[
MicroPubl Biol,
2022]
The Q system is a genetic tool developed to deliver spatiotemporal control over gene expression (Giles et al. 1991; Potter et al. 2010; Wei et al. 2012). Although it has already been adapted for use in C. elegans by Wei et al. in 2012, to date, the Q system has not been applied extensively in this nematode. In the relatively few available reports, it is mainly used to constitutively restrict gene expression in a spatial manner (e.g. Schild et al. 2014; Schild and Glauser 2015; Jee et al. 2016; Tolstenkov et al. 2018; Chiyoda et al. 2021), while but a handful of studies also explore the temporal aspect of the system (Matus et al. 2015; Yuan et al. 2016; Cottee et al. 2017; Hoang and Miller 2017). We aimed to apply this tool in the C. elegans nervous system to gain both spatial and temporal control over expression of a gene encoding a reporter protein that is targeted to the secretory pathway. Despite our efforts, we here report that in our hands, the Q system is not suitable for application in the neurons due to a lack of dynamic range.
[
International Worm Meeting,
2021]
Gaining control over spatiotemporal features of gene expression is useful to improve our understanding of biological regulation. A common approach is to (ir)reversibly switch genes on or off via conditional expression systems. One such genetic toolkit is the Q system, a binary conditional expression system that was originally developed for mammalian cells and D. melanogaster, but has quickly been adapted for use in C. elegans as well. The system consists of three components: QUAS, QF and QS. Expression of a sequence of interest can be controlled by placing it downstream of an enhancer sequence (QUAS) that can be recognized by a transcriptional activator (QF). Advantageous over the canonical Gal4UAS system, the Q system permits to temporally control gene expression in a temperature-independent manner through the addition of quinic acid, which (reversibly) sequesters the transcriptional suppressor QS. Because of this substantial benefit, we turned to the Q system to build a reporter strain to visualize the endocytic capacity of the C. elegans coelomocytes. The objective is to gain reversible temporal control (on and off) over the expression of a fluorescent reporter, mNeonGreen, to generate temporally resolved mNeonGreen secretion by source cells, of which the subsequent degradation by the coelomocytes could then be observed. However, while the Q system has successfully been used to spatially restrict gene expression, there is little support for its performance in terms of temporal control. In an ongoing effort to validate the Q system for the research purpose described above, we encountered several points of attention which we here wish to share with the community. Especially at the level of the QF/QS ratio, there appears to be but a narrow window of opportunity that avoids leaky expression on one hand, vs inefficient de-repression of transcription by quinic acid on the other. We hope these results may engage others using conditional expression systems in a discussion balancing practical challenges and opportunities of these tools.
Driesschaert, Brecht, Dens, Elisabeth, Temmerman, Liesbet, Menschaert, Gerben, Mergan, Lucas, Vandewyer, Elke, Coussement, Louis, Braeckman, Bart P.
[
International Worm Meeting,
2021]
Axenic dietary restriction (ADR) is the most potent form of dietary restriction in C. elegans known to date. Simply removing bacteria and growing worms in a sterile (semi-)defined culture medium results in more than doubling of lifespan, despite an abundance of calories and nutrients. The processes underlying this lifespan extension have remained enigmatic, as the mechanism seems distinct from other forms of DR and does not rely on known longevity pathways. One of the few molecular players that has been linked to ADR is CUP-4, a putative ligand-gated ion channel which when absent significantly reduces the longevity effect of ADR. CUP-4 is expressed solely in the coelomocytes, endocytic cells that have been suggested to serve scavenging, immune or hepatic functions. We wish to understand the underlying mechanisms of ADR longevity, and how the coelomocytes fit into this picture. We performed lifespan assays to determine the effect of coelomocyte ablation or disruption of endocytosis upon longevity under ADR. After optimizing a cell-specific RNA-sequencing set-up for adult C. elegans coelomocytes using fluorescence-activated cell sorting (FACS), we applied this pipeline to differentially analyze bulk coelomocyte transcriptomes under different dietary conditions and endocytic capacities. Our results revealed many differentially regulated immune genes, prompting us to investigate the potential involvement of diverse immune pathways (within the coelomocytes) in axenic dietary restriction. We hope that this will provide new insights into the lifespan-extending mechanisms of ADR, that can potentially be extrapolated to teach us more about the ageing processes at play in animals in general.