dissatisfaction

DEVELOPMENTAL BIOLOGY

Larvae, Pupae and Adults

dissatisfaction is necessary for the expression of appropriate sex-specific behaviors and the formation of specific motoneuronal terminals in both sexes but does not seem to be required for general neurological functions. To determine if dsf is expressed generally or in a limited set of cells, in situ hybridization was used with antisense RNA probes to cryostat sections of wild-type larvae, early pupae, mid-pupae, and pharate adults. RNA expression is detected in only a small subset of neurons at any stage. In wandering larvae of both sexes, there were three labeled groups of cells in the anterior region of the brain. The first group is found at the level where the esophagus passes through the brain hemispheres and the other two groups are located more dorsally. All three groups are visible in a sagittal section through the larval brain. The only other tissue that appears to be faintly labeled in larvae is the salivary gland. In pupal brains, signal is also detected in several small groups of cells in the anterior regions of the protocerebrum but not in other regions of the brain or ventral nerve cord or in other tissues. In pharate adult brains, only a few labeled cells are found. About 20 large neurons, not obviously part of a single cluster of neurons, are located close to the antennal lobe and lateral protocerebral neuropil in bilaterally symmetrical arrays. A cluster of about 10 cells is found just below the chemosensory region of the antennal lobe and near the mechanosensory region of the antennal lobe and subesophageal neuropils. The location of the dsf-hybridizing neurons near the antennal lobes suggests that dsf may function in antennal interneurons or in part of the neuronal pathways that mediate sensory integration of chemical signals. In a small number of cases, pupal and pharate adult brains contain a few labeled neurons at the dorsal-most regions of the brain along the midline. The relationship between cells labeled in third instar larvae and adults has not been determined, but the labeled cells are in relatively the same positions at both stages. These results clearly suggest that dsf identifies a small number of neurons, out of a nervous system of 50,000 neurons, that are likely to be involved in control of the development and function of the sex-specific nervous system. The pattern of labeled neurons is consistent with other data suggesting that these cells may be involved in the control of male and female sexual behavior and focuses attention on small subregions within these larger divisions of the brain. The pattern of cells expressing dsf is different from the fruitless expression pattern in many regions of the brain and ventral nerve cord, consistent with the idea that dsf and fru must act, at least partially, independently of one another. Further reinforcing the idea that dsf and fru act independently, dsf expression has been shown to be normal in fru mutants (Finley, 1998).

Effects of Mutation or Deletion

Few mutations link well defined behaviors with individual neurons and the activity of specific genes. In Drosophila, recent evidence indicates the presence of a doublesex-independent pathway controlling sexual behavior and neuronal differentiation. A gene, dissatisfaction, has been identified that affects sex-specific courtship behaviors and neural differentiation in both sexes without an associated general behavioral debilitation. Homozygous dsf females fail to lay eggs voluntarily or under CO₂ anesthesia. Eggs mature normally in the ovary and pass through the oviducts to lodge within the uterus, where they degenerate. No fertilized eggs are detected among the eggs found lodged in the uterus, even though motile sperm are transferred to the female during copulation and are stored normally in sperm storage organs. Thus, mature eggs are able to reach the uterus from the ovaries, suggesting that the failure to lay eggs results from defects within or distal to the uterus and not in the upper portions of the reproductive tract. The egg-laying defect in females correlates with the absence of motor neuronal innervation on uterine muscles. In dsf female abdomens, the full complement of segmental abdominal muscles and genital muscles is present, and there is no evidence of masculinization of any muscles. No muscles with MOL (a male-specific muscle spanning the fifth abdominal segment in adult males, the "muscle of Lawrence") morphology are found. Innervation of the segmental abdominal muscles and all but one set of genital muscles appear normal. In the single exception, no synapses are detected on the circumferential muscles of the uterus of mutant females, while these muscles in wild-type females are extensively innervated. Only sensory axons appear to contact the uterus in dsf females, which suggests that the two to four motorneurons that innervate the uterus in wild-type females do not reach the uterus in dsf females. Nearby visceral muscles associated with the spermathecae, sperm receptacle, and oviduct are innervated and appear normal. The lack of uterine muscle innervation is likely sufficient to account for the egg-laying deficit. Likewise, examination of XX; tra-; hs-tra females shows that there is a substantial reduction in the number of boutons present on the circular uterine muscles, consistent with the idea that XX; tra; hs-tra- and dsf mutants fail to lay eggs for a similar reason (Finley, 1997).

Male and female mutant animals exhibit abnormalities in courtship behaviors, suggesting a requirement for dsf in the brain. Virgin dsf females resist males during courtship and copulation. dsf males actively court and attempt copulation with both mature males and females but are slow to copulate because of maladroit abdominal curling. Courtship and mating behavior of dsf males was examined to determine if components of their behavior are abnormal. Both dsf / dsf and dsf / Deficiency males actively court mature males even with virgin females present. In tests involving male-only groups, courtship included all behaviors up to and including attempted copulation with the production of short-lived chains of courting males. dsf males court both mutant and wild-type males but are not themselves courted by wild-type males. dsf males were also assayed for courtship of mature wild-type virgin females. Although dsf males initiate courtship as rapidly as wild type, actively court females, and are fertile, single-pair tests demonstrate that dsf males are substantially delayed in their time to copulation. From video observation of courting pairs, it was noted that target females respond normally to dsf courtship by stopping and positioning themselves for copulation, while the dsf males are defective in the final step of courtship, abdominal curling. Males need to bend their abdomen about 180° to make genital-genital contact with females and copulate. From data collected from videotaped records, it is clear that during courtship of females dsf males make fewer bends that fall into the maximum degree category, which would be sufficient for copulation, and more shallow bends than do wild-type males. Since only about 10% of the bends made by dsf males are in the range sufficient for copulation, this inability to produce deep bends likely accounts for the increase in time to copulation noted for dsf males (Finley, 1997).

Structural abnormalities in specific neurons indicate a role for dsf in the differentiation of sex-specific abdominal neurons. The reduced abdominal curling in males correlates with alteration in motor neuronal innervation of male ventral abdominal muscles. The poor abdominal curling of dsf males prompted an examination of their abdominal musculature and innervation. A full complement of abdominal muscles, including normal MOLs and genital muscles, is present in dsf/dsf and dsf/Df males. Examination of muscle innervation with four different anti-neuronal antibodies shows that the nerve terminals, including those on the MOL, are apparently morphologically normal, except for those on one muscle group. Innervation of the ventral longitudinal muscles of abdominal segment 5 (A5) is abnormal in dsf mutants, when compared with other abdominal segments of mutant males and wild-type males. The most striking feature of ventral A5 innervation in mutants is the presence of a few large spherical boutons on each fiber rather than strings of small boutons cascading from the point of nerve contact with the muscle. This mutant phenotype does not appear in dsf females and reveals, for the first time, that innervation of the ventral muscles of A5 is sex-specific, even though the normal pattern of neural connections looks similar between males and females. The improper innervation of ventral A5 is consistent with a causal role for the slow abdominal bends made by dsf males, although additional dysfunction in central nervous system neural connections cannot be ruled out (Finley, 1997).

Epistasis experiments show that dsf acts in a tra-dependent and dsx-independent manner, placing dsf in the dsx-independent portion of the sex determination cascade. Analysis of sex-specific neural and behavioral phenotypes suggests that genes regulating these phenotypes act downstream of tra. If so, XX;dsf- animals masculinized by mutations in tra will have the male-specific ventral neuronal phenotype shown by XY;dsf- males. To test this, ventral abdominal innervation of XX;tra- and XX;dsf-; tra- individuals were examined. XX;dsf-;tra- animals show the dsf phenotype while their dsf⁺; tra- siblings do not. Thus, it is inferred that dsf acts downstream of tra for development of ventral abdominal neuromuscular junctions. At the same time, these data rule out models in which some alternative pathway involving upstream elements such as the X chromosome to autosome ratio or Sxl, which are identical in both tested XX genotypes, independently regulates this process. To test dsf dependence on dsx function, advantage was taken of a gain-of-function dsx mutant, dsx^D, which expresses the male Dsx protein regardless of tra and tra2 activity. In the absence of a wild-type dsx allele, both sexes develop external male morphology. Even so, XX animals express tra and XY flies do not. If dsf acts independently of dsx, it is expected that XX; dsf-; dsx^D/Df (tra ON) animals will have normal ventral innervation and XY; dsf-; dsx^D/Df (tra OFF) animals will have abnormal ventral innervation: the dsf mutant phenotype. If dsf is dependent on the activity of dsx, then XX and XY; dsf-; dsx^D/Df animals should have equivalent and mutant phenotypes. The neurons in ventral A5 were examined for different genotypes of dsx^D/Df mutant animals. XX; dsf/Df; dsx^D/Df are wild type in appearance while XY;dsf/Df; dsx^D/Df have a dsf phenotype. This result is consistent with the idea that dsf is part of a dsx-independent pathway (Finley, 1997).

The isolation and analysis of Drosophila mutants with altered sexual orientation lead to the identification of novel branches in the sex-determination cascade that govern the sexually dimorphic development of the nervous system. One such example is the fruitless (fru) gene, the mutation of which induces male-to-male courtship and malformation of a male-specific muscle, the muscle of Lawrence (MOL). Since the MOL is formed in wild-type flies when the innervating nerve is male, regardless of the sex of the MOL itself, the primary site of Fru function is likely to be the motoneurons controlling the MOL. The fru gene produces multiple transcripts including sex-specific ones. A female-specific mRNA from the fru locus has a putative Transformer (Tra) binding site in its 5' untranslated region, suggesting that fru is a direct target of Tra. The fru transcripts encode a set of proteins similar to the BTB (Bric a brac, Tramtrack and Broad-complex)-Zn finger family of transcription factors. Mutations in the dissatisfaction (dsf) gene result in male-to-male courtship and reduced sexual receptivity of females. The dsf mutations also give rise in males to poor curling of the abdomen during copulation, and in females, to a failure of egg-laying. The latter phenotypes are ascribable to aberrant innervation of the relevant muscles. A genetic analysis reveals that expression of the dsf phenotypes depends on Tra but not on Doublesex (Dsx) or Fru, suggesting that dsf represents another target of Tra. The effect of dsf mutation was determined in chromosomal females after sexual transformation with tra- (Finley, 1997). In the dsf+;tra- XX flies, the ventral abdominal muscles are innervated by normal locking boutons, as is the case in wild-type males (XY dsf+). In contrast, the corresponding muscles in dsf-;tra- XX flies are associated with enlarged round boutons, just like those seen in mutant males (XY dsf-). The expression of the dsf mutant phenotype depends on tra due to the fact that dsf is located downstream of tra in the sex-determination cascade. In accordance with the idea that dsf is downstream of tra, a moderate reduction in tra+ activity in females eliminates motor innervation of the uterine muscle, inducing a dsf phenotype. Taken together, these findings suggest that the sex-determination protein Tra has at least three different targets: dsx, fru and dsf, each of which represents the first gene in a branch of the sex-determination hierarchy functioning in a mutually-exclusive set of neuronal cells in the Drosophila central nervous system (Yamamoto, 1998).

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, 2000).

Loss-of-function mutations affecting the dissatisfaction (Dsf) nuclear receptor alter both sexual behavior and the sex-specific nervous system in Drosophila. As a step toward understanding the way Dsf controls development and function of the nervous system, the regulatory activities of the Dsf protein have been analyzed. Dsf prefers an atypical DNA half site, AAGTCA. Wild-type Dsf, but not the point mutant Dsf⁷, monomerically binds and represses reporter constructs bearing this site. Dsf also contains an atypically long, 356-amino-acid hinge separating its DNA-binding domain (DBD) and ligand-binding domain (LBD). The hinge contains at least two functions: a region that drastically lowers DNA-binding efficiency in vitro, and an amino-terminal repressive domain. The DBD and LBD of Dsf, along with major portions of the hinge, are highly conserved in other insects. Ectopic expression of Dsf in Drosophila imaginal discs results in developmental disruptions in disc-derived tissues, disruptions which are largely suppressed when Dsf is fused to the VP16 activation domain, consistent with a repressive role for Dsf. Finally, when tethered to DNA, Dsf's hinge and LBD regions act as strong transcriptional repressors in multiple larval and pupal tissues, including many Dsf-expressing tissues. These results suggest Dsf can repress transcription in vivo, that repression is largely responsible for its ectopic expression phenotypes, and that repression may be a key component of normal Dsf function (Pitman, 2002).

Finley, K. D., et al. (1997). dissatisfaction, a gene involved in sex-specific behavior and neural development of Drosophila melanogaster. Proc. Natl. Acad. Sci. 94: 913-918.

Finley, K. D., et al. (1998). dissatisfaction encodes a Tailless-like nuclear receptor expressed in a subset of CNS neurons controlling Drosophila sexual behavior. Neuron 21(6): 1363-74.

Pitman, J. L., et al. (2002). DSF nuclear receptor acts as a repressor in culture and in vivo. Dev. Biol. 245: 315-328. 11977984

Yamamoto, D., Fujitani, K., Usui, K., Ito, H. and Nakano, Y. (1998). From behavior to development: genes for sexual behavior define the neuronal sexual switch in Drosophila. Mech. Dev. 73(2): 135-146.

date revised: 10 October 2002

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