/. Embryol. exp. Morph. Vol. 38, pp. 37-48, 1977
Printed in Great Britain
37
In vitro development of palatal tissues from
embryonic mice
III. Interactions between palatal epithelium and
heterotypic oral mesenchyme
By MARY S. TYLER1 AND WILLIAM E. KOCH2
From the Department of Zoology, and Department of Anatomy,
School of Medicine, University of North Carolina
SUMMARY
The epithelium of the secondary palate from embryonic mice (12-14 days of gestation) was
enzymatically separated from the palatal mesenchyme and was cultured in association with
mesenchyme from the embryonic tongue, incisor, and salivary gland. The histological
differentiation of the epithelium in each type of heterotypic recombination was always palatal.
Tongue mesenchyme supported full histodifferentiation of the palatal epithelium, whereas
incisor mesenchyme supported only a limited histodifferentiation in the epithelium. Salivary
gland mesenchyme in direct combination with palatal epithelium influenced the epithelium
to become organized into a system of ducts and acini typical of glandular morphogenesis;
histodifferentiation of the epithelium, however, was palatal. Salivary gland mesenchyme
grown in isolation did not undergo glandular morphogenesis.
The results demonstrate that histodifferentiation of palatal epithelium may be supported
by various types of mesenchyme. That a glandular morphogenesis can be induced in the
epithelium without altering epithelial histogenesis indicates that histogenesis and morphogenesis are not necessarily interdependent parameters of epithelial differentiation. The ability
of palatal epithelium to express its original histogenetic determination when separated from
its mesenchyme as early as 12 days of gestation and recombined with heterotypic mesenchyme
shows that the histogenetic program of the epithelium is relatively stable at an early stage in
palatal development.
INTRODUCTION
In general, an epithelium is dependent upon interactions with an adjacent
mesenchyme in order to differentiate. The nature of these interactions appears
to vary among different organ systems. For example, it has been found that
pancreatic epithelium will differentiate in the presence of a mesenchymal factor
that is common to various types of mesenchyme (Rutter, Pictet & Morris, 1973).
In contrast, salivary gland epithelium exhibits more specific requirements, and
the presence of general mesenchymal factors is not sufficient for supporting its
1
Author's address: Department of Zoology, University of Maine at Orono, Orono,
Maine 04473, U.S.A.
2
Author's address: Department of Anatomy, School of Medicine, University of North
Carolina, Chapel Hill, North Carolina 27514, U.S.A.
38
M. S. TYLER AND W. E. KOCH
differentiation (Golosow & Grobstein, 1962; Rutter, Wessells & Grobstein,
1964; Ball, 1974; Lawson, 1974). In the present investigation, palatal epithelium
is examined to determine the specificity of its requirements for differentiation.
Palatal epithelium was chosen for study since it consists of three distinct regions
with different developmental fates and therefore affords the opportunity of
observing the behavior of several epithelial types within a single experimental
system. These three epithelial regions - the medial, nasal and oral regions - are
defined early in palatal ontogeny (Tyler & Koch, 1977 in press). In the embryonic
mouse, the three epithelial regions are determined as early as 12 days of gestation;
this is the time when palatal processes are visible as two low bilateral ridges on
the maxillary process. Between the 14th and 15th days of gestation, at a time
when the paired palatal processes have assumed a horizontal position above the
tongue and are adhering to one another along their medial surfaces, the medial
epithelium regresses. This regression involves the programmed death of cells
within the epithelium (Shapiro & Sweney, 1969; Tyler & Koch, 1974, 1977).
By 17 days of gestation, the nasal epithelial region has differentiated into
pseudostratified ciliated columnar epithelium, and the oral epithelial region has
become a stratified squamous epithelium which displays birefringence in its
outer squamous cell layers under polarized light. Later in development, alcian
blue-staining cells (mucous cells) differentiate within the nasal region, and
hematoxylin-staining granules (keratohylin granules) become apparent within
cells of the oral region.
Previous tissue isolation and tissue recombination studies have shown that
palatal epithelium exhibits a potential for limited differentiation in the absence
of mesenchyme, but that it requires the presence of mesenchyme in order to
achieve full differentiation (Tyler & Koch, 1974, 1977 in press). In the present
study, palatal epithelium was cultured in the presence of various types of
heterotypic (non-palatal) mesenchyme using direct tissue recombination
techniques.
The results indicate that palatal epithelium does not require a specific type
of mesenchyme in order to differentiate. It was also demonstrated that epithelial
morphogenesis can be modified in the absence of any alteration in epithelial
histogenesis, thereby suggesting that histogenesis and morphogenesis are not
necessarily interdependent parameters of epithelial differentiation. A brief
abstract of certain aspects of this study has been published (Tyler, 1976).
MATERIALS AND METHODS
Tissue preparation
The tissues for this study were taken frome mbryonic mice of strains C57BL
and Brown Belt. Adult animals were mated between 9.00 and 11.00 a.m.; the
day of mating was designated as day zero of gestation. Pregnant females were
killed by cervical dislocation at 9.00 a.m. on the day that tissues were dissected.
Palatal epithelium in heterotypic recombinations
39
Fetuses were removed aseptically from the uterus, and organ dissection was
carried out immediately in a mixture of Tyrode's solution and horse serum
(1:1; v/v). The organ rudiments utilized included: palatal processes from 12-,
13-, and 14-day embryos; tongue from 12-, 13-, and 14-day embryos; mandibular
incisor from 16-day embryos; and submandibular salivary gland from 16-day
embryos; and submandibular salivary gland from 13-day embryos.
The epithelial and mesenchymal tissues of these organs were separated from
one another by treatment with a 3 % trypsin-pancreatin solution (3:1; w/w in
calcium- and magnesium-free Tyrode's solution). Palatal processes and tongue
rudiments were treated enzymatically for 30-60 min at 4 °C. Incisor rudiments
were trimmed to remove their lingual side (Koch, 1967), and the remaining
tissue was then treated with the enzyme solution at room temperature for several
minutes (Auerbach & Grobstein, 1958). Salivary gland rudiments also were
treated enzymatically for several minutes at room temperature. Following
enzymatic treatment, gentle agitation with a small-bore pipette or careful manipulation with cataract knives was sufficient to separate the loosened epithelium
and mesenchyme from one another. Separated tissues were returned to the
dissection solution and stored in an atmosphere of 5 % CO2 in air until used.
Culturing procedures
The separated palatal epithelium was cultured in combination with the
mesenchyme from the tongue, incisor, and salivary gland. For each of the three
tissue combinations, the intact sheet of palatal epithelium which included the
three palatal regions (nasal, oral and medial) was placed in direct contact with
the heterotypic mesenchyme. The tongue mesenchyme was always positioned
with its dorsal surface in association with the palatal epithelium; incisor
mesenchyme was positioned with its labial surface in association with the palatal
epithelium; salivary gland mesenchyme was positioned without a specific
orientation. The palatal epithelium was always placed with its basal surface
facing the mesenchyme.
Tissues were cultured on a Millipore filter disc (0-45 /im porosity and 25 ± 5 jum
thick, from Millipore Filter Corp., Bedford, Massachusetts) cemented to a plexiglass ring; two small glass rods were attached to the ring on the side opposite
the filter (Grobstein, 1956). The epithelium and mesenchyme were placed on the
upper surface of the filter and held in position with a plasma clot. Once the tissues
were in place, the culture apparatus was suspended over the well of a depression
slide containing a complex culture medium (Eagle's basal medium supplemented
with 1 % glutamine, 3 % 11-day chick-embryo extract, 10 % horse serum, and
100 units/ml of penicillin and streptomycin); the medium was in contact with
the lower surface of the filter. Cultures were maintained at 37-5 °C in a humidified incubator gassed with 5 % CO2 in air, and the culture medium was replaced
every 48-72 h. Routine incubation periods were from 3 to 5 days, but occasion-
40
M. S. TYLER AND W. E. KOCH
Table 1. Histogenesis and morphogenesis of palatal epithelium recombined
with heterotypic mesenchyme
Palatal characteristics exhibited by palatal epithelium from 12-14-day
embryonic mice after 5-8 days in culture with heterotypic mesenchyme
Epithelial histogenesis
Characteristics of
oral region
A
>
• Epithelial morphogenesis
A
Birefringent
,
^
outer
Kerato- Flattened
Type of
PseudoStratified
cell
hyalin
cell
Non-palatal
mesenchyme Ciliated stratified Columnar squamous layers
granules sheet
morphogenesis
Tongue
+
+
+
+
+
+
+
Characteristics of
nasal region
,
Incisor
+
—
—
+
+
—
+
—
Salivary
gland
+
+
+
+
+
+
—
Glandular:
ducts and acini
ally cultures were maintained for up to 9 days. Photographs of the living cultures
were taken at 24 h intervals.
Histological procedures
Following incubation, cultures were fixed in Bouin's fluid and embedded in
paraffin blocks. The 4 fim. thick serial sections cut from the embedment blocks
were stained with hematoxylin, eosin, and alcian blue (pH 2-7-3-0) (Pearse,
1960).
The presence or absence of birefringent elements within various epithelia was
detected using a Zeiss polarizing microscope. The sign of birefringence was
determined with a first-order red-retardation plate (Schmitt, 1944), using the
surface of the epithelium as reference for the retardation colors observed. Positive
birefringence denoted an ordered arrangement of elements parallel to the
epithelial surface, a characteristic of keratinized epithelia.
The results are based upon a total of 76 cultures, and are summarized in
Table 1.
RESULTS
Palatal epithelium recombined with tongue mesenchyme
Palatal epithelium grown in contact with tongue mesenchyme spread as
a flattened cell sheet during the culture period (Figs. 1, 2). Histological sections
showed that, for the age groups of epithelium tested (12 and 14 days), the
histodifferentiation of the epithelium was always palatal. The nasal epithelium
differentiated into a pseudostratified ciliated columnar epithelium similar in
height and ciliation to that of the palate in vivo (Fig. 5). The oral epithelium
Palatal epithelium in heterotypic recombinations
41
differentiated into a stratified squamous epithelium (Fig. 6) which exhibited
positive birefringence in its outer squamous cell layers and was similar in
thickness to the oral epithelium of the palate in vivo. Medial-epithelial regression
was difficult to detect, since cellular debris was either sloughed or phagocytized
by the adjacent mesenchymal cells. In cultures that were maintained for an
extended incubation period, alcian blue-staining cells were apparent within the
nasal epithelium (Fig. 7), and there were numerous squamous cell layers which
exhibited positive birefringence within the oral epithelium (Figs. 8, 11). In some
cultures, hematoxylin-staining granules were evident within the oral epithelium.
In all cultures, the timing of epithelial differentiation followed the in vivo
schedule for palatal epithelium.
Palatal epithelium recombined with incisor mesenchyme
Palatal epithelium grown in contact with incisor mesenchyme did not exhibit
any morphogenesis typical of the tooth but instead spread as a flattened epithelial
sheet (Figs. 3, 4). Histological examination revealed that the histodifferentiation of the epithelium was palatal in character, but was not as advanced as
that of the palate in vivo. Also, the degree to which epithelial histodifferentiation
approached that of the palate in vivo depended to a certain extent upon the age
of the epithelium. Twelve-day palatal epithelium recombined with incisor
mesenchyme showed somewhat poorer differentiation than did 14-day palatal
epithelium. The epithelium of the nasal region differentiated into a ciliated layer
of low cuboidal to low columnar cells (Fig. 9), and the epithelium of the oral
region differentiated into a stratified squamous epithelium with fewer squamous
cell layers than that in vivo (Fig. 10). The outer squamous cell layers of the
oral epithelium displayed positive birefringence under polarized light as in vivo
(Fig. 12). Cultures that were maintained for an extended incubation period did
not exhibit alcian blue-staining cells within the nasal epithelium nor were hematoxylin-staining granules observed within the oral epithelium (Figs. 9, 10).
There was no evidence in these cultures of the deposition of dental matrix by
either the palatal epithelium or the dental mesenchyme: the eosin-staining
extracellular material characteristic of enamel matrix and the alcian bluestaining extracellular material characteristic of dentinal matrix were not observed
at either the epithelial or the mesenchymal surfaces (see e.g. Figs. 9, 10).
Palatal epithelium recombined with salivary gland mesenchyme
Palatal epithelium grown in contact with salivary gland mesenchyme did not
remain as a flattened epithelial sheet; instead, it first retracted into a rounded
mass and then underwent a glandular type of morphogenesis (Figs. 13-20). By
the end of the culture period, the epithelium had become organized into a system
of ducts and acini. This particular behavior was exhibited by palatal epithelium
from all age groups tested (12, 13, and 14 days of gestation). Morphogenesis
42
M. S. TYLER AND W. E. KOCH
2 . * . . , ^ " C < "\ . 3
Figs. 1-2. Photographs showing the in vitro development of a direct recombination
between 12-day palatal epithelium and 13-day tongue mesenchyme at 0 and 96 h of
incubation respectively, x 16.
Figs. 3-4. Photographs showing the in vitro development of a direct recombination
between 14-day palatal epithelium and 16-day incisor mesenchyme at 0 and 96 h of
incubation respectively. No tooth-like morphogenesis is evident, x 16.
Figs. 5-6. Photomicrographs of a transverse section through a direct recombination
between 14-day palatal epithelium and 14-day tongue mesenchyme cultured for 72 h.
Epithelial differentiation is palatal: the nasal epithelium (Fig. 5) is a pseudostratified
Palatal epithelium in heterotypic recombinations
43
progressed at a similar rate in all cultures, but the particular branching pattern
of the epithelium varied (cf. Figs. 13-16 with Figs. 17-20).
The palatal epithelium became glandular in its morphology (Figs. 21, 22) but
remained palatal in its histology. The epithelium of the nasal region differentiated into a pseudostratified ciliated columnar epithelium slightly taller than
normal, and the epithelium of the oral region differentiated first into a stratified
cuboidal epithelium (Fig. 21) but later became a stratified squamous epithelium
(Fig. 22) with positive birefringence in its outer cell layers (as in Fig. 25). In
cultures maintained for an extended incubation period, the nasal epithelium
included alcian blue-staining cells (Fig. 23), and cells of the oral epithelium
contained hematoxylin-staining granules (Fig. 24).
Salivary gland mesenchyme cultured in isolation became thinner and did not
undergo glandular morphogenesis (Figs. 26, 27). Histological sections of these
cultures showed no remnants of salivary gland epithelium, indicating that the
glandular morphogenesis seen in direct recombinations of palatal epithelium
and salivary gland mesenchyme was not due to a contaminant of salivary gland
epithelium left with the mesenchyme after the tissue separation procedures.
DISCUSSION
The results of this study demonstrate that palatal epithelium is capable of
attaining full histodifferentiation in the presence of foreign (non-palatal)
mesenchyme. Both tongue and salivary gland mesenchyme support normal
palatal histogenesis in palatal epithelium. In this respect, palatal epithelium is
similar to such epithelia as that of the embryonic pancreas, thymus and liver,
ciliated columnar epithelium, and the oral epithelium (Fig. 6) is a stratified squamous
epithelium. These epithelia are similar to the corresponding epithelia of the differentiated palate in vivo (Tyler, 1975; Tyler & Koch, 1975). x480.
Figs. 7-8. Photomicrograph of a transverse section through a direct recombination
between 12-day palatal epithelium and 13-day tongue mesenchyme cultured for
9 days. Epithelial differentiation is still palatal (cf. Figs. 5, 6). There are alcian bluestaining cells (arrow) in the nasal epithelium (Fig. 7), and numerous squamous cell
layers (several of which are being sloughed) are present within the oral epithelium
(Fig. 8). x 480.
Figs. 9-10. Photomicrographs of a transverse section through a direct recombination
between 12-day palatal epithelium and 16-day incisor mesenchyme cultured for
8 days. Epithelial differentiation, though palatal, is reduced in both the nasal (Fig. 9)
and the oral (Fig. 10) epithelia (cf. Figs. 7, 8). Also, there is no eosin-staining or
alcian blue-staining extracellular matrix at either the epithelial or the mesenchymal
surfaces, indicating that there has been no deposition of dental matrix, x 480.
Fig. 11. Polarization photomicrograph of the oral epithelium shown in Fig. 6. The
squamous cell layers of the oral epithelium are birefringent under polarized light
as they are in vivo, x 480.
Fig. 12. Polarization photomicrograph of the oral epithelium shown in Fig. 10. There
are fewer birefringent squamous cell layers than in vivo (cf. Fig. 11). x 480.
44
M. S. TYLER AND W. E. KOCH
13
Figs. 13-16. A series of photographs showing the in vitro development of a direct
recombination between 14-day palatal epithelium and 13-day salivary gland
mesenchyme at 0,24, 72, and 120 h of incubation respectively. By 24 h of incubation
(Fig. 14), the epithelium has retracted into a rounded mass (arrow); a glandular
morphogenesis then ensues, x 26.
Figs. 17-20. A series of photographs showing the in vitro development of a direct
recombination between 13-day palatal epithelium and 13-day salivary gland
mesenchyme at 0, 24, 72, and 120 h of incubation respectively. The rate of morphogenetic changes is similar to that shown in Figs. 15-18; however, the pattern of
branching is different, x 26.
Palatal epithelium in heterotypic recombinations
45
which, when challenged with various types of mesenchyme, differentiate according to their originally determined histogenetic patterns (Golosow & Grobstein,
1962; Auerbach, 1960; LeDouarin, 1970). Embryonic epidermis, on the other
hand, can be readily altered in its histogenesis by recombining it with heterotypic
mesenchyme (McLoughlin, 1963; Rawles, 1963; Lawrence, 1971). Epithelia,
therefore, vary with respect to their histogenetic stability in heterotypic tissue
recombinations. The nasal and oral epithelial regions of the palate responded
similarly when confronted with heterotypic mesenchyme, to the extent that each
differentiated in accordance with its originally determined histogenetic program.
Not all heterotypic mesenchymes are equivalent in their ability to foster the
histodifferentiation of palatal epithelium: incisor mesenchyme, though it did
not redirect the histodifferentiation of palatal epithelium, did not support full
epithelial differentiation. Since previous studies have shown that the mesenchyme
from embryonic teeth younger than those used here is capable of inducing
amelogenesis in non-tooth epithelia (Kollar & Baird, 1970a, b), further studies
will investigate whether young incisor mesenchyme recombined with palatal
epithelium redirects the differentiation of the epithelium.
The embryonic period over which palatal epithelium was tested, 12-14 days
of gestation, represents the time following epithelial determination during
which overt epithelial differentiation is not yet apparent (Tyler & Koch, 1977).
In most cases, the response of 12-day palatal epithelium to heterotypic mesenchyme did not vary from that of 14-day epithelium. However, in combination
with incisor mesenchyme, 12-day palatal epithelium exhibited less of a potential
for normal palatal histodifferentiation than did 14-day palatal epithelium,
indicating that developmental changes, though not overt, are occurring within
the palatal epithelium between 12 and 14 days of gestation. As in other systems
Figs. 21-22. Photomicrographs of transverse sections through direct recombinations between 12-day palatal epithelium and 13-day salivary gland mesenchyme
(Fig. 21) and between 14-day palatal epithelium and 13-day salivary gland mesenchyme (Fig. 22), each cultured for 120 h. The epithelium, though organized into
a system of ducts and acini, is palatal in its histology. The oral epithelium in Fig. 21
is a stratified cuboidal epithelium, but in Fig. 22 it is seen as a stratified squamous
epithelium. The nasal epithelium in both figures is a pseudostratified ciliated
columnar epithelium, x 190.
Figs. 23-24. Photomicrographs of a transverse section through the culture shown in
Fig. 22 seen at a higher magnification. Cells containing alcian blue-staining
material (arrow) can be seen within the nasal epithelium (Fig. 23), and hematoxylinstaining granules (arrow) are present within the oral epithelium (Fig. 24). x 480.
Fig. 25. Polarization photomicrograph of the oral epithelium shown in Fig. 24. The
squamous cell layers of the epithelium are birefringent under polarized light, x 480.
Figs. 26-27. Photographs of isolated 13-day salivary gland mesenchyme in vitro at
0 and 96 h of incubation respectively, showing that glandular morphogenesis does
not occur in the isolated mesenchyme. x 26.
4
EMB 38
46
M. S. TYLER AND W. E. KOCH
(Rutter et ah 1968), this type of developmental change may be termed
' protodifferentiation'.
The recombinations between palatal epithelium and salivary gland mesenchyme demonstrated that a specific morphogenetic pattern can be imposed
upon palatal epithelium. Although epithelial morphogenesis was dramatically
altered in these recombinations, the histogenetic pattern of epithelial differentiation was not altered, indicating clearly that the morphology of an epithelium
is not necessarily dependent upon the direction of its histodifferentiation. Further
studies are planned which will investigate whether or not palatal epithelium,
once differentiated, is still susceptible to the morphogenetic influence exerted by
salivary gland mesenchyme.
The response of palatal epithelium to salivary gland mesenchyme is similar
to that of epidermis in combination with mammary gland mesenchyme. In
direct recombination studies, it has been shown that embryonic rabbit mammary
gland mesenchyme is able to influence embryonic chick epidermis to become
organized as hollow epithelial invaginations without necessarily eliciting a change
in epidermal histodifferentiation; whether or not the epithelium retained its
epidermal characteristics depended upon the age of the epithelium (Propper,
1969, 1975). Other studies may be cited which also have distinguished between
the morphogenetic and histogenetic aspects of differentiation. In vitro studies
on the thymus have shown that the culture conditions that promote morphogenesis in the thymus gland do not necessarily also promote histogenesis
(Auerbach, 1960; Ball & Auerbach, 1960). Recombination studies using pancreatic epithelium have shown that, when recombined with various types of
mesenchyme (kidney, lung, stomach, and salivary gland), pancreatic epithelium
undergoes morphogenetic branching patterns which vary characteristically with
the type of mesenchyme, but it retains a pancreatic histology (Golosow &
Grobstein, 1962).
It is concluded from this study that the mesenchymal requirements for
histodifferentiation in the palatal epithelium of the embryonic mouse are
relatively non-specific at an early stage in palatal development. The relative
stability of histogenesis in the epithelium does not preclude some lability in its
morphogenesis, and thus histogenesis and morphogenesis, though normally
closely integrated during development, are potentially independent parameters
of epithelial differentiation.
This investigation was supported by USPH Research Grants DE-02863 and DE-02668
from the National Institute of Dental Research, and RR-05333 from the Division of Research
Facilities and Resources National Institutes of Health.
The authors thank Ms Locksley G. Henage and Ms Daynise Skeen for their technical
assistance in this study.
Palatal epithelium in heterotypic recombinations
41
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{Received 9 April 1976, revised 17 November 1976)