Autosomal mutations that interfere with sex determination in somatic cells of Drosophila have no direct effect on the germline

In Drosophila, mutations at the transformer-2, the double-sex, or the intersex loci interfere with the normal sexual development of somatic cells. In order to study the effect of mutations at these loci on germ cell development, mutant germ cells were introduced into normal female or normal male gonads either by pole cell transplantation or by induced mitotic recombination, and their ability to give rise to functional gametes was tested. In contrast to the effects seen in mutant somatic cells, the mutant germ cells ($\text{dsx}\text{dsx}\text{,}\text{dsx}{}^{\mathrm{D}}\text{+}\text{,}\text{dsx}{}^{\mathrm{d}}\text{dsx}\text{,}\text{tra-2}\text{tra-2}\text{,}\text{ix}\text{ix}$) developed normally according to their chromosomal sex, and no sexual transformations of germ cells were observed. In combination with the results of J. L. Marsh and E. Wieschaus (1978, Nature (London)272, 249–251) concerning the transformer locus, it seems that the four autosomal loci known to be involved in sex determination of somatic cells have no important role in the sexual development of the germline. The results are discussed with respect to sex determination in the germline of Drosophila and are compared to observations made on similar mutations in other animal species.     

Sxl in the germline of Drosophila: A target for somatic late induction

In Drosophila, the sex of germ cells is determined by autonomous and inductive signals. Somatic inductive signals can drive XX germ cells into oogenesis or into spermatogenesis. An autonomous signal makes XY germ cells male and unresponsive to sex determination by induction. The elements forming the X:A ratio in the soma and the genes tra, tra2, dsx, and ix that determine the sex of somatic cells have no similar role in the germline. The gene Sxl, however, is required for female differentiation of somatic and germ cells. Inductive signals that are dependent on somatic tra and dsx expression already affect the sex-specific development of germ cells of first instar larvae. At this early stage, however, germline expression of Sxl does not appear to affect the sexual characteristics of germ cells. Since inductive signals dependent on tra and dsx nevertheless influence the choice of sex-specific splicing of Sxl, it can be concluded that Sxl is a target of the inductive signal, but that its product is required late for oogenesis. Other genes must therefore control the early sexual dimorphism of larval germ cells. © 1994 Wiley-Liss, Inc.     

Tribolium castaneum Transformer-2 regulates sex determination and development in both males and females

Tribolium castaneum Transformer (TcTra) is essential for female sex determination and maintenance through the regulation of sex-specific splicing of doublesex (dsx) pre-mRNA. In females, TcTra also regulates the sex-specific splicing of its own pre-mRNA to ensure continuous production of functional Tra protein. Transformer protein is absent in males and hence dsx pre-mRNA is spliced in a default mode. The mechanisms by which males inhibit the production of functional Tra protein are not known. Here, we report on functional characterization of transformer-2 (tra-2) gene (an ortholog of Drosophila transformer-2) in T. castaneum. RNA interference-mediated knockdown in the expression of gene coding for tra-2 in female pupae or adults resulted in the production of male-specific isoform of dsx and both female and male isoforms of tra suggesting that Tra-2 is essential for the female-specific splicing of tra and dsx pre-mRNAs. Interestingly, knockdown of tra-2 in males did not affect the splicing of dsx but resulted in the production of both female and male isoforms of tra suggesting that Tra-2 suppresses female-specific splicing of tra pre-mRNA in males. This dual regulation of sex-specific splicing of tra pre-mRNA ensures a tight regulation of sex determination and maintenance. These data suggest a critical role for Tra-2 in suppression of female sex determination cascade in males. In addition, RNAi studies showed that Tra-2 is also required for successful embryonic and larval development in both sexes.     

The role of the transformer genes in the development of genitalia and analia of Drosophila melanogaster

The autosomal mutations transformer (tra) and transformer-2 (tra-2) of Drosophila convert chromosomal females (X/X) into phenotypical males. Our analysis aims at an understanding of the role which the transformer genes play in the development of the sexually dimorphic genital disc. In each Drosophila embryo, this disc starts development with a male and a female genital primordium, and an anal primordium. Our experiments involved the production of cell clones that were made homozygous for tra or tra-2 at different times of development. Homozygous clones were obtained by inducing mitotic recombination in three types of females heterozygous for tra or tra-2. The cells of the homozygous tra/tra or tra-2/tra-2 clones responded by changing from the female into the male pathway. Male genital structures developed if the clones were induced not later than 81 hr into development. In the analia, male clones appeared up to 120 hr. Our results show that the action of the wild-type alleles of tra⁺ and tra-2⁺ is required until late in larval development to repress the male genital primordium and to support development of the female primordium, as well as to maintain the anal primordium in the female pathway. Our data also suggest that the embryonic genital disc consists of two compartments, one containing the precursors for penis and analia, the other those of the male and female genitalia.     

A Maternal-Effect Sex-Transformation Mutant of the Housefly, MUSCA DOMESTICA L

A maternal-effect sex-transformation mutant, transformer (tra), of the housefly is described. It is located on autosome 4 in close linkage with the Ba locus. Normally, the sex of Musca domestica is determined by the presence or absence of an epistatic factor, M. When produced by tra/tra mothers, a large fraction of the tra/tra genotypic female progeny carrying no M factors are transformed to develop into intersexes or fertile phenotypic males. The tra/+ progeny are also transformed, but less frequently. Aging of the mothers increases the frequency of sex-transformed flies. When produced by tra/+ mothers, tra/tra progeny (but not +/tra) occasionally undergo sex transformation. Thus, tra⁺ is active both maternally and zygotically. Genotypic males carrying the M factor are not affected by the tra mutant. It is concluded that the tra⁺ gene product is required for female determination and/or differentiation. A model is proposed to explain actions of all the known sex-determination genes in M. domestica , and it is discussed in relation to sex-determination mechanisms in several other insect species.     

Genetic control and expression of the major ejaculatory bulb protein (PEB-me) inDrosophila melanogaster

PEB-me is a predominant protein of matureDrosophila melanogaster ejaculatory bulbs. It is resolved into four or five closely spaced subfractions (apparent molecular weight 35–39 kD) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four electrophoretic variants of PEB-me differing in apparent molecular weight by 200–800 daltons were found. These appear to be controlled by four alleles of a gene (peb) located by recombination and deletion mapping to the 60F1-2 region of chromosome 2. A minor ejaculatory bulb protein of ca. 80 kD (hPEB) was found to be immunochemically related to PEB and possibly encoded bypeb. PEB is not detected by immunoblotting techniques in virgin females, in male tissues other than the ejaculatory bulb, or during developmental stages preceding the formation of this organ. The results of transplantations of genital imaginal discs and of immature ejaculatory bulbs between two strains having different PEB alleles suggest that the ejaculatory bulb is the site of PEB synthesis. In flies mutant fortra, tra-2, dsx, orix, tissue specificity of PEB localization is retained and the protein is found whenever the ejaculatory bulb is formed, regardless of the chromosomal sex of the fly. The protein is transferred into the female genital duct during mating, where it can be detected for up to 12 hr. Possible functions of PEB inDrosophila reproduction are discussed.     

The dual functions of a sex determination gene in Drosophila melanogaster

The wild-type function of the sex transforming gene transformer-2 (tra-2) is shown to be required for normal spermatogenesis in XY males. A temperature-shift experiment using the tra-2^ts2 allele suggests that tra-2⁺ must function during the middle stages of spermatogenesis to ensure development of functional sperm. Our results, taken together with those of T. Schüpbach (1982, Dev. Biol.89, 117–127) indicate that the tra-2⁺ gene functions in the male germ line and thus, in contrast to all other sex determination loci examined to date (doublesex, intersex, transformer), its action is not limited to the soma. Orcein-stained testis preparations from tra-2 males reveal a spermiogenic defect similar to that associated with dominant male sterile (X; autosome) translocations.     

Regulation of Sex-Specific Selection of fruitless 5′ Splice Sites by transformer and transformer-2

In Drosophila melanogaster, the fruitless (fru) gene controls essentially all aspects of male courtship behavior. It does this through sex-specific alternative splicing of the fru pre-mRNA, leading to the production of male-specific fru mRNAs capable of expressing male-specific fru proteins. Sex-specific fru splicing involves the choice between alternative 5′ splice sites, one used exclusively in males and the other used only in females. Here we report that the Drosophila sex determination genes transformer (tra) and transformer-2 (tra-2) switch fru splicing from the male-specific pattern to the female-specific pattern through activation of the female-specific fru 5′ splice site. Activation of female-specific fru splicing requires cis-acting tra and tra-2 repeat elements that are part of an exonic splicing enhancer located immediately upstream of the female-specific fru 5′ splice site and are recognized by the TRA and TRA-2 proteins in vitro. This fru splicing enhancer is sufficient to promote the activation by tra and tra-2 of both a 5′ splice site and the female-specific doublesex (dsx) 3′ splice site, suggesting that the mechanisms of 5′ splice site activation and 3′ splice site activation may be similar.     

The temperature-sensitive mutation vir ts(virilizer) identifies a new gene involved in sex determination of Drosophila

Summary When XX animals homozygous for the temperature-sensitive mutation vir ^tsof virilizer (2–103.9) are raised at the restrictive temperature of 29° C, they are transformed into sterile intersexes with a morphology comparable to XX flies mutant at the sex-determining gene doublesex (dsx). The gonads of the vir ^tsintersexes are ovaries in which the germ cells undergo abortive oogenesis. At the permissive temperature of 25° C or below, XX vir ^tsanimals develop into marginally fertile females. The temperature-sensitive period of vir ^tsis within the third larval instar. XY males are not affected by the mutation. Animals that are homozygous for vir ^tsand either transformer (tra) or tra2 develop as pseudomales; on the other hand, constitutive expression of a female-specific tra product rescues XX animals from the effect of vir ^ts, but these females are sterile. The data show that tra and tra2 are epistatic to vir. Animals with only one wildtype copy of either tra or tra2 and mutant for vir ^tsare already transformed into intersexes at 25° C. Conversely, the presence of three copies of the tra ⁺ gene largely prevents the effect of vir ^tsat 29° C; such flies are practically female, but sterile. Animals homozygous for vir ^tsand heterozygous for dsx ^D/+, raised at 29° C, are transformed into severely masculinized intersexes or almost pseudomales. The observations suggest that vir acts above and via tra and tra2 to achieve proper female-specific expression of the dsx gene in XX zygotes. Offprint requests to: R. Nöthiger     

Interaction of the mutants sx,tra and dsx in Drosophila melanogaster. Chaetotaxy of the basitarsus of the forelegs in males and females

The basitarsal bristle pattern of the mutants sx (sexcombless), tra (transformer), and dsx (doublesex), and of the combinations sx-dsx and tra-dsx is described. Epistasis of dsx over both sx and tra for many of the chaetotaxal characteristics was found. The various effects of interaction observed, in individuals of male as well as female chromosomal constitution, are discussed in the light of the levels of action of the mutant genes in modifying the development of sex. It is suggested that intersexes induced by dsx are a class by itself, and that the action of dsx might be at a primary level of sex determination.     

Male- and female-specific variants of doublesex gene products have different roles to play towards regulation of Sex combs reduced expression and sex comb morphogenesis in Drosophila

Sexually dimorphic characters have two-fold complexities in pattern formation as they have to get input from both somatic sex determination as well as the positional determining regulators. Sex comb development in Drosophila requires functions of the somatic sex-determining gene doublesex and the homeotic gene Sex combs reduced. Attempts have not been made to decipher the role of dsx in imparting sexually dimorphic expression of SCR and the differential function of sex-specific variants of dsx products in sex comb development. Our results in this study indicate that male-like pattern of SCR expression is independent of dsx function, and dsx ^F must be responsible for bringing about dimorphism in SCR expression, whereas dsx ^M function is required with Scr for the morphogenesis of sex comb.     

Autoregulatory Functioning of a Drosophila Gene Product That Establishes and Maintains the Sexually Determined State

Sxl appears to head a regulatory gene hierarchy that controls Drosophila sexual dimorphism in response to the X chromosome/autosome balance. Only XXAA cells normally have Sxl⁺ activity. It maintains both the female morphogenetic sequence and a level of X-linked dosage-compensated gene expression compatible with diplo-X cell survival. In the absence of this activity, male sexual development and dosage-compensated gene hyperactivation ensure. Loss-of-function Sxl mutations generally have female-specific lethal effects caused by upsets in dosage compensation. New female-viable Sxl mutant alleles and combinations which lack Sxl's female sex determination function, yet still provide sufficient dosage compensation function for diplo-X survival, are described here. Consequently, such mutants cause genotypic females to develop as phenotypic males. Some of these sex-transforming Sxl mutants do not require the maternally produced da⁺ activity that is normally essential for the functioning of zygotic Sxl alleles. In this paper, products of these unusual alleles are shown to act in trans to induce the expression of zygotic Sxl⁺ alleles that would otherwise be unable to function due to a lack of maternal da⁺ activity. This result indicates a third function for Sxl⁺ product: a positive autoregulatory role. Controls for the autoregulation experiments demonstrated the sex-trans-forming epigenetic effect of the da mutation for the first time in diploids. In these experiments the female-specific zygotic lethal effects that normally would have accompanied loss of maternal da⁺ activity were suppressed by mutations known to block dosage-compensation gene hyperactivation—the autosomal, male-specific lethals. Three types of abnormal sexual phenotypes were produced in the experiments described here, each with important implications for the mechanism of sex determination: (1) a true intersex phenotype produced by one particular Sxl allele shows that Sxl⁺ must be involved in the cellular response to the X/A balance rather than in its establishment; (2) a maternally induced, female-sterile phenotype indicates that either the process of autoregulation or the mutants used to demonstrate it are tissue specific and (3) a mosaic intersexual phenotype whose character implies that the Sxl⁺ activity level is set early in development, both by the da ⁺-mediated X/A balance signal and by autoregulation, and is maintained subsequently in a cell autonomous fashion, independent of the initiating X/A balance signal. Thus, this study supports the view that sex determination is truly determinative in the standard developmental sense, and that Sxl is the carrier of the sexually determined state.     

Unequal Crossing over at the Rrna Tandon as a Source of Quantitative Genetic Variation in Drosophila

Abdominal bristle selection lines (three high and three low) and controls were founded from a marked homozygous line to measure the contribution of sex-linked "mutations" to selection response. Two of the low lines exhibited a period of rapid response to selection in females, but not in males. There were corresponding changes in female variance, in heritabilities in females, in the sex ratio (a deficiency of females) and in fitness, as well as the appearance of a mutant phenotype in females of one line. All of these changes were due to bb alleles (partial deficiencies for the rRNA tandon) in the X chromosomes of these lines, while the Y chromosomes remained wild-type bb⁺. We argue that the bb alleles arose by unequal crossing over in the rRNA tandon.—A prediction of this hypothesis is that further changes can occur in the rRNA tandon as selection is continued. This has now been shown to occur.—Our minimum estimate of the rate of occurrence of changes at the rRNA tandon is 3 x 10^-4. As this is substantially higher than conventional mutation rates, the questions of the mechanisms and rates of origin of new quantitative genetic variation require careful re-examination.     

Genetic Analyses of "Paranoiac" Mutants of PARAMECIUM TETRAURELIA

Six mutants of Paramecium tetraurelia with curious "Paranoiac" phenotypes have been isolated and examined. Instead of the normal transient avoiding reactions in Na⁺ solutions, these mutants show "violent avoidances"—backing continuously for 10 to over 60 sec. This behavior corresponds to prolonged membrane excitation.—Genetic analyses establish five genic loci at which mutations give the "Paranoiac" phenotype. Close linkage between two of these genes occurs. Allelic variants are found for two of the genes. In one case, the two alleles determine very different behavioral phenotypes ("Paranoiac" and "fast-2"). These results show that the mechanism(s) which shuts off excitation in the wild-type membrane is (are) complex, but in the future may be fruitfully pursued in mutants which are defective.     

Novel nematode amber suppressors

Nine amber suppressor mutations were isolated in the nematode Caenorhabditis elegans by reverting amber alleles of a sex-determining gene, tra-3. One suppressor maps to a known locus, sup-5 III , but the other eight map to three new loci, sup-21 X (five alleles), sup-22 IV (two alleles) and sup-23 IV (one allele). Amber alleles of tra-3 and of a dumpy gene, dpy-20, were used to measure the efficiency of suppression; the sup-21 and the sup-22 alleles were both shown to be heterogeneous and generally weaker suppressors than sup-5 alleles, which are homogeneous. The spectrum of mutations suppressed by a strong sup-21 allele, e1957, was investigated and compared to the spectra for the amber suppressors sup-5 III and sup-7 X, using amber alleles in 13 assorted genes. Some of the differences between these spectra may be due to limited tissue specificity in sup-21 expression.—Suppression of dpy-20 was used to show that the sex-linked suppressors sup-7 and sup-21 are not dosage compensated in male (XO) relative to hermaphrodite (XX).—Several uses of amber suppressors are critically discussed: for identifying null mutations, for varying levels of gene activity and for detecting maternal mRNA.     

Sex determination in flies,fruitflies and butterflies

Sex determination mechanisms, differing in their modality, are widely represented in all the various animal taxa, even at the intraspecific level. Within the highly diversified Class Insecta, Drosophila has been used to unravel the mechanistic molecular and genetic interactions that are involved in sex determination. Indeed, the molecularly characterized genes of the Drosophila sex determination hierarchy X:A> Sxl> tra> dsxhave been fruitful starting points in the cloning of homologous genes from other insect species. This cascade seems to control sex determination in all Drosophila species. However, no sex-specific regulatory Sxlhomologues have been isolated from the Mediterranean fruitfly (medfly), Ceratitis capitata, the housefly, Musca domestica, Chrysomya rufifaciesnor from the distantly related phorid fly Megaselia scalaris. Moreover, all these other species use primary signals different from the intricate X:A counting system of Drosophila. However, dsxhomologues isolated from these and other dipteran species as well as from the silkmoth, Bombyx mori, share a conserved sex-specific regulation based on alternative splicing. An understanding of the sex determination mechanisms in insects that are of agricultural or public health importance may help in the development of improved methods for their control using the sterile insect technique.     

A Study of Rdna Magnification Phenomenon in a Repair-Recombination Deficient Mutant of DROSOPHILA MELANOGASTER

The rDNA magnification process consists of a rapid and inheritable rDNA increase occurring in bobbed males: in a few generations the bb loci acquire the wild-type rDNA value and reach a bb⁺ phenotype.—We have analyzed the rDNA magnification process in the repair-recombination-deficient mutant mei9^a, both at the phenotypical and rDNA content levels. In mei9^a bb double mutants the recovery of bb⁺ phenotype is strongly disturbed and the rDNA redundancy value fails to reach the wild-type level. The strong effect of this meiotic mutation on rDNA magnification suggests a close relationship between this phenomenon and the repair-recombination processes.     

Complex Adaptations Can Drive the Evolution of the Capacitor [PSI +], Even with Realistic Rates of Yeast Sex

The [PSI⁺] prion may enhance evolvability by revealing previously cryptic genetic variation, but it is unclear whether such evolvability properties could be favored by natural selection. Sex inhibits the evolution of other putative evolvability mechanisms, such as mutator alleles. This paper explores whether sex also prevents natural selection from favoring modifier alleles that facilitate [PSI⁺] formation. Sex may permit the spread of “cheater” alleles that acquire the benefits of [PSI⁺] through mating without incurring the cost of producing [PSI⁺] at times when it is not adaptive. Using recent quantitative estimates of the frequency of sex in Saccharomyces paradoxus, we calculate that natural selection for evolvability can drive the evolution of the [PSI⁺] system, so long as yeast populations occasionally require complex adaptations involving synergistic epistasis between two loci. If adaptations are always simple and require substitution at only a single locus, then the [PSI⁺] system is not favored by natural selection. Obligate sex might inhibit the evolution of [PSI⁺]-like systems in other species.