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. 2019 Dec 23;9(1):19697.
doi: 10.1038/s41598-019-56171-x.

Terminal neuron localization to the upper cortical plate is controlled by the transcription factor NEUROD2

Gizem Guzelsoy  1   2 , Cansu Akkaya  1 , Dila Atak  1   3 , Cory D Dunn  1   4 , Alkan Kabakcioglu  5 , Nurhan Ozlu  1 , Gulayse Ince-Dunn  6   7
Affiliations

Terminal neuron localization to the upper cortical plate is controlled by the transcription factor NEUROD2

Gizem Guzelsoy et al. Sci Rep. .

Abstract

Excitatory neurons of the mammalian cerebral cortex are organized into six functional layers characterized by unique patterns of connectivity, as well as distinctive physiological and morphological properties. Cortical layers appear after a highly regulated migration process in which cells move from the deeper, proliferative zone toward the superficial layers. Importantly, defects in this radial migration process have been implicated in neurodevelopmental and psychiatric diseases. Here we report that during the final stages of migration, transcription factor Neurogenic Differentiation 2 (Neurod2) contributes to terminal cellular localization within the cortical plate. In mice, in utero knockdown of Neurod2 resulted in reduced numbers of neurons localized to the uppermost region of the developing cortex, also termed the primitive cortical zone. Our ChIP-Seq and RNA-Seq analyses of genes regulated by NEUROD2 in the developing cortex identified a number of key target genes with known roles in Reelin signaling, a critical regulator of neuronal migration. Our focused analysis of regulation of the Reln gene, encoding the extracellular ligand REELIN, uncovered NEUROD2 binding to conserved E-box elements in multiple introns. Furthermore, we demonstrate that knockdown of NEUROD2 in primary cortical neurons resulted in a strong increase in Reln gene expression at the mRNA level, as well as a slight upregulation at the protein level. These data reveal a new role for NEUROD2 during the late stages of neuronal migration, and our analysis of its genomic targets offers new genes with potential roles in cortical lamination.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
NEUROD2 binds to overlapping and unique target sites in embryonic and postnatal cerebral cortex. (A) NEUROD2 binding sites and target genes were compared between embryonic day 14.5 (E14.5) and postnatal day 0 (P0) cerebral cortical tissue. Numbers of genome-wide binding sites and target genes are based upon previously acquired ChIP-Seq data,. Target genes were identified based on the total number of binding sites proximal to the transcription start sites of individual genes. (B) Gene ontology analysis of NEUROD2 target genes from P0 and E14.5 datasets (geneontology.org). Fold enrichment of categories with FDR < 0.01 are plotted. (C) Reactome pathway analysis of NEUROD2 targets from P0 and E14.5 datasets (reactome.org). Fold enrichment of categories with p < 0.05 are plotted.
Figure 2
Figure 2
NEUROD2 is required for normal dendrite development in primary cortical neurons. (A) Primary cortical neurons from E14.5 embryos were transfected with NS (non-silencing) shRNA or shNeurod2-1 at 2 days DIV and fixed at 5 DIV. Images were captured by confocal microscopy, scale bar: 20 µm. (B) Dendrite development was quantified by Sholl analysis. n = 65 neurons per condition derived from two separate cultures. Bars indicate S.E.M. p = 0.0062 by two-way ANOVA.
Figure 3
Figure 3
RNA-seq analysis of primary cortical neurons silenced for Neurod2 expression. (A) RNA-Seq analysis was carried out on primary cortical neurons electroporated with a NS (non-silencing) shRNA or one of the two independent shRNAs against Neurod2. 25 genes were down-regulated and 9 were up-regulated upon knockdown of Neurod2. fold-change > 1.5, FDR < 0.05. (B) A protein interactome network of REELIN revealed additional genetic targets of NEUROD2 that also interact with REELIN. Interactome analysis was based on the String Database,.
Figure 4
Figure 4
NEUROD2 binding to introns along the Reln gene is associated with suppression of Reln gene expression. (A,B) Four prominent intronic NEUROD2 binding sites are plotted on the Reln gene. Ab1, Ab2 and Ab3 represent ChIP experiment carried out with three separate NEUROD2 antibodies. Several different histone modifications corresponding to promoters (H3K4me3), enhancers (H3K4me1), actively transcribed (H3K27ac and H3K36me3) or repressed (H3K9me3 and H3K27me3) chromatin are not enriched in NEUROD2 binding sites. A slight enrichment of CTCF binding within intron 3 of Reln is detected. (C) NEUROD2 binding to all four intronic regions is confirmed by ChIP-qPCR. Immunoprecipitation with an unrelated GFP antibody is used as a negative control. n = 12 (three biological x four technical replicates). Bars represent S.E.M. *p < 0.05, **p < 0.005, ***p < 0.0001 by two-tailed unpaired t-test. (D) RT-qPCR analysis of Reln gene expression in primary cortical neurons reveals significant upregulation upon knockdown of Neurod2 with two separate shRNAs. Gapdh mRNA was used as a normalization control. n = 9 (three biological x three technical replicates). p < 0.0001 by one-way ANOVA. ***p < 0.0001, **p < 0.01 by post hoc Sidak multiple comparison tests. Bars represent S.E.M. (E) Immunoblotting of REELIN from 5 DIV primary cortical cultures electroporated with NS, shNeurod2-1 or with shNeurod2-1 together with a plasmid expressing an shRNA resistant Neurod2 cDNA (resNd2). The protein blot has been probed with REELIN antibody and HSP90 antibody as a loading control. Cropped parts of the gel are indicated with a line. (F) Quantification of REELIN protein amounts from five independent experiments. Data are presented after normalizing to HSP90 loading control. *p = 0.02 by two-tailed unpaired t-test. Abbreviations: NS (non-silencing), shNd2-1 (shNeurod2-1), shNd2-2 (shNeurod2-2), resNd2 (rescue Neurod2), ns (non-significant).
Figure 5
Figure 5
In vivo quantification of REELIN protein levels upon Neurod2 silencing in the embryonic cortex. (A) Embryos were electroporated with non-silencing or shNeurod2-1 shRNA and TdTomato at E14.5, cortices were harvested at E17.5 and confocal images were acquired after REELIN immunofluorescent staining. REELIN levels were not significantly altered within the CP, SVZ-IZ and VZ. However we observed a slight but significant increase in REELIN protein within the MZ. Scale bar: 100 µm. (B) Higher magnification images of those displayed in (A). Scale bar: 50 µm. (C–E) Quantification of images displayed in (A,B). A total of 21 images acquired from n = 3 embryos per group were used for quantification. *p = 0.02 by unpaired two-tailed t-test. Abbreviations: MZ (marginal zone), SVZ-IZ (subventricular-intermediate zone), CP (cortical plate), NS (non-silencing), ns (non-significant).
Figure 6
Figure 6
NEUROD2’s role in neuronal soma positioning to the primitive cortical zone. (A) Embryos (at E14.5) were in utero electroporated with tdTomato fluorescent marker and with NS (non-silencing) shRNA or with shNeurod2-1. Embryos were retrieved at E17.5, coronally sectioned, and tdTomato signal was subsequently imaged by confocal microscopy. VZ (ventricular zone), SVZ-IZ (subventricular-intermediate zone), CP (cortical plate), and MZ (marginal zone) are labeled. Scale bar: 100 µm. (B) Transfected neurons (a total of n = 16,184 for NS and n = 19,339 for shNeurod2-1) derived from in utero electroporation of seven littermate embryo pairs from five independent pregnant mice were counted. Normalized percentages of tdTomato-positive neurons counted in individual zones are plotted. Significantly less neurons are localized to the upper cortical plate in neurons where Neurod2 is knocked down. (C) Higher magnification images of the MZ and the CP in NS and shNeurod2-1 electroporated cortices. White arrow points to occasional neurons that over-migrate into the MZ of the shNeurod2-1 transfected samples. Scale bar: 50 µm. Bars represent S.E.M. p < 0.0001 by two-way ANOVA and *p = 0.0165 and **p = 0.0015 by post hoc Sidak’s test.
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