Fmrp developmentally regulates the expression of inhibitory synaptic proteins. Representative images from immunohistochemistry experiments to detect the vesicular gamma-aminobutyric acid (GABA) transporter [vesicular GABA transporter (vGAT); (A,B,E,F,I,J)] and the postsynaptic scaffold Gephyrin (A’,B’,E’,F’,I’,J’) on transverse trunk spinal cord sections of wild-type and fmr1 mutants at 24 hpf (A,B), 48 hpf (E,F), and 7 dpf (I,J). vGAT (cyan arrows) and Gephyrin expression (red arrows) is most concentrated in the lateral axon tracts. Apposition of vGAT and Gephyrin is shown in third panels (A”,B”,E”,F”,I”,J”), which represent vGAT puncta positioned <250 nm to Gephyrin puncta, and vice versa. Quantification of vGAT (C,G,K) and Gephyrin puncta (D,H,L) normalized to spinal cord area as well as the relative amount of vGAT or Gephyrin apposed to Gephyrin or vGAT, respectively. Cyan numbers in third panels represent the percentage of vGAT puncta apposed to Gephyrin to total vGAT, while red numbers represent the percentage of Gephyrin puncta apposed to vGAT to total Gephyrin. Forth panels (A”’,B”’,E”’,F”’,I”’,J”’) represent a zoom of synaptic protein apposition images (indicated by dashed boxes in third panels). The puncta size color code applies to the first and second panels, not apposition data. Dashed ovals in panel (A–B’) denote the edges of the spinal cord at 24 hpf. Scale bars = 10 μm (2 μm for apposition zoom panels). Aside from a Mann–Whitney test to compare 24 hpf Gephyrin apposition, all other statistical comparisons from unpaired t-tests. P-values indicated in graphs, where p < 0.05 is considered significant. See also Supplementary Figures 1, 2.
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