The long term goal of this project is to understand how trophic interactions regulate neuronal survival and differentiation during development. The present proposal focuses on the mechanisms of neuronal cell death and replacement because the understanding of this process will provide important insights into the progression of human neurodegenerative disease. We have found that neuronal cell death in the CG occurs concurrently with the onset of neural differentiation and that many dying neurons can be observed prior to the differentiation of target tissues. Interestingly, early cell death is balanced by a process of neuronal replacement. The overall goal of our studies is to determine how processes that have an impact on this early phase of cell death can translate to changes that occur in the later phase when actual decline of neuronal number is observed.
The specific aims of this application are: 1) To test the hypothesis that dying CG neurons are replaced by a limited number of neurogenic precursors, non-neuronal cells from E6 quail ganglia will be sorted and back-transplanted into HH St. 10-12 embryos; or grown in cell culture and tested for neural differentiation; 2) To test the hypothesis that neuronal cell replacement is regulated by Notch receptor signalling, the expression patterns of Notch receptor, its ligands Delta and Serrate, and the intracellular Notch inhibitor Numb will be examined in the developing CG by in situ hybridization, and cell-cell interactions will be disrupted by misexpressing Numb, or by overexpressing a constitutively active- Notch with a retroviral vector; 3) To test the hypothesis that newly differentiated CG neurons require CNTF to prevent cell death, CNTF- dependence of CG neurons at E6, 8, 12, and 14 will be assessed by bioassay and by phospho- STAT activation; the sources of CNTF prior to target tissue differentiation will be determined; and the effects of ribozyme-mediated CNTFRa-knock down on cell death and survival at various stages of CG development will be examined; 4) To test the hypothesis that the preganglionic innervation of the CG releases neuregulin, which acts as a trophic molecule for neurogenic precursors in the CG, the oculomotor complex in the midbrain that innervates the CG will be ablated and number of neurons dying and surviving will be assessed at varying stages of development; the effects of overexpression of neuregulin and ribozyme-mediated neuregulin knock down on preganglionically deprived CGs will be tested.
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