We present an immunofluorescence imaging-based method for spatial and temporal localization of active ERK in the dissected C. elegans gonad. The protocol described here can be adapted for visualization of any signaling or structural protein in the C. elegans gonad, provided a suitable antibody reagent is available.
The evolutionarily conserved extracellular signal transducing RTK-RAS-ERK pathway is an important kinase-signaling cascade that controls multiple cellular and developmental processes principally via activation of ERK, the terminal kinase of the pathway. Tight regulation of ERK activity is essential for normal development and homeostasis; overly active ERK results in excessive cellular proliferation, while underactive ERK causes cell death. C. elegans is a powerful model system that has helped characterize the function and regulation of RTK-RAS-ERK signaling pathway during development. In particular, the RTK-RAS-ERK pathway is essential for C. elegans germline development, which is the focus of this method. Using antibodies specific to the active, diphosphorylated form of ERK (dpERK), the stereotypical localization pattern can be visualized within the germline. Because this pattern is both spatially and temporally controlled, the ability to reproducibly assay dpERK is useful to identify regulators of the pathway that affect dpERK signal duration and amplitude and thus germline development. Here we demonstrate how to successfully dissect, stain, and image dpERK within the C. elegans gonad. This method can be adapted for spatial localization of any signaling or structural protein in the C. elegans gonad, provided an antibody compatible with immunofluorescence is available.
The Receptor Tyrosine Kinase (RTK)-RAt Sarcoma (RAS)-Extracellular signal Regulated Kinase (ERK) pathway relays extracellular signals through a conserved kinase cascade that results in the phosphorylation and activation of ERK1-3. ERK proteins are members of the conserved proline-directed serine/threonine MAP (Mitogen Activated Protein) kinase family, and are directly activated by MEK via dual phosphorylation on the threonine (T) and tyrosine (Y) of the conserved TEY motif. Active ERK (referred to as diphosphorylated ERK, or dpERK) then regulates many cellular and developmental processes through its phosphorylation of a battery of downstream substrates1-3. Thus abnormal ERK activity leads 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 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.
C. elegans is a powerful, and genetically malleable model system 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, C. elegans contains one RAS gene (let-60) and one ERK gene (mpk–1), rendering it a genetically more facile system in which to dissect the function of this pathway3,10-14. The C. elegans 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 C. elegans12,13,15,17-19. Specifically, we found that ERK controls and coordinates at least nine distinct biological processes during germline development, ranging from developmental switches such as germ cell apoptosis to cell biological processes such as oocyte growth11,18. Much like in mammalian systems, too much ERK activity in the C. elegans 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 high again in mature oocytes (Zone 2, Figure 1). Recently, we found that nutrition acts through the Insulin-like Growth Factor receptor-1 (daf-2) to activate ERK in Zone 112; prior work showed that the sperm signal (via 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 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.
Active ERK (dpERK) follows a stereotypical spatial and dynamic localization pattern in the C. elegans germline. This stereotyped dpERK spatial pattern (Figure 5), and amplitude in an adult C. elegans 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 mature oocytes (Figure 5). Mating with males' results in effective accumulation of dpERK in Zone 2 in the female germlines11,13, coupled with onset of oocyte maturation and ovulation. Thus analysis of spatial patterns and temporal activation of dpERK upon certain genetic perturbations (such as RNA interference mediated gene inactivation) or chemical treatments allows for identification of factors that regulate ERK signaling and thus ERK-dependent biological processes. Such analyses also enable discovery of novel genetic interactions between signaling pathways.
A critical bottleneck for any imaging based analysis is the availability of functional reagents such as antibodies that are specific to the application. If such a reagent exists then methods such as the one described above can be easily adapted for the analysis of localization pattern for any protein of interest. The application is thus limited by the availability of a functioning antibody. Additionally, because the method describes dissection and staining at a given developmental time, it only enables the capturing of information in one time frame, and does not shed light on the dynamics or protein regulation in real time or rate of protein turnover.
The method described above is adapted from Francis et al.23, which is distinct from the Seydoux and Dunn method24 for gonad extrusion and antibody staining. The method based on Francis et al. will be called the "suspension method" and the Seydoux and Dunn method called the "freeze cracking method" for comparison. The suspension method has been very effective in our hands for obtaining reproducible localization patterns of signaling molecules such as dpERK, that are inherently more sensitive to harsh handling conditions. In the case of dpERK localization, in our experience, the signal in Zone 1 is virtually undetectable with the freeze cracking method. Additionally, sample drying, which can often occur with the freeze cracking method, results in spurious antibody signals. The suspension method minimizes sample drying, since the gonads are suspended in liquid throughout the process. We also find that the suspension method is useful for imaging multiple different genotypes on a single slide. For example, if two genotypes, such as hermaphrodites vs females are to be directly compared for dpERK localization, the two genotypes can be mixed together and processed. This is possible because the phenotypes are distinct and can be distinguished via DAPI morphologies. Staining in the same tube followed by imaging on the same slide allows for direct comparison between the gonads from different genotypes. Alternatively, if the DAPI morphologies are not distinguishable, then a two-step antibody staining can be adopted. First each individual genotype is treated with two distinct antibodies, Antibody A for WT and Antibody B for mutant (both antibodies need to be raised in a host distinct from the dpERK antibody). Once the two genotypes have been stained with the primary antibody, and washed, they can be mixed together in one tube and now stained with the dpERK antibody, followed by a secondary antibody treatment to visualize all the antibodies in the tube. An ability to compare different genotypes on a single slide, especially when deductions of activation patterns are being made ensures accurate interpretations not affected by distinct staining conditions.
While advantageous, there are multiple steps throughout the suspension method that require careful manipulation. It is critical that the dissections occur in a time efficient manner; importantly, the dissections should not take over 5 min. Longer dissection times result in variable dpERK signal, which can often be misinterpreted as regulated changes in ERK activation, rather than as technical failures. It is crucial to start with over 100 – 150 animals for each dissection because throughout the various wash steps the gonads can easily be lost from the tubes due to pipetting errors. Loss of gonads can be reduced either by dissecting in multiple small batches (such as three batches of 50 animals each) that can be combined, or by dissecting one large batch of 150. Another challenge is getting the gonads to lie in a well-spaced formation on the slide so that the entire distal gonad can be imaged. To enable this, use an eyelash on a matchstick or a pulled pipette to space out the gonads. However, care should be taken so as not to damage the gonads during this process. Often with these techniques it takes multiple attempts to master the dissections as well as arrangements of the gonads onto the slides.
Once this technique has been mastered, it serves as a powerful method for varied biological analyses, and can be used to study more than just dpERK levels. For example this method can be used to visualize active p38 levels, or active CDK levels, or any protein for which an antibody reagent exists. Additionally, the method can be coupled with a chemical inhibition screen in whole animals to assay for novel inhibitors or activators of the pathway, via assaying for dpERK levels. This will allow for an in vivo readout of both reaction and sensitivity to any inhibitors that may interact with the RTK-RAS-ERK signaling pathway. Moreover, this method can be used in antibody combinations with multiple signaling outputs that can all be used and visualized at one time. Using multiple antibodies allows correlation between multiple pathways, their activation status, and outcomes all within the same tissue, enabling better understanding of these interactions. These are just a few examples of further applications of this method, but this system can be modified to study multiple research interests.
The authors have nothing to disclose.
Work in the Arur Lab is supported by grants from the National Institutes of Health, NIHGM98200; Cancer Prevention Research Institute of Texas, CPRIT RP160023; the American Cancer Society Research Scholar Award (ACS RSG014-044-DDC); and by the Anna Fuller Funds.
Agarose | Sigma Inc. | A9539 | Dissolve 2 g in 100 ml to make the 2% agarose |
Flat bottom glass watch dish | Agar Scientific | AGL4161 | We use glass, because dissected gonads often stick to plastic |
25 gauge needles | BD PrecisionGlide | 305122 | |
Syringes (could be 1 or 5 ml) | BD Syringes | 1 ml: BD 309659 | |
Microscope slides (25mm x 75mm x 1.0mm) | Fisherbrand | 12-550-343 | |
Coverslips (24x50mm) | Corning | 2935-245 | |
5ml glass conical tube | Corning | 8060-5 | |
9" disposable Pasteur pipette | Fisherbrand | 13-678-20D | |
Clinical bench top centrifuge | |||
Glass tubes (6x50mm) | Fisherbrand | 14-958-A | |
*M9 solution | |||
Levamisole | Sigma | L9756 | |
3% Paraformaldehyde | Electron microscopy services | 17500 | Obtained as 16% solutions in ampoules, and diluted to 3% in Potassium Phosphate Buffer |
**PBS | |||
1X PBST | 1X PBS with 0.1% Tween 20 | ||
Methanol | Electron microscopy services | 18510 | |
Normal goat serum (NGS) | Cell Signaling | 5425 | Diluted to 30% in 1x PBST |
MAPKYT (dpERK) antibody | Sigma Inc. | M9692 | Dilution 1:400 in 30% NGS |
Secondary antibody | Invitrogen | A-11005 | Dilution 1:500 in 30% NGS |
DAPI | Sigma | D9542 | |
Vectashield | Vector Labs | H-1000 | |
*M9 Buffer Recipe | 3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 litre. | ||
**PBS (1X) | 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, 0.24 g KH2PO4, 0.133 g CaCl2.2H2O, 0.10 g MgCl2.6H2O, H2O to 1 litre | ||
Nematode Growth Medium (NGM) | 3 g NaCl, 17 g Agar, 2.5 g Peptone, 975 mL of water in 2 L Erlenmayer Flask. Autoclave for 50 minutes. Cool, and add 1 mL of 1M CaCl2, 1 mL of 5 mg/mL cholesterol, 1 mL of 1M MgSO4 and 25 mL of 1M KPO4. |