WO2005000093A2 - Methods for screening therapeutic agents for serotonin related diseases and other disorders - Google Patents

Abstract

The invention provides methods of screening for compounds which affect the synthesis of serotonin, using the nematode worm C. elegans. The invention further provides methods for screening therapeutic agents for serotonin-­related diseases.

Description

METHODS FOR SCREENING THERAPEUTIC AGENTS FOR SEROTONIN RELATED DISEASES AND OTHER DISORDERS

BACKGROUND OF THE INVENTION

The monoamine 5HT acts as a neurotransmitter and a hormone to induce behavioral and endocrine responses to changes in environmental and physiologic states. A deficit in 5HT is implicated in a broad spectrum of disorders such as depression, eating disorders and type II diabetes (reviewed by Lucki, 1998; Leibowitz and Alexander, 1998; Davidson et al., 2000). In both vertebrates and invertebrates, 5HT is synthesized in a set of neurons with diverse synaptic properties and connectivities. Activation of serotonergic neurons by sensory stimuli was first clearly demonstrated in 1976 through studies in Aplysia, where sensory stimuli increase 5HT signals to induce synaptic facilitation and behavioral sensitization (Brunelli et al., 1976). Long-standing questions are: how do serotonergic neurons integrate sensory signals into 5HT neurotransmission, and are different serotonergic neurons regulated by cell-specific molecular mechanisms? TRP-related proteins are a superfamily of cation channels that share structural homology to the Drosophila transient receptor potential (TRP) protein (reviewed by Clapham et al., 2001; Montell et al., 2002). TRP channels act as molecular integrators of a wide range of chemical and physical stimuli to regulate behavior and physiological function in both vertebrates and invertebrates (reviewed by Scott and ∑uker, 1998; Minke and Cook, 2002). Five genes in the C. elegans genome, ocr-1, ocr-2, ocr-3, ocr-4 and osm-9, encode TRPV subfamily proteins characterized by cytoplasmic N-terminal multiple ankyrin repeats, six transmembrane segments and a non-conserved cytoplasmic C terminus (Harteneck et al., 2000; Tobin et al., 2002). These TRPV genes are expressed in the sensory endmgc of chemosensory neurons and have been shown to regulate sensory functions and social behavior (Colbert et al., 1997; Tobin et al., 2002; de Bono et al., 2002). In mammals, TRPV channels transmit nociceptive stimuli such as pain (Tominaga et al., 1998) and noxious heat (Caterina et al., 1997; Caterina et al., 1999). In addition, mammalian TRPV ion channels also mediate the response to growth factors (Kanzaki et al., 1999). Agonists to TRPV channels induce hypothermia (Meller et al., 1992; Szallasi and Blumberg, 1996) and modulate oxygen consumption (Colquhoun et al., 1995). Mammalian TRPV proteins are expressed in the sensory neurons, as well as in the CNS (Tominaga et al., 1998; Hayes et al., 2000; Mezey et al., 2000; Delany et al., 2001). It has been postulated that TRPV ion channels regulate the release of neural mediators to modulate endocrine activity (Szallasi and Blumberg, 1996), but in vivo evidence has not yet been reported. Herein, we describe the effect of mutations in two TRPV ion channel genes, osm-9 and ocr-2, on biosynthesis of 5HT in C. elegans. Previously, we demonstrated that the tph-1 gene, which encodes the key 5HT biosynthesis enzyme tryptophan hydroxylase, is essential for 5HT biosynthesis and that tph-1 expression is regulated by cell-specific mechanisms (Sze et al., 2000; Sze et al., 2002). Here, we show that a signaling pathway involving the osm-9 and ocr-2 TRPV channel proteins, CaMKII specifically regulates tph-1 expression in the serotonergic chemosensory neurons ADF. Results from this study reveal a remarkably neuron-specific mechanism regulating 5HT biosynthesis and provide genetic insights into how a neuron transduces the events at the cell-surface to 5HT signaling. Thus, in the present invention, the present inventor has used the nematode C. elegans as a model animal system to identify and analyze genes and molecular pathways that control the level of serotonin synthesis in specific serotonergic neurons. This research is motivated by two facts: overwhelming evidence that neuronal components and signaling pathways are highly conserved across phyla, and powerful C. elegans genetics that permits unbiased search and characterizes genes acting in specific serotonergic neurons. Thus, genes identified in C. elegans will generate candidate drug targets for human diseases.

SUMMARY OF THE INVENTION In one embodiment, the present invention relates to a transgenic model animal useful for screening therapeutic agents for serotonin-related diseases, comprising a mutation in a gene encoding a TRPV channel protein and a stably integrated tpbi.vmarker fusion gene, wherein the mutation results in reduced tpbi.vmarker expression. A marker may be any gene whose expression gives rise to a detectable phenotype. In a preferred embodiment, the marker is gjp or other fluorescent protein-encoding gene. Other suitable markers will be apparent to one of skill in the art. Another embodiment of the present invention relates lo a transgenic model animal useful for screening therapeutic agents for serotonin- related diseases, comprising a mutation in osm-9 and a stably integrated tphl:. -marker fusion gene, wherein the mutation results in reduced tphl: /marker expression. A marker may be any gene whose expression gives rise to a detectable phenotype. Another embodiment of the present invention relates to a transgenic model animal useful for screening therapeutic agents for serotonin- related diseases, comprising a mutation in ocr-2 and a stably integrated tphl: .-marker fusion gene, wherein the mutation results in reduced tphl: marker expression. Another embodiment of the present invention relates to a method for screening therapeutic agents for serotonin-related diseases, comprising the steps of: providing a model animal as recited above; dispersing a target drug into agar; culturing the model animal on the agar with drug; and detecting expression of green fluorescent protein (GFP). Another embodiment of the present invention relates to a method for screening therapeutic agents for serotonin-related diseases, comprising the steps of: providing a model animal as recited above; dispersing a target drug into agar; culturing the model animal on the agar with drug; and detecting expression of green fluorescent protein (GFP). Another embodiment of the present invention relates to a method of identifying compounds which enhance or up-regulate the synthesis of serotonin, which method comprises: contacting C. elegans which exhibit reduced serotonin synthesis compared to wild type C. elegans in one or more cell types or tissues with a compound under test; and detecting a phenotypic, biochemical or behavioral change in the C. elegans indicating a reversion towards wild type serotonin synthesis in the one or more cell types or tissues which exhibit reduced serotonin synthesis in the absence of the compound. Another embodiment of the present invention relates to a method of identifying compounds which enhance or up-regulate the synthesis of serotonin, which method comprises: contacting C. elegans which exhibit reduced serotonin synthesis compared to wild type C. elegans in one or more cell types or tissues due to a defective TRPV channel protein with a compound under test; and detecting a phenotypic, biochemical or behavioral change in the C. elegans indicating a reversion towards wild type serotonin synthesis in the one or more cell types or tissues which exhibit reduced serotonin synthesis in the absence of the compound. As used herein, a "defective TRPV channel" is one that results in reduced serotonin synthesis as compared to normal or "wild type" levels of serotonin synthesis, and may result from any alteration in the gene encoding the TRPV channel protein, including but not limited to, base substitution, missense and nonsense mutations, or any posttranslational modification of the TRPV channel protein, so as to result in reduced serotonin synthesis. Still another embodiment of the present invention relates to a transgenic model animal useful for screening therapeutic agents for type II diabetes, comprising ocr-2(yz5), daf- 7(el372) and a stably integrated tphl: .-marker fusion gene, wherein the mutation results in reduced fpbEvmarker expression. daf-7 encodes a TGF-β molecule (Schackwitz et al,Neuron 17, 719-728, 1996; Ren et al, Science 274, 1389-1391, 1996). Still another embodiment of the present invention relates to a method for screening therapeutic agents for type II diabetes, comprising the steps of providing a model C. elegans as recited above; dispersing a target drug into agar; culturing the C. elegans on the agar with drug; and detecting a progression of the development of the C. elegans past the Dauer larval stage in the presence of the target drug. Still another embodiment of the present invention relates to isolated nucleic acids and purified polypeptides for a novel mutant ocr-2(yz5) having a G to A base change at nucleotide position 107 downstream from the translational start, changing amino acid 36 from the wild-type glycine (Gly) to glutamic acid (Glu). This mutant effects tph-1 expression specifically in the ADF chemosensory neurons. The nucleotide sequence of wild type ocr-2 is at NCBI AF047660; the amino acid sequence of the ocr-2(yz5) mutant is shown as SEQ ID NO. 1. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1. Cell-specific effects of yz5 and yz6 mutations on 5HT biosynthesis. (A) The position of serotonergic neurons in the head, and the axon from HSN (shown in red). Also shown are the nonserotonergic amphid chemosensory neurons that express both osm-9 and ocr-2 (in green) (Tobin et al., 2002). The drawing is adapted, with permission, from Starich et al. (Starich et al., 1995). (B) GFP expression of an integrated tph-l::gjp fusion gene in wild-type and mutant animals. In wild type, GFP is strongly expressed in the ADF, NSM and HSN neurons. yz5 and yz6 mutations specifically downregulate the GFP expression in the ADF chemosensory neurons. Note that neither the mutation alone, nor yz5;yz6 double mutation affects GFP levels in NSM and HSN. tph-1: :gfp expression in ADF is restored in the mutant animals carrying a wild-type osm-9 or ocr-2 transgene, respectively. (C) Anti-5HT antibody staining of wild-type and mutant animals. yz5 and the yzό mutant animals show reduction or absence of 5HT immunoreactivity in the ADF neurons. ADF 5HT immunoreactivity is recovered in the mutants carrying a wild-type osm-9 or ocr-2 transgene. All the animals shown are adult hermaphrodites. Anterior is towards the left. Quantification of tph-l::gfp expression in various genetic backgrounds is presented in Table 1, below. (D) A schematic representation of the domain organization of the TRPV subfamily. The approximate site of the yz6 and yz5 mutation in the channel structure is indicated. The drawing is adapted, with permission, from Gunthorpe et al. (Gunthorpe et al., 2002). Fig. 2. Mutations in the TRPY channel proteins do not cause general cell fate transformation of the ADF neurons. (A) Expression of a ocr-2::gfp reporter in a wild- type animal. Both osm-9 and ocr-2 are expressed in ADF (Tobin et al., 2002), but not in other serotonergic neurons. (B-D) Expression of ADF markers in osm-9(yz6) and ocr- 2(yz5) mutants. The GFP reporters were examined in wild-type, yzό and yz5 mutant animals. The expression patterns in wild-type animals have been published: lin-ll:gfp (Hobert et al., 1998), and cat-l::gjp and gtpch-lxgfp (Sze et al., 2002). Representative examples of the reporter expression patterns in mutant animals are shown. (B) The expression of lin-ll::gfp overlaps with osm-9 and ocr-2 in the ADF and ADL chemosensory neurons. Shown is a yzό mutant animal strongly expressing lin-ll::gfp in ADF and ADL. The position of the cell body and the morphology of the axon and dendrite are normal (indicated by arrowheads). (C) A GFP reporter of the CAT-1/vesicular monoamine transporter is localized to the synapses of the serotonergic neurons and dopaminergic neurons (Sze et al., 2002). Unlike the dramatic reduction of tph-l::gjp expression in the ADF neurons (Fig. IB, Table 1), cat-l::gfp can be clearly observed in the ADF neurons of yz5 and yzό mutants. We have repeatedly observed that cat-l::gfp expression in the ADF neurons is relatively weaker in yz5 and yzό mutants than in wild-type animals, but the differences were not statistically significant. The GFP is distributed as punctate pattern, presumably the fusion CAT-1 is associated with synaptic components. (D) A GFP reporter of a probable GTP-cyclohydrolase I gene gtpch-1 is strongly expressed in serotonergic and dopaminergic neurons (Sze et al., 2002). Shown is a yzό animal expressing gtpch-1 ::gfp in ADF and in other serotonergic neurons. Fig. 3. TRPV channel-dependent regulation of tph-1 expression is mediated by a specific cis-regulatory region of tph-1 and is modulated by unc-43 CaMKII. (A)

Expression of a GFP reporter under the control of the sequence -132 to -377 of tph-1 and a pes-10 minimal promoter in wild-type and osm-9 mutant animals. In wildtype animals, GFP is strongly expressed in the ADF neurons, but not in any other serotonergic neuron, indicating that this tph-1 cisregulatory region specifically mediates tph-1 expression in the ADF neurons. The ADF expression of this GFP reporter is significantly reduced in yzό mutant animals; hence, the TRPV signaling stimulates this neuron-specific transcriptional mechanism. In addition to ADF, this GFP reporter is often expressed in two other neurons, which are tentatively identified as the ASI chemosensory neurons based on the relative position of the cell body (White et al., 1986). The GFP intensity in ASI is not significantly affected by the TRPV mutation. (B) tph-l::gjp expression is unaffected by Gα mutations but is modulated by unc-43 CaMKII activity. All the strains bear the same GFP reporter. The average of ADF GFP intensity in wild-type animals is defined as 1, and the average GFP intensity in other strains is normalized against the wild-type average. The data are the summary of four independent trials, and the total number of animals scored for each strain is indicated next to the bar. Error bars indicate the mean of the standard error (s.e.m.). Fig. 4. Effects of the TEPN genes on 5HT-modulated behaviors. (A) Dauer metabolic arrest. Similar to the tph-1 deletion mutation, the TRPV mutations and another ADF-5HT deficit mutation, nss-l(yzl2), enhance Dauer arrest of the daf-7(e!372) mutation al the 15°C growth temperature. This enhanced Dauer phenotype is suppressed by a deletion mutation of the daf-16 gene, which is a negative target of the DAF-2/insulin signaling pathway. We noticed that daf-7; osm-9; daf-16 animals grow slower and sometimes form Dauer-like larvae but then go on to develop to adults, suggesting that the daf-2 pathway may not be the only signaling affected in the mutant. The Plin-ll::osm-9(+) transgene was carried as an extrachromosomal array, only the animals carrying the transgenic marker were scored. The osm-9(yzό) (n=956), or ocr-2(yz5) (n=346) mutants alone do not form Dauers under the assay condition. (B) Egg-laying behavior. Unlike tph-1 mutant animals, the TRPV mutant adults do not accumulate a large amount of fertilized eggs in the uterus. (C) Feeding behavior, tph-1 mutant animals have no detectable 5HT in any serotonergic neuron and exhibit slower pharyngeal pumping rates, whereas animals bearing a mutation in the TRPV genes pump at rates similar to wild-type animals. The feeding and egg-laying behavior represents the summary of two independent trials, ten animals/strain/trial. The Dauer assay is the summary of three independent trials, each in duplicate. Error bars indicate the s.e.m. Fig. 5. A. α-5HT staining (ADF); B. Bar graph showing osmotic avoidance (ASH);

C. Bar graph showing olfactory attraction; D. Bar graph showing diacetyl sensitivity. Fig. 6. Photograph showing both OCR-2: :FLAG and OCR-2(G36E)::FLAG in the plasma membrane and ciliated endings of sensory neurons. Fig. 7. Comparison of the amino acid sequences of OCR3, mTRPV-2, hTRPV-2, and

OCR4. Fig. 8. A. Mouse and human TRPV cDNA tagged with a FLAG epitope under the control of the ocr-2 promoter in ocr-2 deletion mutants. Similar to OCR-2: :FLAG, mouse and human TRPV2::FLAG is distributed in the plasma membrane and ciliated endings; B. ocr-2 mutants expressing either mouse or human TRPV2::FLAT exhibit substantially increased tph-1 expression in ADF; C, D. Bar graphs showing that no improvement in the response of the transgenic animals to diacetyl or high osmolarity solutions was detected.

DETAILED DESCRIPTION OF THE INVENTION Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described. All publications mentioned herein and in any attached documents are incorporated herein by reference for the purpose of describing and disclosing, for example, the compositions and methodologies that are described in the publications which might be used in connection with the presently described invention. The publications listed or discussed above, below and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention. Overview: Regulation of serotonin synthesis in a pair of chemosensory neurons by two TRPV cationic channel proteins. Through a genetic screen for mutations that reduce/eliminate serotonin synthesis in specific serotonergic neurons, the present inventor has isolated a set of five mutant genes that dramatically reduce the transcription of the tph-1 gene, which encodes a key serotonin synthesis enzyme tryptophan hydroxylase, in a pair of the ADF chemosensory neurons. But, these mutations have no effect on tph-l expression in any other serotonergic neurons. Genetic mapping and positional cloning revealed that two mutant genes nss-2 and nss-3 are alleles of two TRPV cationic channel proteins previously identified as osm-9 and ocr-2, respectively: osm-9(yzό) is a nonsense mutation that results in a deletion of the conserved ankyrin motifs and transmembrane regions of the protein, and ocr-2(yz5) is a missence mutation at the front of the ankyrin motifs. Although the Cori Bargmann research group at UC San Francisco first identified the osm-9 and ocr-2 genes encoding TRPV channel proteins, the present inventor has discovered the genetic linkage between the TRPV channel proteins and the regulation of neuron-specific serotonin synthesis. Furthermore, the present inventor has isolated a mutation that specifically affects serotonin synthesis but not other functions of the nss-3/ocr-2 gene. This observation suggests a specific signaling pathway downstream of the TRPV channels regulating serotonin synthesis. Support for this argument is found in our misexpression experiments showing that the osm-9 and ocr-2 TRPV channels act in the ADF serotonergic neurons to regulate the tph-l expression. Another important discovery made by the present inventor is the functional linkage between the osm-9 and ocr-2 TRPV channel proteins and neuroendocrine pathways that control metabolic homeostasis. Both the osm-9(yzό) and ocr-2(yz5) mutations cause animals to arrest at a metabolic inactive larval stage, called dauer. Studies by others have shown that a TGF-beta and an insulin signaling pathway act in parallel to regulate whether an animal enters the reproductive lifecycle or developmentally arrests as a dauer larva; disruption of either pathway is sufficient to cause constitutive dauer arrest. Our genetic analysis indicates that the osm-9 and ocr-2 mutations are likely affecting the insulin but not the TGF-beta pathway, indicating the TRPV channels may be a component in the insulin-mediated neuroendocrine signaling pathway. This work provides the first genetic evidence that serotonin synthesis is regulated by ion channels. This mechanism is very likely conserved in mammals. First, there is a large volume of literature showing that TRPV channel activity and serotonin have similar effects on behavior and physiology. This indicates that TRPV channel and serotonin function in the same physiologic system. Second, the mammalian homologues TRPV1 and TRPVLI channel proteins are expressed in the serotonergic neurons. Thus, TRPV channel activity may also regulate serotonin signaling in mammals. Third, selective agonists of the TRPV 1 channel regulate the transcription of signaling components, such as neuropeptides, and enzyme nitric oxide synthase. These observations indicate that regulation of the expression level of neuronal signaling molecules is a common output of TRPV channel activity. The fact that the function of TRPV channels is phyletically conserved indicates that C. elegans may serve as a simple animal system to explore physiological function of TRPV ion channels and to identify additional components in the signaling pathway. The method of the invention, which will be hereinafter referred to as the 'serotonin assay' is performed using a C. elegans strain which exhibits reduced serotonin activity in one or more cell types or tissues, as compared to the serotonin activity in wild-type C. elegans. In a preferred embodiment of the present invention, the serotonin assay is performed using a C. elegans strain have reduced serotonin activity specifically in ADF neurons. In another preferred embodiment of the present invention, the serotonin assay is performed using a C. elegans strain having a defective TRPV channel protein. It has been observed that worms which exhibit reduced serotonin activity compared to wild-type worms manifest a variety of phenotypic and behavioral defects. The basis of the serotonin assay is therefore to take worms which exhibit defects due to reduced serotonin activity, contact these worms with the compound under test and screen for phenotypic, behavioral or biochemical changes indicating a reversion towards wild-type serotonin activity. For example, worms with reduced serotonin activity show a developmental arrest at the Dauer larval stage. In this case, screening for developmental progression past the Dauer larval stage in the presence of a test compound would indicate a reversion towards wild-type serotonin activity due to the ability of the compound to enhance or up-regulate serotonin. For comparison purposes, an example of a C. elegans strain which exhibits 'wild-type' serotonin activity is the N2 strain (this strain can be obtained from CGC, University of Minn., USA). The N2 strain has been particularly well characterized in the literature with respect to properties such as developmental progression (see Methods in Cell Biology, Volume 48, Caenorhabditis elegans: Modern biological analysis of an organism, ed. by Henry F. Epstein and Diane C. Shakes, 1995 Academic Press; The nematode Caenorhabditis elegans, ed. by William Wood and the community of C. elegans researchers., 1988, Cold Spring Harbor Laboratory Press; C. elegans π, ed. by Donald L. Riddle, Thomas Blumenthal, Barbara J. Meyer and James R. Priess, 1997, Cold Spring Harbor Laboratory Press.). C. elegans which exhibit reduced serotonin activity in one or more cell types or tissues can be obtained in several different ways. In a first embodiment, worms with reduced serotonin activity are obtained by treating a culture of worms with a chemical inhibitor of TRPV channel proteins such as, for example, EGTA. In still further embodiment, the C elegans exhibiting reduced serotonin activity may be a mutant strain in which TPRV function is SPECIFICALLY disrupted in the serotonergic neurons. For example, our ocr-2(yz5) mutations completely abolishes the ocr-2 function in the serotonergic neurons but not in the olfactory neurons. Such mutation is extremely useful in identification of genes and drugs that specifically function and target to the serotonergic neurons, respectively. A reduction-of-function mutant or a knock-out mutant can be isolated using a classical non-complementation screen, starting with a heterozygote C. elegans strain carrying a mutant TRPV allele on one chromosome and a recessive marker close to the wild-type TRPV allele on the other chromosome. The worms are subjected to mutagenesis using standard techniques (EMS or UV-TMP are suitable for this purpose) and the progeny is screened by eye for defects, especially in tissues which express TRPV. Since the screening is performed in the FI generation, mutations will only give rise to a phenotype if the mutation occurs in the TRPV gene (due to non-complementation) or if the mutation is dominant, which does not occur frequently. These two possibilities can be distinguished in subsequent generations. A newly introduced TRPV mutation should be linked to the recessive marker.

As a further control, DNA sequencing can be performed to determine the nature of the mutation. The step of 'detecting a phenotypic, biochemical or behavioral change in the C. elegans indicating a reversion towards wild type serotonin synthesis' may be performed in several different ways. The method of choice is generally dependent upon the phenotype/behavioral characteristics of the starting worm strain, which is in turn generally dependent upon the nature of the cell types or tissues in which serotonin synthesis is reduced. The genes identified from this research are candidates for new therapeutic targets of serotonin-related diseases including but not limited to: respiration and thermoregulation disorders, circadian rhythm entrainment, obesity, high blood pressure, high cholesterol, cardiovascular diseases, type U diabetes, eating disorders such as bulimia nervosa and anorexia nervosa, psychotic disorders such as Schizophrenia, Autism, depression, anxiety disorders, impulsivity, alcoholism, social phobia, chronic neural disorders such as migraine, late-onset Alzheimer's disease, Parkinson's diseases, and defects in a variety of behaviors, for example, circadian rhythm entrainment, sleep-wake cycle, appetite, sexual behavior, sensorimotor reactivity, pain sensitivity, and learning. Also, the animals carrying these mutant genes may be used for drug screens, and for investigating the mechanisms of drug addictions. Specific Techniques: Worm strains. The strains used in this study were wild-type C. elegans Bristol strain (N2) and mutants osm-9(yzό), osm-9(n2743), osm-9(kyl0), osm-9(nl516), ocr-2(yz5), ocr- 2(ak47), odr-10(Icy225), osm-11(nlό04),tph-l(mg280), daf-7(el372), daf-16(mgDf50), nss- l(yzl2), odr-3(n2150); gpa-3(pk35); gpa-13(pl330); ιmc-43(n498)gf, unc-43(e408), and osm-3(nl540). The worms were grown at 20° C and fed with E. coli OP50 as the food. Isolation and characterisation of osm-9(yz6) and ocr-2(yz5) mutants. The yzό and yz5 mutations were isolated based on reduction/absence of GFP in the ADF neurons after ethylmethane sulfonate mutagenesis of wild-type animals carrying an integrated tph-1 r.gfp transgene. The mutagenesis and mutant screens have been described previously (Sze et al., 2002). We screened about 6500 haploid genomes and isolated 24 mutants. None of the mutations completely eliminates tph-1 ::gfp expression in the ADF neurons, but yz!2 (Sze et al., 2002), yz5, yzό and other three mutants showed stronger effects. Genetic mapping and complementation analysis indicated all these six strong mutants as single alleles, thus the screen is probably unsaturated. We mapped yzό between the polymorphisms in the clones C09G12 and M02B7, and yz5 between C49H3 and C01F6. A PCR fragment of the cosmid M57 containing 2.8 kb of the upstream sequence, exons/introns and 1.3 kb downstream sequence of osm-9 restores tph-1 ::gfp expression in ADF of yzό animals. A PCR fragment of the cosmid T09A12 containing 2.5 kb upstream sequence, exons/introns and 1.8 kb downstream sequence of ocr-2 restores tph-1 ::gfp expression in yz5 mutants. The molecular lesion of the mutations was determined by PCR amplification of the exons and exon/intron boundaries from the mutant strains and sequencing the PCR fragments. Expression of osm-9 from heterologous promoters. The osm-9 and ocr-2 genes are co-expressed in six pairs of neurons: ADF, AW A, ADL, ASH, PHA and PHB (Colbert et al.,

1997; Tobin et al., 2002). Chimeric constructs were generated by expressing wildtype osm-9- or ocr-2-coding regions under the control of heterologous promoters that are expressed in a subset of these six pairs of the neurons. The expression pattern of the heterologous promoters overlaps with osm-9 and ocr-2 in the following neurons: odr-7, AWA (Sengupta et al., 1994); osm-10, ASH, PHA and PHB (Hart et al, 1999); tax-2, PHA and PHB (Coburn and Bargmann, 1996); cat-1, ADF (Sze et al., 2002); lin-11, ADF, ADL (Hobert et al., 1998); tph-l( C), ADF (this work). In each case, we first constructed a fusion of the promoter region to the GFP and unc-54 3'-uncoding sequences in the plasmid pPD97.75 (A. Fire) to confirm the expression pattern, then, we replaced the GFP sequence with a genomic sequence encompassing the entire intron and exon regions of osm-9 or ocr-2. Individual constructs were introduced into yzό or yz5 mutants carrying the integrated tph-1 ::gfp reporter. The tph- 1(BC) construct was generated by inserting the sequence from -132 to -377 upstream of the tph-1 translational start [the BC region as described previously (Sze et al., 2002)] to a minimal promoter of the pes-10 gene in the GFP vector pPD122.53 (A. Fire). The plasmid pRF4 containing the dominant Rol-6 gene was co-injected as a transgenic marker for Promoter: :gfp constructs, and a plasmid containing elt-2::gfp (a gift from J. McGhee) as the marker for Promoter: :osm-9 and Promoter:: ocr-2 constructs. GFP expression and Immunoanalysis. The expression pattern of these GFP transgenes in wild-type animals has been published: the tph-1 ::gfp, lin-11 ::gfp and cat-1 ::gfp transgenes were integrated into the chromosomes, and gtpch-1 ::gfp was carried as extra chromosomal arrays (Hobert et al., 1998; Sze et al., 2000; Sze et al., 2002). The individual transgenes were crossed into mutants. Thus, the expression of the same transgene in wild- type and mutant animals was compared. To quantify GFP intensity of tph-1 ::gfp in the ADF neurons, the images were captured with a Zeiss AxioCam digital camera at a fixed exposure time, and the fluorescence within a 25 '25 pixel area of the cell body was scored, hence the same dimension of the ADF neurons in different genetic background was compared. The staining of anti-5HT antibody was performed using the Mclntire-Horvitz whole- mount procedure with modifications as described (Mclntire et al., 1992; Sze et al., 2000). Behavioral assays. Feeding and egg-laying assays were conducted with young adult animals. Well-fed larval stage 4 animals (L4) were picked onto fresh plates seeded with bacteria as the food, and allowed to develop -20 hours at 20°C. Feeding behavior was assayed by measuring the rate of pharyngeal pumping, which was scored by counting pharynx terminal bulb contractions (Duerr et al., 1999; Sze et al., 2000). Egglaying behavior was scored by counting the number of fertilized eggs accumulated inside of the uterus of adults, using DIC optics (Sze et al., 2000). For Dauer assays, 10 young adult animals of a strain were transferred onto a fresh plate and allowed to lay eggs for overnight. The parents were then removed, progeny were allowed to develop at 15°C, and the number of Dauers and L4/adults was scored 5-6 days later. Notice a higher Dauer frequency of tph-l;daf-7 double mutant animals than we previously reported (Sze et al., 2000). This difference is due to different time points at which we scored Dauers. Tph-l;daf-7 mutants grow slower, some of the animals were still at pre- Dauer stages at the earlier time point. Constructs and transgenic lines. All the fusion constructs are generated by PCR. A 2.5 kb sequence upstream from the translational start of the ocr-2 gene was used to direct the expression of all the transgenes, and every construct has the sequence corresponding to the FLAG^® epitope (Sigma) inserted at the end of coding sequence which is fused to unc-54 3'- uncoding sequence in the plasmid pPD97.75 (A. Fire). The coding sequences of in the transgenes are: OCR-2: :FLAG, introns and exons of the coding regions of wild-type (WT) ocr-2; OCR-2(G36E)::FLAG, introns and exons of the cording region from ocr-2(yz5) mutants and the sequence corresponding to the red fluorescence protein in the plasmid JY388; OCR-4: :FLAG, introns and exons of WT ocr-4 and the RPF sequence; OCR-4(G33E) is derived from the same WT ocr-4 sequence using the primers containing the sequence of glutamate substitution for the glyceine at the amino acid 33; OCR-2::OCR-4::FLAG, the genomic sequence from the translational start to the amino acid 186 of ocr-2 fused to the ocr- 4 genomic sequence spanning from the amino acid 145 to the end of the coding region; mTRPV2::FLAG, the cDNA sequence of mouse TRPV2 in a ATCC clone, IMAGE ID 3487527; Htrpv2::FLAG, the cDNA sequence from human TRPV2 in the ATCC clone, IMAGE ID 4298484. Purified PCR products were microinjected into ocr-2(ak47) mutant worms carrying a tph-l::gfp reporter (Sze et al., 2000), and a plasmid containing elt-2::gfp (a gift from J. McGhee) was co-injected as a transgenic marker. For each transgenes, 2 - 5 transgenic lines from at least three generations were examined. To test the effect of the transgenes on sensory behaviors, the tph-l::gfp was outcrossed from the corresponding transgenic lines, i.e. the same transgenes were compared for the effect on OCR-2-dependent functions. Germline transformation. To generate transgenic strains, the constructs of interest were injected into C. elegans (Mello et al., 1991) at a concentration of 70 ng/ul along with the dominant elt-2 r.gfp plasmid as a marker in order to isolate transgenic animals by GFP staining in intestinal nuclei. Multiple independent transgenic lines were established from each injection. Transgenic animals were viewed by fluorescence microscopy. Progeny from the microinjected parents were scored for the presence of enhanced/decreased expression of the tph-l::gfp transgene in the ADF neurons. Cell identifications were made using Nomarski images in well-fed animals raised under uncrowded conditions. GFP expression and Immunoanalysis in Transgenic Strains. Animals of mixed stages were examined using a 40X objective of a Zeiss Axio plan II fluorescence microscope. The expression pattern of the integrated tph-1 ::gfp transgene in WT, and ocr-2 and osm-9 mutant animals has been published (Sze et al, 2000, 2004). The definitions of GFP expression level are: strong, equivalent to GFP in wild-type animals; medium, GFP can be clearly observed at least in the cell body, and very weak/None, GFP was almost visually undetectable. The staining of 5ht antibody was performed using the Mclntire-Horvitz whole- mount procedure with modifications as described (Mclntire et al., 1992; Sze et al., 2000). To conduct the staining with anti-FLAG^® staining, animals were fixed according to the established protocol (Finney et al., 1990), stained with monoclonal anti-FLAG^® antibody M2 ( Sigma) (1:200), and the staining pattern is detected using Alexa Fluor® 568 rabbit anti- mouse antibody (Molecular Probes) at the dilution 1:2000. Behavioral Assays in Transgenic Strains. Behavioral assays were conducted with young adult animals. Chemotaxis assays were performed as described (Bargmann et al., 1993). The chemotaxis index (CI) was determined in the following way: CI = (number of animals at attractant)-(number of animals at diluent)/(total number of animals). Unless specified, the dilutions of the odorants in ethanol are: benzaldehyde, 200x, isoamyl alcohol, 200x, diacetyl, l,0Q0x. Osmotic avoidance was assayed as described (Vowels and Thomas, 1994). Briefly, 10 young adults animals were placed in the center surrounded by a ring of high osmotic strength solution (7M glycerol), and the number of animals that cross the ring during a period of 15 min were scored.

Caenorhabditis elegans TRP¥ ion channel regulates 5HT biosynthesis in chemosensory neurons. yz5 and yz6 mutations specifically affect the production of 5HT in the pair of the chemosensory neurons ADF. Of the 302 neurons present in an adult C. elegans hermaphrodite, nine neurons from five distinct classes are detected by antibodies raised against 5HT (Horvitz et al., 1982) (Fig. 1A); four classes exist as left-right symmetric pairs: the ADF chemosensory neurons, the NSM pharyngeal secretory neurons, the HSN motor neurons, and the AIM interneurons and RIH is a single interneuron. These neurons are generated from different lineages during embryogenesis (Sulston et al., 1983). 5HT immunoreactivity can be detected in the ADF, NSM, AIM and RIH neurons shortly after hatching, and in the HSN neurons only in adults. The ability to monitor identified serotonergic neurons permits the isolation and analysis of mutant genes affecting serotonergic phenotype in specific neurons. Biosynthesis of 5HT in C. elegans requires the tph-1 gene, which encodes the enzyme tryptophan hydroxylase catalyzing the rate-limiting first step of 5HT biosynthesis, tph-1 is expressed in serotonergic neurons, and tph-1 knockout animals have no detectable 5HT (Sze et al., 2000). The present inventor has previously shown that tph-1 expression in different serotonergic neurons is regulated by distinct transcription programs (Sze et al, 2002). To define genes underlying this neuron-specific regulation of 5HT synthesis, the present inventor conducted a genetic screen for neuron-specific serotonin defective (nss) mutants, using a green fluorescent protein (GFP) fusion to tph-1 (tph-1 r.gfp) as a reporter. yz5 and yzό are two of the nss mutations that specifically downregulate tph-1 expression in the ADF neurons. In wildtype animals, tph-1 r.gfp is highly expressed in the ADF, NSM and HSN neurons, but the GFP level in the ADF neurons is dramatically reduced or undetectable in yz5 and yzό mutants (Fig. IB, Table 1). Consistent with the essential role of tph-1 in 5HT biosynthesis, staining of the mutant animals with anti-5HT antibody shows reduced/absence of 5HT immunoreactivity in the ADF neurons (Fig. IC). However, neither the mutation nor yz5;yzό double mutation has a detectable effect on tph-1: :gfp expression or 5HT immunoreactivity in other serotonergic neurons (Table 1; Fig. 1B,C). The ADF neurons are the only serotonergic sensory neurons in hermaphroditic C. elegans, the data suggest that yz5 and yzό specifically regulate the serotonergic phenotype of sensory neurons.

Table 1. osm*9 and atr-2 mutations downregulate fpk-~l::gfp expression In the ADF shemo^enaory neurons of GFP ill Λ.DF Vtϊiy ^w. αi .' strain.. -strung W^'uafc IKIΠU N«M j-føiyi

Ml Uio strains carry the mmz integrated tf.ιA-/:;,g#. transβcm-. Strong, -jquivalαnt to t7FPiιi ¥-M4 |» animals slrasn in l^"i«j. IB; weak, GFP still dctcclablc in tliα cell body; wry weakώoαo, GFP almost visually untM ..b s is the uuπiter αf animals mi med. Mixed staged animals WDIO cbsarved GFP in HSN was sound αnly in adults. Percentage of animal.¹, Lncϋi i cate^αryis shørøi. yz5 and yzό are mutations of the ocr-2 and osm-9 TRPV channel genes, respectively. Genetic mapping and transgene rescue of yz5 and yzό mutations revealed two TRPN channel proteins regulating tph-1 expression in serotonergic chemosensory neurons. The TRPV subfamily is characterized by the cytoplasmic Ν-terminal multiple ankyrin repeats, six transmembrane segments and a cytoplasmic C terminus (Harteneck et al., 2000)

(Fig. ID). Our genetic mapping and sequencing of the mutant genomic DΝA show that yzό is a nonsense mutation that results in a stop codon before the transmembrane domain in the osm-9 TRPN gene, and yz5 is a missense mutation adjacent to the conserved ankyrin motifs in ocr-2 (Fig. ID). To confirm it is the mutation in the TRPN genes that downregulates tph-1 expression in the ADF neurons, we examined tph-1 r.gfp expression and 5HT immunoreactivity in the ocr-2 deletion mutant ak.47 and three osm-9 alleles (kylO, n2743, nl51ό). In every of the mutant strains, tph-lr.gfp expression in ADF is downregulated and 5HT immunoreactivity in ADF is reduced (Table 1; data not shown). Furthermore, yzό and yz5 mutant animals carrying a transgene containing the wild-type osm-9 or ocr-2 gene, respectively, restore tph-l::gfp expression and 5HT immunoreactivity (Fig. 1B,C; Table 1). Thus, yzό is an allele of the osm-9 gene, and yz5 is an allele of ocr-2. TRPV ion channels act cell autonomously to control 5HT biosynthesis. Both osm-9 and ocr-2 are expressed in the ADF neurons (Colbert et al, 1997; Tobin et al., 2002), but not in other serotonergic neurons (Fig. 2A). Beside ADF, osm-9 and ocr-2 also are co- expressed in five pairs of non-serotonergic chemosensory neurons: the AWA, ADL, ASH neurons in the head, and the PHA and PHB neurons in the tail (Tobin el al., 2002). These head neurons, as well as ADF, are component neurons of the amphid sensory organ, and each class senses distinct signals (Bargmann and Horvitz, 1991a). The cell bodies of these head neurons are clustered together, and their processes ran parallel and are interconnected directly or indirectly en passant (While el al., 1986) (Fig. 1A). The TRPV channels could act in the ADF neurons to control tph-1 expression, or they could function in the other chemosensory neurons that regulate ADF neural activity. The AWA neurons detect the attractive odorant diacetyl, and the ASH and ADL neurons sense aversive signals; ocr-2(ak47) and osm-9 mutant animals are defective in sensing these sensory signals (Colbert et al., 1997; Tobin et al., 2002). We tested whether disruption of these sensory signaling is a cause of reducing tph-lr.gfp expression. No GFP reduction was observed in animals with defective diacetyl receptor (odr-10) (Sengupta et al., 1996) or with defective ASH, ADL (osm-11) function (Table 1). Thus, osm-9/ocr-2 function in these sensory signaling is not required to activate tph-1 expression in the ADF neurons. To identify cells in which the TRPV proteins function to upregulate tph-1 expression, we constructed a series of chimeric genes with the osm-9- or ocr-2-coding regions under the control of heterologous promoters and generated transgenic animals expressing wild-type osm-9 or ocr-2 proteins in a subset of these six neuronal types. When osm-9(yzό) and ocr- 2(yz5) mutant animals carry a transgene expressed in the ADF neurons, tph-lr.gfp expression in the ADF neurons is restored, whereas the mutant animals carrying the transgene not expressed in the ADF neurons exhibit reduced tph-lr.gfp expression similar to their non- transgenic siblings (Table 2). These observations collectively argue that the osm-9 and ocr-2 genes act cell-autonomously in the ADF neurons to control the tph-1 expression.

Tiiljle 2. øsm~9 acts in the ADF clienntsensoi-j neurons to upregulate the φA~l expression % aϊtp&-t:%j& in ADF Tran.sg.oiie Transgoπa c-iπric l in αxpressiun. Vtayweult imn-B(vz6) mul-aiits in ADF Strong

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TRPN ion channels regulate specific aspects of serotonergic phenotype. Mutations in OSM-9 and OCR-2 channels may disrupt the regulatory pathway of iph-1 transcription, or they may induce ADF neural degeneration due to ion imbalances. To address these possibilities, we assessed if the yzό and yz5 mutations result in a general morphological transformation of the ADF neurons. The LIM-homeodomain transcription factor lin-11 is co-expressed with osm-9 and ocr-2 in the ADF and ADL chemosensory neurons (Freyd et al., 1990; Hobert et al., 1998). We crossed an integrated lin-llrgfp transgene into osm-9(yzό) and ocr-2(yz5) mutants; no reduction of lin-llrgfp expression levels can be detected in the mutant backgrounds (Fig. 2B). Judged by fluorescence microscopy, the morphology of the cell body and the processes of the ADF neurons are indistinguishable in the mutant and wild-type animals at any developmental stage. Thus, these mutations do not cause the ADF neurons to die prematurely. Rather, these results point to a signaling pathway downstream of the OSM-9 and OCR-2 TRPN channels regulating the transcription of tph-1 in the ADF neurons. Does this TRPN channel signaling specifically regulate tph-1 expression, or does it regulate the expression of all genes involved in 5HT synthesis and neurotransmission? We have explored this question by examining GFP reporters of marker genes. In each case, the GFP reporter construct was first introduced into wild-type animals, and the resulting transgene was then crossed into mutants. Thus, the expression of the same transgene in different genetic backgrounds was compared. CAT-1/vesicular monoamine transporter is required for 5HT neurotransmission (Duerr et al., 1999; Νurish et al., 1999). cat-lrgfp is a functional fusion of GFP to the entire protein coding segment of the cat-1 gene and is localized to the synapses of the serotonergic neurons (Sze et al., 2002). The gene F32G8.6 encodes a probable GTP-cyclohydrolase I (gtpch-1), a co-factor of tryptophan hydroxylase for 5HT biosynthesis, and a gtpch-lrgfp fusion gene also is expressed in the serotonergic neurons (Sze et al., 2002). Unlike the dramatic reduction of tph-lr.gfp in the ADF neurons, there is no significant reduction of cat-lr.gfp or gtpch-lrgfp in yzό and yz5 mutant backgrounds (Fig. 2C,D). These observations are consistent with our previous results that the expression of tph-1, cat-1 and gtpch-1 is differentially regulated in serotonergic neurons (Sze et al., 2002). These data demonstrate a great specificity of the TRPN channel signaling within the ADF neurons and suggest the transcriptional regulation of the tph-1 gene as a major target. The TRPV channel signaling modulates a neuron-specific transcription program. Analysis of the tph-1 promoter has revealed a discrete cisregulatory region essential for tph-1 expression in the ADF neurons (Sze et al., 2002). We investigated whether the TRPN ion channel signaling acts through this neuron-specific transcriptional regulatory mechanism. A GFP reporter under the control of the sequence -132 bp to -377 bp of tph-1 and a minimal promoter from the pes-10 gene is specifically expressed in the ADF neurons of wild-type animals, but the ADF GFP intensity is significantly reduced in yzό mutant background (Fig. 3A). Thus, the 246 bp cis-regulatory region is sufficient to activate tph-1 expression in the ADF neurons, and signaling from the OSM-9 and OCR-2 ion channels regulates the activity of this neuron-specific transcriptional regulatory program. Unlike mutations in the POU-transcription factor UΝC-86 that completely abolish tph-lr.gfp expression in the ΝSM and HSΝ neurons (Sze et al., 2002), none of the mutations in osm-9 or ocr-2, nor the mutation of both eliminates tph-lr.gfp expression in the ADF neurons (Table 1; Fig. 3B), indicating that the TRPN activity modulates tph-1 expression levels but is not essential for the transcription. In yzό background, the GFP reporter under the control of the 246-bp tph-1 cisregulatory sequence fused to a pes-10 minimal promoter is expressed in the ADF neurons at slightly higher levels than the tph-lr.gfp reporter under the control of the 3.1 kb of the tph-1 promoter (Fig. IB, Fig. 3A). This could reflect the difference in the basal expression level of the tph-1 and pes-10 promoter in the reporter constructs. Alternatively, it could be an indication of additional cis-regulatory elements mediating inhibition of tph-1 expression but the elements are not present in the 246 bp tph-1 sequence. unc-43 CaMKII acts downstream or in parallel with the TRPV ion channels to modulate 5HT biosynthesis. The odr-3 Gα protein is essential for osm-9 and ocr-2 function in olfactory, osmosensory and mechanosensory behaviors mediated by the AWA and ASH neurons (Roayaie et al., 1998; Colbert et al., 1997; Tobin et al., 2002). It has been proposed that ODR-3 activity modulates the outputs of the TRPN channel signaling (Roayaie et al., 1998). odr-3 also is expressed in the ADF neurons; however, the odr-3(n2150) deletion mutation does not cause a significant reduction of tph-lr.gfp expression (Fig. 3B). Thus, OSM-9 and OCR-2 can activate tph-1 expression in the absence of odr-3 activity. Beside odr-3, the ADF neurons express two other Gα proteins, gpa-3 and gpa-13 (Jansen et al., 1999). tph-lr.gfp expression is unaffected in gpa-3 or gpa-13 deletion mutants (Fig. 3B). However, animals bearing a double mutation of TRPN and Gα still exhibit reduced tph-lr.gfp expression in the ADF neurons similar to the TRPN mutants. These data indicate that unlike the role of ODR-3 in the sensory behaviors, the Gα proteins do not play an essential role in OSM-9/OCR-2-dependent regulation of tph-1 expression. Because the sensory behaviors and 5HT production are mediated by different neurons, our results suggest that OSM-9 and OCR- 2 TRPN channels in different neurons may be regulated by different mechanisms. CaMKII is a critical mediator of Ca²⁺ signaling. The unc-43 gene encodes the only C. elegans CaMKII (Reiner et al., 1999; Rongo and Kaplan, 1999). An unc-43 loss-of- function mutation causes a twofold reduction of tph-1 ::gfp expression in the ADF neurons (Fig. 3B), but has no effect on other serotonergic neurons (not shown). Conversely, the unc- 43(n498) gain-of-function mutation partially blocks the downregulation of tph-1 expression in yzό (Fig. 3b) and yz5 mutants (data not shown). One simple model to explain these data would be that activation of OSM-9 and OCR-2 channels increases ADF intracellular Ca²⁺ which stimulates UΝC-43 to induce tph-1 transcription, whereas the unc43(n498) mutation, which causes constitutive Ca²⁺-independent activity (Reiner et al., 1999), bypasses the need of the channel function. However, these results do not exclude the possibility that UΝC-43 acts less directly in the TRPN channel signaling pathway. These results suggest that CaMKH acts downstream of or in parallel with the OSM-9 and OCR-2 TRPN channels to control 5HT production in the ADF chemosensory neurons. Dauer phenotype of osm-9 and ocr-2 mutants. We find that both osm-9 and ocr-2 mutants show developmental defects reminiscent of tph-1 deletion mutants (Fig. 4A). The DAF-2/insulin receptor and DAF-7/TGFβ signaling act in parallel to control whether an animal enters the reproductive lifecycle or developmentally arrests at the metabolically inactive Dauer larval stage. Disruption of either pathway causes conditional abnormal arrest at the Dauer stage, but disruption of both the pathways causes constitutive Dauer arrest (Ogg et al., 1997) (reviewed by Riddle, 1997). Similar to the tph-1 deletion mutation, the osm- 9(yzό); osm-9(kyl0) and ocr-2(yz5) mutations enhance the Dauer phenotype of daf-7(el372) mutants (Fig. 4A). At the 15°C growth temperature, about 10% of daf-7 mutant animals arrest as Dauers, but more than 70% form Dauers when daf-7 mutants carry a mutation in osm-9 or ocr-2. Ninety-eight percent of daf-7;tph-l double mutant animals form Dauers, whereas 10-15% tph-1 mutants form Dauers (Sze et al., 2000). None of the TRPN mutants on their own formed Dauers when assayed under the same condition, although they grow slower than wild-type animals. Because el372 is a daf-7-mxll mutation, this enhanced Dauer phenotype of the double mutants implies that the TRPN mutations affect a pathway parallel to daf-7. Enhancement and suppression genetics and other molecular experiments implicate that the TRPN mutations affect 5HT inputs to the insulin pathway. The DAF-16/forkhead transcription factor is a negative target of DAF-2/insulin signaling, and the daf-16(mgDf50) mutation bypasses the need of DAF-2 (Ogg et al., 1997). The Dauer phenotype of daf-7; tph- 1 and daf-7;osm-9 can be suppressed by the daf-lό(mgDfSO) mutation (Fig. 4A), indicating a reduction of DAF-2/insulin signaling in the double mutants that promotes the Dauer formation. Dauer arrest is also enhanced in daf-7(el372) mutants carrying a mutation in the nss-1 gene, which also specifically affects tph-1 expression in ADF (Sze et al., 2002). This raises the possibility that it is the reduction of ADF 5HT signals that downregulates the DAF- 2/insulin pathway. However, expression of the wild-type osm-P-coding sequence under a lin- 11 promoter only mildly suppresses the Dauer phenotype of osm-9(yzό);daf-7(el372) mutants (Fig. 4A), indicating that other ow?.- -expressing cells also contribute to the normal development. osm-9 and ocr-2 mutant animals do not display every deficit observed in mutants with all the serotonergic neurons defective. 5HT regulates several C. elegans behaviors. For example, applying exogenous 5HT to C. elegans stimulates pharyngeal pumping and egg- laying (A ery and Horvitz, 1990; Weinshenker et al., 1995), whereas 5HT-deficient mutants tph-1 and cat-1 exhibit a slower pumping rate and accumulate a large number of fertilized eggs in the uterus (Duerr et al., 1999; Sze et al., 2000). However, osm-9 and ocr-2 mutant animals do not accumulate excess eggs in the uterus and their pharyngeal pumping rates are equivalent to wild-type animals (Fig. 4B,C). Thus, 5HT signals from the other neurons are sufficient for these behaviors. But, we cannot exclude subtle behavioral changes that are difficult to detect visually. The genetic evidence presented here has two major implications. The phenotype of the TRPN mutants represents exquisite specificity in the control of 5HT production, as exemplified by our demonstration that mutations of the osm-9 and ocr-2 TRPN genes specifically downregulate 5HT biosynthesis in the ADF neurons, and this TRPN ion channel regulation of 5HT production is mediated by a neuron-specific transcription program. Our finding that the osm-9 and ocr-2 TRPV channel genes act in the ADF neurons, function upstream of CaMKII to control the key 5HT biosynthesis gene tph-1 provides insights in elucidating the genetic pathway by which a serotonergic neuron couples the activity at the cell surface and 5HT signaling. A TRPV channel-dependent transcription program controls 5HT signaling. It has been demonstrated in many experimental systems that sensory stimuli induce 5HT signals to produce changes in behavior and physiology (e.g. Barzilai et al., 1989; Boadle-Biber, 1993; Milner et al., 1998). Until this study, no endogenous membrane protein has been shown to act in a serotonergic neuron to regulate 5HT signaling. One important finding from this study is the pronounced effect of osm-9 and ocr-2 mutations on the expression of the 5HT synthesis gene tph-1 (Fig. 1). This indicates that the production of 5HT is a site where sensory information is integrated to 5HT signaling. 5HT can be released by controlled exocytosis at the synapses as well as via paracrine 'volume transmission', and even during the controlled exocytosis it is newly synthesized 5HT preferentially released to induce changes in the postsynaptic targets (Attwell et al., 1993; Sanders-Bush, 1982). Hence, the level of 5HT production is one mechanism controlling both forms of 5HT neurotransmission. Transcriptional regulation may represent a general principle of regulation of hormones and neuromodulators. For example, C. elegans' favorite growth environment upregulates the expression of the daf-7/TGFfi and α/-2<-?/insulin genes to induce C. elegans proceeding reproductive development (Schackwitz et al., 1998; Li et al., 2003); in rats, noxious sound stimuli may alter the transcription of their tryptophan hydroxylase gene in a neuron-specific manner (reviewed by Boadle-Biber, 1993); and the expression of tyrosine hydroxylase can be modulated by hormones in mice (Kumer and Nrana, 1996). Transcriptional regulation of these signaling molecules is likely a mechanism to exert a relatively slow but profound effect in the signaling pathways. Mechanisms of TMPY channel action in the ADF neurons. Our genetic results indicate that the osm-9 and ocr-2 channel proteins interact with different signaling transduction pathways to induce different behavioral outputs, osm-9 and ocr-2 are co- expressed in four pairs of the amphid sensory neurons, and are required for a normal response to attractive and aversive odorants mediated respectively by AWA, and ASH and ADL, as well as for ASH-mediated mechanosensory and osmosensory function (Colbert et al., 1997). There are genetic evidences indicating that osm-9 and ocr-2 function in these sensory behaviors requires the odr-3 Gα protein (Roayaie et al., 1998). However, tph-1 expression is unaffected by mutations in odr-3 or in other two Gα proteins expressed in the ADF neurons (Fig. 3B). Although the exact mode of activation of OSM-9 and OCR-2 in any neuron has not yet been defined, our data indicate that the activity of the channels in the ADF neurons is regulated by different signaling molecules. It is interesting to note that osm-9 and ocr-2 also act in the ASH and ADL neurons to regulate social behavior independent of odr-3 activity (de Mono et al., 2002); hence, the OSM-9 and OCR-2 can induce specific behaviors by coupling distinct signaling systems. The reciprocal effects of the unc-43 CaMKII loss- and gain-of-function mutations on tph-1 expression indicate that the amount of Ca²⁺ modulates 5HT biosynthesis (Fig. 3B). The TRP superfamily is Ca²⁺-permeable channels, and CaMKII is known as an important mediator of Ca²⁺ signaling (reviewed by Hanson and Schulman, 1992). Our genetic study shows that the constitutively active, Ca²⁺-independent unc-43(n498) CaMKII (Reiner et al., 1999) can partially activate tph-1 expression in osm-9 deletion mutant animals (Fig. 3B). These results support the model that UNC-43 is a downstream effector of the OSM-9 and OCR-2 channels: Ca²⁺ influx through OSM-9 and OCR-2 activates UNC-43 CaMKII, which induces phosphorylation cascades to activate tph-1 expression. However, unc-43(lf) mutants still express a substantial amount of tph-1, and the unc-43(gf) mutation does not completely bypass OSM-9 activity, indicating that other OSM-9/OCR-2 downstream signaling molecules may act in parallel with UNC-43 to regulate tph-1 expression. The effect of OSM-9 and OCR-2 TRPV ion channels in the ADF neurons is strikingly specific, given the involvement of Ca²⁺ and CaMKII. The general architecture of the ADF neurons is unaffected, we could not detect a significant change in the expression levels of ADF marker genes or genes directly involved in the serotonergic phenotype, nor we could detect an effect of unc-43(lf) or (gf) mutations on tph-1 expression in other serotonergic neurons. This specificity demonstrates that 'multi-functional, widespread' signaling molecules may play a refined role in a particular native cellular setting. It is conceivable that such differential regulation of the serotonergic phenotype genes would allow the ADF neurons to adjust 5HT neurotransmission in response to multiple sensory signals. As TRPV channels are expressed in the serotonergic locus in mammals (Tominaga et al., 1998; Mezey el al., 2000), it would be interesting to determine whether there is a link between TRPV mutations and 5HT deficiency in humans. The role of TMPY channels in a sensoryneuroendocrine signaling pathway. The Dauer phenotype of the osm-9 and ocr-2 mutants demonstrates a genetic link between the TRPV ion channels and endocrine signaling (Fig. 4). C. elegans Dauer/non-Dauer development reflects two alternative metabolic states controlled by sensory inputs to neuroendocrine signaling pathways. The pathways from DAF-7/TGFb and DAF-2/insulin receptor converge to stimulate reproductive growth; harsh environmental conditions transduced by the amphid chemosensory neurons suppress the endocrine signaling to induce Dauer arrest (reviewed by Riddle, 1997). Our enhancement and suppression genetics implies that osm-9 and ocr-2 regulate the DAF-2/insulin-receptor signaling pathway (Fig. 4). The TRPV channels are probably acting in the ADF neurons to modulate endocrine activity: worms bearing defective ADF neurons tend to form Dauers (Shakir et al., 1993), and laser ablation experiments implicate ADF but not the other osm-9 and ocr-2 co-expressing chemosensory neurons in Dauer formation (Bargmann and Horvitz, 1991b). However, expression of the wild-type ø-sτn-9-coding sequence under a lin-11 promoter only partially suppresses daf-7;osm-9 Dauer phenotype, indicating that osm-9 activity in other cells also modulates Dauer phenotype. Alternatively, the lin-11 promoter may not be able to express sufficient amount of osm-9 to induce a wild-type level of ADF 5HT signals (Table 2), or ectopic expression of osm-9 in other Z -ii-expressing cells may interfere with the neuroendocrine signaling cascades for normal development. In mammals, 5HT regulates insulin synthesis, release and response (Breum et al., 1995; Peschke et al., 1997). It has been proposed that a feedback regulatory loop between hypothalamus 5HT and circulating hormones such as insulin, leptin and adipose tissue-derived hormone modulates the satiety and maintains metabolic and energy homeostasis (Leibowitz and Alexander, 1998). Interestingly, mouse TRPV-like channels can be activated by insulin-like growth factors (Kanzaki et al., 1999). We propose that 5HT is one mediator of TRPV channels and endocrine activity.

OCR-2 TRPV channel activity in C elegans serotonergic neurons is mediated by a single nucleotide and can be substituted by human TRPV2

OCR-2 channel protein differentiates sensory functions. In addition to the control of 5HT biosynthesis in the chemosensory neurons ADF, OCR-2 and OSM-9 also act in several non-serolonergic sensory neurons: in the AWA neurons to induce attractive response to the odorant diacetyl, and in the ASH neurons to elicit avoidance to noxious environmental stimuli such as high osmolarity, odor-repellent and nose touch, and animals bearing a deletion mutation of either ocr-2 or osm-9 do not respond to these sensory stimuli (Fig. 5b₅c). Both OCR-2 and OSM-9 proteins are localized to plasma membrane and the ciliated sensory endings of ADF as well as of these non-seroteonergic sensory neurons. Although the exact stoichiometric composition of the channel has not yet been determined, OCR-2 and OSM-9 require each other for routing to the sensory cilia. Consistent with the idea that OCR-2 and OSM-9 assemble to a heteromeric channel, mutant animals with either ocr-2 or osm-9 mutations exhibit as strong a phenotype as those of the double mutation. The essential function of OCR-2/OSM-9 channel in these sensory neuron functions provides a tractable system to investigate the channel molecular mechanisms in different cellular settings within an organism. A first distinction of OCR-2/OSM-9 signaling cascade in the serotonergic neurons is revealed from the study of the Gα protein ODR-3. ODR-3 is expressed in AWA, ASH, as well as in ADF, and is required for OCR-2/OSM-9 function in the AWA and ASH neurons. However, ODR-3 and two other Gα proteins that are expressed in the ADF do not have a detectable effect on OCR-2/OSM-9-dependent activation of tph-1 expression. Thus, OCR-

2/OSM-9 may induce specific outputs by coupling distinct signaling systems. Like OSM-9, OCR-2 is predicted to embody an architecture characteristic of the mammalian vanilliod receptor-related channels of the TRP superfamily - six transmembrane segments, a cytoplasmic N-terminus with three ankyrin motifs, and a cytoplasmic C- terminus. As discussed above, the ocr-2(yz5) allele was identified from a genetic screen for mutations that down regulate tph-lr.gfp expression in the ADF neurons and contains a single nucleotide change that results in a substitution of glutamate for glycine in the N-terminal region (G36E) (Fig. 7). Staining of anti-5HT antibody indicates that ADF 5HT immunoreactivity is nearly eliminated in øcr-2G36E mutants (Fig. 5a). To explore if this glycine underscores an essential element for the general function of OCR-2/OSM-9 channel or if it is a site of integration of the signaling cascade specifically regulating 5HT biosynthesis, we tested the effect of ocr-2G36E on AWA and ASH function. Similar to the ocr-2 deletion allele ocr-2(ak47), ocr-2G36E worms are defective in ASH-mediated avoidance to high osmotic solutions (Fig. 5b). By contrast, whereas ocr-2(ak47) mutants are defective in response to AWA-mediated attraction to the odorant diacetyl, ocr-2G36E mutants responded to diacetyl as well as wild-type animals (Fig. 5c). To determine if the OCR-2(G36E) substitution causes a subtle effect in the sensitivity to diacetyl, we assayed the response to diacetyl in a series of dilutions. There is no significant difference between wild- type and ocr-2G36E worms observed (Fig. 5d). Thus, the G36E substitution disrupts OCR- 2/OSM-9 channel signaling pathways in the ADF and ASH neurons but does not perturb the olfactory sensory transduction. This result indicates that the primary structure at the N- terminus of OCR-2 is a part of determinants of cellular specificity of the channel function. To ascertain the G36E substitution does not alter the expression pattern of OCR-2, we tagged FLAG^® epitope lo the C-lerminus of the wild-type OCR-2 and OCR-2 G36E), introduced the fusion constructs individually into ocr-2(ak47) mutants, and stained the transgenic lines with anti-FLAG antibody. Both OCR-2: :FLAG and OCR-2(G36E)::FLAG were detected in the plasma membrane and ciliated endings of these sensory neurons (Fig. 6). OCR-2: :FLAG but not OCR-2(G36E) transgene can restore ADF to express tph-1 in ocr- 2(ak47) background: 68% ocr-2(ak47) worms carrying OCR-2::FLAG showed strong tph- lrgfp in ADF (n=79), compared to 0% of that carrying OCR-2(G36E) (n = 150) and 0% of non-transgenic ocr-2(ak47) (n = 235). These data indicate that FLAG-tagged OCR-2 is localized to the cellular sites where OCR-2 acts and that G36E substitution does not prevent the subcellular translocation. One possible explanation for this result is that OCR-2 exerts a fundamental role in the assembly of transduction complexes by selectively binding to different signaling components; the yz5 G36E substitution might result in altered affinity for a common component in both ADF and ASH neurons. The immuno-fluorescent straining of OCR-2(G36E)::FLAG is slightly lower than that of OCR-2: :FLAG (Fig. 6). This could be a reflection of the mosaicism of the transgenic arrays. Alternatively it could be an indication of less-stability of OCR-2(G36E) resulted from the failure of binding to the partner or altered structure. Functional determinants in the N-terminal region of OCR-2. The cytoplasmic N- terminal region is crucial for activation of TRPV channels. All TRPV channels contain multiple ankyrin motifs adjacent to the transmembrane domain; changes in this region often eliminate the channel activity. For example, a splicing variant of rat TRPV1 that is lacking of most of the N-terminal region is expressed properly but does not respond to any identified stimulus, and point mutations in ankryin motifs of OSM-9 completely abolish the function. However, our yz5 G36E is located about 220 amino acids upstream from the first ankryin motif and affects a subset of OCR-2 function (Fig. 7). To understand the molecular mechanisms that specify OCR-2 functions, we explored the potential structure from the predicted translational start to upstream of the ankryin motifs of OCR-2. Structural alignments predict that the G36 residue is located in a coil region between two helices and is accessible to the surface, and G36E substitution shifts the start of the C-terminal helical fold and thus alters intra- and inter-molecular interactions in the region. The C elegans genome encodes 5 TRPV genes, ocr-1, ocr-2, ocr-3, ocr-4 and osm-9. The sequence of before the ankryin repeats of ocr-4 shows the least similarity to that of ocr-2 but the region corresponding to the ocr-2 (yz5) point mutation is highly conserved in ocr-4 (Fig. 7). ocr-4 is expressed in the OLQ neurons and its function has yet to be determined. If this region and this glycine is particularly involved in the channel function in the ADF and ASH neurons, ocr-4 might substitute these ocr-2 functions. To test this possibility, we expressed the ocr-4 coding sequence tagged with a FLAG epitope under the control of the ocr-2 promoter and introduced the construct into ocr-2 deletion mutants, ocr-2 deletion mutants expressing OCR-4: :FLAG in ocr-2-expressing cells show substantially increased tph-lr.gfp expression and improved odor and osmo sensory behavior. To test the importance of the glycine residue for the channel function, we introduced the corresponding G-to-E substitution (G33E) to ocr-4. This substitution completely eliminates the ability of ocr-4 to upregulate tph-1 expression in the ADF neurons. This result confirms that the G-to-E substitution causes the yzδ phenotype, and indicates that the glycine residue is likely to underscore a functional moiety conserved among the family members. ocr-4::flag transgene only partially rescues tph-lr.gfp expression in ocr-2 deletion mutants. To test if this lower activity is ascribed to the N-terminal region, we replaced the sequence from the translational start to the predicted first ankyrin motif of ocr-4 with the corresponding region of ocr-2. The OCR-2-OCR-4::FLAG chimera gene rescues tph-lr.gfp expression significantly better than OCR-4: :FLAG, indicating sequence elements within this region of OCR-2 are necessary for an optimal function of channel in the pathway that regulate 5HT biosynthesis. The sequence of this region is quite divergent between ocr-2 and ocr-4, making impossible to guess the critical residues. We constructed a series of small internal deletions within this region of ocr-2 based on the predicted structure. However, all these mini-deletions affect the function as well as the distribution pattern (data not shown). These results suggest that the structure of the N-terminal region of the TRPV channel proteins can determine the specificity as well as the activity of the channel. Although OCR-4: :FLAG can be detected in the plasma membrane and the ciliated endings in OCR-2-expressing cells, OCR-4: :FLAG often aggregate to punctuates on the plasma membrane and is frequently detected in the dendrites, whereas OCR-2: :FLAG exhibits a more uniform distribution on the membrane and is rarely detected in the dendrites, indicating that the interaction between OCR-4 and subcellular localization components are not optimal. Mammalian TRPV2 genes can upregulate tph-1 expression in the ADF neurons. 5HT signaling controls development, metabolism and endocrine activity in disparate phyla. As shown above, worms bearing a mutation in OCR-2/OSM-9 channel are more vulnerable to arrest as metabolically inactive Dauer larvae and this abnormal metabolic arrest is in part due to reduced insulin neuroendocrine signaling. TRPV2 is a mammalian member of TPRV subfamily of ion channels. TRPV2 is expressed in sensory ganglion as well as in most areas of the CNS and can be regulated by insulin growth factors. To explore an ortholog relationship between ocr-2 and TRPV2, we sought to test the ability of TRPV2 to regulate tph-1 expression in the ADF neurons. We expressed mouse and human TRPV cDNA tagged with a FLAG epitope under the control of the ocr-2 promoter in ocr-2 deletion mutants. Similar to OCR-2: :FLAG, mouse and human TRPV2::FLAG is distributed in the plasma membrane and ciliated endings (Fig. 8a). Whereas tph-1 ::gfp expression in the ADF neurons is nearly undetectable in ocr-2 mutants, ocr-2 mutants expressing either mouse or human TRPV2::FLAT exhibit substantially increased tph-1 expression in ADF (Fig. 8b). The GFP intensity in TRPV- 2::FLAG animals is not as strong as in ocr-2 ;OCR-2::FLAG animals: more than 99% of ocr- 2 deletion mutants show very weak nondetectable tph-lr.gfp expression in ADF, 59% (n=82) and 79% (n=95) of ocr-2 deletion mutants bearing respectively mouse and human TRPV2 show medium level of ADF GFP, but 42% (n=273) of ocr-2 deletion mutants bearing OCR- 2::FLAG showed strong and medium GFP, indicating mammalian TRPV2 and OCR-2 are not completely equivalent. Thus far, there is no evidence that another TRPV protein co-express with TRPV2, suggesting that TRPV2 can function as monomeric channel. We have tested this possibility by asking whether TRPV2 genes can restore ADF to express tph-lr.gfp in osm-9 and osm- 9;ocr-2 double mutants. 62% (n= 77) of osm-9 (yzό); mouse TRPV2 and 69% of osm-9(yzό); human TRPV2 animals showed medium level of tph-lr.gfp in the ADF neurons. Do mammalian TRPV2 function in all OCR-2/OSM-9 signaling cascades, or they only retain the function in 5HT signaling? We tested ability of TRPV2::FLAG to rescue the olfactory and osmotic sensation of ocr-2 deletion mutants. We failed to detect any improvement in the response of the transgenic animals to diacetyl or high osmolarity solutions (Fig. 8c,d). These results suggest that the mammalian TRPV2 cannot be integrated into these sensory signaling transduction pathways but can interact with the molecular machinery that controls tph-1 expression in the ADF neurons. Genetics and behavioral analysis of ocr-2/osm-9 mutants congruent with pharmacological and physiological study of mammalian TRPV channels indicating that TRPV channels can mediate the response to multiple sensory modalities, yet, specificity must preserve in vivo. Several lines of evidence suggest that one layer of the specificity is conferred by binding of ligands and intracellular signaling components to specific residues in the intracellular side of TRPV channels. The binding site of vanilloid has been mapped to a segment of TRPVl in the cytoplasmic leaflet between transmembrane segments 2 and 3; a mutation in this site causes TRPVl insensitive to vanilloids but the response to acid, heat retained. Activation of TRPV4 by heat and phorbol derivates requires a tyrosine residue at the predicted N-terminus of the transmembrane segment 3 and is independent of the regulation of the channel by osmotic strength. The results from this study reveal the cytoplasmic N-terminal region upstream of the ankryin repeats of TRPV channels as a part of determinants of cell-specific function of OCR- 2 channels in C. elegans: the yz5 G36E substitution completely disrupts the function of OCR- 2 in osmosensation mediated by ASH and the regulation of tph-1 expression in ADF, but leaves the diacetyl sensation mediated by AWA intact. All Molecular biology work was performed as described by Sambrook et al. Molecular cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, or using minor modifications of the methods described therein. All manipulations of C. elegans worms were performed using techniques described in Methods in Cell Biology, vol 84; Caenorhabditis elegans: modern biological analysis of an organism, ed. Epstein and Shakes, academic press, 1995, or using minor modifications of the methods described therein. Transgenic C. elegans strains were constructed by injection of plasmid DNA into worms using standard techniques known in the art (see Methods in Cell Biology, vol 84 as mentioned above). Although described with reference to a C. elegans model system, it will be appreciated by the skilled artisan that any animal model may be used having a TRPV- encoding gene homologous to those described for C. elegans, wherein a mutation in that gene results in reduced serotonin synthesis. While this invention is described in detail with reference to a certain preferred embodiments, it should be appreciated that the present invention is not limited to those precise embodiments. Rather, in view of the present disclosure which describes the current best mode for practicing the invention, many modifications and variations would present themselves to those of skill in the art without departing from the scope and spirit of this invention. In particular, it is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, constructs, and reagents described as such may vary, as will be appreciated by one of skill in the art.

Claims

WHAT IS CLAIMED IS: 1. A transgenic model animal useful for screening therapeutic agents for serotonin-related diseases, comprising a mutation in osm-9 and a stably integrated tphl: .-marker fusion gene, wherein the mutation results in reduced tphl: .-marker expression.

2. The animal of claim 1, wherein the mutation is osm-9(yzό).

3. The animal of claim 1, wherein the animal is C. elegans.

4. The animal of claim 3, wherein in the marker is a gene for green fluorescent protein (gfp).

5. A transgenic model animal useful for screening therapeutic agents for serotonin-related diseases, comprising a mutation in ocr-2 and a stably integrated tphl: .-marker fusion gene, wherein the mutation results in reduced tphl::marker expression.

6. The animal of claim 5, wherein the mutation is ocr-2(yz5).

7. The animal of claim 5, wherein the animal is C. elegans.

8. The animal of claim 7, wherein in the marker is gfp.

9. A method for screening therapeutic agents for serotonin-related diseases, comprising the steps of: providing a model animal as recited in claim 4; dispersing a target drag into agar; culturing the model animal on the agar with drug; and detecting expression of green fluorescent protein (GFP).

10. A method for screening therapeutic agents for serotonin-related diseases, comprising the steps of: providing a model animal as recited in claim 8; dispersing a target drug into agar; culturing the model animal on the agar with drug; and detecting expression of green fluorescent protein. (GFP)

11. A method of identifying compounds which enhance or up-regulate the synthesis of serotonin, which method comprises: contacting C. elegans which exhibit reduced serotonin synthesis compared to wild type C. elegans in one or more cell types or tissues with a compound under test; and detecting a phenotypic, biochemical or behavioral change in the C. elegans indicating a reversion towards wild type serotonin synthesis in the one or more cell types or tissues which exhibit reduced serotonin synthesis in the absence of the compound.

12. A method as claimed in claim 11 wherein the C. elegans contains a defective TRPV channel protein.

13. A method as claimed in claim 12 wherein the C. elegans contains a missence mutation in osm-9.

14. A method as claimed in claim 12 wherein the C. elegans contains a missence mutation in ocr-2.

15. A method as claimed in claim 12 wherein the C. elegans contains a nonsense mutation in osm-9.

16. A method as claimed in claim 12 wherein the C. elegans contains a nonsense mutation in ocr-2.

17. The method as claimed in claim 12 wherein the C. elegans exhibits reduced synthesis of serotonin specifically in ADF neurons.

18. A method as claimed in claim 11 wherein the C. elegans is a mutant C. elegans which exhibits reduced serotonin activity in one or more cell types or tissues.

19. A method as claimed in claim 18 wherein the C. elegans is a mutant C. elegans which exhibits reduced activity of TRPV channel protein in one or more cell types or tissues.

20. The method as claimed in claim 19 wherein the C. elegans exhibits reduced synthesis of serotonin specifically in ADF neurons.

21. A method as claimed in claim 11 wherein the C. elegans exhibit reduced serotonin synthesis in the ADF neurons, as compared to wild type C. elegans and the step of detecting a phenotypic, biochemical or behavioral change in the C. elegans indicating a reversion towards wild type serotonin synthesis comprises detecting a progression of the development of the C. elegans past the Dauer larval stage in the presence of the compound under test.

22. A method as claimed in claim 20 wherein the C. elegans further contain a transgene comprising a stably integrated tphl: .-marker fusion gene.

23. A method as claimed in claim 22 wherein the marker is a nucleic acid encoding a fluorescent protein.

24. A method as claimed in claim 23 wherein the step of detecting a phenotypic, biochemical or behavioral change in the C. elegans indicating a reversion towards wild type serotonin synthesis comprises comparing the level of fluorescence in the absence of the compound under test and the level of fluorescence in the presence of the compound under test.

25. A model animal useful for screening therapeutic agents for serotonin-related diseases, comprising a mutation in a gene encoding a TRPV channel protein and a stably integrated tphl: .-marker fusion gene, wherein the mutation results in reduced t bLvmarkβr expression.

26. The animal of claim 25, wherein the animal is C. elegans.

27. The animal of claim 25, wherein the gene encoding TRPY channel protein is osm9 or a homolog thereof.

28. The animal of claim 27, wherein the mutation is osm-9(yzό).

29. The animal of claim 25, wherein the gene encoding TRPV channel protein is ocr2 or a homolog thereof.

30. The animal of claim 29, wherein the mutation is ocr-2(yz5).

31. The animal of claim 25, wherein in the marker is a gene for green fluorescent protein (gfp).

32. A transgenic model animal useful for screening therapeutic agents for type II diabetes, comprising ocr-2(yz5), daf-7(el372) and a stably integrated tp/iEvmarker fusion gene, wherein the mutation results in reduced tphl: .-marker expression.

33. The animal of claim 32, wherein the animal is C. elegans.

34. A method for screening therapeutic agents for type II diabetes, comprising the steps of: providing a model C. elegans as recited in claim 33; dispersing a target drug into agar; culturing the C. elegans on the agar with the drug; and detecting a progression of the development of the C. elegans past the Dauer larval stage in the presence of the target drug.

35. A transgenic model animal useful for identifying compounds which enhance insulin signaling in serotonin deficient mutants, comprising ocr-2(yz5), daf-7(e!372).

36. The animal of claim 35, wherein the animal is C. elegans.

37. A method for identifying compounds which enhance insulin signaling in serotonin deficient mutants, comprising the steps of: providing a model C. elegans as recited in claim 36; mutagenizing the C. elegans with EMS; and detecting a progression of the development of the C. elegans past the Dauer larval stage.

38. An isolated nucleic acid comprising a sequence that encodes a polypeptide having the sequence of SEQ. ID NO. 1.

39. An isolated nucleic acid comprising a sequence that encodes a polypeptide having the sequence of SEQ. ID NO. 1 with conservative amino acid substitutions.

40. An isolated nucleic acid comprising a sequence that encodes a polypeptide comprising a fragment of SEQ ID NO. 1 at least 8 residues in length and containing amino acid 36.

41. A purified polypeptide, the amino acid sequence of which consists of SEQ ID NO. 1.

42. A purified polypeptide, the amino acid sequence of which comprises at least ten consecutive residues of SEQ ID NO. 1 and which includes amino acid 36.

43. A purified polypeptide, the amino acid sequence of which comprises residues 1 - 200 of SEQ ID NO. 1.