Mechanisms of Development 73 (1998) 135–146

Review article

From behavior to development: genes for sexual behavior define

the neuronal sexual switch in Drosophila

Daisuke Yamamoto a , c ,*, Kazuko Fujitani a, Kazue Usui a, Hiroki Ito a, Yoshiro Nakano b
a
ERATO Yamamoto Behavior Genes Project, JST, Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194–8511, Japan
b
ERATO Yamamoto Behavior Genes Project, University of Hawaii, Manoa, HI 96822, USA
c
Pioneering Research Division, Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194–8511, Japan

Accepted 13 February 1998

Abstract

The isolation and analysis of Drosophila mutants with altered sexual orientation lead to the identification of novel branches in the sex-
determination cascade which govern the sexually dimorphic development of the nervous system. One such example is the fruitless (fru)
gene, the mutation of which induces male-to-male courtship and malformation of a male-specific muscle, the muscle of Lawrence (MOL).
Since the MOL is formed in wild-type flies when the innervating nerve is male, regardless of the sex of the MOL itself, the primary site of
Fru function is likely to be the motoneurons controlling the MOL. The fru gene produces multiple transcripts including sex-specific ones. A
female-specific mRNA from the fru locus has a putative Transformer (Tra) binding site in its 5′ untranslated region, suggesting that fru is a
direct target of Tra. The fru transcripts encode a set of proteins similar to the BTB (Bric à brac, Tramtrack and Broad-complex)-Zn finger
family of transcription factors. Mutations in the dissatisfaction (dsf) gene result in male-to-male courtship and reduced sexual receptivity of
females. The dsf mutations also give rise to poor curling of the abdomen in males during copulation and failure of egg-laying by females.
The latter phenotypes are ascribable to aberrant innervation of the relevant muscles. A genetic analysis reveals that expression of the dsf
phenotypes depends on Tra but not on Doublesex (Dsx) or Fru, suggesting that dsf represents another target of Tra. Taken together, these
findings suggest that the sex-determination protein Tra has at least three different targets, dsx, fru and dsf, each of which represents the first
gene in a branch of the sex-determination hierarchy functioning in a mutually-exclusive set of neuronal cells in the Drosophila central
nervous system.  1998 Elsevier Science Ireland Ltd. All rights reserved

Keywords: Sex determination; Sexual dimorphism; fruitless; dissatisfaction

1. Introduction
What is the link between development and behavior? A
simple answer to this question is that behavior is generated
by the functional nervous system which develops during the
development of individual organisms based on a precise
‘body’ plan or ‘developmental program’. This means that
animal behavior is a phenotypic reflection of the functional
integrity of the nervous system formed during development.
An obvious prediction deriving from this fact is that factors
* Corresponding author. ERATO Yamamoto Behavior Genes Project at
Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida,
Tokyo 194–8511, Japan. Tel.: +81 427 212334; fax: +81 427 212850;
e-mail: daichan@fly.erato.jst.go.jp
affecting the ‘body’ plan for the development of the nervous
system should affect animal behavior. Indeed, this is the
case.
The neural body plan is programmed in the genome, by
so-called developmental genes, and its execution can be
modulated by environmental conditions. Drosophila mela-
nogaster offers an ideal system for the experimental manip-
ulation of the developmental genes, due to its amenability to
genetic analysis (Lawrence, 1992; Lindsley and Zimm,
1992; Bate and Martinez Arias, 1993; Yamamoto, 1996),
and thus provides a unique opportunity for genetic dissec-
tion of behavior, by mutating such genes (Benzer, 1967;
Hall, 1982, 1985; Davis, 1996; Tully, 1996; Barinaga,
1997). In practice, candidate genes involved in the forma-
tion of the neural systems associated with certain behavior

0925-4773/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved

PII S0925-4773 (98 )0 0042-2
136 D. Yamamoto et al. / Mechanisms of Development 73 (1998) 135–146
are identified either by screening for mutations that lead to
behavioral abnormalities, or by behavioral assays of
mutants known to exhibit disrupted neural structures and/
or functions (Fischbach and Heisenberg, 1984; Buchner,
1991).
When attempting to elucidate the mechanisms underlying
some behavioral patterns rather than those regulating gen-
eral activity levels, our research efforts are concentrated on
the investigation of cellular bases for specific behavioral
acts. A reasonable assumption is that the specificity of beha-
vioral acts derives from the specificity of the neural circui-
try. The specificity of the neural circuitry is determined by
the uniqueness of individual neurons with respect to their
physiology and connectivity.
Such unique properties of neurons are determined and
expressed in discrete steps during development (Goodman
and Spitzer, 1979; Goodman et al., 1980; Bate et al., 1981;
Thomas et al., 1984). At first, cell fate decisions must be
made when each neuron becomes a terminally-differen-
tiated (i.e. non-proliferating) neuron (Hirata et al., 1995;
Knoblich et al., 1995; Doe and Skeath, 1996). The neurons
then have to extend their processes along the correct route
(growth-cone path-finding) (Tessier-Lavigne and Goodman,
1996). Upon reaching the appropriate target cells, further
extension of the growth cone needs to stop (Nose et al.,
1994; Chiba et al., 1995; Matthes et al., 1995; Schneider
et al., 1995) and firm synaptic contacts with the targets need
to be established (Kopczynski et al., 1996; Sone et al.,
1997). Dynamic changes in the structure and function of
synaptic contacts are also required for the plasticity of beha-
vior not only in ‘developing’ animals, but also in adults
(Glanzman et al., 1990; Heisenberg et al., 1995; Singer,
1995; Suhonen et al., 1996; Kempermann et al., 1997). In
the holometabolous insects, such as the fruitfly, behavioral
patterns change dramatically after adult emergence, conco-
mitant with metamorphosis of the body.
A remarkable feature unique to adult behavior is sexual
dimorphism. Sex-specificity in adult behavior should have
its basis in the sexually dimorphic neuronal structure and
function, which are predominantly established during meta-
morphosis in the pupae. All of the basic processes of neu-
rogenesis, such as production of neurons by neuroblasts,
axonal path finding, and formation of synaptic contacts,
need to be specialized according to sex.
This review is intended to address the question of how
such complex neurogenic processes are controlled in a sex-
dependent manner to establish the neuronal circuitry
required for the generation of sexually dimorphic patterns
of behavior in adult Drosophila. Two genes primarily dealt
with are fruitless (fru) and dissatisfaction (dsf), which were
identified by mutations which altered sexual orientation.
2. Neuronal sexual fate and sexual behavior
Mating behavior is initiated by the male which orients
itself to a female and tracks her (Hall, 1994; Greenspan,
1995). Subsequently, the male approaches the female
from behind, tapping the abdomen of the female with its
forelegs. After tapping, the male generates courtship songs
by alternate vibrations of its wings. The courtship songs are
composed of two distinct elements, the pulse song and the
sine song, which are repeated several times alternately in a
single bout. The male then licks the female’s genitalia and
attempts to copulate. If the male successfully mounts the
female, the copulation continues for 15–20 min.
The sexual behavior of females is less evident than that of
males. In response to an attempt at copulation by a male, the
female displays either receptive or rejective actions. When
receptive, the female parts two wings exposing her dorsal
abdomen so that the male can mount her back. Subsequently
the female opens her vaginal plate, allowing the male to
make genital contact. On the other hand, rejection may be
expressed in one of two ways, depending on the physiolo-
gical condition of the female: Non-receptive virgin females
tend to lift up their abdomens so as to physically prevent
genital contact by the male, while non-receptive previously-
fertilized females typically lower their abdominal tips and
extrude their ovipositors and even their eggs (Connolly and
Cook, 1973; Aigaki et al., 1991; Suzuki et al., 1997).
The existence of such an overt sexual difference in beha-
vior implies the sexual dimorphism in the central nervous
system (CNS) areas responsible for mating behavior.
Gynandromorphs have been used to determine the locations
of anatomical foci controlling male-type and female-type
behavior. A sex-linked enzyme marker distinguishes male
tissue from female tissue in sexually mosaic flies. The
results indicated that the posterior dorsal brain near the
mushroom body is indispensable for generating male-type
behavior (Hall, 1979), whereas the dorsal anterior brain is
essential for female-type behavior (Tompkins and Hall,
1983). Thus, according to the gynandromorph studies, the
brain center for male-type behavior is anatomically (geo-
graphically) distinct from that for female-type behavior.
However, subsequent histological analysis of the respective
brain regions did not reveal any obvious sexual differences
in cellular compositions or in neural connectivity.
3. Sex-determination cascade
The analysis of the developmental biology of sexual traits
in D. melanogaster leads to the hypothesis that most of the
sexually dimorphic characteristics are under the control of
the ‘sex-determination cascade’ of genes (Baker, 1989;
Steinmann-Zwicky, 1992; Ryner and Swain, 1995). In Dro-
sophila, the ratio of the number of X chromosomes (X) to
that of the autosomes (A) is the factor determining sex.
When X/A is 0.5, the flies develop as males, while when
it is greater than 1, the flies develop as females. This is
because the master switch gene for female development,
Sex lethal (Sxl), can be activated only when the X/A value
 D. Yamamoto et al. / Mechanisms of Development 73 (1998) 135–146
exceeds 1, the condition in which transcription factors
coded by genes linked to the X chromosome are accumu-
lated to the level sufficient for trans activation of Sxl (Cline,
1993). Lack of Sxl activation results in a fly adopting the
default sexual fate, the male (Fig. 1).
The Sxl gene encodes a splicing inhibitor which binds to
the splice junctions of its own primary transcript, preventing
the use of the exon that contains a stop codon (Sosnowski et
al., 1989; Sakamoto et al., 1992). The Sxl protein also binds
to its downstream target, transformer (tra) mRNA. In this
case, the second exon is skipped as a result of Sxl binding,
allowing the female flies to produce the functional Tra pro-
tein (Inoue et al., 1990). Since Sxl is absent in males, the
second exon with a stop codon contributes to the male
tra transcript, which produces a non-functional protein
(Fig. 1).
Tra is an RNA-binding protein that enhances splicing at
the site it binds to in concert with Tra-2 and RBP1 (Goralski
et al., 1989; Hoshijima et al., 1991; Heinrichs and Baker,
1995, 1997). A classic target of Tra is the doublesex (dsx)
primary transcript, of which the fourth exon bears the bind-
ing sites for Tra. In the presence of a related protein, Tra-2,
Tra induces the female-type splicing which connects the
fourth exon to the third. In the absence of these proteins,
the fourth exon is spliced out and the fifth exon is connected
to the third (the male-type splicing) (Inoue et al., 1992).
Both the female-type and the male-type dsx transcripts
encode functional transcription factors, each of which acti-
vates or represses transcription of a different set of ‘realizer
genes’ required for the development of male-specific or
female-specific structures (Fig. 2).
4. Sexual behavior as a target of the sex-determination
cascade
Sexual behavior, and thus the underlying neural mechan-
isms, are indeed under the control of the sex-determination
cascade. For example, if tra-2 is inactivated in chromosomal
females (XX), which carry a temperature-sensitive tra-2
mutation, by rearing them at a restrictive temperature during
the pupal stage, a large proportion of the females who
emerge display male-typical courtship behavior towards
other females (Belote and Baker, 1987). It is surprising
that there are cases in which the tra-2 females showed
male-like courtship behavior even though they were
exposed to a restrictive temperature only after adult emer-
gence (Belote and Baker, 1987). This might indicate that the
neural system for male behavior and that for female beha-
vior coexist in the brain, and that one of these is activated (or
inactivated) by the constitutive action of the sex-determina-
tion gene products (e.g. Tra and Tra-2). Alternatively, it
might be that the critical period for determination of sexual
behavior types (i.e. male-like or female-like) persists into
early adult life, when practically no new neurons are pro-
duced (Ito and Hotta, 1992). In this case, the sex-determina-
137
tion gene products could determine the sex by the selective
elimination of inadequate neural circuits or the remodeling
of synaptic connections. No evidence is available to support
or reject these possible mechanisms.
5. Identification of a neural center for sexual orientation
by artificial sexual transformation of the brain site
An approach complementary to the use of loss-of-func-
tion mutations in the sex-determination genes is artificial
sexual transformation from male to female by means of
the forced expression of wild-type copies of these genes.
An elegant experiment was performed by Ferveur and his
colleagues (Ferveur et al., 1995) who induced trans-sexual-
ism in restricted regions of the brain with tra + , using the
targeted gene expression system (Brand and Perrimon,
1993). A surprising outcome of the experiment was that
the male flies with trans-sexual neurons display bisexual
courtship only when a given brain region is feminized
(Fig. 3). The critical region in the brain for the induction
of bisexuality is the antennal lobe, which is the primary
olfactory center in the insect’s CNS (Stocker et al., 1990;
Stocker, 1994; Smith, 1996; Tissot et al., 1997). They were
able to identify the glomeruli responsible for changes in
sexual orientation: when the DM2, DA3 and DA4 glomeruli
are feminized by the tra + action, the male flies express
bisexuality in courtship, regardless of the sex of other glo-
meruli in the antennal lobe (Ferveur et al., 1995). Expres-
sion of tra + in a part of the mushroom body to which the
antennal lobe neurons project, also induces bisexual court-
ship in the males (Ferveur et al., 1995; O’Dell et al., 1995).
These results indicate that neurons in the DM2, DA3 and
DA4 glomeruli are different in the two sexes, at least in their
responses to the males (presumably male odors; see Ferveur
et al., 1997) in the context of mating behavior. However, no
differences in the morphology of these glomeruli have been
reported between the sexes, nor has sexual dimorphism in
neurophysiology. It is worth noting that a male-specific
macroglomerulus has been identified morphologically, as
well as physiologically, in the moth antennal lobe. The
macroglomerulus provides the structural basis for the ‘hot-
line’ processing of information related to sex pheromones
(Matsumoto and Hildebrand, 1981). Analysis of cellular
geometry at the single neuron level might resolve morpho-
logical sexual dimorphism even in the antennal lobe of D.
melanogaster.
Bisexual courtship activity by males is known to result
from ubiquitous expression of the white (w) gene under the
control of the heat shock promoter (hs-mini-w + ) (Zhang and
Odenwald, 1995). w + encodes a tryptophan/guanine trans-
porter. The male-male interaction induced by hs-mini-w +
expression is maintained even after amputation of antennae,
maxillary palps or wings, indicating its central origin (Hing
and Carlson, 1996). Zhang and Odenwald (1995) suggested
that deprivation of tryptophan, the precursor of monoamine
138 D. Yamamoto et al. / Mechanisms of Development 73 (1998) 135–146

Fig. 1. The sex-determination genes and their regulation in Drosophila. Transcription of the female determinant, Sxl, is initiated by the ‘early’ promoter (PE)
in the embryo, only when the X chromosome/autosome (X/A) ratio exceeds one. Heterodimers of the transcription factors, e.g. the Daughterless (Da)-
Sisterless-b (Sis-b) complex activate transcription (shown by an arrow). The late promoter (PL) activates thereafter. The Sxl protein thus produced binds to
exon 3 of the Sxl primary transcript so as to be spliced out. The exons are indicated by boxes and those to be adjoined by splicing are connected by thin lines.
Exon 3 contains a stop codon resulting in a non-functional protein when transcribed in males. The Sxl protein also acts on the target gene tra in a similar
manner, leading to production of a Tra protein, functional only in females. The functional Tra protein promotes splicing by binding to the target sites on the
dsx and fru primary transcripts in females. As a consequence of differential splicing, male-specific (Dsx-M, Fru-M) and female-specific (Dsx-F, Fru-F)
proteins are produced. Colored portions indicate protein coding sequences. The existence of alternative usages of coding exons are indicated by different
colors.
 D. Yamamoto et al. / Mechanisms of Development 73 (1998) 135–146 139

Fig. 2. The Dsx proteins (upper panel) determine the sex of the gonads, genitalia, and external sexual characters such as pigmentation of the anal plate and
formation of the sex comb in the male, and some of the abdominal neuroblasts. The Fru proteins (lower panel) control development of the MOL and courtship
behavior through their action in determining the sex of certain neurons.

neurotransmitters, may occur as a consequence of ectopic
expression of w + in the CNS, resulting in homosexuality as
a behavioral phenotype. No evidence supporting or rejecting
this suggestion has been obtained.
6. The fru locus controlling male sexual orientation
There are mutations known to alter flies’ sexual orienta-
tion, a classic example of which is fruitless1 (fru1) discov-
ered by K.S. Gill in his screen for male sterile mutants (Gill,
1963). The males from the fru1 stock court not only females
but also males, although they never copulate with females.
Since 1963, several alleles of fru have been isolated. frusat
males court only males (Ito et al., 1996) whereas males
carrying the other fru alleles court both males and females
(Hall, 1978, 1994; Gailey and Hall, 1989; Taylor et al.,
1994). The fru gene encodes a set of putative transcription
factors with N-terminal BTB (Bric à brac, Tramtrack and
Broad-complex) domains and C-terminal Zn finger motifs,
characteristic of the BTB-Zn finger protein family (Ito et al.,
1996; Ryner et al., 1996; Yamamoto et al., 1996). Some of
the transcripts are present only in males or females. Analysis
of a cDNA corresponding to a female-specific transcript
showed that the open reading frame of the gene encoding
the BTB-Zn finger protein is preceded by a non-coding
140 D. Yamamoto et al. / Mechanisms of Development 73 (1998) 135–146
Fig. 3. Diagram of fru-expressing regions in the pupal CNS. The regions
with high levels of fru expression are shaded. The neural sites implicated
for the determination of male sexual orientation and courtship song gen-
eration, as well as the region at which the somata of MOL-innervating
motoneurons are located, are also indicated. The diagram was drawn based
on previous publications (Hall, 1979; Goralski et al., 1989; Ito and Hotta,
1992; Schneider et al., 1995) and on unpublished data by the authors.
exon, in which three putative Tra-binding sites are located
(Ito et al., 1996; Ryner et al., 1996). The putative Tra-bind-
ing sites contain a conserved 13-nucleotide repeat, which
was originally identified in the fourth exon of the dsx gene
(Burtis and Baker, 1989). The existence of these repeats
suggests that the fru gene is a direct downstream target of
Tra (Fig. 1). In contrast, the male-type counterpart of the
transcript does not contain the 13-nucleotide repeats, as the
exon containing the repeats is spliced out in males at a point
approximately 1600 nucleotides upstream of the splice site
for the female transcript. Due to this alternative splicing, the
male transcript can encode a polypeptide that has an exten-
sion of 101 amino acids beyond the N-terminus of the
female product (Ryner et al., 1996). The functional signifi-
cance of the N-terminal sequence unique to the males
remains to be elucidated.
Tissue localization of the fru transcripts suggests their
functions in the CNS (Fig. 2). Although fru transcripts com-
mon to both sexes are detected in many cells in the CNS and
various non-neural tissues, the expression of the sexually
dimorphic transcript is restricted to a fraction of CNS
cells: in one estimation, only about 500 of the roughly 105
neurons of the CNS give positive signals in in situ hybridi-
zation (Ryner et al., 1996). The cells expressing the sex-
specific transcript are found, for example, in the anterior
ventrolateral as well as in dorsal-posterior areas of the pro-
tocerebrum and in the ventral mesothoracic ganglion. Most
notably, a subset of cells in the antennal lobe shows the
highest level of expression of the sex-specific transcript.
As was already described above, the male antennal lobe
contains a structure that induces homosexual courtship
when feminized by means of GAL4-driven tra + expression
(Ferveur et al., 1995). It is thus tempting to speculate that
the tra+ action of inducing homosexual courtship is
mediated by the fru-expressing cells in the antennal lobe
(Yamamoto et al., 1996). Similarly, it is envisaged that
loss-of-function mutations of the fru locus would perturb
the sex determination of these cells, thereby altering male
sexual orientation (Yamamoto et al., 1996).
7. Development and evolution of the muscle of
Lawrence
In addition to the behavioral phenotype, the fru mutants
exhibit a particular muscular defect (Fig. 4). In wild-type
male flies, a pair of large dorsal muscle bundles known as
the muscle of Lowrence (MOL) is present in the fifth
abdominal segment (Lawrence and Johnston, 1984; Lawr-
ence and Johnston, 1986). The MOL is absent in females
and in the larvae of both sexes. Male flies carrying some of
the fru alleles (e.g. frusat, fru3 and fru4) lack the MOL (Gai-
ley et al., 1991; Taylor, 1992; Hall, 1994; Taylor et al.,
1994; Taylor and Knittel, 1995; Ito et al., 1996). In the
flies carrying the fru1 and fru2 alleles, the muscle fiber length
of the MOL is shortened to half, and the number of nuclei in
the cells of the MOL fibers is greatly reduced.
An interesting feature of the MOL is that not all Droso-
phila species have this muscle: among 95 species of Droso-
philinae examined, 67 lack the MOL (Gailey et al., 1997b).
Since the presence or absence of MOL does not correlate
with the phylogeny of the species and because the ancestral
genera Chymomyza and Scaptodrosophila include species
which have the MOL, it was suggested that the MOL was
an anatomical structure that has undergone phylogeny-inde-
pendent, repeated loss among closely-related evolutionary
lines. Hybrid males generated by interspecific crosses of an
MOL-bearing species (mauritiana) and an MOL-lacking
species (either yakuba or teissieri) possess the MOL, indi-
cating that a genetic factor (or factors) in the mauritiana
autosomes is responsible for the formation of the MOL. An
obvious candidate for such a genetic factor is fru. It remains
to be determined whether any change in fru expression or
Fru function parallels the loss of MOL in Drosophilid spe-
cies. Interestingly, individual variations in MOL develop-
ment were demonstrated in two MOL-bearing species,
Scaptodrosophila lebanonensis and Drosophila miranda,
where a male lacking the MOL and one having a unilateral
MOL, respectively, were found. This type of variability in
MOL development within a species might reflect the weak-
ening of the genetic (fru?) influence on the development of
the MOL in these species.
Male-specific formation of the MOL does not depend on
 D. Yamamoto et al. / Mechanisms of Development 73 (1998) 135–146 141

Fig. 4. The muscle of Lawrence (MOL). Adult dorsal longitudinal muscles are shown for a Canton-S wild-type male (a), a Canton-S wild-type female (b), a
frusat male (c), a fru3 male (d), a frusat/fru3 male (e) and a frusat-rev2 male (f). The MOL is absent in frusat, fru3 and frusat/fru3 males, but is present in frusat-rev2
males. frusat-rev2 is a revertant where the P-element in frusat is removed by precise excision. The existence of the MOL in frusat-rev2 males demonstrates the
causality between the P-element insertion and expression of the muscle phenotype. The lack of the MOL in frusat/fru3 males demonstrates that frusat is indeed
allelic to fru3.

the sex of the myoblasts which give rise to this muscle
(Lawrence and Johnston, 1984, 1986; Kimura et al.,
1994). Three independent experiments demonstrated that
this cell non-autonomous fate decision occurs in the MOL
myoblasts. First, male nuclei transplanted into a female
embryo do not give rise to the formation, in most cases,
of the adult MOL, whereas female nuclei introduced into
a male embryo occasionally give rise to the formation of the
MOL (Lawrence and Johnston, 1986). This result suggests
that some cells other than the myoblasts govern the MOL
fate. Two tissues that are in direct contact with the MOL are
suspected to be the origin of the signal for MOL induction:
the cuticles to which the MOL attaches and the motor nerve
that innervates the MOL. Since the MOL is formed in
gynandromorphs which have the female cuticle at the
MOL attachment site, the cuticles (or the epidermis that
secretes the cuticles) are unlikely to be a source of the
inductive signal for MOL formation (Lawrence and John-
ston, 1986). On the other hand, the sex of the motoneurons is
always male, as judged by a sex-linked enzyme marker,
when the MOL was formed in the nuclear-transplantation
experiment (Lawrence and Johnston, 1986). Based on these
results, Lawrence and Johnston (1986) proposed the sex of
the innervating neurons as the determinant of the MOL fate.
The second experiment that showed the cell non-autonomy
of MOL formation was carried out by Kimura et al. (1994)
who transplanted female myoblasts from the wing disc into
the male abdomen and confirmed that the female cells con-
tribute to the development of the MOL. The third conclusive
experiment, by Currie and Bate (1995), involves denerva-
tion of the MOL-innervating nerve at the onset of metamor-
phosis, resulting in the selective loss of MOL from the
operated male flies whilst allowing other muscles in the
same segment to develop normally. Thus, it is concluded
that the MOL is formed when the innervating motoneurons
are male regardless of the sex of myocytes forming the
MOL.
Since MOL formation primarily relies upon the sex of
innervating motoneurons, it is conceivable that the fru
mutant MOL phenotypes are entirely ascribable to fru’s
effect on the sex determination of the motoneurons. Indeed,
loss-of-function mutations in the upstream elements of the
sex-determination cascade, such as those in tra and tra-2,
lead to ectopic development of the MOL in the mutant
females, suggesting that MOL formation is actively
repressed in the female (Taylor, 1992). In keeping with
the idea that fru and dsx each contributes to a mutually-
exclusive subdivision of the sex-determination cascade
downstream of tra and tra-2, MOL formation is unaffected
by mutations in dsx (Taylor, 1992). These observations are
consistent with the hypothesis that the primary role for fru is
to determine the sexual fate of a subset of neurons, femin-
ization of which in males, as a result of fru mutations, has
phenotypic consequences such as homosexual courtship
(presumably as a result of inadequate sexual determination
of the antennal lobe neurons) and malformation of the MOL
142 D. Yamamoto et al. / Mechanisms of Development 73 (1998) 135–146
(as a result of sexual transformation of abdominal moto-
neurons).
8. fru versus dsx: neuronal sex determination
As in the case of the antennal lobe, the motoneurons that
are subject to fru-dependent sex determination have not
been identified conclusively. It is, however, evident that
not all of the sexually dimorphic neurons in the abdominal
ganglion are under the control of fru (Fig. 2). The most
conspicuous sexual dimorphism in the D. melanogaster ner-
vous system is found in the terminal abdominal region of the
fused ganglion (Taylor, 1989a,b). In total, 12 neuroblasts
are mitotically active in the wandering larvae in this region
(Taylor and Truman, 1992). Four of these display a sexually
dimorphic pattern of proliferation: in females they become
quiescent during the third instar, while in males these neu-
roblasts continue to divide until 12 h after white puparium
formation. Interestingly, this sex-dependent difference in
the mitotic pattern of the terminal neuroblasts depends on
the integrity of the dsx gene (Taylor and Truman, 1992).
Loss-of-function dsx mutations prevent the neuroblasts
from dividing during the third instar, rather than leading
to the ‘intersex’ situation. Regarding the dsxD mutation
placed over a deficiency for the dsx locus, the male-type
dsx transcript is expressed in a chromosomal female in the
absence of the female-type dsx transcript. Under such con-
ditions, the four neuroblasts remain mitotically active even
after pupation, as in the case of males. When the XX
females are heterozygous for dsxD (i.e. dsxD/dsx + ), so that
they produce both male-type and female-type dsx tran-
scripts, four terminal neuroblasts are found, as in wild-
type females. However, one of the four neuroblasts typically
continues to produce neurons after pupation in the dsxD/
dsx + females. In other words, there is a neuroblast which
behaves like a male cell, along with cells that follow the
female-type division cycle, when the male-type dsx tran-
script is expressed in addition to the female-type dsx tran-
script, in females. Based on these findings, it is concluded
that the product of the dsx male transcript masculinizes the
terminal neuroblasts, whereas that of the dsx female tran-
script feminizes them in terms of their division cycle. In the
absence of the expression of either transcript, the terminal
neuroblasts fail to divide.
As expected, loss-of-function mutations in tra and tra-2
induce a male-like division pattern in the female terminal
neuroblasts. What was not expected was the fact that the
tra-2 + product is not necessary during the period when the
sexually dimorphic cell divisions actually take place, but is
required to predispose the neuroblasts to either male-typical
divisions or female-typical divisions early in development
(Taylor and Truman, 1992). For instance, the terminal neu-
roblasts in tra-2ts females exposed to a restrictive tempera-
ture at the embryonic stage follow the male cell-division
program. If the tra-2ts females are reared at a permissive
temperature until the end of the first instar and then sub-
jected to a restrictive temperature, the terminal neuroblasts
adopt the female cell-division program in the third larval
instar. When the tra-2ts females experienced the shift to a
restrictive temperature during the first instar larval stage,
two of the terminal neuroblasts behaved like male cells
and the remaining two like female cells. This means that
the terminal neuroblasts can respond to the sex-determina-
tion signal only before the first instar.
The early determination of sexual fates in these neuro-
blasts is in contrast with the late determination of sexual
behavior types (Belote and Baker, 1987), which are inde-
pendent of dsx but probably depend on fru (Taylor et al.,
1994). It should be pointed out, however, that male sexual
behavior has aspects that are influenced by dsx (Villella and
Hall, 1996): (i) dsx mutant males generate courtship songs
composed only of the pulse song, and (ii) dsx males tend to
court other males more often than wild-type males do. The
male-male interactions occurring between dsx mutants
might be explained by the female-like profile of their sex
pheromone contents, which chemically stimulate them to
court other (male or female) flies. On the other hand, the
reported absence of sine songs in dsx mutant males seems to
reflect the dsx contribution to neural sex-determination in
the song-generating circuit. As it has been observed that
some of the fru alleles are associated with song defects
(Hall, 1994), the neural machinery for the production of
courtship songs appears to be formed by interconnections
between the dsx-dependent neurons and the fru-dependent
neurons.
In this context it should be noted that the existence of
considerable variation in fru-dependent neurogenesis
among species is inferred from comparative studies of
MOL (Gailey et al., 1997b). Inter-species differences are
indeed an important characteristic of the courtship songs
(Wheeler et al., 1991; Tomaru and Oguma, 1994). By ana-
logy with MOL formation, fru might play a role in rendering
the song circuit species-specific. An even more provocative
idea is that dsx contributes to the ‘hard-wired’ sex determi-
nation of vital mechanisms, whereas fru provides flexibility
in the sexual behavior of male flies that might be profitable
for them in sexual selection among conspecific males or in a
competitive interaction among heterospecific males. Studies
on the relative contributions of dsx and fru to the formation
of the song pattern generator in different species would
provide insights into the molecular mechanism whereby
sexually dimorphic species-specific characteristics develop
during evolution.
Recently a new mutation, he’s not interested (hni) was
identified, based on the low level of the courtship of females
by males (Friedman et al., 1995). Interestingly, hni is asso-
ciated with a muscle phenotype, as is fru (Gailey et al.,
1997a): the number of fibers comprising the MOL is
decreased, whereas the fiber number is increased in other
abdominal longitudinal muscles. The hni muscle phenotype
is more marked in heterozygotes than in homozygotes,
 D. Yamamoto et al. / Mechanisms of Development 73 (1998) 135–146
implying that the hni mutation is antimorphic (Gailey et al.,
1997a). Genetic experiments need to be carried out to deter-
mine whether the hni mutation genetically interacts with the
fru mutant.
9. Is sexual satisfaction guaranteed by the sex-
determination genes?
The above observations demonstrate that the sex-deter-
mination cascade has a bipartite pathway downstream of
Tra: one uses Dsx and the other Fru as the transcriptional
regulator of target genes (Ryner and Swain, 1995). How-
ever, a recent study suggests a third branch in the sex-deter-
mination cascade downstream of tra.
The gene functioning in the postulated third branch is
dissatisfaction (dsf), which was recognized as a result of
the aberrant mating behavior of the flies carrying mutations
in this gene (Finley et al., 1997a,b). The dsf mutant pheno-
types manifest themselves in both males and females. First,
the dsf males vigorously court, in addition to females, other
males, forming a short chain of courtees. The dsf males
even show advanced behavior, such as attempted copula-
tion, towards male flies. Second, the time to copulation is
vastly prolonged when the dsf males are involved. This
phenotype appears to result from poor bending of the abdo-
men in mutant males: deep bends of up to 180°, required
for successful copulation, occur three times less often in
dsf than in wild-type males (Finley et al., 1997a). Some
dsf males, however, copulate with females, producing off-
spring.
Female dsf mutants exhibit subnormal receptivity. When
a dsf female is placed with a wild-type male in an observa-
tion chamber, the female displays active resistance to the
courting male, with decamping, wing-flicking and kicking
(Finley et al., 1997a). A similar enhancement of mate refu-
sal in females has been reported to occur in flies carrying
mutations in the spinster (spin) (Suzuki et al., 1997) and
chaste (cht) (Yamamoto et al., 1997) loci, the epistasis of
which with dsf remains to be established. In addition to the
reduced receptivity, the dsf females suffer from a defect in
egg-laying (Finley et al., 1997a). Although eggs are pro-
duced normally and passed down to the uterus, they are
not fertilized with mobile sperm stored in the seminal recep-
tacle and spermathecae in females. Such eggs finally degen-
erate in the uterus.
Although the neurobiological bases for homosexual ten-
dencies in male and reduced receptivity in female dsf
mutants are not determined, insufficient bending of the
male abdomen and the oviposition defect in females are
known to have anatomical correlates in the peripheral ner-
vous system (Finley et al., 1997a). The motoneurons inner-
vating the ventral longitudinal muscles in the A5 segment of
dsf males form a few enlarged spherical boutons that are
distinctly different from the slender, cascading boutons
found in the corresponding synaptic sites in wild-type
143
male flies. Such aberrant synaptic boutons are not observed
on other muscles in dsf males nor on any muscles (of those
in the A5 segment) in dsf females.
In the dsf females, the circumferential muscles of the
uterus lack motor innervation, in contrast with the wild-
type counterparts, which are heavily innervated. The lack
of motor nerve endings on the uterine muscles is probably
responsible for the defective oviposition in dsf females.
Interestingly, the muscles of other reproductive organs,
including the spermathecae, sperm receptacle and oviduct
exhibit normal motor innervation in dsf females.
These phenotypic analyses suggest that requirement of
the wild-type product of the dsf gene is confined to a subset
of neurons in the nervous system, although the cell auton-
omy of dsf function has not been firmly established. It is also
clear that dsf functions in sexually dimorphic structures.
Such sexual phenotypic dimorphism implies that dsf is an
element in the sex-determination cascade. Evidence for this
was provided by the experiment in which the effect of the
dsf mutation was determined in chromosomal females after
sexual transformation with tra − (Finley et al., 1997a). In the
dsf + ;tra − XX flies, the ventral abdominal muscles were
innervated by normal-looking boutons, as was the case in
wild-type males (XY dsf + ). In contrast, the corresponding
muscles in dsf − ;tra − XX flies were associated with enlarged
round boutons, just like those seen in mutant males (XY
dsf − ). The expression of the dsf mutant phenotype depends
on tra due to the fact that dsf is located downstream of tra in
the sex-determination cascade. In accordance with the idea
that dsf is downstream of tra, a moderate reduction in tra +
activity in females (XX; tra − ;hs-tra + ) eliminates motor
innervation of the uterine muscle, inducing a dsf phenotype.
An intriguing observation is that the dsf mutant females
masculinized by dsxD, a mutation whereby only the male-
type transcript is constitutively produced, have normal
motor endings on the abdominal muscles in segment A5
(Finley et al., 1997a). Therefore, the dsf mutation expresses
its phenotype in the absence of the dsx, i.e. dsf is not down-
stream of dsx.
This observation raises the question whether dsf is an
element in the fru-mediated pathway that is tra-dependent
and dsx-independent. The answer to this question is, appar-
ently, no, because the dsf mutations do not affect MOL
formation, which is under fru control. This means that dsf
represents the third branch of the sex-determination cascade
downstream of tra.
10. Conclusions
In this article, we have reviewed recent advances in the
study of some mutations that alter flies’ sexual behavior,
especially male sexual orientation and female sexual recep-
tivity. Two loci, fru and dsf, have been extensively studied.
It has been found that both Fru and Dsf represent novel
elements of the sex-determination cascade, in which they
144 D. Yamamoto et al. / Mechanisms of Development 73 (1998) 135–146
most probably function as transcription factors controlling
the expression of different sets of target genes, depending
on sex (Finley et al., 1997b for Dsf). Contrary to the accu-
mulated knowledge regarding splicing regulation in the sex-
determination cascade upstream of dsx, mechanisms by
which tra and tra-2 control the transcription of fru and dsf
have not been firmly established. In the case of fru, a puta-
tive Tra-binding site is identified in one of the transcripts
which is female specific. Binding of Tra would promote
splicing at this site in females, as is the case for dsx
female-type splicing (Ryner et al., 1996). However, it is
not clear whether sex-dependent splicing is the sole
mechanism for the production of sex-specific transcripts
of fru. Recent analysis of the mechanism of gene dosage
compensation (Kelley et al., 1997) revealed that binding of
Sxl to the target sites in the 5′ and 3′ untranslated exons of
the male specific lethal-2 (msl-2) primary transcript blocks
its translation in females, thereby preventing undesired acti-
vation of X-linked genes. The question remains as to
whether a similar post-transcriptional regulation plays a
role in the production of the sex-specific Fru proteins.
The targets of the Dsf and Fru proteins have not been
identified. The suspected targets include genes encoding
proteins involved in neuronal fate determination, axonal
path finding, and/or recognition and adhesion for formation
of cell-to-cell contacts. Such proteins could be used in the
establishment of the neuronal architecture common to both
sexes. However, those proteins that can be recruited in a
sex-dependent manner when activated (or inactivated) via
the actions of transcription factors in the sex-determination
cascade, such as Fru, remain to be identified.
Although nothing is known about the targets of, and their
regulation by, Fru or Dsf, mechanisms of transcription acti-
vation and repression by Dsx have been studied in some
depth. The best-studied target for Dsx is the yolk protein 1
(Yp1) gene, the expression of which in the fat bodies is
activated by the female-specific DsxF protein and repressed
by the male-specific DsxM protein (Coschigano and Wen-
sink, 1993; An and Wensink, 1995a,b). DsxF and DsxM
interact with the o-r enhancer for Yp1 that is composed of
four binding sites, dsxA, aef1, bzip1 and ref1 (An and Wen-
sink, 1995b). DsxF and DsxM bind to the dsxA site, which is
situated between aef1 and bzip1 with partial overlap, after
forming homodimers via the N-terminal sex-independent
OD1 domain and the C-terminal sex-specific OD2 domain
(An et al., 1996). Binding of DsxF to the dsxA site pushes
out the AEF1 repressor, thereby activating transcription of
Yp1 in females. Binding of DsxM not only excludes the
AEF1 repressor but also the bZIPa activator protein from
the enhancer sequences, keeping Yp1 transcription
repressed. This is the principal mechanism of the sexually
dimorphic transcriptional regulation of Yp1 by Dsx. Inter-
estingly, the tissue specificity of Yp1 expression is also
ascribed to the action of the same enhancer sequences:
although the bzip1 site or the ref1 site alone drives expres-
sion in ovarian somatic cells and all non-gonadal tissues,
together they activate transcription only in fat bodies (An
and Wensink, 1995b).
These facts tell us that a combinatorial nature of tran-
scriptional regulation underlies the complex sex and tissue
specificity seen in the expression of ‘realizer’ genes (e.g.
Yp1). It is anticipated that a strategy similar to the Dsx-
mediated transcriptional regulation would be adopted in
the control of the transcription of downstream genes by
Fru. Identification of the in vivo targets for Fru is indispen-
sable for thorough understanding of the molecular basis of
the sex-specific transcriptional regulation by this protein.
Regulation of transcription of its target genes by Fru
seems to be finely tuned with respect to time and place,
judging by the localized, dynamic pattern of fru expression
and from the very specific phenotypes of fru mutations. The
fru and dsf genes are required only in a limited number of
neurons for determination of their (neuronal) sexual fate and
subsequent sexually dimorphic development during the
pupal and presumably adult stages. Thus analysis of Fru-
and Dsf-mediated transcriptional regulation of the target
genes will provide new information on the developmental
biology of sex-linked traits dealing with the fundamental
questions as to how individually-identifiable neurons in
the nervous system can be generated in a sexually dimorphic
way and how these neurons choose one of the binary pro-
grams that allows the neurons to develop the cellular geo-
metry unique to the two sexes.
Acknowledgements
We thank the members of the Yamamoto laboratory for
their comments on drafts of the manuscript. We also thank
Sachiko Kondo for secretarial assistance.
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