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First published online November 9, 2007
doi: 10.1242/10.1242/dev.009159
1 State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of
Zoology
2 Graduate School, Chinese Academy of Sciences, Beijing, People's Republic of
China.
3 Department of Human Genetics, Emory University School of Medicine, Atlanta,
GA, USA.
Authors for correspondence (e-mails:
pjin{at}genetics.emory.edu)
Accepted 5 September 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Argonaute protein, Ago1, miRNA, GSC self-renewal, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Spradling et al., 2001).
Studies from several laboratories have identified the genes that are essential
for GSC fate determination. Both the bag of marbles (bam)
and benign gonial cell neoplasm (bgcn) genes are known to
act autonomously in the germline and are required for cystoblast (CB)
differentiation (Lavoie et al.,
1999; McKearin and Ohlstein,
1995). By contrast, the BMP ligands glass bottom boat
(gbb) and decapentaplegic (dpp) are expressed in
the niche cap cells and play an essential role in GSC maintenance by silencing
bam transcription to repress GSC differentiation
(Chen and McKearin, 2003a;
Song et al., 2004;
Xie and Spradling, 1998).
Independent of the bam transcriptional silencing pathway, the
self-renewal of GSCs is also controlled by two translational repressors,
Pumilio (Pum) and Nanos (Nos) (Forbes and
Lehmann, 1998; Lin and
Spradling, 1997). Pumilio is not required for bam
silencing, but acts either downstream of bam or parallel to
bam action (Chen and McKearin,
2005; Szakmary et al.,
2005). Recent data also showed that Pelota, another putative
translational repressor, plays a similar role in controlling GSC fate in a
bam-independent manner (Xi et
al., 2005). These findings suggest that cell-autonomous
translational control could contribute a great deal to GSC regulation.
The Argonaute family proteins play the central roles in small-RNA-mediated
gene regulation (Parker and Barford,
2006). In Drosophila, two subfamilies (AGO and PIWI
subfamilies) of Argonaute proteins have been characterized. Piwi, a member of
the PIWI subfamily protein, has been shown to associate with repeat associated
small interfering RNA (rasiRNA or piRNA)
(Vagin et al., 2006) and
exhibit target cleavage RNA activity
(Grivna et al., 2006;
Saito et al., 2006). By
contrast, as members of the AGO subfamily of Argonaute proteins, Argonaute 1
(AGO1) and Argonaute 2 (AGO2) have been shown to be involved in miRNA-mediated
gene regulation and in siRNA-mediated mRNA degradation, respectively
(Lee et al., 2004;
Okamura et al., 2004). In
Drosophila ovary, Piwi has been shown to play essential roles in the
maintenance of GSCs via the bam silencing pathway and the regulation
of GSC division (Chen and McKearin,
2005; Cox et al.,
2000; Szakmary et al.,
2005); however, whether AGO1 is also involved in this biological
process remains unclear. The role of the miRNA pathway in GSC fate
determination has been explored in Drosophila ovary. Previous studies
showed that loss of the loquacious (loqs) gene, which
encodes a partner protein of Dicer1 (Dcr1) in miRNA biogenesis, leads to GSC
maintenance defects (Forstemann et al.,
2005; Jiang et al.,
2005; Park et al.,
2007). However, the role of Dcr1 in the maintenance of GSC is
contradictory (Hatfield et al.,
2005; Jin and Xie,
2007), because an extensive functional analysis of Dcr1
in GSCs suggested that the miRNA pathway plays an important role in the
control of GSC division, rather than GSC self-renewal
(Hatfield et al., 2005). Thus
the role of miRNAs in GSC fate determination is still uncertain and remains to
be elucidated. Here we show that overexpression of AGO1 leads to GSC
overproliferation, whereas loss of Ago1 results in the loss of GSCs.
Combined with germline clonal analyses of Ago1, these findings
strongly support the argument that Ago1 plays an essential and
intrinsic role in the maintenance of GSCs and that an AGO1-dependent miRNA
pathway plays at least a partial instructive role in repressing GSC/CB
differentiation. Furthermore, in contrast to piwi, we show that
Ago1 is not required for bam silencing and probably acts
downstream or parallel of bam in the regulation of GSC
maintenance.
|
MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Ohlstein et al.,
2000). bamBG is a strong allele for
bam as described previously (Chen
and McKearin, 2005). piwiEP and
P{hs-gal4} have been described previously
(Chen and McKearin, 2005;
Cox et al., 2000)
Ago1k08121 has been described previously
(Jin et al., 2004;
Kataoka et al., 2001;
Williams and Rubin, 2002), and
loqsf00791 was a strong allele for loqs from the
Exelixis Collection and described previously
(Forstemann et al., 2005;
Jiang et al., 2005).
P{bamp-gfp} was described by Chen and McKearin
(Chen and McKearin, 2003b), and
P{hs-Ago1} (a gift from Dr T. Uemura, Kyoto University, Japan) was
used for rescuing Ago1 mutants
(Kataoka et al., 2001).
Ago1EMS is generated through an EMS mutagenesis;
sequencing results showed that the Ago1EMS allele contains
a C-T switch, which changes amino acid 212 Q to a stop codon in the AGO1 B
form and changes amino acid 246 Q to a stop codon in the putative AGO1 A and C
forms. The resulting truncated proteins lack both the PAZ and PIWI domains,
which are essential for Argonaute protein function
(Miyoshi et al., 2005;
Okamura et al., 2004;
Shi et al., 2004), indicating
that Ago1EMS is a null allele for the Ago1 gene;
to obtain more mutants for the further analysis of Ago1 function in
GSCs, we performed a mutagenesis through imprecise mobilization of the
P-element in Ago1k08121. Fifteen alleles, which we
referred to as Ago11Ãgo115, were
recovered, and one of them, Ago114, was characterized as
having a 16 kb deletion that covers the Ago1 coding sequence,
indicating that Ago114 is another null allele for the
Ago1 gene.
Immunohistochemistry and microscopy
Ovaries were prepared for reaction with antibodies as described previously
(McKearin and Ohlstein, 1995).
Monoclonal anti-Bam antibody (McKearin and
Ohlstein, 1995) was used at a 1:500 dilution, polyclonal anti-Vasa
antibody (Santa) was used at 1:200 dilution, polyclonal anti-GFP antibody
(Invitrogen) was used at a 1:5000 dilution, monoclonal anti-Hts antibody was
used at a 1:500 dilution, and mouse anti-AGO1 (a gift from Dr M. Siomi and Dr
H. Siomi, Institute for Genome Research, University of Tokushima, Japan)
(Miyoshi et al., 2005) was
used at a 1:200 dilution. Secondary antibodies used were goat anti-mouse Alexa
568, goat anti-rabbit Alexa 488, and goat anti-rat Cy3 (Molecular Probes), all
at 1:200. All samples were examined by Zeiss microscope and images were
captured using the Zeiss Two Photon Confocal LSM510 META system supported by
the State Key Laboratory of Biomembrane and Membrane Biotechnology and
Institute of Zoology, CAS. Images were further processed with Adobe Photoshop
6.0.
Phenotypic assay for quantification of GSC maintenance in mutant adult ovaries
Ovaries isolated from wild-type and different mutant flies of different
ages were incubated with anti-Hts antibody, anti-Vasa antibody and DNA dyes to
identify terminal filament cells, fusomes and germ cells. We scored as GSCs
any Vasa-positive germ cells at the anterior position that appeared close to
cap cells or to the basal cells of terminal filaments and also carried
spherical fusomes at the anterior position or extending fusomes when a GSC was
dividing.
Germline clonal analysis
FLP/FRT-mediated recombination was used to generate Ago1 mutant
GSC and PGC clones. To generate GSC clones, w; FRTG13, Ago1/CyO
(w; FRTG13/CyO as the control) males were crossed to virgin females
of w hsFlp; FRTG13, ubi-gfp, and 3-day-old female progenies lacking
the CyO chromosome underwent heat-shock treatment at 37°C for 60
min twice daily at 12 hourly intervals. Ovaries dissected from {hs-flp;
frtG13,/frtG13, ubi-gfp} or {hs-flp; frtG13, Ago1/frtG13,
ubi-gfp} were stained with anti-GFP and anti-Hts antibodies for
quantification of GSC clones. GSC clones were identified by the lack of GFP
expression and carrying anterior-positioned spectrosome. To analyze GSC
establishment, for adult GSC assay, the heat-shock treatment was started at
the early third larval stage or early to induce PGC clones. GSC clones with
negative GFP in {hs-flp; frtG13, Ago1/frtG13, ubi-gfp} newly eclosed
females were quantified to calculate the rate of GSC clones. In this
experiment, {hs-flp; frtG13,/frtG13, ubi-gfp} was used as the FRT
control. For pupa GSC clonal assay, the progenies from the cross of w;
FRTG13, Ago1/CyO and w hsFlp; FRTG13, ubi-gfp began to be
treated by constitutive heat-shock from the first instar larval stage;
meanwhile, the progenies from the cross of w; FRTG13/CyO and w
hsFlp; FRTG13, ubi-gfp were used as FRT control. After staining with
anti-GFP and anti-Hts antibodies, female gonads containing GFP-negative germ
cells were examined and putative GSC clones were identified by their anterior
position close to the terminal filament and their lack of GFP expression.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In
this study, we distinguished GSCs from differentiated germ cells by using
anti-Vasa and anti-Hts antibodies to visualize germ cells and fusomes,
respectively. The fusome (also called a `spectrosome' in GSCs/CBs) is a
germ-cell-specific organelle that is morphologically spherical in GSCs/CBs or
extends from anterior position to posterior position when a GSC is dividing,
but is branched in differentiated cysts
(Fig. 1A). To assess the
potential role of ectopic AGO1, we scored the number of germ cells carrying
spectrosomes per germarium that came from wild type, and
P{hs-Ago1} flies at 0, 6 and 10 days heat-shock treatment.
As a positive control we used piwiEP; P{hs-gal4}
to induce ectopic GSC-like cells by overexpression of piwi as
described previously (Cox et al.,
2000). As shown in Fig.
1B, before heat-shock treatment, we observed averages of 3.3
(n=128), 3.2 (n=102) and 3.2 (n=95)
spectrosome-containing germ cells per germarium in wild-type,
P{hs-Ago1}, and piwiEP;
P{hs-gal4}, respectively, suggesting that there was no difference in
the number of spectrosome-containing germ cells among wild-type,
P{hs-Ago1}, and piwiEP;
P{hs-gal4} flies without heat-shock treatment. However, after 6 days
of heat-shock treatment (three times per day), we observed averages of 4.9
(n=105) and 5.6 (n=123) spectrosome-containing germ cells
per germarium in P{hs-Ago1} and piwiEP;
P{hs-gal4} ovaries, respectively. We noted that 25.7%
(n=105) of P{hs-Ago1}, and 43.7% (n=145)
of piwiEP; P{hs-gal4}, germaria contained more
than six spectrosome-containing germ cells
(Fig. 1B). As ovaries from
wild-type control females undergoing the same treatment carried only 3.3
spectrosome-containing germ cells per germarium (n=131), we excluded
the possibility that the increase in the number of spectrosome-containing germ
cells could be due to heat-shock treatment. We also found that, by extending
heat-shock induction to 10 days, the average number of spectrosome-containing
germ cells in P{hs-Ago1} and piwiEP;
P{hs-gal4} ovaries was increased to 5.9 (n=176) and 6.5
(n=103) per morphological normal germarium (tumorous germaria found
in P{hs-gal4} were excluded in this quantification). In this case, we
found that 56.5% (n=176) of P{hs-Ago1} and 64.2%
(n=112) piwiEP; P{hs-gal4} germaria
contained more than six spectrosome-containing germ cells. However, for
wild-type control under the same conditions, the number of
spectrosome-containing germ cells was still maintained at 3.2 (n=115)
per germarium. Thus, similar to piwi, the ectopic expression of AGO1
could also increase the number of GSC-like cells. Interestingly, in contrast
to overexpression of piwi, a certain percentage (variable at 5-10%,
n>100) of P{hs-Ago1} germaria were
morphologically tumorous-like, when they were treated with 10 day heat-shock.
As shown in Fig. 1C, GSC-like
cells (Fig. 1C left and right
panels and see Fig. S3 in the supplementary material) and differentiated cysts
(Fig. 1C right panel) were
filled in these tumorous germaria. Thus, the increased AGO1 activity could
also potentially induce the overproliferation of GSC-like cells, probably by
delaying GSC/CB differentiation, and disrupt normal oogenesis.
|
;
Kataoka et al., 2001;
Williams and Rubin, 2002),
leads to a loss of function of Ago1 that causes homozygous mutant
lethality at the embryonic stage. To analyze the specificity of the
Ago1k08121 allele in germ cell development we generated
revertants of the Ago1k08121 allele. We found that the
homozygous animals of precise excision lines were as viable and fertile as
wild type, indicating that, apart from Ago1, no other genetic
background mutations in the Ago1k08121 flies could
possibly be affecting lethality or germ cell development. This finding is also
consistent with previous data showing that Ago1k08121
specifically results in the loss of function of Ago1
(Jin et al., 2004;
Kataoka et al., 2001;
Williams and Rubin, 2002). To
obtain more mutant alleles with different genetic backgrounds for further
analysis of Ago1 function in GSCs, we performed EMS and imprecise
excision mutagenesis to generate an EMS allele (Ago1EMS)
and an imprecise excision line (Ago114) that were null for
Ago1 (see Materials and methods for details). All three mutants of Ago1 (Ago1k08121, Ago1EMS and Ago114) used in this work are lethal at embryonic stage, but homozygous and trans-heterozygous mutant animals could survive to adulthood when the transgene P{hs-Ago1} was introduced and the flies were treated with daily heat shock. Thus, by immediately withdrawing the heat-shock treatment after adult eclosion, the animals became progressively Ago1-deficient as the protein decayed (Fig. 2F). We found that ovaries from 3-day-old Ago1 mutant flies (Fig. 2A) showed no phenotype differences compared to wild-type control flies. Strikingly, however, 15-day-old Ago1 mutant ovaries exhibited strong defects in GSC maintenance. As illustrated in Fig. 2 and Table 1, for 15-day-old Ago1-deficient ovaries (from hs-Ago1; Ago1k08121/Ago1k08121 following heat-shock withdrawal after eclosion), only a few germaria (6.3%, n=79) had a normal-looking structure containing two stem cells, and about 19.0% (n=79) of germaria contained a single stem cell; most of the germaria (74.6%, n=79) contained either differentiated germ cells with branched fusomes only (45.6%) or no germ cells at all (29.1%). In the latter case, germaria composed only of somatic cells were still visible by staining with Hoechst and anti-Hts antibody. We observed similar results with the other two combinations of different Ago1 alleles (hs-Ago1; Ago1k08121/Ago1EMS and hs-Ago1; Ago1k08121/Ago114) undergoing the same treatment (Fig. 2 and Table 1). It would therefore appear that, in contrast to controls, progressively reducing Ago1 activity causes the loss of GSCs. To further exclude the possibility that the phenotype seen in germ cells from these alleles is due to genetic background differences, we treated mutant flies by constitutive daily heat-shock treatment once the fly eclosed. We found that the loss of GSCs and other developmental abnormalities of the germ cells caused by Ago1 deficiency could be rescued by exogenous AGO1 protein (Fig. 2E). Taken together, these results suggest that AGO1 plays an important role in germline stem cell maintenance and possibly controls other aspects of germ cell development as well.
|
|
). The marked mutant GSCs were identified by the
lack of GFP expression and by carrying an anterior-positioned spectrosome. The
three alleles (Ago1k08121, Ago1EMS and
Ago114) of Ago1 were used to generate marked
mutant GSC clones for analysis of Ago1 function in GSCs. The
percentages of Ubi-GFP-marked GSCs were measured at 2, 10 and 20 days after
heat-shock treatment (AHST). As shown in
Fig. 3A-C and
Table 2, for wild-type control
GSC clones, the percentage of marked clones was about 30.0% at day 2, which we
considered the initial percentage, and this was reduced to 19.2% by day 20
AHST, suggesting that the marked clone loss rate was roughly 36% over the 20
day period; however, for Ago1k08121 mutant GSC clones
under the same conditions, the initial percentage of marked clones was
measured at 30.3%, but this fell to 3.9% at day 20 AHST, suggesting a loss
rate for Ago1k08121 of about 90% during the testing period
(Fig. 3D-F and
Table 2). We obtained similar
results with the two other alleles, Ago114 and
Ago1EMS. We observed that all the marked GSC clones were
lost (100% loss rate) in both these alleles over the 20 day testing period, as
shown in Table 2. Taken
together, these findings support the argument that AGO1 is intrinsically
required for GSC maintenance.
|
|
; Zhu and Xie,
2003) were marked (GFP-), in contrast to 24.2% (n=99) of
anterior germ cells (putative GSCs) in FRT controls. Given that the half-life of Ago114 mutant GSCs is no less than 5 days (based on our Ago1 clonal assay), and that very low rate of Ago1 GSC clones from PGC clones were marked compared with FRT controls, we excluded the possibility that Ago1 contributed only to GSC maintenance and proposed that Ago1 could be important for GSC establishment.
To explore whether Ago1 is involved in controlling the rate of GSC
division, we investigated the ability of Ago1 mutant GSC-producing
cysts at day 10 post-heat-shock inductions. For FRT controls, we found 52
germline cyst clones in the presence of 19 GSC clones, whereas for
Ago114 mutants we observed 30 cyst clones in the presence
of 13 GSC clones. As the relative percentages of marked GSCs were 83.3 and
41.6% for FRT controls and Ago114 at day 10 after
heat-shock induction, respectively (shown in
Table 2), this suggests that
83.3% of FRT control cysts and 41.6% of cysts came from the examined GSC
clones. So we deduced that each FRT control GSC clone could produce an average
of 2.3 cyst clones, whereas each Ago114 GSC clone could
produce an average of 0.96 cyst clones. Consistent with the previous findings
of the clonal assay for Dcr1 mutant GSCs
(Hatfield et al., 2005), it
appears that the loss of Ago1 reduces the rate of GSC division.
The microRNA pathway is not required for bam silencing and probably acts downstream of or parallel to bam action
It has been shown previously that BMP/Dpp-dependent bam silencing
represents the primary pathway for GSC self-renewal
(Chen and McKearin, 2003a;
Song et al., 2004). To test
whether Ago1 is involved in bam silencing in GSCs, we
examined BamC expression in both wild-type and marked Ago1 mutant GSC
clones. As shown in Fig. 4A,B,
both wild-type (n>100) and Ago1 GSC clones
(n>100) were BamC-negative, suggesting that Ago1 is not
required for bam silencing. As Ago1 is a key component of
the miRNA pathway, and given the potential role of miRNAs in stem cell
biology, we decided to explore whether other components of the miRNA pathway
might modulate GSC fate in a similar manner as well. One good candidate for
such a modulator is Loquacious (Loqs), which functions together with Dcr1 and
AGO1 to guide miRNA biogenesis (Forstemann
et al., 2005; Jiang et al.,
2005; Saito et al.,
2005). Consistent with two previous studies, we also found that
the loqsf00971 mutant displays defects in GSC maintenance
(see Table S1 in the supplementary material)
(Forstemann et al., 2005;
Jiang et al., 2005). To
determine whether loqs is required for bam silencing, we
then examined the expression of bam reporters in loqs mutant
GSCs, as described previously (Chen and
McKearin, 2005). As shown in
Fig. 4D, 85.5% (n=101)
of newly eclosed loqs-deficient flies carrying P{bamP-GFP}
reporters showed a completely negative GFP pattern in putative GSCs
(Fig. 4D). As loqs
mutants cause complete GSC loss in some cases (about 36.3% at day 2 after
eclosion), we further examined P{bamP-GFP} reporters
(Chen and McKearin, 2003b) in
loqs and bam double mutant flies that preserve GSCs in a
majority of cases. In contrast to what was observed in piwi and
bgcn double mutants (Chen and
McKearin, 2005), our results revealed that, as with GFP patterns
in the wild-type and bam single mutants
(Fig. 4C,E), 87% of GSCs
(n=150 germaria) exhibited a completely GFP-negative pattern in
loqs and bam double mutants
(Fig. 4F), indicating that the
regulation of GSCs mediated by loqs does not require
bam+ activity.
|
|
). In order to further verify this, we next generated
Ago1 and bam double mutant flies by using the transgene
P{hs-Ago1} with daily heat-shock treatment. Consistent with
the analysis of loqs and bam double mutants in this study,
we found that about 80% of tumorous germaria (n=102)
(Fig. 5B) from 15-day-old flies
with {hs-dAgo1; Ago1k08121;
bam86/bamBG} (immediately withdrawing the
heat-shock treatment after adult eclosion) contained differentiated germ cells
with highly branched fusomes (Fig.
5B); by contrast, in ovaries (n>100) from bam
single mutants at the same age {bam86/bamBG},
no differentiated germ cell was observed
(Fig. 5A). Taking these results together, we conclude that Ago1 and loqs are not involved in a bam-silencing pathway to regulate GSC fate. Given that bam+ is dispensable for the loss of GSCs in loqs or Ago1 mutants, we propose that the microRNA pathway probably represses GSC differentiation downstream of or parallel to bam.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). As
members of the PIWI subfamily, Aubergine (Aub) and Piwi play important roles
for pole cell formation (Harris and
Macdonald, 2001; Megosh et
al., 2006). Piwi has been shown to be crucial for the maintenance
of GSCs. A recent study showed that AGO3, another member of PIWI subfamily,
has a similar function to Piwi and associates with rasiRNAs
(Gunawardane et al., 2007).
These findings suggest that PIWI subfamily Argonaut proteins play important
roles in development. In this study, we analyzed the function of AGO1, a
member of the AGO subfamily of Argonaute proteins in GSCs. We showed that
overexpression of AGO1 leads to GSC overproliferation, whereas loss of
Ago1 results in the loss of GSCs. Combined with germline clonal
analyses of Ago1, these findings strongly suggest that AGO1, as a
member of the AGO subfamily also plays an essential role in the maintenance of
GSCs. Given that an AGO1 serves as an important component in the miRNA
pathway, we propose that the AGO1-dependent miRNA pathway plays at least a
partial instructive role in repressing GSC/CB differentiation. Furthermore, in
contrast to previous observations of Piwi function in GSCs, we found that
Ago1 is not required for bam silencing and probably acts
downstream of or parallel to bam action in the regulation of GSC
maintenance.
|
). Based on the data that Loqs functions selectively
in the biogenesis of specific miRNAs (Park
et al., 2007), and the recent results showing that Ago1
and Ago2 act in a partially redundant manner to control key steps in
the midblastula transition and segmental patterning
(Meyer et al., 2006), we
speculate that Dcr1 may have more functions than either loqs or
Ago1 alone (or together). It is possible that Dcr1, loqs and
Ago1 are all required for GSC maintenance; however, in some cases,
even in the absence of Loqs and AGO1, Dcr1 can collaborate with AGO2 to
execute some specific miRNA functions. Recent data have shown that the
Notch/Delta signal plays an important role in controlling both niche and GSC
fates (Ward et al., 2006).
Previous data also demonstrated that Notch signaling was negatively regulated
by the miRNA pathway (Kwon et al.,
2005). Therefore, it is possible that Dcr1 is not only required
for GSC maintenance, but also required for some specific miRNA function to
promote GSC differentiation. In Dcr1-null GSCs, the loss of certain
classes of miRNAs causes GSCs to differentiate; however, the loss of different
miRNAs might lead to the upregulation of Delta activity in GSCs,
which in turn upregulates Notch activity in somatic cells. Conversely, as a
feedback signal, overexpression of Notch in somatic cells represses
or delays GSC differentiation; therefore the determination of
Dcr1-null GSC fate is balanced back to normal. Hence it is likely
that the miRNAs play key roles in GSC maintenance. Importantly in this study, we showed, for the first time, that overexpression of Ago1 can potentially repress GSC/CB differentiation and result in the over-proliferation of GSC-like cells, suggesting that AGO1-dependent miRNAs play at least a partial instructive role in regulating GSC fate. Given the multiple functions of AGO1 in the miRNA pathway, the increase in GSC-like cells could be interpreted to mean that the overexpression of Ago1 probably enhances either the efficiency of specific miRNA(s) production and/or the stability of mature miRNAs to repress the transcriptional or translational activity of the target mRNAs required for the differentiation of pre-cystoblasts (pre-CBs)/CBs, thereby resulting in delayed differentiation of GSCs/CBs.
In the previous model, both BMP/Dpp-dependent bam transcriptional
silencing and the bam-independent pathway are required for GSC
maintenance (Chen and McKearin,
2005; Maines et al.,
2007; Szakmary et al.,
2005; Xi et al.,
2005). Our genetic evidence suggests that the regulation of GSC
self-renewal mediated by the miRNA pathway acts in a
bam-silencing-independent manner. Given the role of miRNAs in
translational regulation, we favor a model in which the translational control
of GSC fate determination may be partially via the miRNA pathway, although the
possibility remains that some selective miRNAs could directly modulate the
stability of specific mRNAs required for GSC/CB differentiation. Similarly,
two other groups reported that Dcr-1 and Loqs, both important components of
the miRNA pathway, are also required for GSC maintenance
(Jin and Xie, 2007;
Park et al., 2007). The
question becomes how the microRNA pathway regulates the fate of GSC. Previous
and current studies showed that Dcr1, loqs and Ago1 are all
not involved in bam transcriptional silencing
(Hatfield et al., 2005;
Park et al., 2007), suggesting
that regulation of GSC fate by microRNAs does not go through a
dpp-dependent bam silencing pathway. A recent study
(Park et al., 2007) showed
that no germ cells can differentiate in loqs and bam;
however, in our study, we observed that at least 10% of germ cells started to
differentiate in loqs; bam double mutants (this study), as well as in
loqs; bgcn double mutant ovaries (data not shown). Consistently, a
similar phenotype was observed in the analysis of Ago1; bam
double mutants, suggesting that Loqs and AGO1 probably act independently of
Bam action.
Given that the Ago1-dependent microRNA pathway plays a major role in translational control, we propose that, aside from the bam silencing pathway, the Ago1 contributes to GSC fate determination either in conjunction (Fig. 6A) or in parallel (Fig. 6B) with the pathway of translational control of Nos/Pum. Overall, our data suggest that miRNA, as an important global regulatory mechanism, plays vital roles in stem cell biology.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/23/4265/DC1
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ACKNOWLEDGMENTS |
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|
Footnotes |
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|
REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Asaoka, M. and Lin, H. (2004). Germline stem
cells in the Drosophila ovary descend from pole cells in the anterior region
of the embryonic gonad. Development
131,5079
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Chen, D. and McKearin, D. (2003a). Dpp signaling silences bam transcription directly to establish asymmetric divisions of germline stem cells. Curr. Biol. 13,1786 -1791.[CrossRef][Medline]
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