Transcriptional Regulation Over Neurogenesis of Cortical Output Neuron Segmental Identity and Diversity - Subcerebral projection neurons (SCPN) reside in the neocortex, and extend axons to subcerebral targets in the brainstem and spinal cord. Corticospinal neurons (CSN), a subclass of SCPN, project to the spinal cord and their axons form the corticospinal tract (CST), a critical circuit for voluntary motor control. In addition, projections from the neocortex to brainstem targets function in parallel with CSN to exert motor control. Degeneration of these projection neurons in amyotrophic lateral sclerosis (ALS), along with degeneration of spinal motor neurons, causes spasticity and paralysis. In humans, damage to the CST after spinal cord injury is a principal cause of loss of voluntary motor control. Further, integrity of corticospinal connectivity is centrally linked to recovery from stroke and cerebral palsy. There have been multiple investigations detailing the role of cortico-brainstem vs. corticospinal projections in both motor control, as well as their distinct contributions to functional recovery in these distinct causes of paralysis. However, it remains unclear when and how these distinct projections are established during development. Understanding the molecular basis of this specification and differentiation during development therefore holds significant promise in establishing approaches that are tailored to enhancing plasticity of these related, yet distinct circuits. A necessary first step toward this ultimate goal is to identify the molecular mechanisms directing SCPN axons to brainstem (cortico-brainstem neurons) versus spinal cord (CSN). Ongoing work in our lab has identified that such projections are initially specified during the process of axon extension during development. We have identified that cortico-brainstem and corticospinal neurons express distinct genes and can be molecularly distinguished in mice by birth. Further our data suggest that the transcriptional regulator Satb2 acts, in part, to specify cortico-brainstem neurons. This proposal investigates the hypothesis that transcriptional regulation controls the development of cortico-brainstem vs. corticospinal projections by late embryonic development in mice. Building on this foundation, we will first identify the time point in development when these projections are specified using knock-in Cre reporter mice (Aim 1). This will be tested using intracerebral injections of AAV-reporters at distinct developmental times in utero with adult analysis of axonal projections. In Aim 2, we will investigate the transcriptional targets of Satb2 in SCPN at later developmental times by profiling all SCPN in Satb2 WT and conditional KO mice, as well as by Satb2 overexpression, at single cell resolution. Finally, in Aim 3 using subpopulation-specific transgenic Cre lines, we will investigate SCPN axon targeting in both Satb2 loss- and gain-of-function. In addition, we will investigate whether misexpression of Satb2 target genes can alter SCPN targeting to the brainstem vs. spinal targets. Together, our work will discern in-depth, the mechanisms of when and how transcriptional regulation controls SCPN segmental “identity” thereby providing a mechanistic framework for subsequent identification of molecules controlling segmentally appropriate SCPN connectivity with subcerebral targets.
