Fezf2 regulates mature neuron maintenance in vitro

Forebrain embryonic zinc-finger family member 2 (FEZF2) is a transcription factor that is expressed at high levels in corticospinal motor neurons and related subcerebral projection neurons from early in development through adulthood. Fezf2 is essential for the development of layer V corticospinal motor neurons, with loss of Fezf2 resulting in a complete absence of corticospinal motor neurons and the corticospinal tract. Despite the continued expression of Fezf2 in the adult brain, a role for Fezf2 in mature neurons has remained elusive. This study primarily aimed to help define the function of Fezf2 in mature projection neuron maintenance in vitro, and identify potential mechanisms of Fezf2 function. To identify FEZF2-related genes, which may contribute to the role of Fezf2 in mature neuron maintenance, microarray analysis was used to identify genes differentially expressed after FEZF2 knockdown in a neural precursor cell line. This analysis revealed potential roles for FEZF2 in neuron projection maintenance and cell survival. Primary neural cultures from transgenic mice expressing GFP under the regulation of the Fezf2 promoter (pFezf2-GFP mice; postnatal day 2) were transduced with one of two Fezf2-shRNAs or non-silencing control viruses co-expressing red fluorescent protein. pFezf2-GFP-positive neurons were analysed by time-lapse imaging or Sholl analysis to quantify projection complexity and length. Fezf2 shRNA expression resulted in at least 53% knockdown of target mRNA, and a significant reduction in projection complexity; both the number of projections and the distance that projections reach from the cell soma. Using time-lapse imaging we found that the majority of cells demonstrating projection retraction died within 96 hours. These results demonstrate that Fezf2 is required for the maintenance of a subset of Fezf2-positive mature cortical projection neurons in vitro. Fezf2 may fulfil this function through potential roles in projection maintenance and/or cell survival. Future experiments to clarify the mechanism of Fezf2 function and test this function in vivo will provide significant new information on the maintenance of cortical neurons in health and disease