Wnt Regulation of Cell Cycle in Cortical Neurogenesis

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DESCRIPTION (provided by applicant) Wnts are extracellular signals known to regulate neurogenesis in regions associated with developmental diseases such as autism and schizophrenia, yet cell cycle mechanisms mediating these effects remain undefined. We aim to study Wnt-3a's role in forebrain neurogenesis and G1 cell cycle machinery regulation in culture, and in vivo, as follows: l) Embryonic rat cortical cell cultures will be treated with media conditioned by fibroblasts transfected with Wnt-3a constructs. S-phase entry will be assessed by [3H]Thymidine incorporation, and characterized cellularly by bromodeoxyuridine labeling. 2) Wnt-3a's effects on cell cycle regulatory protein levels, including D and E cyclins, cyclin dependent kinases (CDKs), and CIP/KIP CDK inhibitors, will be measured by western blotting, and cyclin/CDK complex activity will be assessed by kinase assays. 3) Acute effects of Wnt-3a on proliferation and cell cycle regulation in vivo, will be determined using transuterine intracerebroventricular (ICV) injection. Sustained effects on neurogenesis will be assessed using GFP:Wnt-3a co-transfection by cell electroporation. Defining these mechanisms mediating Wnt signaling in brain development is critical to understanding and treating pervasive neurological diseases.