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. 2016 Feb 19:6:19779.
doi: 10.1038/srep19779.

cGMP Signalling Mediates Water Sensation (Hydrosensation) and Hydrotaxis in Caenorhabditis elegans

Wei Wang  1 , Li-Wei Qin  1 , Tai-Hong Wu  1 , Chang-Li Ge  1 , Ya-Qian Wu  1 , Qiang Zhang  1 , Yan-Xue Song  1 , Yuan-Hua Chen  1 , Ming-Hai Ge  1 , Jing-Jing Wu  1 , Hui Liu  1 , Yao Xu  1 , Chun-Ming Su  1 , Lan-Lan Li  1 , Jing Tang  1 , Zhao-Yu Li  1 , Zheng-Xing Wu  1
Affiliations

cGMP Signalling Mediates Water Sensation (Hydrosensation) and Hydrotaxis in Caenorhabditis elegans

Wei Wang et al. Sci Rep. .

Abstract

Animals have developed the ability to sense the water content in their habitats, including hygrosensation (sensing humidity in the air) and hydrosensation (sensing the water content in other microenvironments), and they display preferences for specific water contents that influence their mating, reproduction and geographic distribution. We developed and employed four quantitative behavioural test paradigms to investigate the molecular and cellular mechanisms underlying sensing the water content in an agar substrate (hydrosensation) and hydrotaxis in Caenorhabditis elegans. By combining a reverse genetic screen with genetic manipulation, optogenetic neuronal manipulation and in vivo Ca(2+) imaging, we demonstrate that adult worms avoid the wetter areas of agar plates and hypo-osmotic water droplets. We found that the cGMP signalling pathway in ciliated sensory neurons is involved in hydrosensation and hydrotaxis in Caenorhabditis elegans.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. N2 worms exhibit hydropreference in multiple behavioural test paradigms.
(a) Scheme of the wedge-shaped agar (WSA) test. Figure not drawn to scale. (b) Water content of the WSA plates. The dashed red line indicates the exponential fitted curve of the water content. (c) Hydroaversive index (H.A.) of the N2 worms in the WSA assay. (d,e) Trajectories of the N2 animals on regular agar (d) and WSA plates (e). (f) Scheme of the quadrant test. Figure not drawn to scale. (g) N2 worm hydrotaxis in the quadrant agar test. (h) Scheme of the four-quadrant agarose assay with equal concentrations of Na+. Figure not drawn to scale. (i) N2 worm hydrotaxis in the quadrant agarose test. (j) The snapshot images show a worm’s aversive response to a water drop. (k) H.A. index of the N2 worms in the drop test in different conditions. WT, wild-type N2. The number of independent tests is indicated for each test on the bar.
Figure 2
Figure 2. cGMP – TAX-2/TAX-4, G-protein-coupled receptor (GPCR) and G-protein signalling is involved in hydrosensation.
(a) The normalised H.A. indexes of the daf-11(m47), tax-2(p671), tax-4(ks28) and genetically rescued worms in the WSA assay. (b) Avoidance ratio of daf-11(m47) in the water drop test. (c,d) Photo-activation of BlgC in daf-11-expressing neurons rescued the hydrotaxis defects in daf-11(m47) mutants in the drop test (c) and the WSA assay (d). (e) The normalised H.A. indexes of the tax-2(ks31) mutants treated with a temperature shift for 24 h at young adulthood after being raised at the permissive or restrictive temperature in the WSA assay. (f) The normalised H.A. indexes of the GPCR and G-protein mutants and worms rescued with full-length genomic DNA in the WSA assay. (g) The normalised H.A. indexes of the daf-11, tax-2, tax-4, grk-2, str-3, gpa-2, and gpa-3 mutants and genetically rescued worms in the four-quadrant agarose (6%-2%) assay with equal concentrations of Na+. The data for each genotype are normalised to the corresponding wild-type N2 control and are shown as only one bar. The number of independent tests for each genotype is indicated on the bar. Statistics: ###P ≤ 0.001 compared with each N2 control; ns, not significant; **P ≤ 0.01 and ***P ≤ 0.001 compared as indicated (Student’s t-test or Mann-Whitney Rank Sum test, depending on the normality of the data distribution). The error bars indicate the SEM. WT, wild-type; H.A., hydroaversive.
Figure 3
Figure 3. Identification of the hydrosensory receptor neurons.
(a) The normalised H.A. index of the osm-6(p811) mutants and neuron-specific rescued worms, as indicated by the WSA assay. (b) The normalised H.A. index of the osm-6(p811) mutants and worms rescued with the full-length osm-6 genomic DNA, as indicated by the four-quadrant agarose assay with equal concentrations of Na+. (c) The normalised H.A. indexes of the daf-11(m47) mutants and worms that were specifically rescued with the full-length daf-11 genomic DNA in the ASI, ASJ and ASK neurons, as tested by the WSA assay. (d) The normalised H.A. indexes of the tax-4(ks28) mutants and worms that were specifically rescued with the tax-4 cDNA in the ASI, ASJ, ASK, and AFD neurons, as tested by the WSA assay. (e) The artificial increase in the cytosolic cGMP levels by photo-activation of BlgC expressed specifically in the ASI, ASJ and ASK neurons remarkably rescued the hydropreference defect of the daf-11(m47) mutants, as assayed by the drop test. The data for each genotype are normalised to the corresponding wild-type N2 control, which is shown as only one bar. The number of independent tests for each genotype is indicated on the bar. Statistics: *P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001 compared to each N2 control (Student’s t-test or Mann-Whitney Rank Sum test, depending on the normality of the data distribution). The error bars indicate the SEM. WT, wild-type; H.A., hydroaversive.
Figure 4
Figure 4. The ASI, ASJ and ASK neurons sense switching between buffer, air and water.
(a–d) Cytosolic Ca2+ signalling in the soma in response to switches between the M13 buffer, air and ultra-pure water, as indicated in the ASJ (a) ASK (b) ASI (c) and AFD (d) neurons. Before and after air and water application, the worms were in the M13 buffer solution. The dark grey and blue shading indicate the challenge of air and water flow, respectively. A summation of the average fluorescence responses in the ASJ, ASK, ASI and AFD neurons is shown in each right-hand panel. The fluorescence signal within the initial 10 s was calculated as the average value for the response of the ASJ neurons in wild-type and rescued animals (a) to water stimulation. (e–h) Cytosolic Ca2+ signals in the soma in response to the switch between the M13 buffer and ultra-pure water in the ASJ (n = 8), ASK (n = 10), ASI (n = 10) and AFD (n = 16) neurons. The blue shading indicates the addition of water. The data are shown as the means ± SEM, as indicated by the solid traces in colours indicated and light grey, respectively. The full-length rescue indicates the transgenes expressing full-length daf-11 genomic DNA. The number of independent tests is indicated for each genotype on the bar. Statistics: ###P ≤ 0.001 compared to each N2 control, ***P ≤ 0.001 compared as indicated (Student’s t-test or Mann-Whitney Rank Sum test, depending on the normality of the data distribution). The error bars indicate the SEM. WT, wild-type.
Figure 5
Figure 5. Working model of hydrosensory information transduction.
Hypothesised model of the hydrosensation signalling pathway, i.e., a transmembrane hydrosensory information transduction pathway. The receptor-like guanylate cyclase DAF-11 may be activated directly or indirectly via G proteins through GPCR signalling, resulting in an increase in cytosolic cGMP levels that opens the CNG channels.
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