The long term goal of the research in my laboratory is to gain an understanding of the molecular and cellular mechanisms involved in the delineation of the neural plate during early mouse embryogenesis and the subsequent restriction of neural epithelial cells to specific precursor fates. The characterisation of three highly homologous SOX transcription factors, SOX1, SOX2 and SOX3, whose expression both reflects and in some cases contributes to neural cell fate, provides us with a unique tool to address this goal. This work involves a genetic approach utilising both the generation and analysis of genetic mutations in the mouse and the establishment of an ex vivo neural cell culture system for isolating neural precursor cells from embryonic stem cells by genetic SOX selection.

SOX domain proteins are a class of developmentally important transcriptional regulators related to the mammalian testis determining factor SRY. The dynamic and diverse patterns of expression of SOX genes and analysis of mutations in humans, mice and Drosophila suggest that SOX factors play key roles in decisions of cell fate during diverse developmental processes, operating in conjunction with co-factors to activate transcription of target genes. SOX domain proteins form a part of a larger family of transcription factors whose DNA binding domains (HMG domains) are related to that of the general chromatin protein HMG-1. In DNA binding studies, SOX proteins exhibit sequence specific binding however, unlike most transcription factors binding occurs in the minor groove resulting in the induction of a bend within the DNA helix. A number of lines of evidence now indicate that SOX proteins by themselves do not exert regulatory potential to activate or repress gene transcription but do so through specific interactions with a partner factor.

The expression profiles of Sox1, Sox2 and Sox3 during mouse embryogenesis suggest these genes could function in the control of neural cell fate. Sox2 and Sox3 begin to be expressed at preimplantation and epiblast stages respectively and are then restricted to the neuroepithelium. The onset of Sox1 expression coincides with induction of the neural ectoderm. Thus, by early gastrulation all three genes are expressed throughout the cells of newly induced neural plate. In light of recent experimental evidence from studies in the chick which suggests that the onset of neural determination may precede gastrulation, in the mouse Sox2 and Sox3 expression may reflect the potential of ectoderm (epiblast) to be neural. After neural induction Sox1, Sox3 expression is confined to proliferating neural precursors, including neural stem cells, along the entire antero-posterior axis of the developing embryo and subsequently in adult neural stem cells. The expression of this Sox gene subfamily has been evolutionarily conserved. The Drosophila, Xenopus , zebrafish and avian putative orthologues of Sox1, Sox2 and Sox3 all show expression throughout the neural primordium. Recent experiments provide compelling evidence for a direct role of SOX-B factors in neural cell fate determination and differentiation. First, Drosophila Dichaete mutants display defects in the specification and differentiation of midline neural cells which can be rescued by murine SOX2. Second, the Xenopus Sox-2 can synergise with FGF signalling to initiate neural differentiation and injection of a dominant interfering forms of Sox2 mRNA inhibits neural differentiation of animal caps caused by attenuation of BMP signals. Finally, through the use of an inducible promoter system which we have established in mouse ectodermal P19 cells, we have shown that mouse SOX1 is sufficient to impart neural fate to P19 cells.

The specific aims that we plan to address are as follows:

To determine whether SOX-B proteins are essential and/or necessary for neural epithelial specification. Through the use of targeted mutagenesis in embryonic stem cells, we will characterise the phenotypic consequences displayed by mice carrying conditional null as well as dominant negative mutations of these genes specifically in neuroepithelial cells.

Generation and characterisation of purified neural precursors from embryonic stem cells by genetic SOX selection. Using a series of antigenic markers with BrDU labelling, we have shown a temporal and spatial correlation between the differentiation of neural cells and downregulation of SOX1, SOX2 and SOX3 expression. Thus, these three SOX genes appear to define proliferating neural precursor of the embryonic Central Nervous System. Furthermore, in vitro clonal analysis of mouse embryonic neuroepithelial cells showed that some of these SOX positive neuroepithelial cells were multipotential and that a single SOX positive cell could generate both neuronal and glial cells. Thus, the restricted expression of SOX-B factors to the neural primordium provides us with a unique tool for the isolation and manipulation of neural precursor cells. We are pursuing a strategy for genetic selection of neural precursors from differentiating embryonic stem (ES) cells. To achieve this goal we have used ES cells in which the geo (lacZ-neo) fusion has been integrated into the Sox2 gene by homologous recombination. When induced to differentiate, under the appropriate conditions, approximately 50% of these cells stained forb-galactosidase activity and for SOX2 and NESTIN, the early neuroepithelial cell marker. When G418 drug was applied to the cultures, selecting for neomycin expressing cells, over 90% of the cells showed b-galactosidase staining and expressed SOX2 and NESTIN. We will further use embryonic stem cells which have various selectable markers targeted into the Sox1 and Sox2 loci (i.e. neomycin, puromycin, TK) to purify populations of neural precursor cells by drug selection. This will provide a culture model for in vivo neural differentiation allowing the process of neural lineage restriction and differentiation to be characterised in detail. Moreover, this system can be used for selectively sorting and amplifying specific neural precursor cell populations for therapeutic use.