- Email: [email protected]
Re-evaluating the role of ELT-3 in a GATA transcription factor circuit proposed to guide aging in C. elegans
Mechanisms of Ageing and Development 133 (2012) 50–53
Contents lists available at SciVerse ScienceDirect
Mechanisms of Ageing and Development journal homepage: www.elsevier.com/locate/mechagedev
Short communication
Re-evaluating the role of ELT-3 in a GATA transcription factor circuit proposed to guide aging in C. elegans Tabitha Tonsaker 1, Ryan M. Pratt 1, James D. McGhee * Department of Biochemistry and Molecular Biology, Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada
A R T I C L E I N F O
A B S T R A C T
Article history: Available online 6 October 2011
Budovskaya et al. (Cell. 134, 291–303, 2008) have proposed that the ELT-3 GATA factor regulates somatic aging genes, including those expressed in the intestine, and participates in a transcription factor circuit that ‘‘guides Caenorhabditis elegans aging’’. We have re-investigated two key features of this proposal: (i) expression of elt-3 in the C. elegans adult intestine where the majority of somatic aging genes are expressed, and; (ii) the ability of elt-3 loss-of-function to revert the extended lifespan of daf-2(e1370) mutants. We find that: (i) in agreement with our previously published results, ELT-3 expression is largely hypodermal and is not expressed at significant levels in the adult C. elegans intestine, and; (ii) the elt3(vp1) zinc-finger deletion mutant does not significantly influence the extended lifespan of daf-2(e1370) mutants. We thus suggest that the role of ELT-3 in C. elegans aging should be re-evaluated. Published by Elsevier Ireland Ltd.
Keywords: C. elegans ELT-3 GATA factor Aging
The small free-living nematode Caenorhabditis elegans is a popular and powerful experimental animal in which to study aging and longevity; (for reviews, see Golden and Melov, 2007; Kim, 2007; Gems and Doonan, 2009; Gruber et al., 2009; Panowski and Dillin, 2009; Kenyon, 2010). A key observation in the field is that C. elegans lifespan and aging are strongly influenced by the insulin/ insulin-like growth factor signalling pathway. In particular, certain mutations in the daf-2 gene (encoding the C. elegans insulin receptor) can extend lifespan by several fold and this lifespan extension is reversed by loss-of-function mutations in the daf-16 gene (encoding a FoxO transcription factor) (Kenyon, 2011). Additional genes involved in C. elegans lifespan and/or aging have been identified, both by classical genetics and by genome-wide RNAi screens (see, for example, Hamilton et al., 2005; Hansen et al., 2005). The role(s) of all these genes is often interpreted in terms of an accumulating damage model of aging; for example, genes that make animals more resistant to stress, both physiological and environmental, might be expected to slow aging and extend lifespan. A different view of C. elegans aging and longevity was provided by Budovskaya et al. (2008), who reported that the GATA-type transcription factor ELT-3 (expressed primarily in the C. elegans hypodermis (Gilleard et al., 1999; Gilleard and McGhee, 2001; Liu et al., 2009)), together with EGL-18 (ELT-5) and ELT-6 (two GATA-type transcription factors that repress elt-3
* Corresponding author. Tel.: +1 403 220 4476. E-mail address: [email protected] (J.D. McGhee). 1 These authors contributed equally to this work. 0047-6374/$ – see front matter . Published by Elsevier Ireland Ltd. doi:10.1016/j.mad.2011.09.006
expression in the hypodermal seam cells (Koh and Rothman, 2001; Koh et al., 2002)), form a ‘‘GATA transcription circuit (that) guides aging in C. elegans’’. The authors interpreted their results in terms of an ‘‘antagonistic pleiotropy’’ model for aging, in which pathways that benefit an organism early in life might well have deleterious consequences post-reproduction. More specifically, the authors suggested that this transcription factor circuit ‘‘is driven by drift of developmental pathways rather than accumulation of damage’’. The two key points of the Budovskaya et al. (2008) argument were: (1) The authors identified 1000 genes either up-regulated or down-regulated in the soma of aging worms and they suggested that these genes are regulated by the ELT-3 GATA factor. The majority of these somatic aging genes (61%) are expressed in the intestine. Indeed, intestinally expressed genes are the only somatic genes enriched in this set of ‘‘aging genes’’; genes expressed in muscle, pharynx and neurons are not enriched, and there was no class listed for aging genes enriched in the hypodermis. (2) The authors reported that loss-of-function of elt-3 was able to reverse the lifespan extension caused by the daf-2(e1370) mutation. This striking observation is reminiscent of daf-16 mutations reversing the extended lifespan of the same daf-2 allele (Kenyon et al., 1993) and was the basis for proposing that ELT-3 down-regulation with age is associated with increased senescence. We wish to re-investigate both of these key points. (1) Does ELT-3 control somatic ‘‘aging genes’’ in the C. elegans adult intestine?
T. Tonsaker et al. / Mechanisms of Ageing and Development 133 (2012) 50–53
In our previous work characterizing the elt-3 gene (Gilleard et al., 1999; Gilleard and McGhee, 2001), we found no evidence that elt-3 is expressed in the C. elegans intestine at any stage of development (cited incorrectly by Budovskaya et al., 2008), as assayed either by ELT-3-specific antibodies or by four different transgenic reporters. elt-3 expression is first and foremost embryonic and hypodermal (except for the lateral seam cells; see also Koh and Rothman, 2001); there is a more enduring but also a more restricted post-embryonic phase of elt-3 expression in a subset of the pharyngeo-intestinal valve cells and intestinal rectal valve cells (but not in the intestine). We verified our previous observations using a new reporter construct (see Fig. 1) that has two additional features: (i) the elt-3 promoter includes the 2.2 kb upstream (up to the nearest upstream gene) of a recently annotated elt-3 alternate transcript (K02B9.4b), and; (ii) a nuclear-localized tdTomato reporter protein was used in order to minimize spectral overlap with intestinal autofluorescence, a common limitation of GFP reporters in C. elegans. Fig. 1 shows examples of young adult hermaphrodites transgenic for this new reporter, dissected in order to extrude the intestine. We focus on the mid-section of the worm (the ‘‘trunk’’) for two reasons:
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(i) Budovskaya et al. (2008) reported that ‘‘in the trunk, elt-3 expression is mostly derived from the hypodermal and intestinal cells’’, and; (ii) the age-associated decrease of elt-3 expression in the trunk is a central point in the overall model of Budovskaya et al. (2008). In agreement with our previous results (Gilleard et al., 1999; Gilleard and McGhee, 2001), reporter expression driven by the elt-3 promoter is overwhelmingly hypodermal and no significant expression can be detected in intestinal nuclei. It is impossible to be certain that a gene is not expressed in a particular tissue, especially based on evidence from C. elegans multi-copy transgenic reporters; these can be highly sensitive but can also show common sites of non-specific expression such as the posterior intestine (Boulin et al., 2006). Thus a suitably cautious conclusion is that the level of elt-3 expression in the midbody section (‘‘trunk’’) of the C. elegans intestine is less than ‘‘a few percent’’ of its level of expression in the hypodermis. This conclusion is supported by completely independent evidence: (i) ectopic expression of ELT-3 in the embryo is able to drive ectopic expression of hypodermal markers but not ectopic expression of intestinal markers (Gilleard and McGhee, 2001), and (ii) elt-3 transcripts were not detected in our previous SAGE analysis of
Fig. 1. The C. elegans elt-3 gene, the elt-3(vp1) deletion allele and the elt-3 expression pattern in transgenic worms. The two variants of the elt-3 gene (K02B9.4a and the more recently annotated longer transcript K02B9.4b) are shown to scale in the middle of the figure. Unfilled boxes correspond to exons; grey boxes correspond to either 50 or 30 UTRs and the black boxes correspond to the ELT-3 zinc-finger DNA binding domain + adjoining basic region. The 2730 bp vp1 deletion completely removes the ELT-3 DNA binding domain. The elt-3 genotype was determined in single worms as described in Gilleard and McGhee (2001); a typical agarose gel shows the elt-3 genotype in the four C. elegans strains used in this study (described in Table 1). As indicated, the new reporter construct (pJM524) used in this study contains 2.2 kb of promoter region upstream of the most upstream elt-3 transcript, fused to tandem-dimer-Tomato (Shaner et al., 2005) and a histone H2B domain that causes tight nuclear localization. The images at the bottom of the figure represent young (1 day past L4 stage) gravid adults, transgenic for pJM524, anaesthetized in cold 50 mM sodium azide and then dissected in order for the intestine and gonads to extrude. Images are overlays of the tdTomato fluorescent signal and differential interference contrast. Scale bars represent 50 microns. In this study, we examined several dozen independently produced transgenic strains, using either the standard dominant rol-6(su1006)-induced Roller phenotype or unc-119(ed3) rescue to identify transgenic animals. pJM524 expression is intense in the hypodermis but is undetectable in the extruded intestine (dashed outline).
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T. Tonsaker et al. / Mechanisms of Ageing and Development 133 (2012) 50–53
Table 1 Genotypes of the four C. elegans strains used in lifespan assay. Strain
Genotype
N2 JG1 CB1370 JM161
daf-2(+); elt-3(+) daf-2(+); elt-3(vp1) daf-2(e1370); elt-3(+) daf-2(e1370); elt-3(vp1)
dissected adult intestines but could easily be detected in intact animal controls (which include hypodermis) (McGhee et al., 2007); (there have been reports of elt-3 transcripts in the intestine (Pauli et al., 2006) but hypodermal contamination has not been ruled out.) In summary, we conclude that it is unlikely that ELT-3 directly controls transcription of aging-associated genes expressed in the C. elegans intestine. We cannot, of course, rule out indirect models in which ELT-3 influences intestinal gene expression from a different tissue. We have argued elsewhere that the GATA factor ELT-2, not ELT-3, is the predominant transcription factor directly driving intestinal gene expression in all stages later than the early embryo (McGhee et al., 2007, 2009). We also note that Budovskaya et al. (2008) used GFP reporter levels (from an undefined transgenic reporter) as a proxy for the levels of endogenous ELT-3 protein, as it changes with age or in response to, for example, egl-18(RNAi) and elt-6(RNAi). However, elt-3-driven reporter proteins (including the present construct used in Fig. 1) appear to perdure and to remain at high levels much later in development than do elt-3 transcripts (detected by Northerns) or endogenous ELT-3 protein (detected by antibodies) (Gilleard et al., 1999). Transgenic elt-3 reporters may be reliable measures of expression patterns but we suggest that they are unreliable measures of expression levels of endogenous ELT-3 protein. (2) Does elt-3 loss-of-function reverse the extended lifespan of daf2(e1370) animals? We now re-investigate the effect of elt-3 on the daf-2(e1370) lifespan extension using a genetically defined elt-3 loss-of-function mutation, rather than anti-elt-3 feeding RNAi as in the majority of the full lifespan curves reported by Budovskaya et al. (2008). We had previously produced the elt-3 mutation vp1, a 2730 bp deletion that removes the ELT-3 zinc-finger DNA-binding domain (see Fig. 1) and that we presume to be a genetic null (Gilleard and McGhee, 2001). We thus measured the lifespans of the four strains (N2, JG1, CB1370 and JM161), whose genotypes are summarized in Table 1. For each strain, the elt-3 genotype was verified by PCR using primers that clearly distinguish the elt-3(+) from the elt3(vp1) deletion allele (see Fig. 1) and the daf-2 genotype was verified by PCR and sequencing (data not shown). On several occasions, we verified strain identity by genotyping the last surviving worms in a lifespan assay. Fig. 2 shows the results of a typical lifespan experiment. We find no evidence that the elt-3(vp1) mutation is able to reverse the lifespan extension induced by the daf-2(e1370) mutation (p > 0.05, Mantel-Cox log-rank test). We have repeated this same full lifespan experiment three independent times (twice double blind) with essentially identical results. In preliminary experiments (with independently constructed strains), essentially the same results were obtained (three repeats, two double blind, with one repeat conducted with added FUdR in the plates to prevent offspring). Overall, our results clearly contradict the results of Budovskaya et al. (2008). In summary, we find no evidence that the elt-3 gene is expressed at a significant level in the C. elegans intestine. We thus suggest that it is unlikely that ELT-3 directly controls the many intestinally expressed genes associated with C. elegans aging, although indirect effects from other expressing tissues cannot be
Fig. 2. Typical lifespan measurements for the four strains used in this study; strain genotypes are summarized in Table 1. Worms that did not respond to persistent prodding were declared dead and then examined at higher power; worms that had ruptured or ‘‘bagged’’ (contained hatched larvae) were censored from the data. At the end of the lifespan assay, 73, 83, 85 and 80 worms had been scored for N2, JG1, CB1370 and JM161 strains, respectively. Temperature = 20.0 8C. As noted in the text, this basic experiment was repeated an additional two times with essentially identical results; (average number worms scored per strain per experiment = 76).
ruled out. Furthermore, we find no evidence that the elt-3(vp1) loss-of-function mutation is able to reverse the extended lifespan conferred by the C. elegans daf-2(e1370) mutation, as reported by Budovskaya et al. (2008). We have no ready explanation for the differences between the lifespans that we determined and those determined by Budovskaya et al. (2008). The answer could be something simple, such as the particular methods of handling worms, or it could be more complex and interesting, such as an unexpected mutation in the genome of one of the strains. In any event, we suggest that the role of ELT-3 in C. elegans aging should be re-evaluated. Acknowledgements The authors should like to thank Barbara Goszczynski for technical assistance and Dr. John Gilleard (University of Calgary, Faculty of Veterinary Medicine) for helpful discussions. We should also like to thank Dr. Joel Rothman (University of California, Santa Barbara) for critically reading the manuscript. Most of this study was performed as an undergraduate research project in the University of Calgary Bachelor of Health Sciences program, and was supported by operating grants from the Canadian Institutes of Health Research (to JDM). JDM is a Medical Scientist of the Alberta Heritage Foundation for Medical Research and a Canada Research Chair in Developmental Biology. References Boulin, T., Etchberger, J.F., Hobert, O., 2006. Reporter gene fusions. In: The C. elegans Research Community (Eds.), WormBook, doi:10.1895/wormbook.1.106.1, http://www.wormbook.org. Budovskaya, Y.V., Wu, K., Southworth, L.K., Jiang, M., Tedesco, P., Johnson, T.E., Kim, S.K., 2008. An elt-3/elt-5/elt-6 GATA transcription circuit guides aging in C. elegans. Cell 134, 291–303. Gems, D., Doonan, R., 2009. Antioxidant defense and aging in C. elegans: is the oxidative damage theory of aging wrong? Cell Cycle 8, 1681–1687. Gilleard, J.S., McGhee, J.D., 2001. Activation of hypodermal differentiation in the Caenorhabditis elegans embryo by GATA transcription factors ELT-1 and ELT-3. Mol. Cell Biol. 21, 2533–2544. Gilleard, J.S., Shafi, Y., Barry, J.D., McGhee, J.D., 1999. ELT-3: a Caenorhabditis elegans GATA factor expressed in the embryonic epidermis during morphogenesis. Dev. Biol. 208, 265–280. Golden, T.R., Melov, S., 2007. Gene expression changes associated with aging in C. elegans. WormBook 1–12. Gruber, J., Ng, L.F., Poovathingal, S.K., Halliwell, B., 2009. Deceptively simple but simply deceptive – Caenorhabditis elegans lifespan studies: considerations for aging and antioxidant effects. FEBS Lett. 583, 3377–3387.
T. Tonsaker et al. / Mechanisms of Ageing and Development 133 (2012) 50–53 Hamilton, B., Dong, Y., Shindo, M., Liu, W., Odell, I., Ruvkun, G., Lee, S.S., 2005. A systematic RNAi screen for longevity genes in C. elegans. Genes Dev. 19, 1544–1555. Hansen, M., Hsu, A.L., Dillin, A., Kenyon, C., 2005. New genes tied to endocrine, metabolic, and dietary regulation of lifespan from a Caenorhabditis elegans genomic RNAi screen. PLoS Genet. 1, 119–128. Kenyon, C., 2011. The first long-lived mutants: discovery of the insulin/IGF-1 pathway for ageing. Philos. Trans. R. Soc. Lond. B Biol. Sci. 366, 9–16. Kenyon, C., Chang, J., Gensch, E., Rudner, A., Tabtiang, R., 1993. A C. elegans mutant that lives twice as long as wild type. Nature 366, 461–464. Kenyon, C.J., 2010. The genetics of ageing. Nature 464, 504–512. Kim, S.K., 2007. Common aging pathways in worms, flies, mice and humans. J. Exp. Biol. 210, 1607–1612. Koh, K., Peyrot, S.M., Wood, C.G., Wagmaister, J.A., Maduro, M.F., Eisenmann, D.M., Rothman, J.H., 2002. Cell fates and fusion in the C. elegans vulval primordium are regulated by the EGL-18 and ELT-6 GATA factors – apparent direct targets of the LIN-39 Hox protein. Development 129, 5171–5180. Koh, K., Rothman, J.H., 2001. ELT-5 and ELT-6 are required continuously to regulate epidermal seam cell differentiation and cell fusion in C. elegans. Development 128, 2867–2880.
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Liu, X., Long, F., Peng, H., Aerni, S.J., Jiang, M., Sanchez-Blanco, A., Murray, J.I., Preston, E., Mericle, B., Batzoglou, S., Myers, E.W., Kim, S.K., 2009. Analysis of cell fate from single-cell gene expression profiles in C. elegans. Cell 139, 623–633. McGhee, J.D., Fukushige, T., Krause, M.W., Minnema, S.E., Goszczynski, B., Gaudet, J., Kohara, Y., Bossinger, O., Zhao, Y., Khattra, J., Hirst, M., Jones, S.J., Marra, M.A., Ruzanov, P., Warner, A., Zapf, R., Moerman, D.G., Kalb, J.M., 2009. ELT-2 is the predominant transcription factor controlling differentiation and function of the C. elegans intestine, from embryo to adult. Dev. Biol. 327, 551–565. McGhee, J.D., Sleumer, M.C., Bilenky, M., Wong, K., McKay, S.J., Goszczynski, B., Tian, H., Krich, N.D., Khattra, J., Holt, R.A., Baillie, D.L., Kohara, Y., Marra, M.A., Jones, S.J., Moerman, D.G., Robertson, A.G., 2007. The ELT-2 GATA-factor and the global regulation of transcription in the C. elegans intestine. Dev. Biol. 302, 627–645. Panowski, S.H., Dillin, A., 2009. Signals of youth: endocrine regulation of aging in Caenorhabditis elegans. Trends Endocrinol. Metab. 20, 259–264. Pauli, F., Liu, Y., Kim, Y.A., Chen, P.J., Kim, S.K., 2006. Chromosomal clustering and GATA transcriptional regulation of intestine-expressed genes in C. elegans. Development 133, 287–295 Epub 2005 Dec. 14. Shaner, N.C., Steinbach, P.A., Tsien, R.Y., 2005. A guide to choosing fluorescent proteins. Nat. Methods 2, 905–909.
Contents lists available at SciVerse ScienceDirect
Mechanisms of Ageing and Development journal homepage: www.elsevier.com/locate/mechagedev
Short communication
Re-evaluating the role of ELT-3 in a GATA transcription factor circuit proposed to guide aging in C. elegans Tabitha Tonsaker 1, Ryan M. Pratt 1, James D. McGhee * Department of Biochemistry and Molecular Biology, Alberta Children’s Hospital Research Institute, University of Calgary, Calgary, Alberta T2N 4N1, Canada
A R T I C L E I N F O
A B S T R A C T
Article history: Available online 6 October 2011
Budovskaya et al. (Cell. 134, 291–303, 2008) have proposed that the ELT-3 GATA factor regulates somatic aging genes, including those expressed in the intestine, and participates in a transcription factor circuit that ‘‘guides Caenorhabditis elegans aging’’. We have re-investigated two key features of this proposal: (i) expression of elt-3 in the C. elegans adult intestine where the majority of somatic aging genes are expressed, and; (ii) the ability of elt-3 loss-of-function to revert the extended lifespan of daf-2(e1370) mutants. We find that: (i) in agreement with our previously published results, ELT-3 expression is largely hypodermal and is not expressed at significant levels in the adult C. elegans intestine, and; (ii) the elt3(vp1) zinc-finger deletion mutant does not significantly influence the extended lifespan of daf-2(e1370) mutants. We thus suggest that the role of ELT-3 in C. elegans aging should be re-evaluated. Published by Elsevier Ireland Ltd.
Keywords: C. elegans ELT-3 GATA factor Aging
The small free-living nematode Caenorhabditis elegans is a popular and powerful experimental animal in which to study aging and longevity; (for reviews, see Golden and Melov, 2007; Kim, 2007; Gems and Doonan, 2009; Gruber et al., 2009; Panowski and Dillin, 2009; Kenyon, 2010). A key observation in the field is that C. elegans lifespan and aging are strongly influenced by the insulin/ insulin-like growth factor signalling pathway. In particular, certain mutations in the daf-2 gene (encoding the C. elegans insulin receptor) can extend lifespan by several fold and this lifespan extension is reversed by loss-of-function mutations in the daf-16 gene (encoding a FoxO transcription factor) (Kenyon, 2011). Additional genes involved in C. elegans lifespan and/or aging have been identified, both by classical genetics and by genome-wide RNAi screens (see, for example, Hamilton et al., 2005; Hansen et al., 2005). The role(s) of all these genes is often interpreted in terms of an accumulating damage model of aging; for example, genes that make animals more resistant to stress, both physiological and environmental, might be expected to slow aging and extend lifespan. A different view of C. elegans aging and longevity was provided by Budovskaya et al. (2008), who reported that the GATA-type transcription factor ELT-3 (expressed primarily in the C. elegans hypodermis (Gilleard et al., 1999; Gilleard and McGhee, 2001; Liu et al., 2009)), together with EGL-18 (ELT-5) and ELT-6 (two GATA-type transcription factors that repress elt-3
* Corresponding author. Tel.: +1 403 220 4476. E-mail address: [email protected] (J.D. McGhee). 1 These authors contributed equally to this work. 0047-6374/$ – see front matter . Published by Elsevier Ireland Ltd. doi:10.1016/j.mad.2011.09.006
expression in the hypodermal seam cells (Koh and Rothman, 2001; Koh et al., 2002)), form a ‘‘GATA transcription circuit (that) guides aging in C. elegans’’. The authors interpreted their results in terms of an ‘‘antagonistic pleiotropy’’ model for aging, in which pathways that benefit an organism early in life might well have deleterious consequences post-reproduction. More specifically, the authors suggested that this transcription factor circuit ‘‘is driven by drift of developmental pathways rather than accumulation of damage’’. The two key points of the Budovskaya et al. (2008) argument were: (1) The authors identified 1000 genes either up-regulated or down-regulated in the soma of aging worms and they suggested that these genes are regulated by the ELT-3 GATA factor. The majority of these somatic aging genes (61%) are expressed in the intestine. Indeed, intestinally expressed genes are the only somatic genes enriched in this set of ‘‘aging genes’’; genes expressed in muscle, pharynx and neurons are not enriched, and there was no class listed for aging genes enriched in the hypodermis. (2) The authors reported that loss-of-function of elt-3 was able to reverse the lifespan extension caused by the daf-2(e1370) mutation. This striking observation is reminiscent of daf-16 mutations reversing the extended lifespan of the same daf-2 allele (Kenyon et al., 1993) and was the basis for proposing that ELT-3 down-regulation with age is associated with increased senescence. We wish to re-investigate both of these key points. (1) Does ELT-3 control somatic ‘‘aging genes’’ in the C. elegans adult intestine?
T. Tonsaker et al. / Mechanisms of Ageing and Development 133 (2012) 50–53
In our previous work characterizing the elt-3 gene (Gilleard et al., 1999; Gilleard and McGhee, 2001), we found no evidence that elt-3 is expressed in the C. elegans intestine at any stage of development (cited incorrectly by Budovskaya et al., 2008), as assayed either by ELT-3-specific antibodies or by four different transgenic reporters. elt-3 expression is first and foremost embryonic and hypodermal (except for the lateral seam cells; see also Koh and Rothman, 2001); there is a more enduring but also a more restricted post-embryonic phase of elt-3 expression in a subset of the pharyngeo-intestinal valve cells and intestinal rectal valve cells (but not in the intestine). We verified our previous observations using a new reporter construct (see Fig. 1) that has two additional features: (i) the elt-3 promoter includes the 2.2 kb upstream (up to the nearest upstream gene) of a recently annotated elt-3 alternate transcript (K02B9.4b), and; (ii) a nuclear-localized tdTomato reporter protein was used in order to minimize spectral overlap with intestinal autofluorescence, a common limitation of GFP reporters in C. elegans. Fig. 1 shows examples of young adult hermaphrodites transgenic for this new reporter, dissected in order to extrude the intestine. We focus on the mid-section of the worm (the ‘‘trunk’’) for two reasons:
51
(i) Budovskaya et al. (2008) reported that ‘‘in the trunk, elt-3 expression is mostly derived from the hypodermal and intestinal cells’’, and; (ii) the age-associated decrease of elt-3 expression in the trunk is a central point in the overall model of Budovskaya et al. (2008). In agreement with our previous results (Gilleard et al., 1999; Gilleard and McGhee, 2001), reporter expression driven by the elt-3 promoter is overwhelmingly hypodermal and no significant expression can be detected in intestinal nuclei. It is impossible to be certain that a gene is not expressed in a particular tissue, especially based on evidence from C. elegans multi-copy transgenic reporters; these can be highly sensitive but can also show common sites of non-specific expression such as the posterior intestine (Boulin et al., 2006). Thus a suitably cautious conclusion is that the level of elt-3 expression in the midbody section (‘‘trunk’’) of the C. elegans intestine is less than ‘‘a few percent’’ of its level of expression in the hypodermis. This conclusion is supported by completely independent evidence: (i) ectopic expression of ELT-3 in the embryo is able to drive ectopic expression of hypodermal markers but not ectopic expression of intestinal markers (Gilleard and McGhee, 2001), and (ii) elt-3 transcripts were not detected in our previous SAGE analysis of
Fig. 1. The C. elegans elt-3 gene, the elt-3(vp1) deletion allele and the elt-3 expression pattern in transgenic worms. The two variants of the elt-3 gene (K02B9.4a and the more recently annotated longer transcript K02B9.4b) are shown to scale in the middle of the figure. Unfilled boxes correspond to exons; grey boxes correspond to either 50 or 30 UTRs and the black boxes correspond to the ELT-3 zinc-finger DNA binding domain + adjoining basic region. The 2730 bp vp1 deletion completely removes the ELT-3 DNA binding domain. The elt-3 genotype was determined in single worms as described in Gilleard and McGhee (2001); a typical agarose gel shows the elt-3 genotype in the four C. elegans strains used in this study (described in Table 1). As indicated, the new reporter construct (pJM524) used in this study contains 2.2 kb of promoter region upstream of the most upstream elt-3 transcript, fused to tandem-dimer-Tomato (Shaner et al., 2005) and a histone H2B domain that causes tight nuclear localization. The images at the bottom of the figure represent young (1 day past L4 stage) gravid adults, transgenic for pJM524, anaesthetized in cold 50 mM sodium azide and then dissected in order for the intestine and gonads to extrude. Images are overlays of the tdTomato fluorescent signal and differential interference contrast. Scale bars represent 50 microns. In this study, we examined several dozen independently produced transgenic strains, using either the standard dominant rol-6(su1006)-induced Roller phenotype or unc-119(ed3) rescue to identify transgenic animals. pJM524 expression is intense in the hypodermis but is undetectable in the extruded intestine (dashed outline).
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T. Tonsaker et al. / Mechanisms of Ageing and Development 133 (2012) 50–53
Table 1 Genotypes of the four C. elegans strains used in lifespan assay. Strain
Genotype
N2 JG1 CB1370 JM161
daf-2(+); elt-3(+) daf-2(+); elt-3(vp1) daf-2(e1370); elt-3(+) daf-2(e1370); elt-3(vp1)
dissected adult intestines but could easily be detected in intact animal controls (which include hypodermis) (McGhee et al., 2007); (there have been reports of elt-3 transcripts in the intestine (Pauli et al., 2006) but hypodermal contamination has not been ruled out.) In summary, we conclude that it is unlikely that ELT-3 directly controls transcription of aging-associated genes expressed in the C. elegans intestine. We cannot, of course, rule out indirect models in which ELT-3 influences intestinal gene expression from a different tissue. We have argued elsewhere that the GATA factor ELT-2, not ELT-3, is the predominant transcription factor directly driving intestinal gene expression in all stages later than the early embryo (McGhee et al., 2007, 2009). We also note that Budovskaya et al. (2008) used GFP reporter levels (from an undefined transgenic reporter) as a proxy for the levels of endogenous ELT-3 protein, as it changes with age or in response to, for example, egl-18(RNAi) and elt-6(RNAi). However, elt-3-driven reporter proteins (including the present construct used in Fig. 1) appear to perdure and to remain at high levels much later in development than do elt-3 transcripts (detected by Northerns) or endogenous ELT-3 protein (detected by antibodies) (Gilleard et al., 1999). Transgenic elt-3 reporters may be reliable measures of expression patterns but we suggest that they are unreliable measures of expression levels of endogenous ELT-3 protein. (2) Does elt-3 loss-of-function reverse the extended lifespan of daf2(e1370) animals? We now re-investigate the effect of elt-3 on the daf-2(e1370) lifespan extension using a genetically defined elt-3 loss-of-function mutation, rather than anti-elt-3 feeding RNAi as in the majority of the full lifespan curves reported by Budovskaya et al. (2008). We had previously produced the elt-3 mutation vp1, a 2730 bp deletion that removes the ELT-3 zinc-finger DNA-binding domain (see Fig. 1) and that we presume to be a genetic null (Gilleard and McGhee, 2001). We thus measured the lifespans of the four strains (N2, JG1, CB1370 and JM161), whose genotypes are summarized in Table 1. For each strain, the elt-3 genotype was verified by PCR using primers that clearly distinguish the elt-3(+) from the elt3(vp1) deletion allele (see Fig. 1) and the daf-2 genotype was verified by PCR and sequencing (data not shown). On several occasions, we verified strain identity by genotyping the last surviving worms in a lifespan assay. Fig. 2 shows the results of a typical lifespan experiment. We find no evidence that the elt-3(vp1) mutation is able to reverse the lifespan extension induced by the daf-2(e1370) mutation (p > 0.05, Mantel-Cox log-rank test). We have repeated this same full lifespan experiment three independent times (twice double blind) with essentially identical results. In preliminary experiments (with independently constructed strains), essentially the same results were obtained (three repeats, two double blind, with one repeat conducted with added FUdR in the plates to prevent offspring). Overall, our results clearly contradict the results of Budovskaya et al. (2008). In summary, we find no evidence that the elt-3 gene is expressed at a significant level in the C. elegans intestine. We thus suggest that it is unlikely that ELT-3 directly controls the many intestinally expressed genes associated with C. elegans aging, although indirect effects from other expressing tissues cannot be
Fig. 2. Typical lifespan measurements for the four strains used in this study; strain genotypes are summarized in Table 1. Worms that did not respond to persistent prodding were declared dead and then examined at higher power; worms that had ruptured or ‘‘bagged’’ (contained hatched larvae) were censored from the data. At the end of the lifespan assay, 73, 83, 85 and 80 worms had been scored for N2, JG1, CB1370 and JM161 strains, respectively. Temperature = 20.0 8C. As noted in the text, this basic experiment was repeated an additional two times with essentially identical results; (average number worms scored per strain per experiment = 76).
ruled out. Furthermore, we find no evidence that the elt-3(vp1) loss-of-function mutation is able to reverse the extended lifespan conferred by the C. elegans daf-2(e1370) mutation, as reported by Budovskaya et al. (2008). We have no ready explanation for the differences between the lifespans that we determined and those determined by Budovskaya et al. (2008). The answer could be something simple, such as the particular methods of handling worms, or it could be more complex and interesting, such as an unexpected mutation in the genome of one of the strains. In any event, we suggest that the role of ELT-3 in C. elegans aging should be re-evaluated. Acknowledgements The authors should like to thank Barbara Goszczynski for technical assistance and Dr. John Gilleard (University of Calgary, Faculty of Veterinary Medicine) for helpful discussions. We should also like to thank Dr. Joel Rothman (University of California, Santa Barbara) for critically reading the manuscript. Most of this study was performed as an undergraduate research project in the University of Calgary Bachelor of Health Sciences program, and was supported by operating grants from the Canadian Institutes of Health Research (to JDM). JDM is a Medical Scientist of the Alberta Heritage Foundation for Medical Research and a Canada Research Chair in Developmental Biology. References Boulin, T., Etchberger, J.F., Hobert, O., 2006. Reporter gene fusions. In: The C. elegans Research Community (Eds.), WormBook, doi:10.1895/wormbook.1.106.1, http://www.wormbook.org. Budovskaya, Y.V., Wu, K., Southworth, L.K., Jiang, M., Tedesco, P., Johnson, T.E., Kim, S.K., 2008. An elt-3/elt-5/elt-6 GATA transcription circuit guides aging in C. elegans. Cell 134, 291–303. Gems, D., Doonan, R., 2009. Antioxidant defense and aging in C. elegans: is the oxidative damage theory of aging wrong? Cell Cycle 8, 1681–1687. Gilleard, J.S., McGhee, J.D., 2001. Activation of hypodermal differentiation in the Caenorhabditis elegans embryo by GATA transcription factors ELT-1 and ELT-3. Mol. Cell Biol. 21, 2533–2544. Gilleard, J.S., Shafi, Y., Barry, J.D., McGhee, J.D., 1999. ELT-3: a Caenorhabditis elegans GATA factor expressed in the embryonic epidermis during morphogenesis. Dev. Biol. 208, 265–280. Golden, T.R., Melov, S., 2007. Gene expression changes associated with aging in C. elegans. WormBook 1–12. Gruber, J., Ng, L.F., Poovathingal, S.K., Halliwell, B., 2009. Deceptively simple but simply deceptive – Caenorhabditis elegans lifespan studies: considerations for aging and antioxidant effects. FEBS Lett. 583, 3377–3387.
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