1 Molecular Embryology Group, MRC Clinical Sciences Centre, Imperial College
London, Hammersmith Hospital Campus, Du Cane Road, London W12 ONN, UK
2 Division of Mammalian Development, National Institute for Medical Research,
Mill Hill, London NW7 1AA, UK
3 Wellcome Trust/Cancer Research UK Institute and Department of Zoology,
University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK
¶ Author for correspondence (e-mail: tristan.rodriguez{at}csc.mrc.ac.uk)
Accepted 15 March 2005
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SUMMARY |
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Key words: Mouse, Extra-embryonic ectoderm, AP patterning, Anterior visceral endoderm, Migration
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Introduction |
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Accumulating evidence indicates that reciprocal interactions between the
epiblast, VE and ExE are essential for establishing both these signalling
centres. For example, recombination experiments
(Yoshimizu et al., 2001),
analysis of mice mutant for the Tgfß factor Bmp4
(Lawson et al., 1999
) and for
its receptor Alk2 (de Sousa Lopes
et al., 2004
), and expression of a constitutively active form of
Alk2 in the VE (de Sousa Lopes et al.,
2004
) have identified a role for the ExE and proximal VE in the
induction of primordial germ cells in the proximal epiblast. Similarly,
analysis of mutations in the Tgfß family member Nodal and its
intracellular signal transducer Smad2, have shown that Nodal signals
from the epiblast are essential for inducing the AVE in the distal tip of the
embryo at 5.0 dpc and for maintaining gene expression in the ExE
(Brennan et al., 2001
).
Lineage analysis (Thomas et al.,
1998) and in vivo time lapse imaging
(Srinivas et al., 2004
) have
shown that shortly after its induction, and in response to unknown cues, the
cells of the AVE migrate from the distal tip to the prospective anterior of
the embryo. Once the AVE has migrated to the prospective anterior, it plays a
crucial role in restricting proximal epiblast markers to the posterior, either
by repressing expression of these markers in the anterior epiblast or by
causing epiblast cells to move to the posterior of the embryo
(Kimura et al., 2000
;
Lu and Robertson, 2004
;
Perea-Gomez et al., 2001
).
Therefore, AVE migration converts the PD axis of the embryo into an AP
axis.
Although Nodal has been shown to be essential for AVE induction
(Brennan et al., 2001), at the
time when the AVE is induced in a localised region of the visceral endoderm,
Nodal is expressed in a widespread fashion throughout the epiblast
and visceral endoderm (Varlet et al.,
1997
). The mechanism by which AVE induction is restricted to the
distal tip of the embryo and what then directs the migration of the AVE cells
is unknown. Similarly, how gene expression in the proximal epiblast is
established is not well understood. In this paper, we identify several
previously unappreciated roles for the ExE: in restricting the induction of
the AVE to the distal tip of the 5.5 dpc embryo, in initiating the migration
of the AVE cells and in inducing mesoderm markers in the proximal/posterior
epiblast. We therefore conclude that by patterning the visceral endoderm and
the epiblast, the ExE plays a crucial role in setting up the future AP axis of
the mouse embryo.
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Materials and methods |
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Microsurgical manipulations and embryo culture
Microsurgical manipulations were carried out as a modification of the
methods of Hogan and Tilly (Hogan and
Tilly, 1981). Forceps or tungsten needles were used to cut the
embryo transversely at the embryonic/extra-embryonic boundary. The embryonic
region and control unmanipulated embryos were cultured in DMEM and 50% rat
serum at 37°C, 5%CO2 overnight or over a 40-hour period as
described (Thomas et al.,
1998
). Embryos were photographed before and after culture using
the fluorescein epifluorescence filter on a Zeiss Axiophot microscope.
Time-lapse imaging of embryos
Embryos were cultured directly on the stage of an Olympus IX70 inverted
microscope as previously described
(Srinivas et al., 2004).
Phase-contrast and epifluorescence digital time-lapse images were acquired
using the Deltavision system from Applied Precision. Images from multiple
focal planes were captured at each time point, deconvolved and an
extended-focus image projected. Where the cultured embryos drifted in the
field of view, projected images from different time points were manually set
in register using Adobe Photoshop. Quicktime movies were compiled from
individual still images using the program Graphic Converter.
Injection of extra-embryonic ectoderm cells or COS-7 cells into 5.5 dpc embryos
To obtain extra-embryonic ectoderm cells for injection, 5.5 dpc embryos
were dissected in M2 medium and washed with PBS. Embryos were then incubated
in trypsin-EDTA for 10 minutes at 37°C and 5%CO2. Visceral
endoderm was dissociated from the rest of the embryo using forceps
(Nagy et al., 2003). The
dissected embryos were then cut transversely at the embryonic/extra-embryonic
boundary and the extra-embryonic ectoderm dissociated into small clumps or
single cells using an injection pipette. Five to 15 cells were then injected
close to the distal tip of 5.5 dpc embryos held securely with a holding
pipette. Injected embryos were cultured in DMEM and 50% rat serum at 37°C,
5%CO2 overnight as described
(Thomas et al., 1998
), fixed
overnight in 4% paraformaldehyde and dehydrated though a graded methanol
series.
Whole-mount in situ hybridization
Embryos for whole-mount in situ hybridization were dissected early in the
day, before the AVE was likely to have started moving anteriorly. Manipulated
and control embryos were cultured for the appropriate time period and then
fixed overnight in 4% paraformaldehyde and dehydrated through a graded
methanol series. Whole-mount in situ hybridization was carried out following
standard procedures (Thomas and
Beddington, 1996). The following probes were used as previously
described: Cer1 (Thomas et al.,
1997
), Lhx1 (Shawlot
and Behringer, 1995
), Afp
(Cascio and Zaret, 1991
),
cripto (Ding et al., 1998
),
Nodal (Conlon et al.,
1994
), T (Wilkinson
et al., 1990
), Pou5f1
(Scholer et al., 1990
),
Sox1 (Wood and Episkopou,
1999
), Hesx1 (Thomas
and Beddington, 1996
) and Six3
(Oliver et al., 1995
).
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Results |
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Embryos were dissected at 5.5 dpc and only those with GFP expression
restricted to the distal tip of the embryo (i.e. prior to the migration of AVE
cells) were selected for this experiment. In control un-manipulated embryos,
after overnight culture, the expression of the Hex-GFP reporter was
restricted to the prospective anterior of the embryo
(Fig. 1A,B) as a result of the
unilateral migration of the AVE cells from the distal tip to the anterior of
the embryo (Srinivas et al.,
2004). In contrast, in embryos in which the extra-embryonic region
had been removed, after overnight culture, the Hex-GFP reporter was
no longer restricted to the anterior but was expressed in a widespread fashion
throughout the visceral endoderm (Fig.
1C-F; n=19/23). This ectopic expression was confirmed to
represent an expansion of the AVE because the AVE markers cerberus-like 1
(Cer1) (Belo et al.,
1997
; Thomas et al.,
1997
) and Lhx1 (previously Lim1)
(Perea-Gomez et al., 1999
)
were also expressed throughout the visceral endoderm of these embryos
(Fig. 3A-D; Cer1 was
expressed ectopically in 17/20 and Lhx1 in 7/7 embryos). Consistent
with this expansion of AVE markers, we also saw a dramatic reduction in the
expression of the proximal/posterior visceral endoderm marker Afp
(Cascio and Zaret, 1991
) in
embryos lacking the extra-embryonic region
(Fig. 3E,F; Afp was
lost or greatly reduced in 19/23 embryos).
|
|
To distinguish between these two alternatives, we examined by time-lapse microscopy 5.5 dpc Hex-GFP embryos in which the extra-embryonic region had been removed. In these embryos, we observed that after 2-3 hours in culture, visceral endoderm cells that would not normally express Hex start to express the Hex-GFP reporter (Fig. 2A-D; see Movies 1 and 2 in the supplementary material; n=3). This indicates that the ectopic expression of the Hex-GFP transgene is due to the de novo induction of Hex within cells of the proximal visceral endoderm.
Previous work has shown that the cells of the AVE move from the distal tip
of the embryo to the boundary between the epiblast and ExE by a process of
migration, and in response to cues from their environment
(Srinivas et al., 2004). We
observed that in embryos where the extra-embryonic region has been removed, in
addition to de novo induction of AVE markers, the original AVE progenitor
cells that are located at the distal tip of the embryo do not migrate
unilaterally to the prospective anterior of the embryo, but instead remain
stationary, in a distal position (Fig.
2A-D; see Movies 1 and 2 in the supplementary material). This
suggests that in addition to being required for the induction of the AVE, the
extra-embryonic region is required for the proper migration of AVE cells.
The extra-embryonic region is required for the expression of proximal/posterior markers in the epiblast
To address how the absence of the extra-embryonic region affects the
patterning of the epiblast, we analysed the expression of the posterior
markers cripto (Ding et al.,
1998), Nodal (Varlet
et al., 1997
) and T
(Wilkinson et al., 1990
). At
5.5 dpc, these genes are expressed throughout the proximal epiblast, in a ring
at the embryonic/extra-embryonic boundary. By 6.5 dpc, their expression
refines to the posterior epiblast, where the primitive streak will form
(Lu et al., 2001
). In control
embryos, after overnight culture, the expression of cripto, Nodal and
T is nearly or completely resolved to the posterior epiblast,
indicating that these embryos have reached a stage between 6.0 and 6.5 dpc
(Fig. 3G,I,K). However, in
embryos lacking the extra-embryonic region, the expression of cripto,
Nodal and T is completely lost from the epiblast after
overnight culture (Fig. 3H,J,L;
cripto was lost in 14/16, Nodal was lost in 7/7 and T was
lost in 9/10 embryos), indicating that induction of posterior epiblast pattern
has not occurred in these embryos. This loss of posterior epiblast markers
suggests that the extra-embryonic region is playing a role in primitive streak
induction (Beddington and Robertson,
1999
). The loss of posterior epiblast markers in our explants is
unlikely to be due simply to the ectopic AVE repressing these markers, because
a similar loss of the posterior marker cripto is observed in epiblast explants
separated from both the ExE and the surrounding visceral endoderm
(Beck et al., 2002
). The
striking absence of any posterior markers in embryos lacking the
extra-embryonic region is also not due simply to a general failure of
patterning in the epiblast, as embryos lacking the extra-embryonic region
express the ectoderm marker Pou5f1 (previously Oct3/4)
(Rosner et al., 1990
) in the
epiblast (Fig. 3N;
n=11/14) at levels comparable with control embryos
(Fig. 3M; n=5/5).
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|
The extra-embryonic ectoderm can repress AVE gene expression and induce proximal/posterior epiblast markers
The upregulation of AVE markers in embryos lacking the extra-embryonic
region suggests that this region may repress AVE gene expression. In order to
identify the specific tissue of the extra-embryonic region that is responsible
for this repression, we tested the ExE for inhibition of AVE formation. For
this purpose, we developed a technique to inject small, dissociated clumps of
extra-embryonic ectoderm cells into the distal region of 5.5 dpc embryos
(Fig. 4A). Consistent with our
previous data, we found that the injection of extra-embryonic ectoderm cells
caused the downregulation of the AVE marker Cer1 in a significant
proportion of embryos (four out of seven;
Fig. 4B-D). Control injection
of COS-7 cells did not cause a similar loss of Cer1 expression,
indicating that this downregulation is not due simply to the injection
procedure every one of the 19 embryos injected with COS-7 cells showed
normal Cer1 expression (Fig.
4E). This observation argues that a signal from the ExE may be
inhibiting the proximal and lateral visceral endoderm from initiating AVE gene
expression and that the ExE is sufficient for this repression.
In recombination experiments, the ExE is capable of ectopically inducing
markers of primordial germ cells in the distal epiblast at 6.5 dpc
(Yoshimizu et al., 2001). To
test whether the ExE could induce proximal/posterior markers at earlier
stages, we analysed the expression of T in embryos injected with ExE
cells using the assay described above. In two out of nine injected embryos, we
observed a significant expansion of the normal domain of T expression
(Fig. 4F-H), while a third
showed ectopic expression of T
(Fig. 4I). This ability of ExE
cells to induce ectopic T expression after their injection into 5.5 dpc
embryos indicates that the ExE may be inducing posterior markers in the
epiblast.
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Discussion |
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Signalling by the Tgfß factor Nodal has been shown to be required for
the induction of the AVE (Brennan et al.,
2001); however, Nodal expression is widespread in the
epiblast and visceral endoderm at the time of AVE induction
(Varlet et al., 1997
),
suggesting that further signals must be required to restrict the formation of
the AVE to the distal tip of the embryo. In our experiments, we observe de
novo ectopic expression of AVE gene markers upon removal of the ExE and a loss
of AVE markers when ExE cells are transplanted adjacent to the presumptive AVE
domain, indicating that the ExE must be acting to restrict AVE induction to
the distal tip of the embryo.
|
The expansion of AVE markers and the loss of proximal visceral endoderm markers after removal of the extra-embryonic region also indicates that, during a specific window of time, all the cells of the visceral endoderm surrounding the epiblast are competent to assume an AVE character. This suggests that the small population of cells at the distal tip of the embryo that normally do become AVE are not exclusively specified for this fate at an early stage. Therefore, at 5.5 dpc the extra-embryonic region mediates the choice of visceral endoderm cells to adopt an AVE or a proximal visceral endoderm cell fate.
Given that, in the absence of the ExE, we observe no migration of AVE
cells, we have identified a second requirement for the ExE in the early
post-implantation embryo, for AVE cell migration. We suggest that the ExE
either directly secretes a factor that initiates AVE cell migration, with the
directional cues being provided by the epiblast
(Srinivas et al., 2004), or,
alternatively, is essential for maintaining the levels of Nodal signalling in
the epiblast that are required for AVE migration to occur
(Norris et al., 2002
;
Yamamoto et al., 2004
). Nodal
signalling has been shown to promote proliferation within the VE and it has
been proposed that increased proliferation within the posterior VE relative to
the anterior VE may drive the anterior movement of the AVE
(Yamamoto et al., 2004
).
However, time-lapse movies of AVE movement show that AVE cells actively
migrate and that this movement is completed relatively quickly, within
4
hours, making it unlikely that differential proliferation is the primary
driving force for the AVE movements
(Srinivas et al., 2004
). To
reconcile these two sets of data, we suggest that AVE movement is achieved by
a rapid migration, but the initial impetus and directionality for this
migration might be provided by Nodal-mediated differential proliferation
between the posterior and anterior VE. As suggested above, removing the ExE
may remove Spc1/Spc4 activity and consequently cause a decrease in the level
of Nodal signalling. This lowering in the level of Nodal signalling could
cause a decrease in proliferation bellow the crucial threshold required for
migration to occur and thereby disrupt AVE cell movements.
Finally, our observation that in the absence of the ExE we lose
proximal/posterior epiblast gene markers indicates that the ExE is required to
induce proximal/posterior cell fates in the epiblast, probably by maintaining
Nodal expression in this tissue. The ability of ExE cells to induce
ectopic T expression after their injection into 5.5 dpc embryos and the
ability of the ExE to ectopically induce primordial germ cells in
recombination experiments between distal epiblast and the ExE
(Saitou et al., 2002;
Ying et al., 2001
;
Yoshimizu et al., 2001
)
supports the view that the ExE may be inducing posterior markers in the
epiblast. The ExE is likely to fulfil this patterning role both directly, by
BMP signalling, and indirectly, by the modulation of Nodal signalling. In
support of a direct role is the fact that analysis of Bmp4 mutants
(Fujiwara et al., 2001
;
Lawson et al., 1999
) has shown
that BMP4 is required in the ExE for extra-embryonic mesoderm and primitive
germ cell development. In support of an indirect role, there is a requirement
for SPC proteases to be secreted from the ExE for the correct processing of
Nodal and the induction of mesoderm markers in the epiblast
(Beck et al., 2002
). We propose
that both the BMP and SPC activity of the ExE are required to maintain
Nodal expression in the proximal epiblast, and in turn this
expression will be essential to induce primitive streak formation. Therefore,
proximal/posterior gene markers will initially become induced all along the
boundary between the epiblast and the ExE before becoming restricted to the
posterior of the embryo by the migrated AVE. By restricting the site of AVE
induction, modulating its migration and inducing proximal/posterior epiblast
markers, the ExE plays a pivotal role in coordinating AP patterning in the
mouse embryo.
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ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/11/2513DC1
* These authors contributed equally to this work
Present address: Department of Human Anatomy and Genetics, University of
Oxford, South Parks Road, Oxford OX1 3QX, UK
Present address: Centre for Diabetes and Endocrinology, University College
London, Rayne Institute, 5 University Street, London WC1E 6JJ, UK
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