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Previous Article | Next Article 
Molecular and Cellular Biology, July 2006, p. 5373-5381, Vol. 26, No. 14
0270-7306/06/$08.00+0 doi:10.1128/MCB.00043-06
Copyright © 2006, American
Society for
Microbiology. All Rights Reserved.
Department of Biological Sciences and Biomedical Research Center, Korea Advanced Institute of Science and Technology, 373-1 Guseoung-dong, Yuseong-gu, Daejeon 305-701, South Korea,1 National Cancer Center, 809 Madu1-dong, Ilsandon-gu, Goyang-si, Gyeonggi-do 410-769, South Korea,2 Department of Pathology, College of Medicine, Chungnam National University, 6 Munwha-dong Jung-gu, Daejeon 301-131, South Korea3
Received 9 January 2006/ Returned for modification 2 February 2006/ Accepted 28 April 2006
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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APC activity is temporally regulated through binding of the complex with either of two activating subunits, Cdc20 or Cdh1 (2, 9). Cdc20 activates APC during early mitosis until anaphase, whereas Cdh1 functions during late mitosis and the G1 phase. APC-Cdc20 activity is regulated via three types of inhibitory proteins or complexes that antagonize Cdc20 function: (i) components of the spindle assembly checkpoint, including at least seven proteins (Bub1, Bub2, Bub3, Mad1, Mad2, Mad3 [BubR1], and Mps1) (16, 22, 40), (ii) the early mitotic inhibitor Emi1 (27, 28), and (iii) the Ras association domain family 1A (Rassf1A) (31, 32). Emi2/Erp1/FBXO43, the Emi1-related protein 1, has recently been reported as another APC-Cdc20 inhibitor during cytostatic factor (CSF) arrest whose inhibitory mechanism remains to be further analyzed (30, 34).
To determine the physiological functions of these APC inhibitors, genetic analyses in mice were performed. The spindle checkpoint proteins Mad2, BubR1, and Bub3 were found to be essential for early development in gene-disrupted mice, as evident from their failure to survive between days 6.5 and 8.5 postcoitus due to extensive apoptosis (4, 14, 37). Null embryos treated with a spindle depolymerization agent failed to arrest in metaphase and displayed increased mitotic abnormalities. Mad2 haploinsufficiency causes premature anaphase and chromosome instability (19), while BubR1 haploinsufficiency induces an increase in the number of splenic megakaryocytes, resulting from spindle checkpoint failure (37). These results confirm that the spindle assembly checkpoint is essential for accurate chromosome segregation in mitotic mouse cells and embryonic viability. rassf1A-targeted mice were viable and fertile but prone to spontaneous tumorigenesis at an advanced age (18 to 20 months) (33, 36). Tumors in rassf1A-targeted mice included lung adenomas, lymphomas, and breast adenocarcinoma. Genetic analyses for Emi1 and Emi2 in mice have not been reported until now. Our present study on emi1-targeted mice is the first account of the physiological importance of Emi1.
Emi1 was initially identified in Drosophila as a gene product, rca1 (regulator of cyclin A), that induces cyclin A accumulation (5). Emi1 inhibits APC-Cdc20 activity at interphase in the Xenopus embryo by blocking binding of the Cdc20 adaptor to its substrates, thus preventing mitotic cyclin destruction (27, 28). The protein additionally regulates the APC-Cdh1 complex in somatic cells to induce the stabilization of S-phase regulators, including cyclin A, at G1-S transition (12). Specifically, Emi1 ensures an interval of cyclin stability from late G1 phase to early mitosis by inhibiting APC-Cdh1 and Cdc20 activity. At the beginning of this interval, Emi1 is upregulated by the E2F transcription factor. At the end, Emi1 is ubiquitinated and destroyed close to the time of cyclin A impairment (12, 18).
Emi1
destruction in early mitosis, mediated by SCFßTrCP
ubiquitin ligase, is required for APC activation
(7,
18). The failure of
ßTrCP-dependent Emi1 destruction stabilizes APC substrates and
results in a mitotic catastrophe, including centrosome overduplication.
On the basis of these findings, it has been proposed that Emi1
destruction relieves a late prophase checkpoint for APC activation.
ßTrCP, a substrate-adapting subunit of the SCF complex,
specifically recognizes a canonical DSGXXS motif within I
B,
ß-catenin, Emi1, and other substrates when both serine residues
are phosphorylated (1,
6,
10,
18,
38). Mitotic
phosphorylation of the Emi1 DSGxxS motif is mediated by MPF and
Polo-like kinase 1 (8,
18,
20). Since
SCFßTrCP is constitutively active, the timing of
Emi1 destruction must be strongly governed by its
phosphorylation.
Emi2/XErp1/FBXO43, a homolog of Emi1, has recently emerged as the most likely candidate to inhibit the APC during CSF arrest in Xenopus eggs (30, 34). Emi2 is destroyed in a process similar to the destruction pathway for Emi1. However, the APC-inhibitory role of the protein in other vertebrates is not yet reported. Also, the physiological function of Emi1 and Emi2 and their functional redundancies remain to be clarified in the whole-mouse system.
To further determine the in vivo function of Emi1, we performed genetic disruption in mice using gene-targeting techniques. We found that Emi1-knockout embryos die at the preimplantation stage and show defects in mitotic progression, chromosomal missegregation, and increased apoptosis. Based on the data obtained, we propose that the late prophase checkpoint function of Emi1 is essential for mitotic fidelity during early embryogenesis.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse ES cells were maintained on a monolayer of puromycin-resistant SNL6 feeders (17) and cultured in conditioned ES medium composed of Dulbecco's modified Eagle's medium (WelGENE, Inc., South Korea) supplemented with leukemia inhibitory factor (1,000 U/ml; Chemicon), 15% heat-inactivated fetal bovine serum (HyClone), 2 mM L-glutamine (GIBCO BRL), 0.1 mM nonessential amino acid solution (GIBCO BRL), penicillin G (100 U/ml), streptomycin sulfate (100 µg/ml), and 50 µM ß-mercaptoethanol (GIBCO BRL).
The Emi1-targeting vector was linearized with KpnI and introduced into ES cells by electroporation using a gene pulser (1 pulse of 0.23 kV and 500 µF; Bio-Rad). Resistant ES cell clones grown in conditioned medium were isolated after an 8-day culture. Homologous recombination was confirmed by Southern blot analysis of genomic DNA prepared from puromycin-resistant ES cell clones using external probes, as indicated in Fig. 1B. The 5' external probe (803 bp) was obtained by PCR using two primers, Pr5, 5'-AAGTTAGGCCGCGCCCTCAG-3', and Pr3, 5'-AGGCGCCGAGCCGCAGCTCT-3' (Fig. 1A). In all colonies with an apparent knockout allele, correct targeting was verified using an internal hybridization probe (a 936-bp BamHI-BglII fragment containing exons 4 and 5) (data not shown).
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In vitro culture and genotyping of preimplantation embryos. All embryos were generated by natural mating of Emi1 heterozygote animals. The morning of the day on which a vaginal plug was detected was designated day E0.5. Embryos were collected on E0.5 or E3.5 by tearing the ampulla of oviduct or flushing uteri with M2 medium (Sigma), followed by culturing in M16 medium (Sigma) for the appropriate times (23).
For genotyping, individual embryos were lysed by incubation at 55°C overnight in 5 µl PCR lysis buffer (10 mM Tris-Cl, pH 8.0, 50 mM KCl, 2 mM MgCl2, 0.45% NP-40, 0.45% Tween 20, 0.2 mg/ml of proteinase K) (15). To detect the wild-type (WT) and mutant Emi1 alleles, amplification reactions were performed (as for Fig. 1C) with the following primers: h3-5 (5'-GGAGTATGGAATTCCTTAATCTGC-3'), h3-3 (5-TCCAAACACACGGGACTGTA), and pgk-3 (5'-GCACGAGACTAGTGAGACGTGCTAC-3'). The amplification protocol included an initial incubation step at 94°C for 5 min, followed by 35 cycles of 30 s of denaturation at 94°C, 30 s of annealing at 55°C, and 50 s of elongation at 72°C with Taq polymerase. WT and mutant alleles yielded products of 959 bp and 1,265 bp, respectively.
Immunocytochemistry.
In vitro-cultured embryos were washed
twice in phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde
in PBS for 30 min at 4°C, and permeabilized for 20 min at room
temperature in PBS containing 0.3% Triton X-100 and 1.0% bovine serum
albumin. For 5-bromo-2-deoxyuridine (BrdU)-treated embryos, DNA was
denatured after permeabilization with 2 N HCl-0.5% Triton X-100 for 20
min at room temperature and washed extensively in PBS with 1.0% bovine
serum albumin. Embryos were incubated with specific primary antibodies
overnight at 4°C. The primary antibodies used in this study
were mouse anti-BrdU (Developmental Studies Hybridoma Bank), rabbit
anti-phosphohistone H3 (Ser-10) (Cell Signaling Technology), mouse
anti-
-tubulin, rabbit anti-cyclin A, and mouse anti-cyclin D1
(Santa Cruz Biotechnology). Incubation with fluorescein isothiocyanate
or rhodamine-conjugated secondary antibodies (Santa Cruz Biotechnology)
and DAPI (4',6'-diamidino-2-phenylindole) was performed
for 1 h at 37°C.
In situ hybridization. A 489-bp HindIII/SacI mouse Emi1 cDNA fragment was cloned in pBluescriptII KS(+) (Clonetech) and employed to generate riboprobes using a digoxigenin RNA-labeling kit (Roche Applied Science). Paraffin sections (4 µm) were mounted on slides coated with 3-aminopropyltriethoxy silane (Sigma) and fixed in 4% paraformaldehyde solution in PBS. In situ hybridization was performed as described previously (39).
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Emi1-targeting construct was electroporated into AK7 (129/Sv) ES cells, and puromycin-resistant clones were screened for homologous recombination by Southern blotting with 5' external probes and BglII digestion, as specified in Fig. 1B. Out of the 192 puromycin-resistant cell lines, 6 (3.1%) displayed the desired target. Mice containing the above-mentioned emi1 mutation were generated with two targeted clones. Both clones were transmitted through germ line after crossing chimeric mice with C57BL/6 females (Fig. 1B).
Emi1 is essential for preimplantation embryo development. Heterozygous Emi1+/– mice were apparently normal, healthy, and fertile with no developmental abnormalities detectable over an 18-month observation period (data not shown). In contrast, no homozygous Emi1-deficient animals were produced out of 185 live births from Emi1+/– intercrosses (Table 1), indicating that one functional emi1 allele is sufficient to support full embryonic and tissue development, whereas inactivation of both alleles leads to embryonic lethality.
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To assess the time of preimplantation developmental failure, we analyzed embryos at different periods of in vitro culture (Fig. 2D). Pronuclei underwent continual cell division with no embryo growth and developed into blastocysts. WT and heterozygote embryos normally developed into blastocysts after 120 h culture, as confirmed by the formation of distinct inner cell mass (ICM), blastocoel cavity, and trophoblasts. Emi1-deficient embryos displayed normal development until the four-cell stage. However, Emi1-deficient embryos at 8- to 16-cell stages appeared to be abnormal and eventually developed into abnormal blastocysts. Most Emi1-null embryos displayed gross cellular degeneration and absence of distinct inner cell mass, blastocoel, or trophoblast. Accordingly, we conclude that Emi1-deficient embryos are defective in preimplantation development.
To further characterize the growth defects of Emi1-null embryos, blastocysts recovered 3.5 days later from heterozygote intercrosses were cultured in vitro for several days (23). After a 5-day culture, spherical blastocysts flatten onto the culture dish and form a multicomponent structure in which the inner cell mass grows as a round shape on top of the extraembryonic trophoblast giant (TG) cells (Fig. 2E). In these cultures, Emi1 heterozygous and WT embryonic cells normally develop into ICM and TG cells. However, only 2 out of 46 cultured embryos were Emi1-null embryos (Table 1) and their ICM displayed severely retarded outgrowth, and very few Emi1–/– ICM cells persisted to day 5. Phase-contrast microscopy revealed similar numbers of TG cells in mutant and control-attached embryos. In addition, BrdU labeling demonstrated that Emi1-deficient trophoblast cells remained alive and underwent DNA replication, even at the point when highly mitotic ICM cells apoptose (data not shown). Cells derived from ICM become highly proliferative, whereas TG cells are mitotically inactive at approximately E4.5 and undergo repeated rounds of S phase, generating a polyploid nucleus and large cytoplasm. These results indicate that Emi1 is not required for survival and proliferation of mitotic inactive TG cells, but it is essential for the continuity of highly proliferative ICM.
S phase of Emi1–/– embryonic cells. Based on our observation of preimplantation developmental failure, we postulate that Emi1-deficient embryos are defective in cell division or cycle progression from G1 phase. Since human Emi1 promotes cyclin A accumulation and S-phase entry in somatic cells, we initially employed the in vitro cultures of pronuclei to ascertain whether the developmental failure of Emi1-deficient embryos resulted from cell proliferation defects. In an initial set of experiments, pronuclei cultured in vitro were pulse labeled for 10 min with BrdU, and BrdU-positive cells were quantitated by indirect immunofluorescence (Fig. 3). As shown in Fig. 3B, we detected 50 ± 5.5% BrdU-positive cells in Emi1-null embryos on average, compared to 45 ± 7.1% in control littermates, signifying no obvious defects in S-phase entry or progression in the former group. Thus, other abnormalities in Emi1-null embryos are prevalent in addition to cell proliferation defects.
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Cells undergoing abnormal mitosis were frequently observed in Emi1-deficient embryos, but rarely in WT and heterozygous embryos. Under normal mitotic conditions, cells contain condensed chromosomes and a bipolar spindle (Fig. 4A, panels a, b, and c). Mitotic cells in prophase/prometaphase, metaphase, anaphase/telophase, and cytokinesis (Fig. 4A, panels d, e, f, and g, respectively) were observed in both WT and heterozygous embryos. In contrast, Emi1-null embryos contained cells undergoing mitotic catastrophe. Some cells exhibited overcondensed chromosomes and abnormal spindle structures at the prometaphase-like stage (Fig. 4B, panels d and e), while others had misaligned chromosomes (Fig. 4B, panels f and g) or were in anaphase with multipolar and tangled spindle structures (Fig. 4B, panel g). Notably, multipolar spindles were additionally observed in nondestructive Emi1-expressing cells (18). The presence of these anaphase-like cells (Fig. 4B, panel g) signifies that Emi1-null cells prematurely undergo anaphase. Abnormal mitotic cells were observed in Emi1-null blastocysts at a high frequency (41 ± 8.4%) but were rarely observed in WT and heterozygous cells (Fig. 4C). These results collectively suggest that Emi1 is required for mitotic progression with fidelity.
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Reduced level of cyclin A in Emi1-deficient embryos. Given that Emi1 inhibits APC activity in prophase, it is likely that its deficiency results in premature activation of the complex, thus prematurely triggering progression into anaphase. However, there are not many cells in early embryo with asynchronous stages; thus, it is technically difficult to directly test whether APC is prematurely activated in Emi1-deficient cells. As cyclin A degradation is inhibited by Emi1 at the S phase and prophase and follows Emi1 degradation at early mitosis, Emi1 deficiency may trigger partial or complete degradation of cyclin A. We performed indirect immunofluorescence experiments for APC substrates, especially cyclin A. Blastocysts recovered at E3.5 from heterozygote intercrosses were fixed and processed for cyclin A and cyclin D1 immunostaining. While cyclin D1 levels were similar in both WT cells and Emi1-deficient cells, relatively small amounts of cyclin A were detected in Emi1-deficient cells (Fig. 5) compared to those in WT cells. Considering that the only function of Emi1 is APC inhibition in S phase and prophase, these results may represent indirect evidence that APC is precociously activated in Emi1-deficient embryonic cells.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S-phase progression of Emi1–/– embryos is rarely defective, as evident from BrdU incorporation experiments (Fig. 3) and growth of blastocysts (Fig. 2E). In previous studies, the depletion of human Emi1 by RNA interference inhibited S-phase entry in somatic cells (12). Normal BrdU incorporation in Emi1-null embryos in this study may be explained by two hypotheses. Since S-phase and mitosis succeed each other repeatedly with no intervening periods of cell growth during embryonic cycles, Emi1 may be dispensable for S-phase entry or progression of embryonic cells. Another possibility is that residual maternal Emi1 sufficiently compensates for loss of the zygotic protein.
Even though only 4% of cultured blatocysts were Emi1–/– in blastocyst outgrowth experiments, apoptosis of Emi1–/– cells is restricted to the rapidly dividing cells of the inner mass. Interestingly, the postmitotic polyploid trophoblast giant cells survive, suggesting that Emi1 is selectively required in mitosis. Abnormalities are additionally observed in approximately 40% of mitotic Emi1–/– cells compared to those in the WT. Mitotic Emi1–/– cells have multipolar spindles and misaligned chromosomes, which are rarely present in WT. Moreover, multipolar spindles are frequently observed in nondestructible Emi1-expressing cells (18).
The cell numbers of Emi1-null embryos during preimplantation development were always lower than those of the WT littermates (Fig. 4). This finding implies that embryos accumulate an abundance of mitotic errors, inevitably resulting in the cessation of development. Consistent with the above data, TUNEL-positive cells were detected readily in Emi1–/– embryos but rarely in heterozygous or WT littermates (Fig. 4D). Alteration of hEmi1 may affect the level and timing of APC activity, leading to genomic instability via several mechanisms (12, 18, 20, 25, 27, 28). Given the functional importance of Emi1 at the late prophase checkpoint, early embryonic death of Emi1–/– mice is possibly due to severe chromosomal instability. As cyclin B1-cdk1 is first activated in the centrosome in which Emi1 is localized (12, 13, 27), the absence of the protein may prematurely activate APC at centrosomes, thereby leading to premature anaphase transition and frequent chromosome misalignment/missegregation. The possibility of premature APC activation in Emi1-null cells was shown, even if indirectly, by immunostaining experiments in which the level of cyclin A, an APC substrate, was lower than that of cyclin D1 in the cells (Fig. 5). However, we cannot rule out the possibility that these defects in mitotic progression in Emi1-null cells are caused by another (unknown) function of the Emi1 protein. Due to the limitation of performing the experiments with embryonic cells and the early lethality of Emi1-deficient embryos, further experiments with cells in which emi1 is conditionally removed are necessary to clarify the presence of premature APC activation in Emi1-deficient cells.
Chromosome misalignment/missegregation caused by Emi1 deficiency is a condition that daughter cells would not tolerate. Developmentally, most cell types are programmed not to allow aneuploidy formation in vivo and would conceivably be eliminated through apoptosis. Thus, it is not surprising that apoptotic cells are abundant in Emi1-deficient embryos (Fig. 4D).
An Emi1 homolog, Emi2/XErp1/FBXO43, which is conserved in amphibian, mouse, and human, has recently been reported as a novel Plx1-regulated inhibitor of APC activity during CSF arrest. However, how Emi2 inhibits APC ligase activity during the cell cycle and whether the two homologs have functional redundancies remain to be demonstrated. Our observations of the embryonic lethality of the Emi1–/– mouse indicate that Emi1 is not functionally redundant with and compensated by Emi2.
Notably, mice from several strains engineered to lack proteins involved in the spindle assembly checkpoint that inhibits APC activity in metaphase die at the peri-implantation stage, with mitotic abnormalities resembling those identified in Emi1-deficient embryos. These strains include Mad2 (4), survivin (35), Bub3 (14), BubR1 (37), Incenp (3), and CENP-E (26) mutants. Genetic data suggest that the late prophase checkpoint by Emi1 is as important as the spindle assembly checkpoint in mitotic progression and chromosome stability. However, unlike Mad- and BubR1-heterozygous mice, Emi1-heterozygous mice display no developmental abnormalities until 18 months, indicating that one functional copy of the Emil gene is sufficient for normal growth.
However, mice lacking Rassf1A, another regulator of APC activity in early mitosis, show no defects in murine embryo development but only tumor susceptibility (33, 36). These Rassf1A genetic data may suggest that the inhibitory functions of the gene product are restricted in unusual or stress conditions, although the proteins potentially display APC regulation. Alternatively, it is possible that other members of the RASSF1 family functionally compensate for the loss of Rassf1A in mice.
Our analyses in mice clearly imply that the Emi1 protein is required for mitotic fidelity. The Emi1 mutation in mice was difficult to study due to the severity of the phenotypes. Thus, tissue-specific or conditional knockout experiments in mice are required for further clarification of Emi1 function.
. . .
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ACKNOWLEDGMENTS |
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This study was supported by the National Research Laboratory Program, the Korea National Cancer Center Control Program (0320370-1 and 0510582-2), and the 21st Century Frontier Functional Human Genome Project of KISTEP (Ministry of Science and Technology of Korea).
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FOOTNOTES |
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Supplemental material for this article may be found at
http://mcb.asm.org/.
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