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.

c-Kit Receptor Maintains Sensory Axon Innervation of the Skin through Src Family Kinases.

Peripheral somatosensory neurons innervate the skin and sense the environment. Whereas many studies focus on initial axon outgrowth and pathfinding, how signaling pathways contribute to maintenance of the established axon arbors and terminals within the skin is largely unknown. This question is particularly relevant to the many types of neuropathies that affect mature neuronal arbors. We show that a receptor tyrosine kinase (RTK), c-Kit, contributes to maintenance, but not initial development, of cutaneous axons in the larval zebrafish before sex determination. Downregulation of Kit signaling rapidly induced retraction of established axon terminals in the skin and a reduction in axonal density. Conversely, misexpression of c-Kit ligand in the skin in larval zebrafish induced increases in local sensory axon density, suggesting an important role for Kit signaling in cutaneous axon maintenance. We found Src family kinases (SFKs) act directly downstream to mediate Kit's role in regulating cutaneous axon density. Our data demonstrate a requirement for skin-to-axon signaling to maintain axonal networks and elucidate novel roles for Kit and SFK signaling in this context. This Kit-SFK signaling axis offers a potential pathway to therapeutically target in sensory neuropathies and to further explore in other neurobiological processes.SIGNIFICANCE STATEMENTThe skin is full of small nerve endings that sense different environmental stimuli. How these nerve endings grow and reach a specific area of the skin during development has been the focus of many studies. In contrast, the cellular and molecular mechanisms required to maintain the function and health of these structures is relatively unknown. We discovered that a specific receptor in sensory neurons, c-Kit, is required to maintain the density of nerve endings in the skin. Furthermore, we found that a molecular target of c-Kit, Src family kinases (SFKs), is necessary for this role. Thus, c-Kit/SFK signaling regulates density and maintenance of sensory nerve endings in the skin and may have important roles in neural disease and regeneration.

Rabconnectin-3a regulates vesicle endocytosis and canonical Wnt signaling in zebrafish neural crest migration.

Cell migration requires dynamic regulation of cell-cell signaling and cell adhesion. Both of these processes involve endocytosis, lysosomal degradation, and recycling of ligand-receptor complexes and cell adhesion molecules from the plasma membrane. Neural crest (NC) cells in vertebrates are highly migratory cells, which undergo an epithelial-mesenchymal transition (EMT) to leave the neural epithelium and migrate throughout the body to give rise to many different derivatives. Here we show that the v-ATPase interacting protein, Rabconnectin-3a (Rbc3a), controls intracellular trafficking events and Wnt signaling during NC migration. In zebrafish embryos deficient in Rbc3a, or its associated v-ATPase subunit Atp6v0a1, many NC cells fail to migrate and misregulate expression of cadherins. Surprisingly, endosomes in Rbc3a- and Atp6v0a1-deficient NC cells remain immature but still acidify. Rbc3a loss-of-function initially downregulates several canonical Wnt targets involved in EMT, but later Frizzled-7 accumulates at NC cell membranes, and nuclear B-catenin levels increase. Presumably due to this later Wnt signaling increase, Rbc3a-deficient NC cells that fail to migrate become pigment progenitors. We propose that Rbc3a and Atp6v0a1 promote endosomal maturation to coordinate Wnt signaling and intracellular trafficking of Wnt receptors and cadherins required for NC migration and cell fate determination. Our results suggest that different v-ATPases and associated proteins may play cell-type-specific functions in intracellular trafficking in many contexts.

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 make up 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 non-overlapping 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.