A FEMALE-SPECIFIC LETHAL LESION IN AN X-LINKED POSITIVE REGULATOR OF THE DROSOPHILA SEX DETERMINATION GENE, SEX-LETHAL

ABSTRACT Characterization of a partial-loss-of-function, female-specific lethal mutation has identified an X-linked genetic element (1-34.3; 10B4) that functions as a positive regulator of Sxl, a central gene controlling sex determination in Drosophila melanogaster. The name, sisterless-a, was chosen both to suggest functional similarities that exist between this gene and another positive regulator of Sxl, the maternally acting gene daughterless (da), and also to highlight an important difference; namely, that in contrast to da, it is the zygotic rather than maternal functioning of sis-a that is involved in its interaction with Sxl. As with da, the female-specific lethal phenotype of sis-a is suppressed both by SxlM #1, a gain-of-function mutant allele of the target gene, and, to a lesser extent, by a duplication of Sxl +. Mutations at sis-a, da and Sxl display female-specific dominant synergism, each enhancing the others' lethal effects. The allele specificity with respect to Sxl of these dominant interactions indicates that sis-a and da affect the same aspect of Sxl regulation. As with previous studies of da and Sxl, the masculinizing effects of loss of sis-a function are generally obscured by lethal effects, presumably related to upsets in dosage compensation. The masculinizing effects can be dissociated from lethal effects by analysis of triploid intersexes (XX AAA) or by analysis of diploid females who are also mutant for autosomal genes known to be required for the transcriptional hyperactivation associated with dosage compensation in males. Analysis of foreleg development shows that intersexuality generated by sis-a is of the mosaic type: At the level of individual cells, only male or female development is observed, never an intermediate sexual phenotype characteristic of true intersexes. Sexual development of diplo-X germline and somatic clones of sis-a tissue generated by mitotic recombination during larval stages is normal, as is the sexual phenotype of homozygous sis-a escapers. Considered in their totality, these results indicate that sis-a functions early in development to help establish the activity state of Sxl and thereby initiate the sexual pathway commitment, rather than functioning later in the processes by which Sxl maintains and expresses the sex determination decision.

Download Full-text

AUTOREGULATORY FUNCTIONING OF A DROSOPHILA GENE PRODUCT THAT ESTABLISHES AND MAINTAINS THE SEXUALLY DETERMINED STATE

Genetics ◽

10.1093/genetics/107.2.231 ◽

1984 ◽

Vol 107 (2) ◽

pp. 231-277

Author(s):

Thomas W Cline

Keyword(s):

Sex Determination ◽

Dosage Compensation ◽

Cellular Response ◽

Regulatory Gene ◽

Activity Level ◽

Loss Of Function ◽

Lethal Effects ◽

Epigenetic Effect ◽

Female Specific ◽

Male Specific

ABSTRACT 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.

Download Full-text

Genetic and molecular analysis of the autosomal component of the primary sex determination signal of Drosophila melanogaster.

Genetics ◽

10.1093/genetics/141.4.1451 ◽

1995 ◽

Vol 141 (4) ◽

pp. 1451-1471 ◽

Cited By ~ 1

Author(s):

D A Barbash ◽

T W Cline

Keyword(s):

Drosophila Melanogaster ◽

Sex Determination ◽

Time Course ◽

Loss Of Function ◽

Lethal Effects ◽

Genome Wide ◽

The Face ◽

Individual Contributions ◽

Female Specific ◽

Inappropriate Expression

Abstract Drosophila sex is determined by the action of the X:A chromosome balance on transcription of Sex-lethal (Sxl), a feminizing switch gene. We obtained loss-of-function mutations in denominator elements of the X:A signal by selecting for dominant suppressors of a female-specific lethal mutation in the numerator element, sisterlessA (sisA). Ten suppressors were recovered in this extensive genome-wide selection. All were mutations in deadpan (dpn), a pleiotropic locus previously discovered to be a denominator element. Detailed genetic and molecular characterization is presented of this diverse set of new dpn alleles including their effects on Sxl. Although selected only for impairment of sex-specific functions, all were also impaired in nonsex-specific functions. Male-lethal effects were anticipated for mutations in a major denominator element, but we found that viability of males lacking dpn function was reduced no more than 50% relative to their dpn- sisters. Moreover, loss of dpn activity in males caused only a modest derepression of the Sxl "establishment" promoter (Sxlpe), the X:A target. By itself, dpn cannot account for the masculinizing effect of increased autosomal ploidy, the effect that gave rise to the concept of the X:A ratio; nevertheless, if there are other denominator elements, our results suggest that their individual contributions to the sex-determination signal are even less than that of dpn. The time course of expression of dpn and of Sxl in dpn mutant backgrounds suggests that dpn is required for sex determination only during the later stages of X:A signaling in males to prevent inappropriate expression of Sxlpe in the face of increasing sis gene product levels.

Download Full-text

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

Development ◽

10.1242/dev.128.6.1015 ◽

2001 ◽

Vol 128 (6) ◽

pp. 1015-1018 ◽

Cited By ~ 1

Author(s):

T.W. Cline

Keyword(s):

Y Chromosome ◽

Rdna Locus ◽

Loss Of Function ◽

Wild Type ◽

Female Development ◽

Regulatory Relationship ◽

Specific Expression ◽

X Chromosomes ◽

Positive Regulator ◽

Female Specific

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.

Download Full-text

The dual role of hermaphrodite in the Drosophila sex determination regulatory hierarchy

Development ◽

10.1242/dev.121.1.99 ◽

1995 ◽

Vol 121 (1) ◽

pp. 99-111 ◽

Cited By ~ 6

Author(s):

M.A. Pultz ◽

B.S. Baker

Keyword(s):

Sex Determination ◽

Dosage Compensation ◽

Regulatory Protein ◽

Female Offspring ◽

Normal Female ◽

Female Development ◽

Salivary Gland Cells ◽

Maternal Function ◽

Different Levels

The hermaphrodite (her) locus has both maternal and zygotic functions required for normal female development in Drosophila. Maternal her function is needed for the viability of female offspring, while zygotic her function is needed for female sexual differentiation. Here we focus on understanding how her fits into the sex determination regulatory hierarchy. Maternal her function is needed early in the hierarchy: genetic interactions of her with the sisterless genes (sis-a and sis-b), with function-specific Sex-lethal (Sxl) alleles and with the constitutive allele SxlM#1 suggest that maternal her function is needed for Sxl initiation. When mothers are defective for her function, their daughters fail to activate a reporter gene for the Sxl early promoter and are deficient in Sxl protein expression. Dosage compensation is misregulated in the moribund daughters: some salivary gland cells show binding of the maleless (mle) dosage compensation regulatory protein to the X chromosome, a binding pattern normally seen only in males. Thus maternal her function is needed early in the hierarchy as a positive regulator of Sxl, and the maternal effects of her on female viability probably reflect Sxl's role in regulating dosage compensation. In contrast to her's maternal function, her's zygotic function in sex determination acts at the end of the hierarchy. This zygotic effect is not rescued by constitutive Sxl expression, nor by constitutive transformer (tra) expression. Moreover, the expression of doublesex (dsx) transcripts appears normal in her mutant females. We conclude that the maternal and zygotic functions of her are needed at two distinctly different levels of the sex determination regulatory hierarchy.

Download Full-text

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

Genetics Research ◽

10.1017/s0016672300030470 ◽

1992 ◽

Vol 59 (3) ◽

pp. 189-198 ◽

Cited By ~ 26

Author(s):

Miguel Torres ◽

Lucas Sanchez

Keyword(s):

Sex Determination ◽

Dosage Compensation ◽

Relative Number ◽

Initial Step ◽

The Other ◽

Loss Of Function ◽

X Chromosomes ◽

Segmentation Gene ◽

Ectopic Activation ◽

Sex Lethal

SummaryIn 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 femalespecific lethality and sexual transformation of XX animals that are heterozygous for Sxl or sis loss-of-function mutations. These interactions are suppressed by SxlMI, 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.

Download Full-text

Genetic and molecular analysis of new female-specific lethal mutations at the gene Sxl of Drosophila melanogaster.

Genetics ◽

10.1093/genetics/129.2.371 ◽

1991 ◽

Vol 129 (2) ◽

pp. 371-383 ◽

Cited By ~ 1

Author(s):

B Granadino ◽

M Torres ◽

D Bachiller ◽

E Torroja ◽

J L Barbero ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Point Mutation ◽

Dosage Compensation ◽

The Other ◽

Initial Activity ◽

Lethal Mutations ◽

Dna Insertion ◽

Complementation Groups ◽

Activity State ◽

Female Specific

Abstract We have isolated three female-specific lethal mutations at the gene Sex-lethal (Sxl): Sxlfb, Sxlfc and Sxlfd. We have carried out the complementation analysis between these mutations and other previously reported Sxlf mutations. It is possible to classify the alleles tested in this report into two complementation groups: the bc group defined by Sxlfb, and Sxlfc, and the LS group defined by SxlfLS. The other alleles tested affect both complementation groups albeit with different degrees. Contrary to what happens with mutations at the LS group, mutations at the bc group do not affect sex determination, nor late dosage compensation nor oogenesis. Both Sxlfb and Sxlfc present a DNA insertion of at least 5 kb between position -10 and -11 on the molecular map, within the fourth intron. On the contrary, Sxlfd, a strong mutation affecting all Sxl functions, is not associated to any detectable DNA alteration in Southern blots, so that it seems to be a "point" mutation. In agreement with their phenotypes, both Sxlfc/SxlfLS and Sxlfc homozygous female larvae express only the late Sxl transcripts characteristic of females, while females homozygous for SxlfLS express only the late Sxl transcripts characteristic of males. Moreover, Sxlfc presents a lethal synergistic interaction with mutations at either da or the X:A ratio, two signals that define the initial activity state of Sxl, while SxlfLS do not. These data suggest that the two complementation groups are related to the two sets of early and late Sxl transcripts, which are responsible for the early and late Sxl functions, respectively: Sxlfb and Sxlfc would affect the early functions and SxlfLS would affect the late Sxl functions.

Download Full-text

A conserved role of the duplicated Masculinizer gene in sex determination of the Mediterranean flour moth, Ephestia kuehniella

10.1101/2021.02.15.431251 ◽

2021 ◽

Author(s):

Sander Visser ◽

Anna Voleníková ◽

Petr Nguyen ◽

Eveline C. Verhulst ◽

František Marec

Keyword(s):

Sex Determination ◽

Early Embryogenesis ◽

Ephestia Kuehniella ◽

Z Chromosome ◽

Female Development ◽

Biased Sex Ratio ◽

The Mediterranean ◽

Female Specific ◽

Mediterranean Flour Moth

AbstractSex determination in the silkworm, Bombyx mori, is based on Feminizer (Fem), a W-linked Fem piRNA that triggers female development in WZ individuals, and the Z-linked Masculinizer (Masc), which initiates male development and dosage compensation in ZZ individuals. While Fem piRNA is missing in a close relative of B. mori, Masc determines sex in several representatives of distant lepidopteran lineages. We studied the molecular mechanisms of sex determination in the Mediterranean flour moth, Ephestia kuehniella (Pyralidae). We identified an E. kuehniella Masc ortholog, EkMasc, and its paralog resulting from a recent duplication, EkMascB. Both genes are located on the Z chromosome and encode a similar Masc protein that contains two conserved domains but has lost the conserved double zinc finger domain. We developed PCR-based genetic sexing and demonstrated a peak in the expression of EkMasc and EkMascB genes only in early male embryos. Simultaneous knock-down experiments of both EkMasc and EkMascB using RNAi during early embryogenesis led to a shift from male- to female-specific splicing of the E. kuehniella doublesex gene (Ekdsx), their downstream effector, in ZZ embryos and resulted in a strong female-biased sex-ratio. Our results thus confirmed the conserved role of both EkMasc and EkMascB genes in masculinization. We suggest that the C-terminal proline-rich domain, we have identified in all functionally confirmed Masc proteins, in conjunction with the masculinizing domain, is important for transcriptional regulation of sex determination in Lepidoptera. The function of the Masc double zinc finger domain is still unknown, but appears to have been lost in E. kuehniella.Author summaryThe sex-determining cascade in the silkworm, Bombyx mori, differs greatly from those of other insects. In B. mori, female development is initiated by Fem piRNA expressed from the W chromosome during early embryogenesis. Fem piRNA silences Masculinizer (Masc) thereby blocking the male pathway resulting in female development. It is currently unknown whether this cascade is conserved across Lepidoptera. In the Mediterranean flour moth, Ephestia kuehniella, we identified an ortholog of Masc and discovered its functional duplication on the Z chromosome, which has not yet been found in any other lepidopteran species. We provide two lines of evidence that both the EkMasc and EkMascB genes play an essential role in masculinization: (i) they show a peak of expression during early embryogenesis in ZZ but not in WZ embryos and (ii) their silencing by RNAi results in female-specific splicing of the E. kuehniella doublesex gene (Ekdsx) in ZZ embryos and in a female-biased sex ratio. Our results suggest a conserved role of the duplicated Masc gene in sex determination of E. kuehniella.

Download Full-text

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

Genetics ◽

10.1093/genetics/144.1.95 ◽

1996 ◽

Vol 144 (1) ◽

pp. 95-108

Author(s):

Helen K Salz ◽

Thomas W Flickinger

Keyword(s):

Sex Determination ◽

Rna Splicing ◽

Loss Of Function ◽

Functional Homology ◽

Snrnp Protein ◽

A Cell ◽

Sex Lethal ◽

Mutant Phenotypes ◽

Female Specific ◽

Male Specific

Abstract The Drosophila snf gene encodes a protein with functional homology to the mammalian UlA 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, snfJA2, is a partial loss-of-function mutation. The other two viable alleles, snf1621 and snfe8H, 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.

Download Full-text

Genetic evidence that the sans fille locus is involved in Drosophila sex determination.

Genetics ◽

10.1093/genetics/120.1.159 ◽

1988 ◽

Vol 120 (1) ◽

pp. 159-171

Author(s):

B Oliver ◽

N Perrimon ◽

A P Mahowald

Keyword(s):

Sex Determination ◽

Dosage Compensation ◽

Germ Line ◽

Ovarian Tumors ◽

Lethal Gene ◽

Nurse Cells ◽

Loss Of Function ◽

Wild Type ◽

Sex Lethal

Abstract Females homozygous for sans fille1621 (= fs(1)1621) have an abnormal germ line. Instead of producing eggs, the germ-line cells proliferate forming ovarian tumors or excessive numbers of nurse cells. The Sex-lethal gene product(s) regulate the branch point of the dosage compensation and sex determination pathways in the soma. The role of Sex-lethal in the germ line is not clear but the germ line of females homozygous for female sterile Sex-lethal alleles or germ-line clones of loss-of-function alleles are characterized by ovarian tumors. Females heterozygous for sans fille1621 or Sex-lethal are phenotypically wild type with respect to viability and fertility but females trans-heterozygous for sans fille1621 and Sex-lethal show ovarian tumors, somatic sexual transformations, and greatly reduced viability.

Download Full-text

Conditional knockdown of transformer in sheep blow fly suggests a role in repression of dosage compensation and potential for population suppression

PLoS Genetics ◽

10.1371/journal.pgen.1009792 ◽

2021 ◽

Vol 17 (10) ◽

pp. e1009792

Author(s):

Megan E. Williamson ◽

Ying Yan ◽

Maxwell J. Scott

Keyword(s):

X Chromosome ◽

Dosage Compensation ◽

Rna Splicing ◽

Pupal Stage ◽

Mrna Levels ◽

Loss Of Function ◽

Blow Fly ◽

Female Development ◽

Australian Sheep ◽

Xx Males

The transformer (tra) gene is essential for female development in many insect species, including the Australian sheep blow fly, Lucilia cuprina. Sex-specific tra RNA splicing is controlled by Sex lethal (Sxl) in Drosophila melanogaster but is auto-regulated in L. cuprina. Sxl also represses X chromosome dosage compensation in female D. melanogaster. We have developed conditional Lctra RNAi knockdown strains using the tet-off system. Four strains did not produce females on diet without tetracycline and could potentially be used for genetic control of L. cuprina. In one strain, which showed both maternal and zygotic tTA expression, most XX transformed males died at the pupal stage. RNAseq and qRT-PCR analyses of mid-stage pupae showed increased expression of X-linked genes in XX individuals. These results suggest that Lctra promotes somatic sexual differentiation and inhibits X chromosome dosage compensation in female L. cuprina. However, XX flies homozygous for a loss-of-function Lctra knockin mutation were fully transformed and showed high pupal eclosion. Two of five X-linked genes examined showed a significant increase in mRNA levels in XX males. The stronger phenotype in the RNAi knockdown strain could indicate that maternal Lctra expression may be essential for initiation of dosage compensation suppression in female embryos.

Download Full-text