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		Transplantation of the eye-forming region in the Axolotl neurula to assess cell specification and differentiation during late-stage gastrulation Anisha Chandra '06 Developmental Biology, Swarthmore College April 1, 2004   Objective and Hypothesis The purpose of this study is to investigate the specification and determination of the cells in the eye-forming region of an Axolotl neurula. This will be done by transplanting this region into the flank of a host embryo of the same species. Isolated eye development in the flank region would indicate that the transplanted cells had already been determined as eye tissue when moved, whereas flank cell development in the graft would imply that the cells had not yet been specified as eye-forming. The eye-forming region of the Axolotl neurula is thought to undergo specification and determination during gastrulation, so this experiment should result in additional eye development on the flank of the host embryo (Gilbert, 2003). Background Amphibian embryos were traditionally favored by experimental embryologists because they were numerous, large, and easy to manipulate. They became less popular with the advent of developmental genetics, because they mature slowly and their chromosomes are often found in several copies (such as the tetraploidal Xenopus), which makes them harder to manipulate. Now, however, new molecular techniques have allowed a return to amphibian models (Gilbert, 2003). Salamander embryos, such as the axolotl Ambystoma mexicanum, are commonly used for experimental study. The following experiment explores cell specification of the eye-forming region of the anterior neural plate. Cell specification in general is thought to occur during gastrulation (Gilbert, 2003). Cell specification in the eye-forming region is linked to neurulation, or the formation of a neural plate and tube - this is the point at which an embryo is called a neurula (Cebra-Thomas, 2004). During neurulation, signals from the underlying dorsal mesoderm and anterior pharyngeal endoderm induce the ectoderm to elongate into columnar neural plate cells. These look different from the flatter epidermal precursor cells surrounding them (Gilbert, 2003). On a molecular level, transcription factors in the ectoderm induce the expression of proteins that activate the neural phenotype, as opposed to the epidermal one (Gilbert, 2003). Additionally, there seems to be a bias in the dorsal cells of the amphibian embryo towards becoming neural, which may be due to signals traveling from the dorsal lip horizontally through the ectoderm. There is correlative evidence that this second signaling center is linked to ß-catenin (Gilbert, 2003). Therefore, in no way is epidermis a 'default' pattern simply because it is simpler than a neural one. Up to fifty percent of the ectoderm becomes the neural plate. Interactions between the neural plate and the epidermal cells (in lens placode formation, for instance) give rise to the structures of the eye. At mid-to-late gastrulation, the epidermal and neural cells of the anterior neural plate should be determined to form eye tissue (Cebra-Thomas, 2004). In Axolotl neurulas, then, the presumptive eye tissue should have been determined. If this region is transplanted to the flank of a host embryo, the endomesoderm should induce proliferation of the graft cells. However, the graft should form eye tissue rather than flank tissue (Cebra-Thomas, 2004)