/ . Embryol. exp. Morph. Vol. 25, l,pp. 85-96, 1971 g 5

Printed in Great Britain

The origin and movementof the limb-bud epithelium and mesenchyme in

the chick embryo as determined byradioautographic mapping

By GLENN C. ROSENQUIST1

From the Department of Pediatrics, The Johns Hopkins Hospital

SUMMARYThe origin of the limb-bud cells was determined by tracing the movements of [3H]thymidine-

labelled grafts excised from late medium-streak to 5-somite stage chick embryos and trans-planted to the epiblast, streak, and endoderm-mesoderm of similarly staged recipientembryos.

Although exact definition of the prelimb areas was not possible because of the smallnumber of grafts placed at each developmental stage, the study showed in general that at thelate medium-streak stage the future limb-bud epithelium is in the epiblast (dorsal) layer nearthe lateral margin of the area pellucida. It moves medially toward the embryonic axis, justlateral to the premesoderm cells which will be invaginated at the primitive streak. Withregression of the streak, the limb-bud epithelium moves relatively anteriorly into a positiondorsal to the limb-bud mesoderm, beginning at least as early as the early head-fold stage.

At the definitive-streak stage, the future limb-bud mesoderm is in the epiblast layer abouthalfway from the streak to the lateral margin of the area pellucida, at a level about halfwaybetween the anterior and posterior ends of the streak. From this position the prelimb meso-derm migrates medially to the streak, and is invaginated into the mesoderm layer at a positionabout halfway between the anterior and posterior ends of the streak; after the head-processstage, it migrates anteriorly and laterally into the somatic layer of the lateral plate, ventral tothe limb-bud epithelium. Mesoderm which will form the anterior limb-bud migrates anteriorto mesoderm which will form the posterior limb-bud; mesoderm which will form the ventralportion of each limb-bud migrates posterolateral to mesoderm which will form the dorsalportion of each limb-bud.

INTRODUCTION

The limb-buds of the chick embryo begin to form at about the 30-somite stage,when the somatic mesoderm along the right and left flanks begins to accumulateinto mounds of mesenchyme, covered by a layer of epidermal ectoderm.

Although previous investigators (Wolff, 1936; Rudnick, 1945; Chaube,1959) have identified areas of limb-bud-forming tissue in the right and leftflanks of embryos prior to the 30-somite stage, the position of this tissue priorto the head-fold stage has not been investigated.

1 Author's address: Department of Pediatrics, The Johns Hopkins Hospital, Baltimore,Maryland 21205, U.S.A.

86 G. C. ROSENQUIST

Using radioautographic analysis the present investigation traces the movementof [3H]thymidine-labelled transplants from their original positions in the epi-blast, streak and endoderm-mesoderm layer of recipient embryos into the limb-buds of these embryos. Although the small number of grafts placed preventsexact definition of the prelimb areas, these regions are more precisely definedthan they were previously, and the mesoderm and ectoderm layers are treatedseparately.

MATERIALS AND METHODS

The methods of preparation of recipient and [3H]thymidine-labelled donorembryos and of transplantation and radioautographic analysis of the graftswere identical to those described in previous publications (Rosenquist, 1966,19706) and the description will not be repeated here.

The late medium-streak to head-process stages of both donor and host embryoshave been described previously (Rosenquist, 1970a) and are shown diagram-matically in Fig. 1A-C. Early head-fold (EHF) stage embryos had an elongatedhead process and a depression at the site of the future head fold, which had not asyet formed a pocket (Fig. 1D). Host embryos fixed prior to the 7-somite stagewere normal when examined with the naked eye or microscopically. In thosehost embryos which survived to the 26-somite to early limb-bud stage the heartwas beating, but extra-embryonic circulation had ceased and the development ofthe embryos was retarded compared to that of similar embryos incubated in ovo.The number of pairs of somites could not always be determined accurately(Fig. 2B, E).

In the older host embryos which had not as yet developed limb-buds, theanterior and posterior limb regions, and their dorsal and ventral portions, weredesignated in cross-sections on the basis of their distance from the anteriorintestinal portal, primitive streak, midline axis and nephrotome in comparisonwith embryos which had already developed limb-buds. This stage is referred toas the 26-30-somite stage in the Table and text, and is equivalent to stage 1(Hamburger, 1938) and stage 15 (Hamburger & Hamilton, 1951).

Host embryos which had developed limb-buds (stages 2-3 of Hamburger,1938; stages 16-17 of Hamburger & Hamilton, 1951) were said to be at the earlylimb-bud (ELB) stage. In these embryos the portion of the ectoderm and somaticmesoderm lateral to the crest of the ectodermal cone is referred to in the textand Table as the future ventral portion of the limb, while the portion of theectoderm and somatic mesoderm medial to the ectodermal cone is called thefuture dorsal portion of the limb.

Although a part of each of the labelled transplants lay in the limb-bud-forming region of its host embryo, the number of embryos investigated wasrelatively small, and each transplant contained cells other than those destinedfor the limb-buds (as indicated in Table 1). Therefore the positions of the trans-plants at each stage in Fig. 1 suggest the location of the limb-bud cells at thatstage, but do not define it precisely.

Origin of limb-bud 87

The mapping of the limb-bud regions is based upon the following assumptions:(1) that since previous studies have established the general position of the pre-mesoderm and pre-ectoderm cells in the epiblast layer of the chick blastodermwithout mapping every part of that layer at each stage (Rosenquist, 1966), asmall number of transplants carefully placed can demonstrate the position ofmore specific portions of the mesoderm and ectoderm, such as the limb-buds.(2) That similar graft positions in different embryos of the same stage are homo-logous even if the embryos were incubated for different lengths of time, and thatthe migration pathways followed by more than one accurately placed graft can becombined to follow movements of a group of cells through several overlappingstages of development. (3) That maps of presumptive organ-forming regions ofthe embryo are valid even if structures in the recipient embryos other than theorgan to be mapped contain labelled cells.

Throughout the text and figures, an asterisk (*) after the embryo numberindicates that the position shown is that of the graft after its migration in thehost embryo.

RESULTS

Limb-bud epithelium

At the late medium-streak stage the cells which would form the epithelium ofthe limb-buds were in the epiblast layer at the lateral edge of the area pellucida,lateral to the primitive streak; in some cases they may have been on or outsidethe boundary between the area pellucida and the area opaca (embryos 1-5,Table 1, Fig. 1A).

At the early head-fold stage, the cells which would form the epithelium of theanterior limb-bud were in the epiblast layer near the anterior half of the streak,about halfway between the streak and the lateral margin of the area pellucida(embryos 6, 7, Table 1, Fig. ID).

By the 4-7-somite stage, the transplant in embryo 6* had migrated anteriorlyand laterally to a position between the anterior end of the streak and the somiteregion (Table 1, Fig. 1E). If embryo 6 had been allowed to develop to the limb-bud stage, labelled cells would have migrated into the anterior limb-bud, as didthose in embryo 7* (Fig. ID, G). Therefore, the transplants in embryos l*-4*,which had reached positions similar to that of the graft in embryo 6* (Fig. 1E),also were considered to be destined for the anterior limb-bud. The transplant inembryo 5* and the posterior end of the transplant in embryo 3* were posteriorto the transplants in embryos l*-4* and 6* at this stage; although there is noevidence from the present study that they would have contributed to the posteriorlimb-bud, they lay in the presumptive posterior limb-bud region as it was definedby Rudnick (1945) and Chaube (1959) (Fig. 3C), and were therefore consideredto be destined for the epithelium of the posterior limb-bud.

At the early limb-bud stage (equivalent to stages 2-3 of Hamburger, 1938 and16-17 of Hamburger & Hamilton, 1951), labelled cells from the transplant in

88 G. C. ROSENQUIST

embryo 7* had formed portions of the epithelium of the right anterior limb-bud,extending along the dorsal side of the ectodermal cone; other labelled cellsfrom this transplant contributed to the epithelial layer of the embryo betweenthe anterior limb-bud and the neural crest, but not to the ectoderm between theanterior and posterior limb-buds, nor to the posterior limb-bud (Table 1,Figs. 1G, 2).

Table 1. Position of labelled cells in recipient embryos carryingtritiated thymidine-labelled grafts*

Embryono.

12345678

/ 9

110111213

I 1 411516

-17

Us1920212223

Stagegrafted

LMSLMSLMSLMSLMSEHFEHFDSHPHPEHFEHFEHFEHFEHFEHFEHFEHFEHFEHF4S4S5S

Incu-bated f

(h)

22232215197

6654

1048

10866

108686668684892566660

Stagefixed

5S7S5S6S6S7SELBEHFEHF4S26-30S26-30S26-30S26-30SELB26-30S26-30SELB26-30SELB26-30SELBELB

Position of labelled cells

Ec: ALBJEc: ALBJEc: ALBJEc: ALBJEc: PLBJEc: ALBJEc: ALB; Px MSt M: PLBJLP M: ALBJ, PLBJ; YS EnLP M: ALBJ, PLBJ; YS EnLPM: ALBJ (dv); YSEnLPM: ALBJ(v); YSEnLPM: ALBJ (d); YSEnLP M: ALBJ (dv), PLBJ (dv); YS EnLP M: ALB (v), PLB (v); YS EnLP M: ALBJ (d), PLBJ (d); YS EnLP M: ALBJ (d), PLBJ (d); YS EnLP M: ALB (dv), PLB (dv); YS EnLP M: ALBJ (d), PLBJ (d); YS EnLP M: ALB (v), PLB (v); YS EnLPM: ALBJ (dv); YSEnLPM: ALB (v); YSEnPx, LPM: PLB (d); YSM

* Key to abbreviations: ALB, anterior limb-bud; d, dorsal; DS, definitive-streak stage;Ec, ectoderm; EHF, early head-fold stage; ELB, early limb-bud stage; En, endoderm;HP, head-process stage; LMS, late medium-streak stage; LP, lateral plate; M, mesoderm;Px, paraxial; PLB, posterior limb-bud; S, somite; St, streak; v, ventral; YS, yolk sac.

t Hours of incubation after placement of the graft.J Presumptive limb-bud region.

Limb-bud mesoderm

At the definitive streak stage, the cells which would form the mesoderm of thelimb-buds were in the epiblast layer on each side of the primitive streak, at alevel about halfway between the anterior and posterior ends of the streak, andabout halfway from the streak to the lateral margin of the area pellucida. Thetransplant in embryo 8 (Table 1, Fig. IB) was within the area destined to be

Origin of limb-bud 89

invaginated at the streak, which was identified in a previous mapping study(Rosenquist, 1966).

By the head-process stage the mesoderm which would form portions of thelimb-buds had been invaginated at the primitive streak and had migrated intothe lateral plate mesoderm on each side of the streak, at a level about 40-50 %of the distance from the anterior to the posterior end of the streak (embryos 9,10, Table 1, Fig. 1C).

At the early head-fold stage, a portion of the mesoderm which would form

A(Ims)

D(ehf)

E (4-7s)

\ a

F (26-30s)

12*. 21"

11*, 14*, 16*-18*

Fig. 1. Drawings (ventral views) illustrating the position of thepresumptivelimb-budmaterial in the epiblast or mesoderm layer. The positions of [3H]thymidine-labelledgrafts (shaded areas) which contributed to the limb-bud epithelium are shown on theleft side of each figure. The positions of labelled grafts (shaded areas) which contri-buted to the limb-bud mesoderm are shown on the right side of each figure. Abbrevia-tions: Ims, late medium-streak stage; ds, definitive-streak stage; hp, head-processstage; ehf, early head-fold stage; s, somite; elb, early limb-bud stage.

As in the text, an asterisk (*) after the embryo number indicates that the positionshown is that of the graft after its migration in the host embryo. Each graft istherefore shown in two positions—immediately after it was placed, and afterincubation.

90 G. C. ROSENQUIST

ec

Origin of limb-bud 91the anterior limb-bud (embryos 9*, 11-13, Table 1, Fig. ID) had migratedanteriorly and laterally from the streak into the strip of somatic mesodermventral to the ectoderm which would form the anterior limb-bud (Rudnick,1945 and embryos 6 and 7, Fig. ID). Additional mesoderm which would formportions of the anterior limb-bud was located closer to the streak (embryos14-20, Table 1; Fig. 1D). Mesoderm which would form the posterior limb-budwas located in the streak or in the nearby mesoderm (embryos 14-20). Mesodermwhich would form the ventral portion of each limb was positioned moreposteriorly in the streak and more posterolaterally in the lateral plate mesoderm(embryos 12, 19, 20, Table 1, Fig. ID) than was mesoderm which would formthe dorsal portions of the limbs (embryos 13, 16-18, Table 1, Fig. ID). Thetransplants in embryos 11,14 and 15 were placed in an intermediate position andcontributed to both dorsal and ventral portions of the somatic layer of meso-derm or the limbs (Table 1, Fig. ID).

At the 4-7-somite stage, the limb-bud mesoderm had migrated further intomore lateral and anterior portions of the lateral plate. The transplants in embryos10*, 21 and 22 indicated the position of the mesoderm which would form theanterior limb-bud, which was in the portion of the embryo identified as destinedfor the anterior limb-bud by Rudnick (1945), Chaube (1959), and by theectodermal grafts in the present study (embryos l*-4*, 6*, 7*, Table 1, Fig. 1D,E, G). The anterior end of this zone was about at the level of the last somitewhich had formed; the posterior end of this zone was anterior to the anterior

Fig. 2. (A) [3H]thymidine-labelled transplant (arrow) was placed in the epiblast layerof an early head-fold stage recipient embryo (embryo 7).(B) After 66 additional h of incubation, the recipient embryo had a well-developedheart. Cellsfromthe transplant formed the dorsal epithelium of theanterior limb-bud.The plane of the cross-section in (C) is shown by the dotted line at the lower right.s — sinoatrium; v = ventricle.(C) Cross-section (x200) through embryo 7 at the plane indicated in (B), illus-trating how the radioautographed cells from the transplant (black grains overnuclei, arrows) formed the epithelium of the trunk of the embryo, extending intothe medial (dorsal) portion of the anterior limb-bud, d — dorsal; ec = ectodermalcone; v = ventral.(D) A [3H]thymidine-labelled transplant (arrow) was placed in the endoderm-mesoderm layer and primitive streak of an early head-fold stage recipient embryo(embryo 20).(E) After 92 additional h of incubation, the recipient embryo had a well-developedheart (Ji) and both anterior (alb) and posterior limb-buds (plb), but was otherwisepoorly developed compared to embryos incubated in ovo. Cells from the transplantcontributed to the mesenchyme of both anterior and posterior limb-buds. The planeof the cross-section in (F) is indicated by the dotted line through the posterior limb-bud.(F) Cross-section (x200) through embryo 20 at the level shown in (E). The radio-autographed cells from the transplant (black grains over nuclei, arrows) contri-buted to the posterior limb-bud mesoderm, which at this level had formed from thesomatic layer of the lateral plate, d = dorsal; ec = ectodermal cone; v = ventral.

92 G. C. ROSENQUIST

end of the streak. The transplant which later formed the mesoderm of theposterior limb-bud was more posteriorly positioned; it was adjacent to theprimitive streak, and extended anteriorly and laterally into the lateral platemesoderm (embryo 23, Table 1, Fig. IE, G). This position corresponded withthe areas previously identified as destined for the posterior limb-bud by Rudnick(1945), Chaube (1959), and an ectodermal graft of the present investigation(embryo 5*, Table 1, Fig. IE). The transplant in embryo 22 contributed to theventral portion of the anterior limb-bud mesoderm and was positioned slightlylateral to the transplant in embryo 21, which would have contributed to bothdorsal and ventral portions of the limb-bud mesoderm. It was in a migrationpathway lateral to the pathway which is believed would be followed by thetransplant which contributed to the dorsal portion of the posterior limb-budmesoderm (embryo 23).

At the 26-30-somite stage (equivalent to stage 1 of Hamburger, 1938, and 15 ofHamburger & Hamilton, 1951), the cells which would form the mesoderm of theanterior limb-bud were located in the lateral portion of the somatic layer of thelateral plate, posterior to the anterior intestinal portal (embryos 12*, 21*,Table 1, Fig. IF). Cells which would form the mesoderm of the posterior limb-bud were even more posteriorly located in the lateral part of the somatic part ofthe mesoderm (embryos 11*, 14*, 16*—18*, Table 1, Fig. IF).

At the early limb-bud stage, the transplants in embryos 13*, 15*, 19*, 20*and 22* all had formed portions of the anterior limb-bud mesoderm (Table 1,Fig. 1G). Of these only the transplants in embryos 22* and 13* did not alsoform portions of the posterior limb-bud mesoderm; they were among the moreanteriorly positioned transplants at the early head-fold and 4-7-somite stages,respectively (Fig. ID, E). The transplant in embryo 23* formed the dorsalportion of the posterior limb-bud mesoderm; it was the most posteriorlypositioned transplant at the 4-7-somite stage (Fig. 1E).

DISCUSSION

Previous attempts to learn the origin and movements of the cells which formthe limbs have utilized several different experimental techniques. Wolff (1936)produced localized injury withX-irradiation to embryos asyoungas9-13-somites,and proposed that the anterior limb-bud regions were at the level of the anteriorend of the rhomboid sinus (i.e. posterior to the last somite), while the posteriorlimb-bud regions were close to the midline and posterior to the anterior end ofthe streak. Rudnick (1945) divided chick blastoderms into full-thickness piecesof various sizes, and transplanted them into the right coelomic space of hostembryos incubated 2\ days; after 8-10 additional days of incubation in thisenvironment, the fragments differentiated into morphologically recognizablelimbs. Using this method, Rudnick demonstrated that the anterior limb-budswould differentiate from a strip of tissue which extended laterally and posteriorly

Origin of limb-bud 93

from the anterior end of the streak at the head-fold stage (Fig. 3 A), and from astrip of tissue which extended laterally from the anterior end of the streaktoward the lateral margin of the area pellucida at the 6-7-somite stage (Fig. 3C).Rudnick did not find posterior limb-bud material at the head-fold stage; how-ever, in 6-7-somite stage embryos, she did find such material slightly posterior tothe prospective anterior limb-bud material, lateral to the anterior end of thestreak (Fig. 3C). Rudnick and Wolff both noted a gradual anterior movement ofboth anterior and posterior limb-bud regions with additional development, butthe presumptive anterior limb-bud remained anterior to the presumptive

A0>f)

Fig. 3. Diagrams illustrating the position of the limb-bud material as determined byprevious investigators. Left side of each figure: Rudnick, 1945, using coelomic graft-ing techniques. Right side: Chaube, 1959, using chalk particles in ovo. Hatchedarea = anterior limb-bud material; shaded area = posterior limb-bud material./;/= head-fold stage; s = somite.

posterior limb-bud in all their preparations. In no instance did limbs differentiatefrom material which originated at a level anterior to the last somite which haddeveloped.

One disadvantage of the studies with grafted blastoderm fragments in theembryonic coelom was that the mesoderm and ectoderm layers contained in thefragments could no longer move independently of each other as they might havedone in the uncut blastoderm. The in ovo studies of Chaube (1959) overcamethis difficulty. With chalk particles on glass needles introduced from the dorsalsurface, Chaube marked adjacent points in the ectoderm and mesoderm layers,beginning at the 2-somite stage. The movement of the chalk particles during thesubsequent development of the embryos showed that although both layers

94 G. C. ROSENQUIST

expanded in an anteroposterior direction and moved anteriorly in relation to thestreak, the points in each layer which were marked with chalk remained adjacentto each other. Chaube thus agreed with Rudnick that the prospective limbmesoderm had completed its gastrulation movements and had reached itsdefinitive position in relation to the ectoderm by the 2-somite stage. Chaube alsonoted stable right-to-left relationships: laterally placed marks assumed ventralpositions in the limb-buds while medially placed marks assumed dorsal positionsin the limb-buds.

Both Rudnick and Chaube attempted to study the limb regions of youngerembryos. Although Rudnick (1945) transplanted portions of embryos as youngas the primitive-streak stage into coelomic cultures, limbs did not form fromfragments excised before the early head-fold stage. The present study indicatesthat at the early stages studied by Rudnick, the prelimb ectoderm is in astable position in the epiblast (ectoderm); however, the prelimb mesodermwhich has been invaginated at this stage has not as yet reached a position directlyventral to the prelimb ectoderm, so that the stable relationship between thetwo which Rudnick found at all later stages had not as yet developed.

Chaube marked a total of thirty-five head-fold stage embryos; a large numberdied soon after they were marked and the remainder 'gave such bizarre results'that they could not be satisfactorily interpreted. From the present study it isclear that at the head-fold stage some of the prelimb mesoderm cells may still bein the epiblast layer, others are in the process of invaginating at the streak, andstill others are migrating anteriorly and laterally from the streak toward theprelimb ectoderm regions. Chalk marks placed in such an environment wouldindeed give bizarre results since only some of the prelimb ectoderm and meso-derm cells have reached a stable position in relation to each other.

The present study indicates that the migration of the prelimb mesoderm is asorderly and highly organized as that noted for mesoderm destined for the splan-chnic layer (Rosenquist, 1970a). After its imagination at the streak, mesodermdestined for the dorsal portion of each limb is anterior to mesoderm destinedfor the ventral portion of each limb. This suggests (but does not prove) thatequally organized relationships exist in the epiblast prior to invagination at thestreak. Premesoderm destined for the dorsal portion of each limb may beanteromedial to premesoderm destined for the ventral portion of each limb.Since the ectoderm which will form the limbs is probably similarly arranged, wemay consider the limb regions of the epiblast as consisting of two parallelbands: the more anteromedial band contains the ectoderm and mesoderm cellswhich will form the dorsal body wall, including the dorsal portion of each limb,while the more posterolateral band contains the ectoderm and mesoderm cellswhich will form the ventral body wall, including the ventral portion of eachlimb. The premesoderm is nearer to the streak than is the pre-ectoderm; itmigrates to a position below the ectoderm, and then the ectoderm and mesodermmigrate together in their respective bands. Such a concept may also apply to the

Origin of limb-bud 9 5

portions of the epiblast destined to form ectoderm and mesoderm of moreproximal and distal portions of the embryo. In future research it should bepossible to place smaller transplants into the epiblast layer at these early stages,and incubate the host embryos to later stages, so that further details of thesemovements may be learned.

RESUME

Uorigine et les mouvements de Vepithelium et du mesenchyme dans le bourgeonde membre du Poulet demontres par le marquage radioautographique

L'origine des cellules du bourgeon de membre a ete mise en evidence en suivant les mouve-ments de greffes marquees par la [3H]thymidine, excisees d'embryons de Poulet aux stadesdepuis la ligne primitive moyenne et avanceejusqu'a celui de 5 somites et transplants dansl'epiblaste, la ligne primitive et l'endo-mesoderme d'embryons notes d'age correspondant.

Malgre qu'une definition exacte des aires presomptives des membres n'ait pas ete possiblevu le nombre limite de greffes executees a chacun des stades, l'etude a pu demontrer de facongenerate qu'au stade de la ligne primitive moyenne et avancee l'epithelium du futur bourgeonde membre se place dans la couche epiblastique (dorsale) proche de la marge laterale de1'area pellucida. II se meut en dedans en direction de l'axe embryonnaire, pour se placer toutjuste en dehors des cellules pre-mesodermiques qui vont s'invaginer dans la ligne primitive.Au cours de la regression de la ligne primitive, l'epithelium du membre se deplace relative-ment vers l'avant, atteignant une position dorsale vis-a-vis du mesoderme de ce membre, etceci au moins depuis le stade du premier repli cephalique.

Au stade de la ligne primitive a son terme, le mesoderme presomptif du bourgeon de membrese trouve dans la couche epiblastique a peu pres a mi-chemin entre la ligne primitiveet la marge laterale de l'area pellucida, et a mi-distance entre les extremites anterieureet posterieure de la ligne primitive. A partir de cette position le mesoderme presomptifdu membre migre vers le dedans et s'invagine dans la ligne primitive a mi-distancede ses deux extremites (anterieure et posterieure); apres le stade du prolongement cephalique,il migre vers l'avant et le dehors dans la couche parietopleurale de la lame laterale,ventralement par rapport a l'epithelium correspondant. Le mesoderme qui formera lemembre anterieur migre en avant de celui qui formera le membre posterieur; le mesodermequi formera la partie ventrale de chaque membre migre en arriere et en dehors de celui quiformera la partie dorsale.

This investigation was supported by USPHS research grants HE 10191 and K3 HE 20074from the National Heart Institute. The author wishes to thank James D. Ebert for hiscontinued interest in this research, Soame D. Christianson for help in the preparation of themanuscript, and Dorothea Rudnick for a critical reading of the manuscript.

REFERENCES

CHAUBE, S. (1959). On axiation and symmetry in transplanted wing of the chick. / . exp. Zool.140, 29-77.

HAMBURGER, V. (1938). Morphogenetic and axial self-differentiation of transplanted limbprimordia of 2-day chick embryos. J. exp. Zool. 77, 379-399.

HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development ofthe chick embryo. /. Morph. 88, 49-92.

ROSENQUIST, G. C. (1966). A radioautographic study of labeled grafts in the chick blastoderm.Development from primitive streak stages to stage 12. Carnegie Instn. Wash. Publ. No. 625,Contr. Embryol. 38, 71-110.

ROSENQUIST, G. C. (1970a). Cardiogenesis in the chick embryo: topology of the precardiacregion from early streak stages until heart formation. Devi Biol. 22, 461-475.

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ROSENQUIST, G. C. (19706). The origin and movement of nephrogenic cells in the chick embryoas determined by radioautographic mapping. / . Embryol. exp. Morph. 24, 367-380.

RUDNICK, D. (1945). Limb-forming potencies of the chick blastoderm: including notes onassociated trunk structures. Trans. Conn. Acad. Arts Sci. 36, 353-377.

WOLFF, E. (1936). Les bases de la teratogenese experimentale des Vertebres amniotes, d'apresles resultats de methodes directes. Arc/is Anat. Histol. Embryol. 22, 1-382.

(Manuscript received 26 June 1970)