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Factors influencing fusion of rat palates grown in vitro.

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Factors Influencing
- Fusion of R a t Palates
Grown in vitro
GORDON S. MYERS? NICHOLAS L. PETRAKIS A N D MELVIN LEES
Graduate Group in Nutrition, Department of Epidemiology and
International Health, University o f California Medical Center,
Sun Francisco, California
ABSTRACT
A technique is described for growth in vitro of embryonic rat palatal
tissue on defined and semi-defined media. Palatal fusion closely resembling that
occurring i n vivo is found in these preparations. Fusion occurs in a large number of
cases even in the absence of an animal extract. However, development is improved if
fetal calf serum is added to the synthetic medium. Using Leibovitz Medium G 1 5 with
20% fetal calf serum, complete fusion was obtained in 87% of the palates, partial
fusion in 9% and no fusion was found in only 4% of the palates cultured.
Incorporation of galactoflavin, a riboflavin antagonist in a riboflavin-deficient diet of
the mother or i n the culture medium results in a decreased number of complete
fusions and corresponding increase in partial fusions. A suggestion is made as to the
manner in which galactoflavin acts i n vivo to produce palatal cleft, based on the incidence and type of clefts and the histological appearance of the palatal tissues.
A method is described in which the tissue sections are sliced prior to culture so that
the behavior of individual palatal shelves can be studied during the fusion process.
Fusion occurs between shelves if the medial margins are apposed, regardless of
whether the two shelves come from the same embryo, from littermate embryos, or
from non-littermate embryos. Some possibilities for application of this technique are
suggested.
The part that nutritional factors play
in normal development of the mammalian
embryo is both basic and complex. Studies
of the effects of nutrients on development
have often made use of agents which are
metabolic inhibitors or nutrient antagonists and which thereby alter the nutritional state of the embryo, (Nelson et al.,
'55, '56; Chamberlain et al., '63). Maternal
dietary deficiencies of riboflavin (Deuschle
et al., 'Sl), folic acid (Asling et al., 5 5 ) ,
niacin (Chamberlain and Nelson, '63), or
vitamin A (Kalter and Warkany, '59), as
well as hypervitaminosis A (Mauer, '64),
have been shown to interfere with normal
palatal fusion as demonstrated by the occurrence of palatal clefts in the young of
rats subjected to these treatments. However, many other factors may produce the
same anomaly. Because the cleft is only
the end-result of an altered morphogenetic
process, little knowledge has been gained
concerning the cell and tissue changes responsible for the occurrence of the defect.
The use of in vitro techniques employing
synthetic or semi-synthetic media may
permit study of the action of specific nutrients on the palatal tissues and show the
ANAT.REC., 162: 71-82.
tissue changes which result in palatal
cleft. Moriarty, Weinstein and Gibson
('63) have reported that the palatal structures of the embryonic rat can be grown in
culture, during the period of normal palatal
fusion, on a plasma clot containing chick
embryo extract. Pourtois ('66) has also reported a procedure for culturing isolated
palatal shelves in vitro. Palatal fusion
closely resembling that seen in viuo was
demonstrated in both instances.
This investigation was undertaken to devise a modification of these methods which
would circumvent the use of the plasma
clot and permit a more precise control of
the nutrients available to the tissues as well
as to facilitate manipulation of the tissue
fragments during the culture period. At the
same time, a high percentage of palatal
fusions under normal conditions of culture
was desirable. We shall describe some of
the conditions found necessary for suc1This research was supported in part by the National Institute of Health Grant DE-20 and by a grant
from the Research Cdmmittee of the. Academic
Senate. Universitv of California. San Francisco.
2 Present address: Faculty of Dentistry, University
of British Columbia Vancouver, British Columbia.
3 Present address: School of Home Economics, University of British Columbia, Vancouver, British
Columbia.
71
72
GORDON S . MYERS, NICHOLAS L. PETRAKIS A N D MELVIN LEE
cessful fusion of the palatal shelves and tioned on the canvas with the mandible
present the results of studies on the effects upward. Iridectomy scissors were used to
of galactoflavin ( a riboflavin antagonist) cut through the ramus of the mandible on
on fusion. We shall also report on experi- either side, allowing the mandible and
ments which indicate that fusion can occur tongue to be removed without disturbing the
between individual palatal shelves from palatal shelves. Further trimming was done
both littermate and non-littermate rat with cataract knives until a section approxiembryos, which may have a bearing on the mately 4 mm wide, 5 mm long and 1 mm
role of genetic constitution in palatal thick was obtained, which consisted of
fusion.
maxillary tissue, palatine shelves and portions of the nasal septum and capsule. The
MATERIALS AND METHODS
two shelves at this time were usually separated by a space of 0.25-0.50 mm although
Experimental animals
Female Sprague-Dawley rats, 100-150 connected anteriorly and posteriorly by the
days of age, were housed two or three to a tissues of the lip and pharynx. The secage and given stock rat pellets and water quence of steps and final preparation are
ad libitum. A timed-pregnancy method was shown in figure 1.
The procedure involves minimal maused with the day of finding sperm in the
nipulation
and disturbance of the palatine
vaginal smear designated as day zero of
pregnancy. With this timing procedure, processes and permits optimum alignment
palatal fusion normally takes place dur- of the shelves during culture.
ing the sixteenth gestational day in this
In vitro culture
strain of rat.
Culture chambers were prepared by placThe pregnant rats were caged individually or in pairs, given water and either ing stainless steel gauze in the form of a
stock diet or an experimental diet ad low platform, in each section of a compartmented disposable Petri dish. A square ( 2
libitum, and weighed twice weekly.
On day 14 8 hours or day 15 8 hours cm x 2 cm) of ethanol-washed rayon acethe female was lightly anesthetized with tate was placed on the wire platform. Culether and killed by cervical dislocation. The ture medium was introduced beneath the
abdomen was swabbed with an alcohol platform only until the rayon acetate square
sponge and the peritoneal cavity opened was wetted. The dissected tissue section
with sterile instruments. The uterus was was lifted from the microscope stage with
removed and rinsed in cold sterile Tyrode’s a small spatula, rinsed in Tyrode’s solution,
solution. The embryos were removed indi- and placed on the rayon acetate square
vidually from the uterus and embryonic with the oral surface upward. In some exmembranes and also rinsed in Tyrode’s so- periments, the rayon acetate was replaced
lution. Strict sterility was maintained in with a Millipore filter6 with no adverse
effect.
these and all subsequent procedures.
The cultures were incubated at 37°C for
72 hours, with partial or complete removal
Palate dissections
and replacement of the medium after 24
The stage of a dissecting microscope was hours. At the end of the incubation period,
covered with a piece of sterile canvas kept
tissue was carefully detached and lifted
moist with Tyrode’s solution. The embryo the
from
the rayon acetate square and placed
was placed on the canvas and an initial
in
Bouin’s
fixative for 24 hours. Following
slice made from the tip of the snout
the section was embedded in parthrough the region of the eye, to the occiput, fixation,
affin, cut serially in a transverse plane at
using a pair of mounted cataract knives
(Beaver-small).6 A parallel slice was made 10 p and mounted sections stained with
through the neck, severing the remainder hematoxylin and eosin.
of the head from the body. The resulting
4 Foodstuff Processing Co., San Francisco, Calitissue section, consisting of the maxillary fornia.
5 Rudolph Beaver, Belmont Massachusetts.
vault and associated structures plus the at6 0.45 B Type SM, 25 mm.,’Millipore Filter Co., Bedtached mandible and tongue, was posi- ford,
Massachusetts.
+
+
73
FACTORS INFLUENCING PALATAL FUSION
Fig. 1 Diagrammatic representation of technique used in obtaining embryonic palatal
tissue.
( a ) Head of embryo illustrating first and second cuts, as described in text.
( b ) Oral view of section as cultured, showing morphology of palatal shelves and surrounding tissues.
Culture media
Two synthetic media were tested: Waymouths Medium MB 752/17 (Waymouth,
'59) which contains a bicarbonate buffer
and was used with a 5% C02-95% compressed air gas phase for maintenance of
pH, and Leibovitz Medium L-15 (Leibovitz, '63), buffered with phosphate and
used in free gas exchange with the atmosphere. Both media contain inorganic salts,
amino acids and water soluble vitamins,
including riboflavin, but neither contain
fatty acids, fat soluble vitamins, or purine
or pyrimidine components. The vitamin
antagonist, galactoflavin? was used only
in conjunction with L-15 medium containing 20% fetal calf serum. Leibovitz medium L-15 normally contains 0.1 pg/ml
riboflavin. Galactoflavin was added to L-15
to produce concentrations of 1, 2 or 3 pg/
ml, an amount believed sufficient to antagonize the riboflavin present in the defined medium as well as the unknown concentration in the fetal calf serum.
galactoflavin was included in the culture
medium as previously noted. Control diets
contained equivalent quantities of riboflavin in place of galactoflavin. The basic
diet was composed of (in gm per 100 g m ) :
vitamin-free casein, 20; powdered sucrose,
43; corn starch, 23; peanut oil, 8; OsborneMendel salt mix, 3; cod liver oil, 2; vitamin
mix (without riboflavin), 1.
The vitamin mix contained (Gms./Kg.) :
choline dihydrogen citrate, 150; inositol,
10; thiamine mononitrate, 0.5; pyridoxine
hydrochloride, 0.6; calcium pantothenate,
0.6; niacinamide, 2; folic acid, 0.2; biotin
( 1% triturate), 1; para-aminobenzoic acid,
0.6; vitamin E (33% triturate), 0.5; menadione, 0.5; vitamin BIZ (0.1% triturate),
20; Mannitol, 813.6.
Experimental diets
For studies on the effect of galactoflavin,
either a powdered riboflavin-deficient diet
containing 55, 100 or 250 mg galactoflavin
per kilogram diet was offered to the pregnant rat from the first day of gestation or
7 Waymouth's Medium was obtained from Hyland
Laboratories Los Angeles California.
8 Leibovitz' L-15 was obtained from Microbiological
Associates. Washington. D. C. L-15 contains 0.1 fig
Shelf repositioning experiments
In order to examine the competency of
the palatal shelves to fuse independently
of.other aspects of palatal development, a
procedure was devised whereby the individual palatal shelves could be repositioned in-
riboflavinjml.
'
9 Galactoflavin was obtained from Microbiological
Associates Inc Bethesda, Maryland, through the
courtesy & the %ancer Chemotherapy National Service
Center, N.I.H., Department of Health, Education and
Welfare, Bethesda, Maryland.
74
GORDON S. MYERS, NICHOLAS L. PETRAKIS AND MELVIN LEE
dependently of each other or recombined
with shelves from other embryos.
Preliminary dissection of the palatal section was carried out as previously described.
The section was then placed, oral surface
upward, on a Millipore filter. With paired
cataract knives, an initial cut was made
through the lip, continued posteriorly between the separated palatal shelves and
carried through the pharyngeal tissues to
the posterior margin of the section (fig. 2).
D i r e c t i o n af
Slice
! i
I
f
I
I
I
I
I
Fig. 2 Diagrammatic representation of slicing
technique used to separate palatal shelves in
shelf-repositioningexperiments.
Extreme care was taken to avoid touching the shelves at any time. In the process,
the filter was also cut in two parts, each
with one-half of the original section. The
halves were moved apart slightly to assure
complete separation, then placed on the
stainless steel platform and positioned so
that the medial borders of the two palatal
shelves appeared to contact. This procedure
permitted culturing together shelves from
the same embryo or shelves from littermate or non-Iittermate embryos. Subsequent handling and examination of these
sections was by the routine used for control
sections.
RESULTS
Figure 3 represents a transverse section
of the palatal region from a 14 + day old
embryo, immediately following dissection.
At this stage the palatine processes were
well formed and consisted of a shelf of
mesenchymal cells and ground substance,
covered by a thin layer of columnar epithelium. Because the preparations are cultured with the oral surface upward, the
shelves tend to assume a horizontal position soon after dissection regardless of the
age of the embryo, Figures 4a and 4b show
a corresponding preparation after growth
for 72 hours on L-15 medium with 20%
fetal calf serum. The shelves had contacted
and fused in the midline, the epithelial
covering in the area of contact had disappeared and there was confluence of the
mesenchymal cells across the midline. The
fusion line was, in some cases, marked by
a slight depression of the epithelium or a
midline groove on the oral surface. Persistence of a laminated epithelial layer in
the midline was rarely found.
The number of fused, partially fused, or
non-fused palates after culture for 72 hours
on Waymouth's Medium or on Leibovitz
Medium L-15 is shown (table 1 ) . Complete
fusion was defined as fusion over more
than two-thirds of the length of the palatai
suture and partial fusion as fusion of onethird to two-thirds of the length of the
suture. Fusion of one or the other type was
obtained in a large number (88% ) of the
palate sections grown on MB752/1 containing no fetal calf serum. In preliminary
studies, M e r e n t concentrations of fetal
calf serum were used with L-15 to determine an optimum concentration for palatal
fusion. The percentage of complete fusions
increased as the concentration of fetal calf
serum was raised from 5% to 20%. Of a
large series of cultures (89) grown on L-15
with 20% fetal calf serum, 96% showed
partial or compIete fusion of the palatal
shelves and this concentration was used for
all subsequent cultures. Although fusion
was obtained with Waymouths Medium
without calf serum and with Leibovitz Medium with 5 and 10% calf serum, the tissues appeared less vital and were smaller
in size after a 72-hour culture period when
compared with sections grown on L-15 with
20% fetal calf serum.
In experiments involving palatal slices
and repositioning of the shelves, fusion was
found in nine of a total of 1 4 palates (64% )
75
FACTORS INFLUENCING PALATAL FUSION
Fig. 3 Transverse section through embryonic rat palate upon removal from 14 + day
embryo. Palatal shelves (PS) and nasal septum (NS) Hematoxylin and eosin. x 20.
TABLE I
Fusion of rut embryo palates cultured on synthetic media with and without
added fetal calf serum 1
Medium
Waymouth’s MB 75211 2
Leibovitz L-15
1
2
3
% Fetal Number of
calf serum palates
Partially
fused
Not
fused
0
26
19 (73% )
4 (15% )
3 (12%)
5
10
20
6
5
89
4 (66% )
4 (80%)
77 (87% )
2 (34% )
1 (20% )
8( 9%)
4 ( 4%)
+
14 + and 15
day palates cultured for 72 hours at 37°C.
Cultured in 5% C02-95% Air.
1% glutamine added.
grown on L-15 with 20% fetal calf serum.
The cutting process usually resulted in a
loss of the nasal septum, making proper
alignment of the shelves for the full length
of the fusion line difficult. In those cultures
in which the medial margin of one shelf
was in contact with an area other than the
medial margin of the other shelf, fusion
did not occur.
When a section containing a palatal shelf
from one embryo was combined with a
similar section from a littermate embryo,
the medial margins fused in a manner
grossly and histologically indistinguishable
from fusion of shelves from the same embryo. Fusion was also found between sections of this kind taken from non-littermate
embryos. A combination of non-littermate
sections is shown in figures 5a and 5b as i t
appeared following a culture period of 72
hours on L-15 medium with 20% fetal calf
serum.
76
GORDON S. MYERS, NICHOLAS L. PETRAKIS AND MELVIN LEE
+
Fig. 4a Transverse section through 14
day embryonic rat palate following growth
in vitro for 72 hours. The fusion line ( F ) is denoted. Hematoxylin and eosin. X 15.
Fig. 4b
Higher magnification of the fusion line shown in figure 4a.
X
100.
77
FACTORS INFLUENCING PALATAL FUSION
+
Fig. 5a Transverse section of 14 day embryonic rat palate consisting of palatal shelves
from two separate non-littermate embryos grown in vitro for 72 hours. Hematoxylin and
eosin. X 10.
Fig. 5b Higher magnification of the fusion line shown in figure 5a.
X
40.
78
GORDON S. MYERS, NICHOLAS L. PETRAKIS AND MELVIN LEE
amounts of galactoflavin in the maternal
diet resulted in a corresponding increase in
the percentage of non-fusions, this relationship did not hold true when galactoflavin
was added to the culture medium.
If palates from mothers fed galactoflavin
(100 mg/Kg of diet) were grown on L-15
medium containing 20% fetal calf serum
Table 2 presents the results obtained
when galactoflavin was included in the maternal diet or when it was incorporated in
the culture medium. In both cases, there
was a decrease in the percentage of complete fusions and a n increase in percentage
of partial fusions or non-fusions when compared with controls. Although increasing
TABLE 2
E f f e c t of galactoflavin on in vitro palatal fusion in Sprague-Dawley rat embryos
Galactoflavin
1
No. palates
cultured
Fused
Partially
fused
Not
fused
42
31
14
32 (76%)
19 (61% )
7 (50% )
9 (21% )
9 (29% )
3 (22% )
1( 3%)
3 (10%)
4 (28% )
11
13
6
6 (55% )
5 (45%)
3 (23% )
2 (33%)
0
9 (69% )
4 (67% )
1( 8%)
0
9
7 (78%)
1 (11% )
l(ll%)
14
13 (93% )
1 ( 7%)
55 mg/kg of diet
100 mg/kg of diet
250 mg/kg of diet
1 pg/ml medium
2 pg/ml medium
3 p g / m l medium
100 mg/kg of diet
+
riboflavin 4 mg/ml in medium
None (control)
1 Cultured
o n Leibovitz Medium L-15 with 20% fetal calf serum for 72 hours at 37°C.
+
day embryonic rat palate grown i n vitro for
Fig. 6 Transverse section through 14
72 hours on medium containing 1 p g/ml galactoflavin. Note persistence of epithelial lamina
in the fusion line. Hematoxylin and eosin. X 100.
FACTORS INFLUENCING PALATAL FUSION
and supplemented with riboflavin, the percentage of complete fusions increased
slightly.
Figure 6 shows a transverse section
through a palate cultured on standard medium with 1 g/ml galactoflavin added.
No generalized histological changes were
evident. The shelves and surrounding tissues appeared well developed, with normal
tissue organization. The principal difference between galactoflavin-treated and
non-treated palates was a persistence of
the epithelial layers in the fusion line of the
treated palates in many instances. Single
and double strands of epithelial cells were
found as well as epithelial remnants.
DISCUSSION
In vitro procedures offer several advantages for study of the effect of environmental factors on developmental processes.
Direct observation is possible during the
process of shelf fusion. Nutrients are directly available to the tissues and are not
subject to maternal alteration or rejection.
The quantity and quality of the available
nutrient factors can be controlled to some
extent. Most important, developmental
changes in the whole embryo do not influence the course of development of particular tissues or organs in vitro. In the case of
palatal fusion, neither the position of the
tongue at the time of closure and fusion of
the shelves nor continued growth in width
of the head need be considered to influence
the fusion process during culture. Therefore one may distinguish between physical
factors which prevent or disrupt seIf apposition and factors inherent in the tissues of
the shelves which inhibit fusion. Finally,
manipulation of the tissue sections is possible in vitro which cannot be carried out in
the whole organism.
On the other hand, no in vitro procedure
can completely reproduce conditions occurring in viva Tissue metabolism is altered by the new environment as well as by
the trauma of dissection. The anatomical
relationships of organs are disrupted so the
influence of morphogenetic movements and
inductive processes may be greatly decreased. In the culture of palatal tissues,
disruption of this kind is produced by shelf
displacement, movement or loss of the
nasal septum, and growth in an inverted
79
position. Swelling of tissues in culture is a
constant finding, and its effects are not subject to interpretation. Increased hydrostatic
pressure in the shelves may affect shelf apposition and must be considered as a factor
in palatal fusion in vitro.
The experiments reported here indicate
that the dissection techniques employed
and the use of a liquid defined or semidefined medium will produce tissues which
are viable and capable of a pattern of development similar to that found in vivo.
Palatal fusion in vitro was obtained in
many cases which was similar, histologically, to that seen in sections from embryos
of the same developmental stage fixed immediately after removal from the uterus.
Cell size and morphology appeared normal
although intercellular spaces were more
evident due to edema of the tissues. Mitotic figures were commonly seen in sections from palates after they had been
grown in culture for 72 hours.
Grooving or lack of fusion was often
found in the anterior third of the fusion
line in control palates. This resulted in
short partial clefts in some preparations.
The cause is not known but may be due to
the fact that this area is in the center of
the tissue section and therefore the least
well nourished. Unphysiological pH might
also occur in this region. These conditions
may not permit completion of the fusion
process during the period of culture.
The studies involving various concentrations of fetal calf serum in the medium indicate that at least 10% serum is required
for optimum growth and development. Mesenchymal fusion was obtained with as little
as 5% calf serum but palatal development
was for the most part unsatisfactory. It
should be noted, however, that a high percentage of fusions was obtained with the
completely defined medium, Waymouths
MB 752/1, although the extent and type of
fusion was not as useful for this study as
that found with L-15 plus 20% fetal calf
serum. The findings are in agreement with
other studies indicating that large tissue
sections cannot be successfully cultured
unless an animal component such as
plasma, embryo extract, or serum is added
to the defined medium.
It has been recognized for many years
that congenital defects of the skeletal sys-
80
GORDON S. MYERS, NICHOLAS L. PETRAKIS AND MELVIN LEE
tem can be produced experimentally by
dietary riboflavin deficiency (Warkany and
Nelson, '40). Soft tissue anomalies in addition to skeletal defects have been produced
by riboff avin-deficient diets to which has
been added the riboflavin antagonist, galactoflavin (Nelson et al., '56). The tissue sections used in the present experiments were
at a developmental stage in which the only
cartilage present occurred in the nasal capsule and septum and therefore the defects
in palatal development represent anomalies
of soft tissue development.
The levels of galactoflavin used by other
workers (Nelson et al., '56) to produce
clefts in the Long-Evans rat did not cause
clefts in 18-day-old Sprague-Dawley embryos and higher levels were consequently
used. A strain difference in degree of susceptibility to the antagonist is indicated by
this finding. Similar strain differences in
response to galactoflavin have been reported
for mice (Kalter and Warkany, '57).
The increase in the incidence of partial
fusions may be significant in view of the
increase also noted in the number of fusions
of the epithelial type. The epithelial fusions
were often incomplete and resulted in deep
grooves which occasionally opened into the
nasopharynx to produce partial clefts. The
incidence of partial clefts and the observed
epithelial fusions suggested that galactoflavin may act in vitro by interfering with
the final stage of palatal fusion; i.e., merging of the mesenchyme. This could involve
a delayed breakdown of the epithelial
lamina which prevents merging of the
mesenchymal celIs during the culture
period. If this were the case, the primary
effect of galactoflavin might be on the
lysosomal or macrophagic processes responsible for epithelial degeneration and
removal. On the other hand, the effect may
be an inhibition of the mesenchymal cells
themselves so that the influence of these
cells on the epithelial lamina is lost. If persistence of the epithelial lamina occurred
in uivo, it seems possible that continued
growth in width of the head could exert
enough tension on the weakened fusion line
that it would rupture and result in palatal
cleft.
It is widely recognized that shelf position
at the normal time of palatal fusion is of
great importance in the production of clefts
of the secondary palate. If the shelves remain in a vertical or semi-vertical position
on either side of the tongue past a certain
developmental stage, growth of the other
cephalic structures wiU make contact (and
therefore fusion) of the shelf borders in the
midline impossible and result in palatal
cleft. At the same time it is suggested that
factors which interfere with the fusion
process itself may produce cleft palate even
though the shelves are in a horizontal position at the proper time and perhaps with
medial borders which contact each other.
The cleft thus produced may be indistinguishable in the newborn animal from one
produced by improper shelf position. The
experiments involving slicing and repositioning of the individual palatal shelves
suggest that the medial borders of the
shelves may have a competency to fuse
which is not present in other surfaces of
the shelves. Other epithelial surfaces of the
palate sections which were forced into close
contact due to conditions of the in vitro
technique (e.g., embryonic lip and future
alveolar ridge) did not fuse even though
the conditions of culture resulted in palatal
fusion. If the medial border of one shelf
was grown in contact with the oral or nasal
surface rather than the medial edge of the
opposing shelf, fusion did not occur. Other
in vitro investigations (Myers et al., '67)
suggest that at least one teratological agent
(6-aminonicotinamide,a niacin antagonist)
can specifically affect the competency of
the shelves to fuse since fusion fails to
occur in the presence of this antimetabolite
even though the medial borders are in close
apposition during the culture period. The
results with galactoflavin indicate that definitive fusion can be inhibited even though
fusion of the surface layers does occur.
Whether the competency to fuse resides in
the surface epithelium or in the underlying
mesenchyme or is the expression of an interaction between the two has yet to be determined. In any case, these studies suggest that palatal cleft may be the result of
a localized alteration of the tissues involved
in fusion as well as more generalized
changes affecting the position or growth of
the shelves as a whole.
The slicing procedure offers a means of
studying the effects of differential treatment of the palatal tissues in that one shelf
FACTORS INFLUENCING PALATAL FUSION
81
may be treated with nutrient antagonists, Deuschle, F. M., E. Takacs and J. Warkany 1961
Postnatal dentofacial changes induced in rats
vitamin excesses, chelating agents, or other
by prenatal riboflavin deficiency. J. Dent. Res.,
substances while the other shelf remains
40: 366-377.
untreated. Vital staining of one shelf would Kalter, H., and J. Warkany 1957 Congenital
malformation in inbred s t r a i n s of mice induced
offer one approach to the problem of morby riboflavin-deficient galactoflavin containing
phogenetic movements of shelf elements
diets. J. Fxp. Zool., 136: 531-565.
during palatal fusion.
- 1959 Experimental production of congenital malformations in mammals by metaThe ability to obtain fusion between
bolic procedures. Physiol. Rev., 39: 69-115.
shelves of W e r e n t embryos offers a tool for Leibovitz,
A. 1963 The growth and maintethe study of genetic influences on palatal
nance of tissue-cell cultures in free gas exfusion. The effect of embryonic age on the
change with the atmosphere. Am. J. Hyg., 78:
173-180.
competency of the shelves to fuse may also
I. 1964 Vitamin A-induced congenital
be investigated by combining shelves from Mauer,
defects in hairless mice. Biol. Neonat., 6: 26.
embryos of various ages.
Moriarty, T. M., S. Weinstein and R. D. Gibson
By utilizing the in vitro technique with
1963 The development in vitro and in vivo
of fusion of the palatal processes of rat emdefined or semi-defined liquid media, varibryos. J. Embryol. Exp. Morph., 22: 605-619.
ous factors of an environmental and heredi- Myers,
G. S., N. L. Petrakis and M. Lee 1967
tary nature may be investigated and comEffect of 6-aminonicotinamide and of added
pared with respect to their influence on
vitamin A on fusion of embryonic rat palates
in vitro. J. Nutr., 93: 252i-262.
fusion of the palate.
ACKNOWLEDGMENTS
Grateful acknowledgment is made to
Miss Hannah Oto for technical assistance
and to Miss Charlott Havemann for preparation of the figures.
LITERATURE CITED
Asling, C. W., M. M. Nelson, H. V. Wright and
H. M. Evans 1955 Congenital skeletal abnormalities in fetal rats resulting from maternal pteroylglutamic acid deficiency during
gestation. Anat. Rec., 121: 775-789.
Chamberlain, J. G., and M. M. Nelson 1963
Multiple congenital abnormalities in the rat
resultine from acute maternal niacin deficiency
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