The Reissner Fiber in the Cerebrospinal Fluid Controls Morphogenesis of the Body Axis.

Organ development depends on the integration of coordinated long-range communication between cells. The cerebrospinal fluid composition and flow properties regulate several aspects of central nervous system development, including progenitor proliferation, neurogenesis, and migration [1-3]. One understudied component of the cerebrospinal fluid, described over a century ago in vertebrates, is the Reissner fiber. This extracellular thread forming early in development results from the assembly of the SCO-spondin protein in the third and fourth brain ventricles and central canal of the spinal cord [4]. Up to now, the function of the Reissner fiber has remained elusive, partly due to the lack of genetic invalidation models [4]. Here, by mutating the scospondin gene, we demonstrate that the Reissner fiber is critical for the morphogenesis of a straight posterior body axis. In zebrafish mutants where the Reissner fiber is lost, ciliogenesis and cerebrospinal fluid flow are intact but body axis morphogenesis is impaired. Our results also explain the frequently observed phenotype that mutant embryos with defective cilia exhibit defects in body axis curvature. Here, we reveal that these mutants systematically fail to assemble the Reissner fiber. We show that cilia promote the formation of the Reissner fiber and that the fiber is necessary for proper body axis morphogenesis. Our study sets the stage for future investigations of the mechanisms linking the Reissner fiber to the control of body axis curvature during vertebrate development.

Pkd2l1 is required for mechanoception in cerebrospinal fluid-contacting neurons and maintenance of spine curvature.

Defects in cerebrospinal fluid (CSF) flow may contribute to idiopathic scoliosis. However, the mechanisms underlying detection of CSF flow in the central canal of the spinal cord are unknown. Here we demonstrate that CSF flows bidirectionally along the antero-posterior axis in the central canal of zebrafish embryos. In the cfap298tm304 mutant, reduction of cilia motility slows transport posteriorly down the central canal and abolishes spontaneous activity of CSF-contacting neurons (CSF-cNs). Loss of the sensory Pkd2l1 channel nearly abolishes CSF-cN calcium activity and single channel opening. Recording from isolated CSF-cNs in vitro, we show that CSF-cNs are mechanosensory and require Pkd2l1 to respond to pressure. Additionally, adult pkd2l1 mutant zebrafish develop an exaggerated spine curvature, reminiscent of kyphosis in humans. These results indicate that CSF-cNs are mechanosensory cells whose Pkd2l1-driven spontaneous activity reflects CSF flow in vivo. Furthermore, Pkd2l1 in CSF-cNs contributes to maintenance of natural curvature of the spine.

The dual developmental origin of spinal cerebrospinal fluid-contacting neurons gives rise to distinct functional subtypes.

Chemical and mechanical cues from the cerebrospinal fluid (CSF) can affect the development and function of the central nervous system (CNS). How such cues are detected and relayed to the CNS remains elusive. Cerebrospinal fluid-contacting neurons (CSF-cNs) situated at the interface between the CSF and the CNS are ideally located to convey such information to local networks. In the spinal cord, these GABAergic neurons expressing the PKD2L1 channel extend an apical extension into the CSF and an ascending axon in the spinal cord. In zebrafish and mouse spinal CSF-cNs originate from two distinct progenitor domains characterized by distinct cascades of transcription factors. Here we ask whether these neurons with different developmental origins differentiate into cells types with different functional properties. We show in zebrafish larva that the expression of specific markers, the morphology of the apical extension and axonal projections, as well as the neuronal targets contacted by CSF-cN axons, distinguish the two CSF-cN subtypes. Altogether our study demonstrates that the developmental origins of spinal CSF-cNs give rise to two distinct functional populations of sensory neurons. This work opens novel avenues to understand how these subtypes may carry distinct functions related to development of the spinal cord, locomotion and posture.

Optimization of a Neurotoxin to Investigate the Contribution of Excitatory Interneurons to Speed Modulation In Vivo.

Precise control of speed during locomotion is essential for adaptation of behavior in different environmental contexts [1-4]. A central question in locomotion lies in understanding which neural populations set locomotor frequency during slow and fast regimes. Tackling this question in vivo requires additional non-invasive tools to silence large populations of neurons during active locomotion. Here we generated a stable transgenic line encoding a zebrafish-optimized botulinum neurotoxin light chain fused to GFP (BoTxBLC-GFP) to silence synaptic output over large populations of motor neurons or interneurons while monitoring active locomotion. By combining calcium imaging, electrophysiology, optogenetics, and behavior, we show that expression of BoTxBLC-GFP abolished synaptic release while maintaining characterized activity patterns and without triggering off-target effects. As chx10(+) V2a interneurons (V2as) are well characterized as the main population driving the frequency-dependent recruitment of motor neurons during fictive locomotion [5-14], we validated our silencing method by testing the effect of silencing chx10(+) V2as during active and fictive locomotion. Silencing of V2as selectively abolished fast locomotor frequencies during escape responses. In addition, spontaneous slow locomotion occurred less often and at frequencies lower than in controls. Overall, this silencing approach confirms that V2a excitation is critical for the production of fast stimulus-evoked swimming and also reveals a role for V2a excitation in the production of slower spontaneous locomotor behavior. Altogether, these results establish BoTxBLC-GFP as an ideal tool for in vivo silencing for probing the development and function of neural circuits from the synaptic to the behavioral level.

Neuronal wiring: linking dendrite placement to synapse formation.

Understanding the processes that drive the formation of synapses between specific neurons within a circuit is critical to understanding how neural networks develop. A new study of synapse formation between motor neurons and pre-synaptic partners highlights the importance of dendrite placement.

Fast Calcium Imaging with Optical Sectioning via HiLo Microscopy.

Imaging intracellular calcium concentration via reporters that change their fluorescence properties upon binding of calcium, referred to as calcium imaging, has revolutionized our way to probe neuronal activity non-invasively. To reach neurons densely located deep in the tissue, optical sectioning at high rate of acquisition is necessary but difficult to achieve in a cost effective manner. Here we implement an accessible solution relying on HiLo microscopy to provide robust optical sectioning with a high frame rate in vivo. We show that large calcium signals can be recorded from dense neuronal populations at high acquisition rates. We quantify the optical sectioning capabilities and demonstrate the benefits of HiLo microscopy compared to wide-field microscopy for calcium imaging and 3D reconstruction. We apply HiLo microscopy to functional calcium imaging at 100 frames per second deep in biological tissues. This approach enables us to discriminate neuronal activity of motor neurons from different depths in the spinal cord of zebrafish embryos. We observe distinct time courses of calcium signals in somata and axons. We show that our method enables to remove large fluctuations of the background fluorescence. All together our setup can be implemented to provide efficient optical sectioning in vivo at low cost on a wide range of existing microscopes.

Hierarchy of neural organization in the embryonic spinal cord: Granger-causality graph analysis of in vivo calcium imaging data.

The recent development of genetically encoded calcium indicators enables monitoring in vivo the activity of neuronal populations. Most analysis of these calcium transients relies on linear regression analysis based on the sensory stimulus applied or the behavior observed. To estimate the basic properties of the functional neural circuitry, we propose a network approach to calcium imaging recorded at single cell resolution. Differently from previous analysis based on cross-correlation, we used Granger-causality estimates to infer information propagation between the activities of different neurons. The resulting functional network was then modeled as a directed graph and characterized in terms of connectivity and node centralities. We applied our approach to calcium transients recorded at low frequency (4 Hz) in ventral neurons of the zebrafish spinal cord at the embryonic stage when spontaneous coiling of the tail occurs. Our analysis on population calcium imaging data revealed a strong ipsilateral connectivity and a characteristic hierarchical organization of the network hubs that supported established propagation of activity from rostral to caudal spinal cord. Our method could be used for detecting functional defects in neuronal circuitry during development and pathological conditions.

ZebraZoom: an automated program for high-throughput behavioral analysis and categorization.

The zebrafish larva stands out as an emergent model organism for translational studies involving gene or drug screening thanks to its size, genetics, and permeability. At the larval stage, locomotion occurs in short episodes punctuated by periods of rest. Although phenotyping behavior is a key component of large-scale screens, it has not yet been automated in this model system. We developed ZebraZoom, a program to automatically track larvae and identify maneuvers for many animals performing discrete movements. Our program detects each episodic movement and extracts large-scale statistics on motor patterns to produce a quantification of the locomotor repertoire. We used ZebraZoom to identify motor defects induced by a glycinergic receptor antagonist. The analysis of the blind mutant atoh7 revealed small locomotor defects associated with the mutation. Using multiclass supervised machine learning, ZebraZoom categorized all episodes of movement for each larva into one of three possible maneuvers: slow forward swim, routine turn, and escape. ZebraZoom reached 91% accuracy for categorization of stereotypical maneuvers that four independent experimenters unanimously identified. For all maneuvers in the data set, ZebraZoom agreed with four experimenters in 73.2-82.5% of cases. We modeled the series of maneuvers performed by larvae as Markov chains and observed that larvae often repeated the same maneuvers within a group. When analyzing subsequent maneuvers performed by different larvae, we found that larva-larva interactions occurred as series of escapes. Overall, ZebraZoom reached the level of precision found in manual analysis but accomplished tasks in a high-throughput format necessary for large screens.