synergistic phenotype was revealed in daf-12(0), as visualized with a nuclear localized SCM::GFP transgene. Furthermore, this synergistic response was also seen when other miRISC genes were down-regulated (Figure 6D). C. elegans larvae hatch with 20 intestinal nuclei, and after 10–14 nuclear divisions in the L1 stage, reach 30–32 nuclei in the adult [53]. We studied postembryonic divisions in intestinal cells as visualized with a nuclear localized elt-2::GFP reporter. As shown in Figure 6E, ain-1 knock-down induced a modest increase in number of intestinal nuclei cells. In contrast, daf-12(0) worms exhibited a synergistic phenotype upon treatment with ain-1 RNAi (Figure 6E) or with RNAi for other miRISC genes (Figure 6F). These results show that daf-12(+) worms can compensate for the loss of miRISC function to ensure normal development. However, daf-12(0) worms are unable to buffer these mutations. Further, previous observations have shown that daf-12(0) worms demon- strate enhanced developmental defects in response to mutations to multiple heterochronic genes that are also DAF-12 targets (Figure S4). Perhaps the combined mis-regulation of heterochronic genes and impaired miRNA activity, due to the control of miRNA [18] and miRISC expression by DAF-12, inhibits the ability of daf- 12(0) worms to buffer genetic mutations during development. These findings point to a role for DAF-12 in ensuring developmental robustness during reproductive development, which we further test below. DAF-12 represses heterochronic and miRISC genes during dauer formation The results presented above show that DAF-12 plays a key role in post-embryonic development and directly regulates the heterochronic circuit, miRISC genes and miRNA expression. We reasoned that these target genes might be repressed in diapause, as DAF-12 binds to the co-repressor DIN-1 to promote dauer formation [11]. To test if DAF-12 target genes are repressed in diapause, synchronized daf-2(e1371) or daf-2(e1371);daf- 12(rh61rh411) worms were grown at 25uC. At this temperature, daf-2 form dauers while daf-2;daf-12 double mutants continue reproductive development. Worms at L3 stage or dauers were collected for mRNA quantification using NanoString analysis. As seen in Figure 7A, both miRISC and heterochronic genes are strongly repressed in dauer larvae relative to L3, suggesting that during reproductive development DAF-12 induces expression of these genes, whereas in dauer formation these genes are instead silenced. To determine if DAF-12 is involved in repression of these genes, we created transgenic worms carrying a 2 Kb promoter region from lin-42A upstream of a GFP reporter. The same promoter region was subjected to mutagenesis to eliminate DAF-12 binding sites, such as DR and M-2 domains (Figure S5) [33,37]. We have chosen to study lin-42A for multiple reasons. DAF-12 regulates lin- 42A expression in every developmental stage (Figure 4C), lin-42 is strongly repressed in diapause (Figure 7A) and down-regulation is necessary for dauer formation [54]. As shown in Figure 7B, in L3 stage both WT and mutant promoters drove GFP expression mainly in the pharynx and the tail, and GFP expression was occasionally observed in the intestine. GFP expression was strongly reduced in dauers when compared with the L3 stage for both reporter constructs (Figure 7B). Consistent with this, endogenous lin-42 was repressed approximately 10006 when compared N2 worms at the L3 or dauer stages (Figure 7D). When endogenous lin-42 and lin-42p::GFP were analyzed by RT-qPCR, GFP and lin- 42 transcripts were similarly repressed when they form dauers (Figure 7B, upper graph). However, when endogenous lin-42 and the lin-42p(mut)::GFP were compared, the GFP transcript retained Identification of DAF-12 Target Genes by ChIP-chip 25-fold more expression than the endogenous lin-42 transcript (Figure 7B, lower graph), suggesting that DAF-12 binding sites are strictly required for a full repression of lin-42 in diapause. This differential GFP expression in dauers was observed at a higher exposure and magnification of the pharynx (Figure 7C). These results show that DAF-12 represses the heterochronic circuit to commit to dauer formation. Interestingly, many heterochronic phenotypes, such as those due to daf-12, dre-1, hbl- 1, lin-4, lin-14, lin-28, lin-42, and lin-58 are suppressed after dauer formation [1,22–25] (Figure S6). Together these results suggest that inhibition of the heterochronic circuit by DAF-12 could be necessary to interrupt development and enter diapause. In addition, this could be necessary to reset the heterochronic circuit to progress through development after worms exit dauer, adding another level of regulation to ensure developmental robustness. DAF-12 acts as a developmental capacitor As daf-12(0) worms are not able to buffer genetic mutations among key developmental genes such as miRISC genes, we hypothesized that this nuclear receptor could have a role as a phenotypic capacitor. Phenotypic capacitors act to minimize the effects of either environmental or internal variations of a stereotyped developmental process. To test whether daf-12 behaves this manner, we exposed WT or daf-12(0) worms carrying the SCM::GFP transgene to environmental fluctuations during development. Developing worms were grown for 72 hours at 20uC (control) or in a variable environment consisting of shifts between 15uC and 25uC every 4 hours. As seen in Figure 8A, while WT worms occasionally show an extra hypodermal cell division, the shifts in temperature during development significantly enhance the heterochronic defects exhibited in daf-12(0) worms. Interestingly, the number of seam cells in both sides is different, suggesting that environmental fluctuation leads to stochastic errors in seam cell division. When compared daf-12(0), stress conditions almost double the number of worms with strong heterochronic pheno- types (Figure 8A and 8B). Together these results suggest that daf- 12(0) worms are sensitized to environmental conditions and confirm that DAF-12 has phenotypic capacitor properties. Discussion Identification of DAF-12 target genes While DAF-12 is necessary for the decision between reproduc- tive development and dauer formation, target genes necessary for these biological functions are still unidentified. In the present work, we identified putative in vivo target genes for DAF-12 in the L2 and adult stages. Several lines of evidence support the specificity and relevance of the binding sites found in our array. First, the highest density of binding sites is found within a 1.5 Kb region upstream of target genes, consistent with DAF-12 often acting by binding to proximal promoter regions. We also found binding sites within genes corresponding to promoter regions of alternative isoforms of the same transcript, such as din-1S and lin-42A. Second, DAF-12 binding sites overlap with RNA Pol II binding sites annotated in an independent ChIP-seq analysis, suggesting that DAF-12 binding sites correspond to regions of active transcription. Third, two independent strains (ALF4 and ALF9) carrying a TAP-tagged daf-12, precipitated either with IgG sepharose or with an anti- DAF-12 antibody, show the same binding regions (Figure 1C and not shown). Fourth, many targets, such as the heterochronic genes dre-1, hbl-1, lin-14, lin-28 and lin-66 have been shown to have genetic interactions with daf-12 [1,11,18,20,24,55]. Moreover, known DAF-12 target genes, such myo-2, ceh-22, lit-1, mir-84 and mir-241 were identified by our ChIP-chip experiments [18,33,37]. PLoS Genetics | www.plosgenetics.org 10 July 2011 | Volume 7 | Issue 7 | e1002179