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

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.

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A transgenic embryonic sexing system for the Australian sheep blow fly Lucilia cuprina

Scientific Reports ◽

10.1038/srep16090 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 23

Author(s):

Ying Yan ◽

Maxwell J. Scott

Keyword(s):

Rna Splicing ◽

Single Gene ◽

Gene Promoter ◽

Pupal Stage ◽

Early Embryo ◽

Lucilia Cuprina ◽

Blow Fly ◽

Tetracycline Transactivator ◽

Australian Sheep ◽

Major Pest

Abstract Genetic approaches, including the sterile insect technique (SIT), have previously been considered for control of the Australian sheep blow fly Lucilia cuprina, a major pest of sheep. In an SIT program, females consume 50% of the diet but are ineffective as control agents and compete with females in the field for mating with sterile males, thereby decreasing the efficiency of the program. Consequently, transgenic sexing strains of L. cuprina were developed that produce 100% males when raised on diet that lacks tetracycline. However, as females die mostly at the pupal stage, rearing costs would not be significantly reduced. Here we report the development of transgenic embryonic sexing strains of L. cuprina. In these strains, the Lsbnk cellularization gene promoter drives high levels of expression of the tetracycline transactivator (tTA) in the early embryo. In the absence of tetracycline, tTA activates expression of the Lshid proapoptotic gene, leading to death of the embryo. Sex-specific RNA splicing of Lshid transcripts ensures that only female embryos die. Embryonic sexing strains were also made by combining the Lsbnk-tTA and tetO-Lshid components into a single gene construct, which will facilitate transfer of the technology to other major calliphorid livestock pests.

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Differential effects of Sex-lethal mutations on dosage compensation early in Drosophila development.

Genetics ◽

10.1093/genetics/136.3.1051 ◽

1994 ◽

Vol 136 (3) ◽

pp. 1051-1061

Author(s):

M Bernstein ◽

T W Cline

Keyword(s):

Sex Determination ◽

X Chromosome ◽

Dosage Compensation ◽

Rna Splicing ◽

Lethal Gene ◽

Loss Of Function ◽

Developmental Pathway ◽

Pathway Choice ◽

Sex Lethal ◽

Blastoderm Stage

Abstract In response to the primary sex determination signal, X chromosome dose, the Sex-lethal gene controls all aspects of somatic sex determination and differentiation, including X chromosome dosage compensation. Two complementary classes of mutations have been identified that differentially affect Sxl somatic functions: (1) those impairing the "early" function used to set developmental pathway choice in response to the sex determination signal and (2) those impairing "late" functions involved in maintaining the pathway choice independent of the initiating signal and/or in directing differentiation. This "early vs. late" distinction correlates with a switch in promoter utilization from SxlPe to SxlPm at the blastoderm stage and a corresponding switch from transcriptional to RNA splicing control. Here we characterize five partial-loss-of-function Sxl alleles to explore a distinction between "early vs. late" functioning of Sxl in dosage compensation. Assaying for dosage compensation during the blastoderm stage, we find that the earliest phase of the dosage compensation process is controlled by products of the early Sxl promoter, SxlPe. Hence, in addition to triggering the sexual pathway decision of cells, products derived from SxlPe also control early dosage compensation, the first manifestation of sexually dimorphic differentiation. The effects of mutant Sxl alleles on early dosage compensation are consistent with their previous categorization as early vs. late defective with respect to their effects on pathway initiation. Results reported here suggest that the dosage compensation regulatory genes currently known to function downstream of Sxl, genes known as the "male-specific lethals," do not control all aspects of dosage compensation either at the blastoderm stage or later in development. In the course of this study, we also discovered that the canonical early defective allele, Sxlf9, which is impaired in its ability to establish the female developmental pathway commitment, is likely to be defective in the stability and/or functioning of products derived from SxlPe, rather than in the ability of SxlPe to respond to the chromosomal sex determination signal.

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Genetic Analysis of X-Chromosome Dosage Compensation in Caenorhabditis elegans

Genetics ◽

10.1093/genetics/117.1.25 ◽

1987 ◽

Vol 117 (1) ◽

pp. 25-41

Author(s):

Philip M Meneely ◽

William B Wood

Keyword(s):

Caenorhabditis Elegans ◽

Genetic Analysis ◽

X Chromosome ◽

Dosage Compensation ◽

Normal Function ◽

Complete Loss ◽

Loss Of Function ◽

Recessive Mutations ◽

Genetic Assay

ABSTRACT We have shown that the phenotypes resulting from hypomorphic mutations (causing reduction but not complete loss of function) in two X-linked genes can be used as a genetic assay for X-chromosome dosage compensation in Caenorhabditis elegans between males (XO) and hermaphrodites (XX). In addition we show that recessive mutations in two autosomal genes, dpy-21 V and dpy-26 IV, suppress the phenotypes resulting from the X-linked hypomorphic mutations, but not the phenotypes resulting from comparable autosomal hypomorphic mutations. This result strongly suggests that the dpy-21 and dpy-26 mutations cause increased X expression, implying that the normal function of these genes may be to lower the expression of X-linked genes. Recessive mutations in two other dpy genes, dpy-22 X and dpy-23 X, increase the severity of phenotypes resulting from some X-linked hypomorphic mutations, although dpy-23 may affect the phenotypes resulting from the autosomal hypomorphs as well. The mutations in all four of the dpy genes show their effects in both XO and XX animals, although to different degrees. Mutations in 18 other dpy genes do not show these effects.

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Transposon insertions causing constitutive Sex-lethal activity in Drosophila melanogaster affect Sxl sex-specific transcript splicing.

Genetics ◽

10.1093/genetics/139.2.631 ◽

1995 ◽

Vol 139 (2) ◽

pp. 631-648

Author(s):

M Bernstein ◽

R A Lersch ◽

L Subrahmanyan ◽

T W Cline

Keyword(s):

Drosophila Melanogaster ◽

X Chromosome ◽

Dosage Compensation ◽

Rna Splicing ◽

Wild Type ◽

Gain Of Function ◽

Positive Regulators ◽

Sex Lethal ◽

Male Specific ◽

Transposon Insertions

Abstract Sex-lethal (Sxl) gene products induce female development in Drosophila melanogaster and suppress the transcriptional hyperactivation of X-linked genes responsible for male X-chromosome dosage compensation. Control of Sxl functioning by the dose of X-chromosomes normally ensures that the female-specific functions of this developmental switch gene are only expressed in diplo-X individuals. Although the immediate effect of X-chromosome dose is on Sxl transcription, during most of the life cycle "on" vs. "off" reflects alternative Sxl RNA splicing, with the female (productive) splicing mode maintained by a positive feedback activity of SXL protein on Sxl pre-mRNA splicing. "Male-lethal" (SxlM) gain-of-function alleles subvert Sxl control by X-chromosome dose, allowing female Sxl functions to be expressed independent of the positive regulators upstream of Sxl. As a consequence, SxlM haplo-X animals (chromosomal males) die because of improper dosage compensation, and SxlM chromosomal females survive the otherwise lethal effects of mutations in upstream positive regulators. Five independent spontaneous SxlM alleles were shown previously to be transposon insertions into what was subsequently found to be the region of regulated sex-specific Sxl RNA splicing. We show that these five alleles represent three different mutant types: SxlM1, SxlM3, and SxlM4. SxlM1 is an insertion of a roo element 674 bp downstream of the translation-terminating male-specific exon. SxlM3 is an insertion of a hobo transposon (not 297 as previously reported) into the 3' splice site of the male exon, and SxlM4 is an insertion of a novel transposon into the male-specific exon itself. We show that these three gain-of-function mutants differ considerably in their ability to bypass the sex determination signal, with SxlM4 being the strongest and SxlM1 the weakest. This difference is also reflected in effects of these mutations on sex-specific RNA splicing and on the rate of appearance of SXL protein in male embryos. Transcript analysis of double-mutant male-viable SxlM derivatives in which the SxlM insertion is cis to loss-of-function mutations, combined with other results reported here, indicates that the constitutive character of these SxlM alleles is a consequence of an alteration of the structure of the pre-mRNA that allows some level of female splicing to occur even in the absence of functional SXL protein. Surprisingly, however, most of the constitutive character of SxlM alleles appears to depend on the mutant alleles' responsiveness, perhaps greater than wild-type, to the autoregulatory splicing activity of the wild-type SXL proteins they produce.

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Sex-biased ZRSR2 mutations in myeloid malignancies impair plasmacytoid dendritic cell activation and apoptosis

10.1101/2020.10.29.360503 ◽

2020 ◽

Author(s):

Katsuhiro Togami ◽

Sun Sook Chung ◽

Vikas Madan ◽

Christopher M. Kenyon ◽

Lucia Cabal-Hierro ◽

...

Keyword(s):

Dendritic Cell ◽

X Chromosome ◽

Rna Splicing ◽

Cell Activation ◽

Intron Retention ◽

Plasmacytoid Dendritic Cell ◽

Sex Bias ◽

Loss Of Function ◽

Dendritic Cell Activation

ABSTRACTBlastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive leukemia of plasmacytoid dendritic cells (pDCs). BPDCN occurs at least three times more frequently in men than women, but the reasons for this sex bias are unknown. Here, studying genomics of primary BPDCN and modeling disease-associated mutations, we link acquired alterations in RNA splicing to abnormal pDC development and inflammatory response through Toll-like receptors. Loss-of-function mutations in ZRSR2, an X chromosome gene encoding a splicing factor, are enriched in BPDCN and nearly all mutations occur in males. ZRSR2 mutation impairs pDC activation and apoptosis after inflammatory stimuli, associated with intron retention and inability to upregulate the transcription factor IRF7. In vivo, BPDCN-associated mutations promote pDC expansion and signatures of decreased activation. These data support a model in which male-biased mutations in hematopoietic progenitors alter pDC function and confer protection from apoptosis, which may impair immunity and predispose to leukemic transformation.STATEMENT OF SIGNIFICANCESex bias in cancer is well recognized but the underlying mechanisms are incompletely defined. We connect X chromosome mutations in ZRSR2 to an extremely male-predominant leukemia. Aberrant RNA splicing induced by ZRSR2 mutation impairs dendritic cell inflammatory signaling, interferon production, and apoptosis, revealing a sex- and lineage-related tumor suppressor pathway.

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Analysis of the circadian clock gene period in the sheep blow fly Lucilia cuprina

Genetics Research ◽

10.1017/s0016672399004425 ◽

2000 ◽

Vol 75 (3) ◽

pp. 257-267 ◽

Cited By ~ 28

Author(s):

G. R. WARMAN ◽

R. D. NEWCOMB ◽

R. D. LEWIS ◽

C. W. EVANS

Keyword(s):

Clock Gene ◽

Lucilia Cuprina ◽

Comparative Approach ◽

Length Variation ◽

Mrna Levels ◽

Cytoplasmic Localization ◽

Repeat Region ◽

Blow Fly ◽

Circadian Clock Gene ◽

Australian Sheep

We have isolated a homologue of the period (per) gene from the Australian sheep blow fly, Lucilia cuprina, as part of a comparative approach to the analysis of dipteran circadian systems. Sequence analysis of the 4 kb per cDNA revealed the conservation of three functional domains, namely the PAS dimerization motif, and the nuclear and cytoplasmic localization domains. A fourth domain, the threonine–glycine (TG) repeat region, is also conserved in L. cuprina per but has been severely truncated. No length variation was found in the TG repeat of L. cuprina or L. sericata collected from several different latitudinal zones. Expression analysis indicated a diel oscillation in per mRNA in LD 12[ratio ]12 with a period of 24 h and a peak at Zt 12. PER-immunoreactive protein oscillations were also demonstrated, with peak immunoreactivity lagging approximately 3 h behind peak mRNA levels. These results show the existence of a Drosophila-like circadian system in a calliphorid fly. They also provide evidence for the conservation of per function across the Diptera, and confirm the relevance of the Drosophila system as a model for fly circadian rhythms.

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

Genetics ◽

10.1093/genetics/113.3.641 ◽

1986 ◽

Vol 113 (3) ◽

pp. 641-663

Author(s):

Thomas W Cline

Keyword(s):

Sex Determination ◽

Dosage Compensation ◽

Loss Of Function ◽

Female Development ◽

Lethal Effects ◽

Larval Stages ◽

Positive Regulator ◽

Sexual Phenotype ◽

Activity State ◽

Female Specific

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.

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A gene-by-gene mosaic of dosage compensation strategies on the human X chromosome

10.1101/2021.08.09.455676 ◽

2021 ◽

Author(s):

Adrianna K. San Roman ◽

Alexander K. Godfrey ◽

Helen Skaletsky ◽

Daniel W Bellott ◽

Abigail F Groff ◽

...

Keyword(s):

X Chromosome ◽

Dosage Compensation ◽

Sex Chromosome ◽

Loss Of Function ◽

Specific Expression ◽

X Chromosomes ◽

Mediated Effects ◽

Multiple Dimensions ◽

Allele Specific ◽

Health And Disease

Dosage compensation in humans - ensuring the viability and fitness of females, with two X chromosomes, and males, with one - is thought to be achieved chromosome-wide by heterochromatinization of one X chromosome during female development. We reassessed this through quantitative gene-by-gene analyses of expression in individuals with one to four X chromosomes, tolerance for loss-of-function mutations, regulation by miRNAs, allele-specific expression, and the presence of homologous genes on the Y chromosome. We found a mosaic of dosage compensation strategies on the human X chromosome reflecting gene-by-gene differences in multiple dimensions, including sensitivity to under- or over-expression. These insights enrich our understanding of Turner, Klinefelter, and other sex chromosome aneuploidy syndromes, and of sex-chromosome-mediated effects on health and disease in euploid males and females.

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