Sonic hedgehog (Shh) is a critical signaling molecule that governs diverse developmental processes as well as postnatal neurogenesis in the brain. The Gli family of transcription factors (Gli1, Gli2, Gli3) are activated or modified in response to Shh activity. 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. My laboratory has investigated the role of Gli3 prior to the onset of Shh signaling in regulation of neural stem/progenitors in developing brain and in postnatal neurogenic niche. The role of Gli3 in cortical development: Although the role of Gli3 in embryonic patterning has been extensively studied, its role in cortical development, especially in the regulation of proliferation and cell fate specification of neural progenitors, is largely unknown. When we conditionally removed Gli3 after the embryonic patterning is complete in mice, resulting Gli3 conditional mutants had enlarged lateral ventricles of the forebrain and thinner cortex compared to the controls. Our results from birth-dating experiments showed that upper layer cortical neurons were not produced properly and there was a prolonged production of deeper layer cortical neurons in Gli3 conditional mutants. Further investigation with in utero electroporation experiments showed that the Gli3, specifically Gli3R, is critical for specifying the fate of cortical neurons that are generated following a stereotypical temporal order. Moreover, we found that Gli3 mutant cells became post-mitotic prematurely, leading to a depletion of proliferating neural progenitors by late gestational stages. Our findings indicate that Gli3 is required for maintaining the cortical progenitors in active cell cycle, suggesting that cells may acquire differentiated status as they turn off Gli3 expression during neurogenesis. The role of Gli3 in postnatal neurogenesis: Around birth, the embryonic neural stem cells (= radial glia cells) undergo a transition to form the postnatal neurognic niche in the subventricular zone (SVZ) of the lateral ventricle. Neural stem cells in the SVZ are in close contact with other types of cells including ependymal cells. While the niche cells are known to provide environmental cues to regulate adult neurogenesis, little is known about how the neurogenic niche is initially established and maintained. Thus, we investigated the signaling mechanism that underpins how distinct cell types are produced in the neurogenic niche. We discovered that Gli3 repressor (Gli3R), a negative regulator of Shh pathway, is critical for establishment and maintenance of the postnatal neurogenic niche in the SVZ in mouse forebrain. We first found that radial glia cells require Gli3R during development to correctly specify postnatal ependymal cells and neural stem cells. Using either genetic mutants or electroporation to remove Gli3 function in vivo, we then showed that proper neurogenesis depends on the continuous presence of Gli3R in ependymal cells - as opposed to neural stem cells. We found that Gli3R maintains an appropriate level of Numb, a negative regulator of the well-characterized juxtacrine signal Notch pathway, in ependymal cells to influence neurogenic activity of neighboring neural stem cells. Our findings highlight the importance of restricted expression of negative regulators of Shh and Notch pathways (Gli3R and Numb, respectively) in ependymal cells for proper neurogenesis and provides a better understanding of the interplay between neural stem cells and their environment. Gene expression profiling of adult neural stem cells and their niche: In order to understand the nature of the niche signals that regulate the behavior of neural stem cells in the subventricular zone (SVZ) of the lateral ventricle, we isolated highly purified neural stem cells and putative niche cells from the SVZ including ependymal cells, astrocytes, and transit-amplifying cells using flow cytometry, and choroid plexus by microdissection. We first validated the identity and purity of sorted cells by immunocytochemisty and confirmed the behavior of neural stem cells in vitro, i.e. self-renewal and multi-potency. Next, we obtained the secretory molecule expression profiling of each NSC niche cell type using Signal Sequence Trap (SST) method. The list contained genes that were previously implicated in the SVZ neurogenesis (cystatin C, ApoE) as well as newly discovered genes with unknown role in the neurogenesis. We confirmed the expression of identified genes both in vitro (freshly sorted cells) and in vivo (tissue sections). We are currently assessing the physiological function of selected genes on neurogenesis in vitro. We are also establishing molecular and genetic network among the SMEP by using bioinformatic tools such as Cytoscape and Ingenuity Pathway Analysis.
