Previously we have shown that adult neural stem cells in mouse forebrain respond to Sonic hedgehog (Shh) signaling using the genetic fate mapping approach in mice. These Shh-responsive NSCs self-renew and generate multiple cell types of the nervous system. We are currently investigating the mechanism by which Shh signaling maintains and regulates proliferation and differentiation of the quiescent NSCs by conditional ablation of major effectors of Shh signaling pathway. Moreover, we are pursuing identification of novel downstream target genes of Shh-signaling in NSCs to further understand the stem cell behavior. Finally, we have undertaken novel genetic approaches to study the biological role of the newly generated neurons in the adult mouse forebrain by analyzing the neural circuits formed by these newborn neurons. These studies will provide the necessary foundation for stem cell biology to develop therapeutic methods in treating various neurodegenerative diseases. ? ? Ahn, S. and Joyner, A.L. (2005) In vivo analysis of quiescent adult neural stem cells responding to Sonic hedgehog. Nature 437, 894-97? ? The molecular mechanism by which Shh acts on neural stem cells? Cheney, Ge, Ahn? ? The Gli2 (activator) and Gli3 (repressor) transcription factors are the major effectors of Shh signaling. In developing neural tube, the dorso-ventral patterning is mediated by Shh-induced activation of Gli2 transcription factor whereas the anterior-posterior patterning of developing limb is mediated through the inhibition of Gli3 repressor (Gli3R) by Shh activity. It is thus possible that proliferation of Shh-responding neural stem cells depend on relative levels of Gli2 and Gli3R. In order to dissect out the distinct contribution of each effector in neural stem cell biology, we are using conditional genetic ablation approaches. First, we have used the Nestin-Cre mice to delete Gli2 or Gli3 from all the neuronal progenitors to investigate the developmental requirements of Gli2 or Gli3 specifically in neuronal populations. Unlike straight Gli2 null mice, Nestin-Cre; Gli2 flox- mice survive to adulthood. The midbrain and cerebellum are greatly reduced in size and complexity in this mutant allele. Moreover, the lateral ventricle of the forebrain is also enlarged in the mutant. We are currently characterizing the changes in the proliferation andor specification of progenitors that arise from the quiescent neural stem cells in these mutant brains. Second, we are using Gli1-CreER mice to delete Gli2 or Gli3 from neural stem cells through Tamoxifen administration in adult. In order to confirm the conditional ablation of Gli2 gene in Tamoxifen treated Gli1-CreER; Gli2 flox-; R26R mice, we isolated the lacZ-positive cells, which indicate that the inducible Cre recombinase has been active to turn on the reporter protein, lacZ. RT-PCR performed on the RNA sample from the FACS-isolated lacZ cells showed that Gli2 expression was greatly reduced compared to lacZ- cells using RT-PCR. Now that we have confirmed that the conditional ablation approach works in the adult mice, we are characterizing the fate of the mutant cells in maintenance and proliferation of neural stem cells.? ? The downstream target genes of Shh signaling in neural stem cells? Ralls, Ahn? ? In order to identify the specific downstream target genes of Shh signaling in the neural stem cells, we have isolated such cells from adult mouse forebrain tissues based on their responsiveness to Shh signaling and on their expression of the putative stem cell marker, GFAP. Initially, we were using the Gli1-EGFP mice, in which green fluorescent protein is expressed from the Gli1 genomic locus in response to Shh signaling. We have found that the fluorescent singaling intensity was too weak for successful isolation based on FACS. Therefore, we have switched to the Gli1-CreER; ZEG system: Tamoxifen is administered in adult mice to conditionally turn on the expression of EGFP reporter protein. We have successfully isolated RNA from the Gli1; GFAP neural stem cells from the subventricular zone and hippocampus of the forebrain. We are currently undertaking the Affymetrix microarray approaches to identify specific genes that are only expressed in the stem cells of SVZ and hippocampus. Identification of downstream target genes will provide an insight into how Shh plays a role in neural stem cell maintenance andor proliferation. ? ? The neural circuit formation by newly generated neurons in the dentate gyrus of the hippocampus? Ahn, Chan; in collaboration with Zervas? ? In the hippocampus, dentate gyrus (DG) granule neurons are continuously generated from the neural stem cells located in the subgranular layer of DG. In order to study the functional role of these newborn neurons in the hippocampal neural circuit, we have started the generation of the reporter mice, which will express the trans-synaptically transferrable fluorescent reporter protein. Currently, we are testing various fluorescent protein and tagging system to select the one with the best fluorescent intensity in mammalian cells. Once the transgenic construct is made, we will produce transgenic mice and select the best line based on the reporter protein expression level. Using this system, we will be analyzing the neural circuit formed by newborn granule neurons in DG to CA3 to CA1. Moreover, we will be able to track the projections that emit from the hippocampus into other regions of the brain.
