Neuronal differentiation of mouse embryonic stem cells induces Npas4 expression

Npas4, also called LE-PAS or Nxf, is a brain specific transcription factor of the basic helix-loop-helix PAS family. Members of this family are typically involved in developmental processes and cellular response to environmental stresses. Npas4 is expressed in neurogenic regions of the adult brain including the hippocampus and olfactory bulb and is up-regulated in response to various stresses such as seizure, cerebral ischaemia and cortical spreading depression. Animal models of brain injury have shown that both seizure and cerebral ischaemia lead to increased neural stem cell proliferation and neurogenesis. Similarly, cortical spreading depression can also stimulate neurogenesis. Most recently, it was shown that mice raised in social isolation had reduced levels of Npas4 expression. These mice had decreased survival of newly differentiated neurons in the hippocampus and impaired learning. Taken together these data suggest a hypothesis that Npas4 may transcriptionally regulate neurogenesis in the adult mammalian brain following environmentally induced stress. We have used neuronal differentiation of mouse embryonic stem (ES) cells as an in vitro model of neurogenesis to investigate the role of Npas4 in this process. ES cells were differentiated as an adherent monolayer in serum-free conditions for ten days and Npas4 expression was measured by quantitative polymerase chain reaction at 48hr intervals. We observed a significant but transient increase of Npas4 expression associated with neuronal differentiation that peaked at four days after the start of differentiation. We next plan to use genetic manipulation to achieve over-expression and knock-down of Npas4 in ES cell cultures to determine the effect of altered Npas4 expression on the process of neuronal differentiation.