FMRP is expressed in migrating neurons of the murine postnatal RMS.

(A) Scheme of a sagittal section of the postnatal RMS connecting the V/SVZ to the OB. V/SVZ, ventricular/sub-ventricular zone; OB, olfactory bulb; RMS, rostral migratory stream. The inset shows the high density of homotypically migrating neurons in the RMS. (B) Representation of cyclic saltatory migration. 1. The neuron is in pause. 2. The leading process extends, and the centrosome moves within a swelling in the leading process. 3. The nucleus moves forward. CK, centrokinesis; NK, nucleokinesis. (C) Scheme of the experimental procedure. 2-day old neonates are intraventricularly electroporated with a GFP-expressing plasmid to label a cohort of migrating neurons that can be subsequently visualized in fixed or acute sections of the RMS. (D) Immunohistochemistry of the RMS showing FMRP expression (magenta) along the stream. The RMS is delineated with dotted lines. Scale bar: 50 μm. (E) Immunohistochemistry of a GFP-positive RMS neuron (cyan) showing FMRP subcellular expression (magenta). The GFP-positive neuron displays a cytoplasmic expression of FMRP around the nucleus (indicated by white arrows). The surrounding GFP-negative neurons express FMRP as well, following the same pattern. Scale bar: 10 μm. (F) Immunostaining for FMRP of a neuroblast migrating away from a V/SVZ explant in Matrigel. The labeling appears granular in the cytoplasm around the nucleus, in the swelling and the leading process. Scale bar: 5 μm.

Migration defects in Fmr1-null neurons.

(A) Migration speed of control (Ctrl) and Fmr1-null neurons. Ctrl: 70.62 (43.32) μm/h; Fmr1-null: 43.34 (25.97) μm/h (Kruskall-Wallis Test: Chi2 = 91.92, p-value < 0.001, df = 3; followed by Dunn’s posthoc test). (B) Percentage of pausing time of control and Fmr1-null neurons. Ctrl: 82 (21.50); Fmr1-null: 93 (9.25) (Kruskall-Wallis Test: Chi2 = 130.61, p-value < 0.001, df = 3; followed by Dunn’s posthoc test). (C) Sinuosity index of control and Fmr1-null neurons. Ctrl: 1.15 (0.26); Fmr1-null: 1.35 (0.66) (Kruskall-Wallis Test: Chi2 = 65.19, p-value < 0.001, df = 3; followed by Dunn’s posthoc test). (D) Migration directionality radar represented in four spatial dials. Percentage of cells migrating in each spatial direction in control and Fmr1-null neurons, relatively to the vector going straight from the SVZ to the OB. (Fisher’s Exact test, p-value < 0.001). (E) NK mean distance of control and Fmr1-null neurons. Ctrl: 11.46 (6.27) μm; Fmr1-null: 9.34 (4.16) μm (Kruskall-Wallis Test: Chi2 = 53.45, p-value < 0.001, df = 3; followed by Dunn’s posthoc test). (F) NK frequency of control and Fmr1-null neurons. Ctrl: 2.5 (2.23) NK/h; Fmr1-null: 1.21 (1.45) NK/h (Kruskall-Wallis Test: Chi2 = 111.53, p-value < 0.001, df = 3; followed by Dunn’s posthoc test). The black line represents the median. Ctrl: N = 3, n = 275; Fmr1-null: N = 3, n = 184. Median (IQR).***p-value < 0.001.

CK defects in Fmr1-null neurons.

(A) CK speed of control and Fmr1-null neurons. Ctrl: 76.90 (46.46) μm/h; Fmr1-null: 49.45 (33.66) μm/h (Mann-Whitney test, p-value < 0.001). (B) CK frequency of control and Fmr1-null neurons. Ctrl: 3.33 (1.71) CK/h; Fmr1-null: 2.33 (1.71) CK/h (Mann-Whitney test, p-value < 0.001). (C) Percentage of efficient CKs in control and Fmr1-null neurons. Ctrl: 54 %; Fmr1-null: 33 % (Chi2 = 57.611, p-value < 0.001). The black line represents the median. Ctrl: N = 3, n = 178; Fmr1-null: N = 3, n = 216. Median (IQR). *** p-value < 0.001.

MAP1B expression in the RMS.

(A) Immunohistochemistry of the RMS showing MAP1B expression (magenta) along the stream. The RMS is delineated with dotted lines. Scale bar: 50 μm. (B) Immunostaining for MAP1B of a neuroblast migrating away from a V/SVZ explant in Matrigel. The labeling is located around the nucleus and in the leading process with occasional bundles of potential microtubules (arrow). Scale bar: 5 μm.

Map1b KD rescues Fmr1-null neurons migration defects.

(A) Migration speed of Fmr1-null neurons expressing MiRNEG and MiRMap1b and control neurons expressing MiRNEG. Fmr1-null neurons + MiRNEG: 45.23 (23.25) μm/h; Fmr1-null neurons + MiRMap1b: 65.72 (24.31) μm/h; control neurons + MiRNEG: 64.54 (21.99) μm/h (Kruskall-Wallis Test: Chi2 = 61.168, p-value < 0.001, df = 2; followed by Dunn’s posthoc test). (B) Percentage of pausing time of Fmr1-null neurons expressing MiRNEG and MiRMap1b and control neurons expressing MiRNEG. Fmr1-null neurons + MiRNEG: 93 (7); Fmr1-null neurons + MiRMap1b: 86 (14); control neurons + MiRNEG: 87.50 (15.67) (Kruskall-Wallis Test: Chi2 = 45.716, p-value < 0.001, df = 2; followed by Dunn’s posthoc test). (C) Sinuosity index of Fmr1-null neurons expressing MiRNEG and MiRMap1b and control neurons expressing MiRNEG. Fmr1-null neurons + MiRNEG: 1.30 (0.45); Fmr1-null neurons + MiRMap1b: 1.28 (0.55); control neurons + MiRNEG: 1.13 (0.16) (Kruskall-Wallis Test: Chi2 = 39.807, p-value < 0.001, df = 2; followed by Dunn’s posthoc test). (D) NK mean distance of Fmr1-null neurons expressing MiRNEG and MiRMap1b and control neurons expressing MiRNEG. Fmr1-null neurons + MiRNEG: 8.93 (3.64) μm; Fmr1-null neurons + MiRMap1b: 9.89 (3.85) μm; control neurons + MiRNEG: 10.23 (3.9) μm (Kruskall-Wallis Test: Chi2 = 11.573, p-value = 0.003, df = 2; followed by Dunn’s posthoc test). (E) NK frequency of Fmr1-null neurons expressing MiRNEG and MiRMap1b and control neurons expressing MiRNEG. Fmr1-null neurons + MiRNEG: 1.18 (1.11)NK/h; Fmr1-null neurons + MiRMap1b:: 2.22(1.95)NK/h; control neurons + MiRNEG1.65(2) NK/h (Kruskall-Wallis Test: Chi2 = 39.272, p-value < 0.001, df = 2; followed by Dunn’s posthoc test). The black line represents the median. Fmr1-null neurons + MiRNEG: N = 6, n = 102; Fmr1-null neurons + MiRMap1b: N = 3, n = 101; control neurons + MiRNEG: N = 3, n = 78. Median (IQR). * p-value < 0.05; *** p-value < 0.001; n.s.

The FMRP/MAP1B duo acts on the microtubular nuclear cage of RMS neurons.

(A) Representative control neurons from MirNeg-GFP (magenta) plus Dcx-RFP (cyan) cp-electroporated V/SVZ explants cultured in Matrigel displaying microtubular bundles enveloping the nucleus. (B) Representative Fmr1 KD neurons from MirFmr1-GFP (magenta) plus Dcx-RFP (cyan) co-electroporated V/SVZ explants cultured in Matrigel. The majority of them display an abnormal cage with disruption (arrows) (46%), sinuous cages (33% or even detached bundle (arrow) (28%). (C) Representative rescued neurons from MirNeg-GFP (magenta) plus Dcx-RFP (cyan) plus MirMAP1B co-electroporated V/SVZ explants cultured in Matrigel displaying microtubular bundles enveloping the nucleus. Scale bars: 5 μm. (D) Quantification of the percentage of disorganized cages in the different conditions: MirNeg 19%, miRFmr1 60%, mirFmr1 + mirMAP1B 28% (Pearson’s χ2 test (2, N=107)= 14.16, p-value < 0.001 with the Benjamini-Hochberg method for correcting multiple testing. MirNeg, N=3, n=37; MirFmr1 N=3, n=35; MirFmr1+MirMAP1B, n=36).