The overall goal of this procedure is to integrate live imaging of fluorescent markers with mouse embryonic, neuro epithelium slice cultures. This is accomplished by first crossing the inducible Cree mouse line with the Cree reporter line to genetically label visibly dividing cells. Next, the whole mouse embryos are dissected and cultured in vitro in the presence of lentivirus.
Then the embryo is sliced and the neural tube section is prepared for live imaging. Finally, the neural tube slice is imaged with time-lapse confocal microscopy. Ultimately, results can be obtained that show direct observation of single cell divisions within the developing neuro epithelium in real time through ex vivo live imaging.
So the strength of this protocol is that we can monitor individual cells in vivo and that enables us to ask questions about each cell's signaling events as well as each cell's behavior. To begin, cross the Tamoxifen inducible Cree mouse line, CAGG CRE ER, with the DS Red Cree reporter line. To monitor the relative timing of sonic hedgehog response in the daughter cells, cross the CAGG CRE ER ds red line with OG two EEG.
FP back transgenic mice dissolved tamoxifen in 100%Ethanol then injected intraperitoneal in pregnant females at E 6.5 to induce CRE expression and DS red labeling in a small subset of embryonic cells. 48 hours later, after dissecting the embryos, use a fluorescent microscope to identify DS red positive embryos. Transfer them to a culture dish and observe single cell divisions during ex vivo imaging.
Dissect E 8.5 embryos into Prewarm Wash medium containing a one-to-one solution of D-M-E-M-F 12 supplemented with 10%newborn calf serum and 1%penicillin streptomycin. Using a fluorescent microscope with a stage heated to 37 degrees Celsius, or working next to a 37 degree hot plate, immediately identify embryos as GFP and or DS RED positive. Transfer up to two embryos into a 500 microliter drop of pre equilibrated culture.Medium.
Apply a thin layer of equilibrated light mineral oil over the medium to prevent evaporation and incubate the culture dish containing the embryos at 37 degrees Celsius and 5%carbon dioxide. To label the cilia in the neuro epithelium, add five to 10 microliters of S sst, R 3G FP lentivirus, approximately 2 million variants to a 500 microliter equilibrated drop of culture medium containing an embryo. After culturing for 18 hours, transfer the infected embryo into several drops of wash medium to wash out the virus and prepare neural tube slices.
Transfer an embryo to a 1%agar coated dish and use a 0.025 millimeter micro knife To dissect the neural tube. Place a 150 microliter drop of equilibrated culture medium without phenol red on a 35 millimeter polyol lysine coated glass bottom dish and place the isolated neural tube ventral side. Down in the drop of medium, put small amounts of a one-to-one mixture of petroleum jelly and melted candle wax around the mounted neural tube and gently press a glass cover slip into the jelly to immobilize the sample.
Cover the dish with a thin layer of equilibrated light mineral oil to image. Use an inverted confocal microscope equipped with an environmental chamber set at 37 degrees Celsius and 5%carbon dioxide. Use a 60 x oil immersion objective to image GFP labeled CLIA and DS red positive cells, and a 40 x oil immersion objective.
To monitor LIC two GFP and DS red positive cells, open the NIS elements software to set up time-lapse imaging conditions. Every 10 minutes, acquire Zacks up to 25 microns thick with a spacing of 1.5 microns. When using a 40 x objective and stacks up to eight microns with a spacing of 0.4 microns for a 60 x objective, refer to the text protocol for further instructions.
After fixing and embedding the tissue in OCT, use a cryostat to prepare sections and perform immunofluorescence according to the text protocol. Here we performed ex vivo live imaging of single cell divisions within the E 8.5 mouse neuro epithelium to label individual cells. We induced CRE recombinase in a subset of cells containing a Cree reporter line that expressed DS red upon recombination as seen here, 48 hours later, single cell divisions were observed through exvivo imaging by including a sonic hedgehog transcriptional reporter, modified back transgenic oli two EGFP line.
We were able to monitor when cells became sonic hedgehog responsive to observe CLIA formation SST R 3G FP lentivirus were generated and used to infect embryos in culture results from time-lapse confocal imaging of the DS Red Cree reporter embryos expressing EG two EGFP or infected with SSTR 3G FP lentivirus during in vitro culture are shown here. One of the powers of this approach is that we can incorporate mutant genetic alleles into the background, which lets us ask what specific genes do to individual cell behavior and signaling events.
