Neural crest cells possess integrin cell surface receptors and migrate along pathways that are lined with extracellular matrix (ECM) molecules. The longterm goals of this proposal are to identify the cell interactions that influence neural crest cell migration. Because it is not yet possible to directly monitor cell-ECM interactions in the embryo, our studies will utilize a quantitative cell adhesion assay to characterize the molecular nature of cell-matrix interactions under defined culture conditions. Using biochemical analysis and cDNA libraries prepared from cranial and trunk neural crest cells, we will identify the repertoire of alpha and beta integrin subunits expressed by neural crest cells. The goal is to characterize and compare the integrins expressed by neural crest cells at different stages and axial levels. With this baseline information, we will look at the effects of over-expression or inappropriate expression of integrins on neural crest cells in situ. Preliminary results demonstrate that microinjection or lipofection of lacZ cDNA constructs into dorsal neural tube cells results in transient expression of E. coli beta-galactosidase in individual neural crest cells in vivo. By introducing cDNA encoding various normal or mutated integrin subunits, we will cause inappropriate expression of integrins in neural crest cells in vivo. By co-injecting a marker gene or using cDNAs from nonavian species, the injected cells are readily identifiable using simple histochemical assays. This approach is particularly advantageous, since we can examine the behavior of identified """"""""mutant"""""""" cells in an otherwise normal background. In addition to integrin-mediated interactions, other cell-matrix or cell-cell interactions may influence the pattern of neural crest migration. Our preliminary studies demonstrate that inhibitory cues, such as those produced by the notochord and the posterior half of the somite, may play an important role in the patterning of neural crest cells. We will characterize the behavior of neural crest cells in these regions of the embryo and identify candidate molecules involved in inhibition of neural crest migration. The proposed experiments will combine tissue culture, experimental embryology, biochemistry and molecular biology to characterize: 1. the function of the alpha1 subunit of integrin in divalent cation independent adhesion of neural crest cells. 2. the repertoire of integrins expressed by cranial and trunk neural crest cells using PCR analysis of neural crest cDNA libraries. 3. the effects of over-expression or inappropriate expression of integrins in neural crest cells. 4. the inhibitory effect of the notochord on neural crest migration 5. the factors controlling the segmented pattern of neural crest cell migration through the somites.
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