Human ciliary epithelia in monolayer culture

Exp.

Eye Res. (1984)

Human KAZUYOSHI Department

38, 423-433

423

Ciliary KONDO,

Epithelia MIGUEL

of Ophthalmology Medicine,

(Received 6 July

in Monolayer

COCA-PRADOS

Culture

AND MARVIN

and Visual Science, Yale University New Haven, CT, U.S.A.

1983 and accepted 21 November

SEARS School of

1983, New York)

The ciliary epithelia of human (one to 12 months old) ciliary processes were isolated by trypsin and EDTA, cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 5% fetal calf serum and examined by phase and electron microscopy. The primary cultures were maintained for three to four months. Only a few non-pigmented epithelia adhered and none of them proliferated. After the first passage the culture seemed to consist of only the pigmented epithelia. Most cells were densely pigmented at first, then became less pigmented during successive proliferations. Half of the cells remained densely pigmented after the first subculture, another half remained less pigmented. The cells started to lose their pigment granules at four to six weeks in culture. After three months of culture, the cell sheets became entirely unpigmented. In thin section, most of the pigment granules in the cells at two weeks in culture were pre-melanosomes, and half of them were at the earliest stage of pre-melanosomes. Monolayer cells possessed basement membranes. At 14 weeks in culture, most cells established an apparent polarity, contained well-devetoped Golgi apparatus and rough endoplasmic reticulum and intermediate filaments, but no pigment granules. A bundle of intermediate filaments was found in the perinuclear cytoplasm. Multilayer cells presented a typical apex-to-apex and base-to-base configuration, and the extracellular material was detected only in the base-to-base intercellular spaces. Our culture system provided differentiated cells derived from the pigment epithelia of human ciliary processes. Key UN&; ciliary epithelium; tissue culture; ultrastructure; human eyes.

1. Introduction The structure of the ciliary processes is characterized by a double layer of epithelium, the non-pigmented epithelium (NPE) and the pigmented epithelium (PE). Numerous gap junctions exist between two layers of epithelium (Ohkuma and Nishiura, 1974; Reale and Spitznas, 1975; Raviola and Raviola, 1978; Ober and Rohen, 1979). In addition to this coupling mechanism, there are also separate functional characteristics to the separate layer of the NPE and the PE. From histochemical studies of ATPase (Kaye and Pappas, 1965), nucleoside phosphatase (Shiose and Sears, 1965), adenylate cyclase (Tsukahara and Maezawa, 1978; Mishima, Sears, Bausher and Gregory, 1982) and carbonic anhydrase (Hansson, 1968 ; Bhattacherjee, 1971) the NPE probably plays a more important role in fluid transport than the PE. Surprisingly little biochemistry is known (Shimizu, Riley and Cole, 1967) about the separate functions of the NPE and PE. The regional differences in the ciliary body as well as the double layer of the ciliary epithelia make it difficult to study their separate functions. Since they are inseparable in vivo, the establishment of a cell culture of either the NPE or PE in vitro may help. To this end, we have studied the behaviour of the cultured ciliary epithelia biochemically (Coca-Prados, Kondo and Sears, 1983) and cytologically (Kondo et al., 1983). In this paper we describe characteristics of the human PE cultured in monolayer. Please address correspondence and reprint Ophthalmology and Visual Science, 310 Cedar 0014.4835/84/040423

+ 11 $03.00/O

requests to: Marvin L. Sears, M.D., Department of Street, P.O. Box 3333, n’ew Haven, CT 06510, U.S.A. 0 1984 Academic

Press Inc. (London)

Limited

Fro. 1. Phase c,ontrast micrographs ~~f‘wlls in monolayer. (a) One week in (.ulturr. h few 1~41s y.\hihit densr pigmentation. most are less pigmented. (h) Three weeks in c,rllturr (third passage). Half of the wlls retain their pigmentation. (c) Ten weeks in culture (sixt.h pansa.ge). Monolayer wlls hecome large in size and irregular in shape. A few cellsstill contain t.he pigment pr:~nules. ((1) Fourteen wrrks in culture (eighth passage). The wll sheets are entirely unpipmented. x 150.

2. Material Cell

and Methods

culture

Human eyes from post-mortem (one to 12 months old, Clonnecticut Eye Bank, Inc.) were used. Eye balls were cut at the equator, lenses and vitreous were removed gently from the anterior half. Approximately 50 tips of diary processes were cut off from each eye in sterile conditions. Ciliary epithelia were dissociated by incubation in a shaking incubator at, 37 “C. The dissociation medium contained 0.05 :b trypsin (Gibco) and 0.02 “I0 EDTA (Gibco) in 3 ml of Ca*+-, Mgz+-free Hank’s BSS. After a 15-60 min incubation, isolated cells were harvested with gentle pipetting and were washed twice in PBS by mild centrifugation. They were incubated in 5 ml of Dulbecco’s Modified Eagle Medium (DMEM) containing n-valine in place of r,-valine and 5’-‘/, fetal bovine serum and 025 mg of gentamycin in a 25 cm2 tissue culture flask. Previous experiments have excluded the possibility that n-valine is toxic to these cells. The cultures were maintained in a humidified atmosphere of 5 O/’ CO, and 95 9~; air at 37°C’. The DMEM and FBS were received from Gibco and the DMEM contained 4.5 g/l-’ and no sodium pyruvate. Costar Polystyrene flasks were used.

Histology Living cultures were observed and photographed under phase contrast microscopy. The monolayer cells grown in polystyrene tissue culture flasks were fixed by 2 o/0 glutaraldehyde in 91 M Millonig’s phosphate buffer for 1 hr at room temperature, postfixed in 1 “; 0~0, for 30 min, and prestained with 2 so aqueous uranyl acetate for 30 min. After fixation and dehydration. cells WCPP embedded in Epon 812 (Kondo, (loca-Prados and Sears. 1983).

MONOLAYER


OF

CILIARY

EPITHELIA

425

FIG. 2. Two weeks in culture. Multilayer ceils contain nume~‘ous pigment granules, scattered rough endoplssmic reticulum and mitochondria. Most of the pigment granules are pre-melanosomes. Asterisks indicate the Stage I pre-melanosomes (Witkop et al.. 1978). The overlying cells present no polarity. The>possess microvilli on both the top and bottom. x 5700. FIG. 3. Two week8 in culture. Gap junctions (arrows) are present between the lower and the upper cells. The lower cell possesses a basement membrane (arrowhead). x lOOO@ Fro. 1. Two weeks in tzult.ure. An apparent polarity is present. (!ellu exhibit a few microvilli at the apex, a basement membrane on the bottom and a series of jumtions at their apical portion (arrows). Various stages of pre-melanosomes and a few mature melanoaomes can he seen in the cytoplasm. An asterisk shows the earliest stage of pre-melanosomes. x 7000. Fm. 5. High magnification of the boxed area in Fig. 4. Arrowheads indicate a basement membrane of the monolayer cells. x 18ooO.

FIG. 6. Six weeks in culture. Stacks of rough endoplasmic reticulum (rER) are seen. Pigment granules are absent. x 10000. FIG. 7. Twelve weeks in culture. Well-developed Golgi apparatus (G) and scattered rough endoplasmic reticulum are present. No pigment granules can be seen. x 6000. Inset: High magnification of the boxed area. Microtubules (arrowheads) and intermediate filaments are present in the perinuclear region. x 17000.

Polymerized Epon was removed from the plastic flask by gentle bending and areas were marked by a diamond pencil under phase contrast microscopy and cut out. Thin se&ions were

stained

with

lead

citrate,

and

were

examined

in a Hitachi

H-600

electron

microscope.

3. Results Isolated cells were spherical and showed a few microvilli. A cell suspension consisted of the NPE and the PE (approximately 1 : 1). At 24 hr of culture more than half of the cells attached to the plastic substrate. The majority of attached cells were the PE and most of them remained spherical at this stage. During the first few days of culture. many PE elongated, flattened and proliferated on the substrate. They were pigmented at first, then monolayer cells became less pigmented during successive proliferations. Free pigment granules were seen in the medium. Epithelial sheets consisted of many lightly pigmented cells and a few densely pigmented cells at one week in culture [Fig. i(a)]. Monolayer cells varied in shape according to cell density, elliposidal or cuboidal shape in crowded area and polygonal shape in less crowded area. Cell sheets usually reached confluency at seven to 10 days. Subculture was carried out every one to two

MONOLAYER

(‘VLTURE

OF

CILIARY

427

EPITHELIA

FIG. 8. Fourteen weeks in culture. A monolayer cell contains no pigment granules. A bundle of intermediate filaments is found in the perinuclear region. One end of the bundle terminates at the nuclear membrane. x 12OOQ. Fro. 9. A part of periphery of the ~11 in Fig. 5. No pigment granules can be seen. Bundles of thin filaments lie in just below the plasma membrane. x 8000.

weeks. When subculture was not done for more than two weeks, growing cells were crowded and overlapped. During several days after the first transfer, growing cells became cuboidal in shape and half of them remained densely pigmented [Fig. l(b)]. They remained pigmented for a few weeks and gradually lost their pigment granules thereafter. After several weeks in culture the cells became larger in size and irregular in shape [Fig. l(c)]. Finally, the cell sheets reached entirely unpigmented after three months of culture [Fig. i(d)]. In thin sections, most, pigment granules in the cells of two-week-old cultures were identified as pre-melanosomes (Figs 2, 3 and 4). Melanosomes were less than 10 oh of the whole pigment granules. On the other hand, the earliest stage of pre-melanosomes (Stage I; Witkop, Quevedo and Fitzpatrick, 1978) occupied as much as half of them. The cells contained scattered rough endoplasmic reticulum, mitochondria and lipoid material. Small numbers of gap junctions were observed between the upper and the lower cells (Fig. 3). The monolayer cells represented an apparent polarity. They possessed a basement membrane on their bottom and the microvilli at their top (Figs 4 and 5). A series of junctions could be seen between the monolayer cells at their apical portion (Fig. 4). In place of multilayers, however, the overlying cells presented no polarity. They possessed the microvilli at both the top and the bottom (Fig. 2). Most of the monolayer cells at six and 12 weeks in culture contained no pigment granules (Figs 6 and 7). They presented stacks of rough endoplasmic reticulum (Fig. 6) and 16

EER 38

Fm. 10. Fourteen weeks in culture. A part of a multilayered area. A cell contains well-developed Golgi apparatus (G) and swollen rough endoplasmic reticulum (rER). Intermediate filaments are found in the cytoplasm (arrowheads). The disposition of multilayer cells is characterized by the apex-to-apex (a) and base-to-base (b) configuration. Extracellular material is present only in the base-to-base intercellular spaces. These cells exhibit few cell-to-cell contacts. x 14000.

well-developed Golgi apparatus (Fig. 7). They were also rich in the microtubules and the intermediate filaments (70-100 A) in the perinuclear region (Fig. 7). After three months of culture, neither monoloayer nor multilayer cells retained their pigment granules (Figs 8,9 and 10). Monolayer cells contained a bundle of intermediate cytoplasm, which terminated at the nuclear filaments (7ClOO A) in the perinuclear membrane (Fig. 8). In the periphery of monolayer cells bundles of thin filaments lay at both the basal and the apical cytoplasm in parallel (Fig. 9). In general, the basal webs were thicker than the apical webs. Multilayer cells also contained the intermediate filaments and the basal and apical webs of thin filaments. They represented a typical apex-to-apex and base-to-base configuration. Small amounts of extracellular material were found in the base-to-base intercellular spaces, whereas they were never seen in the apex-to-apex intercellular spaces. Some ceils contained well-developed Golgi

MONOLAYER

FIG 11. Fourteen x 8400. FTC: 12. A higher

apparatus

cell-to-cell junctions,

weeks

(‘I’L’I‘I’KE

in cultuw

magnification

OF

(‘ILIARY

A gap jundion

(arrow)

view of the gap junrt,ion

F:PI’I’HELIA

is found

in Fig.

b+wxw

the multilayer

cells.

I I. x 8fiOOO.

and swollen rough endoplasmic reticulum. As a whole, there were poor contacts between the upper and the lower cells (Fig. 10). Some gap however, had developed between them (Figs 11 and 12). 4. Discussion

Ln our culture conditions. isolated PE adhered easily to the plastic substrate, proliferated rapidly and retained their pigment granules for as long as two months in culture. On the other hand, isolated NPE rarely adhered to the plastic substrata and any adherent NPE tended to remain spherical in shape. When most PE were removed selectively by a density gradient, a few NPE attached and flattened on the substrate within four to seven days. However, they never proliferated. They detached from the substrate thereafter (unpubl.). In addition, no fibroblast-like cells were seen. Fibroblasts were unable to grow in the medium containing u-valine instead of L-valine (Gilbert and Migeon, 1975). These observations and the observation that cells contained pigment granules indicated that the cells after the first subculture were derived from the PE. When the PE flattened on the substrata at first, the cells were densely pigmented. They became less pigmented during successive proliferations. After the first transfer. half of the cells remained densely pigmented. These cells started to lose their pigment 16.2

430

Ii.

KOiYl)O

P’I’ 1

AI 1

granules at several weeks in culture. It is generally observed in retinal pigment epithelium (RPE) culture of human tissues (Albert, Tso and Rabson. 1972; Mannagh. Arpa and Irvin, 1973), chick embryos (Whittaker, 1963; Rodesrh. 1973) and pig (Franpois, Leuven. Verschraegen-Spae and Netto, 197 1) that the cells tend to lose their pigment granules as tissue culture progressed. We also found in cultured explants of ciliary processes that proliferated PE showed less pigment (Kondo et al.. 19X3). Interestingly, most pigment granules in the growing cells at two weeks in culture were determined to be pre-melanosomes, and half of them were identified as being at the earliest stage (Stage 1; Witkop et al., 1978). This finding agrees with the observation that the ‘pigment granules’ became less pigmented with culture (Mannagh et al.. 1973). In addition, the increase of the ratio of the pre-melanosomes to mature melanosomes was reported in the RPE of human albinism (Fulton, Albert and Craft, 1979) and PTU (phenylthiourea)-treated animals (Eppig, 1970: Mishima, Hasebe, Kondo and Fujita, 1979) in vivo. From these observations and the presence of the pigment granules in the culture medium, it is obvious that the growing cells are likely to release their pigments into the medium on the one hand, yet they try to produce pigment granules on the other. A large proportion of the earliest stage of pre-melanosomes indicates that the growing cells fail to synthesize melanin, possibly caused by limited nutrition, the presence of an inhibitor of pigmentation (Cahn and Cahn, 1966). or scarce cell-to-cell contacts. It is well known in the RPE culture that the pigmentation varies according to culture medium (Cahn and Cahn, 1966; Newsorne, Fletcher, Robison, Kenyon and Chader. 1974) and cell density (Crawford, Cloney and Cahn, 1972; Rodesch, 1973; Crawford, 1979). It is very likely that the decrease in the production of melanin in cultured cells is due to either a decrease in tyrosinase activity or a cessation of tyrosinase synthesis, as suggested in the literature (Whittaker. 1967). than to any ‘melanotoxic’ constituent in the media. The problem of melanogenesis has been more directly addressed in the earlier works cited. Monolayer cells at two weeks in culture possessed a basement membrane (Figs 4 and 5). Some studies presented a proof of basement membrane formation by the epithelial cells cultivated on the frozen-killed lens; cornea1 epithelia (Dodson and Hay, 1971; Hay and Godson, 1973) and neuroepithelia (Cohen and Hay, 1971). Osman and Ruth (1980) reported that the molar epithelia produced a basement membrane only when they grew on the collagenous substrate, and there was no evidence of the basement membrane formation on a plastic culture dish or a Millipore filter. On the other hand, the RPE (Newsome and Kenyon, 1973) and salivary epithelia (Banerjee, Cohn and Bernfield, 1977) cultivated on a plastic substrate, produced a basement membrane. Our observations indicate that the ciliary PE grown on a plastic substrate can produce a basement membrane. In an area of multilayers at three months in culture, the cells formed an apex-to-apex and base-to-base configuration. Small amounts of extracellular tnaterial could be seen only in the base-to-base intercellular spaces (Fig. 10). This observation is similar to the multilayers of the RPE culture (Newsome and Kenyon, 1973). Cultured cells may establish their polarity in response to extracellular material (Hall, Parson and Bissell, 1982). Most cultured cells contain intermediate filaments (Franke, Schmid. Winter, Osborn and Weber, 1979). They are frequently found in the perinuclear region (Haudenschild, Cotran, Gimbrone and Folkman, 1975; Blose and Chacko, 1976; Lazarides, 1980), and may have an important role in the mechanical integration of various cytoplasmic cells at three months in culture organelles (Lazarides, 1980). b‘ome monolayer contained a perinuclear bundle of intermediate filaments (Fig. 8). These filaments were also found in the cytoplasm of the multilayer cells (Fig. 10). The presence of

MONOLAYER

CULTURE

OF

CILIARY

EPITHELIA

43 1

well-developed Golgi apparatus and swollen rough endoplasmic reticulum in t’he cytoplasm of multilayer cells (Fig. 10) suggests that these cells are well differentiated and are in an active state. In general, development of these organelles in the human PE at the crest of the ciliary process is less than the NPE (Hara, Lutjen-Drecoll, Prestele and Rohen, 1977). Although it is unclear whether these cells simply retain their original functions or acquire a new functional property, they may be able to synthesize the intermediate filaments as well as the basement membrane material. The cells showed few cell-to-cell contacts even at a confluent stage. Several gap junctions. however, were detected mainly between the cytoplasmic projections of the upper and the lower cells in the multilayers (Figs 3, 11 and 12). It is well known that a relatively small number of gap junctions develop between the PE of human (Reale and Spitznns. 1975) and monkey and rabbit (Raviola and Raviola, 1978; Townes-Anderson and Raviola, 1981) ciliary processes in vivo. Various types of cells grown in culture develop gap junctions (RPE: Crawford et al., 1972; glial cells: Janka. Latzkovits. Joo and Szentistvanyi, 1979 ; vascular endothelia : Larson and Sheridan, 1982). Only the PE could proliferate and be maintained for longer than four months in our culture system. Most NPE failed to attach to the plastic substrate. The reason for failure of NPE growth in vitro is not yet explained. First, the NPE show no contact with the extracellular matrix except for the zonular apparatus in vivo and for this reason may not attach to the substrate. Second, the NPE ordinarily exhibit an elaborate system of basal and lateral plasma membranes such as basal infoldings and interdigitations. On the other hand. the apical plasma membrane is rather flat. As a result of these constructions, as well as the zonula occludens among the BPE, the greater part of the NPE plasma membrane is in contact with the aqueous humor, and only a small part of the membrane is in touch with ‘serum’ in vivo. Perhaps a conditioned medium which contains some growth factors and either a lower concentration of serum or none at all should be adjusted for promoting NPE growth in cult,ure (Coca-Prados et al., in prep.). Finally, it is obvious that the NPE is homologous to the sensory layers of the ret,ina and we are not aware of any successful attempts to culture these. We have been unahlr to reproduce the results of Runyan, McCombs, Holton, McCoy and Morgan (1983) who showed proliferation of NPE in culture. Since they did not report on standardized controls for possible repigmentation of PE, it is possible chat their cultures were cont,aminated with PE. They do not report any pre-melanosomes or their precursors in their cultures. No attempts to re-establish pigmentation by recognized techniques such as use of conditioned media or peritoneal washes were done by them. The possibility that the cells reported in the paper of Runyan et al. (1983) were cont’aminated with depigmented PE needs to he considered in evaluation of their work. AC’KNOWLEDGMENTS

Gail Chatt was responsible for thr maintenance of’ the tissue culture laboratory in t,hese studies. This work is supported in part by U.S. Public Health Service grants EY 00237. EY 00785. EY 04328. Research to Prevent Blindness. Inc.. Connecticut Lions Eye Research Foundation. Inc. and The New Haven Foundation. REFFREi\‘(:ES 1 Albert, D. M., Tso, M. 0. M. and Rabson, A. S. (1972). In vitro growth of’ pure culturr of retinal pigment epithelium. Arch. Ophthalmol. 88, 63-9. Banrrjer. S. D., Cohn. R. H. and Bernfield. M. R. (1977). Basal lamina ofembryonic salivary

43”

ti

liONI)

ET

.\I,.

epithelia. Production by the eptthelium and role in maintaining lobular morphology. ./. C!ell Biol. 73. 445-63. Bhattacherjee, P. (1971). Distribution ofcarbonic anhydrase in the rabbit eye asdemonstrated histochemically. h’xp. Eye Res. 12, 356-9. Blase. S. H. and Clhacko. 8. (1976). Rings of intermediate (100 A) filament. bundles in the perinuclear region of vascular rndothelial ~~11s. Their mobilization by colcemid and mitosis. J. (‘ell Riol. 70, 459-66. (:ahn, K. D. and (.‘ahn, M. B. (1966). Heritability of cellular differentiation : clonal growth and expression of differentiation in retinal pigment cells in vitro. Proc. Natl Acad. Sri. I’.S.A. 55. 10614. Coca-Prados. M.. Kondo. R. and Sears. M. I,. (1983). Protein phosphorylation in cultured human ciliary epithelia in response to activators of adenylate cyclase, cyclic AMP and analogues. In GZwucoma lipdate II (Eds Krieglstein, G. K. and Leydhecker, H. W.) Pp. 1-6. International Glaucoma Symposium, Carmel. (‘A ,22-27 September 1982. SpringerVerlag, Heidelberg. Cohen, A. M. and Hay. E. D. (1971). Secretion of collagen by embryonic neuroepithelium at the time of spinal cord-somite interaction. Oev. B&Z. 26. 578-605. (‘rawford. B. (1979). Cloned pigmented retinal epithelium. The role of mirrofilaments in the differentiation of cell shape. J. Cell Biol. 81, 301-15. (lrawford, B.. Cloney, R. A. and (!ahn, R. D. (1972). Cloned pigmented retinal cells; the effect of cytochalasin B on ultrastructure and behavior. Z. Zelljorsch. 130. 135-51. Dodson, J. W. andHay, E. D. (1971). S of collagenous stroma by isolated epithelium L eeretion grown in vitro. .Exp. (‘ell Res. 65. 215-60. &pig, Jr.. J. J. (1970). Melanogenesis in amphibians. I. A study of the fine structure of the normal and phenylthiourea-treated pigmented epithelium in Ranu pipienv tadpole eyes. Z. Zelljorsch. 103, 23&46. Franqois, J.. Leuven, M. Th. M.. Verschraegen-Spae, ,&I. R. and Netto, J. A. (1971). Tissue culture study of the retinal pigment epithelium of the adult pig. Ophthalmic Res. 2, 8f.G-95. Franke. W. W., Schmid, IX.: Winter, S., &born. M. and Weber, K. (1979). Widespread occurrence of intermediate-sized filaments of the vimentin-type in cultured cells from diverse vertebrates. Exp. Cell RPR. 123. 2536. Fulton, A. B., albert, I>. M. and Craft. *J. L. (1979). Retinal pigment epithelium and photoreceptors in human albinism. In Pigment Cell. Vol. 5 (Ed. Klaus, S. N.). fathophysiology of Melanacytes. Pp. 915. S. Karger, Basel. Gilbert, S. F. and Migeon, B. R. (1975). n-valine as a selective agent. for normal human and rodent epithelial cell in culture. Cell 5. 1 l-17. Hall. H. G., Farson, D. A. and Bissell. M. ,J. (1982). Lumen formation by epithelial cell lines in response to collagen overlay: a morphogenetic model in culture. Proc. Nutl Acarl. Ski. I:.S.A. 79. 4672-6. Hansson. H. P. J. (1968). Histochemical demonstration of carbonic anhydrase activity in some epithelia noted for active transport. Acta Physiol. Scund. 73, 427-34. Hara. K.. Lutjen-Drecoll. E., Prestele, H. and Rohen, J. W. (1977). Structural differences between regions of the ciliary body in primates. Invest. Ophthulmol. Via. Sci. 16, 912-24. Haudenschild. t’. (1,. Cotran. R.S.. C&mbrone, Jr.. M. A. and Folkman. J. (1975). Fine struct’ure of vascular endothelium in culture. J. r’Ztrasfruct. Res. 50, 22-32. Hay, E. D. and Dodson. .I. W. (1973). Secretion of rollagen by eorneal epithelium. I. Morphology of the collagenous products produced by isolated epithelia grown on frozen-killed lens. J. C’& Biol. 57. 19&213. Janka. Z.. Latzkovits. I,.. Joo. P. and Szentistvanyi, I. (1979). GUI-to-cell contacts in primar.v cultures of dissociated chicken embryonic brain. Cell Tissue Reu. 199, 153-7. Kaye. ($. I. and Pappas. f$. D. (1965). Studies on the ciliarv epithelium and zonule. III. The line structure of the rabbit ciliary epithelium in relation to the localization of ATPase activity. J. ,lilicrosco$e 4. 197-508. Kondo. K.. Cloca-Prados. M. and Sears. M. L. (1983). (‘iliary epithelia of the mammalian eye in cultured explant. (‘~11 Tissue Rex. 233, 629-38. I,arson. 1). M. and Sheridan. J. D. (1982). intercellular junctions and transfer of small molecules in primary vascular rndothelial cultures. J. Cell BioZ. 92, 183-91.

MOKOLAYER~ Lazarides,

K&m

E. (1980).

lntrrmediate

(‘C’LTIlRE filaments

OF

(‘I1,IAK.I

as mechanical

EI’lTHE:LIA integrators

1.13 of cellular

space.

283, 249-56.

Mannagh, J., Arya. D. V. and Irvin. Jr.. A. R. (1973). ‘I&W culture of human retinal pigment epithelium. Imwst. OphthaZwwl. Vis. 8ci. 12, 5%64. Mishima, H.. Haseehe. H., Kondo. K. and Fujita. H. (1979). Fine st’ructural demonstration oftyrosinase activity in the retinal pigment epithelium of normal and PTtT-treated chick embryos. Alhrech,t eon &aefps .4rch. Klin. h’xp. Ophthalmol. 211, l-10. Mishima. H.. Sears. M. I,.. Hausher. L. I’. and Gregory. 1). S. ( 1982). I’ltracytochrnlistr~ ot &okra-toxin binding sites in ciliary processes. (‘r/l Tissccr Kes. 223. U--53. Nrwsomr, I). A.. Fletcher. R. T., Hobison. *Jr., \t’. (i.. Krnyon. K. R. and (Ihader. (:. J (1!)74). Effects of c>c*lic> AMP and sephadex fractions of c.hick rtnbryo extract on cloned retinal pigmented epithrliurn in t.issur c~lturr. ,I. (‘6~11 Hill. 61. Xi$N~. INrwsome, I). A. and Krnyon, K. R. (1973). (‘ollagen production in vitro by the retinal pigmented rpithelium of the chick embryo. L)rr. Hid. 32. 387&kOO. Ohrr. M. and Rohen. .J. w’. (1979). Regional tlif%rrncrs in the fine structure of t,he ciliary rf)it.helium related to accommodation. Lrrwst. Ophthnlmol. l’is. SSci. 18. 655-64. Ohkutua, M. and Il’ishiura, M. (197-L). Intercellular ,junc+.lons of the ciliary epithelium as revealed by freeze-fracture and lunt hanum travel tc~chniqur. .1cftr A’oc. Ophfhnlrnol. Jpn 78. 141!1-30. &man. Xl. and Ruth. *J.-V. (1980). Secretion of basal larnina by trypsin-isolated embryonic mouse molar epithelia cultured in vitro. Dr13. Hiol. 75. 467~-70. Raviola. (4 and Raviola. E. (1978). Int~ercrllular ,junc.tions in t hr ciliary epithrliurn. I,(w.sL.

~~~,htha~md. I’is. A%. 17. 958 -81, Rrale.

E. and Spitznas. M. (1975). Freeze-fract.ure analysis of junctional complexes in human ciliary rpithrlia. ;Ilbwchf wu /kwfrs Arch. Klin. Krp. O~phih.nlmoZ. 195, l-16. Kodrsc+. F. (1973). Differentiation. contact inhibition and int~ercrllular communic.at,ion in retinal pigment, cells. EzI),. (‘rll Krs. 76. 55XQ. Runyan. 1‘. E., Mc(‘ombs. W. 13. 111. Holton. 0 I)., klc(‘oy. (‘. E. and Morgan, M. P. (1983). Human (iliary body rpitheliurn in cult.ure. I~cc~st. O)ph.thnZnwl. l’is. Sci. 24, 687-96. Shimizu. H.. Riley. M. V. and C’ole. 1). F. (1967). The isolation of whole cells from the ciliary q~ithrlium together with some observations on the metabolism of t,he two cell t,ypes. Eq. IC!y:!/rHPS. 6. 14 1 -5 1. Shione. Y. and Sears. M. I,. (1965). Localization and other aspects of the histochrmistry of nuc~lrotidr phosphatasrs in t,he ciliary rpithrlium of albino rabbit. Lnwst. O~~hthulrrtol. 4. Cl-75. Townes-,~rldersorr. E. and Raviola. G. (1981). The format,ion and distribution of intercellular junct.icms in thr rhesus monkey optic cup: thr early dev&~pmrnt of the cili&ridic. and sensory retinas. DIZV. Biol. 85. ~0~~. Tsukahara. S. and Maezawa, R’. (1978). (‘gtochemical localization of adenyl cyclasr in the rabbit ciliary body. h’xp. Eye Rrs. 26, 9!&106. Whittaker. J. R. (1963). Changes in melanogenesis during the dedifferrntiation of chick retinal pigment cells in crll cult&s. I)Pv. Riol. 8. 99-127. Whitt’akrr. .I. R. (1967). Loss of melanotic phenotype in vitro by difYerentiatrd retinal pigment cells : demonstration of mechanisms involved. &I, Hioi. 15. 553-71. Witkop. Jr.. (‘. J.. QUPVP~O. Jr.. W. (‘. and Fitzpatrick. T. R. (197X). Alhinism. In 7% Mrtr~bolic Basis of Inherited IIisPclse (4th edn.) (&Is St,anbury. *I. 13.. Wyngaarden. .J. 12.. Fredrickson. I). S.). Pp. X--3iti. McGraw-Hill. Nrw \‘ork.