The X-linked imprinted gene family Fthl17 shows
predominantly female expression following the
two-cell stage in mouse embryos
Shin Kobayashi1,2,*, Yoshitaka Fujihara2, Nathan Mise3, Kazuhiro Kaseda2, Kuniya Abe3,
Fumitoshi Ishino4and Masaru Okabe2
1Medical Top Track Program, Medical Research Institute, Tokyo Medical and Dental University, Tokyo,
2Research Institute for Microbial Diseases, Osaka University, Osaka,3Technology and Development Team
for Mammalian Cellular Dynamics, BioResource Center, RIKEN Tsukuba Institute, Tsukuba, Ibaraki and
4Department of Epigenetics, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan
Received October 27, 2009; Revised and Accepted February 8, 2010
Differences between male and female mammals are
initiated by embryonic differentiation of the gonad
into either a testis or an ovary. However, this may
not be the sole determinant. There are reports that
embryonic sex differentiation might precede and be
independent of gonadal differentiation, but there is
little molecular biological evidence for this. To test
for sex differences in early-stage embryos, we
newly developed non-invasive sexing methods for
patterns. From this screening, we found that the
Fthl17 (ferritin, heavy polypeptide-like 17) family of
genes was predominantly expressed in female
blastocysts. This comprises seven genes that
cluster on the X chromosome. Expression analysis
based on DNA polymorphisms revealed that these
genes are imprinted and expressed from the
paternal X chromosome as early as the two-cell
stage. Thus, by the time zygotic genome activation
starts there are already differences in gene expres-
sion between male and female mouse embryos. This
discovery will be important for the study of early sex
differentiation, as clearly these differences arise
before gonadal differentiation.
In eutherian mammals, gender is determined genetically at
the time of syngamy and females (XX) have twice as many
X chromosomes as males (XY). However, soon after
fertilization in females, one of the X chromosomes
which is derived from father becomes inactivated and,
after implantation, one of the X chromosomes becomes
inactivated randomly in the embryo proper. This equalizes
the dosage of X-linked genes between sexes (1–3). This is
called ‘X chromosome inactivation’ and demonstrates that
differences in sex chromosome constitution between sexes
start to be compensated prior to embryonic implantation.
Contrary to X inactivation, the presence of the Y chro-
mosome leads to fundamental differences between males
and females. To date, it has been understood that, after
implantation, expression of the Y-linked Sry gene deter-
mines the sex of the gonads (4) and that sex hormones
secreted from the differentiated gonads influence the
fetus and allow various sexual characteristics to become
However, there are some reports that suggest that this
differentiation of gonads is not the sole determinant of all
gender differences. For instance, in several mammalian
embryos prior to implantation (6). Moreover, preimplan-
tation male and female embryos show differences in
glucose metabolism and pentose phosphate pathway
activity (7,8) and female rat neurons harvested and
cultured prior to gonadal differentiation develop more
neurons (9). These early sex differences may have some
effects on sexual differentiation thereafter (10). In spite
of these observations, little molecular biological evidence
about early sex differences has been established so far.
In searching for genetic clues on the nature of sex dif-
ferentiation before gonadal differentiation, we compared
blastocysts. We have already developed a method to sex
blastocysts using a transgenic mouse line in which the X
chromosome is tagged with an enhanced green fluorescent
of maleand female
*To whom correspondence should be addressed. Tel:+81-3-5803-4864; Fax:+81-3-5803-4863; Email: firstname.lastname@example.org
Nucleic Acids Research, 2010, Vol. 38, No. 11 Published online 25 February 2010
? The Author(s) 2010. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
protein (EGFP) transgene (11–13). We then compared
gene-expression patterns between sexed blastocysts using
DNA microarrays. We have reported previously that two
Y-linked genes (Dby and Eif2s3y) are expressed specifi-
cally in male embryos, and that two X-linked genes (Xist
and Rhox5/Pem) are imprinted
predominantly in female blastocysts (14). These genes
were thought to be candidates possibly involved in sex
differentiation. In detail, Dby and Eif2s3y encode an
RNA helicase and a translation-initiation factor, respec-
tively, and are necessary for spermatogenesis, but there is
no report that they are involved in sex differentiation
(15,16). Another gene, Xist, is well known as a non-coding
RNA responsible for X chromosome inactivation that
compensates for dosage differences between sexes (17).
Rhox5 is a homeobox gene (18) and we expected that
Rhox5 would contribute to differentiation between male
and female embryos. However, targeted disruption was
shown to reduce sperm production, but no other
abnormalities have been reported from gene-inactivation
experiments (18,19). Thus, so far there is no gene posi-
tively identified to be involved in early sex differences
and later sex differentiation.
In previous reports (14), we showed that there are
sex-linked differences in gene expression at the blastocyst
stage. However, the arrays we used (Agilent Mouse
Development G4120A) mainly cover postimplantation
stages and do not identify all the known genes. We sus-
pected there might be undiscovered genes showing sex
differences. In this report, to carry out more comprehen-
sive gene-expression analysis, we used arrays capable of
analyzing all the known mouse genes and compared male
and female embryonic gene expression at the blastocyst
stage. From this screening, we found imprinted genes
involved in sex-linked differential expression and deter-
mined the time of onset of differences in the expression
of these genes.
MATERIALS AND METHODS
The handling and surgical manipulation of all experimen-
tal animals were carried out in accordance with the guide-
lines of the Committee on the Use of Live Animals in
Teaching and Research of Tokyo Medical and Dental
University. The B6C3F1 TgN (act EGFP) Osb CX-38
(G38) transgenic mouse strain described in our previous
paper (12) was used to distinguish between male and
Blastocyst collection and RNA extraction
B6C3F1 strain female mice at 8 weeks of age were
gonadotropin followed by 5IU of human chorionic
gonadotropin (hCG) 48h later and were mated with
XGFPY male mice. Four-cell stage embryos were collected
from the oviducts 55h after the hCG injection, placed in
potassium simplex optimization medium (KSOM) and
incubated in a humidified atmosphere of 5% carbon
dioxide (CO2) in air at 37?C for an additional 38h.
of pregnantmare serum
(EGFP-negative) and female (EGFP-positive) embryos
were separated by observing green fluorescence under a
dissecting microscope. To verify differential gene expres-
sion in non-transgenic embryos in vivo, wild-type C57BL/6
blastocysts were obtained from the uteri of superovulated
C57BL/6 females that had been mated with wild-type
C57BL/6 males 92h after hCG injection. Genomic DNA
and RNA were extracted from individual blastocysts.
Male and female blastocysts were pooled separately fol-
lowing the determination of sex by polymerase chain
reaction (PCR) using Ube1x primers as follows: 50-TGG
GCAGCCATCACATAATCCAGATG-30(20). For each
experiment, 30–50 wild-type blastocysts were sexed and
pooled samples were used for expression analysis.
Comparative expression analysis using DNA microarrays
Total RNAs were extracted from over 150 male or female
sexed blastocysts (as determined above). Half of the
isolated total RNA from each sex was labeled with Cy-3
and Cy-5 using a Low Input Fluorescent Linear
Amplification kit (Agilent) following the manufacturer’s
instructions. The Custom Oligo Microarray consisting of
two 22k slides (GEO accession numbers GPL8326 and
GPL8329) was used in this study. To improve accuracy,
samples were collected from three independent prepara-
tions and all experiments were run in triplicate. The
hybridization experiments were duplicated in a reciprocal
labeling manner to reduce dye-integration bias and six
hybridizations were carried out for the entire analysis.
Combining plural array results and statistical analyses
were carried out using Luminator software. The probe
sequence of this array set was designed based mainly on
the representative transcripts of the FANTOM2 database
expressed sequence tag data obtained from a cDNA
library of mouse primordial germ cells (4689 transcripts;
Abe et al., unpublished data) and manually selected
sequences of interest (357 transcripts). The sequences of
all probes were Basic Local Alignment Search Tool
(BLAST) searched against NCBI GenBank (Build 37)
and the top-hit accession number was considered as the
unique ID of the probe. Any alternative transcripts were
regarded as independent and the number of distinct tran-
scripts in our microarray set was then counted.
Reverse transcription PCR of candidate genes for
sexually differential expression
Reverse transcription was carried out with the pooled
total RNA extracted from 100 blastocysts. One percent
of the resulting cDNA samples was amplified by PCR.
Primer sets were as follows: 50-AAGTGTGACGTTGAC
for ?-actin, 50-CTCATCCTCATGTCTTCTCCG-30and
50-GATTCCAGATAGACAGGCTGG-30for Xist and
CTAGAGCCCTGGAG-30for Rhox5, see Supplementary
Table S1 for primers to amplify Fthl17 (ferritin, heavy
polypeptide-like 17) gene. All PCR reactions were
Nucleic Acids Research, 2010,Vol.38, No. 113673
replicated at least once. The same PCR conditions were
used to examine the wild-type blastocysts, except for the
amount of starting materials (30–50 blastocysts).
Verification of imprinting
Two reciprocal sets of F1 hybrid blastocysts, (C57BL/
6?JF1) F1 and (JF1?C57BL/6) F1, were produced by
in vitro fertilization. In each experiment at least 30
blastocysts were sexed by PCR as described above and
pooled according to sex. The pooled RNA was treated
with DNase to eliminate genomic DNA contamination,
and reverse transcription
Superscript III reverse transcriptase (Invitrogen). One-
third of the resulting cDNA samples derived from
pooled RNAs were amplified by PCR using the above
primers. For PCR amplification of each Fthl17 family
gene, r–Taq DNA polymerase (TOYOBO) that does not
have 30!50exonuclease activity was used. In the allelic
expression analysis of Fthl17-L6, pooled 30 blastocysts
were used for each reverse transcription (RT)–PCR
reaction, and the Sty I restriction enzyme was used for
detecting single nucleotide polymorphisms (SNPs). In
the RT–PCR experiments, no genomic DNA contamina-
tion was detected.
In situ hybridization
Whole mount in situ hybridization was performed as
described (21). The probes encompass the whole length
of the Fthl17 sequence (NM_031261).
described (22), with a smaller quantify of starting
samples (600 male blastocysts and 600 female blastocysts).
See Supplementary Table S2 for primers to amplify the
RESULTS AND DISCUSSION
To compare gene-expression patterns between male and
female blastocysts, we used a DNA microarray (22K-1
and 22K-2) that represents all known 41222 mouse
transcripts, whose sequences were mainly obtained from
the Riken FANTOM2 Database (see ‘Materials and
Methods’ section). Using six hybridization experiments
and statistical analysis, 413 transcripts were found to
be expressed to a higher degree in female blastocysts
a higher degree in males (P<0.001). Mapping these
transcripts revealed that many were concentrated on the
X chromosome (Figure 1A). Although most of the differ-
ences were less than 2-fold, there were a few differentially
expressed transcripts showing greater variations (Table 1
for the 22K-1 array and Table 2 for the 22K-2 array). The
candidate 19 probes contained the male-specific genes Dby
and Eif2s3y, and the female-predominant genes Xist and
Rhox5 that we reported previously, confirming the validity
of the experiments. We were interested in discovering
other differentially expressed genes and carried out
RT–PCR to confirm the expression levels of other candi-
date genes. The results showed that the Fthl17 gene was
expressed predominantly in female embryos (Figure 1B).
Furthermore, the real-time PCR analysis revealed that
in female embryos the Fthl17 expression level was 21
times greater than that seen in males (Figure 1C). The
same results were obtained with wild-type blastocysts
not expressing EGFP recovered from the uterus (Sup-
plementary Figure S1), ruling out the possibility that the
differential expression was caused by the artificial condi-
tions. Originally, Fthl17 was reported to be expressed spe-
cifically in spermatogonia (testicular stem cells) (23) as a
gene expected to be a metal ion-binding protein (data
from the NCBI database). In addition, we found here
that it was expressed at the blastocyst stage and showed
predominantly female expression.
Identification of the Fthl17 gene family
Next, to characterize this gene in more detail, we carried
out database searching in GenBank (Build 37.1) and
found that the Fthl17 gene (NM_031261) has seven
highly homologous nucleotide sequences covering approx-
imately 150kb on the X chromosome. Therefore, we
named these genes Fthl17-L1 through Fthl17-L6 and
(Supplementary Figure S2). The expression level of each
gene in this family was examined using specific RT–PCR
primers amplifying individual transcriptional units. All
seven genes were transcribed successfully and showed
predominantly female expression (Figure 1D).
Verification of imprinting of the Fthl17 gene family
How does this family of genes show predominant expres-
sion in females? Because both the X-linked genes Rhox5
and Xist are imprinted and expressed from the paternal X
chromosome, they show a female predominant expression.
Therefore, we examined whether the Fthl17 genes were
also imprinted. First, to distinguish paternal from
maternal expression, we searched for DNA polymor-
Japanese wild mouse, M. mus. molossinus. Southern
blot analysis revealed that this family of genes was
conserved in M. mus. molossinus (JF1 strain) as well as
in M. mus. musculus (B6, C3H and 129sv strains;
Supplementary Figure S3). Therefore, we tried to detect
the B6 and JF1 alleles by PCR using specific primers
for this gene family. However, the B6 allele was
detected, but the JF1 alleles were not, with the exception
of Fthl17-L6 (Figure 2A). This was probably caused by
DNA polymorphisms in
sequences. We then used the B6 allele to examine
whether these genes (Fthl17 and Fthl17-L1-L5) were
active when they were derived paternally. As shown in
Figure 2B, the active alleles of the Fthl17 genes were
examined in F1 blastocysts derived from mating between
C57BL/6 females and JF1 males (B6?JF1) and the
reciprocal cross of JF1 females with C57BL/6 males
the specificPCR primer
3674Nucleic Acids Research, 2010,Vol.38, No. 11
(JF1?B6). The RT–PCR products clearly showed that
these genes (Fthl17 and Fthl17-L1-L5) were active only
when they were derived from the males (Figure 2A).
Furthermore, we also examined allelic expression of
Fthl17-L6 gene using a SNP discovered between B6 and
JF1 alleles (Figure 2C). There was predominant expres-
sion from the paternal allele. All of these results indicate
thatthe Fthl17gene family
Expression of Fthl17 genes in blastocysts
To examine the relationship between Fthl17 expression
pattern and the pattern of X chromosome inactivation,
whole mount in situ hybridization of the Fthl17 gene
family was carried out on blastocysts. The genes were
barely detected in male embryos. However, in female
embryos, strong signals were detected in both the inner
cell mass (ICM) and trophectoderm (TE) with stronger
signals in the ICM than in the TE (Figure 2D). This is
Figure 1. Sex-linked differential gene expression at the blastocyst stage. (A) The chromosomal distribution of differentially expressed genes. Red and
blue bars show genes that were significantly upregulated in female and male embryos, respectively (P<0.001). Dark red and blue bars correspond to
the genes showing >2-fold changes. (B) RT–PCR analysis of the differentially expressed genes. Genes showing >2-fold expression differences were
selected and RT–PCR was carried out using female (GFP-positive) and male (GFP-negative) samples. (C) Real-time PCR measurements of Fthl17
gene expression in female and male samples. (D) RT–PCR analysis of each Fthl17 family gene using specific RT–PCR primers (see Supplementary
Figure S2 and Supplementary Table S1 for primer sequences).
Table 1. List of the differentially expressed genes according to embryo sex (22K-1)
Gene name Accession numberFold changeP Male intensityFemale intensity Map position
Not mapped uniquely
Not mapped uniquely
Nucleic Acids Research, 2010,Vol.38, No. 113675
Figure 2. Verification of imprinting in the Fthl17 gene family. (A) Genomic PCR and RT–PCR amplification for Fthl17. Left two samples: PCR
products of genomic DNA (B6 genome, JF1 genome). Right two samples: RT–PCR products of B6 (C57/BL/6) ?JF1 (JF1/Ms) F1 and JF1?B6 F1
blastocyst samples. (B) Scheme for verification of imprinting in the Fthl17 gene family using intersubspecific hybrid F1 mouse embryos. The active
alleles of the Fthl17 genes were examined in F1 blastocysts derived from mating between C57BL/6 females and JF1 males (B6?JF1) and the
reciprocal cross of JF1 females with C57/BL/6 males (JF1?B6). (C) Allelic expression analysis was carried out using a SNP in the Fthl17-L6 gene.
(D) Whole mount in situ hybridization in female and male blastocysts. ‘S’ indicates the sense strand probe and ‘A’ indicates the antisense strand
Table 2. List of the differentially expressed genes according to embryo sex (22K-2)
Gene name Accession number Fold changeP Male intensity Female intensityMap position
Not mapped uniquely
Not mapped uniquely
All genes showing >2-fold (200% P<0.001) change and <–2.0-fold (50%) change in expression level are listed. Some probes were redundant, but the
results of all probes are listed.
3676Nucleic Acids Research, 2010,Vol.38, No. 11
markedly different from the expression pattern of Xist,
which is a marker of X inactivation. As reported,
Xist was not expressed in the ICM but only in the TE.
We conclude that the Fthl17 family of genes was
mainly expressed from a reactivated paternal X chromo-
some (Xp) in the ICM and was slightly expressed from
TE, indicating escape from imprinted inactivation at this
Expression of Fthl17 genes before and after the
To determine the time of onset of differences in sex-linked
gene expression, we examined the expression levels of
Fthl17 genes before the blastocyst stage. Interestingly,
expression started at the two-cell stage, which corresponds
to the onset of zygotic genome activation (Figure 3A).
Furthermore, allelic expression analysis also revealed
that four highly expressed genes, Fthl17, Fthl17-L1, -L3
and -L5, were imprinted and expressed from the paternal
allele as early as the two-cell stage (Figure 3B). Because of
the low-level expression of Fthl17-L6, allelic expression
analysis using the SNP between B6 and JF1 could not
be done. However, the above results demonstrate that,
at least in M. mus. musculus (C57/BL6), the Fthl17
family of genes, exactly similar to Xist, is expressed from
inactivated Xp chromosomes during preimplantation
stages and thus comprise one of the earliest sets of
imprinted genes that is expressed. It is known that the
fertilized oocyte stores maternally derived mRNA and
that the zygotic genome remains largely inactive. After
fertilization, de novo transcription of embryonic genes
starts mainly after the two-cell stage. Our results clearly
demonstrate that epigenetic gene regulation controls the
differential expression of male and female embryos from
this very early stage of embryonic development. As for
postimplantation stages, we could not detect Fthl17
family gene expression in embryos at day 9.5 and 12.5,
so no information on the imprinting status was obtained
(data not shown).
Effect of X inactivation and genomic imprinting on
X-linked gene expression
We were also interested in the fact that the differentially
expressed 873 transcripts (P<0.001) were concentrated on
the X chromosome (Figure 1A). This suggests that at
preimplantation stages this chromosome is not completely
inactivated. To understand the positional effect of X inac-
tivation, we plotted the female versus male signal ratio of
1021 distinct X-linked transcripts as a fold change (FC) on
a physical map of the X chromosome (Figure 4). All 585
expressed transcripts mapped on chromosome X with a
signal intensity >500 (including genes showing no statis-
tically significant expression differences) could be divided
Figure 3. (A)
(unfertilized oocytes, two-cell, eight-cell, morula and blastocyst). The
RT–PCR primers for Fthl17 were designed to detect all genes in this
family. (B) Allelic expression analysis of the Fthl17 gene family (Fthl17,
Fthl17-L1, -L3 and -L5) at preimplantation stages (two-cell, eight-cell
Expressionof Fthl17 in preimplantationstages
Figure 4. Genomic imprinting of X-linked genes. Fold change (FC) values of transcripts mapped on the X chromosome. Changes in expression of
the transcripts mapped on the X chromosome are plotted along the physical map positions. A set of 586 transcripts showing significant expression
levels (greater than 500 units of signal intensity) was selected from 1021 distinct transcripts mapped uniquely on the X chromosome. Black spots
correspond to transcripts judged to be differentially expressed between male and female blastocysts at a statistically significant level (P<0.001) and
white spots correspond to others not showing statistically significant differences. Besides groups 1–3 (see ‘Results and Discussion’ section), there were
six transcripts showing FC values in the range –1.2 to –1.5, which could be explained by the margin of error in microarray analysis. Spot 1, Fthl17;
spot 2, Rhox5; spot 3, Xist; spot 4, Bex1.
Nucleic Acids Research, 2010,Vol.38, No. 11 3677
into three groups. Group 1 included 512 transcripts
showing no marked differences between male and female
embryos (?1.2<FC <+1.2) indicating that at the
blastocyst stage most, but not all, transcripts had under-
gone X chromosome inactivation. Group 2 contained 63
transcripts that showed greater expression in female than
in male embryos; however, their FC values showed
<2-fold difference (1.2<FC<2.0).
probably had not been inactivated at this stage. As far
as we know, only six mouse genes are reported to escape
X inactivation after implantation (24). Thus, inactivation
during preimplantation stages is not considered as strict as
at postimplantation stages, possibly because of the reacti-
vation of the X chromosome in the ICM or incomplete
inactivation in the TE. At the morula stage there is
reported to be a gradient of gene silencing, with the
lowest degree of silencing near the chromosome ends
and the highest degree of silencing near Xic (1).
However, in our experiments the blastocysts did not
show such a gradient. Instead, the silencing tended to
cover the entire chromosome. Our observation is consis-
tent with a recent report by Patrat et al. (25) on the
(Supplementary Figure S4). Finally, group 3 contained
four transcripts with >2-fold difference between male
and female embryos. This group of genes had not been
predominantly expressed in the female embryos. We
concentrated on these and identified three imprinted
genes: the Fthl17 group, Rhox5 and Xist. These data dem-
onstrate that genomic imprinting could be utilized for
marking differences in gene expression between sexes.
Genomic imprinting on X-linked genes
So far, six imprinted genes (our three identified genes and
Xlr-3b, -4b and -4c) have been identified on the X chro-
mosome (26,27). However, the mechanism of imprinting
on the X chromosome is unknown. For the autosomal
chromosomes, differentially methylated regions (DMRs)
have been discovered that control imprinted expression.
To investigate whether the Fthl17 family of genes is con-
trolled by DMRs, we searched for their sequences for
300kb around the Fthl17 gene. From this we selected
nine areas corresponding to CpG islands and CpG-rich
regions and carried out bisulfite sequencing using male
and female blastocyst genomic DNA samples. As far as
we examined, there was no DMR signal in the Fthl17
locus (Figure 5). However, a control DMR was detected
in the H19-imprinted region, indicating that the bisulfite
sequencing was working well (Supplementary Figure S5).
Although parental allele could not be distinguished using
SNPs, these results suggest that imprinting of the Fthl17
family of genes is maintained by a mechanism independent
from standard DNA methylation. In addition, it was
reported that Xist genes are also not differentially
methylated before implantation
embryos could be controlled in general by methylation-
independent mechanisms. In general, there are different
organizations of the chromatin of the two parental
genomes during the first cell divisions (29,30), but it is
not fully understood how long this differential chromatin
organization remains in later stages. It would be interest-
ing to examine the relationship of this differential
chromatin organization and imprinting on the X chromo-
some in preimplantation-stage
searching for DMRs would be necessary in the Fthl17
region, and the precise mechanisms of imprinting remain
to be elucidated.
The role of sex-linked differentially expressed genes
in preimplantation embryos
It appears likely that sex-linked differentially expressed
genes in preimplantation embryos serve as factors
producing sex differences, which are independent of later
gonadal differentiation. These factors could involve
between the sexes. Concerning the growth rate, it has
been hypothesized that the paternally derived X chromo-
some and/or genes expressed from both X chromosomes
retard development prior to gonadal sex differentiation
(31). Although the growth effects mediated by the X chro-
mosome appear to be complicated,
expressed Fthl17 family of genes (group 3 described
above) and X-linked genes that are not inactivated
(group 2) could serve as good candidates for the cause
of sex differences in the rate of growth. Furthermore,
it has been pointed out that these sex differences arising
prior to implantation might affect later development
and eventually influence the male-to-female ratio at
birth (10). Therefore, analyzing these early sex-linked dif-
ferences will help us to understand other aspects of early
embryonic development that may have effects at later
From our screening of genes that are differentially
embryos, we have identified three imprinted genes.
Rhox5, a homeobox gene, is predominantly expressed
from the eight-cell stage (14). In addition, Xist has been
reported to transcribe a non-coding RNA and to be
expressed from the two-cell stage (32). Here we identified
for the first time the Fthl17 family of genes having a metal
ion-binding motif, which is also expressed from the
two-cell stage. This confirms that there are already differ-
ences in gene expression between sexes at the time of
zygotic genome activation (Figure 6). Previously there
was scarcely any clue to the control of sex differentiation
prior to gonadal differentiation. These X-linked imprinted
genes—Fthl17 and Rhox5—offer clues to deciphering sex
differentiation controlled by epigenetic gene regulation.
Functional analysis of the Fthl17 family of genes using
gene-manipulated mice will provide a novel area of inves-
tigation on the onset of sexual differentiation before
3678 Nucleic Acids Research, 2010,Vol.38, No. 11
Figure 6. Summary of Fthl17 gene family expression in preimplantation stage embryos. The Fthl17 genes are imprinted and expressed from the
paternal X chromosome as early as the two-cell stage. A significant difference in gene expression between male and female embryos appears at the
time of zygotic genome activation (ZGA).
Figure 5. Methylation analysis of Fthl17 genomic sequences at the blastocyst stage. The upper panel indicates the position of CpG islands and a
CpG-rich region in sequences spanning 150kb either side of Fthl17 (NM_031261). The lower panel indicates the results of bisulfite sequence analysis
of male and female blastocyst genomes. Filled ovals indicate methylated CpGs and open ovals indicate unmethylated CpGs. Female samples
contained both the paternal (Xp) and maternal (Xm) alleles, whereas male samples contained only the Xm alleles. CpG-2, ?3, ?4, ?6, ?7, ?8
and ?9 are located inside the Fthl17 family of genes.
Nucleic Acids Research, 2010,Vol.38, No. 113679
The microarray data was deposited in the NCBI database.
The Gene Expression Omnibus (GEO) accession number
Supplementary Data are available at NAR Online.
The authors thank T. Kohda and D. Endo for critical
reading of the manuscript.
Program for Improvement of Research Environment for
Young Researchers from Special Coordination Funds for
Promoting Science and Technology (SCF) commissioned
by MEXT of Japan; a Grant-in-Aid for Scientific
Research from The Ministry of Education, Culture,
Sports, Science and Technology; the 21st Century COE
program from the Ministry of Education, Culture,
Sports, Science and Technology of Japan and the
Mochida Memorial Foundation
Pharmaceutical Research. Funding for open access
charge: Program forImprovement
Technology (SCF) commissioned by MEXT of Japan.
Conflict of interest statement. None declared.
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