J. Embryol. exp. Morph. Vol. 64, pp. 233-250, 1981
Printed in Great Britain © Company of Biologists Limited 1981
233
Presence of serotonin in the palate just prior
to shelf elevation
ERNEST F. ZIMMERMAN 1 , ELIZABETH L. WEE,
NANCY PHILLIPS AND NANCY ROBERTS
From the Division of Cell Biology, Institute for Developmental Research,
Children's Hospital Research Foundation, Cincinnati
SUMMARY
Since serotonin and its antagonists affect shelf rotation in mouse embryo culture, experiments were carried out to determine whether a serotonergic system is present in the palate.
Employing [3H]5-HT, day-14-5 embryos incorporated the monoamine into palates. Active
uptake of [3H]5-HT was shown since excised palates incorporated 9-fold more radioactivity
at 37 °C than at 4 °C. Synthesis of palatal serotonin was measured. Embryos were cultured
in the presence of the serotonin precursor, [3H]5-HTP, and radioactive 5-HT was monitored
in the palate by thin-layer chromatography. Furthermore, excised palates were incubated
with [3H]5-HTP and radioactive 5-HT was measured. Incorporation was linear for about 6 h.
In addition, another radioactive compound was detected which had the same Rf as the
methylated derivative, 5-methoxytryptamine. Synthesis of this compound was appreciable,
about 30% of that of serotonin. Levels of serotonin in the palate were measured by high
pressure liquid chromatography. Palates at day 14-5 of gestation contained 0-40 ng serotonin/
mg protein, which was greater than that of tongue (0-33), body (014) but less than that of
brain (309). Serotonin in palate and other embryonic tissues increased with time of development. Dopamine levels in the palate and other tissues were also determined. The distribution
of serotonin in the palate was analyzed by culturing day-14-5 embryos in the presence of
[3H]5-HTP, and after aldehydefixation,paraffin embedment and sectioning, autoradiography
was performed. Grains were observed throughout the palate in cells of regions 2 and 3,
internal mesenchyme, tooth germ, and epithelium. Surprisingly, the pterygopalatine nerve,
maxillary nerve and pterygopalatine ganglion contained an appreciable concentration of
grains. Thus, the presence of serotonin in the palate is consistent with the neurotransmitter
playing a role in shelf elevation.
INTRODUCTION
Movement by means of the contraction of filopodia has emerged as a major
means of cell locomotion during embryogenesis. Filopodial contraction has
been proposed for the morphological movements associated with flask cell
invagination in amphibian gastrulation (Baker, 1965), the migration of the
deep cells of the teleost blastoderm (Trinkaus, 1973), chick secondary mesenchymal cell movements (Trelstad, Hay & Revel, 1967) and sea urchin gastrula1
Author's address (for reprints): Division of Cell Biology, Institute for Developmental
Research, Children's Hospital Research Foundation, Elland and Bethesda Avenues,
Cincinnati, Ohio 45229, U.S.A.
234
E. F. ZIMMERMAN AND OTHERS
tion (Gustafson & Wolpert, 1967). In this latter developmental process, elongated filopodial projections of secondary mesenchymal cells selectively adhere
to the ectodermal roof of the blastocoel and contract to complete invagination
of the archenteron. In fact, neurotransmitters appear to regulate the mesenchymal cell movements and hence gastrulation; serotonin functions early and
acetylcholine functions on muscarinic receptors later (Gustafson & Toneby,
1970). The biochemical measurement and histochemical localization of serotonin appears to support its role during this process (Buznikov, Chudakova &
Zvezdina, 1964; Buznikov, Sakharova, Manukhin & Markova, 1972). Specific
distribution and temporal appearance of neurotransmitters such as catecholamines and serotonin have been observed during morphogenesis of the neural
tube and gut (Kirby & Gilmore, 1972; Lawrence & Burden, 1973; Wallace,
1979). Evidence for the role of these neurotransmitters in morphogenesis comes
from the pharmacological manipulation of the neurotransmitters leading to
developmental defects of delays in the closure of the neural tube (catecholamines - Lawrence & Burden, 1973; serotonin - Palen, Thorneby & Emanuelsson, 1979) and rotation of the palate (serotonin - Wee, Babiarz, Zimmerman
& Zimmerman, 1979; acetylcholine - Wee, Wolfson & Zimmerman, 1976; Wee,
Phillips, Babiarz & Zimmerman, 1980).
Studies from our laboratory have suggested that palate rotation to the
horizontal position may be, in part, effected by contraction of cytoplasmic
processes and migration of cells, part of the non-muscle contractile systems in
the palate mesenchyme and possibly the epithelium. The non-muscle contractile
systems are located in two areas of the palate mesenchyme: region 2 on the
tongue side extending from the top mid-palate to the posterior limit; and
region 3 along the oral epithelium, extending from the middle two-thirds of
the palate towards the anterior end (Zimmerman, 1979). Since serotonin has
been shown to stimulate palate shelf rotation in embryo culture (Wee et al.
1979) and to stimulate mesenchymal cell movement in culture (Zimmerman,
Wee, Clark & Venkatasubramanian, 1980), the presence of serotonin in the
palate has been sought. It has been shown that serotonin is synthesized in the
palate; the content of serotonin is measured and its distribution described. A
preliminary report of this work has been presented (Zimmerman & Roberts,
1977).
MATERIALS AND METHODS
Due to light sensitivity and labile nature of serotonin and related compounds,
all operations were performed using foil-wrapped containers and a darkroom
facility. Standard solutions were prepared just prior to use and radioisotopes
were chromatographed routinely to monitor radio and chemical decay. Only
chromatographically purified radioisotopes were used in these studies.
Animals. Female A/J mice (Jackson Laboratory) were mated overnight and
the presence of a vaginal plug the following morning was taken as evidence of
Serotin in the palate
235
pregnancy and designated day 0-5 of gestation. Unless otherwise stated,
pregnant mice were killed by cervical dislocation at day 14-5, the time just
prior to shelf rotation (Andrew, Bowen, & Zimmerman, 1973). The gravid
uterus was removed immediately and placed in chilled Dulbecco's Modified
Eagle Medium (DMEM, Gibco,) pregassed with 95 % O2-5 % CO2.
Embryo culture. Explantation and culture of the embryos were carried out
under sterile conditions as previously described (Wee et al. 1979) except that
tongues were not excised. To monitor serotonin uptake, embryos were incubated
with [3H]5-HT at 37 °C for 3 h and then palate and brain excised. Alternatively,
embryos were incubated with [3H]5-hydroxytryptophan([3H]5-HTP,AmershamSearle, 2 Ci/m-mole) for various periods of time and were monitored for their
ability to convert the serotonin precursor to radioactive serotonin (5-HT).
Nialamide (400 /tg, Sigma) was added to 2 ml pregassed culture medium 1 h
before addition of 5 jtiCi of [3H]5-HTP per vial, unless otherwise indicated.
Embryo cultures were terminated by decapitating the heads and rinsing with
three changes of chilled Dulbecco's phosphate-buffered saline (PBS, Gibco).
Palates were excised and analysed for [3H]5-HT as described below.
Tissue culture. Using sterile technique, palates were excised from day-14-5
embryos with the aid of a dissecting microscope and microforceps. Excised
palates (5-10 pairs) were placed on a sterilized Millipore filter (0-22 /*m, 13 mm)
in the centre well of a Falcon organ culture dish. Each dish contained 1 ml of
gassed culture medium with nialamide (400 fig) and was maintained at 37 °C
in a CO2 incubator (Fig. 5). After 1 h pretreatment with nialamide, 5 /*Ci of
[3H]5-HTP was added and culture continued at 37 °C. Alternatively, 5 /id of
[3H]5-HT (New England Nuclear, 21-4 Ci/m-mole) was added and palates
were incubated in the absence of nialamide as indicated in Fig. 2. Incubation
was stopped by rinsing palates with three changes of chilled PBS.
Tissue extraction of serotonin. For extraction of 5-HT, embryonic tissues were
homogenized in cold 80% acetone (e.g. 100/d/palate pair) as described by
Toneby (1973). However, many investigators (e.g. Maickel, Cox, Saillant &
Miller, 1968) have used acidified solvents for extraction of 5-HT from adult
tissues. Since preliminary experiments were carried out which demonstrated
no appreciable difference between the two methods, non-acidified acetone
was routinely employed. Aliquots of the homogenate were taken for protein
estimation (Lowry, Rosebrough, Farr & Randall, 1951). After centrifugation at
20000 xg for 20 min, pellets and aliquots of the supernatant were counted to
estimate recovery.
Thin-layer chromatography. Since overnight storage of extracts at —20 °C
resulted in a lower 5-HT recovery, chromatography was immediately carried
out following tissue preparation. Recovery rates were about 80% using this
precaution. After blowing off the acetone at room temperature with N2, the
remaining extract was applied to a plastic-backed thin-layer chromatogram
(SIL-N-HR-UV254 with 0-2 mm MN silica gel; Brinkmann Instruments) in a
236
E. F. ZIMMERMAN AND OTHERS
\ in. width strip. Precursors and metabolites of 5-HT used as markers (25 jag)
were spotted alongside the radioactive extracts. In addition, marker 5-HT
(25 /*g) was added to each radioactive extract to determine exactly whether
radioactivity peaks had the same mobility as marker 5-HT. Mobility of markers
were monitored with u.v. light.
The solvent routinely employed for TLC was 1-propanol: NH 4 OH: H2O
(8:1:1). Other solvents used were n-butanol:acetic acid:H 2 O (12:3:5) and
methyl acetate:isopropanol:ammonia (9:7:4).
Strips (^ cm) were cut into small pieces and radioactivity was extracted into
0-5 ml Soluene by shaking overnight at 37 °C and counted as before.
Determination of serotonin and dopamine levels. Palates, and other tissues
were excised and immediately frozen in liquid N 2 for assay at a later time or
chilled on ice and assayed immediately. No differences in monoamine levels
were found and the results of both methods of collection were pooled. Adult
brain was that of pregnant dams. Tissues were extracted with 25 mM HC1.
Measurement of serotonin and dopamine were carried out by the method of
Sasa & Blank (1977) using high-pressure liquid chromatography (HPLC) with
electrochemical detection. The tissue suspension (600 /tl) was homogenized
with 25/4 of ascorbic acid (2-75 mg/ml), 50/4 of 0-1 M - E D T A , and 50 /.d of
2-4 /J,M 3,4-dihydroxybenzylamine hydrobromide (DHBA), which was used as
an internal standard. Salt saturation of the homogenate was accomplished by
adding 1 g NaCl. Three ml of butanol were added and the sample was shaken
for 60 min in the cold room. The sample was centrifuged at 400 xg for 15 min
and 2-5 ml of the organic phase was transferred to 4-0 ml heptane and 200 ji\ of
0-01 M-HC1. After shaking for 10 min in the cold room, the mixture was centrifuged at 400 xg for 15 min to separate the layers; most of the butanol/heptane
layer was removed and 50 ju\ of the aqueous layer was injected into the liquid
chromatograph and analysed for serotonin and dopamine.
Distribution of serotonin in the palate. The localization of serotonin in the
palate was sought using the histochemical technique of formaldehyde-induced
fluorescence (Falck, Hillarp, Thieme & Torp, 1962). Temperature, humidity
and time of heating were varied. Nevertheless, specific fluorescence of serotonin
in palate could not be observed, presumably due to low concentration of
monoamine in palate (Table 2). Therefore, localization of serotonin was
performed by autoradiography after incorporation of [3H]5-HTP (Belenky,
Chetverukhin & Polenov, 1979). Day-14-5 mouse embryos were cultured with
5 /tCi of [3H]5-HTP (10~4 M final concentration) for 3 h in the presence of
nialamide (400 /tg/2ml) at 37 °C. Embryos were washed free of radioactive
5-HTP and incubated in radioactive-free medium for one more h. Heads were
removed, washed with cold PBS and fixed for 1 h in Karnovsky's fixative at
room temperature (Karnovsky, 1965) to fix serotonin or its derivatives in dense
core vesicles within cells. Tongue and mandible were removed and heads were
fixed in fresh Karnovsky's solution for an additional h. After paraffin embed-
237
Serotonin in the palate
1
3h
3
•a
200
-
150
-
100
-
50
-
D.
u
a
n
0
[ H]5-HT
-
J
-
-
5-HTL
10
20
Fraction number
Fig. 1. Chromatographic analysis of extracts from palate and brain after 3 h incubation of [3H]5-HT in embryo culture. Each day-14-5 embryo (5) was cultured at
37 °C in 2 ml of medium, including 1 /tCi of [3H]5-HT which was mixed with nonradioactive 5-HT to make 2 x 10~4 M. After a 3 h incubation, palate pairs and brains
were excised, each tissue pooled, extracted with 80% acetone and subjected to
TLC, as described in Materials and Methods.
ment, sections were coated with Kodak NTB2 emulsion and after 1-3-week
incubations in the dark, slides were developed and stained with methyl greenpyronin or bismarck brown. Presence of grains were criteria of 5-HT or its
derivatives in palatal cells. The results presented here are based on histological
observations of six embryos from three litters and counting of grains per
surface area.
RESULTS
Serotonin uptake
The first phase of the work was to determine whether foetal tissues, particularly
palate, could incorporate [3H]5-HT. Embryo culture was employed to monitor
serotonin uptake. As indicated in Fig. 1, a large radioactive peak was observed
in both day-14-5 palate and brain which showed the same Rf as marker 5-HT
(0-47). The radioactivity peak height in embryonic brain (about 200 cpm) was
about 7-fold greater than a palate pair (27 cpm). However, when tissue retention of monoamine was calculated per mass, brain (42-0 n-mole 5-HT/mg
protein) was only about twice that of palate (27-1 n-mole/mg). In addition, both
tissues had apparently metabolized a considerable amount of the [3H]5-HT
238
E. F. ZIMMERMAN AND OTHERS
•
-
1
I
1
—
1
• 37 °C
o 4°C
'2,
1 -
0
0
8
\
i
i
30
60
90
Duration of incubation with [3H]5-HT (min)
i
120
Fig. 2. Uptake of [3H]5-HT into excised palate cultured at various temperatures.
Each palate pair was excised and cultured in 0-5 ml of tissue culture medium in a
5 ml Falcon tube and rotated at 60 rpm at the appropriate temperature. One /*Ci of
[3H]5-HT was mixed with non-radioactive 5-HT in each tube to make 10~5 M. At
various times of incubation, palate pairs were washed three times with cold PBS,
dissolved in Soluene and radioactivity was counted. Each point represents the
mean and the bar the range of the triplicate experiments carried out.
incorporated. In palate, there was much radioactivity at the origin, the
identity of which is not known. In brain, a large radioactive peak at fractions
8-9 was observed which had a mobility of HIAA, the product of monoamine
oxidase. Much less radioactivity with a similar Rf was found in palate (around
fraction 7). A small radioactivity peak at fractions 23-25 of both tissues was
found, which comigrated with melatonin. Finally, a very small peak of radioactivity was seen in palate at fraction 17 which migrated with an Rf similar to
5-methoxytryptamine (5-MT). In contrast, brain did not show a peak of
radioactivity in that area. Since both tissues not only exhibited a significant
[3H]5-HT peak but metabolites as well, it is unlikely that 5-HT was incorporated
into only the extracellular compartment.
Since active uptake of serotonin into cells is a temperature-sensitive process,
excised day-14-5 palates were incubated with [3H]5-HT for various periods of
time at 37 °C and 4 °C. As shown in Fig. 2, there was a 9-fold increase in 5-HT
uptake into palate at 37 °C compared to 4 °C after 120 min incubation. This
result indicates that palate can actively incorporate serotonin.
Serotonin in the palate
239
3000 -
2000 -
1000 -
0
10
20
Fraction number
Fig. 3. Chromatographic analysis of extracts from palate and brain after 18 h incubation of [3H]5-HTP in embryo culture. Embryos (7) were pretreated with nialamide (N) for 1 h and then incubated with [3H]5-HTP.
Serotonin synthesis
Next, experiments were carried out to test whether palate can synthesize 5-HT.
Radioactive 5-HTP was incubated with the appropriate tissue and synthesis of
5-HT was monitored. Since the 5-HT uptake study (Fig. 1) showed significant
metabolism of 5-HT, a monoamine oxidase inhibitor (nialamide) was added to
prevent the further enzymatic conversion of 5-HT to its metabolite, 5-hydroxyindole-3-acetic acid (HIAA).
First, total embryos were cultured in the presence of [3H]5-HTP. Optimal
recovery of labelled 5-HT was obtained with a 1 h preincubation with nialamide
which was subsequently employed. Figure 3 shows the chromatographic separation of radioactive compounds in excised palate and brain after 18 h incorporation of [3H]5-HTP into embryos. In both tissues a radioactivity peak is seen
which comigrates with marker 5-HT; the radioactivity peak was much greater
in brain than in palate. To assure further that the radioactivity was actually
serotonin, thin-layer chromatography of extracts labelled as before was repeated
in other solvent systems. Radioactivity peaks which comigrated with marker
5-HT (as well as 5-HTP) were observed when solvents «-butanol:acetic acid:
H2O and methyl :isopropanol:NH4OH were employed. Little or no HIAA was
observed on these chromatograms. Figure 4 indicates that the incorporation of
labelled 5-HT increases with time of embryo culture in the presence of 3 H-5HTP. When embryos were preincubated for 1 hour with nialamide, the amount
240
E. F. ZIMMERMAN AND OTHERS
4-5
40 -
3-5 -
30
Palate + Nialamide
Palate — Nialamide
1 3
6
Duration of incorporation with
18
Fig. 4. Incorporation of labelled 5-HT into palates of embryos cultured with
[3H]5-HTP. Where indicated, embryos were pretreated with nialamide for one h.
Each time point represents four to eight palate pairs that were analysed for 5-HT
by TLC.
of 5-HT recovered was consistently greater than in the absence of nialamide.
Palates contained a 50 % greater amount of labelled 5-HT when cultured for
18 h in the presence of nialamide.
To show directly that the palate synthesizes serotonin, excised palates were
incubated with [3H]5-HTP for various periods of time. Figure 5 shows the
radioactivity profile from a thin-layer chromatogram after an 18 h incorporation. A large peak of radioactivity migrated with 5-HT indicating that the
palate can synthesize serotonin. Surprisingly, a radioactive peak was also found
that migrated with the same mobility of 5-methyoxytryptamine (5-MT).
Table 1 shows incorporation of [3H]5-HTP in excised palates for various
periods of time. Serotonin and 5-methoxytryptamine peaks were integrated
and cpm per /tg palatal protein calculated. Serotonin was progressively synthesized for 6 h; synthesis at 18 h tailed off. 5-Methoxytryptamine, which was
also proportionally synthesized with time to 6 h, corresponded to a significant
portion of serotonin. Synthesis of 5-MT was about 30 % of 5-HT. The high
Serotonin in the palate
241
Fraction number
Fig. 5. Chromatographic separation of labelled 5-HT after an 18 h incorporation
of [3H]5-HTP into cultured excised palates. Palate pairs (8) were placed on a Millipore filter, overlaid on a wire grid in a small Petri dish containing 1 ml of tissue
culture medium. Palates were preincubated for 1 hr at 37 °C in 5 % CO 2 with
nialamide (400/ig/ml). [ 3 H]5-HTP (2-5 /*Ci) was added and incubated for 18 hr.
The pooled palates were analysed for 5-HT as before.
Table 1. Incorporation of [3H]5-HTP into excised palates
cmp//*g protein
Time-h
n
5-HT
5-MT
5-MT
xlOO
5-HT
0-5
1
3
6
18
3
2
2
2
3
0-24
1-71
2-41
4-30
4-71
0-24
0-62
0-69
112
1-47
100
36
29
26
31
Values are average cpm//*g protein from number of replicate experiments indicated by n
242
E. F. ZIMMERMAN AND OTHERS
Table 2. Content of serotonin and dopamine in various tissues
during development
5-HT
Tissue
Day of
development
ng/mg protein
n
5
4
1-63 ±0-25
100 ±009
0-33 ±008
P < 005
0-81 ±0.23
5
1-84 ±0-26
4
1-77+1-12
014±004
P < 0002
6
100±012
4
2-38 ±0-64
4
3 09 ±0-20
P < 10-6
4-25 ±0-33
4
3-23±O-51
10
26-4 ±5-43
10
250 ±4-98
7
14-5
6
15-5
4
Bodies
14-5
5
Brain
14-5
4
15-5
Tongue
Brain
Adult
ng/mg protein
n
0-40 ±004
0-58 ±006
P < 005
14-5
15-5
Palate
Dopamine
A
4
Values presented are means ± S.E. Significance determined with 2-tailed Student's /-test
relative value of 5-MT at 0*5 h may be inaccurate because of low peak height
values of 5-HT and 5-MT in relation to background values.
Content of serotonin and dopamine
Next, a direct analysis of serotonin in the palate was carried out. Excised
palates and other tissues were extracted and assayed for serotonin by highpressure liquid chromatography. Serotonin levels in the palate at day 14-5 of
gestation were low. This necessitated using pooled samples of 80-100 palate
pairs to obtain adequate peak heights for measurement. For day-15-5 embryos,
30-50 palate pairs were employed. The mean values presented are from four to
seven separate determinations; day-14-5 palate represents seven determinations.
As Table 2 shows, the level of serotonin at day 14-5 of development is 0-40
Fig. 6. Autoradiography of mid-palate after incorporation of [3H]5-HTP in embryo
culture. (A) Low power view of paraffin section of palate indicating regions 2 and 3,
pterygopalatine ganglion (G), maxillary nerve (MN), and tooth germ (TG). Bar,
75/*m. x 115. (B) High concentration of grains in maxillary nerve (MN). Bar,
20 /*m. x 530. (C) Grains are observed in cells including their cytoplasmic processes
of region 2. TE, tongue epithelium. Bar, 20 fim, x 530. (D) Grains are present
throughout region-3 cells. Although relatively few grains are observed in epithelium,
there are proportionately more grains in other sections of the palate (see Fig. 7C).
OE, oral epithelium. Bar, 20 /*m. x 450.
Serotonin in the palate
243
D
244
E. F. ZIMMERMAN AND OTHERS
Serotonin in the palate
245
ng/mg protein, which is significantly greater than that of fetal tongue at that
time of development: 0-33 ng/mg, P < 0-05. Moreover, serotonin was found
in the day-14-5 fetal body (minus head) at 0-14 ng/mg, significantly less than
that of palate (P < 0-002). In contrast, embryonic brain has about 8-fold
greater serotonin than the palate: 3-09 ng/mg, Thus, serotonin is distributed
unequally in the embryo; more in the head than the body, but less in the palate
than the brain (as would be expected). It was of interest to determine if palate
serotonin levels were highest at the time of rotation. This was not the case,
serotonin levels increased in the palate at day 15-5 of development. Serotonin
levels also increased in embryonic brain at the later developmental time. Adult
brain levels were 8-5-fold greater than day-14-5 brain.
The HPLC method employed could simultaneously measure dopamine levels
and these analyses were also carried out (Table 2). Thus dopamine was present
in the day-14-5 palate at 1-63 ng/mg protein, about 4-fold greater than that of
serotonin. Dopamine levels decreased in the day-15-5 palate, and dopamine
was present in the embryonic body in lower concentrations than the palate at
day 14-5 of gestation. Dopamine levels increased in brain with time of development.
Distribution of serotonin
Since serotonin was present and synthesized in the palate, its distribution in
the palate was sought. To observe the localization of serotonin in the palate
when present in very low levels, autoradiography was employed after [3H]5HTP incorporation. Stained paraffin sections showed grains in cells throughout
the palate. Region-3 cells (Fig. 6D) which possess many filopodial processes
did not contain an increased concentration of grains compared to internal
mesenchymal cells (Fig. 7C). Similarly region-2 cells (Fig. 6C), and tooth-germ
epithelium or its mesenchyme (not shown) also contain many grains but their
concentration was not higher than other palatal structures. In addition, grains
could be observed in the epithelium (Fig. 7C). Surprisingly the pterygopalatine
nerve and ganglion showed the presence of grains (Fig. 7B). As Fig. 6B indicates,
Fig. 7. Autoradiography of embryonic palate and eye. (A) Slightly more posterior
palate section than Fig. 6 indicating region 2, pterygopalatine ganglion (G),
pterygopalatine nerve (PN) and oral epithelium (OE). Bar, 75 /*m. xl35. (B)
Higher magnification of area of ganglion and nerve. Much higher concentration of
grains are observed in pterygopalatine nerve (PN) and ganglion (G) than in surrounding mesenchymal cells. Bar, 10 /tm. x 860. (C) Lower area of oral side of
palate shelf. Grains can be seen in mesenchymal cells (M) as well as in epithelium
(OE). Bar, 20/tm. x 450. (D) Section of eye from day-14-5 embryo showing grains
throughout the eye, including less (L). Bar, 15 /.tm. x 670. (E) Higher magnification
of area of eye depicted in square of Fig. 7D. Grains appear to be localized at junction
of inner and outer layer of the optic cup (arrow) as well as in the outer layer. Bar,
15/tm. x720.
246
E. F. ZIMMERMAN AND OTHERS
many grains were localized also in the maxillary nerve. In this palate, the
pterygopalatine nerve was found in a slightly more posterior section (Fig. 7A).
Thus grains appeared throughout the palate including palatal neural tissue.
Other areas of the head including brain were also observed for the presence
of grains. They were found throughout the brain, but were found in highest
concentration in the eye (Fig. 7D). As seen in Fig. 7E, grains were localized
at the junction of inner and outer layer of the optic cup and in the outer layer
which is destined to become the retina. Accumulation of indoleamines in the
developing retina has been reported previously (Baker & Quay, 1969; Ehinger
& Floren, 1978).
DISCUSSION
The experiments carried out in this study were to determine whether serotonin
was present and synthesized in the palate since serotonin appeared to regulate
palate shelf rotation (Wee et al. 1979). Evidence that a serotonergic system
exists in the palate is: (1) Serotonin was actively taken up by excised palates.
(2) Serotonin metabolites were found in the palate. (3) Synthesized serotonin
was located in the palate when embryos or excised palates were cultured with
[3H]5-hydroxytryptophan. (4) Serotonin was detected in excised palates.
Active uptake of serotonin into neurons has been a noted feature of serotonergic systems. This active uptake has been identified by its temperature
dependency. Presumably the cellular uptake process is a method of inactivating
serotonin after its release and subsequent binding to its receptor. Excised palates
when incubated with [3H]5-HT also showed a temperature-sensitive uptake;
9-fold more radioactivity was associated with palate incubated at 37 °C than
at 4 °C (Fig. 2), constituting a criterion for a palatal serotonergic system.
After uptake into cells, serotonin is usually metabolized by monoamine
oxidase to 5-hydroxyindoleacetic acid. When embryos were cultured with
labelled 5-HT, little HIAA was observed in the palate compared to embryonic
brain (Fig. 1). However, incubating embryos with the monoamine oxidase
inhibitor nialamide increased the concentration of serotonin synthesized in the
palate (Fig. 4) suggesting that some monoamine oxidase is present in the palate.
This would seem likely since rat brain at day 15 of gestation had 10% of the
adult monoamine oxidase activity (Saavedra, Coyle & Axelrod, 1974), while
monoamine oxidase activity towards serotonin in chick retina also increased
during development (Suzuki, Noguchi & Yagi, 1977). Low monoamine oxidase
activity in developing palate could allow serotonin to be released from its
sources and diffuse a considerable distance while acting on palate mesenchymal
cells before it was enzymatically inactivated.
Labelled serotonin was observed in the palate when the precursor [3H]5-HTP
was incubated with whole embryos (Fig. 3). Since brain (Baker & Quay, 1969)
or yolk granules in early Ophryotrocha embryos (Emanuelsson, 1974) are
sources of serotonin, it is possible that synthesized serotonin was transported
Serotonin in the palate
247
into the palate from other sites of synthesis via the functioning cardiovascular
system. However, excised palates were also able to convert [3H]5-HTP to
5-HT indicating that synthesis could take place in the palate directly (Fig. 5).
Nevertheless, there has been some controversy on the specificity of the enzyme,
amino acid decarboxylase, that converts the precursor to the monoamine
(Lovenberg, Weissbach & Udenfriend, 1962; Sims & Bloom, 1973).
In any case, the presence of serotonin in the palate has been ascertained by
direct measurement using HPLC. The level of serotonin in the palate at day
14-5 of development is 0-40 ng/mg protein which is significantly greater than
that of the fetal tongue and body at the same time of development. This greater
palatal level of serotonin is consistent with a regulatory role in the palate.
However, palatal serotonin is about 8-fold less than that in brain at this time
in development. In turn, the adult brain level (26-4 ng/mg) is 8-5-fold greater
than brain serotonin at day 14-5 of development. Previously, Saavedra (1977)
has reported a heterogenous distribution of serotonin in discrete areas of adult
rat brain ranging from 2-0 ng/mg protein in the cerebellum to 36-4 in the
nucleus arcuatis of the hypothalamus. Developmental increases in serotonin
have also been reported previously. Kato (1960) indicated that serotonin content
increased about 5-fold from the fetus to the adult in rat brain, while Bourgoin,
et al. (1977) showed a 3-fold increase from the neonatal to the adult stage. The
developmental increase in brain serotonin levels is also mimicked in the palate
which is significantly elevated at day 15-5 of development compared to one day
earlier (Table 2).
In addition, dopamine levels in the \. .«.e were measured which are much
greater than that of serotonin. At day 14-5 of development dopamine levels are
4-fold more than serotonin. However, dopamine as well as norepinephrine have
been tested for their ability to affect palate shelf rotation in embryo culture.
In contrast to serotonin or acetylcholine, these catecholamines did not stimulate
palate rotation employing concentrations as high as 10~4 M (Zimmerman et al.
1980). It may be that these catecholamines are involved in regulating palatal
fusion via the cyclic AMP system; activation of adenylate cyclase (Waterman,
Palmer, Palmer & Palmer, 1976) would then elevate cyclic nucleotide which in
turn would cause programmed cell death and contribute to the fusion process
(Greene & Pratt, 1979).
Since region-3 cells have been implicated in palate reorientation (Zimmerman,
1979), it was expected that serotonin might be preferentially localized in the
area of region-3 cells. However, when autoradiography of sectioned palate
was performed after incorporation of [3H]5-HTP in the embryo and aldehyde
fixation, grains (5-HT or metabolites) were found throughout the palate shelf.
Structures such as region-2 cells, tooth germ, internal mesenchymal cells, and
epithelium all showed many grains. In addition, grains were observed also in
the pterygopalatine ganglion, maxillary nerve and the pterygopalatine nerve.
The presence of grains in the pterygopalatine nerve (Fig. 7B) was surprising
248
E. F. ZIMMERMAN AND OTHERS
since this nerve is sensory, although there may also be parasympathetic
innervation as well. However, electron microscopic analysis of the palate
revealed many dense core vesicles in this nerve (Kuhn, Babiarz, Lessard &
Zimmerman, 1980) which is consistent with serotonin or catecholamine localization. One possibility to explain the presence of grains in the pterygopalatine
nerve is that serotonin is localized in the nerve at day 14-5 of gestation, and at
a later time in development when the nerve assumes its sensory function, the
neurotransmitter composition would change. The concept that neurotransmitter
composition in neurons is developmentally altered as the surrounding target
field changes has been postulated by Bunge, Johnson & Ross (1978). Alternatively, the deep petrosal nerve which is sympathetic could be coursing through
the pterygopalatine nerve to innervate blood vessels. At this time in development
the nerve trunk which courses down into the palate lies adjacent to the palatine
artery. Thus uptake of [3H]5-HTP by sympathetic components in the nerve
could convert the precursor to serotonin by the relatively non-specific amino
acid decarboxylase.
The presence of 5-methoxytryptamine in the palate (Table 1) is of interest
because it has been reported to be the active indoleamine neurotransmitter in
sea urchin development (Renaud, Parisi, Capasso & Monroy, 1979). A function
for palate 5-methoxytryptamine is not known at present.
In summary, serotonin has been shown to be present in the palate at the time
of its elevation which is consistent with the monoamine regulating the morphogenetic process. Alternatively, serotonin may be regulating growth or differentiation of the palate since it has been postulated to regulate these processes in
sea urchin (Buznikov et al. 1972) and mammalian neural development (Vernadakis & Gibson, 1974; Lauder & Krebs, 1978). However, a role for regulating
palate morphogenesis is suggested by the following effects of serotonin: (1)
Stimulation of palate shelf rotation in embryo culture; and blockade of rotation
by its antagonists (Wee et al. 1979). (2) Stimulation of contractility of palate
cells growing in explants (Zimmerman, 1979). (3) Stimulation of palate cell
migration in explants in a collagen gel matrix and cultured cells undergoing
chemotaxis (Zimmerman et al. 1980). (4) Stimulation of palatal protein carboxymethylation (Zimmerman et al 1980), a process associated with cell migration
(O'Dea, etal. 1978).
The presence of serotonin in palatal neural tissues suggest that these structures
could be playing a key role in palate rotation, possibly as a source of extracellular serotonin; nerves have been observed to be active at this time of development (Wragg, Smith & Borden, 1972). The serotonin released from neural tissue
could serve to initiate the secretion of serotonin by mesenchymal and epithelial
cells, and thus act as a trigger for palate rotation.
This research was supported by a grant from the National Institute of Dental Research
(DE 03469) and a Center Grant in Mental Retardation (HD 05221). We thank Dr E Ritter
for carrying out the analysis of serotonin and dopamine.
Serotonin in the palate
249
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