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Female-specific upregulation of insulin pathway activity mediates the sex difference in Drosophila body size plasticity

Jason W. Millington, Chien Chao, Ziwei Sun, Paige J. Basner-Collins, George P. Brownrigg, Lianna W. Wat, Bruno Hudry, Irene Miguel-Aliaga, Elizabeth J. Rideout

Preprint posted on April 24, 2020 https://www.biorxiv.org/content/10.1101/2020.04.22.054239v1

One genotype, two phenotypes: protein-rich environments increase body size plasticity in female flies.

Selected by Vaibhav Menon

Background: Animals exist and thrive across a spectrum of diverse environments. An animal’s survival is largely dependent on unique adaptations to efficiently leverage nutrients from distinct conditions. Animals with the same genotype are able to express distinct phenotypes when exposed to different environments, a phenomenon known as phenotypic plasticity. Body size is one such phenotype subject to nutrient-dependent plasticity in Drosophila melanogaster (1). Interestingly, it has been observed that the extent of nutrient-dependent size plasticity is larger in female flies than in male flies (2, 3). It is known that consumption of nutrient-rich food enhances signaling of the insulin/insulin-like growth factor (IIS) pathway, leading to increased growth. However, the mechanism explaining the sexually dimorphic nature of nutrient-dependent body size plasticity is unclear. In this preprint, the authors convincingly demonstrate that IIS activity is upregulated in females in protein-rich contexts, leading to increased body size plasticity. Mechanistically, they identify several key players that outline a pathway for this sexually dimorphic phenotype. First, females require Drosophila insulin-like peptide dILP2 to upregulate IIS activity in response to protein-rich contexts. Second, female-specific elevated expression of the humoral factor stunted (sun) increases IIS activity. Third, the sex determination gene tra in females mediates a nutrient-dependent increase in sun expression. In illustrating the significant phenotypic and molecular differences between male and female flies in different nutritional contexts, these findings represent an important advancement in an otherwise understudied component of nutrient-processing.

 

Key findings:

Females exhibit increased body size plasticity across different nutrient conditions.

The authors assessed the pupal volume of w1118 (wild-type) males and females reared on diets with different nutrient concentrations (0.5x, 1x, and 2x) and observed that pupal volume of males and females was significantly larger in the 1x diet than in the 0.5x diet. Comparing flies raised on 2x nutrient content to 1x nutrient content, the authors again observed larger pupal volumes in females. In males, the magnitude of pupal volume increase was lower, suggesting different ranges of body plasticity across sexes. To assess the role of dietary protein in driving observed phenotypic plasticity, the authors then analyzed pupal volume in flies raised on diets with different yeast concentrations (0.5Y, 1Y, and 2Y) and observed similar trends, with females showing more body size plasticity across the range of yeast concentrations than males. Additionally, when raised on a 2Y diet supplemented with protease inhibitors to block metabolic protein breakdown, the authors observed a larger decrease in pupal volume in females than in males, suggesting protein may be the macronutrient mediating the difference in body size plasticity between males and females.

Nutrient-dependent elevation of IIS activity is specific to female larvae and required for their body size plasticity

It has previously been observed in mixed-sex populations that IIS signaling activity increases in response to nutrient-rich conditions to promote growth (4). To identify how IIS signaling is affected by changes in nutrient content, the authors measured mRNA expression of InR, brummer (bmm), and 4E-binding protein (4E-BP) – genes whose expression levels are repressed by increased IIS activity (5-9). Indeed, expression of each of these genes was lower in w1118 females raised on 2Y than those raised on 1Y. This was corroborated by analysis of fluorescent expression of GFP fused to a pleckstrin homology domain (GFP-PH). The PH domain binds to PIP3, which is elevated in the plasma membrane when IIS activity increases. The authors observed significantly higher membrane localization of GFP-PH in female flies raised on 2Y than those raised on 1Y. Importantly, in wild-type males, no difference across dietary protein concentrations was observed in either mRNA expression of InR, bmm, and 4E-BP or GFP-PH membrane localization. Taken together, these results strongly suggest IIS signaling activity is higher in flies raised in protein-rich contexts, and this phenomenon is specific to females.

dILP2 and sun are required for female-specific elevation of IIS activity and body size plasticity

dILPs are an important class of peptides that direct nutrient processing and growth-promotion in the fly. Previous findings have identified increased secretion of the dILP2 class of peptides in female larvae in response to increased dietary protein, positioning it as a key potential player in mediating IIS activity (10). Measuring mRNA expression of InR, bmm, and 4E-BP, the authors found that the nutrient-dependent increase in IIS activity observed in wild-type female larvae was abolished in dilp2 mutant female larvae. The nutrient-dependent increase in adult weight and pupal volume was also abolished in dilp2 mutants. Neither mRNA expression of the proxy IIS activity genes nor body size plasticity was significantly affected by mutation of dilp2 in males.

dILP secretion is under the regulatory control of humoral factors whose increased expression changes according to dietary intake of nutrients. Based on prior work in mixed-sex populations that has identified critical humoral factors, the authors were interested in whether any of these factors were critical to controlling dilp2 expression and IIS activity in protein-rich contexts. Indeed, the authors observed in wild-type females a significant upregulation of humoral factor sun in 2Y contexts compared to 1Y while other humoral factors did not change. To probe at its putative upstream role in IIS signaling via regulation of dILP2 secretion, the authors overexpressed a sun-RNAi transgene in larval fat bodies and observed that the nutrient-dependent increase of IIS activity observed in RNAi control female larvae was absent. Correspondingly, the authors also noted reduced body size plasticity in sun-RNAi females. Taken together, these findings suggest a requisite, female-specific role for both dilp2 and sun in elevation of IIS signaling in protein-rich environments, which contributes to increased body size plasticity for females.

Sex determination gene tra is required for nutrient-dependent sun expression, IIS activity and body size plasticity in females

What is driving the sex difference in body size plasticity and upstream IIS-related gene expression profiles? Previous findings have pointed to sex determination gene tra in regulating body size and IIS activity in protein-rich contexts, but its role in driving sex differences for these phenotypes are presently unknown (10). To address this, the authors quantified mRNA expression of 4E-BP and sun in wild-type animals and tra mutants. While wild-type females exhibited a nutrient-dependent decrease in 4E-BP expression(increased IIS activity) and increase in sun expression, tra mutant females failed to recapitulate either of these changes across protein concentrations. These gene expression differences extended to phenotypic plasticity as well, with tra female mutants unable to exhibit nutrient-dependent increases in adult weight or pupal volume.

Importantly, males lack a functional Tra protein, providing a possible mechanistic explanation for their reduced phenotypic plasticity. To address this question, the authors expressed tra ubiquitously in males and observed a significant, nutrient-dependent increase in IIS activity compared to UAS and Gal4 controls. Additionally, they noted in tra-expressing males a corresponding nutrient-dependent increase in sun mRNA and increase in body size compared to control genotypes. These experiments elucidate a novel role for tra in mediating the sex difference observed in protein-dependent body size plasticity.

However, from a mechanistic perspective, it is possible that the Tra protein may alter sun mRNA activity in a nutrient-dependent manner while its regulation of phenotypic plasticity is mediated through an alternative pathway. To identify whether Tra’s control of sun drives phenotypic plasticity, the authors overexpressed tra in males and females mutant for srl, a gene required for normal sun expression. They found that compared to animals overexpressing tra in a control background, srl mutants overexpressing tra did not exhibit protein-dependent body size plasticity. This confirms that the ability of tra to control sun expression is a critical component of its regulation of phenotypic plasticity in a nutrient-rich context.

Body-size plasticity increases female fecundity via IIS activity

The observed increase in body size for wild-type female flies raised on 2Y provided an opportunity to assess a mechanistic link between IIS activity and fecundity, a trait influenced by nutrient availability and body size. The authors quantified egg production by either wild-type or InR mutant females in 1Y or 2Y nutrient conditions. While wild-type flies exhibited a significant increase in eggs produced in 2Y, InR mutants showed no difference between 1Y and 2Y conditions. To further probe the influence of IIS activity on fecundity, the authors targeted sun expression, finding that knocking down sun expression with RNAi abolished the protein-dependent increase in egg production observed in RNAi controls. This suggests that in regulation of IIS activity and body size, sun may also influence fecundity. A role for IIS activity promoting fecundity is consistent with previous findings that point to dILP2’s role in egg production (6).

Why I liked this paper:

This comprehensive preprint identifies in body size a phenotype prone to sexually dimorphic ranges of plasticity and suggests a potential model driving this  plasticity. The authors’ findings represent an important consideration for those studying metabolism and nutrient processing –treating entire genotypes as a monolith without consideration for potentially robust sex-differences may hamper elucidation of key mechanisms. The role of dysregulated insulin signaling and related metabolic homeostasis mechanisms in diseases such as Type II diabetes is well-established, but this work illustrates the need to focus on potential sex differences that may mediate pathology. 

Questions

1. Do you think your findings from flies fed isocaloric sugar diets failing to exhibit increased female body size plasticity are a consequence of sugar-specific dILP2 resistance? Perhaps protein is a better suited macronutrient to “cleanly” elevate IIS activity for growth without inducing epigenetic changes leading to insulin resistance.

2. You have conclusively shown that males fail to display the extent of nutrient-dependent body size plasticity in females show. Do you think non-Tra dependent pathways exist to mediate male body size plasticity, and if so, what kind of change in environmental conditions do you think would prompt this?

 

References

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2.         Alpatov WW. Phenotypical Variation in Body and Cell Size of Drosophila melanogaster. Biological Bulletin. 1930;58(1):19. doi: https://doi.org/10.2307/1537121.

3.         Shingleton AW, Masandika JR, Thorsen LS, Zhu Y, Mirth CK. The sex-specific effects of diet quality versus quantity on morphology in Drosophila melanogaster. R Soc Open Sci. 2017;4(9):170375. Epub 2017/10/11. doi: 10.1098/rsos.170375. PubMed PMID: 28989746; PMCID: PMC5627086.

4.         Grewal SS. Insulin/TOR signaling in growth and homeostasis: a view from the fly world. Int J Biochem Cell Biol. 2009;41(5):1006-10. Epub 2008/11/11. doi: 10.1016/j.biocel.2008.10.010. PubMed PMID: 18992839.

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6.         Gronke S, Clarke DF, Broughton S, Andrews TD, Partridge L. Molecular evolution and functional characterization of Drosophila insulin-like peptides. PLoS Genet. 2010;6(2):e1000857. Epub 2010/03/03. doi: 10.1371/journal.pgen.1000857. PubMed PMID: 20195512; PMCID: PMC2829060.

7.         Junger MA, Rintelen F, Stocker H, Wasserman JD, Vegh M, Radimerski T, Greenberg ME, Hafen E. The Drosophila forkhead transcription factor FOXO mediates the reduction in cell number associated with reduced insulin signaling. J Biol. 2003;2(3):20. Epub 2003/08/12. doi: 10.1186/1475-4924-2-20. PubMed PMID: 12908874; PMCID: PMC333403.

8.         Kang P, Chang K, Liu Y, Bouska M, Birnbaum A, Karashchuk G, Thakore R, Zheng W, Post S, Brent CS, Li S, Tatar M, Bai H. Drosophila Kruppel homolog 1 represses lipolysis through interaction with dFOXO. Sci Rep. 2017;7(1):16369. Epub 2017/11/29. doi: 10.1038/s41598-017-16638-1. PubMed PMID: 29180716; PMCID: PMC5703730.

9.         Puig O, Tjian R. Transcriptional feedback control of insulin receptor by dFOXO/FOXO1. Genes Dev. 2005;19(20):2435-46. Epub 2005/10/19. doi: 10.1101/gad.1340505. PubMed PMID: 16230533; PMCID: PMC1257398.

10.       Rideout EJ, Narsaiya MS, Grewal SS. The Sex Determination Gene transformer Regulates Male-Female Differences in Drosophila Body Size. PLoS Genet. 2015;11(12):e1005683. Epub 2015/12/29. doi: 10.1371/journal.pgen.1005683. PubMed PMID: 26710087; PMCID: PMC4692505.

Tags: drosophila, feeding, fruit fly, insulin, phenotypic plasticity, sexual dimorphism

Posted on: 23rd June 2020

doi: https://doi.org/10.1242/prelights.22091

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