Rereplication in emi1-Deficient Zebrafish Embryos
Occurs through a Cdh1-Mediated Pathway
Mara E. Robu, Yong Zhang, Jennifer Rhodes*
Immune Cell Development and Host Defense Program, Fox Chase Cancer Center, Temple University Health System, Philadelphia, Pennsylvania, United States of America
Disruption of early mitotic inhibitor 1 (Emi1) interferes with normal cell cycle progression and results in early embryonic
lethality in vertebrates. During S and G2 phases the ubiquitin ligase complex APC/C is inhibited by Emi1 protein, thereby
enabling the accumulation of Cyclins A and B so they can regulate replication and promote the transition from G2 phase to
mitosis, respectively. Depletion of Emi1 prevents mitotic entry and causes rereplication and an increase in cell size. In this
study, we show that the developmental and cell cycle defects caused by inactivation of zebrafish emi1 are due to
inappropriate activation of APC/C through its cofactor Cdh1. Inhibiting/slowing progression into S-phase by depleting Cdt1,
an essential replication licensing factor, partially rescued emi1 deficiency-induced rereplication and the increased cell size.
The cell size effect was enhanced by co-depletion of cell survival regulator p53. These data suggest that the increased size of
emi1-deficient cells is either directly or indirectly caused by the rereplication defects. Moreover, enforced expression of
Cyclin A partially ablated the rereplicating population in emi1-deficient zebrafish embryos, consistent with the role of Cyclin
A in origin licensing. Forced expression of Cyclin B partially restored the G1 population, in agreement with the established
role of Cyclin B in mitotic progression and exit. However, expression of Cyclin B also partially inhibited rereplication in emi1-
deficient embryos, suggesting a role for Cyclin B in regulating replication in this cellular context. As Cyclin A and B are
substrates for APC/C-Cdh1 - mediated degradation, and Cdt1 is under control of Cyclin A, these data indicate that emi1
deficiency-induced defects in vivo are due to the dysregulation of an APC/C-Cdh1 molecular axis.
Citation: Robu ME, Zhang Y, Rhodes J (2012) Rereplication in emi1-Deficient Zebrafish Embryos Occurs through a Cdh1-Mediated Pathway. PLoS ONE 7(10):
Editor: Deanna M. Koepp, University of Minnesota, United States of America
Received July 16, 2012; Accepted September 13, 2012; Published October 17, 2012
Copyright: ? 2012 Robu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by a William J. Avery postdoctoral fellowship to MER (http://www.fccc.edu/research/postdoc/fellowships/index.html).
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
Early mitotic inhibitor 1 (Emi1) is a cell cycle regulator that is
essential for proper progression through cell cycle . EMI1 is
regulated in a cell cycle-dependent manner, wherein EMI1 gene
transcription is activated upon entry into S-phase by E2F2 and the
protein is phosphorylated and degraded early in mitosis
[1,2,3,4,5]. As such, EMI1 is present in Ki-67-positive proliferat-
ing cells in a variety of adult murine tissues [6,7]. Studies of the
mammalian and Xenopus homologues of EMI1 have shown that it
inhibits the Anaphase-Promoting Complex/Cyclosome (APC/C),
an ubiquitin ligase complex that targets cell cycle regulated
proteins such as the S- and G2-phase Cyclins A and B, Securin
and Geminin [1,2,8,9]. Thus, the release of APC/C from EMI1
inhibition during mitosis allows for the ubiquitination and
degradation of these key substrates and promotes progression
through mitosis [5,10].
EMI1 is essential to regulate progression through the cell cycle.
Depletion of EMI1 by siRNA knockdown in human cell lines or
immunodepletion in cycling Xenopus extracts results in the untimely
degradation of APC/C substrates, leading to a G2/M arrest and
inducing rereplication [1,8,11,12]. Analysis by microscopy showed
that knockdown of EMI1 in human cell lines prevented
chromosome condensation and nuclear membrane breakdown,
indicating that EMI1-deficient cells are blocked in G2 and do not
proceed into mitosis [1,11].
EMI1-deficient human cell lines and emi1-deficient zebrafish
embryos display an increase in nuclear and whole cell size
[11,12,13]. Flow cytometry analysis revealed that the EMI1
depletion-induced increase in ploidy in cells actively replicating
their DNA correlates with enlarged nuclei . However, it is not
clear whether the increase in cell and nuclear size is a consequence
of rereplication, prolonged cell cycle arrest or the misregulation of
a growth pathway in which the activity of Emi1 has not been
APC/C binds to the cofactor Cdc20 early in mitosis and
transitions to using the Cdh1 cofactor in late mitosis and through
G1 phase ; however, both Cdh1 and Cdc20 promote the
degradation of Cyclins A and B [15,16]. Rereplication in EMI1-
depleted human cell lines was partially inhibited by co-depletion of
APC/C cofactors CDH1, CDC20 or the addition of a non-
degradable form of Cyclin A [11,12]. Similarly, Di Fiore and Pines
examined the progression through a single cell division in
synchronized HeLa cells to show that cells depleted of both
EMI1 and CDH1 progressed through S and G2/M stages with
similar kinetics to control cells, while EMI1-deficient cells were
delayed in G2/M . Interestingly, the cell cycle distribution in
synchronously dividing EMI1-deficient HeLa cells was only
restored back to a wild-type distribution upon depletion of both
PLOS ONE | www.plosone.org1October 2012 | Volume 7 | Issue 10 | e47658
CDC20 and CDH1 , suggesting that in some contexts the
activity of CDC20 may contribute to EMI1 depletion-mediated
defects. It remains to be examined in a more complex biological
system whether Cdh1 and Cyclin A are the key components
regulating events downstream of Emi1 depletion, or if Cdc20 and
Cyclin B are also important contributors. However, these studies
are complicated by the essential nature of cell cycle regulation
during embryonic development.
Mutation in the Drosophila EMI1 homologue rca1 prevents
mitotic entry during early embryonic development and in the
imaginal disk . In vertebrates, Emi1 mutation results in very
early embryonic lethality in mice due to severe mitotic defects and
increased apoptosis prior to zygote implantation . Recent
studies using the zebrafish model system showed that mutation or
antisense morpholino-mediated knockdown of emi1 leads to defects
in morphogenesis and an inhibition of cell division [13,18,19].
However, emi1-deficient zebrafish embryos survive beyond the
stage when body patterning and many organ systems are
established [13,18,19], which is likely due to the rapid develop-
ment of the zebrafish larvae and maternal expression of emi1 .
Embryos homozygous for truncated mutant forms of emi1 (ti121,
ti245, x1) display a loss of phosphorylated-Histone H3 (pH3)-
positive mitotic cells during early gastrulation and have robust
morphological defects [18,19], whereas mutants harboring a
hypomorphic allele (hi2618) displayed less severe developmental
defects, retained pH3 positive cells through somitogenic stages, but
showed decreased numbers of hematopoietic cells and total DAPI-
stained nuclei in the trunk region . Interestingly, both severe
and hypomorphic mutations of emi1 lead to embryos with
increased BrdU incorporation at 24 hours post-fertilization (hpf),
suggesting that even a partial loss of emi1 causes defects in the
regulation of replication [13,18,19]. Rereplication is most likely
the cause of increased ploidy observed in zebrafish emi1 mutant
metaphases , consistent with the flow cytometric detection of
increased DNA content seen in zebrafish cells and human cell lines
depleted of EMI1 [11,12,13].
DNA content has been evaluated as a potential factor
contributing to cell size . Zebrafish embryos deficient in emi1
showed increased cell size by flow cytometry  and increased
nuclear size by DAPI staining [13,18], consistent with previous cell
line data [11,12]. The morphology and cell cycle defects of
emi1hi2648mutants were not altered by the absence of p53 activity
. In total, these data establish that zebrafish is an effective
model to examine the in vivo relationship between emi1 and
In this study, we dissected the genetic pathway through which
loss of emi1 exerts it effects in developing zebrafish embryos. First,
we determined the developmental age at which newly spliced
(predominantly zygotic) emi1 transcripts are required to prevent
cell cycle defects. We then dissected the phenotype of emi1
deficiency by manipulating the levels of factors hypothesized to
have aberrant activity in this cellular context. This analysis makes
three important findings. First, embryos deficient for emi1 can be
restored back to a wild-type phenotype by antisense morpholino
inhibition of the APC/C cofactor cdh1, suggesting that in vivo,
Cdh1-mediated degradation of substrates is responsible for the emi-
deficient phenotype. Second, partially inhibiting origin licensing
by cdt1 knockdown  ablated the rereplicating phenotype of
emi1-deficient cells and normalized the cell size in these embryos,
thereby linking these phenotypes. Lastly, enforced expression of
either Cyclin A or Cyclin B, could partially rescue the
rereplication defects in emi1-deficient embryos, supporting a less
well-known role of Cyclin B in regulating replication in vivo. Given
the role of Cdh1 in targeting Cyclins A and B for degradation ,
this study provides strong evidence that a Cdh1 axis is responsible
for the rereplication and increased cell size in emi1-deficient
Emi1-deficiency-induced Defects are Due to APC/C-Cdh1
To gain insights into the developmental stage at which
depletion of emi1 affects the cell cycle, we used morpholino
oligonucleotide to block emi1 splicing. Following fertilization of
the egg, zebrafish zygotic cells rapidly divide and do not have
gap phases in the cell cycle. Asynchronous cell proliferation
begins during the mid-blastula transition around 3 hpf. During
this period, the cell cycle lengthens and transcription is activated
. Emi1 is maternally expressed and, thus, we used the
morpholino to determine the age at which the embryo becomes
dependent on newly spliced emi1 transcripts and to define the
earliest emi1 depletion-induced defects in the cell cycle. The emi1
morpholino obstructs splicing as expected, although it does not
completely deplete wild-type emi1 transcripts (Fig. S1 A).
Propidium iodide-based analysis of the cell cycle over a
developmental time course showed indistinguishable distribu-
tions in 4 hpf cells from control-injected or emi1 morpholino-
injected (morphant) embryos (Fig. 1). However, by 7 hpf, the
emi1-depleted population showed slightly decreased number of
cells with 2 n content of DNA (G0/G1 cells) and an increased
numbers of cells with 4 n amount of DNA (G2/M cells), in
comparison to the control cell population. Between 10 and 12
hpf, during early somitogenesis, the defects in the emi1
morphant cell cycle become more severe, with a robust increase
in 4 n cells and the accumulation of cells with greater than 4 n
DNA content indicative of rereplication. This result is consistent
with our previous work showing that the hypomorphic emi1
allele hi2648 causes rereplication . The number of cells with
4 n and more than 4 n DNA content in emi1 morphant
embryos decreases over time, likely due to the increase in cell
death of cells undergoing rereplication . Therefore, we
chose to perform the next cell cycle experiments at 10 to 12 hpf
(4–5 somites) when we see the peak of cell cycle defects in emi1
We next examined whether the emi1 deficiency-induced
defects are due to the activity of APC/C. To inhibit APC/C
we used morpholinos to knockdown the APC/C cofactors cdc20
and cdh1. Knockdown of cdc20 was severely lethal to the
zebrafish embryos, alone or in combination with emi1 knock-
down (data not shown), consistent with cdc20 being an essential
gene in yeast [24,25] and mice . On the other hand, cdh1 is
not essential for embryogenesis  and knockdown of this gene
in zebrafish did not result in any overt morphological or cell
cycle defects (Fig. 2). Therefore we focused our analysis on the
described, at 24 hpf the morphological defects caused by emi1
depletion include small heads, with cell death in the head,
abnormal somite structure and ventral tail curvature. Using two
different doses of emi1 morpholino, we found that co-knockdown
of emi1 and cdh1 gave rise to fully or partially rescued embryos
based on visual assessment of embryonic morphology (Fig. 2 A).
Consistent with this finding, we injected control or cdh1
morpholinos into a clutch of embryos generated from breeding
emi1 heterozygous parents and then photographed and geno-
typed each individual embryo. All 20 of the homozygous
mutant embryos (out of a clutch of 77 embryos) exhibited a
wild-type appearance when injected with cdh1 morpholino.
Cdh1 Axis Regulates Defects Due to emi1 Deficiency
PLOS ONE | www.plosone.org2October 2012 | Volume 7 | Issue 10 | e47658
populations, was averaged for 3 independent experiments. There
was no rescue of increased cell size in emi1 morphants by co-
injection of either CYCLIN A-DB or CYCLIN B-DB in any of the
cell cycle phases.
12% polyacrylamide gels, transferred to nitrocellulose and
immunoblotted using a rabbit polyclonal antibody anti-zebrafish
pH2AX (generous gift of Dr. James Amatruda, University of
Texas Southwestern) and anti-actin (AC-40, Sigma; 1:2000
dilution). Detection was performed using horseradish-peroxidase-
conjugated secondary antibodies (Cell signaling; 1:1000 dilution)
and ECL using Immobilon Western Chemiluminescent HRP
Western blots. Samples were separated on 4–
We thank Dr. Greg Enders, Dr. Timothy Yen and Dr. Neil Beeharry for
their critical comments on this manuscript. We gratefully acknowledge the
assistance of Alison Bilbee and Bruce Young from the zebrafish core facility
for their help maintaining the fish, as well as the Flow Cytometry and DNA
Sequencing core facilities at the Fox Chase Cancer Center. We are very
grateful to Dr. James Amatruda (University of Texas Southwestern) for the
zebrafish anti-pH2AX antibody and to Dr. Timothy Yen (Fox Chase
Cancer Center) for the CYCLIN A and CYCLIN B constructs.
Conceived and designed the experiments: MER JR. Performed the
experiments: MER YZ. Analyzed the data: MER JR. Contributed
reagents/materials/analysis tools: MER JR. Wrote the paper: MER JR.
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