Published: August 6th, 2016
This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila.
Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.
A change in intracellular Ca2+ concentration at egg (oocyte) activation is a conserved component of all studied organisms 1,2. This event initiates a wide range of Ca2+-dependent processes, including resumption of the cell cycle and translation of stored mRNAs. Due to this requirement for Ca2+, visualizing the transient changes in intracellular Ca2+ concentrations has been of key interest.
Historically, model organisms were selected for studies on egg activation based on the size and availability of their eggs. Various visualization approaches have been utilized to follow and quantify changes in intracellular Ca2+ concentrations in these systems, including: the photoprotein aequorin in medaka fish 3; Ca2+ sensitive fluorescent dyes such as Fura-2 in sea urchin and hamster 4,5; and calcium green-1-dextran in Xenopus 6.
The generation and improvement of genetically encoded calcium indicators (GECIs) has transformed the ability to visualize Ca2+ dynamics in vivo 7. These genetic constructs are expressed in specific tissues and limit the need for invasive tissue preparations 8.
GCaMPs are a green fluorescent protein (GFP)-based class of GECIs that have been very effective due to their high Ca2+ affinity, signal-to-noise ratio and capacity to be customized 9-11. In the presence of Ca2+, the GCaMP complex undergoes a series of conformational changes, starting with the binding of Ca2+ to calmodulin, that results in an increased fluorescent intensity of the GFP component 9.
GCaMPs have been extensively used in research on Drosophila neurons to visualize changes in intracellular Calcium12. The recent application of GCaMP technology to visualize Ca2+ in mature Drosophila eggs has revealed a single transient Ca2+ wave at egg activation 13,14. The Ca2+ wave can be visualized at low magnification during ovulation in vivo 13 or at higher magnification using an ex vivo activation assay 13,14. In the ex vivo assay, individual mature oocytes are isolated from the ovaries and activated using a hypotonic solution, referred to as activation buffer, which has been shown to recapitulate the events of in vivo activation 15-17.
This ex vivo assay enables easy high resolution visualization of the Ca2+ wave under different experimental conditions including pharmacological disruption, physical manipulations and genetic mutants. This article demonstrates the preparation of mature Drosophila eggs for ex vivo activation and the subsequent microscopy used to visualize the Ca2+ wave using GCaMP. This approach can be used to test the initiation and control of the Ca2+ wave and to probe downstream outcomes.
Note: Carry out all steps at room temperature unless stated otherwise.
1. Preparing for Dissection
Note: The steps described here are in accordance with E. Gavis, Princeton University & Weil et. al. 18. Perform the following steps two days before imaging.
2. Dissecting Drosophila Ovaries
Note: The steps described here are in accordance with Weil et. al. 18.
3. Isolating Individual Mature Eggs
4. Preparing for Imaging
5. Imaging Ex Vivo Egg Activation
6. Post-acquisition Image Processing
Here we have demonstrated how to prepare mature Drosophila eggs for ex vivo activation. Eggs expressing a GECI enable imaging of Ca2+ dynamics at egg activation and the beginning of embryonic development (Figure 1). It should be noted that depending on the GCaMP used, specifically the presence of a myristoyl group, results may have slight qualitative differences13 14. We have also demonstrated a role for functional actin during egg activation by the addition of an inhibitor of actin polymerization, cytochalasin D, to the activation buffer (Figure 2) 14.
A requirement for the cytoskeleton at egg activation is conserved. In C. elegans, following fertilization, cytoskeletal components are reorganized to prepare the one-cell embryo for the first asymmetrical division 21,22. In sea urchin, disruption of cytoskeletal re-organization following activation has been shown to affect the cell cycle by preventing contractile ring formation 23. The role of actin in mediating a Ca2+ wave and its downstream function at egg activation in Drosophila remains to be fully elucidated.
In Drosophila, co-visualization of the Ca2+ wave and actin cytoskeleton can be achieved using this ex vivo activation assay. Time series of multiple channels can be acquired and dynamics of the intracellular Ca2+ can be matched with changes in actin organization in the oocyte (Figure 3).
Figure 1: A Single Ca2+ Wave at Drosophila Egg Activation. Time series of ex vivo mature Drosophila egg expressing UASt-myrGCaMP5 following the addition of activation buffer (A-H). The rise in cytoplasmic Ca2+ originates at the posterior pole (B) and propagates (B-F) with an average velocity of approximately 1.5 µm/sec. Initiation of the wave is typically observed within 3 min of the addition of activation buffer. Following activation, the intracellular calcium levels of the mature egg returns to pre-activation levels (G,H). See supplemental movie 1 (total time 33 min). Scale bars = 50 µm. Max projection = 40 µm. Please click here to view a larger version of this figure.
Figure 2: Addition of Cytochalasin D to Activation Buffer Perturbs Ca2+ Wave Propagation. Time series of ex vivo mature Drosophila egg expressing UASt-myrGCaMP5 following addition of activation buffer containing 10 µg/ml cytochalasin D final concentration (A-H). Whilst the Ca2+ wave is initiated at the posterior pole (A), it is compromised and does not reach the anterior of the egg (F) (white arrowheads denote the front of the wave). No further Ca2+ changes were observed over 30 min). See supplemental movie 2 (total time 30 min). Scale bars = 50 µm. Max projection = 40 µm. Please click here to view a larger version of this figure.
Figure 3: Co-visualization of Actin and Ca2+ at Egg Activation in Drosophila. Time series of ex vivo mature Drosophila egg expressing UASt-myrGCaMP5 (Cyan) and UASp-F-Tractin.tdTomato (Magenta) following addition of activation buffer (A-H). The Ca2+ wave initiates from the posterior pole and recovers as in Figure 1. Actin appears to be changing over time, white arrowheads (A vs H) . The lack of Ca2+ signal detection in the center of the mature egg is due to movement of the sample during image capture. Scale bars = 50 µm. Max projection = 40 µm. Please click here to view a larger version of this figure.
The first critical step in this protocol is isolating the mature eggs without damage. This can be achieved by gentile maneuvering of the eggs with the dissecting probe. Practice will enable this manipulation to be executed without damage to the eggs. A second crucial step is avoiding the loss of the sample when activation buffer is applied to the mature egg in oil. Application of the activation buffer should be done slowly and without contact with the coverslip. This step can be challenging if the microscope set-up does not allow for an easy access to the sample. Moving axillary parts of the microscope, such as a temperature incubator, is advisable.
Modifications to this protocol can be made in order to visualize other fluorescently tagged factors, instead of Ca2+ at egg activation. More generally, different stages of Drosophila development or the results of adding a different buffer could be analyzed using this protocol.
Troubleshooting may be required if the settings on the microscope are not optimal for visualizing the signal from the sample. This can be achieved by increasing or decreasing the laser power, altering capture range and adjusting the Z-stack parameters. Another issue might be eggs consistently moving out of the field of view upon the addition of activation buffer. If this happens regularly, allow the mature eggs to settle on the coverslips for a longer period of time, 15 to 20 minutes. Beware that using a higher viscosity oil will alter the displacement of the oil by activation buffer.
This technique presented here is limited by the working distance of the objective, dehydration of the mature eggs and the ability to dissect the mature eggs without damage. However, when compared to in vivo imaging methods where the mature oocyte passes through the adult female and is deposited 13, our method enables more spatial resolution, the option to physically manipulate the mature egg before activation and the ability to test the role of egg activation without fertilization.
There are many potential future applications of this technique, including testing reporter constructs for cellular components at egg activation and visualizing the Ca2+ wave in mutant backgrounds 14,24. Together with experimental and genetic tools, the ex vivo egg activation assay presented here enables the study of the trigger, propagation and downstream effects of the universally conserved Ca2+ wave.
The authors have nothing to disclose.
We are grateful to Laura Bampton, Alex Davidson, Richard Parton, Arita Acharya for assistance during the preparation of this manuscript; Mariana Wolfner for discussions on egg activation; Matt Wayland for imaging support; and Nan Hu for general support in the laboratory.
This work was supported by the University of Cambridge, ISSF to T.T.W. [grant number 097814].
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