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. Author manuscript; available in PMC: 2010 Jan 11.
Published in final edited form as: Dev Biol. 2008 Dec 7;327(1):97–105. doi: 10.1016/j.ydbio.2008.11.019

Combined informatic and expression screen identifies the novel DAF-16 target HLH-13

Nicole Liachko 1, Rachel Davidowitz 1, Siu Sylvia Lee 1,*
PMCID: PMC2804473  NIHMSID: NIHMS168285  PMID: 19103192

Abstract

Insulin/IGF-signaling (IIS) affects longevity, stress resistance and metabolism in worms, flies, and mammals. The Forkhead transcription factor DAF-16/FOXO is the major downstream effector of IIS and is responsible for the activation and repression of genes that mediate the diverse effects of IIS. We surveyed a set of informatically predicted conserved DAF-16/FOXO target genes and identified the novel DAF-16 direct target hlh-13. hlh-13 is the predicted homolog of the mammalian transcription factor Ptf1a, a critical determinant of pancreatic development. We found that an hlh-13 mutant exits L1 arrest and IIS-dependent dauer diapause faster than control worms, but is not involved in lifespan or resistance to a variety of stresses. Our results have identified a novel DAF-16 target gene and linked its function to known outputs of IIS. Considering the high conservation of IIS in diverse species, our results also hint at an intriguing connection of IIS and Ptf1a in mammals.

Keywords: DAF-2, DAF-16, FOXO, HLH-13, Ptf1a, Dauer, Development, Caenorhabditis elegans, Metabolism, Insulin signaling

Introduction

Insulin and insulin-like growth factor (IGF) signaling (IIS) regulates animal physiology via complex molecular networks. The transcription factor DAF-16 was first identified as the major mediator of IIS in C. elegans (Kimura et al., 1997; Lin et al., 1997; Ogg et al., 1997). The mammalian DAF-16 homologs (FOXO 1, 3, 4, 6) were also subsequently shown to be critical effectors of insulin and IGF signaling in mammals (Arden, 2008; Calnan and Brunet, 2008; Gross et al., 2008). DAF-16 and FOXOs are highly conserved in structure, regulation and function. In response to reduced IIS, DAF-16/FOXO proteins translocate from the cytoplasm into the nucleus (Calnan and Brunet, 2008; Henderson and Johnson, 2001; Lee et al., 2001; Lin et al., 2001) where they regulate the expression of a large number of genes and participate in diverse functions, including cell cycle control, apoptosis, stress response, metabolism and development (Honda and Honda, 1999; Lee et al., 2003; Murphy, 2006; Murphy et al., 2003).

DAF-16/FOXO likely achieves its diverse biological roles by regulating the expression of different subsets of target genes in response to distinct stimuli. In vitro studies have shown that DAF-16 and mammalian FOXO proteins bind an identical 8-base pair DNA consensus sequence (DBE), TTGTTTAC (Furuyama et al., 2000). In vivo, DAF-16 and FOXO3 have been shown to bind to the DBE at the promoters of homologous genes in worms and mammalian cells, respectively, and regulate their expression (Kops et al., 2002; Oh et al., 2006). Recently, the DAF-16 associated element (DAE) CTTATCA has been found at the promoters of a subset of DAF-16 downstream genes (Murphy et al., 2003). The DAE fits well with the consensus sequence motif recognized by GATA transcription factors (Budovskaya et al., 2008), suggesting that a GATA transcription factor may work with DAF-16 to regulate a subset of DAF-16 target genes.

Comprehensive identification and characterization of DAF-16 target genes is critical for a better understanding of how DAF-16/FOXO mediates its diverse biological functions. To further elucidate the transcriptional targets of DAF-16/FOXO, we previously took advantage of the high conservation of the DBE and utilized informatics to predict conserved DAF-16 targets (Lee et al., 2003). The informatic search aimed to identify worm and fly orthologous genes whose promoters contained the DBE in both species. A small subset of the predicted genes were analyzed and several were shown to display expression changes in response to altered IIS and to participate in IIS related functions in worms (Lee et al., 2003), indicating that the informatic strategy was able to predict authentic, novel DAF-16 target genes.

In this study, we employed quantitative reverse transcription PCR (qRT-PCR) to screen the entire predicted set of DAF-16 target genes for regulation by IIS and DAF-16 in C. elegans. We identified 3 genes whose expression was robustly regulated by DAF-16 in response to a short-term reduction in IIS. Using chromatin immunoprecipitation (ChIP), we demonstrated an enrichment of DAF-16 at the promoters of these genes, indicating the genes are likely directly regulated by DAF-16. From these new DAF-16 targets, we further explored possible IIS-related functions of C. elegans HLH-13, a helix-loop-helix transcription factor homologous to the mammalian pancreatic transcription factor 1a (Ptf1a) (Ledent et al., 2002; Ledent and Vervoort, 2001). The results demonstrated roles of HLH-13 in recovery from L1 arrest and IIS-dependent dauer diapause, and moderate resistance to starvation stress, but not lifespan, heat, or oxidative stress. The conservation of the hlh-13 DBE suggests the intriguing possibility that IIS also regulates the critical pancreatic determinant Ptf1a in mammals.

Materials and methods

Worm strains and transgenics

Unless otherwise indicated, worms were cultured using standard procedures (Brenner, 1974) at 16°C. The following strains were obtained from the CGC: wild type (N2), RX39 daf-2(e1370), RX5 daf-16(mgDf47), GR1309 daf-16(mgDf47);daf-2(e1370), DR1572 daf-2(e1368),, TJ1052 age-1(hx546). FX2279 hlh-13(tm2279) was obtained from the National BioResource Project (Japan) and backcrossed 6 times to N2 to generate IU129. IU24 (daf-16(mgDf47);daf-2(e1370);xrIs87[daf-16∷GFP];) was constructed by crossing GR1309 with GR1352 xrIs87. IU137 daf-2(e1370);hlh-13(tm2279) was constructed by crossing IU129 with RX39. IU197 daf-2(e1368);hlh-13(tm2279) was constructed by crossing IU129 with DR1572. IU287 age-1(hx546);hlh-13(tm2279) was constructed by crossing IU129 with TJ1052. IU189 rwIs1[Phlh-13∷GFP∷hlh-13+Pmec-7∷RFP] was generated using PCR fusion (Hobert, 2002). Final construct was injected into early gravid adult worms at a concentration of 10ng/ul, along with 50ng/ul Pmec-7∷RFP co-injection marker and 50ng/ul Bluescript plasmid filler DNA. Integrated lines were made by irradiating L4 worms with 1500 rads gamma radiation and inspection of progeny for 100% transgene transmission. IU285 daf-2(e1370);rwIs1 and IU286 daf-16(mgDf47);daf-2(e1370);rwIs1 were made by crossing IU189 with GR1309 and selecting desired genotypes. IU285 was subsequently outcrossed 5 times to daf-2(e1370) and IU286 was outcrossed 4 times to daf-16(mgDf47);daf-2(e1370). IU219.1–3 daf-2(e1370);hlh-13(tm2279);rwEx5[hlh-13+Pmec-7∷RFP] was generated by injecting PCR amplified genomic hlh-13 and Pmec-7∷RFP co-injection marker as above into IU137 and selecting for RFP+ transmitting lines.

Quantitative RT-PCR

Synchronized populations of worms were grown to the indicated stages of development, harvested by washing with M9, and flash-frozen in liquid N2. RNA was made using TRI Reagent RNA isolation reagent (Molecular Research Center, Inc.) followed by DNAse treatment (Invitrogen), phenol-chloroform extraction, and precipitation. cDNA was made using Superscript III first strand synthesis system for RT-PCR (Invitrogen). qPCR performed using iQ SYBR Green Supermix (BioRad) on a MyiQ Real-Time PCR detection system (BioRad).

Chromatin immunoprecipitation

Synchronized worms were grown at 16°C to L4, and shifted to 25°C for 6 hours immediately before harvesting. Worms were washed with M9 and nuclei isolated as described (Mains and McGhee, 1999) with the following modification. Whole worms were initially broken in a cold Dounce homogenizer using the tight (A) pestle. Isolated nuclei were incubated with 1% formaldehyde for 20 minutes at room temperature, and then quenched with 125mM glycine for 3 minutes. Nuclei were pelleted, resuspended in sonication buffer (0.5% SDS, 20mM Tris pH8, 0.5mM EDTA, 0.5M EGTA, 100mM PMSF, 1x protease inhibitor) at a concentration of approximately 5×10^4 nuclei per microliter, and incubated on ice for 10 minutes. Material was sonicated on a Branson Digital Sonifier for 5- 20-second bursts with 1-minute rest between bursts and then centrifuged for 15 minutes at 15,000g. The supernatant was flash frozen in liquid N2 and stored at −80°C prior to immunoprecipitation (IP). 50μl of supernatant was diluted 1:3 in IP buffer, and pre-cleared with protein-A agarose for 1 hour at 4°C on rotating wheel. Sample was then incubated with anti-GFP antibody (Clontech) overnight at 4°C. Protein-A beads were added to sample and incubated for 2 hours. Beads were washed 3x with low salt wash (0.1% SDS, 1% TritonX-100, 2mM EDTA, 20mM Tris pH7.8, and 150mM NaCl), 3x with high salt wash (as above, with 500mM NaCl), 1x with LiCl wash (1mM EDTA, 10mM Tris pH7.8, 250mM LiCl, 1% NP40, and 1% Sodium Deoxycholate), and 2x with TE. Protein/DNA was eluted from beads with 250μl elution solution (1% SDS and 0.1M NaHCO3) rotating at room temperature for 15 minutes, 2x. Crosslinks were reversed with addition of 5M NaCl and proteinase K to eluted fractions and incubation at 65°C for 4–12 hours. Tris pH8 and glycogen (carrier) were added to each sample, followed by 2 phenol/chloroform extractions, and precipitation of DNA with ethanol and sodium acetate.

Brood size

Worms were separated as L4 onto individual plates at 25°C, and transferred every 24 hours to fresh plates. The numbers of viable and dead embryos were scored under a dissecting microscope (Leica).

Lifespan assay

Worms were grown from a short (4–6 hour) egglay to L4 stage on NGM plates, and then transferred onto NGM plates with added 5-fluorodeoxyuridine (FUDR, 0.05mg/mL) to inhibit growth of progeny. Worms were scored every 1–2 days by gentle touching with a platinum wire. Failure to respond to touch was scored as dead. Statistical analysis was performed using SPSS software.

Heat stress assay

Worms were grown from a short (4–6 hour) egglay to L4 stage on NGM plates at 16°C. FUDR was added to the plates (0.05mg/mL), and worms were transferred to 25°C to inactivate daf-2(e1370) ts allele. At day 3 of adulthood, worms were transferred to 35°C and scored after 8–24 hours by gentle touching with a platinum wire. Failure to respond to touch was scored as dead.

Paraquat assay

The paraquat assay was performed as in (Li et al., 2008) with the following modifications. 150mM paraquat was used, and the worms were incubated in 600uL M9/paraquat for the duration of the experiment.

Starvation assay

Embryos were obtained by bleaching, and allowed to hatch overnight in M9 buffer. The concentration of L1 worms was adjusted to 1 worm/μl in a total volume of 20mL M9, in a 250mL glass flask covered with parafilm. Flasks were maintained in a rotating water bath at 25°C. Every 2–3 days 100μl aliquots were plated onto 3- 60mm NGM plates seeded with OP-50 and allowed to grow. Worms were scored 4–5 days later for survival and development.

Dauer assay, 22.5°C

4–6 L4 worms were picked onto seeded NGM plates and incubated at the experimental temperature 22.5°C overnight. After the worms developed to gravid adults, they were allowed to lay eggs for 4–6 hours, with approximately 50 progeny eggs laid per plate. Parents were picked off and progeny scored for number of worms in dauer versus developing after 4 days at 22.5°C. For hlh-13(+) transgene rescue assay, gravid adult worms were used for the egglay to facilitate picking RFP(+) parents.

Dauer assay 25°C

4–6 gravid adult worms were allowed to egglay onto seeded NGM plates at 25°C for 4–6 hours. Parents were removed, and progeny arrested at dauer stage. After 5 days at 25°C, worms were moved to the permissive temperature, 16°C, and allowed to resume development to adult. Worms were scored after 48 hours at 16°C for number of worms arrested versus developing.

Results

qRT-PCR screen for DAF-16-regulated genes

An informatics survey of C. elegans and D. melanogaster genomes for conserved targets of DAF-16 harboring the DBE identified 116 candidate genes (Lee et al., 2003). To validate the informatic predictions, we surveyed the candidate genes for DAF-16 responsiveness using qRT-PCR. The sensitivity of qRT-PCR allows us to detect expression changes in even low-abundance mRNAs that may not be identified by methods such as microarray or Northern blot. To examine the predicted target genes for IIS-responsive, DAF-16-dependent mRNA changes, we compared mRNA from daf-2(e1370) and daf-16(mgDf47);daf-2(e1370) synchronized L4 worms. daf-2 encodes the insulin/IGF-like receptor in C. elegans (Kimura et al., 1997) and daf-2(e1370) is a temperature sensitive mutant, whereas daf-16(mgDf47) is a null mutant. At the restrictive temperature (25°C), DAF-2 function in daf-2(e1370) worms is reduced, which triggers DAF-16 nuclear translocation and transcriptional activity. When daf-2(e1370) worms are cultured at the permissive temperature, DAF-16 localization, as visualized by a GFP-fused DAF-16 (DAF-16∷GFP), is predominantly cytoplasmic; however, within a 6-hour temperature shift to the restrictive temperature, DAF-16∷GFP becomes strongly localized to the nucleus (data not shown). In addition, within this 6-hour induction, we were able to see robust upregulation of sod-3 (Fig. 1A), a known direct target of regulation by DAF-16 (Oh et al., 2006), indicating that a 6-hour inactivation of daf-2(e1370) is sufficient to detect regulation of DAF-16 target genes.

Fig. 1.

Fig. 1

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A screen of predicted DAF-16 regulated genes identified 3 likely direct targets of DAF-16. (A) qRT-PCR of 116 predicted DAF-16 target genes revealed 3 genes with a fold expression change greater than 2, comparing daf-2(e1370) vs. daf-16(mgDf47);daf-2(e1370) following a 6-hour shift to the restrictive temperature. sod-3 is a known DAF-16 target and is included as a positive control; rpb-2 is a gene not regulated by DAF-16 and is used as a negative control. The expression level of each gene in the two strains was normalized to that of actin. Results of one representative experiment are shown. All genes were tested with at least 3 independent preparations of material and similar results were obtained. (B) ChIP was performed using the daf-16(mgDf47);daf-2(e1370);daf-16∷GFP strain following a 6-hour shift to the restrictive temperature. Results from 3 independent ChIP experiments were pooled and the average ChIP signal relative to input is shown. A statistically significant (*=p<0.05, Student's t-test) enrichment of DAF-16∷GFP at the promoters of sod-3, hlh-13, and K07A1.7 was detected. sod-3 is a gene previously shown to be directly regulated by DAF-16 (Oh et al., 2006). The negative control primers target a region of DNA with no DAF-16 binding sites. Anti-RFP was used as an irrelevant antibody control.

By precisely controlling the temperature of the growing worms, we are able to control the timing of the DAF-16 activation. A previous analysis of the informatics-predicted DAF-16 targets looked at DAF-16-regulated expression changes following a 24-hour induction of DAF-16 at the daf-2(e1370) restrictive temperature 25°C (Lee et al., 2003). In the current study, we chose to survey gene expression following a short, 6-hour incubation at the restrictive temperature to favor identification of early responding genes. We reason that the early responding genes will likely be enriched for direct targets of regulation by DAF-16. To select positives from the qRT-PCR screen, we required genes to show at least a 2-fold expression change in response to DAF-16, with a consistent change in 3 or more independent preparations of RNA. We surveyed a total of 116 predicted target genes using qRT-PCR, and identified three that showed greater than two-fold response to DAF-16 (Fig. 1A). Two of these genes, F02H6.5 and K07A1.7, were also identified in a previous microarray screen for DAF-16-regulated genes (Murphy et al., 2003). The third gene identified, hlh-13, is normally expressed in low levels and was not detected by previous microarray studies (McElwee et al., 2003; McElwee et al., 2004; Murphy et al., 2003), which demonstrates the importance of using multiple strategies to identify the full complement of DAF-16 targets. Interestingly, the DAE sequence (Murphy et al., 2003) is also present in the promoters of both K07A1.7 and hlh-13 (Table 1), perhaps pointing to co-regulation of these genes by DAF-16 and a GATA transcription factor (Budovskaya et al., 2008).

Table 1.

DAF-16-regulated genes identified by qRT-PCR

genea predicted homologyb microarrayc DBEd DAEe
hlh-13 homolog of mammalian Ptf1a ND −764 −1788
F02H6.5 similar to S. pombe HMT2 class 1 −94 NP
K07A1.7 similar to D. melanogaster Headcase class 1 −1319 −636, −6676
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a

3 genes are regulated by DAF-16 from qRT-PCR screen.

b

Predicted functions were assigned by protein-protein BLAST (NCBI).

c

Class 1 gene are upregulated in response to DAF-16 (Murphy et al., 2003).

ND = not detected.

d

DAF-16 binding element (DBE) was identified by searching intergenic DNA sequence from the start site of the gene of interest to the stop codon of the closest upstream gene.

e

DAF-16 associated element (DAE) was identified similarly to DBE.

NP = not present.

We tested whether expression differences were evident in daf-2(e1370) versus daf-16(mgDf47);daf-2(e1370) worms without an incubation at the DAF-2 restrictive temperature. We found that F02H6.5 and K07A1.7 were not expressed differently between the two strains, and hlh-13 was expressed at such a low level that it was undetectable by qRT-PCR (Table 2). This indicates that inactivation of daf-2(e1370) by incubation at the restrictive temperature is necessary for the expression changes detected. Consistent with this, we also did not detect any expression changes of the three genes in wild-type versus daf-16(mgDf47) worms (data not shown), indicating that the induction of these new DAF-16 target genes requires activation of DAF-16 mediated by decreased IIS. We were also interested in whether a longer incubation (12 hours versus 6 hours) at the restrictive temperature would further increase the expression of these genes. We tested their expression following a 12-hour shift to the restrictive temperature, and found that the longer incubation did not further increase the expression of the 3 target genes (Table 2). This indicates that a maximum induction of these genes is likely reached relatively early following DAF-16 activation.

Table 2.

Fold expression changes in response to decreased IIS

genea no TSb 6hrb 12hrb L1c dauer/L2d adultc
hlh-13 ND 33.4 38.6 5.5 10.4 37.6
F02H6.5 1.8 9.2 10.9 3.3 6.5 4.1
K07A1.7 0.9 5.3 4.4 1.8 4.0 11.4
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a

3 genes are regulated by DAF-16 from qRT-PCR screen.

b

Fold mRNA changes comparing daf-2(e1370) vs daf-16(mgDf47);daf-2(e1370) were measured from L4 worms with no, 6-hour, or 12-hour shift to the restrictive temperature.

ND = not detected.

c

Fold mRNA changes in L1 and adult worms following a 12-hour temperature shift.

d

Fold mRNA changes in dauer daf-2(e1370) compared to L2 daf-16(mgDf47);daf-2(e1370) grown from eggprep at the restrictive temperature for 24 hours.

To test whether the developmental stage of the worms affects the DAF-16-dependent expression of these genes, we monitored gene expression in the two strains at the L1, L2/dauer, and adult stages in addition to the L4 stage initially tested (Table 2). We did not test L3 due to the difficulty of accurately timing a temperature shift of the dauer-prone daf-2(e1370) allele at 25°C to obtain a population of L3 daf-2(e1370). We found that F02H6.5 and hlh-13 were upregulated in response to DAF-16 in L1, but K07A1.7 was not. However, all three genes responded to the induction of DAF-16 in dauer, L4, and adult. This indicates that the DAF-16-dependent regulation of these 3 genes functions during development and adulthood.

Direct gene regulation by DAF-16

hlh-13, K07A1.7, and F02H6.5 are regulated by DAF-16 and each contains a canonical DBE within 2kb upstream of their start site (Table 1). By using ChIP, we can assay for the presence of DAF-16 at the promoters of these three genes (Fig. 1B). Because we do not have an anti-DAF-16 antibody that can robustly recognize endogenous DAF-16, we performed ChIP using IU24 (daf-16(mgDf47);daf-2(e1370);daf-16∷GFP) and anti-GFP to immunoprecipitate GFP-fused DAF-16. The DAF-16∷GFP in this strain has previously been demonstrated to be functional (Lee et al., 2001). We paralleled the ChIP conditions to that of qRT-PCR, in which IU24 was shifted to the DAF-2 restrictive temperature, 25°C, for 6 hours. Our positive control for ChIP is the promoter of sod-3, since it has previously been identified as a direct target of DAF-16 using this technique (Oh et al., 2006). Consistent with previous study, the sod-3 promoter was enriched following an IP against DAF-16∷GFP but not in an IP against an irrelevant protein (RFP), indicating DAF-16 is present at its promoter (Fig. 1B). Interestingly, we found the promoter regions of hlh-13 and K07A1.7 were significantly enriched following the DAF-16∷GFP IP compared to the control IP, indicating they are likely direct targets of DAF-16. The promoter of F02H6.5 also showed enrichment at the DAF-16 promoter, but the enrichment was not statistically significant (p>0.05). These results are interesting because only a handful of DAF-16 regulated genes have been shown to be direct targets of regulation (Murphy, 2006; Oh et al., 2006), and these genes may provide further insights into DAF-16 functions.

GFP∷hlh-13 expression and regulation

For further study, we focused on the new DAF-16 target hlh-13. hlh-13 is the predicted homolog of the mammalian pancreatic transcription factor Ptf1a (Ledent et al., 2002; Ledent and Vervoort, 2001), and has a conserved helix-loop-helix domain. Little is known about the biological roles of hlh-13 in C. elegans, and we sought to explore the regulation and function of this novel DAF-16 target gene in connection with insulin signaling.

To monitor the expression and localization of hlh-13, we created transgenic worm strains that express an integrated transgene of full-length hlh-13 tagged with GFP. This full-length hlh-13 transgene contains the endogenous hlh-13 promoter, the genomic sequence of the hlh-13 coding region (including introns) and the flanking 3'UTR of hlh-13 (Fig. 2A). This transgene can functionally replace hlh-13(tm2279) as tested in a dauer recovery assay (Supplemental Fig. 1). We observed the GFP∷hlh-13 reporter in developing and adult worms and found that the full-length GFP∷hlh-13 transgene was expressed in all the hermaphrodite dopaminergic neurons (ADE, CEP, and PDE) throughout development and adulthood, although the expression declineed with age, and in several unidentified neurons in the ventral nerve cord during L2 and L3 developmental stages (Fig. 2, Supplemental Fig. 2, and data not shown).

Fig. 2.

Fig. 2

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Expression of GFP∷hlh-13 is regulated in response to DAF-2/DAF-16 in worms. (A) A schematic representation of the N-terminal GFP-fused hlh-13 transgene is shown, including the endogenous promoter, introns, and 3'UTR. (B–H) GFP∷hlh-13 expression is indicated by white arrows or brackets. Fluorescence not indicated by arrows is autofluorescence in the gut. (B, D, F) GFP∷hlh-13 is upregulated in the hypodermis in the head and tail of L1 (B) or dauer (D, F) daf-2(e1370) worms in response to a 24-hour incubation at 25°C (white arrow). (C, E, G) daf-16(mgDf47);daf-2(e1370) worms do not show increased expression of GFP∷hlh-13 in the hypodermis at the restrictive temperature in L1 (C) or L2 (E, G). White arrows indicate comparable head or tail regions and brackets indicate dopaminergic head neurons (B–G). Similar expression patterns were observed at other developmental stages including adult (Supplemental Fig. 2). (H) GFP∷hlh-13 is expressed in the 4 CEP and 2 ADE dopaminergic neurons in the head (square bracket) and in the 2 PDE dopaminergic neurons (tailed arrow). GFP∷hlh-13 is also expressed in several unidentified ventral nerve cells in L2 and L3 worms (arrow heads). Shown is an L2 worm. Images were captured using the Leica Confocal Software on a Leica TCS SP2 confocal microscope.

To confirm the transcriptional regulation of hlh-13 by DAF-16 in vivo, we compared GFP∷hlh-13 expression in daf-2(e1370) vs. daf-16(mgDf47);daf-2(e1370) worms at the restrictive temperature. Consistent with our qRT-PCR results, the expression of GFP∷hlh-13 was regulated in response to DAF-16 in L1, L2/dauer, L4, and adult worms (Fig. 2B–G, Supplemental Fig. 2A–D, and data not shown). L3 stage worms were not tested due to the dauer-constitutive phenotype of daf-2(e1370) at 25°C. Specifically, the GFP expression in the hypodermis at the head and the tail showed elevated expression in the daf-2(e1370) mutant compared to that in the daf-16(mgDf47);daf-2(e1370) double mutant at 25°C. Also consistent with qRT-PCR, no difference in expression of the GFP∷hlh-13 transgene was observed in wild-type compared to the daf-16(mgDf47) strain (data not shown). In contrast to the hypodermal expression, the neuronal expression of hlh-13 was not responsive to DAF-16 under conditions tested (Fig. 2B–E, Supplemental Fig. 2A and B, and data not shown). These data together indicate that the neuronal expression of hlh-13 is constitutive and is not under transcriptional control of IIS/DAF-16, whereas the hypodermal expression is regulated by IIS/DAF-16. This variation in transcriptional control may indicate multiple functions for hlh-13, both dependent and independent of DAF-16.

hlh-13 mutant exhibits insulin signaling-related phenotypes

Since hlh-13 represents a putative new DAF-16 transcriptional target, we tested whether loss of hlh-13 may result in phenotypes commonly associated with IIS mutants, such as alterations in lifespan, stress resistance and dauer formation. For these assays, we used the mutant allele hlh-13(tm2279) with an in-frame deletion of the entire second exon and part of the first and second introns of hlh-13. This deletion eliminates most of the conserved helix-loop-helix domain (Supplemental Fig. 3A). hlh-13(tm2279) appears superficially normal, with a normal brood size and development rate (Supplemental Fig. 3B and C). We first tested the lifespan of the single mutant hlh-13(tm2279) and the DAF-16-active double mutant daf-2(e1370);hlh-13(tm2279) at three temperatures (16°C, 20°C, 25°C). Neither hlh-13(tm2279) nor daf-2(e1370);hlh-13(tm2279) affected the lifespan of the worms compared to wild-type or daf-2(e1370), respectively, under our test conditions (Supplemental Fig. 4, and data not shown), suggesting that hlh-13 is likely not involved in longevity modulation.

Next, we tested whether hlh-13 functions in other known DAF-16-regulated activities, such as stress response. To assay this, we challenged the hlh-13(tm2279) worms with either heat, oxidative, or starvation stress. For heat stress response, we monitored the survival of hlh-13(tm2279) and daf-2(e1370);hlh-13(tm2279) worms at the heat shock temperature of 37°C. We found that both the hlh-13(tm2279) and daf-2(e1370);hlh-13(tm2279)) worms responded identically to controls over an extended time course tested (Supplemental Fig. 5). Therefore hlh-13 does not appear to affect heat stress survival under our testing conditions.

To assay oxidative stress, we used the free radical generating agent paraquat (Hassan and Fridovich, 1978). hlh-13(tm2279) and daf-2(e1370);hlh-13(tm2279) worms were monitored for survival in the presence of 150mM paraquat, and were found to respond to the acute oxidative stress identically to controls (Supplemental Fig. 6). Thus, under the conditions tested, hlh-13 does not affect resistance to oxidative stress.

We then tested response to starvation stress by hatching worms in M9 without food, allowing them to arrest at L1. To permit normal oxygen flow and prevent hypoxia, the L1 worms were kept in flasks at a density of ~1 worm/μl, sealed with parafilm and gently rotated in a temperature-controlled shaker. The survivors were scored every 1–2 days by plating an aliquot of the starved L1s from each flask onto NGM plates with food. The worms were given time to recover from starvation and develop before scoring both the number of worms alive as well as the number of worms able to develop beyond L1. The hlh-13(tm2279) mutant worms had similar survival kinetics compared to wild-type worms, while daf-2(e1370);hlh-13(tm2279) had slightly extended survival kinetics compared to daf-2(e1370) (Fig. 3A and B). However, we observed that the hlh-13(tm2279) and daf-2(e1370);hlh-13(tm2279) mutants were able to recover from the starvation/L1 arrest and develop beyond L1 better than wild-type or daf-2(e1370), respectively (Fig. 3C and D). We obtained similar observation when hlh-13(tm2279) was combined with another allele of daf-2, daf-2(e1368) (Supplemental Fig. 7).

Fig. 3.

Fig. 3

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hlh-13(tm2279) and daf-2(e1370);hlh-13(tm2279) have enhanced recovery from starvation-induced L1 arrest. (A) hlh-13(tm2279) does not affect starvation survival compared to wild-type (N2). daf-16(mgDf47) worms are known to be hypersensitive to starvation stress and are included as a control. (B) daf-2(e1370);hlh-13(tm2279) show a slight increase in starvation survival compared to daf-2(e1370). (C and D) hlh-13(tm2279) and daf-2(e1370);hlh-13(tm2279) have enhanced recovery from L1 arrest. Results show worms sampled from day 8 and day 12 after starvation, respectively, and the y-axis shows % of worms able to develop beyond L1 after refeeding. Similar differences in recovery between strains were observed for worms sampled from day 6 (hlh-13(tm2279) and day 8 (daf-2(e1370);hlh-13(tm2279) till the end of the assay. Experiments are representative of at least 3 independent trials. *=p<0.05 compared to control worms, Student's t-test.

This interesting result led us to consider whether hlh-13(tm2279) also plays a role in another worm developmental arrest, dauer diapause. We did not observe any dauer-constitutive phenotype with the hlh-13(tm2279) single mutant at 25°C and 27°C (data not shown). We then tested whether hlh-13 may play a role in IIS-dependent dauer formation, by taking advantage of the temperature sensitive dauer-constitutive phenotype of daf-2(e1370). At the restrictive temperature 25°C, daf-2(e1370) worms will arrest as dauer indefinitely, while at the lower temperature 22.5°C daf-2(e1370) will arrest as dauer or L2d for ~3 days and then resume development to fertile adults (Gems et al., 1998; Li et al., 2007). At this sensitive temperature, we can observe mutations that modulate the daf-2(e1370)/IIS-dependent dauer response that may not be noticeable during a stronger dauer arrest at the restrictive temperature of 25°C. We tested the effect of the hlh-13(tm2279) allele in the daf-2(e1370) background, and found that the daf-2(e1370);hlh-13(tm2279) worms entered into dauer/L2d arrest similarly to that of the daf-2(e1370) single mutant (data not shown), but consistently recovered from dauer/L2d and developed into adults earlier at the sensitive temperature of 22.5°C (Fig. 4A). To confirm that the dauer recovery phenotype is specific to the hlh-13(tm2279) allele, we injected a genomic fragment encompassing wild-type hlh-13 into the daf-2(e1370);hlh-13(tm2279) worms, and assayed for rescue of the increased recovery. We studied three independent lines containing extra-chromosomal hlh-13(+) DNA and found that all three rescued the enhanced dauer recovery phenotype of daf-2(e1370);hlh-13(tm2279) (Fig. 4B). Therefore, the phenotype observed is likely due to the mutation in the hlh-13 gene.

Fig. 4.

Fig. 4

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daf-2(e1370);hlh-13(tm2279) recover from dauer faster than daf-2(e1370). (A) daf-2(e1370);hlh-13(tm2279) worms recover from dauer faster at 22.5°C than daf-2(e1370). Worms were scored 4 days after ~20 hour egglay by L4 worms. 52% daf-2(e1370) were developing at the time scored, compared with 88% daf-2(e1370);hlh-13(tm2279) developing at this time. Results shown are combined results from 5 independent trials. *=p<0.05, Student's t-test. (B) Exogenous expression wild-type hlh-13 rescues the dauer phenotype of daf-2(e1370);hlh-13(tm2279). 3 independent lines with extra-chromosomal hlh-13(+) were tested for dauer recovery following a 6-hour egglay by gravid adult worms. RFP(−) are cohort from transgenic lines that lost the extrachromosomal hlh-13(+). RFP(+) are cohort expressing hlh-13(+). Results shown are combination of 4 independent trials. *=p<0.05 compared to daf-2(e1370), **=p<0.05 compared to RFP(−), Student's t-test. (C) daf-2(e1370);hlh-13(tm2279) recover from dauer faster following dauer arrest at 25°C than daf-2(e1370). Worms were incubated at 25°C for 5 days following egglay, allowing a strong dauer arrest. Worms were then shifted to the permissive temperature 16°C, and scored after 2 days. 86% daf-2(e1370) were developing at the time scored, compared with 95% daf-2(e1370);hlh-13(tm2279) developing at this time. *=p<0.05, Student's t-test.

We next tested whether hlh-13(tm2279) affects recovery from a stronger dauer arrest at the restrictive temperature of 25°C. daf-2(e1370);hlh-13(tm2279) worms were induced to arrest as dauers at 25°C for 5 days, and then were shifted back to the permissive temperature of 16°C, allowing development to resume. We observed that daf-2(e1370);hlh-13(tm2279) worms recovered from dauer and developed into adults earlier than daf-2(e1370) worms (Fig. 4C), consistent with our findings at 22.5°C. At the constitutive dauer-forming temperature 25°C, we did not observe any differences in the rate of dauer formation over time (data not shown) between the daf-2(e1370) and daf-2(e1370);hlh-13(tm2279) mutants. Moreover, the morphology of the dauers, such as pharyngeal constriction and alae formation, of the two genotypes was indistinguishable (data not shown). We also utilized a weaker IIS mutant, age-1(hx546), to check for a subtle dauer entry phenotype that may have been masked by the strong dauer phenotype of daf-2(e1370). However, we observed no difference in dauer entry of age-1(hx546);hlh-13(t2279) compared to age-1(hx546) (data not shown). These results indicate that hlh-13 may have a role limited to exit from dauer arrest, and may not affect entry or maintenance of dauer diapause. Since activation of DAF-16 promotes dauer arrest, it is possible that the elevated expression of wild-type hlh-13 in the daf-2(e1370) mutant may be playing a supporting role in preventing dauer exit.

Discussion

In this study, we screened a data set of predicted DAF-16 transcriptional targets and identified 3 direct targets of DAF-16, including one novel DAF-16-regulated gene, hlh-13. Our results highlight the power of informatics to make useful functional predictions about gene regulation in vivo. By limiting the assay conditions to detect genes that show early response to reduced IIS, we enriched our positive candidates for direct targets of DAF-16. If the conditions of the screen are changed, for instance by increasing the length of DAF-16 induction prior to assaying the worms, a greater number of DAF-16 regulated genes will be identified (data not shown), but some of the delayed responding genes may not be direct DAF-16 targets. In addition, other pathways and factors besides IIS, such as sir-2.1 (Tissenbaum and Guarente, 2001) and JNK signaling (Oh et al., 2005), impact the function of DAF-16. Future screening conditions using perturbation of other signaling networks besides IIS will likely yield additional DAF-16 targets from the informatic-predicted candidate set. In addition, microarray analysis suggests that a large number of the DAF-16 downstream genes contain both the DBE and DAE at their promoters (Murphy et al., 2003). Further informatic surveys for genes that harbor the DBE in combination with the DAE may predict DAF-16 regulated genes with greater specificity.

DAF-16/FOXO is emerging as a master regulator of diverse biological functions, and accumulating research indicates that to mediate its diverse functions in lifespan, metabolism, development and stress resistance, DAF-16/FOXO regulates the expression of many different target genes (Halaschek-Wiener et al., 2005; McElwee et al., 2003; Murphy et al., 2003). It is likely the combined effect of expression changes of multiple target genes that contributes to the robust regulation of the different biological outputs by DAF-16/FOXO. This predicts that manipulation of any single DAF-16 target gene would yield only a small effect in a subset of biological phenotypes associated with daf-2/daf-16. In support of this hypothesis, our study of hlh-13 indicates it is a DAF-16 transcriptional target gene with a moderate affect regulating exit from dauer diapause and L1 arrest. The continued characterizations of DAF-16 direct targets and their networks of interactions are necessary for the detailed elucidation of DAF-16/FOXO functions.

We found that the novel DAF-16 target, hlh-13, suppresses recovery from L1 arrest and from IIS-induced dauer arrest. It is interesting that only hypodermal HLH-13 appears to be regulated by IIS/DAF-16. The hypodermis has been shown previously to express a number of dauer and L1 arrest controlling genes. The insulin-like peptides ins-4 and daf-28, which target the daf-2/insulin-like receptor, are expressed in the hypodermis, among other cell types, (Li et al., 2003; Pierce et al., 2001), and ins-4 and daf-28 mutants have been shown to promote dauer exit (Li et al., 2003; Malone and Thomas, 1994). daf-9, a cytochrome p450 likely required for the synthesis of a dauer entry suppressing hormone, is expressed in the hypodermis and other tissues (Gerisch and Antebi, 2004; Jia et al., 2002; Mak and Ruvkun, 2004). Interestingly, hypodermal specific expression of wild-type daf-9 is sufficient to restore reproductive development in a dauer-constitutive daf-9 mutant (Gerisch and Antebi, 2004; Mak and Ruvkun, 2004). In addition, asna-1, a gene that has been shown to interact with IIS to control L1 arrest, is also expressed in the hypodermis, as well as other tissues (Kao et al., 2007). It is probable that the hypodermal tissue is an important center for signals controlling entry into and exit of L1 and dauer arrest.

It is interesting to speculate on the conservation of hlh-13 function and regulation. Based on phylogenetic analysis, hlh-13 is predicted to be the C. elegans homolog of mammalian Ptf1a and Drosophila Fer2 (http://wormbase.org, release WS194). HLH-13 belongs to a large family of helix-loop-helix transcription factors; although it is somewhat difficult to assign functional homologs based solely on sequence, the initial informatic search identified a conserved DBE in the promoter of the Drosophila homolog of hlh-13, Fer2 (Lee et al., 2003). A survey of 12kb of the Ptf1a promoter sequence reveals the presence of 5 potential FOXO binding sites (DBE). In the future, it will be interesting to test whether Fer2 and Ptf1a are also FOXO target genes in Drosophila and mammals respectively. Ptf1a has been studied extensively in its roles during development and adulthood. Ptf1a is necessary for development of the mammalian pancreas and the cerebellum (Kim and MacDonald, 2002; Krapp et al., 1998; Sellick et al., 2004) and is expressed in the developing spinal cord and hindbrain (Glasgow et al., 2005; Hoshino et al., 2005), and the developing and adult pancreas (Krapp et al., 1996). Fer2 is one of three Drosophila genes, Fer1, 2, and 3, with homology to hlh-13 and mammalian Ptf1a (Ledent et al., 2002; Ledent and Vervoort, 2001). All three of these genes are expressed during development; Fer1 is expressed in the epidermis, Fer2 in the ventral nerve cord and brain, and Fer3 in the gut primordia (Moore et al., 2000; Peyrefitte et al., 2001). These expression patterns are consistent with that observed for hlh-13 and Ptf1a. As C. elegans and Drosophila do not have pancrease, and hlh-13 and Fer2 are expressed in the nervous system of worms and flies, respectively, expression in the nervous system may be conserved ancestral features of Ptf1a. In addition to its role during development, mammalian Ptf1a also affects metabolism throughout the life of the animal via its regulation of digestive enzymes expression in the pancreas. Because DAF-16/FOXO regulation of target genes can be highly conserved, the findings reported here suggest the intriguing possibility that FOXO proteins in mammals might also regulate Ptf1a expression. Considering the critical roles of IGF and insulin signaling, as well as Ptf1a, in mammalian development and metabolism, it will be very interesting to investigate the potential links between IIS/FOXO and Ptf1a in mammals in the future.

Supplementary Material

1

Supplemental Fig. 1: The full-length GFP∷hlh-13 transgene rescues the dauer recovery phenotype of daf-2(e1370);hlh-13(tm2279) at 25°C. Worms were incubated at 25°C for 5 days following egglay, allowing a strong dauer arrest. Worms were then shifted to the permissive temperature 16°C, and scored after 2 days. daf-2(e1370);hlh-13(tm2279);gfp∷hlh-13 recovered faster than daf-2(e1370);hlh-13(tm2279). daf-2(e1370);gfp∷hlh-13 recovered slower than daf-2(e1370), consistent with the notion that HLH-13 has a supportive role in preventing dauer recovery.

2

Supplemental Fig. 2: Expression of GFP∷hlh-13 is regulated in response to DAF-2/DAF-16 in adult worms. (A–D) GFP∷hlh-13 expression is indicated by white arrows. Fluorescence not indicated by arrows is autofluorescence in the gut. (A, C) GFP∷hlh-13 is upregulated in the hypodermis in the head or tail, respectively, of adult daf-2(e1370) worms in response to a 24- hour incubation at 25°C (white arrowhead). (B, D) daf-16(mgDf47);daf-2(e1370) worms do not show increased expression of GFP∷hlh-13 in the hypodermis at the restrictive temperature. White arrowhead indicates comparable head region and tailed arrow indicates non-IIS responsive dopaminergic head neurons (A and B) or tail neuron (C and D). Similar expression patterns were observed at other developmental stages (Fig. 2, data not shown).

3

Supplemental Fig. 3: hlh-13(tm2279) has grossly normal development and brood size. (A) Gene schematic of hlh-13(tm2279) shows an in frame deletion of part of introns 1 and 2 and all of exon 2. (B) hlh-13(tm2279) has a similar brood size at 25°C to control worms. (C) hlh-13(tm2279) has a similar developmental rate at 25°C to control worms.

4

Supplemental Fig. 4: The lifespan of hlh-13(tm2279) and daf-2(e1370);hlh-13(tm2279) was measured at least 5 times at 16°C, 20°C, and 25°C. No consistent significant changes were observed from controls.

5

Supplemental Fig. 5: hlh-13(tm2279) and daf-2(e1370);hlh-13(tm2279) respond similarly to control worms when challenged with heat shock at 37°C.

6

Supplemental Fig. 6: hlh-13(tm2279) and daf-2(e1370);hlh-13(tm2279) respond similarly to control worms when challenged with 150mM paraquat.

7

Supplementary Fig. 7: (A) daf-2(e1368);hlh-13(tm2279) has increased survival of starvation compared to daf-2(e1368). (B) daf-2(e1368);hlh-13(tm2279) has enhanced recovery from L1 arrest following starvation compared to daf-2(e1368). *=p<0.05 compared to daf-2(e1368), Student's t-test.

Acknowledgements

We thank the Microscopy and Imaging Facility, Biotechnology Resource Center, Cornell University for assistance with confocal microscopy, and Elisabeth Greer and Kaveh Ashrafi for assistance in identifying the GFP∷HLH-13 expressing dopaminergic neurons. Some of the worm strains used in this study were provided by the Mitani lab (Tokyo Women's Medical University School of Medicine, Japan) and by the C. elegans Genetic Center, which is funded by the NIH National Center for Research Resources (NCRR). We thank members of the Lee, Liu, Kemphues, and Vatamaniuk labs for insightful discussions. This work was supported by a New Scholar Award in Aging from the Ellison Medical Foundation, a R01 grant AG024425-01 from the NIA, and a PCCW Affinito/Stewart Grant awarded to SSL, and a GAANN fellowship and a CVG Scholar Award to NL.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1

Supplemental Fig. 1: The full-length GFP∷hlh-13 transgene rescues the dauer recovery phenotype of daf-2(e1370);hlh-13(tm2279) at 25°C. Worms were incubated at 25°C for 5 days following egglay, allowing a strong dauer arrest. Worms were then shifted to the permissive temperature 16°C, and scored after 2 days. daf-2(e1370);hlh-13(tm2279);gfp∷hlh-13 recovered faster than daf-2(e1370);hlh-13(tm2279). daf-2(e1370);gfp∷hlh-13 recovered slower than daf-2(e1370), consistent with the notion that HLH-13 has a supportive role in preventing dauer recovery.

NIHMS168285-supplement-1.ai (193.3KB, ai)
2

Supplemental Fig. 2: Expression of GFP∷hlh-13 is regulated in response to DAF-2/DAF-16 in adult worms. (A–D) GFP∷hlh-13 expression is indicated by white arrows. Fluorescence not indicated by arrows is autofluorescence in the gut. (A, C) GFP∷hlh-13 is upregulated in the hypodermis in the head or tail, respectively, of adult daf-2(e1370) worms in response to a 24- hour incubation at 25°C (white arrowhead). (B, D) daf-16(mgDf47);daf-2(e1370) worms do not show increased expression of GFP∷hlh-13 in the hypodermis at the restrictive temperature. White arrowhead indicates comparable head region and tailed arrow indicates non-IIS responsive dopaminergic head neurons (A and B) or tail neuron (C and D). Similar expression patterns were observed at other developmental stages (Fig. 2, data not shown).

NIHMS168285-supplement-2.ai (2.1MB, ai)
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Supplemental Fig. 3: hlh-13(tm2279) has grossly normal development and brood size. (A) Gene schematic of hlh-13(tm2279) shows an in frame deletion of part of introns 1 and 2 and all of exon 2. (B) hlh-13(tm2279) has a similar brood size at 25°C to control worms. (C) hlh-13(tm2279) has a similar developmental rate at 25°C to control worms.

NIHMS168285-supplement-3.ai (187.9KB, ai)
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Supplemental Fig. 4: The lifespan of hlh-13(tm2279) and daf-2(e1370);hlh-13(tm2279) was measured at least 5 times at 16°C, 20°C, and 25°C. No consistent significant changes were observed from controls.

NIHMS168285-supplement-4.ai (188.2KB, ai)
5

Supplemental Fig. 5: hlh-13(tm2279) and daf-2(e1370);hlh-13(tm2279) respond similarly to control worms when challenged with heat shock at 37°C.

NIHMS168285-supplement-5.ai (158.5KB, ai)
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Supplemental Fig. 6: hlh-13(tm2279) and daf-2(e1370);hlh-13(tm2279) respond similarly to control worms when challenged with 150mM paraquat.

NIHMS168285-supplement-6.ai (189.5KB, ai)
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Supplementary Fig. 7: (A) daf-2(e1368);hlh-13(tm2279) has increased survival of starvation compared to daf-2(e1368). (B) daf-2(e1368);hlh-13(tm2279) has enhanced recovery from L1 arrest following starvation compared to daf-2(e1368). *=p<0.05 compared to daf-2(e1368), Student's t-test.

NIHMS168285-supplement-7.ai (186.8KB, ai)

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