BIOLOGICAL OVERVIEW

Sex determination in the Drosophila germ line is regulated by both the sex of the surrounding soma and cell-autonomous cues. How primordial germ cells (PGCs) initiate sexual development via cell-autonomous mechanisms is unclear. This study demonstrates that, in Drosophila, the Sex lethal (Sxl) gene acts autonomously in PGCs to induce female development. Sxl is transiently expressed in PGCs during their migration to the gonads; this expression, which was detected only in XX PGCs, is necessary for PGCs to assume a female fate. Ectopic expression of Sxl in XY PGCs was sufficient to induce them to enter oogenesis and produce functional eggs when transplanted into an XX host. These data provide powerful evidence that Sxl initiates female germline fate during sexual development (Hashiyama, 2011).

Primordial germ cells (PGCs) are able to differentiate into eggs or sperm. It is thought that PGCs do not assume a sexual fate until they reach the gonads, where sexual dimorphism is imposed by both the sex of the surrounding soma and cell-autonomous cues. In Drosophila, pole cells or PGCs differentiate to a male fate in response to JAK/STAT signaling from the gonadal soma. The method by which female sexual development is initiated in pole cells, however, has not been elucidated. To clarify the mechanism that initiates a female fate in pole cells, a female-specific marker for this cell type was identified. Although several sex-specific markers, including mgm-1, disc proliferation abnormal, and minichromosome maintenance 5, have been reported, they are all expressed only in male pole cells after gonad formation (stage 15), based on signals from the male gonadal soma. lesswright (lwr), a gene that regulates posttranslational modification of proteins by small ubiquitin-related modifiers, is expressed in pole cells during embryogenesis. lwr is not characterized by sex-specific expression. When a dominant-negative form of lwr (lwr^DN) was expressed in the pole cells of either sex, however, apoptosis was induced only in female (XX) pole cells during migration to the gonads. This effect caused a significant reduction in the number of XX pole cells in the gonads. Introduction of female-specific germline apoptosis induced by a dominant-negative form of lwr (f-gal) provides a previously uncharacterized marker of female sexual identity in migrating pole cells (Hashiyama, 2011).

Sex determination is controlled by the Sex lethal (Sxl) gene, which is first expressed at the blastodermal stage in the embryonic soma. Sxl encodes an RNA binding protein involved in alternative splicing and translation. In the soma of XX embryos, it functions through transformer (tra) and transformer-2 (tra-2), which in turn regulate alternative splicing of the doublesex (dsx) gene to produce a female-specific form of Dsx. In male (XY) embryos, this pathway is turned off, and a male-specific form of Dsx is produced by default. These Dsx proteins determine the sexual identity of somatic tissues. Previous reports, however, suggested that Sxl does not induce female sexual development in the germ line, as it does in the soma. Although Sxl is autonomously required for female sexual development, constitutive mutations in Sxl (Sxl^M) that cause XY animals to undergo sexual transformation from male to female do not necessarily interfere with male germline development. Moreover, tra, tra-2, and dsx are not required for female germline development. Finally, female-specific Sxl expression has been detected later in gametogenesis, but not in early germline development (Hashiyama, 2011).

Contrary to previous observations, this study found that Sxl was expressed in XX but not XY pole cells during their migration to the gonads. In the soma, Sxl transcripts are first expressed from the establishment promoter (Sxl-Pe) in a female-specific manner. Using a probe specific to the early transcript derived from Sxl-Pe, in situ hybridization signals were detected in migrating XX pole cells at around stage 9/10. Transgenic embryos, which expressed enhanced green fluorescent protein (EGFP) under the control of the Sxl-Pe promoter, were used to further confirm this female-specific Sxl-Pe activation. RTPE and sequencing analyses in pole cells were used to detect early Sxl transcripts that had the same sequence as the transcripts expressed in the soma (Hashiyama, 2011).

Next, it was determined whether Sxl feminize early pole cells using f-gal as a marker for female identity. It was found that the loss-of-function mutation Sxl^fP7B0 represses f-gal in XX pole cells. This repression is unlikely to result from sexual transformation of the soma, because an amorphic tra-2 mutation, which alters somatic sex, did not affect f-gal. Conversely, when the expression of Sxl together with lwr^DN is forced in pole cells from stage 9 onward by using nanos-Gal4 and UAS-Sxl, f-gal is ectopically observed in XY pole cells. Sxl alone does not induce apoptosis or developmental defects in pole cells. These observations suggest that female sexual identity of migrating pole cells is regulated cell-autonomously by Sxl (Hashiyama, 2011).

It was then determined whether Sxl induces female development in XY pole cells. Because XY soma produces signals that direct XX germline cells to a male fate, XY pole cells expressing Sxl were transplanted into XX females, and their developmental fate was examined. Even in the presence of a gain-of-function Sxl mutation (Sxl^M1) that causes XY soma to transform from male to female, XY (or XO) pole cells enter the spermatogenic pathway when transplanted into XX females. These results suggest that Sxl is not sufficient to activate female germline development. Sxl^M1 mutations, however, do not affect transcription from the Sxl-Pe promoter, but instead structurally alter the late transcript from the Sxl maintenance promoter (Sxl-Pm), which allows Sxl protein production in both males and females. Consistent with this observation, Sxl transcripts derived from Sxl-Pe were detected in the pole cells of only female Sxl^M1 embryos. Thus, the Sxl^M1 mutation does not result in Sxl expression in XY pole cells as early as in XX pole cells (Hashiyama, 2011).

Instead, nanos-Gal4 and UAS-Sxl were used to induce Sxl expression in XY pole cells. Three types of XY pole cells were transplanted, each characterized by a different duration of Sxl expression: (1) XY pole cells in which Sxl was expressed from stage 9 until stage 16/17 using maternal nanos-Gal4 (XY-mSxl), (2) XY pole cells in which Sxl was expressed from stage 15/16 onward using zygotic nanos-Gal4 (XY-zSxl), and (3) XY pole cells in which Sxl was expressed from stage 9 onward using both maternal and zygotic nanos-Gal4 (XY-mzSxl). XY-mzSxl and XY-mSxl pole cells entered the oogenic pathway and produced mature oocytes in XX females. These oocytes contributed to progeny production. Thus, the XY pole cells produced functional eggs, even though oogenesis and egg production were reduced compared with XX pole cells. In contrast, XY-zSxl pole cells did not enter the oogenic pathway in almost all (92.3%) of the XX female hosts and instead were characterized by a tumorous phenotype, an indication of XY germline cells that have maintained male characteristics. Control XY pole cells from the embryos expressing Sxl only in the soma (XY-nullo-Sxl) showed a similar phenotype to that of XY-zSxl pole cells. These observations demonstrate that Sxl expression in XY pole cells during embryogenesis induces functional egg differentiation in the female soma (Hashiyama, 2011).

Sxl-specific double-stranded RNA (UAS-Sxl^RNAi) under the control of maternal nanos-Gal4 was used to reduce Sxl activity in XX pole cells during embryogenesis. Introducing UAS-Sxl^RNAi resulted in tumorous and agametic phenotypes in female adults, indicating that the XX germ line lost female characteristics. Taken together, these results show that Sxl acts as a master gene necessary and sufficient to induce female development in pole cells (Hashiyama, 2011).

XY-mzSxl pole cells adopted a male fate and executed spermatogenesis when they developed in an XY male soma. This observation suggests that the male soma plays a dominant role in determining the male germline fate, overriding the feminizing effect of Sxl. Another possibility is that the XX female soma plays a critical role in maintaining the Sxl-initiated female germline fate. Indeed, an XX germ line in the male soma shows a male gene-expression profile, whereas an XY germ line in the female soma exhibits a female expression profile, although these germ lines does not execute gametogenesis. Thus, female germline development requires interactions between the germline and somatic cells, in addition to germline-autonomous mechanisms involving Sxl (Hashiyama, 2011).

In mice, germline sexual identity is also regulated by both germline-autonomous and somatic signals. In the coelenterate Hydra, the germline sex is not influenced by the surrounding soma, and the germ line determines the phenotypic sex of the polyp. Thus, germline-autonomous regulation of sex has probably been present throughout the evolution of animals, and somatic control may have evolved with the emergence of mesodermal tissues, including gonadal soma. Sxl does not appear to play a key role in sex determination in non-drosophilid animals. Nevertheless, future studies should determine whether Sxl homologs are expressed in the germ line of non-drosophilids. Moreover, it would be of particular interest to identify downstream targets of Sxl in the Drosophila germ line and to test whether these genes have a widespread role in germline sex determination (Hashiyama, 2011).

The translation initiation factor eIF4E regulates the sex-specific expression of the master switch gene Sxl in Drosophila

In female fruit flies, Sex-lethal (Sxl) turns off the X chromosome dosage compensation system by a mechanism involving a combination of alternative splicing and translational repression of the male specific lethal-2 (msl-2) mRNA. A genetic screen identified the translation initiation factor eif4e as a gene that acts together with Sxl to repress expression of the Msl-2 protein. However, eif4e is not required for Sxl mediated repression of msl-2 mRNA translation. Instead, eif4e functions as a co-factor in Sxl-dependent female-specific alternative splicing of msl-2 and also Sxl pre-mRNAs. Like other factors required for Sxl regulation of splicing, eif4e shows maternal-effect female-lethal interactions with Sxl. This female lethality can be enhanced by mutations in other co-factors that promote female-specific splicing and is caused by a failure to properly activate the Sxl-positive autoregulatory feedback loop in early embryos. In this feedback loop Sxl proteins promote their own synthesis by directing the female-specific alternative splicing of Sxl-Pm pre-mRNAs. Analysis of pre-mRNA splicing when eif4e activity is compromised demonstrates that Sxl-dependent female-specific splicing of both Sxl-Pm and msl-2 pre-mRNAs requires eif4e activity. Consistent with a direct involvement in Sxl-dependent alternative splicing, eIF4E is associated with unspliced Sxl-Pm pre-mRNAs and is found in complexes that contain early acting splicing factors -- the U1/U2 snRNP protein Sans-fils (Snf), the U1 snRNP protein U1-70k, U2AF38, U2AF50, and the Wilms' Tumor 1 Associated Protein Fl(2)d--that have been directly implicated in Sxl splicing regulation (Graham, 2011).

Translation initiation is mediated by the binding of a pre-initiation complex to the 5' cap of the mRNA (reviewed in (Merrick, 1996 ; Gingras, 1999) that in turn recruits the small subunit of the 40S ribosome to the mRNA. The pre-initiation complex consists of the cap binding protein, eIF4E, and a scaffolding protein, eIF4G, which mediates interactions with various components of the 40S initiation complex. In many organisms there is also a third protein in the complex, eIF4A, an ATP dependent RNA helicase. Modulating eIF4E activity appears to be a key control point for regulating translation. One of the most common mechanisms of regulation is by controlling the association eIF4E with eIF4G. Factors such as poly-A binding protein that promote the association between eIF4E and eIF4G activate translation initiation, while factors such as the 4E-binding proteins (4E-BPs; see Drosophila 4E-BP) that block their association, inhibit initiation (Graham, 2011 and references therein).

Although eIF4E's primary function in the cell is in regulating translation initiation, studies over the past decade have revealed unexpected activities for eIF4E at steps prior to translation. Among the more surprising findings is that there are substantial amounts of eIF4E in eukaryotic nuclei. One role for eIF4E in the nucleus is the transport of specific mRNAs, like cyclin D1, to the cytoplasm (Rousseau, 1996). This eIF4E activity is distinct from translation initiation since an eIF4E mutation that prevents it from forming an active translation complex still allows cyclin D1 mRNA transport. The transport function of eIF4E is modulated by at least two other proteins, PML and PRH (Topisirovic, 2002; Topisirovic, 2003). While PML seems to be ubiquitously expressed, PRH is found only in specific tissues. In addition, the intracellular distribution of eIF4E exhibits dynamic changes during Xenopus development (Strudwick, 2002). These observation raise the possibility that eIF4E might have additional functions in the nucleus during development. Consistent with this idea, this study shows that eIF4E plays a novel role in the process of sex determination in Drosophila (Graham, 2011).

Sex determination in the fly is controlled by the master regulatory switch gene Sex-lethal (Sxl). The activity state of the Sxl gene is selected early in development by an X chromosome counting system. The target for the X/A signaling system is the Sxl establishment promoter, Sxl-Pe. When there are two X chromosomes, Sxl-Pe is turned on, while it remains off when there is a single X chromosome. Sxl-Pe mRNAs encode RRM type RNA binding proteins which mediate the transition from the initiation to the maintenance mode of Sxl regulation by directing the female-specific splicing of the first pre-mRNAs produced from a second, upstream promoter, the maintenance promoter, Sxl-Pm. Sxl-Pm is turned on before the blastoderm cellularizes, just as Sxl-Pe is being shut off. In the presence of Sxl-Pe proteins, the first Sxl-Pm transcripts are spliced in the female-specific pattern in which exon 2 is joined to exon 4 (see Model of the alternatively spliced region of Sxl ). The resulting Sxl-Pm mRNAs encode Sxl proteins that direct the female specific splicing of new Sxl-Pm pre-mRNAs and this establishes a positive autoregulatory feedback loop that maintains the Sxl gene in the 'on' state for the remainder of development. In male embryos, which lack the Sxl-Pe proteins, the Sxl-Pm pre-mRNAs are spliced in the default pattern, incorporating the male specific exon 3. This exon has several in-frame stop codons that prematurely truncate the open reading frame so that male specific Sxl-Pm mRNAs produce only small non-functional polypeptides. As a consequence the Sxl gene remains off throughout development in males (Graham, 2011).

In females, Sxl orchestrates sexual development by regulating the alternative splicing of transformer (tra) pre-mRNAs. Like Sxl, functional Tra protein is only produced by female-specific tra mRNAs, while mRNAs spliced in the default, male pattern encode non-functional polypeptides. Sxl also negatively regulates the dosage compensation system, which is responsible for hyperactivating X-linked transcription in males, by repressing male-specific lethal-2 (msl-2). Sxl represses msl-2 by first blocking the splicing of an intron in the 5' UTR of the msl-2 pre-mRNA, and then by inhibiting the translation of the mature mRNA. In addition, there are two other known targets for Sxl translational repression. One is the Sxl mRNA itself. Sxl binds to target sequences in the Sxl 5' and 3' UTRs and downregulates translation. It is thought that this negative autoregulatory activity provides a critical homeostasis mechanism that prevents the accumulation of excess Sxl protein. This is important as too much Sxl can disrupt development and have female lethal effects. The other known target is the Notch (N) mRNA (Penn, 2007). Sxl-dependent repression of N mRNA translation is important for the elaboration of sexually dimorphic traits in females. Like msl-2 and Sxl, translational repression appears to be mediated by Sxl binding to sites in the N UTRs (Graham, 2011).

Translational repression of msl-2 mRNA by Sxl is thought to involve two separate mechanisms acting coordinately. Binding sites for Sxl in the unspliced intron in the 5' UTR and in the 3'UTR of msl-2 are required for complete repression. Sxl binding to the 5'UTR blocks recruitment of the 40S pre-initiation complex (. While factors that act with Sxl at the 5'UTR of msl-2 have yet to be identified, repression by the 3'UTR requires Sxl, PABP and a co-repressor UNR. Somewhat unexpectedly, this complex does not affect recruitment of eIF4E or eIF4G to the 5' end. Instead it prevents ribosomes that do manage to attach to the msl-2 mRNA from scanning (Graham, 2011).

Although eIF4E does not appear to be a key player in the translational repression of msl-2 mRNAs, this study reports that it has an important role in the process of sex determination in Drosophila. eIF4E activity is required in females to stably activate and maintain the Sxl positive autoregulatory feedback loop and to efficiently repress msl-2. Surprisingly, this requirement for eIF4E activity in fly sex determination is in promoting the female-specific splicing of the Sxl and msl-2 transcripts, not in translational regulation (Graham, 2011).

The RNA binding protein Sxl orchestrates sexual development by controlling gene expression post-transcriptionally at the level of splicing and translation. To exert its different regulatory functions Sxl must collaborate with sex-non-specific components of the general splicing and translational machinery. In this study evidence is presented that one of the splicing co-factors is the cap binding protein eIF4E. eif4e was initially identified in a screen for mutations that dominantly suppress the male lethal effects induced by ectopic expression of a mutant Sxl protein, Sx-N, which lacks part of the N-terminal domain. The Sx-N protein is substantially compromised in its splicing activity, but appears to have closer to wild type function in blocking the translation of the Sxl targets msl-2 and Sxl-Pm. As the male lethal effects of Sx-N (in an Sxl^- background) are due to its inhibition of Msl-2 expression, it is anticipated that general translation factors needed to help Sxl repress msl-2 mRNA would be recovered as suppressors in the screen. Indeed, one of the suppressors identified was eif4e. However, consistent with in vitro experiments, which have shown that Sxl dependent repression of msl-2 mRNA translation is cap independent, this study found that eif4e does not function in Sxl mediated translational repression of at least one target mRNA in vivo. Instead, the results indicate that eif4e is needed for Sxl dependent alternative splicing, and it is argued that it is this splicing activity that accounts for the suppression of male lethality by eif4e mutations. In wild type females, Sxl protein blocks the splicing of a small intron in the 5' UTR of the msl-2 pre-mRNA. This is an important step in msl-2 regulation because the intron contains two Sxl binding sites that are needed by Sxl to efficiently repress translation of the processed msl-2 mRNA. When this intron is removed repression of msl-2 translation by Sxl is incomplete and this would enable eif4e/+ males to escape the lethal effects of the Sx-N transgene (Graham, 2011).

Several lines of evidence support the conclusion that eif4e is required for Sxl dependent alternative splicing. One comes from the analysis of the dominant maternal effect female lethal interactions between eif4e and Sxl. The initial activation of the Sxl positive autoregulatory feedback loop in early embryos can be compromised by a reduction in the activity of splicing factors like Snf, Fl(2)d, and U1-70K, and mutations in genes encoding these proteins often show dose sensitive maternal effect, female lethal interactions with Sxl. Like these splicing factors, maternal effect female lethal interactions with Sxl are observed for several eif4e alleles. Moreover, these female lethal interactions can be exacerbated when the mothers are trans-heterozygous for mutations in eif4e and the splicing factors snf or fl(2)d. Genetic and molecular experiments indicate that female lethality is due to a failure in the female specific splicing of Sxl-Pm mRNAs. First, female lethality can be rescued by gain-of-function Sxl mutations that are constitutively spliced in the female mode. Second, transcripts expressed from a Sxl-Pm splicing reporter in the female Sxl^-/+ progeny of eif4e/+ mothers are inappropriately spliced in a male pattern at the time when the Sxl positive autoregulatory loop is being activated by the Sxl-Pe proteins. While splicing defects are evident in these embryos at the blastoderm/early gastrula stage, obvious abnormalities in expression of Sxl protein are not observed until several hours later in development (Graham, 2011).

Though this difference in timing would favor the idea that eif4e is required for splicing of Sxl-Pm transcripts rather than for the export or translation of the processed Sxl-Pm mRNAs, the possibility cannot be excluded that there are subtle defects in the expression of Sxl protein at the blastoderm/early gastrula stage that are sufficient to disrupt splicing regulation during the critical activation phase yet aren't detectable in the antibody staining experiments. However, evidence from two different experimental paradigms using adult females indicates that this is likely not the case. In the first, it was found that reducing eif4e activity in a sensitized snf¹⁶²¹ Sxl^f1/++ background can compromise Sxl dependent alternative splicing even though there is no apparent reduction in Sxl protein accumulation. In this experiment advantage was taken of the fact that once the positive autoregulatory feedback loop is fully activated a homeostasis mechanism (in which Sxl negatively regulates the translation of Sxl-Pm mRNAs) ensures that Sxl protein is maintained at the same level even if there are fluctuations in the amount of female spliced mRNA. While only a small amount of male spliced Sxl-Pm mRNAs can be detected in snf¹⁶²¹ Sxl^f1/++ females, the level increases substantially when eif4e activity is reduced. Since these synergistic effects occur even though Sxl levels in the triply heterozygous mutant females are the same as in the control snf¹⁶²¹ Sxl^f1/++ females, it is concluded that the disruption in Sxl dependent alternative splicing of Sxl-Pm transcripts in this context (and presumably also in early embryos) can not be due to a requirement for eif4e in either the export of Sxl mRNAs or in their translation. Instead, eif4e activity must be needed specifically for Sxl dependent alternative splicing of Sxl-Pm pre-mRNAs. Consistent with a more general role in Sxl dependent alternative splicing, there is a substantial increase in msl-2 mRNAs lacking the first intron when eif4e activity is reduced in snf¹⁶²¹ Sxl^f1/++ females. In the second experiment the splicing was examined of pre-mRNAs from the endogenous Sxl gene and from a Sxl splicing reporter in females heterozygous for two hypomorphic eif4e alleles. Male spliced mRNAs from the endogenous gene and from the splicing reporter are detected the eif4e/+ females, but not in wild type females. Moreover, the effects on sex-specific alternative splicing seem to be specific for transcripts regulated by Sxl as no male spliced dsx mRNAs were seen in eif4e/+ females (Graham, 2011).

Two models could potentially explain why eif4e is needed for Sxl dependent alternative splicing. In the first, eif4e would be required for the translation of some critical and limiting splicing co-factor. When eif4e activity is reduced, insufficient quantities of this splicing factor would be produced and this, in turn, would compromise the fidelity of Sxl dependent alternative splicing. In the second, the critical splicing co-factor would be eif4e itself. It is not possible to conclusively test whether there is a dose sensitive requirement for eif4e in the synthesis of a limiting splicing co-factor. Besides the fact that the reduction in the level of this co-factor in flies heterozygous for hypomorphic eif4e alleles is likely to be rather small, only a subset of the Sxl co-factors have as yet been identified. For these reasons, the first model must remain a viable, but unlikely possibility. As for the second model, the involvement of a translation factor like eif4e in alternative splicing is unexpected if not unprecedented. For this to be a viable model, a direct role for eif4e must be consistent with what is known about the dynamics of Sxl pre-mRNA splicing and the functioning of the Sxl protein. The evidence that the second model is plausible is detailed below (Graham, 2011).

Critical to the second model is both the nuclear localization of eIF4E and an association with incompletely spliced Sxl pre-mRNAs. Nuclear eIF4E has been observed in other systems, and this was confirmed for Drosophila embryos. It was also found that eIF4E is bound to Sxl transcripts in which the regulated exon2-exon3-exon4 cassette has not yet been spliced. In contrast, it is not associated with incompletely processed transcripts from the tango gene that are constitutively spliced. With the caveat that only one negative control is available, it is not surprising that Sxl transcripts might be unusual in this respect. There is growing body of evidence that splicing of constitutively spliced introns is co-transcriptional. However, recent in vivo imaging experiments have shown that the splicing of the regulated Sxl exon2-exon3-exon4 cassette is delayed until after the Sxl transcript is released from the gene locus in female, but not in male cells. These in vivo imaging studies also show that, like bulk pre-mRNAs, the 1^st Sxl intron is spliced co-transcriptionally in both sexes. Consistent with a delay in the splicing of the regulated cassette, it has been previously reported that polyadenylated Sxl RNAs containing introns 2 and 3 can be readily detected by RNase protection, whereas other Sxl intron sequences are not observed. The delay in the splicing of the regulated Sxl cassette until after transcription is complete and the RNA polyadenylated could provide a window for exchanging eIF4E for the nuclear cap binding protein (Graham, 2011).

To function as an Sxl co-factor, eIF4E would have to be associated with the pre-mRNA-spliceosomal complex before or at the time of the Sxl dependent regulatory step. There is still a controversy as to exactly which step in the splicing pathway Sxl exerts its regulatory effects on Sxl-Pm pre-mRNAs and two very different scenarios have been suggested. The first is based on an in vitro analysis of Sxl-Pm splicing using a small hybrid substrate consisting of an Adenovirus 5' exon-intron fused to a short Sxl-Pm sequence spanning the male exon 3' splice site. These in vitro studies suggest that Sxl acts very late in the splicing pathway after the 1^st catalytic step, which is the formation of the lariat intermediate in the intron between exon 2 and the male exon. According to these experiments Sxl blocks the 2^nd catalytic step, the joining of the free exon 2 5' splice site (or Adeno 5' splice site) to the male exon 3' splice site. It is postulated that this forces the splicing machinery to skip the male exon altogether and instead join the free 5' splice site of exon 2 to the downstream 3' splice site of exon 4. Since this study has shown that eIF4E binds to Sxl-Pm pre-mRNAs that have not yet undergone the 1^st catalytic step, it would be in place to influence the splicing reaction if this scenario were correct (Graham, 2011).

The second scenario is more demanding in that it proposes that Sxl acts during the initial assembly of the spliceosome. Evidence for Sxl regulation early in the pathway comes from the finding that Sxl and the Sxl co-factor Fl(2)d show physical and genetic interactions with spliceosomal proteins like U1-70K, Snf, U2AF38 and U2AF50 that are present in the early E and A complexes and are important for selecting the 5' and 3' splice sites. In addition to these proteins, Sxl can also be specifically cross-linked in nuclear extracts to the U1 and U2 snRNAs. Formation of the E complex depends upon interactions of the U1 snRNP with the 5' splice site, and this is thought to be one of the first steps in splicing. The other end of the intron is recognized by U2AF, which recruits the U2 snRNP to the 3' splice site. After the base pairing of the U2 snRNP with the branch-point to generate the A complex the next step is the addition of the U4/U5/U6 snRNPs to form the B complex. However, Sxl and Fl(2)d are not found associated with components of the splicing apparatus like U5-40K, U5-116K or SKIP that are specific for complexes B and B*, or the catalytic C complex. Nor can Sxl be cross-linked to the U4, U5 or U6 snRNAs. If Sxl and Fl(2)d dissociated from the spliceosome before U4/U5/U6 are incorporated into the B complex, then they must influence splice site selection during the formation/functioning of the E and/or A complex. (Since the transition from the E to the A complex has been shown to coincide with an irreversible commitment to a specific 5'—3' splice site pairing, Sxl would likely exerts its effects in the E complex when splice site pairing interactions are known to still be dynamic. If this is scenario is correct, eIF4E would have to be associated with factors present in the earlier complexes in order to be able to promote Sxl regulation. This is the case. Thus, eIF4E is found in complexes containing the U1 snRNP protein U1-70K, the U1/U2 snRNP protein Snf, and the two U2AF proteins, U2AF38 and U2AF50. With the exception of the Snf protein bound to the U2 snRNP, all of these eIF4 associated factors are present in the early E or A complexes, but are displaced from the spliceosome together with the U1 and U4 snRNPs when the B complex is rearranged to form the activated B* complex. This would imply that eIF4E is already in place either before or at the time of B complex assembly. Arguing that eIF4E associates with these E/A components prior to the assembly of the B complex is the finding that eIF4E is also in complexes with both Sxl and Fl(2)d. Thus, even in this more demanding scenario for Sxl dependent splicing, eIF4E would be present at a time when it could directly impact the regulatory activities of Sxl and its co-factor Fl(2)d (Graham, 2011).

Taken together these observations would be consistent with a Sxl co-factor model. While further studies will be required to explain how eIF4E helps promote female specific processing, an intriguing possibility is suggested by the fact that hastening the nuclear export of msl-2 in females would favor the female splice (which is no splicing at all). Hence, one idea is that eIF4E binding to the pre-mRNA provides a mechanism for preventing the Sxl regulated splice sites from re-entering the splicing pathway, perhaps by constituting a 'signal' that blocks the assembly of new E/A complexes. A similar post-transcriptional mechanism could apply to female-specific splicing of the regulated Sxl exon2-exon3-exon4 cassette. The binding of eIF4E (and PABP) to incompletely processed Sxl transcripts after transcription has terminated in females would prevent the re-assembly of E/A complexes on the two male exon splice sites, and thus promote the formation of an A complex linking splicing factors assembled on the 5' splice sites of exons 2 and on the 3' splice site of exon 4 (Graham, 2011).

Sxl_-dependent, tra/tra2-independent alternative splicing of the Drosophila melanogaster X-Linked gene found in neurons

Somatic sexual determination and behavior in Drosophila melanogaster are under the control of a genetic cascade initiated by Sex lethal (Sxl). In the female soma, SXL RNA binding-protein regulates the splicing of transformer (tra) transcripts into a female-specific form. The RNA binding protein TRA and its cofactor TRA2 function in concert in females, whereas SXL, TRA and TRA2 are thought not to function in males. To better understand sex-specific regulation of gene expression, this study analyzed male and female head transcriptome datasets for expression levels and splicing, quantifying sex-biased gene expression via RNA-Seq and qPCR. The data uncouples the effects of Sxl and tra/tra2 in females in the sex-biased alternative splicing of head transcripts from the X-linked locus found in neurons (fne), encoding a pan-neuronal RNA-binding protein of the ELAV family. FNE protein levels are down regulated by Sxl in female heads, also independently of tra/tra2. It is argued that this regulation may have important sexually dimorphic consequences for the regulation of nervous system development or function (Sun, 2015).

The Drosophila sex determination hierarchy is the classical model of developmentally regulated alternative splicing. To identify genes expressed differentially in males and females, head samples were chosen, thereby eliminating large numbers of events restricted to gonadal differentiation. Moreover, the neurons, enriched in heads, are the site of extensive regulation at the level of alternative splicing (Sun, 2015).

In addition to dsx and fru, canonical regulators of Drosophila sex determination, we identified and further characterized the expression of fne and tango13 as genes expressed in a sex-biased manner. This study found that tango13 sex-specific expression responds to tra, tra2, and Sxl mutations in females as expected if under the control of the canonical sex determination pathway. An intriguing feature is the absence of reciprocity in the regulation of the mutually exclusive tango13-a and tango13-b splice forms, because tango13-a levels are reduced in tra, tra2, and Sxl mutants, but tango13-b levels are not. This observation suggests that tra and tra2 could possibly have an impact on the levels/stability of the tango13-a transcript rather than on the alternative splicing of tango13 RNA per se. The impact of tra/tra2 alleles on the expression of tango13-a is similar to that on the expression of dsx-F, consistent with regulation downstream of the sex determination pathway (Sun, 2015).

In contrast to fru, dsx, and tango13, the expression of fne is independent of TRA and TRA2. Crucially, fne splicing nevertheless depends on Sxl function in female heads: Sxl^- pseudomales switch to a male mode of fne alternative splicing, consistent with a role for SXL in promoting, directly or indirectly, the formation of the fne-b isoform at the expense of fne-a in normal females. Further, although fne alternative splicing is male-like in XX Sxl pseudomales, FNE protein levels are also upregulated two-fold to three-fold compared to CS males and females. Both male-like splicing and increased FNE protein levels in the pseudomales are reverted by the introduction of a Sxl⁺ minigene, confirming the specificity of Sxl in the control of both the splicing and protein levels. These data thus show that a Sxl-dependent, tra/tra2-independent mechanism regulates fne expression in females (Sun, 2015).

Canton-S (CS) female and male pools of fne RNA yield similar amounts of FNE protein in the two sexes. However, XX SxlM1,fΔ33 / Sxl^f7M1 pseudomales have a male-like pool of fne RNA and two-fold to three-fold increased FNE protein levels compared to CS. Because fne is an X-linked gene, its expression is presumably influenced by the canonical dosage compensation pathway, which could be responsible for the upregulation of FNE levels in XX Sxl_- pseudomales. However, according to the canonical model, higher fne transcript levels would be expected in pseudomales than in males and females, but that is not the case. Additional mechanisms must be at play (Sun, 2015).

First, increased FNE protein levels in XX Sxl_- pseudomales compared to wild-type males do not result from increased transcript levels. Because males and pseudomales share similar spliced pools of fne RNA, their distinct FNE outputs necessarily result from a regulatory mechanism that operates independently of the effects of Sxl on alternative splicing. Formally, this mechanism appears to stimulate the translation of fne transcripts in XX individuals. Second, increased FNE protein levels, concomitant with changes in alternative splicing but not associated with changes in transcript levels, as in XX Sxl_- pseudomales compared to wild-type females, are consistent with the existence of a Sxl_-dependent mechanism that downregulates FNE protein levels in XX females. Only the XX-dependent upregulation would persist in Sxl_- pseudomales, hence their increased FNE level. It is conceivable that fne regulation by Sxl occurs via direct binding of Sxl to fne transcripts. An interesting alternative as a means to regulate its splicing is the possibility that the impact of Sxl on fne expression occurs indirectly (possibly via an hormonal axis), since the extensive impact of the germline on the expression of somatic genes has been documented (Sun, 2015).

fne encodes an RNA-binding protein concentrated in the soma of neurons and present throughout development. It is necessary for the normal development of the mushroom bodies of males and females, and it is involved in the regulation of male courtship. It is intriguing that the expression of pan-neuronal fne is regulated in a sex-biased manner under the control of Sxl (Sun, 2015).

In addition to its role in the development of the germline, Sxl is involved in several regulatory pathways in the soma. It responds to a cell autonomous signal (number of X chromosomes) and is crucial both for the sexual development of somatic cells and for dosage compensation in males. SXL, but not TRA or TRA2, is also required independently of the somatic sex determination pathway for the development of a subset of sexually dimorphic neurons, with consequences on female ovulation. Additional phenotypes independent of the canonical somatic sex determination pathway but dependent on Sxl are the control of the sexually dimorphic body size of flies and the sex-specific bristle number on the A5 sternite. The latter occurs through general downregulation of the Notch pathway by SXL in multiple tissues . Thus, the Sxl_-regulated expression of fne fits within the context of Sxl acting in parallel with the canonical Sxl_-tra/tra2 cascade, constituting an example of its impact on tissues that do not show obvious sexual dimorphism (Sun, 2015).

fne is a member of a fairly new multigene family restricted to dipterans. The birth of this family predates the role of SXL in sex determination, which is restricted to the drosophilids. Based on RNA-Seq data and the Flybase models, sex-specific alternative splicing has not been reported for either of the other two paralogues in this family, elav (embryonic lethal abnormal visual system, X linked) or rbp9 (RNA binding protein 9, second chromosome). elav is the result of a retrotransposition and is likely to have acquired new cis-regulatory elements in the process. It autoregulates via a posttranscriptional mechanism involving its 3' UTR. It is unclear whether the Sxl_-dependent regulation of fne is an ancestral property that has been lost for rbp9 or was recently acquired. Nevertheless, sex-specific alternative splicing provides fne with the ability to be differentially regulated in females, which may have an important impact on sex-specific nervous system function or development, for which there are numerous instances of a role for Sxl. Within the context of the canonical sex determination pathway, Sxl regulates the expression of fru and dsx, two transcription factors crucial for behavior and nervous system function. SXL also controls, via an independent pathway, specific aspects of female behavior (Evans and Cline 2013). Still outside of the context of the canonical sex determination pathway, Sxl regulates the neurogenic locus Notch. Further, in Drosophila virilis, SXL protein accumulates in the male developing nervous system, consistent with a role there . Thus, the control exerted by Sxl on pan-neuronal fne outside of the context of the canonical sex determination pathway may be part of the heritage of an SXL ancestral function more focused on the nervous system than on sexual differentiation (Sun, 2015).

The sex of specific neurons controls female body growth in Drosophila

Sexual dimorphisms in body size are widespread throughout the animal kingdom but their underlying mechanisms are not well characterized. Most models for how sex chromosome genes specify size dimorphism have emphasized the importance of gonadal hormones and cell-autonomous influences in mammals versus strictly cell-autonomous mechanisms in Drosophila melanogaster. This study used tissue-specific genetics to investigate how sexual size dimorphism (SSD) is established in Drosophila. The larger body size characteristic of Drosophila females is established very early in larval development via an increase in the growth rate per unit of body mass. The female sex determination gene Sex-lethal (Sxl) was shown to function in central nervous system (CNS) neurons as part of a relay that specifies the early sex-specific growth trajectories of larval but not imaginal tissues. Neuronal Sxl acts additively in 2 neuronal subpopulations, one of which corresponds to 7 median neurosecretory cells: the insulin-producing cells (IPCs). Surprisingly, however, male-female differences in the production of insulin-like peptides (Ilps) from the IPCs do not appear to be involved in establishing SSD in early larvae, although they may play a later role. These findings support a relay model in which Sxl in neurons and Sxl in local tissues act together to specify the female-specific growth of the larval body. They also reveal that, even though the sex determination pathways in Drosophila and mammals are different, they both modulate body growth via a combination of tissue-autonomous and nonautonomous inputs (Sawala, 2017).

The sex of an organism has profound effects on its morphology, physiology, and behaviour. It also influences the risk of developing diseases of growth and metabolism such as obesity, metabolic syndrome, cardiovascular disease, and cancer. One important feature of sexual dimorphism is the difference in body size between males and females, called sexual size dimorphism (SSD). SSD is widespread and rapidly evolving across the animal kingdom such that, depending upon the species, the larger sex can either be male or female. It is not yet clear, however, which mechanisms link the chromosomal sex of an organism to specific male and female patterns of growth during development and thus ultimately to adult SSD (Sawala, 2017).

In mammals, the presence or absence of a single gene on the male Y chromosome (Sry) determines gonad differentiation into male testis or female ovary, respectively. The gonads then establish a sex-specific hormonal milieu that controls male and female differentiation, patterns of growth, and metabolism. However, recent studies in mice now demonstrate that the cellular (i.e., chromosomal) sex of nongonadal tissues can also influence growth and metabolism, an effect that is thought to be modified by the action of gonadal hormones. Hence, although many details remain to be explored, it is likely in mammals that patterns of growth relevant to SSD are controlled via both hormonal and cell-autonomous mechanisms (Sawala, 2017).

The fruit fly Drosophila melanogaster has provided insights into many aspects of sexual dimorphism. In Drosophila, sex determination is based on X chromosome dosage, with XX individuals developing as females and XY individuals as males. At the early embryonic stage, the presence of 2 X chromosomes in females activates the expression of sex determination gene Sex-lethal (Sxl), which is thereafter maintained via positive autoregulation. Sxl controls the splicing of multiple downstream targets regulating sexual differentiation as well as X chromosome dosage compensation. A commonly held view is that Drosophila, unlike mammals, deploys sex determination genes in a strictly cell-autonomous manner such that they are required in every somatic cell that is sexually dimorphic. Nevertheless, there are hints that non-cell-autonomous mechanisms regulate at least some aspects of sexual dimorphism. For example, in adult flies, ecdysone and juvenile hormone can act like sex hormones to regulate reproduction and sexual identity (Sawala, 2017).

SSD in Drosophila is present at larval, pupal, and adult stages, with females approximately 30% larger than males. Drosophila larvae are composed of 2 types of tissues: polyploid larval-specific organs that are histolysed during pupation and diploid imaginal tissues, which are the precursors of adult structures. Polyploid tissues comprise the bulk of the larva such that body SSD measured at larval stages reflects their growth rather than that of imaginal tissues. In contrast, body SSD measured at the adult stage reflects the larval/pupal growth of imaginal tissues. SSD appears to be present in most tissues and may be a result of differences in both cell size and number. Signalling pathways important for body growth, such as the insulin/insulin-like growth factor (IGF) and target of rapamycin (Tor) network, are likely to be relevant for SSD but precisely how sex modulates them remains unclear. In terms of sex determination genes, SSD was long thought to be dependent upon Sxl but not on one of its key downstream target genes, transformer (tra), which specifies other aspects of sexual dimorphism. This was challenged recently by a report that tra contributes to SSD, although there is also likely to be a tra-independent mechanism. The same study also reported that tra not only exerts autonomous effects on cell size but also acts in the female fat body to stimulate the secretion of insulin-like peptides (Ilps) from the brain, which in turn promotes larger body size in females (Sawala, 2017).

This study investigated the link between sex determination genes and SSD in Drosophila using cell type-specific genetic manipulations and size measurements of both larval and imaginal tissues. Surprisingly, it was found that Sxl controls SSD by acting in specific subsets of female neurons to increase body growth during larval development. At this stage, neuronal Sxl functions selectively to increase the growth rate of larval but not imaginal tissues. This study reveals that the sex of specific neurons regulates SSD in a non-cell-autonomous manner (Sawala, 2017).

Sex-specific body sizes are established after embryogenesis but early during larval development. SSD is manifested as a higher growth rate in female than male larvae, with a maximal difference occurring during L2 and early L3. Previous SSD studies focused later in development, after critical weight (CW), a key checkpoint in L3 that can influence final body size. The study by Testa (2013) showed that females are larger as they attain a higher CW and then a higher absolute growth rate during subsequent larval development (the terminal growth period). The data are consistent with this but also reveal that the establishment of different growth patterns in females and males involves an early divergence of mass-specific growth rates (mass gain per h per mg of body mass) in L2, well before CW. After SSD has been established in L2/early L3, it is subsequently maintained as larger female larvae gain more absolute mass per hour than smaller males, although both sexes now have very similar mass-specific growth rates (Sawala, 2017).

The insulin signalling pathway is a major stimulator of larval growth in Drosophila and it has been reported that IPC secretion of Ilp2 and InR signalling are both higher in females than in males at the L3 stage (Testa, 2013). However, at earlier stages relevant for the establishment of SSD, this study did not detect any male-female differences in Ilp2 secretion or in insulin signalling. Furthermore, larvae with decreased Ilp production from IPCs or larvae lacking all 3 of the Ilp genes normally expressed in the IPCs are smaller but still retain larval body SSD. Thus, insulin signalling is required for maximal larval growth in both males and females but it does not appear to be a sex-specific regulator of larval SSD. Interestingly, however, SSD of the developing wing disc is decreased by InR knockdown or decreased insulin production from IPCs. Thus, although insulin signalling may not contribute to the SSD of larval tissues, it does regulate that of imaginal tissues. Higher insulin signalling in female imaginal tissues could be driven by local Sxl/tra dependent modulation of insulin sensitivity and perhaps from mid/late L3 stages by increased Ilp production. Very recently, it was reported that another key growth regulator, Myc, is more highly expressed in female than in male larvae and so may contribute to SSD (Wehr Mathews, 2017). Sex differences in myc transcript expression may be the result of this X-linked gene escaping complete dosage compensation rather than via direct Sxl regulation. Interestingly, this study found that the higher expression of myc characteristic of females is fully maintained in elav^c155>Sxl RNAi larvae, even though they are masculinised in terms of larval growth. This indicates that a sex difference in global myc levels is not sufficient to confer SSD. It remains possible, however, that myc in neurons or in another tissue has a role in SSD, potentially downstream of neuronal Sxl (Sawala, 2017).

A central finding of this study is that Sxl is required in the CNS to direct the female-specific growth trajectory of the larval body. By mapping the site of action of Sxl in the CNS using a panel of Gal4 drivers, this study was able to show that it functions additively in 2 nonoverlapping populations of neurons within the CNS: Gad1-Gal4 neurons and IPCs. For the Gad1-Gal4 neurons, it remains to be determined whether GABA+ or GABA- subsets are relevant for SSD and whether or not they functionally interconnect with IPCs in the larval CNS. Nevertheless, it has been reported for the adult CNS that some GABAergic neurons converge on IPCs, which express the metabotropic GABAB receptor and may respond to GABA by decreasing Ilp secretion. Importantly, this study also mapped another critical site of action for Sxl to the IPCs, a cluster of only 7 peptidergic neurons. This finding, together with the evidence that sex-specific IPC secretion of Ilps is unlikely to establish SSD, suggests that one or more of the numerous other neuropeptides/secreted factors expressed in larval IPCs may be relevant (Sawala, 2017).

This study found that neuronal Sxl appears to regulate SSD largely independently of its best-characterised downstream targets, tra and msl-2. The lack of a major neuronal tra input in SSD makes it unlikely that there is a role for its neuronal target fruitless, which regulates many aspects of sex-specific behaviour in adults. In addition, sex-specific isoforms of Fruitless have not been detected until the late L3 stage, long after SSD has been established. New direct Sxl targets have been identified in recent years in several biological contexts and future approaches targeting the early larval nervous system may reveal how neuronal Sxl controls SSD. Importantly, whatever the Sxl targets in neurons relevant for SSD turn out to be, the current results clearly demonstrate that Sxl acts in a remote manner to regulate peripheral tissue growth and overall body size. Identification of the complete remote-control pathway is beyond the scope of this study but, in principle, it could involve the neural secretion of a hormone-like signal and/or innervation of peripheral organ(s) that regulate growth. (Sawala, 2017).

The sex determination gene transformer regulates male-female differences in Drosophila body size

A previous report implicated the Sxl target tra acting in the fat body as an important non-cell-autonomous regulator of female body size (Rideout, 2015). The current study, however, finds that the genetic requirement for Sxl in body size is stronger in neurons (likely acting without tra) than it is in the fat body (acting with tra). Not all of the conclusions made in the two studies are easily reconcilable, but it is noted that the previous report used body size readouts that were adult mass or estimated pupal volume, whereas this study measured larval body mass during L2 and L3. It nevertheless remains possible that Sxl can act in both neurons and fat body to regulate overall body SSD in a non-tissue-autonomous manner. Perhaps the relative contributions of each site of Sxl expression depend upon diet or other factors that varied between the 2 studies. Either way, the current finding that restoration of Sxl expression specifically in neurons is sufficient to increase the body size of Sxl mutant females demonstrates that the neuronal relay mechanism is functional without Sxl in the fat body (Rideout, 2017).

Evidence supporting a neuronal relay model for SSD in Drosophila is discussed. Central to this model is the finding that Sxl in neurons is required to relay a signal for female body size to peripheral larval tissues. Thus, loss of Sxl in neurons abolishes SSD by decreasing the larval body mass of females to that of males. Conversely, neuronal Sxl expression is sufficient for substantial rescue of female body size in Sxl mutant larvae. Neuronal Sxl knockdown decreases SSD in larvae at the level of individual polyploid larval tissues such as the fat body, but, surprisingly, this does not appear to be the case for the diploid wing imaginal disc. Nevertheless, after completion of pupal development, neuronal Sxl depletion does eventually lead to decreased SSD of the adult wings as well as the adult body. So, how is it possible to account for why the neuronal Sxl input affects imaginal tissue SSD at adult but not larval stages? One possible explanation is that neuronal Sxl functions during pupal stages to nonautonomously regulate imaginal tissue growth. Alternative explanations involve neuronal Sxl acting during larval stages to specify pupal resources/signals that themselves regulate the SSD of adult body structures. For example, nutrient resources laid down in larval tissues can be mobilized by the process of histolysis during the pupal period. Hence, the greater mass of the female larva may be needed to sustain the greater final size of the female adult. An important feature of this relay model for SSD is that the Sxl-dependant signal from neurons is integrated with local Sxl/tra inputs in both larval and imaginal tissues. Evidence for these tissue-autonomous Sxl/tra growth inputs comes from previous studies as well as the current study finding that Sxl and tra activities in the larval fat body and wing imaginal disc contribute towards the increased size characteristic of these female tissues. It was also found that Sxl and Tra were unable to increase fat body cell size in a cell-autonomous manner significantly in males or in females lacking neuronal Sxl. Hence, larval tissue-autonomous Sxl/TraF activity may only be able to boost growth efficiently in the presence of the Sxl-dependent signal from neurons. It is therefore proposed that Sxl activity in neurons and in local tissues acts together to maximize female tissue growth (Rideout, 2017).

A widely held textbook view is that somatic sexual dimorphism is regulated very differently in Drosophila and mammals: the former in a cell-autonomous manner, the latter by gonad-derived hormones. However, the discovery that the brain remotely regulates SSD in Drosophila may have its counterpart in mammals. Thus, mammalian gonadal sex steroids are thought to act on neurons in the hypothalamus to regulate growth hormone secretion from the anterior pituitary gland. Male-female differences in the levels and patterns of circulating growth hormone are thought to induce sex-specific insulin-like growth factor 1 (IGF-1) profiles, in turn conferring dimorphic growth patterns. Growth hormone itself does not appear to be conserved in Drosophila, but it is interesting that the pars intercerebralis of the Drosophila brain, harbouring the IPCs, has been likened to the mammalian hypothalamus. Furthermore, IPCs send projections to the ring gland, which has been likened to the pituitary gland in mammals. In a second parallel, the current findings together with several studies in mammals suggest that primary sex determination signals act via a combination of cell-autonomous and non-cell-autonomous mechanisms to control overall SSD. For example, in human and other mammalian embryos, sex differences in body size are noticeable before gonad differentiation, suggesting the existence of gonadal sex hormone-independent mechanisms of SSD. Moreover, an XX rather than an XY chromosome complement in mice can increase adult body mass by approximately 7%, independently of gonadal sex, and this difference can be strongly enhanced by gonadectomy. In conclusion, the upstream sex determination pathways in Drosophila and mammals are very different but the regulatory logic of how they regulate body growth via both cell-autonomous and remote mechanisms appears to be more similar than previously thought (Rideout, 2017).

Tudor-domain containing protein 5-like promotes male sexual identity in the Drosophila germline and is repressed in females by Sex lethal

For sexually reproducing organisms, production of male or female gametes depends on specifying the correct sexual identity in the germline. In D. melanogaster, Sex lethal (Sxl) is the key gene that controls sex determination in both the soma and the germline, but how it does so in the germline is unknown, other than that it is different than in the soma. An RNA expression profiling experiment was conducted to identify direct and indirect germline targets of Sxl specifically in the undifferentiated germline. In these cells, Sxl loss does not lead to a global masculinization observed at the whole-genome level. In contrast, Sxl appears to affect a discrete set of genes required in the male germline, such as Phf7. Tudor domain containing protein 5-like (Tdrd5l) was identified as a target for Sxl regulation that is important for male germline identity. Tdrd5l is repressed by Sxl in female germ cells, but is highly expressed in male germ cells where it promotes proper male fertility and germline differentiation. Additionally, Tdrd5l localizes to cytoplasmic granules with some characteristics of RNA Processing (P-) Bodies, suggesting that it promotes male identity in the germline by regulating post-transcriptional gene expression (Primus, 2019).

Sex determination is an essential process in sexually reproducing species, as the production of eggs and sperm depends on the sexual identity of the germ cells and somatic cells of the gonad. In some animals, such as the medaka fish and the house fly, the sexual identity of the soma determines the sexual identity of the germline. But in other animals, such as fruit flies and mammals, the intrinsic sex chromosome constitution (XX vs. XY) of the germ cells is also important for proper gametogenesis. In such cases, the 'sex' of the germ cells must match the 'sex' of the soma in order for proper gametogenesis to occur. While studies have revealed a great deal about how sex is determined in the soma, how germline sexual identity is determined by a combination of somatic signals and germline autonomous properties is much less well understood (Primus, 2019).

In Drosophila, somatic sexual identity is determined by the X chromosome number, with two X's activating expression of the key sex determination gene Sex lethal (Sxl), promoting female identity. The Sxl RNA-binding protein initiates an alternative RNA splicing cascade to allow female-specific splicing of transformer (tra) and, subsequently, doublesex (dsx) and fruitless (fru). dsx and fru encode transcription factors that control somatic sexual identity. Sxl is also the key gene controlling autonomous sex determination in the germline, as Sxl is expressed in the germline in females, and loss of Sxl causes female (XX) germ cells to develop as germline ovarian tumors, similar to male (XY) germ cells transplanted into a female soma. Further, expression of Sxl is sufficient to allow XY germ cells to make eggs when transplanted into a female soma. However, how Sxl is activated in the female germline and how it regulates female germline identity remain unknown, except for the fact that both are different than in the soma. To understand how Sxl promotes female germ cell identity, it is essential to discover its targets in the germline (Primus, 2019).

This work reports an RNA expression profiling (RNA-seq) experiment conducted to identify genes regulated downstream of Sxl in the germline. A previously uncharacterized tudor domain containing protein, Tudor domain protein 5-like (Tdrd5l), was found to be a target of Sxl in the germline. Tdrd5l is strongly expressed in the Drosophila early male germline and is repressed by Sxl activity in the early female germline. It promotes male identity in the germline, and its loss results in germline maintenance and differentiation defects in males, thus reducing their fertility. Tdrd5l protein localizes to cytoplasmic granules related to RNA Processing (P-) Bodies, suggestive of a function in post-transcriptional regulation of gene expression (Primus, 2019).

It has been known for many years that Sxl is necessary for female germline identity, and Sxl has also been shown to be sufficient to allow XY germ cells to undergo oogenesis. It is likely that Sxl plays multiple roles in the germline, both to promote female identity in the early germline, perhaps as early as in the embryonic germline, and in regulating the differentiation of the germline during oogenesis. This study examined the role of Sxl more specifically in the undifferentiated germline through the use of bam mutants. Principle component analysis indicated that, under these conditions, samples with Sxl function reduced in the germline clustered close to control female samples, and far from male samples. While some of the male/female differences may be contributed by the somatic cells present in these samples, it is concluded that reducing Sxl function in the undifferentiated germline does not lead to a dramatic masculinization at the whole-genome level. In contrast, it is proposed that the role for Sxl in the early germline may be restricted to a relatively small number of changes in sex-specific germline gene expression that are important for female vs. male germline function (Primus, 2019).

Recently, a genomic analysis of ovaries mutant for the RNA splicing factor sans fille (snf) was conducted. This is considered to also be a Sxl germline loss-of-function condition as one important change in snf-mutant ovaries is a loss of Sxl expression and an ovarian tumor phenotype that can be rescued by Sxl expression. In contrast to the current results, an increased expression of spermatogenesis genes was observed in snf tumorous ovaries compared to wild type ovaries. It is likely that changes in these 'differentiation' genes were not observed in bam-mutant samples since germline differentiation is arrested at an earlier stage in bam mutants, allowing this study to focus on the undifferentiated germline. Thus, these two analyses can help separate the role of Sxl in regulating early germline sexual identity vs. later aspects of sex-specific germline differentiation. Interestingly, one 'differentiation' gene was identified in both RNA-seq analyses: the testis-specific basal transcription factor TATA Protein Associated Factor 12L (Taf12L or rye). This may indicate that Taf12L could play a role in the undifferentiated germline as well as the later stages of spermatocyte differentiation. In addition, both analyses found evidence for differential regulation of the important male germline identity factor Phf7 (Yang, 2012), where an upstream promoter is utilized preferentially in the male germline and is repressed downstream of Sxl in females. This indicates a role for Phf7 in both the early and differentiating germline. Finally, strong candidates were not observed for targets of alternative RNA splicing regulated by Sxl. The only strong candidate for alternative RNA splicing was the Sxl RNA itself, where the male-specific exon was retained in the residual Sxl RNA from the Sxl RNAi samples. This provides further evidence that Sxl autoregulation occurs in the germline as it does in the soma, as has previously been proposed. It is likely that Sxl may also act at the level of translational control in the germline, as evidence indicates in this study for regulation of Tdrd5l. Future experiments to identify Sxl-associated germline RNAs will be important for investigating this mechanism of action, as has recently been conducted (Primus, 2019).

In addition to its role in sex determination in the soma, Sxl also acts to initiate global X chromosome gene regulation and dosage compensation through translational control of male-specific lethal-2, and it is possible that Sxl plays a similar role in the germline. Whether or not the germline even undergoes dosage compensation is controversial, and thoughtful work has led to opposite conclusions. Further, if dosage compensation does exist in the germline, it must utilize a separate mechanism from the soma, as the somatic dosage compensation complex members msl1 and msl2 are not required in the germline. However, Sxl could retain an msl-independent role to regulate global X chromosome gene expression in the germline. No evidence was observed for this. First, few X chromosome genes were differentially expressed between Sxl- and control samples (30 genes or 1.2% of X chromosome genes tested). Second, the ratio of average gene expression between X chromsome genes and autosomes was very similar in Sxl- females compared to control females (X/A for controls: 1.24, Sxl-: 1.19) and this was similar when considering only genes in particular expression categories (e.g., all genes with some expression in both samples). Thus, it appears likely that Sxl's role in the germline is distinct from that in the soma; it acts to control sex-specific gene regulation and sexual identity in both the germline and the soma, but acts as a general regulator of X chromosome gene expression and dosage compensation only in the soma (Primus, 2019).

Tdrd5l is both a target for Sxl regulation and is important for male germline identity and spermatogenesis. Tdrd5l expression is highly male-biased, both at the RNA and protein levels. When female germ cells are sensitized by partial loss of female sex determination genes, expression of Tdrd5l exacerbates the masculinized phenotype in these germ cells. Significantly, expression of Tdrd5l is sufficient to promote spermatogenesis in XX germ cells present in a male soma (XX tra-mutant testes). Thus, Tdrd5l clearly has a role in promoting male identity autonomously in the germline, which must coordinate with non-autonomous influences from the soma for proper germline sex determination (Primus, 2019).

Loss of Tdrd5l also has a strong effect on male fecundity, even though it is not absolutely required for spermatogenesis. The 50% reduction in fecundity is a strong effect and indicates that Tdrd5l plays an important role in the male germline. However, the fact that some spermatogenesis still proceeds suggests that other factors act in combination with Tdrd5l to control this process. One good candidate is Phf7 which was previously demonstrated to have a similarly important, but not absolute, requirement for spermatogenesis. However, analyses of Tdrd5l, Phf7 double mutants did not reveal any synergistic effect on male germline development. This suggests that other important players in promoting male germline identity remain to be identified (Primus, 2019).

The data indicate that Tdrd5l regulates male germline identity by influencing post-transcriptional gene regulation. Other tudor-domain containing proteins have been shown to act in RNA-protein bodies to influence RNA stability and translational regulation. Further, Tdrd5l localizes to cytoplasmic punctae, specifically a subset of the punctae that also contain Decapping Protein1 (DCP-1), suggesting that these bodies are related to Processing bodes (P-bodies) that are known to control post-transcriptional gene regulation. Interestingly, Tdrd5l's closest homologs, Tejas in flies and TDRD5 in mice, have been shown to regulate piRNA production and transposon regulation. Further, their localization to VASA-containing nuage is thought to influence transposon control. However, no changes were observed in transposon expression regulated by Tdrd5l, and Tdrd5l does not co-localize with VASA in nuage. No genetic interaction was observed between tejas and Tdrd5l. Thus, it is proposed that Tdrd5l plays a distinct role in regulating male germline identity and spermatogenesis, and this may be in the regulation of mRNAs rather than transposons. One possibility is that the role of mouse TDRD5 in transposon regulation and spermatogenesis has been separated into the roles of Tejas in transposon regulation and Tdrd5l in regulating male identity and spermatogenesis in flies. It is widely known that regulation of germline identity is dependent on post-transcriptional mechanisms involving tudor-domain proteins such as the original Tudor protein, which helps define the germ plasm, an RNA body that regulates germline identity. Further, the regulation of sex-specific gametogenesis is also dependent on RNA bodies and their requisite tudor-domain proteins, such as TDRD5 in mouse. These studies indicate that initial germline sexual identity is similarly regulated by post-transcriptional mechanisms, including RNA bodies containing other, distinct, TUDOR-domain proteins such as Tdrd5l (Primus, 2019).

Antagonism between germ cell-less and Torso receptor regulates transcriptional quiescence underlying germline/soma distinction

Transcriptional quiescence, an evolutionarily conserved trait, distinguishes the embryonic primordial germ cells (PGCs) from their somatic neighbors. In Drosophila melanogaster, PGCs from embryos maternally compromised for germ cell-less (gcl) misexpress somatic genes, possibly resulting in PGC loss. Recent studies documented a requirement for Gcl during proteolytic degradation of the terminal patterning determinant, Torso receptor. This study demonstrates that the somatic determinant of female fate, Sex-lethal (Sxl), is a biologically relevant transcriptional target of Gcl. Underscoring the significance of transcriptional silencing mediated by Gcl, ectopic expression of a degradation-resistant form of Torso (torso(Deg)) can activate Sxl transcription in PGCs, whereas simultaneous loss of torso-like (tsl) reinstates the quiescent status of gcl PGCs. Intriguingly, like gcl mutants, embryos derived from mothers expressing torso(Deg) in the germline display aberrant spreading of pole plasm RNAs, suggesting that mutual antagonism between Gcl and Torso ensures the controlled release of germ-plasm underlying the germline/soma distinction (Colonnetta, 2021).

Following fertilization, a Drosophila embryo undergoes 14 consecutive nuclear divisions to give rise to the cellular blastoderm. While the initial nuclear divisions take place in the center of the embryo, the nuclei begin to migrate toward the periphery around nuclear cycle (NC) 4-6 and reach the cortex at NC9/10. Even before bulk nuclear migration commences, a few nuclei move toward the posterior of the embryo, enter a specialized, maternally derived cytoplasm known as the pole plasm, and induce the formation of pole buds (PBs). The centrosomes associated with these nuclei trigger the release of pole plasm constituents from the posterior cortex and orchestrate precocious cellularization to form the primordial germ cells (PGCs), the progenitors of the germline stem cells in adult gonads. Unlike pole cell nuclei, somatic nuclei continue synchronous divisions after they reach the surface of the embryo until NC 14 when they cellularize (Colonnetta, 2021).

The timing of cellularization is not the only difference between the soma and PGCs. Although newly formed PGCs divide after they are formed, they undergo only one or two asynchronous divisions before exiting the cell cycle. Another key difference is in transcriptional activity. Transcription commences in the embryo during NC 6-7 when a select number of genes are active. Transcription is more globally upregulated when the nuclei reach the surface, and by the end of NC 14, zygotic genome activation (ZGA) is complete. This transition is marked by high levels of phosphorylation of residues Serine 5 (Ser5) and Serine 2 (Ser2) in the C-terminal domain (CTD) of RNA polymerase II. By contrast, in newly formed PGCs, transcription is switched off, and PGC nuclei have only residual amounts of Ser5 and Ser2 CTD phosphorylation. Moreover, and consistent with their transcriptionally quiescent status, other changes in chromatin architecture that accompany ZGA are also blocked in PGCs (Colonnetta, 2021).

Three different genes, nanos (nos), polar granule component (pgc), and germ cell-less (gcl), are known to be required for establishing transcriptional quiescence in newly formed PGCs. The PGCs in embryos derived from mothers carrying mutations in these genes fail to inhibit transcription, and this compromises germ cell specification and disrupts germ cell migration. (As these are maternal effect genes, embryos derived from nos/pgc/gcl mothers display the resulting mutant phenotypes and will be referred to as nos/pgc/gcl here onwards.) Interestingly, these three genes share only a few targets, suggesting overlapping yet distinct mechanisms of action. Nos is a translation factor and thus must block transcription indirectly. Together with the RNA-binding protein Pumilio (Pum), Nos interacts with recognition sequences in the 3'-untranslated regions (3'UTRs) of mRNAs and inhibits their translation. Currently, the key mRNA target(s) that Nos-Pum repress to block transcription is unknown; however, in nos and pum mutants, PGC nuclei display high levels of Ser5 and Ser2 CTD phosphorylation and activate transcription of gap and pair-rule patterning genes and the sex determination gene Sex-lethal (Sxl). pgc encodes a nuclear protein that binds to the transcriptional elongation kinase p-TEFb, blocking Ser5 CTD phosphorylation. In pgc mutant pole cells, Ser5 phosphorylation is enhanced, as is transcription of several somatic genes, including genes involved in terminal patterning (Colonnetta, 2021).

While the primary function of nos and pgc appears to be blocking ZGA in PGCs, gcl has an earlier function, which is to turn off transcription of genes activated in somatic nuclei prior to nuclear migration. Targets of gcl include two X-chromosome counting elements (XCEs), scute (sc/sis-b) and sisterless-a (sis-a), that function to turn on the sex determination gene, Sxl, in female soma. gcl embryos not only fail to shut off sis-a and sis-b transcription in PBs, but also show disrupted PGC formation. In some gcl embryos, PGC formation fails completely, while in other embryos only a few PGCs are formed. In this respect, gcl differs from nos and pgc, which have no effect on the process of PGC formation, but instead interfere with the specification of PGC identity (Colonnetta, 2021).

Studies by Leatherman (2002) suggested that the defects in PGC formation in gcl mutant embryos are linked to failing to inhibit somatic transcription. That study found that when PBs first form during NC 9 in wild-type (WT) embryos, levels of CTD phosphorylation PB are only marginally less than in nuclei elsewhere in the embryo. However, by NC 10, there was a dramatic reduction in CTD phosphorylation even before PBs cellularize. By contrast, in gcl mutant embryos, about 90% of the NC 10 PB nuclei had CTD phosphorylation levels approaching that of somatic nuclei. Moreover, this number showed an inverse correlation with the number of PGCs in blastoderm stage gcl embryos. Whereas WT blastoderm embryos have >20 PGCs per embryo, gcl embryos had on average just three PGCs under their culturing conditions. Interestingly, expression of the mouse homologue of Gcl protein, mGcl-1, can rescue the gcl phenotype in Drosophila (Leatherman, 2000). Supporting the conserved nature of the involvement of Gcl during transcriptional suppression, a protein complex between mGcl-1 and the inner nuclear membrane protein LAP2β is thought to sequester E2F:D1 to reduce transcriptional activity of E2F:D1 (Colonnetta, 2021).

The connection Leatherman postulated between failing to turn off ongoing transcription and defects in PGC formation in gcl mutants is controversial and unresolved. This model predicts that a non-specific inhibition of polymerase II should be sufficient to rescue PGC formation in gcl embryos. However, the PGC formation defects seen in gcl embryos are not rescued after injection of the RNA polymerase inhibitor, α-amanitin. Since α-amanitin treatment disrupted somatic cellularization without impacting PGC formation in WT embryos, it was concluded that it effectively blocked polymerase transcription. On the other hand, subsequent experiments by Pae (2017) raised the possibility that inhibiting transcription in pole cell nuclei is a critical step in PGC formation. The Pae paper showed that Gcl is a substrate-specific adaptor for a Cullin3-RING ubiquitin ligase that targets the terminal pathway receptor tyrosine kinase, Torso, for degradation. The degradation of Torso would be expected to prevent activation of the terminal signaling cascade in PGCs. In the soma, Torso-dependent signaling activates the transcription of several patterning genes, including tailless, that are important for forming terminal structures at the anterior and posterior of the embryo. Thus, by targeting Torso for degradation, Gcl would prevent the transcriptional activation of terminal pathway genes by the MAPK/ERK kinase cascade in PGCs. Consistent with this possibility, simultaneous removal of gcl and either the Torso ligand modifier, torso-like (tsl) or torso resulted in rescue of germ cell loss induced by gcl. Surprisingly, however, Pae (2017) was unable to observe a similar rescue of gcl phenotype when they used RNAi knockdown to compromise components of the MAP kinase cascade known to act downstream of the Torso receptor. Based on these findings, they proposed that activated Torso must inhibit PGC formation via a distinct non-canonical mechanism that is both independent of the standard signal transduction pathway and does not involve transcriptional activation (Colonnetta, 2021).

The current study has revisited these conflicting claims by examining the role of Gcl in establishment/maintenance of transcriptional quiescence. The studies of Leatherman (2002) indicated that two of the key X chromosomal counting elements, sis-a and sis-b, were inappropriately expressed in gcl PBs and PGCs. Since transcription factors encoded by these two genes function to activate the Sxl establishment promoter, Sxl-Pe, in somatic nuclei of female embryos, their findings raised the possibility that Sxl might be ectopically expressed in PBs/PGCs of gcl embryos. This study shows that in gcl embryos, Sxl transcription is indeed inappropriately activated in PBs and newly formed PGCs. Moreover, ectopic expression of Sxl in early embryos disrupts PGC formation similar to gcl. Supporting the conclusion that Sxl is a biologically relevant transcriptional target of Gcl, PGC formation defects in gcl embryos can be suppressed either by knocking down Sxl expression using RNAi or by loss-of-function mutations. As reported by Pae (2017), this study found that loss of torso-like (tsl) in gcl embryos suppresses PGC formation defects. However, consistent with a mechanism that is tied to transcriptional misregulation, rescue is accompanied by the reestablishment of transcriptional silencing in gcl PGCs. Lending further credence to the idea that transcription misregulation plays an important role in disrupting PGC development in gcl embryos, this study found that expression of a mutant form of Torso that is resistant to Gcl-dependent degradation (hereafter referred to as torsoDeg: Pae, 2017) ectopically activates transcription of two Gcl targets, sis-b and Sxl, in PB and PGC nuclei. In addition, stabilization of Torso in early PGCs also mimics another gcl phenotype, the failure to properly sequester key PGC determinants in PBs and newly formed PGCs (Colonnetta, 2021).

gcl differs from other known maternally deposited germline determinants in that it is required for the formation of PBs and PGCs. gcl PGCs exhibit a variety of defects during the earliest steps in PGC development. Unlike WT, gcl PGCs fail to properly establish transcriptional quiescence. While other genes like nos and pgc are required to keep transcription shut down in PGCs, their functions only come into play after PGC cellularization. By contrast, gcl acts at an earlier stage beginning shortly after nuclei first migrate into the posterior pole plasm and initiate PB formation. In gcl PBs, ongoing transcription of genes that are active beginning around nuclear cycle 5-6 is not properly turned off. This is not the only defect in germline formation and specification. As in WT, the incoming nuclei (and the centrosomes associated with the nuclei) trigger the release of the pole plasm from the posterior cortex. However, instead of sequestering the germline determinants in PBs so that they are incorporated into PGCs during cellularization, the determinants disperse into the soma where they become associated with the cytoplasmic territories of nearby somatic nuclei. There are also defects in bud formation and cellularization. Like the release and sequestration of germline determinants, these defects have been linked to the actin cytoskeleton and centrosomes (Colonnetta, 2021).

Two models have been proposed to account for the PGC defects in gcl mutants. In the first, Leatherman (2002) attributed the disruptions in PGC development to a failure to turn off ongoing transcription. The second argues that the role of gcl in imposing transcriptional quiescence is irrelevant. Instead, the defects are proposed to arise from a failure to degrade the Torso receptor. In the absence of Gcl-dependent proteolysis, high local concentrations of the Tsl ligand modifier at the posterior pole would activate the Torso receptor. According to this model, the ligand-receptor interaction would then trigger a novel, transcription-independent signal transduction pathway in PBs and PGCs that disrupts their development. These conflicting models raise several questions. Does gcl actually have a role in establishing transcriptional quiescence in PBs and PGCs? If so, is this activity relevant for PB and PGC formation? Is the stabilization of Torso in gcl mutants responsible for the failure to shut down transcription in PBs and PGCs? If not, does gcl target a novel, transcription-independent but Torso-dependent signaling pathway? Is the stabilization of Torso responsible for some of the other phenotypes that are observed in gcl mutants? These studies have addressed these outstanding questions, leading to a resolved model of Gcl activity and function (Colonnetta, 2021).

Shutting off transcription is, in fact, a critical function of Gcl protein. As previously documented by Leatherman, this study found that several of the key X-linked transcriptional activators of Sxl-Pe are not repressed in newly formed PBs and early PGC nuclei, and Sxl-Pe transcription is inappropriately activated in the presumptive germline. Previous studies found that ectopic expression of Sxl in nos mutants disrupts PGC specification. In this case, the specification defects in nos embryos can be partially rescued by eliminating Sxl activity. The same is true for gcl mutants: elimination or reduction in Sxl function ameliorates the gcl defects in PGC formation/specification. Conversely ectopic expression of Sxl early in embryogenesis mimics the effects of gcl loss on PGC formation. Importantly, the role of Gcl in inhibiting Sxl-Pe transcription is not dependent upon other constituents of the pole plasm. When Gcl is ectopically expressed at the anterior of the embryo, it can repress Sxl. This observation is consistent with the effects of ectopic Gcl on the transcription of other genes reported by Leatherman et al., 2002. Since the rescue of gcl by eliminating the Sxl gene or reducing its activity is not complete, one would expect that there must be other important gcl targets. These targets could correspond to one or more of the other genes that are misexpressed in gcl PB/PGCs. Consistent with this possibility, transcriptional silencing in gcl PBs/PGCs is reestablished when terminal signaling is disrupted by mutations in the tsl gene. On the other hand, it is possible that excessive activity of the terminal signaling pathway also adversely impacts some non-transcriptional targets that are important for PB/PGC formation and that transcriptional silencing in only part of the story (see below) (Colonnetta, 2021).

Pae (2017) showed that mutations in the Gcl interaction domain of Torso (torsoDeg) stabilize the receptor and disrupt PGC formation. Consistent with the notion that Torso receptor is the primary, if not the only, direct target of gcl, they found that mutations in the Torso ligand modifier, tsl, or RNAi knockdown of torso rescued the PGC formation defects in gcl embryos. As would be predicted from those and the current findings, ectopic expression of the TorsoDeg protein induces the inappropriate transcription of sis-b and Sxl-Pe in PBs and newly formed PGCs. Thus, the failure to shut down ongoing transcription in gcl PBs and PGCs must be due (at least in part) to the persistence of the Torso receptor in the absence of Gcl-mediated degradation. Corroborating this idea, the ectopic activation of transcription in gcl PGCs is no longer observed when the terminal signaling pathway is disrupted by the removal of tsl. Taken together, these data strongly suggest that the establishment/maintenance of transcriptional silencing in PBs is a critical function of Gcl (Colonnetta, 2021).

Since RNAi knockdowns of terminal pathway kinases downstream of torso did not rescue gcl mutants, Pae (2017) postulated that the Tsl-Torso receptor interaction triggered a novel, non-canonical signal transduction pathway that disrupted PGC development. If that suggestion is correct, then the activation of sis-b and Sxl-Pe in PBs/PGCs in gcl and torsoDeg embryos would be mediated by this novel terminal signaling pathway. The results of the current study are ambiguous. Consistent with the suggestion of Pae, 2017, GOF mutations in MEK, a downstream kinase in the Torso signaling pathway, did not activate Sxl-Pe transcription in pole cells. However, an important caveat is that the GOF activity of MEK variants that was tested is likely not equivalent to the activity from the normal Torso-dependent signaling cascade. As the pole plasm contains at least two other factors that help impose transcriptional quiescence, the two GOF MEK mutants that were tested may simply not be sufficient to overcome their repressive functions. Two observations are consistent with this possibility. First, like torsoDeg, this study found that MEK^E203K induces Sxl-Pe expression in male somatic nuclei. The same is true for a viable GOF mutation in Torso: it can induce ectopic activation of Sxl-Pe in male somatic nuclei, but is unable to activate Sxl-Pe in PGCs. Second, a key terminal pathway transcription target tailless is not expressed in gcl mutant PBs/PGSs even though the terminal pathway should be fully active. This is also true for embryos expressing torsoDeg and the two GOF MEK proteins. For these reasons, it cannot be unambiguously determined if it is the canonical terminal signaling pathway or another, noncanonical signaling pathway downstream of Torso that is responsible for the expression of sis-b, Sxl-Pe, and other genes in gcl mutant PB/PGCs (Colonnetta, 2021).

There are also reasons to think that the canonical Torso signal transduction cascade must be inhibited for proper PGC formation. One of the more striking phenotypes in gcl mutants is the dispersal of key germline mRNA and protein determinants into the surrounding soma. A similar disruption in the sequestration of pole plasm components is observed not only in torsoDeg embryos but also in MEK^E203K and MEK^F53S embryos. Thus, this gcl phenotype would appear to arise from the deployment of the canonical Torso receptor signal transduction cascade, at least up to the MEK kinase. However, this result does not exclude the possibility that the Tsl->Torso->ERK pathway has other non-transcriptional targets that, like Sxl-Pe expression, can also interfere with PB/PGC formation. If this was the case, it could potentially explain why global transcriptional inhibition failed to rescue the PGC defects in gcl embryos. In this respect, a potential-if not likely-target is the microtubule cytoskeleton. In previous studies, it was found that the PB and PGC formation defects as well as the failure to properly sequester critical germline determinants in gcl arise from abnormalities in microtubule/centrosome organization. Preliminary imaging experiments indicate that centrosome distribution of torsoDeg PBs is also abnormal, suggesting that inappropriate activation of the terminal signaling pathway perturbs the organization or functioning of the microtubule cytoskeleton and/or centrosomes. Such a mechanism would also be consistent with the dispersal of germline mRNA and protein determinants in torsoDeg and GOF MEK embryos. While further experiments will be required to demonstrate microtubule and centrosomal aberrations in torsoDeg and GOF MEK embryos, a role for a receptor-dependent MEK/ERK signaling cascade in promoting centrosome accumulation of γ-tubulin and microtubule nucleation has been documented in mammalian tissue culture cells. It is thus conceivable that MEK/ERK signaling has a similar role in Drosophila PB nuclei and PGCs. It will be important to determine if Torso-dependent activation of MEK/ERK can perturb the behavior or organization of centrosomes and/or microtubules in early embryos, and, if so, whether the influence can alter the pole plasm RNA anchoring and/or transmission. Taken together, the current data reveal a mutual antagonism between the determinants that specify germline versus somatic identity. Future studies will focus on how and when during early embryogenesis such feedback mechanisms are activated and calibrated to establish and/or maintain germline/soma distinction (Colonnetta, 2021).

Role of Hakai in m⁶A modification pathway in Drosophila

N6-methyladenosine (m⁶A), the most abundant internal modification in eukaryotic mRNA, is installed by a multi-component writer complex; however, the exact roles of each component remain poorly understood. This study shows that a potential E3 ubiquitin ligase Hakai colocalizes and interacts with other m⁶A writer components, and Hakai mutants exhibit typical m⁶A pathway defects in Drosophila, such as lowered m⁶A levels in mRNA, aberrant Sxl alternative splicing, wing and behavior defects. Hakai, Vir, Fl(2)d and Flacc form a stable complex, and disruption of either Hakai, Vir or Fl(2)d led to the degradation of the other three components. Furthermore, MeRIP-seq indicates that the effective m⁶A modification is mostly distributed in 5' UTRs in Drosophila, in contrast to the mammalian system. Interestingly, it was demonstrated that m⁶A modification is deposited onto the Sxl mRNA in a sex-specific fashion, which depends on the m⁶A writer. Together, this work not only advances the understanding of mechanism and regulation of the m⁶A writer complex, but also provides insights into how Sxl cooperate with the m⁶A pathway to control its own splicing (Wang, 2021).

There are a variety of chemical modifications on biological macromolecules, such as proteins, nucleic acids, and glycolipids. Like DNA methylation and histone modification, RNA modification represents an extra layer of epigenetic regulatory mechanism. More than 150 chemical modifications in RNA have been discovered, and their biological functions are only starting to be revealed. Chemical modifications of RNA exist in all organisms and for all forms of RNA, including tRNA, rRNA, mRNA, and long noncoding RNA. Common RNA modifications include N6-methyladenosine (m⁶A), N6,2’-O-dimethyladenosine (m⁶A_m), N1-methyladenosine (m¹A), 5-methylcytidine (m⁵C), N4-acetylcytidine (ac⁴C), 7-methylguanosine (m⁷G), and pseudouridine (Ψ). Among them, m⁶A is the most abundant internal modification of mRNA in eukaryotes. Although m⁶A in mRNA was found more than 40 years ago, it was only recently that the field has made extensive progress owing to technological and experimental breakthroughs. By combining m⁶A-specific antibody and high-throughput sequencing, MeRIP-Seq or m⁶A-Seq allows the m⁶A mapping at the whole transcriptome level, thereby providing the possibility to correlate RNA modifications with their biological functions . These and subsequent studies revealed that m⁶A sites contain a consensus motif RRACH (R = G/A; H = U/A/C), and m⁶A peaks are enriched in the 3' untranslated region (UTR) and near the stop codon in yeast and mammals. In Arabidopsis, m⁶A is enriched not only in 3'UTRs and near the stop codon but also in 5'UTRs and around the start codon. In mammalian cells, m⁶A also accumulates in the 5'UTR region in response to stress conditions such as heat shock. The distribution of m⁶A is important since it implies the mechanism by which m⁶A modification regulates its mRNA (Wang, 2021).

Another major breakthrough is the gradual elucidation of the m⁶A modification pathway by biochemical and genetic studies. The m⁶A is deposited by a multicomponent methyltransferase complex ('writers'), mainly recognized by YTH domain-containing 'readers', and can be removed by FTO and ALKBH5 'erasers', although FTO was also indicated as an m⁶A_m demethylase. The key catalytic component of the m⁶A writer complex, Mettl3, was purified and cloned in the 1990s. Since then, studies from yeast, Arabidopsis, Drosophila, and mammalian cells have identified several core components of the writer complex, including Mettl14, WTAP (Fl(2)d), VIRMA (Virilizer), RBM15/15B (Spenito), ZC3H13 (Flacc or Xio), and Hakai. Interestingly, Fl(2)d, Virilizer (Vir), Spenito (Nito), and Xio were first identified from Drosophila sex determination screens and later realized as part of the writer complex. They regulate Drosophila sex determination by controlling the alternative splicing of the master regulatory gene Sex-lethal (Sxl). Recently, Mettl3, Mettl14, as well as the reader Ythdc1, were also shown to be involved in this process. However, the detailed mechanism of how the m⁶A modification cooperates with Sxl protein to modulate its own splicing is still unclear. Thus, Drosophila can serve as a unique system to screen components in the m⁶A pathway and pinpoints a critical role for m⁶A in regulating splicing. Other than Sxl splicing, Drosophila m⁶A genes are highly expressed in the nervous system and exhibit similar wing and behavior defects when mutated. Mutants of several fly m⁶A factors are viable and thus provide an ideal model to study other processes, such as metabolism and immunity, in the future (Wang, 2021).

Hakai, also known as CBLL1, was found as an interacting protein with several m⁶A writer components in proteomic studies. It encodes a RING finger-type E3 ubiquitin ligase and was originally identified as an E-cadherin-binding protein in human cell lines. It was proposed that Hakai ubiquitinates E-cadherin at the plasma membrane and induces its endocytosis, thus playing a negative role post-translationally. Due to the key role of E-cadherin in tumor metastasis, especially epithelial-mesenchymal transition, Hakai has been extensively studied mainly using cell culture and overexpression system, but a previous study using the Drosophila model did not observe an increase of E-cadherin level in Hakai mutants. In Arabidopsis, Hakai mutants show partially reduced m⁶A levels and the mutant phenotypes are weaker than other writer components. Importantly, the in vivo role of Hakai as a core m⁶A writer component has not been studied in any animal species. This study analyzed the role of Hakai in the Drosophila m⁶A modification pathway. The results demonstrated that Hakai is a bona fide member of the m⁶A writer complex, with its mutants showing reduced global m⁶A levels, typical m⁶A mutant phenotypes, and commonly-regulated gene sets. A high-quality fly m⁶A methylome was obtained using stringent MeRIP-seq, discovered a female-specific m⁶A methylation pattern for Sxl mRNA, characterized the role of Hakai in the m⁶A writer complex, and finally revisited the function of Hakai in E-cadherin regulation (Wang, 2021).

m⁶A modification has been known for more than 40 years but has recently gained great attention due to the emergence of technologies to map m⁶A methylome, as well as the identification of the writers, readers, and erasers in this pathway. Since the initial purification of the key methyltransferase Mettl3, other components of the writer complex were gradually identified through biochemical experiments and genetic screens. It is now known that m⁶A writer complex is comprised of multiple components including Mettl3, Mettl14, WTAP, VIRMA, RBM15/15B, ZC3H13. Hakai was first indicated as a WTAP interaction protein and was shown later to be required for full m⁶A methylation in Arabidopsis; however, its role in the m⁶A pathway in animals has not been studied. This study shows that Hakai interacts with other m⁶A writer subunits, and Hakai mutants exhibit characteristic m⁶A pathway phenotypes, such as lowered m⁶A levels in mRNA, aberrant alternative splicing of Sxl and other genes, held-out wings, and flightless flies, as well as reduced m⁶A peaks shared with Mettl3 and Mettl14 mutants in MeRIP-seq. Altogether, these data unambiguously argue that Hakai is the seventh, and likely last core component of the conserved m⁶A writer complex (Wang, 2021).

Each component in the m⁶A writer complex plays a role in mRNA methylation but their exact roles are not well understood. This systematic analysis of several m⁶A writer subunits has provided insights into the mechanism of this important complex. l(2)d, Vir, Hakai, and Flacc were found to form a stable complex, and knocking down either of Fl(2)d, Vir, or Hakai led to the degradation of the other three components. Mettl3, Mettl14, and Nito were not affected by the disruption of Fl(2)d, Vir or Hakai, suggesting that they have separate functions. Knocking down Flacc resulted in less nuclear staining of Fl(2)d, consistent with a role in nuclear localization of the writer complex. Based on these results, a model is proposed for the m⁶A methyltransferase complex. Mettl3 and Mettl14 form a stable heterodimer to catalyze the addition of the methyl group to mRNA. Nito/RBM15 contains three RRM domains and binds to positions adjacent to m⁶A sites, thus may provide target specificity for the m⁶A writer complex. Fl(2)-Vir-Hakai-Flacc form a platform to connect different components and may integrate environmental and cellular signals to regulate m⁶A methylation (Wang, 2021).

Hakai is a potential E3 ubiquitin ligase with an intact C3HC4 RING domain and a C2H2 domain. Its absence led to the degradation, rather than the accumulation of other m⁶A writer subunits, indicating that it may not act as an E3 ubiquitin ligase in this complex. Hakai was initially identified as an E-cadherin binding protein to downgrade its levels CR50, and the role of Hakai in cell proliferation and tumor progression was extensively studied in cell culture. However, the current in vivo analysis using various genetic tools did not find a role of Hakai in E-cadherin regulation. In addition, Hakai appeared as a ubiquitous nuclear protein showing little co-localization with E-cadherin in the membrane. Consistently, Hakai was shown to interact with PTB-associated splicing factor (PSF), a nuclear protein, and to affect its RNA-binding ability. Thus, the role of Hakai in E-cadherin regulation needs to be further investigated using the knockout mouse model and whether Hakai has other substrates for its E3 ligase activity also needs to be determined (Wang, 2021).

Recent emerging studies suggest that m⁶A is involved in numerous developmental processes and human diseases, mainly by regulating mRNA stability, translation, or splicing. Pioneer work has established the framework for the m⁶A pathway in Drosophila. However, only published Drosophila m⁶A methylome was performed in S2R + cells or embryos and was not done against writer mutants. Other than Sxl, few m⁶A target loci have been firmly mapped. By performing MeRIP-seq in wild-type adult flies as well as Mettl3, Mettl14, and Hakai mutants, this study demonstrated that although most m⁶A peaks are distributed in 3'UTRs, the functional peaks responding to the loss of m⁶A writers are mainly located in 5'UTRs. This finding indicates a major difference between Drosophila and mammalian m⁶A methylome, that mainly occurs in 3'UTRs, and is in agreement with a recently published manuscript using miCLIP. Interestingly, LC-MS data show that the overall level of m⁶A modification in Drosophila only accounted for 10-20% of that in mammalian cells. Mettl3 or Mettl14 mutants are embryonic lethal in mice while they develop into adults in flies. It is possible that the m⁶A pathway acquires additional functions during evolution (Wang, 2021).

m⁶A modification in 3'UTRs usually causes mRNA instability and m⁶A in 5'UTRs is linked to translation enhancement. In agreement with the view that functional m⁶A peaks are located in 5'UTRs in Drosophila, this study did not observe an increase in mRNA half-life of m⁶A targets in Mettl3 mutants compared to wild-type. These results imply that the major role of m⁶A modification in Drosophila is not on mRNA degradation, but possibly on translation upregulation, which can be tested by combining ribosome profiling and functional analysis of a single transcript in the future. The current data by combining MeRIP-seq and splicing analysis shed light on how the m⁶A modification contributes to splicing regulation. In all five cases analyzed, four (Dsp1, CG8929, fl(2)d, Aldh-III) in 5'UTRs and one (Sxl) in exon/intron, reduction of m⁶A modification was correlated with enhanced splicing, arguing that the normal role of these modifications might be to repress splicing events nearby (Wang, 2021).

Last but probably the most interesting finding from this work is to demonstrate the female-specific m⁶A modification around Sxl exon3. Sxl is a textbook paradigm to study alternative splicing and has been intensively investigated for more than thirty years. Sxl protein binds to its own mRNA to control the alternative splicing, but its binding sites are located ~200 nucleotides downstream or upstream of the male exon, meaning other regulators should be involved. Recently, the m⁶A modification pathway was shown to modulate Sxl alternative splicing, but the detailed mechanism has not been resolved. The MeRIP-seq data revealed that several m⁶A peaks were deposited only in females on and around Sxl exon3, and they were in the vicinity of Sxl-binding sites. This finding was further validated by independent m⁶A-IP-qPCR and showed that these modifications were reduced in Mettl3 mutant females. This unexpected finding suggests a model that one main function of Sxl may be to recruit the m⁶A writer complex that methylates nearby m⁶A sites. The m⁶A reader Ythdc1 in turn binds to these sites and might interact with the splicing machinery to repress splicing. Future experiments, such as interactions between Sxl and Mettl3/Mettl14, interactions between Ythdc1 and general splicing factors, mapping of the exact m⁶A methylation site in Sxl at the single nucleotide level, comparison of transcriptome-wide binding sites of Sxl with m⁶A modification sites, will be required to firmly prove the model (Wang, 2021).

Earlier treatment of Sxl in The Interactive Fly

Sex determination in the developing fly is like a one ring circus that occasionally complicates an already exciting scene by adding a second and a third ring, providing the audience a variety of different acts taking place at different times, and occasionally, a choice of viewing among simultaneous events (see Schematic of the sex determination hierarchy in Control of male sexual behavior in Drosophila by the sex determination pathway, Billeter, 2006). Whatever goes on, the circus master in charge is Sex lethal (Sxl). At least three acts get top billing in this circus under the direction of Sxl: the development of female somatic fate; the dosage compensation in males during embryonic development, and finally, the sex specific development of female and male germ lines.

Act 1: Immediately after fertilization there is an assessment of the ratio of X chromosomes to autosomes. Three genes are X linked: when there are two X chromosomes, as in females, the ratio of their gene products like Runt (Torres, 1994), Sisterless-A and Sisterless-B (also known as Scute) is higher than in males. These three genes bind the Sex-lethal promoter and induce activation. In the case of males, where there is only one X chromosome, the autosomal proteins Daughterless, Deadpan and Extramachrochaete, absent sufficient activator proteins, act as repressors of Sex lethal. Thus, despite Sex lethal's central position as ringmaster in this developmental circus, it is the crowd of at least a half dozen transcription factors that regulate Sex lethal's activities.

Sex lethal is an RNA splicing enzyme. Sex lethal's immediate target is Transformer mRNA. Since Sex lethal is not transcribed in males, its action on Transformer is restricted to females. Sex lethal acts positively in the functional splicing of Transformer mRNA. Transformer is another splice factor, acting in turn on downstream RNAs that require sex-specific splicing (Sosnowski, 1994). Transformer protein thus determines female developmental fate.

Act 2. In the spotlight here is dosage compensation, regulated by Sex lethal. The immediate target of SXL is male specific lethal-2 (MSL-2), a transcription factor. In the presence of SXL, MSL-2 is spliced into an inactive form, one that cannot function in dosage compensation. In the absence of SXL (in males), MSL-2 splicing is productive, and the active MSL-2 transcription factor effectively carries out dosage compensation.

A word about dosage compensation is in order. The ratio of sex chromosomes to autosomes in females (1:1) is different from the ratio in males (0.5:1). This is because males, by definition, have one X chromosome and not two. This presents a dosage problem. The ratio of gene products coded for by the sex chromosome will be different in males and females, unless some compensatory action is taken. The sex chromosome carries a lot of genes that are simply along for the ride, and the creation of a dosage imbalance spells catastrophe for development. What to do?

Two alternatives are possible. One of the X chromosomes could be shut off, inactivated in females. This is the solution humans and other higher vertebrates have employed. A second option would be to heighten the activity of the single X chromosome in males. This is the route flies take. MSL-2, spliced into a functional form in males, serves to heighten transcriptional activation of the solitary X chromosome. MSL-2 acts in concert with three partners in this task: Maleless, Male-specific lethal-1 and Male specific lethal-3. They bind to about one hundred sites on the male chromosome, modifying the chromatin structure to permit heightened gene activation (Bashaw, 1995). The action of these male specific transcription factors is very similar to proteins of the trithorax complex.

Act 3. In the center ring here is the regulation by Sex lethal of sex specific RNA splicing in ovaries. Ovaries represent a completely different tissue milieu from somatic cells, and consequently Sex-lethal splicing in these tissues will demand a different kind of regulation (Granadino, 1993). Germ line Sex-lethal function requires an XX karyotype, a female soma, and action of the genes ovp, otu and snf (sans fille). SNF is an RNA binding protein and an integral component of the machinery required for splice site recognition (Flickinger, 1994 and Salz, 1996). ovo, a zinc finger protein and presumably a transcription factor, has a higher level of transcription in females than in males, responding to the number of sex chromosomes in the cell (Oliver, 1994). otu (ovarian tumor) gene also acts upstream of Sex lethal (Pauli, 1993 and Bopp, 1993). Thus ovarian Sex lethal activation and splicing is regulated by a completely different set of proteins (with the exception of SNF, whose function is general) from those in the embryo.

Biological systems operate at a level of complexity that continually astounds the student. A tiny fly carries a whole circus at the core of its development, as far as sex determination is concerned.

GENE STRUCTURE

Sex-lethal expresses a set of three early transcripts and a set of seven late transcripts occurring from midembryogenesis through adulthood. Among the late transcripts, male-specific mRNAs have been distinguished from their female counterparts by the presence of an extra exon interrupting an otherwise long open reading frame (ORF). The late transcripts appear to use a common 5' end but differ at their 3' ends by the use of alternative polyadenylation sites. Two of these sites lack canonical AATAAA sequences, and their use correlates in females with the presence of a functional germ line, suggesting possible tissue-specific polyadenylation. A number of non-sex-specific splicing variants have been observed. In females, the various forms of late SXL transcript potentially encode up to six slightly different polypeptides (Samuels, 1991).

Sex lethal has two transcripts, early and late, with different promoters and dramatically different splicing patterns. The Sxl early transcripts are activated transiently in early embryos by a female-specific promoter and have a unique 5' exon (E1) located between late exons 1 and 2. Exon E1 is spliced to exon 4, which is common to all SXL transcripts, skipping both exons 2 and 3 (Keyes, 1992). In contrast, the late SXL transcripts derive from an essentially constitutive promoter but are spliced sex specifically. The male-specific exon, exon 3, is included by default in all male transcripts and contains in-frame nonsense codons that block SXL protein production. In the presence of SXL protein, the late transcripts skip exon 3 and splice in the female pattern. No embryo-specific splicing factors are needed for the early splice. Neither are sex-specific factors required. Instead, the early splicing pattern is dependent on whether the 5' splice site region originates from exon E1 or exon 2 (Zhu, 1997).

PROTEIN STRUCTURE

Amino Acids - 354 for product of female cDNA

Structural domains

The female SXL protein has large duplicate domains each with 74 residues that are 35% identical to one another. These domains are common to RNA binding proteins. SXL shows greatest homology to yeast poly A binding protein (Bell, 1988).

date revised: 10 June 2024

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