Cloning of mouse PSF1.
Although most terminally differentiated somatic cells are not able to proliferate, stem cells and immature progenitor cells are constitutively in cycle to produce mature cells. To elucidate the molecular mechanism regulating mammalian cell division, we constructed a subtraction library from the BM-derived Lin−
hematopoietic stem cell (KSL cells; stem/progenitor cells) fraction as a tester and the Lin+
mature hematopoietic cells as the driver in order to isolate genes encoding proteins that are involved in DNA replication and specifically expressed in immature cells. Among 521 clones that were abundantly expressed in KSL cells, one gene named #e11
was expressed in KSL cells and their progenitor cells (Lin−
) but not in Lin+
mature hematopoietic cells, as confirmed by RT-PCR (Fig. ). This gene corresponded to a hypothetical gene in GenBank (accession no. AK013116
) and was closely related to Psf1
in a budding yeast (partner of sld5-1
), which was shown to encode a protein involved in DNA replication in yeast and in an in vitro model using Xenopus laevis
egg extracts (8
). We identified the binding partner of mouse #e11 to be sld5 by the two-hybrid system (M. Ueno and N. Takakura, unpublished data). Therefore, we considered this #e11
gene to be a mouse ortholog and named it PSF1
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, (more ...) PSF1 is predominantly expressed in highly proliferative organs, especially in the immature cell population.
We analyzed PSF1 expression in several adult tissues. PSF1 expression was predominantly observed in hematopoietic tissues such as the adult BM and thymus on RT-PCR (Fig. ). Moreover, PSF1 expression was observed in reproductive tissues, i.e., the testis and ovary, which have an active stem cell system. In other adult tissues (brain, heart, lung, liver, spleen, and kidney), PSF1 expression was not detectable. These data suggested that PSF1 is expressed specifically in tissues with higher rates of proliferation.
To determine the spatial distribution of PSF1 protein in the adult testis, we generated antibody against PSF1 peptide. Immunohistochemistry on mouse testis sections showed that PSF1 protein is present in immature cells, i.e., spermatogonia (Fig. ). PSF1 is expressed in other immature cell populations including blastocysts (Fig. ), adult thymic progenitor cells, and yolk sac-containing hematopoietic progenitor cells (data not shown). These data suggested that PSF1 is expressed specifically in the immature cell population.
Targeted disruption of the mouse PSF1 gene.
To analyze the function of PSF1, we generated mice lacking a functional PSF1 gene (Fig. ). The mouse PSF1 gene contains seven putative coding exons. The targeting vector was designed by deleting exon 5 and inserting a lacZ-neo cassette. Among the 95 independent ES cell colonies examined, we found two homologous recombinants. Correct targeting was confirmed in these ES clones by Southern blot analyses with a 3′ probe (Fig. ). One ES clone was aggregated with C57BL/6 blastocysts to generate a chimera, which subsequently produced germ line transmission. The PSF1+/− line was established by backcrosses with C57BL/6 mice. To confirm the loss of PSF1 transcript in mutant mice by gene disruption, we performed Northern blot analysis. As expected, PSF1 mRNA was reduced in PSF1+/− testis (Fig. ). PSF1+/− mice were born at Mendelian frequency, and there were no apparent differences between PSF1+/− mice and 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 (Table ). In normal E6.5 embryos, a cylinder-like two-layered cellular structure was observed (Fig. and ). However, PSF1-deficient embryos, which could be identified at this stage by the absence of PSF1 immunoreactivity (Fig. ), lacked such cylinder-like structures (Fig. and ). These data suggested that the PSF1−/− embryos failed to develop past E5.5 and showed disorganized embryonic and extraembryonic structures.
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; (more ...)
The cellular proliferation and differentiation of mutant embryos were investigated in in vitro cultures of blastocysts. On light microscopy, there were no differences among individual blastocysts (data not shown). In PSF1+/+ and PSF1+/− embryos (total n = 104), trophoblasts started to spread over the culture dish after 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. ). These data suggested that the lethality of PSF1−/− embryos in utero was caused at least by the death of ICM cells.
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 +/− (more ...)
To further delineate the proliferation defect of the PSF1−/− blastocysts, we carried out bromodeoxyuridine incorporation assays during blastocyst outgrowth (Fig. ). Vigorous DNA synthesis was observed in PSF1+/− ICM cells by immunostaining with anti-BrdU antibody (Fig. ). However, the presumed ICM cells from the mutants ceased to proliferate, while DNA synthesis was still observed in trophoblasts (Fig. ). Moreover, in the ICM of PSF1−/− blastocyst cultures, TUNEL-positive apoptotic cells appeared on day 5 (Fig. ). Therefore, the PSF1−/− ICM cells were unable to proliferate and underwent apoptosis in culture.
While BrdU+ cells were found in PSF1−/− trophoblasts after 5 days of culturing (Fig. ), PSF1−/− trophoblasts stopped proliferation after 8 days of culturing (Fig. ). BrdU was not incorporated in PSF1−/− trophoblasts beyond 8 days (data not shown), and the number of trophoblasts declined (Fig. ). By contrast, PSF1+/− embryo trophoblasts continuously proliferated after 10 days of culturing (Fig. ). These data indicated that PSF1 is essential for both ICM and trophoblast proliferation.
In this study, we examined the functions of PSF1 in vivo by gene targeting technology. Our results revealed impaired proliferation of the ICM and trophoblasts in PSF1−/−
embryos. Recently, it was reported that Psf1 and CDC45 are involved cooperatively in the initiation of DNA replication in yeast (10
) and that both molecules are prerequisite for DNA replication in yeast (7
). In mice, the phenotype of CDC45
-deficient embryos after uterine implantation (24
) is quite similar to that of PSF1
-null embryos. Mice deficient in the CDC45 ortholog of yeast show a defect in cell proliferation in blastocyst culture (24
). Therefore, the molecular functions of PSF1 and CDC45 for DNA replication may be conserved in mammalian cells.
is indispensable for cell proliferation in yeast (19
), no obvious morphological abnormality was found in PSF1−/−
embryos before implantation (data not shown). This observation raised the possibility of the existence of maternal PSF1
transcript stores and could account for the proliferation of PSF1−/−
embryos through the early developmental stages. To ascertain this, we performed immunostaining on unfertilized eggs with anti-PSF1 antibody which revealed potent expression of PSF1 (data not shown). Because zygotic gene transcription initiates at the two-cell stage and paternal protein contributions are thought to be negligible (16
), we conclude that the timing of PSF1−/−
lethality is due to the loss and/or dilution of maternal PSF1
transcripts around implantation stage.
Most of our knowledge on DNA replication has accumulated from studies on lower eukaryotes. However, it can be argued that DNA replication of higher eukaryotes is more complex. For example, DNA replication is linked to gene transcription in higher eukaryotes including Drosophila melanogaster
), and mice (6
) but not in yeast (14
). Although origin recognition complex (Orc) binds to chromatin at replication origins throughout the cell cycle in yeast (1
), a part of Orc2 localizes at the centrosomes and heterochromatin during the M phase and participates in chromosome segregation in human cells (13
). Mcm10, which is a conserved protein and is involved in initiation of DNA replication in yeast, is required for chromosome condensation in fly cells (3
). Interestingly, in nematodes, loss of PSF1 causes abnormality of chromatin segregation (M. Ueno and N. Takakura, unpublished data). Thus, PSF1 may have pivotal roles in other biological processes. Further analysis on the function of PSF1 will shed light on the complex mechanism of DNA replication in mammalian cells.