All living organisms have the innate need to interact with their environment in order to ensure their survival and reproductive success. Among vertebrates, the somatomotor system plays a critical role in responding to environmental challenges and opportunities by coordinating the movements of the body through the activation of its primary effector organs, the skeletal muscles. However, the execution of somatomotor activities is contingent upon maintaining a controlled internal environment that can adapt to the demands imposed by these actions. In this regard, the endocrine and autonomic nervous systems play vital roles by regulating the activity of internal organs and adjusting the composition of the internal milieu in response to both external and internal perturbations. While the endocrine system acts slowly and exerts long-lasting effects on physiological processes, the autonomic nervous system employs rapid neuronal communication to achieve short-lived adjustments in the activity of its target effector organs. By working in tandem, the autonomic nervous system and the endocrine system maintain the overall physiology of the organism across different timescales, ensuring optimal conditions for survival and reproductive success. The autonomic nervous system comprises two divisions, namely the sympathetic and parasympathetic divisions, which have traditionally been considered to work in opposition to each other. This conceptualization was influenced by the work of physiologists like Walter Cannon, who emphasized the role of the nervous system in maintaining homeostasis. Cannon associated the sympathetic nervous system with the unitary physiological responses observed during fight-or-flight behaviors. However, another group of researchers, primarily interested in understanding the neural control of the sympathetic nervous system, highlighted the diverse patterns of activities generated by this system. They proposed that the sympathetic nervous system is capable of exerting differential control over the effector organs. These findings challenged the notion of a unitary response and gave rise to an alternative perspective on the function of the sympathetic nervous system. As a result of these historical developments, we now have two leading ideas regarding the sympathetic nervous system: the homeostatic view, emphasizing its role in coordinating the body's responses to stress and maintaining physiological balance, and the perspective highlighting the differential control and diverse patterns of activity generated by the sympathetic division. These two ideas reflect different aspects of the complex and multifaceted nature of the sympathetic nervous system. The differential control of the sympathetic nervous system, characterized by the decoupled activation of effector organs, has been a driving force in neuroscience research. While medicine has adapted the homeostatic view, the field of neuroscience has explored the patterns of responses within the sympathetic nervous system. Through behavioral, physiological, neurophysiological, and neuroanatomical studies, researchers have provided evidence for the generation of various activity patterns by the sympathetic nervous system. The remaining challenge has been to uncover the mechanisms underlying this differential control. Neuroanatomical investigations have played a crucial role in understanding the functional organization and the differential control of the sympathetic nervous system. Building upon the ideas of Sherrington, anatomical dissections have revealed a hierarchical structure. At each level of this hierarchy, reflexes are integrated within the central and peripheral nervous system. Each level of the hierarchy has been a focus of research to elucidate its specific role. Anatomically these levels correspond to sympathetic ganglia, sympathetic motor neurons, premotor neurons and the higher centers that have top-down influences on the lower centers of the sympathetic nervous system. This dissertation primarily focuses on the lower centers of the hierarchy and aims to provide molecular and circuit-level dissections of each layer of the sympathetic circuitry, from the sympathetic ganglia to the spinal premotor neurons. Modern tools such as sequencing and tracing techniques, which have been refined over the past two decades, have been employed to gain insights into the functions of these neural components. By leveraging these advanced techniques, this dissertation seeks to enhance our understanding of the lower centers of the sympathetic circuit and their contributions to the overall control of sympathetic activity.

The spinal cord represents the final stage of generating motor behaviors, where descending commands or sensory inputs must be transformed into behaviorally-relevant pattern of motor neuron activity. Networks along the rostrocaudal axis of the spinal cord regulate diverse motor behaviors such as respiration, forelimb, trunk, and hindlimb movements, mediated by stringent innervations of motor neurons to muscle fibers. However, how the network properties of the central nervous system enable these diverse motor outputs remains elusive. To address this question, we set out to investigate whether a cardinal class of spinal cord neurons are diversified in diffeernt spinal segment.

This dissertation describes a series of original work that aims to elucidate the diversification of spinal neurons in different segments of the spinal cord. The first chapter is an introduction into the developmental processes of spinal cord development.

The second chapter proceeds from this review to explore whether spinal neurons are further diversified in different spinal segments to underlie distinct network operation and motor outputs. In particular, we studied V2a interneurons as a model to address this question. Using viral tracing and RNA-sequencing, we uncovered how V2a interneurons exhibit distinct anatomical connectivity schemes and distinct genetic signatures in forelimb regulating- and hidnlimb regulating-segments of the spinal cord. It is my hope that our studies establish a framework of how diversification of spinal neurons along the rostrocaudal axis underlies distinct intrinsic network properties in different spinal segments that ultimately contribute to diverse motor outputs that the spinal cord regulates.

The neuronal subtypes that compose the locomotor central pattern generator (CPG) are found in the ventral horn of the spinal cord. Numerous studies have ablated neuronal subtypes and observed the altered activity in an experimental design called fictive locomotion. These ablation studies have provided great insight into the locomotor CPG, but the remaining network is still complex, making it difficult to obtain deep insight into how the circuit actually functions. To simplify this network, we set out to generate an in vitro stand-in for the locomotor CPG that involved differentiating mouse embryonic stem cells (mESCs) into the neurons that comprise the locomotor CPG.

This dissertation describes a series of original work that aims to elucidate the contributions that different cellular components play in the final output of the locomotor CPG. The first chapter is an introduction into the developmental processes of spinal cord development and the diseases that result when such patterning does not occur properly.

The second chapter proceeds from this review to describe a unique and powerful new technique that allows us to separate the complex locomotor CPG into its cardinal neuronal subtypes. From these component parts, we then generated highly defined de novo networks to determine how a network composed of individual neuronal subtypes behaved. Finally, we mixed one inhibitory neuronal subtype into different pure excitatory neuronal subtype based networks. From these mixing studies we were able to determine that this one inhibitory neuronal subtype has strikingly diverse functions in different excitatory networks.

The third chapter discusses future directions for the work described in Chapter 2. Initially I explain how an extension of the experiments conducted in Chapter 2 could be used to address remaining questions about the locomotor CPG. From there I move on to describe how alterations to the basic experimental designs from Chapter 2 would allow for new, diverse, and exciting experimental questions to be asked. All together, Chapter 3 expands upon the work I have conducted in graduate school and suggests different sets of experiments that I believe would result in many interesting works in their own right.

Evidence is mounting that defective RNA metabolism is central to the pathogenesis of diseases affecting motoneurons (e.g. amyotrophic lateral sclerosis and spinal muscular atrophy). Yet, our understanding of motoneuron-specific gene regulatory pathways is largely limited to those mediated by transcription factors. Investigations into motoneuron-specific, RNA-mediated regulatory pathways (such as those involving microRNAs), may provide novel insights into potential pathogenic mechanisms. In this thesis, I identify a single microRNA (miR-218) that is both highly enriched and abundantly expressed in murine motoneurons. Using a combination of RNA sequencing and mouse genetics, I identify novel alternative promoters embedded within the Slit2/3 genes that contribute to miR-218’s specific expression in brainstem and spinal motoneurons.

My most informative and exciting experiments derive from investigation of miR-218 knockout mice, generated by CRISPR-mediated multiplexed deletions of all four miR-218 alleles. Motoneurons in these mice exhibit dramatic neuromuscular synaptic failure, hyperexcitability, and cellular degeneration – the hallmarks of motoneuron diseases. Without miR-218, mice exhibit flaccid paralysis and neonatal death, firmly demonstrating that this microRNA is indispensable to motoneuron function and survival. How can a single, small non-coding RNA have such a fundamental importance to motoneuron gene regulation? Gene profiling wild type and knockout motoneurons uncovers an impressive network of hundreds of mRNAs that are under miR-218 mediated repression. Using differential expression and unbiased 3’UTR motif-enrichment analysis, I find that miR-218 target genes are expressed lower in motoneurons versus other subpopulations of spinal and cortical neurons. Moreover, I find that miR-218 doesn’t merely reinforce/potentiate target genes’ reduced expression (as has been suggested for microRNAs in general), but instead constitutively and independently drives the repression of its target network in motoneurons.

In summary, this thesis (1) details the identification of one of the most dramatic examples of a neuronal subtype-specific microRNA in mammals, (2) establishes that loss of miR-218 results in neuromuscular failure and motoneuron degeneration, and (3) reveals that motoneurons use miR-218 to tune-down a genetic network expressed across other neuronal cell populations.

Movement is the outcome of signaling between the cortex, brainstem, spinal cord and peripheral nervous system onto the final modes of motor output – motor neurons and their target muscles. Simple motor tasks such as reaching for a cup of coffee involve complex neural calculations to selectively recruit and coordinate appropriate muscle and joint combinations. Spinal interneurons are believed to simplify this process by encoding specific patterns of muscle activation, known as ‘motor synergies.’ The deep dorsal horn of the spinal cord is an important area for generating motor synergies. Interneurons in this region receive descending cortical and peripheral sensory inputs and send outputs to motor neurons, suggesting that they integrate high-level cortical commands and sensory feedback for coordinated movements. Recent studies have demonstrated that spinal interneurons can elicit synergistic patterns of muscle activity in the absence of descending and peripheral inputs. This suggests that the spinal cord contains enough local computing power to generate compound movements. Investigations of these circuits have been insightful, however much remains to be learned of the mechanisms by which they direct motor output.

This dissertation describes a series of original work that aims to identify spinal premotor organization in relation their post-synaptic partners, as well as their transcriptional identities. The first chapter presents a new framework for the anatomical organization of functionally distinct spinal premotor networks. Contrary to previous knowledge, we found that these networks are spatially intermingled rather than segregated. In Chapter 2, we evaluate viral vectors for identifying dual-projecting neurons and conclude that Pseudorabies-Bartha is a suitable tool. In Chapter 3, we build circuit maps of synergistic and antagonistic premotor networks. We find that premotor neurons diverge to form dual projections onto multiple motor pools. Finally, in Chapter 4, we perform single-nuclei RNA sequencing of premotor neurons. We determine that they are predominantly ventral, an equal mix of excitatory and inhibitory, and express group-Z markers, which are indicative long-range projecting neurons.

In summary, this work enhances our knowledge of spinal premotor network organization, composition, and development, providing a new framework for the spatial arrangement of flexor and extensor premotor neurons.

A fundamental question in the study of motor control is how the nervous system accomplishes the complex task of integrating commands from multiple movement systems in order to perform a motor behavior. Even the simplest tasks, such as withdrawing the limb from a painful stimulus or taking a single step, involve multiple areas of the brain and spinal cord. Examples of movement parameters that are encoded within the nervous system include the selection and timing of muscle contraction in order to move a body region to a desired position, and the coordination of multiple body joints and body regions. Significant progress has been made in the field to understand the contribution of individual cell types to motor control, as well as the genetic mechanisms that regulate the development of individual cell types. However, the circuit components that link multiple motor control populations, as well as the mechanisms that regulate the formation of integrative motor networks, are poorly understood.

This dissertation describes a series of original work that aims to elucidate the cellular players and developmental mechanisms that assemble circuit components into functional neural networks. The first chapter provides a framework for understanding how different motor control pathways are organized within the central nervous system. The second chapter describes a group of premotor neurons that are located in the deep dorsal horn of the spinal cord, receive input from multiple motor control pathways, and bind together the activity of multiple motor pools. Importantly, genetic markers (Satb1, Satb2, and tcfAP2β) were identified that, to date, comprise the most significant fraction of molecularly defined premotor neurons in the spinal cord.

The third chapter describes a novel population of cells that express the genetic marker Satb2, spinal interneurons that are located at the intersection of incoming sensory and outgoing motor information. This work examines the role of the Satb2 gene in spinal interneuron development, as well as sensory-motor circuit assembly and function. This work provides an important contribution to our understanding of the organizational logic of integrative spinal networks and the types of movements they control.

Within the nervous system, the spinal cord plays vital roles both at the initial steps of receiving and processing incoming sensory information, and at the final stages of executing appropriate motor plans. Spinal neural networks are sufficient to produce corrective sensorimotor reflexes and locomotor central pattern generator activity, but also must interface with the brain to produce more nuanced and elaborate behavior. Consequently, a diverse array of spinal interneurons mediate these functions. Significant studies have delineated spinal neurons into cardinal classes with distinct developmental origins and functions. However, recent studies have uncovered extensive molecular and circuit diversity within each cardinal class. As a result, because each cardinal class is comprised of a mixture of subpopulations, it has been difficult to interpret which aspects of each cardinal class are most salient to its function. This dissertation describes a series of original work that aims to identify a unifying logic for spinal neuron diversification across cardinal classes. The first chapter consolidates recent work on spinal neuron characterization, with a focus on molecular and circuit diversity within single cardinal classes. The second chapter examines molecular diversity conserved across heterogeneous populations of spinal neurons. We found broad genetic signatures which divide the spinal cord into sensory-motor, local-long range, and inhibitory-excitatory groups of neurons. The local-long range signature corresponded to neuronal birthdate and divided each cardinal class into their local and projection subsets. Thus, many aspects of diversity within each cardinal class can be tracked with the same molecular signatures, greatly simplifying the task of identifying discrete functional cell types. The third chapter provides an initial characterization of transgenic mouse lines developed to label the local and long-range groups of spinal neurons identified in chapter two.

In summary, this work identifies a genetic axis which governs aspects of spinal neuron diversity. This genetic axis is organized temporally, reflecting the birthdate of spinal neurons. It corresponds to whether the neuron projects locally or long-range. This genetic axis is orthogonal to the previously defined cardinal classes, and when used together predict many aspects of spinal neuron attributes, including location, neurotransmitter, and synaptic outputs.

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