1. RESEARCH ARTICLE
Mouse ZAR1-Like (XM_359149) Colocalizes
With mRNA Processing Components and Its
Dominant-Negative Mutant Caused
Two-Cell-Stage Embryonic Arrest
Jianjun Hu,1,2
Fengchao Wang,2
Xiaoquan Zhu,2
Ye Yuan,2
Mingxiao Ding,1
* and Shaorong Gao2
*
Maternal effect genes and encoding proteins are necessary for nuclear reprogramming and zygotic genome
activation. However, the mechanisms that mediate these functions are largely unknown. Here we identified
XM_359149, a Zar1-like gene that is predominantly expressed in oocytes and zygotes, which we designated
Zar1-like (Zar1l). ZAR1L-EGFP formed multiple cytoplasmic foci in late two-cell-stage embryos. Expression
of the ZAR1L C-terminus induced two-cell-stage embryonic arrest, accompanied with abnormal methylation
of histone H3K4me2/3 and H3K9me2/3, and marked down-regulation of a group of chromatin modification
factors including Dppa2, Dppa4, and Piwil2. When ectopically expressed in somatic cells, ZAR1L colocalized
with P-body components including EIF2C1(AGO1), EIF2C2(AGO2), DDX6 and LSM14A, and germline-specific
chromatoid body components including PIWIL1, PIWIL2, and LIN28. ZAR1L colocalized with ZAR1 and
interacted with human LIN28. Our data suggest that ZAR1L and ZAR1 may comprise a novel family of proc-
essing-body/chromatoid-body components that potentially function as RNA regulators in early embryos. De-
velopmental Dynamics 239:407–424, 2010. VC 2009 Wiley-Liss, Inc.
Key words: mRNA processing body; two-cell block; Zar1l; Zar1; zygotic genome activation
Accepted 19 October 2009
INTRODUCTION
The maternal factors that accumulate
during oogenesis play pivotal roles in
nuclear reprogramming, zygotic ge-
nome activation, and preimplantation
embryonic development (Schultz, 1993;
Aoki et al., 1997; Latham, 1999;
Latham and Schultz, 2001; Ma et al.,
2001; Hamatani et al., 2004; Minami
et al., 2007; Stitzel and Seydoux,
2007). Oogenesis involves a number of
critical events because a growing
mouse oocyte is transcriptionally and
translationally active. A large number
of mRNAs are synthesized and stored
to support oocyte maturation and early
preimplantation embryogenesis and
ABBREVIATIONS AAs amino acids C-body chromatoid-body DAB 3,30
-Diaminobenzidine DAPI 40
-6-Diamidino-2-phenylindole EGFP
enhanced green fluorescent protein FBS fetal bovine serum H3K4me1/2/3 Mono-/Di-/Tri-methylated Histone H3 at Lysine 4 H3K9me2/3
Di/Tri- methylated Histone H3 at Lysine 9 HRP horseradish peroxidase mRNP mRNA ribonucleoprotein ORF open reading frame P-body
mRNA processing body PFA paraformaldehyde PHD plant homeodomain PHDL cells pancreatic PNA-HSA double-low cells Phospho-
Rpb1 phosphorylated Rpb1 at C-terminal Ser2/5 repeats RFP red fluorescent protein RT-PCR reverse transcription polymerase chain
reaction.
Additional Supporting Information may be found in the online version of this article.
1
The Department of Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing, People’s Republic of China
2
National Institute of Biological Sciences, Beijing, People’s Republic of China
Grant sponsor: National High Technology 863; Grant number: 2008AA022311; Grant sponsor: National Natural Science Foundation;
Grant number: 30670302.
*Correspondence to: Shaorong Gao, National Institute of Biological Sciences, NIBS, Beijing, 102206, P.R. China. E-mail:
gaoshaorong@nibs.ac.cn or Mingxiao Ding Department of Cell Biology and Genetics, College of Life Sciences, Peking
University, Beijing, 100871, P.R. China E-mail: dingmx01@pku.edu.cn
DOI 10.1002/dvdy.22170
Published online 11 December 2009 in Wiley InterScience (www.interscience.wiley.com).
DEVELOPMENTAL DYNAMICS 239:407–424, 2010
VC 2009 Wiley-Liss, Inc.
DevelopmentalDynamics

2. are not used for immediate translation
(Bachvarova, 1985; Wassarman and
Kinloch, 1992). Mature oocytes arrest
in metaphase during their second mei-
otic division (MII stage), which is asso-
ciated with transcriptional shut-down
and reduced translation. Fertilization
triggers the completion of meiosis,
which is followed by the formation of a
one-cell embryo (zygote) containing
haploid paternal and maternal pronu-
clei (Schultz, 1993; Aoki et al., 1997;
Latham, 1999; Latham and Schultz,
2001; Ma et al., 2001; Hamatani et al.,
2004; Minami et al., 2007; Stitzel and
Seydoux, 2007). Each pronucleus
undergoes DNA replication before
entering the first mitosis to produce a
two-cell embryo.
Global expression profiling revealed
distinct patterns of maternal RNA
degradation and zygotic genome acti-
vation, which includes three transient
waves of de novo transcription: (1) a
minor activation before cleavage
(minor ZGA), (2) a major activation at
the two-cell-stage (major ZGA), and
(3) a major activation preceding the
dynamic morphological and func-
tional changes that occur during the
transition from morula to blastocyst,
which is termed mid-preimplantation
gene activation (MGA) (Hamatani
et al., 2004). The major ZGA promotes
dramatic reprogramming of gene
expression, coupled with the genera-
tion of novel transcripts that are not
expressed in oocytes. Thus, the
genetic program governed by mater-
nal transcripts/proteins must be
switched to one dominated by tran-
scripts/proteins derived from the
newly formed zygotic genome (Schultz,
1993; Aoki et al., 1997; Latham, 1999;
Latham and Schultz, 2001; Ma et al.,
2001; Hamatani et al., 2004; Minami
et al., 2007; Stitzel and Seydoux,
2007).
A great amount of maternal effect
genes produce mRNAs or proteins
that accumulate in the egg during
oogenesis. Nevertheless, a limited
number of maternal-effect genes have
been identified in mice: Nlrp5 (Mater,
maternal antigen that embryos re-
quire) (Tong et al., 2000); Hsf1 (heat-
shock factor 1) (Christians et al.,
2000); Dnmt1 (DNA methyltransfer-
ase 1, oocyte isoform) (Howell et al.,
2001); Npm2 (nucleoplasmin 2)
(Burns et al., 2003; De La Fuente
et al., 2004); Dppa3 (Stella) (Payer
et al., 2003); Zar1 (zygotic arrest 1)
(Wu et al., 2003a); Cdh1 (E-cadherin)
(De Vries et al., 2004); Pms2 (Gurtu
et al., 2002); Ezh2 (enhancer of zeste
2) (Erhardt et al., 2003); Dnmt3a
(DNA methyltransferase 3A) (Kaneda
et al., 2004); Ube2a (HRA6A) (Roest
et al., 2004); and Smarca4 (Brg1)
(Bultman et al., 2006). Apart from
Smarca4, Cdh1, Pms2, Ezh2, Dnmt3a,
and Ube2a, all of these maternal-
effect genes are exclusively expressed
in oocytes. In addition, only Nlrp5
and Ube2a mutants have similar phe-
notypes (two-cell arrest) to that of
Smarca4 maternally depleted embryos.
The other mutants primarily arrest at
the one-cell stage (Npm2, Dppa3, Zar1,
Hsf1), later stages of preimplantation
(Dppa3, Pms2), or during post-implan-
tation (Dnmt3a, Dnmt1o) development.
Ezh2 mutant exhibits a postnatal phe-
notype. Cdh1 mutant appears pheno-
typically normal because of rescuing by
the wild-type paternal allele. Meiotic
maturation triggers the degradation of
maternal transcripts. About 90% of the
maternal mRNAs have been degraded
by the two-cell stage. However, the
mechanisms that regulate the transla-
tion and degradation of maternal tran-
scripts are largely unknown.
In the present study, we identified
XM_359149, a Zar1-like gene that is
predominantly expressed in oocytes
and early preimplantation embryos,
which we named Zar1-like (Zar1l). We
characterized its sub-cellular localiza-
tion and its effect on preimplantation
development. Our data showed that
ZAR1L formed cytoplasmic foci in late
two-cell-stage embryos. Its mutant
form ZAR1L Cter-Flag-EGFP induced
abnormal epigenetic modifications and
gene expression changes in late two-
cell-stage embryos, and finally caused
two-cell-stage arrest. When ectopically
expressed in somatic cells, ZAR1L colo-
calized with P-body components includ-
ing EIF2C1(AGO1), EIF2C2(AGO2),
DDX6, and LSM14A, and germline-
specific chromatoid body components
including PIWIL1, PIWIL2, and
LIN28. ZAR1L colocalized with ZAR1
and interacted with human LIN28.
Our data suggested that ZAR1L and
ZAR1 comprise a novel family of P-
body/C-body-like structure components
in late 2-cell embryos.
RESULTS
Zar1l Gene and Protein
Information and Expression
Pattern
The XM_359419 sequence (GeneID:
545824; LOCUS: XM_359419) was
identified in a search for genes
that are preferentially expressed in
oocytes and early embryos. Protein
blast analysis showed that the
XM_359419 ORF encodes for a ZAR1-
like protein, which we have named
ZAR1L. By genomic analysis and RT-
PCR analysis, we successfully cloned
the full-length ORF of Zar1l (Fig. 1A).
Its orthologs have been found in other
organisms, including humans, dogs,
cows, and rats. Mouse ZAR1L exhibits
greater homology with the predicted
human ZAR1L protein than with the
mouse ZAR1 protein (Fig. 1A and B).
RT-PCR analysis showed that mouse
Zar1l has two transcript isoforms and
is specifically expressed in adult ovar-
ian tissue (Fig. 2A). Moreover, it is
predominantly expressed in oocytes
and zygotes. Cloning and sequencing
of the full-length ORF showed that
one of the mouse Zar1l isoforms is
876 bp (encodes a 291 AA polypeptide)
and the other is 982 bp with out-of-
frame reading (Fig. 2A and data not
shown). RT-PCR analysis showed that
mouse Zar1 gene was also predomi-
nantly expressed in oocytes and
zygotes. In order to determine the
protein levels of ZAR1L, Western blot
was performed. The results showed
that mouse ZAR1L protein was pre-
dominantly expressed in oocytes and
zygotes, and was also maintained at a
certain level in 2-cell- and 4-cell-stage
embryos (Fig. 2B). Only the 876-bp
transcript isoform of Zar1l was char-
acterized in this study.
Sub-Cellular Localization of
Mouse ZAR1L Protein and Its
Mutants
In order to predict protein domain/
structure of ZAR1L, we performed
protein sequence analysis by using
the PreDom 2006.1 and Super-
family 1.73 protein domain prediction
programs, respectively. The PreDom
program analysis showed that mouse
ZAR1L protein might have three
functional domains, one N-terminal
408 HU ET AL.
DevelopmentalDynamics

3. domain (51–103 AAs), one C-terminal
domain (193–241 AAs), and one larger
domain (130–291 AAs, which might
contain the middle region and the C-
terminal region). The Superfamily
1.73 program analysis showed that
mouse ZAR1L protein might contain
one CSE2-like domain (159–190 AAs)
in the middle region, and one atypical
FYVE/PHD zinc finger domain (227–
280 AAs) in the C-terminal. Based on
these protein domain prediction
results, we supposed that mouse
ZAR1L protein should have three
fragments that each contained one
functional domain: a well-conserved
C-terminal region (191–291 AAs, con-
taining the FYVE/PHD zinc finger do-
main), a conserved N-terminal region
(1–111 AAs, containing one functional
unknown domain), and a relatively
conserved middle region (112–190
AAs, containing the CSE2-like do-
main). Various constructs were de-
signed to express wild-type ZAR1L
and dominant-negative mutants of
ZAR1L (Fig. 2C). To study the sub-cel-
lular localization of ZAR1L and its
mutant proteins, as well as their roles
in preimplantation development, in
vitro transcribed mRNAs were micro-
injected into the cytoplasm of GV
oocytes, MII oocytes (followed by
ICSI), and zygotes. The results
showed that the ZAR1L-EGFP signal
formed cloud-like structures in the
cytoplasm of some fully grown GV
oocytes (7.8%, 4/51, Fig. 2E) and
formed cytoplasmic foci in most
(94.9%, 111/117) of the late two-cell-
stage embryos (Fig. 2I). ZAR1L-EGFP
cytoplasmic foci could not be observed
in MI, MII oocytes, and zygotes (Fig.
2D, F–H). The C-terminus fragment,
however, showed similar cytoplasmic
foci localization (99.2%, 118/119; Fig.
2J) with the full-length ZAR1L pro-
tein. The N-terminus deleted form,
ZAR1L DN-EGFP, was observed to
form weak cytoplasmic foci in most of
the late 2-cell-stage embryos (81.5%,
66/81; Fig. 2K). The C249S/C254S-
Flag-EGFP point mutant form com-
pletely lost its capacity to form cyto-
plasmic foci (Fig. 2L). The full-length
ZAR1 predominantly localized to cyto-
plasmic foci in late 2-cell-stage
embryos (Fig. 2M).
ZAR1L C-Terminus
Expression Induced
Embryonic Arrest at the
Two-Cell Stage
In order to study the function of
ZAR1L and its mutants in preimplan-
tation embryonic development, the
mRNAs that encoded full-length and
mutant ZAR1L were microinjected
into zygotes. The results showed that
the zygotes injected with EGFP (Fig.
3A–D), ZAR1L-EGFP (Fig. 3E–H),
ZAR1L C249S/C254S-Flag-EGFP (Fig.
3Q–T), as well as the full-length
ZAR1-EGFP (Fig. 3U–X), could de-
velop to blastocyst stage in vitro.
Fig. 1. Sequence alignment of mouse ZAR1 and ZAR1L proteins, human ZAR1L, and mouse
ZAR1L proteins. A: Sequence alignment of mouse ZAR1 and ZAR1L proteins. The eight well-
conserved cysteines (with # above them) form an atypical plant homeodomain (PHD) zinc finger
domain. The N-terminal sequences exhibit low homology between mouse ZAR1 and ZAR1L pro-
tein (analyzed by DNAMAN). B: Sequence alignment of predicted human and mouse ZAR1L pro-
teins. Sequence analysis showed that the ZAR1L protein contains three domains/motifs: one N-
terminal function unknown domain, one middle CSE2-like domain, and one C-terminal atypical
FVYE/PHD zinc finger domain. The domains/motifs were analyzed by PreDom (2006.1) program
and Superfamily (1.73) program. Based on the domain/motif analysis and the BLASTP alignment
results, we designed ZAR1L mutants with domain/motif containing fragments. The ZAR1L-Cter
fragment contains the well-conserved atypical PHD zinc finger domain (191–291 AAs in mouse
ZAR1L, boxed). The ZAR1L-DN fragment contains the middle domain (112–190 AAs) and C-ter-
minal domain (191–291 AAs), but lacks the N-terminal domain (1–111 AAs, underlined).
ZAR1L REGULATES PREIMPLANTATION DEVELOPMENT 409
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4. Fig. 2. Expression pattern and sub-cellular localization of mouse Zar1l. A: The mouse Zar1l gene is predominantly expressed in the ovary,
oocytes, and zygotes (top). Its mRNA level dramatically decreased from 2-cell-stage embryos. Two transcript isoforms of Zar1l gene have been
found. The mouse Zar1 gene is also predominantly expressed in oocytes and zygotes and then dramatically decreased (bottom). B: Western blot
analysis of ZAR1L protein. ZAR1L is detectable from GV oocytes to 4-cell-stage embryos. C: Design of constructs. The expression constructs
were designed based on the predicted protein sequence, with different tags. D–I: Sub-cellular localization of ZAR1L-EGFP fused protein. ZAR1L-
EGFP localized to the perinucleus region in some full-grown GV oocytes (E, arrow) and formed cytoplasmic foci in most of the late two-cell-stage
embryos (I, arrows). It is distributed predominantly in the cytoplasm in small GV oocytes, MI and MII oocytes, and zygotes. The red signals in E–G
represent the F-actin signal labeled with Rhodamine. J: The ZAR1L-Cter-Flag-EGFP mutant predominantly localizes to the cytoplasm and forms
multiple cytoplasmic foci (arrow). K: The ZAR1LDN-EGFP mutant also forms cytoplasm foci, but with a relative lower level (arrows). L: The ZAR1L-
C292S/C295S-EGFP mutant completely loses the cytoplasmic foci capacity (arrow). M: The full-length ZAR1-EGFP also forms multiple cytoplas-
mic foci (arrows). Scale bars ¼ 20 mm.
DevelopmentalDynamics

5. Fig. 3. Mutant ZAR1L induced cell cycle arrest when injected into zygotes or one blastomere of the two-cell-stage embryos. A–D: The embryos
developed normally when EGFP was injected into the zygotes of the control group. E–H: ZAR1L-EGFP-injected embryos develop normally to the
blastocyst stage. I–P: Most of the ZAR1L zN-EGFP (I–L) and ZAR1L Cter-EGFP (M–P) -injected embryos arrested at a late 2-cell stage. Q–T: Most
of the ZAR1L-C292S/C295S-EGFP-injected embryos can develop to the blastocyst stage. U–X: The ZAR1-EGFP-injected embryos develop to the
blastocyst stage. Panels of each group show embryos at 36, 72, 96, and 120 hr after hCG treatment, respectively. Y, Z: The one blastomere-
injected embryos of the EGFP control group and ZAR1L Cter-EGFP group 120 hr after hCG injection. About half of the cells were EGFP-positive
in the control blastocyst (Y). The ZAR1L Cter-EGFP-injected blastomere arrests after one cell division (arrows) and the other non-injected blasto-
mere develops to form a small blastocyst (Z). The inserted small panel in Z shows the cell cycle of the ZAR1L Cter-EGFP injected blastomere is
delayed. Zygote mRNA injection was performed during 20–22 hr after hCG treatment. Samples were photographed at 48, 72, 96, and 120 hr after
hCG treatment. Scale bars ¼ (A–L, Q–X) 100 mm; (M–P) 150 mm; (Y, Z) 20 mm.
ZAR1L REGULATES PREIMPLANTATION DEVELOPMENT 411
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6. However, most of the zygotes injected
with ZAR1L DN-EGFP (80.2%, 89/
111; Fig. 3I–L) and ZAR1L Cter-Flag-
EGFP (96.9%, 156/161; Fig. 3M–P)
arrested at the two-cell stage. When
EGFP was injected into one blasto-
mere of a two-cell-stage embryo, the
injected blastomere was not affected
and developed to blastocyst normally
(Fig. 3Y). When ZAR1L Cter-Flag-
EGFP was injected into one blasto-
mere of a two-cell-stage embryo, the
injected blastomere arrested after one
cell division. The other non-injected
blastomere, however, grew to form a
small blastocyst (Fig. 3Z).
ZAR1L Cter-Flag-EGFP
Mutant Did Not Affect BrdU
Incorporation But Affected
BrUTP Incorporation
In order to investigate whether DNA
replication was affected by the ZAR1L
Cter-Flag-EGFP, BrdU incorporation
assay was performed after the first cell
cleavage. BrdU staining with anti-
BrdU antibody showed that the BrdU
incorporation in the ZAR1L Cter-Flag-
EGFP group was similar to the NLS-
EGFP control group (Fig. 4A and B).
Further, to investigate whether RNA
synthesis was affected by the ZAR1L
Cter-Flag-EGFP, BrUTP incorporation
assay was performed at late 2-cell
stage (major zygotic genome activa-
tion). BrUTP staining with the anti-
BrdU antibody from middle 2-cell stage
to late 2-cell stage showed that BrUTP
incorporation was moderately down-
regulated by ZAR1L Cter-Flag-EGFP
(Fig. 4I and J) at late 2-cell embryos.
Statistics analysis showed that BrUTP
incorporation levels in Zar1l Cter-
EGFP injected embryos were down-
regulated significantly from middle to
late 2-cell stages, as compared with the
control group (Supp. Fig. 1, which is
available online).
ZAR1L Cter-Flag-EGFP
Reduced H3K4me2 and
H3K4me3 Methylation Levels
and Active RNA Polymerase
II (phosphor-Rpb1) Level
Histones H3K4 and H3K9 methylation
play important roles in regulating ge-
nome structure and gene transcription.
In order to confirm and explain how
the BrUTP incorporation was affected
by the ZAR1L Cter-Flag-EGFP mu-
tant, the RNA transcription-related
histone H3K4 methyl-modifications
were determined by immunostaining.
The results showed that when ZAR1L
Cter-Flag-EGFP mutant was injected
into zygotes, H3K4me2 and H3K4me3
were down-regulated significantly
(Fig. 5C to F), while H3K4me1 was up-
regulated moderately (Fig. 5A and B).
It is interesting that in about half of
the late 2-cell embryos, the H3K4me2
and H3K4me3 level in one blastomere
usually down-regulated earlier/faster
than the other one (Fig. 5G and H).
Quantification and statistics analysis
showed that H3K4me1 levels were low
in 81.8% of the EGFP-injected late 2-
cell embryos. And H3K4me1 levels
were middle or high in 89.9% of the
Zar1l Cter-EGFP-injected late 2-cell
embryos (Fig. 5I). H3K4me2 and
H3K4me3 levels were high in 70.7 and
72.3% of the EGFP injected late 2-cell
embryos (Fig. 5J and K). However,
H3K4me2 and H3K4me3 levels were
high in only 18.2 and 14.1% of the
Zar1l Cter-EGFP injected late 2-cell
embryos (Fig. 5J and K).
Activated RNA polymerase II (phos-
phorylated Rpb1 at C-terminal Ser2/5
repeats, Phospho-Rpb1) was one of
the key factors that represented the
mRNA transcription/synthesis level.
In order to confirm the down-regula-
tion of RNA synthesis (as indicated by
BrUTP incorporation assay), we fur-
ther performed Phospho-Rpb1 immu-
nostaining. The results showed that
the Phospho-Rpb1 signal was also
down-regulated significantly (Fig. 5G
and H). Quantification and statistics
analysis showed that Phospho-Rpb1
levels were high in 82.3% of the
EGFP injected late 2-cell embryos.
However, Phospho-Rpb1 levels were
low in 67.3% of the Zar1l Cter-EGFP-
injected late 2-cell embryos (Fig. 5L).
ZAR1L Cter-Flag-EGFP
Significantly Down-Regulated
H3K9me2 But Up-Regulated
H3K9me3 in Late Two-Cell-
Stage Embryos
To investigate whether Histone H3K9
methylation levels changed according
to the phenotypes observed above,
we performed immunostaining of
H3K9me2 and H3K9me3 at late 2-
cell-stage embryos. The results dem-
onstrated that the H3K9me2 signal
was dramatically down-regulated (Fig.
6A–C) in most of the late 2-cell embryos
that were injected with ZAR1L Cter-
Flag-EGFP. The H3K9me3 signal,
however, was dramatically up-regu-
lated and formed a perinuclear ring
in the ZAR1L Cter-Flag-EGFP group
(Fig. 6D–F). The H3K9me3 signal
was dramatically down-regulated for
a short period of time in normal late
two-cell-stage embryos (Fig. 6D). Sta-
tistics analysis demonstrated that
about 77% of the EGFP control
group embryos showed a high level
of H3K9me2. In contrast, 74% of
the ZAR1L Cter-Flag-EGFP group
embryos showed very weak levels of
H3K9me2. Our results showed that
about 5% (3/57) of the late two-cell-
stage embryos in the control groups
exhibited strong staining of anti-
H3K9me3. However, more than 80%
(83.9 6 6.0%, n ¼ 4; 49/58) of the late
two-cell-stage embryos in the ZAR1L
Cter-Flag-EGFP group had strong
anti-H3K9me3 staining (Fig. 6E and
F). DAPI staining showed that
crescent-like nuclei were observed
in about a half of the ZAR1L Cter-
Flag-EGFP-injected two-cell-stage
embryos (Figs. 5B, D, 6D). The cres-
cent-like nucleus and H3K9me3
perinuclear ring had not been
observed in the control groups (Figs.
5A,C, 6A,D).
ZAR1L Colocalized With
P-Body Components and
Germline-Specific
Chromatoid Body
Components in Somatic Cells
In order to explore the functional
properties and molecular mechanisms
of ZAR1L protein, we over-expressed
ZAR1L-Flag-EGFP in 293T cells and
then performed co-immunoprecipita-
tion with the anti-Flag monoclonal
antibody. A set of widely expressed P-
body components was found in the
immunoprecipitates with ZAR1L-Flag-
EGFP, including EIF2C1, EIF2C2,
DDX6, and LSM14A. To test colocali-
zation between ZAR1L and the P-body
components, we co-expressed ZAR1L
412 HU ET AL.
DevelopmentalDynamics

7. Fig. 4. ZAR1L Cter-EGFP does not affect BrdU incorporation but affects BrUTP incorporation. A, B: BrdU staining of one typical NLS-EGFP-
injected 2-cell embryo (A) and ZAR1L Cter-EGFP-injected 2-cell embryos at 36 hr after hCG treatment. High BrdU staining signal was detected in
both groups (arrows). C–J: BrUTP staining of injected embryos from middle to late 2-cell stage. C, E, G, I: Water-injected control embryos. D, F,
H, J: ZAR1L Cter-EGFP-injected embryos. C–D, E and F, G and H, I and J show the embryos that were injected with BrUTP at 43, 44.5, 46, and
48 hr, respectively, after hCG treatment and collected 90 min after each injection. The BrUTP signals are significantly down-regulated as compared
with the control groups at late 2-cell stage (I and J, arrows). Scale bars ¼ 20 mm.
ZAR1L REGULATES PREIMPLANTATION DEVELOPMENT 413
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8. Fig. 5. ZAR1L Cter-EGFP induces down-regulation of H3K4 methyl-modification and the largest subunit of active RNA polymerase II (phosphor-
Rpb1). A, B: ZAR1L Cter-EGFP moderately induces up-regulation of H3K4me1 level in late two-cell-stage embryos (arrow). C–F: ZAR1L Cter-
EGFP induces significant down-regulation of H3K4me2 (C and D) and H3K4me3 (E and F) in late two-cell-stage embryos (arrows). G, H: ZAR1L
Cter-EGFP severely affects the largest subunit of active RNA polymerase II (phospho-Rpb1) (arrow). Scale bars ¼ 20 mm. I–K: Quantification and
statistics analysis of H3K4me1, H3K4me2, and H3K4me3. H3K4me1 levels were low in 81.8% of the EGFP-injected late 2-cell embryos and its
levels were middle or high in 89.9% of the Zar1l Cter-EGFP-injected late 2-cell embryos (I). H3K4me2 and H3K4me3 levels were high in 70.7 and
72.3% of the EGFP-injected late 2-cell embryos (J and K). However, H3K4me2 and H3K4me3 levels were high in only 18.2 and 14.1% of the
Zar1l Cter-EGFP-injected late 2-cell embryos (J and K). L: Quantification and statistics analysis of Phospho-Rpb1 levels. Phospho-Rpb1 levels
were high in 82.3% of the EGFP-injected late 2-cell embryos. However, its levels were low in 67.3% of the Zar1l Cter-EGFP-injected late 2-cell
embryos (L).
DevelopmentalDynamics

9. Fig. 6. ZAR1L Cter-EGFP down-regulates H3K9me2 but up-regulates H3K9me3 methylation level in late 2-cell embryos. A–C: H3k9me2 is down-regu-
lated significantly by ZAR1L Cter-EGFP mutant in 74.0% of the late 2-cell embryos (arrow). D–F: H3k9me3 is up-regulated significantly by ZAR1L Cter-
EGFP mutant in 83.8% of the late 2-cell embryos. Open arrow indicates the down-regulation of H3K9me3 in the control group (D). Solid arrow indicates
the high level and perinuclear ring formation of H3K9me3 signal (E). One typical crescent-like nucleus is shown (E, arrow). Scale bars ¼ 20 mm.
Fig. 7. ZAR1L colocalizes with P-body components and germline-specific chromatoid-body components in mouse pancreatic PHDL epithelial
cells. A, B: ZAR1L-EGFP colocalizes well with human EIF2C1-myc (A) and human EIF2C2-myc (B), respectively (arrows). C, D: ZAR1L-RFP coloc-
alizes well with DDX6-EGFP (C) and LSM14A-EGFP (D), respectively (arrows). E–G: ZAR1L colocalizes well with germline-specific mouse Piwil1-
Flag (E), Piwil2-Flag (F), and human LIN28-RFP (G), (arrows). H: ZAR1L-RFP colocalizes extensively with ZAR1-EGFP (arrows). Monoclonal anti-
body against Myc tag and Flag tag, and anti-mouse Alex-594 secondary antibody are used. Scale bars ¼ 10 mm.
DevelopmentalDynamics

10. and the P-body components transi-
ently in mouse pancreatic PNA-HSA
double-low (PHDL) cells and human
293T cells. The results showed that
ZAR1L colocalized well with EIF2C1,
EIF2C2, DDX6, and LSM14A in
mouse PHDL cells (Fig. 7A–D) and
human 293T cells (data not shown).
The chromatoid body has a structure
similar to that of the P-body in the
male germline. It was observed in cyto-
plasm as cytoplasmic foci. Because
ZAR1L was a female germline-specific
protein, we assessed whether ZAR1L
colocalized with germline-specific chro-
matoid body components. The results
showed that ZAR1L colocalized exten-
sively with the germline-specific
PIWIL1, PIWIL2, and LIN28 when
they were ectopically expressed in
PHDL cells (Fig. 7E–G) and 293T cells
(data not shown). In addition, ZAR1L
also colocalized extensively with the
germline-specific ZAR1.
Colocalization of ZAR1L,
ZAR1L C-ter-Flag-EGFP,
LIN28, LSM14A, and ZAR1 in
2-Cell Embryos
The protein sequence alignment analy-
sis suggested that mouse ZAR1L pro-
tein might have three functional
domains. In order to characterize
the function properties of different
domains, series mutants have been
designed to analyze their sub-cellular
localization and colocalization with
germline P-body/C-body components.
Our results showed that both ZAR1L-
EGFP and ZAR1L Cter-Flag-EGFP
colocalized extensively with LIN28-
RFP (Fig. 8A and B) and LSM14A-RFP
(Fig. 8C and D) in late 2-cell-stage
embryos. In order to characterize the
colocalization between ZAR1L C-termi-
nus and ZAR1L or ZAR1, we co-
expressed them in zygotes and ana-
lyzed them in late 2-cell embryos. We
found that both ZAR1L-RFP and
ZAR1-EGFP colocalized extensively
with the C-terminus of ZAR1L in
nearly all the 2-cell embryos in the
cytoplasmic foci region (Fig. 8E–H).
However, we also found that ZAR1L
Cter-Flag-EGFP and ZAR1L Cter-RFP
localized to the nucleus in about one
fourth of the late 2-cell embryos
(23.5%, 12/51 and 27.5%, 19/69, respec-
tively, Fig. 8F and H). Further experi-
ment showed that ZAR1L-RFP colocal-
ized extensively with ZAR1-EGFP (Fig.
8I) and ZAR1L Cter-RFP colocalized
well with ZAR1 Cter-EGFP (Fig. 8J).
Serial observation showed that the
ZAR1L C-terminus moved from the nu-
cleus to the cytoplasmic foci in middle
to late 2-cell-stage embryos (Fig. 4D–J,
left panels). The colocalization study of
the Zar1l, Zar1, and Zar1l C-terminus
demonstrated that the full-length Zar1
and Zar1l, as well as Zar1l, C-termi-
nus, could shuttle from the nucleus to
the cytoplasm. Simultaneously, we
found that co-expression of ZAR1L and
C-terminus of ZAR1L could partially
(48.2%, 41/85) rescue the 2-cell-block
phenotype that was induced by the C-
terminus of ZAR1L at 72 hr after hCG
injection (Fig. 8K and L). However,
these embryos still failed to develop to
the blastocyst stage at 120 hr after
hCG injection (Fig. 8M and N).
Mouse ZAR1L Protein
Interacts With Human LIN28
In order to identify some molecular
mechanisms of ZAR1L function, we
performed immunoprecipitation and
silver staining experiments to try to
find out some proteins that might
interact with mouse ZAR1L. Mass
spectrometry analysis revealed that
human LIN28 protein was one of the
ZAR1L immunoprecipitated compo-
nents when mouse ZAR1L and
human LIN28 were co-expressed in
293T cells. Moreover, ZAR1L protein
was rich in the 60-kD band that was
in the lysates generated from LIN28-
Flag-EGFP immunoprecipitation (with
anti-Flag monoclonal antibody) (Fig.
9A). Western blotting analysis further
supported the interaction between
mouse ZAR1L and human LIN28 (Fig.
9B). Further analysis showed that
both ZAR1L-RFP and ZAR1L Cter-
RFP colocalized extensively with
LIN28-EGFP in late two-cell-stage
embryos (Fig. 8A and B).
ZAR1L Cter-Flag-EGFP
Down-Regulated a Set of
Chromatin Modification-
Associated Genes in mRNA
Level
It was well known that P-body compo-
nents played important roles in regu-
lating post-transcriptional mRNA sta-
bility and translation. In order to
characterize whether the ZAR1L Cter-
Flag-EGFP mutant resulted in down-
regulation of some zygote and 2-cell
embryo highly expressed genes, we per-
formed RT-PCR analysis of a group of
selected genes. Our data showed that a
set of chromatin-modification genes,
including Dppa2, Dppa4, and Piwil2
(Mili), were dramatically down-regu-
lated by ZAR1L Cter-Flag-EGFP at an
mRNA level in late two-cell-stage
embryos (Fig. 10A). However, Cbx1,
Dppa3, Oct4, Gata6, and Tbpl1 genes
were not affected significantly (Fig.
10A). In order to confirm the effect of
ZAR1L Cter-Flag-EGFP expression on
DPPA2 in the protein level, we per-
formed immunostaining with DPPA2-
specific antibody. Our results showed
that DPPA2 was dramatically down-
regulated at protein levels in about
90% (90.8 6 2.2%, n ¼ 4; total 42/54) of
the late two-cell-stage embryos that
were injected with ZAR1L Cter-Flag-
EGFP (Fig. 10B–D). Only about 4% (2/
55) of the late two-cell-stage embryos
had a high staining of DPPA2 (Fig.
10D). In the EGFP control group, about
95% (94.7 6 3.8%, n ¼ 4; 50/53) of the
injected embryos had strong staining of
DPPA2 (Fig. 10D). To preliminarily test
whether DPPA2 had important roles
in preimplantation development, the
mRNA corresponding to the N-termi-
nus deleted DPPA2-DN-EGFP mutant
(Fig. 10E) was injected into zygotes.
The DPPA2-DN-EGFP mutant predom-
inantly localized in the nucleus and
induced two-cell-stage arrest (Fig. 10F
and G). To further investigate whether
DPPA2 was mainly responsible for the
ZAR1L Cter-Flag-EGFP-induced 2-cell
arrest phenotype, we performed a
DPPA2 rescue experiment. We found
that over-expression of DPPA2 could
not rescue the 2-cell arrest phenotype
that was induced by ZAR1L Cter-Flag-
EGFP (Fig. 10H and I). Simultane-
ously, we found that, if co-injected with
ZAR1L Cter-Flag-EGFP, ectopically
expressed DPPA2-RFP protein was
also down-regulated significantly at
late 2-cell stage (Fig. 10J and K).
DISCUSSION
Wu et al. (2003a) have reported that
Zar1 (À/À) mice are viable and grossly
normal, but the Zar1(À/À) females are
416 HU ET AL.
DevelopmentalDynamics

11. Fig. 8. Colocalization of ZAR1L, ZAR1L Cter mutant, LIN28, LSM14A, and ZAR1 in 2-cell embryos. A, B: Both ZAR1L-RFP (A) and ZAR1L Cter-
EGFP (B) colocalize extensively with LIN28-EGFP in late two-cell-stage embryos (arrows). C, D: Both ZAR1L-RFP (C) and ZAR1L Cter-EGFP (D)
colocalize extensively with LSM14A-EGFP in late two-cell-stage embryos (arrows). ZAR1L Cter-EGFP shows higher nucleus localization (open
arrows). E: ZAR1L-RFP colocalizes extensively with the ZAR1L Cter-Flag-EGFP in cytoplasmic foci. F: ZAR1L Cter-Flag-EGFP localizes to the nu-
cleus in 23.5% (12/51) of the late 2-cell embryos (open arrows). G: ZAR1-EGFP colocalizes well with ZAR1L Cter-RFP in cytoplasmic foci.
H: ZAR1L Cter-RFP localizes to the nucleus in 27.5% (19/69) of the late 2-cell embryos (open arrows). I, J: ZAR1L-RFP colocalizes extensively
with ZAR1-EGFP, and ZAR1L Cter-RFP colocalizes well with ZAR1 Cter-EGFP (Fig. 8J, arrows). Open arrows in D, F, and H indicate the relatively
high nuclear localization of ZAR1L C-terminus mutant. Scale bars ¼ 20 mm. K–N: Co-injection of Zar1l-EGFP and Zar1l Cter-EGFP could partially
rescue the 2-cell-block phenotype that was induced by Zar1l Cter-EGFP. At 72 hr after hCG injection, EGFP and Zar1l Cter-RFP co-injected
embryos were still blocked at the 2-cell stage (K, open arrows), while 48.2% (41/85) of Zar1l(full)-EGFP and Zar1l Cter-RFP co-injected embryos
were 3-cell or 4-cell embryos (L, solid arrows). At 120 hr after hCG injection, EGFP singly injected embryos developed to blastocyst stage (M).
However, 76.1% (64/84) of Zar1l(full)-EGFP and Zar1l Cter-RFP co-injected embryos developed beyond the 2-cell stage, but were still restrained
to 3-cell to 8-cell embryos (N, open arrows).
ZAR1L REGULATES PREIMPLANTATION DEVELOPMENT 417
DevelopmentalDynamics

12. infertile. They found that ovarian de-
velopment, oogenesis, and fertiliza-
tion are unimpaired in Zar1 (À/À)
mice. Most of the embryos generated
from Zar1(À/À) females, however,
arrest at the one-cell and two-cell
stages (Wu et al., 2003a). They
observed that pronuclei formation
and DNA replication occur, but the
maternal and paternal genomes
remain separate entities in arrested
zygotes. The mechanism by which
null Zar1 expression caused zygote
and two-cell-stage arrest
is unknown. In this study, we charac-
terized a Zar1-like gene,
XM_359149, which encoded a ZAR1-
like protein and we have designated
it as Zar1l. Zar1l ORF sequences
have a poor similarity to the Zar1
ORF. However, they exhibit a high
similarity in protein sequence in
their C-terminus. When our Zar1l
project was ongoing, Sangiorgio
et al. (2008) reported the expression
and preliminary sequence analysis of
Bovine ZAR1-like gene and its ortho-
logs in vertebrates. Unlike bovine
ZAR1-like gene, we found that
mouse Zar1-like gene was predomi-
nantly expressed in ovaries, oocytes,
and early preimplantation embryos.
The differences in expression pat-
terns suggest that ZAR1L may have
species-variant functions. The ZAR1-
like orthologous proteins are con-
served in their predicted N-termi-
nus, middle-region, and C-terminus
domains. The C-terminus zinc-fin-
ger-containing domains are well con-
served from zebrafish to human
(data not shown). These results sug-
gested that the mouse Zar1l gene
may play some important roles in
female germline and/or embryonic
development.
Mutant ZAR1L Induced
Cell Cycle Arrest in
Preimplantation Embryos
Protein sequence analysis suggested
that mouse ZAR1L might contain
three functional domains: an N-termi-
nus unknown domain, a CSE2-like
middle domain, and a C-terminus
atypical PHD zinc finger domain (Wu
et al., 2003b). Series mutations were
designed for functional analysis of
ZAR1L protein in preimplantation
embryos. Our data showed that both
the C-terminus ZAR1L (ZAR1L Cter-
Flag-EGFP) and the N-terminus
deleted ZAR1L (ZAR1L DN-EGFP)
caused two-cell-stage arrest. The
ZAR1L Cter-Flag-EGFP induced a
more severe phenotype. When ZAR1L
Cter-Flag-EGFP was injected into one
blastomere of the two-cell embryos, it
resulted in arrest of the injected blas-
tomere after one time cell division,
whereas the other blastomere grew to
form a small blastocyst. These data
suggested that mouse Zar1l might
play important roles in preimplanta-
tion development in mice.
Mutant ZAR1L Did Not Affect
DNA Replication But Affected
RNA Synthesis
To find out whether DNA replication
and/or RNA synthesis was affected by
the ZAR1L Cter-Flag-EGFP mutant,
we performed BrdU incorporation
assay (Vitale et al., 1989) and BrUTP
incorporation assay (Aoki et al.,
1997). Our results demonstrated that
BrdU incorporation was not affected,
but the BrUTP incorporation was sig-
nificantly down-regulated in late 2-
cell embryos. These data indicated
that RNA synthesis during late 2-cell
embryos was affected by ZAR1L Cter-
Flag-EGFP mutant. RNA synthesis is
one of the key events during major zy-
gotic gene activation. To further con-
firm the influence of ZAR1L Cter-
Flag-EGFP mutant on zygotic gene
activation, we detected the largest
active RNA polymerase II subunit,
Phospho-Rpb1(Ser2/5) (Svejstrup,
2002; Shilatifard et al., 2003). Dra-
matic down-regulation of phosphor-
Rpb1 indicated the RNA synthesis ac-
tivity was affected by ZAR1L Cter-
Flag-EGFP.
ZAR1L Cter-Flag-EGFP
Induced Abnormalities in
H3K4me1/2/3 and H3K9me2/3
Modifications in Two-Cell-
Stage Embryos
It was reported that Histone H3K4
methylation levels correlated with
the genome activation (Lepikhov
and Walter, 2004; Lepikhov et al.,
2008; VerMilyea et al., 2009), while
H3K9me3 methylation (Lachner and
Jenuwein, 2002; Horn and Peterson,
2006; Grewal and Jia, 2007) corre-
lated with genome inactivation. So we
further determined the H3K4me1/2/3
levels and H3K9me2/3 levels. Our
results showed that H3K4me2/3 was
significantly down-regulated, while
H3K4me1 was moderately up-regu-
lated. These data consisted with the
transcription activity as indicated
by a BrUTP incorporation test and
phospho-Rpb1 staining. H3K9me2/3
Fig. 9. Biochemical analysis of interaction between mouse ZAR1L and human LIN28 in 293T
cells. A: Silver staining of samples obtained by immunoprecipitation. Left: ZAR1L Cter-EGFP
immunoprecipitation and silver staining. Middle: ZAR1L-Flag-EGFP was co-transfected with
hLIN28-RFP into 293T cells and immunoprecipitation was performed with anti-Flag monoclonal
antibody. Right: ZAR1L-RFP was co-transfected with hLIN28-Flag-EGFP into 293T cells and
immunoprecipitation was performed with anti-Flag monoclonal antibody. Arrows indicate the
bands of ZAR1L (top) and human LIN28 (bottom). B: Western blotting analysis of samples
obtained by immunoprecipitation. ZAR1L-RFP and LIN28-Flag-EGFP are co-transfected into
293T cells. ZAR1L-RFP and LIN28-Flag-EGFP are used as bait proteins. Before elution, samples
are treated with or without RNaseA (20 mg/ml) for 30 min, respectively. Anti-ZAR1L polyclonal
antibody and anti-Flag monoclonal antibody have been used.
418 HU ET AL.
DevelopmentalDynamics

13. methylation staining results showed
that the H3K9me3 demethylation
during the late 2-cell stage was
severely affected by the ZAR1L Cter-
Flag-EGFP mutant and, correspond-
ingly, the H3K9me2 was at low levels.
The time point of down-regulation of
H3K9me3 at the late two-cell stage in
the EGFP control group is in accord-
ance with the wave of major zygotic
genome activation (embryonic gene
transcription) (Nakayama et al.,
2001; Lachner and Jenuwein, 2002;
Hamatani et al., 2004; Yeo et al.,
2005; Horn and Peterson, 2006;
Grewal and Jia, 2007). The perinu-
clear ring formation of the H3K9me3
signal, as well as the crescent-like nu-
cleus formation, indicated that the
nuclear morphology was changed by
the ZAR1L Cter-Flag-EGFP mutant,
and the chromatin might be in a het-
erochromatin state. These data are
also consistent with the results that
Fig. 10. ZAR1L Cter-EGFP induces dramatic down-regulation of a set of chromatin modification factors. A: RT-PCR analysis. ZAR1L Cter-EGFP
induces dramatic down-regulation of Dppa2, Dppa4, and Piwil2 at the mRNA level. B, C: Immunofluorescent staining. ZAR1L Cter-EGFP induces
dramatic down-regulation of DPPA2 at the protein level (arrow). B is the NLS-EGFP control group. C: The ZAR1L Cter-EGFP group. D: Statistical
analysis of the DPPA2 staining intensity (analyzed by Adobe Photoshop). The DPPA2 staining signal is dramatically down-regulated in 90.8% of
late two-cell-stage embryos in the ZAR1L Cter-EGFP group. E: The construct design for DPPA2 and DPPA2 DN-EGFP. The N-terminus SAP
domain is deleted and the nuclear localization signal remains intact in the mutant construct. F, G: Development of zygotes that were injected
with DPPA2 DN-EGFP, 60 hr after hCG injection. The N-terminus deleted DPPA2 localizes to the nucleus and induces arrest at the 2-cell stage.
H–K: Co-injection of ZAR1L Cter-EGFP and DPPA2-RFP into zygotes. H and J show DPPA2-RFP signals and the inset panels show the bright
field, at 42 and 72 hr after hCG treatment, respectively. Over-expression of DPPA2 does not rescue the 2-cell arrest phenotype that was induced
by ZAR1L Cter-EGFP. I and K show that DPPA2 protein is down-regulated dramatically in co-injected late 2-cell embryos (arrows). Some embryos
occasionally develop beyond the 2-cell stage (arrows in the inset bright field panel in I). Scale bars ¼ (B, C, J, and K) 20 mm; (F–I) 100 mm.
ZAR1L REGULATES PREIMPLANTATION DEVELOPMENT 419
DevelopmentalDynamics

14. significant down-regulation of BrUTP
incorporation and phosphor-Rpb1 lev-
els in the nucleus have been induced
by Zar1l Cter-EGFP.
ZAR1L Colocalized With the
P-body and C-body
Components in Both Somatic
Cells and Late 2-Cell-Stage
Embryos
Many maternal mRNAs accumulate
in growing oocytes and are stored in
MII oocytes. They are translationally
repressed until fertilization. Most of
the maternal mRNAs have been
degraded by the end of the two-cell
stage. Correspondingly, major zygotic
genome activation occurs at the late
two-cell stage. How the maternal
mRNAs are tightly controlled in
terms of their stability, translational
repression, and/or initiation, and deg-
radation is largely unknown. Recent
studies have revealed the existence of
specific mRNA processing bodies (P-
bodies) as multiple cytoplasmic foci in
somatic cells (Hannon, 2002; van Dijk
et al., 2002; Sheth and Parker, 2003;
Cougot et al., 2004; Brengues et al.,
2005; Liu et al., 2005). P-bodies con-
tain untranslated mRNAs and can
serve as sites of mRNA translational
repression and degradation. P-bodies
are highly dynamic structures, and
the components are altered depending
on the cell state. Many proteins have
been reported to be localized to P-
body structures (Hannon, 2002; van
Dijk et al., 2002; Sheth and Parker,
2003; Cougot et al., 2004; Brengues
et al., 2005; Fillman and Lykke-
Andersen, 2005; Liu et al., 2005; Yang
et al., 2006; Eulalio et al., 2007;
Parker and Sheth, 2007; Pressman
et al., 2007). In the male germline,
chromatoid-body (C-body) structures
have been found to be similar to the
P-body structures (Matsumoto et al.,
2005; Kotaja et al., 2006; Kotaja and
Sassone-Corsi, 2007).
The male germline-specific cytoplas-
mic foci (chromatoid body) share com-
ponents found in somatic cell P-bodies,
such as the Agonaute proteins, some of
the RNA enzymes, and ribosomal pro-
teins. We found that the maternal
effect gene Zar1l encoded a female
germline-specific protein ZAR1L,
which localized to the cytoplasmic
foci structures in late two-cell-stage
embryos. Based on the knowledge that
mRNA processing mechanisms are
evolutionarily conserved from germ
cells to somatic cells, we speculated
that ZAR1L might be involved in
P-body- or C-body-like structures.
Indeed, we found that mouse ZAR1L
was colocalized extensively with widely
expressed P-body components, includ-
ing EIF2C1, EIF2C2, DDX6, and
LSM14A and with germline-specific
chromatoid-body components including
PIWIL1, PIWIL2, and LIN28 in so-
matic cells. We also confirmed that
mouse ZAR1L colocalized extensively
with LIN28 and LSM14A in late 2-cell
embryos. Our data further indicated
co-expression of ZAR1L could partially
rescue the 2-cell-block phenotype that
was caused by ZAR1L C-terminus. The
colocalization study of the Zar1l, Zar1,
and Zar1l C-terminus demonstrated
that the full-length Zar1 and Zar1l, as
well as Zar1l C-terminus, could shuttle
from the nucleus to the cytoplasm.
These results indicate that the C-ter-
minus of ZAR1L may induce 2-cell
blocks through a dominant-negative
effect. Because of the lack of commer-
cialized ZAR1L antibody, we custom-
ized peptide antibody against mouse
ZAR1L. It works well for Western blot-
ting but not for immunostaining. We
confirmed the interaction between
mouse ZAR1L and human LIN28 in so-
matic cells using immunoprecipitation
and Western blotting. Our results also
showed that ZAR1L extensively colo-
calized with ZAR1. These data demon-
strated that ZAR1L, as well as ZAR1,
may play some roles in P-body and/or
C-body structures and might have
functions in regulating oocyte-to-
embryo transition.
The ZAR1L Cter-EGFP Down-
Regulated a Set of Chromatin
Modification Factors,
Including Dppa2, Dppa4, and
Piwil2 at the mRNA Level
Nuclear reprogramming is a critical
event that occurs during zygotic ge-
nome activation (Schultz, 1993; Aoki
et al., 1997; Latham, 1999; Latham
and Schultz, 2001; Ma et al., 2001;
Hamatani et al., 2004; Minami et al.,
2007; Stitzel and Seydoux, 2007),
through which the transcriptionally
inactive genome changes into an active
genome. Several maternal factors are
associated with nuclear reprogram-
ming. Depletion of Smarca4 (Brg1), a
chromatin-remodeling factor, caused
arrest at the two-cell stage, which was
accompanied by down-regulation of a
multitude of mRNAs (Bultman et al.,
2006). Dppa3(Stella) is required for
protection of the maternal genome
from DNA-demethylation during early
embryonic development (Nakamura
et al., 2007). Similar to DPPA3, DPPA2
and DPPA4 have one DNA-binding
SAP domain and one uncharacterized
C-terminal domain, and are associated
with chromatin (Aravind and Koonin,
2000; Maldonado-Saldivia et al., 2007;
Masaki et al., 2007). Recent studies
have indicated that mouse DPPA4 pro-
tein associated with transcriptionally
active chromatin in ES cells (Masaki
et al., 2007). Comprehensive ChIP-
on-chip analysis demonstrated that
POU5F1 (OCT-3/4), SOX-2, and
NANOG each bind to the Dppa4 pro-
moter region in human ES cells (Boyer
et al., 2005). However, the roles of
DPPA2 and DPPA4 in early embryonic
development remain largely unknown.
Our results showed that Dppa2 and
Dppa4 were dramatically down-regu-
lated by the mutant ZAR1L Cter-
EGFP in late two-cell-stage embryos.
In order to test whether DPPA2 pro-
tein plays important roles in preim-
plantation development, we designed a
dominant-negative mutant of mouse
DPPA2 and injected it to the zygotes.
Our data showed that deletion of the
N-terminal SAP domain of mouse
DPPA2 caused arrest at the two-cell
stage in vitro. Our data suggested that
DPPA2 may play an important role in
embryonic development. We tried to
rescue the 2-cell arrest phenotype
through over-expression of DPPA2 but
failed. These data indicated that
DPPA2 is one of the important but not
the dominant factors affected by
ZAR1L Cter-Flag-EGFP.
Piwi family members regulate chro-
matin structure, transposon control,
mRNA transcription and translation,
and mRNA degradation through
interactions with piRNAs and associ-
ated complexes (Kuramochi-Miya-
gawa et al., 2004; Parker et al., 2004;
Kavi et al., 2006; Lau et al., 2006;
Aravin et al., 2007; Brower-Toland
et al., 2007; Carmell et al., 2007;
420 HU ET AL.
DevelopmentalDynamics

15. Hartig et al., 2007; Houwing et al.,
2007; Lin, 2007; Klattenhoff and The-
urkauf, 2008). Recently, piRNAs have
been isolated from murine mature
oocytes (Brennecke et al., 2008; Tam
et al., 2008; Watanabe et al., 2008).
Piwil2 (Mili), but not Piwil1 (Miwi),
or Piwil4 (Miwi2) is specifically
expressed in mature oocytes (Wata-
nabe et al., 2008). Piwi family pro-
teins and piRNAs play important
roles in chromatin modification and
genome stability (Aravin et al., 2007,
2008; O’Donnell and Boeke, 2007).
Our data showed that ZAR1L Cter-
EGFP dramatically induced down-
regulation of Piwil2 mRNAs in two-
cell embryos. Down-regulation of
Dppa2, Dppa4, and Piwil2 mRNA by
ZAR1L Cter-Flag-EGFP in vitro in
late two-cell-stage embryos indicated
that ZAR1L may correlate with a set
of mRNAs’ stability or degradation.
In summary, our data demon-
strated that ZAR1L plays important
roles in regulating oocyte-to-embryo
transition and preimplantation devel-
opment. The ZAR1L Cter-Flag-EGFP
mutant induced epigenetic abnormal-
ities and down-regulation of a group
of chromatin modification factors in
late two-cell-stage embryos and
finally caused arrest at the two-cell
stage. ZAR1L colocalized with multi-
ple mRNA chromatoid-body/process-
ing-body components in somatic cells
and late two-cell-stage embryos, and
it interacted with LIN28. ZAR1L
could be the first tissue- and stage-
specific chromatoid-body/processing-
body component that has been identi-
fied in 2-cell-stage mouse embryos.
Zar1l knockout mice will be gener-
ated to analyze the functional role of
ZAR1L in the female germline and
during embryonic development. The
biochemical nature of the domains of
ZAR1L, as well as ZAR1, remains to
be further characterized.
EXPERIMENTAL
PROCEDURES
Animals, Collection of
Oocytes and Embryos
B6D2F1 (C57BL/6JxDBA2) female
mice (8–10 weeks old) were used for
the collection of fully grown germinal
vesicles (GV) and MII oocytes. GV
oocytes were collected according to a
previous study (Wang et al., 2008).
Zygotes were collected from success-
fully mated B6D2F1 females. All
studies adhered to procedures consist-
ent with the National Institute of Bio-
logical Sciences Guide for the care
and use of laboratory animals.
In Vitro Transcription,
Microinjection, and
Preimplantation Embryo
Incubation
The predicted Zar1l ORF was cloned
into pBS-RN3 (Lemaire et al., 1995),
a modified in vitro transcription vec-
tor, in which the wild type and mu-
tant ZAR1L were expressed with Flag
and/or EGFP as fused proteins. In
brief, zygotes were collected from
adult male B6D2F1 mice at 18 hr af-
ter hCG treatment. mRNA injection
was performed during 20–22 hr after
hCG treatment. The surviving zygotes
were cultured in KSOM medium.
Samples were photographed at 48, 72,
96, and 120 hr after hCG treatment
for developmental recording. Late 2-
cell embryos were collected during 48–
50 hr after hCG treatment. Capped
RNAs were transcribed under the con-
trol of a T3 promoter with mMessage
mMachine (Ambion, Austin, TX), as
the protocol dictated. In vitro tran-
scribed mRNAs were injected into GV,
MII oocytes, and zygotes using a
PIEZO micro-injector.
Cell Culture
293T cells were cultured in DMEM-
based medium, which contained 10%
FBS (Hyclone, Logan, UT), 2 mM glu-
tamine, 1Â nucleosides (Gibco, Gai-
thersburg, MD), 1Â nonessential
amino acids (Gibco), 1Â beta-mercap-
toethanol (Gibco), 2 mM glutamine
(Gibco), 100 IU/ml penicilLin, and 100
mg/ml streptomycin (Gibco). Pancre-
atic PNA-HSA double-low cells were
incubated under similar conditions as
described above, with 3% FBS added.
Construction of Transient
Expression Vectors and
Transfection
The mouse Zar1l ORF was obtained
by performing RT-PCR from adult
ovarian tissue. The sequences were
confirmed by sequencing. The DNA
sequences of Zar1l ORF reported in
this study have been deposited in the
Genbank database (www.ncbi.nlm.nih.
gov/Genbank, accession no. FJ858201).
The ORF and mutants were cloned into
the pEGFP-N1 vector for transient
expression. Eukaryotic expression vec-
tors inserted with human EIF2C1,
human EIF2C2, mouse Ddx6, mouse
Lsm14a, mouse Piwil1 (Miwi), mouse
Piwil2 (Mili), and human LIN28 were
co-transfected with Zar1l into PHDL
cells or 293T cells to analyze their coloc-
alization with mouse ZAR1L. Vigofect
reagent was used according to the man-
ufacturer’s protocol (Vigorous). Cells
were collected 24 or 36 hr after trans-
fection. Please see Supp. Table S1 to
view the primer sequences used.
RT-PCR
Total RNA samples were prepared
from adult ovary, testis, liver,
spleen, oocytes, and preimplantation
embryos. The RNA was extracted
using conventional methods for adult
tissues. The PicoPure RNA isolation
kit (Arcturus) was used to extract
RNA from collected oocytes and
preimplantation embryos. Reverse
transcription and PCR were per-
formed by conventional methods
using MMLV reverse transcriptase
(Promega, Madison, WI). Genomic
DNA was extracted by conventional
methods. The RNA was reverse-tran-
scribed by MMLV reverse transcrip-
tase (Promega) and amplified by PCR
for 25 or 30 cycles. Primers were
selected that encompassed the in-
tronic sequences. PCR cycling was
performed at 98
C for 2 min followed
by 98
C for 15 sec, 54–60
C for 15 sec,
72
C for 40 sec, and finally 72
C for 8
min, using the PrimeSTAR HS DNA
polymerase (Takara). Please see
Supp. Table S2 to view the primer
sequences.
Immunofluorescent Staining
and Confocal Microscopy
Conventional immunostaining were
performed for H3K4me1, H3K4me2,
H3K4me3, H3K9me2, H3K9me3,
Phospho-Rpb1, and Dppa2 antibodies.
In brief, samples were fixed by 4%
paraformeldehyde for 20 min. Then,
the samples were permeabilized with
0.5% triton X-100 and blocked with
ZAR1L REGULATES PREIMPLANTATION DEVELOPMENT 421
DevelopmentalDynamics

16. 5% normal horse serum for 2 hr.
The primary antibodies were incu-
bated overnight at 4
C. The antibod-
ies against H3K4me1, H3K4me2,
H3K4me3, H3K9me2, H3K9me3 (all
from Upstate, Billerica, MA), and
Dppa2 (gifted by Dr. Western at the
ARC Centre, Australia) were diluted
at 1:300. Phospho-Rpb1 antibody
(Cell Signaling Technology, Danvers,
MA) was diluted at 1:100. For BrdU
staining, BrdU was added to the me-
dium at 28 hr after hCG treatment.
The BrdU-labeled samples were col-
lected 36 hr after hCG treatment.
Samples were treated with 2 M HCl
for 10 min after permeabilization and
washing. Then, samples were washed
once with pH 8.0 Tris-HCl and then
with pH 7.4 PBS four times after HCl
treatment. Conventional methods
were used for the other staining steps.
For BrUTP staining, a similar
amount of 100 mM BrUTP (Sigma,
St. Louis, MO) was injected into
ZAR1L Cter-EGFP or EGFP pre-
injected (at zygote stage) 2-cell
embryos at 43, 44.5, 46, and 48 hr af-
ter hCG treatment. Samples were col-
lected 90 min after each injection of
BrUTP (Sigma). Both the incorpo-
rated BrdU and BrUTP were stained
with anti-BrdU monoclonal antibody.
The Alexa-594-conjugated secondary
antibodies were incubated for 1 hr at
room temperature. Samples were fur-
ther counterstained with 100 ng/ml of
DAPI. Images were obtained with an
Olympus IX 71 microscope equipped
with a CCD camera (DVC, Austin,
TX), or LSM510 Meta confocal micro-
scope (Zeiss, Oberkochen, Germany).
The staining intensity was analyzed
by Adobe Photoshop.
Immunoprecipitation and
Western Blotting
293T cells were transfected to transi-
ently express ZAR1L Cter-Flag-EGFP,
ZAR1L-Flag-EGFP, ZAR1L-RFP, hu-
man LIN28-Flag-EGFP, and LIN28-
RFP, either independently or in combi-
nation. Samples were collected 36 hr
after transfection. Immunoprecipita-
tions were performed according to the
manufacturer’s protocol (FLAGIPT-1;
Sigma) or with anti-ZAR1L polyclonal
antibody prepared using a specific
polypeptide (134-RRPQDGE DEES-
QEE-147). The final concentration of
the ZAR1L antibody was 1 mg/ml. Pro-
teins were separated in 10% SDS-poly-
acrylamide gels and transferred to
PVDF membrane. Immunoblotting
analysis was performed using anti-
Flag monoclonal and anti-ZAR1L poly-
clonal antibodies. Blots were detected
using ECL (Amersham Biosciences,
Pittsburgh, PA) according to the manu-
facturer’s protocol. Blots were detected
using ECL (GE Healthcare, London,
UK).
Silver Staining and Mass
Spectrometry Analysis
Proteins were separated in 10% SDS-
polyacrylamide gels. Silver staining
was performed according to the man-
ufacturer’s protocol (PROTSIL2-1KT,
Sigma). The bands of interest were
analyzed by a LTQ linear ion trap
mass spectrometer (Thermo Electron,
Waltham, MA).
ACKNOWLEDGMENTS
We greatly appreciate Dr. Gurdon at
the Gurdon Institute for providing the
pBS-RN3 vector. We thank Dr. West-
ern at the ARC Centre (Australia) for
providing the DPPA2 antibody. We
thank Dr. Satomi at Osaka University
for providing the Piwil1 and Piwil2
cDNA. We thank the lab members and
the two anonymous reviewers for help-
ful comments on the manuscript.
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