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TRACNGOFCELLSOFTHEAVIANTHYMUSTHROUGHEMBRYONCLI FE IN I NTERSPEC FICCHMERAS*

BYNCOLELEDOUARNANDFRANCNEV JOTEREAUFromLaboratoi re d Embryol ogi e, Uni uersi te de Nant es, 44037Nantes Cedex, France)

Var i ous t ypes of i mmunol ogi cal def i ci ency di seases are rel ated t o devel op-ment al anomal i es o f pr i mary l ymphoi d organs 1, 2 Si nce t he thymus i s d i r e c t l yconcer ned w t h t he acqui si ti on o f c e l l u l a r i mmuni ty 3 , t he nor mal processes ofthymc di f f erenti ati on have been i nvest i gat ed on many occasi ons The r e s u l t s ,however , have been very cont r over si al concerni ng t he embryol ogi cal o r i g i n oft hymc l ymphocyt es 4 Thi s paper deal s w t h an experi ment al anal ysi s o f t heevol uti on o f t he c el l component s f ound i n t he thymc pri mordi umo f bi rds dur i ngembryoni c l i f e The thymus deri ves f roman epi thel i omesenchymal rudiment or i gi nat i ng f romthe 3rd

and 4th pharyngeal pouches 5-10 I n the chi ckembryo, the thymc endodermseparatesf romthe pharynx duri ng the 5th day of i ncubati on as a cord of epi thel i al cel l s whi chelongates al ong the j ugul ar vei n 9, 10) A thi n mesenchymal capsule surrounds theendodermal pri mordi um and the mesenchymal cel ls penetrate i t , l obul ati on andvascul ari zati on of the organ occurr i ng together Around the 11th day of devel opmentl ymphoi d di f f er ent i at i on of the thymus becomes evident Accordi ng to one vi ewthe l ymphocytes or i gi nate by transformti on of the epi thel i al

cel ls of the ear l y anl age Thi s i dea i n i t i a l l y formulatedby Kol l i ker 11) was support edbyseveral authors who cl aimed t o demonst rate tr ansi t i onal form between thymc epi thel i aland thymc l ymphoi d cel ls 10, 12-17 Froman experimental study of the hi stogeneti ccapaci ti es of the ear l y mouse endodermal thymc rudi ment, Auerbach 18, 19) al so con-cl uded that the epi thel i al component was the source of thymus l ymphocytes Accordi ng t othe substi tuti on theory proposed byHammar 20) and support edbymany subsequentstudi es 9, 21, 22) thymc l ymphocytes were deri ved excl usi vel y f romconnecti ve t i ssuel ymphocytes whi ch i nvaded the ear l y epi thel i al anl age By experi ments carr i ed out i n thechi ckembryo, Moore and Owen 23, 24) demonstrated the exi stence of vascular mgrati onpathways of chromosomal l y l abel edcel l s i nvadi ngthe thymus i n 7- to 8-day-old embryos Accordi ng t o these authors the thymus r ecei ves an i nfl ow of bl ood-borne stemcel l s, pre-sumabl yori gi nati ng f romthebl ood i s lets of theyol k sac, whi chpr ol i f er at e anddi f f er ent i at ei nto l ymphocytes af ter reachi ngthe thymus 25) Snce chromosomal markers gi ve i nformti on onl y about di vi di ng cel l s , i t seemedi nterest i ng t o appl y t o the probl emof thymus hi st ogenesi s, a cel l l abel i ng technique

whi ch, by si mpl e hi stol ogi cal observati ons, al l ows f or the eval uati on of the composi t i onofthewhol e cel l popul ati on of thymc t i ssue at a gi vent i me LeDouari n has recentl ydevi seda newbi ologi cal cel l marki ng techni que 26- 28) whi ch can be employed i n the study ofembryoni c cel l mgrati ons see 29 f or r ef erences) Thi s techni que i s based on structuraldi f f erences i n the i Derphase nucleus of two species of bi rds c losel y rel ated i n taxonomy :

Thi sworkhas been support edbyagrant fromDG. R S . T N 74. 70601 THE JOURNAL OF EXPERMENTAL MEDCNE VOLUME142, 1975 17

Published July 1, 1975


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I H TRACNGOFCELLSOFAVANTHYMUSTHROUGHEM RYON LIFEt he J apanese quai l Coturni x cot urni x j aponi ca) and the chi ck Gal l us gal l us) I n t heJ apanese quai l a l arge amount of het erochromat i c DNAs associ ated t o t he nucl eol us sothat t h i s organel l e i s hypert r ophi ed i n al l emryoni c and adul t c e l l s of t h i s speci es Thi sf eature i s not usual i n bi rds or mammal s 29) and especi all y not i n t he chi ck, i n t h i sspeci es, t he chromti n i s evenl y di spersed i n t he nucl eoplasmw t h some smal lchromocenters and part i ci pates onl y l i t t l e t o nucl eol ar structure As a resul t o f t h i sdi f f erent di sposi ti on of the chr omat i n, quai l andchi ck c e l l s , exper i ment al l y associ ated i nchi meras, can be easi l y di sti ngui shed By the use of het erospeci f i c combi nati ons between quai l and chi ck thymc

rudi mnts the respect i ve contri buti on of endodermal epi thel i um thymcmesenchyme and blood borne extr i ns i c el emnts to the hi st ogenesi s of thethymus hasbeenstudi ed I n the same way i t hasbeen possi bl e t o i nvest i gate theevoluti on of the thymc l ymhocyte populati on and t o f o l l ow the chronology ofstemc el l i nf l ows i n the thymus duri ng embryoni c l i f e

Graf t i ng t echni quesGraf ts of Thymc Pri mordi a

Materi al sandMethodsWhi t e Leghorn chi ck andJ apanese quai l embr yos were used Theeggs were i ncubated a t 38 f I CThe t i mng of t he embr yos was det erm ned accordi ng t o t he numer of somtes i n t he young s t a g e sand t o t he l ength of the i ncubati on peri od l a t e r on I n some exper i mental s e r i e s , stagi ng was doneaccordi ng t o t hedevel opmental st ages def i nedbyHamurger and Haml t on 30 f or t he chi ck and byZacchei 31 f or t he quai l

GRAFTOF QUAIL THYMCENDODERMI NTOTHECHICK SOMATOPLEURE The l aterovent ral wa l l of t heembryoni c phar ynx f romquai l embr yos a t 15 - t o 30- somt e s t a g e s i s treatedbyas ol ut i on of tr ypsi n a t. 1 i n Cal + Mgz+- f reeTyrode f or 20-30 mn a t C Trypsi n d i g es t i o n makes i t po ss i b l e t o separatet he endodermpure f rompharyngeal mesenchyme The endoder mal area whi ch i s sel ected f or t hegr a f t a t di f f e r ent devel opmental stages i s represent ed i n Fi g 1 Theendodermi s i nt roduced i n a s l i t mde i n t hesomat opl eure of a 3- t o 3 . 5 - da y ol d chi ck bet weena nt e r i o r and p os t e r i o r l i mb buds accordi ng t o a t echni que previ ousl y descri bed 32 F i g 2 Thet i s s u e shoul d not be i nt roduced i n the coel omc c a v i t y but l e f t i n contact w th somat opl euralmsenchym whi ch wi l l parti ci pate t o thymc hi stogenesi s I f the expl ant f a l l s i n t o t he coel ome, i tr emai ns purel y e pi t h el i a l mssi ng t he sti mul ati ng i nf l uence exert ed by msenchym on i t s growt hand d i f f e r e n t i a t i o n , i t degenerat es and f a i l s t o g i v e r i s e t o thymc t i s s ue 33 Wen t he endodermgr ows i n cont act w t h somat opl eural mesenchyme, i t s d i f f e r e n t i a t i n g capaci t i es a r e expressed and i tg i v e s r i s e t o t he nor mal deri vati ves of t he t h i r d , fourth pharyngeal pouches, and medi ovent ralendoderm of t he pharynx, i nvol ved i n the gr af t F i g 1 Thus, i n addi ti on t o thymc t i s s ue ,parathyroid, ul t i mobranchi al body, and d i g es t i v e t ube a r e f ound devel opi ng i n t he body wal l of t hehos t I n he t er o spec i f i c associ ati ons bet ween q ua i l endodermand chi ck somatopl eure, al l these organsa r e chi maer i c, t h ei r mesenchymal component bei ng deri ved f rom t he chi ck somatopl eure The hos tembr yo i s s a c r i f i c e d 12 days a f t e r t he i mpl antati on and t he regi on of t he body wa l l contai ni ng thegraf ted t i s s u e i s f i x ed f o r h i s t o l o g i c a l examnati on GRAFTOFCOMPLETETHYMCRUDMENTSENDODERMANDMESENCHYME Thymc pri mordi a of vari ousages were graf ted from quai l i n t o chi ck or r eve r s el y At t he ea r l y s t a ges of devel opment t he areapresumpti ve or t r u e of the 3rd and 4t h branchi al pouches and a r c h e s , i e endoderm msenchymand ectoderm i s di ssectedandgrafted F i g 3 From he6th dayof i ncubati on, t he thymc r udi mentcan be di sti ngui shed al ong the j ugul ar vei nand i s o l a t e d by c a r e f u l d i s s e c t i o n I n t h i s experi ment, t hethymc endodermbei ng associ ated t o i t s homol ogous msenchym i s abl e t o devel op i n t hecoel omc c a v i t y , but a b e t t e r evol ut i on of t he g r a f t s i s observed i n t he somatopl eure, s o t ha t t h i st echni que was extended t o the e n t i r e thymc pri mordi um The g r a f t i s mde i n t o t he somatopl eure of



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A

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NCOLELE DOUARNANDFRANCNE V.JOTEREAU 19

Grafts of Quai l Rhombencephal i cNeural tube i nto Chi ck Embryos

FIG 1 Schemat i c representati onof theendodermal area takenf romquai l embryos at vari ousstages of devel opment i nvol ving the presumpt i ve thymc epi thel i um A) I n 15- to22- somteembryos theventrolateral wal l of thepharynxVLPh) i s cut andthepharyngeal endodermVLPh End) i s i sol ated by trypsi ni zati onof the cardi ac f o l d andgraft edi nto thesomatopl eure ofachi ckembryo B) I n ol der embryos a p ece of the ventrol ateral endodermi ncl udi ng the3rdand4th branchi al pouches 3d, 4th EndP) i s sel ected f or the graft AP, anteri or i ntesti nalportal ; V, ventri cl e

a 3- to3 5-day ol d host I n some experi ments the thymus i s graftedon the chori oal l antoi cmembraneCAM i n 6-day host embryos accordingto aclassi cal techni que 34)

I n order to study the part i ci pati on of the mesenchyme of the 3rd and4th branchial arches t othymus hist ogenesi s, i sochroni c and s ot o pi c gr a f t s of quai l neural rhombencephalonwere carr i ed outi nto chi ckembryos of the 6- t o 9-somte stage Theneural crest or i gi n of t h i s mesenchymehas beendemonstrated i n t hi s l aboratory 35, 36Thegraft i ng techni queprevi ousl y descri bed 26) i s the foll owng I n the f i r s t step the chi ckrhomb-

encephalon i s exci sedby mcrosurgery i n ovo, endoderm notochord, andsomtes being l e f t i n si t uFig 4 Thequai l neural tubeandassociated neural f ol dsare i sol ated by i ncubati on of the adequate



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20 TRACNGOF CELLSOFAVANTHYMUSTHROUGHEMBRYONCLIFE

Fi G 2 Transversesecti on i na3-day ol dchi ckembryo whi ch recei ves the graf t G of aquailthymc endoderml rudimnt i n the somtopl eure S ou r nd endodermCoeC, coel omccavi ty

AFIG 3 Schemti c drawngshowng thearea taken i n the quai l at t o days A andat 5t o5Sz days B of i ncubati oni nvolvi ngtheendomsoderml thymc rudimnt V, ventri cle



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NCOLELE DOUARNANDFRANCNE V JOTEREAUnumerous i n themedul l aandat the peri phery of cor t i cal l obes , are characteri zedby a s i ngl e strongl y Feu gen-posi t i ve centronucl ear condensati on Fi gs 5-6 aApart fromt hi s cent r al mass the nucl eoplasm s onl y weakl y stai ned by theSchi f f reagent I n the chi ck, the nucl eus of re t i cul ar ce l l s shows anetwork ofl i g h t l y stai nedchromati n Thesame character i s t i cs are foundi n connect i vecel l s ,the nucl ei of whi ch i s of ten el ongated Fi g 6 bChronology of Thymc Devel opment i n Quai l and Chi ck Embryos Prel i mnary to theexperimental study of thymusdevel opment i n i nt e r spec i f i c comi na-t i ons, the chronol ogi cal andmorphologi cal evoluti on of thymc pri mordiumwascomared i n quai l andchi ck Our observati ons on thechi ckare i n general agr ee-ment wth the resul ts reportedby previous authors 5, 9, 10 thest ages of s i gn i f i -cant events of thymus hi st ogenes i s i n the two speci es are reported i n Tabl e I Thi s comarat i ve study makes i t c l ear that successi ve steps of thymchi stogenesi s-formti on of epi thel i al cords, l ymhoi d d fferenti ati on-occur al i t t l e ear l i er i n quai l than i n chi ck, but accord ng to a simlar pattern Thusconsi deri ng the c l ose taxonomc vi ci ni t y of the twospecies onecanassume thati nt e r spec i f i c cominat i ons of thymc rudiments can gi ve r i se t o normal l yorgan zed thymuses Experimental anal ysi s of thymus devel opment usi ng quai l -chi ck cominat i onsThrough vari ous t i s sue cominati ons s el ect i ve quai l nucl ear l abel i ng wasachi eved af f ec t i ng one cel l category at a t i me a the endodermal deri vati ves, b themesenchymal comonent c the l ymhoi d cel l popul ati onGraft of quai l presumti ve thymc endoderm nt o the chi ck embryo Presum-

t i v e thymc endodermfrom15- t o 30-somte quai l s was graf ted i n thesomato-pl eure of 3-day chi ckembryos F i gs 1and2 12days after i mlantati on thegraf twasobservedhi s tol ogi cal l y I t hasbeenprevi ousl y demonst rated i nhomospeci fi cassoc i at i ons 33 that the thymc endoderm s determned earl y anddi f f erent i -at es i nt o thymc t i s sue when associ atedto thesomtopleural mesenchyme Thethymus developed i n these cond ti ons has a normal hi stol ogi cal structurewthcortex andmedul l a Fi g 10 Thewhol e l ymhoi d popul at i on i s of chi ck hostor i gi n whi l e re t i cul ar c e l l s show the quai l nucl ear marker Fi g 11 Theperi vascul ar endothel i umandconnect i ve t i s sue ce l l s belong t o the chi ck, andthey are deri ved fromthe somtopleural mesenchyme These resul ts show that thymc endodermi sol ated as earl y as the 15-somtestage i s abl e t o develop i nt o thymc r e t i cul ar ce l l s andt o i nducetheheterologousmesenchyme of thesomatopl eure t o part i cipate i n thymus hi st ogenes i s On theother hand thymus endodermdoes not gi ve r i s e t o l ymhoi d c e l l s , whi ch i n t hi sexperi ment come fromthehost They could deri ve ei t her fromthymc mesen-chyme i . e the thymc pharyngeal mesenchyme i n normal devel opment or theheterologous somatopleural mesenchyme i n the experiment reported above orfromext r i ns i c hemocytobl asts The fol l owngexperiments were devi sedt o deci deon thi s i ssueRol e of themesenchymal comonent i n thymus hi st ogenes i sGRAFTOFCOMPLETEQUALTHYMUSRUDMENT [ENDODERMANDTHYMCPHARYNGEAL

MESENCHYME] INTOTHECHCKEMBRYO Thetota l thymc anlagewas takenfrom



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24 TR CNGOF CELLSOF VI NTHYMUS THROUGHEMBRYONCLIFENUCLE R CH R CTERSTICS OF THE CELLTYPES I N QU L NDCHCKTHYMUSES

FIGS 5-6 Schemati c drawngs 5 andmcrographs 6aand b showngthenuclear f eature ofquai l andchi ck thymc cel l types after Feul gen-Rossenbeck staini ng I n the quai l 6 a thel ymphocyte L nucl ei contai n one l arge heterochromati c mass and several sm l er onesattached to the nuclear membrane I n the chi ck 6 b several smal l chromcenters aredispersed i n the nucl eopl asm

I n the quai l , the l ymphobl asts La show a l arge nucl eus wth a Feulgen-posi ti vecentronucl ear condensati on wh l e i n the ch ck the nucl eus i s onl y c l e ar l y stai ned Reti cul arR andconnecti ve c e l l s of quai l haveusua l y onesi ngl e heterochromati cmass I n thesamecel l typesof thech ck thenucl eus contains af i n e network of evenly distributedchromati n ax 1,450 6bx 1,300

15-somte to 4-dayquai l embryos and grafted i n t o t he somatopl eur e of 3- day ol dchi ck hosts as previ ousl y descri bed The i mplant was f i xed when t he total age oft he thyms was about 14 days I f l ymphoid c e l l s deri ve, even parti al l y, fromthyms mesenchyme proper, l ymphocytes wi l l have t he quai l nucl ear marker Actual l y, a l l t he l ymphoid c e l l s of t he thymc ti ssue came fromt he chi ck host,j ust as i n the f i r s t exper i mental s e r i e s when t he thymcendodermwas graftedal one Reti cul ar c e l l s were of quai l or i gi n Fi g 12 and connect i ve ti ssue c e l l swere of both donor and host t ype, showngthat t he somatopl eural mesenchymepart i ci pates i n t he hi stogenesi s of t he expl ant I n t he cortex, typi cal ret i cul arc e l l s characteri zed by l ong c el l processes, showed t he quai l DN r i ch nucl eol us Large, medi um and smal l l ymphocytes as wel l as l ymphocytobl asts on t hecontrary had t he nucl ear features of t he chi ck Fi g 12Thi s experiment demonst rates that t he l ymphocytes deri ve sol el y fromextr i ns i c el ements absent f rom he quai l thymc rudiment upt o t he 4t h day ofi ncubat i on Nei ther endodermnor mesenchyme of t he thymc primrdi umhavet he abi l i t y t o di f f erent i ate i n t o l ymphocytes Mor eover , these resul ts showthatt he presumpti ve thymc endodermsel ect i vel y attracts t he extr i ns i c l ymphoidstem e l l s evenwhen grafted i n an heterotopi c l ocat i on

Lymphocytes Reti cul ar ce ls Connecti ve ce ls

CHCK

I

i d I I NAnumrous smal l clums Evenly dispersed network

of chromati n of chromti n

i

QU L I I

one l arge clum of heterochro-mati cDNA smal l msses at- One l arge centra mss oftachedtothenuclear membran heterochromati c DN



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26 TR CI NG OF CELLS OF VI N THYMUS THROUGH EMBRYONI C LIFE

FI Gs 8 9 El ectronmcrograph of thecortex i n 16 day ol dquai l thymusFI G Smal l l ymphocyt es show nga l arge centronuclearN mass and several smal l er onesatt ached to the nucl ear membr ane Uranyl acetate l ead i t r t e x 1 5FI G 9 Medi umand l arge l ymphocyt es i n whi ch the centr onucl ear heterochromati c massshows a clear centr al core Uranyl e acetate l ead i t r t e x 10 500



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N COLE M LE DOUARNANDFRANCINE V. JOTEREAU 27TABLE I

Occurrence of theMai nDevel opmental EventsDuri ngThymc H stogenesis i n Quai l andCh ck

STUDYOF THE EVOLUTI ONOFTHETHYMCMESENCHYMAL COMONENTSBYSELECTIVELABELI NG OF BRANCHIAL ARCHES MESENCHYME I t has been previously shown thatthe mesenchyme of the branchal arches deri ves ent i rel y fromtheneural crestexcept f or the muscl e pl atewhi ch i s of mesodermal or i gi n 35-36 I sotopi c andi sochroni c grafts of aquai l rhombencephal i c pri mordi um nt o a ch ckembryo atthe6- t o 9-somte stage resul t i n thei nvasion of the 3rdand4thbranchal archesof the ch ck host by quai l cel l s Fi g 13 Thethymc rudment i s ch mri c fromi t s f i r s t devel opmental steps the host endodermal pouch i s surroundedby quai lmesenchymal cel l s ; l ater on, the ch ckthymc epi thel i al cord i s surroundedbyathi n capsul e of quai l mesenchyme Fi g 14 Thereafter the i nvasion of theepi thel i al anlage by connective cel l s mybe fol l owed and the contri bution ofmesenchymal elements t o the di f f erent i ated thymc t i ssue determned Mesen-chymal cel l s f o l l o w the bl oodvessel s whi ch penetrate i nto the cortex and themdul l a the endothel i umof whi ch i s of host or i gi n Fi g 15 The thymcrudiment i s i nvaded by capi l l ary buds of mesodermal or i gi n wh le thymcconnecti ve cel l s deri ve f rommesenchyme of neural crest or i gi n I n none of the40cases of di f f erent i ated thymuses observed i n thi s experimntal seri es d d thel ymphocytes show the quai l nucl ear marker Th s experiment confi rm theprevious resul t, i . e that thebranchal archmesenchyme whi chpart i ci pates t othymc hi stogenesi s does not gi ve r i se to l ymphoid cel l s Coloni zati on of thethymus rudment by extr i ns i c hemocytoblasts bei ng establ i shed the nextexperimntal st ep, was t o studythetimngof thi s col oni zati on i n quai l andch ck

Timng of Coloni zation of the Thymc Rudiment by Lymphoid StemCel l s Quai l thymc primord aof variousages f rom4 t o 8days of i ncubati onhavebeengraf ted i nto the somatopl eure of 3-daych ck embryos andf i xedwhenthei r t o talage age at thetime of the graf t anddurati onof the graf t reached14days Thereversegraf t of ch ck thymc anlage f rom4- t o 9. 5-day embryos i nt o 3-dayquai l shas al so beencarr i edout i n the samecond tions Thel ymphoidpopul ati on of thethymuses whi chdevel op i n those condi ti ons shouldbe of host or i gi n when theyhave been takenbefore thenormal timeof stemcel l i nvasion Onthecontrary, i f

I s o l a t i o n F i r s t ap - F i r s t ap- Appear anceof v a s c ul a r -o f e pi t h e -l i a l thymc Fusi on of pearanceof basophi - pearance Begi nni ng Lobul ati on i s a t i o n i n Compl e-rudi ment thymc l i e ce l l s i n of l ympho- of l obe of thymc t he e pi t he - l i on o ff romt he r udi ments t he thymc cytes i n f ormati on l o be s l i o-mesen- l obeembryoni c I I I and I V e p i t h e l i a l t he thymc chymal f ormati onphar ynx cord r udi ment thymusanlageQuai lStages of devel op- St 19- 20 St 21 St 20-21 8days of 7 days of 9 1/2 10 9- 10ment accordi ng t o 4 1/2 5 5 1/2 days 5-5 1 /2 i ncu- i ncu- days of days ofZacchei 31 days of i n- o f i ncuba- days of i n- bat i on bat i on i ncubat i on i ncubat i oncubati on cubati on cubati onChi ckSt ages of devel op- St 25 St 28 St 29- 30 9 1 /2 days x8days 8days of 8 days of 15 days ofment accordi ng t 6 4 1/2 5 5 1/2 6 6 1/2 days of i ncu- of i ncu- i ncubat i on i ncubat i on i ncubat i onHamburger and days of i n- days of i n- of i ncuba- ~ bati on bati on

~

Haml ton 30 cubati on cubati on t i o n



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30 TR CI NG OF CELLS OF VI N THYMUS THROUGH EMBRYONI C LIFE

FI G 14 Thymc rudi ment i n a 7-day ol d chi ck embr yo after the graf t of a quai lr hombencephal on accordi ng to the experi mental procedure i ndi cated Fi g The epi thel i alt hymc anl age i s made upof chi ck cel l s whi l e the t hymc mesenchymal capsul e bel ongs to t hequai l species I t ori gi nates from the rhombencephal i c neural crest whi ch gi ves r ise to t hemesenchyme of the 3rd and 4th branchi al arches Feul gen-Rossenbeck x 850FI G 15 Same experi ment as i n Fi g 14 Sect i onof the t hymus at 13 days of i ncubat i on Theconnect i ve cel l s C l i ni ng t he bl ood vessel s deri ve f rom the quai l t hymc mesect odermThymc cortex Co) shows reti cul ar cel ls and l ymphocyt es of host ori gi n No l ymphocyt es showt he quai l nucl ear mar ker and then no l ymphocyt es deri ve from t he mesoder mal t hymccomponent Feul gen-Rossenbeck x 1,450These experi ment s i ndi cate that stem cel l s of l ymphocytes are present i n

t hymus anl age f romthe stages of 5 days i n quai l and 6 5 days i n chi ck embryos Thi s stagecoi nci des w th the t i me at whi ch basophi l i c cel l s become detectabl e i nthe t hymus i n both species Tabl e1 I t seemed i nt eresti ng to f i nd out whetherthese cel l s are the precursors of t hymus l ymphocyt es and t heref ore i f i n ourexperi mental condi ti ons they showthe nucl ear characteri sti cs of the host i n thegraf ted t hymus 6- day ol d chi ck t hymc rudi ments were graf ted f or 3 to 4 daysi nto 3-day ol d quai l embr yos and then f i xed in Zenker s f l ui d and treatedaccordi ngto the Pappenhei ms and Feul gen-Rossenbeck s associ atedtechni ques I n these condi ti ons the epi thel i al chi ck thymc rudi ment s contai ned anumber ofcel l s w th a strongl y basophi l i c cytopl asmshow ng al so the quai l marker Fi g 17 The hypothesi s of Moor e and Owen 24 that the f i rst detectabl e l ymphoi del ements of the embryoni c t hymus are cel l s w th a l arge nucl eus and a strongl ybasophi l i c cytopl asmi s thus conf i rmed



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NCOLELE DOUARNANDFRANCNE V JOTEREAUTABLE I I

Timeof Col oni zati onof Thymc Rudiment by Bl ood-BorneStemCel l sTypeof l ymphocytes i n 14-dayold

Accordi ng t o Zacchei 31Accordi ng t o Hamburger andHaml ton 30

Number of posi ti ve resul ts onnumber of embryos observed

Moreover, i t can be concl uded that i n both speci es at a preci se stage of i t shi st ogenesi s the thymc rudiment recei ves a rapi d i nfl ow of stemcel l s thedurati on of which i s re lat i vel y shor t , i e about 24 h i n thequai l duri ngthe6thdayof i ncubati on and36 h i n thechi ck duri ng thesecond hal f of the7thdayandthewhol e8thday of i ncubati on Af ter thi s stage penetrati onof stemcel l s i nthe thymus i s extremel ysl owi f not t ot al l y nonexistent Further probem ar i se does the thymus cease t o be att ract i ve f or stemcel l s

after thi s st age, or are thymchemcytobasts unavai l ab e i n theembryo duri ngacert ai n peri odof embryoni c l i f e ? On theother hand, does the i n i t i a t i on of stemcel l i nfl ow i nt o the thymus depend ei ther on theappari ti on of i t s capabi l i t y toretain thehemcytobasts or on thestageat whi chthosecel l s becomeavai l abl e i nthe embryo? Toward sol vi ng these probem, stem cel l s wth a capaci ty todi f f erent i ate i nto thymc l ymphocyteswere searched f or i n theembryo at stagesear l i er and l ater thanthat of normal thymc i nvasionPresenceof StemCel l s i n theEmbryoat Vari ousStages of Devel opment I n

order to knowwhether thymc l ymphocytestemcel l s are present i n theembryobefore the stage when they norml l y part i ci pate i n thymus hi st ogenesi s, thefol l owngexperiment hasbeencarr i edout thymc rudimentsweredi ssected f rom4-dayquai l embryos, graftedf or 2days i n 3-dayol dchi cksomtop eureandthenretransp anted i nt o a 3-day ol d quai l f or 8days Fi g 18 At thi s time thequai lthymus aged 14 days total was observedhi s to logi cal l y after Feul gen-Rossenbeckstaini ng

I n t hi s case the l ymphoid popul ati on of the thymc t i ssue showed thecharacteri st i c nucl ei of thechi ckwhi l eret i cul ar andconnecti ve cel l s bel ongedto

Stageof donorembryo attimeof graf t

Days ofi ncubati on

Donor

graf tedthymus

type Host donortype Host type

Quai lSt 18* 4 4/4St 19-20 4 /2 -5 3/3St 20-21 5-5 2 3/6 3/6St 22 6 4/4

Chi ckSt 29t 6-6 1 2 4/4St 29-30 6 2 4/4St 30 6 1/ 2 - 7 4/4St 30-31 7 4/7 3/7St 33-34 8 7/9 2/9St 35 8 /2-9 5/5



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NCOLEMLE DOUARNANDFRANCNEV JOTEREAU 33n

2d

12d

FIG 18 Dagramof the experi mental proceduredevisedto test theavai l abi l i ty of hemopoi -et ic stemce l l s i n thechi ck embryo before thenormal time of thymc col oni zati onAuailthymc rud ment froma4-dayol dembryo i s grafted i nt o a3-dayol d chi ckfor 2days andthentranspl anted i ntoa3-dayquail host embryo whenthetotal ageof thegraftedthymus reached14 days i t i s observed hi s to l ogi cal l y andshows a l ymphoid cel l popul ati on ent i re ly of chi ckor ig in Thi s demonstrates that thestemcel l i nfl owi nto thequai l thymus occurredexclusivel yduri ng thef i r s t transpl antati on i n thechi ckhost

t he quai l speci es These r e s u l t s show t hat hemocyt obl ast s abl e t o respond t ot hym c endodermatt racti onand i nducti onare avai l abl e i n t heembryo as soon a sthe 4th day o f i ncubat i on t hat i s before t he st ageat whi ch t hey normal l y ent er t hethymus pri mordi um Thi s exper i ment a l s o conf i rms that t he durat i on o f t he f i r s tinf lowof c e l l s into t he thymus i s l imted, si nce none or very f ewquai l c e l l s wer ef ound i n t he thymus a f t e r t he second t ranspl antat i on i n q ua i l embryo

I n a second experi ment al s e r i e s t hym c rudi ment s wer e t aken f r om4- day ol dquai l embryos and graf t ed on CAMf 8 . 5 -day ol d chi cks f o r 10 days i n order t osee whet her t hym c hemocyt obl ast s are present i n t he bl ood o f t he chi ck embryoa f t e r t he stage of thymus col oni zati on These g r a f t s showed a nor mal devel op-ment of t hym c ti ssue i n whi ch l ymphoi d c e l l s wer e of host o r i g i n Those exper i ment s demonst rate t hat stem c e l l s compet ent t o respond t ot hym c att racti on are present i n t heembryo bef ore and a f t e r t he st age a t whi chFIG 16 6-dayol dchi ck thymus grafted for 8days i n aquail embryo Mxture of quail QLandchi ck l ymphocytes CL Feulgen-Rossenbeck x 1,280FIG 17 6-day ol d chi ck thymus grafted for days i n a quail embryo a Panopti quetechni que showngbasophi l i c c el l s arrows x1,280 b Thesamesecti on i s post-stai nedwthFeulgen-Rossenbeck techni que Thebasophi l i c c e l l s showthe quai l nucl ear marker Thi sdemonstrates that thehemopoieti c c el l s of thethymus have an extr i nsi c or ig in x1,280FIG 19 Thymus of a8-day ol d quail embryo graftedfor 10 days i nto a chi ck host . I n theexternal regi on of thecortex quai l l ymphocytes have been repl acedby host ones CL Quai ll ymphocytes are present i n the i nternal regi on of the cortex QL Feulgen-Rossenbeck x1,140

VTi me o f coloni za t i onof the Ouo i l thymus

QUALDONOR

1d 2d 3d 6d 7d 8d 9d 10d 11dFPV~ Duration Time of colonizationof t he of the Chick t hymusI st graft1st HOST

CHCK - 1. 1 6 1d 2d 3d d I d 7d ad 10d 11d1

Durati on of t he 2nd graft2ndHOSTQUAL 3rf p i s d 2d 3d 4d Sd 6d 7d ad 9d 10d



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34 TRAC NG OF CELLS OF AVIANTHYMUSTHROUGHEMBRYON C LIFEthey col oni ze the thymus They penetrate i nto the thymc rudi mnt at a preci sestage of i t s organogenesisandduri nga l imtedperi odof ti me after whi chnone ora very sml l i nfl owof cel l s takes pl ace

Duri ng the col oni zat i on peri od the thymc rudi mnt attracts and retai nsbl ood-borne stemcel l s Whenacert ai namount of basophi l i c cel l s are present i nthe epi thel i umthe l at ter l oses at l east par t i al l y i t s capaci ty t o reta i n mrec i r cul at i ng hemcytobl asts Thenext probl ems to knowhow ong thi s nonattracti vi ty or l owatt ract i vi ty

peri od of the emryoni c thymus l as ts I n previ ous experi mnts the graf t peri odended when the total age of thymc rudi mnt was 14 days The het erospeci f i ci ml antati on ti m was protracted i n order to see whether anewnf l owof stemcel l s occurs duri ng emryoni c l i f e

Renewal of Lymhoi d StemCel l s i n t he Thymus at t he end of Emryoni cL i f e Thymc rudi mnts of 8 t o 10day ol dquai l emryoswere graf tedi nto thesomtopl eure of 3-day chi ck emryos f or 8t o 15 days Theproporti on of chi ckandquai l l ymhocytes i n the graf ted thymus was evaluatedby counti ng i n eachcase about 2. 500 cel l s at four di f f erent l evel s of thethymus, i n the external andi nt ernal cortex The resul ts Tabl e I I I showthat a coml ete renewal of the

T LEI I IPercentage of Chi ck Lymphocyt es i n Quai l Graf ted ThymusesDurat i on of t he g r a f t

Two cases were observed f or each experi ment EC external cort ex; I C, i nternal cortex

l ymhocyte popu ati on occurs duri ngtheexperi mnt Ater 8days i n graf t no hostl ymhocyteswere present i n thequai l thymus onl yscatt eredl ymhobl asts cou dbe detected i n the peri pheral cort ex area Chick l ymhocytes f i r s t appeared i nthe ext ernal regi on of the cort ex of the graf ted thymus and progr essi vel y extended i nto the deeper cort i cal l ayers and then i nto the mdu l a Ater 15 daysi n the chi ck whatever the age of the thymus at graf ti ng pract i cal l y the whol el ymhoi dpopu ati on of the i ml ant wasof host or i gi n i n the18 cases observedThe reverse graf t of thymus from9- and 10-day ol d chi ck i nto 3-day quai lemryos was carr i ed out Thedurati on of the graf t was of 8 and 11 days Theresul ts obtainedwere i n agreemnt wth theprevi ous ones rogressi verenewalof donor by host l ymhocytesoccurred i n thegraf tedthymus Thus, the thymus i sthe s i te of successi ve waves of l ymhoi d stemcel l s whi chcoml etel y renewthedi vi di ng l ymhoi d c el l popu ati on of the organ

Age ofthymus 7 days 8days 9 days 10 days 11 days 12days 13 days 14 days 15 daysat t i meof g r a f t EC I C EC I C EC I C EC I C EC I C EC I C EC I C EC I C EC I Cd days 0 0 5 5 0 99 93 69 35 98 93 100 93 83 54 100 100d days 0 0 0 0 10 2 5 83 5 5 5 87 70 5 55 28 99 5 81 99 99 100 100d days 0 0 0 0 38 2 24 7 51 15 83 5 56 5 86 45 100 9 8 , 5 100 10010 days 0 0 10 0 56 5 75 26 98 91 96 5 81 99 6 95 9 100 97 5 100 100



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36 TR CNGOF CELLS OF V NTHYMUS THROUGHEM RYONC LIFEshow that, i nversel y, earl y determned thymc endodermi s abl e t o i nduce anhetero ogous mesenchyme to part i ci pate i n thymus hi st ogenesi s and t o gi ve r i s et o norma l y di f f erenti ated thymc t i ssue

t i s l i k e l y that themechani smof stemcel l s at t ract i on by thethymus i s duetoa di f f us ib le substance to whi ch the hemocytobl asts of the c i r cul at i ng bl ood aresensi t i ve t i s i nteresti ng i ndeed tonoti ce that the penetrati onof hemocytobl astsi nto thymc epi thel i um occurs before the vascul ari zati on of the rudiment Therefore, theymust havemgrated f romthebl oodvessel s i nto the surroundi ngmesenchyme and then penetrated the basement membrane The fact thatthymc endoderm graf ted i n aheterotopic l ocat i on, at t ract s l ymphoi dstemce l l sof the host suggests that the cel l type responsibl e f or the el aborati on of theatt ract i ve substance i s the epi thel i al reti cul um Quai l -chi ck combinati ons ofthymc rudiments performed i n theseexperimentsshowedthat themechani smofat t ract i on of l ymphoi dstemce l l s by thethymus i s not speci es spec i f i c si ncestemce l l s of thechi ck can col oni ze the thymc reti cul umof quai l and i nversel y Becauseof the fact that after bi rth thethymus i s thes i t e of constant i nfl owof

stemce l l s f romthe bonemarrow[ see Metca f andMoore 4) f or r evi ew] , onecanassume that thesamemechani smof at t ract i on i s mai ntai ned i n the adul t organDuri ng embryogenesi s, cert ai n t i ssues apparentl y exert such an at t ract i on onmgratory ce l l s Thi s seem to be thecaseboth f or thegeni tal ri dgewhichattractsandretai ns pri mordi a germce l l s 41, 42 andf or the vari ousorgans col oni zedbyneura crest ce l l s duri ng earl y embryogenesi s [see Horstadi us 43), Wston 44) ,Le Douari n 29) f or revi ew I n al l these cases the att ract i on i s a t r ans i toryphenomenon whi chdi sappears duri ng the process of organogenesi s Thehemo-poi et i c organs seemt o beanexcepti on i n t hi s respect as they re tai n thi s capaci tyto attract stemce l l s f romearl y hi stogeneti c stages throughembryonic andadul tl i f e Cel l tracer techni ques i ncludi ngsex chromosomemarker system and theuseof t r i t i a ted thymdi nehavedemonstrated boththeexistenceandtheimportanceof mgrati on stream of bl ood-borne hematopoieti c ce l l s between di f f erentti ssues i n the course of the ontogeny of the hemopoieti csystem see 4 f or revi ew MooreandOwen 23, 24) using the sex chromosomemarker systemto trace cel lmgrati on i n parabi oti c chi ck embryos showed a chimeri sm n the popul ati on ofdi vi di ng ce l l s not onl y i n the thymus, but al so i n theother hemopoieti corgans,thebursaof Fabri cius 45), thespl een, and the bonemarrow 23) Usi ng the qua l -chi ck marker systemi t was possi bl e to demonstrate that i n

theseorgans [ spl een 46), bursa of Fabri cius 47) ] as i n thethymus, hemopoieti cdi f f erent i at i on i s enti re ly dependent on bl ood-borne i mmgrant stem ce l l s Recent observati ons LeDouari nandJ otereau, unpubl i shed dat a) have revea edthat the hemopoi eti c ce l l s of the bone marrowdo not deri ve f romthe i n s i t umesenchyme of bone rudiment and especi al l y the peri osteal mesenchyme assuggestedbyoom48), but f romi mmgrant ce l l s car r i ed vi a the c i r cul at i on Thus, i t appears that hematopoieti c di f fe rent i at i on i n theembryo empl oys asi ml ar basi c mechanism f or al l bl ood cel l f ormng organs Very earl y i nIMoore,A S andJ J . T Owen 1965 Unpubl i shedobservati onquoted i n Metca f andMore

ref 4)



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N OLELE DOU RNNDFR N NE V JOTERE U 318 Auer bach, R 1960 Mor phogenet i c i nt er acti ons i n t he devel opment of t he mouse

thymus gl and . Dev Bi ol 2: 271 19 . Auerbach, R 1961 Exper i mental anal y si s of t he o r i g i n of c e l l types i n t hedevel opment of t he mouse thymus Dev Bi ol . 3 : 3 3 6 .20 Hammar J 1905 Zur Hi st ogenese and I nvol uti on der Thymusdr uss e nat nz 27 : 23 21 Maximow 1909 Unter suchungen f i b e r Bl ut- and Bi ndegewebe Uber di e Hi s-t ogenese des Thymus be i Saugeti eren rch Mkr nat 74 : 52522 Gr e g o i r e C 1932 . Contr i buti on exper i ment al e a 1 e t u d e duthymus des mamm f er es L acti on compar ee des r ayons Xsur l e t hymus au cours de 1 hi st ogenese et chez 1 a d ul t eenvi sage speci al ement du poi nt de vue de l a r a d i o - s e n s i b i l i t e des p e t i t e s c e l l u l e st hymques , de l a nature du processus de phagocyt ose e t du mecani sme de l ar egenerati on rch I ntern . Med 7 : 5 1 1 23 Moor e, S and J . J . T Owen 1965 Chromosom mar ker st udi es on thedevel opment of the haemopoi et i c system i n the chi ck embryo Natur e (Lond

208 : 95624 Moor e, M S andJ J . TOwen 1967 . Exper i ment a l st udi es on t he devel opment oft he thymus J Exp Med 126: 715 .25 Owen, J J . T andM Ri tt er 1969 Ti ssue i nteracti on i n t he devel opment ofthymus l ymphocyt es J Exp Med 129 : 4 3 1 26 Le Douar i n, N 1969 Part i cul ari tes du noyau i nt erphasi que chez l a C a i l l e j aponai se(Coturni x cot urni x j aponi ca) U t i l i s a t i o n de c e s pa r t i c ul a r i t e s comme mar quagebi ol ogi que dans l es recherches sur l es i nteracti ons t i s s u l a i r e s e t l e s mgrati onsc e l l u l a i r e s au cour s de 1 ontogenbse Bul l . Bi ol Fr . Bel g 103: 435 27 Le Douar i n, N 1971 Caracteri st i ques ul t r a s t r u c t ur a l e s du noyau i nter phasi que chezl C a i l l e et chez l e Poul et et u t i l i s a t i o n de c e l l u l e s de C a i l l e comme mar queur sbi ol ogi ques en Embryol ogi e exper i ment al e nn Embryol e t Morph 4 : 1 2 5 .28 LeDouar i n, N 1973 bi ol ogi cal c e l l l a b e l l i n g t echni que and i t s use i n experi mentalEmbr yol ogy Dev Bi ol . 30: 217 29 Le Douar i n, NM1974 . Ce l l r ecogni t i on based on natural morphol ogi cal nucl earmar ker s Med Bi ol I n press

30 Hambur ger , V and H L Haml t on 1951 s e r i e s of nor mal stages i n t hedevel opment of t he chi ck embr yo . J Morph 88 : 49 31 Zacchei , M1961 Lo svi l uppo embr i onal e d e l l a quagl i a gi apponese (Coturni xcot urni x j aponi caTS Arch nat 66: 3632 Le Douar i n, N Cl Bussonnet , and F Chaumont , 1967 Etude des capaci t es dedi f f erenci ati on e t du r b l e mor phogbne de 1 endoderme phar yngi en chez 1 embr yond O seau nn Embryol Morphol Exp : 29 33 Le Douar i n, N 1967 Determ nati on precoce des bbauches de l thyrdi de e t duthymus chez 1 embr yon de Poul et C RHebdSeances cadSci . 264: 940 34 Wol f f , Et andHLutz 1939 Sur une modi f i cat i on appor t ee a l a t echni que des g r e f f e schori o- al l antdi di ennes chez 1 embryon de Poul et . C R Seances Soc Bi ol F i l 132: 117 35 Le L i e vr e C andNLeDouari n 1973 Cont ri buti on dumes ect oder me a l a genbse desarcs aort i ques chez 1 embr yon d O seau C RHebd Seances cadSci . 276 : 3 8 3 36 . Le Li evre, C andNLeDouar i n 1974 Or i gi ne ectodermque duderme de l a f ace e t ducou, montr ee par des combi nai sons i nters peci f i ques chez 1 embr yon d O seau C RHebd Seances cadSci 278 : 5 1 7 .37 Le Douar i n, N andM T e i l l e t 1970 Sur quel ques aspects de l a mgrati on desc e l l u l e s neur al es chez 1 embr yon de Poul et etudi es par l a methode des g r e f f e sheterospeci f i ques de tube ner veux C R Seances Soc Bi ol F i t 164: 390 .



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0 TR CNGOF CELLSOF V NTHYMUSTHROUGHEM RYONCLIFE38 Feul gen R and HRossenbeck 1924 M kroskopi sch- chem scher Nachwei s e i n er

Nucl ei nsaure vomTypus der Thymonucl ei nsaur e and di e dar auf ber uhende e l e k t i v eFarbung von Zel l kernen i n m kroskopi schen PrAparat en . Hoppe- Seyl er s Z Physi ol hem135: 203 39 Pappenhei m 1910- 1911 . I n Techni ques hi stol ogi ques. MSel on Gabe e di t o r Massoon et Ci e Pari s 40 LeDouari n NM1973 Feul gen- posi ti ve nucl eol us Exp Ce l l Res 77 : 459 41 Si mon D 1960 Contr i buti on a 1 e t u d e de l a ci rcul ati on et du transport des gonocyt espr i mai r es dans l es bl ast odermes d Oi seau c u l t i v e s i n v i t r o Arch Anat M cr osMor phol Exp 40 : 93 42 Duboi s R 1968 La col oni sat i on des ebauches gonadi ques par l es c e l l u l e s germ nal esde 1 embryon de Poul et en cul ture i n v i t r o J Embryol Exp Mor phol 20: 189 43 Horst adi us S 1959 The neural c r e s t I t s propert i es and deri vat i ves i n t he l i g h t ofexper i ment al research Oxf ord Uni versi ty P r e s s London44 West on J 1969 The mgrati on and d i f f e r e n t i a t i o n of neural c r e s t c e l l s dv Morphog 8: 41 .45 Moor e S and J J TOwen 1966 Exper i ment a l studi es on t he devel opment oft he Bur sa of Fabri ci us . Dev Bi ol . 14: 40 46 D eterl en-Li evre F 1974 On t he o r i g i n of haemopoi et i c stem c e l l s i n t he avi anembryo an experi ment al approach J Embryol Exp Morph n press 47 Le Douari n N et Houssai nt E 1974. L or i g i n e des l ymphocyt es de l a bourse deFabri ci us etudi ee sur des chi mbres embr yonnai r es de C a i l l e et de Poul et R HebdSeance s Acad Sci 278: 2975 48 Bl oom W1938 Embr yogenesi s of mammal i an bl ood . I n Handbook of Hemat ol ogy HDowney e di t o r Hoeber NewYork 49 Mar ks P andR Ri f ki nd 1972 Protei n synthesi s t s contr ol i n erythropoi esi s Sci ence Wash DC . 175: 955 50 Mar ks P and RRi f ki nd. 1972 . Feta l i v e r erythropoi esi s and yol k sac c e l l s Sci ence Wash DC . 177: 187 51 Har r i son DE and E S Russel l . 1972 Feta l i v e r erythropoi esi s and yol k sac c e l l s Sci ence WashDC . 177: 187


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