Larysa H. Pevny - US grants

DESCRIPTION (provided by applicant): The vertebrate central nervous system (CNS) is composed of an intricate array of neural cell types that originate from both stem and progenitor cells located within the neural tube. The co-ordinated generation of this complex network of neural cells underlies the ability of the CNS to function properly. Any alteration in the development fate, or the degeneration of any one of these cell types, can lead to CNS dysfunction. The overall objective of the proposed research is to gain insight into the common molecular and cellular mechanisms that are involved in the correct differentiation of neural stem cells both during embryogenesis and in the adult animal. This work will focus on the role of three transcription factors, SOX1, SOX2, and SOX3 (the SOXB1 subfamily) in neural differentiation. In mammals, the onset of SOX1 expression and the concomitant restriction of SOX2 and SOX3 expression to the neural epithelium are coincident with neural induction. The expression of the SOXB1 subfamily is then maintained in proliferating neural epithelial cells throughout embryogenesis and into adulthood. Thus, the SOXB1 subfamily appears to mark a common transcriptional program shared by stage specific neural progenitors and provides a potential means by which to define, isolate, and characterize neural progenitor cells at different developmental stages. Moreover, our past studies provide direct evidence for a role of the SOXB1 subfamily in neural differentiation. The work outlined in this proposal involves a genetic approach in the mouse to assess the function of SoxB1 genes in embryonic and adult neural progenitors. In the long term, results from work will lead to clinical applications such as transplantation therapy in animal models of neurodegenerative diseases. Only through a thorough understanding of the cellular and molecular mechanisms will researchers be able to efficiently direct stem cell differentiation into specific cell types needed for transplantation.

ABSTRACT NOT PROVIDED

[unreadable] DESCRIPTION (provided by applicant): Neural stem cells are self-renewing multipotent progenitors that give rise to neurons, astrocytes and oligodendrocytes in the central nervous system (CNS). However, to date it remains unclear whether there exists a generic neural stem cell, as found in the hematopoietic system. It appears that the CNS consists of heterogenic stem cells that, although restricted in their potency, all retain the ability to self-renew, differentiate, and express of a set of universal markers. To understand exactly what characteristics define a neural stem cell it is first necessary to elucidate the lineage relationship between the various types of stem cells and how they contribute to the formation and maintenance of the central nervous system. In this application we propose genetic and cellular assays to determine the contribution of SOX2-expressing cells and function of SOX2 during adult neurogenesis. The experiments are based on our findings that SOX2 universally marks cells with in vitro stem cell potential isolated from all stages of mouse CNS ontogeny (Ellis, 2004; Brazel, 2005). We will take advantage of this neural stem/progenitor specific SOX2 expression to investigate the cellular identity of adult multipotent neural stem cells (NSCs) directly in vivo using genetic lineage tracing and cell-ablation strategies in the mouse. Furthermore, we have recently demonstrated that SOX2 functions to maintain embryonic and retinal neural progenitor identity (Graham, 2003; Taranova, 2005). We therefore hypothesize that the molecular signaling pathway regulated by SOX2 to define neural stem/progenitor identity during embryogenesis also acts to maintain their cellular and molecular profile throughout ontogeny. To address this we propose a conditional mutagenesis approach to test the role of SOX2 specifically in adult neural progenitors. At the end of the funding period we will have a comprehensive view of fate and role of SOX2 expressing cells and the function of the SOX2 signaling cascade in regulating neural stem cell identity and differentiation in the adult CNS. Understanding the origin, fate and function of NSCs will advance efforts to manipulate NSCs for therapeutic purposes. [unreadable] [unreadable] [unreadable]

The pluripotency of ES cells, combined with their ease of genetic manipulation and selection, has revolutionized gene functional studies in vivo via the generation of transgenic, chimeric and knockout mice. New technologies are evolving that will make the ability to perform appropriate manipulations of ES cells even more important for the next generation of mouse mutants (Draper and Nagy, 2007). These include, for example, the combination of inducible recombinases (Cre and Flp) for regulated temporal and spatial ablation of genes, RNAi, and development of new techniques that accelerate generation of completely ES cell derived FO mice (i.e. tetraploid aggregation and laser-assisted injection). The main service of Core 4 is to provide to NINDS-funded Qualifying Investigators a high level of expertise in mouse ES cell targeting in order to permit the efficient generation of animal models for neurobiological research. In addition to ES cell electroporation and characterization, the staff of the Core 4 will work with the NINDS-funded investigators to establish and characterize ES cell lines from a number of genetic backgrounds (particularly C57BI/6) that allow mice to be generated on a pure genetic background. Finally, Core 4 will maintain a bank of genetically marked ES cell lines. The ES cells and mice generated from these lines will be used for in vivo lineage tracing and in vitro differentiation into neuronal and glial cell lines.

[unreadable] DESCRIPTION (provided by applicant): Specification of neural progenitors in the central nervous system (CNS) and maintenance of their proliferative and differentiation capacities largely depend on the function of transcription factors. One such transcription factor, expressed in all neural progenitors of the CNS, is SOX2. We have shown that SOX2 is required for proliferation and differentiation of retinal neural progenitors. Furthermore, we have shown that lowering expression levels of SOX2 (below 40%) in mice results in restriction in retinal progenitor competence and leads to anophthalmia and microphthalmia--conditions observed in 10% of humans with haploid insufficiency in SOX2. Based on these findings we hypothesize that SOX2 functions to specify retinal neural progenitors and to maintain their proliferative and differentiation capacity during the course of retinogenesis. In this study, we will directly assess the effects of Sox2 ablation and reducing its expression levels on the properties of temporally distinct populations of retinal progenitors. [unreadable] PUBLIC HEALTH REVELANCE: We will utilize mouse genetic tools and cellular assays to dissect the molecular mechanisms affected by the expression levels of SOX2 in retinal progenitors. [unreadable] [unreadable] [unreadable]