The interaction between daughterless and sex-lethal in triploids: a lethal sex-transforming maternal effect linking sex determination and dosage compensation in Drosophila melanogaster

Regulation of Drosophila sex determination and X-chromosome dosage compensation in response to the X-chromosome/autosome (X/A) balance of the zygote is shown to require proper functioning of both the da+ gene in the mother and the Sxl+ gene in the zygote. Previous studies led to the hypothesis that zygotic Sxl+ alleles are differentially active in females (XXAA) vs males (XYAA) in response to the X/A balance, and that maternal da+ gene product acts as a positive regulator in this connection. Sxl+ activity was proposed to impose the female developmental sequence on cells which would follow the male sequence in its absence. Important predictions of this proposal are verified. This study focuses primarily on the phenotype of triploid intersexes (XXAAA, X/A = 0.67). They are shown here to survive effects of da and Sxl mutations that would be lethal to diploids. The ambiguous X/A signal of intersexes normally causes them to develop as phenotypic mosaics of male and female tissue. Loss of maternal da+ or zygotic Sxl+ gene function shifts their somatic sexual phenotype to the male alternative. A gain-of-function mutation at Sxl has the opposite effect, imposing female development regardless of the maternal genotype with respect to da. It also reduces their rate of X-linked gene expression. The effects of a duplication of Sxl+ resemble those of the constitutive Sxl allele, but are less extreme. The role of these genes in the process of X-chromosome dosage compensation is inferred indirectly from the strict dependence of the mutations' lethal effects on the X/A balance in haploids, diploids, and triploids, and more directly from the effects of the mutations on the phenotypes of the X-linked neomorphic mutations, Bar and Hairy-wing. The relationship of da+ and Sxl+ gene functions to those of other sex-specific lethal loci in D. melanogaster, and to sex determination mechanisms in other species, is discussed.     

Production of X0 clones in XX females of Drosophila

The experiments reported here are aimed at determining whether mutations deleting the function of the Sex-lethal (Sxl) gene are able to suppress the lethality of X0 clones, induced in females after the time when the state of activity of Sxl is irreversibly fixed by the ratio of the number of X chromosomes to sets of autosomes (X:A). This analysis was carried out by comparing the frequency of induced male clones (X0 constitution) in SxlfLS/+ and Sxl+/Sxl+ females, following irradiation at blastoderm and larval stages. The genotype used in these experiments, however, could also give rise to 2X; 2A cells homozygous for SxlfLS, and such cells would also differentiate male structures. To minimize this possibility, we have constructed a genotype made up of a ring and a rod X chromosome. In such ring-rod females the production of 2X; 2A clones homozygous for SxlfLS is a rather rare event, if possible at all. X0 male clones were produced in both types of females following irradiation at blastoderm stage, while X0 male clones were only observed in SxlfLS/+ females when irradiation took place at larval stage. In this latter case, the only X0 male clones were those that contained the SxlfLS mutation. These results support the idea of Sánchez & N?thiger (1983) that the X:A signal irreversibly sets the state of activity of Sxl at blastoderm stage, and in addition show that X0 clones generated after that time are viable if they contain a Sxl- mutation. These results are compatible with the idea of Sxl being the only gene that responds to the X:A signal.     

The transformer gene in Ceratitis capitata provides a genetic basis for selecting and remembering the sexual fate

The medfly Ceratitis capitata contains a gene (Cctra) with structural and functional homology to the Drosophila melanogaster sex-determining gene transformer (tra). Similar to tra in Drosophila, Cctra is regulated by alternative splicing such that only females can encode a full-length protein. In contrast to Drosophila, however, where tra is a subordinate target of Sex-lethal (Sxl), Cctra seems to initiate an autoregulatory mechanism in XX embryos that provides continuous tra female-specific function and act as a cellular memory maintaining the female pathway. Indeed, a transient interference with Cctra expression in XX embryos by RNAi treatment can cause complete sexual transformation of both germline and soma in adult flies, resulting in a fertile male XX phenotype. The male pathway seems to result when Cctra autoregulation is prevented and instead splice variants with truncated open reading frames are produced. We propose that this repression is achieved by the Y-linked male-determining factor (M).     

Sex determination in the germ line of Drosophila depends on genetic signals and inductive somatic factors

We have analyzed the mechanism of sex determination in the germ line of Drosophila by manipulating three parameters: (1) the ratio of X-chromosomes to sets of autosomes (X:A); (2) the state of activity of the gene Sex-lethal (Sxl), and (3) the sex of the gonadal soma. To this end, animals with a ratio of 2X:2A and 2X:3A were sexually transformed into pseudomales by mutations at the sex-determining genes Sxl (Sex-lethal), tra (transformer), tra-2 (transformer-2), or dsx (double-sex). Animals with the karyotype 2X;3A were also transformed into pseudofemales by the constitutive mutation SxlM1. The sexual phenotype of the gonads and of the germ cells was assessed by phase-contrast microscopy. Confirming the conclusions of Steinmann-Zwicky et al. (Cell 57, 157, 1989), we found that all three parameters affect sex determination in germ cells. In contrast to the soma in which sex determination is completely cell-autonomous, sex determination in the germ line has a non-autonomous component inasmuch as the sex of the soma can influence the sexual pathway of the germ cells. Somatic induction has a clear effect on 2X;2A germ cells that carry a Sxl+ allele. These cells, which form eggs in an ovary, can enter spermatogenesis in testes. Mutations that cause partial loss of function or gain of function of Sxl thwart somatic induction and, independently of the sex of the soma, dictate spermatogenesis or oogenesis, respectively. Somatic induction has a much weaker effect on 2X;3A germ cells. This ratio is essentially a male signal for germ cells which consistently enter spermatogenesis in testes, even when they carry SxlM1. In a female soma, however, SxlM1 enables the 2X;3A germ cells to form almost normal eggs. Our results show that sex determination in the germ line is more complex than in the soma. They provide further evidence that the state of Sxl, the key gene for sex determination and dosage compensation in the soma, also determines the sex of the germ cells, and that, in the germ line, the state of activity of Sxl is regulated not only by the X:A ratio, but also by somatic inductive stimuli.     

Cell-autonomous and inductive signals can determine the sex of the germ line of drosophila by regulating the gene Sxl

To investigate the mechanism of sex determination in the germ line, we analyzed the fate of XY germ cells in ovaries, and the fate of XX germ cells in testes. In ovaries, germ cells developed according to their X:A ratio, i.e., XX cells underwent oogenesis, XY cells formed spermatocytes. In testes, however, XY and XX germ cells entered the spermatogenic pathway. Thus, to determine their sex, the germ cells of Drosophila have cell-autonomous genetic information, and XX cells respond to inductive signals of the soma. Results obtained with amorphic and constitutive mutations of Sxl show that both the genetic and the somatic signals act through Sxl to achieve sex determination in germ cells.     

Two Closely Linked Mutations in DROSOPHILA MELANOGASTER That Are Lethal to opposite Sexes and Interact with Daughterless

A new spontaneous mutation named Sex-lethal, Male-specific No. 1 (SxlM1) is described that is lethal to males, even in the presence of an Sxl+ duplication. Females homozygous for SxlM1 are fully viable. This dominant, male-specific lethal mutation is on the X chromosome approximately 0.007 map units to the right of a previously isolated female-specific mutation, Female-lethal, here renamed Sex-lethal, Female-specific No. 1 (SxlF1). SxlM1 and SxlF1 are opposite in nearly every repect, particularly with regard to their interaction with maternal effect of the autosomal mutation, daughterless (da). Females that are homozygous for da produce defective eggs that cannot support female (XX) development. A single dose of SxlM1 enables daughters to survive this da female-specific lethal maternal effect. A duplication of the Sxl locus weakly mimics this action of SxlM1. In contrast, SxlF1 and a deficiency for Sxl, strongly enhance the female-lethal effects of da. The actions of SxlM1 and SxlF1 are explained by a model in which expression of the Sxl locus is essential for females, lethal for males, and under the control of a product of the da locus. It is suggested that SxlM1 is a constitutive mutation at the Sxl locus.     

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

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.     

The segmentation gene runt is needed to activate Sex-lethal, a gene that controls sex determination and dosage compensation in Drosophila.

In Drosophila, sex is determined by the relative number of X chromosomes to autosomal sets (X:A ratio). The amount of products from several X-linked genes, called sisterless elements, is used to indicate to Sex-lethal the relative number of X chromosomes present in the cell. In response to the X:A signal, Sex-lethal is activated in females but remains inactive in males, being responsible for the control of both sex determination and dosage compensation. Here we find that the X-linked segmentation gene runt plays a role in this process. Reduced function of runt results in female-specific lethality and sexual transformation of XX animals that are heterozygous for Sxl or sis loss-of-function mutations. These interactions are suppressed by SxlM1, a mutation that constitutively expresses female Sex-lethal functions, and occur at the time when the X:A signal determines Sex-lethal activity. Moreover, the presence of a loss-of-function runt mutation masculinizes triploid intersexes. On the other hand, runt duplications cause a reduction in male viability by ectopic activation of Sex-lethal. We conclude that runt is needed for the initial step of Sex-lethal activation, but does not have a major role as an X-counting element.     

Sex determination in Drosophila: the X-chromosomal gene liz is required for Sxl activity.

In Drosophila, females require products of the gene Sxl for sex determination, dosage compensation and fertility. I show here that the X-chromosomal gene liz, located in 4F1 to 4F11 and previously called fs(1)1621, provides maternal and zygotic functions necessary for Sxl activity in germ line and soma. In XX animals, the mutation SxlM1 which was reported to express the female-specific functions of Sxl constitutively can rescue all phenotypes resulting from lack of liz product. XY animals carrying SxlM1 and lacking maternal or zygotic liz activity survive as males with some female traits. A stock was constructed in which the females are liz SxlM1/liz SxlM1 and males liz SxlM1/Y. This shows that SxlM1 is not truly expressed constitutively in animals with an X:A ratio of 0.5, but requires activity of liz for initiation or maintenance.     

A lesson from flex: consider the Y chromosome when assessing Drosophila sex-specific lethals

Bhattacharya et al. (Bhattacharya, A., Sudha, S., Chandra, H. S. and Steward, R. (1999) Development 126, 5485-5493) reported that loss-of-function mutations in the flex (female-specific lethal on X) gene caused female-specific lethality because flex(+) acts as a positive regulator of the master switch gene Sex lethal (Sxl). Sxl is essential for female development. Key to their conclusion was the ability of flex mutations to suppress the male lethality caused by Sxl(M) mutations, which inappropriately activate Sxl female-specific expression. Here we report our contrary findings that flex mutations fail to suppress even the weakest Sxl(M )alleles, arguing against the proposed regulatory relationship between flex and Sxl. Instead we show that the lethal flex phenotype depends on the absence of a Y chromosome, not on the presence of two X chromosomes. flex lethality is caused by a defect in the functioning of the X-linked rDNA locus called bobbed, since this defect is complemented by the corresponding wild-type rDNA complex on the Y.     

Drosophila melanogaster male somatic cells feminized solely by TraF can collaborate with female germ cells to make functional eggs

Female differentiation of Drosophila germ cells is induced by cell-nonautonomous signals generated in the gonadal soma that work with germ-cell-autonomous signals determined by germ-cell X chromosome dose. Generation of the nonautonomous feminizing signals was known to involve female-specific protein encoded by the master sex-determination gene Sex-lethal (Sxl) acting on its switch-gene target transformer (tra) to produce Tra(F) protein. However, it was not known whether Sxl's action on tra alone would suffice to trigger a fully feminizing nonautonomous signal. We developed a constitutively feminizing tra transgene that allowed us to answer this question. In gynanders (XX//XO mosaics) feminized by this Tra(F) transgene, functionally Sxl- haplo-X (chromosomally male) somatic cells collaborated successfully with diplo-X (chromosomally female) germ cells to make functional eggs. The fertility of such gynanders shows not only that Tra(F) is sufficient to elicit a fully feminizing nonautonomous signal, but also that haplo-X somatic cells can execute all other somatic functions required for oogenesis, despite the fact that their genome is not expected to be dosage compensated for such diplo-X-specific functions. The unexpected observation that some Tra(F)-feminized gynanders failed to lay their eggs showed there to be diplo-X cells outside the gonad for which Tra(F)-feminized haplo-X cells cannot substitute.