83 Ecdysone’s influence: regional intestinal cell population remodeling found in female Drosophila post-mating

After maturation, the body no longer grows linearly, but our organs do not remain static. Instead, they engage in dynamic homeostasis, responding to environmental cues and adapting physiologically. One such cue is pregnancy, where a surge of sex hormones signals all organs to prepare to support new life. This phenomenon is observed throughout the animal kingdom, from humans to Drosophila, operating through evolutionarily conserved pathways. In female Drosophila, the arthropod intestinal organ known as the midgut expands through mating-induced ecdysone influxes to accommodate for a changing metabolism and increased egg production.1 This study researched how the ecdysone influx affected midgut cell populations. We hypothesized the driving agent behind systemic midgut growth would be a known adaption, such as increased cell count and/or larger cells.2

To observe how the midgut grows post mating, we conducted DNA content analyses using microscopy and flow cytometry. For both experiments, we used w1118/esg-Gal4ts and esg- Gal4ts/EcRRNAi crosses. The escargot-GAL4ts line allowed for progenitor cell targeting with GFP and Ecdysone receptor (EcR) RNA interference. Virgins were aged for 5 days at 29°C and then mated for 48 hours at 25°C. Following dissection, midguts were stained with DAPI and GFP. For the microscopy experiments, images were taken with a Zeiss Axio Observer and ZenBlue 3.4. Image analysis was done in ImageJ with the StarDist plug-in to identify individual nuclei and statistical analysis was generated with ANOVAs in GraphPad Prism.3 For the flow cytometry experiments, nuclei were isolated using a nuclear extraction buffer and samples were run on a CytoFLEX LX flow cytometry machine.

The intestinal growth we observed is EcR dependent and regionally distributed, likely correlated to regional function and ovary proximity. Mating has minimal impact on the anterior region: it does not affect proliferative activity of the intestinal stem cells (ISCs), but absorptive enterocyte (EC) DNA endocycling is increased. EC endocycling is amplified when ecdysone cannot bind in progenitors, resulting in higher DNA ploidies of 32C and 64C in ECs. DNA endocycling in response to mating is also observed in the posterior region, and progenitor EcR loss also amplifies this endocycling. In the posterior region exclusively, ecdysone influx significantly upregulates proliferation in ISCs, resulting in more total cells. Whole gut flow cytometry of midguts demonstrated that ecdysone also impacts differentiation. When EcR is knocked down, enteroblasts are unable to differentiate into ECs, leading to an accumulation of progenitor cells and lower EC counts.

Collectively, this study found that mating causes cell population remodeling leading to larger systemic guts. Requirement of the ecdysone receptor implies that the acting agent is ecdysone, a sex hormone that increases in titer post-mating.1 When ecdysone reaches the midgut, it increases proliferation, differentiation, and DNA endocycling simultaneously in different cell types. This effect is regionally distributed: the absorptive posterior region is significantly impacted whereas the digestive anterior region is not. If EcR is lost in progenitor cells, ecdysone is unable to bind there; inhibiting proliferation and differentiation, but with no effect on EC endocycling. Future experiments using the enterocyte driver, Myo1A-Gal4 Tub-Gal80ts, would provide insight on the impact of ecdysone on endocycling: whether it is required or solely a facilitator.

This study contributes to the growing body of work investigating how sex hormones contribute to and change dynamic homeostasis in non-sex organs. Understanding the mechanisms and players in these complex relationships is essential to improving human health treatment and circumventing hormone related disease.