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Heat shock factor functions at the convergence of the stress response and developmental pathways in Caenorhabditis elegans¹

² Correspondence: Veterinary Parasitology, Institute of Comparative Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH. E-mail: e.devaney{at}vet.gla.ac.uk

SPECIFIC AIMS

The heat shock response is an ancient mechanism that evolved to allow adaptation to changing environmental conditions. The aim of this study was to investigate whether heat shock factor (HSF), the key regulator of the stress response in all organisms, functions in pathways other than the stress response. We were particularly interested in determining whether HSF may function in developmental pathways in the nematode Caenorhabditis elegans.

PRINCIPAL FINDINGS

1. Knock down of HSF by RNAi in C. elegans results in a temperature-sensitive developmental arrest
C. elegans wild-type worms (N2) were exposed to hsf(RNAi) using feeding vectors expressing ds RNA constructs that spanned different regions of the gene (Y53C10A.12): the entire predicted open reading frame (2016 bp), a 5' fragment spanning nt 428-1312 (884 bp), and a smaller fragment, nt 1-470, encompassing the 5' end of the gene. Controls were exposed to bacteria expressing empty vector (L4440). Knock down of HSF by RNAi resulted in a developmental arrest at the L2/L3 stage that was more penetrant with increasing size of ds RNA construct and with increasing temperature. We observed a low but significant level of developmental arrest at the L2/L3 stage in worms fed on the two longer constructs grown at 20°C or 25°C. For both constructs, a minimal increase in growth temperature from 25°C to 25.5°C had a much more profound effect on developmental arrest (80% plus of animals arresting) than did an increase in temperature from 20°C to 25°C. Increasing the temperature further to 27°C had no additional effect on the percentage of worms arresting when exposed to the 884 bp and 2016 bp constructs. The arrest phenotype is therefore exquisitely temperature sensitive once a threshold (25°C) is exceeded. The arrested worms were SDS sensitive and were not dauer larvae.

2. hsf(RNAi) results in gro and egl phenotypes
Worms fed on the 884 bp construct that developed to adults were small and scrawny in appearance at both 20°C and 25°C, whereas those fed on the 470 bp construct were less severely affected (see Fig. 1 ). Gonad development was compromised in a significant percentage of worms exposed to the 884 and 2016 bp constructs at 20°C, with the gonad failing to extend laterally. RNAi worms were significantly less fertile than controls (P=0.0263 for the 470 bp construct vs. control worms at 20°C), whereas worms fed on the 884 bp construct were largely infertile (P<0.0001 vs. controls). The reduction in fertility was associated with an increased frequency of egg laying-defective (egl) phenotype or "bagging," in which eggs hatch inside the parent animal.

View larger version (90K): [in this window] [in a new window]   Figure 1. hsf(RNAi) results in a small scrawny phenotype. 72 h-old hermaphrodites of the F1 generation cultured at 20°C and fed on control bacteria (L4440 alone) (A) or bacteria expressing the 470 bp insert (B) or the 884 bp insert (C). Note the small scrawny phenotype of worms treated with the 884 bp construct (C). Scale bar = 100 µm.

3. hsf(RNAi) worms are thermosensitive, display reduced expression of hsp16-2, and have a reduced life span
As HSF is required for the stress response in all organisms, we assessed the thermosensitivity of hsf(RNAi) worms after exposure to heat shock. Animals grown at 20°C or at 25°C were shifted to 35°C and scored for touch-provoked movement and pharyngeal pumping; those failing to display either trait were scored as dead. RNAi with both the 470 bp and the 884 bp constructs resulted in a significant decrease in ability to withstand a thermal stress irrespective of growth temperature (P<0.0001, RNAi treated vs. controls for all conditions). Using a transgenic line expressing GFP under the control of the hsp16-2 promoter (strain CL2070), we examined the effects of hsf(RNAi) in living worms. 44 h-old hermaphrodites grown at 20°C on the appropriate RNAi plates were shifted to 30°C for 1 h to induce GFP expression. Worms were returned to 20°C for 4 h, then GFP expression was examined by UV microscopy. Control worms showed bright fluorescence in the pharynx, gut, and head neurones whereas in hsf(RNAi) worms the fluorescence in the gut was largely ablated, with fluorescence in the pharynx and neurones being less affected. This may reflect tissue-specific differences in the efficacy of RNAi, with expression in neuronal tissue being relatively resistant.

In parallel with the above analysis, we investigated the expression of HSP16 and HSP90 in hsf(RNAi) worms by immunoblotting using either a polyclonal antibody raised against C. elegans HSP16 or a cross-reactive antiserum raised against recombinant HSP90 from the filarial nematode Brugia pahangi. Expression of HSP16 following heat shock was progressively reduced with increasing size of RNAi construct, with exposure to the 2016 bp construct completely ablating HSP16 expression. In contrast, levels of HSP90 were not significantly affected by hsf(RNAi). Consistent with this observation, further analysis showed that HSP90 was expressed at normal growth temperatures and did not vary markedly with heat shock in control N2 worms compared with HSP16, which is strictly stress-inducible.

In C. elegans a direct association between overexpression of HSP16 and longevity has been observed. RNAi with both the 884 bp and 470 bp ds RNAi constructs resulted in a significant reduction in life span at 25°C and 20°C (P<0.0001, 470, and 884 bp constructs vs. controls). As bagging can have a deleterious effect on life span, we also assessed life span in a fer-15/spe-9 mutant background that is 100% infertile. Exposure of these worms to hsf(RNAi) caused a decrease in life span similar to that observed in a wild-type background, indicating that the increased propensity of worms to bag was not directly responsible for the reduced life span. We have also observed a variety of premature ageing phenotypes in hsf(RNAi) worms, but whether these phenotypes relate to reduced levels of HSPs or to other proteins regulated by HSF that are involved in the maintenance of healthy tissue remains to be determined.

4. HSF is required for dauer formation
C. elegans can adopt one of two developmental fates during its life cycle: under optimal conditions, worms develop to reproductive maturity, but when conditions are suboptimal (e.g., lack of food or overcrowding), a stress-resistant long-lived form, the dauer larva, develops. Dauers are formed in response to high concentrations of pheromone (an indicator of population density) and low food, with elevated temperature favoring dauer formation. Mutants that are either dauer-defective (Daf-d, unable to form dauers in response to unfavorable conditions) or dauer-constitutive (Daf-c, form dauers under favorable conditions) have been used extensively to order the genes into three pathways—TGF-ß, cGMP, and insulin signaling—known to lead to dauer formation. To investigate whether HSF functions in the dauer pathway, we exposed a variety of Daf-c and Daf-d mutants in the TGF-ß and insulin signaling pathways to RNAi using the 884 and 470 bp ds RNA fragments. hsf(RNAi) had no effect on the three Daf-d mutants tested. By contrast, in six of seven Daf-c mutant stains tested, RNAi with the 884 bp construct caused a significant reversal in the Daf-c phenotype at 25°C. hsf(RNAi) had a much more pronounced effect on insulin signaling mutants, suggesting a greater sensitivity of this pathway to hsf(RNAi) than the TGF-ß pathway (Table 1 ).

The integrity of sensory amphid neurones is essential for entry into and exit from the dauer pathway and can be assessed by uptake of lipophilic dyes such as DiI. RNAi-treated worms were incubated in 10 ng/mL DiI for 3 h, then examined for uptake of dye. At 20°C, a significant reduction in the percentage of worms staining in both amphidial channels was observed only for worms fed on the 884 bp construct (P=0.0002, 884 bp vs. controls), whereas at 25°C worms fed on both constructs showed a significant decrease in DiI uptake.

CONCLUSIONS AND SIGNIFICANCE

The results of this study demonstrate that HSF is required for normal growth and development in C. elegans in the absence of stress, with hsf(RNAi) affecting development, fertility, and life span under standard conditions of growth. These data are consistent with earlier findings from Drosophila melanogaster demonstrating that flies carrying mutations in hsf arrested at the L1 or L2 instar. Thus, in both C. elegans and Drosophila, HSF may be necessary for the expression of genes required for developmental progression beyond the early larval stages. The L2/L3 arrest observed in C. elegans was exquisitely temperature sensitive, a very small increase in temperature from 25 to 25.5°C having a major effect on the percentage of worms arresting. We conclude that C. elegans responds to subtle variations in temperature with alterations in gene expression that affect developmental decisions.

Our findings highlight the function of HSF in several novel pathways in C. elegans, including dauer formation and life span, and are consistent with a role for HSF in the regulation of multiple genes in addition to HSPs. Many non-HSP genes in C. elegans contain heat shock elements in the upstream region but it remains to be determined whether HSF regulates expression of any of these. The involvement of HSF in dauer formation is intriguing, given that elevated temperature is an important modulator of entry into the dauer pathway. It will be interesting to establish whether temperature sensing is impaired in hsf(RNAi) worms and, if so, to determine whether HSF functions in the thermosensory network, perhaps acting as a "molecular thermometer" directly responding to temperature changes. Worms exposed to hsf (RNAi) had a significantly reduced life span compared with control worms, suggesting that HSF may also regulate expression of genes involved in the maintenance of healthy tissues. As mutations that extend life span in C. elegans frequently affect developmental fate and the ability to resist stress, we propose that HSF provides a link between these pathways. In its natural habitat of the soil, C. elegans is exposed to a constantly changing environment, so that the integration of the stress response with development might be an essential feature of its life history.

View larger version (14K): [in this window] [in a new window]   Figure 2. Genes involved in insulin and TGF-ß signaling (dashed arrows) are known to link the stress response, dauer formation, and aging. Mutations in these signaling pathways that promote dauer development frequently confer stress resistance. Many of these mutations result in an extended life span. HSF (dotted arrows) may be involved in these pathways by monitoring/responding to environmental conditions and integrating inputs from different pathways. By acting as a transcriptional activator or repressor of specific genes, HSF may influence dauer formation, the stress response, and life span.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0164fje; doi: 10.1096/fj.03-0164fje

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This Article Full Text (PDF) All Versions of this Article: 17/13/1960 03-0164fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by WALKER, G. A. Articles by DEVANEY, E. Search for Related Content PubMed PubMed Citation Articles by WALKER, G. A. Articles by DEVANEY, E.