Innervation of Wt1+ neurons and number of commissural neurons in neonatal mice. Innervation of Wt1+ neurons and number of commissural neurons in neonatal.

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Innervation of Wt1+ neurons and number of commissural neurons in neonatal mice. Innervation of Wt1+ neurons and number of commissural neurons in neonatal mice. (A) Spinal cords of Wt1-GFP embryos (stage E16.5) show a Wt1+ interneuron immunopositive for GFP. Wt1 is localized in the nucleus and GFP throughout the cell. Scale bar: 2 μm. (B) Wt1+ neurons (green) receive excitatory, inhibitory, and monoaminergic synaptic contacts. Synaptic terminals are identified with synaptophysin (blue). Glutamatergic terminals were immunolabelled for VGLUT2, inhibitory synapses immunolabelled for VGAT, and monoaminergic terminals immunolabelled for VMAT2. Arrows point to individual synaptic terminals (magenta) present on Wt1+ neurons (green). Boxed areas show higher magnification panels of separated channels. Scale bar: 2 μm. (C) GFP-immunolabelled dI6 neurons in the spinal cord of E16.5 Wt1-GFP embryos. GFP antibody staining (green; left panel) and merging with Hoechst (blue; right panel) is shown. Boxed areas represent location of higher magnification panels shown on the right of each panel. Contralateral projections crossing the midline (dashed line) of the spinal cord are visible (arrow heads in magnified images). Scale bar: 20 μm for overview images and 50 μm for magnified images. (D) Photomicrographs of transverse, 60 μm, lumbar, spinal cord sections with applied fluorescein dextran amine (FDA, green) and rhodamine dextran amine (RDA, red) tracers. Higher magnification images (insets) of wild-type (Nes-Cre;Wt1+/+) and homozygous (Nes-Cre;Wt1fl/fl) segments, showing intersegmental retrograde FDA (white arrow), RDA (open arrow), and double-labelled (triangle arrow) neurons. Scale bar: 200 μm. (E) Schematic illustration of FDA (lumbar [L]1) and RDA (L4/5) application sites tracing descending (green), ascending (red), and bifurcating (yellow) neurons. The area of analysis (L2/L3) is indicated by black dashed line. (F–H) Quantification of descending FDA-labelled neurons (F), ascending RDA-labelled neurons (G), and bifurcating, double-labelled neurons per section (H). Descending, ascending, and bifurcating CINs are significantly fewer in homozygous spinal cords compared with control cords (according to Kruskal–Wallis test and followed by a Dunn’s post hoc test comparing all groups). Data expressed as mean ± SEM (Wt1fl/fl control: 3,975 total cells, 215 sections, and nine spinal cords; Nes-Cre;Wt1fl/fl: 3,421 total cells, 228 sections, and seven spinal cords). Significance level: *P < 0.05, **P < 0.01, ***P < 0.001. Danny Schnerwitzki et al. LSA 2018;1:e201800106 © 2018 Schnerwitzki et al.