Single-cell analysis of Rohon-Beard neurons implicates Fgf signaling in axon maintenance and cell survival [larva]
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 phenotype can be phenocopied by treatment with an FDA-approved Fgf inhibitor dovitinib, which is used in clinic and causes 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. Overall design: About 150 7-day old larva were euthanized in 0.02% tricaine, and individually decapitated behind the hindbrain. They were incubated with 20 mg/ml collagenase (Life Sciences) in a buffer containing 134 mM NaCl, 2.9 mM KCl, 1.2 mM MgCl2, 2.1 mM CaCl2, and 10 mM Na-HEPES (pH 7.8) at 28 °C for 2 hr, with intermittent trituration using a p200 pipette aid at 0, 0.5 hr and 1 hr of the incubation. To release spinal cords from remaining tissue, the final triturations were done using fire-polished Pasteur pipettes with decreased opening sizes (300, 200, 100 µm respectively). Intact spinal cords were transferred to L15 media and washed 3 times with fresh media. The spinal cords were incubated with 0.25% trypsin solution (in 1xPBS containing 1 mM EDTA) at 28 °C for 25 min. The digestion was terminated by adding 500 µl stop solution (L15 with 1% fetal bovine serum). The tissue was collected by spinning at 400 g for 3 min at 4°C, washed once with L15 and resuspended in 200 µl of L15 media. Spinal cord cells were dissociated by triturating the digested tissue with fire-polished Pasteur pipettes with 80-100 µm opening. The solution was filtered through a 35 µm strainer into a siliconized collection tube. The suspension was examined on a microscope for cell count, Trypan blue staining based viability test and proportion of dispersed single cells. Samples with a viability above 70% were used for sequencing