Throughout the developing spinal cord, Olig2+ progenitors in the motor neuron progenitor domain give rise to an impressive array of motor neurons, oligodendrocytes and astrocytes. Motor neurons are further diversified into motor columns and pools based on cell body settling position, general axonal trajectories, and the individual muscles they innervate. Elegant studies have demonstrated that motor neuron columnar and pool diversity, along the rostral-caudal axis of spinal cord, is programmed by extrinsic signals that confer a combinatorial Hox code at each rostral-caudal coordinate. However, we are only beginning to understand the signals that control motor neuron diversification and neuronal versus glial competency within a given rostral-caudal segment level of spinal cord. As a key mediator of cell-to-cell communication, the Notch signaling pathway has been implicated as a primary player in the generation of intra-domain cellular diversity throughout development. Despite this, the role of Notch signaling in contributing to neural diversity within the motor neuron progenitor domain has remained elusive.

The major hurdle to studying the role of Notch in the motor neuron progenitor domain has been the inability to specifically manipulate Notch signaling in motor neuron progenitors. In this dissertation, I use embryonic stem cell (ESC) to motor neuron differentiation technology to demonstrate that Notch signaling has a dual role during motor neuron differentiation. In Chapter 2, I demonstrate that Notch signaling is required for inhibiting motor neuron differentiation and maintaining a subset of progenitors for oligodendrocyte genesis via lateral inhibition. Activation or inactivation of Notch signaling during ESC to motor neuron differentiation is capable of disrupting lateral inhibition and generating homogenous cultures of either glial precursors or motor neurons. Interestingly, induction of Notch signaling during differentiation is sufficient to upregulate glial markers Sox9 and Sox10, suggesting that Notch also plays an instructive role in specifying glial cell fate. In Chapter 3, I show that Notch signaling regulates motor neuron columnar identity.

Specifically, I demonstrate that Notch signaling is required for selection of medial motor column (MMC) identity and that inhibition of Notch signaling during motor neuron differentiation leads to rostral-caudal appropriate conversion of MMC identity into hypaxial motor column (HMC) identity in cervical conditions or lateral motor column (LMC) identity in brachial conditions. I further identify the transition from progenitor to postmitotic motor neuron as the critical period where Notch activity is necessary to select motor neuron columnar identity. Previous studies have proposed that an Olig2/Ngn2 competition model controls motor neuron differentiation. In Chapter 5, I show that contrary to this hypothesis, Olig2 does not inhibit motor neuron differentiation and that Olig2 and Ngn2 largely bind and regulate different sets of genes during motor neuron differentiation. Comparing genome-wide binding and gene expression data after Ngn2 induction, I identify the early gene expression program directly downstream of Ngn2 that drives motor neuron differentiation.