Identifying sexual differentiation genes that affect Drosophila life span

Abstract

Background: Sexual differentiation often has significant effects on life span and aging phenotypes. For example, males and females of several species have different life spans, and genetic and environmental manipulations that affect life span often have different magnitude of effect in males versus females. Moreover, the presence of a differentiated germ-line has been shown to affect life span in several species, including Drosophila and C. elegans.

Methods: Experiments were conducted to determine how alterations in sexual differentiation gene activity might affect the life span of Drosophila melanogaster. Drosophila females heterozygous for the tudor[1] mutation produce normal offspring, while their homozygous sisters produce offspring that lack a germ line. To identify additional sexual differentiation genes that might affect life span, the conditional transgenic system Geneswitch was employed, whereby feeding adult flies or developing larvae the drug RU486 causes the over-expression of selected UAS-transgenes.

Results: In this study germ-line ablation caused by the maternal tudor[1] mutation was examined in a long-lived genetic background, and was found to increase life span in males but not in females, consistent with previous reports. Fitting the data to a Gompertz-Makeham model indicated that the maternal tudor[1] mutation increases the life span of male progeny by decreasing age-independent mortality. The Geneswitch system was used to screen through several UAS-type and EP-type P element mutations in genes that regulate sexual differentiation, to determine if additional sex-specific effects on life span would be obtained. Conditional over-expression of transformer female isoform (traF) during development produced male adults with inhibited sexual differentiation, however this caused no significant change in life span. Over-expression of doublesex female isoform (dsxF) during development was lethal to males, and produced a limited number of female escapers, whereas over-expression of dsxF specifically in adults greatly reduced both male and female life span. Similarly, over-expression of fruitless male isoform A (fru-MA) during development was lethal to both males and females, whereas over-expression of fru-MA in adults greatly reduced both male and female life span.

Conclusion: Manipulation of sexual differentiation gene expression specifically in the adult, after morphological sexual differentiation is complete, was still able to affect life span. In addition, by manipulating gene expression during development, it was possible to significantly alter morphological sexual differentiation without a significant effect on adult life span. The data demonstrate that manipulation of sexual differentiation pathway genes either during development or in adults can affect adult life span.

Figure 1

Drosophila sex determination hierarchy and the tudor mutation. A) The Drosophila somatic sex determination hierarchy. The X to autosome ratio causes Sxl to be "off" in males (indicated in gray color) and to be "on" in females. In males, lack of SXL and TRA activity leads to expression of FRU^M and DSX^M . In females, TRA and TRA2 together direct splicing of downstream targets to produce DSX^F , and fru P1 transcripts that are not translated in females. B) Crossing scheme to produce germ line-ablated flies using tudor[1]. For the experimental group, tudor homozygote females were crossed to wild type Oregon-R males to generate progeny lacking a germ line. For the control group, tudor heterozygote females were crossed to Oregon-R males to generate progeny containing a normal germ line. Both experimental and control groups have the same chromosomal composition. B) Test for Wolbachia. The Wolbachia 16S rDNA sequences were amplified by PCR from the indicated Drosophila lines and controls, and the presence or absence of Wolbachia-specific PCR products was determined by gel electrophoresis and ethidium bromide staining.

Figure 2

Life span assays and mortality rate analysis for germ line-ablated Drosophila. A) Males lacking the germ line. The assay consisted of 125 flies for the experimental group and 122 flies for the control group. B) Mortality rate (Gompertz-Makeham model) of the germ line-ablated and control male groups. C) Redrawn survival curve for males with age-independent mortality removed. D) Females lacking the germ line. The assay consisted of 118 flies for the experimental group, and 124 flies for the control group. E) Mortality rate (Gompertz-Makeham model) of the germ line-ablated and control female groups. F) Redrawn survival curve for females with age-independent mortality removed. G) Males lacking the germ line, repeat assay. The assay consisted of 238 flies for the experimental group and 139 flies for the control group. H) Mortality rate (Gompertz-Makeham model) of the germ line-ablated and control male groups. I) Redrawn survival curve for males with age-independent mortality removed. J) Females lacking the germ line, repeat assay. The assay consisted of 233 flies for the experimental group and 175 flies for the control group. K) Mortality rate (Gompertz-Makeham model) of the germ line-ablated and control female groups. L) Redrawn survival curve with age-independent mortality removed.

Figure 3

Effect of sex differentiation pathway gene mis-expression on survival of male and female adult flies. Sexual differentiation pathway genes or RNAi constructs were over-expressed either during larval development ("L"; gray triangles) or in adults ("A"; solid squares). Open circles represent the no-drug control ("-"). Survival curves are plotted as a function of adult age in days. Median life span and p value for log rank test are indicated in parentheses for each cohort. (A, B) Control flies (progeny of driver crossed to Or-R wild type). (C, D) tra. (E, F) tra2-IR. (G, H) dsxM. (I, J) dsxF. (K, L) fruMA. (M, N) fru-IR.

Figure 4

Effect of tra over-expression during development on sexual differentiation of adults. UAS-tra males were crossed to GS255B virgins, cultured on food with drug (+RU486) to drive the over-expression of tra during development, or cultured on food with ethanol as the control, as indicated. Pictures were taken at the magnification of 100X. (A, B) Female genitalia. (C, D) Male genitalia. (E, F) Male sex comb.