The overall goal of this procedure is to monitor the events of mouse embryo, turning neuro axis elongation and neural tube closure in an extra uterine environment in which conditions can be defined. This is accomplished by first removing the uterine horns from the anesthetized pregnant mouse at gestational or embryonic day 8.5. Next individual mouse embryos are removed from the uterus with their yolk sac intact and much of the decidua.
Then the extra embryonic decidua tissue is trimmed away, leaving the ecto placental cone intact. The final step of the procedure is to transfer embryos in their yolk sac to roller bottles containing media with or without pharmacologic agents being tested and culture embryos for 18 to 24 hours in an incubator with continuous gassing. Ultimately, results can be obtained that show progressive addition of somites, apposition of neural folds, and closure with turning of the embryo into the familiar c curvature of the fetal position.
This method can help answer key questions in the field, such as what molecular pathways are involved in normal neural tube closure. What agents or supplements can help promote normal ululation, especially in embryos that carry genetic mutations that predispose them to neural tube defects or NTDs. Visual demonstration of this technique is critical as the steps in the dissection can be very challenging.
The embryos are small and can be easily damaged, and damage to the yolk sac can impair som might addition, turning and also development of the circulatory system preventing a successful outcome. To begin, prepare culture medium by thawing male rat serum at 37 degrees Celsius. Next e inactivate the rat serum for 30 minutes at 55 degrees Celsius.
Then spin it down at 10, 000 RPM for five minutes. At room temperature, remove the supernatant and mix it in a one-to-one ratio with phenol red free DMEM with high glucose. Sterilize the medium using a 0.45 micron syringe filter.
Isolate the uterus and place it in a Petri dish with saline to remove the embryos. First, rinse the uterus with saline to remove any excess blood and trim away any excess fat from the outside. Transfer it to a clean dish with room temperature, tyro saline to improve visualization under a stereo microscope with hands stabilized on the stage.
Use small scissors or fine forceps to peel the uterus apart and separate each embryo. Insert fine forceps into the space between the uterine wall and the decidua, and peel back the uterine wall using minimal tension to avoid rupturing the yolk sack. Next, orient the deciduous so that the placenta is facing away from you.
Then make an incision with the forceps along the line, separating the dark portion of the decidua from the lighter portion. Being careful not to cut too deeply. Complete the separation of the placenta from the top of the oak sac.
Being careful not to tear the ecto placental cone or EPC. At this point, the riker membrane on the oak sack should be visible. Removing the Riker membrane from the yolk sack is the most difficult part of the procedure, and it requires patience and a steady hand.
In order to delicately remove the riker without damaging the underlying yolk sack, Continue to remove the entire decidua. Being careful not to pierce the yolk sack, gently pinch and remove the riker membrane by peeling it away from the EPC. If the EPC is damaged or separated from the oak sac, embryos will fail to turn normally from E 8.5 to E 9.5.
Once the riker's membrane is removed, the embryo should be easily visible through the oak sac. Stage the embryo by counting the somites along one side, starting beneath the otic vesicle and ending at the tail with scissors. Cut the end of a plastic transfer pipette to use on the embryo without damaging the oak sack.
Fill clean roller bottles with one milliliter of medium per embryo. Depending on the type of roller bottles used, a maximum of three to six embryos can be loaded in each bottle. Using the cut pipette, transfer the embryos to the media filled roller bottle, carrying over as little tyros as possible, so as not to dilute the medium.
Attach the rubber plug to the top of the glass roller bottle. Insert the roller bottles into the roller culture drum so that a tight seal is formed between the plug and the drum. Once all the bottles have been attached, seal any empty slots in the drum with rubber stoppers.
Then activate the roller. Next, open the premix CO2 O2 tank and adjust it to approximately two PSI so that the outlet valve is releasing one bubble per second. For E 8.5, E 10 embryos, 20%O2 and 5%CO2 should be used.
Ensure that the incubator is set to 37 degrees Celsius and cover or close the incubator so that the embryos are shielded from light. To assess embryo development, check them periodically for the addition of somites, the progression of turning and the extent of neural tube closure after two to three hours in culture, if desired, add pharmacological inhibitors or other treatments. When the incubation is complete, turn off the gas, stop the roller and remove the bottles from the incubator.
Transfer the embryos back to tyros or PBS in a Petri dish Under the stereo microscope, the yolk sack should appear balloon like a heartbeat should be visible and the circulation should be evident. Remove the oak sack from the embryo by cutting a hole near the EPC and flipping the yolk sack and amnion over the head and rump of the embryo. Cut the umbilical cord to separate the yolk sac from the embryo.
Finally, examine the embryo for so might number and neural tube defects such as open cranial folds, incomplete closure of the face and or an open coddle neuro pore Embryos can be staged according to the Tyler classification of multiple morphogenetic changes. The appearance of embryos pre and post roller culture is illustrated here at the time of dissection. Embryo should be in an unturned configuration where the tail is behind the head folds.
After 24 hours in culture, embryos should have completed turning so that they are in a C curved fetal position where the tail is in front of the head. This image shows in the top row the development of embryos without inhibitors added. The bottom row shows the effect of pharmacological manipulation with the row A kinase inhibitor Y 2 76 32, a known inhibitor of convergent extension during alation.
This inhibitor results in a shortening of the embryos along the caural axis and inhibits cranial neuro fold closure. Our data show that increasing doses of Y 2 7 6 3 2 progressively impair cranial fold closure and shorten the body axis consistent with the role of downstream row. A signaling in cranial neural and convergent extension Once mastered, the dissection technique can be completed in 60 to 90 minutes for a litter of six to 12 embryos.
After watching this video, you should have a good understanding of the steps required to culture gestation day eight and a half to 10 mouse embryos for the purpose of studying developmental mechanisms at a systems level.
