Lab 4 Gastrulation in Axolotl PDF

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Lab 4 Gastrulation in Axolotl...

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Dev Bio Lab Th 2/19/15 Lab 4: Gastrulation in Axolotl End of the Lab Questions 1. We placed the stain on the blastopore, which would develop into the mesoderm, so the mesodermal cells were labeled. The cells in our amphibian gastrulae could have been stained in both the plasma membrane and cytoplasm. Under certain conditions, Nile red can serve as a vital stain for the detection of cytoplasmic lipid droplets. The vital dye method we used in this lab was placing a dyed agar chip on the surface of the embryo, and the dye could have been absorbed into the yolk platelets within the cell. Depending on the size of the agar chip, some of the staining may have penetrated deeper than the most superficial surface. For example, Nile Blue staining of a 32-cell blastula at the dorsal side of the animal pole yields a blue-stained brain and may also stain the anterior portion of the notochord. 2. The vital dye method we used in this lab transferred the dye to the embryo by contact, meaning that the areas of the embryo in contact with the dyed agar chip would stain (diffusion of the dye may cause residual staining nearby). This dye method relies on careful placement of the agar chip with the surface of the embryo that we wanted to dye. The size of the agar chip is a limiting factor, as it may not accommodate single-cell resolution studies at later stages in development when the cells are smaller. This technique also limits us to observing superficial cells since the dye chip is placed at the surface of the embryo. Solutions to many of these limitations are fluorescent dyes and molecular markers, which can precisely dye cells by binding specifically to molecules in the cell. Fluorescent dyes and molecular markers can be distributed throughout the cell and will only bind to the specific areas for that marker. This gives greater control and selectivity over what cell is labeled and traced. An embryo injected with the fluorescent tracers could also be visualized at multiple time points without fixation. 3. The ideal cell fate markers should not harm cells, be able to be visualized in vivo, and last throughout development (maintaining specificity to the marked cell). 4. Yes. If cells that are still dividing are labeled, the label will be distributed as the cell divides, i.e. the dyed cell gives rise to a large area of dyed cells. If these cells differentiate into different cells, this may affect how you interpret the results. If the cells were not dividing before being labeled, you could have more specificity in following the cell as it develops and determining where a it ends up. 5. Fate maps allow us to trace a cell during development and see where it ends up. However, this information is not enough for us to determine how this occurred, so we cannot distinguish between whether the cell was committed to that particular fate or whether the cell was instructed to its fate by morphogen movements and signals. An experiment that can be done to test the difference between these two possibilities is to take the cell and place it into a new environment (i.e. at the opposite pole). If the transplanted cell develops into the same fate as previous observed, then it can be concluded that the cell was committed to adopt that particular fate. However, if the transplanted cell develops a different fate (i.e. the cell develop a fate like the cells around

Dev Bio Lab Th 2/19/15 it), then it is likely that the cell instructed to its fate by signals and morphogen gradients specific to its microenvironment. 6. a. see attached b. see attached...