BIOLOGICAL OVERVIEW

The dissatisfaction (dsf) gene is essential for many aspects of sexual behavior and neural development in both males and females (Finley, 1997). dsf males are bisexual, courting both males and females, defective in the abdominal bending associated with copulation, and have abnormal synapses on the ventral muscles of abdominal segment 5. dsf females show increased resistance to males during courtship and continued resistance during copulation. These females are sterile because they fail to lay eggs; this failure is associated with loss of motoneuronal innervations on the circular muscles of the uterus (Finley, 1997). The dsf gene is shown to codes for a zinc finger transcription factor more closely related to mammalian Tailless homologs than to Drosophila Tailless itself (Finley, 1998).

Somatic sexual differentiation in Drosophila is well characterized, and is reviewed here briefly. Dissatisfaction functions downstream of Sex lethal (Sxl), Transformer (Tra) and Transformer 2 (Tra2), but independent of Fruitless (Fru) and Doublesex (Dsx). In females, Sex-lethal protein is present and functional. Sxl controls somatic X chromosome dosage compensation and female germline and somatic development. For the somatic sexual pathway, the presence of Sxl protein maintains both the continued production of Sxl protein and the production of active transformer (Tra) protein by Sxl-mediated alternative splicing of SXL and TRA mRNAs. Active Tra protein, in conjunction with Tra2 protein, regulates multiple targets in females. In males, neither Sxl nor Tra proteins are produced. Downstream of tra and tra2, the sex differentiation cascade splits. One branch headed by doublesex controls the development of sex-specific external structures in both sexes, such as the genitalia, and production of yolk proteins but has only a relatively limited role in development of the sex-specific nervous system and male behavior. A second branch defined by fruitless controls aspects of male sexual behavior and nervous system development. Loss of fru in males leads to various sexual abnormalities, including failure to court, bisexual courtship, abnormalities in the courtship ritual, and failure to attempt copulation. Loss of the female-specific portion of fru function in females has no known phenotypic consequences. Sex-specific function of dsx and fru is controlled by Tra and Tra2-mediated alternative RNA splicing, leading to the production of sex-specific RNAs and proteins (Finley, 1998 and references).

Genetic studies show that dsf acts downstream of tra and is necessary for both male and female reproductive functions. dsf is not downstream of the male dsx pathway (Finley, 1997). A comparison of phenotypes between fru and dsf, especially the significant female dsf phenotypes, shows that dsf must act independent of fru, at least in females. From these observations, it has been suggested that dsf defines the existence of a non-fru, non-dsx pathway for the control of some aspects of sexual behavior and neural development (Finley, 1997 and Finley 1998).

Analysis of dissatisfaction expression reveals that it is expressed in a limited number of cells in the central nervous system. Initial attempts at analyzing the dissatisfaction promoter revealed that it targets dsf expression to key cells involved in controlling sexual behavior. This targeting capacity was used to design experiments to discover the neural basis of dsf regulated behavior. dsf enhancers were used to change the sex of subsets of cells in otherwise wild-type males. A series of restriction fragments from the dsf region, covering from the middle of the 11 kb fragment to the 4 kb fragment encoding the ligand binding domain, were cloned upstream of a basal promoter controlling GAL4 expression. The hybrid genes were then transformed into Drosophila. Next, these Enhancer-GAL4 fusion genes were used to drive expression of a female transformer cDNA under the control of a Gal UAS (UAS-tra). In this system, expression of a tra female cDNA under control of the 1.8 kb EcoRI fragment from the middle of the large dsf intron generates interesting behavioral phenotypes in males. Such males are externally normal, court females, and are fertile when they copulate. However, when tested with males, they show significantly elevated levels of male-to-male courtship. Thus, the 1.8 kb EcoRI fragment contains an enhancer that drives expression in some cells that must be male for normal male sex partner choice (Finley, 1998).

As a way of mapping which cells are expressing TRA mRNA female in these animals, and therefore which ones might be responsible for the alteration in behavior, the same 1.8 kb EcoRI-GAL4 construct was used to drive expression of membrane-targeted, modified green fluorescent protein (EGFPF) as part of a UAS-GFP construct. Expression is limited to a small fraction of CNS cells, including cells along the dorsal midline of the brain, in the antennal lobe, mushroom bodies, subesophageal ganglion, and retinal neurons. This GFP pattern shows more cells than are detected by in situ hybridization, but GFP is present in all regions in which dsf expression is seen by in situ hybridization at this and other stages. Notable departures from the dsf in situ pattern include expression in the mushroom bodies, retinal neurons, jump neurons, and some neurosecretory cells. In addition, a small number of cells in the abdominal ganglion express GFP, and labeled neuronal projections onto the female reproductive tract have been observed (Finley, 1998).

The brain regions identified with the UAS-tra system overlap to some extent with the brain areas identified as necessary for male behavior in XX/XO mosaics. The dsf expression pattern is consistent with these results and indicates the importance of even small numbers of cells within these regions. Compared to male behavior, female sexual behavior has been relatively understudied: only one region of the brain, the anterior dorsal protocerebrum, has been identified as a focus for female receptivity. The dsf expression pattern in the protocerebrum of females is within this identified focus, and it is speculated that dsf might be expressed in at least some of the neurons involved in female receptivity (Finley, 1997 and references).

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 dsf function, proper ventral A5 abdominal innervation in males, is downstream of tra but not downstream of dsx. These results lead to the inference 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 MOLs (a male-specific muscle spanning the fifth abdominal segment in adult males, the "muscle of Lawrence"), 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).

GENE STRUCTURE

Examination of the dsf genomic sequence reveals a single copy of the sequence TCATCATCAACAT about 5/7 of the way through the largest intron. This is a variant of the Tra and Tra2 binding sites in dsx and fru, with a single missing base exactly in the middle. Exhaustive experiments rule out the possibility of sex-specific mutually exclusive exon use of the 5' or 3' regions (Finley, 1998).

Bases in 5' UTR - 255

Exons - 5

Bases in 3' UTR - ~707

PROTEIN STRUCTURE

Amino Acids - 693

Structural Domains

There is high sequence similarity between Dsf and the vertebrate Tailless proteins in both the DNA binding domain (DBD) and ligand binding domain (LBD). The greatest similarity between Dsf and vertebrate Tailless is in the DBD and adjacent T box region, with an overall identity of 81%, including 100% identity in the P box and T box sequences. The human, mouse, and chicken Tailless LBD sequences are rated most similar to Dsf, with 44% amino acid identity. The degree of similarity between Dsf and the vertebrate Tailless proteins in the LBD is greater than the similarity between human Tailless and any protein that is not a vertebrate Tailless protein. The LBD of Drosophila Tailless is related at a substantially lower degree (35% identity). In addition to the high degree of similarity in sequence in the DBDs and LBDs, DSF and Tailless proteins are similar in having relatively short amino-terminal and carboxy-terminal sequences preceding the DBDs or following the LBDs. Dsf diverges from all Tailless proteins within the D box region of the DBD and in the A box region that follows the DBD and T box sequences. Dsf also contains a notably longer sequence (380 amino acids) linking the DBD with the LBD that has no similarity to Tailless proteins or to any other proteins in GenBank (Finley, 1998 and references).

date revised: 26 January 98

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