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The EGL-4 PKG acts with the KIN-29 SIK and PKA to regulate chemoreceptor

gene expression and sensory behaviors in C. elegans

Alexander M. van der Linden*, Scott Wiener*, Young-jai You§, Kyuhyung Kim*, Leon

Avery§ and Piali Sengupta*

*Department of Biology and National Center for Behavioral Genomics

Brandeis University

Waltham, MA 02454

§Department of Molecular Biology

University of Texas Southwestern Medical Center

Dallas, TX 75390

Genetics: Published Articles Ahead of Print, published on October 1, 2008 as 10.1534/genetics.108.094771

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Running title: A PKG and SIK regulate sensory gene expression

Keywords: C. elegans; egl-4; kin-29; gene expression; chemoreceptor

Corresponding Author:

Piali Sengupta

Department of Biology and National Center for Behavioral Genomics

MS 008, 415 South Street

Waltham, MA 02454

781-736-2686 (Phone)

781-736-3107 (Fax)

email: sengupta@brandeis.edu

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ABSTRACT

The regulation of chemoreceptor (CR) gene expression by environmental signals and

internal cues may contribute to the modulation of multiple physiological processes and

behavior in C. elegans. We previously showed that KIN-29, a homolog of salt-inducible

kinase, acts in sensory neurons to regulate the expression of a subset of CR genes, as well

as sensory behaviors. Here we show that the cGMP-dependent protein kinase EGL-4 acts

partly in parallel with KIN-29 to regulate CR gene expression. Sensory inputs inhibit

both EGL-4 and KIN-29 functions, and KIN-29 function is inhibited in turn by cAMP-

dependent protein kinase (PKA) activation. EGL-4 and KIN-29 regulate CR gene

expression by antagonizing the gene repression functions of the Class II HDAC HDA-4

and the MEF-2 transcription factor, and KIN-29, EGL-4 and PKA target distinct residues

in HDA-4 to regulate its function and subcellular localization. While KIN-29 acts

primarily via MEF-2/HDA-4 to regulate additional sensory signal-regulated physiological

processes and behaviors, EGL-4 acts via both MEF-2-dependent and independent

pathways. Our results suggest that integration of complex sensory inputs via multiple

signaling pathways allows animals to precisely regulate sensory gene expression, thereby

appropriately modulating physiology and behavior.

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INTRODUCTION

Chemosensory signals regulate multiple aspects of animal development,

physiology and behavior. For example, chemical cues permit successful foraging in

social insects, regulate sexual maturation in rodents, and provide information about

reproductive status to potential mates (DULAC and TORELLO 2003; JACKSON and

RATNIEKS 2006). Food-derived chemosensory cues signal palatability, are critical for the

regulation of appetitive and feeding behaviors, and have been shown to regulate lifespan

in both Drosophila and C. elegans (APFELD and KENYON 1999; SCHWARTZ et al. 2000;

ALCEDO and KENYON 2004; ROLLS 2005; LIBERT et al. 2007). It is, therefore, essential

that animals precisely regulate their ability to sense and respond to environmental

chemicals.

C. elegans provides an excellent system in which to explore the neuronal and

molecular mechanisms by which animals sense and respond to chemical cues. Sensory

signals from food regulate nearly all aspects of C. elegans physiology, behavior and

development including the regulation of entry/exit from the alternate dauer

developmental stage, male mating, body size, locomotion, egg-laying, longevity and fat

storage (GOLDEN and RIDDLE 1984; APFELD and KENYON 1999; SAWIN et al. 2000;

WAGGONER et al. 2000; FUJIWARA et al. 2002; LANJUIN and SENGUPTA 2002; MAK et al.

2006; GRUNINGER et al. 2008). As in other animals, chemosensory behaviors are plastic

and are regulated by prior experience and external and internal cues (SAWIN et al. 2000;

GRAY et al. 2005; ZHANG et al. 2005; SHTONDA and AVERY 2006). Chemicals are sensed

by a small number of chemosensory neurons, each of which expresses multiple candidate

seven transmembrane domain chemoreceptors (CRs) and other signaling proteins

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(BARGMANN 2006). The expression of each CR gene is regulated in a complex manner by

multiple developmental and environmental cues, suggesting that regulated expression of

CR gene subsets may partly underlie the modulation of chemosensory behaviors in C.

elegans (PECKOL et al. 2001; LANJUIN and SENGUPTA 2002; NOLAN et al. 2002; VAN DER

LINDEN et al. 2007).

We previously showed that the KIN-29 Ser/Thr kinase acts in C. elegans sensory

neurons to regulate chemosensory signal-dependent development and behavior (LANJUIN

and SENGUPTA 2002). kin-29 mutants are small, exhibit deregulated entry into the dauer

developmental stage, as well as altered food-regulated foraging behaviors (LANJUIN and

SENGUPTA 2002; MADUZIA et al. 2005). All mutant phenotypes can be rescued by

expression of kin-29 only in sensory neurons (LANJUIN and SENGUPTA 2002). We found

that KIN-29 regulates the expression of a subset of CR genes in multiple chemosensory

neuron types, suggesting that altered expression of CR genes may lead to the inability of

kin-29 mutants to correctly sense and transduce food signals (LANJUIN and SENGUPTA

2002). KIN-29 directly phosphorylates and antagonizes the HDA-4 Class II histone

deacetylase (HDAC) which acts via the MEF-2 transcription factor to repress gene

expression (VAN DER LINDEN et al. 2007). Intriguingly, KIN-29 is a member of the salt-

induced kinase (SIK) family (WANG et al. 1999), members of which have been

implicated in the regulation of metabolism, and gluconeogenic gene expression in

response to fasting and feeding (KOO et al. 2005; DENTIN et al. 2007; WANG et al. 2008).

The related AMP-activated kinases (AMPKs) have also been implicated in similar food-

regulated pathways, and AMPK activity in the hypothalamus has been suggested to

regulate food intake and body weight (MINOKOSHI et al. 2004; KAHN et al. 2005; KIM

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and LEE 2005). Thus, the overall functions of these kinases in the regulation of food-

dependent behaviors may be conserved across phyla.

In addition to KIN-29, the EGL-4 cGMP-dependent protein kinase (PKG) also

regulates chemosensory signal-dependent behaviors in C. elegans. egl-4 mutants exhibit

defects in food-regulated motor behaviors, body size, fat storage, chemosensation and

regulation of dauer entry, and these phenotypes can be largely rescued by EGL-4 activity

in sensory neurons (TRENT et al. 1983; DANIELS et al. 2000; FUJIWARA et al. 2002;

L'ETOILE et al. 2002; HIROSE et al. 2003; NAKANO et al. 2004; RAIZEN et al. 2006).

Recently, EGL-4 has also been shown to act in sensory neurons to regulate quiescence

associated with developmental stage transitions (RAIZEN et al. 2008), as well as food-

induced quiescence behavior proposed to result from satiety (YOU et al. 2008).

Interestingly, PKG function is also required for food-regulated behaviors in Drosophila

and honeybees, suggesting that similar to SIKs, a role for PKG in the sensation and

transduction of food signals may be conserved (OSBORNE et al. 1997; BEN-SHAHAR et al.

2002; KAUN et al. 2007). Targets of EGL-4 required for the regulation of these behaviors

are largely unknown.

Here we show that cGMP signaling acts via EGL-4 to regulate CR gene

expression in C. elegans. We find that cAMP signaling inhibits KIN-29 activity, and that

the cAMP and cGMP signaling pathways act partly in parallel to regulate CR gene

expression. Both KIN-29 and EGL-4 functions are downregulated by sensory inputs.

Loss-of-function (lf) mutations in HDA-4 and MEF-2 fully suppress the CR gene

expression phenotypes of both kin-29 and egl-4 mutants, and we identify potential PKA

and PKG target sites in HDA-4 required for its subcellular localization and function. We

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show that while KIN-29 acts primarily via MEF-2 and HDA-4 in the regulation of

multiple physiological processes, EGL-4 acts via both MEF-2-dependent and

independent pathways. Taken together, our results suggest that regulation of distinct, but

partly overlapping sets of CR and other sensory genes by KIN-29 and EGL-4 may

contribute to the correct modulation of behavior and development by chemosensory cues.

RESULTS

EGL-4 regulates chemoreceptor gene expression:

Since both EGL-4 and KIN-29 act in sensory neurons to regulate multiple aspects

of C. elegans physiology and behavior (FUJIWARA et al. 2002; LANJUIN and SENGUPTA

2002; RAIZEN et al. 2008; YOU et al. 2008), we investigated whether similar to KIN-29,

EGL-4 also regulates the expression of chemoreceptor (CR) genes. Expression of gfp

driven under the regulatory sequences of the candidate CR genes str-1, sra-6 and srh-234

is strongly downregulated in the AWB, ASH and ADL neurons, respectively, in kin-29

mutants, whereas the expression of str-3p::gfp in the ASI neurons is weakly

downregulated (LANJUIN and SENGUPTA 2002). The expression of srd-23, srsx-3 and sru-

38 CRp::gfp fusion genes is unaffected in kin-29 mutants (VAN DER LINDEN et al. 2007).

We found that expression of str-1p::gfp and str-3p::gfp fusion genes was also

downregulated in egl-4(n479) [henceforth referred to as egl-4(lf)] mutants in the AWB

and ASI neurons, respectively (Figure 1A, B; Table 1, Supplemental Table 1), although

expression of additional CRp::gfp fusion genes was unaltered (Supplemental Table 1).

Expression of egl-4 in the AWB neurons under the lim-4 promoter (SAGASTI et al. 1999)

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rescued the altered str-1p::gfp expression phenotype of egl-4(lf) mutants (Table 1),

indicating that EGL-4 acts cell autonomously to regulate CR gene expression.

egl-4(ad450sd) mutants exhibit phenotypes consistent with a gain-of-function (gf)

mutation in egl-4 (RAIZEN et al. 2006). str-1p::gfp expression appeared to be unaffected

in egl-4(ad450sd) [henceforth referred to as egl-4(gf)] mutants (Table 1). Since it is

possible that we were unable to readily quantify upregulated gfp expression perhaps due

to overexpression from multiple copies of the stably integrated str-1p::gfp transgene, we

further examined the levels of endogenous str-1 message using qRT-PCR. Levels of

endogenous str-1 message were significantly downregulated in egl-4(lf) mutants, and

were upregulated in egl-4(gf) mutants (Figure 1C). Taken together, these results imply

that EGL-4 and KIN-29 regulate partly overlapping sets of CR genes.

Chemoreceptor gene expression is regulated by cGMP:

EGL-4 has been shown to act in a pathway with receptor guanylyl cyclases in

mediating olfactory responses, and food-induced quiescence behaviors (L'ETOILE et al.

2002; YOU et al. 2008). Since the AWB olfactory neurons express the daf-11 and odr-1

guanylyl cyclases (BIRNBY et al. 2000; L'ETOILE et al. 2002), we examined whether

mutations in these genes also affect expression of str-1p::gfp. We found that expression

of str-1p::gfp was downregulated in both daf-11 and odr-1 mutants (Figure 1A; Table 1),

with daf-11 mutants exhibiting a more severe phenotype than either egl-4(lf) or odr-1

mutants.

Mutations in daf-11 and odr-1 are predicted to decrease cGMP levels in the AWB

neurons. Addition of the exogenous membrane-permeable cGMP analog 8-Br-cGMP has

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been shown to bypass the requirement for daf-11 and odr-1 in dauer formation and in the

maintenance of ciliary morphology (SCHACKWITZ et al. 1996; MUKHOPADHYAY et al.

2008). Addition of 8-Br-cGMP was sufficient to partly bypass the daf-11 mutant

phenotype in the regulation of str-1p::gfp expression, and enhanced expression in a wild-

type background (Table 1 and Supplemental Figure 1). Thus, cGMP signaling regulates

str-1p::gfp expression.

We next determined whether DAF-11/ODR-1 act via EGL-4 to regulate CR gene

expression. Although we were unable to examine egl-4(lf); daf-11 double mutants since

these animals constitutively entered the dauer stage even under permissive conditions,

egl-4(lf); odr-1 double mutants exhibited CR gene expression phenotypes similar to those

of egl-4(lf) mutants alone (Table 1). Moreover, egl-4(gf) fully suppressed the CR gene

expression phenotype of both daf-11 and odr-1 mutants (Table 1). egl-4(gf) was

previously shown to also suppress the quiescence phenotypes of daf-11 mutants (YOU et

al. 2008). These results suggest that DAF-11 and ODR-1 act non-redundantly to regulate

EGL-4 in the regulation of CR gene expression, but that DAF-11 may also regulate

additional targets.

PKA inhibits KIN-29 to regulate chemoreceptor gene expression:

Although EGL-4 activity is cGMP-dependent, cGMP levels have not been

reported to regulate SIK activity. However, cAMP signaling has been shown to act via

cAMP-dependent protein kinases (PKA) to regulate SIKs. In hepatocytes and muscle

cells, elevated cAMP signaling, for instance during fasting, results in increased SIK

expression (TAKEMORI et al. 2002; KOO et al. 2005; BERDEAUX et al. 2007). In addition,

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PKA-mediated phosphorylation of SIK results in cytoplasmic translocation of SIK and

inhibition of activity (TAKEMORI et al. 2002; OKAMOTO et al. 2004; BERDEAUX et al.

2007; WANG et al. 2008). Thus, PKA acts at both the transcriptional and post-

translational levels to regulate SIK function.

Since KIN-29 encodes a member of the SIK family, we examined whether PKA

regulates KIN-29, and hence str-1p::gfp gene expression. Animals carrying null

mutations in the kin-1 catalytic and kin-2 regulatory subunits of PKA die as embryos; we

therefore used the kin-2(ce179) allele which acts recessively, but results in a holoenzyme

that is hypersensitive to cAMP levels for PKA activation (SCHADE et al. 2005; CHARLIE

et al. 2006). str-1p::gfp expression was severely decreased in kin-2(ce179) mutants

(Table 2 and Figure 1A), and could be rescued by expression of kin-2 in the AWB

neurons (Table 2). PKA activity is regulated by the GSA-1 Gαs subunit and the ACY-1

adenylyl cyclase in the regulation of neurotransmitter release and locomotion (REYNOLDS

et al. 2005; SCHADE et al. 2005). gf mutations in both gsa-1 and acy-1 also

downregulated str-1p::gfp expression, and gsa-1(gf); kin-2 double mutants exhibited str-

1p::gfp expression phenotypes similar to those of kin-2 mutants alone (Table 2). These

results suggest that increased cAMP signaling acts via PKA to downregulate str-1p::gfp

expression.

We next determined whether KIN-2 and KIN-29 act in a linear or parallel

pathway to regulate str-1p::gfp gene expression. Since the tight linkage of kin-2, kin-29

and kyIs104 (stably integrated str-1p::gfp transgene) on LG X prevented the construction

of a kin-2 kin-29 kyIs104 strain, we instead examined gsa-1(gf); kin-29 mutants. The str-

1p::gfp expression phenotype of these double mutant animals was similar to that of kin-

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29 mutants alone (Table 2), suggesting that KIN-29 acts downstream of, and is inhibited

by cAMP signaling. Analysis of the KIN-29 protein sequence revealed eight predicted

PKA phosphorylation sites, three of which are highly conserved in the related SIK1,

SIK2 and/or SIK3 kinases (Supplemental Figure 2A). Of these, the S517 residue in KIN-

29 is analogous to the PKA target sites S577 and S587 in SIK1 and SIK2, respectively

(TAKEMORI et al. 2002) (Supplemental Figure 2A). Mutating Ser577 to Ala results in

nuclear localization of SIK1 (TAKEMORI et al. 2002). However, a GFP-tagged KIN-

29[S517A] protein retained cytoplasmic localization in all cell types (Supplemental

Figure 3), and rescued the kin-29 mutant phenotype of reduced str-1p::gfp expression

(Table 2). Thus, elevated cAMP levels may act via PKA to directly or indirectly target

multiple sites on KIN-29 and inhibit KIN-29 function.

EGL-4 acts downstream of, or partly in parallel to KIN-29 to regulate

chemoreceptor gene expression:

Since lf mutations in both kin-29 and egl-4 downregulate str-1p::gfp expression,

we next determined whether EGL-4 and KIN-29 act in parallel or linear pathways to

regulate gene expression. Double mutants carrying putative null alleles of both kin-29

and egl-4 or daf-11 exhibited str-1p::gfp gene expression phenotypes similar to those of

kin-29 mutants alone (Table 3). egl-4(lf); kin-2 mutant animals were unhealthy, and

exhibited pale bodies, uncoordinated locomotion and slow growth (data not shown).

These animals also exhibited markedly low levels of str-1p::gfp expression similar to

those of kin-29 mutants. However, egl-4(gf) partly suppressed the CR gene expression

defects of kin-29(oy38) mutants, and fully suppressed the CR gene expression phenotypes

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of kin-2(ce179) mutants (Table 3). These observations are consistent with the notion that

EGL-4 acts downstream of, or partly in parallel to KIN-2/KIN-29 to regulate CR gene

expression.

Reduction of sensory activity suppresses the chemoreceptor gene expression defects

of kin-29 and egl-4 mutants:

Chemosensory neurons in C. elegans exhibit ciliated sensory endings which are

essential for correct sensation and transduction of environmental cues (WARD et al. 1975;

WARE et al. 1975; PERKINS et al. 1986). Mutations in genes such as che-3 encoding a

dynein motor, and osm-6 encoding a conserved protein required to build and maintain

cilia result in markedly defective ciliary structures, and altered development and behavior

(PERKINS et al. 1986; COLLET et al. 1998; WICKS et al. 2000; MUKHOPADHYAY et al.

2007). In previous work, it has been suggested that sensory inputs inhibit EGL-4 activity

in the regulation of body size and locomotor activity (FUJIWARA et al. 2002). To

determine whether this is also the case in the regulation of CR gene expression, we

examined str-1p::gfp expression in che-3; egl-4(lf) and che-3; egl-4(gf) mutants.

Surprisingly however, che-3 mutations suppressed the str-1p::gfp expression defects of

egl-4(lf) mutants (Table 4). str-1p::gfp expression was unaffected in che-3; egl-4(gf)

mutants and was similar to the expression levels in either single mutant alone (Table 4).

These results exclude the possibility that sensory inputs act solely via inhibition of EGL-

4 to regulate CR gene expression.

Our genetic epistasis experiments suggest that EGL-4 and KIN-29 may act in

partly parallel pathways to regulate CR gene expression. A simple model based on the

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above results might suggest that sensory inputs inhibit both EGL-4 and KIN-29, such that

upregulation of the KIN-29-regulated pathway in che-3; egl-4(lf) animals is sufficient to

bypass the egl-4(lf) phenotype. If this is the case, reduction of sensory input would be

predicted to also partly bypass the CR expression phenotype of kin-29 mutants due to

activation of the EGL-4-regulated pathway. As shown in Table 4, mutations in che-3 and

osm-6 also partly suppressed the kin-29 mutant phenotypes in the regulation of CR gene

expression. Mutations in the fkh-2 forkhead domain transcription factor gene result in

defects in AWB dendritic extension and ciliary morphology (MUKHOPADHYAY et al.

2007). We observed a significant correlation between the upregulation of str-1::gfp

expression and defective dendritic and/or ciliary structures in the AWB neurons of fkh-

2(ok683) kin-29(oy39) double mutants (95% of AWB neurons with defective

dendritic/ciliary structure expressed str-1::gfp at wild-type levels, as compared to 3% of

AWB neurons with wild-type morphology, n=49).

To further confirm that the observed suppression was due to reduced neuronal

activity as a consequence of altered sensory inputs, we examined str-1p::gfp expression

in animals expressing the rat delayed rectifying voltage-gated potassium channel Kv1.2

(STUHMER et al. 1989; GRISSMER et al. 1994). It has previously been shown that

expression of Kv1.1 or Kv1.2 results in reduction of activity of a subset of sensory

neurons, and downregulation of expression of a subset of KIN-29-independent CR genes

in the ASI chemosensory neurons ( PECKOL et al. 1999; PECKOL et al. 2001). We

expressed a rat Kv1.2 cDNA in the AWB sensory neurons using the lim-4 promoter

which drives expression in the AWB olfactory and additional inter- and motorneurons

(SAGASTI et al. 1999). Transgenic animals expressing this fusion gene phenocopied lim-4

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mutants in their locomotory behavior (SAGASTI et al. 1999) (data not shown), suggesting

that expression of Kv1.2 results in decreased inter- and/or motorneuron function.

Although no effect was observed on str-1 expression upon expression of Kv1.2 in a wild-

type background, we observed partial suppression of the downregulated str-1p::gfp

expression phenotype in kin-29 mutants (Table 4). These results suggest that reduction of

sensory inputs partly bypasses the requirement of either the KIN-29 or the EGL-4

pathway in the regulation of CR gene expression.

If loss of sensory inputs activates both the EGL-4 and KIN-29 pathways to

upregulate str-1p::gfp expression, we would predict that loss of both egl-4 and kin-29

gene functions would fully abolish the sensory input-mediated upregulation of gene

expression. Indeed, we found this to be the case (Table 4). Thus, str-1p::gfp expression

was strongly downregulated in osm-6; egl-4(lf); kin-29 triple mutants, or upon expression

of lim-4p::Kv1.2 in egl-4(lf); kin-29 double mutants. These results further suggest that

reduction of sensory activity bypasses kin-29(lf) or egl-4(lf) mutations by activating the

EGL-4 or KIN-29 pathways respectively, in the regulation of str-1p::gfp expression. No

suppression was observed in animals mutant for the genes unc-13, unc-31 and unc-104

(Table 4), which are required for neurotransmitter release, dense core vesicle fusion, and

trafficking of synaptic vesicles (HALL and HEDGECOCK 1991; MARUYAMA and BRENNER

1991; AHMED et al. 1992; AVERY et al. 1993; AILION et al. 1999; RICHMOND et al. 1999),

implying that inputs from other neurons may not play a role in the regulation of CR gene

expression.

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Mutations in the hda-4 Class II HDAC and the mef-2 transcription factor genes

suppress the egl-4(lf) and kin-2 phenotypes of reduced chemoreceptor gene

expression:

We next turned to identification of molecules that act downstream of EGL-4 and

KIN-29 in the regulation of CR gene expression. We previously showed that lf mutations

in hda-4 and mef-2 suppress all examined phenotypes of kin-29(lf) mutants including

altered CR gene expression, body size and dauer formation (VAN DER LINDEN et al.

2007). If cAMP signaling inhibits KIN-29, we would predict that lf mutations in mef-2

and hda-4 would also bypass the decreased str-1p::gfp expression phenotypes of gsa-

1(gf) and kin-2 mutants. As shown in Table 5, mutations in both mef-2 and hda-4

efficiently suppressed the CR gene expression phenotypes of both gsa-1(gf) and kin-2

mutants. mef-2(gv1) and hda-4(oy57) also fully suppressed the egl-4(lf) phenotype of

downregulated str-1p::gfp gene expression, but had no effect in the egl-4(ad450sd)

mutant background (Table 5). Moreover, mutations in mef-2 suppressed the gene

regulation defects of daf-11 and odr-1 mutants (Table 5). These results indicate that both

KIN-29 and EGL-4 act via MEF-2 and HDA-4 to regulate the expression of str-1p::gfp.

EGL-4 acts via both MEF-2-dependent and independent pathways to regulate body

size:

We next determined whether mef-2 mutations also suppress additional egl-4(lf)

phenotypes. Both kin-29 and egl-4 mutants exhibit defects in body size regulation

although kin-29 mutants are small, whereas egl-4(lf) mutants are long (DANIELS et al.

2000; LANJUIN and SENGUPTA 2002; FUJIWARA et al. 2002; HIROSE et al. 2003; NAKANO

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et al. 2004; MADUZIA et al. 2005) (Figure 2). Both kinases act in sensory neurons to

regulate body size, and mutations in mef-2 and hda-4 fully suppress the body size defects

of kin-29 mutants (FUJIWARA et al. 2002; LANJUIN and SENGUPTA 2002; VAN DER LINDEN

et al. 2007). As expected kin-2(ce179) mutants were also small, and these body size

defects could be partly rescued by mef-2 mutations (Figure 2).

It has previously been shown that mutations in the daf-3 SMAD transcription

factor and the DAF-5 SKI protein suppress the body size defects of egl-4 mutants

(PATTERSON et al. 1997; DANIELS et al. 2000; DA GRACA et al. 2004). Mutations in mef-2

also partly suppressed the body size defects of egl-4(lf) mutants (Figure 2). The body size

of egl-4(lf);kin-29 mutants was similar to that of kin-29 mutants alone; however, the body

size of egl-4(gf); kin-29 mutants was significantly smaller than that of either single

mutant alone (Figure 2). These results suggest that while KIN-29 acts solely through

MEF-2/HDA-4 to regulate body size, EGL-4 acts via both MEF-2-dependent and

independent (and likely DAF-3/5-dependent) pathways in the regulation of body size.

However, we are unable to rule out the possibility of additive effects of egl-4 and mef-2

mutations on the regulation of body size.

The food-induced quiescence behavioral defects of kin-29, but not egl-4 mutants, are

partly suppressed by mutations in mef-2:

When grown on high quality food (SHTONDA and AVERY 2006), at any given

time, over 90% of wild-type animals are found to be in a quiescent state where they cease

locomotion and terminate feeding (YOU et al. 2008). These food-induced quiescence

behaviors have been suggested to be related to satiety behaviors in mammals (SCHWARTZ

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et al. 2000; WOODS et al. 2000). egl-4(lf) mutants exhibit defects in food-induced

quiescence behaviors such that these animals continuously move and feed when grown

on high-quality food (YOU et al. 2008). kin-29 mutants were also found to be defective in

food-induced quiescence behavior (Figure 3A). However the quiescence defects of kin-29

and egl-4 mutants were qualitatively distinct. While egl-4(lf) mutants continued to move

and feed, kin-29 mutants often ceased locomotion but continued to pump when grown on

high quality food. kin-2(ce179) mutants exhibited quiescence defects similar to those of

kin-29 mutants in that kin-2 mutant animals also ceased to move but continued to pump

on high quality food (Figure 3A, B), as might be predicted if PKA inhibits KIN-29

activity. The quiescence defect of kin-29 mutants was partly suppressed by mutations in

mef-2 (Figure 3A), although mef-2 mutations failed to suppress the quiescence defects of

egl-4(lf) animals (data not shown). Thus, both EGL-4 and KIN-29 are defective in a food-

induced behavior, but use distinct pathways to regulate this behavioral phenotype.

Mutations in mef-2 suppress the chemosensory behavior and dauer formation

defects of egl-4 mutants:

Wild-type animals are attracted towards a point source of the volatile odorants

diacetyl and 2,3-pentanedione, which are sensed by the AWA and AWC olfactory neuron

types, respectively (BARGMANN et al. 1993; CHOU et al. 2001). egl-4(lf) mutants exhibit

defects in their responses to both these odorants, although they retain responses to other

odorants sensed by the AWA and AWC neurons (DANIELS et al. 2000) (Figure 4A).

Mutations in daf-3 suppress the chemosensory defects of egl-4(lf) mutants to both

diacetyl and 2,3-pentanedione, while mutations in daf-5 suppress only the behavioral

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defect towards diacetyl (DANIELS et al. 2000). Mutations in mef-2 resulted in weak but

significant defects in the responses to diacetyl, but suppressed the olfactory behavioral

defects of egl-4(lf) mutants to both diacetyl and 2,3-pentanedione (Figure 4A). Thus,

EGL-4 acts via both DAF-3 and MEF-2 to regulate chemosensory behaviors.

In addition to exhibiting chemosensory behavioral defects, egl-4(lf) mutants also

exhibit defects in dauer formation (GOLDEN and RIDDLE 1984b; DANIELS et al. 2000). C.

elegans produces dauer pheromone, a complex mixture of small molecules throughout its

lifecycle (GOLDEN and RIDDLE 1982; JEONG et al. 2005; BUTCHER et al. 2007). High

concentrations of pheromone signal adverse conditions and trigger entry into the dauer

stage. egl-4(lf) mutants are hypersensitive to dauer pheromone, such that they form

dauers at concentrations that are not sufficient to promote dauer formation in wild-type

animals (GOLDEN and RIDDLE 1984b; DANIELS et al. 2000) (Figure 4B). Mutations in

either daf-3 or daf-5 fully suppress dauer formation by egl-4(lf) mutants (DANIELS et al.

2000). Similarly, mutations in mef-2 also suppressed the increased sensitivity of egl-4(lf)

mutants to dauer pheromone (Figure 4B), indicating that EGL-4 acts via DAF-3, DAF-5

and MEF-2 to regulate dauer formation.

Mutations in predicted PKG/PKA phosphorylation sites result in cytoplasmic

localization of HDA-4:

In mammalian cells, phosphorylation of Class II HDACs at a residue analogous to

the S198 residue in HDA-4 (as well as at other residues) results in translocation of

HDACs from the nucleus to the cytoplasm (MCKINSEY et al. 2000a; MCKINSEY et al.

2000b; KAO et al. 2001; LINSEMAN et al. 2003; CHAWLA et al. 2003). Sequestering of

19

HDACs in the cytoplasm alleviates the gene repression functions of these proteins.

However, in C. elegans, phosphorylation of HDA-4 at the S198 residue, likely by KIN-

29, does not alter the subcellular localization of HDA-4, although the gene repression

functions of the protein are abrogated (VAN DER LINDEN et al. 2007). Thus, a mutant

HDA-4[S198A] protein remains nuclear-localized and mediates constitutive repression of

str-1p::gfp expression (VAN DER LINDEN et al. 2007).

We examined whether the localization of functional GFP-tagged HDA-4 was

affected in kin-2(ce179) or egl-4(lf) mutant backgrounds. Localization of HDA-4 was

unaltered in kin-2, kin-29, che-3, egl-4(lf) and egl-4(lf); kin-29 mutant backgrounds

(Figure 5A-E; (VAN DER LINDEN et al. 2007), suggesting that lf of egl-4 or kin-29 alone or

together, or activation of PKA function does not affect HDA-4 localization. We could not

directly examine the requirement of PKA in modulating HDA-4 function due to the

lethality of kin-1 and kin-2 null alleles. Interestingly, however, HDA-4 was

cytoplasmically localized in a subset of neurons in che-3; egl-4 mutants, although HDA-4

localization remained nuclear in non-neuronal cell types (Figure 5F and data not shown).

One interpretation of these results is that PKA activity is downregulated in a subset of

neurons in che-3 mutants, and that downregulation of both PKA and PKG activity results

in cytoplasmic translocation of HDA-4.

PKA and PKG have been shown to target similar consensus sites (KENNELLY and

KREBS, 1991). We examined the HDA-4 protein sequence and identified the S363 and

S462 residues as potential PKG/PKA phosphorylation sites. The S462 residue is highly

conserved in mammalian Class II HDACs (Supplemental Figure 2B). A GFP-tagged

HDA-4[S363A S462A] mutant protein localized to the cytoplasm in wild-type animals

20

and did not affect str-1p::gfp expression (Figure 5G). Moreover, this mutant HDA-4

protein only partly abolished suppression of the str-1p::gfp expression phenotype when

expressed in hda-4 kin-29 mutants (Figure 5H; 32% of hda-4 kin-29 double mutants

expressing this mutant protein retained wild-type levels of str-1p::gfp expression; n =

73). These results suggest that PKA and PKG-mediated phosphorylation of HDA-4 at

S363/462 may be required for nuclear localization of HDA-4. In addition, KIN-29

phosphorylates HDA-4 at S198 to alleviate its gene repression properties.

DISCUSSION

We have described a complex mechanism of regulation of CR gene expression in

C. elegans. In the AWB olfactory neuron type, levels of expression of the str-1p::gfp CR

gene is regulated via integration of signals from multiple pathways. Results described

here suggest that increased cAMP levels downregulate str-1p::gfp expression via

inhibition of the KIN-29 SIK, whereas increased cGMP levels promote str-1p::gfp

expression via activation of the EGL-4 PKG (Figure 6A). These antagonistic effects on

CR gene expression may allow for more precise regulation of CR expression levels than

would be possible via a single pathway alone.

Sensory inputs inhibit both KIN-29 and EGL-4:

Based on our genetic experiments, we suggest that under normal growth

conditions, activity of both the EGL-4 and KIN-29 pathways are required for expression

of str-1p::gfp. Thus, str-1p::gfp expression levels are decreased in either egl-4(lf) or kin-

29 single mutants. However, upon loss of sensory signaling, such as in che-3 or osm-6

21

mutants, activity of both the EGL-4 and KIN-29 pathways may be upregulated, such that

one pathway is now able to bypass the requirement for the second pathway in the

regulation of str-1p::gfp expression (Figure 6A).

Sensory inputs have previously been suggested to inhibit EGL-4 activity likely

via downregulation of cGMP levels (FUJIWARA et al. 2002). In the vertebrate visual

transduction cascade, light activates a cGMP phosphodiesterase, thereby decreasing

intracellular cGMP levels leading to hyperpolarization (ARSHAVSKY et al. 2002).

Similarly, it has recently been shown that the AWC olfactory neurons in C. elegans are

hyperpolarized in the presence of odorants, likely via downregulation of cGMP levels

(CHALASANI et al. 2007). Surprisingly however, our experiments also suggest that

sensory inputs inhibit KIN-29, perhaps via upregulation of cAMP signaling (Figure 6A).

Although we have not formally ruled out the possibility that sensory inputs act via

cAMP-independent pathways to regulate KIN-29, cAMP signaling has also been shown

to regulate SIKs in mammalian cells (TAKEMORI et al. 2002; OKAMOTO et al. 2004; KOO

et al. 2005; BERDEAUX et al. 2007; WANG et al. 2008). Our observations imply that

chemosensory cues may have opposite effects on different signaling pathways in

individual chemosensory neurons in C. elegans. This is not unprecedented, since it has

been shown that different odors can activate or inhibit individual olfactory receptor

neurons in Drosophila, Manduca and rats, and that in Drosophila, a given odor can elicit

either excitatory or inhibitory responses via different receptors (DUCHAMP-VIRET et al.

1999; SHIELDS and HILDEBRAND 2000; DE BRUYNE et al. 2001; HALLEM et al. 2004).

Since each chemosensory neuron in C. elegans expresses multiple CR genes (TROEMEL et

al. 1995), sensory inputs may act via different CR subsets to differentially regulate KIN-

22

29 and EGL-4, thereby allowing for finetuning of gene expression in response to distinct

environmental conditions.

The functions of KIN-29 and EGL-4 are mediated via multiple targets and

mechanisms:

Both KIN-29 and EGL-4 appear to act primarily via MEF-2 and HDA-4 to

regulate str-1p::gfp expression. We showed previously that MEF-2 is not required for

activation of str-1p::gfp expression in the AWB neurons (VAN DER LINDEN et al. 2007).

Instead, in the absence of phosphorylation of HDA-4 at the S198 residue, a MEF-2/HDA-

4 complex interacts directly with cis-regulatory sequences upstream of str-1 to repress

gene expression (VAN DER LINDEN et al. 2007). KIN-29 is able to directly phosphorylate

HDA-4 in vitro at the S198 residue (VAN DER LINDEN et al. 2007), and recently, SIK1 has

been shown to directly target Class II HDACs in skeletal muscle (BERDEAUX et al. 2007).

It is possible that EGL-4 also directly targets HDA-4 at as yet unidentified residues to

alleviate its gene repressive functions.

We have also uncovered a second mode of HDA-4 regulation based on

subcellular localization. Phosphorylation of the conserved putative PKA/PKG target

residues S363 and S462, but not S198, appears to be necessary for nuclear localization of

HDA-4, an essential requirement for its gene repression functions. S363 and S462 may

be targeted redundantly by EGL-4 and PKA (Figure 6A). Although no function has yet

been ascribed to the analogous residues in mammalian HDACs, the phosphorylation state

of the residues analogous to S198 is the primary regulator of the subcellular localization

of Class II HDACs in mammals. Mammalian Class II HDACs are phosphorylated by

23

kinases such as calcium/calmodulin-dependent protein kinases, protein kinase D, and

SIK1 (MCKINSEY et al. 2000a; MCKINSEY et al. 2000b; KAO et al. 2001; CHAWLA et al.

2003; LINSEMAN et al. 2003; DEQUIEDT et al. 2005; PARRA et al. 2005; BACKS et al.

2006; MATTHEWS et al. 2006; BERDEAUX et al. 2007), but unlike the case in C. elegans,

phosphorylated HDACs are localized to the cytoplasm. Thus, in C. elegans, the

subcellular localization and repressive functions of HDA-4 may be regulated via

phosphorylation of different sites by different kinases. Multiple modes of regulation of

HDA-4 function may allow for integration of complex sensory inputs at the level of

regulation of HDA-4 activity, and consequently gene expression. It will be interesting to

determine whether similar mechanisms operate to regulate Class II HDAC functions in a

cell or tissue-specific manner in vertebrates (MARTIN et al. 2007).

While KIN-29 appears to act primarily via MEF-2 and HDA-4 to regulate

multiple behaviors and physiological processes, the case for EGL-4 is more complex

(Figure 6B). EGL-4 acts via MEF-2 to regulate str-1p::gfp expression, and partly via

MEF-2 as well as DAF-3 and DAF-5 to regulate body size, chemosensory behaviors and

dauer formation (DANIELS et al. 2000 and this work). Moreover, while daf-3 mutations

fully suppress all examined egl-4(lf) defects in chemosensory behaviors, daf-5 mutations

suppress only a subset of these phenotypes (DANIELS et al. 2000). One interpretation of

these observations is that EGL-4 acts via distinct effectors in different cell types to

regulate the expression of different gene subsets. The complexity of signaling pathways

mediating EGL-4 function has been noted previously (DANIELS et al. 2000; RAIZEN et al.

2006). An important next step is to define the complete gene sets regulated by KIN-29

and EGL-4 in sensory neurons, and to identify the effectors mediating their functions.

24

Multiple mechanisms regulate chemoreceptor gene expression in C. elegans:

Why must CR gene expression be regulated so precisely in C. elegans? We and

others have previously hypothesized that plasticity in CR gene expression contributes to

behavioral plasticity in C. elegans (PECKOL et al. 2001; LANJUIN and SENGUPTA 2002;

NOLAN et al. 2002; VAN DER LINDEN et al. 2007). Environmental conditions and internal

metabolic state may be translated into defined patterns and levels of expression of CRs

and other chemosensory neuron-expressed genes. The expression of different subsets of

CR genes has been shown to be regulated by population density, food availability,

developmental stage of the animal and neuronal activity (PECKOL et al. 2001; NOLAN et

al. 2002) (A. van der Linden and P.S., unpublished data). Dietary restriction and infection

by pathogenic bacteria have also been shown to regulate gene expression in

chemosensory neuron types (ZHANG et al. 2005; BISHOP and GUARENTE 2007),

indicating that these neurons are able to monitor cellular state to alter gene expression

patterns accordingly.

Although we cannot yet confirm whether altered regulation of CR gene

expression is causal to the physiological phenotypes of kin-29 and egl-4 mutants, we

suggest that regulation of CR gene expression may partly underlie the modulation of

food-seeking and acquisition behaviors, as well as food-regulated physiological processes

such as body size, quiescence and dauer formation in C. elegans. EGL-4 and KIN-29

activity in sensory neurons may therefore integrate multiple chemosensory inputs to

direct precise patterns and levels of expression of distinct, but overlapping sets of CR and

other genes. We have shown that EGL-4 regulates the expression of str-1 and str-3, but

25

not that of five additional CR genes examined. Since the C. elegans genome is predicted

to encode >1,500 CR genes (TROEMEL et al. 1995; ROBERTSON and THOMAS 2006), it is

likely that EGL-4 also regulates additional CR genes. In addition to CR genes, targets of

these kinase pathways may include additional chemosensory neuron-expressed signaling

molecules, which may regulate the activity and neurohormone outputs of these neuron

types (YOU et al. 2008).

The roles of PKGs and SIKs in food-related behaviors are conserved:

The roles of PKGs and SIKs in the regulation of food-related behaviors appear to

be conserved across species. For instance, in Drosophila and honeybees, PKG levels

regulate foraging, a complex food acquisition behavior (OSBORNE et al. 1997; BEN-

SHAHAR et al. 2002). Variations in PKG levels also regulate responses to chemicals such

as sucrose, food acquisition behaviors, and experience-dependent modification of food

intake (SCHEINER et al. 2004; KAUN et al. 2007). Similarly, cAMP signaling via SIKs and

the related AMP-activated kinase have been shown to play critical roles in the regulation

of energy homeostasis, food intake and energy expenditure in Drosophila, C. elegans and

mammals (APFELD et al. 2004; MINOKOSHI et al. 2004; SCREATON et al. 2004; DOWELL

et al. 2005; KAHN et al. 2005; KIM and LEE 2005; KOO et al. 2005; GREER et al. 2007;

WANG et al. 2008). Although SIKs have been shown to act via multiple factors including

CREB, TORC, MEF2 and Class II HDACs (SCREATON et al. 2004; KOO et al. 2005;

BERDEAUX et al. 2007), the regulators, effectors and targets of PKG signaling in food-

related behaviors are largely unknown. We have found that PKG acts with a SIK

signaling pathway to regulate chemoreceptor gene expression and other sensory

26

behaviors, and that PKG function is mediated via multiple downstream factors including

MEF-2. Given the high degree of conservation of signaling pathways across species, we

suggest that SIKs and PKG may act similarly in other species. Identification of additional

targets and regulators of both signaling pathways will lead to a better understanding of

the mechanisms by which animals modulate behavior and development in response to

changing external and internal conditions.

Acknowledgments

We thank Kristin Ma and Rinho Kim for expert technical assistance, the

Caenorhabditis Genetics Center for strains, and Don Katz for help with statistical

analyses. This work was supported by the National Science Foundation (NSF IOS-

0542372 – P.S.) and the National Institutes of Health (NIH DK074065 – L.A.).

27

MATERIALS AND METHODS

Strains: Animals were grown as previously described (BRENNER 1974). The wild-type

strain used was C. elegans variety Bristol, strain N2. The following mutant strains were

obtained from the Caenorhabditis Genetics Center unless indicated otherwise: MT1074

egl-4(n479) IV, DA521 egl-4(ad405sd) IV, JT195 daf-11(sa195) V (from Jim Thomas),

PY1476 kin-29(oy38) X (lab collection), PY1988 kin-29 (oy39) X (lab collection),

KG532 kin-2(ce179) X, DR47 daf-11(m47) V, CX2065 odr-1(n1936), PR811 osm-

6(p811) V, CB1124 che-3(e1124) I, CB1265 unc-104(e1256) II, CB169 unc-31(e169)

IV, KG524 gsa-1(ce94gf) I, KG522 acy-1(md1756gf) III, RB853 fkh-2(ok683) X,

KM134 mef-2(gv1) I, PY4111 hda-4(oy57) X (lab collection). Integrated strains used

were CX3553 lin-15(n765ts); kyIs104[str-1p::gfp+lin-15(+)] X (TROEMEL et al. 1997),

PY1058 lin-15(n765ts); oyIs14[sra-6p::gfp+lin-15(+)] V (SARAFI-REINACH et al. 2001),

CX3596 lin-15(n765ts); kyIs128[str-3p::gfp+lin-15(+)] X (PECKOL et al. 2001); PY3018

oyIs41[kin-29p::kin-29::GFP] IV (LANJUIN and SENGUPTA 2002), PY6220 oyIs68[hda-

4p::hda-4::GFP+unc-122::dsRed] (VAN DER LINDEN et al. 2007). Double mutant strains

were constructed using standard methods, and the presence of each mutation confirmed

by amplification and/or sequencing.

qRT-PCR: Total RNA was isolated from a growth-synchronized population of adult

animals, and reverse-transcribed using oligo-dT primers. Quantitative real-time PCR was

performed with a Corbett Research Rotor-Gene 3000 real-time cycler, Platinum Taq

polymerase (Invitrogen), and primers specific for str-1 and odr-10 (SENGUPTA et al.

1996) coding sequences. Primer sequences for str-1 are:

28

5'-TCAATAACGCCTTCTTCAGG-3’ and 5'-GTGGAATGCTTGGATGTTCA-3’.

Primer sequences for odr-10 are :

5'-GAGAATTGTGGATTACCCTAG-3’ and

5'-CTCAATATGCATTATAGGTCGTAATATG-3’.

Expression constructs and generation of transgenic animals: Site-directed

mutagenesis (Statagene QuikChange site-directed mutagenesis kit) was used to generate

the following constructs: pSL204 odr-4p::kin-29(S517A)::gfp, and pSL287 hda-4p::hda-

4(S363 462A)::gfp. Mutations were confirmed by sequencing. Expression vectors of

Kv1.2, egl-4 and kin-2 were generated by fusing 3.6 and 2.8 kb of upstream regulatory

sequences of lim-4 or srd-23, respectively, to a Kv1.2 cDNA (kind gift from C.

Bargmann) (PECKOL et al. 1999), egl-4 cDNA plus 0.6 kb of 3’-untranslated region, or a

kin-2 cDNA (kind gift from Kenneth Miller). This resulted in the generation of the

following constructs: pSL137 lim-4p::Kv1.2, pSL294 lim-4p::egl-4 and pSL293 srd-

23p::kin-2. In all cases, amplified products were sequenced to confirm the absence of

errors generated via the amplification procedure.

For each promoter-gfp fusion, expression from the same extrachromosomal array

was examined in wild-type, kin-29 or egl-4 mutant animals. Transgenic animals were

generated using either the unc-122p::dsRed (50-100 ng/µl) or pRF4 rol-6(su1006) co-

injection markers injected at at 100 ng/µl. All other plasmids were injected at 20 or 50

ng/µl.

29

Growth in 8-Br-cGMP: NGM agar plates were made containing 8-bromo-cGMP

(Sigma) added to a final concentration of 5 mM from a freshly made 250 mM stock. On

the following day, plates were seeded with 30 µl of a HB101 bacterial suspension and

allowed to dry. ~10 animals were picked to each plate and allowed to lay eggs for 2-3

hours. Parents were then removed and eggs were allowed to develop into adults at 20°C.

str-1p::gfp expression levels in wild-type and daf-11 animals were quantified at 50X

magnification. As shown previously, addition of 8-bromo-cGMP resulted in smaller body

size, and rescued the dauer formation defects of daf-11 mutants (BIRNBY et al. 2000;

NAKANO et al. 2004).

Feeding/quiescence behavior: Quiescence without fasting was measured as described

(YOU et al. 2008). In brief, L4 larvae were placed on a plate seeded with E. coli HB101

for 12 hours before the assay. After 12 hours, each young adult was individually isolated

into HB101-seeded plates and fed for 6 hours without disturbance. 6 hours later, worms

were observed individually through a dissecting microscope at 50X magnification.

Quiescence duration was measured from the time when the worms were found quiescent

until either continuous feeding or continuous locomotion occurred.

Measurement of body size: Body length measurements were carried out as described

(RAIZEN et al. 2006). In short, digital images of adult worms 24 hrs after the L4 larval

molt were acquired at 100x magnification. The length of the worm was traced using four

short line segments using OpenLab software (Improvision), and the sum of the line

30

lengths was calculated. The tail was not included in the measurements (RAIZEN et al.

2006).

Chemosensory behaviors and dauer formation assays: Chemosensory behavioral

assays were carried out essentially as previously described (BARGMANN et al. 1993). For

dauer formation assays, five well-fed and growth-synchronized adult animals were

allowed to lay eggs at 25°C for 4-5 hours on a 3.5-cm assay plate. Adult animals were

then removed, and the plates containing approximately 100 eggs each were placed at

25°C for 48 hr. Dauer and nondauer animals were identified by visual inspection, and

confirmed by selecting for dauer animals that survived 1% SDS treatment for 45 min

(RIDDLE 1988). Assay plates were made with Noble agar (BD Biosciences) lacking

peptone, and seeded with OP50 bacteria. 5 µl crude dauer pheromone extract (VOWELS

and THOMAS, 1994) was added to each plate prior to adding agar. For each experiment,

all strains were assayed in parallel in at least four independent experiments. We note that

the conditions used here for the dauer assay differ from those used previously (DANIELS

et al. 2000), and may account for the observed differences in the overall number of

dauers formed.

Microscopy: Levels of chemoreceptor gene expression were quantified in adult animals

24 hours after the L4 stage and grown at 20ºC under well-fed conditions. Levels of gfp

expression were scored using a dissection microscope equipped with epifluorescence. For

quantitation, gfp expression was scored as ‘strong’ if levels of gfp fluorescence allowed

visualization of the neuronal cell bodies and processes, ‘reduced’ if gfp expression

31

allowed visualization of the cell bodies but not processes, and ‘weak/none’ if gfp

expression could not be detected, or could be detected weakly only under higher

magnifications.

Statistical analyses: Statistical analyses presented in all Tables were performed using the

chi-square test of independence to test for statistically significant differences between

proportions in the categories ‘strong’, ‘reduced’, and ‘weak/none’ for different genotypes

(df = 2). A proportion of 0% was set to a default of 1. Significances presented in Figures

were determined using one-way ANOVA. When appropriate, significances were adjusted

for multiple comparisons using Tukey’s HSD post-hoc test.

32

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39

Table 1. cGMP signaling and EGL-4 regulate str-1p::gfp gene expression. % expressing str-1p::gfp in at least one AWB

neuron at the indicated levelb:

Straina

Strong

Reduced

Weak/ None

P-

valuesc Wild-type kin-29(oy38) PKG mutants egl-4(n479) egl-4(ad450sd) egl-4(n479); Ex[lim-4p::egl-4] Guanylyl cyclase mutants daf-11(m47) daf-11(sa195) 5 mM 8-Br-cGMP daf-11(sa195) + 5 mM 8-Br-cGMP odr-1(n1936) Double mutants egl-4(n479); odr-1(n1936) egl-4(ad450sd); daf-11(sa195) egl-4(ad450sd); odr-1(n1936)

100 0

0 100 94

0 0

100 39 0

0 99

100

0 0

92 0 5 0 0 0

38 53

90 1 0

0 100

8 0 1

100 100 0

23 47

10 0 0

<0.001d

<0.001d d

<0.001e

<0.001d <0.001d d

<0.001f <0.001d

<0.001g <0.001f <0.001g

aAdult animals grown at 20°C were examined in all cases, except for daf-11 mutant animals which were examined at 15°C to permit non-dauer development. All strains contain stably integrated copies of str-1p::gfp fusion genes. bExpression of str-1p::gfp was examined at 50X magnification. Strong expression was defined as expression levels similar to those of wild-type animals allowing visualization of the cell bodies and processes; reduced expression was defined as expression levels that allowed visualization of the cell bodies but not neuronal processes; weak/none was defined as expression that was not visible at 50X magnification but may be observed weakly at higher magnifications. cP-values were determined using a chi-square test of independence (See Materials and Methods). Only differences at P < 0.001 are indicated. dCompared to wild-type. eCompared to egl-4(n479). fCompared to daf-11(sa195). gCompared to odr-1(n1936). n = 100-250 for each.

40

Table 2. cAMP signaling via KIN-2 inhibits KIN-29. % expressing str-1p::gfp in at least one AWB

neuron at the indicated levelb:

Straina

Strong

Reduced

Weak/ None

P-

valuesc Wild-typed kin-29(oy38)d gsa-1 Gsα, acy-1 AC and kin-2 PKA mutants gsa-1(ce94gf) acy-1(md1756gf) kin-2(ce179) kin-2(ce179); Ex[srd-23p::kin-2]e Double mutants with kin-2 and gsa-1 gsa-1(ce94gf); kin-2(ce179) gsa-1(ce94gf); kin-29(oy38) Expression of KIN-29[S517A] kin-29(oy38); Ex[odr-4p::kin-29]e kin-29(oy38); Ex[odr-4p::kin-29(S517A)]e

100 0

0 0 0 93

0 0

95

93

0 0

31 46 3 4 1 6 5 6

0 100

69 54 97 2

99 94h

0 1

<0.001f

<0.001f <0.001f <0.001f <0.001g

g i

<0.001i

<0.001i

aAdult animals grown at 20°C were examined in all cases. All strains contain stably integrated copies of str-1p::gfp fusion genes. bExpression of str-1p::gfp was examined at 50X magnification. Expression levels were defined as in Table 1. cP-values were determined as in Table 1. dData from Table 1. eData shown are from one to two transgenic lines for each. The srd-23 promoter drives expression in the AWB and ASK chemosensory neurons (COLOSIMO et al. 2004). odr-4 regulatory sequences drive expression in a subset of chemosensory neurons (DWYER et al. 1998) fCompared to wild-type. gCompared to kin-2(ce179). hWeak expression of str-1p::gfp in kin-29(oy38) animals can be detected at 400X magnification (LANJUIN and SENGUPTA, 2002; VAN DER LINDEN et al. 2007). This weak expression was not further decreased in gsa-1(gf); kin-29 double mutants. iCompared to kin-29(oy38). n = 30-250 for each.

41

Table 3. EGL-4 may act partly in parallel to KIN-2 and KIN-29 to regulate str-1p::gfp gene expression.

% expressing str-1p::gfp in at least one AWB neuron at the indicated levelb:

Straina

Strong

Reduced

Weak/ None

P-

valuesc Wild-typed kin-29(oy38)d kin-29(oy39) egl-4(n479)d egl-4(ad450sd)d daf-11(sa195)d kin-2(ce179)d Double mutants with kin-29 egl-4(n479); kin-29(oy38) egl-4(ad450sd); kin-29(oy38) daf-11(sa195); kin-29(oy38) Double mutants with kin-2 egl-4(n479); kin-2(ce179) egl-4(ad450sd); kin-2(ce179)

100 0 0 0

100 0 0

0 0 0

0 100

0 0 0

92 0 0 3 0

96 0 0 0

0 100 100 8 0

100 97

100 4

100

100 0

<0.001e <0.001e <0.001e e

<0.001e <0.001e

f

<0.001f f

g

<0.001g aAdult animals grown at 20°C were examined in all cases, except for daf-11 mutants which were grown at 15°C. All strains contain stably integrated copies of str-1p::gfp fusion genes. bExpression of str-1p::gfp was examined at 50X magnification. Expression levels were defined as in Table 1. cP-values were determined as in Table 1. dData from Tables 1 and 2. eCompared to wild-type. fCompared to kin-29(oy38). gCompared to kin-2(ce179) n = 30-250 for each.

42

Table 4. Sensory inputs inhibit both KIN-29 and EGL-4. % expressing str-1p::gfp in at least one AWB

neuron at the indicated levelb:

Straina

Strong

Reduced

Weak/ None

P-

valuesc Wild-typed kin-29(oy38)d kin-29(oy39)d egl-4(n479)d egl-4(ad450sd)d che-3(e1124) osm-6(p811) Double mutants with egl-4 che-3(e1124); egl-4(n479) che-3(e1124); egl-4(ad450sd) Double mutants with kin-29 che-3(e1124); kin-29(oy38) che-3(e1124); kin-29(oy39) osm-6(p811); kin-29(oy38) osm-6(p811); kin-29(oy39) lim-4::Kv1.2 expression Ex[lim-4::Kv1.2]e kin-29(oy38); Ex[lim-4p::Kv1.2]e kin-29(oy39); Ex[lim-4p::Kv1.2]e

egl-4(n479); kin-29(oy38); Ex[lim-4p::Kv1.2]e Triple mutants with kin-29 and egl-4 osm-6(p811); egl-4(n479); kin-29(oy38) Double mutants with unc-13, unc-31 and unc-104 unc-13(e51) unc-13(e51); kin-29(oy39) unc-104(e1265) unc-104(e1265); kin-29(oy39) unc-31(e169) unc-31(e169); kin-29(oy38)

100 0 0 0

100 100 100

82 100

5 90 0 0

100 0 1

0

0

100 0

100 0

100 0

0 0 0

92 0 0 0

18 0

85 10 14 99 0

11 87 0 0

0 0 0 0 0 0

0 100 100 8 0 0 0 0 0

10 0

86 1 0

89 12

100

100

0

100 0

100 0

100

<0.001f <0.001f <0.001f f

f

f

<0.001g f

<0.001h <0.001i <0.001h <0.001i

f

<0.001h <0.001i

h

h

f

i

f

i

f

h

aAdult animals grown at 20°C were examined in all cases. All strains contain stably integrated copies of str-1p::gfp fusion genes.

43

bExpression of str-1p::gfp was examined at 50X magnification. Expression levels were defined as in Table 1. cP-values were determined as in Table 1. dData from Tables 1 and 3. eData shown are from one or two independent transgenic lines for each. fCompared to wild-type. gCompared to egl-4(n479). hCompared to kin-29(oy38). iCompared to kin-29(oy39). n = 80-200 for each.

44

Table 5. Mutations in mef-2 and hda-4 suppress the str-1p::gfp expression phenotypes of egl-4 and kin-2 mutants.

% expressing str-1p::gfp in at least one AWB neuron at the indicated levelb

Straina Strong

Reduced

Weak/ None

P-valuesc

Wild-typed egl-4(n479)d egl-4(ad450sd)d daf-11(sa195)d odr-1(n1936)d gsa-1(ce94gf)d kin-2(ce179)d mef-2(gv1) hda-4(oy57) Suppression of mutations in the kin-2 pathway mef-2(gv1); gsa-1(ce94gf) mef-2(gv1); kin-2(ce179) hda-4(oy57) kin-2(ce179) Suppression of mutations in the egl-4 pathway mef-2(gv1); egl-4(n479) hda-4(oy57); egl-4(n479) mef-2(gv1); egl-4(ad450sd) mef-2(gv1); daf-11(sa195) mef-2(gv1); odr-1(n1936)

100 0

100 0 0 0 0

100 100

97 94 95

97 88 100 98 100

0 92 0 0 53 31 3 0 0

3 6 5

3 12 0 2 0

0 8 0

100 47 69 97 0 0

0 0 0

0 0 0 0 0

<0.001e e

<0.001e <0.001e <0.001e <0.001e e

e

<0.001f <0.001g <0.001g

<0.001h <0.001h e

<0.001i <0.001j

aAnimals were grown at 20°C with the exception of daf-11 mutant animals which were grown at 15°C. All strains carry stably integrated copies of str-1p::gfp fusion genes. bExpression was examined in adult animals at 50X magnification. Expression levels were defined as in Table 1. cP-values were determined as in Table 1. dData from Tables 1 and 2. eCompared to wild-type. fCompared to gsa-1(ce94gf). gCompared to kin-2(ce179). hCompared to egl-4(n479). iCompared to daf-11(sa195). jCompared to odr-1(n1936). n>80 for each.

45

FIGURE LEGENDS

Figure 1. cGMP signaling and EGL-4 regulate chemoreceptor gene expression.

A) Shown is the expression of str-1p::gfp in an AWB neuron of adult animals of the

indicated genotypes. Arrows indicate the AWB cell body. Lateral view; anterior is at left.

Images were acquired using identical exposure times at 400X magnification. Scale – 15

µm. All strains contain stably integrated copies of a str-1p::gfp transgene.

B) Expression of str-3p::gfp in an ASI neuron in wild-type (upper panel) and egl-4(n479)

(lower panel) mutant animals. The ASI cell body is indicated by an arrowhead. Lateral

view; anterior is at left. Images were acquired using identical exposure times at 400X

magnification. Scale – 15 µm. Strains contain stably integrated copies of a str-3p::gfp

transgene.

C) Levels of endogenous str-1 messages are downregulated in egl-4(lf) mutants, and

upregulated in egl-4(gf) mutants. Shown is the ratio of of endogenous str-1 message to

endogenous odr-10 (SENGUPTA et al. 1996) message as quantified by qRT-PCR, in

animals of the indicated genotypes. Expression of odr-10 is unaffected in egl-4 mutants

(DANIELS et al. 2000). The mean of the ratios from two independent experiments is

shown. Error bars denote the SEM. * and ** indicate values that are different from that of

wild-type at P < 0.05 and P < 0.01, respectively.

Figure 2. Mutations in mef-2 partly suppress the egl-4(lf) defects in the regulation of

body size.

Shown is the mean body length of adult animals of the indicated genotypes. The body

lengths of at least 20 animals were measured for each strain. * and ** indicate values that

46

are different at P < 0.05 and P < 0.01, respectively, from that of wild-type; # and ##

indicates values that are different at P < 0.05 and P < 0.01, respectively, between the

values compared by brackets. Statistical analyses were performed using one-way

ANOVA, and adjusted for multiple comparisons using Tukey’s HSD post-hoc test. Error

bars are the SD. All strains contain integrated copies of a str-1p::gfp fusion gene.

Figure 3. kin-29 and kin-2 mutants exhibit defects in food-induced quiescence behavior.

A, B) Quiescence duration of nonfasted animals of the indicated genotypes after feeding

with HB101 bacteria for 6 hours. Quiescence is defined as cessation of both locomotion

and pharyngeal pumping. n = 20 each in two independent assays. Values are the mean ±

SEM. * and ** indicates values that are different at P < 0.05 and P < 0.01, respectively,

from that of wild-type; ## indicates values that are different at P < 0.01 between the

values compared by brackets in A. Statistical analyses were performed using one-way

ANOVA, and adjusted for multiple comparisons in A using Tukey’s HSD post-hoc test.

All strains contain integrated copies of a str-1p::gfp fusion gene.

Figure 4. Mutations in mef-2 suppress the chemosensory behavioral and dauer formation

defects of egl-4(lf) mutants.

A) Responses of adult animals of the indicated genotypes to a point source of 1 µl of a

1:1000 dilution of diacetyl, or 1 µl of a 1:10,000 dilution of 2,3-pentanedione. Each data

point is the average of two independent assays in duplicate with ~100 animals in each

assay.

47

B) Percentage of dauers formed upon exposure of animals of the indicated genotypes to 5

µl of crude pheromone extract. n = 300; two independent experiments.

For both A and B, error bars equal the SEM. ** indicates values that are different at P <

0.01 from that of wild-type; # and ## indicate values that are different at P < 0.05 and P <

0.01 between the values compared by brackets. Statistical analyses were performed using

one-way ANOVA and Tukey’s HSD post-hoc test for multiple comparisons. All strains

contain integrated copies of a str-1p::gfp fusion gene.

Figure 5. Localization of HDA-4 may be regulated by both PKA and PKG-mediated

phosphorylation.

(A-F) Shown is the expression of stably integrated (A-D), or extrachromosomal copies

(E, F) of a functional full-length gfp-tagged hda-4 transgene (VAN DER LINDEN et al.

2007) in the indicated genetic backgrounds. Note cytoplasmic localization in a subset of

cells in F (arrowheads).

(G-H) A GFP-tagged HDA-4[S363A S462A] protein is localized to the cytoplasm

(arrowheads) in the indicated genetic backgrounds. Arrow points to str-1p::gfp

expression in an AWB neuron.

(A-H) Images of L1/L2 larval stage animals were acquired using identical exposure times

at 400X magnification. The area imaged in all panels is indicated by a box in the cartoon

at top. Scale – 20 µm.

Figure 6. Model of KIN-29 and EGL-4-regulated pathways in the modulation of CR

gene expression, and sensory behaviors.

48

A) KIN-29 and EGL-4 act in partly parallel pathways to regulate str-1p::gfp expression.

Increased activity of the GSA-1 Gαs and ACY-1 adenylyl cyclase result in increased

intracellular cAMP levels and activation of PKA. PKA inhibits KIN-29 activity via direct

or indirect phosphorylation. EGL-4 activity is regulated by cGMP levels generated by the

DAF-11 and ODR-1 receptor guanylyl cyclases. Sensory inputs downregulate DAF-

11/ODR-1, but may upregulate ACY-1 activity. KIN-29 and EGL-4 may phosphorylate

HDA-4 at the S198 and other residue(s) to alleviate the gene repression properties of

HDA-4. Nuclear localization of HDA-4 requires phosphorylation at the S363/S462

residues by PKA and EGL-4. Proposed phosphorylation is indicated by dashed arrows.

See Discussion for additional details.

B) KIN-29 acts primarily via MEF-2 and HDA-4 to regulate CR gene expression, body

size, dauer formation and food-induced quiescence behaviors ((VAN DER LINDEN et al.

2007) and this work). EGL-4 acts via MEF-2/HDA-4 to regulate CR gene expression,

and via MEF-2, and the DAF-3 SMAD and DAF-5 SKI proteins to regulate additional

sensory behaviors (DANIELS et al. 2000).

A

van der Linden et al_Figure l

kin-2(ce179)

Wild-type

egl-4(n479lf)

daf-11(sa195)

B

Ratio

of s

tr-1

:odr

-10

mes

sage

WT

egl-4(

n479

lf)

egl-4(

ad40

5gf)

Wild-type

C

Wild-type

egl-4(n479lf)

0

0.4

0.8

1.2

1.6

*

**

van der Linden et al_Figure 2

Mean body-length (µm)

Wild-type

kin-29(oy38)

egl-4(n479lf)

egl-4(ad405gf)

mef-2(gv1)

mef-2(gv1); egl-4(n479lf)

egl-4(n479lf); kin-29(oy38)

egl-4(ad405gf); kin-29(oy38)

kin-2(ce179)

mef-2(gv1); kin-2(ce179)

0 200 400 600 800 1000 1200 1400 1600

**

**

**

**

**

**

**

**

*

####

##

##

#

van der Linden et al_Figure 3

0

5

10

15

20

25

30

35

40

Qui

esce

nce

dura

tion

(sec

)

Wild-type kin-2(ce179)

**

B

0

5

10

15

20

25

30

35

40

Qui

esce

nce

dura

tion

(sec

)

Wild-type kin-29(oy38)

mef-2(gv1); kin-29(oy38)

mef-2(gv1)

A

**

*

##

van der Linden et al_Figure 4

0

0.2

0.4

0.6

0.8

1

0

0.2

0.4

0.6

0.8

1Ch

emot

axis

Inde

x

Wild-type egl-4(n479)

mef-2(gv1)

mef-2(gv1);egl-4(n479)

**

**

Diacetyl 2,3-pentanedione

Wild-type egl-4(n479)

mef-2(gv1)

mef-2(gv1);egl-4(n479)

**

#

A

**

##

0

5

10

15

20

25

30

Wild-type egl-4(n479)

mef-2(gv1)

mef-2(gv1);egl-4(n479)

% d

auer

s for

med

**

##B

van der Linden et al_Figure 5

oyIs68[hda-4::hda-4::gfp]

kin-2(ce179); oyIs68[hda-4::hda-4::gfp]

egl-4(n479); oyIs68[hda-4::hda-4::gfp]

A

B

C G

che-3(e1124); oyIs68[hda-4::hda-4::gfp]

D

egl-4(n479); kin-29(oy38); Ex[hda-4::hda-4::gfp]

str-1p::gfp; Ex[hda-4::hda-4(S363A S462A)]

che-3(e1124); egl-4(n479); Ex[hda-4::hda-4::gfp]

F

E

hda-4(oy57) kin-29(oy38) str-1p::gfp; Ex[hda-4::hda-4(S363A S462A)]

H

HDA-4

MEF-2

EGL-4

Sensory inputs (CHE-3/OSM-6)

S198S363/462

P P

+++str-1

XMEF-2

CR gene expression

DAF-11/ODR-1

HDA-4

P

KIN-2/KIN-1

ACY-1

GSA-1

KIN-29CR gene expression

Sensory inputs

Body-size Dauer formationChemosensation Food-induced quiescence

KIN-29

A B

EGL-4

MEF-2/HDA-4MEF-2/HDA-4 +?

DAF-3/DAF-5? MEF-2/

HDA-4DAF-3/DAF-5 +?

cAMP

cGMP

van der Linden et al_Figure 6

P

derepressionlocalization

derepression

inhibition