Investigating a role for Fezf2 in the mature brain

The excitatory projection neurons (PNs) of the cortex encode the messages that are critical for mediating higher cognitive function. In the primary motor cortex (M1) PNs integrate signals, which regulate sensory driven and volitional motor behaviours. These PNs are hugely diverse in their function and phenotype and each unit contributes a unique role to the M1 circuitry. Maintaining these distinct identities is essential for the healthy brain. Ascertaining the molecular underpinnings that define unique PNs is essential for understanding how their identity is maintained in the adult brain. Forebrain embryonic zinc finger 2 (FEZF2) is a transcription factor essential to the development of PNs in the cortex. Recent work identified expression of Fezf2 in a diverse group of PN types in the mature M1. In particular, the expression of Fezf2 defines a distinct intratelencephalic (IT)-PN type. When compared to Fezf2-negative IT-PNs these neurons display complex morphology of their apical dendrites and a unique electrophysiological phenotype. These findings allude to a broad role for Fezf2 in maintaining the mature PNs of M1. However, a functional role for Fezf2 in the mature brain is yet to be investigated. The aim of this work was to identify the molecular mechanisms that contribute to maintaining Fezf2-expressing neurons of the mature M1. In order to do this a dual approach was applied; first the molecular profiles of Fezf2-positive and Fezf2-negative IT-PNs of M1 were investigated. Here, a transgenic reporter mouse, expressing GFP under the control of Fezf2 regulatory regions (Fezf2-Gfp) and retrograde labelling of PNs were combined with fluorescence-activated cell sorting (FACS) to identify and isolate the Fezf2-positive and Fezf2-negative IT-PNs. Applying low-input RNA-sequencing methods, transcriptome profiles were generated for both IT-PN types. Analysis revealed 199 differentially expressed genes with further bioinformatics analysis identifying functionally intriguing targets with putative roles for the maintenance of these phenotypically distinct neurons. In particular, an enrichment of protein-encoding mRNAs containing a calcium-binding EF hand domain was found amongst the genes increased in Fezf2-positive IT-PNs, suggesting a need for enhanced calcium handling specifically in these neurons. The second approach aimed to investigate a molecular and functional role for Fezf2 in the mature brain. Lentiviral-mediated delivery of a Fezf2 shRNA was utilised to reduce Fezf2 expression in the mature M1, and this led to the differential expression of 756 genes. Further term enrichment analysis of these Fezf2 regulated genes revealed several putative functional roles for Fezf2 in the mature M1. Intriguingly, the regulation of calcium flux was amongst these functional roles, which overlapped with findings from the molecular profiling of Fezf2-positive IT-PNs. Fezf2 regulated genes also associated with directing locomotory behaviour, implicating Fezf2 in the regulation of adult motor output. This role was explored using Drosophila melanogaster; the conditional knockdown of dfezl (Fezf2 homologue) in adult Drosophila causing significant disruption to their startle-induced climbing behaviour. Together the data presented here demonstrated a clear molecular role for Fezf2 in maintenance of the mature brain. Furthermore, the functional effects of knockdown highlight the importance for such regulatory networks in the mature brain