Understanding the intricacies of CNS cell lineage is a major goal of current work in developmental neuroscience. The fundamental mechanisms underlying the processes whereby CNS progenitors are generated and the factors guiding them from multipotent undifferentiated cells to unipotent, terminally differentiated neurons and glia have not been elucidated. Central to the current proposal is that an understanding of the gene expression patterns of CNS progenitor cells and their progeny at different stages is a critical step in forwarding our understanding of these processes. In our preliminary experiments we have used representational difference analysis coupled to microarray screening to identify novel markers of early differentiative events in the embryonic brain, and developed relatively high throughput methods for cataloging expression patterns of these genes. The experiments set forth here will: 1. Create a large cDNA microarray enriched for genes expressed by CNS progenitor cells as well as committed cells at early stages of differentiation. 2. Determine, in a rapid manner, the developmental expression pattern of those mRNAs found to be the most interesting. 3. Test the ability of this newly created resource (the microarray) to determine the differences in gene expression amongst different populations of CNS and other progenitor cells. We anticipate that these studies will not only directly enhance the understanding of CNS progenitor cell biology, but that they will also pave the way for other experiments using similar technology by showing the feasibility of the methodology and provide a microarray resource of early progenitor enriched genes for others to use. The following Specific Aims will be achieved: 1. A 6000 gene microarray will be constructed using cDNAs derived from an RDA subtraction designed to enrich for genes expressed by CNS progenitor and precursor cells at various stages of commitment. 2. Differentially expressed genes will be prioritized for further study based on a stepwise screen designed to confirm microarray expression data and efficiently characterize genes of interest. This screen includes high throughput sequencing, Northern blot, and determination of developmental expression patterns by in situ hybridization. 3. The """"""""neurodevelopmental"""""""" microarray will be used to compare gene expression among progenitor populations derived from different brain regions, time points and different growth factor conditions. We hypothesize that differences in gene expression patterns will identify similarities and differences between these conditions that will have important functional consequences. The proposed research will significantly improve our understanding of CNS gene expression in two ways. First, we estimate that we will determine the expression pattern of nearly 500 novel or previously uncharacterized genes in the developing brain by screening in situ hybridization. Second, our newly created microarray will allow us to test gene expression patterns in a variety of experimental conditions including various cultures containing CNS progenitors, as well as in models of CNS disease or injury.
| Karsten, Stanislav L; Kudo, Lili C; Jackson, Robert et al. (2003) Global analysis of gene expression in neural progenitors reveals specific cell-cycle, signaling, and metabolic networks. Dev Biol 261:165-82 |
| Geschwind, D H; Ou, J; Easterday, M C et al. (2001) A genetic analysis of neural progenitor differentiation. Neuron 29:325-39 |
| Irvin, D K; Zurcher, S D; Nguyen, T et al. (2001) Expression patterns of Notch1, Notch2, and Notch3 suggest multiple functional roles for the Notch-DSL signaling system during brain development. J Comp Neurol 436:167-81 |
| Terskikh, A V; Easterday, M C; Li, L et al. (2001) From hematopoiesis to neuropoiesis: evidence of overlapping genetic programs. Proc Natl Acad Sci U S A 98:7934-9 |
| Kornblum, H; Geschwind, D (2001) The use of representational difference analysis and cDNA microarrays in neural repair research. Restor Neurol Neurosci 18:89-94 |
