Panel A shows representative fluorescence microscopy images of spinal cords from wild type, C3 deficient, and CD47 deficient mice stained for ChAT, CD47, and VGlut1. Panel B shows a fluorescence microscopy image of a somatic synapse and a line intensity profile graph demonstrating overlap of VGlut1 and CD47 signals. Panel C shows a fluorescence microscopy image of a dendritic synapse with a corresponding line intensity profile graph. Panel D shows bar graphs of the percentage of synapses on motor neuron somata tagged by CD47 at postnatal days 1, 5, and 10. Panel E shows bar graphs of CD47 tagged synapses on motor neuron dendrites at the same developmental ages. Panel F shows fluorescence microscopy images of C3 deficient spinal cord stained for ChAT, Iba1, CD47 mRNA, GFAP, and DAPI, with enlarged views of motor neuron somata and surrounding glial structures. Panel G shows a bar graph of CD47 puncta per area at postnatal days 5 and 10. Panel H shows a bar graph of the percentage of synapses on motor neuron somata tagged by C3, comparing wild type, CD47 deficient, and C3 deficient mice.
CD47 tags proprioceptive synapses in spinal sensory-motor circuits. (A) Single-plane confocal images of VGluT1 (red), CD47 (green), and ChAT (blue) immunoreactivity in WT, C3−/−, and CD47−/− mice at P5. Scale bar: 50 μm. (B) VGluT1+ synapse (red) opposed to a motor neuron soma (ChAT, blue) is tagged by CD47 (green). Graphs show that the CD47 signal intensity overlaps with that of VGluT1, indicated by the line profile (arrow, dotted line), for the three antibodies. Scale bar: 5 μm. (C) Similar to B but for a synapse on a motor neuron dendrite. Scale bar: 5 μm. (D) Percentage of somatic synapses tagged by CD47 at P1, P5, and P10. (E) Percentage of dendritic synapses tagged by CD47. Each dot (in grey) corresponds to one motor neuron. Colored data points show average values from a single mouse (N = 3 mice/group). Number of motor neurons analyzed from N = 3 mice (WT: P1, n = 30; P5, n = 24; P10, n = 30) (C3−/−: P1, n = 30; P5, n = 30; P10, n = 30) (CD47−/−: P1, n = 30; P5, n = 20; P10, n = 30). (F) Confocal images showing CD47-mRNA (RNAscope, in green) and immunoreactivity signals with ChAT (blue), Iba1 (red, top row), GFAP (red, bottom row) and DAPI (white) in C3−/− mice at P5. Dotted boxes indicate areas of higher magnification shown on the right. Scale bar: 20 μm. (G) Quantification of CD47 puncta per soma in WT (P5, N = 4 mice; P10, N = 5) and C3−/− (P5, N = 5; P10, N = 5). (H) Percentage of somatic synapses tagged by C3 at P5. Number of motor neurons analyzed from N = 3 mice per genotype (WT: n = 15, CD47−/−: n = 15, C3−/−: n = 15). Statistical comparison was performed across averages from mice. Significance: ns = no significance, *P < 0.01, ***P < 0.001; one-way ANOVA with multiple comparisons using Bonferroni’s test; ns: no significance.