Evolution of the Drosophila feminizing switch gene Sex-lethal

Abstract

In Drosophila melanogaster, the gene Sex-lethal (Sxl) controls all aspects of female development. Since melanogaster males lacking Sxl appear wild type, Sxl would seem to be functionally female specific. Nevertheless, in insects as diverse as honeybees and houseflies, Sxl seems not to determine sex or to be functionally female specific. Here we describe three lines of work that address the questions of how, when, and even whether the ancestor of melanogaster Sxl ever shed its non-female-specific functions. First, to test the hypothesis that the birth of Sxl's closest paralog allowed Sxl to lose essential ancestral non-female-specific functions, we determined the CG3056 null phenotype. That phenotype failed to support this hypothesis. Second, to define when Sxl might have lost ancestral non-female-specific functions, we isolated and characterized Sxl mutations in D. virilis, a species distant from melanogaster and notable for the large amount of Sxl protein expression in males. We found no change in Sxl regulation or functioning in the 40+ MY since these two species diverged. Finally, we discovered conserved non-sex-specific Sxl mRNAs containing a previously unknown, potentially translation-initiating exon, and we identified a conserved open reading frame starting in Sxl male-specific exon 3. We conclude that Drosophila Sxl may appear functionally female specific not because it lost non-female-specific functions, but because those functions are nonessential in the laboratory. The potential evolutionary relevance of these nonessential functions is discussed.

Figure 1.—

Molecular steps in the control of Drosophila sex determination by Sxl. Details of this process are described in the Introduction.

Figure 2.—

The phylogenic relationship among Diptera of Sxl and its closest paralog ssx (CG3056). With a phorid fly (M.sca) Sxl as the outgroup among the “higher” Diptera (Brachycera), we aligned Sxl and CG3056 (sister-of-Sex-lethal) with respect to the two RRMs and 11 residues immediately downstream, as well as the 27 (in melanogaster) C-terminal residues corresponding to the exon 8 isoform of D.mel Sxl. See materials and methods for details. A previous estimate of when the paralog-generating Sxl duplication occurred (traut et al. 2006) could say only that it was sometime after the point indicated by the open arrow. Our analysis placed that duplication later (solid arrow, bootstrap value 99%), closer to the time at which Sxl became a master sex-determining switch gene. The evolutionary divergence scale bar is in substitutions per nucleotide. D.mel, Drosophila melanogaster; D.vir, D. virilis; M.dom, Musca domestica (house fly); Ch.Ruf, Chrysomya rufifacies (blow fly); C.cap, Ceratitis capitata (medfly); M.Sca, Megaselia scalaris (scuttle fly).

Figure 3.—

A positive genetic selection scheme for mutations in D. virilis Sxl as suppressors of XSE-duplication–induced male lethality. Each Minos (Mi) transgene carried a copy of D. melanogaster sc and sisA in tandem,. The w⁺ marker on the X-linked transgene X1 generated orange eyes, while that of the autosomal transgene A1 generated red eyes. F₀ males were exposed to 2700 rad. Of the 39,000 F₁ females, 50% were mated in groups of 10, 20% in groups of 5, and 30% in groups of 2. The attached-X chromosome C(1)w was generated for the purpose of this scheme.

Figure 4.—

Lesions in three new D. virilis Sxl mutant alleles. The exon/intron structure of Sxl is shown. Exon 3 is male specific. Established (exons E1 and 2) and proposed (exons Z and 3) translation start sites are labeled “AUG.” Translation termination sites are labeled S. RRM1 and -2 refer to the two RNA recognition motifs. The frameshifting lesion in vSxl^f1 is predicted to destroy all Sxl functions, and the genetic behavior of this allele is consistent with it being a null.

Figure 5.—

Western blot of Sxl proteins from wild-type and Sxl mutant D. virilis male heads and testes. No Sxl proteins were found in extracts of adult males hemizygous for the predicted null allele, vSxl^f1 (vF1). Sxl proteins from males hemizygous for the intragenic deletion allele vSxl^f2/Y (vF2) matched those from wild-type males (vWT) in mobility, but seemed somewhat more abundant. Note the difference between testes and heads with respect to the male Sxl proteins generated. The doublet for Sxl is likely due to the use of alternative exon 5 3′-splice sites (see Figure 6) (Bopp et al. 1991).

Figure 6.—

DNA sequence conservation near the 5′-splice site of the newly discovered Sxl exon Z. Uppercase letters in boldface type indicate positions completely conserved in a diverse collection of six Drosophila and one Scaptodrosophila species [namely, Scaptodrosophila lebanonensis (leb) and the Drosophila species grimshawi (grm), virilis (vir), wilistoni (wil), pseudoobscura (psu), ananassae (ana), and melanogaster (mel)] (Drosophila 12 Genomes Consortium 2007; Siera and Cline 2008). Uppercase letters in regular type signify positions at which all but one of the seven sequences are identical. For D. wilistoni, the sequence CTCTCTGTAAAGAG was present between the brackets. The boxed ATG is the only potential translation start site for each species that would encode nearly full-length Sxl proteins from mRNAs containing exon Z. A corresponding region was apparent in a cDNA from the scuttle fly, Megaselia scalaris (meg) (Sievert et al. 2000) and in genomic DNA from the housefly, Musca domestica (mus) (our sequence). The splice site shown for Megaselia exon Z is based on the authors' revised genomic sequence (AF110846.1) near exon 4 (only cDNA sequence was available near exon Z). The schematic in the bottom section is for D. melanogaster and shows the sex in which the splice sites for each of the indicated Sxl exons are active. Solid bars indicate protein-coding regions. Note that exon Z mRNAs are made by skipping the male-specific exon 3 in both sexes (see Figure 7).

Figure 7.—

Analysis of D. melanogaster mRNAs showing that exon Z is not sex specific and is disproportionately expressed in heads. Primers for this RT–PCR analysis are positioned on the Sxl schematic and described in materials and methods. The pair used for each gel is indicated. One mRNA preparation was used for the two gels on the left and another for the two gels on the right. Estimated sizes of the bands are consistent with expectations for Exon Z mRNAs being generated in both sexes by a 1-Z-4-5 splicing pattern that skips the male-specific exon 3. Predicted sizes of the various RT–PCR products are presented in materials and methods. The CF primer pair indicated that exon Z mRNA is not present in D. melanogaster ovaries. The same was found to be true for the scuttle fly (Sievert et al. 2000).