J. Embryol. exp. Morph., Vol. 14, Part 3, pp. 223-238, December 1965
Printed in Great Britain
Morphological and autoradiographic studies of
cleft palate induced in rat embryos by maternal
hypervitaminosis A
by DEVENDRA M. KOCHHAR and E . MARSHALL JOHNSON 1
From the Department of Anatomy, College of Medicine, University of Florida
WITH FOUR PLATES
INTRODUCTION
IN the laboratory many teratogenic procedures have been found to produce cleft
palate in conjunction with other congenital malformations (Kalter & Warkany,
1959), but only in embryos of cortisone treated mice has this malformation been
reported to occur singly (Baxter & Fraser, 1950). However, a carefully controlled
treatment with hypervitaminosis A can produce a high incidence of cleft palate
which is only rarely accompanied by other malformations (Giroud & Martinet,
1956). These two teratogenic treatments have other similarities in that both similarly affect the ultimate chemical composition of tissues. That is, both in vivo and
in vitro, cortisone depresses the ability of tissues to synthesize chondroitin sulfate
(Layton, 1951a, 19516; Schiller & Dorfman, 1957) and, in animals receiving
excessive doses of vitamin A, chondroitin sulfate was removed from the matrix of
epiphyseal and articular cartilages (Thomas et ah, 1960). In addition, incorporation of S35-labelled inorganic sulfate into embryonic limbs cultivated in vitro
was inhibited by the presence of vitamin A in the culture medium (Fell, Mellanby
&Pelc, 1956).
It was suggested by Larsson (1960) that the occurrence of cleft palate in response
to cortisone treatment might be due to interference in the synthesis of chondroitin
sulfuric acid. It was demonstrated later by this author (Larsson, 1962) that in
mouse embryos from cortisone treated mothers there was a reduced S35-incorporation (presumably into sulfated-mucopolysaccharides) in the palatine shelves
as well as in other regions of the embryo. Since the morphological sequence of
cortisone induced cleft palate had been described previously (Walker & Fraser,
1957), the present experiment endeavored (1) to compare this sequence with early
1
Authors' address: Department of Anatomy, College of Medicine, University of Florida,
Gainesville, Florida, U.S.A.
224
D. M. KOCHHAR and E. M. J O H N S O N
morphogenesis of cleft palate induced by hypervitaminosis A and (2) to investigate
further the effects of maternal hypervitaminosis A on S35-incorporation into the
rat embryo.
MATERIALS AND METHODS
Eighty proestrus black-hooded female rats ranging from 60-90 days of age and
weighing 150-200 g. were placed overnight with males of the same strain. The
presence of a sperm plug or of free spermatozoa in a smear of vaginal contents the
next morning indicated that mating had occurred and this day was considered as
day 0 of pregnancy. The pregnant animals were divided into two major groups
and maintained by a stock diet and water ad libitum. Commencing on days 9 or
10 of gestation and continuing for 3 successive days, females received 60,000
international units (i.u.) per day of vitamin A acetate in one ml. cottonseed oil by
oral intubation. Control animals were similarly given 1 ml. of pure cottonseed oil.
Experimental and control groups were subdivided for specific experiments as
indicated below.
For study of the genesis of cleft palate and its incidence at term, a series of
treated and control animals were sacrificed at 10.00 A.M. either on day 15, 16, 17
or 20 of gestation corresponding to Witschi's standard stages 31,33,34 and 35 a p
respectively. At autopsy, the fetuses were fixed in either: (1) Bouin's fluid for
measurement and subsequent examination of 1-2 mm. thick freehand frontal
sections or (2) alcohol-formalin for histological study. Sections were stained by
the following procedures: (a) 0 • 1 per cent, toluidine blue in 30 per cent, ethyl
alcohol for identification of acid mucopolysaccharides (Kramer & Windrum,
1955) as contrasted to hyaluronidase treated controls; (b) periodic acid-Schiff
technique (PAS) for identification of glycogen (McManus, 1948) as contrasted to
alpha amylase treated controls; (c) methyl green pyronin for detection of ribonucleoproteins (Bracket, 1942) as contrasted to ribonuclease treated controls
(Pearse, 1960); (d) Feulgen stain for deoxyribonucleic acid (Feulgen & Rossenbeck, 1924 as referred to in Pearse, 1960); (e) iron hematoxylin.
For indication of mucopolysaccharides in normal and abnormal development
of the secondary palate, pregnant females which had been treated as above with
either excess vitamin A or with cottonseed oil were given a single intraperitoneal
dose of carrier free S35-sodium sulfate on either day 13, 14, (10 microcuries/gm.
body weight) or day 15 (5 microcuries/gm. weight) of gestation. Two days after
injection the females were sacrificed and the young were embedded and serially
sectioned for autoradiography (Messier & Leblond, 1957) with Kodak NTB-3
liquid emulsion. In this manner adequate time was allowed for uptake of the
sulfur and the age of the embryo at autopsy was 15, 16 or 17 days of age which
corresponded with the age of the embryos described previously which were
studied by standard and histochemical means. The radioactive sections were
exposed to the emulsion for 12 days in a dry ice chest before the emulsion was
developed and the sections stained with toluidine blue.
Maternal hypervitaminosis A
225
RESULTS
The teratogenic effects of hypervitaminosis A observed on term fetuses are
summarized in Table 1. Among the embryos from treated mothers, 80 per cent,
or more had cleft palate when the three-day treatment period was started on either
day 9 or day 10 of pregnancy. Furthermore, the frequency of eye defects such as
microphthalmia, anophthalmia, open eye, and exophthalmos was similar, but
exencephaly occurred only if vitamin A treatment was instituted on the earlier
day. Intra-uterine mortality was also much higher after earlier treatment. Only
a slight decrease in mean fetal weight and crown rump length was observed in
living fetuses from vitamin A treated mothers.
Normal morphogenesis of the secondary palate
Because palatal closure occurs in these rats on day 16 of gestation, examinations
were made of fetuses of 15, 16 and 17 days gestational age. Data concerning the
reproductive performance and external embryonic morphology at each of these
ages is summarized in Table 2. The embryos as represented in this table had a
pattern of palatal development which was characteristic for each of the three days
selected for examination.
On day 15 the palatine shelves of control embryos were vertically oriented at
either side of the tongue and on the basis of mesenchymal cells, preosteoblasts
and intercellular ground substance, they could be divided into anterior, middle
and posterior thirds. In the anterior one third of the shelf mesenchymal cells were
dispersed uniformly within the metachromatic ground substance to form the
core of the shelf and preosteoblasts with basophilic glycogen-containing cytoplasm were present. In the middle third of the palatine shelf the mesenchymal
cells were closely packed, particularly along the medial border, and the ground
substance was strongly metachromatic. This medial portion of the palatine shelf
was delineated from the rest of the palatal tissue by an epithelial notch. Preosteoblastic tissue extended into both the medial and lateral portions. In the
posterior third of the palatine shelves the epithelial notch became deeper and
consequently separated the shelf from the more laterally placed maxillary areas
and, as a result, in frontal sections the shelf appeared as a vertically oriented plate.
The cell density was still greater than in the middle third and metachromatic
ground substance was abundant. In this region, the preosteoblastic tissue was
assembled at the dorsomedial edge of the palatine shelves which were covered by
a low, simple columnar epithelium. This epithelium was continuous laterally
with the two to three layered columnar epithelium covering the lateral surface of
the shelf as well as the rest of the oral cavity.
On day 16 of gestation the palatine shelf was still divisible into these three
regions (Plate 1, Figs. A-C). On this day evidence of movement by the palatine
shelves from the vertical to the horizontal position was observed at the junction
of the middle and posterior thirds where the osteogenic tissue executed a curve
17th day
16th day
15th day
lABLfc, Z
Eye
0
48
58
defects
0
0
15
Exencephaly
Treatment
f Control
I Vitamin A
f Control
1 Vitamin A
/ Control
1 Vitamin A
37
63
56
86
27
37
Resorbed
10
54
15
62
7
56
Number of
litters
4
11
7
16
3
6
/o
Number of
living
embryos
Average
weights
(g.)
0-263 ±0 030
0-264 ±0-029
0-468 ±0-046
0-467 ±0-039
0-933 ±0026
0-754±0-062
17-4
16-8
130
Average
crown
rump
length (mm.)
10-8
10-2
13-5
61
5-5
7-3
7-2
9-8
8-7
Average
head
length (mm.)
3 •64±0-45
3 •20±0-42
3 •09±0-44
Weights
(g.)
to
X
O
M
ON
a
O
OHNS
Embryonic age
87
9
0
83
22
112
11
12
80
54
114
13
Cleft
palate
Comparison of Control and Hypervitaminotic A Embryos on £teys 15, 16 a/7*/ 17 of Pregnancy
60,000 i.u. Vitamin A
9-11
60,000 i.u. Vitamin A
10-12
Cottonseed Oil
9-11
Treatment and days of
gestation treatment
administered
Teratogenic Action of Hypervitaminosis A Observed in Term Fetuses
Number of
0/
implantation
Number of
% Survivors
/o
sites
litters
Resorbed
malformed
TABLE 1
HAR
Maternal hypervitaminosis A
227
into a medially directed extension of palatal mesenchymal tissue (Plate 1, Fig. D).
This extension progressively enlarged while the vertical portion of the shelf was
gradually withdrawn into the body of the shelf. This movement of the palatine
shelves to the horizontal plane then appeared to extend both anteriorly and
posteriorly and in this manner 35 per cent, of the control embryos on this day
had shelves which had risen above tongue (Plate 1, Fig. E).
At day 17 the palate of control embryos was complete in that the shelves were
horizontal and fused with each other and with the nasal septum. In the anterior
portion of the palate a slight accumulation of preosteoblasts in the midline region
was observed (palatal preosteoblastic area) but the rest of the palate at this level
was composed of loosely arranged mesenchymal cells (Plate 1, Fig. F). At a
slightly more posterior level the palate became highly arched (Plate 1, Fig. G)
and the maxillary bone occupied a small triangular area in the lateral half of the
palate. At this level the medial angle of maxilla was continuous with the palatal
preosteoblastic area and the latter had split into right and left halves (Plate 1,
Fig. G). The stage of osteogenesis in the mandible of these normal control animals
was identical with that observed in the maxilla, and the dental primordia for the
upper and lower molars were considerably advanced in their development and
were deeply lodged within their respective alveoli.
Morphogenesis of cleft palate
Again from Table 2, it can be ascertained that no major difference in mean
embryonic weight and crown rump length was observed between control and
treated embryos on days 15 and 16. The first gross external manifestation of
treatment was seen on day 17, when treated embryos weighed about 200 mg. less
than controls. In marked contrast, however, affected palates in treated embryos
could be recognized in frontal sections of the head on day 15. In comparison with
control embryos, palatine shelves of 72 per cent, of the treated embryos were
abnormal on day 15 (Text-fig. 1). They were either rounded instead of triangular,
reduced in size, or in some instances, completely absent (compare Plate 2, Figs.
H and I). A considerably lesser amount of mesenchymal tissue formed the posterior region of the palatine shelves of treated embryos even if they had a normal
complement of palatal tissue anteriorly (Plate 2, Figs. J and K). The palatine
shelves of treated embryos on day 16 also had a deformed outline (compare
Plate 2, Figs. L, and L2). Surprisingly, however, the movement of palatine shelves
from the vertical to the horizontal position had occurred in 61 per cent, of the
treated embryos on this day compared to only 35 per cent of the controls (Text-fig.
2). In spite of this, only a few of the treated embryos were able to complete successfully the shelf movement as illustrated by the fact that more than 40 per cent of the
treated embryos had palatine shelves so severely malformed that a horizontal or
vertical designation could not be assigned and were therefore designated as being
in an intermediate position (Text-fig. 2).
D. M. KOCHHAR and E. M. JOHNSON
228
Normally shaped
pi*}
llj Abnormally shaped
100-,
75-1
50 -I
25-
0-
Control
Treated
Orientation of palatine shelves
1. Comparison of the percentages of day 15 control and vitamin A treated embryos
on the basis of shape and orientation of their palatine shelves.
TEXT-FIG.
Normally shaped
Abnormally shaped
100-,
3
••5
•-
o
c
2 25-
o-
X
Control
Treated
Orientation of palatine shelves
TEXT-FIG. 2.
Comparison of the percentages of day 16 control and vitamin A treated embryos
on the basis of shape and orientation of their palatine shelves.
The palatine shelves of all control and most treated embryos on day 17 (stage
34 of Witschi, 1956) were horizontal and superior to tongue (Text-fig. 3). The
palatine shelves of treated embryos were, however, widely separated from each
Maternal hypervitaminosis A
229
other and deformed in outline in contrast to controls where the palate was complete by this time (Plate 3, Figs. M & N).
A marked difference between control and vitamin A treated embryos was the
occurrence of chondrogenesis in and around maxillary areas of all treated
embryos. In day 17 embryos from vitamin A treated mothers this heterotopic
cartilage partially or wholly replaced the maxillary bone, and sometimes was
continuous with mandibular bone thereby producing maxillo-mandibular ankylosis (Plate 3, Figs. M and N). Frequently associated with this heterotopic cartilage
were pockets of mesenchymal tissue which were separated from the tissue of
original palatine shelf by infoldings of oral epithelium (Plate 3, Fig. N). This
process of heterotopic chondrogenesis within maxillary preosteoblastic tissue
could also be detected in embryos on days 15 and 16 (Plate 2, Figs. I and L2)
because of the accumulation of a typically metachromatic cartilage matrix around
the cells of the primordial area.
H Normally shaped
| | Abnormally shaped
100-
I
E
o
X
75-
•e 50-
25-
0J
Control
Treated
Orientation of palatine shelves
3. Comparison of the percentages of day 17 control and vitamin A treated embryos
on the basis of shape and orientation of their palatine shelves.
TEXT-FIG.
Because of the above abnormality in the formation of maxillary bone, the
further development of those dental laminae which did not lodge in bone was
arrested (Plate 3, Fig. N) and in some instances the two limbs of the dental laminae
diverged and thus separated the palatal tissue from maxilla (Plate 3, Fig. O).
16
230
D. M. KOCHHAR and E. M. J O H N S O N
Incorporation ofS25 into tissues of normal and cleft palate rat embryos
Autoradiographs of sections from 15 day old normal control embryos showed
incorporation of the label primarily into tissues derived from mesenchyme which
coincided with regions of toluidine blue metachromasia. Palatine shelves and
mesenchymal tissue in other regions of control embryos of this age showed slight
radioactivity (Plate 3, Fig. P), while aggregations of cells in precartilaginous
tissue of nasal septum and Meckel's cartilage showed moderate S35-activity. A
higher density of developed particles was observed in the cartilage of the limb bud,
where considerable metachromatic matrix also was present.
Nasal and limb cartilages in the treated embryo showed S35-incorporation
comparable to that in controls, although mesenchymal tissue in the treated
embryos, including that of palatine shelves, revealed a considerably increased
incorporation above that seen in comparable areas of control embryos. (Compare
Plate 3, Fig. Q with Fig. P). Although the maxillary preosteoblastic tissue in
EXPLANATION OF PLATES
PLATE 1
A-E are frontal sections of the heads from day 16 control embryos, x 40.
F & G are frontal sections of the head from a day 17 control embryo, x 40.
FIG. A. Section through anterior third of vertical palatine shelves (PS). Observe the osteogenic
tissue (OST) within the shelves. Periodic acid-Schiff (PAS) stain.
FIG. B. Section through middle third of vertical palatine shelves. Dense accumulation of
osteogenic tissue (OST) occurs in medial aspect of the shelves. PAS stain.
FIG. C. Section through posterior third of vertical palatine shelves. Aggregation of osteogenic
tissue (OST) occurs on dorsalmost aspect of the shelves. PAS stain.
FIG. D. During shelf movement a bend occurs in the preosteoblastic tissue of the palatine
shelves. (Compare with Fig. B). PAS stain.
FIG. E. Palatine shelves are completely horizontal but unfused. Toluidine blue stain.
FIG. F. Section through anterior region of palate showing normal closure. Toluidine blue stain.
FIG. G. Section through a level posterior to that of Fig. F. Maxilla (M) on each side associates
with palatal osteogenic tissue. Toluidine blue stain.
FIGS.
FIGS.
PLATE 2
FIGS. H-K are frontal sections of the heads from day 15 embryos.
FIGS. L I & L2 are frontal sections of the heads from day 16 embryos.
FIG. H. Control. Section at a level comparable to that of Fig. I. PAS stain, x 40.
FIG. I. Vitamin A treated. Palatine shelves (PS) small or completely absent. Maxilla contains
heterotopic cartilage (HC). (Compare with Fig. H). PAS stain, x 40.
FIG. J. Control. Palatine shelf is composed of a large column of cells. Section at a level
comparable to that of Fig. K. Iron hematoxylin. x 120.
FIG. K. Vitamin A treated. Palatine shelf contains lesser number of cells. (Compare with
Fig. J). Iron hematoxylin. x 120.
FIG. L I . Control. Observe the distribution of osteogenic tissue in maxilla and horizontal
palatine shelves. Section at a level comparable to that of Fig. L2. PAS stain, x 40.
FIG. L2. Vitamin A treated. Heterotopic cartilage (HC) is present within osteogenic tissue
of maxilla and palate. (Compare with Fig. Lx). Palatine shelves are deformed. PAS stain
x40.
Vol. 14, Part 3
J. Embryol. cxp. Morph.
PLATE
D. M. KOCH HAR and E. M. JOHNSON
{Facing page 230)
J. Enibryoi. exp. Morph.
D. M. KOCHHAR and E. M. JOHNSON
Vol. 14, Part 3
J. Embryol. c.xp. Morph.
Vol. 14, Part 3
PLATE 3
D. M. KOCHHAR and E. M. JOHNSON
J. Embryol. exp. Morph.
D. M. KOCHHAR and E. M. JOHNSON
Vol. 14, Part 3
(Facing page 231)
Maternal hypervitaminosis A
231
treated embryos had S35 activity similar to controls, some regions within this
tissue had marked radiotracer incorporation and also toluidine blue metachromasia which indicated the presence of heterotopic cartilage.
By day 16, incorporation of S35 had increased markedly in control embryos
from what it was on the previous day. The most radioactive component was
cartilage (Plate 3, Fig. R), but incorporation into the palatine shelves and mesenchymal tissue of other areas also was increased in embryos of this age. Of especial
interest was the fact that the medial boundary of the shelf had a greater incorporation of tracer than the lateral portion.
Incorporation of S35 into the nasal (Plate 3, Fig. S) and limb cartilages of
treated embryos was very much greater than that exhibited by controls (compare
with Plate 3, Fig. R). Such increased S35-incorporation was also observed in the
mesenchymal tissue of the palatine shelves in treated embryos (Plate 4, compare
Fig. T and U). The presence of heterotopic cartilage in the maxillae of treated
embryos made these areas much more radioactive than the maxillary osteogenic
areas of the control embryos.
EXPLANATION OF PLATES
PLATE 3
FIGS. M-O are frontal sections of the heads from day 17 embryos. PAS stain, x 40.
FIGS. P & Q are autoradiographs of frontal sections of the palatine shelves from day 15 embryos.
Toluidine blue stain, x 1800.
FIGS. R & S are autoradiographs of frontal sections of nasal cartilage of day 16 embryos.
Toluidine blue stain, x 2300.
FIG. M. Control. Completed palate at a level comparable to those of Fig. N and O.
FIG. N. Vitamin A treated. One-half of a section showing: incomplete palate with deformed
palatine shelf (PS). Heterotopic cartilage (HC) and infoldings (IF) of oral epithelium are
present. (Compare with Fig. M).
FIG. O. Vitamin A treated. One-half of a section showing: dental lamina is not lodged in
defective maxilla; epithelium of the lamina has opened up into two separate limbs.
FIG. P. Control. Focus is on particles above the section.
FIG. Q. Vitamin A treated. Compare the labelling with Fig. P.
FIG. R. Control. Note the higher magnification of particles.
FIG. S. Vitamin A treated. Compare the labelling with Fig. R.
PLATE 4
FIGS. T & U are autoradiographs of frontal sections of palatine shelves of day 16 embryos.
Toluidine blue stain, x 1800.
FIGS. V & W are autoradiographs of frontal sections of palatine shelves of day 17 embryos.
Toluidine blue stain, x 1800.
FIGS. X & Y are autoradiographs of frontal sections of limb cartilages of day 17 embryos.
Toluidine blue stain, x 1800.
FIG. T. Control.
FIG. U. Vitamin A treated. Compare the labelling with Fig. T.
FIG. V. Control.
FIG. W. Vitamin A treated. Compare the labelling with Fig. V.
Fig. X. Control.
FIG. Y. Vitamin A treated. Compare the labelling with Fig. X.
232
D. M. KOCHHAR and E. M. J O H N S O N
In contrast, comparison of autoradiographs from day 17 control and treated
embryos showed that tissues in the palatine shelves and neighbouring areas of the
control embryo had a greater S35-incorporation (Plate 4, Fig. V) than palatine
shelves of the incomplete palate in the treated embryo (Plate 4, Fig. W). Of interest
however, was the finding that palatal mesenchymal tissue of treated embryos
which had been entrapped by infoldings of oral epithelium had a greater incorporation than other regions of the palate. Radioactivity in the nasal and limb
cartilages of control embryos was also greater than in the treated embryos (Plate 4,
Figs. X and Y).
DISCUSSION
From the standpoint of morphogenesis, cleft palate can be considered to
develop in several possible ways which have been summarized by Burston (1959),
and Fraser (1955,1960). Since the correct positioning of the left and right palatine
shelves on the superior aspect of tongue is essential for the normal closure of the
secondary palate, the classical concept insisted that the failure on the part of the
tongue to descend from between the vertically disposed palatine shelves may lead
to cleft palate. Some of the modern experimental studies also provide evidence
for this concept, e.g., cleft palate was reported to occur in embryos (1) after forcible
pressing of the head against the chest (Trasler et al., 1956) and (2) in association
with retarded growth of mandible (Asling et al, 1960).
In contrast to previous studies, Walker & Fraser (1956,1957) presented another
concept on the genesis of cleft palate in mouse embryos from cortisone treated
mothers. Observing an active wave-like motion in the palatine shelves which
were preparing to change their position from vertical to horizontal plane, these
authors conceived of some factor or force residing within the palatine shelves
which was responsible for initiation of their movement. Cleft palate in the cortisone treated embryos was assumed to occur as a result of delay in initiation of the
shelf movement. When the vertical palatine shelves ultimately did become horizontal, growth of the head had taken them so far apart that they were unable to
meet and fuse. Support of this concept has been given by authors studying the
teratogenic influence of such agents as hypervitaminosis A (Walker & Crain,
1960; Kamei, 1962), riboflavin deficiency (Walker & Crain, 1961) and X-irradiation (Callas & Walker, 1963).
The present study indicates that cleft palate in rat embryos in response to
maternal hypervitaminosis A is not due to the failure of shelf movement. When
the orientation of the palatine shelves was observed during early developmental
stages it became evident that in a major percentage of the embryos from vitamin A
treated mothers shelf movement was initiated on time and cleft palate developed
in these embryos in spite of the fact that the barrier of the tongue had been crossed
and the palatine shelves were horizontal.
In studies of the morphological and chemical basis for shelf movement,
Walker & Fraser (1956), Walker (1960, 1961) and Larsson (1962) made histo-
Maternal hypervitaminosis A
233
logical and histochemical studies of the palatine shelves of mouse embryos, and
found that prior to initiation of shelf movement the only components making up
the palatine shelves were a core of loose mesenchymal tissue covered by an epithelium. The mesenchymal cells were embedded in a ground substance which
was metachromatically stainable with toluidine blue, and it was suggested that
the shelf movement was initiated by a factor which probably resided in the acidmucopolysaccharides of this intercellular ground substance. Walker & Fraser
(1956) described the occurrence of osteogenesis in maxillary areas proximal to the
base of the shelves. However, in their material no projection of osteogenesis into
the palatine shelves was evident. In the present study of rats, however, osteoblastic and dense mesenchymal tissue was continuous with similarly differentiated tissue of the maxilla and was one of the first components to extend medially
into an outfolding which indicated the beginning of shelf movement from the
vertical to the horizontal position.
Also in the current study several purely morphological factors were encountered
in embryos from hypervitaminotic A rats which were suggestive of participation
in the development of cleft palate. In the first place it was seen that cleft palate
resulted when insufficient mesenchymal tissue outfolded from the maxillary
process into palatine shelf, resulting in the latter either being very small or entirely
absent. In most cases, however, the reduction in size of palatine shelves occurred
only posteriorly while in anterior regions the size was comparable to that in
normal embryos. In contrast cleft palate developed in some embryos in spite of
the apparent presence of sufficient palatal tissue, both anteriorly and posteriorly,
to bridge the gap across the roof of the oral cavity.
In all treated embryos the development of the maxillary bone primordium was
defective at the preosteoblastic stage of organization. Some of the preosteoblastic
cells apparently underwent heterotopic transformation into chondroblasts and
thence into chondrocytes surrounded by a typically metachromatic matrix. In
subsequent development this heterotopic cartilage progressively replaced the
maxillary bone until in 17-day-old embryos the maxilla was represented by
scattered bone trabeculae at the periphery of the heterotopic cartilage and also
some of the palatal mesenchymal tissue became entrapped into abnormal infoldings of oral epithelium. This maxillary cartilage was instrumental in producing maxillomandibular ankylosis. Such heterotopic cartilage was also observed in this region by Deuschle etal. (1959) in newborn rats from hypervitaminotic
A mothers.
Reduction in size of the maxillary bone due to the presence of maxillary cartilage appeared to participate in the production of cleft palate in at least two ways.
Firstly, the development of dental laminae for the upper molars which did not
encounter the maxillary bone became abortive; i.e., the two limbs of the lamina
diverged and thus separated the palatal tissue from the maxilla. Secondly, owing
to the absence of the maxillary bone from its normal position with respect to the
palate, due to its replacement by cartilage, the likelihood exists that normal pres-
234
D. M. KOCHHAR and E. M. J O H N S O N
sure and stress of the developing bone to aid the progression of the horizontal
palatine shelves towards the midline could be partially or completely absent in the
treated embryo. Both these factors could perhaps explain the persistence of widely
separated but horizontal palatine shelves.
Larsson (1962) related the production of cleft palate in embryos from cortisone
treated mice to the inhibition of acid-mucopolysaccharide synthesis within the
palatine shelves which, therefore, failed to develop enough force within themselves
to enable them to move from the vertical to the horizontal position. This conclusion was based on autoradiographic studies which revealed that incorporation
of S35 into palatine shelves and other areas of cleft palate embryos was depressed.
Larsson suggested that other agents like hypervitaminosis A which also possibly
reduced the amount of acid-mucopolysaccharides in tissues might be teratogenic
due to their possession of this biochemical property. Histochemical methods in
the present study, such as metachromatic staining with toluidine blue, revealed no
difference between control and vitamin A treated embryos in the intensity of metachromasia either in the mesenchymal tissues of the palate or in the various cartilaginous structures. Fixation of S35 by tissues of embryos from hypervitaminotic
A rats, however, needs special mention. In 15- and 16-day-old treated embryos,
whose mothers were treated with vitamin A on 9,10 and 11 days of gestation and
were injected with S35-sulfate 48 hrs. before autopsy, the mesenchymal tissue in the
abnormally shaped palatine shelves and in other areas revealed a considerably
higher S35-incorporation than seen in comparable areas of control embryo. The
same occurrence was true of nasal capsule cartilages, Meckel's cartilage and cartilage models in the limb. Heterotopic cartilage within maxilla also activelyfixedS35.
Investigation of fetal stages by Giroud, Gounelle & Martinet (1956, 1957)
demonstrated a slight increase in vitamin A concentration in rat fetuses after
treatment of their mothers by large doses of vitamin A. If such an increase in
vitamin A concentration were present in embryonic tissues in the present study
it might have caused increased incorporation of S35 into the embryo. Increased
rate of S35-uptake following vitamin A administration was also observed by
Dziewiatkowski (1954) in the skeleton and skin of rats which were previously on a
vitamin A deficient diet. He also demonstrated that incorporation of S35 as
sulfate groups had occurred into sulfomucopolysaccharides. Wolf, Varandani &
Johnson (1961) demonstrated that the addition of vitamin A to a homogenate of
colon from vitamin A deficient rats resulted in normal levels of incorporation of
S35-radioactivity into mucopolysaccharides. The present report is the only
known study which examines the in vivo effects of maternal vitamin A treatment on
fixation of S35 by the embryo. Results obtained from studies of tissue culture
however (Fell, Mellanby & Pelc, 1956) demonstrated that excess vitamin A
caused dissolution of embryonic cartilage matrix as evidenced by removal of
previously incorporated S35. These results were substantiated by other studies on
the effects of excess vitamin A on adult cartilage and bone, both in vivo and in vitro
(Thomas et ah, 1960 and Fell & Thomas, 1960). In addition to dissolving the
Maternal hypervitaminosis A
235
cartilage matrix, hypervitaminosis A in intact animals also was reported to impair
the ability of chondrocytes to synthesize chondroitin sulfate in tissue culture
(McElligott, 1962).
Dissolution of cartilage matrix as observed by Thomas et ah (1960) appeared to
result only when the animals were given very large amounts of vitamin A.
Thomas et al. (1960) and McElligott (1962) administered 1 million i.u. of vitamin
A daily to rabbits for 5 to 7 days and brought about dissolution of cartilage matrix.
The dose level of vitamin A employed for teratogenesis in the present study was
not sufficiently high to dissolve the matrix of either maternal cartilage (Kochhar,
1964) or the cartilage of 15- and 16-day fetuses. It was noticed, however, that in
the epiphyseal cartilage of adult rats receiving teratogenic doses of vitamin A
(60,000 i.u. per day for 3 days) the matrix showed no reduction of metachromatic
staining by toluidine blue from that present in rats receiving only cottonseed oil.
Depletion in cartilage matrix of its metachromatically stainable component
was, however, observed when the dosage of vitamin A was increased to 100,000 i.u.
or 200,000 i.u. daily for three days. Dissolution of intercellular matrix may be
responsible for the detection of decreased S35-incorporation observed in the tissues
of day seventeen treated embryos.
It is clear that the effects of hypervitaminosis A on acid-mucopolysaccharide
content of maternal and embryonic organisms are very much different from those
of cortisone, and since morphogenesis of cleft palate with these two agents is also
dissimilar, it may be that two different pathways for the production of cleft palate
are involved.
SUMMARY
1. Oral administration of 60,000 international units of vitamin A acetate to
pregnant rats for 3 consecutive days beginning on either day 9 or 10 of pregnancy
resulted in more than 80 per cent, of their embryos developing cleft palate.
2. The initiation of palatine shelf movement in normal control embryos was
associated with an aggregation of preosteoblastic tissue on the medial aspect of
the vertically oriented shelves at the junction of their anterior two-thirds and
posterior one-third.
3. Delay in the movement of palatine shelves from the vertical to the horizontal
position was not observed in a major percentage of the vitamin A treated embryos.
4. In many vitamin A treated embryos a considerably lesser amount of mesenchymal tissue was contributed by the maxillary processes to form the palatine
shelves.
5. A process of heterotopic chondrogenesis was detected within the preosteoblastic tissues of maxillae and palatine shelves as early as day 15 and by day 17
had replaced a major portion of the maxillary bone.
6. On the basis of the methods employed, more S35 was fixed by the tissues of
treated embryos than by those of normal controls prior to the time of palatal
closure.
236
D. M. KOCHHAR and
E. M. J O H N S O N
RESUME
Recherches morphologiques et autoradiographiques sur la fissuration palatine
induite chez des embryons de rat par hypervitaminose A maternalle.
1. Chez la ratte gestante, des doses de 60.000 U.I. d'acetate de Vitamine A,
administrees par voie orale pendant trois jours, a partir du neuvieme ou du
dixieme jour de la gestation, ont provoque des fissures palatines chez plus de
80 per cent, des embryons.
2. Chez les embryons temoins le mouvement initial du processus palatin a ete
accompagne d'une accumulation de tissu preosteoblastique a la face mediane de
la paroi laterale, au niveau de la jonction des deux tiers anterieurs et du tiers
posterieur.
3. Aucun retard du mouvement des processus palatins n'a ete observe chez
la plupart des embryons soumis au traitement avec vitamine A.
4. Chez un grand nombre des embryons traites, la contribution en mesenchyme
de la part du bourgeon maxillaire au processus palatin a ete considerablement
reduite.
5. Un phenomene de chondrogenese heterotipique dans le tissu preosteoblastique des maxillaires et des processus palatins a ete decele a partir du quin-
zieme jour et cette chondrogenese avait remplace i'os maxillaire en grande partie
lors du dix-septieme jour.
6. D'apres les methodes employes, les tissus des embryons soumis au traitement ont incorpore plus de S35 que ceux des temoins normaux avant le moment ou
intervient la soudure palatine.
ACKNOWLEDGEMENTS
The authors are grateful to Professors James G. Wilson and Donald C. Goodman for their
constructive criticism of the manuscript. This investigation was supported by Public Health
Service Training Grant 3T1 GM 579-0451 and by Research Grant HD 00109.
REFERENCES
ASLING, C. W., NELSON, M. M., DOUGHERTY, H. L., WRIGHT, H. V. & EVANS, H. M. (1960).
The development of cleft palate resulting from maternal pteroylglutamic (folic) acid
deficiency during the latter half of gestation in rats. Surgery Gynec. Obstet. EH, 19-28.
BAXTER, H. &FRASER, F. C. (1950). Production of congenital defects in offspring of female mice
treated with cortisone. McGill Med. J. 19, 245-9.
BRACHET, J. (1942). La localisation des acides pentosenucleiques dans les tissus animaux et
les ceufs d'Amphibiens en voie de developpement. Arch. Bioi, Paris et Liege, 53,207-57.
BURSTON, W. R. (1959). The development of cleft lip and palate. Ann. R. Coll. Surg. 25,225-33.
CALLAS, G. & WALKER, B. E. (1963). Palate morphogenesis in mouse embryos after X-irradiation. Anat. Rec. 145, 61-71.
DEUSCHLE, F. M., GEIGER, J. F. & WARKANY, J. (1959). Analysis of an anomalous oculodentofacial pattern in newborn rats produced by maternal hypervitaminosis A. J.A.Res. 38,
149-55.
DZIEWIATKOWSKI, D. D. (1954). Vitamin A and endochondral ossification in the rat as indicated by the use of sulfur35 and phosphorus32. /. exp. Med. 100,11-24.
Maternal hypervitaminosis A
237
H. B., MELLANBY, E. & PELC, S. R. (1956). Influence of excess vitamin A on the sulfate
metabolism of bone rudiments grown in vitro. J. Physiol., Lond. 134,179-88.
FELL, H. B. & THOMAS, L. (1960). Comparison of the effects of papain and vitamin A on
cartilage. II. The effects on organ cultures of embryonic skeletal tissue. /. exp. Med. Ill,
719-44.
FRASER, F. C. (1955). Thoughts on the etiology of clefts of the palate and lip. Acta genet.
Statist, med. 5, 358-69.
FRASER, F. C. (1960). Some experimental and clinical studies on the causes of congenital clefts
of the palate and of the lip. Archs. Pediat. 11,151-6.
GIROUD, A., GOUNELLE, H. & MARTINET, M. (1956). Concentrationde la vitamine A chez
la mere et le foetus au cours de la teratogenese par hypervitaminose A. C. r. Seanc. Soc.
Biol. 150, 2064-65.
GIROUD, A., GOUNELLE, H. & MARTINET, M. (1957). Donnees quantitatives sur le taux de la
vitamine A chez le rat lors d'experiences de teratogenese par hypervitaminose A. Bull.
Soc. Chim. biol. 39, 331-6.
GIROUD, A. & MARTINET, M. (1956). Teratogenese par hautes doses de vitamine A en fonction
des stades du developpement. Archs. Anat. Microsc. 45, 77-98.
KALTER, H. & WARKANY, J. (1959). Experimental production of congenital malformations in
mammals by metabolic procedure. Physiol. Rev. 39, 69-115.
KAMEI, T. (1962). Embryological and histochemical studies on the artificially induced cleft
palate in mice. Acta anat. nippon. 37, 140-58.
KOCHHAR, D. M. (1964). Effects of maternal hypervitaminosis A on cleft palate formation in
rat embryos. Ph.D. thesis. Gainesville: University of Florida.
KRAMER, H. & WINDRUM, G. M. (1955). The metachromatic staining reaction. /. Histochem.
Cytochem. 3, 227-37.
LARSSON, K. S. (1960). Studies on the closure of the secondary palate. II. Occurrence of
sulpho-mucopolysaccharides in the palatine processes of the normal mouse embryo.
Expl. Cell Res. 21, 498-503.
LARSSON, K. S. (1962o). Studies on the closure of the secondary palate. III. Autoradiographic
and histochemical studies in the normal mouse embryo. Acta morph. neerl.-scand. 4,
349-67.
LARSSON, K. S. (19626). Studies on the closure of the secondary palate. IV. Autoradiographic
and histochemical studies of mouse embryos from cortisone-treated mothers. Acta
morph. neerl.-scand. 4, 369-86.
LARSSON, K. S. (1962C). Closure of the secondary palate and its relation to sulpho-mucopolysaccharides. Acta odont. scand. 20, 1-35.
LAYTON, L. L. (1951a). Cortisone inhibition of mucopolysaccharide synthesis in the intact
rat. Archs. Biochem. 32, 224-6.
LAYTON, L. L. (19516). Effect of cortisone upon chondroitin sulfate synthesis by animal
tissues. Proc. Soc. exp. Biol. Med. 76, 596-8.
MCELLIGOTT, T. F. (1962). Decreasedfixationof sulfate by chondrocytes in hypervitaminosis
A. /. Path. Bact. 83, 347-55.
MCMANUS, J. F. A. (1948). Histological and histochemical uses of periodic acid. Stain Tech.
23, 99-108.
MESSIER, B. & LEBLOND, C. P. (1957). Preparation of coated radioautographs by dipping
sections in fluid emulsion. Proc. Soc. exp. Biol. Med. 96, 7-10.
PEARSE, A. G. E. (1960). Histochemistry, theoretical and applied. Boston: Little, Brown and
Co.
SCHILLER, S. & DORFMAN, A. (1957). The metabolism of mucopolysaccharides in animals:
the effect of cortisone and hydrocortisone on rat skin. Endocrinology, 60, 376-81.
THOMAS, L., MCCLUSKEY, R. T., POTTER, J. L. & WEISSMANN, G. (1960). Comparison of the
effects of papain and vitamin A on cartilage. I. The effects in rabbits. /. exp. Med. I l l ,
705-18.
TRASLER, D. G., WALKER, B. E. & FRASER, F. C. (1956). Congenital malformations produced
by ammiotic-sac puncture. Science, 12A, 439.
WALKER, B. E. (1960). A special component of embryonic mesenchyme. Anat. Rec. 136,298.
FELL,
238
D. M. KOCHHAR and E. M. JOHNSON
B. E. (1961). The association of mucopolysaccharides with morphogenesis of the
palate and other structures in mouse embryos. /. Embryol. exp. Morph. 9, 22-31.
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. & GRAIN, B. (1961). Abnormal palate morphogenesis in mouse embryos induced by riboflavin deficiency. Proc. Soc. exp. Biol. Med. 107, 404-6.
WALKER, B. E. & FRASER, F. C. (1956). Closure of the secondary palate in three strains of
mice. /. Embryol. exp. Morph. 4,176-89.
WALKER, B. E. & FRASER, F. C. (1957). The embryology of cortisone-induced cleft palate.
/. Embryol. exp. Morph. 5, 201-9.
Wrrscffl, E. (1956). The standard stages of rat development (based on the Iowa collection)
figures in: Development of Vertebrates. Philadelphia: W. B. Saunders Company.
WOLF, G., VARANDANI, P. T. & JOHNSON, B. C. (1961). Vitamin A and mucopolysaccharide
synthesizing enzymes. Biochim. biophys. Acta 46, 59-67.
WALKER,
(Manuscript received 15th May 1965)