Synthetic modified Fezf2 mRNA (modRNA) with concurrent small molecule SIRT1 inhibition enhances refinement of cortical subcerebral/corticospinal neuron identity from mouse embryonic stem cells.

Cameron Sadegh,Wataru Ebina,Jeffrey D Macklis,Anthony C Arvanites,Lance S Davidow,Lee L Rubin,Makoto Sato

doi:10.1371/journal.pone.0254113

Abstract

During late embryonic development of the cerebral cortex, the major class of cortical output neurons termed subcerebral projection neurons (SCPN; including the predominant population of corticospinal neurons, CSN) and the class of interhemispheric callosal projection neurons (CPN) initially express overlapping molecular controls that later undergo subtype-specific refinements. Such molecular refinements are largely absent in heterogeneous, maturation-stalled, neocortical-like neurons (termed “cortical” here) spontaneously generated by established embryonic stem cell (ES) and induced pluripotent stem cell (iPSC) differentiation. Building on recently identified central molecular controls over SCPN development, we used a combination of synthetic modified mRNA (modRNA) for Fezf2, the central transcription factor controlling SCPN specification, and small molecule screening to investigate whether distinct chromatin modifiers might complement Fezf2 functions to promote SCPN-specific differentiation by mouse ES (mES)-derived cortical-like neurons. We find that the inhibition of a specific histone deacetylase, Sirtuin 1 (SIRT1), enhances refinement of SCPN subtype molecular identity by both mES-derived cortical-like neurons and primary dissociated E12.5 mouse cortical neurons. In vivo, we identify that SIRT1 is specifically expressed by CPN, but not SCPN, during late embryonic and postnatal differentiation. Together, these data indicate that SIRT1 has neuronal subtype-specific expression in the mouse cortex in vivo, and that its inhibition enhances subtype-specific differentiation of highly clinically relevant SCPN / CSN cortical neurons in vitro.

Highlights

Subcerebral projection neurons (SCPN) are the broad population of cerebral cortex neurons that connect and provide high-level descending control via axonal projections from the neocortex to distal targets in the brainstem and spinal cord [1,2,3,4]
We found that Sirt1 knockdown in primary dissociated E12.5 neocortical neurons recapitulated the effect of small molecule inhibition of Sirtuin 1 (SIRT1), increasing both CTIP2+/SATB2- and CTIP2+/CTIP1- subtype-specific SCPN identity refinements (S4 Fig)
Our data indicate that Sirt1 inhibition or knockdown approaches are effective for the refinement of embryonic stem cell (ES)-derived SCPN/corticospinal neurons (CSN) in vitro, and suggest that SIRT1 is functionally important for the refinement of SCPN/CSN identity in vivo during mouse neocortical development

Summary

Introduction

Subcerebral projection neurons (SCPN) are the broad population of cerebral cortex (cortical) neurons that connect and provide high-level descending control via axonal projections from the neocortex (termed “cortical” here) to distal targets in the brainstem (midbrain, hindbrain) and spinal cord [1,2,3,4]. At later stages of maturation, SCPN discontinue expression of SATB2, and further resolve into diverse subpopulations of FEZF2- and CTIP2-expressing projection neurons with cortical area- and target-specific molecular identities, properties, and functional circuit connectivity [10,11,12]. Multiple epigenetic factors support the post-mitotic identity refinement of contrasting cortical neuron subtypes, such as SCPN and CPN, and enable their maturation [13,14,15,16,17,18]. Together, these reports suggest that chromatin remodeling might contribute to the identify refinement of ES cell-derived cortical neuron subtypes

Methods

Results

Conclusion