Fezf2 expression in layer 5 projection neurons of mature mouse motor cortex

Abstract

The mature cerebral cortex contains a wide diversity of neuron phenotypes. This diversity is specified during development by neuron-specific expression of key transcription factors, some of which are retained for the life of the animal. One of these key developmental transcription factors that is also retained in the adult is Fezf2, but the neuron types expressing it in the mature cortex are unknown. With a validated Fezf2-Gfp reporter mouse, whole-cell electrophysiology with morphology reconstruction, cluster analysis, in vivo retrograde labeling, and immunohistochemistry, we identify a heterogeneous population of Fezf2(+) neurons in both layer 5A and layer 5B of the mature motor cortex. Functional electrophysiology identified two distinct subtypes of Fezf2(+) neurons that resembled pyramidal tract projection neurons (PT-PNs) and intratelencephalic projection neurons (IT-PNs). Retrograde labeling confirmed the former type to include corticospinal projection neurons (CSpPNs) and corticothalamic projection neurons (CThPNs), whereas the latter type included crossed corticostriatal projection neurons (cCStrPNs) and crossed-corticocortical projection neurons (cCCPNs). The two Fezf2(+) subtypes expressed either CTIP2 or SATB2 to distinguish their physiological identity and confirmed that specific expression combinations of key transcription factors persist in the mature motor cortex. Our findings indicate a wider role for Fezf2 within gene expression networks that underpin the diversity of layer 5 cortical projection neurons.

Laminar specific gene expression reveals differences in postnatal laminar maturation in mouse auditory, visual, and somatosensory cortex

“…The antibody recognized 100 kDa and 110 kDa molecular weight on Western blots of mouse brain tissue lysate (manufacturer's technical information). Neurons in layer 5 and layer 6 were immunopositive for this antibody as shown previously by multiple reports in the mouse forebrain (Joshi et al, 2008;McKenna et al, 2011;Tantirigama et al, 2016;Teissier et al, 2010).…”

RNA-Sequencing Analysis Reveals a Regulatory Role for Transcription Factor Fezf2 in the Mature Motor Cortex

“…When injecting the Fezf2 shRNA into the M1 of Fezf2-Gfp mice, no changes in Fezf2 expression were observed (data not shown). We previously demonstrated an increase in Fezf2 CDS expression in the Fezf2-Gfp mouse model (Tantirigama et al, 2016 ), which could have affected the shRNA efficiency. Therefore we chose to complete Fezf2 knockdown experiments in wildtype mice.…”

“…We recently identified Fezf2 expression in diverse pyramidal neuronal subtypes in layer 5 of the mature M1, including PT-PNs and IT-PNs (Tantirigama et al, 2016 ). We therefore wanted to reduce Fezf2 expression globally within the mature M1, in order to target all Fezf2 -expressing neurons (Figure 1A ).…”

“…One such transcription factor, Forebrain embryonic zinc finger 2 ( Fezf2 also known as Zfp312, Fezl, Znf312 ), is expressed in the deep layer PNs (layer 5 and 6), where it is critical to the specification of corticospinal projection neurons (CSpPNs; Chen J.-G. et al, 2005 ; Molyneaux et al, 2005 ). The restricted expression of Fezf2 persists into adulthood (Inoue et al, 2004 ; Ozdinler et al, 2011 ; Tantirigama et al, 2016 ), where high levels of expression in layer 5 were thought to be a CSpPN specific marker (Ozdinler et al, 2011 ). However, recent work found that Fezf2 -expression identifies a diverse group of PNs in layer 5 of the mature primary motor cortex (M1; Tantirigama et al, 2016 ).…”

“…The restricted expression of Fezf2 persists into adulthood (Inoue et al, 2004 ; Ozdinler et al, 2011 ; Tantirigama et al, 2016 ), where high levels of expression in layer 5 were thought to be a CSpPN specific marker (Ozdinler et al, 2011 ). However, recent work found that Fezf2 -expression identifies a diverse group of PNs in layer 5 of the mature primary motor cortex (M1; Tantirigama et al, 2016 ). These comprised of intratelencephalic projection neurons (IT-PNs), which are affected in disorders such as schizophrenia (Shepherd, 2013 ) and pyramidal tract projection neurons (PT-PNs), including CSpPNs that are lost in the progression of amyotrophic lateral sclerosis (ALS; Ozdinler et al, 2011 ).…”