HPL-2 is a regulator of phospholipid metabolism. (a, b) Oil Red O representative images of wild type (WT) (a) and hpl-2(tm1489) (b) animals. Worms are F1 progeny of L4 hermaphrodites shifted to 25°C. Images were taken with a CoolSnap Color Camera with 700 ms exposure time. Scale bar = 50 μm. (c) Simplified overview of phosphatidyl choline (PC) metabolism in C. elegans. P-choline, phosphorylcholine; CDP-choline, cytidyldiphosphate choline; PC, phosphatidylcholine; PS, phosphatidylserine; PS DC, phosphatidylserine decarboxylase; PE, phosphatidylethanolamine; Ptd-MeEtn, phosphatidylmonomethylethylamine. Intermediates whose levels are altered in hpl-2 mutant worms compared to wild type are shown in red. (d) Phospholipid content analysis in wild-type and hpl-2 mutant worms. Bars represent the amount of each lipid species found as a percentage of the total phospholipid content. PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; SM, phingomyelin. Data are the average of two independent cultures. (e) Metabolism of sphingomyelin and ceramide. De novo sphingomyelin synthesis is mediated by sphingomyelin synthase (SMS), which transfers the phosphorylcholine moiety from phosphatidylcholine (PC) onto ceramide, forming sphingomyelin and diacylglycerol (DAG). In C. elegans there are three SMS genes, sms-1, -2, and -3. Ceramide can be generated either by the action of sphingomyelinase (SMase) or by de novo synthesis through the enzyme ceramide synthase (CerS, lagr-1, hyl-1 and hyl-2 in C. elegans). (f) hpl-2 regulates sms-3 mRNA levels. hpl-2/N2 ratios for sphingomyelin synthase sms genes at different developmental stages. Experiments were performed on three independent synchronized L3 cultures and on two adult and embryonic cultures. Reference genes are pmp-3 and cdc-42. Normalization was performed using the mean of both reference genes. Data are the mean of independent biological replicas.