The overall goal of this procedure, is to better understand the cellular shape and movement, during Drosophila gastrulation. Here we provide a robustic measuring method of Drosophila gastrulation, to assay the initiation of morphogenetic cellular movement, during this stage of embryonic development. The main advantage of this technique is that, cell rearrangement at the time of gastrulation, can be identified along with apical constriction, during gastrulation.
To begin the experiment, prepare a mixture of 375 milliliters of water. 125 milliliters 100%commercially available apple juice. 12.5 grams of agar.
12.5 grams of glucose. Next, heat the mixture in a microwave, until the agar gets melted down. And pour it into a three centimeter cell culture dish.
Store the mixture at 4 degrees celsius, for future use. After preparing the apple plate, add a thin layer of kneaded yeast paste on top of it, to allow the flies to lay eggs. Place 25 male and 25 female flies in a bottle on an apple plate covered in kneaded yeast.
Wrap the plastic Drosophilas stock bottles with aluminum foil to prompt the flies to lay more eggs. Keep the bottle upside down in an incubator overnight. The next morning, replace the apple juice plate with a fresh one.
Then wrap the bottle with aluminum foil and allow the flies to lay eggs for three to four hours. After the flies have laid eggs, loosen the eggs from the plate with a cotton bud. Collect the embryos in a strainer, and wash them with phosphate buffered saline, or PBS.
Wash the embryos three more times with PBS, in 12-well culture dishes. Dechorionate the embryos with 50%bleach, in 12-well culture dishes. Following the dechorionation process, wash the embryos three more times with PBS.
Transfer the embryos to a three centimeter cell culture dish containing PBS. And select stage five embryos under the microscope, based on the level of transparency at their borders. Using a 200 microliter pipette tip, collect the staged embryos.
Next, place two selected embryos on a glass cover slip, and remove excess PBS with tissue paper. With a finely twisted tissue paper, orient the embryos dorsal side up to the cover slip. Then attach the embryos to the glass cover slip with silicone grease using a fine needle.
Add a small drop of hollow carbon oil 700 on the embryos, and place the coverslip containing the embryos upside down on the indented slide. Leaving some space at the bottom of the slide. Then stick the cover slip to the slide.
Finally, examine the embryos using a confocal imaging system, an argon 488 laser, and an oil-immersion objective. Time-lapse images were captured from live imaging, using DE-cadherin GFP of cell movement over 500 seconds, to analyze the apical areas of individual cells, of Drosophila during gastrulation. At 500 seconds, apical constriction, and invagination are clearly visible.
By using our method. We can further understand, the molecular mechanisms, of Drosophila gastrulation, as well as, epithelial to mesenchymal transition, involved in cancer metastasis.
