The overall goal of this procedure is to dissect out intact zebra finch embryos at very early embryonic stages for downstream developmental and molecular applications. This is accomplished by first cutting the eggshell lengthwise and gently emptying the egg contents onto WHE paper. Next, the zone of junction is located and the surrounding yolk is cut away, leaving the embryo intact.
Then the embryo is transferred to a Petri dish and washed before being fixed or flash frozen for later analysis. Finally, the vitel membrane is removed from the fixed embryo for increased clarity of embryonic structures. Ultimately in C two hybridization and EDU treatment show gene expression and proliferating cells respectively in early embryonic stages of zebra finch in response to methylmercury treatment.
The main advantage of this technique is that it minimizes embryonic tissue damage. This allows for the investigation of morphology and gene expression throughout all stages of development. This method can help answer key questions in comparative developmental biology and toxicology, such as the effects of various toxins on the earliest stages of development.
Visual demonstration of this technique is critical as locating the zno junction and removing the vilin membrane during dissection can be difficult as early embryonic structures are fragile and difficult to observe After collecting zebra finch eggs and incubating them according to the text protocol, assemble the following materials for dissection. A clean scalpel fine tipped forceps, two extra fine tipped forceps. Three transfer pipettes, one XPBS and 4%para formaldehyde.
If you'll be fixing the embryos, place a piece of 10 by 10 centimeter wave paper on the base of the dissecting scope to provide a clean nonabsorbent surface for dissection. Remove the egg from the incubator at the desired time point as determined by the zebra finch guide. Next, using a fiber optic illuminator lamp, candle the egg by holding it along its vertical axis and shine light through the egg to illuminate the interior.
Place the tip of the light behind the egg and locate the yolk and the developing embryo. Then position the scalpel on the side opposite the yolk and with faint pressure, cut along the egg from tip to base. Remove the contents of the egg by using the thumb and forefinger to carefully apply pressure to the tip end base of the egg, or by using forceps to gently pry apart the eggshell along the cut.
Examine the yolk for the white zone of junction, which is visible as a faint white ring. Encircling the developing embryo and use fine tipped forceps to orient the yolk so that the embryo is located in the center to separate the embryo from the extra embryonic tissue. Puncture the edges of the yolk to relieve the pressure and make single cuts across the length of the yolk diameter alongside the embryonic disc.
Continue making diagonal lines until the section of the yolk with the embryo has been successfully separated. Using a transfer pipette, remove the edges of the yolk, leaving a small amount to lubricate the embryo on the way paper to prevent it from tearing. Wash the embryonic disc by placing the tip of a transfer pipette with one XPBS next to it at a 45 degree angle facing the bottom of the embryo and dispensing the buffer using a small amount of one XPBS.
Transfer the embryo to a small plastic Petri dish. Wash the embryo by adding more PBS into the dish and dripping PBS near the embryo. Swirl the dish to wash and remove residual yolk.
Then tilt the dish to aspirate the waste buffer. If fixing the embryo for in C two hybridization immediately add 4%PFA to submerge the embryo, then drip the PFA directly onto the embryo to flatten it to prevent it from curling. Incubate for 12 hours at four degrees Celsius after dehydrating the embryo in a methanol series.
If the embryo is between stages one through eight, remove the vitel membrane, adhered to the embryo by using extra fine forceps to grip the edges of the membrane that extend beyond the zone of junction. Carefully flip the embryo over multiple times to wash away residual yolk granules and loosen the embryonic disc from the vitel membrane. If a gap does not appear between the zone of junction and the vitel membrane, use extra fine tipped forceps to gently scratch the zone of junction to loosen it from the membrane.
Then grip the viel membrane and the peripheral edge of the embryonic disc at the zone of junction, and gently pull the viel membrane away from the embryo. If flash freezing the embryo following dissection quickly add two to three milliliters of one XPBS to the Petri dish and tilt the Petri dish to separate the embryo from the OK granules. After removing the liquid and repeating the wash two to three times, use fine tipped forceps to transfer the embryo to a pre-labeled micro centrifuge tube and flash freeze in liquid nitrogen before storing at minus 80 degrees Celsius.
To carry out the EDU cell proliferation assay using the fiber optic illuminator lamp, candle, and egg. To locate the embryo or yolk, mark the side of the egg opposite the embryo or yolk to ensure that the embryo is not damaged during the microinjection. Next, with modeling clay line a 60 millimeter plastic dish by molding it in the shape of a bowl to hold the egg in the desired orientation.
Adjust the direction of the marked spot to allow insertion of the microinjection needle. After pulling two glass capillary needles blunt, one for poking a hole in the egg and with mineral oil back, load the second's needle and insert it into the micro injector. Set the volume of solution per injection to 59.8 nanoliters on the micro injector, and then with 10 millimolar stock EDU solution, load the microinjection needle using the blunted glass capillary carefully puncture a hole in the shell on the marked spot, taking care not to shatter the shell or drop pieces of it into the egg cavity.
Orient the egg so that the hole is at a 45 degree angle in alignment with the microinjection needle to be inserted. Then using the micro manipulator, insert the loaded needle approximately one centimeter into the cavity and inject the desired amount of EDU solution into the yolk or embryo. Immediately use multiple layers of plastic to wrap the egg and the clay holder and tape the edges of the wrap to the bottom of the dish to prevent sation to incubation.
Incubate the egg at 37 degrees Celsius on a stable surface until the desired stage is reached before removing the plastic wrap and dissecting the embryo according to the text protocol, this figure shows an NC two hybridization on dissected and fixed embryos exposed to low doses of methyl mercury to detect expression of ortho identical homeo box. As seen in the dorsal and ventral view of the head structures, the treated embryos were developed mentally delayed at stage 11 compared to the untreated embryos. At stage 12, the group of four embryos shown on the left were incubated for the same amount of time.
However, the two untreated embryos on the top left progressed to stage six, while the two mercury treated embryos on the bottom left only progressed to stage five. These embryos were treated with a sense probe to demonstrate a lack of background staining shown here are proliferating cells in a day two embryo that was injected with EDU and then incubated overnight. Click chemistry shows proliferation in the lateral edges of the somites and the tail bud, as well as in the cephalon.
This gel contains RNA samples extracted from dissected stage 16 zebra finch embryos left untreated or treated with 1.2 parts per million of methyl mercury, indicating that the dissection method demonstrated in this video allows for the extraction of high quality RNA Once mastered. The dissection can be done in under a minute if performed properly During this procedure. It's important examine the yolk at an angle to locate the embryo as early embryos can be difficult to observe Following this procedure.
Other methods such as inid hybridization, cell proliferation, assays, and RNA extraction for qualitative analyses can be performed to answer additional questions in developmental and toxicological studies.
