The overall goal of this experimental protocol is to study the role of mechanical forces in early embryonic brain development through ex ovo experiments. This method can help answer key questions in developmental biology and biomechanics, such as the roles of mechanical cues in morphogenesis, which is the generation of biological form. The main advantage of this technique is that it enables the characterization of the role of mechanical forces in morphogenesis at the tissue and organism level.
To begin this procedure, use delicate wipes with 70%ethanol to clean the fertilized specific-pathogen-free White Leghorn chicken eggs. Then, arrange the eggs in a longitudinal orientation on a holder. After that, set the egg incubator at a target temperature of 37.5 degrees Celsius, and maintain the humidity at 48 to 55%The humidity is controlled by adding an appropriate amount of water to the incubator.
Incubate the eggs for approximately 40 to 44 hours to HH11 to 13. Afterward, let the eggs cool down at room temperature for approximately 15 to 30 minutes prior to cleaning with 70%ethanol. In this step, crack the eggs from the bottom, pull apart the shell gently, and carefully deposit the contents into a 150 by 15 millimeter Petri dish.
To ensure the embryo side is up, keep the eggs in the same orientation as they were incubated while cracking them. Remove the thin albumin using a disposable Pasteur pipette. Separate the thick albumin from the yolk using the blunt-ended forceps.
Ensure that the thick albumin has been removed by lightly scraping the top of the yolk. Then, using fine-tipped forceps, place a filter paper ring over the embryo and center it, matching the long axis of the ring with the long axis of the embryo. Subsequently, cut the yolk surrounding the filter paper ring with scissors.
Following that, pull the ring and embryo off the yolk in an oblique direction towards the site where the yolk was first cut. Then, rinse the embryo in two sequential 100-millimeter dishes with room temperature PBS. Next, place a filter paper ring into a 35-millimeter Petri dish.
Place the embryo dorsal side up onto the other filter paper in the 35-millimeter Petri dish. After that, place a stainless-steel ring on top of the filter paper sandwich. Take care not to damage the embryo.
Now, add three milliliters of the previously prepared CCM to each Petri dish. Remove the vitelline membrane of each embryo by lightly skimming the pulled capillary needle across the top of the embryo. Then, peel the vitelline membrane away, starting from the anterior end and proceeding to the notochord.
Following that, place eight 35-millimeter Petri dishes into one 150-millimeter dish lined with water-saturated wipes to maintain humidity. Then, place the 150-millimeter Petri dish into a sealable plastic bag, and fill the bag with a gas mixture comprised of 95%oxygen and 5%CO2. Seal the bag, and place it in a 37.5 degree Celsius incubator.
Now, remove the embryo from the incubator, and use an OCT system to image them and to determine the torsional angle of the neural tube. Transfer the embryos to a light microscope, and visualize at 10X magnification. Use a 200-microliter pipette to incrementally remove 0.2 milliliters of media from the Petri dish.
Take bright-field images following each round of media removal to observe the effect of the media-air interface on the embryo. Continue removing media until the surface tension across the embryo induces torsion. Image the embryo using the OCT system once again to establish a final torsional angle for comparison to control embryos.
To alter the direction of the heart loop, use a pair of forceps to flip the filter paper so that the embryo becomes ventral side up. Then, use the pulled capillary tube to cut open a slit in the splanchnopleure membrane and to exert a mechanical force to push the heart from the right-hand side to the left-hand side. Apply the fluid surface tension to keep the heart on the left-handed side.
Then, incubate the embryo for another 20 hours to see the change in chirality of the torsion. Shown here is a harvested embryo with VM removed at HH11. The same embryo incubated for 27 hours post VM removal showing reduced torsion.
Here is the same embryo that underwent brain torsion upon application of fluid surface tension, and here is the control embryo with normal brain torsion at a comparable stage. While attempting this procedure, it's important to be careful not to damage the embryo. Following this procedure, other methods like finite element analysis can be performed in order to computationally model how physical forces can influence morphology.
Therefore, this technique developed here paved the way for future studies on mechanics of morphogenesis in chick embryos. After watching this video, you should understand how to harvest chick embryos for in vitro culture, micro-surgically remove the vitelline membrane, recapitulate the force provided by the vitelline membrane, and monitor embryonic morphology through OCT and bright-field imaging.
