Abstract
The mature cortex contains hugely diverse populations of pyramidal projection neurons (PNs), critical to normal forebrain circuits. In order to understand the healthy cortex, it is essential to characterize this neuronal complexity. We recently demonstrated different identities for Fezf2-positive (Fezf2+ve) and Fezf2-negative (Fezf2−ve) intratelencephalic-PNs (IT-PNs) from layer 5 of the motor cortex (M1). Comparatively, each IT-PN type has a distinct electrophysiological phenotype and the Fezf2+ve IT-PNs display a unique apical dendritic tuft. Here, we aimed to expand our understanding of the molecular underpinnings defining these unique IT-PN types. Using a validated Fezf2-GFP reporter mouse, retrograde labeling techniques and fluorescence activated cell sorting (FACS), combined with a novel approach for low-input RNA-sequencing, we isolated mature Fezf2+ve and Fezf2−ve IT-PNs for transcriptome profiling. Through the comparison of Fezf2+ve and Fezf2−ve IT-PN gene expression profiles, we identified significant enrichment of 81 genes in the Fezf2+ve IT-PNs and 119 genes in the Fezf2−ve IT-PNs. Term enrichment analysis of these enriched genes demonstrated significant overrepresentation of the calcium-binding EF-hand domain in Fezf2+ve IT-PNs, suggesting a greater importance for calcium handling in these neurons. Of the Fezf2−ve IT-PN enriched genes an unexpected and unique enrichment of genes, previously associated with microglia were identified. Our dataset identifies the molecular profiles of two unique IT-PN types in the mature M1, providing important targets to investigate for their maintenance in the healthy mature brain.
Highlights
Thecerebral cortex of the brain is a layered structure essential for higher cognitive function
We found clear separation of Fezf2+ve and Fezf2−ve IT-projection neurons (PNs) types according to their molecular profiles, rather than the host animal from which they came
We confirmed that injection of the tracer at two sites in M1 could label GFP(Fezf2)+ and GFP(Fezf2)- IT-PNs in the opposite hemisphere of the Fezf2-Gfp M1 (Figure 1)
Summary
Thecerebral cortex of the brain is a layered structure essential for higher cognitive function. PNs are grouped into subtypes according to their projection type (e.g., to the contralateral cortex or corticospinal tract; Molyneaux et al, 2007). Analyses have shown much greater diversity in PN types, both across and Transcriptome Profiling Mouse Projection Neurons within layers of the cortex (Hattox and Nelson, 2007; Oswald et al, 2013; Rouaux and Arlotta, 2013; Tantirigama et al, 2016). Recent analyses of intra-telencephalic projection neurons (IT-PNs) within layer 5 of the mature mouse primary motor cortex (M1) identified two unique subtypes that were defined by the expression or absence of transcription factor Fezf (Tantirigama et al, 2014). Characterization of the unique genetic profiles that underpin such neuronal diversity is vital to our understanding of their maintenance in a healthy adult brain
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