Mendenhall, Alexander et al. (2015) International Worm Meeting "Effects of introns on gene expression."

Mendenhall, Alexander, Brent, Roger, Sands, Bryan

[International Worm Meeting, 2015]

In C. elegans, as in the early days of recombinant DNA in mammalian cells, introns are generally used to increase transgene expression. Specifically, to increase expression, most C. elegans promoter-fused-to-fluorescent-protein reporter genes in use have three synthetic introns developed by the Fire lab inserted into the coding sequence of mCherry or GFP. But exactly how much more expression do those introns result in? Are all three introns necessary for the increased expression? Here, using single copy reporters integrated into the same defined locus, we measured the effects of different numbers, positions and sequences of introns on mean gene expression. We quantified expression from whole animals in flow and individual intestine cells using a point scanning confocal microscope. Preliminary findings suggest that, relative to no intron reporter gene controls, different combinations of the original three synthetic introns can result in increased or decreased mean expression. We intend to use these combinations to set reporter gene expression output to desired levels, for example, so as to be within the dynamic range of measurement of other reporters whose products are expressed in the same cells or in the same visual field. .

Alexander Mendenhall et al. (2007) International Worm Meeting "Sex-Dependent Responses to Oxygen Deprivation in C. elegans."

Pamela Padilla, Michelle LeBlanc, Alexander Mendenhall, Desh Mohan

[International Worm Meeting, 2007]

Oxygen deprivation is a medical, ecological, and economic issue for humans. Identification of the molecular mechanisms animals use to survive oxygen deprivation (anoxia or hypoxia) could result in new medical treatments or environmental policies. We have determined that adult male C. elegans, in comparison to adult hermaphrodites, are able to survive longer periods of anoxic exposure. Briefly, after three days of anoxic exposure males (wild-type or him-8 animals) display a high survival rate (~80%) whereas hermaphrodites have a very low survival rate (~10%). This data lead to the hypothesis that the sex of an organism could influence the response to oxygen deprivation. To test this hypothesis further we are examining genetic and environmental factors that may influence anoxia survival. First, since the daf-2/daf-16 signaling pathway is known to have a role in a variety of stresses including oxygen deprivation we are testing if daf-16 is required for male anoxia survival. Second, we are using RNA interference and genetic mutants to determine if egg production, male genotype or male phenotype influences the ability of an animal to survive anoxia. Third, we are altering animal growth conditions (population density) to analyze the influence pretreatments have on anoxia survival rate. These studies will lead to a greater understanding of the mechanisms nematodes use to survive the stress of anoxia. Furthermore, this work will address the question of whether intrinsic differences that exist between the sexes affect their ability to respond to and survive the stress of oxygen deprivation.

Mendenhall, Alexander R. et al. (2009) International Worm Meeting "Regulation of Gene Expression: Where Did That Noise Come From!?"

Seewald, Alexander K., Tedesco, Patricia M., Mendenhall, Alexander R., Johnson, Thomas E., Cypser, James R.

[International Worm Meeting, 2009]

Isogenic populations of worms show surprising variation in lifespan (Rea et al., 2005). In isogenic worm populations, the level of gene expression of an integrated Phsp-16.2::gfp on the second day of adulthood functions as a very good predictor of subsequent longevity. In an effort to understand how this stochastic variation (also called "noise" and measured as the coefficient of variation [SD/Mean]) in gene expression can correlate with lifespan, we are beginning a systematic study of sources of noise. We have created multiple strains of C. elegans in which we have independently integrated the same Phsp-16.2::gfp reporter gene construct. After heat shock we see considerable variation in the level of GFP expression among both strains and individual worms. In each animal, we observe variation across tissues and among cells within the same tissue (the intestine), indicating that there are factors responsible for gene expression, even within a tissue, which are variable between cells in the same animal. Furthermore, we have constructed strains expressing red and green fluorescent reporter genes both under Phsp-16.2 control in two distinct loci. These strains allow us to understand how different loci containing the same sequence information are, nonetheless, regulated differently, both among individual worms and within an individual. When analyzing whole worms, the brightest red animals are also the brightest green animals, indicating a significant co-specification of overall expression levels. This data suggests that there are common elements (transcription factors, etc.) within individual worms that have an intrinsic effect on level of expression. However, when animals are analyzed in detail under the microscope, some individuals exhibit striking differences in which reporter is activated both within and between tissues, which demonstrates that there is cellular autonomy in determining which locus to express. We are currently working to understand how promoter sequence, transgene copy number, flanking DNA sequence, genetic background, chromatin structure, and other variables contribute to this noise. We are also determining the predictive capabilities of each of the Phsp-16.2 reporter gene strains with regard to frailty and longevity. See Rea et al., 2005. A stress-sensitive reporter predicts longevity in isogenic populations of Caenorhabditis elegans. Nat. Genet. 37: 894-898.

Mendenhall, Alexander et al. (2015) International Worm Meeting "Reproducible quantification of gene expression in single cells of live adult animals."

Tedesco, Patricia, Brent, Roger, Sands, Bryan, Mendenhall, Alexander, Johnson, Thomas

[International Worm Meeting, 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. We developed microscope-based methods to quantify reporter gene expression in cells of Caenorhabditis elegans with low measurement error. 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. We hope they will allow investigators to use changes in reporter expression in single cells in tissues as quantitative phenotypes, and link those to their molecular causes. Moreover, by diminishing measurement error, they should make possible dissection of the causes of the remaining, real, variation in expression. We hope that understanding such variation will help reveal its contribution to differences in complex phenotypic outcomes in multicellular organisms.

Mendenhall, Alexander et al. (2015) International Worm Meeting "Mechanisms of animal-to-animal and cell-to-cell variation in gene expression in adult hermaphrodites."

Mendenhall, Alexander, Brent, Roger, Sands, Bryan, Johnson, Thomas, Tedesco, Patricia

[International Worm Meeting, 2015]

In isogenic populations of C. elegans cultured under homogeneous conditions, differences in the amount of GFP produced under control of the hsp-16.2 promoter predict differences in lifespan. Animals with high expression enjoy longer lifespan, longer healthspan and higher thermotolerance. How do animals end up in this privileged physiological state? We showed previously that variation (here, the coefficient of variation or CV) in hsp-16.2 reporter gene expression was constant among different strains bearing reporter constructs with differences in locus of integration, different copy numbers, and different fluorescent proteins. Here we sought to understand the origins of the variation in expression that results in high expression animals. We used a genetic and cell biological approach to study the origins of variable expression in individual animals and individual cells. Our preliminary results show that, at the level of the whole animal two distinct systems affect variation in gene expression, one of which increases, and the other of which decreases (canalizes) interindividual differences. At the cell level there is not much "intrinsic noise" in reporter expression (that is, fluorescent signal from two different differently colored reporter proteins driven by two different instances of the same promoter is highly correlated). In contrast to some studies in single cell organisms, we find that, when we measure the same cells in different animals, the ratio of fluorescent signal from two different fluorescent proteins driven by two different promoters is almost the same. It is possible that this limitation of variation in gene expression may reflect a need to tightly coordinate expression of different genes during metazoan development and adult life.

Mendenhall, Alexander R. et al. (2011) International Worm Meeting "Factors Affecting the Mean, Variance and Predictive Power of a Lifespan-Predicting Biomarker."

Mendenhall, Alexander R., Lowe, Anita, Tedesco, Pat, Johnson, Thomas E., Taylor, Larry, Brent, Roger, Cypser, James R.

[International Worm Meeting, 2011]

Variation in gene expression among genetically identical individuals grown together in a uniform environment has been largely unexplored in C. elegans. In 2005, Rea et al. showed that the induced expression level of a transcriptional reporter of a small heatshock protein (Phsp-16.2::gfp) in young adult C. elegans could predict lifespan and thermotolerance. However, these studies used a single transgenic insertion (gpIs1), which contains approximately 450 copies of the transgene in a tandem array integrated into chromosome IV. To determine if different transgenic reporter alleles of the same gene show similar predictive capabilities and variance in expression, we generated several new transgenic lines expressing green and red fluorescent proteins under control of the hsp-16.2 promoter; these lines include three new single-copy reporters. Worm-to-worm variance in transgene expression is independent of locus, copy number, and fluor. Variance in GFP signal, measured either as worm-to-worm variance among individual worms or as cell-to-cell variance among matched intestine cells, is not significantly different between strains. The average expression profiles of the single-copy and tandem-array reporters display the most and least GFP in the exact same intestine cells; however, cell-to-cell measures are still preliminary and ongoing. Growth temperature affects the mean and variance of constitutively expressed reporters (Pvit-2::gfp and Pgpd-2::gfp). In contrast, mean and variance of the induced expression of Phsp-16.2::gfp are independent of growth temperature. Interestingly, we observed spontaneous, constitutive Phsp-16.2::gfp expression at 25deg C. Elimination or attenuation of specific signaling systems results in reduced worm-to-worm variance in Phsp-16.2::gfp expression. Thus, worm-to-worm variance in reporter expression is actively increased by intrinsic individual differences in and/or stochastic fluctuation of signal transduction between individuals. Finally, both single copy and tandem array transgenes predict thermotolerance and lifespan. Our studies demonstrate that the predictive capacity of the Phsp-16.2::gfp biomarker is not dependent on any specific transgene configuration. This abstract marks the beginning of a systems biology approach to dissect metazoan gene expression variance at the level of individual animals and cells to determine how single reporters can predict complex phenotype outcomes such as lifespan.

Alexander Mendenhall et al. (2006) Development & Evolution Meeting "Morphological, developmental and genetic analysis of post-embryonic Caenorhabditis elegans exposed to anoxia."

Bobby LaRue, Alexander Mendenhall, Pamela Padilla, Desh Mohan

[Development & Evolution Meeting, 2006]

Organisms have evolved the capacity to survive a variety of environmental stresses such as oxygen deprivation. Metazoan responses to oxygen deprivation include developmental arrest, reduction in developmental trajectory, physiological alterations, metabolic repression, and gene expression changes. Unlike many metazoans, C. elegans are tolerant to severe oxygen deprivation conditions in that all life-cycle stages are able to survive 24 hours of anoxia (< .001kPa O2) by arresting energy requiring processes such as developmental progression and motility. We have found that sex, life cycle stage, and genetics influence the ability to survive long-term anoxia (3-5 days of anoxia at 20 C) or the double stress of high-temperature and anoxia (1 day of anoxia at 28 C). For example, wild-type adult males exposed to anoxia have a significantly higher survival rate in comparison to adult hermaphrodites and the dauer larvae survive several days of anoxia. Furthermore, unlike the wild-type hermaphrodite, the daf-2(e1370) hermaphrodite has the ability to survive long-term anoxia and high temperature anoxia. The goal of this project is to identify mechanisms involved with enhanced anoxia survival. First, we used DIC microscopy to examine and compare tissue structure of wild-type adult hermaphrodites, males and daf-2(e1370) hermaphrodites. We determined, that unlike wild-type hermaphrodites adult, males and daf-2(e1370) hermaphrodites do not accumulate significant tissue damage in long-term anoxia, suggesting that long-term anoxia tolerant animals are better able to maintain tissue structure and function. Second, we used RNA interference to screen DAF-16 regulated genes that suppress the daf-2(e1370) enhanced anoxia-survival phenotype. We found that the enhanced anoxia survival phenotype of males and daf-2(e1370) hermaphrodites is dependent upon the small heat shock protein hsp-12.6. These data suggests that mechanisms have evolved to serve a protective role for animals exposed to severe oxygen deprivation. Identifying the mechanisms regulating oxygen deprivation tolerance in eukaryotic systems may be of interest to further understanding the pathology of environmental stress and genes regulating aging.

Mendenhall A et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Probing Causes and Consequences of Stochastic Variation in C. elegans Gene Expression"

Mendenhall A, Tedesco P, Johnson T, Brent R

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

In Caenorhabditis elegans, development is spatially and temporally determined; adult cell number and anatomy are nearly invariant. Cohorts of isogenic animals synchronously express genes that cause individuals to molt to successive larval stages within a few hours of one other. Yet, even isogenic cohorts cultured in homogenous, liquid environments show 20-fold variation in individual lifespan. Five years ago, we (Johnson lab) found (REA et al. 2005) in isogenic populations that level of expression of a heat-shock promoter-GFP fusion construct (Phsp-16.2::gfp) after mild heat stress predicts subsequent lifespan. Thus, Phsp-16.2 reporter expression on the first day of adult life is a physiological variable (or 'biomarker') predictive of subsequent lifespan. In metazoans, the components and mechanism(s) underlying expression variation of the Phsp-16.2::gfp biomarker (or any other gene) remain mostly unknown. For that reason we have initiated work on Phsp-16.2::gfp expression modeled on previous work in S.cerevisiae. In these yeast studies, we (Brent lab) and others have dissected and quantified different contributions to 'stochastic' variation in gene expression and on cell fate decisions (COLMAN-LERNER et al. 2005). As in these studies, we are quantifying gene expression and variation, dissecting the different contributions to those quantities, and using genetics to understand control of the individual sources of variation. To do so, we measure expression of different-colored fluorescent reporter proteins, under the control of identical or of different promoters, quantify components of the variation based on simple mathematical models, and use the results to guide subsequent hypothesis-directed experiments. At this point, we have some genetic data identifying physiological subsystems that control the amount of worm-to-worm variation in gene expression. We have used defined site, single copy reporter integration technology (MosSCI technology) to determine how variance in gene expression manifests when using single copy reporter genes vs tandem arrays and to minimize variation in gene expression due to chromosomal position and silencing. We are using these reporters to quantify the amount of worm-to-worm variation due to intrinsic noise and that due to the different physiological subsystems. Colman-Lerner,A., A. Gordon,E. Serra, T. Chin, O.Resnekov et al., 2005 Regulated cell-to-cellvariation in a cell-fate decision system. Nature 437: 699-706. Rea, S.L., D. Wu,J. R. Cypser, J. W.Vaupel and T. E.Johnson, 2005 Astress-sensitive reporter predicts longevity in isogenic populations of Caenorhabditis elegans. Nat Genet 37: 894-898.

Alexander R Mendenhall et al. (2005) International Worm Meeting "C. elegans survival guide for anoxia: the requirement of metabolic and heat shock gene products"

Pamela A Padilla, Bobby L LaRue, Alexander R Mendenhall

[International Worm Meeting, 2005]

Oxygen deprivation (hypoxia or anoxia) is central to the pathology of various medical problems leading to severe economic and human health consequences. For many metazoans oxygen deprivation often results in cellular death. However, the absence of oxygen is not always fatal in that some metazoans survive days without oxygen. This raises the hypothesis that protective mechanisms exist in oxygen deprivation tolerant organisms and identification of such will provide insights into understanding or preventing oxygen deprivation induced cell injury. We use C. elegans to study the molecular response to oxygen deprivation. C. elegans survive anoxia by entering into a reversible state of suspended animation, where development, feeding, egg laying and motility arrest. Nematodes recover upon re-exposure to a normoxic environment. Adult hermaphrodites survive at least one day of anoxia with a viability of 90 % or greater, whereas dauer larvae are capable of surviving many days of anoxia. Given this, and the fact that daf-2(e1370ts) adults survive hypoxia (.3% O2) at 28C, we compared the daf-2(e1370ts) and wild-type N2 nematode response to anoxia. We demonstrate that the daf-2(e1370ts) adult hermaphrodites survive long-term anoxic exposure (5 days, 20C), as well as the combined stress of anoxia and high temperature (1 day, 28C). We used time-lapse microscopy to compare the N2 and daf-2(e1370ts) body movement in response to anoxia (20C). N2 adults began to decrease movement after 2 hours of anoxia exposure and arrested between 8-16 hours of anoxia exposure. The daf-2(e1370ts) adults exposed to anoxia also displayed a decrease in movement, yet the percentage of worms that remained motile was significantly higher in comparison to wild type. The daf-2(e1370ts) adults retained the ability to move after 24 hours of anoxia exposure. We used RNA interference to screen through the DAF-16 regulated genes to identify genes that are involved with anoxia response and survival. We determined that gpd-2, which encodes a putative glyceraldehydes-3-phosphate dehydrogenase, and hsp-12.6, which encodes a small heat shock protein, are required for anoxia responses. The gpd-2(RNAi) adult had extensive tissue damage after anoxic exposure, suggesting that GPD-2 has a vital role in the response to anoxia. Furthermore, gpd-2(RNAi) worms become immobile immediately upon anoxia exposure. This data suggests that some DAF-16 regulated genes are required for oxygen deprivation responses and raises the hypothesis that if specific proteins are upregulated prior to anoxic exposure they may serve a protective role in response to oxygen deprivation.

Alexander Mendenhall et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Uncoupling Mechanisms Required for Oxygen Deprivation Survival and Longevity in C. elegans"

Michelle LeBlanc, Alexander Mendenhall, Pamela Padilla, Mary Ladage, Mark Pavlyukovskyy

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Stress resistance and longevity occur from single gene mutations, suggesting that these two traits are regulated by similar mechanisms. Others have shown that mutations in the insulin-like signaling pathway (daf-2(e1370)) or alterations that affect germline (glp-1(q158)) lead to increased lifespan. We show that these same mutations lead to severe oxygen deprivation survival (anoxia), indicating that stress resistance and longevity are under control of DAF-16 or signals involving the reproductive tissue. Further analysis of mutations that enhance long-term anoxia survival indicates that there are distinct genetic and physiological factors affecting anoxia responses that do not affect longevity. For example, the DAF-16-regulated glycolytic genes, gpd-2/3, are required for the daf-2(e1370) animals'' long-term anoxia and high temperature anoxia survival phenotype but not the longevity phenotype. Furthermore, we have identified mutants with reproductive phenotypes, such as the fog-2(q71) animals, which have an enhanced ability to survive long-term anoxia but are known to not be long lived. Additionally, not all daf-2 mutants that are long lived will survive long-term anoxia. Thus, data suggests that modulation of distinct components of the insulin signaling pathway or specific changes in reproductive physiology differentially regulate anoxia survival and longevity. Therefore, we hypothesize that the mechanisms required for anoxia stress responses and longevity are distinct and can be uncoupled. To test this hypothesis we are conducting further analysis on the functional role of gpd-2/3. First, we are determining if gpd-2/3 function is required for other long-term anoxia survival mutants. Second, we are using a transcriptional reporter (Pgpd-2/3::GFP) to analyze gpd-2/3 expression in anoxia exposed animals, aging adults or daf-16(mu86) null mutants. Finally, we tested whether transgenic over expression of GPD-2/3 is sufficient to increase long-term anoxia survival or enhance lifespan. This research will further our understanding of the relationship between oxygen deprivation resistance and longevity.