The absence of Prep1 causes p53-dependent apoptosis of mouse … · Prep1 i/ embryos express ~2% of...

3393DEVELOPMENT AND STEM CELLS RESEARCH ARTICLE

INTRODUCTIONSeveral crucial events take place during early development. In theE3.5 blastocyst, the inner cell mass (ICM) contains progenitorcells, 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 implantationaround E4.5, and from E5.5 to E6.5 forms an epithelium [a processknown as cavitation (Coucouvanis and Martin, 1995)], maintainsits 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 verysensitive to DNA damage (Heyer et al., 2000) and is not protectedby specific G1 and G2 check points (O’Farrell et al., 2004). DNAdamage at this stage leads to p53-dependent apoptosis (Heyer et al.,2000). Formation of the primitive streak (PS) around E6.5 marksthe beginning of gastrulation and requires interactions between theEpi, the extra-embryonic AVE (anterior visceral endoderm) and theExE (extra-embryonic ectoderm). Gastrulation, during which theEpi gives rise to the three embryonic layers (ectoderm, mesodermand definitive endoderm), is followed by the neural tube formationfrom the neural plate (Stern, 2006). After neurulation, the bodyplan of the embryo is established, with distinct antero-posterior,dorso-ventral and left-right axes (Tam and Behringer, 1997).

Prep1 (Pknox1 – Mouse Genome Informatics) homeodomaintranscription factor belongs to the TALE (three amino acids loopextension) superclass of proteins that include Meis1-Meis3,Prep2 and Pbx1-Pbx4. Deletion of Pbx and Meis genes in mouseshows that these genes are essential for organogenesis anddifferentiation. Homozygous Meis1–/– embryos die around E14.5with a severe hematopoietic phenotype (Azcoitia et al., 2005;Hisa et al., 2004) that is similar to that of the hypomorphic Prep1mutants (Prep1i/i) (Di Rosa et al., 2007; Ferretti et al., 2006).Prep1i/i embryos express ~2% of Prep1 mRNA and 2-10% of theprotein, die mostly at E17.5 with liver hypoplasia, anemia,angiogenesis and eye defects (Ferretti et al., 2006). Thehematopoietic phenotype is due to malfunction of long-termrepopulating hematopoietic stem cells (Di Rosa et al., 2007). Noloss of function mutation for Meis2, Meis3 or Prep2 has beendescribed. A compound Pbx1-Pbx2 knockout mouse is lethalaround E12.5 and displays pallor, edema, general organhypoplasia and homeotic features (Capellini et al., 2006; Selleriet al., 2001). While the single Pbx2 knockout mice are viableand fertile (Selleri et al., 2004), Pbx3 null mice are born, but diewithin a few hours owing to central respiratory failure (Rhee etal., 2004). Importantly, Pbx2 partly compensates for Pbx1(Capellini et al., 2006; Selleri et al., 2004).

We have analyzed Prep1–/– embryos in which the expression ofthe protein was eliminated by targeting the DNA-bindinghomeodomain. This mutation leads to early post-implantationlethality. 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 andp53 (Trp53 – Mouse Genome Informatics) as p53 ablation partiallyrescues the Prep1–/– phenotype. Therefore, unlike all other TALEproteins, Prep1 is responsible for protecting the embryo very earlyin development, a unique function within the Meis-Prep familiesof transcription factors.

Development 137, 3393-3403 (2010) doi:10.1242/dev.050567© 2010. Published by The Company of Biologists Ltd

1IFOM, FIRC Institute of Molecular Oncology Foundation, and 2Department ofExperimental 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 BolognettiFoundation and Department of Psychology, Section of Neuroscience, University‘Sapienza’ of Rome, 00185 Rome, Italy. 5Università Vita Salute San Raffaele andIstituto Scientifico San Raffaele, via Olgettina 60, 20132 Milan, Italy.

*Author for correspondence ([email protected])

Accepted 18 August 2010

SUMMARYDisruption of mouse Prep1, which codes for a homeodomain transcription factor, leads to embryonic lethality during post-implantation 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 andepiblast, 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 reducednumber of Oct4-positive cells is constant up to E8.5. At E6.5, Prep1–/– embryos retain a normal mitotic index but show a majorincrease in cleaved caspase 3 and TUNEL staining, indicating apoptosis. Therefore, the mouse embryo requires Prep1 whenundergoing 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 earlylethal phenotype, Prep1 is not essential for ES cell establishment. A differential embryonic expression pattern underscores theunique function of Prep1 within the Meis-Prep family.

KEY WORDS: Prep1 (Pknox1), Embryo development, Epiblast, Gastrulation, p53 (Trp53), Mouse

The absence of Prep1 causes p53-dependent apoptosis ofmouse pluripotent epiblast cellsLuis 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,*

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MATERIALS AND METHODSGeneration of miceExons 7 and 8 of the Prep1 gene were replaced with pSAb-geo vectorcassette (see Fig. S1A-D in the supplementary material) (Hisa et al., 2004)and homologous recombination events tested by Southern blotting of EcoRIdigested DNA with 5� and 3� probes (see Fig. S1E,F in the supplementarymaterial). Of several independent isolated ES cell clones carrying themutation, one was introduced into the germ line using standard techniques.Pure C57/BL6 double KO Prep1-p53 or Prep1-Atm mutant mice wereobtained by standard crossing p53–/– (Jackson Labs, Bar Harbor, ME, USA)or Atm–/– (Borghesani et al., 2000) mice with Prep1+/– females. The Prep1-p53-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 genotypingGenotyping was carried out by Southern blot using standard techniques orby PCR (see Fig. S1 in the supplementary material). The standard protocolwas one cycle of 5 minutes at 94°C, 35 cycles of 30 seconds at 95°C, 30seconds at 55°C and 30 seconds at 72°C. A final elongation step of 5minutes at 72°C was performed. For E8.5 and E7.5 embryos, DNApolymerase concentration was doubled, and the PCR reaction extended to40 cycles. The sequences of the primers for genotyping (arrows in Fig.S1C,D in the supplementary material) were: P12, 5�-GAGAGCTCAAG -GACA GCCAGGCTA-3�; M7, 5�-CCAGGAGATAATGCCTGC GTGA -CC-3�; and T3, 5�-ACCGCGAAGAGTTTGTCCTCAACC-3�.

Reverse transcription-PCRRT-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�-AGA -CAAGCAATGTACCGACTACAG-3�. For the KO mRNA, the primer R2corresponds to M7 and R3 to T3.

ImmunoblottingAntibodies 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 assayElectrophoretic 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�-AAGACAGGGGAGG GAGCCGG -GCGGGAGAGGGAGGGGCGGCGCCGGGGCGGGCCCT-3� (Ibanez-Tallon et al., 2002).

Embryo dissection, whole-mount in situ hybridization and TUNELEmbryos were dissected from the maternal deciduas by standardprocedures and in situ hybridized (including double in situ) as describedpreviously (Liguori et al., 2003). TUNEL was performed using theApopTag Kit (Millipore, Billerica, MA, USA).

RT-PCR of pre-implantation embryosRT-PCR was performed on total RNA (pools of 100 embryos) (TRIzolreagent; Invitrogen, Carlsbad, CA, USA) as described (Fiorenza et al.,2004). Primers used were Prep1, 5�-ATGATGGCG ACACAGACGC -TAAGTATA-3� (sense) and 5�-GGGGTCTG AGACTCGATGGGA GGA -GGACTC-3� (antisense); b-actin, 5�-GGTTC CGAT GCCCTGAGGCTC-3� (sense) and 5�-ACTTGC GGTG CATGGAGG-3� (antisense).

Prep1–/– ES cell derivation and ES cell cultureEmbryonic stem (ES) cells were derived from E2.5-3.5 blastocysts fromPrep1+/– crosses using standard procedures. Mouse ES cells E14Tg2a werecultured without feeders in Glasgow-modified Eagle’s MEM with 15% ES-screened FBS (Hyclone, Logan, UT, USA), under standard conditions with1000 U/ml leukemia inhibitory factor (LIF; Chemicon, CA, USA).Embryoid bodies (EBs) were obtained using the standard protocol ofhanging drops method. One thousand cells per drop were plated in LIF-freeES medium.

ImmunofluorescencePassage 14-16 ES cells were grown on coverslips, fixed inparaformaldehyde, 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-conjugatedantibody (Jackson Labs, Bar Harbor, ME, USA; 1:400), donkey anti-goatA488-conjugated antibody (Invitrogen, Carlsbad, CA, USA; 1:200) andDAPI (1 g/ml). Confocal microscopes are TCS SP2 and TCS SP2 AOBS(Leica Microsystem, Germany). For each embryo, a series of opticalsections (z stacks) was collected. The mitotic index and cleaved caspase 3staining were quantified as the ratio of phospho-histone H3 or cleavedcaspase 3-positive areas compared with the DAPI stained areas, on fivesections per embryo, using ImageJ software. The same procedures wereused for immunostaining EBs with cleaved caspase 3 antibody and signalquantification.

RESULTSElimination of Prep1 function leads to early post-implantation lethalityThe Prep1 homeobox was deleted by replacing exons 7 and 8with a pSAb-geo recombination cassette (Friedrich and Soriano,1991) (see Fig. S1A-D in the supplementary material). Thevector contains a splice acceptor site with stop codons in allreading frames, and b-geo, a fusion lacZ-NeoR gene, with aninternal ATG and a bovine growth hormone polyadenylationsequence (see Fig. S1D in the supplementary material). Onesingle cell line was injected to create chimeric animals andheterozygous germ-line males were backcrossed to C57/BL6-NCr (B6) more than 10 times and are therefore essentiallyC57/BL6 congenic. The insertion was tested by Southernblotting at the 5� (see Fig. S1E in the supplementary material)and 3� (see Fig. S1F in the supplementary material) ends (theexpected fragments are shown as double arrows in Fig. S1A-Bin the supplementary material), and the results confirmed byPCR (data not shown). We have subsequently used a PCR assayto genotype the embryos. Fig. S1G in the supplementary materialshows a typical assay on Prep1+/+, Prep1+/– and Prep1–/–

embryos. Primers are indicated by small arrows in Fig. S1C-Din the supplementary material.

Heterozygous intercrosses did not yield homozygous mutantmice (Table 1). Timed pregnancy analysis placed the death ofPrep1–/– embryos around E7.5. No Prep1–/– homozygous embryo

RESEARCH ARTICLE Development 137 (20)

Table 1. Prep1–/– is lethal at post-implantation stagesEmbryonic day +/+ +/– –/– Absorbed embryos

E7.5 6 21 4 (delayed) –E8.5 7 23 7 (empty yolk sacs) 7E9.5 9 28 6 (empty yolk sacs) 12E10.5 or later 20 28 0 15Born mice 30 73 0 –

Shown are the number of born mice or the number of embryos detected at different days of gestation. DEVELO

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was recovered after E10.5, whereas at E9.5 and E8.5 homozygousstructures mostly looked like empty yolk sacs (Table 1). At E7.5,homozygous embryos appeared mostly small and developmentallydelayed (Table 1 and see below). Prep1 is therefore essential forearly post-implantation mouse development.

Characterization of the Prep1–/– mutationThe approach used to knock out Prep1 might in principle lead totranscription of a di-cistronic mRNA producing two proteins, theN-terminal part of Prep1 with its own ATG and the C-terminalb-geo from an internal ATG (see Fig. S1H in the supplementarymaterial). The fusion di-cistronic mRNA was observed byamplification from Prep1–/– E8.5 cDNA (see Fig. S1H-I in thesupplementary material). Several combinations of primers wereused to test for anomalous Prep1 mRNAs, but no additionalmRNA was found (data not shown). We also sequenced thefusion region of Prep1-b-Geo cDNA (not shown) and found theexpected STOP codons in the three frames, which should preventthe production of a fusion Prep1-b-Geo protein. The fusion

mRNA was also detected in E11.5 heterozygous mouseembryonic 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 findany difference in DNA binding in EMSA performed with Prep1+/+

and Prep1+/– extracts (see Fig. S2D-E in the supplementarymaterial). lacZ was detected by immunoblotting only inheterozygous testis extract (in the cytoplasm, see Fig. S2B in thesupplementary material) and by b-gal staining in the E13.5 retinaof Prep1+/– embryos (see Fig. S2C in the supplementary material)where Prep1 has been shown to be strongly expressed in wild-typeembryos (Ferretti et al., 2006). Finally, western blotting of wild-type, heterozygous and homozygous nuclear extracts of Prep1 EScell lines (see below) (two different clones per genotype) showedno Prep1 in homozygous (see Fig. S1J in the supplementarymaterial) and reduced Prep1 levels in heterozygous nuclear extracts(see Fig. S1J in the supplementary material). We obtained the sameresult with two different monoclonal antibodies (not shown). No

3395RESEARCH ARTICLEPrep1 knockout phenotype

Fig. 1. Absence of PS and lineage differentiation in Prep1–/– mouse embryos. Peri-gastrulation markers analysis at early post implantationstages. Representative data are shown (see Table S1 in the supplementary material for complete data). (A-N)Absence of gastrulation and AVE inPrep1–/– 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). Embryosare 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 anyapparent AP asymmetry. Scale bar: 200m.

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C-terminally truncated Prep1 was observed using antibodiesspecifically recognizing either the N terminus or the C terminus ofPrep1 (see Fig. S3A-C in the supplementary material). The westernblot analysis of the ES cell nuclear extracts using the N terminus-specific antibody allowed us to observe only background stainingat the low molecular weight range of the gel in Prep1–/– ES cells(see Fig. S3D in the supplementary material); this was the caseeven after cultivating the cells with MG132 proteasome inhibitor(see Fig. S3E in the supplementary material). We conclude thatPrep1 is totally absent and hence that Prep1–/– are null embryos.

Onset of gastrulation in the Prep1 KOTable S1 in the supplementary material lists the specific peri-gastrulation markers used and the number of embryos analyzed ineach case. PS formation is evidence of the onset of gastrulationaround 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 werenot (Fig. 1A,B), suggesting that Prep1-deficient embryos lack thePS. To exclude the possibility of a simple delay in T expression, wetested 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 ExEbefore PS formation (Perea-Gomez et al., 2004; Rivera-Perez andMagnuson, 2005) and as we did not observe T in Prep1–/– embryos,we repeated the staining for T and increased the color reaction. Wealso tested for Fgf8 as a second PS marker (Crossley and Martin,1995). Again, Prep1–/– embryos were negative for T (see Fig. S4A-D in the supplementary material) and Fgf8 (Fig. 1C-D). We testedLefty2 as a nascent mesoderm marker (Meno et al., 1999) at E6.75-E7.5 and, in accordance with the absence of PS, Prep1–/– embryoswere also negative for Lefty2 (Fig. 1E,F).

PS induction depends on the crosstalk between the Epi and twoextra-embryonic tissues: AVE and ExE (Ang and Constam, 2004;Tam et al., 2006). In this process, important signaling moleculesinclude Nodal, Wnt3 and Bmp4. While Nodal and Wnt3 areexpressed 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 Wnt3was strongly decreased or absent (Fig. 1I,J); however, Bmp4 wasnormally expressed in the ExE as in wild-type embryos (Fig. 1K-L). The areas of expression of Nodal and Bmp4 in the Epi and theExE, respectively, were reduced in size (Fig. 1G,H,K,L). Finally,we examined the formation of the AVE, a signaling centerimplicated in PS formation (Bertocchini and Stern, 2002; Perea-Gomez et al., 2002) and neural induction (Albazerchi and Stern,2007; Kimura et al., 2000; Perea-Gomez et al., 2001) because itexpresses 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 detectablein Prep1–/– embryos (Fig. 1M,N), suggesting the absence of AVE.Thus, although the Epi and the ExE of Prep1 mutant embryos doexpress 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 embryosDuring gastrulation, between E7.5 and E7.75, Chordin (Chrd) isexpressed in the node and axial mesoderm (Bachiller et al., 2000),Otx2 in the anterior neuroectoderm (Ang et al., 1994; Simeone et

al., 1993), Gbx2 in the posterior neural tube (Wassarman et al.,1997), Cer1 in the definitive endoderm (Belo et al., 1997; Biben etal., 1998; Shawlot et al., 1998) and Sox1 in the neural plate (Woodand Episkopou, 1999). Unlike wild-type and heterozygousembryos, 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), Cer1at E7.5 (Fig. 1U,V) or Sox1 at E7.75 (see Fig. S4E-F in thesupplementary material). Therefore, in agreement with the absenceof PS, Prep1–/– embryos lack mesoderm, endoderm and ectodermpatterning, i.e. establish no embryonic lineage. Next, we looked atRhox5 (Lin et al., 1994; Maclean et al., 2005), Bmp4 (Lawson etal., 1999; Winnier et al., 1995) and Flk1 (Yamaguchi et al., 1993)as extra-embryonic markers. Prep1–/– embryos expressed Rhox5 atE7.5 and E8.5 (Fig. 1W,X and data not shown), most probablymarking the extra-embryonic part of the visceral endoderm.Prep1–/– embryos expressed Bmp4 (Fig. 1Y,Z) that most likelyrepresents the residual expression in the distal ExE. In agreement,Prep1–/– embryos also expressed Cdx2 (not shown), a second ExEmarker (Beck et al., 1995). Finally, Flk1 was almost undetectable(see Fig. S4G,H in the supplementary material) arguing that thereis no formation of endothelial cells in the Prep1 KO. Hence, atE7.5-E7.75 Prep1–/– embryos have no embryonic derivatives andthe extra-embryonic compartments are restricted to residual cellsof the ExE and derivatives of the visceral endoderm.

Size reduction of the epiblast in the Prep1 KOEarly post-implantation Epi cells are pluripotent. Descendants ofsingle early PS Epi cells are able to contribute to more than onetissue type (Lawson et al., 1991; Tam and Behringer, 1997).Moreover, it is possible to extract Epi-stem cells from egg cylinderstage embryos (Brons et al., 2007; Tesar et al., 2007). Hence, theabsence of PS in Prep1–/– embryos might be due to prematuredifferentiation of pluripotent Epi cells. To assess this hypothesis,we examined the expression of Oct4 (Pou5f1), which is widelyaccepted as a marker for pluripotency in post-implantation embryos(Ding et al., 1998; Liguori et al., 2003). Although reduced innumber, Oct4-positive cells were observed in Prep1–/– embryos atE6.75 (Fig. 2A,B), E7.5 (Fig. 2C,D) and E8.5 (Fig. 2E,F). Theexpression of Oct4 in Prep1–/– embryos argues against the abovehypothesis and shows that in Prep1–/– mutants the number ofepiblast cells is reduced.

Altogether, our marker analysis shows that by E7.5-E7.75,Prep1–/– embryos have a reduced number of Epi cells and aresidual ExE all surrounded by derivatives of the visceralendoderm. We have confirmed this point with a double in situagainst Oct4 and Bmp4 (Fig. 2G,H).

Prep1–/– epiblast undergoes apoptosis that isrescued in a p53–/– backgroundThe above results suggest that the phenotype may be due to a basic,general cellular failure, i.e. excessive apoptosis or a block ofproliferation. Epi cells at the egg cylinder stage are highly sensitiveto DNA damage, which induces apoptosis without arresting the cellcycle (Heyer et al., 2000). Therefore, we tested whether apoptosismight account for the decreased number of Epi cells in Prep1–/–

embryos. Whole-mount confocal immunofluorescence of cleavedcaspase 3 showed that, in the absence of Prep1, pluripotent Epicells underwent apoptosis at E6.5 (Fig. 2I,J) and E7.5 (data notshown). A quantification of cleaved caspase 3 staining is shown inFig. 2K. Similar results were obtained in a TUNEL staining at E6.5and E7.5 (see Fig. S4K-O in the supplementary material). Cleavedcaspase 3 and TUNEL staining of Prep1–/– embryos concentrate in


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the Epi region (Fig. 2J, see Fig. S4J-L in the supplementarymaterial), arguing that the apoptotic cells are indeed the Oct4-positive Epi cells.

The high sensitivity of Epi cells to irradiation during the eggcylinder stage is p53 dependent (Heyer et al., 2000). To testwhether the apoptosis we observed in Prep1–/– embryos dependson p53, we crossed Prep1+/– and p53-null mice. Mice withmutations in Prep1 or p53 were both created in a full C57BL6background. 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 adecrease in TUNEL staining in Prep1–/– p53–/– double KOembryos (see Fig. S4K-M in the supplementary material). Cer1was re-expressed in Prep1–/– p53–/– embryos (arrow in Fig. 2O-Q), but in a pattern more similar to that in the AVE than to thepattern in the definitive endoderm. A Prep1–/– p53+/+ embryowas negative for Cer1 (asterisk in Fig. 2P), as expected (see Fig.1V). Moreover, T and Chrd were expressed at E7.5 in double KOembryos, arguing that, unlike Prep1–/– embryos (Fig. 1A-B,O-P),


Fig. 2. Prep1 KO mouse embryos havea smaller epiblast and undergo p53-dependent apoptosis. (A-H)Oct4expression in the Epi of Prep1–/– embryos.Whole-mount in situ hybridization againstOct4 at E6.75 (A,B), E7.5 (C,D) and E8.5(E,F). The arrows in B,D,F show Oct4staining in Prep1–/– embryos. Doublewhole-mount in situ hybridization againstOct4 and Bmp4 (G,H). (I-K)Apoptosis inPrep1–/– embryos. Cleaved caspase 3(Csp3) whole-mount confocalimmunofluorescence at E6.5 (I,J). Leftcolumn shows the DAPI, the middlecolumn shows cleaved caspase 3 and theright column shows the merge. Imagesare at Max Z projection. Caspase 3-positive area normalized by comparisonwith DAPI-positive area (K). (L-W) Whole-mount in situ hybridization on E7.5embryos derived from Prep1+/– p53+/–

intercrosses with Oct4 (L-N), Cer1 (O-Q),T (R-T) and Chrd (U-W) probes. Theasterisks in M,P,S,V show results similar tothose observed in Prep1–/– embryos. Thearrows in N,Q,T,W show the recovery in ap53–/– background.

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Prep1–/– p53–/– embryos formed the PS (Fig. 2R-T), whichreached the most distal part of the embryo (Fig. 2U-W). Finally,Prep1–/– p53–/– embryos were Bmp4-positive and possessed abetter organized ExE than did Prep1–/– embryos (data notshown).

Interestingly, in spite of the recovery in the number of Oct4-positive cells at E7.5 in the two embryos, and the re-expression ofCer1, T and Chr, this recovery was still strongly delayed at E8.5 inPrep1–/– 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 knockoutPrep1-p53 crosses

Prep1+/+ p53+/+ Prep1+/– p53+/+ Prep1+/+ p53+/– Prep1+/– p53+/– Prep1+/+ p53–/– Prep1+/– p53–/– Prep1–/– p53+/+ Prep1–/– p53+/– Prep1–/– p53–/–

Oct4 3 6 3 9 2 3 0 7 2TUNEL 1 5 2 13 1 9 2 4 1Cer1 2 4 2 4 1 3 1 0 1T (E7.5) 2 2 4 12 1 5 1 0 2Chr 2 4 1 5 2 2 0 1 1T (E8.5) 5 3 7 16 3 5 0 3 3

Prep1-Atm crosses

Prep1+/+ Atm+/+ 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 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 Chordinand 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 inparentheses indicate the number of embryos in which a clearly evident phenotype was observed, different from that of Atm+/–.

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 andshape of a Prep1+/– p53–/– embryo at E10.5. (E)Around E10.5 double-mutant embryos are almost reabsorbed. (F)Comparison of the size and shapeof 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-mountimmunofluorescence at E6.5. (I)Mitotic index normalized by comparison with DAPI-positive area.

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reach the anterior part of the embryo (Fig. 3C). We did notprecisely determine the latest stage at which double KO mutantscould 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 thatthe absence of p53 allows the increase in number of epiblast Oct4-positive cells inducing AVE and PS formation, thus only a partialrecovery of the phenotype. This indicates that Prep1 is alsorequired during gastrulation.

Proliferation in Prep1–/– embryosDuring peri-gastrulation stages, the Epi is hyper-proliferative(O’Farrell et al., 2004). First, we tested the proliferation of E6.75Prep1–/– embryos with Ki67 immunostaining, a marker of non-quiescent 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 cellmarker phospho-histone 3 (P-H3) (Gurley et al., 1978), analyzedby confocal microscopy (Fig. 3G-H), showed that embryos werereduced in size, but had a large P-H3-positive signal (Fig. 3H). Themitotic index quantification is shown in Fig. 3I. To complete our

proliferation analysis of the Prep1 KO, we produced a Prep1-p53-p16 triple KO. Ink4Ap16 is an inhibitor of pRb and its absenceconfers a proliferative advantage (Sharpless, 2005). We crossedink4Ap16-null (Krimpenfort et al., 2001) with Prep1+/–p53+/– doubleheterozygous mice. The Ink4Ap16 mice were in a mixed C57BL6-SV129 genetic background. Therefore, in order to exclude that achange in the genetic background would affect the phenotype weverified that the phenotype was conserved in the new background(data not shown). Although we did not yet find a triplehomozygous Prep1–/– p53–/– p16–/– embryo, the Prep1–/– phenotypewas not rescued in p16 Ko (see expression of Oct4 in the p16–/–

double KO embryos) (see Fig. S4N,O in the supplementarymaterial). Thus, despite the reduced size, Prep1–/– embryos retainedtheir proliferation capacity.

Prep and Meis gene expression domainsunderscore the difference in their functionFrom E8.5 to birth, Prep1 is expressed ubiquitously and weakly(Ferretti et al., 1999). We examined the expression pattern of Prep1in wild-type embryos during gastrulation and ES celldifferentiation. Prep1 whole-mount in situ hybridization shows aweak ubiquitous signal from E6.5 to E8.5, in some cases difficult


Fig. 4. Prep1, unlike other MEIS genes, isexpressed ubiquitously during gastrulation,at pre-implantation stages and during ES celldifferentiation. (A-F)Prep1 whole-mountconfocal immunofluorescence at E6.5 (A-B) andE7.5 (D-E). (A,D)DAPI staining (B,E) Prep1staining. The left column shows the negativecontrol (incubated with the secondary antibodyonly), the middle and the right columns show twodifferent mouse embryos. (C,F)Highermagnifications of one of the two analyzedembryos showing the nuclear localization ofPrep1. Left, DAPI staining; middle, Prep1 staining;right, merge. (G)Q-PCR expression analysis ofPrep1 (yellow line), Oct4 (red line), Nanog (blueline) and Nestin (green line) during in vitrodifferentiation of wild-type ES cells. (H)Q-PCRexpression 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 duringwild-type pre-implantation stages normalized tothe ribosomal protein gene S16. (J-L)Whole-mount in situ hybridization of Prep2 (J), Meis1 (K)and Meis2 (L) at E8.5.

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to distinguish from the sense strand control signal, especially atE7.5 (see Fig. S4P-U in the supplementary material). In whole-mount immunofluorescence, however, Prep1 was expressed in thewhole embryo at E6.5 (Fig. 4A,B) and at E7.5 (Fig. 4D,E), and waslocalized to the nucleus (Fig. 4C,F). The specificity of the antibodywas verified using Prep1–/– embryoid bodies (EBs) (data notshown).

We also analyzed the expression of Prep1 at pre-implantationstages by RT-PCR (Fig. 4I) and during ES cell differentiation byqRT-PCR (Fig. 4G). Prep1 was expressed from the one-cell to theblastocyst stage (Fig. 4I). During ES cell differentiation, Prep1 wasnot up- or downregulated, unlike the pluripotent genes Oct4 andNanog (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 Meis2during ES cell differentiation. Unlike Prep1, the levels ofexpression of Prep2, Meis1 and Meis2 increased duringdifferentiation (Fig. 4H). Accordingly, in the E8.5 embryo, whenorganogenesis starts, Prep2 (Fig. 4J), Meis1 (Fig. 4K) and Meis2(Fig. 4L) were not ubiquitously expressed, but had more restrictedexpression patterns.

We conclude that Prep1 is expressed from the earliest stages ofembryogenesis, ubiquitously at gastrulation, and is not regulatedduring ES cells differentiation. These features are unique within theMEIS class of transcription factors.

Prep1–/– ES cell derivationWe 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 expressedOct4 and Nanog (Fig. 5C; data not shown).

Prep1–/– ES cells were able to differentiate using the hangingdrop protocol; however, this gave rise to smaller EBs than in wild-type ES cells, particularly for one of the two Prep1–/– cell lines(Fig. 5D). Similarly to our whole-mount results, the cleavedcaspase 3 staining was more intense in Prep1–/– than in wild-typeEBs (Fig. 5E,F), especially in the smaller EBs. The differenceswere not as pronounced as in embryos (Fig. 2I,J). Analysis ofspecific markers (Oct4 and Nanog for pluripotency, T formesoderm, Fgf5 and Sox1 for neuroectoderm, Gata4 for endodermand Hand1 for trophoectoderm) showed that, although there wasa slight delay, Prep1–/– ES cells were overall capable ofdifferentiation (data not shown).


Fig. 5. Prep1 is not essential for ES cellviability and EBs formation, and thePrep1–/– embryonic phenotype isincreased by Atm decrease. (A-C)Prep1–/–

ES cell line isolation. (A)A wild-type and aPrep1–/– mouse clone. (B)Number andgenotypes of ES clones derived from Prep1+/–

heterozygous intercrosses. (C)Oct4immunofluorescence with wild-type andhomozygous ES cells. (D-F)Different sizesreached by EBs from wild-type and Prep1–/– EScells. (D)EBs formed according to the hangingdrop method with one wild-type and twoPrep1–/– ES cell lines. (E)Cleaved caspase 3whole-mount confocal immunofluorescencewith wild-type and Prep1–/– EBs. (F)Caspase 3-positive area normalized by comparison withDAPI-positive area. (G-J)E7.5 Oct4 whole-mount in situ hybridization in embryos withdifferent doses of Prep1 and Atm genes.(G,H)The Atm-null genotype decreases theOct4+ area in Prep1+/– (arrow) but not inPrep1+/+ embryos. (I,J)The absence of Atm hasa strong effect in the Prep1–/– embryo. Theasterisk in I shows that the Oct4+ area has asize similar to that of homozygous Prep1–/–

embryos. The arrows in J shows the decreasedOct4-staining of Prep1–/– embryos in an Atm–/–

background.

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Altogether, these results confirm the role of Prep1 inhomeostasis in early embryogenesis; Prep1–/– ES cells are extractedin lower ratio than expected (8% instead of the expected 25%). Thetwo viable Prep1–/– ES cell lines phenocopy Prep1–/– embryos,although in a milder fashion, giving rise to small EBs withincreased apoptosis.

Atm downregulation exaggerates the Prep1–/–

phenotypeE6.5 Epi cells do not tolerate double-stranded DNA damage andundergo 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 thebasis of Prep1–/– p53-dependent apoptosis of Epi cells, we crossedPrep1+/– mice with Atm+/– mice (Borghesani et al., 2000), both in afull C57BL6 background. As Atm is important in DNA repair, onewould expect its absence or reduction to worsen the Prep1phenotype. The absence of Atm induced a phenotype in two out offour heterozygous Prep1+/– embryos (Fig. 5G-J), which were delayedin their development, whereas in the other two embryos the effectswere less evident. Moreover, in the single recovered Prep1–/–Atm–/–

double knockout embryo, the phenotype was stronger than in thePrep1–/–Atm+/+, because, by E7.5, the Oct4-positive cells were almostundetectable (Fig. 5G-J). In agreement with the above results,downregulation of Atm with an sh-RNA in Prep1–/– ES EBs induceda further decrease of their already deficient size and increasedcleaved caspase 3 staining, i.e. apoptosis (data not shown).

DISCUSSIONThe absence of Prep1 causes p53-dependent apoptosis of Epi cells,which prevents gastrulation and differentiation. This is probably dueto the accumulation of DNA damage. The absence of Prep1 affectsall cells but apoptosis was mostly observed in the Epi, probablyowing to the tremendous proliferative expansion of the Epi at thisstage. Thus, the role of Prep1 in early embryogenesis is to protectepiblast cells from accumulating damage that induces apoptosis.

Prep1–/– embryos arrest development aroundE5.0-5.25Our data suggest that Prep1–/– embryos arrest around E5.0 orE5.25. Indeed, they die before E5.5, which is when AVE isestablished 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 theposterior Epi at E5.75 (Rivera-Perez and Magnuson, 2005).

Reduction in Epi cell number is the basis for thePrep1 KO phenotypeThe lack of gastrulation is due to the reduced size of the epiblast.Prep1 knockout embryos show a decrease of the pluripotent cellsexpressing Oct4 at E6.5, E7.5 and E8.5 (Fig. 2A-F). The numberof Epi cells is a limiting factor for the initiation of gastrulation(Tam and Behringer, 1997). Elimination of one blastomere from atwo- or four-cell embryo delays gastrulation; gastrulation startsonly when the embryo has accumulated a sufficient number of Epicells (Power and Tam, 1993; Rands, 1986). The size recovery isachieved by increasing cell proliferation prior to and duringgastrulation (Power and Tam, 1993). It is likely that the lownumber of Epi cells explains the absence of the AVE and PS inPrep1–/– embryos, and, therefore, the absence of embryonic

lineages. Nodal signaling from the Epi is essential for specifyingAVE in the most distal part of the embryo (Brennan et al., 2001;Mesnard et al., 2006). The lack of expansion of the Prep1–/– Epimay not keep the distal cells far enough from the negative signalsof the ExE (Mesnard et al., 2006; Richardson et al., 2006;Rodriguez et al., 2005), hence maintaining them under theinhibitory control of the ExE.

Why does Prep1 absence induce p53-dependentapoptosis?The pattern of Prep1 expression during ES cell differentiation andthe ability to harvest Prep1–/– ES cells, which still express Oct4 andcan, on the whole, differentiate (data not shown), suggests thatPrep1 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 withthe increased rate of p53-dependent apoptosis, suggest that Prep1is, however, essential for Epi homeostasis. This is also confirmedby the in vitro analysis of Prep1–/– ES cells. Our currentexplanation 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 induceany response at other stages or in somatic cells undergo p53-dependent apoptosis (Heyer et al., 2000). We hypothesize that inthe absence of Prep1, DNA damage accumulates in the embryoniccells. The DNA damage hypothesis is suggested by the observationthat a decrease in Atm induces a phenotype in the heterozygous,and worsens the phenotype of the homozygous, Prep1 mutantembryos. Likewise, downregulation of Atm in Prep1–/– but not inwild-type ES cells induces apoptosis and inhibits EB growth. Thisargues that the inability to repair DNA synergizes with the absenceof Prep1 and drives Epi cells into apoptosis. Accordingly, inPrep1i/i hypomorphic MEFs, we observed an increase in double-stranded breaks and DNA repair signaling (G.I. and F.B.,unpublished). Why the absence of Prep1 could induce DNAdamage is, at the moment, matter for speculation.

Interestingly, several cancer genes are involved in controllingDNA damage response and DNA repair. Indeed, several pieces ofevidence demonstrate that Prep1 is a novel tumor suppressor(Longobardi et al., 2010).

AcknowledgementsWe are very grateful to Dr Claudio Stern for his interest and advice; to the lateGraziella Persico and to Giovanna Liguori for much advice and reagents; toLuisa Lanfrancone for reagents and helpful discussions; and to Anton Berns forInk4Ap16 mice. We also thank Antonello Mallamaci, Shankar Srinivas, SilviaBrunelli, Miguel Torres, Janet Rossant, Juan Pedro Martinez Barberà, TristanRodriguez, Michael Shen, Daniel Constam, Vania Broccoli, Hans Schoeler, IsaoMatsuo and Robin Lovell-Badge for probes, and Michael Hemann for theshATM 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 statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material for this article is available athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.050567/-/DC1

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