/. Embryol. exp. Morph. Vol. 60, pp. 271-281, 1980
Printed in Great Britain © Company of Biologists Limited 1980
271
Immunohistochemical localization of cyclic AMP in
the developing rodent secondary palate
By ROBERT M. GREENE, ,* JOSEPH L. SHANFELD, 2
ZEEV D AVIDOVITCH 2 AND ROBERT M. PRATT 3
From the Department of Anatomy, Jefferson Medical college,
Philadelphia
SUMMARY
During development of the mammalian secondary palate, medial-edge epithelia (MEE) from
apposing palatal shelves adhere and undergo autolysis allowing palatal mesenchymal regions
to unite. In a prior study (Greene & Pratt, 1979), we reported a transient increase in levels of
cyclic AMP (cAMP) in the mouse and rat palate during epithelial adhesion and cell death.
The objective of this study was to examine the distribution of cyclic AMP in the developing
rodent secondary palate using immunohistochemistry to localize cyclic AMP. Staining for
cAMP was observed in the epithelium just prior to and during epithelial fusion (day .16 in the
rat; day 14 in the mouse). Cyclic AMP was distributed throughout the epithelial cytoplasm
whereas no staining was seen in nuclei. Epithelial staining for cAMP was faint or absent 24
and 48 h prior to epithelial contact. Mesenchymal staining for cAMP was minimal and
associated with the plasma membrane at all stages studied. These results demonstrate that
elevated levels of cAMP in the rat and mouse palate during epithelial adhesion and cell death
are mainly due to increases of the nucleotide in palatal epithelium. This observation suggests
that a transient increase in epithelial cAMP may play a role in palatal epithelial differentiation.
INTRODUCTION
Terminal cellular differentiation is observed during development of the
mammalian secondary palate where the palatal medial edge epithelium (MEE)
undergoes a programmed sequence of events resulting in its death. These events
include cessation of DNA synthesis (Hudson & Shapiro, 1973; Pratt & Martin,
1975), synthesis of cell-surface glycoconjugates which mediate adhesion between
apposing palatal shelves (Greene & Kochhar, 1974; DePaola, Drummond &
Miller, 1975; Pratt & Hassell, 1975; Greene & Pratt, 1977) and synthesis of
lysosomal enzymes (Mato, Smiley & Dixon 1972; Lorente, DePaola, Drummond
& Miller, 1974) requisite for MEE autolysis (Mato, Aikawa & Katahua 1966,
Author's address: Department of Anatomy, Jefferson Medical College, 1020 Locust
Street, Philadelphia, Pa. 19107, U.S.A.
^Author's address: Departments of Orthodontics and Pedodontics, University of Pennsylvania, School of Dental Medicine and Center for Oral Health Research, Philadelphia, Pa.
19104, U.S.A.
^Author's address: Laboratory of Developmental Biology and Anomalies, National
Institute of Dental Research, NIH, Bethesda, Md. 20205, U.S.A.
18-2
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R. M. GREENE AND OTHERS
1967, Smiley, 1970; Smiley & Koch, 1972; Chaudhry & Shah, 1973; Pratt &
Greene, 1976; Greene & Pratt, 1978). After contact of the MEE with the apposing palatal shelf, MEE autolysis allows the mesenchymal portions of the
shelves to unite and form the secondary palate.
Cyclic AMP appears to be involved in the regulation of hormonal effects on
target cells during development of a number of tissues (Weiss & Strada, 1973;
Rogan, Schafer, Anderson & Coggin 1973) and in regulating cellular proliferation and differentiation (Rogan et al. 1973; Creighton & Trevithich, 1974;
Pastan, Johnson & Anderson 1975; Deshpande & Siddique, 1976). Previous
studies (Pratt & Martin, 1975; Olson & Massaro, 1977; Greene & Pratt, 1979)
demonstrated a transient increase in palatal cyclic AMP levels during epithelial fusion which suggests that this cyclic nucleotide may be involved in MEE
differentiation.
The availability of specific anti-cyclic AMP antibodies has allowed immunohistochemical localization of cAMP in tissue sections (Fallon et al. 1974;
Davidovitch, Montgomery, Eckerdal & Gustafson, 1976a, b; Dousa, Barnes,
Ong & Steiner, 1977; Ong & Steiner, 1977; Davidovitch, Montgomery Yost &
Shanfeld, 1978 a, b; Steiner et al. 1978). Such localization of cAMP is particularly useful in heterogeneous tissues containing a number of cell types. This
paper describes the localization and distribution of cAMP in the developing
rodent secondary palate. A preliminary report of these results has already been
presented (Greene, Pratt, Shanfeld & Davidovitch, 1979).
MATERIALS AND METHODS
Animals
Mature female Sprague-Dawley rats were mated overnight to males of known
fertility. The appearance of spermatozoa in vaginal smears taken the following
morning was regarded as evidence of mating and this day was designated as day
0 of gestation. Mature male and female Swiss-Webster mice were also mated
overnight, and the presence of a vaginal plug the following morning (day 0 of
gestation) was considered evidence of mating.
Preparation of anti-cAMP antibodies
Preparation and purification of anti-cyclic AMP antibodies was done according to Davidovitch etal. (1976a, 1977). In brief, 2' 0' succinyl cAMP (SchwartzMann, Orangeburg, N.Y.) was coupled to either bovine or rabbit serum albumin
(Miles Laboratories, Elkhart, Ind.) as described by Steiner, Wehmann, Parker
& Kipnis (1972). Antiserum to cyclic AMP was produced in New Zealand
white rabbits which were immunized against cAMP by repeated injections
of the cAMP-bovine serum albumin complex. Pooled sera obtained from immunized rabbits which displayed no cross reactivity for cyclic GMP was used for
antibody purification. Antibodies were isolated employing a Sepharose 2B-
Cyclic AMP in developing secondary palate
273
rabbit serum albumin-cyclic AMP immunoadsorbant column (Cuatrecasas,
Wilcheik or Anfinsen, 1968). Immune serum was applied to the column, antibodies eluted off with 1 M acetic acid, dialysed against borate buffer, pH 8-3,
concentrated by ultrafiJtration and quantified spectrophotometrically. Immunoelectrophoresis revealed that the purified antibodies were IgG.
Imnumohistochemical procedures
Specific localization of cyclic AMP was performed utilizing the immunoglobulin-enzyme bridge method of Mason et al. (1969) as modified by
Davidovitch et al. (1976a, 1977). Rat (days 14-16 of gestation) and mouse
(days 12-14 of gestation) fetuses were quickly excised, decapitated and intact
fetal heads frozen in pre-cooled hexane at - 7 0 °C. Sections, 6 /an thick, were
then cut at - 2 0 °C in a cryostat, and thawed onto albumin-coated glass slides.
Sections were always taken from the anterior one third of the palatal shelf.
Unfixed frozen sections were incubated at room temperature sequentially
(20 min each) with each of these solutions: (a) rabbit anti-cAMP IgG antibodies
(60/*g/ml); (b) goat anti-rabbit IgG (Cappel Laboratories, Cochranville, Pa.;
antibody protein: lOmg/ml, diluted 1 to 4 in 50 HIM Tris-HCl pH 7-6); (c)
rabbit anti-horseradish peroxidase IgG (Cappel; antibody protein: 2-4 mg/ml,
diluted 1 to 4); (d) horseradish peroxidase (Cappel; 12-5 /tg/ml). The presence
of peroxidase in tissue sections was demonstrated by exposing the sections to
3, 3'-diaminobenzidine (DAB) (3-8 mg/5ml, 50 mM Tris, pH 7-6, containing
H2O2) (Graham & Karnovsky, 1966) for 20 min at 37 °C. A 15 min wash in 50
mM Tris-HCl, pH 7-6, was included between each step in the sequence. Sections
were then dehydrated, mounted beneath coverslips with glycerol/PBS (9 to 1)
and viewed and photographed using a Nikon Biophot photomicroscope.
Specificity tests for cAMP staining in frozen sections
To demonstrate specificity of staining, a series of control experiments were
performed. Some tissue sections were treated directly with peroxidase and DAB
without prior exposure to any immunoglobulins. In another control, an IgG
preparation from a non-immunized rabbit was substituted for the specific anticAMP antibody. Competitive antibody inhibition was performed in other
experiments. The anti-cAMP antibody was incubated for 4 h at 4 °C with the
specific antigen, cyclic AMP, or other nucleotides (5'AMP, ATP or adenosine)
all at 1 x 10~3 M. Following incubation each solution was substituted in the
immunohistochemical procedure as the first step, followed by subsequent
routine steps.
RESULTS
In the Sprague-Dawley rat, palatal shelves undergo reorientation to a horizontal position above the tongue late on day 15 of gestation followed by contact
of the apposing medial-edge epithelia (MEE) and formation of a midline
274
R. M. GREENE AND OTHERS
Fig. 1. Palatal shelf from a day-14 rat fetus. Unfixed frozen, 6 fim coronal section of
a single vertically oriented palatal shelf (P) stained for cAMP. Note that palatal epithelial (arrows) and mesenchymal (arrow heads) staining is faint. Oral epithelium
(OE); nasal epithelium (NE); medial-edge epithelium (ME). Mandible (M); tongue
(T). x50.
Fig. 2. Palatal shelf (P) from a late day-15 rat fetus identically prepared and treated
to that shown in Fig. 1. Note the increased staining intensity for cAMP in the
palatal epithelium (arrows). Mandible (M); tongue (T). x 50.
epithelial seam by day 16 of gestation. This same sequence of events occurs
between gestational days 13 and 14 in the Swiss-Webster mouse. Localization
and distribution of cyclic AMP were visualized by immunohistochemistry during
secondary palate formation in rat palatal processes from day 14 through 16 of
gestation and in mouse palatal processes from days 12 through 14 of gestation.
Figure 1 is a representative frozen, coronal section of a vertically oriented
palatal shelf from a day-14 fetal rat head stained for cAMP. Palatal epithelial
and mesenchymal staining for cAMP is faint. Epithelial staining for cAMP was
most intense just prior to shelf elevation (Fig. 2) and during (Figs. 3 and 5)
epithelial fusion (day 16 in the rat; day 14 in the mouse). Prior to shelf elevation
staining for cAMP was observed in all areas of the palatal epithelium (Fig. 2)
Cyclic AMP in developing secondary palate
275
Fig. 3. Two apposing rat palatal shelves (P) on day 16 of gestation. Coronal section,
6 /tm thick, stained for cAMP. Note the intense staining for cAMP in the epithelial
cells of the intact midline epithelial seam (arrows). Nasal septum (NS). x 70.
Fig. 4. Control section adjacent to that shown in Fig. 3. The anti-cAMP antibody was
pre-incubated with cAMP (4 h at 4 °C). This antibody-hapten complex was substituted in the immunohistochemical procedure in place of the specific anti-cAMP
antibody. Note the elimination of specific staining in the midline epithelial cells, x 70.
Fig. 5. Coronal section, 6 /tm thick, of two apposing mouse palatal shelves (P) on day
14 of gestation showing staining for cAMP in the epithelial cells of the MEE (arrows).
Nasal septum (NS). x 70.
Figs. 6 and 7. Figure 6 is a higher magnification of the palatal midline epithelial seam
shown in Fig. 3 while Figure 7 is a higher magnification of the palatal midline epithelial seam shown in Fig. 5. Note the intense staining for cAMP throughout the
cytoplasm of the midline epithelial cells (arrows). Note also the distribution of
staining along the periphery of epithelial and mesenchymal cells (arrowheads).
xl25.
276
R. M. GREENE AND OTHERS
while MEE appeared to stain most heavily during formation of the midline
epithelial seam (Figs. 3 and 5). This corresponds to the gestational age during
which palatal tissue exhibits a transient increase in the levels of cAMP (Greene &
Pratt, 1979) DAB reaction products were heavily distributed throughout the
epithelial cytoplasm and along the epithelial cell periphery with some diffuse
localization in the mesenchymal cell periphery (Figs. 6 and 7).
In the specificity tests no staining was observed when sections were incubated
with an equivalent concentration of immunoglobulin from a non-immunized
rabbit. Tissue sections treated directly with peroxidase and DAB without prior
exposure to any immunoglobulins also failed to exhibit any staining. Competitive antibody inhibition whereby the antibody-hapten complex was substituted
in the immunohistochemical procedure in place of the specific anti-cAMP antibody eliminated specific staining (Fig. 4). This demonstrated that most, if not all,
the antibody binding sites were occupied by hapten and were unable to react
with the endogenous tissue cAMP in the sections, thus resulting in minimal
staining. No competitive inhibition of staining occurred when the antibody was
preincubated with either 5'AMP, ATP or adenosine. In addition, it is of interest
that this tissue does not show any endogenous peroxidase activity.
DISCUSSION
Cyclic AMP has been proposed to play an important role in cellular differentation in several tissues (Creighton & Trevithich, 1974; McMahon, 1974; Zalin
& Montague, 1974; Zalin & Leaver, 1975; Davidovitch et ah 1976a) including
the developing secondary palate (Pratt & Martin, 1975; Waterman, Palmer,
Palmer & Palmer, 1976, 1977; Greene & Pratt, 1979; Pratt et al. 1979). We have
recently demonstrated a transient increase in palatal cAMP levels as determined by radioimmunoassay (Greene & Pratt, 1979). Maximal values were seen
in the developing rat and mouse palates just prior to and during epithelial
fusion. Since the developing mammalian palate is a heterogeneous tissue comprised primarily of mesenchymal and epithelial cells, it was not known from this
study which cells were responsible for the elevation of cAMP.
In the present study we observed a differential localization of cAMP in
developing rodent palatal tissues. Epithelial staining for cAMP was most
intense just prior to and during epithelial fusion. Cyclic AMP appeared to be
predominantly localized in the epithelium, with lesser amounts observed in the
mesenchyme. Cyclic AMP staining increased dramatically in all areas of the
palatal epithelium just prior to palatal elevation while MEE appeared to stain
most heavily during formation of the midline epithelial seam. This time period
corresponds to the developmental period during which palatal cAMP levels
transiently elevate (Greene & Pratt, 1979). Cyclic AMP was localized throughout
the epithelial cytoplasm with increased intensity near the epithelial as well as
mesenchymal plasma membranes. These observations may reflect binding sites
for cAMP in palatal epithelium, different from those in mesenchymal cells.
Cyclic AMP in developing secondary palate
277
Differential localization of cyclic nucleotides by immunohistochernical procedures has been demonstrated in a number of mammalian tissues (Davidovitch
etal. 1916a, 1978a; Dousa. etal. 1977; Earp, Smith, Ong & Steiner, 1977; Ong&
Steiner, 1977; Steiner et al. 1978). Cyclic AMP is generally found in the cytoplasm. In human lymphocytes (Bloom, Wedner & Parker, 1973) and rat liver
(Steiner, Whitley, Ong & Stowe, 1975), however, distinct plasma membrane
localization has been reported.
The immunohistochemical procedure probably localizes cAMP which is
bound to insoluble tissue receptors since the procedure necessitates frequent
washing of the unfixed tissue sections in aqueous solutions resulting in the loss
of free, unbound water-soluble nucleotides (Steiner, Ong & Wedner, 1976).
Immunohistochemical techniques are basically qualitative and do not allow for
quantification of tissue cAMP concentrations. However, Ortez has recently
demonstrated (Ortez, 1978) that the tissue staining intensity may serve as a
semiquantitative means to assess the relative level of cellular cyclic nucleotide
concentration. Intense staining is representative of a high cellular cAMP content,
while weak staining intensity corresponds with low concentrations of tissue
cAMP.
Using this procedure it is possible to observe differences in the localization of
cAMP in a particular cell type within heterogeneous tissues. Changes in cAMP
involve variations in the degree of staining intensity. The changes shown in this
study correlate temporally with radioimmunologically determined alterations
in palatal cAMP content (Greene & Pratt, 1979). A. correlation also exists in
other systems between the intensity of cellular staining for cyclic nucleotides and
cellular cAMP content (Bloom et al. 1973; Fallon et al. 191 A; Davidovitch et al.
19766; Dousa et al. 1977). For example, reduced levels of cAMPinperiodontal
ligament tension sites (Davidovitch & Shanfeld, 1975) correlated with reduced
immunohistochemical staining for cAMP in these areas (Davidovitch et ah
1916b).
A transient increase in cAMP levels has been causally related to the onset of
myoblast fusion in vitro (Zalin & Montague, 1974; Zalin & Leaver, 1975) thus
demonstrating the importance of cAMP in cellular differentiation. Moreover,
in the present study, the staining of muscle cell clusters in the developing tongue
by anti-cAMP (Fig. 2) may be reflective of cAMP involvement in muscle differentiation. The results of this and a previous study (Greene and Pratt, 1979)
have demonstrated that the cAMP content of rat and mouse palatal epithelial
cells increases during epithelial adhesion and cell death. This suggests that a
transient increase in epithelial cAMP may play a role in palatal epithelial differentiation. This conclusion is also supported by studies showing that dibutyryl
cyclic AMP can prevent the effect of epidermal growth factor which inhibits
differentiation and subsequent death of palatal MEE cells in vitro (Hassell, 1975;
Hassell & Pratt, 1977; Pratt, Figueroa, Nexo & Hollenberg, 1978). Moreover,
addition of dibutyryl cAMP to immature day-14 ra.t palatal shelves in organ
278
R. M. GREENE AND OTHERS
culture (Pratt & Martin, 1975) results in precocious differentiation of MEE with
occurance of several developmental events that normally occur on day 15 and 16
of gestation.
The precise role which cAMP plays in palatal MEE differentiation is not
known. Changes in cyclic nucleotide concentrations have been reported in many
cell culture systems and cAMP has been implicated as a possible regulator in
the control of cell proliferation (Pastan et al. 1975; MacManus, Boynton &
Whitfield, 1978). However, since palatal MEE cease DNA synthesis (Pratt &
Martin, 1975) at least 24 h prior to the reported rise in palatal cAMP levels
(Greene & Pratt, 1979), it is unlikely that this elevation of epithelial cAMP plays
a direct role in cessation of MEE proliferation. The transient elevation of palatal
cAMP is temporally related with palatal epithelial glycoconjugate synthesis and
MEE cell death. Since cell death in the MEE is not a passive event, but an active
process requiring the synthesis of specific proteins (Pratt & Greene, 1976),
palatal cAMP may play a role in the synthesis or activation of proteins requisite
for MEE cell death. In fact, induction of cytolysis of cultured lymphoma cells by
cAMP (Daniel, Litwack & Tompkins, 1973) may require the synthesis of a gene
product involved in cytolysis (Lemaire & Coflino, 1977). The results of the
present study connrm previous observations on transient increases in palatal
cAMP concentrations during the stage of epithelial adhesion and cell death, and
demonstrate that changes in the levels of cAMP occur predominantly in the
palatal epithelium, and not in the adjacent palatal mesenchyme.
This work was supported in part by P.H.S. grants DE-05550 to RMG and DE-03619 to
Z.D.
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{Received 5 February 1980, revised 5 May 1980)