The Pevny Lab

Room 8113 Neuroscience Research Bldg University of North Carolina CB #7250 115 Mason Farm Rd Chapel Hill, NC 27599 Lab Phone: 919-843-4581 Dr. Pevny's Phone: 919-843-5541 larysa_pevny@med.unc.edu

The adult nervous system is composed of a large diversity of cell types that arise from the neural plate, a sheet of morphologically indistinguishable epithelial cells. Once induced, the stem cells of the neural plate undergo a rapid expansion and a combination of epigenetic and genetic mechanisms act to specify regional fate along the future anteroposterior and mediolateral axes of the plate. Coupled with these patterning mechanisms is neurogenesis - the differentiation of neuroepithelial precursors into neurons, oligodendrocytes and astrocytes, the three major postmitotic cell types that constitute the mature CNS. It is now becoming apparent that multipotential neural stem cells, with the capacity for at least limited self-renewal, are present throughout development of the nervous system, initially in the cells of the neural plate and then in the ventricular zone of the neural tube, and persist into adulthood in certain locations. Neural stem cells can also be derived from more primitive embryonic stem cells. The relationship between "stem cell" populations at different stages of development remains unclear. The overall objective of our research is to gain insight into the common molecular and cellular mechanisms that are involved in conferring neural identity to stem cells during embryogenesis and the adult. This work focuses on the role of three transcription factors, SOX1, SOX2, and SOX3 (the SOXB1 subfamily) in this process. We have shown SOXB1 transcription factors to be expressed in neural progenitor cells throughout mouse embryogenesis and into adulthood. Therefore, we have generated transgenic mice expressing green fluorescent protein (EGFP) under the control of the endogenous locus regulatory regions of the Sox2 gene to prospectively identify neural stem/progenitor cells in vivo and in vitro. Fluorescent cells co-express SOX2 protein, and EGFP fluorescence is detected in proliferating neural progenitor cells of the entire anterior-posterior axis of the CNS from neural plate stages to adulthood. SOX2-EGFP cells can form neurospheres that can be passaged repeatedly and can differentiate into neurons, astrocytes and oligodendrocytes. Since the cells expressing SOX2 are marked with EGFP, this allows us to directly and prospectively isolate cells with neural stem cells potential from the embryonic and adult CNS using FACS. To date, our preliminary data illustrates that prospective clonal analysis of SOX2-EGFP positive cells shows that all neurospheres, whether isolated from the embryonic CNS or the adult CNS, express SOX2-EGFP (Ellis et al., 2005).  SOXB1 factors not only mark neural progenitor cells but function to maintain their identity. Our past studies provide direct evidence for a role of the SoxB1 genes in the acquisition of early neural identity by murine ectodermal cells (Pevny et al., 1998). More recently we have shown that SOXB1 factors maintain neural progenitor identity. These data provide evidence that constitutive expression of SOX2 inhibits neuronal differentiation, resulting in the maintenance of progenitor characteristics. Conversely, inhibition of SOX2 signaling results in neural progenitor cells delaminating from the ventricular zone and exiting from the cell cycle. This is associated with both a general loss of pan-neural and regional progenitor markers and the onset of expression of early neuronal differentiation markers (Graham et al., 2003).  Our experiments now aim to directly address the function of SoxB1 genes in neural progenitors. This work involves a genetic approach in the mouse to assess the function of SoxB1 genes in embryonic and adult neural progenitors. Specifically we have generated an allelic series of the Sox2 gene in mice to analyze the effects of graded reduction of SOX2 on neural progenitor differentiation. The direct comparison of SOXB1 function in vivo, in the embryo as well as in the adult, will begin to elucidate similarities and/or differences in the molecular mechanisms during neural progenitor differentiation. Such lessons from the embryo and adult may increase the ability to manipulate neural stem cells for therapeutic applications.