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Previous Article | Next Article
The Journal of Neuroscience, January 15, 2001, 21(2):513-526
Abnormalities of Male-Specific FRU Protein and Serotonin Expression in the CNS of fruitless Mutants in Drosophila
Gyunghee Lee and Jeffrey C. Hall Department of Biology, Brandeis University, Waltham, Massachusetts 02454
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
The fruitless gene in Drosophila produces male-specific protein (FRUM) involved in the control of courtship. FRUM spatial and temporal patterns were examined in fru mutants that exhibit aberrant male courtship. Chromosome breakpoints at the locus eliminated FRUM. Homozygous viable mutants exhibited an intriguing array of defects. In fru1 males, there were 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 exhibited an overall decrement of FRUM expression in all neurons normally expressing the gene. fru4 and frusat mutants only produced FRUM in small numbers 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 exhibited 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.
Key words: fruitless transposons; chromosome aberrations; brain neurons; ventral nerve cord; sexual dimorphism; serotonergic neurons
INTRODUCTION
Courtship in Drosophila melanogaster is regulated by a somatic sex-determination hierarchy. One of the "downstream" genes functioning within this hierarchy is fruitless (for review, see Goodwin, 1999
; Yamamoto and Nakano, 1999
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 (Villella et al., 1997
The fact that several fru-mutant types court other males might be attributable to subnormal levels of serotonin (5-HT) in relevant brain cells. This hypothesis suggested itself because of the anomalous inter-male courtships that are induced by ectopic expression of the white (w+) gene (Zhang and Odenwald, 1995
Along with demonstrating that elements of 5-HT production are downstream of fru functioning (although not in CNS regions that one might have expected), this feature of the study provided the first information on axonal projections of certain FRUM cells. Assessing the subcellular localization of FRUM alone gives no insight into this matter; FRU immunoreactivities are nuclear (Lee et al., 2000
MATERIALS AND METHODS
Strains and culturing. Stocks and progeny from crosses of D. melanogaster were reared in 12 hr light/dark (LD) cycles at 25°C, 70% relative humidity, on a sucrose-cornmeal-yeast medium containing the mold inhibitor Tegosept. A Canton-S strain was used as the wild-type control.
The fruitless mutant stocks fru1, fru2, fru3, fru4, and frusat were maintained as described in Villella et al. (1997)
To obtain 2-d-old pupae homozygous for a fru mutation or carrying a given transheterozygous combination, flies from TM6B-balanced stocks were crossed to each other. This was necessary because of the homozygous lethality or sterility associated with fru variants, with the exception of fru2, which is homozygous-viable and fertile (permitting pupae to be obtained from a true-breeding stock). For the other strains, balancer-over-fru heterozygotes were crossed, and animals not expressing the pupal marker Tb were selected. To stage these developing animals, white prepupae were selected, sexed by gonadal size, and maintained on a Petri dish with a wet filter paper at 25°C, 70% relative humidity, on a 12 hr LD cycle for 2 d. To collect homozygous or transheterozygous fru-mutant adults, TM3- or MKRS-balanced fru stocks were used; progeny not expressing the Sb marker were selected.
Immunohistochemistry and in situ hybridization. Polyclonal anti-FRUM, designed to detect male-specific proteins encoded by male-specific fru transcripts stemming from the action of the sex-specific P1 promoter, was generated in a rat as described in Lee et al. (2000)
To apply anti-5-HT (made in rabbit; Protos Biotech, New York, NY) for immunohistochemistry, CNSs were fixed in a solution of 4% paraformaldehyde including 7.5% picric acid for 1 hr at room temperature; this antibody was used at a dilution of 1:500. The secondary antisera, FITC-conjugated anti-rabbit IgG (made in donkey; Jackson ImmunoResearch), was used at a dilution of 1:200, applying the same procedures used for anti-FRUM immuno-histochemistry.
Immunofluorescent double-labelings were performed on whole-mounted CNSs; anti-FRUM (from rat) and anti-5-HT (from rabbit) were applied to whole-mounted CNSs of wild-type adult males. Dissection, fixation, and washes were performed as described above for anti-5-HT, except for the fixation, which was done on ice. FITC-conjugated anti-rat IgG for anti-FRUM and lissamine rhodamine sulfonyl chloride-conjugated anti-rabbit IgG (from donkey; Jackson ImmunoResearch) for anti-5-HT were used as secondary antisera. Preparations were viewed in the confocal microscope described above, which is equipped with an argon-krypton laser and dual-channel scanning. Colocalization was verified by merging the two channels.
For in situ hybridization with whole-mounted CNSs, an antisense probe from the P1 region of the fru locus (see Fig. 1) was applied to CNSs dissected from 1-d-old Df-fru4-40/Df-sat15 or Df-ChaM5/Df-sat15 (double-deletion) male pupae. The particular probe (called P1.S1; see Fig. 1) and the labeling procedures were as described in Lee et al. (2000)
Scoring of staining intensities. To analyze the levels of FRUM in CNSs of viable fru mutants and wild-type males, fluorescently immunostained signals from various FRUM-expressing neuronal clusters in whole-mounted CNS were quantified as described in Lee et al. (2000)
To assess levels of FRUM expression in CNSs of 2-d-old pupae that carried transheterozygous Dfs or were homozygous for the fru3 mutation, protein expression was scored subjectively in whole-mounts (3-10 individual specimens for each genotype) using a fluorescent microscope at 40× magnification. A representative image for each genotype was obtained using confocal microscopy afterward. To assess staining intensities from CNSs subjected to P1-fru-probe in situ hybridization, at least six specimens were subjectively evaluated using brightfield microscopy. A representative image for an animal of a given fru genotype was obtained at 30× magnification.
5-HT uptake. In attempts to determine whether the lack of 5-HT immunostaining in the abdominal ganglion of the fru3 mutant (see Results) is attributable to the absence of the relevant cells (those that contain signal in wild type) or a dearth of serotonin synthesis, ventral nerve cords (VNCs) of 4- to 5-d-old adult males were dissected and exposed to exogenously applied 5-HT, essentially as described in Vallés and White (1986)
RESULTS
Male-specific fru products in the CNS of chromosome-breakpoint variants
Among the most severely defective fru variants in terms of courtship behavioral subnormalities are those expressing the effects of chromosome breakpoints within the locus. We suspected that animals carrying most or all of these genotypes would lack detectable FRUM. This expectation is based on the molecular characterization of these chromosome aberrations (Fig. 1) against a background of the fru transcript-types that are produced under the control of a given promoter (Ryner et al., 1996
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Figure 1. The fruitless gene and genetic variations at the locus. This large (~130 kb) gene contains at least four promoters (Ps); the 5'-most promoter (P1, left) controls the production of primary transcripts that are sex-specifically spliced with respect to the second exon (Ryner et al., 1996
Drosophila carrying the Df-ChaM5/Df-P14 or Df-fru4-40/Df-P14 deletions develop into viable adults, as can be rationalized by their ability to transcribe fru mRNAs under the control of one or more promoters located downstream of P1 (Fig. 1). These transcripts encode FRU protein isoforms that are produced in both males and females (Lee et al., 2000
Two further double-deletion types cause similar subnormalities of male courtship: Df-ChaM5/Df-sat15 and Df-fru4-40/Df-sat15, whose levels of courtship directed at females are nearly zero (Anand et al, 2001
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Figure 2. Lack of sex-specific fruitless expression in the CNS of fru-breakpoint mutants. A, In situ hybridizations performed on pupal progeny resulting from crosses involving three of the deletions depicted in Figure 1; 1-d-old male pupae had a fru-derived riboprobe (Fig. 1) applied to whole-mounted CNSs of wild type (WT, n = 11) and these two Df/Df types (Df-ChaM5/Df-sat15, n = 6; Df-fru4-40/Df-sat15, n = 6); no signals were elicited by this nucleic-acid probe in any of the 12 double-deletion specimens. B, Anti-FRUM immunohistochemistry performed on pupal progeny resulting from crosses of various deletions and other breakpoint variants (Fig. 1); heterozygotes involving certain of the chromosome aberrations and one of the fru transposon mutants were included as a negative control (compare Fig. 3; Table 2); antibody against the male-specific form of the protein was applied to whole-mounted CNSs dissected from 2-d-old male pupae. Summaries of these immuno-histochemical results (including numbers of samples per genotype) are given in Table 1. Signals (or the absence thereof) were examined by confocal microscopy, and representative images were made for specimens of the various genotypes at 40×. A, B, anterior views of the brains. B, The brain image of fru3/fruw12 (lower left panel) shows a whitish general background staining that was not detected in other specimens of this genotype (cf. Table 1) and bears no relation to the WT pattern. Scale bars: A, 100 µm; B, 50 µm.
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Table 1. Lack of male-specific FRUM protein in severely affected fruitless mutants There is an additional category of intralocus lesions associated with the fru gene: inversion and translocation breakpoints called fruw12 and fruw27. These are located between P1 and the 3'-fru ORF, relatively close to the latter (Ryner et al., 1996
FRUM in the CNS of homozygous-viable fruitless mutants
The other genetic variations involving the fruitless gene are homozygous viable mutants, most of which are caused by transposons inserted within the locus (Fig. 1). The transposons in the four relevant mutants (Ito et al., 1996
However, none of these issues regarding the male-specific FRU protein has been empirically examined in the fru transposon mutants. Thus, in a given mutant, how much, if any, FRUM would be detectable, and where would it be found within the CNS? To address these matters, anti-FRUM immunohistochemistry was performed on the CNSs of 2-d-old male pupae and adults carrying the transposon mutations. A principal goal was to correlate FRUM expression levels with the behavioral impairments of mutants, which are summarized in Table 2. For example, we suspected that this protein would be present at least in fru2 because such males exhibit the mildest courtship abnormalities among the four transposon mutants used in this study (Table 2).
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Table 2. Staining intensities of FRUM-expressing cells in fruitless mutants Homozygous mutants and those heterozygous for a transposon insert and a given recessive-lethal fru variant were tested by CNS dissections and application of anti-FRUM (Figs. 2B, 3, 4; Tables 1, 2). fru2 males exhibited readily detectable staining. In this fertile fru mutant (Gailey and Hall, 1989
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Figure 3. Effects of viable fruitless mutations on FRUM expression in the CNS. Pupal progeny of crosses involving heterozygous male parents (for most of these mutant genotypes, for which homozygosity causes sterility) had CNSs dissected and subjected to immunostaining. All specimens shown are from 2-d-old pupae, the images for which were obtained by confocal microscopy at 20×. The definitions and approximate intra-CNS locations of the FRUM-expressing neuronal clusters designated by white arrowheads (e.g., aSP3, AL, mcAL) are specified in Table 3 (also see Lee et al., 2000
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Table 3. Counts of FRUM-expressing cells in the pupal CNS of fru mutants The other three transposon mutants are sterile and exhibit more severe courtship defects than does fru2 (Villella et al., 1997
The final homozygous-viable fruitless mutant examined was fru1. Such males court females vigorously, although they do not mate with them, and they exhibit by far the most dramatic inter-male courtships of all fruitless mutant types (Table 2). fru1 is caused by an inversion breakpoint within the locus (Gailey and Hall, 1989
Immunohistochemical results from CNSs of 2-d-old pupal males homozygous for fru1 are shown in Figures 3E and 4 and summarized in Tables 2 and 3. Major differences were observed when compared with the wild-type pattern. First, certain clusters, or neurons within a given cluster, were absent in the CNS of the fru1. In particular, regions fru-mAL and aSP1 were devoid of staining. Other brain regions that are stained by anti-FRUM in wild-type males, such as fru-aSP3, Lv, and mcAL, had reasonably clear signals in corresponding portions of fru1 brains (Fig. 4, compare A, C). Second, most of the FRUM neurons of this mutant showed weaker staining intensity when compared with that of wild type. However, few cells, in particular within the fru-aSP3 and fru-Lv brain clusters, exhibited nearly normal levels of FRUM immunostaining (Fig. 4A-D). Third, another difference from the norm involves novel cells within the fru1 brain that express the male-specific protein (Fig. 4H). Such ectopic expression of FRUM within numerous cells of the CNS made it difficult to determine whether certain normal clusters or cells in a given cluster are missing in fru1. The three kinds of fru1 versus wild-type differences just enumerated were also observed in the ventral CNS of male pupae (Fig. 4I,J). Also, fru1-associated reductions in numbers of neurons were discernible in most of the VNC clusters, such as fru-Ab, PrMs, and MsMt (Fig. 4I,J,K). In the fru1 abdominal ganglion, FRUM immunostaining (within the relevant fru-Ab) neurons was diminished (Fig. 4, compare I, J). This is dealt with in more detail in the next section.
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Figure 4. Nonrandom spatial effects of fru1 on expression of FRUM in the brain. Pupal progeny of males heterozygous for this (recessive sterilizing) mutation had CNSs dissected (from 2-d-old pupae) and subjected to whole-mount anti-FRUM histochemistry. A, B, Control brightfield micrographs (from PhotoShop assemblies of four to five consecutive focal-plane images each) obtained from a wild-type (WT) brain, representative of 20 specimens stained by peroxidase-mediated color reactions (Fig. 3, compare A, B). C, D, fru1 anterior- and posterior-brain patterns, respectively, from scrutiny of 12 mutant specimens (processed and photographed as in A and B). Only a few cells with low-intensity staining were detected within the fru-aSP1 and fru-aSP2 clusters of fru1 brains (asterisks, C vs A). C, The boxed area designates the absence of the normally stained fru-mAL cluster (cf. box in A). Certain neurons showed no apparent staining-intensity decrement compared with WT; this is exemplified by neurons pointed to by arrowheads in C (also see E and F). Other FRUM cells or clusters were absent or exhibited significantly decreased staining intensities in this mutant, e.g., in the vicinity of the mushroom-body calyx (D); these qualitative and quantitative anomalies of sex-specific fru expression are consistent with those obtained by in situ hybridizations using later-stage fru1 pupae (Goodwin et al., 2000
These immunohistochemical results from the five viable fru mutants permit certain rationalizations of variations among their extents and types of courtship defects. These mutants can be categorized into two groups. One consists of fru1 and fru2, which court rather vigorously (Table 2). These mutants exhibited decrements in FRUM expression in the CNS, but overall are nowhere near the amorphic state for P1-encoded proteins. fru1 showed strong immunohistochemical decreases in certain FRUM neurons, whereas fru2 was more uniformly hypomorphic. The other mutant group consists of fru3, fru4, and frusat, which exhibit no FRUM expression or immunostaining in very small numbers of neurons. These three sterile mutants court females at lower to much-lower levels than those characteristic of fru1 and fru2 male behavior (Table 2).
Subnormalities of FRUM expression in the VNC of fru mutants are likely to be connected with their courtship-song defects. In this regard, frusat males, which are mute (Goodwin et al., 2000
fru3, fru4, and frusat males do not attempt copulation and lack a male-specific abdominal muscle called the Muscle of Lawrence (MOL; Gailey et al., 1991
Perhaps the most dramatic courtship anomaly exhibited by a fruitless mutant involves the fact that fru1 males court other males in an extremely vigorous manner, compared with the levels of such "courtship chaining" behavior that are caused by any of the other mutations, let alone the complete absence of such behavior in groups of wild-type males (Villella et al., 1997
FRUM in semifertile fru-mutant transheterozygotes
Males homozygous for fru1, fru3, or fru4 do not attempt copulation and are sterile; but fru1/fru3 and fru1/fru4 males are fertile, albeit in lower than normal proportions (Castrillon et al., 1993
Several interesting FRUM immunostaining differences were revealed among these mutant types. First, there was an ~30% decrease in apparent protein expression levels in the transheterozygotes compared with wild type (Fig. 5, examples of quantified results in legend). FRUM levels in the sterile fru1/Df-fruw24 male type were apprehended to be as low as in fru1/fru3 and fru1/fru4 (staining intensities not quantified for fru1/Df-fruw24). Overall expression levels seemed to be uniform throughout CNS in the three heterozygous types just described. Second, there were marked reductions in the numbers of stained cells in several clusters within the CNS of fru1/fru3 and fru1/fru4 males, such as fru-aSP1, aSP2, aSP3, mAL (brain), fru-PrMs, MsMt, and Ab (ventral nerve cord) (Fig. 5). Depending on the neuronal group (among the seven just indicated), the number of FRUM cells decreased approximately two- or threefold (Table 3). In other CNS regions, the numbers of stained neurons were nearly normal (Table 3). The reductions, or lack thereof, were quite consistent when comparing the signals and counts from fru1/fru3 to those from fru1/fru4 (Fig. 5; Table 3). Third, anti-FRUM staining patterns for these fru1/fru3 and fru1/fru4 males were somewhat similar to those of fru1 homozygotes. The distributions of signal-containing neurons in these transheterozygotes resembled those of strongly stained cells in males homozygous for fru1 (Fig. 4, compare with Fig. 5J, K, L). However, weakly stained FRUM cells (and ectopic ones, see below) were not detectable in fru1/fru3 or fru1/fru4 males.
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Figure 5. FRUM expression in the CNS of quasi-fertile fruitless mutant combinations. CNSs from 2-d-old male pupae carrying fru1/fru3 (n = 14), fru1/fru4 (n = 12), wild type (WT, n = 36), and fru1/Df-fruw24 (n = 6) were subjected to anti-FRUM immunohistochemistry. WT and fru1/Df-fruw24 were used as normal and fully mutant controls, respectively. The resulting representative images were prepared with confocal microscopy to show both anterior and posterior views of the brain and whole projection of the VNC for WT (A-C), fru1/fru3 (D-F), fru1/fru4 (G-I), and fru1/Df-fruw24 (J-L). A, D, G, J, White arrows, fru-aSP1 neuronal cluster; black arrows, fru-aSP2; and white arrowheads, fru-mAL. fru1/fru3 and fru1/fru4 samples were stained with relatively low intensities overall; for example, the fru-aSP2 cluster of FRUM-containing brain neurons in fru1/fru3 and fru1/fru4 males (D, G, black arrows) gave staining intensities (see Materials and Methods) of 160 ± 14 (mean ± SEM; n = 3) and 164 ± 9 (n = 3), respectively, whereas the corresponding WT value for aSP2 (black arrow in A) was 217 ± 7 (n = 3). However, the micrographs shown do not reflect such staining-intensity differences, because these confocal images were in saturation to maximize viewability of signal-containing CNS regions. M, N, Drawings of fluorescent signals viewed confocally from the ventral side of the abdominal ganglion to produce both representative diagrams for WT and fru1-homozygous males, respectively. In fru1/fru1, fewer cells than normal stained in a relatively intense WT-like manner (black dots); other neurons, possibly representing further subsets of the normal pattern but including many ectopically expressing cells, stained weakly (gray dots). These numerous ectopic expressing cells could not be revealed in the low-magnification micrographs for this CNS region in this mutant (Figs. 3E, 4J). Scale bars: A-L, 100 µm; M, N, 50 µm.
Now we focus on abdominal ganglion expression of FRUM in fertile fru1/fru3 and fru1/fru4 males compared with sterile fru1/fru1 and fru1/Df-fru24 males, against a background of the likelihood that the male's copulation attempts are controlled by this posterior-most region of the CNS. In the abdominal ganglion, ~50% of the normal numbers of FRUM cells were stained in fru1/fru3 and fru1/fru4, and ~25% in fru1/Df-fruw24 males (Table 3). This difference in the FRUM cell number might be responsible for the lack of attempted copulation by fru1/Df-fruw24 males. However, the twofold decrement in the transheterozygotes (compared with wild-type) is still compatible with routine mating ability. These fru1/fru3 and fru1/fru4 males did not show any ectopic expression of FRUM, of the kind that fru1 homozygotes exhibit in the abdominal ganglion (Fig. 5, compare N,M) as well as in other CNS ganglia (see previous section of Results). This homozygous-sterile mutant type also exhibits a decrement in the number of heavily staining abdominal ganglionic neurons (Fig. 5, N vs M; compare Fig. 3, E vs D, and Fig. 4, J vs I) similar to the paucity shown by fru1/Df-fruw24 males (see above; Fig. 5, compare L,N).
In general, the three male types that each carried only one copy of the fru1 mutation gave protein-expression patterns similar to one another, although fru1/Df-fruw24 hemizygous males were farther from wild type, compared with the fertile but FRUM-subnormal transheterozygotes (Fig. 5; Table 3). Nevertheless, the similarities among fru1/fru3, fru1/fru4, and fru1/Df-fruw24 could be explained by an allele-dosage effect. The Df-fru24 deletion generates no gene product, and homozygosity for fru3 or fru4 eliminates most or all FRUM expression (Fig. 3). Thus, FRUM production in the three heterozygous types being considered would seem mostly to come from the one dose of the fru1 allele in common among them. One reason for the perception of an overall reduction of FRUM, under the influence of this mutation, could be that the weak and ectopically expressing neurons found in fru1 homozygotes are below detection levels in each of the (one-dose) heterozygotes. However, this fru1-dosage effect does not explain why more FRUM cells were observed and counted within certain CNS regions of the fru-mutant transheterozygous types, compared with the near absence of staining in fru1 homozygotes, e.g., for the fru-aSP1 and mAL brain clusters. About one-third to one-half the normal numbers of stained neurons were observed in these locations within the CNS of fru1/fru3 or fru1/fru4 males (Fig. 5; Table 3), whereas almost no cellular signals were detected in the corresponding brain regions of fru1/fru1 males (Fig. 4). Therefore, another factor that may point to an explanation of the differences among these three types, which are most dramatic in terms of fertility, is that there are special kinds of gene interactions between the fruitless alleles themselves when fru1 is heterozygous with fru3 or fru4. This kind of phenomenon would involve something other than the amount of final gene product (in this case male-specific FRU protein) produced according to the dosage of the mutant alleles in question. Instead, as suggested originally by Castrillon et al. (1993)
fru effects on sexually dimorphic serotonergic abdominal cells
To examine the possible relationship between fruitless function and 5-HT (see introduction), we double-labeled whole-mounted CNSs with antibodies against FRUM and the neuromodulator. The results are presented in Figure 6. We found 5-HT-immunoreactive neurons broadly distributed throughout the brain (n = 12, data not shown), the thoracic ganglia, and the abdominal ganglion of adult males (Fig. 6A). Previously, Vallés and White (1986
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Figure 6. Neurons coexpressing FRUM and serotonin in the abdominal ganglion. These cells, dubbed s-Abg, were revealed in the posterior tip of the male VNC by anti-5-HT single-labeling and double-labeling application of that antibody along with anti-FRUM. Twelve 4- to 7-d-old wild-type males were used for anti-5-HT immunohistochemistry alone and 10 separate such males for double-labeling. A, Confocal image showing a dorsal view of serotonergic neurons in the thoracic and abdominal ganglia. Two different sizes of anti-5-HT-labeled neurons appear in the abdominal ganglion; the boxed area shows a cluster of eight s-Abg neurons; the black arrowhead points to the proximal portion of axons projecting posteriorly from these cells into the median trunk nerve. B, C, Neurons expressing 5-HT- and FRUM neurons in an adult-male abdominal ganglion. These sagittal views of the s-Abg neurons show the cell bodies to be located dorsally (toward the right of each panel). D, Combined image of B and C, depicting coexpression of the two antigens. 5-HT immunoreactivity was observed mostly within (and throughout) the cell bodies (red), and FRUM immunoreactivity was detected only in nuclei (green). E, F, Higher-magnification dorsal view of s-Abg neurons labeled by anti-5-HT and anti-FRUM, respectively. Six consecutive 1.2 µm focal planes were combined to show all s-Abg neurons; the dotted line in E was drawn to indicate the symmetrically paired structure of the s-Abg neuronal clusters. G, Combined image of E and F (5-HT in red, FRUM in green). Scale bars: A-D, 100 µm; E-F, 25 µm.
We assume that fruitless mutations and ectopic expression of the white gene (Zhang and Odenwald, 1975
In the course of these double-labeling tests, we scrutinized signals elicited by anti-FRUM and anti-5-HT in all CNS ganglia. Within the male's ventral cord, the great majority of fru-expressing neurons in the four pairs of ganglia (cf. Lee et al., 2000
With regard to the projection patterns of the s-Abg cells that were revealed by 5-HT-immunostaining (Fig. 6A), each neuron appeared to have more than one neurite. In most specimens, the s-Abg neurons were 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 (Hertweck, 1931
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 (Fig. 7, compare A, B). 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 et al., 2000
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Figure 7. Abnormal sex-specific serotonin expression in the abdominal ganglia of fru mutant males. These images are whole (A, B, D, F) or partial (C, E, G) projections of stacked images through a given ganglion, viewed in the confocal microscope from the ventral side. A, B, Abdominal ganglia of 4- to 7-d-old wild-type adult male and female, respectively; in the latter (representative of five female VNCs processed), there were no anti-5-HT-mediated signals like those observed in this region of the male CNS, whose cluster of s-Abg cells (compare Fig. 6) are designated by an asterisk in A (representative of 12 male VNCs observed); the arrow in A points to axonal projections from male s-Abg neurons. C-G, Serotonergic neurons in the abdominal ganglia of fru mutants. C, In fru1 (n = 5), s-Abg cell bodies (asterisk) and processes (arrow) were weakly stained (uniformly among the specimens, in contrast to fru4) (see below). D, In fru2 (n = 8), the s-Abg neuronal cluster (asterisk) and projections from such cells (arrow) were normal or nearly so (in terms of numbers of cell bodies and staining intensities) among the several specimens. The image shown depicts strong signals, although in this one there were weak signals in a serotonergic neuropil that usually gives strong staining (E, G, black arrowheads) in abdominal ganglia of all fru genotypes (including fru2). E, In fru3 (n = 6), no 5-HT immunostaining in s-Abg cells or neurites was observed; the white arrowhead points to where the cell bodies should be. F, In fru4 (n = 5), two specimens showed no 5-HT immunoreactivity in s-Abg neurons, whereas three gave weak staining in one to three s-Abg cell bodies and their processes (as exemplified in the case shown and its asterisk for cell bodies and black arrow for processes). G, In frusat (n = 5), relatively few s-Abg neurons were stained by anti-5-HT (although more than in fru4), and the cell bodies and processes in which signals were elicited (asterisk, arrow) gave weak signals (uniformly among the specimens). Scale bars: A, B, 50 µm; C-G, 100 µm.
In fru-mutant males, anti-5-HT immunoreactivity in the s-Abg neurons as well as the axons projecting from them was absent or defective (Fig. 7). fru1 and frusat showed low levels of transmitter staining in some of the s-Abg neurons and their process (Fig. 7, C and G, respectively).
In fru3, there was no detectable 5-HT immunoreactivity in s-Abg neurons or their axons (Fig. 7E). At best, fru4 mutant males presented weakly detectable 5-HT immunoreactivity in these structures (Fig. 7F). fru2 males were 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 appeared to be lower than in wild type (Fig. 7D).
For fru3, the most severely subnormal mutant 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 expressed late-developmentally lethal Dopa decarboxylase (Ddc) mutations (Vallés and White, 1988
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Figure 8. Serotonin immunoreactivity in the abdominal ganglion resulting from exogenous application of 5-HT. Immunohistochemistry with anti-5-HT was performed with dissected VNCs of fru3 adult males that were incubated in 5-HT solutions. Immunofluorescently labeled tissues were examined by confocal microscopy, and stacked images of abdominal ganglia were obtained from specimens exposed to the range of concentrations indicated. A-D, Representative immunostaining after application of 5-HT at 5 µM (n = 4) (A), 10 µM (n = 5) (B), 100 µM (n = 2) (C), and 500 µM (n = 5) (D). No s-Abg-like 5-HT-immunoreactive neurons or their projections were observed to result from the lowest two concentrations of 5-HT used (A, B). Thus, the thin neurite signal in A, which runs down the center of the median trunk, was traced back to certain cell bodies in the posterior tip of the abdominal ganglion, but they were too small to be s-Abg cell bodies. Another feature of A is a cell body and fiber (white arrowhead) that appears to be outside of the main abdominal nerve. (In association with these signals are varicosities that suggest the structures are neurohemal fibers that could not be projections from s-Abg cells.) B, None of the stained fibers (white arrowheads) in the main nerve could be traced back to s-Abg-like cell bodies. D, At the highest concentration, a few cells that putatively took up exogenous 5-HT appeared to be similar to s-Abg neurons in their size and shape (black arrowhead within the box; image of the box is based on one focal plane); no such cells exhibit intrinsically derived immunostaining in these cells in this mutant (Fig. 7E). The putative s-Abg cell bodies, to which 5-HT was supplied in the fru3 abdominal ganglion shown (D), are in a dorsomedial region of the posterior CNS tip. This is the location of such neurons in more definitively identifiable circumstances (Fig. 7A,C,D). Scale bars, 100 µM.
DISCUSSION
Correlations between FRUM expression and fru-mutant phenotypes
The various fruitless mutants exhibit striking behavioral defects and differences among one another (Goodwin, 1999
