Abstract

In the developing nervous system the Notch signaling pathway has recently been shown to be necessary and sufficient for neural stem cells to differentiate into astroctyes, a process called astrogliogenesis. However, the mechanism of how Notch signaling regulates this process has not yet been characterized. Our lab is examining whether Notch signaling controls the expression of the genes NFIA and Sox9 which are known to regulate astogliogenesis. These candidate genes were selected because their DNA sequences have characteristics that indicate their expression could be controlled by Notch through a transcriptional complex of Notch and other proteins, including the DNA binding protein RBP/J. We developed a low-cost, effective method of qPCR to study the relative abundance of gene expression in mice that underwent selective deletion of the RBP/J gene in neural progenitors (including neural stem cells). We found that disrupting Notch signaling in neural progenitors of the developing central nervous system caused a significant decrease in both NFIA and Sox9 mRNA expression. In order to ensure that this trend was reflected at the protein level we performed fluorescence microscopy utilizing antibodies targeting the NFIA protein. To determine if Notch directly regulates NFIA and Sox9 expression we are currently optimizing a Chromatin immuno-Precipitation assay to establish whether the Notch-RBP/J transcriptional complex binds to the NFIA and Sox9 promoter regions that are important to drive their expression. These novel findings offer insight into this putative signaling pathway, advancing our understanding of how neural stem cell differentiation is regulated.