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

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 II␣-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 anaphasepromoting 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 G 1 /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 (emi1 m/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 mutage...

Ddx18 is essential for cell-cycle progression in zebrafish hematopoietic cells and is mutated in human AML

“…23,[27][28][29] Zebrafish embryos homozygous for the ddx18 hi1727 allele, which results from a viral insertion within the first intron of the ddx18 gene were identified as having a profound reduction in the number of cells expressing mpx at 27 hpf ( Figure 1A-E). Homozygous ddx18 hi1727/hi1727 embryos also showed reduced expression of band3 (slc4a1), a marker of erythroid cells, and the pan-leukocyte marker l-plastin ( Figure 1E, supplemental Figure 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article).…”

Progressive loss of RacGAP1/ ogre activity has sequential effects on cytokinesis and zebrafish development

“…Here we took advantage of a novel cell cycle mutant harpy (hrp; Fig. 6A), carrying a mutation in the gene emi1 (Kane et al, 1996b;Rhodes et al, 2009;Riley et al, 2010;Zhang et al, 2008). Upon running out of maternal product around the onset of gastrulation (6 hours), cells in the homozygous mutant bypass mitosis completely and instead undergo repeated rounds of DNA synthesis.…”

Zebrafish Models for Human Cancer

“…MPNSTs in these mutant lines typically arose in zebrafish older than 1 year. 5,9,85,92 Histologic features of these tumors are similar between mutant models. Neoplastic cells are typically spindloid to variably epithelioid with oval to tapered nuclei, and they are arranged in fascicles, stacks, and whorls (Fig.…”

“…A number of mutant zebrafish lines develop malignant spindle cell tumors that are histologically reminiscent of malignant peripheral nerve sheath tumors (MPNSTs). 5,9,26,85,92 Curiously, these cancers arise with greatest frequency in zebrafish lines that harbor mutations in tumor suppressor genes ( Table 7), suggesting that the cancer spectrum associated with these types of mutations may be more limited in zebrafish compared with humans. MPNSTs in these mutant lines typically arose in zebrafish older than 1 year.…”