The orb gene of Drosophila encodes sex-specific genn-line proteins that contain two RRM-type RNA-binding domains. Here we report the distribution of Orb protein in wild-type, tumorous, and orb mutant ovaries. The wild-type distribution of Orb protein during oogenesis resembles that of its RNA, preferentially accumulating in the cytoplasm of the developing oocyte shortly after the formation of the 16-cell cyst. As anticipated from its germ-line expression, mutations in orb lead to female sterility. Analysis of the effect of orb mutants on the distribution of RNAs known to be required for oocyte differentiation and polarity suggests that orb functions in RNA localization at multiple points during oogenesis. In addition, phenotypic characterization of the orb mutants indicates that the gene is required early in oogenesis for formation of the 16-cell cyst. It then functions in the differentiation of the oocyte and is required for the three-dimensional reorganization of the germ cells in the cyst as well as for the establishment of normal germ-line-soma interactions in the egg chamber.

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The on/off state of the binary switch gene Sex-lethal (Sxl), which controls somatic sexual development in Drosophila melanogaster, is regulated at the level of alternative splicing. In males, in which the gene is off, the default splicing machinery produces nonfunctional mRNAs; in females, in which the gene is on, the autoregulatory activity of the Sxl proteins directs the splicing machinery to produce functional mRNAs. We have used germ line transformation to analyze the mechanism of default and regulated splicing. Our results demonstrate that a blockage mechanism is employed in Sxl autoregulation. However, in contrast to transformer, in which Sxl appears to function by preventing the interaction of splicing factors with the default 3' splice site, a different strategy is used in autoregulation. (i) Multiple cis-acting elements, both upstream and downstream of the male exon, are required. (ii) These cis-acting elements are distant from the splice sites they regulate, suggesting that the Sxl protein cannot function in autoregulation by directly competing with splicing factors for interaction with the regulated splice sites. (iii) The 5' splice site of the male exon appears to be dominant in regulation while the 3' splice site plays a subordinate role.Posttranscriptional regulatory mechanisms play a key role in cell fate decisions in many developmental pathways. In the somatic sexual-development pathway of the fruit fly, Drosophila melanogaster, both the maintenance and elaboration of pathway choice are controlled at the level of alternative pre-mRNA splicing (2, 21). The binary switch gene Sex-lethal (Sxl) sits at the top of the somatic sexualdevelopment pathway and functions in both determination and differentiation. The activity state of the Sxl gene is chosen during early embryogenesis in response to the primary sex determination signal, the ratio of X chromosomes to autosomes (9,14,27,47). Sxl is turned on in females, while it remains off in males. Activation in females triggers an autoregulatory feedback loop in which female Sxl proteins promote their own synthesis by directing the femalespecific splicing of Sxl primary transcripts (3,4 regulates the splicing of transformer (tra) RNA (5). When Sxl is on, it directs the female-specific splicing of tra primary transcripts, producing mRNAs which have an intact open reading frame. The resulting tra protein, together with the constitutively expressed tra-2 protein, then activates the female-specific splice site of doublesex (dsx) to generate female dsx RNA (7,18,24,43). When Sxl is off, the default splicing of tra gives mRNAs with a truncated open reading frame, and in the absence of functional tra protein, dsx is spliced in the male mode.The mechanism for Sxl regulation of tra splicing is now reasonably well understood. As diagrammed in Fig. 1 MATERIALS AND METHODSPlasmid construction and Drosophila transformation. TheSxl miniconstruct diagrammed in Fig. 2 was made by first isolating a 310-bp PstI-HpaII fragment of genomic Sxl which contains exon 4 and upstrea...

Dosage compensation equates between the sexes the gene dose of sex chromosomes that carry substantially different gene content. In Drosophila, the single male X chromosome is hypertranscribed by approximately two-fold to effect this correction. The key genes are male lethal and appear not to be required in females, or affect their viability. Here, we show these male lethals do in fact have a role in females, and they participate in the very process which will eventually shut down their function—female determination. We find the male dosage compensation complex is required for upregulating transcription of the sex determination master switch, Sex-lethal, an X-linked gene which is specifically activated in females in response to their two X chromosomes. The levels of some X-linked genes are also affected, and some of these genes are used in the process of counting the number of X chromosomes early in development. Our data suggest that before the female state is set, the ground state is male and female X chromosome expression is elevated. Females thus utilize the male dosage compensation process to amplify the signal which determines their fate.

In Drosophila melanogaster, sex determination in somatic cells is controlled by a cascade of genes whose expression is regulated by alternative splicing [B. S. Baker, Nature (London) 340: 521-524, 1989; J. Hodgkin, Cell 56:905-906, 1989] [449][450][451][452][453][454][455][456][457][458][459] 1989) have shown that the mechanism for generating female transformer transcripts is not through the activation of the alternative splice site but by the blockage of the default splice site. We have tested whether an activation or a blockage mechanism is involved in Sex-lethal autoregulation. The male exon of Sex-lethal with flanking splice sites was placed into the introns of heterologous genes. Our results support the blockage mechanism. The poly(U) run at the male exon 3' splice site is required for sex-specific splicing. However, unlike transformer, default splicing to the male exon is sensitive to the sequence context within which the exon resides. This and the observation that the splice signals at the exon are suboptimal are discussed with regard to alternate splicing.

Genome analysis in several eukaryotes shows a surprising number of transcripts which do not encode conventional messenger RNAs. Once considered noise, these non-coding RNAs (ncRNAs) appear capable of controlling gene expression by various means. We find Drosophila sex determination, specifically the master-switch gene Sex-lethal (Sxl), is regulated by long ncRNAs (>200 nt). The lncRNAs influence the dose sensitive establishment promoter of Sxl, SxlPe, which must be activated to specify female sex. They are primarily from two regions, R1 and R2, upstream of SxlPeand show a dynamic developmental profile. Of the four lncRNA strands only one, R2 antisense, has its peak coincident with SxlPe transcription, suggesting it may promote activation. Indeed, its expression is regulated by the X chromosome counting genes, whose dose determines whether SxlPe is transcribed. Transgenic lines which ectopically express each of the lncRNAs show they can act in trans, impacting the process of sex determination but also altering the levels of the other lncRNAs. Generally, expression of R1 is negative whereas R2 is positive to females. This ectopic expression also results in a change in the local chromatin marks, affecting the timing and strength of SxlPe transcription. The chromatin marks are those deposited by the Polycomb and Trithorax groups of chromatin modifying proteins, which we find bind to the lncRNAs. We suggest the increasing numbers of non-coding transcripts being identified are a harbinger of interacting networks similar to the one we describe.

The sex determination master switch, Sex-lethal, has been shown to regulate the mitosis of early germ cells in Drosophila melanogaster. Sex-lethal is an RNA binding protein that regulates splicing and translation of specific targets in the soma, but the germline targets are unknown. In an experiment aimed at identifying targets of Sex-lethal in early germ cells, the RNA encoded by gutfeeling, the Drosophila homolog of Ornithine Decarboxylase Antizyme, was isolated. gutfeeling interacts genetically with Sex-lethal. It is not only a target of Sex-lethal, but also appears to regulate the nuclear entry and overall levels of Sex-lethal in early germ cells. This regulation of Sex-lethal by gutfeeling appears to occur downstream of the Hedgehog signal. We also show that Hedgehog, Gutfeeling, and Sex-lethal function to regulate Cyclin B, providing a link between Sex-lethal and mitosis.

The sex determination master switch, Sex-lethal (Sxl), controls sexual development as a splicing and translational regulator. Hedgehog (Hh) is a secreted protein that specifies cell fate during development. We show that Sxl is in a complex that contains all of the known Hh cytoplasmic components, including Cubitus interruptus (