Oxygen deprivation has a role in the pathology of many human diseases. Thus it is of interest in understanding 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 of wild-type and daf-2(e1370) animals 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 hightemperature anoxia, do not accumulate significant tissue damage in either of these conditions, and are motile after 24 hr 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 and gpd-3, but also 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 and gpd-3 may serve a protective role in tissue exposed to oxygen deprivation.

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.

Mendenhall AR, LeBlanc MG, Mohan DP, Padilla PA. Reduction in ovulation or male sex phenotype increases long-term anoxia survival in a daf-16-independent manner in Caenorhabditis elegans. Physiol Genomics 36: 167-178, 2009. First published December 2, 2008 doi:10.1152/physiolgenomics.90278.2008.-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, germ line 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 in 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 were induced by mating with males that have functional or nonfunctional 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 conducive for oxygen deprivation survival. oxygen deprivation; germ line; insulin-like signaling; stress; anoxia METAZOANS INHABITING aquatic or subterranean ecosystems have evolved mechanisms to survive changes in oxygen levels. For example, repression of energy-requiring processes or reallocation of metabolism increases oxygen deprivation survival (21). Although most terrestrial organisms live in environments where the oxygen tension does not fluctuate much, they do have the capacity to sense and respond to oxygen deprivation. In humans, oxygen deprivation is central to various health conditions including myocardial infarction, stroke, blood loss, pulmonary disorders, and solid tumor progression (37, 62). Thus identifying the molecular mechanisms that govern metazoan response to and survival of oxygen deprivation is of great interest.Oxygen deprivation tolerance spans across various phyla (7,20,53,55,68). The comparison of oxygen d...

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.