1. Genetics and Genomics
  2. Neuroscience

Layered roles of fruitless isoforms in specification and function of male aggression-promoting neurons in Drosophila

  1. Margot Wohl
  2. Kenichi Ishii
  3. Kenta Asahina  Is a corresponding author
  1. Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, United States
  2. Neuroscience Graduate Program, University of California, United States
https://doi.org/10.7554/eLife.52702
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  1. Margot Wohl
  2. Kenichi Ishii
  3. Kenta Asahina
(2020)
Layered roles of fruitless isoforms in specification and function of male aggression-promoting neurons in Drosophila
eLife 9:e52702.
https://doi.org/10.7554/eLife.52702
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Abstract

Inter-male aggressive behavior is a prominent sexually dimorphic behavior. Neural circuits that underlie aggressive behavior are therefore likely under the control of sex-determining genes. However, the neurogenetic mechanism that generates sex-specific aggressive behavior remains largely unknown. Here, we found that a neuronal class specified by one of the Drosophila sex determining genes, fruitless (fru), belongs to the neural circuit that generates male-type aggressive behavior. This neuronal class can promote aggressive behavior independent of another sex determining gene, doublesex (dsx), although dsx is involved in ensuring that aggressive behavior is performed only toward males. We also found that three fru isoforms with different DNA binding domains show a division of labor on male aggressive behaviors. A dominant role of fru in specifying sex-specific aggressive behavior may underscore a genetic mechanism that allows male-type aggressive behavior to evolve at least partially independently from courtship behavior, which is under different selective pressures.

Introduction

Aggression is widely observed across animal species, especially in the context of reproductive activities (Huntingford and Turner, 1987). It is mostly males that perform agonistic behaviors, which are often ritualized, to gain access to mating partners. Some species have even developed male-specific weaponry organs to gain advantage (Rico-Guevara and Hurme, 2018). Although these inter-male aggressive behaviors must have evolved to increase fitness, the effect on sexual selection is likely indirect. Unlike courtship behavior, which is selected directly through female preference, females can choose only the end result of inter-male contest and not the aggressive behavior itself. Another distinction of aggressive behavior from courtship behavior is that it can be exhibited by both males and females, but the motor programs are often sexually dimorphic (Hashikawa et al., 2018; Nilsen et al., 2004). Moreover, males are aggressive almost exclusively towards other conspecific males, and not towards females. In other words, aggressive behavior is sexually dimorphic, and its execution is strongly influenced by the sex of the target animal.

The sexually dimorphic nature of aggressive behavior predicts the existence of sexually dimorphic neuronal circuits that support this behavior. Both in mice and flies, mutations in genes with sexually dimorphic expression patterns, including sex-determining genes or steroid sex hormone receptors, can alter aggressive behaviors (Juntti et al., 2010; Ogawa et al., 2000; Vrontou et al., 2006; Wu et al., 2009; Xu et al., 2012). Neurons expressing sex-determining genes also influence aggressive behaviors (Asahina et al., 2014; Chan and Kravitz, 2007; Deutsch et al., 2020; Hashikawa et al., 2017; Hoopfer et al., 2015; Koganezawa et al., 2016; Lee et al., 2014; Palavicino-Maggio et al., 2019; Yang et al., 2013), which is consistent with the above prediction that male-type aggression and female-type aggression may be generated through distinct neural circuits. However, sexual dimorphisms within neurons or circuits that are causally responsible for the generation of sexually dimorphic behavior remain unclear. The genetic and neuronal mechanisms which ensure a male only executes aggressive behaviors towards a male target is not well understood either. Although sex-specific sensory cues seem to play critical roles for both mice (Chamero et al., 2007; Hattori et al., 2016; Stowers et al., 2002) and flies (Fernández et al., 2010; Wang and Anderson, 2010), how the sensory information affects the operation of circuits that generate sexually dimorphic aggressive actions is largely uncharacterized.

Sexual dimorphism of aggressive behavior in the common fruit fly Drosophila melanogaster has been well documented. Males perform aggressive behaviors almost exclusively to one another with a higher intensity than inter-female aggressive behaviors (Chen et al., 2002; Nilsen et al., 2004; Vrontou et al., 2006). Attack actions are also dimorphic: a male fly often perform lunges, in which it raises its front legs and pounces down toward a target fly (Chen et al., 2002; Dow and von Schilcher, 1975; Hoyer et al., 2008; Jacobs, 1960), whereas a female fly performs headbutts, in which it quickly moves its body horizontally and hits a target with its head (Nilsen et al., 2004; Ueda and Kidokoro, 2002). Sex of Drosophila is determined by two genes that encode transcription factors, doublesex (dsx) and fruitless (fru). These two genes undergo sex-specific splicing to produce male-specific (dsxM, fruM) and female-specific (dsxF, fruF) transcripts (Yamamoto and Koganezawa, 2013). fru further undergoes another round of alternative splicing among at least 3 different zing finger DNA binding domains (Usui-Aoki et al., 2000). dsx and fru collectively specify anatomical and behavioral sexual dimorphisms (Marín and Baker, 1998; Yamamoto and Koganezawa, 2013). In Drosophila, several classes of sexually dimorphic neurons, which express dsx or fru (or both), are implicated in aggressive behaviors (Asahina et al., 2014; Chan and Kravitz, 2007; Deutsch et al., 2020; Hoopfer et al., 2015; Koganezawa et al., 2016; Palavicino-Maggio et al., 2019). An outstanding question is which of these neurons are critical for the execution of male-type or female-type aggressive behaviors. Results we present in Ishii et al. (2020) support the hypothesis that dsx specifies an execution mechanism for male courtship behavior, while fru specifies neural circuits underlying sexually dimorphic aggressive actions (Vrontou et al., 2006). Currently, however, it remains unclear which, if any, of the fru-expressing neurons constitutes the execution mechanism for male-type aggressive behavior.

Here, we characterized Tk-GAL4FruM neurons (Asahina et al., 2014) as a part of a fruM-dependent neural circuit that generates male-type aggressive behavior. Tk-GAL4FruM neurons are fru-expressing, male-specific neurons that are not only capable of promoting male-type aggressive behavior, but also are necessary for spontaneous male aggression (Asahina et al., 2014). We first found that Tk-GAL4FruM neurons are specified solely by fru. We then demonstrated that the activation of Tk-GAL4FruM neurons elicited male-type aggressive behaviors irrespective of dsx genotype. However, dsx has a role in refining the application specificity of male-type aggressive behaviors toward a male target fly. We also characterized the impact of different fru isoforms on male aggression. We found that only one of the three fru isoforms is necessary for the specification of Tk-GAL4FruM neurons, while another isoform is necessary to perform spontaneous aggressive behaviors. The dominant role of fru in specifying the circuit for executing sex-specific aggressive behavior is in contrast to the importance of dsx for the execution mechanism for courtship behavior. The distinct roles of dsx and fru on the specification of courtship- and aggression-controlling neurons can be the genetic basis underlying evolution of beneficial behaviors for inter-male contests while preserving species-specificity for courtship behavior.

Results

fruM specifies aggression-promoting Tk-GAL4FruM neurons

Tk-GAL4FruM neurons are male-specific fru-expressing neurons that not only promote male-type aggressive behavior upon activation, but also are necessary for normal levels of male aggression (Asahina et al., 2014). We first characterized the behavioral impact of optogenetic activation of these neurons in the presence of male or female target flies (Figure 1A), because sex of the target fly can be an important biological variable in determining the function of social behavior-controlling neurons. We quantified the time in which a tester fly orients toward a target fly, number of lunges, and duration of unilateral wing extensions (shorthanded as ‘wing extension’ hereafter) using automated behavior classifiers before, during, and after the optogenetic stimulation of Tk-GAL4FruM neurons with programmed LEDs (Figure 1B; see also Materials and methods).

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Tk-GAL4FruM neurons are specified by fru.

(A) Schematic of the design of behavioral assays. (B) Schematic of the optogenetic stimulation paradigm. Time windows 1–4 represent periods in which behavioral parameters are pooled and calculated in subsequent panels. (C, D) Boxplots of time orienting (C1, D1), lunges (C2, D2), and wing extensions (C3, D3) by the tester flies during the time windows 1–4 (see B). Their genotypes and pair numbers are indicated below the plots. Gray lines represent single testers. Target flies are either group-housed wild-type males (C) or mated females (D). In gray: **p<0.01, *p<0.05, n.s. p>0.05 (Kruskal-Wallis one-way ANOVA and post-hoc Wilcoxon signed rank test). In black: **p<0.01, n.s. p>0.05 (Kruskal-Wallis one-way ANOVA and post-hoc Mann-Whitney U-test). (E) Schematics of the sex-determination pathway in Drosophila. (F) Schematic of the four sex genotypes defined by dsx and fru splicing. (G–J) Expression of CsChrimson:tdTomato under the control of Tk-GAL41 and Otd-nls:FLPo (red in G1–J1), black in G2,3-J2,3) in brains of a male (G), fruM female (H), fruF male (I), and female (J) flies is visualized together with a neuropil marker BRP (blue) by immunohistochemistry. Traced Tk-GAL4FruM neurons in a male (G3) and a fruM female (H3) are shown in white. Circle: soma (right cluster is enlarged in G4 and H4). Scale bar: 100 μm (G1–J1), 10 μm (G4, H4). (K) Mean number of cell bodies per hemibrain visualized by anti-DsRed antibody in each genotype represented in G–J) and Figure 1—figure supplement 2J–M. Error bars: S.D. Genotypes and number of hemibrains examined are indicated to the left. Lowercase letters denote significance group (p<0.01, one-way ANOVA with post-hoc Tukey’s honestly significant difference test). (L, M) Z-projection of segmented, registered, and averaged images of CsChrimson:tdTomato expression under the control of Tk-GAL41 and Otd-nls:FLPo (black) in the standard Drosophila brain (gray in L1, (M1). Number of used hemibrains are indicated in L2, M2. N-P: Volumes of medial projection (N), anterior projection (O), and lateral projection (P) of Tk-GAL4FruM neurons in males with genotypes indicated below. Their genotypes and pair numbers are indicated below the plots. n.s. p>0.05 (Kruskal-Wallis one-way ANOVA).

The tester flies in which CsChrimson was expressed under the control of Tk-GAL41 within the brain (which phenocopies the manipulation of Tk-GAL4FruM neurons; Asahina et al., 2014) showed robust aggression toward the target male flies, mainly during LED stimulation (Figure 1 C2, Figure 1—figure supplement 1A2; Watanabe et al., 2017). This coincides with increased time orienting toward the target fly (Figure 1C1, Figure 1—figure supplement 1A1), but wing extension toward males was not affected (Figure 1C3, Figure 1—figure supplement 1A3; see also Video 1 – Part 1). These behavioral effects were qualitatively similar across different stimulation frequencies (Figure 1—figure supplement 1C). In contrast, we observed no lunges toward female target flies after the same optogenetic manipulations (Figure 1D2, Figure 1—figure supplement 1B2). The tester flies increased the time orienting toward the target female during LED stimulations (Figure 1D1, Figure 1—figure supplement 1B1; see also Video 1 – Part 2), suggesting that the activation of Tk-GAL4FruM neurons can promote interaction regardless of the sex of the target fly. Neither did we observe lunges when Tk-GAL41-labeled neurons were optogenetically activated in the absence of any target fly (Figure 1—figure supplement 1D). Thus, the activation of Tk-GAL4FruM neurons induces lunges strictly when a male target fly is present. In addition, we observed that tester flies performed less wing extensions toward target females during LED stimulation than during ISIs (Figure 1D3). However, the total amount of wing extensions was not significantly different from those of the two genetic controls. These phenotypes are largely consistent with those observed when Tk-GAL4FruM neurons were thermogenetically activated (Asahina et al., 2014), and reinforces the idea that the sex of the target fly impacts the behavioral outcome of manipulations of Tk-GAL4FruM neuronal activity.

Video 1
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Representative behavior of a male tester fly that expresses CsChrimson:tdTomato under the control of Tk-GAL41 and Otd-nls:FLPo toward a wild-type male (Part 1) or a wild-type female (Part 2) target fly, at the onset and offset of LED stimulation.

In Part 2, the tester male was not actively performing any behavior toward the target female when the LED turned on.

As was previously shown (Asahina et al., 2014), Tk-GAL4FruM neurons are specified only in males (Figure 1G,J). We next addressed the role of dsx and fru (Figure 1—figure supplement 2A) on specifying Tk-GAL4FruM neurons. Two splicing mutations for fru, fruM and fruF (Demir and Dickson, 2005), forces male-type (fruM) and female-type (fruF) splicing of fru transcripts irrespective of the chromosomal sex composition, which normally determines the sex-determination genetic cascade. As a result, these mutations allowed us to create ‘fruF males’ (expressing dsxM and fruF) and ‘fruM females’ (expressing dsxF and fruM), thus dissociating contributions of dsx and fru on sexual dimorphism at the organismal level (Figure 1—figure supplement 2B). fruM females are reported to show lunges and other male-type aggressive actions (Vrontou et al., 2006), suggesting that fru is the chief architect of neural circuits underlying sexually dimorphism in aggressive behavior. However, two fru-expressing, aggression-promoting neurons (male-type NP2631 ∩ dsxFLP neurons (Koganezawa et al., 2016) and P1a neurons Hoopfer et al., 2015) are not specified in this genotype (see Ishii et al., 2020). These observations strongly suggest that not all fru-expressing neurons underlie the sexual dimorphism of aggressive actions, even if the activation of such neurons in a normal male induces male-type aggressive behavior. Other neuronal populations specified by fru, but not dsx, must constitute a neural substrate for the execution mechanism for male-type aggression.

In contrast to male-type NP2631 ∩ dsxFLP neurons or P1a neurons, Tk-GAL4FruM neurons were present in fruM females (Figure 1H), but not in fruF males (Figure 1I). Consistent with this observation, we detected expression of FruM, but not Dsx, proteins in Tk-GAL4FruM neurons (Figure 1—figure supplement 2C,D). The cell body number of Tk-GAL4FruM neurons in fruM females was indistinguishable from that in males (Figure 1K). To compare Tk-GAL4FruM neuronal morphology in males and fruM females, we first traced Tk-GAL4FruM neurons using a volume visualization software FluoRender (see Materials and methods for details). The distinct neuronal processes of Tk-GAL4FruM neurons allowed us to segment them in an unambiguous manner (Figure 1G3, H3). We then registered the original and segmented confocal images of brains to a unisex standard brain (Bogovic et al., 2018) using non-rigid spatial transformation (Jefferis et al., 2007). The three-dimension average of genetically isolated Tk-GAL4FruM neurons (Figure 1—figure supplement 2E–G; Asahina et al., 2014) almost perfectly overlapped with the average of segmented Tk-GAL4FruM neurons (Figure 1—figure supplement 2I, Video 2), confirming the accuracy of the segmentation. The averaged morphology of registered Tk-GAL4FruM neurons from males (Figure 1L) and from fruM females (Figure 1M) appeared virtually identical (Video 3). To confirm this initial observation, we further segmented three prominent neurites that emanate from the lateral junction (Yu et al., 2010) of Tk-GAL4FruM neurons (Figure 1L2, M2). None of these three processes showed significant difference in volume among fru +/+ control males, fruM/fru4-40 males, and fruM females (Figure 1N–P). While we acknowledge that this approach might miss finer scale differences in neuroanatomy, we conclude that Tk-GAL4FruM neurons are specified predominantly by fru. dsx and fru therefore have clearly separable roles for the specification of neuronal populations that are important for Drosophila aggressive behaviors.

Video 2
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3D-rendered average image of segmented and registered Tk-GAL4FruM neurons (green) and registered Tk-GAL4FruM neurons that are genetically isolated (blue).

Gray represents the standard unisex Drosophila brain (Bogovic et al., 2018).

Video 3
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3D-rendered average image of registered Tk-GAL4FruM neurons in male (green) and in fruM female (blue).

Gray represents the standard unisex Drosophila brain (Bogovic et al., 2018).

Presence of the fru4-40 allele did not affect cell number or overall morphology of Tk-GAL4FruM neurons (Figure 1K, Figure 1—figure supplement 2F–M). Consistent with this, the activation of Tk-GAL41-labeled neurons triggered similar behavioral changes in the backgrounds of +/+, +/fru4-40, or fruM/fru4-40 males (Figure 1—figure supplement 2N). However, heterozygosity of fruM resulted in incomplete specification of Tk-GAL4FruM neurons in a chromosomally female background (Figure 1—figure supplement 3A–C). We also found that fruFLP, a knock-in allele of fru that expresses a DNA recombinase Flippase (Yu et al., 2010), is hypomorphic (Figure 1—figure supplement 3D,E), raising the necessity to use caution when attempting to transform Drosophila sex by manipulating the fru locus.

Tk-GAL4FruM neurons are a part of a fruM-dependent circuit for the execution of male-type aggressive behavior

We next investigated the function of Tk-GAL4FruM neurons in fruM females. Specifically, we wished to obtain insights into the sex recognition mechanism and execution mechanism in the context of aggression. Unfortunately, male ‘target’ flies often perform vigorous courtship toward female tester flies (Figure 2—figure supplement 1B), which interferes with tester females’ social behavior (namely headbutts) towards target males (Figure 2—figure supplement 1A; Vrontou et al., 2006). Following a previous study (Vrontou et al., 2006), we used group-housed fruF males (which are defective at enhancing courtship toward females; Demir and Dickson, 2005; Pan and Baker, 2014; Villella et al., 1997; Figure 2—figure supplement 1B) instead of wild-type male flies as targets when using anatomically female (dsxF) flies as testers.

Optogenetic activation of the Tk-GAL41-labeled neurons in fruM females induced lunges toward a fruF male target for the majority of testers (Figure 2A2, Figure 2—figure supplement 2A2; see also Video 4 – Part 1). Although orienting time increased during LED stimulations compared to during ISIs (Figure 2A1, Figure 2—figure supplement 2A1), no overall change in the amount of wing extensions was observed compared to genetic controls (Figure 2A3). This indicates that Tk-GAL4FruM neurons are part of a fruM-dependent, but dsx-independent, neuronal circuit for male-type aggressive behavior. The motion sequence underlying the lunges observed in this genotype were indistinguishable from wild-type male lunges (Video 5).

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Tk-GAL4FruM neurons in fruM females promote male-type aggressive behaviors.

(A, B) Boxplots of time orienting (A1, B1), lunges (A2, B2), and wing extensions (A3, B3) by the tester flies during the time windows 1–4 (see Figure 1B). Their genotypes and pair numbers are indicated below the plots. Gray lines represent single testers. Target flies are either group-housed fruF males (A) or wild-type mated females (B). In gray: **p<0.01, *p<0.05, n.s. p>0.05 (Kruskal-Wallis one-way ANOVA and post-hoc Wilcoxon signed rank test). In black: **p<0.01, *p<0.05, n.s. p>0.05 (Kruskal-Wallis one-way ANOVA and post-hoc Mann-Whitney U-test). (C) Ratio of male (dataset from Figure 1C, D) and fruM female (dataset from A), (B) tester flies that lunged toward each of the two target sexes (above) in the time window 4. Testers’ genotypes and pair numbers are indicated below the plots. **p<0.01, n.s. p>0.05 (Fisher’s exact test, two genetic controls are pooled). (D) Schematic summary of the roles of sex-determining genes for Tk-GAL4FruM neurons and male-type aggressive behavior.

Video 4
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Representative behavior of a fruM female tester fly that expresses CsChrimson:tdTomato under the control of Tk-GAL41 and Otd-nls:FLPo toward a fruF male (Part 1) or a wild-type female (Part 2) target fly, at the onset and offset of LED stimulation.
Video 5
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A slow-motion comparison of a lunge executed by a male (top left) and fruM female (right) tester fly that express CsChrimson:tdTomato under the control of Tk-GAL41 and Otd-nls:FLPo.

The target fly is a wild-type male (top left), a fruF male (top right), and a wild-type female (bottom right), respectively.

However, we noted two differences compared to the same manipulation in male flies. First, lunges were not induced in fruM females as consistently as in males. Forty-two percent (17/40) of fruM females did not perform a single lunge after LED stimulation (Figure 2C). In contrast, over 96% of male testers lunged toward a male target (Figure 2C). The median of induced lunges observed in fruM females (2) was lower than observed for male testers (58). Second, fruM females occasionally lunged toward a female target (Video 4 – Part 2), in addition to a fruF male target. Even though lunges were induced even less frequently than toward fruF males (Figure 2A2, B2, C, Figure 2—figure supplement 2A2, 2B2), it is still noteworthy because male testers seldom lunge toward a female target, even after optogenetic activation of the Tk-GAL41 neurons (Figure 1D2, Figure 2C). We noticed that headbutts performed by the female target toward these testers often outnumbered the lunges performed by the tester toward the target (Figure 2—figure supplement 3B). This was not the case when the target flies were fruF males (Figure 2—figure supplement 3A). These counter-attacks by female targets may have contributed to the lower number of lunges toward female targets than toward fruF male targets.

We asked whether social isolation, which elevates levels of aggression in males (Hoffmann, 1990; Wang et al., 2008), could enhance the induction of lunges. Interestingly, optogenetic activation of Tk-GAL41-labeled neurons in single-housed fruM females failed to induce a single lunge toward fruF male targets (Figure 2—figure supplement 3C2 F2). Instead, this manipulation resulted in higher levels of orientation and wing extensions than was exhibited by group-housed flies (Figure 2—figure supplement 3C1, C3). These data suggest that the relatively high levels of courtship behavior seen in fruM females interferes with the aggression-promoting function of Tk-GAL4FruM neurons. Consistent with this idea, group-housed fruM females that did not lunge toward a target fly after optogenetic stimulation of Tk-GAL41-labeled neurons tended to show higher levels of wing extensions than testers who lunged (Figure 2—figure supplement 3E, F1). Lastly, optogenetic activation of Tk-GAL4FruM neurons did not induce headbutts (Figure 2—figure supplement 2A3, B3), confirming that this genotype is using a male-type aggressive action exclusively (Vrontou et al., 2006).

Overall, the results above delineate complementary roles of dsx and fru on male aggressive behavior. The activation of Tk-GAL4FruM neurons induces lunges even in the absence of dsx-dependent aggression-promoting neurons. Tk-GAL4FruM neurons are therefore part of a fruM-dependent execution mechanism for male aggressive behavior (Figure 2D). However, the reduced intensity and compromised target sex selectivity of lunges shown by fruM females suggest that a dsx-dependent mechanism may play a role in establishing a target sex-selective application of aggressive behavior. This mechanism can inhibit the execution of lunges specifically toward females, which ensures that lunges are performed exclusively by males towards other males (Figure 2D), or can enhance aggression specifically toward male targets indirectly by inhibiting courtship toward them.

Female-type NP2631 ∩ dsxFLP neurons in fruM females do not promote aggression

Having established that Tk-GAL4FruM neurons can trigger lunges independent of dsx splicing pattern, we wondered whether other sexually dimorphic neurons are involved in the execution of male-type aggressive behaviors in the fruM female brain. One candidate was the NP2631 ∩ dsxFLP neuron group, which was reported to promote aggressive behaviors in both males and females (Koganezawa et al., 2016).

As was discussed above, dsx specifies the sexual dimorphism of NP2631 ∩ dsxFLP neurons, such that the morphology of these neurons in fruM females is largely female-type, and not male-type (Figure 3A; see also Ishii et al., 2020). We confirmed that optogenetic activation of NP2631 ∩ dsxFLP neurons in females robustly increased time orienting (Figure 3C1, Figure 3—figure supplement 1B1) and headbutts (Figure 3C2, Figure 3—figure supplement 1B2) toward female target flies, mostly during LED stimulation (see also Video 6 – Part 1). This behavioral phenotype was consistently observed when the fru locus was either +/+, +/fru4-40 or fruF/fru4-40, similar to the case in males (Figure 3—figure supplement 1C). Interestingly, we observed similar behaviors toward the fruF male targets as well (Figure 3B, Figure 3—figure supplement 1A; see also Video 6 – Part 2). Aggression-promoting neurons that belong to the dsx-expressing ‘pC1’ cluster have been also characterized in the female brain by using different genetic reagents (Deutsch et al., 2020; Palavicino-Maggio et al., 2019). Whether these neurons and the NP2631 ∩ dsxFLP neurons we manipulated belong to the same subtypes remains undetermined. However, we found that NP2631 ∩ dsxFLP neurons did not overlap with neurons labeled by the R71G01-LexA transgene (Figure 3—figure supplement 1D,E). This is consistent with recent findings that female ‘pC1’ neurons are anatomically and functionally heterogeneous (Deutsch et al., 2020; Wang et al., 2020; Wu et al., 2019).

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NP2631 ∩ dsxFLP neurons do not promote either male- or female-type aggressive behavior in fruM females.

(A) Schematics of how Tk-GAL4FruM and NP2631 ∩ dsxFLP neurons (see Ishii et al., 2020 for details) are specified in each genotype. Female-type NP2631 ∩ dsxFLP neurons are specified in fruM females, raising a question about how they contribute to aggression in this genotype. (B–E) Boxplots of time orienting (B1–E1), headbutts (B2–E2), and lunges (D3, E3) by the female (B, C) or fruM female (D, E) tester flies during the time windows 1–4 (see Figure 1B). Their genotypes and pair numbers are indicated below the plots. Gray lines represent single testers. Target flies are either group-housed fruF males (B, D) or mated females (C, E). In gray: **p<0.01, n.s. p>0.05 (Kruskal-Wallis one-way ANOVA and post-hoc Wilcoxon signed rank test), In black: **p<0.01, *p<0.05, n.s. p>0.05 (Kruskal-Wallis one-way ANOVA and post-hoc Mann-Whitney U-test).

Video 6
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Representative behavior of a female tester fly that expresses CsChrimson:tdTomato under the control of NP2631 and dsxFLP toward a wild-type female (Part 1) or a fruF male (Part 2) target fly, at the onset and offset of LED stimulation.

The aggression-promoting function of NP2631 ∩ dsxFLP neurons raises an intriguing question: do female-type NP2631 ∩ dsxFLP neurons promote aggression in fruM females, and if so, are male-type or female-type aggressive behaviors induced (Figure 3A)? If fruM’s role in defining sexual dimorphism in aggression prevails, activation of NP2631 ∩ dsxFLP neurons in fruM may promote male-type aggression (lunges) even though these neurons exhibit female-type morphology. If the morphology of NP2631 ∩ dsxFLP neurons dictates the sexual dimorphic action of aggressive behavior, the same manipulation may promote female-type aggressive actions (headbutts).

Interestingly, we did not observe induction of either lunges or headbutts (Figure 3D2 and E2,3) when we optogenetically activated the NP2631 ∩ dsxFLP neurons in fruM females. Thus, female-type NP2631 ∩ dsxFLP neurons can promote headbutts only if other mechanisms that rely on sexually dimorphic splicing of fru are present in the brain. In addition, this result suggests that NP2631 ∩ dsxFLP neurons with female-like morphology fail to establish functional connections with neurons that are part of the fruM-dependent execution mechanism for male-type aggression (which includes Tk-GAL4FruM neurons; Figure 3A).

We therefore conclude that female-like NP2631 ∩ dsxFLP neurons cannot participate in generation of aggressive behavior (e.g. headbutts) without separate populations of neurons that are specified in the absence of fruM. This finding raises a possibility that the sexual dimorphism of NP2631 ∩ dsxFLP neurons, which is largely specified by dsx, does not determine the sexual dimorphism of aggressive actions. It is also possible that activities of both dsx and fru may be required for the complete functional transformation of NP2631 ∩ dsxFLP neurons. As is shown in Ishii et al. (2020), male-type NP2631 ∩ dsxFLP neurons cannot trigger lunges without a fruM-dependent mechanism, underscoring the dominant role of fru and fru-dependent neural circuits in specifying sexually dimorphic action patterns during aggressive interactions.

Differential roles of fru isoforms on male-male interactions and specification of Tk-GAL4FruM neurons fruM undergoes another layer of alternative splicing to create three transcript isoforms, fruMA, fruMB, and fruMC, (Figure 4A), which encode different zinc finger domains (Usui-Aoki et al., 2000). The loss of each of three fruM isoforms is known to affect male courtship behaviors in distinct manners (Neville et al., 2014; von Philipsborn et al., 2014; Figure 4—figure supplement 1A,B). After finding that fruM is important for specification of Tk-GAL4FruM neurons, as well as for its capacity to execute male-type aggressive actions, we next addressed whether different isoforms of fruM have distinct roles for the specification and function of Tk-GAL4FruM neurons.

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fruMB and fruMC are necessary for male aggression.

(A) Schematics of dsx and fru splicing patterns in a male. (B, C) Boxplots of time orienting (B1, C1), lunges (B2,C2), and wing extensions (B3, C3) by the fru isoform-specific tester mutants (B) or fruMB tester mutants created in trans with the fruF allele (C), along with their genetic controls (single-housed for 6 days), toward group-housed wild-type target males during 30 min assays. Testers’ genotypes of the fru locus and pair numbers are indicated below the plots. Data points that exceed the range (indicated left) are represented by dots, with exact values in parentheses. **p<0.01 (Mann-Whitney U-test with Bonferroni multiple comparison corrections).

First, we characterized how isoform-specific mutations affect spontaneous male-type aggressive behavior after social isolation (Hoffmann, 1990; Wang et al., 2008). In parallel with their previously characterized differential effects on courtship (Neville et al., 2014; von Philipsborn et al., 2014), we found defects of male aggressive behavior in isoform-specific mutants. Loss of fruMA caused no reduction of lunges compared to the fru4-40 heterozygous genetic control (Figure 4B2). In contrast, we observed a dramatic reduction in male-type aggressive behaviors by the loss of fruMB or fruMC isoforms (Figure 4B2). While fruMC mutants showed decreased orientation time toward a male target, orienting times of fruMA and fruMB mutants were comparable to the fru4-40 heterozygous genetic control (Figure 4A1). This reduction in lunges in fruMB mutants was qualitatively recapitulated when the fruF allele was used to create trans-heterozygotes (Figure 4C), and when other mutations affecting FruB zinc finger domains (von Philipsborn et al., 2014) were tested in trans with fru4-40 (Figure 4—figure supplement 1C). We observed no consistent reduction in overall activity levels between fruMB mutants and genetic controls (Figure 4—figure supplement 1D), or in the total amount of sleep exhibited by these flies (Figure 4—figure supplement 1E). Male-male courtship was differentially affected in three mutants (Figure 4A3), reflecting varied degrees of courtship defects toward females (Figure 4—figure supplement 1A,B; Neville et al., 2014; von Philipsborn et al., 2014). These data indicate that each of the three fruM isoforms make different contributions to aggressive behaviors. fruMA had a minimal impact on male aggressive behavior, whereas fruMB and fruMC were both necessary to maintain normal levels of male aggression. Moreover, the decreased aggression of fruMB mutants was not necessarily due to reduced activity levels or opportunities to interact with the target fly.

We next asked how each of the three fruM isoforms contributes to the specification of Tk-GAL4FruM neurons. In fruMC mutants, we found only 1.9 ± 1.1 (mean ± S.D., n = 8; Figure 5D,E) faintly labeled neurons in the brain area where Tk-GAL4FruM neurons are expected to appear, significantly fewer than the number of cells found in genetic controls (5.6 ± 0.5, mean ± S.D., n = 8; Figure 5A,E). Weak staining of the Tk-GAL4FruM neurons in the fruMC mutants prevented us from visualizing their branching pattern. In contrast, fruMA and fruMB mutants did not affect the number of Tk-GAL4FruM neurons (Figure 5E). We compared the neuroanatomy of Tk-GAL4FruM neurons in these genotypes by registering immunohistochemically labeled brains on a standard brain. Tk-GAL4FruM neurons from fruMA (Figure 5G) or fruMB (Figure 5H) mutants are indistinguishable from each other or from fru4-40 heterozygous control males (Figure 5F; see also Video 7). We measured the volumes of three prominent neurites that emanate from the lateral junction (Yu et al., 2010) of Tk-GAL4FruM neurons (Figure 5F2–H2; see also Figure 1L1, M1), and detected no significant differences among fru4-40 heterozygous controls, fruMA mutants, and fruMB mutants (Figure 5I–K). Heterozygosity of all isoform-specific mutations did not change the cell body number or gross morphology of the Tk-GAL4FruM neurons (Figure 5E, Figure 5—figure supplement 1A–D), either.

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Only fruMC is necessary for the specification of Tk-GAL4FruM neurons.

(A–D): Expression of CsChrimson:tdTomato under the control of Tk-GAL41 and Otd-nls:FLPo (red in A1–D1), black in A2,3-D2,3) in male brains is visualized together with a neuropil marker BRP (blue in A1–D1) by immunohistochemistry. Traced Tk-GAL4FruM neurons are shown in white. Circle: soma (right cluster is enlarged in A3-D3). Scale bar: 100 μm (A1–D1), 10 μm (A3–D3). (E) Mean number of cell bodies of Tk-GAL4FruM neurons per hemibrain is visualized by anti-DsRed antibody in each genotype represented in A–D) and Figure 5—figure supplement 1A–D. Error bars, S.D. Lowercase letters denote significance group (p<0.01, one-way ANOVA with post-hoc Tukey’s honestly significant difference test). (F–H) Z-projection of segmented, registered, and averaged images of CsChrimson:tdTomato expression under the control of Tk-GAL41 and Otd-nls:FLPo (black) in the standard Drosophila brain (gray in F1–H1). Number of used hemibrains are indicated in F2–H2). Medial projection (cyan), anterior projection (blue), and lateral projection (purple) are segmented and overlaid in F2–H2). For panels A–H), genotypes of the fru locus are indicated on the left. (I–K) Volumes of medial projection (I), anterior projection (J), and lateral projection (K) of Tk-GAL4FruM neurons in males. Their genotypes of the fru locus and pair numbers are indicated below the plots. n.s. p>0.05 (Kruskal-Wallis one-way ANOVA).

Video 7
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3D-rendered average image of registered Tk-GAL4FruM neurons in fru4-40 heterozygous control male (dark green) and in fruMB mutant (fruΔB/fru4-40) male (bright green).

Gray represents the standard unisex Drosophila brain (Bogovic et al., 2018).

Taken together, we found that fruMA, fruMB and fruMC have distinct effects on male aggression and specification of the Tk-GAL4FruM neurons. fruMA appeared largely dispensable for both, whereas fruMC was necessary for both. Interestingly, fruMB was necessary for normal levels of male-male aggression, but was not required for specification of the Tk-GAL4FruM neurons.

The Tk-GAL4FruM neurons in fruMB mutants can induce male aggression

While the reduction of male aggression in fruMC mutants can be explained by defects in specification of the Tk-GAL4FruM neurons, which are necessary for normal levels of aggression (Asahina et al., 2014), this same logic does not apply for the fruMB mutants, since these mutants have Tk-GAL4FruM neurons that appear to retain their morphology. One possible explanation is that the Tk-GAL4FruM neurons in fruMB mutants have neurophysiological defects and are no longer functional. To address this, we quantified the excitability of Tk-GAL4FruM neurons in fruMB mutants by measuring their intracellular calcium response while optogenetically stimulating them (Figure 6A,B). The overall calcium response dynamics and magnitudes of Tk-GAL4FruM neurons in fruMB mutants were comparable to those seen in the fru4-40 heterozygous controls (Figure 6C,D), suggesting that Tk-GAL4FruM neurons in fruMB mutants are capable of physiologically responding to depolarizing stimuli.

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Tk-GAL4FruM neurons in fruMB mutants can promote male-type aggression.

(A) Schematics of the functional imaging strategy. (B) Fluorescence of GCaMP6f (green) and CsChrimson:tdTomato (magenta), both of which are driven by Tk-GAL41 (top) and pseudocolored intensity of GCaMP6f fluorescence (within the area indicated by the white square in the top panel) before (bottom left), during (bottom middle), and after (bottom right) the LED stimulation, visualized by 2-photon microscopy. (C) Time course of pseudocolored fluorescence intensity (reference: right side) in Tk-GAL41; UAS-GCaMP6f; UAS-CsChrimson male brains, with the fru locus of +/fru4-40 (left) or fruΔB/fru4-40 (right). LED stimulation (pink at the bottom) was for 30 s at 5 Hz. LED powers are indicated on the left. (D) Boxplots of GCaMP6f fluorescence intensity changes in Tk-GAL4FruM neurons (data shown in C). Genotypes at the fru locus, number of brains examined, and LED powers are indicated below. (F–G) Boxplots of lunges by the tester flies under strong (E), medium (F), or weak (G) LED stimulation conditions (indicated in shaded pink boxes) during the time windows 4. Their genotypes and numbers are indicated below the plots. Lowercase letters denote significance groups (p<0.01, Kruskal-Wallis one-way ANOVA and post-hoc Dunn’s multiple comparison test). (H): Schematic summary of the contributions of fru isoforms on male-type aggressive behavior.

Even though Tk-GAL4FruM neurons exhibited normal excitability, it remains possible that neural circuits downstream of the Tk-GAL4FruM do not respond to inputs from Tk-GAL4FruM neurons in fruMB mutants, due to altered development or physiology of these downstream circuits, or inability of Tk-GAL4FruM neurons to excite downstream neurons. To address this possibility, we optogenetically activated Tk-GAL41-labeled neurons in fruMA, fruMB, or fruMC mutants. Optogenetic activation of Tk-GAL41-labeled neurons in fruMB mutants elicited robust levels of male aggression across several LED intensities, as seen in genetic controls (Figure 6E–G, Figure 6—figure supplement 1A–C). This result suggests that Tk-GAL4FruM neurons in fruMB mutants, once activated, can induce male aggression as efficiently as in the fru wild-type males, and argues against the hypothesis that the mutation of fruMB proteins causes defective development of downstream neurons or reduced efficiency of Tk-GAL4FruM neurons to excite downstream neurons. Instead, these results favor the alternative hypothesis that fruMB mutation reduces aggression-promoting input to Tk-GAL4FruM neurons from upstream circuits. This is likely mediated through fruM-dependent mechanisms as well, since activation of Tk-GAL4FruM neurons can completely overcome the lack of such input.

Overall, our results show that three isoforms of fruM assume distinct roles in the specification and function of sexually dimorphic neurons that control male-type aggressive behaviors (Figure 6H). Namely, fruMC is necessary for the specification of Tk-GAL4FruM neurons, whereas fruMB is necessary for a normal level of spontaneous aggression.

Discussion

In this study, we identified Tk-GAL4FruM neurons as part of a fruM-dependent execution mechanism for male-type aggression. The exclusive role of fruM on the specification of a neural circuit for the execution of male-type aggression is in contrast to the circuit for the execution of male-type courtship behavior, for which dsx plays a major role (see Ishii et al., 2020). One role of dsx on male-type aggression appears to be to prevent inappropriate aggression toward a female target, which suggests that dsx may be involved in the sex-recognition mechanism in the context of agonistic interactions. Furthermore, we found that each of three fruM isoforms play separable roles on male-type aggressive behavior. Our finding reveals a layered genetic mechanism of the fru gene for the specification and function of a sexually dimorphic aggression-controlling circuit, which may provide a genetic substrate for the male-specific evolution of contest rituals.

fru specifies a neural circuit for sexually dimorphic aggressive actions

We found that the optogenetic activation of Tk-GAL4FruM neurons in fruM female tester flies induced male-type aggressive behavior. This result indicates that at least Tk-GAL4FruM neurons and downstream populations form a neural circuit that is sufficient for the execution of male-type aggressive behavior. While this does not necessarily mean that fruF males or females lack the capability to execute male-type aggressive behavior, there has been no report that these two sex genotypes perform lunges spontaneously (Vrontou et al., 2006). Moreover, female-type NP2631 ∩ dsxFLP neurons fail to promote aggressive behaviors (lunges or headbutts) in fruM females. NP2631 ∩ dsxFLP neurons were previously proposed to be the ‘aggression output’ of a switch circuit that selects between courtship and aggression (Koganezawa et al., 2016). Since stimulation of NP2631 ∩ dsxFLP neurons in fruF males did not trigger lunges (see Ishii et al., 2020), at least sexual dimorphism in the neuroanatomy of NP2631 ∩ dsxFLP neurons does not correlate with the sexual dimorphism in aggressive motor programs. These observations favor a hypothesis that neurons specified by fru, which include Tk-GAL4FruM neurons, form the execution mechanism for male-type aggressive action, and NP2631 ∩ dsxFLP neurons serve as modulatory neurons that act on this fru-dependent execution mechanism. Sex-specific splicing of fru defines what type of aggressive behavior an animal performs, likely by specifying a mutually exclusive neural circuit that generates either male-type or female-type aggressive actions.

The definitive role of fru in specifying the execution mechanism for aggression does not mean that dsx is irrelevant. In fact, fruM females lunge against females as well as fruF males, suggesting that dsx is important for suppressing inappropriate aggressive behavior toward a female target. Currently, we do not know whether it is the presence of dsxM, or the absence of dsxF, that is critical. Intriguingly, however, dsx-expressing neurons respond to sex-specific sensory cues, such as olfactory, gustatory, and auditory stimuli (Deutsch et al., 2019; Zhou et al., 2014; Zhou et al., 2015). Since thermogenetic activation of Tk-GAL4FruM neurons induces lunges toward a male target that lacks cuticular hydrocarbons (Asahina et al., 2014), one possible scenario is that a dsx-dependent circuit is responsible for detection or transmission of female-specific, aggression-suppressing cues that have yet to be identified. Alternatively, dsx in males may allow execution of male-type aggressive behavior specifically toward male targets indirectly by inhibiting courtship behavior toward males. fruM females that showed high levels of courtship behavior generally showed low levels of male-type aggression. This observation suggests that courtship behavior can interfere with aggressive behavior, possibly through feedforward inhibitory circuits (Clowney et al., 2015; Kallman et al., 2015; Koganezawa et al., 2016). Regardless of the mechanism, our results uncover distinct roles of dsx and fru on male-type aggressive behavior.

The contribution of dsx on the target sex-selective execution of aggressive behavior also implies that information about target sex can be supplied downstream of Tk-GAL4FruM neuronal activities and modify its behavioral influence. Currently, it remains unclear how the stimulation of Tk-GAL4FruM neurons is transformed into lunges, because the downstream partners of Tk-GAL4FruM neurons have not been characterized yet. It is possible that dsx-specified neurons serve as a ‘block’ against neuronal activity generated by Tk-GAL4FruM neurons and other unidentified constituents of the lunge execution mechanism. Since fru-expressing neurons have been comprehensively characterized in the male brain (Cachero et al., 2010; Yu et al., 2010), it may be necessary to establish an equally detailed catalog of dsx-expressing neurons for uncovering the neural mechanism by which male-type aggressive behavior is controlled in a target sex-dependent manner.

The neuronal population that supports execution of female-type aggressive behaviors has begun to be characterized only recently. Consistent with a previous observation (Koganezawa et al., 2016), we observed that NP2631 ∩ dsxFLP neurons in females promote female-type aggression. This population belongs to a cluster of dsx-expressing ‘pC1’ neurons, which is known to promote a variety of female social behaviors including aggression (Deutsch et al., 2020; Palavicino-Maggio et al., 2019; Robie et al., 2017), receptivity (Deutsch et al., 2020; Zhou et al., 2014) or even courtship-like behaviors (Rezával et al., 2016; Wu et al., 2019). In spite of the importance of ‘pC1’ neurons collectively on female social behaviors, female-type NP2631 ∩ dsxFLP neurons are unlikely to be a part of the execution mechanism that generates female-type aggressive actions. Our results suggest that there are uncharacterized sexually dimorphic neurons that are necessary for females to execute headbutts and other female-specific aggressive actions, likely downstream of female-type NP2631 ∩ dsxFLP neurons.

Isoform-specific roles of fru on male-type aggression fru is a complex gene with multiple alternative splicing sites. Male-specific fruM transcripts can encode at least 3 different DNA binding domains (Usui-Aoki et al., 2000). These three isoforms have distinct impacts on gene expression (Dalton et al., 2013; Neville et al., 2014; Vernes, 2015), neuronal specification (Billeter et al., 2006; Ito et al., 2016; Meissner et al., 2016; von Philipsborn et al., 2014), and courtship behavior (Neville et al., 2014; von Philipsborn et al., 2014), prompting us to address whether these isoforms have differential roles on aggression. Indeed, we found that fruMC is important for the specification of Tk-GAL4FruM neurons, whereas fruMB is possibly important for perceiving or transmitting relevant sensory cues about male targets to an aggression execution mechanism. Because optogenetic stimulation of Tk-GAL4FruM neurons in fruMB mutants increases aggression as effectively as the same manipulation in normal males, any role of fruMB on modulating aggression is likely taking place upstream of Tk-GAL4FruM neurons. In order to understand the role of fruMB on the neural circuits controlling male-type aggression, it will be important to characterize the neural inputs to Tk-GAL4FruM neurons and to identify which of such neurons express fruMB (von Philipsborn et al., 2014).

Our result is in line with the previously reported importance of the fruMC isoform in the development of male-specific branching patterns in several fruM-expressing neurons (Billeter et al., 2006; Ito et al., 2016; von Philipsborn et al., 2014). Among them, activities of ‘P1’ neurons (von Philipsborn et al., 2014) and mAL/aDT2 neurons (Ito et al., 2016; von Philipsborn et al., 2014) are necessary for normal levels of courtship behavior (Kallman et al., 2015; von Philipsborn et al., 2011), reflecting the severe courtship defects of fruMC mutant males (Neville et al., 2014; von Philipsborn et al., 2014). Our observation that fruMC is necessary for both male aggression and specification of Tk-GAL4FruM neurons reveals its importance for male-type aggressive behavior as well, at behavioral and circuitry levels. Using an analogy from the function of steroid hormones in mammals (McCarthy, 2008), fruMC can be regarded as exerting ‘organizational’ functions, while fruMB may exert ‘activation’ functions, in the sense that fruMB is dispensable for the specification of a lunge execution mechanism, yet is necessary for induction of spontaneous aggressive behaviors. The additional support of dsx ensures the aggressive behaviors are applied specifically toward male targets.

The segregation of roles within fru isoforms may increase the gene’s flexibility: for instance, change in the circuit connectivity underlying perception of male-specific cues can be independently altered if different isoforms have different roles in the aggression circuit. In mice, there are at least 3 different estrogen receptor genes (Wu et al., 2009). Although the specific function of each receptor on neural circuits that control sexual behaviors remains largely uncharacterized, it is tempting to speculate that a similar type of division of labor may exist among the estrogen receptor genes. For both fru and estrogen receptors, the functional characterization of genes which each isoform regulate will be the first important step to understand how the division of labor is implemented at the molecular level.

fru may allow males to evolve male-type aggressive actions without altering courtship motor programs

In theory, presence or absence of just one gene can specify two sexes. However, most animal species use multiple genes for sex determination, even if one ‘master’ switch gene (such as Sex-lethal in Drosophila) may initiate the sex determination process (Robinett et al., 2010; Williams and Carroll, 2009). Our results suggest that one of the reasons behind this apparent genetic cooperativity may be that each gene controls specific aspects of sexually dimorphic traits, including behaviors (Pereira et al., 2019; Xu et al., 2012). We further speculate that separate genetic mechanisms for establishing sexually dimorphic actions and target sex-dependent action bias may be evolutionarily adaptive.

Courtship and aggression are likely under different types of selection pressures. Courtship behavior is executed by a male toward a female, who either rejects or accepts courtship (Hall, 1994). In Ishii et al. (2020), we showed that dsx plays an important role in specifying the male courtship execution circuit. The dsx gene produces functional proteins in both sexes, and mediates specification of not only male-type circuits for courtship but also female-type circuits that make females receptive to such motor programs (Deutsch et al., 2019; Zhou et al., 2014). This dual function of dsx in males and females may ensure that the species-specificity of courtship rituals are preserved between both sexes even during speciation, which must accompany co-evolution of both a male’s courtship behavior and a female’s choice process through sexual selection (Majerus, 1986). Interestingly, dsx-expressing neurons show high selectivity to courtship pulse song with behaviorally relevant inter-pulse interval frequencies both in males and females (Deutsch et al., 2019; Zhou et al., 2015).

In contrast, aggressive behavior is executed by sexually dimorphic motor programs often between two members of the same sex, most predominantly among males (Chen et al., 2002; Nilsen et al., 2004; Smith and Price, 1973). Consequently, aggressive actions can be under a different selection pressure from courtship behavior. Ample examples of male-specific weaponry organs across a variety of species support the hypothesis that sexually dimorphic traits that bring advantage specifically during inter-male competition can be fixed through evolution even if it is not directly under sexual selection (Rico-Guevara and Hurme, 2018). This hypothesis suggests that such traits, which can be behavioral as well as anatomical, may evolve independently of courtship behavior. Indeed, some closely related Hawaiian Drosophila species evolved species-specific aggressive rituals even though action patterns of courtship behavior as well as morphology of antennae, an organ necessary for successful courtship, remain relatively similar (Kaneshiro and Boake, 1987; Price and Boake, 1995; Spieth, 1981). This example suggests that animals must be equipped with a genetic mechanism that controls motor programs for aggressive behavior at least partially separately from the motor programs for courtship behavior. fru is well situated to be a genetic agent for such a male-specific modification of aggressive behavior (Davis et al., 2000), because it seems dispensable for specifying a neural circuit that generates at least certain aspects of courtship action patterns (see Ishii et al., 2020). At the same time, dsx can ensure that males do not act aggressively toward a conspecific female. Female-type somatic characteristics, including sex-specific chemical cues (Shirangi et al., 2009), are specified by dsx. It is possible that dsxM prevents costly male aggression toward females by helping the brain process female-type sensory cues as an aggression-inhibiting signal (Fernández et al., 2010) in a species-specific manner.

The origin of sexual dimorphism in social behavior is a fundamental neuroscientific and ethological question. While circuit manipulations offer a powerful approach to dissect the neural mechanisms behind sexually dimorphic behaviors, comparative studies of the neural circuits controlling social behaviors in genetic gynandromorphs can serve as a complementary approach to address this question from both developmental, and possibly evolutionary, perspectives. Although sex-determining genetic pathways are diverse across animal species, studying Drosophila sexual dimorphism can lead to logical predictions about the genetic or neuronal mechanisms underlying sexual dimorphism of social behavior in mammals.

Materials and methods

See Supplementary file 1 for details of reagents used in this study.

Fly strains

Request a detailed protocol

See Table 1 for the complete genotypes of Drosophila strains used in each figure panel. Tk-GAL41 (RRID:BDSC_51975) and Otd-nls:FLPo (in attP40) were previously described Asahina et al. (2014). NP2631 (Yu et al., 2010) is a gift from Daisuke Yamamoto (Tohoku University). 20XUAS > myr:TopHAT2 >CsChrimson:tdTomato (in VK00022, VK00005 and attP2) (Duistermars et al., 2018; Watanabe et al., 2017), 20XUAS-IVS-Syn21-GCaMP6f (codon-optimized)-p10 (in su(Hw)attP5), and 13XLexAop2-IVS-Syn21-GCaMP6f (codon-optimized)-p10 (in su(Hw)attP5) were created by Barret Pfeiffer in the lab of Gerald Rubin (HHMI Janelia Research Campus) and kindly shared by David Anderson (California Institute of Technology). fruM (RRID:BDSC_66874), fruF (RRID:BDSC_66873) (Demir and Dickson, 2005), and fruFLP (RRID:BDSC_66870) (Yu et al., 2010) flies are gifts from Barry Dickson (HHMI Janelia Research Campus); dsxFLP (Rezával et al., 2014), fruΔA, fruΔB (Neville et al., 2014), and fruΔC flies (Billeter et al., 2006) are gifts from Stephen Goodwin (University of Oxford); fruP1.LexA (RRID:BDSC_66698) (Mellert et al., 2010) is a gift from Bruce Baker (HHMI Janelia Research Campus); isogenic Canton-S, fruB1, and fruB2 flies (von Philipsborn et al., 2014) are gifts from Anne von Philipsborn (University of Aarhus). fru4-40 (RRID:BDSC_66692) was obtained from Bloomington Drosophila Resource Center in the University of Indiana. See Ishii et al. (2020) for the details of R71G01-LexA.

Table 1
Complete genotypes of Drosophila strains used in this study.
FIGUREPANELABBREVIATED
GENOTYPE		COMPLETE GENOTYPE
(‘Y’ represents the Y chromosome)
Figure 1C, DTk-GAL41, UAS>stop>CsChrimson, Otd-nls:FLPow, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+;20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/+
Figure 1—figure supplement 1A-C
Video 1
Figure 1C, DTk-GAL41, UAS > stop > CsChrimsonw, Tk-GAL41/Y; +/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/+
Figure 1C, DUAS > stop > CsChrimson, Otd-nls:FLPow/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/+
Figure 1G, K, L, N-PTk-GAL41, XY, fru locus: fruM/fru4-40w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruM
Figure 1—figure supplement 2I
Figure 1—figure supplement 3C
Video 2, 3
Figure 1H, K, M-PTk-GAL41, XX, frulocus: fruM/fru4-40w, Tk-GAL41/w, Tk-GAL41; Otd-nls:FLPoin attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruM
Figure 1—figure supplement 3C
Video 3
Figure 1I, KTk-GAL41, XY, fru locus: fruF/fru4-40w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruF
Figure 1J, KTk-GAL41, XX, fru locus: fruF/fru4-40w, Tk-GAL41/w, Tk-GAL41; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruF
Figure 1K, N-PTk-GAL41, XY, fru locus: +/+w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2/+
Figure 1—figure supplement 2C, D, H, J
Figure 1—figure supplement 3C
Figure 1KTk-GAL41, XX, fru locus: +/+w, Tk-GAL41/w, Tk-GAL41; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2/+
Figure 1—figure supplement 2K
Figure 1KTk-GAL41, XY, fru locus: +/fru4-40w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/+
Figure 1—figure supplement 2L
Figure 1KTk-GAL41, XX, fru locus: +/fru4-40w, Tk-GAL41/w, Tk-GAL41; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/+
Figure 1—figure supplement 2M
Figure 1—figure supplement 1DTk-GAL41, UAS > stop > CsChrimson, Otd-nls:FLPo, fru locus: +/+w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2/+
Figure 1—figure supplement 2N
Figure 1—figure supplement 1DTk-GAL41, UAS > stop > CsChrimson, Otd-nls:FLPo, fru locus: +/fru4-40w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/+
Figure 1—figure supplement 2N
Figure 1—figure supplement 1DTk-GAL41, UAS > stop > CsChrimson, Otd-nls:FLPo, fru locus: fruM/fru4-40w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruM
Figure 1—figure supplement 2N
Figure 1—figure supplement 1DTk-GAL41, UAS > stop > CsChrimson, fru locus: fruM/fru4-40w, Tk-GAL41/Y; +/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruM
Figure 1—figure supplement 1DUAS > stop > CsChrimson, Otd-nls:FLPo, fru locus: fruM/fru4-40w/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruM
Figure 1—figure supplement 2F, GTk-GAL41 ∩ fru (genetic intersection)w, Tk-GAL41/Y; +/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2/fruFLP (2 samples for averaged image in F)
Figure 1—figure supplement 2GTk-GAL41 ∩ fru (genetic intersection)w, Tk-GAL41/Y; +/+; 20XUAS>myr:TopHAT2 > CsChrimson:tdTomato in VK00005/fruFLP (three samples) w, Tk-GAL41/Y; 8XLexAop2-FLPL in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2/fruP1.LexA (2 samples)
Video 2
Figure 1—figure supplement 3A, CTk-GAL41, XY, fru locus: fruF/+w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fruF/+
Figure 1—figure supplement 3B, CTk-GAL41, XX, fru locus: fruM/+w, Tk-GAL41/w, Tk-GAL41; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2/fruM
Figure 1—figure supplement 3ETk-GAL41, UAS > stop > CsChrimson (attP2), Otd-nls:FLPo, fru locus: +/+w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2/+
Figure 1—figure supplement 3ETk-GAL41, UAS > stop > CsChrimson (attP2), fru locus: fruFLP/+w, Tk-GAL41/Y; +/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2/fruFLP
Figure 1—figure supplement 3ETk-GAL41, UAS > stop > CsChrimson (VK00005), fru locus: fruFLP/+w, Tk-GAL41/Y; +/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in VK00005, fruFLP/+
Figure 1—figure supplement 3ETk-GAL41, UAS>stop>CsChrimson (VK00005), fru locus:fruFLP/fru4-40w, Tk-GAL41/Y; +/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in VK00005, fruFLP/fru4-40
Figure 2A,-CfruM, Tk-GAL41, UAS > stop > CsChrimson, Otd-nls:FLPow, Tk-GAL41/w, Tk-GAL41; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruM
Figure 2—figure supplement 2A, B
Figure 2—figure supplement 3A, B, E
Video 4, 5
Figure 2A-CfruM, Tk-GAL41, UAS > stop > CsChrimsonw, Tk-GAL41/w, Tk-GAL41; +/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruM
Figure 2—figure supplement 3A, B
Figure 2A-CfruM, UAS > stop > CsChrimson, Otd-nls:FLPow/w; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruM
Figure 2—figure supplement 3A, B
Figure 2CTk-GAL41, UAS > stop > CsChrimson, Otd-nls:FLPow, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/+
Figure 2—figure supplement 3D
Figure 2—figure supplement 1A, BWild type (tester)+/+; +/+; +/+ (Canton-S)
(target)w/Y; +/20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in VK00022; dsxFLP, fru4-40/fruM
fruF (target)w/Y; +/20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in VK00022; dsxFLP, fru4-40/fruF
Figure 2—figure supplement 3C, FfruM, Tk-GAL41 (hetero), UAS > stop > CsChrimson, Otd-nls:FLPow, Tk-GAL41/w; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruM
Figure 2—figure supplement 3CfruM, Tk-GAL41 (hetero), UAS > stop > CsChrimsonw, Tk-GAL41/w; +/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruM
Figure 3B, CNP2631, UAS > stop > CsChrimson, dsxFLPw/w; NP2631/20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in VK00022; dsxFLP, fru4-40/fruF
Figure 3—figure supplement 1A, B
Video 6
Figure 3B, CNP2631, UAS > stop > CsChrimsonw/w; NP2631/20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in VK00022; fru4-40/fruF
Figure 3B, CUAS > stop > CsChrimson, dsxFLPw/w; +/20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in VK00022; dsxFLP, fru4-40/fruF
Figure 3D, EfruM, NP2631, UAS > stop > CsChrimson, dsxFLPw/w; NP2631/20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in VK00022; dsxFLP, fru4-40/fruM
Figure 3D, EfruM, NP2631, UAS > stop > CsChrimsonw/w; NP2631/20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in VK00022; fru4-40/fruM
Figure 3D, EfruM, UAS > stop > CsChrimson, dsxFLPw/w; +/20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in VK00022; dsxFLP, fru4-40/fruM
Figure 3—figure supplement 1CNP2631, UAS > stop > CsChrimson, dsxFLP fru locus: +/+w/w; NP2631/20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in VK00022; dsxFLP/+
Figure 3—figure supplement 1CNP2631, UAS > stop > CsChrimson, dsxFLP fru locus: +/fru4-40w/w; NP2631/20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in VK00022; dsxFLP, fru4-40/+
Figure 3—figure supplement 1CNP2631, UAS > stop > CsChrimson, dsxFLP fru locus: fruF/fru4-40w/w; NP2631/20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in VK00022; dsxFLP,
fru4-40/fruF
Figure 3—figure supplement 1D, ENP2631 ∩ dsxFLP, R71G01-LexAw/w; NP2631/13XLexAop2-IVS-GCaMP6f-p10 in su(Hw)attP5; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in VK00005, dsxFLP/R71G01-LexA in attP2
Figure 4Bfru locus: +/fru4-40+/Y; +/+; +/fru4-40 (F1 hybrid of +; +; fru4-40/TM6B and isogenic Canton-S)
Figure 4—figure supplement 1A-E
Figure 4Bfru locus: fruΔA/fru4-40+/Y; +/+; fruΔA/fru4-40
Figure 4—figure supplement 1A, B
Figure 4Bfru locus: fruΔB/fru4-40+/Y; +/+; fruΔB/fru4-40
Figure 4—figure supplement 1A, B, D, E
Figure 4Bfru locus: fruΔC/fru4-40+/Y; +/+; fruΔC/fru4-40
Figure 4—figure supplement 1A, B
Figure 4Bfru locus: +/++/Y; +/+; +/+ (F1 hybrid of Canton-S and isogenic Canton-S)
Figure 4—figure supplement 1A-E
Figure 4Bfru locus: fruΔA/++/Y; +/+; fruΔA/+
Figure 4—figure supplement 1A, B
Figure 4Bfru locus: fruΔB/++/Y; +/+; fruΔB/+
Figure 4—figure supplement 1A, B, D, E
Figure 4Bfru locus: fruΔC/++/Y; +/+; fruΔC/+
Figure 4—figure supplement 1A, B
Figure 4Cfru locus: +/fruF+/Y; +/+; +/fruF
Figure 4Cfru locus: fruΔB/fruF+/Y; +/+; fruΔB/fruF
Figure 4Cfru locus: +/fruM+/Y; +/+; +/fruM
Figure 4Cfru locus: fruΔB/fruM+/Y; +/+; fruΔB/fruM
Figure 4—figure supplement 1Cfru locus: fruB1/fru4-40+/Y; +/+; fruB1/fru4-40
Figure 4—figure supplement 1Cfru locus: fruB2/fru4-40+/Y; +/+; fruB2/fru4-40
Figure 4—figure supplement 1Cfru locus: fruB1/++/Y; +/+; fruB1/+
Figure 4—figure supplement 1Cfru locus: fruB2/++/Y; +/+; fruB2/+
Figure 5A, E, F, I-KTk-GAL41, XY, fru locus: +/fru4-40w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/+
Video 7
Figure 5B, E, G, I-KTk-GAL41, XY, fru locus: fruΔA/fru4-40w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruΔA
Figure 5C, E, H-KTk-GAL41, XY, fru locus: fruΔB/fru4-40w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruΔB
Figure 5D, ETk-GAL41, XY, fru locus: fruΔC/fru4-40w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruΔC
Figure 5ETk-GAL41, XY, fru locus: +/+w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2/+
Figure 5—figure supplement 1A
Figure 5ETk-GAL41, XY, fru locus: fruΔA/+w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2/fruΔA
Figure 5—figure supplement 1B
Figure 5ETk-GAL41, XY, fru locus: fruΔB/+w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2/fruΔB
Figure 5—figure supplement 1C
Figure 5ETk-GAL41, XY, fru locus: fruΔC/+w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2/fruΔC
Figure 5—figure supplement 1D
Figure 6B-DTk-GAL41, UAS-GCaMP6f, UAS-CsChrimson, fru locus: +/fru4-40w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/20XUAS-IVS-Syn21-GCaMP6f-p10 in su(Hw)attP5; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/+
Figure 6C, DTk-GAL41, UAS-GCaMP6f, UAS-CsChrimson, fru locus: fruΔB/fru4-40w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/20XUAS-IVS-Syn21-GCaMP6f-p10 in su(Hw)attP5; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruΔB
Figure 6E-GTk-GAL41, UAS > stop > CsChrimson, Otd-nls:FLPo, fru locus: +/fru4-40w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/+
Figure 6—figure supplement 1A-C
Figure 6E-GTk-GAL41, UAS > stop > CsChrimson, Otd-nls:FLPo, fru locus: fruΔA/fru4-40w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruΔA
Figure 6—figure supplement 1A-C
Figure 6E-GTk-GAL41, UAS > stop > CsChrimson, Otd-nls:FLPo, fru locus: fruΔB/fru4-40w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruΔB
Figure 6—figure supplement 1A-C
Figure 6E-GTk-GAL41, UAS > stop > CsChrimson, Otd-nls:FLPo, fru locus: fruΔC/fru4-40w, Tk-GAL41/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruΔC
Figure 6—figure supplement 1A-C
Figure 6E-GUAS > stop > CsChrimson, Otd-nls:FLPo, fru locus: +/fru4-40w/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/+
Figure 6E-GUAS > stop > CsChrimson, Otd-nls:FLPo, fru locus: fruΔA/fru4-40w/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruΔA
Figure 6E-GUAS > stop > CsChrimson, Otd-nls:FLPo, fru locus: fruΔB/fru4-40w/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruΔB
Figure 6E-GUAS > stop > CsChrimson, Otd-nls:FLPo, fru locus: fruΔB/fru4-40w/Y; Otd-nls:FLPo in attP40/+; 20XUAS > myr:TopHAT2 > CsChrimson:tdTomato in attP2, fru4-40/fruΔC

To create male flies that lack FruMA, FruMB, or FruMC, we followed approaches taken in previous publications (Neville et al., 2014; von Philipsborn et al., 2014) and created trans-heterozygotes of isoform-specific mutations and fru4-40 or fruF. These two alleles do not transcribe fruM, while an isoform-specific mutation does not transcribe one of fruA, fruB, or fruC. Therefore, each trans-heterozygote is lacking a fru allele that can generate fruMA, fruMB, or fruMC transcripts, respectively.

Immunohistochemistry

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The following antibodies were used for immunohistochemistry with dilution ratios as indicated: rabbit anti-DsRed (1:1,000, Clontech # 632496, RRID:AB_10013483), mouse anti-BRP (1:100; Developmental Studies Hybridoma Bank nc82 (concentrated), RRID:AB_2314866), rabbit anti-FruM (1:10,000, a gift from Barry Dickson; Stockinger et al., 2005), guinea pig anti-FruM (1:100), rat anti-DsxM (1:100) (both gifts from Michael Perry, University of California, San Diego), rat anti-HA (1:100, Roche Cat# 11867423001, RRID:AB_390918), goat anti-rat Alexa 488 (1:100, ThermoFisher Scientific Cat# A11006, RRID:AB_2534074), goat anti-rabbit Alexa 488 (1:100, ThermoFisher Scientific, Cat# A11034, RRID:AB_2576217), goat anti-rat Alexa 488 (1:100, ThermoFisher Scientific Cat# A11006), goat anti-rabbit Alexa 568 (1:100; ThermoFisher Scientific Cat# A11036, RRID:AB_10563566), goat anti-mouse Alexa 633 (1:100; ThermoFisher Scientific Cat# A21052, RRID:AB_2535719), goat anti-guinea pig Alexa 633 (1:100, ThermoFisher Scientific Cat# A21105, RRID:AB_2535757). Immunohistochemistry of the fly brains followed the protocol described in Van Vactor et al. (1991). Briefly, the fly brains are dissected in 1XPBS and fixed in 1XPBS with 2% formaldehyde and 75 mM L-lysine for 75–90 min at room temperature. The brains were then washed in PBST (1XPBS, 0.3% TritonX-100) and were incubated in the blocking solution (10% heat-inactivated normal goad serum, 1XPBS, 0.3% TrintonX-100) for 30 min. Primary antibodies were diluted in the blocking solution and were applied to samples, which were then incubated at 4°C for 2 days. The brains were then washed in PBST and then incubated in the blocking solution for 30 min. Secondary antibodies were diluted in the blocking solution and were applied to the samples, which were then incubated at 4°C overnight. The brains were then washed in PBST, and then either incubated in 1XPBS, 50% glycerol for 2 hr at room temperature before mounted in Vectashield (Vector Laboratories, Cat# H-1000) onto a slide glass, or incubated in FocusClear (CelExplorer Labs, Taiwan, Cat# FC-101) medium for 2 hr at room temperature before being mounted in MountClear (CelExplorer Labs, Taiwan, Cat# MC-301) medium. A small well was made by cutting vinyl tape fixed on a slide glass, and one brain was transferred to each well before a cover slip (#1.5) was placed on the well and was sealed with nail polish. All reactions were carried out in a well of 6 × 10 microwell mini tray (ThermoFisher Scientific Cat# 439225).

For simultaneous detection of DsxM and FruM (Figure 1—figure supplement 2C,D), brains were fixed in 1XPBS with 4% formaldehyde for 15 min at room temperature. Brains were then transferred to a 1.5 mL microtube, in which the remaining steps were carried out.

Z-stack images were acquired by FV-1000 confocal microscopy (Olympus America) except samples for Figure 3—figure supplement 1D, E, which were acquired by a Zeiss 710 confocal microscopy (Carl Zeiss Microscopy) at the Salk Institute Biophotonics Core, and were processed in Fiji software (Schindelin et al., 2012) (RRID:SCR_002285; https://fiji.sc/). The despeckle function was applied before a z-projection image was generated using maximum intensity projection. Minimum or maximum intensity thresholds were adjusted for enhanced clarity. Source image files used in all figures can be found in Source data 1.

Segmentation, registration, and analysis of immunohistochemical samples

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All data points for anatomical quantifications used in all figures, as well as all statistical results with exact p values, can be found in Source data 1.

Parametric tests were applied as indicated in figure legends to compare cell body numbers among different genotypes. All data points have biological replicates of at least 8, which has sufficient power to detect mean changes in cell body number of larger than 1.75 when assuming a mean cell body number of 5 and a standard deviation of 1.5 (which is reasonable based on our data). As indicated in figures, genotype-dependent changes in cell body number had a larger effect size than 2 in all cases.

For segmentation of Tk-GAL4FruM neurons, we first visualized each z-stack in a 3D space using a rendering software FluoRender (Wan et al., 2009) (RRID:SCR_014303; https://github.com/SCIInstitute/fluorender). We then chose the channel corresponding to Alexa 568 (which labels CsChrimson:tdTomato immunohistochemistry signal), and traced neural processes emanating from the cell bodies of Tk-GAL4FruM neurons, which could be identified unambiguously at the lateral side of the brain. The neural processes were then segmented using the Paint Brush function (Wan et al., 2017), and saved as a new. nrrd file.

For registration of brains, we split each channel of a z-stack file and resaved each as an individual. nrrd files in Fiji. We added the newly created. nrrd file that represent Tk-GAL4FruM neurons (see above) into the same folder, and used the Fiji plugin for Computational Morphometry Toolkit (CMTK) (RRID:SCR_002234; https://www.nitrc.org/projects/cmtk; Rohlfing and Maurer, 2003) to register the brain image stacks to the template brain as described in Jefferis et al. (2007) (https://github.com/jefferis/fiji-cmtk-gui). Briefly, for each brain, the image of neuropil visualized by anti-BRP antibody was used to transform the z-stack to the template brain, and the same transformation was subsequently applied to additional channels. Registration was performed by using the same parameters implemented in ‘Cachero et al. (2010)’ (exploration = 26, coarsest = 8, grid spacing = 80, refine = 4, accuracy = 0.4) in CMTK plugin on Fiji. We used JRC2018 INTERSEX (Bogovic et al., 2018) as our template, since we experienced more robust registration result than with the FCWB template brain (Costa et al., 2016) (data not shown).

After the registration of z-stacks, we calculated the average of the images in a hemibrain for each genotype. We did this by horizontally flipping each transformed z-stack images, and calculating the average signal intensity in each voxel. The resulting averaged images are bilaterally symmetrical. Therefore, only the left hemisphere is shown for z-projection images.

To calculate the volumes of specific neuronal structures within Tk-GAL4FruM neurons, we further segmented the target structures using the Paint Brush function of FluoRender, and calculated the volume of each structure using the Volume Size function. Statistical analyses were carried out using MATLAB (The Mathworks, Inc, RRID:SCR_001622). The Kruskal-Wallis test (‘kruskalwallis’) was used to evaluate whether a volume of the given structure was significantly different among different genotypes. When the p-value was below 0.05, the post-hoc the Mann-Whitney U-test (‘ranksum’) was used to detect significant differences between testing and control genotypes. In both cases, Bonferroni correction was applied to p values. Non-parametric tests were applied for volume data since we could not necessarily assume the normal distribution of this data type.

Social behavior analysis

Subject preparation

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Flies were collected on the day of eclosion into vials containing standard cornmeal-based food, and were kept either as a group of up to 16 flies per vial, or singly at 25 °C with 60% relative humidity, and a 9AM:9PM light:dark cycle. For optogenetic experiments, the tester flies were reared on food containing 0.2 mM all-trans retinal (MilliporeSigma, Cat#R2500, 20 mM stock solution prepared in 95% ethanol), and vials were covered with aluminum foil to shield light. Every 3 days, flies were transferred to vials containing fresh food. Tester flies were aged for 5–7 days except tester flies used in Figure 3B–E and Figure 3—figure supplement 1A–C, which were aged for 14–16 days to ensure consistent labeling of targeted neurons (data not shown).

Male target flies were group-reared Canton-S (originally from the lab of Martin Heisenberg, University of Würzberg) virgin males except those used in Figure 4B–C and Figure 4—figure supplement 1C, which were males of an isogenic Canton-S (a gift from Anne von Philipsborn, Aarhus University). They were used as targets in these experiments because we observed that our standard Canton-S target males were sometimes aggressive and dominant towards the tester males even after being reared in groups (data not shown), which we were concerned could obscure the innate level of aggression in the tester male flies. These isogenic males performed few lunges toward the tester male flies (data not shown). To prepare mated wild-type target females, 5 Canton-S males were introduced into vials with 10 virgin females at 4 days old, and were reared for 2 more days to let them mate. At 3 days old, both male and female target flies were briefly anesthetized with CO2, and the tip of either one of their wings were clipped by a razor to create a ‘mark’. This clipping treatment did not reduce the amount of each behavior (lunge, wing extension, and headbutt) detected under our experimental settings (see Ishii et al., 2020 for details).

Behavioral assays

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All behavior assays were conducted in the evening between Zeitberger time (ZT)7 and ZT12 (from 4 to 9PM) at 22–25°C. Social behavior assays were performed in a ‘12-well’ acrylic chamber (Asahina et al., 2014) with food substrate (apple juice (Minute Maid) supplemented with 2.25% w/v agarose and 2.5% w/v sucrose; Hoyer et al., 2008) covering the entire floor of arena. The wall was coated with Insec-a-Slip (Bioquip Products, Inc, Cat# 2871C) and the ceiling was coated with Surfasil Siliconizing Fluid (ThermoFisher Scientific, Cat# TS-42800), both to prevent flies from climbing, as described previously (Asahina et al., 2014; Hoyer et al., 2008). The arenas were lit by LED backlights, which were controlled by a custom-built switch box. For optogenetic experiments, 850 nm infrared LED backlights (Sobel Imaging Systems, CA, Cat# SOBL-150 × 100–850) were used, whereas white backlights (Edmunud Optics, NJ, Cat# 83873) were used for non-optogenetic experiments (Figure 4A,B, Figure 4—figure supplement 1A–C). Flies were introduced into the chamber by gentle aspiration, and were allowed to acclimate for 5 min before recording started.

Recording was done by USB3 digital cameras (Point Grey Flea3 USB3.0, FLIR Inc, Cat# FL3-U3-13Y3M-C) controlled by the BIAS acquisition software (IORodeo, CA; https://bitbucket.org/iorodeo/bias). The camera was equipped with a machine vision lens (Fujinon, Cat# HF35HA1B), and an infrared longpass filter (Midwest Optical Systems, Cat# LP780-25.5) when the infrared light sources were used. Movies were taken at 60 frames per second in the AVI format, either for 10 min in the optogenetic experiments or for 30 min for non-optogenetic experiments. Flies were discarded after each experiment. The food substrates were changed to a new one after 2 recordings for 30 min movies, or after 3 recordings for 10 min movies.

The setup for optogenetic experiments was assembled as described previously (Inagaki et al., 2014). Briefly, the red light (655 nm) LEDs were controlled via an Arduino Uno board (Arduino, Italy) using a custom program. As illustrated in Figure 1B, the stimulation paradigm (10 min in total) consists of 1 min pre-stimulation (time window ‘1’ in Figure 1B), three blocks of 1 min stimulation at an indicated frequency (time window ‘2’, 3 min in total) each followed by 2 min inter-stimulus intervals (ISIs, time window ‘3’, 6 min in total). The recording and LED control were manually started simultaneously.

Quantification of social behavior data

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All behavioral data points used in all figures, as well as all statistical results with exact p values, can be found in Source data 1.

Acquired movies were first processed by the FlyTracker program (Asahina et al., 2014; http://www.vision.caltech.edu/Tools/FlyTracker/), which runs on MATLAB and creates output files which JAABA uses for behavioral classifications. See Ishii et al. (2020) for the creation, validation, and. jaab source files of JAABA-based behavioral classifiers used in this study, as well as the definition of 'time orienting'. The regions of interest were defined as circles corresponding to the chamber of each arena. The identities of tester and target flies were manually confirmed. The fly pair was removed from further analysis when (1) one of the two flies was killed during introduction to the chamber, (2) the wings of either fly was stuck at the extended position, or (3) the discrimination of the two flies was impossible due to wing damage of a tester fly. The amount of behavior is the number of bouts for lunges and headbutts, and the total duration (seconds) for wing extensions. These amounts were binned per minute for quantification. Extremely short bouts detected by a classifier were almost always false positives, and were eliminated from quantification. For lunges and headbutts, events with duration of less than 50 milliseconds were discarded. For wing extensions, events with a duration of less than 100 milliseconds were discarded. The post-processing of data was done using custom MATLAB codes which are available on Github (https://github.com/wohlmp/Ishii_Wohl_DeSouza_Asahina_2019; Wohl, 2019; copy archived at https://github.com/elifesciences-publications/Ishii_Wohl_DeSouza_Asahina_2019).

The frame in which the infrared indicator LED turned on during the first LED stimulation period was used to align frames of movies. Statistical analyses were carried out using MATLAB. After behavior within each time window were calculated (see Figure 1B), the Kruskal-Wallis test (‘kruskalwallis’) was used to evaluate whether a given behavior was significantly different among different illumination periods (periods 1, 2, and 3; Figure 1B) or among different genotypes. When the p-value was below 0.05, the post-hoc Mann-Whitney signed rank test (‘signrank’) was used to detect significant differences between illumination period, and the Mann-Whitney U-test (‘ranksum’) was used to detect significant differences between testing and control genotypes. In both cases, Bonferroni correction was applied to p values. When the uncorrected p value was less than 0.05 but the corrected value did not pass the significant level, the uncorrected value was shown on panels with parenthesis. For Figure 6E–G, Dunn’s multiple comparison test was applied to detect significant groups using a custom code (‘dunn.m’) (Cardillo, 2006; http://www.mathworks.com/matlabcentral/fileexchange/12827).

While it is not feasible to perform power analyses for non-parametric datasets, we performed a bootstrapped power analysis with a sample dataset from 171 single-housed wild-type male fly pairs (which is not included in this manuscript). We asked how many pairs of flies (biological replicates) are necessary to achieve a power of 0.8 when attempting to detect a 50% difference in the number of lunges and found that 22 pairs are sufficient. Thirty pairs of flies would increase the power to 0.9. We tested at least 24 pairs per genotype for all social behavior assays, and many groups contain more than 30 pairs. While the data distribution appears different among different genotypes and behaviors, we are confident that the number of biological replicates used in this study gives us sufficient power to support our conclusions.

Generally, male testers seldom performed lunges toward female targets. Also, noticeably less lunges were detected in fruM females. Behavioral data from these conditions often result in zero-inflated datasets. In such cases, a few false positive incidents can impact the result of statistical tests. To avoid this pitfall, classifier results of lunges from male testers toward female targets and from fruM females were manually validated, and obvious false positives (caused by tracking errors, when a fly was near or on the wall, or when a fly suddenly jumped) were eliminated before statistical tests were applied.

Activity and sleep analysis

Request a detailed protocol

Male flies were collected and singly reared for 6 days as was described in ‘Social behavior analysis’. Experiments were carried out for 3 days at 24°C. Activity and sleep data were acquired using individual Drosophila activity monitors (TriKinetics Inc) under 12 hr:12 hr light:dark cycles, as was described previously (Wu et al., 2014). Counts of beam crosses between Zeitgerber (ZT)8 and ZT12 on day 2 and day 3 were binned per minute (note that ZT8-12 corresponds to the time period when we performed social behavioral assays). Sleep was defined as at least 5 min of inactivity (no beam cross). The amount of activity and sleep were quantified using custom MATLAB scripts provided by William Joiner (University of California, San Diego).

Functional calcium imaging

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Male flies were kept in all-trans retinal food after eclosion and aged 6 days. On the day of the experiment, flies were briefly anesthetized on ice and mounted on a custom chamber using ultraviolet curing adhesive (Norland Optical Adhesive 63, Norland Products, Inc) to secure the head and thorax to a tin foil base. The proboscis was also dabbed with glue to prevent its extension from altering the position of the brain. The head cuticle was removed with sharp forceps in room temperature Drosophila adult hemolymph-like saline (Wang et al., 2003). After cuticle removal, the recording solution was refreshed. Optogenetic stimulation was applied with an external fiber-coupled LED of 625 nm (Thorlabs Inc, Cat# M625F2) controlled by a programmable LED driver (ThorLabs, Cat# DC2200). The end of the LED fiber (Thorlabs, Cat# M28L01) was placed 5 mm from the brain. LED illumination of 5 Hz, 10-millisecond pulses for a 30 s duration was applied for optogenetic activation of CsChrimson. LED power was varied so that each fly received stimulations at 0, 0.02, 0.04, 0.1, and 0.2 mA. The energy from the LED that the neurons receive was estimated from the measurement of the LED power using a photodiode power sensor (Thorlabs, Cat# S130C) 5 mm away from the end of the LED fiber. The energy was read using a digital optical power/energy meter (Thorlabs Cat# PM100D).

The multiphoton laser scanning microscope (FV-MPE-RS, Olympus Corporation), equipped with 25X water immersion objective (Olympus Corporation, Cat# XLPLN25XWMP2), was used for monitoring the fluorescence of GCaMP6f. The recordings began 10 s before stimulation and continued 20 s after stimulation for a total of 1 min. GCaMP6f proteins were excited with a 920 nm laser (Spectra-Physics Insight DL Dual-OL, Newport Corporation). Images were taken at 10 Hz with a 256 × 256 pixel resolution.

Acquired images were converted and analyzed in Fiji with the Olympus ImageJ plugin (http://imagej.net/OlympusImageJPlugin). Imaging windows were chosen that maximally captured the dense set of projections that emanate from Tk-GAL4FruM neurons before they branch out further (lateral junction described in Yu et al., 2010; Figure 6B). Rectangular regions of interest were selected and ∆F/F was calculated using a custom-written MATLAB code. First, the baseline fluorescence value (Fbase) was calculated by averaging the fluorescence for 5 s preceding the stimulation. ∆F/F for each frame (∆F/Fframe=N) was calculated as follows:

(∆F/Fframe=N)=[(Fframe=N)–Fbase]/Fbase

Then, the ∆F/ Fframe=N for frames taken during the 30 s LED stimulation (excluding frames where the light was on) were averaged to calculate the ∆F/F of a given trial. Boxplots of average ∆F/F during stimulation at different LED powers and across flies were generated. Heterozygous controls and fruMB mutants’ fluorescence data were compared across LED powers using the Mann-Whitney U-test (‘ranksum’).

Data availability

All data and statistical results are available in Source data 1.

References

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    2. Turner A
    (1987)
    Animal Conflict
    New York: Chapman and Hall Ltd.
    1. Rico-Guevara A
    2. Hurme KJ
    (2018) Intrasexually selected weapons
    Biological Reviews of the Cambridge Philosophical Society 20:12436.
    https://doi.org/10.1111/brv.12436
    1. Villella A
    2. Gailey DA
    3. Berwald B
    4. Ohshima S
    5. Barnes PT
    6. Hall JC
    (1997)
    Extended reproductive roles of the fruitless gene in Drosophila Melanogaster revealed by behavioral analysis of new fru mutants
    Genetics 147:1107–1130.

Decision letter

  1. Mani Ramaswami
    Reviewing Editor; Trinity College Dublin, Ireland
  2. K VijayRaghavan
    Senior Editor; National Centre for Biological Sciences, Tata Institute of Fundamental Research, India

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Acceptance summary:

The study analyses male-aggression, a sexually dimorphic behaviour. It shows that Fruitless function in a distinct subset of Tk-Gal4 positive neurons is required for male-male aggression independent of Doublesex, which acts in different subset of neuron to enable such aggression to be targeted towards male competitors and not females. The observation that the underlying circuits are different from those involved in courtship has interesting evolutionary implications.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for submitting your work entitled "A genetic code that specifies sexually dimorphic circuits and their control of social behaviors" for consideration by eLife. Your article has been reviewed by a Senior Editor, a Reviewing Editor, and three reviewers. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation between the reviewers. In the discussions, all the referees expressed interest in the main message (dissociating fru and dsx functions and relating these to vertebrate mechanisms) as well as enthusiasm for several very interesting and clear findings from an impressively extensive set of experiments. However, there were also substantial concerns leading to the consensus opinion that additions and revisions required were too extensive to be accomplished in the two months required by eLife.

Based on these discussions and the individual reviews appended below, we regret to inform you that, in present form, your work will not be considered further for publication in eLife. However, if you choose to revise the paper on the lines suggested by the referees and resubmit, then eLife would be happy to consider a fresh submission (or two) that we would endeavour to have reviewed by the same referees.

In addition to the specific comments below, the referees offered some specific overall suggestions for revision that you may find useful. These include: (a) to refocus the paper on solely on the fru/dsx story; (b) to include new rigorous anatomical analysis of the "P1" cluster in P1a and NP2631 flies as requested in the reviews; and (c) providing a detailed discussion of limitation of using systemic/constitutive alleles of fru/dsx in relation to behavior as well as anatomy. The referees also felt that the story be clarified and better communicated by splitting into two separate but simpler manuscripts, with a potentially a separate manuscript on Tk, but you should of course make your own decision on this rather too interventional opinion.

Reviewer #1:In both flies and mammals, multiple sexual differentiation mechanisms guide display of adult social behaviors. In flies it is dsx and fru, whereas in mammals it is largely testosterone and estrogen. Ishii et al. present a wide-ranging set of observations that they use to suggest that various aspects of sexually dimorphic courtship and aggression displays in flies are controlled by either dsx or fru. This seems an over-interpretation of the findings. I am supportive of the study but have a few suggestions.

1) Data from Figure 3 is used to conclude that dsx specifies both P1 and NP2631 neurons. This is inconsistent with the anatomy shown in figure panels, which shows that both dsx and fru contribute. It is true that cell number is dependent on dsx, but the morphology of the arbors indicates that fru is essential as well: E3/J3/K3 are different than C3/H3/I3. Similarly, data from Figure 6 is used to conclude that fruM but not dsx specifies Tk neurons; comparison of E3 and F3 indicates that dsxM is also important. The authors should quantify arborization rather than rely on gross examination for Figure 3 and Figure 6.

Presentation of the histology could be better elsewhere too. For example, from panels Figure 2A,C, I conclude that there is no overlap of R15A01 and R71G01, raising the question of the intersectional identification of P1a neurons.

2) It is clear that FruM is essential for male type lunges toward other males and enhancing wing extension to females. However, FruM is also essential for the pattern of wing extension upon activation of P1a neurons; in controls (Figure 1), wing extensions in phase 2>3 and 2<3 toward males and females, whereas in FruF males (figure 4A-D), wing extensions in phase 2<3 toward males and females. In addition, it is clear that FruM is essential for regulating intensity of wing extensions. It is a gross simplification to conclude that dsxM enables execution of male courtship whereas FruM enforces choice of target sex. Clearly, FruM is also critical for execution of this behavior.

3) Why do males not lunge toward other males in the absence of activation of P1a or NP2631 or Tk neurons (Figure 1, Figure 6)?

4) There is some over-simplification of the literature. It might be true in flies that most studies have only used one sex as a target individual to analyze function of particular neurons (subsection “Effect of a target fly’s sex on optogenetically induced social interactions”), but this is certainly not true in mice (studies from Anderson, Dulac, Lin, Shah).

Rather than draw parallels to the four core genotype in the mouse (using which not a single gene has been identified that regulates social behavior independent of sex hormones, subsection “dsx, and not fru, specifies both P1a and NP2631 ∩ dsxFLP neurons”), it would be more appropriate to discuss the role of estrogen vs. testosterone in regulating male behaviors. It is clear that estrogen masculinizes the brain during a critical window whereas testosterone signaling amplifies male behaviors in adult life. This would be a more instructive analogy when trying to dissect the specific functions of dsx and fru.Reviewer #2:Unfortunately, I have some major technical concerns with the manuscript, and the lack of quantitative anatomical analyses throughout. The rather cursory analysis offered in this manuscript has several critical caveats that are not discussed and undermine the conclusions that can be drawn from the experimental data. Therefore, this manuscript is not suitable for publication without significant experimental clarification of these points.

Subsection “Effect of a target fly’s sex on optogenetically induced social interactions” and throughout the manuscript:

Neither the P1a split-Gal4 nor NP2631 ∩ dsxFLP driver are 'P1/pC1'-specific, some additional neurons in the brain express Gal4 in both cases (see both Hoopfer et al., 2015 and Koganezawa et al., 2016). Therefore, by using these drivers, the authors cannot conclude that the observed behavioral phenotypes were caused purely by the purported 'P1/pC1' neurons. Further intersectional approaches to rigorously target these neurons are essential.

Subsection “dsx, and not fru, specifies both P1a and NP2631 ∩ dsxFLP neurons”:

The expression 'P1a neurons were specified only in the flies that had dsxM' is not accurate and is likely even wrong. Whether the presence of the P1 neurons depends on fru or dsx was originally examined in the very first P1 paper, Kimura et al., 2008. Kimura et al. previously demonstrated by using dsx mutations that the absence of dsxF, but not the presence of dsxM, is the most striking factor for determining the presence of the P1 neurons.

In Figure 3, the dsx locus was always wild type and therefore the authors could not dissociate the presence of dsxM and the absence of dsxF. It is essential for the authors to do the similar experiments in combination with the appropriate dsx mutant alleles.

The authors mentioned that 'processes of P1a neurons in fruF males appeared thinner than those in males'. What does thinner mean? What form of scientific measurement is "thinner"? This needs to be quantified properly. Image co-registration or any other quantitative analyses for the size differences of the neuronal processes between these genotypes must be carried out.

The authors also mention ‘sex differences both in terms of cell body numbers and branching patterns’. If the authors declare the branching patterns were different between genotypes, they must carry out quantitative analyses.

They also declare the neuronal morphology was 'similar' between particular genotypes, but what does 'similar' mean? The authors seem to conclude that the neuronal morphology is different when they expect it is different and that it is similar when they expect it is similar, without any quantitative criteria for measurement.

Subsection “P1a and NP2631 ∩ dsxFLP neurons in fruF males can induce courtship, but not male-type aggressive behaviour”:

The logical link between the anatomy in Figure 3 and behavior in Figure 4 is unclear. In Figure 3, the authors looked at the contributions of the fru and dsx genes in the purported 'P1/pC1'-specific neurons, but in Figure 4, they carried out behavioral assays with the fru mutants, in which fru gene function is disrupted not only in P1/pC1 neurons but also all other FruM-expressing neurons throughout the PNS and CNS, they then base all their conclusions on only the behavioral function of P1/pC1 neurons. These animals will have a plethora of fru mutant phenotypes that are associated with lack of FruM in other neurons in the CNS and PNS.

To make any relevant conclusions the authors need to re-do the behavioral assays in Figure 4 using the flies in which only the P1/pC1 neurons are mutant for fru (e.g., mutant MARCM clones, UAS-Cas9 system, or fru-RNAi).

Subsection “fruM specifies aggression-promoting Tk-GAL4FruM neurons”:

The logic of transition from P1/pC1 to Tk-Gal4 neurons is very difficult to follow. The relationship between the former and the latter need to be explained. Are they completely different stories?

Figure 6:

As a control, the authors should demonstrate that Tk-GAL4 neurons do not express dsx.

Subsection “Differential roles of fru isoforms on male-male interactions and specification of Tk-GAL4FruM neurons”:

The authors showed that the Tk-Gal4 neurons exist in fru∆B mutant males but behavior is affected in these flies. The simplest explanation would be that the neurites are malformed in these flies.

Although the authors mentioned that 'fruMA and fruM mutants did not affect […] their arborization pattern', they didn't seem to carry out any quantitative morphological analyses. Such analyses should be included.

Subsection “The Tk-GAL4FruM neurons in fruMB mutants can induce male aggression”:

The authors mention that 'these mutants have Tk-GAL4FruM neurons that appear to retain their morphology'. But how did they examine this?

Reviewer #3:This paper is a paradox. As far as I can tell it is rigorous on all levels. The scholarship is excellent, deep and well rounded. In addition, the issues are explored thoroughly and there is an enormous amount of data. In addition, an enormous number of fly lines (the table that describes the lines is phenomenal). But on the other hand, it is too much. I would like to discuss the argument and conclusions that surface relating dsx and fru expressing neurons with sexually dimorphic choice vs. sexually dimorphic motor patterns and the interesting differences between reproductive behavior and aggression, but the paper is simply too dense at almost every point. Reading it is a painful experience and this does the work a tremendous disservice. I say this recognizing and admiring the care that has gone into preparing this text. It seems flawless, just impossibly dense.

What to do? Here is a list of suggestions.

1) The text and figures are impeccably constructed but for the average reader they are overly complicated and far too dense. The flow of the manuscript would benefit greatly from severe editing of the text, figures and figure legends. It is with a heavy heart that I ask the authors to do this knowing the amount of effort it took to construct the manuscript.

2) Remove raster plots from figures throughout and move to supplemental material if needed. See Figure 1, Figure 4, Figure 5, Figure 6, Figure 7, Figure 9.

3) Figure 2 is not required. Move to supplemental material and concisely state result in text.

4) Much of the immunohistochemistry could be removed from figures and moved to supplemental material. See Figure 3, Figure 6, Figure 8.

5) The summary panels at the end of some of the figures are helpful. But a single unifying summary/model would be best. Though organized in a systematic and rational way the results still feel piecemeal and it is tiresome to continually have refer back to previous figures or supplemental material to regain your bearing. A single model would help immensely.

Alternatively, it might make sense to turn this into two papers, one about sex, the other about aggression?

[Editors’ note: further revisions were suggested prior to acceptance, as described below.]

Thank you for submitting your article "Layered roles of fruitless isoforms in specification and function of male aggression-promoting neurons in Drosophila" for consideration by eLife. Your article has been reviewed by K VijayRaghavan as the Senior Editor, a Reviewing Editor, and two reviewers. The reviewers have opted to remain anonymous.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

In this follow up paper, the authors analyse male-aggression, a different sexually dimorphic behaviour and that fru function in a distinct subset of Tk-Gal4 positive neurons is required for male-male aggression independent of Doublesex, which acts in different subset of neuron to enable such aggression to be targeted towards male competitors and not females. The observation that the underlying circuits are different from those involved in courtship has interesting evolutionary implications.

Essential revisions:

The issues enumerated below need to be clarified before the paper can be accepted.

1) That activation of Tk-Gal4 neurons in single-housed FruM females does not elicit aggression is at odds with the conclusion that this circuit is sufficient for executing male type aggression. If anything, single housing should have increased aggression. The authors' suggest that the lack of aggression is because of increase in male-type courtship, but this still tacitly indicates that activating the Tk/FruM circuit is insufficient to induce aggression. The authors' conclusions should be tempered accordingly.

2) As with the companion paper (“Sex-determining genes distinctly regulate courtship capability and preference toward females through sexually dimorphic neurons”), it would appear that the effects of Fru isoform on aggression in Tk-GAL4FruM neurons also can only really be assessed by looking at animals were the Tk-GAL4FruM neurons are mutant/ perturbed for fru function (e.g. fru isoform-specific mutant MARCM clones, UAS-Cas9 system, or fru-RNAi). Otherwise, the data would need to be interpreted with the caveat that fru mutant phenotypes could be caused by lack of Fru isoform in other neurons in the CNS and/or the PNS. This issue needs to addressed directly – ideally experimentally given that a lineTK-GAL4/fruP1LexA/LexAop2-FLPL that may allow these neurons to be targeted has been described in Asahina et al., 2014.

3) A key finding arising from optogenetic experiments, artificially activating Tk-GAL4FruM neurons are a refinement of the authors previously published thermogenetic work (Asahina et al., 2014), and the isoform-specific roles of Fru on male behavior and Fru-specified neural circuits, are implied by work from other groups (Neville et al., 2014; von Philipsborn et al., 2014). The discussion must clearly articulate what new information this manuscript provides on Fru isoform function and aggressive behavior.

4) Does Figure 2—figure supplement 2E, F1 (subsection “Tk-GAL4FruM neurons are a part of a fruM-dependent circuit for the execution of male-type aggressive behaviour”) refer to a different figure panel. Please correct.

5) Dsx immunolabeling in Figure 1—figure supplement 2C is poor. Please provide better images.

https://doi.org/10.7554/eLife.52702.sa1

Author response

[Editors’ note: the authors resubmitted a revised version of the paper for consideration. What follows is the authors’ response to the first round of review.]

Our decision has been reached after consultation between the reviewers. In the discussions, all the referees expressed interest in the main message (dissociating fru and dsx functions and relating these to vertebrate mechanisms) as well as enthusiasm for several very interesting and clear findings from a impressively extensive set of experiments. However, there were also substantial concerns leading to the consensus opinion that additions and revisions required were too extensive to be accomplished in the two months required by eLife.

Based on these discussions and the individual reviews appended below, we regret to inform you that, in present form, your work will not be considered further for publication in eLife. However, if you choose to revise the paper on the lines suggested by the referees and resubmit, then eLife would be happy to consider a fresh submission (or two) that we would endeavour to have reviewed by the same referees.

In addition to the specific comments below, the referees offered some specific overall suggestions for revision that you may find useful. These include: (a) 1 to refocus the paper on solely on the fru/dsx story; (b) to include new rigorous anatomical analysis of the "P1" cluster in P1a and NP2631 flies as requested in the reviews; and (c) providing a detailed discussion of limitation of using systemic/constitutive alleles of fru/dsx in relation to behavior as well as anatomy. The referees also felt that the story be clarified and better communicated by splitting into two separate but simpler manuscripts, with a potentially a separate manuscript on Tk, but you should of course make your own decision on this rather too interventional opinion.

We decided to split our original manuscript into two, following two reviewers’ suggestions that this would increase clarity. We agree that our attempts to integrate our findings on 2 genes (dsx and fru), 2 behaviors (courtship and aggression), and 3 neuronal populations (NP2631 ∩ dsxFLP, P1a , and Tk-GAL4FruM neurons) caused difficulties with the flow of logic. We believe that two separate manuscripts allow us to focus on two relatively simpler messages individually, and help us present relevant data without causing confusion. “Sex-determining genes distinctly regulate courtship capability and target preference via sexually dimorphic neurons” also serves to incorporate your first major recommendation: “to refocus the paper on solely on the fru/dsx story”. In line with this recommendation, we now include a new set of data showing that dsx and fru-co-expressing neurons in males can promote both courtship and aggressive behaviors in a target sex-dependent manner (see Figure 1 of that manuscript).

Regarding your second recommendation to provide rigorous neuroanatomical analysis, we thoroughly re-evaluated our anatomical data by registering brains to a common template and analyzing specific volumes with 3-dimentional segmentation. Please see Figure 3L-M, Figure 6H, I, in “Sex-determining genes distinctly regulate courtship capability and target preference via sexually dimorphic neurons”, and Figure 1N-P, and Figure 5I-K in “Layered roles offruitlessisoforms in specification and function of male aggression-promoting neurons in Drosophila” for new quantification of neuronal morphologies. Standardized immunohistochemical data enabled us to directly compare the morphology of specific neurons across multiple genotypes. Moreover, we could detect statistical differences in neuroanatomy by quantitatively comparing segmented volumes of specific neuronal structures. This rigorous quantification revealed differences in P1a neurons between males and fruF males, which compelled us to revise our original conclusions for this class of neurons.

In response to your third recommendation, we added a discussion of the limitations of studying sex-determining genes through constitutive mutants and through cell-specific genetic manipulation (see “An organismal sex and a cellular sex” section in Discussion of “Sex-determining genes distinctly regulate courtship capability and target preference via sexually dimorphic neurons”). We believe that cell-specific manipulation of sex-determining genes is a powerful method that is not without weaknesses, and will serve complimentary roles to constitutive mutants when studying their effects on neuroanatomy and behaviors.

Reviewer #1:

In both flies and mammals, multiple sexual differentiation mechanisms guide display of adult social behaviors. In flies it is dsx and fru, whereas in mammals it is largely testosterone and estrogen. Ishii et al. present a wide-ranging set of observations that they use to suggest that various aspects of sexually dimorphic courtship and aggression displays in flies are controlled by either dsx or fru. This seems an over-interpretation of the findings. I am supportive of the study but have a few suggestions.

1) Data from Figure 3 is used to conclude that dsx specifies both P1 and NP2631 neurons. This is inconsistent with the anatomy shown in figure panels, which shows that both dsx and fru contribute. It is true that cell number is dependent on dsx, but the morphology of the arbors indicates that fru is essential as well: E3/J3/K3 are different than C3/H3/I3. Similarly, data from Figure 6 is used to conclude that fruM but not dsx specifies Tk neurons; comparison of E3 and F3 indicates that dsxM is also important. The authors should quantify arborization rather than rely on gross examination for Figure 3 and Figure 6.

We agree that we should have quantified our findings to draw conclusions about the neuroanatomy of these neurons. Reviewer #2 also raised concerns about our anecdotal statements regarding neuroanatomy. We therefore thoroughly re-analyzed our anatomical data by registering brains to a “template” Drosophila brain, and by using 3-dimentional segmentation and volume measurements. Please see Materials and methods section for technical details. In essence, we used a non-rigid transformation technique described by Jefferis et al., 2007, to register individual sample brains to the standard unisex Drosophila brain described in Bogovic et al., 2018. This allowed us to compare labeling patterns across multiple brains and sexes in the single reference space.

Immunohistochemical labeling can have technical as well as biological variability. Such inter-sample variability might have been perceived as inter-genotype differences. To better represent labeling patterns that are consistent across samples, we averaged the signal intensity of imaged neurons after registration in the standard brain. The resulting images are shown in 2-dimensional projections as well as in 3-dimensional movies. A similar approach was taken to show the neuroanatomy of various fru-expressing neurons (Yu et al., 2010, von Philipsborn et al., 2014), and we feel this is the best approach to concisely summarize our observations from multiple samples.

As a quantification, we compared the volumes of specific neuronal innervations across different genotypes. We used the 3-dimensional rendering program FluoRender to segment structures of interest, and calculated the volumes of each structure.

With these new and rigorous approaches, we are now able to quantitatively support our initial conclusions that (1) NP2631 ∩ dsxFLP neurons are largely specified by dsx, and (2) Tk-GAL4FruM neurons are specified by fru.

In “Sex-determining genes distinctly regulate courtship capability and preference toward females through sexually dimorphic neurons” Figure 3H-K and Video 3, Video 4 and Video 5, we demonstrate that the overall morphology of NP2631 ∩ dsxFLP neurons in males and fruF males overlap well, and that these neurons in females and fruM females are also similar. We quantified three neuropils that show noticeable sexual dimorphism, and in all cases, volumes of these neuropils in fruF males are statistically indistinguishable from those in males. Volumes in fruM females are comparable to values in females except in one neuropil. We acknowledge that our quantification is based on the entire neural population, and that we may not have sufficient sensitivity to detect either subtype-specific contributions of fru, or differences at finer scales such as number or length of branches. We mention this possibility in the main text. Overall, however, our analysis provided us with evidence arguing that sexual dimorphism of NP2631 ∩ dsxFLP neurons is mainly, if not entirely, specified by dsx.

Likewise, data in “Layered roles of fruitless isoforms in specification and function of male-type aggression-promoting neurons in Drosophila” Figure 1L, M and Video 3 clearly show that average Tk-GAL4FruM neurons from males and from fruM females almost perfectly overlap (light cyan in Video 3 indicates the overlap between male (green) and fruM female (dark blue) Tk-GAL4FruM neurons). As is shown in “Layered roles of fruitless isoforms in specification and function of male-type aggression-promoting neurons in Drosophila” Figure 1N-P, three prominent innervations of these neurons show similar volumes across males (both fru +/+ and fruM/fru4-40) and fruM females. We therefore retain our original conclusion that Tk-GAL4FruM neurons are predominantly specified by fru.

In contrast, we found that both dsx and fru are important for specifying P1a neurons. In the previous version, we hastily concluded that dsx specified P1a neurons mainly because P1a neurons appear in fruF male brains, but not in fruM female brains. However, quantification of volumes of two neuropils clearly indicates that P1a neurons in fruF males have distinct morphology from those in males (see “Sex-determining genes distinctly regulate courtship capability and preference toward females through sexually dimorphic neurons”, Figure 6F-I). Results from this and re-analysis of behavioral data (discussed below) compelled us to revise our conclusion about P1a neurons. We now fully acknowledge the role of fru in both specification and function of P1a neurons. We feel this is a striking contrast to NP2631 ∩ dsxFLP neurons, and further supports the idea that these two neurons are distinct subpopulations within what has been collectively described as P1 or pC1 neurons.

We thank the reviewer for encouraging us to take an extra step for verification (which turned out to require substantial manual and computational works), as we feel our revised conclusions are now better supported by the data.

Presentation of the histology could be better elsewhere too. For example, from panels Figure 2A,C, I conclude that there is no overlap of R15A01 and R71G01, raising the question of the intersectional identification of P1a neurons.

We admit that images in our original version were not sufficiently clear, especially on printed media. It is a challenge to show all visible neurons clearly without over-saturating strongly labeled cells, or without dominating the figure panel. We enlarged the images by 20%, and would like to draw attention to original images provided as source data.

Regarding the overlap of R15A01 and R71G01 promoters, our initial submission used the R71G01-LexA transgene in attP40. It is known in the community that transgene expression at the attP40 landing site is not as robust as at the attP2 landing site, which is used for creating the Rubin GAL4 collection. We had created a transgenic animal with R71G01-LexA in attP2, and tested its overlap with NP2631 ∩ dsxFLP and with NP2631 ∩ fruFLP (“Sex-determining genes distinctly regulate courtship capability and preference toward females through sexually dimorphic neurons”, Figure 5C, D and Figure 5—figure supplement 3C, D, respectively). As anticipated, we observed more R71G01-LexA-labeled neurons from the attP2 transgene than from the attP40 transgene. The overlap with NP2631 ∩ dsxFLP or NP2631 ∩ fruFLP neurons is still very small, confirming our initial conclusion that P1a neurons have minimal overlap with these neurons. We strengthen this conclusion by comparing registered and averaged 3D images of NP2631 ∩ dsxFLP and P1a neurons (“Sex-determining genes distinctly regulate courtship capability and preference toward females through sexually dimorphic neurons”, Figure 5E-G). Neuroanatomy of these two populations shows significant differences.

In addition, Hoopfer et al., 2015 showed that optogenetic stimulation of R15A01-LexA neurons recapitulates the behavioural effects of the P1a neuronal activation (see Figure 3H in the citation). This data suggest that R1501-LexA indeed includes P1a neurons. We therefore remain confident with our conclusion which this piece of data is most relevant: NP2631 ∩ dsxFLP and P1a neurons are largely separate subpopulations.

We would like to add that split GAL4 lines are often created from lines labeling seemingly different neurons at a population level, especially in z-projection images. In fact, it may be difficult to discern overlapping neurons from the images of R15A01-GAL4 and R71G01-GAL4 in Figure 1C of Hoopfer et al., 2015 (which described

P1a neurons).

2) It is clear that FruM is essential for male type lunges toward other males and enhancing wing extension to females. However, FruM is also essential for the pattern of wing extension upon activation of P1a neurons; in controls (Figure 1), wing extensions in phase 2>3 and 2<3 toward males and females, whereas in FruF males (Figure 4A-D), wing extensions in phase 2<3 toward males and females. In addition, it is clear that FruM is essential for regulating intensity of wing extensions. It is a gross simplification to conclude that dsxM enables execution of male courtship whereas FruM enforces choice of target sex. Clearly, FruM is also critical for execution of this behavior.

The reviewer was right in pointing out the importance of fru on the function, as well as on neuroanatomy (discussed above), of P1a neurons. We confirmed that optogenetic stimulation of P1a neurons in fruF males induced significantly less wing extensions than in regular males regardless of target sex (“Sex-determining genes distinctly regulate courtship capability and preference toward females through sexually dimorphic neurons”, Figure 6—figure supplement 2). This means that the courtship-executing function, as well as specification, of P1a neurons require the activity of both dsx and fru. Currently, we do not know whether dsx and/or fru is necessary within P1a neurons, or whether the proper function of P1a neurons requires another population of dsx- or fru-dependent neurons (see also discussions regarding cell type-specific manipulation of sex-determining genes). We therefore reasoned that it would be prudent to avoid speculations about the roles of dsx and fru on P1a neurons, and decided to re-frame P1a-related data in the context of discussing the potential diversity of the P1/pC1 neuronal cluster.

On the other hand, optogenetic stimulation of NP2631 ∩ dsxFLP neurons in fruF males induced a similar amount of wing extensions as the same manipulation in regular males did toward male target flies (“Sex-determining genes distinctly regulate courtship capability and preference toward females through sexually dimorphic neurons”, Figure 4C). Together with our observation that the neuroanatomy of NP2631 ∩ dsxFLP neurons in regular males and in fruF males showed little difference, we conclude that (1) fruF males are capable of executing wing extensions, (2) NP2631 ∩ dsxFLP neurons are one neuronal component that belongs to a dsx-dependent wing extension execution circuit, and (3) fruM is important for enhancing courtship behavior specifically toward females.

By referring to the “execution” of courtship behavior, we are specifically focusing on the animal’s capability to express courtship behavior. We aim to uncover a neurogenetic process that enables animals to perform courtship behavior at all, in contrast to the ability to adjust the intensity of courtship behavior toward females. Please note that we do not argue that fru is not required for proper courtship behavior. What we argue is that fru is not necessary for all aspects of male courtship behavior. The importance of fru on male sexual behavior has been extensively demonstrated, leading some to state that fruM specifies all aspects of male sexual behaviors. Here, we wish to shed light on the fundamental characteristics of fru mutants, which is the unusually high level of courtship behavior toward males, not the absence of courtship behavior altogether. This phenotype has been consistently and repeatedly reported (literature include Gailey and Hall (1989), Villella et al. (1997), Ito et al. (1996), Demir and Dickson (2005), Shirangi et al. (2006), Pan and Baker (2014), among others), even though these observations may not have received sufficient attention. Moreover, many of previously characterized fru-expressing neurons are sensory neurons or early-stage interneurons that transmit sensory information. We feel that our conclusions are entirely consistent with past literature. Our novel functional characterization of NP2631 ∩ dsxFLP neurons provides a neuronal substrate that accounts for the courtship behavior-execution capability of fruF males.

3) Why do males not lunge toward other males in the absence of activation of P1a or NP2631 or Tk neurons (Figure 1, Figure 6)?

The tester flies were all housed in groups for 6 days (P1a neurons) or 14 days (for NP2631 ∩ dsxFLP neurons), which potently reduces spontaneous aggressive behavior. Socially isolated animals show high levels of spontaneous aggression, which can cause a ceiling effect when trying to observe further increases of aggression. Both in flies and mice, group-housing is a common treatment to characterize behavioral changes in response to neuronal manipulations.

4) There is some over-simplification of the literature. It might be true in flies that most studies have only used one sex as a target individual to analyze function of particular neurons (subsection “Effect of a target fly’s sex on optogenetically induced social interactions”), but this is certainly not true in mice (studies from Anderson, Dulac, Lin, Shah).

Rather than draw parallels to the four core genotype in the mouse (using which not a single gene has been identified that regulates social behavior independent of sex hormones, subsection “dsx, and not fru, specifies both P1a and NP2631 ∩ dsxFLP neurons”), it would be more appropriate to discuss the role of estrogen vs. testosterone in regulating male behaviors. It is clear that estrogen masculinizes the brain during a critical window whereas testosterone signaling amplifies male behaviors in adult life. This would be a more instructive analogy when trying to dissect the specific functions of dsx and fru.

We appreciate the reviewer’s constructive suggestion to re-frame the roles of dsx and fru. Indeed, studies in mice often use both males (often castrated) and females as intruders. While that these neuronal manipulations may appear to trigger social behaviors largely independent of target sex, we found that some target sex-dependence has been previously reported. For instance, Lin et al. (2010) reported that optogenetic stimulation of the mediolateral part of the ventromedial hypothalamus (VMHml) triggered aggression more consistently toward males than toward females (Figure 4L,M in the citation). Likewise, close examination of data from Yang et al. (2017), which investigated the behavioral changes caused by chemogenetic activation of progesterone receptor expressing neurons in VMHml, suggests that aggression toward males was more consistent that toward females, although it was not explicitly tested (Figure 1F,G in citation). These past observations reinforce our idea that target sex in neuronal manipulation experiments can be an important variable in other experimental models.

We also agree that the “four core genotypes” model has not been particularly successful in providing mechanical insight into the origin of sexual dimorphism. Although the temporal dynamics between mammalian sex hormones and Drosophila sex-determining genes can be quite different, the reviewer’s opinion that the roles of dsx and fru can be analogous to those of estrogen and testosterone is indeed a quite useful framework for our study. We incorporated this suggestion into our Discussion.

Reviewer #2:

Unfortunately, I have some major technical concerns with the manuscript, and the lack of quantitative anatomical analyses throughout. The rather cursory analysis offered in this manuscript has several critical caveats that are not discussed and undermine the conclusions that can be drawn from the experimental data. Therefore, this manuscript is not suitable for publication without significant experimental clarification of these points.

As discussed in response to reviewer #1, we incorporated quantification of our neuroanatomical data. Additional details are provided below, and we hope these new analyses address the reviewer’s concerns.

Subsection “Effect of a target fly’s sex on optogenetically induced social interactions” and throughout the manuscript:

Neither the P1a split-Gal4 nor NP2631 ∩ dsxFLP driver are 'P1/pC1'-specific, some additional neurons in the brain express Gal4 in both cases (see both Hoopfer et al., 2015 and Koganezawa et al., 2016). Therefore, by using these drivers, the authors cannot conclude that the observed behavioral phenotypes were caused purely by the purported 'P1/pC1' neurons. Further intersectional approaches to rigorously target these neurons are essential.

We are a little confused by the reviewer’s assertion that “Neither P1a-GAL4 or NP2631 ∩ dsxFLP are ‘P1/pC1’-specific”, after citing the previous publications. In both cases, we see in citation figures that these drivers in fact label a single cluster of neurons cleanly, both in original publications as well as in our own studies.

For P1a-GAL4 neurons, Figure 1E of Hoopfer et al., 2015 shows clearly that only a single cluster of neurons at the posterior end of a male brain were labeled. We confirmed this specificity repeatedly, as shown in “Sex-determining genes distinctly regulate courtship capability and preference toward females through sexually dimorphic neurons”, Figure 6A, Figure 6—figure supplement 1A, C. NP2631 ∩ dsxFLP neurons described in Figure 4D of Koganezawa et al., 2016 also seems to label a well-defined single cluster of neurons per hemisphere. Again, we observed a bilateral single cluster of neurons at the posterior edge of male brains (“Sex-determining genes distinctly regulate courtship capability and preference toward females through sexually dimorphic neurons”, Figure 3C, Figure 3—figure supplement 1A, C). Please also see the movies showing the 3-dimentional structure of P1a and NP2631 ∩ dsxFLP neurons.

We wondered whether the reviewer was raising the possibility that this seemingly single cluster of neurons may indeed consist of heterogeneous populations. Although the number of cells that are labeled by both drivers is rather small (“Sex-determining genes distinctly regulate courtship capability and preference toward females through sexually dimorphic neurons”, Figure 3G and Figure 6E), further intersection could reveal a specific subset that is responsible for the observed behaviors. We currently do not have a means to reliably separate such subpopulations, and we also feel that such experiments would better belong to a future study. Here, we focus on the existence of dsx-dependent neurons that can execute wing extensions, and a separate, fru-dependent mechanism that enhances wing extensions toward females.

Subsection “dsx, and not fru, specifies both P1a and NP2631 ∩ dsxFLP neurons”:

The expression 'P1a neurons were specified only in the flies that had dsxM' is not accurate and is likely even wrong. Whether the presence of the P1 neurons depends on fru or dsx was originally examined in the very first P1 paper, Kimura et al., 2008. Kimura et al. previously demonstrated by using dsx mutations that the absence of dsxF, but not the presence of dsxM, is the most striking factor for determining the presence of the P1 neurons.

In Figure 3, the dsx locus was always wild type and therefore the authors could not dissociate the presence of dsxM and the absence of dsxF. It is essential for the authors to do the similar experiments in combination with the appropriate dsx mutant alleles.

We admit that we were careless in stating that either dsxM or dsxF is responsible for neuroanatomical or behavioral differences from regular males. With the reagents we used, we could only state whether sexually dimorphic splicing of dsx has an impact on NP2631 ∩ dsxFLP or P1a neurons. We do not know whether altered neuroanatomy of P1a neurons in fruF males is due to the lack of dsxM, or due to the presence of dsxF. As the reviewer points out, we must use dsx mutants (especially dsxDom, which forces male-type splicing of dsx regardless of sex chromosome composition) to gain insight into which of the two isoforms is responsible.

However, we would like to emphasize that it is not the specific role of dsxM or dsxF, but rather the genetic origin of sexual dimorphism, that we wished to investigate in this study. That is, we are interested in addressing which sexually dimorphic characteristics (neuroanatomical and behavioral) are controlled by the dimorphism (e.g., sex-specific splicing) of dsx and fru. As we state in the Discussion, one sex is not a mutant of the other sex, and sex-determining genes often have different functions in each of the two sexes – although fru was often considered silent in females, we presented circumstantial evidence that the fruF allele may not be truly null (“Layered roles of fruitless isoforms in specification and function of male-type aggression-promoting neurons in Drosophila”, Figure 1—figure supplement 3A-C). In this context, splicing mutants such as fruM and fruF provide a useful platform to understand the genetic nature of sex-specific transformation. These two alleles have the added benefit that experiments could be conducted in a symmetrical manner – we can exhaust all four possible combinations of dsx and fru sex-specific isoforms (“Sex-determining genes distinctly regulate courtship capability and preference toward females through sexually dimorphic neurons”, Figure 3B). Currently, no mutation that forces female-type splicing of dsx is known.

We believe our data present strong evidence that dsx and fru have distinct impacts on the specification and function of sexually dimorphic neuronal circuits. We revised our statements and referred to the importance of dsx as a gene, not of sex-specific splicing isoforms.

The authors mentioned that 'processes of P1a neurons in fruF males appeared thinner than those in males'. What does thinner mean? What form of scientific measurement is "thinner"? This needs to be quantified properly. Image co-registration or any other quantitative analyses for the size differences of the neuronal processes between these genotypes must be carried out.

The authors also mention ‘sex differences both in terms of cell body numbers and branching patterns’. If the authors declare the branching patterns were different between genotypes, they must carry out quantitative analyses.

They also declare the neuronal morphology was 'similar' between particular genotypes, but what does 'similar' mean? The authors seem to conclude that the neuronal morphology is different when they expect it is different and that it is similar when they expect it is similar, without any quantitative criteria for measurement.

As stated above in reply to a similar request from reviewer #1, we did an extensive re-analysis of our neuroanatomical data. We followed reviewer #2’s request, and hope that our new set of data will directly address the reviewers’ concerns.

Subsection “P1a and NP2631 ∩ dsxFLP neurons in fruF males can induce courtship, but not male-type aggressive behaviour”:

The logical link between the anatomy in Figure 3 and behavior in Figure 4 is unclear. In Figure 3, the authors looked at the contributions of the fru and dsx genes in the purported 'P1/pC1'-specific neurons, but in Figure 4, they carried out behavioral assays with the fru mutants, in which fru gene function is disrupted not only in P1/pC1 neurons but also all other FruM-expressing neurons throughout the PNS and CNS, they then base all their conclusions on only the behavioral function of P1/pC1 neurons. These animals will have a plethora of fru mutant phenotypes that are associated with lack of fruM in other neurons in the CNS and PNS.

To make any relevant conclusions the authors need to re-do the behavioral assays in Figure 4 using the flies in which only the P1/pC1 neurons are mutant for fru (e.g., mutant MARCM clones, UAS-Cas9 system, or fru-RNAi).

Our experiments address what the NP2631 ∩ dsxFLP or P1a neurons can do in fruF males. We would like to clarify that our original submission did not state that fru is required cell autonomously in either NP2631 ∩ dsxFLP or P1a neurons, precisely because we were aware that we could not attribute the behavioral differences from regular males to the deficit specifically within either neuronal population. We apologize if our wording in the previous submission caused any confusion.

We agree with the reviewer’s critique that the use of constitutive mutants has a limitation. We cannot answer whether the phenotype (anatomical or behavioral) is due to the lack of dsx or fru functions in the neurons of interest, or due to their cell non-autonomous functions (likely in other neurons). However, we would like to reiterate that the cell-autonomous function of dsx or fru is not the focus of this study. We wished to address how sexual dimorphism of dsx and fru (in a form of sex-specific splicing) may contribute to the specification of sexually dimorphic circuits and sexually dimorphic social behaviors. As stated above, simple loss-of-function of dsx or fru does not necessarily distinguish which male-like or female-like characteristics are specified by a given gene, because the loss of a gene is not synonymous to the transformation of sex from one to the other. We respectfully disagree with the reviewer’s assertion that we cannot make “any relevant conclusions” with these mutants. Rather, we believe that systematic investigation into what changes in fruF males and fruM females at circuit and behavioral levels (e.g., comparison of all 4 genotypes shown in “Sex-determining genes distinctly regulate courtship capability and preference toward females through sexually dimorphic neurons”, Figure 3B) is crucial for understanding the transformative nature of sex determination.

We would also like to be mindful of the limitations of cell-type specific manipulations of dsx and fru. For instance, successful gene knockdown using ‘dead Cas9’ (CRISPRi) crucially depends on the sequence of 20-base guide RNA. RNA interference (RNAi) relies on the specificity of ~21-base double-strand RNA sequence. In both cases, off-target effects as well as incomplete (or even ineffective) knockdown of the target gene are prevalent and well documented. Incomplete knockdown can be also caused by varied strengths of GAL4 drivers. To make matters more complicated, dsx and fru are likely crucial during development. This means that a GAL4 driver needs to turn on very early and remain active consistently during development to provide interpretable results. In previous studies, characterization of both the efficacy of fru RNAi knockdown or the temporal activity or GAL4 drivers is disturbingly sparse, raising a question about the interpretability of such data. Presumably because of this limitation, results of RNAi against sex-determining genes are often presented only when such manipulation results in reproduction of phenotypes in systemic mutants (that is, when the result is interpretable). Considering that cell-autonomous role of dsx or fru is not necessarily the focus of our study, we do not think either RNAi or CRISPRi will further support our conclusions.

MARCM can circumvent the problem of timing and strength because the mutations become homozygous at the cell division. However, MARCM also has an often-overlooked weakness that any cell clones in which a GAL4 driver is not active remain undetected. Moreover, creation of animals with mutant cells only in the desired neuronal population is exceedingly difficult, often requiring hundreds of individuals to identify a handful of suitable samples. Because the distribution of cell clones can be analyzed only after behavioral experiments, and since there is substantial inherent variability of behavior, an unreasonable amount of labor would be required to obtain interpretable data using MARCM.

We are aware of the limitation of constitutive mutants, but systematic analysis of such mutants is fundamental to interpret cell-specific manipulations of sex-determining genes. We hope reviewers will understand the value in what we can observe, and allow us to address functions of dsx or fru within specific neuronal populations in future studies.

Subsection “fruM specifies aggression-promoting Tk-GAL4FruM neurons”:

The logic of transition from P1/pC1 to Tk-Gal4 neurons is very difficult to follow. The relationship between the former and the latter need to be explained. Are they completely different stories?

As mentioned at the beginning of this letter, we have split the original submission into 2 parts. This point was also raised by reviewer #3, and we hope the current format better conveys our central messages.

Figure 6:

As a control, the authors should demonstrate that Tk-GAL4 neurons do not express dsx.

Please see Figure 1—figure supplement 2C.

Subsection “Differential roles of fru isoforms on male-male interactions and specification of Tk-GAL4FruM neurons”:

The authors showed that the Tk-Gal4 neurons exist in fru∆B mutant males but behavior is affected in these flies. The simplest explanation would be that the neurites are malformed in these flies.

Although the authors mentioned that 'fruMA and fruM mutants did not affect […] their arborization pattern', they didn't seem to carry out any quantitative morphological analyses. Such analyses should be included.

Subsection “The Tk-GAL4FruM neurons in fruMB mutants can induce male aggression”:

The authors mention that 'these mutants have Tk-GAL4FruM neurons that appear to retain their morphology'. But how did they examine this?

We now show the averaged images of Tk-GAL4FruM neurons in fruMA and fruMB mutants, which is in fact very similar to those in wild type males (“Layered roles of fruitless isoforms in specification and function of male-type aggression-promoting neurons in Drosophila”, Figure 5F-H, Video 7). The volume quantification of three major neural processes in these three genotypes indeed shows that they are indistinguishable from each other.

Again, this level of analysis may not reveal subcellular differences in neuromorphology among regular males, fruMA mutants, and fruMB mutants. However, we would like to point out that our conclusion that fruMB does not affect the specification of Tk-GAL4FruM neurons is based on physiological and behavioral data, as well as neuroanatomical data (“Layered roles of fruitless isoforms in specification and function of male-type aggression-promoting neurons in Drosophila”, Figure 6 and Figure 5, respectively). The fact that optogenetic stimulation of Tk-GAL4FruM neurons in fruMB mutants triggers aggression as robustly as in regular males suggests that the overall integrity of Tk-GAL4FruM neurons remains largely unaffected in fruMB mutants.

Reviewer #3:

This paper is a paradox. As far as I can tell it is rigorous on all levels. The scholarship is excellent, deep and well rounded. In addition, the issues are explored thoroughly and there is an enormous amount of data. In addition, an enormous number of fly lines (the table that describes the lines is phenomenal). But on the other hand, it is too much. I would like to discuss the argument and conclusions that surface relating dsx and fru expressing neurons with sexually dimorphic choice vs. sexually dimorphic motor patterns and the interesting differences between reproductive behavior and aggression, but the paper is simply too dense at almost every point. Reading it is a painful experience and this does the work a tremendous disservice. I say this recognizing and admiring the care that has gone into preparing this text. It seems flawless, just impossibly dense.

We understand a reservation that echoes with other reviewers’ concerns regarding the clarity of our original submission. We would like to address the reviewer’s specific suggestions in the following sections.

What to do? Here is a list of suggestions.

1) The text and figures are impeccably constructed but for the average reader they are overly complicated and far too dense. The flow of the manuscript would benefit greatly from severe editing of the text, figures and figure legends. It is with a heavy heart that I ask the authors to do this knowing the amount of effort it took to construct the manuscript.

We significantly reorganized our original submission. Namely, we first understood the value of the last suggestion and split the original version into two new manuscripts as we already described. We hope this will substantially improve the clarity of our messages.

2) Remove raster plots from figures throughout and move to supplemental material if needed. See Figure 1, Figure 4, Figure 5, Figure 6, Figure 7, Figure 9.

We moved the raster plots of behaviors largely to figure supplement panels.

3) Figure 2 is not required. Move to supplemental material and concisely state result in text.

We feel the evidence suggesting that P1a and NP2631 ∩ dsxFLP neurons are separate populations has significance, especially now that we detected different levels of contribution of fru on these two neuronal populations. We therefore decided to keep the panels in “Sex-determining genes distinctly regulate courtship capability and preference toward females through sexually dimorphic neurons”, Figure 5.

4) Much of the immunohistochemistry could be removed from figures and moved to supplemental material.

See Figure 3, Figure 6, Figure 8.This is in contrast to requirements from reviewers #1 and #2 to perform more rigorous neuroanatomical analyses. We followed a custom for most Drosophila papers and kept representative images, but we would be open to further suggestions for improving clarity.

5) The summary panels at the end of some of the figures are helpful. But a single unifying summary/model would be best. Though organized in a systematic and rational way the results still feel piecemeal and it is tiresome to continually have refer back to previous figures or supplemental material to regain your bearing. A single model would help immensely.

We reduced the number of models to one for “Sex-determining genes distinctly regulate courtship capability and preference toward females through sexually dimorphic neurons” (Figure 4D), and two for “Layered roles of fruitless isoforms in specification and function of male-type aggression-promoting neurons in Drosophila” (Figure 2D and Figure 6H). It was a challenge to summarize our findings in a single model, given that dsx and fru have different roles on 3 populations of neurons and 2 types of social behaviors. We hope the new models make our core finding clear.

[Editors’ note: what follows is the authors’ response to the second round of review.]

Essential revisions:

The issues enumerated below need to be clarified before the paper can be accepted.

1) That activation of Tk-Gal4 neurons in single-housed FruM females does not elicit aggression is at odds with the conclusion that this circuit is sufficient for executing male type aggression. If anything, single housing should have increased aggression. The authors' suggest that the lack of aggression is because of increase in male-type courtship, but this still tacitly indicates that activating the Tk/FruM circuit is insufficient to induce aggression. The authors' conclusions should be tempered accordingly.

We appreciate the reviewer’s attention to point out an imprecise choice of words (“sufficient”). We rephrased the sentence in “Layered roles of fruitless isoforms in specification and function of male-type aggression-promoting neurons in Drosophila”, subsection “Female-type NP2631 ∩ dsxFLP neurons in fruM females do not promote aggression” to more accurately convey our point, as detailed below.

Our conclusion in the original manuscript that the fruM-dependent circuit (which includes Tk-GAL4FruM neurons) is “sufficient for executing male-type aggression” does not mean that the presence and/or the activation of Tk-GAL4FruM neurons and its downstream is “sufficient” to recapitulate all aspects of male-type aggressive behaviors. What we wanted to express is that the activity of Tk-GAL4FruM neurons can generate male-type lunges in the absence of neural components specified by dsx. Although the importance of fruM on organizing male-type aggressive behavior was demonstrated in Vrontou et al., 2006, our point is noteworthy because not all fruM-expressing neurons are required for the animals to perform a lunge. Male-type NP2631 ∩ dsxFLP neurons and P1a neurons are such “non-essential” fru-expressing neuronal components (“Layered roles of fruitless isoforms in specification and function of male-type aggression-promoting neurons in Drosophila”, subsection “fruM specifies aggression-promoting Tk-GAL4FruM neurons”: see also “Sex-determining genes distinctly regulate courtship capability and preference toward females through sexually dimorphic neurons”, Figure 4), even though their activity can also promote lunges in a (normal) male. As we discussed in “Sex-determining genes distinctly regulate courtship capability and preference toward females through sexually dimorphic neurons”, dsx plays an important role in specifying both neuronal populations.

We showed in Figure 1C and D that activation of Tk-GAL4FruM neurons did not elicit aggression toward female target flies (which is consistent with the paper that first described Tk-GAL4FruM neurons for male aggression (Asahina et al., 2014)). To us, the observation that artificial activation of a neuronal population can have different behavioral impacts in a context-dependent manner is an important conclusion (see also subsection “Sex of the target animals is an important biological variable” in the Discussion of “Sex-determining genes distinctly regulate courtship capability and preference toward females through sexually dimorphic neurons”). In fact, our observation that fruM females can attack female targets when their Tk-GAL4FruM neurons were activated suggests that the absence of lunges from a male tester to a female target may be at least partially dependent on the function of dsx (Figure 2D). In this sense, the lack of “sufficiency” that the reviewer pointed out is inherent to the function of Tk-GAL4FruM neurons itself (as stated in subsection “fruM specifies aggression-promoting Tk-GAL4FruM neurons”), and not necessarily an issue specific to fruM females.

2) As with the companion paper (“Sex-determining genes distinctly regulate courtship capability and preference toward females through sexually dimorphic neurons”), it would appear that the effects of Fru isoform on aggression in Tk-GAL4FruM neurons also can only really be assessed by looking at animals were the Tk-GAL4FruM neurons are mutant/perturbed for fru function (e.g. fru isoform-specific mutant MARCM clones, UAS-Cas9 system, or fru-RNAi). Otherwise, the data would need to be interpreted with the caveat that fru mutant phenotypes could be caused by lack of Fru isoform in other neurons in the CNS and/or the PNS. This issue needs to addressed directly – ideally experimentally given that a lineTK-GAL4/fruP1LexA/LexAop2-FLPL that may allow these neurons to be targeted has been described in Asahina et al., 2014.

This is a parallel critique to our Ishii et al., (2020). While we acknowledge that the molecular mechanism by which dsx and fru specified neural sexual dimorphism is an important question, note that “the effects of Fru isoforms on aggression in Tk-GAL4FruM neurons” was not a question we aimed to address in this manuscript. Please see our response to a parallel question for “Sex-determining genes distinctly regulate courtship capability and preference toward females through sexually dimorphic neurons”, which includes the discussion why the interpretation of RNAi or dCas9-mediated gene knockdown (which depends on the GAL4 expression), as well as MARCM analysis, have caveats for studying genes involved in development. We argue that morphological and functional phenotypes of the neurons of interest in a systemic mutant is an important knowledge that deserves attention on its own, precisely because of challenges in addressing functions of these genes within specific cell types.

We would like to point out that Tk-GAL4FruM neurons present an extreme case of this challenge. Since Tk-GAL4FruM neurons are not labeled in the absence of fruM, one likely scenario is that fruM is required cell-autonomously. Neither RNAi or dCas9 would be a valid method to address this possibility because GAL4 must turn on for RNAi or dCas9 to work. This is a classic “catch-22” situation: the very fact that GAL4 turns on likely means that the fate of the cell is already determined before RNAi or dCas9-mediated knockdown of fru has an opportunity to influence its developmental process. In other words, there is no possible way for RNAi or dCas9 to suppress the activity of GAL4 that drives them if fruM is required cell-autonomously to specify Tk-GAL4FruM neurons. If Tk-GAL4FruM neurons disappear like in fruF males – it will be an even bigger mystery that RNAi or dCas9 can exert its effect without GAL4 activity.

It is certainly possible that fru is required outside of Tk-GAL4FruM neurons to specify its sexual dimorphism, but results of RNAi or dCas9 (that is, that Tk-GAL4FruM neurons appear the same as the genetic controls) would not be interpretable. In other words, both RNAi and dCas9 are fundamentally flawed approaches to adequately address this important question.

3) A key finding arising from optogenetic experiments, artificially activating Tk-GAL4FruM neurons are a refinement of the authors previously published thermogenetic work (Asahina et al., 2014), and the isoform-specific roles of Fru on male behavior and Fru-specified neural circuits, are implied by work from other groups (Neville et al., 2014; von Philipsborn et al., 2014). The discussion must clearly articulate what new information thais manuscript provides on Fru isoform function and aggressive behavior.

We would like to emphasize that the previous publication never addressed Tk-GAL4FruM neurons’ specification (Figure 1H, K-P) or functions (Figure 2) in “fruM females”. Also, the contribution of specific fru isoforms on male-male aggression has never been addressed in either of the previous important publications (Neville et al., 2014; von Philipsborn et al., 2014). Therefore, the findings on the differential roles of fru isoforms for male aggression (Figure 4) are entirely new. Also, how the specification of Tk-GAL4FruM neurons (Figure 5) and its function on aggressive behaviors (Figure 6) are affected by fru isoform mutants has never been tested. We therefore state that all key data presented here are novel, and not a mere “refinement of the previous publication (Asahina et al., 2014)” as the reviewer suggested.

Upon the reviewer’s suggestion, we clearly indicated that the courtship defects of fruMB and fruMC mutants were reported in the previous publications (“Layered roles of fruitless isoforms in specification and function of male-type aggression-promoting neurons in Drosophila”, subsections “Female-type NP2631 ∩ dsxFLP neurons in fruM females do not promote aggression” and “Differential roles of fru isoforms on male-male interactions and specification of Tk- GAL4FruM neurons”). Also note that we acknowledged fru isoforms’ functions on gene expression, neuroanatomy, and behavior have been investigated in the previous publications (“Layered roles of fruitless isoforms in specification and function of male-type aggression-promoting neurons in Drosophila”, Discussion section). I hope these clarifications resolve any confusion about what has been known previously. We performed courtship assays on isoform-specific mutants (Figure 4—figure supplement 1A, B) as a control for our genetic manipulations, which we felt was important for demonstrating that fruMB and fruMC mutants are qualitatively different even though their phenotype on aggression (both strong reduction) appears similar.

4) Does Figure 2—figure supplement 2E, F1 (subsection “Tk-GAL4FruM neurons are a part of a fruM-dependent circuit for the execution of male-type aggressive behaviour”) refer to a different figure panel. Please correct.

Thank you for pointing out this mistake. It was supposed to refer to Figure 2—figure supplement 3E, F1, and was corrected accordingly.

5) Dsx immunolabeling in Figure 1—figure supplement 2C is poor. Please provide better images.

Please see the new Figure 1—figure supplement 2C and D for new images, which was generated with a new batch of anti-DsxM and anti-FruM antibodies. As stated in the other letter, these new antibodies coupled with a modified fixation process clearly showed that Tk-GAL4FruM neurons indeed express FruM, but not DsxM.

https://doi.org/10.7554/eLife.52702.sa2

Article and author information

Author details

  1. Margot Wohl

    1. Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, United States
    2. Neuroscience Graduate Program, University of California, San Diego, United States
    Contribution
    Software, Investigation
    Contributed equally with
    Kenichi Ishii
    Competing interests
    No competing interests declared

  2. Kenichi Ishii

    1. Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, United States
    2. Neuroscience Graduate Program, University of California, San Diego, United States
    Contribution
    Investigation
    Contributed equally with
    Margot Wohl
    Competing interests
    No competing interests declared

  3. Kenta Asahina

    Molecular Neurobiology Laboratory, The Salk Institute for Biological Studies, La Jolla, United States
    Contribution
    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Project administration
    For correspondence
    kasahina@salk.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6359-4369

Funding

National Institute of General Medical Sciences (GM119844)

  • Kenichi Ishii
  • Kenta Asahina

National Institute on Deafness and Other Communication Disorders (DC015577)

  • Margot Wohl
  • Kenta Asahina

Naito Foundation

  • Kenichi Ishii

Japan Society for the Promotion of Science

  • Kenichi Ishii

Mary K. Chapman Foundation

  • Margot Wohl

Rose Hills Foundation

  • Margot Wohl

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Drs. David Anderson, Gerald Rubin, and Barret Pfeiffer for sharing unpublished transgenic Drosophila strains with us; Drs. Stephen Goodwin, Daisuke Yamamoto, and Anne von Philipsborn for other Drosophila strains; Eyrun Eyjolfsdottir and Dr. Pietro Perona for developing and improving the FlyTracker program; Dr. Michael Perry for sharing with us anti-DsxM and anti-FruM antibodies; Dr. Yong Wan for development and support for FluoRender; Dr. William Joiner for his help on circadian rhythm and sleep assays; Pavan Nayak and Vivian Shaw for their help on the development of behavior classifiers; Dr. Samuel Pfaff for sharing the Olympus FV-1000 confocal microscopy with us; Drs. Eiman Azim, Weizhe Hong, Samuel Pfaff, Carla Shatz, John Thomas, and members of the Asahina lab for critical comments on the manuscript, and David O’Keefe for scientific editing on the manuscript. The antisera nc82 (anti-BRP), developed by E Buchner, was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242. Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) were used in this study. This work was also made possible in part by software funded by the NIH: FluoRender: Visualization-Based and Interactive Analysis for Multichannel Microscopy Data, 1R01EB023947-01 and the National Institute of General Medical Sciences of the National Institutes of Health under grant number P41GM103545-18.

Senior Editor

  1. K VijayRaghavan, National Centre for Biological Sciences, Tata Institute of Fundamental Research, India

Reviewing Editor

  1. Mani Ramaswami, Trinity College Dublin, Ireland

Version history

  1. Received: October 12, 2019
  2. Accepted: April 3, 2020
  3. Version of Record published: April 21, 2020 (version 1)

Copyright

© 2020, Wohl et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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  1. Margot Wohl
  2. Kenichi Ishii
  3. Kenta Asahina
(2020)
Layered roles of fruitless isoforms in specification and function of male aggression-promoting neurons in Drosophila
eLife 9:e52702.
https://doi.org/10.7554/eLife.52702

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