Extended Data Fig. 3: In vivo characterization of PSD-95-GFP and GFP-gephyrin.
(a) Puncta diameter of PSD-95-GFP (green) and GFP-Gephyrin (magenta) in spinal cord neurons (N) and OPCs from 2-3 dpf larvae. From left, n = 10, 11, 15, and 29 cells from at least 8 fish per condition. (b-c) Representative in vivo images of (b) PSD-95-GFP or (c) GFP-Gephyrin and synaptophysin-RFP (syn.) in spinal cord OPCs at 3 dpf. From left, single plane images and an example of puncta in OPCs at single planes with 3D reconstructed regions, the merged channels, the PSD-95-GFP or GFP-Gephyrin channels, and the syn. channel. In the examples, the alignments are indicated with arrowheads. (d) The % of PSD-95-GFP and GFP-gephyrin puncta in an OPC that align with presynaptic synaptophysin-RFP in the spinal cord in (b-c). n = 13 and 20 cells from at least 10 fish. (e-f) The correlation analysis of puncta number of (e) PSD-95-GFP and (f) GFP-Gephyrin with the corresponding protein overexpression levels at 3 dpf. The linear fit extrapolated to zero overexpression level is at puncta number of 28.6 and 22.9, respectively. (g) Puncta number of PSD-95-GFP (green) and GFP-Gephyrin (magenta) per OPC in the spinal cord at 2 dpf and 3 dpf. From left, n = 16, 18, 29, and 14 cells from at least 5 fish per condition. (h) Representative IHC images of PSD-95-GFP in spinal cord OPCs at 3 dpf with GFP (green) and MAGUK (magenta) antibodies. Left, merged channels; middle and right, single channels. Arrowheads indicate the GFP puncta that colocalize with MAGUK signals; cyan arrows indicate the diffused GFP that does not colocalize with MAGUK signals. (i) % of GFP signal that colocalizes with MAGUK signals from (h) using Imaris. n = 4 and 6 fish per condition. All data are represented as mean ± SEM; N.S., not significant (exact p values are provided in Source Data); (a,g) two-sided Mann-Whitney test; (e,f) two-sided Pearson correlation analysis with linear regression; scale bars 5 μm.
