/ . Embryol. exp. Morph. Vol. 34, 1, pp. 125-154, 1975
Printed in Great Britain
125
Mesenchymal derivatives of
the neural crest: analysis of chimaeric quail and
chick embryos
By C. S. LE LIEVRE 1 AND N. M. LE DOUARIN 1
From the Laboratoire d'Embryologie, Universite de Nantes
SUMMARY
Interspecific grafts of neural tube and associated neural crest (NC) have been made
between quail and chick embryos. Structural differences of the interphase nucleus in the two
species make it possible to identify quail from chick cells in the chimaeras after FeulgenRossenbeck's staining and at the electron microscope level. Owing to the stability of the
natural quail nuclear marker labelling, migration pattern and developmental fate of the
grafted NC cells could be followed in the host embryo. In previous work it has been demonstrated that the visceral skeleton derives entirely from NC mesenchyme and the various
levels of the neural axis from which visceral cartilages and bones originate have been established. In the present work, the contribution to the lower jaw and pharynx of NC mesenchymal derivatives other than bones and cartilages has been studied. It is shown that the
dermis in the face and ventrolateral side of the neck has a neural origin. The wall of the
large arteries deriving from the branchial arches (systemic aorta, pulmonary arteries, brachiocephalic trunks and common carotid arteries) are entirely made up of mesectodermal cells
except for the endothelial epithelium which is mesodermal in origin. The presence in the
wall of the common carotid arteries of fiuorogenic monoamines-containing cells is demonstrated using the formol-induced-fiuorescence technique. Like the secretory cells of the
carotid body, the fluorescent cells of the carotid artery wall originate from the rhombencephalic NC.
Connective tissue of the lower jaw, tongue and ventrolateral part of the neck originate
from the neural crest. Mesectoderm participate in the formation of the glands associated
with the tongue and pharynx (lingual gland, thymus, thyroid, parathyroids) giving their
mesenchymal component. On the other hand, as demonstrated previously by our group,
NC cells are the main cellular component of the UB since they give rise to the calcitoninproducing cells. The wall of the oesophagus and trachea is of mesodermal origin, but adipose
tissue around the trachea and parasympathetic enteric ganglia of the digestive tube derives
from NC. NC cells participate in the formation of striated muscles of the branchial arches
and differentiate there into connective and muscle cells.
It appears from this study that the differentiating capabilities are similar in mesenchymal
and mesectodermal cells with the exception of blood vessel endothelia which in our experiments are always of host origin in mesectoderm-derived tissues.
The capacity of the NC to give rise to mesenchymal derivatives is restricted to the
cephalic neural axis down to the level of the 5th somite in both chick and quail embryos.
1
Authors' address: Laboratoire d'Embryologie, Universite de Nantes, B.P. 1 044, 44037
Nantes Cedex, France.
126
C. S. LE LIEVRE AND N. M. LE DOUARIN
INTRODUCTION
Katschenko (1888) was the first to suggest that some of the mesenchyme of the
head originates from the neural crest. He drew this assumption from the observation of selacian development. Later, Goronowitsch (1892,1893) stated that
in teleosts and birds also the neural crest contributes to the formation of
mesenchyme. At the same time Platt (1893) found that in Necturus embryos the
cartilage of the visceral arches as well as the dentine of the teeth derive from
ectoderm, but she considered the main source of the mesenchyme that gives rise
to these structures to be the lateral ectoderm of the head. She proposed the term
of mesectoderm for the mesenchyme originating from the ectoderm and mesendoderm for the mesodermal mesenchyme. The conclusions of Platt were based
on the fact that the yolk platelets of ectodermal cells were conspicuously smaller
than those of either mesoderm or endoderm, making possible the histological
distinction of migrating cells of the neural crest and placodal origin from other
mesenchymal cells.
Experimental studies on the fate of neural crest cells were later undertaken by
several investigators, mainly utilizing amphibian embryos. By extirpations and
transplantations Stone (1922, 1926, 1929) was able to demonstrate that the
skeletal connective tissues of the upper face and visceral arches were largely of
neural crest origin.
Similar conclusions have been drawn by several authors using interspecific
and intergeneric transplantation techniques (Raven, 1931; Holtfreter, 1933,
1935a, b; Harrison, 1935, 1938; Andres, 1946, 1949; Wagner, 1949, 1955, 1959;
and others: see reviews of Horstadius, 1950 and Weston 1970 for bibliography).
Artificial cell marking techniques such as vital dyes (Stone, 1932; Horstadius &
Sellman, 1946) and tritiated thymidine (Chibon, 1962,1964) were also applied to
this problem in amphibians.
Less numerous are the studies dealing with the contribution of the neural
crest to mesenchymal structures in higher vertebrates. After extirpation experiments in chick embryo, Hammond & Yntema (1953, 1964) observed severe
deficiencies of the nasal septum and visceral skeleton. Johnston (1966) using the
experimental technique of isotopic labelling of nuclei previously developed by
Weston (1962, 1963) to study the migration of spinal neural crest cells in the
chick embryo, carried out an autoradiographic study of the evolution of the
crest at the cranial level. He clearly showed that crest cells contribute extensively
to the mesenchyme of upper facial regions and visceral arches and consequently
that their behaviour is remarkably similar to that of their amphibian counterparts. He was able to demonstrate that labelled crest cells participate in the
formation of cartilage, but due to the instability of the labelling which becomes
diluted through the rapid proliferation of embryonic cells, the isotopic technique cannot provide comprehensive data on the ectodermal mesenchyme
capabilities.
Mesenchymal derivatives of avion neural crest
121
The present report deals with the application of a biological cell marking
technique, which has the advantage of being stable (Le Douarin 1969, 19716,
1973a), to the problem of neural crest cell migration and differentiation. Thus
it is possible to follow the migrating crest cells until they have reached a fully
differentiated state. Cell identification is based on structural differences of the
interphase nucleus in two closely related species of birds, the Japanese quail
(Coturnix cotwnix japonicd) and the chick {Gallus gallus). In the quail, the
nucleus contains one or several large heterochromatic masses associated with the
nucleolar RNA, making the nucleolus considerably enlarged in all embryonic
and adult cell types. In the chick the chromatin is evenly distributed in the
nucleoplasm forming a fine network with some small dispersed chromocenters
and the amount of nucleolar-associated chromatin is small. As a result of this
different disposition of the chromatin material quail and chick cells can easily
be distinguished after the application of the Feulgen-Rossenbeck's specific
staining procedure for DNA. They can also be recognized at the electron
microscope level after routine uranyl acetate-lead citrate staining or by means
of the EDTA preferential staining procedure for RNA according to Bernhard
(1968), in which RNA is stained while DNA and most of the proteins are
unstained (Le Douarin 1971 a, 1913a, 1973b).
The 'quail-chick marking system' has already been applied to the problem of
migration and differentiation of neural crest cells by our group (Le Douarin &
Le Lievre, 1970; Le Douarin & Teillet, 1970; Teillet & Le Douarin, 1970;
Le Douarin, 1971 a, b; Le Douarin & Le Lievre, 1971; Le Douarin & Teillet,
19716; Le Lievre, 1971a, b; Teillet, 1911a, b; Le Douarin & Le Lievre, 1972;
Le Douarin, Le Lievre & Fontaine, 1972; Le Douarin, 1973a; Le Douarin &
Teillet, 1973 a, b; Le Lievre & Le Douarin, 1973; Pearse et al. 1973; Le Douarin,
Fontaine & Le Lievre, 1974; Le Lievre, 1974; Le Lievre & Le Douarin, 1974;
Polak et al. 1974; Teillet & Le Douarin, 1974) and others (Johnston,
Bhakdinaronk & Reid, 1973; Saxod, 1973). In previous papers it was not only
shown that the entire visceral skeleton originates from mesectodermal cells, but
it was also established from what levels of the neural axis the different bones
and cartilages are derived (Le Lievre, 19716, 1974).
The observations reported here concern the capability of neural crest to
give rise, in addition to bone and cartilage, to other mesenchymal derivatives.
Due to the stability of the labelling provided by quail cells, it was possible to
show that the neural crest mesenchyme contributes to various tissues of face and
neck.
MATERIAL AND METHODS
The eggs employed throughout this study were from the White Leghorn
strain of the fowl Gallus gallus and the Japanese quail Coturnix coturnix japonica.
They were incubated in a humidified atmosphere at 38 ± 1 °C. The developmental
stage was determined by the number of somites for the early stages until 2\ days
o
EMB
34
128
C. S. LE LIEVRE AND N. M. LE DOUARIN
of incubation. After this, staging was done by the days of incubation. For the
chick it was useful in some cases to use the staging series of Hamburger &
Hamilton (1951).
Isotopic and isochronic grafts of sections of quail neural primordium into the
chick have been carried out according to the following technique.
In a first step, a piece of neural tube and associated neural crest is surgically
removed at a prescribed transverse level of a chick embryo, between the anterior
limit of mesencephalon to the level of the 9th somite inclusive. The length and
location of the excision varies according to the experiment. The more cranial
the intervention the younger the embryo had to be, since the neural crest
becomes progressively established soon after the closure of the neural tube and
disperses in a cranio-caudal sequence.
In a second step the corresponding transverse part is taken from a quail
embryo at the same developmental stage and immersed in a 0-1 % solution of
trypsin in Ca2+, Mg 2+ free Tyrode solution (10 min at 2 °C) (Moscona &
Moscona, 1952). All tissues adhering to the neural tube are manually removed
with fine dissecting needles. Thus the isolated quail neural rudiment is completely devoid of contamination by non-neurectodermal cells (Fig. 1). The
tissue is then rinsed in Tyrode containing horse serum for 10 min and then in
Tyrode without horse serum for an additional 10 min. After the final rinse the
neural tube is orthotopically grafted into the chick in the space resulting from
the previously excised neural tube.
Four experimental series were made:
(1) In 5- to 7-somite embryos the graft consisted of the mesencephalic primordium (Fig. 2, Expt. 1 a) or the level of the presumptive mesencephalon +
anterior rhombencephalon back to the level of the 1st somite (Fig. 2, Expt. 1 b).
(2) In 6- to 10-somite embryos the intervention consisted of either the anterior
rhombencephalon (from the mesencephalo-rhombencephalic constriction to the
1st somite (Fig. 2, Expt. 2 a), the posterior rhombencephalon from the 1st to the
5th somite (Fig. 2, Expt. 2b), or the whole rhombencephalon (Fig. 2, Expt. 2c).
FIGURE 1
Experimental procedure for isotopic and isochronic transplantations of quail neural
primordium into chick embryo. Ch, Notochord; NC, neural crest.
(A) Transverse section of a 10-somite control embryo at the level of anterior
rhombencephalon. x 180.
(B) Ten-somite chick embryo after removal of the rhombencephalic primordium.
x 250.
(C) Isolated rhombencephalic neural tube and neural folds of a 10-somite quail
embryo, x 380.
(D) Transverse section at the rhombencephalic level in a chick embryo which has
received, 6 h before fixation, the graft of a quail neural primordium. x 250.
The same procedure is used for the reverse graft of chick neural primordium into
quail.
Mesenchymal derivatives of avian neural crest
129
....
50 //m
1B
9-2
130
C. S. LE LIEVRE AND N. M. LE DOUARIN
Exp. 1
Exp.2
Exp. 3
Fig. 2. Levels of heterospecific transplantations of neural rudiment.
Expt. 1. The operations are carried out at 5- to 7-somite stage of host and donor
embryos at the level of mesencephalon (a) or mesencephalon + anterior rhombencephalon down to the 1st somite (b).
Expt. 2. The transplantations are carried out at 6- to 10-somite stage at the level
of anterior rhombencephalon (between mesencephalo-rhombencephalic constriction
and 1st somite) (a), posterior rhombencephalon (from the 1st to the 5th somites)
(b) or concern the whole rhombencephalon (c).
Expt. 3. Isotopic and isochronic transplantations of short fragments of quail
neural tube into chick in order to determine the posterior limit of NC capability
to give rise to mesenchymal derivatives. Stage of intervention: 7—11 somite; graft
of a fragment of neural tube corresponding to the length of 3 somites from the 1st
to the 9th somites.
(3) In a third series of experiments short fragments of the chick neural
primordium were removed and replaced by their quail counterparts at the level
of the first somite pairs in order to find out the location of the posterior limit to
the capabilities of the neural crest cells to give rise to mesenchymal derivatives.
The embryos employed in these latter experiments were at 7- to 11-somite
stages and the length of the neural axis involved in the experiment corresponded
to the extent of three somites (Fig. 2, Expt. 3).
(4) In order to control the validity of the results obtained by grafting quail
neural tube into chick, the reverse operation was carried out in an experimental
series, i.e. heterospecific transplantation of rhombencephalic primordium.
Histological procedures
The operated embryos are sacrificed between 3 and 19 days of incubation.
Up to the 11th day the anterior half of the body (head, neck and thorax down to
and including the anterior limbs) is fixed in Zenker's fluid and cut in serial 5 /.im
thick transverse sections.
Mesenchymal derivatives of avian neural crest
131
Fig. 3. Fixed regions in 11- to 19-day-old chick embryos.
(A) Common carotid artery (c.c.a), vagus nerve (x), carotid body (cb) and pharyngeal glandular derivatives: parathyroids (pt), thymus (tm), thyroids (thy) and
ultimobranchial bodies (r.ub., hub.), tr, trachea; es, oesophagus.
(B) Large vessels, superior part of the ventricles, bulbus and truncus arteriosus.
ao, Systemic artery; /pa, left pulmonary artery; ra, right atrium; rbca, right
brachiocephalic artery; rda, right ductus arteriosus; dao, dorsal aorta.
In the older embryos the following pieces are taken and treated in the way
described above:
(a) The region containing the following tissues: common carotid artery and
carotid body, jugular vein, gastric nerve, nodosum ganglion, thyroid, parathyroids, ultimobranchial body, and the caudal portion of the thymus (Fig. 3 A).
(b) The heart and large arteries according to the scheme of Fig. 3B.
(c) The head and the anterior half of the neck.
Sections are first treated according to Feulgen-Rossenbeck's technique (1924)
and then lightly stained with picroindigocarmine. This staining enabled us to
identify quail and chick cells (Fig. 4).
The formol-induced-fluorescence technique (FIF) which detects histochemically fluorogenic monoamines (Falck, 1962) has been applied to certain pieces
of experimental embryos. After having been observed in u.v. light, sections are
post-fixed in Zenker's fluid and stained by the Feulgen-Rossenbeck procedure
which makes it possible to determine which species, quail or chick, the fluorescent cells represent. The FIF treatment is applied as follows:
The blocks are freeze-dried in a thermoelectric tissue dryer at - 4 0 °C for
18 h. They are then exposed to formaldehyde vapour at 80 °C for 2 h and directly
embedded in vacuo in Epon-Araldite. In each case, part of the freeze-dried
132
C. S. LE LIEVRE AND N. M. LE DOUARIN
6A
FIGURES 4 AND 6
Fig. 4. Quail and chick cells stained with Feulgen-Rossenbeck's reaction.
Mesenchyme of the 1st branchial arch of 4-day-old chick (A) and quail (B) embryos.
In chick nuclei the chromatin is evenly dispersed in the nucleoplasm while quail
nuclei show one or several heterochromatic condensations, x 1440.
Fig. 6. Transverse section of the right maxillary process of a chick host embryo at
4 | days of incubation which has received the graft of a quail mesencephalon and
anterior rhombencephalon at 7-somite stage (Expt. \b of Fig. 2). The mesenchyme originating from the neural crest is made up of quail cells and lined by the
chick host ectoderm (E). (A) General view, x 80. (B) Detail, x 440. FeulgenRossenbeck's staining.
material is embedded without formaldehyde vapour treatment. Those samples
form the control series. Serial sections from all blocks are cut at 5 ju>m, placed in
a drop of distilled water on glass slides, attached through rapid water evaporation, and observed directly without a coverslip. All sections are examined by
fluorescence microscopy using a Leitz Orthoplan microscope fitted with an
HBO 200 W mercury arc lamp. Filters used are BG 12/5 mm, BG 12/3 mm,
BG 12/1-5 mm for excitation with a K 510 barrier filter. Photomicrographs were
taken on Tri-X, Pan film or Rayoscope film.
Mesenchymal derivatives of avian neural crest
133
B
Fig. 5. Diagrammatic representation of the extension of mesencephalic and
rhombencephalic NC cells in facial and pharyngeal structures of 4-day-old embryos.
(A) Lateral view. 1-4: 1st, 2nd, 3rd and 4th branchial arches. 6, 7: Level of the
cross-sections illustrated in Figs. 6 and 7. mp, Maxillary process; h, heart.
(B) Cross-section through the 2nd branchial arch, ao, Dorsal aorta and 2nd aortic
arch; ch, notochord; j , anterior cardinal vein; m, muscle plate; ph, pharynx. NC
cells are located ventrally (stippled area) and extend dorsally to the level of anterior
cardinal vein. The muscle plate is essentially of host origin.
Electron microscopy
For ultrastructural observation tissues are fixed in 6% glutaraldehyde in
0 1 M phosphate buffer, at pH 7-4 for 20 min at 4 °C, and postfixed in 1 %
osmium tetroxide in phosphate buffer for 1 h. The blocks are embedded in
Epon, sectioned, stained by lead citrate and uranyl acetate, and observed in an
Hitachi HS 8 electron microscope.
RESULTS
I. Observation of the host embryos at 3rd and 4th days of incubation
Transverse sections (5 jim) of the anterior part of the embryos were stained
with the Feulgen-Rossenbeck's technique. The neural crest [NC] cells, which
contribute to visceral arch morphogenesis, could be recognized as a result of the
quail nuclear marker.
The results obtained after the graft of mesencephalic and rhombencephalic
primordia (experimental series represented Fig. 2, Expts. 1 and 2) are summarized
in Table 1.
It appears that the mesenchyme of maxillary buds and branchial arches is
composed primarily of mesectodermal cells (Figs. 5-7). Chick cells were found
Exp. 2 c.
Total
rhombencephalon
Exp. 2b.
Posterior part of
rhombencephalon
Exp. 2a
anterior part of
rhombencephalon
Exp. 1 h.
Mesencephalon
+ anterior part of
rhombencephalon
Exp. 1 a.
Mesencephalon
^"""^--^Appearance of heterospecific
Level of ^""""^-^^ mesectodermal
heterospecific
^ ^ ^ c e l l s in the
graft
^"""^^host
Maxillary
process
1st
branchial
arch
2nd
branchial
arch
3rd
branchial
arch
4th branchial
and posterior to
4th pouch
Table 1. Participation of mesectodermal cells in the genesis of facial and pharyngeal structures in a
4-day-old chick embryo
d
o
c!
rtn
%
d
tfl
w
t- 1
p
OJ
Mesenchymal derivatives ofavian neural crest
135
forming the endothelium of the aortic arch and the muscle plate, in which,
however, some quail cells were dispersed.
Similar observations were done whatever the sense of heterospecific combinations was: quail neural tube grafted into chick or inversely. Figs. 7 and 8 show
the distribution of host and donor cells in the 2nd visceral arch in the two kinds
of grafts at rhombencephalic level.
II. Further localization of differentiated mesectodermal cells in facial and
pharyngeal structures
Due to the stability of the nuclear marker provided by quail cells, the ultimate
localization of neurectodermal cells in the various structures of the head and neck
could be determined. From the 6th day onwards, the contribution of quail cells
to the visceral arch derivatives has been studied in chick embryos which had
received various grafts as indicated in Fig. 2.
Mesectodermal cells which colonize the branchial arches give rise to the
entire visceral skeleton as previously reported by Le Lievre (1974). In addition
quail NC cells have been found to differentiate into many other kinds of cells.
A. Contribution of NC cells to the dermis
In the face and neck, mesectodermal cells differentiate into dermis. This
results in the formation of chimaeric feather buds in which the papilla is made
up of quail cells while the epidermis belongs to the host chick (Fig. 9 A). Quail
melanocytes spread dorso-ventrally and cranio-caudally from the grafted neural
tube in the host skin (Teillet & Le Douarin 1970; Teillet 1971a). They are
numerous inside the chick epidermis of the chimaeric feathers (Fig. 9B). Quail
mesectodermal cells form the smooth arrector muscles associated with feathers
(Fig. 9C) and the subcutaneous adipose tissue.
B. Connective tissues deriving from the NC
The connective tissue of the lower jaw, the tongue and the ventral part of the
neck derives from NC mesenchyme. The dorsal limits of the mesectodermal
area reach the level of the auditory pit and the internal carotid arteries. Mixed
with chick host cells, mesectoderm participates in the histogenesis of the wall
of these vessels. No quail cells are encountered dorsally to the notochord.
The loose connective tissue in the tongue and the floor of the mouth is mostly
derived from the NC. Such is the mesenchymal component of salivary glands
(Fig. 10) and the connective stroma in the tongue and lower jaw muscles.
The mesenchymal components of the glandular pharyngeal derivatives are of
NC origin. Quail cells are found in the interlobular spaces and in the medulla of
thymic lobes, forming the parafollicular cells of thyroid gland and the connective
tissue located between the cords of parathyroid glandular cells (Fig. 11). They
are the main cellular component of the ultimobranchial and carotid bodies as
previously described by our group (Le Douarin & Le Lievre, 1970, 1971, 1972;
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C. S. LE LIEVRE AND N. M. LE DOUARIN
8B
Mesenchymal derivatives of avion neural crest
137
Le Douarin et al. 1972; Pearse et al. 1973; Le Douarin et al. 1974; Polak et al.
1974).
The walls of the oesophagus and trachea are primarily derived from chick host
cells with the exception of the enteric parasympathetic ganglia which arise from
the grafted NC when the operation involves the hind brain level, as reported
previously (Le Douarin & Teillet, 19716, 1973a). During the second half of the
incubation period, mesectodermal cells differentiate into adipose tissue in the
vicinity of trachea (Fig. 12).
C. Participation of NC cells in striated muscle differentiation
Mesectodermal cells participate to a certain extent in the histogenesis of the
striated muscles of the tongue, the face and the neck though they mainly derive
from anterior somite mesoderm (Johnston, personal communication). However, as mentioned above, some quail cells are found in the muscle plates of the
branchial arches and are incorporated afterwards in the muscles (Fig. 13).
Striated muscles, which differentiate in the second branchial arch, have been
observed at the electron microscope level and were seen to be a mixture of quail
and chick striated muscle cells (Fig. 14) with a prevalence of host cells. On the
other hand, as mentioned above, the muscle connective tissue derives from NC
mesenchyme.
D. Mesectodermal origin of the arteries deriving from aortic arches
Due to the elongation of the neck and the anteroposterior movement of the
heart during the first half of embryogenesis, the blood vessels which derive from
aortic arches are drawn caudad and incorporated into the thoracic cavity. Except for the endothelium, the wall of the large arteries deriving from the aortic
arches originates entirely from mesectoderm.
When the graft of a quail neural primordium into a chick embryo involves
FIGURES 7 AND 8
Fig. 7. Graft of anterior rhombencephalon of a quail into a chick embryo at
7-somite stage (Expt. 2a). Cross-section through the 2nd branchial arch at 4£ days
of incubation. Feulgen-Rossenbeck's staining.
(A) General view, x 90.
(B) Detail showing that the mesenchyme originates from the grafted quail neural
tissue (Q). The muscle plate (M) is essentially of host origin but contains some
dispersed quail cells. Ectoderm (£) and pharyngeal endoderm (En) are made up of
chick cells. x280.
Fig. 8. Similar experiment as in Fig. 7: chick rhombencephalon is grafted into a
7-somite quail embryo. As in Fig. 7, most of branchial arch mesenchyme is derived
from the graft while the muscle plate and the lining ectodermal and endodermal
epithelia are of host origin. Feulgen-Rossenbeck's staining.
(A) General view, x 220.
(B) Detail, x 580.
138
10 ftm
I I
C. S. LE LIEYRE AND N. M. LE DOUARIN
Mesenchymal derivatives of avian neural crest
139
the whole rhombencephalic primordium (Fig. 2, Expt. 2 c) the wall of the arteries
which arise from the 3rd (brachiocephalic trunks and common carotid arteries),
the 4th (systemic aorta) and the 6th (pulmonary arteries) aortic arches, are
entirely made up of quail cells (Figs. 15, 16). In a transition zone between the
bulbus arteriosus and the aortic trunks arising from the heart, the vessel wall is
formed by a mixture of quail and host cells. The same observation is made in the
distal part of the arch of systemic aorta and in ductus arteriosus corresponding
to the distal portion of the 6th arches. After the 6th day of incubation, quail
mesenchymal cells become distinctly arranged as smooth muscle around the
tube formed by chick endothelial cells lining the aortic arch primordium. Thus
the NC cells undergo smooth muscle cell differentiation and elaborate elastin
fibrils. The chromatin pattern of quail nuclei is modified during this differentiative process. The initial single heterochromatic mass of the nucleus becomes
fragmented in two or three smaller Feulgen-positive patches attached to the
nuclear membrane (Fig. 16 A). The same disposition is observed in vessel walls of
control quail embryos.
The 3rd visceral arch is the site of differentiation of the carotid body in the
close vicinity of the common carotid artery wall as described by Fontaine (1973)
in both quail and chick embryos. The common carotid artery of experimental
embryos (i.e. chick embryos which had received the graft of quail neural
primordium) was sectioned and treated by FIF technique during the second
half of the incubation time. Numerous single or grouped fluorescent cells were
observed randomly distributed throughout its wall (Fig. 16 B). They show the
same greenish fluorescence as do quail carotid body cells, which has been
demonstrated to be due to dopamine (Pearse et al. 1973). If the sections first
observed in u.v. light are then post-stained by Feulgen-Rossenbeck's technique,
fluorescent cells are identified as belonging to quail species, as are the connective
wall of the artery and the carotid body (Le Douarin et al. 1972).
E. Do NC cells participate in endothelial wall of blood vessels?
Various pharyngeal structures in which the connective tissue derive from the
mesectoderm were observed at the electron microscope level in order to find
out whether the endothelial cells of the blood capillaries belonged to mesodermal
or neurectodermal mesenchyme. In all the cases observed (dermis, thymus,
Fig. 9. Experiment \b. Graft of a quail mesencephalon + anterior rhombencephalon
into 6-somite chick embryos.
(A) Transverse section in the neck of the host at 9 days. Feather-bud showing host
ectoderm and quail mesenchyme. x 280.
(B) Feather germ of a 12-day-old host. Quail melanocytes (ra) have invaded the
host ectoderm. Germ pulp is made up of quail cells, x 650.
(C) Longitudinal section of a feather germ in the neck of a 12-day-old host chick
embryo. Arrector feather muscles in the dermis, made up of quail mesectodermal
cells (mp). x 450. Feulgen-Rossenbeck's staining.
140
C. S. LE LIEVRE AND N. M. LE DOUARIN
13
Mesenchymal derivatives of avian neural crest
141
thyroid) none of the endothelial cells have been found to exhibit the quail
nuclear marker. The pericytes, however, which line the external side of the
capillaries, are evidently of quail origin (Fig. 17). Therefore, it must be assumed
that the organs deriving from mesectodermal rudiments are invaded by mesodermal capillary buds during their histogenesis.
Table 2 and Fig. 18 summarize the observations described above, i.e. the
distribution of NC cells in the various structures of the face and neck according
to the level at which the graft of the quail primordium has been made.
III. Posterior limit of mesenchymal potentialities of the NC
Grafts of short fragments of quail neural primordium have been made
in chick according to the schema of Fig. 2, Expt. 3. Ninety embryos
have been operated and 39 survived and could be observed at 6-12 days of
incubation.
When the graft is made behind the level of the 5th somite, no mesenchymal
cells are found in the host embryo. Some dispersed quail cells are encountered in
the 6th aortic arches following a graft involving the levels of 3rd and 4th
somites. In the latter case no quail cells are found in connective tissue of the host,
neither in the dermis. Cells originating from the level of somite 1-3 give rise to
the various kinds of connective cells described above. Thus one can consider
that the posterior limit of the mesenchymal capabilities of the crest corresponds
roughly to the level of the 5th somite.
In the kind of experiments involving the graft of short fragments of quail
neural tube into chick, quail cells are always found mixed to host cells. That
shows that the same transverse level of the pharynx receives NC cells from a
large portion of the neural axis. Thus it appears necessary to perform extensive
FIGURES
10-13
Fig. 10. Experiment 1 b. Graft of a quail mesencephalon + anterior rhombencephalon
into a 7-somite chick embryo. Section through the body of the tongue of the 14-dayold chick embryo. All mesenchymal derivatives (Q) originate from the grafted
neural quail primordium: loose connective tissue (I.e.); pulp of horny papilla (h.p);
lingual gland mesenchyme (l.g.); keratinized epithelium (E) and lingual glandular
cells are of host origin, x 300. Feulgen-Rossenbeck's staining.
Fig. 11. Experiment 2c. Graft of a quail rhombencephalon into a 10-somite chick
embryo. Parathyroid of the 8-day-old chick host embryo. Numerous quail mesenchymal cells (Q) are present between the chick glandular cords (pt). x 510. FeulgenRossenbeck's staining.
Fig. 12. Experiment 2 c. Graft of a quail rhombencephalon into a 7-somite chick
embryo. In the neck of the 12-day-old chick host, quail mesectodermal cells have
differentiated into adipose cells (A), x 920. Feulgen-Rossenbeck's staining.
Fig. 13. Experiment 2c. Graft of a quail rhombencephalon into a 10-somite chick
embryo. Branchiomandibularis muscle (3rd branchial arch) of the 10-day-old host
chick embryo. The muscle is made up of a mixture of host and quail cells, x 920.
Feulgen-Rossenbeck's staining.
142
C. S. LE LIEVRE AND N. M. LE D O U A R I N
Fig. 14. Same experiment as in Fig. 13. Striated muscle cell showing a
characteristic DNA-rich nucleolus of quail, x 27500.
heterospecific transplantation of the neural tube to get a clear view of the normal
distribution and developmental capabilities of crest cells.
DISCUSSION
Interspecific combinations between quail and chick embryos have made it
possible to investigate the normal process of NC cell migration and to follow the
NC derivatives until their completely differentiated state. Unknown derivatives
of this transitory embryonic structure have thus been identified and the extent
of its contribution to cephalic and cervical morphogenesis has been recognized.
1. Reliability of the experimental technique
Experimental techniques devised to investigate embryogenic mapping must
disturb as little as possible the normal developmental conditions of the rudiments. Transplantations of early primordia on the chorioallantois or into the
coelomic cavity have often been used (Willier & Rawles, 1931, Rudnick 1932,
1938; Andrew, 1964, 1969, 1970) but they disturb the micro-environment of the
embryonic tissues and therefore cannot provide really reliable information on
their normal developmental fate during embryogenesis. That is especially true
when the cells concerned undergo extensive migrations in the embryo before
differentiating. As demonstrated for NC cells (Weston, 1963; Weston & Butler,
143
Mesenchymal derivatives ofavian neural crest
CCA
SCA
Fig. .15. Aortic-arch-derived arteries. /////,
the vessel wall is entirely made
up of mesectodermal cells originating from mesencephalic and rhombencephalic
NC, except for the endothelium which is of mesodermal origin.
,
mesectodermal and mesodermal cells are mixed. |
|, the vessel wall is made
up of mesodermal cells in entirety. 3-6, Arteries deriving from the 3rd, 4th
and 6th aortic arches; CCA, common carotid artery; DA, dorsal aorta; PA, pulmonary artery; SCA, subclavian artery; TA, aortic trunk; TP, pulmonary trunk.
1966; Le Douarin & Teillet, 1974), migration pathways are indeed highly
dependent on the structures and substrates encountered.
Isotopic and isochronic grafts of neural primordium between two species of
birds closely related in taxonomy provide almost normal developmental conditions for both host and grafted structures and result in normally developed
embryos, when the operation has been properly done. Compared to extirpation
or electrocauterization of the neural tube (see Horstadius, 1950, and Weston,
1970, for review), this grafting technique has the advantage of precluding regulatory mechanisms due to crest cells from anterior and posterior levels.
The developmental rate being a little faster in the quail than in the chick
embryo, it was important to perform neural tube exchanges between the two
species in both directions, i.e. quail into chick and chick into quail and to compare the distribution of NC cells in the different combinations. When the quail
neural tube is grafted into the chick embryo, the rapidly growing quail cells
might indeed have an advantage on the chick cells and expand more in the host
structures than in normal conditions. However, comparison of the embryos show
a similar pattern of NC cell migration in both kinds of grafts.
In fact, the difference of growth rate is rather slight in the early developmental
10
EMB 34
144
C. S. LE LIEVRE AND N. M. LE DOUARIN
Mesenchymal derivatives of avian neural crest
145
stages when the most significant morphogenetic events occur and becomes significant only during the second half of the incubation period (Hamburger &
Hamilton 1951; Zacchei 1961). Since chick embryos are more resistant than
quail to surgical interventions, most of the results reported in this study have
been obtained on chick hosts grafted with quail NC.
It can be assumed that, in experimental embryos, the structures composed of
cells with the nuclear characteristics of the grafted primordium are of neural
origin. Numerous histological controls of the neural tube before the graft and
after trypsination have shown that the neural rudiment is easily isolated from
neighbouring tissues at the early developmental stages and is, at grafting,
completely devoid of mesenchymal cell contamination.
2. Differentiating capabilities of mesectodermal cells, and role of inductive
influences
Studies on chimaeric quail-chick embryos lead to the conclusion that the
neural primordium contributes extensively to structures of the facial and
pharyngeal regions. Though the interspecific grafts included the whole neural
tube rather than the neural folds alone, it is reasonable to assume that most if
not all mesectodermal derivatives observed in the experimental embryos originate from the NC. Some results obtained previously by Johnston (1966), using
tritiated thymidine-labelled transplants of the NC alone, are in complete agreement with our findings.
Thus it appears that most of the lateroventral part of head and neck is edified
by material originating from the mediodorsat embryonic region, which undergoes extensive migration before reaching its definitive site in the face and
branchial arches.
The results obtained here demonstrate that ectodermal mesenchyme has a
large range of developmental capabilities and appears able to give rise to the
FIGURES 16 AND 17
Fig. 16. Experiment 2c. Graft of a quail rhombencephalic primordium on a
10-somite chick embryo.
(A) Arterial trunk. The endothelium (E) belongs to the host while the musculoconnective tissue of the vessel wall is derived from the quail grafted tissue, x 780.
Feulgen-Rossenbeck's staining.
(B) FIF technique applied to the area represented in Fig. 3 A of a 11-day-old
embryo. Fluorogenic monoamine-containing cells in the wall of the common
carotid artery, as carotid body cells, are derived from rhombencephalic NC. x 140.
Fig. 17. Capillaries in the dermis, connective cells are of NC origin, endothelial
(E) cells belong to the host.
(A) Experiment \b. Graft of quail mesencephalon + anterior rhombencephalon
into a 6-somite chick embryo. Chick host was fixed after 12 days of incubation,
x 1280. Feulgen-Rossenbeck's staining.
(B) Experiment 2c. Graft of a quail rhombencephalic primordium into a 7-somite
chick embryo. Chick host was fixed after 16 days of incubation. P, Pericyte.
x 11600. Uranyl acetate-lead citrate staining.
—
*
(
Differentiation of
4
S
6
7
8
cnmitp
snmitp
somite
somite
somite
1 somite
•
U
U
y
ytJ
Q \W
I somite
-\
2 somite
LJV
r^ \
UJ 1
cephalon
rhomben-
Posterior
ruaicuui
rhombencephalori
I^L
"K
Anterior
1 constriction
1
JI /
\
I
Mesencephalon
^*»»^cells
- \ ^ mesectodermal
>v mesectoderm
Origin of
—^^^
::::::
::::::
;;;;;;
>:::-:'::: *"v.*I*I
^v
Low/er
Skeleton
Hyoid
&£:
M I!
tissue
connective
Loose
Dermis
,
.•.%•.•.•.
artery
Pulmonary
*.*f.'i*t*. I'I'I'I'IVI'.'I'IV*
.: : : : :.: :.: :.:.: :.:.:.:.:.:.:.
art ery
Syste mic
'**"•" *****•****"! *!*•*• *•*•*•*•* I*
carotid
Common
Arterial walls
Table 2. Differentiating capabilities of mesectodermal cells deriving from various levels of
mesencephalic and rhombencephalic neural crest
Mesenchymal derivatives of avian neural crest
147
same differentiated cell types as mesodermal mesenchyme: general and differentiated (elastic and fibrous) connective tissues, adipose cells, dermis,
smooth and striated muscles, bone and cartilage. In addition mesectodermal cells
are able to differentiate into odontoblasts (Horstadius & Sellman, 1946;
Sellman, 1946; De Beer, 1947; Horstadius, 1950; Wagner, 1955; Signoret, 1960;
Koch, 1965; Chibon, 1966). However, the only developmental competency
which neurectodermal mesenchyme does not seem to possess is the formation
of endothelium of the blood vessels. In the branchial and facial regions of
experimental embryos, the vascular endothelia are always host in type, and are
thus of mesodermal origin, NC cells participating only in the formation of the
vessel wall: i.e. pericytes of capillaries and musculo-connective sheath of large
vessels.
During their migration and in their final localization close relationships are
established between NC cells and various mesodermal, endodermal and ectodermal rudiments. The extent to which intertissular inductive reactions between
neurectodermal cells and those tissues control their subsequent differentiation is
not clear. Until now, the normal fate of the NC in the cephalocervical region
being only partially known, this question could not be fully investigated.
Owing to the stability of the quail nuclear marker labelling, the exclusively
neurectodermal origin of the visceral arch cartilages and membrane bones of the
lower jaw is demonstrated. A number of investigators (Horstadius, 1950;
Horstadius & Sellman, 1946; Newth, 1954; Holtfreter, 1968) have shown that
chondrogenesis is induced in NC mesenchyme by pharyngeal endoderm. The
heterotopic transplantations of cephalic neural tube into the trunk, recently
carried out in our laboratory (Le Douarin & Teillet, 1974) show that, in avian
embryos, inductive influences from other sources can play the same role:
cephalic NC cells grafted into the trunk and thus submitted to the rachis
morphogenetic field contribute to vertebral chondrogenesis.
Intimate relationships are established between mesectodermal and mesodermal
cells in striated muscle differentiation in branchial arches. The mesodermal cores
of each visceral arch derive from somitic mesenchyme (Johnston & Listgarten,
1973). From the earliest stages following completion of their migration, crest
cells invade this mesodermal core and become intermingled with the somite
deriving cells essentially concerned with the formation of skeletal muscle. The
ultrastructural study shows that crest cells give rise not only to the connective
tissue elements of muscles but also to striated muscle cells. Mauro (1961), Moss
& Leblond (1971) and others have suggested that the growth process of muscle
fibres involves the addition to the primary myotube of satellite cells. Whether
NC cells play the role of satellite cells in muscles is a hypothesis which would be
worth studying.
NC cell differentiating capabilities include secretory elements such as adrenomedullary (Le Douarin et Teillet, 1971 a), calcitonin producing cells (Le Douarin
& Le Lievre, 1970) and carotid body glandular cells.
148
C. S. LE LIEVRE AND N. M. LE DOUARIN
hv
Fig. 18. For legend see opposite.
Mesenchymal derivatives of avian neural crest
149
In previous work indeed we have demonstrated the neural crest origin of the
carotid body (CB) the cells of which are characterized by a high content of
fluorogenic monoamine (Le Douarin et al. 1972; Pearse et al. 1973). This
glandular structure differentiates in the 3rd branchial arch, in close association
with the common carotid artery. Among the connective cells which form the
vessel wall, some fluorogenic cells of neural origin are present. The nature of the
monoamine they contain seems to be the same as that of CB cells: in the chick
it is serotonin, with a yellow-greenish fluorescence; in the quail, dopamine, with
a greenish fluorescence (Pearse et al. 1973). In chick embryos grafted with a
quail rhombencephalon the monoamine containing cells of CB and common
carotid artery which exhibit the quail nuclear marker after Feulgen-Rossenbeck's staining have a greenish fluorescence, like the homologous structures of
control quail embryos.
3. Extension of NC cell migration in head and neck
In higher vertebrates the competency of neurectoderm to give rise to mesenchyme is restricted to the cephalic region of the neural axis, while in lower forms
mesectoderm also derives from trunk NC (cf. Horstadius, 1950). From our
observations and those of Johnston et al. (1973) based on the use of the quail
chick marker system, it becomes evident that the mesectoderm is quantitatively
a more extensive component of cephalocervical morphogenesis than has preF I G U R E 18
Distribution of NC mesenchymal derivatives in the lower jaw and neck of 9-day-old
chick embryo following the transplantation of quail mesencephalon and rhombencephalon into the chick host.
(I) Distribution of quail cells in the dermis and inner organs of the chick shown
in lateral and ventral views. * + * + * , Mesencephalic NC derivatives. • • • • ,
** w
••••
Rhombencephalic NC derivatives. A, B, C, Levels of the sections which are represented in figures IIA, B and C. cub, ultimobranchial body; hy, hyoid; thym, thymus;
thy, thyroid.
(II) Transverse sections at three different levels of the host showing the distribution
of NC cells.
(A) Lower jaw and tongue. Cells of mesencephalic origin * ^ * ^ * form {me)
Meckel's cartilage and membrane bones (mb) (angular, supra-angular dentary and
operculary); cells of rhombencephalic origin
form the hyoid apparatus
(bb, basibranchial; bh, basihyal; cb, ceratobranchials; eb, epibranchials; and
entoglossum) and both differentiate into loose connective tissue, dermis and muscle
cells; ch, notochord; es, oesophagus; ic, internal carotid. (B) Neck. (C) Level
of carotid bodies.
^ ^ ^ H , Tissues entirely made up of quail cells: d, dermis; c, wall of carotid
arteries; ge, enteric ganglia; cb, carotid body. • • • * » Tissues made up of a
mixture of NC and mesodermal cells: cj, loose connective tissues; x, Schwann cells
of the vagus nerve; gn, glial cells of the nodosum ganglion; connective tissue of thyroid ; p, parathyroid; tm, thymus.
j , Jugular vein; m, tracheohyoideus and tracheolateralis muscles; tr, trachea.
150
C. S. LE LIEVRE AND N. M. LE DOUARIN
viously been estimated. Due to the elongation of the neck, cells originating from
encephalic NC are carried down into the thorax with the heart and the glandular
pharyngeal derivatives. During these morphogenetic movements, mesectodermal rudiments receive mesoderm-deriving cells, making the neck a very
composite structure. Most of its ventral part originates from initially anterior
mediodorsal ectodermal material which migrates ventrad during the early stages
of embryogenesis and caudad during later morphogenesis.
Although these data result from studies on the avian embryo, it is possible
to infer the general picture of NC migration pattern in mammalian embryos.
Thus, a number of spontaneous malformations of the face and brain in the
human can be explained by defective crest cell migration and differentiation.
RESUME
Des greffes interspecifiques d'ebauches neurales rhombencephaliques et mesencephaliques
ont ete realisees entre embryons de Caille et de Poulet. Les caracteres particuliers du noyau
chez la Caille permettent de reconnaitre les cellules de l'hote de celles du greffon dans les
embryons chimeres, et d'etudier les voies de migration et les capacites de differenciation des
cellules issues de la crete neurale (CN) greffee. Le 'marquage' fourni par les cellules de Caille
est en effet stable et permet de suivre les cellules migrantes jusqu'a ce qu'elles aient atteint
un stade de complete differenciation. Dans un travail precedent base sur une experimentation
similaire on avait montre que le squelette visceral derive dans sa totalite des cretes neurales
rhombencephaliques et mesencephaliques. Les recherches rapportees ici concernent les
derives mesenchymateux de la CN autres que les tissus osseux et cartilagineux.
Apres la greffe isotopique et isochronique du rhombencephale et du mesencephale, Je
derme de la face et de la partie ventrolaterale du cou de l'hote est forme de cellules issues du
greffon et par consequent d'origine mesectodermique. II en est de meme de la paroi musculoconjonctive des gros troncs arteriels issus du coeur et derivant des arcs aortiques (troncs
brachiocephaliques, carotides communes, crosse aortique, arteres pulmonaires). Le conjonctif lache du plancher buccal, de la langue, de la face lateroventrale du cou, ainsi que la
trame conjonctive des glandes derivees du pharynx et de la bouche (glandes linguales,
thyroide, parathyroides, thymus) sont formes par du mesectoderme neural.
Les cellules mesenchymateuses originates des CN ont la potentialite de se differencier en
cellules musculaires lisses et participent aussi a l'histogenese des muscles striees des arcs
branchiaux. Elles fournissent du tissu adipeux dans le derme profond et au voisinage de la
trachee. Comme cela a ete precedemment demontre par notre groupe, les CN rhombencephaliques sont a 1'origine des cellules a calcitonine du corps ultimobranchial et des cellules
glandulaires du corps carotidien. Ces dernieres sont caracterisees par une forte teneur en
monoamines fluorigenes. Des cellules similaires ont ete detectees dans la paroi de l'artere
carotide commune par la methode defluorescenceinduite par les vapeurs de formol et nous
avons pu montrer qu'elles derivent egalement de la CN.
II apparait done que les potentialites de differenciation des cellules du mesectoderme sont
variees. Cependant il ne semble pas qu'elles soient a 1'origine de l'endothelium des vaisseaux
sanguins qui, dans nos experiences, derivent toujours de cellules mesodermiques appartenant
a l'embryon hote. La capacite de la crete neurale a fournir des derives mesenchymateux est
limitee a la region du nevraxe anterieure au niveau du 5eme somite.
Mesenchymal derivatives of avian neural crest
151
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(Received 13 December 1974)