/ . Embryo!. exp. Morph. Vol. 24, 3, pp. 455-466, 1970
Printed in Great Britain
455
A radioautographic analysis
of the migration and fate of cells derived from
the occipital somites in the chick embryo with
specific reference to the development of
the hypoglossal musculature
By R. D. HAZELTON 1
From the Research Division, Faculty of Dentistry, University of Toronto
SUMMARY
The 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 the
hypoglossal musculature.
Tritium-labelled thymidine (0-5-10 /tCi per egg) was used as a cell-specific marker. Occipital somites (2-5) with overlying ectoderm were transplanted orthotopically from labelled
donor embryos to unlabelled host embryos (Hamburger & Hamilton, stage 9-10). The embryos
were incubated, for varying lengths of time (24 h-5 days), sacrificed, sectioned and the migration pattern and fate of the labelled cells determined radioautographically.
It appears that the hypoglossal as well as other hypopharyngeal musculature originates
from the occipital somites.
The mesodermal migration pattern extended from the occipital somite region in a ventroposterior direction to the dorsal surface of the pericardial cavity posterior to the expanded
portion of the pharynx. At this position a so-called hypoglossal cord formed on each side
which ran anteriorly to the level of the second pharyngeal pouch where it turned medially and
together with the cord from the other side entered the pharyngeal area of the embryo. This
material apparently forms the intrinsic musculature of the tongue. The mesodermal movements are attributed to differential growth movements of the areas concerned as well as to
active cell mutiplication and migration.
Selective embryonic neuronal staining was undertaken to study the relationship between the
migrating 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 walls
of developing blood vessels, possibly to microglial cells and to the cartilage surrounding the
notocord.
The occipital ectoderm expands dorso-anteriorly and ventro-laterally. In the ventro-lateral
position as contact is made with the pharyngeal endoderm a placode is formed which contributes cells to the nodose ganglion of the tenth cranial nerve. There is no other contribution
of the ectoderm to the underlying tissues.
1
Author's address: Apartment 4, 101 Hazelton Avenue, Toronto 185, Ontario, Canada.
29
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456
R. D. HAZELTON
INTRODUCTION
Morphological approaches used in distinguishing similar embryonic cell
groups have led to difficulties in determining the origin of the hypoglossal
musculature from either somitic or local mesoderm. Experiments using carbon
particle marking techniques by Deuchar (1958) indicated a somitic origin, but
the introduction of cell-specific markers such as tritiated thymidine, has made a
more critical and extensive experimental analysis possible, using the techniques
developed 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 and
overlying ectoderm were transplanted from embryos labelled with tritiated
thymidine to unlabelled host embryos in order to determine the migration pattern
and 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 approximately
30 h by which time most of the embryos had reached stages of development
immediately preceding or just following the onset of somite formation (Hamburger & Hamilton, stage 6-8). They were then prepared for labelling with
tritiated thymidine. After transferring the air space to a position overlying the
embryo, a piece of shell with underlying shell membrane approximately 1 cm
square was removed to provide a 'window' for access to the embryo.
Tritiated thymidine, in saline solution, was deposited on to the vitelline membrane over the embryo. The window was then covered with plastic tape and the
egg returned to the incubator. At least 3-4 h elapsed before transplantations of
the occipital somites and overlying ectoderm to unlabelled host embryos were
carried out. The dose of thymidine used varied between 0-1 and
Operative technique
The second to fourth or fifth occipital somites plus the overlying ectoderm
were removed as a single entity from stage 8-9 unlabelled host embryos and
replaced with comparable segments from the donor embryos labelled with
tritiated thymidine (Figs. 1,2). The ectoderm was included in the grafts to hold
the somites together and facilitate their orientation. The access hole in the vitelline membrane was kept as small as possible. The grafts were cut out with the
use of glass needles and transferred by pipette from the donor to the host
embryos.
Fate of occipital somites
457
Optic
vesicle
Occipital
somite no. 2
Section plane
for figure 2.
Transplanted 2, 3 and 4
occipital somites
and overlying ectoderm
Fig. 1. Stage 9 chick embryo (Hamburger & Hamilton, 1951) to show the
transplanted area of occipital somites and overlying ectoderm (cross-hatch).
Labelled occipital somite
and overlying ectoderm
Myotome
Dermatome
Sclerotome
Somatic mesoderm
Splanchnic mesoderm
Fig. 2. Section of Fig. 1 to illustrate the various parts of somite as well as
transplanted 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 the
oldest embryos reaching approximately stage 30 at the time of fixation. A total
of 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% buffered
formalin (old embryos). Sections were cut at 8 /.i and radioautographs were
prepared using essentially the procedure of W. G. Aldridge (see Gerber,
Aldridge, Koszalka & Gerber, 1962).
Since the original series of transplants involving both the occipital somites
and overlying ectoderm were completed, further experiments involving the
transplant of placodal ectoderm alone have been undertaken. By transplanting
only the ectoderm any contamination from the ectoderm to underlying tissues
was determined.
Hypoglossal nerve
Otic vesicle
and cord
Mandibular
arch
Fig. 3. A stage 38 chick embryo (Hamburger & Hamilton, 1951) to illustrate
the migration pattern of the occipital somites (cross-hatch).
RESULTS
One of the main problems of this type of experimental approach was the
difficulty in keeping the embryos in a viable state following the transplanting
procedures. In order to reduce the fatality rate it is recommended that good
quality eggs be obtained and a high relative humidity (70-80%) maintained
throughout all operative and incubatory procedures. There were seasonal
variations in the success of operations, with the spring and fall being the most
productive 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 25
chick embryo. Hypoglossal cord is illustrated in box. x 100.
(B) Photomicrograph using reflected light of the hypoglossal cord designated in
box of Fig. A. Because of the use of reflected light the labelled cells are white and are
indicated by arrows, x 400.
(C) Photomicrograph through the oral cavity of a stage 30 chick embryo. The
hypoglossal 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 ectoderm
is illustrated in Fig. 3. An active proliferation and ventro-lateral migration of
cells brings the myotomic portion of the somite to the dorso-lateral surface of
the pericardial cavity posterior to the expanded portion of the pharynx. At
this position a so-called hypoglossal cord forms (Fig. 3, Fig. 4A, B) which
runs forward to the level of the second pharyngeal pouch where it turns medially
into the floor of the pharynx and after further forward movement unites with the
cord 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 stage
30 chick embryo. Labelled cells can be observed lateral to the laryngeal-tracheal
groove (L) and are illustrated by a box. x 100.
(B) Photomicrograph of labelled somite cells (arrows) seen in box of A. These cells
will develop into myoblasts of the larynx, x 400.
(C) High-power photomicrograph of a section through the neural tube (N) and
primitive meninx (P) of a stage 25 chick embryo. Labelled cells may be seen in the
primitive meninx (arrows), x 400.
(D) Photomicrograph of labelled cells (arrows) in the endothelial lining of the
anterior 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 the
region 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 stage
32 chick embryo. Labelled cell within the cartilage matrix (M) is indicated by an
arrow. x400.
(B) Photomicrograph taken through the posterior part of the pharynx (P) of a
stage 22 chick embryo to indicate relationship of the hypoglossal nerve (N) to the
hypoglossal 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 developing 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 were
found in the endothelium of the internal carotid artery, aortic arches, the basilar
artery and the anterior cardinal vein (Fig. 5D).
The sclerotomic portion of the somites contributed to the cartilage matrix
surrounding the notocord (Fig. 6 A).
The occipital ectoderm undergoes an antero-dorsal and postero-ventral
expansion and in the postero-ventral region, as contact is made with the
pharyngeal endoderm, placodes form which eventually contribute to the nodose
ganglion of the tenth cranial nerve (Fig. 6C). This is the only ectodermal contribution to the underlying tissues.
Silver staining procedures were used as an additional technique to study the
migratory relationship between the hypoglossal nerve and cord by selective
neuronal 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 otocyst
to the tip of the tail. A central space divides the somite into an inner and outer
region (Fig. 2). Different fates have been attributed to these two regions with
the outer region forming the connective tissue layer of the skin (dermatome)
and the inner region forming both skelatogenous (sclerotome) and voluntary
muscle tissue (myotome). However, the absolute origin of the somite parts is in
dispute with the myotomic portion being the main contributor to the confusion.
Although muscle protein has been demonstrated in the myotome by both
immunofluorescent studies (Holtzer, Marshall & Finck, 1957; Ozawa, 1962;
Ikeda, Abbott & Langman, 1968) and by electron-microscopic identification of
myofilaments (Przybylski & Blumberg, 1966; Allen & Pepe, 1965; Dessouky &
Hibbs, 1965; Obinata, Yamamoto & Maruyama, 1966), the definitive origin of
the myotome has not been established: Williams (1910) and Hamilton, Boyd &
Mossman (1962) state that the myotome originates by a multiplication of cells at
the dorso-medial angle of the somite while Remak (1855), His (1888), Bardeen
(1900) and Langman & Nelson (1968) state that the myotome develops by proliferation and differentiation of the overlying dermatome.
By using the electron microscope the myotomic portion of early chick
embryos can be identified from the other somite parts by the larger size and
lighter 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 occipital
series (Kingsbury, 1915; Hunter, 1935; Goodrich, 1936).
The majority of evidence for either a somitic or local mesodermal origin of the
hypoglossal 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 occipital
somites whereas Lewis (1910) concluded that the hypoglossal musculature
developed 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 marking experiments in the chick Deuchar (1958) concluded the hypoglossal musculature originated from the occipital somites. Although Deuchar's results tended
to confirm earlier descriptive studies, some objections may be made to the
technique: the carbon particles could have been carried to the hypoglossal
musculature by the movements of associated parts or the carbon could have been
engulfed by phagocytic cells which in turn migrated to the hypoglossal area
(Straus & Rawles, 1953; Seno, 1961). However, the present experiments confirm and extend Deuchar's findings.
In order to determine the migration and fate of similar embryonic cell groups
one must first establish their identity. This has been done in the present series
of experiments by specifically marking groups of cells (e.g. occipital somites)
with tritiated thymidine. These tissue segments were then transplanted to unlabelled host embryos and the migration and fate of the labelled cells determined
radioautographically.
The organization of the occipital somites into so-called hypoglossal cords and
the migration of these cords into the hypopharyngeal region has been described
by several investigators (Hunter, 1935; Bates, 1948; Detwiler, 1955; Kallius,
1905). Although differential growth movements of involved and associated
areas are important in the movement of this material, other factors such as cell
proliferation and migration must be considered. If no further cell proliferation
occurs once the myotome has formed (Langman & Nelson, 1968) other processes
must account for the final adult population of cells. This could be attributed to
the hypoglossal cord containing undifferentiated cells which form myotubes
only when a final destination is established or the hypoglossal material could
induce the differentiation of myotubes from local mesoderm.
The role of the hypoglossal nerve in the development of the hypoglossal cord
must also be considered. By selectively staining embryonic tissue for neural
elements the relationship between the nerve and cord was studied, and it was
shown that the muscle cord precedes the nerve along its migratory path. The
same relationship was observed between the sensory nerves of the visceral arches
and their muscle cords.
The expansive movements of the ectoderm in the chick were similar to those
found in Amblystoma by Wilens (1959) and are due to the morphogenetic movements of embryonic parts. The most significant ectodermal finding was its
contribution to the nodose ganglion of the vagus nerve. This finding led to an
investigation of the entire sensory cranial ganglion system, the results of which
will be published in the future.
464
R. D. HAZELTON
RESUME
Analyse radioautographique de la migration et du devenir des cellules
derivees des somites occipitaux chez Vembryon de Poulet en
se referant au developpement de la musculature hypoglosse
Le schema de la migration et le devenir des cellules des somites occipitaux et de l'ectoderme
susjacent sont decrits chez l'embryon de Poulet en se referant au developpement de la musculature 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 a
5 jours), sacrifies et sectionnes. On determine de facon radioautographique la migration et le
devenir 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 direction
postero-ventrale jusqu'a la surface dorsale de la cavite pericardique posterieure a la portion
renflee du pharynx. A cet endroit un cordon, appele cordon hypoglosse, se forme de chaque
cote; 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 materiel
forme apparemment la musculature intrinseque de la langue. Les mouvements mesodermiques
sont aussi bien attribues aux mouvements de croissance differentielle des aires concernees
qu'a l'active multiplication cellulaire et a la migration.
On utilise la coloration neuronale embryonnaire selective pour etudier le rapport entre la
migration 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 endotheliales
des vaisseaux sanguins en developpement, et, peut-etre, aux cellules microgliales et au
cartilage entourant la notocorde.
The author wishes to thank Dr M. C. Johnston for his invaluable guidance both in the
experimental procedures and in the preparation of the manuscript.
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