/ . Embryo!. exp. Morph. Vol. 24, 3, pp. 455-466, 1970 4 5 5

Printed in Great Britain

A radioautographic analysisof the migration and fate of cells derived fromthe occipital somites in the chick embryo with

specific reference to the development ofthe hypoglossal musculature

By R. D. HAZELTON1

From the Research Division, Faculty of Dentistry, University of Toronto

SUMMARYThe migration pattern and fate of cells of the occipital somites and overlying ectoderm have

been described for the chick embryo with particular reference to the development of thehypoglossal musculature.

Tritium-labelled thymidine (0-5-10 /tCi per egg) was used as a cell-specific marker. Occi-pital somites (2-5) with overlying ectoderm were transplanted orthotopically from labelleddonor embryos to unlabelled host embryos (Hamburger & Hamilton, stage 9-10). The embryoswere incubated, for varying lengths of time (24 h-5 days), sacrificed, sectioned and the migra-tion pattern and fate of the labelled cells determined radioautographically.

It appears that the hypoglossal as well as other hypopharyngeal musculature originatesfrom the occipital somites.

The mesodermal migration pattern extended from the occipital somite region in a ventro-posterior direction to the dorsal surface of the pericardial cavity posterior to the expandedportion of the pharynx. At this position a so-called hypoglossal cord formed on each sidewhich ran anteriorly to the level of the second pharyngeal pouch where it turned medially andtogether with the cord from the other side entered the pharyngeal area of the embryo. Thismaterial apparently forms the intrinsic musculature of the tongue. The mesodermal move-ments are attributed to differential growth movements of the areas concerned as well as toactive cell mutiplication and migration.

Selective embryonic neuronal staining was undertaken to study the relationship between themigrating hypoglossal cord and nerve. The cord preceded the nerve in its migration.

There is an occipital somitic contribution to the primitive meninx, to the endothelial wallsof developing blood vessels, possibly to microglial cells and to the cartilage surrounding thenotocord.

The occipital ectoderm expands dorso-anteriorly and ventro-laterally. In the ventro-lateralposition as contact is made with the pharyngeal endoderm a placode is formed which contri-butes cells to the nodose ganglion of the tenth cranial nerve. There is no other contributionof the ectoderm to the underlying tissues.

1 Author's address: Apartment 4, 101 Hazelton Avenue, Toronto 185, Ontario, Canada.

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456 R. D. HAZELTON

INTRODUCTION

Morphological approaches used in distinguishing similar embryonic cellgroups have led to difficulties in determining the origin of the hypoglossalmusculature from either somitic or local mesoderm. Experiments using carbonparticle marking techniques by Deuchar (1958) indicated a somitic origin, butthe introduction of cell-specific markers such as tritiated thymidine, has made amore critical and extensive experimental analysis possible, using the techniquesdeveloped by Weston (1962, 1963), Weston & Butler (1966), Chibon (1962,1964, 1967), Johnston (1966), Hazelton & Johnston (1968) and Langman &Nelson (1968). In the present series of experiments the occipital somites andoverlying ectoderm were transplanted from embryos labelled with tritiatedthymidine to unlabelled host embryos in order to determine the migration patternand fate of the labelled cells.

MATERIALS AND METHODS

Handling of eggs

The eggs employed in this study were from the white leghorn strain of fowl,Gallus domesticus. They were incubated first on their side for approximately30 h by which time most of the embryos had reached stages of developmentimmediately preceding or just following the onset of somite formation (Ham-burger & Hamilton, stage 6-8). They were then prepared for labelling withtritiated thymidine. After transferring the air space to a position overlying theembryo, a piece of shell with underlying shell membrane approximately 1 cmsquare was removed to provide a 'window' for access to the embryo.

Tritiated thymidine, in saline solution, was deposited on to the vitelline mem-brane over the embryo. The window was then covered with plastic tape and theegg returned to the incubator. At least 3-4 h elapsed before transplantations ofthe occipital somites and overlying ectoderm to unlabelled host embryos werecarried out. The dose of thymidine used varied between 0-1 and

Operative technique

The second to fourth or fifth occipital somites plus the overlying ectodermwere removed as a single entity from stage 8-9 unlabelled host embryos andreplaced with comparable segments from the donor embryos labelled withtritiated thymidine (Figs. 1,2). The ectoderm was included in the grafts to holdthe somites together and facilitate their orientation. The access hole in the vitel-line membrane was kept as small as possible. The grafts were cut out with theuse of glass needles and transferred by pipette from the donor to the hostembryos.

Fate of occipital somites 457

Section planefor figure 2.

Opticvesicle

Occipitalsomite no. 2

Transplanted 2, 3 and 4occipital somitesand overlying ectoderm

Fig. 1. Stage 9 chick embryo (Hamburger & Hamilton, 1951) to show thetransplanted area of occipital somites and overlying ectoderm (cross-hatch).

Myotome

Dermatome

Sclerotome

Labelled occipital somiteand overlying ectoderm

Somatic mesoderm

Splanchnic mesoderm

Fig. 2. Section of Fig. 1 to illustrate the various parts of somite as well astransplanted segment of somite and overlying ectoderm (cross-hatch).

Fixation of embryos and preparation of radioautographs

Postoperative incubation periods lasted from 15 min until 5 days, with theoldest embryos reaching approximately stage 30 at the time of fixation. A totalof 200 experiments were carried out, of which 30 were successful. The embryos

29-2

458 R. D. HAZELTON

were fixed in either Bouin's fixative (young embryos) or in 10% bufferedformalin (old embryos). Sections were cut at 8 /.i and radioautographs wereprepared using essentially the procedure of W. G. Aldridge (see Gerber,Aldridge, Koszalka & Gerber, 1962).

Since the original series of transplants involving both the occipital somitesand overlying ectoderm were completed, further experiments involving thetransplant of placodal ectoderm alone have been undertaken. By transplantingonly the ectoderm any contamination from the ectoderm to underlying tissueswas determined.

Hypoglossal nerve

and cordOtic vesicle

Mandibulararch

Fig. 3. A stage 38 chick embryo (Hamburger & Hamilton, 1951) to illustratethe migration pattern of the occipital somites (cross-hatch).

RESULTS

One of the main problems of this type of experimental approach was thedifficulty in keeping the embryos in a viable state following the transplantingprocedures. In order to reduce the fatality rate it is recommended that goodquality eggs be obtained and a high relative humidity (70-80%) maintainedthroughout all operative and incubatory procedures. There were seasonalvariations in the success of operations, with the spring and fall being the mostproductive and the summer and winter least productive.

Fate of occipital somites 459

FIGURE 4

(A) Photomicrograph of a section through the pharyngeal region of a stage 25chick embryo. Hypoglossal cord is illustrated in box. x 100.(B) Photomicrograph using reflected light of the hypoglossal cord designated inbox of Fig. A. Because of the use of reflected light the labelled cells are white and areindicated by arrows, x 400.(C) Photomicrograph through the oral cavity of a stage 30 chick embryo. Thehypoglossal cords of each side have now fused and form a V (arrow), x 100.(D) Higher-power photomicrograph of combined hypoglossal cords seen in C.Labelled cells are indicated by arrows. x400.

460 R. D. HAZELTON

Migration pattern of the occipital somites

The overall migration pattern of the occipital somites and overlying ectodermis illustrated in Fig. 3. An active proliferation and ventro-lateral migration ofcells brings the myotomic portion of the somite to the dorso-lateral surface ofthe pericardial cavity posterior to the expanded portion of the pharynx. Atthis position a so-called hypoglossal cord forms (Fig. 3, Fig. 4A, B) whichruns forward to the level of the second pharyngeal pouch where it turns mediallyinto the floor of the pharynx and after further forward movement unites with thecord of the opposite side to form a V (Fig. 4C, D) and it is in this fused state

FIGURE 5

(A) Photomicrograph passing through the posterior pharyngeal (P) region of a stage30 chick embryo. Labelled cells can be observed lateral to the laryngeal-trachealgroove (L) and are illustrated by a box. x 100.(B) Photomicrograph of labelled somite cells (arrows) seen in box of A. These cellswill develop into myoblasts of the larynx, x 400.(C) High-power photomicrograph of a section through the neural tube (N) andprimitive meninx (P) of a stage 25 chick embryo. Labelled cells may be seen in theprimitive meninx (arrows), x 400.(D) Photomicrograph of labelled cells (arrows) in the endothelial lining of theanterior cardinal vein (AV). x 400.

Fate of occipital somites 461

that the final differentiation into the intrinsic musculature of the tongue occurs.The migration of the occipital material is accompanied by the hypoglossal

nerve.As the myotomic material sweeps ventrally to form the hypoglossal cords,

some myotomic cells migrate to a ventro-lateral pharyngeal position in theregion of the laryngo-tracheal tube to form laryngeal musculature (Fig. 5 A, B).

FIGURE 6

(A) Photomicrograph of a section through the developing notochord (N) of a stage32 chick embryo. Labelled cell within the cartilage matrix (M) is indicated by anarrow. x400.(B) Photomicrograph taken through the posterior part of the pharynx (P) of astage 22 chick embryo to indicate relationship of the hypoglossal nerve (N) to thehypoglossal cord (H). The embryo had been stained with silver, x 100.(C) Photomicrograph of a section through the developing nodose ganglion (N)of a stage 21 chick embryo. Placodal cells (P) are streaming in toward the develop-ing ganglion, x 400.

The contribution of the occipital somites to the primitive meninx (Fig. 5C)adds to the confusion regarding a mesodermal or neural crest origin of this tissue(Harvey & Burr, 1926; Harvey, Burr & van Campenhout, 1933; Raven, 1936;Flexner, 1929; Sensenig, 1951; Triplett, 1958; Johnston, 1966).

Labelled cells were observed entering the substance of the brain just posterior

462 R. D. HAZELTON

to the otic vesicle. These could be microglial cells (Penfield, 1932; Kershmann,1939) or simply endothelial cells of developing blood vessels. Labelled cells werefound in the endothelium of the internal carotid artery, aortic arches, the basilarartery and the anterior cardinal vein (Fig. 5D).

The sclerotomic portion of the somites contributed to the cartilage matrixsurrounding the notocord (Fig. 6 A).

The occipital ectoderm undergoes an antero-dorsal and postero-ventralexpansion and in the postero-ventral region, as contact is made with thepharyngeal endoderm, placodes form which eventually contribute to the nodoseganglion of the tenth cranial nerve (Fig. 6C). This is the only ectodermal contri-bution to the underlying tissues.

Silver staining procedures were used as an additional technique to study themigratory relationship between the hypoglossal nerve and cord by selectiveneuronal staining and it was established that the cord precedes the nerve(Fig. 6B).

DISCUSSION

The somites are among the earliest morphological units to be formed (Hinsch& Hamilton, 1956) and appear in a regular sequential manner from the otocystto the tip of the tail. A central space divides the somite into an inner and outerregion (Fig. 2). Different fates have been attributed to these two regions withthe outer region forming the connective tissue layer of the skin (dermatome)and the inner region forming both skelatogenous (sclerotome) and voluntarymuscle tissue (myotome). However, the absolute origin of the somite parts is indispute with the myotomic portion being the main contributor to the confusion.Although muscle protein has been demonstrated in the myotome by bothimmunofluorescent studies (Holtzer, Marshall & Finck, 1957; Ozawa, 1962;Ikeda, Abbott & Langman, 1968) and by electron-microscopic identification ofmyofilaments (Przybylski & Blumberg, 1966; Allen & Pepe, 1965; Dessouky &Hibbs, 1965; Obinata, Yamamoto & Maruyama, 1966), the definitive origin ofthe myotome has not been established: Williams (1910) and Hamilton, Boyd &Mossman (1962) state that the myotome originates by a multiplication of cells atthe dorso-medial angle of the somite while Remak (1855), His (1888), Bardeen(1900) and Langman & Nelson (1968) state that the myotome develops by pro-liferation and differentiation of the overlying dermatome.

By using the electron microscope the myotomic portion of early chickembryos can be identified from the other somite parts by the larger size andlighter colour of its cells (Hay, 1968).

The first post-otic somite is rudimentary (Patterson, 1907; Hinsch &Hamilton, 1956; Arey, 1938) with the second to fifth making up the occipitalseries (Kingsbury, 1915; Hunter, 1935; Goodrich, 1936).

The majority of evidence for either a somitic or local mesodermal origin of thehypoglossal musculature has been of a descriptive nature. Platt (1891), Neal(1897), Kallius (1905), Hunter (1935) and Bates (1948) stated that the hypoglossal

Fate of occipital somites 463

musculature developed by a ventral and anterior migration of the occipitalsomites whereas Lewis (1910) concluded that the hypoglossal musculaturedeveloped locally from the mesoderm of the floor of the mouth.

The few experimental studies undertaken have not been entirely conclusive.By extirpating the occipital somites in Amblystoma Detwiler (1929, 1937)observed an absence of the hypoglossal musculature. In a series of carbon mark-ing experiments in the chick Deuchar (1958) concluded the hypoglossal muscula-ture originated from the occipital somites. Although Deuchar's results tendedto confirm earlier descriptive studies, some objections may be made to thetechnique: the carbon particles could have been carried to the hypoglossalmusculature by the movements of associated parts or the carbon could have beenengulfed by phagocytic cells which in turn migrated to the hypoglossal area(Straus & Rawles, 1953; Seno, 1961). However, the present experiments con-firm and extend Deuchar's findings.

In order to determine the migration and fate of similar embryonic cell groupsone must first establish their identity. This has been done in the present seriesof experiments by specifically marking groups of cells (e.g. occipital somites)with tritiated thymidine. These tissue segments were then transplanted to un-labelled host embryos and the migration and fate of the labelled cells determinedradioautographically.

The organization of the occipital somites into so-called hypoglossal cords andthe migration of these cords into the hypopharyngeal region has been describedby several investigators (Hunter, 1935; Bates, 1948; Detwiler, 1955; Kallius,1905). Although differential growth movements of involved and associatedareas are important in the movement of this material, other factors such as cellproliferation and migration must be considered. If no further cell proliferationoccurs once the myotome has formed (Langman & Nelson, 1968) other processesmust account for the final adult population of cells. This could be attributed tothe hypoglossal cord containing undifferentiated cells which form myotubesonly when a final destination is established or the hypoglossal material couldinduce the differentiation of myotubes from local mesoderm.

The role of the hypoglossal nerve in the development of the hypoglossal cordmust also be considered. By selectively staining embryonic tissue for neuralelements the relationship between the nerve and cord was studied, and it wasshown that the muscle cord precedes the nerve along its migratory path. Thesame relationship was observed between the sensory nerves of the visceral archesand their muscle cords.

The expansive movements of the ectoderm in the chick were similar to thosefound in Amblystoma by Wilens (1959) and are due to the morphogenetic move-ments of embryonic parts. The most significant ectodermal finding was itscontribution to the nodose ganglion of the vagus nerve. This finding led to aninvestigation of the entire sensory cranial ganglion system, the results of whichwill be published in the future.

464 R. D. HAZELTON

RESUME

Analyse radioautographique de la migration et du devenir des cellulesderivees des somites occipitaux chez Vembryon de Poulet ense referant au developpement de la musculature hypoglosse

Le schema de la migration et le devenir des cellules des somites occipitaux et de l'ectodermesusjacent sont decrits chez l'embryon de Poulet en se referant au developpement de la muscula-ture hypoglosse.

On utilise la thimidine tritiee (0,5-10/*Ci par oeuf) comme marqueur speciflque des cellules.Les somites occipitaux (2-5) et l'ectoderme susjacent sont transplanted, de facon orthotopique,des embryons donneurs marques aux embryons hotes non marques (Hamburger & Hamilton,stade 9-10). Les embryons sont incubes pendant des periodes de temps variables (24 h a5 jours), sacrifies et sectionnes. On determine de facon radioautographique la migration et ledevenir des cellules marquees.

II apparait que la musculature hypoglosse, comme toute musculature hypopharyngienne,a pour origine les somites occipitaux.

La migration mesodermique s'etend depuis la region somitique occipitale en directionpostero-ventrale jusqu'a la surface dorsale de la cavite pericardique posterieure a la portionrenflee du pharynx. A cet endroit un cordon, appele cordon hypoglosse, se forme de chaquecote; ce cordon s'etend anterieurement au niveau de la seconde poche pharyngienne; la,avec le cordon de l'autre cote, il penetre dans l'aire pharyngienne de l'embryon. Ce materielforme apparemment la musculature intrinseque de la langue. Les mouvements mesodermiquessont aussi bien attribues aux mouvements de croissance differentielle des aires concerneesqu'a l'active multiplication cellulaire et a la migration.

On utilise la coloration neuronale embryonnaire selective pour etudier le rapport entre lamigration du cordon et du nerf hypoglosses. Le cordon precede le nerf dans sa migration.

II y a une contribution somitique occipitale a la meninge primaire, aux parois endothelialesdes vaisseaux sanguins en developpement, et, peut-etre, aux cellules microgliales et aucartilage entourant la notocorde.

The author wishes to thank Dr M. C. Johnston for his invaluable guidance both in theexperimental procedures and in the preparation of the manuscript.

REFERENCES

ALLEN, E. R. & PEPE, F. A. (1965). Ultrastructure of developing muscle cells in the chickembryo. Am. J. Anat. 116, 115-148.

AREY, L. E. (1938). The history of the first somite in human embryos. Contr. Embryol. 168,233-269.

BARDEEN, C. R. (1900). The development of the musculature of the body wall on the pig,including its histogenesis and its relation to the myotomes and to the skeletal and nervousapparatus. Johns Hopkins Hosp. Rep. 9, 367-399.

BATES, M. N. (1948). The early development of the hypoglossal musculature in the cat. Am. J.Anat. 83, 329-357.

CHIBON, P. (1962). Etude des cellules migrations de la crete neurale chez Amblystoma mexi-camim Shaw et Pleurodeles waltlii Michah. Bull. Soc. zool. Fr. 87 (Abstract).

CHIBON, P. (1964). Analyse par la methode marquage nucleaire a la thymidine tritiee desderives de la crete neurale cephalique chez l'urodele Pleurodeles waltlii Michah. C.r. hebd.Seanc. Acad. Sci., Paris 259, 3624-3637.

CHIBON, P. (1967). Marquage nucleaire par la thymidine tritiee des derives de la crete neuralechez l'Amphibien Urodele Pleurodeles waltlii Michah. / . Embryol. exp. Morph. 18,343-358.

DESSOUKY, D. A. & HIBBS, R. G. (1965). An electron microscope study of the developmentof the somatic muscle of the chick embryo. Am. J. Anat. 116, 523-566.

Fate of occipital somites 465DETWILER, S. R. (1929) The development of the spinal cord in Amblystoma embryos follow-

ing unilateral myotomectomy. /. exp. Zool. 52, 325-349.DETWILER, S. R. (1937). Observations upon the migration of neural crest cells, and upon the

development of the spinal ganglia and vertebral arches in Amblystoma. Am. J. Anat. 61,63-94.

DETWILER, S. R. (1955). Experiments on the origin of the ventro-lateral trunk musculaturein the Urodele {Amblystoma). J. exp. Zool. 129, 45-75.

DEUCHAR, E. M. (1958). Experimental demonstration of tongue muscle origin in chickembryos. / . Embryol. exp. Morph. 6, 527-529.

FLEXNER, L. B. (1929). The development of the meninges in Amphibia: a study of normaland experimental animals. Contr. Embryol. 20, 31.

GERBER, G. B., ALDRIDGE, W. G., KOSZALKA, T. R. & GERBER, G. (1962). Biochemical andautoradiographic studies on DNA metabolism in vitamin E-deficient Hamster. / . Nutr.78, 397-319.

GOODRICH, E. S. (1936). Studies on the Structure and Development of Vertebrates. London:Macmillan.

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

HAMILTON, J. W., BOYD, J. D. & MOSSMAN, H. W. (1962). Human Embryology. Cambridge:W. Heffer and Sons.

HARVEY, S. C. & BURR, H. S. (1926). The development of the meninges. Archs Neurol.Psychiat. Chicago 15, 545-567.

HARVEY, S. C, BURR, H. S. & VAN CAMPENHOUT, E. (1933). Development of the meninges.Further experiments. Archs Neurol. Psychiat. Chicago 29, 683-690.

HAY, E. D. (1968). Organization and fine structure of epithelium and mesenchyme in thedeveloping chick embryo. Chapter 2. Epithelial Mesenchymal Interactions (ed. R. Fleisch-majer & R. E. Billingham). Baltimore: Williams and Wilkins.

HAZELTON, R. D. & JOHNSTON, M. C. (1968). A radiographic study of the contributions ofneural crest and placodal ectoderm to the sensory cranial ganglia of chick and salamanderembryos. Anat. Rec. 160, 363 (Abstract).

HINSCH, C. W. & HAMILTON, H.L. (1956). The developmental fate of the first somite in chick.Anat. Rec. 125, 225-246.

His, W. (.1888). Untersuchungen iiber die erste Anlage des Wirbeltierleibes. Die ersteEntwicklung des Hunchens im Ei. Bd 16, p. 237. Leipzig: Vogel.

HoLTZER, H., MARSHALL, J. M. & FJNCK, H. (1957). Analysis of myogenesis by the use offluorescent antimyosin. / . biophys. biochem. Cytol. 3, 705-724.

HUNTER. R. M. (1935). The development of the anterior postotic somites in the rabbit. J.Morph. 51, 501-531.

IKEDA, A., ABBOTT, R. L. & LANGMAN, J. (1968). Muscle proteins in the chick myotomeexamined by the immunofluorescent method. / . Embryol. exp. Morph. 19, 193-202.

JOHNSTON, M. C. (1966). A radioautographic study of the migration and fate of cranial neuralcrest cells in the chick embryo. Anat. Rec. 156, 142-156.

KALLIUS, E. (1905). Beitrage zur Entwicklung der Zunge. II. Vogel Anat. Hefte, Abt. 1 28,301-586.

KERSHMANN, J. (1939). Genesis of microglia in the human brain. Archs Neurol. Psychiat.Chicago 41, 24.

KINGSBURY, B. F. (1915). The development of the human pharynx. I. The pharyngeal de-rivatives. Am. J. Anat. 18, 329-397.

LANGMAN, J. & NELSON, G. R. (1968). A radioautographic study of the development of thesomite in the chick embryo. / . Embryol. exp. Morph. 19, 217-226.

LEWIS, N. H. (1910). Chapter 12. Development of the Muscular System in Human Embryology(ed. Keibel & Mall). Philadelphia: J. B. Lippincott.

NEAL, H. V. (1897). The development of the hypoglossus musculature in Petromyzium andSgualis. Anat. Anz. piger 13, 441-463.

466 R. D. HAZELTON

OBINATA, T., YAMAMOTO, M. & MARUYAMA, K. (1966). The identification of randomly formedthin filaments in differentiating muscle cells of the chick embryo. Devi Biol. 14, 192-213.

OZAWA, Y. (1962). Synthesis of skeletal muscle proteins in early embryos and regeneratingtissue of chick and Triturus. Expl Cell Res. 26, 269-274.

PATTERSON, J. T. (1907). The order of appearance of the anterior somites in the chick.Biol. Bull. mar. biol. Lab., Woods Hole 13, 121-133.

PENFIELD, W. (1932). Neuroglia and microglia. The interstitial tissue of the central nervoussystem. Special Cytology, vol. in (ed. E. V. Cowdry), pp. 1447-1482. New York: Hoeber.

PLATT, J. B. (1891). A contribution to the morphology of the vertebrate head, based on a studyof Acanthias vulgaris. J. Morph. 5, 79.

PRZYBYLSKI, R. J. & BLUMBERG, J. M. (1966). Ultrastructural aspects of myogenesis in thechick. Lab. Invest. 15, 836-863.

RAVEN, CHR. P. (1936). Zur Entwicklung der Ganglienleiste. V. Uber die Differenzierung desRumpfganglienleistenmaterials. Wilhelm Roux Arch. EntwMech. Org. 134, 122-146.

REMAK, R. (1855). Untersiichungen uber die Entwicklung der Wirbeltiere. Berlin: Reimer.SENO, T. (1961). An experimental study of the formation of the body wall in the chick. Acta

Anat. 45, 60-82.SENSENIG, E. C. (1951). The early development of the spinal cord meninges in human embryos.

Contr. Embryol. 34, 147.STRAUS, W. N. & RAWLES, M. E. (1953). An experimental study of the origin of the trunk

musculature and ribs in the chick. Am. J. Anat. 92, 471-509.TRIPLETT, E. L. (1958). The development of the sympathetic ganglia, sheath cells and

meninges in amphibians. / . exp. Zool. 128, 283-311.WESTON, J. A. (1962). The migration and localization of neural crest cells labelled with

tritiated thymidine. Am. Zool. 2, 458 (Abstract).WESTON, J. A. (1963). A radioautographic analysis of the migration and localization of trunk

neural crest cells in the chick. Devi Biol. 6, 279-310.WESTON, J. A. & BUTLER, S. L. (1966). Temporal factors affecting localization of neural

crest cells in the chick embryo. Devi Biol. 14, 246-266.WILENS, S. (1959). Endoderm migration during gut morphogenesis. / . exp. Zool. 142,1X2-121.WILLIAMS, L. W. (1910). The somites of the chick. Am. J. Anat. 11, 55-100.

(Manuscript received 11 December 1969)