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:

Article Title:

doi:

Figure Lengend Snippet: (a) Representative confocal images showing KCC2 membrane labelling. CTIP2 + . and NeuN + neurons in the L5-M1 at Week 18. (b) Similar to (a) but showing KCC2 membrane labelling and NeuN + neurons in the L5-VH . (c) Images used to count the number of motor axons from the L5-VR taken with CARs (d) (left - right) Quantification of KCC2 intensity at the membrane, CTIP2 + and NeuN + cell counts from the L5-M1 treated with PCPZ. (e) Similar to (d) but for KCC2 intensity at the membrane. NeuN + cell counts from the L5-VH and motor axon counts from the L5-VR (N = 12-14 forKCC2, CTIP2 + and NeuN + , N = 6 for VR count) . (f & g) Similar to (d & e) but with CLP290 administration. N = 12-18 for KCC2, CTIP2 + and NeuN + , N = 12 for VR count. Also see Supplemental Fig 1. (h) Quantification of KCC2 intensity as a function of distance to the membrane in the presence of PCPZ (left) and CLP290 (right) in the L5-M1. (i) Similar to (h) but in the L5-VH. Dotted grey line denotes membrane. Asterisks denotes significance between vehicle-treated S0D1 and vehicle-treated Ntg. Hashtag denotes significance between vehicle-treated S0D1 and PCPZ-/CLP290-treated Ntg. One-way ANOVA with Tukey’s multiple comparison post hoc. Data are presented as mean ± SEM. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: Slices were then blocked in PBS containing 10% goat serum and 0.1% Triton™ X-100 for 1 h at room temperature, followed by a 16-h incubation in PBS with 0.1% Triton™ X-100 with the following antibodies: mouse monoclonal anti-NeuN (1:500; EMD Millipore, MAB377), rabbit polyclonal anti-KCC2 (1:1000; EMD Millipore; 07-432), rabbit polyclonal anti-MAP2 (1:500; Sigma-Alrich, AB5622), and rabbit polyclonal anti-CTIP2 (1:100, Abcam, ab28448) at 4°C.

Techniques: Membrane, Comparison