Neurogenetic and genomic approaches reveal roles for Dpr/DIP
cell adhesion molecules in
1
Drosophila
reproductive behavior
2
3
Savannah G Brovero
*,1
, Julia C Fortier
*,1
, Hongru Hu
*,1
, Pam
ela C Lovejoy
*,1
, Nicole R
4
Newell
*,1
, Colleen M Palmateer
*,1
, Ruei-Ying Tzeng
*,1
, Pei -Tseng Lee
4
, Kai Zinn
3
, Michelle N
5
Arbeitman
1,2,5
6
* co-first authors
7
8
Orcid IDs:
9
Hongru Hu: (https://orcid.org/0000-0003-0497-4796)
10
Pamela C Lovejoy: (https://orcid.org/0000-0001-7315-5861)
11
Colleen Palmateer: (https://orcid.org/0000-0002-7254-0829)
12
Ruei-Ying Tzeng (https://orcid.org/0000-0002-9009-9483)
13
Michelle Arbeitman: (https://orcid.org/0000-0002-2437-4352)
14
15
Affiliation
16
1.
Department of Biomedical Sciences and Program of Neuroscience, Florida State
17
University, College of Medicine
18
2.
Corresponding author
19
3.
Division of Biology and Biological Engineering, California Institute of Technology
20
4.
Department of Molecular and Human Genetics, Baylor College of Medicine
21
5.
For correspondence: michelle.arbeitman@med.fsu.edu
22
Keywords
23
Drosophila, courtship, reproductive behaviors, cell adhesion molecules (CAMs), single cell
24
RNA-seq, IgSF
25
26
Savannah G Brovero: sbrovero2014@gmail.com
27
Julia C Fortier: julia.c.fortier@gmail.com
28
Hongru Hu: hh17d@my.fsu.edu
29
Pamela C Lovejoy: plovejoy@sjcny.edu
30
Nicole R Newell
: nrnewell@gmail.com
31
Colleen Palmateer: Colleen.palmateer@med.fsu.edu
32
Ruei-Ying Tzeng: rueiying@gmail.com
33
Kai Zinn: zinnk@caltech.edu
34
35
36
.
CC-BY 4.0 International license
(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprint
this version posted October 4, 2020.
.
https://doi.org/10.1101/2020.10.02.323477
doi:
bioRxiv preprint
Abstract
37
38
Drosophila
reproductive behaviors are directed by
fruitless
neurons
(
fru
P1
isoforms). A
39
reanalysis of genomic studies shows that genes encoding
dpr
and
DIP
Immunoglobulin
40
superfamily (IgSF) members are expressed in
fru P1
neurons. Each
fru P1
and
dpr/DIP
(
fru P1
∩
41
dpr/DIP
) overlapping expression pattern is similar
in both sexes, with dimorphism in neuronal
42
morphology and cell number. Behavioral studies of
fru P1
∩
dpr/DIP
perturbation genotypes
43
point to the mushroom body functioning together with the lateral protocerebral complex.
44
Functionally, we find that perturbations of sex hierarchy genes and
DIP-ε
changes sex-specific
45
morphology of
fru P1
∩
DIP-α
neurons. A single-cell RNA-seq analysis shows that the
DIPs
46
have high expression in a restricted set of
fru P1
neurons, whereas the
dprs
are expressed in
47
larger set of neurons at intermediate levels, with a myriad of combinations.
48
49
.
CC-BY 4.0 International license
(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprint
this version posted October 4, 2020.
.
https://doi.org/10.1101/2020.10.02.323477
doi:
bioRxiv preprint
Introduction
50
51
A current goal of neuroscience research is to gain molecular, physiological and circuit-
52
level understanding of complex behavior.
Drosophila melanogaster
reproductive behaviors are a
53
powerful and tractable model, given our knowledge of the molecular-genetic and neural
54
anatomical basis of these behaviors in both sexes. Small subsets of neurons have been identified
55
as critical for all aspects of reproductive behaviors—these neurons express
Drosophila
sex
56
hierarchy transcription factors encoded by
doublesex
(
dsx
) and
fruitless
(
fru
; fru P1
transcripts
57
spliced by sex hierarchy
;
Figure 1A
) (reviewed in D
AUWALDER
2011; Y
AMAMOTO
et al.
2014;
58
A
NDREW
et al.
2019; L
EITNER AND
B
EN
-S
HAHAR
2020). It is clear that these
dsx
- and
fru
P1
-
59
expressing neurons are present in males and females in similar positions, and arise through a
60
shared developmental trajectory (R
EN
et al.
2016), even though these neurons direct very
61
different behaviors in males and females. Males display an elaborate courtship ritual that
62
includes chasing the female, tapping her with his leg, and production of song with wing vibration
63
(reviewed in G
REENSPAN AND
F
ERVEUR
2000). The female decides whether she will mate and
64
then, if mated, she displays post-mating behaviors that includes egg laying, changes in diet, and
65
changes in her receptivity to courtship (see L
ATURNEY AND
B
ILLETER
2014; A
RANHA AND
66
V
ASCONCELOS
2018; N
EWELL
et al.
2020).
67
Sex differences in the nervous system that contribute to reproductive behaviors include
68
dimorphism in
dsx
and
fru P1
neuron number, connectivity, and physiology, with the molecules
69
and mechanisms that direct these differences beginning to be elucidated. Here, through a
70
systematic reanalysis of several genomic studies we show that a set of cell adhesion molecules
71
that are members of the immunoglobulin superfamily (IgSF) are regulated by male-specific Fru
72
(Fru
M
) or are expressed in
fru P1
neurons (
Figure 1B
) (G
OLDMAN AND
A
RBEITMAN
2007;
73
D
ALTON
et al.
2013; N
EVILLE
et al.
2014; V
ERNES
2014; N
EWELL
et al.
2016). This led us to
74
investigate the role of the Dpr (
defective proboscis extension response
) and DIP (
Dpr interacting
75
protein
) IgSF cell adhesion molecules in
fru
P1
neurons and the functions of the neurons in
76
which they are expressed for courtship behavior. Sex
-specific splicing of transcripts produced
77
from the
fru P1
promoter results in production of Fru
M
transcription factors that are members of
78
the BTB-zinc finger family, but no female-specific transcription factors (
Figure 1A
) (I
TO
et al.
79
1996; R
YNER
et al.
1996). The other
fru
transcripts are not sex-specifically spliced and provide
80
.
CC-BY 4.0 International license
(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprint
this version posted October 4, 2020.
.
https://doi.org/10.1101/2020.10.02.323477
doi:
bioRxiv preprint
essential functions (A
NAND
et al.
2001). In addition to the genomic studies, our work showed
81
that
dpr1
, the founding member of the
dpr
family (N
AKAMURA
et al.
2002) has a role in gating
82
the timing of the steps that comprise the male courtship ritual (G
OLDMAN AND
A
RBEITMAN
83
2007). The Dpr and DIP proteins are classified as cell-adhesion molecules, given that they are
84
transmembrane proteins that contain extracellular Ig domains, with short cytoplasmic tails. The
85
Dpr proteins have two extracellular Ig domains, whereas DIPs have three Ig domains (reviewed
86
in Z
INN AND
O
ZKAN
2017; S
ANES AND
Z
IPURSKY
2020). The finding that cell adhesion molecules
87
are regulated by Fru
M
fit well with studies that showed that there are differences in arborization
88
volumes throughout the central nervous system (C
ACHERO
et al.
2010; Y
U
et al.
2010), which
89
would likely be directed by differences in cell adhesion/connectivity properties of the neurons.
90
This led to predictions that differences in neuronal connectivity are important mechanisms to
91
mediate behavioral dimorphism (C
ACHERO
et al.
2010; Y
U
et al.
2010).
92
In-depth
in vitro
analyses of protein-protein interactions have shown that each Dpr has
93
dimeric interactions with specific DIP proteins, with some having multiple DIP interacting
94
partners. Additionally, some Dprs interact dimerically with Dprs through either heterophilic or
95
homophilic interactions, and some of the DIPs interact dimerically through homophilic
96
interactions (O
ZKAN
et al.
2013; C
ARRILLO
et al.
2015; C
OSMANESCU
et al.
2018)(summarized in
97
Supplemental Table 1
). Functional analyses of the Dprs and DIPs have revealed roles in
98
synaptic connectivity and specificity of neuronal targeting in the
Drosophila
neuromuscular
99
junction, visual system and olfactory system (C
ARRILLO
et al.
2015; T
AN
et al.
2015; B
ARISH
et
100
al.
2018; X
U
et al.
2018; A
SHLEY
et al.
2019; C
OURGEON AND
D
ESPLAN
2019; M
ENON
et al.
101
2019; V
ENKATASUBRAMANIAN
et al.
2019; X
U
et al.
2019). Cell adhesion molecules have
102
already been shown to be important for sculpting dimorphism in
fru
P1
neurons, with studies of
103
the IgSF member encoded by
roundabout
(
robo
) shown to be a direct target of Fru
M
and
104
responsible for dimorphic projections and morphology (M
ELLERT
et al.
2010; I
TO
et al.
2016).
105
Thus, the Dprs/DIPs are good candidates for directing sexual dimorphism in connectivity and
106
morphology that underlies differences in reproductive behavior.
107
Our inroad into the study of the role of Dprs/DIPs in
fru P1
neurons came from a
108
systematic reanalysis of several genomic studies that shows that all the
dprs
and
DIPs
examined
109
are potentially regulated by Fru
M
or are expressed in
fru P1
neurons. Additionally, a live tissue,
110
in vivo
staining approach demonstrates that there is sexual dimorphism in the overlap of
fru P1
111
.
CC-BY 4.0 International license
(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprint
this version posted October 4, 2020.
.
https://doi.org/10.1101/2020.10.02.323477
doi:
bioRxiv preprint
neurons that stain with a Dpr or DIP. This prompted us to examine the sets
of neurons that
112
express
fru P1
and one of the
dprs
or
DIPs
, using a genetic intersectional strategy (
fru P1
∩
113
dpr/DIP
;
Figure 1C
), to gain insight into the combinatorial codes of cell adhesion molecules that
114
direct development of
fru
P1
-expressing neurons in males and females. Additionally, we
115
examine the roles of neurons expressing
fru P1
and a
dpr
or
DIP
in reproductive behaviors to
116
gain insight into whether the
dprs/DIPs
expression repertoires provides insights into functions of
117
neuronal subtypes in directing behavior. In addition, this allows us to begin to elucidate which
118
combinations of neurons underlie discrete steps in the courtship ritual. Additional genetic
119
perturbation screens reveal functional roles of the sex hierarchy, and
DIP-ε,
in establishing sex-
120
specific architecture of
fru P1
∩ DIP-α
neurons. A single cell RNA-sequencing analysis
121
demonstrates the myriad, unique combinations of
dprs/DIPs
expressed in individual
fru P1
122
neurons, with overlapping expression of at least one
dpr
or
DIP
in every
fru P1
neuron
123
examined. Additionally, these single cell analyses generally show that
dprs
are expressed in
124
more neurons at intermediate levels, whereas
DIPs
have higher expression in fewer neurons.
125
Taken together, the
dprs
and
DIPs
play critical roles in establishing the
fru P1
neural circuitry in
126
both males and females.
127
128
Results
129
Genome-wide studies provide evidence that
dprs
and
DIPs
function in
fru P1
-expressing
130
neurons
131
Our systematic reanalysis of previous genomic studies shows that
dprs
and
DIPs
likely
132
have a role in
fru P1
neurons (
Figure 1B
), with the majority of the
dpr/DIP
genes in the analysis
133
identified as regulated by Fru
M
or expressed in
fru P1
neurons, in at least three independent
134
genome-wide studies (G
OLDMAN AND
A
RBEITMAN
2007; D
ALTON
et al.
2013; N
EVILLE
et al.
135
2014; V
ERNES
2014; N
EWELL
et al.
2016). Furthermore, a DNA binding site analysis further
136
confirms this regulation. There is alternative splicing at the 3’ end of
fru P1
transcripts that
137
results in one DNA-binding-domain-encoding-exon being retained out of five potential exons.
138
The predominant isoforms of Fru
M
contain either the A, B or C DNA binding domain in the
139
central nervous system (binding sites and genome-wide analysis described in D
ALTON
et al.
140
2013). When we search for the presence of the three sequence motifs near/in the
dpr
/
DIP
loci,
141
Fru
M
binding sites are found near/in all but two
dpr/DIP
loci that are examined (
Supplemental
142
.
CC-BY 4.0 International license
(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprint
this version posted October 4, 2020.
.
https://doi.org/10.1101/2020.10.02.323477
doi:
bioRxiv preprint
Table 1
). Therefore, a systematic reanalysis of genome-wide studies strongly supports a role of
143
dpr/DIPs
in
fru P1
-expressing neurons.
144
145
Live tissue staining shows sexual dimorphism in the number of cells that overlap with
146
Dpr/DIP binding and
fru P1
neurons
147
We perform live tissue,
in vivo
staining, using conditioned tissue culture media that
148
contains the epitope-tagged, extracellular regions of a Dpr or DIP. This allows us to examine
149
binding to their respective Dpr/DIP partners in brain tissues of 48-hour pupae and 0-24 hour
150
adults (as done in F
OX AND
Z
INN
2005; L
EE
et al.
2009; O
ZKAN
et al.
2013). Using this approach,
151
we detect signal for two Dprs and two DIPs in the subesophageal
ganglion of the brain (Dpr3,
152
Dpr16, cDIP, and DIP- γ
; Supplemental Figure 1
). The live staining technique is not effective
153
throughout the adult brain and for all Dprs/DIPs tested
, perhaps due to the inability of the
154
epitope-tagged Dprs/DIPs extracellular regions to penetrate other regions in live brain tissues,
155
which are not permeabilized by detergent, as is done for fixed tissue. The number of neuronal
156
cell bodies with staining is similar in males and females at both time points, in wild type and
fru
157
P1
mutants, with some significant differences with small effect sizes. However, the number of
158
neuronal cell bodies with staining that overlap with
fru P1
is significantly higher in males
159
compared to females at both time points. Given that we
do not see large sex
-specific changes in
160
the number of cells with signal in
fru P1
mutants, suggests that regulation of
dprs/DIPs
is more
161
complex than simple regulation by
fru P1
. Overall, the analysis reveals sexual dimorphism in
162
binding of tagged Dpr/DIP proteins to
fru P1
neurons in the subesophageal
ganglion brain region
163
using a live staining approach (
Supplemental Figure 1
), with more neurons with overlap
164
detected in males.
165
166
A genetic intersectional approach identifies neurons that express both
fru P1
and a
dpr
or
167
DIP
in males and females
168
The above results led us to examine the expression patterns of neurons that express both
169
fru P1
and a
dpr
or
DIP
, using a genetic intersectional approach (
Figure 1C
). This approach
170
restricts expression of a membrane-bound-GFP marker to neurons with intersecting expression
171
of
fru P1
and a
dpr
or
DIP
(
fru P1
∩
dpr/DIP
). This is
accomplished using a UAS-membrane-
172
bound GFP reporter transgene that requires removal of an FRT-flanked stop cassette for
173
.
CC-BY 4.0 International license
(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprint
this version posted October 4, 2020.
.
https://doi.org/10.1101/2020.10.02.323477
doi:
bioRxiv preprint
expression. Removal of the stop cassette is
mediated by
fru P1
driven FLP recombinase (Y
U
et
174
al.
2010). This system is used in combination with a collection of
dpr
- and
DIP
-
Gal4
transgenic
175
strains (
Figure 1C
) (V
ENKEN
et al.
2011; N
AGARKAR
-J
AISWAL
et al.
2015a; N
AGARKAR
-
176
J
AISWAL
et al.
2015b; T
AN
et al.
2015; L
EE
et al.
2018). We primarily focus the analysis on 4-7
177
day adults (
Figures 2 and 3
), which are sexually mature adults, and 0-24 hour adults to
178
determine if the patterns change during early adult stages (
Supplemental Figures 2 and 3
).
179
Additionally, behavioral studies are performed in 4-7 day adults (
Figures 4-6
), so the expression
180
and behavioral data can be co-analyzed (
Figure 7
). At a gross morphological level, the patterns
181
we observe in older 4-7 day old adults are also present in 0-24 hour adults, though in some cases
182
expression in the mushroom was not as robust at the early time point.
183
Based on our examination of the expression patterns in 27 intersecting genotypes, we find
184
that 24 showed clear, membrane-bound GFP expression in the central nervous system at the time
185
points examined. Of these, only two
fru P1
∩
DIP
genotypes have very restricted and unique
186
patterns (
fru P1
∩
DIP
-δ and
fru P1
∩
DIP-
!
), whereas the other genotypes have broader
187
expression, with many in similar regions/patterns (
Figures 2 and 3
). For example, 22
188
intersecting genotypes, in both males and females, have consistent expression in the brain lateral
189
protocerebral complex, including within the arch, ring, junction and crescent (for summary see
190
Figure 7 and Supplemental Table 2
). This region has been shown to have
fru P1
neurons with
191
sexually dimorphic arbor volumes (C
ACHERO
et al.
2010; Y
U
et al.
2010). Furthermore, the
192
lateral protocerebral complex has inputs from sensory neurons and is predicted to be a site of
193
sensory integration, to direct motor output (Y
U
et al.
2010). We find 8 intersecting genotypes
194
have expression in mushroom bodies in both males and females. This region has a well-
195
established role in learning and memory, including learning in the context of courtship rejection
196
(M
C
B
RIDE
et al.
1999; M
ONTAGUE AND
B
AKER
2016; J
ONES
et al.
2018; Z
HAO
et al.
2018).
197
Overall, the majority of
fru P1
∩
dpr/DIP
genotypes are expressed in similar regions, suggesting
198
that some may function in combinatorial manner within a neuron to direct patterning and/or
199
synaptic targeting.
200
We observe sex differences in the presence of morphological features and cell body
201
number in regions we scored (
Figures 2 and 3 and Supplemental Table 2
), which were largely
202
chosen because they were previously reported to display sexual dimorphism (C
ACHERO
et al.
203
2010; Y
U
et al.
2010). For example, 18 intersecting genotypes show consistent presence of signal
204
.
CC-BY 4.0 International license
(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprint
this version posted October 4, 2020.
.
https://doi.org/10.1101/2020.10.02.323477
doi:
bioRxiv preprint
in the mesothoracic triangle neuronal projections in males, but only two
lines do so in females.
205
While both males and female have expression in antennal lobe glomeruli DA1 and V
A1v in
206
several intersecting genotypes, there is also
sexual dimorphism, with four genotypes having
207
consistent expression in only female DA1 glomeruli (
fru P1
∩
dpr3, dpr10, dpr17, DIP-θ
). In
208
the ventral nerve cord, a midline crossing phenotype is consistently observed for the majority of
209
intersecting genotypes only in males, which was previously shown to be a male-specific
210
phenotype for a set of gustatory neurons (M
ELLERT
et al.
2010). For all regions where cell bodies
211
are counted, the trend was that there are more cell bodies in males than females.
Thus, the
212
differences in the patterns of expression between males and females are not large, with several
213
genotypes having quantitative differences in the numbers of cell bodies present, rather than a
214
more complete presence or absence difference. It is possible that there are additional quantitative
215
differences that are not detected based on the resolution of the analyses, including quantitative
216
differences in expression level of
dpr/DIPs
, or their sub-cellular localization, or in
217
regions/features that are not quantified here (
Figures 2 and 3 and Supplemental Table 2
).
218
219
Activation of
fru P1 ∩ dpr/DIP
neurons results in atypical courtship behaviors
220
Substantial progress has been made in showing
fru P1
has a critical role in reproductive
221
behaviors, including determining the function of small
subsets of neurons that are responsible for
222
different aspects of behavior (reviewed in A
UER AND
B
ENTON
2016). The tools in hand can
223
further address if additional combinations or quantitative differences in the number of
fru
P1
224
neurons are important for behavioral outcomes, given the
fru P1
∩
dpr/DIP
subsets and
225
combinations we examine are distinct from those previously studied. We use the genetic
226
intersectional strategy to activate intersecting neurons, by driving expression of TrpA1, a heat
227
activated cation channel (
Figure 1C
) (
VON
P
HILIPSBORN
et al.
2011). This allows for temporal
228
control of neuronal activation by an acute increase of the temperature in the courtship chambers
229
(32°C; controls were at 20°C). We find that neuronal activation resulted in decreases in male
230
following and wing extending towards females for several genotypes (
Figure 4 and 7 and
231
Supplemental Table 3
). We also observe that neuronal activation of
fru P1
∩
dpr
(13/16) and
232
fru P1
∩
DIP
(2/8) genotypes caused atypical courtship behavior towards a female, including
233
double wing extension, and continuous abdominal bending, even if the female had moved away
234
(
Figure 4 and 7
). These atypical behaviors could account for some of the decreases in following
235
.
CC-BY 4.0 International license
(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprint
this version posted October 4, 2020.
.
https://doi.org/10.1101/2020.10.02.323477
doi:
bioRxiv preprint
and wing extension. For example, if a male is locked into abdominal bending, this would reduce
236
courtship following behavior. Additionally, we find that some males ejaculated on the chamber
237
in five intersecting genotypes:
dpr5
(5 /15),
dpr9
(3 /15),
dpr10
(3 /15), and
dpr12
(2 /15), and
238
DIP-θ
(4 /15). Of note,
fru P1
∩
DIP-α
is the only strain that showed a decrease in courtship
239
activities without a concomitant increase in atypical courtship behaviors. This suggests that
fru
240
P1
∩
DIP-α
neurons may normally inhibit courtship behaviors when they are activated.
241
We next determine if the males require females to reach an arousal threshold needed to
242
perform typical and atypical courtship behaviors, given that several of the courtship behaviors
243
described above occur when the male was not oriented towards the female. To address this
244
question, we examine courtship behaviors in solitary males, using the same temporal activation
245
strategy as above. We find that activation of the
fru P1
∩
dpr/DIP
neurons is sufficient to elicit
246
single wing extension, double wing extension, and abdominal bending in
fru P1
∩
dprs
(11/16)
247
and
fru P1
∩
DIPs
(3/8) (
Figure 5, 7 and Supplemental Table 3
). Similarly, activating the
248
intersecting
fru P1
neuronal populations of
fru P1
∩
dpr5
(5 /10),
dpr9
(1/10),
dpr10
(1/10),
249
dpr12
(3/10), and
DIP-θ
(1/10) causes males to ejaculate without a female present. Overall,
250
activation of these subsets of
fru P1
neurons is sufficient to direct reproductive behaviors, even if
251
a female is not present, consistent with other neuronal activation experiments (reviewed in A
UER
252
AND
B
ENTON
2016).
253
254
Silencing
fru P1 ∩ dpr/DIP
neurons result
in courtship changes
255
Given that activation of
fru P1
∩
dpr/DIP
neuronal subsets
resulted in changes in
256
courtship behaviors, we next determine how silencing these neurons impacts male-female
257
courtship, to gain further insight into their roles. To test this we use the genetic intersectional
258
approach with a
UAS
> stop > TNT
transgene (
Figure 1C
) (S
TOCKINGER
et al.
2005). The
259
intersecting genotypes express tetanus toxin light chain, which cleaves synaptobrevin, resulting
260
in synaptic inhibition (S
WEENEY
et al.
1995). As a control we also examine courtship behaviors
261
of flies expressing an inactive form of
TNT
(TNTQ), using the genetic intersectional approach.
262
In addition to scoring courtship behaviors, motor impairment
is also scored (
Figure 6
263
and Supplemental Table 3
). Given that neuronal silencing in several genotypes results in motor
264
impairment, in which the male fell and
is unable to quickly right himself, we quantify the time
265
when the fly could not right himself as “motor defect” and subtract this from the overall
266
.
CC-BY 4.0 International license
(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprint
this version posted October 4, 2020.
.
https://doi.org/10.1101/2020.10.02.323477
doi:
bioRxiv preprint
courtship time for behavioral indices (
Figure 6
). The intersecting genotypes that consistently
267
demonstrate motor defects additionally show decreases in following and wing extension upon
268
silencing, likely due to some motor impairment (
fru P1
∩
dpr1, dpr3, dpr4, dpr5, dpr9, dpr10,
269
dpr11, dpr12, dpr15
and
DIP-η)
. Additional courtship behavioral indices and latencies are
270
quantified and those with motor defects show additional strong courtship phenotypes
271
(
Supplemental Table 3
). However, seven intersecting genotypes have a decrease in
272
following/wing extension indices and only minor or no motor impairment (
fru P1
∩
dpr2, dpr6,
273
dpr17, dpr18, DIP-ε, DIP-θ, and DIP-γ
). One genotype,
fru P1
∩
dpr7
, has an increase in
274
following/wing extension with neuronal silencing.
In the case of
fru P1
∩
dpr7
, we do not detect
275
GFP expression in the central or peripheral nervous system in adults, so the neurons underlying
276
this phenotype remain to be determined. Locomotor activity of the seven intersecting genotypes
277
with no or minor motor defects are further analyzed for motor impairment (p<0.005 for strong
278
motor defects; 0.05>p>0.005 for minor;
Supplemental
Table 3
), along with
fru P1
∩
dpr7
, and
279
fru P1
∩
dpr10
, which has strong motor impairment. If there is a significant difference, the
280
intersecting genotype with neuronal silencing has increased locomotor activity in the activity
281
monitors, suggesting that the courtship phenotypes are not due to overall loss in motor activity
282
(
Supplemental Table 3
).
283
As above in the neuronal activating experiments, silencing
fru P1
∩
Dprs
(13/19) is more
284
likely to cause a courtship defect than silencing
fru P1
∩
DIPs
(4/9). Given the large effect size
285
of the courtship defects compared to the smaller effect size of the motor defect, it is clear that
286
silencing
fru P1
∩
dpr/DIP
neurons in the central nervous system, for most genotypes,
287
suppresses courtship (
Figure 6
). This is consistent with previous studies that have found that
288
silencing
fru P1
neurons in males leads to decreased courtship towards a female (M
ANOLI
et al.
289
2005; S
TOCKINGER
et al.
2005). Interestingly,
fru P1
∩
DIP-α
is the only strain to demonstrate
290
motor defects, but no change in courtship behaviors upon silencing, underscoring the previous
291
hypothesis that these neurons may normally be inhibitory for courtship.
292
293
Meta-analysis of male
fru P1
∩
dpr/DIP
expression patterns and behavioral data
294
Next, we determine if intersecting genotypes with similar expression patterns also have
295
similar behavioral outcomes in the neuronal activating and silencing experiments described
296
above. We use a heuristic approach and generate a heatmap that groups
dprs/DIPs
based on
297
.
CC-BY 4.0 International license
(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprint
this version posted October 4, 2020.
.
https://doi.org/10.1101/2020.10.02.323477
doi:
bioRxiv preprint
similarity of the
fru P1
∩
dpr/DIP
membrane-bound-GFP expression data (
Figure 7A, and
298
Supplemental Table 2
for additional visualizations). At the top of the heatmap is a dendrogram
299
showing the relationships in expression data, grouping those that are most similar together (from
300
data in
Figure 2 and 3 and Supplemental Table 2
). The bottom has colored dots that indicate
301
the behavioral changes observed in the three different behavioral perturbation data sets (from
302
data in
Figures 4-6
). The scoring key for the GFP expression phenotypes is shown (
Figure 7B
303
and Supplemental Table 2
). Only the 24 intersecting genotypes with GFP expression data are
304
included in the heat map.
305
There is a set of eight intersecting genotypes grouped together on the right of the
306
dendrogram that all have expression in the mushroom body and several regions
within the lateral
307
protocerebral complex, but varied expression across the other morphological features (
Figure
308
7A
;
fru P1
∩
dpr4, dpr5, dpr8, dpr9, dpr10, dpr12, dpr14
and
DIP-γ
). Seven have similar types
309
of atypical courtship behaviors in the activating experiments (excluding
fru P1
∩
dpr8
), in the
310
male-female courtship assays. These seven also have similar behavioral phenotypes in the male-
311
alone condition, indicating that the activation threshold in these lines can be achieved without a
312
female present (
Figure 5
).
313
Furthermore, among the eight genotypes, there are four intersecting genotypes that have
314
male ejaculates in the chamber, in both the male-female and male-alone neuronal activation
315
assay
s. All four intersecting genotypes also have relatively high cell body counts in the
316
abdominal ganglion, a region in the ventral nerve cord that has previously been shown to drive
317
ejaculation (
Supplemental Table 2
) (T
AYLER
et al.
2012). However, not all intersecting
318
genotypes with expression in the abdominal ganglion show the ejaculation phenotype, as shown
319
in the heatmap. Furthermore, there is an intersecting genotype that does not have mushroom
320
body expression, but also has the ejaculation phenotype (
fru P1 ∩
DIP-θ
). These results reveal
321
how different combinations and numbers of neurons can direct a similar behavioral outcome.
322
Overall, the results point to a critical role for interactions between the mushroom body and
323
protocerebral complex in directing courtship behaviors, which are modified by being activated in
324
combination with other neuronal populations. This is consistent with an idea put forth previously
325
that posited connections between these two brain regions may integrate diverse external stimuli
326
with internal physiological state and previous behavioral experience (Y
U
et al.
2010).
327
.
CC-BY 4.0 International license
(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprint
this version posted October 4, 2020.
.
https://doi.org/10.1101/2020.10.02.323477
doi:
bioRxiv preprint
Twenty-two intersecting genotypes have expression in different regions of lateral
328
protocerebral complex, but no consistent expression in the mushroom body. An examination of
329
the behavioral phenotypes reveals no consistent behavioral phenotypes, based on the lateral
330
protocerebral complex expression data. While the lateral protocerebral complex is critical for
331
higher order processing, the data further supports the idea that interactions across different
332
combinations of activated neurons, in each intersecting genotype, is critical for the behavioral
333
outcomes and underscores how different patterns of neuronal activity can direct similar
334
behavioral outcomes.
335
336
Correlation of
fru P1
∩
Dpr/DIP
expression patterns
337
As an additional heuristic tool, we plot the correlation of the GFP expression patterns for
338
the male and female data (
Figure 7C and Supplemental Table 2
). One goal is to gain insight
339
into whether Dprs/DIPs with the same interacting partners are co-expressed together. This allows
340
us to gain insight into the mechanisms used by these IgSF molecules to direct cell adhesion and
341
to determine if there are sex differences. Another goal is to determine if the protein-protein
342
interactions may occur through
cis
(within the same neuron) vs
trans
(across neurons)
343
interactions. For example, if protein-protein interactions are in
cis
, then the Dpr/DIP interacting
344
partners will be expressed in the same neurons and have correlated expression patterns. To
345
address these questions, the plots are annotated with DIPs (colored dots) that each Dpr interacts
346
with on the right (based on interactome from C
ARRILLO
et al.
2015).
347
It appears that some Dprs/DIPs that bind the same partner have the most similar
348
expression patterns. For example, in males
fru P1
∩
dpr1
, and
dpr2
have highly correlated
349
expression and both Dpr1 and Dpr2 interact with DIP-η
and DIP-θ. In addition, the male
fru P1
350
∩
DIP- η
expression pattern is highly correlated with
fru P1
∩
dpr1
, and
dpr2
, suggesting that
351
Dpr-DIP protein-protein interactions may also occur in
cis
. Similarly, in females,
fru P1
∩
dpr1
,
352
dpr2
, and
dpr3
have highly correlated expression, with Dpr1, Dpr2 and Dpr3 also all interacting
353
with DIP-η
and DIP-θ. On the other hand, in males,
dpr11
does not have highly correlated
354
expression with
DIP- β
and
DIP-γ
, though Dpr11 interacts with DIP-β and DIP-γ. This is
355
consistent with protein-protein interactions occurring in
trans
. In females,
fru P1
∩
dpr8
,
dpr9
,
356
dpr11
(interact with DIP-β and DIP-γ) have highly correlated expression patterns, which is not
357
observed in males. Therefore, there are sex-differences in the co-expression patterns of Dprs that
358
.
CC-BY 4.0 International license
(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprint
this version posted October 4, 2020.
.
https://doi.org/10.1101/2020.10.02.323477
doi:
bioRxiv preprint
could underlie dimorphism in morphology.
fru P1
∩
DIP-α
and
DIP-δ
have the most restricted
359
expression patterns and they are not highly correlated with the expression patterns of their
360
interacting Dpr partners, in either males or females. Overall, based on the correlation patterns in
361
the expression data, it appears that some protein-protein interactions can occur in
cis
or
trans
.
362
Additionally, some Dpr and DIPs with similar binding partners have correlated expression,
363
which could be a mechanism to mediate the strength of neuronal adhesion. These observations
364
are also supported by the single cell sequencing data (see below).
365
366
A higher resolution analysis of
fru P1 ∩ DIP
-α
reveals additional sexually dimorphic
367
expression patterns
368
To gain insight into mechanisms that generate sexual dimorphism in morphology, we
369
examine the relatively small number of
fru P1
∩ DIP-α
neurons in male and females. Their small
370
number facilitates in-depth analysis, as cell bodies and projection patterns are easier to discern
371
(
Figure 8 and Supplemental Table 4
). While the overall patterns are similar (
Figure 8A
), there
372
are fine-scale differences (
Supplemental Table 4
). There are sex-differences in the superior
373
medial protocerebrum region (SMP;
Figure 8A and B, subpanels I
), where females have a
374
longer (dotted-line) and broader projection (arrowhead), as compared to males. Moreover, in the
375
medial part of midbrain, an “M” shaped peak forms (“M”-like) in males that is not typically
376
observed in females (curved dotted-line,
Figure 8A and B, subpanels II&III
). Additionally, in
377
the ventral lateral protocerebrum region (VLP) there are neuronal cell bodies (arrowhead,
Figure
378
8A and B, subpanels II&III
), and projections in a “square” shaped pattern that are more
379
frequently observed in females (closed dotted-line,
Figure 8A and B, subpanels II&III
). There
380
is also a greater frequency of neuronal cell bodies present in the subesophageal
ganglion (SEG)
381
in females, as compared to males (arrowhead,
Figure 8A and B, subpanels IV
). In the
382
abdominal ganglia (AbG) of the ventral nerve cord there is a higher density of projections in
383
males (
Figure 8A and B, subpanels V
). In contrast, females have a distinct “forceps
” shaped
384
pattern in the AbG region (arrowhead,
Figure 8A and B, subpanels V
). Taken together, it
385
appears that the sex
differences are due to differences in the number of neurons and also
in the
386
morphology of projections and arborizations (
Figure 8
).
387
388
Changing the sex of
DIP-α
neurons
alters the
fru P1 ∩ DIP-α
co-expressing patterns
389
.
CC-BY 4.0 International license
(which was not certified by peer review) is the author/funder. It is made available under a
The copyright holder for this preprint
this version posted October 4, 2020.
.
https://doi.org/10.1101/2020.10.02.323477
doi:
bioRxiv preprint