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Ontogeny of retinal oil droplets in the chick embryo
1Cxptl l~ye Res. (1968) 7, 434-442
O n t o g e n y o f Ret inal Oil D r o p l e t s in the Chick E m b r y o *
De)~(zrt..me~.t. of Che.mGtry, lVa?t~te State U.nicersity, to~d Del)artme~.t. of A.na.to~;¢y, I,t:¢~yne State University School, of Medichu', ])etroit, Michiyan., U.S.A. (Received 20 December 1967', Boston) T h e ¢~t'igiu o f tim e,:~lot'ed oil d r o p l e t s in t h e c h i c k (:ones wr~s invest;its.ted b y m~bjecbing r e t i n a s f r o m st.aged e m b r y o s i~> c o n ' e t a t i v c b i o c h e m i c a l (visible s p e c t r o p h o t - o m e t r i c a s s a y s o f chrom~togreor~hieMly ~c.part~tcd extra.cL,~) ~,nd m i c r o s c o p i c o . m ~ ' s e s . The. earota'.rmid pigment,~ rr.'spons[ble for "t,ho coiorat.ion o f %,he oil d r o p l e t u a r e d e t e c t e d s p e e t . r o p h o t . o m e t r i c M l y b e f o r e %hey e;uz be vistu¢lizod micro.~eopieMly ill ~he f o r m o f c o l o r e d d r o p l e t s . :l?he spcx:trot)hotomet,ric (i~t,t~ wev(:M tt~at e a c h p i g m e n t a r i s e s iu.depot~de~ ~tly in t.ho fi)llowing s e q u e n c e : g o l d e n y e l l o w , y e l l o w i s h g r e e n , a n d red. T i m grdrlen y e l l o w h y d r o c a r b o n is first, de.tt;ct:ed at, s t a g e 27 a t w ] f i e h ~ime She retin-~ con.Mst.~ o1" a. l,hiek. tu~stra.t, ified, n e u r o e p i t , heiitd b~yer wibh ~rt a c b i v e i)rolif(.-ra.tAve z o n e , but, laeRs vimu~.] eell~. (lMIoxr~nt-hin, t, im yellowiM~ g r e e n p i t m e n t , , 'd.riscs t~t, a~age 1¢4. Ti~e regi:ta is st,ilI d e v o i d o[" visua,[ elctnon~s at this stooge, but, is orgt~ttized i n t o sever;.fl n u e l e a r l a y e r s , T h e roJacentrati(m-s o f %he g o l d e n y e t l o w a n d y e l l o w i s h g r e e n efn-of..eJloif[s begirt t o i n c r e a s e r a p i d l y ~t st,~ge ;17 w h e n i n n e r s e g m e n t s a r e differe~ltiating, bu~ oil d r o p l e t , wtc.~ml~,s ha.vt~ rmt, ye~, ~ppe:~red w i t h i n ~hem. (]olorless droplet~s first, e v i d e n t i~i fresh :prep~r~,g~ons u f l, his s t a g e Ju~5" nol; be, oolorless ~% MI, b u t mu, y t e n t h , i n bhe y o l l o w p i g m e n t s i~t u~teh Io,~v eonct;nt, ra{,iox~s ~hat. 6heir eO|Ot~d or%fill.or tit,• diaerimi~i~tx~d m i e r o z e o p i e a l l y . T h e rcti /~igment, as~z~xant, hin, is firs~ e . v i d e u t a~ s t a g e 41 a n d i m m e d i a t e l y e x h i b i t s a st;e:,.dy i n c r e a s e ht e(meent.z'a~ion, '.L'lm regina posses.-ms ,~dtzll; stz.at~ificataioxi a~ t h i s t i m e ; M1 t e n taye, rs are p r e s e n t am.l o i l d r o p l e t , v~z'uoles a.re o b s e r v e d t b r t~he firs~ t i m e . O n t h o baMs o f t,h.e s p e e t r o p b o g o m e t , r i c f i n d i n g s it, .is eont, e n d e d ~:h~ t h e d o u b l e cones ~re
t h e fi~t, c o n e s t o d i f f e r e n t i a t e in tbr.~ c h i c k ret, in(~ ~,nd ~ls~t. t h e c h i e f e o u i p o n o n ~ ( b e a r e r o f t.l~e Igoldon y e l l o w d r o p l e t ) ~rises b e f o r e tJ'~e n.ccessory e l e m e n t (co,riSer o f t.he yollowi.sh green droplet).
1. Introduction The re.rhea of t,h c chickell (Gallus domestieus) eontaims single arm double cones t h a t are characterized b y the presence of a single b r i g h t l y colored oil droplet wit&in t h e distal e x t r e m i t y of each of their i~mer segmettts. Three colored droplets (red,goldert yeUow •rod yellowish green) are discernible, and each is i-est-ricted to a specific cm~e type. R e d droplets ~re housed i~t single coates, whereas t h e golden yellow and yellowish ~.een droplets are confined to t h e ct,ief a n d aecessot T c o m p o n e n t s , respectively, of the dolible cones (Meyer and Cooper, 1966). Chemically, eack colored oil droplel; c o n t a i n s o~m or more ca.rote.noid p i g m e n t s dissQ~Ve.d in lipid m~terial of m i k l m w n composition. :In tel;|hal e x t r a c t s from the chicken t:hree d i s t i n c t l y difl~reItt ea~rotenoid pigme.~Ita are present t h a t reproduce in a p p r o p r i a t e solvezl~s t h e m~t~md colors of ~he oil droplets i~ r i v e . Kfih~ie a n d As~es Tliis inve.~%ig~ion ,,v;~s auppor~ed in p a r t by ]Lifts r(..scar0h gq~ants ( N B 06580 a n d AM[ 0;3334} from the .~at.iorxM rnst.itnt(~s o f I][ealth. stud in p~rt, by r~ J~igh~ for Sig|xt Grant-dmAkl f r o m t h e Grat.ionM Council to Combat, Blilx(hlo.as, Inc., New Y o r k , N.~T. A pre, l i m i n a r y report, o f t.his s t u d y w:~s present, ed at t h e am~nal m e e t i n g of ~he Asaoehttim~ for .Researo, h in OphCAudmology hold in Cle~rrwater, F l o r i d a , 1-3 May 1967. "~ 1Preheat addrens: :Oepar~men~ o f Biok>gicM Seiertces, P u r d u e University, Lo.fas'et,t.e, ~tcl. 47907, U:S.A. 434
ONTOGI~NY
OF
II, l g ¶ I ' I N A L
OIL
DROPLETS
435
(1878) were the first invesi;igators to isOl~tc these pigments, a n d 1.~,5~1d and Zussman (1937, 1938i were t]te first, to crystallize t h e m ~md establish t h e i r carotenoid nature. Weld a.nd Zussman (1938) identiflcd the red pigment as astacene, the golden ~s ~ xal~th.ophyll (htt.ein, m i x e d with its isomer zeaxaltthin), and the yellowish greett pign rant ~s ~m unide~tified hydrocarbol~ with properties Mmost ideuticM with ~arehmne. Cr)~t~ds of e.'~ch pigme:~t were dissolved in suitable solvents, ~.nd ideutified by their .visible ~bsorption spect, ra. Later 'Weld (t948) detected ~ fourth oil dxoplet carot~tmid, which he nm.ne~t gulloxantttin. I{e xw~sramble :o crystMlize this alcoholic pigment b u t recorded visible absorption peM:s a t lower wave2engths than the or,her three carotenoids. W e l d did not associate g a l l o x a n t h i ~ with a n y of the three colored oil droplets; the only reference to the color of this pigment w~,s t h a t it separated as a yellow band[ on a cola:tin of cMcittm carbom~te using benzene as ~ solvent.. Galloxanthi:t was a d s o r b e d j u s t below a s t a x ' m t h i n and ~bove htteln al:d could not be easily separated from either pignmltt. Two subsequent studies, however, have demonst,rated titbit gMloxmtthiL: is the m M n carotenoid co:lstitttel~t of the yellowish green product.: Strot}mr ~md Wolkmt (1960) recognized its spectral characteristics in. i n t a c t yellowish g:'een droplets by me,ms of a microspecgrop]:otomet:~.r :rod 3leyer, Cooper and Gernez (1965) were able t.o separate this pigmcn.t b y mea~:s of t h i n layer ehromatogral.>lty a.nd to record its ahsorptio:~ spectr~ (visible and infrared) irt various solvc,~'..-~. The visible absorption m a x i m ~ closely resembled those reported b y Wakt (1948) [or galloxanthin, a n d the i~ffrared dat.~.~ s u b s t a n t i a t e d its x a n t h o p h y | l -nature. T h u s , . t.he.re appears to be Iitt.le d o u b t t.hut i:~ the chicken retina.galloxa~ttltht represents the yellowish gree:t p i g m e n t theft occupies the accesser Y cone oil droplet. I~ is also now welt established t h a t the coloration of the red oil droplets (single co~ms) i s due to the alcoholic carotenoid, a s t a x a n t h i n , which dm'ing e~.rac~.io~ becomes ~ t t 0 x i d i z e d to its keto form, astaeene (Kuhn, Stene and Strenson, 1939; yon St..l,dnlt, z, N e m n a n n and Loevenich, 194B; WMd, 19t8; Strother and Wolken, 1960; Meyer et el., 1965). The Dosition and ident.ific~tion of the or.her carotenoid p i g m e n ~ detected in re-tinal extracts, however, recluire fm-ther s t u d i e s . . L u t e i n (xa~tthophyll) tbr example, has beer~ associated with the golden yellow.droplet by n ~ : w investigat¢,~ (Weld a n d Z t t s s m a t : , 1937, 1938; Loeve~fich; yon Studnitz a n d Wigger, I9~3; yon St:tdnitz et at., 1948; Weld, 1948; Goodwin, 1954; Strother a n d ~ -:ken, 1960), bug reeen.t Stltdie8. h~ve shown t h a t when t h i s pigm:ent is separated b y m e a n s of ttfin layer e h r o m ~ t o g r a p h y and anaIyzed by visible a.nd infrared stmctroseopy }t is, in reall W, :, h y d r o c a r b o n with visible spectra similar to the nnidentitied hyda'0c:~rbon detected by :V~ld a n d guss~rtal-t (1937, 1938). The d i s t r i b u t i o n of the yello~dsl~ ~-een carotenoid, lacertofulvin, described b y Loevenich et el. (19g3) a n d vo~: Studnit, z et el. (19,t3) is likewise confusing and requires further documentatio~i. We have previotmly reviewed the cont~roversies concerning the n a t u r e of the various ca:'otenoids i:t r e t i n a l oil dropleks a n d their ~bsorption s p e c t r a i a v~riot~s solvents (Meyer et M., 1965). I n a n y case, most- investigators hax:e been: able to separat~ o n l y ~hree earotenoids from retinal e_x~.raehs by chromatogra.phic methods. I n the pre.aent; study t-hese p i g m e n t s wifl be designated :,.s astaxa:tthin (red}, g,~lloxanthin (yetlo~:ish green) artd rm m:ider~tified .hydroea.rbm: (golden yellow). T h e i r ,~bsorption s p e c t r a (~dsibte) in benzene are illustrated in Fig. 1. [.F.or comprehensive s u r v e ~ of carotenoid chemistry co~:stfl~ K a r r e r and Zucker (1950) a n d Goodwin (1954).] I~x the chicken retina the colored oil droplet~ arise during embryo~de life; nil a d u l t colors are discernible h~ t h e newly h,~t.ched chick. Observa;tious on t,he e m b r y o n i c origh: of these hxclusions and their carotenoid content have b e e n m a d e ahno,st
4,26
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eaHrely from the microscopic s t u d y of fresh retin,.s. Since t,h e rellabitity of such (.)bsevvt~)ions d,:pe~tds ~huoat ent, ire ty upon the color discriminM.ory M)ili~ies of ~he observers ~md the quallty eft the optle~l systems employed, their results are quite varial)le, and inconsistent.. The application of more sensit, ive and reliable physieochemical i:~roeedures (e,g. chromatogr;~.phi~ separn.l.io~:t a n d spect.ropho~ometric idmttifieat, ion) fbr the determina.tio~ts of t.he e~n'ly e m b r y o n i c o~'igi.n of the oil d r o p | e t l~ig.mertt.s h a v e seen timit.e.d ~:tse. Coulombre (1.955) employed t h e m for the deteegion of asta.xanthin, bn.t gho other oil droplet, e~rotenoids }rove hog been studied owing to .t,he di{ficatlties incurred, i.tx efl%cting their separation. Advances in se.paration a n d spectra[ mxfltyses, however, ha.re solved ~his problem (Meyer et ai., 1965) a n d now warrant, thei:r appliea.t.ion to t:hese developmentM problems, Irl the present, st, u d y ~he inlgiM ~ppet~ranee ~nd. subsequen~ increase .in eot:teent.ra%iotx of each oil droplet pigqnent h0.s beett ascertained by spee, trophotonxet, ric determ i m ~ i o n s of c h r o m a t o g r a p h i c a l l y separut, ed rethu~l ext,]~&ets ag czech mor phologieM stage from st,a,ge 25 to hr~tehing. :In turn, ~he developmental changes involved in ghe o r i # n of these pigments a n d the formation of oil dropte~ v~.cuoles t h a ; boase t h e m h a v e been studied microscopically in fi'esh ~nd fixed retinas from simih~r stages. 2.
Materials
and
Methods
Twtmty-tbur chick emb~.'yos at, e;~ch morphological st,~ge bebween 25 (approximately t½ d~ys)a.nd 45 (Hambucger a~~d Hamilbon, 1951), iactuding newl.v-Im~hed chicks were ~.unployed. 10"orea,ch stage, 48 whole eyes or ~-etinas-- -depending upon t,lm ease. with which
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~h~.ot. Visible a b s o r p t i o n ~}wctr~L o f the, t h r e e oil d:roplet, cl~robex~oiCLssepara/.ed f r o m chicken re~,im~ by. thin)l'~yer Ohromate~gra.phv: a n d d i s s o l v e d in benzene for spectra.! an~lys~.~ (Meyer, C("~oper m~d ~ m ' n e z , 1985). V e r t i c a l liues h a v e been ~tra~,m at,. 40,f~ ~nd 4,,t9 m/~, t h e most. Sensitive s p e c t r a l r e g i o n s fi~r the differenti~tion of t:he yellowish g r e e n a n d g o h l e n 5-el low earot-enoid :pigmengs, v e s p e o t i v e l y ,
t.he retA'nas could be dissect,ed fi'ee---were removed al,-d pooled for chemical studies, The a~:hole eyes or ret, inas were homogenized in equal mnounbs of beJ~z~me ;.rod 70~/o methanol for 4-0 sc.c and-cent.t:ifuged for 10 rain ,'tt 27,000x g. The. eat'otenoid-eontainiagex'traot (cpiphm~ic aft-~r centr[fugat.ion) was then concent_rated under dry, highly purified nitrogen a~d, depending upon the stage., subjected to the following m a n i p u | a t i o ~ : (1) For the
ON't'OGlgNY OF I ~ E T I N A L O I L DI~.OPLETS
437
early :~tgge,s" (25--40) i~x which only t,wo c;xr6tetmid pi{,nne~t,s (golden yclI(.,w a n d yellowish Vreen) could be detected, t.hc earotenoicl concm~trate was monitored stumt, rophotometrically (Zeiss .PMQ-I[) a t 449 a n d 404 m/z, :respect, ively. These ~wavele.ngtlm (]Pig. I) were ae|eetcd becamse t.hey rl.'present the most: se..usitive spect, rat regions for ~he demonstratio~ of the ve'ry .-'mall alttotutts of carote~mhl pigmm~t i,~ the younger embryos (Fi~ 2 ~nd 3). Sueh
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Stage ]fl(L 2. G r a p h s h o w i n g t h e spectrophot~meta'ic ( l e t e t ; b i o n o f t h e g o l d e n yellmv oil droplet 0e, rot.en~itI { u n i d ~ m t i f i e d h y d r o c a r b o r } ) d u r i n g rui, i ~ a l d e v e l o p m e n t i,x t h e (dfiet¢, ' l ' h o o p t , i c a l d c a s i t , it~s r~rel d e t ~ r ~iline~l i n b e n z e n e a.t 44,q rote.
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F i G . 3- ( A r a p h s h o w i n g t h e s p o c t r o p h o t o m e ~ r i c d(~t.cetion 0i" t&c y d l o w i s h g r e e n o i l drolalet, c a r o t . e t t o i d , g a l l o x a n t h i n , d ~ r l n g r e t l m t l d o v ( ~ l o p m e ~ g i;~ t h e c h i c k . T h v o p t i c a l do~mit-ies w e r e d o * , c r m i , ~ e d i n be~,-zeno ~tt. 4 0 4 m ~ .
small coneent.rations of pigm_~m~t, were nol; amenable .to ch~:omatographic ana.l~sis,.. (2) Su~)sequm~t~ to stage 41 the presence of Lhe ~e~Jt pigments, astaxa.at]fin , comp]eteily ~as:kecl the presence of tlm other two pigmeaat,s ~ d hence ~mcee~sitata~cl i t,s ~ep~ratim, from. ~1i9" lattc:t, two pigm~enlks on K o d a k thin h~yer plate, s deve.!oped Witch chloroform. The s e p a r ~ e d carotenoid:~ were t&en elut, ed fl'om the plates wivh b~nzeae a n d t~e a,bsorptlon of.a~st;~,~zaathin was m~nsured at, 525 m y (Fig. l ). In all (.a.~es a dpectrum e£ t.he:ent.ire benze~,le~extrac~ was t~kmt. :For microscopic stu.dy several retinas.of each utatge wer(; fixed ia Eotme,r's fluicl a n d embedded in pm~flhx ns described.previously (Meyer and. Cooper, 1966). Se~e.ra~-~etinhs of each ~ta.ge .were also e x a m i n e d by m e a n s of b:igh~ field a n d txl/ase c:ontra,~ micr0.a,¢opy
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~,a fresh, unstained (fiat ,tJ|d squashc(t)prepar:.ttio~s h t order to establish visu'~l re.cognit.ion of the oil ¢[rol,let ])igmelltS dttriug t.ltes~.~ critic."d periods. I?m'ticular care was ~aken to vxantine all .areas oI" the. embryoMe ret.itm, ~:speeially t,he posterior fundie region, which cliff's!rent:lares earlier than t.he perildter.'~t cquat-oriat rt;giott (S~dmlt.ze, I8611; Couh,mbre, 1(.)55; Fujita and lh,rii, 1963) 3. R e s u l t s
T}te g(,lde~l ye]l.ow hydrocarbon is the first oil droplet c~u'otexmid t.o be recogtfized spccl;rol)l,otometrically. Ib is initi;~lIy recorded at st.age 27 and does not. begin to iJicreaso, in e o n c e n t r a t i o a until aft.er si:age 30 (Fig. 2). I t thett begins a slow r~te of increase, n.ntil st'~ge 37, when its conceTtt:.rtttion rises sharply. Microscopic sections of reeimts at the bcgimxin.g of carotenoid detectiou (stag4s 27--30) discIose a n immat, ure orgaltization. The inverted ncuroelfithclial h~ycr ('oasist,s of ~m active prolifer¢~t.ive ztme, as indicated by the numerous ntii:otic figures [.Plate l(a)], a n d :~ r e l a t i v e l y thick, unstratitied nuclear l~yer, but ShO~VS nO evidence of immr segmelit format, io~t [Plate (b)]. O
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lqo. 4. G r a p h sho~ving the 6pectropho'~ometric d e t e c t i o n of the r e d oil d r o p l e t caro~.crtoid, ast~cene ( a u t o x i d i z e d a s t a x a n t b i n ) , d u r i a g retin~d d e v e l o p m e n t in t h e chick, T h e opgicM deasities were det.erm i n e d in benzene ~t% 525 mt.¢.
Galloxa.at, hia, the.-yellowish green pigment, is not detected spcctrol)hotometrically un$il st.age 34. (Fig. 3}, ag which ~ime the retim~ has differentia.%ed into several nuclear strgta, but still lacks a. vimm.] ceil lgyer [l)b~.te 2(a)]. Like the golden yellow hydrocarbot~, however, the coneelttratimt of gallo~%nt.hitx r,~pidly increases a t Stage 37 (Fig. 3). Vimtal cell differentiatioll has commenced b y t.his s t a g e. Smelt protoplasmic prot.rtmions are evide.t~t projecting from the cells borderb,g the e x t e r n a l limith~g m e m b r a n e [P.b~te 2(b) a n d (c)]. These cellular exteltsions represent %he prhlfitive imler segment, s of the photoreceptors, which are gn'owiug t o w a r d t h e p i g m e n t epithelium. A s t a x a n t h i t t is initially detected spec)~rophotometrically at, stage 41 (Fig. 4). Unlike the other t w o c~rot.enoids, the concentration of tiffs p i g a m n t m ~ i a t a i n s a s t e a d y r a t e of increase a l m o s t ~ u n e d i a t e t y a f t e r its first ~ppeara.nce (Fig. 4). The
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X-'r.~.T~ I. ~ h o t . o m i c r o g r a p h s o f ckiok e ~ i n ~ a n d .~itja~,rtt ~cler~ (S) trTtd_ ohoroi4 (C) ~t-st/tge 29 (l~atrtb u r g e r antl ]~I.,~n~ilf~m, 1951). (5) T h e stragitic.ation of t-he ro~t.lna, r e s e m b l e s elx~b o b s e r v e d in t h e (',hick r e t i r m at. st.ago 25 (.Fig. 36, O ' l ~ h i t l y ~ud 5[cycr, 1959) anti t i m h u m a a . r e t i n ~ of I]~orizor~ x v (Fig. 27,-O'B.:~hilly, [966). T h e ,xcuro. epitkeliut~a h a s zu)t ycL difi'r, r e u t i a t e d ixtto ib,ttern~l a n d e x t e r n a l aeuroblz~sb'~c .i~3-'ez's.. Pigmetat g r a n u l e s a r e present, in t h e p i g m e n ~ t:pithethtm. Tlxt~kfollo~vi~lg retina.1 lo.yer:~ caJl t)e. easily .distit~gnished*. (I~) c.xtern~d (pig,nenb) layer; i~xvet'te¢[ }~yor (()f o]~tic (;u]?) c o n s i s t i n g (*f tire (EL$[), cxt.e~'nal [iJniting m e m b r a n e ; (M) n. m i t o t i c z o n e ( p r o l i f e r a t i v e htyer); (I) i n t ' c r m i t o t i o 1)b,~so (prh~litivc zc-u.~); &n(1 (-~.Z),. n. ~xuclcar-frcc m ~ r g i u a l zt)ne. (X 900) (b) ~[[igher n~agniticabiolx .~koxx-ing srgifit.'.iaI f,<:l~aration o f p i g m e n t atui h ~ v e r t e d la.ycrs wLticlx ccvett.~ tlxat, visual elcmetxts h ~ v e n o t d e v e l o p e d us y e t . 3lit, erie figurc.~ (arrou-) -tre evide~t~ in t h e t)rolil~rativo zon~,
( x 2~00) [ T o face page 438
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~I?~:.A'J's~ 9. 0~) PlmL~m~h-~rogmph ,~f ehi(;k ret hl,a. ~.t. ,~t.age 34. T i m neur, Jepit-helium ent.i~Lf~(I iDto ~ v ( ~ m ] dis~ret(, ]o.y(:rs. V i s m d (:('.li~ ~tre t~ot, prc.~ent. (.-; :;70}
ha:~ 1,~:e,>am ~]iffer-
(b) Phtm~ e+~nt,r;J,st pho£(~mh:-mgr,plx ~,1' Sh.(: e x t : e r w d porl.ion o f I:~,~: reLim~ a.~ s t a g e ,':I7. 1)(:vel,~ping ~4~ztt~G ceils (arrow) are~ ~vJch-'..tlt us proLoLp!asmie (~.~;t,L'nSiOILXfrc~Jlt thA' exit.ernst[ ~¢:ur~Jt~l~.~l:ie lr~yer (nu~:h'.i o f to[Is alId eone.~). :i:tm v i s w d eell.~ pt'oier't: rms'ard t h e pi.gm(-r~L e|~ith¢~lium Whlel~ Ires he~n ~u'|,iliei,~,tly diat)h~.oe([. ( z 7!0) (:~.'-) .|li;ghar p~)wer, p h a s e (~oni.r~sI, l~hot.~mierogreph a h o w l n g |,he ,tevt, loping vism~.I eelIs (urr0,w) ~tg ~g~,ge g7; oil ~troplef, w~euole..~ a n d (mL/,r segtn.et~t~, h ~ v e lint ul)ipe.ared ~s yet:. ( x lkt00}
(~) ~i.'JL.e pi~m~,ll I; ,,]~it.i~,lium ( 7: I,N(}(~)
I~rt,~ been' (L~{f~.'.i~(~d. I )~.:w,h~.l)i,l ~ ~.i~,,~ ~-~1~.~ ~.r~~ ~.,vid~t,.
( .~ ~I.~1))
(]NTI)t:;I?Nh'
Oil
|fIqT.INAI.
(t|L
I)IL()I'LI",T-'g
4:19
rct.in:~ i~t stage ,It lPhm.'. 3(all res,:mbh:,s c.loscly thin; obsc:rw~d i , t.hc adul.t; ~dt ten ~tcll.nitive layfn.s i~re l~ow ('vide.n.t. tn ]~(>lmo,:-tixed l~h~,.m- micro.~:.oliic prel.~m~ti'm~% v;~(:~.tolc,,i C4rlt b(; St~el't el,.,arly for t:]l(; first, t i m e oc(:'.ttpyiilg t.}t~.: distal ,,xtr,:>mil;y of tim, iniu'r s~,gm(.,n.ts ......tim definitive site ~,t' tit,., oil (h'()pl(,ts [Plate 3(t))J. It.. is not. possible a t 1-ht.,,~e ]'(,lt~tiv(.,.ly y o t m g stages to tliil'~'r~:,~tt.i,~tt. I)etwt:.,.,n single ~llot.h;ss itroplct,s could tju ~tseertidnod J'rol.ll lslieroscot.>ie StilU) + Of .,,:c,r.i ~,l sections. ]),.>.tiniti~ y(.'.llowish gre(.'li ~it(l gol~Ien y e l l o w droph.,ts are ollserved ill fresh pl'e])Aral i o n s (:(~nuni,iitdn. 7 wil, h st.~o ,10; tell dropl(:t..s i~i)iloll, r ~tt st~g(~ ,1'3.
4. Discussion '.l'he existi~lg stati.sticM da.ta ~l~ t,be cnd.~ryoifie dew.'.tol)meiit of ~il dropl¢:t pign~enl~ti~:m..ilt birds haw, b~,~>~t obtail~m| v r i m ~ r i l y f r o m the microscot)ie e x a m i n a t i o l i s of fresh. ret, im~s of t,he ehic.kell, .Iapanese qm~il (Konishi, t965) a n d i;h.e .pigeott {graIts an~t J u d d , 1 .~33), Ilt the dotnest.it~ elli~:k0~l ((l¢t{l.u,<~do~e.st:ie.u.,O t,hese. d a t a h~ve re~-e~t[ed al~ '~lliLOS¢.ctJrltl.)Je~De b~ck of a.gn'~:cmet~t on the ' as evideimed b y our physieo-ch.en:ical (latt~, b u t in mmh d i l u t e a m o u ~ t s t h a t t h e i r Sl.)(.'ci~c eolors canxxot be ¢ilist£rtguished b y n.tic;roscojki¢ ob.~;rvation, In t h e p r e s e n t st.luiy micr¢)seopic examinat.iolxs were mii)I)'lementei~{ With si)c, dti:ol)hotonmtric a,tl.~r[yses of retinal hon'mge.ltates, which provide fi m o r e reliable at~¢t s(,nsit.ivc m e t h o d for t h e i d e n t i i i c a t i o n of th~-se pign~ents At t h e precise "'~ime.-'--'>-,it~ morphologicM stages, of t h e i r initial a p p e a r a n c e . B y tiffs t,echltiq~le it has beer i 8ttow-tl. t.h,~t each carotel~.oid pigmeitt :,rises indepel~dently ag a specific period i~t embr.yo~tie d e v e l o p m e n t a n d in t.h.e following seqtietme: golden yellow (stage 27; approxjm~,t+Jly ;j days), yellowish ~_rre(,rl./stAge 34: APi)r( x i m a el3 8 days), a n d red (stage 4:1 ; l~pprox.i.l n a t e l y 15 <,lays). The iiiitia] a p p e a r a n c e o:f the red oil d r o p l e t (;Aroteltoi.d, asi~xanthin,, eotlfil~ts t h e SlJect.rophotometrit; fimlirtg.~ of" C o u l o m b r e ( I955): I t coincides ~t,h. t h e i.ititiaJ i~t,<'msi6n of mitoehondria< into tht, develot)i~tg i n n e r s e g m e n t (NIeller altd I~rcipohl, t965).,, the oriset of disc forlnati(m in t,Jtt" ollter segmmtt-s ((,~ov~trdovski ~md Kh~trkeevich, 1966), ;~nd the c y t o c h e m i e ~ t detectiol~ of acetyl(-lmliriesteras~- in giie tinter segmettts 0f. rods And cones (Shen, Greenfield a n d Boetl, 1956). R h o d o p s i n al~peArs ~ few d a y s ' l a t e r , a t a b o u t t h e t i m e the first electroi'et.inog.r~m~ can be recorded ( W i t k o v s k y , t963). T h e presence of t h e golden y e l l o w a n d ghe yellowish green carotenoids in r e t i n a s
4..I 0
i F l g l { l ~ A . N i . ' . l e l (;. C O ( . ~ P I ¢ l l .
ANt.)
DAVID
.[~. M b i g I , l l ¢
~l(~voi~l o[' a~tv vi.sun.I elemcnl-s or microscopieMly disucrnil)le drol,b.:.ts imtivat.es that: d.urh~g.t:helr inltial a~pl~eat'a~tce Lhese p.i~nmnt.s n u ~ t be. linetv disperat.,d wit.hi,t the whole t'e!:ht?t...I:]v st:::,.l.,e :~7 (~q.u'()xit]mt.(~ty 11 dtLVs), Imwever, when t:lneir cou(..-e.t.erai.i(nLS mtd.de.~dy begi.~t t.o ris~, s h a r p l y ( Figs 2 ~ut(l 3), inner uegmeltt.s a l r e a d y arc evi.(ten.t, Mt.hough di~crel,e oil ~irople.t vi~t:.tott.s atl'(y still ltot deLet'.t.,M)h', wiLh lighL, or tfln:ts(: IlIit.'.I'OSC.O~.~V. . . . .()ll,', d~l.~," . l~U~0f t/ 1.a ":~ dayS; . a~|.~In'oxi]rtato|v . . st.age ,. ":~8) ". .Le.ptat ( 1.91:;} observ,,l . .. a d.vop or t:wo of Fat. a~t t.ht.' ba.se or ~tt t,he courter of t;he eliipsoidtd mitoelloltd.l:i~t (Lhe elosu. ~ssociat, iott 'th0.l, tyxisL~ betweett oil droph't.,s a.n.d mit.o(:hO[Ldrh~, has l.,rontpted. T..x/g,l. v: [
.:-lye ~ " (i,~- &ut,~. or ~,,o(l~hotoqicat staqe~'), t f i,,.-itiu.! detection a~zd .s'eque,,(,e ~j" a~/,peam .m . e'. . of colo.red o~'! dro)~tet, i n tt~z. .reti.,~a o f the ch.ic.a: embr:~to as revealed b / m i e m s e o I , & : ea'a.ntinatD.,~ a q f J?'cM,. ,retinas Oil d r o p l e t , e o h , ' s [u~*c'3t ig~tt., w~-
.ltteubM.iotl L i m e (days)
Rt'd S e h u l t . z 0 ( ISIRJJ [.{~hu ( 1 9 t 6 ) t D;MIv a n d . J u d d (1933) VVehl a n d Z u , s m a n (11);~7. ti}38) ( ~ o t d u m b r e [ t9&3) Wuki~kt~ ( ! 959) 3.'afar ( t g { i l )
IS 16 = 15 It)' 15 16 1S
Golden ye,!l,w
Yelto~-ish green
1,9 17.- [8 t6 a t7 17 ~ ...... ~0 20 . . . . . . . . . . lS ! .t/~ 17 ~ 14
3 h , r p h u h >gi ca ~ st~gt;s/' t/m, ubt~L.R,n t i m e
(day~F 4a] 17
P r e h e a t . itt ve..st-igators
40/t4
40/14
Al~.o d i ~ , i r . t g u i s h e £ / a t\mrt.h o i l d.vt, plet~ eol<,r: urautge. Dark re(hlislt brown. a ~(e Uo~vi..~i.:b t o w n . a 5.',z..ItowisI, o r a n g e . Pirtk~ a ( i r e e n c,r p a l e b l u e . EeLhm~te.(I f r o m s~egu.s o f t r l a m b u r g e . : ; ~l& . l l a m i i t o : ~ ( l . 9 5 | ) .
•~ , m e :¢esearehers, e.g, Akiyt~ (19113), Ped[6r a n d T~t~le3.- (t963) to a s s u m e a mitoeho~r~:xiM..0rigitt for the oll droplets). Using ~he e[pet-~'o~t,t~ieroseope: o lt drop.lets also have, bee,i~ described at ~ttis time as LrreguIarly Shaped strut;tares of h i g h electron de.nsitv within the de.velop~ugit~er u e g m ~ L , wLtett t.he ~.~}i't.ocho,~dri~ begi,_~ to i_,mrease m n u m b e r (Ueno, 19@), I96'Y). ]Trregttlar o,~nnophfim bodies and vesldpS filled with elec, t ron ~te,~se, . . matea'lal . . also . hay e b~o~, o t 6 s m ' ~ l m':de ~x?lopm,g vlstud eo[ls of yotmger e m b r y o s (9 days;, a p p r o x i m a t e l y st.age 3b~ (Meller, 1964), bug h a v e not been-ide.tzgified gs oil droplet, s. Sil~ce t h e firut two oil drop.!et, pigment~ tb~'appear e m b r y o n i c M l y ~re..t~hi}..golden yellow (.mtknown h y d r o c a r b o n ) a m t yellowish gree,a (g~fllox~nghin) earotenoid.s t.tu~t ~-
'
-
,;r
...
ON'L'I)([;I,'~N'Y t~l,' t k E T I N A L
O I L L)I'OI 1,1.,1~5
,l.iI
ft,,side in the e.hi(,t' a~ul accessory (:otnpo~u;ttts, ceapecLivcly, of the double ,.'o~t,~ (Meyer ~md (:Doper, 1966), it..~eems reasunable to accept tim findings of ()hhashi (L924) a u d .Yat~ (1961) tha-t the double cone is tire first: cone to differe~ttia.tc i~t t.b.e ckick retim~, and t.,, add tJ.mt I:tte chief coml.~onczlt nLa.y develop before the accessory. Our spe.ctro.l,hot.omol, ric ~lata., however, tit, n.ot. sui)port the. tissue cuitttrc st.udies of Y a t a (196l) ~Lll,d Govardovski aml K h a r k e e v i c h (1965) both of which rely on vimtaI observi~tion.~; t,t" oil droph.'t color~iAons in situ. Y a t a (196l) cult.m'cd rcthm.~ from t0-, I3- a.m-I 16-day-okt c.hick embryos and reported t h a t gv(.e~t droplets arc ~hc first ~o apl~ear (in rct.imt~ f r o m 13-th~y-otd e.ntL~ryos ",.fief 4 d~ys in vitro). Since these drc~ple.ts occ~tpy t:o.l~a.~swith barrel shape(i inuer scgnu,.ms, which are c.tuu'acte:'ist, ic of ac.ce.-~,~ory cones, his fi.~M].ugs suggest t h a t the. acee, ssory cone is the iirsI; t,o differentiate. l-[e ol)served yellowisl~ tirallgt; (gohte~l yctlow) and red droplet, s sontt~wll~{) b~ter (ill retittas From 16-day-old embryos ctfltm'ed 2 ~nd 14days, respectively, i~ vitro). According to Gow~rdovsM and K h ~ r k e e v i ( ' h (1965), o~t the othe, r hand, all three colored tirol)lets arise sinuLltatw.ottsly i~t retim~l cttltm'es fi'om chick embryos older t h a a l0 days, As ~:mphasizcd earlier, the mic~'o.,copic delinc~tio~.t of the partitmlur colors of the pig.me~l;s, e.g. goldel~ yellow altd yellowish gre.e~t, i,s severely limited I)y ~he 0p~ical properties of the at).alyzing system a n d visual percepI~ive.~mss of t,h.eobserve.t. [t?hi~ a.pproaeh umloul)tedly .is re,st)oils|hie for the mmierous m m x p l a i n e d discrepat~eies t.iu~i~ a p p e a r i~t bite [iteri~t.m'e with respect, not, only to tim init, ia.i de,toetion, of i)ho oil droplet pigm(ints, but alst~ to ~heir sub.se(ltte]tt seque~xee of ,'~ppearance (T~ble [). Sttch ttiic:ro,geoI)ic. (tat, i~ ('o~lse,qLtelltly ~l'e e.omd,:lered uareliable mdess s~:tpported co~momita)igly by m6rc precise physico-chemica[ deecrmimttions. According to the nmjorit:y of develt~pmentat stttdies (which, u.rtforttttmtely, are predomi.m~lt.ly ndcroatop|e) the firs~ droplets to a p p e a r in the chick ret,im), ~rc colorless. They a r e obsetwe4 i\'om, o.ltt~, to seve.ra[ (t~ys before t.hc first colol'ed dropIcgs (Table I I I , Meyer et al., 1 9 6 5 ) ~m(t ~.re believed to give rise t.o ghem. Ottr speci,vophotomet.ric fixId.ittgs, Ixoweve.r, reveal t,h~t- tim golden yellow ~ml yeliowis!t gree~t pigments ~re present several d a y s before, c.o|orl(.ss d.roph~.ts ;trc reoorde;d iJ.t fresh prcparal;ions (stage 3~ ), imlio~,ti~g t,hag ~he l~rimordi'al d roplet,s nm, y not. be colo.rlass. Dmtead, t h e y m-~y eoutah~ t[~e~: pigme~tts in such low co~tcent.rations t h a t their colors c~mnov be discriati[tated, microscopically. 1RE l~ lg I~]~N C E S .~kiy~., S. (1963). Acts Soc. Ophthain~fl. Jal)o,~. 67, 1 lOS. Coulom[¢r% A. g. (1955). An,. J. A~xt¢. 96, 153. .teujit4~, S. ~md ~[orii, M. (1963). Arch. HistoL (Oka?tama) 23, 359. Goodwln, 9.'-. ~,V. (1954). Carotenoids: Their Comparati'b~ Bi¢~he~'~H.~t13t. ChomicM t?t~blisitialg Co., Ne.w York. Covardov sk i, V. I. ~rltt Kh~rkeovioh, T. A.~ 1965). A rkh..4nat. (~"'~tol. EmbxioL 49, 50, Govardovski, V. I. ~nd Kharke¢;vicb, T_ A. (1966). J, EVOl. Bioche~a. Physiol, (/-2"~.%'/a~D2~ 37. [IM~n, liL (19!6). Z. W/4,'s: Zool. Abt. A. 116, 1. tIan~bnrger, V- and [-[~r_nilgon, I~. L. (195t). J. zlIorptml. 88, 49. K.arrer, t ~. ~n4 Jucker, E. (1950). Carotenoida. Elsevier l=hlblishitlg Co., N e w k'ork. Ko~lkuhi, T. (1065). Dobut.,ugak~ Zas~hi 74, 119. Kuhn, 1~., Stone, J. anti SDrerLso~, xW. A. (1939}..Bet. Deut. Ohem,. (.~nel;lsch. 72~ 1688. Kiihn~, VV. a~td Ayres, W. C. (1878). J. Physiol. 1, 109. ]L~plat, G. (19.13). Anat. A ~ . 45, 2[5. Loevellioh, H. ~K., you StuchLitz, {~, and SViggcr, t[. (1944{). Natur~;L~s, 31, 5B8. hieller, K, ( I 9 6 i ) . Z. ZeLtfor,~k. 64, 788. ,Molle~5 K. arid. tlreipoid, W. (t9~5), Z, Ze!~forscg. 66, 673.
442
T.T~ItRANC, E ,'.], C;OOPER AND DAVID B. MI~YE:I~
M(,yer, D. ]:L and Ccmper, T. (~. (19(16). A,m J. Anal. 1.18, 723. 3[(~3~er, D. B., Ct)~)por, '['. G. ~lrd Gernez, C. ([965). In TI~e Structure oflhe I'Ve., pp. 521-33. (~.'. by R¢)hea, ,I. W.) Schat, t~mor, Stuttg~rb. Ohhashi, Y. (1924). Jcfp. Med. World, 4, 257. O'Rahilty, l{.. (1966). Cor~.Irib. Em.br!/ol. (C;mxcgic ]ast;.) 38, 1. O'llahilty, R.. and MT~;ycr,D. B. (1959)..,lela .d.me:. 36, 20. P~.~dlcr, C, rm4 Tatmtcy, K, (1963). Exptl Eye, ]¢es. 2, 39. Sohulbzc, ~I. (1866). Arch. mi~ro,~kop. A'vv~g.2, 175. Stroghcr, G, :K. a.nd Wolken, J. J. (1960). Exptl Cell Res. 21, 504. Slmtt, S. C., Grccafletd, P. ~uxd Boelt, E. d. (1956). d. Comp, Ne.u~vI. 106, 4J3. yon h.'~udnil.z, G., Neum~nn, H. ,T. a.nd Loe~,er~ioh, H. K. (1943). Pfl'Sgcrs .,4rct~. (~e.~:. ' " l>hysioL 246, 652. Ueno, ]~. (19It0). A eta. S ~ . Ophthalmo~. Ja2~n. 64, 260. Ueno, K. (1961). ,]apart. J, Ophtha/,md. 5, 38. Wakisaka, S. (1959), ,4cUz NOc. Ophth~lmol. Japon. 63, 1137. WMd, G. (1948). J. GeJ~,.PhyMol. 31,377. ~lrald, (~. and Z~lssm~n, H. ([937). Nature ].40, 197. Wald, G, and Znsam~, H.((19~8). J, BR>I. Chem. I22, ,i49. Walls, G. L. ~nd J ndd, H. D. {I933), Brit. J. ())~hthalmol. 17, 641; 705. ~Vi~kovsky, P. (1953). Vi,sioa Be,~. 3~ 341. Yata, M, ([96l). 1Vakuyeu~a Me~. Soc. J° 12~ 819.
O n t o g e n y o f Ret inal Oil D r o p l e t s in the Chick E m b r y o *
De)~(zrt..me~.t. of Che.mGtry, lVa?t~te State U.nicersity, to~d Del)artme~.t. of A.na.to~;¢y, I,t:¢~yne State University School, of Medichu', ])etroit, Michiyan., U.S.A. (Received 20 December 1967', Boston) T h e ¢~t'igiu o f tim e,:~lot'ed oil d r o p l e t s in t h e c h i c k (:ones wr~s invest;its.ted b y m~bjecbing r e t i n a s f r o m st.aged e m b r y o s i~> c o n ' e t a t i v c b i o c h e m i c a l (visible s p e c t r o p h o t - o m e t r i c a s s a y s o f chrom~togreor~hieMly ~c.part~tcd extra.cL,~) ~,nd m i c r o s c o p i c o . m ~ ' s e s . The. earota'.rmid pigment,~ rr.'spons[ble for "t,ho coiorat.ion o f %,he oil d r o p l e t u a r e d e t e c t e d s p e e t . r o p h o t . o m e t r i c M l y b e f o r e %hey e;uz be vistu¢lizod micro.~eopieMly ill ~he f o r m o f c o l o r e d d r o p l e t s . :l?he spcx:trot)hotomet,ric (i~t,t~ wev(:M tt~at e a c h p i g m e n t a r i s e s iu.depot~de~ ~tly in t.ho fi)llowing s e q u e n c e : g o l d e n y e l l o w , y e l l o w i s h g r e e n , a n d red. T i m grdrlen y e l l o w h y d r o c a r b o n is first, de.tt;ct:ed at, s t a g e 27 a t w ] f i e h ~ime She retin-~ con.Mst.~ o1" a. l,hiek. tu~stra.t, ified, n e u r o e p i t , heiitd b~yer wibh ~rt a c b i v e i)rolif(.-ra.tAve z o n e , but, laeRs vimu~.] eell~. (lMIoxr~nt-hin, t, im yellowiM~ g r e e n p i t m e n t , , 'd.riscs t~t, a~age 1¢4. Ti~e regi:ta is st,ilI d e v o i d o[" visua,[ elctnon~s at this stooge, but, is orgt~ttized i n t o sever;.fl n u e l e a r l a y e r s , T h e roJacentrati(m-s o f %he g o l d e n y e t l o w a n d y e l l o w i s h g r e e n efn-of..eJloif[s begirt t o i n c r e a s e r a p i d l y ~t st,~ge ;17 w h e n i n n e r s e g m e n t s a r e differe~ltiating, bu~ oil d r o p l e t , wtc.~ml~,s ha.vt~ rmt, ye~, ~ppe:~red w i t h i n ~hem. (]olorless droplet~s first, e v i d e n t i~i fresh :prep~r~,g~ons u f l, his s t a g e Ju~5" nol; be, oolorless ~% MI, b u t mu, y t e n t h , i n bhe y o l l o w p i g m e n t s i~t u~teh Io,~v eonct;nt, ra{,iox~s ~hat. 6heir eO|Ot~d or%fill.or tit,• diaerimi~i~tx~d m i e r o z e o p i e a l l y . T h e rcti /~igment, as~z~xant, hin, is firs~ e . v i d e u t a~ s t a g e 41 a n d i m m e d i a t e l y e x h i b i t s a st;e:,.dy i n c r e a s e ht e(meent.z'a~ion, '.L'lm regina posses.-ms ,~dtzll; stz.at~ificataioxi a~ t h i s t i m e ; M1 t e n taye, rs are p r e s e n t am.l o i l d r o p l e t , v~z'uoles a.re o b s e r v e d t b r t~he firs~ t i m e . O n t h o baMs o f t,h.e s p e e t r o p b o g o m e t , r i c f i n d i n g s it, .is eont, e n d e d ~:h~ t h e d o u b l e cones ~re
t h e fi~t, c o n e s t o d i f f e r e n t i a t e in tbr.~ c h i c k ret, in(~ ~,nd ~ls~t. t h e c h i e f e o u i p o n o n ~ ( b e a r e r o f t.l~e Igoldon y e l l o w d r o p l e t ) ~rises b e f o r e tJ'~e n.ccessory e l e m e n t (co,riSer o f t.he yollowi.sh green droplet).
1. Introduction The re.rhea of t,h c chickell (Gallus domestieus) eontaims single arm double cones t h a t are characterized b y the presence of a single b r i g h t l y colored oil droplet wit&in t h e distal e x t r e m i t y of each of their i~mer segmettts. Three colored droplets (red,goldert yeUow •rod yellowish green) are discernible, and each is i-est-ricted to a specific cm~e type. R e d droplets ~re housed i~t single coates, whereas t h e golden yellow and yellowish ~.een droplets are confined to t h e ct,ief a n d aecessot T c o m p o n e n t s , respectively, of the dolible cones (Meyer and Cooper, 1966). Chemically, eack colored oil droplel; c o n t a i n s o~m or more ca.rote.noid p i g m e n t s dissQ~Ve.d in lipid m~terial of m i k l m w n composition. :In tel;|hal e x t r a c t s from the chicken t:hree d i s t i n c t l y difl~reItt ea~rotenoid pigme.~Ita are present t h a t reproduce in a p p r o p r i a t e solvezl~s t h e m~t~md colors of ~he oil droplets i~ r i v e . Kfih~ie a n d As~es Tliis inve.~%ig~ion ,,v;~s auppor~ed in p a r t by ]Lifts r(..scar0h gq~ants ( N B 06580 a n d AM[ 0;3334} from the .~at.iorxM rnst.itnt(~s o f I][ealth. stud in p~rt, by r~ J~igh~ for Sig|xt Grant-dmAkl f r o m t h e Grat.ionM Council to Combat, Blilx(hlo.as, Inc., New Y o r k , N.~T. A pre, l i m i n a r y report, o f t.his s t u d y w:~s present, ed at t h e am~nal m e e t i n g of ~he Asaoehttim~ for .Researo, h in OphCAudmology hold in Cle~rrwater, F l o r i d a , 1-3 May 1967. "~ 1Preheat addrens: :Oepar~men~ o f Biok>gicM Seiertces, P u r d u e University, Lo.fas'et,t.e, ~tcl. 47907, U:S.A. 434
ONTOGI~NY
OF
II, l g ¶ I ' I N A L
OIL
DROPLETS
435
(1878) were the first invesi;igators to isOl~tc these pigments, a n d 1.~,5~1d and Zussman (1937, 1938i were t]te first, to crystallize t h e m ~md establish t h e i r carotenoid nature. Weld a.nd Zussman (1938) identiflcd the red pigment as astacene, the golden ~s ~ xal~th.ophyll (htt.ein, m i x e d with its isomer zeaxaltthin), and the yellowish greett pign rant ~s ~m unide~tified hydrocarbol~ with properties Mmost ideuticM with ~arehmne. Cr)~t~ds of e.'~ch pigme:~t were dissolved in suitable solvents, ~.nd ideutified by their .visible ~bsorption spect, ra. Later 'Weld (t948) detected ~ fourth oil dxoplet carot~tmid, which he nm.ne~t gulloxantttin. I{e xw~sramble :o crystMlize this alcoholic pigment b u t recorded visible absorption peM:s a t lower wave2engths than the or,her three carotenoids. W e l d did not associate g a l l o x a n t h i ~ with a n y of the three colored oil droplets; the only reference to the color of this pigment w~,s t h a t it separated as a yellow band[ on a cola:tin of cMcittm carbom~te using benzene as ~ solvent.. Galloxanthi:t was a d s o r b e d j u s t below a s t a x ' m t h i n and ~bove htteln al:d could not be easily separated from either pignmltt. Two subsequent studies, however, have demonst,rated titbit gMloxmtthiL: is the m M n carotenoid co:lstitttel~t of the yellowish green product.: Strot}mr ~md Wolkmt (1960) recognized its spectral characteristics in. i n t a c t yellowish g:'een droplets by me,ms of a microspecgrop]:otomet:~.r :rod 3leyer, Cooper and Gernez (1965) were able t.o separate this pigmcn.t b y mea~:s of t h i n layer ehromatogral.>lty a.nd to record its ahsorptio:~ spectr~ (visible and infrared) irt various solvc,~'..-~. The visible absorption m a x i m ~ closely resembled those reported b y Wakt (1948) [or galloxanthin, a n d the i~ffrared dat.~.~ s u b s t a n t i a t e d its x a n t h o p h y | l -nature. T h u s , . t.he.re appears to be Iitt.le d o u b t t.hut i:~ the chicken retina.galloxa~ttltht represents the yellowish gree:t p i g m e n t theft occupies the accesser Y cone oil droplet. I~ is also now welt established t h a t the coloration of the red oil droplets (single co~ms) i s due to the alcoholic carotenoid, a s t a x a n t h i n , which dm'ing e~.rac~.io~ becomes ~ t t 0 x i d i z e d to its keto form, astaeene (Kuhn, Stene and Strenson, 1939; yon St..l,dnlt, z, N e m n a n n and Loevenich, 194B; WMd, 19t8; Strother and Wolken, 1960; Meyer et el., 1965). The Dosition and ident.ific~tion of the or.her carotenoid p i g m e n ~ detected in re-tinal extracts, however, recluire fm-ther s t u d i e s . . L u t e i n (xa~tthophyll) tbr example, has beer~ associated with the golden yellow.droplet by n ~ : w investigat¢,~ (Weld a n d Z t t s s m a t : , 1937, 1938; Loeve~fich; yon Studnitz a n d Wigger, I9~3; yon St:tdnitz et at., 1948; Weld, 1948; Goodwin, 1954; Strother a n d ~ -:ken, 1960), bug reeen.t Stltdie8. h~ve shown t h a t when t h i s pigm:ent is separated b y m e a n s of ttfin layer e h r o m ~ t o g r a p h y and anaIyzed by visible a.nd infrared stmctroseopy }t is, in reall W, :, h y d r o c a r b o n with visible spectra similar to the nnidentitied hyda'0c:~rbon detected by :V~ld a n d guss~rtal-t (1937, 1938). The d i s t r i b u t i o n of the yello~dsl~ ~-een carotenoid, lacertofulvin, described b y Loevenich et el. (19g3) a n d vo~: Studnit, z et el. (19,t3) is likewise confusing and requires further documentatio~i. We have previotmly reviewed the cont~roversies concerning the n a t u r e of the various ca:'otenoids i:t r e t i n a l oil dropleks a n d their ~bsorption s p e c t r a i a v~riot~s solvents (Meyer et M., 1965). I n a n y case, most- investigators hax:e been: able to separat~ o n l y ~hree earotenoids from retinal e_x~.raehs by chromatogra.phic methods. I n the pre.aent; study t-hese p i g m e n t s wifl be designated :,.s astaxa:tthin (red}, g,~lloxanthin (yetlo~:ish green) artd rm m:ider~tified .hydroea.rbm: (golden yellow). T h e i r ,~bsorption s p e c t r a (~dsibte) in benzene are illustrated in Fig. 1. [.F.or comprehensive s u r v e ~ of carotenoid chemistry co~:stfl~ K a r r e r and Zucker (1950) a n d Goodwin (1954).] I~x the chicken retina the colored oil droplet~ arise during embryo~de life; nil a d u l t colors are discernible h~ t h e newly h,~t.ched chick. Observa;tious on t,he e m b r y o n i c origh: of these hxclusions and their carotenoid content have b e e n m a d e ahno,st
4,26
I.~,IgR.AN(IE
~.
COOPER
ANt)
I)AVll)
:B. ) - [ I ~ Y E I ~
eaHrely from the microscopic s t u d y of fresh retin,.s. Since t,h e rellabitity of such (.)bsevvt~)ions d,:pe~tds ~huoat ent, ire ty upon the color discriminM.ory M)ili~ies of ~he observers ~md the quallty eft the optle~l systems employed, their results are quite varial)le, and inconsistent.. The application of more sensit, ive and reliable physieochemical i:~roeedures (e,g. chromatogr;~.phi~ separn.l.io~:t a n d spect.ropho~ometric idmttifieat, ion) fbr the determina.tio~ts of t.he e~n'ly e m b r y o n i c o~'igi.n of the oil d r o p | e t l~ig.mertt.s h a v e seen timit.e.d ~:tse. Coulombre (1.955) employed t h e m for the deteegion of asta.xanthin, bn.t gho other oil droplet, e~rotenoids }rove hog been studied owing to .t,he di{ficatlties incurred, i.tx efl%cting their separation. Advances in se.paration a n d spectra[ mxfltyses, however, ha.re solved ~his problem (Meyer et ai., 1965) a n d now warrant, thei:r appliea.t.ion to t:hese developmentM problems, Irl the present, st, u d y ~he inlgiM ~ppet~ranee ~nd. subsequen~ increase .in eot:teent.ra%iotx of each oil droplet pigqnent h0.s beett ascertained by spee, trophotonxet, ric determ i m ~ i o n s of c h r o m a t o g r a p h i c a l l y separut, ed rethu~l ext,]~&ets ag czech mor phologieM stage from st,a,ge 25 to hr~tehing. :In turn, ~he developmental changes involved in ghe o r i # n of these pigments a n d the formation of oil dropte~ v~.cuoles t h a ; boase t h e m h a v e been studied microscopically in fi'esh ~nd fixed retinas from simih~r stages. 2.
Materials
and
Methods
Twtmty-tbur chick emb~.'yos at, e;~ch morphological st,~ge bebween 25 (approximately t½ d~ys)a.nd 45 (Hambucger a~~d Hamilbon, 1951), iactuding newl.v-Im~hed chicks were ~.unployed. 10"orea,ch stage, 48 whole eyes or ~-etinas-- -depending upon t,lm ease. with which
1
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-
0,52
-
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70
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80
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90
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4.50 500 Woveteagth
550 ( m14 )
'~-
600
~h~.ot. Visible a b s o r p t i o n ~}wctr~L o f the, t h r e e oil d:roplet, cl~robex~oiCLssepara/.ed f r o m chicken re~,im~ by. thin)l'~yer Ohromate~gra.phv: a n d d i s s o l v e d in benzene for spectra.! an~lys~.~ (Meyer, C("~oper m~d ~ m ' n e z , 1985). V e r t i c a l liues h a v e been ~tra~,m at,. 40,f~ ~nd 4,,t9 m/~, t h e most. Sensitive s p e c t r a l r e g i o n s fi~r the differenti~tion of t:he yellowish g r e e n a n d g o h l e n 5-el low earot-enoid :pigmengs, v e s p e o t i v e l y ,
t.he retA'nas could be dissect,ed fi'ee---were removed al,-d pooled for chemical studies, The a~:hole eyes or ret, inas were homogenized in equal mnounbs of beJ~z~me ;.rod 70~/o methanol for 4-0 sc.c and-cent.t:ifuged for 10 rain ,'tt 27,000x g. The. eat'otenoid-eontainiagex'traot (cpiphm~ic aft-~r centr[fugat.ion) was then concent_rated under dry, highly purified nitrogen a~d, depending upon the stage., subjected to the following m a n i p u | a t i o ~ : (1) For the
ON't'OGlgNY OF I ~ E T I N A L O I L DI~.OPLETS
437
early :~tgge,s" (25--40) i~x which only t,wo c;xr6tetmid pi{,nne~t,s (golden yclI(.,w a n d yellowish Vreen) could be detected, t.hc earotenoicl concm~trate was monitored stumt, rophotometrically (Zeiss .PMQ-I[) a t 449 a n d 404 m/z, :respect, ively. These ~wavele.ngtlm (]Pig. I) were ae|eetcd becamse t.hey rl.'present the most: se..usitive spect, rat regions for ~he demonstratio~ of the ve'ry .-'mall alttotutts of carote~mhl pigmm~t i,~ the younger embryos (Fi~ 2 ~nd 3). Sueh
/
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34
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.
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Stage ]fl(L 2. G r a p h s h o w i n g t h e spectrophot~meta'ic ( l e t e t ; b i o n o f t h e g o l d e n yellmv oil droplet 0e, rot.en~itI { u n i d ~ m t i f i e d h y d r o c a r b o r } ) d u r i n g rui, i ~ a l d e v e l o p m e n t i,x t h e (dfiet¢, ' l ' h o o p t , i c a l d c a s i t , it~s r~rel d e t ~ r ~iline~l i n b e n z e n e a.t 44,q rote.
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34
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40
42
F i G . 3- ( A r a p h s h o w i n g t h e s p o c t r o p h o t o m e ~ r i c d(~t.cetion 0i" t&c y d l o w i s h g r e e n o i l drolalet, c a r o t . e t t o i d , g a l l o x a n t h i n , d ~ r l n g r e t l m t l d o v ( ~ l o p m e ~ g i;~ t h e c h i c k . T h v o p t i c a l do~mit-ies w e r e d o * , c r m i , ~ e d i n be~,-zeno ~tt. 4 0 4 m ~ .
small coneent.rations of pigm_~m~t, were nol; amenable .to ch~:omatographic ana.l~sis,.. (2) Su~)sequm~t~ to stage 41 the presence of Lhe ~e~Jt pigments, astaxa.at]fin , comp]eteily ~as:kecl the presence of tlm other two pigmeaat,s ~ d hence ~mcee~sitata~cl i t,s ~ep~ratim, from. ~1i9" lattc:t, two pigm~enlks on K o d a k thin h~yer plate, s deve.!oped Witch chloroform. The s e p a r ~ e d carotenoid:~ were t&en elut, ed fl'om the plates wivh b~nzeae a n d t~e a,bsorptlon of.a~st;~,~zaathin was m~nsured at, 525 m y (Fig. l ). In all (.a.~es a dpectrum e£ t.he:ent.ire benze~,le~extrac~ was t~kmt. :For microscopic stu.dy several retinas.of each utatge wer(; fixed ia Eotme,r's fluicl a n d embedded in pm~flhx ns described.previously (Meyer and. Cooper, 1966). Se~e.ra~-~etinhs of each ~ta.ge .were also e x a m i n e d by m e a n s of b:igh~ field a n d txl/ase c:ontra,~ micr0.a,¢opy
43S
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] I , t . ~I ~ ~. A N C E
C
COOPER
AND
DAVI])
15. I{]dY.Ii:R
~,a fresh, unstained (fiat ,tJ|d squashc(t)prepar:.ttio~s h t order to establish visu'~l re.cognit.ion of the oil ¢[rol,let ])igmelltS dttriug t.ltes~.~ critic."d periods. I?m'ticular care was ~aken to vxantine all .areas oI" the. embryoMe ret.itm, ~:speeially t,he posterior fundie region, which cliff's!rent:lares earlier than t.he perildter.'~t cquat-oriat rt;giott (S~dmlt.ze, I8611; Couh,mbre, 1(.)55; Fujita and lh,rii, 1963) 3. R e s u l t s
T}te g(,lde~l ye]l.ow hydrocarbon is the first oil droplet c~u'otexmid t.o be recogtfized spccl;rol)l,otometrically. Ib is initi;~lIy recorded at st.age 27 and does not. begin to iJicreaso, in e o n c e n t r a t i o a until aft.er si:age 30 (Fig. 2). I t thett begins a slow r~te of increase, n.ntil st'~ge 37, when its conceTtt:.rtttion rises sharply. Microscopic sections of reeimts at the bcgimxin.g of carotenoid detectiou (stag4s 27--30) discIose a n immat, ure orgaltization. The inverted ncuroelfithclial h~ycr ('oasist,s of ~m active prolifer¢~t.ive ztme, as indicated by the numerous ntii:otic figures [.Plate l(a)], a n d :~ r e l a t i v e l y thick, unstratitied nuclear l~yer, but ShO~VS nO evidence of immr segmelit format, io~t [Plate (b)]. O
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43
45
47
Stage
lqo. 4. G r a p h sho~ving the 6pectropho'~ometric d e t e c t i o n of the r e d oil d r o p l e t caro~.crtoid, ast~cene ( a u t o x i d i z e d a s t a x a n t b i n ) , d u r i a g retin~d d e v e l o p m e n t in t h e chick, T h e opgicM deasities were det.erm i n e d in benzene ~t% 525 mt.¢.
Galloxa.at, hia, the.-yellowish green pigment, is not detected spcctrol)hotometrically un$il st.age 34. (Fig. 3}, ag which ~ime the retim~ has differentia.%ed into several nuclear strgta, but still lacks a. vimm.] ceil lgyer [l)b~.te 2(a)]. Like the golden yellow hydrocarbot~, however, the coneelttratimt of gallo~%nt.hitx r,~pidly increases a t Stage 37 (Fig. 3). Vimtal cell differentiatioll has commenced b y t.his s t a g e. Smelt protoplasmic prot.rtmions are evide.t~t projecting from the cells borderb,g the e x t e r n a l limith~g m e m b r a n e [P.b~te 2(b) a n d (c)]. These cellular exteltsions represent %he prhlfitive imler segment, s of the photoreceptors, which are gn'owiug t o w a r d t h e p i g m e n t epithelium. A s t a x a n t h i t t is initially detected spec)~rophotometrically at, stage 41 (Fig. 4). Unlike the other t w o c~rot.enoids, the concentration of tiffs p i g a m n t m ~ i a t a i n s a s t e a d y r a t e of increase a l m o s t ~ u n e d i a t e t y a f t e r its first ~ppeara.nce (Fig. 4). The
S C
P ELM -M
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X-'r.~.T~ I. ~ h o t . o m i c r o g r a p h s o f ckiok e ~ i n ~ a n d .~itja~,rtt ~cler~ (S) trTtd_ ohoroi4 (C) ~t-st/tge 29 (l~atrtb u r g e r antl ]~I.,~n~ilf~m, 1951). (5) T h e stragitic.ation of t-he ro~t.lna, r e s e m b l e s elx~b o b s e r v e d in t h e (',hick r e t i r m at. st.ago 25 (.Fig. 36, O ' l ~ h i t l y ~ud 5[cycr, 1959) anti t i m h u m a a . r e t i n ~ of I]~orizor~ x v (Fig. 27,-O'B.:~hilly, [966). T h e ,xcuro. epitkeliut~a h a s zu)t ycL difi'r, r e u t i a t e d ixtto ib,ttern~l a n d e x t e r n a l aeuroblz~sb'~c .i~3-'ez's.. Pigmetat g r a n u l e s a r e present, in t h e p i g m e n ~ t:pithethtm. Tlxt~kfollo~vi~lg retina.1 lo.yer:~ caJl t)e. easily .distit~gnished*. (I~) c.xtern~d (pig,nenb) layer; i~xvet'te¢[ }~yor (()f o]~tic (;u]?) c o n s i s t i n g (*f tire (EL$[), cxt.e~'nal [iJniting m e m b r a n e ; (M) n. m i t o t i c z o n e ( p r o l i f e r a t i v e htyer); (I) i n t ' c r m i t o t i o 1)b,~so (prh~litivc zc-u.~); &n(1 (-~.Z),. n. ~xuclcar-frcc m ~ r g i u a l zt)ne. (X 900) (b) ~[[igher n~agniticabiolx .~koxx-ing srgifit.'.iaI f,<:l~aration o f p i g m e n t atui h ~ v e r t e d la.ycrs wLticlx ccvett.~ tlxat, visual elcmetxts h ~ v e n o t d e v e l o p e d us y e t . 3lit, erie figurc.~ (arrou-) -tre evide~t~ in t h e t)rolil~rativo zon~,
( x 2~00) [ T o face page 438
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~I?~:.A'J's~ 9. 0~) PlmL~m~h-~rogmph ,~f ehi(;k ret hl,a. ~.t. ,~t.age 34. T i m neur, Jepit-helium ent.i~Lf~(I iDto ~ v ( ~ m ] dis~ret(, ]o.y(:rs. V i s m d (:('.li~ ~tre t~ot, prc.~ent. (.-; :;70}
ha:~ 1,~:e,>am ~]iffer-
(b) Phtm~ e+~nt,r;J,st pho£(~mh:-mgr,plx ~,1' Sh.(: e x t : e r w d porl.ion o f I:~,~: reLim~ a.~ s t a g e ,':I7. 1)(:vel,~ping ~4~ztt~G ceils (arrow) are~ ~vJch-'..tlt us proLoLp!asmie (~.~;t,L'nSiOILXfrc~Jlt thA' exit.ernst[ ~¢:ur~Jt~l~.~l:ie lr~yer (nu~:h'.i o f to[Is alId eone.~). :i:tm v i s w d eell.~ pt'oier't: rms'ard t h e pi.gm(-r~L e|~ith¢~lium Whlel~ Ires he~n ~u'|,iliei,~,tly diat)h~.oe([. ( z 7!0) (:~.'-) .|li;ghar p~)wer, p h a s e (~oni.r~sI, l~hot.~mierogreph a h o w l n g |,he ,tevt, loping vism~.I eelIs (urr0,w) ~tg ~g~,ge g7; oil ~troplef, w~euole..~ a n d (mL/,r segtn.et~t~, h ~ v e lint ul)ipe.ared ~s yet:. ( x lkt00}
(~) ~i.'JL.e pi~m~,ll I; ,,]~it.i~,lium ( 7: I,N(}(~)
I~rt,~ been' (L~{f~.'.i~(~d. I )~.:w,h~.l)i,l ~ ~.i~,,~ ~-~1~.~ ~.r~~ ~.,vid~t,.
( .~ ~I.~1))
(]NTI)t:;I?Nh'
Oil
|fIqT.INAI.
(t|L
I)IL()I'LI",T-'g
4:19
rct.in:~ i~t stage ,It lPhm.'. 3(all res,:mbh:,s c.loscly thin; obsc:rw~d i , t.hc adul.t; ~dt ten ~tcll.nitive layfn.s i~re l~ow ('vide.n.t. tn ]~(>lmo,:-tixed l~h~,.m- micro.~:.oliic prel.~m~ti'm~% v;~(:~.tolc,,i C4rlt b(; St~el't el,.,arly for t:]l(; first, t i m e oc(:'.ttpyiilg t.}t~.: distal ,,xtr,:>mil;y of tim, iniu'r s~,gm(.,n.ts ......tim definitive site ~,t' tit,., oil (h'()pl(,ts [Plate 3(t))J. It.. is not. possible a t 1-ht.,,~e ]'(,lt~tiv(.,.ly y o t m g stages to tliil'~'r~:,~tt.i,~tt. I)etwt:.,.,n single ~l
4. Discussion '.l'he existi~lg stati.sticM da.ta ~l~ t,be cnd.~ryoifie dew.'.tol)meiit of ~il dropl¢:t pign~enl~ti~:m..ilt birds haw, b~,~>~t obtail~m| v r i m ~ r i l y f r o m the microscot)ie e x a m i n a t i o l i s of fresh. ret, im~s of t,he ehic.kell, .Iapanese qm~il (Konishi, t965) a n d i;h.e .pigeott {graIts an~t J u d d , 1 .~33), Ilt the dotnest.it~ elli~:k0~l ((l¢t{l.u,<~do~e.st:ie.u.,O t,hese. d a t a h~ve re~-e~t[ed al~ '~lliLOS¢.ctJrltl.)Je~De b~ck of a.gn'~:cmet~t on the '
4..I 0
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ANt.)
DAVID
.[~. M b i g I , l l ¢
~l(~voi~l o[' a~tv vi.sun.I elemcnl-s or microscopieMly disucrnil)le drol,b.:.ts imtivat.es that: d.urh~g.t:helr inltial a~pl~eat'a~tce Lhese p.i~nmnt.s n u ~ t be. linetv disperat.,d wit.hi,t the whole t'e!:ht?t...I:]v st:::,.l.,e :~7 (~q.u'()xit]mt.(~ty 11 dtLVs), Imwever, when t:lneir cou(..-e.t.erai.i(nLS mtd.de.~dy begi.~t t.o ris~, s h a r p l y ( Figs 2 ~ut(l 3), inner uegmeltt.s a l r e a d y arc evi.(ten.t, Mt.hough di~crel,e oil ~irople.t vi~t:.tott.s atl'(y still ltot deLet'.t.,M)h', wiLh lighL, or tfln:ts(: IlIit.'.I'OSC.O~.~V. . . . .()ll,', d~l.~," . l~U~0f t/ 1.a ":~ dayS; . a~|.~In'oxi]rtato|v . . st.age ,. ":~8) ". .Le.ptat ( 1.91:;} observ,,l . .. a d.vop or t:wo of Fat. a~t t.ht.' ba.se or ~tt t,he courter of t;he eliipsoidtd mitoelloltd.l:i~t (Lhe elosu. ~ssociat, iott 'th0.l, tyxisL~ betweett oil droph't.,s a.n.d mit.o(:hO[Ldrh~, has l.,rontpted. T..x/g,l. v: [
.:-lye ~ " (i,~- &ut,~. or ~,,o(l~hotoqicat staqe~'), t f i,,.-itiu.! detection a~zd .s'eque,,(,e ~j" a~/,peam .m . e'. . of colo.red o~'! dro)~tet, i n tt~z. .reti.,~a o f the ch.ic.a: embr:~to as revealed b / m i e m s e o I , & : ea'a.ntinatD.,~ a q f J?'cM,. ,retinas Oil d r o p l e t , e o h , ' s [u~*c'3t ig~tt., w~-
.ltteubM.iotl L i m e (days)
Rt'd S e h u l t . z 0 ( ISIRJJ [.{~hu ( 1 9 t 6 ) t D;MIv a n d . J u d d (1933) VVehl a n d Z u , s m a n (11);~7. ti}38) ( ~ o t d u m b r e [ t9&3) Wuki~kt~ ( ! 959) 3.'afar ( t g { i l )
IS 16 = 15 It)' 15 16 1S
Golden ye,!l,w
Yelto~-ish green
1,9 17.- [8 t6 a t7 17 ~ ...... ~0 20 . . . . . . . . . . lS ! .t/~ 17 ~ 14
3 h , r p h u h >gi ca ~ st~gt;s/' t/m, ubt~L.R,n t i m e
(day~F 4a] 17
P r e h e a t . itt ve..st-igators
40/t4
40/14
Al~.o d i ~ , i r . t g u i s h e £ / a t\mrt.h o i l d.vt, plet~ eol<,r: urautge. Dark re(hlislt brown. a ~(e Uo~vi..~i.:b t o w n . a 5.',z..ItowisI, o r a n g e . Pirtk~ a ( i r e e n c,r p a l e b l u e . EeLhm~te.(I f r o m s~egu.s o f t r l a m b u r g e . : ; ~l& . l l a m i i t o : ~ ( l . 9 5 | ) .
•~ , m e :¢esearehers, e.g, Akiyt~ (19113), Ped[6r a n d T~t~le3.- (t963) to a s s u m e a mitoeho~r~:xiM..0rigitt for the oll droplets). Using ~he e[pet-~'o~t,t~ieroseope: o lt drop.lets also have, bee,i~ described at ~ttis time as LrreguIarly Shaped strut;tares of h i g h electron de.nsitv within the de.velop~ugit~er u e g m ~ L , wLtett t.he ~.~}i't.ocho,~dri~ begi,_~ to i_,mrease m n u m b e r (Ueno, 19@), I96'Y). ]Trregttlar o,~nnophfim bodies and vesldpS filled with elec, t ron ~te,~se, . . matea'lal . . also . hay e b~o~, o t 6 s m ' ~ l m':de ~x?lopm,g vlstud eo[ls of yotmger e m b r y o s (9 days;, a p p r o x i m a t e l y st.age 3b~ (Meller, 1964), bug h a v e not been-ide.tzgified gs oil droplet, s. Sil~ce t h e firut two oil drop.!et, pigment~ tb~'appear e m b r y o n i c M l y ~re..t~hi}..golden yellow (.mtknown h y d r o c a r b o n ) a m t yellowish gree,a (g~fllox~nghin) earotenoid.s t.tu~t ~-
'
-
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...
ON'L'I)([;I,'~N'Y t~l,' t k E T I N A L
O I L L)I'OI 1,1.,1~5
,l.iI
ft,,side in the e.hi(,t' a~ul accessory (:otnpo~u;ttts, ceapecLivcly, of the double ,.'o~t,~ (Meyer ~md (:Doper, 1966), it..~eems reasunable to accept tim findings of ()hhashi (L924) a u d .Yat~ (1961) tha-t the double cone is tire first: cone to differe~ttia.tc i~t t.b.e ckick retim~, and t.,, add tJ.mt I:tte chief coml.~onczlt nLa.y develop before the accessory. Our spe.ctro.l,hot.omol, ric ~lata., however, tit, n.ot. sui)port the. tissue cuitttrc st.udies of Y a t a (196l) ~Lll,d Govardovski aml K h a r k e e v i c h (1965) both of which rely on vimtaI observi~tion.~; t,t" oil droph.'t color~iAons in situ. Y a t a (196l) cult.m'cd rcthm.~ from t0-, I3- a.m-I 16-day-okt c.hick embryos and reported t h a t gv(.e~t droplets arc ~hc first ~o apl~ear (in rct.imt~ f r o m 13-th~y-otd e.ntL~ryos ",.fief 4 d~ys in vitro). Since these drc~ple.ts occ~tpy t:o.l~a.~swith barrel shape(i inuer scgnu,.ms, which are c.tuu'acte:'ist, ic of ac.ce.-~,~ory cones, his fi.~M].ugs suggest t h a t the. acee, ssory cone is the iirsI; t,o differentiate. l-[e ol)served yellowisl~ tirallgt; (gohte~l yctlow) and red droplet, s sontt~wll~{) b~ter (ill retittas From 16-day-old embryos ctfltm'ed 2 ~nd 14days, respectively, i~ vitro). According to Gow~rdovsM and K h ~ r k e e v i ( ' h (1965), o~t the othe, r hand, all three colored tirol)lets arise sinuLltatw.ottsly i~t retim~l cttltm'es fi'om chick embryos older t h a a l0 days, As ~:mphasizcd earlier, the mic~'o.,copic delinc~tio~.t of the partitmlur colors of the pig.me~l;s, e.g. goldel~ yellow altd yellowish gre.e~t, i,s severely limited I)y ~he 0p~ical properties of the at).alyzing system a n d visual percepI~ive.~mss of t,h.eobserve.t. [t?hi~ a.pproaeh umloul)tedly .is re,st)oils|hie for the mmierous m m x p l a i n e d discrepat~eies t.iu~i~ a p p e a r i~t bite [iteri~t.m'e with respect, not, only to tim init, ia.i de,toetion, of i)ho oil droplet pigm(ints, but alst~ to ~heir sub.se(ltte]tt seque~xee of ,'~ppearance (T~ble [). Sttch ttiic:ro,geoI)ic. (tat, i~ ('o~lse,qLtelltly ~l'e e.omd,:lered uareliable mdess s~:tpported co~momita)igly by m6rc precise physico-chemica[ deecrmimttions. According to the nmjorit:y of develt~pmentat stttdies (which, u.rtforttttmtely, are predomi.m~lt.ly ndcroatop|e) the firs~ droplets to a p p e a r in the chick ret,im), ~rc colorless. They a r e obsetwe4 i\'om, o.ltt~, to seve.ra[ (t~ys before t.hc first colol'ed dropIcgs (Table I I I , Meyer et al., 1 9 6 5 ) ~m(t ~.re believed to give rise t.o ghem. Ottr speci,vophotomet.ric fixId.ittgs, Ixoweve.r, reveal t,h~t- tim golden yellow ~ml yeliowis!t gree~t pigments ~re present several d a y s before, c.o|orl(.ss d.roph~.ts ;trc reoorde;d iJ.t fresh prcparal;ions (stage 3~ ), imlio~,ti~g t,hag ~he l~rimordi'al d roplet,s nm, y not. be colo.rlass. Dmtead, t h e y m-~y eoutah~ t[~e~: pigme~tts in such low co~tcent.rations t h a t their colors c~mnov be discriati[tated, microscopically. 1RE l~ lg I~]~N C E S .~kiy~., S. (1963). Acts Soc. Ophthain~fl. Jal)o,~. 67, 1 lOS. Coulom[¢r% A. g. (1955). An,. J. A~xt¢. 96, 153. .teujit4~, S. ~md ~[orii, M. (1963). Arch. HistoL (Oka?tama) 23, 359. Goodwln, 9.'-. ~,V. (1954). Carotenoids: Their Comparati'b~ Bi¢~he~'~H.~t13t. ChomicM t?t~blisitialg Co., Ne.w York. Covardov sk i, V. I. ~rltt Kh~rkeovioh, T. A.~ 1965). A rkh..4nat. (~"'~tol. EmbxioL 49, 50, Govardovski, V. I. ~nd Kharke¢;vicb, T_ A. (1966). J, EVOl. Bioche~a. Physiol, (/-2"~.%'/a~D2~ 37. [IM~n, liL (19!6). Z. W/4,'s: Zool. Abt. A. 116, 1. tIan~bnrger, V- and [-[~r_nilgon, I~. L. (195t). J. zlIorptml. 88, 49. K.arrer, t ~. ~n4 Jucker, E. (1950). Carotenoida. Elsevier l=hlblishitlg Co., N e w k'ork. Ko~lkuhi, T. (1065). Dobut.,ugak~ Zas~hi 74, 119. Kuhn, 1~., Stone, J. anti SDrerLso~, xW. A. (1939}..Bet. Deut. Ohem,. (.~nel;lsch. 72~ 1688. Kiihn~, VV. a~td Ayres, W. C. (1878). J. Physiol. 1, 109. ]L~plat, G. (19.13). Anat. A ~ . 45, 2[5. Loevellioh, H. ~K., you StuchLitz, {~, and SViggcr, t[. (1944{). Natur~;L~s, 31, 5B8. hieller, K, ( I 9 6 i ) . Z. ZeLtfor,~k. 64, 788. ,Molle~5 K. arid. tlreipoid, W. (t9~5), Z, Ze!~forscg. 66, 673.
442
T.T~ItRANC, E ,'.], C;OOPER AND DAVID B. MI~YE:I~
M(,yer, D. ]:L and Ccmper, T. (~. (19(16). A,m J. Anal. 1.18, 723. 3[(~3~er, D. B., Ct)~)por, '['. G. ~lrd Gernez, C. ([965). In TI~e Structure oflhe I'Ve., pp. 521-33. (~.'. by R¢)hea, ,I. W.) Schat, t~mor, Stuttg~rb. Ohhashi, Y. (1924). Jcfp. Med. World, 4, 257. O'Rahilty, l{.. (1966). Cor~.Irib. Em.br!/ol. (C;mxcgic ]ast;.) 38, 1. O'llahilty, R.. and MT~;ycr,D. B. (1959)..,lela .d.me:. 36, 20. P~.~dlcr, C, rm4 Tatmtcy, K, (1963). Exptl Eye, ]¢es. 2, 39. Sohulbzc, ~I. (1866). Arch. mi~ro,~kop. A'vv~g.2, 175. Stroghcr, G, :K. a.nd Wolken, J. J. (1960). Exptl Cell Res. 21, 504. Slmtt, S. C., Grccafletd, P. ~uxd Boelt, E. d. (1956). d. Comp, Ne.u~vI. 106, 4J3. yon h.'~udnil.z, G., Neum~nn, H. ,T. a.nd Loe~,er~ioh, H. K. (1943). Pfl'Sgcrs .,4rct~. (~e.~:. ' " l>hysioL 246, 652. Ueno, ]~. (19It0). A eta. S ~ . Ophthalmo~. Ja2~n. 64, 260. Ueno, K. (1961). ,]apart. J, Ophtha/,md. 5, 38. Wakisaka, S. (1959), ,4cUz NOc. Ophth~lmol. Japon. 63, 1137. WMd, G. (1948). J. GeJ~,.PhyMol. 31,377. ~lrald, (~. and Z~lssm~n, H. ([937). Nature ].40, 197. Wald, G, and Znsam~, H.((19~8). J, BR>I. Chem. I22, ,i49. Walls, G. L. ~nd J ndd, H. D. {I933), Brit. J. ())~hthalmol. 17, 641; 705. ~Vi~kovsky, P. (1953). Vi,sioa Be,~. 3~ 341. Yata, M, ([96l). 1Vakuyeu~a Me~. Soc. J° 12~ 819.