Neuroscience

FMRP regulates neuronal migration via MAP1B

S. Messaoudi
J. Stoufflet
C. Fouquet
M. Doulazmi
A. Allam
T. Paillard
A. Trembleau
I. Caillé author has email address

Institut Biologie Paris Seine, Sorbonne University
ULiege
Institut Biologie Paris Seine, Universite Paris Cite

https://doi.org/10.7554/eLife.88782.1

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Figures and data

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 neuroblasts’ cyclic saltatory migration. 1. The neuron is in pause. 2. The leading process (LP) extends, and the centrosome moves within a swelling in the LP. 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, in which a cohort of GFP-positive neurons (cyan) are visible. Scale bar: 100 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), along the leading process and in the growth cone. The surrounding GFP-negative neurons express FMRP as well, according to the same pattern. Scale bar: 10 µm.

Migration defects in Fmr1-null neurons.
(A) Migration speed of control (Ctrl) and Fmr1-null neurons. Ctrl: 76 ± 2 µm/h; Fmr1-null: 47 ± 1 µm/h (Mann-Whitney test, p-value < 0.001). (B) Percentage of pausing time of control and Fmr1-null neurons. Ctrl: 78 %; Fmr1-null: 91 % (Mann-Whitney test, p-value < 0.001). (C) Sinuosity index of control and Fmr1-null neurons. Ctrl: 1.32 ± 0.04; Fmr1-null: 1.93 ± 0.16 (Mann-Whitney test, p-value < 0.001). (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: 12.5 ± 0.3 µm; Fmr1-null: 10.9 ± 0.8 µm (Mann-Whitney test, p-value < 0.001). (F) NK frequency of control and Fmr1-null neurons. Ctrl: 2.9 ± 0.1 NK/h; Fmr1-null: 1.5 ± 0.1 NK/h (Mann-Whitney test, p-value < 0.001). The black line represents the median. Ctrl: N = 3, n = 275; Fmr1-null: N = 3, n = 184. ***p-value < 0.001.

CK defects in Fmr1-null neurons.
(A) Illustration of an RMS-migrating neuron (cyan) co-injected with centrin-RFP (magenta). Scale bar: 5 µm. (B) CK speed of control and Fmr1-null neurons. Ctrl: 82 ± 3 µm/h; Fmr1-null: 54 ± 2 µm/h (Mann-Whitney test, p-value < 0.001). (C) CK frequency of control and Fmr1-null neurons. Ctrl: 3.5 ± 0.1 CK/h; Fmr1-null: 2.6 ± 0.1 CK/h (Mann-Whitney test, p-value < 0.001). (D) 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. *** p-value < 0.001.

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: 47 ± 2 µm/h; Fmr1-null neurons + MiRMap1b: 67 ± 2 µm/h; control neurons + MiRNEG: 64 ± 2 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: 92 %; Fmr1-null neurons + MiRMap1b: 84 %; control neurons + MiRNEG: 84 % (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: 1.77 ± 0.18; Fmr1-null neurons + MiRMap1b: 1.90 ± 0.20; control neurons + MiRNEG: 1.18 ± 0.02 (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: 9.7 ± 0.3 µm; Fmr1-null neurons + MiRMap1b: 11.1 ± 0.4 µm; control neurons + MiRNEG: 10.6 ± 0.3 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: 1.5 ± 0.1 NK/h; Fmr1-null neurons + MiRMap1b: 2.4 ± 0.1 NK/h; control neurons + MiRNEG: 2.5 ± 0.2 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. * p-value < 0.05; *** p-value < 0.001; n.s. (not significant), p-value > 0.05.

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: 47 ± 2 µm/h; Fmr1-null neurons + MiRMap1b: 67 ± 2 µm/h; control neurons + MiRNEG: 64 ± 2 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: 92 %; Fmr1-null neurons + MiRMap1b: 84 %; control neurons + MiRNEG: 84 % (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: 1.77 ± 0.18; Fmr1-null neurons + MiRMap1b: 1.90 ± 0.20; control neurons + MiRNEG: 1.18 ± 0.02 (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: 9.7 ± 0.3 µm; Fmr1-null neurons + MiRMap1b: 11.1 ± 0.4 µm; control neurons + MiRNEG: 10.6 ± 0.3 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: 1.5 ± 0.1 NK/h; Fmr1-null neurons + MiRMap1b: 2.4 ± 0.1 NK/h; control neurons + MiRNEG: 2.5 ± 0.2 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. * p-value < 0.05; *** p-value < 0.001; n.s. (not significant), p-value > 0.05.

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