C. elegans has provided important insights into neuromuscular system function and development. However, the animal's small size limits access to individual neurons and muscle cells for physiological, biochemical, and molecular study. We describe here primary culture methods that allow C. elegans embryonic cells to differentiate into neurons and muscle cells in vitro. Morphological, electrophysiological, and GFP reporter studies demonstrate that the differentiation and functional properties of cultured cells are similar to those observed in vivo. Enriched populations of cells expressing specific GFP reporters can be generated by fluorescence-activated cell sorting. Addition of double-stranded RNA to the culture medium induces dramatic knockdown of targeted gene expression. Primary nematode cell culture provides a new foundation for a wide variety of experimental opportunities heretofore unavailable in the field.

No abstract

CLH-3b is a Caenorhabditis elegans ClC anion channel that is expressed in the worm oocyte. The channel is activated during oocyte meiotic maturation and in response to cell swelling by serine/threonine dephosphorylation events mediated by the type 1 phosphatases GLC-7α and GLC-7β. We have now identified a new member of the Ste20 kinase superfamily, GCK-3, that interacts with the CLH-3b COOH terminus via a specific binding motif. GCK-3 inhibits CLH-3b in a phosphorylation-dependent manner when the two proteins are coexpressed in HEK293 cells. clh-3 and gck-3 are expressed predominantly in the C. elegans oocyte and the fluid-secreting excretory cell. Knockdown of gck-3 expression constitutively activates CLH-3b in nonmaturing worm oocytes. We conclude that GCK-3 functions in cell cycle– and cell volume–regulated signaling pathways that control CLH-3b activity. GCK-3 inactivates CLH-3b by phosphorylating the channel and/or associated regulatory proteins. Our studies provide new insight into physiologically relevant signaling pathways that control ClC channel activity and suggest novel mechanisms for coupling cell volume changes to cell cycle events and for coordinately regulating ion channels and transporters that control cellular Cl− content, cell volume, and epithelial fluid secretion.

Cisplatin is commonly used for chemotherapy in a wide variety of tumors; however, its use is limited by kidney toxicity. Although the exact mechanism of cisplatin-induced nephrotoxicity is not understood, several studies showed that it is associated with DNA fragmentation induced by an unknown endonuclease. It was demonstrated previously that deoxyribonuclease I (DNase I) is a highly active renal endonuclease, and its silencing by antisense is cytoprotective against the in vitro hypoxia injury of kidney tubular epithelial cells. This study used recently developed DNase1 knockout (KO) mice to determine the role of this endonuclease in cisplatin-induced nephrotoxicity. The data showed that DNase I represents approximately 80% of the total endonuclease activity in the kidney and cultured primary renal tubular epithelial cells. In vitro, primary renal tubular epithelial cells isolated from KO animals were resistant to cisplatin (8 M) injury. DNase I KO mice were also markedly protected against the toxic injury induced by a single injection of cisplatin (20 mg/kg), by both functional (blood urea nitrogen and serum creatinine) and histologic criteria (tubular necrosis and in situ DNA fragmentation assessed by the terminal deoxynucleotidyl transferase nick end-labeling). These data provide direct evidence that DNase I is essential for kidney injury induced by cisplatin.

Defecation in the nematode Caenorhabditis elegans is a readily observable ultradian behavioral rhythm that occurs once every 45-50 s and is mediated in part by posterior body wall muscle contraction (pBoc). pBoc is not regulated by neural input but instead is likely controlled by rhythmic Ca 2 ϩ oscillations in the intestinal epithelium. We developed an isolated nematode intestine preparation that allows combined physiological, genetic, and molecular characterization of oscillatory Ca 2 ϩ signaling. Isolated intestines loaded with fluo-4 AM exhibit spontaneous rhythmic Ca 2 ϩ oscillations with a period of ‫ف‬ 50 s. Oscillations were only detected in the apical cell pole of the intestinal epithelium and occur as a posterior-to-anterior moving intercellular Ca 2 ϩ wave. Loss-of-function mutations in the inositol-1,4,5-trisphosphate (IP 3 ) receptor ITR-1 reduce pBoc and Ca 2 ϩ oscillation frequency and intercellular Ca 2 ϩ wave velocity. In contrast, gain-of-function mutations in the IP 3 binding and regulatory domains of ITR-1 have no effect on pBoc or Ca 2 ϩ oscillation frequency but dramatically increase the speed of the intercellular Ca 2 ϩ wave. Systemic RNA interference (RNAi) screening of the six C. elegans phospholipase C (PLC)-encoding genes demonstrated that pBoc and Ca 2 ϩ oscillations require the combined function of PLC-␥ and PLC-␤ homologues. Disruption of PLC-␥ and PLC-␤ activity by mutation or RNAi induced arrhythmia in pBoc and intestinal Ca 2 ϩ oscillations. The function of the two enzymes is additive. Epistasis analysis suggests that PLC-␥ functions primarily to generate IP 3 that controls ITR-1 activity. In contrast, IP 3 generated by PLC-␤ appears to play little or no direct role in ITR-1 regulation. PLC-␤ may function instead to control PIP 2 levels and/or G protein signaling events. Our findings provide new insights into intestinal cell Ca 2 ϩ signaling mechanisms and establish C. elegans as a powerful model system for defining the gene networks and molecular mechanisms that underlie the generation and regulation of Ca 2 ϩ oscillations and intercellular Ca 2 ϩ waves in nonexcitable cells.