Retinoic acid inhibits conversion of dissociated Müller glia into lens-like cells

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L I ~ D A D E ~ E N S ~ B Z N AND A. A. MOscoN-ALazbo Oryfor~Developmenta! ,Biol , :I) ~ leC~tlar" etica a n d Cell Biology; ~ m m ~ ~ Life: Sc, ie=ce or,'U , ~ ¢ r ~ ~ o f C~ ;" i o, 60637, U.S:A. ~ " ..... (R~ived ~

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of glio ~hesivity to neurons; and that, by p~moting ~st0ratiou of gila ~11 con With neu~ns, RA proLec~ the gHoe from phenotyg~v alteration. : noic add; Mfiller gila; 1 ~nmct; ! ~; } n s f o a t i ~ ; ~tina r~s. I. Introduction

In the course o f earlier s t u d i e s on the :retina it w ~ n o ~ d :that; ' u n d e r c e

in

Moscona, n, D e g e n ~ i n , F0X and Sob; 1983; D o P , :Takagi,:Kond0h a Ok a, 1984). Experi ~ s with avian retina have s h ~ n t h ~ ~r mgtion i n ~ 'len~idal' llsisconsis~ntly d rapidly dwith Is s~"- ~" "e retina. I f the retina is dissocia and: the celis i m a i ~ a i ~ d m o nodi~r~d~i~,~ rnonol~yer"cu!ture; M i i H ~ glie~: assume: aniepitheliOid ~/~lagpe. 'begi.n :aRaiV. t~"~diVide, accumu!~te~lens antigens "nd, ' i n 4 - 7 ~ays c 0 n - ~ e r t i n ~ "|entoidal .cells:( .~.9sCO~, ,1i983): The aim of t h e p ent study~w ~ find a g e n ~ t h a t v e n t this cel!:~an r on. We ort here t h a t ~ t i n o i c acid added t o t h e c u ! ~ z e s inhibits the pid C o n v e ~ o n . of Mfiller glia into lentoidal cells.This compou tost~d b e c ~ u ~ , in~.~-arious Othe r

continued~ deprivation ~of normal contacts w~th! o~aL'; s~ This. point¢: t0::~tl~e importance: of cell c o n t a c t ~~ d c o n t a n ~ r a ~ i o ~ : o f , cells~ Ln~C,Sntrol~o f p h e n o ~ p e p mon "~ !and s ~ b i l i t y (Mos~nai":~ 1980):Cell ' P' ~ i t on '; abroga~s';' ~" /~ctions;~odifi t h e c e | l s u r f a c e , a ~ can d'f~:a aad~:f!al~an t; gene e n ~ i d a ] ,i f 6 ~ a t i o h 0 f .:~ " :~'. . .ce[s~m . . . . ~:'"a 'us " e , > ;, exp :~.,. .....slon. ,,:,,., L........ Mfil|er investigati t h e role of cell contact in cell ~ u l a t i o n ; in ~dditiov,, it o t f e r s ~ e ~ g~ into the n a t u ~ of-these final s. Our peri ta/~teml/s neu~ ~~tin~''of'~ " 13=day chick os;. this ~ , t h e ~ t i n e h ~ " ii ar~~ 1 histologie~I/org iza~tion", is:po~t,:initotici-;iboth'"Mi~E ::( of"gliSJ in a v i a n ~ t i ) r~urons ~re p h e ~ ically de ~ d (M ~T o i n v e s ~ i g a ~ t h e e f f e c t s o f tinoic ~id;i,Wei:took ,:~fl!t~yo;im 'of Miiller; giia.~ O n e iS :the: induction byl corti~ol of. glut ne synthet (GS) i n ller: cells ( M o ~ n a ; M o s c o n a : S~enZ;: ! .968;Li rand cona.~1979'i ~ ~ 0 ~ i983)~!:i In the p r ~ n t ! C 0 n ~ x t i ; t h d i m p b r t ~ t p6iht -is~that)GS:i~i~cti0hi:~e~i~i~in;~itibi~::~ ~

L. D E G E N S T E ] N AND A. A. MOSCONA t o the hormone, specific c o n t a c t s between Mfiller cells and n e u r o n s . I f the r e t i n a is d i ~ o c i a ~ d .into single cells, ..... " e d;~ i f the~cells a GS~ c a n n o t be tnduc i edia~ly re d gnd glia2~ur0n t a c t s ~ ~ t d r e d ; ~GS is;again i cible {i~inser and Moscona, 1979; 1983). HOwev~,~ i f She'di i cells m a i n l i n e d disp d in m~olay 1 d t h e gliocy err i n ~ l e ~ o i d a l ceils, t h e y 1o t h e ability to a d h e ~ t~3 neurons a~:d GS is ~ longer inducible (Linser and Moscona, 11)83; Ophir, Moscona, L o y a a n d Be haul,~ 1985). ,T h e ~ f o , w e h a v e u s e d ~GS induction to m o n i t o r t h e effect o f m t i n o i c acid o n , t h e ability of disSoci d ller cells ~o restore functional c o n t a c t s with neurons. A n o t h e r m a r k e r of Mfiller gila is carbonic a n h y d r e-II ( -II; earlier ferred to as 0.4--0; Linser a n d scona, 1981 ; Moscona, 1983). Unlike GS, -II c o n t i n u e s to be in ~o| r c u l t u ~ s (Linser a n d M na, 1983) a n d u h identify MiMler glia-derived cells, including lentoidal cells (Moscona et a l , 1983). I t s expre ion in l e n ~ i d a l l l s is ' e ~ s i s ~ n t with the p r e s e n ~ of this e n z y m e Mso in n~rmal lens (L n r ~d, Moscona, 1 9 8 1 )

2. M a t e t a l s a n d Cell cu

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ginas m 13-d chicken emb s dissociat~ed by t r y p s i n i z a t i o n into cell suspension, in p~v-ious work {Moscona and Moscona, 1952; Moscona, 11961; Moscona and D nstein, 1981). n were ri w~h cMciumagnesiu - e salt solution ( F), incubated in F for 10 rain at 37 °C, then for rain in 0"3 % ~ s t a l l i n trypsin (ICN) in F. The tissue w ~ gently N n ~ d (taking.care not ~ break it u p ) w i t h soybean trypsin inhibitor in ale's(Worthington; 3 ~pg ml-~), then with Medium 199 (M199)~ mining 50 Pg m ]-z (We ing ). The ceils Wer~ di int~ suspension in the l ~ t medium (two ~tin p e r I ml) by: varal ~ n f l o p a ~ s i:~ and out of the tip of a f i ~ - p o | i ~ e d Pasteur pi t ~ . By T -Blue exclusion ~ s ~ a t le t 90% of the l!s viab~. wer

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r e t i n ~ per fl k) ir~ 10 ml M199 m a d e u p with L'amino acids in modified ]s=ank's salts, and peniCilli trep~mycin:: A r a ~2-hr incubation, the medium W~ ca~fully r e p l a ~ d with M199 t~ which 5 ?/~ fetal bovine ~ r u m (FBS)w~s added. Cultur-es were m~in d ~ 4-:or 6 days. Medium w changed e v e ~ other d . Cell a ~ s were p a ~ d fro rachel er cul~res, ~he e u ! ~ ~ , ~ rinsed with F a~d incuba~d for 1"2 rain (37°C1 in I0 m! 0"25 N trypsin in ank's (KC Biological). S was added (1 m] ~per fl ), the medium was ~rdoved, and the flasks were briskly- shaken to dislodge the lls. ~The lls m collec in M199 with 10 % FBS, dispemed by pipetting and pel!e~ed by trifugation (1 ~m 2 rain in 10 ml medium). T h ~ w e ~ resuspended in M199 with DN (50 pg ml?!)and aliquo int~ 25-ml Erlenme r fl~ks, each containing 3 ml M199 with I0 ~o S d the antibiotics. T h e f t s w e ~ ~ t e d s-t 72 ~ m rain- ~ on a r o t a r y incuba.t0 ~ker for 48 hr ( cona, 1961) to aggregate :the cells[ Medium wa~s Changed daily. ' ....... uri~ reti

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all: trains ( S i g m a ) w ~ dissolved, in Ied)~/o ethanol and a d d e d t o c u l t u r e s a t a ntration o f [ , 2 g !0-6 Ni f o r t h e e n t i ~ c u l t u ~ :period. Cent s ~ i d~equivalent amount Of ethanol. " .RA,

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GS iaduetion w ~ With cortisol ( ~ o n a et al.; 196a). hermetic Was added to cu!tures (9× 10-~ ~) ~ described in suits2 Two d r i ~ l addition the eult~a w ~sayed for~ GS:induction,by m e u r i n g G S C/tic ~ N v i t y (Liner: d a,~19791~

INHIBITION muno

O F MOLLER~ O LIA; T R A N ~ F O

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[ ~ 2 l(a)] designated "L~jR cells'"(K~owitz

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into I t0id~l cells. ~ This effec~ o f R A was c o n f i ~ e d by;immunost~inittg ithe~

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96

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L. D E G E N S T E I N AND A ~A. MOSCONA

Fxa. 1. (a) Ur~aymon01ayer Culture of di~ocia neural retina c~lls from 13-day chicken embryofl ned, l a r ~ epithelioid na glio (LER; c,ells)"derived from ~Mfiller cells; Clus~m of neuronal ~lls, and neurons with elongated pro .' Bar 0"08ram. (b) Four-day cultu~ of ~tina lis in mediu m containing ~tinoic acid (RA). Most of the ~ IIS ~ m b l ~ into clus~~ interconnected by long (~ur0nal) pro . only ~w LER ~lls arepresent ( w): ~mpdre~with (a).~Bar 0-08 ram: c o n t i n u e s to be e x p r e s s e d in m o n o l e r c u l t u r e also b y t h e p r o g e of t h e s e ~ l l s ( L E R a n d l e n t 0 i d a l cells) ~:and e n a b :their/identificatiom Thus,:in g a ~ s ~of:: cells n o t a t e d w i t h R A , ~i m u n o s t a i n i n g for - I I is c o n ~ e n t r a in the lentoidal'co [Fig. 3(a)]. I n co t, in a g g r e g a o f R A - t ~ e a t e d cells i m m u n o s t a i n i n g g l i o c y ~ s did n o t semblei~n l l y , , b u t w e r e i n ~ p e ~ e d w i t h n e u r o n s , s i n g l y o r as s m a l l Clusters [Fig.

3(b)lE v i d e n c e ' that:: P~A i n h i b i ~ ~t h e l e n t o i d a l ~modifi t i o n of Mi~ller cells s obtained b y : ~ i ~ m u n o s t a i n i n g : t h e agg a t e s w i t h a n t i b o d i e s to M P 2 6 : : T h i s ~antigens:: i s a charact;eristic m e m b r a n e c o m p o n e n t of.lens ¢~lls; it is n o t d e n o t a b l e in ..the .r e t i n a . 261 b e c o m e s ,expressed;:~in gli0cy~eS t h a t c o n v e r g ~i n ~ l e n ~ i d a l cells a n d i ~ iis a~di o s t i e indic r of their n lens-like p h e n 0 t y p e i ( M o s c o n a e t M.,

INHIBITION~OFM|.~LLER GLI~ TRANSFORMATION

97

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98

L. D E G E N S T E I N

A N D A; A: M O S C O N A

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of RA-tmated ~ l l s [similar to ~t h o ~ in Fig:: 2(c)] immunostain~ f o r -II: Immunostaining gliocytes are scattered throughout the ates, Singly0r as small islets: Bar 0.08 ~nm. (C) L~ent0id-containing ~11 a r e ~ [similar to thc/~ in (a)] immunostain~ with a n t i . r u m t0 M ~ 6 ( a I~S ~ll-membrane anti,n). ~ Cells (lentoid)sh0wstrong immunos~ining ofthe cell membrane: Bar ~ 0 . 0 8 mnn. (d) Part of (c) at a higher magnifi~tion: ~Bar 0 " ~ mm, (ell A g g ~ g a ~ s of RA-t ted c~lls, ~immUn0stained with anti-M 6 anti urn, Only a , of the gli0 sc ~d throug~ut t~ a~ ~ s h 0 w immunostaining, Bar,~ 0:08 rnm. ~ 1983).~ .The . m e m b r a n e ot:: !ent~Mdal ~cens str0ngly~ i m m u n o s t a i n s ~ . w i t h ~ a n t i - M P 2 6 antibodi [Figs 3(e), ~-(d)].: a n t with R A o f m o n o l r cultU s inhibi or

r e a c t i o n w i t h ~t h e a n t i b o d y

INHIBITION OF:Mt~LLER GLIA TRANSFORMATION ~

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~ o . 4 . ( a ) Eflbct of RA on GS induction wi~ co~isol in monol r ltu Of retina ~ells. k bar • GS induction in cultures treated with RA. ire b a r : GS level in cultures not t ~ a ~ d with RA. Co~isoi was added to the cultures on the fourth day. Two days later the ~lls wm~ harves~d e,nd assayed for GS activity. (b) Effect o£ RA and GS induction ~in ~11 a~regah~s' ~ l l s precultured in ~monola~r for 4 days were a ~ m g d byr0tation for 2 d ~ s . Cortisol w ~ added at the beginning Of aggregation: B k ~ r : GS induction in a regals of RA-trea~d cells, ire bar: GS level in r~a~es ofcells not t ted with RA.

T h e a b o v e results i n d i c a t e d t h a t R A p r o m o t e s r e s t o r a t i o n : o f gli n e u r o n c o n t a c t s and, t h e r e b y , i n h i b i ~ glio te modification. This ~c0nclusion w a s f u r t h e r t e s ~ d b y m e a s u r i n g the i n d u c t i o n of g l u t a ~ i n e s y n t h e t a s e (GS) Tl~is e n z y m e ~ m a r k e r of Mfiller glia (Linser a n d Moscona,, 1 9 7 9 ) is i n d u c i b l e With cortisol: :~However, ~as described in the I n t r o d u c t i o n , GS i n d u c t i o n in Mfiller glia also s t r i n g e n t l y r e q u i ~ s specific Cohtacts be een t h e ~ gila cells: a n d n e u s. In onol er ~cultu , dispe ed' glio ~ s , l o s e a d h e s i v i t y t o n e u r o n s as t h e y c h a n g e i n t o lentoi~!al cells, ~a n d can:/no ~longer ~r e s t o r e c o n t a c t s n e c e s s a r y for GS induction! If, as suggested a b o v e ; R A i n h i b i t s t h e s e changeS, t h e n GS should be i n d u c i b l e in R A - t t e d cell ~cultu s. F i g u r e 4 ( a ) s h o w s t h a t a d d i t i o n o f Cortiso! on ~t h e f o u ~ h d a y e l i c i ~ d ~a significant inc ase in GS level in R A - t ~ a c u l t u ~ s , where'as in u n t ~ a ~ d c u l t u r e s GS rose 0 n l y slightly a b o v e t h e basal level A s d e s c r i b e d above, r e t i n a cells plat~ d in R A = c o h t a i n i n g ~ e d i u m s p o n t a n e o u s l y assernble into clusters. G S induction: S t r o n g l y indicates t h a t , in these clusters g l i a e e l l s r e s t o r e c o n t a c t s w i t h r~sbfthe k i n d essentia! for ~t h i S i n d u c t i o n F u r t h e r evidence f o r this i s ' t h e high ~level of G S i n d u c t i o n o b t a i n e d a g g r e g a t e s p r o d u c e d b y r o t a t i o n o f R A - t r e a t e d :cells:: Cell a g g r e g a t i o n b y r o t a t i o n increases t h e q u e n c y of a t t a C h m e n t b e t w e e n 'mUtUally a d h e s i v e Cells: I n a g g r e g a t e s of R A = t r e a t e d Cells c0rtisol i n d u c e d GS to levels 1 0 - 1 5 t i m e s h l g h e r t h a n in agg g a ~ s of U n t r e a t e d cells [Fig. 4(b)]. Tills s t r o n g l y s u p p o r t ' t h e conclusion t h a t R A p r o t e c t s ~ dissociated Mfiller g l i a 'tb-om: r a p i d / l o s s o f adhesive aflinity f o r n ~ r o n s , ( i p r 0 m 0 t e s r e s t o r a t i o n o f g i l a - n e u r o n cell' contacts: and', t h e r e b y ; :inhibits ~ehanges t h a t lead t,J t h e m o d i f i c a t i o n of s e p a r a giiocytes i n t o lentoidalCells. ~ T h e a b o v e t e s ~ ~ e ~ c o n d u c e d w i t h cells c u l t u r e d i n m 0 n o l r f o r 4 d a y s , ~In:longer t e r m ~c u l t U ~ s , t h e inl~ibitory::e ts i~o f R~,_~ :~on .....gliooy~:, ~mOdification ~d i m i n i s h 6 d

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L. DEGENSTEIN ANDA; A. MOSCONA 4, Discussion

The results described here implicate retinoic acid (RA) in regulation of mechanisms of cell adhesion between Mulle glia and neurons. They cor bora~e p vious evidence th Mfill~cellsrequi~ con ,dependent cell i n ~ r tions with neurons for e~p ssion st~bi of phe t y p i c characteristics ( s c o n a , 1983). According to current concepts, specific eel! contaC~ elicit signals in the cell su ce t h a t are communicated i n ~ r n a l l y and (.ontribu~ to regulation o f i n t ~ ~ l l u l proces s; ~ suggea~d earlier, such ~si a|s help ~'t o / m a i n t a i n the p h ~ o t y p i c c|iaracteristics of liar gila. Cell sap tion elicits new signals from the cell surface tha~ m a y al~er ge expression and lead m cell tr tin on (Moseona and Linser, 1983; da and hioseona, 1985); in tile p nt e, to conve ion of Miiller gila into lentoidal cells. We ose t h a t R A p r o ~ c t s sepa ~ d Miiller cells m this conversion by promoting restoration of contacts ~with n e u ~ n s ; i.e. t h a t R A s t i m u l a ~ s in dissociated Miiller cells rapid ~ c ~ v e r y of surface: eehanis s e ntial for adhesion to neurons. Since tina glia contain reti id-binding p r o ~ i n a (Bun i l a and Saari, 1983), these cells can be surned to be subject to RA action (Wiggert, B gsma, wis and Chader, 1977). How RA affects cell adhesion mechanisms has yet to be elucidated. A possible clue be the involve ant of tinoids in glycosylatioaof l! b lecules (De Luca, 1977; Bertram et al., 1981) and in o t h e r aspects of im b ne composition (Lot an, Kramer and Nicholson, 1981; Shidoji, Sasak, Silv an-Jon a n d D e Luca, 1981; sky.and L o t ~ , 1984). This evidence, together wi~h ?,he known role of specific me b ne olecules in cell adhesida ~(Moscona, 1980) su st t h a t tt~e effects of RA described he are due to its influence on biosynthesis of gila,cell membrane component; m cifical~', t h a t RA prevents changes in the cell membrane which result in loss o f g l i o e y ~ adhesi rmss to neurons. P pt ~ s t o tion of contacts with urons safeguards the gila cells from lent~idal cony om I~ is important to n o ~ t h a t tile inhibition by RA of this nversion is not I ring; ber of glioeytes be¢o e ' r e s i s t a n t " s p ~ a d out with cultu time, an i n c r e a s i n ga' and e x p r e ~ l e n dal ~ s.: T h e ason for this is not clear p ntly. The ay exist initial variations in sensiti,(!tv of Mfiller Cells m RA, d ' istan~' cells may ssi iy d o m i n a t e the culture. Another possibility is t h a t the a m o u n t of RASoinding p r o ~ i n s deCmas in Cultu d gliocy~s, rendering t h e m pro ssively 'resis n t ' to R A . . I t is kn n t h a t t h e level of corti~steroid-binding ceptors markedly decreases in monolayer=cultured retina cells (Saad and Moscona, i985). The result~ described hem ~ ise~the.possibility t h a t retinoids also play a role in gli~aeuron 11 adhesion in t h e tina in situ. I f futu s ~ d i e s confirm this, it would suggest that deficiencies or abno ai metabolization (~f ¢i ids ~ i g h t in this Way lead to changes in Miiller is and to retinopathies. ~ The su e s ~ d le o f ~ t i n o i d s in regulation Of I con~c~ and, ~hereby, in phenotype contr91 n provide a unifying rationale for the s i~gly dive e effects of retinoid compounds in various sys~oms- suppression of changes associated witll neoplastic t r a n s / o ~ a t i o , (Meromsky~ an d Lotan, !984), and Promotion of cell d i f f e ~ n t i a t i o n (Stri a n d a n d Mal~d i , : 1 9 7 8 ; S ~ ~ r , ~Shey!ngky,~ Knowles and Strickla , 1979; Fuchs and G n, l!]St; Sher an,: Matthaei and Sclaindler, 198!1. The Underlying common thctor in a t least.some of these effects Could be the critiea| role played b y cell contact in phenotype cont l a~d ~the influence of retinoids on gulation of |l adh ion and cell-contact, s~bilit~r.

I N H I B I T I O I ~ OF ~-I|-~LLER GLIA T R A N S F O R M A T I O N

101

ACKNOWLEDGMENT This work was s u p p o r ~ d by r e a ~ r c h gr~nts N o . P 8408585 rn the Nati0nal Sciene~e undation and No. 1-983 from the March of D i m e ~ B i r t h D cts Found on. REFERENCES Bertram; J. S., Morden, J. L., Blair, S. J. and Hui, S. ~lggl). Effeet~s ofretinoids on neoplastic trunsfi~rmat.ion, cell ~ h e s i o n and membrane ~opography 0f e u l t u ~ d 101T1]2 celia. A . ,V. )~: A c L 359, 218 O. Bollag, W. and Matter, A. (1981). m v i ~ m i n A to r e t i n o i d s in e x ~ r i m e n ~ } and clini l oncology' achievements, failu~a'and outlook. Ann. N, Y. A c ~ : Sci. 359, 9-23. Bun ilam, A. and Saari, J. (198B),Immunocytochemieat locMization oftwo ~tinoid-binding proteins in ..Jer~b ~ ~ t i n ~ , J. Ce//,,~ .97, 703-i2. De Luea, L. M. (1977) e di ct i n v o l v e m e n t o f v i ~ a m i n A i n g l ~ o s y l t n s ~ r ~ Ctions of m malian membranes. ~i n V i t a m i n s and Ho o , Vc~. 35 {Eds Munson; P. L., Diczfalusy, E., Glover, J. and Olson, R. E.). Pp. t-57. AC mic Press" New : f o ~ . De Pomer~i, D . I . , kagi, S., Kondoh, H , and Okada, T. S. I~i(1984). Expression of ~ x i n ~cep~ on cell su ces in t ~ n s d i n~iating cultu s of neu ! ~ t i n a . v. h D~r.

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