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Collagen production in vitro by the retinal pigmented epithelium of the chick embryo
DEVELOPMENTAL
BIOLOGY
Collagen
32, 387-400
(1973)
Production
in Vitro by the Retinal
Epithelium Laboratory
of Vision Research, Development,
of the Chick Embryo
A. NEWSOME
DAVID
National National
Pigmented
AND KENNETH
Eye Institute; Institutes Accepted
R. KENYON
and National Institute of Child Health Bethesda, Maryland 20014
and
Human
of Health,
December
22, 1972
Cloned colonies and explants of embryonic chick retinal pigmented epithelium from donors of various embryonic ages were maintained in culture for different periods and examined by electron microscopy. The cells appeared morphologically differentiated and polarized. Basement-membrane material and striated collagen fibrils were identified as extracellular deposits beneath the basal surfaces of the cells. There appeared to be a distinct spatial and temporal correlation between the production of basement-membrane material and collagen fibrils. Increasing donor age correlated positively with increasing average diameter of the collagenous fibrils produced, as well as a widening of the range of fibril sizes. INTRODUCTION
Collagen production has been detected in extremely young embryos (Green et al., 1966). Recent studies have offered direct evidence that, at least in the developing chick, embryonic epithelia as well as connective tissue cells can synthesize (Goodfellow et al., 1969) and excrete (Trelstad, 1971; Trelstad and Coulombre, 1971; Dodson and Hay, 1971; Cohen and Hay, 1971) collagen. The retinal pigmented epithelium of the embryo of the domestic fowl, a tissue that participates significantly in the development of other tissues of the chick eye (Coulombre, 1965; Reinbold, 1968; Giroud, 1957; Newsome, 1972), also produces collagen (Coon, unpublished observation; Cahn et al., 1970). It is the purpose of this communication to present the first detailed study of the collagen-producing ability of embryonic chick retinal pigmented epithelium grown in vitro. MATERIALS
Embryos
AND
METHODS
of a White
Leghorn cross of all the pigmented epithelium (PE) used in this study. Embryonic donor age is expressed in either stages, as determined by the criteria of Hamburger and Hamilton (1951), or Gallus domesticus supplied
387 Copyright All rights
0 1973 bj Academic Press, Inc. of reproduction in any form reserved.
elapsed days of incubation at 38.0 & 0.5”C. Cultured PE from donors of 14 days of incubation or younger were cloned as follows: PE was dissected from the posterior poles of donor eyes in 0.25% trypsin (GIBCO), and dispersed by incubation for 20 min at 38°C in 0.1% trypsin and 0.02% EDTA in phosphate-buffered Ca2+ and Mg*+ free saline (Dulbecco and Vogt, 1954) at pH 7.8. Single PE cells were identified in this suspension by the presence of pigment. From 1 to 50 cells were pipetted into 60-mm plastic tissue culture dishes (Falcon) containing 3 ml of medium, and propagated by serial passage using standard techniques. Cloned material used in this study was from either the initial platings, which were examined and found free of non-PE cells, or, more usually, the first subcloning passage. Cultures from embryos older than 14 days consisted of explants of posterior pole PE prepared by dissection in 0.25% trypsin, brief washings in a saline G bath (Puck’s N-15 medium minus the amino acids; Puck et al., 1958), and direct placement in culture dishes. Sample explants were examined by electron microscopy and carried no collagenous or fibrillar material. All cultures were maintained at 38°C in a
388
DEVELOPMENTAL
BIOLOGY
5% CO, atmosphere, in Coon’s modification of Ham’s F-12 medium with 5% fetal calf serum and 0.3 pg of penicillin per milliliter (Coon, 1966). The medium was renewed every 3 days. Under these conditions the pH of the medium was 7.2. All fixation and embedding procedures for electron microscopy were performed directly in the tissue culture dishes without manipulation of the colonies in order to preserve spatial relationships. Cultures were rinsed briefly in three changes of saline G, fixed in 1% osmium tetroxide (Verona1 acetate buffered, pH 7.3, 215 milliosmolar) for 15 min at O”C, dehydrated through 5-min changes of graded alcohols at O”C, and embedded in Spurr epoxy resin (Polysciences) . Thin sections of representative PE colonies were cut on a Porter-Blum MT-2 ultramicrotome, care being taken to select central-paracentral areas from cloned colonies, and areas of new growth from explanted tissues. The sections were stained with uranyl acetate and lead citrate in the usual way, and were examined with a JEM 100-B electron microscope. Determinations of fibril diameter and macroperiodicity were made from high magnification electron micrographs (50,000 and 70,000 times) using a calibrated Filar ocular micrometer fitted to a stereomicroscope. For each donor age studied, the cross-sectional diameters of at least 280 fibrils with circular profiles were measured from randomly selected fields. Average fibril diameters were calculated, and frequency distributions were plotted from these measurements. OBSERVATIONS
Under our culture conditions PE cells formed colonies of morphologically welldifferentiated cells as viewed by phase contrast microscopy in the living state. The cultured cells assumed the polygonal array typical of retinal PE in viuo (Fig. 1). Colonies initially consisted of a monolayer of unpigmented cells. After about 10 days
VOLUME
32, 1973
in culture, colonies usually had three morphologically distinct zones: (1) a central area of polygonal, visibly pigmented cells with no (or extremely rare) mitotic figures seen on repeated observation; (2) a paracentral ring of rounded, apparently unpigmented cells, with a few mitotic figures seen in this area; and (3) a peripheral skirt of actively dividing cells with fibrocytic appearance and no visible pigmentation. By 6 weeks in culture, 85-90s of the cells in a typical colony were heavily pigmented, polygonal, and occasionally also binucleate. This sequence was applicable to all clones and explants studied. Cellular
Morphology
The formation of monolayers and sparse pigmentation of PE colonies after a brief time in culture were also evident by electron microscopy. As early as 4 days in vitro the cells exhibited a definite polarity, as junctional complexes had developed between lateral cell membranes at the apical aspects of adjacent cells (Fig. 2). These complexes consisted of cell membranes in close parallel apposition (-200 A gap) with electron-dense granular material aggregated along their cytoplasmic aspects. Tight junctions were never seen. Conventional desmosomal attachments were occasionally present between lateral cell membranes (Figs. 2 and 6). The apical cell membrane was elaborately infolded in numerous pseudopodlike configurations. During the initial days in culture, the plasma membrane limiting the basal surface of the PE cells was free of any extracellular material and was in direct contact with the surface of the culture dish. In rare instances, the readily identifiable apical surfaces of the PE cells lay in direct contact with the dish, demonstrating that the intrinsic polarity of the cells is seemingly independent of their spatial orientation. Basement
Membrane
Formation
After 4 days in vitro, basement membrane formation was first evidenced by the
NEWSOME
AND KENYON
Collagen
Production
389
FIG. 1. As this phase-contrast photomicrograph illustrates, cloned retinal PE cells from a stage 25 donor (5 weeks in vitro) have formed a polygonal array of heavily pigmented cells. Several binucleate cells (*) are evident. x 150.
appearance of short, discontinuous segments of fine granular or fibrillar material beneath the basal surface of the PE cells. By 14 days in culture, these segments had coalesced to form a continuous extracellular lamina of 200-400 A thickness that was separated from the basal plasma membrane by a 300-500 A wide electron-lucent zone. In general, basement membrane formation progressed more rapidly in cultured PE from younger donors, and was complete over the entire basal surface of the epithelial sheet by 3 weeks in vitro. PE explanted from a 21-day donor, in contrast, did not begin to deposit basement membrane until 3 weeks in culture, and required 6 weeks for its completion. Collagen Fibril Deposition
In addition
to basement
membrane,
ran-
domly oriented fibrils of 150-450 A diameter accumulated in quantity beneath the basal surfaces of the several cloned lines and explants of PE studied (Fig. 3). These fibrils exhibited the ultrastructural characteristics of collagen. Although longitudinal macroperiodicity was not clearly discernible in fibrils of small diameter, a definite macroperiod ranging from 550 to 600 A could be resolved in larger fibers (Fig. 3). There was a distinct spatial and temporal correlation between the occurrence of basement membrane and striated fibrils. In particular, PE monolayers without a basement membrane were also devoid of collagen fibrils. Further, prior to the completion of basement membrane formation, fibrils were present only in association with those areas of the basal cell surface subtended by basement-membrane material
~
I
FIG. 2. This electron micrograph of’ retinal PE f’rom a day 21 donor (4 weeks in vitro) shows a monolayer of cells whose polarity is indicated by well-developed apical junctional complexes (JC). The plasma membrane (PM) limiting the basal surface of the cell lies in direct contact with the tissue culture plate (P). Occasional vsomes (circled) are present between lateral cell membranes. Few melanin granules (*I are evident. N, .: M, mitochondrion. A 12,500.
NEWSOME
AND KENYON
Collagen
Production
391
FIG. 3. This electron micrograph of retinal PE from a day 13 donor (14 weeks in uitro) documents the accumulation of collagen fihrils extracellularly beneath the basal surface of the cells. These fibrils measure 220-250 A in diameter, with a longitudinal macroperiodicity of about 550 A (arrowheads). x 80,000.
392
DEVELOPMENTAL
BIOLOGY
(Figs. 4 and 5). None of our specimens showed collagen-containing basal vacuoles, As with basement membrane formation, collagen fibrils initially appeared after 4 days in culture for most donor ages. Cultured day 21 PE showed delayed production of this extracellular product: 7 weeks in uitro (4 weeks after the first appearance of basement membrane) were required before striated fibrils could be visualized. After 6-7 weeks in culture, explanted PE cells often formed single or stacked bilayers that were always organized in an apex-toapex or base-to-base configuration, with large quantities of collagen fibrils lying between their apposed basal surfaces (Fig. 6). Extracellular products were never detected in association with the apical PE cell surfaces. PE colonies cultured for over 6 weeks also exhibited numerous intracellular melanosomes and frequent desmosomes. Bilayering was also seen in some subcloned colonies.
VOLUME
32,1973
striated fibrils deposited by them were quantitatively and qualitatively identical. DISCUSSION
The concept that epithelial cells, as well as fibrocytes, might elaborate collagenous material was current in the early twentieth century (Spuler, 1899; Ladijenski, 1915; Laguesse, 1923, 1926; Baitsell, 1925; review by Hay, 1964), submerged for a few decades, and then resurfaced recently as investigators began to examine the possible roles for extracellular materials in initiating or stabilizing the differentiated state. In the chick embryo, the cornea1 epithelium (Goodfellow et al., 1969; Trelstad, 1971; Trelstad and Coulombre, 1971; Dodson and Hay, 1971), otic epithelium (Coulombre, unpublished observations), and conjunctival papillae (Murray, 1941, 1943; Coulombre, et al., 1962; van de Kamp, 1968) all have been associated with possible collagen production at certain times during embryonic development. In the Collagen Fibril Diameters present study we have direct evidence that Collagen fibrils produced by the PE cells adds the retinal pigmented epithelium of from young donors were smaller on the the chick embryo to this list. Namely, average than those produced by cells from cloned and explanted embryonic chick retiolder donors (Fig. 7 and Fig. 8). Increasing nal PE cells produced extracellular matedonor age and increasing average fibril rial in vitro that exhibited the typical diameter correlated positively (Fig. 9). An ultrastructural appearances of basement membrane and collagen fibrils. Our finding examination of the frequency distribution of fibril diameters showed that, as donor of collagen fibrils confirms that of Cahn et age increased, both the most common fibril al. (1970), who, however, did not note the diameters and the upper range of fibril presence of basement membrane. The diameters increased (Fig. 10). There was characteristic interbanding pattern of native collagen was well defined only in fibrils also a slight increase in average fibril diameter for a particular donor age with larger than 200 A in diameter, but we increasing time in culture (Fig. 11). presume the smaller diameter fibrils are the precursors of the striated fibrils. PreEffect of Serial Passage liminary studies of the hydroxylation of Colonies of the same cloned PE line from radioactively labeled proline and lysine by a stage 26 donor were examined after a cultured PE also indicates that these ceils constant interval of 16 weeks in vitro fol- synthesize collagen in vitro (Newsome and lowing initial plating and either the first, Lichtenstein, unpublished observations). second, or third serial passage. The ultraOur observation that basement-memstructural morphology both of the PE cells brane-material was produced by PE conand of the basement membrane and firms the prior observation of Dodson and
NEWSOME
AND KENYON
Collagen
Production
FIG. 4. This electron micrograph of cloned retinal PE from a stage 26 donor (newer section of a colony established 15 weeks previously) demonstrates the initial appearance of basement membrane (BM) and associated fibrrls (circled), in this case within basal infoldings of the cell membrane (PM). Note that in areas where the basal cell membrane is devoid of basement membrane, no collagen fibrils are evident. P, tissue culture plate. x 14,500.
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BIOLOGY
VOLUME
32, 1973
FIG. 5. A higher magnification electron micrograph of the area shown in Fig. 4 shows surface of a cultured PE cell. Note that fibrils (C) were deposited only along those segments membrane (PM) subtended by basement-membrane material (BM). P, tissue culture granule; M, mitochondrion. x 43,500.
--
the infolded basal of the cell plasma plate; *, melanin
-
FIG, 6. This survey electron micrograph of PE cells cloned from a stage 17 donor (4 weeks in uitro) shows 4 layers of cells, (l-4), organized in typical apex-to-apex and base-to-base configuration. Adjacent cells have developed junctional complexes (JC) at their apical surfaces and are invested by continuous basement membranes (BM) along their basal cell surfaces. Desmosomal attachments (circled) are frequent. In the extracellular spaces between basally-apposed cells, masses of fibrils (C) have been deposited. x 9000.
396
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BIOLOGY
VOLUME
32.1973
FIG. 7. This electron micrograph of PE cloned from a stage 17 donor (4 weeks in vitro; different cell line from that shown in Fig. 6) reveals numerous fibrils measuring about 150 A in diameter. Compare these fibrils with those in Fig. 8 at the same magnification. x 60,000.
Hay (1971) that normal epithelia can deposit basement membrane in culture. At least in the kidney and lens, epithelial basement membrane contains a collagenous component different from interstitial collagen of the same species (Spiro, 1970; Kefalides, 1970; Grant et al., 1972a,b). This information, coupled with our observation of a strict temporal and spatial correlation between the appearance of basement membrane and qtriated collagen fibrils, is in accord with the suggestion of Cohen and Hay (1971) that there may be distinct genetic codes that allow the simultaneous production of multiple forms of a given protein. Analysis of the frequency distribution of
fibril diameters showed that, as embryonic age increased, the size of the most commonly produced fibril also increased. In addition, the range of fibril sizes widened with increasing donor age, indicating the formation of progressively larger fibrils. This effect did not appear to be significantly dependent on the length of time in culture. The slight increase in average fibril diameter that we did see with lengthening time in culture may have been due to the accretion of tropocollagen to existing fibers by lateral polymerization. An alternate possibility is that chick retinal PE from a specific embryonic age may produce fibrils of a characteristic size. For several non-ocular tissues, it has been demon-
NEWSOME
FIG. a larger
8. This electron micrograph diameter (300 A average)
AND KENYON
Collagen
397
Production
of PE from a day 21 donor (8 weeks in uitro) demonstrates than the fihrils produced by younger donors. x 60,000.
strated that the same animal can produce more than one species of collagen molecule, each with particular a-chain composition (Miller and Matukas, 1969) and with distinctive hydroxylation of and glycosidic addition to lysine residues (Spiro, 1970). In the retinal PE, therefore, we could be observing the age-dependent expression of different genetic loci for collagen production. Our data must, of course, be cautiously interpreted because of the potentially large sampling error of our technique, and because of the uncertain relationship between chain composition and fibril size. Does collagen production by the retinal PE also occur in the intact chick embryo? Certain observations indicate that it may. Cleaned, trypsinized retinal PE grown as
310
-
290
-
2
collagen
4 6 s IO I2 14 I6 I8 DONOR AGE OF PIGMENTED EPITHELIUM
fibrils
20
of
22
ldoys)
FIG. 9. This graph illustrates the positive correlation between increasing donor age and increasing average fihril diameter. Each data point represents the average of 280-480 fibrils from 2 or 3 separate specimens. The vertical bars indicate the 95% confidence limits.
398
DEVELOPMENTAL
BIOLOGY
VOLUME
32,1973
1 2 DAY DONOR
3 DAY DONOR
8 DAY
DONOR
110
150
IO
190
230
FIBRIL
270
DIAMETER
310
FIG. 10. The frequency distribution of fibrils by diameters from shown. As donor age increased, so did the range of fibril diameters.
310
-
290
-
’ L
250
a single
DONOR
---“Q_ 270
-
specimen
for each of 4 donor
ages is
AGE
e .-
stage Stoge Stoge
e---
Stoge 34 Day 21
l
-
470
350
(%I
II !-2 days! 17 t-3 days) 23 (4 days) (8 days)
‘/
# H- /- c.
z 230 2 6 210 r 1904’ g 170-
.- ec- /r-.)- ..H”....“,-..yy.
/.c-
*- .*
R +*-
150130 0
I 2
I 4
I4 6
,--., y:... .,;:.r. -.a ..‘.’ .*’ s.d-
II 8 WEEKS
IO IN
..C;:‘i
._..-...
11 12 I4 CULTURE
I6
1 18
I, 20
22
FIG. 11. Companion colonies of cloned PE lines from 2-, 3-, 4-, and 8-day donors and PE explants 21 donors deposited fibrils that seemed to become slightly larger, on the average, with increasing culture.
from day time in
NEWSOME
AND KENYON
chorioallantoic membrane grafts have been reported to be associated with a homogeneous, cell-free layer that exhibited histochemical staining consistent with the presence of collagen (Newsome, 1972). Further, collagen deposits have been reported in another species, man (Lamb, 1935; Font et al., 1972; Tso and Albert, 1972; Wallow and Tso, 1972), and may also be seen in cases of senile maculopathy involving the PE (Hogan, 1972). To summarize, we have demonstrated directly that cultured clones and explants of embryonic chick retinal PE simultaneously produced collagen in two formsbasement-membrane material and striated fibrils. Collagen fibrils were seen only at those times and in those places where basement-membrane material was present. Not only the average fibril diameter, but also the range of diameters, increased with increasing donor age. The possible role(s) that t.his epithelially made collagen may play in the development of the eye remains to be elucidated. The generously given advice of Dr. Hayden G. Coon on the techniques of culturing PE cells is gratefully acknowledged, ;as is the advice and encouragement of Dr. Alfred J. Coulombre.
REFERENCES BAITSELL, G. A. (1925). On the origin of the connective-tissue ground-substance in the chick embryo. Quart. J. Microsc. Anat. 69, 571-589. CAHN, R. D., CRAWFORD, B. and CAHN, M. B. (1970). Differentiation of pigmented retina in clonal culture: Control of morphology and growth. J. Inuest. Dermatol. 54, 5. COHEN, A. M., and HAY, E. D. (1971). Secretion of collagen by embryonic neuroepithelium at the time of spinal cord-somite interaction. Deoelop. Biol. 26, 578-605. COON, H. G. (1966). Clonal stability and phenotypic expression of chick cartilage cells in vitro. Proc. Nat. Acad. Ski. U.S. 55, 66-73. COULOMBRE, A. J. (1965). The eye. In “Organogenesis” (R. L. DeHaan, ed.), pp. 219-251. Holt, Rinehart & Winston, New York. COULOMBRE, A. J., and COULOMBRE, J. L. (1971). Lesions produced in the primary cornea1 stroma of chick embryos by l-azetidine-2-carboxylic acid. Anat. Rec. 169, 301.
Collagen
Production
399
COULOMBRE, A. J., and COULOMBRE, J. L. (1972). Cornea1 development. IV. Interruption of collagen excretion into the primary stroma of the cornea with I-azetidine-2-carboxylic acid. Deuelop. Biol. 28, 183-190. COULOMBRE, A. J., COULOMBRE, J. L., and MEHTA, H. (1962). The skeleton of the eye. I. Conjunctival papillae and scleral ossicles. Deuelop. Biol. 5, 382-401. DODSON, J. W., and HAY, E. D. (1971). Secretion of collagenous stroma by isolated epithelium grown in vitro. Exp. Cell Res. 65, 215-220. DULBECCO, R., and VOGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99, 167-182. FONT, R. L., ZIMMERMAN, L. E., and FINE, B. S. (1972). Adenoma of the retinal pigment epithelium. Histochemical and electron microscopic observations. Amer. J. Ophthalmol. 73, 544-554. GIROUD, A. (1957). Phenomenes d’induction et leurs perturbations chez les mammifbres. Acta Anat. 30, 297-306. GOODFELLOW, R., REVEL, J. P., and HAY, E. D. (1969). Secretion of collagenous connective tissue by corneal epithelium. Anat. Rec. 163, 191. GRANT, M. E., KEFALIDES, N. A., and PROCKOP, D. J. (1972a). The biosynthesis of basement membrane collagen in embryonic chick lens. I. Delay between the synthesis of polypeptide chains and the secretion of collagen by matrix-free cells. J. Biol. Chem. 247, 3539-3544. GRANT, M. E., KEFALIDES, N. A., and PROCKOP, D. J. (1972b). The biosynthesis of basement membrane collagen in embryonic chick lens. II. Synthesis of a precursor form by matrix-free cells and a timedependent conversion to e-chains in intact lens. J. Biol. Chem. 247, 3545-3551. GREEN, H., GOLDBERG, B., and TODARO, G. J. (1966). Differentiated cell types and the regulation of collagen synthesis. Nature (London) 212, 631-633. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92. HAY, E. D. (1964). Secretion of a connective tissue protein by developing epidermis. In “The Epidermis” (W. Montagna and W. C. Lobitz, eds.), pp. 97-116. Academic Press, New York. HOGAN, M. J. (1972). Role of the retinal pigment epithelium in macular disease. Trans. Amer. Acad. Ophthalmol. Otol. 76, 64-80. KEFALIDES, N. A. (1970). Comparative biochemistry of mammalian basement membranes. In “Chemistry and Molecular Biology of the Intercellular Matrix” (E. A. Balazs, ed.), pp. 535-573. Academic Press, New York. LADIJENSKI, V. DE (1915). Sur l’irvolution de la structure fibrillaire de la corn&e chez l’embryon de poulet. C. R. Sot. Biol. 78, 307-308. LAGUESSE, E. (1923). Les lamelles primitives de la
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cornee du poulet sont comme le corps vitre d’origine mesostromale ectodermique. C. R. Sot. Biol. 89, 543-546. LAGUESSE, E. (1926). Development de la corn&e chez le poulet; role du mesostroma; son importance generale; les membranes basales. Arch. Anat. Microsc. Morphol. Exp. 22, 216. LAMB, H. D. (1935). The pathogenesis of some intraocular osseous tissue. True metaplasia in the eye. Amer. J. Ophthulmol. 18, 409-419. MILLER, E. J., and MATUKAS, V. J. (1969). Chick cartilage collagen: A new type of a-1 chain not present in bone or skin of the species. Proc. Nut. Acad. Sci. U.S. 64, 1264-1268. MURRAY, P. (1941). Epidermal papillae and dermal bones of the chick sclerotic. Nature (London) 148, 471. MURRAY, P. (1943). The development of the conjunctival papillae and of the scleral bones in the chick embryo. J. Anat. 77, 225-240. NEWSOME, D. A. (1972). Cartilage induction by the retinal pigmented epithelium of the chick embryo. Develop. Biol. 27, 575-579. PUCK, T., CIECIURA, S., and ROBINSON, S. (1958). Genetics of somatic cells. III. Long-term cultivation of euploid cells from human and animal subjects. J. Exp. Med. 108, 945-956. REINBOLD, R. (1968). Role du tapetum dans la diffe‘renciation de la sclerotique chez l’embryon de poulet. J. Embryol. Exp. Morphol. 19, 43-47.
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SPIRO, R. G. (1970). Biochemistry of basement membranes. In “Chemistry and Molecular Biology of the Intercellular Matrix” (E. A. Balazs, ed.), pp. 511-534. Academic Press, New York. SPULEK (1899). Cited from Hoepke, H., Die Haare. In “Handbuch der mikroskopichen Anatomie des Menschen” (van Miillendorff, ed.), Vol. 3, Part 1, pp. 66-88. Springer-Verlag, Berlin, 1927. TRELSTAD, R. L. (1971). Vacuoles in the embryonic chick cornea1 epithelium, an epithelium which produces collagen. J. Cell Biol. 48, 689-694. TRELSTAD, R. L., and COULOMBRE, A. J. (1971). Morphogenesis of the collagenous stroma in the chick cornea. J. Cell Biol. 50,840-858. Tso, M. 0. M., and ALBERT, D. M. (1972). Pathological condition of the retinal pigment epithelium. Neoplasms and nodular nonneoplastic lesions. Arch. Ophthalmol. 88, 27-38. Tso, M. 0. M., FINE, B. S., and ZIMMERMAN, L. E. (1972). Photic maculopathy produced by the indirect ophthalmoscope. I. Clinical and histopathologic study. Amer. J. Ophthulmol. in press. VAN DE KAMP, M. (1968). Fine structural analysis of the conjunctival papillae in the chick embryo: a reassessment of their morphogenesis and developmental significance. J. Erp. 2001. 169, 447-461. WALLOW, I. H. L., and Tso, M. 0. M. (1972). Pathology of proliferated retinal pigment epithelium in degenerative retinopathy. Amer. J. Ophthalmol. in press.
BIOLOGY
Collagen
32, 387-400
(1973)
Production
in Vitro by the Retinal
Epithelium Laboratory
of Vision Research, Development,
of the Chick Embryo
A. NEWSOME
DAVID
National National
Pigmented
AND KENNETH
Eye Institute; Institutes Accepted
R. KENYON
and National Institute of Child Health Bethesda, Maryland 20014
and
Human
of Health,
December
22, 1972
Cloned colonies and explants of embryonic chick retinal pigmented epithelium from donors of various embryonic ages were maintained in culture for different periods and examined by electron microscopy. The cells appeared morphologically differentiated and polarized. Basement-membrane material and striated collagen fibrils were identified as extracellular deposits beneath the basal surfaces of the cells. There appeared to be a distinct spatial and temporal correlation between the production of basement-membrane material and collagen fibrils. Increasing donor age correlated positively with increasing average diameter of the collagenous fibrils produced, as well as a widening of the range of fibril sizes. INTRODUCTION
Collagen production has been detected in extremely young embryos (Green et al., 1966). Recent studies have offered direct evidence that, at least in the developing chick, embryonic epithelia as well as connective tissue cells can synthesize (Goodfellow et al., 1969) and excrete (Trelstad, 1971; Trelstad and Coulombre, 1971; Dodson and Hay, 1971; Cohen and Hay, 1971) collagen. The retinal pigmented epithelium of the embryo of the domestic fowl, a tissue that participates significantly in the development of other tissues of the chick eye (Coulombre, 1965; Reinbold, 1968; Giroud, 1957; Newsome, 1972), also produces collagen (Coon, unpublished observation; Cahn et al., 1970). It is the purpose of this communication to present the first detailed study of the collagen-producing ability of embryonic chick retinal pigmented epithelium grown in vitro. MATERIALS
Embryos
AND
METHODS
of a White
Leghorn cross of all the pigmented epithelium (PE) used in this study. Embryonic donor age is expressed in either stages, as determined by the criteria of Hamburger and Hamilton (1951), or Gallus domesticus supplied
387 Copyright All rights
0 1973 bj Academic Press, Inc. of reproduction in any form reserved.
elapsed days of incubation at 38.0 & 0.5”C. Cultured PE from donors of 14 days of incubation or younger were cloned as follows: PE was dissected from the posterior poles of donor eyes in 0.25% trypsin (GIBCO), and dispersed by incubation for 20 min at 38°C in 0.1% trypsin and 0.02% EDTA in phosphate-buffered Ca2+ and Mg*+ free saline (Dulbecco and Vogt, 1954) at pH 7.8. Single PE cells were identified in this suspension by the presence of pigment. From 1 to 50 cells were pipetted into 60-mm plastic tissue culture dishes (Falcon) containing 3 ml of medium, and propagated by serial passage using standard techniques. Cloned material used in this study was from either the initial platings, which were examined and found free of non-PE cells, or, more usually, the first subcloning passage. Cultures from embryos older than 14 days consisted of explants of posterior pole PE prepared by dissection in 0.25% trypsin, brief washings in a saline G bath (Puck’s N-15 medium minus the amino acids; Puck et al., 1958), and direct placement in culture dishes. Sample explants were examined by electron microscopy and carried no collagenous or fibrillar material. All cultures were maintained at 38°C in a
388
DEVELOPMENTAL
BIOLOGY
5% CO, atmosphere, in Coon’s modification of Ham’s F-12 medium with 5% fetal calf serum and 0.3 pg of penicillin per milliliter (Coon, 1966). The medium was renewed every 3 days. Under these conditions the pH of the medium was 7.2. All fixation and embedding procedures for electron microscopy were performed directly in the tissue culture dishes without manipulation of the colonies in order to preserve spatial relationships. Cultures were rinsed briefly in three changes of saline G, fixed in 1% osmium tetroxide (Verona1 acetate buffered, pH 7.3, 215 milliosmolar) for 15 min at O”C, dehydrated through 5-min changes of graded alcohols at O”C, and embedded in Spurr epoxy resin (Polysciences) . Thin sections of representative PE colonies were cut on a Porter-Blum MT-2 ultramicrotome, care being taken to select central-paracentral areas from cloned colonies, and areas of new growth from explanted tissues. The sections were stained with uranyl acetate and lead citrate in the usual way, and were examined with a JEM 100-B electron microscope. Determinations of fibril diameter and macroperiodicity were made from high magnification electron micrographs (50,000 and 70,000 times) using a calibrated Filar ocular micrometer fitted to a stereomicroscope. For each donor age studied, the cross-sectional diameters of at least 280 fibrils with circular profiles were measured from randomly selected fields. Average fibril diameters were calculated, and frequency distributions were plotted from these measurements. OBSERVATIONS
Under our culture conditions PE cells formed colonies of morphologically welldifferentiated cells as viewed by phase contrast microscopy in the living state. The cultured cells assumed the polygonal array typical of retinal PE in viuo (Fig. 1). Colonies initially consisted of a monolayer of unpigmented cells. After about 10 days
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32, 1973
in culture, colonies usually had three morphologically distinct zones: (1) a central area of polygonal, visibly pigmented cells with no (or extremely rare) mitotic figures seen on repeated observation; (2) a paracentral ring of rounded, apparently unpigmented cells, with a few mitotic figures seen in this area; and (3) a peripheral skirt of actively dividing cells with fibrocytic appearance and no visible pigmentation. By 6 weeks in culture, 85-90s of the cells in a typical colony were heavily pigmented, polygonal, and occasionally also binucleate. This sequence was applicable to all clones and explants studied. Cellular
Morphology
The formation of monolayers and sparse pigmentation of PE colonies after a brief time in culture were also evident by electron microscopy. As early as 4 days in vitro the cells exhibited a definite polarity, as junctional complexes had developed between lateral cell membranes at the apical aspects of adjacent cells (Fig. 2). These complexes consisted of cell membranes in close parallel apposition (-200 A gap) with electron-dense granular material aggregated along their cytoplasmic aspects. Tight junctions were never seen. Conventional desmosomal attachments were occasionally present between lateral cell membranes (Figs. 2 and 6). The apical cell membrane was elaborately infolded in numerous pseudopodlike configurations. During the initial days in culture, the plasma membrane limiting the basal surface of the PE cells was free of any extracellular material and was in direct contact with the surface of the culture dish. In rare instances, the readily identifiable apical surfaces of the PE cells lay in direct contact with the dish, demonstrating that the intrinsic polarity of the cells is seemingly independent of their spatial orientation. Basement
Membrane
Formation
After 4 days in vitro, basement membrane formation was first evidenced by the
NEWSOME
AND KENYON
Collagen
Production
389
FIG. 1. As this phase-contrast photomicrograph illustrates, cloned retinal PE cells from a stage 25 donor (5 weeks in vitro) have formed a polygonal array of heavily pigmented cells. Several binucleate cells (*) are evident. x 150.
appearance of short, discontinuous segments of fine granular or fibrillar material beneath the basal surface of the PE cells. By 14 days in culture, these segments had coalesced to form a continuous extracellular lamina of 200-400 A thickness that was separated from the basal plasma membrane by a 300-500 A wide electron-lucent zone. In general, basement membrane formation progressed more rapidly in cultured PE from younger donors, and was complete over the entire basal surface of the epithelial sheet by 3 weeks in vitro. PE explanted from a 21-day donor, in contrast, did not begin to deposit basement membrane until 3 weeks in culture, and required 6 weeks for its completion. Collagen Fibril Deposition
In addition
to basement
membrane,
ran-
domly oriented fibrils of 150-450 A diameter accumulated in quantity beneath the basal surfaces of the several cloned lines and explants of PE studied (Fig. 3). These fibrils exhibited the ultrastructural characteristics of collagen. Although longitudinal macroperiodicity was not clearly discernible in fibrils of small diameter, a definite macroperiod ranging from 550 to 600 A could be resolved in larger fibers (Fig. 3). There was a distinct spatial and temporal correlation between the occurrence of basement membrane and striated fibrils. In particular, PE monolayers without a basement membrane were also devoid of collagen fibrils. Further, prior to the completion of basement membrane formation, fibrils were present only in association with those areas of the basal cell surface subtended by basement-membrane material
~
I
FIG. 2. This electron micrograph of’ retinal PE f’rom a day 21 donor (4 weeks in vitro) shows a monolayer of cells whose polarity is indicated by well-developed apical junctional complexes (JC). The plasma membrane (PM) limiting the basal surface of the cell lies in direct contact with the tissue culture plate (P). Occasional vsomes (circled) are present between lateral cell membranes. Few melanin granules (*I are evident. N, .: M, mitochondrion. A 12,500.
NEWSOME
AND KENYON
Collagen
Production
391
FIG. 3. This electron micrograph of retinal PE from a day 13 donor (14 weeks in uitro) documents the accumulation of collagen fihrils extracellularly beneath the basal surface of the cells. These fibrils measure 220-250 A in diameter, with a longitudinal macroperiodicity of about 550 A (arrowheads). x 80,000.
392
DEVELOPMENTAL
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(Figs. 4 and 5). None of our specimens showed collagen-containing basal vacuoles, As with basement membrane formation, collagen fibrils initially appeared after 4 days in culture for most donor ages. Cultured day 21 PE showed delayed production of this extracellular product: 7 weeks in uitro (4 weeks after the first appearance of basement membrane) were required before striated fibrils could be visualized. After 6-7 weeks in culture, explanted PE cells often formed single or stacked bilayers that were always organized in an apex-toapex or base-to-base configuration, with large quantities of collagen fibrils lying between their apposed basal surfaces (Fig. 6). Extracellular products were never detected in association with the apical PE cell surfaces. PE colonies cultured for over 6 weeks also exhibited numerous intracellular melanosomes and frequent desmosomes. Bilayering was also seen in some subcloned colonies.
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striated fibrils deposited by them were quantitatively and qualitatively identical. DISCUSSION
The concept that epithelial cells, as well as fibrocytes, might elaborate collagenous material was current in the early twentieth century (Spuler, 1899; Ladijenski, 1915; Laguesse, 1923, 1926; Baitsell, 1925; review by Hay, 1964), submerged for a few decades, and then resurfaced recently as investigators began to examine the possible roles for extracellular materials in initiating or stabilizing the differentiated state. In the chick embryo, the cornea1 epithelium (Goodfellow et al., 1969; Trelstad, 1971; Trelstad and Coulombre, 1971; Dodson and Hay, 1971), otic epithelium (Coulombre, unpublished observations), and conjunctival papillae (Murray, 1941, 1943; Coulombre, et al., 1962; van de Kamp, 1968) all have been associated with possible collagen production at certain times during embryonic development. In the Collagen Fibril Diameters present study we have direct evidence that Collagen fibrils produced by the PE cells adds the retinal pigmented epithelium of from young donors were smaller on the the chick embryo to this list. Namely, average than those produced by cells from cloned and explanted embryonic chick retiolder donors (Fig. 7 and Fig. 8). Increasing nal PE cells produced extracellular matedonor age and increasing average fibril rial in vitro that exhibited the typical diameter correlated positively (Fig. 9). An ultrastructural appearances of basement membrane and collagen fibrils. Our finding examination of the frequency distribution of fibril diameters showed that, as donor of collagen fibrils confirms that of Cahn et age increased, both the most common fibril al. (1970), who, however, did not note the diameters and the upper range of fibril presence of basement membrane. The diameters increased (Fig. 10). There was characteristic interbanding pattern of native collagen was well defined only in fibrils also a slight increase in average fibril diameter for a particular donor age with larger than 200 A in diameter, but we increasing time in culture (Fig. 11). presume the smaller diameter fibrils are the precursors of the striated fibrils. PreEffect of Serial Passage liminary studies of the hydroxylation of Colonies of the same cloned PE line from radioactively labeled proline and lysine by a stage 26 donor were examined after a cultured PE also indicates that these ceils constant interval of 16 weeks in vitro fol- synthesize collagen in vitro (Newsome and lowing initial plating and either the first, Lichtenstein, unpublished observations). second, or third serial passage. The ultraOur observation that basement-memstructural morphology both of the PE cells brane-material was produced by PE conand of the basement membrane and firms the prior observation of Dodson and
NEWSOME
AND KENYON
Collagen
Production
FIG. 4. This electron micrograph of cloned retinal PE from a stage 26 donor (newer section of a colony established 15 weeks previously) demonstrates the initial appearance of basement membrane (BM) and associated fibrrls (circled), in this case within basal infoldings of the cell membrane (PM). Note that in areas where the basal cell membrane is devoid of basement membrane, no collagen fibrils are evident. P, tissue culture plate. x 14,500.
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FIG. 5. A higher magnification electron micrograph of the area shown in Fig. 4 shows surface of a cultured PE cell. Note that fibrils (C) were deposited only along those segments membrane (PM) subtended by basement-membrane material (BM). P, tissue culture granule; M, mitochondrion. x 43,500.
--
the infolded basal of the cell plasma plate; *, melanin
-
FIG, 6. This survey electron micrograph of PE cells cloned from a stage 17 donor (4 weeks in uitro) shows 4 layers of cells, (l-4), organized in typical apex-to-apex and base-to-base configuration. Adjacent cells have developed junctional complexes (JC) at their apical surfaces and are invested by continuous basement membranes (BM) along their basal cell surfaces. Desmosomal attachments (circled) are frequent. In the extracellular spaces between basally-apposed cells, masses of fibrils (C) have been deposited. x 9000.
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DEVELOPMENTAL
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FIG. 7. This electron micrograph of PE cloned from a stage 17 donor (4 weeks in vitro; different cell line from that shown in Fig. 6) reveals numerous fibrils measuring about 150 A in diameter. Compare these fibrils with those in Fig. 8 at the same magnification. x 60,000.
Hay (1971) that normal epithelia can deposit basement membrane in culture. At least in the kidney and lens, epithelial basement membrane contains a collagenous component different from interstitial collagen of the same species (Spiro, 1970; Kefalides, 1970; Grant et al., 1972a,b). This information, coupled with our observation of a strict temporal and spatial correlation between the appearance of basement membrane and qtriated collagen fibrils, is in accord with the suggestion of Cohen and Hay (1971) that there may be distinct genetic codes that allow the simultaneous production of multiple forms of a given protein. Analysis of the frequency distribution of
fibril diameters showed that, as embryonic age increased, the size of the most commonly produced fibril also increased. In addition, the range of fibril sizes widened with increasing donor age, indicating the formation of progressively larger fibrils. This effect did not appear to be significantly dependent on the length of time in culture. The slight increase in average fibril diameter that we did see with lengthening time in culture may have been due to the accretion of tropocollagen to existing fibers by lateral polymerization. An alternate possibility is that chick retinal PE from a specific embryonic age may produce fibrils of a characteristic size. For several non-ocular tissues, it has been demon-
NEWSOME
FIG. a larger
8. This electron micrograph diameter (300 A average)
AND KENYON
Collagen
397
Production
of PE from a day 21 donor (8 weeks in uitro) demonstrates than the fihrils produced by younger donors. x 60,000.
strated that the same animal can produce more than one species of collagen molecule, each with particular a-chain composition (Miller and Matukas, 1969) and with distinctive hydroxylation of and glycosidic addition to lysine residues (Spiro, 1970). In the retinal PE, therefore, we could be observing the age-dependent expression of different genetic loci for collagen production. Our data must, of course, be cautiously interpreted because of the potentially large sampling error of our technique, and because of the uncertain relationship between chain composition and fibril size. Does collagen production by the retinal PE also occur in the intact chick embryo? Certain observations indicate that it may. Cleaned, trypsinized retinal PE grown as
310
-
290
-
2
collagen
4 6 s IO I2 14 I6 I8 DONOR AGE OF PIGMENTED EPITHELIUM
fibrils
20
of
22
ldoys)
FIG. 9. This graph illustrates the positive correlation between increasing donor age and increasing average fihril diameter. Each data point represents the average of 280-480 fibrils from 2 or 3 separate specimens. The vertical bars indicate the 95% confidence limits.
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DEVELOPMENTAL
BIOLOGY
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1 2 DAY DONOR
3 DAY DONOR
8 DAY
DONOR
110
150
IO
190
230
FIBRIL
270
DIAMETER
310
FIG. 10. The frequency distribution of fibrils by diameters from shown. As donor age increased, so did the range of fibril diameters.
310
-
290
-
’ L
250
a single
DONOR
---“Q_ 270
-
specimen
for each of 4 donor
ages is
AGE
e .-
stage Stoge Stoge
e---
Stoge 34 Day 21
l
-
470
350
(%I
II !-2 days! 17 t-3 days) 23 (4 days) (8 days)
‘/
# H- /- c.
z 230 2 6 210 r 1904’ g 170-
.- ec- /r-.)- ..H”....“,-..yy.
/.c-
*- .*
R +*-
150130 0
I 2
I 4
I4 6
,--., y:... .,;:.r. -.a ..‘.’ .*’ s.d-
II 8 WEEKS
IO IN
..C;:‘i
._..-...
11 12 I4 CULTURE
I6
1 18
I, 20
22
FIG. 11. Companion colonies of cloned PE lines from 2-, 3-, 4-, and 8-day donors and PE explants 21 donors deposited fibrils that seemed to become slightly larger, on the average, with increasing culture.
from day time in
NEWSOME
AND KENYON
chorioallantoic membrane grafts have been reported to be associated with a homogeneous, cell-free layer that exhibited histochemical staining consistent with the presence of collagen (Newsome, 1972). Further, collagen deposits have been reported in another species, man (Lamb, 1935; Font et al., 1972; Tso and Albert, 1972; Wallow and Tso, 1972), and may also be seen in cases of senile maculopathy involving the PE (Hogan, 1972). To summarize, we have demonstrated directly that cultured clones and explants of embryonic chick retinal PE simultaneously produced collagen in two formsbasement-membrane material and striated fibrils. Collagen fibrils were seen only at those times and in those places where basement-membrane material was present. Not only the average fibril diameter, but also the range of diameters, increased with increasing donor age. The possible role(s) that t.his epithelially made collagen may play in the development of the eye remains to be elucidated. The generously given advice of Dr. Hayden G. Coon on the techniques of culturing PE cells is gratefully acknowledged, ;as is the advice and encouragement of Dr. Alfred J. Coulombre.
REFERENCES BAITSELL, G. A. (1925). On the origin of the connective-tissue ground-substance in the chick embryo. Quart. J. Microsc. Anat. 69, 571-589. CAHN, R. D., CRAWFORD, B. and CAHN, M. B. (1970). Differentiation of pigmented retina in clonal culture: Control of morphology and growth. J. Inuest. Dermatol. 54, 5. COHEN, A. M., and HAY, E. D. (1971). Secretion of collagen by embryonic neuroepithelium at the time of spinal cord-somite interaction. Deoelop. Biol. 26, 578-605. COON, H. G. (1966). Clonal stability and phenotypic expression of chick cartilage cells in vitro. Proc. Nat. Acad. Ski. U.S. 55, 66-73. COULOMBRE, A. J. (1965). The eye. In “Organogenesis” (R. L. DeHaan, ed.), pp. 219-251. Holt, Rinehart & Winston, New York. COULOMBRE, A. J., and COULOMBRE, J. L. (1971). Lesions produced in the primary cornea1 stroma of chick embryos by l-azetidine-2-carboxylic acid. Anat. Rec. 169, 301.
Collagen
Production
399
COULOMBRE, A. J., and COULOMBRE, J. L. (1972). Cornea1 development. IV. Interruption of collagen excretion into the primary stroma of the cornea with I-azetidine-2-carboxylic acid. Deuelop. Biol. 28, 183-190. COULOMBRE, A. J., COULOMBRE, J. L., and MEHTA, H. (1962). The skeleton of the eye. I. Conjunctival papillae and scleral ossicles. Deuelop. Biol. 5, 382-401. DODSON, J. W., and HAY, E. D. (1971). Secretion of collagenous stroma by isolated epithelium grown in vitro. Exp. Cell Res. 65, 215-220. DULBECCO, R., and VOGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99, 167-182. FONT, R. L., ZIMMERMAN, L. E., and FINE, B. S. (1972). Adenoma of the retinal pigment epithelium. Histochemical and electron microscopic observations. Amer. J. Ophthalmol. 73, 544-554. GIROUD, A. (1957). Phenomenes d’induction et leurs perturbations chez les mammifbres. Acta Anat. 30, 297-306. GOODFELLOW, R., REVEL, J. P., and HAY, E. D. (1969). Secretion of collagenous connective tissue by corneal epithelium. Anat. Rec. 163, 191. GRANT, M. E., KEFALIDES, N. A., and PROCKOP, D. J. (1972a). The biosynthesis of basement membrane collagen in embryonic chick lens. I. Delay between the synthesis of polypeptide chains and the secretion of collagen by matrix-free cells. J. Biol. Chem. 247, 3539-3544. GRANT, M. E., KEFALIDES, N. A., and PROCKOP, D. J. (1972b). The biosynthesis of basement membrane collagen in embryonic chick lens. II. Synthesis of a precursor form by matrix-free cells and a timedependent conversion to e-chains in intact lens. J. Biol. Chem. 247, 3545-3551. GREEN, H., GOLDBERG, B., and TODARO, G. J. (1966). Differentiated cell types and the regulation of collagen synthesis. Nature (London) 212, 631-633. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92. HAY, E. D. (1964). Secretion of a connective tissue protein by developing epidermis. In “The Epidermis” (W. Montagna and W. C. Lobitz, eds.), pp. 97-116. Academic Press, New York. HOGAN, M. J. (1972). Role of the retinal pigment epithelium in macular disease. Trans. Amer. Acad. Ophthalmol. Otol. 76, 64-80. KEFALIDES, N. A. (1970). Comparative biochemistry of mammalian basement membranes. In “Chemistry and Molecular Biology of the Intercellular Matrix” (E. A. Balazs, ed.), pp. 535-573. Academic Press, New York. LADIJENSKI, V. DE (1915). Sur l’irvolution de la structure fibrillaire de la corn&e chez l’embryon de poulet. C. R. Sot. Biol. 78, 307-308. LAGUESSE, E. (1923). Les lamelles primitives de la
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cornee du poulet sont comme le corps vitre d’origine mesostromale ectodermique. C. R. Sot. Biol. 89, 543-546. LAGUESSE, E. (1926). Development de la corn&e chez le poulet; role du mesostroma; son importance generale; les membranes basales. Arch. Anat. Microsc. Morphol. Exp. 22, 216. LAMB, H. D. (1935). The pathogenesis of some intraocular osseous tissue. True metaplasia in the eye. Amer. J. Ophthulmol. 18, 409-419. MILLER, E. J., and MATUKAS, V. J. (1969). Chick cartilage collagen: A new type of a-1 chain not present in bone or skin of the species. Proc. Nut. Acad. Sci. U.S. 64, 1264-1268. MURRAY, P. (1941). Epidermal papillae and dermal bones of the chick sclerotic. Nature (London) 148, 471. MURRAY, P. (1943). The development of the conjunctival papillae and of the scleral bones in the chick embryo. J. Anat. 77, 225-240. NEWSOME, D. A. (1972). Cartilage induction by the retinal pigmented epithelium of the chick embryo. Develop. Biol. 27, 575-579. PUCK, T., CIECIURA, S., and ROBINSON, S. (1958). Genetics of somatic cells. III. Long-term cultivation of euploid cells from human and animal subjects. J. Exp. Med. 108, 945-956. REINBOLD, R. (1968). Role du tapetum dans la diffe‘renciation de la sclerotique chez l’embryon de poulet. J. Embryol. Exp. Morphol. 19, 43-47.
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SPIRO, R. G. (1970). Biochemistry of basement membranes. In “Chemistry and Molecular Biology of the Intercellular Matrix” (E. A. Balazs, ed.), pp. 511-534. Academic Press, New York. SPULEK (1899). Cited from Hoepke, H., Die Haare. In “Handbuch der mikroskopichen Anatomie des Menschen” (van Miillendorff, ed.), Vol. 3, Part 1, pp. 66-88. Springer-Verlag, Berlin, 1927. TRELSTAD, R. L. (1971). Vacuoles in the embryonic chick cornea1 epithelium, an epithelium which produces collagen. J. Cell Biol. 48, 689-694. TRELSTAD, R. L., and COULOMBRE, A. J. (1971). Morphogenesis of the collagenous stroma in the chick cornea. J. Cell Biol. 50,840-858. Tso, M. 0. M., and ALBERT, D. M. (1972). Pathological condition of the retinal pigment epithelium. Neoplasms and nodular nonneoplastic lesions. Arch. Ophthalmol. 88, 27-38. Tso, M. 0. M., FINE, B. S., and ZIMMERMAN, L. E. (1972). Photic maculopathy produced by the indirect ophthalmoscope. I. Clinical and histopathologic study. Amer. J. Ophthulmol. in press. VAN DE KAMP, M. (1968). Fine structural analysis of the conjunctival papillae in the chick embryo: a reassessment of their morphogenesis and developmental significance. J. Erp. 2001. 169, 447-461. WALLOW, I. H. L., and Tso, M. 0. M. (1972). Pathology of proliferated retinal pigment epithelium in degenerative retinopathy. Amer. J. Ophthalmol. in press.