CSF-contacting neurons respond to Streptococcus pneumoniae and promote host survival during central nervous system infection.

The pathogenic bacterium Streptococcus pneumoniae (S. pneumoniae) can invade the cerebrospinal fluid (CSF) and cause meningitis with devastating consequences. Whether and how sensory cells in the central nervous system (CNS) become activated during bacterial infection, as recently reported for the peripheral nervous system, is not known. We find that CSF infection by S. pneumoniae in larval zebrafish leads to changes in posture and behavior that are reminiscent of pneumococcal meningitis, including dorsal arching and epileptic-like seizures. We show that during infection, invasion of the CSF by S. pneumoniae massively activates in vivo sensory neurons contacting the CSF, referred to as "CSF-cNs" and previously shown to detect spinal curvature and to control posture, locomotion, and spine morphogenesis. We find that CSF-cNs express orphan bitter taste receptors and respond in vitro to bacterial supernatant and metabolites via massive calcium transients, similar to the ones observed in vivo during infection. Upon infection, CSF-cNs also upregulate the expression of numerous cytokines and complement components involved in innate immunity. Accordingly, we demonstrate, using cell-specific ablation and blockade of neurotransmission, that CSF-cN neurosecretion enhances survival of the host during S. pneumoniae infection. Finally, we show that CSF-cNs respond to various pathogenic bacteria causing meningitis in humans, as well as to the supernatant of cells infected by a neurotropic virus. Altogether, our work uncovers that central sensory neurons in the spinal cord, previously involved in postural control and morphogenesis, contribute as well to host survival by responding to the invasion of the CSF by pathogenic bacteria during meningitis.

A lexical approach for identifying behavioural action sequences.

Animals display characteristic behavioural patterns when performing a task, such as the spiraling of a soaring bird or the surge-and-cast of a male moth searching for a female. Identifying such recurring sequences occurring rarely in noisy behavioural data is key to understanding the behavioural response to a distributed stimulus in unrestrained animals. Existing models seek to describe the dynamics of behaviour or segment individual locomotor episodes rather than to identify the rare and transient sequences of locomotor episodes that make up the behavioural response. To fill this gap, we develop a lexical, hierarchical model of behaviour. We designed an unsupervised algorithm called "BASS" to efficiently identify and segment recurring behavioural action sequences transiently occurring in long behavioural recordings. When applied to navigating larval zebrafish, BASS extracts a dictionary of remarkably long, non-Markovian sequences consisting of repeats and mixtures of slow forward and turn bouts. Applied to a novel chemotaxis assay, BASS uncovers chemotactic strategies deployed by zebrafish to avoid aversive cues consisting of sequences of fast large-angle turns and burst swims. In a simulated dataset of soaring gliders climbing thermals, BASS finds the spiraling patterns characteristic of soaring behaviour. In both cases, BASS succeeds in identifying rare action sequences in the behaviour deployed by freely moving animals. BASS can be easily incorporated into the pipelines of existing behavioural analyses across diverse species, and even more broadly used as a generic algorithm for pattern recognition in low-dimensional sequential data.

A norepinephrine-dependent glial calcium wave travels in the spinal cord upon acoustovestibular stimuli.

Although calcium waves have been widely observed in glial cells, their occurrence in vivo during behavior remains less understood. Here, we investigated the recruitment of glial cells in the hindbrain and spinal cord after acousto-vestibular (AV) stimuli triggering escape responses using in vivo population calcium imaging in larval zebrafish. We observed that gap-junction-coupled spinal glial network exhibits large and homogenous calcium increases that rose in the rostral spinal cord and propagated bi-directionally toward the spinal cord and toward the hindbrain. Spinal glial calcium waves were driven by the recruitment of neurons and in particular, of noradrenergic signaling acting through α-adrenergic receptors. Noradrenergic neurons of the medulla-oblongata (NE-MO) were revealed in the vicinity of where the calcium wave started. NE-MO were recruited upon AV stimulation and sent dense axonal projections in the rostro-lateral spinal cord, suggesting these cells could trigger the glial wave to propagate down the spinal cord. Altogether, our results revealed that a simple AV stimulation is sufficient to recruit noradrenergic neurons in the brainstem that trigger in the rostral spinal cord two massive glial calcium waves, one traveling caudally in the spinal cord and another rostrally into the hindbrain.

An in vivo Caenorhabditis elegans model for therapeutic research in human prion diseases.

Human prion diseases are fatal neurodegenerative disorders that include sporadic, infectious and genetic forms. Inherited Creutzfeldt-Jakob disease due to the E200K mutation of the prion protein-coding gene is the most common form of genetic prion disease. The phenotype resembles that of sporadic Creutzfeldt-Jakob disease at both the clinical and pathological levels, with a median disease duration of 4 months. To date, there is no available treatment for delaying the occurrence or slowing the progression of human prion diseases. Existing in vivo models do not allow high-throughput approaches that may facilitate the discovery of compounds targeting pathological assemblies of human prion protein or their effects on neuronal survival. Here, we generated a genetic model in the nematode Caenorhabditis elegans, which is devoid of any homologue of the prion protein, by expressing human prion protein with the E200K mutation in the mechanosensitive neuronal system. Expression of E200K prion protein induced a specific behavioural pattern and neurodegeneration of green fluorescent protein-expressing mechanosensitive neurons, in addition to the formation of intraneuronal inclusions associated with the accumulation of a protease-resistant form of the prion protein. We demonstrated that this experimental system is a powerful tool for investigating the efficacy of anti-prion compounds on both prion-induced neurodegeneration and prion protein misfolding, as well as in the context of human prion protein. Within a library of 320 compounds that have been approved for human use and cross the blood-brain barrier, we identified five molecules that were active against the aggregation of the E200K prion protein and the neurodegeneration it induced in transgenic animals. This model breaks a technological limitation in prion therapeutic research and provides a key tool to study the deleterious effects of misfolded prion protein in a well-described neuronal system.

Spinal sensory neurons project onto the hindbrain to stabilize posture and enhance locomotor speed.

In the spinal cord, cerebrospinal fluid-contacting neurons (CSF-cNs) are GABAergic interoceptive sensory neurons that detect spinal curvature via a functional coupling with the Reissner fiber. This mechanosensory system has recently been found to be involved in spine morphogenesis and postural control but the underlying mechanisms are not fully understood. In zebrafish, CSF-cNs project an ascending and ipsilateral axon reaching two to six segments away. Rostralmost CSF-cNs send their axons ipsilaterally into the hindbrain, a brain region containing motor nuclei and reticulospinal neurons (RSNs), which send descending motor commands to spinal circuits. Until now, the synaptic connectivity of CSF-cNs has only been investigated in the spinal cord, where they synapse onto motor neurons and premotor excitatory interneurons. The identity of CSF-cN targets in the hindbrain and the behavioral relevance of these sensory projections from the spinal cord to the hindbrain are unknown. Here, we provide anatomical and molecular evidence that rostralmost CSF-cNs synapse onto the axons of large RSNs including Mauthner cells and V2a neurons. Functional anatomy and optogenetically assisted mapping reveal that rostral CSF-cNs also synapse onto the soma and dendrites of cranial motor neurons innervating hypobranchial muscles. During acousto-vestibular evoked escape responses, ablation of rostralmost CSF-cNs results in a weaker escape response with a decreased C-bend amplitude, lower speed, and deficient postural control. Our study demonstrates that spinal sensory feedback enhances speed and stabilizes posture, and reveals a novel spinal gating mechanism acting on the output of descending commands sent from the hindbrain to the spinal cord.

Somatostatin 1.1 contributes to the innate exploration of zebrafish larva.

Pharmacological experiments indicate that neuropeptides can effectively tune neuronal activity and modulate locomotor output patterns. However, their functions in shaping innate locomotion often remain elusive. For example, somatostatin has been previously shown to induce locomotion when injected in the brain ventricles but to inhibit fictive locomotion when bath-applied in the spinal cord in vitro. Here, we investigated the role of somatostatin in innate locomotion through a genetic approach by knocking out somatostatin 1.1 (sst1.1) in zebrafish. We automated and carefully analyzed the kinematics of locomotion over a hundred of thousand bouts from hundreds of mutant and control sibling larvae. We found that the deletion of sst1.1 did not impact acousto-vestibular escape responses but led to abnormal exploration. sst1.1 mutant larvae swam over larger distance, at higher speed and performed larger tail bends, indicating that Somatostatin 1.1 inhibits spontaneous locomotion. Altogether our study demonstrates that Somatostatin 1.1 innately contributes to slowing down spontaneous locomotion.

Sensory Neurons Contacting the Cerebrospinal Fluid Require the Reissner Fiber to Detect Spinal Curvature In Vivo.

Recent evidence indicates active roles for the cerebrospinal fluid (CSF) on body axis development and morphogenesis of the spine, implying CSF-contacting neurons (CSF-cNs) in the spinal cord. CSF-cNs project a ciliated apical extension into the central canal that is enriched in the channel PKD2L1 and enables the detection of spinal curvature in a directional manner. Dorsolateral CSF-cNs ipsilaterally respond to lateral bending although ventral CSF-cNs respond to longitudinal bending. Historically, the implication of the Reissner fiber (RF), a long extracellular thread in the CSF, to CSF-cN sensory functions has remained a subject of debate. Here, we reveal, using electron microscopy in zebrafish larvae, that the RF is in close vicinity with cilia and microvilli of ventral and dorsolateral CSF-cNs. We investigate in vivo the role of cilia and the RF in the mechanosensory functions of CSF-cNs by combining calcium imaging with patch-clamp recordings. We show that disruption of cilia motility affects CSF-cN sensory responses to passive and active curvature of the spinal cord without affecting the Pkd2l1 channel activity. Because ciliary defects alter the formation of the RF, we investigated whether the RF contributes to CSF-cN mechanosensitivity in vivo. Using a hypomorphic mutation in the scospondin gene that forbids the aggregation of SCO-spondin into a fiber, we demonstrate in vivo that the RF per se is critical for CSF-cN mechanosensory function. Our study uncovers that neurons contacting the cerebrospinal fluid functionally interact with the RF to detect spinal curvature in the vertebrate spinal cord.

A tensile ring drives tissue flows to shape the gastrulating amniote embryo.

Tissue morphogenesis is driven by local cellular deformations that are powered by contractile actomyosin networks. How localized forces are transmitted across tissues to shape them at a mesoscopic scale is still unclear. Analyzing gastrulation in entire avian embryos, we show that it is driven by the graded contraction of a large-scale supracellular actomyosin ring at the margin between the embryonic and extraembryonic territories. The propagation of these forces is enabled by a fluid-like response of the epithelial embryonic disk, which depends on cell division. A simple model of fluid motion entrained by a tensile ring quantitatively captures the vortex-like "polonaise" movements that accompany the formation of the primitive streak. The geometry of the early embryo thus arises from the transmission of active forces generated along its boundary.

Regulation of the apical extension morphogenesis tunes the mechanosensory response of microvilliated neurons.

Multiple types of microvilliated sensory cells exhibit an apical extension thought to be instrumental in the detection of sensory cues. The investigation of the mechanisms underlying morphogenesis of sensory apparatus is critical to understand the biology of sensation. Most of what we currently know comes from the study of the hair bundle of the inner ear sensory cells, but morphogenesis and function of other sensory microvilliated apical extensions remain poorly understood. We focused on spinal sensory neurons that contact the cerebrospinal fluid (CSF) through the projection of a microvilliated apical process in the central canal, referred to as cerebrospinal fluid-contacting neurons (CSF-cNs). CSF-cNs respond to pH and osmolarity changes as well as mechanical stimuli associated with changes of flow and tail bending. In vivo time-lapse imaging in zebrafish embryos revealed that CSF-cNs are atypical neurons that do not lose their apical attachment and form a ring of actin at the apical junctional complexes (AJCs) that they retain during differentiation. We show that the actin-based protrusions constituting the microvilliated apical extension arise and elongate from this ring of actin, and we identify candidate molecular factors underlying every step of CSF-cN morphogenesis. We demonstrate that Crumbs 1 (Crb1), Myosin 3b (Myo3b), and Espin orchestrate the morphogenesis of CSF-cN apical extension. Using calcium imaging in crb1 and espin mutants, we further show that the size of the apical extension modulates the amplitude of CSF-cN sensory response to bending of the spinal cord. Based on our results, we propose that the apical actin ring could be a common site of initiation of actin-based protrusions in microvilliated sensory cells. Furthermore, our work provides a set of actors underlying actin-based protrusion elongation shared by different sensory cell types and highlights the critical role of the apical extension shape in sensory detection.

Timed Collinear Activation of Hox Genes during Gastrulation Controls the Avian Forelimb Position.

Limb position along the body is highly consistent within one species but very variable among vertebrates. Despite major advances in our understanding of limb patterning in three dimensions, how limbs reproducibly form along the antero-posterior axis remains largely unknown. Hox genes have long been suspected to control limb position; however, supporting evidences are mostly correlative and their role in this process is unclear. Here, we show that limb position is determined early in development through the action of Hox genes. Dynamic lineage analysis revealed that, during gastrulation, the forelimb, interlimb, and hindlimb fields are progressively generated and concomitantly patterned by the collinear activation of Hox genes in a two-step process. First, the sequential activation of Hoxb genes controls the relative position of their own collinear domains of expression in the forming lateral plate mesoderm, as demonstrated by functional perturbations during gastrulation. Then, within these collinear domains, we show that Hoxb4 anteriorly and Hox9 genes posteriorly, respectively, activate and repress the expression of the forelimb initiation gene Tbx5 and instruct the definitive position of the forelimb. Furthermore, by comparing the dynamics of Hoxb genes activation during zebra finch, chicken, and ostrich gastrulation, we provide evidences that changes in the timing of collinear Hox gene activation might underlie natural variation in forelimb position between different birds. Altogether, our results that characterize the cellular and molecular mechanisms underlying the regulation and natural variation of forelimb positioning in avians show a direct and early role for Hox genes in this process.