Molecular Genetics of Neural Stem Cells

Sohyun Ahn, PhD, Head, Unit on Developmental Neurogenetics
Andrea Barrett, PhD, Postdoctoral Fellow¹
Hui Wang, PhD, Visiting Fellow²
Guannan Ge, BS, Postbaccalaureate Student
Lindsay Hayes, BS, Brown-NIH Graduate Partnerships Program Student
Sherry Ralls, BA, Technician-Biologist

In mammals, new neurons are continuously generated in the mature nervous system through the regulated proliferation and differentiation of quiescent neural stem cells (NSCs). One of the signaling molecules that influences the behavior of NSCs is sonic hedgehog (Shh). Using Genetic Inducible Fate Mapping (GIFM), we have shown that Shh-responsive NSCs self-renew and generate several cell types in the nervous system. Using conditional ablation of major effectors of the Shh signaling pathway,we are continuing to investigate the mechanisms by which Shh signaling maintains and regulates proliferation and differentiation of quiescent NSCs. Furthermore, we are pursuing the identification of novel downstream target genes of Shh signaling in NSCs in order to develop a deeper understanding of stem cell behavior. We have also undertaken novel genetic approaches to study the biological role of newly generated neurons in the adult mouse forebrain by analyzing the neural circuits formed by these newborn neurons. Our studies will provide the foundation needed for stem cell biology to develop therapeutic methods for treating various neurodegenerative diseases.

Molecular mechanism by which Shh acts on neural stem cells

Ge, Wang, Ahn

Shh signaling is mediated by the Smo receptor and by the Gli2 (activator) and Gli3 (repressor) transcription factors in responding cells. As in the 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 the distinct contribution of each effector in NSC biology by using conditional genetic ablation approaches both in vitro and in vivo. First, to investigate the early developmental requirements of Gli2 or Gli3 specifically in neuronal populations, we used Nestin-Cre mice to delete Gli2 or Gli3 from all the neuronal progenitors. Unlike Gli2 null mice, Nestin-Cre;Gli2 conditional mutant mice survive to adulthood. As previously reported, the size and complexity of midbrain and cerebellum are greatly reduced in this mutant allele. However, the forebrain structure appears largely intact, as evidenced by histological analysis. Interestingly, Nestin-Cre;Gli3 conditional mutant mice exhibit various forebrain phenotypes, 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 the ependymal wall of the ventricles in the mutant mice. Second, we are administering Tamoxifen™ to adult Gli1-CreER mice to delete conditionally Gli2 or Gli3 in Shh-responding NSCs. Using immunohistochemistry, we are characterizing the consequences of Gli2 or Gli3 deletion in the mice with desired genotypes at both anatomical and molecular levels. Drawing on combinatorial approaches in vivo and in vitro, we will be able to determine how Shh signaling maintains NSCs in their quiescent state or instructs the quiescent NSCs to proliferate and differentiate based on the intracellular balance of Gli activators and repressors.

Downstream target genes of Shh signaling in neural stem cells

Ralls, Ahn

By identifying downstream target genes, we will gain insight into the role played by Shh in NSC maintenance and/or proliferation. We are therefore isolating NSCs from the neurogenic regions of adult mouse forebrain based on their responsiveness to Shh signaling (Gli1-positive) and 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 the EGFP reporter protein in the desired cell population. Using FACS (fluorescence-activated cell sorting), we have successfully isolated Gli1⁺;GFAP⁺ neural stem cells from two neurogenic regions of the forebrain: the subventricular zone (SVZ) and hippocampus. We are currently using an Affymetrix microarray approach to identify genes expressed only in the stem cells of the SVZ and hippocampus. Identification of downstream target genes will shed light on Shh’s role in neural stem cell maintenance and/or proliferation.

Neural circuit formation by newly generated neurons in the dentate gyrus of the hippocampus

Barrett, Chan,³ Ahn

Click for a larger version.
Figure 6.1
New granule neurons are generated from Shh-responding NSCs in the dentate gyrus of hippocampus and send out projections toward the CA3 region.

In the hippocampus, dentate gyrus (DG) granule neurons are continuously generated from NSCs located in the subgranular layer of the DG. By marking and following the projections of newborn granule cells with Tau^mGFP reporter mice (generated in the Arber laboratory) (see Figure 6.1), we are investigating how NSC-derived newborn neurons integrate into existing circuits. To study the formation of the hippocampal trisynaptic circuits from the DG to CA3 to CA1, we 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. By uncovering neural circuit formation by the newly generated neurons, we will be able to gain insights into the function and purpose of continued neurogenesis in normal mice. A parallel study using mutant mice or mouse models of neurological disease will help us understand the consequences of malformed neural circuits for learning and memory.

Genetic lineage of midbrain dopaminergic neurons

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 may be segregated into several clusters that include the substantia nigra pars compacta and the ventral tegmental area. DA neurons in these regions exhibit distinct patterns of axonal projections and innervate specific target areas and their depletion or aberrant projections result in neurological disorders such as Parkinson’s disease or schizophrenia. It has been also shown that Shh signaling is critical in specifying DA neural progenitors during early neural development. Using GIFM, 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. In particular, we are addressing whether Shh-responding and Shh-expressing cells at different stages contribute to distinct DA neuron subpopulations that ultimately innervate functionally segregated target regions.

¹Joined, January 2008
²Joined, December 2007
³Amanda Chan, former Summer High School Student

Collaborators

Jin Woo Kim, PhD, Korea Advanced Institute of Science and Technology, Daejeon, Korea
Cheil Moon, PhD, Kyungpook National University, Daegu, Korea
Mark Zervas, PhD, Brown University, Providence, RI

For further information, contact ahnsohyun@mail.nih.gov.