Additional file 1 of Loss of the neurodevelopmental disease-associated gene miR-146a impairs neural progenitor differentiation and causes learning and memory deficits
Creators: name: Fregeac, Julien
name: Moriceau, Stéphanie
name: Poli, Antoine
name: Nguyen, Lam Son
name: Oury, Franck
name: Colleaux, Laurence
Publisher: name: figshare
Year: 2020
Subjects: subject: Genetics
subject: FOS: Biological sciences
subject: FOS: Biological sciences

description: Additional file 1: Figure S1. The number of cortical neurons is unaltered in the adult Mir146a-/- brain. (a) Cortical slides of WT (top panel) and Mir146a-/- mouse (bottom panel) stained for NeuN to label post-mitotic neurons at P60. Number of NeuN+ cells normalized to area counted (±S.D.) in layer 2 (b), layers 3-4 (c), layer 6 (d) and all layers (e) in the WT and Mir146a-/- cortical slices. At least 2 images at comparable plane per mouse were analyzed. The number of NeuN+ cells at P30 and P60 are indistinguishable, thus, we combined the results for each genotype. Figure S2. Neurogenesis in the mouse neocortex. Neuroepithelial cells (NE) proliferate up to E11 before differentiating into apical radial glia (aRG). aRG are located in the ventricular zone (VZ) where their nucleus undergo interkinetic nuclear migration (INM) and divide at the apical surface. aRG either self-replicate or give rise to a neuron or an intermediate progenitor (IP) after an asymmetric division. IP remain in the subventricular zone (SVZ) and can complete one or two cell cycles before differentiating into neurons. Neurons coming from aRG or IP migrate towards the intermediate zone (IZ) and the cortical plate (CP) where they acquire their final position and identity and start expressing post-mitotic markers such as NeuN. Figure S3. Number of PH3+ cells at apical surface of the E14.5 neocortex is unaltered in the Mir146a-/-. Graph showing the number of PH3+ cells (±S.D.) in images shown in Fig. 1e. NS, not significant by Student’s 2-tailed T-Test. Figure S4. Loss of miR-146a does not affect cortical layers organization. (a) BrdU signal at P7 labeled neurons born at E14.5. The white box shows the analyzed area. (b) Zoom of the white box from (a). The 6 layers of the mature cortex were determined and BrdU+ cells were counted using 3 images from comparable plane. (c) Graph shows the percentage of cells in each layer for the two genotypes, normalized against the total number of labeled cells. Figure S5. Increased hippocampus asymmetry in the Mir146a-/- mice at P60. Volume (±S.D.) of the cortex (a) and the hippocampus (b) of the mutant and WT mice as measured by brain MRI at P60. (c) Scatterplot comparing the ratio between volumes of two hippocampal hemispheres in mutant and WT mice. NS: not significant Student’s unpaired 2-tailed T-Test. On average, one hippocampus side is 15% larger than the other in mutant brains with random distribution (2 right&gt;left and 2 left&gt;right). Figure S6. Locomotion and anxious state of the Mir146a-/- mice are not affected. (a) Open field test (OFT) performed in 3-month old Mir146a-/- mice (n=16) and their control littermates (n=15). Total distance (meter), % of the distance traveled in the center versus periphery, and % of the time spent in the center versus periphery were measured. (b) Light/Dark transition Test (L/DT) performed in 3-month old Mir146a-/- mice (n=16) and their control littermates (n=15). The number of entries in the lit compartment, and % of the distance traveled and time spent in the lit compartment were measured. All behavioral tests were performed on at least two independent experiments, for each experiment: n≥6 mice per group. Results are given as mean ± s.e.m. *P ≤ 0.05, **P≤ 0.01, ***P ≤ 0.001, NS: not significant; by Student’s t-test compared to 3-month-old mice littermates. Figure S7. The Mir146a-/- brain shows no sign of inflammation. Expression level (±S.D.) of inflammatory markers IL1-β (a and b) and TNF- α (c and d) in brain lysates of 8 WT and 8 mutant mice that were used for behavior studies. Technical quadruplicates were performed.