The development of a functional organ system not only entails the determinative events that lead to cell type specificity and appropriate patterning of those cell types, but also the ability to maintain this identity and pattern in the face of geneic and environmental perturbations that occur throughout embryogenesis. This ability to compensate for potentially disruptive alterations in development, which is often referred to regulation, is a near-universal characteristic of embryos, one that is required to ensure normal development. However, there has been relatively little focus on elucidating the mechanisms governing the process of regulation following a developmental perturbation, despite its clear importance for a complete understanding of development as well as its implications for regenerative medicine. Here we employ the classic amphibian embryological system of Xenopus laevis to examine regulative ability during early embryogenesis. Previous experiments from our lab have demonstrated that when the presumptive neural plate is removed from a gastrula-stage embryo, rotated 180o, and transplanted back into a host embryo from which the equivalent region was removed, there is a near total regulation, with the resulting embryo giving rise to a nervous system with appropriate regional gene expression and functional capabilities. The overall goal of this proposal is to examine the mechanisms governing this profound regulative ability. The first specific aim will assess the temporal and spatial expression profile f candidate genes important for the process of regulation, with the goal of obtaining baseline information to determine precisely when and how this regulative process occurs. These genes include: regional markers (XCG-1, Otx-2, En-2, Krox-20, HoxB9);later differentiation genes (GAD, xvGlut1);genes expressed earlier in the neural determination pathway (FoxD5, Geminin, Sox2);and genes encoding key anterior-posterior signaling molecules (Frzb-1, xWnt8, and FGF8).
The second aim will determine if specific signaling cascades mediate the process of regulation by perturbing their function using a targeted morpholino knockdown approach. Finally, unbiased global gene expression approaches, specifically microarray analysis and an RNA-seq approach, will identify additional molecular components of the regulation process. Taken together, the experiments proposed in these three aims will engage an eager cadre of undergraduate students at all levels in an effort to address a fundamental and poorly studied problem in developmental biology that has broader implications for regenerative medicine.
Understanding the mechanisms governing early developmental neural plasticity will not only shed light on diseases in which plasticity is compromised but also provide important information and strategies for regenerative medicine. Knowledge of the mechanisms by which transplants integrate and adapt to new cellular environments will be essential for developing viable stem cell and tissue engineering strategies.
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