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First published online 3 August 2006
doi: 10.1242/dev.02497
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1 Department of Developmental Biology (VIB7), Flanders Interuniversity Institute
for Biotechnology (VIB) and Laboratory of Molecular Biology (Celgen),
University of Leuven, B-3000 Leuven, Belgium.
2 Hubrecht Laboratory, Netherlands Institute of Developmental Biology, Utrecht,
The Netherlands.
3 Thromb-X S.A., Leuven, Belgium.
Author for correspondence (e-mail:
an.zwijsen{at}med.kuleuven.be)
Accepted 15 June 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Allantois, Amnion, Bmp, Chimera, PGC, Smad5/Madh5, Stem cell, Tgfß
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Parameswaran and Tam, 1995;
Kinder et al., 1999). Genetic
evidence and epiblast culture experiments have shown that germ cell competence
and formation of the allantois are induced in the proximal epiblast before
gastrulation, in response to bone morphogenetic proteins (Bmps) produced in
the extra-embryonic ectoderm, but depend also on the presence of visceral
endoderm (reviewed by Zhao,
2003; de Sousa Lopes et al.,
2004).
Bmps are ligands of the transforming growth factor ß (Tgfß)
family that use various receptor complexes to directly activate intracellular
effector proteins (Smad1/5/8). Smads transmit the signal to the nucleus and
participate in the regulation of target gene expression
(Shi and Massagué,
2003). Analysis of conventional knockout mice showed that the
repertoire of Bmp signalling proteins involved in PGC induction comprise Bmps
(Bmp2, -4 and -8b), a type I Bmp receptor (Alk2; Acvr1 - Mouse Genome
Informatics) and two Bmp-Smads (Smad1/5)
(Lawson et al., 1999;
Ying et al., 2000;
Ying et al., 2001;
Ying and Zhao, 2001;
Chang and Matzuk, 2001;
Tremblay et al., 2001;
Hayashi et al., 2002;
de Sousa Lopes et al., 2004;
Okamura et al., 2005).
Recently, it was shown that Blimp1 (Prdm1 - Mouse Genome Informatics), a
transcriptional regulator thought to be induced by Bmp4, is a key regulator of
germ cell specification (Vincent et al.,
2005; Ohinata et al.,
2005). Subsequent PGC localization and survival, as well as
allantois differentiation, also depend on the presence of Bmp4 in the
extra-embryonic mesoderm (Fujiwara et al.,
2001).
Several Bmps have been implicated in the specification and growth of
extra-embryonic mesoderm. The induction of the allantois crucially depends on
Bmp4 (Winnier et al., 1995;
Fujiwara et al., 2001), and on
Alk2-mediated signalling in visceral endoderm
(Mishina et al., 1999;
Gu et al., 1999). Bmp5;Bmp7
double mutant mouse embryos display impaired allantois maturation
(Solloway and Robertson,
1999). Bmp2 plays a unique role in amnion development, as the
pro-amniotic canal does not close in Bmp2 mutant mice, which has been
ascribed to reduced and thus insufficient production of extra-embryonic
mesoderm (Zhang and Bradley,
1996).
Smad1 and Smad5 null embryos die at mid-gestation and
have defects in PGC specification and allocation, and in the development of
extra-embryonic tissues (Chang et al.,
1999; Chang et al.,
2000; Yang et al.,
1999; Lechleider et al.,
2001; Tremblay et al.,
2001; Hayashi et al.,
2002; Umans et al.,
2003). The presence on the amnion of aggregates of cells that
contain ectopic primitive red blood cells, and endothelial and PGC-like cells,
is a unique feature of Smad5 mutants
(Chang et al., 1999;
Chang and Matzuk, 2001).
However, the molecular mechanism underlying this unique phenotype remained
unknown. Our present chimera study reveals that Smad5 functions
non-cell-autonomously in the amnion mesoderm. In addition, we observe ectopic
Bmp expression and signalling in the affected amnion. We therefore propose a
new model in which Smad5 deficiency paradoxically leads to gain of Bmp
function defects. Additional support for this model comes from injection of
rBMP4 protein in the exocoelom of wild-type embryos, which results in abnormal
thickening of the amnion.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) was
outcrossed in C57BL/6J and F2 crosses of F1 (C57BL/6J x CBA) background
for at least five generations. Rosa-ßgeo-26 mice, expressing
Escherichia coli lacZ ubiquitously
(Friedrich and Soriano, 1991),
were maintained in the F2 background. Embryonic stem (ES) cells were
established from blastocysts of Smad5+/m1 (C57BL/6J)
intercrosses as described (Schoonjans et
al., 2003), and genotyped by PCR and Southern blotting
(Chang et al., 1999).
Chimeras between ES cell lines and diploid Rosa-ßgeo-26 morulae were
generated as described (Goumans et al.,
1999). The majority of wild-type
WTlacZ,
Smad5+/m1WTlacZ, and of
Smad5m1/m1WTlacZ chimeric
embryos had a high ES cell contribution (>90%), with only few
acceptormorula derived, ß-galactosidase-positive cells present in hindgut
endoderm. The minority of
Smad5m1/m1WTlacZ embryos (four
out of 18) with a high contribution from the wild-type acceptor morula in
epiblast-derived tissues (20-80% ES cell-derived embryos) are here referred to
as low-percentage chimeras.
Animal care and experiments were approved by the Institutional Ethical Committee of the Catholic University of Leuven.
Collection of post-implantation embryos, in situ hybridization and immunohistochemistry
Embryos from timed matings were dissected in ice-cold PBS at embryonic day
(E) 7.0-9.5 and staged according to Downs and Davies
(Downs and Davies, 1993),
modified for C57BL/6 x CBA embryos (Edinburgh Mouse Atlas Project:
http://genex.hgu.mrc.ac.uk/Databases/Anatomy/MAstaging.shtml),
or on somite number. Radioactive in situ hybridization was performed as
described (Dewulf et al.,
1995). Non-radioactive in situ hybridization [Stella
(Dppa3 -Mouse Genome Informatics), fragilis (Ifitm3 - Mouse Genome
Informatics)] on sections and immunohistochemistry for P-ERK (cat. no 9101,
Cell Signalling Technologies) and P-Smad1/5/8 (cat. no 9511, Cell Signalling
Technologies) were performed with the automated Ventana Discovery system and
Ventana reagents according to the manufacturer's instructions. NBT/BCIP and
DAB were used as chromogens for automated non-radioactive in situ
hybridization and immunohistochemistry, respectively. We used antisense RNA
probes for Bmp2 (Lyons et al.,
1989), Bmp4 (Jones et
al., 1991), periostin/Osf2
(Delot et al., 2003),
Stella and fragilis (Saitou et
al., 2002), and Msx2
(MacKenzie et al., 1992).
Immunohistochemistry for Oct4 (Pou5f1 - Mouse Genome Informatics; sc5279,
Santa Cruz Biotechnology) and SSEA-1 (Fut4 - Mouse Genome Informatics; MC480
DHSB) was performed as described (Van
Eynde et al., 2004). AEC (Sigma) was used as a chromogen.
Detection and counting of PGCs
Dissection of embryos, followed by fixation, and detection of alkaline
phosphatase (AP)-positive PGCs with 
-naphthyl phosphate/Fast Red TR
(Ginsburg et al., 1990) in
whole-mount embryos or with ASMX/Fast Red TR in serial section, were as
described (Lawson et al.,
1999). PGCs were counted on the basis of the strong surface AP
staining and intensely stained cytoplasmic spot
(Ginsburg et al., 1990;
Lawson and Hage, 1994). The
anterior portion of the embryo was retained for genotyping
(Chang et al., 1999).
Measurements
Allantois dimensions were measured on intact embryos after staining for AP.
The axial length of the allantois was measured from the line of intersection
with the yolk sac when viewed posteriorly and from the junctional line between
amnion and yolk sac when viewed laterally. Width at the base was measured in
posterior view along the intersection with the yolk sac.
The length of the anterior-posterior embryonic axis was estimated on images of intact embryos stained for AP and viewed laterally. Axis length was taken as the sum of the three longest cords along the curved axis - from the anterior embryo/amnion junction or the most proximal edge of the neural fold, from the intersection of amnion, allantois and primitive streak to the node, and a cord connecting these two.
Injection of rBMP4 and whole embryo culture
CD1 embryos between neural plate (NP) and headfold (HF) stages were
isolated in 22 mmol/l Hepes and 10% heat-inactivated fetal calf serum (FCS;
Hyclone); Reichert's membrane was removed, but yolk sac integrity with the
ectoplacental cone was maintained. Whole embryo culture and manipulations were
in Dulbecco's modified essential medium (Gibco), 1 mmol/l glutamine,
non-essential amino acids, 0.5 mmol/l Napyruvate, 50% heat-inactivated horse
serum (Gibco) in a rolling culture system at 37°C and 5% CO2
for 15 to 24 hours (0.5 ml medium per embryo). Embryos were either control
injected or injected with 1 µg human rBMP4/ml PBS (R&D Systems) with a
mouth-controlled glass capillary in the exocoelom (1-2 nl; corresponding to
about one-tenth of the luminal volume of the exocoelom). Control injected
embryos developed normally, as judged by embryo viability, somite number,
axial rotation, allantois elongation and fusion, and neural tube closure. Yolk
sac vasculogenesis appeared normal, but the yolk sac was sometimes enlarged
when compared to freshly isolated embryos. Only embryos in which the heart was
beating were assessed for amnion development. Embryos were fixed and processed
for paraffin embedding.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), although
relatively fewer mildly affected embryos were recovered and anterior defects
were more pronounced (data not shown). The very first defects visible in
Smad5 mutants were the delayed appearance of the allantois at late
streak (LS) stages (P<0.01 with Wilcoxon's summed ranks test)
(Fig. 1A) and the relatively
posterior closure of the amnion visible in late streak-early bud (LSEB) to HF
stage embryos (Table 1, data
not shown). The delayed appearance of the allantois was followed initially by
normal extension until the late neural plate (LNP) stage. There was negligible
extension between the LNP and 1-2 somite (S) stage, but cellular material
accumulated into the base of the allantois, expanding its width
(Fig. 1B) and producing
irregularities. Normal extension was resumed after the 1/2S stage until fusion
with the chorion at 
6S stage, which occurred in most mutants with an
irregularly shaped and short allantois
(Fig. 1A,B,D,E). Initiation of
the allantois was also delayed in the heterozygotes at LS stages
(P<0.01) (Fig. 1A);
extension was then parallel to that of the wild type until chorion-allantoic
fusion. This phenotype is very similar to that in Bmp4 heterozygotes
(see Fig. S1A in the supplementary material). The delay in allantoic
initiation and the later growth defect in the Smad5 mutant are not
due to general growth retardation, as shown by relating allantois length to
embryonic axis length (Fig. 1C,
Fig. S1B): the differences remained.
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) (K.A.L.,
unpublished). The amnion was markedly thickened in
Smad5m1/m1 embryos
(Table 1). The amnion of
somite-stage mutants typically contained aggregates of cells
(Table 1) that were often
localized very anteriorly at a remote distance from the allantois. From the 5S
stage onwards, Smad5m1/m1 embryos had an underdeveloped
forebrain and heart when compared with stage-matched control littermates, and
an aggregate of cells was visible on the amnion in more than half of the
mutants (Fig. 1D,E). Failure of
embryo turning, defects in heart looping and dilation of embryonic and yolk
sac blood vessels were as previously described
(Chang et al., 1999;
Chang et al., 2000).
|
) described a Smad5 gene dosage effect, reminiscent
of, although less pronounced than, the Bmp4 null allele, on the size
of the PGC founder population in a 129SvEv/C57 mixed genetic background. We
confirm these observations now in the C57BL/6J and F2 background (see Fig. S2
in the supplementary material), and have determined that the incidence and
number of PGCs is already reduced in Smad5 mutants at the LS stages,
with an intermediate effect in the heterozygotes
(Fig. 1F). Chang and Matzuk
(Chang and Matzuk, 2001)
observed AP- and Oct4+ cells in the aggregate of cells in the
amnion of early somite-stage embryos, and concluded that they are mislocated
PGCs. We explored the timing of appearance of these PGC-like cells in the
amnion. Although half of the NP up to the 4S stage embryos had defects in
allantois development, amnion closure and amnion thickening, no AP+
PGC-like cells were seen in the Smad5m1/m1 amnion at these
stages (Table 1), or elsewhere
outside the normal PGC domain. Only after the 5S stage, did we find
AP+ cells in some of the distinct aggregates of cells on the anmion
(Fig. 1H,I,J;
Table 1). These were located in
the part of the aggregate projecting into the amniotic cavity and therefore
associated with amnion ectoderm (Fig.
1J), unlike the ectopic haematopoietic and endothelial cells that
bulged into the mesoderm side of the amnion facing the exocoelom. By contrast
to previous data (Chang and Matzuk,
2001), we found that AP+ clumps were still prominent at
E9.5 (11/14 embryos: 16-28S) (Fig.
1H,J; Table 1). The
sudden appearance of PGC-like cells in appreciable numbers in the aggregates
(33 to 119 PGC-like cells in 5S-12S embryos; 142 cells in the aggregate of the
26S mutant shown in Fig. 1H,J),
and the anterior position of the aggregates containing them, make it unlikely
that they are PGCs mislocated from the founder population, and supports the
argument for a change in cell fate in situ in the mutant amnion.
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)
(Fig. 2A). Wild-type and
non-affected Smad5m1/m1amnion was always devoid of Oct4
protein (Fig. 2B-D), but the
cell aggregates often contained Oct4+ cells
(Fig. 2C,D). At E6.5, fragilis
(Ifitm3/Mil1) is expressed in proximal epiblast
(Saitou et al., 2002), the
region in which PGC-competent cells reside
(Lawson and Hage, 1994).
During gastrulation, fragilis expression increases within a cluster of cells
at the base of the allantoic bud. At E7.5 (early bud stage) cells with the
highest expression of fragilis initiate at the germ cell-characteristic
expression of Stella/PGC-7/Dppa3
(Saitou et al., 2002;
Tanaka et al., 2004;
Tanaka et al., 2005). Fragilis
expression level in non-thickened amnion was comparable to wild-type amnion.
In the aggregate, an ectopic fragilis expression domain was observed that
contained Oct4+ cells, but exceeded this domain (Fig.
2G,H, compared with
2C,D). Although we observed
Stella-expressing PGCs in the hindgut of wild-type and
Smad5m1/m1 embryos
(Fig. 2I, data not shown), we
did not find any Stella-expressing cell in the cell aggregates in the
Smad5m1/m1 amnion (Fig.
2K,L, data not shown).
Mouse SSEA-1 is expressed in epiblast, postgastrulation ectoderm and
endoderm, and in migrating PGCs (Fox et
al., 1981). We detected expression in visceral and gut endoderm,
and in surface ectoderm (Fig.
2M,N), but at E8.5, PGCs in the hindgut were still SSEA-1 negative
(Fig. 2M)
(Donovan et al., 1986).
Remarkably, Smad5m1/m1 embryos had a robust SSEA-1
staining throughout the entire ectoderm, and sporadically also in the
mesoderm, of the bilayered amnion (Fig.
2N,O). In addition, we observed that SSEA-1+ regions
largely overlapped with fragilis+ cells of the aggregate
(Fig. 2N,O).
Based on this marker analysis we identified several distinct areas in Smad5m1/m1 mutant amnion (Fig. 2Q): (1) SSEA-1+ amnion with normal appearance resembling surface ectoderm; (2) SSEA-1+ and fragilis+ areas in the cell aggregates, and within these areas; (3) SSEA-1+, fragilis+, Oct4+, AP+ cells that did not express Stella. Further co-localization and marker studies may rule out whether cells exist that are either SSEA-1+ or fragilis+, and unravel the precise provenance and identity of the cells. The absence of Stella expression rules out the possibility that the AP+ cells are fully specified PGCs; the expression pattern found is compatible with reversion by the mutant amnion to a pluripotent state. At the mesodermal side of the aggregate facing the exocoelom, cells could be detected that did not express any of the PGC/epiblast/ES cell markers.
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WTlacZ chimeras (14
out of 18) of Smad5m1/m1 ES cells in wild-type
lacZ transgenic (WTlacZ) host embryos were high
chimeras with >90% mutant cells in the epiblast derived tissues, the
low-percentage chimeras (estimated between 20 and 80% chimerism, depending on
the embryo) were very instructive. They had the typical mutant phenotype in
the extra-embryonic tissues, with large cell aggregates in the amnion
(Fig. 3A,B).
Smad5m1/m1 cells made, just like descendants from
wild-type ES cells (Fig. 3C),
an unbiased contribution to the mesodermal and ectodermal components of the
amnion (Fig. 3D,E).
Intriguingly, the aggregates in low-percentage chimeras were not composed
solely of disorganized mesodermal cells, which we anticipated from our earlier
work (Chang et al., 1999), but
the amnion ectoderm was also sometimes thickened, and was even entirely of
wild-type origin in one chimera (Fig.
3E). This suggests that loss of Smad5 in the mesoderm affects not
only the mesodermal but also the ectodermal component of the amnion, and
attributes a non-cell-autonomous function to Smad5 in mesoderm. Based on this
result, we re-evaluated the expression of Twist (amnion mesoderm) and
AP-2 (amnion ectoderm) in Smad5m1/m1
amnion. Aggregates of cells are predominantly of mesodermal origin but
sometimes contain locally a thickened Twist negative and
AP-2 positive ectodermal component
(Fig. 3F,G).
Loss of Smad5 leads to an increase in expression of Bmp genes and Bmp target genes in the amnion
The expansion of the amnion may result from a change in either cell
proliferation or cell shape from a columnar or cuboidal to an extreme squamous
morphology. Bmp2 has been implicated in amniotic expansion, but the mechanisms
remain elusive (Zhang and Bradley,
1996). We evaluated the expression of Bmp genes, and some
of their target genes and modulators. Bmp2 transcript levels were
extremely low in the wild-type amnion at E8.5, if at all above background
(Fig. 4A,B), whereas
Bmp4 expression was detectable in amnion
(Fig. 4E). A dramatic increase
of Bmp2 and Bmp4 transcripts was observed in the
Smad5m1/m1 amnion. Bmp2 was upregulated in the
cells of the aggregates and neighbouring unaffected amnion, but not in
squamous amnion distant from the aggregate
(Fig. 4C,D). Bmp4 was
significantly upregulated, also in squamous amnion
(Fig. 4F), but the expression
of Bmp7 appeared unaffected (data not shown). During gastrulation,
Bmp4 expression is dynamic. Expression persists within the
extra-embryonic ectoderm, and Bmp4 transcripts become detectable in
the extra-embryonic mesodermal components of the amniotic folds, and
subsequently in the amnion, yolk sac and chorion that line the exocoelom, as
well as within the allantoic bud/allantois
(Lawson et al., 1999) (data
not shown). At the NP stage, before detectable thickening of the mutant
amnion, Bmp4 levels appeared already more robust in the amnion of
mutants when compared with control littermates
(Fig. 4G-J). Bmp2
expression was low at this stage of amnion development, without clear
differences between mutants and controls
(Fig. 4K,L).
|
;
Qi et al., 2004). The pattern
of phosphorylated (P)-ERK1/2 staining at E7.5 and 8.5 in wild-type littermates
is very variable and reflects the dynamic nature of phosphorylation-dependent
signalling cascades (data not shown). At E7.5 and 8.5 P-ERK1/2-positive cells
are very rare in the wild-type amnion (Fig.
5A, data not shown). No dramatic increase in P-ERK1/2 staining was
observed in non-thickened amnion (Fig.
5B,C, data not shown); however, manifest ERK signalling was
observed within the aggregates. Likewise, the P-Smad1/5/8 staining pattern was
very dynamic in E7.5 and 8.5 wild-type littermates, and could be observed only
sporadically in amnion (Fig.
5D). In the Smad5m1/m1 amnion enhanced
P-Smad1/8 staining was most evident in the aggregate
(Fig. 5E,F). These results
illustrate enhanced Bmp signalling in the mutant amnion.
We then analysed the expression of acknowledged Bmp target genes in the
Smad5m1/m1 amnion. Periostin/Osf2 is involved in
cell adhesion and spreading
(Kruzynska-Frejtag et al.,
2001), and at E8.5 its only sites of expression are the amnion and
the yolk sac (Fig. 5G). In the
Smad5m1/m1 amnion, periostin expression was more
pronounced, although excluded from the aggregate of cells
(Fig. 5H). Msx2 is
expressed in surface ectoderm (Monaghan,
1991) but barely in amnion
(Fig. 5I). Msx2 is
robustly expressed in the mutant amnion, predominantly in its ectodermal
component (Fig. 5J). This shows
that higher Bmp2/4 levels indeed result in increased expression of
Bmp target genes in the Smad5m1/m1 amnion. Genes encoding
Bmp antagonists like noggin, chordin and Smad7 are expressed at low
levels in the amnion and no significant changes in their expression domain
were detected in Smad5m1/m1 embryos (data not shown).
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The delay in allantoic bud formation in Smad5 mutants, and the
reduced incidence and number of PGCs at the LSEB stage, reflects a well
documented loss of Bmp signalling effect initiated in the extra-embryonic
ectoderm and visceral endoderm (reviewed by
Zhao, 2003). The relatively
short allantois in Smad5 heterozygotes, and in Bmp4
heterozygotes, is a consequence of the slight delay in allantois initiation,
rather than an effect on cell recruitment to, and proliferation in, the bud.
Absence of Smad5 results in abnormal morphogenesis of the allantois after
delayed initiation and early normal extension. Failure to extend further
during the rest of the cell recruitment and proliferation phases
(Downs and Bertler, 2000), and
the accumulation of material in the base of the allantois, suggest that cell
redistribution may be more affected than the addition of cells. The resumption
of extension after the 2S stage coincides with the normal end of recruitment
and rapid proliferation and the onset of extension by cavitation and
vascularization (Downs and Bertler,
2000). This process appears to be Smad5-independent or open to
functional compensation, presumably by Smad1 or Smad8. These
latter Bmp-Smads are also expressed in the amnion and allantois
(Tremblay et al., 2001;
Hayashi et al., 2002) and
could implement its closure, the delay of which is probably caused by reduced
Bmp2 signalling during an earlier phase of amnion development
(Zhang and Bradley, 1996).
A more striking Smad5m1/m1 defect is the ectopic
vasculogenesis, haematopoiesis and development of Oct4+ and
AP+ cells that can be observed in the aggregate of cells in the
Smad5m1/m1 amnion. Previously, we proposed an
exclusive mesodermal origin for these aggregates of cells
(Chang et al., 1999). New
evidence from the low-percentage chimeras showed that the aggregates are not
exclusively comprised of mesodermal cells but occasionally also contain
thickened ectoderm. The mesodermal part of the aggregates in mutants can
result from altered proliferation, disorganization or misdifferentiation of
amniotic mesoderm cells, and/or from the previously proposed mislocation of
allantois tissue (Chang et al.,
1999). These aggregates are remarkably often localized very
anteriorly, at a remote distance from the allantois, but the mesoderm in the
aggregates expresses Tbx2 that is also expressed by allantois tissue
(Chang et al., 1999). However,
in the murine allantois vasculogenesis occurs without haematopoiesis
(Downs et al., 1998), whereas
the Smad5 mutant amnion contains not only flk-1 expressing
endothelial cells, but also 
-globin-expressing primitive blood
cells (Chang et al., 1999).
Hence, the amnion adopts allantois and blood island-like fates in the absence
of Smad5.
|
;
Fujiwara et al., 2001).
Therefore, it is very unlikely that AP+ cells are swept into
ectopic locations immediately after PGC allocation at E7.2 in
Smad5m1/m1 embryos and remain unrecognized. Our analysis
is suggestive of an in situ change in fate of amnion cells.
The AP+ cells are unlikely to be de-novo induced, bona fide PGCs
because no Stella-expressing cells were ever observed in the
aggregates. Although the coexpression of AP, Oct4, and SSEA-1 could indicate
the presence of pluripotent, ES-like cells, the combination of AP, Oct4,
SSEA-1 and fragilis expression is more reminiscent of an earlier developmental
state; PGC-competent epiblast. Fragilis is a direct target gene of
extra-embryonic-derived Bmp4 in the PGC-competent epiblast
(Saitou et al., 2002). The
ectoderm of non-affected Smad5 mutant amnion has characteristics of
surface ectoderm (SSEA-1+ and Msx2+). Despite
this reminiscence of surface ectoderm, the amnion ectoderm expresses also
periostin, a gene normally not expressed in surface ectoderm.
Remarkably, when isolated human amnion membrane cells are cultured, they
also express Oct4 and the epiblast/stem cell/PGC marker
Nanog (Miki et al.,
2005). These isolated human cells have the potential to
differentiate to all three germ layers - endoderm (hepatocytes), mesoderm
(cardiomyocyte) and ectoderm (neural cells) - in vitro
(Sakuragawa et al., 2004;
Tamagawa et al., 2004;
Zhao et al., 2005).
Smad5 deficiency results in gain-of-Bmp-function defects
The Smad5m1/m1 amnion defect is accompanied by a
specific and robust upregulation of Bmp2 and Bmp4, whereas
the expression of Bmp7 appears unaffected. This suggests that Smad5
is normally a predominant Bmp-Smad in the amnion that, directly or indirectly,
negatively regulates Bmp2/4 expression. Ectopic Bmp4
expression is readily visible before local thickening of the amnion at the
LSEB stage, and subsequently in the aggregate of cells, but also in the
neighbouring, seemingly unaffected amnion.
The mutant amnion displays elevated Smad-dependent (Bmp-regulated
phospho-Smad1/5/8) and Smad-independent (P-ERK1/2) signalling. Enhanced Bmp
signalling is also demonstrated by alterations in the expression pattern of
target genes of Bmp. A patent upregulation of periostin can be appreciated in
seemingly unaffected mutant amniotic cells. Tbx2
(Chang et al., 1999), fragilis
and Msx2 are, unlike periostin, highly upregulated in the aggregate
of cells in the mutant amnion. Msx2 is predominantly expressed in the
ectoderm component of the aggregate and non-thickened amnion. The increased or
ectopic expression of these Bmp target genes in distinct but overlapping
domains may indicate that expression of these target genes requires different
dose windows of (combined) Bmp(s), which is reminiscent of the different doses
of Bmp4 signalling that are required for dorsoventral patterning of mesoderm
in Xenopus embryos (Dosch et al.,
1997). A localized source of ligand(s) or antagonists/modulators,
or several sources of ligands (e.g. Bmp2 and Bmp4) could establish graded
signalling. Alternatively or additionally, differences in presence and
concentration of signalling components, but also unexplored endogenous
differences in affinities of Smad1, and Smad8 and non-Smad cascades to
activated receptors, Smad-interacting proteins and their shared target genes,
will determine the outcome of Smad5 deficiency in different regions of the
amnion.
The Smad5m1/m1 amnion acquires features of different
Bmp-sensitive lineages, such as allantois and blood island tissue but also
PGC-competent epiblast (Lawson et al.,
1999) (reviewed by Zhao,
2003; Fujiwara et al.,
2001). In the mutant amnion, epiblast-like cells are localized
near the ectodermal component, whereas ectopic endothelial and haematopoietic
cells are positioned at the exocoelomic side of the aggregate. Concurrent with
the differential ectopic expression patterns of several Bmp target genes, this
regionalization of the aggregates makes it conceivable that cells indeed
interpret different levels of Bmp signalling - whether quantitative and/or
qualitative - that determine their subsequent fate
(Fig. 7). Bmp4 is not only
implicated in differentiation of different cell lineages, but it is also
pivotal in maintaining self-renewal of ES cells
(Ying et al., 2003;
Qi et al., 2004).
Our gain-of-Bmp-function model is especially challenging, as no other mutant mouse model of a Bmp signalling component has been associated so far with ectopic vasculogenesis, haematopoiesis and development of (PGC-competent) epiblast-like cells in the amnion. Evidence that gain of Bmp function may indeed underlie the Smad5 mutant amnion phenotype comes from the induction of amnion thickening in wild-type amnion when rBMP4 is topically administered. Single injection of rBMP4 in the exocoelom compared to constitutive secretion of Bmp4 and Bmp2 in Smad5 mutants is just one out of many explanations why clumps could be significantly smaller in rBMP4-injected embryos than in mutants. The converse experiment, in which Smad5m1/m1 embryos would be rescued by injecting Bmp antagonists such as Noggin and/or Chordin, is appealing. However, it is far more complicated to accomplish, as systemic administration of excess amounts of antagonists would be required to neutralize endogenous Bmps.
Our study has revealed novel aspects of Smad5 signalling, i.e. a non-cell-autonomous function for Smad5 in amnion mesoderm and an in situ change in amnion cell fate upon Smad5 deficiency, and links these to an unexpected gain-of-Bmp-function model that is supported by the exogenous rBMP4 phenocopy experiment. Amnion cultures may be exploited in the future to dissect and to clarify the Bmp signalling pathway that determines change in cell fate of the amnion.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/17/3399/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: MRC Human Genetics Unit, Western General Hospital,
Edinburgh, UK
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REFERENCES |
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