Thesis (Ph.D.)--University of Central Florida, 2009. / Adviser: Kiminobu Sugaya. Includes bibliographical references (p. 45-50).

published_or_final_version / Anatomy / Master / Master of Philosophy

MicroRNAs are short RNA molecules composed of 20-22 nucleotides. They highly accurately indicate cell identity and hence they are useful in labeling cells and tacking lineage commitment. However, this requires accurate microRNA profiling of cells in individual developmental stages. Since microRNAs are important negative regulators of eukaryotic gene expression, microRNA profiling allows better understanding of molecular regulatory networks of important cellular events, such as adult neurogenesis. Adult neurogenesis is the process in which neurons, as well as glia, are generated from neural stem cells. It was found to be responsible for brain regeneration, olfactory discrimination, memory formation and learning. Depression was suggested to be related to dysregulation of neurogenesis. Thus, knowledge in cellular and molecular mechanisms of adult neurogenesis will lay solid foundation to develop therapies to regenerate neural cells after injuries or onsets of neurodegenerative diseases and to understand the cognitive ability, memory formation and learning of the brain. In spite of its importance, investigation into the miRNA profiles and functions in neurogenesis is still infant. This project aimed to establish a preliminary microRNA profile on neurogenesis. Although this was not completed, the project could be extended to a large-scale microRNA profiling in neurogenesis. This would enable future workers to track the lineage commitment, the migration, and the distribution of NSCs and their derived cells accurately by in situ hybridization. Also, the future workers may construct a 2D representation of the changes in miRNA profiles and this may lead to discovery of previously unknown molecular and cellular differences among cells of same cell identity. / published_or_final_version / Anatomy / Master / Master of Medical Sciences

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Neurogenesis occurs in exclusive regions in the adult nervous system, the subventricular zone and dentate gyrus in the brain, and olfactory epithelium (OE) in the periphery. Cell replacement after death or injury, occurs to varying degrees in neural tissue, and is thought to be dependent upon the biological responses of stem and/or progenitor cells. Despite the progress made to identify adult OE and central nervous system (CNS) progenitors and lineage trace their progeny, our spatial and temporal understanding of embryonic OE neuroglial progenitors has been stalled by the paucity of identifiable genes able to distinguish individual candidate progenitors. In the developing CNS, radial glia serve as both neural progenitors and scaffolding for migrating neuroblasts and are identified by the expression of a select group of antigens, including nestin. Here, I show that the embryonic OE contains a novel radial glial-like progenitor (RGLP) that is not detected in adult OE. RGLPs express the radial glial antigens nestin, GLAST and RC2, but not brain lipid binding protein (BLBP), which, distinct from CNS radial glia, is instead found in olfactory ensheathing cells, a result confirmed using lineage tracing with BLBP-cre mice. Nestin-cre-mediated lineage tracing with three different reporters reveals that only a subpopulation of nestin-expressing RGLPs activate the “CNS-specific” nestin regulatory elements, and produce spatially restricted neurons in the OE and vomeronasal organ. The dorsal-medial restriction of transgene-activating cells is also seen in the embryonic OE of Nestin-GFP transgenic mice, where GFP is found in a subpopulation of GFP+ Mash1+ neuronal progenitors, despite the fact that endogenous nestin expression is found in RGLPs throughout the OE. In vitro, embryonic OE progenitors produce three biologically distinct colony subtypes, that when generated from Nestin-cre/ZEG mice, produce GFP+ neurons, recapitulating their in vivo phenotype, and are enriched for the most neurogenic colony subtype. Neurogenesis in vitro is driven by the proliferation of nestin+ progenitors in response to FGF2. I thus provide evidence for a novel neurogenic precursor, the RGLP of the OE, that can be regulated by FGF2, and provide the first evidence for intrinsic differences in the origin and spatiotemporal potential of distinct progenitors during OE development.

Description of the cell cycle features of neural progenitors during late stages of neurogenesis in a gyrencephalic mammal, the ferret.

Our lab has recently found that E2F3, an essential cell cycle regulator, regulates the self-renewal capacity of neural precursor cells (NPCs) in the developing mouse brain. Chromatin immunoprecipitation (ChIP) and immunoblotting techniques revealed several E2F3 target genes, including the polycomb group (PcG) protein, EZH2. Further ChIP and immunoblotting techniques identified the neural stem cell self-renewal regulators p16INK4a and Sox2 as shared gene targets of E2F3 and PcG proteins, indicating that E2F3 and PcG proteins may co-regulate these target genes. E2f3-/- NPCs demonstrated dysregulated expression of EZH2, p16INK4a, and SOX2 and decreased enrichment of PcG proteins at target genes. Restoring EZH2 expression to E2f3+/+ levels restores p16INK4a and SOX2 expression levels to near E2f3+/+ levels, and also partially rescues NPC self-renewal capacity toward E2f3+/+ levels. Taken together, these results suggest that E2F3 controls NPC self-renewal by modulating expression of p16INK4a and SOX2 via regulation of PcG expression, and potentially PcG recruitment.

We sought to determine whether activation of the Wnt signaling pathway altered the function of hNSCs in vitro. We took three approaches to activate Wnt signaling: Wnt3a, constitutively stabilized β-catenin (ΔN90), and the GSK3 inhibitor BIO. While Wnt3a and ΔN90 had no effect on proliferation in both stem cell (+EGF/FGF) and differentiating (-EGF/FGF) conditions, BIO reduced proliferation in both. All methods of Wnt signaling activation promoted neuronal lineage commitment during hNSC differentiation. Furthermore, BIO was able to induce mild neuronal differentiation in stem cell conditions, suggesting that GSK3-inhibition interferes with several pathways to regulate hNSC fate decisions. We also probed BTSC function using BIO-mediated GSK3 inhibition. We found that in stem cell conditions, BIO was able to induce neuronal differentiation, decrease proliferation, and induce cell cycle arrest. Together this data suggests that GSK3-inhibition, possibly through activation of Wnt signaling, may offer a novel mechanism for the differentiation treatment of glioblastomas.

We sought to determine whether activation of the Wnt signaling pathway altered the function of hNSCs in vitro. We took three approaches to activate Wnt signaling: Wnt3a, constitutively stabilized β-catenin (ΔN90), and the GSK3 inhibitor BIO. While Wnt3a and ΔN90 had no effect on proliferation in both stem cell (+EGF/FGF) and differentiating (-EGF/FGF) conditions, BIO reduced proliferation in both. All methods of Wnt signaling activation promoted neuronal lineage commitment during hNSC differentiation. Furthermore, BIO was able to induce mild neuronal differentiation in stem cell conditions, suggesting that GSK3-inhibition interferes with several pathways to regulate hNSC fate decisions. We also probed BTSC function using BIO-mediated GSK3 inhibition. We found that in stem cell conditions, BIO was able to induce neuronal differentiation, decrease proliferation, and induce cell cycle arrest. Together this data suggests that GSK3-inhibition, possibly through activation of Wnt signaling, may offer a novel mechanism for the differentiation treatment of glioblastomas.

Neurogenesis occurs in exclusive regions in the adult nervous system, the subventricular zone and dentate gyrus in the brain, and olfactory epithelium (OE) in the periphery. Cell replacement after death or injury, occurs to varying degrees in neural tissue, and is thought to be dependent upon the biological responses of stem and/or progenitor cells. Despite the progress made to identify adult OE and central nervous system (CNS) progenitors and lineage trace their progeny, our spatial and temporal understanding of embryonic OE neuroglial progenitors has been stalled by the paucity of identifiable genes able to distinguish individual candidate progenitors. In the developing CNS, radial glia serve as both neural progenitors and scaffolding for migrating neuroblasts and are identified by the expression of a select group of antigens, including nestin. Here, I show that the embryonic OE contains a novel radial glial-like progenitor (RGLP) that is not detected in adult OE. RGLPs express the radial glial antigens nestin, GLAST and RC2, but not brain lipid binding protein (BLBP), which, distinct from CNS radial glia, is instead found in olfactory ensheathing cells, a result confirmed using lineage tracing with BLBP-cre mice. Nestin-cre-mediated lineage tracing with three different reporters reveals that only a subpopulation of nestin-expressing RGLPs activate the “CNS-specific” nestin regulatory elements, and produce spatially restricted neurons in the OE and vomeronasal organ. The dorsal-medial restriction of transgene-activating cells is also seen in the embryonic OE of Nestin-GFP transgenic mice, where GFP is found in a subpopulation of GFP+ Mash1+ neuronal progenitors, despite the fact that endogenous nestin expression is found in RGLPs throughout the OE. In vitro, embryonic OE progenitors produce three biologically distinct colony subtypes, that when generated from Nestin-cre/ZEG mice, produce GFP+ neurons, recapitulating their in vivo phenotype, and are enriched for the most neurogenic colony subtype. Neurogenesis in vitro is driven by the proliferation of nestin+ progenitors in response to FGF2. I thus provide evidence for a novel neurogenic precursor, the RGLP of the OE, that can be regulated by FGF2, and provide the first evidence for intrinsic differences in the origin and spatiotemporal potential of distinct progenitors during OE development.