Differential expression patterns of occ1-related genes in adult monkey visual cortex.

Abstract

We have previously revealed that occ1 is preferentially expressed in the primary visual area (V1) of the monkey neocortex. In our attempt to identify more area-selective genes in the macaque neocortex, we found that testican-1, an occ1-related gene, and its family members also exhibit characteristic expression patterns along the visual pathway. The expression levels of testican-1 and testican-2 mRNAs as well as that of occ1 mRNA start of high in V1, progressively decrease along the ventral visual pathway, and end of low in the temporal areas. Complementary to them, the neuronal expression of SPARC mRNA is abundant in the association areas and scarce in V1. Whereas occ1, testican-1, and testican-2 mRNAs are preferentially distributed in thalamorecipient layers including "blobs," SPARC mRNA expression avoids these layers. Neither SC1 nor testican-3 mRNA expression is selective to particular areas, but SC1 mRNA is abundantly observed in blobs. The expressions of occ1, testican-1, testican-2, and SC1 mRNA were downregulated after monocular tetrodotoxin injection. These results resonate with previous works on chemical and functional gradients along the primate occipitotemporal visual pathway and raise the possibility that these gradients and functional architecture may be related to the visual activity-dependent expression of these extracellular matrix glycoproteins.

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Cerebral Cortex August 2009;19:1937--1951
doi:10.1093/cercor/bhn220
Advance Access publication December 10, 2008
Differential Expression Patterns of occ1-
Related Genes in Adult Monkey Visual
Cortex
Toru Takahata1,3, Yusuke Komatsu1, Akiya Watakabe1,
Tsutomu Hashikawa2, Shiro Tochitani1,4and
Tetsuo Yamamori1
1Division of Brain Biology, National Institute for Basic Biology,
Okazaki, Aichi 444-8585, Japan,2Laboratory for Neural
Architecture, Brain Science Institute, RIKEN, Wako, Saitama
351-0198, Japan,3Department of Psychology, Vanderbilt
University, Nashville, TN 37240, USA and4Department of
Anatomy and Developmental Neurobiology, Institute of Health
Biosciences, The University of Tokushima Graduate School,
Tokushima 770-8501, Japan
We have previously revealed that occ1 is preferentially expressed
in the primary visual area (V1) of the monkey neocortex. In our
attempt to identify more area-selective genes in the macaque
neocortex, we found that testican-1, an occ1-related gene, and its
family members also exhibit characteristic expression patterns
along the visual pathway. The expression levels of testican-1 and
testican-2 mRNAs as well as that of occ1 mRNA start of high in V1,
progressively decrease along the ventral visual pathway, and end of
low in the temporal areas. Complementary to them, the neuronal
expression of SPARC mRNA is abundant in the association areas
and scarce in V1. Whereas occ1, testican-1, and testican-2 mRNAs
are preferentially distributed in thalamorecipient layers including
‘‘blobs,’’ SPARC mRNA expression avoids these layers. Neither SC1
nor testican-3 mRNA expression is selective to particular areas, but
SC1 mRNA is abundantly observed in blobs. The expressions of
occ1, testican-1, testican-2, and SC1 mRNA were downregulated
after monocular tetrodotoxin injection. These results resonate with
previous works on chemical and functional gradients along the
primate occipitotemporal visual pathway and raise the possibility
that these gradients and functional architecture may be related to
the visual activity--dependent expression of these extracellular
matrix glycoproteins.
Keywords: extracellular matrix, follistatin-related protein/TSC-36/FSTL1, in
situ hybridization, monocular deprivation, RLCS
Introduction
Recent genome-wide analysis of gene expression patterns in the
mouse brain has unveiled the variety of genes that are
expressed in distinct parcellation of neuronal anatomical
architecture (Lein et al. 2007). It has been suggested that these
genes play roles in the formation or function of such structure
(Pimenta et al. 1995; Job and Tan 2003; Hamasaki et al. 2004).
Our own effort to identify area-selective genes in the adult
monkey neocortex by differential display analysis has demon-
strated that occ1 mRNA is preferentially expressed in the
primary visual area (V1) (Tochitani et al. 2001). Examination of
the occ1 mRNA expression by in situ hybridization (ISH)
revealed the following anatomical features: occ1 mRNA
expression 1) is dependent on neuronal activity, 2) is
preferentially localized in thalamorecipient layers including
‘‘blobs’’ in layers II/III, and 3) is also localized in thalamor-
ecipient layers of the extrastriate, somatosensory, and auditory
cortices, although the expression levels are much lower than
that in V1. These characteristics are restricted to the
expression in excitatory neurons, whereas the expression is
activity independent in parvalbumin (PV)-containing c-amino-
butyric acidergic (GABAergic) inhibitory interneurons that are
? 2008 The Authors
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broadly distributed throughout the neocortex (Tochitani et al.
2001; Komatsu et al. 2005; Takahata et al. 2006).
Our continued analysis is to search for area-selective genes
using restriction landmark cDNA scanning (RLCS) technique,
a large-scale analysis of differential gene expression. By this
means, we identified testican-1 as a V1-enriched gene (Fig. 1A).
The domain structure of the gene product of testican-1 is
closely related to that of occ1. Both are characterized by the
presence of one follistatin domain (FS domain) followed by one
extracellular calcium-binding domain (EC domain). At least 4
other genes are known to belong to this family (Yan and Sage
1999) (Fig. 1B). They are secreted glycoproteins and are
considered as part of the extracellular matrix (ECM). Although
their roles in anticell proliferation and anticell adhesion have
been reported (Yan and Sage 1999), the physiological roles of
those genes in the central nervous system remain to be
elucidated. Recent studies suggest that the ECM is involved in
the maturation and plasticity of the synapse (Pizzorusso et al.
2002; Dityatev and Schachner 2006). Thus, occ1-related genes
may have significant roles in regulating synaptic plasticity in the
visual area in the primate neocortex.
Here, we performed a comprehensive expression analysis of
the members of this family, namely, testican-1, testican-2,
testican-3 (also referred to as SPOCKs), secreted protein acidic
and rich in cystein (SPARC: also referred to as BM-40 or
osteonectin), and SPARC-like1 (SC1: also referred to as hevin).
We found both similar and complementary expression patterns
of the members of this family to that of occ1 in terms of area
difference, particularly the V1-temporal association area (TE)
gradient, lamina preference, blob preference, cell-type speci-
ficity, and sensory input dependence.
Materials and Methods
Animals and Sample Preparation
For RLCS analysis and quantitative real-time reverse transcriptase--
polymerase chain reaction (RT-PCR), postmortem brain tissues of
African green monkeys (Cercopithecus aethiops) were obtained from
the Japan Poliomyelitis Research Institute and processed as previously
described (Watakabe et al. 2001). For ISH, ten Japanese macaque
monkeys (Macaca fuscata, adult, either sex, 2.9--9.1 kg) were used. Six
of the Japanese macaques were subjected to monocular deprivation
(MD) during 5--21 days before sacrifice as follows: 10 ll of tetrodotoxin
(4.7 mM in saline) was slowly microinjected into the left eyeball twice
a week under ketamine anesthesia. The pupil of the injected eye was
dilated after operation. A part of these samples were used in our
previous studies (Tochitani et al. 2001; Takahata et al. 2006). Four of
the 6 MD samples (5, 10, 14, and 21 days MD each) were used for
quantification of MD effect on gene expression (Fig. 9). All the animals
were administered an overdose of pentobarbital (at least 100 mg/kg

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body weight) and perfused intracardially with 4% paraformaldehyde
(PFA) in 0.1 M phosphate buffer (PB). The brain was then removed
from the skull, postfixed for 3--6 h at room temperature in the same
fixative, cut into several blocks, and cryoprotected in 30% sucrose in
0.1 M PB at 4 ?C. The block samples were frozen and stored at –80 ?C.
The frozen blocks were cut on a sliding microtome into 15-lm thick
sections for fluorescence ISH or into 35-lm thick sections for
colorimetric ISH and immunohistochemistry (IHC). The sections were
maintained in a cryoprotectant solution (30% glycerol, 30% ethylene
glycol, 40% 0.1 M phosphate-buffered saline [PBS]) at –30 ?C until
further processing when not used within 24 h after sectioning.
The protocols used in this study were approved by the Animal
Research Committee of the National Institute for Basic Biology (NIBB)
and the National Institute for Physiological Science, Japan; they were in
accordance with the animal care guidelines of the National Institutes of
Health, United States.
RLCS
Differential gene expression among 4 cortical areas (V1, TE, the
primary motor area [M1], and Brodmann’s area 46) of African green
monkey was comprehensively identified by RLCS method as previously
described (Suzuki et al. 1996; Shintani et al. 2004). Total RNAs were
extracted separately from the cortical areas as described previously
(Watakabe et al. 2001). Poly (A)+RNA was purified with BioMag Oligo
(dT)20(PerSeptive Biosystems, Framingham, MA) following manufac-
turer’s instructions. Double-stranded cDNAs were synthesized with an
anchor primer targeting poly (A)+tail and SuperScript Choice System
(Invitrogen, Carlsbad, CA). An overhang was created at the 5#-terminal
of the cDNA using ApaLI, and the restriction cut site was radiolabeled
by a full up reaction with [a-32P] deoxynucleotides. Labeled fragments
containing the 3#-terminal were collected using Dynabeads Oligo
(dT)25(Dynal, Oslo, Norway) and cut away from the 3#-terminal by NotI
digestion. The fragments were then subjected to agarose gel
electrophoresis. After electrophoresis, the cDNA fragments in gel were
digested with HinfI, in situ, and then subjected to acrylamide-gel
electrophoresis. The cDNA fragments labeled and separated by the
2-dimensional (2D) electrophoresis were displayed as spots on X-ray
films.
The spot intensity was carefully compared among the 4 cortical areas
and the DNA fragments corresponding to the differential spot were
eluted from the cutout gel and ligated with 2 adapters; one for the
overhang site of ApaLI digestion and the other for the overhang site of
HinfI digestion, and the ligated fragments were amplified by polymerase
chain reaction (PCR) using the adapter sequence. The amplified
fragments were cloned as described previously (Komatsu et al. 2005).
A total of 30 colonies per one cloned spot were picked up and
subjected to PCR using the plasmid vector sequence. To select
a positive clone corresponding to a differential spot, the fragments
were subjected to restriction fragment length polymorphism (RFLP)
analysis using a 4-base cutter, AluI. The inserted sequence that showed
the most frequent pattern in RFLP was determined for each spot.
Real-Time RT-PCR
To quantify the difference in the mRNA expression levels among areas,
real-time RT-PCR was performed. Total RNA was isolated from tissues
of 5 cortical areas; V1, TE, M1, the primary somatosensory area (S1), and
area 46. In all, 20 lg of each was subjected to RNase-free DNase treat-
ment, followed by another round of acid guanidinium thiocyanate--
phenol--chloroform extraction to completely remove the contaminat-
ing genomic DNA. Two micrograms of such RNA was converted to
cDNA using random nonamer and MMLV reverse transcriptase
(Invitrogen) and used as the template for real-time RT-PCR. Prepared
cDNAs were reacted with specific primers for each gene (Supplemen-
tary Table 1) and SYBR Premix Ex Taq (Takara, Otsu, Japan) under the
control of OPTICON2 (Bio-Rad, Hercules, CA). Only single bands were
obtained when each reacted sample was electrophoresed, and the
sequences of those bands were confirmed as derived from the targeted
sequences of each gene. Lines of diluted solutions of brain samples
were examined to determine amplification efficiencies for each primer
set. The amount of each mRNA in each area was calculated by cycle
Figure 1. (A) The 2D electrophoresis in RLCS analysis in V1, TE, M1, and area 46. A
more intense spot of the testican-1 gene was observed in V1 than in the other 3
areas (shown by a circle). (B) Schematic domain structures of occ1-related proteins.
Testican-1 (*), which was identified as a V1-enriched gene, has a domain structure
partially homologous to this group of proteins. They are characterized by one FS
domain (FS) and a following EC domain (EC). Testicans have their unique domain both
in the N-terminal (TES) and the C-terminal (C). TY denotes a thyroglobulin-like domain.
Black arrowheads indicate the site of the glycosylated serine in Testicans (Alliel et al.
1993). SPARC has the highest homology to SC1 (ca. 70%) (Soderling et al. 1997).
Open bars represent unidentified unique domains for each protein (Maurer et al.
1995). (C) Quantification of area difference in mRNA expression of occ1-related
genes among V1, TE, M1, S1, and area 46 by real-time RT-PCR analysis. The amount
of each mRNA was normalized as a ratio to the amount of the internal standard,
g3pdh mRNA. occ1, testican-1, and testican-2 mRNAs were most abundant in V1
among these 5 areas.
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Expression Patterns of occ1-Related Genes
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Takahata et al.

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numbers, fluorescence intensities, and amplification efficiencies. The
amount of each mRNA was expressed as the ratio to that of the
glyceraldehyde 3-phosphate dehydrogenase (g3pdh) mRNA, which
was used as an internal standard.
ISH
For colorimetric ISH, digoxigenin (DIG)-labeled antisense and sense
riboprobes were prepared using a DIG-dUTP labeling kit (Roche
Diagnostics, Indianapolis, IN). Riboprobes were newly prepared from
the cDNA library of each animal by RT-PCR and conventional TA
cloning techniques. The primer sequences are listed in Supplementary
Table 1. The sense probes did not detect signals stronger than the
background signal (data not shown). ISH was carried out as described
previously (Tochitani et al. 2001; Takahata et al. 2006). Briefly, free-
floating sections were soaked in 4% PFA/0.1 M PB (pH 7.4) overnight at
4 ?C and treated with 10 lg/ml proteinase K for 30 min at 37 ?C. After
acetylation, the sections were incubated in the hybridization buffer (53
standard saline citrate [SSC; 13 means 150 mM NaCl, 15 mM Na citrate,
pH 7.0], 50% formamide, 2% blocking reagent, 0.1% N-lauroylsarcosine
[NLS], 0.1% sodium dodecyl sulfate, 20 mM maleic acid buffer; pH 7.5)
containing 1.0 lg/ml DIG-labeled riboprobe at 60 ?C overnight.
Hybridized sections were washed by successively immersing in the
washing buffer (23 SSC, 50% formamide, 0.1% NLS; 60 ?C, 20 min,
twice), RNase A buffer (10 mM Tris--HCl, 10 mM ethylenediaminetetra-
acetic acid, 0.5 M NaCl; pH 8.0) containing 20 lg/ml RNase A (37 ?C, 15
min), 23 SSC/0.1% NLS (37 ?C, 20 min, twice), and 0.23 SSC/0.1% NLS
(37 ?C, 15 min, twice). Hybridization signals were visualized by alkaline
phosphatase (AP) immunohistochemical staining using a DIG detection
kit (Roche Diagnostics). Because ISH signals were so strong for testican-
2 and SC1, we shortened their reaction time (to 6--8 h) with nitro blue
tetrazolium/5-Bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) from
the original reaction time (16--18 h) to prevent the saturation of ISH
signals. Sections were mounted onto glass slides and dehydrated through
a graded series of increasing ethanol concentration followed by xylene
and then coverslipped with Entellan New (Merck, Tokyo, Japan).
Fluorescence double-labeling ISH was performed as described
previously (Takahata et al. 2006). Fluorescein isothiocyanate (FITC)-
labeled riboprobes for glutamic acid decarboxylase 67 (GAD67),
vesicular glutamate transporter 1 (VGluT1), and glial fibrillary acidic
protein (GFAP) were prepared. Brain sections were hybridized with
both the DIG-labeled and the FITC-labeled probes. The hybridization
protocol was the same as that of colorimetric single ISH. DIG was
detected by staining with AP-conjugated anti-DIG antibodies and using
an HNPP Fluorescence Detection kit (Roche Diagnostics). FITC was
detected by horseradish peroxidase--conjugated anti-FITC antibodies
(Roche Diagnostics) followed by enhancement using a TSA-Plus DNP
system (Perkin Elmer Life Sciences, Boston, MA) and staining with
Alexa 488--conjugated anti-DNP antibodies (Molecular Probes, Eugene,
OR). The sections were then counterstained with Hoechst 30442
(Molecular Probes) diluted in PBS to 1:1000. After mounting onto glass
slides, sections were air dried and coverslipped with the PermaFluor
Aqueous mounting medium (Thermo, Pittsburgh, PA).
The brain regions of macaques were identified using brain atlases
(Paxinos et al. 2000). Images of the ISH sections were captured with an
SZX12 or BX50 microscope (Olympus, Tokyo, Japan) using a 3CCD
color video camera, DP50 (Olympus), and processed using Photoshop
CS3 Extended (Adobe, San Jose, CA). The scale bars in the figures are
corrected for shrinkage caused by ISH.
IHC
Free-floating sections were immersed into the blocking buffer (2%
bovine serum albumin [BSA], 0.5% Triton X-100 in tris-buffered saline
[TBS] for Testican-1 IHC; 10% normal goat serum, 2% BSA, 0.5% Triton
X-100 in TBS for SPARC IHC) for 1 h at room temperature. The sections
were then reacted with the primary antibodies, goat anti-Testican-1-
antiserum AF2327 (R&D systems, Minneapolis, MN, 2 lg/ml IgG), or
mouse anti-SPARC-monoclonal antibody PP16 (Santa Cruz Biotechnol-
ogy, Santa Cruz, CA, 1 lg/ml IgG) in the same blocking buffer overnight
at 4 ?C. The sections were then washed in TBS 3 times and reacted with
the secondary antibodies conjugated with biotin for 2 h at room
temperature. Finally, immunoreactivity was detected using an avidin/
biotin/peroxidase detection kit (Vectastain, CA) and diaminobenzinine
as a substrate, with nickel enhancement. The sections were dehydrated
through a graded series of increasing ethanol concentrations followed
by xylene and coverslipped with Entellan New. Sections developed
without the primary antibodies showed no detectable signal above the
background level (data not shown).
Quantification
The number of cells those contain fluorescent signal in double-labeling
ISH was manually counted in V1 and TE. The images taken in 3 channels
(ISH signals in red [occ1-related genes]/green [cell-type markers]
channels and Hoechst nuclear staining in the blue channel) were
layered into a single file. First, cortical layer boundaries were
determined in reference to Hoechst nuclear staining. Then, the cells
positive for green ISH fluorescent signal were plotted and counted in
each cortical layer. Those cells that contain signals intense enough to
be distinguished from background level were counted as positive cells.
Finally, those cells that have the identical shape of red and green ISH
fluorescent signals were counted as double-positive cells. Using the
Hoechst nuclear staining, we confirmed that each of them has a single
nucleus and the red and green signals are not derived from side by cells.
Cells that had unclear ISH fluorescent signal due to the crowdedness of
cells were excluded from the analysis.
To quantify the effect of MD on gene expression, the relative optical
densities (RODs) of ISH signals in the bright field microscope were
determined for the deprived or nondeprived ocular dominance
column. We mainly used tangential/semitangential sections or coronal
sections that showed wide ocular dominance columns for the analysis,
in order to identify regions of each ocular dominance column. The
shapes of each ocular dominance column were delineated with
reference to the occ1 ISH staining pattern in V1 tangential or coronal
sections from 4 MD monkeys and applied to adjacent ISH sections for
the other genes (see Fig. 10). The gray level of ISH signal for each gene
and each column was converted into ROD using the following
equation, ROD = log10(255/observed gray levels).
The background ROD was subtracted from each ROD. The value of
each ROD was then expressed as mean ± standard error of the mean,
and the difference between deprived and nondeprived columns was
determined by paired Student’s t-test (P < 0.05 was considered
significant).
Results
Identification of testican-1 as a V1-Enriched Gene by
RLCS
To systematically identify area-specific molecules in the
monkey neocortex, we carried out large-scale screening by
RLCS. In this analysis, mRNAs were purified from 4 distinct
cortical areas V1, TE, M1, and area 46, converted to cDNA by
reverse transcription, and digested with a pair of restriction
enzymes for 2D analysis. In ApaLI and HinfI enzyme combina-
tion, approximately 850 spots were obtained, and we found
86 apparent differential spots among the 4 cortical areas.
Among them, one spot was particularly intense in the visual
area (Fig. 1A). This spot was cloned and identified as the
testican-1 gene, a member of the gene family to which occ1
belongs (Fig. 1B).
Expression Patterns of occ1-Related Genes in Macaque
Neocortex
The identification of testican-1 as a V1-enriched gene
prompted us to carry out the comparative expression analysis
of all the family genes of occ1 in the monkey neocortex.
Besides occ1 and testican-1, there are 4 genes reported to
belong to this family, namely, testican-2, testican-3, SPARC, and
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SC1 (Vannahme et al. 1999; Nakada et al. 2001) (Fig. 1B). To
estimate the differences in expression levels of those genes
among different cortical areas, we carried out quantitative real-
time RT-PCR using specific primers for occ1-related genes on
cDNAs prepared from cortical samples of V1, TE, M1, S1, and
area 46. We confirmed that occ1 mRNA is particularly abundant
in V1, compared with other areas (Fig. 1C), with occ1 mRNA in
V1 being about 3 times as abundant as that in M1 and
association areas (TE and area 46). The expression level of occ1
mRNA in S1 was approximately 1.5 times higher than those in
M1 and association areas. The testican-1 mRNA was also most
abundant in V1, and its level was 2 times higher than that in
association areas, validating the result of the RLCS analysis.
Similarly, we observed that testican-2 mRNA was also most
abundant in V1, and its level was 1.2--1.4 times higher than that
in other areas. No clear area difference was observed for
testican-3, SPARC, and SC1 in this analysis.
We further examined their mRNA expression in detail using
ISH histochemistry. Figure 2 shows their expression patterns in
4 coronal sections of the macaque neocortex in low-magni-
fication photographs. Consistent with the RT-PCR analysis
results, we confirmed that both the occ1 and testican-1 ISHs
exhibited a V1-enriched pattern. Similarly, testican-2 mRNA
signals were also observed to be abundant in V1, although the
area difference was less conspicuous than those of occ1 and
testican-1. Both in S1 and A1, the mRNA signals of occ1,
testican-1, and testican-2 were also relatively strong. The
mRNA signals of testican-1 and testican-2 were moderate in
the higher association areas compared with those in V1 and
other primary sensory areas. To our surprise, the staining
pattern of ISH for SPARC showed clear area difference.
Complementary to those of occ1, testican-1, and testican-2,
the mRNA signals of SPARC were weak in V1 and strong in the
higher association areas. The SPARC mRNA signals were also
scarce in S1 and A1. Both testican-3 and SC1 ISH exhibited
moderate or strong signals in the macaque neocortex, re-
spectively, but the area differences in their expression were
not observed.
Figure 3 shows magnified views of ISH sections for the occ1
family genes in V1 and extrastriate visual cortices. As reported
previously (Tochitani et al. 2001; Takahata et al. 2006), layer
IVCb was most strongly labeled by the occ1 probe among the
layers in V1. Strong signals were also observed in layers II/III of
V1. Due to the strong V1 expression of occ1 mRNA, the V1/V2
boundary was distinct (arrow in Fig. 3A). In extrastriate
cortices, the occ1 mRNA signals were localized mainly in pyra-
midal neurons that were present in the deeper part of layer III,
and the signal intensity reduced along the visual ventral
pathway (asterisks in Fig. 3A--D). Only faint occ1 mRNA signals
were observed in TE. The expression patterns of testican-1 and
testican-2 mRNAs were quite similar to each other, and their
area preferences were similar to that of occ1; that is, the
testican-1 and testican-2 mRNA signals were strongest and
densest in layer IVC of V1 (Fig. 3E,I), and thus, the V1/V2
boundary was clearly observed in their ISH. Pyramidal neurons
in the deeper part of layer III were strongly labeled by the
testican-1 and testican-2 probes in V2, and the signal intensity
gradually decreased along the visual ventral pathway (Fig. 3E--
L). Compared with those of occ1, the testican-1 and testican-2
mRNA signals in the visual cortices were stronger and more
broadly distributed to other layers. Layer VIa also showed
strong testican-1 and testican-2 mRNA signals and layers II,
upper III, IV, and V of extrastriate cortices exhibited moderate
testican-1 and testican-2 mRNA signals as well. The difference
in the expression pattern between testican-1 and testican-2
was that the laminar specificity and area preference of the
testican-1 mRNA signal distribution were clearer than those of
testican-2 (Fig. 3E--L). The testican-3 mRNA signals were
sparsely distributed all across the visual cortices without
apparent difference among layers and areas, except for layer
I, which showed a low level of testican-3 mRNA signal. The
granule layer (layer IV) showed slightly strong testican-3
mRNA signals (Fig. 3M--P). SPARC-mRNA-positive cells were
observed on the surface of layer II and deeper layer VI (VIb) in
V1, and little signal was observed in the other layers (Fig. 3Q).
The SPARC ISH pattern in V2 was similar to that in V1, and thus,
the V1/V2 boundary was not apparent in the ISH section for
SPARC. The only difference in the SPARC mRNA expression
between V1 and V2 was that the width of the SPARC mRNA
Figure 2. Expression patterns of occ1-related genes in coronal sections of adult
monkey neocortex. For occ1, testican-1, and testican-2, note that the strongest
signals were observed in V1, and moderate signals were observed in S1 and A1 as
well (see the annotation in sections for testican-1 ISH). Conversely, the expression of
SPARC mRNA was strong in higher-order association areas, such as in TE, whereas
weak in V1, S1, and A1. These sections corresponded to Bregma levels 8.5 (section
1), ?14.4 (section 2), ?32.8 (section 3), and ?45.5 mm (section 4). ca/calcarine
sulcus, cgs/cingulate sulcus, cs/central sulcus, eca/external calcarine sulcus, ios/
inferior occipital sulcus, itp/intraparietal sulcus, lf/lateral fissure, lu/lunate sulcus, ots/
occipitotemporal sulcus, and sts/superior temporal sulcus; scale bar 5 10 mm.
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signals in layer II was slightly broadened into deeper layers in
V2 compared with that in V1. Along the visual ventral pathway,
the width of the SPARC mRNA signals in the supragranular
layers further extended into deeper layers, and it almost
covered the entire layers II/III in TE (brackets in Fig. 3Q--T).
The SPARC mRNA signals also gradually increased along the
pathway in layer V of extrastriate visual cortices. This
expression pattern of the SPARC mRNA is very reminiscent
of that of retinal-binding protein (Rbp), which we have
identified as an association area--enriched gene in the adult
monkey neocortex (Komatsu et al. 2005). The abundance of
the SPARC mRNA signals in layer VIb was not apparently
different throughout the visual cortices. In addition, the SPARC
mRNA signals were most strongly observed on the pial surface
throughout the visual cortices, where neuronal cells are not
present. The SC1 mRNA signals were very strong throughout
the neocortex and layers, including layer I (Fig. 3U--X). The SC1
mRNA signals were more intense in layers IVC and VI of V1
than the other layers in V1, and signals in layers III, IV, and VI of
extrastriate visual cortices were also strong.
We have already reported that the patchy distribution of the
occ1 mRNA signals in layers II/III of V1 coincides with CO-
dense puffs or blobs (Takahata et al. 2006). We examined in V1
tangential sections whether other genes of this family were also
enriched in blobs. We found that both the testican-1 and
testican-2 mRNA signals were broadly distributed in tangential
sections of layers II/III, but their expression levels were slightly
higher in CO-rich blobs than in the remaining (interblob)
regions (Fig. 4A--D). Notably, the distribution of the SC1 mRNA
signals exhibited clear enrichment in the CO-dense blobs (Fig.
4E,F). The testican-3 and SPARC mRNA signals showed no such
correlation with blobs (data not shown).
As ISH for occ1-related genes showed characteristic expres-
sion patterns in the visual circuit, we also examined their ISH in
Figure 3. Higher magnification of coronal ISH sections for occ1-related genes in the visual cortices. The mRNA signals of occ1, testican-1, and testican-2 were strongest in V1.
Their signals in deeper layer III of the extrastriate cortices gradually decreased along the visual pathway (asterisks). Conversely, the SPARC mRNA expression was weak in V1 and
gradually broadened in superficial layers and layer V along the visual pathway (brackets). WM/white matter; scale bar 5 1.0 mm.
Cerebral Cortex August 2009, V 19 N 8 1941

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a distinct area of the primate visual system, the middle
temporal visual area (MT), which is a highly myelinated
compartment and the center of motion processing in the
visual dorsal pathway (Kaskan and Kaas 2007). However, the
expression patterns of occ1-related genes in MT were similar to
those in extrastriate visual cortex and did not show clear
demarcation with surrounding areas (Fig. 5).
Cell-Type Specificity in Expression of occ1-Related Genes
in Visual Cortex
To identify the type of the cells that express occ1-related genes
in the macaque neocortex, we carried out fluorescence
double-labeling ISH with an excitatory neuron marker, VGluT1,
and a GABAergic neuron marker, GAD67, as we had done to
analyze occ1-mRNA-positive neurons in the previous study
(Takahata et al. 2006). Furthermore, we used in this study an
astrocyte marker, GFAP, because SPARC and SC1 proteins are
reported to localize in glial cells in the brain (Johnston et al.
1990; Mendis et al. 1995; Mendis, Ivy, and Brown 1996). Below
we first describe the double-labeling ISH experiments on occ1-
related genes in V1. The testican-1 mRNA signals colocalized
with VGluT1 (Fig. 6A) and GAD67 mRNA signals (Fig. 6B) but
not with the GFAP mRNA signals (Fig. 6C). This suggests that
testican-1 mRNA is expressed in both excitatory neurons and
GABAergic interneurons but not in astrocytes in V1. Similarly,
the testican-2 mRNA signals colocalized with both VGluT1 and
GAD67 mRNA signals but not with the GFAP mRNA signals in
V1 (data not shown). In contrast to those of testican-1 and
testican-2, the testican-3 mRNA signals predominantly colo-
calized with the GAD67 mRNA signals, and colocalization with
the VGluT1 mRNA signals was rare, although low levels of
testican-3 mRNA signals colocalized with the VGluT1 mRNA
signals in layer IV (Fig. 6D,E). Colocalization of the testican-3
mRNA signals with the GFAP mRNA signals was not observed
(Fig. 6F). The SPARC mRNA signals in layer VIb predominantly
colocalized with the VGluT1 mRNA signals (Fig. 7A). The
strong SPARC mRNA signals on the pial surface corresponded
to the GFAP mRNA signals (Fig. 7C). Given that there were few
SPARC-mRNA-positive cells, little colocalization of the SPARC
mRNA signals with any markers was observed in the remaining
layers of V1 (Fig. 7A--C). The SC1 mRNA signals colocalized
with both the VGluT1 and GAD67 mRNA signals in the granular
and infragranular layers of V1 (Fig. 7D,E). The SC1 mRNA
signals also colocalized with the GFAP mRNA signals in
superficial layers (Fig. 7F), although they appeared a different
population from the SPARC--GFAP-double-positive cells.
In higher visual cortices, the cell-type specificity of testican-
1 (Fig. 8A--C), testican-2, testican-3, and SC1 (data not shown)
Figure 4. Tangential sections of ISH for testican-1 (B), testican-2 (D), and SC1 (F)
and adjacent sections reacted for CO enzymatic activity (A, C, E) in layers II/III of V1.
Note that their expression patterns, particularly that of SC1, coincide with the CO-
dense blobs of each respective panel on the left. Arrowheads indicate the same blood
vessels. Scale bar 5 500 lm.
Figure 5. Expression patterns of occ1-related genes in coronal sections of MT. Their
expression patterns were similar to those of the extrastriate cortex. Scale bar 5 500
lm.
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mRNA expression was essentially the same as that in V1, with
the exception of SPARC. SPARC mRNA signals were rarely
observed in layers II--V of V1; however, there were high levels
of SPARC mRNA signals in layers II/III and V of TE. They
colocalized well with both the VGluT1 and GAD67 mRNA
signals (Fig. 8D,E). This indicated that SPARC mRNA expres-
sions in excitatory and GABAergic neurons are similar for
a particular cortical area. The SPARC mRNA signals on the pial
surface in TE colocalized with the GFAP mRNA signals and
those in layer IVb colocalized with the VGluT1 mRNA signals
(Fig. 8D,F) as well as in V1.
To calculate the proportion of either testican-1- or SPARC-
mRNA-positive neurons to excitatory or GABAergic neurons of
V1 and TE, we counted the number of cells positive for each
mRNA (Table 1). The proportions of testican-1-mRNA-positive
cells to VGluT1-mRNA-positive cells were high in layers of V1
and TE (>75%), except for infragranular layers of V1 (41.8%),
where the expression is low in layers V and VIb. The
proportions of testican-1-mRNA-positive cells to GAD67-
mRNA-positive cells were approximately half (42.9--69.2%) in
each layer of V1 and TE. On the other hand, the proportions of
SPARC-mRNA-positive cells to VGluT1-mRNA-positive cells
were very low in each layer of V1 (<15%), whereas those
were remarkably higher in TE, especially in supragranular
layers (3.6% in V1 and 71.0% in TE), which is consistent with
our observation described above. The proportions of SPARC-
mRNA-positive cells to GAD67-mRNA-positive cells were also
higher in TE (19.8--38.5%) than in V1 (1.1--24.1%) throughout
all the layers.
Immunohistochemical Identification of OCC1-Related
Proteins
We examined IHC using anti-Testican-1 and anti-SPARC anti-
bodies to reveal their protein localizations. In Testican-1 IHC,
a consistently similar pattern to the ISH pattern was observed
(Fig. 9A--F). There were abundant Testican-1 signals in layers II--
IVA, IVC, and VI of V1 (Fig. 9A,F). IHC signals were also
abundantly observed in layers deeper III, IV, and VI of
extrastriate visual cortices and consistent with ISH data,
Testican-1 protein signals in the thalamorecipient layers
progressively decreased along the occipitotemporal visual
pathway (Fig. 9A,B). At higher magnification, Testican-1 protein
signals were detected in the neuronal cell bodies and proximal
dendrites (Fig. 9C--F). It may be noted that a secreted form of
Figure 6. Coronal sections for double-labeling fluorescence ISH for occ1-related genes (red) and cell-type marker genes (green) in V1. (A--C) The testican-1 mRNA signals
colocalized well with both VGluT1 (A) and GAD67 (B) mRNA signals but not with GFAP mRNA signals (C). (D--F) The testican-3 mRNA signals more predominantly colocalized with
GAD67 mRNA signals (E) than with VGluT1 mRNA signals (D). They did not colocalize with GFAP mRNA signals (F). Small squares indicate the region where higher magnification
photos, which are shown under each panel, were taken. Scale bar 5 200 lm.
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Testican-1 protein, if it exists, may be difficult to detect using
IHC. In SPARC IHC, intense signals were detected only in the
pial surface in all the cortical areas that were examined, which
most likely corresponded to SPARC mRNA signals detected in
astrocytes of the pial surface. At higher magnification, IHC
signals were observed in glia-like small cells in the pial surface,
and processes that are considered to be derived from astr-
ocytes (Fig. 9G--J).
Activity Dependence in Expression of occ1-Related
Genes in V1
We observed strong expression of testican-1 and testican-2
mRNAs in layer IVC and in blobs of V1 and in the deeper layer
III of the extrastriate cortex. SC1 mRNA expression was also
strong in blobs. These observations raised the possibility that
the transcriptions of those genes are driven by neuronal
activity because those regions show more activity, as revealed
by CO enzymatic reactivity, than others (Horton and Hubel
1981; Wong-Riley et al. 1989). Thus, we examined whether the
expression of occ1-related genes, as with occ1, is dependent on
sensory activity in V1 (Tochitani et al. 2001). ISH for occ1
revealed periodical light and dark columns in the coronal
sections of V1 in macaques of 5--21 days MD (Fig. 10A). In the
adjacent coronal sections of ISH for testican-1, testican-2, and
SC1, there was a partial decrease of the signal intensities in
apparent deprived ocular dominance columns (Fig. 10B--D). To
confirm that this reduction occurred in the deprived column,
we examined ISH in the tangential sections of V1 in MD
macaques. In both layers II/III and IVC, the decrease in the
occ1 mRNA expression was clearly observed, and the charac-
teristic pattern of ocular dominance column staining was
observed (Fig. 10E,I). In the ISH for testican-1, testican-2, and
SC1 in adjacent tangential sections of around layer IVC, the
same pattern of ocular dominance column staining to that of
occ1 ISH was observed, presumably because of a significant
decrease in their signals in the deprived columns (Fig. 10J--L),
whereas such patterns were not observed in around layer III
(Fig. 10F--H). To quantify the effects of MD on their expression,
we measured their RODs in deprived and nondeprived columns
and compared them between the 2 (Fig. 10M--P). The occ1
mRNA expression showed a large and significant decrease in
the ROD of the deprived columns both in layers III and IVC
(23.5 ± 5.2%, P < 0.05 and 48.0 ± 2.9%, P < 0.005, respectively;
n = 4 each). The testican-1 and testican-2 mRNA expressions
also exhibited large and significant decreases in RODs owing to
MD in layer IVC (16.4 ± 1.7%, P < 0.05 and 20.3 ± 1.7%,
P < 0.05, respectively; n = 4 each). The reduction in the SC1
Figure 7. Continuation of Figure 6. (A--C) As SPARC mRNA signals were scarce in V1, they rarely colocalized with VGluT1 (A) and GAD67 (B) mRNA signals, except for the
deepest layer (VIb), where colocalization between SPARC and VGluT1 mRNA signals was observed. SPARC mRNA signals colocalized well with GFAP mRNA signals on the pial
surface (C). (D--F) SC1 mRNA signals colocalized well with both VGluT1 and GAD67 mRNA signals. They also colocalized with GFAP mRNA signals in superficial layers. Scale
bar 5 200 lm.
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mRNA signals was small (5.7 ± 1.3%) but statistically significant
(P < 0.05; n = 4) in layer IVC. Significant reduction in mRNA
expression of testican-1, testican-2, and SC1 was not observed
in layer III (4.2 ± 3.1%, P = 0.41, 5.7 ± 2.9%, P = 0.15 and
2.0 ± 1.9%, P = 0.48, respectively; n = 4 each). The effects of
MD in the mRNA expression of testican-3 and SPARC were not
detected (data not shown).
Discussion
In this study, we examined the expression of occ1-related
genes in cortical areas, particularly in the visual areas of
macaque monkeys. We found that the area and laminar
preferences of the testican-1 and testican-2 mRNA expressions
were similar to that of occ1, whereas that of SPARC mRNA was
complementary to them. Neurons in the blob regions exhibited
high levels of testican-1, testican-2, and SC1 mRNA expres-
sions. These suggested that their expressions are linked to
neuronal connectivity. The activity-dependent regulation of
testican-1, testican-2, and SC1 mRNA expression in V1 also
raised the possibility of a functional involvement in synaptic
plasticity. In earlier studies, we reported unique area- and
lamina-associated expression of occ1 and Rbp and their
complementary patterns in the monkey neocortex (Yamamori
and Rockland 2006). The novelty of the present study is the
finding that a particular paralogous group of genes exhibit
similar or complementary expressions, in relation to the
anatomical architecture of the sensory and association cortex
Figure 8. Coronal sections for double-labeling fluorescence ISH for occ1-related genes (red) and cell-type marker genes (green) in TE. (A--C) testican-1 mRNA signals colocalized
well with both VGluT1 (A) and GAD67 (B) mRNA signals but not with GFAP mRNA signals (C). (D--F) SPARC mRNA signals also colocalized with both VGluT1 (D) and GAD67 (E)
mRNA signals. SPARC mRNA signals colocalized well with GFAP mRNA signals on the pial surface (F) as well as in V1. Scale bar 5 200 lm.
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in an orderly manner, and that this closely resembles the
laminar and area specificity of occ1 and Rbp.
Anatomical Architecture and Molecular Distribution
The occ1, testican-1, and testican-2 genes are abundantly
expressed in layer IVC of V1, blobs and deeper layer III of the
extrastriate visual cortex, and scarce in higher-order areas. This
graded distribution pattern resembles the immunoreactivities
of PV, which reveal the distribution of thalamocortical pro-
jection fibers (Hashikawa et al. 1995; Levitt et al. 1995;
Rockland et al. 1999). The same kind of graduation is also
exhibited by CO enzymatic activity (Wong-Riley 1994). On the
other hand, the expression of SPARC, as well as Rbp, seems to
avoid the thalamorecipient layers, and thus, this expression
pattern also resembles the graded distribution of zinc-enriched
terminations, which represent a subset of nonthalamocortical
glutamatergic synapses (Ichinohe and Rockland 2005). Similar
increasing chemical gradients of GluR2/3 and calbindin D-28K
outside the granular layer along the visual pathway have also
been reported (Kondo et al. 1994; Xu et al. 2003).
Overall, there seems to be 2 opposing patterns of neurochem-
ical gradient between V1 and TE, which are represented by the
occ1 and Rbp patterns. It has been reported that there are
gradients intiming of maturation,dendritic field sizeofpyramidal
neurons, and susceptibility of synaptic plasticity among occipito-
temporal visual pathways (Murayama et al. 1997; Elston and Rosa
1998; Bourne and Rosa 2006). These features may be due to the
molecules that exhibit gradual distribution along the visual
pathway of the neocortex, by regulating cell maturation, arbor
extension, and physiological properties.
Unlike the correspondence of occ1 mRNA expression with
blobs to which we described as activity-dependent regulation
(Takahata et al. 2006), this is not the case for SC1. Interestingly,
SC1 mRNA was also selectively expressed in the koniocellular
layers of the dorsal lateral geniculate nucleus (dLGN) (Takahata
and Yamamori, unpublished data), which presumably send
thalamocortical axons to blob regions (Ding and Casagrande
1998). The occ1 mRNA is only weakly expressed in the
magnocellular layers of dLGN (Tochitani et al. 2001), which
mainly send axons to layer IVCa of V1 (Callaway 1998). Thus,
the SC1 mRNA expression may reflect a specific type of
projections rather than neuronal activity.
Cell-Type Specificity in Expression of occ1-Related Genes
We have already demonstrated that the pronounced area-
specific expressions of occ1 and Rbp are primarily due to their
Table 1
The estimation of percentage of testican-1- or SPARC-mRNA-positive cells to VGluT1- or
GAD67-mRNA-positive cells in V1 and TE
V1TE
testican-1 Layer
II/III
IVC
V/VI
Double/VGluT1
75.4% (1284/1743)
96.0% (1084/1139)
41.8% (846/2431)
Layer
II/III
IV
V/VI
Double/VGluT1
78.2% (1855/2396)
88.5% (724/824)
87.5% (1349/1568)
Layer
II/III
IVC
V/VI
Double/GAD67
42.9% (323/972)
64.4% (338/556)
52.3% (239/491)
Layer
II/III
IV
V/VI
Double/GAD67
69.2% (768/1151)
55.9% (157/295)
55.0% (183/347)
SPARCLayer
II/III
IVC
V/VI
Double/VGluT1
3.6% (49/1479)
0.3% (3/1003)
13.0% (187/1488)
Layer
II/III
IV
V/VI
Double/VGluT1
71.0% (1567/2209)
16.0% (148/936)
48.3% (792/1707)
Layer
II/III
IVC
V/VI
Double/GAD67
24.1% (164/680)
1.1% (6/530)
5.5% (17/310)
Layer
II/III
IV
V/VI
Double/GAD67
38.5% (414/1098)
19.8% (49/258)
22.2% (91/409)
Note: Figures in the brackets indicate numbers of counted cells those are double positive for
testican-1 or SPARC and cell-type marker (VGluT1 or GAD67) out of numbers of counted cells
those are positive for particular cell-type marker (VGluT1 or GAD67).
Figure 9. Immunohistochemical analysis of Testican-1 (A--F) and SPARC (G--J) expression in the monkey neocortex. (A, B) IHC for Testican-1 in V1 (A) and TE (B) showed
patterns very similar to those obtained by ISH at a glance. The arrow in (A) shows the V1/V2 boundary. (C--E) High power images of Testican-1-positive neurons in layer III of V2
(C) and layer V of V2 (D) and TE (E). The immunoreactivity was mainly observed in the cell body and proximal neurite. (F) Immunostaining with anti-Testican-1-antiserum in V1. (G,
H) IHC for SPARC in V1 (G) and TE (H). In this staining, SPARC signals were only observed in the astrocyte-like cells. (I, J) High power images of SPARC immunoreactivity in the
pial surface (I) and layer III (J) of V1. Scale bar in (A, B) 5 400 lm, scale bar in (C--E) 5 10 lm, scale bar in (F) 5 80 lm, scale bar in (G, H) 5 80 lm, and scale bar in (I, J) 5
10 lm.
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expressions in excitatory neurons (Komatsu et al. 2005;
Takahata et al. 2006). The pronounced expression of occ1 is
observed in excitatory neurons of thalamorecipient layers in
V1, whereas the occ1 expression in inhibitory neurons is
observed throughout all the layers of the neocortical areas
examined (Takahata et al. 2006). As we have shown above,
testican-3 mRNA, which does not show clear area difference or
activity dependence, is expressed predominantly in GABAergic
inhibitory interneurons. This difference supports our idea that
the gene expression in excitatory neurons mainly comprises of
area-selective and activity-related patterns.
The proportions of testican-1 mRNA expression to either
excitatory or GABAergic neurons were not evidently different
between V1 and TE, which suggest that it is the difference in
the level of expression per cell, rather than in the probability
whether or not a cell is determined to express the gene, is
a main factor in the area selectivity of testican-1 mRNA
expression. In this regard, it is unclear whether the area
selectivity of testican-1 and testican-2 mRNA expressions is
exclusively due to the expression in excitatory neurons. In case
of SPARC, it is obvious that the expressions in excitatory and
GABAergic neurons both contribute to the overall area
selectivity of SPARC mRNA expression. This observation was
unexpected because excitatory glutamatergic neurons and the
majority of GABAergic interneurons are presumably different in
their developmental origins (Kornack and Rakic 1995; Tan et al.
1998). Therefore, the expression pattern of SPARC mRNA may
be established after the formation of cortical areas and the
tangential migration of GABAergic interneurons. Alternatively,
SPARC-mRNA--containing GABAergic neurons may have similar
origin to excitatory neurons. In humans, it is reported that
more than half of GABAergic neurons originate from progen-
itors in the neocortical ventricular and subventricular zones of
the dorsal forebrain (Letinic et al. 2002). In either case, the
establishment of area-selective gene expression might be
initially regulated by several differential mechanisms and finally
tuned in a coordinated manner.
Consistent with previous studies (Johnston et al. 1990; Mendis
et al. 1995; Mendis, Ivy, and Brown 1996; Liu et al. 2005; Vincent
et al. 2008), we found that SPARC and SC1 mRNA expressions
are observed not only in neurons but also in glial cells. We have
demonstrated that their heterogeneous distributions in the
Figure 10. Coronal (A--D) and tangential (E--L) sections of ISH for occ1-related genes in the MD monkey V1. (E--H) are from layer III, and (I--L) are from layer IVC. The expressions
of occ1 (A, I), testican-1 (B, J), testican-2 (C, K), and SC1 (D, L) mRNA were significantly downregulated in the deprived columns by MD in layer IVC. In layer III, whereas
occ1 mRNA expression was significantly downregulated (E), the expressions of testican-1 (F), testican-2 (G), and SC1 (H) were not affected. (M--P) Measurement of RODs for
each gene in deprived and nondeprived columns in layers III and IVC. */P \ 0.05; **/P \ 0.01 (n 5 4, each data); scale bar in (A) 5 500 lm for (A--D), scale bar in (E) 5 1.0
mm for (E--L).
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neocortex are only observed in the neurons and that no
particular area preference in glial expression was found for these
genes. Their expressions in glial cells would obscure the regional
preference of neuronal expressions both in the real-time PCR
and histochemistry. The SPARC or SC1 mRNA expressions in
neurons showed clearer structure-related boundaries than the
overall appearance that includes those of glial cells.
IHC revealed that the protein localization of Testican-1
parallels that of testican-1 transcripts. This observation
evidently demonstrates the existence of the same type of
heterogeneity in the macaque neocortex in the protein level as
in the mRNA level. This is inconsistent with the report that the
Testican-1 protein exclusively localizes in the postsynaptic
region of neurons in the mouse cortex (Bonnet et al. 1996).
Although the exact reason for this discrepancy is not known,
one possible explanation is that posttranscriptional and/or
posttranslational modifications of testican-1 gene are different
between mice and macaques. We also confirmed the reduction
of immunoreactivity of Testican-1 after MD (data not shown).
SPARC IHC only showed signals in the astrocytes and their
processes in the pial surface and not in cortical neurons. This
inconsistent observation of IHC signals with ISH signals is likely
attributable to the differential detection sensitivity between
the 2 methodologies, that is, the neuronal expression of SPARC
is too weak to be detected in IHC (also note that testican-1 ISH
signals are much stronger than Testican-1 IHC signals). The
strength of IHC signals can be weakened due to low specificity
of antibody, inappropriate fixation, and diffusion of targeted
protein. Nonetheless, it is possible that the neuronal translation
of SPARC is indeed considerably weak or SPARC proteins are
substantially secreted from neurons, and they are too diffusely
localized to be sensitive to IHC.
Possible Function of OCC1-Related Proteins in Visual
Processing and Plasticity
The OCC1-related proteins are all multifunctional secreted
glycoproteins and considered to be components of the ECM
(Yan and Sage 1999). They are classified on the basis of their FS
and EC domain structures. In addition to these common motifs,
each protein of the OCC1 family has their original domains in
the N-terminal or both the N- and C-terminals (Fig. 1B)
(Vannahme et al. 1999). Other than the genes described in
this paper, the structures of SMOC-1 and SMOC-2 have a partial
similarity with those of OCC1-related proteins (Vannahme et al.
2003). However, their expressions did not show any clear area
preference (data not shown). It is reported that Testican-3 has
an alternative splicing variant form, N-Tes, which lacks the C-
terminal domain of Testican-3 (Nakada et al. 2001). The
expression level of N-tes mRNA was too low to be detected
by ISH and RT-PCR (data not shown).
Lines of evidence suggest that ECM proteins modulate
neuronal development and plastic changes. Several groups have
proposed that ECM limits plasticity in the rodent neocortex and
ECM degeneration is required to implement ocular dominance
plasticity (Pizzorusso et al. 2002; Oray et al. 2004). Secreted
glycoproteins, such as Reelin, regulate both neuronal positioning
in the developing nervous system and synaptic plasticity in the
adult (Bock and Herz 2003; Dityatev and Schachner 2006).
Although it is not as plastic as in the nervous systems of
juvenile animals, matured nervous systems also respond to
sensory and motor experiences by reorganizing cortical
circuitry (Sawtell et al. 2003; Giannikopoulos and Eysel 2006;
O’Shea et al. 2007), and spines are dynamic even in matured
neocortex (Majewska et al. 2006). In addition, there is evidence
that the characteristics of plasticity are different among cortical
areas. For example, it has been reported that the dendritic
spine motility level is low in V1 among cortical areas in adult
mice (Majewska et al. 2006). Discontinuous brief high-fre-
quency electrical stimulation of the horizontal pathway in
layers II/III pyramidal neurons induces long-term potentiation
of extracellular field potentials in layers II/III of TE in adult
macaques, whereas the same treatment conversely induces
long-term depression in layers II/III of V1 (Murayama et al.
1997). Together with the area selectivity and activity de-
pendence in expression of occ1-related genes, these observa-
tions raise a possibility that OCC1-related proteins modulate
synaptic plasticity in the adult cerebral cortex.
As an intriguing hypothesis, the activity of matrix metal-
loproteinases (MMPs), which play crucial roles in neuronal
plasticity through degradation of ECM and synapse apparatus in
brains (Dzwonek et al. 2004; Monea et al. 2006), may be re-
gulated in different ways among areas by OCC1-related
proteins. OCC1-related proteins have a Kazal-like motif that is
associated with serine protease inhibition in their FS domains
(Alliel et al. 1993). More directly, the N-domains of Testican-1
and Testican-3 are shown to inhibit protease activity of MT1-
MMP in cancer tissues (Nakada et al. 2001). Conversely, Tre-
mble et al. (1993) suggested that SPARC induces MMP
expression in fibroblasts. If OCC1-related proteins regulate
the MMP activity in macaque neocortex, then they may play
a role in stabilizing synapses through the suppression of MMPs
in active neurons of V1 and allow plasticity to occur in higher-
order areas. Once retinal activity is silenced, the expressions of
occ1-related genes may be downregulated, allowing neuronal
remodeling of visual areas where the silenced retina had
projected. Additionally, the idea that OCC1-related proteins are
involved in stabilization of ECM may also explain the fact that
the dendritic field area of layer III pyramidal neurons, as well as
their horizontal axon field size, progressively increases along
the visual ventral pathway (Elston and Rosa 1998; Tanigawa
et al. 2005) because ECM can limit the neurite extension and
MMP can release it (Hayashita-Kinoh et al. 2001). Consistently,
Testican-1 and Testican-2 have been shown to inhibit neurite
outgrowth in cultured cells (Marr and Edgell 2003; Schnepp
et al. 2005).
Glial expressions of SPARC and SC1 are well studied in
rodents. Their expressions are similar to those in macaques as
described here, suggesting that their function has been
conserved through the course of evolution. Brown’s group
suggested the possibility that the pial SPARC regulates
angiogenesis from the pia, interacting with platelet-derived
growth factor, thrombospondin, basic fibroblast growth factor,
and others (Mendis and Brown 1994). In addition, they have
also hypothesized that SC1 is involved in synaptogenesis, based
on its expression pattern and protein localization in post-
synapse apparatus and perisynaptic glial processes, and its
anticell adhesion ability (Lively et al. 2007; Lively and Brown
2008). Both SPARC and SC1 expressions in the brain are
induced under stressful conditions such as seizure, brain injury,
morphine intoxication, and axon deafferentation (Mendis et al.
1998, 2000; Ikemoto et al. 2000; Liu et al. 2005; Lively and
Brown 2007), suggesting that they have protective roles against
harmful assaults to the brain. This kind of glial expression
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under gliosis has also been reported for human Testican-1
(Marr et al. 2000).
The significance of the differential expression among occ1-
related genes is still unclear. As such, questions include: 1) Why
are the expression patterns of occ1, testican-1, and testican-2
genes alike in spite of their functional differences? That is, it has
been suggested that Testican-2 interacts with other Testican
family proteins (Nakada et al. 2003), and OCC1 has lost its ability
to bind extracellular calcium in the course of molecular
evolution (Hambrock et al. 2004). 2) Why is testican-3 mRNA
predominantly expressed in GABAergic interneurons? 3) Why is
SC1 mRNA expression in blobs not downregulated after MD?
Modification of the basic functions may result in significantly
heterogeneous extracellular environments among distinct cor-
tical areas. Detailed functional assays are required to elucidate
the roles of OCC1-related proteins in the cerebral cortex.
Significance of occ1-Related Genes in Brain Evolution
One of our primary motivations in the investigation of area-
specific genes is what they can tell us about the evolution of
the primate neocortex. We previously showed that the
characteristic V1-enriched expression pattern of occ1 is
conserved between macaques and marmosets but is absent in
mice, rabbits, and ferrets (Takahata et al. 2006). Moreover, the
expression of the orthologue gene of occ1, occ1/Frp, in mouse
sensory domains is not downregulated by sensory ablation
(Takahata et al. 2008). Thus, we currently consider that the V1-
selective and activity-dependent occ1 mRNA expression was
uniquely acquired by the primate lineage during the course of
evolution.
The testican-1 and testican-2 mRNA expressions have been
shown to be distributed strongly and evenly throughout the
adult mouse neocortex (Bonnet et al. 1996; Vannahme et al.
1999). According to Mendis and Brown (1994; Mendis, Shahin,
et al. 1996), the expression intensities of SPARC and SC1, at
both the mRNA and protein levels, do not differ among cortical
areas in mice, although the SPARC expression shows an
anterior--posterior gradient in the entire brain. Consistent with
these reports, there is no evidence for area-related differential
expression of occ1-related genes in adult mice (either from the
database [http://www.brain-map.org/welcome.do] or from our
preliminary experiments). However, the results of RNA dot blot
analysis in the human neocortex suggested that testican-1
mRNA is most abundantly expressed in the occipital lobe and
most scarcely expressed in the frontal lobe (Marr et al. 2000)
(ca. 1.4 times richer in the occipital lobe than in the frontal
lobe). Our preliminary experiment suggested that testican-1
and testican-2 mRNAs are preferentially expressed in V1 of
both marmosets and squirrel monkeys (data not shown). These
are consistent with our present data in macaques and in view of
these findings, we consider that the area-selective expression
patterns of occ1-related genes may also be present exclusively
in the primate lineage.
All the members of the occ1 family of genes are present in
the genome of various vertebrates and their amino acid
sequences are well conserved, suggesting an ancient origin
and a functional importance of these genes. Nevertheless, their
transcriptional regulatory systems in neuronal cells appear
markedly altered in the cerebral cortex in the primate lineage.
We suggest that this alteration is tightly and specifically
correlated with the evolution of the architecture in primates.
In this sense, further studies on occ1-related genes will provide
molecular clues to understanding the basis of the anatomical
heterogeneity of the primate cerebral cortex.
Supplementary Material
Supplementary
oxfordjournals.org/.
Table1 can befound at: http://www.cercor.
Funding
A Grant-in-Aid for Scientific Research on Priority Areas
(Molecular Brain Science) from the Ministry of Education,
Culture, Sports, Science, and Technology of Japan to T.Y.
Funding to pay the Open Access publication charges of this
article was provided by a Grant-in-Aid for scientific research on
priority areas (Molecular Brain Science) from the Ministry of
Education, Culture, Sports, Science, and Technology of Japan
(#17024055).
Notes
We thank Dr Takehiko Kobayashi, National Institute of Genetics, for his
help in the real-time PCR experiment and Kaoru Sawada, NIBB, for her
preparation of monkey cDNAs. We also thank Dr Kathleen S. Rockland,
RIKEN, for her critical reading of this manuscript, Dr Hiroshi Sato,
Kanazawa University, for his informative discussion about the function
of Testican family genes, and Peiyan Wong, Vanderbilt University,
for her proofreading of the manuscript. Conflict of Interest: None
declared.
Address correspondence to Tetsuo Yamamori, Division of Brain
Biology, National Institute for Basic Biology, 38 Nishigonaka, Myodaiji,
Okazaki, Aichi 444-8585, Japan. Email: yamamori@nibb.ac.jp.
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