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Articles

Emi1 Maintains Genomic Integrity during Zebrafish Embryogenesis and Cooperates with p53 in Tumor Suppression

Jennifer Rhodes, Adam Amsterdam, Takaomi Sanda, Lisa A. Moreau, Keith McKenna, Stefan Heinrichs, Neil J. Ganem, Karen W. Ho, Donna S. Neuberg, Adam Johnston, Yebin Ahn, Jeffery L. Kutok, Robert Hromas, Justin Wray, Charles Lee, Carly Murphy, Ina Radtke, James R. Downing, Mark D. Fleming, Laura E. MacConaill, James F. Amatruda, Alejandro Gutierrez, Ilene Galinsky, Richard M. Stone, Eric A. Ross, David S. Pellman, John P. Kanki, A. Thomas Look
Jennifer Rhodes
1Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
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  • For correspondence: Jennifer.Rhodes@fccc.edu
Adam Amsterdam
2Massachusetts Institute of Technology, Cambridge, Massachusetts 02139
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Takaomi Sanda
3Dana-Farber Cancer Institute, Boston, Massachusetts 02115
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Lisa A. Moreau
3Dana-Farber Cancer Institute, Boston, Massachusetts 02115
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Keith McKenna
3Dana-Farber Cancer Institute, Boston, Massachusetts 02115
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Stefan Heinrichs
3Dana-Farber Cancer Institute, Boston, Massachusetts 02115
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Neil J. Ganem
3Dana-Farber Cancer Institute, Boston, Massachusetts 02115
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Karen W. Ho
3Dana-Farber Cancer Institute, Boston, Massachusetts 02115
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Donna S. Neuberg
3Dana-Farber Cancer Institute, Boston, Massachusetts 02115
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Adam Johnston
3Dana-Farber Cancer Institute, Boston, Massachusetts 02115
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Yebin Ahn
3Dana-Farber Cancer Institute, Boston, Massachusetts 02115
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Jeffery L. Kutok
4Brigham and Women's Hospital, Boston, Massachusetts 02115
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Robert Hromas
5University of New Mexico Cancer Center, Albuquerque, New Mexico 87131
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Justin Wray
5University of New Mexico Cancer Center, Albuquerque, New Mexico 87131
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Charles Lee
4Brigham and Women's Hospital, Boston, Massachusetts 02115
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Carly Murphy
4Brigham and Women's Hospital, Boston, Massachusetts 02115
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Ina Radtke
6St. Jude Children's Research Hospital, Memphis, Tennessee 38105
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James R. Downing
6St. Jude Children's Research Hospital, Memphis, Tennessee 38105
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Mark D. Fleming
7Children's Hospital, Boston, Massachusetts 02115
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Laura E. MacConaill
3Dana-Farber Cancer Institute, Boston, Massachusetts 02115
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James F. Amatruda
8University of Texas Southwestern Medical Center, Dallas, Texas 75390
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Alejandro Gutierrez
3Dana-Farber Cancer Institute, Boston, Massachusetts 02115
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Ilene Galinsky
3Dana-Farber Cancer Institute, Boston, Massachusetts 02115
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Richard M. Stone
3Dana-Farber Cancer Institute, Boston, Massachusetts 02115
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Eric A. Ross
1Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111
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David S. Pellman
3Dana-Farber Cancer Institute, Boston, Massachusetts 02115
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John P. Kanki
3Dana-Farber Cancer Institute, Boston, Massachusetts 02115
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A. Thomas Look
3Dana-Farber Cancer Institute, Boston, Massachusetts 02115
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DOI: 10.1128/MCB.00558-09
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ABSTRACT

A growing body of evidence indicates that early mitotic inhibitor 1 (Emi1) is essential for genomic stability, but how this function relates to embryonic development and cancer pathogenesis remains unclear. We have identified a zebrafish mutant line in which deficient emi1 gene expression results in multilineage hematopoietic defects and widespread developmental defects that are p53 independent. Cell cycle analyses of Emi1-depleted zebrafish or human cells showed chromosomal rereplication, and metaphase preparations from mutant zebrafish embryos revealed rereplicated, unsegregated chromosomes and polyploidy. Furthermore, EMI1-depleted mammalian cells relied on topoisomerase IIα-dependent mitotic decatenation to progress through metaphase. Interestingly, the loss of a single emi1 allele in the absence of p53 enhanced the susceptibility of adult fish to neural sheath tumorigenesis. Our results cast Emi1 as a critical regulator of genomic fidelity during embryogenesis and suggest that the factor may act as a tumor suppressor.

Successful cell division requires faithful replication of the genome, and defects in this process can contribute to genomic instability and subsequent malignant transformation (23). A key regulator of the normal cell cycle is the early mitotic inhibitor 1 (EMI1/FBXO5), a zinc finger protein expressed by a variety of adult tissues and especially in proliferating Ki-67-positive cells (39). Studies of the mammalian and Xenopus homologues of EMI1 have shown that it inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase complex that targets cell cycle-regulated proteins, such as the S- and M-phase cyclins (A and B), securin, and geminin (13, 25, 31). Depletion of EMI1 by small interfering RNA (siRNA) knockdown in cell lines or immunodepletion in cycling Xenopus extracts results in the untimely degradation of APC/C substrates, delaying G1/S- and M-phase progression and inducing rereplication (6, 21, 25, 31). Such rereplication is a consequence of decreased levels of the APC/C substrates cyclin A and geminin, which are regulators of replication licensing (6, 21). The result of EMI1 depletion in some cell lines is senescence (39).

Despite these insights into the molecular underpinnings of EMI1 function, little is known about the role of this protein in development. Knockout of murine Emi1 results in an embryonic-lethal phenotype prior to implantation, while a deficiency of Emi1 in cultured pronuclear zygotes leads to multipolar and tangled spindle structures, orphan chromosomes, large nuclei, and apoptosis by the 16-cell stage (17). Otherwise, the dynamic influence of EMI1 on early vertebrate development remains undefined. We sought to close this gap by taking advantage of the zebrafish model system. Zebrafish embryos harboring homozygous mutations of emi1 (emi1m/m) develop beyond the onset of circulation, providing a unique opportunity to examine the developmental roles of Emi1 in vivo. The zebrafish emi1 mutant (hi2648) line was originally identified by a proviral insertional mutagenesis screen designed to identify genes that are necessary for normal morphological development in embryos (1, 8, 9). Subsequent studies showed that the insertion was located between the first and second exons of the emi1 gene (2). The morphological defects in emi1m/m embryos at 2 days postfertilization (p.f.) are described in the public access zebrafish model organism database (http://zfin.org ). Briefly, abnormalities in emi1m/m embryos can be identified as early as 20 h p.f. and include slightly smaller heads and a lack of ventral curving of the posterior presomitic mesoderm. By 25 h p.f., the tail is more ventrally curved than in normal embryos, and increased cell death is observed throughout the central nervous system. Mutant embryos have circulating blood cells, although the onset of circulation is delayed. We became interested in this mutant because it harbors defects in the numbers and morphology of granulocytes, an important myeloid cell type within the innate immune system.

There is evidence that EMI1 may function in cancer pathogenesis, and a variety of human tumors express this factor very highly, although in some cases this may be a consequence of elevated proliferation rates (11, 18). In fact, the human homologue of emi1 resides within chromosome 6q25, a region often deleted in leukemia, which, together with the cell cycle-regulatory role of EMI1, suggested that this factor may also function as a tumor suppressor whose loss of function could promote genetic instability. Thus, in addition to investigating the role of zebrafish emi1 in zebrafish development, with particular emphasis on hematopoiesis, we examined mammalian cells to identify mechanisms that may be important in EMI1-related malignant transformation and explored a putative tumor suppressor role for this cell cycle-regulatory protein.

MATERIALS AND METHODS

Zebrafish.Wild-type AB stocks of Danio rerio and transgenic and mutant lines were maintained by standard methods and staged as previously established (16, 40). The transgenic zpu.1-EGFP, emi1m (emi1hi2648/fbxo5hi2648), and p53m (tp53zdf1/p53e7) lines have been previously described (2, 4, 14).

Imaging.Images were obtained using a Nikon SMZ-1500 zoom microscope or a Zeiss Axiovert upright microscope, and color images were captured with Openlab software (Improvision). When necessary, multiple focal layers from separate Openlab images were combined to make a single Photoshop figure; however, we did not enhance, obscure, move, or remove any specific feature within an image. Confocal images were captured with the Zeiss LSM 510 META NLO laser scanning microscope and a Zeiss LD 40× 0.6NA Achroplan objective lens. A multitrack line scan configuration allowed line-by-line pseudosimultaneous capture of red, green, and blue channels. Imaging was performed at room temperature with a Hamamatsu Orca or Nikon DS-Ri1 digital camera. All figures show sibling embryos, or cells are shown at equal magnifications.

Embryo injection experiments.Antisense morpholino oligonucleotides (Gene Tools) were designed to target the emi1 translational start site (ATG) and the splice donor site of exon 2. The ATG morpholino sequence is 5′-GTTTGGACACTTCATATTGAGGAGA-3′, the exon 2 splice donor morpholino sequence is 5′-ATTGTCGTTTCACCTCATCATCTGA-3′, and the exon 2 splice donor mismatch control morpholino is 5′-ATTcTCcTTTCAgCTCATgATgTGA-3′ (lowercase letters indicate mismatched base pairs). The efficacy of the emi1 exon 2 splice site-targeting morpholino was tested by single-embryo reverse transcription (RT)-PCR using primers in exon 1 (forward) and exon 3 (reverse). The zebrafish emi1 gene was cloned from wild-type 24-h p.f. zebrafish using a Qiagen OneStep RT-PCR kit, and DNA sequencing confirmed its identity with published mRNA sequences (accession number NM_001003869). Human emi1 was cloned from a cDNA library made from a CD34+ cell fraction of normal human bone marrow, and the DNA sequence was identical to that of a published mRNA (NM_012177). Both EMI1 genes were subcloned into the pCS2+ vector. Zebrafish transcripts were transcribed in vitro for antisense (XhoI and SP6 RNA polymerase) or sense (ClaI and T3 RNA polymerase) mRNA or probes. Human mRNA was prepared in a similar manner (sense, KpnI and SP6; antisense, BamHI and T3). Full-length mRNAs were in vitro transcribed using mMessage Machine kits (Ambion), and Ambion labeling reagents were used to generate all in situ probes. Embryos obtained from crosses of adult fish were injected at the one- or two-cell stage with 200 pl of sense or antisense mRNA (100 ng/μl, with phenol red) or 0.5 mM morpholinos with an injection volume equal to 1 ng of morpholinos per embryo. For the hi2648 rescue studies, the primers used for emi1 hi2648 allele genotyping following mRNA injection were 5′-GCTACCACTGCAATAGCGACAAG-3′ (emi1 sequence) and 5′-GCCAAACCTACAGGTGGGGTC-3′ (viral sequence), and primers to exon 1 and intron 1, corresponding to the region of viral insertion, were used to identify the wild-type allele. The methods for genotyping the wild-type and e7 mutant p53 alleles and use of the p53 morpholino were published previously (4, 34).

Immunohistochemistry and in situ hybridization.Embryos obtained from crosses of adult fish were raised in E3 medium containing 0.2 mM N-phenylthiourea (40). Embryos were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) overnight at 4°C and dehydrated in 100% methanol at −20°C. To examine cells undergoing DNA replication, live dechorionated embryos were incubated with 10 mM 5-bromo-2-deoxyuridine (BrdU) (Sigma) on ice for 30 min, and then the BrdU was removed and the embryos were transferred to a 28°C incubator for 1 h. The embryos were fixed as described above and permeabilized with an acetone treatment for 7 min at −20°C, followed by a 6-min incubation with 0.25% trypsin in PBS. To allow the detection of BrdU, the embryos were treated with 2 N HCl for 1 h at room temperature, and incorporation was detected using a mouse BrdU antibody (1/100; Roche) and an Alexa-conjugated secondary antibody (1:1,000; Invitrogen). Zebrafish phosphorylated histone H2AX (p-H2AX) was detected using a polyclonal anti-p-H2AX antibody (1:1,000) that was generously provided by James Amatruda, University of Texas Southwestern, and an Alexa-conjugated secondary antibody (1:1,000; Invitrogen). To permeate the embryos for p-H2AX detection, fixed embryos in methanol were treated with acetone. Blocking and antibody incubations were performed in PBS with 0.1% Tween 20, 2% blocking reagent (Roche), 5% fetal calf serum, and 1% dimethyl sulfoxide (DMSO). Using a similar protocol, zebrafish phosphorylated histone H3 (p-H3) was detected using a polyclonal anti-p-H3 antibody (1:1,000; Santa Cruz Biotechnology). The protocols for generating antisense RNA probes to detect mpo, l-plastin, pu.1, and α-globin expression by whole-mount RNA in situ hybridization (WISH) were published previously (32). We examined the difference between the average numbers of myeloid cells in emi1m/m versus wild-type homozygous (emi1wt/wt) or heterozygous (emi1wt/m) siblings using a two-tailed t test and standard error (P < 0.001). Hemoglobin was detected by incubating live embryos in o-dianisidine/H2O2 (Sigma) for 5 to 10 min at room temperature. Using Photoshop, individual o-dianisidine-positive cells were selected, and the area was quantified in pixels. The emi1m/m versus emi1wt/(wt or m) cell size distribution and averages were examined by using a two-tailed t test and standard error (P < 0.001). The protocols for terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining of fixed embryos and acridine orange staining of live embryos have been previously established (34).

FACS and cytology.Cells were sorted from zpu.1-EGFP transgenic embryos and stained with May-Grunwald-Giemsa stain as previously described (32). Propidium iodide-based cell cycle analysis was performed as published previously (34) with slight modifications. The modified protocol included the fixation of cell suspensions in 2% glucose and 3% PFA in PBS, which were then dehydrated by dropwise addition of 80% ethanol and resuspended in PBS with 20 mM HEPES, 0.25% NP-40, and 0.1% bovine serum albumin.

Zebrafish tumor experiments.Adult zebrafish harboring p53 and emi1 mutations were generated by standard mating practices. Adult progeny from a single cross were genotyped, segregated based on genotype, and examined weekly for the duration of their life spans; thus, all the fish examined were siblings. Fish were kept at a density of no more than 29 fish per 9 liters of water, with some variation in density between the genotypes. Fish with tumors comprising about 5% to 10% of their total body mass were culled with ice, their abdomens were punctured, and the fish were submerged in 4% PFA in PBS. Tissue processing, paraffin embedding, sectioning, and hematoxylin/eosin staining were performed by standard procedures within the Dana-Farber Harvard Cancer Center's Specialized Histopathology Core Laboratory, and the designation of the tumor type was determined by Jeffery Kutok. The tumor incidences were plotted using inverse Kaplan-Meier curves, and the two-sided P values were obtained by a log rank test. A total of 98 adult fish generated from a sibling incross were used in this experiment (see Fig. 9C).

Cytogenetics.Metaphase chromosome spreads were obtained from 28-h p.f. embryos using standard cytogenetic procedures (33). Cells were arrested at metaphase with colcemid, subjected to hypotonic treatments, and fixed onto glass slides with 3:1 methanol-acetic acid, and the chromosomes were visualized with Wright's stain (Sigma). Experiments performed in duplicate had similar results (see Fig. 7 for representative chromosomes and counts from a single experiment).

Fluorescence in situ hybridization of tumor sections was performed as previously published (33). Fluorescently labeled probes included a green ch211-114c12 bacterial artificial chromosome (BAC) DNA probe for zebrafish chromosome 5, encompassing the p53 gene, and a yellow chromosome 5 centromere probe, using BAC zc150k20.

Cell line experiments.Cell lines were cultured using standard media and practices. Knockdown and Western blot analyses were performed as published previously (21, 30, 34, 39). For knockdown analysis, control siRNA and Emi1 siRNA were purchased from Dharmacon (On-Target plus siControl nontargeting siRNA and Emi1/Fbxo5 On-Target plus Smartpool, respectively). Each siRNA pool was transfected at a final concentration of 20 nM using Hiperfect reagent (Qiagen) according to the manufacturer's instructions. Emi1 protein was detected using a polyclonal antibody (1:1,000; Zymed) and a horseradish peroxidase-conjugated secondary antibody (1:2,000; Cell Signaling Technology). Decatenation assays were performed as previously described with P values determined using Student's t test (41).

RESULTS

emi1 m/m zebrafish have developmental defects and hematopoietic abnormalities.As part of an analysis of insertional zebrafish mutants in the collection identified by Nancy Hopkins, we found that 2 days p.f., emi1m/m embryos have decreased numbers of myeloperoxidase (mpo)-expressing cells, or granulocytes (Fig. 1A and B). Further experiments using WISH analysis showed decreased numbers of mature myeloid cells expressing mpo or l-plastin in emi1m/m embryos at 24 h p.f. (Fig. 1C and D and data not shown). Furthermore, there were fewer pu.1-expressing myeloid progenitor cells in emi1m/m embryos (Fig. 1E and F). By manually quantifying the WISH-positive cells, we confirmed that the average number of cells per embryo was significantly decreased in emi1m/m embryos compared with wild-type emi1wt/(wt or m) siblings (Fig. 1G) (P < 0.001 for each comparison).

FIG. 1.
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FIG. 1.

Decreased myelopoiesis in zebrafish hi2648 mutants. (A to F) RNA WISH of wild-type siblings (homozygous emi1wt/wt or heterozygous emi1wt/m) or mutant embryos (homozygous emi1m/m) to examine mpo (A to D) or pu.1 (E and F) RNA expression. Representative embryos are shown in lateral views at 48 h p.f. (A and B) or 30 h p.f. (C to F). (G) The mean numbers of myeloid cells per embryo (error bars indicate standard errors of the mean) are shown for pu.1-, mpo-, and l-plastin-expressing cells in wild-type (black) or mutant (blue) sibling embryos (>5 embryos were counted for each condition). The asterisks indicate mean values that were different from those of the wild-type controls at a P value of <0.001.

The WISH analysis shown in Fig. 1 also revealed that the residual emi1m/m myeloid cells appeared larger than normal, leading us to examine mutants carrying the zpu.1-EGFP transgene, in which the zebrafish pu.1 promoter drives expression of the gene encoding enhanced green fluorescent protein (EGFP; referred to here as GFP) (14). We observed fewer live GFP+ cells in emi1m/m transgenic embryos (data not shown), consistent with the WISH data. High-magnification microscopy of live GFP-expressing mutant cells showed that these cells were indeed quite large but maintained normal cell shapes (Fig. 2A and B). We then quantified the myeloid progenitor cell size by FACS analysis of GFP+ cells from emi1m/m or emi1wt/(wt or m) siblings at 21 h p.f. (Fig. 2C and D). The forward scatter (x axis) showed a shift to the right, indicating that the mutant cell population contained cells that were on average twice as large as those in the wild-type population. Cytospins of the GFP+ cell populations at 21 h p.f. and 48 h p.f. (data not shown and Fig. 2E and F) confirmed that the emi1m/m myeloid cells were much larger but shared similar morphological features with emi1wt/(wt or m) myeloid cells.

FIG. 2.
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FIG. 2.

emi1 m/m embryos show increased myeloid cell size. zpu.1-EGFP transgenic emi1wt/(wt or m) or emi1m/m embryos were used to analyze GFP+ myeloid cells. (A and B) Live fluorescence microscopy images of representative normal (A) or mutant (B) cells at 48 h p.f. at the same magnification (bar, 5 μm). (C and D) FACS analysis of the GFP+ cell populations from wild-type (C) or mutant (D) embryos at 21 h p.f. showing the side scatter (SSC) (y axis; granularity) and the forward scatter (FSC) (x axis; cell size). These plots include 6,795 wild-type GFP+ cells and 8,073 mutant cells obtained from 30 to 40 embryos per genotype. The red vertical lines at 102 on the x axes serve as reference values for comparing the relative sizes of the GFP+ populations. (E and F) May-Grunwald-Giemsa staining for morphological analysis of FACS-sorted GFP+ populations (bar, 10 μm).

As previously noted, emi1m/m embryos exhibit widespread developmental abnormalities (Fig. 3A and B), suggesting that many tissues may be affected, including additional hematopoietic lineages. A comparison of embryos with circulating erythrocytes showed that emi1m/m mutants had reduced circulating blood (data not shown) and fewer α-globin-expressing cells than their wild-type siblings (Fig. 3C and D). Interestingly, in contrast to normal erythrocytes, the emi1m/m erythroid cells appeared to vary widely in size. Whole-mount o-dianisidine staining of hemoglobin-containing cells further demonstrated the variation in emi1m/m erythroid cell size (Fig. 3E to H). By quantifying the area of o-dianisidine-positive cells from images of stained embryos, we confirmed that the distribution of emi1m/m erythroid cell sizes was significantly different from that of the emi1wt/(wt or m) erythroid population, with emi1m/m erythrocytes having an average size that was twofold larger than that of wild-type cells (Fig. 3K) (P < 0.001). Furthermore, FACS analysis showed that the total cell population for mutants was larger than that for wild-type cells (Fig. 3I and J), indicating that Emi1 has a functional role affecting many different cell types throughout the developing embryo.

FIG. 3.
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FIG. 3.

Developmental defects in emi1m/m mutants. (A and B) Phase-contrast analysis of embryos at 48 h p.f. emi1m/m mutants have small heads and eyes and ventrally curved bodies. (C and D) α-globin WISH analysis of 48-h p.f. embryos showing uniform erythroid cell size in wild-type siblings (C) in contrast to mutant cells (D), which have abnormal variations in cell size. The insets are enlargements at equal magnifications of the boxed regions. (E to H) o-Dianisidine-stained whole-mount siblings at 48 h p.f. Whole-mount (E and F) and magnified (G and H) views show variations in cell size in emi1m/m mutants (F and H) in comparison to wild-type siblings (E and G). Bars, 20 μm. (I and J) FACS analysis of cells from wild-type (I) or mutant (J) disaggregated embryos at 21 h p.f., showing the side scatter (SSC) (y axis), indicating granularity, and the forward scatter (FSC) (x axis), indicating cell size. The red vertical lines at 102 on the x axes serve as reference values for comparing the relative sizes of the total cell populations. (K) The distribution of o-dianisidine-positive (o-D+; o-Dianisidine+) cell sizes and average cell size (error bars indicate standard errors of the mean) are shown. The area of stained cells was quantified in pixels. These plots include 138 total cells from five emi1m/m embryos and 152 total cells from five emi1wt/(wt or m) siblings. The asterisks indicate a P value of <0.001.

Zebrafish emi1 is expressed maternally and in a ubiquitous pattern throughout the developing embryo (data not shown and Fig. 4B) but was not detected in mature myeloid cells, as indicated by the lack of emi1-positive cells on the yolk of 21-h p.f. embryos, where differentiated granulocytes are normally found. This pattern is consistent with the reported expression of emi1 in proliferating cells (39). At 21 h p.f., emi1m/m embryos showed a severe reduction of emi1 expression (Fig. 4C). We verified that the hematopoietic and morphological phenotypes of emi1m/m embryos were due to the loss of emi1 expression by phenocopying the mutant with morpholinos targeting either an emi1 splice site (Fig. 4A and D to F) or the emi1 ATG codon (data not shown) and also by rescuing emi1m/m embryos by the forced expression of zebrafish emi1 mRNA (Fig. 4G to I). Importantly, the injection of human EMI1 mRNA into emi1m/m mutants rescued the development of normal-size mpo-positive cells and could return their numbers to wild-type levels (data not shown), indicating that these gene orthologs are functionally conserved in vivo.

FIG. 4.
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FIG. 4.

The phenotype of emi1m/m embryos is due to decreased emi1 expression. (A) Diagram of the zebrafish emi1 locus indicating the locations of the hi2648 insertion, morpholino (MO) targeting sites, and the RT-PCR primer positions. (B and C) emi1 WISH on emi1wt/(wt or m) (B) or emi1m/m (C) embryos at 21 h p.f. The embryos are shown in whole-mount lateral views. Note the decreased emi1 expression in emi1m/m mutant siblings. (D and E) Wish analysis of mpo expression in control MO-injected (D) or exon 2 splice site (ex2sd) MO-injected (E) embryos at 24 h p.f. Representative embryos are shown after injection with 1 ng of MO. (F) RT-PCR analysis of emi1 splicing in control (c) MO-injected or exon 2 splice site (sd) MO-injected individual embryos at 21 and 48 h p.f. Ex2sd MO-injected embryos had weak signals for the normal transcript (arrow) and alternate, abnormally spliced transcripts (arrowheads). (G to I) Rescue of myeloid cell numbers in homozygous mutant embryos injected with zebrafish emi1 mRNA (zemi1). Uninjected emi1wt/(wt or m) (G) and emi1m/m (H) embryos are shown for comparison. (I) A representative emi1m/m embryo that was partially rescued with zemi1 RNA, showing normal numbers of mpo-expressing cells and normal head morphology, with some abnormal morphological features in the trunk region.

emi1 m/m embryos have defects in genomic integrity.Depletion of EMI1 in human cell lines results in rereplication (6, 21, 39), suggesting that an essential role of the protein is to preserve genomic integrity by blocking rereplication. To address whether the EMI1 function is conserved in zebrafish, we performed propidium iodide-based FACS analysis of cells from 21 h p.f. emi1m/m or emi1wt/(wt or m)zpu.1-EGFP transgenic siblings. At this age, hematopoietic progenitors and more differentiated cells are present in the developing embryos, and this is the earliest age at which we could morphologically distinguish emi1m/m mutants from emi1wt/(wt or m) siblings. The total cell population and the GFP+ subset from wild-type embryos showed typical distributions of cycling normal cells (Fig. 5A) in G1 (64% and 61%, total and GFP+, respectively), S (31% and 33%), and G2/M (5% and 6%). In contrast, the distributions of emi1m/m total and GFP+ cycling cells were G1, 36% and 31%; S, 33% and 30%; and G2/M, 31% and 38%, with a large increase in both a sub-G1 population and a cell fraction greater than the 4 N complement of DNA (Fig. 5B). Previous studies of mammalian cell lines showed that severe siRNA depletion of EMI1 caused rereplication, which is consistent with our findings, and prevented cells from entering into mitosis, as indicated by a very low percentage of cells with nuclear envelope degradation or p-H3 (6, 21). Interestingly, using confocal microscopy to assess p-H3 staining, we found that emi1m/m mutants and emi1wt/(wt or m) siblings had identical mitotic indexes, in that a wild-type sibling contained 10 p-H3+ nuclei per 198 DAPI+ nuclei that were analyzed (∼5% mitotic cells) whereas an emi1m/m mutant embryo showed 5 p-H3+ nuclei out of a total of 98 DAPI+ nuclei enumerated (∼5% mitotic cells). Representative images are shown in Fig. 5C to H, illustrating that in the trunks of the embryos, especially in the p-H3+ and DAPI merged panels (E and H), the DAPI-stained emi1m/m mutant nuclei are larger and fewer in number but the proportion of p-H3+ nuclei is the same as in emi1wt/(wt or m) siblings. In previous studies (6, 21), severe siRNA depletion of EMI1 prevented cells from entering into mitosis, suggesting that the cells entering mitosis in emi1m/m mutant zebrafish have residual maternal Emi1 or low levels of zygotic Emi1 that allow mitotic entry. However, when viewed in the context of the FACS analysis of nuclear DNA content, most of the increased cells with a 4 N or greater complement of DNA in emi1m/m mutant zebrafish represent G2-phase cells or cells continuing in cycle with rereplicated DNA.

FIG. 5.
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FIG. 5.

Decreased emi1 results in cell cycle defects. (A and B) FACS scanning of propidium iodide (PI)-stained total cells (top) or the myeloid GFP+ cell fraction (bottom). Cells with 2 N DNA content (G1), cells replicating their DNA (S), and cells with 4 N DNA content (G2/M) are indicated, as well as cells with a less than 2 N (subG1) or greater than 4 N amount of DNA. Note that mutant cells have greatly increased populations of cells with <2 N DNA and >4 N DNA. (C to H) Confocal analysis of whole-mount embryos at 21 h p.f.; lateral views of the trunk are shown. Representative embryos show immunohistochemistry of phosphorylated p-H3 (red) and DAPI (blue) in emi1wt/(wt or m) (C to E) and emi1m/m (F to H) siblings. The emi1wt/(wt or m) embryo displays 10 pH 3+ nuclei with condensed DNA out of a total of 198 DAPI+ nuclei compared with 5 pH 3+ out of 98 DAPI+ nuclei in the emi1m/m mutant. E and H show magnified merged views of the boxed areas. Bars, 10 μm.

Published studies using human cell lines show that loss of EMI1 leads to an increase in double-strand DNA breaks, as indicated by analysis of p-H2AX (6, 21, 39). We therefore evaluated the levels of p-H2AX in zebrafish embryos and detected increased levels of this phosphorylated protein in emi1m/m embryos by Western analysis (Fig. 6Q) and whole-mount immunohistochemistry (Fig. 6R and S). Using confocal microscopy, we found that the levels of p-H2AX in zpu.1-GFP myeloid cells were strikingly increased in emi1m/m embryos (Fig. 6I to P) relative to emi1wt/(wt or m) siblings (Fig. 6A to H).

FIG. 6.
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FIG. 6.

Increased DNA damage in emi1m/m cells. zpu.1-EGFP transgenic emi1wt/(wt or m) (A to H) and emi1m/m (I to P) embryos were examined by confocal microscopy at 30 h p.f. Immunohistochemistry of p-H2AX is shown in red. Bars, 5 μm. Note the punctate nuclear p-H2AX in mutant myeloid cells (compare panels J and N with B and F). (Q) Western analysis of p-H2AX and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) loading control from emi1m/m or emi1wt/(wt or m) embryo lysates. (R and S) Whole-mount immunohistochemical analysis of p-H2AX in emi1wt/(wt or m) (R) and emi1m/m (S) siblings. Shown are lateral views of the posterior trunk region; the images were captured using identical exposure settings.

We consistently observed increased BrdU incorporation, reflecting increased DNA replication, in cells throughout emi1m/m embryos at 24 h p.f. (Fig. 7C and D). We next examined whether emi1m/m embryos manifested changes in chromosome numbers by analyzing metaphase spreads of cells from emi1wt/(wt or m), and emi1m/m siblings. In multiple independent experiments, metaphase spreads from wild-type embryos had a modal chromosome count of 50 chromosomes per cell, which is normal for zebrafish cells (Fig. 7E, H, and K). In contrast, almost half of the metaphases from emi1m/m mutants showed rereplicated, unsegregated chromosomes (Fig. 7F, I, and K) or polyploidy (Fig. 7G, J, and K). These data indicate that zebrafish cells lacking emi1 exhibit aberrant DNA rereplication during embryogenesis.

FIG. 7.
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FIG. 7.

Rereplicated, unsegregated chromosomes and polyploidy in emi1m/m embryos. (A to D) DAPI staining (blue) (A and B) and BrdU DNA labeling (red) (C and D) in a lateral view of the posterior trunk reveals larger nuclei in emi1m/m cells than in emi1wt/(wt or m) cells undergoing replication (bars, 20 μm). The images were captured using identical exposure settings. (E to J) Metaphase spreads on zebrafish cells, genotypes, and chromosome numbers (c) are indicated in each panel. Twofold enlargements of the boxed regions are shown in panels H to J. (K) Quantitation of the numbers of chromosomes in cells from emi1wt/(wt or m) or emi1m/m embryos. Twenty-one-hour p.f. embryos were treated for 8 h with colcemid, followed by metaphase analysis. The modal chromosome numbers are shown for 25 metaphases from emi1wt/(wt or m) cells and 16 metaphases from emi1m/m mutant cells. Zebrafish normally have 50 metaphase chromosomes. In emi1m/m cells, 3/12 metaphases had 50 centromeres containing four chromatids each, termed rereplicated. (L) Quantitative analysis of metaphase cells following ICRF-193 treatment. HCT116 cells transfected with control siRNA (C-siRNA) or EMI1 siRNA were incubated with DMSO or Topo IIα inhibitor (ICRF-193) for 18 h at the indicated concentrations. The average values are the numbers of cells minus the average values for vehicle control (DMSO), average values for five determinations, and standard error. **, P < 0.01.

We hypothesized that rereplication in the absence of chromatid disjunction might result in highly intertwined, or catenated, DNA. Decatenation, or untangling of the chromosomes, requires topoisomerase (Topo) IIα, and this process can be blocked by ICRF-193, a chemical inhibitor of Topo IIα (3, 15). Hence, we used HCT116 human colon carcinoma cells to test whether EMI1 depletion affects the proportion of cells that are dependent on Topo IIα to progress through mitosis, thus indirectly assessing the percentage of mitotic cells harboring catenated DNA. EMI1 was partially depleted with an siRNA specific to human EMI1 mRNA (Fig. 8T). Catenation can inhibit cell cycle progression at two points, preventing progression from G2 to M (7) and progression within M phase from metaphase to anaphase, ultimately blocking cell division if the chromosomal junctions are unresolved (5, 24, 35, 38). We studied the percentage of cells in metaphase by immunofluorescence microscopy of asynchronous cells stained with DAPI and a tubulin antibody, a control for spindle assembly (data not shown). Equal percentages of metaphase cells were identified in control and EMI1-depleted cells following exposure to DMSO, which is consistent with the normal mitotic index in emi1m/m embryos and probably reflects the residual EMI1 protein evident by Western blot analysis in the EMI1-siRNA-treated HCT116 human colon carcinoma cells (Fig. 8T). Following ICRF-193 exposure, EMI1-depleted cells showed a statistically significant increase in the percentage of cells in metaphase in comparison with control cells (Fig. 7L) (P < 0.01). Thus, the mitotic obstruction that occurs when Topo IIα is challenged with ICRF-193 is exacerbated by EMI1 depletion, indicating that decreased EMI1 results in an increased proportion of cells with mitotic catenated DNA, which may contribute to genomic instability and the production of cells with greater than 4 N DNA content.

FIG. 8.
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FIG. 8.

Decreased emi1 induces defects that are independent of p53-mediated apoptosis. Apoptosis was indicated by TUNEL staining (A and B) or acridine orange staining (C to F) of 28-h p.f. embryos. Levels of apoptosis in emi1m/m embryos (B and D) are sharply increased compared with emi1wt/(wt or m) siblings (A and C). p53 morpholino (MO) knockdown blocks apoptosis in emi1m/m embryos (compare D and F). (G to I) mpo WISH analysis shows the lack of rescue of decreased numbers and enlarged myeloid cells in emi1m/m mutant embryos harboring a homozygous p53m/m mutation (I) in comparison to embryos with normal p53wt/wt (H). (J) Mean numbers (± standard errors of the mean) of mpo-expressing cells are not affected by the p53 genotype. The black bars represent emi1wt/(wt or m) embryos, and the blue bars represent emi1m/m mutants. (K to S) Bright-field microscopy and propidium iodide-based cell cycle FACS analysis of control (C) or EMI1 siRNA-transfected HCT116 cells with wild-type p53 (p53wt/wt) or null p53 (p53−/−). The experiments were performed in triplicate at 48 h posttransfection, and representative data are shown. Panel S shows the percentage of cells in each phase of the cell cycle for each condition. (T) Western analysis of EMI1, p53, p-H2AX, and α-tubulin proteins in the indicated lysates.

Mutation of emi1 cooperates with p53 loss to promote adult tumors.When analyzed by FACS, emi1m/m embryos showed an increased population of sub-G1 DNA content cells indicative of apoptotic cells, which we confirmed by TUNEL (Fig. 8A and B) and acridine orange staining (Fig. 8C and D). Cell death was most prevalent in the central nervous system within the head and spinal cord of mutant embryos but was also observed in cells on the yolk and in the tail. We could block cell death in emi1wt/(wt or m) and emi1m/m embryos by p53-morpholino knockdown or in a p53 mutant line (Fig. 8E and F and data not shown) (4). However, the aberrant granulocytic cell phenotype of emi1m/m embryos was p53 independent by morphology and mpo WISH analysis (Fig. 8G to J). Furthermore, EMI1 knockdown in HCT116 cells in the presence or absence of p53 resulted in identical cell cycle distributions, indicating that the cell cycle defects were not caused by p53 activation (Fig. 8O to T).

Zebrafish embryos heterozygous for emi1 mutations appear to develop normally, and the adult fish do not develop tumors at a higher rate than wild-type strains. To determine whether emi1 haploinsufficiency could synergize with p53 mutations in tumorigenesis, we bred this line into a p53 mutant zebrafish line in which the adult fish develop peripheral neural sheath tumors (4). We analyzed tumors formed in adults harboring mutations in p53 (p53wt/m or p53m/m) with wild-type alleles of emi1 (emi1wt/wt) or emi1 haploinsufficiency (emi1wt/m). The tumors identified in p53 null fish were consistent with previous studies and were predominantly malignant peripheral neural sheath tumors of the spindle cell type (Fig. 9A and B), with a single case showing a more epithelioid morphology (data not shown). Fish heterozygous for the p53 mutation also developed malignant peripheral neural sheath tumors, although other tumor types, such as rosette-bearing neural tumors with morphology consistent with peripheral primitive neuroectodermal tumors, were occasionally observed. Fish heterozygous for both the p53 mutation and the emi1 mutation displayed decreased tumor incidence compared to p53 heterozygotes with wild-type emi1wt/wt (P = 0.007), indicating that emi1 haploinsufficiency in this context did not promote tumorigenesis and suggesting that heterozygous emi1 mutation does not promote the loss of the wild-type allele of p53. To verify this finding, we examined the regions on zebrafish chromosome 5 encompassing the zebrafish p53 gene and the centromeric region, using fluorescently labeled BACs in tumors arising from double-heterozygous fish (Fig. 9D and E). The fish analyzed were not siblings of those in Fig. 9C. Paraffin sections of the double-heterozygous tumors from two fish were probed, and in every cell examined from both tumors (cell count, >100 for each tumor), there were two signals for p53, suggesting that the p53 wild-type allele is retained, although we cannot rule out the possibility that the wild-type allele had not been inactivated either by gene conversion from the mutant allele or by de novo mutation.

FIG. 9.
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FIG. 9.

Emi1 functions as a tumor suppressor. (A) Bright-field microscopy of a spontaneous tumor arising in an emi1wt/mp53wt/m double-heterozygous adult fish. Adults that were p53wt/m or p53m/m showed similar gross features upon tumor formation. The arrow indicates the tumor. (B) Representative hematoxylin- and eosin-stained paraffin section of an emi1wt/mp53m/m peripheral neural sheath tumor (magnification, ×400). (C) Tumor incidence in adult fish siblings that were generated by standard breeding methods. Genotypes and P values are indicated. (D and E) Fluorescence in situ hybridization of paraffin sections of tumors from two different emi1wt/mp53wt/m fish (tumor 1/fish no. 1 and tumor 2/fish no. 2). These fish were not siblings of the cohort in panel C. BACs were used to detect chromosome 5 regions encompassing the p53 gene (green) and the centromere (yellow). More than 100 cells per tumor were analyzed, but no deletions of p53 were found.

Turning to the p53m/m fish, we found a significant increase in the rapidity of onset and the overall cumulative incidence of tumors in emi1wt/mp53m/m fish compared with emi1wt/wtp53m/m or emi1wt/wtp53wt/m fish (P = 0.047) (Fig. 9C). Thus, emi1 functions as a haploinsufficient tumor suppressor that synergizes with loss of p53 function, which is found in at least 50% of human cancers overall.

DISCUSSION

We have shown that inactivation of emi1 in the zebrafish germ line has marked developmental defects, including multilineage hematopoietic abnormalities. Human EMI1 mRNA rescued the phenotype of zebrafish emi1m/m embryos, and depletion of EMI1 in zebrafish or human cells led to similar cell cycle defects, indicating that the function of this mitotic regulator is conserved from zebrafish to humans. EMI1 depletion in human cells and zebrafish leads to increased cell size, while FACS analysis of human cell lines, as well as zebrafish cells, in the present study indicated that this factor is essential for inhibiting rereplication (6, 21, 39, 42). It is likely that our observation of rereplicated and unsegregated chromosomes or polyploid karyotypes has not been reported before because knockdown of EMI1 in human cell lines induces senescence and the murine Emi1 knockout has a very early lethal phenotype (17, 39). Nonetheless, we have shown that emi1 depletion is a mechanism for the genesis of near-tetraploid cells, suggesting that emi1m/m zebrafish may be a useful model for examining the molecular mechanism regulating this process and the ensuing cellular consequences, such as the derivation of aneuploid cells, as well as identifying additional mechanisms through which emi1 may contribute to leukemogenesis. Of note, increased ploidy has been associated with an increase in cell size (10, 33, 36), suggesting that these two phenotypes may be inherently linked in our mutant.

To account for the presence of metaphase spreads with polyploidy or rereplicated, unsegregated chromosomes in emi1m/m cells, we asked whether EMI1 depletion affected the percentage of cells that are dependent on Topo IIα in order to progress beyond metaphase, thus indirectly assessing the percentage of mitotic cells harboring catenated DNA. Our results showed that loss of EMI1 in human cells enhanced the percentage of cells in metaphase when Topo IIα was inhibited, suggesting that these cells retained mitotic catenated DNA and were unable to undergo chromosome segregation. Thus, loss of EMI1 resulted in an increased population of cells with catenated DNA, a defect that may be due to rereplication. Interestingly, Emi1-deficient mouse pronuclei showed an abundance of abnormal mitoses, which may be due in part to catenation-related defects in chromosome disjunction (17). Our working hypothesis is that unresolved DNA catenation in emi1-deficient cells may prevent cells from completing cell division or facilitate unbalanced chromosomal segregation, resulting in aneuploid cells, a process that may be facilitated by a cellular environment that precedes apoptosis.

Zhang et al. recently described an alternate allele of emi1 (emi1tiy121) in which a premature stop codon near the amino terminus results in a truncated protein, and the resultant zebrafish mutant appears to have more severe morphological defects than does our mutant (42). The emi1tiy121 studies focused on the morphologically abnormal somites, while we observed normal-appearing chevron-shaped somites. The emi1tiy121 mutant cells did have larger nuclei than wild-type cells, consistent with our studies. In that study, as well as in our own, homozygous emi1 mutations could be rescued by forced expression of emi1 mRNA, indicating that both mutant phenotypes are due to the loss of emi1 expression. A small amount of emi1 mRNA can be detected by WISH in our insertional mutant, which may account for the less severe phenotype of our mutant in comparison to the emi1tiy121 mutant and the ability of emi1hi2648 mutant cells to enter into mitosis. Furthermore, heterozygous mutation of zebrafish or murine emi1 did not give rise to obvious defects during embryogenesis, and the adults remained healthy, indicating that heterozygous mutation of emi1 alone is not sufficient to disturb essential developmental pathways (17).

A striking phenotype of our emi1m/m embryos is robust apoptosis, which is evident from acridine orange- and TUNEL-positive cells in the central nervous system, on the yolk, and in posterior trunk regions consistent with apoptosis in hematopoietic cells. Apoptosis in myeloid cells was confirmed by FACS analysis, showing an increase in the sub-G1 (apoptotic) population of the emi1m/m myeloid cell population (Fig. 5). The acridine orange-positive apoptotic cells in emi1m/m embryos could be blocked by p53 depletion, indicating that this apoptosis is p53 dependent in all tissues. However, the emi1m/m myeloid cell phenotype of reduced cell numbers and the emi1 siRNA-induced cell cycle defects observed in human cell lines are p53 independent. We interpret this to mean that the cell cycle defects in the emi1m/m myeloid cells are primarily responsible for the myeloid cell phenotype, which is p53 independent and likely reflects the known effects of loss of Emi1-mediated inhibition of the activity of the APC/C in rapidly dividing myeloid cell progenitors (21, 25, 31).

Our results led us to hypothesize that cooperation between the heterozygous loss of emi1, or haploinsufficiency, and p53 mutation might play a role in cancer pathogenesis. Genome-wide approaches have recently revealed cryptic chromosomal changes and unexpected gene mutations in leukemia, suggesting that there are many as yet unidentified factors that can contribute to this disease (19, 27, 29). EMI1 has been shown to be oncogenic, as evidenced by proviral insertion into the Emi1 locus that accelerated lymphomagenesis in mice transgenic for the c-myc gene under the control of the immunoglobulin heavy-chain enhancer (E mu-myc) (12). In humans, high levels of EMI1 protein and RNA have been found in a variety of malignant tumors (11, 18). In contrast, deletion of the long arm of chromosome 6 encompassing the EMI1 gene has been observed in primary human leukemias and as an abnormality associated with progression of myelodysplastic syndrome to acute myeloid leukemia (20, 22, 26, 37). In addition, haploinsufficiency or complete deficiency of E2F2, a transcriptional activator of the EMI1 gene, also accelerated the onset and progression of lymphomas in E mu-myc transgenic mice (28), indirectly suggesting that EMI1 may function as a tumor suppressor. We found that compound homozygous p53 mutant and heterozygous emi1 mutant adults developed tumors at an accelerated rate and with higher penetrance than did fish with wild-type emi1, establishing that emi1 functions as a haploinsufficient tumor suppressor when combined with loss of p53 in zebrafish. This finding suggests that haploinsufficiency for the EMI1 gene is likely to be associated with inactivation of p53 in leukemia and other human cancers.

In summary, our data support an essential and evolutionarily conserved function for emi1 in regulating the integrity of the genome. Our studies are in agreement with previous reports showing that EMI1 depletion leads to rereplication and the activation of p53 (6, 21, 39). In addition, we showed that the EMI1 depletion is associated with mitotic catenated DNA and that the cell cycle defects are independent of p53. Similarly, the decrease in myeloid cell numbers and increase in myeloid cell size in zebrafish embryos are also p53 independent, even though apoptosis is markedly decreased in p53-depleted embryos. Importantly, we have provided the first evidence that emi1 acts as a tumor suppressor in p53 mutant zebrafish, and we hypothesize that emi1 may have a similar role in human malignancy.

ACKNOWLEDGMENTS

We thank Nancy Hopkins for generously providing the emi1hi2648 mutant zebrafish line. Thanks go to John Gilbert for manuscript editing and helpful comments. We thank members of the Look laboratory for discussions and reagents, especially Samuel Sidi, Raymond Hoffmans, and Hui Feng. We acknowledge Tingxi Liu and Min Deng for the zebrafish emi1 clone and Adolfo Ferrando for use of his panel of T-ALL patient sample DNAs. We are grateful to Thomas Diefenbach and Lihong Bu for their assistance in the Mental Retardation and Developmental Disabilities Research Center's imaging facility at Children's Hospital, Boston, and Tianyu Li in the FCCC Biostatistics and Bioinformatics Facility for her help analyzing the zebrafish tumor data.

This work was supported by NIH grants R01 CA93152 (A.T.L.), K01 DK69672 (J.R.), P01 CA66996-11A1 (R.M.S. and J. D. Griffin), R01 CA111560 (C.L.), and R01 RR12589 (A.A. and Nancy Hopkins).

FOOTNOTES

    • Received 29 April 2009.
    • Returned for modification 30 June 2009.
    • Accepted 13 August 2009.
    • Accepted manuscript posted online 24 August 2009.
  • Copyright © 2009 American Society for Microbiology

REFERENCES

  1. 1.↵
    Amsterdam, A., S. Burgess, G. Golling, W. Chen, Z. Sun, K. Townsend, S. Farrington, M. Haldi, and N. Hopkins. 1999. A large-scale insertional mutagenesis screen in zebrafish. Genes Dev.13:2713-2724.
    OpenUrlAbstract/FREE Full Text
  2. 2.↵
    Amsterdam, A., R. M. Nissen, Z. Sun, E. C. Swindell, S. Farrington, and N. Hopkins. 2004. Identification of 315 genes essential for early zebrafish development. Proc. Natl. Acad. Sci. USA101:12792-12807.
    OpenUrlAbstract/FREE Full Text
  3. 3.↵
    Andoh, T., M. Sato, T. Narita, and R. Ishida. 1993. Role of DNA topoisomerase II in chromosome dynamics in mammalian cells. Biotechnol. Appl. Biochem.18:165-174.
    OpenUrlWeb of Science
  4. 4.↵
    Berghmans, S., R. D. Murphey, E. Wienholds, D. Neuberg, J. L. Kutok, C. D. Fletcher, J. P. Morris, T. X. Liu, S. Schulte-Merker, J. P. Kanki, R. Plasterk, L. I. Zon, and A. T. Look. 2005. tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors. Proc. Natl. Acad. Sci. USA102:407-412.
    OpenUrlAbstract/FREE Full Text
  5. 5.↵
    Clarke, D. J., R. T. Johnson, and C. S. Downes. 1993. Topoisomerase II inhibition prevents anaphase chromatid segregation in mammalian cells independently of the generation of DNA strand breaks. J. Cell Sci.105:563-569.
    OpenUrlAbstract/FREE Full Text
  6. 6.↵
    Di Fiore, B., and J. Pines. 2007. Emi1 is needed to couple DNA replication with mitosis but does not regulate activation of the mitotic APC/C. J. Cell Biol.177:425-437.
    OpenUrlAbstract/FREE Full Text
  7. 7.↵
    Downes, C. S., D. J. Clarke, A. M. Mullinger, J. F. Gimenez-Abian, A. M. Creighton, and R. T. Johnson. 1994. A topoisomerase II-dependent G2 cycle checkpoint in mammalian cells. Nature372:467-470.
    OpenUrlCrossRefPubMedWeb of Science
  8. 8.↵
    Gaiano, N., A. Amsterdam, K. Kawakami, M. Allende, T. Becker, and N. Hopkins. 1996. Insertional mutagenesis and rapid cloning of essential genes in zebrafish. Nature383:829-832.
    OpenUrlCrossRefPubMedWeb of Science
  9. 9.↵
    Golling, G., A. Amsterdam, Z. Sun, M. Antonelli, E. Maldonado, W. Chen, S. Burgess, M. Haldi, K. Artzt, S. Farrington, S. Y. Lin, R. M. Nissen, and N. Hopkins. 2002. Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development. Nat. Genet.31:135-140.
    OpenUrlCrossRefPubMedWeb of Science
  10. 10.↵
    Gregory, T. R. 2001. Coincidence, coevolution, or causation? DNA content, cell size, and the C-value enigma. Biol. Rev. Camb. Phil. Soc.76:65-101.
    OpenUrlCrossRefPubMed
  11. 11.↵
    Gutgemann, I., N. L. Lehman, P. K. Jackson, and T. A. Longacre. 2008. Emi1 protein accumulation implicates misregulation of the anaphase promoting complex/cyclosome pathway in ovarian clear cell carcinoma. Mod. Pathol.21:445-454.
    OpenUrlCrossRefPubMed
  12. 12.↵
    Haupt, Y., W. S. Alexander, G. Barri, S. P. Klinken, and J. M. Adams. 1991. Novel zinc finger gene implicated as myc collaborator by retrovirally accelerated lymphomagenesis in E mu-myc transgenic mice. Cell65:753-763.
    OpenUrlCrossRefPubMedWeb of Science
  13. 13.↵
    Hsu, J. Y., J. D. Reimann, C. S. Sorensen, J. Lukas, and P. K. Jackson. 2002. E2F-dependent accumulation of hEmi1 regulates S phase entry by inhibiting APC(Cdh1). Nat. Cell Biol.4:358-366.
    OpenUrlCrossRefPubMedWeb of Science
  14. 14.↵
    Hsu, K., D. Traver, J. L. Kutok, A. Hagen, T. X. Liu, B. H. Paw, J. Rhodes, J. N. Berman, L. I. Zon, J. P. Kanki, and A. T. Look. 2004. The pu.1 promoter drives myeloid gene expression in zebrafish. Blood104:1291-1297.
    OpenUrlAbstract/FREE Full Text
  15. 15.↵
    Ishida, R., T. Miki, T. Narita, R. Yui, M. Sato, K. R. Utsumi, K. Tanabe, and T. Andoh. 1991. Inhibition of intracellular topoisomerase II by antitumor bis(2,6-dioxopiperazine) derivatives: mode of cell growth inhibition distinct from that of cleavable complex-forming type inhibitors. Cancer Res.51:4909-4916.
    OpenUrlAbstract/FREE Full Text
  16. 16.↵
    Kimmel, C. B., W. W. Ballard, S. R. Kimmel, B. Ullmann, and T. F. Schilling. 1995. Stages of embryonic development of the zebrafish. Dev. Dyn.203:253-310.
    OpenUrlCrossRefPubMedWeb of Science
  17. 17.↵
    Lee, H., D. J. Lee, S. P. Oh, H. D. Park, H. H. Nam, J. M. Kim, and D. S. Lim. 2006. Mouse emi1 has an essential function in mitotic progression during early embryogenesis. Mol. Cell. Biol.26:5373-5381.
    OpenUrlAbstract/FREE Full Text
  18. 18.↵
    Lehman, N. L., R. Tibshirani, J. Y. Hsu, Y. Natkunam, B. T. Harris, R. B. West, M. A. Masek, K. Montgomery, M. van de Rijn, and P. K. Jackson. 2007. Oncogenic regulators and substrates of the anaphase promoting complex/cyclosome are frequently overexpressed in malignant tumors. Am. J. Pathol.170:1793-1805.
    OpenUrlCrossRefPubMedWeb of Science
  19. 19.↵
    Ley, T. J., E. R. Mardis, L. Ding, B. Fulton, M. D. McLellan, K. Chen, D. Dooling, B. H. Dunford-Shore, S. McGrath, M. Hickenbotham, L. Cook, R. Abbott, D. E. Larson, D. C. Koboldt, C. Pohl, S. Smith, A. Hawkins, S. Abbott, D. Locke, L. W. Hillier, T. Miner, L. Fulton, V. Magrini, T. Wylie, J. Glasscock, J. Conyers, N. Sander, X. Shi, J. R. Osborne, P. Minx, D. Gordon, A. Chinwalla, Y. Zhao, R. E. Ries, J. E. Payton, P. Westervelt, M. H. Tomasson, M. Watson, J. Baty, J. Ivanovich, S. Heath, W. D. Shannon, R. Nagarajan, M. J. Walter, D. C. Link, T. A. Graubert, J. F. DiPersio, and R. K. Wilson. 2008. DNA sequencing of a cytogenetically normal acute myeloid leukaemia genome. Nature456:66-72.
    OpenUrlCrossRefPubMedWeb of Science
  20. 20.↵
    Look, A. T. 2005. Molecular pathogenesis of MDS. Hematol. Am. Soc Hematol. Educ. Program2005:156-160.
    OpenUrl
  21. 21.↵
    Machida, Y. J., and A. Dutta. 2007. The APC/C inhibitor, Emi1, is essential for prevention of rereplication. Genes Dev.21:184-194.
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    Maciejewski, J. P., and C. Selleri. 2004. Evolution of clonal cytogenetic abnormalities in aplastic anemia. Leuk. Lymphoma45:433-440.
    OpenUrlCrossRefPubMedWeb of Science
  23. 23.↵
    Maser, R. S., B. Choudhury, P. J. Campbell, B. Feng, K. K. Wong, A. Protopopov, J. O'Neil, A. Gutierrez, E. Ivanova, I. Perna, E. Lin, V. Mani, S. Jiang, K. McNamara, S. Zaghlul, S. Edkins, C. Stevens, C. Brennan, E. S. Martin, R. Wiedemeyer, O. Kabbarah, C. Nogueira, G. Histen, J. Aster, M. Mansour, V. Duke, L. Foroni, A. K. Fielding, A. H. Goldstone, J. M. Rowe, Y. A. Wang, A. T. Look, M. R. Stratton, L. Chin, P. A. Futreal, and R. A. DePinho. 2007. Chromosomally unstable mouse tumours have genomic alterations similar to diverse human cancers. Nature447:966-971.
    OpenUrlCrossRefPubMed
  24. 24.↵
    Mikhailov, A., M. Shinohara, and C. L. Rieder. 2004. Topoisomerase II and histone deacetylase inhibitors delay the G2/M transition by triggering the p38 MAPK checkpoint pathway. J. Cell Biol.166:517-526.
    OpenUrlAbstract/FREE Full Text
  25. 25.↵
    Miller, J. J., M. K. Summers, D. V. Hansen, M. V. Nachury, N. L. Lehman, A. Loktev, and P. K. Jackson. 2006. Emi1 stably binds and inhibits the anaphase-promoting complex/cyclosome as a pseudosubstrate inhibitor. Genes Dev.20:2410-2420.
    OpenUrlAbstract/FREE Full Text
  26. 26.↵
    Mori, N., R. Morosetti, E. Hoflehner, M. Lubbert, H. Mizoguchi, and H. P. Koeffler. 2000. Allelic loss in the progression of myelodysplastic syndrome. Cancer Res.60:3039-3042.
    OpenUrlAbstract/FREE Full Text
  27. 27.↵
    Mullighan, C. G., L. A. Phillips, X. Su, J. Ma, C. B. Miller, S. A. Shurtleff, and J. R. Downing. 2008. Genomic analysis of the clonal origins of relapsed acute lymphoblastic leukemia. Science322:1377-1380.
    OpenUrlAbstract/FREE Full Text
  28. 28.↵
    Opavsky, R., S. Y. Tsai, M. Guimond, A. Arora, J. Opavska, B. Becknell, M. Kaufmann, N. A. Walton, J. A. Stephens, S. A. Fernandez, N. Muthusamy, D. W. Felsher, P. Porcu, M. A. Caligiuri, and G. Leone. 2007. Specific tumor suppressor function for E2F2 in Myc-induced T cell lymphomagenesis. Proc. Natl. Acad. Sci. USA104:15400-15405.
    OpenUrlAbstract/FREE Full Text
  29. 29.↵
    Paulsson, K., J. B. Cazier, F. Macdougall, J. Stevens, I. Stasevich, N. Vrcelj, T. Chaplin, D. M. Lillington, T. A. Lister, and B. D. Young. 2008. Microdeletions are a general feature of adult and adolescent acute lymphoblastic leukemia: unexpected similarities with pediatric disease. Proc. Natl. Acad. Sci. USA105:6708-6713.
    OpenUrlAbstract/FREE Full Text
  30. 30.↵
    Pinkel, D., R. Segraves, D. Sudar, S. Clark, I. Poole, D. Kowbel, C. Collins, W. L. Kuo, C. Chen, Y. Zhai, S. H. Dairkee, B. M. Ljung, J. W. Gray, and D. G. Albertson. 1998. High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays. Nat. Genet.20:207-211.
    OpenUrlCrossRefPubMedWeb of Science
  31. 31.↵
    Reimann, J. D., E. Freed, J. Y. Hsu, E. R. Kramer, J. M. Peters, and P. K. Jackson. 2001. Emi1 is a mitotic regulator that interacts with Cdc20 and inhibits the anaphase promoting complex. Cell105:645-655.
    OpenUrlCrossRefPubMedWeb of Science
  32. 32.↵
    Rhodes, J., A. Hagen, K. Hsu, M. Deng, T. X. Liu, A. T. Look, and J. P. Kanki. 2005. Interplay of pu.1 and gata1 determines myelo-erythroid progenitor cell fate in zebrafish. Dev. Cell8:97-108.
    OpenUrlCrossRefPubMedWeb of Science
  33. 33.↵
    Shepard, J. L., J. F. Amatruda, D. Finkelstein, J. Ziai, K. R. Finley, H. M. Stern, K. Chiang, C. Hersey, B. Barut, J. L. Freeman, C. Lee, J. N. Glickman, J. L. Kutok, J. C. Aster, and L. I. Zon. 2007. A mutation in separase causes genome instability and increased susceptibility to epithelial cancer. Genes Dev.21:55-59.
    OpenUrlAbstract/FREE Full Text
  34. 34.↵
    Sidi, S., T. Sanda, R. D. Kennedy, A. T. Hagen, C. A. Jette, R. Hoffmans, J. Pascual, S. Imamura, S. Kishi, J. F. Amatruda, J. P. Kanki, D. R. Green, A. A. D'Andrea, and A. T. Look. 2008. Chk1 suppresses a caspase-2 apoptotic response to DNA damage that bypasses p53, Bcl-2, and caspase-3. Cell133:864-877.
    OpenUrlCrossRefPubMedWeb of Science
  35. 35.↵
    Skoufias, D. A., F. B. Lacroix, P. R. Andreassen, L. Wilson, and R. L. Margolis. 2004. Inhibition of DNA decatenation, but not DNA damage, arrests cells at metaphase. Mol. Cell15:977-990.
    OpenUrlCrossRefPubMedWeb of Science
  36. 36.↵
    Torres, E. M., T. Sokolsky, C. M. Tucker, L. Y. Chan, M. Boselli, M. J. Dunham, and A. Amon. 2007. Effects of aneuploidy on cellular physiology and cell division in haploid yeast. Science317:916-924.
    OpenUrlAbstract/FREE Full Text
  37. 37.↵
    Tosi, S., E. Ballabio, A. Teigler-Schlegel, J. Boultwood, J. Bruch, and J. Harbott. 2005. Characterization of 6q abnormalities in childhood acute myeloid leukemia and identification of a novel t(6;11)(q24.1;p15.5) resulting in a NUP98-C6orf80 fusion in a case of acute megakaryoblastic leukemia. Genes Chromosomes Cancer44:225-232.
    OpenUrlCrossRefPubMedWeb of Science
  38. 38.↵
    Toyoda, Y., and M. Yanagida. 2006. Coordinated requirements of human topo II and cohesin for metaphase centromere alignment under Mad2-dependent spindle checkpoint surveillance. Mol. Biol. Cell17:2287-2302.
    OpenUrlAbstract/FREE Full Text
  39. 39.↵
    Verschuren, E. W., K. H. Ban, M. A. Masek, N. L. Lehman, and P. K. Jackson. 2007. Loss of Emi1-dependent anaphase-promoting complex/cyclosome inhibition deregulates E2F target expression and elicits DNA damage-induced senescence. Mol. Cell. Biol.27:7955-7965.
    OpenUrlAbstract/FREE Full Text
  40. 40.↵
    Westerfield, M. 1995. The zebrafish book. University of Oregon Press, Eugene.
  41. 41.↵
    Williamson, E. A., K. K. Rasila, L. K. Corwin, J. Wray, B. D. Beck, V. Severns, C. Mobarak, S. H. Lee, J. A. Nickoloff, and R. Hromas. 2008. The SET and transposase domain protein Metnase enhances chromosome decatenation: regulation by automethylation. Nucleic Acids Res.36:5822-5831.
    OpenUrlCrossRefPubMedWeb of Science
  42. 42.↵
    Zhang, L., C. Kendrick, D. Julich, and S. A. Holley. 2008. Cell cycle progression is required for zebrafish somite morphogenesis but not segmentation clock function. Development135:2065-2070.
    OpenUrlAbstract/FREE Full Text
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Emi1 Maintains Genomic Integrity during Zebrafish Embryogenesis and Cooperates with p53 in Tumor Suppression
Jennifer Rhodes, Adam Amsterdam, Takaomi Sanda, Lisa A. Moreau, Keith McKenna, Stefan Heinrichs, Neil J. Ganem, Karen W. Ho, Donna S. Neuberg, Adam Johnston, Yebin Ahn, Jeffery L. Kutok, Robert Hromas, Justin Wray, Charles Lee, Carly Murphy, Ina Radtke, James R. Downing, Mark D. Fleming, Laura E. MacConaill, James F. Amatruda, Alejandro Gutierrez, Ilene Galinsky, Richard M. Stone, Eric A. Ross, David S. Pellman, John P. Kanki, A. Thomas Look
Molecular and Cellular Biology Oct 2009, 29 (21) 5911-5922; DOI: 10.1128/MCB.00558-09

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Emi1 Maintains Genomic Integrity during Zebrafish Embryogenesis and Cooperates with p53 in Tumor Suppression
Jennifer Rhodes, Adam Amsterdam, Takaomi Sanda, Lisa A. Moreau, Keith McKenna, Stefan Heinrichs, Neil J. Ganem, Karen W. Ho, Donna S. Neuberg, Adam Johnston, Yebin Ahn, Jeffery L. Kutok, Robert Hromas, Justin Wray, Charles Lee, Carly Murphy, Ina Radtke, James R. Downing, Mark D. Fleming, Laura E. MacConaill, James F. Amatruda, Alejandro Gutierrez, Ilene Galinsky, Richard M. Stone, Eric A. Ross, David S. Pellman, John P. Kanki, A. Thomas Look
Molecular and Cellular Biology Oct 2009, 29 (21) 5911-5922; DOI: 10.1128/MCB.00558-09
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KEYWORDS

Cell Cycle Proteins
Embryonic Development
Genome
Neoplasms
Tumor Suppressor Protein p53
zebrafish
Zebrafish Proteins

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