This well-defined protocol looks at primary neural crest migration in mammals. We use primary neural crest migration in order to study birth defects that affect the neural crest. This approach allows us to take a closer look at the neural crest cells as they emerge out of the neural plate.
Testing for cell behaviors in tissues carrying mutant genes allures or after pharmaceutical treatment, allows health screening for the effects of drug treatment or environmental changes during embryonic development. A similar approach can be used to study neural crest cells that migrate to the heart or the gut or to study metastatic tumor cells. Guessing the anatomy of the embryo right is challenging.
Its tissues change very rapidly during early development. We suggest practicing microdissection before undertaking a big experiment. Because the embryos are alive and three dimensional, speed and precision are crucial.
At embryonic day 8.5, use sterile tools and solutions to harvest the uterus into a container of ice cold PBS. Cut the mesometrium to separate each embryo. Dissect the uterus carefully, separating each decidual swelling.
Harvesting the embryo at the right developmental stage is critical to the reducing variability and increasing the success in the adhesion of the explant to the matrix as well as cell migration. Slide forceps between the muscle layer and the decidual tissue of the embryo and use a second pair of forceps to remove the muscle layer. Use the forceps to pierce the decidua at the edges of the mesometrial pull and use the second pair of forceps to tear the tissue open perpendicularly to the pull.
Peel back the decidual tissue with the forceps to visualize the ricarts membrane before carefully removing the membrane. The visceral yolk sak will become visible and the embryo will be observed inside. Remove the visceral yolk sak and the anion, and position the embryo to allow visualization of the head fold.
Cut the head fold above the heart and scrape away the underlying mesoderm to obtain a clean neural plate. Neural plate borders should then be dissected. Using a glass Pasteur pipette, transfer the dissected neural plate onto a hydrogel coated dish filled with conditioned neural crest medium and gently swirl the dish to position the neural plate in the middle of the well.
Then incubate the neural plate at 37 degrees Celsius and 5%carbon dioxide for the appropriate experimental time period. At 24 hours post explanting, place the tissue culture dish into the specimen holder of a face contrast microscope, and tape down the plate lid and carbon dioxide needle to prevent shaking during multi-well acquisition. Then focus on the cranial neural crest cells at a 10 times magnification with a matching phase ring in the condenser selected.
To quantify the neural crest cell migratory capacity, set the microscope to acquire one frame every five minutes at a 10 times magnification. To quantify cell morphology, set the microscope to acquire one frame per minute at a 40 times magnification. To quantify lamellipodia dynamics, set the microscope to acquire one frame every 10 seconds for 10 minutes at a 40 times magnification.
For multi-well imaging, set the mechanical stage to move between selected X, Y positions of interest, and confirm that the cranial neural crest cells are in focus and that the stage positions are correct. Then click acquire to start the time lapse imaging. For single cell tracking, open image J and import the data as tiff stack files.
Change the orientation and brightness and contrast levels and click analyze and set scale to calibrate the dot stk files in pixel micrometers according to microscope settings. Click plug-ins, tracking and manual tracking to open the image J manual cell tracking plugin and select add track to begin cell tracking. Then track cells through all frames of time-lapse movies using the nucleus as a reference point.
To assess the neural crest migratory capacity, open a suitable migration analysis software program and click the import data tab to import the cell tracking data as a dot txt file. Under data sets and initialization, select the number of slices or frames to be analyzed and set the X Y calibration and time interval between frames. Select apply settings to save the settings.
Then select the plot data symbol to form trajectory plots and select the statistic symbol to quantify the distance and speed measures. To quantify neural crest cell area and circularity, open image J and under analyze and set measurements, click to select the cell shape parameters, cell area, perimeter and shape descriptor. Use the free hand selection tool to manually draw an outline around each cell using the cell membrane boundaries as a guide.
Press Control+B keys on the keyboard to maintain the cell outline overlay on the image and repeat for each cell over each time lapse frame. Click image overlay and to reach of interest manager and select the cells of interest. Then click measure to obtain the values for the selected cell shape parameters of interest.
Within 24 hours of explant and culture, a region of the pre migratory epithelial cranial neural crest can clearly be observed to surrounding the neural plate explant. Furthermore, a subpopulation of neural crest cells will have undergone epithelial to mesenchymal transition and appear fully mesenchymal. Explant cultures plated on either an extracellular matrix-based hydrogel or fibronectin, form similar explant structures comprised of neural plate premigratory neural crest and migratory neural crest cell populations.
However explants plated on fibronectin produce cells with more prominent lamellipodia at the cell leading edge seemingly more polarized in the direction of migration. Time lapse microscopy allows the tracking of X, Y coordinates of individual cells and the analysis of neural crest cell migration and the analysis of cranial neural crest cell morphology dynamics over time. By outlining individual cell membranes, cell area and perimeter measurements can be calculated from all frames of the movies to allow the subsequent quantification of the cell area and circularity of individual and groups of cells.
Note that as the cells migrate away from the explant, the cell area significantly increases while the cells circularity remains relatively constant, suggesting that as cells depart from the epithelial edge and lose cell to cell contacts, they show increased cell spread area. Pay particular attention to scraping away the underlying mesoderm to obtain a clean neural plate, as this promotes the migration of neural crest cells away from the tissue. Many other techniques such as immunostaining, RTPCR, or single cell analysis can be performed to analyze different proteins or genes of interest among different populations of culture cells.
We are interested in the developmental rolls of some key neuroblastoma genes that might be involved in neural crest cell migration and in using this approach to study human disease genes.
