Journal: The Journal of Neuroscience

Article Title: Cenpj Regulates Cilia Disassembly and Neurogenesis in the Developing Mouse Cortex

doi: 10.1523/JNEUROSCI.1849-18.2018

Figure Lengend Snippet: Cenpj was specifically depleted in the dorsal telencephalon of CenpjCKO mice. A, Schematic of Cenpj-trapped mice construction strategy. A LacZ reporter cassette and a neo tag, flanked by two FRT sequences, were introduced between the fourth exon and the fifth exon of Cenpj gene locus. The fifth exons were flanked by two LoxP sites in CenpjLacZ mice. LacZ reporter cassettes can be removed by Flippase splicing and the Cenpj gene can be knocked out by tissue-specific Cre recombinase splicing. B, Western blot analysis for Cenpj +/+ and CenpjCKO cerebral cortex lysate at E15.5. C, Quantification of Cenpj knock-out by Western blot. Histogram shows the mean ± SD; ****p < 0.0001 as determined by a t test; n = 3. D, Brain sections were immunostained by anti-Pericentrin (green) antibody and anti-Cenpj (red) antibody on the ventricular surface in Cenpj+/+ and CenpjCKO mice. Scale bar, 10 μm. E, Representative whole-mount images of Cenpj+/+ and CenpjCKO brains at E15.5. Scale bar, 0.5 mm. F, Quantification of cerebral hemisphere circumference at E15.5. Data are presented as the mean ± SD; ***p = 0.0004 as determined by a t test; n = 3. G, Representative images of Cenpj+/+ and CenpjCKO brain coronal sections at E15.5 by Nissl staining. Right, Magnified cortical column. Scale bars, 500 μm (left) and 100 μm (right). H, Histogram of the cerebral cortex thickness. Data are presented as the mean ± SD; ****p < 0.0001 as determined by a t test; n = 3; 3 brain slices per experiment. I, Body size of Cenpj+/+ and CenpjCKO mice at P7. J, Representative images of the Cenpj+/+ and CenpjCKO brain size at P7. Scale bar, 1 mm. K, Quantification of P7 cerebral hemisphere circumference. Data are presented as the mean ± SD; ****p < 0.0001 as determined by a t test; n = 3. L, Representative images of Ctip2 (green) and Satb2 (red) expressions in the Cenpj+/+ and CenpjCKO cortex at P7. High-magnification images show the cortical column. Scale bar, 30 μm. M, Quantification of Ctip2+ cells and Satb2+ cells ratios in the neocortex. Data are presented as the mean ± SD; *pCtip2 = 0.0335, **pSatb2 = 0.0011 as determined by a t test; n = 3; 3 brain slices per experiment. N, Representative images of Foxp2 and Cux1 expressions in the Cenpj+/+ and CenpjCKO cortex at P7. Scale bar, 30 μm. O, Quantification of Foxp2+ cells ratios in the neocortex. Data are presented as the mean ± SD; *p = 0.0175, as determined by a t test; n = 3; 3 brain slices per experiment. P, Quantification of Cux1+ cells ratios in the neocortex. Data are presented as the mean ± SD; ***p = 0.0001 as determined by a t test; n = 3; 3 brain slices per experiment.

Article Snippet: Primary antibodies used were as follows: Zo-1 (1:300, 339100; Invitrogen), γ-Tubulin (1:5000, T3559; Sigma-Aldrich), γ-Tubulin (1:5000, T5326; Sigma-Aldrich), Acetylated Tubulin (1:30000, T7451; Sigma-Aldrich), Cenpj (1:50, 11517-1-AP; Proteintech), Pericentrin (1:300, 611814; BD Biosciences), GFP (1:500, GFP-1020; Aves Laboratories), Cleaved Caspase 3 (1:300, 9664; Cell Signaling Technology), Tbr2 (1:300, ab23345; Abcam), Satb2 (1:300, ab34735; Abcam), Ctip2 (1:300, ab18465; Abcam), Ki67 (1:300, ab9260; Millipore), BrdU (1:300, ab6326; Abcam), Pax6 (1:300, PRB-278P; Covance), Sox2 (1:300, sc-17320; Santa Cruz Biotechnology), EdU (Click-iT EdU Alexa Fluor 594 Imaging Kit, {"type":"entrez-nucleotide","attrs":{"text":"C10339","term_id":"1535410","term_text":"C10339"}} C10339 ; Thermo Scientific), GFAP (1:500, G9269,Sigma-Aldrich), and β-Catenin (1:300, 610153; BD Biosciences).

Techniques: Western Blot, Knock-Out, Staining

Journal: bioRxiv

Article Title: Pathogenic MAST3 variants in the STK domain are associated with epilepsy

doi: 10.1101/2021.03.09.434675

Figure Lengend Snippet: (A) Heatmap of bulk RNA-seq data from the Brainspan atlas of the developing human brain (see list of URLs). MAST3 expression begins at about 26 weeks in prenatal development. In contrast, expression of MAST1 is highest early in pretnatal development (9 weeks) and then is reduced throughout the lifespan. This pattern is similar in other DEE-associated genes, SCN1A and SCN3A , where only the latter is associated with malformation of cortical development . Key neuronal and developmental markers NESTIN, EOMES, SOX2 and TUBB3 ) are also shown. FPKM = Fragments Per Kilobase of transcript per Million mapped reads. (B) Heatmap of single-nucleus RNA-seq data from post-mortem adult human motor cortex for MAST3 and MAST1 (see list of URLs). MAST3 is expressed exclusively in excitatory neurons, while MAST1 is expressed in both inhibitory and excitatory neurons in the cortex. (C) Immunofluorescence in 11-week-old cerebral organoids shows the presence of MAST3 in a fraction of excitatory CTIP2-positive neurons (upper panels, arrows) and in the majority of CUX1-positive neurons (lower panels, arrowheads). (D) Immunofluorescence performed in E14.5 (left panels) and E16.5 (right panels) mouse coronal brain sections confirm MAST3 expression in postmitotic upper layer neurons, co-markers evolving as cerebral development progresses. TBR1 and CTIP2 are neuronal markers specific to deep cortical layers. SATB2 is a neuronal marker for upper cortical layers. Scale bars = 50 µm.

Article Snippet: Sections were permeabilized and blocked for 1-hour at room temperature with 0.3% Triton X-100 and 10% normal donkey serum, respectively, and then overnight at 4°C with the following primary antibodies: MAST3 (Novus Biologicals, Cat # NBP1-82993), CUX1 (Santa Cruz Biotechnology, sc-514008), SATB2 (Abcam, ab51502), TBR1 (Proteintech Group, 66564-1-Ig) and CTIP2 (Abcam, ab18465).

Techniques: RNA Sequencing, Expressing, Immunofluorescence, Marker