Components of the RNAi Machinery That Mediate Long-Distance Chromosomal Associations Are Dispensable for Meiotic and Early Somatic Homolog Pairing in Drosophila melanogaster
Homolog pairing is indispensable for the proper segregation of chromosomes in meiosis but the mechanism by which homologs uniquely pair with each other is poorly understood. In Drosophila, somatic chromosomes also undergo full homolog pairing by an unknown mechanism. It has been recently demonstrated that both insulator function and somatic long-distance interactions between Polycomb response elements (PREs) are stabilized by the RNAi machinery in Drosophila. This suggests the possibility that long-distance pairing interactions between homologs, either during meiosis or in the soma, may be stabilized by a similar mechanism. To test this hypothesis, we have characterized meiotic and early somatic chromosome pairing of homologous chromosomes in flies that are mutant for various components of the RNAi machinery. Despite the identification of a novel role for the piRNA machinery in meiotic progression and synaptonemal complex (SC) assembly, we have found that the components of the RNAi machinery that mediate long-distance chromosomal interactions are dispensable for homologous chromosome pairing. Thus, there appears to be at least two mechanisms that bring homologous sequences together within the nucleus: those that act between dispersed homologous sequences and those that act to align and pair homologous chromosomes.
Copyright Ó2008 by the Genetics Society of America
Components of the RNAi Machinery That Mediate Long-Distance
Chromosomal Associations Are Dispensable for Meiotic and
Early Somatic Homolog Pairing in Drosophila melanogaster
Justin P. Blumenstiel,*
William E. Theurkauf
and R. Scott Hawley*
*Stowers Institute for Medical Research, Kansas City, Missouri, 64110,
Program in Molecular Medicine and Program
in Cell Dynamics, University of Massachusetts Medical School, Worcester, Massachusetts 01605,
School of Medicine,
University of Missouri, Kansas City, Missouri 64110 and
Department of Physiology, University of Kansas
Medical Center, Kansas City, Kansas 66160
Manuscript received June 14, 2008
Accepted for publication September 8, 2008
Homolog pairing is indispensable for the proper segregation of chromosomes in meiosis but the
mechanism by which homologs uniquely pair with each other is poorly understood. In Drosophila,
somatic chromosomes also undergo full homolog pairing by an unknown mechanism. It has been recently
demonstrated that both insulator function and somatic long-distance interactions between Polycomb
response elements (PREs) are stabilized by the RNAi machinery in Drosophila. This suggests the
possibility that long-distance pairing interactions between homologs, either during meiosis or in the soma,
may be stabilized by a similar mechanism. To test this hypothesis, we have characterized meiotic and early
somatic chromosome pairing of homologous chromosomes in ﬂies that are mutant for various
components of the RNAi machinery. Despite the identiﬁcation of a novel role for the piRNA machinery
in meiotic progression and synaptonemal complex (SC) assembly, we have found that the components of
the RNAi machinery that mediate long-distance chromosomal interactions are dispensable for
homologous chromosome pairing. Thus, there appears to be at least two mechanisms that bring
homologous sequences together within the nucleus: those that act between dispersed homologous
sequences and those that act to align and pair homologous chromosomes.
HOMOLOGOUS chromosome pairing—deﬁned
here as the close physical association of homolo-
gous loci—mediates a host of important biological
phenomena. Perhaps most importantly, the proper
segregation of chromosomes during the reductional
division of meiosis depends on chromosomes becoming
uniquely paired with their homolog. In several species,
meiotic pairing has also been recruited as a scanning
and defense mechanism; in Neurospora, meiotic chro-
mosome pairing limits the proliferation of transposons
through a mechanism known as meiotic silencing of
unpaired DNA (MSUD) (Shiu et al. 2001; Shiu and
Metzenberg 2002) and in mice and worms unpaired
sex chromosomes are silenced (Bean et al. 2004; Turner
et al. 2005).
Outside of the germline, somatic pairing between
homologs also plays an important role in the control of
gene expression. For example, transient associations
between the X chromosomes during early female mam-
malian development aid the establishment of the inactive
X chromosome (Bacher et al. 2006; Xuet al. 2006).
Somatic homolog pairing has also been implicated in the
action of enhancers in trans—a phenomenon known as
transvection (Lewis 1954; Wuand Morris 1999;
Duncan 2002)—and pairing-sensitive silencing (Kassis
1994; Americo et al. 2002). Moreover, somatic homolog
pairing can facilitate repair of DNA damage. In G1,
prior to S phase when a sister chromatid is unavailable as
a template, DNA damage can effectively be repaired off
the homolog (Esposito 1968; Fabre 1978; Kadyk and
Hartwell 1992; Rong and Golic 2003).
Despite the important nature of these interactions
between homologs, the mechanism by which pairing is
achieved is incompletely understood. During meiosis in
yeast (Bhuiyan and Schmekel 2004; Henderson and
Keeney 2004), synapsis—the formation of the protein-
aceous synaptonemal complex (SC) between the ho-
mologs—depends on double-stranded breaks (DSBs)
that are repaired off the homolog. This also appears to
be the case in mice (Mahadevaiah et al. 2001) and Ara-
bidopsis (Grelon et al. 2001). However, DSB-independent
mechanisms are important in the establishment of
pairing prior to synapsis. Cha et al. (2000), Nabeshima
et al. (2001), and multiple studies have demonstrated
pairing in the absence of recombination (Weiner and
Kleckner 1994; Nag et al. 1995). Moreover, in some
species, both meiotic pairing and synapsis are entirely
independent of DSB formation. For example, Drosophila
Corresponding author: 1200 Sunnyside Ave., Department of Ecology and
Evolutionary Biology, University of Kansas, Lawrence, KS 66045.
Genetics 180: 1355–1365 (November 2008)
melanogaster (McKim et al. 1998) and Caenorhabditis
elegans (Dernburg et al. 1998) undergo perfectly good
synapsis in the absence of programmed DSBs. In fact, in
the somatic tissues of broader Dipterans, homologous
chromosomes are paired (Metz 1916; Hiraoka et al.
1993; Fung et al. 1998). Thus, it has been proposed that
somatic and meiotic pairing in Drosophila are achieved
by the same mechanism and that meiotic pairing is
simply an extension of pairing established in the mi-
totically dividing germline (Stevens 1908; Brown and
Stack 1968; Vazquez et al. 2002; Gong et al. 2005;
Sherizen et al. 2005). The mechanism underlying the
pairing interaction is thought to be mediated either
directly by nucleic acid homology [through either direct
DNA–DNA interactions (i.e.,Wilson 1979, McGavin
1989) or interactions that may include transcribed
RNA] or indirectly through protein–protein interac-
tions (Comings and Riggs 1971; Gemkow et al. 1998;
Phillips et al. 2005; Fritsch et al. 2006; Phillips and
Aside from full homolog pairing, another sort of
chromosomal interaction has relevance to the pairing
problem. Dispersed sequences residing at heterologous
sites also demonstrate long-range physical interactions
within the nucleus and these interactions play an impor-
tant role in gene regulation. In mice, the imprinted Igf2
locus is regulated through its physical interaction with the
H19 locus (Murrell et al. 2004) and the Wsb1/Nf1 locus is
also regulated by its physical interaction with Igf2 (Ling
et al. 2006). In ﬂies, regulatory elements know as Poly-
comb response elements (PREs) mediate the physical
clustering ofdispersed sequences within the nucleus and
this physical clustering governs the expression of neigh-
boring genes (Bantignies et al. 2003). A similar cluster-
ing can be seen between gypsy insulators and this
clustering is proposed to mediate insulator function
through the formation of looped chromosome domains
(Gerasimova et al.2000;Byrd and Corces 2003).
Interestingly, both PRE clustering in wing imaginal
discs and insulator function have also been shown to be
dependent on the RNAi machinery (Grimaud et al.
2006; Lei and Corces 2006b). The involvement of the
RNAi machinery in the promotion of long-distance
chromosome interactions is particularly intriguing as
it suggests a model for the association of dispersed ho-
mologous sequences within the nucleus (Figure 1). In
particular, since it is known that the RNAi machinery
can load onto chromosomal sites in complexes that
contain small RNAs (Noma et al. 2004; Verdel et al.
2004; Brower-Toland et al. 2007), these complexes
could conceivably promote or stabilize pairing by captur-
ing RNA transcripts in trans (Lei and Corces 2006a).
It has been suggested that the mechanism that leads
to the clustering of dispersed sequences is mechanisti-
cally related to the mechanism of homolog pairing. In
fact, the observation that loss of Su(Hw) protein leads to
reduced homolog pairing in wing imaginal discs sup-
ports this (Fritsch et al. 2006). If the mechanism
underlying homolog pairing and the association of
dispersed sequences were in fact identical, one would
expect that the same components of the RNAi machin-
ery that affect clustering would also be expected to
affect homolog pairing. For this reason, we have tested
this hypothesis by characterizing meiotic and early em-
bryonic somatic pairing in such ﬂies. By focusing on
these tissues, rather than wing imaginal discs as other
studies have done, we speciﬁcally ask whether defects in
the same RNAi components lead to defects in pairing
establishment or meiotic pairing, which is essential for
proper segregation. We show here that the RNAi defects
that have been previously shown to lead to defects in
PRE clustering and insulator function show no obvious
defects in meiotic or early somatic homolog pairing,
despite other defects in synaptonemal complex forma-
tion and meiotic progression. These results indicate
that either the mechanism of homolog pairing and
dispersed sequence interactions are fundamentally
different or homolog pairing can also be achieved by
MATERIALS AND METHODS
Fly stocks and culture: All ﬂies were maintained at 25°on
standard food. Stocks with the following alleles were used: dcr-
(Lee et al. 2004), ago2
(Okamura et al.
(Xuet al. 2004), aub
and Wieschaus 1991), spn-E
and Berg 1995), piwi
(Cox et al. 1998) and mnk(lok)
et al. 2004). Embryos from piwi
homozygous clones were gen-
erated using the FLP-DFS system (Chou and Perrimon 1992,
1996). Speciﬁcally, piwi
FRT40 larvae between 0 and 5 days old were heat shocked
at 38°for 1 hr and females were collected and allowed to
lay embryos. A near 0% hatch rate indicated that embryos were
in fact defective for embryonic function of piwi (Cox et al.
Figure 1.—Model for RNAi-mediated homolog pairing.
Dispersed sequences, perhaps middle repeat or intercalary
heterochromatin that function as a source of small RNA could
function as distributed attachment points that mediate or sta-
bilize homolog pairing. Since small RNAs are known to assem-
ble on chromatin, small RNA/protein complexes could
potentially mediate pairing by capturing nascent transcripts
in trans. In this cartoon, a nascent transcript on one homolog
is captured by small RNA/protein complexes assembled on
the other homolog.
1356 J. P. Blumenstiel et al.
FISH and immunostaining: Prior to dissection of ovaries,
female ﬂies were kept for 2–3 days after eclosion in yeasted
vials with males. Immunostaining was performed as described
elsewhere (Page and Hawley 2001) with a cocktail of the
following C(3)G mouse monoclonal antibodies: 1G5-2F7,
1A8-1G2, and 5G4-1F1, each used at 1:500. Either Alexa-
488 or Alexa-555 conjugated anti-mouse IgG secondary anti-
bodies (Invitrogen–Molecular Probes) were also used at 1:500
and ring canals were visualized by staining with rhodamine-
phalloidin at 1:200 during staining with the secondary
antibody. FISH, followed by immunostaining, was performed
on germaria with a slightly altered procedure. Germaria were
ﬁxed for 4 min in 3.7% formaldehyde in 13ﬁx buffer (100 mm
potassium cacodylate, 100 mmsucrose, 40 mmsodium ace-
tate, and 10 mmEGTA). After ﬁxation, ovaries were rinsed
three times in 23SSCT and hybridization was performed as
previously described (Sherizen et al. 2005). Subsequent
to hybridization and after the ﬁnal 23SSCT rinse, ovaries
were rinsed two times in PBST and the immunostaining
protocol (above) was resumed. FISH on 2- to 4-hr-old embryos
was performed without immunostaining according to a pre-
viously described protocol (Fung et al. 1998). From sets of
2- to 4-hr-old embryos, pairing was assayed in embryos that
had completed cellularization and just begun gastrulation
(3 hr old).
FISH probes: All probes were generated by conjugating
ARES Alexa ﬂuors (either 488 or 647) with amine-labeled
DNA probes. For euchromatic probes from region 25, DNA was
prepared and aggregated from three overlapping BACs (Fly-
Base names: BACR05M06, BACR13M11, and BACR 06K07).
For the histone probe, a portion of the histone cluster was PCR
ampliﬁed with the two following primer pairs: 59-aacctcagcggc
tatggtg-39/59-aattattccgcgtcatctgc-39. For BAC- and PCR-based
probes, DNA was digested into smaller fragments with the
following four-cutters prior to tailing with amino-allyl-UTP
with terminal transferase (Roche): AluI, HaeIII, MseI, MspI,
RsaI, and Sau3AI. For the dodeca probe, an amino-conjugated
oligonucleotide corresponding to the 59-cccgtactggtcccgtact
ggtcccgtactcggtcccgtactcggt-39dodeca satellite sequence was
purchased from IDT. Amino-labeled DNA (BAC, PCR product,
or oligo) was dye-conjugated according to manufacturer’s
Microscopy: Images were acquired on a DeltaVision micros-
copy system (Applied Precision) equipped with an Olympus
1370 inverted microscope and CoolSnap CCD camera. All
germarium images were acquired at 1003with auxiliary 1.53
magniﬁcation and all embryo images were acquired at 603
with auxiliary 1.53magniﬁcation. Zslices were captured with
a 0.2-mm step size and deconvolved using SoftWorx v2.5 soft-
ware. All images are shown as either single z-slices or maximum
intensity projections of deconvolved images, aside from ring
canal images, which are single z-slices that were not decon-
volved. Distances between FISH foci were measured by hand in
three dimensions using the SoftWorx Explorer package.
Meiotic pairing is independent of RNAi components
that mediate insulator function and somatic interac-
tions between PREs: Recent work has shown that the
RNAi machinery plays an important role in mediating
heterologous interchromosomal interactions that are
important for proper gene regulation. Speciﬁcally,
Aubergine (Aub) and PIWI, two components of the
piRNA/rasiRNA pathway, Dicer-2 (Dcr-2), a compo-
nent of the siRNA pathway, and Argonaute1 (AGO1), a
component of the miRNA pathway, all contribute to
long-distance chromosomal interactions between Poly-
comb response elements (PREs) (Grimaud et al. 2006).
Furthermore, Aub and PIWI also play a role in the
function of insulators, which are proposed to mediate
intrachromosomal associations that lead to chromo-
some looping and the formation of unique chromo-
some regulatory domains (Lei and Corces 2006b).
These results suggest that DSB independent pairing
between homologs during meiosis in Drosophila could
be mediated by a related mechanism. To determine
whether this is the case, we assayed pairing in meiotic
nuclei of the germarium in female ﬂies that were mu-
tant for two components of the siRNA machinery, dcr-2
and ago-2, as well as aubergine. In addition, we also
examined pairing in ﬂies defective for spindle-E (spn-E),
also known to be important for piRNA/rasiRNA func-
tion. Meiotic pairing in females mutant for piwi was not
characterized due to gross defects in the germarium
that made identiﬁcation of meiotic nuclei difﬁcult.
Pairing was assayed for three unique loci. For a
heterochromatic sequence, we used the dodeca satel-
lite, residing in the pericentric heterochromatin of
chromosome 3. For euchromatic regions, we used the
histone locus, which demonstrates an especially strong
afﬁnity for its homolog (Hiraoka et al. 1993) and
resides near the base of 2L in region 39, and a normal
euchromatic locus on the arm of 2L in region 25. This
set of loci was chosen to exhibit a range of characteristics
with regard to chromatin state and nature of repetitive-
ness. Meiotic nuclei within the germarium were identi-
ﬁed by performing immunoﬂuorescence with antibodies
to C(3)G—a protein that forms the transverse ﬁlaments
within the synaptonemal complex (Page and Hawley
2001) (Anderson et al. 2005). Synapsis is known to
maintain pairing in the meiotic nuclei of Drosophila.
Thus, our meiotic pairing assay speciﬁcally determined
whether the pairing that is maintained by synapsis is
disrupted. Figure 2 shows results of pairing experiments
in the germarium of control w
ﬂies with C(3)G
staining indicating meiotic nuclei. Figure 3 shows the
distribution of distance between foci in meiotic nuclei,
as deﬁned by the presence of synaptonemal complex
visualized by immunostaining of C(3)G, in regions 2a
and 2b. In control w
ﬂies, the vast majority of histone
foci and euchromatic foci are within 0.5 mm of each
other. Interestingly, dodeca foci are typically farther
apart—a large fraction of foci are between 0.5 and 1 mm
apart. This is consistent with the fact that the synapto-
nemal complex does not appear as robust in the centro-
meric heterochromatin (Carpenter 1979; Mehrotra
and McKim 2006). Thus, while heterochromatic regions
are paired in meiosis, this loose SC structure may not
facilitate as tight an association of homologous sequen-
ces in the centromere.
Pairing in Drosophila 1357
Overall, we found a striking consistency in the dis-
tribution of pairing distances for all loci across mutants
for different RNAi mutants. This was the case for ﬂies
that were defective for components of the siRNA ma-
chinery as well as the piRNA/rasiRNA machinery. If
anything, dodeca foci are closer together in ﬂies that are
RNAi defective, especially in the case of the piRNA/
rasiRNA mutants. This is consistent with the observation
that defects in the RNAi machinery can reduce chro-
matin compaction of heterochromatic sequences (Peng
and Karpen 2007)—reduction in chromatin compac-
tion of heterochromatic sequences may increase the
degree to which homologous sequences tightly pair
with one another.
Embryonic establishment of somatic pairing is also
independent of RNAi components that mediate in-
sulator function and somatic interactions between
PREs: In addition to examining pairing in meiotic
tissues, we also examined pairing in embryonic nuclei
just as pairing is established. Somatic pairing is estab-
lished in early embryogenesis on a locus-by-locus basis
soon after the early rapid mitotic divisions begin to
slow—around cycle 13 (Fung et al. 1998). This occurs
at about the same time as robust zygotic transcription
begins, suggesting that pairing may be mediated by re-
sidual maternally contributed factors. Thus, it was nec-
essary to examine pairing in embryos whose mothers
were also homozygous for the given mutation. Compo-
nents of the siRNA machinery are dispensable for both
fertility and viability but defects in the piRNA/rasiRNA
pathway lead to sterility. Thus, pairing in embryos of
dcr-2 or AGO2 mothers was directly assayed but pairing
in piRNA/rasiRNA defective embryos was assayed using
additional measures (see below).
Pairing state is substantially more transparent and
binary in the nuclei of the embryo than in meiotic
nuclei. For example, paired histone loci are virtually
always seen as a single dot, whereas unpaired foci are
seen as two (Figure 2). As others have shown (Hiraoka
et al. 1993; Fung et al. 1998; Gemkow et al. 1998), this
allows for a binary classiﬁcation of whether loci were
paired or unpaired within each nucleus (though it
should be stated that for the dodeca satellite, this was
achieved with some additional difﬁculty due to the fact
that sister chromatids often appeared separated, giving
the occasional appearance of three or four foci). The
distribution of distances between foci is included as
supplemental material and shows similar results (sup-
plemental Figure S2).
To determine whether there were any pairing defects
in ﬂies whose mothers were defective for components of
the siRNA pathway, FISH was performed in 2- to 4-hr-old
embryos. To ensure consistency of staging for these
embryos whose age ranged over a total of 2 hr, pairing
was assayed in a subset of these embryos—speciﬁcally,
those that were cellularized and just prior to or at the
beginning of gastrulation. In each of the multiple
embryos, at least 50 nuclei were counted. Pairing of
canonical euchromatic regions is very low at this de-
velopmental stage (Fung et al. 1998, data not shown).
Since the power to detect reduced pairing levels is weak
when wild-type pairing levels are already low, we only
analyzed pairing state for the histone locus and dodeca
satellite (Figure 2). Furthermore, while pairing for
canonical euchromatic regions is higher at larval stages,
embryos that are defective for components of the
piRNA/rasiRNA machinery do not progress to this stage.
Thus, this study of pairing establishment in embryos is
limited to loci that are not representative of canonical
euchromatic regions. Interestingly, as suggested by
others (Hiraoka et al. 1993), we found a nonrandom
clustering of nuclei that shared the same pairing state
(data not shown), suggesting that either local domains
within the embryo can effect pairing state (perhaps
based on relative timing of nuclear division) or neigh-
boring nuclei are more likely to share pairing state since
Figure 2.—FISH pairing assay in germa-
ria and embryos of control w
Cartoon diagram of the germarium. Pair-
ing was assayed collectively in C(3)G stain-
ing nuclei of regions 2a and 2b. (B) Paired
dodeca foci (green), partially separated be-
tween a strand of SC, indicated by immuno-
ﬂuorescence (red) to C(3)G in region 2a.
(C) A single focus (green) using a region
25 BAC probe indicating pairing in a mei-
otic nucleus of 2a. (D) A single focus
(green) using a histone cluster probe indi-
cating pairing in a meiotic nucleus of 2a. (E
and F) FISH in early embryos. FISH experi-
ments were performed on 2- to 4-hr-old
embryos. Pairing was assayed in embryos
that had completed cellularization and just
begun gastrulation (3 hr). (E) Dodeca
probe. (F) Histone cluster probe. In all
panels blue indicates DAPI. Each panel is
a single z-section.
1358 J. P. Blumenstiel et al.
Figure 3.—Distribution of distances between foci (in mm) of C(3)G staining nuclei of regions 2a and 2b of the germarium
, siRNA defective and piRNA/rasiRNA defective females. Rows 2, 3, and 4 indicate females that were homozygous for
defects in the siRNA machinery. Rows 5 and 6 indicate females that were trans-heterozygous for defects in the piRNA/rasiRNA
Pairing in Drosophila 1359
they are more likely derived from the same mother
nucleus (indicating that pairing status can be trans-
mitted through nuclear division).
As in the case of meiotic pairing, we found no obvious
tive in components of the siRNA machinery (Figure 4).
If anything, pairing frequencies for the dodeca satellite
are higher for ago-2
and, for the histone locus, higher
While mothers that are defective for components of
the piRNA/rasiRNA machinery are sterile, associated
defects can be partially rescued by a mutation in mnk,
the ﬂy homolog of the DNA damage signaling Chk2
(Klattenhoff et al. 2007). In particular mnk
mothers lay embryos that partially de-
velop, enough to potentially assay pairing state. Although
these embryos are defective, with grossly misshapen
nuclei (Figure 5), pairing can be assayed in embryos that
partially escape this phenotype. Upon ﬁrst observation,
it appeared that pairing frequencies were substantially
greater for the dodeca satellite in such embryos. How-
ever, after quantifying the ﬂuorescence intensity of the
dodeca probe in embryos that appeared to have high
levels of pairing, it was clear that the apparent high
numbers of nuclei with a single focus was associated with
a substantial reduction in ﬂuorescence intensity (sup-
plemental Figure S1). It is known that defects in the
RNAi machinery can lead to decondensation of hetero-
chromatin and this decondensation can lead to ectopic
exchange and loss of genetic material (Peng and
Karpen 2007). Thus, it appears that for embryos that
show a large number of nuclei with single dodeca foci,
this can be attributed to either loss of a single third
chromosome or loss of a substantial portion of the
dodeca satellite, perhaps during an early embryonic
nuclear division. In embryos that do not show this excess
of single dodeca foci (cutoff .60% nuclei showing a
single dot), pairing rates are not reduced (Figure 4).
Together, these data indicate that even maternally sup-
plied Aubergine protein is not necessary for the
establishment of chromosome pairing in embryos. To
assay pairing in piwi
embryos we generated germ-
line clones using the FLP-DFS method (Chou and
Perrimon 1992, 1996). As in mnk,aub, embryos were
highly defective with many misshapen nuclei. Nonethe-
less, in embryos for which this phenotype did not
prohibit analysis, pairing of the histone and dodeca
sequences was not reduced (Figure 4).
Mutations in aubergine lead to defects in SC assembly
and oocyte determination: Although we found no
obvious defects in either somatic or meiotic full homo-
log pairing in ﬂies defective for components of the
RNAi machinery, we did observe two obvious meiotic
defects. For example, in meiotic nuclei of aub
females, C(3)G displays the normal threadlike mor-
phology, suggesting that gross SC structure is normal.
However, large donut-shaped aggregations of C(3)G
protein were often observed ﬂanking the meiotic nuclei
(Figure 6). While these donut structures are reminis-
cent of ring canals, they do not colocalize with them
(supplemental Figure S3). In wild-type ﬂies, the pro-
portion of cysts in region 3 of the germarium with
aggregates was 10% (n¼21) and in stage 2, they were
observed 14% (n¼21) of the time. In aub ﬂies, the
Figure 4.—Pairing frequencies for the histone
cluster and dodeca satellite in 3-hr-old siRNA
and piRNA/rasiRNA defective embryos. siRNA
defective embryos were the progeny of homozy-
gous mothers and fathers. Piwi
generated with the FLP-DFS technique (see text).
were the product of mutant females mated with
males. Each point indicates the pairing fre-
quency of a single embryo in which the pairing
state in at least 50 nuclei was determined. Error
bars are standard errors for estimates of the pair-
1360 J. P. Blumenstiel et al.
proportion of region 3 and stage 2 cysts with aggregates
was 80% (n¼15) and 56% (n¼18), respectively (P,
0.01 and P,0.01, Fisher’s exact test comparing
respective regions). These aggregates of C(3)G resem-
ble the polycomplexes observed when the C(3)G pro-
tein lacks the C terminus that mediates attachment to
the lateral elements of the SC ( Jeffress et al. 2007).
However, the complexes seen here differ from those by
Jeffress et al. (2007) in that they were seen outside of
the DAPI staining nuclei. The fact that normal SC
structure is observed alongside these complexes sug-
gests that a mutation in aubergine leads to an accumu-
lation of free C(3)G protein in the cytoplasm rather
than simply prohibiting incorporation into the SC. In
turn, this accumulation of cytoplasmic protein forms
aggregates. Interestingly, the presence of these com-
plexes depended on activation of the Chk2 DNA
damage response. In mnk
the occurrence of these aggregates was equal to that in
wild-type ﬂies (region 3: 10%, n¼21; stage 2: 0%, n¼
15). Thus, in mnk ﬂies where the DNA damage response
fails to proceed, C(3)G protein no longer accumulates
and forms aggregates outside the nucleus. This suggests
that activation of the DNA damage checkpoint governs
either the production of C(3)G protein or access of
C(3)G protein to the meiotic nucleus. Furthermore,
since a large excess of DSBs are observed to form in aub
ﬂies (Klattenhoff et al. 2007), DSB excess may also
play a role in regulating C(3)G function or expression.
Mutations in aubergine also lead to a delay in ﬁnal
oocyte determination (Figure 7). In wild-type ﬂies,
region 3 cysts of the germarium typically display a single
oocyte nucleus, as deﬁned by threadlike C(3)G staining.
Occasionally two nuclei show threadlike C(3)G staining,
but in one nucleus the threadlike structure is midway
toward disassembly. We established an ‘‘oocyte index’’ by
counting within cysts the number of nuclei that show
maintenance of threadlike C(3)G staining and desig-
nating those nuclei that have partially dissembled SC as
0.5 nuclei. In wild-type ﬂies, this gave an oocyte index of
1.40 (n¼21) in region 3 and 1.05 (n¼21) in stage 2.
However, aub ﬂies frequently display two to four oocytes
in region 3 of the germaria (oocyte index: 2.40, n¼15)
and frequently two oocytes in stage 2 egg chambers
(oocyte index: 1.5, n¼18). Since aub germaria are
much reduced in size, it is conceivable that region 3 cysts
in aub ﬂies may be actually younger and less develop-
mentally mature than region 3 cysts from wild-type ﬂies.
Thus, a direct comparison between oocyte indices of
region 3 cysts may not be reasonable. Nonetheless, stage
2 egg chambers in aub ﬂies show a higher oocyte index
than region 3 wild-type cysts, indicating that this delay
in oocyte determination is in fact real. And while a
mutation in mnk rescues the formation of the C(3)G
aggregates, a similar delay in ﬁnal oocyte determination
Figure 5.—Embryos of mnk
ers are highly disorganized: DAPI staining of 2- to 4-hr-old em-
bryos. The exact developmental point in early embryonic
development is difﬁcult to determine due to gross defects.
(A) DAPI nuclei show looped-out circles of DNA. (B) A highly
disorganized ﬁeld of nuclei.
Figure 6.—Aggregates of C(3)G (red) that resemble poly-
complexes form ﬂanking the meiotic nuclei within the ger-
marium of aub
ﬂies. (A and B) Donut-shaped
aggregates of C(3)G in aub
ﬂies. (C and D) Aggre-
gates fail to form in mnk
Pairing in Drosophila 1361
is also observed in mnk
(region 3 oocyte index: 2.0, n¼21; stage 2 oocyte index:
1.41, n¼16). This indicates that while activation of the
DNA damage checkpoint promotes the formation of
C(3)G aggregates, it does not lead to the delay in oocyte
determination in aubergine ﬂies.
In sexual species, proper segregation of chromo-
somes during meiosis requires homologs to become
paired. Ironically, sexual reproduction itself fosters the
conditions for proliferation of selﬁsh genetic elements,
such as transposons (Hickey 1982) that might pose a
challenge for the pairing process due to their dispersed
repetitive nature. The RNAi machinery is thought to
have evolved to limit the spread of such selﬁsh repeats
and viruses (Matzke et al. 2000; Plasterk 2002; Zamore
2002; Cerutti and Casas-Mollano 2006; Ding and
Voinnet 2007) and subsequently, the RNAi machinery
has also evolved a wide range of secondary functions.
In Drosophila, the RNAi machinery has been shown
to have several different roles in producing the higher
order structure of chromosomes in the nucleus (Pal-
Bhadra et al. 2004; Matzke and Birchler 2005; Kavi
et al. 2006; Savitsky et al. 2006; Peng and Karpen 2007).
In particular, the RNAi machinery mediates both in-
sulator function and the long-distance clustering of
Polycomb response elements (Grimaud et al. 2006; Lei
and Corces 2006b). These observations have suggested
a mechanism for how long-range chromosomal associ-
ations are mediated by the RNAi machinery. An impor-
tant feature of this model is that insulator looping and
PRE clustering depends on both the RNAi machinery
and proteins that are speciﬁc to particular loci. In the
case of insulator looping, a complex of CP190/Mod
(mdg4)2.2/Su(HW) proteins is proposed to bind to
insulator sequences and loops may be stabilized by the
small RNAs that are recruited to the complex. In the
case of Polycomb response elements it is proposed that
bound Polycomb proteins, together with small RNAs,
stabilize long-range interactions.
Nonetheless, we have shown that defects in the RNAi
machinery that mediate these functions have little effect
on pairing establishment both in the embryo and in
the germline. This is consistent with work that has
shown no reduction in crossing over frequency in ﬂies
defective in piRNA/rasiRNA components (Cross and
Simmons 2008), contrary to what would be expected if
there were a pairing defect. In light of Su(Hw)’s effect
on homolog pairing in the soma (Fritsch et al. 2006),
the association of dispersed sequences may in fact
facilitate whole chromosome pairing, but this function
appears to be independent of the RNAi machinery.
Altogether, this suggests a hierarchical model for the
association of homologous sequences—between homo-
logs and at heterologous locations—within the nucleus.
First, particular chromatin-bound proteins dispersed
along the chromosomes may facilitate chromosome
pairing on a locus-by-locus basis. For example, the his-
tone cluster, which shows great afﬁnity between the
homologs, may pair in a very efﬁcient manner due to the
fact that it is uniquely contained within the histone locus
body (Liu et al. 2006; Liu and Gall 2007). Full-length
homolog pairing may thus be mediated by a large
assemblage of various chromatin-bound proteins, in-
cluding insulator and Polycomb proteins, distributed
along the length of the chromosome arms. Overlaid on
this system, the RNAi machinery may contribute, in a
cooperative fashion, to long-distance interactions by
binding distinct classes of chromatin-bound proteins
into larger domains. Consistent with this hierarchical
model, Grimaud et al. (2006) found that the localiza-
tion of Polycomb proteins to endogenous PREs did not
depend on components of the RNAi machinery and
that the RNAi machinery more likely plays a role in
stabilizing long-distance interactions rather than initi-
ating them. A hierarchical model is also suggested by
the observation that the RNAi machinery, in conjunc-
tion with heterochromatic proteins, has an inhibitory
effect on ectopic exchange between dispersed hetero-
chromatic repeat sequences (Peng and Karpen 2007).
In a combinatorial fashion, the RNAi machinery, inter-
acting with one class of chromatin-bound proteins, may
Figure 7.—A defect in aub leads to a delay in meiotic progression. White bars indicate region 3 of the germarium. (A) w
control ﬂies show one C(3)G staining nucleus in region 3. (B and C) aub
ﬂies frequently show two or more C(3)G
staining nuclei in region 3, indicating a delay in the decision to form a single oocyte. (D) This defect is not alleviated in
1362 J. P. Blumenstiel et al.
thus mediate dispersed interactions, but in the context
of another set of chromatin-bound proteins, lead to the
inhibition of dispersed interactions, presumably by
leading to condensation of heterochromatin and exclu-
sion from the homology search initiated after DNA
One caveat to this interpretation is that functional
redundancy between different components of the RNAi
machinery may limit our conclusion that the RNAi
machinery does not play a role on meiotic or somatic
homolog pairing. Our results show that the same com-
ponents that mediate long-range interactions are not
individually necessary for homolog pairing. However, full
proof of a complete lack of involvement of the RNAi
machinery in pairing requires an analysis of pairing in a
genetic background that is simultaneously defective for
primary components of each of the three branches of
the RNAi system—the miRNA, siRNA, and piRNA/
rasiRNA pathways. Unfortunately, such analysis is difﬁ-
cult since these components are essential for viability
In spite of these results, we have uncovered two new
functions for the RNAi machinery in oocyte determina-
tion and regulation of synaptonemal complex forma-
tion during Drosophila female meiosis. Components of
the piRNA/rasiRNA machinery—aubergine, piwi, spindle-
E, and armitage—have previously been shown to be
essential for silencing transposons in the germline. In
the face of transposon mobilization, DNA damage will
lead to activation of the ATR/Chk2 DNA damage
checkpoint, leading to the classic spindle defects that
include fused dorsal appendages and abnormal kary-
some formation (Chen et al. 2007; Klattenhoff et al.
2007). We have now found that defects in the piRNA/
rasiRNA machinery also lead to defects in the assembly
of the synaptonemal complex as measured by C(3)G
immunostaining. In particular, a mutation in the piRNA/
rasiRNA component aubergine leads to the formation of
extranuclear C(3)G aggregates as well as a delay in
resolving the identity of the single oocyte among the 16
mitotic nuclei. The C(3)G aggregates resemble those
polycomplexes observed in ﬂies whose sole sources of
C(3)G protein is truncated at the C-terminal end that
interacts with the lateral elements of the synaptonemal
complex ( Jeffress et al. 2007). However, the fact that
the aubergine C(3)G aggregates reside outside the
nucleus and occur alongside SC with normal C(3)G
morphology indicates that they likely arise due to a
failure for all C(3)G protein to enter the nucleus rather
than any intrinsic failure to form SC. Interestingly, the
formation of the extranuclear C(3)G aggregates is
rescued by a mutation in mnk/Chk2. This suggests a
potential feedback between activation of the DNA
damage checkpoint and regulation of C(3)G.
Unlike C(3)G aggregate formation, the delay in
oocyte determination in aubergine ﬂies is not dependent
on activation of the Chk2 DNA damage cascade—simi-
lar numbers of two-oocyte region 3 germarium were
found in aub and aub,mnk ﬂies. This two-oocyte pheno-
type has also been observed in ﬂies defective for the
rasiRNA component spn-E, as well as mus301/spn-C
(Huynh and StJohnston 2000; McCaffrey et al.
2006). Mus301 has been shown to play a role in DSB
repair, but the associated two-oocyte phenotype in
mus301 ﬂies was also observed in ﬂies that lack mei-
otic double-stranded breaks programmed by mei-W68
(McCaffrey et al. 2006). This result suggested that
mus301 had a role in oocyte speciﬁcation independent
of programmed DSBs. It remains to be determined
whether the mechanism underlying the two-oocyte
phenotype is the same in the case of DNA repair de-
fective ﬂies that lack programmed DSBs and piRNA/
rasiRNA defective ﬂies with abundant DSBs that arise in
the face of transposon mobilization and are thus W68
Many thanks to Jack Bateman for discussions, suggestions, and
insight and Danny Stark for assistance in microscopy. Flies were kindly
provided by the Carthew, Gao, Schedl, Elgin, and Berg laboratories.
This work was funded by an American Cancer Society Postdoctoral
Fellowship to J.P.B. and an American Cancer Society Professor Award
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Communicating editor: T. C. Kaufman
Pairing in Drosophila 1365