Protein Expression Profiles Characterize Distinct Features of Mouse Cerebral Cortices at Different Developmental Stages

<div><p>The proper development of the mammalian cerebral cortex requires precise protein synthesis and accurate regulation of protein expression levels. To reveal signatures of protein expression in developing mouse cortices, we here generate proteomic profiles of cortices at embryonic and postnatal stages using tandem mass spectrometry (MS/MS). We found that protein expression profiles are mostly consistent with biological features of the developing cortex. Gene Ontology (GO) and KEGG pathway analyses demonstrate conserved molecules that maintain cortical development such as proteins involved in metabolism. GO and KEGG pathway analyses further identify differentially expressed proteins that function at specific stages, for example proteins regulating the cell cycle in the embryonic cortex, and proteins controlling axon guidance in the postnatal cortex, suggesting that distinct protein expression profiles determine biological events in the developing cortex. Furthermore, the STRING network analysis has revealed that many proteins control a single biological event, such as the cell cycle regulation, through cohesive interactions, indicating a complex network regulation in the cortex. Our study has identified protein networks that control the cortical development and has provided a protein reference for further investigation of protein interactions in the cortex.</p></div

Gene Ontology (GO) biological process analyses for proteins expressed in cortices at E13.5 (A), E15.5 (B), and P1 (C).

<p>The default setting for Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to perform the analysis. Only top 10 enriched terms were shown in the pie chart.</p

Functional protein association network of differentially expressed proteins in cortices at E13.5 and P1.

<p>Different line colors represent the types of evidence for the association: green, neighborhood; red, gene fusion; blue, co-occurrence; black, co-expression; purple, experiments; turquoise, database; yellow, text mining; and aqua, homology. The default setting for SPRING 9.1 was used to perform the analysis.</p

Generate protein expression profiles of the developing mouse cerebral cortex using tandem mass spectrometry (MS/MS).

<p>(A) A flow chart showing protein identification process using MS/MS. Protein expression was analyzed by MS/MS. To identify proteins with significant expression levels in the cortex, the ‘protein score’ with the threshold > 35 was applied. As a result, 1351, 1380, and 1327 proteins were identified in cortices at E13.5, E15.5 and P1, respectively. (B) Diagram summarizing the pattern of proteins found in mouse cortices at three developmental stages. Each sphere indicates proteins expressed at one stage. The overlapping part of the spheres indicates proteins identified at both stages. The numbers of proteins identified are labeled accordingly. (C) Frequency distribution of identified proteins by MS/MS. Identified proteins by MS/MS were assigned into different bins (from < -2 to > 8) by their protein expression value (see details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125608#sec002" target="_blank">Materials and Methods</a> for the definition of protein expression value). Briefly, protein matches were normalized and log2-transformed to generate protein expression value. The number of protein ID in each bin was summed as the frequency of this bin. (D) Proportional frequency distribution of identified proteins by MS/MS. Identified proteins by MS/MS were assigned into different bins by their protein expression value and the number of protein ID in each bin divided by the number of the total protein ID was summed as the proportional frequency of this bin.</p

Proteins differentially expressed during the cortical development and their potential functions in the nervous system.

<p>Proteins differentially expressed during the cortical development and their potential functions in the nervous system.</p

Functional protein association network of differentially expressed proteins in cortices at E13.5 and E15.5.

<p>Different line colors represent the types of evidence for the association: green, neighborhood; red, gene fusion; blue, co-occurrence; black, co-expression; purple, experiments; turquoise, database; yellow, text mining; and aqua, homology. The default setting for SPRING 9.1 was used to perform the analysis.</p

Analyses of biological process (A), molecular function (B), and cellular component (C) for proteins differentially expressed in cortices between E13.5 and E15.5.

<p>The default setting for Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to perform the analysis. Only top 10 enriched terms were shown in the pie chart.</p

Hierarchical clustering for differentially expressed proteins in cortices at three stages.

<p>(A) Differentially expressed proteins between E13.5 and E15.5. (B) Differentially expressed proteins between E13.5 and P1. Protein expression value was used to represent the individual protein expression and was used in the hierarchical analysis.</p

Increased cell death and decreased proliferation of neural progenitors in the E18.5 <i>Nestin-Dicer cko</i> hippocampus.

<p>(A) A coronal section with Nissl staining of the E18.5 wild type hippocampus. The CA1, CA3, and dentate gyrus (DG) regions are labeled. (B) Increased apoptotic cells in the hippocampus of <i>Nestin-Dicer cko</i> brains (arrowheads) compared to controls (<i>Ctrl</i>), as detected by TUNEL assays. (C) The number of hippocampal progenitors was decreased in <i>Nestin-Dicer cko</i> brains. Progenitors were detected with anti-Ki67, anti-BrdU, and anti-PH3 antibodies. (D-G) Quantification of TUNEL⁺, Ki67⁺, BrdU⁺ and PH3⁺ cells in the <i>Nestin-Dicer cko</i> and control hippocampus. At least three <i>Nestin-Dicer cko</i> and three control animals were used for all statistical analysis. n>3, <i>**</i>, <i>P<0.01</i>; <i>***</i>, <i>P<0.001</i>.</p

Abnormal production and migration of Cajal-Retzius neurons in <i>Dicer</i> conditional knockout mouse brains.

<p>(A, B) <i>Lhx2</i> expression was reduced in the E13.5 (arrowheads) and E18.5 (arrows) hippocampus in <i>Emx1-Dicer cko</i> and <i>Nestin-Dicer cko</i> mice. (C, D) <i>Reelin</i> expression, which labels Cajal-Retzius neurons (arrows), was normal in the E13.5 <i>Emx1-Dicer cko</i> cortex, and E15.5 and E18.5 <i>Nestin-Dicer cko</i> cortices. However, ectopic <i>Reelin</i> expression was detected in the E18.5 <i>Emx1-Dicer cko</i> cortex (arrowheads).</p