fruitless
Fruitless has been postulated to be a neural sex determination factor that directs development of the central nervous system (CNS), thereby producing male-typical courtship behaviour and inducing male-specific muscle. Male-specific Fru protein is expressed in small groups of neurons scattered throughout the CNS of male, but not female. Collectively, these observations suggest that Fru 'masculinizes' certain neurons, thereby establishing neural substrates for male-typical behaviour. However, specific differences between neurons resulting from the presence or absence of Fru are unknown. Previous studies have suggested that Fru might result in sexual differences in the CNS at the functional level, because no overt sexual dimorphism in CNS structure was discernible. This study identifies a subset of fru-expressing interneurons in the brain that show marked sexual dimorphism in their number and projection pattern. This study also demonstrates that Fru supports the development of neurons with male-specific dendritic fields, that are programmed to die during female development as a result of the absence of Fru. Thus, Fru expression can produce a male-specific neural circuit, probably used during heterosexual courtship, by preventing cell death in identifiable neurons (Kimura, 2005).
Assuming that Fru is involved in development of neural circuits crucial for the generation of male-typical behaviour, it is plausible that Fru-expressing neurons show some degree of sexual dimorphism structurally and/or functionally. Unambiguous identification of Fru-expressing neurons is necessary for evaluating this possibility. In a search for enhancer trap lines that express Gal4 in Fru-expressing cells, a conspicuous sex difference was found in the localization of the Gal4 reporter mouse (m)CD8-GFP (green fluorescent protein) in the NP21 strain. The NP21 strain has a P-element insertion in the second intron of the fru gene (Kimura, 2005).
The following histological experiments were carried out primarily with flies heterozygous for the described P-element insertion. Sexually dimorphic reporter expression in NP21 flies was found in two discrete locations: the optic lobe and the region dorsal to the antennal lobe. In the optic lobe, a subset of neurons in the distal medulla expressed mCD8-GFP only in males. In the region dorsal to the antennal lobe, approximately 30 neurons expressed mCD8-GFP in males, compared with approximately 5 neurons in females. The somata of these neurons form a cluster called 'neurons medially located, just above antennal lobe', or mAL (Kimura, 2005).
To identify projection patterns of mAL neurons without interference from
surrounding neurons, the MARCM method, which allows mCD8-GFP to be expressed in a single precursor cell and/or its progeny, was used. Somatic chromosomal recombination is induced during development by the stochastic action of a heat-shock-inducible flippase. By changing the timing of the heat-shock, chromosomal recombination can be induced at different stages of development, resulting in a different number of mCD8-GFP-expressing cells (Kimura, 2005).
Early heat-shock resulted in mCD8-GFP expression in about 30 cells in male flies and 5 cells in female flies, indicating the clonal nature of the mAL cluster. In males, the mAL neurons extended neurite projections bilaterally. In females, the neurons had only contralateral projections. In both sexes, the contralateral neurite bifurcated after crossing the midline, with one neurite extending dorsally and terminating in the superior lateral protocerebrum, and the other neurite extending ventrally, terminating in the suboesophageal ganglion. The male-specific ipsilateral neurite also terminated in the suboesophageal ganglion. When single-cell labelling was carried out, the 5 cells in females were devoid of this ipsilateral projection. A further difference between the sexes was observed in the branching pattern of these neurons in the suboesophageal ganglion: the mAL neurons had forked arborization only in females. The forked arborization typical of female mAL neurons was never observed in males. However, the male mAL had two classes of neurons: one with bilateral projections and the other with only contralateral projections. It was possible to differentiate 8 types among male neurons on the basis of their shape (Kimura, 2005).
The terminal structures in the superior lateral protocerebrum were decorated with many varicosities that were nearly absent from the terminal structures of branches in the suboesophageal ganglion. This arrangement suggests that the superior lateral protocerebrum is the output site of these interneurons, and the suboesophageal ganglion is the input site. This hypothesis is supported by the observation that a presynaptic marker (synaptotagmin-HA) was transported to the terminals in the superior lateral protocerebrum but not to those in the suboesophageal ganglion (Kimura, 2005).
How the timing of somatic recombination by heat-shock application affected the number of labelled neurons was investigated. When a heat-shock was applied to the embryo, the maximal number of mAL neurons was labelled (that is, about 30 in males and 5 in females, indicating that the neuroblast that produced the sexually dimorphic neurons was proliferating in the embryonic stage in both sexes. In contrast, heat-shock activation at the late third instar larval to early pupal stage (that is, 4-5 days after egg collection) failed to label any mAL cells in females, but did result in labelling in males. The number of labelled cells in these cases was very low (typically one cell), but indicates that the neuroblast continued to proliferate in males at this stage. These observations are compatible with any of three alternative explanations for the production of fewer neurons in females compared with males: (1) the female neuroblast stops dividing and becomes quiescent during the late third instar larval to early pupal stage; (2) the female neuroblast dies earlier than the male neuroblast; (3) the female neuroblast continues to proliferate as the male counterpart does, but the daughter cells are eliminated by programmed cell death only in females. To differentiate the second and third possibilities from the first, the effect of mutations that block cell death was examined (Kimura, 2005).
head involution defective (hid, also known as Wrinkled or W), grim and reaper (rpr) are the predominant cell-death genes in Drosophila. They are aligned in tandem in the genome, and therefore can be removed by a single deficiency, Df(3L)H99. In viable rpr mutants of Df(3L)H99/Df(3L)XR38, the mAL cluster had, on average, 10 mAL neurons in females and 30 in males. This result is consistent with the idea that, in females, rpr-mediated cell death eliminates some cells that otherwise contribute to the mAL cluster. In contrast, viable hidA206/Df(3L)H99 mutants had approximately 5 mAL neurons in females and 30 in males. To observe the effect of removing hid, grim and rpr simultaneously, cells homozygous for Df(3L)H99 were generated in a heterozygous background by the MARCM method [a homozygous Df(3L)H99 deficiency causes lethality]. Now the mutant females had as many as 29 mAL neurons. Again, the number of mCD8-GFP-positive cells in mutant males was about 30. These results show that programmed cell death occurs specifically in female flies, reducing the number of mAL neurons (Kimura, 2005).
It was predicted that the cell death programme was probably operating in the daughter cells rather than in the neuroblast itself. This is because Df(3L)H99 homozygous neurons could be produced and labelled by heat-shock-induced activation of recombination at the late larval to early pupal stage even in females, which normally reveal no sign of labelling at this stage. Thus, in wild-type females, the cell death mechanism eliminates late-born neural progeny, whereas in neural progeny homozygous for Df(3L)H99, these cells survive and differentiate into neurons (Kimura, 2005).
When cell death was inhibited at the embryonic stage by heat-shock-induced recombination of the Df(3L)H99-harbouring chromosome, the number of mAL neurons was increased in adult females. Such neurons had the contralateral forked arborization typical of females. Notably, the ipsilateral projections never seen in normal females were formed in those female mAL neurons protected by the Df(3L)H99 deletion. A single neuron with bilateral projections typical of males was stained in a female mAL cluster homozygous for Df(3L)H99. The contralateral projection of the neuron showed neither the typical female nor the typical male arborization pattern. This indicates that sex-specific cell death is not the sole mechanism for establishing sexually dimorphic neuronal projections, since the pattern of H99-protected neurons in the suboesophageal ganglion was not necessarily male-like (Kimura, 2005).
The NP21 strain has a P-element insertion in the second intron of the fru gene. Across the entire male brain, >80% of Gal4-expressing cells expressed Fru. All of the mAL neurons expressing mCD8-GFP in males were Fru-positive. NP21 homozygous males and NP21/frusat transheterozygous males showed similar abnormalities: loss of the male-specific muscle of Lawrence, no expression of Fru and no detectable expression of intact male-specific fru transcripts by polymerase chain reaction with reverse transcription (RT-PCR) analysis. Markedly reduced courtship behaviour towards males as well as females is also observed. These results show that NP21 is a loss-of-function allele of fru (Kimura, 2005).
Whether mutations in fru could affect the sexually dimorphic development of the mAL cluster was examined. NP21/frusat males had only about 5 mAL neurons, a number comparable with that in females. The males of two other fru mutants also showed a significant reduction in the number of mAL neurons. The similar reduction in the number of mAL cells in fru mutant males was confirmed by cell labelling using the MARCM method. Single-progeny labelling was performed in NP21/NP21 flies, and about 5 mAL neurons were counted in male flies (Kimura, 2005).
In NP21 homozygous males, no neurons with ipsilateral projection were observed. Instead, all mAL neurons in NP21 homozygous males had contralateral, bifurcating projections reminiscent of their female counterparts. For males of other heteroallelic fru mutants, most mAL neurons had 'female-like' unilateral projections, but there were also neurons that had the extended ipsilateral projection typical of males. It should be emphasized that the forked projections typical of females were formed in male fru mutants. To determine whether the Fru protein is required cell-autonomously in mAL neurons for male-typical development, NP21/NP21 homozygous cells were generated in male flies heterozygous for NP21/ + . mAL neurons with the NP21/NP21 genotype had forked projections typical of females, indicating that cell-autonomous fru+ function is necessary for male-type development of mAL neurons (Kimura, 2005).
According to the hypothesis that Fru suppresses cell death specifically in males, Fru expression in female mAL neurons should inhibit the death of mAL neurons in females. The experiments show that this is indeed the case: tra1 homozygous females express Fru in mAL neurons with male-like bilateral projections, which also have male-like arborizations in the suboesophageal ganglion. Thus, the tra1 mutation not only blocks cell death but also masculinizes the neuron branching pattern, presumably by inducing Fru expression in female mAL neurons. Such tra1 mutant females vigorously courted partner females (Kimura, 2005).
This study has demonstrated that Fru expression is essential for the establishment of a 'male-typical' neural network. The observations support the model that those neurons programmed to extend male-typical projections are eliminated in females by cell death. Fru inhibits cell death in male neurons at the pupal stage, allowing neurons to develop male-typical projections. A similar mechanism would operate in the formation of sexually dimorphic neurons in the optic lobe. Fru could thus be considered to function as a male-female switch in the CNS neural circuit (Kimura, 2005).
The male-typical projections of mAL neurons in the suboesophageal ganglion have terminals that probably function as input sites. The suboesophageal ganglion is known to include the association centre for gustation, and the central projections of both tarsal and labellar gustatory neurons terminate in the suboesophageal ganglion. Contact (gustatory) pheromones are also known to be involved in partner choice in Drosophila mating. Thus, an intriguing possibility is that the male-typical projections of mAL neurons in males act as the input sites of pheromonal information conveyed by gustatory neurons. The enhanced homosexual preference observed in fru mutant males might be due to a failure in integrating the pheromonal information crucial for partner recognition in mating behaviour, as a consequence of the loss of ipsilateral projections from the mAL neurons. The study of Fru-expressing neurons and their circuits may therefore help to identify fundamental principles about the construction of sex-specific structures and functions in the CNS (Kimura, 2005).
In both sexes, only about 500 of the roughly 100,000 neurons of the CNS label with an mRNA probe: there is no labeling in other tissues of the body. In both sexes, labeled neurons are found most commonly as small groups and less frequently as single cells; most labeled cells are distributed in similar areas of the brain and ventral nerve cord of males and females. A set of nine groups of labeled neurons, ranging from 10-30 cells is detected in the CNS of males in positions likely to be involved in particular male courtship behaviors. Comparably positioned cells are found in six of these locations in females.
A set of two groups of labeled neurons is detected in areas that must have a male identity for flies to perform particular steps in male courtship. One is a prominently labeled group of cells in the dorsal-posterior protocerebral region; early steps in courtship (following and wing extension) map to this region using gynandromorphs. The second is a group of cells located in the ventral mesothoracic ganglion in males (but not females), an area to which courtship song has been mapped. FRU positive neurons are also found in areas not known to be involved in sexual behavior, such as three groups of neurons in the anterior ventrolateral protocerebrum. These are found associated with higher order sensory processing neuropils of the CNS. There is also a male-specific neuronal cluster in the abdominal ganglion, a ganglion that controls abdominal movements during copulation (Ryner, 1996).
The small number and the locations of the neurons expressing male-specific transcripts suggest that fru is directly involved in only some of the sensory and motor systems necessary for male sexual behavior. For example, the sex-specific transcripts of FRU are abundantly expressed in a group of primary sensory interneurons in the antennal lobe involved in the processing of chemosensory information. These appear to be relay interneurons, known to project to higher brain centers such as the calyx of the mushroom body.
One unexpected finding is that some fru expressing cells are detected only in females (for example, in the subesophageal ganglion), yet no female behavioral phenotypes of extant fru mutations have been detected. Often the intensity of hybridization also differs between the labeled neurons in male versus female CNSs. Other tissues labeled by a common coding-region probe are a subset of direct flight muscle cells, gonadal nurse cells, spermatocytes, fat body cells, gut epithelium, and ectodermal epithelium, including the internal and external genitalia (Ryner, 1996).
The Drosophila fruitless gene product Fru has been postulated to be a neural sex-determination factor that directs the development of at least two male-specific characteristics, namely courtship behavior and formation of the muscle of Lawrence (MOL). The fru gene possesses consensus Tra-binding sequences, and encodes a putative transcription factor that has a BTB domain and two zinc-finger motifs. The binding of Tra to the Tra-binding sequences results in sex-specific alternative splicing of the FRU mRNA, leading to production of the 'male-type' or 'female-type' Fru protein. The Fru protein is not detected in the female central nervous system (CNS), despite the similar level of expression of FRU mRNA in both male and female CNS. Since ectopic expression of both the 'male-type' (with the sequence for the amino-terminal extension) and 'female-type' (without the sequence for the amino-terminal extension) fru cDNA can induce formation of the MOL in females, the presence or absence of the Fru protein, and not its sex-specific structure, seems to be responsible for the sexually dimorphic actions of the fru gene (Usui-Aoki, 2000).
The fru gene has been reported to have four promoters, two of which
generate the 5' ends of the different transcripts identified in this
study. The most distal promoter is required for the transcription of the exon
that undergoes sex-dependent splicing. As a result of the sex-dependent splicing,
the N terminus of the male-specific protein is expected to have a unique extension
of 101 amino acids, followed by the BTB domain, which is
shared by all Fru isoforms. This is because the female exon
II has a stop codon that prematurely truncates the open reading frame (ORF),
which begins near the 5' end of exon II, whereas this stop codon is
spliced out in the male exon 2. There
is no evidence for or against the possibility that the truncated ORF is transcribed
to produce a peptide of 94 amino acids in females. The possibility cannot be excluded that translation starts from the second AUG that is present 113 base
pairs (bp) downstream of the first AUG of exon 2, allowing the transcript
to encode an ORF of 81 amino acids, with a one-base frameshift relative to
the ORF defined by the first AUG. In addition to the N-terminal variants,
five Fru isoforms with variations in their carboxy terminus have been identified. Three of these contain two zinc-finger motifs,
each of which is encoded by a different exon. The remaining two do not have
any zinc-finger motifs. Northern blot analysis of the wild-type head extracts
with a DNA probe for the BTB domain detects at least three sets of transcripts
differing in size between males and females, and two transcripts that show
no such sex difference. In contrast,
using the same DNA probe, this sexually dimorphic pattern of transcripts is
not detected in fru-mutant homozygotes (Usui-Aoki, 2000).
frusat and fru3 represent strong
viable alleles, which in males lead to flies showing male preference in courtship
and complete lack of formation of the MOL. When the second exon sequence is
used as a probe, a sex difference in the size of the transcripts has been
demonstrated, even in frusat homozygotes. Since the frusat mutation was induced
by a P-element insertion into the second large intron, the aberrant transcripts
found in frusat homozygotes appear to represent chimaeric
products of the fru gene and the P-element vector sequence; this was
subsequently confirmed by reverse transcription polymerase chain reaction
(RT-PCR). The second exon of the fru
gene, which undergoes sexually dimorphic splicing, is spliced so as to fuse
with the P-element sequence, resulting in the termination of transcription
in the vector. The second aberrant mRNA in frusat
is transcribed from the mini-white promoter in the vector, and spliced
to the downstream fru exon that encodes the BTB domain. Due to the
dicistronic organization, however, translation of functional
proteins from this aberrant mRNA cannot be expected. Thus, P-element insertion
into the second intron inactivates the sexually dimorphic transcripts in the
frusat mutant (Usui-Aoki, 2000).
To determine the expression pattern of the fru gene product in the
nervous system by immunohistochemical analysis, anti-Fru antibody was generated.
Attention was focused on the expression of the Fru protein during the
pupal stage, during which the protein is required for normal courtship after
emergence, as well as for the development of the MOL. The antibody
stains a proportion of the cellular nuclei in the brain and ventral ganglia
in wild-type males. A significant proportion of the Fru-expressing cells in the CNS are neurons, since they were also immunopositive for the anti-Elav antibody, a neuron-specific marker. Fru-expressing neurons form discrete clusters in various regions of the brain. In the mid-pupal stage (72 h after puparium formation, APF), 59-69
cells per hemisphere are stained by the anti-Fru antibody in the posterior
brain. These dorsal neurons do not contribute to the structure of the mushroom
body, as revealed by a double-labelling experiment using the enhancer-trap
reporter specific for the mushroom body, and the anti-Fru antibody. In the anterior brain, the largest cluster, composed of 43-44 cells per hemisphere, is found in the anterolateral protocerebrum. Another large cluster containing 34-39 cells per hemisphere is observed in the medio-dorso-anterior protocerebrum. The third largest cluster is formed by 19-20 cells per hemisphere, and is located in the medio-anterior protocerebrum. In addition, the suboesophageal ganglion has a cluster composed of 16-20 cells per hemisphere. The localization of these immunoreactive cells coincides with that of cells expressing FRU mRNA. In addition to these areas, a few previously undescribed clusters of Fru-expressing cells were identified, including a cluster of 31-32 cells per hemisphere in the dorso-anterior-lateral protocerebrum at 15 h APF. The Fru immunoreactivity in this region decays thereafter, and becomes undetectable at 72 h APF. In the ventral ganglia, Fru-positive neurons are found in three thoracic segments as well as in all the abdominal segments. The same sets of neurons, with the exception of those in the dorso-anterior-lateral protocerebrum as mentioned above, continue to express the Fru protein throughout the pupal stage. After adult eclosion, the immunoreactivity of these neurons with the anti-Fru antibody decreases markedly (Usui-Aoki, 2000).
Surprisingly, the Fru antibody does not stain any neurons in the CNS of
wild-type females, even though it stains the eye disc when the Fru protein is expressed ectopically in the disc in females. Furthermore, the antibody does not stain any neurons in the CNS of
frusat homozygotes, regardless of sex, reflecting the absence
of any functional Fru protein in this mutant.
Lack of staining by the anti-Fru antibody in the wild-type female CNS indicates
that the Fru protein is not present here. fru mRNA is, however, detected
by in situ hybridization in the CNS of female wild-type pupae, at a
level similar to that in the male wild-type pupae (Usui-Aoki, 2000).
These observations raise the intriguing possibility that although the fru gene is transcribed, the mRNA is not translated to produce the Fru protein in female neurons. Because the sex-dependent actions of the Fru protein are under the control of Tra, this sex-specific repression of the translation
of fru mRNA might be mediated by the binding of Tra to the fru
mRNA in females. Indeed, Tra binding appears to repress
the translation of FRU mRNA in females. Significant reporter activity
is, however, detected, even in the presence of Tra and Tra-2, unlike in the
case of the female CNS, where the Fru protein is undetectable. This observation
suggests that an additional mechanism is involved in the translation block
in females in vivo (Usui-Aoki, 2000).
If Tra and Tra-2 function to directly or indirectly repress translation
in females, then this repression should be alleviated or removed by tra
or tra-2 mutations. In keeping with this expectation, anti-Fru
antibody-reactive neurons are clearly observed in tra mutant females
in a similar pattern to that in wild-type males. These observations collectively indicate that Tra represses the Fru protein expression in females in vivo (Usui-Aoki, 2000).
These observations suggest the interesting possibility that the presence or absence of Fru determines the sexual fate of certain neurons. This consideration led the authors to examine whether ectopic expression of fru in females can induce male-like characteristics,
in particular, the formation of the MOL. It first had to be determined which isoform
of the Fru protein is responsible for the formation of the MOL in males. To
address this issue, the ability of heat-shock promoter-driven
transgenes to rescue the MOL phenotype in frusat-homozygous
males was evaluated. When either type A or type B transgene expression was induced during the third instar larval to pupal stage, the MOL is restored in a proportion of the fru sat mutant males, some of whom have one or two MOL(s), but only unilaterally. A few other rescued males exhibited one of
the restored MOLs ectopically in the abdominal segment A4 rather than in segment
A5 where it is normally formed. However, similar
heat-shock treatment applied from the embryonic through the third instar larval
stages has no effect. Types C, D and E transgenes
are unable to rescue the MOL phenotype of frusat
males, irrespective of heat-shock treatment. On the basis of these results, type A and type B transcripts appear
to be essential for MOL formation in males. Another important finding is that
the 'female-type' transcripts devoid of the male-specific 5'
extension are able to produce the male-specific MOL in frusat
males. Thus, the male-specific N-terminal polypeptide does not appear to be
necessary to rescue the MOL phenotype of frusat
males (Usui-Aoki, 2000).
In another series of rescue experiments, the GAL4-UAS system
was used for induction instead of heat-shock promoter-mediated induction.
As the Gal4 source, the D42-GAL4 strain which expresses
Gal4 in almost all motor neurons but not in muscles was tested. Motor nerve endings on the MOL in the D42-GAL4 strain express Gal4. The basis for this
experiment resides in the knowledge that the motor neurons innervating the
MOL must be male, while the sex of the myocytes that form the MOL does not
affect the muscle development. Using the D42-GAL4 line, it was confirmed that the type B transcript can rescue the MOL phenotype
of frusat mutant males. The possibility,
however, that the male-specific N-terminal extension enhances the activity
of the Fru protein, even though it does appear to be unnecessary, cannot be excluded (Usui-Aoki, 2000).
To determine whether ectopic expression of the Fru protein leads to the
formation of the MOL in females, UAS-fru+ type
B was expressed, driven by D42-GAL4, in either the wild-type
background or the frusat mutant background. In both
cases, it was found that the MOL is formed ectopically in most of these female
flies; 92% of wild-type females and 91% of frusat homozygous females with ectopic fru+ develop the MOL.
Some of these females had one to three MOL(s) in the A4 (and A3) segments
in addition to a pair of MOLs in the A5 segment of the abdomen. This is in
contrast to the results obtained using hs-fru+ cDNA,
which did not induce the formation of the MOL in any females.
This result shows unequivocally that the absence or presence of the Fru protein
determines whether the MOL is formed (Usui-Aoki, 2000).
Several possible mechanisms are proposed for the sex-specific expression
of Fru. One possibility is that the Fru protein is translated only in males,
even though its mRNA is transcribed in both males and females. The second
is that the Fru protein is rapidly degraded in females whereas its durability
is significantly higher in males. The third possibility is that the primary
mRNA is not processed to give the mature fru transcript in the female,
resulting in the absence of the Fru protein. This last possibility is the
least likely, because sex-specific splicing by Tra and Tra-2 of the primary
fru transcript has been shown both in vivo and
in vitro. Sex-specific control of Fru protein synthesis
is a more plausible hypothesis, in which Tra binding to the primary fru
transcript in females is postulated to repress translation. Only female-type
mature mRNA possesses the Tra-binding site, which lies 61 bp upstream of the
translation start site in females. In fact, in transfection assays, the TraB
sequence of the fru female transcript placed 5' to the translation
start site of the luciferase gene inhibits translation from this reporter
in the presence of Tra and Tra-2 (Usui-Aoki, 2000).
The translational regulation of sex-dependent processes in Drosophila
is not without precedent. In Drosophila males, the activity of
the X chromosome is doubled by a mechanism for gene dosage compensation, mediated
by Male-specific lethal (Msl) proteins. Msl-2, one of the
Msl proteins, is translated only in males because binding of the Sxl protein
to the msl-2 mRNA represses its translation in females.
The Sxl protein also functions as a splicing inhibitor when it binds to the
same site. By analogy with Sxl, the Tra protein may
act as both a translational repressor and a splicing regulator when bound
to the fru mRNA. Note, however, that Tra and Tra-2 only partially repress
translation from the reporter mRNA with TraB in transfected S2 cells, in contrast
to the case in the female fly CNS, where the Fru protein is undetectable.
A large amount of the reporter mRNA might have titrated Tra and Tra-2, resulting
in unbound mRNA that contributes to residual translation. Alternatively, a
separate mechanism could be involved in in vivo repression of fru
translation. In this regard, the dicistronic organization of the female
FRU mRNA merits discussion. The long ORF encoding
the functional Fru protein is preceded by a short ORF for 94 amino acids in
females. If this short ORF is translated, the second long ORF is likely not
to be translated. A comparable mRNA organization has been found in the male form
Sxl transcript, in which the first ORF yields only a non-functional protein
because of the presence of a stop codon in male-specific exon III, yet the
second ORF starting in exon IV is translated at extremely low levels and thus
has no biological effect on sex determination (Usui-Aoki, 2000).
If the absence of Fru proteins in female CNS is entirely due to this mechanism,
then the effects of tra mutation on the induction of Fru expression
in females are solely due to male-type splicing of the FRU mRNA precursor
in female flies. To evaluate this possibility, it is crucial to determine
whether or not the 94-amino-acid peptide encoded by the short ORF is present
in females (Usui-Aoki, 2000).
The prevailing thought behind the sexually dimorphic function of fru is that the male-type Fru protein and the female-type Fru protein, each
derived from the sex-specific mRNA produced by Tra-mediated alternative splicing,
probably have different target specificities, and thus activate distinct sets
of genes required for either male or female development (Usui-Aoki, 2000).
The 'female-type' Fru protein can support the
formation of the male-specific MOL, indicating that the male-specific function
of the fru product cannot be ascribed to the 'male-specific'
structure of the protein. It should be noted, however, that the sex-specific
difference in the Fru protein structure deduced from the cDNA structure needs
to be verified experimentally. The size difference of 101 amino acids could
be easily resolved by Western blotting, if the antibody were reactive to denatured
Fru protein. Whereas the male-type and female-type cDNAs are equally effective
in rescuing the MOL phenotype when they are expressed by D42-GAL4
, their effects are different when driven by the hsp 70 promoter:
the rescuing ability of the male-type cDNA can not be assessed, because
of its lethal effect, whereas the female-type cDNA supports the formation
of the MOL. Although the lethality seems to reflect the non-physiological
interference of an unknown process by misexpression of the protein in inappropriate
tissues, one might argue, on the basis of this observation, that the male-type
protein has higher activity than the female-type protein. To better understand
the functional significance of the sexual differences in the Fru protein structure,
the female- and male-type proteins must be compared for their ability to bind
to putative target genes and their transcriptional regulatory activity (Usui-Aoki, 2000).
The fruitless gene of Drosophila
produces both sex-specifically and non-sex-specifically
spliced transcripts. Male-specific fru products are believed
to regulate male courtship. To further an understanding
of this gene's behavioral role, the
central nervous system (CNS) was examined for temporal, spatial, and
sexually dimorphic expression patterns of sex-specific
fru products by in situ hybridization and immunohistochemistry.
For the latter, antibodies were designed to
detect only male-specific forms of the protein (FRUM) or
amino acid sequences that are in common among all
translated products (FRUCOM). Sex-specific mRNAs
and male-specific proteins are first observed in mature
larvae and peak in their apparent abundances during
the first half of the pupal period. At later stages and in
adults, faint mRNA signals are seen in only a few
neural clusters; in contrast, relatively strong FRUM signals persist into adulthood. Twenty neuronal groups composed of 1700 fru-expressing neurons were identified in the midpupal CNS. These groups overlap most of the
neural sites known to be involved in male courtship.
Staining with anti-FRUCOM leads to widespread labeling of neural and
nonneural tissues in both sexes, but in the female CNS,
this occurs only in developing ganglia, and in a pattern different from
that of the male's FRUM cells. Expression of sex-specific
fru mRNAs in the CNS of males analyzed from the
earliest pupal stages indicates that sex-specific alternative
splicing is not the exclusive mechanism regulating
expression of fruitless transcripts (Lee, 2000a).
The spatial distribution of fru products overlaps
with most brain regions inferred to be involved in
male sexual behaviors from analysis of sex mosaics. The so-called foci underlying the
sequential steps of male courtship include a dorsal-brain
region associated (in genetically male form)
with orientation, following, and wing extension. Five
groups of FRUM neurons that have now been specified (fru-aSP1,
fru-aSP2, fru-aSP3, fru-pSP1, fru-pSP2) are
within dorsal brain regions determined from mosaics
to be involved in early stages of the behavioral sequence.
Additional brain regions, in the antennal lobe and
within the mushroom body, are involved in sexual
recognition;
and clusters of FRUM neurons (fru-mAL and fru-AL)
located near the antennal lobe might be involved in
this process. This hypothesis is supported by the absence
of sex-specific fru transcripts (or the cells that
usually express them) in the antennal lobe of the fru1
mutant: fru1males exhibit dramatic defects in sex recognition (Lee, 2000a).
Given the courtship-song defects exhibited by these and
other fru mutants and that a portion
of the VNC -- possibly the mesothoracic ganglion --
needs to be genetically male for normal courtship song to be produced, it is
interesting that FRUM-containing neurons (fru-PrMs,
fru-MsMt) are found in this region of the CNS (Lee, 2000a).
Two sex-specific characteristics are associated
with the abdominal ganglion, probably through the
action of certain abdominal motorneurons: the formation
of a male-specific abdominal muscle and the
male's abdominal bending that occurs during copulation
attempts, both of which are defective to varying
degrees in fruitless mutants. Many
FRUM neurons (fru-Ab) are present in this posteriormost ganglion. It is hypothesized that certain subsets of the fru-Ab cluster are involved in these two sexually dimorphic phenotypes (Lee, 2000a).
An unexpected result in this study was the presence of fru gene products in the optic lobes. Although vision is not essential for courtship and mating in this Drosophila species, visual cues are important for rapid recognition and sustained tracking of a sexual partner. However, no particular significance to fru expression in the visual system has yet been found. For example, it is not known whether male-specific female tracking behavior is impaired in these mutants. Yet it is interesting that fru3 and fru4 mutant males initiate subnormal frequencies of courtship following bouts and that the turning responses and locomotion necessary to sustain such bouts are relatively brief (Lee, 2000a).
The antibodies generated against a stretch of
amino acids common to all forms of FRU (which lack the N-terminal, male-specific amino acids) led to immunostaining in the CNS of both males and females. It is inferred that anti-FRUCOM elicited staining of both non-sex-specific anti-FRUCOM proteins and FRUM in males and in females, only of anti-FRUCOM proteins produced under the control of promoters other than P1. Genetic-mosaic studies point to portions
of the CNS involved in female-specific sexual behaviors, including an anterior brain region that perforce is included within the broad expression pattern
of Fru in females. However, such female neural foci, including the anterior one that underlies receptivity to male mating attempts, may not require fruitless in order to form or function (Lee, 2000a).
In the CNS of females there should be no FRUM
for anti-FRUM or anti-FRUCOM to detect,
consistent with the patterns observed. Anti-FRUCOM was labeled by the latter reagent in many neural tissues of both sexes, but only during
development. Here it is assumed, from the absence of
anti-FRUM-like anti-FRUCOM immunostaining in the
CNS of animals carrying fru deletions, that the neural
signals labeled by anti-FRUCOM in adult males stem from FRUM alone. That protein type almost certainly is involved in
regulating the behavior of adult males. However, the
larval- and pupal-specific anti-FRUCOM proteins in the
CNS could also have behavioral significance with
respect to features of the CNS that have formed after
either males or females have progressed through these
developmental stages (Lee, 2000a).
This supposition implies that the reproductive behavior
of females as well as males would be abnormal in
fruitless mutants; however, no impairments of female
courtship have been detectable, including that the mating
receptivity of these mutants is normal. Yet, all the fru mutants tested as females can be inferred primarily to affect P1-promoted transcripts, either in terms of severe quantitative
reductions (transposon mutants) or qualitative
spatial-expression abnormalities (the effect of a chromosome
breakpoint near the P1 promoter in the fru1 mutant). Those
mRNAs encode FRUM, which is undetectable in females. At any developmental stage, no anti-FRUCOM-containing female counterparts could be detected of the CNS cells that express P1 transcripts with sharply defined, relatively high-level signals. Such mRNAs are translatable on paper, provided that the relatively downstream start codon is used in females. That this appears not to be the case (or that
translated products of ORF-2 do not accumulate) suggests
that P1-controlled female forms of Fru are not
candidates for proteins that would be affected by the
viable fru mutations in flies of this sex (Lee, 2000a).
Females are affected by certain genetic variations
at the fru locus, in that the chromosomal breakpoints
in question kill them, along with their brothers, during
late developmental stages. These
near-lethal effects are likely to be associated with the
broad and non-sex-specific spatial patterns of
anti-FRUCOM proteins. Indeed, the chromosomal lesions
that cause severe decrements in viability eliminate all detectable Fru proteins, including the common forms produced under the control of
three promoters located downstream of P1. Eliminating the P1 promoter from
chromosomes [Df(3R)ChaM5 or Df(3R)4-40] that otherwise
can encode some of the anti-FRUCOM proteins leads
to the expected retention of such material.
This rationalizes the viability of M5/P14 and 4-40/P14 flies, bearing in mind that the P1-retaining chromosome in these variants [Df(3R)P14] is missing
most of fru's coding region. That these
Df/Df males and females show much lower-than-normal
levels of anti-FRUCOM-stained tissues is
probably due to the fact that the M5 and 4-40 fru
deletions are missing certain of the downstream promoters. In particular 4-40 is deleted of P2 and may possess a damaged P3; M5 could lack
P2 or possess only a portion of that gene-regulatory
region. At least one fru promoter (P4) is
likely to be intact in both the M5 and 4-40 chromosomes (Lee, 2000a).
That regulatory region is far downstream of
the P1 promoter, thus rather close to the bulk of fru's ORF. P4's action appears sufficient to allow for normal viability of the Df/Df flies, their reduced
levels of anti-FRUCOM notwithstanding.
Within the CNS of both sexes, the spatial distributions
of fru transcripts are complex. There are so
many ganglia and cells involved that is it difficult to determine whether the 'high-mRNA' cells in females have exact counterparts in males (Lee, 2000a).
Are the similarly labeled cells in males, which presumably correspond to FRUM-expressing neurons, present at all in females? In this
regard, throughout the pupal period sexually
dimorphic expression of mRNAs were detected whose production
is controlled by the P1 promoter. These
in situ hybridizations involved male-exclusive neural
signals. Such sexually dimorphic features of the RNA
spatial pattern arise just after the time when sex-specific
forms of the gene products are first observed.
These findings reveal that there is more to sex-specific
expression of fruitless than the alternative RNA splicing
of P1 transcripts that occurs under the
control of the sex-determination hierarchy. One possibility
is that the gene's transcriptional control is an
additional component of its sex-specific regulation.
For example, P1-promoted expression patterns could
be different in males and females from the earliest
stages of pupal development because of gene activation
that would occur in the relevant CNS cells of males only. Alternatively, transcriptional activation of fru may be initiated identically in both sexes, with the sex-specific pattern being generated by repression
of P1 transcription in certain cells of females
or by selective cell death in the early-pupal CNS.
These two possibilities are subsumed under the
broader question of whether fruitless gene action is
primarily involved in the structure of the CNS (including
early aspects of sex-specific pattern formation)
or the subcellular quality of its components
(aspects of terminal cell differentiation). A further
subsidiary question involves how the fruitless mutants
might differ from wild type: are the former only
devoid of the gene's expression in many or all of the
normal CNS locations, or are they neuroanatomically
defective as well? The inferred absence of P1-promoted,
FRUM-encoding mRNA in some of the most
severely affected but viable fru mutants (such as those
carrying transposon inserts within the locus) makes an answer to this question by
application of anti-FRU reagents problematical. If no
FRUM protein are immunohistochemically detectable
in a mutant such as fru3 (which is nearly null for
P1-promoted mRNA), this could be because the relevant
neurons did not form or underwent cell death
during development. Alternatively, all the relevant
neurons could be present in this mutant, but their
connectivity or neurochemical qualities might be aberrant (Lee, 2000a).
Given the prominent expression of fruitless
during the earliest stages of imaginal development, it is suspected
that fruitless is crucially involved in 'early-formative'
features of sex-specific neural development (whether
or not the gene products also play a role at later
stages). This conjecture includes the possibility that
fru influences the presence versus absence of a CNS
component in one sex or the other, as opposed to
controlling only the fine features of neuronal differentiation.
In either case, sex-specific anatomy in Drosophila
needs to be understood at levels deeper than those involving certain gross ganglionic differences. How the reproductively relevant structures are formed and take on their detailed features must be apprehended by higher resolution
examinations of females and males and of fru mutants (Lee, 2000a).
Male-specific fruitless (fru) products (FruM) are both necessary and sufficient to 'hardwire' the potential for male courtship behavior into the Drosophila nervous system. FruM is expressed in ~2% of neurons in the male nervous system, but not in the female. The insertion of GAL4 was targeted into the fru locus, allowing visualization and manipulation of the FruM-expressing neurons in the male as well as their counterparts in the female. Evidence suggests that these neurons are directly and specifically involved in male courtship behavior and that at least some of them are interconnected in a circuit. This circuit includes olfactory neurons required for the behavioral response to sex pheromones. Anatomical differences in this circuit that might account for the dramatic differences in male and female sexual behavior are not apparent (Stockinger, 2005).
An important step toward understanding the neural basis of any behavior is to trace the neural circuits involved. This study characterizes at the level of single identifiable neurons, the circuitry that governs Drosophila male courtship behavior. These neurons are defined by their expression of fruGAL4, created by the targeted insertion of GAL4 into the fru locus. It is believed that fruGAL4 identifies most if not all of the neurons with sex-specific functions in courtship because male-specific FruM proteins are necessary and sufficient for courtship (Demir, 2005) and fruGAL4 includes all the neurons that express FruM. Synaptic silencing of these neurons impairs courtship behavior but leaves unrelated behaviors intact. Thus, the fruGAL4 neurons function directly in male courtship, and are largely dedicated to this behavior (Stockinger, 2005).
Of course, this is not to say that courtship only involves fruGAL4 neurons, nor that these neurons function only in courtship. Clearly, courtship also involves many neurons that do not express fruGAL4. However, these neurons most likely have more general functions, common to many behaviors and to both sexes. It is also possible that fruGAL4 neurons function in other behaviors that have not yet been examined, in particular other sex-specific behaviors such as aggression. Also, almost all of the fruGAL4 neurons have counterparts in females. The functions of these neurons in females are unknown (Stockinger, 2005).
How many of the fruGAL4 neurons are actually involved in male sexual behavior? At one extreme, just one or a few of the fruGAL4 neurons might be critical, with most fruGAL4 neurons having nothing to do with courtship. Alternatively, most or even all of the fruGAL4 neurons might be directly involved, each contributing in some way to the behavior. The latter scenario is favored. (1) There are no obvious examples of cells that express fruGAL4 but are clearly not involved in courtship. fruGAL4 is not detected at all in embryos nor during larval stages until shortly before pupariation, and even in adults it is confined to only a small fraction of neurons. (2) Distinct roles in sexual behavior have already been defined, or seem likely, for several subsets of these fruGAL4 neurons, such as the fruGAL4 ORNs and the fruGAL4 motor neurons that innervate the penis and ejaculatory bulb. It thus appears that male sexual behavior involves the contributions of many different fruGAL4 neurons, including neurons at each of the sensory, central, and motor levels (Stockinger, 2005).
Dissecting out the contributions of each of these neurons will require a means to manipulate defined subsets of fruGAL4 neurons, as was done in this study for the fruGAL4 ORNs by using antenna-specific FLP expression and FLP-dependent silencers. With the appropriate FLP reagents, it might be possible to extend this approach to other parts of the nervous system. Similarly, expression of GAL80, a GAL4 repressor, should allow the selective exclusion of specific subsets of fruGAL4 neurons. These approaches could also be combined, providing logical AND and NOT operations that might ultimately allow the operation of the entire fruGAL4 circuit to be examined, piece by piece (Stockinger, 2005).
fruGAL4 neurons are dispersed throughout the nervous system, generally comprising a small subset of neurons at each successive neural level. For example, fruGAL4 labels subsets of both first-order olfactory neurons (ORNs) and second-order olfactory neurons (probably PNs). Remarkably, the fruGAL4 ORNs innervate the same three glomeruli in the antennal lobe as the fruGAL4 PNs, indicating that they are most likely synaptic partners. Third-order olfactory neurons are located in the superior protocerebrum and mushroom bodies, and here too subsets of neurons express fruGAL4. Thus, it is possible that a “fruGAL4 connects to fruGAL4” principle might even extend into higher brain centers, and perhaps even continue through to the descending pathways and motor neurons that express fruGAL4. Clearly, fruGAL4 neurons also make synaptic contact with neurons that do not express fruGAL4, many of which will also have important (but general) roles in the neural processing that drives male courtship. It is speculated that many if not all of the neurons with sex-specific roles in courtship express fruGAL4 (and normally FruM), and that these neurons may be directly interconnected in a circuit that extends from sensory input through to motor output. A precedent for this organization, albeit for a much simpler behavior, is the connectivity of sensory and motor neurons that express the same Ets transcription factors in the vertebrate monosynaptic spinal reflex circuit (Stockinger, 2005).
In >Drosophila, as in many other animals, male mating behavior is triggered by sex pheromones emitted by the female. Drosophila females produce both volatile (long-range) and nonvolatile (contact) sex pheromones. Volatile pheromones are thought to stimulate courtship behavior, whereas nonvolatile pheromones may facilitate sex and species discrimination. The major nonvolatile female pheromones are 7,11-heptacosadiene and 7,11-nonacosadiene, for which Gr68a is a candidate receptor. The volatile pheromones have not yet been identified nor have the receptors or neurons that detect them. It is postulated that these pheromones are detected by the fruGAL4 ORNs. This conclusion rests on two main lines of evidence. (1) Sensory stimuli of particular significance often have enlarged representations in the brain, and so the fact that the fruGAL4 ORNs innervate glomeruli that are larger in males than in females suggests that the odors they detect are more important to males than to females. (2) Selective silencing of these fruGAL4 ORNs severely impairs courtship behavior, both in males exposed to normal females, and in fruM females exposed to males that emit female pheromones (Stockinger, 2005).
Just as the fruGAL4 ORNs appear to comprise a distinct class of 'specialist' ORNs involved in pheromone detection, the fruGAL4 PNs are also distinct from the 'generalist' PNs, many of which are labeled by GH146-GAL4. This suggests that the processing of pheromones and general odors is anatomically segregated in the Drosophila brain, just as it is for example in rodents and fish. This segregation may not be complete, however, as the glomeruli targeted by fruGAL4 ORNs and PNs are also innervated by GH146-GAL4 PNs, and silencing the GH146-GAL4 neurons also inhibits male courtship (as well as other olfactory behaviors) (Stockinger, 2005).
What is the role of fru in this olfactory circuit? fru is not required for the synaptic specificity of fruGAL4 ORNs and PNs, nor for the expression of putative pheromone receptors in the fruGAL4 ORNs. fru is however responsible for the sex differences in the size of each of the three glomeruli targeted by fruGAL4 ORNs and PNs. This sexual dimorphism is probably due to fru function in the ORNs rather than the PNs, since genetic feminization of ORNs reduces the size of at least two of these glomeruli in males. Similarly, for the sphinx moth Manduca sexta, the enlargement of pheromone-processing glomeruli in males also depends on the sex of the antenna, not of the brain (Stockinger, 2005).
Behavioral differences between males and females reflect sex differences in neural function. An important question, for any species, is whether the essential difference between the sexes lies primarily in their neuroanatomy or their neurophysiology. In some species, sexually dimorphic behaviors correlate with striking differences in neuroanatomy. For example, in some songbirds, such as the zebra finch, only males sing, and brain regions involved in the acquisition and performance of the song are much larger in males than females. However, in many other species, including humans and mice, sex differences in neuroanatomy are much more subtle, and their functional significance, if any, is still unknown (Stockinger, 2005).
For Drosophila sexual behavior, the reason why males court but females do not must reflect some sex-specific property of the fruGAL4 neurons. It is thought that it is not their gross anatomy. With the trivial exception of neurons innervating the reproductive organs, only subtle differences are detected in the numbers of these neurons and no differences at all in their morphologies or projections (see however Kimura, 2005). Pending further studies at higher resolution, it is tentatively concluded that sex differences in courtship behavior do not rest on differences in the production, survival, or connectivity of the neurons involved (Stockinger, 2005).
This conclusion offers a rather sobering perspective on the considerable effort that continues to be devoted to identifying and characterizing sexual dimorphisms in the mammalian brain. In Drosophila, the sexual behaviors of males and females are dramatically different and highly stereotyped; this difference can be attributed to a single splicing event in a single gene, and the neurons that express this gene can be examined at single-cell resolution. Yet even under these ideal circumstances, anatomical differences that might account for the dramatically different sexual behaviors of males and females have not yet been found. This suggests that differences in neural chemistry, rather than gross neuroanatomy, might underlie the profound differences in behavior between males and females in Drosophila, and surely in many other species as well (Stockinger, 2005).
If the essential difference between the sexes in Drosophila lies in the physiology of the fruGAL4 neurons, then it is necessary to begin to search for this difference. fruGAL4 itself will be a powerful tool in this endeavor. Coupled with optical indicators of neuronal activity and tools such as FLP and GAL80 to highlight specific subsets of fruGAL4 neurons, it should now be possible to look for sex differences in the patterns of neuronal activity elicited by sexual stimuli. Thus, by defining the neural circuit that governs male sexual behavior and providing a tool for its manipulation, this work paves the way for a mechanistic investigation of a complex innate behavior. It is now possible to begin to explore how this circuit operates, why it operates differently in males and females, and how this difference is programmed during development (Stockinger, 2005).
The fruitless (fru) gene functions in Drosophila males to establish the potential for male sexual behaviors. fru encodes a complex set of sex-specific and sex-nonspecific mRNAs through the use of multiple promoters and alternative pre-mRNA processing. The male-specific transcripts produced from the distal (P1) fru promoter are believed to be responsible for its role in specifying sexual behavior and are only expressed in a small fraction of central nervous system (CNS) cells (Goodwin, 2000).
The fru locus spans at least 140 kb and produces a complex array of transcripts due to the use of four promoters and alternative splicing. Transcripts from the distal promoter (P1) are alternatively spliced near their 5' termini to generate sex-specific transcripts. It is thought that the male-specific P1 transcripts encode fru's sex determination function, whereas the transcripts produced from the more proximal promoters encode fru's vital function(s). Alternative splicing at the extreme 3' end of the fru transcripts leads to the inclusion of one of three mutually exclusive exons that encode alternative pairs of zinc fingers. Fifteen transcript classes are possible if the five identified 5' ends (two are produced by sex-specific splicing from P1) are combined with all the identified 3' ends; cDNAs corresponding to seven of these classes have been identified. However, the potential transcript diversity may be more extensive, due to the discovery of additional fru transcripts containing one of several micro-exons. The transcripts from all fru promoters have open reading frames that encode proteins related to the BTB-ZF protein family (Goodwin, 2000 and references therein).
When a probe common to all known classes of fru cDNAs was hybridized to Northern blots of poly(A)+ RNA from sexed wild-type adult heads, three female-specific (9.0, 8.0, and 7.4 kb), three male-specific (7.9, 6.4, and 5.4 kb), and one common (4.4 kb) transcripts were detected. Sex-specific fru transcripts are generated by the sex-specific usage of alternative 5' splice sites that are 1590 nt apart in the P1-derived pre-mRNA (Ryner, 1996). It has been suggested that the three male-specific and three female-specific transcripts are generated from P1-derived transcripts by sex-specific splicing at their 5' ends and by sex-nonspecific alternative splicing to exons containing three alternative zinc-finger pairs at their 3' ends. This predicts that the three classes of female-specific transcripts would have common sequences at their 5' ends, as should the three male-specific forms. To test this, a fru probe from upstream of the female-specific 5' splice site was used to probe Northern blots of poly(A)+ RNA from sexed adult heads. Only the three female-specific (9.0, 8.0, and 7.4 kb) transcripts seen previously were detected; no signals were observed in male head RNA, even with long autoradiographic exposures. These results are consistent with the interpretation that all three female-specific fru transcripts share common 5' female-specific sequences (Goodwin, 2000).
The sequences of the sex-specific fru transcripts revealed that there are no sequences unique to the male transcripts: both male- and female-specific fru transcripts share common sequences from upstream of the male-specific 5' splice site (Ryner, 1996). Therefore a probe from this region was used on Northern blots of poly(A)+ RNA from sexed wild-type heads; only the three female sex-specific transcripts (9.0, 8.0, and 7.4 kb) and three male sex-specific transcripts (7.9, 6.4, and 5.4 kb) were observed. This set of results provides strong support for the conclusion that these six sex-specific fru transcripts arise from sex-specific splicing at the 5' end of transcripts produced from the distal (P1) fru promoter (Goodwin, 2000).
An important feature of P1-derived transcripts is that they are expressed in several hundred neurons in the CNS of adults. The spatial pattern of P1 expression was analyzed in wild type in more detail and, at this higher level of resolution, an examination was made to determine whether any of the viable fru alleles alter the gene's spatial expression pattern. Nine groups emerged from this analysis: six in the brain and three in the ventral nerve cord. In the brain, the locations of the six groups are as follows: (1) a large group in the dorsal posterior protocerebrum, medial and ventral to the mushroom bodies; (2) a lateral group in the protocerebrum, anterior to the medullary division of the optic lobes; (3-5) three anterior groups in the protocerebrum, subdivided into lateral (3), intermediate (4), and medial (5) sets; and (6) one anterior group near the mechanosensory part of the antennal lobe. In the ventral nerve cord, the three groups of male-specific fru P1-expressing neurons are as follows: (7) a lateral group between the prothoracic and mesothoracic neuromeres and ventral to the wing neuromere; (8) a ventro-medial group in the mesothoracic ganglion; and (9) a ventral group in the abdominal ganglion. In addition to these groups of cells, both sexes have a small population of labeled neurons that are found as singletons or are too widely separated from other labeled cells to recognize them as belonging to a group (Goodwin, 2000).
In contrast to the restricted pattern of expression of P1 fru transcripts, a probe (C1) that detects the protein-coding region common to all fru transcripts has revealed expression in virtually all neurons in the CNS and in several other tissues. Most cells in the CNS have a relatively low level of expression, whereas a small number of neurons display markedly higher levels of fru expression. By their location and number, the subsets of neurons labeled by the C1 probe that have relatively high levels of fru expression appear to correspond to the nine groups of neurons detected by the S1 probe (Goodwin, 2000).
To determine whether there were changes in the cellular pattern of fru expression in the five viable fruitless mutants, analogous in situ hybridizations were undertaken with both the S1 and C1 probes. Only the presence and relative size of the aforementioned nine groups of labeled neurons were analyzed, because these groups of fru-expressing cells are distinct and could be unambiguously identified in tissue sections (Goodwin, 2000).
Examination of the expression pattern of P1-derived transcripts in fru1 males revealed distinct groups of labeled neurons in only four of the nine regions where cells expressing these transcripts are found in wild-type males. The four groups of neurons detected were those in the dorsal posterior protocerebrum (1), the optic lobe (2), the antennal lobe, and the ventro-medial mesothoracic groups. The numbers of labeled neurons in the antennal lobe and dorsal posterior groups in fru1 males are not significantly different from wild type (Goodwin, 2000).
The other five groups of cells detected in wild-type males are very difficult to detect in fru1 males. In the anterior protocerebrum, the normal pattern for three groups of neurons (3-5) is not observed. In the dorsal anterior protocerebral region, only about one-quarter of the expected number of labeled neurons are detected. These labeled neurons are distributed throughout the medial, intermediate, and lateral subdivisions, suggesting that fru1 leads to a reduced number of cells expressing P1 transcripts in all three regions. It was not possible to identify definitively the remaining labeled neurons as belonging to one of the three anterior protocerebral groups. Likewise, labeled neurons are difficult to detect in two of the male-specific neuronal groups in the ventral nerve cord of fru1. A small cluster of neurons, ~15% of the expected number, is found in the ventral area, between the prothoracic and mesothoracic neuromeres. By contrast, almost no labeled cells are found in the ventral abdominal region, where group 9 neurons are observed in wild-type males. The reduced numbers of cells expressing P1 transcripts in fru1 males may be caused by a reduction in transcription from the P1 promoter, instability of these transcripts, or the loss of neurons that normally express these transcripts (Goodwin, 2000).
When a probe (C1) common to all fruitless transcripts is used, all neurons in fru1 male CNSs are labeled at a low level, comparable to what is seen in the wild-type CNS. In addition, subsets of neurons show relatively high levels of fru expression. Neurons with such heavy labeling are detected in the dorsal posterior protocerebrum (1), optic lobe (2), antennal lobe (6), and mesothoracic groups (8); these are the same regions in which the neurons expressing the sex-specific transcripts are abundant in fru1 males. In fru1, fewer-than-normal numbers of heavily labeled neurons are detected with the C1 probe in the anterior protocerebrum (3-5) as well as within the thoracic (7) and the abdominal ganglion (9) groups. These regions are the same as those in which fewer or no labeled neurons are found by in situ hybridization with the S1 probe (Goodwin, 2000).
in situ hybridization was used to determine whether the spatial expression of P1-derived transcripts was affected in the transposon mutants. In all four mutants, the S1 probe detects groups of labeled neurons in the nine locations where P1-expressing neurons are found in wild-type males. For example, labeled neurons were found in the antennal lobe, the dorsal anterior protocerebrum, the thoracic ganglion, and the abdominal ganglion. The numbers of neurons labeled in these groups are similar to those found in wild-type males. However, there was an overall increase in the number of heavily labeled neurons in the brains of frusat males compared to the other fru mutant or wild-type males. In wild-type males, cells labeled with the S1 probe often have darkly stained dots within the nucleus, as well as cytoplasmic staining. In a similar fashion, in all the P-element-mutant males, neurons labeled with the S1 probe often have very darkly stained dots in the nucleus in addition to weak cytoplasmic staining (Goodwin, 2000).
The loss of fruitless expression in the regions described likely accounts for the striking courtship abnormalities exhibited by fru1 males. Sequences in the transposons in the four P-element mutants lead to nonproductive splicing of fru transcripts. This fact offers a first-order explanation for how
the insertions of these transposons into the large fru intron generate mutant phenotypes. However, what is not accounted for by these findings is that these four
insertion mutations differ in the severity of their phenotypic effects. One possible explanation for these phenotypic differences is
that low levels of normal FRU mRNAs are generated from the P1 promoter in these mutants. Indeed, RT-PCR experiments using poly(A)+ RNA from heads are
able to detect normally spliced products from the P1 promoter in all four of the transposon mutants. These RT-PCR experiments are not quantitative, allowing for
the possibility that there are different residual levels of normal, P1-derived mRNAs in the mutants; this could account for the different severities of these alleles. That
some of the transposons are not only intrinsically different, but also are inserted at different intronic positions, could contribute to the probability of a given transcript
being subject to aberrant splicing during its processing. Thus, it is suggested that the mutant phenotypes in fru2, fru3, fru4, and frusat animals are due to a failure to appropriately splice P1 transcripts, whereas the mutant phenotype of fru1 animals is due to the reduction or absence of P1 transcripts within specific regions of the CNS (Goodwin, 2000).
Various fru mutations have varying effects on male sexual behavior, Muscle of Lawrence development and locomotion. Many mutant alleles result in male flies that court indiscriminately, fail to copulate and have MOL defects. Some mutant males show very little wing extension; during wing displays, they generate no song pulse signals. This defect is specific to courtship, as these mutants are normal for flight and are able to flick their wings when rejecting advances made by another male. Further reductions in sexual behavior are seen with other alleles or combinations. More severly impaired males barely court, yet they exhibit essentially normal locomotor activity. When fru mutant males are grouped together, they form male-male courtship chains in which each male is simultaneously both courting and being courted. All mutant combinations show some male-male chaining. Thus, the early steps of courtship (orientation, following, and wing extension) as well as the later steps (courtship song and attempted copulation) are disrupted in various mutants and mutant combinations (Ryner, 1996).
A genetically defined element of the fruitless locus regulates the development of a male-specific
muscle spanning the fifth abdominal segment in adult males, the "muscle of Lawrence" (MOL). The region is defined by two
cytological deletions, each with a breakpoint that co-maps with previously described mutant courtship phenotypes at cytogenetic
interval 91B on the third chromosome. Flies that carry both of these deletions are viable, and males express abnormalities of courtship
similar to those caused by the fru inversion breakpoint at 91B. In addition, these double-deletion males show the complete absence of
the MOL, suggesting that they have little or no gene expression of a postulated MOL determinant; the musculature in the fifth
abdominal segment of these mutants to indistinguishable from that of a normal female. Other mutant combinations that produce
fruitless courtship phenotypes--including deletion and inversion breakpoints, and a marked transposon inserted at 91B--produce
intermediate forms of the MOL. A new genetic variant, induced by imprecise excision of the marked transposon, is homozygous lethal
and disrupts fru functions related to courtship and the MOL. The MOL is shown to be dispensable for fertility and is therefore not the
causative factor of fru-induced behavioral sterility (Gailey, 1991).
Muscle of Lawrence (MOL) development, which is induced by male-specific
innervation, was studied in a variety of genotypes: in wild-type males, in males fed hydroxyurea to ablate the muscle precursors and in
fruitless mutants, in which the MOL develops aberrantly. One striking feature of MOLs in wild-type males is the presence
of additional muscle nuclei compared with neighboring muscles or MOL-homologs in females. Does muscle length and
the sex-specific expression of a reporter gene depend critically on the number of nuclei present within a MOL fiber? MOL fibers
developing from a reduced myoblast pool in hydroxyurea-affected hemisegments are recognizable by their attachment points and still
contain more nuclei than do neighboring medial fibers, suggesting that these MOL fibers are able to actively recruit myoblasts
nearly as well as wild-type MOLs. However, many of the hydroxyurea-affected MOL fibers are incapable of the normal
male-specific expression of a muscle-specific reporter gene. It is suggested that early events in MOL development, such as finding the
correct muscle attachment points, are relatively insensitive to the number of MOL nuclei, as compared with later events, such as the
sex-specific expression of a reporter gene. In fruitless mutant males, MOL-position fibers are smaller and have substantially fewer
nuclei, when compared to wild-type MOLs. Since the number and distribution of muscle precursors is the same in fruitless mutant and
wild-type animals, it is proposed that one fru+ function is to direct the male-specific recruitment of myoblasts into MOL-myotubes.
However, fruitless+ must have more than one role in MOL fiber development, since simple reduction in the number of muscle nuclei,
as demonstrated by the hydroxyurea ablations, is insufficient to account for all of the MOL muscle phenotypes in fruitless mutant
males (Taylor, 1995).
The muscle of Lawrence (MOL) is a bilaterally symmetrical muscle spanning the tergite of the fifth abdominal segment of adult male
Drosophila melanogaster. It is not, however, a general feature of male-specific development within the subfamily Drosophilinae. Of 95
species surveyed within this subfamily, 67 exist with no MOL at all. By drawing comparisons with published cladograms of species
relatedness, three conclusions have been reached regarding the evolutionary history of the MOL: (1) it predates the major radiations of
the genus Drosophila, given its presence in earlier-branching Chymomyza and Scaptodrosophila; the MOL has been subsequently
excluded in at least one present species of each of these two primitive genera. (2) Within the genus Drosophila the MOL is present
sporadically in the radiation of the subgenus Sophophora, showing repetitive loss even in very close evolutionary lineages. (3) The
MOL may have been entirely excluded from the prolific radiation of the subgenus Drosophila. Thus the MOL shows a uniquely
incongruous pattern of presence or absence relative to accepted Drosophilid phylogeny (Gailey, 1997).
The fruitless mutants fru3 and fru4 were assessed for sex-specific reproductive-behavioral phenotypes
and compared to the previously reported fru mutants. Among the several behavioral anomalies
exhibited by males expressing these relatively new mutations, some are unique. fru3 and fru4 males are
less stimulated to court females than fru1 and fru2. No courtship pulse song is generated by either fru3
or fru4 males, even though they perform brief wing extensions. fru3 and fru4 males display significantly
less chaining behavior than do fru1 males. The hierarchy of courtship responses by fru males directed
toward females vs. males, when presented with both sexes simultaneously, is that fru1 males perform
vigorous and indiscriminant courtship directed at either sex; fru4 males are similarly indiscriminant, but
courtship levels are lower than fru1; fru2 males prefer females and fru3 males show a courtship bias
toward males. fru3 and fru4 males essentially lack the Muscle of Lawrence (MOL). On several
reproductive criteria, there was no difference between fru- variant females and fru+ (Gailey, 1997).
There exists in Drosophila a tra-dependent,
dsx-independent mechanism for the control of aspects of male and female sexual behavior and neural development. One dissatisfaction function, proper ventral A5
abdominal innervation in males, is downstream of tra but not
downstream of dsx. These results lead to the inferrence that
dsf functions in a tra-dependent and
dsx-independent process. Another gene, fruitless (fru), has also
been postulated to be part of a dsx-independent pathway
controlling behavior and neuronal development. Males with
strong fru mutations (1) fail to differentiate tissues known as the muscle of Lawrence (MOL); (2) show abnormal courtship partner choice,
courting both mature males and females; (3)
are sterile as a result of an inability to curl their abdomens into a
copulatory position, and (4) generate an abnormal
courtship song. Mutant phenotypes have not been reported for
fru females (Finley, 1997 and references).
The identification of dsf as a second
dsx-independent gene controlling sexual behavior and neural
development raises questions about whether dsf and
fru are part of a single regulatory pathway or are parts of two
different regulatory pathways. There are both substantial
similarities and substantial differences in phenotype between these two
genes. dsf and fru are similar in that mutations in both genes lead to male by male courtship and to abnormality (dsf) or failure (fru) in
abdominal curling during copulation. Yet,
there are behavioral and neurological differences between
dsf and fru mutants. The most notable among these
is the lack of any reported abnormalities for fru females in
either courtship or fertility relative to the substantial abnormalities
exhibited by dsf females. There are also phenotypic
differences between dsf and fru males. Males
with strong fru alleles do not produce male-like MOLs, while dsf males produce normal MOLs with normal innervation. In addition, the failure of abdominal bending is absolute in males carrying strong fru alleles and only partial in
dsf mutants, while ventral abdominal muscles are innervated
normally in fru males and
abnormally in dsf males. The
multiple differences between the fru and dsf
phenotypes lead to the conclusion that these genes act in separate
regulatory pathways, each of which is required for appropriate sexual
behavior (Finley, 1997 and references).
Male sexual behavior is regulated by the sex-determination hierarchy (SDH) in Drosophila melanogaster. The fruitless gene, one of the regulatory factors functioning downstream of other SDH factors, plays a prominent role in male sexual behavior. fru mutations cause a previously unappreciated behavioral anomaly: high levels of head-to-head interactions between mutant males. These apparent confrontations between males are exhibited by all of the homozygous-viable fru mutants (expressing the effects of a given allele, among the four tested). Mutant dissatisfaction (dsf) males also exhibit this behavior at higher-than-normal levels, but it was barely displayed by doublesex or intersex mutants. For fru, a social component is involved in the head-interaction phenotype, while increasing age is a modifying factor for the behavior of dsf males. It is suggested that head-to-head interactions, especially those performed by fru males, are instances of putative aggression analogous to those exhibited by wild-type males and that head interactions are, to an extent, operationally separable from courtship behavior (Lee, 2000b).
The fruitless gene produces male-specific protein (FRUM) involved in the control of courtship. One approach toward understanding how fru regulates male courtship is to compare patterns of FRUM expression in the CNS of various fruitless mutants that display behavioral phenotypes ranging from mildly to severely defective. The courtship subnormalities and bisexual behavior caused by fru mutations could be understood in terms of where FRUM is expressed in the CNS (or not expressed, as the case may be) in a given mutant. FRUM spatial and temporal
patterns were examined in fru mutants that exhibit aberrant male courtship. Chromosome breakpoints at the locus eliminate FRUM. Homozygous viable mutants exhibit an intriguing array of defects. In fru1
males, there are absences of FRUM-expressing
neuronal clusters or stained cells within certain clusters, reductions
of signal intensities in others, and ectopic FRUM
expression in novel cells. fru2 males exhibit an overall decrement of FRUM expression in all neurons normally expressing the gene.
fru4 and frusat mutants produce
FRUM in only a small number of neurons at extremely low
levels, and no FRUM signals were detected in
fru3 males. This array of abnormalities was inferred to correlate with the varying behavioral defects exhibited by these mutants. Such abnormalities include courtship among males, which has been hypothesized to involve anomalies
of serotonin (5-HT) function in the brain. However, double-labeling uncovered no coexpression of FRUM and 5-HT in brain neurons. Yet, a newly identified set of sexually dimorphic FRUM/5-HT-positive neurons was identified
in the abdominal ganglion of adult males. These sexually dimorphic neurons (s-Abg) project toward regions of the abdomen involved in male reproduction. The s-Abg neurons and the proximal extents of their axons were unstained or absent in wild-type females and exhibit subnormal or no 5-HT immunoreactivity in certain
fru-mutant males, indicating that fruitless controls the formation of these cells or 5-HT production in them (Lee, 2001).
To examine the possible relationship between fruitless
function and 5-HT, whole-mounted CNSs were double-labeled with antibodies against
FRUM and the neuromodulator. 5-HT-immunoreactive neurons are broadly distributed throughout the brain, the thoracic ganglia, and the
abdominal ganglion of adult males. Nine groups
of serotonergic neurons have been reported in the adult brain and five groups in the
ventral nervous system of Drosophila. A comparison was made of of serotonergic neurons of wild type and fru mutant adult flies (Lee, 2001).
It was assumed that fruitless mutations and ectopic
expression of the white gene (Zhang, 1995) cause males to court other flies because of anomalous brain function (possible involvement of the VNC is counterintuitive). Ectopic expression (and probably overexpression) of
w+ in the brain could deplete
5-HT levels in cells that normally express the fru gene,
mutations of which can easily be found to cause a similar neurochemical
deficit. Thus FRUM and 5-HT would be coexpressed in at least some of the neurons that normally contain these substances. However, within the brain of wild-type males, no FRUM neurons whatsoever were
double-labeled with anti-5-HT. The usual locations of cells and processes immunoreactive for this substance were observed. The number of 5-HT neurons is not
particularly large, reinforcing the possibility that global uptake of a
serotonin precursor throughout the brain could deplete levels of this
substance in their usual locations. However, if ectopic expression of
w+ is mechanistically related to fru-mutational effects via 5-HT, the current results indicate that there is a need to formulate a hypothesis different from one involving direct intracellular effects of the latter genotypes.
Perhaps white and the tryptophan transporter it encodes
cause this neuromodulator to be anomalously present in
FRUM cells or other neurons that directly
interact with them; such effects might derange
fru-controlled brain functions insofar as sex recognition is
regulated. Another possibility, not mutually exclusive, is that ectopic
w+ leads to anomalous 5-HT
levels in cells that interact with FRUM
neurons, deranging brain functions that are not directly
controlled by fruitless but are components of the neural
substrates for courtship. In any case, the lack of a simple
relationship between fruitless gene products and
serotonergic neurons, which would have bolstered the notion that both
abnormal genotypes cause their courtship effects via 5-HT depletion in
the same key brain cells, suggests that ectopic-white males
are made to behave in a manner that caricatures the phenotype of fruitless mutants (Lee, 2001).
In the course of these double-labeling tests, signals
elicited by anti-FRUM and anti-5-HT were scrutinized in all
CNS ganglia. Within the male's ventral cord, the great majority of
fru-expressing neurons in the four pairs of ganglia contain no detectable 5-HT. There was, however, an exception within one VNC region. It involves certain newly identified serotonergic cells in the abdominal ganglion. For these
neurons, coexpression of FRUM and 5-HT was
observed in a total of eight cells at the posterior tip of the VNC. These serotonergic-abdominal giant neurons (s-Abg) are
located close to one another in a relatively dorsal side of the
abdominal ganglion and have conspicuously large cell bodies. Larval serotonergic neurons in the developing nervous system are reorganized during metamorphosis. In this respect, putative precursors of the s-Abg neurons were not detected in the third-instar larval CNS or in the abdominal ganglion of 2-d-old male
pupae. Therefore, these s-Abg neurons in Drosophila may form during metamorphosis, or they may have been born earlier and only take
on their final neurochemical quality during late stages of
development (Lee, 2001).
With regard to the projection patterns of the s-Abg cells that were
revealed by 5-HT-immunostaining, each neuron appears to have more than one neurite. In most specimens, the s-Abg neurons are closely clumped together. A few preparations exhibited fairly clear bilaterality of these cell bodies and their posterior projections. These 5-HT-immunoreactive neurites also appear
to be within the median trunk (which is known to innervate posterior
abdominal segments), genital segments, and internal reproductive organs (Lee, 2001).
The putatively fru-related function of these cells and their
processes would seem to involve aspects of male reproduction because
the patterns of 5-HT immunoreactivity being described were not observed
in or posterior to the abdominal ganglion of adult females. Whether these cells exist in females, as opposed to
being present but devoid of 5-HT, is unknown. In this regard,
bear in mind that there is no FRUM
immunostaining anywhere in the CNS of females (Lee, 2001).
In fru-mutant males, anti-5-HT immunoreactivity in the s-Abg
neurons as well as the axons projecting from them was either absent or
defective. fru1 and frusat show low levels of transmitter staining in some of the s-Abg neurons and their processes (Lee, 2001).
In fru3, there is no detectable 5-HT
immunoreactivity in s-Abg neurons or their axons.
At best, fru4 mutant males present
weakly detectable 5-HT immunoreactivity in these structures. fru2 males are normal with respect to numbers of s-Abg neurons and their projections
(as stained by anti-5-HT), although the levels of staining intensity in
both subcellular compartments of these neurons appears to be lower
than in wild type (Lee, 2001).
For the most severely subnormal
mutant (fru3) in terms of FRUM and 5-HT
expression in the abdominal ganglion, it was not immediately possible
to determine whether the general absence of both kinds of
immunoreactivity is caused by an absence of s-Abg neurons or by the
lack of serotonin production in these cells. To address this question,
5-HT-uptake experiments were performed. These were based on the fact that exogenously applied 5-HT was found to be absorbed selectively by serotonergic neurons in the CNSs of third-instar larvae that express late-developmentally lethal Dopa decarboxylase (Ddc) mutations; relatively
severe (but viable) Ddc variants cause severe decrements in
5-HT synthesis. Ventral nerve cords from
adult fru3 males were exposed to a series
of 5-HT-creatinine concentrations. The resulting immunostaining led to the
following patterns. In wild-type VNCs, lowered endogenous 5-HT levels were observed in the serotonergic neurons that are undisturbed by this fru mutation; it was inferred that the incubation procedure necessary for 5-HT uptake is the major cause of this depletion. In fru3 VNCs, at
the lowest concentration applied to fru3
specimens, neither s-Abg-like cell bodies nor neurites could be recognized; but as
incubations with increasing 5-HT concentrations were performed, there
were increasing numbers of immunostained cells along with stronger
signal strengths. At the highest concentration of 5-HT applied, a subset of these structures in the ganglion of the mutant exhibited what appeared to be the appropriate immunoreactivity. The signals associated with the VNC cell bodies and processes in question appeared similar to those of genetically normal s-Abgs in their size, shape, and intraganglionic location. Thus, it seems as if at least some of these VNC cells are retained in this mutant and able to take up serotonin.
However, it was not possible to determine unambiguously whether the
normal fru/5-HT-expressing cells and their projections were
labeled in the fru3 ganglia. Therefore,
it remains an open question as to whether these neurons are eliminated by a developmental effect of this mutation, or whether, if present, the cells are unable to absorb exogenously applied 5-HT under the conditions used (Lee, 2001).
The discovery of sexually dimorphic s-Abg neurons in the abdominal
ganglion could provide an anatomical link to fru+-dependent sex-specific phenotypes not yet known to be influenced by this gene. The s-Abg
neurite signals elicited by anti-5-HT also provide the first
information on a projection pattern for fru-expressing
cells. These findings indicate that the formation of the s-Abg neurons
or production of 5-HT in them is male-specific and under fru
control. The 5-HT-uptake results suggest that s-Abg cells are
present but are unable to synthesize this transmitter in the
FRUM-less fru3 mutant (Lee, 2001).
Innervation by 5-HT/FRUM neurons of
abdominal muscles influenced by fru-gene action is unlikely.
This is because glutamate is the canonical neuromuscular transmitter in
Drosophila, although comprehensive information is understandably lacking as to whether this molecule is responsible for intercellular communication at all such
synapses. In addition to the muscles in this body region, there are
male-specific organs that have better-appreciated structures (compared
with unknown muscles hypothetically devoted to attempted copulation)
and functions (compared with the MOL) for which reproductive
significance is unknown. In this regard, the s-Abg neurons seem to send
their axonal projections into the median-trunk nerve, the terminal branches of which innervate the genital segments and internal reproductive organs as well as certain abdominal muscles (Lee, 2001 and references therein).
5-HT has been suggested to play a role in altered sexual orientation in
Drosophila (Zhang, 1995). A potentially close
connection between the action of FRUM and
serotonin in terms of inter-male courtship would have been worthy of
deeper consideration if these factors had been found to be coexpressed.
But the current results uncovered no overt 5-HT link to
fru-expressing brain neurons. The fact that there is a
distinctly separate colocalization of FRUM
and serotonin at the opposite end of the CNS suggests that
regulation of the presence and action of this neurotransmitter can be a
downstream target of fru function, an unexpected connection between the control of sexual differentiation and this piece of Drosophila neurochemistry. This bonus was but one of the
results of being able to monitor the presence of fruitless
gene products at high resolution (Lee, 2001).
A multibranched hierarchy of regulatory genes controls all aspects of somatic sexual development in Drosophila melanogaster. One branch of this hierarchy is headed by the fruitless (fru) gene and functions in the central nervous system, where it is necessary for male courtship behavior as well as the differentiation of a male-specific abdominal structure, the muscle of Lawrence (MOL). A preliminary investigation of several of the mutations described here has shown that the fru gene also has a sex-nonspecific vital function. The fru gene produces a complex set of transcripts through the use of four promoters and alternative splicing. Only the primary transcripts produced from the most distal (P1) promoter are sex-specifically spliced under direction of the sex-determination hierarchy. Eight new fru mutations, created by X-ray mutagenesis and P-element excision, have been analyzed to try to gain insight into the relationship of specific transcript classes to specific fru functions. Males that lack the P1-derived fru transcripts show a complete absence of sexual behavior, but no other defects besides the loss of the MOL. Both males and females that have reduced levels of transcripts from the P3 promoter develop into adults but frequently die after failing to eclose. Analysis of the morphology and behavior of adult escapers shows that P3-encoded functions are required for the proper differentiation and eversion of imaginal discs. Furthermore, the reduction in the size of the neuromuscular junctions on abdominal muscles in these animals suggests that one of fru's sex-nonspecific functions involves general aspects of neuronal differentiation. In mutants that lack all fru transcripts as well as a small number of adjacent genes, animals die at an early pupal stage, indicating that fru's function is required only during late development. Thus, fru functions both in the sex-determination regulatory hierarchy to control male sexual behavior through sex-specific transcripts; fru also functions sex-nonspecifically to control the development of imaginal discs and motorneuronal synapses during adult development through sex-nonspecific transcript classes (Anand, 2001).
It has been proposed that fru's role in controlling male sexual behavior is through the products of the distal (P1) fru promoter. Briefly, the reasoning is as follows. Genetic analysis has shown that while all aspects of male somatic sexual differentiation are controlled by the transformer (tra) and transformer-2 (tra-2) sex determination genes, doublesex (dsx), the only known sex determination regulatory gene below tra and tra-2 in the sex determination hierarchy, does not control two aspects of male sexual differentiation (the MOL and male sexual behavior). These results indicated that there is a previously unrecognized branch of the sex determination hierarchy below tra and tra-2 and has lead to the molecular search for other genes that are direct targets of tra and tra-2. That search identified fru as a direct target of tra and tra-2 because the transcripts from the P1 fru promoter are sex-specifically alternatively spliced under the control of the TRA and TRA-2 proteins.
fru function is known to be required for some aspects of male sexual behavior and the formation of the MOL, and the fru P1 transcripts are expressed in regions of the CNS previously implicated in certain aspects of male sexual behavior. This has provided a strong case for the products of the P1 fru promoter functioning to control aspects of sexual differentiation in the CNS needed for male sexual behavior and the MOL formation. However compelling that case may seem, it falls short of establishing that the products of the fru P1 promoter have the proposed functions, since there has no demonstration that mutations that impair P1 fru function alone have the expected phenotypes. Recent data consistent with the proposed function of P1 fru products have come from the following findings: (1) the fru1 allele, which affects male sexual behavior, is an inversion broken 5' to the P1 promoter and alters the spatial pattern of P1 expression in the CNS (and based on its location probably does not affect expression from the other fru promoters, and (2) the fru3 and fru4 alleles, which have strong effects on male sexual behavior, result in a substantial reduction in the amount of normal P1 and P2 (but not P3 and P4)-derived transcripts (Anand, 2001 and reference therein).
Further support for the proposed roles of the P1 fru transcripts is provided by various genotypes involving the new fru alleles reported here. The findings that males, which express P3 and P4 transcripts, but no P1 or P2 transcripts, are sterile and exhibit little or no male-specific reproductive behaviors in single-pair tests strongly support the hypothesis that the products encoded by fru's P1 (and possibly P2) transcripts control male sexual behavior. Flies of some of the these genotypes show reduced general activity and thus one might argue that this contributes to their reduced sexual behavior. However, as quantified in this study, there is in general only a weak correlation between the scores on courtship tests and the test of general activity. Moreover, for several genotypes, males had moderate levels of activity, indicating that for these genotypes the absence of male sexual behavior is unrelated to any general behavioral deficit (Anand, 2001).
Males having P1-encoded functions that are either lacking or seriously impaired show as severe decrements in their sexual behavior as males lacking both P1- and P2-encoded functions. This suggests that fru's role in sexual behavior may be entirely attributed to its P1-encoded functions. In single-pair tests of courtship behavior, males lacking or severely subnormal for P1 functions show essentially no sexual behavior. Moreover, all types of males show either normal or moderate levels of general activity. Since a genotype that lacks only P2-encoded functions is not available, the possibility that P2-encoded functions are also important for male sexual behavior cannot be excluded. However, all of the data currently available can be explained by the proposal that fru's P1-encoded functions are responsible for fru's role in male sexual behavior (Anand, 2001).
The new fru genotypes analyzed also provide some insight into which fru products are necessary for fru's vital function. With respect to whether P2 encodes a vital function, two aberrations, Df(3R)fru4-40 and In(3R)fruw27, both of which lack P1 and P2 transcripts and produce P3 and P4 transcripts, give completely discordant results when used in complementation tests with other fru alleles that lack fru's vital function. It is thought that this disparity is due to In(3R)fruw27 being defective in more than just fru's P1 and P2 functions (Anand, 2001).
In summary, these studies implicate the products of the P1 fru promoter as being responsible for fru's control of male sexual behavior and the products of the P3 fru promoter for carrying out fru's vital function. The functions of the products of the P2 and P4 promoters are currently unclear. However, it should be noted that genotypes that individually remove the functions encoded by P2-, P3-, or P4-derived transcripts are unavailable. Thus conclusions as to the functions of P1- and P3-derived transcripts are the simplest ones compatible with the data. Ghe possibilities that P2-, P3-, or P4-derived products may play some role in male sexual behavior or that P4-encoded products may also carry out a vital function cannot be excluded. Moreover, the findings that flies lacking P1 and P2 promoter-derived products are viable, whereas flies lacking P1-, P2-, and P3-derived products are lethal are most simply compatible with the proposal that P3 products carry out a vital function. These data do not preclude some models in which there is redundancy between the P1, P2, and P3 products in providing fru's vital function (Anand, 2001).
From a detailed analysis of the new fru alleles it has been shown that wild-type fru function is necessary for the production of nearly all aspects of male sexual behavior. fru mutant males lacking P1-encoded products no longer performs any courtship behavior with either single male or female partners in standard courtship tests. The observed failure of males of various fru genotypes to court might reflect a specific defect in sexual behavior, or alternatively it might be a by-product of a general reduction in activity. Although males of several fru genotypes did have reduced levels of short-term activity, regression analysis has shown that, overall, there is only a poor correlation between the level of activity and male courtship behavior; this indicates that low activity by these males could at most account for only a small part of the variation in male courtship behavior. Moreover, the reduction in courtship in single-pair tests occurs in some males that have moderate to high levels of activity. Further evidence indicating that reduced courtship with a single partner is a specific defect is shown by a number of fru genotypes in which males do not court a male or female partner but do engage in male-male group courtship as measured by the ChI. This dichotomy in CI vs. ChI shows that males are capable of generating at least some courtship behaviors in one situation but do not express that similar behavior in a different setting. Thus, the elimination of male courtship to both males and females in a subset of fru mutant genotypes reflects a sex-specific courtship effect of the loss of fru+ function (Anand, 2001).
There are two ways these results could be interpreted in terms of the wild-type function of fru. First, since male courtship is a dependent action pattern, with the occurrence of one step in the courtship sequence generally requiring the completion of preceding steps, these results could simply mean that these fru males are blocked in some way prior to the very earliest steps of courtship. Alternatively these results could mean that fru+ function is necessary for each step of courtship. The data support the latter alternative. Using several hypomorphic alleles, previous studies have shown that fru mutant males courted males and females but were blocked at certain individual steps occurring during courtship. For example, fru3 and fru4 males are blocked in the middle of the courtship behavioral sequence such that although they court they do not vibrate their wings to generate the courtship song and do not attempt copulation. By comparison, fru1 males are blocked only in the penultimate step of attempted copulation. These studies have provided strong evidence that male-specific fru+ function is involved with the specific neuronal circuitry required for the wing vibration needed for courtship song and the abdominal bending for copulation. Various heteroallelic combinations tested also show similar defects at intermediate to late stages of courtship. Thus many of the combinations of new fru alleles with fru1 or fru2 show significant levels of courtship activity as measured by either the CI or the WEI yet are sterile or have greatly reduced fertility due to failures to copulate. Similarly, a number of the fru genotypes tested fail to produce a courtship song during the reduced period that they are extending their wings. Taken together, these findings suggest that fru functions to establish the potential for essentially all aspects of male sexual behavior (Anand, 2001).
The exact molecular role that the P1 products play in controlling male-specific behaviors is not known, but they are likely to affect the development and differentiation of particular male-specific neurons and neuronal circuits. Of the clusters of P1-expressing neurons that have been identified, focus was placed on nine different clusters of P1-expressing neurons that have been identified by in situ hybridization in late stage pharate male brains and are prominent in sectioned preparations. Mutant males of a genotype that did not produce any courtship behavior appear to retain many or most of these neurons in the CNS even though these animals are not producing any functional P1 or P2 transcripts. This finding strongly suggests that the primary role of the P1 transcripts is to direct sex-specific differentiation and not direct the production or facilitate the survival of these neurons (Anand, 2001).
One aspect of the courtship phenotypes of the fru mutant combinations that requires special consideration is what might appear to be somewhat discordant results between single-pair courtship tests (both male-male and male-female) and the male-male group courtship (chaining) test. Of the 10 genotypes that were tested that lacked P1 function, all displayed little or no courtship in single-pair tests, but 6 of these genotypes displayed significant levels of male-male group courtship. In addition, of the 15 genotypes tested in which one of the new alleles was heterozygous with either fru2, fru3, or fru4, 12 genotypes displayed little or no courtship in single-pair tests, yet 13 of these genotypes had significant, often substantial, levels of male-male group courtship. In thinking about these data it is important to recall that although single-pair male-female courtship, single-pair male-male courtship, and male-male group courtship are all referred to as courtship, male-male group courtship is a mutant phenotype, whereas single-pair male-female courtship is the wild-type phenotype in D. melanogaster. Mature males courting each other in pairs essentially never occurs in a sustained manner in this species, so single-pair male-male courtship might also be viewed as a 'mutant' phenotype. Thus one might expect a priori that there would not be parallel dependencies of these three different 'courtship' phenotypes on fru function. In that regard it is worth recalling that the levels of courtship as measured by the CIs of males of a given genotype with single males or single females are highly correlated. Strikingly, a similar linear regression analysis of the mean CIm-m and the ChI across all fru genotypes shows a similarly strong positive correlation. These high degrees of concordance suggest that these three behavioral assays are measuring behaviors that are largely equivalent in their fru dependency. The difference between wild type and any of the fru mutants that have been tested to date would be that in the fru mutant situations males as well as females would be seen as appropriate partners for whatever level of courtship males of a particular genotype can attain (Anand, 2001).
As was just noted for a number of genotypes, males show little or no courtship in single-pair tests but are able to carry out the very first steps of courtship (orientation and following), but not later steps, of courtship in group tests. These differences within a genotype are likely related to the different ways single-pair and group tests are done. In particular, male-male group tests are carried out by placing a group of males together for 3-4 days prior to testing, whereas males used in single-pair courtship tests were stored individually until tested. The richer environment experienced by males used in male-male group courtship tests (more stimulation through dynamic interactions between animals and a longer time for association between animals) is likely the reason for the different levels of courtship seen in single-pair and group tests. The preceding interpretation is suggested by the prior observation that fru2, fru3, and fru4 homozygous males chained little, if at all, when initially placed together in groups, but that the level of chaining increased over the next couple of days. The finding that fru mutant animals perform some courtship steps under group conditions also shows that the appropriate parts of the brain for at least the very initial steps of courtship are still present and can be activated in these genotypes but that the conditions of the test for pairwise courtship do not lead to the activation of these same centers and the expression of orientation and following (Anand, 2001).
Another striking aspect of these data is that 6 of the 10 genotypes that completely lack P1 promoter-encoded functions showed significant levels of male-male group courtship. The finding that 4 of these 10 genotypes showed no courtship in either single pairs or as groups of males is consistent with the conclusion that it is the products of the P1 fru promoter that establish the potential for male sexual behavior. However, the results from the 6 genotypes that show significant male-male group courtship suggest that other factors are involved that are not understood. There does not seem to be an obvious genetic explanation for these data, since genotypes that should be identical in terms of their arrays of fru products do not have equivalent phenotypes (Anand, 2001).
It is suggested that the products of the P1 fru promoter are responsible for both MOL development and male sexual behavior. An obvious question is whether this fru-regulated male-specific muscle has any connection with fru-regulated male-specific courtship behavior. To date, the physiological function of the MOL is unknown. It has been demonstrated that the MOL is not necessary for copulation to occur in this species, and, in fact, the males of most Drosophila species do not even develop a MOL (Anand, 2001).
Given the data indicating that the P1-derived male-specific FRU proteins govern MOL development, a recent report comes as a surprise: it found that expression of a fru cDNA construct encoding a protein lacking the 101-amino-acid male-specific N terminus could partially rescue the MOL phenotype in frusat homozygous males and lead to MOL development in wild-type females was surprising. The fact that a Fre protein can induce MOL development in an otherwise wild-type female provides striking evidence that Fru function is sufficient to trigger MOL development. With respect to the question of which class of Fre proteins controls MOL development in wild type, the following is noted. The Fru protein shown to be capable of producing a MOL is identical to a P1-derived Fru protein, except for the absence of the male-specific 101-amino-acid N terminus. It would not be surprising if there were some overlap in the functionality of these two proteins. The heat-shock and GAL4 expression systems used for Fru expression are likely to produce Fru protein at significantly higher levels than are present for that protein in wild type. Thus the production of MOLs in their experiments may be being brought about by the overexpression of a protein whose function partially overlaps that of P1-encoded proteins. Alternatively, it could be that these proteins are functionally fully synonymous, and these functions are biologically determined by the locations and levels at which they are expressed (Anand, 2001).
Studies of the phenotypes of lethal fru mutant combinations provide insights into the nature of fru's essential function. Genotypes that lack P3 (and P1 and P2) promoter function have lethal phases in the mid- to late-pupal period, but in all cases a substantial fraction of these individuals are viable if assisted in emerging from the pupal case. Thus the vital function encoded by P3 is only essential very late in development (Anand, 2001).
P3-encoded functions appear to be necessary for the differentiation of imaginal-disc derivatives, such as legs and wings. The rare survivors with fru lethal genotypes show defects in their external morphology and have specific sensory bristle organs duplicated. Moreover, imaginal discs frequently failed to evert in adult escapers of genotypes that lacked P3 function. These observations suggest that there is a role for fru in imaginal disc development or differentiation. The gross morphological defects produced in adult derivatives of imaginal discs makes it likely that the nearly lethal phenotype of fru genotypes lacking P3 function arises from these defects (Anand, 2001).
In addition, many of the adult escapers that lack P3 function have neuroanatomical defects in the motor innervation to the abdominal muscles. These defects in the motorterminals may explain part of the inability of many of these fru mutant genotypes to successfully eclose from the pupal case. All abdominal muscles appear to be innervated, suggesting that the defects in these fru mutants are not due to abnormalities in neuronal pathfinding but rather some feature of synaptogenesis once the motorneuronal axons reached their target muscles. Changes in the branching of motorneurons at the neuromuscular junction in larval Drosophila have been described for various mutants that affect neuronal excitability. In other respects the development of the nervous system of fru mutant genotypes appears normal. No obvious change in the number or patterns of divisions of postembryonic neuroblasts was found in either a null fru genotype or a genotype lacking P1, P2, and P3 functions, suggesting that the normal complement of adult-specific neurons is likely produced in the complete absence of fru function. The apparently normal level of postembryonic divisions of neuroblasts in the larval CNSs of mutants which are destined to die within a day, makes it unlikely that the loss of adult neurons is responsible for the death of these animals (Anand, 2001).
While there is currently not a fru genotype that is null for all fru functions and wild type for neighboring genes, it was possible to place a limit on how extreme a completely null fru genotype might be by examining the lethal phase of mutant combinations that are null for fru as well as a small number of adjacent genes. The development of these individuals is arrested in early pupal development, around the time of pupal ecdysis. The morphology of the pupal case in these genotypes is similar to the phenotypes of animals mutant for the ecdysone receptor and the crooked legs gene. However, the fru genotypes lack not only all fru function but also the function of at least four, and possibly six, neighboring genes. Thus it is not clear if the earlier lethal phase caused by these genotypes -- as compared to fru genotypes that are lacking just P1, P2, and P3 function -- is a consequence of the absence of P4 function or the absence of one of the neighboring genes. In any case, these two null genotypes establish that a complete absence of fru function does not result in a lethal phase prior to the beginning of the pupal period (Anand, 2001).
In summary, these results establish that fru functions sex-specifically in the sex-determination regulatory hierarchy to control male sexual behavior and sex-nonspecifically to control the development of imaginal discs and motorneuronal synapses during development. Perhaps most importantly these results show that just the absence of the products of the P1 fru promoter results in the loss of all normal male sexual behavior and thus provide strong support for the proposal that these proteins function to establish the potential for nearly all components of a Drosophila male's sexual behavior (Anand, 2001).
The fruitless (fru) gene acts sex-nonspecifically in the development of the embryonic central nervous system (CNS) and has sex as well as sex-nonspecific functions in the development of the adult CNS. In the embryo, sex-nonspecific fru mRNAs and proteins are widely expressed during neurogenesis and present in both neurons and glia. To assess whether the fru gene plays any role in fate determination of neuronal precursors and neurons, the development of Eve-positive [Eve(+)] GMCs and neurons was examined in fru mutants. In fru mutant embryos in which most or all fru transcripts are eliminated, the normal complement of Eve(+) neurons is present initially, but some neurons are unable to maintain their Eve-expression. Concomitantly, a subset of Eve(+) neurons also showed inappropriate expression of the glial marker, Reversed polarity. In addition, neurons that normally do not express Eve became Eve(+) in these fru mutants. These defects are rescued in fru mutant embryos expressing specific fru transgenes under the control of the sca-GAL4 and elav-GAL4 drivers. These phenotypic analyses and rescue experiments provide evidence that one of the sex-nonspecific functions of the fru gene is the maintenance of neuronal identity rather than establishment of a neuron's initial fate (Song, 2003)
The fruitless gene in Drosophila is a multifunctional gene that has sex-specific functions in the regulation of male sexual behavior and sex-nonspecific functions affecting adult viability and external morphology. While much attention has focused on fru's sex-specific roles, less is known about its sex-nonspecific functions. This study examines fru's sex-nonspecific role in embryonic neural development. fru transcripts from sex-nonspecific promoters are expressed beginning at the earliest stages of neurogenesis, and Fru proteins are present in both neurons and glia. In embryos that lack most or all fru function, FasII- and BP102-positive axons have defasciculation defects and grow along abnormal pathways in the CNS. These defects in axonal projections in fru mutants were rescued by the expression of specific UAS-fru transgenes under the control of a pan-neuronal scabrous-GAL4 driver. These results suggest that one of fru's sex-nonspecific roles is to regulate the pathfinding ability of axons in the embryonic CNS (Song, 2002).
To determine the spatial and temporal distribution of embyonic fru transcripts, in situ hybridizations were carried out with antisense-BTB and antisense-Com riboprobes, which detect most or all fru transcripts. fru mRNAs are expressed in a dynamic temporal and spatial pattern from the beginning of embryogenesis until stage 16. fru transcripts are uniformly distributed in very early embryos and become incorporated into segregating pole cells. At the start of gastrulation (stage 6), heavily labeled cells are found in the ventral and cephalic furrows. In slightly older embryos (stages 7-9) the most prominent distribution of fru transcripts is found in the developing CNS within mesectodermal and ventral neuroectodermal cells. Transcripts become localized to delaminating neuroblasts and after stage 10, fru transcripts are detected in medial but not in lateral neuroblasts. Following expression in the progeny of neuroblasts, the level of fru expression in the CNS continues to decline until becoming undetectable at stage 16. Thus, fru transcripts are expressed throughout the development and early differentiation of the CNS but become undetectable at later stages. Cells in some non-CNS tissues, the amnioserosa and tracheal placodes, also expressed fru transcripts (stages 9-11), but no in situ hybridization signal was detected in other tissues, such as the PNS or body wall muscles (Song, 2002).
The fru locus encodes a complex set of transcripts. To better understand the embryonic pattern of fru transcripts, in situ hybridization was performed with a set of 5' end riboprobes to distinguish transcripts made from different fru promoters. No P1 or P2 transcripts were detected at any embryonic stage using riboprobes specific to these transcripts. Thus, P1 transcripts, which encode the male-specific fru proteins, are not expressed in the embryo. Transcripts from P3 and P4 promoters were expressed during embryogenesis in a temporal and spatial pattern that mirrored that of fru transcripts detected by antisense-BTB or -Com riboprobes. In the developing CNS, both P3- and P4-specific riboprobes labeled mesectodermal and neuroectodermal cells followed by labeling of delaminating neuroblasts. At slightly later stages (stages 9-11), medial neuroblasts continued to express P3 and P4 transcripts, but lateral neuroblasts no longer had detectable transcripts. In stages 7-12 embryos, the in situ hybridization signal for P4 transcripts was less intense, suggesting that the level of P4 transcripts was lower than that of P3 transcripts in these stages. However, P3 transcript levels became undetectable in all tissues after stage 12, whereas P4 transcripts were still detectable up to stage 16. In summary, the in situ hybridization data show that both P3 and P4 fru transcripts are expressed in the developing CNS and, very likely, in the same cells. Furthermore, the higher level of fru transcripts detected with riboprobes to fru common sequences in stages 9-12 CNSs is likely due to the presence of both P3 and P4 transcripts whereas the lower level of fru transcripts in stages 12-16 CNSs reflects the presence of only P4 transcripts (Song, 2002).
An additional complexity in fru transcripts reflects alternative splicing at the 3' end, which generates transcripts containing one of three different pairs of Zn-finger domains. While it is known that P1 transcript isoforms are spliced to each of the three alternative 3' ends, the full complexity of the 3' alternative splicing of transcripts produced from the P2, P3, and P4 promoters is not known. Embryos were examined by in situ hybridization with three different 3' end riboprobes to detect transcripts having the A, B, or C 3' ends. Overall, the temporal and spatial expression pattern of transcripts containing the A, B, and C 3' ends was consistent with the pattern found for fru transcripts labeled with antisense-BTB and antisense-Com riboprobes. In the developing CNS, transcripts containing the C 3' end appeared to be more abundant than those containing the A or B 3' ends. In addition, some tissues, such as the tracheal placodes and amnioserosal cells, were labeled only when riboprobes to the C 3' end were used, suggesting that there is some tissue-specific regulation of the 3' alternative splicing of P3 and or P4 fru primary transcripts. The localization of Fru proteins during embryogenesis is largely consistent with the spatial and temporal RNA distribution. All Elav-positive cells in the CNS (neurons) express Fru proteins, even though there were variable levels of Fru protein expression in individual cells. Likewise, all Repo-positive cells in the CNS (glia) were also Fru positive. In summary, all neurons and glia appear to express Fru proteins. All neurons and some lateral glia contain Fru isoforms having at least two different carboxy termini, but some lateral glia and midline cells contain Fru proteins that may have only one type of carboxy terminus (Song, 2002).
Given the findings that the fru gene is widely expressed in the embryonic CNS, fru mutant embryos are likely to have defects in CNS development: two antibodies, anti-FasII and mAb BP102, were used to assay axonal projections within the CNS. FasII is a neural adhesion molecule expressed on the cell surface of axons forming specific longitudinal fascicles or tracts running throughout the entire ventral nerve cord and into the brain. In whole-mount or flat-dissected preparations of stage 16 and 17 wild-type embryos, three tracts, medial, intermediate, and lateral, are visible in the CNS (Song, 2002 and references therein).
FasII-positive tracts are abnormal in fru mutants lacking all or most fru transcripts. Between 12% and 25% of fru null mutant embryos have abnormal FasII-positive