Paraneoplastic antigen-like 5 gene (PNMA5) is preferentially expressed in the association areas in a primate specific manner

Abstract

To understand the relationship between the structure and function of primate neocortical areas at a molecular level, we have been screening for genes differentially expressed across macaque neocortical areas by restriction landmark cDNA scanning (RLCS). Here, we report enriched expression of the paraneoplastic antigen-like 5 gene (PNMA5) in association areas but not in primary sensory areas, with the lowest expression level in primary visual cortex. In situ hybridization in the primary sensory areas revealed PNMA5 mRNA expression restricted to layer II. Along the ventral visual pathway, the expression gradually increased in the excitatory neurons from the primary to higher visual areas. This differential expression pattern was very similar to that of retinol-binding protein (RBP) mRNA, another association-area-enriched gene that we reported previously. Additional expression analysis for comparison of other genes in the PNMA gene family, PNMA1, PNMA2, PNMA3, and MOAP1 (PNMA4), showed that they were widely expressed across areas and layers but without the differentiated pattern of PNMA5. In mouse brains, PNMA1 was only faintly expressed and PNMA5 was not detected. Sequence analysis showed divergence of PNMA5 sequences among mammals. These findings suggest that PNMA5 acquired a certain specialized role in the association areas of the neocortex during primate evolution.

Figure 1.

Identification of PNMA5 as a gene preferentially expressed in the association areas. (A) The left hemisphere of the macaque neocortical areas is illustrated. Anterior is to the left and posterior to the right. Major sulci are indicated by lower case letters: p, principal sulcus; as, arcuate sulcus; ce, central sulcus; ip, intraparietal sulcus; ts, superior temporal sulcus; l, lunate sulcus. (B) RLCS analysis of the macaque neocortex. This panel shows an example of an autoradiography of a 2D gel of RLCS analysis in area 46, digested with BclI and HinfI. Four distinct regions of the macaque neocortex depicted in panel A were analyzed. The area corresponding to the white box is shown at higher magnification in panel C. (C) Higher magnification of the boxed region in panel B in 4 distinct areas of panel A. Arrows indicate the spot of PNMA5 mRNA. These spots were strong in area 46 and temporal area, and relatively weak in the primary motor and lowest in the primary visual area.

Figure 2.

Distribution of PNMA5 mRNA in macaque brains. Coronal sections were prepared from the positions depicted on the brain diagram in H as A–G, and PNMA5 mRNA was detected by ISH. This figure is comprised of the sections from 3 macaques. Panel C is from 1 macaque, panel E is from another one, and the other panels are from a third macaque. Some of the representative areas magnified in Figure 3 are shown. Abbreviations: ps, principal sulcus; as, arcuate sulcus; ce, central sulcus; sf, sylvian fissure; sts, superior temporal sulcus; ls, lunate sulcus; cal, calcarin sulcus.

Figure 3.

Expression pattern of PNMA5 mRNA in the macaque cortex. The cortical areas and hippocampus depicted in Figure 2 are magnified. The layers indicated on the left side of each panel were identified according to the Nissl staining of the nearby section. Left of each panel shows Nissl staining and right shows the expression patterns of PNMA5 mRNA by ISH. Abbreviations: DG, dentate gyrus; S, subiculum.

Figure 4.

Double ISH of PNMA5 and VGLUT1 or GAD67 mRNAs in area TE. (A) PNMA5 mRNA (left, red) was expressed in most of the VGLUT1-mRNA-positive cells (middle, green) in layer II (bottom: higher magnification). (B) PNMA5 mRNA was barely expressed in GAD67-mRNA-positive cells (middle, green).

Figure 5.

Comparison of expression patterns of PNMA5 and RBP mRNAs in macaque brains. (A) Coronal sections at positions A, C, D, E, and F in Figure 2 are shown. (B) Higher magnification views of several areas. The expression patterns of PNMA5 and RBP mRNAs are shown, together with the laminar profiles of ISH signals quantified by measuring the optical density (red, PNMA5 mRNA; green, RBP mRNA). These profiles show the averages of the normalized values of 2 or 3 different macaques. The cortical layers were identified in reference to the Nissl staining of the nearby section. (C) Double ISH of PNMA5 and RBP mRNAs were performed to examine the coexpression of these mRNAs. Note that PNMA5 mRNA (left, red) is expressed in a large population of RBP-mRNA-positive cells (middle, green) in layer II.

Figure 6.

Expression analyses of PNMA5 gene and the PNMA family genes in various tissues by northern hybridization and RT-PCR. (A,C) Northern hybridization of PNMA family genes of African green monkeys (A) and mice (C). A single membrane blotted with various poly(A) mRNAs was serially probed with 5 PNMA family genes and GAPDH gene with repeated stripping and hybridization. No hybridization signals were detected after each stripping. The positions of the size marker are indicated on the left. (B) Quantification of northern hybridization in the neocortical areas of the African green monkey. The X-ray films shown in panel A were scanned and digitized for quantification. This panel shows the results of quantification of the lanes for 5 neocortical areas of the African green monkey for each gene. The expression levels were normalized to the ratio between the PNMA and GAPDH mRNA expression levels in area 46. The expression levels of PNMA3 mRNA are indicated by the averages of the upper and lower bands. Abbreviations: S1, primary somatosensory area; TE, temporal area; 46, area 46; V1, primary visual area; M1, primary motor area. (D–F) RT-PCR analyses of the PNMA family genes expressed in developmental brains and various adult tissues of mice (D), brain and testis of the rat (E), and human brain (F). PCR using reverse-transcribed cDNAs from the same amount of total RNAs was performed with the predetermined optimal cycle for each primer set. RT-PCR products obtained with the same primer sets for RNAs derived from the same species were loaded and separated on the same agarose gel. The GAPDH gene was used as control.

Figure 7.

Expression patterns of the family genes of PNMA5 in the macaque neocortex. (A) Coronal sections at positions A, E, and G in Figure 2 are shown. (B) Higher magnifications of area 46, area 3b, TE and V1. The layers indicated on the left side of each panel were identified according to the Nissl staining of the nearby section.

Figure 8.

ISH of PNMA5 and the PNMA family genes in the mouse brains. (A) Coronal sections at several different planes for each gene are shown. The boxed region (V1) of each gene is shown in B at higher magnification. (B) The layers indicated on the left side are identified according to the Nissl staining of the nearby section.