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Aberrant Splicing and Altered Spatial Expression Patterns in fruitless Mutants of Drosophila melanogaster
Stephen F. Goodwina,d, Barbara J. Taylorb, Adriana Villellaa, Margit Fossb, Lisa C. Rynerc, Bruce S. Bakerc, and Jeffrey C. Hallaa Department of Biology, Brandeis University, Waltham, Massachusetts 02454,
b Department of Zoology, Oregon State University, Corvallis, Oregon 97331,
c Department of Biological Sciences, Stanford University, Stanford, California 94305
d Division of Molecular Genetics, University of Glasgow, Glasgow G11 6NU, United Kingdom
Corresponding author: Stephen F. Goodwin, Division of Molecular Genetics, Anderson College, University of Glasgow, Glasgow G11 6NU, United Kingdom., stephen{at}molgen.gla.ac.uk (E-mail)
Communicating editor: T. SCHÜPBACH
| ABSTRACT |
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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. To understand the molecular etiology of fruitless mutant phenotypes, we compared wild-type and mutant transcription patterns. These experiments revealed that the fru2, fru3, fru4, and frusat mutations, which are due to P-element inserts, alter the pattern of sex-specific and sex-nonspecific fru RNAs. These changes arise in part from the P-element insertions containing splice acceptor sites that create alternative processing pathways. In situ hybridization revealed no alterations in the locations of cells expressing the P1-fru-promoter-derived transcripts in fru2, fru3, fru4, and frusat pharate adults. For the fru1 mutant (which is due to an inversion breakpoint near the P1 promoter), Northern analyses revealed no significant changes in fru transcript patterns. However, in situ hybridization revealed anomalies in the level and distribution of P1-derived transcripts: in fru1 males, fewer P1-expressing neurons are found in regions of the dorsal lateral protocerebrum and abdominal ganglion compared to wild-type males. In other regions of the CNS, expression of these transcripts appears normal in fru1 males. The loss of fruitless expression in these regions likely accounts for the striking courtship abnormalities exhibited by fru1 males. Thus, we suggest 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.
THE fruitless (fru) gene of Drosophila functions at the head of a recently identified branch of the sex-determination hierarchy (![]()
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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 (Figure 1A and Figure B; ![]()
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Phenotypic analyses of fruitless mutants have extended the previously known roles of fru in male courtship behavior (![]()
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fruitless exerts its effects on male sexual behavior through its expression in a limited portion of the central nervous system (CNS; ![]()
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Here, we focus on the molecular etiology of the phenotypes produced by fru alleles that affect male sexual behavior, but not fruitless's vital function. These include four P-element mutations (fru2, fru3, fru4, and frusat), which are inserted between the P1 promoter and the common coding region, and the fru1 mutation, which is due to an inversion breakpoint just distal to the P1 promoter (Figure 1A). Northern analysis shows that the four P-element mutations alter the size of the fru transcripts produced from the promoters located distal to the inserts. These changes arise in part from aberrant splicing into acceptor sites present within the P-element inserts. Not all transcripts produced from the distal promoters are abnormal in these mutants: reverse transcription (RT)-PCR experiments revealed low levels of RNA species spliced in a normal manner. The finding of some wild-type transcripts in the P-element mutations can account for the fact that they differ in the severity of their phenotypes. In situ hybridization to tissue sections revealed that the spatial pattern of expression in the P-element fru mutants was similar to the wild-type male pattern. In contrast, males homozygous for fru1 make a normal set of sex-specific transcripts, but exhibit an altered spatial pattern of fruitless expression: a subset of the cells that express fru's sex-specific transcripts in wild type do not express these transcripts in fru1 homozygotes. These data strongly support the suggestion (![]()
| MATERIALS AND METHODS |
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Drosophila strains and culture conditions:
Cultures of Drosophila melanogaster were reared in 12:12-hr light:dark cycles at 25° and 70% relative humidity on a sucrose-cornmeal-yeast medium containing the mold inhibitor Tegosept. Bottles were cleared of parents after 5 days. Males were collected under mild ether anesthetization 06 hr after eclosion and then aged individually on unyeasted autoclaved food (see below). Wild-type Canton-S virgin females were collected 06 hr after eclosion, grouped in food vials, and then aged for 15 days.
fruitless stocks:
fru1 was balanced with In(3LR)TM3, Sb (TM3). fru2 contains a w+, ry+-marked P element, P{(w+, ry+)A} (![]()
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Courtship observations and recordings:
For single-pair observations, individual frusat males (710 days old) were placed in a recording chamber (cf. ![]()
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For male chaining observations, males were collected at eclosion, aged individually for 67 days, and then eight males were grouped per food vial (![]()
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To test male fertility, males were collected at eclosion, grouped (<10 males per food vial), and aged for 58 days. Individual males were placed in a food vial with three to five wild-type virgin females. The presence or absence of progeny was scored 710 days later. Vials with no progeny and in which the male was dead at the time of scoring were excluded.
Molecular biology:
RNA isolation, purification, and Northern analyses were as described previously (![]()
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RT-PCR:
These reactions were performed as described previously (![]()
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Orientation and position of the P-element transposons:
fru3 and fru4 are P{PZ} transposons inserted within the fru locus in unknown orientations (Figure 1A and Figure D; ![]()
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In situ hybridizations to tissue sections:
Flies were cryostat sectioned at 20 µm in the horizontal plane; the sections were transferred to slides, fixed for 1 hr in RNase-free 4% paraformaldehyde, rinsed in RNase-free PBS-Tx (140 mM NaCl, 50 mM phosphate buffer, pH 7.4, and 0.1% Triton X-100), dehydrated through a methanol series (25100%), and rehydrated with PBS-Tx. Sections were acetylated with acetic anhydride in 0.1 M triethanolamine (pH 8.0) and hybridized overnight at 55° with 15100 ng of labeled riboprobe in 150 µl hybridization buffer (50% formamide, 5x SSC, 0.01% Tween-20, 0.01% tRNA, and 0.005% RNase-free heparin per slide). The sections were washed in buffer (600 mM NaCl, 1 mM EDTA, pH 8.0, 10 mM Tris, pH 8.0, 50% formamide) at 55° and incubated at 37° with RNaseT1 and RNaseA. For signal visualization, the sections were blocked for 1 hr at 25° in 5% nonfat powdered milk in PBS-Tx, incubated for 1 hr at 25° with antidigoxygenin Fab fragments coupled to alkaline phosphatase (1:1000; Roche Molecular Biochemicals, Indianapolis, IN) in blocking solution and developed with NBT/BCIP (GIBCO BRL, Grand Island, NY) in NTMT (100 mM NaCl, 50 mM MgCl2, 100 mM Tris, 0.1% Triton X-100, and 0.015% levamisole, pH 9.5). The sections were mounted in 80% glycerol and 1% propyl-gallate.
Standard protocols (Roche Molecular Biochemicals) were used to synthesize riboprobes from fru cDNA and DNA templates [subcloned into pBluescript SK(+) (Stratagene, La Jolla, CA) or pGEM-T Easy (Promega)]. Linearized probes were synthesized using T3, T7, or SP6 polymerase with digoxygenin-coupled nucleotides (DIG RNA Labeling Kit; Roche Molecular Biochemicals). Two riboprobes were made that detect P1-promoter-derived transcripts: probe S1, derived from a genomic clone (coordinates 1261 with respect to GenBank accession no. U72491), includes sequences from probe S and an extra 340 nt of genomic sequence. Two common coding-region riboprobes were made: probe C1 (coordinates 27833607 with respect to GenBank accession no. U72492) and the overlapping probe C (see above). The S and S1 probes gave identical results, as did the C and C1 probes. For the analysis of in situ hybridization patterns, the following numbers of animals were examined: Canton-S males (S1 probe, n = 18; C1 probe, n = 10), females (S1, n = 10; C1, n = 11); fru1 males (S1, n = 4; C1, n = 2), females (S1, n = 7, C1, n = 2); fru2 males (S1, n = 5; C1, n = 8), females (S1, n = 3; C1, n = 3); fru3 males (S1, n = 3; C1, n = 14), females (S1, n = 3; C1, n = 9); fru4 males (S1, n = 4; C1, n = 14), females (S1, n = 4; C1, n = 8); and frusat males (S1, n = 9; C1, n = 14), females (S1, n = 6; C1, n = 4).
Statistical analyses:
For the courtship data (see legend to Table 1), nonparametric Kruskal-Wallis pairwise comparisons (JMP, Statistics and Graphics Guide, version 3; SAS Institute Inc., Cary, NC) were performed to determine whether there were differences among the CIs or ChIs recorded for frusat males, or between mutant and fru+-associated CIs. For the in situ hybridizations (see legend to Table 5), the cell counts were analyzed by one-way ANOVA (Statgraphics Version 5.0; Manugistics Inc., Rockville, MD) using 95% Tukey honestly significant difference (HSD) for contrast to compare numbers of labeled neurons between mutant and wild-type animals of the same sex for a particular neuronal group.
| RESULTS |
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frusat behavior:
One objective of these studies was to elucidate the molecular basis for courtship defects caused by viable fru mutations. Because frusat has not been as well characterized behaviorally as the other fru mutants (![]()
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frusat males were completely sterile when heterozygous with the deletions fruw24 (n = 20) or P14 (n = 16). However, 1 frusat/ChaM5 male out of 39 was fertile. frusat/fru1 males were also weakly fertile (14/30 males); this proportion is similar to those observed when fru3 or fru4 are heterozygous with fru1 (~65% in both cases; ![]()
Origins of normal fruitless transcripts:
When a probe common to all known classes of fru cDNAs (Figure 1, probe C) 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 (Figure 2A; ![]()
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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 (![]()
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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 (![]()
Molecular etiology of fru mutant phenotypes:
Because the viable fru alleles are due to lesions outside of coding sequences (Figure 1), we suspected that they might have effects that would be detectable at the transcript level. Northern blots hybridized with the common coding region probe (Figure 1, probe C) showed that in the case of the fru1 mutant, the relative levels of the male- and female-specific transcripts were not appreciably different from those of wild-type controls (Figure 3). Northern blots involving RNA from the four P-element mutants, hybridized with the common coding region probe (C), revealed that sex-specific differences in the pattern of fru expression were no longer detected (Figure 3). Instead, transcripts of ~7.4 and 4.4 kb were detected in both males and females (Figure 3, AC). In addition to the anomalous 7.4-kb transcript detected in all four mutants, a novel 3.0-kb transcript was observed in fru3, fru4, and frusat. These results suggest that, in the four P-element mutants, sequences from the distal P1 promoter are no longer sex-specifically spliced to the common-coding region.
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To characterize further the effects of these mutations, we rehybridized the Northern blots shown in Figure 3 (AC) with a probe from the 5' end of P1-derived transcripts (Figure 1, probe S). Again, in the fru1 mutant, the fru transcript pattern appears to be normal (Figure 3D). In the four P-element mutants, two patterns of transcripts were seen. First, in fru2 and frusat, there is an abundant 4.0-kb transcript in males and an abundant 6.0-kb transcript in females (Figure 3E and Figure F; longer exposures revealed a faintly hybridizing band at 1.35 kb in frusat); none of these corresponds to a transcript size seen in wild type. Second, fru3 and fru4 also produce apparently identical arrays of transcripts; these include two major RNA species, a male-specific 2.0- and a 4.0-kb transcript, found in both sexes (Figure 3D). In addition, there are three minor bands common to fru3 and fru4 males and two different minor bands common to fru3 and fru4 females. Taken together, the above findings indicate that the array of transcripts in fru1 males and females is normal, but a novel array of P1-derived transcripts is produced in all the P-element mutants.
The finding of a transcript apparently common to the two sexes in fru3 and fru4 is surprising, because all other transcripts detected in these mutants, as well as wild-type with this probe, are sex specific. We therefore wondered whether the 4.0-kb transcripts seen in fru3 and fru4 males and females are really identical, or whether they were only fortuitously of the same size. We therefore reanalyzed the Northern shown in Figure 3 with a fru probe from upstream of the female-specific 5' splice site (Figure 1, probe B), which in the wild type detected only the three female-specific transcripts (Figure 2B). In the fru3 and fru4 mutants, this probe hybridized only to a single female-specific transcript of 4.0 kb; no transcripts were detected in males. These results indicate that the 4.0-kb transcripts observed in fru3 and fru4 females and males are two molecularly distinct species.
There are several striking features of these Northern results:
- P elements inserted at different locations within a 60-kb region of the large fru intron all lead to the production of novel fruitless transcripts.
- The fru2 and frusat mutants produce identical arrays of novel fru transcripts despite the fact that these insertions are ca. 40 kb apart (Figure 1).
- Similarly, the fru3 and fru4 mutations also generate identical arrays of novel fru transcripts. A possible basis for the different effects of these two pairs of fru mutants is that fru3 and fru4 are due to P-element insertions of the same transposon, P{PZ}, while fru2 and frusat are due to the insertion of different P-element transposons [P{(w+, ry+)A} and P{lwB}, respectively] that share some sequences in common.
RT-PCR analysis of the transcripts generated in P-element fru mutants:
We used RT-PCR to investigate whether the four transposon inserts lead to splicing between fru sequences transcribed from a given fru promoter and sequences in the respective downstream transposon. Because potential splice-acceptor sites within the inserted elements depend on the orientation of the transposon relative to fru, we first determined the orientations of the fru3 and fru4 inserts (see MATERIALS AND METHODS). The PZ elements in fru3 and fru4 are oriented such that the rosy+ marker gene is expressed from the same strand as fru. In the case of fru2 and frusat, the P element inserted in these mutants carries part of the white gene, including its first intron and the splice sites that flank it. We confirmed previous reports that these two elements are inserted such that the sense strands of fru and the white gene in the P elements are the same (cf. ![]()
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The positions of the fru promoters relative to the P-element insertions (Figure 1A) suggest which transcripts might be affected in each mutant. For example, sequences from frusat, which is located between P2 and P3 (Figure 1C), would be expected to be included in transcripts originating from the P1 and P2, but not the P3 and P4 promoters, and, thus, might alter the processing of P1- and P2-derived transcripts. Because the Northern results led us to expect the effects of fru2 and frusat to be similar to one another, and different from those of fru3 and fru4, we describe results from these two pairs of mutants separately.
To determine whether there are transcripts being produced in fru2 and frusat that fuse fru sequences with white sequences from within these transposons, RNA was isolated from these mutants and subjected to RT-PCR; the primer-set combinations represented each fru promoter (P1P4) as well as nested white-gene-specific primers (see MATERIALS AND METHODS). PCR products were sequenced directly. The nucleotide sequences of such products showed that fru P1 transcript sequences are spliced to those of white in both mutants in each sex (Figure 4A). These splices utilize the normal fru+ male- and female-specific 5' splice donor sites, which are spliced to a common acceptor site >70 kb downstream in wild-type P1 transcripts. However, in fru2 and frusat mutants, these 5' splice sites are joined to the normal acceptor site in the first intron of the white gene of the P{(w+, ry+)A} and P{lwB) inserts, respectively (![]()
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We also detected aberrant transcripts derived from the other fru promoters located distal to these inserts. In fru2 mutants, transcripts from P2 and P3 promoters are spliced via the normal fru 5' splice donor sites (L. C. RYNER and S. F. GOODWIN, unpublished results) to the normal acceptor site in the first intron of the white gene of the P{(w+, ry+)A} (Figure 4B and Figure C), whereas P4 transcripts are normal. In frusat mutants, as predicted from its enhancer trap location between P1 and P2, P2 transcripts are spliced to white sequences from the P{lwB} insert (Figure 4B), whereas P3 and P4 transcripts appear to be normal.
Both the fru3 and fru4 inserts are located between the P2 and P3 promoters and, thus, might be expected to affect transcripts from the P1 and P2 promoters. ![]()
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We also examined the effects of the inserts in fru3 and fru4 on the processing of transcripts from the other fru promoters. Aberrant transcripts derived from the P2 fru promoter were detected in both fru3 and fru4. Transcripts from the P2 promoter are spliced via the normal fru 5' splice donor site (L. C. RYNER and S. F. GOODWIN, unpublished results) to the previously identified 3' splice site in l(3)S12 of the PZ element present in these two mutants (Figure 4E). In both fru3 and fru4, processing of transcripts from the P3 and P4 promoters is normal, as predicted from the locations of these promoters downstream of the insertion sites in these two mutants. Thus, in fru3 and fru4, as in fru2 and frusat, processing of all transcripts initiating upstream of a P-element insertion site is disrupted, whereas processing of transcripts initiated from downstream promoters is normal.
The findings that sequences in all four transposons lead to nonproductive splicing of fru transcripts offers a first-order explanation for how transposon inserts generate fru-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 there are varying levels of normal fru transcripts generated from the P1 promoter in these mutants. Although normal fru P1 transcripts were not detected in our Northern analysis (see above), it is possible that such transcripts are present, but at levels below those detectable by Northern analysis (~110 pg of transcript; cf. ![]()
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RT-PCR experiments utilizing total RNA failed to detect wild-type fru transcripts from the P1 or P2 promoters in the P-element mutants, in contrast to wild-type controls (data not shown; see MATERIALS AND METHODS). In addition, P4 sex-nonspecific transcripts were detectable in all the mutants tested; P3 transcripts were detected in fru3, fru4, and frusat males, but not in fru2 males. This experiment was repeated twice using different RNA isolates, and the RNA's integrity in the RT-PCR reaction was checked using primers derived from the rp49 gene (see MATERIALS AND METHODS); in all samples, we observed amplification of rp49 sequences. In a further effort to identify normal fru transcripts in these mutants, we used head poly(A)+ mRNA isolated from wild-type male and female heads (separately)and from sexed heads of fru2, fru3, fru4, and frusat fliesfor RT-PCR analysis. Sequencing of PCR products was used to confirm their identity. Although these RT-PCR experiments were not quantitative, we were able to detect the normal array of spliced fru products from all four promoters in all the P-element mutants (data not shown).
CNS expression patterns in fru mutants:
An important feature of P1-derived transcripts is that they are expressed in several hundred neurons in the CNS of adults (![]()
In situ hybridization to P1 transcripts (using the S1 probe; Figure 1) revealed that P1 expression in wild-type pharate adults was largely localized to nine neuronal clusters in the brain and ventral nerve cord (Figure 5A and Figure B). Labeled neurons were considered part of a group if the signal-containing cells were adjacent to or within a few cell diameters of other similarly sized labeled cells. By these criteria, we identified six neuronal groups, ranging from 10 to 50 neurons per group, which are found in similar locations within male and female brains (Figure 5A), and three neuronal groups in the ventral nerve cord that are male specific (Figure 5B). All nine groups are present as bilateral pairs.
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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; 35, three anterior groups in the protocerebrum which are subdivided into lateral (3), intermediate (4), and medial (5) sets; and 6, one anterior group near the mechanosensory part of the antennal lobe (Figure 5A; cf. ![]()
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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 (Figure 1) revealed expression in virtually all neurons in the CNS and in several other tissues (cf. ![]()
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To determine whether there were changes in the cellular pattern of fru expression in the five viable fruitless mutants, we carried out analogous in situ hybridizations 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.
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 (Figure 6A and Figure B, group 6), and the ventro-medial mesothoracic groups (Figure 5A, group 8). The numbers of labeled neurons in the antennal lobe and dorsal posterior groups in fru1 males were not significantly different from wild type (Table 2).
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The other five groups of cells detected in wild-type males were very difficult to detect in fru1 males. In the anterior protocerebrum, the normal pattern for three groups of neurons (35) was not observed (Figure 6F and Figure G). In the dorsal anterior protocerebral region, only about one-quarter of the expected number of labeled neurons was detected (Table 2). These labeled neurons were 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 were 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, was found in the ventral area, between the prothoracic and mesothoracic neuromeres (Figure 6K and Figure L; Table 2). By contrast, almost no labeled cells were found in the ventral abdominal region, where group 9 neurons are observed in wild-type males (Figure 6O; Table 2). 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.
When a probe (C1) common to all fruitless transcripts was used, all neurons in fru1 male CNSs were labeled at a low level, comparable to what is seen in the wild-type CNS. In addition, subsets of neurons showed relatively high levels of fru expression. Neurons with such heavy labeling were 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 (see above). In fru1, fewer-than-normal numbers of heavily labeled neurons were detected with the C1 probe in the anterior protocerebrum (35) as well as within the thoracic (7) and the abdominal ganglion (9) groups (data not shown). These regions are the same as those in which fewer or no labeled neurons were found by in situ hybridization with the S1 probe (Figure 5A and Figure B, and Figure 6A, Figure B, Figure F, Figure G, Figure K, Figure L, Figure O).
We also used in situ hybridization to determine whether the spatial expression of P1-derived transcripts was affected in the transposon mutants. In all four mutants, the S1 probe detected groups of labeled neurons in the nine locations where P1-expressing neurons are found in wild-type males (see the schematic representations in Figure 5A and Figure B; because no differences among the expression patterns for fru2, fru3, fru4, and frusat were detected, only the locations of labeled neurons for a fru4 brain and thoracic/abdominal ganglia are shown). For example, labeled neurons were found in the antennal lobe (Figure 6, CE, group 6), the dorsal anterior protocerebrum (Figure 6, HJ, groups 3 and 4), the thoracic ganglion (Figure 6M and Figure N, group 7), and the abdominal ganglion (Figure 6Q and Figure R, group 9). The numbers of neurons labeled in these groups were similar to those found in wild-type males (Table 2). 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 (Table 4). In wild-type males, cells labeled with the S1 probe often had darkly stained dots within the nucleus, as well as cytoplasmic staining (cf. ![]()
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In situ hybridization to the four P-element mutants with the C1 probe detected heavily labeled cells only in fru2 males. These neurons were in the same positions as the nine groups labeled by the S1 probe (e.g., group 6, cf. Figure 6C and Figure Y; group 7, cf. Figure 6Z and Figure M). These neurons had the same darkly stained dots in the nucleus, as was seen with the S1 probe (see above), in addition to having a higher level of cytoplasmic expression than the surrounding neurons (Figure 6X and Figure Y). Thus, the expression pattern in fru2 males appears to be similar to that of wild-type males.
In contrast, in the CNS of fru3, fru4, and frusat males, no heavily expressing neurons were detected with the C1 probe, although there was a general low level of CNS staining. In these sections, only ~10 neurons in total had evidence of nuclear dots. These findings suggest that detectable levels of transcripts containing normal P1-derived transcripts are not found in the CNS neurons of fru3, fru4, and frusat males. Because a population of heavily labeled neurons was detected using the S1 probe in these mutants, the fru-expressing neurons are present, but it does not appear that intact P1 transcripts can be detected in these cells by in situ hybridization. This finding is in accord with the RT-PCR results presented above, in which it was necessary to use poly(A)+ RNA from heads to detect normally spliced P1-derived transcripts in these mutants.
Using both the S1 and C1 probes, we also examined the effects of all five fru mutations on fruitless expression in females. In the four P-element mutants, the cellular distributions and expression levels of transcripts from the P1 promoter (Figure 5A and Figure B, S1 probe; Table 3) were comparable to those of wild-type females (data not shown). With the C1 probe, only fru2 females showed heavily expressing neurons; these neurons were in the same locations as the cells detected by the S1 probe, suggesting that the same neurons are labeled with both probes (cf. ![]()
Examination of the distribution of P1-derived transcripts (S1 probe) in fru1 and frusat females revealed a substantial change in the distribution of labeled neurons. In fru1 and frusat females, the overall staining intensity and number of labeled neurons were increased in the CNS compared to wild-type females (Figure 5A and Figure B, and Figure 6, SU; Table 3). The normal pattern of six groups of labeled neurons was still present in fru1 and frusat female brains, and the numbers of labeled neurons were not significantly different from those in wild-type or other P-element mutants (Figure 5A; Table 3). The increase in the number of labeled neurons appeared in regions where labeled neurons are not found in wild-type males and females, e.g., in ventral regions of the thoracic ganglion (Figure 5B and Figure 6U). This increased expression of transcripts from the P1 promoter was confined to the CNS, because no expression was detected in the non-neural tissues of fru1 and frusat females (data not shown). The difference between fru1 and wild-type females in the numbers of heavily labeled neurons in the ventral thoracic CNS was statistically significant; although the numbers in the brain were also larger than normal, this difference was not statistically significant (Table 4).
When the C1 probe was used on fru1 females, we observed heavily labeled neurons as well as widespread low-level neuronal expression in the CNS. As was found with the S1 probe, more neurons gave strong signals with the C1 probe in fru1 than in wild-type females.
| DISCUSSION |
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The primary purpose of this study was to gain insights into the molecular basis of the phenotypes of the five viable fruitless mutants, all of which result from lesions in noncoding regions of fru (![]()
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fru expression in wild-type:
Our analysis of normal fruitless expression focused on the transcripts produced by the P1 promoter, as we believe it is through the products of this promoter that fru controls male sexual behavior. Previous RT-PCR experiments showed that P1-derived transcripts are sex-specifically spliced near their 5' end to generate male- and female-specific products (![]()
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We also extended the analysis of wild type with respect to the distributions of P1-promoter-derived transcripts within the CNS (cf. ![]()
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Comparing such behavioral analyses of genetic mosaics to the patterns of fruitless expression indicates that all CNS regions in which the P1 fru transcripts are found correspond to sites within the brain and the ventral nervous system whose male genotype is correlated with the performance of various steps within the courtship sequence.
Effects of P-element mutations on fru expression:
Northern analysis, RT-PCR experiments, and in situ hybridization to tissue sections all led to a consistent view of the effects of the P-element fru alleles on gene expression.
Northern analysis showed that in the four P-element mutants, the normal array of six sex-specific P1 transcripts and one sex-nonspecific fru transcript was entirely absent; instead, novel arrays of transcripts were produced. Strikingly, the novel transcripts produced by fru3 and fru4 were equivalent, as were those observed in fru2 and frusat. Two further findings suggest that these novel arrays of transcripts come from the effects of these transposon inserts on the processing of fruitless transcripts. First, the transposons are all inserted in the large fru intron at locations well removed from the P1 promoter (Figure 1); our quantitative analysis of the cellular expression patterns of the P1 promoter in these mutants revealed no differences between these four mutants and wild type in the number or locations of cells expressing P1 transcripts. Second, the pairs of transposons that produced similar arrays of novel fru transcripts were either identical in terms of the sequences that they contained (fru3 and fru4), or they shared some sequences in common (fru2 and frusat).
That effects of these mutations on the pattern of fru transcripts were due to sequences in these transposons providing splice-acceptor sites that are used in aberrant splicing events, as is known to occur for analogous transposon mutants in Drosophila (![]()
Additional RT-PCR experiments showed that the effects of fru2, fru3, fru4, and frusat are not restricted to P1-derived transcripts. We found that transcripts from all fru promoters located 5' to a given transposon insert (Figure 1) can be aberrantly spliced to an acceptor site in the transposon, whereas splicing from fru promoters located 3' to the insertion sites are unperturbed.
The findings that sequences in the transposons in the four P-element mutants lead to nonproductive splicing of fru transcripts offer 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 (Table 1; ![]()
Additional data bearing on the above points come from our in situ hybridizations to fruitless RNA. With respect to fru3, fru4, and frusat, these experiments also suggest that there is little normal splicing of P1 transcripts in these mutants: high levels of expression of fru common coding sequences were not detected in the cells in which the P1 promoter is expressed. There is a full complement of the neurons that normally express the P1 promoter in these three mutants despite the fact that there is little or no P1-promoter-derived mRNA that encodes fru proteins in these cells; this suggests that sex-specific fru expression is not necessary for the generation (during development) and subsequent survival of these cells in adults. A related point is that the expression of fruitlessin the context of fru mutation effects on behaviormay be involved in regulating the ongoing function of the nervous system in such flies. Indeed, all of our Northern analyses (Figure 2 and Figure 3) involved detection of fru transcripts in mature adults.
In the case of fru2, in situ hybridization detected high levels of expression of fru common coding sequences in the cells in which the P1 promoter is normally expressed, suggesting that there are significant levels of normally spliced P1-derived transcripts produced. This finding may account for the nearly normal behavioral phenotype of fru2 males compared to the other P-element mutants (![]()
One feature of the data from the P-element mutants that is not entirely accounted for are the specific arrays of novel transcripts detected on Northerns. In particular, if the only abnormal event in the processing of P1 transcripts in these mutants involves a splice from the male or female donor site to an acceptor site in the transposon, then a probe from the 5' end of the P1 transcripts should detect one novel transcript in each sex. Whereas this is what was found in the cases of fru2 and frusat, in the fru3 and fru4 mutants, more complex arrays of novel transcripts were detected by this probe. Possible explanations for the latter results are that these hybrid transcripts terminate at multiple sites, or that there are additional novel splices from the transposon sequences to sequences further downstream. If there are additional novel splicing events involved in the production of such RNAs, they are not to the normal acceptor site at the beginning of the fru common coding region, as a probe to that region does not detect these transcripts. Thus, whatever their exact nature, these RNA species are not likely to be a source of fru+ function. In addition, it should be noted that a novel transcript of unknown origin is detected with the common region probe in the fru2 mutant; whether this transcript encodes a functional fru product is unknown.
Behavioral effects of viable fru mutations:
The molecular findings on the origins of P-element fru alleles should be considered in the context of phenotypic differences among these mutants. The effects of fru1, fru2, fru3, and fru4 on male sexual behaviors have been well characterized (![]()
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The molecular results discussed above establish that the P-element alleles of fruitless are associated with high levels of aberrant splicing events in which fru sequences are spliced into sequences in the transposons. The associated reductions in the levels of wild-type fru transcripts are the likely cause of the mutant phenotypes in these mutants, and differences among these mutants in the severity of their phenotypes are likely due to differences in the levels of residual normal transcripts.
There is an alternative interpretation of certain aspects of the fru2 and frusat phenotypes that is worth commenting upon. These two mutants contain white genes in their transposon, and it has been shown that ectopic expression of the white gene product induces courtship among D. melanogaster males (![]()
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Effects of fru1 mutation on fruitless expression:
The fru1 mutation does not cause any detectable change in the array of fru transcripts. However, fru1 exhibits changes in the numbers of neurons expressing detectable levels of P1-derived transcripts. In fru1 males, there are marked reductions in the numbers of cells expressing P1-derived transcripts in five of the nine groups of cells in which these transcripts are expressed in the wild type. The groups affected include three groups in the anterior protocerebrum, a male-specific neuronal group in the thoracic ganglia, and a male-specific neuronal group in the abdominal ganglia. In the remaining four groups, there were normal numbers of neurons expressing P1-derived transcripts. That the expression pattern affected by fru1 involves neurons in only certain regions of the CNS leads us to suggest that the behaviors defective in this mutant are controlled, at least in part, by specific subsets of the neurons expressing P1-derived transcripts. In particular, the reduction in the number of fruitless-expressing neurons in a thoracic-ganglionic cluster of fru1 males may account for the defect in fru1's singing behavior (e.g., ![]()
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On the control of male sexual behavior by fru:
We previously proposed that the male-specific products of the P1 fru promoter are responsible for establishing the potential for male sexual behaviors (![]()
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Two features of our findings with respect to the viable fruitless mutants are relevant to the idea that the P1 fru products govern male sexual behavior. First, the finding that all four of the P-element alleles we have analyzed result in reduced levels of wild-type P1 transcripts is consistent with the suggested role of these transcripts in male behavior. However, the additional findings that each of these alleles also interferes with the processing of transcripts from other fru promoters make it likely
at there are reduced levels of wild-type products from these promoters. This raises the question of whether the products of these other promoters are needed in conjunction with the products of the P1 promoter to establish the potential for male sexual behavior. Second, our results showing that fru1 leads to the absence of P1-derived transcripts in a subset of the CNS cells in which the P1 transcripts are normally expressed provide compelling evidence for the role of P1-derived transcripts in male sexual behavior. Moreover, these data also provide strong support for the notion that the cells expressing P1 transcripts in the CNS, as identified by in situ hybridization, govern male sexual behavior.
| ACKNOWLEDGMENTS |
|---|
We thank Daisuke Yamamoto for the frusat stock, Gail Fasciani and Julia Becker for helping with fly collections, Celeste Berg for providing the pPZ plasmid, Becky Meyers for automated sequencing, Edward Dougherty for photographic assistance, Laura Wilson and Randy Bender for in situ hybridizations, and Frank Moore for access to a cryostat. We appreciate comments on the manuscript from Ralph J. Greenspan, Troy Carlo, Michael Rosbash, Jean-Christophe Billeter, Megan C. Neville, Ravi Allada, and Donald A. Gailey. This work was supported by National Institutes of Health grants NS-33252 and GM-21473. Current financial support to S. F. Goodwin, from the Wellcome Trust and Division of Molecular Genetics, University of Glasgow, is gratefully acknowledged.
Manuscript received May 18, 1999; Accepted for publication October 14, 1999.
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