/. Embryo/, exp. Morph. Vol. 53, pp. 75-90, 1979
Printed in Great Britain @ Company of Biologists Limited 1979
75
Palate Morphogenesis
IV. Effects of serotonin and its antagonists on rotation
in embryo culture
By ELIZABETH L. WEE,1 BRUCE S. BABIARZ2,
STEPHEN ZIMMERMAN AND ERNEST F.ZIMMERMAN 1 ' 3
From the Institute for Developmental Research, Children's Hospital Research
Foundation, and Departments of Pediatrics and Pharmacology, University of
Cincinnati
SUMMARY
Previous studies have localized non-muscle contractile systems in the posterior (region 2)
and the anterior (region 3) ends of mouse palates at the time of shelf movement. In order to
determine whether these contractile systems function in shelf rotation, effects of pharmacologic agents have been analyzed in embryo culture. First, it was shown that the posterior end
of the palate rotates before the anterior end, and its rotation in culture was proportionally
greater as development of the embryo progressed. Generally, the posterior end of the palate
was more easily inhibited in embryo culture than the anterior end. Serotonin at 10~8 M to
10~5 M was shown to significantly stimulate rotation at the anterior end of the palate after
2 h in embryo culture. The effect on the posterior palate was less pronounced. To investigate
further the role of this neurotransmitter on palate shelf rotation, serotonin antagonists
were employed. Methysergide (10~ 4 M) inhibited anterior shelf rotation to 12% of control
values (/* < 0005), while not significantly affecting the posterior end. Ergotamine (10~6 M)
significantly inhibited the stimulation induced by 10~5M serotonin (P < 0025). Cyproheptadine (10 ° M) partially inhibited anterior and posterior shelf rotation in embryo culture.
When injected into the pregnant dam, cyproheptadine partially inhibited shelf rotation
and fusion. The palate was examined histologically after embryo culture. In the presence
of 10~4 M methysergide, the elongated contractile cells in region 3 at the anterior and midpalatal mesenchyme were prevented from rounding. Thus, serotonin may be regulating
rotation of the anterior end of the palate by an effect on a cell-mediated process.
INTRODUCTION
Development of the secondary palate in mice involves elevation of the shelves
between days 14-5 and 15-5 of gestation, and shortly thereafter the shelves fuse
to complete palate formation (Walker & Fraser, 1956). Uncertainty still exists
concerning the mechanisms which are responsible for the elevation of the palatal
shelves. It has been postulated that the force moving the palate is external:
1
Authors' address: Children's Hospital Research Foundation, Elland and Bethesda
Avenues, Cincinnati, Ohio 45229, U.S.A.
2
Author's address: Department of Developmental Genetics, Sloan-Kettering Institute,
New York, New York .10021, U.S.A.
3
Reprint requests to Dr Ernest F. Zimmerman.
76
E. L. WEE AND OTHERS
straightening of the cranial base (Verrusio, 1970), and non-palatal muscular
movements, such as neck flexion, swallowing and descent of tongue and lower
jaw (Humphrey, 1969; Walker, 1969). Alternatively, an intrinsic shelf force
(Walker & Fraser, 1956) has been postulated to move the shelf. One candidate
would derive from the rapid synthesis of glycosaminoglycans in the palate
(Larsson, 1962; Ferguson, 1978). Hyaluronic acid, which is rapidly synthesized
in the palate (Pratt, Goggins, Wilk & King, 1973), is hydrophilic, binds considerable quantities of water and causes an increase in osmotic pressure
(Laurent, 1970). As a result, a force is thought to be produced to elevate the
shelves. Initial studies in our laboratory have shown that the contractile proteins, actin and myosin, are synthesized in day-14.5 mouse palates at a rate
equal to that of the tongue (Lessard, Wee & Zimmerman, 1974). Morphological
studies using myosin ATPase histochemistry, electron microscopy, and immunofluorescence with actin and myosin antibodies have localized the contractile
systems into three regions: region 1, a skeletal muscle system on the oral side
of the far posterior palate; region 2, a non-muscle contractile system on the
tongue side extending along the top of the shelf from mid-palate to the posterior
limit; and region 3, another non-muscle contractile system along the oral epithelium from the mid-posterior palate extending into the anterior end (Babiarz,
Allenspach & Zimmerman, 1975; Kuhn, Babiarz, Lessard & Zimmerman, 1977).
To determine whether these putative contractile systems function in shelf
rotation, an embryo culture system in which palate shelves rotate has been
developed (Wee, Wolfson & Zimmerman, 1976). In that study it was shown
that a cholinergic system was involved in rotation of the posterior end of the
palate shelf.
This present study investigates the role of serotonin on palate shelf rotation
since this neurotransmitter has been implicated in morphogenetic movements
of cellular processes, such as sea urchin gastrulation (Gustafson & Toneby,
1970), myofibroblast contraction in wound healing (Ryan et al. 191 A), and
contraction of platelets (Boullin & Orr, 1976). Furthermore, it has been shown
that palate shelves take up and synthesize serotonin during the period just prior
to rotation (Zimmerman & Roberts, 1977). In the present investigation it is
shown that serotonin predominantly stimulates rotation of the anterior palate
in the embryo culture system and antagonists of serotonin inhibit the morphogenetic movement. These results support the hypothesis that serotonin plays a
role in regulating palate morphogenesis through a non-muscle contractile
system observed in the palate.
MATERIALS AND METHODS
Materials. Dulbecco's Modified Eagle medium (DMEM) was purchased
from Grand Island Biological Company. Gentamicin (40 /^g/ml) was added to
DMEM. The following pharmacological agents were used: 5-hydroxytrypt-
Serotonin on palate rotation
11
amine (serotonin) creatinine sulfate (Sigma); ergotamine tartrate (Sigma);
cyproheptadine HC1 (Merck, Sharp and Dohme); methysergide maleate
(Sandoz). All drug solutions were freshly prepared.
Animals. A/J mice were purchased from the Jackson Laboratory. Female
mice were mated overnight and the presence of a vaginal plug the following
morning was taken as evidence of pregnancy and designated day 0-5 of gestation. Pregnant mice were killed by cervical dislocation at day 14-75 of gestation,
the time just prior to shelf rotation (Andrew, Bowen & Zimmerman, 1973). The
gravid uterus was removed immediately and placed in a sterile dish containing
cold DMEM that was pregassed with 95 % O2-5 % CO2.
In the experiment in which cyproheptadine was administered in vivo (Table 3),
3-24 mg/kg was injected twice i.p. on days 14-5 and 14-75 and fetal palates were
analyzed at day 15-5 of gestation. Embryos were fixed in Bouin's solution for
24 h and palate gap was measured under a dissecting microscope with an ocular
micrometer, as described in Wee et al. (1976).
Embryo culture. Culture medium contained 25 % human serum (immediately
centrifuged fresh serum; Steele & New, 1974) and DMEM, unless otherwise
specified. Serum was stored at —20 °C for no longer than 2 weeks to be considered fresh. Explantation and culture of the embryos were as previously
reported (Wee et al. 1976). Briefly, individual day-14-75 conceptuses were
removed from the uterine horn and placed in cold DMEM. Reichert's membrane
was removed and a slit was cut in the yolk sac. The embryo was slipped out of
the sac and the tongues were carefully excised to allow palate rotation in vitro.
It is widely appreciated that embryos vary widely in their development, either
when they are present in the same litter or in other litters. Previously we
employed crown-rump length as a basis for selection of embryos within a
specific developmental time (Wee et al. 1976). In the present study, we have
employed a morphological rating (MR) of the embryo based on external
features (Walker & Crain, 1960) for each embryo suspended in medium. Values
were arbitrarily assigned for each of five developmental features: forefeet,
hindfeet, ears, hair follicles and eyes. A morphological rating for an embryo
was calculated by adding up the numerical values. For example, a typical day14-75 embryo with a morphological rating of 7 would have 1/2 webbed forefeet
(numerical values = 2), 3/4 webbed hindfeet (2), 1/3 closed ears (1), hair
follicles on body and a few on side of head (2) and opened eyes (0). Only embryos
with MR from 5-7 were employed in the experiments, unless otherwise
indicated.
After determining the MR, only single embryos with beating hearts were
placed into glass vials (19x48 mm, Kimble Opticlear) that were filled with
2 ml of culture medium. Each vial was gassed with a mixture of 95% O 2 5 % CO2, tightly closed with a silicone rubber stopper, and sealed with tape.
The explanted embryos were cultured at 37 °C under sterile conditions, for the
time specified, by rotation of the vials at 60 rev./min in a Bellco variable speed
6
EMB
53
78
E. L. WEE AND OTHERS
roller drum. As previously described, palate rotation in vitro was complete
after 6 h at which time heart beat was normal. After overnight culture, heartbeat usually had ceased (Wee et al. 1976). After incubation, embryos were fixed
in Bouin's solution for 24 h at which time heads were cut off and mandibles
removed to observe palate rotation and fusion.
Quantitative assay of movement at the anterior and posterior ends of the
palates was performed as previously described (Wee et al. 1976). Blocks of
fixed heads, cut coronally half-way through the anterior and posterior ends,
were placed on end, and the angle of the anterior or posterior shelves to the
nasal septa was estimated with an ocular micrometer. Values of 1-5 were
arbitrarily assigned for the palate shelf index (P.S.I.). A number 1 was assigned
for a completely vertical shelf; 5 for a completely horizontal shelf; 3 for a shelf
at 45° (Fig. 4A); 2 for a shelf between 1 and 3; and 4 for a shelf position
between 3 and 5. Computing a mean position from these numerical values
allowed a statistical comparison of the treatment groups, employing the Student's
t test. Since there is a certain degree of rotation before embryo culture, this was
taken into account in expressing the results as % control movement where:
n,
. ,
. treatment group
P.S.I.-fixed group P.S.I. 1/xn
% control movement =
—r- 5
x 100.
z—-—'
control group p.s.i.-nxed
group p.s.i.
Light microscopy. After embryo culture, whole fetal heads were fixed for 1 h
at room temperature in phosphate-buffered (pH 7-3) Karnovsky's fixative
(Karnovsky, 1965). After the top of the head was dissected away, the heads
were fixed for another hour at room temperature in Karnovsky solution.
Following dehydration in a graded series of acetone (50 %, 70 %, 85 %, 95 %,
and 100%), the heads were infiltrated with an acetone and epon mixture overnight and then embedded in a soft epon resin (Luft, 1961), employing three
parts of solution A and one part solution B. Blocks were then polymerized at
60 °C for 48-60 h. These heads were serially cross-sectioned at 1 /tm on a
Reichert OMU-3 ultramicrotome using glass knives. Sections, on gelatincoated slides, were stained in 0-1 % toiuidine blue (Trump, Smuckler & Bend,
1961) and photographed on a Zeiss photomicroscope with bright field optics
and Kodak Panatomic-X film.
RESULTS
Palate shelf rotation: before and after embryo culture
In the present study, we have correlated the stage of palate movement (palate
shelf index, P.S.I.) with morphological development (morphological rating).
A comparison was carried out with embryos that had been cultured and those
that had not (Fig. 1). It is observed that, in embryos not cultured (fixed), little
anterior palate rotation had occurred during development delineated by a
morphologic rating of 5-12. Similarly, the posterior palate did not move when
development was staged to a MR of 7. Thereafter, a progressive increase in
Serotonin on palate rotation
7
10
79
11
12
MR
Fig. 1. Palate shelf rotation at various stages of development with embryos before
and after embryo culture. Embryos were removed from uterine horns, tongues
excised and morphological rating (MR) determined as described in Materials and
Methods. Thereafter, embryos were either fixed or cultured overnight and then
fixed. Values presented are the means of the palate shelf index (p.s.r.) at the anterior
and posterior ends of palates computed from 14 to 95 embryos at the indicated
MR. Bars indicating S.E. are presented for all values except those from the anteriorcultured group since the S.E. in all cases were 000. • , ANT. fixed; O—O, POST,
fixed; A - - A , ANT. cultured; A - - A , POST, cultured.
elevation of the posterior end of the palate shelf occurred up to a MR of 12,
the last stage that was monitored. These results indicate that the posterior end
of the palate appreciably elevates before the anterior end during development.
When embryos were removed at various developmental stages (MR 3-12)
and then cultured overnight, the anterior end of the palate elevated completely
(P.S.I. of 5). This is in marked contrast to the posterior end. In general, the later
developmental^ that the embryo was cultured, the greater the degree of palate
elevation. Since the posterior shelves were already moving in vivo at the later
stage in development (MR 8-12), subsequent experiments employed embryos
at a morphologic rating of 5-7; stages when the posterior end rotated in culture
without having appreciably moved in vivo before culture.
Embryo culture conditions
Different culture conditions were tested for their effects on palate shelf
rotation (Table 1). Substituting aged human serum for fresh serum did not
alter palate shelf rotation. The use of Dulbecco's Modified Eagle Medium or
saline without serum caused an inhibition of posterior shelf rotation. Substituting N 2 for O2 in the gassing procedures also caused a slight inhibition of
posterior shelf rotation (71 % of control). Under all of these experimental
conditions, anterior ends of the shelves completely rotated after overnight
culture.
6-2
80
E. L. WEE AND OTHERS
Table 1. Palate shelf rotation \in embryo culture*
% Control movement
P.S.lt.
Condition
n
Fixed
134
Control
180
+ Aged serum, - serum 30
- Serum
40
- O 2 , +N 2
26
-Serum, -Dulbecco's
10
medium, + saline
Anterior
Posterior
Anterior
Posterior
2-61 ±005
500 ±000
500 ±000
500 ±000
500 ±000
500 + 000
1.22 ±0.04
3-23±O13
3-40 ±0-34
1.98 ±0-23
2-65 ±0-33
2.40 ±0-50
(100)
100
100
100
100
(100)
108
38
71
59
* Overnight culture, morphological rating (MR) = 5-7. n equals the total number of
palate shelves monitored, P.S.I. values are means ± S.E.
|
350
| Anterior
R53 Posterior
300
?
in
o
v -
250
x
V
S 200
<u
o
E
o 150
Control i—i
100
50
JQ-10
(48)
iQ-9
I
10 -8
\
I
10" 7
10" 6
(38)
(30)
M 5-HT
1
I
10"
(48)
Fig. 2. Palate shelf rotation in embryo culture with various concentrations of
serotonin (5-HT). Day 14-75 embryos whose morphological rating were between
5 and 7 were cultured for 2 h. Values represent % of control movement by 5-HTtreated groups as described in Materials and Methods. Number of palates employed
is indicated in parentheses for the indicated concentration of 5-HT. P values for
statistical significance using Student's / test are also indicated.
81
Serotonin on palate rotation
|
150 i—
| Anterior
Posterior
Control
S 100
(94)
1
50
l0"s
(44)
10' 7
10' 6
(34)
10"5
(50)
J
10"
(32)
Methysernide (M )
Fig. 3. Palate shelf rotation in embryo culture with various concentrations of
methysergide. Methods are the same as in Fig. 2.
Pharmacological effects of serotonin and its antagonists
To test for any possible stimulatory effect of serotonin on palate shelf
rotation, embryos were cultured for a short period in which anterior shelf
elevation was not complete. Therefore, embryos were cultured for only 2 h ;
a period of time in which both anterior and posterior shelf elevation was comparable (Wee et al. 1976). The effects of adding increasing concentrations of
serotonin (5-HT) to embryo cultures are seen in Fig. 2. Serotonin at 3 x 10~10 M
did not affect anterior shelf rotation whereas inhibition was observed at the
posterior end. It was shown that serotonin significantly stimulated palate shelf
rotation at concentrations between 10~8 M and 10~5 M at the anterior end and
produced less effect at the posterior end; with 10~5M serotonin, anterior
movement was 319% of the control value (P < 5 x 10~5). A slight, but significant effect was observed at the posterior end (141 % of control, P < 0-025).
To investigate further the role of this neurotransmitter on palate shelf
rotation, serotonin antagonists were next employed: methysergide, ergotamine
and cyproheptadine. Figure 3 shows a dose-response study with methysergide
on palate shelf rotation. A concentration of 3 x 10~7 M methysergide inhibited
both anterior and posterior shelf rotation to about 50 % of control value. While
10~3 M antagonist produced little effect on rotation, 10~4 M markedly inhibited
rotation at the anterior end (12% of control, P < 0-005); inhibition at the
posterior end was not significant.
Table 2 shows that ergotamine (10~6 M) alone did not produce a significant
inhibitory effect on palate shelf rotation. In fact at a low concentration (10~9 M),
ergotamine stimulated anterior shelf rotation to a significant degree (183% of
control movement, P < 0-05). Nevertheless, ergotamine significantly inhibited
82
E. L. WEE AND
OTHERS
Table 2. Effect of serotonin and its antagonists on palate shelf rotation
in mouse embryo culture
% Control movement
P.S.I.
n
Condition*
Fixed
Control
5-HT(10-5M)
Ergotamine (10~9 M)
Ergotamine (10~6 M)
5-HT(10~5M) +
134
94
48
14
30
26
ergotamine (10~6 M)
Cyproheptadine(10~9M)28
Anterior
Posterior
Anterior
Posterior
2-61 ±005 1-22 ± 0 0 4
—
3-02 ±0-07 2-35±011
(100)
3-92±0-12 2-81 ±0-21 319 (P <5 x 10~5)
3-36±O13 2-57±0-37 183 (P < 005)
3O3±O13 2.07±0-21
102
3-31 ±009 2-31 ±0-27 171 + (P < 0025)
2-79 + 011
l-89±0-17
—
(100)
141 (P < 0025)
119
75
96
44(i><005)
59 (P < 0025)
* Two h culture, MR = 5-7.
f Significant at P < 001 when compared to 5-HT (10~5 M).
Table 3. Effect on fetal palate by administration of cyproheptadine
to pregnant mice
Palate gap (mm)
% Fused palate
A
n
Control
Cyproheptadine
55
42
Anterior
0-20 ± 0 0 4
0-46 + 006
(/><5xlO- 4 )
Posterior
Anterior
Posterior
014 + 003
0-24 + 0-03
(P < 001)
60
24
42
17
(P < 0-01) the stimulation at the anterior end of the palate induced by 10~5 M
serotonin.
Cyproheptadine, a serotonin antagonist with also antihistaminic, anticholinergic and antidopaminergic properties (Ferrari et al. 1976), partially
inhibited anterior and posterior shelf rotation at 10~9 M (Table 2).
To obtain further evidence that the effects of serotonin antagonists on palate
shelf rotation were not due to artifacts of the embryo culture system, cyproheptadine was injected into pregnant dams and the effect on palate closure was
monitored the next day (Table 3). There is a significant increase in the distance
of the palate gap of embryos from mice treated with cyproheptadine which is
another manifestation of an inhibition of palate shelf rotation (Wee et al.
1976). This effect was more pronounced at the anterior end of the palate.
Palate fusion at the anterior end was inhibited also in the drug-treated group
(24 % fusion) compared to control embryos (60 % fusion). Cyproheptadine also
caused a comparable decrease in palate fusion at the posterior end.
Serotonin on palate rotation
83
Fig. 4. Histology of palate shelves after 2 h in embryo culture. (A) Anterior palate
of embryo cultured in presence of 10~5 M serotonin, P.S.I. = 3. x 110. (B) Anterior
palate from embryo cultured with 10~4 M methysergide. P.S.I. = 1. x l l O . (C)
Posterior palate of control embryo, P.S.I. = 3. x l 3 5 . (D) Posterior palate after
methysergide incubation, P.S.I. = 2. X 135. Region 3 in mesenchyme adjacent to the
oral epithelium at the anterior and region 2 at the tongue side at the posterior ends
are indicated. Bars are 50/tm.
Light microscopy of palate shelves after pharmacologic agents
Embryos cultured for 2 h with and without serotonin show no apparent
difference in the histological sections of the palate shelves, although a significantly higher mean palate shelf index for embryos cultured with serotonin was
observed (Table 2). The histological sections also show that the 2 h culture did
not produce any necrotic change in the palate shelves. Figure 4A shows the
histology of the anterior palate shelf from an embryo cultured with 10~5 M
serotonin (P.S.I. = 3). In contrast, an anterior section of an embryo cultured
84
E. L. WEE AND OTHERS
Fig. 5. Histology of mid-palate after 2 h in embryo culture and rotation. (A) Midpalate of control embryo. Horizontalization of the oral epithelium (short arrow)
is observed with a loss of orientation of region-3 cells (see Fig. 6). In addition, a bend
at the tongue side (long arrow) is formed coincident with an orientation of elongate
mesenchymal cells at region 2. N, nerves; G, pterygopalatine ganglion; TG, tooth
germ. Bar, 50 jtim. x 120. (B) Mid-palate of embryo cultured with methysergide
(10~4 M) for 2 h reveals an abnormal shape of the shelf. Mesenchymal cell elongation
is observed in region 2 and along the tongue side epithelium (TE). Arrow, major
palatane artery; G, pterygopalatine ganglion; N, nerves; TG, tooth germ. Bar,
50/*m. x 120. (C) Tongue side bend of shelf from control embryo. The mesenchymal cells in region 2 are beginning to show an elongate morphology (arrows).
OS, ossification center. Bar is 20 /tm. x 480. (D) Along the tongue side epithelium
(TE) of methysergide-treated embryo, elongate mesenchymal cells (arrows) are
observed. This area is opposite region 3. Bar is 20/tm. x480.
Serotonin on palate rotation
85
4
with 10~ M methysergide is shown in Fig. 4B. Shelf rotation has been completely
inhibited in this preparation (P.S.I. = 1) and the morphology of the palate shelf
appears abnormal. Figure 4C shows an elevated posterior shelf in a control
embryo cultured for 2 h (P.S.I. = 3). Region 3 in the anterior end (Fig. 4A,B)
and region 2 in the posterior shelf (Fig. 4C, D) are indicated, which have been
described as non-muscle contractile systems (Babiarz et al. 1975; Kuhn et al.
1977). Shelf rotation and cell morphology at the posterior end appear to be
less affected by methysergide (Fig. 4D).
Figure 5 shows the effects of methysergide on the mid-palate where both
regions 2 and 3 overlap and are present in a single section. This portion of the
shelf also shows an abnormal shape (Fig. 5B) compared to a control (Fig. 5 A).
When the embryo is cultured for 2 h, palate shelf rotation is associated with
characteristic cell shape changes (Babiarz, Wee & Zimmerman, 1977). Region-3
cells which are elongated and arranged perpendicular to the oral epithelium
before rotation at day 14-5 of gestation (Fig. 6A), round up and lose their
orientation to the oral epithelium as it elevates (Fig. 6B). However, the cells in
the medial and tongue side mesenchyme underlying the epithelium, which
showed no orientation now assumed an elongate shape and were aligned
perpendicular to the epithelium. Region-2 cells, which are located at the tongue
side bend and peripheral to the ossification center, also began to assume an
elongate shape and become aligned perpendicular to the epithelium (Fig. 5C).
As rotation proceeded these cells become more elongated and finally round up
as the shelf is horizontalized (Babiarz etal. 1977). Methysergide did not affect
the normal process of elongation and orientation of cells of region 2 and those
extending down along the tongue side mesenchyme during shelf elevation
(Fig. 5D). However, region-3 cells appear to have rounded less (Fig. 6C) than
in the 2 h incubated control (Fig. 6B) commensurate with the oral epithelium
(Fig. 5B) not elevating as much as the control (Fig. 5 A). Also the morphology
of the region-3 cells was altered (Fig. 6C). Similar changes in the morphology
of region-3 cells are also seen in the anterior end. However, in other embryos
treated with methysergide in which rotation has not been as profoundly inhibited, region-3 cells appear less distorted.
DISCUSSION
Quantitation of anterior and posterior palate shelf rotation separately by a
measurement of palate shelf index and its correlation with morphological
ratings of day-14-75 mouse embryos reveal several features of the development
of the palate shelf. Although the anterior palate was slightly more ventromedial
(P.S.I. = 2) than the posterior end (P.S.I. = 1) and showed a slight initial
elevation, the anterior shelf remained unchanged from a morphological rating
of 4-10. On the other hand, the posterior end showed a progressive horizontalization of the shelf as the embryo became more developed. This result is in
86
E. L. WEE AND OTHERS
Fig. 6. Area of region-3 cells located in mesenchyme adjacent to oral epithelium.
(A) Control cells before rotation show an elongate morphology, contain many
filopodial processes and are oriented perpendicular to the oral epithelium (OE).
(B) After 2 h in embryo culture, region-3 cells show a loss in orientation along the
oral epithelium (OE) and have rounded. Less filopodial processes are observed and
cells appear less dense. BV, blood vessel. (C) The region-3 cells after methysergide
treatment appear to have rounded less and to be slightly distorted in shape. BV, blood
vessel; OE, oral epithelium; N, nerve. Bars are 20/«n. x 500.
Serotonin on palate rotation
87
accord with the observations of Walker & Fraser (1956) that in the mouse the
posterior palate rotates before the anterior end. When the embryos were cultured
overnight, the anterior end completely rotated to a horizontal position (P.S.I.
= 5) at all stages in development observed. Conversely, the posterior end
rotated in culture in proportion to embryo development. One possible explanation is that the force in the posterior end is weaker. Thus the motive components
in the posterior end may have to be synthesized or positionally arranged during
development to allow this end to rotate.
Further support that the force in the anterior end of the palate is greater than
that in the posterior end comes from the observations that the posterior palate
is much more sensitive to culture perturbations (Table 1). Deleting serum or
replacing N 2 for O2 in the gas mixture depressed posterior shelf movement while
having no effect on the anterior end.
To study further the mechanism of palate shelf rotation, embryos were
cultured for 2 h in the presence and absence of serotonin, since this neurotransmitter has been shown to be involved in the regulation of contraction of
other processes containing similar non-muscle contractile systems. In. embryo
culture, it was observed that serotonin-stimulated palate rotation, predominantly
at the anterior end of the shelf (Fig. 2). In addition, the stimulation induced by
serotonin was shown to be inhibited by the serotonin antagonist, ergotamine.
Two other serotonin antagonists (methysergide and cyproheptadine), when
used alone in the culture system, inhibited palate shelf rotation at certain concentrations. However, methysergide at 10~4 M predominantly inhibited anterior
shelf rotation (12% of control movement). When dose-response experiments
were carried out with serotonin and methysergide, the shape of the stimulation
curve with serotonin (Fig. 2) and the inhibition curve with methysergide (Fig.
3) was biphasic. Failure to obtain a simple dose-response pattern for these
agents may be attributed to their paradoxical effects previously observed
in other systems: serotonin can act as an antagonist, as well as an agonist
(Allen, Gross, Henderson & Chou, 1976); methysergide shows a 'biphasic
effect' on canine nasal circulation (Schonbaum etal. 1975); methysergide shows
mixed agonist-antagonist activity on dopamine and serotonin receptors (Creese,
Burt & Snyder, 1975). In addition, the stimulation of 10~ 9 M ergotamine on
palate shelf rotation and its inhibition of serotonin's stimulatory effects may
also be attributed to the known agonist-antagonist activity of this drug
(Frankhuijzen, 1975).
Even though the pharmacological activities of serotonin and its antagonists
are not specific, the results would suggest that serotonin plays a role in palate
morphogenesis, predominantly at the anterior end of the shelf. The effect of
the serotonin neurotransmitter on the palate shelf would be consistent with an
effect on a muscle or non-muscle contractile system.
Our previous studies have indicated that there are mesenchymal and epithelial cell movements in the palate associated with shelf rotation (Babiarz et al.
88
E. L. WEE AND OTHERS
1977). In the posterior end, cell movements are localized at the bend between
the nasal septum and the tongue side of the palate. The mass of mesenchymal
cells, termed region-2 cells, contains an osteogenic site in its center which can
be identified by its alkaline phosphatase reaction (Shapiro & Sweney, 1969;
Pourtois, 1972) and filopodial-rich cells on the periphery (Babiarz et ah 1975;
Kuhn et ah 1977). It would seem possible that the contraction and movement of
these mesenchymal cells and associated epithelial cells play a role in posterior
palate rotation. The sum total of their movement could produce the 'remodeling' of the posterior shelf during rotation frequently described (Coleman,
1965; Greene & Kochhar, 1973; Larsson, 1962; Pons-Tortella, 1937). Subsequent formation of bone from the osteogenic center could serve to fix the
posterior end in the horizontal position. The observation that cholinergic agents
stimulate posterior shelf rotation is consistent with an involvement of a cellmediated process (Wee et ah 1976). Such an involvement does not preclude
the production of a physico-chemical force, such as mucopolysaccharide
hydration and elevated osmotic pressure, which could also help elevate the
shelf.
Movement of the anterior palate has been described as 'barndoor rotation'
(Coleman, 1965; Lazarro, 1940; Walker & Ross, 1972). Ferguson (1978) has
indicated that there is greater Alcian blue staining of the anterior end than in
the posterior end, suggesting a greater concentration of mucopolysaccharides,
which would produce a greater physico-chemical force to move the shelf. In the
mid and anterior end of the palate, another putative non-muscle contractile
system (region 3) has been observed (Babiarz et ah 1975; Kuhn et ah 1977;
Innes, 1978). However, this mass of mesenchymal cells lacks a comparable
osteogenic site, as found in the posterior region 2. Rotation of the anterior to
mid area of the palate has been shown to correlate with a prior elongation of
the region-3 mesenchymal cells and their perpendicular alignment to the
epithelium before rotation, and a subsequent rounding of these cells after
rotation (Babiarz et ah 1977; see also Figs. 5 and 6). These results suggest that
contraction and movement of cells may, in addition to any physico-chemical
force (due to mucopolysaccharide hydration), aid in rotation of the anterior
palate. It was observed that, in those shelves markedly inhibited in rotation by
the serotonin antagonist (methysergide) in embryo culture, region-3 cells in the
anterior to mid-palate appear to have rounded less than the control. Also the
morphology of the region-3 cells appeared to be slightly distorted. These effects
suggest that serotonin may be regulating a cell-mediated process in palate
rotation which is inhibited by methysergide. The role of serotonin in palate
rotation is further supported by the observation that palate shelves take up and
synthesize serotonin during this period of palate rotation (Zimmerman &
Roberts, 1977). However, whether serotonin can elicit its effects on palate
rotation by a direct effect on palatial cells or indirectly by a neural mechanism
cannot yet be determined.
Serotonin on palate rotation
89
This research was supported by a grant from the National Institute of Dental Research
(DE 03469), a Center Grant in Mental Retardation (HD 0522.1), and a gift from the
Jacob G. Schmidlapp trust.
This paper represents part of the Ph.D. dissertation of B. S. Babiarz submitted to the
Graduate Program in Developmental Biology, University of Cincinnati.
REFERENCES
ALLEN, G. S., GROSS, C. J., HENDERSON, L. M. & CHOU, S. N. (1976). Cerebral arterial
spasm. Part 4: In vitro effects of temperature, serotonin analogues, large non-physiological
concentrations of serotonin and extracellular calcium and magnesium on serotonininduced contractions of the canine basilar artery. / . Neurosurg. 44, 585-593.
ANDREW, F. D., BOWEN, D. & ZIMMERMAN, E. F. (1973). Glucocorticoid inhibition of RNA
synthesis and the critical period for cleft palate induction in inbred mice. Teratology 7,
.167-175.
BABIARZ, B. S., ALLENSPACH, A. L. & ZIMMERMAN, E. F. (1975). Ultrastructural evidence of
contractile systems in mouse palates prior to rotation. Devi Biol. 4,1, 32-44.
BABIARZ, B. S., WEE, E. L. & ZIMMERMAN, E. F. (1977). Palate morphogenesis. II. Changes
in cell shape and orientation during shelf elevation. Fifth International Conference on Birth
Defects, No. 426 (J. W. Littlefield, editor), 183 (abstract). Amsterdam, Oxford: Excerpta
Medica.
BOULLIN, D. J. & ORR, M. W. (1976). Effects of aspirin and prostaglandin E3 on secondary
phase of aggregation response of schizophrenic patients treated with chlorpromazine.
Brit. J. Clinical Pharm. 3, 929-933.
COLEMAN, R. D. (1965). Development of the rat palate. Anat. Rec. 151, 107-118.
CREESE, I., BURT, D. R. & SNYDER, S. H. (1975). The dopamine receptor: differential binding
of D-LSD and related agents to agonist and antagonist states. Life Sciences 17, 1715-1719
FERGUSON, M. W. J. (1978). Palatal elevation in the Wistar rat fetus. /. Anat. 125, 555-577.
FERRARr, C , PARACCHr, A., RONDENA, M., BECK-PECCOZ, P. & FAGLIA, G. (1976). Effect of
two serotonin antagonists on prolactin and thyrotrophin secretion in man. Clin. Endocrinol. 5, 575-578.
FRANKHUIJZEN, A. L. (1975). Analysis of ergotamine-5-HT interaction on the isolated rat
stomach preparation. Eur. J. Pharmacol. 30, 205-212.
GREENE, R. M. & KOCHHAR, D. M. (1973). Palatal closure in the mouse as demonstrated in
frozen sections. Am. J. Anat. 137, 477-482.
GUSTAFSON, T. & TONEBY, M. (1970). On the role of serotonin and acetylcholine in sea urchin
morphogenesis. Expl Cell Res. 62, 102-117.
HUMPHREY, T. (1969). The relation between human fetal mouth opening reflexes and closure
of the palate. Am. J. Anat. 125, 317-344.
INNES, P. B. (1978). The ultrastructure of the mesenchymal element of the palatal shelves
of the fetal mouse. /. Embryol. exp. Morph. 43, 185-194.
KARNOVSKY, M. J. (1965). A formaldehyde-glutaraldehyde fixative of high osmolality for
use in electron microscopy. /. Cell Biol. 27, 137 A.
KUHN, E. M., BABTARZ, B. S., LESSARD, J. L. & ZIMMERMAN, E. F. (1977). Palate morphogenesis. I. Localization of non-muscle contractile systems by immunofiuorescence and
electron microscopy. Fifth International Conference on Birth Defects, No. 426 (J. W.
Littlefield, editor), 114 (abstract). Amsterdam, Oxford: Excerpta Medica.
LARSSON, K. S. (1962). Closure of the secondary palate and its relation to sulfomucopolysaccharides. Acta Odontal. Scand. 20, Suppl. 31, 1-35.
LAURENT, T. C. (1970). Structure of hyaluronic acid. In Chemistry and Molecular Biology of
the Intercellular Matrix, vol. 2 (ed. E. A. Balazs), pp. 703-732. New York: Academic Press.
LAZARRO, C. (1940). Sul meccanismo di chiusura del palato secondairo. Monitore zool. Ital.
51, 249-273.
LESSARD, J. L., WEE, E. L. & ZIMMERMAN, E. F. (1974). Presence of contractile proteins in
mouse fetal palate prior to shelf elevation. Teratology 9, 113-125.
90
E. L. WEE AND OTHERS
LUFT, J. H. (1961). Improvement in epoxy resin embedding methods. /. biophys. biochem.
Cytol. 9, 409-414.
PONS-TORTELLA, E. (1937). Ober die Bildingsweise des sekundaren Gauments. Anat. Anz. 84,
13-17.
POURTOIS, M. (1972). Morphogenesis of the primary and secondary palate. In Developmental
Aspects of Oral Biology, chap. 5 (eds. H. C. Slavkin & L. A. Bavetta), pp. 81-108. New
York and London: Academic Press.
PRATT, R. M., GOGGINS, J. F., WILK, A. L. & KING, C. T. G. (1973). Acid mucopolysaccharide synthesis in the secondary palate of the developing rat at the time of rotation
and fusion. Devi Biol. 32, 230-237.
RYAN, G. B., CLIFF, W. J., GABBIANI, G., IRLE, C, MONTANDON, D., STATKOV, P. R. &
MAJNO, G. (1974). Myofibroblasts in human granulation tissue. Human Path. 5, 55-67.
SCHONBAUM, E., VARGAFTIG, B. B., LEFORT, J., LAMAR, J. C. & HASENACK, T. (1975). An
unexpected effect of serotonin antagonists on the canine nasal circulation. Headache 15,
180-231.
SHAPIRO, B. L. & SWENEY, L. (1969). Electron microscopic and histochemical examination
of oral epithelial-mesenchymal interaction (programmed cell death). /. dent. Res. 48,
652-660.
STEELE, C. E. & NEW, D. A. T. (1974). Serum variants causing the formation of double
hearts and other abnormalities in explanted rat embryos. /. Embryol. exp. Morph. 31,
707-719.
TRUMP, B., SMUCKLER, E. & BEND, H. S. (1961). A method for staining epoxy sections for
light microscopy. /. Ultrastruc. Res. 5, 343-347.
VERRUSIO, A. C. (1970). A mechanism for closure of the secondary palate. Teratology 3,17-20.
WALKER, B. E. (1969). Correlation of embryonic movement with palate closure in mice.
Teratology!, 191-198.
WALKER, B. E. & CRAIN, B. (1960). Effects of hypervitaminosis A on palate development in
two strains of mice. Am. J. Anat. 107, 49-58.
WALKER, B. E. & FRASER, F. C. (1956). Closure of the secondary palate in three strains of
mice. J. Embryol. exp. Morph. 4, 176-189.
WALKER, B. E. & Ross, L. M. (1972). Observations of palatine shelves in living rabbit
embryos. Teratology 5, 97-102.
WEE, E. L., WOLFSON, L. G. & ZIMMERMAN, E. F. (1976). Palate shelf movement in mouse
embryo culture: evidence for skeletal and smooth muscle contractility. Devi Biol. 48,
91-103.
ZIMMERMAN, E. F. & ROBERTS, N. (1977). Palate morphogenesis. IV. Synthesis of serotonin.
Fifth International Conference on Birth Defects, No. 426. (J. W. Littlefield, editor), 327
(abstract). Amsterdam, Oxford: Excerpta Medica.
{Received 13 July 1978, revised 30 March 1979)