? DESCRIPTION (provided by applicant): Vertebrate neurogenesis is a highly regulated process during which a limited number of neural stem cells produce a highly diverse number of glial and neural cell types. Understanding the molecular mechanisms of vertebrate neurogenesis has important implications in human brain evolution as well as several developmental disease states. During neurogenesis, neural stem cells self-renew and produce more committed progeny through asymmetric cell division, which involves coordinated cell polarization and spindle orientation. Recent attempts to determine the contribution of spindle orientation to neocortical neurogenesis, especially the genetic manipulation of mammalian Inscuteable (mInsc) and its binding protein LGN (mammalian Pins homologue) in mice, have led to conflicting results. While it is clear that both LGN and mInsc control cleavage plane orientation, their mutant phenotypes are different. Loss of LGN induces more oblique divisions, but does not obviously affect the rates of neuronal production. Overexpression of mInsc also induces oblique divisions; however, neuronal production is significantly increased in this case. These results call into question the exact role of spindle orientation during neurogenesis. Furthermore, Notch signaling has been shown to be differentially regulated in neural stem cells versus intermediate progenitors in the developing neocortex. How Notch signaling is coupled with spindle orientation and asymmetric cell division in mammalian systems remains largely unknown. In preliminary studies, we discovered a direct physical interaction between LGN and the intracellular domain of Notch (N-ICD). We found that LGN negatively regulates N-ICD-mediated transcriptional activation and that slight overexpression of LGN results in enhanced neuronal differentiation of pluripotent neural progenitor cells in vitro. We propose that in additin to directing spindle orientation, mammalian Pins (including LGN and its homologue AGS3) contribute to cell fate specification by direct modulation of Notch signaling activity. The goal of the proposed research is to establish a novel function of mammalian Pins as intrinsic modulators of Notch signaling activity that may link asymmetric cell division to cell fate specification during neocortical neurogenesis. Through combined molecular, cellular, biochemical and in utero electroporation approaches, we will 1) further characterize the mammalian Pins-Notch interaction and determine how mammalian Pins regulate Notch signaling activity; 2) demonstrate the significance of the mammalian Pins-Notch interaction for cell fate specification during mouse neocortical neurogenesis. Recently, mutations of human LGN gene have been causally linked to the Chudlley-McCullough syndrome (CMS). CMS is characterized by congenital sensorineural hearing loss and different forms of brain abnormalities, indicating that LGN is critical for normal human brain development. Studying the mechanisms by which LGN contributes to neurogenesis will provide insights into common CMS-like human brain abnormalities.
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