cell differentiation

Proc. Nati. Acad. Sci. USA Vol. 77, No. 1, p. 490-493, January 1980

Developmental Biology

Lentropin: A factor in vitreous humor which promotes lens fiber cell differentiation (chicken embryo)

DAVID C. BEEBE, DOUGLAS E. FEAGANS, AND HOLLY ANN H. JEBENS Department of Anatomy, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20014

Communicated by James D. Ebert, October 9, 1979

ABSTRACT An activity has been identified in chicken vitreous humor which stimulates embryonic chicken lens epithelial cells to elongate and specialize for lens crystallin synthesis. The activity is heat-labile and is destroyed by treatment with tsin or agents that reduce disulfides. Gel filtration and ultrafiltration analyses indicate that it has an apparent molecular weight of -60,OOO. Its properties differ from those of an activity present in serum which also can promote lens fiber cell formation in vitro. We call this material "lentropin" and suggest that it is responsible for stimulating lens fiber cell formation in vivo and, consequently, plays an important role in determining the shape and polarity of the lens.

The lens of the eye consists of two cell types: epithelial cells, which form a monolayer covering the anterior surface of the lens, and elongated fiber cells, which make up the bulk of the lens. Lens epithelial cells proliferate and differentiate into lens fiber cells; these processes are largely responsible for lens growth. Fiber cell differentiation occurs only in the posterior portion of the lens and the lens is therefore polarized, with the epithelium occupying the anterior pole and the fiber cells occupying the posterior pole. Evidence has been presented which indicates that some influence is present in the posterior portion of the eye which induces equatorial lens epithelial cells to form fiber cells and is therefore responsible for establishment of lens polarity (1-3). When the central portion of an early embryonic chicken lens epithelium is cultured in medium supplemented with fetal calf serum (4-6) or insulin (1 ttg/ml) (7, 8), the epithelial cells elongate and become similar to lens fiber cells (3, 5, 6, 9). Central lens epithelial cells cultured in unsupplemented medium do not resemble lens fiber cells in either shape or pattern of protein synthesis (4, 10, 11). In vitro culture of 6-day embryonic lens epithelia has been used in the present investigation to identify and partially characterize an activity present in adult and embryonic chicken vitreous humor which causes lens epithelial cells to elongate and differentiate into cells that resemble lens fiber cells. MATERIALS AND METHODS White Leghorn strain embryos and adult chickens were obtained from Truslow Farms (Chestertown, MD). Eggs were incubated in a Humidaire incubator at 37.50C. Lens epithelia were obtained from 6-day embryos (9) and cultured as described (12). Measurement of Cell Length. Central lens epithelia were dissected and cultured as explants in 35-mm Falcon plastic The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 490

tissue culture dishes (9). Cell length was measured, with a Zeiss inverted microscope with Nomarski interference contrast optics by focusing on the upper and lower surfaces of the epithelial monolayer (12). The distance between these focal planes was determined with a micrometer built into the focusing mechanism (Baltimore Instrument Company, Baltimore, MD) and was confirmed as an accurate measure of cell length by directly measuring epithelial cell length in fixed, embedded, sectioned cultures. Preparation of Vitreous Humor. Vitreous bodies were removed from decapitated adult and embryonic chickens by making a 900 lateral incision at the corneal limbus, expressing the vitreous substance with gentle pressure to the outside of the eye and grasping the vitreous with fine forceps. If the lens remained attached to the vitreous body, it was carefully removed intact. Only the gel portion of the vitreous body was used (13). Prior to removal of the vitreous body from the eye, the heads were thoroughly washed with 0.01 M phosphate-buffered saline (pH 7.4) to minimize contamination of the vitreous substance with blood. Pooled vitreous bodies were centrifuged for 10 min at 40C at 26,000 X g in a Sorvall RC-5 centrifuge and the supernatant was saved (six 15-day vitreous bodies t1 ml). This preparation is referred to as "vitreous humor" and does not contain the cells or fibrous elements of the vitreous body. For culturing lens epithelia, small molecules were removed from the vitreous humor by centrifugal desalting over Sephadex G-25 (14). Fine or superfine Sephadex was swollen in distilled water, and the excess water was removed by centrifugation of the gel bed in plastic columns (Bio-Rad polypropylene Econo-Columns) at 400 X g in a Sorvall HS-4 rotor. Vitreous humor (up to 25% of the bed volume) was applied to the top of the resulting gel cake and allowed to soak into the gel. Centrifugation at 400 X g for 5 min forced the large molecules of the vitreous humor (Mr > 5000) through the gel, and these were collected at their original concentration; molecules smaller than Mr 5000 were retained on the column. Nine volumes of desalted vitreous humor was mixed with 1 vol of 10X Ham's F-10 medium (15) (GIBCO) for most culture experiments ("90% vitreous humor"). For some experiments, vitreous humor that had not been desalted was supplemented -with sodium bicarbonate (1.2 mg/ml) or, in other experiments, lower concentrations of vitreous humor in Ham's F-10 medium were used to culture lens epithelia. Gel Filtration Chromatography. Untreated vitreous humor (10 ml) was lyophilized, dissolved in 2 ml of Ham's F-10 medium, and chromatographed on Sephadex G-70 or G-100 (Sigma) or Ultrogel AcA 34 (LKB) in Ham's F-10 medium supplemented with 2% horse plasma. Bed volumes of 30-40 ml in 1.5-cm-diameter columns were used (flow rate, 0.05 ml/min) and molecular weight standards (rabbit pyruvate kinase, bovine

Developmental Biology: Beebe et al.

Proc. Natl. Acad. Sci. USA 77 (1980)

serum albumin, and horse cytochrome c, all from Sigma) were run prior to each separation. Column fractions (3 ml) were filter-sterilized and assayed directly for their ability to stimulate lens epithelial cell elongation. Measurement of Epithelial Protein Synthesis. Groups of four central lens epithelia were incubated for 1 hr in 0.25 ml of Ham's F-10 medium containing 15% fetal calf serum and 200,uCi of [a5S]methionine (New England Nuclear; 500-600 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels). After labeling they were washed twice in Puck's saline G (GIBCO), dissolved in 50 ,gl of electrophoresis sample buffer [1% sodium dodecyl sulfate/1% 2-mercaptoethanol/8 M urea/10% (wt/vol) glycerol/0. 125 M TrisIHCl, pH 6.9] and heated at 100'C for 3 min. Lysates were electrophoresed for 3.5-4 hr at 120 V on a discontinuous acrylamide slab gel containing 0.1% sodium dodecyl sulfate (separating gel: 4% C, 10.4% T) (16) in a Bio-Rad model 220 electrophoresis apparatus. Gels were stained by immersion in 0.2% Coomassie blue R in 45% methanol/10% acetic acid, electrophoretically destained (E-C Apparatus) in 5% acetic acid, dried, and autoradiographed (Kodak, X-Omat) and the autoradiographs were scanned with a Quick Scan Jr. densitometer

491

RESULTS Cell Elongation and Crystallin Synthesis. Lens epithelial cells from lenses of 6-day chicken embryos elongated when cultured in vitreous humor-supplemented medium. In 90% vitreous humor, their length increased from a mean (+SEM) of 10.0 ± 0.5 Aim to 19.1 ± 0.5 pm after 5 hr of culture and 32.4 + 1.2 pm after 24 hr. Elongation continued for at least 72 hr. Cell elongation was dose-dependent, with maximal elongation occurring at approximately 25% (vol/vol) vitreous humor in Ham's F-10 medium (Fig. 1). Vitreous humor from 6- to 19-day chicken embryos and adults also contained the activity (data not shown). Embryonic lens fiber cell differentiation in vivo and in vitro is characterized by increased synthesis of the lens protein 6crystallin (9, 17, 18). When central lens epithelia were cultured in 90% vitreous humor and their newly synthesized proteins were labeled with [35S]methionine, b-crystallin increased from 6-8% of total protein synthesis in freshly explanted epithelia to nearly 25% after 48 hr of culture (Fig. 2).

(Helena).

a

Trypsin Treatment. Desalted vitreous humor was incubated with trypsin (10 Aig/ml, Worthington) at 370C for 24 hr. Control desalted vitreous humor was incubated at 37°C for 24 hr, and trypsin was added at the end of the incubation. To both treated and control vitreous humors was added soybean trypsin inhibitor (25 ,g/ml; Worthington), and they then were mixed 1:9 (vol/vol) with 1OX concentrated Ham's F-10 medium and used for culture of lens epithelia. Other Treatments. In order to test the chemical stability of the vitreous humor activity, vitreous humor was incubated for 1 hr at 37°C with one of the following additions: 1% 2-mercaptoethanol, 10 mM dithiothreitol, 8 M urea, or 3M guanidine hydrochloride. After treatment, the vitreous humor was desalted twice (see above), mixed 9:1 (vol/vol) with 1oX concentrated Ham's F-10 medium, and used for culture of lens

b

epithelial explants.

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20

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FIG. 1. Elongation of 6-day embryo chicken lens epithelial cells cultured for 5 hr in Ham's F-10 medium supplemented with different amounts of vitreous humor from 15-day chicken embryos. Each point is the mean of five measurements on a single lens epithelium. 0, Untreated vitreous humor; 0, vitreous humor incubated at 37°C for 24 hr.

FIG. 2. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of proteins synthesized in 6-day embryo chicken lens epithelia at the time of explantation (lane a) or after culture for 24 hr in Ham's F-10 medium supplemented with 15% fetal calf serum (lane b) or 90%1 vitreous humor (lane c). The position of the b-crystallin band is indicated. Groups of four epithelia were incubated in medium containing [35S]methionine for 1 hr, and the labeled proteins were separated and autoradiographed.


Developmental Biology: Beebe et al.

492

order to facilitate rapid analysis of column fractions, the separations were performed in columns equilibrated with Ham's F-10 medium containing 2% horse plasma and fractions were collected, filter-sterilized, and used immediately for culture of 6-day embryo lens epithelia. Fig. 3 shows the elution profile, from Ultrogel AcA 34, of material absorbing at 280 nm. The elution volumes of marker proteins are shown, and the bars indicate the presence of elongation-promoting activity. The peak of activity corresponds to Mr -60,000.

Table 1. Effect of various treatments on lens cell elongation activity Cell length after 5-hr

Cultured in

culture, ,um*

90% vitreous humor 15% fetal calf serum

19.1 ± 0.5 19.1 + 0.4 10.0 i 0.2

Treatment

None

Unsupplemented Ham's F-10 90% vitreous humor 11.8 i 0.3 15% fetal calf serum 21.6 ± 1.0 90% vitreous humor 10.4 i 0.1 15% fetal calf serum 18.1 ± 0.5 90% vitreous humor 11.9 ± 0.3 90% vitreous humor 10.4 ± 0.3 90% vitreous humor 18.2 ± 0.6 90% vitreous humor 16.2 * 0.4

1000C, 10 min

Mercaptoethanol (1%, 1 hr) Trypsin (10 ,g/ml, 24 hr) Dithiothreitol (10 mM, 1 hr) Urea (8 M, 1 hr) Guanidine-HCl (3 M, 1 hr) * Mean + SEM.

Characterization of the Vitreous Humor Activity. Experiments were designed to determine some of the physical characteristics of the vitreous activity. There was a 50% decrease in elongation-stimulating activity after incubation of the vitreous humor at 37'C for 24 hr (Fig. 1). Heating fresh vitreous to 1000C for 10 min completely destroyed the activity (Table 1). Similar heat treatment of fetal calf serum did not diminish its ability to promote lens cell elongation. Trypsin treatment (10 jig/ml, 24 hr) destroyed the ability of vitreous humor to stimulate cell elongation (Table 1). Epithelial cells grown in control vitreous humor containing tryspin immediately inactivated with soybean trypsin inhibitor (25 gtg/ml) elongated and were similar in length to untreated controls after 24 hr of culture. Epithelia cultured in trypsintreated vitreous humor lost many cells and showed evidence of cell death after 24 hr of culture, a result similar to that seen in epithelia cultured for 24 hr in Ham's F-10 medium alone. Treatment with mercaptoethanol (1%, 1 hr) or dithiothreitol (10 mM, 1 hr) eliminated the elongation-promoting activity of the vitreous humor, whereas similar treatment with 8 M urea or 3 M guanidine hydrochloride only slightly diminished this activity. Treatment of fetal calf serum with 1% mercaptoethanol did not affect its ability to cause cell elongation. The vitreous activity passed through Amicon ultrafiltration membranes with a pore size which retained molecules of Mr 100,000 or larger (XM-100) and did not pass through membranes that were impermeable to molecules larger than Mr 10,000 (UM-10). Gel filtration analysis of the vitreous activity was hindered by rapid loss of activity during chromatography unless 2% horse plasma was included in the column buffer. In 68

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FIG. 3. Gel filtration chromatography of 15-day embryo chicken vitreous humor on Ultrogel AcA 34. Elongation-promoting activity was found primarily in fractions 10-13 corresponding to Mr of 760,000. Bars show cell length as mean + SEM; numerals are Mr X 10-3.

DISCUSSION We have presented evidence for a factor in chicken embryo and adult vitreous humor that stimulates cell elongation and specialization for b-crystallin synthesis in cultured embryonic chicken lens epithelial cells. Cell elongation and specialization for b-crystallin synthesis are characteristic of lens fiber cell differentiation from lens epithelial cells in chicken embryos (5, 6, 11), and fiber cell differentiation in the lens normally occurs in those cells exposed to vitreous humor (1-2). The cellular source of this factor is not known with certainty. Philpott and Coulombre (3) have shown that embryonic mesenchyme and neural retina can stimulate limited lens fiber cell differentiation in precultured 6-day embryo central lens epithelial cells. It is also possible that the activity is derived from the vascular system because plasma proteins have been reported in vitreous humor (19-21) and are the most abundant soluble proteins in chicken embryo vitreous humor as early as 6 days of development (unpublished data). Our results suggest, however, that the differentiation-promoting activity in vitreous humor is not the same as that present in fetal calf serum (4) because these substances had different sensitivities to heating and treatment with

2-mercaptoethanol. Our results suggest that the vitreous factor is a protein of Mr t60,000. Its activity was destroyed by trypsin, heat, 2-mercaptoethanol, and dithiothreitol. The susceptibility of the vitreous humor activity to reducing agents suggests that the factor contains one or more disulfide bonds responsible for maintaining an active conformation of the protein or for crosslinking subunits. The activity cochromatographed on Ultrogel AcA 34 columns with proteins of Mr a60,000. This Mr should be considered as tentative because the activity could have been aggregated or bound to other molecules in the vitreous humor or to components of the horse plasma present in the column buffer. There is little information concerning the identification of differentiation-promoting substances in vitreous humor or other ocular tissues, although Arruti and Courtois (22) have recently reported an activity in bovine retina, iris, and vitreous body that stimulates DNA synthesis, cell division, and altered cellular morphology in a cultured bovine lens epithelial cell line. This activity is trypsin sensitive and similar in size to the chicken vitreous humor activity (Y. Courtois, personal communication). It is not known whether this activity will induce differentiation of embryonic lens epithelial cells as we have described for chicken vitreous humor. Identification of the substance from the eye that is normally responsible for causing lens fiber cell differentiation would provide a significant tool for studying the morphogenesis of the lens and the control of cell differentiation. Lens fiber cell differentiation involves extensive cell elongation by a mechanism that appears to involve an increase in cell volume (ref. 12; unpublished data). Numerous investigators have also demonstrated that lens fiber cell differentiation involves accumulation of large amounts of lens crystallins. Because the vitreous humor activity we have described is found in proximity to those lens cells that are forming or have formed lens fibers and stimulates

Developmental Biology: Beebe et al. both lens cell elongation and increased crystallin synthesis, we propose that it is the substance that promotes lens fiber cell differentiation in the eye and suggest the name "lentropin." We thank Lillian Magruder for preparing the manuscript and Martha Johnson, Pamela Compart, Joram Piatigorsky, Peggy Zelenka, and A. J. Coulombre for their helpful comments. D. B. extends special thanks to his wife, Betsy, for her support and for suggesting the name "lentropin." 1. Coulombre, A. J. & Coulombre, J. L. (1963) Science 142, 1489-1490. 2. Coulombre, A. J. & Coulombre, J. L. (1969) Invest. Ophthalmol. 8,251-257. 3. Philpott, G. W. & Coulombre, A. J. (1968) Exp. Cell Res. 52, 140-146. 4. Philpott, G. W. & Coulombre, A. J. (1965) Exp. Cell Res. 38, 635-644. 5. Piatigorsky, J., Webster, H. deF? & Wollberg, M. (1972) J. Cell Biol. 44, 82-92. 6. Piatigorsky, J., Rothschild, S. S. & Milstone, L. (1973) Dev. Biol. 34,334-345. 7. Piatigorsky, J. (1973) Dev. Biol. 30, 214-216.


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8. Milstone, L. M. & Piatigorsky, J. (1977) Exp. Cell Res. 105, 9-14. 9. Piatigorsky, J., Beebe, D. C., Zelenka, P., Milstone, L. M. & Shinohara, T. (1976) INSERM 60,85-112. 10. Philpott, G. W. (1970) Exp. Cell Res. 59,57-68. 11. Beebe, D. C. & Piatigorsky, J. (1977) Dev. Biol. 59, 174-182. 12. Beebe, D. C., Feagans, D. E., Blanchette-Mackie, E. J. & Nau, M. (1979) Science 206, 836-838. 13. Balazs, E. A., Toth, L. Z. J., Jutheden, G. M. & Collins, B-A. (1965) Exp. Eye Res. 4, 237-248. 14. Neal, M. W. & Florini, J. R. (1973) Anal. Biochem. 55, 328330. 15. Ham, R. G. (1963) Exp. Cell Res. 29,515-526. 16. Maisel, J. V. (1971) in Methods Virology, eds. Marmarosch, K. & Koprowski, H., (Academic, New York) Vol. 5, p. 179. 17. Piatigorsky, J., Webster, H. deF. & Craig, S. P. (1972) Dev. Biol. 27, 176-189. 18. Milstone, L. M. & Piatigorsky, J. (1975) Dev. Biol. 43,91-100. 19. Fayet, M. T. (1959) Bull. Soc. Chim. Biol. 41, 1189. 20. Laurent, V. B. G., Laurent, T. C. & Howe, A. F. (1962) Exp. Eye Res. 1, 276. 21. Cooper, W. C., Halbert, S. P. & Manski, W. J. (1963) Invest. Ophthalmol. 2, 369. 22. Arruti, C. & Courtois, Y. (1978) Exp. Cell Res. 117,283-292.