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. 2021 Dec 13;10:e68224. doi: 10.7554/eLife.68224

Figure 2. Molecular classification of neuromodulator-type neurons.

(A) The schematic showing the procedure of collecting single-cell transcriptomes of neuromodulator neurons with fluorescence-activated cell sorting (FACS). Using Tg (ETvmat2:GFP) fishline, we could isolate dopaminergic (DA), serotonergic (5-HT), and norepinephrinergic (NE) neurons. (B) The t-distributed stochastic neighbor embedding (t-SNE) plot of 5368 cells obtained from Tg (ETvmat2:GFP) fishline expressing monoaminergic neuromodulators, showing 22 clusters, each marked by a number, color-coding brain regions and cell type of each cluster. (C) Dot plot showing the expression of glutamatergic marker (vglut2a/vglut2b/vglu1/vglu3) and GABAergic marker (gad1b/gad2) in each of the 22 vmat2+ clusters in B. The neurotransmitter phenotypes were color-coded. Empty squares depict the ones with undefined neurotransmitter phenotypes. The average expression levels of these genes for all cells in each cluster were coded by the gray level. The percentage of cells expressing each gene within each cluster was coded by dot size.

Figure 2—source data 1. The annotation of neuromodulator-type neuronal types with well-known markers.

Figure 2.

Figure 2—figure supplement 1. Molecular classification of neuromodulator neurons.

Figure 2—figure supplement 1.

(A) The JaccardRainCloundPlot showing the stability of vmat2+ neuromodulator clusters in Figure 2B. Red line indicated the 0.6 cutoff of Jaccard index to evaluate the cluster stability. Red arrowhead indicated unstable clusters. (B) Representative image of fishline Tg (ETvmat2:GFP). The fishline labeled different monoaminergic neuronal types. Scale bars: 100 μm. (C) Dot plot showing the cell-type-specific markers of 22 vmat2+ neuromodulator clusters, with the average level of their expressions in all cells of each cluster was color-coded, percentage of gene expression was indicated by dot size. (D) t-Distributed stochastic neighbor embedding (tSNE) plots showing the expression of different brain region-specific markers (dlx5a, otpa, nr4a2b, en2a) in 22 vmat2+ neuromodulator clusters. (E) Representative confocal images of 8 dpf Tg (ETvmat2:GFP) and Tg (gad1b:gal4::uas:mcherry) zebrafish. Scale bars: 100 μm. Right: magnified images showing localization of vmat2 and gad1b in hindbrain (Hind), and the regions underneath the optic tectum (sub-OT). Scale bars: 20 μm.