Cell dynamics are most easily understood with live imaging. This protocol enables live imaging of the developmental stage of gonadogenesis that was previously inaccessible to visualization. The fine dissections necessary to isolate the gonad require delicate and precise manipulations under high magnification.
Only a visual demonstration can convey how to execute each step. Someone new to this technique might struggle with rapid timing and delicate tissue manipulations required at certain steps. We recommend practicing sections of this protocol separately while learning.
Begin by cutting a 22-by-22-millimeter cover slip into four equally sized strips using a diamond-tipped knife. Pick up one strip using forceps, and spread 30 microliters of heptane glue solution on both sides of the strip. Tilt the strip at various angles to achieve an even layer of glue residue as the heptane evaporates.
Store the strip in an empty cover slip box. Set a slanted but upright position to minimize contact with the box and to maintain its stickiness. Then close the box.
Transfer aged dechorionated embryos to a small watch glass filled with 500 to 750 microliters of heptane. Draw the embryos into the narrow portion of the micro pipette and slowly aggregate them near the inner tip of the pipette before gently pipetting them onto the slide. Twist the corner of a tissue wipe into a fine tip and wick away heptane from the embryos on the slide, making it easier to capture them on a glue-covered strip.
Pick up a glue-covered strip using forceps and gently touch it to the embryos. Place the strip in the imaging dish, just outside the Poly-D-Lysine-coated center with embryo side up. Press the strip bound to the dish to ensure that it is fixed in place.
Immediately flood the dish with two to 3.5 milliliters of Ringer's solution to submerge the embryos and prevent them from drying. During the dissection, tissue debris may stick to the dissecting needle. To get rid of this debris, periodically raise the needle just above the surface of the Ringer's solution.
Select stage 16 embryos with three gut constrictions that create four stacked gut segments. To begin devitellinization, pierce the selected embryo at the anterior end with a tungsten needle and peel the vitelline membrane off the embryo. Hook the needle through the embryo in a region far from the gonads and transfer the embryo to the polylysine-coated region of the cover slip.
Drag it against the bottom until it adheres. Similarly, arrange all the devitellinized embryos in a row along the top of the cover slip, leaving plenty of space below. Filet the embryo to expose its interior and slice through the embryo from its center by moving the needle posteriorly between the gonads.
Tease out some internal tissue to reveal the gonad and allow it to adhere to the cover slip. Using a needle, slice a piece of tissue, including the gonad, and separate it from the remaining carcass. Then, draw this tissue to a fresh region of the coated cover slip to adhere it to the dish.
Remove as much of the surrounding tissue from the gonad as possible by carefully guiding away the extraneous tissue and adhering the gonad to a fresh sticky region of the dish. Make sure not to touch the goad directly to avoid damaging it. Following dissection, add registration marks to the outside of the dissecting dish to help with locating gonads at the confocal microscope later.
Remove the glue-coated strip from the dish by gently inserting the bottom prong of a pair of forceps under the strip, clasping the forceps, and then tilting the strip upwards to free it from the dish minimizing disturbance of the adhered gonads. Place the imaging dish in the stage holder using the registration marks to place the dish in the same approximate orientation as during dissection. Use Brightfield microscopy and a lower power objective to identify and focus on any piece of tissue adhered to the cover slip.
Switch the IP settings to reveal fluorescence, and use the binocular eye pieces to systemically scan the dish, marking the position of each gonad within the imaging software. Gently remove the entire stage holder assembly with the imaging dish still in its holder and place the assembly on the work bench. Remove some of the Ringer's solution with a P1000 micropipette from the inside upper ledge of the imaging dish.
Then remove approximately 50 to 100 microliters of the solution from under the surface using a P200 micropipette, making sure not to remove all solution. Add approximately 200 microliters of imaging media containing insulin under the surface of the remaining Ringer's solution. Add the remaining imaging media to the dish, starting at the outermost rim of the upper ledge.
While pipetting, move toward the central dome of the fluid and eventually merge the tube by brushing the pipette tip across both. Switch the microscope to a higher power objective and apply proper immersion fluid. Then replace the stage holder assembly.
Adjust the focus to the marked gonad position and begin imaging. Bright field visualization of an isolated gonad on a confocal microscope reveals a dark shadow surrounding the gonad periphery, which is the extracellular matrix that maintains the integrity of gonads during imaging. An example of a healthy well-cultured gonad expressing labeled and somatic gonadal cells and histone markers is shown here.
If the gonads are not sufficiently hydrated during imaging, the tissue shrivels. A gonad whose extracellular matrix is damaged during dissection has a compromised boundary, which is evident by the presence of gonad-specific cells outside of the confines of the gonad. Prior to compaction, the forming stem cell niche in the X-VIVO cultured gonad initially appears as a loose aggregate of somatic cells at the gonad anterior.
The niche cells are surrounded by germline stem cells. The niche aggregate initially exhibits an irregular boundary. Throughout the course of imaging, the individual niche cells rearrange their positions such that the niche aggregate acquires smoother borders, resulting in a decrease of the niche area.
When attempting this protocol it is important to work quickly when transferring embryos to a coated dish and to be delicate when dissecting the gonad away from the surrounding tissue. X-VIVO culture can be combined with both genetic and pharmacological manipulations, allowing for multifaceted approaches to investigate events occurring during male or female gonadogenesis. This technique made it possible for us to visualize how the stem cell niche acquires its final architecture.
Consequently, we can now form and test hypotheses about underlying cell dynamics that drive its morphogenesis.
