The scute (T4) gene acts as a numerator element of the X:a signal that determines the state of activity of sex-lethal in Drosophila.

The ratio of X chromosomes to sets of autosomes (X:A) is the primary genetic signal that determines sex and dosage compensation in Drosophila. The gene Sex-lethal (Sxl) receives this signal and is responsible for the execution of the alternative developmental programmes of males and females. We have found that the scute (T4) gene, which is involved in neurogenesis, also plays a role in the activation of Sxl. The following results suggest that scute (T4) may be a numerator element of the X:A signal: scute (T4) mutations show female-specific lethality. There are female-specific lethal synergistic interactions between sis-a, a previously described numerator element, and mutants for T4. The female lethality is suppressed by SxlM1, a constitutive allele which expresses an active Sxl product independently of the X:A ratio. The Hw685 mutation, which overexpresses T4, is lethal to males with a duplication of sis-a. This lethality is suppressed by either Sxlf1, or the T4 point mutation sc10-1. There are female-specific lethal interactions between sc10-1 and daughter-less (da), a gene needed maternally for Sxl to become active. The sc10-1 mutation masculinizes triploid intersexes.     

X-linked gene expression and sex determination in Caenorhabditis elegans

The signal for sex determination in the nematode Caenorhabditis elegans is the ratio between the number of X chromosomes and the number of sets of autosomes (the X/A ratio). Animals with an X/A ratio of 0.67 (a triploid with two X chromosomes) or less are males. Animals with an X/A ratio of 0.75 or more are hermaphrodites. Thus, diploid males have one X chromosome and diploid hermaphrodites have two X chromosomes. However, the difference in X-chromosome number between the sexes is not reflected in general levels of X-linked gene expression because of the phenomenon of dosage compensation. In dosage compensation, X-linked gene expression appears to be 'turned down' in 2X animals to the 1X level of expression. An intriguing and unexplained finding is that mutations and X-chromosome duplications that elevate X-linked gene expression also feminize triploid males. One way that this relationship between sex determination and X-linked gene expression may be operating is discussed.     

Evidence That Sisterless-a and Sisterless-B Are Two of Several Discrete ``numerator Elements'''' of the X/a Sex Determination Signal in Drosophila That Switch Sxl between Two Alternative Stable Expression States

The primary signal for Drosophila sex determination is the number of X chromosomes relative to the number of sets of autosomes. The present report shows that the numerator of this X/A signal appears to be determined by the cumulative dose of a relatively limited number of discrete X-linked genetic elements, two of which are sisterless-a and sisterless-b. This discovery regarding the nature of the sex determination signal grew out of previous studies of both the likely X/A signal target (the feminizing switch gene, Sex-lethal) and two positive regulators of that target gene (sis-a and daughterless). Combinations of genetic perturbations in these three genes had been shown to have synergistic effects. A model proposed in part to account for these interactions generated a large variety of strong predictions for sex-specific synergistic interactions that would be diagnostic for X/A numerator elements and could distinguish them from other components of the sex determination system. All these predictions, as well as other predictions for X/A numerator elements, are shown here to be fulfilled. The most compelling observations involve sexually reciprocal viability effects of duplications of wild-type genes: combinations of sis-a+, sis-b+ and/or Sxl+ duplications are lethal to males but rescue females from the otherwise lethal effects of changes in other components of the sex determination machinery. The many interactions described here illustrate an important principle that may seem counter-intuitive: perturbations of the sex determination signal for Drosophila generally will not appear to affect adult sexual phenotype. This principle follows from the fact that Sxl is involved in dosage compensation as well as sex determination, and from important aspects of the nature and timing of Sxl's regulation both by the X/A signal and by Sxl's own products (positive autoregulation). These factors mask potential effects on adult sexual differentiation by causing the premature death of cells and/or individuals. The fact that the vast array of results presented here conform to this principle is strong evidence in favor of a "binary state" model for Sxl regulation by the X/A signal. This model is favored over an alternative "multiple state" hypothesis that was proposed by others in a different study of the X/A signal. In that same study it was concluded that region 3E8-4F11 of the X chromosome contained especially potent X/A numerator elements.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic Evidence That the Ovo Locus Is Involved in Drosophila Germ Line Sex Determination

Zygotically contributed ovo gene product is required for the survival of female germ cells in Drosophila melanogaster. Trans-allelic combinations of weak and dominant ovo mutations (ovoD) result in viable germ cells that appear to be partially transformed from female to male sexual identity. The ovoD2 mutation is partially suppressed by many Sex-lethal alleles that affect the soma, while those that affect only the germ line fail to interact with ovoD2. One of two loss-of-function ovo alleles is suppressed by a loss-of-function Sex-lethal allele. Because ovo mutations are germ line dependent, it is likely that ovo is suppressed by way of communication between the somatic and germ lines. A loss-of-function allele of ovo is epistatic to germ line dependent mutations in Sex-lethal. The germ line dependent sex determination mutation, sans fille, and ovoD mutations show a dominant synergistic interaction resulting in partial transformation of germ line sexual identity. The ovo locus appears to be involved in germ line sex determination and is linked in some manner to sex determination in the soma.     

The Drosophila segmentation gene runt has an extended cis-regulatory region that is required for vital expression at other stages of development.

The Drosophila runt gene functions in several developmental pathways during embryogenesis. This gene was initially characterized due to the pivotal role that it plays in the genetic regulatory network that establishes the segmented body pattern. Recently it was found that this X-chromosome-linked gene is one of several dosage-sensitive, X-linked components that is involved in activating the Sex-lethal gene in blastoderm stage female embryos. Finally, this gene is also extensively re-expressed in later stages of embryogenesis in the developing nervous system where it plays an important role in the development of specific neural lineages. We have initiated an analysis of the runt cis-regulatory region in order to investigate runt's roles in these (and other) developmental pathways. Analysis of both the function and the expression patterns of runt genes with truncated cis-regulatory regions indicates that there are multiple elements that make quantitative contributions to runt regulation during segmentation. We find that sequences that are more than 8.5 kb upstream of the runt promoter are necessary for normal expression during the post-blastoderm stages of embryogenesis. Genetic experiments indicate that the post-blastoderm expression of runt is vital to the organism.     

Dosage compensation: making 1X equal 2X

Animals that have XX females and XY or XO males have differing doses of X-linked genes in each sex. Overcoming this is the most immediate and vital aspect of sexual differentiation. A number of systems that accurately compensate for sex-chromosome dosage have evolved independently: silencing a single X chromosome in female mammals, downregulating both X chromosomes in hermaphrodite Caenorhabditis elegans and upregulating the X chromosome in male Drosophila all equalize X-linked gene expression. Each organism uses a largely non-overlapping set of molecules to achieve the same outcome: 1X = 2X.     

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.     

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.     

A role for the Drosophila fragile X-related gene in circadian output

Mutations that abolish expression of an X-linked gene, FMR1, result in the pathogenesis of fragile X syndrome, the most common form of inherited mental retardation. To understand the normal function of the FMR1 protein, we have produced fly strains bearing deletions in a Drosophila homolog of FMR1 (dfmr1). Since fragile X patients show a number of abnormal behaviors including sleep problems, we investigated whether a loss-of-function mutation of dfmr1 affect circadian behavior. Here we show that under constant darkness (DD), a lack of dfmr1 expression causes arrhythmic locomotor activity, but in light:dark cycles, their behavioral rhythms appear normal. In addition, the clock-controlled eclosion rhythm is normal in DFMR1-deficient flies. These results suggest that DFMR1 plays a critical role in the circadian output pathway regulating locomotor activity in Drosophila.     

The relationship between gene dosage,gene expression,and sex in Drosophila

In Drosophila, the ratio of the number of X chromosomes to sets of other chromosomes initiates a series of events which result in sexual differentiation. In addition, this ratio establishes dosage compensation, a mechanism which equalizes the products of X-linked genes in males and females. The present review discusses possible genetic entities responsible for the interpretation of chromosomal sex and subsequent sex-mediated regulation during development.     

The Drosophila melanogaster sex determination gene sisA is required in yolk nuclei for midgut formation

During sex determination, the sisterlessA (sisA) gene functions as one of four X:A numerator elements that set the alternative male or female regulatory states of the switch gene Sex-lethal. In somatic cells, sisA functions specifically in sex determination, but its expression pattern also hints at a role in the yolk cell, a syncytial structure believed to provide energy and nutrients to the developing embryo. Previous studies of sisA have been limited by the lack of a null allele, leaving open the possibility that sisA has additional functions. Here we report the isolation and molecular characterization of four new sisA alleles including two null mutations. Our findings highlight key aspects of sisA structure-function and reveal important qualitative differences between the effects of sisA and the other strong X:A numerator element, sisterlessB, on Sex-lethal expression. We use genetic, expression, clonal, and phenotypic analyses to demonstrate that sisA has an essential function in the yolk nuclei of both sexes. In the absence of sisA, endoderm migration and midgut formation are blocked, suggesting that the yolk cell may have a direct role in larval gut development. To our knowledge, this is the first report of a requirement for the yolk nuclei in Drosophila development.     

Both Loss-of-Function and Gain-of-Function Mutations in Snf Define a Role for Snrnp Proteins in Regulating Sex-Lethal Pre-mRNA Splicing in Drosophila Development

The Drosophila snf gene encodes a protein with functional homology to the mammalian U1A and U2B" snRNP proteins. Studies, based on the analysis of three viable alleles, have suggested a role for snf in establishing the female-specific splicing pattern of the sex determination switch gene, Sex-lethal. Here, we show that the non-sex-specific lethal null allele is required for female sex determination, arguing against the formal possibility that the viable alleles disrupt a function unrelated to snf's wild-type function. Moreover, we find snf is required for normal cell growth and/or survival, as expected for a protein involved in a cell-vital process such as RNA splicing. We also show that of the three viable alleles only one, snf(JA2), is a partial loss-of-function mutation. The other two viable alleles, snf(1621) and snf(e8H), encode antimorphic proteins. We find the antimorphic proteins are mislocalized and correlate their mislocalization with their molecular lesions and mutant phenotypes. Finally, we provide genetic evidence that the antimorphic alleles interfere with the autoregulatory splicing function of the Sex-lethal protein. Based on these studies we suggest a model in which the snRNP protein, Snf, functions with Sex-lethal to block recognition of the regulated male-specific exon.