Melanocytes contribute to the vasculature of the choroid

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 determines 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, leptomeninges, etc (Plonka et al., 2009).
One of intriguing questions is whether those melanocytes that localize in different tissues and organs play the same rolls.For example, it is well known that melanocytes in the skin produce melanin that provides protection from damage by ultraviolet radiation.Then, what are these extracutaneous melanocytes doing in such sun-protected habitats, still producing melanin?Here we focus on melanocytes in the eye to observe the structural significance contributed by them.Melanocytes in the eye are localized in the 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 nutriments 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 vessels in the retina (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 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, Mitf mi-bw , to focus on the structures of their habitats without melanocytes.Mitf mi-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 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, Mitf mi-bw homozygous mice lack mature melanocytes all over 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 Mitf mi-bw homozygous melanocyte-deficient mice show a much thinner choroidal layer and an abnormal choroidal vasculature formation suggesting that choroidal melanocytes contribute to support the normal vasculature structure.Our structural observations provide a helpful cue to infer the functional evolution of melanocytes scattered throughout the body.

Mice
The mutant mouse strain carrying the Mitf mi-bw allele (ID: MGI:1856089) was obtained as described previously (Yajima et al., 1999).Mitf mi-bw mice were maintained on a C57BL/6N background by backcrossing them more than 6 times with C57BL/6NJcl mice (purchased from CLEA Japan, Inc, Tokyo).C57BL/6N-Mitf mi-bw /Mitf mi-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 experiments were carried out at the Nagahama Institute of Bioscience and Technology and were performed in accordance with the guidelines outlined in the Guidelines of Animal Experimentation, Nagahama Institute of Bioscience and Technology, Japan.All experiments were carried out using 4-week-old mice unless otherwise noted.
Paraffin section and hematoxylin-eosin (HE) stain Mice were sacrificed by decapitation, after which their eyes were removed and scratched on the cornea, then fixed with 2.5% glutaraldehyde in phosphate buffered saline (PBS, pH 7.3) for 2 hours 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.Those eyecups were then dehydrated, embedded in paraffin (Sakura, Tokyo, Japan), and sectioned at 7 m 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 Mitf mi-bw /Mitf mi-bw (N=5) mice were 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 mentioned above and were fixed with 4% paraformaldehyde in PBS (pH 7.3) for 2 hours at room temperature.All specimens were then washed with PBS, bleached with 3% H2O2 diluted with 0.05 M phosphate buffer (PB, pH 7.4) for 2 hours at 55°C, dehydrated, treated with embedding medium (20% sucrose, 33% OCT compound (Sakura, Tokyo, Japan) in PBS) overnight at 4°C, and then embedded in the same medium and frozen at −80°C.
Sections were mounted in VECTASHIELD (Vector Laboratories, Burlingame, USA) and photographed using a BX51fluorescence microscope (Olympus).
Three-dimensional vascular structural analysis of blood vessels in the choroid Microfil (Flow Tech, Inc., Carver, MA, USA) is a radio-opaque silicone rubber containing lead chromate and lead sulfate particles.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 followed by perfusion with 10% formalin in PBS using a P-1 peristaltic pump (GE Healthcare UK Ltd, Amersham, England) with a flow rate of 2.5 ml/min.PBS and 10% formalin were prewarmed at 37°C.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 Co., Ltd., Kanagawa, Japan).Some eyeballs were treated with 3% H2O2 diluted with 0.05 M PB (pH 7.4) for 2 hours in order to bleach the melanin pigments to allow cells and tissues covered with pigmented tissues (layers) to be observed.Bleached eyeballs were washed two times 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, VT, USA) perfusion system was applied in a similar way, but was partly modified as that used in the Microil perfusion system.In brief, Mercox (20 ml) of blue color and the catalyst (0.15 g) were mixed just before the injection to each mouse.After removing the blood and fixative, mice were perfused with Mercox at flow rate of 2 ml/min.To polymerize completely, mice were warmed up and incubated at 50°C for about 12 hours.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, spattered with 15 mA containing with tungsten for 15 sec using an E-1045 Ion sputter (Hitachi, Tokyo, Japan) and examined with a S-3400 scanning electron microscope (Hitachi) at an accelerating voltage of 1.5 kV and an emission current of 37 A.

Histological analysis of the 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 the choroidal tissue structure between 4-week-old wild-type mice and Mitf mi-bw /Mitf mi-bw mice.Wild-type mice have black hairs and eyes (Fig. 1A).On the other hand, Mitf mi-bw /Mitf mi-bw melanocytedeficient 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, Mitf mi-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 Mitf mi-bw /Mitf mi-bw mice.The cut planes of choroidal blood vessels of mutant mice were flattened out (Fig. 1F).
To identify histochemical changes that could account for the observed structural abnormalities in Mitf mi-bw homozygous mutant mice, we carried out an immunohistochemical study to observe histologic 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 Mitf mi-bw mutant mice is much thinner than the choroid of wild-type mice and multiple layers of blood vessels are not developed (Fig. 1H).As mentioned above for the usual HE-stained specimens, many of the cut planes of the blood vessels of mutant mice are much thinner and flattened out (Fig. 1G, 1H).The choroidal thickness of wild-type mice measured was significantly thicker than that of Mitf mi-bw /Mitf mi-bw mice (Fig. 1I and1 J).

Three-dimensional vascular structural analysis using micro-CT
To detect the influence of melanocyte defects that might not be obvious by 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 Mitf mi-bw /Mitf mi-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 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 Mitf mi-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 mice show their vessels are overlapped as if each of them were crushed in the vortex vein (Fig. 2C).Furthermore, inferior branch blood vessels are not well developed in Mitf mi-bw homozygous melanocyte-deficient mice (Fig. 2E, magenta arrow).In the temporal view, irregularities at the level of the collected venules are seen in melanocytedeficient Mitf mi-bw homozygous mice that were absent in wild-type animals (Fig. 2F, 2G, white arrowheads).

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 our naked eye using a binocular microscope.Therefore, to further understand the morphological changes of the choroid in Mitf mi-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 Mitf mi-bw homozygous mice are easily observed because there are no melanocytes in the choroid (Fig. 3B).The black background of homozygous Mitf mi-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 Mitf mi-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 Mitf mi-bw homozygous melanocyte-deficient mice that were absent in wild-type animals (Fig. 3F).

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 Mitf mi-bw / Mitf mi-bw mice, we prepared vascular corrosion casts of Mitf mi-bw / Mitf mi-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 Mitf mi-bw homozygous mice (Fig. 4A, 4B).Although wild-type animals exhibited a regular network of collected venules (Fig. 4A, black arrowheads), the choroid of Mitf mi-bw / Mitf mi-bw mice reduced the number of branches of the collected venules (Fig. 4B black arrowheads).
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-Tyr c-2J / Tyr c-2J homozygous albino mice (data not shown).Interestingly, this albino strain showed somewhat larger blood vessels in diameters than wild-type mice.In recent reports, less pigmented melanocytes, such as those in albino mice, express high levels of fibromodulin (an extra cellular matrix protein) and monocyte chemotactic protein-1 (MCP-1), factors that promote an angiogenic microenvironment (Adini et al., 2014(Adini et al., , 2015)).Those angiogenic factors may, in part, explain our preliminary observation of larger choroidal blood vessels in diameters in C57BL/6-Tyr c-2J / Tyr c-2J albino mice.In any case, the presence (albino mice do have melanocytes) or the absence of melanocytes (this study, melanocyte-deficient Mitf mi-bw / Mitf mi-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 malanocytes specifically expressed glutathione S-transferase alpha 4 (Gsta4) in the stria vascularis, which encodes one of the cytosolic glutathione S-transferases (GSTs) playing an important role in detoxification process (Uehara et al., 2009).Interestingly, neither follicular melanocytes nor choroidal melanocytes expressed Gsta4 (Uehara et al., 2009).Our study suggests that melanocytes may differentiate to express tissue-specific function(s) depending on their habitats (microenvironments).Especially, choroidal melanocytes may contribute to visual function by supporting the normal vasculature structure that may be independent of their melanogenic function.

It remains unknown whether the choroidal structural phenotype observed in
Mitf mi-bw homozygous mice in this study, only one of the melanocyte-deficient black-eyed white mouse mutants available in 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 can't be discussed separately here.Although there is another black-eyed white mouse phenotype caused by a mutation at the W locus, such as Kit W-v ∕ Kit W compound-heterozygous mice, still, the signal transduction pathway of Kit overlaps that of Mitf.To clarify this issue, we may have to utilize white-spotting alleles of these loci to observe 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, the ductus arteriosus of the heart, etc., the unknown function(s) of melanocytes including their contribution to the morphogenesis and/or maintenance of the structure of tissues are waiting to be uncovered.Research on molecular mechanisms underlying a wide range of abilities expressed by melanocytes depending on the habitats should be continuously conducted.
Comparing their gene expression profiles is one of necessary steps to be carried out.
Such discoveries may also emerge from research using other animal models and wild animals.These lines of research will inevitably elucidate the functional evolution of melanocytes.A wild-type mouse with a black coat and black eyes (A).A melanocyte-deficient Mitf mi-bw / Mitf mi-bw homozygous mouse with black eyes and a white coat (B).Wild-type eye with a normal RPE and a pigmented choroidal layer (C, enlarged in E).Mitf mi-bw / Mitf mi-bw eye with an apparently normal RPE but a thinner choroidal layer due to the deficiency of melanocytes (D, enlarged in F).PECAM-1 antigen localization in the choroid of wild-type (G) and melanocyte-deficient (H) mice.PECAM-1 in blood vessels is expressed in vascular endothelial cells and is used to visualize blood vessels.

Figure legends
Choroidal blood vessels of wild-type mice are restricted in the melanocyte abundant layer.The thickness of RPE and choroidal layers of wild-type and Mitf mi-bw homozygous melanocyte-deficient mice were measured (n=5) (I).The ratio of the thickness of the choroid to that of the RPE of wild-type and Mitf mi-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,).