Fig. 4: Q86Pfs inhibits neuronal migration, and promotes self-renewing RGC fate.

The in vivo activity of Q86Pfs was monitored in mouse brains electroporated with GFP or Q86Pfs-ires-GFP constructs. a, d Schematics of in utero electroporation at E15.5 and brain analysis at E18.5 (a–c) or E17.5 (d–h). GFP labels the electroporated cells expressing Q86Pfs (Q86Pfs-ires-GFP) or GFP. a–c Analysis of GFP⁺ neuronal migration. The proportion of GFP⁺ cells was increased in the IZ and decreased in the CP in Q86Pfs-expressing brains compared to GFP-alone-expressing brains, indicating that Q86Pfs suppresses neuronal migration. BCL11B marks deep-layer neurons and demarcates the CP. The immunostaining with Flag antibody shows the subcellular localization pattern of Flag-tagged Q86Pfs protein. The magnified view of the Flag staining depicts the speckle-like distribution of Q84Pfs. Red-arrow, GFP⁺ RGC cell bodies at the ventricular surface; blue-arrow, GFP⁺ RGC’s glial processes; green-arrow, GFP⁺ RGC’s basal endfeet at the pial-surface; red-line, ventricular surface. VZ, ventricular zone; IZ, intermediate zone; SP, subplate; CP, cortical plate. c The percentage of GFP⁺ cells in each area. n = 5 mice/condition. The error bars represent SEM. VZ: p = 0.5377, IZ: **p = 0.0027, SP: p = 0.0650, CP: **p = 0.0045 in two-tailed Mann-Whitney test. e, f Analysis of RGC fate using the RGC marker NESTIN. g, h Analysis of proliferating cells using BrdU labeling. f, h Quantification of the number of GFP⁺NESTIN⁺ RGC scaffold processes (f) or GFP⁺BrdU⁺ proliferating cells (h) in 100μm-wide vertical areas encompassing VZ to CP of the cortex. The number of GFP⁺ RGC’s glial scaffold (f) and BrdU⁺ proliferating cells (h) was increased in Q86Pfs-expressing brains relative to GFP-expressing brains. n = 5 mice/condition, ****p < 0.0001 (f) or n = 8 mice for GFP alone and n = 13 mice for Q86Pfs-ires-GFP, **p = 0.0028 (h). Scale bars, 100 μm (low magnification images in b), 10 μm (magnified view of Flag immunostaining, b), 50 μm (low magnification images in e, g), 20 μm (magnified images in e, g). Yellow-line, ventricular surface (g). In the box-plots (f, h) the whiskers show minimum and maximum values, the box represents the 25th and 75th percentile of the data, and the center line indicates the median. Only the representative images are shown. i, Model for Q84Pfs actions to block neuronal migration and promote RGC fate.