Single-cell RNA-sequencing analysis of the developing mouse inner ear identifies molecular logic of auditory neuron diversification.

Different types of spiral ganglion neurons (SGNs) are essential for auditory perception by transmitting complex auditory information from hair cells (HCs) to the brain. Here, we use deep, single cell transcriptomics to study the molecular mechanisms that govern their identity and organization in mice. We identify a core set of temporally patterned genes and gene regulatory networks that may contribute to the diversification of SGNs through sequential binary decisions and demonstrate a role for NEUROD1 in driving specification of a Ic-SGN phenotype. We also find that each trajectory of the decision tree is defined by initial co-expression of alternative subtype molecular controls followed by gradual shifts toward cell fate resolution. Finally, analysis of both developing SGN and HC types reveals cell-cell signaling potentially playing a role in the differentiation of SGNs. Our results indicate that SGN identities are drafted prior to birth and reveal molecular principles that shape their differentiation and will facilitate studies of their development, physiology, and dysfunction.

Schwann cell precursors represent a neural crest-like state with biased multipotency.

Schwann cell precursors (SCPs) are nerve-associated progenitors that can generate myelinating and non-myelinating Schwann cells but also are multipotent like the neural crest cells from which they originate. SCPs are omnipresent along outgrowing peripheral nerves throughout the body of vertebrate embryos. By using single-cell transcriptomics to generate a gene expression atlas of the entire neural crest lineage, we show that early SCPs and late migratory crest cells have similar transcriptional profiles characterised by a multipotent "hub" state containing cells biased towards traditional neural crest fates. SCPs keep diverging from the neural crest after being primed towards terminal Schwann cells and other fates, with different subtypes residing in distinct anatomical locations. Functional experiments using CRISPR-Cas9 loss-of-function further show that knockout of the common "hub" gene Sox8 causes defects in neural crest-derived cells along peripheral nerves by facilitating differentiation of SCPs towards sympathoadrenal fates. Finally, specific tumour populations found in melanoma, neurofibroma and neuroblastoma map to different stages of SCP/Schwann cell development. Overall, SCPs resemble migrating neural crest cells that maintain multipotency and become transcriptionally primed towards distinct lineages.

Genome-wide association meta-analysis identifies 48 risk variants and highlights the role of the stria vascularis in hearing loss.

Hearing loss is one of the top contributors to years lived with disability and is a risk factor for dementia. Molecular evidence on the cellular origins of hearing loss in humans is growing. Here, we performed a genome-wide association meta-analysis of clinically diagnosed and self-reported hearing impairment on 723,266 individuals and identified 48 significant loci, 10 of which are novel. A large proportion of associations comprised missense variants, half of which lie within known familial hearing loss loci. We used single-cell RNA-sequencing data from mouse cochlea and brain and mapped common-variant genomic results to spindle, root, and basal cells from the stria vascularis, a structure in the cochlea necessary for normal hearing. Our findings indicate the importance of the stria vascularis in the mechanism of hearing impairment, providing future paths for developing targets for therapeutic intervention in hearing loss.

Theory of branching morphogenesis by local interactions and global guidance.

Branching morphogenesis governs the formation of many organs such as lung, kidney, and the neurovascular system. Many studies have explored system-specific molecular and cellular regulatory mechanisms, as well as self-organizing rules underlying branching morphogenesis. However, in addition to local cues, branched tissue growth can also be influenced by global guidance. Here, we develop a theoretical framework for a stochastic self-organized branching process in the presence of external cues. Combining analytical theory with numerical simulations, we predict differential signatures of global vs. local regulatory mechanisms on the branching pattern, such as angle distributions, domain size, and space-filling efficiency. We find that branch alignment follows a generic scaling law determined by the strength of global guidance, while local interactions influence the tissue density but not its overall territory. Finally, using zebrafish innervation as a model system, we test these key features of the model experimentally. Our work thus provides quantitative predictions to disentangle the role of different types of cues in shaping branched structures across scales.

Correction: Human bone marrow harbors cells with neural crest-associated characteristics like human adipose and dermis tissues.

[This corrects the article DOI: 10.1371/journal.pone.0177962.].

Involvement of Scratch2 in GalR1-mediated depression-like behaviors in the rat ventral periaqueductal gray.

Galanin receptor1 (GalR1) transcript levels are elevated in the rat ventral periaqueductal gray (vPAG) after chronic mild stress (CMS) and are related to depression-like behavior. To explore the mechanisms underlying the elevated GalR1 expression, we carried out molecular biological experiments in vitro and in animal behavioral experiments in vivo. It was found that a restricted upstream region of the GalR1 gene, from -250 to -220, harbors an E-box and plays a negative role in the GalR1 promoter activity. The transcription factor Scratch2 bound to the E-box to down-regulate GalR1 promoter activity and lower expression levels of the GalR1 gene. The expression of Scratch2 was significantly decreased in the vPAG of CMS rats. Importantly, local knockdown of Scratch2 in the vPAG caused elevated expression of GalR1 in the same region, as well as depression-like behaviors. RNAscope analysis revealed that GalR1 mRNA is expressed together with Scratch2 in both GABA and glutamate neurons. Taking these data together, our study further supports the involvement of GalR1 in mood control and suggests a role for Scratch2 as a regulator of depression-like behavior by repressing the GalR1 gene in the vPAG.

Distinct subtypes of proprioceptive dorsal root ganglion neurons regulate adaptive proprioception in mice.

Proprioceptive neurons (PNs) are essential for the proper execution of all our movements by providing muscle sensory feedback to the central motor network. Here, using deep single cell RNAseq of adult PNs coupled with virus and genetic tracings, we molecularly identify three main types of PNs (Ia, Ib and II) and find that they segregate into eight distinct subgroups. Our data unveil a highly sophisticated organization of PNs into discrete sensory input channels with distinct spatial distribution, innervation patterns and molecular profiles. Altogether, these features contribute to finely regulate proprioception during complex motor behavior. Moreover, while Ib- and II-PN subtypes are specified around birth, Ia-PN subtypes diversify later in life along with increased motor activity. We also show Ia-PNs plasticity following exercise training, suggesting Ia-PNs are important players in adaptive proprioceptive function in adult mice.

Single cell RNA sequencing identifies early diversity of sensory neurons forming via bi-potential intermediates.

Somatic sensation is defined by the existence of a diversity of primary sensory neurons with unique biological features and response profiles to external and internal stimuli. However, there is no coherent picture about how this diversity of cell states is transcriptionally generated. Here, we use deep single cell analysis to resolve fate splits and molecular biasing processes during sensory neurogenesis in mice. Our results identify a complex series of successive and specific transcriptional changes in post-mitotic neurons that delineate hierarchical regulatory states leading to the generation of the main sensory neuron classes. In addition, our analysis identifies previously undetected early gene modules expressed long before fate determination although being clearly associated with defined sensory subtypes. Overall, the early diversity of sensory neurons is generated through successive bi-potential intermediates in which synchronization of relevant gene modules and concurrent repression of competing fate programs precede cell fate stabilization and final commitment.

Molecular design of hypothalamus development.

A wealth of specialized neuroendocrine command systems intercalated within the hypothalamus control the most fundamental physiological needs in vertebrates1,2. Nevertheless, we lack a developmental blueprint that integrates the molecular determinants of neuronal and glial diversity along temporal and spatial scales of hypothalamus development3. Here we combine single-cell RNA sequencing of 51,199 mouse cells of ectodermal origin, gene regulatory network (GRN) screens in conjunction with genome-wide association study-based disease phenotyping, and genetic lineage reconstruction to show that nine glial and thirty-three neuronal subtypes are generated by mid-gestation under the control of distinct GRNs. Combinatorial molecular codes that arise from neurotransmitters, neuropeptides and transcription factors are minimally required to decode the taxonomical hierarchy of hypothalamic neurons. The differentiation of γ-aminobutyric acid (GABA) and dopamine neurons, but not glutamate neurons, relies on quasi-stable intermediate states, with a pool of GABA progenitors giving rise to dopamine cells4. We found an unexpected abundance of chemotropic proliferation and guidance cues that are commonly implicated in dorsal (cortical) patterning5 in the hypothalamus. In particular, loss of SLIT-ROBO signalling impaired both the production and positioning of periventricular dopamine neurons. Overall, we identify molecular principles that shape the developmental architecture of the hypothalamus and show how neuronal heterogeneity is transformed into a multimodal neural unit to provide virtually infinite adaptive potential throughout life.

Muscle-selective RUNX3 dependence of sensorimotor circuit development.

The control of all our motor outputs requires constant monitoring by proprioceptive sensory neurons (PSNs) that convey continuous muscle sensory inputs to the spinal motor network. Yet the molecular programs that control the establishment of this sensorimotor circuit remain largely unknown. The transcription factor RUNX3 is essential for the early steps of PSNs differentiation, making it difficult to study its role during later aspects of PSNs specification. Here, we conditionally inactivate Runx3 in PSNs after peripheral innervation and identify that RUNX3 is necessary for maintenance of cell identity of only a subgroup of PSNs, without discernable cell death. RUNX3 also controls the sensorimotor connection between PSNs and motor neurons at limb level, with muscle-by-muscle variable sensitivities to the loss of Runx3 that correlate with levels of RUNX3 in PSNs. Finally, we find that muscles and neurotrophin 3 signaling are necessary for maintenance of RUNX3 expression in PSNs. Hence, a transcriptional regulator that is crucial for specifying a generic PSN type identity after neurogenesis is later regulated by target muscle-derived signals to contribute to the specialized aspects of the sensorimotor connection selectivity.

A cell fitness selection model for neuronal survival during development.

Developmental cell death plays an important role in the construction of functional neural circuits. In vertebrates, the canonical view proposes a selection of the surviving neurons through stochastic competition for target-derived neurotrophic signals, implying an equal potential for neurons to compete. Here we show an alternative cell fitness selection of neurons that is defined by a specific neuronal heterogeneity code. Proprioceptive sensory neurons that will undergo cell death and those that will survive exhibit different molecular signatures that are regulated by retinoic acid and transcription factors, and are independent of the target and neurotrophins. These molecular features are genetically encoded, representing two distinct subgroups of neurons with contrasted functional maturation states and survival outcome. Thus, in this model, a heterogeneous code of intrinsic cell fitness in neighboring neurons provides differential competitive advantage resulting in the selection of cells with higher capacity to survive and functionally integrate into neural networks.

PRDM12 Is Required for Initiation of the Nociceptive Neuron Lineage during Neurogenesis.

The sensation of pain is essential for the preservation of the functional integrity of the body. However, the key molecular regulators necessary for the initiation of the development of pain-sensing neurons have remained largely unknown. Here, we report that, in mice, inactivation of the transcriptional regulator PRDM12, which is essential for pain perception in humans, results in a complete absence of the nociceptive lineage, while proprioceptive and touch-sensitive neurons remain. Mechanistically, our data reveal that PRDM12 is required for initiation of neurogenesis and activation of a cascade of downstream pro-neuronal transcription factors, including NEUROD1, BRN3A, and ISL1, in the nociceptive lineage while it represses alternative fates other than nociceptors in progenitor cells. Our results thus demonstrate that PRDM12 is necessary for the generation of the entire lineage of pain-initiating neurons.

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.

Neuronal heterogeneity and stereotyped connectivity in the auditory afferent system.

Spiral ganglion (SG) neurons of the cochlea convey all auditory inputs to the brain, yet the cellular and molecular complexity necessary to decode the various acoustic features in the SG has remained unresolved. Using single-cell RNA sequencing, we identify four types of SG neurons, including three novel subclasses of type I neurons and the type II neurons, and provide a comprehensive genetic framework that define their potential synaptic communication patterns. The connectivity patterns of the three subclasses of type I neurons with inner hair cells and their electrophysiological profiles suggest that they represent the intensity-coding properties of auditory afferents. Moreover, neuron type specification is already established at birth, indicating a neuronal diversification process independent of neuronal activity. Thus, this work provides a transcriptional catalog of neuron types in the cochlea, which serves as a valuable resource for dissecting cell-type-specific functions of dedicated afferents in auditory perception and in hearing disorders.

Human bone marrow harbors cells with neural crest-associated characteristics like human adipose and dermis tissues.

Adult neural crest stem-derived cells (NCSC) are of extraordinary high plasticity and promising candidates for use in regenerative medicine. Several locations such as skin, adipose tissue, dental pulp or bone marrow have been described in rodent, as sources of NCSC. However, very little information is available concerning their correspondence in human tissues, and more precisely for human bone marrow. The main objective of this study was therefore to characterize NCSC from adult human bone marrow. In this purpose, we compared human bone marrow stromal cells to human adipose tissue and dermis, already described for containing NCSC. We performed comparative analyses in terms of gene and protein expression as well as functional characterizations. It appeared that human bone marrow, similarly to adipose tissue and dermis, contains NESTIN+ / SOX9+ / TWIST+ / SLUG+ / P75NTR+ / BRN3A+/ MSI1+/ SNAIL1+ cells and were able to differentiate into melanocytes, Schwann cells and neurons. Moreover, when injected into chicken embryos, all those cells were able to migrate and follow endogenous neural crest migration pathways. Altogether, the phenotypic characterization and migration abilities strongly suggest the presence of neural crest-derived cells in human adult bone marrow.

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.

miR-183 cluster scales mechanical pain sensitivity by regulating basal and neuropathic pain genes.

Nociception is protective and prevents tissue damage but can also facilitate chronic pain. Whether a general principle governs these two types of pain is unknown. Here, we show that both basal mechanical and neuropathic pain are controlled by the microRNA-183 (miR-183) cluster in mice. This single cluster controls more than 80% of neuropathic pain-regulated genes and scales basal mechanical sensitivity and mechanical allodynia by regulating auxiliary voltage-gated calcium channel subunits α2δ-1 and α2δ-2. Basal sensitivity is controlled in nociceptors, and allodynia involves TrkB+ light-touch mechanoreceptors. These light-touch-sensitive neurons, which normally do not elicit pain, produce pain during neuropathy that is reversed by gabapentin. Thus, a single microRNA cluster continuously scales acute noxious mechanical sensitivity in nociceptive neurons and suppresses neuropathic pain transduction in a specific, light-touch-sensitive neuronal type recruited during mechanical allodynia.

Hippocampal Transcriptome Profile of Persistent Memory Rescue in a Mouse Model of THRA1 Mutation-Mediated Resistance to Thyroid Hormone.

Hypothyroidism due to THRA1 (gene coding for thyroid hormone receptor α1) mutation-mediated Resistance to Thyroid Hormone (RTH) has been recently reported in human and is associated with memory deficits similar to those found in a mouse model for Thra1 mutation mediated RTH (Thra1(+/m) mice). Here, we show that a short-term treatment of Thra1(+/m) mice with GABAA receptor antagonist pentylenetetrazol (PTZ) completely and durably rescues their memory performance. In the CA1 region of the hippocampus, improvement of memory is associated with increased in long-term potentiation (LTP) and an augmentation of density of dendritic spines (DDS) onto the apical dendrites of pyramidal cells reflecting an increase in the local excitatory drive. Unbiased gene profiling analysis of hippocampi of treated Thra1(+/+) and Thra1(+/m) mice were performed two weeks and three months post treatment and identified co-expression modules that include differentially expressed genes related with and predicting higher memory, LTP and DDS in the hippocampi of PTZ-treated animals. We observed that PTZ treatment changed similar sets of genes in both Thra1(+/+) and Thra1(+/m) mice, which are known to be involved in memory consolidation and neurotransmission dynamics and could participate in the persistent effects of PTZ on memory recovery.

A role for the canonical nuclear factor-κB pathway in coupling neurotrophin-induced differential survival of developing spiral ganglion neurons.

Neurotrophins are key players of neural development by controlling cell death programs. However, the signaling pathways that mediate their selective responses in different populations of neurons remain unclear. In the mammalian cochlea, sensory neurons differentiate perinatally into type I and II populations both expressing TrkB and TrkC, which bind respectively brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3). How these two neuronal populations respond differentially to these two neurotrophins remains unknown. Here, we report in rat the segregation of the nuclear factor-κB (NFκB) subunit p65 specifically within the type II population postnatally. Using dissociated cultures of embryonic and postnatal spiral ganglion neurons, we observed a specific requirement of NFκB for BDNF but not NT3-dependent neuronal survival during a particular postnatal time window that corresponds to a period of neuronal cell death and hair cell innervation refinement in the developing cochlea. Consistently, postnatal p65 knockout mice showed a specific decreased number in type II spiral ganglion neurons. Taken together, these results identify NFκB as a type II neuron-specific factor that participates in the selective survival effects of BDNF and NT3 signaling on developing spiral ganglion neurons.

A local source of FGF initiates development of the unmyelinated lineage of sensory neurons.

The principle by which unmyelinated primary sensory neurons transducing thermal, itch and pain perception are specified in early development is unknown. These classes of sensory neurons diversify from a common population of late-born neurons, which initiate expression of Runt homology domain transcription factor RUNX1 and the nerve growth factor receptor TrkA. Here, we report that signals emanating from within the mouse dorsal root ganglion mediated partly by early-born neurons destined to a myelinated phenotype participate in fating late-born RUNX1(+)/TrkA(+) neurons. Inductive factors include FGFs via activation of FGF receptor 1 (FGFR1). Consistently, FGF2 is sufficient to induce expression of RUNX1, and Fgfr1 conditional mutant mice display deficits in fating of the common population of late-born RUNX1(+)/TrkA(+) neurons that develop into unmyelinated neurons. Thus, the distinct lineages of sensory neurons are acquired in response to increasing FGF levels provided by a rising number of born neurons.