Gene expression analysis of neuronal precursors from adult mouse brain and differential screen for neural stem cell markers

In the adult mouse brain, neuronal precursor cells continuously emanate from neural stem cells (NSC) in the subventricular zone (SVZ) and migrate into the olfactory bulb (OB) where they differentiate to serve as replenishment for GABAergic interneurons. During the migration process, PSA-NCAM (Polysialic acid-Neural cell adhesion molecule) specifically marks the neuronal precursors (PSA+ cells). This phenomenon was exploited in the framework of this doctoral thesis to isolate a homogeneous cell population of neuronal precursor cells using Fluorescence-activated cell sorting. Here, the first comprehensive picture of the gene expression in PSA+ precursors was generated using Serial Analysis of Gene Expression (SAGE). Comparison of SAGE data for PSA+ cells and for adult total brain (ATB) led to the identification of precursor-enriched genes. For selected genes, the results were validated using cDNA microarrays and quantitative real-time PCR, and the expression was analyzed at the cellular level in mouse brain using in situ hybridizations. Genes previously described in this context like the proliferation inhibitor CD24, the sialyltransferase STX and the Reelin receptor ApoER2 confirmed the identity of the precursor cells and the accuracy of the SAGE. Individual characterized genes that were so far unknown in the PSA+ cell population were identified as well as functional groups of genes by means of cluster analysis of SAGE data. The presence of transcription factors of the Sox and Dlx families, Pax6 and Meis2 indicated that secondary neurogenesis might be largely controlled by the same factors that are active during development. Clusters for apoptosis and proliferation are both upregulated. The high expression of chemotactic factors in the neuronal precursors suggests that they might be involved in neuronal cell migration. In addition, novel genes like RIKEN 3110003A17 were observed. First functional data based on the SAGE are being generated in the framework of our collaboration with the Developmental Biology Institute of Marseille. Given that a lack of markers for NSC considerably impedes progress in NSC biology, the second part of this work aimed at identifying potential NSC markers by comparing SAGE data for embryonic stem (ES) cells, PSA+ cells and ATB. The selection strategy was based on two assumptions. First, in a hierarchical order of developmental potential, ES cells are positioned above NSC, which are above restricted precursors that in turn are above adult neurons and glia. Second, the genetic programs of ES cells and NSC overlap. Thus, genes that are highly expressed in ES cells and downregulated or absent in PSA+ neuronal precursors and ATB should in part also be expressed by the few stem cells in the adult brain. Eight candidates coding for cell surface proteins were identified from the resulting list of candidates and were investigated. Due to a public database mistake in situ hybridizations were performed for the glutamate transporter GLT1 and demonstrated expression in embryoid bodies, neurospheres and, strikingly, in the SVZ, the neurogenic area of the mouse forebrain. Taken together, this doctoral thesis generated the first gene expression profile for PSA+ neuronal precursors, which -together with the SAGE library for Bruce-4 ES cells- will serve as a starting basis for future functional analysis.