/ . Embryol. exp. Morph. Vol. 58, pp. 251-264, 1980
Printed in Great Britain © Company of Biologists Limited 1980
251
Tissue interactions and the initiation
of osteogenesis and chondrogenesis in the neural
crest-derived mandibular skeleton of the
embryonic mouse as seen in isolated murine
tissues and in recombinations of murine
and avian tissues
By BRIAN K. HALL 1
From the Department of Biology, Life Sciences Centre,
Dalhousie University, Halifax
SUMMARY
Mandibular processes from 9- to 13-day-old embryonic mice formed both bone and
cartilage when grafted to the chorioallantoic membranes of host embryonic chicks. Isolated
ectomesenchyme, taken from 9-day-old embryos did not form bone or cartilage, while older
ectomesenchyme formed both. Recombination of the epithelial and ectomesenchymal components confirmed that the presence of the epithelium was a sufficient stimulus for the
initiation of both chondro- and osteogenesis. Recombinations between components of mouse
and chick mandibular processes showed that 9-day-old mouse ectomesenchyme could respond to chick epithelium but that, although older murine epithelia could initiate osteogenesis from the avian ectomesenchyme, epithelia from 9-day-old embryos could not. These
results indicated that an epithelial-ectomesenchymal interaction was responsible for the
initiation of both osteo- and chondrogenesis within the mandibular arch of the mouse;
that the interaction began at 10 days of gestation; that the ectomesenchyme was capable of
responding at 9 days, but that the epithelium did not acquire its ability to act on the ectomesenchyme until 10 days of gestation.
INTRODUCTION
Both the cartilaginous and the bony elements in the mandibular skeletons of
vertebrate embryos form from ectomesenchymal cells derived from the embryonic neural crest. These cells have their origin within the crests of the neural folds.
During neurulation these neural crest cells migrate away from the neural folds.
Those cells which originate in the cranial neural crest become preferentially
localized within the primordia of the cranio-facial skeleton. The ability of these
cells to form the cartilages and bones of the craniofacial skeleton depends upon
1
Author's address; Department of Biology, Life Sciences Centre, Dalhousie University,
Halifax, Nova Scotia, Canada, B3H 4J1.
17
EMB 58
252
B. K. HALL
interactions which they undergo, either during or after, their migration. Thus,
in the embryo of the lamprey, Lampetrafluviatilis L., contact with the endoderm
is required before chondrogenesis can be initiated (Damas, 1944; Newth, 1951,
1956). While it is known that cranio-facial skeletal elements are of neural crest
origin in the fishes (Jollie, 1971; Berkovitz & Moore, 1974; Schaeffer, 1977), the
role of tissue interactions in their genesis has not been studied. Interaction of
neural crest cells with pharyngeal endoderm is a prerequisite for the initiation of
chondrogenesis in the visceral skeleton of the urodele amphibians (Wagner, 1949;
Wilde, 1955; Cusimano-Carollo, 1963, 1972; Drews, Kocher-Becker & Drews,
1972; Epperlein, 1974; Corsin, 1975). However, although neural crest cells are
known to form bone in the urodeles (de Beer, 1947; Cassin & Capuron, 1977),
tissue interactions have not been studied. Nor is there any information for the
anuran amphibians (see Horstadius, 1950 and Hall, 1978a for a discussion).
The initiation of intramembranous osteogenesis within the scleral, maxillary
and mandibular skeletons of the domestic fowl results from inductive interactions between scleral, maxillary, or mandibular epithelia and post-migratory
ectomesenchymal cells from the neural crest (Murray, 1943; Tyler & Hall, 1977;
Tyler, 1978; Hall, 1978c, b, 1980; Bradamante & Hall, 1980; Tyler & McCobb,
1980). Pre-migratory neural crest cells must interact, (a) with cranial epithelial
ectoderm if they are to be able to form Meckel's cartilage later in development
(Hall & Tremaine, 1979; Bee & Thorogood, 1980), or (b) with the pigmented
retinal epithelium if they are to form scleral cartilage (Reinbold, 1968; Newsome,
1972, 1976; Hall, 1978a).
Although the details vary, epithelial-mesenchymal interactions emerge as
important prerequisites for the initiation of osteogenesis and chondrogenesis in
the sub-mammalian vertebrates. The possible existence of similar interactions
in the mammalian embryo has not been explored. Indeed, even the origin of the
mammalian cranio-facial skeleton from neural crest cells has not been substantiated experimentally. There is histological evidence for spurs of ectodermal
cells leading into the visceral arches (Bartelmez, 1952; Mulnard, 1955; O'Rahilly,
1965; Mayer, 1973); histochemical visualization of presumed neural crest cells
(Milaire, 1959; 1974); indications that high doses of vitamin A both arrest
migration of cranial neural crest cells (Morriss & Thorogood, 1978) and produce first arch defects such as Treacher Collins syndrome (Poswillo, 1974), and
the evidence that ectoderm or teratocarcinomas isolated from rat embryos form
both cartilage and bone (Levak-Svajger & Svajger, 1971; Damjanov, Solter
& Serman, 1973) - all of which provide circumstantial evidence for contributions to the facial skeleton from the neural crest.
The only study known to the author which explores the role of tissue interactions in the development of the mammalian mandibular skeleton is that of
Svajger and Levak-Svajger (1976). These workers separated first branchial arch
mesenchyme of 13-day rat embryos from their epithelia, organ cultured the
mesenchyme, and observed the subsequent development of bone and cartilage.
Skeletogenesis in the murine mandible
253
If an epithelial-mesenchymal interaction was involved, it must have taken place
before thirteen days of gestation.
The aim of the present study was to determine whether an epithelial-mesenchymal (ectomesenchymal) interaction was involved in the initiation ofchondrogenesis and/or osteogenesis within the mandibular skeleton of the embryonic
mice. To this end, whole mandibular processes, and mandibular ectomesenchyme, separated from the mandibular epithelium by treatment with proteolytic enzymes, were grafted to the chorioallantoic membranes of host embryonic
chicks. (This vascularized site is preferred over organ culture when studying
osteogenesis.) The separated components were also either recombined with one
another or combined with components isolated from the mandibular processes
of embryonic chicks and grafted to chorioallantoic membranes. Histological
analysis was used to document the presence or absence of cartilage and bone in
the recovered grafts.
MATERIALS AND METHODS
Animals
ICR Swiss albino mice obtained from Bio-breeding Laboratories of Canada,
Ottawa, Ontario, were used as the experimental animal. Mice were weaned at
4 weeks of age, housed with three or four of the same sex per cage, and fed
Purina lab. chow and water ad Hbidum. Matings were carried out using animals
which were at least 8 weeks old. Each male was placed with from one to three
females overnight (16.00 to 08.00 hours), 08.00 hours was designated as the
commencement of day zero of the pregnancy. Pregnancy was determined by
presence of a vaginal plug and by weight gain, as measured on alternate days.
Embryos
Pregnant females were killed by an overdose of ether, the embryos removed by
dissection under sterile conditions and aged using the morphological series of
stages of Griineberg (1943). Embryos of 9-13 gestational days were used in this
study. The 9-day-old embryos had well developed mandibular processes, lacked
posterior limb buds, and had unsegmented posterior somitic mesoderm. By 13
days of gestation the embryos were recognizably murine, with five rows of
whiskers containing hair follicles, footplates on the limbs and marked segmentation to the distal tips of the tails.
Mandibular processes
Mandibular processes were dissected from these embryos and either grafted
intact, or separated into their epithelial and ectomesenchymal components and
then grafted. Separation was achieved by placing the processes into a solution of
trypsin and pancreatin in calcium- and magnesium-free Tyrode's solution
(257 mg bovine pancreatic trypsin + 43 mg porcine pancreatic pancreatin/10
17-2
254
B. K. HALL
ml. both obtained from BDH Chemicals, Toronto, Ontario) for 2 h at 4° C.
The mandibular processes were then placed into a solution of the synthetic
culture medium BGJb (GIBCO, Grand Islands, N.Y.) and Horse Serum (1:1) to
stop the enzymic digestion and the epithelia separated from the ectomesenchyme
by microdissection using sharpened hypodermic needles. A similar separation
was performed on mandibular processes obtained from embryonic chicks which
had been incubated for 4 days and attained Hamburger & Hamilton (1951)
stage 22.
Grafting
Whole mandibular processes, recombined components from the mouse, or
components combined with epithelia or ectomesenchyme from mandibular
processes of the chick, were grafted to the chorio-allantoic membranes of 8- or
9-day-old embryonic chicks following the procedure outlined by Hall (1978 c).
Briefly, the mandibular process or epithelium was placed onto a square of sterile,
black, Millipore filter (0-45/*m porosity, 125-150/mi thick, Millipore Filter
Corp., Montreal, Quebec), mandibular ectomesenchyme was placed in direct
contact with the epithelium and the intact, or recombined processes, grafted
for 7 days.
Histology
Grafts were recovered, dissected away from the chorioallantoic membranes,
fixed in neutral buffered formal saline, processed histologically, serially sectioned
and stained with haematoxylin, alcian blue and chlorantine fast red (modified
from Lison, 1954).
RESULTS
Intact mandibular processes
The chorioallantoic membrane (CAM) has previously been shown to be a
graft site which supports the initiation and maintenance of chondrogenesis and
osteogenesis from avian mandibular processes (Tyler & Hall, 1977; Hall, 1978£,
1980). To determine whether the CAM would support cartilage and bone
formation from murine tissue, and to determine whether the initiation of either
tissue was dependent upon the age of the donor embryo, whole mandibular
processes from mouse embryos of 9-13 gestational days were grafted to the
CAMs of host embryonic chicks. Osteogenesis and chondrogenesis were initiated in at least 75 % of the grafts, irrespective of the ages of the mouse embryos
(column I, Table 1). Typically, the cartilage consisted of one or two rods of
hypertrophic cartilage, with scant amounts of extracellular matrix and an
ill-defined perichondrium. The bone typically consisted of one or two ossicles of
trabecular, membrane bone, usually separated from the cartilage by intervening connective tissue and often resting on the Millipore Filter substrate.
3
7
10
18
7
4
6
6
16
6
Bone
4
7
10
21
8
0
13
5
14
18
12
16
13
5
1
Cartilage Bone
8
17
6
15
18
Cartilage Bone
A
III
Recombined
epithelium and
ectomesenchyme
A
4
5
3
7
10
Cartilage Bone
I
IVf
Mouse ectomesenchyme +
chick epithelium
9
7
4
7
10
vt
6
7
12
6
10
0
5
12
4
6
Cartilage Bone
Chick ectomesenchyme + mouse
epithelium
6
7
12
6
11
* n, number of grafts analysed.
t The chick tissues were obtained from embryos of H. H. stage 22 (4 days of incubation). When chick mandibular ectomesenchyme was recombined
with chick epithelium and grafted, all grafts formed both cartilage and bone.
9
10
11
12
13
Age rembryo Cartilage
(days)
Intact mandibular
processes
II
Isolated ectomesenchyme
Table 1. Number of grafts forming cartilage and/or membrane bone
N
256
B. K. HALL
Fig. 1. Membrane bone (B) developed when mandibular ectomesenchyme from a
10-day-old embryo was grafted to the chorioallantoic membrane of a host embryonic
chick. O, osteocyte; M, Millipore filter substrate. Alcian blue, chlorantine fast red
and haematoxylin.
Both the large percentage of grafts forming skeleton tissues and the advanced
state of differentiation attained by the tissues, indicated that the chorioallantoic
membrane was a suitable graft site for these mouse cells. The mandibular
epithelium also continued to differentiate and formed a multi-layered squamous
epithelia, with numerous buds extending into the underlying ectomesenchyme.
Isolated mandibular ectomesenchyme
Mandibular ectomesenchyme which had been dissected away from the
mandibular epithelium after treatment of the intact mandibular process with
trypsin and pancreatin was then grafted. Provided that the embryo was at least
10 days old, cartilage and membrane bone formed in a similar percentage of
grafts to that seen when intact mandibular processes were grafted, viz. at least
75 % (see column II, Table 1, Fig. 1). The lack of murine epithelial cells in these
grafts attested to the adequacy of the tissue separations. Although the amount
of cartilage and bone present was not quantified, it was not noticeably less than
that obtained from intact mandibular processes.
The ectomesenchyme isolated from the 9-day-old embryos behaved quite
differently. Chondrogenesis was not initiated in any graft, and osteogenesis
only in one out of the eight grafts. The grafts consisted of a loose meshwork of
etomesenchymal cells (Fig. 6).
Thus, mandibular ectomesenchyme would only form bone and cartilage if the
epithelium was present until 10 days of gestation.
Skeletogenesis in the murine mandible
257
Recombined epithelia and ectomesenchyme
To confirm that the inability of ectomesenchyme from 9-day-old embryos to
form cartilage and bone was a consequence of its isolation from mandibular
epithelium, these two components were recombined and grafted to the CAMs of
host embryonic chicks. Recombinations of components from 10- to 13-day-old
embryos served as a control for the procedure. In these specimens chondrogenesis and osteogenesis were initiated at the same, on a higher rate, than when
intact mandibular processes or isolated ectomesenchyme were grafted (column
III, Table 1, Fig. 2).
Having confirmed that the procedure of recombination did not inhibit
skeletogenesis in these older tissues, epithelia and ectomesenchyme from mandibular processes of 9-day-old embryos were recombined and grafted. Both
osteogenesis and chondrogenesis were initiated in such grafts (column III,
Table 1), indicating that the lack of cartilage and bone in the isolated ectomesenchymes was attributable to the absence of the mandibular epithelia.
Heterospecific recombinations
Mandibular processes from embryonic mice and from H. H. (Hamburger &
Hamilton, 1951) stage-22 embryonic chicks were separated and recombined
heterospecifica lly.
It is known that mandibular epithelium from the H. H. stage-22 embryonic
chick will allow chick mandibular ectomesenchyme to initiate osteogenesis
(Tyler & Hall, 1977; Hall, 1978Z?). Would chick mandibular epithelium substitute for murine epithelium in initiating osteogenesis? What of the initiation of
chondrogenesis? Chondrogenesis in the chick mandible is not dependent on
the presence of the mandibular epithelium. Would the chick epithelium allow
the murine ectomesenchyme to form cartilage?
In the first recombination, mouse mandibular ectomesenchyme was recombined with chick mandibular epithelium and grafted. Ectomesenchymes from
10- to 13-day-old mice were used as controls. Both bone and cartilage formed in
these recombinants (column IV, Table 1, Figs. 3, 4). Cartilage and bone also
formed in four out of the nine grafts containing 9-day murine ectomesenchyme
(Fig. 7). (In assessing these results all recovered grafts were counted. Given that
the amount of epithelial-ectomesenchymal contact varied from graft to graft,
the positive results reported represent a minimal estimate.) This recombination
indicated that mandibular ectomesenchyme from 9-day-old embryonic mice
could respond to chick mandibular epithelium by initiating both chondrogenesis and intramembranous osteogenesis.
In the second recombination, H. H. stage-22 chick mandibular ectomesenchyme was recombined with mouse mandibular epithelium of various ages
(column V, Table 1). As noted above, the initiation of chondrogenesis in such
avian ectomesenchyme is not dependent upon the presence of mandibular
258
B. K. HALL
Skeletogenesis in the marine mandible
259
Fig. 5. Cartilage (C) and bone (B) both formed when mandibular ectomesenchyme
from an H. H. stage-22 chick embryo was combined with mandibular epithelium from
a 13-day-old mouse embryo. Contrast the cartilage with that in Figs. 2 and 3. The
murine epithelium (e) has also continued to differentiate. M, Millipore filter substrate. Alcian blue, chlorantine fast red, haematoxylin.
epithelium. Therefore virtually all of these grafts contained nodules or rods of
cartilage (Table 1, Fig. 5). Osteogenesis was also initiated in the ectomesenchyme,
provided that the murine epithelium had been obtained from embryos of at
least 10 days of gestation (column V, Table 1, Fig. 5). The chick ectomesenchyme also maintained the continued, normal differentiation of the mouse
epithelium (Fig. 5).
Epithelium from 9-day-old embryos was not able to induce bone formation
from the avian ectomesenchyme, but epithelial differentiation was maintained.
Therefore (a) the epithelial-ectomesenchymal interaction is neither species
nor class-specific; (b) ectomesenchyme from all aged mice is able to respond to
chick epithelia by initiating both osteogenesis and chondrogenesis, and (c) the
epithelium from the mouse does not acquire the ability to induce osteogenesis
F I G U R E S 2-4
Figs. 2, 3. Cartilage (C) and bone (B) formed when mandibular epithelium and
ectomesenchyme from this 12-day-old embryo were recombined (Fig. 2) and when
mouse mandibular ectomesenchyme was combined with chick mandibular epithelium
(Fig. 3). Note the similarity of the cartilages in the two combinations. Alcian blue,
chlorantine fast red and haematoxylin.
Fig. 4. A higher magnifications of the bone seen in Fig. 3 to show the trabeculae of
bone (B), osteocytes (O), osteoblasts (ob) and flattened ectomesenchymal cells (e)
presumed to be osteoprogenitor cells. Alcian blue, chlorantine fast red, haematoxylin.
260
B. K. HALL
Fig. 6. Ectomessnchyme isolated from 9-day-old embryos and grafted to the
chorioallantoic membrane formed a loose meshwork of cells. Neither chondrogenesis nor osteogenesis is seen. Alcian blue, chlorantine fast red and haematoxylin.
Fig. 7. Bone formed when mandibular ectomesenchyme from a 9-day-old mouse
embryo was combined with mandibular epithelium from an H. H. stage-22 embryonic
chick and grafted. Alcian blue, chlorantine fast red and haematoxylin.
Skeletogenesis in the marine mandible
261
or chondrogenesis until 10 days of gestation. The lack of bone or cartilage
development from ectomesenchyme isolated from 9-day-old embryos reflected
the absence of an epithelial signal rather than the inability of the ectomesenchyme
to respond to such a signal.
DISCUSSION
Although a considerable amount is known about chondrogenesis, surprisingly
little information is available on the microenvironmental control of the initiation of intramembranous osteogenesis. It is known (a) that calvarial osteogenesis is only initiated after pre-osteogenetic cells have undergone an inductive
interaction with the brain or spinal cord (Schowing, 1968); (b) that scleral bones
only form in the avian eye after ectomesenchymal cells have interacted with
epithelial scleral papillae (Coulombre, Coulombre & Mehta, 1962); (c) that the
sub-periosteal bone around the shafts of developing long bones only forms after
perichondrial cells have been in contact, either with hypertrophic chondrocytes
or with their extracellular products (Shimomura, Yoneda & Suzuki, 1975); and
(d) that membrane bones of the avian mandible and maxilla only form after
ectomesenchymal cells have interacted with the appropriate epithelia (see
Introduction). The dentary of the mouse may now be added to this list.
The present study demonstrates that cells of the mandibular process must
interact with the mandibular epithelium if they are to initiate the sequence of
differentiative events which culminate in the differentiation of osteoblasts and
osteocytes, the deposition of osteoid and the mineralization of that osteoid to
form bone. That this developmental interaction had not occurred by 9 days
of gestation was shown by the fact that ectomesenchyme isolated from such
aged embryos failed to form bone (except in one case (column II, Table 1), where
epithelial cells were found to have been left on the ectomesenchyme). The reinitiation of osteogenesis in recombinants confirmed that presence of the epithelium was a sufficient osteogenetic stimulus for these ectomesenchymal cells.
Therefore, the murine dentary resembles the membrane bones of the avian
mandible (Tyler & Hall, 1977) in requiring a mandibular epithelial-ectomesenchymal interaction for its initiation.
Unlike Meckel's cartilage in avian embryos, initiation of the murine cartilage
was dependent upon the presence of the mandibular epithelium. In the chick the
ectomesenchymal cells are already able to form cartilage before they reach the
mandibular arch (Tyler & Hall, 1977; Hall & Tremaine, 1979), apparently
because of prior interactions with cranial epithelial ectoderm (Bee & Thorogood,
1980). In the urodele amphibians the equivalent interaction is between cells
derived from the neural crest and the pharyngeal endoderm (see Introduction).
Thus, these three classes of vertebrates (Amphibia, Aves, Mammalia) share
epithelial-ectomesenchymal interactions as a common component for the
initiation of chondrogenesis in the mandibular skeleton, differing only in the
nature of the signalling epithelium. Whether these interactions are mediated via
262
B. K. HALL
epithelial cell products, as is the initiation of scleral chondrogenesis (Newsome,
1972, 1976), or via direct cell-to-cell contact, remains to be determined. The
osteogenetic interaction in the chick requires (a) that the epithelium be vital,
and proliferating (Hall, 1980), and (b) the presence of epithelially-derived
collagen and glycosaminoglycans (Bradamante & Hall, 1980).
It was further shown that these interactions, like those which are responsible
for the formation of epidermal structures such as hairs, feathers and teeth
(Kollar, 1972; Dhouailly, 1975), were neither species nor class specific. Mouse
ectomesenchyme of any age responded to chick epithelium by forming both
bone and cartilage. Chick ectomesenchyme responded to mouse epithelium
older than 9 days by forming bone and cartilage. From these results I concluded
that mouse mandibular ectomesenchyme was capable of responding to epithelial influences at 9 days of gestation but that the murine epithelium was not
active until 10 days of gestation. A qualitatively similar temporal pattern has
been shown for inductively active epithelia from the chick (Hall, 1978 b). In
each combination the bone and cartilage formed were typical of the species
providing the ectomesenchyme (cf. Fig. 4 and 5), indicating that, while the
epithelium was required to initiate osteo- or chondrogenesis, it played no role
in determining the type of cartilage or bone which formed.
The identification of these tissue interactions provide a new basis for the study
of malformations affecting the mammalian facial skeleton. Failure of these
tissues to interact, interaction with fewer than normal ectomesenchymal cells,
or delayed interactions, could all alter craniofacial growth and/or morphogenesis. Mutant mice, or embryos from females given teratogens, could be used
to provide epithelia or ectomesenchyme for recombination with components
from wild-type or untreated embryos to further explore the role of such tissue
interactions in development.
This research has been supported by the National Sciences and Engineering Research
Council of Canada (Grant no. A5O56). The Dalhousie University Research Development
Fund in the Sciences provided funds to purchase the CO2 Incubator. Pilot grafts of whole
mandibular process and ectomesenchyme from the older embryos were performed by P.
Elliott. Sharon Brunt provided expert technical assistance.
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{Received 9 January 1980, revised 14 March 1980)