In Vivo Visualization of Calcium Transients during Fertilization and Early Development in C. elegans

The production of viable embryos is a complex process. Our overall goal is to define the cellular signals that coordinate the transition from an oocyte to an embryo. The CaFE reporter is a new tool developed to visualize calcium signals that occur upon fertilization and during early embryogenesis.

Using the CaFE reporter, we have discovered a delayed calcium wave in oocytes that are prematurely ovulated. We have also discovered the presence of single-cell calcium transients during early embryogenesis. Both findings illustrate the utility of a genetically-encoded calcium indicator during early development.

Previous techniques to visualize calcium oocytes relied upon dye injection into the gonad. This technique is labor-intensive and requires specialized micro-injection equipment. The CaFE reporter enables easy visualization of the calcium wave in oocytes and embryos with widely available equipment.

The CaFE Reporter facilitates investigation into the timing and regulation of calcium signaling in oocytes and embryos. This reporter can be easily combined with existing mutants, markers and RNAi knockdowns to uncover calcium's behavior relative to other landmark events during early embryogenesis, such as myotic maturation. To begin, hold the worm-pick handle and loosen the silver collar towards its head so that the four sections come apart.

Place a 1.5-inch platinum wire in the middle of the four sections. Hold the wire in the center while tightening the collar to bring the four sections together. Using a pair of flat-nose pliers, carefully shape the wire, and repeatedly clamp down the end of the wire to create about a two-to-three-millimeter foot.

Use this foot to pick up the worms. Prepare a solution of 3%agarose in water and heat it in a microwave until agarose dissolves completely. Place a blank microscope slide between two other microscope slides that have a single layer of lab tape across the long axis.

Using a one-milliliter pipette, drop the heated 3%agarose solution onto the center of the blank slide. Quickly place a new microscope slide onto the agarose droplet, perpendicular to the blank slide, with the droplet resting across the adjacent taped slides, then gently separate the slides, keeping the pad intact. Using a 20-microliter pipette, add a small drop of one-millimolar levamisole in M9 buffer to the center of the agarose pad to paralyze the worms.

Pick at least five synchronized worms and gently touch the foot of the worm-pick handle to the levamisole droplet to transfer the worms. Carefully cover the agarose pad with a cover slip. Check the location of the worms, as the cover slip may cause the dispersal of the worms.

Label the microscope slide with appropriate details, such as the name of the worm strain. Locate the worms using a low-powered objective on the microscope using bright-field illumination. Use a minimum of a 20x air objective or an immersion objective of 40x for clearly visualizing the calcium wave in oocytes.

For optimal visualization of the fluorescent reporters, adjust the gain or exposure time for the GFP and RFP channels. Examine each minus-one oocyte in the gonad arms of the worm to determine if a fertilization event is imminent. After nuclear envelope breakdown, the cytoskeleton of the most proximal minus-one oocyte will rearrange, take on a rounded appearance, and separate from the minus-two oocyte.

When the ovulation occurs imminently, start recording the video in a wide-field setting immediately. The proximal oocyte is denoted by the minus-one directly before the spermatheca, where the sperm are stored. The most recent embryo is located closest to the spermatheca, but inside the uterus.

The calcium wave displayed a biphasic pattern, starting with a rapid burst followed by a wave of fluorescence moving from the entry point toward the opposite pole. A calcium signal was observed immediately upon entry into the spermatheca in the leading minus-one oocyte, but not in the trailing minus-two oocyte. Embryos in-utero and ex-utero displayed a fluorescence signal in a single cell during embryogenesis.