The discovery of sex-biased proliferation in the intestinal stem cells of fruit-fly midguts reveals that the organ's size is determined by a previously undefined, sex-specific molecular pathway. See Letter p.344
Within-species genetic differences are central to many biological phenomena, from evolution to disease susceptibility. One of the most under-studied intraspecies differences is sex. We tend to think of sex in terms of characteristics that are related to reproduction, but differences between sexes extend to many parts of the body. For instance, the midgut (an absorptive organ similar to the small intestine) of the fruit fly Drosophila melanogaster is longer in females than in males — especially after mating, when females produce many eggs that are replete with proteins and lipids1. On page 344 of this issue, Hudry et al.2 report that a previously unidentified branch of the sex-determination pathway underlies this dynamic difference in organ size, by controlling the proliferation of stem cells in the midgut.
The classic view of sex determination in fruit flies involves a regulatory cascade in which genes are spliced into different forms in a sex-specific manner3. These early steps in fly sex determination differ from those in the mammalian set-up, but the ultimate outcomes are similar. In flies with two X chromosomes, the protein Sex lethal (Sxl) splices an RNA called transformer (tra) into a protein-coding isoform. The TRA protein, acting with its cofactor TRA2, binds to both the RNA produced from the doublesex (dsx) gene, splicing it into a female-specific isoform, and to the RNA of fruitless (fru), blocking production of a male-specific isoform. In XY flies, the gene Sxl is not expressed. As such, dsx and fru are spliced into male-specific isoforms by default. Through another pathway, Sxl prevents dosage compensation in females (in males, transcription of genes on the X chromosome is upregulated, to compensate for the fact that females have two copies of X).
Hudry et al. investigate sex-specific differences in gene expression in the fruit-fly midgut. They report that genes involved in cell division are preferentially expressed in females. Furthermore, they find that Sxl acts to enhance the proliferative capacity of intestinal stem cells in the female midgut. These differences from the male midgut help to explain the larger, more-plastic guts found in females.
The authors go to great lengths to demonstrate that these sex-specific differences are not regulated by the classic splicing pathway. They rule out a role for dsx and fru in female gut growth and plasticity and provide evidence that TRA2 might also be dispensable. However, there is a caveat to this suggestion — although mutation of tra2 had no effect on proliferation, there was still TRA2 activity in these flies. By contrast, tra expression is required for proliferation to be enhanced in females. Thus, sex differences in the fruit-fly midgut are regulated by a previously unidentified branch of the sex-determination pathway, one that is downstream of Sxl and TRA.
Plasticity in the female gut is blocked by downregulating Sxl, but Hudry and colleagues show that plasticity can be rescued by the expression of TRA. These data make a convincing case that sex differences in the gut are mediated by tra rather than by dosage compensation. Moreover, misexpression of tra in intestinal progenitors enhances proliferation in the male midgut. Thus, unlike differentiated sex-specific organs such as the male accessory gland (the equivalent of the human prostate), the sex effects on intestinal stem cells are fully reversible.
Next, the authors look for direct and indirect targets of TRA in intestinal stem cells by expression profiling. They find 72 genes for which RNA splicing or steady-state expression levels are modulated by TRA, three of which encode proteins that regulate proliferation of the cells (Fig. 1). TRA is thought of as an RNA-binding protein and splicing factor4, so direct targets would be expected to be regulated post-transcriptionally. However, in these three targets, TRA modulates transcript abundance. It is possible that these genes are indirect targets of TRA and are regulated by an unidentified downstream transcription factor. Alternatively, TRA might have a direct effect on transcript stability, or even on the regulation of transcription.
Cell-autonomous events (those that occur on a cell-by-cell basis, rather than affecting neighbouring cells) have received much attention in sex-determination research in D. melanogaster, whereas vertebrate work has focused mainly on the influence of hormones such as testosterone. However, coordinating sex-biased expression between cells and organs in fruit flies clearly requires broader control. Indeed, although tra is required only in the intestinal stem cells in which it is expressed, two of its three proliferation-regulating targets — Imaginal disc growth factor 1 (Idgf1) and Serpin 88Eb (Spn88Eb) — encode secreted proteins. Idgf proteins are known to increase proliferation5 and might bind to receptor proteins on the intestinal stem cells from which they are secreted to promote proliferation directly. Serpin proteins can also bind to a variety of protein types, including hormones, which they may escort around the body6.
In addition to having longer guts, female fruit flies are larger than males. Recent work7 shows that fat-cell expression of tra, but not of dsx or fru, contributes autonomously to cell size and systemically to female body size, by promoting insulin secretion from the brain. Idgf1 and Spn88Eb are highly expressed in fat cells8 — could they be TRA targets there, too? Of note, the protein Serpinb1 mediates plasticity in insulin-producing cells in mice9, suggesting that this pathway might be evolutionarily conserved. There is still much to learn, but the sex-biased sizing of organisms and organs seems likely to be under the control of an entirely unknown branch of the sex-determination cascade, with both cell-autonomous and non-cell-autonomous functions.
In a final series of experiments, Hudry and colleagues demonstrate that sex-specific organ plasticity alters susceptibility to disease, because female flies are more sensitive than males to tumours that are induced through genetic changes. Little is known about sex-biased disease susceptibility in D. melanogaster, although sex-specific interactions are implicated in a host of disease-related traits10, suggesting that sex is an important consideration for understanding disease in flies. Sex bias is seen in many human diseases11, including several digestive-tract cancers. Furthermore, although the authors show substantial sex-biased gene expression in the fruit-fly gut, many of the studies on digestive-tract tumour development in D. melanogaster looked only at females. Thus, the current study highlights how influences on disease can be missed by ignoring sex chromosomes and sexual identity, even in non-mammalian model organisms.Footnote 1
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Sex-Specific Among-Individual Covariation in Locomotor Activity and Resting Metabolic Rate in Drosophila melanogaster
The American Naturalist (2019)