The organization of spinal neurons: Insights from single cell sequencing.

To understand how the spinal cord enacts complex sensorimotor functions, researchers have studied, classified, and functionally probed it's many neuronal populations for over a century. Recent developments in single-cell RNA-sequencing can characterize the gene expression signatures of the entire set of spinal neuron types and can simultaneously provide an unbiased view of their relationships to each other. This approach has revealed that the location of neurons predicts transcriptomic variability, as dorsal spinal neurons become highly distinct over development as ventral spinal neurons become less so.

Molecular and spatial profiling of the paraventricular nucleus of the thalamus.

The paraventricular nucleus of the thalamus (PVT) is known to regulate various cognitive and behavioral processes. However, while functional diversity among PVT circuits has often been linked to cellular differences, the molecular identity and spatial distribution of PVT cell types remain unclear. To address this gap, here we used single nucleus RNA sequencing (snRNA-seq) and identified five molecularly distinct PVT neuronal subtypes in the mouse brain.

A cellular taxonomy of the adult human spinal cord.

The mammalian spinal cord functions as a community of cell types for sensory processing, autonomic control, and movement. While animal models have advanced our understanding of spinal cellular diversity, characterizing human biology directly is important to uncover specialized features of basic function and human pathology. Here, we present a cellular taxonomy of the adult human spinal cord using single-nucleus RNA sequencing with spatial transcriptomics and antibody validation.

The neurons that restore walking after paralysis.

A spinal cord injury interrupts pathways from the brain and brainstem that project to the lumbar spinal cord, leading to paralysis. Here we show that spatiotemporal epidural electrical stimulation (EES) of the lumbar spinal cord applied during neurorehabilitation (EES) restored walking in nine individuals with chronic spinal cord injury. This recovery involved a reduction in neuronal activity in the lumbar spinal cord of humans during walking.

Single cell atlas of spinal cord injury in mice reveals a pro-regenerative signature in spinocerebellar neurons.

After spinal cord injury, tissue distal to the lesion contains undamaged cells that could support or augment recovery. Targeting these cells requires a clearer understanding of their injury responses and capacity for repair. Here, we use single nucleus RNA sequencing to profile how each cell type in the lumbar spinal cord changes after a thoracic injury in mice.

Intersectional genetic tools to study skilled reaching in mice.

Reaching to grasp is an evolutionarily conserved behavior and a crucial part of the motor repertoire in mammals. As it is studied in the laboratory, reaching has become the prototypical example of dexterous forelimb movements, illuminating key principles of motor control throughout the spinal cord, brain, and peripheral nervous system. Here, we (1) review the motor elements or phases that comprise the reach, grasp, and retract movements of reaching behavior, (2) highlight the role of intersectional genetic tools in linking these movements to their neuronal substrates, (3) describe spinal cord cell types and their roles in skilled reaching, and (4) how descending pathways from the brain and the sensory systems contribute to skilled reaching.

A harmonized atlas of mouse spinal cord cell types and their spatial organization.

Single-cell RNA sequencing data can unveil the molecular diversity of cell types. Cell type atlases of the mouse spinal cord have been published in recent years but have not been integrated together. Here, we generate an atlas of spinal cell types based on single-cell transcriptomic data, unifying the available datasets into a common reference framework.

Confronting false discoveries in single-cell differential expression.

Differential expression analysis in single-cell transcriptomics enables the dissection of cell-type-specific responses to perturbations such as disease, trauma, or experimental manipulations. While many statistical methods are available to identify differentially expressed genes, the principles that distinguish these methods and their performance remain unclear. Here, we show that the relative performance of these methods is contingent on their ability to account for variation between biological replicates.

Selecting single cell clustering parameter values using subsampling-based robustness metrics.

Background: Generating and analysing single-cell data has become a widespread approach to examine tissue heterogeneity, and numerous algorithms exist for clustering these datasets to identify putative cell types with shared transcriptomic signatures. However, many of these clustering workflows rely on user-tuned parameter values, tailored to each dataset, to identify a set of biologically relevant clusters. Whereas users often develop their own intuition as to the optimal range of parameters for clustering on each data set, the lack of systematic approaches to identify this range can be daunting to new users of any given workflow.

Cell type prioritization in single-cell data.

We present Augur, a method to prioritize the cell types most responsive to biological perturbations in single-cell data. Augur employs a machine-learning framework to quantify the separability of perturbed and unperturbed cells within a high-dimensional space. We validate our method on single-cell RNA sequencing, chromatin accessibility and imaging transcriptomics datasets, and show that Augur outperforms existing methods based on differential gene expression.

Cerebellospinal Neurons Regulate Motor Performance and Motor Learning.

To understand the neural basis of behavior, it is important to reveal how movements are planned, executed, and refined by networks of neurons distributed throughout the nervous system. Here, we report the neuroanatomical organization and behavioral roles of cerebellospinal (CeS) neurons. Using intersectional genetic techniques, we find that CeS neurons constitute a small minority of excitatory neurons in the fastigial and interpositus deep cerebellar nuclei, target pre-motor circuits in the ventral spinal cord and the brain, and control distinct aspects of movement.

Decoding Cell Type Diversity Within the Spinal Cord.

To understand fundamental mechanisms of mammalian spinal cord function, it is necessary to reveal the diverse array of constituent spinal "cell types" - populations that can be consistently identified because they share a unique and cohesive set of characteristics. Many parameters can contribute to the definition of a spinal cord cell type, including location, morphology, lineage, electrophysiological properties, circuit features, gene expression patterns, and behavioral contribution. While it is not necessary for all of these features to align completely at all times to identify an individual cell type, a correlation of these characteristics paints a rich portrait of cell identity.

Isolation of Adult Spinal Cord Nuclei for Massively Parallel Single-nucleus RNA Sequencing.

Probing an individual cell's gene expression enables the identification of cell type and cell state. Single-cell RNA sequencing has emerged as a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissues such as the central nervous system. However, dissociation methods required for single cell sequencing can lead to experimental changes in the gene expression and cell death.

Massively Parallel Single Nucleus Transcriptional Profiling Defines Spinal Cord Neurons and Their Activity during Behavior.

To understand the cellular basis of behavior, it is necessary to know the cell types that exist in the nervous system and their contributions to function. Spinal networks are essential for sensory processing and motor behavior and provide a powerful system for identifying the cellular correlates of behavior. Here, we used massively parallel single nucleus RNA sequencing (snRNA-seq) to create an atlas of the adult mouse lumbar spinal cord.

Graded Arrays of Spinal and Supraspinal V2a Interneuron Subtypes Underlie Forelimb and Hindlimb Motor Control.

The spinal cord contains neural networks that enable regionally distinct motor outputs along the body axis. Nevertheless, it remains unclear how segment-specific motor computations are processed because the cardinal interneuron classes that control motor neurons appear uniform at each level of the spinal cord. V2a interneurons are essential to both forelimb and hindlimb movements, and here we identify two major types that emerge during development: type I neurons marked by high Chx10 form recurrent networks with neighboring spinal neurons and type II neurons that downregulate Chx10 and project to supraspinal structures.

Satb2 Is Required for the Development of a Spinal Exteroceptive Microcircuit that Modulates Limb Position.

Motor behaviors such as walking or withdrawing the limb from a painful stimulus rely upon integrative multimodal sensory circuitry to generate appropriate muscle activation patterns. Both the cellular components and the molecular mechanisms that instruct the assembly of the spinal sensorimotor system are poorly understood. Here we characterize the connectivity pattern of a sub-population of lamina V inhibitory sensory relay neurons marked during development by the nuclear matrix and DNA binding factor Satb2 (ISR(Satb2)).

Identification of a cellular node for motor control pathways.

The rich behavioral repertoire of animals is encoded in the CNS as a set of motorneuron activation patterns, also called 'motor synergies'. However, the neurons that orchestrate these motor programs as well as their cellular properties and connectivity are poorly understood. Here we identify a population of molecularly defined motor synergy encoder (MSE) neurons in the mouse spinal cord that may represent a central node in neural pathways for voluntary and reflexive movement.

Spatial organization of cortical and spinal neurons controlling motor behavior.

A major task of the central nervous system (CNS) is to control behavioral actions, which necessitates a precise regulation of muscle activity. The final components of the circuitry controlling muscles are the motorneurons, which settle into pools in the ventral horn of the spinal cord in positions that mirror the musculature organization within the body. This 'musculotopic' motor-map then becomes the internal CNS reference for the neuronal circuits that control motor commands.

GDF3 is a BMP inhibitor that can activate Nodal signaling only at very high doses.

Within the TGF-beta superfamily, there are approximately forty ligands divided into two major branches: the TGF-beta/Activin/Nodal ligands and the BMP/GDF ligands. We studied the ligand GDF3 and found that it inhibits signaling by its co-family members, the BMPs; however, GDF3 has been described by others to have Nodal-like activity. Here, we show that GDF3 can activate Nodal signaling, but only at very high doses and only upon mRNA over-expression.

Proposal of a model of mammalian neural induction.

How does the vertebrate embryo make a nervous system? This complex question has been at the center of developmental biology for many years. The earliest step in this process - the induction of neural tissue - is intimately linked to patterning of the entire early embryo, and the molecular and embryological of basis these processes are beginning to emerge. Here, we analyze classic and cutting-edge findings on neural induction in the mouse.

GDF3 at the crossroads of TGF-beta signaling.

Embryonic stem (ES) cells present an excellent system for addressing the relevance of our current knowledge about how cell fate is determined and how cells integrate multiple signals into a single outcome as a function of time. Many of the factors that mediate these phenomena have been discovered through classical embryological experiments and are organized into several major signal transduction pathways including TGF-beta/BMP, Jak-STAT, Hedgehog, Wnt, Notch and FGF/MAPK. This review will summarize the current understanding of TGF-beta signaling in ES and focus on early embryological roles of the TGF-beta member, GDF-3.

GDF3, a BMP inhibitor, regulates cell fate in stem cells and early embryos.

The TGFbeta superfamily of ligands plays key functions in development and disease. In both human and mouse embryonic stem cells, a member of this family, GDF3, is specifically expressed in the pluripotent state. We show that GDF3 is an inhibitor of its own subfamily, blocks classic BMP signaling in multiple contexts, interacts with BMP proteins and is expressed specifically in the node during gastrulation in a pattern consistent with BMP inhibition.