Regulated disruption of inositol 1,4,5-trisphosphate signaling in Caenorhabditis elegans reveals new functions in feeding and embryogenesis

Abstract

Inositol 1,4,5-trisphosphate (IP(3)) is an important second messenger in animal cells and is central to a wide range of cellular responses. The major intracellular activity of IP(3) is to regulate release of Ca(2+) from intracellular stores through IP(3) receptors (IP(3)Rs). We describe a system for the transient disruption of IP(3) signaling in the model organism Caenorhabditis elegans. The IP(3) binding domain of the C. elegans IP(3)R, ITR-1, was expressed from heat shock-induced promoters in live animals. This results in a dominant-negative effect caused by the overexpressed IP(3) binding domain acting as an IP(3) "sponge." Disruption of IP(3) signaling resulted in disrupted defecation, a phenotype predicted by previous genetic studies. This approach also identified two new IP(3)-mediated processes. First, the up-regulation of pharyngeal pumping in response to food is dependent on IP(3) signaling. RNA-mediated interference studies and analysis of itr-1 mutants show that this process is also IP(3)R dependent. Second, the tissue-specific expression of the dominant-negative construct enabled us to circumvent the sterility associated with loss of IP(3) signaling through the IP(3)R and thus determine that IP(3)-mediated signaling is required for multiple steps in embryogenesis, including cytokinesis and gastrulation.

Figure 1

IP₃ sponge constructs used in this study. Site-directed mutagenesis was used to abolish and to increase the affinity of IP₃ binding. (A) Schematic representation of an IP₃R subunit, showing domain organization. (B) In vitro binding to [³H]IP₃ of the following fusion proteins: GST-BD, GST fusion protein expressing the wild-type IP₃ binding domain; and GST-BD R511C, derivative of GST-BD with the mutation indicated (i.e., super-sponge). Binding was measured by the inhibition of specific [³H]IP₃ binding to purified fusion protein by various concentrations of IP₃. Specific binding was calculated by subtraction of nonspecific binding and normalized by expression as a percentage of maximal binding. (C) IP₃ binding to GST fusion proteins in vitro. GST-BD K579Q, R582Q, derivative of GST-BD with the mutations indicated (i.e., control-sponge). Error bars are SD.

Figure 2

Disruption of IP₃ signaling results in increased defecation cycle length and increased variation in cycle length. Defecation was observed on food at 20°C, ∼6 h (worms 1–6) or 18 h (worms 7–12) after onset of heat shock. Worms were recorded for 10 cycles. Squares are mean; bars are minimum-maximum, for individual worms. Mean ± SD is shown above each graph. ∗, worms failed to defecate in >10 min and are not included in the mean.

Figure 3

Disruption of IP₃ signaling or IP₃R expression results in slower and more erratic pharyngeal pumping. (A) Pharyngeal pumping in transgenic strains expressing IP₃ sponge constructs was observed on food or in the absence of food at 20°C, ∼6 or 18 h after heat shock [wild-type and dec-4 (sa73) animals were not heat shocked]. The effects of serotonin were assayed after 1–2 h on NGM, 7.5 mM 5-hydroxytryptamine in the absence of food. Mean calculated from five observations for 12 worms. Error bars are SD. (B) Pharyngeal pumping in RNAi-treated animals. Assays were carried out as described above; pumping on food was assayed after 1–2 h on OP50. Mean calculated from five observations for 10 worms.

Figure 4

Disruption of IP₃ signaling results in embryonic arrest. (A) Percentage of eggs laid that were arrested. (B–D) Early cell divisions generally proceed as normal. Typical embryo from an adult expressing the super-sponge. Arrow indicates an example of the large vesicle-like structures. (E) In some cases, defects are seen in cytokinesis. One-cell embryo from an adult expressing the super-sponge, showing failure of the pronuclei (arrows) to separate fully before formation of the cleavage furrow (c). (F) Typical embryo from a control-sponge transgenic adult. (G) Immunofluorescence labeling with anti-PGL-1 polyclonal antibody (green), and propidium iodide staining of DNA (red) of an embryo illustrating the most common arrest phenotype of embryos from adults expressing the super-sponge. The most commonly observed terminal phenotype (H and I) and another terminal phenotype of embryos from adults expressing the super-sponge (J and K). Nomarski images, except H and J, which are fluorescence images (showing rhabditin granules). ELT-2::GFP fluorescence (L) and corresponding transmission image of typical adult expressing the super-sponge (M). ELT-2::GFP fluorescence (N) and corresponding transmission image of typical adult expressing the control-sponge (O).