Single-Cell Analysis of Rohon-Beard Neurons Implicates Fgf Signaling in Axon Maintenance and Cell Survival.

Peripheral sensory neurons are a critical part of the nervous system that transmit a multitude of sensory stimuli to the central nervous system. During larval and juvenile stages in zebrafish, this function is mediated by Rohon-Beard somatosensory neurons (RBs). RBs are optically accessible and amenable to experimental manipulation, making them a powerful system for mechanistic investigation of sensory neurons. Previous studies provided evidence that RBs fall into multiple subclasses; however, the number and molecular makeup of these potential RB subtypes have not been well defined. Using a single-cell RNA sequencing (scRNA-seq) approach, we demonstrate that larval RBs in zebrafish fall into three, largely nonoverlapping classes of neurons. We also show that RBs are molecularly distinct from trigeminal neurons in zebrafish. Cross-species transcriptional analysis indicates that one RB subclass is similar to a mammalian group of A-fiber sensory neurons. Another RB subclass is predicted to sense multiple modalities, including mechanical stimulation and chemical irritants. We leveraged our scRNA-seq data to determine that the fibroblast growth factor (Fgf) pathway is active in RBs. Pharmacological and genetic inhibition of this pathway led to defects in axon maintenance and RB cell death. Moreover, this can be phenocopied by treatment with dovitinib, an FDA-approved Fgf inhibitor with a common side effect of peripheral neuropathy. Importantly, dovitinib-mediated axon loss can be suppressed by loss of Sarm1, a positive regulator of neuronal cell death and axonal injury. This offers a molecular target for future clinical intervention to fight neurotoxic effects of this drug.

Rohon-beard neurons do not succumb to programmed cell death during zebrafish development.

During neural development, sculpting of early formed circuits by cell death and synaptic pruning is necessary to generate a functional and efficient nervous system. This allows for the establishment of rudimentary circuits which necessitate early organism survival to later undergo subsequent refinement. These changes facilitate additional specificity to stimuli which can lead to increased behavioral complexity. In multiple species, Rohon-Beard neurons (RBs) are the earliest mechanosensory neurons specified and are critical in establishing a rudimentary motor response circuit. Sensory input from RBs gradually becomes redundant as dorsal root ganglion (DRG) neurons develop and integrate into motor circuits. Previous studies demonstrate that RBs undergo a dramatic wave of cell death concurrent with development of the DRG. However, contrary to these studies, we show that neurogenin1+ (ngn1) RBs do not undergo a widespread wave of programmed cell death during early zebrafish development and instead persist until at least 15 days post fertilization (dpf). Starting at 2 dpf, we also observed a dramatic medialization and shrinkage of ngn1+ RB somas along with a gradual downregulation of ngn1 in RBs. This alters a fundamental premise of early zebrafish neural development and opens new avenues to explore mechanisms of RB function, persistence, and circuit refinement.

Complete persistence of the primary somatosensory system in zebrafish.

The somatosensory system detects peripheral stimuli that are translated into behaviors necessary for survival. Fishes and amphibians possess two somatosensory systems in the trunk: the primary somatosensory system, formed by the Rohon-Beard neurons, and the secondary somatosensory system, formed by the neural crest cell-derived neurons of the Dorsal Root Ganglia. Rohon-Beard neurons have been characterized as a transient population that mostly disappears during the first days of life and is functionally replaced by the Dorsal Root Ganglia. Here, I follow Rohon-Beard neurons in vivo and show that the entire repertoire remains present in zebrafish from 1-day post-fertilization until the juvenile stage, 15-days post-fertilization. These data indicate that zebrafish retain two complete somatosensory systems until at least a developmental stage when the animals display complex behavioral repertoires.

Long-lived zebrafish Rohon-Beard cells.

During normal development of the nervous system, extensive neuronal proliferation as well as death occurs. The extent of development death varies considerably between neuronal populations from little to almost 100%. Early born somatosensory neurons, known as Rohon-Beard cells, have served as an example of neurons that disappear during early developmental stages, presumably as their function is taken over by later developing dorsal root ganglion neurons. However, recent studies have raised questions about the extent to which zebrafish Rohon-Beard cells die during embryogenesis. While Rohon-Beard cells have distinguishing morphological features during embryonic stages development, they subsequently undergo substantial changes in their shape, size and position that hinder their unambiguous identification at later stages. To overcome this obstacle, we identify Rohon-Beard cells at one day, and using a combination of mosaic and stable transgenic labeling and repeated observation, follow them for 13-16 days post fertilization. We find that about 40% survive to late larval stages. Our studies also reveal that Rohon-Beard cells display an unusual repertoire of cell death properties. At one day, about 25% Rohon-Beard cells expose phosphatidyl serine at the surface membrane, but less than one Rohon-Beard cell/embryo expresses activated-caspase-3. Further, the temporal delay between detection of cell death markers and loss of the soma ranges from

From neuron to behavior: Sensory-motor coordination of zebrafish turning behavior.

Recent development of optogenetics brought non-invasive neural activation in living organisms. Transparent zebrafish larva is one of the suitable animal models for this technique, which enables us to investigate neural circuits for behaviors based on a whole individual nervous system. In this article we review our recent finding that suggests sensory-motor coordination in larval zebrafish escape behavior. When water vibration stimulates mechanosensory Rohon-Beard (RB) neurons, intra-spinal reflex circuit launches contralateral trunk muscle contraction that makes rapid body curvature for turning. In addition, positional information of the stimulus is conveyed to supra-spinal circuits, and then regulates the curvature strength for appropriate escape pathway from the threat. Sensory-motor coordination is a fundamental feature to adapt behaviors to environment, and zebrafish larvae would be an excellent model for elucidating its neural backbones.

Activation of α2A-containing nicotinic acetylcholine receptors mediates nicotine-induced motor output in embryonic zebrafish.

It is well established that cholinergic signaling has critical roles during central nervous system development. In physiological and behavioral studies, activation of nicotinic acetylcholine receptors (nAChRs) has been implicated in mediating cholinergic signaling. In developing spinal cord, cholinergic transmission is associated with neural circuits responsible for producing locomotor behaviors. In this study, we investigated the expression pattern of the α2A nAChR subunit as previous evidence suggested it could be expressed by spinal neurons. In situ hybridization and immunohistochemistry revealed that the α2A nAChR subunits are expressed in spinal Rohon-Beard (RB) neurons and olfactory sensory neurons in young embryos. To examine the functional role of the α2A nAChR subunit during embryogenesis, we blocked its expression using antisense modified oligonucleotides. Blocking the expression of α2A nAChR subunits had no effect on spontaneous motor activity. However, it did alter the embryonic nicotine-induced motor output. This reduction in motor activity was not accompanied by defects in neuronal and muscle elements associated with the motor output. Moreover, the anatomy and functionality of RB neurons was normal even in the absence of the α2A nAChR subunit. Thus, we propose that α2A-containing nAChRs are dispensable for normal RB development. However, in the context of nicotine-induced motor output, α2A-containing nAChRs on RB neurons provide the substrate that nicotine acts upon to induce the motor output. These findings also indicate that functional neuronal nAChRs are present within spinal cord at the time when locomotor output in zebrafish first begins to manifest itself.

Spatiotemporal expression of the cocaine- and amphetamine-regulated transcript-like (cart-like) gene during zebrafish embryogenesis.

The cocaine- and amphetamine-regulated transcript (CART) genes are involved in the neural regulation of energy homeostasis; however, their developmental expressions and functions are not fully understood in vertebrates. We have identified a novel zebrafish cart-like gene that encodes a protein of 105 amino acids possessing sequence similarity to zebrafish and mammalian CART proteins. RT-PCR analysis revealed that the cart-like transcripts were maternally supplied and gradually decreased during the cleavage, blastula and gastrula stages; then, transcripts subsequently reaccumulated at the segmentation, pharyngula and hatching stages. Based on a whole-mount in situ hybridization analysis using an antisense cart-like RNA probe, we found that the cart-like transcript was predominantly expressed in both the Rohon-Beard neurons and trigeminal ganglia, suggesting the involvement of the cart-like gene in zebrafish neural development.

Acetyl L-carnitine protects motor neurons and Rohon-Beard sensory neurons against ketamine-induced neurotoxicity in zebrafish embryos.

Ketamine, a non-competitive antagonist of N-methyl-D-aspartate (NMDA) type glutamate receptors is commonly used as a pediatric anesthetic. Multiple studies have shown ketamine to be neurotoxic, particularly when administered during the brain growth spurt. Previously, we have shown that ketamine is detrimental to motor neuron development in the zebrafish embryos. Here, using both wild type (WT) and transgenic (hb9:GFP) zebrafish embryos, we demonstrate that ketamine is neurotoxic to both motor and sensory neurons. Drug absorption studies showed that in the WT embryos, ketamine accumulation was approximately 0.4% of the original dose added to the exposure medium. The transgenic embryos express green fluorescent protein (GFP) localized in the motor neurons making them ideal for evaluating motor neuron development and toxicities in vivo. The hb9:GFP zebrafish embryos (28 h post fertilization) treated with 2 mM ketamine for 20 h demonstrated significant reductions in spinal motor neuron numbers, while co-treatment with acetyl L-carnitine proved to be neuroprotective. In whole mount immunohistochemical studies using WT embryos, a similar effect was observed for the primary sensory neurons. In the ketamine-treated WT embryos, the number of primary sensory Rohon-Beard (RB) neurons was significantly reduced compared to that in controls. However, acetyl L-carnitine co-treatment prevented ketamine-induced adverse effects on the RB neurons. These results suggest that acetyl L-carnitine protects both motor and sensory neurons from ketamine-induced neurotoxicity.