Identification of guanylyl cyclases that function in thermosensory neurons of Caenorhabditis elegans

Abstract

The nematode Caenorhabditis elegans senses temperature primarily via the AFD thermosensory neurons in the head. The response to temperature can be observed as a behavior called thermotaxis on thermal gradients. It has been shown that a cyclic nucleotide-gated ion channel (CNG channel) plays a critical role in thermosensation in AFD. To further identify the thermosensory mechanisms in AFD, we attempted to identify components that function upstream of the CNG channel by a reverse genetic approach. Genetic and behavioral analyses showed that three members of a subfamily of gcy genes (gcy-8, gcy-18, and gcy-23) encoding guanylyl cyclases were essential for thermotaxis in C. elegans. Promoters of each gene drove reporter gene expression exclusively in the AFD neurons and, moreover, tagged proteins were localized to the sensory endings of AFD. Single mutants of each gcy gene showed almost normal thermotaxis. However, animals carrying double and triple mutations in these genes showed defective thermotaxis behavior. The abnormal phenotype of the gcy triple mutants was rescued by expression of any one of the three GCY proteins in the AFD neurons. These results suggest that three guanylyl cyclases function redundantly in the AFD neurons to mediate thermosensation by C. elegans.

Figure 1.

gcy-8, gcy-18, and gcy-23 form a subfamily of gcy genes and encode receptor-type guanylyl cyclases in C. elegans. (A) Phylogenetic tree of C. elegans gcy genes encoding receptor-type guanylyl cyclases. Sequences of C. elegans guanylyl cyclases (gene name or GenBank cosmid designation in italics) were compared using the ClustalW alignment program and the tree was constructed using TreeView. A subfamily consisting of gcy-8, gcy-18, and gcy-23 is circled. (B) Protein domains of receptor-type guanylyl cyclases. Receptor-type guanylyl cyclases appear to form a homodimer or a homotetramer in mammals. Receptor-type guanylyl cyclases consist of three domains: an extracellular domain, a transmembrane region, and an intracellular region containing a kinase homology domain, a hinge region, and a cyclase domain. (C) Multiple alignment of amino acid sequences of three C. elegans guanylyl cyclases (GCY-8, GCY-18, and GCY-23) and four human guanylyl cyclases (Hs_GC-A, Hs_GC-B, Hs_GC-C, and Hs_GC-F). Conserved cysteine residues in both C. elegans and humans (solid circle), in C. elegans (shaded circle), and in humans (open circle) are indicated. The kinase homology domain is boxed. Conserved serine and threonine residues are indicated by an asterisk (*). The transmembrane region and cyclase domains are indicated by an open bar and a solid bar, respectively. DDBJ/EMBL/GenBank and Swiss-Prot accession numbers for sequences used in the alignment are AB201388, (GCY-8), AB201389 (GCY-18), AB201390 (GCY-23), P16066 (Hs_GC-A), P20594 (Hs_GC-B), P25092 (Hs_GC-C), and P51841 (Hs_GC-F).

Figure 2.

Gene structures of gcy-8, gcy-18, and gcy-23 genes and schematics of promoter fusions or full-length genomic fusions to reporter genes. Exons are boxed and numbered. Solid bar indicates a deleted region.

Figure 3.

Expression analysis of gcy-8, gcy-18, and gcy-23 genes. (A–C) GFP fluorescence images of gcy-8p∷GFP (A), gcy-18p∷GFP (B), and gcy-23p∷GFP (C) transgenes. (D–F) DsRed fluorescence images of gcy-8p∷DsRed transgene as an AFD specific marker. (G–I) Merged images of top and middle. (J–L) Intracellular localization of products of genomic gcy-8∷GFP fusion (J), genomic gcy-18∷GFP fusion (K), and genomic gcy-23∷GFP fusion (L).

Figure 4.

Thermotaxis phenotypes of gcy single and double mutants cultivated at three different temperatures. (A–C) Thermotaxis phenotypes of wild-type animals and gcy single mutants cultivated at 15° (A), 20° (B), or 25° (C). (D–F) Thermotaxis phenotypes of wild-type animals and gcy double mutants cultivated at 15° (D), 20° (E), or 25° (F). For each genotype, 59–199 animals were assayed individually. Phenotypic categories are described in materials and methods.

Figure 5.

Behavioral phenotypes of gcy triple mutants and tax-4; gcy-23 gcy-8 gcy-18 quadruple mutants. (A) Thermotaxis phenotypes of wild-type animals and gcy triple mutants cultivated at different temperatures. Wild-type animals (n = 90) and gcy triple mutants (n = 357–359) were assayed individually. Phenotypic categories are described in materials and methods. (B) Thermotaxis phenotypes of wild-type animals, gcy triple mutants, tax-4 mutants, and tax-4; gcy-23 gcy-8 gcy-18 quadruple mutants cultivated at 20°. For each genotype, 40–177 animals were assayed individually. Phenotypic categories are described in materials and methods. (C) Chemotaxis phenotypes of wild-type animals and gcy triple mutants toward four volatile odorants: diacetyl (dia) and pyrazine (pyr) sensed by the AWA olfactory neurons and isoamyl alcohol (iaa) and benzaldehyde (benz) sensed by the AWC olfactory neurons. Each data point represents the average of 8–24 assays. The error bar indicates standard deviation. (D) Chemotaxis phenotypes of wild-type animals and gcy triple mutants toward NaCl. Wild-type animals (n = 50) and gcy triple mutants (n = 100) were assayed individually. gcy triple mutants showed no difference from wild type in a chi-square test.

Figure 6.

TTX plot of wild-type animals and gcy mutants cultivated at three different temperatures. Wild-type animals and gcy mutants cultivated at 15° (A), 20° (B), or 25° (C) were assayed and the TTX plot values were calculated as described in materials and methods . Horizontal axis indicates a value of (20 − 17/25). Vertical axis indicates a value of (25 − 17).

Figure 7.

Rescue experiments using full-length gcy∷GFP fusions. Animals were cultivated at 20°. Transgenic animals carrying full-length gcy∷GFP fusions, Ex[gcy-8∷GFP], Ex[gcy-18∷GFP], or Ex[gcy-23∷GFP], also carry the ges-1∷GFP transgene. For each genotype, 59 ∼ 359 animals were assayed individually. Phenotypic categories are described in materials and methods.

Figure 8.

Effect of application of 8-bromo-cGMP on thermotaxis behaviors of wild-type animals and gcy triple mutants. Animals were cultivated at 20°. For each condition, 19 or 20 animals were assayed individually. Phenotypic categories are described in materials and methods.

Figure 9.

A molecular model thermosensory signal transduction in AFD. Temperature sensed by a thermoreceptor leads to changes in intracellular cGMP concentration via the function of three guanylyl cyclases, GCY-8, GCY-18, and GCY-23. cGMP regulates activity of the TAX-4 CNG channel, resulting in changes in AFD membrane potential.