dissatisfaction, a gene involved in sex-specific behavior and neural development of Drosophila melanogaster

Abstract

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. We have identified a gene, dissatisfaction (dsf), that affects sex-specific courtship behaviors and neural differentiation in both sexes without an associated general behavioral debilitation. 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 and fail to lay mature eggs. dsf males actively court and attempt copulation with both mature males and females but are slow to copulate because of maladroit abdominal curling. Structural abnormalities in specific neurons indicate a role for dsf in the differentiation of sex-specific abdominal neurons. The egg-laying defect in females correlates with the absence of motor neuronal innervation on uterine muscles, and the reduced abdominal curling in males correlates with alteration in motor neuronal innervation of male ventral abdominal muscles. 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.

Figure 1

Regulation of the sex determination cascade. The top line shows a schematic diagram of the regulatory relationships of the sex differentiation cascade. Solid lines represent definitively characterized interactions. Dotted lines represent possible additional relationships (2, 24, 25). Genetic data imply the existence of other genes that control behavior and neural development, which are under the control of tra and tra2 but not regulated by dsx (24, 26). One gene proposed as a candidate for such regulation is fruitless (fru; refs. , , and 25). The work in this paper suggests dissatisfaction (dsf) as a second candidate for a member of a dsx-independent pathway controlling behavior.

Figure 2

dsf behavioral phenotypes. The genotypes examined are (genotype 1) wild type (+/+), (genotype 2) dsf/+, (genotype 3) Df/+, (genotype 4) dsf/dsf, and (genotype 5) dsf/Df. (A) dsf females resist male courtship. Individual aged virgin females of the genotypes shown were paired with wild-type (Canton-S) males, and the time from the initiation of singing to copulation was measured. +/+, n = 30; dsf/+, n = 14; Df/+, n = 9; dsf/dsf, n = 19; and dsf/Df, n = 18. Mutants are significantly different from wild type (dsf/dsf, P < 0.0001; dsf/Df, P < 0.0001). (B and C) dsf females resist during copulation, as judged by excess movement during copulation. (B) The number of times the mating pair made a 180° turn was determined from the videotapes. +/+, n = 15; dsf/dsf, n = 11, P = 0.0037; and dsf/Df, n = 14, P = 0.0004. (C) The number of times the mating pair crossed a predetermined center line of the chamber was scored from the same videotapes as in B. +/+, n = 15; dsf/dsf, n = 11, P = 0.0009; and dsf/Df, n = 14, P = 0.0072. (D) Three homozygous dsf males forming a short courtship chain, including a copulation attempt by the middle male. Both homozygous dsf and dsf/deletion males demonstrate this phenotype but with incomplete penetrance. dsf males spend a substantial amount of time orienting and following other males, but dsf chains contain few individuals and are not continued over extended periods of time, probably due to the robust rejection response exhibited by dsf males. dsf males court wild-type males, but are not themselves courted by wild-type males. (E) dsf males are delayed in copulation. Single males of the genotypes shown were paired with aged wild-type (Canton-S) virgin females. The time from initiation of courtship to copulation was measured. +/+, n = 30; dsf/+, n = 13; Df/+, n = 10; dsf/dsf, n = 13, P < 0.0001; and dsf/Df, n = 17, P = 0.0003. (F) Abdominal curling is altered in dsf males. Courtship events involving males of the genotypes shown, and wild-type (Canton-S) females were recorded on videotape. The magnitude of abdominal bending for each observable bend was determined from the videotaped images, with ≤45° being the minimal bend observable and 180° being the maximum magnitude bend observable. Bending at 180° is necessary to achieve the genital–genital contact associated with copulation. Intermediate bending categories (data not shown) have generally intermediate percentages of total bends.

Figure 3

Motor neuronal innervation of the female reproductive tract. Synaptic boutons have been stained with anti-synaptotagmin (37). All views are side views. U, uterus, SR, sperm receptacle. (A) Wild-type female internal genitalia (n = 11). Synaptic boutons are present on the muscles of the oviduct, sperm receptacle, spermathecae, and uterus and on other muscles associated with the external genitalia. (B) Higher power photomicrograph of wild-type uterine muscles and their innervation. (C) Internal genitalia from a dsf female. There are no boutons on the uterine muscles, but boutons are visible on the muscles of the oviduct, sperm receptacle, and spermathecae and on other muscles associated with the external genitalia. This phenotype is seen in both dsf/dsf (n = 16) and dsf/Df (n = 11). (D) Higher power photomicrograph of the uterine musculature of a dsf female (arrows). (E) Internal genitalia (side view) from an XX; tra⁻; hs-tra female (n = 32). There are few boutons on the uterine muscles, but boutons are visible on the the oviduct, sperm receptacle, and spermathecae and on other muscles associated with the external genitalia. (F) Higher power photomicrograph of the uterine musculature of an XX; tra⁻; hs-tra female. Dorsal is to the left. (Bar = 20 μm.)

Figure 4

Motor neuron innervation of the ventral abdominal muscles in wild-type and mutant males. Nerve terminals are stained with anti-synaptotagmin (37). (A) The third abdominal segment in a wild-type male. (B) The fifth abdominal segment in a wild-type male. (C) Morphologically normal third abdominal segment in a dsf male. (D) The fifth abdominal segment of a dsf/Df male. Fewer and enlarged boutons are found on the muscles. This phenotype is seen in both dsf/dsf and dsf/Df. (E) Morphologically normal third abdominal segment in a dsf female. (F) Morphologically normal fifth abdominal segment in a dsf female. Wild-type male, n = 12. XY; dsf/dsf, n = 10. XY; dsf/Df, n = 11. XX; dsf/dsf, n = 7. XX; dsf/Df, n = 12. Anterior is to the top. (Bar = 20 μm.)

Figure 5

dsf acts downstream of tra but independently of dsx. (A and B) dsf acts downstream of tra. Motor neuron innervation of the ventral muscles of abdominal segment 5 in XX animals transformed to maleness by a transformer mutation. Nerve terminals are stained with anti-synaptotagmin (37). (A) Abnormal innervation in XX; dsf⁻; tra⁻ pseudomales (n = 4). (B) Morphologically normal innervation in XX; dsf⁺; tra⁻ pseudomales (n = 3). (C–E) dsf is not under the control of dsx. Motor neuron innervation of the ventral muscles of abdominal segment 5 in XX and XY dsx^D/Df animals. Such animals differentiate as normal-looking males without regard for chromosomal sex or the action of tra and tra2. Nerve terminals are stained with anti-synaptotagmin (37). (C) Abnormal innervation in an XY; dsf⁻; dsx^D/Df male (n = 11). (D) Normal innervation in an XX; dsf⁻; dsx^D/Df pseudomale (n = 6). (E) Normal innervation in an XX; dsf⁺; dsx^D/Df pseudomale (n = 23). (Bar = 20 μm.)