Larysa Pevny

Each cell lineage specified in the preimplantation mammalian embryo depends on intrinsic factors for its development, but there is also mutual interdependence between them. OCT4 is required for the ICM/epiblast lineage, and at transient high levels for extraembryonic endoderm, but also indirectly through its role in regulating Fgf4 expression, for the establishment and proliferation of extraembryonic ectoderm from polar trophectoderm. The transcription factor SOX2 has also been implicated in the regulation of Fgf4 expression. We have used gene targeting to inactivate Sox2, examining the phenotypic consequences in mutant embryos and in chimeras in which the epiblast is rescued with wild-type ES cells. We find a cell-autonomous requirement for the gene in both epiblast and extraembryonic ectoderm, the multipotent precursors of all embryonic and trophoblast cell types, respectively. However, an earlier role within the ICM may be masked by the persistence of maternal protein, whereas the lack of SOX2 only becomes critical in the chorion after 7.5 days postcoitum. Our data suggest that maternal components could be involved in establishing early cell fate decisions and that a combinatorial code, requiring SOX2 and OCT4, specifies the first three lineages present at implantation.

Multipotent neural stem cells are present throughout the development of the central nervous system (CNS), persist into adulthood in defined locations and can be derived from more primitive embryonic stem cells. We show that SOX2, an HMG box transcription factor, is expressed in multipotent neural stem cells at all stages of mouse ontogeny. We have generated transgenic mice expressing enhanced 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 coexpress 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. Moreover, 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. In contrast, the pattern of SOX2-EGFP expression using randomly integrated Sox2 promoter/reporter construct differs, and neurospheres are heterogeneous for EGFP expression. These studies demonstrate that SOX2 may meet the requirements of a universal neural stem cell marker and provides a means to identify cells which fulfill the basic criteria of a stem cell: self-renewal and multipotent differentiation.

Approximately 10% of humans with anophthalmia (absent eye) or severe microphthalmia (small eye) show haploid insufficiency due to mutations in SOX2, a SOXB1-HMG box transcription factor. However, at present, the molecular or cellular mechanisms responsible for these conditions are poorly understood. Here, we directly assessed the requirement for SOX2 during eye development by generating a gene-dosage allelic series of Sox2 mutations in the mouse. The Sox2 mutant mice display a range of eye phenotypes consistent with human syndromes and the severity of these phenotypes directly relates to the levels of SOX2 expression found in progenitor cells of the neural retina. Retinal progenitor cells with conditionally ablated Sox2 lose competence to both proliferate and terminally differentiate. In contrast, in Sox2 hypomorphic/null mice, a reduction of SOX2 expression to <40% of normal causes variable microphthalmia as a result of aberrant neural progenitor differentiation. Furthermore, we provide genetic and molecular evidence that SOX2 activity, in a concentration-dependent manner, plays a key role in the regulation of the NOTCH1 signaling pathway in retinal progenitor cells. Collectively, these results show that precise regulation of SOX2 dosage is critical for temporal and spatial regulation of retinal progenitor cell differentiation and provide a cellular and molecular model for understanding how hypomorphic levels of SOX2 cause retinal defects in humans.