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. 2020 Apr 3;11:1674. doi: 10.1038/s41467-020-15412-8

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 8 — a Neocortical E13 VZ of WT (n = 6 animals) and Celf1 KO (n = 2 animals). IHC for Cdp (red). DAPI shown in blue. Scale bar: 100 μm. b (left) Representative IHC of three days in vitro primary neocortical neuronal cultures. Transfected neurons are expressing GFP (Ctrl shRNA) or RFP (Celf1 shRNA) and neuronal identity marker Cdp (white). Scale bar: 40 μm. (right) Quantification of Cdp colocalization. Data represent the mean and SEM. N = 1 litter (14 embryos). Statistics: Students t-test. ***p < 0.001. c Schematic: Celf1 translationally represses Elavl4-v3, which decreases Cdp+ identities. Once Celf1 is silenced, Elavl4-v3 translation is derepressed, increasing Cdp+ identities. d P0 representative confocal images of E13 IUE neocortices electroporated with CAG-GFP and either Ctrl shRNA (n = 5 animals) or Celf1 shRNA (n = 4 animals) at E13. Ventricular zone/subventricular zone (VZ/SVZ) shown underneath. GFP is green. DAPI shown in blue. Scale bar: 100 μm. UL = upper layers, LL = lower layers. e (left) P0 representative confocal images of transfected neocortices in d, immunostained for cortical neuronal identity markers Cdp and Ctip2 in CP. Arrowheads indicate colocalization. Scale bar: 40 μm. (right) Quantification of Cdp and Ctip2 colocalization. Data represent the mean and SEM. Statistics: Student’s t-test. ***p < 0.001. f (left) Representative confocal images of transfected neocortices in e, immunostained for cortical neuronal identity marker Cdp in VZ/SVZ. Arrowheads indicate colocalization. Scale bar: 40 μm. (right) Quantification of Cdp colocalization. Data represent the mean of sections and SEM. Statistics: Student’s t-test. ***p < 0.001. g (left) P0 representative confocal images of E13 IUE neocortices electroporated with CAG-GFP and either Ctrl shRNA (n = 5 animals), Celf1 shRNA and Celf1 OE (n = 3 animals), or Celf1 shRNA and Elavl4-v3 shRNA (n = 3 animals). IHC for GFP (green) and layer marker Cdp (red). Arrowheads indicate colocalization. Scale bar: 40 μm. UL = upper layer. (right) Quantification of Cdp colocalization. Data represent the mean of sections and SEM. Statistics: one-way ANOVA with Tukeyʼs. *p < 0.05, ***p < 0.001, n.s. = not significant. h (left) P0 representative confocal images of E13 IUE neocortices electroporated with GFP and either Ctrl (n = 4 animals), Celf1 OE (n = 3 animals), or Celf1 OE and 5′ UTR-Elavl4-v3 (n = 3 animals) OE plasmids IHC for GFP (green), and cortical layer identity markers Cdp (red). Arrowheads indicate colocalization. DAPI shown in blue. Scale bar: 40 μm. (right) Quantification of Cdp colocalization. Data represent the mean and SEM. Statistics: one-way ANOVA with Tukeyʼs. ***p < 0.001. i, j Representative confocal images of the corpus callosum, contralateral neocortex, and striatum of P0 neocortices that underwent IUE with GFP and either Ctrl shRNA (n = 5 animals) or Celf1 shRNA (n = 4 animals) (i) or either Ctrl OE (n = 3 animals), Celf1 OE (n = 3 animals), or Celf1 OE and 5′ UTR-Elavl4-v3 (j). Scale bar is 200 μm.