1 Department of Biological Sciences, Columbia University, New York, NY 10027
USA
2 Tufts University School of Veterinary Medicine, North Grafton, MA 01536
USA
3 Laboratory of Molecular Oncology, MGH Cancer Center, Charlestown, MA 02129,
USA
* Author for correspondence (e-mail: ly63{at}columbia.edu)
Accepted 20 December 2002
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SUMMARY |
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Key words: E2F transcription, Trophectoderm, Mouse
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INTRODUCTION |
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Members of the pRB family restrict cell cycle progression and apoptosis, to
a large part, through the direct repression of the E2F/DP transcription factor
family (Dyson, 1998;
Trimarchi and Lees, 2002
). Six
E2F family members have been cloned, five of which form active repressor
complexes with pRB family members (E2F1-3 with pRB, E2F4-5 with p107 or p130).
By contrast, only two DP family members (DP1 and DP2) have been cloned, which
interact with all E2F family members equally well, and subsequently are found
in trimeric complexes with all three pRB family members. Although mutations in
genes encoding pRB or upstream regulators of pRB are frequently found in human
tumors, intragenic mutations in the genes encoding the E2F and DP
transcription factor families have not been isolated. This may be due to the
dual nature of E2F/DP heterodimers, which act as bifunctional transcriptional
switches to drive cell cycle-dependent target gene activation when free, and
otherwise repression of target genes when complexed to pRB family members
(Dyson, 1998
). Moreover, free
E2F/DP heterodimers stimulate S-phase entry and either subsequent
proliferation or apoptosis, depending on environmental signals
(Yamasaki, 1999
). Given the
paradoxical functions of E2F/DP heterodimers, it has been predicted that
mutations in E2F or DP genes simply may be too pleiotropic for organismal
survival.
Inactivation of E2F family members results in a range of diverse
phenotypes. We and others have reported that E2f1-deficient mice
display tissue-specific atrophy (e.g. testes, thyroid) and tumor
predisposition (e.g. lymphoma, lung adenocarcinoma, uterine sarcoma)
(Field et al., 1996;
Yamasaki et al., 1996
).
However, loss of E2f1 can also reduce the pituitary and thyroid
tumorigenesis that develops in Rb+/- mice
(Yamasaki et al., 1998
), and
reduce the nervous system and erythropoietic defects seen in the
Rb-/- embryos (Tsai et
al., 1998
). Taken together, it is clear that E2f1 acts as
a tissue-specific growth regulator, ranging from an oncogene to a tumor
suppressor. Inactivation of E2f2 results in viable adults that, when
crossed to E2f1-deficient mice, are highly tumor prone
(Zhu et al., 2001
). Loss of
E2f3 in mice results in strain-dependent embryonic lethality and
congestive heart failure in those surviving E2f3-deficient adults
without obvious tumor predisposition (Cloud
et al., 2002
; Humbert et al.,
2000b
). Loss of E2f3 lessens the nervous system and
erythropoietic defects in Rb-deficient embryos
(Ziebold et al., 2001
), while
a combination of E2f3-deficiency with E2f1-deficiency
accentuates the phenotype of either single mutant
(Cloud et al., 2002
). MEFs that
are triply deficient for E2f1-E3f3 are unable to proliferate,
demonstrating the importance of those E2Fs with high affinity to pRB for cell
cycle progression (Wu et al.,
2001
). Loss of E2f4 leads to neonatal death with abnormal
hematopoiesis and intestinal defects
(Humbert et al., 2000a
;
Rempel et al., 2000
), while
loss of E2f5 in mice leads to juvenile hydrocephaly because of a
defect in the production of cerebral spinal fluid by the choroid plexus
(Lindeman et al., 1998
).
Finally, the simultaneous inactivation of E2F4 and E2F5 in mice results in
neonatal lethality (Gaubatz et al.,
2000
). Clearly, E2F family members have unique roles in vivo, and
loss of any single member still allows at least part of each mutant population
to survive until birth.
Much less is known about the roles of the DP1 and DP2 in vivo. DP1 is
ubiquitously expressed at high levels in tissues and in cell lines
(Gopalkrishnan et al., 1996;
Wu et al., 1995
). By contrast,
DP2 is expressed at low levels with alternative splicing in a restricted set
of tissues and cell lines (Ormondroyd et
al., 1995
; Rogers et al.,
1996
; Wu et al.,
1995
; Zhang and Chellappan,
1995
). Despite their distinct patterns of expression, DP1 and DP2
function indistinguishably in in vitro assays, such as those for
heterodimerization, DNA binding and transactivation, when overexpressed with
various E2F partners and pRB family members. Overexpression of DP1 or DP2
cooperates with activated Ras to transform fibroblasts
(Jooss et al., 1995
) and a
dominant-negative DP1 mutant arrests cells in G1
(Wu et al., 1996
).
Importantly, E2Fs require prior dimerization with a DP member for all
functions except pRB family interaction.
To delineate a role for DP1 in vivo, we inactivated the Dp1 locus in mice by homologous recombination. This paper reports that Dp1 deficiency results in embryonic lethality prior to 12.5 days of gestation, owing to a failure of extra-embryonic tissues to develop properly.
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MATERIALS AND METHODS |
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Production of F1 and F2 animals and genomic PCR genotyping
F1 Dp1+/- animals were genotyped by Southern analysis
and genomic PCR. These Dp1+/- animals were then intermated
to produce the F2 offspring. F2 Dp1+/- animals were used
to maintain the mutant line, with the inclusion of one backcross to C57BL/6
females during the course of this study. To genotype the animals by PCR, tail
DNA was added to PCR cocktails containing the common L48 intron primer
(5'-GACTCATCACAAGACTAGCGTGACC-3') and either the Dp1
exon-specific L43 primer (5'-GACATTGAGGTGCTCAAGCGCATGG-3') to
amplify the wild-type allele (350 bp) or the neoR-specific L28
primer (5'-CTACCCGGTAGAATTGACCTGCA-3') to amplify the mutant
allele (
300 bp). Reactions were run at 64°C annealing temperature for
39 cycles and electrophoresed on a 1.6% TAE-agarose gel to visualize products
with ethidium bromide. For small amounts of DNA from manually dissected or
laser-captured embryos, a different set of primers (L75, L78 and L79) was used
in a combined reaction. For manually dissected embryos, yolk sacs were removed
to genotype the embryo, using PCR cocktails with the common L75 intron primer
(5'-GACACGTCTGATTGTTGTGAAT-3'), the Dp1 exon-specific L78
primer (5'-ACTGGGGTGGGGGTGCTCACCC-3') to amplify the wild-type
allele (
180 bp) and the neoR-specific L79 primer
(5'-CCTCTGTTCCACATACACTTCAT-3') to amplify the mutant allele
(
150 bp). Reactions were run at 58°C annealing temperature for 29 to
39 cycles and electrophoresed on a 2% TAE-agarose gel to visualize products.
For genotyping of the p53;Dp1-double mutant animals, PCR reactions
for the p53 status were performed as previously described
(Jacks et al., 1994
).
Western blotting of embryo lysates
Manually dissected embryos (E9.5) were genotyped from yolk sacs and lysed
in 2x Laemmli Buffer (10 volumes) with repeated rounds of
sonication and boiling until completely solubilized. Equal amounts of total
proteins (as judged by Coomassie staining) were separated on 10% SDS-PAGE and
semi-dry transferred to Immobilon P for immunoblotting. The immunoreactivity
of the monoclonal antibodies used were first verified as specific for each DP
family member using recombinant mouse DP1 protein or human DP2 protein in
western blotting experiments (data not shown). Monoclonal antibodies against
DP1 (1DP06, Labvision), DP2 (G-12, Santa Cruz) and PCNA (PC10, Zymed) were
used at 1µg/ml, and visualized with an HRP-sheep antimouse IgG secondary
antibody (Amersham). A rabbit polyclonal antibody against actin (RBI/Sigma)
was used at a 1:250 dilution with an HRP-donkey anti-rabbit IgG secondary
antibody (Amersham). Western blots were developed with an ECL kit (Amersham)
and exposed to autoradiographic film.
In situ embryo analysis
Pregnant females were injected i.p. with BrdU/FdU mixtures (100 µg
5-bromo-2' deoxyuridine and 6 µg 5-fluoro-2' deoxyuridine per
gram of body weight) 30 or 120 minutes before sacrifice. Decidua with embryos
were isolated from the uterine horn, fixed briefly in buffered formalin and
then serially sectioned. Sections were then used for Hematoxylin/Eosin
staining and immunohistochemical detection of trophectoderm-derived tissues
with TROMA1 (Developmental Studies Hybridoma Bank, NICHD and University of
Iowa), using a biotin-goat anti-rat IgG secondary antibody and
streptavidin-peroxidase for amplification. Immunohistochemical detection of
DP1 was performed using a monoclonal against DP1 (1DP06 from LabVision) or
mouse IgG at 1 µg/ml for E8.5 and 3 µg/ml for E7.5 and developed with a
mouse-on-mouse detection kit (LabVision). For detection of proliferation in
utero, sections were measured for BrdU incorporation using a biotin-anti-BrdU
antibody kit (Zymed). Slides were counterstained briefly in Hematoxylin
following development with 3,3'-diaminobenzidine (DAB, Vector Labs).
Laser capture microdissection
Sections were stained with Hematoxylin, and dehydrated finally with
HistoClear II (National Diagnostics). Microdissection was performed on an
Arcturus Pixcell station on sections of decidua containing target conceptuses.
Genomic DNA was recovered from the microdissected conceptuses using 0.04%
proteinase K digestion in 10 mM Tris, 1 mM EDTA and 0.5% Tween 20 for 4 hours
at 50°C. After inactivation of proteinase K, genomic PCR was performed as
described above.
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RESULTS |
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Timing of Dp1-deficient embryonic lethality
To identify the developmental window in which the
Dp1-/- embryos die, we intercrossed
Dp1+/- animals and harvested embryos from pregnant females
from E15.5 to E7.5. After dissection and removal of all maternal tissues, the
embryos were inspected morphologically and the yolk sacs were genotyped using
PCR primers specific for the wild-type or mutant allele
(Fig. 1C;
Table 1).
Dp1-/- embryos die by E12.5, and are only rarely viable (1
out of 3) and petite at E11.5. Although non-Mendelian numbers of
Dp1-/- embryos were recovered from manual dissections at
E15.5 to E10.5, the expected number of Dp1-/- embryos was
found by E9.5, when they appeared viable. None of the recovered
Dp1-/- embryos were normal in size or appearance at E10.5
or E9.5 compared with the wild-type and Dp1+/- embryos
(Fig. 2A,B).
Dp1-/- embryos are smaller and developmentally delayed
with regard to morphological staging criteria (e.g. turning, somite number),
and substantial variability in the degree of growth retardation was observed
at E11.5-E9.5. This miniaturization occurred in the extra-embryonic tissues,
as well as the embryonic tissues of the Dp1-/- conceptuses
recovered. Upon manual dissection, we have always observed an abnormally small
yolk sac associated with a petite Dp1-/- embryo,
suggesting that the requirement for Dp1 affects the development of
both extraembryonic and embryonic compartments.
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Expression of DP1 and DP2 in utero
To verify the loss of expression of DP1 protein in
Dp1-/- embryos and to assess the levels of DP2 in these
mutant embryos, we genotyped E9.5 embryos obtained from intercrossing
Dp1+/- animals, and then subjected embryo lysates to
immunoblotting analysis with specific monoclonal antibodies. DP1 protein is
not expressed in the Dp1-/- embryo extracts, but is
clearly present in the wild-type embryo extracts
(Fig. 2C, top panel). By
contrast, all of the embryos expressed DP2 protein
(Fig. 2C, second panel). Thus,
the embryonic lethality, miniaturization and developmental delay of the
Dp1-deficient embryos occur despite the expression of DP2,
demonstrating that DP1 and DP2 proteins fulfill distinct roles during
development. These experiments revealed that Dp1-/-
embryos did not express more DP2 than that expressed in wild-type embryos,
using actin as a loading control (Fig.
2C, bottom panel). In all of the embryo extracts, high levels of
PCNA were detected (Fig. 2C, third panel), suggesting that embryonic lethality occurred despite obvious
embryonic proliferation. Using western blotting, we can also detect the
expression of both DP1 and DP2 proteins in lysates of manually dissected
wild-type embryos from E8.5 to E12.5 (Fig.
2D). Thus, the developmental window in which
Dp1-/- embryos are clearly compromised, is a period in
which both DP family members are expressed.
Additionally, we detected DP1 protein expression in wild-type conceptuses
immunohistochemically at E7.5 and E8.5 in situ using a monoclonal antibody
specific for DP1 (Fig. 3). DP1
is expressed in both the extra-embryonic and embryonic tissues at E7.5
(compare (Fig. 3A with 3B).
Expression of DP1 also occurs in the E8.5 embryo
(Fig. 3C,F), and within
extraembryonic tissues (Fig.
3C,E,F) such as the chorion, ectoplacental cone and trophoblast
giant cells. At E8.5, there is a strong background of cytoplasmic staining
with mouse IgG in the trophoblast giant cells
(Fig. 3D,G,H); however,
specific nuclear staining is evident in these trophoblast giant cells with the
DP1 monoclonal antibody. Furthermore, DP1 mRNA expression using in situ
hybridization has previously been documented in embryonic (E8.5-E18.5) as well
as extraembryonic tissues (E8.5), including trophoblast giant cells
(Gopalkrishnan et al.,
1996).
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Dp1-deficient extra-embryonic defects at E8.5
To examine how loss of Dp1 affected the development of earlier
embryos, we analyzed in situ serially sectioned decidua containing E8.5
embryos from Dp1+/- intermatings. To genotype the embryos
in situ and identify Dp1-/- embryos, we used laser-capture
microdissection followed by genomic PCR
(Fig. 1C). Using this method,
we recovered 31 microdissected E8.5 embryos with the following genotypes:
eight Dp1-/-, 18 Dp1+/- and five
wild-type embryos. Histological inspection showed that the
Dp1-/- conceptuses at E8.5 were thinner and less developed
than the wild-type conceptuses, appearing as the E7.5-E8.0 conceptuses would
(compare Fig. 4A with 4B,C). At
the distal pole of these conceptuses, Dp1-/- embryos were
smaller and less developed than were the wild-type E8.5 embryos.
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To determine whether extra-embryonic tissues were formed correctly in the
absence of Dp1, we examined the trophectoderm-derived tissues
surrounding each conceptus. The ectoplacental cone (EPC) and both the primary
and secondary trophoblastic giant cells arise from the trophectoderm of the
implanted blastocyst. While the EPC proliferates markedly at E8.5, the primary
and secondary trophoblast giant cells instead undergo successive rounds of
endoreduplication reaching up to 500 times the normal content of genomic DNA
(Hogan et al., 1994). All of
these trophectoderm derivatives can be visualized using the trophoblast marker
TROMA1 (Brulet and Jacob,
1982
). Immunohistochemical detection of the EPC and trophoblast
giant cells using the TROMA1 antibody revealed the abnormal formation of these
structures surrounding the Dp1-/- embryos (compare
Fig. 4D with 4E,F). Although
present, the EPC and trophoblast giant cells were vastly under-represented in
the Dp1-/- mutant conceptuses. Normally, it is the EPC and
trophoblast giant cells that must establish the implantation site in the
uterine crypt, forming the essential connection to the maternal circulation
through the placenta and the parietal yolk sac, which ultimately are
responsible for nutrient acquisition and filtering of fetal wastes.
To assess whether loss of Dp1 influenced the level of proliferation occurring in these E8.5 conceptuses in situ, we injected 5-bromo-2' deoxyuridine (BrdU) into these pregnant Dp1+/- females (30 minutes before sacrifice) and measured BrdU incorporation immunohistochemically in serial sections (Fig. 4G-L). Remarkably, this analysis demonstrated that all of the extra-embryonic, trophectoderm-derived tissues of the Dp1-/- conceptuses display dramatic DNA replication defects. The EPC and both the primary and secondary trophoblast giant cells surrounding the implantation site incorporated little if any BrdU in the Dp1-/- conceptuses (Fig. 4H-I), yet incorporated high levels of BrdU in the wild-type conceptuses (Fig. 4G). At higher magnification, the primary trophoblast giant cells are much smaller and incorporate very little BrdU in the Dp1-/- conceptuses compared with those in the wild-type conceptuses (compare Fig. 4K-L with 4J, arrowheads). Surprisingly, at E8.5 no obvious differences in BrdU incorporation were seen in the embryonic compartment of the Dp1-/- conceptuses (Fig. 4J versus 4K-L; serpentine-shaped embryos are present).
Dp1-deficient extra-embryonic defects at E7.5 and E6.5
To further characterize the failure of the Dp1-deficient
extra-embryonic tissue observed at E8.5
(Fig. 4), we expanded our
analysis to include Dp1-deficient embryos resulting from
Dp1+/- intercrosses at E7.5
(Fig. 5) and E6.5
(Fig. 6). By examining earlier
timepoints, we attempted to distinguish between two possibilities of how
trophectoderm-derived tissues are compromised by the loss of Dp1. The
first possibility is that loss of Dp1 limits DNA replication and
thereby proliferation of the diploid chorion and EPC (from which secondary
trophoblast giant cells arise), and additionally limits endoreduplication of
the primary and secondary trophoblast giant cells. The second possibility is
that loss of Dp1 results in a deficit in the number of trophoblast
precursors, which give rise to trophoblast giant cells, the EPC and the
chorion. This possibility could involve the premature differentiation of
trophoblast precursors. Both possibilities would affect lineages derived from
both the mural and polar trophectoderm.
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As the majority of secondary trophoblast giant cells arise from the EPC prior to E8.5, scoring BrdU incorporation at earlier timepoints can help visualize defects specifically in the EPC and secondary trophoblast giant-cell population. Normal morphology and BrdU incorporation of the EPC at earlier timepoints would suggest that no defect in the trophoblast precursor population exists. Normal morphology but abnormal BrdU incorporation in the EPC would suggest that only the proliferation of trophoblast precursors is defective. Alternatively, normal BrdU incorporation in the EPC with abnormal morphology would suggest that the trophoblast precursor population is deficient, perhaps prematurely differentiated. Finally, abnormal morphology and abnormal BrdU incorporation at earlier timepoints would prevent the assignment of the defect to only one of these two possibilities.
In contrast to our analysis of embryos at E8.5, we were not able to assign genotypes to embryos in situ at E7.5 or E6.5 by laser-capture microdissection. Although we could use immunohistochemical detection of DP1 to classify expressing versus non-expressing embryos at E8.5 (data not shown), we were unable to use this methodology at E7.5 and E6.5, either due to lower levels of DP1 expression or the possible persistence of maternally encoded DP1. Instead we relied on our morphological analysis of serial sections at E7.5 and E6.5 to find the small, developmentally delayed and abnormal embryos, which comprised approximately one-quarter of embryos analyzed and were the presumptive Dp1-deficient mutants (Fig. 5, embryos shown in B-D are presumptive mutants at E7.5; Fig. 6, embryos in B,C are presumptive mutants at E6.5). At least for E7.5, the abnormal morphology of these presumptive Dp1-deficient mutants agreed with the abnormal morphology we observed for sets of manually dissected Dp1-deficient embryos, which we genotyped unequivocally by genomic PCR.
At both E7.5 and E6.5, we continued to observe impaired BrdU incorporation in the EPC and trophoblast giant cells of presumptive Dp1 mutant embryos compared with that in normal conceptuses (Fig. 5I-L; Fig. 6G-I). In addition, the EPC appears abnormally small in presumptive Dp1 mutant conceptuses compared with normal conceptuses, as judged by histological staining (Fig. 5A-D for E7.5; Fig. 6A-C for E6.5) and by TROMA1 staining at E7.5 and E6.5 (Fig. 5E-H for E7.5; Fig. 6D-F for E6.5). These qualitative results do not allow us to distinguish easily between the two possibilities outlined above (defective trophoblast proliferation or decreased numbers of trophoblast precursors). As we always observe presumptive mutants with small EPCs, which incorporate BrdU very poorly, loss of Dp1 appears to affect the number of trophoblast precursors (possibly by differentiation), as well as their ability to replicate DNA appropriately.
To quantify the Dp1 deficiency in extra-embryonic tissues, we counted the total number of trophoblast giant cells or EPC cells, and scored the fraction of which were BrdU positive for embryos at E8.5 (those from Fig. 4 and two additional sets of embryos at E8.5) as well as for those at E7.5 (Fig. 5) and at E6.5 (Fig. 6). What is clear from this quantitation (Table 2) is that the total number of trophoblast giant cells and total number of EPC cells differ greatly between the wild-type and Dp1 mutant conceptuses for all timepoints, arguing that the number of trophoblast precursors contributing to the mural and polar trophectoderm derivatives is reduced substantially with the loss of Dp1.
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Interestingly, the percentage of trophoblast giant cells surrounding Dp1 mutant embryos that are BrdU positive at E6.5-E8.5 is moderately reduced or unchanged compared with that for wild-type embryos (Table 2). However, for all Dp1 mutant conceptuses, the extent of BrdU incorporation in trophoblast giant cells (as indicated by the intensity of the BrdU labeling per cell in Figs 4,5,6) is greatly diminished. This argues that the fraction of Dp1 mutant trophoblast giant cells engaged in DNA replication is the same as that in wild-type conceptuses, but the rate of DNA replication (30 minutes of BrdU labeling for Figs 4, 5) is much slower in Dp1 mutant conceptuses. As trophoblast giant cells are exclusively engaged in successive rounds of DNA replication without mitosis (endoreduplication), the Dp1-deficient defect results in fewer and smaller trophoblast giant cells with only minimal nuclear enlargement.
In contrast to the situation in trophoblast giant cells, the percentage of EPC cells that are BrdU-positive in the wild-type conceptus is very different from that in the Dp1 mutant conceptus (Table 2). At E8.5, a 17- to 1.5-fold greater percentage of EPC cells are incorporating BrdU in wild-type versus mutant conceptuses. At E7.5, a 1.8-fold greater difference is seen in the percentage of EPC cells that are incorporating BrdU in wild-type conceptuses than in Dp1 mutant conceptuses. At E6.5, no difference is seen between wild-type and Dp1 mutant conceptuses; however, this could be due to the longer labeling period used (2 hours of BrdU labeling for Fig. 6) or the absence of a BrdU defect at E6.5. Nevertheless, the decreased percentages of EPC cells incorporating BrdU in the Dp1 mutant conceptuses at E8.5 and E7.5 argue that in addition to arising from reduced numbers of trophoblast precursors, Dp1 mutant EPC cells proliferate poorly by E7.5.
To evaluate how the reduced BrdU incorporation in Dp1-deficient conceptuses affected trophoblast giant cell maturation over time, we extended the BrdU analysis to serially sectioned extra-embryonic tissue from E8.5 and E9.5 conceptuses. The inability of Dp1-deficient trophoblast giant cells to replicate their DNA at E8.5 is even more apparent at E9.5 (data not shown), and resulted in many fewer trophoblast giant cells (in each serial section) with markedly smaller nuclei, presumably owing to fewer rounds of endoreduplication. Clearly, the malformation and defective proliferation of the EPC, combined with the failure of the trophoblast giant cells to endoreduplicate in the Dp1-/- embryos, result in the petite size and subsequent failure of the Dp1-deficient mutant embryos between E8.5 and E11.5.
Loss of p53 fails to rescue Dp1-deficient embryonic
lethality
During the course of these studies, we considered an alternative mechanism
for embryonic lethality of Dp1-deficient embryos: that loss of
Dp1 may have increased apoptosis in utero. Although we observed
numerous TUNEL-positive cells in the EPC and at the distal tip of the
implantation site on all conceptuses, we have seen few if any apoptotic cells
in the embryonic region of any conceptus at E8.5. So, if increased apoptosis
were responsible for Dp1-deficient embryonic lethality, it is more
likely that it results from apoptosis in the extra-embryonic region of the
conceptus or in the surrounding deciduum. Presumably, such extra-embryonic or
decidual apoptosis would have to be distinct from the p53-mediated apoptosis
induced by ionizing radiation, which is restricted to only the embryonic
region of the fetus (Heyer et al.,
2000). Importantly, it is well established that overexpression of
E2F1/DP1 in tissue culture is associated with induction of apoptosis, which
can be either p53 dependent or p53 independent (reviewed by
Ginsberg, 2002
;
Melino et al., 2002
), and
which can be suppressed by co-expression of pRB. E2F1/DP1-mediated apoptosis
can occur via the induction of specific target genes, such as Arf
(Bates et al., 1998
;
DeGregori et al., 1997
), which
stabilizes p53 by inhibiting Mdm2-mediated degradation, and/or p73, the p53
homolog with apoptotic capabilities (Irwin
et al., 2000
; Stiewe and
Putzer, 2000
). Theoretically, loss of Dp1 could lead to
increased apoptosis by preventing pRB-mediated repression of p19ARF or
p73.
To determine whether p53-dependent apoptosis (e.g. at the implantation
site) played a role in the death of the Dp1-deficient embryos, we
crossed Dp1+/- animals to p53 mutant animals. If
the lethality of the Dp1-deficient embryos were due to excessive
apoptosis in utero, then inactivation of p53 could reduce this
apoptosis leading to improved viability, as has been shown previously for
Mdm2-deficient and XRCC4-deficient embryos
(Gao et al., 2000;
Jones et al., 1995
;
Montes de Oca Luna et al.,
1995
). Inactivation of p53 failed to produce any adult
animals that lacked Dp1 (Table
3). Analysis of five p53-/-;Dp1-/- or
p53+/-;Dp1-/- embryos from double mutant crosses at
E10.5 revealed that inactivation of p53 clearly did not rescue the
Dp1-deficient embryonic lethality even partially in utero (data not
shown). Furthermore, from intercrossing
p53+/-;Dp1+/- double mutants, we also obtained
p53-/-;Dp1+/- adult animals
(Table 3). However, this
genotype was represented at only half the expected frequency (observed=27,
expected=52), a highly significant reduction (P=0.0005,
2 test). The cohort of viable male and female
p53-/-;Dp1+/- adults was recovered without
obvious perinatal lethality, suggesting that lethality of approximately half
of this cohort occurred sometime prior to birth. Analysis of three
p53-/-;Dp1+/- embryos from a double mutant
cross at E10.5 did not reveal any differences relative to
p53+/-;Dp1+/- or
p53+/-;Dp1+/+ embryos that might have contributed
to this reduced viability. These data indicate that the cause for this
decreased recovery of p53-/-;Dp1+/-
animals is distinct from the extra-embryonic mechanism responsible for the
death of the Dp1-/- embryos. However, 3 (2 females and 1
male) out of 13 p53-/-;Dp1+/- embryos
recovered at E13.5 displayed exencephaly, which has been described previously
for a subset of the p53-/- females
(Sah et al., 1995
). In fact,
we recovered fewer female versus male
p53-/-;Dp1+/- adult animals
(Table 3, 8 females versus 19
males).
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DISCUSSION |
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Consistent with the well-documented role of E2F/DP heterodimers in
stimulating S-phase entry by transactivating the ensemble of E2F target genes
required for DNA replication (DeGregori et
al., 1997; Muller et al.,
2001
), the greatly reduced BrdU incorporation in the
Dp1-deficient EPC (percentage of cells) and Dp1-deficient
trophoblast giant cells (extent per cell) strongly suggest that at least one
major mechanism for embryonic lethality is the failure to replicate
extra-embryonic DNA appropriately. Presumably, this is due to the inability of
the trophectoderm-derived tissues of the Dp1-/-
conceptuses to induce the large number of E2F target gene products necessary
for S-phase entry and successive rounds of endoreduplication.
Interestingly, the endoreduplication failure of the mouse trophoblast giant
cells reported here is reminiscent of the inability of Drosophila E2F
and DP mutants to amplify the chorion gene as part of the normal
polytenization observed in ovarian follicle cells
(Royzman et al., 1999).
However, in that case, E2F appears to associate with ORC complexes suggesting
a direct role may exist for RBF and E2F/DP heterodimers in endoreduplication
(Bosco et al., 2001
).
Currently, we have no such evidence for the direct participation of mammalian
E2F/DP1 heterodimers in the replicative machinery of the extra-embryonic
genomes.
The requirement for DP1 in the Dp1-deficient extra-embryonic tissues correlates with growth retardation for the embryo. Developmental delay and runting is at least partly due to the progressive failure of the extra-embryonic tissues to form the placenta correctly, which must develop properly to sustain the growing embryo with nutrients and prevent hypoxia and waste accumulation. At this time, however, it is impossible to rule out the possibility that DP1 also has embryonic roles, which are masked in this study by the overwhelming failure of the extra-embryonic compartment. By E8.5, the expansion and massive endoreduplication seen in the extra-embryonic compartment (the EPC, chorion and the trophoblast giant cells) may exhaust the maternally encoded E2F target gene products for S-phase entry and DNA replication faster than the requirement for these same maternally encoded products in the embryonic compartment. Accordingly, it is likely that proliferation defects within the Dp1-deficient embryos would become apparent if the placental defect from Dp1-deficiency were rescued, revealing later roles for Dp1 in the embryo proper.
The death of Dp1-deficient embryos in utero is in stark contrast
to the much less severe adult and neonatal phenotypes seen with the loss of
E2F family members. Thus, it must be concluded that the requirement for one
half of the heterodimer, DP1, greatly exceeds the necessity for the E2F half
of the complex during embryonic development. This is due in part to the fact
that the DP family differs in many respects from the E2F family
(Dyson, 1998). The E2F family
is larger (E2F1-6) and has partial biochemical redundancy built into it
(E2F1-3 bind pRB versus E2F4-5 bind p107 or p130). Inactivation of E2F1
compromises transcriptional activation through itself and transcriptional
repression due to complex formation of E2F1 with pRB. By contrast, the DP
family is small (DP1 and DP2), and biochemically redundant; yet, DP1 is the
major family member expressed. Thus, loss of Dp1 potentially
compromises transactivation as well as repression through all pRB family
members, leading to a more pleiotropic phenotype. The death of the
Dp1-deficient embryos underscores the vital importance of E2F/DP
heterodimers for DNA replication during embryonic development.
Effect of p53-deficiency on Dp1 mutants
Loss of p53 failed to rescue Dp1-deficient embryonic lethality.
Minimally, this means that p53-dependent apoptosis is not involved in the
failure of the Dp1-deficient extra-embryonic tissues to develop.
Formally, it is still possible that p73-dependent apoptosis may be involved in
the Dp1-deficient phenotype. A much more likely scenario is that loss
of Dp1 compromises the number of trophoblast precursors, as well as
their proliferation.
Only half the expected number of
p53-/-;Dp1+/- animals survived past
birth. The absence of any obvious deformities or defects in
p53-/-;Dp1+/- embryos at E10.5 when
Dp1-deficient embryos are severely compromised, indicates that the
mechanism for the death of
p53-/-;Dp1+/- animals cannot be the
same as the extra-embryonic failure responsible for the death of the
Dp1-deficient embryos. Instead, these animals die of exencephaly
similar to that reported previously for the p53-deficient females
(Sah et al., 1995).
Conclusions
This study defines a crucial role for DP1 in vivo, the proper development
of the extra-embryonic lineages arising from the trophectoderm. The
participation of DP1 in this process demonstrates its significant biological
importance. Unexpectedly, the nature of the Dp1-deficient
extra-embryonic phenotype demonstrates that the role of DP1 in vivo can be
highly lineage specific. At this time, it is impossible to rule out that DP1
plays additional later or more-subtle roles in the embryo. To some degree, the
lineage specificity of the DP1 mutant phenotype reflects the existence and
possible reliance of the mouse on DP2 for overlapping functions in most
tissues. Assessment of the potentially essential nature of DP2 awaits its
targeted inactivation.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Bates, S., Phillips, A. C., Clark, P. A., Stott, F., Peters, G., Ludwig, R. L. and Vousden, K. H. (1998). p14ARF links the tumour suppressors RB and p53. Nature 395,124 -125.[CrossRef][Medline]
Bosco, G., Du, W. and Orr-Weaver, T. L. (2001). DNA replication control through interaction of E2F-RB and the origin recognition complex. Nat. Cell Biol. 3, 289-295.[CrossRef][Medline]
Brulet, P. and Jacob, F. (1982). Molecular cloning of a cDNA sequence encoding a trophectoderm-specific marker during mouse blastocyst formation. Proc. Natl. Acad. Sci. USA 79,2328 -2332.[Abstract]
Clarke, A. R., Maandag, E. R., van Roon, M., van der Lugt, N. M., van der Valk, M., Hooper, M. L., Berns, A. and te Riele, H. (1992). Requirement for a functional Rb-1 gene in murine development. Nature 359,328 -330.[CrossRef][Medline]
Cloud, J. E., Rogers, C., Reza, T. L., Ziebold, U., Stone, J.
R., Picard, M. H., Caron, A. M., Bronson, R. T. and Lees, J. A.
(2002). Mutant mouse models reveal the relative roles of E2F1 and
E2F3 in vivo. Mol. Cell. Biol.
22,2663
-2672.
Cobrinik, D., Lee, M. H., Hannon, G., Mulligan, G., Bronson, R. T., Dyson, N., Harlow, E., Beach, D., Weinberg, R. A. and Jacks, T. (1996). Shared role of the pRB-related p130 and p107 proteins in limb development. Genes Dev. 10,1633 -1644.[Abstract]
Dannenberg, J. H., van Rossum, A., Schuijff, L. and te Riele,
H. (2000). Ablation of the retinoblastoma gene family
deregulates G(1) control causing immortalization and increased cell turnover
under growth-restricting conditions. Genes Dev.
14,3051
-3064.
DeGregori, J., Leone, G., Miron, A., Jakoi, L. and Nevins, J.
R. (1997). Distinct roles for E2F proteins in cell growth
control and apoptosis. Proc. Natl. Acad. Sci. USA
94,7245
-7250.
Dyson, N. (1998). The regulation of E2F by
pRB-family proteins. Genes Dev.
12,2245
-2262.
Field, S. J., Tsai, F. Y., Kuo, F., Zubiaga, A. M., Kaelin, W. G., Livingston, D. M., Orkin, S. H. and Greenberg, M. E. (1996). E2F-1 functions in mice to promote apoptosis and suppress proliferation. Cell 85,549 -561.[Medline]
Gao, Y., Ferguson, D. O., Xie, W., Manis, J. P., Sekiguchi, J., Frank, K. M., Chaudhuri, J., Horner, J., DePinho, R. A. and Alt, F. W. (2000). Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development. Nature 404,897 -900.[CrossRef][Medline]
Gaubatz, S., Lindeman, G. J., Ishida, S., Jakoi, L., Nevins, J. R., Livingston, D. M. and Rempel, R. E. (2000). E2F4 and E2F5 play an essential role in pocket protein-mediated G1 control. Mol. Cell 6,729 -735.[Medline]
Ginsberg, D. (2002). E2F1 pathways to apoptosis. FEBS Lett 529, 122.[CrossRef][Medline]
Gopalkrishnan, R. V., Dolle, P., Mattei, M. G., la Thangue, N. B. and Kedinger, C. (1996). Genomic structure and developmental expression of the mouse cell cycle regulatory transcription factor DP1. Oncogene 13,2671 -2680.[Medline]
Harrison, D. J., Hooper, M. L., Armstrong, J. F. and Clarke, A.
R. (1995). Effects of heterozygosity for the Rb-119neo
allele in the mouse. Oncogene
10,1615
-1620.[Medline]
Heyer, B. S., MacAuley, A., Behrendtsen, O. and Werb, Z.
(2000). Hypersensitivity to DNA damage leads to increased
apoptosis during early mouse development. Genes Dev.
14,2072
-2084.
Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994). Manipulating the Mouse Embryo A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Hu, N., Gutsmann, A., Herbert, D. C., Bradley, A., Lee, W. H. and Lee, E. Y. (1994). Heterozygous Rb-1 delta 20/+mice are predisposed to tumors of the pituitary gland with a nearly complete penetrance. Oncogene 9,1021 -1027.[Medline]
Humbert, P. O., Rogers, C., Ganiatsas, S., Landsberg, R. L., Trimarchi, J. M., Dandapani, S., Brugnara, C., Erdman, S., Schrenzel, M., Bronson, R. T. et al. (2000a). E2F4 is essential for normal erythrocyte maturation and neonatal viability. Mol. Cell 6,281 -291.[Medline]
Humbert, P. O., Verona, R., Trimarchi, J. M., Rogers, C.,
Dandapani, S. and Lees, J. A. (2000b). E2f3 is critical for
normal cellular proliferation. Genes Dev.
14,690
-703.
Irwin, M., Marin, M. C., Phillips, A. C., Seelan, R. S., Smith, D. I., Liu, W., Flores, E. R., Tsai, K. Y., Jacks, T., Vousden, K. H. et al. (2000). Role for the p53 homologue p73 in E2F-1-induced apoptosis. Nature 407,645 -648.[CrossRef][Medline]
Jacks, T., Fazeli, A., Schmitt, E. M., Bronson, R. T., Goodell, M. A. and Weinberg, R. A. (1992). Effects of an Rb mutation in the mouse. Nature 359,295 -300.[CrossRef][Medline]
Jacks, T., Remington, L., Williams, B. O., Schmitt, E. M., Halachmi, S., Bronson, R. T. and Weinberg, R. A. (1994). Tumor spectrum analysis in p53-mutant mice. Curr. Biol. 4,1 -7.[Medline]
Jones, S. N., Roe, A. E., Donehower, L. A. and Bradley, A. (1995). Rescue of embryonic lethality in Mdm2-deficient mice by absence of p53. Nature 378,206 -208.[CrossRef][Medline]
Jooss, K., Lam, E. W., Bybee, A., Girling, R., Muller, R. and la Thangue, N. B. (1995). Proto-oncogenic properties of the DP family of proteins. Oncogene 10,1529 -1536.[Medline]
Lee, E. Y., Chang, C. Y., Hu, N., Wang, Y. C., Lai, C. C., Herrup, K., Lee, W. H. and Bradley, A. (1992). Mice deficient for Rb are nonviable and show defects in neurogenesis and haematopoiesis. Nature 359,288 -294.[CrossRef][Medline]
Lee, M. H., Williams, B. O., Mulligan, G., Mukai, S., Bronson, R. T., Dyson, N., Harlow, E. and Jacks, T. (1996). Targeted disruption of p107: functional overlap between p107 and Rb. Genes Dev. 10,1621 -1632.[Abstract]
Lindeman, G. J., Dagnino, L., Gaubatz, S., Xu, Y., Bronson, R.
T., Warren, H. B. and Livingston, D. M. (1998). A specific,
nonproliferative role for E2F-5 in choroid plexus function revealed by gene
targeting. Genes Dev.
12,1092
-1098.
Melino, G., de Laurenzi, V. and Vousden, K. H. (2002). p73: Friend or foe in tumorigenesis. Nat. Rev. Cancer 2,605 -615.[CrossRef][Medline]
Montes de Oca Luna, R., Wagner, D. S. and Lozano, G. (1995). Rescue of early embryonic lethality in mdm2-deficient mice by deletion of p53. Nature 378,203 -206.[CrossRef][Medline]
Muller, H., Bracken, A. P., Vernell, R., Moroni, M. C.,
Christians, F., Grassilli, E., Prosperini, E., Vigo, E., Oliner, J. D. and
Helin, K. (2001). E2Fs regulate the expression of genes
involved in differentiation, development, proliferation, and apoptosis.
Genes Dev. 15,267
-285.
Ormondroyd, E., de la Luna, S. and la Thangue, N. B. (1995). A new member of the DP family, DP-3, with distinct protein products suggests a regulatory role for alternative splicing in the cell cycle transcription factor DRTF1/E2F. Oncogene 11,1437 -1446.[Medline]
Palmero, I. and Peters, G. (1996). Perturbation of cell cycle regulators in human cancer. Cancer Surv. 27,351 -367.[Medline]
Peeper, D. S., Dannenberg, J. H., Douma, S., te Riele, H. and Bernards, R. (2001). Escape from premature senescence is not sufficient for oncogenic transformation by Ras. Nat. Cell Biol. 3,198 -203.[CrossRef][Medline]
Rempel, R. E., Saenz-Robles, M. T., Storms, R., Morham, S., Ishida, S., Engel, A., Jakoi, L., Melhem, M. F., Pipas, J. M., Smith, C. et al. (2000). Loss of E2F4 activity leads to abnormal development of multiple cellular lineages. Mol. Cell 6, 293-306.[Medline]
Rogers, K. T., Higgins, P. D., Milla, M. M., Phillips, R. S. and
Horowitz, J. M. (1996). DP-2, a heterodimeric partner of E2F:
identification and characterization of DP-2 proteins expressed in vivo.
Proc. Natl. Acad. Sci. USA
93,7594
-7599.
Royzman, I., Austin, R. J., Bosco, G., Bell, S. P. and
Orr-Weaver, T. L. (1999). ORC localization in Drosophila
follicle cells and the effects of mutations in dE2F and dDP. Genes
Dev. 13,827
-840.
Sage, J., Mulligan, G. J., Attardi, L. D., Miller, A., Chen, S.,
Williams, B., Theodorou, E. and Jacks, T. (2000). Targeted
disruption of the three Rb-related genes leads to loss of G(1) control and
immortalization. Genes Dev.
14,3037
-3050.
Sah, V. P., Attardi, L. D., Mulligan, G. J., Williams, B. O., Bronson, R. T. and Jacks, T. (1995). A subset of p53-deficient embryos exhibit exencephaly. Nat. Genet. 10,175 -180.[Medline]
Sherr, C. J. (1996). Cancer cell cycles.
Science 274,1672
-1677.
Stiewe, T. and Putzer, B. M. (2000). Role of the p53-homologue p73 in E2F1-induced apoptosis. Nat. Genet. 26,464 -469.[CrossRef][Medline]
Trimarchi, J. M. and Lees, J. A. (2002). Sibling rivalry in the E2F family. Nat. Rev. Mol. Cell Biol. 3,11 -20.[CrossRef][Medline]
Tsai, K. Y., Hu, Y., Macleod, K. F., Crowley, D., Yamasaki, L. and Jacks, T. (1998). Mutation of E2f-1 suppresses apoptosis and inappropriate S phase entry and extends survival of Rb-deficient mouse embryos. Mol. Cell 2,293 -304.[Medline]
Wu, C. L., Classon, M., Dyson, N. and Harlow, E. (1996). Expression of dominant-negative mutant DP-1 blocks cell cycle progression in G1. Mol. Cell Biol. 16,3698 -3706.[Abstract]
Wu, C. L., Zukerberg, L. R., Ngwu, C., Harlow, E. and Lees, J. A. (1995). In vivo association of E2F and DP family proteins. Mol. Cell Biol. 15,2536 -2546.[Abstract]
Wu, L., Timmers, C., Maiti, B., Saavedra, H. I., Sang, L., Chong, G. T., Nuckolls, F., Giangrande, P., Wright, F. A., Field, S. J. et al. (2001). The E2F1-3 transcription factors are essential for cellular proliferation. Nature 414,457 -462.[CrossRef][Medline]
Yamasaki, L. (1999). Balancing proliferation and apoptosis in vivo: the Goldilocks theory of E2F/DP action. Biochim. Biophys. Acta 1423,M9 -M15.[Medline]
Yamasaki, L., Bronson, R., Williams, B. O., Dyson, N. J., Harlow, E. and Jacks, T. (1998). Loss of E2F-1 reduces tumorigenesis and extends the lifespan of Rb1+/- mice. Nat. Genet. 18,360 -364.[Medline]
Yamasaki, L., Jacks, T., Bronson, R., Goillot, E., Harlow, E. and Dyson, N. J. (1996). Tumor induction and tissue atrophy in mice lacking E2F-1. Cell 85,537 -548.[Medline]
Zhang, Y. and Chellappan, S. (1995). Cloning and characterization of human DP2, a novel dimerization partner of E2F. Oncogene 10,2085 -2093.[Medline]
Zhu, J. W., Field, S. J., Gore, L., Thompson, M., Yang, H.,
Fujiwara, Y., Cardiff, R. D., Greenberg, M., Orkin, S. H. and DeGregori,
J. (2001). E2F1 and E2F2 determine thresholds for
antigen-induced T-cell proliferation and suppress tumorigenesis.
Mol Cell Biol 21,8547
-8564.
Ziebold, U., Reza, T., Caron, A. and Lees, J. A.
(2001). E2F3 contributes both to the inappropriate proliferation
and to the apoptosis arising in Rb mutant embryos. Genes
Dev. 15,386
-391.