Sohyun Ahn, PhD, Head, Unit on Developmental Neurogenetics
Jihyun Yoon, PhD, Visiting Fellow
Brian Cheney, BS, Postbaccalaureate Fellow

Sonic hedgehog (Shh) signaling is involved in various developmental processes, such as patterning, cell specification, and proliferation. Mutations in Shh signaling pathway have been associated with numerous human diseases, including cancer. Recently, Shh signaling has also been implicated in promoting the proliferation of neural stem cells (NSCs) in the forebrain. Using the genetic fate mapping approach in mice, we have discovered that adult NSCs in the forebrain respond to Shh signaling; they also self-renew and generate several cell types found in the nervous system. These results provided the first in vivo evidence of an endogenous cell population that meets all criteria of NSCs. We are interested in elucidating the mechanism by which Shh signaling maintains and regulates proliferation and differentiation of quiescent NSCs during normal and pathological conditions in adult mouse. Moreover, discovery of novel downstream target genes of Shh-signaling in NSCs will provide further understanding of stem cell behavior. We also plan to investigate the biological role of newly generated neurons in the adult mouse forebrain by analyzing the neural circuits formed by such neurons. The studies will provide the foundation needed for stem cell biology to develop therapeutic methods for treating various neurodegenerative diseases.

The molecular mechanism by which Shh acts on neural stem cells

Ahn, Cheney

The Gli2 (activator) and Gli3 (repressor) transcription factors are the major effectors of Shh signaling. In the developing neural tube, dorso-ventral patterning is mediated by Shh-induced activation of the Gli2 transcription factor, whereas the anterior-posterior patterning of developing limb is mediated through inhibition of the Gli3 repressor (Gli3R) by Shh activity. It is thus possible that proliferation of Shh-responding NCSs depends on the relative levels of Gli2 and Gli3R. We have begun analyzing the requirement for Gli2/3 by determining the fate of adult NCSs (Gli1-CreER-derived cells) in Gli2 and Gli3 mutant backgrounds, using conditional mutant alleles of Gli2 and Gli3 mice (generated in the Joyner laboratory). In addition to Gli1-CreER mice, we are using 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.

The downstream target genes of Shh signaling in neural stem cells

Yoon, Ahn

Despite considerable interest in identifying novel genes specifically expressed in NCSs, the lack of specific markers has made it difficult to isolate a pure population of endogenous stem cells. We can, however, isolate NCSs from adult mouse forebrain tissue based on their responsiveness to Shh signaling and their expression of the putative stem cell marker GFAP. We can also isolate Shh-responding cells from Gli1-lacZ or Gli1-EGFP mice in which the reporter genes encoding beta-galactosidase or green fluorescent protein are expressed from the Gli1 genomic locus in response to Shh signaling. After isolating NCSs based on their coincidental expression of GFAP and Gli1 (EGFP+ or lacZ+) cells from the SVZ and DG of adult mouse forebrain, we plan to investigate gene expression profiles by using Affymetrix microarray. Identification of downstream target genes will provide insight into the role played by Shh in NCS maintenance and/or proliferation.

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

Ahn, Cheney; in collaboration with Zervas

New granule neurons are continuously generated throughout the life of an animal. These neurons may modulate synaptic activity within hippocampal neural circuits, which may serve as an underlying mechanism for learning and short-term memory formation. However, only a limited number of newly generated neurons survives and integrates into the granular layer of dentate gyrus of hippocampus. We plan to investigate how these newborn neurons integrate into existing circuits. More specifically, we will mark and follow the projections of newborn granule cells by using, for example, Z/EG reporter mice in which green fluorescence protein (EGFP) is expressed in the fate-mapped cells (Shh-responding, Gli1-CreER-marked cells). We are also investigating the establishment of second-order synaptic connections from DG to CA3 to CA1 by using reporter mice in which the tagged protein will be trans-synaptically transferred (wheat germ agglutinin reporter mice), allowing us to visualize changes in synaptic connections from the newly generated granule cells of DG to CA1. By establishing neural circuit formation by these newly generated neurons, we will be able to gain insight into the function and purpose of continuous neurogenesis in normal mice. A parallel study using mutant mice or mouse models of neurological disease will enable us to understand the consequences of malformed neural circuits in behavioral outputs such as defects in learning and memory.

COLLABORATOR

Mark Zervas, PhD, Brown University, Providence, RI

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