/. Embryol. exp. Morph. 88, 265-279 (1985)
265
Printed in Great Britain © The Company of Biologists Limited 1985
The influence of the epithelium on palate shelf
reorientation
ROBERT F. BULLEIT AND ERNEST F. ZIMMERMAN
Division of Basic Science Research, Children's Hospital Research Foundation,
Cincinnati, Ohio 45229, U.S.A.
SUMMARY
The intrinsic forces necessary for directing the reorientation of the secondary palate appear to
reside in the anterior two thirds of the palate or presumptive hard palate. The hard palate could
reorient regardless of whether it was intact or separated from the posterior third or presumptive
soft palate. The soft palate could only reorient if the palate shelves are left intact. These intrinsic
forces, within the hard palate, may be mediated by the mesenchymal cells, their extracellular
matrix, or the epithelium surrounding the shelves. This latter possibly was tested by removing
the epithelium, from either the presumptive oral or nasal surface followed by measurement of
reorientation in vitro. Only after removal of the oral epithelium was a significant inhibition in
reorientation observed. The treatment used to remove the epithelium, EDTA and scraping, was
shown to remove 41 % of the oral epithelium leaving the majority of the basement membrane
intact. The observed inhibition of reorientation did not appear to be a consequence of wound
healing. Creation of wounds twice the area that was observed after treatment with EDTA and
scraping inhibited reorientation minimally. These results suggest that the epithelium and
particularly the anterior oral epithelium plays a major role in the reorientation of the murine
secondary palate.
INTRODUCTION
One aspect of the morphogenesis of the mammalian secondary palate involves
the reorientation of the two apposing palate shelves. The shelves move from a
vertical position on either side of the tongue to a horizontal position above the
tongue separating the oral and nasal cavities. The process of reorientation has
been studied in vitro using murine embryos (Brinkley, 1975; Wee et al. 1976).
Foetal mouse heads cultured following removal of the tongue and brain showed
similar reorientation to that observed in situ (Brinkley et al. 1975, 1978). These
results suggest that structures such as the tongue and brain as well as circulation
are unnecessary for the process of reorientation. The observations are consistent
with an intrinsic shelf force directing reorientation (Walker & Fraser, 1956). The
intrinsic components in the shelves which could be involved in directing this
morphogenesis may be associated with the mesenchyme and its extracellular
matrix, or the epithelium surrounding the shelves (Larsson, 1960; Pourtois, 1972;
Pratt et al. 1973; Babiarz et al. 1975; Ferguson, 1977; Brinkley, 1980). The
epithelium is of particular interest because epithelia define the shape and structure
Key words: palate, epithelium, reorientation, mouse embryo.
266
R. F. BULLEIT AND E. F. ZIMMERMAN
of tissues and appear to be morphogenetically active in a number of developing
systems (Grobstein, 1967; Karfunkel, 1974; Brady & Hilfer, 1982; Brun & Garson,
1983). The evidence to support a role for the epithelium in palate shelf
reorientation is still inconclusive. Epithelial cell shape, distribution and/or
layering changes, as well as rugae development and an increase in mitotic figures in
the presumptive oral epithelium do correlate with the timing of reorientation
(Brinkley, 1984; Luke, 1984). What is uncertain is whether these changes are
necessary for reorientation to occur.
In the present study we examined the effects of epithelial removal on
reorientation in vitro to determine more directly whether the epithelium plays a
role in this process. Preliminary observations indicated that disruption of the
presumptive oral surface could inhibit reorientation in vitro (Bulleit &
Zimmerman, 1984). The results presented in this paper substantiate those findings
and demonstrate that removal of the epithelium on the anterior oral aspect of the
palate and not wounding is primarily responsible for the observed inhibition.
These findings are consistent with the epithelium, in particular the anterior oral
epithelium, directing secondary palate morphogenesis.
MATERIALS AND METHODS
Animals and culture system
A/J mice (Jackson Laboratories) were used in these studies. We have also used CFW mice
(Charles River), a random-bred strain, and have obtained similar results. Matings were carried
out by placing individual females with males overnight. The presence of a vaginal plug the
following morning was taken as evidence for pregnancy and designated day zero. On day 14 of
gestation animals were sacrificed by cervical dislocation and gravid uteri were placed on ice. This
is approximately 24 h prior to in situ shelf reorientation (Andrew et al. 1973). Embryos were
removed and examined, those with cleft lip were eliminated. The remaining embryos were
staged using a morphologic rating system described by Walker & Crain (1960). Embryos with a
rating of 5-7 were used for culture.
Heads were removed followed by excision of mandibles and tongues. This allowed access to
palates for mechanical manipulation of the epithelial layers. A cut was also made down the
midline of the cranium which allowed better access of medium to tissues. For most experiments
a transverse cut was made in the palate two thirds the distance from the anterior end to separate
the presumptive hard and soft palate (Diewert, 1978). This is indicated in Fig. 1 by the dashed
line (H/S). Heads were then placed in 15 x 45 mm vials containing 3 ml Dulbecco's Modified
Eagles Medium (DMEM, Gibco) and 10 % foetal bovine serum (FBS, M.A. Bioproducts). The
vials were gassed with a mixture of 95 % O 2 ,5 % CO2 and sealed with rubber stoppers. The vials
were placed in a rotating culture apparatus (approximately 30 r.p.m.) for up to 6 h. In one set of
experiments cultures were maintained for 24 h.
Epithelial removal and wounding
Heads were treated prior to culture in phosphate-buffered saline (PBS) without Ca2+ and
Mg2"1" containing 10mM-EDTA for lOmin at 37 °C. The tongue remained between the palate
shelves during this treatment. Following treatment, the palates on either the presumptive oral or
nasal surface were scraped using the edges of finely sharpened curved forceps to tease the
epithelium from the underlying basement membrane.
Wounding of the palate surface was performed by making a tear in the oral surface running
the length of the hard palate and projecting into the underlying mesenchyme. This was
accomplished using the sharpened point of a pair of forceps. Following epithelial removal or
Epithelial influence on palate reorientation
267
Fig. 1. The dashed line H/S indicates the transverse cut made in the palate shelves to
separate the presumptive hard (H) and soft (S) palate. The solid lines indicate where
razor blade sections were cut to measure anterior (A), mid (B) or posterior (C) PSI.
wounding the tongue was removed and the presumptive hard and soft palate were separated.
The heads were then cultured as described previously.
Quantitation of reorientation in vitro
The amount of reorientation occurring during culture was measured using palate shelf index,
PSI (Wee et al. 1976). Following culture, heads were fixed in Bouin's solution for 24 h. Fixed
heads were cut coronally using a razor blade. Razor blade sections were made by cutting the
palate shelves in the middle of each third of the palate, allowing for measurement of anterior,
mid, and posterior PSI, as indicated in Fig. 1 by the lines A, B, and C respectively. The
transverse sections were placed on end and visualized using a dissecting microscope. Values of
1-5 were assigned to express the degree of palate shelf reorientation. A number of 1 was
assigned for a completely vertical and a 5 for a completely horizontal palate. Numbers 2, 3 or 4
were designated intermediate elevations of one-quarter, one-half or three-quarters of the way to
the horizontal position. Experimental results were expressed as mean PSI ± standard error.
Statistical analysis of the difference between the mean PSI of treatment groups and their
respective controls was performed using a two-tailed Student's t-test. The number of palate
shelves evaluated in each treatment group is indicated in the tables.
Scanning electron and light microscopy
For SEM heads were fixed in 3 % glutaraldehyde in 0-163 M-sodium cacodylate buffer for
4-5 h at 25 °C followed by washing in buffer. The heads were then dehydrated in a graded series
of ethanol (30-100%) and critical-point dried using carbon dioxide as a transitory fluid.
Thereafter heads were mounted, coated with gold, and examined using a scanning electron
microscope (ETEC Autoscan).
For light microscopy, heads were fixed in modified Karnofsky's fixative, as modified by
Singley & Solursh (1980). Isotonic sodium phosphate buffer (0-2 M) was used instead of sodium
cacodylate buffer. Heads were fixed overnight followed by dehydration in a graded series of
ethanol (50-100 %) with a final passage through 100 % propylene oxide. The heads were then
embedded in Poly/Bed 812 (Polyscience, Inc.). Sections were cut at 1-2/mi, stained in
Richardson's stain (Richardson et al. 1960) and examined with a Zeiss photomicroscope.
Immunofluorescence
Heads were fixed for 4h in Hank's balanced salt solution without Ca2+ and Mg2+ containing
2% paraformaldehyde and 0-32M-sucrose. Heads were removed and dehydrated through a
graded series of ethanol (50-100 %) followed by two changes of xylene. The heads were then
embedded in paraffin. Sections were cut at 6^m, placed on glass slides and rehydrated prior to
antibody staining. Slides were incubated sequentially with rabbit antiserum and rodamineconjugated goat anti-rabbit IgG diluted from 1:50 to 1:100. Rabbit antisera to laminin (Engvall
268
R. F. BULLEIT AND E. F. ZIMMERMAN
et al. 1983), heparan sulphate proteoglycan BM1 (Hassell et al. 1980), and type IV collagen
(Timpl et al. 1978) were gifts of Erkki Ruoslahti, John Hassell and George Martin, respectively.
Morphometric analysis
Palates were analysed morphometrically. The degree of epithelial removal was quantitated
from scanning electron micrographs of heads treated with EDTA and scraping of the oral
surface. From these micrographs three different areas on the oral surface were defined and
measured using computer-assisted image analysis (Videoplan, Zeiss). The areas measured were
(1) total oral surface of the palate, (2) area of epithelial removal, (3) area in which the basement
membrane had been damaged exposing the underlying mesenchyme, defined as the area of
wounding. Thirty palates were measured in this experiment and the mean, standard error, and
percent of total oral surface were determined for each defined area.
RESULTS
Reorientation of segmented palate shelves in vitro
The separation of the presumptive hard and soft palate allowed reorientation of
the hard palate to occur while reorientation of the soft palate was inhibited. The
measurement of palate shelf index showed that in fact the anterior and mid palate
were enhanced in their degree of reorientation following separation of the palate
shelves (Table 1). This was observed at all times of culture. Particularly apparent
was a 0-6 PSI unit difference following l h of culture between controls and
segmented palates in both anterior and mid palate. By 6 h of culture the anterior
palate had reached a PSI of 4-5 or 90 % movement toward the horizontal position.
The mid palate had not yet reached the same degree of movement (75 % of
horizontal). There was also observed in both anterior and mid palate a reduced
gap between the separated palate shelf compared to the control shelf (Fig.
2A,B>E),E)- in contrast the posterior palate was inhibited from reorienting
following separation of the hard and soft palate. By 6 h of culture there was a
Table 1. The effects of separation of the hard and soft palate on reorientation ofpalate
shelves in vitro
Palate
segment
measured
Anterior
Mid
Posterior
PSI±S.E.M.(n)
Protocol
0
Control
H/S
Control
H/S
Control
H/S
2•4 ± 0-09 (28)
1•9 ± 0-05 (28)
1•1± 0-07 (28)
Time in culture (h)
1
3
3-4 ± 0-09 (30)
4-0 ± 0-08 (34)a
2-6 ± 0-09 (30)
3-2 ± 0-09 (34)a
1-4 ±0-09 (30)
1-2 ±0-06 (36)
H/S: palate shelves with hard and soft palate separated.
Different from control values:
a
P< 0-0001
b
P < 0-001
c
P<0-02
d
P<0-05
3-9 ± 0-07 (36)
4-2 ± 0-07 (30)d
2-8 ± 0-07 (36)
3-3±0-ll(30) b
1-7 ±0-09 (36)
1-2 ± 0-09 (30)b
6
4-2 ± 0-09 (30)
4-5 ± 0-07 (48)c
3-0 ±0-13 (30)
3-7 ± 0-09 (48)a
1-9 ±0-10 (30)
1-3 ± 0-06 (48)a
Epithelial influence on palate reorientation
269
difference of 0-6 PSI unit between controls and segmented palate shelves (Table
1). In comparison, segmented palates had a larger gap between the posterior
shelves and they appeared more vertical (Fig. 2C,F). When heads were cultured
for 24 h, the anterior palate could fully reorient and fuse, while the soft palate was
still markedly inhibited. However some movement of the soft palate toward the
horizontal position took place (Fig. 3B). In contrast, when the hard and soft palate
were left intact both regions of the palate could reorient and fuse (Fig. 3A).
In all further experiments, culture periods of 24 h were not employed because of
poor survival of the inner cranial tissue. Cultures were maintained for 1 to 6h
following separation of the hard and soft palate. In this way the effect of epithelial
removal on reorientation of the hard palate could be examined separate from
influences of the soft palate.
Fig. 2. Coronal section of palate shelves following 6 h of culture (x60). Shelves were
cultured with the full shelf intact (A,B,C) or following separation of the presumptive
hard and soft palate (D,E>F)- Sections were made in the anterior (A,D), mid (B,E) or
posterior (C,F) palate. Bar equals 100/im.
270
R. F. BULLEIT AND E. F. ZIMMERMAN
Fig. 3. Scanning electron micrographs showing the oral surface of palate shelf
following 24 h of culture (x35). Palate shelves were cultured with the full shelf intact
(A) or following separation of the anterior two-thirds or presumptive hard palate (H)
from the posterior one-third or presumptive soft palate (S). pm, premaxilla; arrow,
rugae. Rugae were observed on the oral surface of all palate shelves before and after
culture. Bar equals 100[Am.
The effects of epithelial removal on reorientation in vitro
Measurements of PSI showed no consistent significant difference in the degree
of reorientation from control values following treatment with EDTA alone or
EDTA followed by scraping of the nasal surface. EDTA alone inhibited
reorientation to a small degree which was only significant in the anterior palate
following 3h of culture. Nasal epithelial disruption showed inhibition only in the
mid palate after 1 h of culture.
In contrast, treatment with EDTA followed by scraping of the oral surface
significantly inhibited palate shelf reorientation when compared to control or
EDTA treatment alone. This result was observed in both the anterior and mid
palates at all time intervals measured (Table 2). However the anterior palate
appeared to be more affected by this treatment. The inhibition did not appear to
be specific to the A/J strain of mouse. CFW, a random bred strain of mouse,
showed a similar pattern of inhibition. When treated with EDTA alone the PSI in
the anterior end was 4-5 ±0-11 and the mid palate was 3-7 ±0-11 after 6h of
culture. In contrast, if they were treated with EDTA followed by scraping of the
oral epithelium, inhibition was observed in both anterior (PSI = 3-0 ± 040) and
mid (2-7 ± 0-13) palates. In all of these studies the posterior or soft palate did not
reorient and was unaffected by these treatments.
The morphology of the palate shelves after 6h of culture in control, EDTA
treatment alone, or EDTA treatment followed by nasal epithelial disruption, was
very similar (Fig. 4B-D). After nasal surface disruption, removal of the
epithelium was observed as well as some minor disruption of the underlying
Epithelial influence on palate reorientation
271
structures (Figs 4D, 5C). In contrast the morphology of palate shelves treated with
EDTA followed by disruption of the oral epithelium was altered (Fig. 4E). The
shape of the palates appeared expanded compared to controls or EDTA treatment
alone. Also observed was an increase in the gap between the two apposing palate
shelves. At higher magnification, the oral surface was devoid of epithelium and
the mesenchyme directly beneath this removed epithelium appeared relatively
undisturbed (Fig. 5D).
Analysis of epithelial removal
The degree of epithelial removal and wounding following treatment with EDTA
and scraping was quantitated using computer-assisted image analysis. It was
observed that on the average 41 % of the oral epithelium had been removed while
only 4% of the oral surface showed evidence of wounding (Table 3). Scanning
electron micrographs showed areas in which the epithelium was still intact and
other areas where the epithelium had been teased away exposing a non-cellular
structure (Fig. 6A,B). In some places the non-cellular structure had been torn
away to expose underlying mesenchymal cells, which indicate some wounding
(Fig. 6B). These observations suggested that the non-cellular structure was
basement membrane apposed between epithelium and mesenchyme. This
Table 2. The effect of EDTA treatment and mechanical disruption of the palate
epithelium on reorientation of palate shelves in vitro
~
segment
measured
Anterior
Mid
Posterior
Treatment
0
Control
2-4 ±0-09 (28)
EDTA
EDTA,N/E
EDTA,O/E
Control
1-9 ±0-05 (28)
EDTA
EDTA,N/E
EDTA,O/E
Control
1-1 ±0-07 (28)
EDTA
EDTA,N/E
EDTA,O/E
-
PSI±s.B.M.(n)
Time in culture (h)
1
3
4-0 ±0-08 (34) 4-2 ±0-07 (30)
3-8 ±0-10 (18) 3-9 ± 0-14 (20)d
4-0 ±0-11 (18) 4-2 ±0-11 (20)
2-8 ± 0-13 (18)a 2-9 ± 0-15 (20)a
3-2 ±0-09 (34) 3-3 ±0-11 (30)
3-2 ±0-10 (18) 3-3 ±0-12 (20)
2-8 ± 0-10 (18)c 3-3 ±0-14 (20)
2-7 ± 0-11 (18)c 2-6 ± 0-11 (20)b
1-2 ±0-06 (34)
1-2 ±0-09 (30)
1-1 ±0-06 (18)
1-2 ±0-10 (20)
1-2 ±0-10 (18)
1-3 ±0-10 (20)
1-2 ±0-09 (18)
1-3 ±0-10 (20)
6
4-5 ±0-07 (48)
4-3 ±0-11 (30)
4-4 ±0-13 (20)
3-3 ± 0-10 (32)a
3-7 ±0-09 (48)
3-5 ±0-14 (30)
3-5 ±0-11 (20)
2-8 ± 0-11 (32)a
1-3 ±0-06 (48)
1-2 ±0-07 (30)
1-2 ±0-09 (20)
1-1 ±0-11 (32)a
Palate shelves were pretreated with 10mM-EDTA for lOmin. Mechanical disruption was
performed by scraping the presumptive nasal (N/E) or oral (O/E) surface withfinelysharpened
forceps. Following this treatment the palate shelves were separated into hard and soft palate and
cultured in DMEM plus 10 % FBS.
Different from values for EDTA treatment alone:
a
P < 0-0001
b
P < 0-001
C
P< 0-005
Different from control values:
d
P<0-05
272
R. F. BULLEIT AND E. F. ZIMMERMAN
conclusion was confirmed by performing immunofluorescence studies using
antisera to two basement membrane antigens, type IV collagen and heparan
sulphate proteoglycan. Both antisera showed immunoreactivity with a structure
apposed between epithelium and mesenchyme, as well as remaining attached to
areas of mesenchyme after epithelial removal (Fig. 7A,B)- A similar pattern of
immunoreactivity was also observed with antisera to the basement membrane
glycoprotein laminin (result not shown).
The effects of wounding on reorientation in vitro
Wounds were made on the oral surface running the length of the hard palate and
covering approximately 9 % of the oral surface, or greater than twice the area of
Fig. 4. Coronal section through palate shelves following Oh (A) or 6h (B,C,D,E) of
culture (x60). Palate shelves were cultured following treatment with PBS with Ca2+
and Mg2+ (B), PBS without Ca2+ and Mg2+ containing 10mM-EDTA alone (C),
followed by scraping the presumptive nasal surface (D), or scraping the presumptive
oral surface (E). Bar equals 100 ^m.
Epithelial influence on palate reorientation
273
Fig. 5. The epithelial surface of palate shelves cultured for 6h (xl50). Oral epithelial
surface of a control palate shelf following treatment in PBS with Ca2+ and Mg2+ (A).
Oral epithelial surface of a palate shelf treated in PBS without Ca2+ and Mg2*
containing 10mM-EDTA (B). Nasal epithelial surface of a palate shelf treated in PBS
without Ca2+ and Mg2* containing 10 mM-EDTA followed by scraping of the nasal
surface (C). Oral epithelial surface of a palate shelf treated in PBS without Ca2+ and
Mg2+ containing 10 mM-EDTA following by scraping of the oral surface (D). Bar
equals 100 fim.
wounding observed after EDTA and oral surface disruption (Fig. 8A). The
wounds protruded deep into the underlying mesenchyme and the surface of the
wound appeared devoid of epithelium (Fig. 8B). Following 6h of culture, wound
healing could be observed in the palate shelves (Fig. 8C). The mesenchyme in the
area of the wound was condensed and contracted with some areas of the
mesenchyme still remaining separated. The epithelium at this time had not yet
covered the wound. The palate shelves also showed substantial movement toward
Table 3. Morphometric analysis of the oral surf ace following EDTA and mechanical
treatment
Area measured
Total oral surface
Area of epithelial removal
Area of basement membrane disruption
% of total oral
Mean area of
micrographs (mm2) ± S.E. surface area
3323-3 ±89-1
1363-8 ± 67-8
130-6 ± 20-7
100
41
4
Analysis was performed on scanning electron micrographs of 30 palates immediately
following treatment with EDTA for lOmin and mechanical scraping of the oral surface.
Micrographs were magnified x85.
274
R. F. BULLEIT AND E. F. ZIMMERMAN
Fig. 6. Scanning electron micrographs of palate shelves immediately following
treatment in PBS without Ca2+ and Mg2+ containing 10 mM-EDTA and scraping of the
oral surface A (x 143), B (x715). e, epithelial cells; m, mesenchymal cells; b, basement
membrane; arrow, wounded surface. Bar equals 10/mi.
the horizontal position. Measurement of PSI showed that wounding could inhibit
reorientation only to a small degree and this inhibition was significantly less than
that observed following treatment with EDTA and oral epithelial removal (Table
4).
DISCUSSION
The process of palate shelf reorientation may be directed by forces intrinsic to
the palate shelves (Walker & Fraser, 1956). Consistent with this idea is the ability
of the palate shelves to reorient in culture in the absence of the tongue, brain and
circulation (Brinkley, 1975, 1978). Experiments presented in this paper as well as
observations made previously (Brinkley & Vickerman, 1979) suggest that this
intrinsic force is particularly associated with the anterior two thirds or presumptive
hard palate. The hard palate could reorient whether intact or separated from the
posterior third or presumptive soft palate, while the soft palate failed to reorient
(Table 1). This result was also observed even after an extended culture period,
although some minimal movement was apparent (Fig. 3). This result might imply
that soft palate reorientation requires extrinsic factors provided by the hard
palate. This process could be accomplished by an anterior to posterior 'zippering'
effect of adhesion and fusion along the medial edge (Brinkley & Vickerman,
1979). Alternatively, hard palate reorientation may provide a pulling force for
directing the soft palate to a horizontal position. This latter interpretation is
supported by observations that the soft palate provides resistance to reorientation
of the hard palate. When the two segments were separated, hard palate
Epithelial influence on palate reorientation
275
reorientation was accelerated while the soft palate failed to reorient. The
reorientation of the isolated soft palate may require factors that cannot be
expressed in this culture system. The soft palate has been described as remodelling
around the tongue. This remodelling may require the presence of the tongue for
full expression. However, the importance of the tongue and these other factors
appear to be minimal in this culture system since the intact shelf showed
substantial reorientation and fusion of the soft palate (Fig. 3).
The major emphasis of this paper was to examine the role of the epithelium in
generating the intrinsic force for reorientation. The results observed suggest that
epithelium on the oral aspect of the hard palate is a major factor in generating this
intrinsic force. Removal of the oral epithelium significantly inhibited the pattern of
reorientation in vitro. In contrast, disruption of the epithelium on the presumptive
nasal surface had no effect on the degree of movement. The treatment used to
remove the epithelium, EDTA and scraping, removed primarily epithelium
leaving the majority of the basement membrane intact. This observation was
7A
m
Fig. 7. Immunofluorescence photographs of palate shelves treated in PBS without
Ca2+ and Mg2+ containing lOmM-EDTA and scraping of the oral surface (x500).
Palate shelf was fixed immediately following treatment for histology (A) or
immunofluorescence (B,C,D). Immunofluorescence was carried out using rabbit
antiserum to type IV collagen (B), antiserum to heparan sulphate proteoglycan (C), or
with a control rabbit serum (D). e, epithelium; m, mesenchyme. Bar equals O
276
R. F . BULLEIT AND E. F. ZIMMERMAN
Fig. 8. Palate shelves following wounding of the oral surfaces. The scanning electron
micrograph taken immediately following wounding shows the wound (wd) running the
length of the hard palate, mag. x35. Light micrograph of coronal section through the
palate immediately following wounding shows the wound (wd) running deep into the
mesenchyme, mag. X60 (B). Coronal section through wounded palate shelf following
6h of culture. Arrows indicate area of wound healing, mag. x60 (C). Bar equals
100 ^m.
Table 4. The effect of wounding the oral surface of the palate on reorientation of palate
shelves in vitro
Palate
segment
measured
PSI-SE (n)
Treatment
Time in <;ulture (h)
0
6
Anterior
Control
Wounded
2-4 ± 0-09 (28)
4-5 ± 0-07 (48)
4-3 ± 0-08 (52)a
Mid
Control
Wounded
1-9 ±0-05 (28)
3-7 ± 0-09 (48)
3-4 ± 0-07 (52)b
Posterior
Control
Wounded
1-1 ±0-07 (28)
1-3 ±0-06 (48)
1-2 ± 0-06 (52)
Palate shelves were wounded on the presumptive oral surface usingfinelysharpened forceps.
Following this treatment the palate shelves were separated into hard and soft palate and
cultured in DMEM plus 10 % FBS.
Different from control values:
a
P<0-05
b
P<0-02
Epithelial influence on palate reorientation
277
supported by both morphological (Hall & McSween, 1984) and immunohistochemical criteria (Table 3, Fig. 7). However this treatment also created some
wounding. It is possible that contraction of such wounds during wound healing
may have created a force to overcome the force necessary for reorientation and
was responsible for the observed inhibition. This interpretation appears unlikely
for two reasons. First, the degree of wounding observed after treatment with
EDTA and scraping was minimal, covering only on the average 4 % of the oral
surface. Secondly, when wounds were created covering two times this area only a
small degree inhibition of palate reorientation was observed. It could not be
determined whether this small degree of inhibition was due to wound healing or
the removal of the. epithelium over the wound. These results are consistent with
the removal of the oral epithelium and not wounding as being responsible for the
observed inhibition of palate shelf reorientation.
Brinkley & Vickerman (1979) have previously suggested that reorientation of
the hard palate may actually occur by expression of a pre-existing infrastructure.
The oral epithelium or an interaction between the oral epithelium and the
mesenchyme may help define this infrastructure. Alternative mechanisms for
controlling the process of reorientation include the expression of an active
contractile system within the mesenchyme (Babiarz et al. 1975) or the accumulation of a hyaluronic acid (HA)-rich extracellular matrix (Larsson, 1960; Pratt et
al. 1973; Furguson, 1977; Brinkley, 1980). It appears most likely that HA plays a
role in the reorientation of the mid palate. Enhanced degradation of glycosaminoglycans including HA by chlorcyclizine was able to inhibit reorientation in
this area while reorientation of the anterior palate was unaffected (Brinkley,
1982). This is in contrast to removal of the oral epithelium which inhibited
reorientation in the anterior palate more effectively (Table 2). It is possible that
the intrinsic force for reorientation is generated not by simply one element but by
the interaction of a number of elements including the oral epithelium, mesenchyme and a HA-rich extracellular matrix.
Our results suggest that the oral epithelium is particularly important in
generating the force for reorientation. How this is accomplished is unclear. The
control of growth and differentiation of this epithelium may be responsible for
generating a particular structure which is necessary for expression of this force.
Luke (1984) has observed an unequal cell division within palate epithelium. The
oral epithelium appears to be dividing more rapidly than the nasal epithelium. He
has suggested that this differential in cell division and development of rugae on the
oral surface may be responsible for palate shelf reorientation. The rugae may be
particularly important since they are exclusively associated with the hard palate
and their development correlate with reorientation in a number of mammals
(Waterman & Meller, 1974; Meller et al 1980). The formation of rugae on the oral
surface may stiffen the structure of the oral epithelium forcing the palate shelf
toward the horizontal position. Interference with the development of the
epithelium and rugae may then lead to the induction of cleft palate.
278
R. F. BULLEIT AND E. F. ZIMMERMAN
This work represents part of the Ph.D. dissertation of Robert Bulleit to the Graduate
Program in Developmental Biology, University of Cincinnati.
This study was supported by a NIEHS Training Grant (ESO7051).
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(Accepted 21 February 1985)