Mendenhall AR et al. (2006) Genetics "Glyceraldehyde-3-phosphate dehydrogenase mediates anoxia response and survival in Caenorhabditis elegans."

Padilla PA, Larue B, Mendenhall AR

[Genetics, 2006]

Oxygen deprivation has a role in the pathology of many human diseases thus it is of interest to understand the genetic and cellular responses to hypoxia or anoxia in oxygen deprivation tolerant organisms such as Caenorhabditis elegans. In C. elegans the DAF-2/DAF-16 pathway, an IGF-1/insulin-like signaling pathway, is involved with dauer formation, longevity and stress resistance. In this report we compared the response wild- type and daf-2(e1370) animals have to anoxia. Unlike wild-type animals, the daf-2(e1370) animals have an enhanced anoxia-survival phenotype in that they survive long-term anoxia and high-temperature anoxia, do not accumulate significant tissue damage in either of these conditions and are motile after 24 hrs of anoxia. RNA interference was used to screen DAF-16 regulated genes that suppress the daf-2(e1370) enhanced anoxia-survival phenotype. We identified gpd-2 and gpd-3; two nearly identical genes in an operon that encode the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. We found that not only is the daf-2(e1370) enhanced anoxia phenotype dependent upon gpd-2/3 but the motility of animals exposed to brief periods of anoxia is prematurely arrested in gpd-2/3(RNAi) and daf-2 (e1370);gpd-2/3(RNAi) animals. These data suggest that gpd-2/3 may serve a protective role in tissue exposed to oxygen deprivation.

Mendenhall A et al. (2017) J Gerontol A Biol Sci Med Sci "Environmental Canalization of Life Span and Gene Expression in Caenorhabditis elegans."

Sutphin G, Tedesco PM, Mendenhall A, Leiser S, Johnson TE, Brent R, Crane MM, Kaeberlein M

[J Gerontol A Biol Sci Med Sci, 2017]

Animals, particularly poikilotherms, exhibit distinct physiologies at different environmental temperatures. Here, we hypothesized that temperature-based differences in physiology could affect the amount of variation in complex quantitative traits. Specifically, we examined, in Caenorhabditis elegans, how different temperatures (15C, 20C, and 25C) affected the amount of interindividual variation in life span and also expression of three reporter genes-transcriptional reporters for vit-2, gpd-2, and hsp-16.2 (a life-span biomarker). We found the expected inverse relationship between temperature and average life span. Surprisingly, we found that at the highest temperature, there were fewer differences between individuals in life span and less interindividual variation in expression of all three reporters. We suggest that growth at 25C might canalize (reduce interindividual differences in) life span and expression of some genes by eliciting a small constitutive heat shock response. Growth at 25C requires wild-type hsf-1, which encodes the main heat shock response transcriptional activator. We speculate that increased chaperone activity at 25C may reduce interindividual variation in gene expression by increasing protein folding efficiency. We hypothesize that reduced variation in gene expression may ultimately cause reduced variation in life span.

Mendenhall AR et al. (2011) J Gerontol A Biol Sci Med Sci "Genetic dissection of late-life fertility in Caenorhabditis elegans."

Park SK, Link CD, Mendenhall AR, Tedesco PM, Wu D, Johnson TE, Cypser JR, Phillips PC

[J Gerontol A Biol Sci Med Sci, 2011]

The large post-reproductive life span reported for the free-living hermaphroditic nematode, Caenorhabditis elegans, which lives for about 10 days after its 5-day period of self-reproduction, seems at odds with evolutionary theory. Species with long post-reproductive life spans such as mammals are sometimes explained by a need for parental care or transfer of information. This does not seem a suitable explanation for C elegans. Previous reports have shown that C elegans can regain fertility when mated after the self-fertile period but did not report the functional limits. Here, we report the functional life span of the C elegans germ line when mating with males. We show that C elegans can regain fertility late in life (significantly later than in previous reports) and that the end of this period corresponds quite well to its 3-week total life span. Genetic analysis reveals that late-life fertility is controlled by conserved pathways involved with aging and dietary restriction.

Mendenhall A et al. (2017) J Gerontol A Biol Sci Med Sci "Caenorhabditis elegans Genes Affecting Interindividual Variation in Life-span Biomarker Gene Expression."

Brent R, Tedesco PM, Johnson TE, Mendenhall A, Crane MM

[J Gerontol A Biol Sci Med Sci, 2017]

Mendenhall AR et al. (2012) J Gerontol A Biol Sci Med Sci "Expression of a single-copy hsp-16.2 reporter predicts life span."

Cypser JR, Mendenhall AR, Lowe A, Johnson TE, Taylor LD, Tedesco PM

[J Gerontol A Biol Sci Med Sci, 2012]

The level of green fluorescent protein expression from an hsp-16.2-based transcriptional reporter predicts life span and thermotolerance in Caenorhabditis elegans. The initial report used a high-copy number reporter integrated into chromosome IV. There was concern that the life-span prediction power of this reporter was not attributable solely to hsp-16.2 output. Specifically, prediction power could stem from disruption of some critical piece of chromatin on chromosome IV by the gpIs1 insertion, a linked mutation from the process used to create the reporter, or from an artifact of transgene regulation (multicopy transgenes are subject to regulation by C elegans chromatin surveillance machinery). Here we determine if the ability to predict life span and thermotolerance is specific to the gpIs1 insertion or a general property of hsp-16.2-based reporters. New single-copy hsp-16.2-based reporters predict life span and thermotolerance. We conclude that prediction power of hsp-16.2-based transcriptional reporters is not an artifact of any specific transgene configuration or chromatin surveillance mechanism.

Mendenhall AR et al. (2009) Physiol Genomics "Reduction in ovulation or male sex phenotype increases long-term anoxia survival in a daf-16-independent manner in Caenorhabditis elegans."

Leblanc MG, Padilla PA, Mohan DP, Mendenhall AR

[Physiol Genomics, 2009]

Identifying genotypes and phenotypes that enhance an organism''s ability to survive stress is of interest. We used Caenorhabditis elegans mutants, RNA interference (RNAi) and the chemical 5-fluorodeoxyuridine (FUDR) to test the hypothesis that a reduction in progeny would increase oxygen deprivation (anoxia) survival. In the hermaphrodite gonad, germline processes such as spermatogenesis and oogenesis, can be simultaneously as well as independently disrupted by genetic mutations. We analyzed genetic mutants (glp-1(q158), glp-4(bn2ts), plc-1(rx1), ksr-1(ku68), fog-2(q71), fem-3(q20), spe-9(hc52ts), fer-15(hc15ts)) with reduced progeny production due to various reproductive defects. Furthermore, we used RNAi to inhibit the function of gene products in the RTK/Ras/MAPK signaling pathway, which is known to be involved with a variety of developmental processes including gonad function. We determined that reduced progeny production or complete sterility enhanced anoxia survival except in the case of sterile hermaphrodites (spe-9(hc52ts), fer-15(hc15ts)) undergoing oocyte maturation and ovulation as exhibited by the presence of laid unfertilized oocytes. Furthermore, the fog-2(q71) long-term anoxia survival phenotype was suppressed when oocyte maturation and ovulation was induced by mating with males that have functional or non-functional sperm. The mutants with a reduced progeny production survive long-term anoxia in a daf-16 and hif-1 independent manner. Finally, we determined that wild-type males were able to survive long-term anoxia in a daf-16 independent manner. Together, these results suggest that the insulin signaling pathway is not the only mechanism to survive oxygen deprivation and that altering gonad function, in particular oocyte maturation and ovulation, leads to a physiological state conductive for oxygen deprivation survival. Key words: oxygen deprivation, germline, insulin-like signaling, gonad, anoxia.

Mendenhall AR et al. (2015) PLoS One "Single Cell Quantification of Reporter Gene Expression in Live Adult Caenorhabditis elegans Reveals Reproducible Cell-Specific Expression Patterns and Underlying Biological Variation."

Johnson TE, Tedesco PM, Sands B, Mendenhall AR, Brent R

[PLoS One, 2015]

In multicellular organisms such as Caenorhabditis elegans, differences in complex phenotypes such as lifespan correlate with the level of expression of particular engineered reporter genes. In single celled organisms, quantitative understanding of responses to extracellular signals and of cell-to-cell variation in responses has depended on precise measurement of reporter gene expression. Here, we developed microscope-based methods to quantify reporter gene expression in cells of Caenorhabditis elegans with low measurement error. We then quantified expression in strains that carried different configurations of Phsp-16.2-fluorescent-protein reporters, in whole animals, and in all 20 cells of the intestine tissue, which is responsible for most of the fluorescent signal. Some animals bore more recently developed single copy Phsp-16.2 reporters integrated at defined chromosomal sites, others, "classical" multicopy reporter gene arrays integrated at random sites. At the level of whole animals, variation in gene expression was similar: strains with single copy reporters showed the same amount of animal-to-animal variation as strains with multicopy reporters. At the level of cells, in animals with single copy reporters, the pattern of expression in cells within the tissue was highly stereotyped. In animals with multicopy reporters, the cell-specific expression pattern was also stereotyped, but distinct, and somewhat more variable. Our methods are rapid and gentle enough to allow quantification of expression in the same cells of an animal at different times during adult life. They should allow investigators to use changes in reporter expression in single cells in tissues as quantitative phenotypes, and link those to molecular differences. Moreover, by diminishing measurement error, they should make possible dissection of the causes of the remaining, real, variation in expression. Understanding such variation should help reveal its contribution to differences in complex phenotypic outcomes in multicellular organisms.

Seewald AK et al. (2010) PLoS One "Quantifying phenotypic variation in isogenic Caenorhabditis elegans expressing Phsp-16.2::gfp by clustering 2D expression patterns."

Cypser J, Mendenhall A, Seewald AK, Johnson T

[PLoS One, 2010]

Isogenic populations of animals still show a surprisingly large amount of phenotypic variation between individuals. Using a GFP reporter that has been shown to predict longevity and resistance to stress in isogenic populations of the nematode Caenorhabditis elegans, we examined residual variation in expression of this GFP reporter. We found that when we separated the populations into brightest 3% and dimmest 3% we also saw variation in relative expression patterns that distinguished the bright and dim worms. Using a novel image processing method which is capable of directly analyzing worm images, we found that bright worms (after normalization to remove variation between bright and dim worms) had expression patterns that correlated with other bright worms but that dim worms fell into two distinct expression patterns. We have analysed a small set of worms with confocal microscopy to validate these findings, and found that the activity loci in these clusters are caused by extremely bright intestine cells. We also found that the vast majority of the fluorescent signal for all worms came from intestinal cells as well, which may indicate that the activity of intestinal cells is responsible for the observed patterns. Phenotypic variation in C. elegans is still not well understood but our proposed novel method to analyze complex expression patterns offers a way to enable a better understanding.

Crane MM et al. (2019) Sci Rep "In vivo measurements reveal a single 5'-intron is sufficient to increase protein expression level in Caenorhabditis elegans."

Brent R, Johnson B, Mendenhall A, Battaglia C, Yun S, Kaeberlein M, Sands B, Crane MM

[Sci Rep, 2019]

Introns can increase gene expression levels using a variety of mechanisms collectively referred to as Intron Mediated Enhancement (IME). IME has been measured in cell culture and plant models by quantifying expression of intronless and intron-bearing reporter genes in vitro. We developed hardware and software to implement microfluidic chip-based gene expression quantification in vivo. We altered position, number and sequence of introns in reporter genes controlled by the hsp-90 promoter. Consistent with plant and mammalian studies, we determined a single, natural or synthetic, 5'-intron is sufficient for the full IME effect conferred by three synthetic introns, while a 3'-intron is not. We found coding sequence can affect IME; the same three synthetic introns that increase mcherry protein concentration by approximately 50%, increase mEGFP by 80%. We determined IME effect size is not greatly affected by the stronger vit-2 promoter. Our microfluidic imaging approach should facilitate screens for factors affecting IME and other intron-dependent processes.

Burnaevskiy N et al. (2018) Elife "Reactivation of RNA metabolism underlies somatic restoration after adult reproductive diapause in C. elegans."

Mendenhall A, Chen S, Kaeberlein M, Burnaevskiy N, Reynolds A, Karanth S, Mailig M, Van Gilst M

[Elife, 2018]

The mechanisms underlying biological aging are becoming recognized as therapeutic targets to delay the onset of multiple age-related morbidities. Even greater health benefits can potentially be achieved by halting or reversing age-associated changes. <i>C. elegans</i> restore their tissues and normal longevity upon exit from prolonged adult reproductive diapause, but the mechanisms underlying this phenomenon remain unknown. Here, we focused on the mechanisms controlling recovery from adult diapause. Here, we show that functional improvement of post-mitotic somatic tissues does not require germline signaling, germline stem cells, or replication of nuclear or mitochondrial DNA. Instead a large expansion of the somatic RNA pool is necessary for restoration of youthful function and longevity. Treating animals with the drug 5-fluoro-2'-deoxyuridine prevents this restoration by blocking reactivation of RNA metabolism. These observations define a critical early step during exit from adult reproductive diapause that is required for somatic rejuvenation of an adult metazoan animal.