Sex lethal: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

Gene name - Sex lethal

Synonyms -

Cytological map position - 6F4-7B3

Function - splice factor

Keyword(s) - sex determination

Symbol - Sxl

FlyBase ID:FBgn0264270

Genetic map position - 1-19.2

Classification - RNA-binding protein

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene


Recent literature
Fear, J. M., Arbeitman, M. N., Salomon, M. P., Dalton, J. E., Tower, J., Nuzhdin, S. V. and McIntyre, L. M. (2015). The Wright stuff: Reimagining path analysis reveals novel components of the sex determination hierarchy in Drosophila melanogaster. BMC Syst Biol 9: 53. PubMed ID: 26335107
Summary:
This study used a structural equation modeling approach, leveraging natural genetic variation from two studies on Drosophila female head tissues to expand understanding of the sex hierarchy gene regulatory network (GRN). The GRN was expanded adding novel links among genes, including a link from fruitless (fru) to Sex-lethal (Sxl). This link is further supported by the presence of fru binding sites in the Sxl locus. 754 candidate genes were added to the pathway, including the splicing factors male-specific lethal 2 and Rm62 as downstream targets of Sxl. Independent studies of doublesex and transformer mutants support evidence for a link between the sex hierarchy and metabolism, via Insulin-like receptor. The genes added in one population were enriched for genes with sex-biased splicing and components of the spliceosome. Using natural alleles this approach not only identifies novel relationships, but using supervised approaches can order genes into a regulatory hierarchy.

Sun, X., Yang, H., Sturgill, D., Oliver, B., Rabinow, L. and Samson, M. L. (2015). Sxl-dependent, tra/tra2-independent alternative splicing of the Drosophila melanogaster X-Linked gene found in neurons.G3 (Bethesda) [Epub ahead of print]. PubMed ID: 26511498
Summary:
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.

Li, F. and Scott, M. J. (2015). CRISPR/Cas9-mediated mutagenesis of the white and Sex lethal loci in the invasive pest, Drosophila suzukii. Biochem Biophys Res Commun [Epub ahead of print]. PubMed ID: 26721433
Summary:
Drosophila suzukii (commonly called spotted wing Drosophila) is an invasive pest of soft-skinned fruit (e.g. blueberries, strawberries). A high quality reference genome sequence is available but functional genomic tools, such as used in Drosophila melanogaster, remain to be developed. This study used the CRISPR/Cas9 system to introduce site-specific mutations in the D. suzukii white (w) and Sex lethal (Sxl) genes. Hemizygous males with w mutations develop white eyes and the mutant genes are transmissible to the next generation. Somatic mosaic females that carry mutations in the Sxl gene develop abnormal genitalia and reproductive tissue. The D. suzukii Sxl gene could be an excellent target for a Cas9-mediated gene drive to suppress populations of this highly destructive pest.
Ote, M., Ueyama, M. and Yamamoto, D. (2016). Wolbachia protein TomO targets nanos mRNA and restores germ stem cells in Drosophila Sex-lethal mutants. Curr Biol [Epub ahead of print]. PubMed ID: 27498563
Summary:
Wolbachia, endosymbiotic bacteria prevalent in invertebrates, manipulate their hosts in a variety of ways: they induce cytoplasmic incompatibility, male lethality, male-to-female transformation, and parthenogenesis. However, little is known about the molecular basis for host manipulation by these bacteria. In Drosophila melanogaster, Wolbachia infection makes otherwise sterile Sex-lethal (Sxl) mutant females capable of producing mature eggs. Through a functional genomic screen for Wolbachia genes with growth-inhibitory effects when expressed in cultured Drosophila cells, this study identified the Wolbachia gene WD1278 encoding a novel protein called toxic manipulator of oogenesis (TomO), which phenocopies some of the Wolbachia effects in Sxl mutant D. melanogaster females. TomO enhances the maintenance of germ stem cells (GSCs) by elevating Nanos (Nos) expression via its interaction with nos mRNA, ultimately leading to the restoration of germ cell production in Sxl mutant females that are otherwise without GSCs.
Kan, L., Grozhik, A. V., Vedanayagam, J., Patil, D. P., Pang, N., Lim, K. S., Huang, Y. C., Joseph, B., Lin, C. J., Despic, V., Guo, J., Yan, D., Kondo, S., Deng, W. M., Dedon, P. C., Jaffrey, S. R. and Lai, E. C. (2017). The m6A pathway facilitates sex determination in Drosophila. Nat Commun 8: 15737. PubMed ID: 28675155
Summary:
The conserved modification N6-methyladenosine (m6A) modulates mRNA processing and activity. This study establish the Drosophila system to study the m6A pathway. miCLIP was applied to map m6A across embryogenesis, characterize its m6A 'writer' complex, validate its YTH 'readers' CG6422 and YT521-B, and generate mutants in five m6A factors. While m6A factors with additional roles in splicing are lethal, m6A-specific mutants are viable but present certain developmental and behavioural defects. Notably, m6A facilitates the master female determinant Sxl, since multiple m6A components enhance female lethality in Sxl sensitized backgrounds. The m6A pathway regulates Sxl processing directly, since miCLIP data reveal Sxl as a major intronic m6A target, and female-specific Sxl splicing is compromised in multiple m6A pathway mutants. YT521-B is a dominant m6A effector for Sxl regulation, and YT521-B overexpression can induce female-specific Sxl splicing. Overall, the transcriptomic and genetic toolkit reveals in vivo biologic function for the Drosophila m6A pathway.
Moschall, R., Strauss, D., Garcia-Bayert, M., Gebauer, F. and Medenbach, J. (2018). Drosophila Sister of Sex-lethal is a repressor of translation. RNA 24(2):149-158. PubMed ID: 29089381
Summary:
The RNA-binding protein Sex Lethal (Sxl) is an important post-transcriptional regulator of gene expression in female Drosophila. To prevent the assembly of the dosage compensation complex in female flies, Sxl acts as a repressor of msl-2 mRNA translation. It employs two distinct and mutually reinforcing blocks to translation that operate on the 5' and 3' untranslated regions (UTRs) of msl-2 mRNA, respectively. While 5' UTR-mediated translational control involves an upstream open reading frame, 3' UTR-mediated regulation strictly requires the co-repressor protein Upstream of N-ras (Unr) which is recruited to the msl-2 mRNA by Sxl. This study has identified the protein Sister of Sex Lethal (Ssx) as a novel repressor of translation with Sxl-like activity. Sxl and Ssx have a comparable RNA-binding specificity and can associate with Uracil-rich RNA regulatory elements present in msl-2 mRNA. Moreover, both repress translation when bound to the 5' UTR of msl-2 However, Ssx is inactive in 3' UTR-mediated regulation as it cannot engage the co-repressor protein Unr. The difference in activity maps to the first RNA-recognition motif (RRM) of Ssx. Conversion of three amino acids within this domain into their Sxl counterpart results in a gain-of-function and repression via the 3' UTR, allowing detailed insights into the evolutionary origin of the two proteins and into the molecular requirements of an important translation regulatory pathway.
Ota, R., Morita, S., Sato, M., Shigenobu, S., Hayashi, M. and Kobayashi, S. (2017). Transcripts immunoprecipitated with Sxl protein in primordial germ cells of Drosophila embryos. Dev Growth Differ 59(9):713-723. PubMed ID: 29124738
Summary:
In Drosophila, Sex lethal (Sxl), an RNA binding protein, is required for induction of female sexual identity in both somatic and germline cells. Although the Sxl-dependent feminizing pathway in the soma was previously elucidated, the downstream targets for Sxl in the germline remained elusive. To identify these target genes, transcripts associated with Sxl in primordial germ cells (PGCs) of embryos were selected using RNA immunoprecipitation coupled to sequencing (RIP-seq) analysis. A total of 308 transcripts encoded by 282 genes were obtained. Seven of these genes, expressed at higher levels in PGCs as determined by microarray and in situ hybridization analyses, were subjected to RNAi-mediated functional analyses. Knockdown of Neos, Kap-alpha3, and CG32075 throughout germline development caused gonadal dysgenesis in a sex-dependent manner, and Su(var)2-10 knockdown caused gonadal dysgenesis in both sexes. Moreover, as with knockdown of Sxl, knockdown of Su(var)2-10 in PGCs gave rise to a tumorous phenotype of germline cells in ovaries. Because this phenotype indicates loss of female identity of germline cells, Su(var)2-10 is considered to be a strong candidate target of Sxl in PGCs. These results represent a first step toward elucidating the Sxl-dependent feminizing pathway in the germline.
Guo, J., Tang, H. W., Li, J., Perrimon, N. and Yan, D. (2018). Xio is a component of the Drosophila sex determination pathway and RNA N(6)-methyladenosine methyltransferase complex. Proc Natl Acad Sci U S A. PubMed ID: 29555755
Summary:
N(6)-methyladenosine (m(6)A), the most abundant chemical modification in eukaryotic mRNA, has been implicated in Drosophila sex determination by modifying Sex-lethal (Sxl) pre-mRNA and facilitating its alternative splicing. This study identified a sex determination gene, CG7358, and renamed it xio according to its loss-of-function female-to-male transformation phenotype. xio encodes a conserved ubiquitous nuclear protein of unknown function. Xio was shown to colocalize and interacts with all previously known m(6)A writer complex subunits (METTL3, METTL14, Fl(2)d/WTAP, Vir/KIAA1429, and Nito/Rbm15) and that loss of xio is associated with phenotypes that resemble other m(6)A factors, such as sexual transformations, Sxl splicing defect, held-out wings, flightless flies, and reduction of m(6)A levels. Thus, Xio encodes a member of the m(6)A methyltransferase complex involved in mRNA modification. Since its ortholog ZC3H13 (or KIAA0853) also associates with several m(6)A writer factors, the function of Xio in the m(6)A pathway is likely evolutionarily conserved.
Morita, S., Ota, R. and Kobayashi, S. (2018). Downregulation of NHP2 promotes proper cyst formation in Drosophila ovary. Dev Growth Differ. PubMed ID: 29845608
Summary:
In Drosophila ovary, germline stem cells (GSCs) divide to produce two daughter cells. One daughter is maintained as a GSC, whereas the other initiates cyst formation, a process involving four synchronous mitotic divisions that form 2-, 4-, 8-, and 16-cell cysts. This study found that reduction in the level of NHP2, a component of the H/ACA small nucleolar ribonucleoprotein complex that catalyzes rRNA pseudouridylation, promotes progression to 8-cell cysts. NHP2 protein was concentrated in the nucleoli of germline cells during cyst formation. NHP2 expression, as well as the nucleolar size, abruptly decreased during progression from 2-cell to 4-cell cysts. Reduction in NHP2 activity in the germline caused accumulation of 4- and 8-cell cysts and decreased the number of single cells. In addition, NHP2 knockdown impaired the transition to 16-cell cysts. Furthermore, a tumorous phenotype caused by Sex-lethal (Sxl) knockdown, which is characterized by accumulation of single and two-cell cysts, was partially rescued by NHP2 knockdown. When Sxl and NHP2 activities were concomitantly repressed, the numbers of four- and eight-cell cysts were increased. In addition, Sxl protein physically interacted with NHP2 mRNA in ovaries. Thus, it is reasonable to conclude that Sxl represses NHP2 activity at the post-transcriptional level to promote proper cyst formation. Because NHP2 knockdown did not affect global protein synthesis in the germarium, it is speculated that changes in NHP2-dependent pseudouridylation, which is involved in translation of specific mRNAs, must be intact in order to promote proper cyst formation.
Sawala, A. and Gould, A. P. (2018). Sex-lethal in neurons controls female body growth in Drosophila. Fly (Austin): 1-9. PubMed ID: 30126340
Summary:
Sexual size dimorphism (SSD), a sex difference in body size, is widespread throughout the animal kingdom, raising the question of how sex influences existing growth regulatory pathways to bring about SSD. In insects, somatic sexual differentiation has long been considered to be controlled strictly cell-autonomously. This paper discusses the surprising finding that in Drosophila larvae, the sex determination gene Sex-lethal (Sxl) functions in neurons to non-autonomously specify SSD. Sxl was found to be required in specific neuronal subsets to upregulate female body growth, including in the neurosecretory insulin producing cells, even though insulin-like peptides themselves appear not to be involved. SSD regulation by neuronal Sxl is also independent of its known splicing targets, transformer and msl-2, suggesting that it involves a new molecular mechanism. Interestingly, SSD control by neuronal Sxl is selective for larval, not imaginal tissue types, and operates in addition to cell-autonomous effects of Sxl and Tra, which are present in both larval and imaginal tissues. Overall, these findings add to a small but growing number of studies reporting non-autonomous, likely hormonal, control of sex differences in Drosophila, and suggest that the principles of sexual differentiation in insects and mammals may be more similar than previously thought.
BIOLOGICAL OVERVIEW

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.

Drosophila Sex lethal gene initiates female development in germline progenitors

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 (lwrDN) 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 (SxlM) 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 SxlfP7B0 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 lwrDN 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 (SxlM1) 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. SxlM1 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 SxlM1 embryos. Thus, the SxlM1 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-SxlRNAi) under the control of maternal nanos-Gal4 was used to reduce Sxl activity in XX pole cells during embryogenesis. Introducing UAS-SxlRNAi 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 Sxlf1/++ 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 Sxlf1/++ 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 Sxlf1/++ 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 Sxlf1/++ 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 1st 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 1st 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 2nd 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 1st 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).

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 elavc155>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).


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).


Sex lethal: Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

date revised: 2 December 2018

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