MOLECULAR GENETICS OF NEURAL STEM CELLS
, Head, Unit on Developmental Neurogenetics
Brian Cheney, BS, Postbaccalaureate Student1
Guannan Ge, BS, Postbaccalaureate Student2
Lindsay Hayes, BS, Brown-NIH Graduate Partnerships Program Student2
Sherry Ralls, BA, Technician-Biologist
Amanda Chan, Summer Student
Using the genetic fate mapping approach, we have shown that adult neural stem cells (NSCs) in mouse forebrain respond to Sonic hedgehog (Shh) signaling. The Shh-responsive NSCs self-renew and generate several cell types of the nervous system. Using conditional ablation of major effectors of the Shh signaling pathway, we are currently investigating the mechanism by which Shh signaling maintains and regulates proliferation and differentiation of quiescent NSCs. Moreover, we are pursuing identification of novel downstream target genes of Shh signaling in NSCs in order to understand stem cell behavior. Finally, we have undertaken novel genetic approaches to study the biological role of newly generated neurons in the adult mouse forebrain by analyzing the neural circuits they form. The 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
The Gli2 (activator) and Gli3 (repressor) transcription factors are the major effectors of Shh signaling. In the developing neural tube, Shh-induced activation of the Gli2 transcription factor mediates dorso-ventral patterning, whereas inhibition of the Gli3 repressor (Gli3R) by Shh mediates the anterior-posterior patterning of developing limb. It is thus possible that proliferation of Shh-responding NSCs depends on the relative levels of Gli2 and Gli3R. To dissect the distinct contribution of each effector in NSC biology, we are using conditional genetic ablation approaches. First, we used Nestin-Cre mice to delete Gli2 or Gli3 from all neuronal progenitors in order to investigate the developmental requirements for Gli2 or Gli3 specifically in neuronal populations. Unlike Gli2 null mice, Nestin-Cre;Gli2flox/− 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 null mice forebrain is enlarged. In Nestin-Cre;Gli3flox/− mice, the patterning of forebrain structure seems to be affected to varying degrees; some of the mutant brains have a thinner cortical layer, an enlarged ventricle, and much-reduced hippocampal formation. We are currently characterizing changes in the proliferation and/or specification of progenitors that arise from quiescent NSCs in the mutant brains. Second, we are using Gli1-CreER mice to delete Gli2 or Gli3 from neural stem cells through tamoxifen administration to the adult. To confirm the conditional ablation of the Gli2 or Gli3 gene in tamoxifen-treated Gli1-CreER/+;Gli2flox/−;R26R/+ or Gli1-CreER/+;Gli3flox/−;R26R/+ mice, we isolated lacZ-positive cells, which indicate that the inducible Cre recombinase was active to turn on the reporter protein lacZ. RT-PCR performed on an RNA sample from lacZ+ cells isolated by fluorescence-activated cell sorting (FACS) showed that Gli2 or Gli3 expression was much lower than in lacZ− cells. 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 the maintenance and proliferation of neural stem cells.
Downstream target genes of Shh signaling in neural stem cells
To identify the specific downstream target genes of Shh signaling in NSCs, we isolated such cells from adult mouse forebrain tissues based on the cells’ responsiveness to Shh signaling and their expression of the putative stem cell marker glial fibrillary acidic protein (GFAP). Initially, we used Gli1-EGFP mice, in which green fluorescent protein is expressed from the Gli1 genomic locus in response to Shh signaling. We found that the fluorescent signaling intensity was too weak for successful isolation using FACS. Therefore, we switched to the Gli1-CreER/+;Z/EG system and administered tamoxifen to adult mice to turn on the expression of the enhanced green fluorescent protein (EGFP) reporter protein conditionally. We successfully isolated RNA from Gli1+;GFAP+ NSCs in the subventricular zone and hippocampus of the forebrain. We are currently using an Affymetrix® microarray approach to identify specific genes that are expressed only in the stem cells of the subventricular zone and hippocampus. Identification of downstream target genes will help us understand the role played by Shh in neural stem cell maintenance and/or proliferation.
Neural circuit formation by newly generated neurons in the dentate gyrus of the hippocampus
In the hippocampus, dentate gyrus (DG) granule neurons are continuously generated from neural stem cells located in the subgranular layer of the DG. To study the function of these newborn neurons in the hippocampal neural circuit, we have begun to generate reporter mice that express a trans-synaptically transferable fluorescent reporter protein. Currently, we are testing various fluorescent protein and tagging systems to select the one with the best fluorescence intensity in mammalian cells. Once we complete the transgenic construct, we will produce transgenic mice and select the best line based on the reporter protein expression level. Using this system, we will analyze neural circuits formed by newborn granule neurons connecting the DG to regions CA3 and CA1. Moreover, we will be able to track the projections that emerge from the hippocampus into other regions of the brain.
1 Left, July 2007.
2 Joined, July 2007.
COLLABORATOR
Mark Zervas, PhD, Brown University, Providence, RI
For further information, contact ahnsohyun@mail.nih.gov.
