Sonic hedgehog (Shh) promotes proliferation of neural stem cells (NSCs) in adult brain. However, Shh signaling does not act on NSCs until late gestational stages, suggesting that embryonic NSCs (= radial glia cell) and postnatal NSCs are differentially regulated for their proliferation. Furthermore, postnatal neurogenic niche contains various cell types such as ependymal cells that are also derived from embryonic NSCs around birth. Yet, how the distinct niche cell types are specified remains unclear. We focused on the Gli family of transcription factors (Gli1, Gli2, Gli3), which are activated or modified in response to Shh activity to better understand the molecular regulatory mechanism of Shh signaling. In particular, Gli3 is processed into a repressor form (Gli3R) in the absence of Shh signal and acts as the major negative transducer of the pathway. We have investigated the role of Gli3 as a repressor in two systems in which Shh activity is lacking: the developing dorsal forebrain and the embryonic NSCs. Our findings demonstrate the novel role of Gli3R in regulation of neural stem/progenitors in developing brain and in postnatal neurogenic niche. The role of Gli3 in postnatal neurogenesis: NSCs reside in neurogenic niches and continuously produce new neurons. While niche cells are known to provide environmental cues that regulate adult neurogenesis, how distinct niche cells are produced remains unclear. We demonstrated that embryonic radial glia cells require Gli3R to correctly specify postnatal ependymal cells and NSCs. Moreover, postnatal ependymal cells require Gli3R to maintain their cell identity whereas Gli3R is not required in postnatal NSCs. In addition to the niche structure, the proper neurogenesis depends on the continuous presence of Gli3R in ependymal cells, but not NSCs, suggesting a cell non-autonomous requirement of Gli3R in the SVZ. Finally, we reported in vivo evidence that Gli3R maintains an appropriate level of Numb, a negative regulator of the well-characterized juxtacrine signal Notch pathway, in ependymal cells. The loss of Numb in postnatal ependymal cells led to the compromised neurogenic activity of neighboring NSCs, thus providing the basis for cell non-autonomous role of Gli3 in SVZ neurogenesis. Our findings highlight the importance of the restricted expression of negative regulators of Shh and Notch pathways (Gli3R and Numb, respectively) in the SVZ cell types. Taken together, we provide a novel mechanism of the establishment of the SVZ niche structure and neurogenesis through the interplay between NSCs and environment. Gene expression profiling of adult neural stem cells and their niche: In order to expand the molecular repertoire that controls NSC behavior in the SVZ, we carried out the systematic profiling of signaling molecules present in distinct niche cell types. We focused on the identification of autocrine/paracrine signaling molecules as well as cell-cell contact mediated signaling molecules. We took the advantage of Genetic Inducible Fate Mapping system and transgenic mice and specifically marked and isolated the NSCs and SVZ niche cells including ependymal cells, transit amplifying progenitors (TAPs), astrocytes, and vascular endothelial cells. We then validated the identity and purity of sorted cells and confirmed the behavior of purified NSCs for their ability to self-renew and produce multiple neural cell types in vitro. We obtained the Secretory Molecule Expression Profile (SMEP) of each cell type plus choroid plexus using the Signal Sequence Trap method 28 and identified a total of 151 genes encoding the secretory or membrane proteins. In addition, we obtained the potential SMEP of NSCs using the cDNA microarray technology. We identified and tested the physiological function of novel niche-related genes including Enpp2, Ttr, Sparcl1, and CPE on NSCs in vitro. Together, we provide the first systematic profiling of signaling molecules that are present in distinct or overlapping cell types in the SVZ niche cells (Lee et al., submitted). Now that we have identified novel components related to the niche, we are currently pursuing the physiological in vivo function of these molecules in regulation of NSC behaviors.
