Figure 4. FMRP Controls RNA Transport to Radial Glia Basal Endfeet.
(A) Cartoon representation shows an EGFP+ RGC, with a red box highlighting the regions shown in (B)–(G).
(B–G) Kif26a smFISH in EGFP+ RGCs of control (B, C, and F) and Fmr1−/− brain sections with (F) and (G) showing close-up views of regions highlighted in (C) and (E), respectively. Dotted line, basement membrane. CP, cortical plate.
(H) The smFISH signal intensity analyses of Apc and Kif26a show the decrease in pial Kif26a, but not Apc, smFISH signal in Fmr1−/− (red) versus WT (black) E15.5 neocortices (n = 3).
(I) qPCR analyses of Apc and Kif26a transcripts in WT (black) and Fmr1−/− (red) E15.5 neocortices (n = 4).
(J) Approach used for imaging mRNA, in which CFP is followed by a Kif26a-3′ UTR with Ms2-binding sites for the MCP-EGFP protein, as shown.
(K and L) Boxplots show velocities (K) and run lengths (L) quantified for the indicated genotypes. WT, n = 171 RNA movements from n = 10 embryos; Fmr1−/−, n = 119 movements from n = 6 embryos.
(M–R) Examples of live imaging of mRNAs in RGCs of brain slices from WT (M–O) and Fmr1−/− (P–R), E15.5 cortices. Shown are the schematic representations of the regions imaged (N and Q), outlines and still images acquired prior to live imaging (N and Q), and kymographs of Ms2-Kif26a-3′ UTR (O and R) RNAs moving apically (red arrowhead) or basally (blue arrowhead).
(S) Cartoon model represents the study findings. RNA is transported bi-directionally within RGC basal processes and translated in RGC endfeet.
Scale bars, 20 (B–E) and 5 µm (F and G). (N, O, Q, and R) y axis, length, 5 µm; x axis, time, 5 s. Boxplots represent median; bottoms and tops of boxes represent 25th and 75th percentiles, and whiskers represent median ± 1.5 × interquartile range. ***p < 0.001; ns, non-significant. Error bars, SD. See also Figure S4.