Institute of Genetics, University of Nottingham, Queen's medical Centre, Nottingham NG7 2UH, UK
Author for correspondence (e-mail:
roger.patient{at}nottingham.ac.uk)
Accepted 23 September 2002
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SUMMARY |
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Key words: Blood, Endothelium, Haemangioblast, BMP, Transcription, Xenopus
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INTRODUCTION |
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The origins of blood in vertebrate embryos have been controversial.
Evidence has been produced supporting both yolk sac and intra-embryonic
origins for the adult blood stem cell
(Keller, 2001).
Transplantation studies in Xenopus embryos have demonstrated that the
precursors of adult and embryonic blood are located in the dorsal lateral
plate (DLP) mesoderm and the ventral blood island (VBI), respectively, at
neurula stages (Kau and Turpen,
1983
). Early fate maps, which concentrate on primitive (embryonic)
blood, traced it to a region of the gastrula embryo called the ventral
marginal zone (VMZ), although a small dorsal contribution was reported but
ignored (Dale and Slack, 1987
;
Moody, 1987
). Confirmation
that embryonic blood also derives from dorsal marginal zone (DMZ) cells came
from the study of the transcription factor Xaml, the Xenopus
homologue of the haematopoietic progenitor gene, AML1/Runx1, which is normally
expressed in the VBI at tadpole stages
(Tracey et al., 1998
). This
study showed that isolated DMZ explants are able to express Xaml and that, in
the intact embryo, when Xaml expression was suppressed in the DMZ by injection
of a dominant negative form, the anterior blood island no longer expressed the
blood marker
-globin. Subsequently, the fate map was redrawn by Lane
and Smith, who looked at the contributions of 32-cell stage blastomeres to
primitive circulating blood, monitored at later stages, and found that all
blastomeres around the meridian of the embryo (tiers C and D) contributed to
primitive blood (Lane and Smith,
1999
). These authors, however, used irregularly cleaving embryos,
and a subsequent refinement of this fate map using regularly cleaving embryos
showed that primitive blood in the anterior VBI derives from the dorsal
blastomeres, C1 and D1, while the posterior VBI primitive blood arises from
the ventral D4 blastomere (Ciau-Uitz et
al., 2000
). This study was the first to monitor the origins of the
adult DLP blood and, importantly, found that this derives from blastomere C3.
Although a very recent study using a different lineage tracer has raised the
possibility that the original lacZ tracer may have labelled only a
subset of the derivatives of the C3 blastomere
(Lane and Sheets, 2002
), the
ability to label the adult and embryonic blood compartments separately
demonstrates that their origins are distinct in Xenopus, whether at
the 32-cell stage or slightly later.
The blood in the adult and embryonic compartments develops in close
association with the endothelium of the early vasculature in Xenopus
embryos. Expression of blood (SCL and GATA2) and endothelial (Xfli1)
markers is detected in the DLP during tailbud stages, although double
labelling to confirm co-expression has not been carried out
(Ciau-Uitz et al., 2000). At
stage 27 (tailbud), a subpopulation of these cells not expressing blood genes
begins to migrate to the midline towards the hypochord, an endoderm-derived
structure that secretes VEGF, where they form the dorsal aorta
(Cleaver and Krieg, 1998
;
Ciau-Uitz et al., 2000
). One
day later blood gene expression is once again detected in and beneath the
floor of the dorsal aorta (Ciau-Uitz et
al., 2000
). Lineage labelling of the C3 blastomere shows that
derivatives of this blastomere are found in the DLP at tailbud stage and then
in the dorsal aorta, as well as in mesenchymal cells below the dorsal aorta,
suggesting that the haematopoietic cells associated with the floor of the
dorsal aorta, thought to be the first adult blood stem cells, derive from the
DLP (Ciau-Uitz et al., 2000
).
No similar analysis of blood and endothelium in the embryonic (VBI)
compartment has been carried out.
The distinct origins and migration pathways of embryonic and adult blood
suggest that they may come under the influence of different embryonic signals
during their ontogeny. Indeed it has been demonstrated that ventral mesoderm
transplanted into the DLP region contributed to adult haematopoiesis, and,
conversely, that DLP mesoderm transplanted to the VBI region contributed to
embryonic blood (Turpen et al.,
1997). These results indicate that haematopoietic precursors
located in both the VBI and the DLP have the potential to produce both blood
lineages, and suggest that micro-environmental signals restrict
differentiation into one or other of the lineages. The close association of
blood and endothelial precursors suggests that endothelium may also respond to
similar signals.
BMP4 has long been associated with VBI induction and the formation of
blood. Thus, ectopic expression of BMP4 in intact Xenopus embryos, or
in explants of prospective ectoderm, stimulates blood cell and globin
production (Dale, 1992; Jones et al.,
1992; Maeno et al.,
1994b
), and injection of a dominant-negative receptor for BMP
results in a reduction of blood and globin
(Graff et al., 1994
;
Maeno et al., 1994a
). The role
of BMP in endothelial development has not been studied in the embryo. The
distinct origins of the blood compartments in Xenopus has allowed us
to investigate their requirements for BMP signalling by targeting the relevant
regions of the early cleaving embryo. Using endothelial markers as well as
those for blood, in conjunction with lineage tracing, we show that both blood
compartments derive from populations of cells that co-express blood and
endothelial genes, which also give rise to the vasculature, and we have looked
at their dependence on BMP signalling. We conclude that while all blood and
endothelial development requires BMP, the specific molecular responses to this
signal differ between the adult and embryonic compartments.
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MATERIALS AND METHODS |
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ß-gal staining and in situ hybridisation procedures
For ß-gal analysis, embryos were fixed and stained as previously
described (Ciau-Uitz et al.,
2000). Whole mount in situ hybridisation was performed according
to Bertwistle et al. (Bertwistle et al.,
1996
). When double in situ hybridisation was performed, embryos
were hybridised with probes differentially labelled with flouresceine and
digoxigenin. The first colour was developed using BCIP (Boehringer Mannheim)
then BM Purple (Boehringer Mannheim) was used for the second colour reaction.
The gene with the strongest expression was always chosen for first staining.
In situ hybridisation to wax sections was performed according to Ciau-Uitz et
al. (Ciau-Uitz et al., 2000
).
For the details of the preparation of labelled probes see the following
publications: GATA2 and
T4-globin
(Walmsley et al., 1994
); GATA3
(Bertwistle et al., 1996
); Xaml
(Tracey et al., 1998
); XHex
(Jones et al., 1999
); Xfli1
(Meyer et al., 1995
); SCL
(Mead et al., 1998
); Xlim1
(Taira et al., 1994
); Xmsr
(Devic et al., 1996
); Nkx2.5
(Tonissen et al., 1994
); and
CG-1 (Sive et al., 1989
).
Histological analysis
For histological analysis embryos were embedded in wax and 10 µm
sections cut in a microtome as described
(Ciau-Uitz et al., 2000).
Sections of whole mounted embryos (10 µm) were obtained by wax embedding
and cutting in a microtome as indicated previously
(Ciau-Uitz et al., 2000
).
Sections were dewaxed and rehydrated to water through an ethanol series,
counterstained for 30 minutes with 0.1% Eosin Y in 70% ethanol, dehydrated to
100% ethanol, cleared in xylene and finally mounted in DPX or mounted in 80%
glycerol without counterstaining. For cryostat sectioning, ß-gal stained
embryos stored in 100% methanol were rehydrated to PBS-Tween through a
methanol series. After rehydration, embryos were washed twice with 30% sucrose
in PBS for 30 minutes before an overnight infiltration in OCT:30% sucrose in
PBS (1:4). Embryos were then placed in plastic embedding moulds containing
fresh OCT:30% sucrose in PBS (1:1), oriented and snap frozen in liquid
nitrogen. Frozen blocks were attached to cryostat holders and 10 µm
sections cut in a cryostat. Sections were collected on
3-aminopropyltriethoxy-silane coated slides and left to dry overnight.
Sections were washed three times in PBS-Tween for 5 minutes and then mounted
in 80% glycerol.
Ribonuclease protection assay analysis
RNA was prepared from frozen embryos using the SV Total Isolation System
(Promega). RNase protection assay for ß-globin UTRs and EF1- was
performed as described (Gove et al.,
1997
), except that hybrids were digested with T1 RNase
(Calbiochem) using 10 units/sample for 30 minutes at 37°C.
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RESULTS |
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At earlier stages of development, co-expression of SCL and Xfli1 is also
detected in a group of cells located immediately posterior to the cement gland
(Fig. 1C, stages 17, 20 and 22
and see Fig. 1A,B, stages 17
and 20a for expression of each gene alone). The overlap of SCL and Xfli1
expression is greatest at stage 17, with cells expressing only SCL (turquoise,
arrowheads) becoming increasingly separated from the Xfli1 expressing cells
(purple) as development proceeds (Fig.
1C, stages 17-22b). The anterior VBI, which contains the anterior
components of the embryonic blood and associated vitelline veins, derives from
the mesoderm of the dorsal marginal zone of the early gastrula embryo
(Ciau-Uitz et al., 2000;
Lane and Smith, 1999
;
Tracey et al., 1998
). These
cells travel the length of the embryo during gastrulation and enter the
anterior VBI after passing behind the future cement gland. Thus, the cells
detected posterior to the cement gland at stage 17, co-expressing SCL and
Xfli1, may well be progenitors for the blood and endothelial cells of the
anterior VBI.
Cells co-expressing blood and endothelial genes in stage 17 embryos
are likely progenitors for the anterior VBI
In order to further characterise this putative aVBI progenitor population,
we probed for additional genes expressed in blood [GATA2
(Walmsley et al., 1994) and
Xam1 (Tracey et al., 1998
)] or
endothelium [Xhex (Jones et al.,
1999
)], either singly (Fig.
2A, parts a,d,e) or in combination
(Fig. 2A, parts f and g) and
found that all these markers were expressed in the region immediately
posterior to the cement gland. To explore this population at higher
resolution, we carried out in situ hybridisation on 10 µm sections of
neurula stage embryos with the same battery of probes
(Fig. 2B). All of these genes
are clearly expressed in the mesodermal layer immediately posterior to the
cement gland (CG, Fig. 2B, part
a) and marked by expression of the gene CG1
(Sive et al., 1989
)
(Fig. 2B, part g). GATA2 is
also expressed in the endodermal and ectodermal layers at this stage
(Fig. 2A, part a,
Fig. 2B, part f), while XHex is
also expressed in the liver diverticulum
(Fig. 2B, part e). However, the
locations of the positive signals for all of these genes suggest a significant
degree of overlap (Fig. 2Bi),
which is borne out by double-labelled in situ hybridisation
(Fig. 2A, parts f, g) and
hybridisation to alternate sections (Fig.
2B, parts b-h). Thus, all the blood and endothelial genes tested
are co-expressed in the mesoderm posterior to the cement gland at stage 17
(orange cells in Fig. 2B, part
i). Anterior to these cells are a small group of cells expressing all the
blood and endothelial genes except XHex (green in
Fig. 2B, part i), while
posteriorly there are cells expressing only blood genes (GATA2, SCL, Xaml; red
in Fig. 2B, part i).
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In order to explore the fate of cells in this region, we carried out
lineage tracing experiments. The progeny of the ß-gal RNA-injected D1
blastomere of the 32-cell embryo (Fig.
2C, part a) populate the anterior VBI of tail bud (stage 26)
embryos (Ciau-Uitz et al.,
2000), leading to overlap between ß-gal staining and blood
and endothelial gene expression in all embryos examined (Xfli1 and SCL, 12/12
embryos; XHex, Xam1 and
T4-globin, nine out of nine embryos; data not
shown). At earlier stages of development, ß-gal-labelled derivatives of
the D1 blastomere are found posterior to the cement gland in the position of
the cells co-expressing blood and endothelial genes
(Fig. 2C, parts b,c, arrow).
The other D1 derivatives are predominantly endoderm, including the liver
diverticulum. The more anterior medial cells express Nkx2.5 (data not shown)
but none of the blood or endothelial genes, making them unlikely contributors
to the VBI. Thus, the cells co-expressing blood and endothelial genes that are
located posterior to the cement gland in stage 17 embryos very probably
represent progenitors of the anterior VBI cells expressing blood or
endothelial genes at stage 26. Similar results were obtained for blastomere
C1, which also contributes to the aVBI
(Ciau-Uitz et al., 2000
): the
only lineage labelled cells co-expressing blood and endothelial genes were the
putative progenitor population posterior to the cement gland (data not shown).
To confirm that anatomically identifiable blood and endothelial cells are
indeed formed from D1-labelled cells, we monitored embryos at much later
stages when vasculogenesis has taken place and blood circulation has
commenced. ß-gal staining was found quite clearly in both endothelial
cells of the vasculature (Fig.
2C, parts d,e, arrowheads) and in circulating blood cells
(Fig. 2C, part e, arrow) in
seven out of seven embryos in each case. Labelling was also detected in
endocardium in all these embryos (data not shown). Thus, the extensive
co-expression of endothelial and haematopoietic genes in the lineage labelled
cells at stage 17, together with the later appearance of lineage label in
blood and endothelial cells and their precursors, suggest that the cells
posterior to the cement gland are progenitors of the blood and vasculature of
the aVBI and also heart-associated vessels.
Cells co-expressing blood, endothelial and pronephric duct genes in
the DLP at tailbud stages are likely progenitors of adult blood and the major
vessels
Previous lineage labelling studies have shown that the cells of the DLP
region can be labelled separately from the VBI by injection of ß-gal RNA
into the C3 blastomere of the 32-cell stage embryo
(Ciau-Uitz et al., 2000). The
lineage label is later found in the endothelial cells of the dorsal aorta (see
also Fig. 3A, arrowhead) and
the posterior cardinal veins (Ciau-Uitz et
al., 2000
). That the DLP gives rise to the dorsal aorta is
supported by direct labelling of the DLP region
(Cleaver and Krieg, 1998
).
C3-injected lineage label is also found in sub-aortic mesenchymal cells
expressing blood genes (Ciau-Uitz et al.,
2000
) and in clusters of cells attached to the floor of the dorsal
aorta (Fig. 3A, arrow). Such
cells are currently the best candidates for the first adult haematopoietic
stem cells in developing vertebrate embryos
(Keller, 2001
). Thus, the DLP
region contains progenitors of both blood and endothelium. Cells in the DLP
region co-expressing haematopoietic and endothelial genes would be strong
candidates for these progenitors. However, thus far, co-expression has only
been explored for one blood gene and one endothelial gene. We therefore set
out to determine the extent to which co-expression could be observed in this
region with a view to defining more precisely the putative progenitors there.
The close proximity of the pronephric duct cells and their derivation from the
C3 blastomere (Ciau-Uitz et al.,
2000
) led us to monitor genes expressed in these cells also.
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The genes monitored were SCL and GATA2 for blood, Xfli1, XHex and Xmsr for endothelium and Xlim1 and GATA3 for pronephric duct. A careful time course of expression in the DLP region showed that Xlim1 is present in a broad area, including the DLP from neurula stages with DLP-restricted Xfli1 and GATA3 expression detected from stage 20, followed by GATA2 from stage 22, and then SCL, XHex and Xmsr from stage 24 (Fig. 3B). The blood genes, SCL and GATA2 become undetectable in the DLP region after stages 33 and 34, respectively, while the endothelial and pronephric genes continue to be expressed beyond stage 36, reflecting the formation of the posterior cardinal veins and the pronephric ducts in this location.
Relative spatial expression of the various genes in tailbud embryos was established by double in situ hybridisation (Fig. 3C). Whole-mounted embryos were sectioned transversely at two points in the anteroposterior axis posterior to the forming pronephros. Wholemounts, anterior sections and posterior sections are presented left to right for each probe combination. Probe combinations that could be used were restricted by the fact that only strong hybridisation signals could be detected using the BCIP technique (turquoise, see Materials and Methods). Thus, panels a-l and bb-dd were hybridised first with Xlim1, the pronephric marker (turquoise) and secondly (purple), with endothelial (Xfli1, XHex, Xmsr), blood (SCL) or pronephric (GATA3) markers. Panels p-aa and ee-gg were hybridised first with the pronephric marker, GATA3 (turquoise) and secondly (purple) with endothelial (Xfli1, XHex, Xmsr) and blood (SCL, GATA2) markers. The only combination of endothelial and blood markers strong enough to give a clear result was Xfli1 (turquoise) followed by SCL (purple) (panels m-o).
The pronephric genes Xlim1 and GATA3 and the endothelial gene Xfli1 show
complete overlap (Fig. 3C,
parts a-c,j-l,p-r). Together these genes span the whole of the DLP region
dorsoventrally (Fig. 3C, blue
and red areas in summary). The expression of the blood genes SCL and GATA2
(Fig. 3C, parts
d-f,m-o,s-u,y-aa) along with the endothelial markers XHex and Xmsr
(Fig. 3C, g-i,v-x,bb-gg) is
restricted to a few cell layers at the dorsalmost end of the DLP region (red
area in summary, Fig. 3C).
Thus, the only cells expressing all the blood and endothelial genes tested,
and therefore the best candidates for a progenitor population, are located at
the dorsal extent of the DLP region (red area in summary,
Fig. 3C). However, at this
stage of their development, these cells are also expressing genes that are
later restricted to pronephric duct cells (Xlim1 and GATA3). These two
pronephric duct genes are also expressed in cells located more ventrally in
the DLP region, in this case without the majority of the blood and endothelial
genes (blue area in summary, Fig.
3C). It seems likely, therefore, that these cells will go on to
form the pronephric duct. The only blood or endothelial gene expressed in
these cells at this stage is Xfli1; however, this expression is not maintained
in differentiated pronephric duct cells. The significance of this early Xfli1
expression for pronephric duct development is at present unknown. Temporal
gene expression in the zebrafish indicates a similar dorsoventral arrangement
of cell fates in the lateral plate mesoderm, with pronephric duct gene
expression initially overlapping with blood and endothelial gene expression,
with subsequent sorting into adjacent domains
(Brown et al., 2000;
Gering et al., 1998
). Of note
in the Xenopus DLP is the absence of Xaml expression. This is an
important distinction between the adult/DLP and embryonic/ventral blood and
endothelial progenitor populations. Xaml is not expressed in the adult DLP
lineage until haematopoietic precursor cells appear 2 days later associated
with the floor of the dorsal aorta
(Ciau-Uitz et al., 2000
).
BMP signalling is required for both anterior and posterior VBI
formation
Bone morphogenetic protein (BMP) signalling is required for ventral
development and its suppression is essential for dorsal development (reviewed
by Dale and Jones, 1999). We
have shown that the two blood and endothelial progenitor populations described
here derive from both ventral and dorsal regions of the early embryo: the
anterior ventral population from the dorsal marginal zone (DMZ), and the DLP
population from the ventral marginal zone (VMZ) (this study)
(Ciau-Uitz et al., 2000
). The
blood and endothelium of the posterior VBI also derive from the VMZ. To
explore its role in the specification of all these blood and endothelial cell
populations, BMP signalling was inhibited separately in derivatives of the VMZ
and DMZ. Inhibition was achieved by injection of RNA coding for a dominant
negative, truncated form of the BMP type I receptor, tBR
(Northrop et al., 1995
;
Suzuki et al., 1994
).
Four-cell embryos were injected either dorsally or ventrally in the
marginal zone with RNA encoding tBR, allowed to develop to stage 26 equivalent
(tailbud) and then examined for blood and endothelial gene expression
(Fig. 4 and
Table 1). At this stage of
development, globin is normally expressed throughout the VBI, displaying the
characteristic V-shape at the anterior end
(Fig. 4, row 1, Control). When
tBR RNA was injected into the VMZ, a partial second embryonic axis was formed
as reported previously (Graff et al.,
1994; Northrop et al.,
1995
; Suzuki et al.,
1994
), and in the primary axis only the V-shaped anterior
globin-expressing cells remained (Fig.
4, row 1, VMZ; Table
1, tBR VMZ). Thus, the more posterior cells failed to form blood
in the absence of BMP signalling. To confirm that the absence of globin
expression was due to the presence of tBR, embryos were co-injected with
ß-gal RNA. Fig. 5A
confirms that when embryos were injected in the VMZ with ß-gal RNA alone,
the posterior region was labelled but globin expression in the VBI was
unaffected. However, when tBR was co-injected along with the lineage tracer,
the progeny of the injected VMZ became dorsalised and ß-gal was now found
in the neural tube and somites of the second axis, with concomitant loss of
globin expression from the posterior VBI
(Fig. 5B). The cells that now
populated the posterior VBI region (arrowheads in
Fig. 5B) were derived from
cells that had not experienced the block to BMP signalling. Nonetheless these
cells were unable to express globin, presumably because they were derived from
a position in the embryo that had not been exposed to the correct signalling
environment required to set up the blood programme.
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When tBR RNA was injected into the DMZ, little alteration to the gross
phenotype was observed because this region is already low in BMP signalling.
However, the characteristic V-pattern of globin expressing cells in the
anterior blood island was lost and, at best, only a more diffuse patch typical
of the posterior VBI remained (Fig.
4, row 1, DMZ; Fig.
5C, lower embryo, arrowhead;
Table 1, tBR DMZ). Some
injected embryos lost globin expression in the posterior VBI completely
(Fig. 5C, upper embryo, arrow;
Table 1, tBR DMZ), however this
effect was less apparent with earlier blood markers and its significance is
currently not understood. Inclusion of the ß-gal lineage tracer confirmed
the presence of the dominant-negative BMP receptor in dorsal and anterior
structures including the anterior VBI (Fig.
5C). A reduction in globin production has been reported previously
following injection of tBR at the 32-cell stage into both C1 or C4
blastomeres, which are central to the DMZ or VMZ injected here
(Kumano et al., 1999). We
conclude that both component populations of the VBI require BMP signalling if
they are to develop as red blood cells.
In order to determine when in the primitive blood specification pathway BMP signalling is required, we monitored markers for early blood precursors, namely Xaml, SCL and GATA2. Similar results to those seen with globin were obtained with VMZ and DMZ injections, resulting in loss of posterior or anterior expression, respectively (Fig. 4, rows 2-4; Table 1; data not shown for GATA2). Thus, BMP signalling is required at an early stage in the specification of primitive blood. Furthermore, similar results were obtained with markers for endothelial cells, namely Xfli1 and XHex (Fig. 4, rows 5-8; red arrows identify the vitelline veins which link up anteriorly with the endocardium; black arrowheads indicate XHex expression in the liver). Thus, BMP appears to be essential for the specification of both the primitive blood and endothelium from mesoderm. We therefore conclude that both dorsal and ventral marginal zones fail to produce blood and endothelium in the VBI region when BMP signalling is blocked.
Xfli1 expression in embryonic/aVBI progenitors is not dependent on
BMP
We have presented evidence to indicate that blood and endothelium of the
aVBI derive from a population of progenitors located near the cement gland at
neurula stages. The absence of blood or endothelium in tailbud stage embryos
injected with tBR in the DMZ predicts that this population of progenitors
would be absent at neurula stages. To monitor this, we injected tBR RNA along
with the lineage tracer into the DMZ and probed the embryos at stage 18
(neurula) for SCL and Xfli1 expression. When the ß-gal lineage tracer was
located in the progenitor region posterior to the cement gland, SCL expression
was absent or strongly reduced (Fig.
5, compare E with D, 24/25 embryos). By contrast, Xfli1 expression
was retained in the absence of BMP signalling, with clear overlap between the
purple in situ hybridisation stain and the turquoise lineage tracer
(Fig. 5, compare F with G,
33/33 embryos). Thus, Xfli1 expression is turned on in a BMP-independent
manner. However, although inhibition of BMP signalling has no effect on Xfli1
expression at stage 18, the normal expression of Xfli1 at stage 26 in the
vitelline veins is not observed (Fig.
4, rows 5 and 6, DMZ; Table
1, tBR DMZ). Overall, these data suggest that, in the absence of
BMP signalling, normal progenitors for embryonic blood and endothelium
co-expressing SCL and Xfli1 are not formed, and that these cells are obligate
precursors to the blood and endothelium of the anterior VBI. BMP is required
in this progenitor population to activate SCL expression but not Xfli1.
In a parallel set of experiments, Walters et al.
(Walters et al., 2002)
concluded that inhibition of BMP signalling in DMZ derivatives has no effect
on SCL expression, as measured by northern blot. They therefore interpreted
the requirement for BMP to be in the differentiation of erythroid cells rather
than the initial formation of haematopoietic tissue. In our hands, SCL
expression does not survive the blockade to BMP signalling. Furthermore we
monitored the survival of the injected tBR RNA using the ß-globin UTRs as
tags in an RNase protection assay (Fig.
6). We found that injected RNA remains abundant through
gastrulation but during neurula stages undergoes significant turnover, so that
by stage 18 it has essentially disappeared. This implies that its effect
precedes the appearance of the embryonic blood and endothelial progenitors.
The loss of endothelial cells as well as blood at tailbud stages is also
consistent with a failure to make these precursors.
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BMP signalling is required for DLP formation
The DLP blood and endothelial progenitor compartment derives from the VMZ
along with the pVBI (Ciau-Uitz et al.,
2000). Whereas tBR injected into VMZs eliminated blood and
endothelial expression in the pVBI, expression of all these markers in the DLP
was unaffected (black arrows in Fig.
4, rows 2-7, VMZ and
Table1, tBR VMZ). However, when
we injected a lineage label along with tBR into the VMZ, the label was located
in the neural tube and somites, and did not extend more ventrolaterally into
the DLP region (Fig. 5B). Thus
the progenitor population in the DLP appeared not to have experienced an
inhibition of BMP signalling. When we injected the tBR and ß-gal RNA more
laterally into the ventrolateral marginal zone (VLMZ), we were able to deliver
the lineage label and dominant negative receptor into the region of the DLP.
With this more lateral injection, double axes were still generated
(demonstrating that active tBR RNA had spread to the ventral midline) but
lineage label was often found in both axes of the embryo instead of solely in
the second axis, as seen with VMZ injections. In addition the embryos were
usually very foreshortened lacking posterior structures. At stage 28,
expression of SCL in the DLP was compromised in all embryos (n=10).
Six out of ten embryos lost all signal for SCL in the DLP
(Fig. 5H, arrowheads) and pVBI,
while retaining expression in the aVBI
(Fig. 5H, arrow). In the
remaining four embryos, as well as a robust SCL signal in the aVBI, a few
SCL+ cells remained in the DLP region. However, when these embryos
were sectioned, we found no overlap of SCL signal and lineage label in the DLP
region (data not shown), indicating that, as tBR acts cell autonomously, those
SCL expressing cells that remained had not experienced a block in BMP
signalling. When we probed embryos injected in the VLMZ with tBR and
ß-gal RNA for expression of the endothelial marker Xfli1, we found no
Xfli1 signal overlapping the ß-gal signal (23/24 embryos,
Fig. 5I, arrowheads). As for
SCL, the aVBI acted as a positive control
(Fig. 5I, arrow). Similar loss
of expression in the DLP was observed for the pronephric duct gene, Xlim1
(Fig. 5J, arrowheads, 24/24
embryos). Whenever the lineage label missed the DLP, expression of all these
genes occurred normally (see, for example,
Fig. 5K, arrowheads). Thus, we
conclude that, as for the embryonic lineage, the blood and endothelial
progenitor population in the DLP, along with the associated pronephric duct
progenitors, is dependent on BMP signalling for its integrity. However, in
contrast to the embryonic population, progenitors in the DLP are dependent on
BMP signalling for expression of both Xfli1 and SCL.
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DISCUSSION |
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Both populations of progenitors can differentiate into endothelium in situ but migrate to different locations before differentiating into haematopoietic cells. Thus, a subpopulation of the embryonic progenitors stay where they are and differentiate into the endothelial cells of the endocardium and vessels in this region, while the remainder of the population migrate posteriorly into the aVBI and give rise to the embryonic blood and the vitelline veins. Similarly, a subpopulation of the adult progenitors in the DLP stay lateral giving rise to the posterior cardinal veins, while the remainder migrate to the midline and form the dorsal aorta and the haematopoietic clusters. Thus, it appears that the micro-environments in which the two populations of progenitors are formed are unsuited to haematopoietic differentiation.
Distinct programming of the VBI and DLP progenitors
Analysis of gene expression in the two progenitor populations investigated
here reveals differences between them in the temporal appearance of the genes
tested. Thus, whereas GATA2 appears to be the earliest gene expressed in the
mesoderm giving rise to the VBI (Bertwistle
et al., 1996), it is preceded by Xfli1 in the DLP (this study).
Furthermore, although XHex is expressed before SCL in the precursors of the
aVBI (Jones et al., 1999
), it
comes on at the same time as SCL in the DLP (this study). Finally, Xaml is
expressed early in the aVBI precursors (this study), but only in the
derivatives of the DLP after they have migrated to the midline and the dorsal
aorta has been formed (Ciau-Uitz et al.,
2000
). Therefore, the temporal cascades of gene expression in the
adult/DLP and embryonic/aVBI progenitor populations suggest that distinct
programming is taking place.
The separate origins of the adult/DLP and embryonic/VBI progenitors in the
embryo (Ciau-Uitz et al., 2000)
also raised the possibility that the cells might come under the influence of
distinct signalling regimes as they move through the embryo to their sites of
differentiation. To test this, we perturbed a signal thought to be important
in blood formation: BMP. We found programming differences between the adult
and embryonic populations in terms of the dependence of Xfli1 expression on
BMP signalling. Thus, while expression of this gene requires BMP signalling in
the adult/DLP progenitors, it is activated without BMP in the embryonic/aVBI
progenitor population. Its later expression in the endothelial derivatives of
the embryonic/aVBI progenitors, however, is not maintained in the absence of
BMP. Although the significance of this difference is not currently known, it
is intriguing to note that the cloche mutation in zebrafish, which
affects both blood and endothelium and has not yet been identified
(Stainier et al., 1995
),
similarly fails to prevent the initial expression of Fli1 while abolishing its
later expression (Brown et al.,
2000
). This similarity raises the possibility that cloche
encodes a protein in the BMP signalling pathway. The observation that
cloche behaves both cell-autonomously and cell non-autonomously
(Stainier et al., 1995
) could
be consistent with this notion if one takes account of the positive-feedback
loop involved in maintenance of BMP expression
(Jones et al., 1992
). Finally,
we have found that the enhancer that drives expression of the mouse
Scl gene in blood progenitors and endothelial cells, drives
expression of a reporter gene in transgenic Xenopus embryos in
adult/DLP progenitors (Gottgens et al.,
2002
). This enhancer has binding sites for the transcription
factors Fli1, GATA2 and Elf1 in multipotential blood progenitors, and all of
these sites are required for the activity of the enhancer in the DLP. In
adult/DLP progenitors, the loss of Xfli1 expression as a consequence of
blocking BMP signalling is therefore a likely explanation for the loss of SCL
expression. Such a scenario is supported by the timing of expression of these
two genes in the DLP with Xfli1 preceding the appearance of SCL
(Fig. 3B). If SCL expression is
driven by the same enhancer in embryonic/aVBI progenitors, its loss at neurula
stages when BMP signalling is blocked cannot be explained by an effect on
Xfli1 whose expression is maintained. However, activity of the enhancer also
depends on GATA2 binding, the expression of which in the aVBI progenitors does
require BMP signalling (data not shown).
Timing of the BMP requirement and the role of dorsoventral
patterning
A number of observations suggest that the BMP requirement for all three
populations of blood and endothelial cell precursors is during gastrulation.
Firstly, Kumano et al. demonstrated a profound inhibition of globin expression
in the VBI as a result of injection of BMP antagonists into the A4 blastomere
at the 32 cell stage (Kumano et al.,
1999). This blastomere contributes to ventral anterior ectoderm, a
rich source of BMP. The gastrulating leading edge mesoderm, which gives rise
to the VBI, migrates towards the ventral midline finally contacting the
ventral anterior ectoderm at the end of gastrulation
(Keller, 1991
;
Bauer et al., 1994
). Second,
our studies of the DLP progenitor population show that it develops in close
association with precursors of the pronephric duct, which also require BMP
signalling. The pronephric duct has been shown to be specified no later than
stage 14, which is soon after gastrulation, suggesting that BMP is required
earlier (Brennan et al., 1998
;
Brennan et al., 1999
). Finally,
our time course on the stability of injected dominant-negative BMP receptor
RNA is consistent with a requirement for BMP during or soon after
gastrulation.
We suggest that the dorsoventral gradient of BMP activity thought to exist
during gastrulation (Dosch et al.,
1997) is important in specifying the different blood and
endothelial compartments (Fig.
7A). At the highest level of BMP signalling the posterior VBI is
specified; at an intermediate level BMP specifies DLP precursors. Both of
these suggestions are consistent with the gradient model and, in the case of
the posterior VBI, fit the many experimental results linking blood formation
with high BMP (Dale, 1992; Jones et al.,
1992
; Dosch et al.,
1997
; Maeno et al.,
1994b
). However, the specification of the aVBI requires more
explanation. These cells derive from the dorsal marginal zone, specifically
blastomeres C1 and D1, which also give rise to Spemann's organiser. The cells
of the organiser are characterised by the expression of low levels of BMPs and
high levels of BMP antagonists such as chordin and noggin. However, this
region contains the first cells to involute during gastrulation
(Keller, 1991
) and, therefore,
at an early stage during gastrulation, they will be proximal to high levels of
BMP signalling in the animal cap (Fainsod
et al., 1994
; Maeno et al.,
1994b
). We suggest that it is at this stage that C1 and D1 progeny
become committed to the blood and endothelial programmes
(Fig. 7B).
|
In addition to this exposure to BMP in the animal cap soon after the start
of migration, the initial location of the aVBI precursors in the low BMP
organiser territory may also be important for their specification. Three lines
of evidence support this. First, Tracey et al. showed that treatment of
Xenopus single cell embryos with ultraviolet light, which prevents
cortical rotation and establishment of the Spemann organiser, resulted in the
loss of the aVBI (Tracey et al.,
1998). Second, Walters et al. have shown that the injection of
BMP4 RNA into the DMZ can lead to a decrease in globin expression as well as
an increase depending on the dose (Walters
et al., 2002
). Thus, at high BMP4, as found by others
(Maeno et al., 1994b
;
Hemmati-Brivanlou and Thomsen,
1995
; Maeno et al.,
1996
), all the mesoderm, including axial, paraxial and
dorsolateral plate was converted to ventral mesoderm (which, in this case, is
represented by the pVBI) and therefore globin production was increased.
However, when a moderate dose of BMP4 was injected, reflected by an
intermediate dorsoanterior index in the resulting embryos, the aVBI was
converted to more lateral mesoderm, possibly including DLP, which either does
not express globin or does so much later. Finally, the three blood
compartments seen in the early Xenopus embryo have possible parallels
in the zebrafish in the head, the trunk and the region posterior to the anus,
as defined by SCL expression (Gering et
al., 1998
). In the chordino mutant, which lacks the product of the
BMP antagonist, chordin, the anterior haemangioblast compartment is reduced
while the posterior compartment is expanded (M. Gering and R. P.,
unpublished). In addition, overexpression of BMP leads to a complete loss of
this anterior population (Liao et al.,
2002
). Thus, as in Xenopus, the anterior population in
zebrafish appears to require conditions of low or zero BMP at some time in its
ontogeny.
Conclusions
In conclusion, we have presented combined gene expression and lineage
tracing evidence that both adult and the anterior component of embryonic blood
in Xenopus embryos derive from populations of progenitors that also
give rise to endothelial cells. By targeting a dominant-negative BMP receptor
RNA to distinct regions of the embryo that give rise to these progenitor
populations, we have shown that BMP is needed for the formation of blood and
endothelium in both embryonic and adult compartments. Programming by BMP of
these blood and endothelial precursor populations most probably occurs during
gastrulation. Expression of the endothelial gene, Xfli1, is not BMP dependent
in embryonic progenitors but is dependent on BMP in adult progenitors, while
the blood gene, SCL, is dependent on BMP signalling in both compartments.
These data identify for the first time differential programming by embryonic
signals of the adult and embryonic blood and endothelial lineages.
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ACKNOWLEDGMENTS |
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Footnotes |
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