1
Genetic control of a sex
-
specific piRNA program
Peiwei Chen
1
&
and
Alexei A
.
Aravin
1
&
1
California Institute of Technology,
Division of Biology and Biological Engineering
Pasadena, CA 91125, USA
&
To whom correspondence should be
addressed:
Alexei
A.
Aravin
aaa@caltech.edu
Peiwei Chen
peiweitc@gmail.com
.
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;
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2
Abstract
Sexually dimorphic traits in morphologies are widely studied, but those in essential molecular
pathways remain largely unexplored.
Previous work showed
substantial
sex
differences in
Drosophila
gonadal piRNA
s
,
which
guide
PIWI
proteins
to silence
selfish genetic elements
thereby safeguarding fertility
.
However, the
genetic control
mechanisms
of piRNA
sexual
dimorphism
remain
unknown
.
Here, we showed that most sex differences in the piRNA program
originate from the germline
rather than
gonadal
somatic
cells.
Building on this
,
we
dissected
the
contribution of sex chromosome and cellular sex
ual
identit
y
towards
the
sex
-
specific
germline
piRNA program
. We
found that
the
presence of
the
Y
chromosome
is sufficient to recapitulate
some
aspects of the
male piRNA
program
in
a female cellular environment
. Meanwhile, sexual
identit
y
control
s
the
sexually
divergent piRNA
production from
X
-
linked and autosomal loci
,
revealing
a
crucial
input from
sex determination
into piRNA biogenesis.
S
exual
identity
regulates
piRNA biogenesis
through
Sxl
and
this effect is
mediated in part through
chromatin proteins
Phf7
and
Kipferl.
Together, our work delineated the genetic control of
a sex
-
specific piRNA program
,
where sex chromosome and sexual identit
y
collectively sculpt an essential molecular trait.
.
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The copyright holder for this preprint
this version posted October 26, 2022.
;
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3
Main
Sexual dimorphi
sm
,
where a trait is modified by the
biological sex to manifest in distinct ways
between
males and females
, is pervasive in nature
.
While
sexually dimorphic traits
in
morphologies
have
been widely studied
1
–
4
,
those
in essential molecular pathways remain largely
unexplored.
In
Drosophila
melanogaster
gonads, the piRNA program
executes a
critical
function
by
guid
ing
the PIWI
-
clade Argona
u
te proteins
to
silence
selfish genetic elements
such as
transposons
5
–
7
,
thereby safeguarding fertility. To
pass on the transgenerational memory
of
proper
piRNA targets,
mother
s
deposit piRNAs to
the
embryo
,
instructing
the zygotic genome
to mount
a homologous piRNA program
in the next generation
that reflects
the
maternal
response
to
genomic parasites
8
–
11
.
However, males
implement
a piRNA program distinct from
their
female
siblings
12
, the underlying mechanism
of which
is
elusive.
We previously found evidence for both
differential
transcription
of piRNA
loci
in the nucleus
13
and differential processing
of piRNA
precursor
transcripts
in the cytoplasm
12
betwee
n
the two sexes
,
but
the upstream control
of these
sexually dimorphic molecular events
is
unknown
.
In this work, we
sought to decipher
the genetic
control of
piRNA sexual dimorphism
, in order to gain
insights into
the mechanisms by which
sexual
dimorphism
in
essential molecular trait
s
is sculpted
.
Results
Prior work compared the male and female piRNA
profiles
from two different
D. melanogaster
lab
strains
12
,
where
distinct genetic backgrounds confound
ed
the
characteriz
ation
of piRNA sexual
dimorphism.
In addition,
the sex
of
D. melanogaster
is determined
independent
ly
of the presence
of
the
Y
chromosome
(both XY and
XO flies are phenotypic males
,
while XX and XXY flies are
phenotypic
females)
14
–
16
,
so the
morphology
-
based identification of males and females
does
not
directly translate to
an interpretation of
Y chromosome
status
.
Given that several
piRNA
-
producing loci reside on the Y
12
,
the inability to
infer
Y chromosome content from
the
phenotypic
sex
complicates
the
characterization
of
piRNA sexual dimorphism.
To circumvent these issues,
we
introduced
a Y chromosome marked by
y
+
and
w
+
genes
(hereafter
y
+
w
+
Y)
into
an
inbred
yw
stock
and backcrossed
it
to
yw
for
multiple
consecutive
generations
(
see methods
)
.
This line
allowed us to
unequivocally identify XY males (
red
-
eyed
flies with
black body
color
and
male
genitalia
)
and XX females (
white
-
eyed flies with yellow body
color
and female
genitalia)
(
Fig. 1a
)
,
from which we
profiled the
gonadal
piRNA
s
in
each of
the
two sexes.
Analysis of
the
piRNA
libraries showed
substantial intersexual differences
in
the abundance of piRNAs targeting
different transposon
families
(
Fig. 1b
)
and expression levels of
individual major
piRNA
loci
in the
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4
genome
(
Fig. 1c
)
, largely
in agreement
with our previous
study
12
.
Having excluded the possible
confounding effects of
genetic backgrounds
and Y chromosome
status
, we
confirmed
that
the
piRNA program in
D. melanogaster
gonads
is sexually dimorphic.
Germline is the major cell type origin of piRNA sexual dimorphism.
In
D. melanogaster
gonads, t
he piRNA program operates in both the germline and
gonadal
somatic cells,
but
piRNA
biogenesis and targets differ between
the
two
cell types
9,17
.
Thus, t
he
male
-
female differences seen in gonad
-
wide piRNA quantification could reflect sexual
dimorphism in either
germline
or
gonadal soma
tic cells
, or
both
cell types
,
which
could be
further
skewed
by distinct
germline
-
soma ratios in testis and ovary. To distinguish these
possibilities, we
isolate
d
the somatic piRNA
s
in the gonad,
by
immunoprecipitat
ing
Piwi upon
germline
-
specific
piwi
knock
-
down
(
see methods
)
and
then sequencing
the small RNA
s
associated with Piwi
in
gonadal somatic cells
(
Fig.
1d
)
.
This
allowed us to profile the gonadal somatic piRNA program i
n
each of the
two
sexes.
E
xperimentally isolated
gonadal
somatic piRNA
s
from
test
e
s and ovar
ies
(
Fig. 1d
)
allowed
us to directly compare the piRNA program
in gonadal soma
between sexes
.
We found that t
he
flamenco
piRNA
locus
shows
a similar
piRNA coverage profile
and produces piRNAs that take up
comparable fractions of total piRNAs
in
testicular and ovarian soma
(
Fig.
1e
)
.
M
ost
of the
highly
expressed piRNAs
in gonadal soma
are antisense to transposons
in both
males and females
,
and
piRNAs targeting different transposon families display
a
strong
positive correlation
between
the
two
sexes (Pearson’s r = 0.91,
p
< 0.0001
; Spearman’s r = 0.
89
,
p
< 0.0001
;
Fig.
1f
)
.
When
normalized
to
flamenco
,
a few
transposons
are
targeted
more in
either
males (e.g.,
idefix
) or
females (e.g.,
gtwin
and
mdg1
)
, but these biases are
relatively
mild (
Fig.
1f
).
To
examine piRNA
production across the genome
, we defined piRNA
-
producing loci in gonadal soma and
measured
their expression levels
(
see methods
)
.
Akin to
piRNA
quantification based on
their
transposon
targets,
quantif
ying piRNA
s
based on their genomic origin
s also revealed
a strong positive
correlation
between
the
two sexes
(Pearson’s r = 0.89,
p
< 0.0001
; Spearman’s r = 0.82,
p
<
0.0001
;
Fig. 1g
).
We did
, however,
note
an
exception: a
novel
piRNA locus
we identified
in
the
gonadal soma
,
77B
(
Fig. 1h
),
produces more piRNAs in males than females
(
Fig. 1g
).
This
locus
resembles
flamenco
,
as it
makes piRNAs from one genomic strand
downstream of a
prominent
RNA
p
ol II peak
that is
indicative of
a
promoter
, producing antisense piRNAs against transposons
active in the gonadal soma
(e.g.,
idefix
;
Fig. 1h
).
Nevertheless, the
genome
-
wide view of
the
piRNA production in gonadal soma highly correlate
s
between
sexes.
T
he 3’ UTR of some genes
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(e.g.,
tj
) is known to produce piRNAs in ovarian soma
18
, and the same holds in the male
counterpart
(
Fig. 1g
).
Overall
,
the piRNA program operat
ing
in gonadal soma
shows
very
few
sex
differences
.
Taking advantage of the fact that the
flamenco
piRNA locus is active
exclusively in the
gonadal soma but not in the germline of both testis (
Extended Data Fig. 1
) and ovary
6,19
, we
inferred that germline piRNAs make up about 97% and 79% of the gonadal piRNAs in testis and
ovary, respectively (
see methods
). Because germline piRNAs dominate the whole gonad piRNA
pool to comparable extents in both
sexes (97% / 79% = 1.2
-
fold difference), gonad
-
wide piRNA
quantification is a close approximation of the germline piRNA program when studying male
-
female differences.
These results
s
uggest
that
the piRNA sexual dimorphism
we
observed
in
the
whole
gonads
originate
s
from the germline rather than gonadal soma.
For the rest of this work,
we use
d
the gonad
-
wide
piRNA
sexual dimorphism
to approximate germline
piRNA
sexual
dimorphism, as germline piRNAs account for comparable fractions of total piRNAs
in
whole
gonads of males and females.
Y chromosome is necessary and sufficient to recapitulate aspects of male piRNA program.
Having
found
that
germline
is the major cell type origin of
piRNA sexual dimorphism,
we
aimed to
dissect
its underlying
genetic control mechanisms
(
Fig. 2a
)
.
Distinct sex chromosome contents
between sexes, specifically
, the
presence of Y chromosome in males,
could
in theory explain
some sex differences
in piRNAs
.
On the other hand
,
distinct sexual identities
could
lead to
differential piRNA production
even
from
identical
piRNA
loci located
outside the Y
.
Importantly,
t
he
sex determination in
D. melanogaster
does not
involve
the Y
14
,
which provides
us with a
unique opportunity to
manipulate
the
Y chromosome
without perturbing sexual identit
ies
.
To pin
point the contribution of the Y
chromosome
to
sex
differences in
the
piRNA
program,
we
first
generated
XY and XO
male
sibling flies
that
only
differ in the Y chromosome content
but
are otherwise genetically identical
. This is
d
one by
using
spontaneous sex chromosome
nondisjunction that occurs
at about
10% frequency
in
X^XY
females carry
ing
the
compound X
chromosome,
C(1)A
(
Fig. 2c
).
When compared to their XY brothers, XO males
lose
piRNAs
targeting several transposon
families
(
Fig. 2d
)
,
indicating
that
the
Y chromosome
is a
source of
transposon
-
targeting piRNAs
in the male.
For example
,
the
absence of
the
Y chromosome
causes
decrease
s
in
piRNAs
against
nomad
and
invader2
(
Fig. 2d
)
–
two transposons that are normally
targeted by more piRNAs in males
than
females (
Fig. 1b
)
,
suggesting that these sex differences
.
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could be largely explained
by the Y chromosome
alone
.
We also note that
,
piRNAs
targeting
I
-
element
appear
upregulated in males
lacking
the Y chromosome
(
Fig. 2
d
)
, which
warrants future
investigation.
R
emoving
the
Y
also
led to
a specific
loss of piRNAs from two Y
-
linked
loci
–
Su(Ste)
and
petrel
(
Fig. 2b
)
–
while leaving
the
piRNA
production
from
other
loci
on X and autosomes
unperturbed
(
Fig. 2
b,
e
)
.
Therefore,
Y chromosome
contributes to the male piRNA program via
production of
piRNAs
from two loci
on the Y
,
Su(Ste)
and
petrel
,
as well as
piRNAs
targeting
a
select group of transposons
.
Complementing
the male experiment,
we
generated
XX and XXY
female sibling flies that
only differ in their Y chromosome content
s
but are otherwise
genetically identical
.
This is achieved
by
first obtaining an exceptional XXY
female
from
primary
sex chromosome nondisjunction that
occurs
naturally
about 1 in 2,000
wildtype
flies
14
,
and then
crossing
this
XXY female
with
XY males
to
sire
XX and
XXY females
through
secondary sex chromosome nondisjunction
(
Fig. 2f
)
.
T
he
extra Y chromosome
barely
alter
ed
t
he overall transposon
-
targeting piRNA program
in females
(
Fig. 2g
)
.
Nonetheless,
the
pr
esence of
the
Y chromosome in
females
trigger
s
piRNA
biogenesis
from
Su(Ste)
and
petrel
(
Fig. 2h
),
two
loci
that reside
on
the Y
chromosome
.
Furthermore
, t
he
se
two
Y
-
linked piRNA loci exhibit
comparable activities to other top piRNA loci in females, including
42AB
,
38C
,
and
80F
(
Fig. 2h
)
,
suggesting that
the
Y is
an active and productive piRNA source in
a
female cellular environment.
We
generate
d
genetically identical
male and female siblings that only differ in their Y
chromosome content
s
,
however,
these cross
es
necessitated
the
employ
ment of
mothers
carrying
a Y chromosome
(
Fig. 2c,f
)
.
Given that
m
aternally deposited piRNAs
instruct
piRNA biogenesis
in the
progeny
8,10,11
,
Y
-
bearing
mothers
might
creat
e
a permissive
environment
to produce
Y
piRNAs
in the offspring
by
deposit
ing
Y
-
derived piRNAs to the embryo
.
Consequently
, it is
unclear
whether
the effects of Y chromosome on male and female piRNA production we observed
(
Fig.
2d,e,g,h
)
depends
on
mothers
carrying
a
Y chromosome.
To empirically
test the role of Y
chromosome in piRNA production with
out
mothers
bearing
a
Y
,
we
devised
a
strategy to generate
half siblings
of both sexes
that
share similar, albeit not identical,
genetic backgrounds
with and
without
Y chromosome
from
XX mothers
(
Fig. 2i,l
)
.
We observed similar
effects of the Y
chromosome on piRNA production when mothers do not have a Y:
Y
chromosome
is an important
source of
transposon
-
targeting piRNAs in males but not
in
females (
Fig. 2j,m
)
,
and
the two Y
-
linked piRNA loci
,
Su(Ste)
and
petrel
,
produce
piRNAs
in both
sexes
,
irrespective
of the cellular
sexual identity
(
Fig. 2k,n)
.
We noticed that
the Y
exerts a slightly greater effect
on
the transposon
-
targeting piRNA
program
in th
is latter
cross scheme
(
Fig. 2i,l
)
compared to the former
one
.
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;
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7
involving
Y
-
bearing mothers
(
Fig. 2c,f
),
which likely result
s
from having different fathers and thus
distinct paternally inherited haploid genome.
Nevertheless
,
the
results
between mothers
with and
without a Y chromosome
are
qualitatively
very
similar
.
We conclude that
the
presence of
the
Y
chromosome
alters
the piRNA
profiles
independently of its presence in
the
mothers.
Y chromosome
in
D. melanogaster
is
known to
exhibit
imprinting effects
20
–
22
, that is, Y
can
behave differently when inheri
ted from
the
mother or
the
father.
To test if
Y
-
linked piRNA loci
show
parent
-
of
-
origin effects,
we
designed crosses that allow females to inherit
a
Y chromosome
from either
parent
.
I
n the case of paternally inheriting the Y,
we
also designed
crosses
either with
or without mothers bearing a Y
(
Fig. 3a top
)
. In all cases, we detected nascent transcripts
from
Su(Ste)
and
petrel
piRNA
loci located on
the
Y chromosome
(
Fig. 3a middle
), indicating that
piRNA loci on the Y are transcriptionally active
in
the female germline
when inherited from either
parent.
We
also
observed similar behaviors of the Y
-
linked
piRNA
loci in the male counterpart
(
Fig. 3b
)
–
w
hen inherited from either
parent
, with or without mothers carrying a Y, Y chromosome
activates both
Su(Ste)
and
petrel
loci in the male germline
.
Thus,
there
are
no obvious imprinting
effects of the
two
Y
-
linked piRNA loci
,
and
the
mere
presence of
the
Y
can translate to an effect
on the
germline
piRNA program
in
both
sex
es
.
Interestingly, in our cross scheme of passing the
Y from mothers to daughters,
Su(Ste)
piRNA precursor transcription is also activated in the follicle
cells (a gonadal somatic cell type), an unexpected finding that calls for future studies
.
Whereas
Su(Ste)
piRNAs silence
the
Stellate
genes that are only active in the male
germline
,
petrel
piRNAs silence the
pirate
gene
that is ubiquitously expressed in all
tissues
including female germline
,
which
allows us
to
explore
if
activating
petrel
piRNA biogenesis in
the
female germline
lead
s
to
pirate
silencing
.
In wildtype XX female germline,
the
pirate
gene is
active,
and its transcripts can be readily detected
by RNA
in situ
HCR
(
Fig. 3a bottom
)
. However,
introducing
a Y into the female germline from either
parent
led to a
marked
silencing of the
pirate
gene
(
Fig. 3a bottom
)
, suggesting that
making
petrel
piRNAs in
female germline
has a direct
functional outcome.
M
eanwhile,
the presence of
Y
chromosome in
mothers was
neither necessary
nor sufficient
for the silencing of
pirate
in the female
progeny. Thus,
pirate
silencing
in
the
female
germline
require
s
the
presence of the
Y chromosome
,
regardless of the
parental origin
of the Y
.
Similarly, in the male
germline,
h
aving a Y
-
bearing mother is neither sufficient nor required for the
male germline to tame
Stellate
and
pirate
,
and
the presence of
Y chromosome
triggers
silencing
of
both
Stellate
and
pirate
genes
regardless of the
Y’s
inheritance path
(
Fig.
3b
)
.
Taken together,
the differences in piRNA profiles caused by the presence of
Y
chromosome
directly
translates to
differential
silencing
of several targets
,
without obvious parent
-
of
-
origin
effect
s
.
.
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;
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Cellular sexual identity provides a key input
into piRNA biogenesis.
T
hough necessary, t
he presence of
the
Y chromosome in males is not sufficient to explain
sex
differences in
the piRNA program
, as piRNA loci
outside the Y
are
also
differentially expressed in
two sexes (
Fig.
1
c
)
.
What underlies
piRNA
sexual dimorphism
outside the Y
?
In
D. melanogaster
,
a cascade of molecular switches
take
s
place
after counting the
number of X
,
culminat
ing
in
either
male or female
cellular
sex
ual
identity
14
–
16,23
–
28
(
Fig. 4a
)
.
To
examine
the contribution of sexual
identit
ies
to
germline piRNA sexual dimorphism
without confounding impact
s
of
the
Y
chromosome,
we
sought to masculinize XX female germline and compare it to XO male
germline
that lacks a Y
(
Fig. 4
b
)
.
Unlike the
Drosophila
soma
, where
sex determination
occur
s
cell
-
autonomous
ly
,
Drosophila
germline
receives
an additional input from the som
a
on top of its own
chromosomal
content
to determine
the
germline
sex
24,25
.
When the
germline sex
does not match
the
somatic sex,
germline
either
dies or becomes tumor
ous
29,30,23
–
25,31,32
,
so
a
productive
germline
sex
reversal requires
perturbing both
the
germline and soma
tic sex
.
Given that
there is
very
little
sexual dimorphism in the
gonadal soma
tic piRNAs
(
Fig. 1e,f,g,h
)
,
reversing the somatic sex
should not confound
our study of germline piRNA sexual dimorphism
.
Hence,
our germline sex
reversal
was
done in sex
-
reversed
soma
,
which allowed us to
interrogate
the effect of sexual
identities on germline piRNA sexual dimorphism.
To
explore
if and how sexual identities
impact
germline
piRNA
profiles
,
we
masculinized
XX female germline by
germline
-
specific
knock
-
down
of
Sex
lethal
(
Sxl
)
,
the
major factor
that
govern
s
the
female
identity
in germline
23
–
26
,
in
the
tra
nsformer
(
tra
)
mutant background that
has
a masculinized
soma
29
(
Fig. 4c
)
.
Strikingly,
masculiniz
ing
the
XX
female germline
converted
its
transposon
-
targeting piRNA program to
a state
that
closely resemb
les
the
XO male germline
(
Fig.
4d
left
)
.
When
quantif
ied
,
the
median
extent of
masculinization
for
piRNAs targeting
different
transposon families
is
99%
(
see methods
;
Fig. 4d right
)
.
Similarly,
for major piRNA loci outside
the Y,
their
expression
levels and piRNA coverage profiles
also
switched from an XX female state
to an XO
male
state
upon
masculinization
of
the
XX germline
(
Fig. 4e
,f
)
.
For example,
abundant
piRNA
s are made
from both proximal and distal ends of the
42AB
piRNA locus
in XX
females,
but
masculinized
XX
germline
only
make
some
piRNAs from the distal end
of
42AB
and
barely
any
from the proximal side,
reminiscent of the
XO male germline (
Fig. 4f left
).
In sum,
reversing
the germline sexual identity
is sufficient to
switch
the
germline piRNA program from one sex to
the other, suggesting that the cellular sexual identity provides
a
key
input into piRNA biogenesis.
.
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9
How does
the germline interpret its sexual identity
to
elicit a sex
-
specific piRNA program
?
We
showed
that this sexual
-
identity
effect on piRNA biogenesis is governed by the major switch
protein Sxl
(
Fig. 4d,e,f
)
,
which is active in the female, but not male, germlin
e. Next,
we
looked into
how Sxl
orchestrates a female
-
specific
piRNA program
in the germline
.
Sxl is known to
regulate
two target genes that
exhibit
sex
-
specific expression pattern
s
in the germline
33
: Tdrd5l
28
, a
cytoplasmic protein that forms granules distinct from
the
piRNA
processing
sites,
and Phf7
34
,
a
chromatin
reader
protein that binds H3K4me2/3
.
Both
Tdrd5l and Phf7 promote a male identity,
and
Sxl
represses
th
es
e two factors to
bolster a
female identity
.
Since
Tdrd5l and Phf7
act
genetically redundantly to
support a male identity
28
, we
focused on Phf7
for this study
and aske
d
whether
and
, if so,
to what extent
Phf7 mediates
the sexual
-
identity effect on piRNA biogenesis.
E
xpressing Phf7
in the female germline
accompanied
by somatic masculinization
partially
masculinize
d
the XX female germline
,
leading to a 29% median extent
of masculinization of
transposon
-
targeting piRNA
s
(
Fig. 4d
)
.
For many transposon
-
targeting
piRNAs
(e.g.,
those
targeting
copia
and
burdock
)
,
the
ectopic expression of Phf7
in XX germline
shifted the piRNA
profile from a female state
towards a male state,
but
not
as completely as
losing
Sxl
did
(
Fig. 4d
right
)
.
This partial
reversal of the piRNA program from one sex to the other
by Phf7
activation
is
also
obvious
when
examining the
expression of major piRNA loci
in the genome
.
E
ach of the
major piRNA loci
in Phf7
-
expressing
XX
female germline
resume
d
an activity
somewhere in
between the
wildtype
XX female and XO male
,
for both male
-
and female
-
biased loci
(
Fig. 4e,f
)
.
For instance,
Phf7
dampened the activity of
42AB
, a female
-
biased piRNA locus
,
and
enhanced
the activity of
38C
, a male
-
biased piRNA locus (
Fig. 4e,f
).
These observations
indicate
that
Phf7
promotes
a
male piRNA program,
and
Sxl
supports
a female piRNA program
in part
through
repressing Phf7
.
Thus, Phf7
mediates
part of the sexual
-
identity effect on piRNA biogenesis
,
acting downstream of Sxl.
Recently,
a female
-
specific
piRNA
biogenesis factor, the zinc
-
finger protein Kipferl,
was
described
to drive
a subset of
piRNA
production
in the
female germline
35
.
In
particular,
piRNA
production from
80F
, a
sex
-
specific
piRNA locus only active in
the
female germline
(
Fig. 4e
)
,
depends on Kipferl
35
(
Fig
. 4g
)
,
indicating
that Kipferl is directly responsible for
some
of the
germline piRNA sexual dimorphism.
As
Kipferl
appears dedicated to
the piRNA pathway and is
absent in the male germline, we hypothesized that
Kipferl is an effector
protein
that acts
downstream of the sex determination pathway
to
elaborate
a
female
piRNA program
.
Indeed,
knocking
-
down
Sxl
in the female germline is sufficient to abrogate
the expression of
Kipferl
in
XX
germline
, suggesting that
Kipferl expression
depends on Sxl
(
Fig. 4
h
)
.
On the other hand
,
expressing Phf7
in the
XX
female germline did not perturb
Kipferl
expression
(
Fig. 4
h
)
,
suggesting
.
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that
Phf7 and Kipferl act
in parallel, both
downstream of Sxl,
to promote male and female piRNA
programs, respectively
(
Fig. 4i
)
.
Taken together, we elucidate
d
a
genetic
circuit
that connects the sex determination
pathway to
germline piRNA sexual dimorphis
m
(
Fig. 4i
)
.
XX germline activates
Sxl,
which
positively regulates Kipferl to
produce
female
-
specific piRNAs and negatively
regulates
Phf7
to
suppress a male piRNA program.
On the contrary, XY germline lacks
functional
Sxl to activate
Kipferl and instead
expresses Phf7 to
elaborate a male piRNA program.
We conclude that
male
and female
sexual identities
enable
divergent piRNA
production programs
,
sculpting
a sexually
dimorphic molecular trait
alongside the
male
-
specific
Y chromosome.
Discussion
In this work,
we
identified the germline as the
source
of
piRNA
sexual dimorphism
in fly gonads.
Building on this
, we
deciphered
the
genetic control
underlying
the
sex
-
specific
piRNA program
.
We
characterized the contribution of the Y chromosome to the male piRNA program, and we
showed that
the
presence of
the
Y is sufficient
to recapitulate
some
aspects of the male piRNA
program in
a female cellular environment
.
In fact, t
he effect of
the
Y is independent
of
its
parental
origin and mother
s’
sex chromosome content
s
.
T
he ability of Y
-
linked piRNA loci to act
in both
male and female cellular environments
independent
ly
of its inheritance path implies
unique
regulatory mechanisms
36
employed by
the Y
and
distinctive
evolutionary forces acting on the Y
.
Meanwhile,
we
showed
that sexual identit
y is a
nother
major determinant of
the
piRNA
program
that
regulates piRNA biogenesis outside the Y
.
Specifically, s
exual identit
y
shape
s
piRNA sexual
dimorphism
un
der the control of Sxl,
which
relays
the sexual identity of a cell
to piRNA biogenesis
through
the
histone reader
protein
Phf7 and
the
zinc
-
finger protein Kipferl
.
We speculate that the
sex determination pathway
has
been hijacked by transposons to facilitate their sex
-
biased
germline
invasion
12
,
so
integrating the information of
germline sexual identit
ies
into piRNA
biogenesis provides a means to
directly
couple
the
sex
-
specific
piRNA defense program with sex
-
specific transposon threats.
Together,
our work revealed that s
ex chromosome and sexual identit
y
control
different facets of piRNA sexual dimorphism, and it is
the
ir
collective action that
sculpts
the
sex
-
specific piRNA
program
in fly germline
.
It is very likely that
other
sexually dimorphic
traits
are under the control of both sex chromosome and sexual identit
y
, and disentangling the effects
of the two promises to offer new insights into
how a molecular pathway can be
modified by each
of the two sexes to execute essential functions
.
.
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Acknowledgement
We thank Grace YC Lee,
Felipe Karam Teixeira
,
Justin Blumenstiel
, Katalin Fejes Toth, and
members of the Aravin Laboratory for discussion
,
Jim Kennison for advice on se
x chromosome
nondisjunction
,
Mark Van Doren and Helen Salz for advice on sex determination
, and Yukiko
Yamashita for sharing unpublished results
.
We thank Angela Stathopoulos
, Ellen Rothenberg,
and
Henry Chung
for comments on the manuscript.
We are grateful to
Liz Gavis,
Bloomington
Drosophila Stock Center
and
Vienna Drosophila Resource Center
for fly
lines
. We appreciate
the help of Igor Antoshechkin (Millard and Muriel Jacobs Genetics and Genomics Laboratory,
Caltech) with sequencing,
the help of Fan Gao (Bioinformatics Resource Center,
Caltech
) with
bioinformatic analysis, the help of Grace Shin
(Molecular Technologies, Caltech)
with
in situ
HCR,
and
the help of Giada Spigolon and Andres Collazo (Biological Imaging Facility,
Caltech
)
with microscopy. This work was supported
by grants from
the National Institutes of Health (R01
GM097363) and by the HHMI Faculty
Scholar Award to A.A.A.
.
CC-BY-NC-ND 4.0 International license
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted October 26, 2022.
;
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doi:
bioRxiv preprint
12
Figure Legends
Fig
. 1 |
Germline is the major cell type origin of piRNA sexual dimorphism in
D. mel
gonads.
a
,
Genotype and
phenotype of
males and females that can be identified with definite
chromosome content, employing
an X chromosome lacking
y
and
w
genes
as well as
a Y
chromosome that carries the
wildtype
y
+
and
w
+
genes.
b
,
Comparison of the abundance of piRNAs targeting individual transposon families in XY testis
versus
XX ovary, normalized to the expression of
20A
piRNAs.
Sex
-
biased transposon
-
targeting
piRNAs are color coded
and listed on the right
.
c
,
Comparison of the expression of major piRNA loci in the genome
12
in XY testis versus XX
ovary, normalized to the expression of
20A
piRNAs.
Each locus is marked by a different color.
d
, Illustration
of
the experimental strategy to isolate somatic piRNAs in the gonad.
Left: cartoon
showing the cell type composition of t
estis and ovary, with germline having a blue outline and
gonadal somatic cells having a red outline.
Both germline and gonadal soma
tic cells
express
Piwi, which is marked by yellow
.
Right:
cartoon showing
Piwi
expression in testis and ovary
upon
efficient,
germline
-
specific
knock
-
down of
piwi
that completely depletes Piwi
in the
germline, leaving the somatic cells as the only source of Piwi in the gonad
.
Gonadal somatic
piRNAs
are isolated by
immunoprecipitating
Piwi from
the
se
gonads that
lo
se
germline Piwi.
e
,
UCSC genome browser view of the
flamenco
piRNA locus
showing
flamenco
piRNAs
take up
similar fractions of gonadal somatic piRNAs
in testis and ovary with
similar coverage profiles.
f
,
Comparison of
the abundance of
transposon
-
antisense piRNAs
in testicular and ovarian
soma, normalized to the expression of
flamenco
piRNAs
.
Sex
-
biased piRNAs are color coded in
the same way as in
b
and the correlation coefficients are reported.
g
,
Comparison of the expression of different piRNA loci in testicular and ovarian soma,
normalized to the expression of
flamenco
piRNAs. Sex
-
biased piRNAs are color coded in the
same way as in
b
and the correlation coefficients are reported.
h
, UCSC genome b
rowser view of the
77B
piRNA locus
, showing its flanking protein
-
coding
genes,
its
transposon content
s
and
piRNA coverage profiles in two sexes. Note the difference in
y
-
axis scale
s
that
reflects a higher relative activity of
77B
in the testicular soma
than the female
counterpart.
A putative promoter marked by an RNA pol II peak likely drives the expression of
piRNAs
from the plus strand
that are antisense to two transposons
, 176
and
idefi
x
.
.
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available under a
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The copyright holder for this preprint
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;
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Fig. 2 | Y chromosome produces piRNAs in both males and females.
a
,
L
eft: l
isted are factors that might explain piRNA sexual dimorphism.
Right: cartoon showing
different sex chromosome contents and respective sexual identities.
X and Y chromosomes
are
depicted
in different ways, and
sexual identities are color coded with
the
male
identity
being
green and
the
female
one
being orange. Note that the Y chromosome
of
D. melanogaster
does
not participate in sex determination, and the sex
instead depends on the number of X. Hence,
XY and XO are both males,
and
XX and XXY are both females.
b
, Illustration showing the karyotype
of
D. melanogaster
with five chromosomes
–
X, Y, 2, 3,
and 4, as well as the rough genomic location
s
of major piRNA loci
in the germline.
c
, Cross scheme of the generation of XY and XO brothers.
d
,
T
he abundance of transposon
-
targeting piRNAs
in
XO males compared to their XY brothers
,
showing the loss of
piRNAs targeting several transposon families.
e
,
T
he expression of major
germline
piRNA loci
in XO males compared to their XY brothers
,
showing a sp
ecific loss of piRNAs from two Y
-
linked loci,
Su(Ste)
and
petrel
.
f
, Cross scheme of the generation of XX and XXY sisters.
g
,
The
abundance of transposon
-
targeting piRNAs
in
XXY
females compared to their XX
sisters, showing very limited differences.
h
,
The
expression of major
germline
piRNA loci
in XXY females compared to their
XX sisters,
showing piRNA
production
from two Y
-
linked loci,
Su(Ste)
and
petrel
.
i
, Cross scheme of the generation of XY and XO half
-
brothers, with
the same
XX mothers.
j
, The abundance of transposon
-
targeting piRNAs in XO males compared to their XY half
-
brothers,
both of which are sired by XX mothers,
showing
similar
loss of piRNAs ta
rgeting
several transposon families
as seen in
d
.
k
, The expression of major germline piRNA loci in XO males compared to their XY
half
-
brothers,
both of which are sired by XX mothers,
showing a
similar
loss of piRNAs from two Y
-
linked loci,
Su(Ste)
and
petrel
, as seen in
e
.
l
, Cross scheme of the generation of
XX
and X
XY
half
-
sisters
, with the same XX mothers.
m
, The abundance of transposon
-
targeting piRNAs in XXY females compared to their XX half
-
sisters, both of which are sired by XX mothers, showing
very
few differences similar to that
in
g
.
.
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n
, The expression of major germline piRNA loci in
XXY females compar
ed to their XX half
-
sisters, both of which are sired by XX mothers,
showing piRNA production from two Y
-
linked
loci,
Su(Ste)
and
petrel
, as seen in
h
.
Fig. 3 | Y
-
linked piRNA loci are active and functional in both sexes, when inherited from
either parent, regardless of whether mothers carry a Y chromosome.
a
,
Top:
Cross schemes that generate
:
XX females without mothers bearing a Y (column 1),
XX
females
with Y
-
bearing mothers (column 2), XXY females without mothers bearing a Y (column
3), XXY females with Y
-
bearing mothers but inhering the Y from the father (column 4), and XXY
females with Y
-
bearing mothers and inheriting the Y from the mother (column 5)
.
Middle: cartoon
showing the genotype
of each
kind
of
female
s
generated
and whether their mothers carry a Y is
reflected by whether they receive maternally deposited
Y
-
derived
piRNAs
. Bottom: RNA
in situ
HCR detecting
Su(Ste)
piRNA precursors (row 1),
petrel
piRNA precursors (row 2)
,
and
pirate
mRNAs (row 3)
in stage 6
-
7
egg chambers.
Scale bar:
5
μm.
b
,
Top: Cross schemes that generate
:
X
O
males
without mothers bearing a Y (column 1), X
O
males with Y
-
bearing mothers (column 2), XY males without mothers bearing a Y (column 3), XY
males with Y
-
bearing mothers but inhering the Y from the father (column 4), and XY males with
Y
-
bearing mothers and inheriting the Y from the mother (column 5). Mi
ddle: cartoon showing the
genotype of each kind of males generated and whether their mothers carry a Y is reflected by
whether they receive maternally deposited
Y
-
derived
piRNA
s
. Bottom: RNA
in situ
HCR detecting
Su(Ste)
piRNA precursors (row 1),
Stellate
mRNAs (row
2
)
,
petrel
piRNA precursors (row
3
)
,
and
pirate
mRNAs (row
4
)
and
at
the
apical tips of
the
testes
.
Scale bar:
1
0μm.
Fig. 4 | Sexual identity provides a
key
input into piRNA biogenesis and is a major
determinant of the piRNA program.
a
,
A simplified model of the sex determination pathway in germline and soma.
On top of
its own
chromosome content, germline receives an additional input from the soma to determine
its
sex.
b
,
A c
omparison scheme that
uses sex reversal to examine the
effect
s
of sexual identit
ies
on
the
piRNA
program
.
XX female germline is masculinized and compared to both
wildtype
XX female
s
(the origin state) and XO male
s
(the target state).
Thus, any differences
observed
would reveal
the effect
s
of sexual identit
ies on piRN
A
s
, without confounding impact
s
of the Y chromosome.
.
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c
,
Cartoon showing the masculinization of the XX female germline by genetic perturbations,
without any chan
g
es to the sex chromosome content.
To facilitate germline masculinization, the
soma is masculinized by mutating
tra
.
In addition, germline
-
specific knock
-
down of
Sxl
near
completely masculinizes the female germline, while ectopic expression of
Phf7
in the germline
led to partial masculiniza
tion.
d
,
Left: a
heatmap showing the abundance of piRNAs targeting
different
transposons in XX female,
XO male, or XX
masculinized
by perturbing either
Phf7
or
Sxl
expression
(in the
tra
mutant
background)
.
Each row represents piRNAs that target a
different transposon, and their expression
levels are color coded.
Middle:
Quantification of the extent of masculinization for piRNAs targeting
individual transposon
familie
s.
For each transposon family, the abundance of corresponding
antisense piRNAs
in XX female is scaled to 0% and that in XO male is scaled to 100%, so the
levels of transposon
-
targeting piRNAs
in masculinized XX
can be normalized to reflect the extent
of mascu
linization.
Shown are
the summary statistics (
median and interquartile range
)
of
the
antisense piRNAs targeting different transposon families.
Right:
piRNA abundance upon XX
masculinization for
four
examples of male
-
biased and female
-
biased transposon
-
targ
eting
piRNAs
, respectively
. In these examples
as well as the overall summary statistics
, perturbing
Sxl
led to a stronger masculinization of the piRNA
program
than perturbing
Phf7
.
e
,
A heatmap showing the expression of major germline piRNA loci (
located
outside Y) in XX
female, XO male, or XX
masculinized
by perturbing either
Phf7
or
Sxl
expression
(
in the
tra
mutant
background
)
.
f
,
UCSC genome browser view of the piRNA coverage profile
s
over the locus
42AB
(left) and the
locus
38C1
(right) in
X
X female, XO male, or XX
masculinized
by perturbing either
Phf7
or
Sxl
expression
(
in the
tra
mutant background
)
.
g
,
F
emale
-
specific expression of Kipferl and Kipferl
-
dependent piRNAs.
h
,
RNA
in situ
HCR detecting
Kipferl
mRNA in XX female, XO male, or XX masculinized by
perturbing either
Phf7
or
Sxl
expression (
in the
tra
mutant background
).
i
. A genetic circuit
that connects the sex determination pathway and piRNA biogenesis.
.
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16
Extended Data Fig. 1 | The
flamenco
piRNA locus is only active in the gonadal soma in
testis.
RNA
in situ
HCR detecting
flamenco
piRNA precursors
in testes expressing mCherry
-
Vasa
and
Tj
-
GFP
under endogenous regulatory elements
, stained for DAPI.
flamenco
transcripts are only detected in
gonadal
somatic cells
(
including both
early cyst cells marked by
Tj expression and hub cells marked by
the
lack of Tj
and Vasa expression
)
, but not in germline
cells (marked by
Vasa expression
)
. Scale bar: 10μm.
.
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available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted October 26, 2022.
;
https://doi.org/10.1101/2022.10.25.513766
doi:
bioRxiv preprint
17
Methods
Fly stocks
and crosses
Stocks and crosses were raised at 25 °C. The following stocks were used:
yw
(BDSC
6599
),
y
+
w
+
Y
(
BDSC
7060
),
C(1)A
(BDSC
2950
),
C(1)RM
(BDSC
9460),
C(1;Y)
(BDSC
9460)
,
nos
-
Gal4
(BDSC
4937
),
bam
-
Gal4
(
BDSC 80579
),
sh
-
piwi
(BSDC
33724
),
sh
-
Sxl
(BDSC
38195
)
,
UAS
-
Phf7
(
BDSC 15894
),
tra
1
(BDSC
675
) and
Df(tra)
(BDSC
5416
)
were obtained from Bloomington
Drosophila Stock Center
,
and
Tj
-
GFP
(
VDRC
318066
) was obtained from
Vienna Drosophila
Resource Center
.
GFP
-
Piwi
(BAC)
and
mCherry
-
Vasa
w
ere
previously describe
d
37,38
.
To
minimize genetic background differences,
yw /
y
+
w
+
Y was backcrossed to
the
inbred
yw
line
for
six consecutive generations
, via a single male at every generation
.
Similarly,
after
generating
C(1)A / y
+
w
+
Y
females,
we backcrossed them
to
yw /
y
+
w
+
Y
males for six consecutive generations
,
via 2
-
3 females at every generation.
To obtain an XXY
exceptional female,
we looked for
a female
carrying
the
marked Y chromosome (
y
+
w
+
Y)
in
the
yw /
y
+
w
+
Y stock
,
which typically took
no more
than
two
months
.
To
deplete germline Piwi
,
we
expressed
sh
-
piwi
using both
nos
-
Gal4
and
bam
-
Gal4,
which
led to
efficient
knock
-
down of
Piwi in
the
germline as
evidenced
by the
loss
of
germline
GFP
-
Piwi
expression
in both testis and ovary
.
For
sex reversal experiments, a Y
chromosome marked by
B
S
(present in the
Df(tra)
stock
)
that alters the eye shape
was employed,
such that
the
sex chromosome content
could be inferred independent
ly
of the morphological sex.
RNA
in situ
hybridization
and RNA
in situ
hybridization
chain reaction (HCR)
For
RNA
in situ
HCR,
prob
es
,
amplifiers
and buffers were purchased from Molecular Instruments
(molecularinstruments.org) for
flam
(
3893/E046
),
petrel
(
3872/E024
),
pirate
(
3916/E064
)
,
Stellate
(
4537/E832
)
and
Kipferl
(
4708/E1062
)
transcripts.
RNA
i
n situ
HCR v3.0
39
was
done
according
to manufacturer’s recommendations for g
eneric samples in solution.
To detect
Su(Ste)
transcripts
,
we
did
conventional
RNA
in situ
hybridization using DNA probes
(75bp, position 994
-
1068 of
Su(Ste): CR42424
, sense direction)
directly conjugated with fluorophore
purchased from
IDT
.
Image acquisition and
processing
Confocal images were acquired with Zeiss LSM 800
or LSM 980
using a 63x oil immersion
objective (NA=1.4) and processed using Fiji
40
. Si
ngle focal planes were shown in all images,
where dotted outlines were
manually
drawn for illustration purposes.
.
CC-BY-NC-ND 4.0 International license
available under a
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The copyright holder for this preprint
this version posted October 26, 2022.
;
https://doi.org/10.1101/2022.10.25.513766
doi:
bioRxiv preprint
18
Small
RNA
sequencing
Argona
u
te
-
associated small RNAs were isolated
from ovaries
(20 pairs per sample)
or testes
(30
pairs per
s
ample)
using TraPR columns
41
.
Purified small RNA was subject to library prep using
NEBNext Multiplex Small RNA Sample Prep Set for Illumina (NEB E7330).
Adaptor
-
ligated,
reverse
-
transcribed, PCR
-
amplified samples were purified again by PAGE (6% polyacrylamide
gel)
, from which
w
e cut out the
band
within
the
desir
ed
size range
.
This additional size selection
by PAGE
eliminated
other
, longer
RNAs
(>30 nt)
captured
by TraPR columns.
To isolate Piwi
-
associated small RNAs in gonadal soma,
we
first
immunoprecipitated
GFP
-
Piwi from gonads
lacking germline Piwi
(
see
above for fly crosses
)
using GFP
-
Trap (ChromoTek) magnetic agarose
beads
, as described before
42
.
Small RNAs associated with gonadal somatic Piwi are then purified
by TraPR columns
and
library
-
prepared
,
as described above for all Argonaute
-
associated small
RNAs.
Two biological replicates per genotype were sequenced on
Illumina HiSeq 2500.
Analysis of
transposon
-
targeting piRNAs
To
computationally extract
piRNAs
from
all
Argonaute
-
associate
d
small RNAs
, adaptor
-
trimmed
small RNAs were size
-
selected for 23
-
29nt (cutadapt 2.5) and those mapped to rRNA,
tRNA,
snRNA, snoRNA
, miRNA, hpRNA
and
7SL RNA
were discarded (bowtie 1.2.2 with
-
v 3). piRNAs
were
then
mapped to RepBase
25
.08
(manually curated)
and
those antisense to transposon
consensus sequences with ≤3 mismatches
are designated as transposon
-
targeting
piRNAs.
For
LTR transposons,
reads mapping to
the
LTR and internal sequences
of a given transposon family
were merged for
quantification,
given thei
r well
-
correlated behaviors.
All quantification was done
using
the
mean
of two biological replicates.
Analysis of the expression of major piRNA loci
piRNAs were computationally extracted as described above.
piRNAs were mapped to the dm6
genome using a previously described algorithm
12
that
considers both
piRNAs that map to single
unique positions in the genome
as
well as
those
that map to
“
local repeats
”
(
defined as repeats
that are contained within a genomic
window <2Mb in length
in
dm6
reference genome
). Major
piRNA loci
, their coordinates and quantification method were
described before
12
.
The average of
two biological replicates
was shown in
all figures.
.
CC-BY-NC-ND 4.0 International license
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted October 26, 2022.
;
https://doi.org/10.1101/2022.10.25.513766
doi:
bioRxiv preprint
19
Definition of piRNA
-
producing loci in gonadal soma
piRNA
-
producing loci in gonadal soma were defined as previously described for piRNA
-
producing
loci (also
known as
“piRNA clusters”) in whole gonads
12
.
Briefly,
piRNAs
isolated from gonadal
soma
were mapped to the genome and those
that
map uniquely or to local repeats were kept and
quantified over 1Kb windows that tile the entire genome.
Neighboring 1Kb widows within 3Kb
were merged. If merged windows were ≥5Kb, they were merged again within 15Kb
, and this
process was repeated
twice
.
Thi
s
de novo
method recapitulated the
flamenco
locus
and
the
3’UTR of the
tj
gene
–
two loci that are known to make
abundant
piRNAs in ovarian soma
–
confirming its utility.
Inference of
germline contribution to
whole gonad
piRNAs
Given that
flamenco
is
only active in the gonadal soma but not in the germline,
flamenco
piRNAs
found in whole gonad piRNAs must come from somatic cells in the gonad.
Experimentally isolated
gonadal somatic piRNA
s
revealed the contribution of
flamenco
piRNAs to total piRNAs in the
gonadal soma
(e.g., 25%)
, so if
flamenco
takes up 5% of whole gonad piRNAs
, gonadal soma
will
contribute to 20% (5% / 25% = 20%) of whole gonad piRNAs.
Then, the germline contribution
to whole gonad piRNAs is 100%
-
20% = 80%.
When calculating actual contribution
s
of
flamenco
piRNAs
to gonadal soma and whole gonads of both sexes, the mean of two re
plicates was used.
Data visualization and statistical analysis
All
data visualization and statistical analysis were done in Python 3 via JupyterLab with the
following software packages: numpy
43
, pandas
44
and altair
45
. The UCSC Genome Brower
46
and
IGV
47,48
were used to explore sequencing data and to prepare
genome
browser track panels
shown.
Two biological replicates show
ed
similar coverage profiles on the genome browser,
so
one of the two replicate
s
was randomly selected to be shown in
the
figures.
Data availability
Sequencing data
will be uploaded to
NCBI SRA
.
.
CC-BY-NC-ND 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted October 26, 2022.
;
https://doi.org/10.1101/2022.10.25.513766
doi:
bioRxiv preprint