Journal of Molecular Biology
Regular articleDiverse Caenorhabditis elegans genes that are upregulated in dauer larvae also show elevated transcript levels in long-lived, aged, or starved adults a
Introduction
Development of Caenorhabditis elegans proceeds through four larval stages (L1–L4), punctuated by molts, to the reproductively mature adult. Larvae at the late L1 stage can respond to adverse environmental stimuli (food restriction and/or crowding) by pursuing an alternative developmental pathway that arrests at a modified third larval stage, the dauer or “enduring” larva. Dauer larvae are specialized for stress-resistance, extended survival, and dispersal (Wadsworth & Riddle, 1989); when favorable conditions return, however, they can resume development to the adult. Dauer larvae cease feeding entirely, are intermittently motile, and have reduced metabolic activity Klass and Hirsh 1976, Riddle 1988, Wadsworth and Riddle 1988, Anderson 1978.
Several physiological and metabolic changes have been demonstrated to be associated with entry into and exit from the dauer stage, including substantial changes in intracellular pH and energy metabolism Wadsworth and Riddle 1988, Wadsworth and Riddle 1989. After the L1 molt, metabolic pathways diverge between predauer (L2d) and non-dauer (L2) worms. Whereas L2 larvae committed to growth switch from the glyoxylate pathway to the citric acid cycle, and show marked increases in isocitrate dehydrogenase activity and in levels of ATP and other high-energy phosphates, this transition does not occur in L2d larvae destined to form dauers (Wadsworth & Riddle, 1989).
Genetic studies of dauer formation have focused primarily on the identification of genes that affect the switch in L1, initially directing development to the dauer pathway, and genes influencing morphogenesis of dauer larvae. Two classes of recessive daf (dauer formation) mutants have been analyzed: dauer-defective (daf-d), which fail to enter the dauer pathway despite induction by pheromone, and dauer-constitutive (daf-c), which commit to dauer-formation at restrictive temperatures even in the absence of induction (Riddle, 1988). A branched dauer-formation pathway has been inferred from epistatic interactions among mutants, and comprises alternating stimulatory and inhibitory effectors Vowels and Thomas 1992, Thomas et al 1993, Gottlieb and Ruvkun 1994, Larsen et al 1995. Certain daf gene products have been shown to participate in signal transduction Georgi et al 1990, Estevez et al 1993, while temporal aspects of dauer formation are regulated by heterochronic genes (Liu & Ambros, 1989).
Dauer arrest, over periods of 0–60 days, does not reduce adult life-span following resumption of development (Klass & Hirsh, 1976). Since starvation of larvae at other stages can also arrest development for more limited periods (Johnson et al., 1984), such experiments have long been used as evidence that aging commences only after completion of development. More recently, however, mutations in the genetic program that initiates dauer formation have been shown to greatly extend the longevity of adult worms without developmental arrest. Several dauer-constitutive mutants (e.g. daf-2, age1 (previously known as daf-23)), which at 25.5 °C form dauer larvae even without pheromone induction, develop at a permissive temperature (15–20 °C) into adults that survive for twice the normal life-span Kenyon et al 1993, Larsen et al 1995, Morris et al 1996. Moreover, certain double mutants have life-spans extended by at least threefold Larsen et al 1995, Lakowski and Hekimi 1996. Thus, the same molecular mechanisms that exempt the dauer larvae from aging appear to govern adult longevity. The age-1 mutation, which confers stress resistance and increased life-span, encodes a PI-3 kinase (Morris et al., 1996). This functions just downstream of daf-2, identified as a nematode homologue of human genes encoding receptors for insulin and insulin-like growth factors (Kimura et al., 1997), and upstream of daf-16, which encodes a Fork head transcription factor Lin et al 1997, Ogg et al 1997. These observations suggest that metabolic attenuation in daf-2 and age-1 mutants (both as dauer larvae and as unarrested adults) results from interruption of a signal transduction pathway: ligand-activated daf-2 receptor acting via the age-1 PI-3 kinase to generate PIP3, which then engages metabolic upregulation through multiple mechanisms including a kinase cascade and transcriptional activation Kimura et al 1997, Lin et al 1997, Ogg et al 1997.
To identify gene-expression changes in dauer larvae, which may underlie both their long-term survival and the extended longevity of daf-mutant adults, we prepared a subtractive library of cDNAs expressed at much higher levels in the dauer stage than in L3. Six genes were found to be markedly upregulated in dauer larvae, in terms of steady-state mRNA levels, and their expression was also monitored during aging and starvation of adults. These dur (dauer-upregulated) cDNA clones comprise genes encoding poly(A)-binding protein, heat-shock proteins hsp70 and hsp90 and three novel genes. The novel genes encode a homologue of human activating signal cointegrator 1 (ASC-1; Kim et al., 1999), a GTP-binding protein homologous with a ribosomal protein, and an SH3-domain protein. Five of the dur genes showed elevated transcript levels during adult aging, and four were also upregulated to some degree following starvation. Two long-lived strains bearing mutations in the dauer formation pathway, daf-2 and daf-2; daf-12, show greater adult-stage expression of all six dur genes than is seen in wild-type or daf-16; daf-2 worms of normal longevity. This suggests that products of the dur genes may play protective roles, contributing to survival of the adult as well as the dauer larvae.
Section snippets
Preparation of a subtracted cDNA library for dauer-upregulated mRNA clones
A dauer cDNA library was enriched for mRNA species that are much more abundant in dauer larvae than in L3 larvae, by two cycles of subtractive hybridization (Sive & St. John, 1988) with excess L3 mRNA. This procedure effectively depletes sequences from the dauer library that are present at comparable or greater transcript levels in L3 larvae. Of 240 clones analyzed on duplicate dot-blot arrays, 25 produced markedly and reproducibly greater hybridization to dauer [32P]cDNA than to L3 probe,
Normalization of transcript levels
The evaluation of mRNA steady-state levels by Northern-blot analysis usually involves normalization of hybridization signals with respect to a control RNA species. Ideally, results would be corrected to reflect mRNA molecules per cell, but this is an elusive goal in practice. Rehybridization of filters to cloned cDNA probes representing “housekeeping” genes such as β-actin is a common procedure (Sambrook et al., 1989) to normalize loads of total RNA or of partially purified poly(A)+ mRNA.
Accession numbers of new sequence data
The sequences referred to herein as dur123, dur135, and dur191 have been assigned GenBank accession numbers U52070, U52071, and U52072, respectively.
Strains utilized
Caenorhabditis elegans wild-type strain (var. Bristol-N2, wild-type), and the dauer-constitutive ts-mutant strain CB1372 daf-7 (e1372) were obtained from the Caenorhabditis Genetics Center (St. Paul, MN). Strain TJ1061 was provided by Thomas E. Johnson (University of Colorado, Boulder, CO). Strains JT5488 daf-2(e1370); daf-12(m20) and JT7098
Acknowledgements
This work was supported by grant R01-AG09413 from the National Institute on Aging. Several worm stocks were supplied by the Caenorhabditis Genetics Center, funded by the NIH National Center for Research Resources. We thank Sergey Sokol (Harvard Medical School, Boston MA) for kindly providing laboratory space for several experiments; Thomas E. Johnson (University of Colorado, Boulder) for providing strain TJ1061; James Thomas for strains JT7098 and JT5488; Gary Ruvkun for strain CB1391; and
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Present addresses: V. Cherkasova, Harvard University School of Medicine, Dept. of Biological Chemistry & Molecular Pharmacology, 240 Longwood Avenue, Boston, MA 02115, USA; N. Egilmez, Department of Molecular Immunology, Roswell Park Cancer Institute, Elm & Carlton Streets, Buffalo NY 14263, USA.