Fig 1.
Morphology of REN, ROLE and RELL neurons.
(A) Schematic view of the projection paths and neuromast locations of the zebrafish Anterior Lateral Line and Posterior Lateral Line at 5 dpf (adapted from Manuel and colleagues [11]). (B) Top view of the rhombomere region of 5 dpf old Tg(dmrt3a:GAL4;UAS:Tomato) larvae, where a single inhibitory efferent neuron (REN, ROLE, RELL) or single neurons that do Not Project to the lateral line (NP) can be seen (magenta). Lateral line sensory afferent projections (cyan) marked by Tg(Hgn39D) were included for orientation and determining efferent neuron positions. White arrows indicate axon projection towards the lateral line. (C) Schematic overlay of REN, ROLE, RELL and NP neurons showing their position in relation to each other. Dashed lines represent the midline. Scale bar = 50 µm.
Fig 2.
Expression of genes related to neurons and glia.
(A) Number of reads for each sample. Line represents the mean for each group. (B) Number of detected genes in each sample. Line represents the mean for each group. (C) Dot-plot of genes related to neurons, inhibitory efferent neurons, and glia. (D) Box and whiskers plot (median with 2.5-97-5 percentile) of the neuron/glia expression ratios for REN, ROLE, RELL NP, and HB. Numbers above the plots indicate median values per cell group, higher ratios indicate stronger neuron marker expression compared to glia marker expression. p = 0.01 (**).
Fig 3.
Differentially expressed genes lateral line inhibitory efferent neurons.
(A) PCA representation based on the top 2000 expressed genes in the cells retained in the dataset. HB samples clustered separate from the other samples. Two REN were plotted as outliers (bottom right). (B) Heatmap of differentially expressed genes when comparing NP vs. REN, ROLE, RELL. (C) Heatmap of differentially expressed genes comparing REN vs. ROLE, RELL. (D-F) Heatmaps of differentially expressed genes comparing: REN vs. ROLE (D), REN vs. RELL (E), or ROLE vs. RELL (F). Go Analysis results for the DEGs: B: none; C: 14 processes, including tripeptide transport, oligopeptide transport, maturation of lsu-rRNA and 5.8s rRNA (nGenes 1 each); D: 15 processes, including myelination periphery axons, Schwann cell development, lateral line glia cell development, oligodendrocyte development (nGenes 2 each) and 1 process involves Membrane organization (nGenes 4); E: none; F: substrate adhesion-dependent cell spreading (nGenes 4) and cell substrate adhesion (nGenes 4)..
Fig 4.
Expression of neurotransmitters and their signalling pathways.
(A) Dot-plot of genes related neurotransmitter phenotype. (B) Dot-plot of genes related to glutamate, glycine, and GABA signalling. Note that gls2b (<1.0 scaled expression) is not plotted. (C) immunoreactivity for Gad1b antibody (Green; C’) in Tg(dmrr3a:GAL4,UAS:mKate2) marking neurons in the REN and CEN, including the REN, ROLE, and RELL inhibitory efferent neurons (magenta. C”). Arrows indicate efferent axons projecting towards the lateral line, asterisks mark a selection of Gad1b-positive neurons near mKate2-postive REN and CEN neurons. Scale bar = 10 µm.
Fig 5.
Expression of genes related to rhombomeres and axon guidance.
(A) Dot-plot of genes related to rhombomere position. (B) Dot-plot of genes related to axon guidance and those enabling axons to exit the nervous system.
Fig 6.
Expression of genes related to gap-junctions and membrane potential.
(A) Dot-plot of genes related to gap-junctions and their support proteins. Note that gjd2 (<1.0 scaled expression) is not plotted. (B) Dot-plot of selected genes related to glutamate sensitivity and membrane potential.
Fig 7.
Neuromast innervation by ROLE and RELL neurons.
(A) Schematic outline of the lateral line projections and neuromasts on a 5 dpf zebrafish larvae (adapted from Manuel and colleagues [11]). The P1 and P2 neuromast were selected based on their flow polarity: anterior-posterior (A-P) for P1, and dorsal-ventral (D-V) for P2. Circles contain representative confocal images of neuromasts in Tg(myo6b:hs:eGFFP,dmrt3a:GAL4,UAS:Tomato) larvae, showing individual hair cells (grey) and inhibitory efferent innervation (magenta). (B) Quantification of GFP-positive hair cells in the P1 and P2 neuromasts of Tg(myo6b:hs:eGFP) larvae (10 dpf). Values represent the average (± standard deviation) number of hair cells found among quantified neuromasts. (C) Number of synaptic boutons observed in neuromasts innervated by ROLE or RELL in Tg(dmrt3a:GAL4,UAS:Tomato) or all inhibitory efferent neurons (FULL) in Tg(dmrt3a:GAL4,UAS:mKate2). Data is plotted as the average (± standard deviation) number of boutons found in maximum projections of innervated neuromasts. (D-F) Statistical analysis of the number of boutons observed in the P1 and P2 neuromasts. (G-H) Quantification of the percentage of hair cells in the P1 and P2 neuromast in close proximity of synaptic boutons from ROLE, RELL, or FULL innervations. Assessment of proximity was done in 3D rendered confocal images. (I) Overlay of the position-maps of synaptic boutons from ROLE, RELL, or FULL innervation of neuromasts. (J) Overlay of the position maps of hair cells connected by ROLE, RELL, or FULL innervation of a neuromast. Percentages reflect the observations for connected hair cells in that segment of the neuromast. N = number of neuromasts for each group. Statistics: p < 0.05 (*), p < 0.01 (**).