Drosophila rasiRNA Pathway Mutations Disrupt
Embryonic Axis Specification through Activation
of an ATR/Chk2 DNA Damage Response
Carla Klattenhoff,1Diana P. Bratu,1,3Nadine McGinnis-Schultz,1Birgit S. Koppetsch,1Heather A. Cook,2
and William E. Theurkauf1,*
1Program in Molecular Medicine and Program in Cell Dynamics, University of Massachusetts Medical School,
Worcester, MA 01605, USA
2Department of Biological Sciences, Wagner College, Staten Island, NY 10301, USA
3Present address: Department of Biological Sciences, Hunter College, New York, NY 10021, USA
Small repeat-associated siRNAs (rasiRNAs)
mediate silencing of retrotransposons and the
Stellate locus. Mutations in the Drosophila
disrupt embryonic axis specification, triggering
defects in microtubule polarization as well as
asymmetric localization of mRNA and protein
determinants in the developing oocyte. Muta-
tions in the ATR/Chk2 DNA damage signal
transduction pathway dramatically suppress
these axis specification defects, but do not
restore retrotransposon or Stellate silencing.
Furthermore, rasiRNA pathway mutations lead
to germline-specific accumulation of g-H2Av
foci characteristic of DNA damage. We con-
clude that rasiRNA-based gene silencing is
not required for axis specification, and that
RNA interference (RNAi) and related processes utilize
short RNAs to direct protein complexes to chromatin
and RNA,triggering heterochromatin formation, transcrip-
tional silencing, translational repression, or RNA destruc-
negger, 2005). Mutations that disrupt small RNA functions
affect a remarkable range of processes, including early
embryogenesis in mice (Bernstein et al., 2003), embryonic
morphogenesis in zebrafish (Giraldez et al., 2005), chro-
mosome segregation in cultured chicken cells (Fukagawa
et al., 2004) and yeast (Provost et al., 2002; Volpe et al.,
2003), and developmental timing in worms (Grishok
et al., 2001). In Drosophila, RNAi-related functions are
required for stem cell division, stem cell maintenance,
and viral immunity (Forstemann et al., 2005; Hatfield
et al., 2005; Galiana-Arnoux et al., 2006; Wang et al.,
2006). However, the full scope of biological functions
controlled by small RNAs is only beginning to emerge,
and the targets for most small RNAs have not been
Mutations in the Drosophila armitage (armi), spindle-E
(spn-E), and aubergine (aub) genes disrupt siRNA-guided
RNA cleavage and assembly of the RNA-induced silenc-
ing complex (RISC) in ovary extracts and the production
of 24–30 nt repeat-associated siRNAs (rasiRNAs), which
are linked to retrotransposon and Stellate locus silencing
(Aravin et al., 2004; Tomari et al., 2004a; Vagin et al.,
2006). Strong loss-of-function mutations in these genes
disrupt embryonic axis specification, triggering defects
in microtubule organization and microtubule-dependent
localization of mRNA and protein determinants in the de-
in argonaute-2 (ago-2) and dicer-2 (dcr-2) that disrupt the
siRNA pathway, but do not block rasiRNA production, are
viable and fertile (Deshpande et al., 2005; Lee et al., 2004;
Okamura et al., 2004; Tomari et al., 2004b; Vagin et al.,
2006). The rasiRNA pathway thus appears to have an
essential function in embryonic axis specification; how-
ever, the critical developmental targets for this pathway
have not been defined.
Mutations in the armi, aub, and spn-E genes lead to
premature expression of Oskar (Osk) protein during early
sion of axis specification genes could lead to the pattern-
ing defects associated with rasiRNA pathway defects.
However, here we show that the axis specification defects
by null mutations in mei-41 and mnk, which respectively
encode ATR and Chk2 kinases that function in DNA
double-strand break (DSB) signaling. We also show that
rasiRNA pathway mutations lead to germline-specific
accumulation of g-H2Av foci, characteristic of DNA
DSBs. Significantly, the ATR/Chk2 mutations do not sup-
press the defects in retrotransposon and Stellate silenc-
ing. We therefore conclude that rasiRNA-based gene
silencing is not required for axis specification, and that
the critical developmental function for the Drosophila
rasiRNA pathway is to suppress DNA damage signaling
in the germline.
Developmental Cell 12, 45–55, January 2007 ª2007 Elsevier Inc. 45
ATR and Chk2 Mutations Suppress armi
and aub Axis Specification Defects
The armi, spn-E, and aub genes are required for produc-
tion of rasiRNAs, and mutations in these genes lead to
Stellate overexpression during spermatogenesis and pre-
mature Osk protein expression during oogenesis (Aravin
et al., 2001; Cook et al., 2004; Vagin et al., 2006). These
mutations also lead to female sterility and disrupt embry-
onic axis specification, suggesting that rasiRNAs control
expression of genes involved in patterning the oocyte
(Cook et al., 2004). However, mutations in the meiotic
DSB repair pathway also lead to axis specification
defects, andthesedefects resultfromactivationof adam-
age-signaling pathway that includes the ATR and Chk2
kinases (Bartek et al., 2001; Abdu et al., 2002; Ghabrial
and Schupbach, 1999). These findings raised the alterna-
tive possibility that rasiRNA pathway mutations disrupt
axis specification by activating ATR and Chk2.
To genetically test the role of DNA damage signaling in
the rasiRNA pathway mutant phenotype, we analyzed
double-mutant combinations with mei-41 or mnk, which
encode the Drosophila ATR and Chk2 homologs, respec-
tively. We were unable to recover mnk; spn-E double
mutants, and it is unclear if this reflects a significant neg-
ative genetic interaction between these genes or the pres-
ence of background mutations on the mnk or spn-E chro-
mosome. Our analyses thus focused on armi and aub,
which we were able to combine with both mei-41 and
mnk. If armi and aub mutations block axis specification
through ATR/Chk2 activation, the patterning defects
associated with these mutations will be suppressed in
the double mutants. Initial suppression analysis focused
on the dorsal appendages, which are easily scored egg-
shell structures that are induced through Gurken (Grk) sig-
naling from the oocyte to the somatic follicle cells during
midoogenesis (Schupbach, 1987). Appendages do not
form in the absence of Grk, a single appendage forms
with low Grk levels, and two appendages form when sig-
naling is normal (Gonzalez-Reyes et al., 1995; Roth
ically suppress the appendage defects associated with
armi and aub. Two appendages are present on 100% of
the embryos derived from wild-type and mei-41 females,
and on 94% of the embryos derived from mnk single
mutants (Table 1). By contrast, only 3.5% of the embryos
derived from armi72.1/armi1mutant females have two dor-
sal appendages. Strikingly, 92% of the embryos derived
from mnk; armi72.1/armi1double mutants show wild-type
appendage morphology. Similarly, two appendages are
present on 48% of the embryos derived from aub single
mutants, and 98% of the embryos from mnk, aub double
mutants have two appendages.
Mutations in mei-41 also suppressed the eggshell pat-
terning defects associated with armi and aub, although
suppression by mei-41 was consistently less dramatic
than suppression by mnk. A total of 56% of the embryos
from mei-41; armi72.1/armi1double mutants show normal
appendages. The mei-41 mutation was also less effective
than mnk in suppressing appendage defects associated
with homozygous armi1(data not shown) and aub (Table
1). Therefore, partial suppression of the patterning defects
by mei-41 is not allele or gene specific. Chk2 can be acti-
vated by both ATR and ATM kinases (Bartek et al., 2001;
Bartek and Lukas, 2003; Hirao et al., 2002), and the lower
level of suppression by mei-41/ATR relative to mnk/Chk2
may therefore reflect redundant Chk2 activation by the
Table 1. mnk and mei-41 Mutations Suppress Dorsal-Ventral Patterning Defects in rasiRNA Mutants
Dorsal Appendage (%) Phenotype
Hatch Rate (%)N 2 (Wild-Type) 1 (Fused)0 (Absent)
94.1 2.33.6 73.9827
94.341.7 67.2 1281
56 38.45.60 575
3.6 37.9 58.50280
mnkp6, aubHN2/mnkp6, aubQC42
46 Developmental Cell 12, 45–55, January 2007 ª2007 Elsevier Inc.
DNA Damage Signaling in rasiRNA Pathway Mutants
Drosophila ATM homolog. However, null alleles of the
Drosophila atm gene are lethal (Oikemus et al., 2004),
making direct tests of this hypothesis difficult. Nonethe-
less, these initial observations indicated that the axis
specification defects associated with rasiRNA pathway
mutations result from activation of an ATR/Chk2 kinase
DNA damage signal.
The axis specification defects associated with repair
mutations are suppressed by mutations in mei-W68,
which encodes the Drosophila homolog of the Spo11 nu-
clease that catalyzes meiotic DSB formation (McKim and
Hayashi-Hagihara, 1998). By contrast, mei-W68 has no
effect on the dorsal appendage defects associated with
armi (Table 1). Meiotic breaks thus do not appear to be
the source of damage in armi mutations.
Localization of Axis Specification Determinants
During early oogenesis, the TGFa homolog Grk localizes
to the posterior of the oocyte and signals to the overlying
oogenesis, Grk signals from the oocyte to the dorsal folli-
cle cells to generate the dorsal-ventral axis (Gonzalez-
Reyes et al., 1995; Roth et al., 1995). Mutations in armi
and aub disrupt Grk protein localization at both stages,
leading to posterior and dorsal-ventral axis specification
defects (Cook et al., 2004). To determine if the DNA
Figure 1. mnk Suppresses Gurken Protein Localization Defects in rasiRNA Mutants
(A) (a) In wild-type stage-6 egg chambers, Gurken (Grk) protein (green) accumulates at the posterior cortex (arrowhead). (a0) By stage 9, Grk is local-
ized at the dorsal anterior cortex. Actin filaments (red) mark the cell boundaries. (b and b0) In mnkp6oocytes, Grk localization is the same as in wild-
type. (c and c0) In armi72.1/armi1egg chambers, only low levels of Grk are present, and the protein is dispersed throughout the oocyte-nurse cell com-
chambers, Grk localization is similar to that of armi72.1/armi1oocytes. (e0) At stage 9, Grk is localized correctly in aubQC42/aubHN2, but not at wild-type
levels. (f and f0) In mnkp6, aubQC42/mnkp6, aubHN2egg chambers, the Grk localization level is restored. Images were acquired under identical condi-
tions for either stage. Projections of three serial 0.6 mm optical sections are shown. The oocyte nucleus is indicated (asterisk). Scale bars are 10 mm
and 25 mm for stage-6 and -9 egg chambers, respectively.
(B) Quantification of Grk localization in stage-6 oocytes. The average fluorescence intensity along a line beginning in the nurse cell cytoplasm and
extending through a cross-section of the oocyte is shown (inset).
Developmental Cell 12, 45–55, January 2007 ª2007 Elsevier Inc. 47
DNA Damage Signaling in rasiRNA Pathway Mutants
damage signaling mutations suppress the Grk localization
tion of this protein by indirect immunofluorescence and
laser-scanning confocal microscopy (Figure 1). For these
studies, Grk protein levels within cross-sections of
stage-6 oocytes were measured, and an average fluores-
cence intensity profile for each genotype was generated
(Figure 1B, inset). In wild-type stage-6 oocytes, Grk pro-
tein accumulates near the posterior cortex (Figure 1Aa).
In armi and aub single mutants, by contrast, low levels of
Grk protein are uniformly distributed in the oocyte and
nurse cells (Figures 1Ac and 1Ae). However, Grk shows
almost wild-type accumulation near the posterior cortex
1Ad and 1Af). The defects in dorsal-anterior localization
of Grk during midoogenesis (Figures 1Ac0and 1Ae0) are
also restored in the mnk double mutants (Figures 1Ad0
and 1Af0). Weaker suppression is observed with mei-41,
consistent with our analysis of the dorsal appendages
(Figure S1; see the Supplemental Data available with this
To determine if mnk and mei-41 suppress the armi- and
aub-induced defects in posterior morphogen localization
(Cook et al., 2004), we analyzed the distribution of the
pole plasm proteins Vasa (Vas) and Osk during midoogen-
esis. Osk localizes to the posterior in only 10% of stage-9
and -10 oocytes from armi females (2 of 23), and there is
no detectable localization in the remaining egg chambers
(Figure 2C). By contrast, Osk shows wild-type posterior
accumulation in over 80% of stage-9 and -10 mnk; armi
double mutants (27 of 33; Figure 2D). Vas localization
to the posterior pole is similarly restored in the double
mutants (not shown). mei-41 leads to a less dramatic
suppression of the posterior patterning defects (not
shown). Osk and Vas localization is also disrupted in aub
mutants (Figure 2E and data not shown), and localization
is restored in double mutants with mnk and mei-41
(Figure 2F). The defects in posterior and dorsal-ventral
morphogen localization associated with both armi and
aub thus require ATR and Chk2, which function in DNA
damage signal transduction.
Microtubule Organization and Vas Phosphorylation
Specification of the posterior pole is initiated during early
oogenesis, when the microtubule cytoskeleton reorga-
nizes to form a polarized scaffold in the oocyte-nurse
cell complex. While these complexes are in the germa-
rium, a prominent microtubule-organizing center (MTOC)
forms at the anterior pole of the oocyte, and this MTOC
appears to be required for oocyte differentiation (Fig-
ure 3Aa) (Theurkauf et al., 1993). After cysts bud from
the germarium, a posterior MTOC is established (Fig-
ure 3Aa0). This asymmetric microtubule array directs Grk
to the posterior pole of the oocyte, which signals to the
overlying somatic follicle cells to induce posterior differen-
tiation (Gonzalez-Reyes et al., 1995; Roth et al., 1995).
In armi mutants, both the early anterior and later posterior
MTOCs are much less prominent than in wild-type (Cook
et al., 2004) (Figures 3Ab and 3Ab0; Figure S2). By con-
trast, egg chambers double mutant for armi and mnk
show near-wild-type anterior and posterior MTOCs (Fig-
ures 3Ac and 3Ac0). Restoration of normal microtubule
organization correlates with suppression of the Grk local-
ization defects (Figure 1Ad). Eggchambers double mutant
for armi and mei-41 show a phenotype intermediate be-
tween the armi mutants and wild-type controls, consistent
with partial suppression of posterior patterning defects
later in oogenesis (Figure S2). The microtubule organiza-
tion defects in aub are also strongly suppressed by mnk,
and they are more weakly suppressed by mei-41 (Fig-
ure S2). Mutations in armi and aub thus trigger Chk2-
dependent defects in microtubule organization. These
cytoskeletal defects are likely to contribute to the loss of
axial patterning later in oogenesis.
Figure 2. Oskar Protein Localization Defects Associated with
rasiRNA Mutations Are Suppressed by mnk
(A–F) Egg chambers were fixed and labeled against Oskar (Osk) pro-
tein (green) and Actin (red). In stage-9 to -10 (A) wild-type and (B)
mnkp6mutant oocytes, Osk localizes tightly to the posterior cortex.
In similarly staged (C) armi72.1/armi1and (E) aubQC42/aubHN2oocytes,
Osk fails to localize to the posterior pole. Osk localization is restored
in the double mutants (D) mnkp6; armi72.1/armi1and (F) mnkp6,
aubQC42/aubHN2.Egg chambers areorientedwithposterior totheright.
Images were acquired under identical conditions. Single optical sec-
tions are shown. The scale bar is 20 mm.
48 Developmental Cell 12, 45–55, January 2007 ª2007 Elsevier Inc.
DNA Damage Signaling in rasiRNA Pathway Mutants
The axis specification defects associated with muta-
tions that disrupt meiotic DSB repair are also suppressed
by mnk. To determine if Chk2-dependent disruption of the
oocyte cytoskeleton contributes to these defects, we
analyzed microtubule organization in ovaries mutant for
spn-D, which encodes a rad51C homolog required for
DSB repair (Abdu et al., 2003). Mutations in spn-D, like
mutations in armi and aub, disrupt both the prominent
MTOC at the anterior of stage-1 egg chambers and the
posterior MTOC during stages 2–6 (Figures 3Ad and
3Ad0). These defects are suppressed in mnk; spn-D dou-
ble mutants (Figures 3Ae and 3Ae0), suggesting that DSB
Chk2-dependent pathway that disrupts microtubule
DSB repair mutations induce Chk2-dependent phos-
phorylation of Vas, a conserved RNA helicase required
for posterior and dorsal-ventral patterning (Ghabrial and
Schupbach, 1999; Styhler et al., 1998). To determine if
rasiRNA mutations also trigger Chk2-dependent Vas
phosphorylation, we probed western blots of armi and
mnk; armi double mutants for Vas protein. Vas protein
levels are also somewhat lower in the armi mutant egg
chambers, but this may reflect differences in egg cham-
ber-stage distribution in the isolated ovaries (Figure 3B).
More significantly, a lower-electrophoretic mobility spe-
cies is observed in ovaries homozygous for a strong
loss-of-function allele, armi72.1, and both species are ob-
served with a weaker allelic combination, armi72.1/armi1.
Only the faster-migrating species is present in mnk;
armi72.1/armi1double mutant extracts. After phosphatase
treatment, the lower-mobility species present in armi mu-
tant extracts disappears, and the faster-migrating species
increases in intensity (not shown), indicating that the
Figure 3. mnk Suppresses Microtubule Organization Defects and Vasa Phosphorylation in rasiRNA Pathway Mutants
(A) Microtubules were labeled with an anti-a-tubulin antibody. (a) A bright microtubule organizing center (MTOC) is localized to the anterior pole of the
oocyteinwild-typestage-1 egg chambers (arrowhead).(a0)Bystage 6,theMTOCis localized along theposterior cortex (arrowhead).Inarmi72.1/armi1
armi72.1/armi1and mnkp6;spn-D2egg chambers, (cand e) anterior MTOC during stage 1 (arrowheads)and the (c0and e0) posterior MTOC during stage
6 (arrowheads) are restored. Stage-1 oocytes are outlined. Images were acquired under identical conditions. Projections of four serial 0.6 mm optical
sections are shown. Posterior is oriented to the right. The scale bar is 10 mm.
(B) Western blot analysis of Vasa (Vas) protein in wild-type, armi1, armi72.1, armi72.1/armi1, and mnkp6; armi72.1/armi1ovary extracts. Vas from homo-
are present in armi72.1/armi1ovary extracts. Only the faster-migrating form is present in mnkp6; armi72.1/armi1extracts (arrow).
Developmental Cell 12, 45–55, January 2007 ª2007 Elsevier Inc. 49
DNA Damage Signaling in rasiRNA Pathway Mutants
lower-mobility band is a phosphorylated form of Vas. Mu-
tations in armi, like meiotic DSB repair mutations, thus
trigger Chk2-dependent phosphorylation of Vas. While
the physiological significance of Vas phosphorylation
has not been established, these findings support the
hypothesis that armi mutations lead to Chk2 kinase
rasiRNA Pathway Mutations Lead to Germline
The observations described above indicate that the axis
specification defects associated with armi and aub are
mediated by ATR and Chk2 kinases, which are normally
activated by DNA DSBs. To determine if armi and aub
lead to DSB accumulation, we labeled mutant ovaries
for the phosphorylated form of the Drosophila histone
H2AX variant (g-H2Av), which accumulates on chromo-
somes near break sites (Modesti and Kanaar, 2001; Re-
don et al., 2002). After chromosome breakage, Drosophila
H2Av, like H2AX, is phosphorylated at a conserved SQ
motif within an extended C-terminal tail (Madigan et al.,
2002; Rogakou et al., 1998). We therefore used an anti-
phosphoprotein antibody specific for g-H2Av (Gong
et al., 2005). In wild-type ovaries, g-H2Av foci are re-
stricted to region 2 of the germarium, where meiotic
DSBs are formed (Figure 4A) (Jang et al., 2003). Consis-
tent with earlier observations, this labeling is significantly
reduced in mei-W68 mutants,which do not initiate meiotic
breaks (Figure 4G). In armi and aub mutants, prominent
Unlike wild-type, these foci persist and increase in inten-
sity as cysts mature and bud to form stage-2 egg cham-
bers (Figures 4C and 4E). g-H2Av foci persist in double
mutants with mnk, indicating that suppression of the pat-
terning defects by mnk is not the result of enhanced DNA
g-H2Av foci also persist in egg chambers mutant for
a third rasiRNA gene, spn-E (Figure S3). The pattern of
germline-specific g-H2Av accumulation in armi, aub, and
spn-E is similar to the pattern of g-H2Av accumulation in
mutants for the DNA repair gene spn-D, although the
foci appear to arise at somewhat earlier stages in the
rasiRNA mutants (Figures 4C, 4E, and 4I). Accumulation
of g-H2Av foci in spn-D mutants is suppressed by mei-
W68, consistent with a function for this gene in meiotic
DSB repair (Abdu et al., 2002). By contrast, g-H2Av foci
persist in mei-W68; armi double mutants (Figure 4H). We
have not yet assayed mei-W68 double mutants with aub
or spn-E, but the observation described above suggests
that the g-H2Av foci in rasiRNA pathway mutations are
independent of meiotic DSB formation.
The observations presented above strongly suggest that
the axial patterning defects associated with armi and
aub are a consequence of DNA damage signaling, and
that rasiRNA-based gene silencing is not directly involved
in embryonic patterning. However, the mnk and mei-41
mutations could suppress the defects in rasiRNA function
associated with armi and aub. We therefore analyzed
Figure 4. g-H2Av Foci Accumulate in
armi and aub Mutant Ovaries
(g-H2Av) accumulates near double-strand
break sites. (A and B) In wild-type and mnkp6
mutants, g-H2Av foci are restricted to region
2 of the germarium, where meiotic DSBs
form. (C–F) In (C) armi72.1/armi1, (D) mnkp6;
armi72.1/armi1, (E) aubQC42/aubHN2, and (F)
the germarium and persist and increase in
intensity as cysts bud from the germarium to
form egg chambers. (I) A similar pattern is
observed in ovaries mutant for spn-D2, which
is required for DSB repair. (G) Mutations in
encodes the Spo11 nuclease that initiates mei-
otic DSBs, suppress formation of g-H2Av foci
in region 2 of the germarium. (H) However,
mei-W68 does not suppress g-H2Av focus
formation in armi mutants (mei-W681/mei-
W68k05603; armi72.1/armi1). (J) A schematic rep-
resentation of the regions of the germarium
and a developing egg chamber. Projections
of six serial 1 mm optical sections are shown.
Posterior is to the right. The scale bar is 20 mm.
50 Developmental Cell 12, 45–55, January 2007 ª2007 Elsevier Inc.
DNA Damage Signaling in rasiRNA Pathway Mutants
rasiRNA-dependent silencing of both the Stellate gene
during spermatogenesis and the HeT-A retrotransposon
during oogenesis in single and double mutants. The Stel-
late gene is repressed during spermatogenesis, appar-
ently through mRNA turnover guided by rasiRNAs derived
from the Suppressor of Stellate locus (Aravin et al., 2001;
Gvozdev et al., 2003). Mutations in armi and aub lead to
accumulation of full-length Stellate mRNA and Stellate
protein overexpression, which leads to the assembly of
Stellate crystals in mutant testes (Aravin et al., 2004;
Forstemann et al., 2005; Stapleton et al., 2001; Tomari
et al., 2004a) (Figures 5Ab and 5Ac). Stellate crystals are
present in both mnk; armi and mnk, aub double mutant
testes (Figures 5Ae and 5Af). Stellate overexpression is
also linked to male sterility, and mnk; armi males are
also sterile (data not shown).
HeT-A is a retrotransposon that contributes to telomere
formation in Drosophila (Pardue et al., 2005), and HeT-A
expression is dramatically derepressed in armi, aub, and
spn-E mutant ovaries (Aravin et al., 2001; Vagin et al.,
2004, 2006). HeT-A is not expressed at detectable levels
on northern blots of wild-type or mnk RNAs. However,
HeT-A transcripts are abundant in armi and aub mutants
(Figure 5C). Significantly, HeT-A is also overexpressed in
mnk; armi and mnk, aub double mutants (Figures 5B and
5C). In fact, HeT-A expression is higher in the double mu-
tants than in the single mutants. FISH analyses indicate
that this reflects increased expression in the germline
Figure 5. The mnk Mutation Does Not Suppress Defects in rasiRNA Function
(A) Silencing of the Stellate locus during spermatogenesis. Stellate is not expressed in (a) wild-type or (d) mnkp6mutant testes. However, Stellate is
overexpressed, and the protein assembles into crystals in testes from (b) armi72.1/armi1, (c) aubQC42/aubHN2, (e) mnkp6; armi72.1/armi1, and (f) mnkp6,
aubQC42/mnkp6, aubHN2males. DNA (red) was labeled with TOTO3, and Stellate protein (green) was detected with anti-Stellate antibody. Projections
of five serial 1 mm optical sections are shown. The scale bar is 20 mm.
(B) FISH analysis of HeT-A retrotransposon silencing. (a and a0) In wild-type ovaries, only background levels of HeT-A expression are detected. (b–c0)
By contrast, HeT-A is expressed at high levels in the germline and somatic follicle cells of early and midoogenesis-stage armi72.1/armi1and mnkp6;
armi72.1/armi1egg chambers. Panels (a), (b), and (c) are projections of 12–15 serial 1.5 mm optical sections. Panels (a0), (b0), and (c0) are single optical
sections. Posterior is oriented to the right. The scale bar is 20 mm for the left panels and 50 mm for the right panels.
HeT-A transcript. HeT-A transcripts are undetectable in wild-type and mnkp6samples, but they are abundant in RNA derived from armi1, armi72.1/
armi1,mnkp6;armi72.1/armi1,aubQC42/aubHN2,and mnkp6,aubQC42/aubHN2mutant ovaries. Ribosomal protein49(rp49)wasusedas aloading control.
Developmental Cell 12, 45–55, January 2007 ª2007 Elsevier Inc. 51
DNA Damage Signaling in rasiRNA Pathway Mutants
and somatic cells of the ovary, during both early and mid-
oogenesis (Figure 5B). Therefore, the mnk mutation does
not suppress defects in rasiRNA-based gene silencing
during spermatogenesis or oogenesis, leading us to con-
clude that rasiRNA-based silencing is not required for axis
Mutations in the Drosophila armi, aub, and spn-E genes
disrupt oocyte microtubule organization and asymmetric
localization of mRNAs and proteins that specify the poste-
rior pole and dorsal-ventral axis of the oocyte and embryo
(Cook et al., 2004). Mutations in these genes block ho-
mology-dependent RNA cleavage and RISC assembly in
ovary lysates (Tomari et al., 2004a), RNAi-based gene
silencing during early embryogenesis (Kennerdell et al.,
2002), rasiRNA production, and retrotransposon and Stel-
late silencing (Aravin et al., 2001; Vagin et al., 2006). Muta-
tions in dcr-2 and ago-2 genes, by contrast, block siRNA
function (Okamura et al., 2004; Tomari et al., 2004b), but
specification (Vagin et al., 2006). The rasiRNA pathway
thus appears to be required for embryonic axis specifica-
tion. However, the function of rasiRNAs in the axis specifi-
cation pathway has not been previously established.
Here, we show that the cytoskeletal polarization, mor-
phogen localization, and eggshell patterning defects
associated with armi and aub are efficiently suppressed
by mnk and mei-41, which respectively encode Chk2
and ATR kinase components of the DNA damage signal-
ing pathway (Figures 1–3; Table 1). In addition, we show
that armi and aub mutants accumulate g-H2Av foci
characteristic of DNA DSBs (Figure 4) and trigger Chk2-
dependent phosphorylation of Vas (Figure 3B), an RNA
helicase required for posterior and dorsal-ventral specifi-
cation (Styhler et al., 1998). Mutations in spn-E also
disrupt the rasiRNA pathway (Aravin and Tuschl, 2005;
Vagin et al., 2006), trigger axis specification defects
(Cook et al., 2004), and lead to germline-specific accumu-
lation ofg-H2Avfoci(see FigureS3).Significantly,themnk
and mei-41 mutations do not suppress Stellate or HeT-A
overexpression, indicating that axis specification does
not directly require rasiRNA-dependent gene silencing.
Based on these findings, we conclude that the rasiRNA
pathway suppresses DNA damage signaling in the female
germline, and that mutations in this pathway disrupt axis
specification by activating an ATR/Chk2 kinase pathway
that blocks microtubule polarization and morphogen
localization in the oocyte (Figure 6).
The cause of DNA damage signaling in armi, aub, and
spn-E mutants remains to be established. In wild-type
ovaries, g-H2Av foci begin to accumulate in region 2 of
the germarium (Jang et al., 2003), when the Spo11 nucle-
ase (encoded by the mei-W68 gene) initiates meiotic
breaks (McKim and Hayashi-Hagihara, 1998). The axis
specification defects associated with DNA DSB repair
mutations are efficiently suppressed by mei-W68 muta-
tions, indicating that meiotic breaks are the source of
DNAdamage in thesemutants(Abduetal., 2002;Ghabrial
specification defects and g-H2Av focus formation asso-
ciated with armi, by contrast, are not suppressed by
mei-W68 (Figure 4; Table 1). We have not yet analyzed
mei-W68 double mutants with aub or spn-E, but this
observation strongly suggests that meiotic DSBs are not
Retrotransposon silencing is disrupted in armi, aub, and
spn-E mutants (Aravin et al., 2001; Vagin et al., 2006),
and transcription of LINE retrotransposons in mammalian
cells leads to DNA damage and DNA damage signaling
(Belgnaoui et al., 2006; Gasior et al., 2006). Loss of retro-
transposon silencing could therefore directly induce the
DSBs in rasiRNA pathway mutants. However, DNA dam-
age can also lead to loss of retrotransposon silencing
(Bradshaw and McEntee, 1989; Farkash et al., 2006;
Rudin and Thompson, 2001). Mutations in the rasiRNA
pathway could therefore disrupt DNA repair and thus
induce DNA damage, which, in turn, induces loss of retro-
transposon silencing. Finally, the HeT-A retrotransposon
is associated with telomeres, and overexpression of this
element could reflect a loss of telomere protection and
could damage signaling by chromosome ends in the
rasiRNA pathway mutants. The available data do not
distinguish between these alternatives.
In mouse, the piwi-related Argonauts Miwi and Mili bind
piRNAs, 30 nt RNAs derived primarily from a single strand
Figure 6. Model for rasiRNA Control of Axis Specification
The rasiRNA pathway and meiotic DSB repair machinery function
independently to suppress DNA damage signaling in the female germ-
line. Mutations that disrupt either pathway activate a common DNA
damage response, mediated by the ATR and Chk2 kinases. Chk2
activation blocks axis specification by disrupting microtubule organi-
zation and phosphorylating Vas, an RNA helicase required for axis
specification that has been implicated in grk mRNA translation.
52 Developmental Cell 12, 45–55, January 2007 ª2007 Elsevier Inc.
DNA Damage Signaling in rasiRNA Pathway Mutants
that appear to be related to rasiRNAs (Aravin et al., 2006;
Girard et al., 2006; Grivna et al., 2006). Mutations in these
genes disrupt spermatogenesis and lead to germline
apoptosis (Deng and Lin, 2002; Kuramochi-Miyagawa
et al., 2001), which can be induced by DNA damage
signaling. Mammalian piRNAs and Drosophila rasiRNAs
may therefore serve similar functions in suppressing
a germline-specific DNA damage response.
All animals were raised at 25?C on standard food. Oregon R was used
for wild-type control. The following alleles were used: mnkp6(Brodsky
et al., 2004; Takada et al., 2003); armi72.1and armi1(Cook et al., 2004);
mei-41D3(Hari et al., 1995; Hawley and Tartof, 1983); aubQC42, aubHN2
(Schupbach and Wieschaus, 1991); spn-E1(Gillespie and Berg, 1995;
Gonzalez-Reyes et al., 1997); spn-D2(Abdu et al., 2003); and P[lacW]-
mei-W68k05603, mei-W681(McKim and Hayashi-Hagihara, 1998). The
mnkp6allele was kindly provided by M. Brodsky (Brodsky et al.,
Stock Center (Drysdale et al., 2005; http://flybase.org/). Standard ge-
netic procedures were used to generate double mutant combinations.
Primers annealing to each of the translation start and stop sites of
a Stellate cDNA (Bozzetti et al., 1995) were designed (Integrated
DNA Technologies, Inc.) with attached Gateway (Invitrogen) se-
quences. The resulting PCR product wasused to make aDONR clone,
which, in turn, was used to subclone into the 6X-His-tagged Gateway
vector pDest17, yielding a 29 kDa 6X-His-tagged Stellate fusion
protein.Thefusion protein waspurifiedon aProbondNimatrix (Invitro-
gen) under denaturing conditions, isolated by SDS-PAGE, and used to
immunize two rabbits (Pocono Rabbit Farm and Laboratory, Inc.) by
following standard protocols for antibody production. Antiserum was
used at 1:1000 for immunohistochemistry.
Egg chamber fixation and whole-mount antibody labeling were
performed as previously described (Theurkauf, 1994). Microtubules
were labeled with FITC-conjugated mouse monoclonal anti-a-tubulin
(Sigma Chemical Co.) used at 1:200. Osk protein was labeled with rab-
bit polyclonal anti-Osk antibody (Vanzo and Ephrussi, 2002) at 1:1000.
Vas protein waslabeled withrabbit polyclonalanti-Vasantibody (Liang
et al.,1994)at 1:1000.Grkprotein waslabeledwithmouse monoclonal
anti-Grk antibody (obtained from the Developmental Studies Hybrid-
oma Bank, University of Iowa) at 1:10. Antibody against g-H2Av was
kindly provided by Kim McKim (Gong et al., 2005), and egg chambers
were labeled as described previously (Belmont et al., 1989). Rhoda-
mine-conjugated phalloidin (Molecular Probes) was used at 1:100 to
stain F-actin, and TOTO3 (Molecular Probes) was used at 1:500
(0.2 mM final concentration) to visualize DNA.
Fluorescence In Situ Hybridization
An antisense HeT-A digoxygenin (DIG)-labeled RNA probe was
synthesized in vitro from a 500 bp PCR-amplified cDNA fragment car-
rying a T7 promoter (generously provided by P. Zamore) with a DIG
RNA Labeling Kit by following the manufacturer’s instructions (Roche).
Whole-mount in situ hybridization was performed as described previ-
ously (Tautz, 1988; Tautz and Pfeifle, 1989; Cha et al., 2001). Tyramide
signal amplification (TSA) was performed by following the manufac-
turer’s instructions (Perkin Elmer).
Fly ovaries were dissected in 13 Robb’s medium (55 mM potassium
acetate, 40 mM sodium acetate, 100 mM sucrose, 10 mM glucose,
1.2 mM MgCl2, 1 mM CaCl2, and 100 mM HEPES [pH 7.4]). Total
RNA was isolated from ?30 mg ovaries by using the RNeasy Mini Kit
by following the manufacturer’s instructions (Qiagen). Approximately
rose/formaldehyde gel. RNA was transferred to a positively charged
nylon membrane (Roche) by standard capillary transfer. After transfer,
RNA was fixed to the membrane via UV crosslinking (Stratalinker UV
Crosslinker 2400). After prehybridization, blots were probed with
DIG-labeled RNA by following the manufacturer’s recommendations
(Roche). rp49 was used as a loading control. Blots were developed
with CDP-Star (Tropix) according to the manufacturer’s directions.
Images were acquired with the Kodak 4000MM Image Station.
Western Blot Analysis
The western blot was performed as described (Ghabrial et al., 1998;
Ghabrial and Schupbach, 1999); the rabbit polyclonal anti-Vas
antibody was used at 1:5000.
enediamine (Sigma). Samples were analyzed with a Leica TCS-SP
inverted laser-scanning microscope with 633 NA 1.32 PlanApo oil
and 403 NA 1.25 Planapo oil objectives. Identical imaging conditions
were used for each set of wild-type and mutant samples. Images were
processed with Image J software.
Supplemental Data include three figures and are available at http://
We thank Kim McKim and Anne Ephrussi for antibodies, and Alla
Sigova, Vasia Vagin, and Phil Zamore for the HeT-A PCR probe. We
also thank Beatrice Benoit and Hanne Varmark for helpful comments
on the manuscript; Phil Zamore, Vasia Vagin, Klaus Forstemann, and
Yuki Tomari for stimulating discussion and for sharing data prior to
publication; and Beatrice Benoit for technical support. The Grk mono-
clonal antibody 1D12 was obtained from the Developmental Studies
Hybridoma Bank developed under the auspices of the National Insti-
tute of Child Health and Human Development (NICHD) and maintained
by the University of Iowa, Department of Biological Sciences. This
work was supported by a grant to W.E.T. from the NICHD, National
Institutes of Health (R01 HD049116).
Received: June 30, 2006
Revised: November 10, 2006
Accepted: December 2, 2006
Published: January 8, 2007
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