FMRP regulates multipolar to bipolar transition affecting neuronal migration and cortical circuitry

Abstract

Deficiencies in fragile X mental retardation protein (FMRP) are the most common cause of inherited intellectual disability, fragile X syndrome (FXS), with symptoms manifesting during infancy and early childhood. Using a mouse model for FXS, we found that Fmrp regulates the positioning of neurons in the cortical plate during embryonic development, affecting their multipolar-to-bipolar transition (MBT). We identified N-cadherin, which is crucial for MBT, as an Fmrp-regulated target in embryonic brain. Furthermore, spontaneous network activity and high-resolution brain imaging revealed defects in the establishment of neuronal networks at very early developmental stages, further confirmed by an unbalanced excitatory and inhibitory network. Finally, reintroduction of Fmrp or N-cadherin in the embryo normalized early postnatal neuron activity. Our findings highlight the critical role of Fmrp in the developing cerebral cortex and might explain some of the clinical features observed in patients with FXS, such as alterations in synaptic communication and neuronal network connectivity.

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Figure 1: Fmrp is involved in neuronal cortical positioning.
Figure 2: Fmr1−/− mice do not show differences in progenitor proliferation nor radial migration of excitatory neurons.
Figure 3: Fmr1−/− neurons have a delayed exit from the multipolar stage.
Figure 4: Fmrp regulates neuronal cortical positioning by stabilizing Cdh2 mRNA.
Figure 5: Fmrp regulates N-cadherin during multipolar-to-bipolar transition.
Figure 6: Postnatal brain networks are affected in the developing Fmr1−/− mouse.
Figure 7: Excitatory and inhibitory networks are affected in the developing Fmr1−/− mouse.
Figure 8: DTI studies show neuronal connectivity impairments in juvenile Fmr1−/− mice.

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Acknowledgements

We are very grateful to E. Lemmens for administrative support, K. Jonckers, J. Royaert and I. Beheydt for their help and assistance with primary neurons and mouse colonies. We are thankful to T. Voets and S. Munck (coordinator of LiMoNe) for providing us excellent suggestions on the calcium imaging, P. Fazzari for helping with the synapses quantification, and M. Regoli for suggestions on the statistical analysis. G.L.F., N.D.-I. and M.A. were supported by grants FWO-G.0705.11 and FWO-G.0667.09 to C.B. T.A. was supported by a VIB grant to C.B. A.G. was partially supported by a VIB grant to C.B. and C.G.D. as well by an FWO grant (ZKC6058-00-W01) to A.G. U.H. and T.D. received financial support from the KUL-program financing IMIR (PF10/017). T.D. was supported by FWO-ZKC5858. R.M.M. J.D. and R.B.P. were supported by grants from FRAXA and Dutch Medical Research Council NWO VIDI (#917.10.372). G.L.F. is part of the Brain Train network (http://www.brain-train.nl/braintrain-program/partners/). This work was supported by grants from VIB, SAO, FWO-G.0705.11 and FWO-G.0667.09, and Associazione Italiana Sindrome X Fragile to C.B., and FWO-G0.666.10N, NEUROBRAINNET IAP 7/16, Flemish Methusalem grant, and Innovation Ingenio-Consolider, CSD2010-00045 to C.G.D.

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G.L.F., A.G., C.G.D., T.A. and C.B. designed the experiments and wrote the manuscript. A.G. and C.B. supervised the project. G.L.F. and A.G. performed a major part of the experiments and analyzed the data. N.D.-I. contributed substantially to the experiments and data analysis during the entire review process. M.A. contributed to some experiments. T.D. and U.H. performed and analyzed the MRI scans. R.M.M., J.D. and R.B.P. performed and analyzed the initial calcium imaging experiments and A.G., N.D.-I. and T.A. performed and analyzed the electroporation with calcium imaging experiments.

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Correspondence to Claudia Bagni.

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Integrated supplementary information

Supplementary Figure 1 Expression of Fmrp in early cortical development.

a) Representative western blot showing Fmrp expression during two developmental stages (E14 and E17) in WT and Fmr1−/− embryonic somatosensory cortex. Ribosomal protein S6 (rpS6) was used as loading control b) Confocal images showing the distribution of WT and Fmr1−/− neuronal cells, upon IUE with the pCAG-EGFP plasmid at E14.5, in mouse cortices at P0. EGFP fluorescence (green) and DAPI staining (blue). Right histogram shows the frequency distribution and quantification of EGFP positive cells in ten equal bins (VZ 1 to CP 10) in WT (n = 6), Fmr1−/− (n = 5), Two-way ANOVA; F (10, 165) = 51.97, Values: mean ± SEM. Scale bars = 50 μm.

Supplementary Figure 2 Absence of Fmrp does not affect proliferation of precursor cells at E14.5.

a) Left, confocal images from WT and Fmr1−/− E14.5 mouse cortices stained with markers for the intermediate (Tbr2-positive) and radial (Sox2-positive) progenitor populations (anti-Tbr2, green; anti-Sox2, red). Right, the graph represents the ratio of Tbr2-positive to Sox2-positive in WT and Fmr1−/− cortices. Scale bars = 20 μm. (Values are mean ± SEM; two tailed unpaired t-test: P = 0.1503, t=1.776, df = 4, n = 3 WT animals and n = 3 Fmr1−/− animals). b) Histogram represents the percentage of cells transfected at E14.5 with pCAG-EGFP that are Ki67-positive in the VZ/SVZ/IZ of WT and Fmr1−/− cortices. (Values are mean ± SEM; two tailed unpaired t-test: P = 0.423, t = 0.86, df = 6, n = 3 WT animals and n = 3 Fmr1−/− animals).

Supplementary Figure 3 Morphology and positioning of radial glia cells are not affected in Fmr1−/− cortices.

a) Individual radial glia cells in E15.5 WT and Fmr1−/− embryos were labeled, by IUE, using a BLBP-EGFP (Brain Lipid Binding Protein promoter specific for radial glia cells) construct. Brain sections were analysed at E16.5. Fmr1−/− radial glia cells display normal morphology spanning the entire cortex (green arrow-heads) and have apical (yellow arrow-heads) and basal (red arrow-heads) endfeets as observed in WT. b) Coronal section from E17.5 WT and Fmr1−/− littermates labeled with anti β-catenin (upper panel) and γ-tubulin antibodies (lower panels). Scale bars = 50 μm.

Supplementary Figure 4 Migration in vitro of the Fmr1−/− neurons is not affected.

WT and Fmr1−/− cells were isolated from E14.5 embryonic cortices, left to form aggregates and embedded in Matrigel 18 h later. Sequential images show four time points of WT and Fmr1−/− neurons migrating away from the in vitro explants. Both average velocity and directionality (total path length from time 0 to time x/ net distance time 0 to time x) were not different between WT and Fmr1−/−­­ neurons: Average velocity: WT = 0.1537 μm/min ± 0.0226, n = 6 (71 neurons); Fmr1−/− = 0.1609 μm/min ± 0.01687, n = 5 (59 neurons); unpaired two tailed t-test: P = 0.8122, t = 0.2447, df = 9, n = number of explants. Directionality: WT 0.6629 ± 0.03070, arbitrary units; Fmr1−/− 0.6642 ± 0.06615 arbitrary units, P = 0.9857, t=0.01844, df=9. Scale bar = 10 μm

Supplementary Figure 5 Absence of Fmrp does not alter cortical layering.

Left, immunohistochemistry showing E17.5 confocal images of coronal sections from WT and Fmr1−/− cortices labeled with anti-COUP-TF-interacting protein 2 antibody (Ctip2, transcription factor) for lower layer neurons and anti-Cut homeobox 1 gene antibody (Cux1, transcription factor) for differentiated neurons in the upper layer. Scale bars = 50 μm. Right, quantification of the average line profiles throughout the cortex (n = 3). Normalization was performed taking into account the slice thickness. Displayed is the average plot profile.

Supplementary Figure 6 Activity-driven calcium events were correlated to action potential firing of individual neurons.

a) Cell numbers within networks in WT and postnatal (P0-P7) Fmr1−/− brains (values are mean ± SEM; ANOVA: age 0 (P < 0,0001), genotype (p = 0.64), genotype*age interaction (p = 0.504) F(3,88)=8,618, n = 21 pus and 53 slices from WT and n = 21 pups and 44 slices from Fmr1−/− animals) b) Individual events from sample traces during aCSF baseline and following TTX application. Grey bar indicates events in aCSF condition. c) Cell-attached patch recording showing correlated synchronization of events to AP spiking in cell and neighbor. d) Raster plot for corresponding slice showing network activity across all cells for baseline and TTX perfusion in 4 min recording blocks. e) TTX blockage effect on cell activity across the network.

Supplementary Figure 7 Representative Western blotting.

a) Representative Western blotting (entire membrane) showing N-cadherin expression in E14.5 embryonic somatosensory cortex lysates from WT and Fmr1−/− mice. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) has been used as the internal control for normalization. This entire gel refers to the experiment described in Figure 4a. b) Representative Western blotting (entire membrane) showing Fmrp immunoprecipitation performed from E17 WT and Fmr1−/− cortical extracts. This entire gel refers to the experiment described in Figure 4b.

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La Fata, G., Gärtner, A., Domínguez-Iturza, N. et al. FMRP regulates multipolar to bipolar transition affecting neuronal migration and cortical circuitry. Nat Neurosci 17, 1693–1700 (2014). https://doi.org/10.1038/nn.3870

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