2 Neuroscience Department, Oberlin College, Ohio
3 Neuroscience Program, University of Illinois Urbana-Champaign
Positions are available at all levels for enthusiastic, collaborative scientists interested in both basic research and translating these findings to combating neglected tropical diseases. Write to Jordan D. Ward if interested: email@example.com
These findings suggest that IIS is reduced comparably at 15°C and 25°C and argues against the assumption that e1370 is necessarily a temperature-sensitive allele in the classic sense where temperature affects protein folding and activity. Interestingly, about 10% wild-type worms form dauers when grown at 27°C under otherwise standard culturing conditions (ample food and not overcrowded; (Ailion & Thomas 2000 PMID:11063684)). Furthermore, it has been observed that null mutants in other abnormal-DAuer-Formation (daf) genes than daf-2, only show the dauer phenotype at higher temperatures, suggesting that the dauer phenotype itself is temperature sensitive. This observation was published over 30 years ago (Golden & Riddle 1984) and is discussed in (Wood 1988).
We are motivated to write to the WBG community by the assumption that during adulthood daf-2(e1370) mutants improperly reactive phenotypes that are reminiscent of dauer larvae (reduced mobility, reduced brood-size, short body size; (Gems et al. 1998)). Recently, we have shown that daf-2(e1370) mutants form dauers independent of SKN-1/NRF1,2,3 and that SKN-1 is only required for the daf-2(e1370)-longevity phenotype when these dauer-related phenotypes are not activated during adulthood (Ewald et al. 2015). Furthermore, comparing expression profiles of daf-2(e1370) mutants with dauer larvae revealed that they share only a common overlap under conditions that reactivate these dauer phenotypes (e.g., daf-2(e1370) at higher but not at lower temperatures; (Ruzanov et al. 2007 PMID:17543485; McElwee et al. 2004 PMID:15308663; Ewald et al. 2015)). Finally, treating wild-type worms with dauer pheromone was sufficient to increase lifespan (Kawano et al. 2005 PMID:16377915; Ewald et al. 2015). This suggests that there might be a dauer program activated in daf-2(e1370) mutants at higher temperatures that masks other underlying mechanisms of reduced IIS on lifespan. Further research is required, however, these findings suggest that reduced IIS can extend lifespan via dauer-dependent and dauer-independent mechanisms and this changes the interpretation of experiments performed with daf-2(e1370) mutants under conditions that promote these dauer-associated phenotypes.
Golden, J.W. & Riddle, D.L., 1984. A pheromone-induced developmental switch in Caenorhabditis elegans: Temperature-sensitive mutants reveal a wild-type temperature-dependent process. Proceedings of the National Academy of Sciences of the United States of America, 81(3), pp.819–823.
Wood, W.B. ed., 1988. The Nematode Caenorhabditis elegans. 1st ed., Cold Spring Harbor Monograph Series.
The C. elegans che-12 gene is expressed in a subset of ciliated sensory neurons in the head and tail of the animal, including the amphid neurons with simple rod like cilia (Bacaj et al., 2008). CHE-12 localizes to the cilia in these neurons which form a bundle in the amphid channel. Previously, we observed CHE-12::GFP puncta along the amphid cilia suggesting coupling to intraflagellar transport machinery, however, the arrangement of amphid cilia in a bundle inside the amphid channel presented a challenge to follow a single fluorescent particle continuously from the cilia base to the distal tip (Das et al., 2015). In order to better visualize CHE-12 dynamics in a single amphid cilium, we devised a strategy to fluorescently tag the endogenous che-12 gene in a single amphid neuron, the ASER neuron.
Our cell-specific tagging cassette (CTC) includes a Stop codon and a strong terminator sequence (let-858 3’UTR) bounded at either end by two LoxP sites within short synthetic introns, followed by the coding sequence of any fluorescent protein (Figure 1A). We inserted a hygromycin resistance gene in the inter-genic region between the che-12 and B0024.4 genes for the selection of positive knock-in candidates. The CTC is introduced between the end of the coding sequence of the gene of interest and its stop codon using Cas9-targeted homologous recombination methods described in Dickinson et al., 2013. Animals bearing a correct insertion of the CTC at the selected genetic locus express the untagged version of the protein. A cell/tissue specific promoter is then used to drive the expression of Cre-recombinase in the specific cell/tissue where expression of the fluorescently tagged endogenous protein is desired. Cre-recombinase will excise out the region flanked by the LoxP sites in these specific cells, creating a fusion of the gene of interest and the fluorescent protein coding sequence, leading to cell/tissue-specific tagging of endogenous proteins. See Figure 1A an illustration of this strategy.
We introduced a CTC as outlined in Figure 1A at the 3’-end of the che-12 coding sequence using Cas9-targeted homologous recombination. We then used a ubiquitous or cell/tissue-specific promoter-driven Cre-recombinase, expressed from an extra-chromosomal array to allow selective recombination in those specific cells and fuse the che-12 coding sequence with mNeonGreen coding sequence. This strategy was successful in selectively labeling CHE-12 in a single neuron when we used gcy-5 promoter-driven Cre-recombinase (Figure 1B). In contrast, when we used a ubiquitous promoter such as Peft-3 to drive Cre-recombinase expression, we observed CHE-12::mNeonGreen signal in all amphid neurons (Figure 1C). The tagged CHE-12::mNeonGreen fusion protein localized correctly to the ASER cilium (Figure 1B and 1D).
This cell-specific tagging strategy is applicable for cell and tissue types for which strong cell/tissue-specific promoters have been characterized. This strategy can also be modified to delete domains within a protein or the entire coding sequence of a gene in a desired cell type by introducing synthetic introns containing embedded LoxP sites at the boundaries of a segment targeted for deletion.
In our initial attempts to use video-tracking to measure basal slowing and thrashing, we encountered two main obstacles: using a camera mounted on a dissecting microscope resulted in too small of a field of view, such that worms could enter and exit the field during the assay, and using a brightfield illuminator made it difficult to have sufficient contrast between the worms and the bacteria. To overcome these limitations we developed a microscope-free system for measuring movement (see Fig 1A for image). To expand the field of view, we have utilized a macro lens (Navitar Zoom 7000) attached directly to a FireWire camera (Allied Vision Technologies Stingray F-504B 2/3” CCD Monochrome Camera), both mounted on a weighted boom stand. To achieve increased contrast, we employed a LED darkfield base illuminator (Nikon Model P-DF).
For those who have not measured basal slowing before, we provide our standard protocol. Approximately 50 worms (day 3 of adulthood) are washed in 1.5 ml of M9 buffer to remove any residual bacteria, and allowed to sink to the bottom by gravity. The 50 worms are then transferred in 250 µl of liquid to 60 mm NGM plates that are either unseeded or seeded completely with OP50 (seeded plates are covered completely with bacteria and allowed to dry and grow for 48 hours). The 250 µl of liquid is spread out over the plate and the plate is left uncovered to facilitate drying. After 5 minutes of acclimation, videos of the largest square that would fit inside the edges of a plate were recorded for 1 min using and the MATLAB image acquisition tool (see Fig 1B for typical zoom). Videos were recorded in 8-bit at 2452×2056 resolution with a frame rate of 9fps (540 frames for the 1 minute video).
For analysis, videos were imported to imageJ as AVI files using the wrMTrck plugin. This plugin is publically available at http://www.phage.dk/plugins. The website also includes detailed instructions for thresholding and analysis. Briefly, videos are thresholded and, after defining min/max pixel size for a worm, quantification of crawling speed on seeded plates and unseeded plates is performed. Basal slowing is then calculated as the difference in rate of movement on unseeded plate versus seeded plates divided by the rate of movement on unseeded plates (See Fig 1C for typical data). Thrashing rate, the rate of movement in liquid, can also be measured using this set-up. For this assay, approximately 50 worms are transferred to an unseeded 60 mm NGM plate to which 1 ml of M9 buffer is added. Videos are recorded and analyzed as with basal slowing although we typically zoom in further to measure the thrashing rate.