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First published online 30 November 2006
doi: 10.1242/dev.02697
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1 Department of Genetics, National Institute of Genetics, SOKENDAI, 1111 Yata,
Mishima, Shizuoka 411-8540, Japan.
2 Division of Mammalian Development, National Institute of Genetics, SOKENDAI,
1111 Yata, Mishima, Shizuoka 411-8540, Japan.
3 Core Research for Evolutional Science and Technology (CREST), Japan Science
and Technology Corporation, 2-6-15, Shibakoen, Minato-ku, Tokyo 105-0011,
Japan.
Author for correspondence (e-mail:
ysaga{at}lab.nig.ac.jp)
Accepted 16 October 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PE
promoter and examined Nanos2 function in a Nanos3-null genetic
background. We find that ectopically produced Nanos2 protein rescues the
Nanos3-null defects, because the germ cells fully develop in both
sexes in the transgenic mice. This result indicates that Nanos2 can substitute
for Nanos3 during early PGC development. By contrast, our current data show
that Nanos3 does not rescue the defects in Nanos2-null mice. Our
present findings thus indicate that there are redundant functions of the Nanos
proteins in early PGC development, but that Nanos2 has a distinct function
during male germ cell development in the mouse.
Key words: Primordial germ cell, Transgenic mouse, Oct4 enhancer, Antibody, Genetic rescue, Spermatogenesis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Fujii et al., 2006;
Lehmann and Nusslein-Volhard,
1991; Pilon and Weisblat,
1997; Subramaniam and Seydoux,
1999) and vertebrates
(Koprunner et al., 2001;
MacArthur et al., 1999;
Tsuda et al., 2003). Although
the expression of the Nanos genes is confined to the germ cell lineage in most
instances, it has also been shown that Nanos mRNA and protein are supplied as
germ-plasm components in Drosophila oocytes. In mouse, three Nanos
genes, Nanos1-3, have now been identified
(Haraguchi et al., 2003), among
which Nanos2 and Nanos3 are expressed in the embryonic germ
cells, and a deficiency in these genes results in the loss of germ cells
(Tsuda et al., 2003). The
initial expression of Nanos3 is detectable in the early PGCs that
have emerged from the base of the allantois (our unpublished observation), and
this expression continues during the migration of these cells. After entering
the genital ridge, the expression of Nanos3 in male PGCs is retained
for a while, but disappears by embryonic day 15.5 (E15.5), whereas, in PGCs
entering into the female gonad, this expression disappears prior to E13.5.
Nanos3 expression then restarts in a small population of
spermatogonia after birth (our unpublished data). In contrast to the
Nanos3 expression profile that initiates in early PGCs prior to
sexual specification, Nanos2 expression is male-PGC specific and
commences only in PGCs that have entered the male genital ridge. All male PGCs
express Nanos2, and this pattern continues during the embryonic stage
when these cells are arrested in the G0-G1 phase. However, the expression of
Nanos2 decreases just before birth and only a small number of
spermatogonia express this gene after birth, similar to Nanos3
(Tsuda et al., 2006).
We have previously reported the phenotypes of Nanos2-null and
Nanos3-null mice (Tsuda et al.,
2003), and our findings indicate that both genes are essential for
the maintenance of PGCs. Nanos3-null PGCs are lost during the
migration stage, irrespective of the sex of the animal, whereas
Nanos2-null PGCs die in only the male gonads and show no evident
defects in females. Because Nanos2 and Nanos3 share a conserved zinc-finger
domain that is implicated in their functional roles
(Curtis et al., 1997), we
speculated that these proteins have equivalent functions and that any
differences in their associated null-phenotypes may depend on their expression
pattern only. However, PGCs are lost in Nanos2-null mice, although
both Nanos2 and Nanos3 transcripts can be observed in the
wild-type embryos, which may suggest that a nonredundant role exists for
Nanos2 during the maintenance of male PGCs. Moreover, because
Nanos3-null PGCs are lost before entry into the gonads, analysis of
the compound knockout mice provides no information concerning the impact upon
PGC development at later stages. To overcome this problem, we adopted a
transgenic strategy in our current study that will enable us to determine
whether these two proteins share any functional similarities. For this
purpose, we employed Oct4 enhancer elements, because they have been fully
characterized in previous transgenic strategies. In particular, Oct4PE
shows both broad and low levels of activity in the epiblasts of
egg-cylinder-stage mouse embryos, but the expression of genes under its
control becomes restricted in the PGCs that form at the base of the allantois
at E7.25, and is continuously observed in the PGCs during the migration stage
and in the male gonads (Yeom et al.,
1996; Yoshimizu et al.,
1999). In addition, the ectopic expression of
Oct4PE-controlled genes in the PGCs upon entry into the female gonads
is repressed. Significantly, these expression profiles are very similar to
endogenous Nanos3, and Oct4PE thus serves as an ideal enhancer
to drive the Nanos2 gene in a Nanos3-expressing lineage, as
we have not yet identified the Nanos3 enhancer elements. Permanent
transgenic mouse lines were therefore established using Oct4PE to drive
a Nanos2 transgene in a Nanos3-null background to determine
whether Nanos2 can functionally substitute for the loss of Nanos3. We further
generated transgenic mice in a Nanos2-null background that drive
Nanos3 expression in a Nanos2-expressing PGC lineage. Our
results indicate that Nanos2 can replace Nanos3 function, but that Nanos3 does
not rescue Nanos2-null defects.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Western blotting
Embryonic gonads were sonicated with an ultrasonic processor (Vibra Cell,
Sonics and Material Inc.) in 2xSDS-PAGE sample buffer (200 mM Tris-HCl,
pH 8.3, 4% SDS, 400 mM DTT, 20% glycerol, 2 mM EDTA, 0.05% bromophenol blue).
Extracts were then resolved on 15% SDS-PAGE gels and electroblotted onto
nitrocellulose membrane (BioTrace NT, Pall Corporation). Western blotting was
performed using the primary antibodies anti-Nanos2 (1:200), anti-Nanos3
(1:200) and anti-actin (1:2000, Sigma). This was followed by incubation with
swine anti-rabbit IgG conjugated with AP (1:2000, DAKO) as the secondary
antibody for anti-Nanos2 and anti-Nanos3, and goat anti-mouse IgG conjugated
with AP (1:2000, Novagen) for the anti-actin primary antibody. The detection
of immunoreactivity was performed using a BCIP/NBT Phosphatase Substrate kit
(KPL) according to the manufacturer's instructions.
Generation of transgenic mouse lines
The Oct4PE-FLAG-Nanos2 construct was generated by ligating the
Oct4PE promoter-enhancer (Yoshimizu
et al., 1999) with a 3xFLAG-tagged Nanos2 cDNA
insert containing a 3'-UTR. To construct Nanos2E-FLAG-Nanos3, a
BamHI fragment (9.2 kb) containing the upstream region of the mouse
Nanos2 gene was ligated with 3xFLAG-tagged Nanos3 cDNA
followed by the 3'-UTR of the Nanos2 gene. These transgenes
(schematically shown in Figs 2
and 5) were injected into
fertilized eggs and two independent transgenic lines were subsequently
established for each construct. The specific expression of Nanos2 and
Nanos3 in the embryonic male germ cells was confirmed by western blot
analysis using polyclonal antibodies. Genotyping was performed using the
primers FLAG-F (5'-CTACAAAGACCATGACGGTG-3') and
Nanos2-3'UTR-R (5'-CCCGAGAAGTCATCACCAG-3')
for Oct4-PE-FLAG-Nanos2 and Nanos2E-FLAG-Nanos3.
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Nanos2-F: 5'-AACTTCTGCAAGCACAATGG-3';
Nanos2-R: 5'-CCGAGAAGTCATCACCAG-3';
Nanos3-F: 5'-TCCCGTGCCATCTATCAG-3';
Nanos3-R: 5'-GGATGTTGAGGCAACACC-3'.
Control PCR-amplification reactions were performed using primers for glyceraldehyde-3-phosphate dehydrogenase (G3PDH):
G3PDH-F: 5'-ACCACAGTCCATGCCATCAC-3';
G3PDH-R: 5'-TCCACCACCCTGTTGCTGTA-3'.
Histological methods
For histological analysis, samples (testes, ovaries and male gonads) were
fixed in Bouin's solution and embedded in paraffin. Sections (6 µm) were
stained with hematoxylin and Eosin. For immunohistochemical detection of male
germ cells, sections were incubated with TRA104 (1:10) or TRA98 (1:9000)
(Tanaka et al., 1997) followed
by subsequent detections using the Vectastain ABC kit (Vector Laboratories).
Such sections were counterstained with hematoxylin prior to microscopic
analysis. Both TRA antibodies specifically recognize germ cells, but TRA98
detects only a part of spermatogonia after birth. For embryonic female gonads,
samples were directly embedded into OCT compound (Tissue-Tek, SAKURA) and
sectioned by cryostat. The frozen sections were incubated with TRA98 antibody
(1:9000) followed by Alexa-488 conjugated goat anti-rabbit IgG (Molecular
Probes) and observed using a fluorescent microscope (Olympus BX61). The method
for whole-mount detection of PGCs by alkaline phosphatase staining has been
described previously (Tsuda et al.,
2003).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). However, our reverse transcription (RT)-PCR analysis
indicated that Nanos3 was also expressed during male germ cell
development (Tsuda et al.,
2003). To investigate the functional relationship between these
proteins in our current study, we first attempted to generate polyclonal
antibodies and successfully obtained anti-Nanos2 and anti-Nanos3 antibodies
that specifically recognize the endogenous proteins (Nanos2, approximately 18
kDa; Nanos3, approximately 27 kDa; Fig.
1A and see Fig. S1 in the supplementary material). We then
examined the expression profiles of these two factors in embryonic male gonads
by western blot analyses using extracts derived from one-half of each single
testis. In the wild-type mouse embryo, Nanos2 expression was undetectable at
E12.5 but was found to increase at E13.5 and continue until E17.5
(Fig. 1B). Because male germ
cells are arrested in the G0-G1 stage during E13.5-E17.5, the change in the
amount of the protein reflects the expression level in germ cells, whereas the
change in the amount of actin is mainly due to the increase in somatic cell
number. By comparison, Nanos3 was already detectable by E12.5 but became
undetectable by E15.5. We additionally examined Nanos3 expression in
a Nanos2-null embryonic gonad, as we expected that Nanos3 may be
downregulated in Nanos2-null mice and thus account for the PGC
defects in the gonad. Interestingly, however, Nanos3 expression appeared to be
up-regulated in the Nanos2-null germ cells, in which large amounts of
Nanos3 could be detected until E16.5 (Fig.
1B). We speculate that this may be due to transcriptional
regulation, because RT-PCR analysis revealed that Nanos3 mRNA is also
up-regulated in the Nanos2-/- mouse
(Fig. 1C).
Transgenic strategy
Our protein-expression data indicated that Nanos2 and Nanos3 may not have
redundant functions, because Nanos3 was found to be abundantly expressed in
the Nanos2-null embryonic gonad, in which germ cells are lost by
programmed cell death. However, during the early stages of PGC development,
only Nanos3 is expressed. To examine whether Nanos2 could substitute
for the loss of functional Nanos3 in early-stage PGCs, we adopted a transgenic
strategy that used an Oct4 enhancer. Because the Nanos3-promoter
region has not yet been identified, we employed the Oct4-PE enhancer
because the expression profiles of the transgenes driven by this element
resemble the Nanos3 expression pattern. It has been shown previously
that this Oct4 enhancer is active in early PGCs and maintains its activity
during the migration stage, and also in the male gonads
(Yoshimizu et al., 1999).
Moreover, this expression continues after birth in the small population of
spermatogonia that contribute to spermatogenesis. We expressed FLAG-tagged
Nanos2 under the control of Oct4-PE in PGCs. Among the three transgenic
lines that we established in these experiments, we confirmed the expression of
the transgene in embryonic gonads in two lines. Western blot analysis also
revealed that both of the transgenic mice that we generated produced an
appreciable quantity of FLAG-tagged Nanos2, in addition to endogenous Nanos2
(Fig. 2A). However, it is
noteworthy that the endogenous levels of Nanos2 were decreased in these
transgenic mice, which may indicate the presence of some regulatory mechanism
that controls the Nanos2-protein levels. Further analysis was conducted using
a transgenic line (line #5), which showed a higher expression of the
transgene. The transgenic mice showed no apparent defects as a result of the
ectopic expression of FLAG-tagged Nanos2 in the early-stage embryos and the
transgene was successfully transmitted via both males and females.
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) (Fig. 2D).
However, we observed an increased testis size in these knockout mice following
the expression of the Nanos2 transgene
(Fig. 2B,F), and a subsequent
histological study of the transgenic tissues revealed the presence of normal
spermatogonia, spermatocytes and spermatids in the mature seminiferous tubules
(Fig. 2E). We also confirmed
that these mice are fertile, indicating that the FLAG-tagged Nanos2 protein is
functional. However, we did observe the presence of morphologically abnormal
tubules in the Nanos2-null testis that expressed the transgene
(Fig. 2E, asterisk), and that
the relative size of the testis in the transgenic mouse remained smaller than
the Nanos2+/- mouse. This may be due to the lower expression of the
Nanos2 transgene, or might reflect the differences between the activities of
the Oct4-PE and endogenous Nanos2 enhancers in the testis of adult
mice.
Nanos2 rescues the Nanos3 deficiency in both male and female mice
We next introduced our FLAG-tagged Nanos2 transgene into a
Nanos3-null genetic background to test whether this would rescue the
Nanos3-null defect. Consistent with earlier reports
(Tsuda et al., 2003),
Nanos3-null male mice showed no detectable germ cells at any of the
developmental stages examined after birth
(Fig. 3C,G). However, we could
detect significant increases in the sizes of the testes as a result of
transgene expression (Fig.
3A,E,I). Immunohistological examination of these tissues further
revealed that TRA104- and TRA98-positive germ cells were present in the
Nanos3-/- testes harboring the Nanos2 transgene
(Fig. 3D,H). In 2-week-old
Nanos3-/- testes expressing exogenous Nanos2, the number of germ
cells was lower, reflecting their smaller size (compare
Fig. 3B with 3D). However,
normal maturation of the spermatocytes could be observed in 6-week-old testes
from both the normal and transgenic animals (compare
Fig. 3F with 3H), although the
relative sizes of these rescued Nanos3-/- testes were still less
than wild type (Fig. 3I). This
defect could be due to the incomplete rescue during embryonic development (see
below) or, alternatively, could result from the difference in expression
caused by the heterologous Oct4 promoter and enhancer. Nevertheless, we
confirmed that the transgenic mice were fertile and could transmit their germ
lines to the next generation. These data indicate that Nanos3 is not required
for spermatogenesis and that any role it may play in this process can be
substituted for by Nanos2.
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Nanos2 partially rescues the loss of Nanos3 function in early-stage embryos
To investigate the precise extent to which Nanos3-null germ cells
are rescued by Nanos2-transgene expression, including the timing, we
examined germ cell development at different embryonic stages. In the
Nanos3-null embryo, the germ cells are gradually lost during the
migration stage and only a few PGCs reach the genital ridge at E11.5
(Fig. 4B). However, many PGCs
were found to have reached the genital ridge in the Nanos3-null
embryos expressing the Nanos2 transgene
(Fig. 4C,D). Although their
number was still lower than in wild type
(Fig. 4A), these PGCs also
appeared to be maintained in the developing gonads in both the male and female
transgenic embryos (Fig. 4E-J).
We could also detect an appreciable number of TRA104- or TRA98-positive PGCs
in both the E14.5 male (Fig.
4G) and E16.5 female (Fig.
4J) gonads in Nanos3-null mouse embryos harboring the
Nanos2 transgene.
Nanos3 does not rescue the defects in Nanos2-null PGCs
The finding that Nanos2 rescues the function of Nanos3 in early-stage mouse
embryos indicates that functional redundancy exists between these two
proteins. However, we speculated as to whether this was really the case for
Nanos3; defects could still be observed in Nanos2-null PGCs, in which
we observed an increase in Nanos3 expression. To examine this
further, we generated transgenic lines expressing a FLAG-tagged
Nanos3 transgene under the control of the Nanos2 enhancer. We had
already confirmed that the 8.6 kb upstream region of the Nanos2 gene
is sufficient to facilitate Nanos2 expression in both embryonic and
postnatal germ cells (unpublished data). Two transgenic lines were
subsequently established and one of these expressed a good level of exogenous
Nanos3 in the embryonic male gonads (Fig.
5A). This transgene was then introduced into a
Nanos2-null background and the development of germ cells was examined
in both the embryonic (Fig.
5B-D) and adult (Fig.
5E-G) testes. In the Nanos2-null testes, PGCs begin to
undergo apoptosis from E15.5 and most of the gonocytes disappear at birth. The
small population of gonocytes remaining after birth is eventually lost within
4 weeks (Fig. 5F). At first, we
examined E16.5 gonads, in which Nanos2-null PGCs begin to migrate out
from the testis cords (Fig.
5C). We did not find any differences in the dynamics of the
TRA104-positive PGCs irrespective of the presence or absence of the transgene
in the Nanos2-null background (compare
Fig. 5C with 5D). Apoptotic
cell death was detected in E18.5 testes from both Nanos2-/- and
Nanos2-/- containing the Nanos3 transgene (see Fig. S2 in
the supplementary material). Furthermore, no germ cells were evident in
Nanos2-null testes expressing the Nanos3 transgene in
4-week-old mice (Fig. 5F,G).
These results indicate that Nanos3 can not compensate for the loss of Nanos2
function in male mouse PGCs.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). If the
suppression of this apoptotic response was the primary function of the Nanos
proteins, we would expect that one could rescue the defects caused by a
deficiency in another. The function of Nanos3 in early PGCs that were null for
this gene was indeed found to be substituted for by Nanos2, emphasizing that
functional redundancy does exist between these two proteins. However, we show
that Nanos3 does not rescue the Nanos2 defect in Nanos2-knockout male
PGCs, indicating that there are distinct functions of Nanos2 that cannot be
complemented for by Nanos3. Hence, we conclude that Nanos3 does not have a
similar role to Nanos2 in male PGCs in the mouse. The function of Nanos in the
suppression of apoptosis has been shown previously in Drosophila
development, and the direct target is indicated to be involved in this
apoptotic response pathway (Hayashi et al.,
2004) (S. Kobayashi, personal communication). However, neither the
target RNA molecules nor the mechanisms underlying Nanos function during mouse
PGC development are known. Our present study suggests the presence of
different targets that may be specifically recognized by Nanos2 or that a
distinct mechanism may be mediated via Nanos2 in male PGCs. However, we can
not exclude the possibility that the phenotype of the PGCs in
Nanos2-null embryos reflects the activity of a functional Nanos3 in
these cells and may in fact be a rescued state. This possibility is suggested
by the fact that the male germ cell-less phenotype became severe after birth
when the Nanos3 gene dosage was reduced in the Nanos2-null
genetic background (see Fig. S3 in the supplementary material). It will be
necessary to generate and analyze a Nanos2-Nanos3 double
knockout in male PGCs to elucidate this fully, and conditional knockout mice
will be available for future experiments to address this crucial issue. One of remaining questions regarding the function of the Nanos proteins is their precise roles during spermatogenesis. Because both Nanos2 and Nanos3 are required during the embryonic stages of development in the mouse, we cannot yet address this issue, but will employ conditional knockout strategies in future studies to examine this important issue.
Regulation of Nanos expression
Our current western blot analyses have revealed the presence of two
interesting regulatory mechanisms that are involved in the Nanos pathways. In
the absence of Nanos2 in embryonic male PGCs, expression of Nanos3 is
up-regulated, and this is accompanied by an increase in its transcript levels
also. This is unlikely to be the result of a direct function of the loss of
Nanos2, however, because the Nanos2 protein is most likely to be present in
the cytoplasm and not in the nucleus. One possible mechanism could be the
negative regulation of transcriptional activators by Nanos2. Our preliminary
GeneChip analysis indicates that the levels of other mRNA species are
increased in the absence of Nanos2. We thus speculate that one of major the
functions of Nanos2 might well be translational repression of specific
transcriptional activators.
Another noteworthy observation that has emerged from our current data is
the possible auto-regulation of Nanos2. In our transgenic mice harboring
enhanced exogenous Nanos2 expression, the endogenous levels of the Nanos2
protein were found to be decreased. This observation indicates the presence of
a mechanism that maintains Nanos2-protein expression at constant levels. This
contention is further supported by our analysis of Nanos2-heterozygous
embryos, in which the levels of Nanos2 protein are equivalent to the wild-type
embryos, although the transcript levels are decreased to about 50% of that of
wild type. One possible explanation for this is translational repression of
Nanos2 by Nanos2 itself. We expect that excess Nanos2 protein may negatively
affect its own translational efficiency, but we have shown that the
Nanos2-3'-UTR might be required for efficient translation also
(Tsuda et al., 2006). Because
the 3'UTR elements within the Nanos family of mRNAs have also been shown
to be regulated by several proteins
(Dahanukar and Wharton, 1996;
de Moor et al., 2005;
Duchow et al., 2005;
Nelson et al., 2004), these
factors may also affect the translational efficiency of Nanos2 in combination
with other mechanisms.
Taken together, it is most likely that the Nanos proteins function as a cellular component of RNA metabolism. Further studies in our laboratory will therefore focus upon the mechanisms that regulate Nanos2 and Nanos3 expression, as well as the functions of these two proteins themselves.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/1/02697/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Department of Physiology and Biological Information,
Dokkyo University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi
321-0293, Japan
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