Molecular Architecture of the Mouse Nervous System.

The mammalian nervous system executes complex behaviors controlled by specialized, precisely positioned, and interacting cell types. Here, we used RNA sequencing of half a million single cells to create a detailed census of cell types in the mouse nervous system. We mapped cell types spatially and derived a hierarchical, data-driven taxonomy. Neurons were the most diverse and were grouped by developmental anatomical units and by the expression of neurotransmitters and neuropeptides. Neuronal diversity was driven by genes encoding cell identity, synaptic connectivity, neurotransmission, and membrane conductance. We discovered seven distinct, regionally restricted astrocyte types that obeyed developmental boundaries and correlated with the spatial distribution of key glutamate and glycine neurotransmitters. In contrast, oligodendrocytes showed a loss of regional identity followed by a secondary diversification. The resource presented here lays a solid foundation for understanding the molecular architecture of the mammalian nervous system and enables genetic manipulation of specific cell types.

Postnatal Sox6 Regulates Synaptic Function of Cortical Parvalbumin-Expressing Neurons.

Cortical parvalbumin-expressing (Pvalb+) neurons provide robust inhibition to neighboring pyramidal neurons, crucial for the proper functioning of cortical networks. This class of inhibitory neurons undergoes extensive synaptic formation and maturation during the first weeks after birth and continue to dynamically maintain their synaptic output throughout adulthood. While several transcription factors, such as Nkx2-1, Lhx6, and Sox6, are known to be necessary for the differentiation of progenitors into Pvalb+ neurons, which transcriptional programs underlie the postnatal maturation and maintenance of Pvalb+ neurons' innervation and synaptic function remains largely unknown. Because Sox6 is continuously expressed in Pvalb+ neurons until adulthood, we used conditional knock-out strategies to investigate its putative role in the postnatal maturation and synaptic function of cortical Pvalb+ neurons in mice of both sexes. We found that early postnatal loss of Sox6 in Pvalb+ neurons leads to failure of synaptic bouton growth, whereas later removal in mature Pvalb+ neurons in the adult causes shrinkage of already established synaptic boutons. Paired recordings between Pvalb+ neurons and pyramidal neurons revealed reduced release probability and increased failure rate of Pvalb+ neurons' synaptic output. Furthermore, Pvalb+ neurons lacking Sox6 display reduced expression of full-length tropomyosin-receptor kinase B (TrkB), a key modulator of GABAergic transmission. Once re-expressed in neurons lacking Sox6, TrkB was sufficient to rescue the morphologic synaptic phenotype. Finally, we showed that Sox6 mRNA levels were increased by motor training. Our data thus suggest a constitutive role for Sox6 in the maintenance of synaptic output from Pvalb+ neurons into adulthood.SIGNIFICANCE STATEMENT Cortical parvalbumin-expressing (Pvalb+) inhibitory neurons provide robust inhibition to neighboring pyramidal neurons, crucial for the proper functioning of cortical networks. These inhibitory neurons undergo extensive synaptic formation and maturation during the first weeks after birth and continue to dynamically maintain their synaptic output throughout adulthood. However, it remains largely unknown which transcriptional programs underlie the postnatal maturation and maintenance of Pvalb+ neurons. Here, we show that the transcription factor Sox6 cell-autonomously regulates the synaptic maintenance and output of Pvalb+ neurons until adulthood, leaving unaffected other maturational features of this neuronal population.

Transcription and Signaling Regulators in Developing Neuronal Subtypes of Mouse and Human Enteric Nervous System.

BACKGROUND & AIMS: The enteric nervous system (ENS) regulates gastrointestinal function via different subtypes of neurons, organized into fine-tuned neural circuits. It is not clear how cell diversity is created within the embryonic ENS; information required for development of cell-based therapies and models of enteric neuropathies. We aimed to identify proteins that regulate ENS differentiation and network formation. METHODS: We generated and compared RNA expression profiles of the entire ENS, ENS progenitor cells, and non-ENS gut cells of mice, collected at embryonic days 11.5 and 15.5, when different subtypes of neurons are formed. Gastrointestinal tissues from R26ReYFP reporter mice crossed to Sox10-CreERT2 or Wnt1-Cre mice were dissected and the 6 populations of cells were isolated by flow cytometry. We used histochemistry to map differentially expressed proteins in mouse and human gut tissues at different stages of development, in different regions. We examined enteric neuronal diversity and gastric function in Wnt1-Cre x Sox6fl/fl mice, which do not express the Sox6 gene in the ENS. RESULTS: We identified 147 transcription and signaling factors that varied in spatial and temporal expression during development of the mouse ENS. Of the factors also analyzed in human ENS, most were conserved. We uncovered 16 signaling pathways (such as fibroblast growth factor and Eph/ephrin pathways). Transcription factors were grouped according to their specific expression in enteric progenitor cells (such as MEF2C), enteric neurons (such as SOX4), or neuron subpopulations (such as SATB1 and SOX6). Lack of SOX6 in the ENS reduced the numbers of gastric dopamine neurons and delayed gastric emptying. CONCLUSIONS: Using transcriptome and histochemical analyses of the developing mouse and human ENS, we mapped expression patterns of transcription and signaling factors. Further studies of these candidate determinants might elucidate the mechanisms by which enteric stem cells differentiate into neuronal subtypes and form distinct connectivity patterns during ENS development. We found expression of SOX6 to be required for development of gastric dopamine neurons.

Mutations in DMRT3 affect locomotion in horses and spinal circuit function in mice.

Locomotion in mammals relies on a central pattern-generating circuitry of spinal interneurons established during development that coordinates limb movement. These networks produce left-right alternation of limbs as well as coordinated activation of flexor and extensor muscles. Here we show that a premature stop codon in the DMRT3 gene has a major effect on the pattern of locomotion in horses. The mutation is permissive for the ability to perform alternate gaits and has a favourable effect on harness racing performance. Examination of wild-type and Dmrt3-null mice demonstrates that Dmrt3 is expressed in the dI6 subdivision of spinal cord neurons, takes part in neuronal specification within this subdivision, and is critical for the normal development of a coordinated locomotor network controlling limb movements. Our discovery positions Dmrt3 in a pivotal role for configuring the spinal circuits controlling stride in vertebrates. The DMRT3 mutation has had a major effect on the diversification of the domestic horse, as the altered gait characteristics of a number of breeds apparently require this mutation.

Ascl1 Is Required for the Development of Specific Neuronal Subtypes in the Enteric Nervous System.

The enteric nervous system (ENS) is organized into neural circuits within the gastrointestinal wall where it controls the peristaltic movements, secretion, and blood flow. Although proper gut function relies on the complex neuronal composition of the ENS, little is known about the transcriptional networks that regulate the diversification into different classes of enteric neurons and glia during development. Here we redefine the role of Ascl1 (Mash1), one of the few regulatory transcription factors described during ENS development. We show that enteric glia and all enteric neuronal subtypes appear to be derived from Ascl1-expressing progenitor cells. In the gut of Ascl1(-/-) mutant mice, neurogenesis is delayed and reduced, and posterior gliogenesis impaired. The ratio of neurons expressing Calbindin, TH, and VIP is selectively decreased while, for instance, 5-HT(+) neurons, which previously were believed to be Ascl1-dependent, are formed in normal numbers. Essentially the same differentiation defects are observed in Ascl1(KINgn2) transgenic mutants, where the proneural activity of Ngn2 replaces Ascl1, demonstrating that Ascl1 is required for the acquisition of specific enteric neuronal subtype features independent of its role in neurogenesis. In this study, we provide novel insights into the expression and function of Ascl1 in the differentiation process of specific neuronal subtypes during ENS development. SIGNIFICANCE STATEMENT: The molecular mechanisms underlying the generation of different neuronal subtypes during development of the enteric nervous system are poorly understood despite its pivotal function in gut motility and involvement in gastrointestinal pathology. This report identifies novel roles for the transcription factor Ascl1 in enteric gliogenesis and neurogenesis. Moreover, independent of its proneurogenic activity, Ascl1 is required for the normal expression of specific enteric neuronal subtype characteristics. Distinct enteric neuronal subtypes are formed in a temporally defined order, and we observe that the early-born 5-HT(+) neurons are generated in Ascl1(-/-) mutants, despite the delayed neurogenesis. Enteric nervous system progenitor cells may therefore possess strong intrinsic control over their specification at the initial waves of neurogenesis.

DCC mediated axon guidance of spinal interneurons is essential for normal locomotor central pattern generator function.

Coordinated limb rhythmic movements take place through organized signaling in local spinal cord neuronal networks. The establishment of these circuitries during development is dependent on the correct guidance of axons to their targets. It has previously been shown that the well-known axon guidance molecule netrin-1 is required for configuring the circuitry that provides left-right alternating coordination in fictive locomotion. The attraction of commissural axons to the midline in response to netrin-1 has been shown to involve the netrin-1 receptor DCC (deleted in Colorectal Cancer). However, the role of DCC for the establishment of CPG coordination has not yet been resolved. We show that mice carrying a null mutation of DCC displayed an uncoordinated left-right activity during fictive locomotion accompanied by a loss of interneuronal subpopulations originating from commissural progenitors. Thus, DCC plays a crucial role in the formation of spinal neuronal circuitry coordinating left-right activities. Together with the previously published results from netrin-1 deficient mice, the data presented in this study suggest a role for the most ventral originating V3 interneurons in synchronous activities over the midline. Further, it provides evidence that axon crossing in the spinal cord is more intricately controlled than in previously suggested models of DCC-netrin-1 interaction.

Hop Mice Display Synchronous Hindlimb Locomotion and a Ventrally Fused Lumbar Spinal Cord Caused by a Point Mutation in Ttc26.

Identifying the spinal circuits controlling locomotion is critical for unravelling the mechanisms controlling the production of gaits. Development of the circuits governing left-right coordination relies on axon guidance molecules such as ephrins and netrins. To date, no other class of proteins have been shown to play a role during this process. Here, we have analyzed hop mice, which walk with a characteristic hopping gait using their hindlimbs in synchrony. Fictive locomotion experiments suggest that a local defect in the ventral spinal cord contributes to the aberrant locomotor phenotype. Hop mutant spinal cords had severe morphologic defects, including the absence of the ventral midline and a poorly defined border between white and gray matter. The hop mice represent the first model where, exclusively found in the lumbar domain, the left and right components of the central pattern generators (CPGs) are fused with a synchronous hindlimb gait as a functional consequence. These defects were associated with abnormal developmental processes, including a misplaced notochord and reduced induction of ventral progenitor domains. Whereas the underlying mutation in hop mice has been suggested to lie within the Ttc26 gene, other genes in close vicinity have been associated with gait defects. Mouse embryos carrying a CRISPR replicated point mutation within Ttc26 displayed an identical morphologic phenotype. Thus, our data suggest that the assembly of the lumbar CPG network is dependent on fully functional TTC26 protein.

Diversification of molecularly defined myenteric neuron classes revealed by single-cell RNA sequencing.

Autonomous regulation of the intestine requires the combined activity of functionally distinct neurons of the enteric nervous system (ENS). However, the variety of enteric neuron types and how they emerge during development remain largely unknown. Here, we define a molecular taxonomy of 12 enteric neuron classes within the myenteric plexus of the mouse small intestine using single-cell RNA sequencing. We present cell-cell communication features and histochemical markers for motor neurons, sensory neurons and interneurons, together with transgenic tools for class-specific targeting. Transcriptome analysis of the embryonic ENS uncovers a novel principle of neuronal diversification, where two neuron classes arise through a binary neurogenic branching and all other identities emerge through subsequent postmitotic differentiation. We identify generic and class-specific transcriptional regulators and functionally connect Pbx3 to a postmitotic fate transition. Our results offer a conceptual and molecular resource for dissecting ENS circuits and predicting key regulators for directed differentiation of distinct enteric neuron classes.

Netrin-1-dependent spinal interneuron subtypes are required for the formation of left-right alternating locomotor circuitry.

Neuronal circuits in the spinal cord that produce the rhythmic and coordinated activities necessary for limb movements are referred to as locomotor central pattern generators (CPGs). The identities and preceding development of neurons essential for coordination between left and right limbs are not yet known. We show that the ventral floor plate chemoattractant Netrin-1 preferentially guides dorsally originating subtypes of commissural interneurons, the majority of which are inhibitory. In contrast, the excitatory and ventralmost V3 subtype of interneurons have a normal number of commissural fibers in Netrin-1 mutant mice, thus being entirely independent of Netrin-1-mediated attraction. This selective loss of commissural fibers in Netrin-1 mutant mice resulted in an abnormal circuitry manifested by a complete switch from alternating to synchronous fictive locomotor activity suggesting that the most ventral-originating excitatory commissural interneurons are an important component of a left-right synchrony circuit in the locomotor CPG. Thus, during development, Netrin-1 plays a critical role for the establishment of a functional balanced CPG.

Schwann Cell Precursors Generate the Majority of Chromaffin Cells in Zuckerkandl Organ and Some Sympathetic Neurons in Paraganglia.

In humans, neurosecretory chromaffin cells control a number of important bodily functions, including those related to stress response. Chromaffin cells appear as a distinct cell type at the beginning of midgestation and are the main cellular source of adrenalin and noradrenalin released into the blood stream. In mammals, two different chromaffin organs emerge at a close distance to each other, the adrenal gland and Zuckerkandl organ (ZO). These two structures are found in close proximity to the kidneys and dorsal aorta, in a region where paraganglioma, pheochromocytoma and neuroblastoma originate in the majority of clinical cases. Recent studies showed that the chromaffin cells comprising the adrenal medulla are largely derived from nerve-associated multipotent Schwann cell precursors (SCPs) arriving at the adrenal anlage with the preganglionic nerve fibers, whereas the migratory neural crest cells provide only minor contribution. However, the embryonic origin of the ZO, which differs from the adrenal medulla in a number of aspects, has not been studied in detail. The ZO is composed of chromaffin cells in direct contact with the dorsal aorta and the intraperitoneal cavity and disappears through an autophagy-mediated mechanism after birth. In contrast, the adrenal medulla remains throughout the entire life and furthermore, is covered by the adrenal cortex. Using a combination of lineage tracing strategies with nerve- and cell type-specific ablations, we reveal that the ZO is largely SCP-derived and forms in synchrony with progressively increasing innervation. Moreover, the ZO develops hand-in-hand with the adjacent sympathetic ganglia that coalesce around the dorsal aorta. Finally, we were able to provide evidence for a SCP-contribution to a small but significant proportion of sympathetic neurons of the posterior paraganglia. Thus, this cellular source complements the neural crest, which acts as a main source of sympathetic neurons. Our discovery of a nerve-dependent origin of chromaffin cells and some sympathoblasts may help to understand the origin of pheochromocytoma, paraganglioma and neuroblastoma, all of which are currently thought to be derived from the neural crest or committed sympathoadrenal precursors.

Multipotent peripheral glial cells generate neuroendocrine cells of the adrenal medulla.

Adrenaline is a fundamental circulating hormone for bodily responses to internal and external stressors. Chromaffin cells of the adrenal medulla (AM) represent the main neuroendocrine adrenergic component and are believed to differentiate from neural crest cells. We demonstrate that large numbers of chromaffin cells arise from peripheral glial stem cells, termed Schwann cell precursors (SCPs). SCPs migrate along the visceral motor nerve to the vicinity of the forming adrenal gland, where they detach from the nerve and form postsynaptic neuroendocrine chromaffin cells. An intricate molecular logic drives two sequential phases of gene expression, one unique for a distinct transient cellular state and another for cell type specification. Subsequently, these programs down-regulate SCP-gene and up-regulate chromaffin cell-gene networks. The AM forms through limited cell expansion and requires the recruitment of numerous SCPs. Thus, peripheral nerves serve as a stem cell niche for neuroendocrine system development.

Neurodevelopment. Parasympathetic neurons originate from nerve-associated peripheral glial progenitors.

The peripheral autonomic nervous system reaches far throughout the body and includes neurons of diverse functions, such as sympathetic and parasympathetic. We show that the parasympathetic system in mice--including trunk ganglia and the cranial ciliary, pterygopalatine, lingual, submandibular, and otic ganglia--arise from glial cells in nerves, not neural crest cells. The parasympathetic fate is induced in nerve-associated Schwann cell precursors at distal peripheral sites. We used multicolor Cre-reporter lineage tracing to show that most of these neurons arise from bi-potent progenitors that generate both glia and neurons. This nerve origin places cellular elements for generating parasympathetic neurons in diverse tissues and organs, which may enable wiring of the developing parasympathetic nervous system.

Genetic analysis of left-right coordination of locomotion .

While there is a rather large amount of data from pharmacological and anatomical studies of the murine locomotor CPG network, comprehensive information regarding the cellular and functional properties of the neuronal populations is lacking. Here we describe concepts arising from genetic studies of the locomotor network with a focus on commissural interneurons regulating left-right coordination. In particular, this involves several families of axon guidance molecules relevant for midline crossing. We also describe recent advances within the field of neural circuit analysis, including imaging, genetic inactivation and optogenetic strategies, which are applicable to locomotor circuits. Such efforts, for example by using available genetic markers, should substantially increase our possibilities to decipher the functionality of spinal cord neuronal networks.

Identification of novel spinal cholinergic genetic subtypes disclose Chodl and Pitx2 as markers for fast motor neurons and partition cells.

Spinal cholinergic neurons are critical for motor function in both the autonomic and somatic nervous systems and are affected in spinal cord injury and in diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy. Using two screening approaches and in situ hybridization, we identified 159 genes expressed in typical cholinergic patterns in the spinal cord. These include two general cholinergic neuron markers, one gene exclusively expressed in motor neurons, and nine genes expressed in unknown subtypes of somatic motor neurons. Further, we present evidence that chondrolectin (Chodl) is expressed by fast motor neurons and that estrogen-related receptor beta (ERRbeta) is a candidate marker for slow motor neurons. In addition, we suggest paired-like homeodomain transcription factor 2 (Pitx2) as a marker for cholinergic partition cells.