Maintaining Sufficient Nanos Is a Critical Function
for Polar Granule Component in the Specification
of Primordial Germ Cells
Girish Deshpande, Emma Spady,1Joe Goodhouse, and Paul Schedl2
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544
ABSTRACT Primordial germ cells (PGC) are the precursors of germline stem cells. In Drosophila, PGC
specification is thought to require transcriptional quiescence and three genes, polar granule component
(pgc), nanos (nos), and germ cell less (gcl) function to downregulate Pol II transcription. While it is not
understood how nos or gcl represses transcription, pgc does so by inhibiting the transcription elongation
factor b (P-TEFb), which is responsible for phosphorylating Ser2 residues in the heptad repeat of the C-
terminal domain (CTD) of the largest Pol II subunit. In the studies reported here, we demonstrate that nos
are a critical regulatory target of pgc. We show that a substantial fraction of the PGCs in pgc embryos have
greatly reduced levels of Nos protein and exhibit phenotypes characteristic of nos PGCs. Lastly, restoring
germ cell–specific expression of Nos is sufficient to ameliorate the pgc phenotype.
The germline of Drosophila arises from a special group of primordial
germ cells (PGC). PGCs are formed during nuclear cycles 9 and 10
when nuclei migrate from the center of the embryo into the posterior
pole plasm. These nuclei induce cellularization, incorporating the
maternal germline determinants that are assembled in the pole plasm
during oogenesis. In addition to precocious cellularization, PGCs differ
from the surrounding soma in a number of important respects [re-
viewed in Santos and Lehmann (2004), Seydoux and Bruan (2006), and
Wylie (1999)]. One of these is transcription. Whereas somatic nuclei
turn on transcription, it is shut down in PGCs, and they remain
transcriptionally quiescent until after they exit the gut much later in
development (Zalokar 1976; Seydoux and Dunn 1997). Global down-
regulation of transcription in PGCs correlates with the phosphoryla-
tion status of the heptad repeats in the C-terminal domain (CTD) of
the largest Pol II subunit. There are two Serine (Ser) residues in each
heptad, Ser2 and Ser5, which are phosphorylated at different steps.
Ser5 phosphorylation is coordinated with initiation, and Ser2 phos-
phorylation accompanies elongation (Phatnani and Greenleaf 2006;
Hirose and Ohkuma 2007). While both of these modifications are
elevated in somatic nuclei of blastoderm embryos, this is not true in
PGCs: the elongation phosphorylation, PSer2, is absent, and there are
only low levels of the initiation phosphorylation, PSer5 (Seydoux and
Dunn 1997; Deshpande et al. 2005). Flies are not the only organism
in which primordial germ cells downregulate transcription. Transcrip-
tional quiescence is also a hallmark of germline progenitors in C. elegans
and Xenopus (Seydoux and Dunn 1997; Lai et al. 2012).
The establishment of transcriptional quiescence in fly PGCs is
mediated by at least three maternally deposited pole plasm de-
terminants, germ cell-less (gcl), polar granule component (pgc), and
nanos (nos) (Asaoka et al. 1998, 1999; Deshpande et al. 1999, 2004;
Leatherman et al. 2002; Martinho et al. 2004). These three maternal
factors have different (though potentially overlapping) gene targets for
downregulation, act at slightly different times, and use different mech-
anisms. gcl functions when PGCs are formed and targets genes that are
activated prior to the mid-blastula transition. pgc and nos are required
later, and while they both prevent transcription of somatic mid-
blastula transition genes, there are differences in their targets. pgc
blocks zen and tailess, whereas nos is required to prevent pair rule
genes like even-skipped from being activated. Blocking Pol II activity
appears to be important for PGC development. While PGCs from gcl
and pgc mothers can go on to form functional germline stem cells
(GSC), the number of PGCs in coalesced stage 14–15 mutant gonads is
substantially reduced. Even more drastic effects are evident in nos
embryos. nos PGCs fail to maintain PGC identity, and unlike either
Copyright © 2012 Deshpande et al.
Manuscript received June 7, 2012; accepted for publication September 10, 2012
This is an open-access article distributed under the terms of the Creative
Commons Attribution Unported License (http://creativecommons.org/licenses/
by/3.0/), which permits unrestricted use, distribution, and reproduction in any
medium, provided the original work is properly cited.
Supporting information is available online at http://www.g3journal.org/lookup/
1Present address: Swarthmore College, 500 College Ave., Swarthmore, PA
2Corresponding author: Lewis Thomas Labs, Washington Road, Princeton
University, Princeton, NJ 08544. E-mail: email@example.com
Volume 2|November 2012|
gcl or pgc PGCs, they never develop into functional GSCs (Kobayashi
et al. 1996; Sato et al. 2007). Further supporting the importance of
nos-dependent transcriptional quiescence, nos PGCs can be partially
rescued by mutations in one of the nos target genes Sex-lethal
(Deshpande et al. 1999).
As for mechanisms, nothing is known about how gcl blocks tran-
scription, whereas nos is thought to act by repressing the translation of
an unknown general transcription factor. For pgc, the mechanism is
well understood (Hanyu-Nakamura et al. 2008). Pgc protein interacts
with transcription elongation factor b (P-TEFb), which is responsible
for phosphorylating Ser2 residues in the CTD heptad repeat. The
association of Pgc with P-TEFb prevents P-TEFb from being recruited
to sites of paused polymerase. Consistent with this biochemical mech-
anism, PGCs in blastoderm-stage pgc mutants have high levels of
CTD-PSer2. Moreover, targeting transcriptional elongation seems to
be a conserved mechanism for imposing transcriptional quiescence, as
the C. elegans PIE-1 protein is also thought to arrest transcription by
inhibiting P-TEFb (Seydoux et al. 1996; Batchelder et al. 1999;
Nakamura and Seydoux 2008).
Although it has been suggested that Pgc and its target P-TEFb are
the central players in establishing transcriptional quiescence in newly
formed PGCs (Cinalli et al. 2008; Nakamura and Seydoux 2008), it is
striking that loss of pgc does not fully disrupt the specification of PGC
fate or their eventual transition into functional GSCs. This would
suggest either that the establishment and/or maintenance of transcrip-
tional quiescence is not a necessary step in PGC development in
Drosophila or that pgc has an important, but not absolutely essential,
role in this process. For these reasons, we have re-examined the func-
tioning of pgc in germline development and explored its relationship
to the PGC/GSC-determinant nos.
MATERIALS AND METHODS
Fly stocks and culture
Flies were grown at room temperature (22?) on standard medium.
The following stocks obtained from Bloomington Stock Center were
used for analysis: nanos-Gal4:VP16, twist-Gal4, nosBN, nosRC. Also
used were two different extensively characterized loss-of-function
alleles of pgc: pgc[EY09338]and pgc[EY07794](Martinho et al. 2004).
The loss of Nos protein in pgc PGCs was confirmed using AS-26,
antisense pgc transgene (Nakamura et al. 1996). Nos-tubulin 39 UTR
transgenes were a kind gift from Liz Gavis, Princeton University.
Typically, virgin females homozygous for the Gal4 transgene were
mated with males carrying the UAS transgene (Brand and Perrimon
1993). Embryos from these crosses were fixed and stained for
The stainings were performed essentially as described in Deshpande
et al. (1995). For immunofluorescence-based stainings, to begin with,
the optimal settings were determined using wild-type control em-
bryos. After the initial optimization, the alterations were kept to
a minimum during the experiment. Multiple embryos and/or PGCs
were imaged to assess the relevant differences in concentrations. Also,
to minimize the variation, wherever possible, alterations in the levels
were assessed using the immediately adjacent PGCs belonging to the
same embryo. Vasa antibody was either a rat or rabbit polyclonal used
at 1:500 dilution. Anti-Nanos is a rabbit polyclonal antibody used at
1:500 dilution (Hanyu-Nakamura et al. 2008). Alpha-Spectrin and
Hu-li tai shao mouse monoclonal antibodies were used at 1:4 dilution
(from Developmental Studies Hybridoma Bank).
RESULTS AND DISCUSSION
Subset of pgc pole cells displays loss of Nos protein
Although Nakamura et al. (1996) have reported that nos mRNA levels
in PGCs are reduced when pgc activity is compromised by an antisense
pgc transgene, it has been argued that this diminution of nos mRNA is
too slight and takes place too late to be relevant for pgc function
(Martinho et al. 2004). However, Nos protein accumulation in PGCs
compromised for pgc was not examined, and it seemed possible that
the loss of pgc activity might have a greater effect on protein expres-
sion than it does on nos message levels. For this reason, we examined
Nos accumulation in syncytial and cellular blastoderm stage in both
antisense embryos (see supporting information, Figure S1) and
embryos produced by two different pgc mutant alleles (Figure 1). In
all three cases, we found that PGCs compromised for pgc activity
exhibit an unusual, heterogeneous pattern of Nos accumulation. As
illustrated for a stage 4 pgc embryo in Figure 1, the pattern of Nos
protein accumulation in pgc PGCs is quite different from that in wild-
type. In wild-type embryos, Nos protein accumulates to essentially the
same level in most all PGCs (Figure 1; see also Figure 2 and Figure S1).
By contrast, Nos levels are quite variable in pgc PGCs (Figure 1; see
also Figure 2 and Figure S1). Some PGCs have near wild-type levels of
Nos, others have intermediate amounts, and still others have little or
no Nos. This highly heterogeneous pattern of Nos accumulation is
evident soon after PGC formation and continues beyond the cellular
blastoderm stage. Overall, over half of the pgc PGCs (58%; 75 out of
126 PGCs) in syncytial and cellular blastoderm-stage embryos have
clearly reduced levels of Nos protein, whereas in wild-type, Nos levels
are reduced in less than 10% (7%; 6 out of 91 PGCs) of the PGCs.
From these findings, we conclude that the previously reported dimi-
nution of nos mRNA impacts Nos accumulation, but significantly, it
does so only in a subset of the PGCs.
To determine whether the effects on Nos are specific, we examined
the accumulation of another germline-specific translation factor, Vasa.
As shown in Figure 1, Vasa levels in blastoderm-stage pgc PGCs are
unaffected and resemble wild-type PGCs. However, later in develop-
ment, Vasa levels are often substantially reduced in pgc PGCs that are
Initiation CTD phosphorylation PSer5 is upregulated
in a subset of pgc PGCs
An intriguing question is whether the reduction in Nos protein in
a subset of the pgc PGCs has any impact on the development of these
cells. If it does, one would expect to find that some pgc PGCs exhibit
characteristic nos-like phenotypes, while others do not. Although both
pgc and nos are known to play important roles in establishing tran-
scriptional quiescence, they interfere with polymerase activity at dif-
ferent steps. Elegant studies by Hanyu-Nakamura et al. (2008) have
shown that Pgc imposes transcriptional quiescence by specifically
inhibiting the P-TEFb–dependent transcriptional elongation CTD
phosphorylation, PSer2. By contrast, nos appears to downregulate
transcription in PGCs at an earlier step in the transcription cycle, as
the levels of both the initiation CTD phosphorylation, PSer5, and the
elongation CTD phosphorylation, PSer2, are elevated in nos mutant
PGCs (Deshpande et al. 2005).
If the loss of Nos in pgc PGCs affects their specification, then we
would expect to find that PSer5 is upregulated in cells that have re-
duced amounts of Nos. Before testing this possibility, we first con-
firmed previous reports that PSer2 is elevated in all pgc PGCs (data
not shown). We next examined PSer5 in wild-type, nos, and pgc PGCs.
As shown for one of the pgc alleles in Figure 2, although PSer5 is
| G. Deshpande et al.
clearly present in wild-type PGCs, the level of this CTD phosphory-
lation is substantially reduced compared with nearby somatic nuclei
(n = 70). Similar results were obtained for the other pgc allele and for
the pgc antisense. By contrast, in nos embryos, essentially all PGCs
have levels of PSer5 approaching that in the surrounding somatic
nuclei (see Figure S2). As was observed for Nos protein, there is a quite
heterogeneous pattern of PSer5 in pgc PGCs. As shown in Figure 2,
some pgc PGCs resemble wild-type and have little PSer5. However,
a subset of the PGC nuclei have levels of PSer5 approaching that
found in somatic nuclei. A careful analysis of multiple pgc embryos
indicates that the extent of upregulation of PSer5 is variable and that
elevated levels of this CTD phosphorylation are seen unambiguously
in only about 50% of stage 4 or 5 PGCs (n = 100). In the remaining
PGCs, there was either no increase or only a marginal increase.
We next asked whether the pgc PGCs with elevated nuclear PSer5
are the ones with reduced amounts of Nos. To test this possibility, we
examined PGCs in stage 5 (cellular blastoderm embryos) coimmu-
nostained with Pser5 and Nos antibodies. Figure 2 shows that the
subset of PGCs with reduced Nos corresponds closely to the subset
with elevated PSer5. In this experiment, we found that 61% (32 out of
53) of the pgc pole cells had reduced amounts of Nos. Of the pole cells
with reduced Nos, PSer5 was elevated in nearly 90% (27 out of 30).
Significantly, none of the PGCs with normal levels of Nos had elevated
PSer5. We next examined stage 4 embryos to test whether Nos protein
Figure 1 Nos protein levels are dimin-
ished in pgc- PGCs. Wild-type (panels A–
C and G–I) and pgc (panels D–F and J–L)
blastoderm-stage embryos were probed
with Vasa (green) and Nos (red) antibod-
ies. Panels A, D, G, and J show the
merged images; panels B, E, H, and K
show Vasa alone; and panels C, F, I,
and L show Nos alone. Although levels
of Nos are reduced, sometimes substan-
tially in a subset of pgc PGCs, there is no
apparent alteration in the levels of Vasa
protein at the blastoderm stage. The dif-
ference and heterogeneity in Nos level is
apparent in panels G–L, which show
magnified images of two pole cells each.
Similar results were obtained in three in-
dependent trials. The numbers in the text
are from one of these experiments.
Figure 2 Loss of Nanos protein is in pgc-PGCs is cor-
related with increased CTD PSer5. Stage 5 embryos
from either wild-type mothers (A–C) or pgc mothers
(D–F) were coimmunostained with Nos (red) and Pser5
(green) antibodies. Many pgc-PGCs have reduced lev-
els and/or uneven distribution of Nos (compare panels E
and K). PGCs with reduced Nos have elevated Pser5
(panels F and L). One of the two different pgc pole cells
displays higher levels of Nos protein and corresponding
decreases in Pser5 levels. By contrast, wild-type PGCs
show only low levels of signal compared with surround-
ing somatic nuclei (panels D–F). Similar results were
obtained in two independent experiments. The num-
bers in the text are from one of the experiments. Arrows
point to pgc PGC with reduced Nos and elevated
Volume 2 November 2012| Nanos Is a Critical Target for PGC|
levels are diminished in pgc PGCs in stage 4 embryos. As can be seen
from embryo shown in Figure S3, Nos protein levels are non-uniform
in pgc PGCs even at this earlier stage. Moreover, in the PGCs with
reduced Nos, there is concomitant increase in PSer5 levels.
Does loss of Nos in pgc PGCs have other
The results described above argue that the CTD initiation phosphor-
ylation is upregulated in a subset of pgc mutant PGCs because they
lack sufficient Nos. If this conclusion were correct, then we would
expect an approximately similar fraction of the pgc PGCs to exhibit
other phenotypes characteristic of nos PGCs, such as migration
defects, upregulation of Cyclin B, and premature division, cell death,
and a failure to initiate the transition from PGC to GSC identity.
Consistent with these expectations, Nakamura et al. (1996) reported
that a subset of the PGCs in progeny of antisense pgc mothers
exhibited migration defects and died. We confirmed the migration
defects in progeny pgc mutant mothers. We also found that the co-
alesced gonads of stage 14 mutant embryos had fewer PGCs. Whereas
wild-type have 9.5 PGCs/gonad (n = 100), pgc embryos have on
average 3.5 PGCs/gonad (n = 75). To address this issue further, we
determined whether pgc PGCs exhibit other nos-like phenotypes.
Premature cell division in nos PGCs is due to the inappropriate
expression of the mitotic cyclin Cyclin B (Asaoka et al. 1999). In wild-
type PGCs, Nos together with Pumilio repress the translation of
cyclinB mRNA and PGCs arrest the mitotic cycle in G2. To determine
whether loss of Nos in pgc PGCs results in the premature expression
of Cyclin B protein, we probed wild-type and pgc embryos with Cyclin
B antibodies. As shown in Figure 3, a substantial fraction of pgc PGCs
in stage 10 embryos (70%; .100 PGCs) have Cyclin B, whereas Cyclin
B is infrequently detected in PGCs of wild-type embryos (5%; .100
PGCs) of the same stage. In fact, in wild-type embryos, Cyclin B is not
upregulated until much later in development after gonad coalescence
(Asaoka et al. 1999).
Cell death in nos PGCs is due to activation of the head involution
defective apoptosis pathway Sato et al. (2007) and Maezawa et al.
(2009) have shown that over 20% of the PGCs in stages 12–16 nos
embryos express the cell death marker cleaved Caspase3. We used
antibodies specific for cleaved Caspase3 to test whether this pathway
is also activated in pgc PGCs. As Figure 4 shows, all pgc embryos
examined had at least one PGC that was positive for cleaved Caspase
3. Many of the activated Caspase3-positive PGCs also had greatly
reduced Vasa (arrows). As Vasa differed from Nos in that it was
not lost in cellular blastoderm pgc PGCs, we suspect that this is
a consequence of cell death rather than some more direct function
of pgc in sustaining Vasa protein levels.
The transition from PGC to GSC identity begins with assembly of
a germ cell–specific organelle called the spectrosome (Lin et al. 1994;
Lin and Spradling, 1995). Spectrosome-like structures can first be
detected at stage 11, just after the PGCs exit the midgut and start
migrating through the mesoderm. Between stages 11 and 15 of
embryogenesis as PGCs complete their migration and coalesce into
the embryonic gonad, the spectrosome enlarges progressively. By stage
15, it is spherical in shape and closely resembles the structure found in
Wawersik and Van Doren (2005) have shown that nos is required
to initiate and maintain the assembly of spectrosomes in migrating
PGCs of stages 11 and 12 embryos, and in nos mutants, spectrosomes
are not detected in more than 90% of PGCs. To test whether
spectrosome assembly is also disrupted in the progeny of pgc mothers,
we probed wild-type and pgc embryos with spectrin antibodies. We
found that newly formed spectrosomes in PGCs of stages 11 and 12
pgc embryos are typically smaller than the spectrosomes in wild-type
embryos of the same age (not shown). As shown in Figure 5,
abnormalities in spectrosome assembly were even more apparent after
gonad coalescence. In wild-type stage 14 gonads (Figure 5A), all PGCs
have a large, brightly stained spherical spectrosome (Figure 5, arrows).
In contrast, in pgc mutants spectrosomes are missing altogether
(Figure 5, B and C) or not fully developed (Figure 5F, arrow). About
50% (n = 100) of the surviving PGCs lack spectrosomes altogether;
however, as with the other nos-like pgc phenotypes, we observe PGCs
that have “wild-type” spectrosomes (not shown). (Note that it was not
possible to draw the correlation between lack of Nos and missing
spectrosomes, activated Caspase 3, or premature Cyclin B expression,
as we were unable to label PGCs after they were carried inside the gut
during gastrulation with the Nos antibody).
Is nos a critical pgc target?
The results described above indicate that in addition to inappropri-
ately upregulating CTD initiation phosphorylation, several nos phe-
notypes evident in PGCs of older embryos are recapitulated in pgc
mutants. One hypothesis to explain this connection is that a critical
function of pgc in PGC specification and development is to ensure
proper Nos accumulation. In this scenario, PGCs in pgc embryos that
have reduced Nos would not be properly specified, have defects in
migration, and undergo apoptosis. Moreover, all of these phenotypes
would be the consequence of failing to maintain sufficient amounts of
Nos. In contrast, the pgc PGCs that are able to coalesce properly with
the somatic gonad and develop into functional GSCs would be limited
(at the minimum) to those that maintain sufficient levels of Nos. If
this hypothesis were correct, it should be possible to rescue pgc PGCs
by providing Nos protein.
Figure 3 Cyclin B is prematurely expressed in pgc PGCs. Wild-type
(A, B) and pgc (C, D) embryos were probed with Vasa (red) and Cyclin
B (green) antibodies. Panels A and C show the merged image; panels
B and E show Cyclin B protein alone; and panels C and F show Vasa
protein only. Shown here are stage 10 wild-type and pgc embryos.
Whereas Cyclin B is infrequently observed in wild-type stage 10 PGCs
(5%; 1 out of 19), the majority of the pgc PGCs express detectable
levels of Cyclin B at this stage of development (68%; 17 out of 25).
Three independent experiments yielded similar results. Also, note that
inappropriate expression of Cyclin B can be detected in pgc PGCs
from younger blastoderm-stage embryos; however, Cyclin B–positive
PGCs are much less frequent at earlier stages. Arrows in panels E and F
indicated PGCs with elevated levels of Cyclin B, while the arrowhead
indicates a PGC with little or no Cyclin B.
|G. Deshpande et al.
For this purpose, we used the Gal4/UAS system. Because the nos
39UTR contains elements that control localization, translation, and
stability, we used an UAS transgene carrying the nos coding sequence
fused to the tubulin 39 UTR (Bergsten and Gavis 1999). To drive
expression specifically in the germline, we used a nos:Gal4 transgene.
As the nos promoter is not activated in PGCs until after they exit the
midgut and begin migrating toward the somatic gonad, we anticipated
that if supplying Nos rescues the pgc PGCs, rescue should be at best
incomplete because of this delay. Figure 6 shows that the effects of pgc
on the migration and viability of PGCs can be partially rescued by
providing Nos. While embryonic gonads of pgc mutants have on
average 3.5 PGCs (n = 32), pgc mutants carrying the nos: Gal4/
UAS: nos-Tublin 39 UTR transgene combination had on average 7
(n = 35; P value = 1.918 · 1025). The effects of supplying Nos can also
be seen in the upward shift in the number of PGCs in embryonic
gonads (e.g. nearly 40% of the pgc embryos have 3 or fewer PGCs/
embryonic gonad, whereas about 90% of the rescued embryos had 4
PGCs or more).
The finding that PGCs in pgc mutant embryos can be rescued by
supplying Nos indicates that one critical pgc function is to ensure that
there are sufficient amounts of Nos. This conclusion fits with the nos-
like phenotypes evident in a subset of pgc mutant PGCs. At the
blastoderm stage, the subset of PGCs that lose Nos also fail to prevent
upregulation of the CTD initiation phosphorylation PSer5, which is
the target for nos-dependent transcriptional quiescence. Although it
wasn’t possible to draw a similar connection in the older pgc embryos,
we nevertheless found that a substantial fraction of the PGCs exhibit
phenotypes characteristic of nos mutations (migration defects, pre-
mature Cyclin B expression, failure to initiate spectrosome assembly,
and activation of apoptosis). Because rescue in our experiment was
incomplete, it could be argued that pgc has functions that are impor-
tant for the specification and development of PGCs in addition to
maintaining high levels of Nos. On the other hand, not all pgc PGCs
lose Nos (at the blastoderm stage). Consequently, it remains possible
that all that is needed for pgc PGCs to form fully functional GSCs is
that they retain sufficient levels of Nos. If this were true, then the only
important (but not absolutely essential) function of pgc would be to
ensure that the levels of Nos in PGCs remain high enough so that it
can properly specify PGC/GSC fate.
While our findings argue that maintaining full nos activity is a crit-
ical function of pgc, it is not clear which of the known nos regulatory
Figure 4 pgc-pole cells undergo apoptosis. Stages 13–15 wild-type
(not shown) and pgc-embryos were probed with activated Caspase3
(green) and Vasa (red) antibodies. Activated caspase3 is only present in
cells undergoing apoptosis. Panels A, C, and G show both Vasa and
activated Caspase3; panels B, D, and F show only activated Caspase3.
Arrows indicate Vasa-positive cells with activated Caspase3. In some
cases, little Vasa remains. No activated Caspase3 was detected in
PGCs of similarly staged wild-type embryos (n = 20 embryos; .100
PGCs). For this reason, the control is not shown here. The experiment
was repeated twice; numbers in the text represent a single trial.
Figure 5 Defective spectrosomes in pgc-PGCS. Wild-type and pgc-
embryos were probed with alpha-Spectrin (green) and Vasa (red) anti-
bodies. Panels A, C, and E show both the signals, whereas panels B, D,
and F show only the Spectrin-specific signal. Wild-type germ cells
in coalesced embryonic gonads have characteristic large, spherical,
GSC-like spectrosomes (panel B), whereas pgc-germ cells either lack
spectrosomes altogether (panel D) or have incompletely formed spec-
trosomes (panel F). Arrows indicate GSC-like spectrosomes in wild-
type and pgc-PGCs.
Volume 2 November 2012| Nanos Is a Critical Target for PGC|
targets is primarily responsible for the detrimental effects on PGC
development. Previous studies indicate that an important function
for nos in PGC development is to maintain transcriptional quiescence
(Deshpande et al. 1999). Moreover, as is observed in nos mutant
PGCs, the initiation CTD phosphorylation PSer5 is elevated in pgc
PGCs that have reduced levels of Nos protein but not in PGCs that
have wild-type levels of Nos. Although this correlation would seem to
point to nos-dependent transcriptional quiescence, misexpression of
two known nos transcriptional targets, Sxl and even-skipped, was not
detected in pgc PGCs (Deshpande et al. 2004; Martinho et al. 2004).
Thus, it is possible that the detrimental effects of reduced Nos in pgc
PGCs arises from a failure to regulate one of the other nos targets, for
example Cyclin B mRNA translation. On the other hand, while Nos
protein is reduced in amount in a subset of blastoderm-stage pgc
PGCs, it is not completely eliminated at this point in development,
and further reductions might occur as embryogenesis proceeds. In this
case, it is possible that some of the nos transcriptional targets become
activated a bit later in development. An additional caveat is that the
methods used to assay Sxl and even-skipped expression were not the
most sensitive, and a low level of expression of these genes in a subset
of the blastoderm pgc PGCs could have been missed.
The idea that pgc might play a subordinate role to nos in germline
development and that its primary function is to ensure that Nos can
specify PGC/GSC fate would dovetail nicely with recent studies of Lai
et al. (2012) on the development of the primordial germline in the
vertebrate Xenopus. As has been previously reported for the function-
ing of nos during germline development in the Drosophila embryo, Lai
and coauthors found that Xenopus nos is required for transcriptional
quiescence, for germ cell survival and migration to the somatic gonad,
and for the process of germ cell specification. In this context, it is also
notable that the nos gene is widely conserved across the animal
kingdom, as is its function in the process of PGC/GSC specification
[for example, see Suziki et al. (2007) and Tsuda et al. (2003)]. By
contrast, nos function in abdominal segmentation in Drosophila
appears to be a specialized adaption that is likely restricted to insects.
Similarly, the pgc gene is not well conserved. These differences would
be consistent with the speculation that a pgc-like activity evolved in
flies (and presumably other insects) to ensure that the functioning of
nos in germline development is not compromised by the quite differ-
ent requirements for its activity and its regulation in the development
of the posterior soma.
We would like to acknowledge Yu-Chiun Wang and Liz Gavis for
discussions and advice on this project. For antibodies and stocks, we
thank Liz Gavis, Akira Nakamura, Paul Lasko, Paul Macdonald,
and Bloomington Stock Center. Gordon Gray provided the fly food.
Research was supported by a grant from the National Institutes of
Asaoka, M., H. Sano, Y. Obara, and S. Kobayashi, 1998
regulates zygotic gene expression in germline progenitors of Drosophila
melanogaster. Mech. Dev. 78(1–2): 153–158.
Asaoka, M., M. Yamada, A. Nakamura, K. Hanyu, and S. Kobayashi, 1999
ternal pumilio acts together with Nanos in germline development in Drosophila
embryos. Nat. Cell Biol. 1: 431–437.
Batchelder, C., M. A. Dunn, B. Choy, C. Cassie, E. Y. Shim et al., 1999
scriptional repression by the Caenorhabditis elegans germ-line protein PIE-1.
Genes Dev. 13: 202–212.
Bergsten, S. E., and E. R. Gavis, 1999
ytanslational activation but not spatial restriction of nanos RNA. Devel-
opment 126: 659–669.
Brand, A. H., and N. Perrimon, 1993
of altering cell fates and generating dominant expression. Development
Cinalli, R. M., P. Rangan, and R. Lehmann, 2008
Cell 132: 559–562.
Deshpande, G., J. Stukey, and P. Schedl, 1995
Drosophila sex determination. MCB 15: 4430–4440.
Deshpande, G., G. Calhoun, J. Yanowitz, and P. Schedl, 1999
tions of nanos in downregulating mitosis and transcription during the
development of Drosophila germline. Cell 99: 271–281.
Deshpande, G., G. Calhoun, and P. D. Schedl, 2004
nisms function to establish transcriptional quiescence in the embryonic
Drosophila germline. Development 131: 1247–1257.
Deshpande, G., G. Calhoun, T. M. Jinks, A. D. Polydorides, and P. Schedl,
2005 Nanos downregulates transcription and modulates CTD phos-
phorylation in the soma of early Drosophila embryos. Mech. Dev. 122:
Hanyu-Nakamura, K., H. Sonobe-Nojima, A. Tanigawa, P. Lasko, and A.
Nakamura, 2008Drosophila Pgc protein inhibits P-TEFb recruitment
to chromatin in primordial germ cells. Nature 451: 730–733.
Hirose, Y., and Y. Ohkuma, 2007Phosphorylation of the C-terminal do-
main of RNA polymerase II plays central roles in the integrated events of
eukaryotic gene expression. J. Biochem. 141: 601–608.
Kobayashi, S., M. Yamada, M. Asaoka, and T. Kitamura, 1996
of the posterior morphogen nanos for germline development in Dro-
sophila. Nature 280: 708–711.
Lai, F., A. Singh, and M. L. King, 2012
prevent endoderm gene expression and apoptosis in primordial germ
cells. Development 139: 1476–1486.
Leatherman, J. L., L. Levin, J. Boero, and T. A. Jongens, 2002
acts to represses transcription during the establishment of the Drosophila
germ cell lineage. Curr. Biol. 12: 1681–1685.
Lin, H., and A. C. Spradling, 1995Fusome asymmetry and oocyte deter-
mination in Drosophila. Dev. Genet. 16: 6–12.
Lin, H., L. Yue, and A. C. Spradling, 1994
agermline-specific organelle, contains membrane skeletal proteins and
functions in cyst formation. Development 120: 947–956.
Maezawa, T., K. Arita, S. Shigenobu, and S. Kobayashi, 2009
the apoptosis inducer gene head involution defective in primordial germ
cells of the Drosophila embryo requires eiger, p53, and loki function. Dev.
Growth Differ. 51: 453–461.
Martinho, R. G., P. S. Kunwar, J. Casanova, and R. Lehmann, 2004
coding RNA is required for the repression of RNA pol II-dependent
transcription in primordial germ cells. Curr. Biol. 14: 159–165.
Role for mRNA localization in
Targeted gene expression as a means
Germ cells are forever.
scute (sis-b) function in
Xenopus Nanos1 is required to
The Drosophila fusome,
Figure 6 Germ-cell loss in pgc mutants is partially rescued by ectopic
Nos. Blue and red bars correspond to PGCs/gonad in pgc and pgc;
nos: Gal4/UAS nos: tubulin 39 UTR embryos, respectively. The em-
bryonic gonads were classified based on total number of surviving
PGCs at stage 13 and beyond. This experiment was done twice. Both
experiments gave similar distributions and are tabulated together in
|G. Deshpande et al.
Nakamura, A., and G. Seydoux, 2008
germline by transcriptional repression. Development 135: 3817–3827.
Nakamura, A., R. Amikura, M. Mukai, S. Kobayashi, and P. F. Lasko,
1996Requirement for a noncoding RNA in Drosophila polar granules
for germ cell establishment. Science 274: 2075–2079.
Phatnani, H. P., and A. L. Greenleaf, 2006
of the RNA polymerase II CTD. Genes Dev. 20: 2922–2936.
Santos, A. C., and R. Lehmann, 2004
in Drosophila and beyond. Curr. Biol. 14: R578–R589.
Sato, K., Y. Hayashi, Y. Ninomiya, S. Shigenobu, K. Arita et al.,
2007Maternal Nanos represses hid/skl-dependent apoptosis to main-
tain the germ line in Drosophila embryos. Proc. Natl. Acad. Sci. USA 104:
Seydoux, G., and R. E. Bruan, 2006Pathway to totipotency: lessons from
germ cells. Cell 127: 891–904.
Seydoux, G., and M. A. Dunn, 1997
lack a subpopulation of phosphorylated RNA polymerase II in early
Less is more: specification of the
Phosphorylation and functions
Germ cell specification and migration
Transcriptionally repressed germ cells
embryos of Caenorhabditis elegans and Drosophila melanogaster. De-
velopment 124: 2191–2201.
Seydoux, G., C. C. Mello, J. Pettitt, W. B. Wood, J. R. Priess et al., 1996
pression of gene expression in the embryonic germ lineage of C. elegans.
Nature 382: 713–716.
Suzuki, A., M. Tsuda, and Y. Saga, 2007
Nanos proteins and a distinct role of Nanos2 during male germ cell
development. Development 134: 77–83.
Tsuda, M., Y. Sasaoka, M. Kiso, K. Abe, S. Haraguchi et al., 2003
role of nanos proteins in germ cell development. Science 301: 1239–1241.
Wawersik, M., and M. Van Doren, 2005
the spectrosome, a germ cell specific organelle. Dev. Dyn. 234: 22–27.
Wylie, C., 1999 Germ cells. Cell 96: 165–174.
Zalokar, M., 1976Autoradiographic study of protein and RNA formation
during early development of Drosophila eggs. Dev. Biol. 49: 425–437.
Functional redundancy among
Nanos is required for formation of
Communicating editor: B. J. Andrews
Volume 2 November 2012| Nanos Is a Critical Target for PGC|