The overall goal of the techniques presented here is to label and culture embryonic tissues for measuring morphogenetic deformation during early development for culture of the early chick embryo, begin by removing the embryo from the egg using a filter paper carrier method. Depending on the experimental application, label the extracted embryo with fluorescent lipophilic dyes delivered via magnetic iron particles or with polystyrene microspheres. Next, track the labels during culture using fluorescence microscopy or optical coherence tomography imaging.
Imaging resulting morphogenetic strain maps indicate regions of tissue elongation, shortening and shear in both two and three dimensions. Hi, my name's Ben Philis and I'm a graduate student in the lab of Dr.Larry Tabor in the Department of Biomedical Engineering at Washington University in St.Louis. Before beginning these types of experiments, it is important to consider relevant tissue culture techniques in your model system, imaging modalities to which you might have access, and also the type of data that you desire from your experiments.
For example, are you interested in tissue analyzing tissue deformations in two or three dimensions? We address some of these questions here today by showing two complimentary labeling techniques in the early chick embryo. Hi, my name is Victor Varner, and I work in the laboratory of Dr.Larry Tabor at Washington University in St.Louis.
In both of these techniques, we quickly and simultaneously label hundreds of cells in the early Chick embryo. Tracking the motion of these labels then allows us to not only determine the tissues into which these cells eventually take up residence, but also to quantify the tissue level deformation that mold the early embryo. First, incubate the eggs in a longitudinal orientation to the desired stage as defined by hamburger and Hamilton.
Crack the bottom of the egg on the side of a 150 millimeter Petri dish. Pull apart the shell from the bottom, empty the contents of the egg into the dish, and using blunt forceps, remove the thicker viscous albumin from the yolk. Now punch holes through the middle of a circular filter paper.
Place one of the filter paper rings on the yolk such that the embryo is visible through the ring's central hole. Trim around the outer perimeter of the filter paper ring with micro scissors. Then remove the paper with attached embryo from the yolk with fine forceps and gently soak the embryo in a bath of PBS To remove adherent yolk particles, transfer the filter paper assembly to a 35 millimeter culture dish.
Placing it ventral side up, send which the embryo with a second filter paper ring. Secure it with a metal ring and submerge the embryo in culture medium. If the embryo is not completely submerged, tissue geometry and subsequent morphogenesis can be greatly altered by abnormally high surface tension loads.
During culture, using a brightfield microscope, verify proper morphology and embryonic stage. First, take a small quantity of iron powder and add a few drops of saturated dye and alcohol and air dry with a tip of a pulled micro pipette. Break up the clumped iron particles and transfer them to a micro centrifuge tube.
After several rinses covered with the ionized water position, the harvested embryo dorsal site up in a 35 millimeter culture dish covered with a generous layer of culture medium. Under a dissecting microscope, use the glass needle to remove the vilin membrane and expose the electrodermal cells. Now draw a column of labeled iron particles into a peor pipette.
Using the dissecting microscope submerge the pipette to position just above the embryo. Roll the pipette slowly back and forth between the fingertips to sprinkle particles across the embryo. After a 10 minute incubation at 37 degrees Celsius, remove the particles using a strong magnet.
If necessary, attach the magnet to a magnetic pair of fine forceps and use the magnetized forceps to carefully remove any residual iron particles. Proceed to acquire both brightfield and fluorescent images of the embryo. With a 22 gauge needle.
Pull a solution of black 10 micrometer diameter microspheres in PBS into a 10 cc syringe. Then bevel the tips of pulled glass needles using a computer hard drive and fill with bead solution. Lightly flick the micro pipette for the solution to reach the tip using a micro manipulator.
Place the glass needle in the same field of view as the embryo. Confirm the flow of beads out of the pipette tip. Now insert the pipette tip into the lumen of the brain tube.
Taking care not to pierce the ventral floor of the tissue. Monitor the transient swelling of the tissue as beads and fluid enter the lumen. Then quickly remove the glass needle.
Finally, verify a sufficient bead density in the inner lumen of the brain tube. Using a bright field microscope, let the beads settle for 20 to 30 minutes before acquiring bright field images or an optical coherence tomography dataset. After acquiring the first images, place up to 7 35 millimeter culture dishes of samples in a 150 millimeter Petri dish and place the entire assembly in a small plastic bag.
Add drops of deionized water to the bag for humidification, and then fill the bag with a mixture of 95%oxygen and 5%carbon dioxide. Place the embryos into a 37 degree Celsius incubator until the next imaging time point place labeled embryos into a delta T dish containing 1.2 milliliters of culture medium and cover the dish with a glass lid kept at 37 degrees Celsius Super fuse the embryo with a 95%O2 5%CO2 gas mixture supplied from a mini pump variable flow device. Now set up the software to automatically acquire images that desire time intervals.
Over the course of the experiment, labels can be tracked automatically or manually, and morphogenetic strain maps can be calculated for marker coordinates. Here, the iron particle technique was used to label and track the motion of electrodermal cells during head fold formation. In the early chick embryo, fluorescently labeled cells were distributed across the entire embryo.
Brightfield and fluorescent images of the embryo were captured at different intervals during ex ovo culture. To monitor motions of the tract labels. Cumulative data allows calculation of the evolving morphogenetic strain distributions during head fold formation.
Similarly, the polystyrene microsphere technique was used to track tissue movements at the mid hind brain boundary of the early chick brain. Note that this method is capable of handling distinctly 3D deformation as beads tend to stick to all sides of the inner lumen of the brain. After tracking bead motions for six hours, marker coordinates allow calculation of strains, characterizing tissue deformation in the longitudinal and circumferential directions.
After watching this video, you should have a good idea of some of the experimental steps required to implement your own tissue culture and labeling. Protocol techniques presented here are particularly advantageous in that they're minimally invasive to the native tissues and that they also involve low cost and readily available materials. These experiments are particularly useful for studies on the mechanics of embryonic development.
Since the deformation fields computed using the tracked motion of labeled cells can be used to both test and construct physical models of morphogenesis.
