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First published online 3 March 2004
doi: 10.1242/dev.01047

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Caenorhabditis elegans TRPV ion channel regulates 5HT biosynthesis in chemosensory neurons

Department of Anatomy and Neurobiology, College of Medicine, University of California Irvine, Irvine, CA 92697-4040, USA

View larger version (79K): [in a new window]   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-1::gfp 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 yz6 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-1::gfp expression in various genetic backgrounds is presented in Table 1. (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).

View larger version (74K): [in a new window]   Fig. 2. Mutations in the TRPV 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, yz6 and yz5 mutant animals. The expression patterns in wild-type animals have been published: lin-11:gfp (Hobert et al., 1998), and cat-1::gfp and gtpch-1::gfp (Sze et al., 2002). Representative examples of the reporter expression patterns in mutant animals are shown. (B) The expression of lin-11::gfp overlaps with osm-9 and ocr-2 in the ADF and ADL chemosensory neurons. Shown is a yz6 mutant animal strongly expressing lin-11::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-1::gfp expression in the ADF neurons (Fig. 1B, Table 1), cat-1::gfp can be clearly observed in the ADF neurons of yz5 and yz6 mutants. We have repeatedly observed that cat-1::gfp expression in the ADF neurons is relatively weaker in yz5 and yz6 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 yz6 animal expressing gtpch-1::gfp in ADF and in other serotonergic neurons.

View larger version (41K): [in a new window]   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 wild-type animals, GFP is strongly expressed in the ADF neurons, but not in any other serotonergic neuron, indicating that this tph-1 cis-regulatory region specifically mediates tph-1 expression in the ADF neurons. The ADF expression of this GFP reporter is significantly reduced in yz6 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-1::gfp expression is unaffected by G 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.).

View larger version (23K): [in a new window]   Fig. 4. Effects of the TRPV 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-1(yz12), enhance Dauer arrest of the daf-7(e1372) mutation at 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-11::osm-9(+) transgene was carried as an extrachromosomal array, only the animals carrying the transgenic marker were scored. The osm-9(yz6) (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.