Journal: eLife

Article Title: Single-cell RNAseq analysis of spinal locomotor circuitry in larval zebrafish

doi: 10.7554/eLife.89338

Figure Lengend Snippet: ( A ) Dot plot for synaptic genes differentially enriched in PMns compared to SMns. Both percentage of cells with expression and average expression levels are shown. Examples of synaptic genes that are expressed at comparable levels between the two Mn types are shaded in gray. ( B ) A similar comparison for differentially expressed ion channel genes as shown in ( A ). ( C ) Feature plots for three top differentially expressed ion channel genes, cacna1ab , scn4ba, and kcna3a, shown in the Mn t-distributed stochastic neighbor embedding (t-SNE) projection (left). The assignment of Mn type identity was duplicated from for reference (right graphs). The proportion of cells in each Mn type expressing individual cassette member (top) and cassette member combinations (bottom). ( D ) Representative in situ hybridization images with scn4ba probes in Tg(mnx1:GFP) (left) and Tg(SAIG213A;GFP) (right) transgenic fish. Each image shows approximately two segments of the spinal cord in the middle trunk of 4 days post fertilization (dpf) fish. Arrows indicate the PMns in Tg(mnx1:GFP) and CaP in Tg(SAIG213A;GFP) fish (n = 15–18 fish). Two commissural local (CoLo) interneurons labeled with scn4ba probes are also indicated (arrowhead). ( E ) Expression of scn4ba in the MiP and RoP PMns. The morphology of GFP-labeled MiP and RoP in an injected fish shown (left). In situ hybridization images with scnba probes in this fish showed colocalization with GFP labeling (right). n = 7–10 cells. Scale bar 20 μm; white dashed line indicates the boundary of spinal cord; dorsal is up. ( F ) Validation of kcnc3a enrichment in PMns by immunohistochemistry staining. ( F1 ) KillerRed-mediated photoinactivation of CaP. Representative fluorescent images showing approximately two segments of a Tg(SAIG213A;EGFP) fish with a single CaP (arrow) expressing KillerRed, before photo illumination at 2 dpf (top), and ~40 hr after inactivation (bottom). Both the soma (the location indicated by an arrow) and periphery branches (see also F2 leftmost panel) are absent after the ablation. ( F2 ) Immunohistochemical staining of the same fish with a Kcnc3-specific antibody. GFP expression is revealed by anti-GFP antibody staining, and the location of synapses labeled by α-Btx. Top panels represent a maximal intensity projection of a stacked of z-plane images, while the bottom shows a single focal plane of the CaP target field (indicated by a white box). Scale bar 20 μm. n = 5 fish.

Article Snippet: We transiently expressed the phototoxic KillerRed protein in Tg(SAIG213A:EGFP) fish by injecting a plasmid expressing KillerRed driven by UAS promoter (Addgene plasmid #115516; a gift from Marco Morsch).

Techniques: Expressing, Comparison, In Situ Hybridization, Transgenic Assay, Labeling, Injection, Biomarker Discovery, Immunohistochemistry, Staining, Immunohistochemical staining