Channel nuclear pore protein 54 directs sexual differentiation and neuronal wiring required for female reproductive behaviors in Drosophila

The post-mating response induced by male-derived sex-peptide in Drosophila females is a well-established model to elucidate how complex innate behaviors are hard-wired into the brain. Here, we found that the channel nuclear pore protein Nup54 is essential for the sex-peptide response as viable mutant alleles do not lay eggs and reduce receptivity upon sex-peptide exposure. Nup54 directs correct wiring of few adult brain neurons that express pickpocket and are required for egg laying, but channel Nups also mediate sexual differentiation and male X-chromosome dosage compensation. Consistent with links of Nups to speciation, the Nup54 promoter is a hot spot for rapid evolution and Nup54 is differentially expressed in the brain. These results implicate altered expression of Nup54 to the onset of speciation processes leading to changes in neuronal wiring and sexual differentiation as a response to sexual conflict arising from male-derived SP to direct the female post-mating response.


Introduction
Female reproductive behaviors and physiology change profoundly after mating. Control of pregnancyassociated changes in physiology and behaviors are largely hard-wired into the brain to guarantee reproductive success, yet the gene expression programs that direct neuronal differentiation and wiring at the end of the sex determination pathway are largely unknown.
In most insects, male-derived substances transferred during mating direct female physiology and postmating behaviors [1][2][3]. Drosophila females display a repertoire of sex specific behaviors after mating including reduced receptivity (readiness to mate) and increased egg laying [4,5]. The main trigger of these post-mating behaviors is male-derived sex-peptide (SP) [6][7][8][9]. SP is a 36 amino acid peptide that is transferred during mating to the female [10]. Besides reducing receptivity and increasing egg laying, SP induces a number of other behavioral and physiological changes including increased egg production, feeding, a change in food choice, sleep, constipation and stimulation of the immune system [11][12][13][14][15][16][17][18]. In addition, SP binds to sperm and acts as a sperm sensor, is required for the release of stored sperm and imposes costs of mating [19][20][21].
Many of the phenotypic effects of SP on female physiology are known, but central aspects of the neuronal circuitry governing regulation of the main post-mating behaviors like reduced receptivity and increased oviposition are unclear. First insights came from the analysis of egghead mutant alleles that are insensitive to SP [22]. In these mutants neuronal connections from the ventral nerve cord to the central brain show defects resulting in an egg retainer phenotype and non-responsiveness to SP in reducing receptivity. This suggests that receptor signaling is disconnected from the motor output programs. Other attempts to map the circuitry mediating the post-mating response used expression of membrane-tethered SP or RNAi knockdown of a receptor for SP, SPR, in distinct neuronal expression patters [9,[23][24][25][26][27]. These screens identified pickpocket (ppk), fruitless (fru) and dsx expressing neurons mediating the post-mating switch via expression of membrane-tethered SP in multiple pathways [9].
The ability of males to manipulate post-mating responses of females, for example, by SP in Drosophila, can promote sexual conflict and trigger an arms-race between the sexes [28][29][30]. This arms-race drives rapid evolution and can fuel speciation. Two essential nuclear pore proteins, Nup96 and Nup160, that are part of the outer ring of the pore and interact with each other, trigger inviability in hybrids between species through adaptive divergence in these two proteins [31][32][33][34]. The megadalton nuclear pore complex constitutes a bidirectional gateway connecting the nucleus and cytoplasm to control transport of all macromolecules [35], but how nuclear pore proteins can drive speciation is unknown.
Here, we have identified a role for the essential Nuclear pore protein 54 (Nup54) gene, that is part of the nucleo-cytoplasmic transport channel, in constituting the SP response through an allele displaying egg retention and insensitivity to SP with regard to receptivity. Nup54 is a core protein of the nuclear pore and localizes to the transport channel connecting the nucleus with the cytoplasm. Despite being a constitutive part of the nuclear pore, Nup54 is differentially expressed in the brain and required early in neuronal development to direct proper wiring of few neurons in the adult brain that express ppk and are part of the neuronal circuit directing egg laying. In addition, more general roles for the nuclear pore in regulating sexual differentiation are indicated by RNAi knockdown of channel Nups, which results in differentiation defects of internal and external sexual features in males and females.

Mutant females for Nup54 alleles display an egg retainer phenotype and are insensitive to sexpeptide for reducing receptivity
To identify novel genes involved in specifying the SP response, we screened females from egg retainer lines that have normal oogenesis, but are unable to lay eggs, for their ability to reduce receptivity [22].
Using this approach, we identified a homozygous viable EMS induced line, QB62 [36], that did not reduce receptivity upon injection of SP ( Figure 1A-D). We mapped this allele by meiotic recombination based on the egg retention phenotype to 2-69.9, corresponding approximatively to chromosome position 50C. Using overlapping deficiencies this allele could then be mapped to chromosome section 49A4 by deficiencies Df(2R)BSC305 and Df(2R)Exel16061 ( Figure 1A). To further restrict the number of genes in this chromosomal area we generated a smaller deficiency, Df(2R)9B4, by FRT mediated recombination between transposons P{XP}CG8525 d06853 and PBac{RB}DUBAI e00699 ( Figure 1A). We then identified a Mi{ET1}transposon insert in the Nup54 gene, Nup54 MB03363 , that was allelic to QB62 with respect to both egg retention and reduction of receptivity after SP injection ( Figure 1B-D). Nup54 localizes to the transport channel of the megadalton nuclear pore complex that constitutes a bidirectional gateway connecting the nucleus and cytoplasm to control transport of all macromolecules [35]. The Nup54 MB03363 transposon insert leads to a truncation of the Nup54 ORF and removes the C-terminal alpha helical domain that connects Nup54 with Nup58, but leaves the core part containing the FG repeats composing the inner nuclear channel and the interaction of Nup54 with Nup62 via the alpha/beta helical region intact ( Figure 1E) [35,37,38]. Sequencing of the Nup54 QB62 allele identified a small deletion (CATTAGAGAGGAGGCGTAA) in the promoter region 186 nt upstream of the first transcribed nucleotide suggesting that this allele impacts on the expression of Nup54 ( Figure 1A), possibly only in a subset of cells, because we did not detect differences by qPCR ( Figure S1A). These two alleles represent hypomorphic mutations as channel Nups are essential genes [35].
A genomic rescue using a construct with a deletion of the first intron containing the overlapping gene CG432041 (gNup54) further confirmed that the egg retention and SP insensitivity in receptivity maps to the Nup54 gene ( Figure 1B and C). We then analyzed the expression of Nup54 from gNup54 via the Cterminal HA tag. In salivary glands, Nup54 localizes to the nuclear membrane. Intriguingly, in both the larval ventral nerve cord and the adult brain, Nup54 expression is dynamic and levels are higher in some cells ( Figure S1B-D).

Nup54 is required before neuronal maturation for establishing the post-mating response
Next, we expressed Nup54 from a UAS construct in a Nup54 MB03363 /Df(2R)9B4 background to identify its temporal and spatial requirement for rescuing the egg retainer and SP insensitivity phenotype in receptivity. Global expression with tubGAL4 in Nup54 MB03363 /Df(2R)9B4 completely rescued these phenotypes after injection of SP. We then expressed UASNup54 in all differentiated neurons with elavGAL4 C155 or in subsets using ppkGAL4, fruGAL4 and dsxGAL4 to test whether they can rescue the post-mating response in Nup54 MB03363 /Df(2R)9B4 after SP injection ( Figure S2A and B). These experiments revealed that fruGAL4 and dsxGAL4, which express earlier than elav, can rescue the SP insensitivity and suppress remating, but only dsxGAL4 can marginally rescue egg laying ( Figure S2A and B).
Taken together these data indicate that Nup54 is required early in development before neurons are fully differentiated to specify the neuronal circuits required for the post-mating response.

Nup54 QB62 displays defects in neuronal wiring of pickpocket expressing neurons
Expression of membrane-bound SP can induce the post-mating response in ppk expressing neurons [23][24][25][26]. To examine whether Nup54 function is required for neuronal wiring relevant for the post-mating response, we visualized neuronal projections of ppk neurons in wild type and Nup54 QB62 /Df(2R)9B4 mutant females by expression of CD8::GFP from UAS under the control of ppkGAL4. In the larval brain of control females ppk sensory neuronal projections display a regular pattern of connectives and commissures, while in Nup54 QB62 /Df(2R)9B4 mutants many connectives and commissures are disrupted ( Figure 2A-F). In the adult brain, projections in the central brain were dramatically reduced in Nup54 QB62 /Df(2R)9B4 females compared to control females ( Figure 2G and H). So far it has been shown that ppk expresses in sensory neurons projecting to the brain [23][24][25][26]. A closer examination of ppk expression in the adult female brain, however, revealed four paired neurons consistently expressing a nuclear histon2B::YFP marker expressed from UAS with ppkGAL4. Strikingly, these neurons lacked projections in Nup54 QB62 /Df(2R)9B4 females ( Figure 2G-I).
ppk neurons in the brain are part of the circuit required for egg laying To test whether ppk expressing neurons in the brain are involved in the post-mating response we used an intersectional gene expression approach based on brain-specific expression of orthodentical (otd) to specifically inhibit neuronal transmission in brain ppk neurons by expression of tetanus-toxin (TNT) [39]. To achieve restricted expression of TNT in ppk brain neurons, we crossed otd-flipase (otd::flp) flies with ppkGAL4, UAS FRTGFPstopFRT TNT flies and analysed the female progeny for PMRs ( Figure 3A).
These experiments revealed that females with inhibited ppk neurons in the brain displayed a compromised response to SP as they did not fully reduce receptivity and did not lay the eggs stored in the ovaries after injection of SP ( Figure 3B-D). In contrast, inhibition of all ppk neurons partially reduced receptivity in SP injected females. In addition, these females display increased egg laying as virgins, but SP does not further enhance egg laying ( Figure 3B-D). Control females of UAS TNT without a GAL4 driver showed a normal response to SP (data not shown).
Taken together, these results demonstrate an essential role for ppk neurons in the brain in inducing egg laying, and a partial requirement for reducing receptivity in response to SP.

Specification of neuronal circuits for receptivity and egg laying are separable
To further test the role of Nup54 in the post-mating response we used UASRNAi knock-down of Nup54 in either all neurons using elavGAL4 C155, or in subsets of neurons using ppkGAL4, fruGAL4 and dsxGAL4. RNAi knock-down of Nup54 with elavGAL4 C155 , ppkGAL4 and fruGAL4 did not affect receptivity, or egg laying after SP injection further demonstrating that Nup54 is required early in neuronal development ( Figure S3A and B). In contrast, RNAi knock-down of Nup54 with dsxGAL4, oviposition was not induced upon SP injection while receptivity was reduced normally ( Figure S3A and B).
In summary, the combined results from experiments in Figure S2A and B, and Figure S3A and B indicate that Nup54 is required early in neuronal development and that the neuronal circuits for egg laying and receptivity are specified by distinct neuronal populations.

Channel Nups have a role in sex determination and dosage compensation
When knocking down Nup54 with RNAi using dsxGAL4, we noticed that females laid eggs that did not develop. In addition, male survival was low (20%, n=107, P{TRIP.HMC04733}) or males were completely absent (n=107, P{GD14041}v42153; P{GD14041}v42154), indicating that Nup54 has additional roles in dosage compensation [40,41]. Therefore, we hypothesized that the other two channel Nups, Nup58 and Nup62, that form a complex with Nup54, also have roles in specifying the post-mating response and/or in sexual differentiation and dosage compensation. RNAi knockdown of Nup58 (P{TRIP.HMC05104}) with dsxGAL4 resulted in lethality with only few very weak female escapers.
These females, however, were sterile due to the lack of ovaries and deformed genitals. Similar results were obtained by RNAi knock-down of Nup62 with dsxGAL4 resulting in male lethality (9%, n=81 and 2%, n=45 escapers with P{TRIP.GLV21060} and P{TRIP.HMC03668}Since ) and females without ovaries (100%, n=74 and 43%, n=35 with P{TRIP.GLV21060} and P{TRIP.HMC03668}). In addition, these females also had deformed genitals and males displayed underdeveloped sex combs indicating a role for channel Nups in sexual differentiation ( Figure S3C-F). Consistent with a regulatory role in development, the channel Nups display dynamic expression during development and in different tissues ( Figure S4A-F).

The role of insertion/deletion (indels) during the evolution of Nup54 function
Adaptation to new situations drives the evolution of hybrid incompatibility genes Nup96 and Nup160 [31-34]. Here, we used the combined analysis of polymorphism within D. melanogaster and divergence between D. melanogaster and D. simulans within the coding region of Nup54 to test for deviations from expectations under the neutral model of molecular evolution. We found no deviation from the null neutral model hypothesis, as the ratio of nonsynonymous to synonymous polymorphisms did not significantly differed from the ratio of nonsynonymous to synonymous fixed differences between species (MK test: χ 2 = 0.09; P= 0.923). The test was also non-significant when we partition the analysis by exons.
Also, the upstream noncoding region did not significantly depart from neutrality (Fisher's exact test P= 0.135), but there was a trend towards an excess of upstream noncoding relative to silent (synonymous and intron) substitutions between species (dNC/dS=3.1) relative to polymorphisms within D. melanogaster (PNC/PS=2.0) (neutrality index: NI = 0.65).
Indels are a common form of genetic variation, being able to affect gene function and their pattern of evolution being subjected to selective forces [42][43][44]. We noticed that a stretch of amino acids of increasing length in more distantly related species is inserted in the FG repeat region ( Figure S5A). We therefore hypothesized that this indel could alter the function of Nup54 in specifying the post-mating response. To test this hypothesis, we replaced the FG-repeat region from D. melanogaster with the region from D. elegans in the genomic rescue construct gNup54ele and tested its capacity to rescue viability of Df(2R)9B4 and the post-mating response defects. Both the D. melanogaster gNup54 and the chimeric D.
elegans construct gNup54ele rescued viability of Df(2R)9B4 to 91% (n=242) and 95% (n=381) without sex-bias, respectively. After injection of SP, females rescued with the gNup54 or the gNup54ele construct showed normal post-mating responses in receptivity and oviposition arguing that this indel is not the source for an altered post-mating response ( Figure S5B and C).
In the Nup54 promoter region rapid evolution is identifiable by the presence of four hotspots for substitutions between species, all within 1 kb upstream of the Nup54 transcription start site ( Figure 4A and B). Interestingly, one substitutional hotspot (-359 to -295) includes an indel that overlaps with the location of the characterized Nup54 QB62 regulatory allele ( Figure 4B). The deletion in the Nup54 QB62 allele in fact is to a large extent a wild type condition in sister species ( Figure 4A). While it is unclear whether this change has been driven by selection, it has clearly evolved in the context of the post-mating response to SP.

Discussion
Here we show that Nup54 which is part of the central transport channel of the nuclear pore exerts a role in specifying the neuronal circuits involved in mediating the SP induced post-mating switch. Viable alleles of these genes display an egg retainer phenotype and are insensitive to SP with regard to reducing receptivity. Nup54 is required for wiring of ppk expressing neurons in the brain that constitute part of the circuitry required for egg laying. The role of Nup54 and the other two channel Nups, Nup58 and Nup62, in sexual differentiation, however, seems to be more fundamental as reducing their expression levels impacts on general sexual differentiation of external and internal sexual features, such as female genitals and ovaries, but also on dosage compensation in males.

Roles for Nups in development
A key role for Nups in sexual differentiation seems rather unexpected as the function of channel Nups in directing constitutive export of mRNAs and proteins from the nucleus to the cytoplasm has been viewed as static for a long time. However, a number of recent studies have brought about developmental and neuronal roles for the nuclear pore and nucleocytoplasmic transport reflected in a number human diseases including neurodegeneration associated with specific Nups [45][46][47]. A central aspect of these more profound roles for Nups lay in their varying expression levels between different cell types, tissues and in development. Nup210's key role in muscle and neuronal differentiation are associated with tissuespecific expression [48]. Likewise, high expression levels of Nup153 are critical for maintenance of ESC pluripotency as reducing levels results in neuronal differentiation [49]. Also, channel Nup62 shows increased expression in various epithelia and is required for proliferation [50]. In Drosophila, channel Nups 54, 58 and 62 show increased expression in the larval brain and adult gonads [51]. Our findings that channel Nups have a role in sexual differentiation add to this view that the nuclear pore has key roles in differentiation and likely also neuronal function.

Specification of neuronal circuits for receptivity and egg laying are separable
It has previously been argued that the neurons co-expressing fru, dsx and ppk in the genital tract sense SP and direct the SP induced post-mating [24][25][26]. However, our data from expressing a Nup54 rescue construct and from RNAi knockdown in various cell types suggests a are more complex picture including separation of the paths leading to reduced receptivity and increased egg laying. Here, expression of Nup54 in fru and dsx neurons, but not ppk neurons rescues receptivity while egg laying is not rescued in all three expression patterns. Also, RNAi of Nup54 in dsx neurons leads to inhibition of egg laying, but receptivity is reduced normally. RNAi in fru and ppk neurons has no effect on SP induced changes in receptivity and egg laying. Although the latter effect could be explained by different expression levels of fru, dsx and ppk GAL4 lines, this is not observed and they express strongly. Consistent with a requirement for Nup54 early in development, dsx is expressed earlier than fru and ppk. Differential effects on receptivity, but not egg laying by dsx driven rescue or knock-down by Nup54, however, more likely indicates that receptivity and egg laying are governed by different neuronal circuits. These results are supported by previous observations, that G(o) is not required in fru, but needed in dsx and ppk neurons to reduce receptivity [9]. Likewise, membrane-tethered SP can only induce oviposition in the absence of SPR in dsx, but not fru and ppk neurons [9].

Roles of Nups in speciation
New species can arise through selection of new features enhancing display of sexual attributes and altered courtship behavior. A driving force to speciation can be sexual conflict imposed by male-directed post-mating responses by females [28][29][30]. In Drosophila melanogaster, females respond to malederived SP by reducing receptivity and increasing egg laying, but need to adapt their physiological status to environmental conditions. Thus, the possibility to limit the male influence imposed by SP is in the female interest of optimizing reproductive success when resources to produce eggs are scarce. In this context, Nup54 regulation could impact on egg laying in dedicated circuits such as brain ppk neurons.
Consistent with this interpretation, the Nup54 promoter region has undergone a dramatic change compared to closely related species, while no adaptive changes were detected in the protein coding part.
Likewise, exchanging the FG repeat of Nup54 mediating nucleo-cytoplasmic transport, which shows most variability between D. melanogaster, D. elegans and other closely related species had little impact on the post-mating response. Since Nup54 is required in all cells, varying its concentration could have profound effects on general expression of genes and select for a specific compensatory genetic element from the mating partner to prevent deleterious effects on the fitness of the progeny[31-34]. Such interpretation is well in line with roles of essential Nup96 and Nup160 in speciation as they have been identified in causing hybrid sterility or lethality. Intriguingly however, Nup96 and Nup160 are part of the larger NUP107 subcomplex and four out of eight proteins from this complex are under adaptive selection in D. simulans [32]. The driving forces behind the role of Nups in speciation and the molecular mechanism leading to speciation, however, need to be further explored.
Here we have identified a few ppk expressing neurons in the brain as a cellular focus preventing female escape from male manipulation. Changes in neuronal wiring likely directed in response to sexual conflict arising from male-derived SP to direct the female post-mating response marks an early event in the splitting of species and links differentiation of key neurons involved in female control of reproduction to fitness as a result of sexual conflict. Our results indicate a central role for the nuclear pore in implementing alterations of gene expression impacting on neuronal wiring at the onset of speciation processes.
Flies were kept on standard cornmeal-agar food (1%industrial-grade agar, 2.1% dried yeast, 8.6% dextrose, 9.7% cornmeal and 0.25% Nipagin, all in (w/v)) in a 12 h light : 12 h dark cycle. Sexually mature 3-5 day-old virgin females were injected and examined for their post-mating behaviors as described previously [12,22]. For injections, virgin female flies were cooled to 4º C and 3 pmol SP in 50 nl Ringer's solution was injected. Ovaries were analyzed as previously described [12]. ANOVA followed by planned pairwise comparisons with Fisher's protected least significant difference was done for statistical analysis using STATVIEW or Graphpad prism.
The UAS construct with a N-terminally HA-tagged Nup54 was generated in a three way ligation with fragments of Nup54 amplified from cDNA by RT-PCR that were cloned into a modified pUC, pUC 3GLA UAS HA, with NheI and Acc65I with a blunt site in between. The 5' part was amplified using primers 8831F1 (CATCGCTAGCGCCTGCAGGATCGTTCTTCGGATCCAACACGTCGCTGG) and transposon insertion lines, P{XP}CG8525 d06853 and PBac{RB}DUBAI e00699 as described [53,54]. The tubGal4 line was a third chromosomal insert (Bloomington #5138), UAS FRTGFPstopFRT TNT and the other lines have been previously described [9,55,56]. Antibody stainings were done as previously described [57,58].