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Analysis of area-specific expression patterns of RORbeta, ER81 and Nurr1 mRNAs in rat neocortex by double in situ hybridization and cortical box method.

Hirokawa J, Watakabe A, Ohsawa S, Yamamori T - PLoS ONE (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Brain Biology, National Institute for Basic Biology, Okazaki, Japan.

ABSTRACT

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.

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Cortical box method reveals the area-specific expression of RORbeta mRNAs.(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 Materials and Methods 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.
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pone-0003266-g004: Cortical box method reveals the area-specific expression of RORbeta mRNAs.(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 Materials and Methods 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.

Mentions: As mentioned in the introduction, it is difficult to accurately identify cortical areas without clear-cut landmarks. To circumvent this problem, we applied a standardization and reconstruction procedure for the ISH samples of the serially prepared coronal sections of the posterior part of the rat cortex as follows (see also [31]). In the reconstruction, the shape of the cortex was transformed to fit into a rectangle, as illustrated in Fig. 4A. The left and right borders of the rectangle correspond to the medial ends of the cortex and the rhinal fissure, respectively, both of which can be easily determined. We also normalized the level of ISH signals so that the relative strength of the ISH signals at a given location can be compared across different data sets (Fig. 4A). Figure 4B illustrates the standardization process from the ISH data of the RORbeta gene. As shown in this figure, seventeen ISH coronal sections in total were used to cover the posterior part of one rat brain hemisphere (−2.1 to −6.3 mm from Bregma) with 280 µm intervals (Fig. 4B; original images). It was already evident from the original images that there are three distinct clusters of high RORbeta signals, which roughly corresponded to the somatosensory, auditory and visual areas (delineated by yellow, red and blue lines, respectively). The middle panel of Fig. 4B shows the images transformed into seventeen rows of cortical rectangles. In these rows of images, the three clusters of high RORbeta ISH signals were now more clearly visualized (“Representative”). Importantly, once the staining intensity was standardized, we could easily integrate multiple sets of data. In the right panel of Fig. 4B, the average of six sets of samples from three rats is shown. Note that the pattern of a single set of sample (“Representative”) was very similar to that of the average. The characteristic expression pattern of RORbeta mRNA is therefore reproducibly captured across different animals.


Analysis of area-specific expression patterns of RORbeta, ER81 and Nurr1 mRNAs in rat neocortex by double in situ hybridization and cortical box method.

Hirokawa J, Watakabe A, Ohsawa S, Yamamori T - PLoS ONE (2008)

Cortical box method reveals the area-specific expression of RORbeta mRNAs.(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 Materials and Methods 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2533703&req=5

pone-0003266-g004: Cortical box method reveals the area-specific expression of RORbeta mRNAs.(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 Materials and Methods 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.
Mentions: As mentioned in the introduction, it is difficult to accurately identify cortical areas without clear-cut landmarks. To circumvent this problem, we applied a standardization and reconstruction procedure for the ISH samples of the serially prepared coronal sections of the posterior part of the rat cortex as follows (see also [31]). In the reconstruction, the shape of the cortex was transformed to fit into a rectangle, as illustrated in Fig. 4A. The left and right borders of the rectangle correspond to the medial ends of the cortex and the rhinal fissure, respectively, both of which can be easily determined. We also normalized the level of ISH signals so that the relative strength of the ISH signals at a given location can be compared across different data sets (Fig. 4A). Figure 4B illustrates the standardization process from the ISH data of the RORbeta gene. As shown in this figure, seventeen ISH coronal sections in total were used to cover the posterior part of one rat brain hemisphere (−2.1 to −6.3 mm from Bregma) with 280 µm intervals (Fig. 4B; original images). It was already evident from the original images that there are three distinct clusters of high RORbeta signals, which roughly corresponded to the somatosensory, auditory and visual areas (delineated by yellow, red and blue lines, respectively). The middle panel of Fig. 4B shows the images transformed into seventeen rows of cortical rectangles. In these rows of images, the three clusters of high RORbeta ISH signals were now more clearly visualized (“Representative”). Importantly, once the staining intensity was standardized, we could easily integrate multiple sets of data. In the right panel of Fig. 4B, the average of six sets of samples from three rats is shown. Note that the pattern of a single set of sample (“Representative”) was very similar to that of the average. The characteristic expression pattern of RORbeta mRNA is therefore reproducibly captured across different animals.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Brain Biology, National Institute for Basic Biology, Okazaki, Japan.

ABSTRACT

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.

Show MeSH