Analysis of Area-Specific Expression Patterns of RORbeta, ER81 and Nurr1 mRNAs in Rat Neocortex by Double In Situ Hybridization and Cortical Box Method

BACKGROUND: The mammalian neocortex is subdivided into many areas, each of which exhibits distinctive lamina architecture. To investigate such area differences in detail, we chose three genes for comparative analyses, namely, RORbeta, ER81 and Nurr1, mRNAs of which have been reported to be mainly expressed in layers 4, 5 and 6, respectively. To analyze their qualitative and quantitative coexpression profiles in the rat neocortex, we used double in situ hybridization (ISH) histochemistry and cortical box method which we previously developed to integrate the data of different staining and individuals in a standard three-dimensional space. PRINCIPAL FINDINGS: Our new approach resulted in three main observations. First, the three genes showed unique area distribution patterns that are mostly complementary to one another. The patterns revealed by cortical box method matched well with the cytoarchitectonic areas defined by Nissl staining. Second, at single cell level, RORbeta and ER81 mRNAs were coexpressed in a subpopulation of layer 5 neurons, whereas Nurr1 and ER81 mRNAs were not colocalized. Third, principal component analysis showed that the order of hierarchical processing in the cortex correlates well with the expression profiles of these three genes. Based on this analysis, the dysgranular zone (DZ) in the somatosensory area was considered to exhibit a profile of a higher order area, which is consistent with previous proposal. CONCLUSIONS/SIGNIFICANCE: The tight relationship between the expression of the three layer specific genes and functional areas were revealed, demonstrating the usefulness of cortical box method in the study on the cerebral cortex. In particular, it allowed us to perform statistical evaluation and pattern matching, which would become important in interpreting the ever-increasing data of gene expression in the cortex

Spatial distributions of RORbeta, ER81 and Nurr1 in the standardized cortical map.

<p>(A) Average standardized layer maps (layers 2/3, 4, 5 and 6) for in situ hybridization immunohistochemistry (ISH) of RORbeta, ER81 and Nurr1. Black lines indicate the cytoarchitectonic borders of the cortical area defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003266#pone-0003266-g005" target="_blank">Fig. 5A</a>. (B) Coefficient of variance (CV) of standardized layer maps (layers 2/3, 4, 5 and 6) for in situ hybridization immunohistochemistry (ISH) of RORbeta, ER81 and Nurr1. White color represents the CV values of the pixels with low average values (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003266#s4" target="_blank">Materials and Methods</a>).</p

Coexpression of RORbeta, ER81 and Nurr1 genes.

<p>(A)–(C) Double ISH of RORbeta (red) and ER81 (green) mRNAs in a somatosensory area. Note the extensive coexpression of the two genes (denoted by the arrows). (D)–(F) Double ISH of RORbeta (red) and Nurr1 (green) mRNAs in a laterocaudal area. (G)–(I) Double ISH of ER81 (red) and Nurr1 (green) mRNAs in a laterocaudal area. Note that the two genes in (D)–(F) and (G)–(I) are not coexpressed. Scale bar: 50 µm.</p

Primers used to clone RORbeta gene segments.

<p>Primers used to clone RORbeta gene segments.</p

Cortical box method reveals the area-specific expression of RORbeta mRNAs.

<p>(A) Example of a cortical section of RORbeta ISH image processed for the cortical box standardization procedure. The mediodorsal end (MD), lateroventral end (LV), inner contour (IC), and outer contour (OC) were manually determined to select the part of the cortex for further processing. The selected cortical region was converted into a standard rectangle (a standardized cortical section). The intensity of the ISH signals was normalized and pseudocolored, so that the mean +1 SD becomes 0% and the mean +3 SD becomes 100% (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003266#s4" target="_blank">Materials and Methods</a> for details). Scale bar, 2 mm. (B) Example of one series of coronal sections (from the Bregma distance of −2.1 to −6.2 mm, number 1 to 17) of RORbeta ISH (left). These images of cortical sections were standardized as displayed on the right side of the original images (representative). We performed the same processing for six series of such samples (right and left hemispheres from three rats) and averaged them. Note that the patterns of the representative and the average data are quite similar. We also performed the same procedure for the Nissl-stained samples and determined the cytoarchitectonic borders for primary somatosensory (Par1, yellow), visual (OC1, blue) and auditory (Te1, red) areas. (C) Layer distributions of RORbeta, ER81 and Nurr1 (left axis) as well as the Nissl-gray level index (GLI, right axis) from the pial surface (0% of cortical depth) to the cortex/white matter border (100%). Each line plot shows the average signal intensity at a given cortical depth. The entire cortical regions except the mediodorsal and laterocaudal 10% were used to calculate the average. Green, orange, blue and red lines represent RORbeta, ER81, Nurr1 ISHs and Nissl staining, respectively. (D) Conceptual figure to illustrate the construction of the cortical box. In this example, the layer 4 fraction (30–50% cortical depth) was extracted to demonstrate the area distribution pattern of RORbeta mRNA in a two-dimensional map.</p

Principal component analysis (PCA) of standardized cortical map.

<p>Cortical areas were divided into 340×100 points so that the ISH signal values could be represented by a matrix of 34000 data points. The data of five layer maps (layer 4 of RORbeta, layers 4 and 5 of ER81, and layers 5 and 6 of Nurr1) were analyzed for PCA to extract two primary components, PC1 and PC2. (A) Pseudocolored layer maps (top) and eigenvectors (bottom) of PC1 and PC2. The layer maps indicate the PC scores plotted in the two dimensional space. The eigenvectors below the layer maps show the contributions of the five layer maps in constructing each PC. (B) The averaged PC1 and PC2 scores of each cortical area (shown in the left panel) were plotted in the PC1–PC2 space. The cortical areas represented by the dots in the right panel are grouped by modality and connected by colored lines (yellow, red and blue for somatosensory, auditory and visual, respectively) in the order of PC1 scores. Gray represents other areas, including motor, limbic and paralimbic areas. The nomenclature of each area is the same as that in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003266#pone-0003266-g005" target="_blank">Fig. 5</a>.</p

Area differences in gene expressions.

<p>The regions denoted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003266#pone-0003266-g001" target="_blank">Fig. 1</a> are magnified. In these figures, the contrast was adjusted simultaneously so that the area differences can be directly compared across different areas. The layers denoted on the left side of each panel were determined in reference to the Hoechst 30442 nuclear staining. These panels are considered to correspond to cytoarchitectonic areas as follows: a; Oc2MM, b; Par1, c; Par2, d; Oc1, e; Te3R, f; Ect, a'; DZ, b'; Par1/Par2 border, c'; Par2, d'; Oc1, e'; Oc2L, f'; Oct. S; subiculum. Scale bar: 100 µm.</p

Double in situ hybridization histochemistry (ISH) of RORbeta/ER81 (A) and RORbeta/Nurr1 (B).

<p>Signals in red are for RORbeta and those in green are for ER81 (A) or Nurr1 (B). The arrowheads indicate the area borders that were deduced by comparing the gene expression patterns shown by double ISH and those revealed by the cortical box method. Par1, Par2, Oc1, Oc2L and Te1 correspond to the primary and secondary somatosensory areas (Par1 and Par2), the primary and secondary visual areas (Oc1 and Oc2L) and the primary auditory area (Te1). Ectorhinal cortex (Ect) is also indicated. The white bars denoted as a–e and a'–e' are the regions magnified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003266#pone-0003266-g002" target="_blank">Fig. 2</a>. This figure is a montage of several images. Although the lighting condition of the original images was not even at this low resolution, we adjusted the contrast of each component image manually so that the montage appeared to be consecutive. Scale bar, 2 mm.</p