The method shown in this video utilizes fate maps of the cleavage stage opus embryo to identify single blaser's culture in isolation to assess whether embryonic sulfate is determined by intrinsic or extrinsic instructions, first two cell embryos are collected in which the first cleavage furrow bisects the lightly pigmented area of the animal hemisphere. These embryos are cultured when the embryos reach 16 to 32 cells, the vitel membrane is then removed and neighboring cells are dissected away from the cell chosen. For explan, the dissected blaster is then transferred to a depression in an auger coated culture, well and cultured.
Once the explan reaches the desired stage, it is harvested to assess gene or protein expression using in situ hybridization QPCR or immunohistochemistry. The major advantage of this technique over others such as culturing, large groups of embryonic cells or organ on login, is that single embryonic blasphemers whose developmental fate is known from lineage tracing studies can be cultured without added growth factors or the presence of neighboring cells. Thus, one can directly test whether factors intrinsic to the cell instruct that blaspheme to become the precursor of a specific tissue or whether it requires signals from surrounding cells.
Visual demonstration of this method is critical as the dissection of Blairs and their transfer to culture wells requires delicate manipulations under the stereo microscope. In this video, I will be using this method to demonstrate whether maternal derived mRNAs bias cells towards a neural fate Working under a dissection microscope. Sharpen four sets of forceps using Illumina abrasive film.
One pair of forceps serves as a backup in case a tip is damaged during the procedure, autoclave all four forceps and store in a sterile container make 500 milliliters each of 0.1 x and 1.0 x of modified barth saline or MBS and filter sterilize them. Store these solutions at 14 to 18 degrees Celsius for months in a glass screw cap bottle, prepare 2%electrophoresis grade aros in 100 milliliters of one X culture. Medium autoclave to dissolve the aros microwave, to liquefy the aros when it is next needed while the aros is liquid.
Pour about 0.5 milliliters onto the bottom of each well of a sterile 24 well culture plate. This will prevent the implants from sticking to the plastic. Then pour about two to three milliliters onto the bottom of two to 360 millimeter Petri dishes and swirl gently to distribute.
These will serve as dissection dishes and the aros prevents embryos from sticking to the plastic. Once their membranes are removed, when the aros has cooled and hardened flame, the tip of a six inch pasture pipette until it melts into a ball. Reframe the ball briefly, then lightly touch it on the surface of the aros in a well of the culture plate.
This will create a shallow depression into which the X explan will be placed. Repeat for each well in the plate. Next, fill each well of the culture plate with one milliliter of one x sterile culture medium.
Then fill the dissection dish with 0.1 XMBS to facilitate blaster separation. Once the dissection dishes are prepared, transfer fertilized eggs with the jelly coats removed to 100 millimeter Petri dishes containing 0.5 x culture medium. Those that are not immediately used can be cultured at 14 degrees Celsius while the first dish is surveyed for two cell embryos.
Survey the embryos in the first Petri dish using a dissection microscope using a plastic transfer pipette transfer about 101 cell and two cell embryos to a separate dish filled with 0.5 x culture.Medium. Place the Petri dish in a 14 degree Celsius incubator. The ability of this assay to accurately assess the developmental potential of a cell relies upon dissecting out the correct blasphemy based on fate maps.
Therefore, it is crucial to choose embryos with the correct pigmentation pattern at the two cell stage that consequently conform to regular cleavage patterns. As illustrated here. Regular cleavages often occur only on one side of the embryo.
These embryos can still be used as donors if the donor cell originates from the regular side. When embryos reach the two cell stage, sort those in which the first cleavage furrow bisects the lightly pigmented area in the animal hemisphere into a separate dish, sort about five times as many embryos as will be dissected. These will be the donors for the explan.
Keep the remaining two cell embryos in a separate dish next to the donor embryos throughout the procedure to serve as sibling controls to stage the explan. After the embryos are sorted and dissected, return to the dishes in the incubator to find more one cell and two cell embryos. Since it takes one cell embryos about 90 minutes to divide, this can be done up to two hours after egg collection.
To prepare the explan place five to 10 embryos in a dissection dish. Then position the first embryo so the transparent vitel membrane can be grasped with forceps. It usually cannot be seen, but can be grasped at the animal pole where it is separated by a clear peral space above the surface of the embryo.
Using a sharpened forceps in one hand, grasp at a distance from the blaster that is to be dissected to avoid damaging it. Then with sharpened forceps, in the other hand, grasp the membrane close to the tip of the other forceps and gently pull in opposite directions to peel the membrane away. The embryo will flatten when the membrane has been removed.
Next, grab a neighboring cell with the forceps and use this cell as a handle so the cell to be dissected is not directly touched with the forceps in the other hand, gently pulled the remaining neighboring cells away from the desired blaspheme. If midline cells which share the same fate are targeted, they can be removed together as a pair. Finally, dissect away the handle cell.
Pick up the blaspheme or amere pair with a sterile glass past pipette, avoiding air bubbles and excessive suction. Place the tip of the pasture pipette under the surface of the culture medium in a well of the X explan culture dish, and gently expel the blaster. It should slide into the shallow depression by gravity.
Blasphemers from more than one embryo can be combined into a single explan by depositing them in the same depression in a culture. Well repeat this procedure with the remaining embryos in the dissection dish one by one. After about 10 embryos, the dissection dish will be filled with cellular debris.
When this occurs, change to the next dissection dish. After all dissections are done, check whether the explan are in the shallow depressions. If not, they can be gently pushed into the depression with a hair loop that has been sterilized in 70%Ethanol and air dried incubate implants at room temperature on the bench about an hour after the last dissection.
Remove debris surrounding the healed explan with a sterile glass pasture pipette culture. The plate of explan at 14 to 20 degrees Celsius next to the Petri dish containing the sibling control embryos. Sibling embryos will indicate the stage of development of the blaspheme explan.
After about one to two hours in culture, the explanted blastomeres will have divided about four times and sloughed off most of the debris remaining from damaged neighboring cells. After 20 hours, they form small sturdy balls of cells. When the siblings reach the desired developmental stage, harvest the explan.
These explan survive quite well, even if some of the cells disintegrate. Therefore, if there is a cloudy mass in the culture, well explore it with a hair loop or forceps to determine if a healthy explan is buried within. Pick up the explan in a small volume of culture solution with a glass past your pipette and gently expel them into a fixative or lysis buffer appropriate for the assay to be conducted.
Two dorsal blasphemers or two ventral blasphemers were dissected at the 16 cell stage and cultured in one XMBS at 14 degrees Celsius until sibling embryos reached early neural plate stages. Each sample was assayed for the expression of a zygotic neural plate. Gene Sox two by in situ hybridization, which is seen as the blue reaction product.
The results of this assay demonstrate that the majority of dorsal animal blasphemers, which are precursors of the neural plate, can express SOX two autonomously. That is without additional signaling from other regions of the embryo. Those explan that do not express SOX two may have been incorrectly identified at the time of dissection.
In contrast, none of the ventral animal blasphemers, which are precursors of the ventral epidermis express SOX two. These data indicate that dorsal animal blasphemers have an intrinsic ability to form neural tissue, whereas the ventral animal blasphemers do not note. Some of the dorsal explan have also elongated a morphology indicative of dorsal mesoderm formation.
These blastomeres also are faded to form the Noor in the intact embryo and an explan culture express a Noor marker protein to test the effect of altering the endogenous levels of a maternal expressed neural gene. Fox D five mRNAs were injected into the eight cell ventral precursor blasphemers. As shown here, the majority of the ventral implants expressed SOX two.
Conversely, when the endogenous level of Fox D five was reduced by injection of antisense morph oligonucleotides into the eight cell dorsal precursor blasters, only a few dorsal explan expressed SOX two. These experiments indicate that the maternal expression of Fox D five plays a role in the intrinsic ability of dorsal blasphemers to acquire a neural fate Prior to this procedure. Other methods such as injecting into the Blairs to be dissected either mRNAs for gain of function or antisense morpho oligonucleotides for loss of function can be performed to answer additional questions.
For example, what genes are involved in the intrinsic ability of cells to give rise to a specific developmental fate? In addition, one can test known signaling factors by adding purified factors to the culture medium. This approach can also be used to transplant individual blasphemers to novel locations in the intact embryo.
This test whether the transplanted cell is committed to its original developmental fate, this information is critical for defining the cellular mechanisms by which a pluripotent embryonic cell becomes committed to becoming the precursor of a defined tissue.
