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white regulates proliferative homeostasis of intestinal stem cells during ageing in Drosophila

Abstract

Tissue integrity is contingent on maintaining stem cells. Intestinal stem cells (ISCs) over-proliferate during ageing, leading to tissue dysplasia in Drosophila melanogaster. Here we describe a role for white, encoding the evolutionarily conserved ATP-binding cassette transporter subfamily G, with a particularly well-characterized role in eye colour pigmentation, in ageing-induced ISC proliferation in the midgut. ISCs increase expression of white during ageing. ISC-specific inhibition of white suppresses ageing-induced ISC dysregulation and prolongs lifespan. Of the proteins that form heterodimers with White, Brown mediates ISC dysregulation during ageing. Metabolomics analyses reveal previously unappreciated, profound metabolic impacts of white inhibition on organismal metabolism. Among the metabolites affected by White, tetrahydrofolate is transported by White, is accumulated in ISCs during ageing and is indispensable for ageing-induced ISC over-proliferation. Since Thomas Morgan’s isolation of a white mutant as the first Drosophila mutant, white mutants have been used extensively as genetic systems and often as controls. Our findings provide insights into metabolic regulation of stem cells mediated by the classic gene white.

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Fig. 1: ISCs over-proliferate in both males and females in a white-dependent manner during ageing.
Fig. 2: White regulates ISC homeostasis cell autonomously during ageing.
Fig. 3: white has profound effects on metabolism.
Fig. 4: mini-white does not restore normal metabolism but has pleiotropic effects on ISC proliferation during ageing.
Fig. 5: brown is necessary for ageing-induced ISC over-proliferation.
Fig. 6: Folate metabolism regulates white-mediated ISC proliferation during ageing.

Data availability

Source data are provided with this paper. All other data that support the findings are available within the paper.

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Acknowledgements

We thank H. Jasper, K. Akagi, L. Partridge, N. Perrimon, S. Hou, I. Hariharan, E. Kuranaga, S. Hayashi and S. Davies, and TRiP at Harvard Medical School, the Bloomington Stock Center and the Kyoto Stock Center for transgenic fly stocks. We thank the members of the Yoo laboratory, L. Yang and Y.-C. Wang for helpful comments on the manuscript. This work was supported by AMED-PRIME (17939907) and JSPS KAKENHI (JP16H06220) to S.K.Y. and JSPS KAKENHI (JP17H03658) to T.N.

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S.K.Y. conceived and oversaw the project. A.S., T.N., T.T., S.N. and S.K.Y. executed the experiments. A.S., T.T., S.N. and S.K.Y. contributed expertise in ISC regulation and ageing. T.T. and T.N. contributed expertise in metabolic analysis. S.K.Y. wrote the paper with input from all authors.

Corresponding author

Correspondence to Sa Kan Yoo.

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The authors declare no competing interests.

Additional information

Peer review information Nature Metabolism thanks Bart Deplancke and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Pooja Jha; Isabella Samuelson.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 ISCs overproliferate in both males and females in a white-dependent manner during ageing.

a, ISC proliferation during ageing in males. b, ISC proliferation during ageing in females. Note that ISCs in females proliferate more than those in males regardless of age and genetic background. c, Females have more mitotic ISCs per gut (left), bigger guts (middle) and more mitotic ISCs per unit area (right) than males regardless of age. Progeny from crosses between female Oregon R and male w1118 were used for this analysis. d, Male progeny from crosses between Oregon R and w1118 have the same autosomes but different sex chromosomes. e, The male progeny with endogenous white exhibit ageing-induced ISC proliferation but those without white do not. f, Male progeny from crosses between Oregon R and w1 were used to examine ISC proliferation during ageing. g, w1 Oregon R flies do not demonstrate ISC dysregulation during ageing. h, Paraquat or DSS induces comparable ISC proliferation in Oregon R and wOregon R flies. Statistical significance was determined using two-tailed unpaired t-test (a, b, e, f, g), two-tailed Mann Whitney test (c) and one-way ANOVA with Dunnett’s post-test (h).

Extended Data Fig. 2 white regulates ISC homeostasis cell autonomously during ageing.

a, RT-qPCR of white transcript from Oregon R and w1118 demonstrates specificity of the designed primers for white. b, RT-qPCR of white transcript in the midgut of Oregon R shows that white transcription is not induced by paraquat treatment. c, Ubiquitous knockdown of white using act-Gal4, UAS-white RNAis shows that only one RNAi (BL33623) among 9 RNAi examined makes the eyes white, demonstrating an efficient knockdown of white. This effective RNAi (BL33623) was used to knockdown white throughout this paper. d, white knockdown in ISCs/EBs suppresses the ageing-induced increase of the number of ISCs. e, Representative pictures of esg>GFP positive cells (ISCs and EBs) in the midgut. Scale bar, 50 um. f, Ectopic expression of white does not promote ISC proliferation in young flies. g, Lifespan of Oregon R and wOregon R. In contrast to the effect of ISC-specific white knockdown (Fig. 2i), white knockout in the whole body does not extend lifespan. h, Lifespan of flies with ubiquitous knockdown of white. Consistent with the knockout phenotype, ubiquitous knockdown of white does not extend lifespan. Statistical significance was determined using two-tailed unpaired t-test (a, b, d) and Log-rank (Mantel-Cox) test (g, h). The experiments were repeated independently at least twice with similar results (e).

Extended Data Fig. 3 white has profound effects on metabolism.

a, Two-way ANOVA analyses of the data in Fig. 3d and Extended Data Fig. 3b. b, The amount of methylhistidine decreases during ageing regardless of genetic background.

Extended Data Fig. 4 Metabolomics of the carcass and the midgut.

a, Principal component (PC) analysis of water-soluble metabolites from the carcass (whole body without the gut) and the gut of Oregon R and wOregon R. The age of young (YG) and old male flies are 7 days and 1.5 months old, respectively. Ellipses of clusters show the 95% confidence regions for each sample group. b, Representative white-related metabolites are presented. Source data

Extended Data Fig. 5 brown affects ISC homeostasis during ageing.

a, In contrast to Oregon R and st1, bw1 mutants do not exhibit ageing-induced ISC proliferation. b, RU486 extends lifespan of flies with 5961-GS, UAS-brown RNAi. Statistical significance was determined using two-tailed unpaired t-test (a) and Log-rank (Mantel-Cox) test (b).

Extended Data Fig. 6 Purine metabolism does not affect ISC homeostasis.

a, Purine catabolism pathway. Hydrogen peroxide is generated during conversion of hypoxanthine to xanthine and xanthine to uric acid. b, ISCs proliferate during ageing in rosy mutant rosy506 flies. c, Feeding xanthine or guanosine does not promote ISC proliferation in young flies. Statistical significance was determined using two-tailed unpaired t-test (b) and one-way ANOVA with Dunnett’s post-test (c).

Extended Data Fig. 7 Folate metabolism regulates white-mediated ISC proliferation during ageing.

a, Yeast depletion through feeding only sucrose lowers THF signals in the gut while feeding exogenous THF increases them. b, Quantification of THF signals in the gut of flies fed with normal food (yeast food), yeast depletion (sucrose only) and exogenous THF (sucrose + THF). c, LC-MS-based measurement of THF shows that Oregon R maintains the THF amount in the gut during ageing, but wOregon R does not. d, Feeding folates does not promote ISC proliferation in young Oregon R. e, Feeding THF promotes ISC proliferation in old wOregon R flies. f, Endogenous expression of White using GFP knock-in flies. White shows vesicular localization in principal cells of the Malpighian tubules. g, RT-qPCR of white or brown transcript from the carcass of Oregon R shows that there is no transcriptional change of these genes during ageing. Statistical significance was determined using one-way ANOVA with Dunnett’s post-test (b, d) and two-tailed unpaired t-test (c, e). The experiments were repeated independently at least twice with similar results (a, f). Scale bars, 10 μm.

Supplementary information

Reporting Summary

Supplementary Table 1

Drosophila stocks used in this study.

Source data

Source Data Fig. 3

Metabolomics data for Fig. 3a,b.

Source Data Fig. 4

Metabolomics data for Fig. 4f,g.

Source Data Fig. 5

Metabolomics data for Fig. 5e.

Source Data Extended Data Fig. 4

Metabolomics data for Extended Data Fig. 4a.

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Sasaki, A., Nishimura, T., Takano, T. et al. white regulates proliferative homeostasis of intestinal stem cells during ageing in Drosophila. Nat Metab 3, 546–557 (2021). https://doi.org/10.1038/s42255-021-00375-x

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