Investigation
of
Drosophila
fruitless
neurons that express Dpr/DIP cell adhesion molecules
1
2
Savannah G Brovero
*,1
,
Julia C Fortier
*,1
,
Hongru Hu
*,1
,
Pamela C Lovejoy
*,1
,5
,
Nicole
R
3
Newell
*,1
,
Colleen M Palmateer
*,1
,
Ruei
-
Ying Tzeng
*,1
,
Pei
-
Tseng Lee
4
,
Kai Zinn
3
,
Michelle N
4
Arbeitman
1,2
,
6
5
* co
-
first authors
6
7
Orcid IDs:
8
Hongru Hu: (https://orcid.org/0000
-
0003
-
0497
-
4796)
9
Pamela C Lovejoy: (
https://orcid.org/0000
-
0001
-
7315
-
5861
)
10
Colleen Palmateer: (
https://orcid.org/0000
-
0002
-
7254
-
0829
)
11
Ruei
-
Ying Tzeng (
https://orcid.org/0000
-
0002
-
9009
-
9483
)
12
Kai Zinn (https://orcid.org/0000
-
0002
-
6706
-
5605)
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,
Cal
ifornia Institute of
Tech
nology
20
4.
Departmen
t
of Molecular and Human Genetics,
Baylor
College of Medicine
21
5.
Current Address: Department of Biology, St. Joseph’s College, New York
22
6.
For correspondence: michelle.a
rbeitman@med.fsu.edu
23
Keywords
24
Drosophila, courtship,
reproductive behaviors,
cell adhesion
molecules (CAMs)
, single cell
25
RNA
-
seq,
IgSF
26
27
Savannah G Brovero
:
sbrovero2014@gmail.com
28
Julia C Fortier
:
julia.c.fortier@gmail.com
29
Hongru Hu
:
hh17d@my.fsu.edu
30
Pam
ela C
Lovejoy
:
plovejoy@sjcny.edu
31
Nicole
R
Newell
:
nrnewell@gmail.com
32
Colleen Palmateer: Colleen.palmateer@med.fsu.edu
33
Ruei
-
Ying Tzeng
:
rueiying@gmail.com
34
Kai Zinn:
zinnk@caltech.edu
35
36
Abstract
37
38
Drosophila
reproductive behavior
s
are directed by
fruitless
neurons
.
A
re
analysis of
genomic
39
studies show
s
that genes
encoding
dpr
and
DIP
Immunoglobulin superfamily (IgSF)
members
40
are expressed in
fru P1
neurons
.
We find that e
ach
fru P1
and
dpr/DIP
(
fru P1
∩
d
pr/DIP
)
41
overlapping expression
pattern
is
similar
in both sexes
,
but
there are
dimorphism
s
in neuronal
42
morphology and cell number.
Behavioral studies of
fru P1
∩
d
pr/DIP
perturbation genotypes
43
indicates
that the
mushroom body
function
s
together with the lateral protocerebral complex
to
44
direct courtship behavior
.
A
single
-
cell RNA
-
seq
analysis
of
fru P1
neurons
shows that
many
45
DIPs
have high expression in a
small set of neurons
, whereas the
dprs
are
often
expressed in
a
46
larger set of neurons at intermediate levels
, with a myriad of
dpr/DIP
expression combinations.
47
Functionally, we
find that perturbations of sex hierarchy genes
and
of
DIP
-
ε
change
the
sex
-
48
specific morpholog
ies
of
fru P1
∩
DIP
-
α
neurons
.
49
50
Introduction
51
A goal of neuroscience research is
to gain
molecular
, physiological
and circuit
-
level
52
understanding of complex behavior.
Drosophila melanogaster
reproductive behaviors are a
53
powerful
and tractable
model, given
our knowledge of the molecular
-
genetic and neur
al
54
anatomical
basis
of
these behaviors
in both sexes
.
S
mall
subset
s
of
neurons
have
b
een identified
55
as critical for all aspects of reproductive behaviors
. T
hese neurons
express sex
-
specific
56
transcription factors encoded by
doublesex
(
dsx
)
and
fruitless
(
fru
; fru P1
transcripts
are
splic
ed
57
under
sex hierarchy
regulation
;
Figure 1A
)
(reviewed in
D
AUWALDER
2011;
Y
AMAMOTO
et al.
58
2014;
A
NDREW
et al.
2019;
L
EITNER AND
B
EN
-
S
HAHAR
2020)
.
ds
x
-
and
fru
P1
-
expressin
g
59
neurons are present in males and females in similar positions
,
and arise
through a shared
60
developmental trajectory
(
R
EN
et al.
2016)
,
al
though
these neurons direct very different
61
behaviors in males and females.
Males display an elaborate courtship ritual
that includes chasing
62
the female, tapping her
with his leg
,
and
p
roduction of
song
with wing vibration
(reviewed in
63
G
REENSPAN AND
F
ERVEUR
2000)
. T
he female
decide
s whether she will
mate and then
, if mated,
64
displays post
-
mating behaviors
that include egg laying
, changes in diet,
and changes in
65
receptivity to courtship
(see
L
ATURNEY AND
B
ILLETER
2014;
A
RANHA AND
V
ASCONCELOS
2018;
66
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
. The
molecules and
69
mechanisms that direct these differences
are
beginning to be elucidated.
Several genome
-
wide
70
studies have been performed to find genes
that are
regulated by
male
-
specific Fru (Fru
M
) or are
71
expressed
in
fru
P1
neurons
. These
independent
studies
examine
d
fru P1
loss
-
of
-
function and
72
gain
-
of
-
function gene
expression changes,
fru P1
cell
-
type
-
specific gene expression
,
and the
73
direct targets
for
Fru
M
binding
(
G
OLDMAN AND
A
RBEITMAN
2007;
D
ALTON
et al.
2013;
N
EVILLE
74
et al.
2014;
V
ERNES
2014;
N
EWE
LL
et al.
2016)
. A reanalysis of these genomic studies
75
demonstrates that cell adhesi
on molecules that are members of the immunoglobulin superfamily
76
(IgSF) are regulated by male
-
specific Fru (Fru
M
) or are expressed in
fru
P1
neurons (
see
Figure
77
1B
).
In this study we focus on an interacting set of IgSF molecules encoded by
dpr
s
/DIPs
.
78
The
Dpr and DIP proteins are membrane
-
linked cell adhesion molecules with N
-
terminal
79
extracellular Ig domains and C
-
terminal glycosyl
-
phosphatidylinositol (GPI) linkage sequences
80
or transmembrane domains. The Dpr proteins have two extracellular Ig domains, whe
reas DIPs
81