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Cold Shock Recovery has diverse phenotypes. a–b 12 h post 4 h cold shock (HpCS), late stage embryos accumulate in the gonads, eventually causing a bag of worms phenotype. c–f 24 HpCS the intestine has drastically decreased in width and pigmentation and the gonad arms have shrunk. Braces indicate intestine width; brackets indicate the posterior gonad arm. g–j Cold-shocked worms that survive to 72 h largely regain their intestinal pigmentation and size; however, they exhibit a protruding vulva phenotype. Braces indicate intestine; arrow indicates vulva
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Animals are exposed to a wide range of environmental stresses that can cause potentially fatal cellular damage. The ability to survive the period of stress as well as to repair any damage incurred is essential for fitness. Exposure to 2 °C for 24 h or longer is rapidly fatal to the nematode Caenorhabditis elegans, but the process of recovery from a...
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... and the worm's internal organs begin to shrink (Fig. 3b, g). In particular, the gonads appear to wither and there is a distinct dis- ruption of the developing oocytes as well as a decrease in their number relative to controls. Adult worms that had already produced a few embryos before the cold shock accumulate additional embryos within the uterus (Fig. 4a, b, Additional file 1: Figure S1). The intestinal cells have less pigmentation and decreased width along much of the worm's length. Large gaps begin to appear in the worm's body cavity, which contribute to the over- all appearance of clearing and are likely caused by the shrinking of the worm's internal ...
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... day after the cold shock, the damage is more pro- nounced, with even larger gaps in the worm's bodies Fig. 3 Cold shock recovery progression after 4-hour cold shock. Control a-e and cold-shocked f-j worms after 4, 12, 24, 48, and 72 h of recovery, respectively (Figs. 3c, h and 4c-f ). The intestinal cells are still further reduced in width, and have continued to lose pigmenta- tion, in many cases losing it entirely (Fig. 4d, f ). The posterior gonad arm has usually completely collapsed or occasionally ruptured at this point (Figs. 3h and 4f ). The anterior arm is often less damaged, though still severely disrupted, ...
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... gaps in the worm's bodies Fig. 3 Cold shock recovery progression after 4-hour cold shock. Control a-e and cold-shocked f-j worms after 4, 12, 24, 48, and 72 h of recovery, respectively (Figs. 3c, h and 4c-f ). The intestinal cells are still further reduced in width, and have continued to lose pigmenta- tion, in many cases losing it entirely (Fig. 4d, f ). The posterior gonad arm has usually completely collapsed or occasionally ruptured at this point (Figs. 3h and 4f ). The anterior arm is often less damaged, though still severely disrupted, ranging from a reduction in size and disrup- tion of developing oocytes to complete collapse (Fig. ...
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... cold-shocked f-j worms after 4, 12, 24, 48, and 72 h of recovery, respectively (Figs. 3c, h and 4c-f ). The intestinal cells are still further reduced in width, and have continued to lose pigmenta- tion, in many cases losing it entirely (Fig. 4d, f ). The posterior gonad arm has usually completely collapsed or occasionally ruptured at this point (Figs. 3h and 4f ). The anterior arm is often less damaged, though still severely disrupted, ranging from a reduction in size and disrup- tion of developing oocytes to complete collapse (Fig. ...
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... days after cold shock, nearly all live worms have regained much of their pigmentation (Figs. 3e, j and 4g, h). The majority of these worms have a prominent protruding vulva phenotype, and while their gonads are still partially disrupted, they resume production of oocytes and embryos (Fig. 4i, j). Control non-cold- shocked worms contain a large number of unfertilized and very few embryos, suggesting that by this time point many of them have ...
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... days after cold shock, nearly all live worms have regained much of their pigmentation (Figs. 3e, j and 4g, h). The majority of these worms have a prominent protruding vulva phenotype, and while their gonads are still partially disrupted, they resume production of oocytes and embryos (Fig. 4i, j). Control non-cold- shocked worms contain a large number of unfertilized and very few embryos, suggesting that by this time point many of them have depleted their stores of self- produced sperm (Fig. 3e). In contrast, cold-shocked worms that have recovered at 20 °C for 72 h contain em- bryos that appear to be fertilized and viable (Fig. ...
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... in the survival of heavy metal and oxidative stress, as well as the innate immune re- sponse. Worms containing null mutations in fshr-1 have wild-type longevity under normal growth conditions, but have increased sensitivity to oxidative, heavy metal, and pathogen stresses [15,17] . Surprisingly these mutant worms are resistant to 2 °C cold shock (Fig. ...
Citations
... A recent investigation of the contribution of the reproductive system to oxygen consumption in C. elegans utilized 2,3-BDM, stating that this method of inhibition is "not increasing ATP synthesis or mitochondrial oxygen consumption" [21]; however, to our knowledge, no studies have empirically tested the effect of 2,3-BDM on ATP levels. 2,3-BDM induces flaccid paralysis, such that muscles are not contracted during imaging, but this effect appears to be relatively slow; previous work often allows the worms a full hour to paralyze prior to imaging [21][22][23]. ...
... Exposure to cold temperature slows metabolic processes in the worm, reducing movement. Cold shock at 2 C was only lethal after 12-hour exposures or longer, though cold shocked worms show decreased gonad size, loss of pigmentation, and increased vulval abnormalities after recovery [23]. Cold shock at 4 o C for 16 hours is capable of inducing blebbing of dopaminergic neurons, suggesting it may induce dopaminergic neurodegeneration [27]. ...
One aspect of Caenorhabditis elegans that makes it a highly valuable model organism is the ease of use of in vivo genetic reporters, facilitated by its transparent cuticle and highly tractable genetics. Despite the rapid advancement of these technologies, worms must be paralyzed for most imaging applications, and few investigations have characterized the impacts of common chemical anesthetic methods on the parameters measured, in particular biochemical measurements such as cellular energetics and redox tone. Using two dynamic reporters, QUEEN-2m for relative ATP levels and reduction-oxidation sensitive GFP (roGFP) for redox tone, we assess the impact of commonly used chemical paralytics. We report that no chemical anesthetic is entirely effective at doses required for full paralysis without altering redox tone or ATP levels, and that anesthetic use alters the detected outcome of rotenone exposure on relative ATP levels and redox tone. We also assess the use of cold shock, commonly used in combination with physical restraint methods, and find that cold shock does not alter either ATP levels or redox tone. In addition to informing which paralytics are most appropriate for research in these topics, we highlight the need for tailoring the use of anesthetics to different endpoints and experimental questions. Further, we reinforce the need for developing less disruptive paralytic methods for optimal imaging of dynamic in vivo reporters.
... Acute cold shock has been shown to be harmful to worms, with various negative phenotypes being reported [47,48]. Previous experiments have shown the prevalence of dendritic blebbing in the PVD neurons of worms following cold shock [22]. ...
Caenorhabditis elegans (C. elegans) has served as a simple model organism to study dopaminergic neurodegeneration, as it enables quantitative analysis of cellular and sub-cellular morphologies in live animals. These isogenic nematodes have a rapid life cycle and transparent body, making high-throughput imaging and evaluation of fluorescently tagged neurons possible. However, the current state-of-the-art method for quantifying dopaminergic degeneration requires researchers to manually examine images and score dendrites into groups of varying levels of neurodegeneration severity, which is time consuming, subject to bias, and limited in data sensitivity. We aim to overcome the pitfalls of manual neuron scoring by developing an automated, unbiased image processing algorithm to quantify dopaminergic neurodegeneration in C. elegans. The algorithm can be used on images acquired with different microscopy setups and only requires two inputs: a maximum projection image of the four cephalic neurons in the C. elegans head and the pixel size of the user's camera. We validate the platform by detecting and quantifying neurodegeneration in nematodes exposed to rotenone, cold shock, and 6-hydroxydopamine using 63x epifluorescence, 63x confocal, and 40x epifluorescence microscopy, respectively. Analysis of tubby mutant worms with altered fat storage showed that, contrary to our hypothesis, increased adiposity did not sensitize to stressor-induced neurodegeneration. We further verify the accuracy of the algorithm by comparing code-generated, categorical degeneration results with manually scored dendrites of the same experiments. The platform, which detects 20 different metrics of neurodegeneration, can provide comparative insight into how each exposure affects dopaminergic neurodegeneration patterns.
... To ensure that physiological changes occurred during cold shock treatment, we performed survival assays to track the fates of C. elegans in the post-cold shock (PCS) stage. Within the first 96 h of the PCS stage, 59% of the worms subjected to cold shock treatment died whereas those raised at 20 • C experienced negligible mortality (Fig. 5A), which was in line with the findings of a previous study [49]. This assay affirmed the occurrence of physiological changes of worm populations under an acute cold shock treatment. ...
Although next-generation sequencing technology has been used to delineate RNA modifications in recent years, the paucity of appropriate converting reactions or specific antibodies impedes the accurate characterization and quantification of numerous RNA modifications, especially when these modifications demonstrate wide variations across developmental stages and cell types. In this study, we developed a high-throughput analytical platform coupling ultra-performance liquid chromatograph (UPLC) with complementary mass spectrometry (MS) to identify and quantify RNA modifications in both synthetic and biological samples. Sixty-four types of RNA modifications, including positional isomers and hypermodified ribonucleosides, were successfully monitored within a 16-min single run of UPLC–MS. Two independent methods to cross-validate the purity of RNA extracted from Caenorhabditis elegans (C. elegans) were developed using the coexisting C. elegans and Escherichia coli (E. coli) as a surveillance system. To test the validity of the method, we investigated the RNA modification landscape of three model organisms, C. elegans, E. coli, and Arabidopsis thaliana (A. thaliana). Both the identity and molarity of modified ribonucleosides markedly varied among the species. Moreover, our platform is not only useful for exploring the dynamics of RNA modifications in response to environmental cues (e.g., cold shock) but can also help with the identification of RNA-modifying enzymes in genetic studies. Cumulatively, our method presents a novel platform for the comprehensive analysis of RNA modifications, which will be of benefit to both analytical chemists involved in biomarker discovery and biologists conducting functional studies of RNA modifications.
... Interestingly, PA28γ/psme-3 overexpression slightly decreased lifespan at standard temperature (20 °C) and was strongly detrimental at warmer temperature (Fig. 3h,i). On the contrary, PA28γ/psme-3 overexpression further extends longevity at 15 °C (Fig. 3j) 39 . To examine whether PA28γ/PSME-3 can confer resistance to acute cold shock, we exposed worms to extreme low temperature (4 °C) for 12 h and then shifted them back to 20 °C. ...
Aging is a primary risk factor for neurodegenerative disorders that involve protein aggregation. Because lowering body temperature is one of the most effective mechanisms to extend longevity in both poikilotherms and homeotherms, a better understanding of cold-induced changes can lead to converging modifiers of pathological protein aggregation. Here, we find that cold temperature (15 °C) selectively induces the trypsin-like activity of the proteasome in Caenorhabditis elegans through PSME-3, the worm orthologue of human PA28γ/PSME3. This proteasome activator is required for cold-induced longevity and ameliorates age-related deficits in protein degradation. Moreover, cold-induced PA28γ/PSME-3 diminishes protein aggregation in C. elegans models of age-related diseases such as Huntington’s and amyotrophic lateral sclerosis. Notably, exposure of human cells to moderate cold temperature (36 °C) also activates trypsin-like activity through PA28γ/PSME3, reducing disease-related protein aggregation and neurodegeneration. Together, our findings reveal a beneficial role of cold temperature that crosses evolutionary boundaries with potential implications for multi-disease prevention.
... Acute cold shock has been shown to be harmful to worms, with various negative phenotypes being reported (47,48). Previous experiments have shown the prevalence of dendritic blebbing in the PVD neurons of worms following cold shock (22). ...
Caenorhabditis elegans ( C. elegans ) has served as a simple model organism to study dopaminergic neurodegeneration, as it enables quantitative analysis of cellular and sub-cellular morphologies in live animals. These isogenic nematodes have a rapid life cycle and transparent body, making high-throughput imaging and evaluation of fluorescently tagged neurons possible. However, the current state-of-the-art method for quantifying dopaminergic degeneration requires researchers to manually examine images and score dendrites into groups of varying levels of neurodegeneration severity, which is time consuming, subject to bias, and limited in data sensitivity. We aim to overcome the pitfalls of manual neuron scoring by developing an automated, unbiased image processing algorithm to quantify dopaminergic neurodegeneration in C. elegans . The algorithm can be used on images acquired with different microscopy setups and only requires two inputs: a maximum projection image of the four cephalic neurons in the C. elegans head and the pixel size of the user’s camera. We validate the platform by detecting and quantifying neurodegeneration in nematodes exposed to rotenone, cold shock, and 6-hydroxydopamine using 63x epifluorescence, 63x confocal, and 40x epifluorescence microscopy, respectively. Analysis of tubby mutant worms with altered fat storage showed that, contrary to our hypothesis, increased adiposity did not sensitize to stressor-induced neurodegeneration. We further verify the accuracy of the algorithm by comparing code-generated, categorical degeneration results with manually scored dendrites of the same experiments. The platform, which detects 19 different metrics of neurodegeneration, can provide comparative insight into how each exposure affects dopaminergic neurodegeneration patterns.
... Prior studies involving cooling of C. elegans indicate that cooling for shorter periods of time does not appear to impact the animal health, 5 but longer periods (>4 h) cause an immediate or progressive decline in health. 10,15 We assayed viability and fecundity of animals immobilized by cooling for 1 h. First, we immobilized animals at 6 C (slow cooling) and quantified their lifespan and number of offspring ( Figure S3). ...
Despite its profound impact on biology, high-resolution in vivo microscopy largely remains low throughput because current immobilization techniques require substantial manual effort. We implement a simple cooling approach to immobilize entire populations of the nematode Caenorhabditis elegans directly on their cultivation plates. Counterintuitively, warmer temperatures immobilize animals much more effectively than the colder temperatures of prior studies and enable clear submicron-resolution fluorescence imaging, which is challenging under most immobilization techniques. We demonstrate 64× z-stack and time-lapse imaging of neurons in adults and embryos without motion blur. Compared to standard azide immobilization, cooling immobilization reduces the animal preparation and recovery time by >98%, significantly increasing experimental speed. High-throughput imaging of a fluorescent proxy in cooled animals and direct laser axotomy indicate that the transcription factor CREB underlies lesion conditioning. By obviating individual animal manipulation, our approach could empower automated imaging of large populations within standard experimental setups and workflows.
... Moreover, the lifespan extension seen in ets-4(−) mutants, as is the case with insulin pathway mutants, depends on the transcription factor DAF-16/ FOXO 21,25 . These and additional reports, that insulin pathway mutants display cold resistance depending on DAF- 16 15,19 , prompted us to examine the genetic relationship between ets-4(−) and the insulin pathway mutants in the context of cold resistance. First, using a lossof-function allele of the insulin-like receptor, daf-2(e1370) 26 , we observed that these mutants survived cold even better than ets-4(−) mutants (Fig. 2a). ...
How animals rewire cellular programs to survive cold is a fascinating problem with potential biomedical implications, ranging from emergency medicine to space travel. Studying a hibernation-like response in the free-living nematode Caenorhabditis elegans, we uncovered a regulatory axis that enhances the natural resistance of nematodes to severe cold. This axis involves conserved transcription factors, DAF-16/FoxO and PQM-1, which jointly promote cold survival by upregulating FTN-1, a protein related to mammalian ferritin heavy chain (FTH1). Moreover, we show that inducing expression of FTH1 also promotes cold survival of mammalian neurons, a cell type particularly sensitive to deterioration in hypothermia. Our findings in both animals and cells suggest that FTN-1/FTH1 facilitates cold survival by detoxifying ROS-generating iron species. We finally show that mimicking the effects of FTN-1/FTH1 with drugs protects neurons from cold-induced degeneration, opening a potential avenue to improved treatments of hypothermia. Strategies to improve cold resistance are of potential biomedical interest. Here the authors demonstrate that ferritin-mediated detoxification of iron, preventing the generation of reactive oxygen species, promotes cold survival in both Caenorhabditis elegans and cultured mammalian neurons.
... We have previously shown that acute cold shock at 2 °C causes loss of intestinal pigmentation, immobility, and reproductive disruption in wild-type hermaphrodite worms. In many cases, this phenotypic program results in lethality 15 . Here we extend this work by characterizing the induction of these phenotypes via the neuronal TAX-2/TAX-4 thermotransduction channel, mediated by the canonical stress response regulator SKN-1/Nrf2. ...
... Acute cold shock causes drastic phenotypic alterations. The duration of cold exposure for young adult hermaphrodite C. elegans at 2 °C is negatively correlated to post-shock survival rates 15 . Wild-type hermaphrodite worms exposed to a 4-h cold shock (CS) do not initially display high mortality rates (Fig. 1a); this allows observation of a range of phenotypic transitions as they recover from the limited-duration cold stress at their preferred temperature of 20℃. ...
... Wild-type hermaphrodite worms exposed to a 4-h cold shock (CS) do not initially display high mortality rates (Fig. 1a); this allows observation of a range of phenotypic transitions as they recover from the limited-duration cold stress at their preferred temperature of 20℃. One of the most striking phenotypes exhibited in post-cold shock (post-CS) animals during the recovery period is a dramatic decrease in pigmentation in the normally highly pigmented intestine, so that the body becomes almost entirely clear (Fig. 1b, c) 15 . This is often accompanied by motor and reproductive disruptions such as mobility loss, withering of the gonad arms, decreased number of internal embryos, and the eventual death of about 30% of the population (Fig. 1a-d) 15 . ...
Challenges from environmental stressors have a profound impact on many life-history traits of an organism, including reproductive strategy. Examples across multiple taxa have demonstrated that maternal reproductive investment resulting from stress can improve offspring survival; a form of matricidal provisioning when death appears imminent is known as terminal investment. Here we report a reproductive response in the nematode Caenorhabditis elegans upon exposure to acute cold shock at 2 °C, whereby vitellogenic lipid movement from the soma to the germline appears to be massively upregulated at the expense of parental survival. This response is dependent on functional TAX-2; TAX-4 cGMP-gated channels that are part of canonical thermosensory mechanisms in worms and can be prevented in the presence of activated SKN-1/Nrf2, the master stress regulator. Increased maternal provisioning promotes improved embryonic cold shock survival, which is notably suppressed in animals with impaired vitellogenesis. These findings suggest that cold shock in C. elegans triggers terminal investment to promote progeny fitness at the expense of parental survival and may serve as a tractable model for future studies of stress-induced progeny plasticity.
... Additionally, worms stop thrashing upon cold exposure via ice or cooled liquids (e.g., a cold hydrogel), which allows for manipulation and placement of the worm within the hydrogel (Chung et al. 2008). However, both ice and cold hydrogels can trigger acute cold shock, which is known to trigger a stress response that has negative consequences in worms, including morphological changes within the germline and gut (Robinson and Powell 2016). Therefore, this method is not an option if the worms need to be exposed to continuous elevated temperatures either to maintain the mutant phenotype or to study the heat shock stress response pathway. ...
The visualization of biological processes using fluorescent proteins and dyes in living organisms has enabled numerous scientific discoveries. The nematode Caenorhabditis elegans is a widely used model organism for live imaging studies since the transparent nature of the worm enables imaging of nearly all tissues within a whole, intact animal. While current techniques are optimized to enable the immobilization of hermaphrodite worms for live imaging, many of these approaches fail to successfully restrain the smaller male worms. To enable live imaging of worms of both sexes, we developed a new genetic, conditional immobilization tool that uses the auxin-inducible degron (AID) system to immobilize both adult and larval hermaphrodite and male worms for live imaging. Based on chromosome location, mutant phenotype, and predicted germline consequence, we identified and AID-tagged three candidate genes (unc-18, unc-104, and unc-52). Strains with these AID-tagged genes were placed on auxin and tested for mobility and germline defects. Among the candidate genes, auxin-mediated depletion of UNC-18 caused significant immobilization of both hermaphrodite and male worms that was also partially reversible upon removal from auxin. Notably, we found that male worms require a higher concentration of auxin for a similar amount of immobilization as hermaphrodites, thereby suggesting a potential sex-specific difference in auxin absorption and/or processing. In both males and hermaphrodites, depletion of UNC-18 did not largely alter fertility, germline progression, nor meiotic recombination. Finally, we demonstrate that this new genetic tool can successfully immobilize both sexes enabling live imaging studies of sexually dimorphic features in C. elegans.
... Coldshock has been previously studied as a stressor for C. elegans [62][63][64][65][66][67][68][69][70][71][72][73]. Robinson and Powell identified that animals can survive short (4 h) exposures to acute coldchock (2°C), but longer exposures (24 h) result in death for a fraction of the population [74]. Furthermore, Ohta et al. showed that the pre-cold-shock culture temperature is inversely correlated with survival rate (more animals survive cold-shock if previously cultured at lower temperatures) [75]. ...
Background:
Access to quantitative information is crucial to obtain a deeper understanding of biological systems. In addition to being low-throughput, traditional image-based analysis is mostly limited to error-prone qualitative or semi-quantitative assessment of phenotypes, particularly for complex subcellular morphologies. The PVD neuron in Caenorhabditis elegans, which is responsible for harsh touch and thermosensation, undergoes structural degeneration as nematodes age characterized by the appearance of dendritic protrusions. Analysis of these neurodegenerative patterns is labor-intensive and limited to qualitative assessment.
Results:
In this work, we apply deep learning to perform quantitative image-based analysis of complex neurodegeneration patterns exhibited by the PVD neuron in C. elegans. We apply a convolutional neural network algorithm (Mask R-CNN) to identify neurodegenerative subcellular protrusions that appear after cold-shock or as a result of aging. A multiparametric phenotypic profile captures the unique morphological changes induced by each perturbation. We identify that acute cold-shock-induced neurodegeneration is reversible and depends on rearing temperature and, importantly, that aging and cold-shock induce distinct neuronal beading patterns.
Conclusion:
The results of this work indicate that implementing deep learning for challenging image segmentation of PVD neurodegeneration enables quantitatively tracking subtle morphological changes in an unbiased manner. This analysis revealed that distinct patterns of morphological alteration are induced by aging and cold-shock, suggesting different mechanisms at play. This approach can be used to identify the molecular components involved in orchestrating neurodegeneration and to characterize the effect of other stressors on PVD degeneration.
