The transcription factor DAF-16 regulates a plethora of phenotypes in C. elegans, including longevity, stress tolerance and pathogen resistance. WormBase (WS212) predicts seven alternative isoforms of DAF-16, but little is known about the differential expression or regulation of these isoforms.

We set out to analyse the expression of the DAF-16 isoforms using quantitative real-time PCR (qRT-PCR). We were able to design isoform-specific primer sets for six of the seven isoforms; the seventh (R13H8.1e.1) is identical to R13H8.1e.2 with the exception of one missing exon and therefore cannot unambiguously be identified. We isolated RNA from mixed stage populations grown at 20°C on OP50, checked for genomic DNA contamination and linearly converted RNA to cDNA using reverse transcriptase. Isoform specific PCR on this cDNA pool detected isoforms R13H8.1a, R13H8.1b, R13H8.1c and R13H8.1d. Additional PCR investigations and cDNA sequencing demonstrated that isoforms R13H8.1e.2 and R13H8.1f are expressed but, under the conditions we tested, both isoforms retain introns currently annotated as being spliced out. R13H8.1e.2 retains the intron in the 5’UTR, whilst R13H8.1f retains the intron between exons one and two. We have submitted a revision of this annotation to WormBase for inclusion in WS215.

Using these splice form-specific primer sets, we determined the expression level of each of the six isoforms relative to the housekeeping gene gpd-3 under resting (20°C) and heat-shock (37°C for three hours immediately prior to RNA extraction) conditions (Table 1). Under both conditions, isoform R13H8.1b is by far the most strongly expressed and is the only isoform to significantly increase its expression upon heat-shock. This isoform was also the only variant to show increased expression in response to heavy metal stress (7mM CuCl₂) or pathogen challenge (Staphylococcus aureus exposure). Thus, the dominant DAF-16 form likely to be present in wildtype C. elegansis R13H8.1b. Determining the role(s) of the alternative isoforms of this key transcription factor will be of great relevance for our understanding of longevity and stress response in C. elegans.

Table 1. Expression of daf-16 isoforms at 20°C and following 3hr heat-shock at 37°C. Expression levels were normalised to gpd-3 and then presented as fold-expression relative to R13H8.1a under resting (20°C) conditions. *Note that isoforms R13H8.1e.2 and R13H8.1f retain introns currently annotated as spliced out – see text for details.

C. elegans EMS mutagenesis screens are an important tool for identifying novel components of biological processes. Here, we report a screen to identify novel gain-of-function alleles promoting EGF pathway activity in vulval development. Simultaneous loss of the lin-15A and lin-15B loci confers a strong herniated multivulva (Muv) defect consistent with over-induction of 1° vulval fates, and lin-15(n765ts) animals are null for one locus and temperature sensitive mutant for the other (Huang et al., 1994). This hyper-induced vulval phenotype is due to de-repressed LIN-3/EGF expression in the hyp7 hypodermis surrounding the vulval precursor cells (VPCs), such that all VPCs receive an inappropriately high EGF signal (Cui et al., 2006). We reasoned that using temperature-sensitive lin-15(n765) mutant animals, we could titrate EGF activity to the level just barely insufficient for ectopic vulval induction. In this background an F₁ screen should allow screening through tens of thousands of haploid genomes to uncover heterozygous activating mutations (gain-of-function alleles) sufficient to induce ectopic vulval formation.

To identify a non-inducing condition, we first assayed lin-15(ts) animals at a number of temperatures. We found that at 15°C, lin-15(ts) animals were not Muv, while Muv animals were observed at higher temperatures. We applied the standard 50 mM EMS mutagenesis protocol to late L4 lin-15(n765ts) animals, plated mutagenized P₀ animals at 15°C, and screened F₁ adult progeny for the Muv phenotype. We found an extremely high rate of positives, with 44/455 (9.7%) Muv F₁ animals. Many putative mutants (19/44, 43.2%) segregated >5 Muv progeny, indicating that picked animals did not represent background noise, but rather were bona fide enhancer mutations that conferred a dominant Muv phenotype in this background at 15°C. Given this exceptionally high positive rate, we are probably identifying genes that are haploinsufficient for lin-3/EGF repression or EGFR pathway inhibition, which is similar to the sensitized Drosophila enhancer screen for haploinsufficiency in sevenless function, the E(sev) screen (Simon et al., 1991). Based on the estimated identifiable loss-of-function mutation rate of 5X10^-4 for this EMS mutagenesis protocol, we estimate the gene target size of our screen to be roughly 84 genes.

Clearly this screen is unsuitable for our purposes. We speculate that we have identified a very large group of genes required for the transcriptional repression of lin-3/EGF, the synMuv genes, or another repressive function in vulval induction. Increased stringency should be achieved by using a lower temperature, thus making the screen less sensitive.

I would like to let our community to know that I am back in Hungary in a new position at Pannonia University in Keszthely, Hungary, where I am resuming my research on C. elegans, C. briggsae and other nematodes. I would like to thank Ann Rougvie, Aric Daul and Theresa Stiernagle from the CGC and Prof. Ralf-Udo Ehlers from the University of Kiel, Germany, for providing strains and protocols. It is great to be back, and I am proud to be teaching real Mendelian-Brennerian Genetics to my students in my Homeland.