Role of Doublesex-Dependent Phosphodiesterase 1c Expression in Gustatory Receptor Neurons in Male Courtship Behavior of Drosophila Melanogaster
Hattori M, Takase D, Aoki F and Suzuki MG
Published on: 2024-05-01
Abstract
Objective: Doublesex (dsx) is known to control sexual dimorphism in male and female morphology and sexual behaviors, such as courtship, in Drosophila melanogaster. However, Dsx target genes involved in male courtship behavior have yet to be identified. This study was performed to examine the molecular mechanisms underlying the neural circuitry driving male courtship behavior by identifying novel target genes of Dsx that are involved in male sexual behavior.
Methods: We screened 6230 genes predicted to be direct targets of Dsx by previous ChIP- seq analyses for the word “mating” in the GO term and reported to have functions related to courtship behavior. We investigated the expression patterns of the resulting candidate Dsx target genes in the central nervous system and the effects of their functional depletion on male mating behavior.
Results: The results identified two genes, both of which encode phosphodiesterases, which fulfilled both of the above conditions. One of the selected genes, phosphodiesterase 1c (Pde1c), was found to be coexpressed with dsx in several neuronal subsets, including the gustatory receptor neurons (GRNs), which are involved in male courtship behavior and are also known as dsx-expressing neurons. Pde1c knockdown in dsx-expressing cells caused significant delay in the time to initiation of copulation and significantly decreased the mating rate. Furthermore, these abnormalities in mating behavior were partially reproduced by knockdown of Pde1c expression in GRNs.
Conclusion: Pde1c expression in GRNs is involved in male courtship behavior and its specific expression in GRNs may be under the control of Dsx.
Keywords
Doublesex; Phosphodiesterase 1c; Male courtship behavior; Gustatory receptor neuronIntroduction
In sexually reproducing animals, sexual behavior determines reproductive success or failure and is essential for producing offspring. Sexual behaviors include courtship and mating behaviors exhibited by males toward females of the same species, behaviors in which females accept or reject mating in response to courtship by males of the same species, and egg-laying behaviors in which females lay eggs at the appropriate place and time for survival of their offspring. As sexual behavior shows distinct sexual dimorphism, the nerves that regulate sexual behavior should also be sexually dimorphic. The developmental mechanisms that give rise to sexual dimorphism in the nervous system and the genes that regulate such neuronal development have long been of interest in attempts to understand sexual differences in behavior [1].
Drosophila melanogaster is an excellent model organism for neurogenetic and neuroanatomical studies, and has long been used to study sexual behavior. Sex in Drosophila is determined by the ratio of the number of sets of autosomes (A) to the number of X chromosomes (X) (X/A ratio) [2], with X/A = 1.0 corresponding to female and X/A = 0.5 corresponding to male because the sex lethal (Sxl) gene is expressed in the early embryo only when X/A = 1.0 [3]. Sxl protein binds to the transformer (tra) gene pre- mRNA and induces its female-specific splicing, resulting in production of a functional TRA protein. Subsequently, TRA induces female-specific splicing of the doublesex (dsx) pre-mRNA via the transformer-2 (TRA-2) protein to produce the female-specific isoform of Dsx protein (DsxF) [4,5]. On the other hand, Sxl protein is not produced when X/A = 0.5, so no functional TRA protein is produced and dsx pre-mRNA undergoes default splicing to produce the male isoform of Dsx protein (DsxM) [4,5].
DsxF and DsxM are transcription factors containing a DNA-binding domain (DBD) called the Doublesex and Mab-3 (DM) domain. Dsx has a DM domain in its N-terminal region, which is common to both males and females [6,7]. On the other hand, the C-terminal amino acid sequence differs between DsxF and DsxM [8,9], which is thought to be responsible for the sex-related differences in expression of Dsx target genes. The results of a previous study using ChIP-seq predicted that Dsx binds as many as 6250 genes [10]. Therefore, the target genes of Dsx are expected to be extremely diverse. However, only seven genes have been identified as direct targets of Dsx by genetic and biochemical methods to date. The first of these genes to be identified were yolk protein 1 (yp1) and yolk protein 2 (yp2), which are located next to each other in the genome and have a Dsx- binding cis-element in the intergenic region [11,12]. In females, DsxF binds to this cis- element and promotes transcription of yp1 and yp2 via recruitment of bZiPa, which is encoded by the slowborder cells (slbo) gene. In males, on the other hand, yp1 and yp2 transcription is repressed due to inhibition of bZiPa recruitment by binding if the male- specific region of DsxM to the cis-element [11,12].
DsxF and DsxM are transcription factors that positively or negatively regulate the expression of downstream target genes and induce differentiation of various sexually dimorphic traits. The sexually dimorphic traits under the control of Dsx are not only morphological and physiological but also behavioral. For example, among the genes identified as targets of Dsx, leucine-rich repeat G protein-coupled receptor 3 (Lgr3) is expressed in the nervous system and has been implicated in female mating receptivity [13]. Therefore, the search for novel target genes of Dsx is likely to provide insights into the mechanisms that give rise to sexual dimorphism in behavior. However, direct target genes of Dsx involved in the regulation of male courtship and mating behavior have not yet been identified.
In Drosophila, male courtship behavior is primarily innate, with males exhibiting typical courtship behavior as soon as they are placed with a virgin female. Males generally chase after the female, touch the female with their forelegs, lick the female’s genitalia with their proboscis, sing a species-specific courtship song, and bend their abdomen to copulate [14]. The successful accomplishment of these behavioral sequences requires the integration of a variety of sensory stimulus information continuously emitted from the female. For example, gustatory receptor neurons (GRNs) in the forelegs detect nonvolatile female pheromones when the male touches the female’s abdomen with his forelegs, facilitating mating behavior [15]. Any partial disruption in the sequence of behaviors from courtship to completion of copulation prevents successful mating and consequently hinders reproduction [16]. The male-specific isoform of Dsx, DsxM, is expressed in these neurons involved in controlling the precise execution of male courtship behavior [17,18]. Artificial stimulation of these DsxM-expressing neurons can alter courtship and mating behavior in males [19-24]. Approximately 650 neurons in the male central nervous system express Dsx, and inhibition of the activity of all of these neurons disrupts all courtship and mating behaviors [18]. These findings show that Dsx-expressing neurons are involved in the control of male courtship and mating behavior. However, it remains unclear what genes are regulated by Dsx to produce male courtship behavior.
Based on the above background, this study was performed to identify novel target genes of Dsx that are expressed and function in the nervous system to gain insight into the regulatory mechanism of male courtship behavior. For this purpose, we searched for genes reported to be expressed in the nervous system and involved in male courtship behavior among those predicted to be targets of Dsx in a previous study using ChIP-seq analysis [10]. Phosphodiesterase 1c (Pde1c) encoding a phosphodiesterase (PDE) that degrades cAMP and cGMP, mutation of which has been shown to result in male infertility and reduced mating rate [25], was identified as a strong candidate. We identified expression of Pde1c in gustatory receptor neurons (GRNs), which are well known as dsx-expressing neurons and are important for distinguishing species and sex in male courtship behavior. We further examined the effects of knockdown of Pde1c expression in either dsx- expressing cells or GRNs on male courtship behavior. We observed delayed initiation of copulation and reduced mating rate in the former and delayed initiation of copulation in the latter. These results indicated that Pde1c expression in GRNs is required for sufficient activation of male mating behavior and that its expression in GRNs is under the control of dsx. To our knowledge, this is the first report identifying a strong candidate target gene for Dsx involved in male mating behavior.
Materials and Methods
Experimental Animals and Rearing Conditions
Flies (Drosophila melanogaster) were reared on standard medium at 25 °C in vials (Chiyoda Science) with an inner diameter of 22 mm and a height of 96 mm. The Drosophila strains used in this study—Pde1c-EGFP (#63195), UAS-myr::tdTomato (#32222), UAS-mCD8::GFP (#32185), UAS-Pde1c IR (HMC) (#55925), and UAS-Pde1c IR (JF) (# 28728), poxn-GAL4 (#66685), and y1w1118 (#6598)—were all purchased from the Bloomington Drosophila Stock Center. The y1w67c23 strain was purchased from BestGene. dsx-GAL4(Δ2) was kindly provided by Dr. Carmen C. Robinnet (Stanford University). Oregon-R was obtained from the Kyoto Stock Center.
Immunohistochemistry
Adults 4 days after hatching were used for immunohistochemical analysis based on the method described previously [26]. For dissection, individual flies were anaesthetized with CO2, immersed in 70% ethanol, and transferred to 1× phosphate-buffered saline (PBS). The excised central nervous system (CNS) was immersed in 4% paraformaldehyde (PFA)/1× PBS and allowed to stand at room temperature for 25 min, followed by fixation. The fixed CNS samples were washed six times with 0.3% Triton X-100/1× PBS (PBST) for 15 min each time at room temperature, and then blocked with 5% normal goat serum (NGS) (Sigma-Aldrich)/PBST for 1 h at room temperature with gentle shaking. The blocking solution was then removed, and primary antibody diluted in 5% NGS/PBST was added and shaken at 4 °C for at least 48 h. After washing six times with 0.3% Triton X-100/1× PBS (PBST) for 15 min each time at room temperature, secondary antibody diluted with 5% NGS/PBST was added and shaken at 4 °C for at least 48 h. The CNS samples were washed six times with 0.3% Triton X-100/1× PBS (PBST) for 15 min each time at room temperature, immersed in Vectashield (Vector Laboratories), and shaken for 1 h at room temperature for encapsulation. The resulting CNS was observed using an LSM5 EXCITER (Carl Zeiss) or FV3000 (Olympus) confocal microscope and a BZ- X700 (Keyence) fluorescence microscope. Image processing was performed using ImageJ Fiji (https://imagej.net/software/fiji/downloads). The primary antibodies used in this study were rabbit anti-GFP (1:1000, catalogue #A-11122; Invitrogen), mouse anti- Bruchpilot, nc82 (1:30; Developmental Studies Hybridoma Bank), chicken anti-GFP (1:1500, catalogue #ab13970; Abcam), and rabbit anti-DsRed (1:1000, catalogue #632496 Clontech). The secondary antibodies were fluorescein (FITC)-conjugated AffiniPure goat anti-rabbit IgG (H+L) (1:400, catalogue #111-095-003; Jackson Immuno Research), Alexa Fluor 647 goat anti-mouse IgG (1:400, catalogue #A-21236; Invitrogen), Alexa Fluor 488 goat anti-chicken IgG (1:400, catalogue #A-11039; Invitrogen), and Alexa Fluor 568 donkey anti-rabbit IgG (1:400, catalogue #A-10042; Invitrogen).
Male Behavioral Experiments
Flies were collected within 8 h of emergence at 25 °C. Males were isolated and females were collected before mating and reared at 25 °C under a 12 h light/12 h dark cycle. Males were reared in 5-mL tubes (Sarstedt) with an inner diameter of 12 mm and a height of 75 mm. Females were reared in vials (Chiyoda Science) as described above. Males within 3 to 5 days after emergence and virgin females within 3 to 8 days after emergence (yw strain) were used for behavioral experiments. These males and females were paired and placed in a chamber with an inner diameter of 8 mm and height of 3 mm using a suction tube, and the behavior of the paired flies was recorded for 1 h. Copulation latency was defined as the time required for the test male to successfully copulate. The time for which the male exhibited courtship behaviors (localization, tapping, courtship song, licking, mating attempt, and successful mating) within 10 min after the beginning of the recording was measured and used as the courtship index. The percentage of test males that successfully mated within 10 min after the start of recording was calculated as the copulation index.
Results
Pde1c Shows Sexually Dimorphic Expression in the Central Nervous System
In our previous studies, we screened for novel Dsx target genes expressed in the nervous system [27] (Figure 1).
A total of 6230 genes predicted to be potential target genes of Dsx by ChIP-seq analysis were subjected to screening. Among these genes, we selected those that were orthologous to 1439 genes previously identified in mice as potential targets of Dmrt1[28], the mammalian orthologue of Dsx, to search for plausible Dsx target genes. Of the 585 resulting genes, 10 genes had GO terms that included “mating” and were previously reported to have functions related to mating behavior. Of these genes, Pde1c and dunce (Pde4) were the only genes known to show sex-related differences in expression in the central nervous system [25,27].
A total of 6230 potential Dsx target genes identified previously by ChIP-seq analysis were subjected to screening. Doublesex and MAB-3-related transcription factor 1 (Dmrt1) is the mouse orthologue of dsx, and 1439 potential Dmrt1 target genes have been identified previously [28]. We found 585 genes that were common targets for both Dsx and Dmrt1. Furthermore, 10 of these genes have GO terms that include “mating” and have been reported to have functions related to mating behavior (Table 1). Among these 10 genes, we focused on Pde1c, which has been reported to exhibit sex-related differential expression in the CNS [25].
Figure 1: Screening for novel Dsx target genes expressed in the nervous system.
Table 1: 10 genes selected by our screening illustrated in Table 1.
Gene |
References |
Tiwaz |
10 |
DILP-receptor |
28 |
Lnk |
28 |
ether-a-go-go |
14 |
Moe |
15 |
alan shepard |
16 |
muscleblind |
15 |
Pde1c |
25 |
dunce |
26,27 |
doublesex |
22,19,18, 20, 29,6,23 |
Pde1c encodes a PDE that degrades the second messengers cAMP and cGMP, thereby regulating intracellular signal transduction [25]. Mutations in Pde1c in D. melanogaster have also been reported to cause abnormal male courtship behavior [25]. We first examined whether Pde1c shows sexually dimorphic expression in the CNS.
As antibodies against Pde1c protein were not available, we used the Pde1c protein trap line, in which the open reading frame (ORF) encoding enhanced green fluorescent protein (EGFP) was inserted within the intron of the Pde1c gene [29] (Figure 2A). Sites with Pde1c expression were then detected by immunostaining with anti-EGFP antibody. As shown in Figure 2B, sexually dimorphic expression was observed in T1 and T3 thoracic neuromeres of the ventral nerve cord (VNC). Neurons that showed sexually dimorphic expression of Pde1c in T1 thoracic neuromeres were morphologically very similar to GRNs (Figure 2B, dotted box). The formation of GRNs is regulated by both dsx and another important sex-determining gene, fruitless (fru), and is characterized by the presence of crossed axon projections in the midline only in males (Montell, 2009). Consistent with this observation, Pde1c-expressing neurons showed a pattern of axon projection crossing in the midline only in the male VNC (Figure 2B, arrow). These results suggested that Pde1c is expressed in GRNs, which are known as dsx-expressing neurons. On the other hand, the level of Pde1c expression was higher in the abdominal ganglion (Abg) of females than males.
Figure 2: Analysis of Pde1c expression in the central nervous system using Pde1c protein trap line.
(A) Structure of the Pde1c gene in the Pde1c protein trap line. Black squares indicate exons and straight lines indicate introns. Nine splice variants produced from the Pde1c gene are also shown. The blue triangle indicates the approximate insertion site of the EGFP reporter construct.
SA, splice acceptor sequence; SD, splice acceptor sequence; EGFP, EGFP ORF. (B) The left panel shows a schematic diagram of the central nervous system (CNS) in the adult fly.
VNC, ventral nerve cord; T1-T3, 1st to 3rd thoracic neuromeres; Abg, abdominal ganglion.
(C) Pde1c expression in the central nervous system of male and female adults of the Pde1c protein trap line [29] was detected by immunostaining using anti-EGFP antibody. Neurons that were likely to be gustatory receptor neurons (GRNs) are surrounded by a dotted box. Arrows indicate areas of axon projection crossing between male and female GRNs in the midline. Scale bar: 100 µm.
Pde1c is Coexpressed with Dsx in Gustatory Receptor Cells
If Pde1c is indeed under transcriptional control of Dsx, then Pde1c and Dsx should be expressed in the same cells. To test this hypothesis, we examined whether Pde1c and Dsx were coexpressed in the CNS. Expression of dsx in the nervous system was examined using dsx-GAL4(Δ2), a GAL4 driver that expresses GAL4 protein in dsx-expressing cells [3]0, and UAS-tdTomato as a reporter. Immunostaining with anti-DsRed antibody demonstrated that Pde1c and Dsx were coexpressed in several neuronal subsets in the VNC (Figure 3A). Coexpression of Pde1c and Dsx was also observed in neurons that appeared to be GRNs (Figure 3A, dotted box). These results indicated that Pde1c and Dsx function in the same cells, suggesting that Pde1c expression may be under the control of Dsx.
GRNs are important for male courtship behavior to identify the species and sex of the mate, and are known to express dsx [15]. To confirm whether Pde1c is indeed expressed in GRNs, we detected GRNs using the poxn-GAL4 driver, which enables induction of reporter expression almost specifically in these neurons [31], and examined whether Pde1c expression could be observed in these cells. The results showed that poxn-GAL4 was coexpressed with Pde1c, indicating unambiguously that Pde1c is expressed in GRNs (Figure 3B, dotted line box).
As Pde1c was coexpressed with dsx in CNS neurons, including GRNs that are closely related to male sexual behavior, it was considered likely that Pde1c expression is under the control of Dsx and involved in the regulation of male sexual behavior.
Figure 3: Coexpression of Pde1c with Dsx in GRNs.
(A) Pde1c (green) and Dsx- expressing cells (red) in the adult CNS and merged image. Pde1c was detected by immunostaining as shown in Figure 1B. Dsx-expressing cells were detected by immunostaining of UAS-tdTomato expression driven by dsx-GAL4 (Δ2) with anti-DsRed antibody. (B) Pde1c (green) and GRNs (red) and merged image. Pde1c was detected by the same method as described in A. Expression of UAS-tdTomato driven by poxn-GAL4 was detected by immunostaining using anti-DsRed antibody to visualize GRNs. The upper panel shows the brain, and the lower panel shows the ventral nerve cord (VNC). T1-T3, 1st to 3rd thoracic neuromeres. GRNs are surrounded by a dotted box. T2-T3, 2nd to 3rd thoracic neuromeres; GRNs are surrounded by a dotted box. Scale bar: 100 µm.
Knockdown of Pde1c in Dsx-Expressing Cells Resulted in Male Courtship Defects
UAS-taiman inverted repeat (UAS-IR) lines with a transgene designed to produce double- stranded RNAs (dsRNAs) of target genes in a driver expression-dependent manner are commonly used for RNA interference (RNAi)-mediated gene expression knockdown in Drosophila. Two UAS-IR lines are available for knockdown of Pde1c expression called Pde1c IR-HMC and Pde1c IR-JF, which express short hairpin RNA and long dsRNA targeting Pde1c mRNA, respectively [32]. The dsx-GAL4(Δ2) driver-dependent knockdown of Pde1c was observed with Pde1c IR-HMC (Figure 4A). As shown in Figure 4B, the level of Pde1c expression in GRNs was significantly reduced in knockdown males to less than 1/3 of that in control males.
We next examined the effects of the same knockdown on male courtship behavior. For this purpose, we paired knockdown males with virgin females and measured copulation latency (time required for the male to initiate copulation), courtship index (percentage of time that the male exhibited courtship behavior per unit time), and copulation index (percentage of males that successfully copulated with the female per unit time). The results showed that knockdown of Pde1c expression resulted in significant prolongation of copulation latency and significant decreases in copulation index and courtship index in males (Figure 4C–E). These results were consistent with previously reported abnormalities in courtship behavior in Pde1c mutant males [25].
Based on these results, the expression of Pde1c in dsx-expressing cells was expected to play an important role in inducing courtship behavior and mating success in males.
Knockdown of Pde1c in Poxn-Expressing Cells Causes Courtship Defects in Males
Next, to investigate the function of Pde1c in GRNs, we examined the effects of pox-neuro (poxn)-expressing cell-specific Pde1c knockdown on male courtship behavior using Pde1c IR-HMC driven by the GRN-predominant driver, poxn-GAL4. As shown in Figure 4A, poxn-GAL4 driver-dependent knockdown of Pde1c was observed with Pde1c IR- HMC (Figure 4A). The level of Pde1c expression in GRNs was significantly reduced in poxn-GAL4 > Pde1c IR-HMC males in comparison with control males (Figure 4B).
Quantitative evaluation of male courtship behavior using the same method as described above revealed significant prolongation of copulation latency in Pde1c knockdown males (Figure 4C). On the other hand, there were no significant differences in courtship index or copulation index between knockdown and control males (Figure 4D, E). These results suggested that Pde1c expression in GRNs may play some role in inducing courtship behavior in males.
Figure 4: Effects of Pde1c knockdown in dsx-expressing cells and GRNs on male courtship behavior.
(A) Pde1c expression in male VNCs when RNAi was performed using UAS-Pde1c IR-HMC as UAS-IR. dsx-GAL4(Δ2) and poxn-GAL4 were used to specifically induce RNAi in dsx-expressing cells and GRNs, respectively. Males without a driver were used as negative controls (indicated as “+” in the figure). The upper panel is an image of the VNC and the lower panel is a magnified image of the area surrounded by squares (corresponding to T1) in the upper panel. Pde1c expression was detected by the same method as described in Figure 1AUAS-Pde1c IR-HMC driven by the same driver used in A. (C) Copulation latency (time required for males to initiate copulation). *** P < 0.001, Dunnett’s test. (D) Copulation index (percentage of males that successfully copulated with females per unit time). *** P< 0.001, Chi-squared test. (E) Courtship index (percentage of time that males exhibited courtship behavior per unit time). *** P < 0.001, Dunnett’s test. Numbers in parentheses indicate the number of individual flies examined.
Discussion
Sexually Dimorphic Expression of Pde1c in the CNS and Sexual Behavior
This study demonstrated sexually dimorphic expression of Pde1c in the GRNs of the VNC, and it was also highly expressed in Abg in females compared to males. Neural sexual dimorphism underlies many sex-specific behaviors. GRNs, which are involved in taste reception, project axons from the forelegs to the VNC in both males and females, but their projections cross the midline of the VNC only in males. Taste reception is important in male courtship behavior to discriminate mates, i.e., to determine whether a potential mate is a female of the same species [33]. On the other hand, dsx-expressing neurons have been shown to have important roles in male courtship behavior [18]. Therefore, our observation that Pde1c shows sexually dimorphic expression in GRNs among dsx-expressing neurons is noteworthy.
This study also showed that the Pde1c expression level in Abg was higher in females than in males. This was reminiscent of our previous observation that Dunce (Dnc), which is a member of the PDE family similar to Pde1c, is highly expressed in the female Abg and is involved in female postmating behavior [27]. It has been reported that virgin females injected with sex peptide (SP), a component of semen that promotes the female postmating response, show decreased receptivity to copulation, while SP injection into dnc mutant virgin females does not decrease receptivity to copulation [34]. The intracellular cAMP level is known to be decreased when SP is received by the sex peptide receptor (SPR) [35]. Our previous study suggested that dnc may regulate female postmating behavior by degrading cAMP in SPR-expressing cells [27]. Pde1c, which was highly expressed in the female Abg, may also be involved in female mating behavior similar to dnc.
Relation between dsx and Pde1c
Only seven genes have been identified as direct targets of Dsx, of which only Lgr3 was reported to be expressed in the nervous system and to be involved in female sexual behavior [13]. In this study, we focused on Pde1c, which was a strong candidate as a novel Dsx target gene. Our genomic sequence analysis of Pde1c revealed the presence of 28 Dsx-binding cis-elements. In addition, data from ChIP-seq analysis performed by Clough [10] demonstrated significant Dsx enrichment in eight of these regions (Figure 5).
Figure 5: Predicted Dsx-binding sites in the genomic sequence of Pde1c.
The upper panel shows the schematic gene structure of Pde1c. The lower panel shows a diagram visualizing the Dsx-binding sites at the Pde1c locus using Integrated Genome Browser [41] with the previously reported ChIP-seq data [9]. The blue peak indicates DsxM enrichment and the orange peak indicates DsxF enrichment. Black squares indicate exons and horizontal lines indicate introns. The yellow vertical lines indicate sites of the Dsx- binding core consensus sequence (ACAATGT) in the Dsx enrichment region.
This study demonstrated that Pde1c showed sexually dimorphic expression in the CNS and that Pde1c expression in the CNS was colocalized with many Dsx-expressing neurons. In addition, we found that knockdown of Pde1c in dsx-expressing cells caused abnormal courtship behavior in males.
Much of the research on male sexual behavior has focused on fru, a well-known sex- determining gene expressed in the nervous system. It has been reported that fru mutant males show not only reduced courtship index, but also altered sexual orientation with males courting other males [36]. On the other hand, dsx mutant males are known to show abnormalities in courtship song [37]. In addition, DsxM, a male-specific isoform of Dsx, is expressed in neurons involved in controlling the precise performance of courtship behavior in males. Inhibition of the activity of Dsx-expressing neurons in the male CNS causes a significant increase in the time required for initiation of courtship and decreases in mating rate and courtship index [18]. In the present study, males with knockdown of Pde1c expression in dsx-expressing cells exhibited behavioral abnormalities similar to males with inhibition of the activity of dsx-expressing neurons. Taken together, the results of this study and the previous findings suggest that Pde1c is likely to be under direct control of Dsx.
Knockdown of Pde1c in dsx-Expressing Cells Causes Abnormal Courtship Behavior in Males
This study showed that knockdown of Pde1c in dsx-expressing cells caused abnormalities in male courtship behavior. As the abnormalities in male courtship behavior observed in the present study were consistent with a previous study indicating that Pde1c mutant males show delayed copulation latency and reduced mating rate [25], it is reasonable to assume that the abnormalities in courtship behavior of Pde1c mutant males may be due to functional defects in Pde1c in dsx-expressing neurons. On the other hand, as dsx is also expressed in nonneuronal cells, it is also possible that the abnormal courtship behavior observed in males in this study was due to dysfunction of Pde1c in such cells. However, this possibility could be excluded based on a previous report in which the delayed copulation latency and reduced mating rate of Pde1c mutant males were completely rescued by the neuron-specific driver, ELAV-GAL4 [25].
Although a previous study suggested that the number of Pde1c-expressing cells differs between males and females, the expression of Pde1c in GRNs has not been reported [25]. A GAL4 driver using the upstream sequence of Pde1c as a promoter was combined with a UAS-EGFP reporter to identify Pde1c-expressing cells. However, it has not been confirmed whether the driver indeed reflects the expression pattern of endogenous Pde1c. In contrast, our study used a protein trap line with incorporation of the EGFP ORF into the Pde1c gene, which is likely to reflect the endogenous Pde1c expression pattern. As protein trap lines have been reported to closely recapitulate endogenous gene expression patterns [38], the Pde1c expression pattern identified in the present study was likely to be more reliable than that in the previous study.
Abnormalities in Male Courtship Behavior Caused By Pde1c Knockdown in Poxn-Expressing Cells (GRNs)
Knockdown of Pde1c in poxn-expressing cells resulted in a delay in copulation latency, but no abnormalities were observed in courtship index or mating rate. These results suggested that Pde1c expression in GRNs may play an important role in inducing male courtship behavior. GRNs have been reported to be involved in male courtship behavior [15]. It was reported that the crossing of axonal projections of GRNs in males depends on expression of the sex-determining genes, fru and dsx [25]. When a male courts a female, the male identifies a suitable mate by tapping the abdomen of the female with its forelegs and exchanging gustatory information. The number of gustatory sensilla in the first to fourth tarsal segments of the forelegs is greater in males than in females, and this sexual dimorphism is regulated by dsx [25]. Therefore, it is possible that the knockdown of Pde1c by poxn-GAL4 caused abnormalities in male courtship behavior because of some impairment of the tapping step.
The binding of SP by SPR-expressing cells in females results in a decrease in concentration of intracellular cAMP, which is important for triggering female postmating behavior [35]. Similarly, when gustatory sensory receptor cells recognize females, the concentration of cAMP in the cells decreases, which may be an important signal for eliciting courtship behavior in males. Pde1c may be important for degrading and maintaining low levels of cAMP in gustatory sensory receptor cells.
Pde1c Expression and Male Courtship Behavior in dsx-Expressing Cells other than GRNs
Unlike dsx-expressing cells, the knockdown of Pde1c in GRNs did not result in abnormalities in courtship index or mating rate. This suggested that courtship index and mating success may be regulated by Pde1c expression in dsx-expressing neurons other than GRNs. In this regard, dsx/glutaminergic and dsx/GAVA agonist neurons have been shown to control genital coupling and disconnection during mating, respectively [16]. Among these neurons, dsx/glutaminergic neurons are localized to GRNs and Abg, while dsx/GAVAergic neurons are localized to Abg and parts of the brain. Suppression of the activity of these neurons was shown to reduce both courtship index and mating rate [16]. Therefore, the abnormalities in courtship index and mating rate associated with Pde1c knockdown in dsx-expressing cells observed in the present study may have been due to impaired function of these neurons as a result of Pde1c knockdown.
Conclusion
This study demonstrated that Pde1c expression in GRNs is required for sufficient activation of male mating behavior in Drosophila and that its expression in GRNs is under the control of Dsx. To our knowledge, this is the first study to identify a strong candidate Dsx target gene involved in male mating behavior in Drosophila. However, to determine whether Pde1c is indeed a direct target gene of Dsx, further experiments are needed to examine the expression pattern of Pde1c in dsx mutants and to identify the enhancer region of Pde1c that is directly regulated by Dsx.
Our results showed that Pde1c is strongly expressed in GRNs, cells in which fru is highly expressed and plays an essential role in sexual dimorphism (i.e., left-right axon contacts only in males) [39]. In a previous study that comprehensively searched for Fru target genes by DamID-seq, Pde1c was identified as a potential target gene for Fru [40]. Lgr3, which has been identified as a Dsx target gene acting in the central nervous system, is known to be regulated by both Dsx and Fru [13]. Further studies are needed to determine whether Pde1c is under the control of not only dsx but also fru.
Acknowledgments
The author would like to express sincere thanks to Dr. Tokai Sawamoto for advice on this research, experimental instruction, and support.
Funding Information
This work was supported by JSPS KAKENHI Grant Number JP22K19166.
Funding Information
The authors have declared that no competing interests exist.
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