Genes & Genetic Systems
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Melanocytes contribute to the vasculature of the choroid
Hirotoshi ShibuyaRyutaro WatanabeAkiteru MaenoKaoru IchimuraMasaru TamuraShigeharu WakanaToshihiko ShiroishiKoji OhbaKazuhisa TakedaHiroshi TomitaShigeki ShibaharaHiroaki Yamamoto
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2018 年 93 巻 2 号 p. 51-58

詳細
ABSTRACT

Melanocytes develop from the vertebrate embryo-specific neural crest, migrate, and localize in various organs, including not only the skin but also several extracutaneous locations such as the heart, inner ear and choroid. Little is known about the functions of extracutaneous melanocytes except for cochlear melanocytes, which are essential for hearing ability. In this study, we focused on the structure of the choroid, in which melanocytes are abundant around the well-developed blood vascular system. By comparing structural differences in the choroid of wild-type and melanocyte-deficient Mitfmi-bw/Mitfmi-bw mutant mice, our observations suggest that choroidal melanocytes contribute to the morphogenesis and/or maintenance of the normal vasculature structure of that tissue.

INTRODUCTION

Mammalian pigment cells produce melanin, a biopolymer that is synthesized in specialized organelles called melanosomes. The quality and quantity of melanin pigments primarily determine skin, hair and eye color. In mammals, there are two embryonic origins for melanin pigment cells. One is destined for retinal pigment epithelium (RPE) cells that are derived from the optic cup, and the other is for melanocytes derived from the neural crest that migrate out of the dorsal margin of the neural tube. Melanocyte precursors (melanoblasts) migrate and settle into cutaneous locations such as the basal layer of the epidermis and hair follicles, and also into extracutaneous locations, such as the eye, inner ear, heart and leptomeninges (Plonka et al., 2009).

One intriguing question is whether those melanocytes that localize in different tissues and organs play the same roles. For example, it is well known that melanocytes in the skin produce melanin that provides protection from damage by ultraviolet radiation. What, though, are the extracutaneous melanocytes doing in such sun-protected habitats, where they still produce melanin? Here, we focus on melanocytes in the eye to assess the structural significance of their contribution. Melanocytes in the eye are localized in a melanin-rich layer called the uvea, which covers the outside of the eye and consists of the choroid, the iris and the ciliary body. The choroid is a layer of highly vascularized tissue at the back side of the retina. Choroidal endothelial cells originate from the paraocular mesenchyme. All other cells of the choroid, such as stromal cells, melanocytes and pericytes, develop from the cranial neural crest (Torczynski, 1982; Saint-Geniez and D’Amore, 2004).

Choroidal blood vessels are surrounded by highly pigmented melanocytes. The supply of oxygen and nutrients to the retina greatly depends on choroidal blood vessels. In the eye, there are two sources of blood supply to the retina, one from choroidal blood vessels through the RPE and the other from retinal blood vessels that lead from the center of the optic nerve. Choroidal blood vessels are different from most others in that they have fenestrations, like the kidney. Furthermore, choroidal blood vessels form a vasculature that is also observed in the lung, heart and dorsal aorta (Pardanaud et al., 1989). These vessels can be expected to have an increased circulatory capacity. Several reports have suggested that choroidal blood flow helps to maintain a stable temperature environment for the outer retinal layers, especially in the macular area (Bill et al., 1983; Parver et al., 1983). If that is true, choroidal blood vessels have a much more important physiological role than expected to maintain normal eye conditions, with both well-known and unknown functions.

To analyze the effects of the presence or absence of melanocytes on the structure of their niches, we used a mouse Mitf mutant allele, Mitfmi-bw, to focus on the structures of their habitats without melanocytes. Mitfmi-bw homozygous mice have black eyes and a white coat color due to the deficiency of melanocytes in their skin and hair bulbs; it should be noted that these mutant mice have no melanocytes in their choroid and that their eyes are black because of the normally developed and pigmented RPE. This recessive allele has an insertion of a 7.2-kb novel L1 element into the third intron, which abolishes expression of the Mitf-M isoform that is indispensable for melanocyte development. Thus, Mitfmi-bw homozygous mice lack mature melanocytes throughout the body (Yajima et al., 1999). In this study, we observed the fine structure of the choroid of mice with or without melanocytes. We found that Mitfmi-bw homozygous melanocyte-deficient mice show a much thinner choroidal layer and abnormal choroidal vasculature formation, suggesting that choroidal melanocytes contribute to supporting normal vasculature structure. Our structural observations provide a helpful cue to infer the functional evolution of melanocytes scattered throughout the body.

MATERIALS AND METHODS

Mice

A mutant mouse strain carrying the Mitfmi-bw allele (ID: MGI:1856089) was obtained as described previously (Yajima et al., 1999). Mitfmi-bw mice were maintained on a C57BL/6N background by backcrossing them more than six times with C57BL/6NJcl mice (purchased from CLEA Japan, Tokyo). C57BL/6N-Mitfmi-bw/Mitfmi-bw mice were used as melanocyte-deficient individuals. C57BL/6N mice homozygous for the wild-type Mitf allele with a normal black coat color were used as controls. All animal experiments were carried out at the Nagahama Institute of Bio-Science and Technology in accordance with the Guidelines of Animal Experimentation, Nagahama Institute of Bio-Science and Technology. All experiments were carried out using 4-week-old mice unless otherwise noted.

Paraffin section and hematoxylin-eosin stain

Mice were sacrificed by decapitation, after which their eyes were removed and scratched on the cornea, and then fixed with 2.5% glutaraldehyde in phosphate-buffered saline (PBS, pH 7.3) for 2 h at room temperature. After fixation, each eye was cut so that the lens and vitreous body could be removed, leaving the eyecup for further treatment. These eyecups were then dehydrated, embedded in paraffin (Sakura, Tokyo, Japan), and sectioned at 7 μm with a RM2125RTS microtome (Leica, Wetzlar, Germany). Retinal cross-sections were stained with hematoxylin (Merck, Darmstadt, Germany) and eosin (Sigma-Aldrich, St. Louis, USA). The choroidal thickness of wild-type (N = 5) and Mitfmi-bw/Mitfmi-bw (N = 5) mice was measured using cellSens imaging software (Olympus, Tokyo, Japan). Data are reported as the average thickness of each field ± standard error of the mean. Statistical differences between wild-type and mutant mice were analyzed using Student’s t-test.

Immunohistochemistry

Immunohistochemical staining was carried out as reported previously (Uehara et al., 2009). In brief, eyecups were prepared as described above and were fixed with 4% paraformaldehyde in PBS for 2 h at room temperature. All specimens were then washed with PBS, bleached with 3% H2O2 in 0.05 M phosphate buffer (PB, pH 7.4) for 2 h at 55 ℃, dehydrated, treated with embedding medium (20% sucrose, 33% OCT compound (Sakura, Tokyo, Japan) in PBS) overnight at 4 ℃, and then embedded in the same medium and frozen at −80 ℃. Mounted retinal cross-sections (each 7 μm thick) were prepared with a CM3500 cryostat (Leica) and washed three times with Tris-buffered saline containing Tween 20 (TBST; 0.15 M NaCl, 0.1 M Tris-HCl, 0.1% Tween 20, pH 7.5) for 5 min each at room temperature, and then blocked with 0.5% skim milk in TBST. To visualize blood vessels, each specimen was incubated with rat IgG anti-mouse PECAM-1 (platelet endothelial cell adhesion molecule-1, Dianova, Hamburg, Germany) diluted 1:300 with 3% BSA in PBS overnight at 4 ℃, and then washed three times with TBST for 5 min each at room temperature. Anti-rat IgG Alexa Fluor 594 (abcam, Cambridge, UK) diluted 1:500 with 3% BSA in PBS was used as a secondary antibody. Sections were mounted in VECTASHIELD (Vector Laboratories, Burlingame, USA) and photographed using a BX51 fluorescence microscope (Olympus).

Three-dimensional vascular structural analysis of blood vessels in the choroid

Microfil (Flow Tech, Carver, USA) is a radio-opaque silicone rubber. Under sodium pentobarbital (50 mg/kg, i.p.) anesthesia, mice were transcardially perfused with 20 ml heparinized (5 U/ml) PBS to replace the blood and then by perfusion with 10% formalin in PBS, using a P-1 peristaltic pump (GE Healthcare, Amersham, UK) with a flow rate of 2.5 ml/min. PBS and 10% formalin were prewarmed at 37 ℃. The 10% formalin in PBS was replaced with 5 ml Microfil. After polymerization, each eyeball was removed and scanned with Scan X mate E090S, a micro-CT scanner (Comscantecno, Kanagawa, Japan). Some eyeballs were treated with 3% H2O2 in 0.05 M PB for 2 h to bleach the melanin pigments so that cells and tissues covered with pigmented tissues (layers) could be observed. Bleached eyeballs were washed twice with PB for 5 min each at room temperature and were then photographed with a SZX16 stereomicroscope (Olympus).

Corrosion casting

The Mercox (Ladd Research Industries, Williston, USA) perfusion system was applied in a similar way to the Microfil perfusion system, with partial modification. In brief, 20 ml of blue Mercox and 150 mg of catalyst were mixed just before injection into each mouse. After removing the blood and fixative, mice were perfused with Mercox at a flow rate of 2 ml/min. For complete polymerization, the mice were incubated at 50 ℃ for about 12 h. After polymerization, the eyeballs were dissolved in 30% KOH for 7 days at room temperature and the resultant samples were then washed with distilled water. Specimens were freeze-dried, coated with tungsten for 15 sec at 15 mA using an E-1045 ion sputter (Hitachi, Tokyo, Japan) and examined with an S-3400 scanning electron microscope (Hitachi) at an accelerating voltage of 1.5 kV and an emission current of 37 μA.

RESULTS

Histological analysis of mouse choroidal tissue structure

To analyze the effects of the presence or absence of melanocytes on the structure of mouse choroidal tissue, we observed and compared choroidal tissue structure between 4-week-old wild-type mice and Mitfmi-bw/Mitfmi-bw mice. Wild-type mice have black hairs and eyes (Fig. 1A). On the other hand, Mitfmi-bw/Mitfmi-bw melanocyte-deficient mutant mice have a white coat and black eyes because they have a normal RPE but have lost the neural crest-derived melanocytes in their hair follicles (Fig. 1B). Wild-type mice show a normally pigmented choroid and retina (Fig. 1C, 1E). In contrast, Mitfmi-bw homozygous melanocyte-deficient mice are devoid of pigmentation and show a thinner choroid (Fig. 1D, 1F). The RPE layer thickness of wild-type mice was not significantly different from that of Mitfmi-bw/Mitfmi-bw mice. The cut planes of choroidal blood vessels of mutant mice were flattened out (Fig. 1F).

Fig. 1.

Phenotype of the mouse melanocyte-deficient Mitfmi-bw allele. A wild-type mouse with a black coat and black eyes (A). A melanocyte-deficient Mitfmi-bw/Mitfmi-bw homozygous mouse with black eyes and a white coat (B). Wild-type eye with a normal RPE and a pigmented choroidal layer (C; indicated region enlarged in E). Mitfmi-bw/Mitfmi-bw eye with an apparently normal RPE but a thinner choroidal layer due to the deficiency of melanocytes (D; indicated region enlarged in F). PECAM-1 antigen localization in the choroid of wild-type (G) and melanocyte-deficient (H) mice. PECAM-1 is expressed in vascular endothelial cells and is used to visualize blood vessels. Choroidal blood vessels of wild-type mice are restricted to the melanocyte-abundant layer. The thickness of RPE and choroidal layers of wild-type and Mitfmi-bw homozygous melanocyte-deficient mice was measured (N = 5) (I). Ratio of the thickness of the choroid to that of the RPE of wild-type and Mitfmi-bw homozygous melanocyte-deficient mice (N = 5) (J). Data are expressed as mean ± S.E. An asterisk denotes a significant difference (Student’s t-test, P < 0.01). R: retina; ON: outer nuclear layer; IS: inner segment; OS: outer segment; RPE: retinal pigment epithelium; CH: choroid. Scale bars = 200 μm (C, D), 50 μm (E – H).

To identify histochemical changes that could account for the observed structural abnormalities in Mitfmi-bw homozygous mutant mice, we carried out an immunohistochemical study to observe histological cross-sections of the choroid at 1 month of age (N = 5). Wild-type mice normally have a thick choroid wherein two or three layers of blood vessels develop (Fig. 1G). On the other hand, the choroid of melanocyte-deficient Mitfmi-bw mutant mice is much thinner than that of wild-type mice and multiple layers of blood vessels are not developed (Fig. 1H). As mentioned above for the usual hematoxylin-eosin-stained specimens, many of the cut planes of the blood vessels of mutant mice are much thinner and flattened out (Fig. 1G, 1H). The choroid of wild-type mice was significantly thicker than that of Mitfmi-bw/Mitfmi-bw mice (Fig. 1I and 1J).

Three-dimensional vascular structural analysis using micro-CT

To detect the influence of melanocyte defects that might not be obvious from analyzing choroidal cross-sections, we performed three-dimensional vascular structural analysis using micro-CT. This technique provides information on the three-dimensional structural integrity of choroidal reconstruction images. We focused on the choroidal vasculature of 4-week-old wild-type and Mitfmi-bw/Mitfmi-bw mice perfused with Microfil (N = 5 each). Enucleated eyeballs were micro-CT-scanned and their images were reconstructed with an isotropic cubic voxel size of 6 μm using OsiriX. The main blood supply to the mouse choroid is the terminating posterior ciliary artery (PCA, Fig. 2A), which branches off the central retinal artery and the long posterior ciliary artery (LPCA, Fig. 2A). The temporal LPCA sends off the inferior branch to inferior regions of the choroid. Choroidal blood vessels collect into the vortex vein. Each quadrant has one vortex vein (Fig. 2A). In micro-CT images, the LPCA diverges into the temporal and nasal sides observed both in wild-type and in melanocyte-deficient Mitfmi-bw homozygous mice (Fig. 2B, 2C). The inferior branch and vortex veins, which are seen in each quadrant, were also found in all micro-CT images of the mutant mice. In the sclera view, mutant mouse vessels overlap, as if each of them were crushed in the vortex vein (Fig. 2C). Furthermore, inferior branch blood vessels are not well developed in Mitfmi-bw homozygous melanocyte-deficient mice (Fig. 2E, magenta arrow). In the temporal view, irregularities at the level of the collected venules are seen in melanocyte-deficient Mitfmi-bw homozygous mice that were absent in wild-type animals (Fig. 2F, 2G, white arrowheads).

Fig. 2.

Three-dimensional reconstruction images of the structure of choroidal blood vessels. Schematic drawing of the choroidal vasculature (A). Sclera views of the choroidal vasculature in wild-type (B) and mutant (C) eyes. In each choroid, the temporal (T) and nasal (N) LPCAs (long posterior ciliary arteries, white arrows) that branch off from the PCA (posterior ciliary artery, black arrows) are developed. The white arrowheads and magenta arrows indicate vortex veins and the inferior branch (IF), respectively. D and E are magnified images of the areas surrounded with dashed lines in B and C, respectively. Temporal views of the choroidal vasculature in wild-type (F) and mutant (G) eyes. S: superior; I: inferior. Scale bars = 500 μm (B, C, F, G), 250 μm (D, E).

Stereomicroscopic analysis of the mouse choroidal vasculature

Although micro-CT images uncovered a structural difference in vascularization between wild-type and melanocyte-deficient mutant mice, we sometimes lost the continuity of blood vessels when we tried to trace the vasculature, especially of very small capillaries. Because micro-CT images are just reconstructed images, we decided to look at differences with the naked eye using a binocular microscope. Therefore, to further understand the morphological changes of the choroid in Mitfmi-bw homozygous melanocyte-deficient mice, we performed a stereomicroscopic analysis. Because the choroid of wild-type mice contains densely populated melanocytes around its vasculature, as expected, choroidal blood vessels can barely be seen without bleaching the melanin (Fig. 3A). In fact, they are thickly surrounded by melanocytes. On the other hand, the blood vessels of melanocyte-deficient Mitfmi-bw homozygous mice are easily observed because there are no melanocytes in the choroid (Fig. 3B). The black background of homozygous Mitfmi-bw mutant mice is due to the existence of the RPE (Fig. 3B). Accordingly, we depigmented the eyes with bleach to expose and detect the choroidal blood vessels (Fig. 3C–3F), which allowed us to compare the structure of the blood vasculature of wild-type and the mutant mouse eyes. In stereomicroscope images, an abnormal inferior branch pattern (orientation) of choroidal blood vessels was detected in Mitfmi-bw homozygous melanocyte-deficient mice (Fig. 3D, magenta arrow). In the temporal view, seemingly normal development of the vortex vein was also observed in mice lacking melanocytes. In contrast, irregularities at the level of the collected venules were seen in Mitfmi-bw homozygous melanocyte-deficient mice that were absent in wild-type animals (Fig. 3F).

Fig. 3.

Stereomicroscopic view of the choroidal vasculature perfused with Microfil. Wild-type (A) and mutant (B) eyes before bleaching. Note that the choroidal vasculature of the wild-type eye is hardly visible because of the presence of choroidal melanocytes surrounding the vasculature. The background in black of the mutant eye is due to melanin granules in the normally developed RPE cells. Bleaching allows the Microfil-cast vasculature to be clearly seen in the choroidal vasculature in wild-type (C) and Mitfmi-bw homozygous mutant (D) eyes. An abnormal inferior branch pattern (orientation) of choroidal blood vessels is detected in Mitfmi-bw homozygous melanocyte-deficient mice (D, magenta arrow). Temporal view of the choroidal vasculature in wild-type (E) and Mitfmi-bw homozygous mutant (F) eyes. Irregularities at the level of the collected venules are seen in Mitfmi-bw homozygous melanocyte-deficient mice (F) that are absent in wild-type animals (E). Yellow tubes show polymerized Microfil in the blood vessels. White arrows: LPCAs; magenta arrows: inferior branches; white arrowheads: vortex veins; black arrows: PCA. Scale bars = 1 mm.

Corrosion casts

Because it is not easy to observe the vascular pattern of the collected venules in the choroid with micro-CT and stereomicroscope images, to further examine the structural pattern (abnormalities) of the collected venules in Mitfmi-bw/Mitfmi-bw mice, we prepared vascular corrosion casts of Mitfmi-bw/Mitfmi-bw and wild-type eyes and analyzed them using scanning electron microscopy (N = 3). In the corrosion casts, choriocapillaries were well developed both in wild-type and in melanocyte-deficient Mitfmi-bw homozygous mice (Fig. 4A, 4B). Although wild-type animals exhibited a regular network of collected venules (Fig. 4A, black arrowheads), the choroid of Mitfmi-bw/Mitfmi-bw mice displayed a reduced number of branches of the collected venules (Fig. 4B, black arrowheads).

Fig. 4.

Scanning electron micrographs of vascular corrosion casts. Corrosion cast of vortex veins of wild-type (A) and mutant (B) mice. The collecting venules (black arrowheads) converge into the vortex vein (black arrow). Scale bars = 500 μm.

DISCUSSION

Cross-sections of the eyes of wild-type mice show pigmented choroids and normal vascular layers (Fig. 1C, 1E and 1G). In contrast, the eyes of Mitfmi-bw/Mitfmi-bw mice show melanocyte-deficient thinner choroids and their vascular layers are narrower than those of wild-type mice (Fig. 1D, 1F and 1H). In previous studies, the choroidal vasculature was observed in wild-type C57BL/6 mice and other wild-type animals having melanocytes (Bhutto and Amemiya, 2001; Ninomiya and Inomata, 2006). In three-dimensional views, the micro-CT observations of normal choroids with melanocytes in the present study yield choroidal blood vessel images similar to those obtained previously with other methods, such as scanning electron microscopy (Ninomiya and Inomata, 2006). In micro-CT images, the inferior branched blood vessels look poorly developed in Mitfmi-bw homozygous melanocyte-deficient mice (Fig. 2E). Furthermore, the choroids of Mitfmi-bw/Mitfmi-bw mice had a reduced number of branches of the collected venules (Fig. 4B, black arrowheads). Our observations suggest that choroidal melanocytes contribute to underpinning the morphogenesis and/or maintenance of the normal vasculature structure of the choroid.

The blood vessels in the choroid supply the outer retina segment with oxygen and metabolites. Choroidal blood flow, which is as great as in any other organ, may also cool the retina (Parver et al., 1983). Changes in the vascular structure due to melanocyte deficiency might indirectly affect the physiological function of choroidal vessels. If this is the case, it is vital that melanocytes developmentally migrate and localize in this area.

Concerning the effects of melanin pigments on the structure of choroidal blood vessels, we could not observe any thinner or flattened blood vessels in C57BL/6-Tyrc-2J/Tyrc-2J homozygous albino mice (data not shown). Interestingly, this albino strain showed somewhat larger-diameter blood vessels than wild-type mice. In recent reports, less pigmented melanocytes, such as those in albino mice, express high levels of fibromodulin (an extracellular matrix protein) and monocyte chemotactic protein-1, factors that promote an angiogenic microenvironment (Adini et al., 2014, 2015). These angiogenic factors may, in part, explain our preliminary observation of larger-diameter choroidal blood vessels in C57BL/6-Tyrc-2J/Tyrc-2J albino mice. In any case, the presence (albino mice do have melanocytes) or absence of melanocytes (this study, melanocyte-deficient Mitfmi-bw/Mitfmi-bw mice) differently affects the three-dimensional choroidal structure.

It should be noted that, in the inner ear, cochlear melanocytes are essential for normal hearing acuity via maintenance of the endolymphatic potential at the scala media (Tachibana, 1999). It has also been reported that pigmentation is not essential for hearing ability. Nevertheless, we have suggested that melanogenesis is required to respond to stressful conditions, such as toxic conditions and intense noise exposure, because the mouse cochlear melanocytes specifically expressed glutathione S-transferase alpha 4 (Gsta4), in the stria vascularis, which encodes one of the cytosolic glutathione S-transferases playing an important role in the detoxification process (Uehara et al., 2009). Interestingly, neither follicular melanocytes nor choroidal melanocytes expressed Gsta4 (Uehara et al., 2009). Our study suggests that melanocytes differentiate to express tissue-specific function(s) depending on their habitats (microenvironments). In particular, choroidal melanocytes may contribute to visual function by supporting the normal vasculature structure, a role that may be independent of their melanogenic function.

It remains unknown whether the choroidal structural phenotype observed here in Mitfmi-bw homozygous mice, one of the melanocyte-deficient black-eyed white mouse mutants available at this locus, is caused solely by the lack of melanocyte development from the neural crest and/or the specific loss of the Mitf-M isoform (Yajima et al., 1999; Hozumi et al., 2012) among several Mitf isoforms expressed in wild-type mice. The Mitf-M isoform is indispensable for the development of neural crest-derived melanocytes (Yajima et al., 1999; Hozumi et al., 2012). Therefore, the loss of Mitf-M isoform expression and the impossibility of melanocyte development cannot be discussed separately here. Although there are other black-eyed white mouse phenotypes caused by a mutation at the W locus, such as KitW-v/KitW compound-heterozygous mice, the signal transduction pathway of Kit nevertheless overlaps that of Mitf. To clarify this issue, we may have to utilize white-spotting alleles of these loci to determine whether the number of melanocytes in the choroid correlates with the structural phenotypic severity of the tissue. Conditional KO mice that lose Mitf expression in the developed choroid will be also useful.

Finally, thinking about the localization of melanocytes in microenvironments not exposed to the sun, such as the stria vascularis of the cochlea and the ductus arteriosus of the heart, unknown function(s) of melanocytes including their contribution to the morphogenesis and/or maintenance of the structure of tissues remain to be uncovered. Research on molecular mechanisms underlying a wide range of abilities expressed by melanocytes depending on their habitats should be continuously conducted. Comparing melanocyte gene expression profiles is one of the necessary experiments to be carried out. Such discoveries may also emerge from studies using other animal models and wild animals. These lines of research will inevitably elucidate the functional evolution of melanocytes.

ACKNOWLEDGMENTS

We thank Drs. Sei-ichi Ishiguro, Makoto Tamai, Hiroyuki Ide and Koji Tamura for helpful discussions and for allowing us to use their facilities at Tohoku University in the early stages of this study when H. Y. belonged to that university. We also thank Drs. Takahiro Kunisada and Hitomi Aoki at the Gifu University Graduate School of Medicine for supplying us with specimens prepared from their own unique transgenic mice at very early stages of this study. We are also grateful to Ms. Yukiko Yano and Ms. Yuki Iwabuchi for their preliminary research. Our study is indebted to Drs. Shintaro Nomura, Nobuo Nagai and the late Akitsugu Yamamoto at the Nagahama Institute of Bio-Science and Technology for their technical advice. This work was partly supported by a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan, to H. Y. (24650239), and by the NIG Collaborative Research Program (2010-B6, 2011-B6 and 2012-B5) to H. Y.

REFERENCES
 
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