Pioneer neurons are molecularly distinct, and their axon targeting is regulated by retinoic acid signaling (WT 30 h)

During nervous system development, pioneer neurons are the first to extend their axons into target tissues, creating a scaffold for follower neurons. Despite years of study, whether pioneer neurons are a molecularly distinct population is unknown. Analysis of zebrafish posterior lateral line (pLL) sensory neurons during axon growth using single-cell RNA sequencing (scRNA-seq) revealed that pioneer and follower neurons are transcriptionally distinct. Expression profiling of differentiating pLL progenitors defined follower as the ground state, whereas “pioneer” is a later developmental state. The scRNA-seq data revealed active retinoic acid (RA) signaling in followers, but not in pioneers. Modulation of RA signaling within single pLL neurons showed that its downregulation in pioneers is necessary for expression of neurotrophic factor receptor ret, which is required for correct targeting of pioneer axons. Our study provided insights into the molecular landscape of pioneer neurons and revealed the regulatory role of RA signaling in their development. Overall design: Thirty hour old TgBAC(neurod1:EGFP)nl1 zebrafish embryos were collected and euthanized in 1.7 ml microcentrifuge tubes. Embryos were deyolked using a calcium-free Ringer's solution (116 mM NaCl, 2.6 mM KCl, 5 mM HEPES pH 7.0), by gently pipetting up and down with a P200 pipet. Embryos were incubated for 5 minutes in Ringer's solution. Embryos were transferred to pre-warmed protease solutions (0.25% trypsin, 1 mM EDTA, pH 8.0, PBS) and collagenase P/HBSS (100 mg/mL) was added. Embryos were incubated at 28° C for 15 minutes and were homogenized every 5 minutes using a P1000 pipet. The Stop solution (6X, 30% calf serum, 6 mM CaCl2, PBS) was added and samples were centrifuged (350xg, 4° C for 5 minutes). Supernatant was removed and 1 mL of chilled suspension solution was added (1% FBS, 0.8 mM CaCl2, 50 U/mL penicillin, 0.05 mg/mL streptomycin, DMEM). Samples were centrifuged again (350g, 4° C for 5 minutes) and supernatant was removed. 700 µl of chilled suspension solution was added and cells were resuspended by pipetting. Cells were passed through a 40 µm cell strainer into a FACs tube and kept on ice. GFP and RFP+ cells were FAC sorted on a BD Symphony cell sorter into sorting buffer (50 µl PBS/ 2% BSA) in a siliconized 1.5mL tube.