Mak, Jennifer Chen Hei
(2020).
Deciphering the mechanism of adult reproductive diapause in C. elegans.
PhD thesis, Universität zu Köln.
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DECIPHERING THE MECHANISM OF ADULT REPRODUCTIVE DIAPAUSE IN C.pdf - Accepted Version Download (23MB) | Preview |
Abstract
The discovery of novel longevity genes, through the use of model organisms has revealed different conserved molecular pathways involved in the ageing process. RNAi or mutagenesis based screens are powerful tools however both possess limitations. To ease the challenge of mutagenesis screens we incorporated ARD as tool to allow direct selection of longevity mutants. Starvation conditions encountered at the late larval stage of wild type C. elegans induces ARD. This delays reproduction and extends the lifespan compared to animals grown in ad libitum (AL) conditions, to survive more than 80 days without food. Known longevity mutants displayed additive ARD lifespan extension, therefore, we hypothesised the selection of longevity mutants through an ARD mutagenesis screen would also show longevity in AL conditions. This allowed us to directly select for longevity mutants. We performed four different screens and obtained longevity mutants through this method. However, we discovered the method requires optimisation to obtain a higher number of longevity mutants with more robust phenotypes to discover novel longevity genes. Upon re-evaluation, we observed a high number of our mutants were long lived under ARD and decided to focus on deciphering the mechanism driving ARD longevity. Since previous work from our laboratory discovered signalling pathways known to regulate longevity are additive to ARD lifespan, this suggests an independent mechanism for survival. We discovered mutants of upp-1, encoding uridine phosphorylase, to extend ARD lifespan as well as improve recovery from ARD. upp-1 is a key regulator of pyrimidine metabolism, catalysing the conversion of uridine to uracil and ribose phosphate. In C. elegans, upp-1 exhibits uridine and thymine phosphorylase activity. We performed metabolomic analysis of upp-1 mutants after 10 days of ARD and discovered elevated levels of uridine and thymine compared to wild type. Interestingly, supplementation of thymine increased ARD lifespan in wild type animals but not upp-1 mutants, revealing that thymine enhanced ARD survival through a similar mechanism as upp-1 mutants. Altogether, we discovered a role for pyrimidine metabolism in regulated ARD longevity. Our laboratory recently identified HLH-30/TFEB as a master regulator of ARD. hlh-30 mutants drastically reduces ARD lifespan, depletes fat stores, rapidly reduces body length, reduces oxygen consumption and fails to recover from ARD upon refeeding. This reveals HLH-30 to be essential for regulating ARD survival, morphogenesis and recovery. To investigate the mechanism by which HLH-30 modulates ARD survival, we performed an unbiased suppressor screen to identify mutants rescuing hlh-30 ARD shortevity. We discovered mutations on daf-1 (TGF-β receptor subtype 1 of the TGF-β pathway) and pdk-1 (phosphoinositide dependant protein kinase of the IIS pathway) to not only increase hlh-30 ARD lifespan but also prevented the rapid body shrinkage and decreased fat content of hlh-30 mutants in ARD. We also discovered other components of these pathways, daf-7 (TGF-β ligand) and daf-2 (insulin receptor) to show similar effects to daf-1 and pdk-1 respectively. We performed a second screen and discovered other components of the TGF-β pathway, daf-3, further validating our first screen, as well as some new interesting candidates such as mxl-2, part of the Myc superfamily, let-363, the C. elegans orthologue of mammalian mTOR, and ceh-60, a homeodomain transcription factor. Taken together, these results suggest a role of the TGF-β pathway in regulating hlh-30 ARD longevity and survival.
| Item Type: | Thesis (PhD thesis) | ||||||||
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| URN: | urn:nbn:de:hbz:38-527668 | ||||||||
| Date: | 2020 | ||||||||
| Language: | English | ||||||||
| Faculty: | Faculty of Mathematics and Natural Sciences | ||||||||
| Divisions: | Außeruniversitäre Forschungseinrichtungen > MPI for Biology of Ageing | ||||||||
| Subjects: | Life sciences | ||||||||
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| Date of oral exam: | 30 November 2020 | ||||||||
| Referee: |
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| Refereed: | Yes | ||||||||
| URI: | http://kups.ub.uni-koeln.de/id/eprint/52766 |
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