is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. to many cell and developmental defects4-7. Stringent regulation of ERK activity is critical for normal development: in mammalian systems too much ERK activity leads to excessive cellular proliferation leading to oncogenic growth; too little VPS33B activity leads to cell death4,6. Additionally, changes in the duration of ERK activity can also lead to distinct outcomes: in PC12 cells, ERK activation for 30 min or less induces cell proliferation, but ERK activation for 60 min or more induces neuronal differentiation8,9. Tight regulation of ERK activity is thus clearly essential for normal development and homeostasis. is a powerful, and genetically malleable model system Epipregnanolone to dissect the function and regulation of the RTK-RAS-ERK signaling pathway3,10-15. Relative to mammalian systems, which contain multiple genes for RAS and ERK, contains one RAS gene (germline is essentially a tube that consists of mitotic stem cells at its distal end and mature oocytes at its proximal end (Figure 1)16. Germ cells initiate meiosis just proximal to the distal mitotic zone, and progress through an extended meiotic prophase (pachytene), after which they begin to form oocytes in the loop region, finally undergoing oocyte maturation in the proximal region16. Genetic studies from multiple labs, including our own, have shown that the RTK-RAS-ERK pathway is essential for germline development in germline results in production of multiple small oocytes, while too little activity results in one large oocyte11. Thus tight regulation of dpERK is essential for normal germline development. The active form of ERK, as visualized by an antibody specific to dpERK, displays a stereotypical, dynamic, bimodal localization pattern: dpERK is high during mid-pachytene (Zone 1, Figure 1), low in the loop region and Epipregnanolone high again in mature oocytes (Zone Epipregnanolone 2, Figure 1). Recently, we found that nutrition acts through the Insulin-like Growth Factor receptor-1 (the Ephrin receptor tyrosine kinase) activates ERK in Zone 213. Given that active ERK functions as a rheostat in the germline to regulate oocyte growth,?spatial and temporal localization, as Epipregnanolone well as amplitude of dpERK, is key to understanding its normal signaling outcome. Using the method described here, changes in the stereotypical localization of dpERK can be easily monitored and predictions derived on the impact of the environmental or genetic perturbations on ERK activity and thus function. Thus, assaying for dpERK enables a comprehensive understanding of its role during germline development. Protocol The protocol described here is primarily for the invertebrate model system working stock cultures on Nematode Growth Medium20,21 (NGM, see Materials Table) agar seeded with OP50. Maintain worm stocks at temperatures between 16 C and 25 C. NOTE: Culturing worms at higher temperatures results in faster growth rate, often, aberrant germline development and lower brood sizes. Additionally, temperature sensitive genotypes will display different phenotypes at different temperatures. Transfer worms to new NGM plates every 2 – 3 days Epipregnanolone depending on their growth rate so as to maintain a constant supply of well-fed worms. NOTE: For any given experiment involving dpERK levels, worms should NOT be obtained from a starved or crowded plate. Crowding or starvation impacts insulin signaling in worms22, and insulin signaling regulates ERK12 activity. Thus worms from starved or overcrowded plates will result in variable and difficult-to-reproduce staining patterns. 2. Dissection of Adult Worms for Obtaining Gonads Pick 100 – 150 WT (N2) or desired genotype of worms at the desired and specific developmental stage (L1, L2, L3, L4, adult, female germlines (at 8 h?past mid-L4 stage of development) stained for DNA (E, DAPI) and dpERK (F). Young female germlines display weak dpERK in Zone 1, and in a single oocyte in a sperm independent manner. Zone 2 is sperm dependent, and thus absent in the female germline. Scale bar: 20 m. Please click here to view a larger version of this figure. Discussion Active ERK (dpERK) follows a stereotypical spatial and dynamic localization pattern in the germline. This stereotyped dpERK spatial pattern (Figure 5), and amplitude in an adult gonad, can be effectively correlated with the many biological processes that ERK regulates. For example, an inability to activate ERK in Zone 2 results in an arrest in oocytes in prophase of meiosis, a phenotype observed in female germlines that do not specify sperm, and thus do not activate ERK in.