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 2ϩ 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. Inositol 1,4, ) is an important and widely used second messenger in animal cells. IP 3 is produced by the hydrolysis of phosphatidylinositol 4,5-bisphosphate after the activation of phospholipase C (PLC). The best understood routes of PLC activation are through the stimulation of G protein-coupled receptors or tyrosine kinase-linked receptors at the cell surface, although the presence of additional isoforms of PLC suggests other possible routes (Rhee and Bae, 1997;Katan, 1998). The only known action of IP 3 is to induce Ca 2ϩ release from intracellular stores through activation of IP 3 receptors (IP 3 Rs) (Berridge, 1993). This pathway and the Ca 2ϩ signals derived from it are central to a wide range of cellular responses (Berridge, 1993(Berridge, , 1997Clapham 1995). INTRODUCTIONTwo challenges in the study of this signaling network are to unravel the different functions played by IP 3 signaling in animals and to develop methods for manipulating these processes and thus advance our understanding of how the specificity of IP 3 signaling is achieved. Recent genetic approaches have provided new insights into IP 3 function in animals. The central role played by IP 3 Rs in a wide range of cellular responses is reflected by the high level of lethality resulting from gene knockout observed in both Drosophila (Acharya et al., 1997;Venkatesh and Hasan, 1997) and mice (Matsumoto et al., 1996). Nevertheless, careful analysis has enabled, for example, the functions of IP 3 Rs in cerebellum in mice (Inoue et al., 1998) and phototransduction in Drosophila to be studied (Acharya et al., 1997;Raghu et al., 2000). Caenorhabditis elegans is also proving to be a powerful system for understanding IP 3 signaling function. IP 3 Rs i...
Complex behavior requires the coordinated action of the nervous system and nonneuronal targets. Male mating in Caenorhabditis elegans consists of a series of defined behavioral steps that lead to the physiological outcomes required for successful impregnation. We demonstrate that signaling mediated by inositol 1,4,5-trisphosphate (IP 3 ) is required at several points during mating. Disruption of IP 3 receptor (itr-1) function results in dramatic loss of male fertility, due to defects in turning behavior (during vulva location), spicule insertion and sperm transfer. To elucidate the signaling pathways responsible, we knocked down the six C. elegans genes encoding phospholipase C (PLC) family members. egl-8, which encodes PLC-, functions in spicule insertion and sperm transfer. itr-1 and egl-8 are widely expressed in the male reproductive system. An itr-1 gain-of-function mutation rescues infertility caused by egl-8 RNA interference, indicating that egl-8 and itr-1 function together as central components of the signaling events controlling sperm transfer.
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