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Crystallin synthesis by Chick Lens
Exp. Eye Res. (1971) 11, 184-194
C ystallin Synthesis by Chick Lens II. Changes in Synthetic Activities of Epithelial and Fiber Cells during Embryonic Development KAZUO ~ O S H I D A AND AI,~'['I-iUR. KATOH
Onc~)logy Laboratory, Division of Radiotherapy, Mercy Hospital, Pittsburgh, P~n~ylvania 15219, U.S.A. (Received 3 August 1970, Be,~ton) C r y s t a l l i n %vnthesis b y epithelial a n d fiber cobs f r o m 7- t o 18-d,~y-old c h i c k e n e m b r y o lenses was i n v e s t i g a t e d b y cellulose at~otate m e m b r a n e electrophore~is. E p i t h e l i a l cells o f 7- t o 12-d~y-old e m b r y o lenses s y n t h e s i z e m a i n l y 8 eryst~llin a n d a small a m o u n t o f ~ - a n d fl.eryatallins; t h e s e are all s e n s i t i v e t o a c t i n o m y c i n D (30,ug/ml). As t h e lens d e v e l o p s f r o m 12 t o 18 d a y s , epithelial cells b e c o m e i n s e n s i t i v e t o a e t i n o m y e i u D in r e s p e c t t o t h e svn.thesis o f ~- a n d fl-eryst~llins. A t t h e s a m e t i m e , t h e s y n t h e s i s o f 6-cryst~llin ceases. On t h e o t h e r h a n d , t h e fiber cell s y n t h e s i z e s p r i m a r i l y $-cryst~llin w i t h a d d i t i o n a l small a m o u n t s o f ~.a n d fl-eryst~llins; those a r e all i n s e n s i t i v e t o a v t i n o m y c i u D d u r i n g all stages o f t h e d e v e l o p m e n t o f t h e lenu f r o m 7 to 18 d~vs.
1. Introduction Tile v e r t e b r a t e lens, derived exclusively from ectoderm, is a co~nposite of three m~jor zones: the outer, anterior, single layer of epithe!ial cells; the t ~ n s i t i o n a l cuboidal (:ells of the a n n u l a r pad, which are in the process of elongation a t the equatorial region; and t h e fully elongated fiber cells .~n the lens body. Cell division is restricted to a nan'ow b a n d of epithelial cells at, the pre-equatorial region. The resulting, d a u g h t e r cells elongate a n d enter the body of the lens where the d.ifl~rentiation into secondary fiber cells is completed. :Fl~rther g r o w t h of the lens takes place by t h e continual, conc~entric addition of elongated fiber cells a t t h e periphery (Coulombre, 1965). The conversion of the lens epithelial cells into the fiber cell provides a suitable system, for s t u d y i n g cell differentiation, in t h a t it represents a progressive diversification of morphological structure associated w i t h t h e acquisition of synthetic capa c i t y for tissue-specific proteins ( P a p a c o n s t a n t i n o u , 1967). As such it has been utilized b y S t e w a r t a n d F a p a c o n s t a n t i n o u (1967) who showed t h a t a stabiliz~tion of m R N A t e m p l a t e s occurs during the conversion of epithelial into fiber cells in the bovine lens. This wins established b y t h e use of actinomycin :D a t doses inhibiting ~ll R h e a syn thesis, which resulted in abolishing crystallin synthesis in epithelial cells b u t h a d no effect on t h e fiber cell's capacity for protei~ synthesis. Using wotinomycin D on t h e 12-day-old chick embxTo lens, Reeder a n d Bell (1967) :~howed t h a t m o s t epithelial cell proteins are m a d e on short-lived m_RNA t e m p l a t e s . As t h e epit2aclial cell enters t h e body region and. elongates to fiber cell, actinomycin D-resist~nt protein synthesis, which is restricted t o two proteins, is directed b y longlived m.R2NA. U n f o r t u n a t e l y , t2ae specific lens proteins concerned were n o t identified in tJaese experiments. Recently, we introduced a t e o ~ i q u e utilizing cellulose acetate m e m b r a n e electrophoreais f o r the analysis a n d identification of radioactive chicken lens oryst~llins 184
CRY S T A L L I N SY~'TI-IESIS BY C H I C K LENS
185
(Yoskida a n d K a t o h , 197i). I a this p a p e r , we p r e s e u t s t u d i e s or: t h e effect of a c t i n o my'tin l0 on c r y s t a l i i n s y n t h e s i s duxing lens d e v e l o p m e n t . I n t h e 7- to 12-day-old c h i c k e m b r y o lens, e p i t h e l i a l cell. p r o ~ i n s y n t h e s i s , w h i c h is d o m i n a t e d b y $-, a l o n g w i t h s m a l l amomxt~ of a- a n d fl-crystallins, is d i r e c t e d by s h o r t - l i v e d m R N A s . As the lens d e v e l o p s f r o m 12 to 18 d a y s , t h e e p i t h e l i a l cells s y n t h e s i z e s i g n i f i c a n t a m o u n t s of ~- mxd fl-crystallins, w h i c h are n o w botJa m a d e on Iong-llved m R N A s . Conconfikuntly', t h e s y n t h e s i s of 8 - c r y s t a l l i n coo.sos. The fiber cells, o n t h e ot]mr h a n d , are e n g a g e d p r i m a r i l y i n t h e s y n t h e s i s of ~c r y s t a l l i n a l o n g w i t h s m a l l a m o u n t s of ~- a n d f l - e r y s t a l l i n s d a r i n g ~1I !ztages of e m b r y onto d e v e l o p m e n t b e t w e e n 7 a n d 18 d a y s . F u r t h e r m o r e , t h e s ~ fiber cell p r o t e i n s are all directed b y long-lived nLRNAs. T h e r e l a t i o n s h i p b e t w e e n t h e s y n t h e s i s of c r y s t a l l i n s and t h e d e v e l o p m e n t o f t h e lens is discussed. 2. M a t e r i a l s and M e t h o d s
Isotope i.ncorporation Lenses of "White Leghorn chicken embryos, r~,.nging from 7 to 18 d,~ys of incubation, were used t h r o u g h o u t this study. For the incorporation of radioactive a,aino acids into le~s proteins, 25-30 lenses were gently r o t a t e d in 20-ml Erlenmeyer flasks :containing 10 ml of Eagle's m i n i m u m essential medium (ME.3~), which was supplemented v~th 50/xCi; of ltC-reconstituted protein h y d r o l y s a t e (I'tC-RPH) (Schwarz BioResea~ch, Orangeburg, N.Y., U.S.A.) and penicillin a n d streptomycin, 100 u~lits/ml each (Difco, .Detroit, MicMgan, U.S.A.). The flasks were incubated a t 37°C in a hmnidified atmosphere of 5~/o CO.. in 95:/0 air. A_ftcr the culture period, which ranged from 1 to 23 hr: the lenses were washed w i t h MEM, I t a n k ' s balanced salt solution (BSS), a n d t h e n homogenized w i t h BSS. E q u a l volumes of cold 10°/,~ trichloroacetic acid (TCA) were added to the homogenaVes. T h e y were t h e n i n c u b a t e d a t 90°0 for 15 miu. The TCA precipitates were separated from the supernatauts by centrifugation. The pellets a n d s u p e r n a t a u t s were collected o~ glass membrane filter discs a n d prepared for scintillation counting as those of fz'ee amino acids and proteins. The il.~corporation of [3H]uridinc (20 ~LOi/nll, Schwarz BioResearch) was measured with a.ud withotlt a c t i n o m y c i n D (30 #,g/ml, Calbiochem., Los Angeles, Calif., U.S.A.). Aft~r the culture, the lenses were washed as above a n d homogenized with 5 % cold TC.4,. The homogenates were centrifuged a t 10,000 g for 20 rain a n d the radioacti~-itiee of the supel:n~ttants and pellets counted as those of free uriclines a n d RNAs.
Preparation of chicke~ l~z~.sextracts for electroplmresis Lenses of 7-18 d a y s of incubation were g e n t l y r o t a t e d i n 10 nfl of ~ E b l containing: 50 ~Oi of I*C-:RPtI a n d antibiotics a t 37°0 as described above. K c t i n o m y c i n D was added at a concentration of 30/~g/ml to the t r e a t e d cultures. After th~ culture period, the lenses were washed succe~ssively w i t h MEM, and w i t h BSS. Lenses were t h e n dissected into epitheli,~l and fiber cells w i t h w a t c h m a k e r ' s forceps under a Zeiss stereoscopic microscope. The lenses or dissected cells were homogenized alld their proteins were extracted a n d applied to Sepraphore FII cellulose acetate membranes for electrophoresis as described previously (Yoshida a n d K a t o h , 1971). P i e r , in content of epithelial a n d fiber cell e x t r a c t s was d e t e r m i n e d as follows: t h e ceil extracts were m a d e up to 5~/o TCA a n d i n c u b a t e d a t 90°G for 15 rnin. The h o t TGAlarecipitares were washed ~-ith ethanol, e t h a n o l :ether {3:1 v/v), and w i t h ether. The washed laellet~ were t h e n dissolved in 1 ~¢ N a O H . The protein conVent of the solutions was e s t i m a t e d b y Lo~wry's m e t h o d (Lowry, Rosebrough, iTarr a n d Randall, !951), a n d i t s r a d i o a c t i v i t y we.~ cornered in a P a c k a r d scintiUation caimt~r to determine the specific a ~ t i v i t y of the protein sample.
186
~,:. Y O S H I D A
AND
A. ] K A T O H
3. Results
Crystallin synthesis during tlw. d~velopment of the lens Inv.orpara~ion of amino .avids into lens and stability of lens proteins. Radioactive a m i n o acids were l i n e a r l y incorporated into proteins of the 7-day-old e m b r y o lens for at least 23 h r w i t h o u t significant lag time, as shown in Fig. l(a). B y this criterion t h e lens appears to sl~rvive the in vitro culture conditions of our experiments. :Fignre l(b) shows t h a t radioactive a m i n o acids are r a p i d l y incorporated into the pool of the lens w i t h i n t h e first hour, a n d t h a t t h e pool was a h n o s t saturated w i t h i n 2 hr. In lenses of 10-, 12- ~nd 17-day-old embryos, similar incorporation curves of radioactive a m i n o acids were obtained. The s t a b i l i t y of lens proteins was checked b y labeling lenses w i t h ~C-t2.PH for 1 or {o)
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~ G . l. (a), (b) I n c o r p o r a t i o n o f r a d i o a c t i v e a m i n o acids i n t o c h i c k e m b r y o lens, L e n s e s o f 7 - d a y - o k t c h i c k e m b r y o s w e r e c u l t u r e d in 10 m l of ~ I E M , w h i c h w a s s u p p l e m e n t e d xvigh 1.dO-I~PH (50/~Ci). A t i n t e r v a l s , lenses ~,ere w i t h d r a w n f r o m t h e c u l t u r e m e d i u m , a n d s a m p l e d for r a d i o a o t i v i b y m c a s u r c m e n t ~ . I n t h e p u l s e - c h a ~ e x p e r i m e n t s , lenses l a b e l e d w i t h ~¢C-RPH for 1 h r or 5 h r (arrows) were w a s h e d e x t e n s i v e l y w i t h M E M a n d t r a n s f e r r e d i n t o fresh M E M . AS i n t e r v a l s , lenses w e r e w i t h d r a w n f r o m t h e c u l t u r e raerlium a n d s e p a r a t e d i n t o (a) p r o t e i n a n d (b) a c i d soluble f r a c t i o n s . A, c o n t i n u o u s label; t], 5 h r label-18 h r chase; a n d C, 1 b r label-22 h r chase.
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.Fz(~. 2, (a), (b) I n c o r p o r a t i o n o f r a d i o a c t i v e a m i n o a c i d s i n t o epiShelial a n d fiber cells o f chick e m b r ) , o l e n s . L e n s e s f r o m v a r i o u s a g e s o f c h i c k 6 m b r y o s w e r e c u l t u r e d w i t h z~C-I~PH (5 iLCi/ml) fox" 5 hr. Af~e.r t h e c u l t u r e , l e n s e s , w e r e w a s h e d a n d d i s s e c t e d i n t o epithelial a n d fiber~cells, a n d t h e p r o t e i n s e x t r a c t e d . T o t a l r a d i o i s o t o p e ixlcorporatiou i n t o p r o t e i n s o f epithelial a n d .fiber cells p e r lens (a), a n d specific a c t i v l k y o f t h e larotein'~ 114) w e r e d e t e r m i n e d a s d e s c r i b e d in i~Iaterials a n d ~ [ e t h o d s . A, ~Fibcr cells; B, epithelial cells.
CRYSTALLI:K
SYi~T]-IESIS
BY CHICK
187
LENS
5 hr, washing thoroughly with MEM, and chasing in radioisotope-free 5'IE~ for 22 or 18 hr, respectively. As shown in :Fig. 1(~), the radioactivity that was incorporated int~) lens proteins was Mmost completely retained. On the other hand, the radioactivity t h a t entered the amino acid pool d~s'~ppeared ahnost instantaneously '~luring the wash and chase periods. (el
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l ~ o . 3 . ( a ) , ( b ) , ( e ) , ( d ) , ( e ) , ( f ) ~leot..~)phoret,]c ~ ' " profiles o f e x t r a c t s ot" epitheliM cells o f chick e m b r y o lens. 3 $¢g P r o t e i n of epithelial cell e x t r a c t s p r e p a r e d in Fig. 2~vere a p p l i e d o n t o 'cellulose a~et~t~, m e m b r a n e electrophoresis. A n o d e is o n t h e r i g h t side o f t h e Bgeres, 0 is origin. (a) 7 - d a y . o l d e m b r y o lens; (b) 10-d~yo l d e m b r y o lens; (c) 12-day-old e m b r y o lens; (d) 16-day-old e m b r y o lens; (e) 17-day-old e m b r y o lens ; (f) 18-day-01d e m b r y o l e ~ / C) O , R i g h t o r d i n a t e ; Q - - O , lef6 o r d i n a t e .
188
K. YOSI-][II)A
AND
A. KATOtl
Synthesi.s. of c'rystalIins du:ri~vj the develoTment of the Imz~. In order to follow the patterns of cryst, Mlin synghesis b y epithelial and fiber cells, lenses Of embryos ranging from 7 to 18 d a y s of incubation were ¢~uI~uxed wi~h ~ C - R P H for 5 hr. After the culgure 'period, lenses were washed,'diss.e.cged int~ epighelial a n d fiber ceils, a n d tKeix' proteins extracted. Total isotope incorporation into epigheliaI a n d fiber cells and ghe specific (o1~
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3 ~g Protein of fib~ cell extract~ prepared in Fig. 2 wer~ applied onto cellulose acetate m e m b r a n e eloctrophoreMs.'A~dde is on the righ~ of the figures. 0 iv,origin. (a) 7-day-old embryo lens; (b) IO-day-old e m b r y o l e n s ; (o)'.'~!'.~';day-old e m b r y o l e n s ; (d) 1 6 - d a y - o ! d e m b r y o l e n s ; (e) IT-davy-old e m b r y o l e n s ; (f) 1 8 . d a y - o l f l e m b r y o lens.
CYCYSTALLI:N
SYNTHESIS
BY
CHIOK
189
LENS
a c t i v i t y of their e x ~ a e t e d proteins were determined before application onto cellulose acetate m e m b r a n e s for electrophoresis. Isotope incorporation into proteins of the epithelial a n d fiber cells increases linearly w i t h age of the lens [:Fig. 2(a)]. On the other hand, the specific a c t i v i t y of their proteins decreases linearly with age of the lens, as shown in Fig. 2(b). This is consistent with the results of the previous section where it was shown t h a t lens proteins are very stable, and theft newly synthesized proteins are diluted with accumulated stable proteins.
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F l a . 5. (a), (b) C h a n g e s o f s y n t h e t i c a c t i v i t y o f crs, st.allins b y e p i t h e l i a l a n d f i b e r cells d u r i n g t h e (level,,i)men$ o f t h e lens. (a) R a t i o s ( ~ f i n e o r p o r a t i o t l o f r a x l i o a e t i v e a m i n o a c i d s i n t o ¢t-, fl- a n d $ - c r y s t a l l i i m t o t o t a l i n c o r p o r a t i o n i n t o p r o t e i n s o f f i b e r cells for 5 h r w e r e c a l c u l a t e d f r o m t h r e 6 d i f f e r e n t e x p e r i m m ~ t a i d a t a , o n e o f w h i c h is s h o w n in F i g , 4. T h e v a l u e s w e r e p l o t t e d a g a i n s t t h e ag(~ o f t h e l(:nsea. (b) S a m e c a l c u l a t i o n a s (a) w a s e ~ r r i e d o u t f o r c l e c t . r o p h o r e t i c p r o f i l e s o f e p i t h e l i a l cells. ['_]~f-q, $ - C r 3 - s t a l l i n ; ® ~ O, fi-cry~tallin ; A--A, ~-erystallin.
Electrophoretic :profiles of epithelial cell extracts are shown in :Fig. 3. L1 epithelial ceils of the 7-.day-old embryo lens, ~-crystallin picks up most of the r a d i o a c t i v i t y [Fig. 3(a)] hut dm.ing further development, of the lens up to 18 days, this a c t i v i t y is gr~:[ua, lly lost,. Dm'ing the same time period, a- and fl-crystallins show a -,radual increase of synthetic activity [see Fig. 3(c), (d), (e), (f)]. Note t h a t the broken lines of Fig. 3(c)-(f) are drawn to a (lifferen£ scale in order to magni£y the activities of ae.nd fl-crystallins and to facilitate the comparison of their synthetic ratios. I n the fiber cells on the other hand, most of the radioactivity is found in 8-cry-stal!in, ~nd only a small a m o u n t of r a d i o a c t i v i t y is evident in ~.- and fl-crystallin regions during all stages of the development of the lens studied here [Fig. 4(a)-(f)]. The resu/~s also show t h a t the specific a c t i v i t y of the crystallins from both epithelial a n d fiber cells decreases w i t h age, because the same a m o u n t of protein samples (3 fig) was applied onto electrophoresis (see solid lines of :Figs 3 a n d 4). This is consistent with the results described in the previous section. Ck-ystMlins are stable proteins, as described above. Therefore, the synthetic a c t i v i t y of the ¢rystallins of epithe!i,~l and fiber cells can be expressed b y ~he ratio of the incorporation of radioactive amido acids into crystallins to t h a t intm lens proteins for 5 in-. The m e a n values are c~[culated from three different experimental data, one of which is shown in F i g s 3 and 4. This is t h e n plotted aga;mst t h e age of the lenses " (Ii'ig. 5). The results indicate t h a t fiber cells m a i n t a i n t h e same capaqity for crystalhn
190
K. YOSHIDA
AND
A. K & T O H
synthesis ~nd primarily synthesize 8-oryst~Ilia ~]~roughout the development of l;he lens [Figs 5(~) and 4]. On tim other h~md, epitholiM cells gradually lose t.hoir capacity for 8-crysttdlin syntltesis while g,~ining that for ct- ~nd fl-eryst,~llin [Figs 5(b) ~nd 3]. EinMly, epitimlbd cells primarily syut.hesize ~- ~nd fl-erystMlins, whereas fiber cells prim~rily synthesize 8-cryst,allin. I
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J"[o. 6. (a), (b), (e) Electrophoretio profiles of epithelia.l cells from !cases l.rcated with actinomycia D. Lenses prepared from e m b r y o s of various age.s were cultured under the same conditions as described in Fig. 2, except, th.'~t. actinomycin D was pre~sent a t a coneeatr~tti.or~ of 30 pg/ml. T h e extracts from epithelial cells were applied onto cell ul f,so acetate m e m b r a n e s for eleetrophorcsi~. Anode is on tim right side of the figures. O is origin. (a) 7-thKy-old e m b r y o lens. (b) 12-day-old e m b r y o Ions; (c) 16-d~y-old e m b r y o lens. O . . . . O , R;.ght ordinate; O ~ @ , I~eft ordinat.e.
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Fro. 7. (a), (b), (e) Electrophoretie profiles of extracts of fiber cells from lenses t r e a t e d with ac4inomyein D. The extraet~ from fiber cells, which svero p r e p a r e d as indies.ted in Fig. 6, were applied onto cellr:!ose acetate mem'branea for eleetrophore~is. Anode is on the right of the figures. 0 is origin. (a) 7.day-old e m b r y o lens; (b) 12-day-ohl e m b r y o lens; (c) 16.d~y-old e m b r y o lens.
CI'¢YSTALLI-.-N SYNTI~IESIS
BY C}IICI(
LENS
191
Effects of actinomycin D on crystallin synthesis Zen~ses labeled with laO-am.ino acids in the presence of actinomycin D. In order to study the effects of actinomycin D on crystallin zyntlmsis, lenses from 7- to 18-day-old embryo.~ were cultured with I'~C-]~PH: and their epithelial and fiber cell extracts were analyzed by electrophoresis, as described in Fig. 2, with the exception t h a t actinomyein D (30 Fg/ral) was present during the culture ~ime. Crystallin synthesis by epithelial cells of 7- and 12-day-old embryo lens was almost completely inhibited by actinomyein D [Fig. 6(~) and (b); compare Fig. 3(a) and (c) as controls], t:[owever, crystallin synthesis by fiber cells was not arrested by actinomyci~ D [Fig. 7(a) and (b); compare Fig. 4(a) and (c) as controls]. On the other han~l, fin lenses of 16-day-old embryos, aetinomycin D did not inhibit the synathesis of cry~tallins by both fiber and epithelial ceils, but rather showed a stimulatory effect [Figs 6(c) and 7(c) ; compare to Figs 3(d) and 4(4)]. The effects of actinomycin D on crystallin synthesis by 17- and 18-day-old embryo lenses were similar to those shown by the 16-day-old embryo lens. The resu/ts of Fig. 6(a) and (b) also indicate that the separation of epithelial and fiber cells was clcanly executed since no contaminating radidactivity of 8-crystallin from fiber cells appeared in the electrophoretic profile of the epithelial cells treated with ac~inomycin D. Incorporation of radioactive uridine into R N A of le.n.ses in the wesence of acti~wmycin D. To check whether actinomycin D really arrestc~I RNA synthesis of lens under our culture conditions, lel~.es of 1.7-day-old embryos were cultured with [aId"Juridine in the presence or absence of actinomycin D (30/zg/ml). As shown in .Fig. 8(a) radioactive uridine is rapidly incorporated into the pool of l~ridine and R N A ibr 5 hr. ~kfter 5 hr of cultl~re, the incorporation r~te levels off but nonetheless remains constant for at least 25 b.r. The effect of acCinomycin D on incorporation of [aH]uridine into 17-day'-old embryo lens is sltown in Fig. 8(b). It is evident tLtat aetinomycin D almost completely
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F*G. 8. (a), (b) I n c o r p o r a t i o n o f [aH]uridino i n t o lens in the presence (b) or a b s e n c e (a) o f a e t i n o m y c i n D. Lenses p r e p a r e d f r o m 17.day-o~d e m b r y o ~ were c u l t u r e d with [3H]uridino (20/xCi/ml) in t h e presence or absence o f a e t i n o m y c i n D (30 btg/ml). A f t e r t h e eul~ur~ interval, lenses were w i t h d r a w n tkom t h e m e d ium, w a s h e d with I~,IEM, a n d w i t h BSS t t i o r o u g h l y . T.he lens h o m o g e n a t e s were m i x e d w i t h e q u a l v o l u m e o f cold 10°/o TCA. T h e h o m o g e n a t c s were s e p a r a t e d into pellets a n d s u p e r n a t ~ n t , s b y oe.ntrifugation, a n d collected on glass m e m b r a n e .filter discs a n d their radioaetivitiea c o u n t e d . Q - - @ , Pellets ( R N A ) ; O - - - C), s u p e r n a t a n t ~ (,~eid soluble fraction).
192
K. YOSHII)A
AND
A. KATOH
arrests the synthesis o f R N A in lenses u n d e r our e x p e r i m e n t a l conditions. However, a c t i n o m y c i n D does not influence the incorporation of [~H]uridino into the pool of the lens. Similar results were obtained for t h e 7-day-old e m b r y o lens. 4. Discussion
Caution m u s t be exercised in the use of" a c t i n o m y c i n D since it call be v e r y toxic to presumptive lens explants treated w i t h low concentrations such as 0-20/~g/ml for 1 h r (Katoh, B r a v e r m a n a n d Yo~hido,, u n p u b l i s h e d data). However, t h e effects of actinom y e i n D on the lens in this study are of a different natm'e, because morphological integrity and isoix)pic incorporation of a m i n o acids and l~ridlne into pools of the lenses were m a i n t a i n e d as described above. This indicates a~ least t h a t a c t i n o m y e i n D has not d a m a g e d cell m e m b r a n e s . Furthermore, the d a t a presented here show t h a t aotin om y e i n D suppresses R N A synthesis in epithelial .cells from 7- to 12-day-old e m b r y o lenses. .We described in the Results ~hat the separation of epithelial cells ~zom fiber cells was cleanly exec~uted. However, a n n u l a r p a d cells were not removed from both groups. Therefore, epithelial cells, as d~seribed in this paper, p r o b a b l y correspond to the replicating cells which consist of central epithelial, peripheral epithelial, a n d outer a n n u l a r p a d cells, as described b y Persons a n d Modak (1970). The fiber cells correspond to the non-replicating cells, which include middle annular, inner annu]~.r pad ancl fiber cells. As shown here, the stabilization of m R N A for crystallin synthesis occurs in b o t h epithelial a n d fiber cells. The stabilization of mRN2L in t h e fiber cell has a l r e a d y occurred in the 7-day-old e m b r y o lens. On the other hand, m R N A for crystallil~ synthesis b y epi~heliaI cells is stabilized between 12 a n d 16 days. More correctly, the stabilization occurs during the development of the lens between 12-13 d a y s according to our most recent results, which aze n o t included in this paper. Therefore, the stabilization m e c h a n i s m m a y be in ol~eration dm~h~g t h a t critical period. As described in t h e Introduction, t h e stabilization of m R l ~ A has been considered to be one of the characteristics of tezmlinal cellular differentiation (Reeder a n d Bell, 1965; P~paconstantinou, 1967). Stewart and P a p a c o n s t a n t i n o u (1967) h~ve suggested t h a t the stabilization of m R N A is associated w i t h loss of mitotic or replicating P~tivity of cells. I n order to check tb]s possibility, we e x a m i n e d in detail the data of Persons ~nd Mod~k (1970) a n d plotted the mitotic a c t i v i t y of epithelial cells versus the age of the lens (:Fig. 9). There appears to be no s i g h t , c a n t difference between the mitotic or replicating a c t i v i t y of epithelial cells from 12-, 13-, 14- a n d 16-day-old e m b r y o lenses, which still r e t a i n signi~cant mitotic activity. This suggests t h a t the stabilization of r a R N A is not entirely associated w i t h t h e loss of mitotic activ~ity, as suggested b y Stewart a n d P a p a c o n s t a n t i n o u (1967). Thus, the association of the stabilization of m R N A w i t h t h e loss of mStotic a c t i v i t y appears to need careful re-examination. Reeder ~nd Bell (1967) analyzed lens proteins of the l ~ d ~ y - o l d chicken e m b r y o b y p o l y a c r y l a m i d e gel electrophoresis i n t h e pzesence of urea, w h i c h is a strong denaturation agent of p~0tein. T h e y f o u n d thxee p r e d o m i n a n t p r o t e i n b a n d s (1, 2, 3) in the lens extracts. I n comparison w i t h our results, b a n d 3 is p r o b a b l y a s u b u n i t of c~and/or fl-crystallins. B a n d s 1 a n d 2 a~e p r o b a b l y subunits of 8-erystallin. Our d a t a indicate t h a t there are at least two kinds o£ c¢-, fl- a n d 8-crystallins, respectively. One i s m a d e on long:lived m R N A , ~nd the other is m a d e on s h o ~ - l i v e d mRI~IA. T h i s raises t h e question whetJaer or not the two types of each of the c r y s ~ l l i n s
CRY'STALLIN
SYNTIIESIS
BY CHICK
LENS
193
are the same proteins. ~Vo were u n a b l e to de~ect differences of eleotrophoreti,~ m o b i l i t y between the 3-crystallins fl~)m the fiber cells of the 7- a n d 17-day-old e m b r y o lens (m~published data). Nevertheless, t h e possibility r e m a i n s t h a t one s u b u n i t of ~crystallin is m a d e on long-lived m R N i a n d the other s u b u n i t is made on short-lived mRNA. The results shown iu :Fig. 2(a) show t h a t the synthetic a c t i v i t y of lens proteins per Ions increases with age. However, this does not always m e a n an increase of s y n t h e t i c ~l"~-
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l"tO. 9. (a) T h e c h a n g e of mit()tic a c t i v i t y o f epithelial ceils d u r i n g d e v e l o p m e n t , kNumbers o f D ~ A syathe.~izing cells a m o n g r e p l i c a t i n g coils (epithelial a n d o u t e r a m a u l a r p a d ceils) w e r e c a l c u l a t e d f r o m thB d a t a o f l)ersons a n d 5$odak (1970). h i i t o t i c r, etivi~y is e x p r e s s e d b y t h e r a t i o o f D ~ A s y n t h e s i z i n g ceils to t h e t o t a l n u m b e r oi cells, a n d t h e n p l o t t e d a g a i n s t t h e a g e o f t h e lenses. (b) T h e c h a n g e o f cell p o p u l a tion (luring d c v e l o p r a e n : . P o p u l a t i o n s o f r e p l i c a t i n g cells (epithelial a n d o u t e r a n n u l a r p a d ceils) a n d nonr e p l i c a t i n g cells (middle, inner a l m u l a r p a d , a n d fiber coils), w h i c h wore c~lculated f r o m t h e d a t a of t~erson8 a n d 5Ioctak (I 970) were p l o t t e d a g a i n s t t h e a g e of t h e leases. A, :Non-replicating cells; B, replicating eel[~ T ~ BL~- T
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194
K. Y O S H I D A A N D A. K A T O H
activity of lens p~oteins per cell bee:~use an increase of cell number also accompanies age, as sho~'n in Fig. 9(b) the data of which came ~ o m experiments of Persons and Modak (1970). The synthetic activity for lens proteins of epithelial and fiber cells is sttown in Table I. WtfilCthe synthetic activity of the fiber eelI is three to four times higher than t h a t of the epithelial cell, there is no remarkable change of synthetic activities b y epithelial and fiber cells during bhe developmental period studied here. Epithelial cells gr~ctually gain the ability to synthesize ~ - a n d fl-crystallins, and lose their ability to synthesize 3-crystallins during the development ¢,f the lens. However, fiber cells maintain their capacity for synthesizing 8-cryBtallins at 65% or more dtwing the development of the lens. A small amolmt of a - a n d fi-crystallins are always detected in fiber cell extracts during the development of the lerm. These crystal/ins probably come from contaminated epithelial cells or annular pall cells that ~re in a transit, ioual stage from epithelial to fiber ceils. Therefore, it is very likely t h a t fibe~ cells synthesize only 8-crystallin. The above results suggest the existence of a r%-nflatx~ry mectmnism for 8-crystallin s)mthesis, which is antagonistic to t h a t for ~.and fl-cryst~.llin synthesis, in the lens. There are only small amounts of radioactivities in the fast moving fl-crystallin regions compared with the stained bands of electrophoresis of adult lens extracts [see Plate 2(a) of the paper by Yo.~hida and K a t o h (1971)]. I t is probable that the fastmoving fl-crystallins are actively synthesized by epithe]-al cells after hat~hbag. Genis-Galvez, Maisel and Castro (1968) estimated the proportion ef erystallins in the chicken lens of late embryonic stages and the adult lens. According to their results, 3-crystallin is primarily found in fiber cells. On the other hand, ~- and fl-crystallins are mainly fomad in epithelial ~nd annular pad cells. In general, their results are consistent with the data presented in this paper. The hfitiation o~ 8-, fl- and ~.-crustallin synthc.~is, however, occurs in primordia of 2-, 2½- and 3-day-old ernbryos, respectively, actor(ring to the imnmnofluorescent data of Zwaan (1968). At present, the relat.ionship between the onset of crv-stallin synthesis and the activation or de-repressioD of ~.- and flcrystallin synthesis in epithelial ceils described in ~his paper is not. clear. ACKNOWLEDGMENTS
We-gratefully acknowledge the technical assistance of Cindy Hambrick, Mona Tu and Betty Ford. The continued interest and encouragement of Dr Car! Kap!an, Director of the Division of'Radiotherapy, is acknowledged. Supported in part by Atomic Energy Commission (contract AT(30--1)3825. REPE~ENCES,
Coulombre, A. J. (1965). I n Organogcnesis, pp. 227-232. (Ed. by DeH~an, R. L. and Urspr~ang, H.) Holt, R i n e h a r t a n d Winston, iN*ewYork. Genis-Ga/verz, J. 1~[., Maisel, H. and'Castro, J. (1968). Exp. Eye Res. 7, 593. Lowry, 0 . H., Rosebrough, N. J., Farr, R. J. and R~ndall, I~. J. (1951). J. Biol. Chem. 19S, 2 6 5 Papaconsea~tinou, J. (1967). Science 156, 338. Persons, B. J. and Modak, S. P. (1970)..Exp. Eye Res. 9, 144. l ~ e d e r , IR.. ~ . a n d 3Bell, E . (1965). ~v/en~e 150, 71. tleeder, 1%. :It. and Bell, E. (1967). J . Mol. 2~ol. 23, 577. St~war~, J. A. azxd Papacor~stantinou, J. (1967). J. Mol. Biol. 29, 357. Yoshida, K . ~ n d K a t o h , A. (1971). Exp. Eye ~es. 11, 122. Zwaan, J-. (1968). J. Cell P h y S . 72, supp. 1, 47.
C ystallin Synthesis by Chick Lens II. Changes in Synthetic Activities of Epithelial and Fiber Cells during Embryonic Development KAZUO ~ O S H I D A AND AI,~'['I-iUR. KATOH
Onc~)logy Laboratory, Division of Radiotherapy, Mercy Hospital, Pittsburgh, P~n~ylvania 15219, U.S.A. (Received 3 August 1970, Be,~ton) C r y s t a l l i n %vnthesis b y epithelial a n d fiber cobs f r o m 7- t o 18-d,~y-old c h i c k e n e m b r y o lenses was i n v e s t i g a t e d b y cellulose at~otate m e m b r a n e electrophore~is. E p i t h e l i a l cells o f 7- t o 12-d~y-old e m b r y o lenses s y n t h e s i z e m a i n l y 8 eryst~llin a n d a small a m o u n t o f ~ - a n d fl.eryatallins; t h e s e are all s e n s i t i v e t o a c t i n o m y c i n D (30,ug/ml). As t h e lens d e v e l o p s f r o m 12 t o 18 d a y s , epithelial cells b e c o m e i n s e n s i t i v e t o a e t i n o m y e i u D in r e s p e c t t o t h e svn.thesis o f ~- a n d fl-eryst~llins. A t t h e s a m e t i m e , t h e s y n t h e s i s o f 6-cryst~llin ceases. On t h e o t h e r h a n d , t h e fiber cell s y n t h e s i z e s p r i m a r i l y $-cryst~llin w i t h a d d i t i o n a l small a m o u n t s o f ~.a n d fl-eryst~llins; those a r e all i n s e n s i t i v e t o a v t i n o m y c i u D d u r i n g all stages o f t h e d e v e l o p m e n t o f t h e lenu f r o m 7 to 18 d~vs.
1. Introduction Tile v e r t e b r a t e lens, derived exclusively from ectoderm, is a co~nposite of three m~jor zones: the outer, anterior, single layer of epithe!ial cells; the t ~ n s i t i o n a l cuboidal (:ells of the a n n u l a r pad, which are in the process of elongation a t the equatorial region; and t h e fully elongated fiber cells .~n the lens body. Cell division is restricted to a nan'ow b a n d of epithelial cells at, the pre-equatorial region. The resulting, d a u g h t e r cells elongate a n d enter the body of the lens where the d.ifl~rentiation into secondary fiber cells is completed. :Fl~rther g r o w t h of the lens takes place by t h e continual, conc~entric addition of elongated fiber cells a t t h e periphery (Coulombre, 1965). The conversion of the lens epithelial cells into the fiber cell provides a suitable system, for s t u d y i n g cell differentiation, in t h a t it represents a progressive diversification of morphological structure associated w i t h t h e acquisition of synthetic capa c i t y for tissue-specific proteins ( P a p a c o n s t a n t i n o u , 1967). As such it has been utilized b y S t e w a r t a n d F a p a c o n s t a n t i n o u (1967) who showed t h a t a stabiliz~tion of m R N A t e m p l a t e s occurs during the conversion of epithelial into fiber cells in the bovine lens. This wins established b y t h e use of actinomycin :D a t doses inhibiting ~ll R h e a syn thesis, which resulted in abolishing crystallin synthesis in epithelial cells b u t h a d no effect on t h e fiber cell's capacity for protei~ synthesis. Using wotinomycin D on t h e 12-day-old chick embxTo lens, Reeder a n d Bell (1967) :~howed t h a t m o s t epithelial cell proteins are m a d e on short-lived m_RNA t e m p l a t e s . As t h e epit2aclial cell enters t h e body region and. elongates to fiber cell, actinomycin D-resist~nt protein synthesis, which is restricted t o two proteins, is directed b y longlived m.R2NA. U n f o r t u n a t e l y , t2ae specific lens proteins concerned were n o t identified in tJaese experiments. Recently, we introduced a t e o ~ i q u e utilizing cellulose acetate m e m b r a n e electrophoreais f o r the analysis a n d identification of radioactive chicken lens oryst~llins 184
CRY S T A L L I N SY~'TI-IESIS BY C H I C K LENS
185
(Yoskida a n d K a t o h , 197i). I a this p a p e r , we p r e s e u t s t u d i e s or: t h e effect of a c t i n o my'tin l0 on c r y s t a l i i n s y n t h e s i s duxing lens d e v e l o p m e n t . I n t h e 7- to 12-day-old c h i c k e m b r y o lens, e p i t h e l i a l cell. p r o ~ i n s y n t h e s i s , w h i c h is d o m i n a t e d b y $-, a l o n g w i t h s m a l l amomxt~ of a- a n d fl-crystallins, is d i r e c t e d by s h o r t - l i v e d m R N A s . As the lens d e v e l o p s f r o m 12 to 18 d a y s , t h e e p i t h e l i a l cells s y n t h e s i z e s i g n i f i c a n t a m o u n t s of ~- mxd fl-crystallins, w h i c h are n o w botJa m a d e on Iong-llved m R N A s . Conconfikuntly', t h e s y n t h e s i s of 8 - c r y s t a l l i n coo.sos. The fiber cells, o n t h e ot]mr h a n d , are e n g a g e d p r i m a r i l y i n t h e s y n t h e s i s of ~c r y s t a l l i n a l o n g w i t h s m a l l a m o u n t s of ~- a n d f l - e r y s t a l l i n s d a r i n g ~1I !ztages of e m b r y onto d e v e l o p m e n t b e t w e e n 7 a n d 18 d a y s . F u r t h e r m o r e , t h e s ~ fiber cell p r o t e i n s are all directed b y long-lived nLRNAs. T h e r e l a t i o n s h i p b e t w e e n t h e s y n t h e s i s of c r y s t a l l i n s and t h e d e v e l o p m e n t o f t h e lens is discussed. 2. M a t e r i a l s and M e t h o d s
Isotope i.ncorporation Lenses of "White Leghorn chicken embryos, r~,.nging from 7 to 18 d,~ys of incubation, were used t h r o u g h o u t this study. For the incorporation of radioactive a,aino acids into le~s proteins, 25-30 lenses were gently r o t a t e d in 20-ml Erlenmeyer flasks :containing 10 ml of Eagle's m i n i m u m essential medium (ME.3~), which was supplemented v~th 50/xCi; of ltC-reconstituted protein h y d r o l y s a t e (I'tC-RPH) (Schwarz BioResea~ch, Orangeburg, N.Y., U.S.A.) and penicillin a n d streptomycin, 100 u~lits/ml each (Difco, .Detroit, MicMgan, U.S.A.). The flasks were incubated a t 37°C in a hmnidified atmosphere of 5~/o CO.. in 95:/0 air. A_ftcr the culture period, which ranged from 1 to 23 hr: the lenses were washed w i t h MEM, I t a n k ' s balanced salt solution (BSS), a n d t h e n homogenized w i t h BSS. E q u a l volumes of cold 10°/,~ trichloroacetic acid (TCA) were added to the homogenaVes. T h e y were t h e n i n c u b a t e d a t 90°0 for 15 miu. The TCA precipitates were separated from the supernatauts by centrifugation. The pellets a n d s u p e r n a t a u t s were collected o~ glass membrane filter discs a n d prepared for scintillation counting as those of fz'ee amino acids and proteins. The il.~corporation of [3H]uridinc (20 ~LOi/nll, Schwarz BioResearch) was measured with a.ud withotlt a c t i n o m y c i n D (30 #,g/ml, Calbiochem., Los Angeles, Calif., U.S.A.). Aft~r the culture, the lenses were washed as above a n d homogenized with 5 % cold TC.4,. The homogenates were centrifuged a t 10,000 g for 20 rain a n d the radioacti~-itiee of the supel:n~ttants and pellets counted as those of free uriclines a n d RNAs.
Preparation of chicke~ l~z~.sextracts for electroplmresis Lenses of 7-18 d a y s of incubation were g e n t l y r o t a t e d i n 10 nfl of ~ E b l containing: 50 ~Oi of I*C-:RPtI a n d antibiotics a t 37°0 as described above. K c t i n o m y c i n D was added at a concentration of 30/~g/ml to the t r e a t e d cultures. After th~ culture period, the lenses were washed succe~ssively w i t h MEM, and w i t h BSS. Lenses were t h e n dissected into epitheli,~l and fiber cells w i t h w a t c h m a k e r ' s forceps under a Zeiss stereoscopic microscope. The lenses or dissected cells were homogenized alld their proteins were extracted a n d applied to Sepraphore FII cellulose acetate membranes for electrophoresis as described previously (Yoshida a n d K a t o h , 1971). P i e r , in content of epithelial a n d fiber cell e x t r a c t s was d e t e r m i n e d as follows: t h e ceil extracts were m a d e up to 5~/o TCA a n d i n c u b a t e d a t 90°G for 15 rnin. The h o t TGAlarecipitares were washed ~-ith ethanol, e t h a n o l :ether {3:1 v/v), and w i t h ether. The washed laellet~ were t h e n dissolved in 1 ~¢ N a O H . The protein conVent of the solutions was e s t i m a t e d b y Lo~wry's m e t h o d (Lowry, Rosebrough, iTarr a n d Randall, !951), a n d i t s r a d i o a c t i v i t y we.~ cornered in a P a c k a r d scintiUation caimt~r to determine the specific a ~ t i v i t y of the protein sample.
186
~,:. Y O S H I D A
AND
A. ] K A T O H
3. Results
Crystallin synthesis during tlw. d~velopment of the lens Inv.orpara~ion of amino .avids into lens and stability of lens proteins. Radioactive a m i n o acids were l i n e a r l y incorporated into proteins of the 7-day-old e m b r y o lens for at least 23 h r w i t h o u t significant lag time, as shown in Fig. l(a). B y this criterion t h e lens appears to sl~rvive the in vitro culture conditions of our experiments. :Fignre l(b) shows t h a t radioactive a m i n o acids are r a p i d l y incorporated into the pool of the lens w i t h i n t h e first hour, a n d t h a t t h e pool was a h n o s t saturated w i t h i n 2 hr. In lenses of 10-, 12- ~nd 17-day-old embryos, similar incorporation curves of radioactive a m i n o acids were obtained. The s t a b i l i t y of lens proteins was checked b y labeling lenses w i t h ~C-t2.PH for 1 or {o)
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~ G . l. (a), (b) I n c o r p o r a t i o n o f r a d i o a c t i v e a m i n o acids i n t o c h i c k e m b r y o lens, L e n s e s o f 7 - d a y - o k t c h i c k e m b r y o s w e r e c u l t u r e d in 10 m l of ~ I E M , w h i c h w a s s u p p l e m e n t e d xvigh 1.dO-I~PH (50/~Ci). A t i n t e r v a l s , lenses ~,ere w i t h d r a w n f r o m t h e c u l t u r e m e d i u m , a n d s a m p l e d for r a d i o a o t i v i b y m c a s u r c m e n t ~ . I n t h e p u l s e - c h a ~ e x p e r i m e n t s , lenses l a b e l e d w i t h ~¢C-RPH for 1 h r or 5 h r (arrows) were w a s h e d e x t e n s i v e l y w i t h M E M a n d t r a n s f e r r e d i n t o fresh M E M . AS i n t e r v a l s , lenses w e r e w i t h d r a w n f r o m t h e c u l t u r e raerlium a n d s e p a r a t e d i n t o (a) p r o t e i n a n d (b) a c i d soluble f r a c t i o n s . A, c o n t i n u o u s label; t], 5 h r label-18 h r chase; a n d C, 1 b r label-22 h r chase.
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.Fz(~. 2, (a), (b) I n c o r p o r a t i o n o f r a d i o a c t i v e a m i n o a c i d s i n t o epiShelial a n d fiber cells o f chick e m b r ) , o l e n s . L e n s e s f r o m v a r i o u s a g e s o f c h i c k 6 m b r y o s w e r e c u l t u r e d w i t h z~C-I~PH (5 iLCi/ml) fox" 5 hr. Af~e.r t h e c u l t u r e , l e n s e s , w e r e w a s h e d a n d d i s s e c t e d i n t o epithelial a n d fiber~cells, a n d t h e p r o t e i n s e x t r a c t e d . T o t a l r a d i o i s o t o p e ixlcorporatiou i n t o p r o t e i n s o f epithelial a n d .fiber cells p e r lens (a), a n d specific a c t i v l k y o f t h e larotein'~ 114) w e r e d e t e r m i n e d a s d e s c r i b e d in i~Iaterials a n d ~ [ e t h o d s . A, ~Fibcr cells; B, epithelial cells.
CRYSTALLI:K
SYi~T]-IESIS
BY CHICK
187
LENS
5 hr, washing thoroughly with MEM, and chasing in radioisotope-free 5'IE~ for 22 or 18 hr, respectively. As shown in :Fig. 1(~), the radioactivity that was incorporated int~) lens proteins was Mmost completely retained. On the other hand, the radioactivity t h a t entered the amino acid pool d~s'~ppeared ahnost instantaneously '~luring the wash and chase periods. (el
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l ~ o . 3 . ( a ) , ( b ) , ( e ) , ( d ) , ( e ) , ( f ) ~leot..~)phoret,]c ~ ' " profiles o f e x t r a c t s ot" epitheliM cells o f chick e m b r y o lens. 3 $¢g P r o t e i n of epithelial cell e x t r a c t s p r e p a r e d in Fig. 2~vere a p p l i e d o n t o 'cellulose a~et~t~, m e m b r a n e electrophoresis. A n o d e is o n t h e r i g h t side o f t h e Bgeres, 0 is origin. (a) 7 - d a y . o l d e m b r y o lens; (b) 10-d~yo l d e m b r y o lens; (c) 12-day-old e m b r y o lens; (d) 16-day-old e m b r y o lens; (e) 17-day-old e m b r y o lens ; (f) 18-day-01d e m b r y o l e ~ / C) O , R i g h t o r d i n a t e ; Q - - O , lef6 o r d i n a t e .
188
K. YOSI-][II)A
AND
A. KATOtl
Synthesi.s. of c'rystalIins du:ri~vj the develoTment of the Imz~. In order to follow the patterns of cryst, Mlin synghesis b y epithelial and fiber cells, lenses Of embryos ranging from 7 to 18 d a y s of incubation were ¢~uI~uxed wi~h ~ C - R P H for 5 hr. After the culgure 'period, lenses were washed,'diss.e.cged int~ epighelial a n d fiber ceils, a n d tKeix' proteins extracted. Total isotope incorporation into epigheliaI a n d fiber cells and ghe specific (o1~
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3 ~g Protein of fib~ cell extract~ prepared in Fig. 2 wer~ applied onto cellulose acetate m e m b r a n e eloctrophoreMs.'A~dde is on the righ~ of the figures. 0 iv,origin. (a) 7-day-old embryo lens; (b) IO-day-old e m b r y o l e n s ; (o)'.'~!'.~';day-old e m b r y o l e n s ; (d) 1 6 - d a y - o ! d e m b r y o l e n s ; (e) IT-davy-old e m b r y o l e n s ; (f) 1 8 . d a y - o l f l e m b r y o lens.
CYCYSTALLI:N
SYNTHESIS
BY
CHIOK
189
LENS
a c t i v i t y of their e x ~ a e t e d proteins were determined before application onto cellulose acetate m e m b r a n e s for electrophoresis. Isotope incorporation into proteins of the epithelial a n d fiber cells increases linearly w i t h age of the lens [:Fig. 2(a)]. On the other hand, the specific a c t i v i t y of their proteins decreases linearly with age of the lens, as shown in Fig. 2(b). This is consistent with the results of the previous section where it was shown t h a t lens proteins are very stable, and theft newly synthesized proteins are diluted with accumulated stable proteins.
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F l a . 5. (a), (b) C h a n g e s o f s y n t h e t i c a c t i v i t y o f crs, st.allins b y e p i t h e l i a l a n d f i b e r cells d u r i n g t h e (level,,i)men$ o f t h e lens. (a) R a t i o s ( ~ f i n e o r p o r a t i o t l o f r a x l i o a e t i v e a m i n o a c i d s i n t o ¢t-, fl- a n d $ - c r y s t a l l i i m t o t o t a l i n c o r p o r a t i o n i n t o p r o t e i n s o f f i b e r cells for 5 h r w e r e c a l c u l a t e d f r o m t h r e 6 d i f f e r e n t e x p e r i m m ~ t a i d a t a , o n e o f w h i c h is s h o w n in F i g , 4. T h e v a l u e s w e r e p l o t t e d a g a i n s t t h e ag(~ o f t h e l(:nsea. (b) S a m e c a l c u l a t i o n a s (a) w a s e ~ r r i e d o u t f o r c l e c t . r o p h o r e t i c p r o f i l e s o f e p i t h e l i a l cells. ['_]~f-q, $ - C r 3 - s t a l l i n ; ® ~ O, fi-cry~tallin ; A--A, ~-erystallin.
Electrophoretic :profiles of epithelial cell extracts are shown in :Fig. 3. L1 epithelial ceils of the 7-.day-old embryo lens, ~-crystallin picks up most of the r a d i o a c t i v i t y [Fig. 3(a)] hut dm.ing further development, of the lens up to 18 days, this a c t i v i t y is gr~:[ua, lly lost,. Dm'ing the same time period, a- and fl-crystallins show a -,radual increase of synthetic activity [see Fig. 3(c), (d), (e), (f)]. Note t h a t the broken lines of Fig. 3(c)-(f) are drawn to a (lifferen£ scale in order to magni£y the activities of ae.nd fl-crystallins and to facilitate the comparison of their synthetic ratios. I n the fiber cells on the other hand, most of the radioactivity is found in 8-cry-stal!in, ~nd only a small a m o u n t of r a d i o a c t i v i t y is evident in ~.- and fl-crystallin regions during all stages of the development of the lens studied here [Fig. 4(a)-(f)]. The resu/~s also show t h a t the specific a c t i v i t y of the crystallins from both epithelial a n d fiber cells decreases w i t h age, because the same a m o u n t of protein samples (3 fig) was applied onto electrophoresis (see solid lines of :Figs 3 a n d 4). This is consistent with the results described in the previous section. Ck-ystMlins are stable proteins, as described above. Therefore, the synthetic a c t i v i t y of the ¢rystallins of epithe!i,~l and fiber cells can be expressed b y ~he ratio of the incorporation of radioactive amido acids into crystallins to t h a t intm lens proteins for 5 in-. The m e a n values are c~[culated from three different experimental data, one of which is shown in F i g s 3 and 4. This is t h e n plotted aga;mst t h e age of the lenses " (Ii'ig. 5). The results indicate t h a t fiber cells m a i n t a i n t h e same capaqity for crystalhn
190
K. YOSHIDA
AND
A. K & T O H
synthesis ~nd primarily synthesize 8-oryst~Ilia ~]~roughout the development of l;he lens [Figs 5(~) and 4]. On tim other h~md, epitholiM cells gradually lose t.hoir capacity for 8-crysttdlin syntltesis while g,~ining that for ct- ~nd fl-eryst,~llin [Figs 5(b) ~nd 3]. EinMly, epitimlbd cells primarily syut.hesize ~- ~nd fl-erystMlins, whereas fiber cells prim~rily synthesize 8-cryst,allin. I
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J"[o. 6. (a), (b), (e) Electrophoretio profiles of epithelia.l cells from !cases l.rcated with actinomycia D. Lenses prepared from e m b r y o s of various age.s were cultured under the same conditions as described in Fig. 2, except, th.'~t. actinomycin D was pre~sent a t a coneeatr~tti.or~ of 30 pg/ml. T h e extracts from epithelial cells were applied onto cell ul f,so acetate m e m b r a n e s for eleetrophorcsi~. Anode is on tim right side of the figures. O is origin. (a) 7-thKy-old e m b r y o lens. (b) 12-day-old e m b r y o Ions; (c) 16-d~y-old e m b r y o lens. O . . . . O , R;.ght ordinate; O ~ @ , I~eft ordinat.e.
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Fro. 7. (a), (b), (e) Electrophoretie profiles of extracts of fiber cells from lenses t r e a t e d with ac4inomyein D. The extraet~ from fiber cells, which svero p r e p a r e d as indies.ted in Fig. 6, were applied onto cellr:!ose acetate mem'branea for eleetrophore~is. Anode is on the right of the figures. 0 is origin. (a) 7.day-old e m b r y o lens; (b) 12-day-ohl e m b r y o lens; (c) 16.d~y-old e m b r y o lens.
CI'¢YSTALLI-.-N SYNTI~IESIS
BY C}IICI(
LENS
191
Effects of actinomycin D on crystallin synthesis Zen~ses labeled with laO-am.ino acids in the presence of actinomycin D. In order to study the effects of actinomycin D on crystallin zyntlmsis, lenses from 7- to 18-day-old embryo.~ were cultured with I'~C-]~PH: and their epithelial and fiber cell extracts were analyzed by electrophoresis, as described in Fig. 2, with the exception t h a t actinomyein D (30 Fg/ral) was present during the culture ~ime. Crystallin synthesis by epithelial cells of 7- and 12-day-old embryo lens was almost completely inhibited by actinomyein D [Fig. 6(~) and (b); compare Fig. 3(a) and (c) as controls], t:[owever, crystallin synthesis by fiber cells was not arrested by actinomyci~ D [Fig. 7(a) and (b); compare Fig. 4(a) and (c) as controls]. On the other han~l, fin lenses of 16-day-old embryos, aetinomycin D did not inhibit the synathesis of cry~tallins by both fiber and epithelial ceils, but rather showed a stimulatory effect [Figs 6(c) and 7(c) ; compare to Figs 3(d) and 4(4)]. The effects of actinomycin D on crystallin synthesis by 17- and 18-day-old embryo lenses were similar to those shown by the 16-day-old embryo lens. The resu/ts of Fig. 6(a) and (b) also indicate that the separation of epithelial and fiber cells was clcanly executed since no contaminating radidactivity of 8-crystallin from fiber cells appeared in the electrophoretic profile of the epithelial cells treated with ac~inomycin D. Incorporation of radioactive uridine into R N A of le.n.ses in the wesence of acti~wmycin D. To check whether actinomycin D really arrestc~I RNA synthesis of lens under our culture conditions, lel~.es of 1.7-day-old embryos were cultured with [aId"Juridine in the presence or absence of actinomycin D (30/zg/ml). As shown in .Fig. 8(a) radioactive uridine is rapidly incorporated into the pool of l~ridine and R N A ibr 5 hr. ~kfter 5 hr of cultl~re, the incorporation r~te levels off but nonetheless remains constant for at least 25 b.r. The effect of acCinomycin D on incorporation of [aH]uridine into 17-day'-old embryo lens is sltown in Fig. 8(b). It is evident tLtat aetinomycin D almost completely
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F*G. 8. (a), (b) I n c o r p o r a t i o n o f [aH]uridino i n t o lens in the presence (b) or a b s e n c e (a) o f a e t i n o m y c i n D. Lenses p r e p a r e d f r o m 17.day-o~d e m b r y o ~ were c u l t u r e d with [3H]uridino (20/xCi/ml) in t h e presence or absence o f a e t i n o m y c i n D (30 btg/ml). A f t e r t h e eul~ur~ interval, lenses were w i t h d r a w n tkom t h e m e d ium, w a s h e d with I~,IEM, a n d w i t h BSS t t i o r o u g h l y . T.he lens h o m o g e n a t e s were m i x e d w i t h e q u a l v o l u m e o f cold 10°/o TCA. T h e h o m o g e n a t c s were s e p a r a t e d into pellets a n d s u p e r n a t ~ n t , s b y oe.ntrifugation, a n d collected on glass m e m b r a n e .filter discs a n d their radioaetivitiea c o u n t e d . Q - - @ , Pellets ( R N A ) ; O - - - C), s u p e r n a t a n t ~ (,~eid soluble fraction).
192
K. YOSHII)A
AND
A. KATOH
arrests the synthesis o f R N A in lenses u n d e r our e x p e r i m e n t a l conditions. However, a c t i n o m y c i n D does not influence the incorporation of [~H]uridino into the pool of the lens. Similar results were obtained for t h e 7-day-old e m b r y o lens. 4. Discussion
Caution m u s t be exercised in the use of" a c t i n o m y c i n D since it call be v e r y toxic to presumptive lens explants treated w i t h low concentrations such as 0-20/~g/ml for 1 h r (Katoh, B r a v e r m a n a n d Yo~hido,, u n p u b l i s h e d data). However, t h e effects of actinom y e i n D on the lens in this study are of a different natm'e, because morphological integrity and isoix)pic incorporation of a m i n o acids and l~ridlne into pools of the lenses were m a i n t a i n e d as described above. This indicates a~ least t h a t a c t i n o m y e i n D has not d a m a g e d cell m e m b r a n e s . Furthermore, the d a t a presented here show t h a t aotin om y e i n D suppresses R N A synthesis in epithelial .cells from 7- to 12-day-old e m b r y o lenses. .We described in the Results ~hat the separation of epithelial cells ~zom fiber cells was cleanly exec~uted. However, a n n u l a r p a d cells were not removed from both groups. Therefore, epithelial cells, as d~seribed in this paper, p r o b a b l y correspond to the replicating cells which consist of central epithelial, peripheral epithelial, a n d outer a n n u l a r p a d cells, as described b y Persons a n d Modak (1970). The fiber cells correspond to the non-replicating cells, which include middle annular, inner annu]~.r pad ancl fiber cells. As shown here, the stabilization of m R N A for crystallin synthesis occurs in b o t h epithelial a n d fiber cells. The stabilization of mRN2L in t h e fiber cell has a l r e a d y occurred in the 7-day-old e m b r y o lens. On the other hand, m R N A for crystallil~ synthesis b y epi~heliaI cells is stabilized between 12 a n d 16 days. More correctly, the stabilization occurs during the development of the lens between 12-13 d a y s according to our most recent results, which aze n o t included in this paper. Therefore, the stabilization m e c h a n i s m m a y be in ol~eration dm~h~g t h a t critical period. As described in t h e Introduction, t h e stabilization of m R l ~ A has been considered to be one of the characteristics of tezmlinal cellular differentiation (Reeder a n d Bell, 1965; P~paconstantinou, 1967). Stewart and P a p a c o n s t a n t i n o u (1967) h~ve suggested t h a t the stabilization of m R N A is associated w i t h loss of mitotic or replicating P~tivity of cells. I n order to check tb]s possibility, we e x a m i n e d in detail the data of Persons ~nd Mod~k (1970) a n d plotted the mitotic a c t i v i t y of epithelial cells versus the age of the lens (:Fig. 9). There appears to be no s i g h t , c a n t difference between the mitotic or replicating a c t i v i t y of epithelial cells from 12-, 13-, 14- a n d 16-day-old e m b r y o lenses, which still r e t a i n signi~cant mitotic activity. This suggests t h a t the stabilization of r a R N A is not entirely associated w i t h t h e loss of mitotic activ~ity, as suggested b y Stewart a n d P a p a c o n s t a n t i n o u (1967). Thus, the association of the stabilization of m R N A w i t h t h e loss of mStotic a c t i v i t y appears to need careful re-examination. Reeder ~nd Bell (1967) analyzed lens proteins of the l ~ d ~ y - o l d chicken e m b r y o b y p o l y a c r y l a m i d e gel electrophoresis i n t h e pzesence of urea, w h i c h is a strong denaturation agent of p~0tein. T h e y f o u n d thxee p r e d o m i n a n t p r o t e i n b a n d s (1, 2, 3) in the lens extracts. I n comparison w i t h our results, b a n d 3 is p r o b a b l y a s u b u n i t of c~and/or fl-crystallins. B a n d s 1 a n d 2 a~e p r o b a b l y subunits of 8-erystallin. Our d a t a indicate t h a t there are at least two kinds o£ c¢-, fl- a n d 8-crystallins, respectively. One i s m a d e on long:lived m R N A , ~nd the other is m a d e on s h o ~ - l i v e d mRI~IA. T h i s raises t h e question whetJaer or not the two types of each of the c r y s ~ l l i n s
CRY'STALLIN
SYNTIIESIS
BY CHICK
LENS
193
are the same proteins. ~Vo were u n a b l e to de~ect differences of eleotrophoreti,~ m o b i l i t y between the 3-crystallins fl~)m the fiber cells of the 7- a n d 17-day-old e m b r y o lens (m~published data). Nevertheless, t h e possibility r e m a i n s t h a t one s u b u n i t of ~crystallin is m a d e on long-lived m R N i a n d the other s u b u n i t is made on short-lived mRNA. The results shown iu :Fig. 2(a) show t h a t the synthetic a c t i v i t y of lens proteins per Ions increases with age. However, this does not always m e a n an increase of s y n t h e t i c ~l"~-
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l"tO. 9. (a) T h e c h a n g e of mit()tic a c t i v i t y o f epithelial ceils d u r i n g d e v e l o p m e n t , kNumbers o f D ~ A syathe.~izing cells a m o n g r e p l i c a t i n g coils (epithelial a n d o u t e r a m a u l a r p a d ceils) w e r e c a l c u l a t e d f r o m thB d a t a o f l)ersons a n d 5$odak (1970). h i i t o t i c r, etivi~y is e x p r e s s e d b y t h e r a t i o o f D ~ A s y n t h e s i z i n g ceils to t h e t o t a l n u m b e r oi cells, a n d t h e n p l o t t e d a g a i n s t t h e a g e o f t h e lenses. (b) T h e c h a n g e o f cell p o p u l a tion (luring d c v e l o p r a e n : . P o p u l a t i o n s o f r e p l i c a t i n g cells (epithelial a n d o u t e r a n n u l a r p a d ceils) a n d nonr e p l i c a t i n g cells (middle, inner a l m u l a r p a d , a n d fiber coils), w h i c h wore c~lculated f r o m t h e d a t a of t~erson8 a n d 5Ioctak (I 970) were p l o t t e d a g a i n s t t h e a g e of t h e leases. A, :Non-replicating cells; B, replicating eel[~ T ~ BL~- T
Protein synthetic activity of e?ithelial and fiber cells t~C-amino a c i d i n c o r p o r a t i o n into proteins (cts/min × 10-*)t
A~e o f lens (days)
Cell n u m b e r ( × 10-~) *
7
3-0
1.6
0.053
10
6-0
4.0
0.067
12
8-2
4.6
0.056
16
12.2
%0
0-057
17 18
13-2
7-4
0.056
14.2
8-2
0.058
7 I0 12 16
2-2 3-8 5-0 7-2
F i b e r cells 4.3 6-0 6.4 9-3
Synthetic activity (cts/min/ce|l)
:Epithelial cells
0.19 0-16 0.13 0-13
17
7-7
10-0
0-14
18
8-3
14.0
0-17
* Culer~ated f r o m F i g . 9(b); t C a l e u l a ~ d f r o m Fig. 2(a).
194
K. Y O S H I D A A N D A. K A T O H
activity of lens p~oteins per cell bee:~use an increase of cell number also accompanies age, as sho~'n in Fig. 9(b) the data of which came ~ o m experiments of Persons and Modak (1970). The synthetic activity for lens proteins of epithelial and fiber cells is sttown in Table I. WtfilCthe synthetic activity of the fiber eelI is three to four times higher than t h a t of the epithelial cell, there is no remarkable change of synthetic activities b y epithelial and fiber cells during bhe developmental period studied here. Epithelial cells gr~ctually gain the ability to synthesize ~ - a n d fl-crystallins, and lose their ability to synthesize 3-crystallins during the development ¢,f the lens. However, fiber cells maintain their capacity for synthesizing 8-cryBtallins at 65% or more dtwing the development of the lens. A small amolmt of a - a n d fi-crystallins are always detected in fiber cell extracts during the development of the lerm. These crystal/ins probably come from contaminated epithelial cells or annular pall cells that ~re in a transit, ioual stage from epithelial to fiber ceils. Therefore, it is very likely t h a t fibe~ cells synthesize only 8-crystallin. The above results suggest the existence of a r%-nflatx~ry mectmnism for 8-crystallin s)mthesis, which is antagonistic to t h a t for ~.and fl-cryst~.llin synthesis, in the lens. There are only small amounts of radioactivities in the fast moving fl-crystallin regions compared with the stained bands of electrophoresis of adult lens extracts [see Plate 2(a) of the paper by Yo.~hida and K a t o h (1971)]. I t is probable that the fastmoving fl-crystallins are actively synthesized by epithe]-al cells after hat~hbag. Genis-Galvez, Maisel and Castro (1968) estimated the proportion ef erystallins in the chicken lens of late embryonic stages and the adult lens. According to their results, 3-crystallin is primarily found in fiber cells. On the other hand, ~- and fl-crystallins are mainly fomad in epithelial ~nd annular pad cells. In general, their results are consistent with the data presented in this paper. The hfitiation o~ 8-, fl- and ~.-crustallin synthc.~is, however, occurs in primordia of 2-, 2½- and 3-day-old ernbryos, respectively, actor(ring to the imnmnofluorescent data of Zwaan (1968). At present, the relat.ionship between the onset of crv-stallin synthesis and the activation or de-repressioD of ~.- and flcrystallin synthesis in epithelial ceils described in ~his paper is not. clear. ACKNOWLEDGMENTS
We-gratefully acknowledge the technical assistance of Cindy Hambrick, Mona Tu and Betty Ford. The continued interest and encouragement of Dr Car! Kap!an, Director of the Division of'Radiotherapy, is acknowledged. Supported in part by Atomic Energy Commission (contract AT(30--1)3825. REPE~ENCES,
Coulombre, A. J. (1965). I n Organogcnesis, pp. 227-232. (Ed. by DeH~an, R. L. and Urspr~ang, H.) Holt, R i n e h a r t a n d Winston, iN*ewYork. Genis-Ga/verz, J. 1~[., Maisel, H. and'Castro, J. (1968). Exp. Eye Res. 7, 593. Lowry, 0 . H., Rosebrough, N. J., Farr, R. J. and R~ndall, I~. J. (1951). J. Biol. Chem. 19S, 2 6 5 Papaconsea~tinou, J. (1967). Science 156, 338. Persons, B. J. and Modak, S. P. (1970)..Exp. Eye Res. 9, 144. l ~ e d e r , IR.. ~ . a n d 3Bell, E . (1965). ~v/en~e 150, 71. tleeder, 1%. :It. and Bell, E. (1967). J . Mol. 2~ol. 23, 577. St~war~, J. A. azxd Papacor~stantinou, J. (1967). J. Mol. Biol. 29, 357. Yoshida, K . ~ n d K a t o h , A. (1971). Exp. Eye ~es. 11, 122. Zwaan, J-. (1968). J. Cell P h y S . 72, supp. 1, 47.