Sexual Dimorphism of Body Size Is Controlled by Dosage of the X-Chromosomal Gene Myc and by the Sex-Determining Gene tra in Drosophila

Abstract

Drosophila females are larger than males. In this article, we describe how X-chromosome dosage drives sexual dimorphism of body size through two means: first, through unbalanced expression of a key X-linked growth-regulating gene, and second, through female-specific activation of the sex-determination pathway. X-chromosome dosage determines phenotypic sex by regulating the genes of the sex-determining pathway. In the presence of two sets of X-chromosome signal elements (XSEs), Sex-lethal (Sxl) is activated in female (XX) but not male (XY) animals. Sxl activates transformer (tra), a gene that encodes a splicing factor essential for female-specific development. It has previously been shown that null mutations in the tra gene result in only a partial reduction of body size of XX animals, which shows that other factors must contribute to size determination. We tested whether X dosage directly affects animal size by analyzing males with duplications of X-chromosomal segments. Upon tiling across the X chromosome, we found four duplications that increase male size by >9%. Within these, we identified several genes that promote growth as a result of duplication. Only one of these, Myc, was found not to be dosage compensated. Together, our results indicate that both Myc dosage and tra expression play crucial roles in determining sex-specific size in Drosophila larvae and adult tissue. Since Myc also acts as an XSE that contributes to tra activation in early development, a double dose of Myc in females serves at least twice in development to promote sexual size dimorphism.

Keywords: Drosophila melanogaster; Myc; dosage compensation; sexual dimorphism; tra.

Figure 1

Regions of interest (regions A–D) and gaps (gap 1–5) not covered in Dp(1;Y) screen were examined using smaller X duplications, Dp(1;3), translocated to the third chromosome. (A) Males with X duplications that span regions A–D were crossed with w females (allele w¹¹¹⁸), sons with the duplication were again crossed with w females and F₂ sibling males were analyzed. Bars represent weight ratios of third instar wandering w males with a duplication to w males with no duplication. Control lacZ transgene-expressing lines were examined in the same manner. (B) Males with X duplications spanning gaps 1–5 were examined as described for regions A–D above. The absence of bars for duplication lines Dp(1;3)DC068 and Dp(1;3)DC232 reflect persistent gaps in coverage, resulting from reduced viability and fertility in males with these duplications. Error bars indicate where experiments were repeated.

Figure 2

Fine-scale mapping of duplication-sensitive regions using overlapping Dp(1;3) lines. Strong growth-promoting regions are highlighted in blue, regions with minor or no growth effects are shaded in pink. (A) Only the Myc locus is fully contained in growth-promoting duplications Dp(1;3)DC059 and Dp(1;3)DC060. (B) Candidates in region C narrowed to tay, MSBP, CG15916, shi, and cycD. (C) In region D, four candidates remain: CanA-14F, SMC3, Ubc7, and CG9784. (D) Three genes in Dp(1;3)RC026 are candidates for dose-sensitive growth promoters: CG15211, Ant2, and sesB. Maps were derived from GBrowse representations in FlyBase.

Figure 3

(A) Effect of mutations in a candidate gene on male larvae with a duplication that induces or restricts growth. In each case, animals were heterozygous for the mutations described. The strong hypomorphic Myc mutation, Myc^P0, obliterates the growth promoting effects of Dp(1;3)DC060, indicating that duplication of Myc underlies the weight increase in Dp(1;3)DC060 males. The converse negative effect on growth imposed by Dp(1;3)DC463 is alleviated through a mutation of Mnt, implicating this gene as the growth suppressor. A partial suppression of the Dp(1;3)DC133’s growth promotion was observed by the null mutation cycD¹. A second factor in Dp(1;3)DC133 likely contributes to the observed increase in male size. A partial suppression of the growth induced by Dp(1;3)DC316 was also found through elimination of CanA-14F (CanA-14F^KO). Combined removal of CanA-14F and the neighboring gene calcineurin Pp2B-14D (CanA^dKO) resulted in complete eradication of the duplication’s effects. (B) The effects of dosage of candidate genes were studied in females. Wandering female larvae heterozygous for mutations in candidate genes in a w background (allele w¹¹¹⁸) were weighed. A significant weight reduction compared to w was only observed in females heterozygous for the hypomorphic Myc mutation, Myc^P0, or for a deficiency in Myc.

Figure 4

Transcript levels of Myc and related genes were assessed by qRT-PCR in feeding third instar larvae. (A) Myc transcript levels in whole larvae, as well as in isolated fat body tissue, are 1.7 times higher in female than in sibling male larvae. No significant differences in cycD or CanA14-F/Pp2B-14D transcript levels were observed between the sexes. (B) Transcripts of neither Mnt nor Max, the two other components of the Myc-Mnt-Max interactome, amass in a sexually dimorphic manner. Transcripts for the ribosomal proteins Rpl1 and Rpl135, known targets of Myc, are expressed equally in males and females as well. (C) Overall levels of Desat1 RNA are the same in both sexes but transcript levels are 1.5 times higher in male fat-body tissue compared to that of sibling females. (D) Male and female larvae with Cg-Gal4-driven expression of Desat1 in fat-body tissue are not significantly different in size than cocultured w¹¹¹⁸ control animals. * P < 0.02, *** P < 0.001; P-values were calculated using Student’s t-test. Error bars represent the standard deviation. n.s., not significant.