DEVELOPMENT AND STEM CELLS
RESEARCH ARTICLE 3393
Development 137, 3393-3403 (2010) doi:10.1242/dev.050567
© 2010. Published by The Company of Biologists Ltd
The absence of Prep1 causes p53-dependent apoptosis of
mouse pluripotent epiblast cells
Luis C. Fernandez-Diaz1, Audrey Laurent1, Sara Girasoli1, Margherita Turco2, Elena Longobardi1,
Giorgio Iotti1, Nancy A. Jenkins3, Maria Teresa Fiorenza4, Neal G. Copeland3 and Francesco Blasi1,5,*
SUMMARY
Disruption of mouse Prep1, which codes for a homeodomain transcription factor, leads to embryonic lethality during postimplantation stages. Prep1–/– embryos stop developing after implantation and before anterior visceral endoderm (AVE) formation.
In Prep1–/– embryos at E6.5 (onset of gastrulation), the AVE is absent and the proliferating extra-embryonic ectoderm and
epiblast, marked by Bmp4 and Oct4, respectively, are reduced in size. At E.7.5, Prep1–/– embryos are small and very delayed,
showing no evidence of primitive streak or of differentiated embryonic lineages. Bmp4 is expressed residually, while the reduced
number of Oct4-positive cells is constant up to E8.5. At E6.5, Prep1–/– embryos retain a normal mitotic index but show a major
increase in cleaved caspase 3 and TUNEL staining, indicating apoptosis. Therefore, the mouse embryo requires Prep1 when
undergoing maximal expansion in cell number. Indeed, the phenotype is partially rescued in a p53–/–, but not in a p16–/–,
background. Apoptosis is probably due to DNA damage as Atm downregulation exacerbates the phenotype. Despite this early
lethal phenotype, Prep1 is not essential for ES cell establishment. A differential embryonic expression pattern underscores the
unique function of Prep1 within the Meis-Prep family.
INTRODUCTION
Several crucial events take place during early development. In the
E3.5 blastocyst, the inner cell mass (ICM) contains progenitor
cells, including epiblast (Epi) precursors (Chazaud et al., 2006),
which generate embryonic stem (ES) cells (Evans and Kaufman,
1981; Martin, 1981). The Epi is established during implantation
around E4.5, and from E5.5 to E6.5 forms an epithelium [a process
known as cavitation (Coucouvanis and Martin, 1995)], maintains
its pluripotent state (Niwa, 2007) and proliferates actively
[undergoing a major expansion with a cell cycle as short as 2 hours
(O’Farrell et al., 2004; Snow, 1977)]. At this time, the Epi is very
sensitive to DNA damage (Heyer et al., 2000) and is not protected
by specific G1 and G2 check points (O’Farrell et al., 2004). DNA
damage at this stage leads to p53-dependent apoptosis (Heyer et al.,
2000). Formation of the primitive streak (PS) around E6.5 marks
the beginning of gastrulation and requires interactions between the
Epi, the extra-embryonic AVE (anterior visceral endoderm) and the
ExE (extra-embryonic ectoderm). Gastrulation, during which the
Epi gives rise to the three embryonic layers (ectoderm, mesoderm
and definitive endoderm), is followed by the neural tube formation
from the neural plate (Stern, 2006). After neurulation, the body
plan of the embryo is established, with distinct antero-posterior,
dorso-ventral and left-right axes (Tam and Behringer, 1997).
1
IFOM, FIRC Institute of Molecular Oncology Foundation, and 2Department of
Experimental Oncology, European Institute of Oncology (IEO), IFOM-IEO Campus,
via Adamello 16, 20130 Milan, Italy. 3Institute of Molecular and Cell Biology,
61 Biopolis Drive, Proteos, 138673, Singapore. 4Pasteur Institute, Cenci Bolognetti
Foundation and Department of Psychology, Section of Neuroscience, University
‘Sapienza’ of Rome, 00185 Rome, Italy. 5Università Vita Salute San Raffaele and
Istituto Scientifico San Raffaele, via Olgettina 60, 20132 Milan, Italy.
*Author for correspondence ([email protected])
Accepted 18 August 2010
Prep1 (Pknox1 – Mouse Genome Informatics) homeodomain
transcription factor belongs to the TALE (three amino acids loop
extension) superclass of proteins that include Meis1-Meis3,
Prep2 and Pbx1-Pbx4. Deletion of Pbx and Meis genes in mouse
shows that these genes are essential for organogenesis and
differentiation. Homozygous Meis1–/– embryos die around E14.5
with a severe hematopoietic phenotype (Azcoitia et al., 2005;
Hisa et al., 2004) that is similar to that of the hypomorphic Prep1
mutants (Prep1i/i) (Di Rosa et al., 2007; Ferretti et al., 2006).
Prep1i/i embryos express ~2% of Prep1 mRNA and 2-10% of the
protein, die mostly at E17.5 with liver hypoplasia, anemia,
angiogenesis and eye defects (Ferretti et al., 2006). The
hematopoietic phenotype is due to malfunction of long-term
repopulating hematopoietic stem cells (Di Rosa et al., 2007). No
loss of function mutation for Meis2, Meis3 or Prep2 has been
described. A compound Pbx1-Pbx2 knockout mouse is lethal
around E12.5 and displays pallor, edema, general organ
hypoplasia and homeotic features (Capellini et al., 2006; Selleri
et al., 2001). While the single Pbx2 knockout mice are viable
and fertile (Selleri et al., 2004), Pbx3 null mice are born, but die
within a few hours owing to central respiratory failure (Rhee et
al., 2004). Importantly, Pbx2 partly compensates for Pbx1
(Capellini et al., 2006; Selleri et al., 2004).
We have analyzed Prep1–/– embryos in which the expression of
the protein was eliminated by targeting the DNA-binding
homeodomain. This mutation leads to early post-implantation
lethality. Apoptosis reduces the number of pluripotent Epi cells,
which, hence, fail to form the AVE, PS and differentiated lineages.
This phenotype uncovers a genetic interaction between Prep1 and
p53 (Trp53 – Mouse Genome Informatics) as p53 ablation partially
rescues the Prep1–/– phenotype. Therefore, unlike all other TALE
proteins, Prep1 is responsible for protecting the embryo very early
in development, a unique function within the Meis-Prep families
of transcription factors.
DEVELOPMENT
KEY WORDS: Prep1 (Pknox1), Embryo development, Epiblast, Gastrulation, p53 (Trp53), Mouse
3394 RESEARCH ARTICLE
Development 137 (20)
Prep1–/– ES cell derivation and ES cell culture
Generation of mice
Exons 7 and 8 of the Prep1 gene were replaced with pSAb-geo vector
cassette (see Fig. S1A-D in the supplementary material) (Hisa et al., 2004)
and homologous recombination events tested by Southern blotting of EcoRI
digested DNA with 5⬘ and 3⬘ probes (see Fig. S1E,F in the supplementary
material). Of several independent isolated ES cell clones carrying the
mutation, one was introduced into the germ line using standard techniques.
Pure C57/BL6 double KO Prep1-p53 or Prep1-Atm mutant mice were
obtained by standard crossing p53–/– (Jackson Labs, Bar Harbor, ME, USA)
or Atm–/– (Borghesani et al., 2000) mice with Prep1+/– females. The Prep1p53-ink4Ap16 mutant line was obtained by crossing an ink4Ap16-null
(Krimpenfort et al., 2001) with a Prep1+/–p53+/– double heterozygous mouse.
Southern blot and PCR genotyping
Genotyping was carried out by Southern blot using standard techniques or
by PCR (see Fig. S1 in the supplementary material). The standard protocol
was one cycle of 5 minutes at 94°C, 35 cycles of 30 seconds at 95°C, 30
seconds at 55°C and 30 seconds at 72°C. A final elongation step of 5
minutes at 72°C was performed. For E8.5 and E7.5 embryos, DNA
polymerase concentration was doubled, and the PCR reaction extended to
40 cycles. The sequences of the primers for genotyping (arrows in Fig.
S1C,D in the supplementary material) were: P12, 5⬘-GAGAGCTCAAGGACAGCCAGGCTA-3⬘; M7, 5⬘-CCAGGAGATAATGCCTGCGTGACC-3⬘; and T3, 5⬘-ACCGCGAAGAGTTTGTCCTCAACC-3⬘.
Reverse transcription-PCR
RT-PCR was performed with the Superscript II (Invitrogen, Carlsbad, CA,
USA) (see Fig. S1H in the supplementary material). Primers used were:
F1, 5⬘-GACACCGTGTGCTTCTCGCTCAAG-3⬘; and R1, 5⬘-AGACAAGCAATGTACCGACTACAG-3⬘. For the KO mRNA, the primer R2
corresponds to M7 and R3 to T3.
Immunoblotting
Antibodies used were Pbx1 (Abcam, Cambridge, UK); Pbx2 and b-Actin
(Santa Cruz Biotec, CA); b-Gal (Promega, Madison, WI, USA); and Prep1
(Ferretti et al., 2006).
Electrophoretic mobility shift assay
Electrophoretic mobility shift assay was carried out as described previously
(Berthelsen et al., 1996) with 20,000 cpm 32P-labeled oligonucleotides:
B2PP2, 5⬘-GGAGCTGTCAGGGGGCTAAGATTGATCGCCTCA-3⬘
(Ferretti et al., 2000); and SP1, 5⬘-AAGACAGGGGAGGGAGCCGGGCGGGAGAGGGAGGGGCGGCGCCGGGGCGGGCCCT-3⬘ (IbanezTallon et al., 2002).
Embryo dissection, whole-mount in situ hybridization and TUNEL
Embryos were dissected from the maternal deciduas by standard
procedures and in situ hybridized (including double in situ) as described
previously (Liguori et al., 2003). TUNEL was performed using the
ApopTag Kit (Millipore, Billerica, MA, USA).
RT-PCR of pre-implantation embryos
RT-PCR was performed on total RNA (pools of 100 embryos) (TRIzol
reagent; Invitrogen, Carlsbad, CA, USA) as described (Fiorenza et al.,
2004). Primers used were Prep1, 5⬘-ATGATGGCGACACAGACGCTAAGTATA-3⬘ (sense) and 5⬘-GGGGTCTGAGACTCGATGGGAGGAGGACTC-3⬘ (antisense); b-actin, 5⬘-GGTTCCGATGCCCTGAGGCTC3⬘ (sense) and 5⬘-ACTTGCGGTGCATGGAGG-3⬘ (antisense).
Embryonic stem (ES) cells were derived from E2.5-3.5 blastocysts from
Prep1+/– crosses using standard procedures. Mouse ES cells E14Tg2a were
cultured without feeders in Glasgow-modified Eagle’s MEM with 15% ESscreened FBS (Hyclone, Logan, UT, USA), under standard conditions with
1000 U/ml leukemia inhibitory factor (LIF; Chemicon, CA, USA).
Embryoid bodies (EBs) were obtained using the standard protocol of
hanging drops method. One thousand cells per drop were plated in LIF-free
ES medium.
Immunofluorescence
Passage 14-16 ES cells were grown on coverslips, fixed in
paraformaldehyde, permeabilized and blocked in PBS/10% calf serum/1%
BSA/0.1% Triton X-100, and incubated with anti-Oct3/4 antibody (1/500,
Santa Cruz). For whole-mount immunofluorescence, the antibodies were:
rabbit anti-cleaved caspase 3 (Cell Signaling, Boston, MA, USA; 1:50);
rabbit anti-phospho-histone H3 (Upstate, Billerica, MA, USA; 1:1000);
goat anti-Oct4 (Abcam, 1:500); donkey anti-rabbit CY3-conjugated
antibody (Jackson Labs, Bar Harbor, ME, USA; 1:400), donkey anti-goat
A488-conjugated antibody (Invitrogen, Carlsbad, CA, USA; 1:200) and
DAPI (1 g/ml). Confocal microscopes are TCS SP2 and TCS SP2 AOBS
(Leica Microsystem, Germany). For each embryo, a series of optical
sections (z stacks) was collected. The mitotic index and cleaved caspase 3
staining were quantified as the ratio of phospho-histone H3 or cleaved
caspase 3-positive areas compared with the DAPI stained areas, on five
sections per embryo, using ImageJ software. The same procedures were
used for immunostaining EBs with cleaved caspase 3 antibody and signal
quantification.
RESULTS
Elimination of Prep1 function leads to early postimplantation lethality
The Prep1 homeobox was deleted by replacing exons 7 and 8
with a pSAb-geo recombination cassette (Friedrich and Soriano,
1991) (see Fig. S1A-D in the supplementary material). The
vector contains a splice acceptor site with stop codons in all
reading frames, and b-geo, a fusion lacZ-NeoR gene, with an
internal ATG and a bovine growth hormone polyadenylation
sequence (see Fig. S1D in the supplementary material). One
single cell line was injected to create chimeric animals and
heterozygous germ-line males were backcrossed to C57/BL6NCr (B6) more than 10 times and are therefore essentially
C57/BL6 congenic. The insertion was tested by Southern
blotting at the 5⬘ (see Fig. S1E in the supplementary material)
and 3⬘ (see Fig. S1F in the supplementary material) ends (the
expected fragments are shown as double arrows in Fig. S1A-B
in the supplementary material), and the results confirmed by
PCR (data not shown). We have subsequently used a PCR assay
to genotype the embryos. Fig. S1G in the supplementary material
shows a typical assay on Prep1+/+, Prep1+/– and Prep1–/–
embryos. Primers are indicated by small arrows in Fig. S1C-D
in the supplementary material.
Heterozygous intercrosses did not yield homozygous mutant
mice (Table 1). Timed pregnancy analysis placed the death of
Prep1–/– embryos around E7.5. No Prep1–/– homozygous embryo
Table 1. Prep1–/– is lethal at post-implantation stages
Embryonic day
+/+
+/–
–/–
Absorbed embryos
E7.5
E8.5
E9.5
E10.5 or later
Born mice
6
7
9
20
30
21
23
28
28
73
4 (delayed)
7 (empty yolk sacs)
6 (empty yolk sacs)
0
0
–
7
12
15
–
Shown are the number of born mice or the number of embryos detected at different days of gestation.
DEVELOPMENT
MATERIALS AND METHODS
Prep1 knockout phenotype
RESEARCH ARTICLE 3395
was recovered after E10.5, whereas at E9.5 and E8.5 homozygous
structures mostly looked like empty yolk sacs (Table 1). At E7.5,
homozygous embryos appeared mostly small and developmentally
delayed (Table 1 and see below). Prep1 is therefore essential for
early post-implantation mouse development.
Characterization of the Prep1–/– mutation
The approach used to knock out Prep1 might in principle lead to
transcription of a di-cistronic mRNA producing two proteins, the
N-terminal part of Prep1 with its own ATG and the C-terminal
b-geo from an internal ATG (see Fig. S1H in the supplementary
material). The fusion di-cistronic mRNA was observed by
amplification from Prep1–/– E8.5 cDNA (see Fig. S1H-I in the
supplementary material). Several combinations of primers were
used to test for anomalous Prep1 mRNAs, but no additional
mRNA was found (data not shown). We also sequenced the
fusion region of Prep1-b-Geo cDNA (not shown) and found the
expected STOP codons in the three frames, which should prevent
the production of a fusion Prep1-b-Geo protein. The fusion
mRNA was also detected in E11.5 heterozygous mouse
embryonic fibroblasts (MEFs), liver, kidney, lung and spleen
(not shown).
At the protein level, we never found any truncated form of Prep1
(see Fig. S2A in the supplementary material). We also did not find
any difference in DNA binding in EMSA performed with Prep1+/+
and Prep1+/– extracts (see Fig. S2D-E in the supplementary
material). lacZ was detected by immunoblotting only in
heterozygous testis extract (in the cytoplasm, see Fig. S2B in the
supplementary material) and by b-gal staining in the E13.5 retina
of Prep1+/– embryos (see Fig. S2C in the supplementary material)
where Prep1 has been shown to be strongly expressed in wild-type
embryos (Ferretti et al., 2006). Finally, western blotting of wildtype, heterozygous and homozygous nuclear extracts of Prep1 ES
cell lines (see below) (two different clones per genotype) showed
no Prep1 in homozygous (see Fig. S1J in the supplementary
material) and reduced Prep1 levels in heterozygous nuclear extracts
(see Fig. S1J in the supplementary material). We obtained the same
result with two different monoclonal antibodies (not shown). No
DEVELOPMENT
Fig. 1. Absence of PS and lineage differentiation in Prep1–/– mouse embryos. Peri-gastrulation markers analysis at early post implantation
stages. Representative data are shown (see Table S1 in the supplementary material for complete data). (A-N)Absence of gastrulation and AVE in
Prep1–/– embryos at E6.75. Whole-mount in situ hybridization against T (A,B), Fgf8 (C,D), Lefty2 (E,F), Nodal (G,H), Wnt3 (I,J), Bmp4 (K,L) and Cer1
(M,N). (O-Z)Gastrulation stage markers at E7.5-E7.75. Whole-mount in situ hybridization with probes recognizing embryonic markers Chrd (O-P),
Otx2 (Q-R), Gbx2 (S-T) and Cer1 (U,V). Whole-mount in situ hybridization against extra-embryonic markers Rhox5 (W,X) and Bmp4 (Y,Z). Embryos
are oriented with the anterior part to the left and the posterior to the right, as shown in A, except Prep1–/– embryos, which do not show any
apparent AP asymmetry. Scale bar: 200m.
C-terminally truncated Prep1 was observed using antibodies
specifically recognizing either the N terminus or the C terminus of
Prep1 (see Fig. S3A-C in the supplementary material). The western
blot analysis of the ES cell nuclear extracts using the N terminusspecific antibody allowed us to observe only background staining
at the low molecular weight range of the gel in Prep1–/– ES cells
(see Fig. S3D in the supplementary material); this was the case
even after cultivating the cells with MG132 proteasome inhibitor
(see Fig. S3E in the supplementary material). We conclude that
Prep1 is totally absent and hence that Prep1–/– are null embryos.
Onset of gastrulation in the Prep1 KO
Table S1 in the supplementary material lists the specific perigastrulation markers used and the number of embryos analyzed in
each case. PS formation is evidence of the onset of gastrulation
around E6.5 (Tam and Behringer, 1997). The T gene (Brachyury)
marks the PS and axial mesoderm (Wilkinson et al., 1990). Prep1+/+
and Prep1+/– embryos were T positive but Prep1–/– embryos were
not (Fig. 1A,B), suggesting that Prep1-deficient embryos lack the
PS. To exclude the possibility of a simple delay in T expression, we
tested its expression at E7.5 and E8.5; at neither stage did Prep1–/–
embryos express T (not shown). As T is expressed in the distal ExE
before PS formation (Perea-Gomez et al., 2004; Rivera-Perez and
Magnuson, 2005) and as we did not observe T in Prep1–/– embryos,
we repeated the staining for T and increased the color reaction. We
also tested for Fgf8 as a second PS marker (Crossley and Martin,
1995). Again, Prep1–/– embryos were negative for T (see Fig. S4AD in the supplementary material) and Fgf8 (Fig. 1C-D). We tested
Lefty2 as a nascent mesoderm marker (Meno et al., 1999) at E6.75E7.5 and, in accordance with the absence of PS, Prep1–/– embryos
were also negative for Lefty2 (Fig. 1E,F).
PS induction depends on the crosstalk between the Epi and two
extra-embryonic tissues: AVE and ExE (Ang and Constam, 2004;
Tam et al., 2006). In this process, important signaling molecules
include Nodal, Wnt3 and Bmp4. While Nodal and Wnt3 are
expressed in the posterior embryonic/extra-embryonic junction,
next to where the PS arises (Brennan et al., 2001; Liu et al., 1999),
Bmp4 is expressed in the distal ExE (Coucouvanis and Martin,
1999; Lawson et al., 1999). In Prep1–/– embryos at E6.5-E6.75,
Nodal was indeed expressed in the Epi (Fig. 1G,H), whereas Wnt3
was strongly decreased or absent (Fig. 1I,J); however, Bmp4 was
normally expressed in the ExE as in wild-type embryos (Fig. 1KL). The areas of expression of Nodal and Bmp4 in the Epi and the
ExE, respectively, were reduced in size (Fig. 1G,H,K,L). Finally,
we examined the formation of the AVE, a signaling center
implicated in PS formation (Bertocchini and Stern, 2002; PereaGomez et al., 2002) and neural induction (Albazerchi and Stern,
2007; Kimura et al., 2000; Perea-Gomez et al., 2001) because it
expresses antagonists of Nodal (Meno et al., 1999; Takaoka et al.,
2006), Bmp (Belo et al., 2000) and Wnt (Kimura-Yoshida et al.,
2005) genes. The AVE marker Cerberus-like (Cer1) (Belo et al.,
1997; Biben et al., 1998; Shawlot et al., 1998) was not detectable
in Prep1–/– embryos (Fig. 1M,N), suggesting the absence of AVE.
Thus, although the Epi and the ExE of Prep1 mutant embryos do
express important signaling molecules such as Nodal and Bmp4,
Prep1–/– embryos at the onset of gastrulation are reduced in size,
do not express Wnt3 and form no AVE, PS or mesoderm.
Gastrulation in Prep1 KO embryos
During gastrulation, between E7.5 and E7.75, Chordin (Chrd) is
expressed in the node and axial mesoderm (Bachiller et al., 2000),
Otx2 in the anterior neuroectoderm (Ang et al., 1994; Simeone et
Development 137 (20)
al., 1993), Gbx2 in the posterior neural tube (Wassarman et al.,
1997), Cer1 in the definitive endoderm (Belo et al., 1997; Biben et
al., 1998; Shawlot et al., 1998) and Sox1 in the neural plate (Wood
and Episkopou, 1999). Unlike wild-type and heterozygous
embryos, Prep1–/– embryos did not express Chrd at E7.5 (Fig.
1O,P), Otx2 at E7.75 (Fig. 1Q,R), Gbx2 at E7.75 (Fig. 1S,T), Cer1
at E7.5 (Fig. 1U,V) or Sox1 at E7.75 (see Fig. S4E-F in the
supplementary material). Therefore, in agreement with the absence
of PS, Prep1–/– embryos lack mesoderm, endoderm and ectoderm
patterning, i.e. establish no embryonic lineage. Next, we looked at
Rhox5 (Lin et al., 1994; Maclean et al., 2005), Bmp4 (Lawson et
al., 1999; Winnier et al., 1995) and Flk1 (Yamaguchi et al., 1993)
as extra-embryonic markers. Prep1–/– embryos expressed Rhox5 at
E7.5 and E8.5 (Fig. 1W,X and data not shown), most probably
marking the extra-embryonic part of the visceral endoderm.
Prep1–/– embryos expressed Bmp4 (Fig. 1Y,Z) that most likely
represents the residual expression in the distal ExE. In agreement,
Prep1–/– embryos also expressed Cdx2 (not shown), a second ExE
marker (Beck et al., 1995). Finally, Flk1 was almost undetectable
(see Fig. S4G,H in the supplementary material) arguing that there
is no formation of endothelial cells in the Prep1 KO. Hence, at
E7.5-E7.75 Prep1–/– embryos have no embryonic derivatives and
the extra-embryonic compartments are restricted to residual cells
of the ExE and derivatives of the visceral endoderm.
Size reduction of the epiblast in the Prep1 KO
Early post-implantation Epi cells are pluripotent. Descendants of
single early PS Epi cells are able to contribute to more than one
tissue type (Lawson et al., 1991; Tam and Behringer, 1997).
Moreover, it is possible to extract Epi-stem cells from egg cylinder
stage embryos (Brons et al., 2007; Tesar et al., 2007). Hence, the
absence of PS in Prep1–/– embryos might be due to premature
differentiation of pluripotent Epi cells. To assess this hypothesis,
we examined the expression of Oct4 (Pou5f1), which is widely
accepted as a marker for pluripotency in post-implantation embryos
(Ding et al., 1998; Liguori et al., 2003). Although reduced in
number, Oct4-positive cells were observed in Prep1–/– embryos at
E6.75 (Fig. 2A,B), E7.5 (Fig. 2C,D) and E8.5 (Fig. 2E,F). The
expression of Oct4 in Prep1–/– embryos argues against the above
hypothesis and shows that in Prep1–/– mutants the number of
epiblast cells is reduced.
Altogether, our marker analysis shows that by E7.5-E7.75,
Prep1–/– embryos have a reduced number of Epi cells and a
residual ExE all surrounded by derivatives of the visceral
endoderm. We have confirmed this point with a double in situ
against Oct4 and Bmp4 (Fig. 2G,H).
Prep1–/– epiblast undergoes apoptosis that is
rescued in a p53–/– background
The above results suggest that the phenotype may be due to a basic,
general cellular failure, i.e. excessive apoptosis or a block of
proliferation. Epi cells at the egg cylinder stage are highly sensitive
to DNA damage, which induces apoptosis without arresting the cell
cycle (Heyer et al., 2000). Therefore, we tested whether apoptosis
might account for the decreased number of Epi cells in Prep1–/–
embryos. Whole-mount confocal immunofluorescence of cleaved
caspase 3 showed that, in the absence of Prep1, pluripotent Epi
cells underwent apoptosis at E6.5 (Fig. 2I,J) and E7.5 (data not
shown). A quantification of cleaved caspase 3 staining is shown in
Fig. 2K. Similar results were obtained in a TUNEL staining at E6.5
and E7.5 (see Fig. S4K-O in the supplementary material). Cleaved
caspase 3 and TUNEL staining of Prep1–/– embryos concentrate in
DEVELOPMENT
3396 RESEARCH ARTICLE
Prep1 knockout phenotype
RESEARCH ARTICLE 3397
the Epi region (Fig. 2J, see Fig. S4J-L in the supplementary
material), arguing that the apoptotic cells are indeed the Oct4positive Epi cells.
The high sensitivity of Epi cells to irradiation during the egg
cylinder stage is p53 dependent (Heyer et al., 2000). To test
whether the apoptosis we observed in Prep1–/– embryos depends
on p53, we crossed Prep1+/– and p53-null mice. Mice with
mutations in Prep1 or p53 were both created in a full C57BL6
background. Embryos from double-heterozygous Prep1+/– p53+/–
intercrosses were analyzed with specific markers.
Thirty-five E7.5 embryos were extracted and hybridized with
an Oct4 probe (Table 2). Among them, the two Prep1–/– p53–/–
double homozygous embryos showed a strong increase in the
size of the Oct4 expression domain (arrow in Fig. 2L-N).
Prep1–/– embryos heterozygous for p53 (asterisk in Fig. 2M)
were indistinguishable from Prep1–/– embryos (see Fig. 2D).
Thus, the absence of p53 rescues the number of Epi cells.
Indeed, the increase in Oct4 staining was accompanied by a
decrease in TUNEL staining in Prep1–/– p53–/– double KO
embryos (see Fig. S4K-M in the supplementary material). Cer1
was re-expressed in Prep1–/– p53–/– embryos (arrow in Fig. 2OQ), but in a pattern more similar to that in the AVE than to the
pattern in the definitive endoderm. A Prep1–/– p53+/+ embryo
was negative for Cer1 (asterisk in Fig. 2P), as expected (see Fig.
1V). Moreover, T and Chrd were expressed at E7.5 in double KO
embryos, arguing that, unlike Prep1–/– embryos (Fig. 1A-B,O-P),
DEVELOPMENT
Fig. 2. Prep1 KO mouse embryos have
a smaller epiblast and undergo p53dependent apoptosis. (A-H)Oct4
expression in the Epi of Prep1–/– embryos.
Whole-mount in situ hybridization against
Oct4 at E6.75 (A,B), E7.5 (C,D) and E8.5
(E,F). The arrows in B,D,F show Oct4
staining in Prep1–/– embryos. Double
whole-mount in situ hybridization against
Oct4 and Bmp4 (G,H). (I-K)Apoptosis in
Prep1–/– embryos. Cleaved caspase 3
(Csp3) whole-mount confocal
immunofluorescence at E6.5 (I,J). Left
column shows the DAPI, the middle
column shows cleaved caspase 3 and the
right column shows the merge. Images
are at Max Z projection. Caspase 3positive area normalized by comparison
with DAPI-positive area (K). (L-W) Wholemount in situ hybridization on E7.5
embryos derived from Prep1+/– p53+/–
intercrosses with Oct4 (L-N), Cer1 (O-Q),
T (R-T) and Chrd (U-W) probes. The
asterisks in M,P,S,V show results similar to
those observed in Prep1–/– embryos. The
arrows in N,Q,T,W show the recovery in a
p53–/– background.
3398 RESEARCH ARTICLE
Development 137 (20)
Fig. 3. Prep1-p53 double null mouse embryos are almost reabsorbed at E10.5; Prep1-null embryos retain proliferation capacity.
(A-F)Prep1-p53 double KO lethality. (A-C)Whole-mount in situ hybridization with a T probe on Prep1-p53 double KO embryos at E8.5. (D)Size and
shape of a Prep1+/– p53–/– embryo at E10.5. (E)Around E10.5 double-mutant embryos are almost reabsorbed. (F)Comparison of the size and shape
of a Prep1+/– p53+/– (right) with a Prep1–/– p53–/– (left) embryo at E10.5. (G-I)Proliferation in Prep1–/– embryos. (G,H)Oct4 and P-H3 whole-mount
immunofluorescence at E6.5. (I)Mitotic index normalized by comparison with DAPI-positive area.
Prep1–/– p53–/– embryos formed the PS (Fig. 2R-T), which
reached the most distal part of the embryo (Fig. 2U-W). Finally,
Prep1–/– p53–/– embryos were Bmp4-positive and possessed a
better organized ExE than did Prep1–/– embryos (data not
shown).
Interestingly, in spite of the recovery in the number of Oct4positive cells at E7.5 in the two embryos, and the re-expression of
Cer1, T and Chr, this recovery was still strongly delayed at E8.5 in
Prep1–/– p53–/– embryos. The expression of T in E8.5 Prep1–/–
p53–/– embryos is still evident (Fig. 3A-C) but the staining does not
Table 2. The Prep1-p53 and Prep1-Atm double knockout
Oct4
TUNEL
Cer1
T (E7.5)
Chr
T (E8.5)
Prep1+/– p53+/+
3
1
2
2
2
5
6
5
4
2
4
3
Prep1+/+ p53+/– Prep1+/– p53+/– Prep1+/+ p53–/–
3
2
2
4
1
7
9
13
4
12
5
16
Prep1+/– p53–/–
Prep1–/– p53+/+
Prep1–/– p53+/–
Prep1–/– p53–/–
3
9
3
5
2
5
0
2
1
1
0
0
7
4
0
0
1
3
2
1
1
2
1
3
2
1
1
1
2
3
Prep1-Atm crosses
Prep1+/+ Atm+/+ Prep1+/– Atm+/+ Prep1+/+ Atm+/– Prep1+/– Atm+/– Prep1+/+ Atm–/– Prep1+/– Atm–/– Prep1–/– Atm+/+ Prep1–/– Atm+/–
Oct4
2
4
7
9
3
4 (2)
2
7
Prep1–/– Atm–/–
1 (1)
The top part shows genotype and number of embryos derived from Prep1+/– p53+/– intercrosses analyzed by whole-mount in situ hybridization for Oct4, Cer1, T and Chordin
and by whole-mount TUNEL at E 7.5. T was analyzed also at E8.5.
The bottom part shows genotype and number of embryos derived from Prep1+/– Atm+/– intercrosses analyzed by whole-mount in situ hybridization for Oct4. The numbers in
parentheses indicate the number of embryos in which a clearly evident phenotype was observed, different from that of Atm+/–.
DEVELOPMENT
Prep1-p53 crosses
Prep1+/+ p53+/+
Prep1 knockout phenotype
RESEARCH ARTICLE 3399
reach the anterior part of the embryo (Fig. 3C). We did not
precisely determine the latest stage at which double KO mutants
could be recovered. However, E9.5 and E10.5, Prep1–/– p53–/–
embryos are almost reabsorbed (Fig. 3D-F and data not shown).
We conclude that Prep1 is required to prevent p53-dependent
apoptosis in the embryonic Epi at peri-implantation stages and that
the absence of p53 allows the increase in number of epiblast Oct4positive cells inducing AVE and PS formation, thus only a partial
recovery of the phenotype. This indicates that Prep1 is also
required during gastrulation.
Proliferation in Prep1–/– embryos
During peri-gastrulation stages, the Epi is hyper-proliferative
(O’Farrell et al., 2004). First, we tested the proliferation of E6.75
Prep1–/– embryos with Ki67 immunostaining, a marker of nonquiescent cells, and found no difference between Prep1+/+, Prep1+/–
and Prep1–/– embryos (data not shown). Moreover, at E6.75,
whole-mount immunofluorescence for Oct4 and for the mitotic cell
marker phospho-histone 3 (P-H3) (Gurley et al., 1978), analyzed
by confocal microscopy (Fig. 3G-H), showed that embryos were
reduced in size, but had a large P-H3-positive signal (Fig. 3H). The
mitotic index quantification is shown in Fig. 3I. To complete our
proliferation analysis of the Prep1 KO, we produced a Prep1-p53p16 triple KO. Ink4Ap16 is an inhibitor of pRb and its absence
confers a proliferative advantage (Sharpless, 2005). We crossed
ink4Ap16-null (Krimpenfort et al., 2001) with Prep1+/–p53+/– double
heterozygous mice. The Ink4Ap16 mice were in a mixed C57BL6SV129 genetic background. Therefore, in order to exclude that a
change in the genetic background would affect the phenotype we
verified that the phenotype was conserved in the new background
(data not shown). Although we did not yet find a triple
homozygous Prep1–/– p53–/– p16–/– embryo, the Prep1–/– phenotype
was not rescued in p16 Ko (see expression of Oct4 in the p16–/–
double KO embryos) (see Fig. S4N,O in the supplementary
material). Thus, despite the reduced size, Prep1–/– embryos retained
their proliferation capacity.
Prep and Meis gene expression domains
underscore the difference in their function
From E8.5 to birth, Prep1 is expressed ubiquitously and weakly
(Ferretti et al., 1999). We examined the expression pattern of Prep1
in wild-type embryos during gastrulation and ES cell
differentiation. Prep1 whole-mount in situ hybridization shows a
weak ubiquitous signal from E6.5 to E8.5, in some cases difficult
DEVELOPMENT
Fig. 4. Prep1, unlike other MEIS genes, is
expressed ubiquitously during gastrulation,
at pre-implantation stages and during ES cell
differentiation. (A-F)Prep1 whole-mount
confocal immunofluorescence at E6.5 (A-B) and
E7.5 (D-E). (A,D)DAPI staining (B,E) Prep1
staining. The left column shows the negative
control (incubated with the secondary antibody
only), the middle and the right columns show two
different mouse embryos. (C,F)Higher
magnifications of one of the two analyzed
embryos showing the nuclear localization of
Prep1. Left, DAPI staining; middle, Prep1 staining;
right, merge. (G)Q-PCR expression analysis of
Prep1 (yellow line), Oct4 (red line), Nanog (blue
line) and Nestin (green line) during in vitro
differentiation of wild-type ES cells. (H)Q-PCR
expression analysis of Prep1 (yellow line), Prep2
(red line), Meis1 (blue line) and Meis2 (green line)
during in vitro differentiation of wild-type ES cells.
(I)RT-PCR analysis of Prep1 expression during
wild-type pre-implantation stages normalized to
the ribosomal protein gene S16. (J-L)Wholemount in situ hybridization of Prep2 (J), Meis1 (K)
and Meis2 (L) at E8.5.
3400 RESEARCH ARTICLE
Development 137 (20)
Fig. 5. Prep1 is not essential for ES cell
viability and EBs formation, and the
Prep1–/– embryonic phenotype is
increased by Atm decrease. (A-C)Prep1–/–
ES cell line isolation. (A)A wild-type and a
Prep1–/– mouse clone. (B)Number and
genotypes of ES clones derived from Prep1+/–
heterozygous intercrosses. (C)Oct4
immunofluorescence with wild-type and
homozygous ES cells. (D-F)Different sizes
reached by EBs from wild-type and Prep1–/– ES
cells. (D)EBs formed according to the hanging
drop method with one wild-type and two
Prep1–/– ES cell lines. (E)Cleaved caspase 3
whole-mount confocal immunofluorescence
with wild-type and Prep1–/– EBs. (F)Caspase 3positive area normalized by comparison with
DAPI-positive area. (G-J)E7.5 Oct4 wholemount in situ hybridization in embryos with
different doses of Prep1 and Atm genes.
(G,H)The Atm-null genotype decreases the
Oct4+ area in Prep1+/– (arrow) but not in
Prep1+/+ embryos. (I,J)The absence of Atm has
a strong effect in the Prep1–/– embryo. The
asterisk in I shows that the Oct4+ area has a
size similar to that of homozygous Prep1–/–
embryos. The arrows in J shows the decreased
Oct4-staining of Prep1–/– embryos in an Atm–/–
background.
We conclude that Prep1 is expressed from the earliest stages of
embryogenesis, ubiquitously at gastrulation, and is not regulated
during ES cells differentiation. These features are unique within the
MEIS class of transcription factors.
Prep1–/– ES cell derivation
We have derived ES cells from E3.5 blastocysts of Prep1+/–
intercrosses. The derivation yield of Prep1–/– ES cell lines was low
(2/26) (Fig. 5A,B; data not shown). However, these lines expressed
Oct4 and Nanog (Fig. 5C; data not shown).
Prep1–/– ES cells were able to differentiate using the hanging
drop protocol; however, this gave rise to smaller EBs than in wildtype ES cells, particularly for one of the two Prep1–/– cell lines
(Fig. 5D). Similarly to our whole-mount results, the cleaved
caspase 3 staining was more intense in Prep1–/– than in wild-type
EBs (Fig. 5E,F), especially in the smaller EBs. The differences
were not as pronounced as in embryos (Fig. 2I,J). Analysis of
specific markers (Oct4 and Nanog for pluripotency, T for
mesoderm, Fgf5 and Sox1 for neuroectoderm, Gata4 for endoderm
and Hand1 for trophoectoderm) showed that, although there was
a slight delay, Prep1–/– ES cells were overall capable of
differentiation (data not shown).
DEVELOPMENT
to distinguish from the sense strand control signal, especially at
E7.5 (see Fig. S4P-U in the supplementary material). In wholemount immunofluorescence, however, Prep1 was expressed in the
whole embryo at E6.5 (Fig. 4A,B) and at E7.5 (Fig. 4D,E), and was
localized to the nucleus (Fig. 4C,F). The specificity of the antibody
was verified using Prep1–/– embryoid bodies (EBs) (data not
shown).
We also analyzed the expression of Prep1 at pre-implantation
stages by RT-PCR (Fig. 4I) and during ES cell differentiation by
qRT-PCR (Fig. 4G). Prep1 was expressed from the one-cell to the
blastocyst stage (Fig. 4I). During ES cell differentiation, Prep1 was
not up- or downregulated, unlike the pluripotent genes Oct4 and
Nanog (the expression of which decreased during differentiation),
and the neural marker Nestin [the expression of which increased
(Fig. 4G)].
We have also studied the expression of Prep2, Meis1 and Meis2
during ES cell differentiation. Unlike Prep1, the levels of
expression of Prep2, Meis1 and Meis2 increased during
differentiation (Fig. 4H). Accordingly, in the E8.5 embryo, when
organogenesis starts, Prep2 (Fig. 4J), Meis1 (Fig. 4K) and Meis2
(Fig. 4L) were not ubiquitously expressed, but had more restricted
expression patterns.
Altogether, these results confirm the role of Prep1 in
homeostasis in early embryogenesis; Prep1–/– ES cells are extracted
in lower ratio than expected (8% instead of the expected 25%). The
two viable Prep1–/– ES cell lines phenocopy Prep1–/– embryos,
although in a milder fashion, giving rise to small EBs with
increased apoptosis.
Atm downregulation exaggerates the Prep1–/–
phenotype
E6.5 Epi cells do not tolerate double-stranded DNA damage and
undergo p53 dependent apoptosis (Heyer et al., 2000). In Prep1i/i
hypomorphic MEFs, we observed an increase of basal apoptosis
(Micali et al., 2009) and increased double strand break response (G.I.
and F.B., unpublished). To test whether DNA damage was at the
basis of Prep1–/– p53-dependent apoptosis of Epi cells, we crossed
Prep1+/– mice with Atm+/– mice (Borghesani et al., 2000), both in a
full C57BL6 background. As Atm is important in DNA repair, one
would expect its absence or reduction to worsen the Prep1
phenotype. The absence of Atm induced a phenotype in two out of
four heterozygous Prep1+/– embryos (Fig. 5G-J), which were delayed
in their development, whereas in the other two embryos the effects
were less evident. Moreover, in the single recovered Prep1–/–Atm–/–
double knockout embryo, the phenotype was stronger than in the
Prep1–/–Atm+/+, because, by E7.5, the Oct4-positive cells were almost
undetectable (Fig. 5G-J). In agreement with the above results,
downregulation of Atm with an sh-RNA in Prep1–/– ES EBs induced
a further decrease of their already deficient size and increased
cleaved caspase 3 staining, i.e. apoptosis (data not shown).
DISCUSSION
The absence of Prep1 causes p53-dependent apoptosis of Epi cells,
which prevents gastrulation and differentiation. This is probably due
to the accumulation of DNA damage. The absence of Prep1 affects
all cells but apoptosis was mostly observed in the Epi, probably
owing to the tremendous proliferative expansion of the Epi at this
stage. Thus, the role of Prep1 in early embryogenesis is to protect
epiblast cells from accumulating damage that induces apoptosis.
Prep1–/– embryos arrest development around
E5.0-5.25
Our data suggest that Prep1–/– embryos arrest around E5.0 or
E5.25. Indeed, they die before E5.5, which is when AVE is
established in the distal tip of the embryo (Tam and Loebel, 2007).
However, death must occur after the time of expression of Oct4
(which is present at implantation in the Epi) (Pfister et al., 2007).
This timing coincides with the loss of Wnt3 expression in the
posterior Epi at E5.75 (Rivera-Perez and Magnuson, 2005).
Reduction in Epi cell number is the basis for the
Prep1 KO phenotype
The lack of gastrulation is due to the reduced size of the epiblast.
Prep1 knockout embryos show a decrease of the pluripotent cells
expressing Oct4 at E6.5, E7.5 and E8.5 (Fig. 2A-F). The number
of Epi cells is a limiting factor for the initiation of gastrulation
(Tam and Behringer, 1997). Elimination of one blastomere from a
two- or four-cell embryo delays gastrulation; gastrulation starts
only when the embryo has accumulated a sufficient number of Epi
cells (Power and Tam, 1993; Rands, 1986). The size recovery is
achieved by increasing cell proliferation prior to and during
gastrulation (Power and Tam, 1993). It is likely that the low
number of Epi cells explains the absence of the AVE and PS in
Prep1–/– embryos, and, therefore, the absence of embryonic
RESEARCH ARTICLE 3401
lineages. Nodal signaling from the Epi is essential for specifying
AVE in the most distal part of the embryo (Brennan et al., 2001;
Mesnard et al., 2006). The lack of expansion of the Prep1–/– Epi
may not keep the distal cells far enough from the negative signals
of the ExE (Mesnard et al., 2006; Richardson et al., 2006;
Rodriguez et al., 2005), hence maintaining them under the
inhibitory control of the ExE.
Why does Prep1 absence induce p53-dependent
apoptosis?
The pattern of Prep1 expression during ES cell differentiation and
the ability to harvest Prep1–/– ES cells, which still express Oct4 and
can, on the whole, differentiate (data not shown), suggests that
Prep1 is not a reprogramming or pluripotency-establishing gene
(Avilion et al., 2003; Chambers et al., 2003; Mitsui et al., 2003;
Nichols et al., 1998).
The decreased number of Oct4-expressing cells, together with
the increased rate of p53-dependent apoptosis, suggest that Prep1
is, however, essential for Epi homeostasis. This is also confirmed
by the in vitro analysis of Prep1–/– ES cells. Our current
explanation for the p53-dependent apoptosis of the Prep1–/–
epiblast is DNA damage.
The early post-implantation embryo is very sensitive to UV
irradiation, and Epi cells irradiated with doses that do not induce
any response at other stages or in somatic cells undergo p53dependent apoptosis (Heyer et al., 2000). We hypothesize that in
the absence of Prep1, DNA damage accumulates in the embryonic
cells. The DNA damage hypothesis is suggested by the observation
that a decrease in Atm induces a phenotype in the heterozygous,
and worsens the phenotype of the homozygous, Prep1 mutant
embryos. Likewise, downregulation of Atm in Prep1–/– but not in
wild-type ES cells induces apoptosis and inhibits EB growth. This
argues that the inability to repair DNA synergizes with the absence
of Prep1 and drives Epi cells into apoptosis. Accordingly, in
Prep1i/i hypomorphic MEFs, we observed an increase in doublestranded breaks and DNA repair signaling (G.I. and F.B.,
unpublished). Why the absence of Prep1 could induce DNA
damage is, at the moment, matter for speculation.
Interestingly, several cancer genes are involved in controlling
DNA damage response and DNA repair. Indeed, several pieces of
evidence demonstrate that Prep1 is a novel tumor suppressor
(Longobardi et al., 2010).
Acknowledgements
We are very grateful to Dr Claudio Stern for his interest and advice; to the late
Graziella Persico and to Giovanna Liguori for much advice and reagents; to
Luisa Lanfrancone for reagents and helpful discussions; and to Anton Berns for
Ink4Ap16 mice. We also thank Antonello Mallamaci, Shankar Srinivas, Silvia
Brunelli, Miguel Torres, Janet Rossant, Juan Pedro Martinez Barberà, Tristan
Rodriguez, Michael Shen, Daniel Constam, Vania Broccoli, Hans Schoeler, Isao
Matsuo and Robin Lovell-Badge for probes, and Michael Hemann for the
shATM vector. This work was funded by grants from TELETHON Onlus (Italy)
and Ministero della Salute to F.B., and from FIRC to G.I.
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.050567/-/DC1
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Prep1 knockout phenotype