| The process of cell fate specification within the developing vertebrate nervous system is highly complex and involves both positive and negative regulatory molecules. Positively acting factors including the proneural basic helix-loop-helix (bHLH) proteins function to activate neuronal differentiation while negative factors including Notch, the enhancer of split genes, and the related molecule hairy block neuronal differentiation. We examined the interactions between positively acting bHLH factors encoded by Neurogenin (X-NgnR-1), Xenopus atonal homologue 3 and 5 (Xath3 and Xath5), and NeuroD and the negative regulators encoded by Xhairy1, Notch, enhancer of spit related 1 (ESR1), and the kinase GSK3beta during development of the neural plate and retina of Xenopus laevis.; Previous studies have indicated that overexpression of the bHLH genes encoded by X-NgnR-1, Xath3 and Xath5, and XNeuroD can promote differentiation during neural plate and retinal stages however the mechanisms that regulate these factors during these events are not well characterized. In contrast, Notch and hairy related genes in other species have been demonstrated to inhibit neuronal differentiation in both the developing CNS and retina. These results led us to hypothesize that the molecules encoded by negative and positive factors interact to regulate neuronal differentiation. To test this, we overexpressed Xhairy1, Notch, and ESR1 in Xenopus embryos and analyzed the ability of these molecules to regulate proneural bHLH activity.; Our results indicate that Xhairy1 functions as a negative regulator of primary neurogenesis by inhibiting the activity and expression of X-NgnR-1, XNeuroD, and Xath3 in a Notch-independent manner and that Xhairy1 function can be attenuated by the kinase GSK3b. We also demonstrate that during neural plate and retinal stages, overexpression of Notch and ESR1 can block the ability of Xath5 to promote neuronal differentiation in the neural plate and developing retina and that the related factor XNeuroD is not regulated by Notch, but like Xhairy1, is instead regulated by the kinase GSK3beta. Together, these studies indicate that differentiation in both the CNS and retina is produced by a balance between interacting positive and negative factors. |
