Sci. Aging Knowl. Environ., 5 May 2004
Vol. 2004, Issue 18, p. pe18
[DOI: 10.1126/sageke.2004.18.pe18]

PERSPECTIVES

Come One, Come All

Siu Sylvia Lee

The author is in the Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14850, USA. E-mail: SSL29{at}cornell.edu

http://sageke.sciencemag.org/cgi/content/full/2004/18/pe18

Key Words: C. elegansdaf-2 • gene expression • microarrays

Microarray analysis of global gene expression is a particularly powerful approach for dissecting complex biological processes such as aging. Microarray experiments require a large amount of RNA and thus a large amount of starting material. This requirement poses a problem, because variation within a sample increases with sample size and can easily skew the results.

In their recent Aging Cell paper (1), Golden and Melov describe their attempt to circumvent this sample size problem by analyzing the gene expression profiles of individual nematodes as they aged, comparing wild-type and long-lived mutant daf-2/InR worms; these mutant worms live twice as long as wild-type animals [see Johnson Review (2)]. Synchronized populations of worms were obtained by collecting eggs laid within a 2.5-hour window. These worms were allowed to develop and age. At the desired time points, individual worms were picked, and each was placed in a small volume of water and immediately frozen. Total RNA for each worm was prepared, and two rounds of linear amplification were performed to obtain antisense RNA for microarray hybridization. For each of the genotypes, the authors monitored the gene expression profiles of individual nematodes at four different time points, from day 4 to day 19. In their experiments, day 4 corresponded to the early gravid adult stage, whereas day 19 probably corresponded to the old adult stage just before death for wild-type animals. (The precise life stages of the animals are unclear, because the life-span distribution of their population of assayed animals was not shown in the paper.) They surveyed a set of 921 genes (4.5% of the worm genome), which were selected mostly because they encode proteins that either localize to mitochondria or are related to the worm's stress response. They then identified groups of genes in both wild-type and daf-2 worms whose expression changes correlate with chronological age. For example, the expression of multiple heat-shock genes increases with chronological age regardless of the genotype. They also identified groups of genes whose age-related changes in expression differed significantly between the two genotypes. This latter class of genes may represent interesting candidate biomarkers for physiological age or may represent genes that are important for modulating the Caenorhabditis elegans life span.

This study is of great importance because it represents the first microarray analysis of individual nematodes over the worm's life span. As the authors appropriately point out, previous microarray analyses have depended on collecting a large number of synchronized worms. The isolation of a homogenous population of worms is challenging when aging animals are considered. This is because individuals within a population of isogenic C. elegans, cultured under the same conditions, age at different rates and show significant variation regarding the deterioration of their cellular and tissue integrity and the time of death (3, 4). Although gene expression profiling of populations of young versus old worms (5) and age mutants versus controls (6, 7) have provided valuable information, it is possible that some of the gene expression changes detected previously are either exaggerated or masked as a result of this variation within a population. Furthermore, the need to recover a large number of nematodes often requires culturing animals under conditions that are stressful for them and thus may induce gene expression changes that are not typical of normal aging. The ability to perform gene expression profiling using a small number of worms should bypass many of these caveats.

However, precisely because aging is stochastic, the gene expression profile of a small number of worms may not appropriately reflect the "typical" profile of aging worms. Arguably, if a tightly synchronized population of C. elegans can be obtained, expression profiling of a large number of worms may in fact eliminate some of the variation between individuals that might simply represent noise in the system. To test this possibility, expression profiling of individual worms will have to be performed in many replicates in order to examine whether variations in gene expression among different individuals exist. The authors attempted to address this issue by examining four or five different individual worms for each test condition, and no wide-spread differences in gene expression were detected among the worms tested. Among the 912 genes surveyed, T12A2.3 (which encodes a translation initiation factor) and F25H2.13 (which encodes a helicase) are the only genes that showed significant expression differences among individual worms of the same genotype at the same time point. T12A2.3 exhibited the highest variance among wild-type worms at day 14, and F25H2.13 showed the greatest variance among daf-2 worms at day 4.

A limitation of this paper is that although the authors hypothesize that the stochastic nature of the aging process in C. elegans has the potential to render the expression profile of a large population of aged worms unreliable, they did not address this issue specifically in their experiments. For example, the authors compared the expression profiles of individual worms according to their chronological age, but did not attempt to monitor the morphological and cellular deterioration associated with each aging worm (3, 4), which is probably a more accurate indicator of the "physiological age" of each of the animals. In future experiments, it will be critical to compare the gene expression differences between worms that are the same chronological age but exhibit differential tissue and cellular deterioration. One would expect that the individuals with similar tissue and cellular deterioration would share common gene expression changes, whereas individuals of distinct tissue and cellular integrity would exhibit gene expression differences.

Upon validating the feasibility of profiling the transcriptional changes within individual worms, the authors went on to compare the gene expression differences in wild-type worms at several time points, from young adult to very old, as well as the corresponding time points for the daf-2 mutant worms. Whereas the expression profiles of aging wild-type worms (5), as well as those of daf-2 versus daf-2;daf-16 mutant worms (6, 7) (see Larsen Perspective), have been previously investigated, the interesting comparison between gene expression profiles of aging wild-type worms versus aging daf-2 worms has not been examined until this paper. This comparison may identify gene expression changes that are a signature of physiological aging. Presumably, some of the gene expression changes that appear as wild-type worms age and die will manifest either with much slower kinetics in the long-lived daf-2 mutant or at a later time when the daf-2 worms finally age and die.

In the Golden/Melov paper (1), the genes that showed age-related changes in expression that differed the most between wild-type and daf-2 worms are (i) a thaumatin family member, which may be involved in antimicrobial defense (there was a similar expression trend in wild-type and daf-2 worms but slower kinetics in daf-2 worms); (ii) an electron transport chain component (an increase in expression with age in wild-type worms but a decrease in expression with age in daf-2 mutant worms); (iii) a proteasome component (increase in expression with age in wild-type worms); (iv) actin (decrease in expression with age in wild-type worms but increase in expression with age in daf-2 mutant worms); and (v) the Akt/PKB kinase (increase in expression with age in wild-type worms but decrease in expression with age in daf-2 mutant worms). Because Akt/PKB is a component of the daf-2 pathway, the authors propose the hypothesis that the shorter wild-type life span may be related to heightened insulin signaling with advanced age. Additional analyses of the expression differences between the wild type and the long-lived daf-2 mutant may shed further light on the molecular mechanisms that mediate the longevity effect of daf-2/insulin signaling.

Unfortunately, the authors only carried out their experiments to day 19 of the worm's life, which is close to the end of the wild-type life span but is only about 40% of the daf-2 mutant life span (2, 8). In the future, it would be very interesting to continue to monitor the expression profile of the daf-2 mutant worms until just before the end of their life span. The later time points for daf-2 worms may be critical for the identification of gene expression changes that reveal molecular markers of aging. In addition, as a previous study indicates, the first 4 days of adulthood are the critical window of time in which insulin signaling appears to have a lasting effect on the life span of C. elegans (9); thus, it would also be interesting to determine the expression profile of individual wild-type and daf-2 worms at multiple time points during this early adult period. Results from such experiments would also provide an extremely valuable data set to compare with presently available expression profile data obtained with large populations of wild-type and daf-2 worms spanning similar time points (7). Such an empirical comparison would be helpful in assessing whether expression profiling of a single worm as it ages represents is a more appropriate approach as compared to expression profiling of a large population of aging worms.

The ability to profile the gene expression changes of individual worms has important implications for microarray studies in C. elegans. Future experiments that survey the expression changes of all genes in the C. elegans genome and that span more time points throughout the life of a nematode, together with a more precise correlation of gene expression with the physiological state of an aging worm, promise to reveal exciting new findings that will likely advance our understanding of the aging process in C. elegans and perhaps in other species.


May 5, 2004
  1. T. R. Golden, S. Melov, Microarray analysis of gene expression with age in individual nematodes. Aging Cell, 29 April 2004 (doi:10.111/;1474-9728.2004.00095.x). [Abstract]
  2. C. Kenyon, J. Chang, E. Gensch, A. Rudner, R. Tabtiang, A C. elegans mutant that lives twice as long as wild type. Nature 366, 461-464 (1993).[CrossRef][Medline]
  3. D. Garigan, A. L. Hsu, A. G. Fraser, R. S. Kamath, J. Ahringer, C. Kenyon, Genetic analysis of tissue aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation. Genetics 161, 1101-1112 (2002).[Abstract/Free Full Text]
  4. L. A. Herndon, P. J. Schmeissner, J. M. Dudaronek, P. A. Brown, K. M. Listner, Y. Sakano, M. C. Paupard, D. H. Hall, M. Driscoll, Stochastic and genetic factors influence tissue-specific decline in ageing C. elegans. Nature 419, 808-814 (2002).[CrossRef][Medline]
  5. J. Lund, P. Tedesco, K. Duke, J. Wang, S. K. Kim, T. Johnson, Transcriptional profile of aging in C. elegans. Curr. Biol. 12, 1566-1573 (2002).[CrossRef][Medline]
  6. J. McElwee, K. Bubb, J. H. Thomas, Transcriptional outputs of the Caenorhabditis elegans forkhead protein DAF-16. Aging Cell 2, 111-121 (2003).[CrossRef][Medline]
  7. C. T. Murphy, S. A. McCarroll, C. I. Bargmann, A. Fraser, R. S. Kamath, J. Ahringer, H. Li, C. Kenyon, Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature 424, 277-283 (2003).[CrossRef][Medline]
  8. K. D. Kimura, H. A. Tissenbaum, Y. Liu, G. Ruvkun, daf-2, an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans [see comments]. Science 277, 942-946 (1997).[Abstract/Free Full Text]
  9. A. Dillin, D. K. Crawford, C. Kenyon, Timing requirements for insulin/IGF-1 signaling in C. elegans. Science 298, 830-834 (2002).[Abstract/Free Full Text]
Citation: S. S. Lee, Come One, Come All. Sci. Aging Knowl. Environ. 2004 (18), pe18 (2004).








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