1 Department of Molecular Genetics, The Weizmann Institute of Science, Rehovot
76100, Israel
2 Department of Cell and Molecular Biology, Lund University, Box 94, S-221 00,
Lund, Sweden
3 Department of Human Anatomy and Cell Biology, University of Liverpool,
Liverpool L69 3G3E, UK
* Author for correspondence (e-mail: peter.lonai{at}weizmann.ac.il)
Accepted 20 August 2004
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SUMMARY |
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Key words: FGF signalling, GATA6, GATA4, Basement membrane, Polarization, Early development, ES cells
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Introduction |
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Embryonic stem (ES) cell derived embryoid bodies (EBs) are similar to the
egg cylinder embryo, but, in contrast to it, they can be grown in large
quantities, providing a useful model for early embryogenesis. In their
classical paper, Coucouvanis and Martin set out the mechanism of EB
differentiation as a model for pregastrulation development and tube formation
by cavitation. EBs have an external endoderm that is similar to the primitive
or visceral endoderm of the embryo and is separated from the inner columnar
ectoderm by a basement membrane (BM). Using a genetically undefined
spontaneous mutation, which fails to form the columnar ectoderm layer, it was
proposed that cavitation is regulated by two signals: one emanating from the
outer endoderm layer was thought to be responsible for the apoptotic signal/s
of cavitation; the second, originating in the BM, was considered necessary for
the maintenance and survival of the columnar ectoderm
(Coucouvanis and Martin,
1995).
The work to be described here started as a study of the role of FGF
signalling in EB differentiation and lead to questions regarding BM assembly
that were investigated using ES cells that express truncated Fgfr2 cDNA as a
dominant-negative mutation (Chen et al.,
2000). We reported that ES cells expressing dnFgfr fail to develop
the two characteristic cell layers of the EB. They display a homogenous
aggregate of non-polar cells and form no endoderm or ectoderm-like elements,
but survive for weeks during cultivation
(Chen et al., 2000
). We
observed that EBs formed by dnFgfr ES cells fail to synthesize laminin and
collagen IV isotypes, which supply the protein network of the BM.
Co-cultivating wild-type and dnFgfr ES cells rescued EB differentiation,
suggesting that an FGF-controlled extracellular substance, subsequently
identified as laminin 1, is required for epiblast differentiation. Exogenously
added laminin 1 partially rescued the EB phenotype and induced epithelial
transformation, demonstrating that laminin 1 produced by the endoderm
(Hogan, 1980
;
Leivo et al., 1980
) is
necessary and sufficient to induce epiblast polarization
(Li et al., 2001a
).
Other reports also demonstrated that laminin 1 is required for EB
differentiation. Targeted disruption of ß1-integrin, which inhibits
laminin 1 synthesis, interferes with epiblast differentiation
(Aumailley et al., 2000
).
Disruption of Lamc1 encoding laminin
1, one of the three
polypeptides of the laminin 1 heterotrimer, leads to a similar phenotype
(Smyth et al., 1999
).
Significantly, defective epiblast differentiation caused by loss of either
gene was rescued by exogenously added laminin 1
(Murray and Edgar, 2000
),
which in turn could be inhibited by the E3 fragment of laminin
1
containing the heparin and sulfatide binding site of the LG4 globular domain
of the laminin
1-chain (Li et al.,
2002
). Recognising the potential importance of these findings for
understanding epithelial differentiation and early development, we assumed
that it would help their analysis if we defined the succession and main
intermediates of EB differentiation.
In the present study, we aimed to obtain a comprehensive view of the
developmental interactions that precede gastrulation. To achieve this, several
specific questions had to be answered. Is FGF signalling required for the
differentiation of both epithelia and the pattern of their arrangement in the
EB, or for only an initial step that is necessary for later events? Defective
FGF signalling could be partially restored by exogenous laminin 1
(Li et al., 2001a). The next
question is can the same effect be obtained by laminin 1 presented by the BM
in a physiological cell-matrix interaction? It was also important to determine
whether laminin affects the stem cell directly, or whether it activates
precursors after they reached a specific stage of FGF dependent
differentiation. To answer these questions, we used mutant and wild-type ES
cell lines, and studied their behaviour as an effect of chemical inhibitors
and co-cultivation experiments between mutant and wild-type cells.
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Materials and methods |
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Embryoid bodies were cultured as described before
(Chen et al., 2000). Briefly,
ES cells grown on irradiated embryonic fibroblasts were harvested by
trypsinization and plated on tissue culture plates for two consecutive 2 hour
periods to remove feeder cells. They were then were incubated overnight, when
the aggregates formed were removed and transferred to bacteriological plates
to be grown as suspension without the addition of LIF. GATA6 and GATA4
transformed cells were grown in ES cell medium, without LIF. Medium was
changed every second day. Mixed cultures between GATA transformed ES cells and
dnFgfr or Lamc1 mutant ES cells were prepared by mixing single cell
suspensions. Cells from each cell line (2x106) were mixed in
a 9 cm bacteriological plate and grown as described before
(Li et al., 2001a
).
Cytology
For morphological detail paraformaldehyde fixed JB4 plastic embedded 1-4
µm sections were stained with Toluidine Blue. For chimera experiments, the
ES cell pellet was stained for ß-galactosidase and the sections were
counterstained with neutral red. Confocal analysis was as described before
(Li et al., 2001a).
Expression studies
Western blotting was as described before
(Chen et al., 2000). Microarray
data were from an unpublished study using Affymetrix MG-U74Av2 chips. Total
RNA was isolated by the RNAzol B of Tel-Test from 1C6 dnFgfr and R11 wild-type
embryoid bodies at culture day 0, 2 and 4. cRNA for hybridization and scanning
was prepared as described by others (Clark
et al., 2000
). The following genes and accession numbers of the
probe sets were used to detect them: Gata4, M98339; Gata6,
U51335; Rac1, X57277; Cdc42, L78074; RhoC, X80638;
Fog1, AF0066492; HNF3b, L10409; Sox17, D49473. Each
analysis was repeated twice.
Antibodies
Monoclonal antibody MAB200, which is specific for LG4 of laminin 1
was described before (Kadoya et al.,
1995
). Laminin 1 antibody was from Sigma (L9393); collagen IV
antibody (AB756); antibody to perlecan (MAB #1948) and antibody to ZO1 (Mab
1520) were from Chemicon International. Antibodies to ROCKI and II were from
BD Transduction Laboratories (611136 and 610623). Fluoresceinated phalloidin
was from Sigma-Aldrich (P-1951); Cy-3- or FITC-labelled secondary antibodies
were from Jackson ImmunoResearch Laboratories.
Microscopy and image analysis
For bright-field images a Nikon E800 microscope with a Nikon DXM1200
digital camera and a 20x (N.A., 0.75) or a 40x (D.A., 0.95)
objective was used. Confocal microscopy was in a Zeiss LSM5 instrument using a
20x (N.A., 0.5) or 40x (N.A., 1.20) objective. The images were
processed by Photoshop 5.5 or 7.0.
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Results |
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GATA factor-transformed endoderm-like cells secrete BM proteins
Most GATA6 or GATA4 transformed ES cell clones displayed endoderm like
epithelial morphology, although the periphery of larger colonies contained a
few fat cell-like and neuron-like elements (not shown). The endoderm-like
cells underwent further differentiation from primitive endoderm-like cells
containing BM proteins in their cytoplasm to cells surrounded by extracellular
BM components similar to the visceral endoderm. Staining with antibody to
laminin 1 or collagen IV revealed that these proteins are retained in
the cytoplasm during the first days of culture
(Fig. 3A). Extracellular
secretion became apparent by the third day
(Fig. 3B) and by the fourth
day, the BM proteins formed a lattice surrounding groups of endoderm-like
cells, which did not contain cytoplasmic laminin 1 or collagen IV
(Fig. 3C) [although while both
laminin and collagen IV could be detected in the culture supernatant by
western blotting (not shown)]. Suspension cultures of GATA6-transformed
endoderm-like cells grown on bacteriological dishes formed cysts, which after
3-4 days of incubation were filled with a mixture of laminin
1,
collagen IV and perlecan (Fig.
3D-L). It follows that GATA6 transformed endoderm-like cells
synthesize and secrete large amounts of multiple BM components. It is worth
noting that the
1LG4-specific antibody, used to detect laminin
1, did not stain the surface of mature GATA4/6 transformed visceral
endoderm-like cells (Fig.
3C,F), suggesting that these cell do not exhibit the appropriate
laminin receptor or laminin anchorage site.
|
1C6 dnFgfr ES cells expressing the ß-galactosidase reporter were co-cultivated with ß-galactosidase-negative GATA4-transformed endoderm-like cells of AB2.2 origin. After 5 days of culture, the EBs were fixed, stained for ß-galactosidase, embedded in plastic and counterstained with neutral red (Fig. 4A-D). Wild-type AB2.2 cells developed into cystic EBs with external endoderm and inner columnar ectoderm (Fig. 4A). ß-Galactosidase-positive cells of the dnFgfr mutant (1C6) line exhibited no signs of endoderm or ectoderm differentiation (Fig. 4B). The endoderm-like wild-type AB2.2-GATA4 cells, formed cysts bordered by a single endoderm layer with no obvious epiblast differentiation (Fig. 4C). By contrast, co-cultivation of 1C6 and AB2.2-GATA4 cells resulted in chimeric EBs with ß-galactosidase-negative endoderm derived from the GATA transformed cell line and ß-galactosidase-positive columnar ectoderm derived from the dnFgfr mutant 1C6 cell line (Fig. 4D).
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Pluripotent stem cells are the target for epiblast epithelialization
Pluripotent ES cells are induced directly to an endoderm-like phenotype by
forced GATA4 or GATA6 expression (Fujikura
et al., 2002). Although cystic embryoid bodies frequently contain
non-polar stem cell-like elements, it was not clear whether the columnar
ectoderm of the epiblast develops from the same stem cell pool that gives rise
to the endoderm. To address this issue, we used dnFgfr ES cells of the 1C6
line. Although these cells do not undergo EB differentiation, when aggregated
with four-cell stage wild-type embryos and transplanted into pseudopregnant
females they developed into most cell types in the chimeric embryo
(Fig. 5). When such dnFgfr ES
cells were treated with 30-40% conditioned medium from GATA6 transformed
Lamc1+/- cells, 70-80% of the resulting EBs differentiated
into single cavitated columnar epithelia without an endoderm layer
(Fig. 6A). By contrast, no
differentiation could be observed when supernatants derived from GATA6
transformed Lamc1-/- cells were used
(Fig. 6B). It follows that
ectodermal epithelialization and cavitation is due to endodermal laminin 1 and
its target cells are undifferentiated pluripotent ES cells.
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Endoderm and epiblast polarization follows distinct ROCK-dependent mechanisms
Both ectodermal and endodermal epithelialization of the EB involves
extensive cytoskeletal rearrangements. To investigate this aspect of EB
development, our attention was turned to the Rho family of small GTPases that
are important mediators of cytoskeletal and cell shape changes
(Etienne-Manneville and Hall,
2002). To obtain preliminary information, we inspected data from a
DNA microarray experiment (L.L. and P.L., unpublished) that compared wild-type
and dnFgfr EB cultures (Fig.
7A). All three major members of the Rho family were detected in
the developing EB. Rac1 and Cdc42 transcripts were displayed at similar levels
in undifferentiated ES cells and during EB development, showing no obvious
dependence on FGF signalling. By contrast, the C isotype of Rho was most
prominently expressed in the wild type with its peak at day 4, when epiblast
polarization takes place (Fig.
7A).
|
Immunofluorescence analysis revealed the familiar structure of wild-type
cavitated EBs with robust F-actin accumulation at the apical domain of the
columnar cells (Fig. 8A). The
morphology of defective ES cell differentiation induced by ROCK inhibitors
(Fig. 8B-D) could be separated
into two groups. They exhibited either an interior aggregate of round
non-polar stem cells (Fig.
8B,C), or if any cavitation took place, non-polar, non-columnar
cells surrounded the cavity and no significant F-actin accumulation could be
observed in their apical domain (Fig.
8B,D). In this latter group cavitation could be observed without
epithelialization. Both variants displayed well-formed subendodermal BMs as
detected by antibodies to laminin 1
(Fig. 8A,B), collagen IV
(Fig. 8C) or perlecan (not
shown). According to these findings, ROCK is required for epiblast
polarization, but is not essential for endoderm differentiation. This
assumption was supported by similar expressions of the tight junction protein
ZO-1 characteristic for the endoderm of the EB, in the lateral aspect of both
wild type (Fig. 8E) and
H1152-treated endoderm (Fig.
8F). It follows that the cytoskeletal rearrangement of the
endoderm and ectoderm is under separate and distinct control.
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Discussion |
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Endoderm differentiation is induced by FGF and GATA4/6 signalling
Endoderm differentiation depends on FGF signalling, as demonstrated by the
targeted disruption of Fgf4
(Wilder et al., 1997).
Fgf4 is expressed in the ICM and contributes to the maintenance of
the endoderm (Goldin and Papaioannou,
2003
), where the multiple FGF receptors that read its signals are
localized (Chen et al., 2000
).
Expression of GATA4 and GATA6, where GATA4 is regulated by GATA6, is
controlled by FGF signalling (Morrisey et
al., 1998
). Nevertheless, the immediate downstream elements of FGF
signalling are insufficiently understood in EB differentiation. In vitro
evidence suggests that most FGF dependent signals go through Frs2a, a
docking protein (Lax et al.,
2002
), which communicates with the Grb2 adaptor.
Interestingly although null mutants of Fgf4
(Feldman et al., 1995
) or
Grb2 (Cheng et al.,
1998
) die with defective endoderm development shortly after
implantation, Frs2a null embryos survive till advanced gastrulation
(Hadari et al., 2001
),
indicating that FGF signalling may exhibit unique characteristics in the early
embryo. Analysis of signal transduction in dnFgfr ES cells revealed that
PI3K-Akt/PKB rather than MAPK-ERK signalling is affected by defective FGF
activity (Chen et al., 2000
).
In agreement, we also found that constitutively active Akt/PKB enhances
endoderm development and the synthesis of laminin and collagen IV isotypes,
indicating that the PI3K-Akt/PKB pathway predominates in FGF-dependent
endoderm differentiation (Li et al.,
2001b
).
We showed here that GATA6 is an intermediary of FGF signalling. GATA6,
which is transcribed already in the ICM
(Koutsourakis et al., 1999),
behaves as a master gene for endoderm differentiation
(Fujikura et al., 2002
). GATA6
activates the synthesis of all three polypeptide chains of laminin 1, which
together with collagen IV, nidogen and perlecan assemble into the
sub-endodermal BM. We found that GATA factors induce endoderm differentiation
and BM assembly even in dnFgfr ES cells, indicating that once activated, these
transcription factors induce endoderm differentiation independently from FGF
signalling. Because endoderm differentiation requires GATA6 (Morrissey et al.,
1998) and because cysts of GATA6 transformed cells contain only endoderm-like
elements, we conclude that GATA factors are required and sufficient to induce
endoderm development and deposition of the subendodermal BM.
Additional elements of this pathway are the transcription factors COUP-TFs
I and II, which are upregulated by GATA4/6 during endoderm development
(Fujikura et al., 2002) and
induce Lamc1 and Lamb1 expression
(Murray and Edgar, 2001
). It
follows that minimal elements of this interaction are, sequentially,
Fgf4 (Wilder et al.,
1997
), multiple Fgfr (Chen et
al., 2000
), PI3K and AKT/PKB
(Chen et al., 2000
;
Li et al., 2001b
), GATA6 and
GATA4 (Fujikura et al., 2002
),
COUP-TFs I and II (Murray and Edgar,
2001
), as well as the genes encoding the three polypeptide chains
of laminin 1.
Evidence mainly from the Kemler laboratory demonstrates that E-cadherin is
also required for early EB differentiation. E-cadherin-null ES cells fail to
aggregate, do not form a normal ectoderm and do not undergo EB differentiation
(Larue et al., 1994).
Therefore, E-cadherin-dependent ES cell aggregation may be a prerequisite for
the restriction of FGF signalling to the outer cells of the developing EB.
E-cadherin is connected to the ß-catenin-GSK3-wnt pathway
(Huber et al., 1996
).
Patterning events involving cadherin-Wnt/ß-catenin interactions have been
shown to be controlled by FGF signalling
(Ciruna and Rossant, 2001
;
Kawakami et al., 2001
).
Directional cell-to-matrix, matrix-to-cell signalling activates epiblast polarization
There is strong evidence for the epithelialization of ES cells by exogenous
laminin 1 (Murray and Edgar,
2000; Li et al.,
2001a
; Li et al.,
2002
). Here, we demonstrate that laminin 1 can induce epiblast
differentiation as part of the BM that mediates the physiological interaction
of the endoderm with the epiblast. We also show that while laminin 1 binds to
ES cells and their ectodermal derivatives, it does not associate with the
primitive endoderm. Thus, the cell-binding domains of the laminin
1
chain determine the location of the subendodermal BM by interacting with their
receptors displayed by the stem cells localized below the endoderm layer
(Li et al., 2002
). This
therefore defines the direction of laminin-mediated signalling, thereby
determining the topographical relationship of endoderm and ectoderm.
Besides inducing epiblast polarization, the BM affects the simple two-cell
layer pattern of the EB and egg cylinder embryo. As cell-to-matrix
interactions take place through direct contact, epithelialization of residual
stem cells is precluded, and a single epiblast monolayer develops from cells
immediately adjacent to the BM. It has been proposed that the residual stem
cells are removed by programmed cell death induced by factors derived from the
endoderm, to form a central cavity
(Coucouvanis and Martin, 1995).
Investigation of the role of BMP signalling in cavitation indicated that BMP2
synthesized in the endoderm, and BMP4 in the primitive ectoderm can both
contribute to cavitation, although BMP4 is expressed only for a short period
(Coucouvanis and Martin, 1999
).
Our data indicate that cavitation and columnar ectoderm differentiation does
not require the endoderm, provided that exogenous laminin 1 is presented. It
is therefore possible that the developing ectoderm itself secretes the
necessary apoptotic factors, such as BMP4, although inhibition of ROCK
activity uncouples cavitation from full epithelialization of the primitive
ectoderm and argues that cavitation may be either not different from necrosis,
or it might be due to mechanical separation of the columnar ectoderm from the
residual stem cells. This issue requires further study.
Rho kinase is required for epiblast polarization
Dominant-negative ROCK abolishes epiblast polarization without affecting
endoderm differentiation, suggesting that it may be regulated separately in
the two cell lineages. This assumption was supported by observing that ROCK
expression and epiblast polarization does not require the endoderm for the
laminin-induced differentiation of dnFgfr ES cells. Although ROCK is required
for the epithelialization of the primitive ectoderm, it is not sufficient to
induce this process, as suggested by our observation that dominant-active ROCK
does not rescue dnFgfr differentiation (L.L. and P.L., unpublished). Although
in the epiblast ROCK activity may be induced by laminin, in the endoderm it
appears to be under FGF control and the resistance of endodermal
differentiation to ROCK inhibition is consistent with the possibility that
RAC1 or Cdc42, which are co-expressed in the endoderm, may have a role in
endodermal differentiation.
Separation of endoderm and epiblast differentiation has been repeatedly observed in this study. We show that FGF signalling is required for endoderm differentiation but not for epiblast polarization, which is independently induced by laminin 1 of the sub-endodermal BM. The two lineages are also distinguished by laminin binding. ES cells and their ectodermal derivatives bind laminin, while the primitive and visceral endoderm do not, which defines the direction of laminin-induced differentiation. It follows that the extra-embryonic and embryonic epithelium of the EB and egg cylinder embryo develop by distinct mechanisms, which are connected by the inductive activity of the laminin component of their common BM. Future research will have to clarify whether other epithelial transitions are also controlled by laminin-dependent mechanisms.
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ACKNOWLEDGMENTS |
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