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Fine structure of the epithelial dental organ in the frog during early odontogenesis.

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Fine Structure of the Epithelial Dental Organ in the
Frog during Early Odontogenesis '
Department of Histology, College of Dentistry, University of Illinois,
P . 0. Box 6998, Chicago, Illinois 60680
A total of 227 tooth germs in different developmental stages were dissected from the maxillae of 58 leopard frogs ( R a n a pipiens). Five fixatives and three
buffers were used and optimum preservation of the odontogenic epithelium was observed i n specimens fixed in cold 4% glutaraldehyde at pH 7.3 for three hours followed
by 1% osmium tetroxide for one hour. The inner dental epithelium showed mitotic figures limited to the initial stage of odontogenesis. The cells contained numerous free
ribosomes, tonofdaments, few mitochondria and sporadic cisternae of endoplasmic reticulum. Their distal portions were drawn into processes studded with numerous hemidesmosomes. At a later stage of development, yet prior to amelogenesis, these processes
disappeared. The preameloblasts followed two pathways of histodifferentiation depending on their location. Ventrolabially the columnar cells exhibited progressive differentiation in contrast to the cuboidal cells on the ventrolingual surfaces of the cusps.
The external dental epithelial cells were flattened, closely packed and connected by
numerous desmosomes. Their cytoplasm contained mainly mitochondria and tonofilaments, as well as few Golgi vesicles. With the beginning of amelogenesis large intercellular spaces and lysosomal-like bodies were frequently observed and the Golgi complex is more prominent.
We concluded that at early odontogenesis the epithelio-mesodermal interaction in
the frog's dental organ is similar to that of mammalian species but the frog's internal
epithelium shows a different pattern of histodifferentiation.
The purpose of the present investigation
was to study the fine structure of the dental
epithelial organ and to clarify the interrelationship between the epithelial and mesodermal components of the amphibian tooth
germ during the initial stages of odontogenesis.
Gillette ('55) reported that in the frog
only the internal dental epithelial cells of
the ventrolabial surfaces of the cusps produce enamel, whereas the cells of the ventrolingual surface do not. The present
study was concerned with the fine structural differences in histodifferentiation
within the internal dental epithelium
which lead to the two types of cells.
The histology of the dental epithelium in
the frog during tooth development was f i s t
reported by Hertwig (1874). He described
the external and internal epithelial layers
as well as an extension of both layers at
the base of the dental organ. The latter
structure, which surrounds the subcoronal
part of the tooth, has become known as the
epithelial sheath of Hertwig. Kerr ('60)
described the epithelial dental organ in
ANAT. REC., 168: 79-92.
urodeles as consisting of an inner enamel
epithelium and outer epithelium with a few
cells in between which show no tendency
to form a stellate reticulum. Goin and Hester ('61 ) investigated tooth development
in the frog Hyla cinerea and observed the
proliferating epithelial cells which form
the dental organ to differentiate into distinct but continuous layers. The inner
enamel epithelium is formed of columnar
cells and the outer enamel epithelium is
formed of squamous cells.
The fine structural investigations of the
epithelial dental organ have been almost
exclusively confined to observations in certain mammalian species and only a few reports were primarily concerned with early
developmental stages (Nylen and Scott,
'58; Quigley, '59; Pannese, '62). Fearnhead
('61) observed mitotic activity in the peripheral epithelium at the base of the rat
tooth germ and suggested that such activReceived Feb. 27, '70.Accepted Apr. 23, '70.
1 This investigation was supported in part by USPHS
research grant DE 02201-03and USPHS-GRSG-FR5309.
2 Present address: Department of Oral Blology, University of Connecticut Health Center, Hartford Plaza,
Hartford, Connecticut 06105.
ity added new cells to the inner dental epi- 7.3 or phosphate buffer at pH 7.2 (Pease,
thelium. Pannese ('62) described a con- '62).
tinuous basement membrane separating
2. Four per cent paraformaldehyde in
the internal and external enamel epithelia phosphate buffer at pH 7.2 (Pease, '62).
of the cat tooth germ from the surrounding
3. Veronal-buffered 1% osmium tetroxmesenchyme. He claimed that the mem- ide (Palade, '52).
brane persists next to the external epithe4. Potassium permanganate, 1.2% in
lium although it is no longer observed next veronal buffer (Luft, '56).
to the internal epithelium in subsequent
5. Chrome osmium (Dalton, '55).
stages of differentiation. Moreover, he obAdditional variables tested were : cacoserved microvesicles in the external epi- dylate versus phosphate buffers and imthelium at the side next t a the dental sac mediate dissection of tooth germs as comas well as in the dental sac which he sug- pared to dissection following three hour
gested might be pinocytotic vesicles. A period of furation. In the latter case, the
peculiar finding by Pannese was the occa- erupted teeth were cut off with a Bardsional presence of desmosomes between the Parker blade as a single wedge to provide
interdigitating apical zones of the internal for more accessibility of the fixative soluenamel epithelial cells and the odonto- tion to the area of developing teeth.
Evaluation of the results of these experiblasts. Decker ('63) emphasized the presence of free RNP particles in the cytoplasm ments led to the following conclusions :
1. The pH and osmolarity of the furaof all cells in the enamel organ. He observed hemidesmosomes along the outer tives are of crucial importance.
2. Six per cent glutaraldehyde and 4%
dental epithelial cell surface bordering the
basement membrane, but they were lack- paraformaldehyde caused marked cellular
ing at the corresponding site of the inner shrinkage.
3 . There was no appreciable difference
dental epithelium. However, the latter cells
to the type of buffer, or the time of
showed more desmosomes than the former.
final dissection of tooth germs.
4. The best results obtained in these
comparative experiments were observed in
A total of 227 tooth germs in different specimens fixed in cold 4% glutaraldehyde
developmental stages were dissected from in cacodylate buffer for three hours fol58 frogs (Rana p i p i e n s ) . The animals were lowed by veronal-buffered 1% osmium
sacrificed by decapitation and the skin on tetroxide for one hour. Hence this method
the labial side of the right or left maxilla was adopted for subsequent fixation of the
was stripped. This was done cautiously in specimens.
view of the continuity of the epithelium of
5. Potassium permanganate was particthe skin, oral mucosa, and dental lamina to ularly effective in preservation of the memwhich the tooth buds are attached. The branous structures as well as the interface
stripped maxilla was excised at its suture between the epithelial dental organ and the
with the premaxilla and transferred to the mesodermal dental papilla.
stage of a dissecting microscope in a pool
The specimens were dehydrated in
of cold fixative. A ledge of the lingual oral graded series of alcohol and embedded in
mucosa at the level of the tooth germs was Araldite (Fluka A. G., Chemische Fabrik
trimmed and small cross sections 0.5 mm Buch S. C., Switzerland). During embedthick were cut. These usually contained ding care was taken to orient the specimens
two adjacent tooth germs of a chosen area so that sectioning could be done with
following the classification of Gillette ('55).
proper reference to the tooth germ position.
A series of experiments was designed to This was facilitated by the use of previously
compare the results of five commonly used prepared embedding capsules. A central
fixatives relative to the specific cells being hole approximately 1 mm in diameter was
investigated. These fixatives are :
drilled in a 2-3 mm thick layer of polymer1. Six and 4% glutaraldehyde (Biologi- ized embedding medium at the bottom of
cal grade 50% -Fisher
Scientific Com- the capsule. This hole not only accommopany) in 0.08 M cacodylate buffer at pH dated the specimen in the desired position
but also prevented any subsequent change
until polymerization was completed. Undemineralized tooth germs were sectioned
on a Sorvall Porter-Blum microtome
equipped with a diamond knife.
Thick sections (0.5 thick) were cut
and stained with crystal violet and counterstained with basic fuchsin for examination in the light microscope (Moore,
Mumaw and Schoenburg, '60). The study
of these sections and the subsequent examination of adjacent thin sections (500750 A ) in the electron microscope provided
for a direct correlation of identical areas.
The thin sections were collected on
parlodion-carbon coated grids (200 mesh
type) and stained with 8% uranyl magnesium acetate and lead citrate (Reynolds,
'63) then examined in Phillips EM75D and
RCA-EMU 3D or 3H electron microscopes.
thickness ran parallel to the distal boundaries of the cells and separated them from
the cell-free zone of the dental papilla.
Later in development, as these cells increased in height, their distal portions were
drawn into long pointed processes of variable length that projected into the cell-free
zone. The distal cell membranes limiting
these processes showed numerous hemidesmosomes (fig. 3). The basal lamina has
increased in thickness and is about 300400 A thick and folded to follow closely
the contour of the epithelial cell processes
but separate from them by a space of about
800 A in width. These processes contained
mainly bundles of tonofilaments that seem
to converge on the dense thickened plaques
of the hemidesmosomes. Finer and less
dense filaments connected the dense
plaques to the underlying basal lamina. At
high magnification (fig. 3, insert) the
dense plaques appeared closely packed and
Internal dental epithelium
varied in size with an average length of
At the early stage of tooth formation, the 1000 A. The fine filaments extending from
internal dental epithelium was seen to con- the plaques to corresponding thickenings
sist of a single layer of cuboidal cells about in the basal lamina did not seem to be
7-8 p wide (fig. 1 ) . The cells were closely continuations of the tonofilaments. The
packed and each cell was limited by a fairly former were of smaller diameter and were
smooth membrane. The limiting mem- perpendicularly arranged in the space bebranes of the adjacent cells were connected tween the hemidesmosomes and the basal
by small desmosomes and showed very few lamina while the tonofilament bundles
wide intercellular spaces. An exception was were almost parallel to this space. At a
seen at the proximal ends where intercellu- subsequent stage of development the inlar spaces were more frequently found be- ternal dental epithelial cells continued to
tween the internal and external dental increase in length and the nuclei remained
epithelia. The cytoplasm contained few centrally located and fairly large in size.
scattered round or oval mitochondria and The predominant organelles were the mitoonly a few elements of the granular endo- chondria, while the Golgi complex and the
plasmic reticulum were observed in the endoplasmic reticulum were not promiform of dispersed ribosome-studded tubules nent; bundles of tonofilaments were readily
and vesicles. Free ribosomes and tonofila- observed. The distal cell processes and the
ments were present, the latter were espe- folding of the basal lamina observed earlier
cially prominent in permanganate-fixed disappeared at this stage and the boundary
specimens. At the early stage of tooth de- between the epithelium and dental papilla
velopment mitotic figures were observed at was far less irregular (fig. 4). Hemidesmothe base of the tooth germ and in the in- somes were still present but fewer in numternal dental epithelial extension which ber and appeared more widely spaced.
contributes to the formation of Hertwig's
In the early stage of tooth development
sheath (fig. 2). The boundary between the the cuboidal cells of the internal dental
internal dental epithelium and the dental epithelium transformed into short colpapilla showed a marked structural change umnar preameloblasts. With further develduring development. Initially the distal opment the preameloblasts exhibited differlimiting membranes of the internal dental ent morphology according to their location.
epithelial cells were fairly smooth (fig. 2). Only the cells on the ventrolabial surfaces
A basal lamina of about 100-150A in of the cusps showed further differentiation
into columnar cells with basally located nuclei and increased organelle content. The
cells on the ventrolingual surface of the
cusps did not follow this pathway of differentiation and were cuboidal in outline with
centrally located nuclei (fig. 5).
External dental epithelium
The external dental epithelium in the
frog consists of several rows of flattened
cells extending from the internal dental
epithelium up to the dental sac. These flattened cells were closely packed and their
adjacent limiting membranes closely approximated and connected by numerous
desmosomes (fig. 1 ) . The external dental
epithelium varied in thickness being widest
over the cusps and tapering towards the
cervical area where it was confluent with
the internal dental epithelium. There were
no observable structural differences between the inner layer of the external dental
epithelium, i.e., those cells abutting on the
internal dental epithelium, and the outer
layer of the external epithelium next to
the mesodermal dental sac.
A peculiar feature observed only in specimens fixed in potassium permanganate
was a deep invagination of the nuclear
envelope of the external epithelial cells at
the early stage of tooth development.
Before enamel formation each external
cell was oval in longitudinal section and
round in cross section and a spherical large
nucleus. occupied most of the cell. The
cells were orientated perpendicular to the
long axis of the cuboidal preameloblast and
later maintained this orientation in respect
to the columnar ameloblast. The main cytoplasmic components were mitochondria
and tonofilaments. No elements of the endoplasmic reticulum were observed and
few Golgi vesicles were present.
With the beginning of odontogenesis, the
limiting membranes of adjacent cells that
were previously closely approximated were
now often separated by large spaces (fig.
6). The nuclei were still large, occupying
most of the cell space. The cytoplasm
showed more mitochondria with a moderate number of cristae and a few intramitochondria1 granules. Occasionally a
few sparsely scattered tubules or vesicles
of granular endoplasmic reticulum were ob-
served, however a well-developed reticulum
was not seen.
At a later stage, a Golgi complex consisting of small vesicles and three to four
flattened cisternae was seen situated in the
juxta-nuclear zone. Vesicles of similar appearance and size to those of Golgi complex
were found in other areas of the cytoplasm
and in close proximity to the cell membrane (fig. 7).
There were also numerous tonofilaments,
60-90 A thick, which were sometimes isolated and at other times clustered in groups.
These groups were of more frequent occurrence with the progress of enamel formation. Numerous long and slender cell processes extended in the wide intercellular
spaces and processes of adjacent cells
seemed to come in contact along their
broad surfaces. Smooth vesicles and tonofilaments were the main contents of these
processes. The adjacent cells made contact
in small areas where typical desmosomes
were often observed.
Moreover, large membrane-bound dense
bodies with variable content were observed
especially in the Golgi region. Most frequently these bodies contained a homogeneous very dense material; less frequently, these bodies contained a moderately dense matrix with numerous very
dense particles of different sizes. These
dense bodies resembled lysosomes.
At all stages of development, a basal
lamina, 200-300 A thick, separated the
external dental epithelium from the surrounding dental sac. The epithelial cell
membrane facing the basal lamina showed
typical hemidesmosomes.
In the amphibian dental organ only two
distinct layers can be discerned, the internal dental epithelium and the external
dental epithelium. The term external
dental epithelium carries different connotations when applied to the mammalian or
amphibian dental organs. The mammalian
external dental epithelium is the one-cell
thick layer external to the stellate reticulum and next to the mesodermal dental sac.
The amphibian external dental epithelium
is a multiple-cell layer external to the internal dental epithelium and extending to
the basal lamina which delineates its outer
findings of such desmosomes in the cat by
Pannese ('62). Scott and Nylen ('60) mentioned the irregularity in the distal surface
of the internal dental epithelial cells of
mammalian teeth and claimed its relation
to the scalloping observed at the future
amelodentinal junction. This is not the case
in frog teeth as the epithelial cell processes
disappear and the distal cell membranes
seem to be fairly regular before the apposition stages of development. During early
Internal dental epithelium
odontogenesis the fine structure of the preThe fine structure of the internal dental ameloblasts reflects variations depending
epithelial cells in the frog is essentially in on the location of the cells and their relaagreement with the report of Pannese ('62) tionship to enamel formation. Thus the
of the same layer in the cat. It is signifi- cells on the ventrolabial surface of the
cant that in these cells the endoplasmic cusps, where enamel is to be formed, show
reticulum and the Golgi complex are not progressive histodifferentiation while the
well developed, while a considerable con- cells on the ventrolingual surfaces, where
tent of free ribosomes is present. This indi- no enamel is formed, are morphologically
cates that while the internal epithelial cells similar to the less differentiated cells of the
may be capable of protein synthesis, any early inner epithelium.
formed protein is for intracellular conExternal dental epithelium
sumption and not for export. This assumption is substantiated by the observation
The present study confirms the reported
that these cells indeed grow progressively absence in the frog's teeth of a stellate reticin length and are not engaged in any ap- ulum or stratum intermedium similar to
parent secretory activity at this early stage that of mammalian dental organ (Gillette,
of development.
'55; Kerr, '60; Goin and Hester, '61; LawThe mitotic figures observed in the frog son, '66; Soule. '66). Since the stellate reinternal dental epithelium are in agree- ticulum and the stratum intermedium are
ment with similar observations in mam- claimed to perform different functions remalian tooth development (Fearnhead, '61; lated to mammalian amelogenesis (JohnTonge, '67). This is to be expected in view son and Bevelander, '57; Sicher, '66), it
of the proliferative nature of the internal was thought that the different layers of
dental epithelium at the early develop- the amphibian external dental epithelium
mental stage. In addition, no mitotic figures might show morphological differences corwere observed in any of the subsequent related with their location in respect to
stages which indicates that the differentia- the inner dental epithelium. The observation of the internal dental epithelial cells tions in this study show no such differis accompanied by a concomitant loss of ences. At the early developmental stage,
mitotic activity. This morphologic observa- morphology of the external dental epithetion is supported by autoradiographic study lium is compatible with the proliferative
of McBride and Gillette ('65) who demon- nature of this stage. However, no mitotic
strated mitotic activity only during early activity was observed, probably due to midevelopment of frog teeth.
tosis occurring at an earlier stage of develThe changes observed in the boundary opment. The structural modifications of
between the internal dental epithelium and the external dental epithelium concomitant
the mesodermal dental papilla in the frog with amelogenesis indicate a transport
are similar to those observed in human function from the blood vessels in the
teeth germs (Ronnholm, '62) and in the dental sac. Pannese ('62) suggested that
cat (Pannese, '62). However, no desmo- exchange of material occurs through the
some-like attachments are present between external dental epithelium, close to the
the internal dental epithelial cells and the capillaries in the mesodermal dental sac
odontoblasts in the frog contrary to the of the cat. The increased number of mitoborder from the mesodermal dental sac.
The cells of the amphibian external dental
epithelium share the same morphological
features and no layers corresponding to the
mammalian stratum intermedium or stellate reticulum are present. Thus, on the
basis of location, it seems appropriate to
designate those cells external to the internal dental epithelium collectively as the
external dental epithelium.
chondria in the external dental epithelium
may play a role in this transport function.
Dalton, A. J. 1955 A chrome-osmium fixative
for electron microscopy. Anat. Rec., 121: 281.
Decker, J. D. 1963 A light and electron microscope study of the rat molar enamel organ.
Arch. Oral Biol., 8: 301-310.
Fearnhead, W. 1961 Secretory products of
ameloblasts. In: Electron Microscopy in Anatomy. J. D. Boyd, F. R. Johnson and J. D. Lever,
eds. The Williams and Wilkens Co., Baltimore,
pp. 241-260.
Gillette, R. 1955 The dynamics of continuous
succession of teeth in the frog (Rana pipiens).
Am. J. Anat., 96: 1-36.
Goin, J., and M. Hester 1961 Studies o n the
development, succession and replacement of
teeth i n the frog H y l a cinerae. J. Morph., 309:
Hertwig, 0. 1874 Uber das zahnsystem der Amphibien und seine bedeutung f i r die genese des
skelets der mundhole. Arch. F. mikr. Anat., 11,
supplement: 1-208.
Johnson, P. L., and G. Bevelander 1957 The
role of the stratum intermedium in tooth development. Oral Surg., 10: 437-443.
Kerr, T. 1960 Development and structure of
some actinopterygian and urodele teeth. Proc.
2001. SOC.(London), 133: 401-424.
Lawson, R. 1965 The teeth of hypogeophis rostratus (Amphibia apoda) and tooth structure
i n the amphibia. Proc. Zool. SOC.,145: 321-325.
Luft, J. H. 1956 Permanganate-A
new fixative for electron microscopy. J. Biophys. Biochem. Cytol., 2: 799-801.
McBrjde, L., and R. Gillette 1965 Correlation
of mitosis and development i n the tooth cycle
of the frog. Abstract no. 317. International Association for Dental Research.
Moore, R. D., V. Mumaw and M. D. Schoenberg
1960 Optical microscopy of ultrathin tissue
sections. J. Ultrastruct. Res., 4: 113-116.
Nylen, M. U.,and D. B. Scott 1958 An electron
microscopic study of the early stages of dentinogenesis. Public Health Service Publication no.
Palade, E. 1952 A study of fixation for electron
microscopy. J. Exp. Med., 95: 285-298.
Pannese, E. 1962 Observations i n the ultrastructure of the enamel organ. 111. Internal and
external enamel epithelia. J. Ultrastruct. Res.,
6: 186-204.
Pease, D. C. 1964 Histological techniques for
electron microscopy. Second edition, Academic
Press, New York, p. 52.
Quigley, M. B. 1959 Electron microscopy of the
amelodentinal junction during early development of the molars of hamsters. J. Dent. Res.,
38: 558-568.
Reynolds, E. S. 1963 The use of lead citrate
at high pH as an electron opaque stain in electron microscopy. J. Cell Biol., 17: 208-212.
Ronnholm, E. 1962 An electron microscopic
study of the amelogenesis in human teeth. I.
The fine structure of the ameloblasts. J. Ultrastruct. Res., 6: 229-248.
Scott, D. B., and M. U. Nylen 1960 Changing
concepts in dental histology. Ann. N.Y. Acad.
of Sci., 85: 133-144.
Sicher, H. 1966 ed. Orban’s oral histology and
embryology. Sixth edition. The C. V. Mosby Co.,
St. Louis, pp. 70-72.
Soule, J. D. 1966 Origin of the enamel matrix
in developing amphibian teeth. Bull. Southern
Calif. Acad. of Sci., 65: 193-201.
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Res. (Suppl.), 46: 876-878.
BL, Basal Lamina
CFZ, Cell Free Zone
Cp, Cell process
cv, Coated vesicle
Dp, Dental papilla
d, Desmosome
EDE, External dental epithelium
G, Goigi complex
hd, Hemidesmosome
ICS, Intercellular space
IDE, Internal dental epithelium
L, Lysosome-likebody
m, Mitochondria
n, Nucleus
0, Odontoblast
PA, Preameloblast
r, Ribosome
rer, Rough (granular) endoplasmic reticulum
sv, Smooth surface vesicle
tf, Tonofilaments
1 Electron micrograph of internal and external dental epithelia in the
early stage of tooth development. Note the cuboidal shape and the
large central nucleus in the internal epithelium; the cytoplasm contains mainly mitochondria and free ribosomes. The external epithelial
layer is formed of flattened cells connected by numerous desmosomes
(arrows) and showing few intercellular spaces. x 4,600.
Electron micrograph of the inner and outer dental epithelia next to
the dental papilla at the cervical region of a developing tooth germ.
Note the internal dental epithelial cell in mitosis and the basal lamina
(arrow ). x 4,500.
A. E. Zaki, James A. Yaeger and Roy Gillette
3 Electron micrograph of transverse section i n the internal dental epithelium and the cell free zone of the dental papilla. Note the projections of the epithelial cells into the cell free zone. The basal lamina is
infolded and following closely the contour of the cellular projections.
x 7,000.
Insert: Higher magnification to show the connecting filaments between the hemidesmosomes and corresponding thickenings of thc
basal lamina. Note the granular nature of the latter structure.
x 34,500. (Potassium pernanganate fixation.)
Eltctron micrograph of the boundary between preameloblasts and
odontoblasts. Note the fewer hemideqmosomes i n the preameloblast
distal cell membrane which shows a n almost straight course. Also
note the abundant ribosomes and several mitochondria, one of which
is i n close relation to the nuclear envelope (arrow). x 19,500.
A. E. Zaki, James A. Yaeger and Roy Gillette
Electron niicrograph of the tip of the cusp in early tooth development.
Note the contrast between the columnar preameloblasts o n the ventrolabial surface of the cusp (left) and the cuboidal preameloblasts on
the vmtrolingual surface (right). At this stage the epitheliomesencliymal interface is straight and shows filamentous aggregations on
the mesenchymal side. x 4,000.
Electron micrograph of the external dental epithelial cells during early
odontogenesis. Note the wide intercellular spaces and the cell processes abutting. on each other along their wide surfaces. The approximated cell surfaces are connected by desmosomes. The cytoplasm
shows few organelles but many smooth vesicles particularly next to
the cell membrane and tonofilaments grouped into bundles. x 10,000.
Electron micrograph of external dental epithelium at a later stage of
odontoqenesis. The cytoplasm shows a prominent Golgi complex, many
smooth vesicles and few coated ones as well as bundles of tonofilaments. Note the dense content of lysosomal-like bodies and the lack
of the endoplasmic reticulum. X 17,000.
A. E. Zaki, James A. Yaeger and Roy Gillette
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structure, epithelium, dental, odontogenesis, organy, fine, frog, early
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