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Insulin initiation of lens fiber differentiation in culture: Elongation of embryonic lens epithelial cells
DEVELOPMENTAL
BIOLOGY
Insulin
30, 214-216
Initiation Elongation
(1973)
of Lens Fiber Differentiation
in Culture:
of Embryonic
Cells
Lens Epithelial
JORAM PIATIGORSKY Zabomtory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, U.S. Department of Health, Education, and Welfare, Bethesda, Maryland 20014 Accepted July 17, 1972
Insulin replacesserum as a stimulant of cell elongation in the cultured 6-day-old embryonic chick lens epithelium. Thus, cell elongation, which is characteristic of lens fiber differentiation, can now be studied under chemically defined conditions. This finding should facilitate further analysesof the mechanismof cell elongation. INTRODUCTION
The developing vertebrate lens is a useful system for studying cellular differentiation (Papaconstantinou, 1967; Clayton, 1970). The anterior surface of the embryonic lens consists of a dividing population of epithelial cells which elongate at the lens equator as an initial step in lens fiber formation. Cells in the excised lens epithelium of the 6-day-old chick embryo elongate in tissue culture if the medium is supplemented with fetal calf serum (Philpott and Coulombre, 1965). In the absence of serum, the cells survive for at least 10 days but fail to elongate. The lens cell elongation which takes place in culture resembles fiber differentiation in uiuo with respect to morphology (Philpott and Coulombre, 1965), nucleic acid synthesis (Philpott, 1970; Piatigorsky and Rothschild, 1971, 1972), protein synthesis and ultrastructure (Piatigorsky et al., 1972a). The present report shows that the addition of insulin
to a chemically
defined
medium
lacking serum promotes cell elongation in the cultured lens epithelium of the chick embryo.
from the embryos with sharpened jeweler’s forceps, placed into culture dishes (Falcon Plastic, 60 mm in diamter x 15 mm deep, 2 lenses per dish) containing 5 ml of Ham’s F-10 medium (Ham, 1963), and cleaned of the vitreous body and other adhering debris. The fiber mass was removed from each lens through a tear in the capsule at the lens posterior. A central square with a side of approximately 0.7 mm2 was cut from the epithelium; the peripheral regions of the epithelium were discarded. As the cuts were being made, the edges of the explants were pushed into the surface of the culture dish with the scalpel. Thus, the central regions of the lens epithelium remained anchored to the dish, with the lens capsule situated between the epithelial cells and the culture dish. The explants were cultured either in Ham’s F-10 alone, in Ham’s F-10 supplemented with insulin (Eli Lilly and Company; crystalline porcine zinc-insulin; potency 21.7 units/ml; glucagon content <0.005%; zinc content 0.49%) or in Ham’s F-10 supplemented with
MATERIALS AND METHODS
Fertilized eggs of White Leghorn chickens (obtained from Truslow Farms, Inc., Chestertown, Maryland) were incubated at 37°C in a humidified air environment. At 6 days of age, 120 lenses were removed
carbon
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
dioxide.
Protein
Biologi-
determinations
were performed calorimetrically et al., 1951).
(Lowry
The tissues were fixed for 3 min at ambient temperature with Carnoy’s fixative (3 214
Copyright All rights
fetal calf serum (Baltimore
cal Laboratories), at the concentrations given below, for 24 hr at 37°C in a water saturated air atmosphere containing 5%
215
BRIEF NOTES
parts absolute ethanol, 1 part acetic acid), embedded in Paraplast, sectioned serially at 5 p in a plane normal to the surface of the epithelium and stained with Ehrlich’s hematoxylin and eosin Y. Cell lengths were measured to the nearest 2.5 p with a calibrated ocular micrometer; the mean cell length per epithelium was obtained by averaging the length of the cells in the central third of each explant in 5 sections, spaced 6 to 8 sections apart. Photomicrographs were taken with 35 mm Panatomic X film at a magnification of 160 x. RESULTS
AND
TABLE 1 MEAN CELL LENGTH IN ~-DAY-OLD LENS EPITHELIA
CULTURED
EMBRYONIC CHICK FOR 24 HR
Insulin concentration hdml)
No. of epithelia examined
Mean cell length (p + standard error)
None
10 6
9.0 =t 0.8 9.3 f 0.4 13.8 zt 0.8
10-Z 10-Z 10-l 1 10
10 10 9
6
19.0
* 1.2
30.0 f 0.7 26.0 ziz 0.6
A
DISCUSSION
The mean cell lengths in the explanted lens epithelia were longer after 24 hr of cultivation in Ham’s F-10 medium supplemented with insulin than after culture in Ham’s F-10 alone (Table 1). The lengths of the cells in epithelia fixed immediately after explantation (not shown in the table) were not statistically different from those fixed after 24 hr of culture without insulin. The extent of elongation during 24 hr of cultivation increased with the concentration of insulin, reaching a maximum at 1 pg/ml; 10 pg/ml of insulin did not produce more elongation; rather, it appeared to be less effective than 1 pg/ml. Photomicrographs show the typical histological appearance of the lens epithelial cells after 24 hr of cultivation in Ham’s F-10 alone (Fig. 1A) or in Ham’s F-10 supplemented either with 15% fetal calf serum (Fig. 1B) or with 1 pg/ml of insulin (Fig. 1C). The nuclei were ovoid and tended to align in the center of the elongated cells after cultivation in either serum or insulin. This also occurs during fiber formation in the intact lens. The mean cell lengths in epithelia cultured with serum or with insulin were 30 CL,which represents an approximate 3-fold increase over the initial length. This is similar to the results of a previous study in which the mean cell length in 43 epithelia cultured for 24 hr with 15% fetal calf serum was 28 p (Pi-
I
I 2w
FIG. 1. Histological sections of 6-day-old embryonic chick lens epithelia cultured for 24 hr. (A) Ham’s F-10 alone. (B) Supplemented with 15% fetal calf serum. (C) Supplemented with 1 pglml (0.02 unit/ ml) of insulin. No cells were seen in mitosis after cultivation in Ham’s F-10 alone, and only about 2% of the cells contained mitotic figures after cultivation in serum or insulin.
atigorsky et al., 197213).The length and appearance of cells cultured with 1 pug/ml of insulin and 15% fetal calf serum together were the same as those cultured with insulin or serum alone. The effect of insulin on cell elongation in the cultured lens epithelium is not due to the trace amounts of glucagon or zinc in
216
DEVELOPMENTALBIOLOGY
the insulin preparation. These compounds did not stimulate elongation when applied individually or together at the same concentration as present in 1 pg/ml or 10 pug/ml of insulin. In addition, insulin is not acting simply as a nonspecific protein supplement, because elongation did not occur when epithelia were cultured with fetal calf serum diluted to 1 pg/ml of protein (0.0025% serum), or with 1 pg/ml of cytochrome c. Cytochrome c, like insulin, is a relatively low molecular weight protein (about 12,300 daltons). It is not known whether insulin is responsible for the serum-induced lens cell elongation, or if insulin and serum stimulate cell elongation by similar mechanisms. Appreciable elongation was obtained at a serum concentration as low as 40 pg/ml of protein (0.1% serum). This argues against insulin being the serum factor promoting cell elongation, since 0.1% serum would be expected (Cummingham, 1962) to contain, at most, 400 times less insulin than is required for minimal elongation when insulin alone is used as a supplement. However, serum factors could lower the concentration of insulin needed for cell elongation in culture by acting synergistically with insulin or by protecting insulin from degradation or adsorption to the culture dishes. Although the mechanism by which insulin stimulates cell elongation is not known, there are considerable data indicating that lens cell elongation in uiuo and in culture depends upon microtubule assembly (see Piatigorsky et al., 1972a, for further references). The cultured lens cells can double in length and assemble microtubules even in the absence of protein synthesis (Piatigorsky et al., 1972b). Recently, we have shown that insulin also causes microtubule assembly in the cultured lens epithelial cells (Piatigorsky, Rothschild and Wollberg, in preparation), as it does in cultured rat adipocytes (Soifer et al., 1971). Disruption of the microtubules with colchicine inhibits cell elon-
VOLUME 30. 1973
gation promoted by insulin or serum. Thus, the present evidence is consistent with the possibility that insulin initiates elongation of the cultured lens cells by stimulating the assembly of microtubules. I thank Mrs. Miriam Wollberg for expert technical help and Dr. J. M. McGuire of Eli Lilly and Company for a gift of crystalline insulin. REFERENCES CLAYTON, R. M. (1970). Problems of differentiation in the vertebrate lens. Curr. Top. Deuelop. Biol. 5, 115-180. CUNNINGHAM, N. F. (1962). The insulin activity of bovine and ovine blood plasma. II. The insulinlike action of extracts of bovine and ovine blood plasma. J. Endocrinol. 245, 43-52. HAM, R. G. (1963). An improved nutrient solution for diploid Chinese hamster and human cell lines. Exp. Cell Res. 29, 515-526. LOWRY, 0. H., ROSEBROUCH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. PAPACONSTANTINOU,J. (1967). Molecular aspects of lens cell differentiation. Science 156, 338-346. PHILPOTT, G. W. (1970). Growth and cytodifferentiation of embryonic chick lens epithelial cells in
oitro. Exp. Cell Res. 59,57-68. PHILPOIT, G. W., and COULOMBRE, A. J. (1965). Lens development. II. Differentiation of embryonic chick lens epithelial cells in vitro and in duo.
Exp. Cell Res. 38, 635-644. PIATIGORSKY, J., and ROTHSCHILD, S. S. (1971). Effect of serum on the synthesis of RNA and of DNA in the cultured lens epithelium of the chick embryo: initiation of lens fiber formation in uitro. Biochim. Biophys. Acta 240, 86-98. PIATIGORSKY, J., and ROTHSCHILD, S. S. (1972). Loss during development of the ability of chick embryonic lens cells to elongate in culture: inverse relationship between cell division and elongation. Deuelop. Biol. 28,382-389. PIATIGORSKY, J., WEBSTER, H. DEP., and CRAIG, S. P. (1972a). Protein synthesis and ultrastructure during the formation of embryonic chick lens fibers in uivo and in vitro. Develop. Biol. 27, 176189. PIATIGORSKY, J., WEBSTER, H. DEF., and WOLLBERG, M. (1972b). Cell elongation in the cultured embryonic chick lens epithelium with and without protein synthesis: involvement of microtubules. J. Cell Biol. 55, 82-92. SOIFER, D., BRAUN, T., and HECHTER, 0. (1971). Insulin and microtubules in rat adipocytes. Science 172, 269-271.
BIOLOGY
Insulin
30, 214-216
Initiation Elongation
(1973)
of Lens Fiber Differentiation
in Culture:
of Embryonic
Cells
Lens Epithelial
JORAM PIATIGORSKY Zabomtory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, U.S. Department of Health, Education, and Welfare, Bethesda, Maryland 20014 Accepted July 17, 1972
Insulin replacesserum as a stimulant of cell elongation in the cultured 6-day-old embryonic chick lens epithelium. Thus, cell elongation, which is characteristic of lens fiber differentiation, can now be studied under chemically defined conditions. This finding should facilitate further analysesof the mechanismof cell elongation. INTRODUCTION
The developing vertebrate lens is a useful system for studying cellular differentiation (Papaconstantinou, 1967; Clayton, 1970). The anterior surface of the embryonic lens consists of a dividing population of epithelial cells which elongate at the lens equator as an initial step in lens fiber formation. Cells in the excised lens epithelium of the 6-day-old chick embryo elongate in tissue culture if the medium is supplemented with fetal calf serum (Philpott and Coulombre, 1965). In the absence of serum, the cells survive for at least 10 days but fail to elongate. The lens cell elongation which takes place in culture resembles fiber differentiation in uiuo with respect to morphology (Philpott and Coulombre, 1965), nucleic acid synthesis (Philpott, 1970; Piatigorsky and Rothschild, 1971, 1972), protein synthesis and ultrastructure (Piatigorsky et al., 1972a). The present report shows that the addition of insulin
to a chemically
defined
medium
lacking serum promotes cell elongation in the cultured lens epithelium of the chick embryo.
from the embryos with sharpened jeweler’s forceps, placed into culture dishes (Falcon Plastic, 60 mm in diamter x 15 mm deep, 2 lenses per dish) containing 5 ml of Ham’s F-10 medium (Ham, 1963), and cleaned of the vitreous body and other adhering debris. The fiber mass was removed from each lens through a tear in the capsule at the lens posterior. A central square with a side of approximately 0.7 mm2 was cut from the epithelium; the peripheral regions of the epithelium were discarded. As the cuts were being made, the edges of the explants were pushed into the surface of the culture dish with the scalpel. Thus, the central regions of the lens epithelium remained anchored to the dish, with the lens capsule situated between the epithelial cells and the culture dish. The explants were cultured either in Ham’s F-10 alone, in Ham’s F-10 supplemented with insulin (Eli Lilly and Company; crystalline porcine zinc-insulin; potency 21.7 units/ml; glucagon content <0.005%; zinc content 0.49%) or in Ham’s F-10 supplemented with
MATERIALS AND METHODS
Fertilized eggs of White Leghorn chickens (obtained from Truslow Farms, Inc., Chestertown, Maryland) were incubated at 37°C in a humidified air environment. At 6 days of age, 120 lenses were removed
carbon
0 1973 by Academic Press, Inc. of reproduction in any form reserved.
dioxide.
Protein
Biologi-
determinations
were performed calorimetrically et al., 1951).
(Lowry
The tissues were fixed for 3 min at ambient temperature with Carnoy’s fixative (3 214
Copyright All rights
fetal calf serum (Baltimore
cal Laboratories), at the concentrations given below, for 24 hr at 37°C in a water saturated air atmosphere containing 5%
215
BRIEF NOTES
parts absolute ethanol, 1 part acetic acid), embedded in Paraplast, sectioned serially at 5 p in a plane normal to the surface of the epithelium and stained with Ehrlich’s hematoxylin and eosin Y. Cell lengths were measured to the nearest 2.5 p with a calibrated ocular micrometer; the mean cell length per epithelium was obtained by averaging the length of the cells in the central third of each explant in 5 sections, spaced 6 to 8 sections apart. Photomicrographs were taken with 35 mm Panatomic X film at a magnification of 160 x. RESULTS
AND
TABLE 1 MEAN CELL LENGTH IN ~-DAY-OLD LENS EPITHELIA
CULTURED
EMBRYONIC CHICK FOR 24 HR
Insulin concentration hdml)
No. of epithelia examined
Mean cell length (p + standard error)
None
10 6
9.0 =t 0.8 9.3 f 0.4 13.8 zt 0.8
10-Z 10-Z 10-l 1 10
10 10 9
6
19.0
* 1.2
30.0 f 0.7 26.0 ziz 0.6
A
DISCUSSION
The mean cell lengths in the explanted lens epithelia were longer after 24 hr of cultivation in Ham’s F-10 medium supplemented with insulin than after culture in Ham’s F-10 alone (Table 1). The lengths of the cells in epithelia fixed immediately after explantation (not shown in the table) were not statistically different from those fixed after 24 hr of culture without insulin. The extent of elongation during 24 hr of cultivation increased with the concentration of insulin, reaching a maximum at 1 pg/ml; 10 pg/ml of insulin did not produce more elongation; rather, it appeared to be less effective than 1 pg/ml. Photomicrographs show the typical histological appearance of the lens epithelial cells after 24 hr of cultivation in Ham’s F-10 alone (Fig. 1A) or in Ham’s F-10 supplemented either with 15% fetal calf serum (Fig. 1B) or with 1 pg/ml of insulin (Fig. 1C). The nuclei were ovoid and tended to align in the center of the elongated cells after cultivation in either serum or insulin. This also occurs during fiber formation in the intact lens. The mean cell lengths in epithelia cultured with serum or with insulin were 30 CL,which represents an approximate 3-fold increase over the initial length. This is similar to the results of a previous study in which the mean cell length in 43 epithelia cultured for 24 hr with 15% fetal calf serum was 28 p (Pi-
I
I 2w
FIG. 1. Histological sections of 6-day-old embryonic chick lens epithelia cultured for 24 hr. (A) Ham’s F-10 alone. (B) Supplemented with 15% fetal calf serum. (C) Supplemented with 1 pglml (0.02 unit/ ml) of insulin. No cells were seen in mitosis after cultivation in Ham’s F-10 alone, and only about 2% of the cells contained mitotic figures after cultivation in serum or insulin.
atigorsky et al., 197213).The length and appearance of cells cultured with 1 pug/ml of insulin and 15% fetal calf serum together were the same as those cultured with insulin or serum alone. The effect of insulin on cell elongation in the cultured lens epithelium is not due to the trace amounts of glucagon or zinc in
216
DEVELOPMENTALBIOLOGY
the insulin preparation. These compounds did not stimulate elongation when applied individually or together at the same concentration as present in 1 pg/ml or 10 pug/ml of insulin. In addition, insulin is not acting simply as a nonspecific protein supplement, because elongation did not occur when epithelia were cultured with fetal calf serum diluted to 1 pg/ml of protein (0.0025% serum), or with 1 pg/ml of cytochrome c. Cytochrome c, like insulin, is a relatively low molecular weight protein (about 12,300 daltons). It is not known whether insulin is responsible for the serum-induced lens cell elongation, or if insulin and serum stimulate cell elongation by similar mechanisms. Appreciable elongation was obtained at a serum concentration as low as 40 pg/ml of protein (0.1% serum). This argues against insulin being the serum factor promoting cell elongation, since 0.1% serum would be expected (Cummingham, 1962) to contain, at most, 400 times less insulin than is required for minimal elongation when insulin alone is used as a supplement. However, serum factors could lower the concentration of insulin needed for cell elongation in culture by acting synergistically with insulin or by protecting insulin from degradation or adsorption to the culture dishes. Although the mechanism by which insulin stimulates cell elongation is not known, there are considerable data indicating that lens cell elongation in uiuo and in culture depends upon microtubule assembly (see Piatigorsky et al., 1972a, for further references). The cultured lens cells can double in length and assemble microtubules even in the absence of protein synthesis (Piatigorsky et al., 1972b). Recently, we have shown that insulin also causes microtubule assembly in the cultured lens epithelial cells (Piatigorsky, Rothschild and Wollberg, in preparation), as it does in cultured rat adipocytes (Soifer et al., 1971). Disruption of the microtubules with colchicine inhibits cell elon-
VOLUME 30. 1973
gation promoted by insulin or serum. Thus, the present evidence is consistent with the possibility that insulin initiates elongation of the cultured lens cells by stimulating the assembly of microtubules. I thank Mrs. Miriam Wollberg for expert technical help and Dr. J. M. McGuire of Eli Lilly and Company for a gift of crystalline insulin. REFERENCES CLAYTON, R. M. (1970). Problems of differentiation in the vertebrate lens. Curr. Top. Deuelop. Biol. 5, 115-180. CUNNINGHAM, N. F. (1962). The insulin activity of bovine and ovine blood plasma. II. The insulinlike action of extracts of bovine and ovine blood plasma. J. Endocrinol. 245, 43-52. HAM, R. G. (1963). An improved nutrient solution for diploid Chinese hamster and human cell lines. Exp. Cell Res. 29, 515-526. LOWRY, 0. H., ROSEBROUCH,N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. PAPACONSTANTINOU,J. (1967). Molecular aspects of lens cell differentiation. Science 156, 338-346. PHILPOTT, G. W. (1970). Growth and cytodifferentiation of embryonic chick lens epithelial cells in
oitro. Exp. Cell Res. 59,57-68. PHILPOIT, G. W., and COULOMBRE, A. J. (1965). Lens development. II. Differentiation of embryonic chick lens epithelial cells in vitro and in duo.
Exp. Cell Res. 38, 635-644. PIATIGORSKY, J., and ROTHSCHILD, S. S. (1971). Effect of serum on the synthesis of RNA and of DNA in the cultured lens epithelium of the chick embryo: initiation of lens fiber formation in uitro. Biochim. Biophys. Acta 240, 86-98. PIATIGORSKY, J., and ROTHSCHILD, S. S. (1972). Loss during development of the ability of chick embryonic lens cells to elongate in culture: inverse relationship between cell division and elongation. Deuelop. Biol. 28,382-389. PIATIGORSKY, J., WEBSTER, H. DEP., and CRAIG, S. P. (1972a). Protein synthesis and ultrastructure during the formation of embryonic chick lens fibers in uivo and in vitro. Develop. Biol. 27, 176189. PIATIGORSKY, J., WEBSTER, H. DEF., and WOLLBERG, M. (1972b). Cell elongation in the cultured embryonic chick lens epithelium with and without protein synthesis: involvement of microtubules. J. Cell Biol. 55, 82-92. SOIFER, D., BRAUN, T., and HECHTER, 0. (1971). Insulin and microtubules in rat adipocytes. Science 172, 269-271.