Download Spatial organization of the epithelium and the role of neural crest

155
Development 103 Supplement, 155-169 (1988)
Printed in Great Britain © The Company of Biologists Limited 1988
Spatial organization of the epithelium and the role of neural crest cells
in the initiation of the mammalian tooth germ
A. G. S. LUMSDEN
Division of Anatomy,
United Medical and Dental Schools, Guy's Hospital, London SEI 9RT
Summary
Teeth develop from composite organ rudiments that
are formed through the interaction of oral epithelium
and mesenchyme of the first branchial arch; cells of
the former differentiate into enamel-secreting ameloblasts whereas those of the latter differentiate into
dentine-secreting odontoblasts. Experimental analysis
of odontogenic tissue interactions in mammalian embryos has focused on the late developmental stages of
morphogenesis and cytodifferentiation; little is known
about initial pattern-forming events, during which
presumptive tooth-forming cells are specified and the
sites of tooth initiation become established. It requires
to be shown, for example, whether the mesenchymal
cells of mammalian teeth are derived, like those of
amphibians, from the cranial neural crest, and if so,
whether these form a specified subpopulation in the
neural folds. Alternatively, are they specified after
migration into the mandibular arch, possibly by
interaction with the oral epithelium?
The developmental potentials of mouse embryo
premigratory cranial neural crest cells (CNC explanted from the caudal mesencephalic and rostral
metencephalic neural folds) have been studied in intra-
ocular homograft recombinations with various regions
of embryonic surface ectoderm. Cartilage, bone and
neural tissue developed in all combinations of CNC
and epithelium. Teeth formed in combinations of CNC
with mandibular arch epithelium but not in combinations of CNC with limb bud epithelium. Teeth also
formed in combinations of mandibular arch epithelium with neural crest explanted from the trunk
level. These results indicate that mammalian neural
crest has an odontogenic potential but that this is not
restricted to the crest of presumptive tooth-forming
levels. Normal migration appears not to be a prerequisite for expression of odontogenic potential but this
does require an interaction with region-specific epithelium. It is reasonable to infer that during normal
development the neural crest that enters the mandibular arch is odontogenically unspecified before or
during migration and that the oral epithelium is the
earliest known site of tooth pattern.
Introduction
Lumsden, 1979). Morphogenetic movements of the
apposed epithelial and mesenchymal layers generate
a spatial configuration of their interface which varies
with tooth position, being simply spatulate or conical
in the case of incisors or canines and folded into a
more complicated shape of elevations and depressions in the case of molars. Specialized secretory
cells then differentiate from the single layers of
precursors on either side of the folded basement
membrane; odontoblasts differentiate from mesenchyme cells and secrete dentine matrix, ameloblasts
differentiate from epithelial cells and secrete enamel
matrix. The two matrices are deposited back to back
The dentition is not only a major component of the
mammalian craniofacial complex but also it provides
us with a valuable model of development; combined
in a single organ system are the phenomena of spatial
organization, symmetry, acquisition of complex form
and organ-specific cytodifferentiation.
Development of an individual tooth (Fig. 1) is
characterized by an extensive series of reciprocal
epithelial-mesenchymal interactions which lead the
organ rudiment from its initiation through morphogenesis to ultimate cytodifferentiation (Kollar &
Key words: neural crest, tooth germ, odontogenesis,
amelogenesis, tissue interaction.
156
A. G. S. Lumsden
EK
Cranial
neural
E9
crcst
migralion ?
Heterologous^
E10 mesenchvme
JAW
MESENCHYME
^
|
^
Heterologous
epithelium
.,
Dental
mesenchyme
Ell
ORAL
ECTODERM
\•
Dental
epithelium-
f
E12
E13
Dental
papilla
—
E14
Dermis
Enamel
organ
E15
E16
Preodontoblasts
Prcameloblasts
E17
Odontoblasts
EI8
E19
Predentine
Dentine
Ameloblasts
Epidermis
Enamel
Fig. 1. Scheme of events during tooth development in the mouse embryo. Postconception age (plug = day 0) in lefthand column. Tissue interactions are shown by dashed arrows.
against the template formed originally by soft tissue,
the shape of which becomes effectively fossilized by
subsequent mineralization of dentine and enamel.
In few other epithelial-mesenchymal organs do
both component tissues ultimately contribute organspecific materials and none displays the complex
organization of extracellular matrices that characterizes the tooth. For these reasons, and because late
stages of development are easily accessible to experimentation, interest in the developing tooth as a
model system has focused almost exclusively on
differentiation events (reviewed by Thesleff & Hurmerinta, 1981; Ruch, 1985). This paper, however, is
concerned with the initiation period, during which
presumptive dental epithelium and mesenchyme become specified and the pattern of odontogenic loci
around the jaws is established.
Experiments with amphibian embryos (Platt, 1893,
1897; Landacre, 1921; Adams, 1924; Stone, 1926;
Raven, 1931; Sellman, 1946; de Beer, 1947; Chibon,
1966, 1970) have revealed that dental mesenchyme is
derived from the cranial neural crest and that enamel
organs of premaxillary, maxillary and dentary teeth
develop from ectoderm. The respective roles played
by (ecto)mesenchyme and epithelium in the initiation
of tooth development have been investigated by
Wagner (1949, 1955), in a series of xenoplastic transplants between urodele and anuran larvae; experiments that depended on the fact that whereas the
former possess true teeth the latter do not. When
urodele (Triturus) larvae received unilateral orthotopic transplants of anuran (Bombina) cranial neural
crest, the chimaeric larvae developed teeth in which
the dental papilla cells derived from the frog (Fig. 2).
Although it is not expressed during normal larval
development, the anuran neural crest has the capability both of inducing enamel organ formation and of
being induced to form dentine. In a complementary
experiment, urodele neural crest was grafted orthotopically into anuran hosts; a urodele-type visceral
skeleton was formed but no teeth developed. Henzen
(1957) replaced a region of the presumptive stomatodeal ectoderm of anuran larvae with orthotopic
transplants from urodele larvae; in the area of the
graft, normal urodele-type teeth developed from
chimaeric tooth germs.
Taken together, these results suggest that the
stomatodeal ectoderm provides a signal for the initiation of tooth development and that this signal is
lacking from the oral epithelium of larval anurans.
The competence of anuran neural crest to form teeth,
normally only revealed at metamorphosis, was revealed prematurely by the inductive influence of
urodele epithelium. The reported ability of excised
cranial neural folds of Ambystoma to form teeth in
ectopic sites (Avery, 1954) indicates, however, that a
degree of determination has already been acquired by
the crest prior to migration but the requirement for
Initiation of mammalian tooth germ
Fig. 2. Xenoplastic orthotopic graft of anuran (Bombina)
neural crest into urodele (Triturus) neurula (B) gave rise
to a larva (C) whose branchial skeleton derived from the
donor. The larva subsequently developed chimaeric teeth
(D) with enamel organs derived from the host and dental
papillae from the donor (after Wagner, 1949).
interaction with specific epithelium suggests that
determination is incomplete (i.e. cells are not committed).
In the mouse embryo (Fig. 1), the initiation period
extends from embryonic day 8 (E8), when crest cells
first emerge from the cranial neural folds, to Ell
when definitive tooth germs appear. Between E12
and E15, the precise form of the individual tooth
(incisor, first or second molar) is established.
Although some preliminary data suggested that tooth
type might be dictated by the epithelium (Dryburgh,
1967; Miller, 1969) this has not been substantiated;
rather, it has become clear that after Ell the dental
papilla mesenchyme is not only capable of instructing
157
the precise form of morphogenesis in dental epithelia
(Kollar & Baird, 1969; Heritier & Deminatti, 1970;
Kollar, 1972; Ruch, 1984) but it is also able to induce
enamel organ morphogenesis (with subsequent
ameloblast differentiation and amelogenesis) in heterologous epithelia, for example, the presumptive
plantar epidermis of homologous embryos (Kollar &
Baird, 1970; Ruch, Karcher-Djuricic & Gerber, 1973)
and chick embryo oral epithelium (Kollar & Fisher,
1980).
Thus one of the principal outcomes of development
during the initiation period is the acquisition by
dental mesenchyme of a degree of determination
which is manifested in its ability to instruct competent
epithelia to participate in enamel organ morphogenesis. The patterning processes involved in initiation,
controlling the timing and positioning of tooth primordia, however, must precede the morphological
appearance of the germ. Although selfevident, this is
often overlooked; the ability of E l l + dental mesenchyme to instruct nondental epithelia has been
assumed also to be a property of the presumptive
dental mesenchyme and adduced as evidence that it is
in the mesenchyme, and not in the epithelium, that
the pattern resides (Ruch etal. 1973; Ruch, 1984). Yet
there is no direct evidence for mammals that could
implicate either the epithelium or the mesenchyme as
the primary site of pattern. Because the teeth of
mammals and amphibians are regarded as homologous it has been assumed that the mesenchyme of
mammalian teeth also is contributed by the neural
crest, and assertions to this effect appear, often
without qualification, in the literature. Notwithstanding recent discoveries that the greater part of avian
craniofacial mesenchyme is crest derived (Le
Douarin, 1982; Noden, 1984), the neural crest origin
of odontoblasts and dental pulp cells, or indeed any
cranial mesenchyme derivative, has yet to be directly
demonstrated in mammals. It is important to know
whether the dental mesenchyme of mammalian teeth
is indeed derived from the cranial neural crest and to
know which layer, epithelium or mesenchyme, first
acquires tooth-specific properties; is there a specified
subpopulation in the neural folds (as suggested by
Ruch, 1984, 1985)? Are crest cells odontogenically
specified during migration? Or do they only become
specified after migration as a result of an interaction
with the epithelium, in which tooth-specific inductive
activity has already been prepatterned?
Development of the cranial neural crest in
mammals
Attempts at labelling the premigratory cranial crest
and orthotopic grafting of extrinsically marked crest
158
A. G. S. Lumsden
3A
Fig. 3. Transverse lj.im sections through an E8 7-somite mouse embryo at midbrain (A) and hindbrain (B) levels
showing premigratory and emerging neural crest cells. The regions excised as CNC explants are delineated (compare
region shown in A with Fig. 5A). Bars, 100^m.
Section through
rhombomere A
mm
E8
6-12 somites
cells have met with only limited success in mammalian embryos (Johnston & Hazelton, 1972; Tan &
Morriss-Kay, 1986).
Our knowledge of the mammalian crest consists
mainly of observations on normal mouse and rat
development which are limited, by the lack of crestspecific markers in these species, to the periods of
emergence and early migration during which crest
cells can be distinguished by topography and morphology. Even during this brief period, however,
rodent development has been observed to differ from
chick, particularly as regards the onset of cranial crest
cell migration. In the avian embryo, crest cells
emigrate from the neuroepithelium at or shortly after
neural tube closure (Johnston, 1966; Bancroft &
Bellairs, 1976). In rodent embryos, cranial crest
migration begins when the neural folds are open and,
at mesencephalic and rostral metencephalic (trigem-
Fig. 4. Summary of
experimental procedure; for
explanation see text. CNC,
cranial neural crest; TNC, trunk
neural crest; ME, mandibular
arch epithelium; MM,
mandibular arch mesenchyme;
LE, fore limb bud epithelium;
LM, fore limb bud mesenchyme.
Bars, 1 mm.
inal) levels (Fig. 3A), when the surface of the neural
plate is still convex (Nichols, 1986). At the caudal otic
level (vagal crest) cells emigrate from a closing or
already closed neural tube whereas at the rostral otic
level (hyoid crest), the last cranial region to commence migration, cells emigrate from still open Vshaped neural folds (Fig. 3B). In mammals, therefore, cranial crest cells begin to emigrate before,
during and after neural tube closure depending on
their location on the neuraxis, but not in a simple
rostrocaudal sequence nor in simple relation to the
caudorostral closure of the neural tube (Tan &
Morriss-Kay, 1985).
Although the neuroectoderm is classically regarded
as the source of neural crest cells in all vertebrates
(Adelman, 1925), Verwoerd & van Oostrom (1979)
considered the origin in the mouse to be the epidermal ectoderm lateral to the margins of the neural
Initiation of mammalian tooth germ
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....
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159
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5 A.,
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•
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.
i
Fig. 5. (A) Transverse 1 ^m section through a CNC explant from a 7-somite embryo at the rostral hindbrain level
(compare with region delineated in Fig. 3B). Neural crest cell staining has been enhanced by cetyl pyridium chloride
fixation (Nichols, 1981). (B) Transverse lfOTi section through the open posterior neuropore region of a 6-somite embryo
showing premigratory trunk level neural crest which, on the left side, has been excised as TNC explant. e, surface
ectoderm; NC, neural crest cells; ne, neuroepithelium. Bars, 20^m.
plate. Nichols (1981, 1986) used a fixative containing
cetyl pyridium chloride to enhance the light microscope visibility of emerging crest cells and has described an initial period of emigration from the lateral
ectoderm followed by migration from the neuroectoderm. Ectomesenchyme that comes to occupy the
mandibular arch is thought to originate from the
lateralmost region of the midbrain (maxillary process) and rostral hindbrain (mandibular process)
neural folds, where later more medial emigration
contributes cells to the trigeminal ganglion (Nichols,
1986).
The earliest crest cells leave the ectoderm at the 4+
somite stage (E8 in the mouse) from mesencephalic
and rostral metencephalic levels (Nichols, 1981).
Because the neuroepithelium is still wide open at this
time, these cells emerge at a level which is approximately lateral to the dorsal margin of the pharynx,
near the root of the mandibular arch (Nichols, 1986).
By the 6-somite stage, the mesencephalic crest has
formed a diffuse array in the future periocular region,
but the rostral metencephalic crest has formed a
compact column which stretches from the edge of the
neural plate ventrad through a narrow transient
subectodermal space into the first pharyngeal arch.
By the 7- to 8-somite stage, the first arch becomes
discernible as it swells with immigrating ectomesenchyme. Migration from the neuroepithelium at the
metencephalic level continues through the 11-somite
stage (E8-5 in the mouse) thereafter dwindling as the
mandibular arch increases in prominence (Tan &
Morriss-Kay, 1985). Milaire (1959) and Pourtois
(1964) used histochemical methods to distinguish
ectomesenchyme (on account of its purportedly
higher RNA content) and noted that immigration
into the mandibular arch is complete by E9-5-E10 in
the mouse, whereas recent studies of the osteogenic,
chondrogenic and odontogenic potential (Hall, 1980;
Lumsden, 1984a; Lumsden & Buchanan, 1986) of
mandibular arches explanted from early mouse embryos, suggest that the full complement of ectomesenchyme is already present by E9.
Ontogenlc potential of the cranial neural crest
Although we are not yet able to define the normal
migration paths and fates of neural crest cells in
mammals, it is possible to explore the potentials of
neural crest populations and the nature of tissue
interactions leading to morphogenesis and differentiation of tissues derived from ectomesenchyme. This
approach (Lumsden, 1984b,c, 1987) involves explanting premigratory neural crest from mouse embryos
and growing these cells as homografts either alone or
in association with a variety of explanted ectodermal
epithelia (Fig. 4).
Cranial neural crest (CNC) was obtained from E8
(6- to 12-somite) CD1 albino mouse embryos by
excision of the free margins of the neural plate at
posterior mesencephalic and metencephalic levels
rostral to the preotic sulcus. The crest in this region is
demarcated both medially and laterally by sulci and is
thereby clearly distinguishable under the dissecting
160
A. G. S. Lumsden
t ~
w \ . ...
-w
. . . •'>•*&.' "-V
Initiation of mammalian tooth germ
microscope and separable from adjacent neuroectoderm. Since neural crest cells are thought to emerge
also from the lateral ectoderm during early stages
(Nichols, 1981, 1986) this was included in the explant
(Fig. 5A). Explants from early embryos (6-somite)
would have included presumptive ectomesenchyme
of the mandibular arch, whereas explants from later
embryos would have included presumptive crest only
of more dorsal regions.
Ectodermal epithelia were obtained from the mandibular arches (ME) of both E9 and ElO embryos and
from the forelimb buds (LE) of ElO embryos by
treatment of the explanted organ rudiments either
with 0-5-1 % crude trypsin at 4°C (Kollar & Baird,
1969) for 45min or with 0-05 % collagenase at 37°C
(Kratochwil, 1969) for 20min at 4°C followed by
20min at 37°C. The mesenchymal cores of these
organ rudiments (MM and LM) were also used in
recombination experiments. Enzymic digestion was
continued long enough for the epithelia to float freely
away from the mesenchyme either spontaneously or
with gentle flushing through a siliconized pipette, a
procedure that ensured that epithelia were not contaminated by adherent mesenchyme cells. Mandibular arch epithelium was taken from a later developmental stage than the CNC explants to allow for the
migration period. Emergence begins at 4 somites and
continues in the region rostral to the preotic sulcus
until 11 somites; although there are no data for
migration rates in the mouse, rates for chick crest of
40jumh~' in vitro (Rosavio, Delouvfe, Timpl,
Yamada & Thiery, 1982) and 20-30^mh~' in vivo
(Loring & Erickson, 1987) suggest that migrating
crest would normally encounter and interact with
mandibular epithelia between 6 and 24 h following
first emergence from the ectoderm.
Fig. 6. Paraffin-wax-embedded 7^m sections of grafts
recovered after 12-14 days intraocular development. (A)
Mandibular epithelium (ME) graft showing
differentiation of keratinized, cyst-like epithelium. Phase
contrast. (B) Cranial neural crest (CNC) graft showing
differentiation of cartilage. (C) CNC + limb bud
epithelium (LE) graft showing differentiation of
membrane bone. (D) CNC + mandibular arch epithelium
(ME) graft showing formation of sinus hairs and tooth.
(E) CNC + ME graft with multiple tooth formation; all
are embedded in membrane bone. (F) CNC+ME graft; a
crypt-like capsule of membrane bone (arrowed) has
developed beneath and around the sides of the tooth, but
not over the cuspal region. (G) Detail of Fig. 6F showing
enamel and dentine matrices and periodontal tissues.
Lison's alcian blue-chlorantine fast red. Bars, 100^m.
ace, anterior chamber; b, bone; c, cartilage; co, cornea;
/, iris; Ic, lens capsule; /, tooth germ; h, sinus hair; e,
enamal matrix; d, dentine matrix; a, ameloblast layer; o,
odontoblast layer.
161
Explants were grafted singly and, in the case of
enzymically separated tissues also in normal, reciprocal and abnormal recombinations, to the anterior
chambers of homologous adult male mice eyes
(Lumsden, 19846). The anterior chamber of the eye
provides near optimal conditions for development
and growth; for example, tooth germs explanted at
very early stages develop in oculo at the same or a
faster rate than in situ (Lumsden, 1984a) to form teeth
of near normal size and shape, with normally deposited and mineralized dentine and enamel matrices
(Lumsden, 1979). Furthermore, grafting to the anterior chamber is quick, bloodless and amongst the
least harmful types of experimental surgery that can
be performed (Olson, Seiger & Stromberg, 1983). All
grafting operations were performed under deep nembutal anaesthesia.
Recombination of heterotypic tissues was performed in oculo: freshly explanted and separated
tissue fragments were implanted individually into the
anterior chamber by injection though a small incision
in the cornea and moved into juxtaposition on the
surface of the iris by gentle massage of the cornea
before being finally lodged together in the iridocorneal angle. Although this procedure incurred losses
through failure of the explants to remain contiguous,
over 50 % of intraocular recombinations underwent
subsequent tissue interactions and differentiated epitheliomesenchymal structures in oculo. Grafts
became vascularized by ciliary vessels within 1-2 days
and grew to fill the chamber within 12-14 days.
Mandibular arch epithelium
Grafts of E9 or ElO mandibular epithelium grew little
and differentiated into keratinizing surface epithelium (Fig. 6A). The lack of mesenchymal development in enzymically separated and grafted epithelia
indicated that these were uncontaminated by mesenchyme cells and that epithelial-mesenchymal interactions in recombination grafts of epithelia with CNC
involved only mesenchyme derived from the latter.
Cranial neural crest
Grafts of cranial neural crest did not form odontoblasts and no evidence of tooth morphogenesis was
visible in the grafts, but neural tissue and rods or
small nodules of cartilage were formed (Fig. 6B).
Cranial neural crest-limb epithelium recombinations
In grafts of CNC combined with LE, perichondral
bone formed around cartilage nodules and islands of
woven membrane bone developed (Fig. 6C). No
invasive patterns of epithelial morphogenesis were
observed.
162
A. G. S. Lumsden
Table 1. Results of various experimental and control grafts showing the number recovered (n) and the
number with teeth displaying both enamel and dentine matrices (T)
T/n
(a) Experimental recombinations
Cranial NC (E8) + limb epithelium (E9-11)
Cranial NC (E8) + mandibular epithelium (E9)
18
117
Mandibular mesenchyme + limb epithelium (E9)
Mandibular mesenchyme + limb epithelium (E10)
Mandibular mesenchyme + limb epithelium ( E l l )
18
12
24
—
36
9
31%
37%
Limb mesenchyme + mandibular epithelium (E9)
22
-
Trunk NC (E8) + mandibular epithelium (E9)
Trunk NC (E8) + limb epithelium (E9)
40
5
5
-
12-5%
Rostral
Caudal
Rostral
Caudal
8
8
15
18
7
2
2
7
87-5 %
25%
13%
39%
(b) Controls
Mandibular epithelium
Cranial NC (E8)
10
32
-
Mandibular mesenchyme + mandibular epithelium (E9)
Mandibular arch entire (E9)
12
20
5
14
half mandibular arch (E9)
half mandibular arch (E9)
mesenchyme + caudal epithelium (E9)
mesenchyme + rostral epithelium (E9)
Cranial neural crest-mandibular arch epithelium
recombinations
In the majority of recombination grafts of CNC with
both E9 and E10, ME invasive patterns of epithelial
morphogenesis abounded; hair and glandular (alcian
blue positive) structures developed (Fig. 6D) and in a
number of grafts, teeth were formed and reached
advanced stages of development. These had welldifferentiated odontoblast and ameloblast layers juxtaposed to thin layers of dentine and enamel (Figs
6E,F; Table 1). Their crown shape was, without
exception, molariform. In most instances, tooth morphogenesis had been accompanied by periodontal
differentiation, with the formation of follicular tissue
and woven alveolar bone beneath or encapsulating
the teeth (Fig. 6F,G).
Mandibular arch mesenchyme-limb epithelium
recombinations
Cartilage and bone formed in these grafts but no
teeth developed from either E9 or E10 recombinations. The limb bud epithelium differentiated into
keratinocytes and formed keratocysts but did not
undergo enamel organ or glandular morphogenesis.
In Ell recombinations, teeth did form (see also Mina
& Kollar, 1987).
These findings provide the first direct evidence that
the mammalian neural crest, like that of urodele
amphibians, has the potential for participating in the
initiation and morphogenesis of tooth germs and for
ultimately differentiating as secretory odontoblasts.
They also demonstrate that ectomesenchyme can
contribute the entire cell population of the dental
42%
70%
pulp and progenitor cells for the periodontium,
including alveolar osteoblasts (Ten Cate & Mills,
1972). These potentials are expressed when cranial
neural crest cells are associated with an ectodermal
epithelium which itself is induced to form an enamel
organ and to differentiate into ameloblasts and other
tooth-specific epithelial cellular phenotypes, i.e. stellate reticulum and stratum intermedium cells. It
seems that neither normal crest cell migration nor
itinerant contact with pharyngeal endoderm (Sellman, 1946) are absolute requirements for odontogenic development by mammalian cranial neural crest.
A comparison between the results obtained with
mandibular arch epithelium and limb bud epithelium
reveals that the cranial neural crest would have been
odontogenically uncommitted prior to migration and,
as has been shown for osteogenic differentiation
(Hall, 1983), odontogenic differentiation of neural
crest cells appears to be specified by an interaction
with epithelium at their destination rather than some
aspect of their migration route to that site. But
whereas bone formation depends on a permissive
interaction (any epithelium, whether it is appropriate
or not, will induce bone; Tyler & Hall, 1977), tooth
formation appears to depend on a site-specific epithelium (Lumsden, 19846,c; Mina & Kollar, 1987).
Neither presumptive (E8CNC) nor definitive
(E10MM) mandibular arch ectomesenchyme has the
ability to interact odontogenically with a heterologous but isochronic epithelium (E10LE). This
ability, which has been demonstrated in Ell mandibular arch mesenchyme (Ruch et al. 1973; Ruch,
1984) and E13 or later dental papillae (Kollar &
Initiation of mammalian tooth germ
Baird, 1969, 1970; Kollar & Fisher, 1980), therefore
appears only when tooth development is already
manifestly underway and must be acquired as a
consequence of a prior interaction with a specific
epithelium. The regional specificity of mandibular
epithelium together with the lack of odontogenic
inductive activity in presumptive tooth-forming mesenchyme suggests that tooth development might normally be initiated by the specific action of mandibular
arch epithelium on competent ectomesenchyme. This
does not imply that the initiating odontogenic interaction would necessarily be instructive (Wessels, 1977;
Saxe"n, 1977), an appropriate permissive epithelial
signal may be restricted topographically to the
specific region of epithelium. It remains to be shown
whether or not mandibular arch epithelium can form
teeth when it is combined with mesenchyme from a
source other than the cranial neural folds or the
mandibular arch.
Odontogenic potential of postcranlal
mesenchyme
The sources of mesenchyme chosen for this test were
limb bud cores from E10 embryos (see above) and
trunk level neural crest from E8 (6+ somite) embryos. Trunk crest (TNC) was excised from the
margins of the open posterior neuropore at the level
of the future thoracic somites (Figs 4, 5B); in this
region, as in the head, the crest is demarcated
medially by a sulcus and is clearly distinguishable
(Schoenwolf & Nichols, 1984). Although crest mesenchyme does not emerge in the trunk region until
after the neural folds approach and fuse with one
another (Nichols, 1986), these explants would have
contained the precursors of the definitive trunk crest.
Limb mesenchyme-mandibular arch epithelium
In recombinations of E10LM+E10ME, the mesenchyme developed limb-specific rather than tooth or
mandibular-arch-specific structures and tissues. Cartilages formed in rod shape but these ossified endochondrally and perichondrally in the manner of the
appendicular skeleton (Fig. 7A; Table 1). At certain
regions, chondrocytes formed columnar arrays resembling the maturational and hypertrophic zones in
epiphyseal plates. These epiphysis-like areas were
associated with zones of calcification, vascularization
and ossification. The epithelium formed sinus and
pelage hairs but not enamel organs in association with
limb bud mesenchyme.
Trunk neural crest-mandibular arch epithelium
A small minority of these grafts formed mandibular
arch structures including teeth and bone (Fig. 7B-D;
Table 1). Unlike CNC+ME grafts, in which bone
163
formed both in periodontal locations and in isolated
patches, the bone in TNC+ME grafts was found only
in association with teeth.
The inability of the mandibular arch epithelium to
induce participation in odontogenic development by
limb bud mesenchyme immediately suggested that
the initial odontogenic interaction is permissive - that
in normal development, the role of the epithelium is
to allow cranial ectomesenchyme to engage in a
particular developmental pathway which has already
been chosen, by some other mechanism, from a wider
range of possibilities. The development of teeth in
recombinations of E9 mandibular arch epithelium
with trunk level neural crest cells, however, suggests
either that this epithelium may have a more instructive influence or that, of the tissues tested, only the
neural crest is competent to respond to a local
permissive epithelial signal.
A possible interpretation of this somewhat surprising result could be that the epithelium had been
incompletely separated from its original mesenchyme
and that normal jaw development had merely continued in the graft. However, ME explants were
discarded unless they had separated either spontaneously or with only the most gentle mechanical
separation following enzyme treatment, and all ME
explants were checked for visual signs of contamination by mesenchymal cells. Furthermore, ME control grafts rarely showed any evidence of related
mesenchymal development and this never achieved
the prominence displayed by mesenchyme in the
TNC+ME grafts; similarly, morphogenesis of the
TNC+ME type was not observed in LM+ME associations. A possible source of contamination could
have been the unsegmented paraxial mesoderm that
lies in ventral contiguity with the trunk neural crest.
Contamination could only be avoided by careful
dissection, but the possibility of chance inclusion of
mesoderm in TNC explants cannot be ruled out.
It is not known whether trunk neural crest in
mammals normally contributes to connective tissues,
as it does in amphibians (forming trunk dermis in
Ambystoma; Raven, 1931, 1936, and the median fin
fold, all of the dorsal and lateral mesenchyme and
part of the meninges in Pleurodeles larvae, Chibon,
1966), or whether, as in avian embryos, the thoracolumbar neural crest does not normally form ectomesenchyme (Le Douarin & Teillet, 1974; Le Douarin,
1982). In the avian embryo, ectomesenchymal capabilities are normally expressed only by crest cells
rostral to the 5th somite (Le Lievre & Le Douarin,
1975). Thoracolumbar crest may nonetheless have an
ectomesenchymal potential; the heterotopic replacement of cranial neural crest by trunk level neural crest
(Nakamura & Ayer-Le Lievre, 1982) resulted in the
differentiation of connective tissues from the trunk
164
A. G. S. Lumsden
*•»»
Initiation of mammalian tooth germ
cells, although such phenotypes were only acquired
when the grafted cells were able to mix with cranial
neural crest cells that had migrated either from the
contralateral neural fold or from the fringes of the
graft. Tissue interactions in the cranial environment
elicited some developmental,potencies that are not
normally expressed by avian trunk level crest.
If it is assumed that the crest contribution to trunk
connective tissues is diminished in mammals as in
birds, then the developmental repertoire of murine
trunk crest may have been similarly expanded by the
abnormal tissue association in the grafts. In neither
the avian nor the amphibian heterotopic grafts did
trunk-derived ectomesenchyme form cartilage or
bone, but odontoblastic differentiation by labelled
amphibian trunk-level neural crest cells was noted in
3 % of Chibon's grafts; this, and the results presented
above, indicate that, whereas chondrogenic potential
of the crest may be confined to the cranial region,
odontogenic potential may not be confined to its
presumptive region of expression.
Since neural crest cells migrate and differentiate
according to site of grafting rather than their original
position along the rostrocaudal axis (Le Douarin,
1982), it is generally believed that they are not
committed prior to migration. This is not to say,
however, that the neural crest is necessarily a homogeneous population of pluripotent cells from which
specific environmental cues induce specific cell
phenotypes. An alternative possibility exists, namely
that the crest constitutes a mosaic of developmentally
distinct subpopulations with already restricted potentials; interaction with specific environmental factors
could lead to differential promotion of their survival
or proliferation or to modulation of their phenotypic
expression (Cohen & Konigsberg, 1975; Cohen, 1977;
Le Douarin, 1984). Because heterotopic grafting
involves whole populations of cells it is operationally
unable to reveal the existence of heterogeneous
subpopulations if these were each present at all levels
on the rostrocaudal axis. Cloning experiments have
demonstrated bipotentiality for certain crest phenotypes (Seiber-Blum & Cohen, 1980) but whether the
Fig. 7. Histology of graft development, as Fig. 6. (A)
Limb bud mesenchyme (LM) + ME graft showing sinus
hair development and limb-specific skeletal structure;
proliferation, hypertrophying and ossifying regions of
cartilage and bone in the form of a shaft. (B) Trunk
neural crest (TNC) + ME graft with tooth development.
A small amount of alveolar bone has formed (arrowed).
(C,D) Other TNC + ME grafts with tooth development.
(E) RMA graft displaying normal mandibular
development. (F) RME + CM graft with tooth
development. Bars, 100fim. dp, dental papilla;/,
follicular mesenchyme; oee, outer enamel epithelium; sr,
stellate reticulum.
165
E9-E10
13-34 somites
CM
CE
Fig. 8. Scheme of experiments in which the mandibular
arch was bisected into rostral and caudal halves, which
were either grafted or separated into epithelial and
mesenchymal components which were reciprocally
recombined.
crest is homogeneous or heterogeneous and whether
the environment acts inductively or selectively remain areas of uncertainty. Recent single-cell-marking
experiments in the chick (Bronner-Fraser & Fraser,
1988), however, have shown that the progeny of
individual neural crest cells differentiate into a wide
range of differentiated cells, suggesting that the
environment induces specific differentiation of pluripotent cells (but see Bee & Newgreen, this volume).
The formation of teeth by both CNC and TNC
explants, but not by LM explants, suggests that the
potential to form odontoblasts (following interaction
with mandibular arch epithelium) may be a neural
crest property which is tissue-specific but not axial
level-specific. Localized expression of this potential in
an initially homogeneous crest cell population may be
mediated by focal instructive tissue interactions restricted to this specific region of the head.
Site specificity in the mandibular arch
epithelium
Neural crest cells may arrive in the mandibular arch
in an uncommitted state and require a local signal
from the epithelium in order to engage in odontogenesis. Is this signal confined broadly to mandibular
epithelium (as opposed to limb bud epithelium, for
example) or is it topographically more restricted,
166
A. G. S. Lumsden
perhaps to those regions of the mandibular epithelium where, 1-2 days later, separate incisor and
molar tooth germs develop? Dental pattern formation could thereby be a function of a prepattern in the
oral epithelium which is transferred to adjacent
ectomesenchyme through the initiating tissue interaction.
The possibility that the tooth-forming loci are
defined by the epithelium has been approached in two
ways; first, the sites of future tooth development were
located by testing the tooth-forming potential of
sectors of the arch obtained by subdivision across its
mesiodistal axis (Lumsden, 1982; Lumsden & Buchanan, 1986). Second (Fig. 8), the regional inductive
potency of the epithelium around the central (dorsoventral) axis of the mandibular arch has been assessed
in reciprocal recombination grafts of epithelia and
mesenchyme obtained from hemisectioned mandibular arches. These two sets of experiments will be
discussed in turn.
Whereas both incisor and molar teeth with near
normal crown shapes developed in intraocular homografts of complete mandibular arches explanted at E9
and E10, arches that had been divided in the midline
gave rise only to molars (Lumsden & Buchanan,
1986). The ventral midline region of the arch produced incisors at E10 but not at E9. These results
indicated that the mandibular incisor primordium is
initiated in or very close to the median epithelial
isthmus and that incisors are not determined until
E10. During E10 in normal development, the median
isthmus region fills with ectomesenchyme and this
region becomes competent to form incisors. By Ell,
however, the ventral midline region no longer produces incisors when isolated and grafted (Lumsden,
1982) and median section of the arch no longer
suppresses incisor formation in the isolated hemimandible. At this time, incisors are formed in grafts
of laterally adjacent sectors where, one day later in
normal development, incisor rudiments first become
visible (Ruch, 1984).
In the normal E9 mandibular arch, ectomesenchyme has yet to complete its ventrad migration into
proximity with epithelium in the presumptive incisor
region. By this time, however, the crest has already
reached proximity with epithelium in the molar
region. During E9, therefore, the posterior (molar)
region of the arch would contain ectomesenchyme
which is presumptive for both molars and incisors yet
only molars and not incisors develop when the entire
region is grafted. This is evidence that E9 mandibular
ectomesenchyme is equipotential with respect to
tooth development and that it could not, therefore,
have been specified earlier, during its migration into
the arch. These findings also suggest that the absence
of incisors is due to damage or destruction of the
incisor epithelium when the arch is divided at the
midline. Destruction of the incisor epithelium at
explantation could also account for the exclusively
molariform shape of teeth formed in CNC+ME and
TNC+ME grafts in the present study.
In the second set of experiments (Fig. 8), the
mandibular arches of E9 embryos were sectioned by a
single cut in the dorsoventral plane to give approximately equal oral (rostral-half mandibular arch,
RMA) and aboral (caudal-half mandibular arch,
CMA) regions. These half arches were grafted entire
or, following treatment with collagenase to separate
their epithelia and mesenchyme, in various recombinations between the rostral epithelium (RE), rostral
mesenchyme (RM), caudal epithelium (CE) and
caudal mesenchyme (CM).
Development of halved mandibular arches
Grafts of rostral half-mandibles developed a spatially
organized set of teeth, incisors and molars, surrounded by alveolar bone. Where the isthmus region
connecting the two mandibular processes had been
preserved intact during dissection (as in Fig. 7E), the
sectioned graft presented an appearance which is
similar to that of a normal mouse jaw. Grafts of
caudal half-mandibles developed cartilage and sinus
hairs but neither teeth nor bone.
Development of reciprocally recombined halfmandibular arch tissues.
Teeth were formed in both sets of recombinations,
but the incidence in RE+CM grafts (Fig. 7F) was
substantially higher than in CE+RM grafts (Table 1).
The results from intraocular homografting reciprocally recombined half-mandibular tissues indicate
that regions of the oral epithelium differ in their
capacity to initiate tooth development in association
with mandibular arch mesenchyme. The pattern of
development in half-mandibles shows that tooth development is restricted to the rostral (oral) half and
that the caudal (aboral) half does not regulate in its
absence. Rostral tissue can therefore be regarded as
normal tooth-forming, and caudal tissue as normal
nontooth-forming. The finding that RE + CM grafts
formed teeth at a markedly higher incidence than
CE+RM indicates that the epithelium (RE), rather
than the mesenchyme (RM) is regionally specified
and suggests, again, that mandibular arch mesenchyme as a whole is equipotential with respect to
tooth development.
Taken together, the results of these experiments
allow the following conclusions. First, tooth initiation
involves an inductive interaction between regionspecific epithelium and competent but unspecified
mesenchyme cells normally derived from the cranial
neural crest. Second, a prepatterned distribution of
Initiation of mammalian tooth germ
inductive potency may exist in the epithelium of the
early mandibular arch, restricting this inductive event
to individual loci around the stomatodaeum and thus
controlling the spatial organization of the dentition as
a whole.
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