of 35
*For correspondence:
michelle.arbeitman@med.fsu.edu
These authors contributed
equally to this work
Present address:
Department
of Biology, St. Joseph’s College,
New York, United States
Competing interests:
The
authors declare that no
competing interests exist.
Funding:
See page 29
Received:
17 September 2020
Accepted:
22 February 2021
Published:
22 February 2021
Reviewing editor:
Michael B
Eisen, University of California,
Berkeley, United States
Copyright Brovero et al. This
article is distributed under the
terms of the
Creative Commons
Attribution License,
which
permits unrestricted use and
redistribution provided that the
original author and source are
credited.
Investigation of
Drosophila fruitless
neurons that express Dpr/DIP cell
adhesion molecules
Savannah G Brovero
1†
, Julia C Fortier
1†
, Hongru Hu
1†
, Pamela C Lovejoy
1†‡
,
Nicole R Newell
1†
, Colleen M Palmateer
1†
, Ruei-Ying Tzeng
1†
, Pei-Tseng Lee
2
,
Kai Zinn
3
, Michelle N Arbeitman
1
*
1
Department of Biomedical Sciences and Program of Neuroscience, Florida State
University, College of Medicine, Tallahassee, United States;
2
Department of
Molecular and Human Genetics, Baylor College of Medicine, Houston, United
States;
3
Division of Biology and Biological Engineering, California Institute of
Technology, Pasadena, United States
Abstract
Drosophila
reproductive behaviors are directed by
fruitless
neurons. A reanalysis of
genomic studies shows that genes encoding
dpr
and
DIP
immunoglobulin superfamily (IgSF)
members are expressed in
fru P1
neurons. We find that each
fru P1
and
dpr/DIP
(
fru P1
T
dpr/DIP
)
overlapping expression pattern is similar in both sexes, but there are dimorphisms in neuronal
morphology and cell number. Behavioral studies of
fru P1
T
dpr/DIP
perturbation genotypes
indicate that the mushroom body functions together with the lateral protocerebral complex to
direct courtship behavior. A single-cell RNA-seq analysis of
fru P1
neurons shows that many
DIPs
have high expression in a small set of neurons, whereas the
dprs
are often expressed in a larger set
of neurons at intermediate levels, with a myriad of
dpr/DIP
expression combinations. Functionally,
we find that perturbations of sex hierarchy genes and of
DIP-
"
change the sex-specific
morphologies of
fru P1
T
DIP-
a
neurons.
Introduction
A goal of neuroscience research is to gain molecular, physiological and circuit-level understanding of
complex behavior.
Drosophila melanogaster
reproductive behaviors are a powerful and tractable
model, given our knowledge of the molecular-genetic and neural anatomical basis of these behaviors
in both sexes. Small subsets of neurons have been identified as critical for all aspects of reproductive
behaviors. These neurons express sex-specific transcription factors encoded by
doublesex
(
dsx
) and
fruitless
(
fru; fru P1
transcripts are spliced under sex hierarchy regulation;
Figure 1A
) (reviewed in
Dauwalder, 2011
;
Yamamoto et al., 2014
;
Andrew et al., 2019
;
Leitner and Ben-Shahar, 2020
).
dsx
- and
fru P1
-expressing neurons are present in males and females in similar positions, and arise
through a shared developmental trajectory (
Ren et al., 2016
), although these neurons direct very
different behaviors in males and females. Males display an elaborate courtship ritual that includes
chasing the female, tapping her with his leg, and production of song with wing vibration (reviewed
in
Greenspan and Ferveur, 2000
). The female decides whether she will mate and then, if mated,
displays post-mating behaviors that include egg laying, changes in diet, and changes in receptivity
to courtship (see
Laturney and Billeter, 2014
;
Aranha and Vasconcelos, 2018
;
Newell et al.,
2020
).
Sex differences in the nervous system that contribute to reproductive behaviors include dimor-
phism in
dsx
and
fru P1
neuron number, connectivity, and physiology. The molecules and mecha-
nisms that direct these differences are beginning to be elucidated. Several genome-wide studies
Brovero, Fortier, Hu,
et al
. eLife 2021;10:e63101.
DOI: https://doi.org/10.7554/eLife.63101
1 of 35
RESEARCH ARTICLE
have been performed to find genes that are regulated by male-specific Fru (Fru
M
) or are expressed
in
fru P1
neurons. These independent studies examined
fru P1
loss-of-function and gain-of-function
gene expression changes,
fru P1
cell-type-specific gene expression, and the direct targets for Fru
M
binding (
Goldman and Arbeitman, 2007
;
Dalton et al., 2013
;
Neville et al., 2014
;
Vernes, 2015
;
Newell et al., 2016
). A reanalysis of these genomic studies demonstrates that cell adhesion mole-
cules that are members of the immunoglobulin superfamily (IgSF) are regulated by male-specific Fru
(Fru
M
) or are expressed in
fru P1
neurons (see
Figure 1B
). In this study, we focus on an interacting
set of IgSF molecules encoded by
dprs/DIPs
.
The Dpr and DIP proteins are membrane-linked cell adhesion molecules with N-terminal extracel-
lular Ig domains and C-terminal glycosyl-phosphatidylinositol (GPI) linkage sequences or transmem-
brane domains. The Dpr proteins have two extracellular Ig domains, whereas DIPs have three
Female
Male
XX
XY
Sxl
Sxl
Sxl
tra
tra-2
Tra
F
Tra-2
+
dsx
fru P1
Dsx
F
tra
tra-2
Tra-2
dsx
fru P1
Dsx
M
Fru
M
STOP
frt
frt
UAS
Effector/Marker
Gene
fru P1
flippase
dpr/DIP
Gal4
UAS
Effector/Marker
Gene
1
2
3
A
B
C
fru P1
regulatory regions drive
flippase
expression
Flippase mediated excision of stop cassette in
fru P1
neurons
dpr/DIP
regulatory regions drive
Gal4
expression
Gal4
Effector/Marker
gene expressed
in intersecting neurons
Goldman Dalton
Neville
Vernes
Newell
dpr1
dpr2
dpr3
dpr4
dpr5
dpr6
dpr7
dpr8
dpr9
dpr10
dpr11
dpr12
dpr13
dpr14
dpr15
dpr16
dpr17
dpr18
dpr19
dpr20
DIP-
Į
DIP-
ȕ
DIP-
Ȗ
DIP-
į
DIP-
İ
DIP-
ȗ
DIP-
Ș
DIP-
ș
DIP-
Ț
DIP-
ț
DIP-
Ȝ
dpr/
DIP
fru P1
Figure 1.
Overview of sex hierarchy and experimental design. (
A
) The
Drosophila
somatic sex determination hierarchy is an alternative pre-mRNA
splicing cascade (reviewed in
Andrew et al., 2019
). The presence of two X chromosomes in females results in splicing of
Sxl
pre-mRNA, such that
functional Sxl is produced. Sxl regulates
Sxl
and
tra
pre-mRNA splicing, resulting in continued production of functional Sxl and Tra in females. Tra and
Tra-2 regulate the pre-mRNA splicing of
dsx
and
fru P1
in females, whereas in males
dsx
and
fru P1
are spliced by the default pre-mRNA splicing
pathway. The sex-specific splicing results in production of sex-specific Dsx and Fru transcription factors (
Burtis and Baker, 1989
;
Ito et al., 1996
;
Ryner et al., 1996
).
dsx
regulates sex differences that lead to both dimorphic behavior and gross anatomical morphological differences, whereas
fru P1
regulates sex differences that lead to dimorphic behaviors. (
B
) Previous genome-wide studies found that
dpr/DIPs
are regulated downstream of
fru P1
,
Fru
M
, and/or are expressed in
fru P1
neurons (
Goldman and Arbeitman, 2007
;
Dalton et al., 2013
;
Neville et al., 2014
;
Vernes, 2015
;
Newell et al.,
2016
). (
C
) A genetic intersectional strategy was used to express marker or effector genes in
fru P1
T
dpr/DIP
neurons. This strategy takes advantage of
the two-component Gal4/UAS expression system, and Flippase-mediated removal of a stop cassette within an expression vector. Expression of the
marker/effector gene requires both removal of the stop cassette via
fru P1-flippase
(
flp
) expression and expression of Gal4 via
dpr/DIP
regulation.
Therefore, only neurons that express both
fru P1
and one of the
dpr/DIPs
have expression of the effector or marker (shown on right).
The online version of this article includes the following source data for figure 1:
Source data 1.
Data table of Fru
M
binding sites in
dpr
and
DIP
genes for three Fru
M
isoforms.
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et al
. eLife 2021;10:e63101.
DOI: https://doi.org/10.7554/eLife.63101
2 of 35
Research article
Genetics and Genomics
Neuroscience
(reviewed in
Zinn and O
̈
zkan, 2017
;
Sanes and Zipursky, 2020
). In addition to the genomic studies,
our previous work showed that
dpr1
, the founding member of the
dpr
family (
Nakamura et al.,
2002
), has a role in gating the timing of the male courtship steps (
Goldman and Arbeitman, 2007
).
The finding that cell adhesion molecules are regulated by Fru
M
fits well with studies that showed
that there are sex-differences in arborization volumes throughout the central nervous system
(
Cachero et al., 2010
;
Yu et al., 2010
). Thus, it was predicted that differences in neuronal connectiv-
ity are one mechanism to generate behavioral dimorphism (
Cachero et al., 2010
;
Yu et al., 2010
).
In-depth, in vitro analyses of Dpr/DIP protein-protein interactions have shown that most DIPs
interact with multiple Dprs, and vice versa. Additionally, 4 of the 11 DIPs and 2 of the 21 Dprs dis-
play homophilic interactions, and there are two heterophilic interactions between Dprs
(
O
̈
zkan et al., 2013
;
Carrillo et al., 2015
;
Cosmanescu et al., 2018
). Functional analyses of the
Dprs and DIPs have revealed roles in synaptic connectivity and specificity of neuronal targeting in
the
Drosophila
neuromuscular junction, visual system, and olfactory system (
Carrillo et al., 2015
;
Tan et al., 2015
;
Barish et al., 2018
;
Xu et al., 2018
;
Ashley et al., 2019
;
Courgeon and Desplan,
2019
;
Menon et al., 2019
;
Venkatasubramanian et al., 2019
;
Xu et al., 2019
). Additionally, cell
adhesion molecules have already been shown to be important for sculpting dimorphism in
fru P1
neurons, with studies of the IgSF member encoded by
roundabout
(
robo
) shown to be a direct tar-
get of Fru
M
and responsible for dimorphic projections and morphology (
Mellert et al., 2010
;
Ito et al., 2016
). Thus, the Dprs/DIPs are good candidates for directing sexual dimorphisms in con-
nectivity and morphology that may underlie differences in reproductive behavior.
Here, we further examine the expression repertoires of
dprs/DIPs
in
fru P1
neurons using immu-
nofluorescence analyses. We examine the sets of neurons that express
fru P1
and one of the
dprs
or
DIPs
, using a genetic intersectional strategy (
fru P1
T
dpr/DIP
;
Figure 1C
). Additionally, this genetic
strategy provides a method to examine the roles of neurons expressing
fru P1
and a
dpr
or
DIP
in
directing male reproductive behaviors. Here, we elucidate which combinations of neurons lead to
atypical courtship behaviors when activated or silenced. A single-cell RNA-seq analysis shows the
myriad and unique combinations of
dprs/DIPs
expressed in individual
fru P1
neurons, with expres-
sion of at least one
dpr
or
DIP
in every
fru P1
neuron examined. Additionally, the single-cell RNA-
seq analyses show that many
dprs
are expressed in a large number of neurons at intermediate levels,
whereas most
DIPs
have higher expression in fewer neurons. Genetic perturbation screens reveal
functional roles of the sex hierarchy, and
DIP-
"
,
in establishing sex-specific architecture of
fru P1
T
DIP-
a
neurons.
Results
dprs
and
DIPs
are expressed in
fru P1
neurons, with expression
regulated by Fru
M
A reanalysis of previous genomic studies shows that
dprs
and
DIPs
are regulated by Fru
M
and are
expressed in
fru P1
neurons (
Figure 1B
). These independent studies examined
fru P1
loss-of-function
(
Goldman and Arbeitman, 2007
) and Fru
M
gain-of-function/overexpression (
Dalton et al., 2013
)
gene expression changes,
fru P1
cell-type-specific gene expression in males and females
(
Newell et al., 2016
), and identified direct genomic targets of Fru
M
(
Neville et al., 2014
;
Vernes, 2015
). The majority of the
dpr/DIP
genes are identified as regulated by Fru
M
or expressed
in
fru P1
neurons in at least three of these independent genome-wide studies (
Figure 1B
). Further-
more, a computational DNA-binding site analysis confirms Fru
M
regulation. There is alternative splic-
ing at the 3’ end of
fru P1
transcripts that results in one DNA-binding-domain-encoding-exon being
retained out of five potential exons. The predominant isoforms of Fru
M
contain either the A, B, or C
DNA-binding domain that each bind a different DNA sequence motif (genome-wide analysis
described in
Dalton et al., 2013
). When we search for the presence of the three DNA sequence
motifs near/in the
dpr
/
DIP
loci, Fru
M
binding sites were found near/in all but two
dpr/DIP
loci that
are examined (
Figure 1—source data 1
). Further evidence that
dprs/DIPs
have a role in
fru P1
neu-
rons comes from a live-tissue staining approach, using epitope-tagged, extracellular regions of a
Dpr or DIP to examine binding (as done in
Fox and Zinn, 2005
;
Lee et al., 2009
;
O
̈
zkan et al.,
2013
). This revealed sexual dimorphism in binding of tagged Dpr/DIP proteins to
fru P1
neurons in
the subesophageal ganglion brain region, with more neurons with overlap detected in males
Brovero, Fortier, Hu,
et al
. eLife 2021;10:e63101.
DOI: https://doi.org/10.7554/eLife.63101
3 of 35
Research article
Genetics and Genomics
Neuroscience
(
Brovero et al., 2020
). Together, these results demonstrate that every
dpr
and
DIP
gene is either
regulated by Fru
M
or expressed in
fru P1
neurons in either males and females.
A genetic intersectional approach identifies neurons that express both
fru P1
and a
dpr
or
DIP
in males and females
The above results led us to examine the expression patterns in the central nervous system of neurons
that express both
fru P1
and a
dpr
or
DIP
, using a genetic intersectional approach (
Figure 1C
). This
approach restricts expression of a membrane-bound-GFP marker to neurons with intersecting
expression of
fru P1
and a
dpr
or
DIP
(
fru P1
T
dpr/DIP
). This is accomplished using a UAS-mem-
brane-bound GFP reporter transgene that requires removal of an FRT-flanked stop cassette for
expression. Removal of the stop cassette is mediated by
fru P1-
driven FLP recombinase (
Yu et al.,
2010
). This system is used in combination with a collection of
dpr
- and
DIP-Gal4
transgenic strains
(
Figure 1C
;
Venken et al., 2011
;
Nagarkar-Jaiswal et al., 2015a
;
Nagarkar-Jaiswal et al., 2015b
;
Tan et al., 2015
;
Lee et al., 2018
). We primarily focused the analysis on 4- to 7-day-old
adults (
Figure 2
and
Figure 3
), which are sexually mature, and 0- to 24-hr adults to determine if the
expression patterns change during adult stages (
Figure 2—figure supplement 1
and
Figure 3—fig-
ure supplement 1
). Additionally, behavioral studies were performed on 4–7 day adults (
Figure 4
,
Figure 5
,
Figure 6
), so the expression and behavioral data can be co-analyzed (
Figure 7
). At a gross
morphological level, the patterns we observe in older 4- to 7-day-old adults are also present in 0–24
hr adults, though in some cases expression in the mushroom body was not robust at the earlier 0–24
hr time point.
Based on our examination of the expression patterns in 27 intersecting genotypes, we find that
24 genotypes showed clear and consistent, membrane-bound GFP expression in the central nervous
system. Of these, only two
fru P1
T
DIP
genotypes have restricted and unique patterns (
fru P1
T
DIP
-
d
and
fru P1
T
DIP-
a
), whereas the other genotypes have broader expression, with many in simi-
lar regions/patterns (
Figures 2
and
3
).
fru P1
T
DIP
-
d
neuronal projections are near the anterior sur-
face of the protocerebrum and appear to be near the
g
-lobe of the mushroom body, based on
visual inspection (
Aso et al., 2014
). The
fru P1
T
DIP-
a
expression pattern is described in detail
below. The 22 intersecting genotypes with broad expression, in both males and females, have con-
sistent expression in the brain lateral protocerebral complex, including within the arch, ring, junction,
and crescent (for summary see
Figure 7
and
Source data 1
). This region has been shown to have
fru
P1
neurons with sexually dimorphic arbor volumes (
Cachero et al., 2010
;
Yu et al., 2010
). Further-
more, the lateral protocerebral complex has inputs from sensory neurons and is predicted to be a
site of sensory integration, to direct motor output necessary for coordinating courtship activity
(
Yu et al., 2010
). We find eight intersecting genotypes have expression in mushroom bodies in both
males and females. This region has a well-established role in learning and memory, including learn-
ing in the context of courtship rejection (
Jones et al., 2018
;
McBride et al., 1999
;
Montague and
Baker, 2016
;
Zhao et al., 2018
). Overall, the majority of
fru P1
T
dpr/DIP
genotypes have expres-
sion in similar regions, suggesting that some may function in a combinatorial manner within a neuron
to direct patterning and/or synaptic targeting, consistent with the single-cell RNA-sequencing data
presented below.
We observe sex differences in morphological features and cell body number in regions we scored
(
Figures 2
and
3
). These regions were largely chosen because they were previously reported to
have
fru P1
neurons that display sex differences downstream of sex hierarchy genes
transformer
and
fru P1
(
Cachero et al., 2010
;
Yu et al., 2010
). For example, 18 intersecting genotypes show consis-
tent presence of signal in the mesothoracic triangle neuronal projections in males, but only two gen-
otypes do so in females. While both males and female have expression in the DA1 and VA1v
antennal lobe glomeruli in several intersecting genotypes, we also observe sexual dimorphism, with
four genotypes having consistent expression in only female DA1 glomeruli (
fru P1
T
dpr3, dpr10,
dpr17, DIP-

). In the ventral nerve cord, neurons that cross the midline are consistently observed
only in males and not females. Previous work found a midline crossing phenotype that was also
male-specific for a set of gustatory neurons (
Mellert et al., 2010
). For all regions where cell bodies
are counted, the trend was that there are more cell bodies in males than females. Thus, the differen-
ces in the patterns of expression between males and females are not large, with several genotypes
having quantitative differences in the numbers of cell bodies present. It is possible that there are
additional quantitative differences that are not detected based on the resolution of the analyses,
Brovero, Fortier, Hu,
et al
. eLife 2021;10:e63101.
DOI: https://doi.org/10.7554/eLife.63101
4 of 35
Research article
Genetics and Genomics
Neuroscience
Figure 2.
Visualization of
fru P1
T
dpr
neurons. Maximum intensity projections of brain and ventral nerve cord tissues from 4- to 7-day-old male and
female flies. The
fru P1
T
dpr
intersecting neurons are labeled with green (rabbit
a
-GFP Alexa Flour 488), and neuropil are labeled with magenta
(mouse
a
-nc82, Alexa Flour 633). The genotype is
dpr-Gal4/10xUAS > stop > GFP.Myr; fru P1
FLP
, except for
dpr4
,
dpr14
,
dpr18,
and dpr19
. These
Gal4
transgenic strains were generated using a CRISPR-mediated insertion of the
T2A-Gal4
with the dominant 3xP3-GFP marker. For
this strain,
10xUAS > stop > myr::smGdP-cMyc
was used and
fru P1
T
dpr
intersecting neurons are labeled with red (rabbit
a
-Myc, Alexa Flour 568) and
then false-colored to green. The neuropil are labeled with magenta (mouse
a
-nc82, Alexa Flour 633). These Gal4s did not show consistent
fru P1
intersecting expression:
dpr7
,
dpr13
, and
dpr19
. The
dpr7
and
dpr13
Gal4s have expression with 10xUASmCD8gfp, confirming the Gal4s can
drive expression outside of
fru P1
neurons.
dpr19
was tested with 10xUAS-RFP and did not show expression outside of
fru P1
neurons.
The online version of this article includes the following figure supplement(s) for figure 2:
Figure 2 continued on next page
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et al
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Research article
Genetics and Genomics
Neuroscience
including quantitative differences in expression level of
dpr/DIPs
, or their subcellular localization, or
in regions/features that are not quantified here.
Activation of
fru P1
T
dpr/DIP
neurons results in atypical courtship
behaviors
fru P1
neurons have critical roles in reproductive behaviors. Studies have already determined the
function of small subsets of neurons that are responsible for different aspects of behavior (reviewed
in
Auer and Benton, 2016
). The
dpr/DIP
tools in hand can further address if additional combinations
and/or quantitative differences in the number of
fru P1
neurons are important for behavioral out-
comes, given the
fru P1
T
dpr/DIP
subsets and combinations we examine are distinct from those
previously studied. We used the genetic intersectional strategy to selectively activate intersecting
neurons by driving expression of TrpA1, a heat-activated cation channel
(
Figure 1C
;
von Philipsborn et al., 2011
). This allows for temporal control of neuronal activation by
an acute increase of temperature in the courtship chambers (32 ̊C; controls at 20 ̊C). We note that
quantitative differences in TrpA1 expression levels may account for some behavioral differences, in
addition to the differences in the spatial expression patterns observed (
Figures 2
and
3
).
We find that neuronal activation resulted in decreases in male following and wing extending
toward females for several genotypes (
Figure 4
and
Source data 2
). We also observe that neuronal
activation of
fru P1
T
dpr
(13 of 16) and
fru P1
T
DIP
(2 of 8) genotypes caused atypical courtship
behavior toward a female, including double wing extension, and continuous abdominal bending,
even if the female had moved away (
Figures 4
and
7
; for abdominal bending phenotype see
Fig-
ure 4—video 1
). These atypical behaviors could account for some of the decreases in following and
wing extension. For example, if a male is locked into abdominal bending, this would reduce court-
ship following behavior. Additionally, we found that some males ejaculated on the chamber in five
intersecting genotypes:
dpr5
(5 of 15 animals),
dpr9
(3 of 15 animals),
dpr10
(3 of 15 animals),
dpr12
(2 of 15 animals), and
DIP-

(4 of 15 animals). Of note,
fru P1
T
DIP-
a
is the only strain that showed
a decrease in courtship activities without a concomitant increase in atypical courtship behaviors. This
suggests that
fru P1
T
DIP-
a
neurons may normally inhibit courtship behaviors when they are
activated.
We next determined if the males require females to reach an arousal threshold needed to per-
form typical and atypical courtship behaviors, given that several of the courtship behaviors described
above occur when the male was not oriented toward the female. To address this question, we exam-
ine courtship behaviors in solitary males, using the same temporal activation strategy as above. We
find that activation of the
fru P1
T
dpr/DIP
neurons is sufficient to elicit single wing extension, dou-
ble wing extension, and abdominal bending in
fru P1
T
dprs
(11 of 16 genotypes) and
fru P1
T
DIPs
(3 of 8 genotypes) (
Figures 5
and
7
). Similarly, activating the intersecting
fru P1
neuronal popula-
tions of
fru P1
T
dpr5
(5 of 10 animals),
dpr9
(1 of 10 animals),
dpr10
(1 of 10 animals),
dpr12
(3 of
10 animals), and
DIP-

(1of 10 animals) causes males to ejaculate without a female present. Overall,
activation of these subsets of
fru P1
neurons is sufficient to direct reproductive behaviors, even if a
female is not present, consistent with other neuronal activation experiments (reviewed in
Auer and
Benton, 2016
).
Silencing
fru P1
T
dpr/DIP
neurons result in courtship changes
Given that activation of
fru P1
T
dpr/DIP
neuronal subsets resulted in changes in courtship behav-
iors, we next determine how silencing these neurons impacts male-female courtship, to gain further
insight into their roles. To test this, we use the genetic intersectional approach with a
UAS > stop >
TNT
transgene (
Figure 1C
;
Stockinger et al., 2005
). The intersecting genotypes express tetanus
toxin light chain, which cleaves synaptobrevin, resulting in synaptic inhibition (
Sweeney et al.,
1995
). For the control conditions, we also examine courtship behaviors of flies expressing an inactive
form of
TNT
(TNTQ), using the genetic intersectional approach.
Figure 2 continued
Figure supplement 1.
Visualization of
fru P1
T
dpr
neurons from 0-to 24-hour adults.
Brovero, Fortier, Hu,
et al
. eLife 2021;10:e63101.
DOI: https://doi.org/10.7554/eLife.63101
6 of 35
Research article
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Neuroscience
Figure 3.
Visualization of
fru P1
T
DIP
neurons. Maximum intensity projections of brain and ventral nerve cord
tissues from 4- to 7-days old male and female flies. The
fru P1
T
DIP
intersecting neurons are labeled with green
(rabbit
a
-GFP Alexa Flour 488), and neuropil are labeled with magenta (mouse
a
-nc82, Alexa Flour 633). The
genotype is
DIP-Gal4/10xUAS > stop > GFP.Myr; fru P1
FLP
, except for
DIP-
i
. This
Gal4
transgenic strains was
Figure 3 continued on next page
Brovero, Fortier, Hu,
et al
. eLife 2021;10:e63101.
DOI: https://doi.org/10.7554/eLife.63101
7 of 35
Research article
Genetics and Genomics
Neuroscience