PSF1 Is Essential for Early Embryogenesis in Mice

Psf1 (partner of sld five 1) forms a novel heterotetramer complex,GINS (Go, Ichi, Nii, and San; five, one, two, and three, respectively,in Japanese), with Sld5, Psf2, and Psf3. The formation of thiscomplex is essential for the initiation of DNA replication inyeast and Xenopus laevis egg extracts. Although all of the componentsare well conserved in higher eukaryotes, the biological functionin vivo is largely unknown. We originally cloned the mouse orthologof PSF1 from a hematopoietic stem cell cDNA library and foundthat PSF1 is expressed in blastocysts, adult bone marrow, andtestis, in which the stem cell system is active. Here we usedthe gene-targeting technique to determine the physiologicalfunction of PSF1 in vivo. Mice homozygous for a nonfunctionalmutant of PSF1 died in utero around the time of implantation.PSF1^–^/^– blastocysts failed to show outgrowth inculture and exhibited a cell proliferation defect. Our dataclearly indicate that PSF1 is required for early embryogenesis.

INTRODUCTION

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Introduction

Eukaryotic chromosomal replication is tightly regulated to maintainthe integrity of genomic information. In Saccharomyces cerevisiae,Orc (origin recognition complex) is bound to replication originsthroughout the cell cycle (1, 4, 21). From late M to G₁ phase,MCM (minichromosome maintenance) protein is loaded onto theorigin, marked by Orc, Cdc6, and Cdt1, and forms the prereplicationcomplex (pre-RC) (2, 9). On activation and recruitment of additionalfactors, such as CDC45, the pre-RC is converted to the preinitiationcomplex, which is the complex essential for the transition toDNA replication (2, 23). In yeast, CDC45 is essential for theinitiation and elongation of DNA replication (7, 20).

Recently, a novel multiprotein complex, GINS, was identified.The GINS complex contains Psf1 (partner of sld five 1), Psf2,Psf3, and Sld5 and forms a ring-like structure (8, 10, 19).During the S phase, the GINS complex is loaded onto chromatinafter the formation of pre-RCs and then tightly associates withthe replication origin. This binding is suppressed by p21 andgeminin by inhibition of the loading of CDC45 onto chromatin(11) and the pre-RC formation by binding to Cdt1 (23). Moreover,the chromatin binding of GINS complex and that of CDC45 aremutually dependent processes, but they do not associate witheach other. The association of PSF1 and Dpb11/Cut5 with theorigins is also mutually dependent. All genes encoding GINScomponents are evolutionarily conserved and are essential forcell growth (22). However, the functions of the GINS complexin mammalian cells have not been reported.

In this study, we isolated the mouse ortholog of PSF1 and generatedPSF1-deficient mice by a gene-targeting technique. In mice,PSF1 is specifically expressed in proliferating immature cells.Loss of PSF1 causes embryonic lethality around the implantationstage. PSF1^–^/^– embryos revealed impaired proliferationof the inner cell mass (ICM) and trophoblasts. Our data suggestthat PSF1 is essential for mouse embryogenesis.

MATERIALS AND METHODS

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Materials and Methods

Mice. C57BL/6 mice were purchased from SLC. All animal studies wereapproved by the Animal Care Committee of Kanazawa University.

Construction and screening of a stem cell-specific cDNA library. Preparation of bone marrow (BM) cells and fluorescence-activatedcell sorting analysis were performed as described previously(17, 18). The antibodies used in flow cytometric analysis forlineage marker (Lin) were phycoerythrin-conjugated Mac-1 (M1/70),Gr-1 (RB6-8C5), B220 (RA3-6B2), TER119, anti-CD4 (GK1.5), andanti-CD8 (53-6.72). Allophycocyanin-conjugated anti-c-kit (ACK2)and biotin-conjugated anti-Sca-1 were also applied. Biotinylatedanti-Sca-1 was visualized with peridinin chlorophyll protein-streptavidin.These antibodies were purchased from BD Pharmingen. Lin⁺ cellsand Lin^– c-kit⁺ Sca-1⁺ cells were purified from BM cellsthat had been obtained from an 8-week-old C57BL/6 mouse by cellsorting. The stained cells were analyzed with a FACSCalibur(Becton Dickinson) and sorted with an EPICS ALTRA (Beckman Coulter)instrument. Total RNAs were extracted from these cell populationsand were subjected to cDNA synthesis and amplification usinga SMART PCR synthesis kit (BD Clontech) according to the manufacturer'sprotocol. Subtractive cloning was performed using a PCR-SelectcDNA subtraction kit (BD Clontech).

Immunohistochemistry and fluorescence-activated cell sorting analysis. Tissue fixation, preparation of tissue sections, and stainingof sections with antibodies were performed as described previously(18). For immunohistochemistry, anti-PSF1 (see below) and antibromodeoxyuridine(anti-BrdU) antibodies (Zymed) were used. Cy3- or horseradishperoxidase (HRP)-conjugated secondary antibodies were obtainedfrom Jackson ImmunoResearch.

To obtain a specific antibody against PSF1, a rabbit was immunizedwith a synthetic peptide (CEQLIRQGVLEH) derived from the C-terminalregion of PSF1. Antisera were affinity purified with the samepeptide. Preimmunized rabbit immunoglobulins were used as anegative control to confirm specific staining.

RT-PCR analysis. Reverse transcription-PCR (RT-PCR) was performed as previouslydescribed (18). We used the following primer sets: 5'-TTA AGAAAT AGA CGC TGC ACG A-3' and 5'-TGC CAT CAT CAA CTT CAA ATTC-3' (for PSF1) and 5'-ACC ACA GTC CAT GCC ATC AC-3' and 5'-TCCACC ACC CTG TTG CTG TA-3' (for glyceraldehyde-3-phosphate dehydrogenase[GAPDH]).

Gene targeting. We isolated genomic clones encoding mouse PSF1 from the mouse129Sv/J library (Stratagene) by using mouse PSF1 cDNA as a probe.In the targeting construct, exon 5 was replaced with the ß-galactosidasegene and a pGK-neomycin resistance gene (see Fig. 2). We linearizedthis construct with NotI and electroporated it into 129Sv/JE14.1 embryonic stem (ES) cells. We selected G418-resistantclones and screened them by PCR and Southern blot analysis forthe correct recombination (see Fig. 2). Chimera mice were generatedby the aggregation method (12). We mated chimera males withC57BL/6J females and screened the offspring by Southern blottingand PCR analyses for mice bearing the PSF1^+/^– genotype.

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FIG. 2. Targeted disruption of mouse PSF1. Generation of PSF1-deficient mice. (A) Exons are represented by vertical bars and introns by intervening horizontal lines. Exon 5 of PSF1 was replaced by homologous recombination with a ß-galactosidase gene (lacZ) and a neomycin resistance gene driven by the Pgk1 prompter (neo). E, EcoRI; B, BamHI. This increased the size of the EcoRI restriction fragment. (B) Southern blot analysis of wild-type (E14) and targeted (#34 and #38) ES cells demonstrated homologous recombination in PSF1. (C) Northern blot analysis of 30 µg of total RNA isolated from wild-type (wt) and heterozygous PSF1 mutants hybridized with a PSF1 cRNA probe. Heterozygous PSF1 mutants had decreased PSF1 mRNA in the testis (top). GAPDH mRNA signals were used as an internal standard (bottom). (D and E) PSF1 expression was analyzed by immunohistochemical staining using anti-PSF1 antibody in PSF1^+/+ (D) and PSF1^–^/^– (E) embryos at E6.5. Original magnification, x100.

Northern blot. Total RNA was extracted from testis and liver of 8-week-oldmice. Thirty-microgram aliquots of total RNA were separatedon 1% formaldehyde-agarose gels and transferred onto nylon membranefilters by capillary transfer using 20x SSC (1x SSC is 0.15M NaCl plus 0.015 M sodium citrate) buffer. Filters were hybridizedovernight at 68°C in DIG Easy Hyb (Roche) with a digoxigenin-labeledPSF1 cRNA probe. Hybridized probe was detected with alkalinephosphatase-conjugated antidigoxigenin antibodies using theDIG luminescent detection kit (Roche), following the manufacturer'sinstructions. GAPDH mRNA was used as an internal standard toensure that equal amounts of RNA were loaded in each lane.

In vitro culture. Embryos (E3.5) were flushed out from the uteri of pregnant miceand individually cultured in standard ES medium with leukemiainhibitory factor (Invitrogen) for 10 days. DNA synthesis wasassessed by BrdU (10 µM; Sigma) incorporation in culturedblastocysts from days 4 to 5; on day 5 of culturing, the blastocystswere fixed with 4% paraformaldehyde in phosphate-buffered saline,stained with anti-BrdU antibody (1 µg/ml; Zymed), andcounterstained with hematoxylin. The TUNEL (terminal deoxynucleotidyltransferase-mediateddUTP-biotin nick end labeling) assay for detection of apoptoticcells was performed using the In Situ Cell Death Detection kit(Roche) according to the manufacturer's instructions.

RESULTS AND DISCUSSION

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Results and Discussion

Cloning of mouse PSF1. Although most terminally differentiated somatic cells are notable to proliferate, stem cells and immature progenitor cellsare constitutively in cycle to produce mature cells. To elucidatethe molecular mechanism regulating mammalian cell division,we constructed a subtraction library from the BM-derived Lin^–c-kit⁺ Sca-1⁺ hematopoietic stem cell (KSL cells; stem/progenitorcells) fraction as a tester and the Lin⁺ mature hematopoieticcells as the driver in order to isolate genes encoding proteinsthat are involved in DNA replication and specifically expressedin immature cells. Among 521 clones that were abundantly expressedin KSL cells, one gene named #e11 was expressed in KSL cellsand their progenitor cells (Lin^– c-kit⁺ Sca-1^–)but not in Lin⁺ mature hematopoietic cells, as confirmed byRT-PCR (Fig. 1A). This gene corresponded to a hypothetical genein GenBank (accession no. AK013116) and was closely relatedto Psf1 in a budding yeast (partner of sld5-1), which was shownto encode a protein involved in DNA replication in yeast andin an in vitro model using Xenopus laevis egg extracts (8, 10).We identified the binding partner of mouse #e11 to be sld5 bythe two-hybrid system (M. Ueno and N. Takakura, unpublisheddata). Therefore, we considered this #e11 gene to be a mouseortholog and named it PSF1.

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FIG. 1. PSF1 expression in adult tissues. (A and B) RT-PCR of fractionated adult BM cells (A) and adult tissues (B). KSL, Lin^– Sca-1⁺ c-kit⁺ cells; KL, Lin^– Sca-1^– c-kit⁺; LINK, Lin⁺ c-kit⁺; LIN, Lin⁺. Br, brain; Hr, heart; Ln, lung; Th, thymus; Lv, liver; Sp, spleen; Kd, kidney; Ts, testis; Ov, ovary. (C and D) Immunostaining of sections of the testis (C) and blastocysts (E3.5) (D) with anti-PSF1 antibody labeled with HRP (C) or Cy3 (D). Sections were counterstained with hematoxylin or 4',6'-diamidino-2-phenylindole (DAPI). Color staining: HRP, brown; Cy3, red; DAPI, blue. Bar, 50 µm.

PSF1 is predominantly expressed in highly proliferative organs, especially in the immature cell population. We analyzed PSF1 expression in several adult tissues. PSF1 expressionwas predominantly observed in hematopoietic tissues such asthe adult BM and thymus on RT-PCR (Fig. 1B). Moreover, PSF1expression was observed in reproductive tissues, i.e., the testisand ovary, which have an active stem cell system. In other adulttissues (brain, heart, lung, liver, spleen, and kidney), PSF1expression was not detectable. These data suggested that PSF1is expressed specifically in tissues with higher rates of proliferation.

To determine the spatial distribution of PSF1 protein in theadult testis, we generated antibody against PSF1 peptide. Immunohistochemistryon mouse testis sections showed that PSF1 protein is presentin immature cells, i.e., spermatogonia (Fig. 1C). PSF1 is expressedin other immature cell populations including blastocysts (Fig.1D), adult thymic progenitor cells, and yolk sac-containinghematopoietic progenitor cells (data not shown). These datasuggested that PSF1 is expressed specifically in the immaturecell population.

Targeted disruption of the mouse PSF1 gene. To analyze the function of PSF1, we generated mice lacking afunctional PSF1 gene (Fig. 2). The mouse PSF1 gene containsseven putative coding exons. The targeting vector was designedby deleting exon 5 and inserting a lacZ-neo cassette. Amongthe 95 independent ES cell colonies examined, we found two homologousrecombinants. Correct targeting was confirmed in these ES clonesby Southern blot analyses with a 3' probe (Fig. 2B). One ESclone was aggregated with C57BL/6 blastocysts to generate achimera, which subsequently produced germ line transmission.The PSF1^+/^– line was established by backcrosses with C57BL/6mice. To confirm the loss of PSF1 transcript in mutant miceby gene disruption, we performed Northern blot analysis. Asexpected, PSF1 mRNA was reduced in PSF1^+/^– testis (Fig.2C). PSF1^+/^– mice were born at Mendelian frequency, andthere were no apparent differences between PSF1^+/^– miceand wild-type mice.

PSF1 is required for cell proliferation, and loss of PSF1 leads to early embryonic lethality. We analyzed the PSF1 gene in neonates resulting from PSF1^+/^–intercrosses and did not obtain any homozygous offspring (Table1). In normal E6.5 embryos, a cylinder-like two-layered cellularstructure was observed (Fig. 2D and 3A). However, PSF1-deficientembryos, which could be identified at this stage by the absenceof PSF1 immunoreactivity (Fig. 2E), lacked such cylinder-likestructures (Fig. 2E and 3B). These data suggested that the PSF1^–^/^–embryos failed to develop past E5.5 and showed disorganizedembryonic and extraembryonic structures.

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TABLE 1. Progeny from PSF1 heterozygotes

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FIG. 3. Histological analysis of PSF1^–^/^– embryos. Hematoxylin- and-eosin-stained, longitudinal sections of E6.5 embryos in decidua. (A) Wild-type embryo; (B) mutant PSF1^–^/^– embryo. epc, ectoplacental cone; exe, extraembryonic ectoderm; ee, embryonic endoderm. Bars, 100 µm.

The cellular proliferation and differentiation of mutant embryoswere investigated in in vitro cultures of blastocysts. On lightmicroscopy, there were no differences among individual blastocysts(data not shown). In PSF1^+/+ and PSF1^+/^– embryos (totaln = 104), trophoblasts started to spread over the culture dishafter hatching and supported robust ICM outgrowths after 2 days.While the trophoblasts from PSF1^–^/^– blastocysts(n = 32) also attached and spread over the dish, PSF1^–^/^–ICM cells failed to form outgrowths (see Fig. 4). These datasuggested that the lethality of PSF1^–^/^– embryosin utero was caused at least by the death of ICM cells.

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FIG. 4. Defective growth of PSF1^–^/^– blastocysts in vitro. Blastocysts (E3.5) were recovered from heterozygous intercrosses, cultured individually for a period of 6 days as indicated, and genotyped by PCR. Cultures of representative +/– (A to D) and –/– (E to H) blastocysts are shown. Arrow, ICM; arrowhead, trophoblast. (I and J) DNA synthesis in cultured blastocysts (BrdU incorporation). Immunostaining with anti-BrdU antibody was performed (brown). (I) PSF1^+/^– blastocyst; (J) PSF1^–^/^– blastocyst. Inset in panel I shows a different field in this culture plate. Arrow, ICM; arrowhead, labeled trophoblast. (K and L) Apoptosis of cells in cultured ICM from blastocysts (TUNEL assay; green, apoptotic cells). (K) PSF1^+/^– blastocyst; (L) PSF1^–^/^– blastocyst. Nuclei were counterstained with propidium iodide (red). Bars, 100 µm. (M to P) Blastocysts were cultured for 8 days (M and O) and 10 days (N and P). (M and N) PSF1^+/^– blastocyst; (O and P) PSF1^–^/^– blastocyst.

To further delineate the proliferation defect of the PSF1^–^/^–blastocysts, we carried out bromodeoxyuridine incorporationassays during blastocyst outgrowth (Fig. 4I and J). VigorousDNA synthesis was observed in PSF1^+/^– ICM cells by immunostainingwith anti-BrdU antibody (Fig. 4I). However, the presumed ICMcells from the mutants ceased to proliferate, while DNA synthesiswas still observed in trophoblasts (Fig. 4J). Moreover, in theICM of PSF1^–^/^– blastocyst cultures, TUNEL-positiveapoptotic cells appeared on day 5 (Fig. 4K and L). Therefore,the PSF1^–^/^– ICM cells were unable to proliferateand underwent apoptosis in culture.

While BrdU⁺ cells were found in PSF1^–^/^– trophoblastsafter 5 days of culturing (Fig. 4J), PSF1^–^/^– trophoblastsstopped proliferation after 8 days of culturing (Fig. 4O). BrdUwas not incorporated in PSF1^–^/^– trophoblasts beyond8 days (data not shown), and the number of trophoblasts declined(Fig. 4P). By contrast, PSF1^+/– embryo trophoblasts continuouslyproliferated after 10 days of culturing (Fig. 4M and N). Thesedata indicated that PSF1 is essential for both ICM and trophoblastproliferation.

In this study, we examined the functions of PSF1 in vivo bygene targeting technology. Our results revealed impaired proliferationof the ICM and trophoblasts in PSF1^–^/^– embryos.Recently, it was reported that Psf1 and CDC45 are involved cooperativelyin the initiation of DNA replication in yeast (10, 19) and thatboth molecules are prerequisite for DNA replication in yeast(7, 20). In mice, the phenotype of CDC45-deficient embryos afteruterine implantation (24) is quite similar to that of PSF1-nullembryos. Mice deficient in the CDC45 ortholog of yeast showa defect in cell proliferation in blastocyst culture (24). Therefore,the molecular functions of PSF1 and CDC45 for DNA replicationmay be conserved in mammalian cells.

Although Psf1 is indispensable for cell proliferation in yeast(19), no obvious morphological abnormality was found in PSF1^–^/^–embryos before implantation (data not shown). This observationraised the possibility of the existence of maternal PSF1 transcriptstores and could account for the proliferation of PSF1^–^/^–embryos through the early developmental stages. To ascertainthis, we performed immunostaining on unfertilized eggs withanti-PSF1 antibody which revealed potent expression of PSF1(data not shown). Because zygotic gene transcription initiatesat the two-cell stage and paternal protein contributions arethought to be negligible (16), we conclude that the timing ofPSF1^–^/^– lethality is due to the loss and/or dilutionof maternal PSF1 transcripts around implantation stage.

Most of our knowledge on DNA replication has accumulated fromstudies on lower eukaryotes. However, it can be argued thatDNA replication of higher eukaryotes is more complex. For example,DNA replication is linked to gene transcription in higher eukaryotesincluding Drosophila melanogaster (15), Xenopus (5), and mice(6) but not in yeast (14). Although origin recognition complex(Orc) binds to chromatin at replication origins throughout thecell cycle in yeast (1, 4, 21), a part of Orc2 localizes atthe centrosomes and heterochromatin during the M phase and participatesin chromosome segregation in human cells (13). Mcm10, whichis a conserved protein and is involved in initiation of DNAreplication in yeast, is required for chromosome condensationin fly cells (3). Interestingly, in nematodes, loss of PSF1causes abnormality of chromatin segregation (M. Ueno and N.Takakura, unpublished data). Thus, PSF1 may have pivotal rolesin other biological processes. Further analysis on the functionof PSF1 will shed light on the complex mechanism of DNA replicationin mammalian cells.

ACKNOWLEDGMENTS

We thank Y. Shimizu, K. Ishida, M. Sato, and Y. Nakano for technicalassistance.

This work was partly supported by the Japanese Ministry of Education,Culture, Sports, Science and Technology and the Japan Societyfor Promotion of Science.

FOOTNOTES

* Corresponding author. Mailing address: Department of Stem Cell Biology, Cancer Research Institute, Kanazawa University, 13-1, Takara-machi, Kanazawa 920-0934, Japan. Phone: 81-76-265-2751. Fax: 81-76-234-4513. E-mail: ntakaku{at}kenroku.kanazawa-u.ac.jp.

REFERENCES

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