The longterm goals of this proposal are to establish the role of cell adhesion molecules in initiation of migration and in the formation of neural crest-derived ganglia. Because it is not yet possible to directly monitor cell-cell interactions in the embryo, our studies will utilize perturbation approaches to disrupt normal interactions and examine the subsequent abnormalities in neural crest migration. We will use an injection technique to introduce antibodies and enzymes onto neural crest pathways. By blocking the function of N-CAM or N-cadherin molecules, we aim to determine the importance of these molecules during normal neural crest development. We will also look at the effects of inappropriate expression of cell adhesion molecules in neural crest cells. We have prepared a variety of constructs with full length N-CAM cDNA linked to the beta-actin or SV40 promoter and will prepare similar constructs coding for N-cadherin. By microinjecting the these constructs into premigratory neural crest cells within the dorsal neural tube, these will be used to cause inappropriate expression of adhesion molecules in neural crest cells in vivo. We can identify injected cells using a simple histochemical assay by co-injecting cDNA encoding the Escherichia coli beta-galactosidase gene. In addition, we will examine the behavior of cell lines transfected with N- CAM or N-cadherin after injection onto neural crest pathways of the embryo to see whether these cells migrate and localize in normal neural crest sites. These experiments will test if ectopic expression of a cell adhesion molecule is sufficient to impede migration and/or cause premature gangliogenesis. The proposed experiments combine molecular biology, experimental embryology, and tissue culture to explore: 1. the distribution and developmental regulation of N-CAM and N-cadherin during neural crest cell migration and gangliogenesis. 2. the effects on neural crest cell emigration from the neural tube and ganglion formation of immunologically or biochemically perturbing the function of N-CAM and N-cadherin. 3. the behavior of N-CAM or N-cadherin-transfected cell lines implanted onto neural crest pathways. 4.the effects of over-expression of N-CAM and N-cadherin on neural crest cells in situ. The over-expression experiments allow one to study altered, identifiable cells in an otherwise normal embryonic environment. These studies will clarify normal mechanisms of neural crest cell emigration from the neural tube and formation of neural crest-derived ganglia. In addition, they may shed light on birth defects such as tuberous sclerosis which may be related to abnormal expression or regulation of N-CAM resulting in tumorous growths in many neural crest-derived tissues.
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