
that at T1, proliferation along the epithelium remained unchanged including
the newly formed DML. An exception to this behavior was the lateral fifth of
the DM where an average of 27% of nuclei in the S phase of the cell cycle was
measured. This local value further decreased at T2 to 25% in the lateralmost
epithelium and attained only 10% in the VLL itself
(Fig. 2C,D arrows, and E middle
and lower panels). Surprisingly, these low levels of cell proliferation
remained at least until E4.5, when the VLL had already entered the
somatopleura (not shown). A slightly decreased proportion of BrdU-positive
nuclei could also be detected at T2 in the center of the DM when compared with
earlier stages (Fig. 2E, lower
panel). Taken together, all precursors along the mediolateral DM actively
proliferate despite local quantitative differences. Hence, if considering cell
proliferation as a major factor driving epithelial growth, the expansion of
the DM in the mediolateral direction cannot be accounted for by regionalized
proliferative centers restricted to the extreme DML and VLL.
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These results show that morphogenetic changes occur everywhere along the growing DM. In the transverse plane, this reorganization defines three distinct and dynamic regions: medial, central and lateral, rather than the two recognized epaxial and hypaxial domains. Moreover, these changes occur with a distinctive timing, comprising two discrete and separable phases, a first phase from T0 to T1 when the lateral and central domains change their shape and proliferative behavior, and a second phase from T1 to T2 when the medial third flattens and extends.
The overall mediolateral growth pattern of the DM between T0 and T2
is proportional
To understand how the observed temporally and spatially restricted changes
in proliferation and cell rearrangements integrate to affect overall growth of
the DM, lineage tracing with CM-DiI was performed. Discrete injections were
made at T0 and embryos were reincubated till T2.
Fig. 4 shows three
representative segments that received the dye at medial, central and lateral
positions of the epithelial somite, respectively
(Fig. 4A,C,E). Notably, upon DM
development, the initial spots of dye-labeled cells spread due to both
proliferation and extension movements. Yet, medially directed labeling
remained medial in the DM (n=6,
Fig. 4A,B). Likewise, central
injections at T0 projected to a central site at T2 (n=7,
Fig. 4C,D), and lateral
injections projected onto lateral regions of the DM (n=6,
Fig. 4E,F), suggesting an
overall coherent pattern of DM growth between the two stages considered.
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The dorsal dermis derives both from medial as well as lateral somite
halves
Observation of transverse sections at stages corresponding to dermis
formation reveals that dermal cells first dissociate from the center of the DM
epithelium and then the process spreads both medially and laterally involving
most of the DM, except for the extreme DML and VLL, which remain epithelial
for a few additional days. Notably, initial establishment of the subectodermal
mesenchyme occurs while the DM epithelium is still largely confined to the
epaxial domain, except for the VLL tip which begins extending beyond the
ectodermal notch to enter the somatopleura
(Fig. 6A-C between arrowheads).
In addition, we have shown that the mediolateral extent of the dorsal
epithelial somite projects proportionally onto the DM (Figs
4 and
5). As a consequence of the two
latter observations, the dorsal dermis would be expected to arise from both
medial as well as lateral halves of the epithelial somite. Yet, based on
quail-chick grafts of half-somites of either medial or lateral type, it was
suggested that the dorsal dermis derives entirely from the medial but not the
lateral half of the somite
(Olivera-Martinez et al.,
2000). In light of this discrepancy, we reinvestigated the
question of the mediolateral origin of the somite-derived dermis.
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Dermal cells derived from the lateral half of the somite are included
within the Sim-1-expressing domain
The Sim-1 transcription factor was found to be sequentially expressed in
the lateral domains of the epithelial somite, then of the dermomyotome and
finally of the somite-derived dermis, respectively
(Pourquie et al., 1996;
Olivera-Martinez et al.,
2002
). To examine the topographical relationship between dermal
cells derived from the lateral region of the epithelial somite
vis-à-vis the Sim-1-positive domain of the dermis, the lateral aspect
of somites was labeled with CM-DiI at T0 and embryos were further incubated
till T3. The localization of DiI-positive cells in the dermis was analyzed
first in serial sections, which were subsequently subjected to in situ
hybridization as described under Materials and Methods. At T3, Sim-1
transcripts were localized to the lateral third of the dermis in addition to
the epithelial remnant of the lateral dermomyotome excluding the VLL itself
(Fig. 7E,F). At flank levels of
the axis, the lateral (or ventral) limit of Sim-1 expression in the dermis
corresponded to the ectodermal notch (large arrow), the limit between somite
and LPM-derived dermis (Olivera-Martinez
et al., 2002
; Huang and
Christ, 2000
; Nowicki et al.,
2003
). Lateral somitic cells labeled with CM-DiI at T0 developed
into dermal cells that were included within the Sim-1-positive territory
(Fig. 7G, thin arrows and 7F,
n=4). Moreover, we also observed CM-DiI-labeled dermal cells
localized medial to the Sim-1 domain (Fig.
7F,G arrowhead, and data not shown). The relative abundance of
such cells varied according to the mediolateral extent of labeling. These
results confirm that the lateral half of epithelial somites gives rise to
dermis whose lateralmost cells express the Sim-1 transcription factor. Taken
together with our fate mapping experiments revealing a coherent and regionally
restricted projection of lateral epithelial somite progenitors onto the
corresponding lateral DM and then onto lateral dermal cells, the sequential
lateral patterns of Sim-1 expression at corresponding stages indicates that
the Sim-1-expressing dermal cells are lineally related with their somitic
ascendants.
Origin of the dorsomedial mesenchyme
We next examined the origin of the dorsomedial mesenchyme present between
DML and dorsal neural tube. Recent data had suggested that this mesenchyme
derives from the DML and is, therefore, of a dermal nature
(Olivera-Martinez et al.,
2002). Labeling of the medial DM region including the DML at T1
with CM-DiI, gave rise at T3 to fluorescent cells in the myotome, in the
dorsal dermis lateral to the DML (arrows in
Fig. 8A) and in the DML itself,
but not in the dorsomedial mesenchyme (asterisk in
Fig. 8A), results which are at
variance with the previous suggestion. Therefore, we further explored this
question using quail-chick chimeras. Grafting dorsal halves of quail
epithelial somites into the equivalent place of chick counterparts, resulted
at E5 in the development of dorsal somite derivatives bearing the quail
marker; these included muscle, dorsal dermis and the residual DML. In
contrast, the dorsomedial mesenchyme (asterisk in
Fig. 8B) was of the host
(chick) type like the sclerotome, suggesting it derived from the latter. It
was only when whole chick epithelial somites were replaced by their quail
counterparts that the dorsomedial mesenchyme was of the quail type (data not
shown). These results suggest that the dorsomedial mesenchyme derives from the
sclerotome rather than from the DML. To directly examine this issue, CM-DiI
was directly applied to the nascent sclerotome as described under Material and
Methods. At E5, labeled cells were found both in the sclerotome as well as in
the dorsomedial mesenchyme (n=4,
Fig. 8C) confirming their
sclerotomal derivation.
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DISCUSSION |
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In contrast to thinning of the medial and lateral domains, a condensation
of epithelial cells is apparent in the central domain of the DM, which causes
a local thickening of the structure. As this area is the first to dissociate
into dermis, it is tempting to speculate that this process forecasts
subsequent cell mesenchymalization. Furthermore, at variance with previous
findings suggesting that the central domain of the DM is quiescent relative to
the extreme regions (Denetclaw and Ordahl,
2000), direct measurements of cell proliferation and lineage
tracing here presented show the dynamic nature of this epithelial subdomain.
Along the same line, it has been proposed that the myotome subjacent the
central region of the DM contains the oldest fibers and remains static; growth
being driven by incremental cell addition lateral and medial to this region
(Denetclaw and Ordahl, 2000
).
Our previous studies have documented instead, that the myotome underlying the
central DM region equally expands when compared with adjacent regions by
continuous addition of myofibers from the rostral and caudal lips of the DM
among pre-existing pioneers, hence containing both old and newly added cells
altogether (Kahane et al.,
2002
).
A coherent pattern of mediolateral development of all dorsal
derivatives of the somite
In previous studies, we have found that growth of the myotome in both
mediolateral and transverse planes is accounted for by addition of precursors
that emanate from all four lips of the overlying DM. The latter progressively
intercalate among the pioneer fibers, that originated earlier in the medial
epithelial somite (Kahane et al.,
1998a; Kahane et al.,
1998b
; Kahane et al.,
2001
; Cinnamon et al.,
1999
; Cinnamon et al.,
2001
). The integration between these successive waves of myogenic
precursors results in an even and proportional pattern of mediolateral growth
of the developing myotome (Kahane et al.,
2002
).
Likewise, direct tracing of the projection of dorsal epithelial somite
precursors onto the mature DM revealed an overall pattern of coherent and
proportional expansion of this structure along the mediolateral extent. These
results are consistent with the outcome predicted from calculating the pattern
of DM growth between T0 and T2 based solely on the measured morphogenetic
changes (R.B.-Y. and C.K., unpublished). Hence, the present results further
strengthen the topographical association between DM and myotome, suggesting
that cells directly translocate from the DM into the corresponding region of
the myotome (Fig. 9). Our
results are consistent with those of Eloy-Trinquet and Nicolas
(Eloy-Trinquet and Nicolas,
2002) who reported, based on clonal analysis in the mouse, on a
direct relationship between myotome precursors in the DM and their daughter
cells in the myotome. This view therefore refutes the alternative model which
suggests that two opposite stem cell systems located in the DML and VLL drive
incremental growth. The latter model does not consider the contributions of
similar stem cells localized in the extreme rostral and caudal lips of the DM
that contribute both to DM and myotome growth and of those progenitors located
within the entire DM sheath that actively proliferate and contribute, at
least, to continuous expansion of the DM and to subsequent formation of the
dermis (Fig. 9).
Consistent with the coherent mediolateral growth of the myotome and DM, the
present data using lineage tracing and GFP electroporation of intact segments,
demonstrate that the dermis originates from both medial as well as lateral
domains of the dorsal epithelial somite which, at an intermediate stage,
project into corresponding regions in the DM. In line with our results, a
previous study has found that both medial as well as lateral half-somites have
the potential to contribute to dermis under experimental conditions
(Houzelstein et al., 2000).
Our results, however, contrast with previous data that proposed that the
dorsal dermis derives entirely from the medial but not the lateral half of the
somite (Olivera-Martinez et al.,
2000
). The latter study was based on quail-chick grafts of
half-somites of either medial or lateral type. Studies performed in our
laboratory (N.K. and C.K., unpublished) revealed that under such conditions,
many grafts resulted in formation of two half-somites that did not fuse and
remained separated by adjacent lips (see also
Olivera-Martinez et al.,
2000
). At later stages, we observed that the grafted quail
moieties sometimes took over the formation of most of the myotome and dermis
(Olivera-Martinez et al.,
2000
), regardless of their origin as medial or lateral and only
when keeping their original proportions was the dermis composed of both quail
and chick cells. This variability in the experimental outcomes suggested that
the problem of the precise mediolateral origin of the dermis had to be
re-examined in intact embryos.
Notably, flank-level DM was also shown to participate to the formation of
the scapular blade in avian embryos (Huang
et al., 2000a). Although most likely, cells emanating from
intermediate and lateral regions of the DM are at the origin of this ossifying
structure, the precise mediolateral origin of the scapular blade progenitors
was not determined. It is therefore possible that lateral DM-derived
mesenchyme also contributes to the scapular blade. Yet, as labeling of the
dorsolateral parts of cervical somites, which do not form cartilaginous
structures, also led to the appearance of labeled cells in the subectodermal
mesenchyme, we maintain that lateral somite cells are destined to develop into
the lateral domain of the dorsal dermis.
Taken together, lineage analysis of the formation of the myotome, DM and
dermis consistently suggests that the development of all dorsal derivatives of
the somite follows a coherent and continuous mediolateral pattern in which
there is, on the one hand, a direct relationship between the epithelial
progenitors in the DM and their ascendants in the dorsal somite, and, on the
other hand, with their corresponding progeny in the myotome and dermis
(Fig. 9). The only exception so
far characterized is the development of the pioneer myoblasts which originate
in the medial domain of the epithelial somite but upon differentiation into
myofibers end up spanning the entire mediolateral extent of the segment
(Kahane et al., 1998a).
Consequently, this view refutes previously accepted models sustaining that the
medial but not the lateral half of the somite gives rise to the whole
repertoire of epaxial derivatives up to the lateral somitic boundary
separating the somite from LPM derivatives.
Fate of distinct areas of the DM
Progenitors localized in the DML and VLL
(Denetclaw et al., 2001;
Venters and Ordahl, 2002
) as
well as in the rostral and caudal lips (our results) give rise to both DM and
myoblast descendants. Yet, recent data suggested that the DML also gives rise
to the dorsomedial mesenchyme that is apposed to the neural tube at E4-4.5 of
avian development (Olivera-Martinez et
al., 2002
). Based on this putative DML origin and on the
expression of the Wnt11 gene, this mesenchyme was interpreted to become
dermis. Although it is tempting to speculate that expression of Wnt11 in both
the DML and in this mesenchyme reflects a direct lineage relationship between
the two, our present DiI labeling results clearly show that the DML does not
participate in the formation of the dorsomedial mesenchyme localized between
the DML and dorsal neural tube. Consistent with this observation, interspecies
chimeras in which the dorsal half of a chick somite was replaced by its quail
counterpart, thus giving rise to a DM (including the DML) composed of quail
cells and a sclerotome with chick nuclei, revealed that the dorsomedial
mesenchyme was composed of chick nuclei. Our results thus confirm previous
data based on quail/chick analysis (Huang
and Christ, 2000
; Christ et
al., 2000
). It was only when sclerotomal cells were of quail type
(Huang et al., 2000b
) or when
sclerotomal cells were directly labeled with DiI (our results) that the
dorsomedial mesenchyme was positively labeled with quail nuclei or DiI,
respectively. Hence, the dorsomedial mesenchyme derives from sclerotome and
not from DML. Consequently, this mesenchyme develops into a vertebral fate,
perhaps as part of the vertebral arch, rather than into dermis. In line with
such a possibility, this mesenchyme was shown to participate in the formation
of the spinous process of the vertebrae under the influence of BMP4
(Takahashi et al., 1992
;
Monsoro-Burq et al., 1996
;
Watanabe et al., 1998
). The
transcription factor Msx1, a target of BMP4 signaling, is indeed expressed in
the dorsomedial mesenchyme (Houzelstein et
al., 2000
). Using mouse-chick chimeras in which an nlacZ
reporter gene was integrated into the mouse Msx1 locus, Houzelstein et al.
(Houzelstein et al., 2000
)
have confirmed that the dorsomedial mesenchyme expressing Msx1 indeed derives
from the somite. However, as grafts were of whole somites it was not possible
in this study to discriminate between a sclerotomal versus DM origin.
What is then the origin of the dorsomedial dermis forming the dorsal pterylae? This dermis, which develops only at later stages (after E7) is likely to arise from secondary rearrangements of dermal cells that derived initially from the medial DM region and subsequently relocate towards the dorsal midline of the embryo, in a similar way to which sclerotomal cells migrate dorsomedially to form the neural arch.
Our results therefore suggest that the somite-derived dermis arises from
the entire DM sheath, including medial and lateral domains. Each region of the
DM originates, in turn, at corresponding regions in the epithelial somite
(Fig. 9). Consistent with our
fate-mapping experiments is the conserved lateral expression of the Sim-1
transcription factor at these three consecutive stages
(Fig. 7)
(Pourquie et al., 1996;
Olivera-Martinez et al.,
2002
). Surprisingly, we have observed that labeling of the central
domain of the dorsal epithelial somite or of the DM sheath also gives rise to
cells that colonize the myotome and appear mesenchymal rather than fibers (see
for example Fig. 6E,F and
Fig. 7B,D). These cells cannot
result from accidental labeling of the myotome as no myotome is present at the
epithelial somite stage. Moreover, if myotome labeling occurred in injections
performed at T1, then these cells would have been detected in segments fixed
at T2. However, they become apparent only at E4-4.5 (T3 and older). Therefore,
epithelial progenitors from the non-lip regions of the DM may be late
contributors of the third wave category of mitotically active progenitors that
initially invade the myotome from the rostral and caudal lips
(Kahane et al., 2001
). The
properties and fate of these cells are now under investigation. Hence, the
possibility remains open that by the time of delamination, the DM epithelium
produces mesenchymal cells which migrate bidirectionally; both superficially
to colonize the subectodermal space and give rise to dermis, and also towards
the myotome to further contribute to its growth.
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ACKNOWLEDGMENTS |
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