Title: Subtype diversification and synaptic specificity of stem cell-derived spinal inhibitory interneurons
Description: During nervous system development, thousands of distinct neuronal cell types are generated and assembled into highly precise circuits. The proper wiring of these circuits requires that developing neurons recognize their appropriate synaptic partners. Analysis of a vertebrate spinal circuit that controls motor behavior reveals distinct synaptic connections of two types of inhibitory interneurons, a ventral V1 class that synapses with motor neurons and a dorsal dI4 class that selectively synapses with proprioceptive sensory neuron terminals that are located on or in close proximity to motor neurons. What are the molecular and cellular programs that instruct this remarkable synaptic specificity? Are only subsets of these interneurons capable of integrating into this circuit, or do all neurons within the same class behave similarly? The ability to answer such questions, however, is hampered both by the complexity of the spinal cord, where many different neuronal cell types can be found synapsing in the same area; as well as by the challenge of obtaining enough neurons of a particular subtype for analysis.

Meanwhile, pluripotent stem cells have emerged as powerful tools for studying neural development, particularly because they can be differentiated to produce large amounts of diverse neuronal populations. Mouse embryonic stem cell-derived neurons can thus be used in a simplified in vitro system to study the development of specific neuronal cell types as well the interactions between defined cell types in a controlled environment. Using stem cell-derived neurons, I investigated how the V1 and dI4 cardinal spinal classes differentiate into molecularly distinct subtypes and acquire cell type-specific functional properties, including synaptic connectivity. In Chapter Two, I describe the production of lineage-based reporter stem cell lines and optimized differentiation protocols for generating V1 and dI4 INs from mouse embryonic stem cells, including confirming that they have molecular and functional characteristics of their in vivo counterparts. In Chapter Three, I show that a well-known V1 interneuron subtype, the Renshaw cell, which mediates recurrent inhibition of motor neurons, can be efficiently generated from stem cell differentiation.

Importantly, manipulation of the Notch signaling pathway in V1 progenitors impinges on V1 subtype differentiation and greatly enhances the generation of Renshaw cells. I further show that sustained retinoic acid signaling is critical for the specific development of the Renshaw cell subtype, suggesting that interneuron progenitor domain diversification may also be regulated by spatially-restricted cues during embryonic development. In Chapter Four, using a series of transplantation, rabies virus-based transsynaptic tracing, and optogenetics combined with whole-cell patch-clamp recording approaches, I demonstrate that stem cell-derived Renshaw cells exhibit significant differences in physiology and connectivity compared to other V1 subpopulations, suggesting that synaptic specificity of the Renshaw cell-motor neuron circuit can be modeled and studied in a simplified in vitro co-culture preparation. Finally, in Chapter Five, I describe ongoing investigations into molecular mechanisms of dI4 interneuron subtype diversification, as well as approaches to studying their synaptic specificity with proprioceptive sensory neurons.

Overall, my results suggest that our stem cell-cell based system is well-positioned to probe the functional diversity of molecularly-defined cell types. This work represents a novel use of embryonic stem cell-derived neurons for studying inhibitory spinal circuit assembly and will contribute to further understanding of neural circuit formation and function during normal development and potentially in diseased states.