C. Elegans Lifespan Regulation By Spatiotemporal Activity of DAF-16

Several environmental factors affect longevity in C. elegans. Most of these function through the DAF-16/FOXO transcription factor, which regulates expression of genes involved in aging and stress response [1]. We aim to elucidate how the lifelong molecular activity of daf-16, driven by environmental interventions, determines lifespan in C. elegans. Current methods to track DAF-16 activity have a destructive nature (microarrays, RT-PCR), and utilized strains with multiple copies of daf-16. Thus, we created a single copy GFT-tagged strain at the endogenous locus of daf-16. This strain, along with quantitative analysis of fluorescence imaging enables lifelong tracking of DAF-16 activity in response to environmental perturbations in vivo. Thus, the relationships between environmental perturbations, longitudinal gene activity, and lifespan can be explored. This will enable elucidating how the lifelong history of these perturbations determines lifespan in C. elegans.

Using a custom image-processing algorithm, we tracked DAF-16 activity under various dietary restriction regimes. In particular, the image processing approach enabled evaluation of complex patterns of DAF-16 nuclear migration, a hallmark of active DAF-16, at the tissue and cellular levels. To characterize DAF-16 activity, we developed a strain that labels the endogenous DAF-16 protein with GFP using the CRISPR/Cas9 system [2]. We observed increased migration of DAF-16 to cell nuclei in tissues (intestine, hypodermis, muscles, and neurons) of nematodes under conditions of reduced food intake. This pattern increased the longer the animals were under this condition, reaching a peak after approximately 12 hours of dietary restriction and decreasing thereafter. Moreover, under repeated and intermittent exposure of the same C. elegans population to dietary restriction, we identified a decreasing activity of DAF-16 in subsequent days (Fig. 1A). Additionally, we have observed migration of DAF-16 to nucleoli, a phenomena not described previously, and an increased response of DAF-16 in neurons when compared to other tissues (Fig. 1B). We aim to quantify how tissue specific activity of DAF-16 contributes to longevity in this nematode. Lifespan measurements were performed for animals under the same dietary restriction regimes, obtaining a correlation between DAF-16 levels of expression, measured as cumulative intensity at the cellular level, and average lifespan.

We have developed an experimental platform for the characterization of lifespan in C. elegans, while correlating this metric to quantifiable endogenous activity of DAF-16 under various dietary restriction regimes. Furthermore, our findings show that DAF-16 activity is tissue specific and its life-long activity is correlated with longevity. These results will help understand the fundamental mechanisms by which this transcription factor regulates the aging process.

References:

1. K. Lin, H. Hsin, N. Libina, and C. Kenyon, “Regulation of the Caenorhabditis elegans longevity protein DAF-16 by insulin/IGF-1 and germline signaling,” Nature Genetics, vol. 28, no. 2. pp. 139–145, 2001.
2. D. J. Dickinson, A. M. Pani, J. K. Heppert, C. D. Higgins, and B. Goldstein, “Streamlined genome engineering with a self-excising drug selection cassette,” Genetics, vol. 200, no. 4, pp. 1035–1049, 2015.

Figures:

Figure 1: A) DAF-16 activity decreases after exposure to dietary restriction in subsequent days. B) DAF-16 migrates to cell nuclei under reduced food intake. Scale bar 25 μm.