© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 1073-1082 doi:10.1242/dev.116590
RESEARCH ARTICLE
STEM CELLS AND REGENERATION
Maintenance of Drosophila germline stem cell sexual identity in
oogenesis and tumorigenesis
ABSTRACT
Adult stem cells maintain tissue homeostasis by balancing selfrenewal and differentiation. In Drosophila females, germline stem
cells (GSCs) require Sex lethal (Sxl) to exit the stem cell state and to
enter the differentiation pathway. Without Sxl GSCs do not
differentiate and instead form tumors. Previous studies have shown
that these tumors are not caused by a failure in the self-renewal/
differentiation switch. Here, we show that Sxl is also necessary for the
cell-autonomous maintenance of germ cell female identity and
demonstrate that tumors are caused by the acquisition of male
characteristics. Germ cells without Sxl protein exhibit a global
derepression of testis genes, including Phf7, a male germline
sexual identity gene. Phf7 is a key effector of the tumor-forming
pathway, as it is both necessary and sufficient for tumor formation. In
the absence of Sxl protein, inappropriate Phf7 expression drives
tumor formation through a cell-autonomous mechanism that includes
sex-inappropriate activation of Jak/Stat signaling. Remarkably, tumor
formation requires a novel response to external signals emanating
from the GSC niche, highlighting the importance of interactions
between mutant cells and the surrounding normal cells that make up
the tumor microenvironment. Derepression of testis genes, and
inappropriate Phf7 expression, is also observed in germ cell tumors
arising from the loss of bag of marbles (bam), demonstrating that
maintenance of female sexual identity requires the concerted actions
of Sxl and bam. Our work reveals that GSCs must maintain their
sexual identity as they are reprogrammed into a differentiated cell, or
risk tumorigenesis.
KEY WORDS: Sxl, Oogenesis, Germline tumors, Jak/Stat, Phf7
INTRODUCTION
Homeostasis of adult tissues depends on a stable population of stem
cells that have the capacity to give rise to both self-renewing and
differentiating daughter cells. Many cancers exhibit stem cell-like
phenotypes, suggesting a direct link between aberrant stem cell
behavior and tumor formation (Friedmann-Morvinski and Verma,
2014). The mechanisms involved, however, remain largely unknown.
Drosophila oogenesis is a powerful model system for the study of
adult stem cells and their connection to cancer stem cells (Hudson and
Cooley, 2014; Spradling et al., 2011; Tipping and Perrimon, 2014).
Adult ovaries are composed of individual strands of developing
egg chambers called ovarioles. Each ovariole maintains a steady
population of two to three germline stem cells (GSCs) located at the tip
in a structure called the germarium. The GSCs divide asymmetrically
to give rise to one daughter that remains a stem cell and another
Department of Genetics and Genome Sciences, Case Western Reserve University
School of Medicine, Cleveland, OH 44106-4955, USA.
*Author for correspondence ([email protected])
Received 12 August 2014; Accepted 28 January 2015
daughter, called a cystoblast (CB), that commits to differentiation.
The CB then undergoes four mitotic divisions to form an
interconnected 16-cell cyst. Only one of these 16 cells differentiates
into an oocyte, while the remaining 15 cells develop as polyploid
nurse cells. An egg chamber is formed as the somatic follicle cells
surround the 16-cell cyst and bud off from the germarium (Fig. 1A).
The decision between stem cell maintenance and differentiation
is controlled by both intrinsic and extrinsic mechanisms. The GSCs,
which are located at the anterior end of the germarium, receive
differentiation-inhibiting signals from their somatic neighbors. For
example, somatic cells at the tip of the germarium activate BMP
signaling in GSCs to directly repress transcription of the
differentiation-promoting gene bag of marbles (bam) (Chen and
McKearin, 2003a,b, 2005; Song et al., 2004). Cell-intrinsic
programs, such as those carried out by the translational repressor
Nanos, are also required for stem cell maintenance (Forbes and
Lehmann, 1998; Gilboa and Lehmann, 2004; Harris et al., 2011;
Wang and Lin, 2004). Following an oriented cell division, the GSC
daughter that is specified as a CB displays an increase in Bam
protein levels and a decrease in self-renewal factors, including
Nanos protein (Chau et al., 2009, 2012; Li et al., 2009).
We recently found that the switch to the CB fate requires the
female-specific RNA-binding protein Sex lethal (Sxl) (Chau et al.,
2009, 2012). Germ cells without Sxl protein form tumors that
comprise a few bona fide GSCs located at the tip of the germarium
and cells that coexpress both Bam protein and a set of GSC markers,
including Nanos protein. Studies, showing that nanos is an Sxl target
gene and that Nanos downregulation in CB cells is controlled at the
level of translation, indicate that Sxl enables the GSC-to-CB switch by
directly inhibiting nanos translation (Chau et al., 2009, 2012; Li et al.,
2009). Although nanos is clearly necessary for tumor growth, both
loss- and gain-of-function studies indicate that the failure to regulate
nanos is not a trigger for tumorigenesis (Chau et al., 2012; Harris
et al., 2011; Li et al., 2009). Consequently, the mechanism driving
tumor formation in the absence of Sxl protein remains unknown.
Previous studies have shown that Sxl also functions in somatic
cells, where its activation in early embryogenesis is the central female
determining event (Salz, 2011; Salz and Erickson, 2010). Sxl,
however, is not essential for establishing sexual identity in the female
germline. In the absence of Sxl, germ cells develop normally and
exhibit the appropriate female-specific behaviors and expression
patterns through the end of the larval period (Casper and van Doren,
2009; Chau et al., 2009; Steinmann-Zwicky, 1994). Germ cells,
unlike somatic cells, acquire their female identity by a non-cellautonomous mechanism, as demonstrated by studies showing that the
gene expression program and behavior of embryonic XX germ cells is
masculinized if placed in a male somatic environment (Casper and
van Doren, 2009; Horabin et al., 1995; Staab et al., 1996; Wawersik
et al., 2005). Dictation by somatic signals continues through the larval
stages, after which point XX germ cells control their own sexual
development in a cell-autonomous manner.
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DEVELOPMENT
Laura Shapiro-Kulnane, Anne Elizabeth Smolko and Helen Karen Salz*
RESEARCH ARTICLE
Development (2015) 142, 1073-1082 doi:10.1242/dev.116590
In this study, we establish that, although Sxl does not play a
cell-autonomous role in germ cell sex determination, it is
necessary for the maintenance of female identity. We show that,
when germ cells lack Sxl protein, tumor formation is accompanied
by a global derepression of testis genes, including aberrant
expression of the male germline sexual identity gene PHD finger
protein 7 (Phf7) (Yang et al., 2012). We further ascertain that Phf7
drives tumor formation through a mechanism that includes the sexinappropriate activation of Janus kinase/Signal transducer and
activator of transcription (Jak/Stat) signaling in XX germ cells.
Notably, the tumor phenotype depends on paracrine signals from
neighboring somatic gonadal cells. Together with previous studies
showing that male, but not female, germ cells are able to activate
the Jak/Stat pathway in response to signals emanating from the
somatic niche (Decotto and Spradling, 2005; Kiger et al., 2001;
Leatherman and DiNardo, 2008, 2010; López-Onieva et al., 2008;
Tulina and Matunis, 2001; Wang et al., 2008), our work suggests
that tumors in this model system form because mutant germ cells
respond to their environment as if they were male germ cells.
Remarkably, derepression of testis genes, including inappropriate
Phf7 expression, is also observed in bam ovarian tumors. This
work demonstrates that female GSCs must maintain their female
sexual identity, through a mechanism that requires the concerted
actions of Sxl and bam, as they differentiate or risk tumor
formation.
RESULTS
snf148 ovarian tumors inappropriately express a large
number of testis transcripts
Here, as in our earlier studies, we investigate Sxl function in germ
cells by taking advantage of the viable sans fille (snf148) allele,
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which interferes with Sxl regulation in germ cells without disrupting
Sxl regulation and expression in the somatic cells of the ovary (Chau
et al., 2009, 2012). In wild-type females Sxl protein is present in all
female somatic cells and accumulates to high levels in the cytoplasm
of only a few germ cells located at the tip of the germarium (Chau
et al., 2009) (Fig. 1B). By contrast, in snf148 mutants the ovary is
filled with proliferating undifferentiated germ cells that fail to
accumulate Sxl protein even though Sxl protein is clearly detectable
in the surrounding somatic cells (Chau et al., 2009) (Fig. 1C).
Importantly, all aspects of the snf148 germline tumor phenotype
described to date can be restored by expression of P{otu::SxlcDNA},
a transgene that expresses Sxl cDNA under the control of a
germline-specific promoter (Chau et al., 2009, 2012; Nagengast
et al., 2003).
To gain a better understanding of why the loss of Sxl protein in the
germline leads to tumors, we used high-throughput sequencing
(RNA-seq) to compare the transcriptomes of 3-day-old snf148
tumorous ovaries with ovaries from 0- to 24-h-old virgin wild-type
females (Fig. 1D,E). Because ovaries from young wild-type females
lack late-stage egg chambers, we reasoned that this comparison would
minimize the identification of gene expression changes unrelated to
the mutant phenotype. Visualization of our RNA-seq data on the
UCSC genome browser (UCSCdm3/FlyBase r5) showed that malespecific Sxl RNA products are detectable in tumors, but not in wild
type (Fig. 1F). Sxl transcripts are sex-specifically spliced to produce
mRNAs with different coding potentials (Salz, 2011; Salz and
Erickson, 2010). In males, all transcripts include the translationterminating third exon and encode truncated, inactive proteins. In
females, the third exon is always skipped to generate proteinencoding mRNAs. Our finding that tumors contain male-specific
transcripts is consistent with earlier studies documenting that loss of
DEVELOPMENT
Fig. 1. snf148germline tumors. (A) Schematic of a wild-type Drosophila germarium. Germline cells are in shades of green. Cells of somatic origin are in shades of
blue, red and gray. Spectrosomes and fusomes are yellow. GSC, germline stem cell; CB, cystoblast; sp, spectrosome; TF, terminal filament. (B,C) Germaria
from wild-type and snf148 ovaries stained for Sxl (green) and DNA (red). The prominent cytoplasmic Sxl staining observed in wild-type GSCs and their daughter
cells is absent in snf148 tumors. Arrows mark the anterior tip of the germarium. Scale bar: 50 µm. (D) DAPI-stained ovariole from a 0- to 1-day-old wild-type female.
The white bracket indicates the germarium. The yellow bracket indicates the egg chambers. (E) DAPI-stained tumor from a 3-day-old snf148mutant female.
The arrow (D,E) marks the anterior tip of the germarium. (F) UCSC genome browser view of a portion of the Sxl locus. The screen shot is reversed so that the start
of transcription is on the left, and tracks are viewed at the same scale. Wild-type reads are in blue and snf148 reads in red. The reads that are unique to snf148
are highlighted with gray shading. RefSeq annotations (beneath) indicate that the unique read aligns to exon 3. Gray boxes indicate the untranslated exons and
black boxes represent the open reading frame. Exon 3, which includes multiple in-frame stop codons, does not produce a functional protein.
RESEARCH ARTICLE
Development (2015) 142, 1073-1082 doi:10.1242/dev.116590
Fig. 2. snf148tumors ectopically express testis-enriched genes.
(A) Examples of genes overexpressed in snf148 mutant ovaries. (B) Of the 483
genes with increased expression, 204 were not expressed in wild-type ovaries
(red), and 139 of those transcripts were annotated as testis-enriched by
ModENCODE (light blue).
The germ cell male sex determining gene Phf7 is
inappropriately expressed in tumors
Since male identity in germ cells is controlled by Phf7 (Yang
et al., 2012), we asked whether it is inappropriately expressed
in snf148 tumors. Visualization of our RNA-seq data on the
UCSC genome browser (UCSCdm3/FlyBase r5) reveals that the
Phf7-RC transcript is expressed in snf148 tumors but not in
wild-type ovaries, whereas Phf7-RA is expressed in both
samples (Fig. 3A). Published modENCODE RNA-seq analysis
of adult tissues indicates that Phf7-RC is transcribed from an
Fig. 3. The Phf7 expression pattern is altered in snf148ovarian tumors.
(A) UCSC genome browser view of the Phf7 gene locus. The screen shot is
reversed so that the start of transcription is on the left, and all tracks are viewed
at the same scale. Wild-type RNA-seq reads are in blue and snf148 RNA-seq
reads are in red. The reads that are unique to snf148 are highlighted by gray
shading. Beneath is the RefSeq gene annotation of the two Phf7 transcripts,
Phf7-RC and Phf7-RA. Gray boxes indicate the 5′ and 3′ UTRs and black
boxes represent the open reading frame. Note that both transcripts have the
potential to encode identical proteins, but differ in their 5′ UTRs. Primers used
to detect Phf7-RC by RT-qPCR are indicated by red arrowheads; primers used
to determine the total amount of Phf7 transcripts are in blue. (B) RT-qPCR
analysis of the Phf7-RC transcript in gonads isolated from wild-type females
(blue), snf148/snf148females (red) and snf148/snf148; P{otu::SxlcDNA} females
(green). Expression, normalized to the total level of Phf7, is shown as fold
change relative to snf148/snf148. Error bars indicate s.d. of three biological
replicates. (C,D) Ovaries from wild-type and snf148 animals carrying an
HA-Phf7 transgene stained for HA (red), α-Spectrin (green) to visualize
spectrosomes (a germ cell-specific organelle), and DNA (white). The
prominent HA staining observed in mutant germ cells is absent from wild type.
(E,F) Ovaries from wild-type and snf148 animals carrying an HA-Phf7
transgene stained for HA (red), Fas3 (green) to highlight the somatic follicle cell
membranes, and DNA (white). Scale bars: 50 µm.
upstream testis-specific transcription start site (TSS) (Brown
et al., 2014).
Using RT-qPCR, we confirmed that Phf7-RC RNA expression
is sexually dimorphic, with higher levels in testis than in ovaries
(supplementary material Fig. S1). Moreover, there is a significant
increase in Phf7-RC levels in tumors as compared with wild-type
ovaries, whereas the level of Phf7-RC is once more comparable
to that of wild-type ovaries in a snf148; P{otu::SxlcDNA} mutant
background (Fig. 3B). Based on these data, we conclude that
Phf7-RC misexpression is due to the lack of Sxl protein in the
snf148 mutant germline.
Although Phf7-RC has the same coding potential as Phf7-RA,
recent studies have shown that Phf7 protein expression is limited to
testis (Yang et al., 2012). There is no available antibody against
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DEVELOPMENT
Sxl activity in the snf148 germline is caused by a splicing defect that
ultimately prevents Sxl protein production (Nagengast et al., 2003).
We identified 804 genes that change expression at least twofold in
snf148 tumorous ovaries relative to wild type (P<0.05). Of these, 483
are upregulated and 321 are downregulated (supplementary material
Tables S1 and S2). Interestingly, Gene Ontology (GO) analysis
identified a set of ‘male-meiosis’ (GO:0007140) genes that are
significantly over-represented in the upregulated gene set (Fig. 2A).
Among these genes are two testis-specific transcription factors, aly
(a mip130 paralog) and rye (Taf12L – FlyBase; a Taf12 paralog),
that are required for expression of the spermatocyte transcription
program (White-Cooper, 2010). A visual survey of the most highly
overexpressed genes in tumors revealed several more genes known
to be specifically expressed in spermatocytes, such as the testisspecific transcription factor TfIIA-S2 (TFIIA gamma subunit)
(Aoyagi and Wassarman, 2000), the bromodomain-containing
proteins mitoshell (mtsh) (Bergner et al., 2010) and tbrd-1 (Leser
et al., 2012), as well as the eIF4E-3 translation initiation factor
(Hernández et al., 2012).
These observations suggested that the expression pattern of snf148
tumors includes the anomalous activation of the testis gene expression
program. Indeed, of the 483 upregulated genes, 42% (204/483) are
expressed in tumors but not in wild type (FPKM<1; Fig. 2B;
supplementary material Table S3). Moreover, an examination of the
expression data provided by the modENCODE_mRNA-seq_tissues
data sets (Brown et al., 2014) reveals that 68% (139/204) of these
uniquely expressed tumor genes are expressed in testis (RPKM≥1).
Together, these findings indicate that the tumor cells are trending
towards a male fate.
RESEARCH ARTICLE
Development (2015) 142, 1073-1082 doi:10.1242/dev.116590
Phf7. We therefore used a functional HA-tagged transgene (Yang
et al., 2012) to follow Phf7 protein expression. This analysis
confirmed that Phf7 protein expression is sex-specifically regulated,
with expression detectable in testis but not in ovary (supplementary
material Fig. S2). In contrast to wild-type ovaries, HA-Phf7 protein
is detectable in snf148 ovaries (Fig. 3C-F). In both testis and snf148
mutant ovaries, expression of the tagged protein is detectable in the
cytoplasm and in the nucleus, raising questions about whether the
localization of the tagged protein accurately reflects the distribution
of the endogenous protein. Nevertheless, our data strongly suggest
that Phf7 protein is ectopically expressed in snf148 tumors.
Inappropriate expression of Phf7 is responsible for the tumor
phenotype
Next, we tested whether snf148 tumors are dependent on the sexinappropriate expression of Phf7, using RNA interference (RNAi)
to knock down Phf7 expression. We achieved efficient depletion of
Phf7 (>99% efficiency, assayed by RT-qPCR) by driving
expression of a short hairpin RNA under the control of the
upstream activating sequence (UAS) with the germ cell-specific
nanos (nos)-Gal4 driver (van Doren et al., 1998). Remarkably,
germline-specific knockdown of Phf7 in the snf148 mutant
background suppressed the tumor phenotype (Fig. 4). Control
experiments, in which Phf7 was knocked down in the germline of
wild-type females (nos>Phf7RNAi) with comparable efficiency, had
no effect on oogenesis (data not shown), confirming previously
published data (Yang et al., 2012).
In contrast to snf148 females, which are sterile with germline
tumors, the majority of snf148; nos>Phf7RNAi females lay eggs and
many, but not all, of their ovaries contain late stage egg chambers,
indicating that inhibition of Phf7 expression suppresses the tumor
phenotype. To quantitate the efficiency of suppression, we stained
ovaries with an antibody against Orb, a marker for oocyte
determination. In the wild-type ovary, Orb protein concentrates in
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the cytoplasm of the oocyte (Fig. 4A). In the majority of snf148
mutant egg chambers, Orb staining remains diffuse, consistent with
earlier findings that the tumor is filled with germ cells that are
blocked prior to oocyte determination (Chau et al., 2009).
Similarly, Orb staining is diffuse in the majority of control
ovaries from snf148 females carrying the driver alone (Fig. 4B). On
occasion, however, we observe Orb protein accumulation in the
cytoplasm of a mutant germ cell (2.5%, n=277), even though
differentiation is clearly blocked and the ovariole remains tumorous
(supplementary material Fig. S3A). In sharp contrast, 42% (n=196)
of ovarioles obtained from snf148; nos>Phf7RNAi mutant females
contained at least one egg chamber with localized Orb staining
(Fig. 4C). Based on these data, we conclude that sex-inappropriate
expression of Phf7 is a key cause of the snf148 ovarian tumor
phenotype.
Finding that snf148 tumor formation is dependent on anomalous
Phf7 expression suggests that forcing Phf7 expression in wild-type
female germ cells might be sufficient for tumorigenesis. Previous
studies have reported that forced Phf7 expression can be achieved
by driving the expression of Phf7EY03023, an EP insertion line that
contains a UAS sequence inserted within the first intron of Phf7,
with the germline-specific nos-Gal4 driver (Yang et al., 2012).
However, it was reported that forced expression leads to an empty
gonad phenotype due to germ cell death during the larval period
(Yang et al., 2012). To bypass this early germ cell lethality, we
induced ectopic expression in the adult using a temperature shift
protocol in which tub-gal80ts; nos>Phf7EY03023 flies were reared at
the restrictive temperature (18°C) and then shifted as adults to the
permissive temperature (27°C) for 10 days to allow for significant
expression of Phf7. We found that 18% (n=156) of the Phf7expressing ovarioles showed evidence of tumor formation, based
on the number of round spectrosome-like structures present in
the germarium (Fig. 4F,G). The spectrosome is a spherical
α-Spectrin-containing structure that is normally found only in
DEVELOPMENT
Fig. 4. Ectopic Phf7 expression is
responsible for the tumor phenotype.
(A-C) Knockdown of Phf7 suppresses the
snf148 tumor phenotype. Ovarioles from
wild-type, snf148and snf148; nos>Phf7RNAi
females stained for Orb (green) and DNA
(red). In wild type the Orb protein
accumulates around the presumptive
oocyte. In snf148 tumors Orb protein does
not accumulate in any one location. In
snf148; nos>Phf7RNAi at least one site of
localized Orb accumulation was observed
in 42% (n=196) of all ovarioles scored.
Arrows mark the anterior end of the
germarium. (D-G) Forced Phf7 expression
in the female germline elicits tumor
formation. Germaria from wild-type,
snf148/snf148and nos>Phf7EY females
stained for α-Spectrin (green) to visualize
spectrosomes (sp) and fusomes (fu), and
DNA (red). Note that α-Spectrin also
stains somatic cell membranes.
(G) Quantification of the number of round
spectrosome-like structures per
germarium in wild type (WT), nos>Phf7EY
and snf148/snf148 (snf ). Scale bars: 50 µm.
RESEARCH ARTICLE
GSCs and CB cells (∼5 per ovariole). During the subsequent
divisions, the round spectrosome elongates and branches out to
form a fusome that connects the synchronously dividing germ cells
(Fig. 4D). Tumors in nos>Phf7EY03023 ovaries are composed of
cells that contain round spectrosomes or abnormal fusome-like
structures, resembling snf148 tumors (Fig. 4E,F). The low
penetrance of the tumor phenotype could be due to the
inefficient production of Phf7 protein or might indicate that
other key downstream targets in addition to Phf7 are needed to
foster tumorigenesis.
Phf7 drives tumor formation through aberrant Jak/Stat
activation
Interestingly, our RNA-seq data reveal that the levels of two Jak/
Stat pathway activating cytokines, unpaired 2 (upd2) and
unpaired 3 (upd3) (Agaisse et al., 2003; Gilbert et al., 2005;
Hombría et al., 2005), and of a downstream effector of the Jak/Stat
signaling pathway, chronologically inappropriate morphogenesis
(chinmo) (Flaherty et al., 2010), are significantly increased in
snf148 ovarian tumors (supplementary material Table S2). Jak/Stat
activity in adult germ cells is sexually dimorphic, being required in
male but not female cells (Decotto and Spradling, 2005; Kiger
et al., 2001; Leatherman and DiNardo, 2008, 2010; Tulina and
Matunis, 2001). Consistent with these earlier studies, we find that
germline-specific knockdown of Stat92E or hopscotch (hop), the
sole Jak kinase in the Drosophila genome, does not disrupt
oogenesis (supplementary material Fig. S3B,C). Together, these
Development (2015) 142, 1073-1082 doi:10.1242/dev.116590
data suggest the possibility that Jak/Stat is aberrantly activated in
the snf148 mutant germline.
Using RT-qPCR, we confirmed that there is a significant increase
in upd2, upd3 and chinmo levels in tumors as compared with wildtype ovaries (Fig. 5A). The levels of these three transcripts are once
more comparable to those of wild type in a snf148; P{otu::SxlcDNA}
mutant background, confirming that the effects that we are
observing are due to the absence of Sxl protein in the mutant
germline. Notably, we also observed a genetic dependence on
inappropriate Phf7 expression, as the levels of these three transcripts
were restored to near wild-type levels in a snf148; nos>Phf7RNAi
mutant background. These data suggest a genetic pathway in which
the lack of Sxl protein leads to anomalous Phf7 expression, which in
turn results in increased upd2 and upd3 production, inappropriate
Jak/Stat signaling, and chinmo expression (Fig. 5B).
In agreement with these findings, Jak/Stat activity, as assayed by
staining with an antibody against Stat92E, is detectable in snf148
tumors (Fig. 5C,D). Stat92E encodes the only Stat transcription
factor in the Drosophila genome, and activation of the pathway is
accompanied by increased levels or stability of Stat92E protein
(Chen et al., 2003). In wild-type ovaries, Stat92E protein is highly
expressed in the somatic cells of the germarium, but is not
detectable in the germline of wild-type ovaries (Fig. 5C). By
contrast, Stat92E is detectable throughout the snf148 tumor
(Fig. 5D).
These observations, together with studies that have connected
both aberrant Jak/Stat signaling and ectopic chinmo expression to
tumor formation in other contexts (Flaherty et al., 2010), suggest
that anomalous activation of this pathway might play a role in the
formation of snf148 germline tumors. Consistent with this
hypothesis, we find suppression of the tumor phenotype upon
inactivation of key components of the Jak/Stat pathway (Fig. 6).
When Stat92E was knocked down in a tumor background, 12%
(n=254) of the snf148; nos>Stat92ERNAi mutant ovarioles contained
at least one egg chamber with localized Orb staining (Fig. 6A).
Similarly, when hop was knocked down, 16% (n=178) of the snf148;
nos>hopRNAi mutant ovarioles contained at least one egg chamber
(Fig. 6B).
Fig. 5. Ectopic Phf7 expression leads to aberrant Jak/Stat activity. (A) RTqPCR analysis of chinmo, upd2 and upd3 transcripts in gonads isolated from
wild-type (blue), snf148/snf148(red), snf148/snf148; P{otu::SxlcDNA} (green) and
snf148/snf148; nos>Phf7RNAi ( purple) females. Expression, normalized to rp49,
is shown as fold change relative to wild type. Error bars indicate s.d. of three
biological replicates. (B) The genetic pathway leading to tumorigenesis.
(C,D) Ovarioles from wild type and snf148 tumors stained for Stat92E (green)
and DNA (red). Whereas Stat staining is limited to somatic cells in wild-type
ovaries, staining is observed throughout the snf148/snf148 mutant ovariole.
Arrows mark the anterior end of the germarium. Scale bars: 50 µm.
In wild-type ovaries, the Upd2 and Upd3 cytokines are thought to
be produced by the somatic terminal filament (TF) cells located at
the tip of the germarium (López-Onieva et al., 2008). It is possible,
therefore, that the snf148 mutant germ cells have acquired a novel
ability to receive signals from the surrounding somatic cells that
normally produce these signals. On the other hand, the increased
level of upd2 and upd3 observed in tumors is dependent on aberrant
Phf7 expression in germ cells (Fig. 5A), raising the possibility that
snf148 germ cells produce these cytokines and activate Jak/Stat in an
autocrine manner. These two possibilities are not mutually
exclusive, as the tumor phenotype might reflect a novel response
to paracrine signals as well as an additional production and response
to autocrine signals.
These scenarios may be distinguished by determining the cellular
source of the cytokines important for snf148 tumor formation. We
therefore performed tissue-specific knockdown of upd2 and upd3
in the snf148 mutant germline (with the nos-Gal4 driver), TF cells [with
the bab1-Gal4 driver (Cabrera et al., 2002)] and escort cells [with the
c587-Gal4 driver (Song and Xie, 2003)]. Interestingly, we observed
that knockdown in the germline and in TF cells, but not in escort cells,
led to suppression of the tumor phenotype (Fig. 6C). In germ cells,
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Rescue of sterility phenotypes reveals a role for both
autocrine and paracrine signaling
RESEARCH ARTICLE
Development (2015) 142, 1073-1082 doi:10.1242/dev.116590
Fig. 6. Knockdown of Jak/Stat pathway components
suppresses the snf148tumor phenotype. An ovariole was
scored as rescued when at least one site of localized Orb
accumulation was observed. (A) snf148/snf148;
nos>Stat92ERNAi and (B) snf148/snf148;
nos>hopRNAiovarioles stained for Orb (green) and DNA
(red). Arrows mark the anterior end of the germarium. Scale
bar: 50 µm. (C) Frequency of rescued snf148/snf148
ovarioles upon knocking down upd2 or upd3 in the
germline, somatic TF cells or somatic escort cells.
snf and bam ovarian tumors express a common subset of
testis transcripts
bam mutant ovaries, like snf148 ovaries, are highly enriched for
proliferating germ cells that fail to differentiate (McKearin and
Ohlstein, 1995; McKearin and Spradling, 1990). We, and others,
have previously noted the anomalous expression of several testis
transcripts in bam ovarian tumors (Chau et al., 2009; Kai et al.,
2005; Wei et al., 1994). To determine whether there is a global
depression of testis genes in bam ovarian tumors, we examined the
previously published RNA-seq analysis in which the transcriptomes
of bam tumorous and wild-type ovaries from 0- to 24-h-old females
were compared (Gan et al., 2010). From this analysis we identified
2661 genes that are upregulated at least twofold in bam tumors.
Of these, 48% (1278/2661) are expressed in tumors but not in wild
type (RPKM<1; Fig. 7A). Furthermore, 54% (692/1278) of these
uniquely expressed tumor genes are expressed in testis (RPKM≥1).
Our finding that the bam mutant gene expression program is
masculinized is further supported by RT-qPCR analysis showing
aberrant expression of the male-specific transcript Phf7-RC in
bamΔ86 ovarian tumors (Fig. 7B).
1078
Together, these expression studies suggest that the failure to
maintain sexual identity, as indicated by the global depression of
testis genes, is a common feature of both snf and bam tumors.
Interestingly, although there is a strong overlap between the
uniquely expressed genes in snf and bam tumors, the overlap is
not complete (Fig. 7C; supplementary material Table S4). We do
not yet know whether these differences are important, but our
finding that the bamΔ86 tumor phenotype is not suppressed by
germline-specific knockdown of Phf7 (supplementary material
Fig. S4) suggests that there are fundamental biological differences
between bam and snf tumors.
DISCUSSION
When housed in a normal ovary, female germ cells lacking Sxl are
unable to differentiate and instead form tumors. Although Sxl has a
pivotal role in the cell fate switch from a self-renewing GSC to a
differentiation-competent CB, tumor formation is not simply the
result of the failure of this switch (Chau et al., 2009, 2012). By
taking advantage of a snf mutant allele that specifically eliminates
Sxl protein expression in the germline, we have shown that it is the
loss of female sexual identity and the acquisition of male
characteristics that leads to tumor formation. Our finding that Sxl
jointly controls exit from the stem cell state and the maintenance of
sexual identity provides the basis for a model (Fig. 8A) in which Sxl
safeguards the previously made sexual decision as the stem cell
reprograms itself towards a differentiated fate.
Taking a genome-wide approach, we establish that Sxl is a potent
repressor of testis genes. This global analysis supports and extends
earlier studies that identified several inappropriately expressed testis
genes in ovarian tumors (Chau et al., 2009; Wei et al., 1994).
Although these mutant germ cells are not sex reversed, as they
continue to express ovary-specific genes, the degree to which they
are in fact masculinized is illustrated by our key observation that
Phf7, a gene known to be required for male germline sexual identity
(Yang et al., 2012), is expressed in the absence of Sxl. This suggests
that Sxl maintains female identity by silencing Phf7. Sex- and tissue-
DEVELOPMENT
knockdown of upd2 effectively suppressed the tumor phenotype, with
24% (n=333) of ovarioles obtained from snf148; nos>upd2RNAi
females containing at least one egg chamber with localized Orb
staining. Knockdown of upd3 in the germline was significantly
weaker, with only 2% (n=410) of snf148; nos>upd3RNAi mutant
ovarioles showing evidence of Orb protein localization. In TF cells,
both upd2-RNAi and upd3-RNAi effectively suppressed the tumor
phenotype; 41% (n=90) of snf148; bab1>upd2RNAi ovarioles and 46%
(n=166) of snf148; bab1>upd3RNAi ovarioles showed evidence of Orb
localization. By contrast, knockdown in escort cells had no effect on
the tumor phenotype; no Orb localization was observed in snf148;
c587>upd2RNAi ovarioles (n=210) or in snf148; c587>upd3RNAi
ovarioles (n=212). Overall, these studies demonstrate the importance
of both autocrine and paracrine signaling in establishing and/or
maintaining the tumor phenotype.
Fig. 7. bam and snf148 tumors ectopically express an overlapping set of
genes. (A) Analysis of previously published RNA-seq data (Gan et al., 2010)
reveals that, of the 2661 genes with increased expression in bam tumors, 1278
are not expressed in wild-type ovaries (red), and 692 of these uniquely
expressed genes are expressed in testis (blue). (B) Phf7-RC expression as
assayed by RT-qPCR in gonads isolated from wild-type, snf148 and bamΔ86
mutant females. Phf7-RC expression, normalized to the total level of Phf7, is
shown as fold change relative to snf148 animals. Error bars indicate s.d. of three
biological replicates. (C) The overlap in genes uniquely expressed by bam and
snf tumors.
specific regulation of Phf7 appears to be achieved by a mechanism
that involves alternative TSSs and translational regulation.
Although Sxl is known to inhibit translation in other contexts
(Salz and Erickson, 2010), the lack of putative Sxl binding sites
(multiple polyuridine runs of seven or more nucleotides) in Phf7
transcripts argues that Sxl is likely to regulate Phf7 expression
indirectly.
Interestingly, Phf7 encodes a methyl-histone-binding protein that
preferentially recognizes lysine 4-dimethylated histone H3
(H3K4me2) (Yang et al., 2012). We therefore propose that Sxl
maintains germline sexual identity by impacting epigenetic
regulation. In this model, the global depression of testis genes
observed in the absence of Sxl would be due to unscheduled
epigenetic changes. In this regard, we find it intriguing that
disrupting the H3K4 methylation pathway in female germ cells by
knocking down Set1, the H3K4 di- and trimethyltransferase, also
leads to germ cell tumors (Yan et al., 2014). A future challenge will
be to understand the molecular connection between Sxl, which is
an RNA-binding protein, and sexually dimorphic epigenetic
programming.
Through our pathway analysis (Fig. 8B) we establish that tumor
formation is caused by the activation of a sex-inappropriate gene
expression network that is unleashed by the loss of Sxl protein. Phf7
is a key effector of this pathway, as we have shown that it is both
necessary and sufficient for tumor formation. Our findings indicate
that Phf7 drives tumor formation through inappropriate activation of
the Jak/Stat pathway. It is intriguing that activation of Jak/Stat
signaling leads to tumor formation because in male GSCs activity is
only needed for adhesion to the somatic hub cells (Leatherman and
DiNardo, 2010). It is possible, therefore, that the tumors express a
Stat-regulated pathway that is unrelated to the pathway expressed in
the male germline. Our data also suggest that Phf7 drives tumor
formation through a mechanism that leads to increased levels of two
of the Upd cytokine family members known to activate the Jak/Stat
pathway. How inappropriate Phf7 expression leads to upregulation
of Upd cytokines remains an open question. One possibility,
Development (2015) 142, 1073-1082 doi:10.1242/dev.116590
Fig. 8. Model for the integration of maintenance of female sexual identity
with the self-renewal/differentiation switch in adult GSCs. (A) The genetic
pathways employed by a Sxl-bam partnership to control the GSC/CB switch
(left) while maintaining female sexual identity (right). (B) The genetic pathway
leading to tumor formation in germ cells lacking Sxl protein. In the absence of
Sxl protein, the presumptive GSCs located at the tip of the ovariole respond
appropriately to signals emanating from the somatic niche (black arrow). Upon
division they fail to exit the stem cell stage, continue to proliferate and form a
tumor. The tumor cells co-express GSC markers (including Nanos protein), the
differentiation marker Bam and the male germline sexual identity gene Phf7.
Ectopic Phf7 expression causes the tumor cells to interpret the signals
emanating from the somatic niche (red arrow) as if they were male germ cells,
leading to the sex-inappropriate Jak/Stat activation.
suggested by our transcriptional profiling experiments, is that Phf7
expression leads to the derepression of genes that include, but is not
limited to, testis-specific genes. Irrespective of the mechanism, the
finding that depletion of Upd2, and to a lesser extent Upd3, in germ
cells reverts the tumor phenotype indicates a requirement for
autocrine signaling. Furthermore, aberrant autocrine signaling is
consistent with data emerging from numerous studies connecting
hyperactive Jak/Stat signaling to other Drosophila tumor models
and human cancers (Amoyel et al., 2014).
A remarkable and unexpected aspect of our analysis is that
tumor formation also depends on Upd2 and Upd3 produced by
somatic cells in the neighboring microenvironment. The somatic
microenvironment at the tip of the germarium consists of three
cooperating cell types: TF cells, cap cells and escort cells. In wild
type, secretion of the Upd family of cytokines from TF cells
activates Jak/Stat signaling in cap and escort cells, but not in the
adjacent germ cells (Decotto and Spradling, 2005; López-Onieva
et al., 2008; Wang et al., 2008). The situation is different in
males, where somatic cytokine production activates Jak/Stat
in neighboring somatic and germ cells (Kiger et al., 2001;
Leatherman and DiNardo, 2008, 2010; Tulina and Matunis, 2001).
Because germ cells lacking Sxl protein are masculinized, we
hypothesize that mutant germ cells interpret the information
provided by the microenvironment as if they were male, leading to
sex-inappropriate Jak/Stat activation. Although we do not
understand how these mutant cells acquired the ability to receive
activating signals from the surrounding somatic cells, these results
highlight the importance of interactions between mutant cells
and the surrounding normal cells that make up the tumor
microenvironment in driving tumor formation.
Masculinization of the gene expression program is also observed
in bam ovarian tumors, suggesting that maintenance of sexual
identity requires both Sxl and bam. A Sxl-bam partnership is
1079
DEVELOPMENT
RESEARCH ARTICLE
RESEARCH ARTICLE
strongly supported by genetic epistasis experiments, which show
that the two proteins jointly repress expression of the stem cell
maintenance factor nos (Chau et al., 2009, 2012; Li et al., 2013).
Based on these observations we propose that Sxl and bam coregulate at least two independent pathways, one of which leads to
germ cell differentiation whereas the other maintains female identity
(Fig. 8A).
Altogether, our studies establish that sexual identity must be
maintained in an adult stem cell lineage, or risk tumor formation.
Although the gene regulatory networks that specify sex vary between
species, the need for a germ cell to remember its sexual identity is
likely to extend beyond Drosophila. In humans, germ cell tumors
occur most frequently in individuals with intersex disorders (Hersmus
et al., 2012; Kraggerud et al., 2013; Pleskacova et al., 2010). There is
also increasing evidence of a connection between testicular germ cell
tumors and the disruption of sex-specific processes (Kanetsky et al.,
2011; Koster et al., 2014; Matson et al., 2011; Turnbull et al., 2010).
Non-reproductive cells and tissues also exhibit sexually dimorphic
gene expression programs, suggesting that the failure to maintain this
dimorphism could have a role in cancer and other diseases (Bellott
et al., 2014; Nakada et al., 2014; Ronen and Benvenisty, 2014). It will
be important, therefore, to determine whether there is a mechanism to
maintain sexual identity in tissues other than gonads and whether the
failure in this process leads to disease.
MATERIALS AND METHODS
Fly stocks and culture conditions
Flies were reared on standard media at 25°C unless otherwise indicated.
Mutant alleles, transgenic, enhancer trap and Gal4 drivers used in this study
include snf148 [Bloomington Drosophila Stock Center (BDSC) #7398
(Nagengast et al., 2003)], P{HA-Phf7} (Yang et al., 2012), Phf7EY03023
(BDSC #15894), nanos-Gal4 [BDSC #4937 (van Doren et al., 1998)],
c587-Gal4 (Song and Xie, 2003), bab1-Gal4 [BDSC #6802 (Cabrera et al.,
2002)], tub-Gal80ts [BDSC #7019 (McGuire et al., 2003)] and bamΔ86
[BDSC #5427 (McKearin and Ohlstein, 1995)]. Additional information
about the Drosophila strains used in this study is available from FlyBase
(http://flybase.org).
Knockdown studies with UAS-RNAi were carried out at 27°C with the
following lines from the Drosophila Transgenic RNAi Project (TRiP; Ni
et al., 2011): Phf7-P{TRiP.GL00455} (BDSC #35807), stat-P{TRiP.
GL00437} (BDSC #35600), hop-P{TRiP.GL00305} (BDSC #35386),
upd2-P{TRiP.HMS00948} (BDSC #33988) and upd3-P{TRiP.HMS00646}
(BDSC #32859). The HMS RNAi lines are constructed in the VALIUM20
vector (upd2, upd3) and are expressed in both somatic and germline cells. The
GL RNAi lines are constructed in the VALIUM22 vector [Phf7, stat
(Stat92E), hop] and show strong expression in the female germline and weak
expression in the soma. The Gal4/Gal80ts system was used to force Phf7
expression in adult germ cells. Animals were raised at 18°C to maintain the
Gal80-dependent repression of Gal4 until adulthood. To induce Gal4 activity,
adult females were transferred to 27°C for 10 days before immunostaining.
Development (2015) 142, 1073-1082 doi:10.1242/dev.116590
discovery rate (FDR) with the Benjamini and Hochberg method and a twofold
or higher change were considered significant. GO term enrichment analysis
was performed separately on the upregulated and downregulated gene sets
using High-Throughput GoMiner (Zeeberg et al., 2005).
Gene expression analysis by RT-qPCR
Total RNA was isolated using TRIzol (Invitrogen) and treated with DNase
(Promega). RNA yield and quality were assessed with a NanoDrop
spectrophotometer (NanoDrop Technology), followed by reverse
transcription using random hexamers with the SuperScript First-Strand
Synthesis System Kit (Invitrogen). Transcript levels were measured with
Quanta PerfeCTa SYBR Fastmix (VWR) in an MJ Research PTC-200
gradient cycler. Initial activation was performed at 95°C for 30 s, followed
by 39 cycles of: 95°C for 5 s, 60°C for 15 s, 70°C for 10 s. The melting
curve was generated ranging from 50°C to 95°C with an increment of
0.5°C each 5 s. Primers used for RT-qPCR are listed in supplementary
material Table S5. Measurements were performed on biological
triplicates, with technical duplicates of each biological sample. chinmo,
upd2 and upd3 RNA levels were normalized to rp49 (RpL32 – FlyBase).
Phf7-RC levels were normalized to the total level of Phf7 using a primer
set that detects both Phf7-RA and Phf7-RC. The relative transcript levels
were calculated using the 2−ΔΔCT method (Livak and Schmittgen, 2001).
Immunofluorescence and image analysis
Ovaries and testes were fixed and stained by standard methods. The following
primary antibodies were used: mouse Sxl-M18 [1:100, Developmental
Studies Hybridoma Bank (DSHB)], mouse Orb-4H8 (1:50, DSHB), mouse
Orb-6H4 (1:50, DSHB), α-Spectrin (1:100, DSHB), rat HA high affinity
(1:500, Roche, 11867423001), rat Vasa (1:100, DSHB), rabbit Stat92E
[1:1000 (Chen et al., 2002)] and mouse Fas3-7G10 (1:50, DSHB). Secondary
antibodies conjugated to Alexa Fluor 555 (Life Technologies) or FITC
(Jackson ImmunoResearch Labs) were used at 1:200. Images were acquired
on a Leica TCS SP8 confocal microscope and assembled using Photoshop
(Adobe) and PowerPoint (Microsoft). A full description of these data can be
found at http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE65932
Acknowledgements
We thank M. Van Doren, J. McDonald, the TriP at Harvard Medical School, the
Bloomington Drosophila Stock Center and the Iowa Developmental Studies
Hybridoma Bank for fly stocks and antibodies; the CWRU GTSC sequencing core
for RNA-seq library generation and sequencing; A. Miron and S. Bai for help with the
bioinformatic analysis; and R. Conlon, I. Greenwald, J. McDonald, M. Van Doren and
M. Wawersik for helpful discussions.
Competing interests
The authors declare no competing or financial interests.
Author contributions
L.S.-K., A.E.S. and H.K.S. conceived, designed and performed the experiments.
H.K.S. wrote the paper.
Funding
This work was supported by the National Institutes of Health (NIH) [R01GM102141]
and a CTSC Core Utilization Grant [funded under NIH UL1TR000439]. Imaging was
performed using equipment purchased through NIH S10OD016164. Deposited in
PMC for release after 12 months.
Gene expression analysis by RNA-seq
1080
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.116590/-/DC1
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