© 2014. Published by The Company of Biologists Ltd | Development (2014) 141, 259-268 doi:10.1242/dev.101410
RESEARCH ARTICLE
STEM CELLS AND REGENERATION
Zfrp8/PDCD2 is required in ovarian stem cells and interacts with
the piRNA pathway machinery
ABSTRACT
The maintenance of stem cells is central to generating diverse cell
populations in many tissues throughout the life of an animal.
Elucidating the mechanisms involved in how stem cells are formed
and maintained is crucial to understanding both normal
developmental processes and the growth of many cancers.
Previously, we showed that Zfrp8/PDCD2 is essential for the
maintenance of Drosophila hematopoietic stem cells. Here, we show
that Zfrp8/PDCD2 is also required in both germline and follicle stem
cells in the Drosophila ovary. Expression of human PDCD2 fully
rescues the Zfrp8 phenotype, underlining the functional conservation
of Zfrp8/PDCD2. The piRNA pathway is essential in early oogenesis,
and we find that nuclear localization of Zfrp8 in germline stem cells
and their offspring is regulated by some piRNA pathway genes. We
also show that Zfrp8 forms a complex with the piRNA pathway protein
Maelstrom and controls the accumulation of Maelstrom in the nuage.
Furthermore, Zfrp8 regulates the activity of specific transposable
elements also controlled by Maelstrom and Piwi. Our results suggest
that Zfrp8/PDCD2 is not an integral member of the piRNA pathway,
but has an overlapping function, possibly competing with Maelstrom
and Piwi.
KEY WORDS: Somatic stem cells, Germline stem cells, piRNA
pathway, Drosophila
INTRODUCTION
Gene expression in multicellular organisms is regulated at many
levels. Complex transcriptional control is followed by processing,
transport and translation of all mRNAs. Small RNAs function in all
of these steps by guiding the regulatory protein complexes to
specific RNA and DNA targets. One class of small RNAs is the
repeat-associated small interfering RNAs (rasi-RNAs) or, more
commonly, Piwi-interacting RNAs (piRNAs) (Saito et al., 2006;
Vagin et al., 2006; Brennecke et al., 2007). These 23-31 nt small
RNAs are produced by Dicer-independent mechanisms and
associate with Piwi family Argonaute (AGO) proteins. Both RNAs
and protein components of the piRNA pathway are most abundantly
expressed in the gonads of all animals, but recent studies suggest
that the pathway may similarly function in other somatic stem and
progenitor cells (reviewed by Juliano et al., 2011; Siddiqi and
Matushansky, 2012; Mani and Juliano, 2013; Peng and Lin, 2013).
Much of the progress in understanding piRNA pathway
mechanisms comes from Drosophila ovaries, where piRNAs
Rutgers University, Department of Molecular Biology, Waksman Institute, Cancer
Institute of New Jersey, 190 Frelinghuysen Road, Piscataway, NJ 08854, USA.
*These authors contributed equally to this work
‡
Authors for correspondence ([email protected];
[email protected])
Received 17 July 2013; Accepted 15 October 2013
suppress the activity of transposable elements (TEs) and protect
genome integrity during germ stem cell (GSC) differentiation and
oocyte development (reviewed by Siomi et al., 2011; Guzzardo et
al., 2013; Peng and Lin, 2013). Different sets of TEs are active in
the ovarian germline and surrounding somatic cells and are
regulated by piRNA mechanisms that partially overlap (Malone et
al., 2009). The pathway used in the somatic cells is called the
primary piRNA processing pathway. The primary single-stranded
RNAs are transcribed from piRNA clusters and exported into the
cytoplasm. Their maturation requires the RNA helicase Armitage
(Armi) (Klattenhoff et al., 2007; Olivieri et al., 2010; Saito et al.,
2010; Qi et al., 2011), co-chaperone Shutdown (Shu) (Munn and
Steward, 2000; Olivieri et al., 2012; Preall et al., 2012),
endoribonuclease Zucchini (Zuc) (Pane et al., 2007; Nishimasu et
al., 2012), and soma-specific Tudor domain-containing RNA
helicase, Yb [Fs(1)Yb – FlyBase] (Olivieri et al., 2010; Saito et al.,
2010; Qi et al., 2011). Then primary piRNAs complex with Piwi and
are targeted to the nucleus (Cox et al., 2000; Ishizu et al., 2011;
Darricarrère et al., 2013). Current studies suggest that Piwi silences
TEs at the transcriptional level by inducing chromatin changes at
genomic TE sites (Brower-Toland et al., 2007; Klenov et al., 2007;
Sienski et al., 2012; Huang et al., 2013; Le Thomas et al., 2013;
Rozhkov et al., 2013).
TE silencing in the germline requires two additional Piwi family
proteins, Aubergine (Aub) and Argonaute 3 (AGO3) (Harris and
Macdonald, 2001; Vagin et al., 2004; Brennecke et al., 2007;
Gunawardane et al., 2007; Li et al., 2009). Unlike Piwi, Aub and
AGO3 are cytoplasmic proteins. They mainly localize to the
germline-specific perinuclear structure called the nuage (Harris and
Macdonald, 2001; Brennecke et al., 2007; Lim and Kai, 2007; Patil
and Kai, 2010). The nuage is thought to serve as a docking site for
assembly of the piRNA machinery and as a site of ‘ping-pong’
piRNA amplification (Gunawardane et al., 2007; Lim and Kai,
2007; Ishizu et al., 2011; Siomi et al., 2011). The nuage contains
many other conserved components of the piRNA pathway, including
Vasa (Vas), Spindle E (Spn-E) and Maelsrom (Mael) (Findley et al.,
2003; Vagin et al., 2004; Klenov et al., 2007).
Zinc finger protein RP-8 (Zfrp8), PDCD2 in vertebrates, is a
conserved protein with unknown molecular function (Minakhina et
al., 2007). All Zfrp8/PDCD2 proteins share a zinc finger, Myeloid,
Nervy and Deaf1 (MYND) domain, present in a large group of
proteins and involved in protein-protein interactions (Matthews et
al., 2009). Mammalian PDCD2 is most prevalent in the cytoplasm,
but is also detected in the nucleus, where it is associated with
chromatin (Scarr and Sharp, 2002; Mu et al., 2010).
We showed previously that Zfrp8/PDCD2 is essential in fly
hematopoietic stem cells (HSCs), but is largely dispensable in more
mature cells (Minakhina and Steward, 2010). PDCD2 is highly
expressed in human HSCs and precursor cells (Kokorina et al.,
2012; Barboza et al., 2013). Zfrp8/PDCD2 is also essential in mouse
embryonic stem cells (Mu et al., 2010), and profiling of mouse
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Svetlana Minakhina*,‡, Neha Changela* and Ruth Steward‡
embryonic, neural and hematopoietic stem cells showed an
enrichment of PDCD2 mRNA (Ramalho-Santos et al., 2002).
To investigate if the requirement of Zfrp8 is restricted to
Drosophila hematopoiesis and to obtain insight into the molecular
function of the gene, we studied the Zfrp8 phenotype in ovaries and
found that loss of Zfrp8 protein results in the abnormal development
of germline and somatic stem cell-derived cells. Importantly, we
found that Zfrp8 is essential in stem cells, as both somatic and
germline mutant stem cells stop dividing and are ultimately lost. The
phenotype can be rescued by the expression of human PDCD2,
demonstrating that the molecular function of Zfrp8/PDCD2 is
conserved. We discovered genetic interactions of Zfrp8 with piRNA
pathway genes and confirmed their close connection at the cellular
and molecular levels. We show that Zfrp8 complexes with Mael and
is required for perinuclear localization of Mael in GSCs and
cystoblasts. In turn, the nuage components Spn-E, AGO3 and Vas
are required for nuclear localization of Zfrp8. Zfrp8 regulates a
subset of germline-specific TEs, which are also regulated by Piwi
and Mael. Although Zfrp8 has weaker effects on transposon
expression than piwi and mael, it causes stronger phenotypes,
especially in stem cells. We propose that Zfrp8, though not an
integral component of the piRNA pathway, does have overlapping
function with Piwi and Mael and is particularly important in the
maintenance of stem cell identity and survival.
RESULTS
Zfrp8 is essential in follicle stem cells
Zfrp8 was originally identified by its strong hematopoietic
phenotype and lethality (Minakhina et al., 2007). Clonal analysis in
lymph glands showed that Zfrp8 is essential in stem cells, as no
persistent clones were recovered, but is dispensable in more mature
cells, as transient Zfrp8 clones had the same developmental potential
as wild-type clones (Minakhina and Steward, 2010), for clone
nomenclature see Fox et al. (Fox et al., 2008). To investigate
whether Zfrp8 may be required in other stem cells, we studied its
function in the Drosophila ovary, which contains two distinct
populations of stem cells: GSCs and somatic or follicle stem cells
(SSCs or FSCs) located in the germarium (reviewed by Kirilly and
Xie, 2007; Morrison and Spradling, 2008; Losick et al., 2011;
Spradling et al., 2011).
To determine the function of Zfrp8 in follicle stem cells, we
induced mosaic analysis with a repressible cell marker (MARCM)
clones in wild type and Zfrp8/+ (see Materials and Methods) (Lee
and Luo, 2001), and analyzed persistent clones in adult females 10
and 20 days after induction (ACI) (Fig. 1) when transient clones
were no longer detectable in the ovary (Margolis and Spradling,
1995). In wild-type ovaries 10 days ACI, we observed persistent
clones in nearly 60% of ovarioles (Fig. 1E), whereas the number of
ovarioles with Zfrp8 clones was only ~20%. Twenty days ACI, 38%
of ovarioles contained wild-type clones but only 2% contained Zfrp8
clones. Wild-type persistent clones usually included one stem cell
and ~50% of the follicle cells in one ovariole (Fig. 1A,B, arrow). By
contrast, mutant clones were often found in only few egg chambers
and were smaller (Fig. 1D), suggesting that mutant FSCs initially
undergo a few divisions but that their progeny cannot sustain normal
development. The absence of mutant stem cells or persistent clones
in older flies demonstrates that Zfrp8 stem cells stop dividing and
are lost.
Interestingly, in the absence of Zfrp8 persistent clones Zfrp8
mutant escort cells were observed with similar frequency as wildtype controls [~70% of ovarioles 10 days ACI, and ~40% of
ovarioles 20 days ACI (Fig. 1B′,C′ arrowheads, 1E)]. The location,
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Development (2014) doi:10.1242/dev.101410
morphology and numbers of wild-type and Zfrp8 escort cell clones
were similar to those observed previously (Fig. 1A-C,E) (Kirilly et
al., 2011). The presence of Zfrp8 escort cells suggests that in
contrast to the FSCs, Zfrp8 function is dispensable in these somatic
self-duplicating cells. Using the same approach we also looked for
transient Zfrp8 clones 2 and 5 days ACI. At each time point, the
number and morphology of wild-type and mutant clones were
similar. Together, our results show that loss of Zfrp8 only affects
cells derived from mutant FSCs and the FSCs themselves. The
requirement of Zfrp8 in ovarian stem cells is in agreement with our
prior observations that the gene is required in lymph gland stem
cells but not in prohemocytes (Minakhina and Steward, 2010).
Zfrp8 function in the germline
To investigate the requirement of Zfrp8 in the germline and GSCs,
we induced clones by flippase recognition target (FRT)-mediated
site-specific mitotic recombination (see Materials and Methods) (Xu
and Rubin, 1993). Phenotypes were analyzed 10 and 20 days ACI.
At both time points, we detected similar numbers of wild-type and
Zfrp8 clones (45% of ovarioles 10 days ACI and 15% of ovarioles
20 days ACI). But Zfrp8 clones showed strong phenotypes that
became more pronounced in older clones. We did not detect Zfrp8
egg chambers older than stage 8 (Fig. 2A-D) and ovarioles
containing only mutant germ cells accumulated several egg
chambers of similar size (stage 4 to 5; Fig. 2A, all egg chambers in
2D). In mosaic ovarioles, smaller Zfrp8 egg chambers were located
posterior to developmentally more advanced heterozygous cysts
(Fig. 2B). These phenotypes were observed in 40% of ovarioles 10
days ACI and 35% 20 days ACI (small egg chambers in Fig. 2D).
Twenty days ACI, 7% of Zfrp8 clones were restricted to the
germarium compared with none in control GSC clones. Thus,
similar to the phenotype observed in the soma, depletion of Zfrp8 in
the germline stem cells results in germline cysts that cannot support
normal development.
To investigate the function of Zfrp8 in GSCs further, we obtained
a short hairpin RNAi line (TRiP at Harvard Medical School, Boston,
MA, USA) and expressed Zfrp8 RNAi under the nos-Gal4 germlinespecific driver (see Materials and Methods). Ovaries from flies
carrying either the RNAi or the driver alone did not show any
phenotype, but the Zfrp8 knockdown (KD) ovaries displayed an
array of phenotypes that worsened with the age of the flies (Fig. 3).
In young flies (<2 days old) the organization of the germarium was
close to normal and contained GSCs that gave rise to a few egg
chambers (Fig. 3H,J). Ovarioles of older flies (7-10 days) either
contained a large cyst of cells instead of a well-organized germarium
and egg chambers (Fig. 3A) or ovarioles that lost most or all of the
germline cells (Fig. 3A, arrow).
To verify that the observed phenotype was specific to the loss of
Zfrp8, we complemented the nos-Gal4 Zfrp8 KD by expressing the
green fluorescent protein (GFP)-tagged human homolog of Zfrp8,
PDCD2, under the same driver. The rescued GSCs gave rise to
normal egg chambers, and the flies were fertile (Fig. 3C,D). These
results underline the strong functional conservation of the fly Zfrp8
and human PDCD2 proteins.
In both Zfrp8 germline clones and Zfrp8 KD ovaries, we observed
abnormalities in the stem cells and disruption of egg chamber
development, but depletion of Zfrp8 due to KD had a stronger
phenotype. To assess the effect of loss of Zfrp8 on GSCs, we stained
ovaries with P-MAD, a marker for TGFβ signaling between the niche
and GSCs (Kai and Spradling, 2003), and an antibody (1B1) that
stains spectrosomes and fusomes (de Cuevas et al., 1996). In wildtype GSCs round spectrosomes are located on the anterior side next
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Development (2014) doi:10.1242/dev.101410
to the cap cells that form the niche, and in dividing GSCs it forms an
asymmetric extension reminiscent of an ‘exclamation mark’
(Fig. 3E,E′, arrows). The majority of Zfrp8 GSC clones (60% in 65
total mutant stem cells analyzed) contained morphologically abnormal
spectrosomes. Frequently, there was no spectrosome visible on the
cap-side, and only a short dumbbell-shaped spectrosome connecting
the GSC to a daughter cystoblast was detected (Fig. 3F′, arrow). We
did not detect the same abnormalities in wild-type GSCs. In Zfrp8 KD
GSCs the spectrosomes were also abnormal and frequently replaced
by a fusome connecting several cells, a hallmark of more advanced
cysts (Fig. 3B,D). Interestingly, these abnormal GSCs kept their
connection to the cap cells and were positive for P-MAD, indicating
that TGFβ signaling between the niche and GSCs was not affected
(Fig. 3B, arrowhead) (Kai and Spradling, 2003).
To explore how Zfrp8 affects egg chamber formation and oocyte
development, we studied the distribution of Bicaudal D (BicD) and
Orb in early oogenesis. In normal ovarioles, both proteins become
localized to the oocyte as soon as the 16-cell cyst is formed and
control the establishment of the polar axes (Lantz et al., 1994; Oh
and Steward, 2001). In Zfrp8-depleted egg chambers BicD failed to
localize to the oocyte and remained distributed throughout all 16
cyst cells, forming aggregates, whereas Orb accumulated and was
abnormally distributed within the oocyte. Oocyte specification and
polarity is disrupted in a similar way in ovaries with defects in the
dynein motor complex and in piRNA pathway mutants, such as spnE, aub, armi, vas and mael (Paré and Suter, 2000; Navarro et al.,
2009). The abnormal distribution of the BicD and Orb proteins was
reminiscent of our findings several years ago in shutdown (shu)
mutants when we identified shu as a gene regulating BicD
localization within the egg chamber and the oocyte (Munn and
Steward, 2000). Shu protein has recently been shown to be a part of
the piRNA biogenesis machinery, acting as a co-chaperone (Olivieri
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Development
Fig. 1. Zfrp8 is required for somatic
stem cell maintenance. Wild-type
MARCM FSC persistent clones (green) in
ovariole (A,A′) and germarium (B,B′). The
number of Zfrp8 FSC clones (C-D′) is
strongly reduced 10 and 20 days ACI (E),
whereas the number of wild-type and Zfrp8
escort cell clones (arrowheads in A′-C′) is
similar (E). Abnormal, persistent Zfrp8
clones are observed 10 days ACI. FSC
clones encompass less than 50% of the
follicle cells (compare D and A). See also
inset (D′ and D″, MAX projection). The
number and appearance of wild-type and
Zfrp8 transient clones (2 and 5 days ACI)
are similar (F). MARCM clones are labeled
with CD8-GFP (green), 1B1 (red) stains
follicle cells, fusomes and spectrosomes,
DNA is shown in blue. Scale bars: 10 μm.
WT, wild type.
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Development (2014) doi:10.1242/dev.101410
Fig. 2. Zfrp8 function in germline. Germline
Zfrp8 FRT clones (A-B′) in Zfrp8/Ubi-GFP.nls
background. Mutant stem cells and their progeny
cysts are recognized by the absence of GFP.
Similar numbers of wild-type and mutant clones
were observed 10 and 20 days ACI (C), but Zfrp8
mutant cysts show delay in growth and
development that worsen with time (D). In both
mosaic ovarioles and ovarioles in which all egg
chambers were mutant, cyst development
stopped before stage 8. Undersized mutant cysts
were frequently observed posterior to more
mature wild-type cysts (B, arrows in B′, ‘Small egg
chambers’ in D). In ovarioles with all mutant egg
chambers (‘All egg chambers’ in D), cysts
appeared to reach mostly stage 4 to 5 (A,A′). 20
days ACI 7% (compare with 0% in wild type) of
mutant clones were confined to the germarium
(‘Germarium only’ in D) and showed a strong
GSC phenotype (see Fig. 3F,F′). 1B1 (red), Vasa
(red), DNA (blue). Scale bars: 10 μm.
et al., 2012; Preall et al., 2012). In addition, a number of piRNA
pathway mutants [piwi, fs(1)Yb, zuc, cuff] cause severe
abnormalities in germline development, reduction in GSC divisions
and eventual GSC loss (Cox et al., 1998; King et al., 2001;
Szakmary et al., 2005; Chen et al., 2007; Klenov et al., 2011). These
observations lead us to investigate a possible function of Zfrp8 in
the piRNA pathway.
(supplementary material Table S1A,B). The egg phenotypes of vas,
AGO3 and spn-E mutants were notably enhanced by lack of one
copy of Zfrp8, consistent with their effect on Zfrp8 nuclear
localization. These results suggest that Zfrp8 functions in the
nucleus and that its function is regulated at least in part by piRNA
pathway genes.
Nuage components alter Zfrp8 protein distribution
We further defined the function of Zfrp8 within the piRNA pathway
by looking at the distribution of piRNA pathway proteins in Zfrp8
ovary clones and Zfrp8 KD germaria. We did not observe notable
changes in Piwi nuclear localization or in the cytoplasmic distribution
of Armi (data not shown; supplementary material Fig. S3). Three
cytoplasmic proteins, Vas, AGO3 and Aub, which normally
accumulate in the nuage of nurse cells, also show similar perinuclear
localization in GSCs and cystoblasts. In Zfrp8 KD germaria their
distribution was not disrupted (Fig. 5A-B′, supplementary material
Fig. S3) suggesting that Zfrp8 does not affect the formation of nuagelike structures in the germarium. By contrast, the distribution of Mael
was clearly changed in both Zfrp8 KD and Zfrp8 GSC clones
(Fig. 5D-E′″, white arrows). In normal germaria, Mael shows strong
perinuclear accumulation (Fig. 5C,C′,E-E′″, green arrows) and is also
found in the cytoplasm and the nucleus. The nuage-like localization
of Mael was lost in Zfrp8 GSCs and cystoblasts and Mael protein
distribution appeared more uniform, reminiscent of Mael distribution
previously observed in vas and spn-E mutant ovaries, where the nuage
is disrupted (Findley et al., 2003).
In contrast to vas and spn-E, the egg phenotype of mael and the
ovary phenotype of piwi are suppressed by loss of one copy of Zfrp8
(supplementary material Table S1), suggesting that Zfrp8 has an
opposite function to that of Mael and Piwi and is required
downstream of or in parallel with the two proteins.
Zfrp8, Piwi and Mael are all found in the cytoplasm and the
nucleus. This led us to investigate whether the three proteins are
physically associated. Co-immunoprecipitation with anti-Mael
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Development
Zfrp8 is expressed at all stages of development (supplementary
material Fig. S1B; FlyBase data) (Tweedie et al., 2009; Marygold et
al., 2013). To study the tissue-specific expression and subcellular
localization of Zfrp8 we raised antibodies that specifically recognize
the protein on western blots and in tissues (see Materials and
Methods; supplementary material Fig. S1). The protein was present
in all cells of the ovary, distributed throughout the cytoplasm and
nucleus (Fig. 4A,A′).
A large number of piRNA pathway proteins are enriched in the
cytoplasm, often in the nuage (Lim and Kai, 2007; Pane et al., 2007;
Patil and Kai, 2010). By contrast, Piwi and Mael proteins are
detected in the cytoplasm, the nuage and the nucleus (Findley et al.,
2003; Klenov et al., 2011; Sienski et al., 2012), and their subcellular
distribution is regulated by the cytoplasmic factors Armi, Aub, SpnE and Vas (Findley et al., 2003; Sienski et al., 2012). To investigate
the possible connection between Zfrp8 and the piRNA pathway we
tested the subcellular localization of Zfrp8 in a number of mutants.
We found that the nuclear localization of Zfrp8 was most strongly
reduced in vas, AGO3, spn-E and squ GSCs (Fig. 4; for all tested
mutants see supplementary material Fig. S2).
To study the relationship between Zfrp8 and piRNA pathway
factors we tested whether lack of one copy of Zfrp8 can modify the
phenotypes of mutants with an established function in piRNA
biogenesis, even though Zfrp8/+ ovaries do not show a notable
phenotype. Zfrp8 dominantly enhanced or suppressed the ovary or
egg phenotypes of the majority of piRNA pathway mutants
Mael and Zfrp8 interaction
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Development (2014) doi:10.1242/dev.101410
antibody (Fig. 5F; see Materials and Methods) shows that indeed
Zfpr8 protein interacts with Mael, but no interaction between Zfrp8
and Piwi was observed in co-immunoprecipitation experiments
using anti-Piwi antibody (data not shown). Taken together, our data
show that Zfrp8 and Mael are found in the same complex, and that
perinuclear accumulation of Mael depends on Zfrp8. They have
antagonistic functions but do not control each other’s nuclear
targeting (Fig. 4E; Fig. 5E′″).
Zfrp8 and transposon activity
Of all the functions associated with the piRNA pathway, its role in
the repression of TEs has been most intensively studied. In many
piRNA pathway mutants the RNA levels of TEs is strongly
increased. We have chosen nine representatives of different TE
classes mainly expressed in somatic cells (mdg1, Gypsy), germline
cells (G-element, roo, HeT-A and TART-A) or in both soma and
germline (F-element, 297 and 412) for quantitative reverse
transcription polymerase chain reaction (RT-PCR) analysis (Fig. 6;
see Materials and Methods).
For these experiments, we used RNA prepared from wild-type
and mutant ovaries of newly eclosed flies (>10 hours old). At this
stage, Zfrp8 KD and wild-type ovaries are both small and contain
similar numbers of egg chambers. The levels of TE RNAs in these
young wild-type ovaries were 3-5 times higher (P<0.01) than those
in ovaries of 5-day-old flies that are usually the source for this type
of experiment (supplementary material Fig. S5) (Li et al., 2013).
Consequently, piRNA pathway mutants did not show as strong an
increase in the levels of expression of several TEs in our
experiments as noted by others (Klenov et al., 2011) (Fig. 6A). Lack
of one copy of Zfrp8 did not significantly affect the level of
expression of TEs in piwi, mael and aub (P>0.05 in Fig. 6A) mutant
ovaries, suggesting that the phenotypic suppression by Zfrp8 we
described above did not depend, or only partly depended, on
reduction of transposon activity.
To assess the germline-specific effects of Zfrp8 on TE expression,
we used nos-Gal4 Zfrp8 KD that strongly depleted the protein in the
germline (supplementary material Fig. S1D,D′). Out of the seven
TEs typically expressed in germline, RNA levels of two TEs, TARTA and HeT-A, were significantly increased in Zfrp8 KD ovaries (six
to eight times, P<0.01). HeT-A and TART-A are non-long-terminalrepeat (LTR) elements that constitute Drosophila telomeres
(Danilevskaya et al., 1997; Pardue et al., 2005; Shpiz and
Kalmykova, 2011; Shpiz et al., 2011). Unlike roo and most other
germline TEs, they have a clear cellular role in maintaining genome
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Fig. 3. Loss of Zfrp8 results in abnormal GSCs
and failed oogenesis. The array of phenotypes
observed in Zfrp8 KD driven by nos-Gal4 (A,B,H,J)
ovaries was rescued by expression of human Zfrp8
homolog, PDCD2 (37% identical aa sequence) in
the same genetic background (nos-Gal4, UASZfrp8 RNAi, UAS-GFP-PDCD2 C,D, green). In 10day-old females Zfrp8 KD (A,B) leads to fused
cysts and eventual loss of germline (arrow in A).
Zfrp8 KD GSCs were P-MAD (red) positive (B,
arrowhead), indicating that normal niche signaling
has occurred. However, instead of normal
spectrosomes (red in D), the Zfrp8 KD stem cells
contain fusome-like structures (green in B) typical
of wild-type cysts. Similarly, Zfrp8 mutant stem
cells were often found connected to the cystoblast
with symmetric fusomes; compare the dumbbellshaped spectrosome/fusome in Zfrp8 GSC clone
(red in F,F′, arrow) to normal round and
exclamation-mark-shaped spectrosomes (arrow
and arrowhead) in wild-type clones (red in E,E′).
Clones are recognized by the absence of GFP
(green). In younger ovaries (<24 hours from
eclosure), depletion of Zfrp8 results in abnormal
localization of BicD and Orb (G-J). In nos-Gal4
Zfrp8 KD egg chambers BicD (green) is distributed
and forms aggregates in the nurse cells instead of
accumulating in the oocyte (compare G and H).
Orb is mislocalized to the oocyte anterior (green
in J) instead of sharp lateral and posterior
localization observed in wild type (green in I). nosGal4/+ was used as a wild-type control. DNA (blue)
Scale bars: 10 μm.
Fig. 4. Zfrp8 localization in pi-RNA pathway mutants. In the wild-type
germarium (A,A′) Zfrp8 is present in all cells and evenly distributed
throughout nuclei and cytoplasm. In vas (B,B′), AGO3 (C,C′) and spnE (D,D′)
mutants, the nuclear localization of Zfrp8 is strongly reduced. mael (E,E′)
does not affect Zfrp8 nuclear localization in GSCs. Lamin (green) is used to
visualize the nuclear envelope, Zfrp8 is shown in red and as a separate
channel (white). Mutant alleles are indicated. w118 is used as a wild-type
control. Scale bars: 10 μm. WT, wild type. For complete germaria and more
mutants see supplementary material Fig. S2.
integrity, and at early stages of oogenesis both are mainly regulated
by Piwi and Mael rather than by Aub (Fig. 6A).
In the Zfrp8 somatic KD ovaries (tj-GAL4 Zfrp8RNAi) the RNA
levels of soma-specific TEs were not specifically affected, but it
must be noted that in these ovaries the level of protein expression is
not strongly reduced (supplementary material Fig. S1E). We did
observe increased expression of one germline element, roo, about
twice (P<0.003). Furthermore, in nos-GAL4 Zfrp8 ovaries the
expression of one soma-specific element, mdg1, was increased
threefold (P<0.001). These results suggest that Zfrp8 has a non-cellor non-tissue-autonomous effect on the expression of these TEs.
DISCUSSION
Our studies on Zfrp8 requirement in the Drosophila ovary show that
the gene is essential in stem cells. Our results suggest that Zfrp8 is
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Development (2014) doi:10.1242/dev.101410
not required in cells with limited developmental potential, as
transient wild-type and mutant clones were similar in number and
size. We also found no difference in Zfrp8 and wild-type escort cell
clones, indicating that Zfrp8 is not required in these cells that
multiply by self-duplication (Margolis and Spradling, 1995; Kirilly
et al., 2011). Furthermore, Zfrp8 and wild-type MARCM clones
induced in third instar larvae were indistinguishable in the adult
antenna and legs 20 days ACI (supplementary material Fig. S5).
These results support the conclusion that Zfrp8 function is primarily
required in stem cells.
Despite this functional requirement, Zfrp8 protein was not
enriched in Drosophila GSCs and FSCs. This is surprising, because
in mice Zfrp8/PDCD2 is enriched in several types of stem cell
(Ramalho-Santos et al., 2002; Mu et al., 2010). Zfrp8/PDCD2 is
also highly expressed in human bone marrow and cord blood stem
and precursor cells with protein levels decreasing significantly as
these cells differentiate (Kokorina et al., 2012; Barboza et al., 2013).
We observed that loss of Zfrp8 in the Drosophila germline did not
affect signaling from the niche to the stem cells. But the stem cells
themselves are highly sensitive to loss of Zfrp8. In both
Zfrp8germline stem cell clones and Zfr8 KD germaria we observed
abnormal spectrosomes reminiscent of fusomes. These phenotypes
suggest that these germline stem cells are losing stem identity and
show features of a stem cell and a more advanced cystocyte.
Germline and somatic stem cells and their daughter cells ultimately
stop dividing when depleted of Zfrp8 but continue to survive for
several days, as evident from the phenotype of the persistent stem
cell clones. Similarly, in leukemia and in cancer cell lines that
initially have high levels of the protein, reduction of Zfrp8/PDCD2
correlates with delay or arrest of the cell cycle rather than cell death
(Barboza et al., 2013).
The most severe abnormalities were observed 10-20 days ACI in
Zfrp8 GSC clones induced in larvae (Fig. 2) and adults (data not
shown). The phenotype of Zfrp8 KD ovarioles also became more
pronounced with age, starting from a relatively normal-looking
germarium and a few egg chambers (Fig. 3H,J) in young flies, to
ovarioles made up of disorganized cysts (Fig. 3A), and finally, to
ovarioles in which germ cells were almost entirely absent (Fig. 3A,
arrow). The temporal change in phenotype can be explained in two
ways. First, it is possible that Zfrp8 levels are initially high enough
in mutant and KD stem cells to support a few divisions and the
formation of mutant cysts. However, as Zfrp8 is gradually depleted
the cells stop dividing and are eventually lost. Alternatively, lack of
Zfrp8 may induce changes in parental cells that affect the
developmental potential of the daughter cells. For instance,
chromatin modifications could be affected in the absence of Zfrp8,
but it could take several cell generations for these changes to have
a phenotypic effect. In both these scenarios, loss of Zfrp8 would
predominantly affect cells undergoing constant or rapid divisions,
such as stem cells and cancer cells.
The loss of asymmetry in the stem cells, the mislocalization of
BicD and Orb proteins to and within the oocyte, the mislocalization
of Zfrp8 protein in GSCs of several piRNA pathway mutants, and
the genetic interaction of Zfrp8 with piRNA pathway genes
suggested a connection between Zfrp8 and the piRNA pathway. The
de-repression of the subset of transposons in Zfrp8 KD ovaries
further links the gene with the piRNA pathway.
We have tested several LTR and non-LTR retroelements that
represent three major TE classes based on their tissue-specific
activity in the germline, soma or in both tissues (intermediate)
(Malone et al., 2009; Sienski et al., 2012; Rozhkov et al., 2013).
When Zfrp8 is depleted in the germline, two out of seven
Development
RESEARCH ARTICLE
RESEARCH ARTICLE
Development (2014) doi:10.1242/dev.101410
Fig. 5. Zfrp8 and Mael interaction. (A-E′″) The
distribution of AGO3 (red in A,B, white in A′,B′) shows that
depletion of Zfrp8 (nos-Gal4 Zfrp8 KD, B,B′) does not
disrupt nuage formation in GSCs and cystoblasts.
Localization of Mael (red in C,D, white in C′,D′) to nuage
of GSCs and cystoblasts in control germaria (C,C′, green
arrows), is not observed in Zfrp8 KD germaria (D,D′).
Similarly, Mael was mislocalized in Zfrp8 GSC clones 10
days ACI (E-E′″). Compare mutant clone GSC and
cystoblast (white arrows) and corresponding
heterozygous cells (green arrows). Clones are recognized
by absence of GFP (green). Mael is shown in red; DNA is
in blue (each channel is shown separately in E′-E′″).
(F) Immunoprecipitation from wild-type ovary extracts with
anti-Mael antibody show that Zfrp8 is found in a complex
with Mael. The upper panel shows western blot of extracts
(Input) and co-immunoprecipitates (IP) probed with antiMael antibody. The lower panel shows the same western
blot probed with anti-Zfrp8. For the negative control, coimmunoprecipitation without antibody (no AB) was
performed.
are also their targets for repression. By contrast, the majority of
primary piRNAs are derived from piRNA clusters and target TEs
dispersed throughout the genome (Brennecke et al., 2007; Yin and
Lin, 2007). Furthermore, the repression of TART and HeT-A in the
germline involves an unusual combination of piRNA factors. We
found that at early stages of oogenesis they appear to be regulated
by piwi and mael, but not by the germline-specific Piwi family
member Aub (Fig. 6A). This result is in agreement with recent
studies on piwi function in the soma and germline that showed that
Fig. 6. Zfrp8 is involved in TE repression. Quantitative RT-PCR of fold accumulation of TE transcripts in young mutant ovaries relative to w118 controls
(log2 scale, mean ± s.d.; n≥3, normalized to RP49/RPL32). (A) Mutations in piwi (piwi06843/piwi2), mael (maelr20/Df(3L)ED230) and aub (aubQC42/aubHN2) leads
to TE de-repression. As expected, soma specific TEs (blue) were not affected by aub, but de-repressed in piwi. Germline-specific (yellow) and intermediate
(green) TEs were differentially affected in different mutants. Germline-specific telomeric elements HeT-A and TART-A (black and gray) are strongly derepressed in piwi and mael, but not in aub mutant ovaries. Mutants lacking one copy of Zfrp8 show insignificant changes of TE levels compared with mutants in
Zfrp8+ background (P≥0.05). (B) Fold accumulation of transcripts in young ovaries with Zfrp8 KD in the germline (nos>Zfrp8 KD), soma (tj>Zfrp8 KD) and in
controls (average nos-Gal4/+ tj-Gal4/+ UAS-Zfrp8 RNAi/+) relative to w118 ovaries (Log2 scale, mean ± s.d.; n≥6, normalized to RP49/RPL32). Major changes
were observed for HeT-A and TART-A TEs in nos>Zfrp8 KD ovaries (six to ten times, P<0.01). Other TEs, with the exception of mdg1 in nos>Zfrp8 KD and roo
in tj>Zfrp8 KD, were not significantly changed (P≥0.1).
265
Development
intermediate and germline elements tested, HeT-A and TART, show
significant de-repression. These elements are different from the
majority of Drosophila TEs. The HeT-A, TART and the TAHRE
elements are integral components of fly telomere. Their activity is
tightly regulated and is required to protect chromosome ends
(Danilevskaya et al., 1997; George et al., 2010; Shpiz and
Kalmykova, 2011; Shpiz et al., 2011). These elements, like other
TEs, are controlled by the piRNA machinery, but their primary
piRNAs are likely to be derived from the same telomeric loci that
RESEARCH ARTICLE
MATERIALS AND METHODS
Fly lines and genetic interactions
We obtained a Zfrp8 RNAi construct in the VALIUM22 vector (line
GL00541) from the TRiP at Harvard Medical School, Boston, MA, USA.
The soma-specific Zfrp8RNAi line (P{GD4600}v11521) was obtained from
the Vienna Drosophila RNAi Center (Dietzl et al., 2007). The nos-Gal4
driver (P{GAL4::VP16-nos.UTR}), alleles of aub, cuff, tud, vas and piwi2
were obtained from T. Schupbach (Princeton, NJ, USA), piwi1 and piwi2
alleles from H. Lin (New Haven, CT, USA) and tj-GAL4 (tj-GAL4/Cyo; tubGAL80ts/MKRS) from S. DiNardo (Philadelphia, PA, USA). Other mutant
alleles, balancer and deficiency stocks were from the Bloomington Stock
Center.
UAS-GFP-PDCD2 was made by RT-PCR amplification of the human
PDCD2 coding region (Barboza et al., 2013). The gene was cloned into the
Gateway vector pDONR4 (Life Technologies) and subsequently transferred
266
into pPGW (Gateway). Transgenic fly lines were created following standard
protocols (Rubin and Spradling, 1982; Brand and Perrimon, 1993). For
RNAi experiments, flies were raised at 29°C.
For genetic interaction experiments, the Zfpr8null allele (Minakhina et al.,
2007) was recombined/combined with alleles of each gene tested
(supplementary material Table S1A,B). Eggs were collected from 5- to 7day-old females and allowed to develop for 2 days at 25°C for examination
of egg phenotypes. Mutant females that produced few or no eggs were
dissected and 10-15 ovaries of 2-, 5- and 14-day-old females were analyzed.
Clonal analysis
The fly stocks used to induce MARCM clones have been previously
described (Lee and Luo, 2001; Minakhina and Steward, 2010). To induce
clones, third instar larvae and 3-day-old adults of the appropriate genotype
were exposed to 38°C for 60 minutes in the morning of two consecutive
days. Ovaries were dissected, fixed stained and analyzed 2 and 5 days ACI,
for transient clones, and 10 and 20 days ACI, for persistent clones.
The FLP/FRP site-specific recombination system was used to generate
mutant clones with a heat-shock promoter (Xu and Rubin, 1993). We
induced mutant clones in FRTG13 UAS-mCD8-GFP/FRTG13 Zfrp8null (Zfrp8)
larvae. More than 200 ovarioles of each genotype were analyzed on a Zeiss
Axioplan-2 microscope.
Zfrp8 antibody
The C-terminal Zfrp8 sequence coding for amino acids 168 to 347 of Zfrp8
was cloned into the pET15 vector expressed, and purified following the
standard protocols of the Northeast Structural Genomics (NESG) consortium
(Acton et al., 2011). Polyclonal antibodies were raised in rabbits and affinity
purified at Pocono Rabbit Farm and Laboratory (Canadensis, PA, USA).
Antibodies were tested on western blots and by immunostaining ovaries
(supplementary material Fig. S1).
Co-immunoprecipitation
Co-immunoprecipitation with monoclonal mouse anti-Mael and anti-Piwi
antibody (Saito et al., 2006; Sato et al., 2011) was performed in the presence
of RNase inhibitor RNaseOUT (Invitrogen) as previously described
(Brennecke et al., 2007). Ovaries from 50 young flies (<20 hours old) were
used for each experiment. Proteins were analyzed by western blotting using
rabbit polyclonal anti-Mael, anti-Piwi and anti-Zfrp8.
Antibodies and microscopy
Rat anti-Vas antibody (1:20) developed by A. Spradling and D. Williams
and mouse anti-1B1 antibody (1:20) developed by H. Lipshitz were obtained
from the Developmental Studies Hybridoma Bank (Iowa University, Iowa
City, IA, USA). Rabbit anti P-MAD (anti-phosphorylated Smad1, PS1) used
at 1:3500 was given to us by N. Yakoby (Rutgers University, Camden, NJ,
USA) (Persson et al., 1998; Niepielko et al., 2011). Monoclonal mouse antiMael (1:200), anti-Armi (1:200) and anti-Piwi (1:500) were a gift from M.
C. Siomi (University of Tokyo, Tokyo, Japan) (Saito et al., 2006; Sato et al.,
2011). Polyclonal rabbit anti-Aub (1:2000); anti-AGO3 (1:250) and antiPiwi (1:500) were from T. Schupbach (Princeton University, Princeton, NJ,
USA) and G. Hannon (Cold Spring Harbor Laboratory, Cold Spring Harbor,
NY, USA) (Brennecke et al., 2007). Polyclonal rabbit anti-Mael (1:200) was
a gift from H. Ruohola-Baker (University of Washington, Seattle, WA,
USA) (Findley et al., 2003). Phalloidin-546 (Invitrogen) and secondary
antibodies (Jackson Laboratories) were used at 1:300. Hoechst 33258
(1:5000) was used to stain DNA. Images were captured using a Leica DM
IRBE SP2 or Leica TSC SP5 laser scanning confocal microscope (objectives
40× and 63× oil), analyzed with Leica Microsystems software and further
processed using Adobe Photoshop.
Quantitative RT-PCR
The level of TE transcripts depends on age and may be dependent on the
stage of egg chambers (supplementary material Fig. S4). For comparative
analysis, we used young virgin ovaries (<10 hours old). RNA was isolated
from 10-20 ovaries using RNeasy Plus Mini kit (Qiagen). Quantitative RTPCR was performed as described in the manufacturer’s instructions using
Development
HeT-A and TART elements are among the TEs most strongly
regulated by Piwi in the germline (Rozhkov et al., 2013). Thus,
Zfrp8 may target the same TEs as Piwi and Mael but not those
regulated by Aub.
De-repression of TEs caused by Zfrp8 KD could be responsible
for the enhancement of developmental defects seen in piRNA
pathway mutants. For instance, the increase of TE transcripts may
enhance dorsoventral patterning defects in armi, AGO3, aub, spnE
and vas because of the competition between TE transcripts and
oocyte polarity factors for the same RNA transport machinery (Van
De Bor et al., 2005; Chen et al., 2007; Klattenhoff et al., 2007;
Navarro et al., 2009). However, the interaction of Zfrp8 with the
piRNA pathway machinery seems to be more complex. Zfrp8
enhanced the egg phenotype of only three mutants, spnE, AGO3 and
vas, and in these mutants the nuclear localization of Zfrp8 protein
was also affected. These results suggest that Zfrp8 functions
downstream of the three factors.
Both piwi and mael are dominantly suppressed by Zfrp8. Both
these factors have important nuclear functions, regulating chromatin
modifications and controlling TEs at the transcriptional level, and
both are required to repress HeT-A and TART-A elements. Zfrp8
could suppress piwi or mael by inducing a competing chromatin
modification at the genomic loci targeted by Piwi or Mael.
Chromatin modifications are generally stable through several cell
generations (Trifonov, 2011; Rando, 2012). Such a function would
therefore be consistent with the temporal changes of phenotypes in
Zfrp8 ovarian clones and KD ovarioles.
Although Piwi and Mael target the same genomic loci, no
interaction between the two proteins have been detected (Sato et al.,
2011; Le Thomas et al., 2013). Our co-immunoprecipitation
experiments suggest that Zfrp8 complexes with Mael but not with
Piwi, indicating that the observed genetic interaction between Zfrp8
and piwi may be mediated by mael. Mael is one of the most
enigmatic proteins in the piRNA pathway. It is found in the
cytoplasm, nuage and nucleus, and has been implicated in diverse
cellular processes including the ping-pong piRNA amplification
cycle in the germline (Findley et al., 2003; Aravin et al., 2009),
MTOC assembley in the oocyte (Sato et al., 2011) and Piwidependent chromatin modification in somatic cells (Sienski et al.,
2012). Zfrp8/PDCD2 is also required in the soma and germline and
may function both in the cytoplasm and in nuclei (Scarr and Sharp,
2002; Mu et al., 2010). However, in contrast to mael, Zfrp8
homozygous mutants are lethal and Zfrp8 ovaries show a stronger
phenotype. Based on the observation that Mael and Zfrp8 are found
in the same complex and that Zfrp8 dominantly suppresses Mael, we
propose that they act in opposite fashion on a common target,
whether during piRNA biogenesis or chromatin modification.
Development (2014) doi:10.1242/dev.101410
the QuantiTect SYBR Green kit (Qiagen), Smart cycler II (Cepheid) and the
relative standard curve method. Primers used for RT-PCT are listed in
supplementary material Table S2. Transcript levels were normalized to those
of Rp49. nos-GAL4/+, tj-GAL4/+ and UAS-Zfrp8RNAi/+ were used as
control ovaries, and all data were normalized to the transcript levels in w118
ovaries (baseline=1). At least two biological and two technical replicates
were performed for each genotype. Statistical significance (P-value) was
determined using two-tailed Student’s t-test.
Acknowledgements
We thank T. Schupbach, G. Desphande and E. Reilly for helpful comments on the
manuscript. The TRiP at Harvard Medical School (NIH/NIGMS R01-GM084947) is
acknowledged for providing the transgenic Zfrp8RNAi fly stock. We also thank L.
Nguyen and J. Sussman for expert fly food preparation and stock maintenance, B.
MacTaggart for the help with the genetic screen, G. Hannon, H. Ruohola-Baker, T.
Schupbach, M. C. Siomi, and N. Yakoby for antibodies, and H. Lin, S. DiNardo and
T. Schupbach for fly stocks.
Competing interests
The authors declare no competing financial interests.
Author contributions
S.M. and N.C. prepared the manuscript and performed experiments and data
analysis. R.S. conceived the studies and prepared and edited the manuscript.
Funding
This work was supported by National Institutes of Health (NIH) [NIHD018055 and
5R01GM089992]; and by the Goldsmith Foundation. Deposited in PMC for release
after 12 months.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.101410/-/DC1
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