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Molecular Genetics of Neural Stem Cells

Sohyun Ahn, PhD, Head, Unit on Developmental Neurogenetics
Lindsay Hayes, BS, Predoctoral Fellow: Brown-NIH Graduate Partnerships Program
Sherry Ralls, BA, Technician-Biologist
Hui Wang, PhD, Visiting Fellow
Cheol Lee, PhD, Visiting Fellow
Anna Kane, Predoctoral Intramural Research Training Award Fellow
Emma Karey, Postbaccalaureate Intramural Research Training Award Fellow

The laboratory continues to investigate the role of Sonic hedgehog (Shh) and its downstream signaling components in the developing and mature nervous system. We demonstrated a requirement for Gli3, a major negative regulator of Shh pathway, in intermediate progenitors during embryonic cortical development. We are currently investigating the role of Shh downstream effectors in adult neurogenesis using conditional gene ablation approaches in mouse. In addition, we have identified several genes that are specifically expressed in adult neural stem cells and are in the process of validating their expression and function. We are also investigating the distinct spatial and temporal contribution of Shh and Gli1 lineage cells in dopaminergic neuron development in ventral midbrain to better understand the origin of diversity among dopamine neurons and their circuit formation.

The role of Gli3 in neurogenesis

Gli3, one of three vertebrate Gli transcription factors in the Hedgehog (Hh) pathway, is processed into a repressor form (Gli3R) in the absence of Hh signal and acts as the major negative transducer of the pathway. Although the role of Gli3 in embryonic patterning has been extensively studied, its role in cortical neurogenesis, especially in the regulation of neural progenitors in proliferation and cell fate specification, is largely unknown. In order to bypass the patterning defects caused by loss of Gli3, we conditionally deleted Gli3 after patterning is complete in mouse (Gli3^cko). Our results from birthdating and in utero electroporation experiments demonstrate that the Gli3, specifically Gli3R, is critical for specifying the fate of cortical neurons that are generated according to a stereotypical temporal order. Moreover, Gli3 is required for maintaining the cortical progenitors in active cell cycle, suggesting that cells may acquire differentiated status in response to the loss of Gli3 during neurogenesis. Around birth, embryonic neural stem cells (NSCs) undergo a transition to form the postnatal neurogenic niche in the subvenricular zone (SVZ) of the lateral ventricle. The cytoarchitecture of the SVZ is arranged in a pinwheel structure, in which multi-ciliated ependymal cells surround a central cluster of postnatal NSCs. Given our previous results showing that Gli3, a negative transducer of Sonic hedgehog (Shh) pathway, is required for maintenance of cortical progenitors, we investigated whether Gli3 plays a similar role in the postnatal NSCs. We found that the establishment of pinwheel structures in the SVZ was disrupted in Gli3cko mice where Gli3 is conditionally deleted with Nestin-Cre mice. Specifically, the cells in the SVZ of Gli3^cko mice displayed characteristics of both ependymal cells and NSCs: multi-cilia and GFAP expression. Since both ependymal cells and NSCs originate from embryonic NSCs, our findings suggest that Gli3 is critical for their fate specification and establishment of postnatal cytoarchitecture. In addition, ependymal cell-specific deletion of Gli3 using FoxJ1-Cre also resulted in upregulation of NSC markers, suggesting that Gli3 is also required for maintenance of ependymal cell characteristics and suppression of NSC features.

The genetic lineage of midbrain dopaminergic neurons

Dopamine neurons (DNs) in the ventral midbrain (vMb) play diverse roles in cognitive function, emotion, addiction, and motor control. However, the origin of such diversity has not been extensively studied. We obtained evidence for the importance of the early genetic lineages of DNs that may determine one aspect of such diversity, the spatial distribution within the vMb. Specifically, we investigated the contribution of cells derived from progenitors expressing Sonic hedgehog (Shh) and Gli1, the downstream target gene of Shh signaling. Using Genetic Inducible Fate Mapping (GIFM) approaches, we found that Shh and Gli1 lineages contribute to a spatially overlapping population of DNs with little distinction. However, the temporal contribution of Gli1 genetic lineage preceded the Shh lineage by one day in development, suggesting that the Shh-response may pre-set the future Shh-expression domain within the vMb. In addition, the spatial contribution of Gli1 lineages changed from medial to lateral DNs reflecting Gli1's dynamic expressions between E8.5-E11.5. In contrast, Shh lineages contributed to a broader domain encompassing both medial and lateral DNs. Interestingly, we found that there is a progression from anterior to posterior contribution of both lineages, suggesting important differences between anterior and posterior DNs. In addition, we found that Shh is expressed approximately a day later in Gli1-lineage cells, which had turned off Gli1 expression. Thus, there is a wave of medial to lateral shift in the Gli1 expression domain that is taken over by Shh expression in vMb. In order to understand whether the inhibition of autocrine Shh signaling is a prerequisite for induction and maintenance of Shh expression within vMb and proper DN development, we are performing gain-of-function and loss-of-function of Shh signaling in Shh- or Gli1-expression domains. Taken together, our results should enhance our understanding of the temporal sequence of events necessary for proper development of DNs of vMb.

Hippocampal neurogenesis

Temporal and spatial control of marking cells of specific genetic lineages provides valuable insights into gene function during developmental and maturation processes. However, the current GIFM approaches utilize only one recombination event that results in an irreversible marking. We developed a dual GIFM system that allows us to control recombination events at two distinct time points with CreER(T2) and FlpE:PR, which are tightly regulated with tamoxifen and RU486, respectively. In addition, we have developed two universal reporter mouse lines that conditionally express fluorescent protein–tagged wheat germ agglutinin (tWGA:FP), which is transferred across synapses during neural activities. Combinatorial use of our dual GIFM with the novel trans-synaptic reporter system enables us to trace the neural circuits that are formed between adult-born granule neurons from Shh-responding neural stem cells and pyramidal neurons of CA3 and subsequently of CA1.

Gene expression profiling of adult neural stem cells and their niche

Stem cells reside in the "niche," a sheltering microenvironment that is thought to coordinate normal homeostatic production of functional mature cells. We investigated the nature of the niche signals responsible for the unique properties of neural stem cells (NSCs) in the subventricular zone (SVZ) of the lateral ventricle. While several studies have linked known signaling molecules, including Shh and Wnts, to adult neurogenesis, a systematic profiling of autocrine/paracrine signaling molecules in the neurogenic regions involved in maintenance, self-renewal, proliferation, and differentiation of NSCs has not been done. We took advantage of the GIFM system and transgenic mice to isolate distinct cell types of the neurogenic SVZ including NSCs, ependymal cells, astrocytes and transit-amplifying cells. We are currently validating the secretory molecule expression profiling (SMEP) of each cell type using the Signal Sequence Trap method.

Additional Funding

DOD Grant (2009-2011)

Publications

Carney RSE, Mangin J-M, Hayes L, Mansfield K, Sousa V, Fishell G, Machold R, Ahn S, Gallo V, Corbin J. Sonic hedgehog expressing and responding cells generate neuronal diversity in the medial amygdala. Neural Dev 2010;5:14.
Boudin CM, Gritli-Linde A, Ahn S, Harfe BD. Shh pathway activation is present and required within the vertebrate limb bud apical ectodermal ridge for normal autopod patterning. Proc Natl Acad Sci U S A 107;5489-5494.

Collaborators

Regina Armstrong, PhD, Uniformed Services University of the Health Service, Bethesda, MD
Joshua Corbin, PhD, Children's National Medical Center, Washington, DC
Brian Harfe, PhD, University of Florida, Gainesville, FL
Yosuke Mukoyama, PhD, Genetics and Developmental Biology Center, NHLBI, Bethesda, MD
Mark Zervas, PhD, Brown University, Providence, RI

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