1 Baylor College of Medicine, Department of Molecular and Cellular Biology, One
Baylor Plaza, Houston, TX 77030, USA
2 Harvard Medical School, Department of Genetics, 200 Longwood Avenue, Boston,
MA 02115, USA
* Author for correspondence (e-mail: mzhang{at}bcm.tmc.edu)
Accepted 15 December 2003
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SUMMARY |
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Key words: Maspin, Mouse, Endoderm, Homozygous lethality, Embryonic development
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Introduction |
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Maspin (Serpinb5 Mouse Genome Informatics) is a member of the
serine protease inhibitor family with tumor suppressing activity
(Zou et al., 1994). Initially
identified from normal mammary epithelial cells, the maspin gene is neither
mutated nor deleted, but it is transcriptionally downregulated or silenced by
epigenetic changes in breast cancer
(Futscher et al., 2002
;
Zhang et al., 1997a
). Maspin
protein, made in either E. coli, yeast, or insect cells, inhibits
breast tumor cell migration and invasion
(Sheng et al., 1996
;
Zhang et al., 1997b
) in vitro.
It also inhibits mammary tumor progression and metastasis in maspin transgenic
mice (Zhang et al., 2000a
) and
in a syngeneic mammary tumor model (Shi et
al., 2001
). Maspin has also been shown to inhibit angiogenesis in
the rat cornea and xenograft models (Zhang
et al., 2000b
). Like many other serpins, maspin exerts its
inhibitory role against cell migration and invasion through a functional
domain named the reactive site loop (RSL). However, as a member of the
non-inhibitory serpin family, maspin acts in the absence of a protease
inhibitor activity (Bass et al.,
2002
). Evidence from recent studies have indicated a role for
maspin in cell adhesion (Abraham et al.,
2003
; Ngamkitidechakul et al.,
2003
; Seftor et al.,
1998
).
The processes of tumor cell invasion and metastasis shares many features with early embryonic development. To understand the role of maspin in normal embryonic development, we disrupted the maspin gene by a gene targeting strategy. Homozygosity for the maspin mutant resulted in an embryonic lethality in the mouse that occurred at the peri-implantation stage. The presence of empty decidua indicated that the Mp/ embryos were implanted into the uterine wall, but failed to develop into gastrulated embryos thereafter, resulting in embryonic death at the peri-implantation stage. Further experiments proved that the organization of the endodermal layer and specification of ectoderm cells were affected in Mp/ embryoid bodies. In vitro embryo outgrowth studies showed that the inner cell mass from the Mp/ embryos failed to grow appropriately. Endoderm development requires the attachment of endodermal cells to the extracellular matrix. This interaction was compromised in the absence of maspin. These results indicate that maspin plays an essential role in early embryonic development and it does so by controlling the function of the extra-embryonic endoderm, thereby affecting epiblast morphogenesis.
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Materials and methods |
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To select for Mp/ ES cells, Mp+/ ES cells were cultured in ES medium with a high concentration of G418 (3 µg/µl). ES cell clones were isolated and transferred to 24-well plates without a feeder cell layer for genotyping purposes. Two Mp+/+ and two Mp/ ES clones were used for EB formation. The genotype analysis of the animals, embryos and embryo outgrowths were carried out by PCR using the following primers: wild-type sense 5'-gatggtggtgagtccatc-3' and antisense 5'-tgacaaatgaagagcac-3'; knockout sense 5'-gccttcttgacgagttct-3' and antisense 5'-tgacaaatgaagagcac-3'. RT-PCR was performed using the sense primer 5'-gcttttgctgttgacttgttc-3' (exon 2) and the antisense primer 5'-ttggtgtcttctgtcttgctgatt-3' (exon 5).
Embryo recovery and outgrowth
Superovulated females were caged overnight with males and plugs were
checked the following morning. Fertilization was assumed to occur at midnight
and embryos were staged accordingly (the time of noon on day 1 is termed
E0.5). Embryos at the two-cell stage were flushed from the oviducts of the
superovulated females at E1.5 and blastocysts were flushed from uteri at E3.5.
The embryos were cultured in a few microdrops of M2 medium (Sigma) and covered
with mineral oil (Fisher Scientific). For the outgrowths, the flushed
blastocysts were cultured on 60 mm dishes. After a few days in culture, the
hatched embryos attached to the dishes and the ICM continued to proliferate
and form large cell masses.
Isolation of endoderm cells from blastocysts
The blastocysts from the heterozygous intercrosses were collected at E3.5
and cultured in ES medium with a feeder cell layer for 7-8 days. The cell
masses were picked, trypsinized with 0.25% trypsin, and transferred
individually to 96-well plates with feeder cell layers. These cells were
cultured for 3-4 days before being trypsinized and transferred to a new plate.
After several passages on a feeder cell layer, these blastocyst cells
frequently developed into endoderm cells rather than ES cells. These small,
round endodermal cells grew quickly and spread throughout the plates. At this
point, some cells were harvested for an endoderm adhesion assay. Aliquots of
the cells were passed to dishes without feeder cell layers for three times and
DNAs were made from the Mp+/+ and
Mp+/ genotypes. RNAs were also isolated and
analyzed by RT-PCR for the expression of VE and PE markers, HNF4, and
follistatin.
ES cell in vitro differentiation
ES cells were cultured on a layer of feeder cells for 3-4 days before they
were trypsinized and transferred to petri dishes for EB formation.
Adenovirus-maspin or control adenovirus was added to the ES cells before they
were transferred to the petri dishes for EB formation. The adenovirus was
diluted to 5 MOI (multiplicities of infection) with serum-free DMEM medium and
incubated with the ES cells for 1 hour at 37°C. The cells were fed with
new ES medium after the adenovirus infection. For the maspin antibody blocking
experiment, anti-maspin serum (1:100 dilution) was added to the medium either
at the beginning or during the process of EB formation. After 8-12 days in
culture, the embryoid bodies were fixed in 4% PBS (pH 7.4) buffered
formaldehyde for 1-2 hours, embedded with 2% agarose and sectioned.
Histology: in situ hybridization and immunohistochemistry
Implanted uteri at 4.5-7.5 days of gestation (E4.5-E7.5) were fixed in 4%
PBS (pH 7.4) buffered formaldehyde for 20-24 hours and embedded in paraffin
wax. The embryoid bodies were fixed in 4% PBS (pH 7.4) buffered
polyformaldehyde for 1-2 hours and embedded in paraffin wax. Blocks were
serially sectioned at 5 µm and mounted on poly-l-lysine-coated slides. The
sections were blocked with 5% normal goat serum before staining with antibody.
Primary antibodies to maspin (1:400), GATA4 (Santa Cruz Biotechnology, CA,
1:100), laminin 1 (NeoMarkers, CA, 1:2000), fibronectin (Novus Biologicals,
CA, 1:200), and Oct-4 (Santa Cruz Biotechnology, CA, 1:1000) were incubated
for 1 hour, followed by three washes with PBS. Samples were incubated with a
1:400 dilution of biotin conjugated secondary antibody (Vector Laboratories)
for 45 minutes, washed in PBS, and incubated with the avidin and biotin
solution for 45 minutes. The color was developed with a DAB Kit (Vector
Laboratories) and images were captured with a Leica microscope equipped with a
Spot digital camera. For in situ hybridization, the HNF4 probe (from Dr Fred
Pereira, Baylor) was labeled by digoxigenin according to the manufacturer's
instructions (Roche, Germany). Hybridization was carried out according to the
conditions described by Duncan et al.
(Duncan et al., 1997). The
mitotic index for proliferation was analyzed using samples from embryo
outgrowths and EBs. The embryo outgrowths were fixed with 4% polyformaldehyde
for 1-2 hours at 4°C and then incubated with 0.1% Triton X-100 for 10
minutes. Both the outgrowth slides and EB sections were blocked with 5% normal
serum before being incubated with the anti-phosphorylated histone 3 antibody
(Upstate Biotech, NY, 1:200) for 1 hour. The images were captured with a Leica
microscope equipped with a Spot digital camera.
TUNEL assay
The TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP nick end
labeling) assay was performed according to the manufacturer's specifications
(Roche). Briefly, slides were incubated with 50 µl of the TUNEL reaction
mixture for 30 minutes at 37°C. The slides were then rinsed three times
with PBS and counterstained with DAPI. The mounted slides were analyzed using
fluorescence microscopy. Apoptosis was quantified by counting the number of
apoptotic positive cells in four slides with a 20xobjective.
Endoderm cell adhesion and growth on ECM
Ninety-six-well plates were coated with 5 µg/cm2 of
fibronectin or 25 µg/cm2 of laminin for 1 hour at room
temperature as described by others
(Streuli and Gilmore, 1999).
After washing with PBS, the coated wells were treated with 10 mg/ml BSA for 1
hour. Equal numbers of Mp+/+ or
Mp+/ endoderm cells were seeded in each well. For
the antibody blocking experiments, anti-maspin or preimmune antiserum was
added to the wells at a 1:100 dilution. After 1 hour incubation, the plates
were washed with PBS, trypsinized and counted with a hemacytometer. For the
endoderm cell growth assay, the 96-well plates were coated with 25
µg/cm2 of laminin in PBS overnight at 4°C. A total number of
10,000 cells (Mp+/+ or Mp+/)
were seeded in each well. The cells were cultured for 5 days and counted
daily.
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Results |
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Mp/ blastocysts display an inner cell mass failure during outgrowth and differentiation
The Mp/ embryos may die of a generalized
growth failure due to the lack of an organized layer of VE cells. To determine
the extent to which mutant preimplantation embryos can proliferate, E3.5
blastocysts derived from Mp+/ matings were cultured
individually in vitro. During the outgrowth process, blastocysts undergo both
cell growth and differentiation. Trophoblasts from
Mp/ embryos differentiated and expanded
without any noticeable defects. Among the 30 embryos plated, all of the
Mp/ embryos developed a trophoblast region
equal to the size of the Mp+/+ embryos, suggesting that
the initial trophoblast differentiation, as well as the morphogenesis, was
normal in the Mp/ embryos. In contrast to
the trophoblast, the morphogenesis and survival of the ICM in blastocyst
outgrowths of Mp/ and
Mp+/+ embryos showed a significant difference after 3 days
of cell culture. Although the ICM regions of the
Mp/ and Mp+/+ embryos
were indistinguishable up to 48 hours of culturing (data not shown), a clear
difference was observed in the growth rate of the ICM regions between these
two embryo types by 96 hours (Fig.
3). This difference became more dramatic between the fourth and
eighth day in culture as the ICM cells from the
Mp/ embryos failed to proliferate while the
wild-type ICM cells continued to expand. The inner cell mass outgrowth failure
occurred in 100% of the embryos identified by PCR as being
Mp/. Outgrowth penetration did not occur in
the Mp/ blastocysts; thus supporting the
observed lethality of the Mp/ homozygous
embryos in vivo. Morphologically, most of the Mp+/+
outgrowths were spherical while the Mp/
outgrowths were irregular in shape (Fig.
3). Immunostaining with the GATA4 antibody confirmed that the
Mp+/+ embryos had more GATA4-positive endoderm cells than
the Mp/ embryos had at day 4 of culture
(data not shown). Under the microscope, the Mp+/+ inner
cell mass showed a continuous layer of small and round endodermal cells during
days 4-8 of culture. However, this monolayer of endodermal cells was not
obvious in any of the Mp/ ICM regions
(Fig. 3). To examine whether
the inner cell mass failure resulted from a defect in proliferation, embryo
outgrowths from days 6 and 8 of culture were harvested and stained with the
mitotic marker, phosphorylated histone 3. The cells from the
Mp/ blastocysts had a dramatic reduction in
their rate of proliferation compared to that of the wild type blastocysts
(data not shown).
|
Defective apoptosis pattern and reduced rate of proliferation in Mp/ EBs
To further characterize the defect in VE development in
Mp/ EBs, the TUNEL (TdT-mediated dUTP-biotin
nick end labeling) assay was carried out using both the
Mp+/+ and Mp/ embryoid
bodies. As shown in Fig. 5A,
apoptotic cells were observed in the center of the Mp+/+
embryoid bodies where the presumed lumens would be formed. By contrast, the
TUNEL-positive cells were scattered throughout the
Mp/ embryoid bodies. In fact, the lumen
frequently did not form in the Mp/ EBs.
However, statistical analysis did not show a significant difference in the
rate of apoptosis between the Mp+/+ and
Mp/ EBs
(Fig. 5A).
|
Gain and loss of maspin function in the embryoid body
To prove that the EB formation defects in the
Mp/ ES cells are maspin specific, the maspin
gene was introduced into Mp/ ES cells by
adenovirus infection. After adenovirus-maspin infection, ES cells were induced
to form embryoid bodies. As shown in Fig.
6A, embryoid bodies with small lumens in the center were formed.
This is in contrast to the Mp/ EBs, which
were never found to contain a lumen structure
(Fig. 4, Fig. 6A). Additionally, some of
the GATA4-positive endoderm cells were re-organized into a layer outside of
the embryoid body (Fig. 6B).
Concurrently, Oct4 positive ectoderm cells were able to align directly under
the VE layer, confirming that the lack of maspin resulted in a defect in
ectoderm development (Fig. 4).
This partial rescue of the defect in the Mp/
EBs by adenovirus-maspin infection indicates that maspin is directly involved
in the organization of the endoderm layer as well as the development of the
embryonic ectoderm.
|
Maspin-expressing endoderm cells had increased binding to laminin 1 and a higher cell growth rate
As maspin acts in an extracellular manner, it may regulate cell attachment
to the extracellular matrix. To test whether maspin regulates endodermal cell
adhesion, the components of the extracellular matrix that surround the VE
cells in embryos and EBs were examined
(Fig. 7A, parts a-f). As shown
in Fig. 7A, both laminin 1 and
fibronectin were present in the basement membrane adjacent to the VE layer
(Fig. 7A, parts a-f). The VE
cells made and secreted these components to deposit them on the basement
membrane to set the boundary between the VE and the ectoderm cells. Although a
large amount of fibronectin and laminin 1 were present in the
Mp/ EBs, a normal, continuous layer of
basement membrane was not formed, but rather the basement membrane layer was
spread throughout the scattered endodermal cells
(Fig. 7A, parts g,h). We then
tested the ability of the endoderm cells to adhere to the matrix of either
laminin 1 or fibronectin. Because it was impossible to isolate
Mp/ endoderm cells from blastocyst
outgrowths, owing to the inner cell mass failure, we isolated
Mp+/+ and Mp+/ endoderm cells
for the cell adhesion analysis. In the presence of fibronectin, both
Mp+/+ and Mp+/ endoderm cells
displayed a similar ability to attach to the matrix
(Fig. 7B, part a). However,
significant differences were observed when the cells were plated on the
laminin 1 matrix (Fig. 7B, part
b). Mp+/+ endoderm cells attached to laminin
better than Mp+/ cells, indicating that maspin
selectively increases VE attachment to laminin. To prove that this increased
adhesion to laminin is maspin specific, we treated Mp+/+
endoderm cells with an anti-maspin antibody. This treatment significantly
decreased cell adhesion to laminin (P<0.01). Treatment with
preimmune serum had no adverse effect on cell adhesion (P>>0.05).
To test whether this increased attachment resulted in an increase in cell
survival and growth, endoderm cells were plated on laminin to observe the
growth rate. As shown in Fig. 7B (part
c), Mp+/+ endoderm cells had a significant
growth advantage over the Mp+/ cells.
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Discussion |
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How does maspin regulate the development of the embryonic ectoderm?
Apparently, maspin expression is restricted to the visceral endoderm in the
wild-type EB but not in the E5.5 epiblast and the PE cells (Figs
2,
4). Our data indicate that
maspin exerts its effect through the VE cells. Numerous reports demonstrate
that the three early embryonic lineages depend on each other for survival,
patterning and differentiation. BMP molecules have been shown to mediate the
signaling cascade between the endoderm and ectoderm cells
(Coucouvanis and Martin,
1999). Loss of several these BMP transcription factors expressed
in the VE result in an altered epiblast morphology
(Ang et al., 1996
;
Dufort et al., 1998
;
Tremblay et al., 2001
). The
defect in the Mp/ embryos is more severe in
comparison to the transcription factor mutants. The development of VE cells
was clearly affected by the lack of maspin in the
Mp/ embryos. The VE cells in both the
Mp/ EBs and blastocyst outgrowths had
decreased proliferation (Fig.
5). The isolated Mp+/ endoderm cells
also proliferated slower than the Mp+/+ cells
(Fig. 7B, part c). In addition,
unlike the Mp+/+ EBs, the Mp+/
EBs did not form an organized VE cell layer. Rather, the cells were scattered
and surrounded by ECM molecules (Fig. 7A,
parts g,h). The disordered formation of the VE layer upon the
basement membrane is indicative of incomplete differentiation of the VE at a
late stage of embryogenesis (Duncan et
al., 1997
). For example, the early specification for VE is normal
in the Hnf4/ EBs, but further
differentiation of the VE is defective
(Duncan et al., 1997
). The VE
from the Mp/ EBs might not be able to
undergo further differentiation. The gradual disappearance of the ectoderm
cells could contribute to the defect for further VE development because of the
lack of cell signaling between the VE and the ectoderm. Conversely, defective
VE cells could then send destructive signals to the remaining ectoderm cells,
eventually resulting in the elimination of the ectoderm cells. In this regard,
although maspin is not essential for early endoderm specification, the
possibility for its involvement in late VE differentiation cannot be
excluded.
As the formation of wild-type EBs depends on the VE cells lying on an
intact layer of basement membrane and the maspin antibody treatment was able
to block normal EB formation, we suspect that maspin exerts its function at
least partially through a cell-ECM interaction. We showed that the VE cells
from the Mp+/ embryos had a significant reduction
in their adhesion and growth rate in comparison to the
Mp+/+ VE cells when plated on laminin. This reduction
caused by the loss of one copy of maspin seems to affect embryonic development
in vivo, as we observed that some of the heterozygous mice die at certain
stage(s) of embryonic development (see Table S1 at
http://dev.biologists.org/supplemental).
Therefore, defective VE development could result from reduced cell adhesion to
matrix and reduced proliferation. The effect on adhesion and proliferation may
not be mutually exclusive as many cells require appropriate cell adhesion to
unique ECM components for survival. ECM proteins are generally assembled in
basement membranes and recent data indicates that the regulation of this
process is crucial for embryo development
(Colognato and Yurchenco, 2000;
Li et al., 2002
). The basement
membranes formed between the visceral endoderm, the developing epiblast and
the parietal endoderm (Reichert's membrane), which extends over the
trophectoderm, are the first membranes formed during embryogenesis
(Leivo et al., 1980
). Although
differentiation of the primitive endoderm cells precedes basement membrane
assembly, the processes of epiblast differentiation and proamniotic cavitation
require the completion of basement membrane assembly to be entirely functional
(Murray and Edgar, 2000
;
Murray and Edgar, 2001
).
Recent experiments have demonstrated that the laminin 1-null and
ß1-integrin-null cells are unable to form basement membranes or undergo
epiblast differentiation and cavitation
(Li et al., 2003
;
Li et al., 2002
). Not
surprisingly, in the Mp+/+ EBs and embryos that stained
positive for laminin and fibronectin expression, the basement membrane layers
were intact (Fig. 7A). However,
this distinctive basement membrane layer as well as the visceral endoderm
layer was diminished in the Mp/ EBs (Figs
4,
6). Therefore, maspin produced
in the VE may be involved in the ECM assembly, which controls further
embryonic development.
The importance of cell-ECM interactions during early embryonic development
is highlighted by several mouse mutants that lack integrin molecules, the
cell-surface adhesion receptors for ECM. One of these integrin molecules,
V integrin, is present in blastocysts and associates with the ß3
subunit. Through its interactions with the other ECM components, the
Vß3 integrin mediates the initial blastocystuterine interaction
during implantation (Cross et al.,
1994
). In addition, deletion of either the
5 or the
4 integrin subunits results in an embryonic lethality because of a
defect in mesoderm development (Yang et
al., 1993
; Yang et al.,
1995
). The defects observed in the integrin ß1 mutants during
embryonic development because of the similarity to the maspin mutant. In the
ß1-null embryos, endoderm morphogenesis was defective and the embryo died
at E5.5 (Fassler and Meyer,
1995
; Stephens et al.,
1995
). Blastocyst outgrowths for the ß1-null embryos were
blocked because of an inner cell mass failure. However, the trophoblast
function in the ß1-null embryos was largely normal; both the decidual
reaction was induced and outgrowths on fibronectin coated substrates were
observed. These features were faithfully duplicated in the maspin null
embryos. Current investigations are focusing on whether the functional
similarities of these two deletion mutants are the result of an interaction
between these two molecules during embryonic development.
As maspin was discovered as a putative tumor suppressor gene, we have
carried out a series of animal experiments demonstrating that maspin is
capable of inhibiting mammary tumor growth and metastasis. The observed
suppression of primary tumor growth is probably due to the inhibition of
angiogenesis and an increase in apoptosis. However, the mechanism responsible
for the inhibition of metastasis is not fully understood. Initially, it was
thought that maspin might inhibit tumor metastasis by inhibiting certain
proteases. However, recent evidence has suggested that maspin functions
independent of protease inhibition (Bass et
al., 2002; Zhang et al.,
1999
). The study of maspin in early embryonic development provides
a definitive answer to one of these mechanisms of maspin action. Tumor
metastasis requires the detachment of tumor cells from the extracellular
matrix as well as extensive invasion through the basement membrane and stroma
(Liotta et al., 1991
;
Stetler-Stevenson, 1993). The increased cell adhesion caused by maspin could
hinder such a process and thereby prevent tumor metastasis. Further
experiments on the role of maspin in endoderm differentiation and cell-cell
interactions during embryonic development will probably shed more light on our
understanding of its role in tumor invasion and metastasis.
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ACKNOWLEDGMENTS |
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Footnotes |
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