New neurons are continuously generated in the mature nervous system through the regulated proliferation and differentiation of quiescent neural stem cells (NSCs) in mammals. Among signaling molecules that influence the behavior of NSCs is Sonic hedgehog (Shh). Using the Genetic Inducible Fate Mapping (GIFM), we have shown that Shh-responsive NSCs self-renew and generate multiple cell types of the nervous system. We are continuing the investigation of the mechanisms 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 analysing 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. ? ? The molecular mechanism by which Shh acts on neural stem cells? Ge, Wang, Ahn? Shh signaling is mediated via Smo receptor and the Gli2 (activator) and Gli3 (repressor) transcription factors in the responding cells. As in developing neural tube and limb, the relative levels of Gli2 and Gli3R may play a critical role in determining whether quiescent NSCs exit the cell cycle to generate proliferative precursors. Therefore, we are dissecting out the distinct contribution of each effector in NSC biology by using conditional genetic ablation approaches both in vitro and in vivo. First, we have used the Nestin-Cre mice to delete Gli2 or Gli3 from all the neuronal progenitors to investigate the early developmental requirements of Gli2 or Gli3 specifically in neuronal populations. Unlike straight Gli2 null mice, Nestin-Cre; Gli2 conditional mutant mice survive to adulthood. The midbrain and cerebellum are greatly reduced in size and complexity in this mutant allele as previously reported. However, the forebrain structure appears largely intact evidenced by histological analysis. Interestingly, Nestin-Cre; Gli3 conditional mutant mice exhibit a varying degree of forebrain phenotype including thinner cortical layers, enlarged lateral ventricles, and reduced hippocampal formation. We are currently characterizing the changes in the proliferation and/or specification of progenitors at different developmental stages and the integrity of ependymal wall of the ventricles in these mutant mice. In addition, we are using Gli1-CreER mice to conditionally delete Gli2 or Gli3 in the Shh-responding NSCs through Tamoxifen (TM) administration in adult. Consequences of Gli2 or Gli3 deletion are characterized in the mice with desired genotypes at the anatomical level as well as at molecular level using the immunohistochemistry. Using combinatorial approaches in vivo and in vitro, we will be able to determine how Shh signaling maintains NSCs in their quiescence or instructs the quiescent NSCs to proliferate and differentiate based on the intracellular balance of Gli activators and repressors.? ? The downstream target genes of Shh signaling in neural stem cells? Ralls, Ahn? Identification of downstream target genes will provide an insight into how Shh plays a role in NSC maintenance and/or proliferation. Therefore we are isolating NSCs from the neurogenic regions of adult mouse forebrain based on their responsiveness to Shh signaling (Gli1-positive) and on their expression of the putative stem cell marker, GFAP. Specifically, we are labeling Shh-responding NSCs with Tamoxifen in Gli1-CreER/+; Z/EG mice, which express EGFP reporter protein in desired cell population. We have successfully isolated the Gli1+; GFAP+ neural stem cells from two neurogenic regions of the forebrain, the subventricular zone (SVZ) and hippocampus, using FACS. We are currently undertaking the Affymetrix microarray approache 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 and/or proliferation. ? ? The neural circuit formation by newly generated neurons in the dentate gyrus of the hippocampus? Barrett, Ahn? In the hippocampus, dentate gyrus (DG) granule neurons are continuously generated from the NSCs located in the subgranular layer of DG. As the first step, we are investigating how the NSC-derived newborn neurons integrate into the existing circuits by marking and following the projections of newborn granule cells using the reporter mice TaumGFP (generated in the Arber lab). We have also generated novel WGA (wheat germ agglutinin) reporter mice, in which a trans-synaptically transferable fluorescent protein is expressed in the Shh-responding NSCs and their progeny in order to study the formation of the hippocampal tri-synaptic circuits from the DG to CA3 to CA1. By establishing the neural circuit formation by these newly generated neurons, we will be able to gain insights into the function and purpose of the continued neurogenesis in normal mice. Also a parallel study using mutant mice or neurological disease mouse model will enable us to understand consequences of the malformed neural circuits in behavioral outputs such as defects in learning and memory.? ? The genetic lineage of midbrain dopaminergic neurons? Foresee (Hayes), Ahn: in collaboration with Zervas? Dopaminergic (DA) neurons in the ventral midbrain are involved in various neurological processes including the control of voluntary movements and the regulation of emotion. Midbrain DA neurons can be segregated into several clusters that include the Substantia Nigra pars compacta (SNc) and the Ventral Tegmental Area (VTA). DA neurons in these regions have distinct patterns of axonal projections and innervate specific target areas. Depletion or aberrant projections of DA neurons in these areas result in neurological disorders such as Parkinsons disease or Schizophrenia. It has been also shown that Shh signaling is critical in specifying DA neural progenitors during early neural development. We are testing our hypothesis that specific subclasses of DA neurons are specified by the genetic lineage at distinct developmental stages of their precursors in the ventral midbrain using GIFM. Specifically, we are addressing whether the Shh-responding and Shh-expressing cells from different stages contribute to distinct DA neuron subpopulations that ultimately innervate functionally segregated target regions.
