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First published online 4 July 2007
doi: 10.1242/dev.02877
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Institute for Stem Cell Research, School of Biological Sciences, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh EH9 3JQ, UK.
* Author for correspondence (e-mail: v.wilson{at}ed.ac.uk)
Accepted 15 May 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Mouse, Rostrocaudal axis, Axial elongation, Stem cells, Node, Primitive streak, Chordoneural hinge
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In the mouse, this process lasts for around 6 days,
eventually producing some 60 somites - segmented blocs of mesoderm that
contain progenitors of the axial skeleton and musculature. These flank the
neural tube - the progenitor of spinal cord - and the notochord, a mesodermal
tissue that patterns the neural tube and somites
(Kaufman, 1992). Clonal
lineage analyses in mouse and chick have indicated that at least some axial
progenitors are present for long periods of axial elongation (here termed
long-term axial progenitors), giving rise to descendants that contribute to
differentiated axial tissues over large rostrocaudal distances
(Mathis and Nicolas, 2000;
Nicolas et al., 1996;
Selleck and Stern, 1992a)
Thus, these long-term axial progenitors might be stem cells (cells that can
self-renew as well as differentiate). Consistent with these data, a series of
transplantation experiments has suggested the presence of self-renewing cells
in a region of the tail bud termed the chordoneural hinge (CNH)
(Cambray and Wilson, 2002).
When CNH cells were transplanted from embryonic day (E)10.5-12.5 (35-55-somite
stage) tail buds into the primitive streak region of E8.5 (2-6-somite stage)
embryos that were then cultured for 48 hours, they contributed to rostral
somites, notochord and neural tube. Therefore, progenitors of the caudal axis
retain the capacity to produce more rostral axial tissues. Moreover,
descendants of these cells were also retained in the CNH and could be passaged
through successive embryos, and thus could repeatedly incorporate in the same
segments of the axis. Together, these lineage analyses and transplantation
studies provide evidence that at least some long-term axial progenitors are
stem cells. In the above transplantation studies, all donor tissues were grafted to the junction between the node (which gives rise to notochord) and the rostral primitive streak (which generates somites) to provide a standard environment to compare different graft types. In these experiments, grafting the node to the junction of the node and streak showed two distinct patterns of axial colonisation: some grafts gave rise exclusively to axial tissues (the ventral neural tube and notochord), whereas others additionally colonised the somites. The grafts that colonised somites also gave rise to cells in the tail bud that were subsequently transplanted for several generations. Although the number of grafts was too small to distinguish a significant effect, it suggested that grafts that included somite progenitors might contain a localised source of axial stem cells, whereas those that contained only notochord and neural tube did not.
Several features of the E8.5 mouse embryo make a more careful analysis
possible. The node and rostral streak are initially not covered by the ventral
part of the hindgut tube, and are therefore accessible to manipulation.
Culture of E8.5 embryos is straightforward over the ensuing 48-hour period
when around half of all somites are made
(Copp and Cockroft, 1990).
Furthermore, at E8.5, the node is a morphologically distinct structure. It
consists of a group of ventrally located notochord progenitors
(Beddington, 1994;
Wilson and Beddington, 1996)
that are transiently inserted in the endoderm layer and are bounded laterally
and caudally by a semicircular raised edge of radially oriented cells
(Sulik et al., 1994)
[elsewhere termed the `crown' (Bellomo et
al., 1996)]. The caudal aspect of this edge delineates the
boundary between the node and primitive streak. This ventral region of the
node and the ectoderm immediately above it form a smooth convex shape when
viewed from the dorsal side. The junction with the streak forms an abrupt
transition to the concave shape of the streak, and is thus morphologically
distinct. Later, as the hindgut invaginates and the notochord separates from
the endoderm layer between E8.5-E9.5, these morphological distinctions
disappear.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) (`GFP
transgenic') mice were maintained on a 14-hour light, 10-hour dark cycle. Noon
on the day of finding a vaginal plug was designated E0.5. Dissection and
culture was performed as described (Copp
and Cockroft, 1990)
In situ hybridisation
Embryos, tails and tissue fragments were subjected to whole-mount in situ
hybridisation as described previously
(Wilkinson, 1992) except that
proteinase K treatment was empirically adjusted between 5-18 minutes according
to embryo size and stage. The riboprobes used were: T (brachyury)
(Herrmann, 1991),
Fgf8 (Mahmood et al.,
1995), Cdx2 (Tanaka
et al., 1998), Wnt3a
(Takada et al., 1994),
Evx1 (Dush and Martin,
1992), Foxa2 (Sasaki
and Hogan, 1993) and Sox1
(Aubert et al., 2003). Embryos
were then dehydrated via an ethanol series, processed for paraffin wax
histology and sectioned at 7 µm in a microtome (Anglia Scientific
0325).
Embryo dissection and grafting
GFP transgenic x MF1 litters were dissected as described
(Cambray and Wilson, 2002). The
node and primitive streak areas were dissected using fine glass needles by
making longitudinal lateral cuts, isolating two adjacent 
100 µm-wide
strips of tissue containing either the entire midline - including the rostral
node, border and streak 1-5 regions - or the area lateral to the streak -
including the lateral border and lateral 1-5 regions
(Fig. 1A). The regions were
further dissected by making transverse cuts with glass needles. Dissection of
wild-type MF1 recipient embryos, grafts and culture were performed as
described (Cambray and Wilson,
2002).
Embryo processing
At the end of the culture period, green fluorescence was assessed in a
Zeiss Stemi SV11 dissecting microscope with fluorescence attachment. Images
were captured using Improvision Openlab software and processed using Adobe
Photoshop. Grafted embryos and Sox1-GFP embryos were
embedded in 10% gelatin; 10% albumin in PBS, fixed in 4% PFA, then coated with
a 0.05% gelatin, 15% BSA; 20% sucrose; 10% glutaraldehyde solution to create a
tear-resistant outer layer. Transverse sections were cut in a Series 1000
Vibratome at 100 µm and then incubated with TO-PRO-3 (Molecular Probes) for
30 minutes before being mounted under coverslips in Vectashield (Vector
Laboratories) for immediate confocal microscopy.
Assessing the contribution of GFP-labelled cells to axial tissues
In cases in which the contribution of cells from adjacent graft types (e.g.
lateral regions 1,2,3 or streak regions 2,3,4) was indistinguishable, results
were pooled for scoring in Table
1. To quantify the contribution of grafted cells in each tissue,
the number of sections that showed integration in a given tissue was expressed
as a proportion of the total number of sections that contained any labelled
cells. Labelling in more than 50% of the sections was considered `high level',
corresponding to at least 10 out of a total of 20 scorable sections.
Integration in 20-50% of sections was considered `low level'. Integration in
less than 20% of the sections was scored as zero, because this usually
corresponds to a small clump of cells in the area around the original graft
site. The position of the tissue scored as `CNH' is indicated in
Fig. 1B. Mesenchyme caudal to
the CNH was scored as tail bud mesoderm (TBM). In the tail bud, where there
are relatively few sections, any integration was scored as high level for
either CNH or TBM.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), we performed a careful gene expression study on a selection
of genes known to be expressed in the primitive streak and tail bud.
Consistent with the expression patterns previously reported for these genes
(Takada et al., 1994;
Crossley and Martin, 1995;
Herrmann, 1991;
Kispert and Herrmann, 1994;
Dush and Martin, 1992;
Ang et al., 1993), we observed
Wnt3a, Fgf8, T (brachyury) and Evx1 expression in the E8.5
primitive streak and emergent mesoderm
(Fig. 2A,C,E,G), and
Foxa2 and T expression in the ventral layer of the node
(Fig. 2G,G',I,I').
Cdx2 was expressed broadly in the streak and tail bud, encompassing
all tissues in the streak and node at E8.5, as well as the entire CNH and tail
bud mesoderm (TBM) at E10.5 (Fig.
3I and data not shown). However, somewhat surprisingly, the
expression domain of Wnt3a, Fgf8, T and Evx1 in the streak
terminated not at the streak-node junction, but in the dorsal (ectoderm) layer
of the caudal half of the node itself, overlying a domain of Foxa2
and T expression in the ventral (mesoderm) layer
(Fig.
2A',C',E',G',I'). The expression of
Wnt3a, Fgf8, T and Evx1 appeared to be complementary to a
further Foxa2 expression domain in the ectoderm of the rostral half
of the node. An additional novel expression domain was seen at the caudal tip
of the ventral node, which expressed Fgf8
(Fig. 2A'). These
expression domains were mirrored in the tail bud, because ectoderm cells
expressing Wnt3a, Fgf8, T and Evx1 overlaid cells in the
ventral, emergent notochord that expressed Foxa2 and T, and,
at the caudal notochord tip, Fgf8
(Fig. 2B,D,F,H,J). This region
of apposed gene expression corresponded to the CNH, which we have previously
shown contains long-term axial progenitors, raising the possibility that the
node-streak junction at E8.5 contains similar axial progenitors.
Sox1 expression was examined using in situ hybridisation and a GFP
transgene targeted to the Sox1 locus
(Ying et al., 2003). GFP
protein was found at high levels in E8.5 embryos in all neurectoderm as far
caudally as the rostral half of the node
(Fig. 2K and data not shown).
Because Sox1 is thought to be a neural-specific marker, it was somewhat
surprising that it was detected at relatively high levels in the ectoderm of
the CNH (Fig. 2L,N). It was
also present at lower levels in the TBM
(Fig. 2L,L',O), which we
have previously shown to be the immediate descendant of CNH ectoderm
(Cambray and Wilson, 2002). The
expression of Sox1 mRNA was essentially the same as that of GFP
protein, except that Sox1 mRNA was almost undetectable in the TBM
(Fig. 2M), suggesting that
expression is quickly downregulated on mesoderm formation, whereas GFP protein
persists in this tissue. Intriguingly, cells strongly positive for
Sox1-GFP were present in the caudal notochordal territory
(Fig. 2N,N'), and some
were continuous with two ventrolateral horns of the neural tube, suggesting
that cells from the Sox1-GFP-positive neurectoderm can
intermingle with notochord, at least at its caudal and lateral extremities.
Thus, axial progenitors upregulate Sox1 during their transition from primitive
streak to tail bud.
To determine whether the progenitors remained unchanged during axis elongation, we also examined the expression of T, Wnt3a, Fgf8 and Cdx2 in E11.5-E13.5 tail buds. The late expression domains of genes in the tail bud were similar to those at earlier stages, confirming a similar topology of the tail bud as axial elongation progresses (Fig. 3 and see Fig. 1 in the supplementary material). However, at these late stages, the expression levels changed dramatically. Fgf8 and Wnt3a were expressed strongly throughout the tail bud until E10.5 (Fig. 3A,E) but, thereafter, their expression declined (see Fig. S1 in the supplementary material). Analysis of sections shows that this is due to both a decrease in the number of expressing cells and in the levels of expression per cell. Expression essentially disappeared by E13.5, when elongation ceased (Fig. 3B-D,F-H'). Similarly, Cdx2 expression was strong until E11.5 (Fig. 3I,J and see Fig. 1 in the supplementary material), at which point its expression declined, becoming undetectable by E13.5 (Fig. 3L). T transcripts were present at high levels until E12.5 (Fig. 3M-O and see Fig. 1 in the supplementary material), and were still detectable at lower levels at E13.5 (Fig. 3P). Thus, the expression of several genes declines 48 hours or less before the end of axis elongation.
Fate mapping and commitment to given fates
To examine the fate and commitment to these fates in the above regions, and
in particular their relationship with later axial progenitors in the CNH, we
manually microdissected regions of the E8.5 streak, node and lateral areas
according to the scheme shown in Fig.
1. Thus, rostral and caudal regions of the node (`rostral node'
and `border', respectively, the latter containing the crown at the node-streak
border), five successively more caudal pieces of the primitive streak (`streak
1-5'), and the regions lateral to the border and streak (lateral border,
lateral 1-5 regions) (Fig. 1A,
Fig. 4A-C) were isolated. To
assess accurate dissection, rostral node, border and streak 1 pieces were
subjected to in situ hybridisation for Fgf8 or Foxa2
(Fig. 4D-I). All fragments
showed the expected expression patterns: rostral node expressed only
Foxa2 and streak 1 expressed only Fgf8, whereas the border
expressed both markers. Lateral pieces expressed primitive streak markers
(Fig. 4J-M). A representative
sample of six dissected pieces were dissociated and the cells counted. Each
contained 100-230 cells, with an average of 149 cells.
Fragments from GFP transgenic embryos were grafted homotopically or heterotopically to stage-matched host embryos, which were then cultured for 48 hours. Embryos developed around 30-35 somites as expected, and all except one that received a graft contained labelled cells at the end of the experiment (n=84). In 12/40 embryos in which streak was either the donor or host, small self-contained clumps were observed in addition to well-integrated stretches of cells in the axis, typically attached to the dorsal neural tube. They probably resulted from early expulsion of excess grafted tissue and, because they did not perturb axial development, they were not considered further. All grafted embryos were sectioned and their contribution to the axis and tail bud scored (see Materials and methods for the method of scoring). Section (i) below describes the fates of different regions of the primitive streak and node, and their commitment to these fates is detailed in sections (ii)-(iv).
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We examined the rostral limit of labelling in each embryo, using the somite
level as a morphological landmark. In general, a more rostral position in the
streak correlated with a more rostral limit of labelling
(Table 1) and corresponded well
to that seen when labelling the equivalent regions in situ using DiI
(Wilson and Beddington, 1996).
Thus, grafted cells generally incorporate quickly in the host embryo. One
exception was that rostral node integration in notochord occurred around the
level of somite 18, suggesting there was a delay in integration of this
tissue. The rostral limit of the short unilateral somite stretch derived from
lateral 1-3 grafts (somite 13) corresponded almost exactly to that from the
adjacent midline streak 1-4 grafts (somite 13-14), even though, as a more
differentiated derivative of streak, it would be expected to lie more
rostrally. This implies that some cells exit rapidly from the midline to the
adjacent presomitic mesoderm. The rostral limit of the predominantly neural
lateral border derivatives was at somite 7-8
(Fig. 5H,
Table 1) and the total labelled
region was no more than 9 somite lengths. Neural cells derived from lateral
1-4 grafts stretched from the somite 22 level caudally, although this anterior
limit was variable (range: somite 15-27). The lack of overlap between lateral
border and lateral 1-4 derivatives suggests that there is little mixing
between long-term neural progenitors and their more differentiated progeny
located further rostrally. Finally, streak 5 and lateral 5 grafts colonised
much more caudal regions (around the level of somite 21-22) than streak 1-4
and lateral 1-4 grafts (around somite 13). Because somite 20 is derived from
progenitors in the streak at E9.5, it appears that the lateral mesoderm is
delayed in its exit from the streak or lateral regions by about 24 hours
relative to the presomitic mesoderm.
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(ii) The border can produce rostral node and rostral streak derivatives
The border, although it integrated in all dorsal axial derivatives, did not
contribute extensively to trunk notochord or to lateral regions of the trunk
somites. To test whether this was because descendants were already committed
to form medial somites and posterior notochord, the border was transplanted to
the rostral node or streak 1 position. In the rostral node, border cells
formed large stretches of trunk notochord
(Fig. 6G,M,
Table 2), whereas, in the
streak 1 position, they contributed much more prominently to the whole somite
(Fig. 6H,N,T,
Table 2). This implies that it
is the position of border cells, and not their intrinsic differentiation
capacity, that directs their low contribution to notochord and lateral regions
of the trunk somites in situ. However, in both sets of grafts, there was a
consistent low-level contribution to tissues that were not colonised in node
or streak homotopic grafts, including the CNH, suggesting that the border
retained some of its original characteristics in these ectopic environments
(Table 2). Thus, border cells
can change fate in response to node or streak environments, although they form
a broader spectrum of differentiated cell types than node or streak cells in
situ. Notably, border descendants were retained in the CNH in these
heterotopic grafts.
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(iv) The rostral primitive streak has a limited differentiation capacity
Whereas primitive streak cells in situ populated the entire mediolateral
extent of the somite, streak 1 cells grafted to the border environment tended
to colonise medial positions in a pattern characteristic of border homotopic
grafts (Fig. 6K). However, they
did not generate notochord, and only a very small number of cells was present
in caudal ventral neural tube and in the CNH
(Fig. 6W). In striking contrast
to all other graft types, streak 1 cells grafted to the node showed a very
poor level of incorporation (Table
2). In a high proportion of embryos, these unincorporated cells
also formed ectopic neural-like structures that joined and separated from the
endogenous neural tube along the axis (Fig.
6F,L, inset). Of the cells that incorporated well in the axis,
most formed medial paraxial mesoderm (Fig.
6L), although some examples of apparently bona fide incorporation
in the other axial tissues were present. In the five embryos that showed
incorporation at some level along the axis, three colonised the CNH in the
tail bud (Fig. 6R,
Table 2). In summary, the
streak shows the least plasticity on ectopic grafting, but instead tends to
form ectopic neural-like structures. However, cells were sometimes present in
the CNH after culture. Retention in the CNH is thus a property of all ectopic
grafts.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Gene expression in the streak and tail bud
Our expression study showed that E8.5 streak and node expression domains
and their relative orientation are preserved in the CNH, highlighting a
similarity in the general layout of the streak and tail bud, and particularly
between the border and CNH (Fig.
2P). This similarity in gene expression is reflected in the unique
ability of these two regions to give rise to notochord, somites and ventral
neural tube (Cambray and Wilson,
2002) (this study). In later axis elongation, the levels of gene
expression decline (Fig. 3 and
see Fig. 1 in the supplementary
material). Cdx2 and Wnt3a are essential for complete axis
elongation, because mutants lacking wild-type levels of these genes have short
tails (Takada et al., 1994;
Chawengsaksophak et al., 2004).
Despite this requirement, Wnt3a expression decreases between E10.5
and E11.5, with the progenitors of a further 20-25 somites still present. Its
expression is almost absent in the progenitors of the last 10 somites, which
also express very little Cdx2. Fgf8 levels follow a similar schedule
to Wnt3a. Fgf8 has not been shown to be essential for this phase of
axis elongation because null mutations terminate at the gastrulation stages
(Meyers et al., 1998), but FGF
signalling is essential for this process
(Partanen et al., 1998).
Therefore, it is possible that lowered levels of Fgf, Wnt and
Cdx2 transcripts lead to the termination of axis elongation after a
delay, perhaps due to a slow decline in FGF/Wnt signalling levels. If this is
true, the supply of axial progenitors might be exhausted by E13.5 as a direct
consequence of an earlier loss of essential components needed for maintenance
of the progenitors. However, axial progenitor capacity in E12.5 and E13.5 tail
bud cells can recover when grafted to younger embryos
(Cambray and Wilson, 2002;
Tam and Tan, 1992), and so
these cells must remain responsive to signals from their environment,
presumably including FGF/Wnt signalling from neighbouring cells.
A new fate map for the late mouse primitive streak and surrounds
The fate map that we have deduced from homotopic grafting at E8.5
(Fig. 7) confirms and extends
that of Wilson and Beddington (Wilson and
Beddington, 1996) and is similar in essence to that of other
vertebrates, in particular, the chick
(Psychoyos and Stern, 1996).
However, our results reveal a number of interesting differences between the
mouse and chick. In chick, at equivalent somite stages [Hamburger and Hamilton
(HH)7-9], although the rostrocaudal arrangement of progenitors is the same as
that in mouse, the proportion of the streak fated for the different mesoderm
types differs. Somites are produced from a much higher proportion of the mouse
streak than the chick (approximately 80% versus 20% of streak length). In the
chick, the caudal 50% of the streak length contains extraembryonic mesoderm
progenitors, whereas, in mouse, all extraembryonic mesoderm has exited the
streak by the early headfold stage. Lateral mesoderm is produced between
approximately 50-80% of the streak length in the chick (where 90-100% is the
node), whereas, in mouse, we found that lateral mesoderm progenitors are
confined to the caudal 20% of its length. In this respect, the fate map of
chick at 7 somites resembles the fate map of the mouse E7.5 streak (late
streak, equivalent to approximately stage 4+ in chick)
(Smith et al., 1994). In
agreement with data in chick, we found that cells in or very close to the node
exclusively produce the medial part of the somite in the trunk
(Selleck and Stern, 1991;
Freitas et al., 2001).
However, fate maps of the primitive streak in chick suggest that descendants
of the rostral streak behind the node are also at least partially restricted
to the medial somite, whereas regions located further caudally contain at
least some laterally restricted progenitors
(Psychoyos and Stern, 1996).
In our experiments, cells abutting the border (streak 1) generally produced
the entire somite, whereas some streak 4-derived cells extended into medial
regions, although there was a tendency for a more lateral somite contribution
in more caudal streak grafts. The overlap of streak-derived cells with the
domain populated by border descendants raises the possibility that the
somite-organising capacity of medial paraxial mesoderm
(Freitas et al., 2001) is
initiated by a subpopulation of medial progenitors, rather than the entire
domain.
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;
Wilson and Beddington, 1996),
generating cells that will pass through the presomitic mesoderm to the
somites. Although it is known that axial progenitors at E8.5 reside in or
close to the streak (Snow,
1981), it is not known how close to the midline these progenitors
are. We have shown that there is significant movement from lateral ectoderm to
the midline at this stage, consistent with results in the chick
(Iimura and Pourquié,
2006). Meanwhile, streak cells exit completely from the midline
ectoderm, so that cells from the border and lateral ectoderm are the only
occupants of the E10.5 CNH. Within the mesoderm, some cells exit quickly from
the midline to presomitic mesoderm, as judged by the identical rostral limits
of streak 1-4 and lateral 1-4 grafts in mesoderm. However, because some
streak-derived cells at E8.5 are found in the TBM 48 hours later, the period
of exit from midline mesoderm to presomitic mesoderm is relatively
prolonged.
Commitment of border cells to their fates
The border contains both neural and somite progenitors. These are most
probably located in the ectoderm layer, because labelling the ventral layer of
the node (including the cells in the ventral part of the border) shows no
neural or somitic descendants (Wilson and
Beddington, 1996; Cambray and
Wilson, 2002). However, the border additionally contains notochord
progenitors. Labelling the ectoderm of the chick node
(Selleck and Stern, 1991)
shows that cells from this layer can colonise notochord. Therefore, at least
some of the material for the mouse notochord might be derived from the
ectoderm. Interestingly, in the border ectoderm, a few cells express
T very highly (Fig.
2G' and inset). Because ectoderm cells in the node can
produce notochord (and not vice versa), these ectoderm cells that strongly
express notochord markers might therefore be in transit to the notochord. The
apparent continuity between Sox1-positive cells in the CNH ectoderm
and the notochord (Fig.
2N,N') suggests that, here too, cells marked by
Sox1 continue to exit the CNH ectoderm to notochord.
It is interesting that progenitors of notochord, neural tube and somites
are contained in this small region. Selleck and Stern
(Selleck and Stern, 1991)
showed that single cells in the chick node can produce more than one tissue
type: namely, notochord and neural tissue or notochord and somites. Because
the ectoderm layer can produce all three tissues, the border ectoderm could
comprise a population of multi-fated or multipotent cells, although a mixture
of cells each fated for one tissue type cannot be ruled out from our
experiments. On ectopic grafting, border cells can either produce
predominantly somites or notochord, depending on their location
(Fig. 6). These ectopic grafts
show few unincorporated cells, suggesting that most cells of the graft could
participate in the development of either somites or notochord. The results of
these ectopic grafting experiments thus imply that at least some border cells
can change fate in response to environmental cues and therefore might be
multipotent. A similar study on earlier-stage chick embryos
(Selleck and Stern, 1992b)
also shows that a region of the node containing both notochord and somite
precursors (the lateral sector) can adapt to heterotopic grafting to regions
of prospective notochord (medial sector) or somites (streak) by producing more
or less somitic mesoderm.
Commitment of rostral node and streak 1 cells to their fates
Heterotopic grafts of rostral node and streak 1 showed that they are less
potent than the border, because grafting them ectopically did not confer the
ability to produce the full range of border-derived tissues. Indeed,
heterotopic streak 1 grafts to the node showed a reduced level of graft
incorporation and, in the embryos that maintained the graft, many of the
resulting cells formed ectopic structures, showing that streak cells are not
readily adaptable to the node environment
(Fig. 6). However, rostral node
cells, which contain no neural progenitors in situ, were capable of converting
to neural progenitors on heterotopic grafting. Most strikingly, there was
significant neural contribution when rostral node was grafted to streak 1
(Fig. 6J,P). Because neither
donor nor host contained any neural progenitors, the streak 1 environment
unveiled a neural progenitor capacity inherent in (but not exhibited by) the
rostral node ectoderm. It is likely that the node environment represses neural
progenitor identity, because border cells grafted to the node showed a reduced
neural fate compared with border homotopic grafts, and also produced only
notochord and not ectoderm in the CNH
(Table 2). Interestingly,
heterotopic grafts of node and streak all contributed to the CNH. This might
result from a delay in the integration of an ectopic piece of tissue, leading
to delayed exit from the progenitor region. Because the rostral limit of
grafted cell contribution is generally slightly more caudal than that of the
corresponding homotopic graft (Fig.
5H, Table 2), this
might be the case. However, it is also possible that, in some cases, the
ectopic tissue combination imposes progenitor status upon grafted cells that
would otherwise have been en route for axial differentiation.
The latter possibility is consistent with the results of our previous study
(Cambray and Wilson, 2002),
because some node grafts that produced no somites and thus probably excluded
the border region could be retransplanted once, giving rise to both axis and
CNH. These must therefore have re-acquired at least some aspects of progenitor
status, and suggests that there might be some plasticity in the rostral node
and rostral streak populations. The normal encroachment of lateral ectoderm
cells on the CNH would then be consistent with an ability of cells to acquire
long-term progenitor status based on their position, and not on an inherent
property of the cells. Joubin and Stern
(Joubin and Stern, 1999)
showed that the early chick node normally receives incoming lateral cells up
to stage 3+ and, therefore, until this stage, the node consists of a transient
population defined by molecular interactions. However at later stages, they
observed little movement of lateral cells towards the node. Our study shows
that some cells still move towards the border descendant after the mouse
equivalent of stage 4 (mid-late streak, E7.5) but from more caudal positions.
Therefore, the imposition of progenitor status by the border and CNH might be
a continuous process throughout axis elongation.
Interchangeability of neurectoderm and somite progenitors
Neural progenitors in the lateral ectoderm are closely associated with
mesoderm progenitors, because lateral grafts give rise to neurectoderm and
somites at similar (
3 somites distant) axial levels
(Fig. 5Fa,Fb, inset; and data
not shown). Three embryos receiving a graft in the lateral 1-3 position were
particularly informative. In two of these, no somitic contribution was
evident, and the third showed no neural contribution. This suggests that,
although neural and somitic progenitors overlap in the lateral ectoderm, they
do not exactly coincide. It is therefore possible that position in the
ectoderm determines a neural or somitic fate. However, we could not
distinguish neural and somitic progenitors on the basis of the gene expression
markers used, suggesting they are related cell types.
Apparent interchangeability of neural and somitic progenitors was seen in
embryos lacking Wnt3a, its target (brachyury; T), or
Tbx6. Here, cells normally destined for mesoderm differentiated
inappropriately as neural tissue
(Yoshikawa et al., 1997;
Yamaguchi et al., 1999;
Chapman and Papaioannou, 1998).
Signalling via Fgfr1 is also required for Wnt3a-mediated mesoderm
differentiation and Tbx6 expression
(Ciruna and Rossant, 2001).
Furthermore, ectopic expression of T, Fgf and Wnt family members in
Xenopus can drive mesoderm formation in naïve ectoderm
(Cunliffe and Smith, 1992;
Slack et al., 1990;
Sokol, 1993). Thus, activation
of this cascade in prospective mesoderm appears sufficient to divert cells
from a neural to mesodermal lineage, suggesting that the two progenitor types
have a related differentiation capacity.
Resident cells and stem cells
Clonal analysis in the chick has indicated a stem cell population for
somites and notochord in the node (Selleck
and Stern, 1992a). In the mouse, retrospective clonal lineage
analyses also point to a stem cell progenitor of the myotome
(Nicolas et al., 1996) and
postcranial spinal cord (Mathis and
Nicolas, 2000). The primitive streak and tail bud are the only
known locations for axial progenitors during axial elongation. Therefore,
these putative stem cells would have been located there at some point in their
history, although this was not directly shown because only differentiated
cells, and not the progenitors, were scored. Can these myotome and spinal cord
stem cells be linked with the border?
The E8.5 border is the major contributor to the CNH, the only region of the
tail bud that later shows stem cell-like properties
(Cambray and Wilson, 2002).
Therefore, the border, as the progenitor of a candidate stem cell-containing
region, might itself contain stem cells. In this context, it is interesting
that clonal prospective fate mapping a day earlier, at E7.5, has identified
apparently resident cells in a small region at the caudal end of the node,
apparently corresponding to the border
(Forlani et al., 2003).
Indeed, early in gastrulation (E6.5), the incipient node is the only region to
contain apparently resident cells (Lawson
et al., 1991). Therefore, prospective fate mapping studies
consistently point to the node region (and when it appears, the border), as a
site for resident cells throughout axis elongation. Although residence in the
progenitor region does not by itself demonstrate that cells are axial
progenitors or stem cells, it is a prerequisite for such cells. Thus, although
each study only deals with a short period in axis elongation, stem cells
similar to those proposed by Selleck and Stern
(Selleck and Stern, 1992a) in
the chick might be present from the beginning to end of axis elongation,
localised successively in the incipient node, border and CNH region of the
mouse.
The above lineage studies, together with our previous serial transplantation of CNH cells, suggest that axial progenitors can behave as stem cells. However, our data show that these progenitors undergo changes in gene expression and, therefore, they are not strictly self-renewing in vivo, but are maturing. Thus, experimental perturbation might enable self-renewal of progenitors that would normally evolve progressively.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/15/2829/DC1
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ACKNOWLEDGMENTS |
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